Cordyceps militaris Induces Immunogenic Cell Death and Enhances Antitumor Immunogenic Response in Breast Cancer

Cordyceps militaris has been widely used as a traditional medicine in East Asia. Its effects against breast cancer have been reported previously. However, whether C. militaris-induced breast cancer cell death is immunogenic remains unelucidated. This study aimed to determine whether ethanolic extracts of C. militaris (CM-EE) could induce immunogenic cell death (ICD) in breast cancer immunotherapy to improve the efficacy of immune checkpoint inhibitors. Human and mouse breast cancer cells were treated with various concentrations of CM-EE for 72 h, and cytotoxicity was measured using the sulforhodamine B assay. Flow cytometry was used to assess cell death with annexin V/7-AAD staining and measure the surface exposure of damage-associated molecular pattern (DAMP) molecules including calreticulin, HSP70, and HSP90. Western blot for cleaved poly (ADP-ribose) polymerase (PARP) was used to confirm apoptotic cell death. The immunogenicity of CM-EE-induced dead cells was evaluated using the CFSE dilution assay. CM-EE reduced the viability of human (MCF7, MDA-MB-231, HS578T, and SKBR3) and mouse (4T1-neu-HA, TUBO-HA, and TUBO-P2J-HA) breast cancer cells. The IC50 was 25–50 µg/ml in human breast cancer cells and 10–50 µg/ml in mouse breast cancer cells at 72 h. CM-EE-treated breast cancer cells were positively stained by annexin V, cleaved PARP, and cleaved caspase 3/7 which were increased upon CM-EE treatment. Surface exposure of DAMP molecules was increased in dose- and time-dependent manners. The CFSE dilution assay revealed that dendritic cells fed with CM-EE-treated breast cancer cells successfully stimulated tumor-specific T cell proliferation without inhibiting DC function and T cell proliferation. The expression of PD-L1 mRNA and protein level was increased in dose-dependent manners. In addition, CM-EE also potentiated the cytotoxic activity of tumor-specific T cells. CM-EE can induce immunogenic and apoptotic cell death in breast cancer cells, and it is a good candidate for cancer immunotherapy and may improve the efficacy of immune checkpoint inhibitors.

Hematopoietic Stem Cells Increase Quiescence during Aging

Introduction: Definitive hematopoietic stem cells (HSCs) sustain blood production from fetal development throughout life. In mice, most of steady state, young adult HSCs are in the G0 phase of cell cycle (quiescence), and are estimated to divide roughly once a month. Daily hematopoietic production is thus mainly sustained by highly proliferative downstream hematopoietic progenitor cells (HPCs). Aged haematopoiesis was demonstrated to be distinct from young haematopoiesis in various aspects such as i) a shift from lymphopoiesis to myelopoiesis, ii) functional decline of HSCs (self-renewal, homing), and iii) HSCs pool expansion. While several studies attempted to address whether changes in HSCs turnover during aging can account for the distinct aging associated phenotype and function, it remained to be determined whether aged HSCs overall cycle more or less frequently than young HSCs. Methods: To construct data-based, quantitative models, we measured turnover rates and compartment sizes of populations of HSCs, HSPCs and granulopoiesis/granulocytes, i.e. a post-mitotic mature hematopoietic linage with a short half-life. We examined four age groups: 3 week, 2 month, 1 year and 2 year old mice. Mice in each group were i.p. injected every 4 hours with 1 mcg EdU up to a maximum time of 48 hours. HSC, HSPC and granulopoiesis/granoulocyte compartment sizes and snapshot cell-cycle analysis was performed by FACS at multiple sampling points in BM and peripheral blood (PB), respectively. Based on this data, we built a mathematical model of HSC turn-over and HSPC differentiation during ageing. Moreover, we evaluated HSC cycling by CFSE dilution in steady-state transplantation experiments (as described before; Takizawa et al., J Exp Med 2011). Results: In line with previous reports, the HSCs compartment size gradually increased with age from 3wk old mice to 2 year old mice. In sharp contrast, cycling activity of HSCs as determined by EdU incorporation decreased gradually and significantly with increasing age. This was driven by decreased activation from the quiescent state, while the time that actively cycling HSCs require to progress through cell-division remains constant with age. Multipotent Progenitor (MPP) cycling showed a non-significant trend towards slower turn-over. These results were confirmed by complementary CFSE-dilution experiments. Mathematical modeling of HSC proliferation and differentiation revealed a higher probability of self-renewing divisions in 3 week old mice as compared to 2 month, 1 and 2 year old mice, with the latter both having nearly equal chances of self-renewing versus differentiating divisions. Conclusions: Our data clarifies the long-standing question, how the HSC pool increases with age. Instead of an increase in active cycling, an increase in HSC quiescence is responsible for the increased size of the HSCs pool in aging. Disclosures No relevant conflicts of interest to declare.

Functional Characterization of a T Cell Stimulation Reagent for the Production of Therapeutic Chimeric Antigen Receptor T Cells

Adoptive cell therapy using gene-modified T cells has demonstrated promising clinical outcomes in hematologic malignancies. Production of gene-modified T cells involves the selection of patient T cells, activation via stimulation through the endogenous T cell receptor (TCR) complex and a costimulatory domain, followed by introduction of a tumor antigen-specific TCR or chimeric antigen receptor (CAR) through gene modification. Here we characterize a soluble T cell stimulation reagent, known as an ExpamerTM reagent, in the production of therapeutic CAR T cells. The Expamer reagent used in these studies is designed to be a late-stage clinical and commercial manufacturing ancillary material with two important attributes that make it highly attractive from a manufacturing and regulatory standpoint; it is a soluble and dissociable reagent. These attributes increase the ease of both introduction and removal from the manufacturing process, giving products manufactured with this reagent consistent product quality and purity. This reagent activates T cells through the simultaneous engagement of the TCR-CD3 complex and the costimulatory receptor CD28 and is compatible with manufacturing of both current and next-generation therapeutics. Purified healthy donor T cells cultured in the presence of the Expamer reagent rapidly fluxed Ca2+, demonstrating the capacity to induce early TCR signaling. Activation through this reagent additionally promotes upregulation of the cell surface activation marker CD25 and proliferation as measured by CFSE dilution. Following stimulation with this reagent, T cells are readily transduced with a CD19-specific CAR construct. The function of CAR T cells generated with this reagent was measured by effector cytokine production, proliferation, and cytolytic activity in the presence of CD19 expressing and control target cells in vitro. CAR T cells robustly produced IFN-ɣ and IL-2 after activation with a CAR specific antigen. In addition, proliferation in the presence of CD19 expressing target cells was observed as measured by CFSE dilution. Finally, significant cytolytic activity against CD19-expressing target cells was observed. Collectively, these data provide evidence that functional engineered T cells can be manufactured using the Expamer reagent and support implementation into the production of both current and next-generation therapeutic gene-modified T cells. The first two authors contributed equally to this work. Disclosures Bashour: Juno Therapeutics: Employment. Larson:Juno Therapeutics: Employment. Graef:Juno Therapeutics: Employment. Stemberger:Juno Therapeutics: Employment. Lothar:Juno Therapeutics: Employment. Odegard:Juno Therapeutics: Employment. Ramsborg:Juno Therapeutics: Employment.

Selective Photodepletion of Recipient-Alloreactive T-Cells Enables Safe and Efficacious Haploidentical HSCT: Initial Results from a Phase 2 Trial in Patients with AML, ALL, and MDS

Introduction For patients in need of a hematopoietic stem cell transplant (HSCT) but lacking an HLA matched donor, a haploidentical family donor is a particularly appealing alternative. However, to prevent graft-versus-host disease (GVHD), haploidentical HSCT necessitates intensive in vivo or ex vivo T-cell depletion that results in frequent and often lethal infectious complications and/or high relapse rates, thus decreasing overall survival. To overcome this limitation, we have developed a strategy that photodepletes host-reactive cells from the donor T cell graft, while preserving anti-infection and anti-leukemia reactivity. Patients and Methods In an open-label, multi-center phase 2 clinical trial (CR-AIR-007; NCT01794299), 12 of a planned 23 patients with high-risk hematologic malignancies were treated to date with this immunotherapy approach consisting of donor lymphocytes selectively allodepleted of host-reactive T-cells using photodynamic therapy (ATIR). ATIR was infused 28-32 days after haploidentical CD34-selected HSCT. No post-transplant GVHD prophylaxis was used. These patients were compared to a control group of 28 patients treated in a previous Phase 2 study with an investigational product manufactured using a process different from the Phase 1 trial and resulting mainly in dead and inactive cells instead of ATIR (CR-AIR-004). Results Twelve patients, mean age of 45 (range 21-64), 6 females/6 males with AML (n=9) and ALL (n=3) were treated with ATIR so far. ATIR consisted mainly of T-cells (>90%), with residual B and NK cells (≤10%). Selective depletion of recipient-reactivity in each ATIR cell graft was assessed using a CFSE-based proliferation assay. Cell division numbers upon stimulation were analyzed using Modfit LT software (Fig 1A), which generated a proliferation index representing viable/reactive T-cells in donor cells (blue) and final ATIR product (green)(Fig 1B). Selective depletion of recipient-reactive T-cells with preservation of reactivity towards 3rd party antigens and anti-CD3/CD28 was observed in all ATIR cell grafts and used as a release criteria in the 007 study. Figure 1: A) CFSE-dilution pattern in Modfit LT software of ATIR stimulated with 3rd party cells. B) CFSE-based proliferation confirmed selective depletion of recipient-reactive T-cells in all grafts (representative depiction). Figure 1:. A) CFSE-dilution pattern in Modfit LT software of ATIR stimulated with 3rd party cells. B) CFSE-based proliferation confirmed selective depletion of recipient-reactive T-cells in all grafts (representative depiction). Preparative regimen consisted of A) FTBI (1200 cGy; n=5) or B) melphalan (120 mg/m2; n=7), along with thiotepa (10 mg/kg), fludarabine (30 mg/m2 x5 d) and ATG (2.5 mg/kg x4 d). Neutrophil and platelet engraftment was achieved in all patients at a median of 12 days (range: 9-35). No patient experienced graft rejection. Patients (n=28) in the 004 control group, mean age of 42 (range 18-61), 13 females/15 males had AML (n=19), ALL (n=6) or MDS (n=3). CFSE proliferation in T-cell grafts could not be assessed a posteriori due to low cell viability. These 004 patients received the same A) FTBI- (n=14), B) melphalan- (n= 10) based preparative regimen as 007 patients, except for 4 patients receiving single fraction (800 cGy) TBI. Neutrophil and platelet engraftment was achieved at a median of 16 days (range: 7-54). Three patients showed secondary graft rejection. Two patients in study CR-AIR-007 developed acute GVHD grade I (skin only) approximately 130 days post HSCT, which was of short duration, (18 and 41 days). Two patients died of infection and no patient relapsed at a mean follow-up of 8 months post HSCT (range 1-14 months). In the CR-AIR-004 control group, 2 patients developed grade I, 1 patient grade II and 3 patients grade III GvHD, none of these cases were lethal. Seventeen patients died of transplant related complications and 2 patients of relapse/disease progression. TRM is 20% in 007 group vs 63% in the 004 control group and OS is 80% in 007 group vs 35% in the 004 control group at 9 months post-transplant (Figures 2A and 2B). Figure 2A Kaplan Meier Transplant Related Mortality: 004 vs 007 (p=0.06) Figure 2B Kaplan Meier Overall Survival (OS): 004 vs 007 (p=0.03) Conclusions These data confirm that a novel immunotherapy strategy consisting of donor lymphocytes selectively photodepleted of alloreactive cells (ATIR) can be manufactured consistently and reproducibly. Results to date show that ATIR is safe and does not cause any grade III/IV GvHD. Moreover, haploidentical HSCT patients treated with ATIR demonstrate very promising TRM and OS rates when compared to the control group. Disclosures Roy: Kiadis Pharma: Consultancy, Research Funding. Foley:Hoffman-LaRoche: Advisory Board/Lectures Other, Honoraria; Lundbeck: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Sanofi: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Celgene: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Pfizer: Advisory Board/Lectures Other, Honoraria; Novartis: Advisory Board/Lectures Other, Honoraria; Jansen: Advisory Board/Lectures Other, Honoraria; Alexion: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Roche Canada: Honoraria, Research Funding, Unrestricted educational grant, Unrestricted educational grant Other. De Jong:Kiadis Pharma: Employment. Velthuis:Kiadis Pharma: Employment. Gerez:Kiadis Pharma: Employment. Reitsma:Kiadis Pharma: Employment. Wagena:Kiadis Pharma: Employment.

Low Dose Interleukin-7 Supplementation Increases Intrinsic Cord Blood T Cell Survival without Enhancing Proliferative Alloresponses

Abstract 4353 Background: Umbilical cord blood (CB) transplantation is associated with delayed and defective immune reconstitution, in part because recent thymic emigrants, the most abundant subset among CB T cells, have limited intrinsic survival capacity. Interleukin-7 has been reported to increase the initial recovery of the graft-derived T cell compartment. The aim of this in vitro study was to define the optimal supplementation with recombinant human IL-7 (rhIL-7) than can promote the survival of CB T cells without enhancing allogeneic reactions, so as to limit the risk of eliciting an uncontrolled acute GVHD in vivo. Methods: Twenty-six CB were obtained immediately after normal-term delivery, using the same procedure as for CB banking, and a freeze-thawing step in order to recapitulate the clinical procedure. CB T cells were cultured for one week alone or with HLA-mismatched monocytes from healthy adults (MLR conditions), in medium supplemented or not with rhIL-7. Cell viability was assessed by flow cytometry by scatter analysis and 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] and propidium iodide staining. CB T cell proliferation was assessed by CFSE dilution. Results: Under basic culture conditions, a unique high dose of rhIL-7 (1 ng/mL) added on day 0 improved CB T cell survival but also triggered their proliferation. A daily addition of a low dose of rhIL-7 (final concentration of 100 pg/mL) reduced CB T cell apoptosis and increased significantly cell survival after 1 week of culture (p <0.001), without inducing any cell proliferation. After one week of culture in allogeneic condition, small and large CB T cells were individualized on the cytogramm. Small cells corresponded to nonresponder CB T cells (CFSE dilution < 6,3%), while large CB T cells were allogeneic responder cells which underwent cell division. When allogeneic cultures were run with daily addition of rhIL-7 (100 pg/mL), the number of viable quiescent small CB T cells rose markedly (median 59,279 versus 36,726 without IL-7 supplementation, p=0.01), but the number of dividing cells among the large CB T cells did not increase significantly (median 29, 437 versus 24,471, p=0.19). Conclusion: These experimental data show that repeated exposition to low-doses of rhIL-7 can preserve a viable CB T cell compartment, potentially useful in CB transplantation both for T cell reconstitution and T cell recruitment after primary infections. In these IL-7 exposition conditions, recent thymic emigrants could survive better without undergoing an uncontrolled expansion that would be deleterious in increasing the risk of acute GVHD. These results indicate that clinical low dose IL-7 administration in umbilical cord blood transplanted patients, could improve post-transplant immune reconstitution, without potentiating risk of acute GVHD. Disclosures: No relevant conflicts of interest to declare.

Abstract 90: The Atheroprotective Effects of Id3 Are Linked to B-1a Cell Proliferation and Plasma Levels of E06

Background: The HLH transcription factor, Id3, is essential for B cell-mediated atheroprotection in mice and a functionally significant single nucleotide polymorphism in the human ID3 gene at rs11574 is associated with carotid intimal medial thickness in humans. Yet, the mechanisms mediating Id3 atheroprotection are poorly understood. Recent studies provide clear evidence that B cell effects on atherosclerosis are subset dependent; with B2 cells promoting and B-1a cells attenuating atherosclerosis in a sIgM-dependent manner. Innate, natural IgM antibodies, such as E06, recognize oxidized phospholipids, are produced by B-1a cells, and inhibit atherogenesis. Therefore, we hypothesized that Id3 would regulate plasma levels of E06 IgM and atheroprotective B-1a cells. Methods & Results: Id3 -/- ApoE -/- mice (n = 9) at 8 weeks old had lower plasma levels of E06 compared to control Id3 +/+ ApoE -/- mice (n = 9) (1500 vs. 2750 RLU, p < 0.05) as measured by ELISA. This was not due to lower total IgM (270 vs. 100 ug/mL respectively, p < 0.05). ApoB-100 levels were not different (1500 vs 1600 RLU, n.s.). Consistent with plasma data, the number of B-1a cells, assessed by flow cytometry, was lower in Id3 -/- ApoE -/- (n = 5) compared to Id3 +/+ ApoE -/- mice (n = 4) (0.8 x10 5 vs 3.0 x10 5 , p < 0.05). There was no difference in transcript levels, as measured by real-time PCR, of E06 or sIgM in fluorescence-activated cell sorted B-1a cells. Furthermore, we found decreased homeostatic proliferation, measured by in situ CFSE dilution, of B-1a cells in Id3 -/- ApoE -/- mice (n = 5) compared to control mice (n = 5) (30.1% vs. 50.0%, p = 0.001). Additionally, humans with the functionally significant polymorphism in ID3 had lower plasma levels of IgM to MDA-LDL (n = 97, trend with p = 0.08) and no difference in IgG as measured by ELISA. Conclusion: Taken together, these data suggest a novel role for Id3 in regulating B-1a cell proliferation, resulting in reduced plasma levels of the atheroprotective natural antibody, E06. Moreover, the data suggest that the ID3 polymorphism in humans at rs11574 may link protective IgM to modified lipids with vascular disease.

Absence of STAT1 Signaling in Host Hematopoietic Cells Leads to Enhanced Gvhd Induction and Involves Increased Expression of MHC Class II and Reduced PD-L1 Expression on Dendritic Cells,

Abstract 4026 Interferon-g/STAT1 signaling plays a critical role in regulating dendritic cell activation and function. Blockade of IFN-g signaling leads to reduced DC activation and impaired anti-tumor and acquired adaptive immunity. We recently reported that lack of IFN-g/STAT1 in donor lymphocytes leads to reduced GVHD induction in both MHC- and mHA-mismatched mouse BMT models. In this study, we addressed the role of host STAT1 in the regulation of GVHD. Wildtype or STAT1-deficient 129 mice (H2b) underwent allogeneic Bone Marrow Transplantation (BMT) following lethal irradiation (1044 rad). GVHD was induced using either BALB/c or B6 donor spleen cells. We unexpectedly observed that absence of STAT1 in recipient mice led to increased GVHD-associated mortality in both MHC-mismatched (MST 5 vs. 8, p=0.01) and mHA-mismatched (MST 11 vs. 23, p<0.01) BMT settings. The enhanced GVHD induction was found to be associated with increased activation (expression of CD69 and CD25) and allo-antigen driven proliferation of donor CD4 and CD8 T cells as determined by CFSE-dilution. As host APCs have been reported to being crucial for induction of GVHD, we phenotypically and functionally characterized STAT1 deficient DCs. Our studies revealed that STAT1-deficient bone marrow-derived dendritic cells (BMDCs) which were maturated in the presence of LPS showed significantly increased MHC class II, CD86, CD80 and CD40 expression compared with wildtype BMDCs. Furthermore, STAT1-deficient BMDC showed significantly increased direct allo-stimulatory capacity resulting in increased responder cell proliferation as determined by standard MLR assays using 3H-Thymidine uptake assays as well as CFSE-dilution studies. STAT1−/− BMDCs significantly promoted CD44+CD62L- expression in responder CD4 and CD8 T cells compared to wild type BMDCs (all p<0.001). The increased MHC II expression in STAT1-deficient DC was further confirmed in host CD11b+ and CD11c+ cells following GVHD induction in vivo. To determine whether non-hematopoietic cells in STAT1−/− host contribute to the increased GVHD induction, we created radiation chimeras in which STAT1 was only deficient in the hematopoietic compartment by transplanting 129.STAT1−/− BMC into 129.STAT1+/+ recipients following lethal irradiation. 120 days later GVHD was induced using fully MHC-mismatched BALB/c donor splenocytes. Similar to STAT1-deficient recipients STAT1−/− ®WT chimeras showed enhanced GVHD induction compared to STAT1+/+®WT chimeras (MST 11 vs. 5, p<0.05). To determine the mechanism underlying the enhanced expansion of donor T cells in response to stimulation with STAT1-deficient APC, we hypothesized that STAT-deficiency may impair expression of the T cell inhibitory molecules Programed Cell Death-Ligand1 or-2 (PD-L1,-L2) on APC. We therefore studied the expression of PD-L1 and PD-L2 expression on wildtype and STAT1-deficient DC. Indeed, were able to demonstrate that absence of STAT1 significantly suppressed PD-L1 expression on BMDCs upon in vitro LPS stimulation (Mean Fluorescence Intensity 167.2± 15.9 vs. 532.5±7.6, p<0.001) and also in vivo tested on day+ 6 post-BMT in the mHA-mismatched setting. In line with these results using in vitro stimulation we could demonstrate significantly reduced Activation Induced Cell Death (AICD) in activated B6.SJL CD69+ CD4 and CD8 cells stimulated with 129.STAT1−/− BMDCs compared to cells stimulated with 129.STAT1+/+ BMDCs (10.6±1.5% vs. 28.2±1.9 % for CD4; 13.0±0.7% vs. 30.5±1.1% for CD8 respectively, p<0.001 for all). Importantly, blocking IFN-g with neutralizing antibodies significantly increased MHC class II, CD86 expression and reduced reduced PD-L1 expression on BMDCs upon LPS stimulation. In summary, our data suggest two mechanisms how the absence of STAT1 signaling in host hematopoietic cells may promote the development of GVHD: First, increased expression of MHC II and co-stimulatory molecule in STAT1-deficient APC may lead to enhanced activation and proliferation of donor lymphocytes. Second, absence of STAT1 in maturated host DC inhibits PD-L1 expression thus leading to reduced AICD of activated donor lymphocytes. These findings suggest that STAT1-signaling modulates host APC function and shapes the GVH-response by causing increased allo-antigen-specific donor T cell activation, survival and proliferation. Disclosures: Lentzsch: Centocor Ortho Biotech: Research Funding; Genzyme: Consultancy; Onyx: Consultancy; Celgene: Consultancy, Research Funding.

Engraftment of CLL-Derived T Cells in NSG Mice Is Feasible, Can Support CLL Cell Proliferation, and Eliminates the Need for Third Party Antigen Presenting Cells

Abstract 975 We recently described a xenograft model of chronic lymphocytic leukemia (CLL) using NOD/SCID/γcnull (NSG) mice. Adoptive transfer of primary patient PBMCs into these mice results in engraftment and proliferation of CLL cells if autologous activated T cells are present. To date, such CLL-derived T cell proliferation has been achieved by co-transfer of third party antigen presenting cells (APCs). Unfortunately, in this setting, mice succumb to graft-versus-host disease, due to complex interactions between CLL cells, alloAPCs, T cells, and the xenogeneic host. We hypothesized that alternative strategies of autologous T cell activation might refine the model, ultimately providing longer CLL cell engraftment and animal survival. We describe 3 approaches to achieving engraftment and activation of CLL-derived T cells that support B cell growth in vivo. First, we activated CLL CD3+ cells isolated from PBMCs of 4 patients with anti-CD3/28 beads for 72 hours in vitro. Cells were then mixed with CFSE-labeled PBMCs from the same patient at varying ratios (1:50 to 1:1000 CD3+ cells:CLL PBMCs) and injected intraorbitally (io) into a total of 17 mice. CD4, CD8 and CD19 cell engraftment, identified by a human CD45 lymphocyte gate (hCD45), and proliferation, assessed by CFSE dilution of labeled cells, were monitored weekly. All mice demonstrated detectable CD3+ and CD5+CD19+ cells from week (wk) 1. The percent (%) CD3+ cells, as a proportion of hCD45, increased weekly in all mice receiving anti-CD3/28-activated cells. While overall % CD5+CD19+ cells decreased weekly, the % proliferating increased and strongly correlated with increasing % of T cells (r2=0.7799, p<0.0001, 45 evaluable data pairs). At the time of reporting (up to 5 wks follow up), 4 animals have died. Death correlated with high % circulating CD8+ (mean 57.7% vs. 14.2% prior to death, p<0.0001), but not CD4+ cells (mean 31.0% vs 36.3%, p=n/s). Spleen immunohistochemistry analyses revealed follicles containing CD20+ CLL cells, based on L chain restriction and RT-PCR for the leukemic IGHV-D-J. Follicles were infiltrated with both CD4+ and CD8+ cells. These findings were the same as found with our published model. Our second approach involved engraftment of solely CLL-derived peripheral blood T cells, without associated autologous PBMCs or alloAPCs. Positively selected CD3+ populations from 2 patients were activated with anti-CD3/28 beads, expanded in vitro, and 5–10×106 such cells injected into mice io. Cells from both patients yielded engraftment of CD4+ and CD8+ cells by wk 2. We then utilized two mice engrafted with cells from one patient, 1 with 70% and 1 with 30% CD4+ cells to compare the effect of CD4+ T cells in CLL cell growth. Injection of CFSE-labeled PBMCs into the mouse with high % of CD4 cells showed a clear circulating CFSE+CD5+CD19+ population at wk 1. By wk 2, 95% of the leukemic B cells had divided with 60% showing ≥6 divisions. In the mouse with low % CD4, CD5+CD19+ cell engraftment was suboptimal, never being more than 100 detectable events in analysis from any single bleed. No clear CFSE dilution pattern was apparent. Finally, we have assessed the development of CLL-derived T cells from autologous bone marrow CD34+ cells of 4 CLL cases. 48hr old neonatal NSG mice received CD34+ cells (≤1×106) intrahepatically. In 1 of the 4 animals, CD19+ cells emerged at 2 months but by month 4 the predominant hCD45+ population was CD3+ (60% of total hCD45). Subsequent io injection of CFSE-labeled PBMCs from the same subject demonstrated engraftment and immediate proliferation (detected by wk1 following injection) of both the io transferred CD3+ and CD5+CD19+ populations. In conclusion, we demonstrate that engraftment and expansion of CLL-derived T cells is feasible in NSG mice both as in vitro activated cells or as descendants of CD34+ cells and that the presence of CLL cells and third party APCs is not required. Such T cells are able to support engraftment and proliferation of CLL B cells from the same CLL subject. Strategies to reduce excessive numbers of T-cell subsets which may prevent early death of animals are underway. Elimination of third party APCs will permit easier dissection of the pathophysiology within this model, allowing for a better understanding of CLL cell kinetics and mechanism of action of novel therapeutics to treat this disease. Disclosures: No relevant conflicts of interest to declare.