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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 Figure 1. PD-L1-/- vs. WT donor T cells lessen GVHD lethality, independent of donor Treg function. (A) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells. (B) Survival of B10.BR mice with WT B6 or PD-L1-/- T cells. (C) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells, or with WT B6 or PD-L1-/- CD25 depleted T cells (recipients of WT T cells vs. WT CD25-depleted T cells, P = 0.0003; recipients of PD-L1-/- T cells vs. PD-L1-/- CD25-depleted T cells, P = 0.2306; recipients of WT vs. PD-L1-/- T cells, P < 0.0001). (D) BALB/c mice transplanted with WT B6 or PD-L1-/- T cells. Mice were killed on d7 post-BMT and splenocytes were analyzed for LPAM-1, CCR9, and CXCR3 expression (not shown) on donor T cells. (E-F) BALB/c mice were transplanted with B6 Ly5.2 T cells plus PD-L1-/- T cells. Mice were killed on d3 post-BMT and splenocytes were analyzed for CTLA-4 and Lag-3 expression on donor T cells. Figure 1. PD-L1-/- vs. WT donor T cells lessen GVHD lethality, independent of donor Treg function. (A) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells. (B) Survival of B10.BR mice with WT B6 or PD-L1-/- T cells. (C) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells, or with WT B6 or PD-L1-/- CD25 depleted T cells (recipients of WT T cells vs. WT CD25-depleted T cells, P = 0.0003; recipients of PD-L1-/- T cells vs. PD-L1-/- CD25-depleted T cells, P = 0.2306; recipients of WT vs. PD-L1-/- T cells, P < 0.0001). (D) BALB/c mice transplanted with WT B6 or PD-L1-/- T cells. Mice were killed on d7 post-BMT and splenocytes were analyzed for LPAM-1, CCR9, and CXCR3 expression (not shown) on donor T cells. (E-F) BALB/c mice were transplanted with B6 Ly5.2 T cells plus PD-L1-/- T cells. Mice were killed on d3 post-BMT and splenocytes were analyzed for CTLA-4 and Lag-3 expression on donor T cells. Disclosures Aoyama: CHUGAI PHAMACEUTICAL CO.,LTD: Honoraria; Mochida Pharmaceutical Co.,Ltd: Honoraria; Kyowa Hakko Kirin Company,Limited: Honoraria. Milone:Novartis: Patents & Royalties, Research Funding. Miller:Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau. Sharpe:Costim Pharmaceuticals: Patents & Royalties.

10052 Background: Sirolimus, an oral mTOR inhibitor, has anti-tumor effects mainly by blocking signals which drive cells from G1 to S phase. It has antiangiogenic activity and has shown antitumor a...

Given the significant morbidity associated with current post-transplant immunosuppressive regimens, induction of immune tolerance continues to be an important goal of clinical organ transplantation. While many strategies for inducing tolerance have been successfully applied in murine models, significant barriers are faced when translating these approaches to the clinic. This has necessitated pre-clinical studies in the more closely related model system, the non-human primates (NHP). In this review, we will discuss the four most prominent strategies for inducing transplantation tolerance and highlight their relative success and shortcomings in NHP. These strategies are: (1) T-cell costimulation blockade (2) mixed chimerism induction (3) T-cell depletion and (4) tolerance induction through regulatory T-cells. After discussing the progress that has been made with each of these strategies, we will identify this field's most pressing unmet needs and discuss how we may best overcome the resulting barriers to tolerance induction.

Kidney transplantation remains limited by toxicities of calcineurin inhibitors (CNIs) and steroids. Belatacept is a less toxic CNI alternative, but existing regimens rely on steroids and have higher rejection rates. Experimentally, donor bone marrow and sirolimus promote belatacept's efficacy. To investigate a belatacept-based regimen without CNIs or steroids, we transplanted recipients of live donor kidneys using alemtuzumab induction, monthly belatacept and daily sirolimus. Patients were randomized 1:1 to receive unfractionated donor bone marrow. After 1 year, patients were allowed to wean from sirolimus. Patients were followed clinically and with surveillance biopsies. Twenty patients were transplanted, all successfully. Mean creatinine (estimated GFR) was 1.10 ± 0.07 mg/dL (89 ± 3.56 mL/min) and 1.13 ± 0.07 mg/dL (and 88 ± 3.48 mL/min) at 12 and 36 months, respectively. Excellent results were achieved irrespective of bone marrow infusion. Ten patients elected oral immunosuppressant weaning, seven of whom were maintained rejection-free on monotherapy belatacept. Those failing to wean were successfully maintained on belatacept-based regimens supplemented by oral immunosuppression. Seven patients declined immunosuppressant weaning and three patients were denied weaning for associated medical conditions; all remained rejection-free. Belatacept and sirolimus effectively prevent kidney allograft rejection without CNIs or steroids when used following alemtuzumab induction. Selected, immunologically low-risk patients can be maintained solely on once monthly intravenous belatacept.

1). To study the mechanisms of anemia, erythroid hyperplasia, and red blood cell (RBC) clearance in murine models of sickle cell disease (Sickle) and beta-thalassemia (Th1/Th1); 2) To determine the contribution of the phospholipid scramblase enzyme to phosphatidylserine (PS) exposure and RBC death in Sickle and Th1/Th1 mice.We used a combination of flow-cytometric analysis and assays for phospholipid remodeling to determine the extent and sites of erythroid hyperplasia, PS exposure, and cell death.1) Sickle RBCs have a much shorter half-life than Th1/Th1 RBCs (0.8 days vs. 11 days). A significant proportion of Th1/Th1 peripheral reticulocytes mature into erythrocytes, however, approximately fivefold fewer Sickle reticulocytes mature. While erythroid hyperplasia exists in both Sickle and Th1/Th1 mice, Th1/Th1 produce fourfold more RBCs than necessary to maintain steady state, while Sickle produce no excess RBCs. 2) 61% of Sickle and 34% of Th1/Th1 RBCs are scramblase(+) as measured by internalization assays of the fluorescent phospholipid NBD-PC. The majority of NBD-PC(+) RBCs are also annexin-V(+), supporting a mechanistic link between scramblase activity and PS exposure. A proportion of both reticulocytes and older RBCs in Sickle and Th1/Th1 mice have active scramblase, and the degree of scramblase activation in these strains correlates with the propensity for RBC death.Sickle and Th1/Th1 mice are both anemic, with significant erythroid hyperplasia. Th1/Th1 mice display ineffective erythropoiesis while Sickle mice show rapid peripheral destruction of RBCs. PS exposure and phospholipid scramblase activity serve as markers of RBCs with altered phospholipid asymmetry and greater propensity for cell death.

Introduction: We have previously shown that blocking CD28:CD80/86 costimulation with CTLA4-Ig can be a successful strategy to inhibit GVHD. However, since CTLA4-Ig targets CD80/86, there remain concerns of off-target, immune activating effects, given that it can inhibit both positive signaling through CD28 and negative signaling through CTLA4. In order to specifically target CD28, we have developed a humanized anti-CD28 monovalent Fab antibody (FR104) and have tested its ability to protect against GVHD. Methods: NHP underwent MHC-mismatched transplantation after myeloablative radiation-based pre-transplant conditioning. They were transplanted with GCSF-mobilized PBSCs (4.2x10^8 +/- 1.1x10^8 TNC/kg and 1.9 x 10^7 +/- 0.5 x 10^7 CD3+ T cells/kg. GVHD prophylaxis consisted of either monotherapy with FR104 (5mg/kg given weekly) or with combination FR104 and rapamycin (rapamycin trough 5-15 ng/ml). These were compared to three control groups: no GVHD prophylaxis, rapamycin monotherapy and autologous HSCT. Clinical and histologic GVHD was monitored using our NHP grading scale, and longitudinal immunologic analysis was performed. Treatment was continued as tolerated with planned terminal analysis at 35 days in all survivors. Results: Untreated controls (n = 5) developed rapid, severe multi-organ GVHD (MST=7 days). Rapamycin monotherapy (n=6) partially protected against GVHD (MST =14 days). Monotherapy with FR104 showed a significant prolongation in survival compared to untreated animals (MST = 21 days, p=0.02 n = 3), with breakthrough GVHD (liver, skin predominant) ultimately occurring. In contrast to all other groups, combination therapy with FR104 and rapamycin (n=4) resulted in striking protection from GVHD with all animals reaching planned terminal analysis with minimal clinical disease (p=0.01 vs. untreated controls, p=0.01 vs. rapamycin monotherapy, Fig. 1). We have documented a major role for both T cell proliferation (Ki-67) and Granzyme B (Gran-B) overexpression in GVHD, with untreated controls demonstrating rampant CD8+ proliferation (81% + 5.4% Ki-67+ at day 7 vs. 4% pre-transplant) and Gran-B overexpression (81% + 2.7% Gran-Bvery high at day 7 vs. 0.5% + 0.5% pre-transplant). Monotherapy with either rapamycin or FR104 partially controlled proliferation (53% + 23% and 30% + 21.7%, respectively) and Gran-B overexpression (21% + 8.7% and 11% + 5%, respectively) on day 7. Combination therapy with FR104 + rapamycin resulted in synergistic control of both proliferation and Gran-B overexpression (0.6% + 0.2% Ki67+ and 2.3% + 1.3% Gran-B overexpression on Day 7), reverting the CD8+ activation phenotype to that observed in auto-HSCT controls (5.6% + 2.1% Ki67+, 9% + 3.2% Gran-B overexpression, Fig. 2). This reversion to the auto-HSCT phenotype was maintained until the terminal analysis, and the control of Day 7 proliferation and Gran-B overexpression correlated closely with GVHD-free survival. The control of CD28-mediated T cell activation in FR104-treated recipients resulted in significant preservation of the naïve CD8+ T cell (CD8 Tn) phenotype in these animals. Thus, in untreated controls and rapamycin monotherapy groups, the CD8 Tn population virtually disappeared post-transplant (2.1% + 0.7% and 6.6% + 1.5% at day 7, respectively compared to 33.6%+ 4.1% pre-transplant), consistent with widespread allo-stimulation and costimulation. In contrast, FR104 monotherapy resulted in partial preservation of CD8 Tn (24.3% + 2.8% at Day 7) and FR104 + rapamycin combination therapy promoted nearly complete preservation of CD8 Tn (48.3% + 0.9% at day +7), with maintenance of this naïve population for the duration of therapy (Fig. 3). Discussion: These results show, for the first time, that specific blockade of CD28 can inhibit NHP GVHD, and when coupled with rapamycin it can effectively control both the clinical and immunologic sequelae of this disease. CD28 blockade with FR104 + rapamycin significantly inhibited both CD8+ T cell proliferation and Granzyme B overexpression, and lead to robust preservation of the naïve T cell phenotype, consistent with efficient inhibition of allo-activation. Our results suggest that targeting the CD28:CD80/86 costimulation axis by selective CD28 blockade may be effective in inhibiting the T cell activation associated with GVHD, and is deserving of clinical evaluation for safety and efficacy in patients undergoing HCT. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Vanhove: Effimune: Research Funding.

While novel therapeutics for graft versus host disease (GvHD) are desperately needed, new prevention and treatment strategies for this disease have been extremely slow to reach the clinic. One of the major barriers has been the historic lack of a powerful translational system capable of both (1) interrogating the clinical efficacy of novel approaches and (2) discovering the underlying mechanisms of GvHD prevention. Our lab has addressed this unmet need with the development of a non-human primate (NHP) model of GvHD, and the first therapeutic approach developed with the NHP model, costimulation blockade with CTLA4-Ig, is currently in clinical trials for GvHD prevention (Clinical Trials.gov #NCT01743131). While the first-in-disease trials of GvHD prevention add CTLA4-Ig to standard tacrolimus/methotrexate, previous work has suggested that mTOR inhibition with sirolimus is more pro-tolerogenic than calcineurin inhibition when paired with costimulation blockade. We have now addressed the possibility of pairing CTLA4-Ig with sirolimus to prevent GvHD in the NHP model, and have interrogated the outcomes using a systems-based approach. Our new experiments provide strong clinical, immunologic and trancriptomic evidence for potent synergy when CTLA4-Ig and sirolimus are combined for GvHD prevention. In this study, we determined the impact of the following treatment groups on GvHD after high-risk haplo-identical HSCT using T cell-replete peripheral blood stem cell transplantation: 1) no therapy (n=4) 2) CTLA4-Ig monotherapy (using the second generation CTLA4-Ig formulation, belatacept, n=3) 3) sirolimus monotherapy (n=4) 4) combination belatacept and sirolimus (n=3). To determine the relative impact of each therapeutic approach, we monitored clinical GvHD, GvHD-free survival, and flow cytometric signs of immune activation post-transplant. Moreover, to create a comprehensive molecular map of their impact on GvHD, we performed transcriptomic analysis, on CD3+/CD20- T cells that were purified on day +14 post-transplant and analyzed using the Affymetrix microarray platform. The synergistic impact of combined belatacept +sirolimus was evidenced through all analyses techniques: Thus, GvHD-free survival with belatacept + sirolimus was prolonged compared to all other groups (MST belatacept + sirolimus = 33d, p < 0.02 compared to MST for untreated controls (7.5d), belatacept monotherapy (9d, p < 0.03) and sirolimus monotherapy (12d) (Figure 1a). GvHD clinical scores mirrored the clinical survival (Figure 1b). In addition, canonical flow cytometric signs of CD8+ T cell activation (proliferation, measured by Ki-67, and excessive cytotoxicity measured by granzyme B overexpression) also mirrored the clinical synergy we measured with combined belatacept +sirolimus compared to all other groups (Figure 1c). Comparing the expression profile of T cells during acute GvHD in the four cohorts allowed examination of treatment synergy at an unprecedented level of molecular detail. Unsupervised analysis of transcriptomic signatures as a whole revealed clustering of principal component projections from belatacept + sirolimus to be strikingly similar to a large comparative healthy control cohort (n=28), underscoring the high degree of control alloreactivity with this regimen. Moreover, the comparison of differentially expressed genes from animals receiving belatacept + sirolimus revealed significant divergence from monotherapy with either belatacept or sirolimus, again underscoring the profound control of allo-activation that was observed with belatacept + sirolimus (Figure 2a). The mirror-image analysis provided additional support or this synergy, with transcriptional analysis of T cells from belatacept + sirolimus-treated animals most dissimilar from the transcriptome of unprophylaxed GvHD (Figure 2b). Those genes for which expression was significantly normalized by combination therapy showed pathway enrichment prominently in T cell effector function (prominently including granzyme signaling – Figure 2c), cytokine networks (prominently IL2, IL12 and IL-18 – Figure 2d), as well as in proliferation and cell cycle pathways (Figure 2e). These data reveal a treatment synergy between T cell costimulation blockade with CTLA-4-Ig and mTOR inhibition and suggest that this combination of therapy will be useful for acute GvHD immunoprophylaxis in humans. Disclosures No relevant conflicts of interest to declare.

Graft versus host disease (GvHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate targeted strategies for prevention and treatment. To address this, we determined the gene expression profile of non-human primate (NHP) T cells during active and partially controlled acute GvHD (aGvHD), in order to accomplish two goals: 1) uncover important genetic drivers of aGvHD and 2) identify novel, targetable pathways for optimal aGvHD prevention. Utilizing microarray technology, we measured the gene expression profiles of flow cytometrically purified CD3+ T cells from NHP recipients of MHC partially-matched HCT in three treatment cohorts resulting in increasing degrees of survival: 1) no immunoprophylaxis (No Rx, MST = 7.5); 2) sirolimus monotherapy (MST = 17) tacrolimus-methotrexate (Tac-Mtx) dual prophylaxis (MST = 49). Arrays were performed on T cells purified on Day +14 post-transplant (unless terminal analysis occurred earlier due to severe disease). This comparison allowed us to determine the impact of both mTOR and calcineurin inhibition on the molecular pathways dysregulated during GvHD, and to determine which genes and pathways remained dysregulated despite prophylaxis. Pathways identified by this strategy may contain new therapeutic targets unaffected by current immunoprophylactic approaches. We found that the transcriptional profile of donor T cells from HCT recipients with unprophylaxed GvHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function and cytokine synthesis (Figure 1a). By identifying pathways unaffected by sirolimus or tac-mtx therapy (Figure 1b), we discovered multiple potentially druggable targets not previously implicated in the pathophysiology of aGvHD. These targets prominently included the hedgehog and the aurora kinase A pathways. Utilizing a murine aGvHD model, we demonstrated that pharmacologic inhibition of these pathways could mitigate disease and improve survival (Figure 2a,b). These data provide the first identification of the T cell transcriptome of primate acute GvHD and the hedgehog and aurora kinase A pathways as novel potential targets for prevention of this disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (γ-irradiation, depletion of the peripheral immune system) or administration of “mega” doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-γ-producing T cells re-emerged and skin grafts were rejected at ∼100 days. When applied to a murine β-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.

A National Non-Human Primate (NHP) DNA bank has been established by the National Primate Research Centers and the National Center for Research Resources, NIH, providing a new resource for comparative genomic studies. The collection includes genomic DNA samples from macaques, chimpanzees, baboons, vervets, marmosets, sooty mangabeys and titi monkeys. The repository includes DNAs from 697 unrelated animals, suitable for comparing allele representation within and between species. Another 474 DNAs are derived from family-trios (dam, sire, off spring), and are useful for verifying the segregation of genetic variants. The National NHP DNA Bank includes specified holdings within each of the eight National Primate Research Centers, though detailed information on the entire collection is available through a common website.

Ex-vivo expanded natural regulatory T cells (nTregs) are emerging as promising cellular therapeutics for the prevention of allograft rejection and graft-versus host disease. However, their widespread translation to the clinic has not yet occurred, in part because of the lack of a clear understanding of their in-vivo quantitative and functional dynamics. Here we have used our translational non-human primate model to answer some of the critical questions related to the survival, stability and immunologic compatibility of Tregs with other immunosuppressive therapies. By infusing escalating doses of CFSE-labeled, autologous CD4+CD25++CD127-/lowFoxP3+ Tregs, we determined, for the first time, that in primates, the accessible, peripheral Treg pool is quite large: ∼74± 13 x106 cell/kg: > 20-times larger than the number of Tregs that have previously been infused into patients (Figure 1). Tregs infused in the absence of concomitant immunosuppression had a surprisingly short in-vivo half-life, even when transferred into autologous recipients (T1/2=2.1± 0.3 days): Tregs infused at dose of 20x106 cell /kg were undetectable in either the blood, bone marrow or lymph nodes within 2 weeks after their infusion, despite their initial trafficking to all three sites early after infusion. In addition to their short half-life, infused Tregs quickly underwent rapid phenotypic alteration, with significant loss of both CD25 and FoxP3 expression within 6 days of transfer, suggestive of a potential concomitant loss of suppressive function (Figure 2). By Day +10, only 28.0± 2.7% of infused CD4+CFSE+ cells were CD25+FoxP3+ compared to 74.2± 6.6% on the day of infusion (p< 0.01). While treatment with tacrolimus was able to rescue the loss of CD25 expression, it did not improve the rapid loss of FoxP3 expression that occurred in the infused cells, resulting in a half-life that was not improved compared to Tregs infused without immunosuppression (2.6± 0.2 days, p = ns compared to no immunosuppression) as well as a similarly rapid loss of FoxP3 expression (25± 9 % FoxP3 expression on day +10 post-infusion). In striking contrast, rapamycin extended the persistence of the infused Tregs beyond day 35 (T1/2 = 3.9± 0.8 days, p< 0.01 compared to no immunosuppression) and significantly improved the CD25+FoxP3+ phenotype of the adoptively transferred cells, with 70± 6% retaining expression of both CD25 and FoxP3 at day +10, p= 0.01 compared to no immunosuppression) (Figure 2). In addition, compared to animals receiving Tregs either without immunosuppression or in the presence of tacrolimus, rapamycin treatment resulted in significantly more infused Tregs in the bone marrow and lymph nodes when measured one week after transfer (in the bone marrow, 6± 0.7 % vs 1± 0.6 % without immunosuppression and 2.4± 0.4 % on tacrolimus, p< 0.01 compared to either treatment). These results provide the first quantitative evaluation of the availability of the accessible Treg pool in primates, and suggest that Tregs undergo a significant and potentially deleterious phenotypic degradation after transfer. While tacrolimus was unable to fully rescue this degradation, rapamycin significantly improved both the survival and phenotypic stability of these cells. These results suggest that even natural Tregs may be phenotypically and functionally unstable after transfer, and that adjunctive strategies to stabilize function and half-life, including mTOR inhibition, should be strongly considered when using these cells as an adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Background Sensitization to MHC antigens because of transfusion, pregnancy, and previous grafts is among the most critical challenges to clinical transplantation. The presence of preformed donor-specific antibody increases the risk for bone marrow and solid organ graft rejection. It is well documented that some microbial molecules and microorganisms can directly induce the proliferation and differentiation of antibody-secreting cells from different B cells, regardless of their antigen specificity. The presence of a strong polyclonal B cell response resulting in hypergammaglobulinemia secretion has been described in several animal models of infection with viruses, parasites, and bacteria. Recent experiments in our lab showed that pathogen infection in naïve animals induces activation of naïve allospecific B cell and formation of de novo alloantibody. This prompted us to analyze the effect of pathogen infection induced alloantibody on hematopoietic cell transplantation. Herein, these studies show that preformed donor specific antibody induced by pathogen infection in the absence of donor-antigen is a major barrier to bone marrow engraftment. Methods /results: LCMV infection is a model system of viral infection and also an established system to examine virus nonspecific, polyclonal hypergammaglobulinemia. Naïve C57BL/6 (H2b) mice were infected with acute and chronic strain of LCMV (Armstrong and clone 13, n=10 each). Virally infected mice were bled weekly to analyze total IgG and formation of de novo alloantibody in the serum. Viral infection induced both hypergammaglobulinemia and the formation of de novo alloantibody (anti-H2d). Peak levels of donor-specific antibody in acute and chronically infected mice were increased to approximate 3 fold when compared to uninfected controls as assessed by flow cytometric crossmatch. The results were consistent with formation of anti-H2d antibody when measured by anti-H2d ELISA with peak level of approximately 336.3 ± 51.84 units/ml when compared to 2.179 ± 0.4597 units/ml in uninfected mice (p Next we analyzed if virally induced preformed de novo alloantibody would represent a barrier to bone marrow engraftment. Naïve C57Bl/6 mice were irradiated with 950 rads and received 15x106BALB/c bone marrow. BALB/c bone marrow cells were either incubated ex vivo with 1:6 diluted sera from naïve B6 mice or with the sera from clone 13 infected mice (d28 post-infection). After incubation, bone marrow was washed and complement inactivated prior to infusion. All of the 10 mice receiving bone marrow incubated with naïve mice serum engrafted and survived, while only 1/10 mice survived receiving bone marrow incubated with serum from infected mice (Mean survival = >100 vs 24.5 days respectively, p < 0.0067, figure 2). We also examined the effects of virally induced preformed de novo alloantibody in a costimulation blockade based regimen to induce mixed allogeneic chimerism and transplant tolerance. Naïve C57BL/6 mice received 30x106 BALB/c bone marrow infusion along with costimulation blockade treatment (250ug each of CTLA4-Ig and anti-CD40L (MR1) at 0,2,4,6 days post transplant) after nonirradiation-based conditioning with busulfan (-3,-2 days. 20mg/kg IP injection). BALB/c bone marrow cells were either incubated ex vivo with 1:6 diluted sera from naïve B6 mice or with sera from clone 13 infected mice (d28 post-infection) as described above. Consistent with the experiments using the irradiation based conditioning regimen, mice receiving bone marrow incubated with naïve mice serum engrafted and established stable donor chimerism while none of mice receiving bone marrow incubated with serum from infected mice showed any donor cell engraftment Discussion: Allogeneic hematopoetic cell is used as therapy for hematologic malignancies and for non-malignant hematologic diseases. Our results indicate that preformed donor specific antibody induced by viral infection in the absence of previous exposure to donor-antigen is a major barrier to bone marrow engraftment. These results suggest that an individual’s prior immune history to pathogens may significantly impact subsequent humoral responses upon bone marrow transplantation. Disclosures: No relevant conflicts of interest to declare.

Introduction Patients with sickle cell disease (SCD) are at high risk for graft rejection after hematopoietic stem cell transplant (HSCT) especially when using reduced intensity conditioning (RIC). Alloimmunization from red blood cell (RBC) transfusions, critical to the management of SCD, may predispose these patients to HSCT rejection. Alloimmunization to major or minor histocompatibility antigens (mHA) may occur after RBC transfusion due to residual white blood cells or platelets present in RBC units even after leukoreduction. In mice, despite extensive leukoreduction, RBC transfusions caused alloimmunization and subsequent HSCT rejection after RIC. We conducted a cross-sectional study to determine if RBC transfusions are associated with alloantibody formation to human leukocyte antigens (HLA) and mHA, the target of the host versus graft rejection in HLA-identical transplantation. For mHA, we assessed immunity to H-Y antigens, mHA encoded on the Y chromosome. We have previously demonstrated the importance of H-Y antibodies in a variety of settings including chronic graft versus host disease (in male recipients of female grafts) and rejection of renal transplants (in female recipients of male grafts). Methods We enrolled 114 nulliparous pediatric SCD (SS or Sβ0) patients: 58 females on chronic transfusion, 32 males on chronic transfusion, and 24 females who had never been transfused. Serum was evaluated for HLA antibodies using the FlowPRA® (panel reactive antibody) screening test which detects IgG antibodies to the majority of HLA class I and II antigens. Serum was also tested for IgG antibodies against 5 H-Y antigens (DDX3Y [DBY], EIF1AY, RPS4Y1, UTY, ZFY) using protein microarray technology with each H-Y-seropositive threshold determined as a microarray mean fluorescence intensity (MFI) > the median MFI + 2.5 quartiles measured in 60 adult healthy males. Results Chronically transfused patients had a higher prevalence of HLA class I antibodies than never transfused patients (33.3% vs. 12.5%, p=0.074). This increased prevalence of HLA class I antibodies in the chronic transfusion group was significant when examining the number of patients who tested positive to more than 25% of the panel (17.8% vs. 0%, p=0.022). Few patients had detectable HLA class II antibodies with no significant differences between chronically transfused and non-transfused patients. Few SCD patients had detectable H-Y alloantibodies with no significant differences in chronically transfused females, chronically transfused males, and non-transfused females. Conclusions Leukocyte-reduced RBC transfusions appear to engender immunization to class I HLA but not class II HLA or H-Y antigens in pediatric SCD patients. Since hematopoietic progenitor cells express class I HLA, transfusion-related immunity to these antigens could pose a barrier to engraftment in HLA-disparate transplants. Since these antigens are also expressed on platelets, class I HLA antibodies in SCD could have important implications for platelet transfusion support during HSCT. While RBC transfusion does not appear to be an adequate stimulus for H-Y alloantibody formation, it is possible that exposure to mHA during transfusion could cause a cellular rather than a humoral immune response that may contribute to future HSCT graft rejection. Our result that few (6%) nulliparous female children with SCD had detectable H-Y alloantibodies is also a significant finding considering that 19-41% of healthy adult females had antibodies to at least one H-Y antigen in previous studies. Finally our finding that 25% of never transfused children with SCD had a positive HLA PRA is unexpected for an “unsensitized” group; its significance is being further investigated. Disclosures: No relevant conflicts of interest to declare.

Abstract 741 We have previously shown that a costimulation blockade-containing regimen could provide effective protection against acute GvHD (aGvHD) in a non-human primate model. We therefore designed a first-in-disease trial of in vivo CD28:CD80/86-directed costimulation blockade with CTLA4-Ig (abatacept) to prevent aGvHD after unrelated-donor HSCT for patients > 12y. (Clinical Trials.Org #NCT01012492). In this trial, 10mg/kg abatacept was administered IV on day −1, +5, +14, +28 in addition to standard prophylaxis with cyclosporine + MTX. Enrollment of all patients is complete, and the study is evaluable for feasibility, toxicity, engraftment, and the primary immunologic outcome: the incidence of Grade III-IV aGvHD by d+100. Patient Characteristics and Survival: 10 patients, with a median age of 44.5 y (17–74) were enrolled and treated. 6 patients received HLA-mismatched grafts (matched at 7/8 alleles) and 4 received 8/8 HLA-matched URD grafts. 8 received PBSCs and 2 received BM. All received high-intensity conditioning (Bu/Cy, TBI/Cy or Flu/Mel). With a median follow-up of 367 days (262–680), 7 patients are alive and in remission. 2 patients died of relapse (Day +121 and Day +147). One patient died, in remission, of multi-organ failure on day +453 post-transplant. Feasibility, Pharmacokinetics and Pharmacodynamics of Abatacept in HSCT: All 10 patients received all 4 scheduled abatacept doses, without infusion reactions. The average peak (230.9 +/− 7.4 mg/ml) and trough (45.9 +/− 2.8 mg/ml) abatacept levels, as well as the terminal T1/2 (19.6 +/− 1.9 days) were similar to that observed previously. Importantly, as has been previously established in vitro, patients receiving abatacept demonstrated significant inhibition of post-transplant CD4+ T cell proliferation (with >80% reduction in the accumulation of Ki-67+ proliferating CD4+ T cells at d+14 and +28 compared to a control cohort who received cyclosporine + methotrexate without abatacept, Figure 1). This data establishes, for the first time, the feasibility of giving abatacept to this new patient population, and that the PK and PD parameters in HSCT closely mirror those previously shown to effectively control immune-mediated diseases. Engraftment: All patients achieved neutrophil engraftment (median d+16.5) and donor engraftment (100% CD33 chimerism at d+30). Lymphocyte recovery was rapid: Day +100 mean peripheral blood counts showed ALC = 1053 +/− 259 cells/ml, total T cells = 741 +/− 208 cells/ml, and CD8+ T cells = 381 +/− 99 cells/ml. The Day +100 CD4+ T cells = 285 +/− 105 cells/ml, not significantly different from historical controls (n = 43) that received CNI/MTX aGvHD prophylaxis without abatacept (262 +/−26 cells/ml). GvHD: Patients receiving the abatacept-containing regimen had encouragingly low rates of early severe aGvHD: Two patients developed aGvHD before day +100, with one of these patients (Gr II) progressing to steroid-dependent cGvHD of the liver and one patient (Gr III) with complete resolution of aGvHD (and currently off steroids). The cumulative incidence of grade II-IV and III-IV aGvHD by day 100 was thus 20% and 10%, respectively (Figure 2). Importantly, there was no Gr IV aGvHD, no patient received salvage therapy for aGvHD, and there were no deaths from aGvHD. After day +100, one patient developed late acute GvHD (currently off all immunosuppression), and two patients developed overlap syndrome. Both had responsive disease and are currently either weaning or off corticosteroids. No other patient developed late aGvHD, and no other patient has developed moderate or severe cGvHD. Infection: No life-threatening infections occurred. 5 patients developed CMV reactivation, all responsive to antivirals. No patient developed CMV disease. No EBV viremia >1000 copies/ml occurred. One patient developed EBV+ plasmacytic hyperplasia of the tongue, which resolved without intervention. No other EBV-related disease occurred. Conclusions: This trial demonstrates, for the first time, the feasibility of adding in vivo T cell costimulation blockade with abatacept for aGvHD prevention. The decreased CD4+ T cell proliferation post-transplant and the encouragingly low rates of early, severe aGvHD suggest that costimulation blockade may be an effective agent for aGvHD prophylaxis and support the conduct of a larger, randomized phase 2 study. Disclosures: Off Label Use: Abatacept was used in this trial. It is a costimulation blockade agent which was tested for its ability to prevent aGvHD.

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 Implications: This is the first description of the primate GvHD transcriptome. This network approach has identified previously unappreciated genes and pathways associated with GvHD, for which several novel therapeutic strategies are immediately available for pre-clinical and clinical evaluation. Disclosures: No relevant conflicts of interest to declare.

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.

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 10 fold compared to controls, p 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.

Abstract 1008 Regulatory T cells (Tregs) have been shown to be potent inhibitors of autoimmunity, and to be capable of suppressing alloimmune responses that occur during both allograft rejection and graft-versus host disease. However, they have yet to gain widespread use clinically, due in part to the fact that it remains extremely costly and difficult to produce them in sufficient numbers and with sufficient suppressive capacity to significantly impact the alloimmune response. Here we have used our established non-human primate model to demonstrate that significant Treg expansion (up to 600-fold in 21 days) can be maintained, and suppressive capacity enhanced by exposing Treg cultures to a short burst of sirolimus at the end of the culture period. Using a highly sensitive and specific in vitro CFSE-MLR assay we show that Tregs significantly inhibit allo-proliferation of multiple T cell subpopulations including both CD4+ and CD8+ T cells (3.2 and 2.7-fold inhibition of proliferation, respectively), as well as their CD28+CD95+ and CD28-CD95+ subpopulations (2.2 and 2.1 and 1.9 and 2.7-fold inhibition of CD4+ and CD8+ subpopulation proliferation, respectively). Tregs were able to combine in vitro with the newly FDA-approved CTLA4-Ig analog belatacept to enhance the inhibition of alloproliferation that occurred with either agent alone (4.8-fold inhibition of CD8 T cell proliferation with Tregs + belatacept, compared to 3.0-fold or 1.9-fold inhibition of CD8 T cell proliferation with Tregs or belatacept alone, respectively). Importantly, we have found that the suppressive activity of ex-vivo expanded Tregs could be further enhanced by pulsing with sirolimus. Thus, while long-term culture of Tregs in the presence of sirolimus (1–1000 nM) profoundly inhibited Treg expansion (50–800 fold inhibition of expansion when cultured in the presence of 1–1000 nM sirolimus), a 48 hour pulse of sirolimus (100 nM) on days 20–21 of culture completely preserved Treg yields while doubling their suppressive function against CD8 proliferation when compared to unpulsed Tregs, p Our results suggest that the creation of SPTs may provide a novel avenue by which to achieve enhanced Treg-based suppression of alloimmunity, in a manner that is amenable to large-scale ex-vivo expansion and to combinatorial therapy with novel, costimulation-blockade-based immunosuppression strategies. Disclosures: No relevant conflicts of interest to declare.

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.

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.

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.

Abstract 1461 Latent herpesvirus infections are ubiquitous and are important causes of transplant complications mediated by both direct viral pathology and anti-viral-related immune dysregulation. An emerging paradigm, heterologous immunity, suggests that alloreactivity arises in a subset of anti-viral memory T cells during infection, putting patients at increased risk for rejection. Murine γ-herpesvirus 68 (MHV68) provides a rodent model of human herpesvirus infections, including EBV, allowing the study of herpesvirus infections in transplantation. We have previously shown that latent infection with wild type (WT) MHV68 induces resistance to engraftment in a non-myeloablative, costimulation-blockade- (CoB) based bone marrow transplant model. Resistance to donor-engraftment was not observed in mice infected with a mutant virus lacking the viral M1 gene. (Stapler, et al. J Immunol 2008). Based on these findings, we sought to determine the mechanism(s) by which MHV68 induces heterologous alloimmunity and transplant rejection. B6 mice were infected i.p. with WT MHV68 or M1 mutant MHV68 and viral latency was defined as 6–8 weeks post infection (p.i.) after which some mice received allogeneic (BALB/c) BMT or skin grafts (SG). Transplanted mice received 0.5 mg each of anti-CD154 and CTLA4-Ig on days 0, 2, 4, and 6 relative to transplantation. BMT mice additionally received 0.6 mg busulfan on the day before transplant. Analysis of graft survival and multicolor flow cytometric analysis of immune phenotype was then performed. In addition to inducing rejection of donor bone marrow, as we previously observed, our new results indicate that latent infection with WT MHV68 decreases the length of allogeneic SG survival. While SG in non-infected, CoB-treated recipients have a mean survival time (MST) of 26 days (n = 5), MHV68 decreases this to 13 days (n=10, p=.0004*), similar to that in non-CoB treated recipients (11 days, n=5). This suggests a broad impact of latent MHV68 infection on transplant rejection. Flow cytometric analysis has revealed a unique cell population that may increase the risk of transplant rejection in MHV68-infected mice. MHV68 infection is associated with the expansion of a distinct CD8 T cell population with decreased per-cell CD8 expression and a unique phenotype, CD8Int. In non-infected mice, CD8Int are uncommon (11% of CD8s), but are highly induced by WT MHV68 (76%) and less so by an M1 mutant strain (35%, p165 days p.i. In infected mice, T cells specific for two MHV68 epitopes (p56 and p79) are exclusively CD8Int by tetramer staining, identifying the CD8Int compartment as containing anti-viral T cells. Given the correlation of high-level induction of CD8Int and costimulation blockade-resistant transplant rejection, we sought to determine the mechanisms by which these cells might heterologously induce graft rejection. After infection with WT MHV68, CD8Int are significantly more activated vs CD8Nml (CD8 T cells with unchanged CD8 expression) as assessed by increased expression of CD44 (mean fluorescence intensity [MFI] 2746 vs 1640, p=.0025*) and KLRG1 (2553 vs 1003, p=.004*). Despite high KLRG1 expression, a significant fraction of CD8Int also express the memory marker CD127 suggesting that CD8Int consists of both effector and effector memory T cells. CD8Int exhibit decreased expression of the costimulatory molecules ICOS (MFI Int vs Nml, 171 vs 329, p Disclosures: No relevant conflicts of interest to declare.

Abstract 2455 Poster Board II-432 Introduction: While hematopoietic stem cell transplantation (HSCT) offers a cure for many hematologic diseases, it remains plagued by often fatal graft-versus-host disease (GvHD). Despite the inadequacy of current GvHD prevention strategies, especially for MHC-mismatched HSCT, the pace of the clinical introduction of novel therapeutics has been slow, likely due to the lack of a suitable translational model to rigorously test the immunologic and clinical impact of novel biologic therapies. Among the most promising of these therapies include those that block T cell costimulation blockade. While they have been used for both autoimmune disease and to prevent rejection of solid organ transplants, costimulation blockade reagents have not yet been evaluated for efficacy in preventing clinical GvHD. Here we describe a novel primate model of MHC-mismatched GvHD, that has allowed us, for the first time, to evaluate the mechanisms controlling GvHD in a primate translational system, and to evaluate the efficacy of costimulation blockade for the prevention of primate GvHD, even across haplo-MHC barriers. Methods: Using DNA microsatellite-based pedigree analysis and MHC haplotype determination, we have developed the first MHC-defined Rhesus macaque HSCT system. MHC haplo-identical transplant pairs were chosen, and recipients prepared for transplant with TBI (8 Gy, as a single dose, with lung shielding to 6 Gy). Animals were either treated with no immunosuppression post-transplant (controls) or with a costimulation blockade-based regimen which included CD28/B7 blockade with abatacept (20mg/kg every 7 days), CD40/CD154 blockade with the 3A8 anti-CD40 monoclonal antibody (maintenance dosing at 5mg/kg twice weekly) as well as sirolimus to maintain serum trough levels between 5-10 ng/mL. Either leukopheresis-derived peripheral blood stem cells or bone marrow was used for transplant (average total nucleated cell dose = 9.3 +/-2.7×108/kg; average CD3+ cell dose = 1.1 +/- 0.88 ×108/kg) Donor engraftment was measured by microsatellite analysis, and GvHD was graded clinically using standard scales. The immune phenotype after transplant was determined by multicolor cell- and serum-based flow cytometric analyses. Results: Seven haploidentical transplants have been completed. Three controls received no immunosuppression. These animals demonstrated rapid and complete donor engraftment, with donor T cell activation and proliferation occurring within one week of transplant, coincident with the onset of severe clinical GvHD, which predominantly targeted the GI tract. Flow cytometric analysis showed loss of CD127 expression on both CD4+ and CD8+ T cells, consistent with their rapid clonal expansion and differentiation. Multiplexed luminex cytokine analysis demonstrated high-level secretion of the inflammatory cytokines IFNγ, and IL18, as well as the counter-regulatory cytokine IL-1RA. Importantly, no rise in TNF, IL-1b, nor IL17 was measured despite severe GvHD. In contrast, four treated animals received a haplo-identical BMT in the setting of abatacept/anti-CD40 and sirolimus for GvHD prophylaxis. All of these recipients demonstrated rapid donor engraftment, but, unlike the controls, they were protected against clinical GvHD—they displayed neither the skin rash nor the profuse diarrhea noted in the control animals. Flow cytometric analysis demonstrated maintenance of CD127 expression on both CD4+ and CD8+ T cells. Furthermore, luminex analysis revealed that expression of IFNγ, IL18 and IL-1RA were all normal in the setting of GvHD prophylaxis with costimulation blockade and sirolimus. Conclusions: We have established a robust model of haplo-identical HSCT and GvHD using an MHC-defined Rhesus macaque colony. This model has allowed us to begin to determine the mechanisms underlying GvHD during primate haplo-identical BMT and to assess the efficacy of novel regimens to prevent this disease. We find that unprotected primate GvHD is characterized by rapid T cell proliferation, with concomitant loss of expression of CD127 on both CD4+ and CD8+ T cells. In addition, it is associated with a cytokine storm, including high level secretion of IFNγ, IL18 and IL-1RA into the serum. Finally, we find that CD28/CD40-directed costimulation blockade in combination with sirolimus can effectively inhibit both the clinical and cellular hallmarks of GvHD during haplo-identical BMT, and thus may deserve close clinical scrutiny as a possible prophylaxis strategy during these high risk transplants. Disclosures: No relevant conflicts of interest to declare.

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.

In order to understand the mechanisms underlying the increased rates of stem cell transplant rejection in patients with sickle cell disease (SCD) we have developed a mouse model of transplant rejection utilizing the Berkeley SCD mouse. We find that Berkeley SCD mice exhibit a significantly higher rate of transplant rejection when transplanted with fully allogeneic bone marrow from an SJL donor after non-myeloablative conditioning and peri-transplant T cell costimulation blockade. Thus, while control C57BL/6 (BL/6) mice or hemizygous litter-mates exhibit 80–85% engraftment rates, only 20% of Berkeley SCD mice engraft with donor marrow. Transplant rejection in the SCD mice is mediated by CD8+ and/or NK1.1+ cells, as depletion with antibodies directed at either of these cell-surface molecules increases engraftment rates to 75–90%. To dissect the mechanistic underpinnings of these observations, we performed a multiparameter phenotypic and functional flow-cytometric analysis of leukocytes from the peripheral blood, lymph nodes and spleen from SCD mice, and compared them to the two non-SCD controls. We observed that SCD mice displayed striking phenotypic differences compared to normal mice, but that their hemizygous litter-mates were indistinguishable from the BL/6 controls, underscoring the sickle-specific nature of our observations. SCD mice showed minimal phenotypic variation in the peripheral blood, but the spleens and lymph nodes from SCD mice were highly abnormal. Sickle mice exhibited ∼10-fold more splenic NK and NK-T cells than either the BL/6 or hemizygous controls. These cells produced large quantities of both TNF-α and IFN-γ, potentially contributing to the inflammatory milleu that is associated with SCD. Furthermore, while absolute numbers of splenic T cells were not different in sickle mice compared to controls, their phenotype was skewed toward a highly activated state. Thus, while many (40%) splenic memory T cells from normal controls exhibited a Central Memory (Tcm) phenotype, there were fewer Tcm cells in SCD mice (6%), and a skewing of the memory repertoire towards a more activated Intermediate Memory phenotype (71% in SCD mice compared to 25% in controls). Finally, in ELISpot analysis, lymph node cells from naive sickle mice, having never been previously exposed to allo-antigen, were able to secrete IFN-γ when briefly co-cultured with donor SJL stimulators, a phenomenon not observed with either control BL/6 or hemizygous controls, again, pointing to the increased inflammatory milieu and potential for rejection in the sickle mice. In summary, we have observed evidence for a multi-faceted increase in immune activation in SCD mice, which correlates with their increased transplant rejection. These results point both toward possible therapeutic targets aimed at decreasing SCD-mediated transplant rejection and toward future studies in SCD patients, in order to determine whether similar mechanisms of immune activation also occur in the patient population.

Gene therapy and stem cell transplantation are attractive potential therapies for sickle cell disease (SCD). Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS), but have not addressed whether or not the increased ROS may alter the bone marrow (BM) microenvironment or affect stem cell function. Using the Berkeley sickle mouse model, we examined the effects of sickle cell disease on hematopoietic stem cell function and the bone marrow microenvironment. We transplanted C57BL/6 (control) BM into C57BL/6 and homozygous sickle mice. Recipients received 2 × 106 BM cells and a conditioning regimen consisting of busulfan, anti-asialo GM1, and co-stimulation blockade (anti-CD40L and CTLA4-Ig). Following transplantation, sickle mice demonstrated increased donor cell engraftment in the peripheral blood compared to normal mice (58.3% vs. 33.1%, respectively). Similarly, BMT in a fully allogeneic system also resulted in enhanced engraftment in sickle recipients. Next we analyzed whether or not engraftment defects exist within the BM stem cell population of sickle mice. In vitro colony forming assays showed a significant decrease in progenitor colony formation in sickle compared to control BM. By flow cytometry, we determined that there was a significant decrease in the KSL (c-Kit+, Sca-1+, Lineage−) progenitor population within the BM of sickle mice. Cell cycle analysis of the KSL population demonstrated that significantly fewer sickle KSL cells were in G0 phase compared to control, suggesting that there are fewer quiescent stem cells in the BM of sickle mice. To assess the potential role of ROS and glutathione depletion in sickle mice, we tested the engraftment efficiency of KSL cells from untreated and n-acetyl-cysteine (NAC) treated control, hemizygous sickle (hemi), and sickle mice in a competitive repopulation experiment. Peripheral chimerism showed an engraftment defect from both hemizygous and homozygous sickle mice such that control KSL cells engrafted > hemi > sickle at a ratio of 1 : 0.4 : 0.25. Treatment with NAC for four months prior to transplantation partially restored KSL engraftment (control : hemi : sickle; 1 : 0.97 : 0.56 ). We have demonstrated that congenic and allogeneic BMT into sickle mice result in increased donor cell engraftment in the sickle recipients. Both the decreased number of KSL cells and the decreased percentage of quiescent KSL cells in the sickle mice indicate that more stem cells in the transgenic sickle mouse model are mobilized from the BM environment. The engraftment defect of sickle KSL cells that was partially ameliorated by NAC treatment suggests that an altered redox environment in sickle mice may contribute to the engraftment deficiencies that we observed.

Introduction: Immunologic tolerance remains an elusive goal of transplantation. In mice, mixed-chimerism and donor-specific tolerance can be induced by blocking the CD28/CD40L T-cell costimulatory pathways after bone marrow transplant (BMT). However, large doses of marrow (~1x109 cells/kg) are required, and these regimens have not yet been successfully translated to clinical practice. There is a growing body of evidence that NK cells may play a central role in the failure of low doses of donor bone marrow to engraft, but the mechanisms underlying NK alloreactivity remain to be determined. Methods: (1) BMT in the presence of CD28/CD40L T cell costimulation blockade was performed using C57BL/6 (B6) recipients and Balb/C donor bone marrow. The role of host-anti-donor NK alloreactivity in preventing engraftment was determined by specifically depleting B6 NK cells. The contribution of the NK cell-surface receptor, LFA1 to NK alloreactivity was determined with the anti-LFA1 blocking antibody M17/5.2. (2) An in vivo NK alloreactivity assay was developed that should allow the investigation of the mechanism of NK alloreactivity and the molecular mediators of this process. In this assay, CFSE-labeled B6 splenocytes were adoptively transferred into B6xBalbC F1 progeny. As such, alloreactivity was specifically mediated by NK cells. NK alloreactivity was measured flow-cytometrically by the disappearance of the CFSE-labeled B6 population. Results: Transient depletion of recipient NK cells resulted in increased donor stem cell survival and the induction of stable mixed-chimerism and tolerance despite BMT with low doses (≤2x106 cells) of donor bone marrow. This effect was specific to allogeneic donor cells: depletion of NK cells did not increase engraftment of syngeneic bone marrow. Blocking the adhesion molecule, LFA-1 recapitulated the effects of whole-scale NK depletion. Newly emergent NK cells exhibited significantly lower expression of the donor-specific activating receptor, Ly49D, and these NK cells did not exhibit in vivo alloreactivity. These results suggest that the NK repertoire in the mixed-chimeric setting exhibited donor-specific tolerance. Using the in vivo hybrid resistance NK alloreactivity assay, we measured 80% NK-specific target killing 8 days after adoptive transfer. Significantly less killing occurred at 2, 4, and 6 days. Pre-sensitizing the recipient for 4 days increased the efficiency of killing—from 50% to 80%, suggesting a potent activation phenomenon required for efficient NK allorecognition and/or cytotoxicity. Implications: These results reveal the importance of NK alloreactivity in the acquisition of mixed-chimerism after BMT at limiting stem cell doses, and suggest that clinical approaches to tolerance-induction transplantation may require mechanisms to control NK alloreactivity.

While hematopoietic stem cell transplantation (HSCT) represents the only curative therapy for sickle cell disease, sickle patients undergoing HSCT face many complications, including an increased risk of graft rejection compared to non-sickle patients. We have used the Berkeley sickle mouse model to study the potential mechanisms underlying this increased risk of rejection. Using a CD28/CD40 costimulation-blockade-based non-myeloablative HSCT regimen, we transplanted Berkeley sickle mice with fully allogeneic SJL bone marrow. While the vast majority (>85%, n=25) of control C57BL/6 animals became stably chimeric and immunologically donor-tolerant with this transplant regimen, sickle mice were much more prone to reject the transplant (~20% graft acceptance, n=25). Both CD8+ cells and NK1.1+ cells were found to contribute to this rejection, as depletion of either of these cell populations led to a marked increase in the percent of engrafted mice (>85% graft acceptance, n=15–25), while depletion of CD4+ cells led to the opposite effect, with 0% (n=25) animals engrafted in this depletion cohort. The increased propensity of HSCT rejection in the Berkeley sickle mice may, in part, be explained by the presence of increased numbers of donor-reactive T cells (5–10-fold compared to C57BL/6 controls) in naïve sickle mice, despite their lack of exposure to donor antigens, and their housing in a Specific-Pathogen-Free environment. We speculate that these increased numbers of anti-donor T cells may occur as a result of heightened inflammation in the context of active sickle cell disease, which could lead to increased expansion and persistence of a T cell repertoire containing anti-donor heterologous T cell immunity. This heterologous immunity may have a profound effect on the success of HSCT for sickle cell disease, especially when non-myeloablative regimens are employed.

The last several decades have brought significant immunologic advances in the fields of inflammation, infection, and transplantation tolerance. Heretofore, our understanding of how complex immune interactions occur has been limited to static in situ tissue analysis and in vitro dynamic studies using isolated cells devoid of stromal elements typically present in vivo. Recent advances in molecular, flow cytometry, and intravital imaging have provided new insight into the dynamic interactions occurring among a variety of cells within the bone marrow (BM) and immune systems, ranging from undifferentiated hematopoietic progenitors to fully committed effector memory cells, which will likely have direct clinical and translational implications. In this review we highlight how the application of these cutting-edge technologies will sculpt the landscape of the next generation of immunologic advances.