ALBERT

Description: Abstract 2998 Introduction: GvHD remains the most deadly complication of HSCT despite current prevention strategies. To address the unmet need for better GvHD control, we have created a non-human primate (NHP) model with which to rigorously test mechanism and efficacy of novel therapeutics. In this study, we determined whether a novel combination of mTOR inhibition (with sirolimus) and CD28:CD80/86 costimulation blockade (with belatacept) could control GvHD. Here we show for the first time that these two agents combine synergistically to prevent both the clinical and immunologic manifestations of primate aGvHD. Methods: Rhesus macaque recipients were irradiated (9.6 Gy in 2 fractions at 7cGy/min), and then transplanted with G-CSF-mobilized PBSC from a haplo-identical donor (1–5×108 TNC/kg). Recipients were treated with either sirolimus alone (n = 4, troughs targeted at 5–10 ng/mL), belatacept alone (receiving weekly doses of 20 mg/kg), or combination therapy. Clinical GvHD was monitored using our previously described NHP grading scale (Miller et al., Blood 2010), and multiparameter flow cytometric analysis was performed. Results: Untreated controls (n = 5) developed rapid, severe histopathologically-proven aGvHD and succumbed rapidly (MST = 7 days). Recipients treated with either sirolimus or belatacept alone were partially protected from the clinical manifestations of GvHD. Sirolimus-treated recipients (n = 6) developed predominantly GI disease (with diarrhea but no elevation of bilirubin) and had an MST of 14 days (Figure 1). Recipients treated with belatacept alone (n = 3) developed primarily liver aGvHD (bilirubin rapidly rising to 6–30 × normal with histologically-confirmed lymphocytic infiltration) and an MST of 11 days. In striking contrast, recipients treated with combined sirolimus + belatacept (n = 5) demonstrated neither uncontrolled diarrhea nor hyperbilirubinemia at the timed terminal analysis (1 month post-transplant). We employed multiparameter flow cytometry to determine the immunologic consequences of sirolimus and belatacept on T cell proliferation (using Ki-67 expression) and cytotoxity (using granzyme B expression). We found that the clinical synergy observed with combined therapy was recapitulated immunologically. Thus, while untreated aGvHD was associated with rampant CD8+ proliferation (with 83 +/− 14% Ki-67+ CD8+ vs 4.7 +/− 0.6% pre-transplant), sirolimus or belatacept as monotherapy both partially controlled proliferation (35 +/− 3% and 65 +/− 23% Ki-67+ CD8+ with sirolimus or belatacept, respectively). Combined sirolimus + belatacept dramatically reduced proliferation (to 8 +/− 3%, favorably comparing with 13% Ki-67+ CD8+ T cells using standard Calcineurin Inhibitor/Methotrexate (CNI/MTX) prophylaxis). Sirolimus and belatacept both also partially controlled GvHD-related T cell cytotoxicity. Thus, while untreated aGvHD was associated with excessive granzyme B expression in CD8+ T cells (82 +/− 2% granzyme Bvery high CD8+ cells vs 0.3 +/− 0.2% pre-transplant) sirolimus or belatacept monotherapy also partially controlled cytotoxicity (8 +/− 1% and 35 +/− 1% granzyme Bvery high with sirolimus or belatacept, respectively). Combination therapy dramatically reduced the proportion of these cells, to 1.5 +/− 0.8 % granzyme Bvery high, favorably comparing with 4% granzyme Bvery high using CNI/MTX. The ability of sirolimus, belatacept, or the combination to control Ki-67 and Granzyme B expression closely correlated with survival (Figure 2A, B) supporting a pathogenic role for these highly proliferative and cytotoxic cells in aGvHD pathology. Moreover, significant co-expression of granzyme B in the Ki-67+ cells was observed (Figure 2C) suggesting that dual-positive Ki-67/Granzyme B cells may mark a pathogenic population, amenable to tracking in the peripheral blood. Implications: These results reveal a previously undiscovered synergy between sirolimus and belatacept in the control of primate aGvHD, and provide support for future clinical investigation of this novel prevention strategy. They also identify CD8+/Ki-67+/Granzyme Bvery high dual-positive T cells as a potentially sensitive biomarker of GvHD pathogenesis, amenable to monitoring in either the blood or in GvHD target organs. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2550 We have developed a novel, MHC-defined rhesus macaque model of total body irradiation-based haploidentical hematopoietic stem cell transplantation. This model has permitted us, for the first time, to perform a rigorous study of the cellular and molecular basis of uncontrolled primate GvHD, and to evaluate the efficacy of a novel, clinically-relevant T cell costimulation blockade-based immunosuppressive regimen to control this disease. We have found that after unprophylaxed haploidentical transplant, severe GvHD developed, which was characterized by rapid clinical decline, and widespread T-cell infiltration and organ damage, with histopathologic evidence of disease in the lungs, the liver, and the GI tract. Mechanistic analysis revealed activation as well as possible counter-regulation, with rapid, CD8-predominant T-cell expansion and accumulation of both CD8+ and CD4+ granzyme B+ effector cells as well as FoxP3pos/CD27high/CD25pos/CD127low CD4+ T-cells. In addition, CD8+ cells downregulated CD127 and BCl-2 and upregulated Ki-67, consistent with a highly activated, proliferative profile. A cytokine storm also occurred, with GvHD-specific secretion of IL-1Ra, IL-18, and CCL4. The combination of CD40/CD28 costimulation blockade (using a monoclonal antibody against CD40 and the CTLA4Ig fusion protein) and mTOR inhibition with sirolimus (Costimulation Blockade and Sirolimus, “CoBS”) resulted in striking protection against GvHD. Thus, at the 30-day primary end-point, CoBS-treated recipients demonstrated 100% survival compared to no survival in untreated recipients. Long-term analysis revealed that CoBS treatment resulted in mean survival increasing from 11.6 to 62 days (p

Description: Abstract 2549 Introduction: Given the emerging importance of sirolimus as a therapuetic for graft-versus host disease (GvHD), it is critical to rigorously define the mechanisms by which this agent impacts T cell immunity after hematopoietic stem cell transplantation (HSCT). Therefore, we have used our novel rhesus macaque model of haploidentical HSCT and GVHD to probe the mechanisms of sirolimus-mediated GvHD prevention when given as a monotherapy. The insights gained from this study will facilitate the rational design of sirolimus-containing combinatorial therapies to maximize immunosuppressive efficacy. Methods: Transplant recipients were prepared with 8Gy total body irradiation and were then infused with MHC-mismatched donor leukopheresis products(n=3, avg. 6.5×108 TNC/kg, 3.4×107 total T cells/kg). Recipients received sirolimus monotherapy (serum troughs 5–15 ng/mL) alone as post-transplant immunosuppresson. Clinical GvHD was monitored according to our standard primate GvHD scoring system and flow cytometric analysis was performed to determine the immune phenotype of sirolimus-treated recipients compared to a cohort of recipients (n= 3) that were given no GvHD immunoprophylaxis. Results: Sirolimus modestly prolonged survival after MHC-mismatched HSCT compared to no immunosuppression (〉19 days versus 6.5 days in the untreated cohort, with GvHD confirmed histopathologically at the time of necropsy). We found that sirolimus significantly inhibited lymphocyte proliferation in transplant recipients: The ALC remained suppressed post-transplant (eg ALC of 0.46 × 106/mL on day 15 post-transplant versus 4.3 × 106/mL pre-transplant, with recovery of other leukocytes: WBC=5.1 × 106/mL, ANC=2.6 × 106/mL). These results suggest that sirolimus can have a profound impact on lymphocyte proliferation, inhibiting GvHD-associated lymphocyte expansion by as much as 200–300-fold compared to untreated controls. Sirolimus had a similar impact on CD4+ and CD8+ subpopulation expansion. Thus, while CD4+ T cells and CD8+ T cells expanded by as much as 300-fold and 2000-fold, respectively, without sirolimus, the expansion of these cells was significantly blunted with sirolimus, with maximal expansion of CD4+ and CD8+ T cells being 4- and 3.6-fold, respectively compared to the post-transplant nadir. Sirolimus-treated recipients also better controlled the upregulation of the proliferation marker Ki-67 on CD4+ or CD8+ T cells. Thus, while untreated recipients upregulated Ki-67 expression by as much as 10-fold after engraftment, (with 〉80-98% T cells expressing high levels of Ki-67 post-transplant versus 5–10% pre-transplant) sirolimus-treated recipients better controlled Ki-67 expression (17-40% Ki-67-high CD4+ and CD8+ T cells post-transplant). While the impact of sirolimus on T cell proliferation was profound, it failed to completely inhibit activation of T cells, as measured by both Granzyme B and CD127 expression. Thus, when effector CD4+ and CD8+ T cell cytotoxic potential was measured by determining expression levels of granzyme B, we found that sirolimus could not downregulate this key component of immune function and GvHD-mediated target organ damage: Granzyme B expression in both CD4+ and CD8+ CD28-/CD95+ effector T cells was unchanged despite sirolimus monotherapy. Down-regulation of CD127 expression, which identifies activated CD8+ T cells in both humans and rhesus macaques, also demonstrated resistance to sirolimus treatment. Thus, while a cohort of recipients that were treated with combined costimulation blockade and sirolimus maintained stable CD127 levels post-transplant, and untreated animals demonstrated total loss of CD127, up to 60% of CD8+ T cells in sirolimus-treated recipients down-regulated CD127, consistent with breakthrough activation of these cells despite mTOR inhibition. Discussion: These results indicate that while the predominant effect of sirolimus during GvHD prophylaxis is its striking ability to inhibit T cell proliferation, sirolimus-based immunosuppression spares some cellular signaling pathways which control T cell activation. These results imply that therapies that are combined with sirolimus during multimodal GvHD prophylaxis should be directed at inhibiting T cell activation rather than proliferation, in order to target non-redundant pathways of alloimmune activation during GvHD control. Disclosures: No relevant conflicts of interest to declare.

Description: We have developed a major histocompatibility complex–defined primate model of graft-versus-host disease (GVHD) and have determined the effect that CD28/CD40-directed costimulation blockade and sirolimus have on this disease. Severe GVHD developed after haploidentical transplantation without prophylaxis, characterized by rapid clinical decline and widespread T-cell infiltration and organ damage. Mechanistic analysis showed activation and possible counter-regulation, with rapid T-cell expansion and accumulation of CD8+ and CD4+ granzyme B+ effector cells and FoxP3pos/CD27high/CD25pos/CD127low CD4+ T cells. CD8+ cells down-regulated CD127 and BCl-2 and up-regulated Ki-67, consistent with a highly activated, proliferative profile. A cytokine storm also occurred, with GVHD-specific secretion of interleukin-1 receptor antagonist (IL-1Ra), IL-18, and CCL4. Costimulation Blockade and Sirolimus (CoBS) resulted in striking protection against GVHD. At the 30-day primary endpoint, CoBS-treated recipients showed 100% survival compared with no survival in untreated recipients. CoBS treatment resulted in survival, increasing from 11.6 to 62 days (P 〈 .01) with blunting of T-cell expansion and activation. Some CoBS-treated animals did eventually develop GVHD, with both clinical and histopathologic evidence of smoldering disease. The reservoir of CoBS-resistant breakthrough immune activation included secretion of interferon-γ, IL-2, monocyte chemotactic protein-1, and IL-12/IL-23 and proliferation of cytotoxic T-lymphocyte–associated antigen 4 immunoglobulin-resistant CD28− CD8+ T cells, suggesting adjuvant treatments targeting this subpopulation will be needed for full disease control.

Description: A strategy for producing high-level hematopoietic chimerism after non-myeloablative conditioning has been established in the rhesus macaque. This strategy relies on hematopoietic stem cell transplantation after induction with a non-myeloablative dose of busulfan and blockade of the IL2-receptor in the setting of mTOR inhibition with sirolimus and combined CD28/CD154 costimulation blockade. Hematopoietic stem cells derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level chimerism. When transplants were performed between animals that were totally unmatched at the MHC, mean peripheral blood peak donor chimerism was 81% with a median chimerism duration of 145 days. Additional immune modulation strategies, such as pre-transplant CD8 depletion, donor-specific transfusion, recipient thymectomy or peritransplant deoxyspergualin treatment did not improve the level or durability of chimerism in the setting of these MHC-unmatched transplants. Recipient immunologic assessment suggested that chimerism occurred amidst donor-specific down-regulation of alloreactive T cells, and the reappearance of vigorous T-mediated alloreactivity accompanied rejection of the transplants. Furthermore, viral reactivation constituted a significant transplant-related toxicity and may have negatively impacted the ability to achieve indefinite survival of transplanted stem cells. In order to address the dual complications of lack of indefinite chimerism and viral reactivation, we sought to increase the level of MHC matching between donor and recipient transplant pairs. This necessitated a large-scale analysis of an NIH-sponsored rhesus macaque colony in order to determine the familial relationships and MHC-disparity between potential transplant pairs. We have analyzed over 500 animals from this colony, which has resulted in the first primate transplant series between donors and recipients with known degrees of MHC disparity. Our results suggest that viral reactivation is vastly improved when transplant occurs with even one shared MHC haplotype, but that the current immunomodulation strategy is insufficient to achieve immune tolerance in these haplo-identical transplants. However, we have observed long-term mixed chimerism in fully MHC matched transplant pairs that is stable even years after withdrawl of costimulation blockade-based immunomodulation. These results suggest that costimulation blockade may be an important addition to current immunomodulation strategies for producing stable engraftment after nonmyeloablative pre-transplant preparation, with potential application to non-malignant hematologic diseases and genetic disorders.

Description: Abstract 1888 Introduction: There is a critical unmet need to devise effective strategies to prevent GvHD. However, the best combinatorial therapies remain undetermined, and the identification of new targeted approaches to GvHD prevention remains a challenge. To address this, we have developed a genome-wide approach to studying GvHD, using whole-transcriptome analysis of pathogenic T cells in a clinically-relevant non-human primate (NHP) model. Using computational approaches, we have identified, for the first time, the transcriptional networks that drive primate GvHD, and that lead to its partial control with sirolimus. Methods: CD3+/CD20- T cells were purified flow cytometrically from 4 cohorts: (1) Healthy Controls (“HC” n = 15); (2) Recipients of an autologous HSCT (“Auto” n = 3); (3) Haplo-identical allogeneic HSCT recipients without GvHD prophylaxis, who developed histopathologically confirmed severe aGvHD (“GvHD” n = 4); and (4) Allo-HSCT recipients who received sirolimus alone, and were partially protected from aGvHD (“Sirolimus” n = 4). Purification of T cells after allo-HSCT occurred 1–2 weeks post-transplant. RNA was purified (Qiagen), and rhesus macaque-specific Affymetrix Gene Arrays were performed. Computation: Gene array signals were processed and normalized using the Robust Multichip Averaging Method and ComBat. Principal Component Analysis (PCA) was applied to summarize modes of gene array variance. Importantly, PCA revealed that variation was primarily determined by the experimental cohort (Figure 1). This result was critical, and confirmed that transcriptomics could be applied to identify genes and pathways controlling GvHD. Differentially expressed genes (“DE”, fold change 〉 2) were defined between cohorts, yielding unique and overlapping gene signatures. We found that 775 annotated genes were DE between GvHD and HC and 286 were DE between Sirolimus and HC (Figure 2A, B). Importantly, a subset of the GvHD and Sirolimus DE gene sets were overlapping, indicating incomplete control of T cell activation with sirolimus (Figure 2B), and identifying pathways that could be targeted in combination with sirolimus for improved GvHD control. To further define genes by their individual expression profiles using an unbiased approach, we applied Class Neighbor Analysis (GenePattern, Figure 3A). Finally, using Ingenuity Pathway Analysis (IPA) we characterized gene signatures according to molecular pathways (using right-tailed Fisher's Exact test and FDR correction, Figure 3B). Results: T cells from animals with severe aGvHD demonstrated transcriptional signs of rampant proliferation and cytotoxicity as well as potentially counter-regulatory cell death pathways. IPA identified highly statistically significant upregulation of Cell Cycle and Cellular Movement networks (Figure 3B, p〈 0.001) as well as Cell Trafficking and Inflammatory Response Networks (Figure 3B, p 〈 0.001). These networks contained some expected genes and some surprises. Thus, as previously documented, GvHD was associated with upregulation of JAK and IFN signaling (p 〈 0.001). Unexpectedly, GvHD was also associated with upregulation of the Sonic Hedgehog and Aurora Kinase A Pathways (p 〈 0.01). Both of these represent targetable pathways for which novel therapeutics are currently available. Sirolimus resulted in significantly different gene expression patterns compared to uncontrolled GvHD. This included partial downregulation of the proliferation marker Ki-67 and the cytotoxicity gene, Granzyme B. However, there were many genes, pathways and networks that were shared between the Sirolimus and GvHD cohorts. These prominently included upregulation of the FOXM1 and IRF8 transcription factors, involved in cell cycle progression (p