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Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2–containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation “armored” chimeric antigen receptor (CAR) engineering of cord blood–derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15–secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy. Key Points: • CRISPR-Cas9 CISH deletion enhances the metabolic fitness and antitumor activity of armored IL-15–secreting CB-derived CAR-NK cells.

Farrerol, a dihydroflavone isolated from Rhododendron dauricum L., can inhibit vascular smooth muscle cell (VSMC) proliferation and exert a protective effect on H2O2-induced vascular endothelial cells injury. In this study, we investigated the effects of farrerol on VSMC phenotypic modulation and balloon injury-induced vascular neointimal formation and explored the underlying mechanisms. Serum-starved rat thoracic aorta SMCs (RASMCs) were first pretreated with farrerol (3, 10, and 30 μM, respectively), U0126 (a MEK kinase inhibitor), and SB203580 (a p38 kinase inhibitor), and followed by treatment with serum (10% FBS). The expression of several VSMC-specific markers, including α-SMA, SM22α, and OPN, were analyzed by western blot. Phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and p38 mitogen-activated protein kinase (MAPK) was also investigated. Farrerol inhibited the serum-induced transition of RASMCs from the contractile to the synthetic phenotype, and this was associated with a decrease in α-SMA and SM22α expression, and an increase in OPN expression. Farrerol also inhibited serum-induced phosphorylation of ERK1/2 and p38MAPK in RASMCs. Moreover, U0126 and SB203580 both inhibited the serum-induced phenotypic transition of RASMCs. These findings indicate that farrerol can maintain the contractile phenotype of VSMCs partly via inactivating the ERK1/2 and p38 MAPK signaling pathways. Using a rat model of carotid artery balloon injury, inhibition of VSMC phenotypic transition and suppression of neointimal formation were confirmed in vivo following the perivascular application of farrerol. Our results suggested that farrerol could be a promising lead compound for the treatment of vascular proliferative diseases.

Oxidative stress-mediated endothelial injury is considered to be involved in the pathogenesis of various cardiovascular diseases. Farrerol, a typical natural flavanone from the medicinal plant Rhododendron dauricum L., has been reported to show protective effects against oxidative stress-induced endothelial injuries in our previous study. However, its action molecular mechanisms and targets are still unclear. In the present study, we determined whether farrerol can interact with glycogen synthase kinase 3β- (GSK-3β-) nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling, which is critical in defense against oxidative stress. Our results demonstrated that farrerol could specifically target Nrf2 negative regulator GSK-3β and inhibit its kinase activity. Mechanistic studies proved that farrerol could induce an inhibitory phosphorylation of GSK-3β at Ser9 without affecting the expression level of total GSK-3β protein and promote the nuclear translocation of Nrf2 as well as the mRNA and protein expression of its downstream target genes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in EA.hy926 cells. Further studies performed with GSK-3β siRNA and specific inhibitor lithium chloride (LiCl) confirmed that GSK-3β inhibition was involved in farrerol-mediated endothelial protection and Nrf2 signaling activation. Moreover, molecular docking and molecular dynamics studies revealed that farrerol could bind to the ATP pocket of GSK-3β, which is consistent with the ATP-competitive kinetic behavior. Collectively, our results firstly demonstrate that farrerol could attenuate endothelial oxidative stress by specifically targeting GSK-3β and further activating the Nrf2-ARE signaling pathway.

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone (TDD), possess anti‐atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD‐treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS‐induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose‐dependently inhibited the production or expression of IL‐6, IL‐1β, MCP‐1, TNF‐α, VCAM‐1, ICAM‐1 and E‐selectin as well as ROS in LPS‐stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti‐inflammatory and anti‐oxidative effects of TDD. Furthermore, TDD inhibited LPS‐induced NF‐κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS‐induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox‐sensitive NF‐κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.

The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4+ T cells are not well-defined. Here we identify a subset of memory CD4+ T cells capable of effluxing cellular toxins, including rhodamine (Rho), through the multidrug efflux protein MDR1 (also known as P-glycoprotein and ABCB1). Drug-effluxing CD4+ T cells were characterized as CD161+CD95+CD45RA-CD127hiCD28+CD25int cells with a distinct chemokine profile and a Th1-polarized pro-inflammatory phenotype. CD4+CD161+Rho-effluxing T cells proliferated vigorously in response to stimulation with anti-CD3/CD28 beads and gave rise to CD161- progeny in vitro. These cells were also capable of self-renewal and maintained their phenotypic and functional characteristics when cultured with homeostatic cytokines. Multidrug-effluxing CD4+CD161+ T cells were enriched within the viral-specific Th1 repertoire of healthy donors and patients with acute myeloid leukemia (AML) and survived exposure to daunorubicin chemotherapy in vitro. Multidrug-effluxing CD4+CD161+ T cells also resisted chemotherapy-induced cytotoxicity in vivo and underwent significant expansion in AML patients rendered lymphopenic after chemotherapy, contributing to the repopulation of anti-CMV immunity. Finally, after influenza vaccination, the proportion of influenza-specific CD4+ T cells coexpressing CD161 was significantly higher after 2 years compared with 4 weeks after immunization, suggesting CD161 is a marker for long-lived antigen-specific memory T cells. These findings suggest that CD4+CD161+ T cells with rapid efflux capacity contribute to the maintenance of viral-specific memory T cells. These data provide novel insights into mechanisms that preserve antiviral immunity in patients undergoing chemotherapy and have implications for the development of novel immunotherapeutic approaches.

Regulatory T cells (Tregs) play a fundamental role in the maintenance of self-tolerance and immune homeostasis. Defects in Treg function and/or frequencies have been reported in multiple disease models. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting upper and lower motor neurons. Compelling evidence supports a neuroprotective role for Tregs in this disease. Indeed, rapid progression in ALS patients is associated with decreased FoxP3 expression and Treg frequencies. Thus, we propose that strategies to restore Treg number and function may slow disease progression in ALS. In this study, we developed a robust, Good Manufacturing Practice (GMP)-compliant procedure to enrich and expand Tregs from ALS patients. Tregs isolated from these patients were phenotypically similar to those from healthy individuals but were impaired in their ability to suppress T-cell effector function. In vitro expansion of Tregs for 4 weeks in the presence of GMP-grade anti-CD3/CD28 beads, interleukin (IL)-2 and rapamcyin resulted in a 25- to 200-fold increase in their number and restored their immunoregulatory activity. Collectively, our data facilitate and support the implementation of clinical trials of adoptive therapy with ex vivo expanded and highly suppressive Tregs in patients with ALS.

Cord blood (CB) offers a number of advantages over other sources of hematopoietic stem cells, including a lower rate of chronic graft-versus-host disease (cGVHD) in the presence of increased HLA disparity. Recent research in experimental models of autoimmunity and in patients with autoimmune or alloimmune disorders has identified a functional group of interleukin-10 (IL-10)-producing regulatory B cells (Bregs) that negatively regulate T-cell immune responses. At present, however, there is no consensus on the phenotypic signature of Bregs, and their prevalence and functional characteristics in CB remain unclear. Here, we demonstrate that CB contains an abundance of B cells with immunoregulatory function. Bregs were identified in both the naive and transitional B-cell compartments and suppressed T-cell proliferation and effector function through IL-10 production as well as cell-to-cell contact involving CTLA-4. We further show that the suppressive capacity of CB-derived Bregs can be potentiated through CD40L signaling, suggesting that inflammatory environments may induce their function. Finally, there was robust recovery of IL-10-producing Bregs in patients after CB transplantation, to higher frequencies and absolute numbers than seen in the peripheral blood of healthy donors or in patients before transplant. The reconstituting Bregs showed strong in vitro suppressive activity against allogeneic CD4(+) T cells, but were deficient in patients with cGVHD. Together, these findings identify a rich source of Bregs and suggest a protective role for CB-derived Bregs against cGVHD development in CB recipients. This advance could propel the development of Breg-based strategies to prevent or ameliorate this posttransplant complication.

The ability of cord blood transplantation (CBT) to prevent relapse depends partly on donor natural killer (NK) cell alloreactivity. NK effector function depends on specific killer-cell immunoglobulin-like receptors (KIR) and HLA interactions. Thus, it is important to identify optimal combinations of KIR-HLA genotypes in donors and recipients that could improve CBT outcome. We studied clinical data, KIR and HLA genotypes, and NK-cell reconstitution in CBT patients (n = 110). Results were validated in an independent cohort (n = 94). HLA-KIR genotyping of recipient germline and transplanted cord blood (CB) grafts predicted for large differences in outcome. Patients homozygous for HLA-C2 group alleles had higher 1-year relapse rate and worse survival after CBT than did HLA-C1/C1 or HLA-C1/C2 (HLA-C1/x) patients: 67.8% vs 26.0% and 15.0% vs 52.9%, respectively. This inferior outcome was associated with delayed posttransplant recovery of NK cells expressing the HLA-C2-specific KIR2DL1/S1 receptors. HLA-C1/x patients receiving a CB graft with the combined HLA-C1-KIR2DL2/L3/S2 genotype had lower 1-year relapse rate (6.7% vs 40.1%) and superior survival (74.2% vs 41.3%) compared with recipients of grafts lacking KIR2DS2 or HLA-C1. HLA-C2/C2 patients had lower relapse rate (44.7% vs 93.4%) and better survival (30.1% vs 0%) if they received a graft with the combined HLA-C2-KIR2DL1/S1 genotype. Relapsed/refractory disease at CBT, recipient HLA-C2/C2 genotype, and donor HLA-KIR genotype were independent predictors of outcome. Thus, we propose the inclusion of KIR genotyping in graft selection criteria for CBT. HLA-C1/x patients should receive an HLA-C1-KIR2DL2/L3/S2 CB graft, while HLA-C2/C2 patients may benefit from an HLA-C2-KIR2DL1/S1 graft.

Immune checkpoint-based therapies, which target the regulatory pathways of immunocompetent cells to enhance anti-tumor responses, have been at the heart of many recent clinical advances and have led to long-term remissions and possible cures. Most of this success was achieved with T-cells, but there are compelling reasons to predict that checkpoint ablation could modify NK cells in ways that would facilitate their antitumor activity. The suppressor of cytokine signaling (SOCS) family of proteins plays an important role in NK cell biology by attenuating cytokine signaling and effector function against cancer. One of its members, cytokine inducible SH2 containing protein (CIS), encoded by the CISH gene, is as an important checkpoint molecule in NK cells and is upregulated in response to IL-15. We hypothesized that CIS may act as a potent checkpoint in our iC9/CAR19/IL15 NK cells given the fact that they continuously produce IL-15, and that targeting this pathway would enhance their potency against B cell malignancies. In a series of in vitro studies, we showed that CISH is induced in iC9/CAR19/IL15 NK cells in a time dependent manner. To examine the functional consequences of CISH deletion in our CAR-NK cells, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing to silence CISH and retroviral transduction with the iC9/CAR19/IL15 construct. On day 7 we nucleofected the CAR transduced NK cells with Cas9 alone (Cas9 control) or Cas9 pre-loaded with crRNA:tracrRNA duplex targeting CISH exon 4. Gene editing efficiency was >90% as quantified by PCR and western blot. CISH knockout induced a phenotype characterized by the increased expression of markers of activation and cytotoxicity. These included granzyme-b, perforin, TRAIL and CD3z; transcription factors such as eomesodermin and T-bet; adaptor molecules such as DAP12; and activating coreceptors/proliferation markers such as DNAM, CD25 and Ki67. CISH knockout resulted in significantly enhanced function of iC9/CAR19/IL15 NK cells against Raji lymphoma evident by increased cytokine production (TNFa p=0.007, IFNg p=0.033) and degranulation (CD107a p=0.003) compared to Cas9 control cells. Moreover, CISH KO iC9/CAR.19-IL15 NK cells killed Raji lymphoma more efficiently than Cas9 control cells and formed a stronger immunologic synapse (p=0.037). RNA sequencing with gene set enrichment analysis (GSEA) confirmed enrichment of JAK/STAT signaling, TNFα and IFN-γ inflammatory response, mTORC1, and MYC hallmark pathways in CISH KO iC9/CAR19/IL15 NK cells compared to Cas9 control counterparts, providing a molecular mechanism for their enhanced effector function. Moreover, in an in-vivo NSG mouse model of Raji lymphoma, the antitumor activity of a single dose of CISH KO iC9/CAR19/IL15 transduced CB NK cells was significantly better than that of Cas9 control cells leading to a significant survival advantage (p=0.003) without evidence of increased toxicity. Thus, we demonstrate for the first time, that silencing a critical checkpoint in CAR-NK cells improves their potency, permitting greater cytotoxic effector function than seen with unmodified CAR-NK cells. Our data support the merging of CAR-engineering and immune checkpoint gene editing to enhance the therapeutic potential of NK cells. We are in the process of scaling up this approach in our GMP facility for translation to the clinic for the treatment of relapsed/refractory B cell hematologic malignancies. Disclosures Konopleva: Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding. Champlin:Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.

Introduction: A number of Clinical trials have demonstrated the feasibility, safety and efficacy of cell and gene therapy for cancer, autoimmune disorders and infectious disease. Strategies that enhance the function and survival of immune cells are critical for the success of immunotherapy. We have developed a strategy for the ex vivo expansion of off-the-shelf viral-specific T cells (VSRs) from healthy donor buffy coat which have been extremely effective in eradicating refractory cytomegalovirus (CMV), polyomavirus and adenovirus infections in immunocompromised patients. Glucocorticoids commonly used to treat graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are a common cause of iatrogenically-induced immunosuppression and contribute the risk of life-threatening viral-infections. To render VSTs resistant to the lymphocytotoxic effect of glucocorticoids, we have developed a novel strategy to silence the expression of the glucocorticoid receptor using RNA-guided endonucleases CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) 9 gene editing.. Methods: The technique involves the expansion from donor blood of CMV, BKV or adenoviral-specific T cells using peptide libraries from the immunodominant viral proteins followed by CRISPR knockout of exon 2 of the GR gene on chromosome 5 of the human NR3C1 gene. Cells are electroporated with the RNP (Cas9 plus guide RNA) complex (IDT pre-designed alt-R crispr Cas9 platform) using Neon electroporation and the Amaxa 4-D nucleofector system. Results: GR knockout efficiency in ex vivo expanded virus-specific T cells was consistently > 90%. In vitro experiments confirmed the resistance of VSTs to corticosteroid treatment as assessed by annexin V assay. GR KO VSTs maintained potent antiviral activity as assessed by their ability to proliferate and release effector cytokines in response to viral antigens. Conclusions: CRISPR gene-editing to knock-out the glucocorticoid receptor gene in viral-specific T cells can preserve the activity of VSTs in the presence of corticosteroid-induced immunosuppression. Engineering runs using GMP-compliant Cas9 protein and gRNA are underway in anticipation of a clinical trial. Disclosures Champlin: Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.

NK cells are potent cellular immunotherapeutic agents against a wide array of human malignancies. Ex vivo expansion of NK cells to achieve clinically relevant numbers must overcome poor in vitro growth kinetics, low starting percentages within the mononuclear cell fraction (especially from autologous donors with active disease), and limited in vivo life span. We targeted three universally critical NK signaling pathways, namely IL-21, 4-1BB, and SLAM family member 4 (SLAMF4), to increase NK cell proliferation and enhance survival. We genetically-engineered HLA-A-ve and -B-ve K562 cells to enforce expression of membrane-bound IL-21 (mbIL21), 4-1BB-L, and CD48, forming a universal antigen presenting cell (UAPC) to generate highly potent clinical-grade umbilical cord blood (CB-NK) or peripheral blood NK cells (PB-NK). While the mbIL21 and 4-1BB signaling nexuses have been utilized previously, we highlight here SLAMF-mediated immunological sculpting of NK cells for clinical applications. SLAMF triggering of co-receptors modulate NK cell activation, in particular through high-avidity interactions between SLAMF4 (2B4/CD244) and its heterophilic, robust affinity, and physiological ligand CD48, a glycosyl-phosphatidyl-inositol (GPI)-anchored cell surface protein. Upon ligand binding and receptor phosphorylation, SLAMF4 recruits PTPN11/SHP-2 and SH2D1A/SAP for downstream signaling, including significant increases in NK cell-mediated cytotoxicity, granule exocytosis, and production of IFN-γ and IL-2. Other than a subset of low surface density expressers from aging subjects consistently associated with inefficient and impaired activating signal transduction, the majority of NK cells express SLAMF4. The functional prominence of SLAMF4, presently recognized as an activating co-receptor, is also evidenced by loss-of-function mutations associated with X-linked lymphoproliferative (XLP) disease. We achieved log-scale expansion of NK cells with UAPC (>1000 fold in 2 weeks), with excellent purity (>99% CD56+ve/CD3−ve and < 1% CD3+ve cells), without indications of senescence/exhaustion, even after 4 weeks of culture. Surface molecular phenotypes of UAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded without SLAMF4. Synergistic signals from IL-21/STAT3, 4-1BB/4-1BBL, and SLAMF4 drive tonic NK propagation, supporting their clinical application. Our novel and clinically accessible platform technology for generation of high purity CB-NKs, a promising source of fresh and cryopreserved allogeneic NK cells, is well-suited for adoptive cancer immunotherapy. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

Mesenchymal stromal cells (MSCs) have been infused in hundreds of patients to date, with minimal reported side effects. However, follow-up is limited and long-term side effects are unknown. Because several animal models have raised safety concerns, we sought to develop a system allowing control over the growth and survival of MSCs used therapeutically. We have previously described a suicide system based on an inducible caspase-9 (iCasp9) protein that is activated using a specific chemical inducer of dimerization (CID), analogs of which have been safely tested in a phase I study. Here, we show that MSCs can be easily transduced with this system and selected to high purity (greater than 97%) with clinical grade immunomagnetic procedures. The transduced cells maintain their basic physiology, including expression of surface antigens (such as positivity for CD73, CD90, and CD105, and negativity for hematopoietic markers) and their potential to differentiate into diverse connective tissue lineages (adipocytes, osteoblasts, and chondroblasts). Those cells and their differentiated progeny can be selectively eliminated in vitro or in vivo within 24 hours after exposure to pharmacological levels of CID, with evidence of apoptosis in more than 95% of iCasp9-positive cells. In conclusion, we have developed directed MSC killing to provide a necessary safety mechanism for therapies using progenitor cells. We believe that this approach will become of increasing value as clinical applications for MSCs develop further.

Objectives A G>T transversion in a tyrosine kinase JAK2 ( V617F ) was reported in over 80% of patients with polycythemia vera (PV). Current evidence suggests that JAK2 V617F somatic mutation is involved in the pathogenesis of PV, as it confers erythropoietin-independent proliferation to erythroid progenitor cells. However, several unanswered questions regarding the essential role of JAK2 V617F arose as 1) it is not a dominant mutation, 2) it is not PV-specific as it is found in several myeloproliferative disorders, and 3) some (∼20%) PV patients lack the JAK2 V617F mutation. We investigated the relative frequency of JAK2 V617F in in vitro–expanded PV progenitors. Methods In vitro expansion of erythroid progenitors from mononuclear cells was optimized. Frequency of JAK2 V617F allele was measured by using allele-specific real-time polymerase chain reaction. Clonality was performed using established procedure. Results In vitro expansion of PV erythroid progenitors and differentiated dendritic cells resulted in a decrease of the frequency of JAK2 V617F allele compared with granulocytes or CD235 + erythroid progenitors. Clonality analysis demonstrated that although granulocytes of these PV patients were clonal, expanded erythroid cells were polyclonal. However, in vitro–expanded PV erythroid progenitors still had approximately a twofold increased proliferative capacity in comparison with erythroid progenitors from healthy individuals. Erythropoietin favors the cells without JAK2 V617F allele. Dendritic cells in one out of three patients remained clonal. Conclusion JAK2 V617F mutation does not provide a proliferative/survival advantage to the PV clone during in vitro expansion. These data suggest that the JAK2 V617F mutation plays an important role in the biology of PV, yet it may not be the PV-initiating event.

Self-tumor Ags that elicit antitumor immune responses in responses to IFN-α stimulation remain poorly defined. We screened a human testis cDNA library with sera from three polycythemia vera patients who responded to IFN-α and identified a novel Ag, MPD6. MPD6 belongs to the group of cryptic Ags without conventional genomic structure and is encoded by a cryptic open reading frame located in the 3′-untranslated region of myotrophin mRNA. MPD6 elicits IgG Ab responses in a subset of polycythemia vera patients, as well as patients with chronic myelogenous leukemia and prostate cancer, suggesting that it is broadly immunogenic. The expression of myotrophin-MPD6 transcripts was up-regulated in some tumor cells, but only slightly increased in K562 cells in response to IFN-α treatment. By using bicistronic reporter constructs, we showed that the translation of MPD6 was mediated by a novel internal ribosome entry site (IRES) upstream of the MPD6 reading frame. Furthermore, the MPD6-IRES-mediated translation, but not myotrophin-MPD6 transcription, was significantly up-regulated in response to IFN-α stimulation. These findings demonstrate that a novel IRES-mediated mechanism may be responsible for the translation of unconventional self-Ag MPD6 in responsive to IFN-α stimulation. The eliciting antitumor immune response against unconventional Ag MPD6 in patients with myeloproliferative diseases suggests MPD6 as a potential target of novel immunotherapy.

Idiopathic myelofibrosis (IM) is characterized by the constitutive mobilization of CD34+ cells. IM peripheral blood (PB) CD34+ cells had a reduced cloning efficiency and a lower frequency of cobblestone areas compared with normal granulocyte colony-stimulating factor (G-CSF)-mobilized PB CD34+ cells. IM CD34+ cells engrafted nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, demonstrating that they contain bone marrow (BM)-repopulating cells. G-CSF-mobilized CD34+ cells produced multiple hematopoietic lineages within the NOD/SCID mice with a predominance of CD19+ cells. By contrast, IM CD34+ cells produced predominantly CD33+ cells, increased numbers of CD41+ cells, but fewer CD19+ cells. Transcriptional clonality assays of the engrafted human IM cells demonstrated their clonal origin. CD34+ cells from one patient isolated prior to leukemic transformation were capable of generating acute leukemia in NOD/SCID mice. The engrafted human cells exhibited the same abnormal karyotype as primary cells in a portion of the population. These findings demonstrate that BM-repopulating cells and more differentiated progenitor cells are constitutively mobilized into the PB in IM, and that their differentiation program is abnormal. In addition, the NOD/SCID model may be useful in gaining an understanding of the events occurring during the transition of IM to acute leukemia. (Blood. 2005;105:1699-1705)

The first congenital defect of hypoxia-sensing homozygosity for VHL 598C>T mutation was recently identified in Chuvash polycythemia. Subsequently, we found this mutation in 11 unrelated individuals of diverse ethnic backgrounds. To address the question of whether the VHL 598C>T substitution occurred in a single founder or resulted from recurrent mutational events in human evolution, we performed haplotype analysis of 8 polymorphic markers covering 340 kb spanning the VHL gene on 101 subjects bearing the VHL 598C>T mutation, including 72 homozygotes (61 Chuvash and 11 non-Chuvash) and 29 heterozygotes (11 Chuvash and 18 non-Chuvash), and 447 healthy unrelated individuals from Chuvash and other ethnic groups. The differences in allele frequencies for each of the 8 markers between 447 healthy controls (598C) and 101 subjects bearing the 598T allele (P < 10–7) showed strong linkage disequilibrium. Haplotype analysis indicated a founder effect. We conclude that the VHL 598C>T mutation, the most common defect of congenital polycythemia yet found, was spread from a single founder 14 000 to 62 000 years ago.

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD, IDS, and MPP1 genes, which together were informative in about 65% of female subjects. To increase our ability to detect clonality, we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these, all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis, whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly, interferon α therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET, and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus, these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels.

The congenital polycythemic disorders with elevated erythropoietin (Epo) have been until recently an enigma, and abnormality in the hypoxia-sensing pathway has been hypothesized as a possible mechanism. The tumor suppressor von Hippel-Lindau (VHL) participates in the hypoxia-sensing pathway, as it binds to the proline-hydroxylated form of the hypoxia-inducible factor 1α (HIF-1α) and mediates its ubiquitination and proteosomal degradation. The loss of VHL function may result in the accumulation of HIF-1α and overproduction of HIF-1 downstream target genes including Epo. VHL syndrome is an autosomal dominant disorder predisposing to the development of tumors, due to inherited mutations in the VHL gene. Some rare patients with VHL syndrome have polycythemia, which has been attributed to Epo production by a tumor. It was recently found that homozygosity for theVHL Arg200Trp mutation is the cause of Chuvash polycythemia, an autosomal recessive polycythemic disorder characterized by elevated serum Epo and hypersensitivity of erythroid cells to Epo. We evaluated the role of VHL in 8 children with a history of polycythemia and an elevated serum Epo level and found 3 different germline VHL mutations in 4 of them. One child was homozygous for the Arg200Trp VHL mutation, and another compound heterozygous for the Arg200Trp and the Val130Leu mutations. Two children (siblings) were heterozygous for an Asp126Tyr mutation, one of them fulfilling some criteria of VHL syndrome. We propose that mutations of the VHL gene represent an important cause of pediatric sporadic polycythemias with an inappropriately high serum Epo concentration.

A subset of regulatory B cells (Bregs) in mice negatively regulate T-cell immune responses through the secretion of regulatory cytokines such as IL-10 and direct cell-cell contact and have been linked to experimental models of autoimmunity, inflammation, and cancer. However, the regulatory function of Bregs in human disease is much less clear. Here we demonstrate that B cells with immunoregulatory properties are enriched within both the CD19(+)IgM(+)CD27(+) memory and CD19(+)CD24(hi)CD38(hi) transitional B-cell subsets in healthy human donors. Both subsets suppressed the proliferation and interferon-γ production of CD3/CD28-stimulated autologous CD4(+) T cells in a dose-dependent manner, and both relied on IL-10 secretion as well as cell-cell contact, likely mediated through CD80 and CD86, to support their full suppressive function. Moreover, after allogeneic stem cell transplantation, Bregs from patients with chronic graft-versus-host disease (cGVHD) were less frequent and less likely to produce IL-10 than were Bregs from healthy donors and patients without cGVHD. These findings suggest that Bregs may be involved in the pathogenesis of cGVHD and support future investigation of regulatory B cell-based therapy in the treatment of this disease.

Adoptive transfer of T lymphocytes expressing a CD19-specific chimeric antigen receptor (CAR.CD19) induces complete tumor regression in patients with lymphoid malignancies. Although in vivo persistence of CAR-T cells correlates with clinical responses, it remains unknown whether specific cell subsets within the CAR-T-cell product correlate with their subsequent in vivo expansion and persistence. We analyzed 14 patients with B-cell malignancies infused with autologous CAR.CD19-redirected T cells expanded ex vivo using IL-2, and found that their in vivo expansion only correlated with the frequency within the infused product of a CD8(+)CD45RA(+)CCR7(+) subset, whose phenotype is closest to "T-memory stem cells." Preclinical models showed that increasing the frequency of CD8(+)CD45RA(+)CCR7(+) CAR-T cells in the infused line by culturing the cells with IL-7 and IL-15 produced greater antitumor activity of CAR-T cells mediated by increased resistance to cell death, following repetitive encounters with the antigen, while preserving their migration to secondary lymphoid organs. This trial was registered at www.clinicaltrials.gov as #NCT00586391 and #NCT00709033.

Patients with CLL experience generalized immune suppression, susceptibility to infections and secondary malignancies that likely involve complex bi-directional interactions between leukemic cells, components of the tumor microenvironment and immune effectors. CLL cells are capable of secreting IL-10 and exhibit regulatory functions comparable to that of normal B10 cells, a regulatory B cell subset that suppresses effector T-cell function through STAT3-mediated production of IL-10. However, the underlying mechanisms governing IL-10 production by CLL cells are not fully understood. The chemokine CXCL12 is constitutively secreted by bone marrow stroma in CLL, and binds CXCR4 to direct chemotaxis, support tumor survival and activate various signaling pathways, including STAT3. Thus, we investigated if CXCL12 can enhance IL-10 production by activating the STAT3 pathway in CLL. Using peripheral blood mononuclear cells (PBMC) from 24 CLL patients who had not received therapy for ≥2 years, we showed that CXCL12 can enhance IL-10 production by CLL cells by activating S727-STAT3. This effect was CXCR4-mediated since blocking the CXCR4-CXCL12 interaction with a blocking antibody abolished CXCL12-induced IL-10 production. Addition of the STAT3 inhibitor curcubitacin to the culture also abrogated CXCL12-induced IL-10 production, confirming an important role for S727-STAT3 as a mediator of CXCL12-CXCR4-induced IL-10 production by CLL cells. We next determined if activation of the CXCR4-CXCL12-STAT3-IL10 pathway in CLL is important in mediating their immunoregulatory function. Culture of primary CLL withCXCL12 induced significantly more suppression of CD3+ T cell function, including TNF-α, IFN-γ and IL-2 production, and CD107a degranulation, compared to CD3+ T cells cultured with untreated CLL cells or with CXCL12 alone. The addition of IL-10 blocking antibody to the co-culture completely reversed T cell dysfunction, supporting an important for IL-10 in CLL-mediated T-cell suppression. IL-10 has been reported to induce T cell suppression through phosphorylation of Y705-STAT3.. Blocking IL-10 also prevented CLL-induced phosphorylation of Y705-STAT3 in T cells, confirming an important role for CLL-derived IL-10 in activation of Y705-STAT3 and induction of T cell dysfunction. Lenalidomide is an immune-modulatory drug with clinical efficacy in CLL and was recently reported to inhibit STAT3 phosphorylation. To investigate if lenalidomide can inhibit CXCL12-induced STAT3 phosphorylation, we treated CLL cells with lenalidomide and measured p-S727-STAT3 levels. Exposure of CLL cells to 10µM/ml lenalidomide prevented CXCL12-induced increase in p-S727-STAT3 and resulted in significant reduction in the IL-10 response by CLL cells. Lenalidomide also suppressed IL-10-induced Y705-STAT3 phosphorylation in healthy T cells, thus reversing CLL-induced T cell dysfunction. We next confirmed the in vivo relevance of our findings using PBMC cryopreserved from patients treated with lenalidomide monotherapy (NCT00535873). When compared to pretreatment samples, CLL cells from on-treatment samples produced less IL-10 and showed significantly improved T cell function. We thus conclude that the capacity of CLL to produce IL-10 is mediated by the CXCL12-CXCR4-STAT3 pathway and may contribute to immunodeficiency in patients. Lenalidomide can reverse CLL-induced immunosuppression through multiple mechanisms that involve abrogation of the CXCL12-CXCR4-S727STAT3-mediated IL-10 response by CLL B cells and prevention of IL-10-induced phosphorylation of Y705-STAT3 in T cells. Disclosures Estrov: incyte: Consultancy, Research Funding. Wierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Rezvani:Pharmacyclics: Research Funding.

INTRODUCTION: The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is a covalent inhibitor of BTK, a member of the B-cell receptor (BCR) signaling pathway and induces objective clinical responses in the majority of CLL patients (Byrd et al., NEJM 2013). Interestingly this drug also inhibits L2-inducible T cell kinase (ITK), an essential enzyme for the development and effector function of Th2 and Th17 cells, and has been shown to shift the balance towards a Th1 response. The purpose of the study was to determine how ibrutinib influences the Th1/Th17/T regulatory cell response, expression of immune checkpoint blockade molecules on T cells and the functional pathogen-specific T cell recovery. METHODS: Here we present data from a clinical trial of ibrutinib versus ibrutinib + rituximab in previously treated patients (NCT02007044). Peripheral blood and serum were collected at baseline, 3 months and 6 months during therapy. Multicolor flow cytometry was used to characterize B cell subsets, T-cell subsets, expression of PD-1, PD-L1 and CTLA-4 and T-cell effector function. For statistical analysis of pre-treatment to on-treatment measurements the paired Student t-test was used. RESULTS: Here we report on the phenotypic and functional recovery of immune subsets in 41 CLL patients treated with ibrutinib (n=17) or ibrutinib + rituximab (n=25). Both PD-1 and PD-L1 were expressed at high levels on CLL cells. Interestingly, by 3 and 6 months, there was a significant decrease in PD-1 expression from a pre-treatment median of 15% to 4% (at 3 months) and 3% (at 6 months; P CONCLUSION: Based on these data we propose that ibrutinib therapy can modulate the T cell response through multiple mechanisms which include (i) direct inhibition of ITK and skewing of the T cell response toward a Th1 profile; (ii) reduction in PD1/PDL1 expression on B and T cells and (iii). suppression of Tregs. It is unclear how ibrutinib influences the PD1/PDL1 interaction and whether this is a direct effect of the drug on essential components of B and T cell-receptor signaling or whether it is an indirect effect related to a reduction in the leukemia burden. Mechanistic studies to understand how ibrutinib modulates the PD1/PL1 axis are currently underway. Figure 1. Ibrutinib therapy is associated with a reduction in PDL1 expression on the surface of CD19+ B cells. Figure 1. Ibrutinib therapy is associated with a reduction in PDL1 expression on the surface of CD19+ B cells. Figure 2. Ibrutinib therapy is associated with a reduction in PD1 expression on the surface of CD3+ T cells. Figure 2. Ibrutinib therapy is associated with a reduction in PD1 expression on the surface of CD3+ T cells. Disclosures Burger: Pharmacyclics LLC, an AbbVie Company: Research Funding. Wierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Rezvani:Pharmacyclics: Research Funding.

Adoptive cellular immunotherapy for Hodgkin lymphoma (HL) associated with EBV infection has had considerable success, inducing >50% complete and sustained remission rates in patients with relapsed/resistant disease. However, only 40% of HL patients express EBV-associated antigens. By contrast, almost all HL and some non-Hodgkin lymphomas (NHL), such as anaplastic large cell lymphoma (ALCL), express the CD30 antigen both at diagnosis and relapse, and monoclonal antibodies targeting CD30 produce objective antitumor responses. Monoclonal antibodies (mAb), however, have limited bio-distribution and their effects may be limited in duration. We therefore expressed the antigen binding domain of a CD30 mAb as part of a chimeric antigen receptor (CAR) on T cells, coupled to the CD28 and ζ chain endodomains, in an effort to ensure prolonged persistence, active penetration of tumors and activation of multiple lytic components of the immune system. We report here an initial analysis of our phase I dose escalation study of activated autologous CD30.CAR-T cells (CD30.CARTs) infused in patients with relapsed/refractory EBV-negative CD30+ HL or NHL. We manufactured CD30.CARTs for 18 patients using retroviral transduction. Starting from a median of 2.4×107 PBMCs (range 3.6×106 to 4.9×107), we obtained 9.0×108 CD30.CARTs (range 2.8×108 to 2.9×109) in 15±3 days of culture, with a transduction efficiency of 89%±1%. The cell products comprised >99% T cells and phenotypic analysis showed 58%±29% CD8+ T cells, with a majority of them being effector T cells (CD45RO+ 94%±7%). 51 Cr-release cytotoxicity assays confirmed that patients' CD30.CARTs lysed a CD30+ tumor line, HDLM-2 (60%±13% killing at a 20:1 effector:target ratio), with negligible effects on CD30− target cells ( Nine patients (7 with HL and 2 ALCL) have received CD30.CARTs. Eight of these had relapsed or progressed after treatment with the CD30 mAb brentuximab. Two patients were treated on dose level (DL) 1 (2×107 CD30.CAR+ T cells/m2), 2 patients on DL2 (1×108) and 5 patients on DL3 (2×108). None of the patients received any conditioning regimen before CART infusion. CART infusions produced no attributable adverse events; in particular, no patient had evidence of cytokine release syndrome. As CD30 can be transiently expressed by activated T cells, for example during infection with viruses, we monitored antiviral immunity in CART recipients. The frequency of T cells responding to CMV, adenovirus, influenza virus and EBV (assessed using stimulation with viral peptides in IFNγ ELIspot assays) remained unchanged by treatment. The molecular signal from CARTs, assessed by Q-PCR in peripheral blood, peaked at 1 week following infusion, but decreased to near background by 4 weeks post infusion. The signal level was dose dependent, with an mean of 7020 copies/μg DNA in patients treated on DL3, in whom CAR-T cells were detectable by flow cytometry in the peripheral blood (~5% of PBMCs), versus 60 copies/μg for DL1. At 6 weeks after treatment, 1 patient had a CR, 1 patient had a very good PR, and 4 patients had stable disease (persisting for 1½ to 8 months), while 3 patients had disease progression. Having completed the dose escalation and found that DL3 is safe and associated with significant in vivo expansion, we will now explore the use of these cells after autologous stem cell transplantation in patients at high risk of relapse. Disclosures Off Label Use: Adoptively transferred T cells administered under an IND. Rooney:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement. Brenner:Bluebird Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cell Medica: Other: Licensing Agreement; Celgene: Other: Collaborative Research Agreement. Heslop:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement.

Background: Multiple myeloma (MM) is an incurable disease thought to be characterized by immune dysregulation and exhaustion, whereby proliferation of malignant plasma cells is not checked by the native immune system. Long term remissions in some patients after allogeneic stem cell transplant (SCT) suggest a graft versus myeloma effect; however the treatment-related toxicity limits the widespread use of this modality. Allogeneic natural killer (NK) cells are active in various hematologic malignancies and may have a role against MM, without concomitant graft versus host disease (GVHD). Umbilical cord blood is a potential source for allogeneic NK cells and ex vivo expanded umbilical cord blood-derived NK (CB-NK) cells demonstrate activity comparable to that of peripheral blood-derived NK cells. We describe here the results of a phase I, first-in-human study of ex vivo expanded allogeneic CB-NK cells in conjunction with high dose chemotherapy and autologous SCT. Methods: Patients with symptomatic MM who were appropriate candidates for high dose chemotherapy and autologous SCT were eligible. CB units with at least 4/6 match at HLA-A, B and DR were chosen for each patient. When possible, CB units with potential NK alloreactivity were prioritized. On day (-19) CB units were thawed and mononuclear cells (MNCs) were isolated by ficoll density gradient centrifugation. MNCs were cultured in a gas permeable bioreactor with irradiated (100 Gy) K562-based aAPCs expressing membrane bound IL-21 "Clone 9.mbIL21" (2:1 feeder cell:MNC ratio) and IL-2 (100 IU/mL). On day 7, cells were CD3-depleted via immunomagnetic depletion and remaining cells were re-stimulated with aAPC feeder cells and cultured for an additional 7 days. NK cell purity was determined after 14 days of culture. Due to pre-clinical data demonstrating synergy between lenalidomide and NK cells, patients received lenalidomide (10 mg orally daily) from days (-8) to day (-2). Melphalan 200 mg/m2 was given intravenously on day (-7). Freshly expanded CB-NK cells were infused on day (-5). Autologous peripheral blood progenitor cells (PBPC) were infused on day (0). Results: 12 patients have been enrolled thus far with 3 patients each on the following CB-NK cell dose levels: 5 e6 NK cells/kg, 1 e7 NK cells/kg, 5 e7 NK cells/kg and 1 e8 NK cells/kg. 11/12 patients had at least 1 high-risk feature of progressed/relapsed disease (n=7), high-risk cytogenetics (n=2) or International Staging System III disease (n=3). Successful NK expansion to target dose was achieved in all patients with median NK purity (CD56+/CD16+/CD3-) of 98.9% (96.8-99.7). Expanded cells demonstrated cytotoxicity against classic K562 and MM cell line targets. There were no infusional toxicities and no occurrence of GVHD. One patient (1 e8 NK cells/kg) failed to engraft due to a poor PBPC graft quality; this patient was rescued with a back-up autologous PBPC graft. There have been no other significant adverse events and no second primary malignancies. 11/12 patients are evaluable beyond day 100. Best response has been 8/12 nCR or better, 2/12 VGPR and 1/12 PR. 4/12 patients have progressed at a median of 330 days. By DNA microsatellite chimerism analysis, donor CB-NK cells were detected in 2 patients at the 1 e7 NK cells/kg dose and all 3 patients in the 1 e8 NK cells/kg dose, for at least 5 days after infusion. By a more sensitive flow cytometric chimerism assay using HLA class I-specific antibodies for donor or recipient, donor CB-NK cells were detected in 3 evaluable patients at doses of 1 e7 NK cells/kg, 5 e7 NK cells/kg and 1 e8 NK cells/kg for at least 12 days after infusion. Further analysis of these cells indicated persistence of an activated phenotype (NKG2D+/NKp30+) in vivo. Conclusion: CB-NK cells can be activated and expanded to clinical scale. This is the first clinical study of CB-NK cells for MM. When infused in the setting of myeloablative chemotherapy, up to 1 e8 allogeneic CB-NK cells/kg are well tolerated with no infusional toxicities or GVHD. These cells can persist for at least 12 days in vivo and demonstrate an active phenotype. Though clinical data are early, responses are encouraging in this high-risk patient population. Further updated data will be presented at the annual meeting. Disclosures Off Label Use: Lenalidomide with high dose chemotherapy and autologous stem cell transplantation. Kaur:UT MD Anderson Cancer Center: Employment. Orlowski:Millennium Pharmaceuticals: Consultancy, Research Funding; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Genentech: Consultancy; Onyx Pharmaceuticals: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding; Acetylon: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding; Forma Therapeutics: Consultancy. Cooper:Intrexon: Equity Ownership, Patents & Royalties, Research Funding; ZIOPHARM Oncology: Employment, Equity Ownership, Patents & Royalties, Research Funding. Lee:Cyto-Sen: Equity Ownership; Ziopharm: Equity Ownership; Intrexon: Equity Ownership.

We generated MHC-independent chimeric antigen receptors (CARs) directed to the GD2 antigen expressed by neuroblastoma tumor cells and treated patients with this disease. Two distinguishable forms of this CAR were expressed in EBV-specific cytotoxic T lymphocytes (EBV-CTLs) and activated T cells (ATCs). We have previously shown that EBV-CTLs expressing GD2-CARs (CAR-CTLs) circulated at higher levels than GD2-CAR ATCs (CAR-ATCs) early after infusion, but by 6 weeks, both subsets became low or undetectable. We now report the long-term clinical and immunologic consequences of infusions in 19 patients with high-risk neuroblastoma: 8 in remission at infusion and 11 with active disease. Three of 11 patients with active disease achieved complete remission, and persistence of either CAR-ATCs or CAR-CTLs beyond 6 weeks was associated with superior clinical outcome. We observed persistence for up to 192 weeks for CAR-ATCs and 96 weeks for CAR-CTLs, and duration of persistence was highly concordant with the percentage of CD4+ cells and central memory cells (CD45RO+CD62L+) in the infused product. In conclusion, GD2-CAR T cells can induce complete tumor responses in patients with active neuroblastoma; these CAR T cells may have extended, low-level persistence in patients, and such persistence was associated with longer survival. This study is registered at www.clinialtrials.gov as #NCT00085930.

The canonical Wnt/β-catenin signaling pathway plays an important role in thymocyte development and T cell migration, but little is known about its role in naive-to-effector differentiation in human peripheral T cells. We show that activation of Wnt/β-catenin signaling arrests human peripheral blood and cord blood T lymphocytes in the naive stage and blocks their transition into functional T effector cells. Wnt signaling was induced in polyclonally activated human T cells by treatment either with the glycogen synthase kinase 3β inhibitor TWS119 or the physiological Wnt agonist Wnt-3a, and these T cells preserved a naive CD45RA+CD62L+ phenotype compared with control-activated T cells that progressed to a CD45RO+CD62L− effector phenotype, and this occurred in a TWS119 dose-dependent manner. TWS119-induced Wnt signaling reduced T cell expansion, as a result of a block in cell division, and impaired acquisition of T cell effector function, measured by degranulation and IFN-γ production in response to T cell activation. The block in T cell division may be attributed to the reduced IL-2Rα expression in TWS119-treated T cells that lowers their capacity to use autocrine IL-2 for expansion. Collectively, our data suggest that Wnt/β-catenin signaling is a negative regulator of naive-to-effector T cell differentiation in human T lymphocytes. The arrest in T cell differentiation induced by Wnt signaling might have relevant clinical applications such as to preserve the naive T cell compartment in Ag-specific T cells generated ex vivo for adoptive T cell immunotherapy.

We studied 25 patients with myelofibrosis with myeloid metaplasia and 19 patients with secondary myelofibrosis associated with pulmonary hypertension (PH). In these 2 groups, we compared the peripheral-blood CD34 count, the clonality of granulocytes and platelets in peripheral blood, the mutational status of the JAK2 kinase gene, and the morphology of the peripheral blood and bone marrow. We found that the following were distinctive features of myelofibrosis with myeloid metaplasia but not of secondary myelofibrosis due to PH: high circulating CD34 cell count, the presence of clonal platelets and granulocytes and of peripheral-blood dacrocytes, and a JAK2 1849G>T (V617F) mutation. We conclude that these are intrinsic features of clonal progenitors present in patients with myelofibrosis due to myeloproliferative disorders and that these features are not due to the abnormal marrow architecture seen in secondary myelofibrosis.

Adoptive transfer of T cells with a CD19-specific chimeric antigen receptor (CAR) to treat B-cell malignancies shows remarkable clinical efficacy. However, long-term persistence of T cells targeting CD19, a pan-B cell marker, causes sustained depletion of normal B cells and consequent severe hypogammaglobulinemia. In order to target B-cell malignancies more selectively, we exploited the clonal restriction of mature B-cell malignancies, which express either a κ or a λ-light immunoglobulin (Ig) chain. We generated a CAR specific for κ-light chain (CAR.κ) to selectively target κ+ lymphoma/leukemia cells, while sparing the normal B cells expressing the reciprocal λ-light chain, thus minimizing the impairment of humoral immunity. After preclinical validation, we designed a phase I clinical trial in which patients with refractory/relapsed κ+ non-Hodgkin lymphoma (NHL) or chronic lymphocytic leukemia (CLL) are infused with autologous T cells expressing a CAR.κ that includes a CD28 costimulatory domain. The protocol also included patients with multiple myeloma with the aim of targeting putative myeloma initiating cells. Three dose levels (DL) are being assessed, with escalation determined by a continual reassessment method: 0.2 (DL1), 1 (DL2) and 2 (DL3) ×108 T cells/m2. Repeat infusions are allowed if there is at least stable disease after treatment. End points being evaluated include safety, persistence of CAR+T cells and antitumor activity. T cells were generated for 13 patients by activating autologous PBMC with immobilized OKT3 (n=5) or CD3/CD28 monoclonal antibodies (n=8). In 2 patients with >95% circulating leukemic cells, CD3 positive selection was performed using CliniMACS. After transduction, T cells (1.2×107±0.5×107) were expanded ex vivo for 18±4 days in the presence of interleukin (IL)-2 to reach sufficient numbers for dose escalation. CAR expression was 81%±13% by flow cytometry (74,112±23,000 transgene copy numbers/mg DNA). Products were composed predominantly of CD8+ cells (78%±10%), with a small proportion of naïve (5±4%) and memory T cells (17%±12%). CAR+ T cells specifically targeted κ+ tumors as assessed by 51Cr release assays (specific lysis 79%±10%, 20:1 E:T ratio) but not κ–tumors (11%±7%) or the NK-sensitive cell line K562 (26%±13%). Ten patients have been treated: 2 on DL1, 3 on DL2 and 5 on DL3. Any other treatments were discontinued at least 4 weeks prior to T-cell infusion. Patients with an absolute leukocyte count >500/µL received 12.5 mg/kg cyclophosphamide 4 days before T-cell infusion to induce mild lymphopenia. Infusions were well tolerated, without side effects. Persistence of infused T cells was assessed in blood by CAR.κ-specific Q-PCR assay and peaked 1 to 2 weeks post infusion, remaining detectable for 6 weeks to 9 months. Although the CAR contained a murine single-chain variable fragment (scFv), we did not detect human anti-mouse antibodies following treatment and CAR.κ+T cell expansion continued to be observed even after repeated infusions. We detected modest ( All 10 patients are currently evaluable for clinical response. Of the patients with relapsed NHL, 2/5 entered complete remission (after 2 and 3 infusions at dose level 1 and 3, respectively), 1/5 had a partial response and 2 progressed; 3/3 patients with multiple myeloma have had stable disease for 2, 8 and 11 months, associated with up to 38% reduction in their paraprotein; and 2/2 patients with CLL progressed before or shortly after the 6-week evaluation. In conclusion, our data indicate that infusion of CAR.κ+ T cells is safe at every DL and can be effective in patients with κ+ lymphoproliferative disorders. Disclosures: Savoldo: Celgene: Patents & Royalties, Research Funding. Rooney:Celgene: Patents & Royalties, Research Funding. Heslop:Celgene: Patents & Royalties, Research Funding. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

CD4+ T cells are important in the establishment of long-lived pathogen-specific immunity. However, the mechanisms by which antigen specific CD4 T resist insult by lymphocytotoxic agents and are sustained long-term is not well defined. A recent report described the existence of a subset of long-lived CD8+ memory T cells with stem-like properties (Turtle et al, 2009), including the ability to efflux cellular toxins through the ABC–superfamily multidrug efflux protein ABCB1. We hypothesized that a similar subset of T cells with drug-effluxing properties also exists within the CD4+ T cell compartment. We used multiparameter flow cytometry to measure the capacity of CD4+ T cells from donors to efflux the fluorescent substrate Rh123. We identified a subset of memory CD4+ T cells with rapid drug-effluxing ability, defined as CD161+CD95+CD45RA-CD127hiCD28+CD25int that shared remarkable phenotypic similarity to CD8+drug-effluxing memory T cells. The stem cell marker c-kit was preferentially expressed on Rh123 effluxing CD4+CD161+ T cells, whereas CD57, a marker of terminal differentiation, was exclusively expressed on non-effluxing CD4+CD161+ T cells. Rh123 effluxing CD4+CD161+ T cells also displayed differential expression of CD31, CD38, CD58, CD122 and IL-18RA. Rh123 effluxing CD4+ CD161+ T cells were undetectable in cord blood, but found in adult blood, consistent with the emergence of this subset of memory T cells as a consequence of antigen exposure during childhood and adult life. We reasoned that this subset may be enriched within the viral-specific T cell repertoire. Indeed, CMV-specific CD4+ T cells were found to share the same phonotypic markers as Rh123 effluxing CD4+CD161+ T cells. We purified CMV-specific CD4+ T cells using the interferon gamma capture assay (Miltenyi), and showed that CMV-enriched CD4+T cells preferentially and rapidly efflux Rh123. The high ABCB1-mediated drug efflux capacity of CD4+ CD161+ memory cells also facilitated their in vitro resistance to daunorubicin, which was abrogated by competitive inhibitors of ABCB1. In keeping with the in vitro data, we found a significant increase in the frequencies of CMV-specific CD4+ T cells in the peripheral blood of patients with AML after recovery from remission induction chemotherapy, suggesting that CMV-specific CD4+ T cells can preferentially survive and proliferate following chemotherapy. Since interleukin (IL)-7 and IL-15 drive the proliferation of T cells during lymphopenia to restore homeostasis, we assessed the response of CD4+CD161+ T cells to stimulation with CD3/CD28 +IL7 and IL15. Both effluxing and non-effluxing sort-purified central and effector memory CD4+CD161+ T cells proliferated and upregulated Ki67 in vitro. Whereas CD4+CD161+ T cells were able to differentiate into CD4+CD161- T cells, a subset retained CD161 expression. These data suggest that although CD4+CD161+ T cells share phenotypic similarities with terminally differentiated cells, they are able to fully proliferate, differentiate to CD161-ve cells and self-renew to preserve the pool of memory T cells CD161 is also a hallmark of Th17 cells. We examined the cytokine profile of CD4+CD161+ T cells stimulated with a pool of overlapping MHC class II CMV pp65 peptides. After 6 and 24 hrs of in vitro stimulation we failed to detect significant IL-17 production. Furthermore, by real time qPCR, the Th1 transcription factor Tbet, rather than RORC2 (a Th17 hallmark), was found to be preferentially expressed in CMV enriched CD4+CD161+ T cells, indicating that CMV-specific CD4+CD161+T cells in fact represent a unique subset of Th1 cells, distinct from Th17 cells. Our data delineate novel findings related to a distinct subset of drug-effluxing CD4+CD161+ viral-specific memory T cells. Signaling pathways leading to CD4+CD161+ABCB1+ differentiation, the role of this subset in drug resistance and the presence or absence of “stemness” which may impart this subset with extended longevity are being explored. †Muharrem Muftuoglu and Abdullah Alsuliman contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

The immunosuppressive function of IL10 producing regulatory B cells (Bregs) has been shown in several murine models of inflammation and autoimmune disease. However, there is a paucity of data regarding the existence of an equivalent regulatory B cell subset in healthy individuals and their potential role in the pathogenesis of chronic graft-versus-host disease (cGVHD) remains unknown. Here, we examined the functional regulatory properties of peripheral blood (PB)-derived human B cell subsets from healthy individuals. In addition, we carried out studies to explore their role in cGVHD, using B cells from patients following allogeneic stem cell transplantation (HSCT). We first determined whether human IL-10 producing B cells are enriched within any othe previously described human B cell subsets: CD19+IgM+CD27+ IgM memory, CD19+IgM-CD27+ switched memory, CD19+IgM+CD27- naive, and and transitional CD19+CD24hiCD38hi. Following in vitro stimulation with CD40 ligand, the majority of IL-10 producing B cells were found within the CD24hiCD38hi transitional and CD19+IgM+CD27+B cell subsets. We next assessed the regulatory properties of the PB-derived B cell subsets, by sort-purifying IgM memory (CD19+IgM+CD27+), switched memory (CD19+IgM-CD27+), naïve (CD19+IgM+CD27-) and transitional (CD19+CD24hiCD38hi) B cells from healthy controls, and cultured them 1:1 with autologous magnetic-bead purified CD4+ T cells. CD3/CD28 stimulated CD4+ T cells cultured with either CD19+IgM+CD27- naïve or CD19+IgM-CD27+ switched memory B cells proliferated to the same extent and produced equivalent amounts of IFN-γ to cultures containing CD4+ T cells alone. In contrast, culture of CD4+ T cells with IgM memory and transitional B cells significantly suppressed CD4+ T cell proliferation [median percent proliferating CD4+ T cells 52.5%; (33%-75%)] and 51% (25%-63%)], respectively when compared with CD3/CD28 stimulated CD4+ T cells (positive control) [89.5% (75%-92%], p=0.0001. The inhibitory effect of IgM memory and transitional B cells on CD4+ T cell proliferation was cell dose dependent with the highest suppression observed at a ratio of 1:1. These data suggest that human PB transitional and IgM memory B cells are endowed with regulatory function. We next examined if the in vitro suppressive effect of transitional and IgM memory B cells is mediated by regulatory T cells (Tregs). For this purpose, CD4+ T cells were depleted of CD127lo CD25hi CD4+ T cells by magnetic cell purification. B cell subsets were cultured with CD3/CD28 stimulated CD4+ CD25- T cells at a ratio of 1:1. IgM memory and transitional B cells were able to significantly suppress the proliferation and Th1 cytokine response by CD4+ CD25- T cells compared to cultures containing CD4+ CD25-T cells alone, indicating that the suppressive activity of Bregs is independent of Tregs. To further understand the underlying mechanims though which Bregs exert T-cell suppression, we used antibody blockade experiments and showed that this suppressive effect was mediated partially via the provision of IL-10, but not TGF-ß. Using transwell experiments, we further determined that the suppressive function of Bregs is also partly dependent on direct T cell/B cell contact. We next assessed whether the activity of Breg cells might be altered in patients with cGVHD. B cells from patients with cGVHD were refractory to CD40 stimulation and produced less IL-10 when compared to patients without cGVHD post-SCT and healthy controls, [1.02% (0.22-2.26) vs.1.72% (0.8-5.52) vs. 2.16 (1.3- 5.6), p=0.001]. Likewise, the absolute number of IL-10 producing B cells was significantly lower in cGvHD patients compared to patients without cGVHD and healthy controls (p=0.007), supporting both a qualitative and quantitative defect in IL-10 producing B cells in cGvHD. Our combined studies provide important new data defining the phenotype of B cell populations enriched in regulatory B cells in healthy humans and provide evidence for a defect in the activity of such cells in patients with cGVHD post-SCT. In association with previous reports showing defects in Treg cell activity in GVHD, our results suggest the existence of a broad range of deficiencies in immune regulatory cell function in cGvHD patients. * Both Anushruti Sarvaria and Ahmad K contributed equally. Disclosures: No relevant conflicts of interest to declare.

Donor lymphocyte infusions (DLI) following hematopoietic stem cell transplantation may reduce or control opportunistic infections and leukemia/lymphoma relapse, but the associated graft versus host disease (GvHD) limits the clinical success of this procedure. Since T cell immunotherapy may be a safer alternative to DLI we have now used a single T cell platform that mediates both antileukemic and antiviral activity. Autologous T cells modified to express CD19-specific chimeric antigen receptors (CD19.CAR) have had clinical activity against CD19-expressing malignancies, but it is unknown if similarly modified allogeneic T cells will be equally effective. Allogeneic virus specific T cells (VSTs) directed to cytomegalovirus (CMV), adenovirus (Adv), and Epstein Barr virus (EBV) have been shown to be safe and effective in preventing and treating life-threatening viral infections post HSCT. Therefore, we sought to determine whether allogeneic VSTs could be engineered to express CD19.CAR and would retain the safety and effectiveness of unmodified VSTs whilst gaining anti-tumor activity. VSTs were expanded ex vivo using antigen presenting cells engineered to express adenovirus and cytomegalovirus (using an Ad5f35 adenoviral vector expressing the CMV pp65 gene), and Epstein Barr virus (using EBV-infected lymphoblastoid cell lines) antigens. After 3 stimulations, the VST’s were modified to express CD19.CAR.28ζ using a retroviral vector encoding the CAR-CD19 receptor coupled to the CD28 co-stimulatory molecule and the T cell receptor zeta (ζ) chain. Nine CD19.CAR-modified virus specific T cell (CD19.CAR-VSTs) products were generated for infusion. All VST lines recognized at least one viral antigen as determined by Elispot or chromium release assays and 20% to 48% of cells expressed the CD19.CAR. All lines killed CD19-expressing cells in vitro. We treated nine patients with these CD19.CAR-VSTs, 3 months to 13 years after HSCT. Six patients received CD19.CAR-VSTs for relapsed disease and 3 patients received the T cells as adjuvant therapy to prevent viral infection and relapse after HSCT. Safety. There were no infusion-related toxicities. One patient presented with gastrointestinal symptoms following infusion subsequently determined to be unrelated to the T cells. Persistence. VSTs persisted a median of 8 weeks in the peripheral blood and up to 9 weeks at disease sites. In three patients (#1, #3 and #5), CD19.CAR signals were detectable in the bone marrow or the lymph nodes (44.8, 25.85, and 32 copies/1000 ng DNA) even when no signal was measurable in peripheral blood, indicating preferential accumulation of the infused T cells at the disease site. Anti-Tumor Activity. During the period of CD19.CAR-VST persistence, objective anti-tumor activity was evident in 2/6 patients with relapsed disease (patient # 1 had detectable blasts in the peripheral blood which disappeared within 1-2 weeks following infusion, patient # 2 had 16% circulating CLL cells which decreased within 2 weeks of T cell infusion) but disease recurred after 3 and 2 months, respectively. The two patients who received cells while in remission remain disease-free >3 and >9 months later. Anti-Viral Activity. In two patients with EBV reactivation, donor CD19.CAR-VSTs expanded concomitant with an increase in virus-specific T cell responses, and decreased viral load. A third patient had a rise in adenovirus specific VSTs during an episode of adenovirus associated diarrhea. Although the infection was controlled, there was no concomitant rise in CD19-CAR expressing T cells in this patient. No other patient had viral disease. In conclusion, allogeneic CD19.CAR-VSTs administered after allogeneic HSCT are safe and can exert both anti-tumor and anti-viral activity in the absence of GvHD. Earlier administration of CD19.CAR-VSTs after HSCT, when the host is lymphodepleted and the incidence of viral infection is higher, may allow these cells to better capture the potential advantages of native TCR stimulation (and associated co-stimulation) for expansion and persistence, and thereby produce a higher frequency of sustained tumor responses. Alternatively, intentional stimulation of the native TCRs by viral vaccines may produce equal benefit, with greater predictability. Disclosures: Savoldo: Celgene: Patents & Royalties, Research Funding. Heslop:Celgene: Patents & Royalties, Research Funding; Cell Medica: Patents & Royalties. Rooney:Cell Medica: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Patents & Royalties, Research Funding. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

Abstract 3949 B cell lymphomas have been effectively treated by immunotherapy, including monoclonal antibodies (MAbs) and adoptive T cell transfer. To extend this success, investigators have genetically modified T cells to express a B cell specific antibody incorporated in an artificial chimeric antigen receptor (CAR), essentially combining antibody and cell-based approaches. Early B cell directed CARs combined the antigen binding domains of the variable regions of a CD19 or CD20 MAb (scFv) with the CD3ζ endodomain of the TCR/CD3 complex. Although such CARs confer potent cytotoxic function to T cells, initial clinical trials showed that T cells modified to express CARs engineered in this way had limited in vivo persistence, apparently receiving insufficient costimulation following CAR engagement. To overcome the above limitations, a multiplicity of costimulatory endodomains, including CD28, 41BB or OX40, have been incorporated into the CAR molecule. Because of patient heterogeneity, it has proved difficult to draw definitive conclusions about the relative expansion, persistence and effectiveness of cells with each modification, so that their comparative value in human subjects remains speculative. We therefore designed a phase I, dose escalation clinical trial in which patients with refractory/relapsed B cell malignancies were simultaneously infused with two autologous T cell products. Both express a CAR with an identical CD19-specific exodomain, but one CAR also has a CD28-ζ endodomain while the other expresses only a ζ endodomain. With this study design, each patient acts as a “self-control”, allowing us to directly discover the consequences of CD28 costimulation for the fate of the T cells in vivo. We enrolled two patients at each of the three cell dose levels (1st level = 2×107/m2 of each product; 2nd level = 1×108/m2; 3rd level = 2×108/m2). End points of the study were safety, persistence of each of the two generations of CAR-modified T cells, and assessment of antitumor activity. T cell products were generated by activation of autologous PBMC with immobilized OKT3 and gene modified with retroviral vectors encoding either CAR.19ζ or CAR.19-28ζ. After transduction, T cells were expanded ex vivo for a median of 14 days (range 6–18) in the presence of IL-2. CAR expression was 42%±18% and 49%±16% for CAR.19ζ and CAR.19-28ζ, respectively. This corresponded to 51,246±16,795 and 18,283±9,484 transgene copy numbers/μg DNA, respectively, as measured by Q-PCR. Products contained both CD8+ cells (CAR.19ζ = 49%±22%, CAR.19-28ζ = 48%±22%). Few naïve T cells were present in either transduced population (CD45RA+ = 6%±5% and 6%±6%, respectively), and memory T cells predominated in both (CD45R0+CD62L+ = 50%±24% and 47%±66%, respectively). Both T cell components equally and specifically targeted CD19+ tumors in vitro as assessed by 51Cr release assays (specific lysis was 53%±10% for CAR.19ζ and 65%±19% for CAR.19-28ζ at a 20:1 E:T ratio). All infusions were well tolerated in all patients. Persistence of CAR+ T cell was assessed in peripheral blood by Q-PCR assays specific for CAR.19ζ and CAR.19-28ζ. Molecular signals for CAR.19-28ζ began at a low level after infusion, but progressively increased (7 to 63 fold) to peak at 1–2 weeks post infusion, before declining to background levels over the ensuing 8 to 13 weeks. By contrast, molecular signals corresponding to CAR.19ζ+ cells were barely detectable after infusion, showed no expansion, and rapidly disappeared. Currently 4 patients are evaluable for disease response; 2 had stable disease for up to 6 months and 2 had progressive disease. Hence, infusion of both CAR.19ζ and CD19-28ζ T cells has been safe at current doses. Direct comparison of each cell product in individual patients indicates that inclusion of the CD28 costimulatory endodomain (2nd generation CAR) enhances expansion and persistence. Nonetheless, both the limited expansion and persistence and the modest clinical effects suggest that additional modifications will need to be made to CAR endodomains to optimize the benefits of this therapy. We suggest our approach will allow these modifications to be evaluated systematically and directly even in small-scale clinical studies. Disclosures: Off Label Use: T cell products in studies conducted under INDs.

Abstract 560 EBV-associated Hodgkin's Lymphoma (HL) and some non-Hodgkin's lymphomas (NHL) express EBV proteins, which are potential targets for immunotherapy. Lymphomas arising in the immune-competent host, however, utilize multiple immune evasion strategies to evade host T cell responses. These include limited expression of EBV antigens, so that only the subimmunodominant EBNA1 and LMP1 and 2 antigens are expressed (type II latency); and production of immunosuppressive cytokines, such as TGF-ß. We first tested our hypothesis that cytotoxic T lymphocytes (CTL) enriched for specificity to LMP antigens would have efficacy in patients with EBV+ lymphoma. 36 patients with EBV+ HL and NHL received LMP CTLs generated using antigen-presenting cells genetically modified to overexpress either LMP2 alone (n=16), or LMP2 and inactive LMP1 (ΔLMP1) (n=20). No immediate toxicity was observed following infusion. Of the evaluable patients who have completed 8 weeks of follow-up, 17 were high-risk and/or multiply relapsed patients who received LMP-CTL as adjuvant treatment. 16 of 17 remain in remission for a median of 2.5 years (range 6 months to >5 years). Another 16 patients had detectable disease at the time of CTL infusion, and 11 had clinical responses (78%), including 8 (57%) complete responses. The median duration of the clinical responses is 1.5 years. However, 36% of patients with active disease were either resistant to CTL therapy (n=3) or relapsed/progressed >9 months after CTL therapy (n=2), suggesting the importance of additional tumor immune escape mechanisms. TGF-ß inhibits the growth and function of effector T cells and is secreted both by lymphoma cells and by infiltrating T regulatory cells. To test our hypothesis that the infused CTL can be made resistant to the TGF-ß produced by these tumors, LMP-CTL from 7 patients with relapsed EBV+ve HL, were transduced with a retrovirus vector encoding the dominant-negative TGF-ß type II receptor (DNR). Unlike non-transduced CTL, DNR-transduced CTL were resistant to the anti-proliferative effects of recombinant TGF-ß in vitro. These 7 CTL lines were predominantly CD45RAneg/CD62Lneg (mean 43%;range 1–86%) with a mean of 20% (range 1–48%) of T cells showing a CD45RAneg/CD62Lpos phenotype. Transduction efficiency ranged from 11–30% (median 20%). 3 patients have been treated with DNR-transduced LMP-CTL receiving a dose range of 4 to 10 × 107CTLs/m2 without toxicity. A 2–7 fold increase in EBV/LMP2 –specific T cells was seen by 3 months post CTL infusion, measured as increased numbers of T cells responding in IFN-γ Elispot assay. DNR-transduced T-cells have been detected by quantitative real-time PCR amplification in the peripheral blood of all patients for a median of 7 months (range 2 to >12) post infusion. One patient who had an incomplete response to LMP-CTL alone attained a sustained complete remission, one patient had a partial response and one patient a mixed clinical response. Hence, immunotherapy with CTL targeting LMP antigens is well tolerated in patients with EBV+ lymphoma and can induce durable clinical responses. Furthermore, for patients resistant or with incomplete responses to CTL therapy, the use of DNR-transduced CTL may be beneficial. Disclosures: No relevant conflicts of interest to declare.

Abstract 559 Adverse effects from cellular therapies may be prolonged and progressively worsen as cells expand and persist long-term, unlike most small molecule toxicities, which diminish following drug withdrawal. “Suicide genes” can be introduced into administered cells and activated – generally by a small molecule pro-drug - in the event of adverse effects. Although one such approach using a transferred Herpes Simplex Virus thymidine kinase (HSV-tk) gene may provide effective control, its mechanism of action requires interference with DNA synthesis. As a consequence, cell killing may be protracted over several days, with an even longer delay in clinical benefit. Moreover, an anti-viral therapeutic pro-drug (such as ganciclovir) is required for cell elimination, removing this class of agents from the therapeutic repertoire. Finally, HSV-tk is virus-derived and hence potentially immunogenic. We now report the clinical evaluation of a human suicide gene, inducible Caspase-9 (iCasp9), which is designed to interface with the physiological apoptotic pathway. To generate the iCasp9 molecule we modified human caspase 9 to dimerize and activate upon exposure to a synthetic, otherwise bioinert, small molecule dimerizing agent, AP1903. We infused iCasp9-expressing T lymphocytes, in an effort to enhance immune recovery and reduce infection/relapse following transplantation of HLA-haploidentical hemopoietic (CD34+) stem cells as treatment for high-risk, relapsed leukemia. The infused T cells were first depleted of alloreactive progenitor cells after stimulation with recipient irradiated, EBV-transformed lymphoblastoid cells (40:1) for 72 hrs, and subsequent exposure to a CD25-directed immunotoxin (RFT5-dgA). The allodepleted cells were then transduced with a retroviral vector encoding the iCasp9 suicide gene and a selection marker (DCD19), which allowed enrichment to >95% purity. Four subjects received 1–3 x106 gene-modified T cells/kg. Forced expression of a transgenic caspase-derived molecule did not preclude in vivo survival or expansion of infused T cells, which became detectable by flow cytometry (CD3+DCD19+cells) and by Q-PCR (for iCasp9) within 7 days of infusion. By day 14 these cells expanded to a median cell number/μ l of 175 (range 2–348) (dose level 1) and 49 (range 4–93) (dose level 2). The iCasp9+ T cells contained both CD4+ and CD8+ subsets, were viral-reactive (CMV, EBV, and ADV) and polyclonal, and have now persisted beyond 240 days. Three of the 4 subjects subsequently developed grade I/II acute GvHD of skin, one of whom also had a rising bilirubin, attributed to liver GvHD. As per protocol, these subjects received a single dose of dimerizer agent. Within 30 minutes of completing AP1903 administration, we observed a circa 90% reduction of transgenic T cells, as assessed by flow cytometry for CD3+CD19+ T cells and by Q-PCR amplification for iCasp9. This effect was followed within 36 hrs by resolution of all aGvHD, showing that the iCasp9 transgene can be functional in vivo and can rapidly deplete sufficient T cells to control GvHD. Of note, the residual allodepleted T cells were no longer associated with GvHD but were still able to re-expand within 21 days (median CD3+CD19+ cells/μ l: 77 (range 38–87)), and contained subpopulations that preserved reactivity to viruses (CMV) and fungi (Aspergillus fumigatus), as assessed by IFN-γ production. In conclusion, administration of small numbers of iCasp9+ allodepleted T cells may produce CD4+ and CD8+ T-cell reconstitution after haplo-identical, CD34+ SCT, while administration of a small molecule dimerizer agent has rapidly ablated residual allo-reactive T cells and abrogated early GvHD, whilst preserving anti-viral specificity. Supported by NIH-NHLBI U54HL081007 Disclosures: No relevant conflicts of interest to declare.

Objective The somatic JAK2 V617F mutation is seen in most polycythemia vera (PV) patients; however, it is not clear if JAK2 V617F is the PV-initiating mutation. Methods In order to examine this issue, we developed a novel real-time quantitative allele-specific PCR, in which allelic discrimination is enhanced by the synergistic effect of a mismatch in the −1 position, and a locked nucleic acid (LNA) nucleoside at the −2 position. Results Determination of allelic frequencies was reproducible (SD = 1.5%) and sensitive—0.1% mutant allele detected in 40 ng of DNA. The JAK2 V617F frequency in clonal granulocytes from 3 PV females was less than 50% (27.5 ± 11) and in 7 females greater than 50% (75 ± 10.5). We also found that wild-type JAK2 BFU-E colonies from PV patients can grow without erythropoietin. The identification of the primary genetic lesion resulting in PV is essential for the development of novel therapeutic strategies. Conclusion Our studies correlating the frequency of JAK2 V617F mutant allele and clonality, as well as the presence of homozygous wild-type JAK2 erythropoietin-independent erythroid colonies, provide compelling evidence that the JAK2 V617F is not the PV-initiating mutation. This supports a model wherein the JAK2 V617F mutation arises as a secondary genetic event. Furthermore, our results indicate that an undefined molecular lesion, preceding JAK2 V617F , is responsible for clonal hematopoiesis in PV. We conclude that development of therapeutic strategies that target the JAK2 V617F clonal cells may not be sufficient for eradication of PV.

Self-tumor antigens that elicit anti-tumor immune/autoimmune responses in responses to interferon-a (IFN-a) stimulation remain poorly defined. We screened a human testis cDNA library with sera from three polycythemia vera (PV) patients who responded to IFN-a and identified a novel antigen, MPD6. MPD6 belongs to the group of cryptic antigens without conventional genomic structure and is encoded by a cryptic open reading frame located in the 3′untranslated region (3′UTR) of myotrophin mRNA. MPD6 elicits IgG antibody responses in a subset of PV patients, as well as patients with chronic myelogenous leukemia and prostate cancer, suggesting that it is broadly immunogenic. The expression of myotrophin-MPD6 transcripts in tumor cells was upregulated in some tumor cells, but only slightly increased in K562 cells in response to IFN-a treatment. By using bicistronic reporter constructs, we showed that the translation of MPD6 was mediated by a novel internal ribosome entry site (IRES) upstream of the MPD6 reading frame. Furthermore, the MPD6-IRES mediated translation, but not myotrophin-MPD6 transcription, was significantly upregulated in response to IFN-a stimulation. These findings demonstrate for the first time that a novel IRES-mediated mechanism is responsible for the translation of unconventional self-antigen MPD6 in responsive to IFN-a stimulation. The eliciting anti-tumor immune response against unconventional antigen MPD6 in patients with myeloproliferative diseases suggests MPD6 as a potential target of novel immunotherapy. Our results also suggest that the enhancement of IRES-mediated translation of unconventional self-antigen(s) by IFN-a is a novel mechanism of IFN-a enhanced anti-tumor immune/autoimmune responses.

We were asked to investigate a 54 year old male who was undergoing evaluation for sleep apnea. The patient had a low resting O2 saturation at 91%, but no signs of cardiac or pulmonary disease as indicated by a normal echocardiogram, normal spirometry, and normal high resolution CT of the chest. Arterial blood gases revealed a pO2 of 79 mm Hg on room air, but pCO2 was normal. The patient’s hemoglobin concentration was 13.7 g/dL. The possibility of a mutant hemoglobin was raised and HPLC revealed HbA2 = 2.6%, HbA = 51.4% and an unidentified mutant hemoglobin of 46%. Oxygen dissociation testing demonstrated a P50 of 39 mm Hg (normal 24–32) consistent with a low affinity hemoglobin. The isopropanol test showed that the hemoglobin variant was unstable. Trypsin digestion of the abnormal hemoglobin resulted in a smaller than normal HPLC peak for the β9 segment of β-globin, which is composed of amino acids 67 through 82, and the appearance of a new peak at 193 min. This peak did not match any of the known mutant hemoglobins contained in our reference bank. We PCR amplified and sequenced the β-globin gene. The patient was heterozygous for an A to C mutation in the second position of codon 66. This mutation changed the lysine to threonine and revealed that the hemoglobin variant was Hb Chico. This result was consistent with the loss of ~50% of the β9 peak in the trypsin digest due to loss of a cleavage site after amino acid 66, and the appearance of a new peak which was a hybrid segment of β8 and β9. This represents the first DNA sequence of hemoglobin Chico a mildly unstable hemoglobin variant with decreased oxygen affinity.

Myelofibrosis with myeloid metaplasia (MMM) is a clonal myeloproliferative disorder, characterized by splenomegaly and myelofibrosis. Like all myeloproliferative disorders, it is due to an acquired somatic mutation of a hematopoietic progenitor resulting in clonal erythrocytes, platelets, and granulocytes. A single acquired point mutation of JAK2 1849G>T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, occurs in 50%–97% of patients with MMM, essential thrombocythemia (ET) and polycythemia vera (PV). We studied 25 myelofibrosis patients with myeloid metaplasia and myelofibrosis complicating PV or ET. These were compared to 19 patients with secondary myelofibrosis of equivalent severity associated with pulmonary hypertension (PH) treated by epoprostenol. In these two groups we compared peripheral blood CD34 counts, clonality of granulocytes and platelets in peripheral blood, mutational status of JAK 2 kinase gene, and morphology of peripheral blood and bone marrow. Heterozygosity for JAK 2 1849G>T somatic mutation was seen in 17 of 23 (74%) patients with MMM including 10 of 15 patients (67%) with idiopathic myelofibrosis and 7 of 8 patients (88%) with myelofibrosis complicating PV or ET, but was absent in all 19 patients with myelofibrosis associated with PH (P We conclude that a high circulating CD34 count, clonal platelets and granulocytes, presence of dacrocytes, and JAK2 1849G>T (V617F) mutation of clonal progenitors are the intrinsic features present in patients with myelofibrosis due to myeloproliferative disorders and that these features are not due to the abnormal marrow architecture that is seen in secondary myelofibrosis.

A single acquired point mutation of JAK2 1849G>T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, is found in 70%–97% of patients with polycythemia vera (PV). In the studies of tyrosine kinase inhibitors on JAK2 1849G>T (see Gaikwad et all abstract at this meeting) we decided to study the possible therapeutic effect of these agents using native in vitro expanded cells from peripheral blood. To our surprise, the in vitro expansion of PV progenitors preferentially augmented cells without JAK2 1849G>T mutation. We used a 3 step procedure to amplify erythroid precursors in different stages of differentiation from the peripheral blood of 5 PV patients previously found to be homozygous or heterozygous for the JAK2 1849G>T mutation. In the first step (days 1–7), 106/ml MNCs were cultured in the presence of Flt-3 (50 ng/ml), Tpo (100 ng/ml), and SCF (100 ng/ml). In the second step (days 8–14), the cells obtained on day 7 were re-suspended at 106/ml in the same medium with SCF (50 ng/ml), IGF-1 (50 ng/ml), and 3 units/ml Epo. In the third step, the cells collected on day 14 were re-suspended at 106/ml and cultured for two more days in the presence of the same cytokine mixture as in the step 2 but without SCF. The cultures were incubated at 37oC in 5% CO2/95% air atmosphere and the medium renewed every three days to ensure good cell proliferation. The expanded cells were stained with phycoerythrin-conjugated anti-CD235A (glycophorin) and fluorescein isothiocyanate-conjugated anti-human-CD71 (transferrin receptor) monoclonal antibodies and analyzed by flow cytometry. The cells were divided by their differential expression of these antigens into 5 subgroups ranging from primitive erythroid progenitors (BFU-Es and CFU-Es) to polychromatophilic and orthochromatophilic erythroblasts; over 70% of harvested cells were early and late basophilic erythroblasts. The proportion of JAK2 1849G>T mutation in clonal PV granulocytes (GNC) before in vitro expansion and in expanded erythroid precursors was quantitated by pyrosequencing (Jelinek, Blood in press) and is depicted in the Table. These data indicate that in vitro expansion of PV progenitors favors expansion of erythroid precursors without JAK2 V617F mutation. Since three PV samples were from females with clonal granulocytes, erythrocytes, and platelets, experiments were underway to determine if the in vitro expanded erythroid cells were clonal PV cells without JAK2 V617F mutation, or derived from polyclonal rare circulating normal hematopoietic progenitors. The Proportion of JAK2 T Allele Patients GNC T Allele (%) Expanded Cells T Allele (%) PV1 (Female) 81 10 PV2 (Male) 77 28 PV3 (Male) 44 42 PV4 (Female) 78 19 PV5 (Female) 78 28

Secondary bone marrow fibrosis can be seen in several conditions such as hematological malignancy, metastatic malignancy and autoimmune disorders like lupus. We report here a 50 year old Middle Eastern female with a history of pulmonary sarcoidosis diagnosed by lung biopsy in January of 2004, beta thalassemia trait, hypertension and hypothyroidism. At the time of diagnosis she was started on bronchodilating inhalers. Nine months later she was referred to hematology for left lower quadrant pain and possible enlarged spleen on examination. CT scan revealed extensive mediastinal, retroperitoneal and inguinal adenopathy but no splenomegaly. Hemoglobin was 12.3 with a hematocrit of 38 and an MCV of 61, WBC was 7.8with a normal differential and Platelet count was 238. Her peripheral blood smear revealed moderate anisocytosis, polychromasia and some teardrops. Bone marrow biopsy showed significant fibrosis confirmed by reticulin staining. Biopsy of an inguinal node was consistent with her lung biopsy showing granulomatous disease. In the course of follow up she developed fever, fatigue, anemia, and splenomegaly proven by ultrasound (16cm). Her labs also revealed increased ACE and ESR levels. We started her on prednisone at 1mg/kg and her hemoglobin rose from 8.0 to 12.5. X-chromosome transcription-based clonality assays were employed to deteremine whether this patient had primary myelofibrosis with myeloid metaplasia, a myeloproliferative disorder characterized by clonal hematopoiesis or secondary myelofibrosis which is characterized by polyclonal hematopoiesis. Six exonic X-chromosome polymorphisms were tested to determine the clonality of the patient’s platelets and granulocytes (Liu, BLOOD 2003). Unfortunately, this patient was not informative in any of six exonic X-chromosome polymorphism markers, and we could not determine the clonality of hematopoiesis. Peripheral blood CD34 level; however, was low normal, consistent with reactive fibrosis rather than a primary myeloproliferative disease. To our knowledge this is the first report of secondary bone marrow fibrosis in sarcoidosis.

Therapeutic responses to interferon-a (IFN-a) therapy in patients with malignancies are at least in part from the effects of IFN-a on the enhancement of anti-tumor immune responses. Antigens responsible for immune responses enhanced by IFN-a remain largely unknown. Polycythemia vera (PV) is a myeloproliferative disease (MPD) resulting from the clonal expansion of a pluripotential hematopoietic precursor cell. IFN-a therapy induces a complete remission in ~50% of PV patients, thus making PV a good model to study the molecular mechanism of the cytokine-enhanced immune responses against tumors. We hypothesized that IFN-a enhances anti-PV immune responses by modulating the expression of non-mutated self-antigens. We identified three novel MPD antigens by applying a technique, termed serological identification of tumor antigens by expression cDNA cloning (SEREX), to screen a human testis cDNA library with sera from three PV patients who responded to IFN-a therapy. Of these three novel SEREX antigens, MPD5 belongs to the newly identified group of unconventional cryptic antigens. These unconventional antigens are encoded by either the introns of genes, or the untranslated regions of mRNAs. MPD5 is the antigen encoded by the complementary strand of the intron 1 region of NEK-6 gene. Two additional MPD antigens, MPD13 and MPD67, are conventional antigens, which are encoded by the genes with conventional exon/intron structures. Our results showed that these novel MPD antigens elicit potent IgG antibody immune responses in the sera of patients with PV, as well as patients with chronic myelogenous leukemia who responded to IFN-a treatment and in patients with prostate cancer, suggesting that these antigens are broadly immunogenic. Previous reports showed that unconventional antigens can elicit cellular T cell mediated responses. Our data demonstrated that these antigens also elicit humoral IgG antibody responses, which can be further enhanced by IFN-a therapy. These findings provide new insights into the molecular mechanism underlying the regulation of the self-antigen repertoire including both conventional antigens and unconventional antigens, in eliciting anti-tumor immune responses in responses to IFN-a therapy, and suggest their therapeutic potential as the targets of novel immunotherapy for MPD.

Idiopathic myelofibrosis (IMF) is a fatal clonal myeloproliferative disorder, characterized by variable pancytopenia, splenomegaly, leukoerythroblastic blood smear, marrow fibrosis (MF), and myeloid metaplasia. Like all myeloproliferative disorders, it is due to an acquired somatic mutation of a single hematopoietic progenitor. MF has also been reported in systemic lupus and metastatic malignancies involving bone marrow. We have recently demonstrated the occurrence of MF in 85% patients with pulmonary hypertension (PH), which (unlike IMF) is associated with polyclonal hematopoiesis and normal number of circulating CD34+ cells. The pathophysiological mechanisms resulting in IMF and secondary MF (SMF) in PH remain still unclear. To date, 52 heterozygous germline mutations in the gene ( BMPR2 ) encoding the bone morphogenetic protein type II receptor (BMPR-II) have been found to characterize many cases of familial (Lane, 2000) and sporadic primary PH (PPH) (Thomson, 2000). Recently, increased activity of arginase in pulmonary tissue of patients with PH was found. This may contribute to the pathogenesis of pulmonary hypertension by decreasing the substrate for NO synthesis. We examined BMPR2 and Arginase II mRNA levels by quantitative real-time RT-PCR in mononuclear cells (MNC), platelets (PLT) and granulocytes (GNC) from IMF patients and PH patients with secondary MF. We show for the first time that the mRNA level of BMPR2 is increased in PLT and GNC of IMF compared with normal controls. Arginase II mRNA was significantly increased in GNC of IMF as well as PH patients with SMF. Rearrangement and over expression of high-mobility group AT-hook2 ( HMGA2 ) gene in myeloid progenitors in two patients with IMF was recently reported. This gene is known to play a major role in fetal growth and development, and is expressed in trace amount in adult lung, kidney, and synovia. We found significantly increased HMGA2 mRNA expression in MNC of IMF patients. In summary, the biomarkers studied here can be abnormal in both IMF and PH with secondary MF and may not be useful to discriminate between the two types of MF. Dysregulation of BMPR2 , Arginase II and HMGA2 expression may play a pathophysiological role in MF of both IMF and PH. Expression of BMPR2 , Arginase II and HMGA2 in Blood Cells of IMF and SMF | Gene | Cells | Control ( Δ CT) | Idiopathic MF ( Δ CT) | Secondary MF ( Δ CT) | |:----------------------------------------------------------------------------------------------------------------------------------------------------------------:| ----- | --------------- | --------------------- | -------------------- | | *The P value represents a significant difference of gene expression in IMF or SMF compared with normal controls. A lower ΔCT indicates a higher gene expression. | | BMPR2 | PLT | 18.5±1.2 | 16.7±1.4 (p=0.038) * | 18.4±2.2 (p=0.881) | | | GNC | 19.2±1.0 | 18.3±1.3 (p=0.009) * | 19.1±1.2 (p=0.893) | | | MNC | 18.3±1.2 | 19.2±1.8 (p=0.059) | 19.0±2.7 (p=0.554) | | Arginase II | GNC | 24.5±0.6 | 21.9±1.7 (p=0.000) * | 22.6±1.2 (p=0.004) * | | | MNC | 24.4±3.5 | 24.4±3.9 (p=0.965) | 22.5±2.4 (p=0.222) | | HMGA2 | MNC | 32.4±1.6 | 29.3±1.7 (p=0.000) * | 30.8±1.8 (p=0.085)

Idiopathic myelofibrosis (IF) is due to an acquired somatic mutation(s) of a single hematopoietic multipotent progenitor and thus circulating myeloid, erythroid, megakaryocyte, B and T cells (Reeder et al Blood. 2003;101: 1981–1983) are clonal. It has been suggested that fibrosis may occur secondary to cytokines secreted by the malignant clone. Although marked elevation of circulating CD34 cells occurs in patients with IF, it is not known whether it is a consequence of the primary defect of the hematopoietic stem cell, or secondary to fibrosis and impaired marrow microenvironment. To answer this question, we studied CD34 count in peripheral blood of patients with IF and secondary myelofibrosis due to pulmonary hypertension. Nineteen patients with either primary or secondary pulmonary hypertension due to connective tissue disease and 11 patients with IF were studied. Bone marrow biopsy revealed fibrosis in all 19 patients with PH: 11 severe, 3 moderate, and 5 mild. All eleven patients with IF had severe fibrosis. Clonality studies in 13/14 informative patients with PH showed polyclonal platelets and granulocytes in contrast to monoclonal platelets and granulocytes in patients with IF. Peripheral blood was assayed for CD34+ cells using a three-color direct immunofluorescent staining method. Mean absolute CD34 count was significantly (P=0.02) lower - 6/ul (range: 1–18/ul) in patients with PH compared to 142 /ul (range 4–833/ul) in patients with IF. We therefore conclude that high CD34 count is not seen in patients with PH with secondary myelofibrosis; furthermore, elevated CD34 in patients with IF is not due to marrow fibrosis and altered microenvironment.

Autologous T cells expressing a CD19-specific chimeric antigen receptor (CD19.CAR) are active against B-cell malignancies, but it is unknown whether allogeneic CD19.CAR T cells are safe or effective. After allogeneic hematopoietic stem cell transplantation (HSCT), infused donor-derived virus-specific T cells (VSTs) expand in vivo, persist long term, and display antiviral activity without inducing graft-vs-host disease; therefore, we determined whether donor VSTs, engineered to express CD19.CAR, retained the characteristics of nonmanipulated allogeneic VSTs while gaining antitumor activity. We treated 8 patients with allogeneic (donor-derived) CD19.CAR-VSTs 3 months to 13 years after HSCT. There were no infusion-related toxicities. VSTs persisted for a median of 8 weeks in blood and up to 9 weeks at disease sites. Objective antitumor activity was evident in 2 of 6 patients with relapsed disease during the period of CD19.CAR-VST persistence, whereas 2 patients who received cells while in remission remain disease free. In 2 of 3 patients with viral reactivation, donor CD19.CAR-VSTs expanded concomitantly with VSTs. Hence CD19.CAR-VSTs display antitumor activity and, because their number may be increased in the presence of viral stimuli, earlier treatment post-HSCT (when lymphodepletion is greater and the incidence of viral infection is higher) or planned vaccination with viral antigens may enhance disease control. This study is registered at clinicaltrials.gov as #NCT00840853.