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Cancer treatment has been transformed by checkpoint blockade therapies, with the highest anti-tumor activity of anti-programmed death 1 (PD-1) antibody therapy seen in Hodgkin lymphoma. Disappointingly, response rates have been low in the non-Hodgkin lymphomas, with no activity seen in relapsed/refractory chronic lymphocytic leukemia (CLL) with PD-1 blockade. Thus, identifying more powerful combination therapy is required for these patients. Here, we preclinically demonstrate enhanced anti-CLL activity following combinational therapy with anti-PD-1 or anti-PD-1 ligand (PD-L1) and avadomide, a cereblon E3 ligase modulator (CELMoD). Avadomide induced type I and II interferon (IFN) signaling in patient T cells, triggering a feedforward cascade of reinvigorated T-cell responses. Immune modeling assays demonstrated that avadomide stimulated T-cell activation, chemokine expression, motility and lytic synapses with CLL cells, as well as IFN-inducible feedback inhibition through upregulation of PD-L1. Patient-derived xenograft tumors treated with avadomide were converted to CD8+ T cell-inflamed tumor microenvironments that responded to anti-PD-L1/PD-1-based combination therapy. Notably, clinical analyses showed increased PD-L1 expression on T cells, as well as intratumoral expression of chemokine signaling genes in B-cell malignancy patients receiving avadomide-based therapy. These data illustrate the importance of overcoming a low inflammatory T-cell state to successfully sensitize CLL to checkpoint blockade-based combination therapy.

Introduction: Revlimid (Rev), binds to CRL4 CRBN E3 ligase leading to recruitment and proteasomal degradation of transcription factors Aiolos and Ikaros. This inhibits proliferation of malignant B cells and stimulates activity of T, NK and macrophage cells, thereby providing clinical activity of Rev as a single agent and in combination with CD19/CD20 antibodies in DLBCL and FL. Iberdomide (Iber), a new CELMoD with enhanced substrate degradation compared to Rev, is currently being studied in clinical trials for B-NHL and MM. Presented here is extensive in vitro and in vivo characterization of immune enhancement and antitumor effects of Iber with direct comparison to Rev. Results: In a panel of DLBCL cell lines, comprising ABC and GCB-DLBCL models, Iber degraded Aiolos/Ikaros with faster kinetics and to a greater depth than Rev, which led to enhanced antiproliferative and cytotoxic effects. Iber acted in a cell of origin independent manner, whereas Rev is preferentially active in ABC-DLBCL. To examine the molecular effects of Iber and Rev in immune cells, we performed RNAseq and proteomic based analyses on Iber and Rev treated T, NK and monocyte cell populations. These experiments revealed a complex series of immunomodulatory activities including promotion of pro-inflammatory cytokine production, activation marker expressions and migratory machinery with a trend of Iber exhibiting greater enhancements. We confirmed these findings by demonstrating that secretion of chemoattractants for T cells, NK cells and monocytes including CXCL9, 10 and 11 (10-90% increase) and CCL8 (30% increase, p Conclusion: Our data demonstrate that Iber is more potent in substrate degradation and functionality in anti-proliferative activity against DLBCL cell line models and at triggering immunostimulatory activities in multiple lymphoid and myeloid populations. Additionally, we generated a humanized CRBN mouse model that revealed the ability of CELMoDs in inducing immune-rich TMEs supporting rational combination strategies with immune focused agents being explored in lymphoma such as SIRPα blockade, CAR T and CD3xCD20 bispecifics. Figure 1 Figure 1. Disclosures Nakayama: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Narla: Bristol Myers Squibb: Current Employment. Shakya: Bristol Myers Squibb: Current Employment. Gamez: Bristol Myers Squibb: Current Employment. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gandhi: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

BACKGROUND. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T, NK and NKT cells, sharing structural and mechanistic similarities with PD-1 and CTLA-4. TIGIT competes with CD226, its partner receptor, for the binding to CD155 ligand: signaling triggered upon CD155 binding to CD226 potentiates T cell receptor (TCR) signaling and CD8 + T cell cytotoxicity against tumor cells (positive signaling). On the contrary, concomitant TIGIT expression on the cell surface prevents CD226 activation either by sequestering CD155 or by impeding CD226 homodimerization and phosphorylation (negative signaling). Recently, TIGIT was shown to be expressed on the surface of normal memory B cells, where it could directly act to suppress T cell responses. No data are available on TIGIT or CD226 expression by chronic lymphocytic leukemia (CLL) cells. AIM AND METHODS. Our aim was to investigate expression of the TIGIT and CD226 receptors and of the CD155 ligand in a cohort of clinically and molecularly annotated CLL patient samples. To this end, we designed a multiparametric panel of antibodies for flow cytometry and examined expression of the TIGIT/CD226/CD155 axis in peripheral blood mononuclear cells (PBMC) from our patient cohort. To investigate the impact of TIGIT/CD226 engagement on B cell responses, purified leukemic B cells were activated either through the B cell receptor (BCR) using an αIgM polyclonal antibody or with CpG oligonucleotide and interleukin 15 (IL-15) to induce proliferation. In selected experiments, we added recombinant human (Rh) TIGIT-Fc or CD155-Fc chimeras and αTIGIT or αCD226 blocking antibodies to interfere with this axis. RESULTS. Surface expression of TIGIT, CD226 and CD155 was evaluated in a cohort of 115 CLL samples and compared to age- and sex-matched healthy subjects. Both TIGIT and CD226 were upregulated on leukemic B cells compared to normal B lymphocytes, while CD155 was expressed at lower levels. A similar trend was observed on CD4 + and CD8 + T lymphocytes. High-risk CLLs (unmutated IgV genes, unfavorable cytogenetics and advanced stage) were predominantly TIGIT low and CD226 high, indicating an unbalance towards "positive signaling". Results were confirmed by confocal microscopy analyses on lymph node (LN) biopsies, which showed i) an overall higher TIGIT expression in CLL compared to reactive LNs and ii) among CLL LNs a stronger TIGIT positivity in mutated vs unmutated cases, confirming flow cytometry data. In line with these findings, Richter's syndrome samples and patient-derived xenografts models showed the lowest TIGIT and the highest CD226 levels. We next examined TIGIT axis expression during the follow up of CLL cases who underwent treatment with BTK inhibitor (BTKi). While CD226 levels remained unmodified upon treatment, a sharp decrease in surface TIGIT was detected soon after BTKi initiation. Since TIGIT acts by decreasing TCR signaling to shut down T cell responses, we hypothesized similar functions in B cells. By crosslinking the BCR with an αIgM antibody in a selected cohort of IGHV UM CLL cells, we found that BTK phosphorylation was induced to a lesser extent in TIGIT high compared to TIGIT low samples, suggesting that TIGIT is a marker of CLL cell anergy. Accordingly, interruption of receptors/ligand interactions with RhTIGIT-Fc chimera or with αTIGIT or αCD226 blocking antibodies, modulated BCR signaling capacity. Specifically, in TIGIT high samples, preventing receptor engagement by CD155 increased αIgM-induced BTK phosphorylation; in contrast, in TIGIT low samples, blocking CD155 interaction affected mostly CD226 signaling, thereby depotentiating BCR activation. Similar results were obtained when stimulating CLL cells with CpG/IL-15. Interestingly, we observed a significant upregulation of surface CD226 in CLL cells cultured for 6 days in the presence of CpG/IL-15. CONCLUSIONS. These results show for the first-time expression of TIGIT by CLL cells. Furthermore, they indicate that TIGIT is a marker of CLL cells anergy, whereas activated CLL cells express high levels of CD226. Inhibition of TIGIT binding to CD155 partially restores B cell signaling and activation. Future studies are needed to gain insights on the mechanisms behind its deregulation and to obtain a complete functional characterization of the axis. Disclosures Coscia: AbbVie: Honoraria, Other; Janssen: Honoraria, Other, Research Funding; AstraZeneca: Honoraria; Gilead: Honoraria. Gaidano: Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astrazeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Allan: Genentech: Consultancy, Research Funding; Epizyme: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celegene: Research Funding; AstraZeneca Pharmaceuticals LP, Genentech, a member of the Roche Group, Janssen Biotech Inc, TG Therapeutics Inc.: Research Funding; AbbVie Inc, AstraZeneca Pharmaceuticals LP, BeiGene, Janssen Biotech Inc, Pharmacyclics LLC: Consultancy; AbbVie Inc, Ascentage Pharma, Epizyme, Genentech, a member of the Roche Group, Janssen Biotech Inc, Pharmacyclics LLC: Other: Advisory Committee; TG Therapeutics: Research Funding. Furman: Oncotracker: Consultancy; Verastem: Consultancy; Abbvie: Consultancy, Honoraria, Other: Expert testimony; Sunesis: Consultancy; Incyte: Consultancy; Beigene: Consultancy; Acerta/AstraZeneca: Consultancy; Loxo Oncology: Consultancy; Genentech: Consultancy; Morphosys: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; TG Therapeutics: Consultancy; X4 Pharmaceuticals: Consultancy; Janssen: Consultancy, Honoraria; AstraZeneca: Honoraria. Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

CLL cells proliferate in secondary lymphatic tissues where the CLL cells interact with T cells, monocyte-derived nurselike cells and mesenchymal stromal cells, collectively referred to as the tissue microenvironment. The CLL lymph node morphology with proliferation centers resembles normal B cell follicles, suggesting that mechanisms regulating the expansion of antigen-selected normal B cells during the germinal center (GC) reaction also regulate CLL cell expansion. Normal GC reactions require specialized T follicular helper (Tfh) cells, which provide T cell help to antigen-stimulated B cells. Another important T cell subset are Tregs which regulate immune tolerance and homeostasis. NLC co-culture have been used to dissect cellular and molecular interactions between CLL cells and the lymph node microenvironment in vitro. To gain insight into the role of T cell subsets in CLL, we utilized the NLC model to characterize T cell subsets and their dynamics in NLC co-cultures. We quantified CD4 + and CD8 + T cells in co-cultures from 18 different patients, observing that CD4 + T cell counts remained stable over time (449±62 at baseline to 443±80 cells/µl after 14 days). In contrast, numbers of CD8 + T cells declined from 180±42 cells/µl to 98±16 cells/µl on day 14 (p Disclosures Ferrajoli: Janssen: Other: Advisory Board ; AstraZeneca: Other: Advisory Board, Research Funding; BeiGene: Other: Advisory Board, Research Funding. Wierda: Oncternal Therapeutics, Inc.: Research Funding; Sunesis: Research Funding; Gilead Sciences: Research Funding; GSK/Novartis: Research Funding; Acerta Pharma Inc.: Research Funding; Genentech: Research Funding; Karyopharm: Research Funding; KITE Pharma: Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Cyclacel: Research Funding; Miragen: Research Funding; Loxo Oncology, Inc.: Research Funding; Janssen: Research Funding; Xencor: Research Funding; AstraZeneca: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding. Patten: ABBVIE: Honoraria; ASTRA ZENECA: Honoraria; GILEAD SCIENCES: Honoraria, Research Funding; JANSSEN: Honoraria; NOVARTIS: Honoraria; ROCHE: Research Funding. Burger: Novartis: Other: Travel/Accommodations/Expenses, Speakers Bureau; Beigene: Research Funding, Speakers Bureau; Pharmacyclics LLC: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Gilead: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; AstraZeneca: Consultancy; Janssen: Consultancy, Other: Travel/Accommodations/Expenses, Speakers Bureau.

Introduction Our understanding of 'immunome" and its role in the pathogenesis and clinical outcomes of hematological diseases has significantly improved in recent years, which highlights the importance of multidimensional cytometry techniques in investigating immune response both in clinical and research settings. Liquid mass cytometry (LMC), imaging mass cytometry (IMC) and flow cytometry (FC) are powerful techniques for immunophenotyping, biomarker discovery, and patients' immune-monitoring. These techniques provide the ability to profile over 40 markers per cell, resulting in large amounts of data which could be challenging to analyze and interpret. Therefore, we developed ImmunoCluster, an easy-to-use open-source computational pipeline, to explore high dimensionality single-cell cytometry datasets. Case studies Here we describe three examples of the implementation of the ImmunoCluster tool, which is a self-contained R package (accessed via GitHub: https://github.com/kordastilab/ImmunoCluster). Previously published LMC data from 15 leukemia patients 30 and 90 days after bone marrow transplantation (BMT) were used to test ImmunoCluster's ability to reproduce results. Post-BMT 3/15 patients suffered acute graft versus host disease (GvHD). For IMC data a lymph node section from a diffuse large B-cell lymphoma (DLBCL) patient. Finally, FC data from BM of 7 healthy donors (HDs) taken during hip surgery. The pipeline provides tools to allow researchers to follow a workflow which guides them through experimental design, data analyses and interpretation, to publishable graphics identifying differences in phenotype and abundance of cells between conditions (Figure 1A). ImmunoCluster comprises of three core computational stages: Stage 1. Data import and quality control In the experimental design stage, the high dimensional dataset is imported into ImmunoCluster with an associated metadata file, this included timepoints, and response to treatment. All data was stored within a SingleCellExperiment (SCE) object, a data container in which you can store/retrieve information such as metadata and UMAP/tSNE coordinates (Figure 1B). Initial exploratory visualization of the data, e.g. Multidimensional scaling (MDS) plots, and heatmaps showing marker expression for each patient were created and metadata used for annotation. Stage 2. Dimensionality reduction and unsupervised clustering We used three dimensionality reduction tools: MDS, uniform manifold approximation and projection (UMAP), and t-Distributed Stochastic Neighbor Embedding (tSNE). Two clustering algorithms: an ensemble clustering method of FlowSOM and Consensus clustering; and PhenoGraph. The aim of these algorithms were to assign all cells to clusters corresponding to true cell types. Stage 3. Annotation and differential testing Tools exploring cluster marker expression via projection onto UMAP/tSNE, or heatmaps, aided the identification of cell types and phenotypically distinct clusters. Metadata were used to annotate figures, allowing for visualization of the distribution of cell islands between different conditions/timepoints. Tools such as median marker expression, hierarchical clustered heatmaps, and box plots of cell cluster abundance were applied. Statistically significant differences between conditions were identified. Results We successfully replicated the findings from Hartmann et al., 24 cell populations were identified (Figure 2A-B). Significant differences between memory B-cells (FDR p=4.38 x 10-3), naïve B-cells (FDR p=1.35 x 10-2), and naïve CD4+ T-cells (FDR p=3.47 x 10-2) were identified between the GvHD and none (Figure 2C). From the FC HD CD4+ BM population data we were able to identify regulatory T cells (Tregs), including subpopulations, Treg A and Treg B (as low as 0.7% (0.1-2.0) and 0.9% (0.2-2.4), respectively). A marker expression ranking tool was applied to the DLBCL patient IMC data. We identified the majority of Ki-67 high population were proliferating tumor cells (84%), and the Ki-67 low population consisted of a heterogeneous collection of immune cell populations (Figure 3). These case studies show that ImmunoCluster could help clinicians and researchers with varying experience in computational biology to drive their projects from experimental design, wet lab/clinical trial, through to the data analysis process and visualization. Disclosures McLornan: JAZZ PHARMA: Honoraria, Speakers Bureau; NOVARTIS: Honoraria, Speakers Bureau; CELGENE: Honoraria, Speakers Bureau. Harrison:Gilead Sciences: Honoraria, Speakers Bureau; Incyte Corporation: Speakers Bureau; Janssen: Speakers Bureau; Sierra Oncology: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau; AOP Orphan Pharmaceuticals: Honoraria; Shire: Honoraria, Speakers Bureau; Promedior: Honoraria; Roche: Honoraria; Celgene: Honoraria, Research Funding, Speakers Bureau; CTI Biopharma Corp: Honoraria, Speakers Bureau. Kordasti:Celgene: Research Funding; Novartis: Research Funding; Alexion: Honoraria.

Immunotherapy has emerged as a viable clinical strategy to harness endogenous antitumor T-cell immunity. Lenalidomide is an oral immunomodulatory drug that repairs antitumor T-cell function and is showing efficacy in ongoing chronic lymphocytic leukemia (CLL) and lymphoma clinical trials. This article focuses on advances in our understanding of its mechanism of action in the tumor microenvironment and provides a clinical update in CLL. Challenges associated with this drug and its potential use in the targeted drug treatment era are discussed.

The leukocyte adhesion cascade is important in chronic lymphocytic leukemia (CLL), as it controls migration of malignant cells into the pro-survival lymph node microenvironment. Circulating trisomy 12 CLL cells have increased expression of the integrins CD11a and CD49d, as well as CD38, but the tissue expression of these and other molecules, and the functional and clinical sequelae of these changes have not been described. Here, we demonstrate that circulating trisomy 12 CLL cells also have increased expression of the integrins CD11b, CD18, CD29, and ITGB7, and the adhesion molecule CD323. Notably, there was reduced expression of CD11a, CD11b, and CD18 in trisomy 12 cases with NOTCH1 mutations compared with wild type. Trisomy 12 cells also exhibit upregulation of intracellular integrin signaling molecules CALDAG-GEFI, RAP1B, and Ras-related protein ligand, resulting in enhanced very late antigen-4 [VLA-4] directed adhesion and motility. CD38 expression in CLL has prognostic significance, but the increased CD38 expression in trisomy 12 CLL cells must be taken into account in this subgroup, and the threshold of CD38 positivity should be raised to 40% for this marker to retain its prognostic value. In conclusion, trisomy 12 CLL cells exhibit functional upregulation of integrin signaling, with β2-integrin expression being modulated by NOTCH1 mutation status.

Despite the success of autologous anti-CD19 CAR T cell therapy in B-Acute lymphoblastic leukaemia (B-ALL) and Diffuse Large B Cell Lymphoma (DLBCL), treatment failures occur. One contributing factor may be the intrinsic T cell fitness of the CAR T cell product that is influenced by the underlying malignancy and prior treatments. With the advent of gene editing, 'off the shelf' non-HLA matched healthy donor (HD) CAR T cells are under investigation for the treatment of patients (pts) in clinical trials. UCART19 (S68587) is a first-in-class allogeneic CAR T cell product expressing a second generation anti-CD19 CAR with TALEN®-mediated gene knockouts of T cell receptor alpha chain (TRAC) and CD52 to prevent graft versus host disease and to render them resistant to anti-CD52 antibody used for lymphodepletion. Preliminary clinical trial data on the use of UCART19 in B-ALL was previously reported at ASH (Benjamin et al, 2018). The phenotypic and functional characteristics of CAR T cell products manufactured from B-ALL, Chronic Lymphocytic Leukaemia (CLL) and DLBCL pts were compared to young adult healthy donor (HD) CAR T cell products. In addition, potential effects related to knocking out TRAC in HD TCR-CAR T cells were examined. Thawed PBMCs from B-ALL, CLL, DLBCL pts and HDs underwent T cell enrichment, activation with anti-CD3/CD28 beads and IL-2, followed by transduction with anti-CD19 4-1BB CD3ζ lentiviral CAR construct and expansion. HD TCR- CAR T cells were manufactured by electroporation of HD CAR T cells with mRNA coding for TRAC TALEN® and residual TCRαβ+cells were removed by magnetic bead selection. CAR expression levels, T cell subsets, and exhaustion markers were examined by flow cytometry. Expression of activation markers CD25 and CD69 was measured in response to co-culture with the CD19+cell line NALM-6. Cytotoxicity against NALM-6 and Raji was assessed and antigen-mediated proliferation measured over 14 days. HD CAR T cells (n=11) expanded significantly more during manufacture than CAR T cells derived from B-ALL (n=9), CLL (n=8) or DLBCL (n=8) pts. As expected, the electroporation step resulted in a transient decrease in viability which recovered over time in culture (n=10). Median CAR expression level was higher on CLL CAR T cell products compared to those from B-ALL pts and HDs, thought to be due to a higher CD4:CD8 ratio in some CLL products. As a consequence of TCR knockout, CD3 expression was lost on HD TCR- CAR T cells (n=10), apart from a small population of γδ CAR T cells. CLL and DLBCL CD8+CAR+cells expressed higher levels of PD1 than HD CD8+CAR+cells. DLBCL CD4+CAR+cells also expressed significantly higher levels of PD1 than HD or HD TCR-CD4+CAR+T cells. CAR+CD8+CD27+PD1- T cells have been previously described as a functionally important population that correlated with clinical outcome in pts who received CLL CAR T cells (Fraietta et al, 2018). We found HD (n=13) and HD TCR- (n=10) CAR T cells had significantly more CD8+CD27+PD1- CAR T cells compared to those derived from CLL (n=8) and DLBCL (n=6) pts, but similar levels to B-ALL pts (n=10). In the absence of CD19 antigen, DLBCL CAR+CD8+ T cells (n=6) had greater expression of CD25 and CD69. However, in response to stimulation with CD19+ NALM-6 cells, HD (n=12), HD TCR- (n=10) and B-ALL (n=10) CAR T cells had higher fold increase in CD69+ cells compared to DLBCL (n=6) CAR T cells. On paired analysis (n=6), no difference was seen in activation in response to CD19 antigen on HD compared to HD TCR- CAR T cells. All CAR T cell products demonstrated comparable cytotoxicity against NALM-6 and Raji cell lines in short term in vitro assays. However, long-term cytotoxicity will be evaluated in a murine model. We performed a detailed comparison of the phenotypic and functional characteristics of CAR T cells derived from pts with B-cell malignancies and HDs. DLBCL CAR T cells showed lower antigen specific activation but higher baseline activation which could lead to more differentiated exhausted T cells. CAR T cells derived from HDs show a higher proportion of the therapeutically relevant CAR+CD8+CD27+PD1- cells compared to patients with mature B cell malignancies (CLL and DLBCL), which is maintained after TRAC knockout. This suggests allogeneic CAR T cells, such as UCART19, may provide a more effective product for pts with T cell dysfunction. Disclosures Graham: Gillead: Other: Funding to attend educational meeting; Servier: Research Funding. Jozwik:Servier: Research Funding. Metelo:Pfizer: Research Funding; Allogene: Research Funding. Almena-Carrasco:Servier: Employment. Peranzoni:Servier: Employment. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Dupouy:Servier: Employment. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Patten:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Roche: Honoraria, Research Funding. Benjamin:Amgen: Honoraria; Allogene: Research Funding; Gilead: Honoraria; Servier: Research Funding; Eusapharm: Consultancy; Pfizer: Research Funding; Takeda: Honoraria; Novartis: Honoraria.

Bruton's tyrosine kinases inhibitors (BTKis) represent major advances in CLL therapy. However resistance to this form of therapy is emerging, and such patients often progress more rapidly. Hence there is an important need for therapies that address resistance. Microenvironmental input like IL-4 is critical for CLL disease progression. Compared with normal B cells, CLL cells exhibit significantly higher levels of surface membrane (sm) IL-4 receptor (IL4-R) and contain increased amounts of pSTAT6, a downstream mediator of IL-4R signaling. IL-4 stimulation of CLL B cells suppresses smCXCR4 and increases smIgM, thus promotes CLL cell retention and expansion. In this study, we aimed to examine if smIL-4R expression, IL4R signaling, and IL-4-producing cells are altered in patients sensitive or resistant to BTKis. To do so, T and B cell subset changes were studied overtime in 12 acalabrutinib-treated CLL patients, 6 zanubrutinib-treated CLL patients, 30 ibrutinib-sensitive and 5 ibrutinib-resistant CLL patients, 4 of which exhibited BTK mutations. Consistent with only ibrutinib inhibiting T-cell kinase (ITK), T-cell subset analyses revealed no changes in Th1, Th2, Th17, Th9, and Th22 cells after zanubrutinib or acalabrutinib treatment. In contrast, a Th1-biased T-cell immunity was observed in patients responsive to ibrutinib. In patients progressing on ibrutinib, significantly reduced Th2 T cells were found during the resistant as well as sensitive periods. In an in vitro T-cell function assay using T cells collected before and after the treatment with each BTKi, only ibrutinib treated patients exhibited a reduced ability of T cells to support CLL B cell survival. We next studied changes in CLL B cells, including numbers of IL-4, -10 and -13 producing B cells after BTKi treatment. IL-13 producing CLL B cells were not changed. IL-10 producing CLL B cells were reduced in both ibrutinib sensitive and resistant patients, but not in zanubrutinib or acalabrutinib treated patients. Importantly, IL-4 producing CLL B cells were significantly decreased in patients treated with all 3 BTKi. Significantly reduced smIL-4R levels, impaired IL-4R signaling, decreased smIgM and increased smCXCR4 were also seen in patients treated with each BTKi. To understand the mechanism responsible for inhibition of IL-4 production in CLL cells treated with BTKis, we stimulated CLL cells through IgM, Toll-like receptor and CD40L, finding that only anti-IgM stimulation significantly increased IL-4 production and p-STAT6 induction. We then explored the function of IL-4. IL-4 enhanced CLL B cell survival in vitro and this action was blocked by all 3 BTKis. Moreover, adhesion of CLL B cells to smIL-4R expressing stromal cells was decreased by IL-4 and IL-4R neutralizing antibodies, especially in M-CLL cases. In in vivo studies transferring autologous T cells and CLL PBMCs into alymphoid mice, we found less CLL B cells in mouse spleens post ibrutinib than zanubrutinib or acalabrutinib treatment. This might be due to the suppressed Th2 cells found only in ibrutinib, while IL-4 producing B cells were reduced in all 3 BTKi treated mice. These results support the idea that IL-4 promotes CLL B cell adhesion and growth in tissues. Finally, we investigated the IL-4/IL-4R axis in ibrutinib-resistant patients. Although IL-4 producing T cells remain reduced during the sensitive and resistant phases, CLL B cell production of IL-4 and expression of and signaling through smIL-4R returned when patients developed ibrutinib-resistance. When comparing paired ibrutinib-sensitive and -resistant CLL B cells collected from 3 patients in a xenograft model that requires T cell help, we found ibrutinib-resistant CLL B cells grew in vivo with only minimal (~15%) numbers of autologous T cells compared to B cells collected from ibrutinib-sensitive phase; this suggested a reduced requirement for T-cell help for growth of ibrutinib-resistant CLL cells. In summary, we found IL-4 is a key survival factor in the CLL microenvironment that also improves leukemia cell adhesion to stromal cells expressing smIL-4R. IL-4 production and signaling can be stimulated in CLL B cells through the B-cell receptor, and are consistently blocked by BTKis. Moreover, the recovered ability of ibrutinib-resistant CLL B cells to produce and respond to IL-4 leads to disease progression, suggesting blocking the IL-4/IL-4R axis is a potential treatment for ibrutinib-resistant CLL patients. Disclosures Chen: Pharmacyclics: Research Funding; Beigene: Research Funding; Verastem: Research Funding; ArgenX: Research Funding. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Kolitz:Boeringer-Ingelheim: Research Funding; Roche: Research Funding; Astellas: Research Funding. Zhou:BeiGene: Employment. Barrientos:Genentech: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Rai:Pharmacyctics: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees.

In this issue of Blood, Paggetti et al present novel findings that chronic lymphocytic leukemia (CLL)-derived exosomes and their molecular cargo are actively transferred to stromal cells that reside in the lymphoid tumor microenvironment (TME), promoting the reprogramming of these cells into cancer-associated fibroblasts (CAFs).

Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4(dim)CD5(bright) phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D-related (DR). In addition, LN-CLL cells have higher expression of α4β1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4(dim)CD5(bright) with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d(hi) CLL cells showed an enhanced capacity to activate T cells compared with CD49d(lo) subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4(+) T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells.

Immunotherapy has demonstrated potential to reactivate or transfer T cell immunity and regress tumors, offering hope to relapsed or refractory diffuse large B-cell lymphoma (DLBCL) patients. However, many DLBCL patients do not experience therapeutic benefit, likely owing to a lack of pre-existing anti-tumor immunity and/or poorly understood immunosuppressive mechanisms in the tumor microenvironment (TME). Understanding the different obstacles that cytotoxic T cells face in the DLBCL TME will help the development of novel therapeutic approaches to overcome them and optimize immunotherapy. Stroma-associated gene signatures reflecting fibroblast and immune cell activity as well as angiogenesis have been associated with outcome in DLBCL but the biology underlying these signatures has been understudied. Here we have examined beyond tumor 'effacement' and hypothesized that, rather than being sparse bystanders, lymph node stromal cells may be important players in driving immune suppression in lymphoma. Multiplex immunofluorescence (IF) confocal microscopy analysis of the major stromal cell subsets revealed a marked expansion and remodeling of podoplanin, PDPN+ fibroblastic reticular cells (FRCs) in DLBCL lymph node tissue TME biopsies across GCB and ABC subtypes (n=40) compared to reactive control tissues (n=10). FRC myofibroblasts were similarly remodeled in tumors from the transgenic mouse model of DLBCL (Iμ-HABCL6, n=10) compared to wild type littermates (n=5). These altered PDPNhigh, αSMAhigh FRC networks were interspersed within effaced lymph node tissues in close proximity to DLBCL tumor cells. To model the interactions between tumor cells and FRCs, we established 2D and 3D co-culture platforms that combined DLBCL cells (or non-malignant control B-cells) and PDPN+ FRCs derived from human or murine lymph node tissues. These quantitative assays have shown that tumor cells activate FRCs promoting their proliferation, increased expression of PDPN, marked elongation/stretching and subsequent reduced ability to contract 3D collagen matrix (non-contractile) (P An important function of FRCs in regulating immunity is attracting and maintaining T cells by secreting chemokines and promoting their migration along the network. Functional assays revealed that T cells show significantly reduced chemotaxis as well motility (quantitative time-lapse movies) across 2D and 3D lymphoma FRC networks compared to control FRCs (P In conclusion our data indicate that DLBCL tumor cells convert FRCs into immunosuppressive CAFs, which exhibit altered immumomodulatory activities at different levels that we believe has important implications for the regulation of anti-tumor immunity as well as response to immunotherapy. Disclosures Vardi: Gilead: Research Funding; Janssen: Honoraria. Ramsay:Roche Glycart AG: Research Funding; MedImmune: Research Funding; Celgene Corporation: Research Funding.

The targeted therapy ibrutinib inhibits B cell receptor signaling (BTK inhibitor) and has yielded high response rates and durable remissions in patients with chronic lymphocytic leukemia (CLL). However, it is widely believed that the addition of immune therapies to targeted drugs will be required to activate anti-tumor immunity and work towards curative therapy. Identifying effective combinations of targeted drugs and/or standard chemotherapy with immunotherapy is a priority research area and particularly relevant for CLL, as patients' T cells have been shown to exhibit profound tolerance/exhaustion and notably, no activity was reported in a recent trial of anti-PD-1 immunotherapy for relapsed disease. Ibrutinib has shown beneficial immunomodulatory activity in CLL by inhibiting IL-2-inducible T cell kinase (ITK) as well BTK that is associated with increased effector CD4+and CD8+ T cell numbers and decreased expression of inhibitory checkpoint receptors such as PD-1 on patient T cells. Here we have performed comparative immune bioassays from a randomized phase III trial comparing ibrutinib-based therapy to traditional FCR chemoimmunotherapy to assess the effects of treatments on anti-tumor T cell function. Viable peripheral blood mononuclear cell samples were collected serially (baseline, 6 months and 12 months) from CLL patients on the randomized phase III E1912 trial of ibrutinib and rituximab versus FCR for previously untreated disease to allow longitudinal batched immune analysis. Cytotoxicity assays revealed that highly purified CD3+ T cells from the FCR treated patients at 6 and 12-month time-points did not change their activated killing function against autologous baseline CD19+ CLL tumor B cells (acting as target antigen-presenting cells pulsed with superantigen, sAg) compared to pre-treatment/baseline exhausted T cells (n=22). In contrast, patients treated with ibrutinib-based therapy had a significant increase in activated anti-tumor T cell killing function (P Flow cytometric analysis of circulating immune subsets revealed that the percentage of PD-1 and PD-L1 positive cells among CD8+ and CD4+ T cells (particularly effector compartments) were reduced with ibrutinib-based therapy, whereas only a partial reduction was detected following FCR treatment. However, patients' T cells from both treatment arms responded normally to T cell receptor engagement by upregulating these checkpoint molecules. This led us to explore ex vivo treatment of highly purified CD3+ T cells and CD19+ CLL B cells from both treatment arms with anti-PD-L1 or anti-PD-1 immunotherapy prior to cytotoxicity assays. Our functional data revealed that the T cells from both FCR time-points (6 and 12-months) were not sensitive to either anti-PD-L1 (n=14) or anti-PD-1 (n=14) treatment. In contrast, ibrutinib-based treatment sensitized anti-tumor T function (23% increase in killing) following anti-PD-L1 treatment (n=14) at the 6-month time-point only (P To investigate the mechanism underlying these effector function differences, we compared the ability of highly purified CD3+ T cells from each treatment arm (n=45) time-point to form F-actin immunological synapses with baseline autologous CLL tumor B cells. Quantitative confocal image analysis revealed that ibrutinib-based therapy significantly (P We believe this data supports the concept of incorporating functional bioassays to immune-monitoring assays associated to clinical trials that should aid knowledge-led design of future combination immunotherapy. Disclosures Kay: Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Shanafelt:Pharmacyclics: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding; Jansen: Research Funding. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding; MedImmune: Research Funding.

Immune checkpoint blockade has demonstrated potential to reactivate anti-tumor immunity and regress tumors. However, response rates in B-cell non-Hodgkin lymphoma patients have been lower compared to Hodgkin lymphoma, with no activity reported in a recent trial of anti-PD-1 immunotherapy in relapsed CLL. This suggests that certain lymphoma subtypes may harbor non-immunogenic tumor microenvironments (TME). Our preliminary studies revealed that avadomide (CC-122), a cereblon modulator, can enhance T cell immune synapse signaling with autologous CLL cells, resulting in a concomitant increase in PD-1/PD-L1 expression at repaired synapses, indicating that avadomide may represent a complementary treatment partner for checkpoint inhibition. Here, we have extended our pre-clinical studies to investigate how these immunotherapy drugs alter the function and gene signatures of previously exhausted T cells from treatment naïve CLL patients (representing disease heterogeneity). Cytotoxicity assays revealed that treating primary T cells and autologous CLL cells with avadomide (1 μM, 48h) activated anti-tumor T cell killing function (P We next performed RNA sequencing on highly purified T cells from treatment naïve CLL patients, representing extremes of prognosis (n=6 good and n=6 poor including disease harboring TP53 abnormalities), following 18 h treatment with avadomide (100 nM) or anti-PD-1 (nivolumab) or PD-L1 (durvalumab) alone (10 μg/ml) or in combinations. Differential expression pathway analysis revealed that the top functional gene categories common for all the avadomide and combination treated samples (independent of anti-PD-1 or anti-PD-L1 monotherapy) were related to the response to type I and II IFN signaling, as well as inflammatory/stimulatory cytokine TNF-α, proliferative IL-6/JAK/STAT3, and IL-2/STAT5 responses. Type I IFN drives expression of chemokines CXCL10 and CXCL9, which are linked to enhanced tumor-infiltrated lymphocyte recruitment, and these chemoattractant genes were significantly upregulated in the avadomide and combination treated patient samples. In addition to their immunostimulatory roles, type II IFN (IFN-γ) and chronic type I IFN signaling have been linked to T cell resistance/exhaustion. We detected upregulated PD-L1 transcript in avadomide and combination treated T cells - supporting blockade of this inhibitory ligand. To investigate the ability of avadomide to modulate T cell migration, we performed comparative quantitative time-lapse microscopy analysis of pretreated patient T cells (n=12). These assays revealed that avadomide, as well as anti-PD-1 or anti-PD-L1 monotherapies, significantly enhanced (P Patient-derived xenograft models demonstrate that the therapeutic treatment of established tumors (3 weeks post-engraftment) with avadomide treatment (0.5 mg/kg) can activate anti-tumor T cells and significantly reduce disease volume (P In conclusion, our findings support the concept that PD-L1/PD-1 blockade in CLL could be enhanced when combined with avadomide through the promotion of immunogenic IFN signaling that enhances T cell infiltration and function. We believe these data support the rationale for combination immunotherapy that could convert a non-immunogenic TME into a 'hot' T cell inflamed TME which would be sensitive to checkpoint blockade. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Patten:L Hoffman La Roche: Honoraria, Research Funding; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, Research Funding; AbbVie Inc: Honoraria, Other: travel; Janssen: Honoraria, Other: travel. Vardi:Gilead: Research Funding; Janssen: Honoraria. Ramsay:MedImmune: Research Funding; Roche Glycart AG: Research Funding; Celgene Corporation: Research Funding.

Follicular lymphoma has considerable clinical heterogeneity, and there is a need for easily quantifiable prognostic biomarkers. Microvessel density has been shown to be a useful prognostic factor based on numerical assessment of vessel numbers within histologic sections in some studies, but assessment of tumor neovascularization through angiogenic sprouting may be more relevant. We therefore examined the smallest vessels, single-staining structures measuring less than 30 μm2 in area, seen within histologic sections, and confirmed that they were neovascular angiogenic sprouts using extended focal imaging. Tissue microarrays composing diagnostic biopsies from patients at the extremes of survival of follicular lymphoma were analyzed with respect to numbers of these sprouts. This analysis revealed higher angiogenic activity in the poor prognostic group and demonstrated an association between increased sprouting and elevated numbers of infiltrating CD163+ macrophages within the immediate microenvironment surrounding the neovascular sprout.

Introduction MicroRNAs (miRNAs) are short endogenous non-coding RNAs consisting of 18-25 nucleotides in length which influence gene expression and play pivotal roles in a diverse range of cellular processes. Aberrant miRNA expression has been implicated in a variety of cancers, including haematological malignancies. The miR-181 family plays a crucial role in haematopoiesis, including megakaryocytic, erythroid and myeloid differentiation and both B and T cell development and differentiation. We therefore focused our study on validating novel downstream targets of miR-181. Methods A novel functional assay utilising an optimised 3'UTR enriched library and a dual selection strategy (Gäken et al., 2012) was performed to identify biologically relevant targets of miR-181c. BRK1 (BRICK1, SCAR/WAVE Actin Nucleating Complex Subunit) was identified as a potential target and validation was performed by quantitative real time PCR and western blot analysis. Given the potential role of BRK1 in the Wiskott-Aldrich Syndrome Protein Family Verprolin-Homologous Protein-2 (WAVE2) complex and actin polymerisation in T cells, we investigated the influence of the miR-181c-BRK1 axis on T cell function. Knockdown of BRK1, using short hairpin RNA (shRNA) lentiviral vectors, and overexpression of miR-181c, via transfection with miR-181c expression vectors, were performed in Jurkat and primary T cells. T cell activation was examined by measurement of CD69 and CD154 expression and actin polymerisation was quantified by total cellular F-actin content. Immune synapse formation was studied by conjugate formation between T cells and antigen-pulsed B cells. Lastly, lamellipodia formation was investigated by assessing the ability of T cells to spread on anti-CD3 coated slides. Results Target genes downregulated by miR-181c were identified. One such target was BRK1, a component of the WAVE2 complex that has been shown to play a pivotal role in actin polymerisation. Validation experiments showed that overexpression and inhibition of miR-181c had no impact on BRK1 mRNA expression but did in fact modulate protein expression, suggesting that miR-181c regulates BRK1 at the translational level. We demonstrated that primary T cell activation resulted in downregulation of miR-181c and upregulation of BRK1 protein expression, further strengthening our hypothesis that the miR-181c-BRK1 axis may play an important role in T cell activation. Next, we found that loss of BRK1 resulted in reduced T cell activation as shown by decreased expression of CD69 and CD154. Furthermore, we showed that downregulation of BRK1 expression by shRNA resulted in reduced actin polymerisation after T cell stimulation. Reduced expression of BRK1 led to a marked reduction in the total area (in square micrometers) of F-actin accumulation at T cell contact sites and synapses with B cells indicating defective immune synapse formation. Moreover, reduced BRK1 expression resulted in defect in lamellipodia formation in response to T cell receptor stimulation. Similarly, ectopic expression of miR-181c in Jurkat T cells also led to a reduction in T cell activation and actin polymerisation coupled with defects in immune synapse and lamellipodia formation, hence confirming the important role of the miR-181c-BRK1 axis in T cell activation. Lastly, we demonstrated that suppression of BRK1 induced reduced expression of other pivotal proteins in the WAVE2 complex including WAVE2, Abi1 and Sra1. This suggests that impairment of actin polymerisation-dependent T cell functions were a result of instability of the WAVE2 complex following BRK1 suppression. Conclusion For the first time, we hereby demonstrate that BRK1 is a target of miR-181c. Moreover, we have highlighted the potential role of the miR-181c-BRK1 axis in impaired actin polymerisation-dependent T cell function and immune synapse formation. Deregulation of the miR-181c-BRK1 axis requires further evaluation in haematological malignancies. Disclosures No relevant conflicts of interest to declare.

Immunomodulatory drugs (IMiDs®) such as lenalidomide and immune checkpoint blockade (ICB) antibodies can enhance autologous anti-tumor T cell immunity and have the potential to elicit durable control of disease in B cell malignancies. These immunotherapies are likely to be most effective when employed in treatment combinations. Thus, the goal of pre-clinical research should be to reveal mechanisms of action (MOA) in the tumor microenvironment (TME) and identify biomarkers to guide development of combination therapy for patients. CC-122 is a novel first-in-class pleiotropic pathway modifier (PPM®) that has potent anti-proliferative, anti-angiogenic and immunomodulatory activities and is currently in Phase I clinical trials for lymphoma and chronic lymphocytic leukemia (CLL). Here, we have utilized the immunological synapse bioassay to examine T cell interactions with CLL tumor cells (modeling anti-tumor T cell responses in the TME) following CC-122 treatment and measure the expression of co-signaling complexes at the synapse. Conjugation assays and confocal imaging were used to visualize intercellular conjugate interactions and F-actin polymerization at the immune synapse between CD4+ and CD8+ T cells and autologous CLL tumor cells pulsed with superantigen (acting as antigen-presenting cells, APCs). Peripheral blood was obtained from treatment naive CLL patients (n=40) representative of disease heterogeneity. Treatment of both purified CLL cells and CD4+ or CD8+ T cells with CC-122 (0.01 - 1 μM for 24h) dramatically enhanced the number of T cells recognizing tumor cells (% conjugation) and increased the formation of F-actin immune synapses (area, μm2) compared to vehicle treated cells (P In conclusion, our results demonstrate for the first time that CC-122 can activate T cell immune synapse signaling against autologous CLL tumor cells and this immunomodulatory capability is more potent than lenalidomide. We further show that CC-122 activation of T cells is associated with enhanced expression of the co-stimulatory receptor ICOS and co-inhibitory checkpoints CTLA-4 and PD-1 at the synapse site. Importantly, our pre-clinical data demonstrates that this regulatory feedback inhibition can be exploited by the addition of anti-PD-L1, anti-PD-1 or anti-CTLA-4 ICB to CC-122 to more optimally stimulate T cell activity against immunosuppressive tumor cells. Disclosures Hagner: Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment. Gandhi:Celgene: Employment, Equity Ownership. Ramsay:MedImmune: Research Funding; Celgene: Research Funding.

There is a clinical need to identify novel treatments for relapsed/refractory DLBCL. Cancer cells engage in novel associations with stromal and immune cells in the TME that provide crucial contributions to disease progression, immune evasion and therapeutic response. However, these hallmark capabilities have been understudied in DLBCL. Given tumor cell genetic complexity, targeting the TME has become a compelling therapeutic strategy. Immune checkpoint blockade therapy (ICB) (e.g. anti-PD-1), which can activate anti-tumor immunity, has provided a new weapon against cancer and serves as an illustrative example of therapeutically re-educating the TME. Clinical results indicate that only a fraction of DLBCL patients currently respond to ICB. Understanding ill-defined TME-driven immune suppression should help optimise ICB and identify novel therapeutic opportunities. Gene expression studies of DLBCL have identified molecular signatures present in both GCB and ABC subtypes related to the TME that correlated with outcome. The prognostically favorable stromal-1 signature reflects reprogrammed stromal cells, extracellular matrix (ECM) and an active immune response. The less favorable stromal-2 signature indicates elevated angiogenesis and blood vessel density. CAFs promote ECM remodelling and angiogenesis in solid cancers. We hypothesized that CAFs play an important role in the pathogenesis of DLBCL including the regulation of subverted host anti-tumor immunity. To assess whether DLBCL tumor cells induce a CAF phenotype in previously healthy stromal cells, we established a co-culture system with subsequent imaging of conditioned cells. Primary human lymphatic fibroblasts (HLFs) were co-cultured for 5 days in direct contact with a panel of GCB (SU-DHL4, SU-DHL6, DOHH2) and ABC (OCI-LY10, RIVA, U2932) DLBCL cell lines or healthy control B-cells. Quantitative analysis revealed a strong induction of CAF molecular marker expression including FAPα and α-SMA in all DLBCL-educated stromal cells compared to healthy B-cell exposed fibroblasts (P In conclusion, our results establish the ability of DLBCL tumor cells to reprogram HLFs into CAFs that acquire functional capabilities to modulate the TME. Notably, activated CAFs show a compensatory inhibitory response by up-regulating PD-L1 expression that may represent an important TME-driven immunosuppressive mechanism. We believe this data contributes to the understanding of the biology that underlies stromal signatures in the DLBCL TME, in particular the contribution of CAFs to immune privilege. Disclosures Ramsay: Celgene: Research Funding; MedImmune: Research Funding.

An important hallmark of cancer progression is the ability of tumor cells to evade immune recognition. Understanding the relationship between neoplastic cells and the immune microenvironment should facilitate the design of improved immunotherapies. Here we identify impaired T-cell immunologic synapse formation as an active immunosuppressive mechanism in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). We found a significant reduction in formation of the F-actin immune synapse in tumor-infiltrating T cells (P < .01) from lymphoma patients compared with age-matched healthy donor cells. Peripheral blood T cells exhibited this defect only in patients with leukemic-phase disease. Moreover, we demonstrate that this T-cell defect is induced after short-term tumor cell contact. After 24-hour coculture with FL cells, previously healthy T cells showed suppressed recruitment of critical signaling proteins to the synapse. We further demonstrate repair of this defect after treatment of both FL cells and T cells with the immunomodulatory drug lenalidomide. Tissue microarray analysis identified reduced expression of the T-cell synapse signature proteins, including the cytolytic effector molecule Rab27A associated with poor prognosis, in addition to reduced T-cell numbers and activity with disease transformation. Our results highlight the importance of identifying biomarkers and immunotherapeutic treatments for repairing T-cell responses in lymphoma.

In this issue of Blood , Lapalombella and colleagues present evidence that the immunomodulatory drug lenalidomide (Revlimid) may repair and reverse the humeral defect in CLL.[1][1] Clinical response rates have improved significantly for chronic lymphocytic leukemia (CLL) patients treated with chemo

Chronic lymphocytic leukemia (CLL) is a disease of considerable clinical and genetic heterogeneity. Trisomy of chromosome 12 is the third-most common cytogenetic abnormality and has several distinguishing features including abnormal morphology, high prevalence of NOTCH1 mutations, high expression of CD11a and no additional clinical benefit from the addition of rituximab to fludarabine and cyclophosphamide. Previous studies have also reported a higher prevalence of trisomy 12 in patients with small lymphocytic lymphoma (SLL). The heterodimeric integrins CD18/CD11a (LFA-1), CD18/CD11b (Mac-1) and CD49d/CD29 (VLA-4) are cell surface transmembrane proteins involved in the inducible adhesion of leukocytes to vascular walls during the process of transendothelial migration from the bloodstream into the tissues. This process is particularly important in CLL as it allows the malignant cells to enter lymphoid organs where they receive growth and survival signals and are protected from chemotherapy. Multi-color flow cytometry was used to assess the cell surface expression of a panel of proteins involved in cell adhesion, motility and transendothelial migration. Compared to control B cells from the peripheral blood of age-matched healthy donors (n=25), CLL cells from peripheral blood of untreated CLL patients (n=101) had significantly reduced expression of CD11a, CD11b, CD18, CD29 and CD49d (p To examine the functional significance of the altered integrin expression on CLL cells, time-lapse microscopy was used to investigate the behaviour of tumor cells from CLL patients with or without trisomy 12, as well as B cells from healthy donors. Plates were coated with intercellular adhesion molecule (ICAM)-1 (LFA-1 ligand), and the cells stimulated with the chemokine CXCL12 (SDF-1). To measure cell motility, images were acquired using a Nikon Biostation IM microscope using a 20x objective lens at 30 second intervals for 40 minutes. A minimum of 50 individual cells per patient were tracked and their average velocity calculated using NIS-Element AR software. Although the average velocity of healthy B cells was significantly greater than CLL cells from all cytogenetic groups (mean 0.06119 µm/s, n=3 versus 0.03081 µm/s, n=8, p In conclusion, CLL cells from patients with trisomy 12 have a unique phenotype with relatively preserved expression of CD11a, CD11b, CD18, CD29 and CD49d. However, this increased integrin expression compared to non-trisomy 12 CLL cells does not result in improved LFA-1-mediated motility on ICAM-1. The motility defect of CLL cells may thus be indicative of a more general cytoskeletal defect that is independent of cell surface integrin expression. We continue to study mechanisms whereby altered expression of integrins on trisomy 12 CLL cells is associated with increased resistance to anti-CD20 mAb therapy, or whether this is instead mediated by differences in the tumor microenvironment. Disclosures: Gribben: Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria. Riches:Celgene: Research Funding.

Chronic lymphocytic leukemia (CLL) is associated with profound defects in immune function, resulting in failure of anti-tumor immunity and increased susceptibility to infection. We have previously demonstrated alterations in gene expression profiles of T cells from CLL patients, which translate into functional defects in T-cell immune synapse formation, motility and cytotoxicity (Gorgun et al. JCI 2005; Ramsay et al. JCI 2008, Blood 2013). However a comparison of the transcriptome of natural killer (NK) cells from CLL patients and controls has not been investigated. NK cells were isolated from the peripheral blood of patients with CLL and healthy donors, followed by gene expression profiling using the Affymetrix U133Plus2.0 platform. 117 probes showed a >2-fold decrease in expression while only 18 probes showed a >2-fold increase in expression (adjusted p-value < 0.05) in CLL NK cells compared to healthy donor NK cells. Strikingly, 52 out of the 117 significantly down-regulated probes (44.4%) were for interferon-inducible genes including STAT1 (Signal Transducers and Activator 1), SOCS1 (Suppressor of cytokine signaling 1), interferon regulatory factor genes IRF7 and IRF9, and oligoadenylate synthetase genes OAS1, OAS2, and OAS3. The majority of these genes were inducible by both type 1 and type 2 interferons. Many of these genes have been implicated in host immunity to viral infections, and so it is possible that decreased NK-cell responsiveness to interferon contributes to the increased susceptibility of CLL patients to viruses. Notably, there was also altered expression of signaling pathways in common with T cells from CLL patients, with dysregulation of the cytoskeleton genes RAB3GAP1, RAB38, and EPHA1 and down-regulation of JUN mirroring the dysregulated JNK-signaling and the altered actin cytoskeleton pathways we have found in T cells from CLL patients. These changes were not due to differences in the relative frequencies of CD56DIM and CD56BRIGHTNK cells. Lenalidomide has significant clinical activity in CLL. It is not directly toxic to tumor cells in vitro, but instead is thought to activate anti-tumor immunity and block pro-tumor micro-environmental factors. NK-cell proliferation has been shown to correlate with clinical response to lenalidomide in CLL (Chanan Khan et al. BJ Haem 2011). Therefore, we investigated the effect of lenalidomide treatment on the gene expression profiles of NK cells from CLL patients in comparison to healthy controls. PBMCs from CLL patients or healthy controls were cultured in the presence of 1μM lenalidomide or vehicle control for 48 hours, followed by NK-cell isolation, RNA extraction and gene expression profiling. There were striking differences in the effect of lenalidomide on NK cells from CLL patients compared with healthy NK cells. In CLL NK cells, lenalidomide repaired the down-regulation of interferon-inducible genes, by increasing the expression of genes such as OAS3, IFIT1, IFI44L, IFIT3, OAS1, PDK4, and ACTN1. Pathway analysis highlighted the effect of lenalidomide on inducing interferon signaling, showing significant activation of interferon α, γ, and λ as upstream regulators. While many of the interferon-inducible genes were up-regulated >3-fold in CLL NK cells, only OAS3 was significantly up-regulated in healthy NK cells with lenalidomide. Furthermore, the gene for IFNγ, IFNG, was actually significantly down-regulated in healthy NK cells by this agent. Lenalidomide also significantly down-regulated the expression of 5 genes encoding killer-cell immunoglobulin-like receptors (KIRs): KIR2DL1, KIR2DL2, KIR2DS3, KIR2DS4, and KIR3DL2, in healthy NK cells, but did not significantly down-regulate KIR genes in the CLL NK-cell dataset. Lenalidomide treatment did have some overlapping effects on CLL and healthy NK cells, including up-regulation of genes ARL11, CYFIP, and CORO1B that regulate the actin cytoskeleton pathway. In conclusion, NK cells from CLL patients have down-regulation of interferon response genes and pathways known to regulate normal immune function in response to bacteria and viruses. Lenalidomide has a differential effect on CLL and healthy NK cells: in CLL NK cells it repairs defective interferon responses, whereas in healthy NK cells it down-regulates inhibitory pathways. Disclosures: Riches: Celgene: Research Funding. Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Abstract 313 Background: We have previously demonstrated that CD4 and CD8 T-cells from CLL patients show profound dysfunctions in multiple gene pathways, including the actin cytoskeleton, which impairs the formation of functional immunologic synapses between T cells and APCs. Functional screening assays on Mec-1 cells have identified CD200, CD270, CD274, and CD276 as inhibitory ligands which induce impaired actin synapse formation in both allogeneic and autologous T cells. We also demonstrated that the Eμ-TCL1 transgenic mouse model of CLL closely resembles the T-cell defects observed in humans, validating it as a valuable preclinical tool to examine changes in the microenvironment alongside the development of leukaemia. The aim of the current study is to investigate the role of CD200, CD270, CD274, and CD276 in the Eμ-TCL1 model. Methods: We used multiparameter flow cytometry to establish the expression of inhibitory ligands on CD19+/CD5+ unpurified splenocytes from Eμ-TCL1 mice on both the C57Bl/6 (B6) and the C3HB6-F1 background and compared this to unpurified splenocytes from age matched wild-type (WT) controls of the respective coisogenic strain. Results: A total of 19 leukemic Eμ-TCL1 (n=10 C57Bl/6 and n=9 C3HB6-F1 background) and 11 WT mice (n=6 C57Bl/6 and n=5 C3HB6-F1 background) were examined. CD19+/CD5+ CLL cells constituted 92% (range 62–97%) of the DAPI-negative lymphocyte population. On CD19+/CD5+ CLL cells, CD274 (mean 98% ± SEM 0.4) and CD200 (mean 84% ± SEM 2.9 were uniformly strongly expressed, while CD270 (mean 74% ± SEM 4.7) and CD276 (mean 50% ± SEM 6.6) showed a weaker and more diverse expression, with no significant differences between the two backgrounds (all p>.05). Similar expression patterns were observed in Eμ-TCL1 mice with spontaneously occurring CLL and transplanted transgenic mice, with no differences between spontaneous and induced CLL (all p>.05). We then compared transgenic CD19+/CD5+ CLL cells to the WT CD19+ and the WT CD19+/CD5+ B1a-like cell population. Eμ-TCL1 CLL splenocytes showed a significant higher expression of CD274 and CD276 compared to expression on WT CD19+ (p In previous experiments, we used the Eμ-TCL1 model to investigate the polarization of F-actin and phosphotyrosine at the immune synapse between splenic autologous T-cells and APCs and subsequent effector function. Age-matched WT mice had a significantly higher accumulation than transgenic mice. To assess the functional role of inhibitory ligands, knock-down experiments using lentiviral shRNA and blocking antibodies are currently under way to assess if this restores immune synapse formation and T cell effector function in vivo. Conclusions: The inhibitory ligands CD200, CD270, CD274 and CD276 are expressed in vivo and appear to be of functional relevance for the anti-cancer immune response. They therefore represent attractive targets to restore T-cell effector function, which might be achieved by gene therapy approaches and blocking antibodies. Disclosures: Gribben: Celgene: Honoraria.

Abstract 564 The ability to evade immune destruction is increasingly being recognised as a crucial feature of cancer cells. Chronic lymphocytic leukemia (CLL) is associated with profound defects in T-cell function, resulting in failure of anti-tumor immunity and increased susceptibility to infections. T cells from CLL patients exhibit functional defects and alterations in gene expression, that show similarities to exhausted T cells in chronic viral infections. However, it is unclear whether CLL T cells are truly exhausted, or whether these defects are restricted to expanded populations of CMV specific T cells. We investigated the phenotype and function of CD8+ T cells from CLL patients and controls matched for age and CMV-serostatus. We demonstrate an increased proportion of CCR7- effector T cells in both CLL patients and CMV-seropositive individuals (p Lenalidomide has recently been demonstrated to have significant clinical activity in CLL. Its mechanism of action in this disease is not well understood, but it thought to act primarily by a combination of CLL cell and immune cell activation. We therefore examined the ability of lenalidomide to repair the observed T cell defects by investigating the impact of this agent on the gene expression profiles and function of CLL T cells. Treatment of CLL CD8+ T cells with lenalidomide increased the expression of 137 genes, while 34 genes were downregulated. The most prominent changes in expression were of genes involved in cytoskeletal signaling including WASF1 (Wiskott-Aldrich syndrome protein, family member 1), and TPM2 (tropomyosin 2). There was also upregulation of genes involved in lymphocyte activation, including TNFSF4 (Tumor necrosis factor ligand superfamily, member 4: OX40L), LAG3 (Lymphocyte-activation gene 3), and TNF, and genes involved in cell proliferation such as IKZF1 (Ikaros) and GRN (Granulin). Although lenalidomide treatment or anti-CD3 stimulation alone had no impact on T-bet expression, co-treatment with both anti-CD3 stimulation and lenalidomide resulted in significantly enhanced T-bet expression and increased production of interferon-γ. In contrast, lenalidomide treatment alone was able to improve T cell cytotoxic function, associated with repair of trafficking of granzyme B into the immunological synapse. In conclusion, T cells from CLL patients exhibit features of T-cell pseudo-exhaustion that are present irrespective of CMV serostatus. Treatment of CLL T cells with lenalidomide results in upregulation of genes involved in proliferation, activation, and cytoskeletal pathways, resulting in repair of the functional T cell defects. Table 1. Comparison of the phenotypic and functional defects of T cells from CLL patients with T-cell “exhaustion” in chronic viral infections Exhausted T cells in chronic viral infections T cells from CLL patients Increased expression of inhibitory receptors Yes Yes Abnormal transcription factor profile Yes Yes Reduced proliferative potential Yes Yes Decreased expression of IL-7R (CD127) Yes No Decreased cytokine production ↓IL-2, ↓IFN-γ Yes No Impaired cytotoxicity Yes Yes Disclosures: Riches: Celgene: Research Funding. Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

Abstract 246 Natural killer (NK) cells are an innate component of immune system that can produce a graft vs. leukemia (GVL) effect after stem cell transplantation. NK cells derived from acute myeloid leukemia (AML) patients are defective in their cytolytic function against leukemic cells. In order to better understand the mechanism of this defect, we performed functional assays examining immunological synapse formation of AML patient NK cells with autologous and allogeneic primary AML cells acting as antigen-presenting cells (APCs). Confocal microscopy was used to image and score F-actin polymerization at the immunological synapse between patient NK cells and leukemic cells. Accumulation of F-actin beneath the area of the NK: APC contact site is a hallmark of NK lytic synapses and allows signaling molecules to regulate appropriate activation and effector function. AML patient derived NK cells (AML-NK cells) formed significantly fewer synapses with autologous leukemia cells than healthy donor NK cells (12% versus 30%, n = 16. p > 0.001). Moreover, AML-NK cells were defective in their ability to recruit the key receptor NKG2D and the signaling molecule phosphotyrosine to immunological synapse contact sites. Signaling through the costimulatory ligand4-1BB-L (CD137L) has been shown to activate T cells, enhance antitumor responses and has multiple immunomodulatory effects on dendritic cells and NK cells. We postulated that AML-NK cells could be activated for enhanced cytolytic activity using artificial APCs generated to express CD137L. To test this, we setup co-culture assays using AML-NK cells and artificial CD137L-APCs before subsequent examination of immunological synapse function with AML blasts. Stimulated AML-NK cells that formed cell conjugate interactions with AML blasts, showed a significant increase in formation of immunological synapses compared to unstimulated AML-NK cells. The number of AML-NK/AML blast immunological synapses increased 16 hours after stimulation and peaked at approximately 72 hours. CD137L stimulation of AML-NK cells was also associated with increased cytotoxic function against primary AML cells (n = 6, p Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Abstract 802 We have previously demonstrated that impaired formation of the T cell immunological synapse in response to autologous (auto) antigen-presenting cells (APCs) is a global immunosuppressive mechanism in chronic lymphocytic leukemia (CLL) (J Clin Invest. 2008;118(7):2427-2437). Polymerization of F-actin beneath the area of the T cell:APC contact site generates a structural support for signaling molecules to assemble and regulate appropriate CD4+ T cell activation and cytolytic CD8+ T cell (CTL) effector function. Importantly, direct contact interaction with tumor cells was shown to induce defective actin polarization at the synapse in previously healthy allogeneic (allo) peripheral blood (PB) T cells. Here we have extended our functional screening coculture assays and show that CD200, CD270 (TNF receptor, TNFR-superfamily 14, SF14), CD274 (programmed death ligand 1, PD-L1), and CD276 (B7-H3) are co-opted by primary CLL cells (n=25) to induce impaired actin polymerization at the CD3+ T cell synapse. Antibody neutralizaton of these CLL ligands significantly increased allo T cell synapse actin polymerization with APCs compared to isotype control treated cells (P Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Abstract 1794 Immune dysfunction is a hallmark of chronic lymphocytic leukemia (CLL) including suppressed humoral and cell-mediated immune responses. The immunomodulatory agent lenalidomide has shown effective clinical activity against CLL, but its mechanism of action is poorly understood. Previous work has demonstrated that the T cell immunological synapse and functional defects in CLL can be reversed following lenalidomide treatment (J Clin Invest. 2008; 118). Polymerization of F-actin at the NK cell immunological synapse with tumor cells is required for signaling molecules to assemble and regulate NK cell activation and effector function. Confocal microscopy was used to visualize and analyze F-actin polymerization at the immune synapse between NK cells and CLL cells. The impaired immune synapse defect identified in CLL could result from not only the defects of CLL B cells but also defects in the CLL NK cells or a combination of both factors. To investigate the contribution of each factor, we examined synapse formation in experiments using CLL B cells with autologous CLL NK cells or healthy allogeneic NK cells. Conjugates formed with healthy NK cells and CLL B cells exhibited a strong band of F-actin at the immune synapse. In contrast, significantly less actin polymerization at the synapse was observed in autologous CLL NK cells and CLL B cells (P < 0.01). These results indicate CLL B cells, together with CLL NK cells contributed to the immune dysfunction in CLL. As autologous NK cell function in CLL is suppressed, we investigated the utility of CB as a potential functional source of NK cells for CLL immunotherapy. We examined the effect of lenalidomide on NK cell immune synapse function with CLL B cells acting as APCs. We demonstrated that ex vivo treatment of CLL cells with lenalidomide (500 ng/ml) for 48 hours caused a significant increase in the ability of autologous CLL NK cells to form F-actin immune synapses with CLL B cells. The same treatment of CLL B cells also significantly increased the ability of CB-NK cells to form F-actin immunological synapses with these treated CLL B cells compared to untreated CLL B cells (33.6% to 67.3%, P < 0.01, n=6). Our results also show that lenalidomide treatment of autologous NK cells from CLL patients enhanced synapse formation with treated CLL cells compared to experiments using untreated NK cells, but with reduced function compared to CB NK cells. Of note, lenalidomide treatment was shown to increase the recruitment of the signaling molecule Lck to NK cell:CLL cell synapse site, that is known to regulate lytic synapse function. Importantly, lenalidomide treatment significantly increased CB-NK killing of CLL B cells compared to untreated CLL B cells (20.5% versus 48.2%, E:T ratio of 10:1, n = 6, p < 0.001). These results provide insight into the potential mechanism of action of lenalidomide's anti-leukemic function – priming CLL tumor cells for enhanced NK cell lytic synapse formation and effector function. In addition, the data suggests that immunotherapeutic strategies utilizing a combination of CB-NK cells and lenalidomide has an enhanced clinical efficacy in CLL. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Abstract 976 Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL. Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B). In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Abstract 141 Patients with follicular lymphoma (FL) have an extremely variable clinical course. Although clinical parameters can be used to define prognostic subgroups, there is a need to identify robust biomarkers that not only aid in prognosis, but also help define the underlying pathophysiology of the disease. Previous gene expression profiling studies demonstrated that among the most important prognostic markers were the molecular features of nonmalignant tumor-infiltrating immune cells present in the tumor at diagnosis (Sandeep et al. NEJM 2004). To investigate the molecular mechanisms whereby T cells are altered in the FL microenvironment we studied highly purified, sorted infiltrating CD4 and CD8 T cells from previously cryopreserved single cell suspensions of lymph node (LN) biopsies at the time of diagnosis in patients with FL (n=12) and compared them to those isolated from reactive tonsils (n=7) as well as from peripheral blood (PB) (n=10) of age matched healthy individuals. These tumor infiltrating T cells (TILs) have impaired proliferative and cytotoxic responses and impaired capacity to form immunologic synapses with antigen presenting cells. Co-culture of FL cells in contact with healthy allogeneic T cells induces similar T cell functional defects, demonstrating that it is the FL cells that drive the altered T cell function. To investigate the molecular mechanisms for this, we performed global gene expression profiling using Affymetrix U133Plus2 chips of highly purified (>95% purity) CD4 and CD8 cells. For both CD4 and CD8 cells, unsupervised analyses distinguished healthy and FL T cell subsets. Using a fold change cut off > 2 and false discovery rate of 5%, 280 genes were differentially expressed for CD4 and 717 genes for CD8, with109 genes overlapped for both subsets. The gene array data was validated for RNA using Real-Time Quantitative-PCR and for protein by flow cytometry and immunohistochemistry. Pathway analysis indicated disruption in multiple pathways including cytokine signaling, T cell differentiation, cell proliferation and, actin-based motility/cytoskeleton formation. In both CD4 and CD8, among the most downregulated genes in FL TILs were ACTN1 and IL17A, and the most upregulated genes were PMCH and ETV1. Using Tissue Microarray (TMA) we demonstrate that the intensity of expression in TILs in FL was significantly higher for PMCH (p Disclosures: Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

Abstract 914 T lymphocytes have an essential role in adaptive immunity and rely on tightly regulated signaling through integrin lymphocyte function-associated antigen (LFA)-1 to migrate into lymph nodes and interact with antigen-presenting cells. Malignant cells modify their immune microenvironment to prevent effective host anti-tumor responses, promote tumor progression, and suppress the therapeutic benefit of immunotherapy treatments. Here we assessed LFA-1-mediated cell migration of highly purified T cells from treatment naïve chronic lymphocytic leukemia (CLL) patients compared to age-matched healthy donor T cells using CXCL12 stimulation and immobilized ICAM-1, the principal integrin ligand. Video microscopy with motility tracking analysis identified that both CD4 and CD8 T cells from CLL patients (n=14) exhibited significantly reduced migration rates (P < .01) compared to healthy donor T cells (5.5 ± 0.3 (SEM) μm/min and 4.4 ± 0.2 μm/min compared to 8.2 ± 0.3 μm/min and 7.5 ± 0.3 μm/min respectively). We further identified that direct CLL cell contact, and not soluble factors alone, induced similar T cell motility dysfunction in previously healthy CD3 T cells. Primary co-culture of healthy donor T cells with CLL cells caused a significant decrease in the speed of migration on ICAM-1 compared to coculture with control healthy B cells (6.2 ± 0.3 μm/min versus 9.5 ± 0.6 μm/min) (n=9) (P < .05). Next we sought to repair this T cell defect in CLL using a clinically relevant agent. We identify that treatment of CLL patient T cells (n=9) with lenalidomide restores rapid LFA-1 mediated migration on ICAM-1. Ex vivo treatment of CLL T cells with lenalidomide (1μM for 24 hours) significantly increased the speed of T cell migration compared to untreated patient T cells (7 ± 0.4 μm/min versus 2.5 ± 0.7 μm/min) (P < .05) and the rescued T cell migratory function of lenalidomide exposed patient T cells was comparable to healthy donor T cells treated with or without drug. Interference reflection microscopy (IRM) examining the contact zone between migrating T cells and ICAM-1 identified a significant CLL patient T cell adhesion defect (P < .05) with reduced spreading area and strength of adhesive contacts (pixel density) compared to healthy donor T cells. IRM was further utilized with pharmacological inhibitors to demonstrate that exposure to lenalidomide rescued CLL T cell adhesion by acting on the Rho family GTPases that are dysregulated in cancer patient T cells. Lenalidomide significantly increased (P < .05) levels of active RhoA in CLL patient T cells compared to untreated cells. In addition, untreated CLL patient T cells adhering to ICAM-1 exhibited significantly reduced expression levels of phosphorylated myosin light chain (MLC) compared to healthy donor T cells (P < .05) and this defect was repaired following lenalidomide treatment. MLC is normally phosphorylated by MLC kinase at the T cell leading edge and by the RhoA target, ROCK at the trailing edge, and is an important downstream signaling molecule during LFA-1-mediated T cell motility. Further expression analysis identified that lenalidomide significantly increased (P < .01) ICAM-1-engaged high-affinity LFA-1 in CLL patient T cells to levels comparable to healthy donor T cells. Overall, our results show that T cells in CLL patients have dysfunctional tumor-induced cytoskeletal signaling via the Rho GTPase signaling pathway, and this is reversed by lenalidomide, rescuing dynamic LFA-1 mediated outside-in signalling and migration. Lenalidomide's immunomodulatory activity was highly cancer T cell specific: rescuing defective LFA-1 migration and signaling in CLL T cells, but with no detectable effects on healthy donor T cells. These findings provide important mechanistic insight into the action of lenalidomide, and highlight the potential clinical utility of immunomodulatory drugs to rescue normal immune function in cancer. Disclosures: Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

Abstract 696 The ability of cancer cells to modulate the immune microenvironment is now recognized as an important hallmark of disease pathophysiology. Identifying the molecular mechanisms of cancer immune suppression in the laboratory is key to the design of more effective immunotherapeutic treatment strategies. We previously demonstrated that chronic lymphocytic leukemia (CLL) cells induce alterations in global gene expression profiles in patient CD4 and CD8 T cells, and a profound T cell immunological synapse formation defect that can be reversed with lenalidomide (J Clin Invest. 2005;115(7):1797-1805, and 2008;118(7):2427-2437). Here we used small interfering RNA (siRNA) with a 2-part functional screen to identify key CLL cell molecules inducing T cell immune suppression. siRNA treated tumor cells were cocultured in direct contact with healthy allogeneic T cells for 24 hours, T cells purified from coculture and used in cell conjugation immune synapse assays with superantigen-pulsed third party B cells as antigen-presenting cells (APCs). Confocal microscopy and image analysis software was used to quantify the mean area of T cell F-actin immune synapse formation events from each experimental cell population. Treatment of the CLL cell line MEC-1 with either TNFα, TGFβ, IL-10, or IL-6 siRNA identified no gain in subsequent CD3 T cell immune synapse function compared to control non-targeting siRNA or untreated CLL cells. However, CD200 or programmed death 1 (PD1) ligand 1 (PD-L1, CD274) siRNA treatment significantly enhanced (P < .01) subsequent T cell synapse formation events with APCs (comparable to positive control experiments blocking tumor cell:T cell direct contact with ICAM-1 siRNA, or primary coculture of T cells with allogeneic healthy donor B cells). Primary CLL patient cells (n=10) were treated with individual or pooled neutralizing antibodies, or siRNA, targeting PD-L1, CD200, or cytokines. This analysis revealed that counteracting the combined activity of PD-L1, CD200 and TGFβ exhibited the most pronounced repair of subsequent T cell synapse function compared to control treated tumor cells (P < .01). These data suggest that CLL-released cytokines such as TGFβ contribute to, but are not essential for the T cell synapse defect. We also identified that blocking the T cell receptors PD-1, CD200-R and TGFβ-R1 with neutralizing antibodies prevents CLL inhibitory signaling (P < .01) compared to isotype control IgG treated T cells in contact with tumor cells. We further show that knock-down of PD-L1, CD200 and TGFβ on ex vivo CLL cells prevents inhibitory CD4 and CD8 T cell synapse function compared to control siRNA (P < .01) using the Eμ-TCL1 mouse model of CLL. The addition of lenalidomide (1μM) in ex vivo CLL cell:T cell coculture assays significantly increased (P < .01) subsequent T cell synapse function compared to untreated vehicle control experiments. Flow cytometric analysis identified that lenalidomide down-regulates both CLL expressed PD-L1 and CD200 ligands, and T cell cognate receptor PD1 and CD200R expression during intercellular contact interactions. Moreover, subsequent effector T cell killing function was significantly enhanced (P < .05) following antibody blockade of CLL cell PD-L1 and CD200 with or without lenalidomide treatment during primary coculture with CD8 T cells. We are currently investigating the expression and activity of PD-L1, CD200, and other co-inhibitory molecules in CLL and other haematological and solid malignancies, using patient tissue microarray analysis and confocal co-localization analysis. This work is identifying common inhibitory ligands utilized by tumor cells to suppress T cell synapse function. These results provide important mechanistic insight into immune suppression in CLL and the action of lenalidomide, and identify co-inhibitory ligands as potential immunotherapeutic targets to repair T cell function. Disclosures: Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

Abstract 2370 Poster Board II-347 Adoptive transfer of NK cells in human B-CLL has been limited by the lack of suitable animal models to test the clinical efficacy of this immune therapy strategy. Primary patient B-CLL cells are difficult to engraft in NOD-SCID mice as these mice lack an immune microenvironment that provides essential accessory cells for tumor development. In the current study, we utilize a novel 2-step engraftment protocol using NOD-SCID γnull mice (NSG). Firstly, human cord blood (CB) derived CD34+ stem cells were engrafted to generate humanized chimeric mice capable of supporting B-CLL cells. By week 12, the human engraftment level reached 30% to 60% as detected in peripheral blood. Secondly, these mice were infused with human primary B-CLL cells labeled with CFSE. Our results show that following tail vein injection of 5E7 primary patient CLL cells, chimeric mice exhibited proliferation of CFSE+CD5+ B cells in the spleen and bone marrow, with disease development resembling human CLL. This allowed us to investigate the efficacy of ex vivo IL-2 expanded CB NK cell therapy using this novel mouse model system. As early as 24 hours post-infusion of IL-2 expanded human CD56+CD3- CB NK cells (10E7 per mouse) there was a detectable reduction of CD5+ leukemia cells in the peripheral circulation of CLL-engrafted mice by flow cytometry analysis. Moreover, by day 7 there was a dramatic 99.5 % reduction of CD5+ B-CLL cells in the blood, bone marrow, and spleen of experimental animals compared to the non-treated control group (P Disclosures: No relevant conflicts of interest to declare.

Abstract 3029 Poster Board II-1005 Donor peripheral blood (PB) natural killer (NK) cell have shown clinical promise in cancer immunotherapy. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. Umbilical cord blood (CB) represents an important alternative source of NK cells for adoptive immune therapy. We first demonstrated that cord blood (CB) derived NK cells have poor cytolytic activity and deficiency in the formation of the F-actin immunological synapse with HLA class I deficient target K562 cells and primary AML blasts compared to PB-NK cells. In this study, we explored the cellular mechanism of these dysfunctions. We hypothesized that adhesion and signaling molecules may be defective in unmanipulated CB NK cells. Activating receptor Both CD2 and the integrin lymphocyte function-associated antigen (LFA-1) play important roles in both T lymphocyte and NK cell immune synapse formation and their trafficking to the immune synapse regulates both T and NK cell function. We now show that unmanipulated CB NK cells exhibit reduced LFA-1 mediated adhesion to mobilized ICAM-1 compared to IL-2 expanded CB NK cells (CB NK 29.7+/- 3.2 %, vs expanded CB NK 78.5+/- 6.1%, n=6). Moreover, unmanipulated CB-NK cells demonstrated reduced surface expression of CD2, and high affintyLFA-1 detected by the specific antibody (MHM24). There was decreased recruitment of CD2 and LFA-1 to the NK cell immune synapse site as quantified by confocal microscope analysis (RRI CD2 CB NK 2.02 vs PB NK 4.98, n=3). Furthermore, defective LFA-1 trafficking lead to a decrease in downstream cytotoxic granules that traffic to the immunological synapse as demonstrated by decreased perforin trafficking to the CB-NK synapse site (> 60% reduction).We next wanted to confirm that CD2 or LFA-1 play a role in restoring the immune synapseformation for IL-2 expanded CB NK cells. We incubated expanded CB NK cells with blocking antibodies specific for LFA-1 or CD2 prior to conjugation to the K562 target cells. After CD2 or LFA-1 blocking there was decreased synapse formation, with a resultant decrease in cytotoxic function. When monoclonal antibodies against both CD2 and LFA-1 were used there was significant blockade of the formation of the immune synapse, and a marked reduction of CB NK cell cytolytic activity (Mean specific lysis of K562 targets at E:T ratio 20:1 was 81% IgG control vs 22% with anti-CD2; and 29% with anti-LFA-1, n=6, P Disclosures No relevant conflicts of interest to declare.

Abstract 2663 Poster Board II-639 Natural killer (NK) cells are an important component of the innate immune surveillance of tumor cells. Defective NK cell function has been correlated with poor prognosis in acute myeloid leukemia (AML). It is well established that NK cell-mediated cytolytic activity is significantly diminished in AML patients; the mechanisms of this hypo-function are not well understood. Identifying mechanisms of tumor-induced immune suppression of lymphocytes function will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired NK cell responses in AML and demonstrate impaired NK cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between CD56+ CD3- NK cells and autologous AML blasts. We identified a significant reduction in formation of the NK cell immune synapse (NKIS) (p 70% reduction). This defect was induced by direct tumor contact since NK cell defects were induced in healthy NK cells when they were co-cultured (in direct contact) for 48 hr with allogeneic AML blasts, but not with healthy allogeneic monocytes (P < 0.01). In control transwell co-culture experiments, where the NK cells and AML blast were not in direct contact, we did not observe the induced defect. We examined the molecular nature of the AML blast induced defect by quantifying recruitment of a number of these NK cell adhesion and cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with AML blasts, healthy NK cells showed significantly reduced recruitment of integrin LFA-1, CD2, Lck, WASP, and tyrosine-phosphorylated protein to the NK-AML target interactions synapse (P < 0.001). These studies demonstrate a role for the tumor induced immune suppression of NK cells and will aid in the development of immunotherapeutic approaches targeting AML. Disclosures: No relevant conflicts of interest to declare.

Contrary to expectation, we have shown that the absolute number of T cells in the peripheral blood of patients presenting with AML is higher than that of healthy individuals (p=0.0006). Previous publications have demonstrated that in vitro the microenvironment in AML is immunosuppressive to healthy T cell function (Buggins et al. 2001). To analyze the molecular nature of T cell defects in patients with AML, we performed global gene expression profiling of peripheral blood CD4 and CD8 T cells from 10 AML patients compared to that of 21 healthy volunteers. The purity of the CD4 T cells was 86% in AML and 94% in healthy donors and 92% for AML and 87% in healthy donors for CD8 T cells. cRNA was hybridised on Affymetrix U133Plus2 chips using standard protocols. Samples were normalised using the MAS5 algorithm and subsequent analysis using Bioconductor software indicated marked differences between the gene expression profiles of CD4 and CD8 T cells from AML patients compared with normal individuals. Using a false discovery rate of 0.01 and fold change greater than 2, 1407 genes were found to be differentially regulated for CD4 and 779 genes for CD8. There was a large overlap in the two gene lists. The microarray data was validated using real-time quantitative PCR. Initial pathway analysis using Ingenuity software indicated many genes involved in cell proliferation, cell death and gene expression. Molecular defects in T cells from AML patients show marked differences from those we have already noted in T cells from patients with CLL. Only 2% of genes for CD4 and 6% of genes for CD8 are differentially regulated in T cells from both AML and CLL patients. These data suggest a different molecular basis for the T cell defects in these disease types. However, 2 genes downregulated in CD8 T cells in AML patients, ACTN1 and FILIP1 suggested impaired actin cytoskeletal activity may contribute to immune dysfunction in AML T cells as we have already demonstrated in CLL. We therefore tested the ability of AML CD4 and CD8 T cells to form intact immunological synapses (IS) with autologous AML blasts in the presence or absence of superantigen. F-actin was visualised with rhodamine phalloidin and recruitment of activated T cell receptor mediated signalling molecules was detected using an anti-phosphotyrosine antibody. Conjugate and synapse formation and phosphotyrosine signalling was assessed by confocal microscopy with at least 50 random conjugates analyzed. Unlike CLL B cells, AML blasts can act as antigen presenting cells (APCs) for conjugate and IS formation with healthy T cells. Again, in contrast to CLL, AML T cells are able to form conjugates with autologous tumour cells but demonstrate impaired IS formation and, in CD8 T cells, decreased phosphotyrosine signalling at the synapse site. This data indicates that T cells in patients presenting with AML are abnormal in terms of their gene expression profile and although they retain the ability to conjugate with autologous blasts, reduced polarisation of F-actin to form immune synapses is seen. We are currently investigating if these changes are induced directly by tumour cells and the functional consequences of the gene expression changes identified. The ability of AML blasts to function as APCs for IS formation is in keeping with the observed GVL effect in this disease. Understanding the nature of T cell defects in patients presenting with AML is fundamental before successful autologous immunotherapeutic strategies can be implemented.

Natural killer (NK) cells play an important role in host immunity by eradicating pathogen-infected and tumor cells. Peripheral blood-derived NK cell therapy has shown promise in clinical trials for acute myeloid leukemia. Cord blood (CB) is another potentially rich source of NK cells. However NK cells isolated directly from CB have poor cytolytic activity. We investigated the mechanism for the low cytolytic activity of CB NK cells and whether the defect could be overcome by ex vivo expansion. NK cell killing of the target cells is achieved by formation of a mature immune synapse, followed by secretion of lytic granules containing perforin and granzymes. F-actin polymerization at the NK-tumor cell conjugates is a hallmark of immune synapse formation which can be detected and quantitated by confocal microscopy. We hypothesized that CB NK cells exhibit low cytotoxicity against leukemia target cells due to a defect in the formation of the immune synapse. We confirmed that unmanipulated CB NK cells exhibit low cytotoxicity against AML blasts (5% at an E:T ratio 20:1) in comparison to peripheral blood (PB) NK cells (35% at an E:T ratio 20:1). Evaluation of the natural cytotoxicity receptors (NCRs) showed normal expression of the NKp30 and the NK46 receptors. We then investigated whether this poor cytolytic capability is accompanied by poor immune synapse formation with tumor cells. Surprisingly, both CB and PB NK cells expressed comparable levels of perforin and demonstrated similar perforin polarization to the immune synapse. F-actin polarization was observed in only 12% (range 9– 21%) of the CB NK cell/K562 conjugates versus 85% (range 68–87%) of PB NK cell/K562 conjugates (P

Understanding the elusive mechanisms of tumor-driven immune evasion will aid the refinement of existing cancer immunotherapy strategies and identify novel treatments. To date, pre-clinical animal models that closely model human cancer, including the immune suppressive mechanisms utilized by cancer cells, have been under-characterized. The identification and use of such models should allow better predictions of successful human responses to immunotherapy. As a model for changes induced in non-malignant cells by cancer, we examined T cell function in Eμ-TCL1 transgenic mice as they developed leukemia from 12-months of age. Transgenic expression of TCL1 in B cells had no demonstrable effect on T cells, however, mice with leukemia had decreased in vivo antigen specific T cell activation, suppressed T cell mitogenic proliferation and impaired induction of idiotype specific CD8 T cells capable of killing CLL cells compared to control WT mice (age-matched throughout study) or Eμ-TCL1 transgenic mice without CLL. Leukemic mice also had dysfunctional T cell lymphokine production (Th2-preponderant). To understand the molecular basis for the observed functional defects and to compare changes seen in mice and patients with CLL we performed gene expression profiling. Analysis of highly purified CD4 and CD8 T cells in CLL mice demonstrated altered gene expression profiles compared to WT mice or to young Eμ-TCL1 mice without disease. Of note, infusion of CLL cells into young Eμ-TCL1 mice induced gene expression changes comparable to those seen in mice with developed leukemia, demonstrating a causal relationship between leukemia and the T cell defects. Analysis of gene expression changes in T cells in CLL mice compared with those in patients was performed using RESOURCERER, a database for annotating and linking microarray resources within and across species and identified 50 overlapping genes in CD4 T cells and 45 overlapping genes in CD8 T cells. The majority of differentially expressed genes in CD4 T cells from both mice and patients with CLL were involved in cell proliferation and activation pathways with increase in Lck. Multiple defects within the actin cytoskeletal formation pathways were identified in both CD4 and CD8 T cells including Cdc42. Integrity of the T cell cytoskeleton is essential to regulate the dynamic signaling required for T cell activation and effector function in response to immunological recognition of antigen-presenting cells (APCs). T cell conjugates from mice with leukemia had suppressed antigen-dependent F-actin accumulation and early T cell signaling at the immune synapse with CLL cells (APCs) compared to WT mice conjugates. Moreover, we have demonstrated that infusion of CLL cells into young mice induces this T cell defect, demonstrating an in vivo immunomodulating mechanism utilized by tumor cells. Treatment of both CLL cells and autologous T cells from leukemic mice with lenalidomide (0.5 μM for 24 h) enhanced the formation of the F-actin immune synapse and recruitment of tyrosine-phosphorylated proteins irrespective of the presence of exogenous antigen. Of note, the capacity to repair immunological recognition with this agent was associated with increased recruitment of the cytoskeletal signaling molecules Lck and Cdc42 to the immunological synapse, regardless of whether the gene was increased or decreased on gene expression profiling. These results demonstrate that leukemia cells induce changes in multiple T cell pathways regulating antigen recognition and effector function. The similarities with human CLL including reversible immunological synapse dysfunction with an immunomodulating drug validates the use of Eμ-TCL1 mice as a model for further analyses of ways to prevent and reverse cancer-induced immune dysfunction. The use of this model to understand and reverse the molecular changes in T cells induced by leukemia will likely have broad applications to maximize immune responses in patients.

Following allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion (DLI) from adult peripheral blood (APB), chronic lymphocytic leukemia (CLL) cells are good targets of a graft-versus-leukemia effect. However, some patients eligible for this treatment do not have a suitable allogeneic donor and CLL B cells have been shown to be dysfunctional antigen-presenting cells (APCs) for allogeneic APB T cells. As a result, allogeneic APB T cells show suppressed immunological synapse formation with CLL cells. Umbilical cord blood (CB) is a promising source of hematopoietic cells for allogeneic transplantation and can be obtained from matched unrelated donors with greater tolerance for incompletely HLA-matched recipients. Moreover, we have successfully expanded CB T cells ex vivo (anti-CD3/CD28 beads and rIL-2) using a protocol that retains a naïve and diverse immune population including central memory cells. In this present study we used confocal microscopy to visualize F-actin polymerization to assess immunological synapse formation of CB T cells compared to APB T cells with CLL B cells with and without superantigen as APCs. Our results identify the ability of unexpanded and expanded CB CD4 and CD8 T cells to form F-actin immune synapses with CLL B cells and of note, CB was more effective than unexpanded or expanded APB T cells (p

Identifying mechanisms of tumor-induced immune suppression will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired T cell responses in follicular lymphoma (FL) and demonstrate impaired T cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between tumor-infiltrating CD4 and CD8 T cells and autologous FL tumor cells with and without superantigen as antigen-presenting cells (APCs). We identified a significant reduction in formation of the T cell immune synapse in both CD4 (p 50% reduction). Comparable immunological defects were also identified in CD4 and CD8 T cells from transformed-FL (t-FL) patients. This defect was induced by tumor contact since T cell defects were induced in healthy T cells when they were were co-cultured for 48 hr with either allogeneic FL or t-FL cells, but not with healthy allogeneic B cells (P < 0.01). Of interest there was enhanced suppression of CD4 T cell synapses following FL co-culture assays and CD8 T cells in the t-FL setting. Since previous gene expression profile studies (Dave et al. NEJM, 2003) demonstrated prognostic significance of altered expression of immune signature genes, we examined the molecular nature of the FL-induced T cell defect by quantifying recruitment of a number of these T cell cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with FL cells, previously healthy T cells showed suppressed recruitment of integrin LFA-1, Lck, Itk, Rab27A and filamin-A to the synapse in subsequent T cell:APC interactions (P < 0.01). We further demonstrate that the immune synapse defects were repaired in part by treatment of the cells with the immunomodulatory drug lenalidomide. Of note, treatment of both FL B cells and autologous T cells with lenalidomide (1 μM for 24h) was required to enhance formation of the F-actin synapse and recruitment of tyrosine-phosphorylated protein and filamin-A irrespective of the presence of exogenous antigen (P < 0.01). We validated the altered expression of a number of these molecules including Itk, Rab27A and filamin-A at the protein level in FL tissue microarrays (TMA). Of note, elevated expression of intrafollicular Rab27A, that mediates targeted secretion of CD8 T cell cytolytic granules, was found in samples from a long-survival group (FL patients who lived more than 15 years). These results provide mechanistic insight into the demonstrated activity of lenalidomide in relapsed or refractory aggressive FL (Wiernik et al. JCO, 2008). These studies demonstrate the role of the tumor in the observed T cell defects in FL, and the molecular basis of these defects and suggest that immunotherapeutic approaches including the use lenalidomide offer the exciting prospect of repairing T cell suppression in B cell malignancies to enhance immune-mediated immunological responses against lymphoma cells.

We have previously demonstrated that multiple gene expression abnormalities are induced in T cells from chronic lymphocytic leukemia (CLL) patients including defects within the actin cytoskeleton signaling pathways that control immune recognition and motility (Gullu et al. JCI, 2005). T cell immune surveillance requires rapid migratory responses and LFA-1 (CD11a/CD18; αLβ2) is a promigratory receptor that engages the cytoskeleton to control migration. We hypothesized that CLL T cells may exhibit dysfunctional migration in response to ICAM-1, the principal ligand for LFA-1. Using time lapse microscopy, we observed significantly reduced chemokine SDF-1 (CXCL12) induced migration on ICAM-1 of CLL CD4 and CD8 T cells compared to age-matched healthy donor T cells. Healthy T cells tracked for 45 min displayed a random course of migration with an average speed of ~ 8 μm/min, whereas CLL T cells were slower ~ 5 μm/min (n=14, ~ 30% reduction, p

Cancer is associated with immune deficiency, but the molecular basis for this is poorly defined. We have previously demonstrated that multiple gene expression abnormalities are induced in patients with chronic lymphocytic leukemia (CLL) including defects within the actin cytoskeleton formation pathways. Based on this data, we hypothesized that failure of actin polymerization would result in defects in the formation of the immunological synapse (IS) which is critical for T cell activation and effector function. To assess this, actin polymerization at the IS in T cells in response to superantigen-pulsed B cells (APCs) was visualized using confocal microscopy. We observed significantly reduced ability to polymerize actin at the IS (> 50% reduction) in autologous CD4 and CD8 T cells from previously untreated CLL patients compared to age-matched healthy donors (p