ALBERT

Description: Abstract 350 Introduction: Leukapheresis is a widely utilized modality for collecting hematopoietic stem cells (HSCs). While collection of CD34+ cells with stem-cell activity is the primary goal of most mobilization and leukapheresis procedures, these cells only represent ∼1% of most leukapheresis products. The profile of the non-CD34+ cells is likely influenced by the choice of mobilization strategy, and has the potential to profoundly impact the post-transplant immune milieu of the transplant recipient. Two of the most critical of the CD34-negative cell populations that are collected during leukapheresis include effector and regulatory T cells. Thus, in evaluating mobilization regimens, the impact on these regimens on the mobilization of each of these T cell populations into the peripheral blood should be rigorously evaluated. Methods: We used a rhesus macaque model to determine the impact that mobilization with AMD3100 (a.k.a., Plerixafor or Mozobil®)+ G-CSF (“A+G”) had on peripheral blood CD4+ and CD8+ effector T cell populations as well as on FoxP3+/CD4+ T cells. Three rhesus macaques were mobilized with 10ug/kg SQ of G-CSF for five consecutive days prior to leukapheresis. AMD3100 was administered at 1mg/kg SQ in combination with the last dose of G-CSF two hours prior to leukapheresis. Leukapheresis procedures were performed for two hours using a modified CS3000 Plus cell separator. A peripheral blood sample was taken before cytokine therapy, just prior to leukapheresis following mobilization, one hour during leukapheresis, and at the end of the procedure. These samples were analyzed by multicolor flow cytometry using a BD LSRII flow cytometer. Results: Bulk, effector, and regulatory T cell subpopulations were analyzed flow cytometrically. The proportion of total CD3+ T cells remained stable during mobilization and apheresis: Thus, CD3+ T cells represented 77% of peripheral blood lymphocytes prior to mobilization, and 69% post-apheresis). The balance of CD4+ to CD8+ T cells was also relatively stable. Thus, for one of the three animals tested, the CD4+ and CD8+ proportions remained unchanged after apheresis. For two animals, the average CD4+ % decreased from 67% prior to mobilization to 52% post-apheresis. In these two animals, there was a reciprocal increase in the % of CD3+ T cells that were CD8+ (28% pre-G+A to 40% post-apheresis). The CD28+/CD95- naïve (Tn), CD28+/CD95+ central memory (Tcm) and CD28-/CD95+ effector memory (Tem) subpopulation balance of CD4+ and CD8+ T cells was also determined, by comparing the relative percentages of each subpopulation post-apheresis with their relative percentages prior to mobilization. Compared to their pre-G+A percentages, the post-apheresis CD4+ percentages of Tn, Tcm and Tem were 92%, 93% and 160%, respectively. Thus, the relative proportions of Tn and Tcm CD4+ cells decreased post-apheresis, while the relative proportion of CD4+ Tem increased compared to cytokine administration. For CD8+ T cell subpopulations, the post-apheresis proportions of Tn, Tcm, and Tem compared to their pre-G-CSF proportions were 99%, 70% and 130%, respectively–thus demonstrating the same direction of change as observed for CD4+ T cells. The most striking change in T cell subpopulations occurred in the CD4+/FoxP3+ compartment. The proportion of CD4+ T cells expressing FoxP3 increased by an average of 600% when post-apheresis samples were compared to pre-mobilization samples (FoxP3+ cells were 9.6% of CD4+ T cells post-apheresis versus 1.5% pre-GCSF). An average of 32% of these FoxP3+ CD4+ T cells expressed high levels of CXCR4. CXCR4 expression has been previously documented on human FoxP3+ T cells (Zou et al., Cancer Res, 2004), but this is the first observation of high level expression of CXCR4 on macaque FoxP3+ CD4 T cells, or of their ability to be efficiently mobilized with AMD3100. Discussion: These results suggest that treatment with AMD3100 and G-CSF may mobilize T cell subsets into the peripheral blood that could have beneficial effects during allo-transplantation. The combination of an increase in Tem cells, which have been observed to have decreased ability to cause GvHD (Zheng et al., Blood 2008), along with FoxP3+/CD4+ T cells, which may have regulatory functions, suggests that A+G mobilization could produce an apheresis product with a beneficial CD34-negative cell profile for allogeneic transplantation. 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: In this study, we used the rhesus macaque model to determine the impact that AMD3100 has on lymphocyte mobilization, both alone and in combination with G-CSF. Our results indicate that, unlike G-CSF, AMD3100 substantially mobilizes both B and T lymphocytes into the peripheral blood. This led to significant increases in the peripheral blood content of both effector and regulatory T-cell populations, which translated into greater accumulation of these cells in the resulting leukapheresis products. Notably, CD4+/CD25high/CD127low/FoxP3+ Tregs were efficiently mobilized with AMD3100-containing regimens, with as much as a 4.0-fold enrichment in the leukapheresis product compared with G-CSF alone. CD8+ T cells were mobilized to a greater extent than CD4+ T cells, with accumulation of 3.7 ± 0.4-fold more total CD8+ T cells and 6.2 ± 0.4-fold more CD8+ effector memory T cells in the leukapheresis product compared with G-CSF alone. Given that effector memory T-cell subpopulations may mediate less GVHD compared with other effector T-cell populations and that Tregs are protective against GVHD, our results indicate that AMD3100 may mobilize a GVHD-protective T-cell repertoire, which would be of benefit in allogeneic hematopoietic stem cell transplantation.

Description: 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

Description: 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.