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Introduction: The routine incorporation of proteasome inhibitors, immunomodulatory drugs (IMIDs), and monoclonal antibody drugs (mAbs) in managing multiple myeloma (MM) has improved patients' prognosis. Unfortunately, triple refractory patients continue to do poorly. Immunotherapeutic approaches (mAbs, CAR-T cells) have shown the most promise in these patients. However, the efficacy of these therapies depends on a functional tumor immune microenvironment (iTME), which is extensively reshaped by existing MM therapies and disease evolution. Understanding how the iTME and malignant clone co-evolve over the disease course is crucial to optimize the use of immune-based treatments and understand their mechanisms of action. Methods: Newly diagnosed MM patients (NDMM), MM patients relapsing prior to anti-CD38 antibody exposure (RMM), and triple refractory MM patients (TRMM) were identified from the Mayo Clinic biospecimen database. We performed CyTOF (with a 37 marker lymphoid panel on CD138- bone marrow [BM] mononuclear cell samples) to assess the cellular iTME, Luminex (using BM plasma) to assess the humoral iTME (cytokines and chemokines) and RNAseq analysis (using BM CD138+ cells) to assess the malignant clone transcriptome. Astrolabe was used to identify immune cell clusters. Gene set enrichment analysis (GSEA) was used to identify gene pathways with differential expression across groups. JMP was used to perform unsupervised hierarchical clustering and descriptive statistics. An FDR corrected p value of Results: This study included 39 patients (13 NDMM, 11 RMM, and 15 TRMM). Samples were hierarchically clustered by cellular iTME, humoral iTME and malignant cell transcriptome, separately. The cellular iTME best recapitulated known disease biology and was used as the "anchoring" variable for subsequent analyses. Three distinct immune clusters were identified (figure 1): cluster 1, comprised mainly of NDMM and RMM patients; and clusters 2 and 3, comprised primarily of TRMM patients. In cluster 2, 4/5 patient samples were collected immediately after progression on daratumumab-IMID therapy, whereas all 7 TRMM patient samples in cluster 3 were collected at a median of 2.8 (range 0-17.2) months after last daratumumab exposure (table 1). Most naïve T cell subsets (including the TIGIT+ T5 and the C38/CD127+ subsets T1, T2, and T12) were highest in cluster 1 compared to clusters 2 and 3. Several unfavorable T cell subsets were elevated in Cluster 2: 1) CD161+ T6, T7 and T17 subsets, which are thought to be Th17 polarized cells that promote MM growth; 2) T7, T9 CCR5+ subsets, which are thought to represent tumor-specific exhausted (PD-1+) T cell subsets; 3) senescent subsets (T-10, T14, T16,T18, T19); and 4) TCR-gd subsets with features of senescence (KLR, CD57), exhaustion (PD-1, TIGIT) and Th17 polarization (CD161). There were no significant differences in cytokine and chemokine expression based on disease status (NDMM, RMM, or TRMM) or iTME (immune clusters 1, 2, or 3). GSEA was performed to compare clusters 2 and 3 (TRMM enriched) with cluster 1. The most up-regulated gene pathways in clusters 2 and 3 related to cell cycle targets of E2F transcription factors and MYC proliferation pathways. The most down-regulated gene pathways in clusters 2 and 3 were IFN-gamma, IFN-alpha and TNF-alpha response and signalling. Conclusion: Changes in the cellular iTME mirror the disease course better than changes in the malignant clone or humoral iTME. Patients with more refractory MM have a smaller pool of naïve T cells which express TIGIT or CD127 and could be "revived" with anti-TIGIT antibodies or IL-7 prior to manufacturing CART cells or administering BCMA-based therapies. Significant heterogeneity exists within the more refractory subgroups, with some patients having more dysfunctional T cell subsets which may be less responsive to immunotherapeutic approaches. The malignant transcriptome of samples in clusters 2 and 3 parallels the aggressive clinical course, with myc upregulation and downregulation of cytokine pathways that mediate an antitumor immune response. This study suggests that the MM iTME becomes more dysfunctional with therapy and may explain the differential sensitivity of patients to immunotherapeutic approaches. Disclosures Kumar: MedImmune: Research Funding; Janssen Oncology: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; BMS: Consultancy, Research Funding; Tenebio: Other, Research Funding; Genecentrix: Consultancy; Sanofi: Research Funding; Genentech/Roche: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Oncopeptides: Consultancy, Other: Independent Review Committee; IRC member; Adaptive Biotechnologies: Consultancy; Takeda: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Merck: Consultancy, Research Funding; AbbVie: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Dr. Reddy's Laboratories: Honoraria; Amgen: Consultancy, Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments, Research Funding; Carsgen: Other, Research Funding; Novartis: Research Funding; Cellectar: Other; Kite Pharma: Consultancy, Research Funding; Karyopharm: Consultancy; Celgene/BMS: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments.

Background Young donors are reported to be associated with better transplant outcomes than old donors in allo-HSCT, but the underlying mechanism is still uncertain. Successful allo-HSCT relies on the rapid reconstitution of donor-derived haematopoietic and immune systems in the recipient. Therefore, characterizing the differences in percentages of HSCs and progenitors and immune cell subtypes between young and old donors may help explain the disparities in transplant outcomes. In humans, HSCs give rise to multipotent progenitors (MPPs) that further segregate into either common myeloid progenitors (CMPs) or multipotent lymphoid progenitors (MLPs), which in turn segregate into either common lymphoid progenitors (CLPs) or granulocyte-macrophage progenitors (GMPs). CMPs further segregate into either megakaryocyte-erythroid progenitor (MEPs), or GMPs. However, differences in the frequencies of HSCs and their progenitors between young and old adults remain uncertain. In addition, aGVHD is generally considered to be associated with increased ratios of donor Th1/Th2, Tc1/Tc2 and M1/M2 macrophage. However, little is known about the cytokine-producing T cell subsets and macrophage subsets in BM between young and old donors. Aims To evaluate the different subsets of HSCs and their progenitors and immune cells among young (aged 45 years). Moreover, to analyze the association between donor characteristics and HSC frequency. M ethods In this prospective study, a total of 60 healthy adult donors, including 20 young donors, 20 middle-aged donors, and 20 old donors were enrolled. The frequencies and ROS levels of BM HSCs(CD34+CD38−CD90+CD45RA−) and progenitors including MPPs(CD34+CD38−CD90−CD45RA−), MLPs(CD34+CD38−CD45RA+), CLPs(CD34+CD38+CD7−CD10+CD45RA+), GMPs(CD34+CD38+CD7−CD10−CD45RA+), CMPs(CD34+CD38+CD7−CD10−CD135+CD45RA+), and MEPs(CD34+CD38+CD7−CD10−CD135−CD45RA−) were quantified by flow cytometry. Furthermore, T cell and macrophage subsets were analyzed in young, middle-aged and old donors by flow cytometry. Effector T cells, naïve T cells, effector memory T cells and central memory T cells were identified as CD45RA+CCR7−, CD45RA+CCR7+, CD45RA−CCR7−, and CD45RA−CCR7+. Th1, Th2, Tc1 and Tc2 were identified as CD3+CD8−IFN-γ+, CD3+CD8−IL-4+, CD3+CD8+IFN-γ+ and CD3+CD8+IL-4+, respectively. In addition, M1 and M2 were identified as CD14+CCR2+CD68+ and CD14+CX3CR1+CD163+. Moreover, the association of donor characteristics with HSC frequency was analysed by univariate and multivariate analysis. Results To determine the differences in HSCs and progenitors in different age donors, HSCs and six subpopulations were compared among young, middle-aged and old donors. The frequencies of HSCs and myeloid progenitors, including CMPs and MEPs in CD34+ cells were significantly lower and the frequencies of lymphoid progenitors including MLPs and CLPs in CD34+ cells were higher in the BM of young donors than in that of old donors. Significantly lower levels of ROS in HSCs and progenitors were observed in young donors than in the other donors. Furthermore, to investigate the differences in the differentiation potential from HSCs to immune cells in different age donors, T cell and macrophage subsets were compared among the three donor age groups. Young donors demonstrated a lower CD4+/CD8+ T cell ratio, lower memory T cell frequency and higher naïve T cell frequency in both CD4+ cells and CD8+ cells. Importantly, BM immune cells from young donors polarized towards less pro-inflammatory T cells characterized by Th1 and Tc1, and more immune suppressor cells, such as M2, than those from old donors. As a result, young donors had lower ratios of Th1/Th2, Tc1/Tc2 and M1/M2 in BM. In addition, multivariate analysis showed that age≥37 was independently correlated with a higher HSC frequency. Conclusion BM HSCs from young donors exhibited a lower frequency, balanced myeloid-lymphoid differentiation potential, lower ROS level and produced more immune suppressors and fewer immune effector cells than those from old donors. Donor age might be a good predictor of HSC frequency. Although further validation is required, the differences in the frequency and immune differentiation potential of HSCs in BM between young and old donors may partly explain the different outcomes of allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Introduction: CD6 is a T-cell costimulatory receptor that has been implicated in the pathogenesis of multiple autoimmune and inflammatory (AI) diseases. In GVHD, CD6 is expressed on reconstituting T cells soon after transplant (Rambaldi et al., 2019), including Th1 and Th17 cells which are both implicated in the induction and pathogenesis of acute GVHD (aGVHD). CD6 is highly expressed on these cells and promotes immune synapse formation, T-cell activation, and T-cell migration via interaction with its ligand activated leukocyte cell adhesion molecule (ALCAM). Furthermore, studies have demonstrated that ex-vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998), highlighting the importance of CD6. While the contribution of CD6 to T cell activation has been well described, less is known regarding the expression levels and role of CD6 on effector and memory T cells (Teff) which are prominent in all diseases including aGVHD. Consequently, the aim of this study was to determine the role of CD6 specifically on effector T cells, and further illuminate the mechanism of itolizumab, an anti-CD6 monoclonal antibody. Methods: Naïve T cells were enriched from frozen PBMCs via a naïve T cell magnetic separation kit (Stemcell). Naïve T cells were polarized towards a Th1 phenotype for 6 days with a Th1 differentiation cocktail (Stemcell) and CD3/CD28 T cell activator (Stemcell) and rested overnight prior to entering restimulation conditions. Naïve T cells were polarized towards a Th17 phenotype for 8 days with IL-6, IL-1β, TGF-β, IL-23, anti-IL-4 and IFN-γ, the CD3/CD28 T cell activator was used to activate the T cells. To re-stimulate differentiated T cells, anti-CD3 mAb and ALCAM-Fc or anti-CD3 mAb alone were coated on 48-well plates overnight at 4oC. Th1 or Th17 T cells were labeled with CFSE and seeded with isotype control or itolizumab for 72hrs. Cells were collected for flow cytometry analysis and supernatant collected for relevant cytokine detection. To assess surface levels of CD6, cryopreserved PBMCs were thawed and incubated with itolizumab or isotype at 37oC for specific timepoints. Following incubation, cells were washed and stored at 4oC for subsequent staining. All samples were stained at the same time and surface levels of CD6 was detected using a monoclonal anti-CD6 antibody that does not compete with itolizumab. Results: Blockade of the CD6 pathway, using itolizumab during restimulation of differentiated Teff cells in the presence of ALCAM, inhibited multiple effector functions including proliferation and changes in cell size. An average of a 40% decrease in CFSE proliferation was observed across multiple donors. Furthermore, treatment of Teff cells with itolizumab resulted in a significant decrease in expression level of T cell markers of activation and exhaustion such as CD25, PD-1 and Tim3. This effect was exclusively in the presence of ALCAM, indicating that the effect was specific to blockade of the CD6-ALCAM pathway. When levels of CD6 were assessed, CD45RO+ (Teff/mem) cells expressed higher levels than CD45RA+CD45RO- (Tnaive). Itolizumab treatment of in vitro generated CD45RO+ T cells inhibited this stimulation-induced increase in CD6 in a dose-dependent manner, suggesting that the drug may modulate surface CD6 expression as a mechanism separate from physical blockade of CD6. Conclusions: These findings are the first to characterize the CD6-ALCAM pathway as a key regulator of differentiated effector T-cell function. Modulation of activation markers and CD6 itself by itolizumab, suggest modulation of Teff activity by both direct and indirect inhibition of CD6 signaling. These data further support targeting the CD6-ALCAM pathway to inhibit both naïve and effector T cell populations in aGVHD. Disclosures Ampudia: Equillium: Current Employment, Current equity holder in publicly-traded company. Chu:Equillium: Current Employment, Current equity holder in publicly-traded company. Doherty:Equillium Inc.: Research Funding. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company.

Introduction Regulatory T cell (Treg) based therapies are a promising approach for graft-versus-host disease (GvHD) prevention and treatment. However, mechanisms of Treg suppression of alloreactive conventional T cells (Tcon) in GvHD are incompletely understood. In this study, we performed paired RNA and T cell receptor (TCR) sequencing analysis on Tcon and Treg before and after transplantation to further elucidate Treg suppressive function during in vivo suppression of acute GvHD in an MHC major-mismatch mouse model. Methods CD45.2 Thy1.2 BALB/c mice were lethally irradiated (8.8 Gy) and transplanted with 5x106 T cell- depleted bone marrow cells (TCD-BM) from CD45.1 Thy1.2 C57Bl/6 mice alone or together with CD45.2 Thy1.2 C57Bl/6 FoxP3/GFP+ Treg (1x106) on day 0. On day 2, CD45.1 Thy1.1 C57Bl/6 Tcon (1x106; CD4:CD8 ratio = 2:1) were injected to induce GvHD. Irradiated (11 Gy) syngeneic C57Bl/6 recipients receiving C57Bl/6 TCD-BM and CD45.1 Thy1.1 Tcon alone were used as controls. On day 8, donor Thy1.1+ CD45.1+ CD4 and CD8 Tcon and Thy1.2+CD45.2+ FoxP3/GFP+ Treg were isolated by FACS from spleens and lymph nodes. T cell subsets before injection and recovered at day 8 were analyzed by paired RNA and TCR sequencing. Results The transcriptomic analysis revealed a dominant effect of the allogeneic transplant procedure on the clustering of the different T cell populations. Principal component analysis (PCA) of the top 1000 most differentially expressed genes revealed that 68% of the variance was explained by PC1, which clearly segregated CD4and CD8Tcon recovered at day 8 from allogeneic recipients from cells before injection or recovered from syngeneic recipients (Fig. 1). PC1 was mainly driven by naïve T cell genes (Ccr7, Sell, Il6ra, Il6st, Foxo1) that were progressively downregulated along PC1. Analysis of the TCR repertoire based on sequencing of the TCR alpha chain revealed a progressive clonal restriction along PC1 in CD4 and CD8 T cells (Fig. 1). Accordingly, clonal overlap between cells collected at day 8 and cells analyzed before injection were reduced in allogeneic recipients (Morisita Index [MI], CD4: 0.06±0.01; CD8: 0.02±0.01) compared to syngeneic controls (MI, CD4: 0.23±0.26; CD8: 0.57±0.05). Treg administration did not affect CD4 or CD8 T cell segregation along PC1, suggesting that they minimally interfered with cell activation and differentiation during GvHD. Accordingly, Treg did not inhibit clonal restriction (Fig.1) nor the reduction in clonal overlap (MI, CD4: 0.04±0.01; CD8: 0.01±0.01), indicating that Treg did not inhibit the initial activation of alloreactive T cells clones. Treg impact on CD4 but not CD8 Tcon transcriptome was revealed by PC2 (Fig.1). Treg induced the downregulation of TH1-signature genes (Tbx21, Il12rb1, Il12rb2, Stat4) and proinflammatory genes (Il18rap) while promoting up-regulation of anti-inflammatory genes (Il18bp), TH2 signature genes (Ccr4, Il4) and Il2 in CD4Tcon. Moreover, gene set enrichment analysis (GSEA) revealed that Treg treatment significantly impacted gene sets involved in metabolic processes in CD4and CD8Tcon, leading to a global up-regulation of genes encoding for enzymes involved in oxidative phosphorylation (OXPHOS) and downregulation of genes encoding for enzymes contributing to glycolysis (Slc2a1, Hk1, Pfkl, Pfkp, Pkm). Treg recovered at day 8 preserved a distinct transcriptomic signature observed before injection and further enhanced by the up-regulation of genes involved in Treg activation and suppressive function (Gata3, Tnfrsf18, Tnfrsf4, Icos, Ccr1, Ccr4, Il9r). GSEA in Treg revealed significant up-regulation of genes in the OXPHOS signature. TCR repertoire analysis showed clonal restriction of Treg during GvHD. Direct comparison of clone frequencies in Treg and CD4Tcon showed smaller clonal overlap on day 8 (MI=0.005±0.006) compared to day 0 (MI= 0.11±0.004) suggesting that Treg and CD4Tcon responses during GvHD are engaging different cell clonotypes triggered by different epitopes or antigens. Conclusion Our results indicate that Treg treatment did not interfere with Tcon activation and differentiation of alloreactive Tcon clones in a model of acute GvHD. Treg predominantly affected CD4Tcon and to a lesser extent CD8Tcon transcriptome, modulating the transcription of genes encoding pro- and anti-inflammatory molecules as well as enzymes involved in metabolic processes. Disclosures Turkoz: Adicet Bio: Current Employment, Current equity holder in private company. Negrin:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; BioEclipse Therapeutics: Current equity holder in private company; Amgen: Consultancy; KUUR Therapeutics: Consultancy; Biosource: Current equity holder in private company; UpToDate: Honoraria.

Background Foxp3+ regulatory T cells (Tregs) comprise of natural (n) and induced (i) Treg subsets play an important role in immune system. Currently, isolation of nTregs and in vitro-expanded nTregs was shown to be an effective therapy to GVHD patients. However, shortage of nTregs in peripheral blood and time consumption of expansion in vitro may eventually limit the clinical application. Conversely, iTregs can be generated in vitro from naïve T cells and to a large number of iTregs in short time. As we known, regulatory T cells would decay after a period of time, in vivo or in vitro. Keeping a certain number of iTregs during the GVHD treatment is necessary, it should be the best to provide iTregs to the patient more than single usage. Aim Manipulated supplements of TGF-β1-induced Foxp3+ regulatory T cells should be a good way for prevention from acute graft-versus-host disease within a short time. Investigation was performed via animal model. Methods Splenocytes from C57BL/6 mice were used as a source of naïve T cells by a CD4+ naïve T cell isolation Kit. To induce Foxp3+ regulatory T cells (iTregs), the CD4+ naïve T cells were incubated with anti-CD3/CD28 coated 24-well plate in the presence of IL-2 (20U/ml) and TGF-b1 (50ng/ml) for 3 days. Foxp3+iTregs were harvested and identified as the expressions of CD4+/CD25+/FoxP3+/CD127- via flow cytometry (Fig.1). In this experiment, recipients (BALB/c) were irradiated with 800cGy and then infused with donor (C57BL/6) bone marrow cells with (TCD-BM+CD4T) or without donor T cells (TCD-BM) by intravenous injection. TCD-BM+CD4T cells mice would appear aGVHD phenotype. 8x106 Foxp3+ iTregs were injected into the TCD-BM with donor T cell mouse one or twice (TCD-BM+CD4 T +iTreg) for immunosuppression assay as shown in Fig.2. Mouse GVHD phenotype, body weights and survival rates were investigated lasting for over 90 days. Tissue sections were stained with haematoxylin-eosin. Results According to our preliminary data, it indicated the injection of iTregs in the prevention of aGVHD should be feasible (Fig.3). Consequently, we have tried to investigate preventative efficiency of repeated iTregs supplements in TCD-BM mice. First of all, we compared the single-dose of iTregs with the repetition-dose of iTregs in aGVHD prevention. The data showed in Fig.4. The data showed that the survival rate was 73.3% in repeated treatment in mice, however, the survival rate was only 45.8% in single-dose of iTregs mice within 24 days. As the TCD-BM survival rate was 76.1%. It indicated that the repetition-dose of iTregs would prevent the occurrence of aGVHD, and the survival rate was similar as the bone marrow transplantation mice. The BM-CD4T mice with aGVHD phenotype could survive no more than 10 days. Furthermore, we investigated the survival time of the continual iTreg supplements mice. The data showed in Fig.5. After 90 days later, the body weight of iTregs treated mice could maintain the recovery efficiency to 83.8±2.1% and the survival rate to 78%, comparing with the TCD-BM mice was 88.8±0.6% and 73%. All of these mice could keep alive more than 90 days. Using histographic staining, we confirmed the aGVHD prevention with repeated supplement of Foxp3+iTregs to the CD4T mice (Fig.6). The mice, administration of CD4T cells with bone marrow cells, failed to survival for the serious damage of intestine villi (Fig.6A) and Peyer's patches (Fig.6B). In contrast, CD4T mice with Foxp3+-iTregs (iTregs) could survival more than 90 days and intestine villi were recovered after 90 days (Fig.6A). Peyer's patches are an important gut associated lymphoid tissue in small intestine and play a crucial role in immune response. Therefore, we have investigated the changes of Peyer's patches (Fig.6B). As the recovery of mice with iTregs for twice, the Peyer's patches reappeared after 90 days later. It indicated that keeping more iTregs in vivo could more efficient on prevention of aGVHD. It indicated that more alive iTregs to prevent GVHD occurrence more efficient and may provide the information pre-clinically. Conclusion We showed that repetition supplement of iTreg cells to TCD-BM+CD4T-treated mice, could maintain the mice in high survival rate. Therefore, we may provide more of the functional iTregs to GVHD patients, continuously. It's a good way to prevent the occurrence of GVHD. The result should develop a novel-cell based approach for potentially reducing the risk of acute GVHD clinically. Disclosures No relevant conflicts of interest to declare.

The molecular mechanisms that underlie T-cell quiescence are poorly understood. In the present study, we report a primary immunodeficiency phenotype associated with MST1 deficiency and primarily characterized by a progressive loss of naive T cells. The in vivo consequences include recurrent bacterial and viral infections and autoimmune manifestations. MST1-deficient T cells poorly expressed the transcription factor FOXO1, the IL-7 receptor, and BCL2. Conversely, FAS expression and the FAS-mediating apoptotic pathway were up-regulated. These abnormalities suggest that increased cell death of naive and proliferating T cells is the main mechanism underlying this novel immunodeficiency. Our results characterize a new mechanism in primary T-cell immunodeficiencies and highlight a role of the MST1/FOXO1 pathway in controlling the death of human naive T cells.

Follicular lymphoma (FL) is an indolent, but incurable malignancy as most patients eventually experience progressive disease. We hypothesized that clonal heterogeneity and patient-specific immune responses would contribute to variable clinical outcomes and that understanding the complexity of the entire tumor "ecosystem" would allow us to better match patients with specific types of tumor- and immune-targeted therapies. In this study, we performed 38-dimensional single-cell phenotyping by mass cytometry (CyTOF) to simultaneously characterize both the substructure of malignant B cell populations as well as the T cell microenvironment in a cohort of 77 diagnostic patient FL biopsies and 35 benign reactive LN (rLN) biopsies. We first applied the t-distributed Stochastic Neighbour Embedding (t-SNE) algorithm to explore intra- and inter- tumoral heterogeneity among malignant B cell populations. t-SNE mapping of individual samples showed that more than a third of FL samples contain at least two phenotypically distinct tumor subpopulations, supporting the notion of multi-clonal tumor architectures presumably due to ongoing clonal evolution. Batched analysis combining all 77 FL cases together with 35 rLN samples revealed two distinct tumor subtypes comprising about 25% (type "A") and 10% (type "B") of total FL samples, respectively, with individual tumors within each subtype showing highly similar and partially overlapping phenotypes. Mapping the same data using Uniform Manifold Approximation and Projection (UMAP), a dimensional reduction algorithm similar to t-SNE but preserves global structure more accurately, revealed that type A tumors localized in close proximity to normal germinal center (GC) B cells, thus fulfilling conventional expectations as to the histogenesis of FL. In contrast, type B tumors localized more closely to pre-GC B cells, implying the existence of an alternate histogenic path in FL. Importantly, we also performed single-cell RNA-Seq on a subset of FL cases which independently confirmed the type A vs type B distinction in whole transcriptomic space. We next analyzed matching T cell data using a modified Statistical Scaffold algorithm in order to place distinct subsets in context with conventionally defined normal T cell populations. Clustering analysis using multi-layer phenograph performed on T cells from all FL and rLN samples combined yielded hundreds of small, but phenotypically distinct populations that were then annotated according to the nearest conventionally defined T cell subset. These imputed designations were used as features to perform hierarchical clustering of samples which revealed 3 major clusters. Cluster1 was characterized by mostly naive T cell populations and contained the majority of rLN samples. Cluster2 was characterized by more differentiated effector T cell populations and was dominated by FL samples. Samples within Cluster2 could be further divided into Tfh, Treg and Th1-rich subgroups. Cluster3 was characterized by a diverse T cell environment including naive, memory and differentiated effector subsets and contained a mixture of rLN and FL samples. Integrative analysis correlating B- and T- cell features revealed type B FL tumors were associated with a Tfh-rich immune landscape. Taken together, these data reveal pervasive phenotypic heterogeneity in both malignant and immune cell compartments of patient FL samples and suggest that defining tumoral subtypes as well as the status of the local immune response within individual samples will support more refined diagnostic classification and highlight functional interactions most amenable to therapeutic targeting. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Celgene: Consultancy, Honoraria; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Janssen: Research Funding. Steidl:Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Nanostring: Patents & Royalties: patent holding; Bristol-Myers Squibb: Research Funding; Tioma: Research Funding; Roche: Consultancy.

The naive phenotype of cord blood (CB) T cells may reduce graft-versus-host disease after umbilical cord blood transplantation, but this naivety and their low absolute numbers also delays immune reconstitution, producing higher infection-related mortality that is predominantly related to CMV, adenovirus (Adv), and EBV. Adoptive immunotherapy with peripheral blood-derived virus-specific cytotoxic T lymphocytes (CTLs) can effectively prevent viral disease after conventional stem cell transplantation, and we now describe the generation of single cultures of CTLs from CB that are specific for multiple viruses. Using EBV-infected B cells transduced with a clinical-grade Ad5f35CMVpp65 adenoviral vector as sources of EBV, Adv, and CMV antigens, we expanded virus-specific T cells even from CB T cells with a naive phenotype. After expansion, each CTL culture contained both CD8+ and CD4+ T-cell subsets, predominantly of effector memory phenotype. Each CTL culture also had HLA-restricted virus-specific cytotoxic effector function against EBV, CMV, and Adv targets. The CB CTLs recognized multiple viral epitopes, including CD4-restricted Adv-hexon epitopes and immunosubdominant CD4- and CD8-restricted CMVpp65 epitopes. Notwithstanding their naive phenotype, it is therefore possible to generate trivirus-specific CTLs in a single culture of CB, which may be of value to prevent or treat viral disease in CB transplant recipients. This study is registered at www.clinicaltrials.gov as NCT00078533.

What are the rules that govern a naive T cell's prospects for survival or division after export from the thymus into the periphery? To help address these questions, we combine data from existing studies with robust mathematical models to estimate the absolute contributions of thymopoiesis, peripheral division, and loss or differentiation to the human naive CD4+ T-cell pool between the ages of 0 and 20 years. Despite their decline in frequency in the blood, total body numbers of naive CD4+ T cells increase throughout childhood and early adulthood. Our analysis shows that postthymic proliferation contributes more than double the number of cells entering the pool each day from the thymus. This ratio is preserved with age; as the thymus involutes, the average time between naive T-cell divisions in the periphery lengthens. We also show that the expected residence time of naive T cells increases with time. The naive CD4+ T-cell population thus becomes progressively less dynamic with age. Together with other studies, our results suggest a complex picture of naive T-cell homeostasis in which population size, time since export from the thymus, or time since the last division can influence a cell's prospects for survival or further divisions.

Exosomes are secreted vesicles formed in late endocytic compartments. Immature dendritic cells (DCs) secrete exosomes, which transfer functional major histocompatibility complex (MHC)–peptide complexes to other DCs. Since immature and mature DCs induce different functional T-cell responses (ie, tolerance versus priming), we asked whether DC maturation also influenced the priming abilities of their exosomes. We show that exosomes secreted by lipopolysaccharide (LPS)–treated mature DCs are 50- to 100-fold more potent to induce antigen-specific T-cell activation in vitro than exosomes from immature DCs. In vitro, exosomes from mature DCs transfer to B lymphocytes the ability to prime naive T cells. In vivo, only mature exosomes trigger effector T-cell responses, leading to fast skin graft rejection. Proteomic and biochemical analyses revealed that mature exosomes are enriched in MHC class II, B7.2, intercellular adhesion molecule 1 (ICAM-1), and bear little milk-fat globule–epidermal growth factor–factor VIII (MFG-E8) as compared with immature exosomes. Functional analysis using DC-derived exosomes from knock-out mice showed that MHC class II and ICAM-1 are required for mature exosomes to prime naive T cells, whereas B7.2 and MFG-E8 are dispensable. Therefore, changes in protein composition and priming abilities of exosomes reflect the maturation signals received by DCs.

Allogeneic hematopoietic stem cell transplantation (AHSCT) leads to a prolonged state of immunodeficiency characterized by low peripheral naive T-cell counts. To identify the mechanisms leading to this defect we quantitatively and qualitatively analyzed thymic function through quantification of T-cell receptor excision circle (TREC) frequencies (both the signal-joint TREC [sjTREC] and 6 different DbetaJbeta TRECs, by-products of T-cell receptor [TCR] alpha and beta gene rearrangement, respectively), in conjunction with immunophenotype and spectratype analyses in a cohort of patients sampled from 1 to 10 years following AHSCT. In this cohort, reduced thymic function was associated only with ongoing clinical chronic graft-versus-host disease (cGVHD). Nonetheless, the diversity of thymic production remained unchanged irrespective of the patient's cGVHD status. Interestingly, increased homeostatic proliferation was found in the naive T-cell compartment of cGVHD- patients who underwent transplantation. However, reduced expression of both the interleukin-7 receptor alpha (IL-7Ralpha) (CD127) chain and the antiapoptotic protein Bcl-2 was observed. Taken together, these data indicate that the inability to reconstitute the naive T-cell compartment for several years after AHSCT, in the absence of cGVHD, is a consequence of impaired naive T-cell survival rather than thymic dysfunction.

Background Allogeneic hematopoietic stem cell transplantation (HSCT) has been used to treat some of hematological malignancies and inherited or acquired non-malignant diseases. Unfortunately, graft-versus-host disease (GVDH) occurred approximately 15% in transplant recipients and decreases the success of allogeneic HSCT. Currently, isolation of natural regulatory T cells (nTregs) and in vitro-expanded nTregs was shown to be an effective therapy to GVHD patients. However, shortage of nTregs in peripheral blood and time consumption of expansion in vitro may eventually limit the clinical application. Conversely, induction regulatory T cells (iTregs) can be generated in vitro from naïve T cells and to a large number of iTregs in short time. It may improve the GVHD therapeutic efficiency. The stability of FoxP3 in iTregs is a crucial factor for suppression of GVHD. Besides, a functional iTreg cell should be CD127 negative. Because of that, we should provide a large number of effective iTreg cells, the CD4+CD25+FoxP3+CD127-iTregs, to ensure the successful therapy. Aim In order to harvest the large number of effective iTregs for preventing the GVHD, we try to develop a method for induction of more iTreg cells via expansion and repeated activation of naïve T cells. Methods Human PBSC were prepared from peripheral blood of health donor by Ficoll-Hypaque density gradient centrifugation. Naïve T cells were isolated by negative selection. The harvested naïve T cells were cultured and stimulated under cytokines-containing RPMI1640 medium. The flow chart was shown in figure. For cell proliferation, the cells were cultured under IL-2 supplemented medium. The harvested cells were analyzed by flow cytometry with fluorescence-conjugated CD antibodies, including CD4, CD25, CD127 and FoxP3. Results With our purpose, harvest more iTreg cells should be efficient for clinical application. Amplification of the T cell number can obtain more iTreg cells; therefore, cultivation of the T cells under IL-2-containing medium would stimulate the cell proliferation to about 4-fold on the 10th day. After the first cytokines stimulation, we harvested the iTregs, and then, keep the cells in IL-2 only medium for another 5 days to amplify the T cells. On the 10th day, naïve T cell reactivation would be performed with anti-CD3/CD28 antibodies and with cytokines supplement for the second induction. The number of iTregs would increase to about 20-40%. It indicated that we would harvest more iTreg cells for application. If we cultured the naïve T cells under IL-2 medium in the beginning, we would not obtain much more iTregs, comparing with the reactivation method. Conclusion Our study showed that after the first activation and induction of naïve T cells, we could expand more cells under IL-2 containing medium. Then, after the second activation and induction, we could harvest more iTreg cells markedly. The result should develop a novel-cell based approach for potentially reducing the risk of GVHD within short time. Figure Figure. Disclosures No relevant conflicts of interest to declare.

In acute lymphoblastic leukaemia (ALL), T cell anergy induced by tumour antigen presentation without co-stimulation contributes to immunological escape and disease progression. Cross priming of T cells by dendritic cells (DCs) may overcome this escape mechanism, restoring T cell activation and promoting a successful anti-tumour immune response. Remission induction chemotherapy induces rapid cytoreduction of leukaemia and has the modifying potential to affect effector-target cell ratios and tumour cell recognition. DCs may take up necrotic and apoptotic tumour cells, process tumour antigens, and present these to T cells alongside the costimulatory molecules required for effective priming. Further, lymphocyte recovery during remission induction chemotherapy has been linked with improved prognosis, leading to postulation that selective lymphocyte expansion may occur as an immune response against tumour cells. In this study, we extensively characterised paediatric B cell ALL patient immune cells and plasma cytokines, at diagnosis and throughout remission induction chemotherapy in peripheral blood (PB) and the bone marrow (BM) tumour microenvironment. 17 paediatric patients diagnosed with precursor B cell ALL were enrolled in this study. Matched PB and BM aspirate tissue samples were collected at time points co-ordinating with treatment protocol (diagnosis, and induction days 8 and/or 15 and 29). Mononuclear cells and plasma samples were obtained by density gradient centrifugation. DCs, monocytes, B cells, natural killer (NK) cells, T cells, invariant natural killer T cells, cytokine induced killer cells and leukaemic blasts were extensively characterised by flow cytometry in matched PB and BM aspirate samples at diagnosis, day 8, day 15 and day 29 of induction therapy. Plasma cytokines from diagnosis and day 29 samples were analysed by Luminex multi-plex immunoassay. PB absolute lymphocyte counts (ALCs) at induction days 8 and 15 were lower than those recorded at diagnosis and lymphocyte recovery was observed at day 29. An equivalent pattern of lower mononuclear cell yields at days 8 and 15 with increased yields at day 29 was recorded in PB and BM samples processed for flow cytometry. All major immune cell populations were identified in PB and BM at all time points. BM DCs were significantly increased at day 29 compared to earlier time points and compared to matched day 29 PB DCs (Figure 1A). Naïve (CCR7+, CD45RA+) T cells dominated the peripheral and BM T cell pools at all time points with only low numbers of effector (CCR7-, CD45RA+), effector memory (CCR7-, CD45RA-) and central memory (CCR7+, CD45RA-) T cells recorded. However, activated (HLA-DR+) naïve CD4 T cells were significantly increased in the BM at day 29 compared to matched PB (Figure 1B) concordant to increased BM DCs. BM soluble cytokine analysis confirmed the presence of IL-1β, TNFα, IL-2, IL-4, IL-12p70 and IFNα in day 29 samples which may provide stimulus in the BM microenvironment for maturation of DCs and subsequent priming and activation of T cells. Immunological alterations in the BM tumour microenvironment may be reflective of an altered capacity to mount an effective anti-tumour immune response. Particularly, T cell reconstitution following induction chemotherapy may have important implications in childhood leukaemia, especially if recovering T cells are able to mediate anti-leukaemia effects. Rapid lymphocyte recovery has previously been associated with improved survival, and has been shown to be accounted for by increasing T cell numbers following induction chemotherapy in paediatric ALL. Here, while ALC recovery was recorded at day 29, extensive immune phenotyping of PB samples confirmed that recovery of T cell numbers is related to an increase in naïve T cells. T cell expansion associated with a specific immune response would be expected to occur in the effector and memory T cell compartments and the increased naïve T cell numbers observed here likely relate to homeostatic proliferation or increased thymic output. However, BM DCs were increased at day 29 alongside increased activation in naïve CD4 BM T cells, which may represent initiation of an anti-leukaemia T cell response. In vitro studies to investigate patient T cell response to autologous tumour cells are required as confirmation and are currently ongoing. Disclosures No relevant conflicts of interest to declare.

Background Graft-versus-host disease (GVHD) frequently causes morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT) as a result of organ damage and infections. In HLA-identical HCT, GVHD results from recognition by donor T cells of minor histocompatibility (H) antigens on recipient tissues. Complete T cell depletion (TCD) of donor hematopoietic cell products is more effective than pharmacologic immunosuppression for preventing GVHD, but is complicated by delayed immune reconstitution and consequent life-threatening infections.Approaches to HCT which preferentially deplete the T cells that primarily cause GVHD and preserve pathogen-specific T cells may improve HCT outcomes. Mature CD3+ CD8+ and CD3+ CD4+ T cells can be classified into CD45RA+ CD62L+ naïve (TN) and CD45RO+ memory (TM) subsets, the latter of which includes effector memory (TEM) and central memory (TCM) cells. Murine studies in which allogeneic TCD bone marrow (BM) is transplanted with purified T cells from individual T cell subsets to irradiated minor H antigen disparate recipients have demonstrated that the most severe GVHD results from transplanting T cells of the TN subset. Purified TCM causes mild GVHD and TEM do not cause detectable GVHD and can transfer immunity to pathogens.In vitro studies have similarly demonstrated that human donor CD8+ T cells specific for recipient minor H antigens are found predominantly within the TN cell subset, suggesting selective TN cell depletion may alter the GVHD incidence and/or severity in human HCT. Methods and results We developed an effective process for engineering human peripheral blood stem cell (PBSC) grafts that depletes CD45RA+ TN cells and retains CD34+ stem cells and functional CD45RO+ TM cells specific for a broad range of opportunistic pathogens (Bleakley BBMT 2014). We are conducting clinical trials to evaluate the selective depletion of TN cells from HLA-matched allogeneic PBSC grafts for the prevention of GVHD in patients with acute leukemia, the first of which has been published (Bleakley JCI 2015, N=35). Seventy patients have now been treated on three consecutive phase II trials. The median age was 34 years (1-56 years), 56% of patients had a diagnosis of ALL, 46% had previously relapsed or had detectable disease (MRD or relapse) at the time of HCT, and 23% had unrelated donor (URD) grafts. Intensive myeloablative, TBI-containing (13.2Gy) conditioning was used for 63 patients, whilst 7 patients received a medium intensity 'midi' preparative regimen, including 4Gy of TBI. The TN-depletion procedure was successfully performed on URD PBSC products shipped overnight from donor centers throughout the US, as well as on MRD PBSC collected at our centers. Reliable engraftment with high-level donor chimerism was observed in recipients of 'midi' as well as intensive myeloablative conditioning. The 2-year estimates of overall survival, disease-free survival, survival free of relapse and chronic GVHD (CRFS) and survival free of relapse, grade II-IV acute GVHD, and chronic GVHD (GRFS) are 79%, 73%, 69% and 63% respectively. Median follow-up among survivors is 26 months. The frequency and severity of chronic GVHD is remarkably low (5%) compared to historical rates of 40-60% chronic GVHD in HLA-matched PBSC transplantation with conventional calcineurin inhibitor-based immunosuppression. Relapse and non-relapse mortality (NRM) are acceptably low at 19% and 8%, respectively. No NRM occurred in patients Conclusions The outcomes of recipients of TN-depleted PBSC grafts compare very favorably to published results of HCT for patients with acute leukemia. For example, the 69% incidence of CRFS at 2 years in TN-depleted recipients compares with reported 2-year GRFS rates of 37% and 17% in recipients of allogeneic PBSC from HLA-matched related donors with or without ATG (Kroger et al. NEJM 2016). Our results suggest that TN-depletion of PBSC grafts may reduce the risk of chronic GVHD without negatively impacting other important HCT outcomes. Disclosures Riddell: Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Consultancy, Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria.

S.K and H.-J.S. contributed equally for this works Dendritic cell-derived exosome (DEX) has been known as an efficient stimulator of T cells. However, the production of sufficient DEX remains a barrier to broad utility for immunotherapy. In this study, we engineered K562 cells expressing triple-co-stimulatory signals (CD80, 4-1BBL, and CD83) with HLA-A2 as an AAPC. Specifically, CD137L (4-1BBL) is an ideal signaling molecule for long-term propagation of CD8+ T cells, and the addition of other co-stimulatory molecules, such as CD80 and CD83, is used to support the expansion of naive T cell subsets. DC-derived exosomes display immunologically important molecules such as HLA and co-stimulatory molecules. Likewise, CoEX-A2 expressed high levels of HLA-A2, CD80, CD83, and CD137L (41BBL) and mediate strong, antigen-specific CD8+ T lymphocyte activation. The stimulation of freshly isolated peripheral CD8+ T cells with the appropriate antigen specificity observed here was likely made possible by the use of the sensitive ELISPOT assay. Viral or tumor protein-pulsed exosomes can directly stimulate CD8+ T cell proliferation and differentiation into CTLs in vitro. In addition, exosomes can be taken up by both CD8+ T cells and K562 cells. Meanwhile, K562 cells that have taken up exosomes can also stimulate CD8+ T cells, which may be due to the higher levels of HLA-A2, CD80, CD83, and 41BBL expression observed on exosomes. Therefore, the CD8+ T cell antigen-specific expansion observed in our cultures is likely the result of coated CoEX-A2s working directly or in a cross-dressed manner. The results suggest that these novel exosomes may provide a crucial source to generate antigen-specific CD8+ T cells for adoptive cell therapy against viral infection and tumors. Disclosures No relevant conflicts of interest to declare.

The polarization of naïve CD4+ T cells may initiate multiple reactions in immune system. The balance between Th1 and Th2 cells is critical for innate and acquired immune reactions. But the exact mechanism of its polarization is still unclear. IL-4 is specifically produced by Th2 cells, and regulates Th2 differentiation. Once IL-4 binds to IL-4 receptor (IL-4R), the Th2 polarization signal is activated by phosphorylation of STAT6 (recruited by IL-4Rα), its relocation to nucleus, activation of STAT6 downstream genes (gata3, il-4 and il-4rα etc) and consequent Th2 polarization. CD44 an important T cell activation and T helper cell differentiation gene participates in the regulation of Th1 and Th2 differentiation. Furthermore, CD44 variant isoforms produced by alternative RNA splicing, have different physiological and pathological functions including tumor metastasis, drug resistance and anti-apoptosis effect in tumor cells. Here we report hitherto unknown specific CD44 variant isoforms involved in T helper cell differentiation and functional regulator of CD4+ T cell polarization. We developed various PCR primer sets able to distinguish different CD44 isoforms in human and mouse Th2 cells. We found higher expression of CD44 variant 4 (CD44v4) and CD44v5 in both human and mouse Th2 cells compared with Th1 cells, indicating their role in Th2 cell differentiation. In order to investigate the role of CD44v4 and CD44v5 in Th2 cells polarization, we treated human naïve CD4+ T cells with CD44v4 or CD44v5 antibody separately for 3 days in polarizing condition. We observed that CD44v5 antibody treatment dramatically decreased the level of phospho-JAK1 and pSTAT6 compared to control cells treated with same amount of normal mouse IgG. At the same time, the expression of GATA3 detected by western blot and the secretion of IL-4 measured by ELISA decreased. Notably, the phosphorylation of STAT1 in Th1 cells was not inhibited by CD44v5 blocking. There is a significant decrease in GATA3 and IL4 expression with CD44v5 antibody but not in CD44v4 antibody treated group, which indicated that Th2 polarization was mainly influenced by CD44v5. In order to verify our finding, CD44v5 specific siRNA were used and we observed similar result in CD4+ T cells. Interestingly, we found that the degradation of IL-4Rα increased after treatment of Th2 cells with CD44v5 antibody compared with control group. Using confocal microscopy of single cell, we observed that CD44v5 co-localized with IL-4Rα. Importantly, CD44v5 antibody treatment could interrupt the CD44v5 and IL-4Rα interaction and also the co-localization of T cell receptor (TCR). On Th1 cells, we didn't find the co-localization between IFNgR and CD44v5. The IFN-γ secretion in Th1 cells were not influenced by either CD44v5 blocking or CD44v5-deficient CD4+ T cells indicating that CD44v5 only influenced the differentiation of Th2 cell, but not Th1 cells. In conclusion, CD44v5 plays an important role in naive T cell differentiates into Th2 cells. We hypothesis that CD44v5 can bind to IL-4Rα through CD44v5 variant domain and stabilize the IL-4Rα, blocking CD44v5 induced IL-4R degradation and reduce the Th2 cell differentiation. CD44v5 antibody treatment inhibiting Th2 differentiation without affecting Th1 development provides a potential novel immuno-therapy target. Disclosures No relevant conflicts of interest to declare.

Introduction: We have previously shown that blocking CD28:CD80/86 costimulation with CTLA4-Ig can be a successful strategy to inhibit GVHD. However, since CTLA4-Ig targets CD80/86, there remain concerns of off-target, immune activating effects, given that it can inhibit both positive signaling through CD28 and negative signaling through CTLA4. In order to specifically target CD28, we have developed a humanized anti-CD28 monovalent Fab antibody (FR104) and have tested its ability to protect against GVHD. Methods: NHP underwent MHC-mismatched transplantation after myeloablative radiation-based pre-transplant conditioning. They were transplanted with GCSF-mobilized PBSCs (4.2x10^8 +/- 1.1x10^8 TNC/kg and 1.9 x 10^7 +/- 0.5 x 10^7 CD3+ T cells/kg. GVHD prophylaxis consisted of either monotherapy with FR104 (5mg/kg given weekly) or with combination FR104 and rapamycin (rapamycin trough 5-15 ng/ml). These were compared to three control groups: no GVHD prophylaxis, rapamycin monotherapy and autologous HSCT. Clinical and histologic GVHD was monitored using our NHP grading scale, and longitudinal immunologic analysis was performed. Treatment was continued as tolerated with planned terminal analysis at 35 days in all survivors. Results: Untreated controls (n = 5) developed rapid, severe multi-organ GVHD (MST=7 days). Rapamycin monotherapy (n=6) partially protected against GVHD (MST =14 days). Monotherapy with FR104 showed a significant prolongation in survival compared to untreated animals (MST = 21 days, p=0.02 n = 3), with breakthrough GVHD (liver, skin predominant) ultimately occurring. In contrast to all other groups, combination therapy with FR104 and rapamycin (n=4) resulted in striking protection from GVHD with all animals reaching planned terminal analysis with minimal clinical disease (p=0.01 vs. untreated controls, p=0.01 vs. rapamycin monotherapy, Fig. 1). We have documented a major role for both T cell proliferation (Ki-67) and Granzyme B (Gran-B) overexpression in GVHD, with untreated controls demonstrating rampant CD8+ proliferation (81% + 5.4% Ki-67+ at day 7 vs. 4% pre-transplant) and Gran-B overexpression (81% + 2.7% Gran-Bvery high at day 7 vs. 0.5% + 0.5% pre-transplant). Monotherapy with either rapamycin or FR104 partially controlled proliferation (53% + 23% and 30% + 21.7%, respectively) and Gran-B overexpression (21% + 8.7% and 11% + 5%, respectively) on day 7. Combination therapy with FR104 + rapamycin resulted in synergistic control of both proliferation and Gran-B overexpression (0.6% + 0.2% Ki67+ and 2.3% + 1.3% Gran-B overexpression on Day 7), reverting the CD8+ activation phenotype to that observed in auto-HSCT controls (5.6% + 2.1% Ki67+, 9% + 3.2% Gran-B overexpression, Fig. 2). This reversion to the auto-HSCT phenotype was maintained until the terminal analysis, and the control of Day 7 proliferation and Gran-B overexpression correlated closely with GVHD-free survival. The control of CD28-mediated T cell activation in FR104-treated recipients resulted in significant preservation of the naïve CD8+ T cell (CD8 Tn) phenotype in these animals. Thus, in untreated controls and rapamycin monotherapy groups, the CD8 Tn population virtually disappeared post-transplant (2.1% + 0.7% and 6.6% + 1.5% at day 7, respectively compared to 33.6%+ 4.1% pre-transplant), consistent with widespread allo-stimulation and costimulation. In contrast, FR104 monotherapy resulted in partial preservation of CD8 Tn (24.3% + 2.8% at Day 7) and FR104 + rapamycin combination therapy promoted nearly complete preservation of CD8 Tn (48.3% + 0.9% at day +7), with maintenance of this naïve population for the duration of therapy (Fig. 3). Discussion: These results show, for the first time, that specific blockade of CD28 can inhibit NHP GVHD, and when coupled with rapamycin it can effectively control both the clinical and immunologic sequelae of this disease. CD28 blockade with FR104 + rapamycin significantly inhibited both CD8+ T cell proliferation and Granzyme B overexpression, and lead to robust preservation of the naïve T cell phenotype, consistent with efficient inhibition of allo-activation. Our results suggest that targeting the CD28:CD80/86 costimulation axis by selective CD28 blockade may be effective in inhibiting the T cell activation associated with GVHD, and is deserving of clinical evaluation for safety and efficacy in patients undergoing HCT. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Vanhove: Effimune: Research Funding.

The processes of ex vivo transduction of T cells to express chimeric antigen receptors (CARs) and of CAR+ T cell expansion influence the phenotype, function and ultimate fate of the final CAR+T cell product infused into patients. CAR constructs, despite expression of endogenous activation signals, require exogenous T cell activation during CAR transduction to allow optimal lenti-viral or retroviral-mediated integration of the CAR gene of interest into T cells. Clinical CAR therapy trials utilize anti-CD3 antibody-mediated activation or combined CD3 and CD28 stimulation using CD3, CD28 specific magnetic beads. We introduce novel magnetic nanoparticle beads generated from iron oxide nanoparticles conjugated to streptavidin and bound to biotinylated T cell activating antibodies for the purpose of CAR transduction. The small size of these nanobeads confers the advantage of decreased steric hindrance and enhanced capability of bead surface antibodies to access T cell surface antigen for binding and stimulation. We achieve efficient CAR transduction using anti-CD3 nanobead-mediated T cell stimulation and demonstrate CD19 specific CAR-mediated cytotoxicity of CD19+ tumor using an annexin V and 7AAD cytotoxicity assay. Evaluation of T cell phenotype following anti-CD3 nanobead-mediated T cell activation demonstrates preferential activation of naïve T cells as compared to central and effector memory cells. Addition of anti-CD28 costimulation is not necessary to achieving or inhibiting this preferential naïve T cell activation. Naïve T cells exhibit greater replicative capacity and anti-tumor function as compared to both effector and central memory T cells for adoptive transfer. We anticipate that preferential generation of naïve T cell derived CAR+ T cells achieved by introducing anti-CD3 nanobead stimulation can further improve the outcomes of clinical trials using CAR therapy. Disclosures Fu: NVIGEN Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Introduction Acute graft versus host disease is a frequent and life threatening complication following HSCT. Some predictive factors have been identified in the last decades. Experimental studies in mice suggest that the naïve cytotoxic T cells (CD3+/CD8+/CD45RA+/CD62L+) are the major mediators of acute GVHD and that removing this subset of the donor T cells, called ‘naïve T cells’, before transplant may reduce the frequency and intensity of GVHD. Detailed immunophenotyping of the graft including naïve and memory (CD3+/CD8+/CD45RA-/CD62L- ; CD3+/CD8+/CD45RA+/CD62L- ; CD3+/CD8+/CD45RA-/CD62L+ ) T-cell contents have never been explored in human GVHD. We studied the correlation between memory and naive T cell in bone marrow and peripheral blood grafts and development of acute GVHD after hematopoietic stem cell transplant. Methods We analyzed by detailed immunophenotyping, the grafts of a cohort of 210 patients among 402 patients who received an allogeneic stem cell transplantation from bone marrow and peripheral blood between January 2009 and June 2012 at a single center. There were no differences between the 210 studied patients and the other 192 in whom grafts were not studied. Characteristics of patients investigated for naïve and memory T cells were compared using Wilcoxon rank-sum tests and Fisher’s exact tests. The main outcome was occurrence of acute GVHD grade II – IV. Cumulating incidence of acute GVHD was estimated using usual methods and compared according to tertiles of T cytotoxic lymphocytes subpopulations using Gray’s test. Adjusted analyses were performed using Fine- Gray proportional hazards models. All tests were two - sided and p-values ≤ 0.05 were considered as indicating significant association. T cytotoxic lymphocytes were typed in all 210 grafts using CD3, CD8, CD45 RA and CD62L four colors immuphenotyping. Clinical and histological characteristics of patients were recorded. Including age, gender, ABO group and rhesus, viral serology of both the donor and the patient, characteristic of the grafts including HLA compatibility, bone marrow or peripheral blood, lymphocytes and nucleated cell and CD34 numeration, conditioning regimens, GVHD prophylaxis, characteristics of GVHD (date of onset, organs involved, stage and grade). Results Median follow up from transplant was 18 months. Cumulative incidence of acute GVHD was 59% (95% CI range 45 to 59) overall, and 49% (95% CI 42 to 56) at 100 days. In univariate analysis increased absolute counts of memory T cell subtypes were significantly correlated with the onset of an acute GVHD grade II – IV. Risk factors for acute GVHD (multivariate analysis) were use of an unrelated donor, positive CMV donor for a negative recipient, and use of TBI 12Gy. In a multivariate analysis the subtype CD3+/CD8+/CD45 RA-/CD62L- was associated with the onset of acute GVHD grade II-IV (adjusted Hazard Ratio = 1.26 and 1.98, p=0.02). Adjusting analysis on the total number of total nucleated cells infused did not affect the results. Restricting analyses to patients receiving peripheral blood stem cells also provided same conclusions. Conclusion This first study on the relation between rate of memory T cell and GVHD revealed that CD3+/CD8+/CD45 RA-/CD62 L- T-cells numbers and percentage were associated with acute GVHD grade II – IV. In contrast to murine models we did not find evidence for a link between naïve T-cells and GVHD risk Disclosures: Robin: novartis: Research Funding.

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells (HSC) for allogeneic HSC transplantation but reconstitution of T cell immunity remains problematic even with double UCB transplantation (dUCBT). This is related to the small T cell numbers of the UCB graft, the naïve nature of these T cells and often the use of ATG as part of the conditioning regimen. Consequently, UCBT recipients are susceptible to viral infections that require intact T cell immunity for their containment. Epstein Barr virus (EBV) is a gammaherpes virus that is transmitted orally. EBV initially replicates in a lytic cycle in the epithelium of the oropharynx and then establishes a latent state in memory B cells. Reactivation of EBV from latent to lytic cycle is mediated by physiologic stress of the memory B cell pool and by defects in antiviral T cell immunity, and can cause aggressive lymphomas known as post transplant lymphoproliferative disorder (PTLD) in immunocompromised hosts. The specific immunological correlates of EBV reactivation in adult UCBT recipients have not been examined. In the present study we attempted to distinguish immune reconstitution profiles in UCBT recipients who developed EBV viremia from those who did not, in order to better understand the immune profile of UCBT recipients who control gammaherpes virus reactivation. Thirty-one patients with hematologic malignancies received dUCBT with melphalan, fludarabine, ATG conditioning and tacrolimus plus sirolimus for GvHD prophylaxis. EBV viral load was determined on a weekly basis after dUCBT. Immunophenotype of peripheral blood lymphocytes, serum cytokine levels and T cell receptor excision circle analysis (TREC) values, were examined prior to and at 1, 2, 3, 6 and 12 months after UCBT and were compared between EBV viremic and non-viremic patients using the Wilcoxon-rank sum test. During the first 12 months after dUCBT, 14 of 31 (45%) patients developed EBV viremia and four (13%) developed PTLD. At one month after dUCBT, patients who developed EBV viremia displayed higher numbers of CD19+ B cell numbers (p=0.04) and CD4+CD25+ T regulatory cells (p=0.03) compared with patients who never became viremic. Surprisingly, development of EBV viremia correlated with increased numbers of CD3+ (p=0.04), CD4+ (p=0.015) and CD8+ (p=0.021) T cells. This finding was counterintuitive, as one might expect better quantitative peripheral T cell reconstitution in patients with recovering immunity and no EBV reactivation. One mechanism of impaired immune reconstitution after UCBT is skewing towards a late effector memory T cell phenotype, a stage in which T cells are incapable of mounting protective immune responses. We examined whether development of EBV viremia was associated with altered naïve versus memory T cell distribution. We determined that patients who developed EBV reactivation had higher numbers of memory cell subsets (CD4+CD45RO+, p=0.0023; CD8+CD45RO+, p=0.019) at two months after dUCBT. Neither development nor control of EBV viremia correlated with TREC recovery, which occurred after the time period of EBV viremia. We were unable to identify EBV-specific T cells in any patient group due to the very low T cell numbers during the time of viremia. However, analysis of repertoire diversity by deep-sequencing on PCR-amplified CDR3 regions of the TCRb gene using the ImmunoSEQ assay showed a more diverse TCR repertoire, as determined by higher entropy (p=0.03) and lower clonality (p=0.03), among patients who did not develop EBV reactivation, compared with those who developed EBV viremia and PTLD. Assessment of serum levels of SCF (a c-kit ligand), IL-7, thrombomodulin, VEGF and angiopoetin-1 showed that patients without EBV viremia had significantly higher levels of SCF (p=0.0001) and IL-7 (p=0.05), at 1 and 2 months after dUCBT, compared with patients who developed EBV reactivation and PTLD. SCF-mediated signaling via c-kit is integral to the homing and longevity of uncommitted hematopoietic stem cells whereas IL-7 is a critical factor for survival and homeostasis of naïve T cells. Together our findings suggest that control of EBV reactivation after dUCBT might be linked to the support of naïve T cell homeostasis, which enables maintenance of a diverse TCR repertoire. Disclosures: No relevant conflicts of interest to declare.

Abstract 1890 Stem cell transplantation has become a widely used procedure in the treatment of haematological and non-haematological clinical disorders. Unfortunately, cure is often hampered by relapse of the underlying disease, graft-versus-host disease (GVHD), or severe opportunistic infections. Slow T-cell reconstitution is regarded as primarily responsible for infections, GVHD, and relapse, therefore, enhancing immune reconstitution is important. Glycogen synthase kinase-3β (GSK3β) was recently identified as an important regulator of T cell function acting through the Wingless (Wnt) pathway. The effect of in vivo administration of GSK3β inhibitor 6-Bromoindirubin 3'-oxime (BIO) was examined in a humanised mouse model. Mice transplanted with highly purified cord blood CD34+ stem cells demonstrated efficient multilineage reconstitution including myeloid, B and T cell lymphoid compartments. The presence of human CD4 and CD8 single positive human T cells was abundant in peripheral blood (PB). De novo generated T cells exhibited low CD31 expression in the naïve CD4+ T cells suggesting prolonged post-thymic proliferative history of these cells. This is not completely surprising considering that graft recipient mice are characterized by impaired thymopoiesis following irradiation. Human T cells at various stages of differentiation including late effector T cells were recorded by detecting the expression of CD62L, CD45RA and CD45RO. Late memory T cell skewing was observed in PB and spleen of graft recipient mice. Activation of human T cells expressing CD25 was registered in the spleen, however, the recipients of the graft did not exhibit any signs of GVHD suggesting normal positive and negative selection occurring in the thymus during human T cell development in this mouse model. Human T cells isolated from the spleen of transplanted mice exhibited strong proliferative responses to mitogenic and allogeneic stimulation, however, they did not demonstrated any CTL activity tested following vaccination with human leukaemia U937 cells. In vivo administration of GSK3β inhibitor promoted T cell reconstitution in mice transplanted with human CD34+haematopoietic progenitor cells. Per cell output of T cells from CD34+ and CD34+CD38- primitive bone marrow (BM) progenitor cells was higher in BIO-treated mice while CD19+ B cell output was reduced suggesting T-cell developmental skewing in expense of B cell development. In vitro analysis of CD34+ progenitor cells co-cultured with bone marrow stroma MS5 cells has demonstrated inhibited B-cell development following BIO-treatment. CD31 expression in naïve CD4+ T cells was not up-regulated by BIO suggesting that GSK3β inhibition does not act to increase thymic output of T cells. GSK3β inhibition also increased naïve/memory T cell ratio in reconstituted mice. A similar effect was observed in mice transplanted with mature cord blood (CB)-derived T cells. delayed naive to memory T cell transition is likely related to decreased T cell activation and proliferation demonstrated ex vivo. BIO reduced IFNγ and TNFα production in human T cells. BIO increased naïve T cell production in mitogenically stimulated T cells and in mixed lymphocyte cultures. GSK3β inhibition preserved naïve T cell gene expression profile and suppressed the expression of genes activated during effector T cell differentiation. BIO actrivated β-catenin sigbnaling and up-regulated IL7Rα expression. IL7 signalling prevents activated T cell death following effector differentiation suggesting that the mechanism triggered by BIO may act through the inhibition of activated T cell death. In addition, BIO down-regulated negative regulator of IL7Rα SOCS1 as well as CTLA4 and PDCD1 both up-regulated during effector differentiation. Thus clinically GSK3β inhibition acting to prevent late memory T cell skewing and preserving a subset of naïve T cells may increase T cell diversity and improve T cell responses in the recipients of CB transplant particularly in adult patients with impaired thymic function. Disclosures: No relevant conflicts of interest to declare.

Abstract 835 The establishment of the thymic microenvironment early in life is crucial for the production functional T cells. Conversely, thymic involution results in a decreased T cell output. Thymic involution has important health implications especially following bone marrow transplant. Our objective is to determine molecular and cellular mechanisms that will allow for regeneration of involuted thymic tissue, restore production of naïve T cells, and improve immune function while improving our understanding of immunobiology. In this pursuit, we have focused on the Retinoblastoma family of tumor suppressor proteins. The main function of the RB pathway is to restrict passage through the G1/S transition of the cell cycle. RB and its two family members, p107 and p130, mediate the action of a broad range of cellular signals to control the proliferation, survival, and differentiation status of a large number of mammalian cell types. We found that inactivation of the RB pathway in the thymus by early deletion of RB family genes prevents thymic involution, promotes expansion of functional thymic epithelial cells (TECs), and increases thymic T cell output. Moreover, we have identified a direct regulatory relationship between RB and the Foxn1 transcription factor Via E2F transcription factors, where RB/E2F complexes directly repress the Foxn1 promoter, thereby promoting involution. Thus, the RB family is a critical mediator of extra- and intra-cellular signals to regulate thymic epithelial cells and thymus function, and decreasing RB pathway function may promote regeneration of the involuted thymus and restoration of naïve T cell production in patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 226 Umbilical cord blood transplantation (UCBT) has extended the availability of hematopoietic stem cell transplantation (HSCT) to patients without compatible adult stem cell donors. However, prolonged time to engraftment, delayed immunologic reconstitution and late memory T cell skewing compromise the favorable outcome of UCBT. Studies in zebrafish and mouse models have shown that the prostaglandin compound, 16,16 dimethyl prostaglandin E2 (PGE2) increases HSC number, homing and engraftment by modifying the Wnt signaling cascade through cAMP/PKA-mediated stabilization of β-catenin and expression of Wnt target genes. We performed a Phase Ib clinical trial of double UCBT (dUCBT), using one untreated and one ex vivo PGE2-treated UCB unit, to determine safety of this uproach. 12 subjects with hematologic malignancies were enrolled (median age 58.1 years (19.8–66.5)). The median time to engraftment was 17.5 days, superior to a 21 day median for a historic control group (n=53; p=0.045). The PGE2-UCB was the dominant source of hematopoiesis in 10 of 12 subjects (p=0.039) with full T cell chimerism as early as 13 days after dUCBT, suggesting that PGE2 regulated the outcome of UCBT by T cell-dependent mechanisms. Here we examined whether PGE2 might alter the properties of UCB T cells thereby affecting T cell reconstitution after UCBT. We determined that EP2 and EP4 receptors were constitutively expressed in UCB T cells and mediated increase of intracellular cAMP in response to PGE2. PGE2 modulated the Wnt/β-catenin pathway in UCB T cells as determined by upregulation of β-catenin and expression of Wnt target genes including Lef1, Tcf7 and Runx1. Similarly to pharmacologic activation of Wnt/β-catenin signaling by the Gsk3β inhibitor TWS119, PGE2 inhibited proliferation of UCB T cells in response to stimulation via TCR/CD3 and CD28. Under these conditions, a significant proportion of T cells expressed naïve immunophenotypic features with high levels of CD45RA, CD62L and CD127. To examine whether these PGE2-mediated effects on UCB T cells might have in vivo implications we assessed reconstitution of T lymphocytes in PGE2-UCBT recipients and compared the pattern of recovery with that of dUCBT recipients without PGE2 treatment. The numbers of total CD3+ T lymphocytes, CD4+ and CD8+ T subsets remained significantly lower (p=0.036) in PGE2-UCBT recipients during the first year after UCBT. In contrast, T cell receptor excisions circles (TRECs), a marker of naïve post-thymic T cells and indicator of thymic function, were substantially higher in PGE2-UCBT recipients and never reached the nadir observed in dUCBT recipients without PGE2, who had values below the limit of detection through 100 days. Importantly, PGE2-UCBT recipients displayed potent antiviral immunity as determined by the reduced incidence of CMV viremia and no cases of EBV-mediated posttransplant lymphoproliferative disorder (PTLD), which caused significant morbidity and mortality in dUCBT recipients without PGE2. In mouse models Wnt/β-catenin signaling promotes the generation of a central memory/stem cell memory T cell population with naïve immunophenotypic features but potent immune function. To examine whether such PGE2-mediated Wnt/β-catenin imprinting might be evident in PGE2-UCBT recipients, we examined expression of the Wnt/β-catenin target Eomes, a transcription factor that links the long-term memory CD8+ T cells to effector potency and protective immunity. Real time qPCR revealed that expression of Eomes was significantly elevated in the PBMCs of PGE2-UCBT recipients compared to control UCBT recipients. Consistent with the role of Wnt/β-catenin to maintain a central memory/stem cell memory phenotype in CD8+ cells, there was an increased fraction of CD62L+ cells within the CD8+ populations in recipients of PGE2-UCBT. Our in vitro and in vivo findings indicate that PGE2-UCB treatment improves HSC engraftment while preserving a naïve T cell compartment and favoring the generation of long-lived memory CD8+ cells via Wnt-mediated gene programming. This novel treatment might significantly improve the outcome of UCBT where delayed engraftment and impaired immunity are serious causes of morbidity and mortality. Disclosures: No relevant conflicts of interest to declare.

Abstract 791 Background: Myelodysplastic syndromes (MDS) are characterized by dysregulated myelopoiesis and peripheral cytopenias with enormous disease heterogeneity owing to diverse molecular pathobiology. The early manifestations of MDS, however, are relatively well conserved and include increased apoptosis coupled to excessive proliferation of myeloid progenitors. In addition to myeloid abnormalities, repertoire contraction and memory expansion is demonstrable in T cells. The notion that apoptosis of hematopoetic cells may be triggered through an immune–mediated mechanism arose from similarities with aplastic anemia (AA). Our recent data showed that MDS responsive to immunosuppressive therapy has accelerated naïve T cell turnover (ie, high proliferative index plus excessive cell death) which led us to hypothesize the presence of an inherent T cell abnormality impairing homeostatic regulation. AA can be caused by somatic mutations within telomere repair components. T-cells are one of a few somatic cells that retain telomerase function to control naïve T-cell survival, replication potential, and antigenic diversity. To this end, we examined telomere function and replicative burst capacity of MDS T cells as a possible mechanism for immune dysregulation. Methods: Primary specimens from MDS (n=37), AA (n=8), and controls (n=42) were investigated. Peripheral blood mononuclear cells were isolated from patient blood or buffy coats by Ficoll-Hypaque gradient centrifugation. Purified CD3+ T cells were isolated using negative selection and then stimulated with anti-CD3/anti-CD28 T cell activator beads (Dynabead®) for 3 days. Telomere length was assessed by quantitative PCR (q-PCR) and telomerase enzymatic function measured by Telomere Repeat Amplification Protocol (TRAP) assays. Results: Mean telomere length in purified T cells was significantly shorter among MDS patients compared to controls after adjusting for age and sex (p Next, to determine the functional consequences of the disturbance in telomere repair in MDS, the ability of T cells to enter S-phase and to undergo an antigen-induced proliferative burst were examined. TCR signaling was shown to be preserved, evidenced by induction of an early activation antigen CD69. Although some cells were capable of entering S-phase, the replicative burst potential was severely impaired in T cells form all patients. Telomere repair is exclusively present in naïve T cells and progressively declines after memory transition. TCR triggered telomerase activity was measured in sorted naïve (CD45RA+, CD45RO-) and memory (CD45RO+, CD45RA-) T cells. The telomere length in naïve cells was shorter in MDS patients compared to controls (p=0.018) and the telomerase activity was suppressed in naïve MDS T cells (p=0.0207) indicating that telomere dysfunction underlies the altered homeostasis of naïve T cells in MDS, a feature mechanistically akin to AA and other telomere repair disorders. Conclusion: Results of this study indicate that there is loss of telomere maintenance in naïve T cells due to a defect in hTERT transcription is associated with impaired replicative potential. This abnormality in naïve T cell homeostasis represents an inherent defect that contributes to a memory cell growth advantage and repertoire contraction associated with autoimmunity in AA and MDS. Disclosures: No relevant conflicts of interest to declare.

Abstract 1124 CD81 is a widely expressed tetraspanin molecule that physically associates with CD4 and CD8 on the surface of human T cells. Coengagement of CD81 and CD3 results in the activation and proliferation of T cells. CD81 also costimulated mouse T cells that lack CD28, suggesting either a redundant or a different mechanism of action. Here we show that CD81 and CD28 have a preference for different subsets of T cells - primary human naïve T cells are better costimulated by CD81, while the memory T cell subsets and Tregs are better costimulated by CD28. The more efficient activation of naïve T cells by CD81 was due to prolonged signal transduction compared to that by CD28. We found that IL-6 played a role in the activation of the naïve T cell subset by CD81. Combined costimulation through both CD28 and CD81 resulted in an additive effect on T cell activation. Thus, these two costimulatory molecules complement each other both in the strength of signal transduction and in T cell subset inclusions. Costimulation via CD81 might be useful for expansion of T cells for adoptive immunotherapy to allow the inclusion of naïve T cells with their broad repertoire. Disclosures: No relevant conflicts of interest to declare.

Abstract 3002 Adoptive transfer of CMV-specific T cells derived from adult CMV-seropositive (CMVpos) donors can effectively restore antiviral immunity after stem cell transplantation. However due to the absence of CMV antigen-specific memory T cells in cord blood (CB) and adult CMV-seronegative (CMVneg) donors, different culture systems are required to generate virus-specific T cells for adoptive transfer. With a novel protocol we have generated CMVpp65-specific T cells from CB and found that 15/15 CB T cell lines recognized atypical epitopes of pp65. We then explored the generation of CMV-specific CTL from CMVneg donors using a GMP-compliant methodology and studied the epitopes recognized. CD45RA+ naive T cells were selected from the peripheral blood of CMVneg donors and stimulated with pp65-Pepmix-pulsed dendritic cells with supplemented with IL-7, IL-12, and IL-15. For subsequent stimulations T cells were stimulated with pp65-Pepmix-pulsed EBV-LCL and IL-15 or IL-2. CMVpp65-specific T cells (CMV-CTL) expanded from 8 of 11 CMVneg donors were primarily CD8+ T cells (mean 71%). Naïve donor CMV-CTL secreted IFN- γ in response to pp65 peptides (mean 224; range: 38–611 SFC/1×105 cells) compared to irrelevant peptides (mean 12;Range 3–37) as measured in Elispot assays and lysed pp65-pulsed target cells (mean :48; range :15–70%) but not negative controls (mean 22; range 4–40%). These CMV-CTL derived from naive (but not memory) T cells recognized only novel and atypical pp65 epitopes (such as the HLA-A2-restricted epitopes LQTGIHVRV and MLNIPSINV) but not the typical HLA-A2-restricted epitope NLVPMVATV as confirmed by ELISPOT and multimer analysis. These results are similar to CB-derived CTL. Analysis of the avidity of naïve donor CTL specific for the atypical CMV epitopes revealed that the 1/2 maximum effective concentration was similar (mean: 600 pM) to CMVpos CTL recognizing typical epitopes (mean: 300 pM), and more avid than CMVpos CTL recognizing atypical epitopes (mean: 4 nM), highlighting the difference between naïve-derived and memory-derived CTL. TCR sequencing performed on T cells specific for typical (CMVpos) and atypical (CMVpos, CMVneg, and CB) epitopes revealed that CMVpos donor CMV-CTL recognizing typical epitopes were markedly more oligoclonal than CTL recognizing the atypical epitopes derived from CB, CMVpos, or CMVneg donors. To address the concern that atypical epitopes might not be naturally presented by CMV-infected cells and therefore not recognized by in vitro generated CTL, we tested whether CMV CTL (from CB, CMVpos, CMVneg) generated using CMV AD169-infected fibroblasts or CMV VR1814-infected DCs would recognize the same epitopes. As before, CMVpos CMV CTL recognized typical epitopes of pp65 while CB and CMVneg CMV CTL recognized only atypical epitopes, suggesting that the epitopes are naturally processed and presented by APCs, and that the atypical epitopes observed are not an artifact of using exogenous antigens like the pp65 Pepmix. Thus, despite their unusual repertoire, T cells derived from CB or CMVneg donors are likely to control CMV infection. These results reveal major differences in the naïve and memory CMV specific T cell repertoire that merits further exploration. Nevertheless, we demonstrated that atypical epitopes are naturally presented by CMV infected cells and we are now evaluating the clinical efficacy of these CTL in recipients of CBT. These studies should determine if naive T cells primed in vitro are able to persist and establish memory and virus protection in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 2982 In clinical trials of adoptive T cell therapy, the persistence of transferred cells correlates with therapeutic efficacy. The engraftment fitness of ex vivo propagated T cells is in part pre-determined by the differentiation status of precursor T cells from which effector cell products are derived. In peripheral blood, T cells are comprised of three major subsets- naïve, central memory (Tcm) and effector memory (Tem), each having distinct phenotypic and functional attributes. We have previously shown that human CD8+ effector cells derived from Tcm were superior to Tem in their ability to engraft and persist following adoptive transfer (Wang, X. 2011). In this study, we set out to compare the engraftment fitness of CD8+ effector cells derived from naïve precursors with that of Tcm-derived effectors. FACS sorted CD8+ CD45RA+CD62L+ naïve (Tn) and CD8+CD45RO+CD62L+ Tcm cells from healthy donors were expanded in vitro for 14 days in the presence of OKT3, feeder PBMC and IL-2 at 50U/mL. Despite the comparable levels of CD28 expression on unstimulated CD8+ Tn and CD8+Tcm (91±2% for CD8+Naive and 94±2% for CD8+Tcm), Tcm-derived effector T cells (CD8+TCM/E) displayed significantly higher levels of CD28 expression with corresponding high levels of cytoplasmic phosphorylated-AKT as compared to Tn-derived cells (CD8+TN/E) (47±5% for CD8+TN/E vs. 83±4% for CD8+ TCM/E). After the initial 14 days of in vitro expansion, CD8+TCM/E were able to persist in vitro for more than 50 days in the presence of gamma-c cytokines (IL-2 50/ml or IL-15 10ng/ml) while CD8+TN/E failed to survive in either condition, which is consistent with the observed higher expression levels of IL-15R α and IL-2R β by CD8+TCM/E. After engraftment in huIL-15 replete immunodeficient (NSG) mice, we observed that CD8+TCM/E exhibited higher levels engraftment and mounted more robust proliferative responses to in vivo challenge with immunostimulatory vaccines. In contrast to CD8+TN/E, engrafted CD8+TCM/E reaquired/sustained high frequency of cells expressing IL-7Ralpha. To re-direct T cell specificity against B-lymphoid malignancies, purified CD8+Tn and CD8+Tcm from healthy donors were engineered with lentiviral vector encoding CD19R-CD28-EGFRt-epHIV7. CD19-specific CD8+TCM/E persist after adoptive transfer into the human IL15 reconstituted NSG mice, whereas no engraftment was detected in the CD19-specific CD8+TN/E infused NSG mice. These studies using human T cells support the use of central memory derived effector cells for adoptive therapy of cancer and infectious disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 648 Donor lymphocyte infusion (DLI) can be a curative treatment for patients with relapsed hematological malignancies after HLA matched allogeneic stem cell transplantation (alloSCT). However, curative responses in patients with acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphoid blastic phase (CML-BP) are infrequent after HLA matched DLI. This may be partly explained by the poor immunogenicity of these malignancies, since we previously demonstrated efficient induction of Graft-versus-Leukemia (GvL) immune responses in vitro and in vivo upon modification of ALL and CML-BP cells into leukemic antigen presenting cells (APC). Leukemic-APC may be particularly relevant for efficient generation of GvL immune responses after HLA matched DLI, since T cells recognizing allo-antigens in matched HLA molecules are known to reside in the naïve T cell compartment. In contrast, T cells recognizing allo-antigens in mismatched HLA molecules reside in the memory T cell compartment as well. Since memory T cells can also be activated by non-professional APC, HLA mismatched alloSCT and DLI may particularly be considered as a treatment modality for induction of GvL reactivity against poorly immunogenic malignancies. However, T cell responses across HLA barriers can induce severe Graft-versus-Host Disease (GvHD). Mismatched HLA class I molecules, which are broadly expressed on all nucleated cells, are frequent targets of alloreactive T cells. Since HLA class II molecules are predominantly expressed on hematopoietic cells, HLA class II mismatched alloSCT and DLI may more selectively induce GvL reactivity without inducing severe GvHD. In this study, we investigated the in vivo immunogenicity of established B-ALL or CML-BP by comparing the anti-tumor responses after fully HLA matched versus HLA class II mismatched DLI in a NOD/scid mouse model. Mice engrafted with primary B-ALL and CML-BP were treated with DLI from HLA matched (12/12 match) or HLA class II mismatched, but HLA class I matched donors. In mice engrafted with B-ALL or CML-BP, treatment with HLA matched DLI induced expansion of human CD4+ and CD8+ T cells in peripheral blood, but leukemic cells were only delayed in growth, and not eliminated. In contrast, after HLA class II mismatched DLI, leukemic cells rapidly disappeared upon emergence of human CD4+ and CD8+ T cells in peripheral blood. To analyze the specificity of the T cells, we clonally isolated CD4+ and CD8+ T cells from bone marrow and spleens of mice after treatment with DLI. All T cell clones were tested for recognition of patient leukemic cells, donor EBV transformed B cells (EBV-LCL) and murine bone marrow derived dendritic cells in IFNg ELISA. Isolated CD8+ and CD4+ T cell clones recognized either patient leukemic cells or murine cells, indicating that the T cell clones were either leukemia-reactive or xeno-reactive. After HLA matched DLI, only 2 of the 106 CD4+ T cell clones, and none of the 183 CD8+ T cell clones, recognized patient leukemic cells. The majority of isolated CD4+ and CD8+ T cell clones were xeno-reactive, as demonstrated by specific recognition of murine bone marrow derived dendritic cells, or non-reactive against any of the tested target cells. In contrast, after HLA class II mismatched DLI, 95 of the 322 CD4+ T cell clones specifically recognized patient leukemic cells. These leukemia-reactive CD4+ T cell clones were shown to be restricted by the mismatched HLA-DRB3, -DQB1 and –DPB1 alleles of the patient. None of the 49 CD8+ T cell clones were leukemia-reactive, but a significant number of CD8+ T cell clones and remaining CD4+ were xeno-reactive. In conclusion, our data show that HLA class II mismatched, but HLA class I matched, DLI is far more effective in inducing anti-tumor reactivity as compared to HLA matched DLI, whereas the in vivo capacity of both DLI's to induce allo-immune reactivity based on the induction of xeno-reactive T cells was similar. Our study emphasizes the necessity of HLA class II disparity for efficient in vivo induction of HLA class II mediated anti-tumor immunity against poorly immunogenic B-ALL and CML-BP in NOD/scid mice. We therefore hypothesize that use of HLA class II mismatched as compared to HLA matched alloSCT and DLI, despite an increased risk for GvHD, may improve the outcome for patients with HLA class II positive high risk acute lymphoblastic leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 282 RhoA GTPase is an intracellular signal transducer capable of regulating a wide range of cell functions including cytoskeleton dynamics, proliferation, and survival. In lymphocytes, studies by using dominant negative mutant or C3 transferase expressing transgenic mice suggest that RhoA is involved in TCR and BCR signaling and related T cell functions such as polarization, migration, survival, and proliferation. To date, the physiological role of RhoA in lymphocyte development remains unclear. In this study, we have achieved T cell, B cell, and hematopoietic stem cell-specific deletion of RhoA by conditional gene targeting with CD2, CD19 and Mx1 promoter-driven Cre expression, respectively, in the RhoAloxP/loxP mice. First, we found that RhoA gene disruption in early T cells caused a drastic decrease in thymocyte cellularity, with the numbers of CD4−CD8− double negative (DN), CD4+CD8+ double positive (DP), CD4+CD8− single positive (SP), and CD4−CD8+ SP T cells decreased by 88.8% ± 6.0%, 99.4% ± 1.0%, 99.3% ± 1.2%, and 98.6% ± 2.0%, respectively. Among DN subpopulations, CD44+CD25− (DN1), CD44+CD25+ (DN2), CD44−CD25+ (DN3), and CD44−CD25− (DN4) cells were reduced by 91.7% ± 6.0%, 54.9% ± 27.7%, 50.9% ± 33.3%, and 96.7% ± 3.4%, respectively. Further, RhoA knockout led to a significant loss of DP thymocytes at the initial stage (CD69highTCRint) of positive selection, suggesting that RhoA is required for positive selection. The decreased thymocyte cellularity in mutant mice is associated with increased apoptosis of all thymic T lineages. RhoA deficiency also resulted in a perturbation in thymocyte cell cycle progression as manifested by increased BrdU incorporation in DN1 and DN2 cells and decreased BrdU incorporation in DN4 and DP cells. Concomitantly, RhoA-deficient thymocytes showed a 59.8% ± 26.3% reduction in proliferative potential in response to TCR crosslinking. Western blot analysis revealed that the activities of ZAP70, LAT, Akt, Erk, and p38 were impaired in RhoA-/- thymocytes. In periphery, spleens of the RhoA null mice contained 7.4% ± 8.0% of CD4+ T cells and 3.7% ± 2.7% of CD8+ T cells compared with that of wild type (WT) mice. Loss of peripheral mature T cells in mutant mice is reflected by a marked reduction of naive T cells, whereas effector and memory phenotype cells were marginally affected by RhoA deficiency. RhoA-deficient naïve T cells were more susceptible to apoptosis, suggesting that homeostatic defect of naïve T cells in RhoA-/- mice is attributed to impaired cell survival. Abrogation of RhoA caused an increased in vivo BrdU incorporation in naïve T cell compartments. Thus, RhoA deficiency induces naïve T cell homeostatic proliferation, possibly due to a compensatory effect of lymphopenia. In contrast to that in thymocytes, Erk was constitutively activated in RhoA-deficient splenic T cells. These observations implicate RhoA in the multiple stages of T cell development and the proper assembly of early TCR signaling complex. Second, deletion of RhoA in pre-proB cells had no effect on early B cell development in bone marrow but significantly inhibited late B cell development in spleen, resulting in 78.2% ± 13.6%, 78.6% ± 16.9%, and 93.2% ± 3.4% reduction in transitional, follicular, and marginal zone B cells, respectively. Plasma cells in spleen were decreased by 50.9 % ± 25.9% in RhoA null mice. However, we did not detect any changes in survival of in vivo RhoA-/- B cells or RhoA-/- B cells cultured in vitro with survival factor BAFF. Distinct from previously characterized Cdc42 knockout mice, BAFF-R expression was not altered in RhoA-/- B cells. Moreover, RhoA-/- B cells appeared to be normal in proliferation and Akt and Erk activation in response to BCR crosslinking. These data suggest that RhoA is important for late B cell development through regulation of differentiation but not cell survival or proliferation. Finally, deletion of RhoA from hematopoietic stem cells did not affect common lymphoid progenitor production, indicating that RhoA is not required for early lymphoid progenitor commitment. Taken together, these lineage-specific mouse genetic studies demonstrate that RhoA critically regulates T and B cell development by distinct cellular mechanisms at multiple stages of lymphopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 959 Positive and negative selection of developing thymocytes is mediated primarily by cortical and medullary epithelial cells (CEC and MEC), respectively, in the thymus. Tolerance to peripheral tissue antigens (PTA) not normally expressed in the thymus can result when PTA expression is induced by AIRE in MEC or when corticomedullary dendritic cells (DC) present endogenous antigens, both mechanisms contributing to deletion of potentially auto-reactive T cells. However, in some instances, negative selection may be incomplete, leading to the release of autoreactive cells into the periphery. Indeed, T cells specific for PR1, the HLA-A2-restricted self-peptide derived from proteinase 3 (P3) and neutrophil elastase (NE), are present in peripheral blood of healthy adults, albeit at extremely low frequency of fewer than 0.0005% of CD8+ T cells. This suggests there is strong thymic central tolerance to PR1. However, PR1-CTL can increase from 0.1 to 2% of peripheral blood CD8 T cells in CML patients treated with interferon or stem cell transplant, and similar levels can be achieved after PR1 peptide vaccination, which suggests that under some circumstances this central tolerance can be reversed. Because of the increasing use of umbilical cord blood (UCB) as an alternative donor source and because incomplete graft-versus-leukemia (GVL) immunity after UCB transplant contributes to relapse, we sought to determine whether PR1-CTL can be expanded from UCB. We hypothesized that PR1-CTL frequency should be low in UCB due to central tolerance. Surprisingly, we found PR1-CTL at a frequency ranging from 0.007 to 0.345% (mean 0.117%) of CD8+ cells in 57 HLA-A2+ cord blood units, similar to what is observed in immunologically responsive vaccine patients and 100- to 1000-fold higher than in healthy adults. The PR1-CTL were predominantly CCR7+CD45+CD28+ and did not efficiently expand ex vivo following peptide stimulation and low dose IL-2, which was consistent with a naive T cell subset. Therefore, this data suggests that central tolerance to PR1 is incomplete. To study whether PR1 is expressed in human thymus, we used the PR1/HLA-A2-specific antibody 8F4 to study PR1 expression. Thymic CEC and MEC expressed no P3, NE, or PR1 by flow cytometry or immunofluorescence imaging of sectioned fetal thymus, which is consistent with previous reports showing absence of P3 or NE induction by AIRE in MEC. Interestingly, by flow cytometric analysis, we found that PR1 is expressed on the surface of thymic dendritic cells (DC) exclusively. This selective expression of PR1 was further confirmed via immunoflourescent staining of sectioned fetal thymus, and the PR1-expressing DC were localized to the corticomedullary junction and medulla. Thus it appears that PR1 expression by DC in the thymus is insufficient for complete central tolerance to PR1. Furthermore, because we have previously shown that PR1-overexpressing CML cells can induce apoptosis of high affinity PR1-CTL, it is possible that peripheral tolerance mechanisms are most critical for preventing autoreactivity to PR1 in humans. These observations suggest possible strategies to overcome tolerance to PR1 by modifying DC uptake and cross-presentation of soluble P3 and NE, when selective autoimmunity may be desirable for leukemia patients or detrimental for patients with vasculitis such as Wegener's granulomatosis. Disclosures: No relevant conflicts of interest to declare.

Abstract 2659 Poster Board II-635 Introduction: Cord blood transplantation (CBT) are being more and more used both for children and adults. However, one of the major limitation remains the low virus- CTL (cytotoxic T lymphocyte) response associated with the delayed immune recovery. Because the large majority of cord blood lymphocytes are naïve and so are not able to rapid clonal expansion, the monitoring of patients undergoing CBT provides a unique opportunity to 1) better understand the development of innate and adaptive human immune system and 2) identify the immune parameters associated to the viral response especially innate immunity (NK and gammadelta T cells) as well as T cells. Specifically, we have analysed their status of activation, function, and the expression of receptors (PD-1, 2B4, BTLA) recently demonstrated to be associated to immune escape. Materials and Methods: We investigated children with severe CMV or EBV disease following CBT and compared them both with children who controlled viral-reactivation following CBT or allogenic bone marrow transplantation (BMT) and with healthy donors. All patients received myeloablative conditioning including ATG and achieved full donor chimerism. Moreover, none of the selected patients received corticosteroid at the time of disease. CMV and EBV-specific CD8 T cells were investigated using HLA class I pentamers or tetramers together with extensive phenotype analyses by multi parametric flow cytometry and functional assay were performed using IFN gamma ELISPOT assay. Results: Kinetic of immune recovery showed a dramatic increase of CD8 but no CD4 T cells in patients with CMV or EBV disease. All CD8 T cells were highly activated (CD69high, CD57 high, PD-1 high, BTLAlow, 2B4 high, CD28 high). Virus-specific CTL were present in variable proportion. The major population of EBV-CTL were differentiated in CD45RA-CD27-CD28- late effector memory (EM) with almost no effector memory CD45RA+ cells (TEMRA) nor EBV specific activated naïve T cell. This is in sharp contrast with EBV-CTL of either: 1) healthy volunteers with controlled viral infection, who were mainly in a CD45RA-CD27+CD28+ Central Memory (CM) and CD45RA-CD27+CD28- early EM stage of differentiation; 2) patients with EBV infection during post transplantation immunosuppression who have activated naïve and TEMRA EBV-CTL. CMV-CTL were also mainly in late EM but no TEMRA differentiation stages in contrast to healthy volunteers and patients who controlled their CMV infection after CBT. Virus- CTL were highly activated (High expression of 2B4, CD28, PD-1, DR) with an overexpression of inhibitory receptors as PD-1 and 2B4 but no BTLA. The T cell response against viruses was highly variable in regard to different virus and patients as analysed by IFNg ELISPOT assay. CD4 T cells although low ( Conclusion: Thus, these reports illustrate a full activation of innate effectors as well as virus-CTL during viral disease after CBT, despite lack of viral clearance. We have identified at least three mechanisms that could explain this apparent immune failure: Perspectives: These data support for the first time the hypothesis that after CBT, the immune system although quantitatively insufficient, is highly activated and so that in-vitro expansion of patient self-lymphocytes or means to expand CD4 and CD8 T cells pools are a potential way to cure patients suffering from uncontrolled viral replication after CBT together with blockade of inhibitory pathways. Disclosures: No relevant conflicts of interest to declare.

Abstract 709 The differentiation and functional specialization of effector T cells allows for effective immune response to diverse insults. However, tight regulation of effector T cell responses is required for effective control of infections and avoidance of autoimmunity. Naïve CD4 T cells can differentiate into IFN-γ-secreting type I (Th1) cells and IL-4-secreting type II (Th2) cells. Recently, the Th1/Th2 paradigm of T helper (Th) cells differentiation has been expanded following the discovery of a third subset of effector Th cells that produce IL-17 (Th17). Regulatory T (Treg) cells have a remarkable ability to prevent naïve T cell differentiation into Th1 and Th2 cells and to suppress immune responses driven by Th1 and Th2 effector cells. The role of Treg cells in regulating IL-17 production remains undetermined. Some studies suggest that Treg cells may promote differentiation of naïve T cells into Th17 cells in the context of inflammatory cytokine milieu. The aim of our present study was to determine the role of Treg cells and conventional CD4+ T cells (Tconv) in the differentiation of IL-17 producing cells in the absence of exogenous cytokines and insults. Naïve Tconv cells stimulated with anti-CD3 mAb in the presence of antigen presenting cells (APCs) secreted significant amounts of IFN-γ and IL-4 but no detectable levels of IL-17, whereas Treg cells were incapable of producing any of these cytokines under the same culture conditions. Production of IFN-γ and IL-4 was significantly reduced by addition of Treg cells in the cultures of Tconv cells with anti-CD3 mAb and APC. In contrast, production of IL-17 was considerably enhanced in these co-culture conditions and the level of IL-17 displayed a positive correlation with the number of Treg cells added in the culture. To evaluate whether TCR-mediated stimulation of both Treg and Tconv cells was required for IL-17 production, we used Tconv cells and Treg cells from two different TCR transgenic mouse strains in H-2b background, 2D2 (MOG35-55-specific) and OT-II (OVA323-339-specific), respectively, and co-cultured them in the presence of APCs (H-2b). Production of IL-17 was not observed when either MOG peptide or OVA peptide alone was added in the cultures. In contrast, addition of both MOG and OVA resulted in production of IL-17, suggesting that simultaneous activation of Tconv and Treg cells was essential for induction of IL-17. To determine the source of IL-17 during co-culture of Treg and Tconv cells, we purified Treg cells from C57/B6 mice and co-cultured them with Tconv cells from the B6 congenic mouse strain B6.PL, which carry the Thy1a (Thy1.1) allele and can be easily recognized by flow cytomeric analysis using a Thy1.1-specific mAb. Detailed evaluation during co-culture revealed that a significant proportion of Thy1.1- T cells (the source of Treg) gradually downregulated expression of Foxp3 while obtaining expression of IL-17. In contrast, there was no significant change in the expression of either Foxp3 or IL-17 in the Thy1.1+ population (the source of Tconv), suggesting that Treg was the main source of IL-17 when stimulated in the presence of antigen and activated Tconv cells. Several cytokines have been implicated in the induction of IL-17, in particular, TGF-β. For this reason, we investigated the potential involvement of TGF-β in this conversion process. Addition of TGF-β to Tconv cultured with APCs and anti-CD3 mAb in the absence of Treg cells resulted in upregulation of Foxp3 but not IL-17. In contrast, addition of TGF-β neutralizing antibody to Tconv cultured with APC and anti-CD3 mAb in the presence of Treg, suppressed IL-17 production. Moreover, assessment of TGF-β signaling in Tconv and Treg cells revealed a dramatically increased level of Smad3 phosphorylation in Treg compared to Tconv cells, indicating a reduced threshold of TGF-β mediated signaling in Treg cells. Taken together, our data indicate that reciprocal interactions of Treg and Tconv cells are required for conversion of Treg into IL-17 producing cells and that TGF-β-mediated signaling is required for this process. In addition, our results provide evidence that Treg may convert into proinflammatory effectors producing IL-17, under conditions that promote Tconv differentiation into Treg cells. These observations provide a new dimension to our understanding of Treg cells functions and may have important implications in therapeutic strategies using Treg cells. Disclosures: No relevant conflicts of interest to declare.

Acute graft-versus-host disease (aGVHD) occurs in approximately 20–50% of matched-related stem cell transplants (HCT). The presence of increased frequencies of CD4+CD25+ regulatory T cells (Treg) in murine donor grafts has been shown to ameliorate aGVHD. In similar models, several groups have demonstrated that the transfer of equivalent numbers of naïve T cells (vs. various memory subsets) confers a relatively greater risk for aGVHD. To test the hypothesis that natural variations in donor graft naïve and Treg content would be associated with the incidence of aGVHD in graft recipients, we conducted a detailed analysis of donor graft T cells within unmanipulated grafts of 70 SCT recipients transplanted for AML/MDS following the administration of a uniform fludarabine/busulfan conditioning regimen. Median follow up time was 38 months. 19% (n=13) of recipients experienced grades II–IV aGVHD (n=3 grades III–IV). Cryopreserved donor grafts samples were analyzed using 9-color flow cytometry (with a panel including a viability dye and MAbs recognizing CD4, CD8, CD25, CD127, CD45RA, CD27, Foxp3, and Ki-67). Tregs were defined as being CD4+CD25+CD127loFoxp3+. CD45RA and CD27 were used to measure naïve (CD45RA+CD27+) and memory conventional CD4+ and CD8+ T cells. In addition, the proliferating fraction of all cells was defined by Ki-67 nuclear-antigen expression. We conducted analyses based on donor graft T cells assessed both continuously and by quartiles, with similar results. No significant associations were found between aGVHD incidence and total Tregs (HR 1.01, 95%CI 0.9–1.2, p 0.8), nor with proliferating Tregs (HR 1.2, 95%CI 0.2–5.9, p 0.8). Since murine models typically fix the dose of Tregs vs. conventional T cells (Tcon), we also analyzed GVHD risk by donor graft Treg:Tcon ratio, finding no association (HR 1.03, 95%CI 0.8–2.1, p 0.3). Although patients in the highest Treg:Tcon quartile received Treg doses known in vitro to induce suppression (>1:21.5), there was no reduction in aGVHD (HR 1.5 Quartile 4 vs others, 95%CI 0.5–4.9, p 0.5). Additionally, no significant association with aGVHD was found with total naïve T cells (HR 1, 95%CI 0.9–1.0, p 0.9), naïve CD4+ (HR 1, 95%CI 0.9–1.03, p 0.6), or naïve CD8+ cells (HR 0.98, 95%CI 0.9–1.03, p 0.6). Because naïve:memory cell ratios were also fixed in murine models demonstrating the importance of naïve T cells in GVHD, we also assessed naïve:memory T cell ratios within donor grafts with respect to recipient aGVHD. No reduction in aGVHD was observed considering naïve:memory ratios analyzed continuously (HR 1.1, 95%CI 0.3–4.2, p 0.8) or in the quartile receiving the lowest naïve:memory ratio (HR 0.5 Quartile 1 vs others, 95%CI 0.1–2.2, p 0.3). Our results demonstrate that donor graft Treg and naïve T cell content, assessed in the context of unmanipulated PBSC transplantation in matched siblings, does not predict recipient aGVHD incidence. These data suggest that the therapeutic efficacy of Treg enrichment and/ or naïve T cell depletion may require grafts to be manipulated to supraphysiologic levels for maximal therapeutic benefit.

BACKGROUND: A subset of patients with Myelodysplastic Syndrome (MDS) responds well to immunosuppressive therapy (IST) and the only validated predictor of response is age, with younger patients faring much better than older patients. Hematologic improvement on immunosuppressive therapy is associated with a survival benefit with response rates ranging from 15% to 50%, clearly comparable or better than results with other existing therapies in MDS. Despite progress in the basic understanding of immune pathobiology of MDS and a clear therapeutic value, including improved long-term survival, IST including anti-thymocyte globulin (ATG) and/or cyclosporine A (CyA) is rarely offered to MDS patients in the U.S. due to uncertain criteria for selection of patients and potential toxicities. In addition, there is an underlying concern that inappropriate use of immunosuppressive therapy may negatively impact risk for leukemia progression, which occurs in 30–40% of MDS cases. The long-term goal of this study is to identify an immune signature that has postive predictive power for IST responsiveness. METHODS: To determine the effect of age on T-cell homeostasis and function and IST response, we performed a study of 54 MDS patients compared to 37 healthy controls. In a pilot study, T cell abnormalities associated with response to equine anti-lymphocyte globulin (eATG, lymphoglobulin, Pfizer, Inc) and/or CyA was studied in 12 younger MDS patients composed of 6 responders and 6 non-responders. RESULTS: CD4+ T-cells are normally present in the peripheral blood lymphocyte pool at 2 to 4 times greater than that of CD8+ T-cells, and diminished CD4:CD8 ratio has been previously shown to correlate with poor survival outcome in MDS. Similar to previous reports, we found that the age-adjusted CD4:CD8 ratio was reduced in MDS patients compared to healthy controls (p-value CONCLUSIONS: Association between the T-cell abnormalities reported in this study and response to IST strongly suggests that aberrant T-cell homeostasis may represent a critical determinant of autoimmunity in MDS that may have positive predictive power for response to IST.

Our group has published encouraging results in adult patients who underwent full haplotype mismatched related graft after a novel chemotherapy-alone conditioning regimen (Lacerda et al, Biol Blood Marrow Transplant9:633, 2003 and Biol Blood Marrow Transplant11:399–400, 2005). With a longer follow-up, patients with acute myeloid leukemia transplanted in 1st, 2nd, or 3rd remission have a 57% probability of disease free survival at 8 years. In the current study, we assessed the degree of long-term immune reconstitution in patients submitted to a haploidentical HSCT more than 5 years ago. A total of 5 patients (mean age 25 years) at a median of 7 years post-transplant were compared with their parental donors (mean age 48 years) and 5 age-matched controls (AMC) (mean age 29.5 years). There were no significant differences in absolute counts of CD3+, CD4+, CD8+, CD19+ and CD56+CD16+ cells. Within the CD4+ population, there was a trend for an increased percentage of naïve cells (CD45RA+CD62L+CD27+) in patients (57.8%) as compared to donors (46.1%) and AMC (53.4%). This was associated with a decreased percentage in late memory CD4+ cells (CD45RO+CD45RA-CD27-) in the patients (4.3%) as compared to donors (9.1%) and AMC (7.1%). In the CD8+ population, patients had a significantly higher percentage of naïve (CD45RA+CD45RO-CD27+) cells than their donors (67.9% versus 29.6%, p=0.002) (also significant in absolute number counts) and a lower percentage of late memory CD8+ (CD45RA+CD45RO-CD27-) cells than both their donors (2.2% versus 28.6%, p=0.0001) (also significant in absolute number counts) and AMC (2.2% versus 9.2%, p=0.05). With respect to the evaluation of the relative contributions of de novo thymopoiesis and peripheral expansion to the maintenance of the naïve T cell pool, patients had a significantly greater proportion of recent thymic emigrants within the CD4+ T cell population (CD45RA+CD62L+CD31+) than both their donors (40.8 versus 20.7%, p=0.019) and AMC (40.8% versus 17.5%, p=0.0006). In agreement with these observations, patients had higher levels of TRECs than both their donors and AMC, although this did not reach statistical significance. Telomeres of naïve CD4+ and CD8 + cells were shorter in patients as compared to AMC, but longer than their older donors. In CD4+ naïve T cells, the difference in telomere length reached statistical significance when patients and AMC (p=0.03), and when donors and AMC were compared (p=0.03); whole differences in CD8+ T cells did not reach statistical significance. We also found decreased serum IL-7 levels in patients as compared to donors (5 pg/ml versus 6.8 pg/ml, p=0.04) and AMC (5 pg/ml versus 9.5 pg/ml, p=0.005). Furthermore, our patients also had normal proportions of regulatory T cells and within this population we found a significantly higher percentage of cells with a naïve phenotype (CD45RO-CD62L+) when compared to donors (37.9% versus 18.8%, p=0.02) and AMC (37.9% versus 18.4%, p=0.01). Together, our data suggest that despite the HLA mismatch, there is a major contribution of thymic output to the reconstitution of the T cell pool, resulting in a normal balance of naïve and memory T cell subsets in these patients.

Besides its role in hemato- and lymphopoiesis, bone marrow (BM) has emerged as a secondary lymphoid organ with important roles in both T cell priming and memory responses. Due to these properties, non-malignant T cells persisting within the BM of patients with acute leukemias may be involved in the immune response to leukemia and the control of minimal residual disease. Here, we investigated the phenotypic signature of residual T cells present at diagnosis in 25 pediatric patients (age 2–16 years) with B cell precursor ALL. Patients with high risk disease including Philadelphia chromosome-positive or MLL-rearranged leukemias were excluded from this analysis. Mononuclear cells were isolated from freshly aspirated BM by density gradient centrifugation and analyzed by six-color-flow cytometry using monoclonal antibodies directed towards various T-cell associated surface and intracellular markers, including CD3/CD56/TCRαβ/TCRγδ/CD86/HLA-DR (Panel 1); CD3/CD4/CD8/CCR7/CD45RA/ CD45RO (Panel 2); CD4/FoxP3/CD25/CD45RA/CD45RO/CCR7 (Panel 3). For each sample, ≥15,000 CD3+ cells (Panel 1, 2) or ≥8,000 CD4+ cells (Panel 3) were analyzed with FACS Canto and Diva Software. The Student’s t test was used to determine statistical significances between individual subgroups, and correlations were performed using the Pearson test. Consistent with published data on BM T cell subsets in healthy donors, the CD4+/CD8+ T cell ratio was 1.32±0.41. The predominant subset among CD8+ T cells (55.2±17.6%) had a naïve T cell phenotype (CD45RA+CCR7+), while 20.7±11.5% were effector memory T cells (TEM; CD45RA-CCR7-), and 7.1±6.2% were central memory T cells (TCM; CD45RA-CCR7+). No differences were found between TEL/AML1 positive or negative leukemias, or between patients stratified into standard (SR) vs. medium risk (MR) groups according to the criteria of the ALL-BFM 2000 study group. T cells bearing γδ T cell receptors have been attributed important roles in the primary immune defense against microbes and in immune control of cancer. We found that 6.9±3.0% (1.8 to 11.8%) of BM T cells were γδTCR+ (Vγ9Vδ2). A statistically significant (p

T cell depletion (TCD) of donor allogeneic blood or marrow grafts can reduce the incidence and severity of acute graft-versus-host disease (GvHD) but may place the patient at risk for disease relapse. This multicenter randomized trial examined the impact of selective depletion of αβ T cells (αβTCD) + cyclosporine immunosuppression in recipients of unrelated marrow grafts vs. T-replete marrow grafts + methotrexate and cyclosporine immunosuppression. Previously reported data from this trial have shown that 3-year disease-free survival (DFS) did not differ between these groups, although the αβTCD group showed improved neutrophil recovery, decreased incidence of acute GvHD, and reduced grade III-IV toxicities. In order to further clarify these findings, we examined the impact of each graft manipulation and immunosuppression strategy on lymphocyte subset recovery and associated outcomes. Lymphocyte recovery was assessed at 1, 3, 6, 12, 18, and 24 months post-SCT. Of all patients accrued, only patients that had survived for at least one year and who had at least four separate immunophenotyping assessments were included in this analysis (unmanipulated grafts = 66/206 32.0%; αβTCD grafts 57/203 28.1%). Non-parametric pair wise tests were used to compare recovery of absolute cell counts at each measurement period. We also fit a mixed model to each individual’s data to estimate the rate of change in cell counts in the two groups. The slope estimate for each subject obtained from the mixed model was then used as a variable in models to assess DFS. The Cox proportional hazards test was used to examine associations between recovery of individual cell subsets and DFS. Recovery of total absolute CD3, αβ, γδ, CD3+CD4+, CD3+CD8+ T cell count as well as the absolute B cell count did not significantly differ between groups for at each measurement period. CD3+CD4+CD45RA naive T cell recovery remained below the expected adult normal range for all time points in both groups but was improved in patients who received unmanipulated grafts. Interestingly, NK cell absolute count recovery was significantly improved in patients that received TCD grafts over those who received unmanipulated grafts (p = 0.001) through the first year post-SCT. Analysis of the impact of individual cell subset recovery on DFS revealed that an absolute γδ T cell count greater than 1.75 x 105/ml at any time during the first year predicted for improved DFS regardless of treatment group (p = 0.05) consistent with previous single-institution reports. Although the αβTCD regimen did not result in improved DFS in this trial, these findings show that αβTCD is a reasonable graft engineering strategy that can reduce the incidence of acute GvHD without producing a significant negative impact on lymphocyte subset recovery. Indeed, patients in the αβTCD group showed markedly improved NK recovery. Approaches that are currently in early-phase trials such as post-SCT targeted T and/or NK cell therapies could be used to supplement the αβTCD regimen, potentially improving relapse-free survival.

Defining the epigenetic mechanisms (e.g. chromatin modifications) that underlie T cell fate decisions is a major challenge. The transcriptional coactivators CREB binding protein (CBP) and the closely related p300 comprise a two-member family of histone/protein acetyltransferases that interact with over 50 T lymphocyte-essential transcriptional regulators. Rather than having distinct regulatory roles, CBP and p300 are often thought to confer utilitarian transactivation and histone modifying functions to transcription factors that mediate T cell fate. In contrast to this view, we show here that CBP acts uniquely in conventional T cell development. Inactivation of CBP, but not p300, starting at the double negative stage of T cell development yielded thymocytes with partial activation of an effector/memory- or innate-T cell program. CD8SP thymocytes from CBP mutant mice expressed genes that define professional CD8 cells such as Il-2/Il-15 receptor β chain, granzyme A, interferon γ (Ifnγ), Fas ligand, perforin, and the chemokine receptors Ccr5, and Cxcr3. CD4SP thymocytes from CBP mutant mice also expressed effector genes such as Ifnγ, Il-4, and Ccr5. In addition, CD8SP and CD4SP thymocytes from CBP mutant mice produced Ifnγ protein when the cells were stimulated with phorbol ester and ionomycin. Mechanistically, loss of CBP acted cell non-autonomously to induce the expression of the CD8 T cell master regulatory transcription factor eomesodermin (Eomes). This suggests that CBP in thymocytes or T cells controls an extracellular factor that helps demarcate conventional naïve T cell development in the thymus from effector/memory T cell differentiation in the periphery.

INTRODUCTION: Graft-versus-host disease (GVHD) is a major complication following allogeneic bone marrow transplantation. Despite advances in understanding the etiology of GVHD it remains a formidable obstacle to the widespread application of BMT. T regulatory (Treg) cells have been shown to inhibit GVHD while preserving the beneficial graft-versus-leukemia (GVL) effect. Published studies by numerous groups, including our own, have shown the importance of Treg homing molecule expression in preventing GVHD. Of note, the Treg population that expressed high levels of the adhesion molecule L-Selectin (CD62L) was shown to be capable of promoting BM engraftment and inhibiting lethal acute GVHD, whereas the CD62Llo population was not. While these studies provided evidence that the phenotype of the CD62Lhi Treg population was responsible for inhibition of GVHD they did not directly assess the role of CD62L. METHODS: To investigate the importance of CD62L in Treg mediated protection from GVHD we utilized a well-described parent into F1 model. We transferred Tregs (2×106) from CD62L deficient mice (CD62L−/−) or wild type (WT) C57BL/6 mice with WT naïve T cell effectors (5×106) into lethally irradiated B6D2 recipients. Our results demonstrated similar survival of recipient mice receiving CD62L−/− Tregs (40% survival) compared to those receiving WT Tregs (60% survival: p =0.66). In addition fluorescence microscopy revealed no difference in GFP+ T cell effector migration/accumulation in GVHD target organs (liver, lung, GI tract) or secondary lymphoid organs (spleen, mesenteric lymph node, inguinal lymph node, Peyers Patches) in the presence of CD62L−/− or WT Tregs. Also, we tracked the migration of GFP+ CD62L−/− Tregs in vivo by fluorescence microscopy and found they were able to efficiently migrate to secondary lymphoid organs and GVHD target organs. Surprisingly, GFP+ CD62L−/− Tregs were also able to home to peripheral lymph nodes. CONCLUSIONS: These results suggest CD62L is dispensable for Treg mediated suppression of GVHD and that the function of the L-selectinhi Treg fraction may be due to the difference in the expression of other homing/migration molecules necessary for Treg migration into lymphoid and GVHD target organs. Figure Figure

T cell homeostasis is regulated by several molecules among which Interleukin 7 (IL-7) plays an essential role for survival and homeostatic proliferation of peripheral naive T cells. In a previous study we demonstrated whether human mesenchymal cells could be engineered with IL-7 gene to produce functional level of this cytokine. Now we analysed the impact of different quantities of IL-7 produced by mesenchymal cells on survival and proliferation of a negative immunoselected naive (CD3+/CD45RA+) T cell population. Co-cultivation of peripheral naive T cells with mesenchymal cells producing low (16 pg/ml) or high (1000 pg/ml) IL-7 levels or in presence of exogenous IL-7 (0,01 ng/ml and 100 ng/ml) maintained the CD3+/CD45RA+ naive T cell phenotype. The chemokine receptor CCR7+ expression was also maintained among this T cell population. Naive T cell molecular characteristics were maintained as assessed by the Vβ spectratyping complexity score which shows the maintenance of a broad T cell repertoire. No Th1 or Th2 differentiation was observed as assessed by IFNγ or IL-4 accumulation. In contrast only mesenchymal cells producing high IL-7 amount caused increases in the activation (CD25 31.2%±12 vs 10%±3.5, p0.05) and phase S cell cycle (15% vs 6.9%, p>0.05). Exogenous IL-7 did not exert any significant effect. In conclusion, we demonstrated that IL-7 produced by mesenchymal cells has a dose-independent effect on naive T cell survival while exerts a dose-dependent effect on activation/proliferation. Due to continuous production of IL-7 by engineered cells, our system emerge as more efficacious than the exogenous IL-7.

Background: Nucleoside analogs have potent anti-tumor activity, especially against lymphoid malignancies, with immuno-suppression being a significant toxicity. We have previously described the immunosuppressive effects of fludarabine on murine T-cells, and found selective cytotoxicity against naïve subsets that reduced their allo-reactivity in a GvHD model of murine BMT (Giver et al 2003 BBMT 9:616). In this study we tested the cytotoxicity of fludarabine and pentostatin against human and murine T-cell subsets to determine their immune-suppressive activity, mechanism of action, and their potential utility as agents to decrease the allo-reactivity of allogeneic donor lymphocyte infusions. Methods: Peripheral blood mononuclear cell samples from normal donors were incubated ex vivo ex vivo treatment with Flu at doses of 5, 50, and 250 mcg/ml or pentostatin at doses of 1, 10, and 100 mcg/ml at 0, 4, 24, and 48-hour time points following 1 or 24 hour drug exposure. All samples that obtained dCF also received 25 mcg/ml of deoxyadenosine (dAdo), which enables dCF to mediate cytotoxicity against lymphocytes. Control samples for fludarabine and pentostatin treatment were untreated or treated with dAdo only, respectively. The total number of viable cells and the fractions of the CD4 and CD8 memory and naive T cell subsets that survived nucleoside exposure were determined by multi-parameter flow cytometry following staining with antibodies to CD3, CD4, CD8, CD45RA, CD6L, Annexin, and 7-AAD. Results: The decrease in the viability of the human naïve CD4 T cells, whether treated with fludarabine or pentostatin, was rapid and most notably observed after the 24-hour time point. On the other hand, the human memory CD4 T cells displayed a more gradual decline in percent viability in both treatment with fludarabine and pentostatin. Furthermore, the potency of pentostatin was exhibited in that it achieved a comparable effect to fludarabine while at substantially lower doses. Data obtained from the murine model also demonstrated a relative a relative sensitivity of the CD4 naïve T cells as oppesed to the CD4 memory to purine analog exposure. Conclusions: Future work will determine the optimal dosage and period of incubation for pentostatin and fludarabine that can utilized to induce immunosuppresion ex vivo in donor lymphocytes. SELECTIVE DEPLETION OF NAÏVE CD4+ T-CELLS FOLLOWING IN VITRO EXPOSURE TO PURINE ANALOGS SELECTIVE DEPLETION OF NAÏVE CD4+ T-CELLS FOLLOWING IN VITRO EXPOSURE TO PURINE ANALOGS

Graft-versus-host-disease (GVHD) remains the major complication of allogeneic stem cell transplantation (SCT). While pharmacological approaches have long been the mainstay of GVHD prevention, recent strategies have focused on cellular approaches, specifically the use of regulatory cell populations to prevent or mitigate the severity of GVHD. Gr-1+Mac-1+ myeloid-derived cells, termed myeloid suppressor cells (MSCs), are a regulatory cell population that has been shown to play an important role in the suppression of tumor and anti-infectious immunity. In these studies, we examined whether these cells were detectable in GVHD recipients and played any role in the suppression of alloreactive T cell responses in vitro and in vivo. Lethally irradiated Balb/c (H-2d) mice were transplanted with MHC-incompatible C57BL/6 (H-2b) bone marrow and spleen cells to induce GVHD. Immature myeloid cells with a Gr-1+Mac-1+ phenotype were found to constitute 60% of all splenocytes at day 10 post-SCT thereafter declining to 30% by day 21. MSCs isolated from the spleens of mice with GVHD expressed high levels of class I and II, but low levels of the costimulatory molecules CD80, CD86, CD40 and were negative for CD11c. Highly purified Gr-1+ Mac-1+ cells obtained from the spleens of GVHD mice 10 or 21 days post-SCT potently suppressed naive T cell proliferation in a standard MLC by 70% at a responder to suppressor ratio of 1:1. Notably, MSCs could also be generated in vitro from normal B6 BM after 6 days in culture with G-CSF and were equally potent at suppressing T cell alloreactivity. Suppression was partially mediated by nitric oxide (NO) as addition of the NO inhibitor L-NMA reversed 50% of the inhibitory effect. To determine whether MSCs exerted a suppressive effect in vivo, lethally irradiated Balb mice were transplanted with B6 BM plus naïve spleen cells with or without MSCs obtained from the spleens of GVHD animals 10 or 21 days post-BMT. The adoptive transfer of MSCs failed to protect mice from GVHD when assessed by overall survival, serial weight measurements or histological analysis. As an alternative approach to augment GVHD protection, G-CSF was administered for 14 days beginning at the time of BMT to enhance the in vivo survival of MSCs. G-CSF administration in both irradiated and non-irradiated MHC-incompatible GVHD mouse models, however, also had no protective effect. We reasoned that a possible explanation for the lack of an effect in vivo was that MSCs did not appropriately localize to nodal sites where GVHD is initiated. To examine this question, B6-green fluorescent protein (GFP) mice were used as donor animals to permit the detection and trafficking of MSCs in vivo. Adoptive transfer of B6 GFP-MSCs from the spleens of GVHD mice 10 days after SCT demonstrated that these cells could be detected in the secondary lymphoid organs of recipient mice 20 hours after BMT, but were completely absent by day 3, supporting the premise that the lack of protection was attributable, at least in part, to the inability of these cells to persist at nodal sites where T cell priming against host alloantigens was occurring. These results indicate that MSCs, which are present in the spleen of GVHD recipients, show potent T cell suppressive capacity in vitro, but fail to mediate a similar effect in vivo. Lack of protection appears to be due to the inability of these cells to localize to all critical secondary lymphoid sites underscoring the importance that the capability of regulatory cells to migrate to and persist at appropriate tissue sites plays in the design of cellular strategies to prevent GVHD.

Treatment of patients with relapsed or persistent acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL) after allo-SCT with donor lymphocyte infusions (DLI) is frequently unsuccessful, probably due to the limited immunogenicity of these malignant B cells, and the lack of specificity of the T cells. In this study, we generated a broadly applicable stimulation and isolation procedure for the induction of leukemia-reactive T cells under good manufacturing practice (GMP) conditions. First, B-cell leukemias were modified into professional antigen presenting cells (APC). We previously demonstrated that CD40 crosslinking was required for the production of appropriate malignant B cell-APCs. CD40L expressing mouse fibroblasts were potent activators of CD40, but not GMP approved. CD40L trimers and CD40 antibodies crosslinked to plates or beads only minimally triggered CD40 on malignant B cells. As previously demonstrated, activated T cells briefly express CD40L. We first determined the dynamics and kinetics of CD40L expression on peripheral blood (PB) donor T cells after stimulation with CD3/28 Dynal T cell expansion-beads added in a ratio of 1 bead/10 PBMC. The kinetics were highly variable, with the optimal surface expression of CD40L on a median of 20% of the T cells (range 10–35%, n=6) being detectable between day 1 and 5. We next investigated the capacity of these activated T cells to stimulate malignant B cells in PB from patients with ALL or CLL containing 60–95% leukemic cells. We developed a two-step strategy using special CD3/28 Dynal isolation beads applicable not only for T cell activation, but also for T cell isolation. Using these beads, the T cells were depleted from the PB of the patient, directly irradiated and added back to the B cell cultures. After 3–7 days all B cells displayed a good APC phenotype with expression of CD80, CD86, and CD83 in the patient samples containing >5% T cells. If insufficient T cell numbers were present, donor T cells could be used as source of CD40L. Leukemia-reactive T cells could be reproducibly generated after two stimulations of fully HLA matched donor T cells with the leukemic APC under mild stimulatory conditions, followed by isolation of the responding T cells based on their production of interferon gamma (IFNg). Next, we investigated whether the leukemia-reactive T cells were the result of the successful induction of a primary immune response or that recognition of mimicry epitopes by previously activated T cells was underlying the activity. We separated unmodified donor T cells into CD45 RO+ memory cells, CD45RA+/CD27+ naive cells, and CD45RA+/CD27− effector cells by MACS CD45RO depletion, followed by FACS sorting on CD27, and stimulated these fractions with malignant APCs. Whereas high frequencies of IFNg producing T cells (5–15%) with cytotoxic activity against the primary leukemia (20–50% lysis) were induced from the naive T cell population, no leukemia-reactive T cells could be isolated from the memory or effector T cell populations (n=3). In conclusion, T cells stimulated with CD3/28 Dynal beads have a transient expression of CD40L and can be used as an alternative source of CD40L to generate good APC of malignant B cells under GMP conditions. These malignant B cell APCs were capable of inducing efficient priming of primary anti-leukemic immune responses by activating naive donor T cells.

The Rho family GTPases are increasingly implicated for their important roles in T cell development and function. We have found that RhoH, a hematopoietic-specific member of this family is essential for thymocyte development (Gu et al, Nat Immunol, in press). Further, Rhoh−/− mice showed T cell lymphopenia. In particular, naïve T cells were reduced in secondary lymphoid organs and blood both in unchallenged and LCMV-challenged Rhoh−/− mice compared to WT controls. These findings suggest a possible defect in T cell emigration from thymus and peripheral T cell homeostasis in Rhoh−/− mice. To study the role(s) of RhoH in T cell migration and homeostasis, purified splenic T cells were adoptively transferred into common γ−/−; Rag2−/− mice. T cells were then recovered from spleens 3d and 8d post-transplantation. The recovery of Rhoh−/− T cells was decreased by 60% compared with WT cells. In vitro migration of Rhoh−/− T cells towards SDF-1α, a homeostatic chemokine for T cells, in a transwell migration assay was significantly reduced compared to WT cells. Flow analysis showed decreased number of CXCR4 expressing and reduced expression levels of CXCR4 on Rhoh−/− T cells. Furthermore, apoptotic T cells were increased twofold in Rhoh−/− mice compared to controls. CFSE staining of adoptively transferred T cells demonstrated a comparable proliferation rate between Rhoh−/− and WT cells in the recipient mice. Our data suggest that RhoH plays an important role in T cell homeostasis via regulating cell survival and SDF-1α-mediated migration. To further investigate how RhoH regulates T cell survival, we focused on IL-7, an essential factor for prolonged survival of naïve T cells. One way IL-7 exerts its effect is through upregulating the Bcl-2 family of antiapoptotic proteins. Our data showed that in vitro survival of Rhoh−/− T cells in response to IL-7 was impaired, and expression of IL-7Rα and Bcl-2 were both decreased in Rhoh−/− T cells. To further study a potential role of RhoH in regulation of IL-7R expression, FACsvantage sorted naïve T cells (CD4+CD44low, or CD8+CD44low) were cytokine starved overnight, then cultured with or without the addition of IL-7 for 6 hrs, and analyzed for IL-7Rα expression by flow cytometry. In WT cells, during cytokine starvation CD4 and a subset of CD8 naïve cells upregulated IL-7Rα expression, but this upregulation was reduced followed IL-7 stimulation. In contrast, Rhoh−/− T cells failed to show either up- or subsequent down-regulation of IL-7Rα in response to cytokine starvation and IL-7 exposure. These data may indicate a role for RhoH in regulating IL-7R expression in naïve CD4 T cells. Taken together, these findings suggest that RhoH is required for IL-7-mediated T cell survival and SDF-1α-mediated homing and/or emigration from thymus. Thus, deficiency of naïve T cells in Rhoh−/− mice likely results from combined defects in T cell migration and homeostasis.

MT103 (MEDI-538) is a bispecific single-chain antibody construct directed at CD3 on human T cells and CD19 on human B lymphoma and normal B cells. Transient linkage of B and T cells by MT103 provides T cells with a T cell receptor (TCR)-like signal leading to redirected lysis of B cell targets without apparent need of costimulation and inducing T cells to proliferate, secrete cytokines and upregulate surface activation markers. TCR-like signalling by MT103 is strictly dependent on the presence of target cells. Redirected lysis of CD19-positive cells by MT103 is seen at low picomolar concentrations and at low effector-to-target ratios. The in-vivo half-life of MT103 is approximately two hours. In the ongoing dose escalation study MT103-104, patients with relapsed B-NHL have so far received continuous infusion of MT103 at maintenance flow-rates of 0.5, 1.5, 5 and 15 μg/m2/24h for 4 or 8 weeks following a 3+3 dose escalation design. Serum concentrations of MT103 remained constant over the entire treatment period at a level depending on the respective maintenance flow-rate. Depletion of circulating B (lymphoma) cells could be observed more frequently with increasing dose levels (DL) from DL1 to DL3, and in all evaluable patients at DL4. Three of six evaluable patients at DL4 showed clinical responses (2 PR, 1 CR) according to standardized Cheson criteria, but no patient of DL1-3. The time courses of absolute CD4 and CD8 T cell counts in peripheral blood were determined by flow cytometry. CD8 T lymphocytes were further subdivided for analysis into naïve T cells, TCM (central memory T cells), TEM (effector memory T cells) and TEMRA (non-proliferating terminally differentiated CTL), and CD4 T lymphocytes into naïve T cells, TCM and TEM. Activation of CD4 and CD8 T cell subsets was determined by measuring upregulation of CD69, CD25 and HLA-DR. Serum levels of cytokines were determined as additional biomarkers for T cell activation. In 50% of patients at DL1 to DL3, CD4 and CD8 T cell counts increased during the course of treatment - over pre-treatment levels. The TEM subset from both CD4 and CD8 T cells accounted for most of the observed increases, while the naïve T cell subsets showed no increase but also no signs of apoptosis. The non-proliferative TEMRA subset of CD8 T cells also remained unchanged in most patients. This indicated that the selective increase of proliferation-competent TEM subsets was attributed to MT103-induced T cell proliferation. At DL4, all evaluable patients showed signs of T cell expansion after 2 weeks of MT103 infusion, which was most pronounced in those who developed a partial or complete remission. The increase of CD8 T cell counts was more pronounced than that of CD4 T cells. T cell expansion was accompanied by upregulation of T cell activation markers as well as by increases in serum concentrations of cytokines like IFN-γ. T cell expansion and activation reverted in all cases when the infusion of MT103 was stopped. In summary, MT103 induced a reversible secondary T cell response involving T cell activation and proliferation as well as T cell cytotoxicity against circulating B cells and lymphoma tissue. The dose-dependent T cell expansion observed during long-term infusion of MT103, particularly within the cytotoxic TEM subset of CD8 T cells, appears to play a key role for clinical activity.

Background: Maintaining T cell function and survival after ex vivo manipulation remains a major challenge in adoptive immunotherapy. We previously demonstrated that murine T cells activated and expanded ex-vivo with anti-CD3 and anti-CD28 antibody-coated magnetic beads (CD3/CD28 beads) exhibit reduced GVHD-inducing potential compared to naïve (unmanipulated) T cells in a murine allogeneic BMT model. Blood diagnostics are commonly used to help identify patterns of biomarkers in multiple disease states. Objective: Determine if plasma profiles of 59 different analytes are altered in murine allogeneic BMT recipients who receive ex-vivo activated, suicide gene transduced and selected (Td) T cells compared with animals that received naïve suicide gene expressing T cells. Methods: Murine T cells were Td with a chimeric CD34-thymidine kinase (CD34-TK) fusion suicide gene. High efficiency (70%) gene transfer of CD34-TK to C57BL/6 (B6) murine T cells was accomplished 24 h after CD3/CD28 bead activation and gene-modified cells were purified to > 96% by CD34 immunomagnetic selection 2 days post-infection. Naïve B6 CD34-TK-expressing T cells were purified from the spleens of CD34-TK transgenic mice the day of BMT. To induce GVHD, lethally irradiated BALB/c allogeneic recipients were given T cell depleted (TCD) B6 BM supplemented with or without B6 CD34-TK purified naïve or Td T cells. Animals were bled 1 week after BMT and EDTA plasma samples were prepared and frozen. Quantitative measurements of 59 analytes were obtained using a rodent multi-analyte profile test (MAP test; Charles River Lab) on the plasma of 2 mice that received naïve T cells and died from GVHD on days 16 and 21 after BMT, 2 mice that received Td T cells and died from GVHD on day 34 after BMT, and 2 mice that received TCD BM only and survived > 100 days. Results: As before, we found that ex vivo activation of the donor T cells before BMT significantly prolonged the survival of mice transplanted with the Td T cells compared with mice receiving naïve T cells (p = 0.0028). Eighteen analytes, including IFN-γ, IL-2, IL-3, IL-4, IL-7, IL-11, and TNF-α were below the detection limits of the rodent MAP test for all samples analyzed. Twenty-six analytes, including VEGF, SCF, MIP-2, MIP-1α, MIP-3β, MCP-5, IL-1β, IL-18 and GM-CSF were detectable but not significantly different than the BM only controls for either the naïve or Td T cell groups. Nine analytes, including eotaxin, MIP-1γ, MCP-3, MCP-1, and IL-10 were similarly increased 2- to 3-fold in both the Td and naïve T cell groups compared to the BM only control. Interestingly, recipients of naive T cells exhibited increased plasma levels of growth hormone (≥14-fold), MIP-1β (4.6-fold), IP-10 (2.3-fold), and lymphotactin (2-fold), as well as decreased levels of leptin (≥14-fold), compared to both the BM only and Td T cell groups. This extreme modulation of growth hormone and leptin levels is particularly interesting given the fact that leptin influences local growth hormone secretion from lymphocytes and that both of these analytes have multiple biologic effects on T cells. Finally, only a single analyte, monocyte derived chemokine (MDC), was increased (4-fold) in mice that received Td T cells compared to the naïve T cell recipients. Conclusion: These results indicate that several plasma analytes with important immunological functions are altered when donor T cells are manipulated ex-vivo. These alterations may account for the decreased GVHD-inducing potential of ex vivo manipulated T cells after allogeneic BMT.

Background: Current SCT approaches consistently achieve rapid donor myeloid engraftment, but delayed immune recovery remains a significant obstacle and results in increased risk of infection and relapse. T cells are regenerated via 2 pathways, thymus-derived and peripheral expansion, processes for which IL-7 is critical. We postulated that non-myeloablative pre-transplant conditioning might preserve thymic function in pediatric SCT recipients thus enhancing thymus-derived naïve T cell regeneration. Methods: We analyzed T cell subsets, T cell receptor excision circles (TREC), and IL-7 levels in peripheral blood after SCT in 21 pediatric pts with high-risk malignancies (median age 14, range 4–21). Fludarabine-based induction chemotherapy was administered for disease control and targeted CD4 count reduction. Pre-transplant conditioning consisted of cyclophosphamide (1,200 mg/m2/day) and fludarabine (30 mg/m2/day) × 4 days plus melphalan (100 mg/m2 × 1 dose in sarcoma pts). Grafts consisted of G-CSF mobilized unmodified peripheral blood stem cells from 5–6/6 HLA-matched first-degree relatives (median CD34 dose 11.7 × 10E6/kg, range 4.4–19.1; median CD3 dose 416 × 10E6/kg, range 228–815). Cyclosporine was used for GVHD prophylaxis. Results: Donor-derived engraftment was rapid (absolute neutrophil count > 500/uL median day 9, range 8–11). Complete donor lymphoid chimerism (>95% by VNTR-PCR on CD3 sorted peripheral blood) was achieved in all by day 28. Immune recovery was brisk and sustained. Substantial numbers of naïve (CD45RA+/CD62L+) CD4+ and CD8+ T-cells were detected at day 28 (Fig 1). There was a steady increase in TREC from 3 to 12 months consistent with early, robust thymic-dependant T cell generation (Fig 2). This was not seen in adult pts treated on a parallel trial (data not shown). IL-7 levels were elevated and inversely correlated with T cell counts (r=−0.56, p Conclusions: Targeted immune depletion and NMSCT results in rapid, sustained immune reconstitution in pediatric pts with malignancy. Preserved thymic function appears to contribute to naïve T cell recovery in this setting. We postulate that non-myeloablative conditioning is thymus sparing and that this, in combination with immune depletion-induced IL-7 elevation, promotes early thymic-derived lymphoid recovery. This approach may serve as a strategy to overcome the prolonged immunodeficiency commonly encountered after allogeneic SCT in pediatrics and might be used as a platform to direct allogeneic anti-tumor immune responses in high-risk childhood cancers. Figure 1 Figure 1. Figure 2 Figure 2.

CD4(+)CD25(+)FoxP3(+) T regulatory (T reg) cells constitute 1–2% of peripheral blood cells in adults and function prevalently as immunosuppressors. 70% of T reg are memory/effector cells with a CD45RO+ phenotype. The other 30% have a naive T cell phenotype (CD45RA+). Both sub-populations exert a regulatory function and when sorted and/or purified each inhibits a mixed lymphocyte culture. In the present study, we investigated the effects of human recombinant IL-7 (rIL-7), human mesenchymal cells (hMSC) and hMSC engineered with the IL-7 gene (hMSC/IL-7) on regulatory T cells expressing a memory (CD4/CD25/CD45RO) or naive (CD4/CD25/CD45RA) phenotype. T cells from healthy donors were enriched by immnuselection to provide populations of CD45RA+ cells (95 % ± 2.9) and CD45RO+ cells (97 % ± 0.25). Enriched naive and memory cells were cultured in presence rIL-7 (100 ng/ml), hMSC (ratio 5:1) or hMSC/IL-7 (ratio 5:1). After 7 days’ culture we examined the CD4/CD25+ cells within the naive and memory populations. In the naive T cell population the T reg starting fraction of 0.05 % ± 0.01 of CD4/CD25 positive cells, did not change in the presence of rIL-7 while it rose to 0.2 % ± 0.14 in presence of human MSC and more interestingly reached 1.67 % ± 0.6 in presence of IL-7 engineered human MSC. In the memory T cell population the T reg starting fraction of 0.3 % ± 0.05 of CD4/CD25 positive cells, did not change in presence of rIL-7 while it rose 1.5 % ± 0.9 in the presence of human MSC and more interestingly reached 11.55 % ± 7.5 in the presence of human IL-7 engineered-MSC. We analyzed CD127 expression within different groups. In the naive T reg starting fraction 3 % ± 1.2 expressed CD127 which was down-regulated to 0.96 % ± 0.5 in the presence of rIL-7, to 0.29 % ± 0.2 with human MSC and to 0.37 % ± 0.02 with human IL-7engineered-MSC. Memory T reg cells expressed CD127 in 15% ± 1.2 of the starting fraction which was down-regulated to 1.2 % ± 0.45 in the presence of rIL-7, to 1.32 % ± 0.34 with hMSC and to 4.01% ± 0.74 in presence of hMSC/IL-7. FoxP3 expression was measured by real time quantitative PCR in sort-purified subsets of peripheral blood, identified by staining with a combination of CD4, CD25, CD45RA or CD45RO. In naive T reg FoxP3 expression was increased 1.15 fold in the presence of hMSC and 2.7 fold in presence of hMSC/IL-7. In memory T reg FoxP3 expression was increased 1.14 fold in the presence of hMSC and 2.67 fold in presence of hMSC/IL-7. In conclusion naive T reg cells are IL-7 independent and up-regulated by human MSC. Engineering human MSC with the IL-7 gene enhanced up-regulation. Memory T reg cells are also IL-7 independent and are up-regulated by human MSC. Engineering human MSC with the IL-7 gene markedly increased up-regulation. The different regulatory systems may underlie different functions within the T reg sub-populations.

CD4+CD25+ regulatory T cells (Treg) are essential negative regulators of immune responses. However, the mechanisms of immune suppression and the spectrum of cells they target remain incompletely defined. In particular, although Treg directly suppress conventional T cells in vitro, they might also affect antigen presenting cells (APC). Here, we studied the maturation of human myeloid (mDC) and plasmacytoid (pDC) dendritic cells activated with Toll-like receptor (TLR) ligands in the presence of CD4+ CD25high regulatory T cells in vitro. T cells and DC subsets were purified from normal human peripheral blood. LPS, CpG ODN 2216 and R-848 were used to trigger the maturation of mDC, pDC or both through TLR4, TLR9 and TLR7/8 respectively. Preactivated CD4+ CD25high Treg had no effect on the maturation of pDC. Conversely, they strongly suppressed TLR-triggered mDC costimulatory molecules up-regulation, pro-inflammatory cytokines secretion and their antigen presentation capacity, as opposed to conventionnal T cells (Tconv). At a ratio of 3 Treg for 1 DC, the percentage of mDC acquiring CD80 was reduced 5 fold (from 75% to 16%) while the Mean Fluorescence Intensity was decreased by approximately 65% for CD80 and 35% for CD86 after LPS stimulation and by 50% and 20% after R-848 stimulation. Furthermore, Treg dramatically decreased the secretion of IL-12p40, TNF-α, and IL-6 by mDC (95%, 93% and 50% average inhibition respectively) after LPS activation and to a lesser but still significant extent (38%, 35%, and 38% average inhibition respectively) after R848 stimulation. Finally, we found that Treg-conditionned mDC had a reduced ability to trigger naïve T cell proliferation in a mixed leukocyte reaction. Suppression of mDC activation by Treg appeared to require cell-cell contact. Moreover, the inhibition of pro-inflammatory cytokines secretion, but not of phenotypic maturation, was almost completely restored using an anti-IL10 receptor monoclonal antibody, but not anti-TGFβ nor anti-CTLA-4 blocking antibodies. Those data suggest that Treg prevent the co-stimulatory molecules up-regulation on mDC through contact dependent mechanisms, while the modulation of cytokines secretion appears to be largely mediated by IL-10. Overall, our results provide the first evidence of a direct inhibition of human mDC but not pDC maturation by CD4+ CD25high Treg. Therefore, by restraining the maturation of mDC, human Treg may enlist those cells for the initiation and the amplification of tolerance in vivo.

One of the major problems following allogeneic stem cell transplantation (allo-SCT) is the inability to reconstitute an adequate immune system for an extended period. T-cell reconstitution is also delayed for years, especially in CD4+ T cells. In addition to impaired thymic function, shortened Naive T cell survival due to altered T cell homeostasis is reported to be responsible for delayed immune reconstitution. To further investigate the mechanisms of delayed immune recovery after allo-SCT, we focused on the frequencies of effector CD4+ T cells, because according to the previous reports, progressive linear differentiation model of CD4+ T cell predicts the accumulation of terminally differentiated effector cells when transition from naïve to memory T cells and memory to effector cells are accelerated. By flowcytometric analyses we confirmed that CD27−CD4+ T cells from allo-SCT recipients uniformly express CD95, with negative expression of CCR7 and CD62L. They also produce g-interferon (IFNg) in response to the immobilized anti-CD3 and soluble anti-CD28 stimulation, which is consistent with previous reports insisting that CD27−CD4+ T cells are functionally differentiated effector T cells. Measuring the ratio of CD27−CD4+ T cells among CD4+ T cells revealed that, although healthy donors and patients received allo-SCT within a year had comparable CD27+CD4+T-cell rate (90% vs. 83%, P=0.4436), significantly decreased rate was observed in patients transplanted more than 1 year before (55% vs. 83%, P=0.0005). The ratio of CD27+CD4+ T cells kept low during the first 5 years after allo-SCT, and then it slowly begun to increase. In addition, in patients who received stem cell grafts more than 1 year before, the ratio of CD27+CD4+ T cells were significantly higher in patients transplanted from HLA-matched siblings than in those received unrelated grafts (69% vs. 42%, P=0.0002). Other factors, such as stem cell source (BM or PBSC), patient age, and the presence of chronic GVHD did not influence the ratio of CD27+CD4+ T cells. To further investigate the characteristics of CD27−CD4+ T cells in post-transplant periods, peripheral CD4+ T cells from patients who had received allo-SCT more than 1 year before as well as healthy volunteers were sorted into CD27− and CD27+ fractions, stained with CFSE, and stimulated with immobilized anti-CD3 and soluble anti-CD28 antibodies. CD27−CD4+ T cells proliferated more vigorously at 3 days after stimulation, though after another 2-day culture, there was no difference in cell divisions between both cell groups. In addition, CD27+ cells from transplanted patients lost their expression more frequently than those from volunteers, while none of the CD27− cells stored its expression. The fact of one-way transition from CD27+ to CD27− also supported that CD27−CD4+ T cells are terminally differentiated T cells. The finding that the frequencies of CD27−CD4+ T cells begin to elevate at 1 year after allo-SCT indicates that T cells infused with allograft do not easily lose the surface expression of CD27, while T cells derived from donor’s stem cells do. Considering the fact that ratio of CD27−CD4+ T cells is much higher in recipients of unrelated grafts, and it gradually begin to decrease at 5 years after allo-SCT, the increased ratio of CD27−CD4+ T cells may reflect altered T cell homeostasis. The serial monitoring of the ratio of CD27−CD4+ T cells after allo-SCT may be useful in evaluating immune reconstitution status.