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Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8

Summary Repulsive guidance molecule A (RGMa) is a potent inhibitor of neuronal regeneration and a regulator of cell death, and it plays a role in multiple sclerosis (MS). In autopsy material from progressive MS patients, RGMa was found in active and chronic lesions, as well as in normal-appearing gray and white matter, and was expressed by cellular meningeal infiltrates. Levels of soluble RGMa in the cerebrospinal fluid were decreased in progressive MS patients successfully treated with intrathecal corticosteroid triamcinolone acetonide (TCA), showing functional improvements. In vitro, RGMa monoclonal antibodies (mAbs) reversed RGMa-mediated neurite outgrowth inhibition and chemorepulsion. In animal models of CNS damage and MS, RGMa antibody stimulated regeneration and remyelination of damaged nerve fibers, accelerated functional recovery, and protected the retinal nerve fiber layer as measured by clinically relevant optic coherence tomography. These data suggest that targeting RGMa is a promising strategy to improve functional recovery in MS patients.

Induction of mixed hematopoietic chimerism results in donor-specific immunological tolerance by apoptosis-mediated deletion of donor-reactive lymphocytes. A broad clinical application of this approach is currently hampered by limited predictability and toxicity of the available conditioning protocols. We developed a new therapeutic approach to induce mixed chimerism and tolerance by a direct pharmacological modulation of the intrinsic apoptosis pathway in peripheral T cells. The proapoptotic small-molecule Bcl-2 inhibitor ABT-737 promoted mixed chimerism induction and reversed the antitolerogenic effect of calcineurin inhibitors by boosting the critical role of the proapoptotic Bcl-2 factor Bim. A short conditioning protocol with ABT-737 in combination with costimulation blockade and low-dose cyclosporine A resulted in a complete deletion of peripheral donor-reactive lymphocytes and was sufficient to induce mixed chimerism and robust systemic tolerance across full major histocompatibility complex barriers, without myelosuppression and by using moderate doses of bone marrow cells. Thus, immunological tolerance can be achieved by direct modulation of the intrinsic apoptosis pathway in peripheral lymphocytes-a new approach to translate immunological tolerance into clinically applicable protocols.

Summary Apoptosis controls the adaptive immune system through regulation of central and peripheral lymphocyte deletion. Therefore, substances that selectively interact with the intrinsic apoptosis pathway in lymphocytes offer unexplored opportunities to pharmacologically modulate the immune response. Here, we present evidence that the BH3-mimetic ABT-737 suppresses allogeneic immune responses. In vitro, ABT-737 prevented allogeneic T-cell activation, proliferation, and cytotoxicity by apoptosis induction, but without impairing the physiological functions of remaining viable T cells. In vivo, ABT-737 was highly selective for lymphoid cells and inhibited allogeneic T- and B-cell responses after skin transplantation. The immunosuppressive effect of ABT-737 was markedly increased in combination with low-dose cyclosporine A, as shown by the induction of long-term skin graft survival without significant inflammatory infiltrates in 50% of the recipients in an MHC class I single antigen mismatched model. Thus, pharmacological targeting of Bcl-2 proteins represents a novel immunosuppressive approach to prevent rejection of solid organ allografts.

Cell therapies have demonstrated clinical benefit for some patients; however, in more resistant tumors, efficacy, persistence, and cell expansion may be improved with co-administration of an inflammatory cytokine such as interleukin 15 (IL-15). Clinical use of IL-15 has been limited due to systemic toxicity resulting in a narrow therapeutic index. Torque has advanced a “backpack” technique to attach IL-15 to the surface of T cells for adoptive transfer. IL-15 backpacks are ~120 nanometer particles consisting of multiple chemically crosslinked human IL-15/IL-15Rα/Fc heterodimers. Following crosslinker cleavage the IL-15 fusion proteins are released and drive T cell division in an autocrine fashion, providing a targeted, controllable and time-dependent immune stimulus which we term Deep Priming. Torque's lead program, TRQ15-01, consists of antigen-directed, autologous T cells carrying IL-15 backpacks. To explore the effects of IL-15 backpacks on T cells, we characterized backpack-carrying capacity of T cells, IL-15 heterodimer release kinetics, and T cell expansion by incubating IL-15 backpacks with human T cells activated by CD3/CD28 beads. Across 4 donors, T-cells were loaded in a reproducible manner proportional to the backpack labeling solution concentration. Backpacks promote the exponential expansion of activated T-cells for over one week through sustained release of IL-15 into the extracellular space. To explore the effect of IL-15 backpacks on CAR-T cell activity, IL-15 backpacks were loaded onto anti-EGFR CAR-expressing human CD3 cells. NSG mice bearing human H1299 tumor xenografts received CAR-T therapy with and without backpacks 7 days after tumor implantation. IL-15 backpacks significantly improved in vivo expansion of CAR-T cells (p < 0.008, at Day 10) and improved tumor growth inhibition (p < 0.003, 2-way ANOVA). To enable exploration of the clinical utility of backpacking using antigen-directed T-cells, we developed a novel, fully-closed, semi-automated cell manufacturing process with a yield of up to several billion antigen-directed cells. This process utilizes monocyte-derived dendritic cells pulsed with peptides from multiple tumor-associated antigens to expand cytotoxic T lymphocytes (CTLs). In a final step the antigen-directed CTLs are loaded with IL-15 backpacks to generate the TRQ15-01 cell product. Characterization of TRQ15-01 reveals controllable, dose-dependent loading of IL-15 backpacks on the antigen-directed T cells. Compared to non-backpacked antigen-directed cells, TRQ15-01 products displayed significantly higher expansion levels both in vitro (10-fold increase) and in NSG mice (20-fold increase). IL-15 backpack loading on human T cells is tunable and produces slow IL-15 release with sustained cell expansion and enhanced anti-tumor activity. Clinical trials with TRQ15-01 will be initiated in 2018. Citation Format: Pengpeng Cao, De-Kuan Chang, Andy Rakestraw, Amy Shaw, Ferdinando Pucci, Fabio Fachin, Christine McInnis, Shawn Carey, Austin Boesch, David Chirgwin, Carlos Tassa, Aaron Handler, Khristianna Jones, Philip D. Bardwell, Elena Geretti, Jon Nardozzi, Doug Jones, Jesse Lyons, Jonathan B. Fitzgerald, Becker Hewes, Ulrik B. Nielsen, Thomas Andresen. Application of deep IL-15 backpacks to human T cells demonstrates tunable loading with enhanced cell proliferation and antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3577.

BACKGROUND Studies of bone marrow transplantation (BMT) from wild-type mice or rats to alpha1,3-galactosyltransferase (GalT) knockout mice have demonstrated that induction of mixed chimerism tolerizes not only T cells, but also natural antibody-producing B cells, even across xenogeneic barriers. Given that rodent cells express lower levels of the alphaGal epitope than the more clinically relevant porcine species, the consequences of exposure to cells expressing high levels of alphaGal on the ability to induce B-cell tolerance are unknown. METHODS The effects on chimerism and anti-alphaGal B-cell tolerance of an i.p. injection of 10(9) porcine RBC were evaluated in GalT knockout mice receiving wild-type allogeneic BMT after non-myeloablative conditioning with T-cell depleting monoclonal antibodies, thymic irradiation, and low-dose total body irradiation. RESULTS Achievement of mixed chimerism and tolerance of anti-alphaGal-producing B cells was not affected by exposure to high-density alphaGal at the time of BMT. The absence of induced anti-alphaGal or anti-pig antibody responses in conditioned control mice suggested that the B-cell xeno-response to pig is T-cell-dependent. CONCLUSION High alphaGal density on pig cells might not preclude the ability to achieve tolerance of pre-existing alphaGal-reactive human B cells via induction of mixed chimerism. This strategy has the potential to induce B-cell tolerance to non-alphaGal epitopes, against which natural antibodies have been found in the sera of healthy humans.

Purpose of review Preformed and induced antibodies pose a major barrier to xenotransplantation. Recent experience involving allotransplantation in the presence of preformed anti-blood group antibodies and induced anti-human leukocyte antigen antibodies provides information that is relevant to tolerance induction in xenotransplantation. This review summarizes recent advances in our understanding of B cell tolerance in rodent models and patients. Recent findings In the setting of pediatric heart and liver transplantation, evidence has been obtained for tolerization of anti-blood group antigen natural antibody-producing cells. In animal models, tolerance of preexisting xenoantibody-secreting cells by solid organ transplantation with immunosuppression or through the establishment of mixed chimerism has been achieved. Advances in our basic understanding of B cell tolerance mechanisms and those applying to the various transplant settings are also described. Summary B cell tolerance is a critical goal in xenotransplantation. Strategies for tolerance induction developed in the animal models may be applied clinically as an understanding of their mechanisms emerges.

Presensitization to HLA antigens limits the success of organ transplantation. The achievement of donor-specific tolerance via mixed chimerism could improve outcomes of transplantation in presensitized patients. In presensitized B-cell-deficient μMT B6 mice, we developed nonmyeloablative bone marrow transplantation (BMT) regimens that successfully tolerized presensitized T cells, achieving long-term (LT) multilineage chimerism and tolerance to donor-type skin. To apply these regimens in wild-type (WT) animals while avoiding antibody-mediated destruction of donor bone marrow cells, presensitized WT B6 mice were rested >2 years to allow alloantibody clearance. However, chimerism and tolerance were not reliably achieved in LT presensitized WT B6 mice in which alloantibody had declined to minimal or undetectable levels before BMT. Strong antidonor memory T-cell responses were detected in LT presensitized WT B6 mice after rejection of donor bone marrow (BM) occurred, whereas levels of alloantibody remained consistently low. In contrast, presensitized μMT B6 mice had diminished memory T-cell responses compared to WT B6 mice. These data implicate T-cell memory, but not alloantibody, in rejection of donor BM in LT presensitized WT mice.

B-1b cells produce IgM natural antibodies against α1-3Galβ1-4GlcNAc (αGal). These can be tolerized by nonmyeloablative induction of mixed chimerism using αGal-positive (αGal+) donor marrow. We assessed the role of CR1/2 in this model for induction of tolerance of B-1b cells. Mixed hematopoietic chimerism was induced in α1-3galactosyltransferase (GalT−/−) and GalT−/−Cr2−/− mice with αGal+ BALB/c marrow donors. Anti-αGal Ab and anti-αGal Ab–producing B cells became undetectable in GalT−/− chimeras, whereas they persisted in chimeric GalT−/−Cr2−/− mice. To determine whether CR1/2 expression on stromal cells and/or hematopoietic cells was critical for B-1–cell tolerance, we generated GalT−/− radiation chimeras in which CR1/CR2 was expressed on either stromal cells, hematopoietic cells, neither, or both. After induction of mixed chimerism from αGal+ allogeneic bone marrow (BM) donors, anti-αGal–producing B cells were rendered tolerant in reconstituted recipients expressing only stromal CR1/CR2. Our results suggest a possible role for follicular dendritic cells that pick up immune complexes via CR1/CR2 receptors in the tolerization of B-1b cells.