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Inflammatory signals arising from the microenvironment have emerged as critical regulators of hematopoietic stem cell (HSC) function during diverse processes including embryonic development, infectious diseases, and myelosuppressive injuries caused by irradiation and chemotherapy. However, the contributions of cellular subsets within the microenvironment that elicit niche-driven inflammation remain poorly understood. Here, we identify endothelial cells as a crucial component in driving bone marrow (BM) inflammation and HSC dysfunction observed following myelosuppression. We demonstrate that sustained activation of endothelial MAPK causes NF-κB-dependent inflammatory stress response within the BM, leading to significant HSC dysfunction including loss of engraftment ability and a myeloid-biased output. These phenotypes are resolved upon inhibition of endothelial NF-κB signaling. We identify SCGF as a niche-derived factor that suppresses BM inflammation and enhances hematopoietic recovery following myelosuppression. Our findings demonstrate that chronic endothelial inflammation adversely impacts niche activity and HSC function which is reversible upon suppression of inflammation., Myelosuppressive injuries lead to chronic MAPK activation and impair blood reconstitution. Here, the authors show that chronic activation endothelial MAPK impairs hematopoietic stem cell (HSC) function through NFkB signaling, and that post-myelosuppressive HSC defects can be reversed by administration of Stem Cell Growth Factor SCGFa.

The thymus is not only extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood, and this capacity diminishes considerably with age. We show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration via their production of bone morphogenetic protein 4 (BMP4) ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signaling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance, and regeneration, and its downstream targets such as Dll4, a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.

FDA approval of chimeric antigen receptor T cells (CART cells) is the culmination of several decades of technology development and interrogation of the properties of these gene therapies. CART cells exist as personalized “living drugs” and have demonstrated astounding anti-tumor efficacy in patients with leukemia and lymphoma. However, the future promise of CART efficacy for solid tumors, the greatest unmet burden, is met with a number of challenges that must be surmounted for effective immune responses. In this review, we discuss the next-generation developments of CARs to target solid tumors, including fine-tuned and combinational-targeting receptors. We consider the structural intricacies of the CAR molecules that influence optimal signaling and CART survival, and review pre-clinical cell-intrinsic and cell-extrinsic combinational therapy approaches.

Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation. Key points Live imaging of endothelial to hematopoietic conversion identifies distinct subpopulations of hESC-derived hemogenic endothelium. Expression of the Notch ligand DII4 on vascular ECs drives induction of myeloid fate from hESC-derived hematopoietic progenitors.

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv–based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII + glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).

The recent efficacies demonstrated using Chimeric Antigen Receptor (CAR) mediated immunotherapy to treat hematological malignancies have been met with great enthusiasm as holding great potential for the eradication of hematological malignancies. Unfortunately, the ability to target metastatic solid tumors like prostate cancer with CAR T cells has been less successful. The major parameter to achieve in using CAR T cells to treat prostate cancer is overcoming the immunosuppression that is created by the tumors to inhibit CAR T cells. Our efforts have aimed to create PSMA specific CAR T cells that are resistant to the TGFBeta induced suppression extensively demonstrated to exist in prostate cancer. Upon binding PSMA, our second-generation CAR supplies 4-1BB and CD3zeta signaling previously demonstrated to allow for long-term T cell persistence and eradication of leukemia when targeted to CD19. We therefore created anti-PSMA CAR T Cells that coexpress the dominant negative TGFBeta receptor II (dnTGFRBetaII).Having focused on safety and efficacy of these anti-PSMABBz CAR T cells that express dnTGFRBetaII (dnTGFRBetaII-T2A-PBBZ), we demonstrate evidence of dnTGFRBetaII functionality. The dnTGFRBetaII functions to prevent SMAD signaling induced by TGFBeta, therefore resisting upregulation of CD25 and CTLA-4 by T cells. When co-cultured with tumor cells in vitro, efficient antigen specific lysis is induced by the PBBZ CAR and the dnTGFRBetaII-T2A-PBBZ CAR T cells exhibit up to 15 fold overall proliferation than PBBZ alone CAR T cells over 42 days. This allows for superior levels of T cell persistence in the peripheral blood of NSG mice after 3-4 weeks post infusion when compared to T cells expressing the anti-PSMA CAR alone. Most importantly, these CAR T cells are very effective at eradicating systemic PSMA+ PC3 prostate cancer cells in vivo demonstrated in two animal experiments at three different doses of CAR T cells. These studies suggest proper resistance to TGFBeta by CAR modified T cells that show great promise to eradicate metastatic prostate cancer in the clinic.Disclosure of Conflict of Interest:C.C.K. reports having ownership interest in patents owned by Memorial Sloan-Kettering Cancer Center and licensed to Juno Therapeutics and is a consultant for 121 Bio, LLC. J.L. reports no conflicts. C.H.J. reports receiving commercial research grants from Novartis and has ownership interest in patents owned by University of Pennsylvania and licensed to Novartis.Funding Acknowledgments:This study was supported by the Prostate Cancer Foundation All-Star Killer T-Cell Special Challenge Award 2014 - 2016.

T cell therapies utilizing engineered T cells show great promise for cancer immunotherapy, as illustrated by the CD19 paradigm. Much of the excitement about this approach, and second generation CARs in particular, is due to the dramatic clinical results recently reported by a few centers, especially in acute lymphoblastic leukemia, and the applicability of this approach, in principle, to a wide range of cancers. Extending the use of CAR therapies to cancers other than B cell malignancies will require selective tumor targeting with minimal or acceptable “on-target, off-tumor” effects. The identification of new CAR target antigens is thus one of the next big challenges to address. Recognizing the paucity of currently available tumor-specific targets, we have developed broadly applicable approaches to enhance the tumor selectivity and safety of engineered T cells. Here we review two promising concepts. One is to improve tumor targeting based on combinatorial antigen recognition. The other utilizes receptors that provide antigen-specific inhibition, which we named iCARs, to divert T cells from the normal tissues one wants to protect.

Progress in adoptive T-cell therapy for cancer and infectious diseases1–2 is hampered by the lack of readily available, antigen-specific, human T lymphocytes. Pluripotent stem cells could provide an unlimited source of T lymphocytes, but the therapeutic potential of human pluripotent stem cell–derived lymphoid cells generated to date remains uncertain3–6. Here we combine induced pluripotent stem cell (iPSC)7 and chimeric antigen receptor (CAR)8 technologies to generate human T cells targeted to CD19, an antigen expressed by malignant B cells, in tissue culture. These iPSC-derived, CAR-expressing T cells display a phenotype resembling that of innate γδ T cells. Similar to CAR-transduced, peripheral blood γδ T cells, the iPSC–derived T cells potently inhibit tumor growth in a xenograft model. This approach of generating therapeutic human T cells ‘in the dish’ may be useful for cancer immunotherapy and other medical applications.

Although many adults with B cell acute lymphoblastic leukemia (B-ALL) are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs) and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL.

Current T-cell engineering approaches redirect patient T cells to tumors by transducing them with antigen-specific T-cell receptors (TCRs) or chimeric antigen receptors (CARs) that target a single antigen. However, few truly tumor-specific antigens have been identified, and healthy tissues that express the targeted antigen may undergo T cell-mediated damage. Here we present a strategy to render T cells specific for a tumor in the absence of a truly tumor-restricted antigen. T cells are transduced with both a CAR that provides suboptimal activation upon binding of one antigen and a chimeric costimulatory receptor (CCR) that recognizes a second antigen. Using the prostate tumor antigens PSMA and PSCA, we show that co-transduced T cells destroy tumors that express both antigens but do not affect tumors expressing either antigen alone. This 'tumor-sensing' strategy may help broaden the applicability and avoid some of the side effects of targeted T-cell therapies.

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Moreover, despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. We have recently described a central role for group 3 innate lymphoid cells (ILC3) in a complex cellular and molecular network that drives endogenous thymic regeneration (Dudakov 2012 Science 336:91). Although IL-22 contributes considerably towards thymic regeneration and mice deficient for IL-22 lag behind WT controls in recovery of thymic function, there is still some tissue regeneration in these mice, suggesting that other regeneration pathways also play a role. Unlike other lymphoid cells, ILC3 were extremely radio-resistant with little if any depletion of cells after even lethal doses of total body irradiation (TBI). However, comprehensive analysis of all thymus-resident cell subsets revealed that ILCs were not the only damage-resistant population in the thymus as endothelial cells (ECs) were also remarkably resistant to damage in multiple clinically relevant models of acute tissue injury including corticosteroids, chemotherapy and sublethal total body irradiation (SL-TBI, 550cGy) (Fig. 1a). Thymopoiesis is dependent on the close interaction between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) and ECs. While the role of TECs has been well studied, the contribution of ECs to thymopoiesis and thymic regeneration remains largely unclear. Here we demonstrate that rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate thymic repair. Using a technique whereby ECs are transduced with the adenoviral gene E4ORF1 - ECs could be expanded ex vivo (exEC) and, when administered to mice after SL-TBI, significantly boost recovery of thymic function (Fig. 1b). Intriguingly, this trophic effect was only observed when exEC were derived from the thymus but not when they were derived from heart or kidney (Fig. 1b). Mechanistically, in vivo administration of exEC(Thy) induced the expression by TECs of Foxn1 (Fig. 1c), a key transcription factor required for thymus organogenesis, maintenance and regeneration. In vitro co-culture assays revealed that conditioned media (CM) from exEC derived from the thymus, but not the heart or the kidney, could induce Foxn1 expression by TECs (Fig. 1d), in addition to the FOXN1 target genes Kitl and Dll4; a Notch ligand itself critical for T cell development. These findings suggest that thymus-derived exEC produce a soluble factor that contributes toward thymic regeneration via activation of Foxn1. Transcriptome analysis of highly purified thymic ECs after SL-TBI identified that, among other things, expression of Bmp4 was significantly increased, offering a potential mechanism by which thymic ECs mediate their regeneration. Consistent with this hypothesis, not only could recombinant BMP4 promote the expression of Foxn1 by TECs in vitro, induction of Foxn1 by CM from exEC(Thy) was abrogated by Noggin, an inhibitor of BMP4 signaling (Fig. 1e). Moreover, exEC(Thy) produced significantly more BMP4 compared to exEC derived from the heart or kidney; and silencing Bmp4 expression by shRNA within exEC(Thy) limited their capacity to mediate exogenous thymic regeneration and failed to induce expression of Foxn1 and Dll4. Finally, strengthening its role in endogenous regeneration, administration of a pharmacologic BMP inhibitor inhibited thymic regeneration after SL-TBI; and inducible deletion of Bmp4 specifically in ECs reduced thymic regeneration after SL-TBI (Fig. 1f). These studies not only detail a novel pathway promoting endogenous thymic regeneration, but also offer an innovative clinical approach to enhance T cell immunity in recipients of allo-HSCT and for individuals with T cell deficiencies due to aging, infectious disease, and common cancer treatments such as chemo- and radiation-therapy. Disclosures Rafii: Angiocrine Bioscience: Other: Founder of Angiocrine Biosceince, which is developing the technology behind endothelial cell propagation. van den Brink:Boehringer Ingelheim: Consultancy, Other: Advisory board attendee; Regeneron: Honoraria; Merck: Honoraria; Tobira Therapeutics: Other: Advisory board attendee; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Generation of vascular endothelial cells (EC) from human embryonic stem cells (hESC) is a vital component of cell-based strategies for treatment of cardiovascular disease. Before hESC-derived ECs can be administered in therapeutic modalities, however, chemically defined culture conditions must be developed that reproducibly and robustly induce vascular differentiation. One approach to screening for culture conditions that support differentiation of hESCs to any cell type is their genetic modification with exogenous DNA sequence comprising a tissue-specific gene promoter driving reporters such as fluorescent protein or antibiotic drug resistance. The protocols herein provide instructions for the generation of clonal hESC lines containing a reporter transgene that is specifically expressed in vascular endothelial derivatives. Additionally, they demonstrate the methodology employed to assess vascular differentiation from clonal lines. Together, these protocols provide a solid foundation for study of vascular differentiation, and may also be applied, in principle, to studies of other specialized cell types derived from hESCs.

Mycobacterium avium subspecies paratuberculosis (MAP) is a significant concern to the American and European dairy industries and possibly to human health. MAP possesses the rare ability to survive and replicate in infected macrophages, cells that are typically able to destroy pathogens. Little is known about what changes occur in MAP-infected macrophages that prevent phagosome maturation and lead to intracellular survival of the bacteria. In this study, a bovine immunologically specific cDNA microarray was used to study genes whose expression was altered in monocyte-derived macrophages (MDM) when these cells were infected with 10 different strains of MAP bacteria. Although we used MAP strains isolated from four different host species, cluster analysis of each strains influence in infected MDMs showed no species of origin specific MAP alterations in the host transcriptome. However, MAP strain K10 was observed as a clear outlier in the cluster analysis. Additionally, we observed two SuperShedder MAP strains clustering very closely together compared to the other strains in this study. Overall, microarray analysis yielded 78 annotated genes whose expression was altered by MAP infection, regardless of strain. Few of these genes have been previously studied in the context of Johne’s disease or other mycobacterium-caused diseases. Large groups of apoptosis genes, transcription factors and cytokines were found to be differentially expressed in infected monocyte-derived macrophages as well as several genes not previously linked to MAP-host interactions. Identifying novel host genes affected by MAP infection of macrophages may lead to a more complete picture of this complex host^ pathogen interaction.

Thymopoiesis is a complex process involving crosstalk between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes thymic epithelial cells (TECs), fibroblasts and endothelial cells (ECs). Despite its importance, the thymus is exquisitely sensitive to cellular insults, including cytoreductive chemo- and radiation therapy required for successful hematopoietic stem cell transplantation; therefore identification of thymic repair mechanisms will offer promising therapeutic targets for immune regeneration. Recent studies in tissues such as liver, lung and bone marrow have revealed that ECs not only passively deliver oxygen and nutrients to tissues, but also actively produce distinct paracrine factors that can orchestrate their repair. The role of thymic ECs in thymopoiesis beyond their contribution of local circulation and the importation of lymphoid progenitors has not been comprehensively studied. In order to evaluate the role of ECs in thymic regeneration, we closely examined the kinetics of thymic recovery following total body irradiation (TBI, 550cGy) in young C57BL/6 mice. Although we observed a dramatic decline in total thymic cellularity, we found no significant change in the number of thymic ECs, suggesting they are extremely radio-resistant (Fig. 1A). Interestingly, although thymic ECs appeared to be resistant to the cytoreductive effects of TBI, we revealed an increase in their proliferation as measured by expression of Ki67 shortly after injury, indicating a role in the endogenous regeneration of the thymus (Fig. 1B). Given their radioresistance and cycling after TBI, we hypothesized that thymic ECs can provide instructive signals supporting thymic regeneration. To explore potential regenerative signals stemming from ECs during thymic regeneration, we performed a transcriptome analysis of highly purified ECs of untreated mice and at days 4 and 7 following TBI. Among the 288 genes that were altered in ECs after TBI (131 upregulated and 157 downregulated), we found significant upregulation in the expression of BMP4 (which has previously been implicated in thymic regeneration) and was validated using quantitative PCR (Fig 1C). In addition, we have developed a novel light sheet fluorescence microscopy approach to visualize the thymic vasculature in 3D at high resolution (Fig. 1D). Consistent with their potential role in aiding thymic regeneration, administration of ex vivo expanded thymic ECs (ex-EC) could significantly enhance thymic regeneration when given after TBI. Intriguingly, this regenerative effect of ex-ECs was tissue specific as ex-EC derived from heart or kidney did not exhibit a similar regenerative effect (Fig. 1E). Similar to our findings in the endogenous regeneration setting, we found that thymic ex-ECs demonstrated a significantly higher BMP4 expression compared to cardiac and kidney ex-ECs (Fig. 1F). Previous reports have suggested that BMP4 signaling is capable of directly regulating the key transcription factor of TEC function, FOXN1. We found that incubation of the TEC cell line C9 with thymic ex-EC conditioned medium induced an increase in FOXN1 expression but was abrogated in the presence of Noggin, a potent BMP signaling inhibitor (Fig. 1G). These findings support the hypothesis that BMP4 mediates the regenerative effect of ECs in thymic regeneration. In summary, we found that thymic ECs are capable of mediating endogenous thymic regeneration, likely via their expression of BMP4. Adoptive transfer of ex-ECs leads to enhanced thymic regeneration after immune injury. Thus, adoptive transfer of thymic ECs represents a novel strategy to improve immune function in immunocompromised patients. Figure 1 Figure 1. Disclosures Ginsberg: Angiocrine Bioscience: Employment. Rafii:Angiocrine Biosciences: Founder Other.

Understanding the intricate cellular components of the bone marrow microenvironment can lead to the discovery of novel extrinsic factors that are responsible for the initiation and progression of leukemic disease. We have shown that endothelial cells (ECs) provide a fertile niche that allows for the propagation of primitive and aggressive leukemic clones. Activation of the ECs by vascular endothelial growth factor (VEGF)-A provides cues that enable leukemic cells to proliferate at higher rates and also increases the adhesion of leukemia to ECs. Vascular endothelial growth factor A-activated ECs decrease the efficacy of chemotherapeutic agents to target leukemic cells. Inhibiting VEGF-dependent activation of ECs by blocking their signaling through VEGF receptor 2 increases the susceptibility of leukemic cells to chemotherapy. Therefore, the development of drugs that target the activation state of the vascular niche could prove to be an effective adjuvant therapy in combination with chemotherapeutic agents.

Abstract 2613 Recent work by our group and others demonstrates the therapeutic potential of CD19-targeted T cells to treat patients with indolent B cell malignancies. These studies make use of T cells that are genetically engineered with chimeric antigen receptors (CARs) comprising an scFv fused to various T cell activating elements. Whereas firs-generation CARs only direct T cell activation, second-generation CARs include two signal elements, such as CD3z and CD28 signaling domains (19–28z). We and our colleagues at MSKCC are currently evaluating the safety of 19–28z-transduced T cells in patients with acute leukemia (B-ALL) in a Phase I protocol (NCT01044069). Pre-clinical studies performed to date have mostly relied on xenogeneic models utilizing immunodeficient animals, which enable the evaluation of human engineered T cells but do not recapitulate all the interactions that may affect tumor eradication by CAR-modified T cells. We have therefore developed a pre-clinical immunocompetent mouse model of B-ALL, and addressed therein the impact of conditioning and T cell dose on the eradication of leukemia by syngeneic, CAR-targeted T cells. To establish an immunocompetent mouse model of B cell leukemia, we generated a clone from the lymph node of an Eμ-myc B6 transgenic mouse. The immunophenotype and gene-expression profile of clone Eμ-ALL01 is consistent with a progenitor B cell origin. Syngeneic B6 mice inoculated with this clone develop florid acute leukemia and die approximately 2–4 weeks after injection from progressive bone marrow infiltration. We created an anti-mouse CD19 CAR comprising all murine elements, including the CD8 signal peptide, a CD19-specific single chain variable fragment, the CD8 transmembrane region, and the CD28 and CD3z signaling domains. Transduction of the murine 19–28z CAR into mouse T cells was robust and successfully retargeted the T cells to B cells. In vitro assays demonstrated that m19–28 z transduced T cells mediated effective killing of CD19-expressing target cells and the production of effector cytokines such as IFNγ and TNFα. Cyclophosphamide either alone or in combination with control syngeneic T cells is insufficient to eradicate established Eμ-ALL01 in B6 mice. However, treatment with cyclophosphamide and m19–28z-transduced T cells cured nearly all mice. Mice sacrificed six months after treatment exhibited a dramatic reduction of B cells in the bone marrow (BM), blood, and spleen. The few remaining B lineage cells found in the BM had a phenotype consistent with early pro-B cells, suggesting that endogenous reconstitution of the B cell compartment was thwarted by persisting, functional m19–28z+ T cells. Thus, T cells are retained at the site of antigen expression, which is maintained through regeneration of progenitor B cells. The persisting CD19-targeted T cells in the BM exhibited a cell surface phenotype consistent with effector and central memory cells. Using B cell aplasia as a surrogate endpoint for assessing in vivo T cell function and persistence, we evaluated how conditioning chemotherapy and T cell dose determine the level of B cell depletion induced by adoptively transferred CD19-targeted T cells. Overall, increasing the cyclophosphamide or T cell dose, increased the degree and duration of B cell depletion and the number of persisting CAR-modified T cells. Significantly, increasing the T cell dose at a set cyclophosphamide level had a lesser impact than increasing the conditioning intensity for a given T cell dose. In summary, the new Eμ-ALL01 syngeneic, immunocompetent B-ALL model we describe here is a valuable tool for modeling CD19 CAR therapies. Our results indicate that m19–28z transduced T cells are effective at eradicating B-ALL tumor cells and persist long-term, preferentially in bone marrow. Our findings further establish that conditioning intensity and T cell dose directly determine B cell elimination and long-term T cell persistence. These studies in mice will serve as an important framework to further model and perfect our studies in patients with B-ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 451 Chimeric antigen receptors (CAR) represent a potent strategy to target T cells against selected tumor antigens. Ongoing clinical trials indicate that autologous T cells expressing CARs targeting CD19, a B cell-associated antigen, can induce complete remission and B cell aplasia in patients with B cell malignancies. Donor CD19-CAR+ T cells could potentially be used to treat recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the risk of alloreactivity mediated by endogenous T cell receptors (TCR) triggering an acute GVHD is not known. This is partly due to the absence of in vivo models to study the relative effects of CAR and endogenous TCR signaling. For the first time, we have evaluated the relative effects of CD19-targeted donor T cells on the elimination of CD19+ B cells and endogenous TCR-mediated alloreactivity in mouse models of allo-HSCT. We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: as a control, CD19-delta, a tail-less CAR lacking the CD3ζ signaling domain; CD19z1, which signals through its CD3ζ endodomain; and CD19-28z, which signals through CD28 and CD3ζ (Figure 1A). CD19z1+ and CD19-28z+ T cells mediated specific lysis of CD19-expressing tumors in vitro, while CD19-delta+ T cells did not. In order to assess the anti-tumor capacity of CD19-CAR+ T cells in vivo, we transferred the transduced B6 donor T cells into lethally irradiated BALB/c recipients that were administered T cell-depleted allografts and CD19+ lymphoma A20-TGL (B6–> BALB/c+A20-TGL). CD19-CAR+ T cells (CD19z1 and CD19-28z) mediated clearance of A20 tumor cells visualized by in vivo imaging of luciferase-expressing tumor cells (Figure 1B and data not shown) and significantly improved tumor free survival. CD19-CAR+ B6 T cells could sustain prolonged B cell hypoplasia when adoptively transferred into lethally irradiated haploidentical CBF1 recipients of T cell-depleted allografts (B6–> CBF1, Figure 1C). These data indicate that under alloreactive conditions, donor CD19-CAR+ T cell signaled through the CAR leading to specific elimination of CD19+ tumors and B lineage cells. In order to determine the risk of GVHD, we transferred the donor CD19-CAR+ T cells into haploidentical HSCT recipients. Interestingly, CD19-CAR+ T cells mediated significantly less acute GVHD, resulting in improved survival and lower GVHD scores (Figure 1D). Donor CD19-delta+ T cells however mediated lethal GVHD, indicating that the endogenous TCR mediated strong alloreactivity in the absence of CAR signaling. Similar results were obtained from experiments using MHC-mismatched (B6–> BALB/c) models. It is known that signaling through endogenous TCR is accompanied by down-regulation of surface TCR expression. We found significant decreases in surface CD3ϵ, TCRβ and CD90 expressions in donor CD19-delta+ T cells under alloreactive conditions. In contrast, donor CD1928z+ T cells failed to down-regulate surface TCR expression under similar conditions, suggesting that endogenous TCR function was altered in CAR-activated T cells. In the context of allo-HSCT, preferential CAR signaling at the expense of alloreactive endogenous TCR signaling may thus lead to reduced alloreactivity and attenuation of GVHD. These results provide the first pre-clinical evidence suggesting that CAR-modified, unselected donor T cells may be safely applied in an allogeneic context. Disclosures: No relevant conflicts of interest to declare.

SummaryT cell engineering is a powerful means to rapidly generate anti-tumor T cells. The costimulatory properties of second-generation chimeric antigen receptors (CARs) determine the overall potency of adoptively transferred T cells. Using an in vivo “stress test” to challenge CD19-targeted T cells, we studied the functionality and persistence imparted by seven different CAR structures providing CD28 and/or 4-1BB costimulation. One configuration, which uses two signaling domains (CD28 and CD3ζ) and the 4-1BB ligand, provided the highest therapeutic efficacy, showing balanced tumoricidal function and increased T cell persistence accompanied by an elevated CD8/CD4 ratio and decreased exhaustion. Remarkably, induction of the IRF7/IFNβ pathway was required for optimal anti-tumor activity. Thus, 1928z-41BBL T cells possess strikingly potent intrinsic and immunomodulatory qualities.