Although there is evidence linking hematopoietic chimerism induction and solid organ transplant tolerance, the mechanistic requirements for chimerism-induced tolerance are not clearly elucidated. To address this, we used an MHC-defined primate model to determine the impact of impermanent, T cell-poor, mixed-chimerism on renal allograft survival. We compared two cohorts: one receiving a bone marrow and renal transplant ("BMT/renal") and one receiving only a renal transplant. Both cohorts received maintenance immunosuppression with CD28/CD40-directed costimulation blockade and sirolimus. As previously demonstrated, this transplant strategy consistently induced compartmentalized donor chimerism, (significant whole-blood chimerism, lacking T cell chimerism). This chimerism was not sufficient to prolong renal allograft acceptance: the BMT/renal mean survival time (MST, 76 days) was not significantly different than the renal transplant alone MST (85 days, p = 0.46), with histopathology documenting T cell mediated rejection. Flow cytometric analysis revealed significant enrichment for CD28-/CD95+ CD4+ and CD8+ Tem cells in the rejected kidney, suggesting a link between CD28-negative Tem and costimulation blockade-resistant rejection. These results suggest that in some settings, transient T cell-poor chimerism is not sufficient to induce tolerance to a concurrently placed renal allograft and that the presence of this chimerism per se is not an independent biomarker to identify tolerance.

Abstract Abstract 1054 While hematopoietic stem cell transplantation (HSCT) is a curative therapy for individuals with sickle cell disease (SCD), these patients are at increased risk of graft rejection, especially after non-myeloablative HSCT. This suggests that SCD patients may exhibit immune activation at baseline. However, a rigorous analysis of the extent and character of this immune activation, using newly available multiplexed flow cytometric techniques, has not been previously reported. The objective of this study was to describe the extent of immune deviation in a cohort of pediatric subjects with SCD during steady state, given that this age group represents the majority of SCD patients undergoing HSCT. Methods: An IRB-approved prospective cross-sectional study design was used to compare patients, aged 10 –16y with SCD (homozygous SS or Sb0-thalassemia) during steady state (at least 21 days from an acute SCD exacerbation or other illness including infections requiring antibiotics, and, at least 8 weeks from any RBC transfusion) with an ethnic and age-matched control group of healthy individuals without SCD. Patients were recruited from the Aflac Comprehensive Sickle Cell Clinic at Children's Healthcare of Atlanta. Controls were recruited using fliers from general pediatric clinics and other community centers in the metro Atlanta area. Inclusion criteria included a confirmed documented diagnosis of homozygous SS or sickle b0 thalassemia for cases and the absence of any sickle hemoglobinopathy for controls. Subjects could only participate once in this study, were excluded if they had renal disease, any significant illness that might be associated with an immune defect such as SLE, were on oral/parenteral corticosteroids, had liver disease (AST/ALT >3ULN), or were unable to give informed consent or complete all study procedures. We have currently enrolled 23/40 SCD patients and 18/30 controls. All patients and controls were African American. Other baseline demographic characteristics of both groups were similar with regards to gender and age (p=0.775 and 0.8314 respectively). Following informed consent, each subject had blood drawn for quantitative immune analysis: total WBC, ANC, ALC, total T cell count (CD4/CD8 T cell subsets), total B cell and NK cell count, and enumeration of CD4 and CD8 T cells for their memory subpopulations. Functional immune assessment was done on a subset of samples (18 patients, 6 controls) using multiplexed enumeration of 25 serum cytokines using the Invitrogen 25-plex human cytokine panel. Quantitative and qualitative flow cytometric and cytokine analysis was performed using FloJo software and the Prism software statistical package. Results: Our results provide evidence for quantitative and functional immune deviation in SCD patients compared to controls. In addition to the well-documented increases in total WBC and ANC (1.9-fold and 1.7-fold compared to controls, respectively, p <0.05), SCD patients also demonstrated significantly higher total lymphocytes, monocytes and both cytotoxic and cytokine-secreting NK cells (2.9, 2.4, and 2.2-fold compared to controls, p <0.01 for all). Circulating B cells were significantly higher in SCD patients (3.3 fold, p= 0.0005). Circulating T cells were also increased in SCD patients, but this effect was specific to CD4+ T cells (1.7-fold, p = 0.002). There was no difference in CD8+ T cells (p= 0.4). The increased CD4+ T cell count predominantly involved memory CD4+ T subpopulations, with both central and effector memory CD4+ T cells significantly increased compared to controls (1.9-fold and 2.5-fold, respectively, p< 0.01). SCD patients also showed evidence of functional immune activation at steady state; plasma cytokine analysis revealed higher circulating CXCL10, CCL4 and IL-15 (2.4, 2.1, and >10 fold compared to controls, p <0.05 for all). These cytokines, which are secreted by activated endothelial cells and monocytes, are implicated in promoting the proliferation (IL-15) and the activation (CXCL10 and CCL4) of both T cells and NK cells. Implications: Our results confirm that even during steady state, pediatric patients with SCD exhibit evidence of significant quantitative and functional immune deviation. This observation suggests that targeting immune pathways impacting NK cell, B cell, and CD4 T cell function may be required for successful engraftment of SCD patients during non-myeloablative HSCT. Disclosures: No relevant conflicts of interest to declare.

In murine models, T-cell costimulation blockade of the CD28:B7 and CD154:CD40 pathways synergistically promotes immune tolerance after transplantation. While CD28 blockade has been successfully translated to the clinic, translation of blockade of the CD154:CD40 pathway has been less successful, in large part due to thromboembolic complications associated with anti-CD154 antibodies. Translation of CD40 blockade has also been slow, in part due to the fact that synergy between CD40 blockade and CD28 blockade had not yet been demonstrated in either primate models or humans. Here we show that a novel, nondepleting CD40 monoclonal antibody, 3A8, can combine with combined CTLA4Ig and sirolimus in a well-established primate bone marrow chimerism-induction model. Prolonged engraftment required the presence of all three agents during maintenance therapy, and resulted in graft acceptance for the duration of immunosuppressive treatment, with rejection resulting upon immunosuppression withdrawal. Flow cytometric analysis revealed that upregulation of CD95 expression on both CD4+ and CD8+ T cells correlated with rejection, suggesting that CD95 may be a robust biomarker of graft loss. These results are the first to demonstrate prolonged chimerism in primates treated with CD28/mTOR blockade and nondepletional CD40 blockade, and support further investigation of combined costimulation blockade targeting the CD28 and CD40 pathways.

Abstract Abstract 4078 Background: Acute GvHD remains the major cause of complications and death following unrelated-donor HSCT. In a non-human primate model, we have previously shown that in vivo costimulatory blockade of donor T-cells could provide effective protection against GVHD. To begin to explore its clinical utility, we are conducting a trial (Clinical Trials.Org # NCT01012492) to determine the feasibility of combining abatacept (CTLA4-Ig) with cyclosporine and methotrexate as acute GVHD prophylaxis for patients undergoing unrelated marrow and peripheral blood stem cell transplants for hematologic malignancies. Methods: Patients older than 12 with advanced hematologic malignancies, conditioned with either TBI/Cytoxan, Busulfan/Cytoxan or Fludarabine/Melphalan are eligible. Abatacept is administered IV on days −1, +5, +14, and +28 at 10 mg/kg in addition to standard GvHD prophylaxis consisting of cyclosporine (day −2 to day 100), and methotrexate (15 mg/m2 on day +1 and 10 mg/m2 on days +3, 6 and 11). Patients are then followed for clinical outcomes and immunologic reconstitution through day +365. Results: 9 patients (planned enrollment = 11 patients) have thus far been enrolled on the study of which 5 are evaluable for engraftment, toxicity and acute GvHD. The other four patients consist of 2 who are currently receiving abatacept, 1 who was discovered to have an ongoing viral infection at the start of the first abatacept infusion so was removed from the treatment regimen, and 1 who is awaiting transplant. The median age for the 5 evaluable patients is 47 years (17–74 years). 3 patients had AML and 2 had ALL. Patients were conditioned with Bu/Cy (n=1), TBI/Cy (n=2) and Flu/Melphalan (n=2). 4 donor-recipient pairs were allele matched at 9 of 10 loci (A, B, C, DRB1 and DQB1), while 1 was fully matched. Four of the 5 patients are currently alive and in remission and 1 relapsed at day +98 (and died on day +121 with refractory AML). The four other patients are surviving without relapse with a follow-up of 155–313 days. All 5 patients received the 4 scheduled abatacept doses. No infusional side effects were noted. All patients achieved neutrophil engraftment (median day +20 (11–47). 4 of 5 patients have achieved platelet engraftment (median day +27 (14–35). Donor engraftment (100% CD33 and 99–100% CD3 at Day +30) occurred in all cases. All patients have demonstrated rapid lymphocyte engraftment, with the mean ALC reconstituting to >500 cells/μL by day +21 post-transplant. At day +100, the mean CD3+ count was 673 +/− 251 cells/μL. Both CD8+ and CD4+ T cells reconstituted by day 100, with the mean CD8+ count = 384 +/− 148 cells/μL and the mean CD4+ count = 229 +/− 119 cells/μL. T cell reconstitution was accompanied by a shift away from naïve (Tn, CCR7+/CD45RA+) toward a CCR7-/CD45RA- effector memory (Tem)-predominant phenotype. Thus, the average proportion of CD4+ Tem cells in the recipient increased from 22 +/− 6% pre-transplant to 46 +/− 7% at day +100 with a concomitant loss of CD4+ Tn cells. Likewise, the proportion of CD8+ Tem also significantly increased, from an average of 15 +/− 4% pre-transplant to 32 +/− 7% at day +100, also with a reciprocal decrease in CD8+ Tn cells. One patient developed steroid responsive grade 3 acute GVHD involving the skin and the liver, followed by steroid responsive liver chronic GvHD. This patient is currently weaning corticosteroids. Another patient developed steroid responsive late-onset (day +217) acute GVHD (liver and GI) during cyclosporine weaning, which was also steroid responsive, and is also currently weaning corticosteroids. No other systemic acute or chronic GvHD has occurred. No unexpected complications or life-threatening infections were observed. 3 patients have experienced 5 episodes of CMV reactivation, all responsive to antiviral therapy. One patient developed polyclonal EBV-related PTLD (plasmacytic hyperplasia) in the absence of EBV viremia, which regressed without intervention. No other EBV-related disease has occurred. Conclusions: These preliminary data suggest that abatacept can be safely added to cyclosporine and methotrexate for GVHD prophylaxis in recipients of hematopoietic grafts from unrelated donors, with encouraging rates of acute GVHD. As such, they support the conduct of a larger, randomized phase 2 study. Disclosures: Off Label Use: Abatacept: It is an immunosuppressive agent that targets the CD28/B7 T cell costimulation pathway. It is approved for use in Rheumatoid arthritis.

In murine models, mixed hematopoietic chimerism induction leads to robust immune tolerance. However, translation to primates and to patients has been difficult. In this study, we used a novel MHC-defined rhesus macaque model to examine the impact of MHC matching on the stability of costimulation blockade-/sirolimus-mediated chimerism, and to probe possible mechanisms of bone marrow rejection after nonmyeloablative transplant. Using busulfan-based pretransplant preparation and maintenance immunosuppression with sirolimus, as well as CD28 and CD154 blockade, all recipients demonstrated donor engraftment after transplant. However, the mixed chimerism that resulted was compartmentalized, with recipients demonstrating significantly higher whole blood chimerism compared to T cell chimerism. Thus, the vast majority of T cells presenting posttransplant were recipient-rather than donor-derived. Surprisingly, even in MHC-matched transplants, rejection of donor hematopoiesis predominated after immunosuppression withdrawal. Weaning of immunosuppression was associated with a surge of antigen-experienced T cells, and transplant rejection was associated with the acquisition of donor-directed T cell alloreactivity. These results suggest that a reservoir of alloreactive cells was present despite prior costimulation blockade and sirolimus, and that the postimmunosuppression lymphocytic rebound may have lead to a phenotypic shift in these recipient T cells towards an activated, antigen-experienced phenotype, and ultimately, to transplant rejection.