ALBERT

Description: Key Points CMV reactivation fundamentally resets posttransplant CD8 reconstitution, resulting in massive expansion of CMV-specific CD8 Tem. CMV reactivation is associated with defects in the underlying TCRβ immune repertoire.

Description: T stem cell memory (Tscm) represents a subset of T cells whose phenotype sits between those conventionally thought of as naive and those designated central memory. Functionally, they exhibit the capacity for self-renewal and can generate other memory cell subsets. As such, Tscm cells replenish and support the memory cell pool during infections, and contribute to immune reconstitution in patients following hematopoietic cell transplantation (HCT). Their fate and biological role in Graft-Versus-Host Disease (GVHD) is not yet understood. To address this question, we determined the dynamics and tissue-specific homeostasis of Tscm following allogeneic HCT in the translational non-human primate model of GVHD. In this study, we tracked Tscm cells by flow cytometry at baseline, and longitudinally after MHC haploidentical HCT. Recipients were divided into 2 large groups based on clinical outcome: "Primary GVHD": Animals receiving subtherapeutic GVHD prophylaxis (either no prophylaxis, or monotherapy with either Rapamycin or CTLA4-Ig; neither of which significantly prevented GVHD); "Breakthrough GVHD": Animals receiving combined Rapamycin/CTLA4-Ig or Tacrolimus/Metotrexate (both of which prevented primary GVHD but were eventually associated with breakthrough disease). We have previously shown that reconstituting T cells in animals with primary versus breakthrough GVHD display highly significant differences in their immunophenotype and transcriptome profiles. We have now discovered that primary GVHD is associated with a robust expansion of Tscm cells that peaks at day 5 post HCT (Fig 1A and B) and is associated with a decrease in the number of naive T lymphocytes. The Tscm compartment subsequently exhibited a contraction that accompanied an increase in the number of differentiated memory/effector T cell subsets (not shown). In contrast, animals that initially controlled GVHD maintained a delayed and prolonged expansion of Tscm cells (Fig 1A and B) that was associated with the significant preservation of naive T lymphocyte counts. Importantly, upon development of breakthrough GVHD, recipients demonstrated a transition of Tscm cells toward effector cells (Fig 1C). At the time of necropsy, animals with both primary and breakthrough GVHD displayed similar Tscm tissue distribution profiles, with these cells residing in the peripheral blood 〉 lymph nodes 〉 spleen 〉 bone marrow 〉 colon ~ liver ~ lungs. These data suggest that during GVHD, naive T cells transit to memory/effector lymphocytes via Tscm, and that one of the key functions of GVHD immunoprophylaxis may be to limit the rate at which naive T cells enter into the Tscm and subsequent effector T cell pool. Figure 1. T stem cell memory dynamics during acute GVHD. A-B) The frequencies (A) and absolute numbers (B) of CD8 T stem cell memory cells identified as CD3+CD14/20-CD8+CD45RA+CCR7+CD95+ lymphocytes and tracked longitudinally by flow cytometry following allogeneic HCT in primary (red circles) and breakthrough (black open circles) GVHD. Data display mean ± SEM. *p

Description: The PD-1/PD-L1 pathway plays an important role in regulation of alloimmune responses and in induction and maintenance of peripheral tolerance. Because GVHD is driven by donor T cells and PD-L1 expression can be markedly elevated on T cells during activation, we investigated the functional significance of PD-L1 expressed by donor T cells in regulating murine models of acute GVHD. PD-L1 expression was up-regulated on donor CD4 and CD8 T cells during GVHD. We considered the possibility that PD-L1 expression on activated donor T cells might inhibit GVHD by down regulating donor anti-host T cell responses, consistent with PD-L1 co-inhibitory activity when expressed on host parenchymal cells during GVHD. Surprisingly, T cell mediated GVHD lethality was markedly reduced in recipients of PD-L1-/- compared to WT donor T cells in both B6 to BALB/c model of GVHD(P

Description: Acute graft-versus-host disease (aGVHD) develops in more than half of patients after allogeneic hematopoietic cell transplantation (allo-HCT) despite poly-pharmacy immunoprophylaxis. Importantly, the dysregulated pathways responsible for this breakthrough disease remain largely unidentified. Thus, the discovery of these pathways represents one of the critical challenges for the field of allo-HCT. To address these needs, we have developed a model of aGVHD in rhesus macaques, which allows us to study the mechanisms of aGVHD both in its untreated state and in a variety of immunoprophylactic settings. Using a systems-based approach, we have created both a multiparameter flow cytometric and transcriptomic map of the immune landscape of aGVHD in allo-HCT recipients, in comparison to two critical control groups: (1) healthy untransplanted controls, and (2) those receiving autologous transplantation. We find that recipients of allo-HCT receiving 1) no immunoprophylaxis 2) monotherapy with CTLA4Ig or 3) monotherapy with sirolimus develop early fulminant aGVHD with multi-organ disease (Figure 1a-grouped as 'Primary GVHD'). The immunophenotype of T cells from the Primary GVHD cohort exhibits an effector/memory phenotype with robust proliferation and acquisition of cytotoxic function. Transcriptomic analysis reveals enrichment of Th1-associated transcripts (IL12RB2, CCR5, CXCR3) as well as programs of proliferation early in the post transplant period (Figure 1b). Flow cytometric data confirms an increase in the number of CD4 and CD8 T cells producing the Th1 cytokine, IFN-g at this time-point (Figure 1c). In contrast, standard-of-care Tacrolimus/Methotrexate (Tac/Mtx) as well as novel CTLA4Ig/sirolimus combination immunoprophylaxis (CoBS) both significantly improved survival of animals after allo-HCT. However, similar to human patients undergoing allo-HCT, these recipients often developed clinical signs of breakthrough aGVHD (starting around day 30 post-transplant) characterized by both gastrointestinal and skin pathology. This cohort was thus termed the "Breakthrough GVHD" cohort (Figure 1a). Unexpectedly, despite the presence of breakthrough clinical aGVHD, the Tac/Mtx and CoBS cohorts were still able to control programs of T cell proliferation, effector phenotype acquisition and Th1 cytokine skewing. However, both transcriptional and flow cytometric profiles demonstrated enrichment for molecules that reflect Th17/Th22 skewing (RORC, IL17A, AHR, and IL22) (Figure 2a) and production of IL17a (Figure 2b). These results suggest that while current methods of immunoprophylaxis are able to limit both T cell proliferation and Th1 polarization, breakthrough Th17/Th22 pathway activation occurs despite these therapies. These data suggest that emphasis should be placed on exploration of pharmacologic inhibitors of IL17/IL22 for the prevention/treatment of breakthrough aGVHD. Disclosures No relevant conflicts of interest to declare.

Description: The Notch signaling pathway is an evolutionarily conserved, cell-cell communication system with critical functions in organogenesis and tissue homeostasis, including hemato- and immuno-poiesis. Recent data have revealed important roles for Notch in the regulation of mature T cell differentiation and function. Studies in mouse models have identified Notch as a critical regulator of pathogenic T-cell responses during acute GVHD (aGVHD) (Zhang Y, 2011, Blood). However, the exact biological effects and the therapeutic potential of Notch pathway manipulation in clinical settings remains unclear. To address this question, we tested the activity of Notch pathway blockade in a non-human primate (NHP) aGVHD model, previously shown to exhibit donor T cell-intrinsic activation of the Notch pathway during aGVHD (Furlan SN, 2015, Sci Transl Med). To inhibit the Notch pathway, we used a blocking mAb to the Notch ligand DLL4, identified as the dominant ligand in a mouse aGVHD model (Tran, 2013, JCI; Chung, 2017, JCI). Prophylactic treatment regimens with either a single administration of anti-DLL4 mAb on day 0 (3 mg/kg), or with 3 doses (3 mg/kg each) on days 0, 7 and 14 significantly improved GVHD-free survival of allo-HCT recipients (median survival time (MST) = 26.5 days for the single dose regimen, and MST = 26 days for the triple dose regimen) in comparison with unprophylaxed controls (MST = 8 days, p

Description: As the field of regulatory T cell (Treg) adoptive therapy develops, questions concerning how to best to deploy these cells in patients must be addressed. Prominent among these questions is which adjunctive therapies will pair most synergistically with the transferred cells. Most ideally, a therapy that could prolong persistence and stabilize function of the cells is sought. Prior work has shown that mechanistic target of rapamycin (mTOR) inhibition can afford both stability and a degree of increased persistence to ex-vivo expanded thymic Tregs (tTregs) upon adoptive transfer. In this study, we investigated whether the addition of low-dose interleukin-2 (IL2) to the mTOR inhibitor rapamycin could impart additional persistence to ex-vivoexpanded tTregs after adoptive transfer. Using a non-human primate model of CFSE-labeled autologous tTregs, we found that the addition of IL2 to rapamycin supported a near 10-fold increase in the half-life of adoptively transferred tTregs, effectively doubling the cells in the tTreg compartment for the first month after adoptive transfer. Using a combination of single cell approaches, we were then able to show that transferred tTregs, in the setting of IL2 and rapamycin adjunctive therapy, retain high levels of Treg-specific genes, including FOXP3, after adoptive transfer. Additionally, we found that adoptively transferred tTregs are remarkably homogenous and become more transcriptionally similar to endogenous tTregs with time in vivo. Together these preclinical data support the use of combination IL2 and rapamycin as adjunctive therapy forex-vivo expanded adoptively transferred tTregs. Disclosures Tkachev: Regeneron Pharmaceuticals, Inc.: Research Funding. Blazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Kean:Regeneron Pharmaceuticals, Inc.: Research Funding.

Description: Key PointsThe transcriptional networks controlling breakthrough acute GVHD can be mapped, and correlate closely with clinical disease. Breakthrough acute GVHD is transcriptionally controlled by T-cell persistence, inflammation, and Th/Tc17 skewing.

Description: Graft-versus-host disease (GVHD) remains the major source of morbidity and transplant-related mortality after allogeneic hematopoietic cell transplant (alloHCT). Since the mid-1980s, the standard prophylaxis for GVHD has been a calcineurin inhibitor plus methotrexate (MTX). Subsequent clinical trials designed to intensify GVHD prophylaxis have shown an increased risk of relapse, thus negating survival benefit. The ideal GVHD prophylaxis regimen is therefore associated with a high graft-versus-host disease-free, relapse-free survival (GRFS), a composite endpoint that reflects survival without morbidity. A recent 3-arm randomized phase II study conducted through the Blood and Marrow Transplant Clinical Trials Network (BMT CTN 1203) showed a benefit in GRFS using post-transplant cyclophosphamide, tacrolimus, and mycophenolate mofetil (PTCy/Tac/MMF) with a hazard ratio of 0.72 (90% confidence interval 0.54 - 0.94, p-0.044) compared to contemporary non-randomized CIBMTR Tac/MTX controls. Based upon these results, the BMT CTN has launched 1703, a multicenter randomized phase III study of standard Tac/MTX vs PTCy/Tac/MMF in the setting of reduced intensity conditioning, matched peripheral blood stem cell transplantation (RIC PBSCT). The primary endpoint of 1703 is GRFS at 1 year, and several secondary endpoints, including patient-reported outcomes, will be analyzed. A series of recent, predominantly single-center studies have shown an association with the composition of intestinal microbiota and the development of acute GVHD, which suggests that this may be a potential modifiable biomarker of disease. The generalizability of findings from these studies is yet unknown, and no large multi-center studies on the intestinal microbiota of HCT patients has yet been carried out. Patients enrolled in 1703 will be eligible to co-enroll in BMT CTN 1801, the Mi-Immune study, detailing microbiota and immune reconstitution in this cohort. The primary endpoint of 1801 is 1-year non-relapse mortality by tertiles of stool microbial diversity at engraftment. Secondary objectives, including effect of GVHD prophylaxis on diversity, oligodomination and risk of bloodstream infection, volume of antimicrobial exposure, and T-cell receptor diversity, will be analyzed. The combined studies of BMT CTN 1703/1801 will define the standard of care for GVHD prophylaxis in the RIC PBSCT setting and vastly expand our knowledge on the impact of intestinal microbiota in critical outcomes after alloHCT. This clinical trial is registered at ClinicalTrials.gov NCT03959241. Figure Disclosures Holtan: Incyte: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; CSL Behring: Consultancy. Bhatt:ArcBio: Other: Scientific Advisory Board; January.ai: Other: Scientific Advisory Board; Caribou Bioscience: Other: Scientific Advisory Board; Kaleido Biosciences: Consultancy, Other: Paid Consultant; Janssen Human Microbiome Institute: Consultancy, Other: Paid Consultant; Illumina: Honoraria; 10x Genomics: Other: Research collaboration without funding support; Illumina: Other: Research collaboration without funding support; Agilent: Research Funding; Global Oncology, Inc: Other: Nonprofit Board. Perales:Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; MolMed: Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Medigene: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Kyte/Gilead: Research Funding; Miltenyi: Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Kymab: Consultancy; Jazz: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding. Hamadani:Medimmune: Consultancy, Research Funding; Takeda: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Merck: Research Funding; Celgene: Consultancy; Otsuka: Research Funding; ADC Therapeutics: Consultancy, Research Funding; Sanofi Genzyme: Research Funding, Speakers Bureau. Bolaños-Meade:Incyte Corporation: Other: DSMB fees.

Description: The advent ofadoptive T-cell therapy using CD19 Chimeric Antigen Receptor (CAR) T cells has revolutionized the treatment of relapsed and refractory acute lymphoblastic leukemia (ALL). CAR T cells have shown encouraging results in clinical trials, with complete remissions in 90% of patients with refractory B-cell ALL. However, CD19 CAR T cell therapy is associated with significant side effects, including cytokine release syndrome (CRS), encompassing fevers, myalgias, hypotension, respiratory distress, coagulopathy as well as neurologic toxicity, ranging from headaches to hallucinations, aphasia, seizures and fatal cerebral edema. Our understanding of CRS and neurologic toxicity has been significantly limited by the lack of animal models that faithfully recapitulate these symptoms. We chose the non-human primate (NHP), Macaca mulatta, given that it closely recapitulates the human immune system, to create an animal model of B-cell-directed CAR T cell therapy targeting CD20. Rhesus macaques (n=3) were treated with 30-40mg/kg cyclophosphamide followed 3-6 days later by an infusion of CAR T cells at a dose of 1x107 transduced cells/kg. Recipient animals were monitored for clinical signs and symptoms of CRS and neurotoxicity, and data were collected longitudinally to determine CAR T cell expansion and persistence, B cell aplasia, as well as clinical labs of CRS and cytokine levels. Prior to testing the CD20 CAR T cells, we performed a control experiment, in which 1x107/kg control T cells, transduced to express GFP only (without a CAR construct), were infused following cyclophosphamide conditioning. This infusion resulted in short-lived persistence of the adoptive cellular therapy, with disappearance of the cells from the peripheral blood by Day +14 (Figure 1, green traces) and no clinical signs of CRS (Figure 2) or neurologic toxicities. In contrast, recipients of 1x107 cells/kg CD20 CAR-expressing T cells (n = 3) demonstrated significant expansion of the CAR T cells, and persistence for as long as 43days post-infusion, which corresponded to concurrent B cell aplasia (Figure 1). These recipients also developed clinical signs and symptoms of CRS as well as neurologic toxicity which was manifested by behavioral abnormalities and extremity tremors, beginning between days 5 to 7 following CAR T cell infusion, with the onset of clinical symptoms coinciding with maximum CAR T cell expansion and activation. The neurologic symptoms were responsive to treatment with the anti-epileptic medicationlevetiracetam. The clinical syndrome was accompanied by elevations in CRP, Ferritin, LDH and serum cytokines, including IL-6, IL-8 and ITAC (Figure 2 A and B), recapitulating data from clinical trials using CD19 CAR T cells. An expansion of CD20 CAR T cells on day 7 following infusion was also observed in the CSF in the animals, and coincided with the onset of neurotoxicity. Strikingly, we also detected CD20 CAR T cells in multiple regions of the brain via flow cytometry, including the frontal, parietal, and occipital lobes, as well as the cerebellum, and demonstrated an increased number of infiltrating T cells by immunofluorescence in the brains of animals treated with CD20 CAR T cells when compared to healthy controls. These data demonstrate the successful establishment of a large animal model of B-cell directed CAR T cell therapy that recapitulates the most significant toxicities of CAR T cell therapy, including CRS and neurotoxicity. This model will permit a detailed interrogation of the mechanisms driving these toxicities as well as the pre-clinical evaluation of therapies designed to prevent or abort them after CAR T cell infusion. Figure 1. Absolute numbers of GFP T cell (n=1) and CD20 CAR T cell (n=3) expansion and persistence in rhesus macaques (top graph). Maximum CD20 CAR T cell expansion occurred between day 7 and day 8 following CAR T cell infusion. Absolute numbers of B cells in rhesus macaques following GFP T cell (n=1) and CD20 CAR T cell (n=3) infusion (bottom graph). Figure 1. Absolute numbers of GFP T cell (n=1) and CD20 CAR T cell (n=3) expansion and persistence in rhesus macaques (top graph). Maximum CD20 CAR T cell expansion occurred between day 7 and day 8 following CAR T cell infusion. Absolute numbers of B cells in rhesus macaques following GFP T cell (n=1) and CD20 CAR T cell (n=3) infusion (bottom graph). Figure 2. A. CRP, Ferritin and LDH levels were elevated following CD20 CAR T cell infusion, their peaks closely correlated with maximum CAR T cell expansion. No elevation of CRP, Ferritin or LDH was observed in Animal 1 which received GFP T cells. B. Elevations in IL-6, IL-8 and ITAC levels following CD20 CAR T cell infusion were highest surrounding the time of maximum CAR T cell expansion. Figure 2. A. CRP, Ferritin and LDH levels were elevated following CD20 CAR T cell infusion, their peaks closely correlated with maximum CAR T cell expansion. No elevation of CRP, Ferritin or LDH was observed in Animal 1 which received GFP T cells. B. Elevations in IL-6, IL-8 and ITAC levels following CD20 CAR T cell infusion were highest surrounding the time of maximum CAR T cell expansion. Disclosures Kean: Juno Therapeutics, Inc: Research Funding. Jensen:Juno Therapeutics, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: While calcineurin inhibition (CNI)-based strategies remain the mainstay for GVHD prevention, CNI are notoriously antagonistic to immune tolerance induction. Rapamycin (Rapa) has been shown to be more pro-tolerogenic; however, the best agents to combine with Rapa are still undetermined, and it remains a second-line GVHD prevention strategy without clear superiority over CNI. Finding tolerogenic partners for Rapa, therefore, represents a critical unmet need in the field. Of the possible partners for Rapa, the OX40/OX40L pathway represents an important target. OX40 is a costimulatory receptor expressed on activated human T cells, which, upon interaction with OX40L delivers activation signals to conventional T cells (Tconv) promoting their proliferation, survival and clonal expansion. Notably, these same OX40/OX40L signals may either inhibit or promote Treg functions, depending on context, suggesting that blockade of this pathway may simultaneously control Tconv activation while permitting Treg homeostasis. During GVHD in non-human primates (NHP), we found OX40L upregulation on myeloid dendritic cells and OX40 upregulation on activated T cells in recipients treated with multiple immunosuppressive agents, including Rapa (Fig 1). These data provided strong rationale for testing KY1005, a novel human monoclonal antibody that binds to OX40L and blocks its interaction with OX40, as a potential partner with Rapa. We tested the outcomes of prophylactic blockade of this pathway on NHP GVHD, using KY1005 alone and in combination with Rapa. These experiments utilized our previously published NHP GVHD model, in which GVHD is studied after T cell-replete haplo-identical HCT. KY1005 was dosed at 10mg/kg weekly from days -2ˆ+54 and Rapa was continued through Day +100. Prophylaxis with KY1005 alone provided initial evidence for its in vivo activity, with control of CD4〉CD8 T cell proliferation and mitigation of the expansion of CD4〉CD8 T effector/memory cells. Consistent with the partial control of T cell activation, these recipients demonstrated improved GVHD-free survival versus unprophylaxed controls, but disease ultimately broke through (Median Survival Time (MST) = 19.5 days with KY1005 (n=4) compared to 8 days in unprophylaxed recipients (n= 10, Fig 2)). We next investigated the impact of OX40L blockade + Rapa. We have published that Rapa as a monotherapy minimally controlled both immunologic and clinical disease, with an MST = 14 days (n=6). Combined prophylaxis was striking: recipients given KY1005+Rapa (n=5) maintained robust health throughout the entire experiment (MST 〉100d), and demonstrated high levels of donor T cell chimerism (86 +/- 3% at Day 100), rapid hematopoietic reconstitution, and had a terminal GVHD Grade of 0, compared to a Grade of III-IV in both KY1005- and Rapa-monotherapy cohorts. Immunologic analysis demonstrated synergistic control of both CD4 and CD8 T cell proliferation, restoring it to the level observed during autologous immune reconstitution, and resulting in a concomitant abrogation of CD4 and CD8 memory/effector expansion while preserving T cells with a na•ve phenotype. In striking contrast to the inhibition of Tconv activation by KY1005+Rapa, recipients of dual therapy demonstrated intact Treg reconstitution post-HCT, which resulted in a favorable Treg:Tconv ratio of 5.4 vs 1.4:100 in KY1005+Rapa treated compared to untreated recipients (p 〈 0.05). Transcriptomic analysis confirmed the unique immunologic state conferred by KY1005+Rapa on purified T cells, with gene arrays from these recipients demonstrating separation from all other transplant cohorts in Principal Component space (Figure 3A) and Class Neighbor Analysis identifying unique expression modules that tracked with KY1005 + Rapa prophylaxis (Figure 3B red and blue boxes). These results underscore the critical role of OX40/OX40L signaling in the development of GVHD and demonstrate the striking control of GVHD in KY1005+Rapa recipients. They represent the first demonstration of uniform, long-term GVHD-free survival in the primate model of high-risk haplo-identical HCT, and the first therapeutic strategy that simultaneously controls Tconv activation while supporting Treg homeostasis in this model. They suggest that OX40L blockade + Rapa is a novel, evidence-based combinatorial strategy to control GVHD that is an exceptional candidate regimen for clinical translation. Disclosures Tkachev: Kymab Ltd: Patents & Royalties: US Patent 9,382,325, Research Funding. Casson:Kymab Ltd: Employment. Kirby:Kymab Ltd: Employment, Patents & Royalties: US Patent 9,382,325. Bland-Ward:Kymab Ltd: Employment, Patents & Royalties: US Patent 9,382,325. Kean:Juno Therapeutics, Inc: Research Funding.