ALBERT

Description: Background A critical barrier to progress in allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been a lack in understanding regarding why some transplant recipients of HLA-matched transplant grafts develop severe graft-versus-host disease (GvHD) while other recipients have relapse of their cancer without GvHD. Patients who develop a modest degree of acute and/or chronic GvHD have less relapse and optimal survival after allogeneic BMT. Thus, a mechanistic understanding of regulation of donor T-cell activation after allo-HSCT is needed. Using mouse models, Desmarets et al. have shown that pre-transplant leukoreduced RBC transfusions can cause recipient immunization against minor histocompatibility antigens (miHA) and activation and expansion of recipient T-cells that recognize donor miHA, contributing to rejection of subsequent allo-HSCT (Blood. 2009; 114:2315). Preliminary data from our lab suggest that leukoreduced RBC transfusions given concurrently with allo-HSCT can also increase post-transplant activation and expansion of donor T-cells, an effect which may lead to increased GvHD after transplant. Here, we have conducted a retrospective study of post-transplant RBC transfusions and acute GvHD (aGvHD) in allo-HSCT patients. We hypothesized that increased numbers of transfusions during the 30-day post-transplant period would be correlated with increased severity of aGvHD in these patients. Methods We conducted a retrospective analysis of RBC transfusion records and aGvHD data collected for 181 adult allo-HSCT patients who received their transplants at Emory University Hospital (EUH) between 2004 and 2009. Nine patients were excluded who died 〈 50 days post-transplant without developing aGvHD, since this was too early to determine aGvHD occurrence. Of the remaining 172 patients studied (median age 48 yrs at time of transplant, range 18-72), 88 (49%) were male and 84 (51%) were female. Patients had received either matched related HSCT (n=69, 40%) or matched unrelated HSCT (n=103, 60%) for treatment of SAA (n=7), BAL (n=2), ALL (n=18), AML (n=69), hemolytic anemia (n=2), CLL (n=6), CML (n=8), HD (n=5), MDS (n=23), myelofibrosis (n=6), MM (n=7) or NHL (n=19). For pts who developed aGvHD, the onset time ranged from 1 to 139 days post-transplant, with a median of 30 days. No aGvHD (grade 0) was diagnosed in 58 pts (34%), while 37 pts (21%) developed grade 1 aGvHD and 77 pts (45%) developed grade 2-4 aGvHD. The number of ABO matched, irradiated RBC units transfused 0 - 30 days post-transplant was tallied for each patient, ranging from 0 (no transfusions, n=13, 7.6% of pts) to 26 units, with an average of 5.6 and median of 4 units. All transfusions during this timeframe were administered at EUH. The median follow up time was 22 months post-transplant (range, 1.1 – 96.1 months). Results Pts were assigned to two groups, those who developed grade 0-1 aGvHD (n=95, 55%) or grade 2-4 aGvHD (n=77, 45%) within 140 days post-transplant. This study did not include analysis of late-onset aGvHD or chronic GvHD past this time point. Patients with grade 2-4 aGvHD had a higher average number of transfusions 0 - 30 days post-transplant compared with patients having grade 0-1 aGvHD (6.5 vs. 4.9 units, p = 0.02). Receiver-operator characteristics (ROC) analysis showed that a cutoff value of 〉 4 transfusions 0 - 30 days post-transplant had 56% sensitivity and 65% specificity to predict development of grade 2-4 aGvHD. When tested by logistic regression in a multivariate model, this cutoff value had a highly significant correlation with grade 2-4 aGvHD, with an odds ratio of 2.83 and p value = 0.0024. Other covariates including patient age, gender, and type of transplant (related vs. unrelated) were not significantly associated with aGvHD outcome. Conclusion Our retrospective analysis identified a significant positive correlation between the number of post-transplant RBC transfusions and severity of aGvHD after allo-HSCT. Additional studies are planned to determine whether RBC transfusions 0 - 30 days post-transplant stimulate allo-reactive T-cells via allo-antigen presentation or by otherwise promoting inflammation, and if one or both of these mechanisms contribute to increased GvHD. If so, it may be possible to develop strategies for optimization of RBC transfusion practices to reduce the risk of severe aGvHD after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Background Graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplant (allo-HSCT) is caused by CD4+ and CD8+ donor T cells directed against mismatched recipient antigens, presented in the context of donor MHC-II (indirect pathway) and recipient MHC-I (direct pathway). Recently, the presence of 'cross-dressed' CD11c+ antigen presenting cells (APCs) expressing both donor and recipient type MHC-I molecules has been demonstrated in animal organ and HSCT transplant models supporting 'semi-direct' pathway of allo-activation (Wang et al, Blood. 2011).These APCs can efficiently present allo-antigens to both CD4+ and CD8+ T cells and activate immune responses that could lead to allograft rejection or GVHD. Exchange of membrane fragments and associated proteins between cells, termed trogocytosis, generates cross-dressed APCs.We sought to test whether cross-dressed APCs facilitate antigen presentation to donor T cells and initiate GVHD following allo-HSCT. Further, we tested an array of drugs as inhibitors of trogocytosis, to interrupt the semi-direct pathway of allo-antigen presentation. Methods In vivo experiments used a B6(H2Kb) ˆ B10.BR(H2Kk) murine transplant model. Spleens of transplanted mice were analyzed on days 10, 15, 20 post-transplant for presence of cross dressed CD11c+cells, and their expression of CD80, CD86 and MHC-II by flow cytometry. Cross dressed donor CD11c+ FACS sorted cells from recipient spleens were co-cultured with CFSE labeled donor type T-cells for 6 days, and T-cell proliferation was measured as dilution of CFSE by flow cytometry. In vitro experiments used primary MLR consisting of CFSE labeled B6 bone marrow cells co-cultured with PKH26 (membrane dye) labeled B10.BR splenocytes. B6 antigen presenting cells were analyzed by flow cytometry for the presence of CFSE+PKH26+ double positive cells generated by trogocytosis. Pharmacological inhibitors of cytoskeleton function were added to the primary MLR and their effect on trogocytosis as well as T cell proliferation was assessed. Results Cross-dressed donor CD11c+ APCs were generated in vivo following allo-HSCT (Figure 1). Recipient spleens showed that 50%, 28.6% (p=0.01) and 12% (p=0.02) of donor type CD11c+ cells were cross dressed on days 10, 15 and 20 respectively post transplant (n=5). These cross dressed APCs expressed higher levels of co-stimulatory molecules CD80 (p

Description: Background: In allogeneic BMT patients, the presence of allo-reactive donor CD4+ T cells in the graft were reported to be the primary cause of GvHD. Moreover, donor T-cells are required to promote the stem cell engraftment and to decrease the disease relapse. A number of studies also reported that a subset of CD4+CD25+ T cells usually generated de novo from the thymus that expressed FoxP3 regulate the T cells allo-reactivity in vivo. Thus, to establish a therapeutically useful adoptive T-cells immunotherapy, we depleted the CD4+ T cells from the graft and transplanted along with T cell depleted (TCD) BM cells in clinically relevant parent to F1 experimental allogeneic BMT model. Our hypothesis is that CD4-depleted graft will not cause GvHD, preserve the thymic function, homeostatically produce donor BM-derived CD4+ T cells along with FoxP3+CD4+CD25+ regulatory T cells with beneficial anti-opportunistic infection and anti-tumor effects. Methods: We used a parent (C57BL/6) to (C57BL/6 X BALB/c)CB6F1 allogeneic BMT model with a combination of TCD BM and splenocytes as the hematopoietic graft. CD4+ or CD8+ cells were selectively depleted from the splenocytes of C57BL/6 donor mice using MACS column. 1×106 CD4-depleted splenocytes or a mixture of 2×106 CD8-depleted and 1×106 CD4-depleted splenocytes and/or grafts containing 10×106 unfractionated splenocytes along with 5×106 TCD BM cells harvested from the congeneic C57BL/6 donor mice, were adoptively transferred to lethally irradiated (11Gy) CB6F1 mice. GvHD was monitored twice weekly by weight loss and other clinical signs. After 50 days post transplant recipients mice were bled or sacrificed and lymphocytes isolated from blood and different organs were analyzed by multicolor FACS. Results: Within 50 days of transplant the recipients of CD4-depleted splenocytes had 100% survival without GvHD whereas recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes suffered from severe GvHD (%weight loss below 20%) with 50% survival. Surprisingly, very significantly expansion of total CD4+ T cells (37% ± 7% of lymphocytes, CD4:CD8 ratio 6:1) occurred in the blood of recipients of CD4-depleted splenocytes. In contrast the recipients of mixture of CD4- and CD8-depleted splenocytes DLI or whole splenocytes had only few CD4+ T cells (~2% ± 2% of lymphocytes, CD4:CD8 ratio 1:2). Over 90% of the CD4+ T cells in the blood of recipients of CD4-depleted splenocytes were from the donor BM and included significantly higher number of CD25+CD4+ T cells compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes. Similarly, significantly increased numbers of FoxP3+CD25+CD4+ regularity T cells were also found in the spleen and thymus of recipients of CD4-depleted splenocytes compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes (p

Description: The intestinal microbiota in allogeneic bone marrow transplant (allo-BMT) recipients modulates graft-versus-host disease (GVHD), a systemic inflammatory state initiated by donor T cells that leads to colitis, a key determinant of GVHD severity. Indole or indole derivatives produced by tryptophan metabolism in the intestinal microbiota limit intestinal inflammation caused by diverse stressors, so we tested their capacity to protect against GVHD in murine major histocompatibility complex–mismatched models of allo-BMT. Indole effects were assessed by colonization of allo-BMT recipient mice with tryptophanase positive or negative strains of Escherichia coli, or, alternatively, by exogenous administration of indole-3-carboxaldehyde (ICA), an indole derivative. Treatment with ICA limited gut epithelial damage, reduced transepithelial bacterial translocation, and decreased inflammatory cytokine production, reducing GVHD pathology and GVHD mortality, but did not compromise donor T-cell-mediated graft-versus-leukemia responses. ICA treatment also led to recipient-strain-specific tolerance of engrafted T cells. Transcriptional profiling and gene ontology analysis indicated that ICA administration upregulated genes associated with the type I interferon (IFN1) response, which has been shown to protect against radiation-induced intestinal damage and reduce subsequent GVHD pathology. Accordingly, protective effects of ICA following radiation exposure were abrogated in mice lacking IFN1 signaling. Taken together, these data indicate that indole metabolites produced by the intestinal microbiota act via type I IFNs to limit intestinal inflammation and damage associated with myeloablative chemotherapy or radiation exposure and acute GVHD, but preserve antitumor responses, and may provide a therapeutic option for BMT patients at risk for GVHD.

Description: Background: Therapeutic options for steroid-refractory chronic graft-versus-host-disease (cGVHD) are limited. Extracorporeal photopheresis (ECP) is a photoimmune therapeutic modality to treat cGVHD that is tolerated relatively well, but its mechanism has not been fully defined. One model for the mechanism of ECP in cGVHD is dendritic cell (DC) depletion and T-cell modification (Alcindor, T, et al., BLOOD2001, 98:1622). We tested this hypothesis by determining the numbers of circulating DCs and T-cells prior to ECP and during therapy in patients with cGVHD, and correlating cell numbers with response. Methods: This study was IRB approved. We studied 25 adult pts (median age 43 yrs, range 23–71) with histories of hematological malignancies including NHL (n=7), AML (n=5), CML (n=5), ALL (n=3), MDS (n=3), Hodgkin’s lymphoma (n=1), and CLL (n=1), who developed cGVHD after allogeneic, HLA-matched HPCT. Ten pts had progressive, 9 pts had de novo, and 6 pts had interrupted cGVHD. Initial treatment of cGVHD included corticosteroids in all pts. At the time of ECP initiation, pts were either dependent upon corticosteroids for control of cGvHD (21 pts), or steroid-intolerant (4 pts). No pts had received ECP prior to this study. ECP was administered 2 consecutive days every week for the first 2 months, two times a week every other week for 2 months, and then two times a week once a month. In addition to ECP, pts received steroids (21), MMF (n=13), FK506 (n=15), cyclosporine (n=3), MTX (n=3), rapamycin (n=1), rituximab (n=1) or pentostatin (n=1). Sites of cGVHD included skin (n=25), oropharynx (n=7), liver (n=5), gut (n=4), lung (n=1), and eye (n=1). A good response was defined as having 〉 50% reduction in the corticosteroid dose within 4 months of starting ECP, with improved or stable lesions on skin and other sites. For steroid-intolerant pts, clinical parameters such as improvement in skin condition were used to identify responders. Peripheral blood mononuclear cells were analyzed before ECP began and every 2 months during ECP therapy. The numbers of plasmacytoid DCs (pDC, Lin− CD123+ CD11c− HLA-DR+), myeloid DCs (mDC, Lin− CD123− CD11c+ HLA-DR+), and CD4+ and CD8+ T-cells in blood were determined by flow cytometry. Results: Median follow up of the 25 pts was 47.1 months (range, 8.6–90.9) from the time of transplant. The median number of ECP treatments was 26 (range 2–68). Fourteen pts (56%) had good response, and 11 were non-responders. The median time between HPCT and onset of cGVHD was similar for responders (8.6 months, range 3.3–34.7) and non-responders (6.1, range 3.4–43.8, p=0.52). The median time between HPCT and ECP was also similar for the two groups (32.3 months, range 13.1–60.0, vs. 21.9 months, range 4.1–47.5, respectively, p=0.12). Responders had an estimated 2-yr survival of 88% after starting ECP, vs 18% for non-responders (p=0.004). Two responders died at 11.2 and 31.2 months after starting ECP, compared with 7 non-responders (median 4.4 months, range 2.8–22.1). Non-responders had a relative risk of death of 11.6 compared with responders (p=0.022). Average prednisone doses for responders and non-responders were comparable, averaging 24.3 and 41.8 mg/day, respectively (p=0.11). Responders had higher baseline numbers of pDCs (average 5.8 vs. 0.6 cells/mcL, p=0.025) and mDCs (average 15 vs. 3.8 cells/mcL, p= 0.01) compared with non-responders. Baseline CD4+ T-cell numbers were higher in responders compared with non-responders (average 623 vs. 178 cells/mcL, p=0.005), as were CD8+ T-cell numbers (712 vs. 251 cells/mcL, p=0.047). Contrary to the original hypothesis, there were no consistent changes in the numbers of circulating DCs and T-cells among responders over a 12-month period. Receiver-operator characteristics (ROC) analysis showed that baseline numbers of blood mDCs of 〉3.7 cells/mcL prior to ECP had 79% sensitivity and 82% specificity to predict response of cGvHD patients to ECP. Conclusion: Our results demonstrate that higher numbers of circulating DCs and T-cells predict response to ECP in pts with cGVHD. Response to ECP was significantly associated with improved survival in univariate and multivariate analyses (p

Description: We are investigating methods to reduce the graft-versus-host disease (GVHD) potential of donor T-cells while retaining graft-versus-leukemia (GVL) activity in allogeneic HSCT. Previous investigations by our group and others in have shown that naive CD4 T-cells induce severe acute GVHD, while memory CD4 T-cells do not induce GVHD but retain GVL activity in murine transplant models. These findings have led to studies for the development of methods to increase the number of memory T-cells available for transplant. The calcium ionophore, ionomycin, is a T-cell activating agent and mitogen. By increasing intracellular Ca2+ levels, ionomycin is induces T-cell activation through signaling mechanisms including phospholipase C activation, hydrolysis of phosphoinositides, and activation of protein kinase C. Differences in memory and naive T-cell responses to ionomycin have been attributed to resistance of memory T-cells to increases in Ca2+. Memory T-cells lack intracellular Ca2+ stores, and are also resistant to influx of Ca2+. Brief low dose ionomycin exposure (20min, 2μM) of T-cells, leading to increased density of naive T-cells, has previously been exploited as a method for separating memory and naive T-cells by Percoll gradient separation. Since ionomycin exposure induces T-cell activation through native Ca2+ dependent signaling mechanisms, we hypothesized that ionomycin-treated T-cells would shift to an activated/memory T-cell phenotype. Murine splenic T-cells were treated with 1.3μM ionomycin for 4hr. Memory and naive T-cell subsets and activation markers were analyzed by flow cytometry. 75% and 85% of untreated CD4 and CD8 T-cells, respectively, had the CD62L+ naive phenotype. These numbers were dramatically reduced to 7% and 17% after ionomycin exposure, representing a shift to the memory T-cell phenotype. Viability of T-cells was not significantly affected. The majority of remaining CD62L+ naive T-cells expressed activation markers CD25 and CD69. The fraction of CD4+CD25+Foxp3+ regulatory T-cells was also determined by intracellular staining of the transcription factor and co-expression of surface markers. CD4+CD25+Foxp3+ regulatory T-cells represented 4% of untreated CD4 T-cells and 3% of ionomycin-treated CD4 T-cells. While ionomycin has been used for many years in studies of T-cell activation, to our knowledge this is the first demonstration of a rapidly-induced shift of naive T-cells to a memory phenotype. A pilot experiment was conducted testing the GVHD activity of ionomycin-treated splenocytes (SP) in B6→ (B6 × Balb/C)CB6F1 recipients. 5 × 106 T-cell depleted bone marrow cells (TCD-BM) were transplanted along with 10 × 106 treated or untreated SP. Mice that received untreated SP all died from acute GvHD by 34 days after transplant, while all recipients of ionomycin-treated SP survived until the experiement was terminated at day 49 (average weight loss was 25%, data not shown). Continuing experients will refine the dose to further reduce GVHD symptoms and also test GVL activity of the treated cells. Treatment of donor T-cells with ionomycin may represent a clinically applicaple method to engineer donor lymphocyte infusions that are safer for HSCT patients. Figure 1. Survival of CB6F1 recipients after transplant with 5 million B6 TCD-BM and 10 million B6 splenocytes that were either untreated or stimulated ex-vivo with a combination of PMA, ionomycin and brefeldin-A for 4 hours. 5 recipient animals per group. The experiment was terminated at day 49. Figure 1. Survival of CB6F1 recipients after transplant with 5 million B6 TCD-BM and 10 million B6 splenocytes that were either untreated or stimulated ex-vivo with a combination of PMA, ionomycin and brefeldin-A for 4 hours. 5 recipient animals per group. The experiment was terminated at day 49.

Description: Abstract 4909 Introduction: The proteasome inhibitor bortezomib and the histone deacetylase inhibitor (HDACi) romidepsin are both able to induce tumor cell death in single-agent clinical studies. In vitro studies using the combination of bortezomib and romidepsin have demonstrated synergistic activity against tumor cell lines, and clinical trials using both classes of agents have demonstrated efficacy. Bortezomib and romidepsin induce thrombocytopenia that is transient and reversible. It is thought that these agents temporarily disrupt the process of platelet budding from megakaryocytes, in contrast to long-term thrombocytopenia induced by classical cytotoxic chemotherapeutic agents that deplete the megakaryocyte population. However, thrombocytopenia has been a dose-limiting factor when proteasome inhibition is combined with HDACi. We have previously shown that platelet recovery after bortezomib exposure in mice results in transient high elevation of circulating platelets and that the megakaryocyte population is preserved, with increased numbers of immature forms suggesting a rebound effect (Lonial et al., Blood, 2005). Here, we conducted additional murine studies to determine whether combination bortezomib + romidepsin induces increased thrombocytopenia compared to the single-agent drugs, examine platelet recovery kinetics, and test effects on bone marrow megakaryocytes. Methods: Six groups of 12 female BALB/c mice were used: Control, 1mg/kg bortezomib, 2mg/kg bortezomib, 1mg/kg romidepsin, 2mg/kg romidepsin, and the combination of 1mg/kg bortezomib + 1mg/kg romidepsin. Only one injection timepoint was used (d1, via tail vein) to narrow the focus of thrombocytopenic effects. Three mice per group were bled on subsequent days (to d11) for CBC including WBC and platelet counts. Plasma samples were frozen for batch analysis of thrombopoietin (TPO) levels by ELISA. Subgroups of 2–3 mice were sacrificed at d3, 5, and 8 for analysis of bone marrow megakaryocyte ploidy. Bone marrow cells were fixed in ethanol and analyzed by flow cytometry to determine the representation of CD41-positive megakaryocytes in the different 7-AAD ploidy categories based upon 7-AAD-stained DNA content. Bone marrow H&E histological analysis of marrow megakaryocyte content is ongoing. Results: WBC counts for controls and mice treated with bortezomib alone were at baseline values or greater during the course of the experiment. Mice treated with romidepsin, or the combination of bortezomib + romidepsin, showed an initial drop in d2 WBC counts to 50% of baseline. The platelet nadir for most groups was at d3, with very similar counts for all single-agent groups near 500 × 103 per mcL (Figure 1, bars represent standard deviation). Counts for the groups treated with 2mg/kg bortezomib or romidepsin were only slightly lower than for groups treated with 1mg/kg. However, the average day 3 platelet count for the combination group was significantly lower at 250 × 103 per mcL (Figure 1, * indicates p=0.0018). Platelet recovery was very rapid in the mice that received bortezomib alone, with d6 levels 150% of baseline. Platelets remained low in romidepsin-treated mice until d5 and then began increasing. Interestingly, platelet counts in the combination group also remained low until d5, similar to romidepsin alone, but then rose rapidly with kinetics reminiscent of the rebound seen in the bortezomib-treated groups (Figure 1). On d3, thrombocytopenia was associated with an increase in plasma TPO, especially in the bortezomib + romidepsin group, indicating that platelet reduction induced by the combination of both agents is not the result of TPO suppression. At d5 and d8, drug-treated groups had lower percentages of 8N/16N megakaryocytes and corresponding increases in the 2N/4N category, suggesting a megakaryocyte rebound effect in response to thrombocytopenia. Conclusions: The combination of bortezomib + romidepsin induces more profound thrombocytopenia in mice compared to either drug alone, but platelets recover to baseline levels or greater by 7 days after treatment. The platelet recovery phase is characterized by a rapid increase in platelet counts exceeding baseline values, similar to the rebound effect induced by bortezomib alone. This suggests that the platelet-producing megakaryocyte population is maintained after bortezomib + romidepsin treatment. Disclosures: Nix: Gloucester, Celgene: Consultancy, Employment. Lonial: Gloucester, Celgene: Consultancy, Research Funding.

Description: Introduction Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1 immunity and inhibits antiviral immunity. Decreased Th1 immunity is problematic for allogeneic bone marrow transplant (allo-BMT) patients requiring T-cell immunity against blood cancers (Graft-versus-Tumor) and against secondary infections such as CMV. VIPhyb, a modified VIP peptide, is a VIP receptor antagonist that decreases VIP signaling. VIP-knockout mice and mice treated with VIPhyb after allo-BMT are known to have better antiviral immunity and survival after CMV infection without increasing GvHD (Li et al. PLoS One. 2013 May 27;8(5):e63381) (Li et al. Blood. 2013 Mar 21;121(12):2347-51.), thus making VIPhyb of interest for pharmacological use in humans to improve the efficacy of allo-BMT The effects of VIPhyb on T-cell immunity are not yet fully profiled. This study aimed to analyze the effects of VIPhyb on CD4+ and CD8+ T-cell proliferation and activation in order to better understand the mechanistic implications of VIP inhibition on T-cell adaptive immunity. This study also aimed to show that mixed lymphocyte reactions (MLRs), an in vitro allo-BMT model, could be used to provide rapid and reliable results that are consistent with in vivo data. It was hypothesized that VIPhyb would increase T-cell immunity as profiled by: increased T-cell proliferation, CD69 and PD1 co-upregulation in early T-cell activation, and PD1 downregulation in T-cells after initial activation. Methods Splenocytes from two histoincompatible mice were cultured together at 37°C in a 1:1 ratio in a one-way MLR. BALB/c splenocytes (stimulators) were irradiated at 20Gy, and Pepboy splenocytes (responders) were labeled with CFSE to trace proliferation. VIPhyb was added daily to the cell cultures in doses of 0.1μM, 0.3μM, 1μM, or 3μM. Treatment groups were compared to a PBS control. Proliferation, CD69, and PD1 were assessed by flow cytometry on the BD FACSAria. All results are shown as mean ± SEM (n=3). One-way ANOVA tests with Dunnett post-tests were calculated using Prism software. *p 〈 0.05; **p 〈 0.01; ***p 〈 0.001 Results VIPhyb increased CD4+ and CD8+ T-cell proliferation: 3, 5, and 7 days after initiating a one-way MLR, CFSE expression of Pepboy responder T-cells was assessed using flow cytometry (Figure 1). As the VIPhyb dose increased, the percentage of initial splenocytes that underwent proliferation increased in both CD4+ and CD8+ T-cells. VIPhyb increased early T-cell CD69 expression and abrogated later PD1 upregulation in CD8+ T-cells: 3, 5, and 7 days after initiating a one-way MLR, expression levels of CD69 and PD1 on Pepboy responder T-cells were assessed by flow cytometry. Significant upregulation of CD69 on CD4+ and CD8+ T-cells on day 3 occurred with increasing VIPhyb doses (Figures 2A and 2B). PD1 was co-upregulated with CD69 during early activation, and VIPhyb significantly decreased PD1 expression on CD8+ T-cells on days 5 and 7 (Figures 2C and 2D). Conclusions VIPhyb increased T-cell proliferation; CD8+ T-cells were affected more significantly. VIPhyb increased early co-upregulation of CD69 and PD1 in all T-cells and significantly decreased later CD8+ T-cell PD1 expression, indicating that VIPhyb increases T-cell activation. We hypothesize that the decreased PD1 expression will be critical for understanding the pathways involved in VIP inhibition. Importantly, since it has been shown in vivo that VIPhyb does not increase GvHD, then it can be assumed that the VIPhyb-induced T-cell proliferation and activation will increase GvL and adaptive immunity without increasing alloreactivity. Notably, these results are consistent with published in vivo data, which demonstrates that the MLR can be used as a faster method of analyzing pharmacological compounds than in vivo experiments. Given these results, VIPhyb is still of interest as a potential therapy for allo-BMT patients. Disclosures: No relevant conflicts of interest to declare.

Description: The uncontrolled proliferation of genetically mutated cells is the commonly understood mechanism for cancer growth and invasion, with accumulation of new mutations in daughter cells leading to clonal diversity of cancer derived from a single founding event. The genetic alterations are passed to new generations by cell division and vertical gene transfer. Viral transmission of oncogenes represents a known mechanism of lateral gene transfer in cancer initiation. Some experimental systems have also suggested that circulating DNA or micro-vesicles may contribute to lateral oncogene transfer in tumorigenesis. We hypothesized that interactions between leukemic cells and adjacent normal hematopoietic stem or progenitor cells may provide an alternative mechanism for the accumulation of mutated genes and the multiplicity of distinct clones in leukemia. To test this hypothesis, we performed experiments to determine whether tumorigenic properties could be transferred from a tumor cell line to normal mouse bone marrow cells using both in vivo and in vitro and systems. B6-GFP+ mice were injected i.v. with 200,000 C1498-Luc cells (a B6-derived NKT-cell-like mouse tumor cell line expressing luciferase and DSRed). Bioluminescent imaging was used to monitor the progression of tumor cell growth in recipients. At 1 month after tumor-cell inoculation, marrow from these mice was harvested and FACS-sorted for GFP+ cells (to eliminate C1498 cells), and then cultured on irradiated stromal cell layers in 96-well plates in a limiting dilution analysis for Poisson analysis of GFP+ clonogenic precursor frequency on day 9. On day 10, cells were harvested from culture and GFP+ cells resorted onto fresh stromal layers for second and third determinations of GFP+ clonogenic precursor frequency on days 15 and 18. As shown in Figure 1, the frequency of clonogenic precursors increased with each successive determination for marrow from C1498-injected mice, while control cultures from non-injected mice showed no increase in precursor frequency, suggesting that exposure to C1498 cells conferred a growth advantage to the marrow cells in the tumor-cell injected mice. Similar results were obtained using an in vitro system of co-culture using C1498 cells and GFP+ bone marrow cells, followed by serial rounds of GFP+ sorting and Poisson analysis, showing increases in clonogenic frequency over 5 successive sorts and re-cultures over a 2-month period, while control cultures showed decreased clonogenic frequencies over the course of the experiment. To confirm these observations in vivo, B6-GFP mice were injected with C1498-Luc and marrow was harvested after a month and sorted for GFP+ cells. The sorted marrow was transplanted into 11Gy-irradiated (FVB x B6albino)F1 recipients (5 x 106 cells per recipient, n=5). Control recipients were irradiated and transplanted with GFP+ marrow from non-injected donors. All recipients developed full hematopoietic engraftment with GFP+ cells. At 6 months post-transplant, a tumor was observed near the left shoulder of one of the recipients of C1498-exposed GFP+ marrow. Figure 2 shows IVIS GFP imaging of this mouse with the GFP+ tumor along with control animals. The tumor was not positive for luciferase expression. The mouse was sacrificed and the tumor excised and a portion was dissociated for flow cytometric analysis and culturing (with other segments reserved for subsequent histological and genetic analysis). Both GFP+ and non-GFP cells were found in the dissociated tumor cell suspension. The GFP+ cells were hematopoietic in origin (CD45+) and exhibited a mixed phenotype containing markers expressed on C1498 (DX5+) and myeloid lineage cells (CD11b+) as well as Sca-1, a stem cell marker. Cultures of the GFP+ tumor yielded a population of GFP+ mononuclear cells. These data are consistent with a model in which growth-promoting or transforming genes from cancer cells become incorporated within a healthy hematopoietic stem or progenitor cell, which contributes to the genetic diversity of the cancer through the initiation a new transformed clone. Genetic analysis with deep sequencing will compare the DNA sequences between the parental C1498 cell line, sorted populations of clonogenic GFP+ cells obtained from the in vitro and in vivo experiments, and the GFP+ tumor cells to confirm the transformation of healthy bone marrow hematopoietic stem cells with genetic sequences derived from the C1498 cells. Disclosures No relevant conflicts of interest to declare.

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