ALBERT

Description: Abstract 817 Strategies to suppress GVHD are often associated with broader suppression of the immune system leading to a compromised GVT effect. Using experimental models, we have demonstrated a novel strategy to enhance GVT effects and explicitly suppress GVHD using genetically engineered T lineage cells over-expressing TNF-Related Apoptosis Inducing Ligand (TRAIL). TRAIL can induce apoptotic signals through death receptor (DR) 4 and 5 molecules (only DR5 in mice) expressed on target cells. Expression of DR5 is higher on certain tumors and can be enhanced on others using small molecules rendering them susceptible to TRAIL mediated killing. TRAIL is therefore an attractive candidate for genetic engineering of donor T cells to enhance their GVT potential. We evaluated the effect of TRAIL over-expression (TRAIL+) in donor T cells (mature and precursor) on GVHD and GVT. Mature T cells derived from donor B6 splenocytes were transduced with a lentiviral TRAIL expression vector. The transduced TRAIL+ T cells were adoptively transferred on day 0 into lethally irradiated CBF1 recipients of T cell depleted allografts and LB27.4 tumor (B6 ^ CBF1+LB27.4) to assess their GVHD and GVT activity. TRAIL+ T cells displayed significantly enhanced antitumor immunity compared to T cells transduced with a control vector against LB27.4 tumor cell lines in vitro and upon transfer into tumor bearing allo-BMT recipients (p

Description: We have previously reported on the efficacy of the JAK1/2 inhibitor ruxolitinib in corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) in 95 patients (pts) (Leukemia 2015;29(10):2062-8). To assess long-term follow-up results, we collected data from the same pts treated in 19 centers in Europe and the US. Pts were classified as SR-aGVHD (n=54, all grade III or IV) or SR-cGvHD (n=41, all moderate or severe). Median numbers of pre-ruxolitinib GVHD treatment lines were 3 (1-7) and 3 (1-10) for SR-aGVHD and SR-cGvHD, respectively. The median follow-up was 19 and 24 months for aGVHD and cGVHD, respectively. The 1-year overall survival (OS) from was 62.4% (CI: 49.4%-75.4%) and 92.7% (CI: 84.7%-100%) for SR-aGVHD and SR-cGvHD, respectively. The estimated median OS (50% death) was 18 months for aGVHD and not reached for cGVHD patients. The median duration of ruxolitinib treatment was 5 and 10 months for patients with SR-aGVHD and SR-cGVHD, respectively reflecting the different biology of the diseases. At follow-up, 22/54 (41%) of SR-aGVHD patients and 10/41 (24%) of SR-cGVHD patients have an ongoing response and are free of any immunosuppression. GVHD relapse or progression after achieved PR/CR was observed in 14/45 (31%) and 13/36 (36%) patients with SR-aGVHD and SR-cGVHD, respectively. Response to re-treatment with Ruxolitinib or any immunosupressive therapy was seen in 11/14 (78%) and 11/13 (86%) patients with SR-aGVHD and SR-cGVHD, respectively. Cytopenia (any grade) and CMV-reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. These findings extend our previous report by showing that patients with SR-aGVHD and SR-cGVHD may benefit long-term from ruxolitinib treatment with an OS that is relatively high for steroid-refractory GVHD. GVHD-relapse or GVHD-progression rates were moderate and more than 75% of the relapse/progression patients responded to re-treatment with ruxolitinib or other immunosuppression. Disclosures Meyer: Stanford University: Patents & Royalties. Marks:Pfizer: Honoraria. Lübbert:Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding; Janssen-Cilag: Other: Travel Funding, Research Funding. Scheid:Novartis: Other: funding outside this work; Celgene: Other: funding outside this work; Janssen: Other: funding outside this work. Kobbe:Celgene: Honoraria, Other: travel support, Research Funding; Jansen: Honoraria, Other: travel support. Negrin:Stanford University: Patents & Royalties. Brune:Meda Pharma: Consultancy. Mielke:JAZZ Pharma: Speakers Bureau; Novartis: Consultancy; MSD: Consultancy, Other: Travel grants; Gilead: Other: Travel grants; Celgene: Other: Travel grants, Speakers Bureau. Kuball:Gadeta B.V,: Membership on an entity's Board of Directors or advisory committees. Kröger:Sanofi: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Riemser: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Peschel:MophoSys: Honoraria. von Bubnoff:BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding.

Description: Neo-vascularization has been implicated in a number of inflammatory diseases as well as tumor growth. Both angiogenesis, the sprouting of resident tissue endothelial cells (ECs), and vasculogenesis, the recruitment of bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs), are thought to participate in neo-vascularization. EPCs have been implicated in tumor growth, however, the biologic significance of EPCs during inflammation is unclear. We studied neo-vascularization and the role of EPCs during inflammation in well-characterized murine models of graft-versus-host disease (GVHD). We found a significantly increased number of donor BM-derived EPCs in peripheral blood and BM in allogeneic bone marrow transplantation (allo-BMT) recipients with GVHD at different time points after BMT. We next quantified neo-vascularization during inflammation in GVHD target organs by immunofluorescence microscopy and by flow cytometry. We found significantly increased numbers of donor-derived ECs in the liver as well as a significantly higher vessel density in the liver, illeum and colon. We adoptively transferred selected GFP+ EPCs and observed incorporation into the neo-vasculature of the inflamed intestines (Fig. 1A) and liver during GVHD. Taken together, these data suggest that neovascularization during GVHD is due to vasculogenesis from donor EPCs. Next we used an antibody (E4G10) against the vascular endothelial adhesion molecule VE-cadherin, which recognizes a terminal epitope that is exposed on circulating EPCs, but is masked in the established vasculature, and found a significant reduction of EPCs in the peripheral blood and BM. We observed that depletion of EPCs was associated with a significant inhibition of donor BM-derived neo-vascularization in the liver, illeum and colon during GVHD. E4G10 treated recipients had significantly better survival and lower clinical GVHD scores in all tested models (B6BALB/c [1×106 T], B6B6D2F1 [1×106 T], B6B6D2F1 [2×106 T], B6B6D2F1 [3×106 T]). We found significantly reduced numbers of allo-reactive donor T cells in secondary lymphoid organs during GVHD, but no changes in the expression of activation markers and homing molecules, as a consequence of E4G10 administration. In blinded histopathological analyses we found significantly less GVHD and reduced numbers of tissue-infiltrating CD3+ T cells in the liver, illeum and colon in E4G10-treated allo-BMT recipients. To better emulate the clinical setting, we first assessed the role of EPCs in tumor growth in allo-BMT recipients. We transferred sorted GFP+ EPCs as well as renal carcinoma (RENCA) cells to BALB/c recipients and found that GFP+ EPCs were recruited to the neo-vasculature of lung metastases. We detected a significant inhibitory effect of E4G10 administration on tumor growth, as determined by in vivo bioluminescence imaging, in both tumors tested (RENCA, A20 lymphoma) as well as a significant survival prolongation in tumor-bearing mice that were treated with E4G10 in the RENCA and C1498 (AML) model. Finally, we performed experiments in which tumor-bearing allo-BMT recipients received allogeneic T cells, which mediate the favorable graft-versus-tumor (GVT) effect but also cause inflammation in GVHD target organs. We found that administration of E4G10 led to a significantly higher rate of tumor-free survival in all models (B6'BALB/c [1×105 B6 T and 2×105 RENCA], B10BR'B6 [1×105 B10BR T and 2×105 C1498], B6'BALB/c [2×105 B6 T and 5×105 A20]), which was due to both attenuation of GVHD as well as inhibition of tumor growth (Fig. 1B). We conclude that depletion of EPCs is a strategy to simultaneously ameliorate inflammatory disease and tumors, providing a new approach to improve therapeutic outcome of allogeneic hematopoietic stem cell transplantation. This study demonstrates the biological significance of EPCs for neo-vascularization during inflammation and identifies the specific targeting of EPCs to disrupt neo-vascularization as a novel therapeutic concept to decrease inflammation. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group.

Description: Abstract 1335 Poster Board I-357 Alloreactive T cells are crucial for graft-versus-host-disease (GVHD) pathophysiology, and we hypothesized that controlling their trafficking can ameliorate GVHD. P-selectin is a dimeric glycoprotein found on most inflamed endothelium, which interacts with multiple lectin-type molecules on leukocytes, including T cells. We used murine allogenienc BMT models to study GVHD and found that P-selectin−/− recipients exhibited significantly less GVHD mortality and morbidity, as well as decreased GVHD of the skin, liver and small bowels. However, WT and P-selectin−/− allo-BMT recipients had comparable large bowel GVHD. This decrease in target organ and systemic GVHD was associated with diminished infiltration of alloactivated T cells into the Peyer's Patches and small bowels, coupled with increased numbers of donor T cells in the spleen and secondary lymphoid organs (SLO) on day 14 and day 35 post-transplant. However, donor alloreactive T cells in WT and P-selectin−/− allo-BMT recipients had similar alloactivation and apoptosis, and donor alloactivated T cells from WT and P-selectin−/− allo-BMT recipients with GVHD showed similar proliferation in vitro in a mixed leukocyte reaction, suggesting that the inflammatory environment in WT and P-selectin−/− recipients was comparable. Finally, non-transplanted P-selectin−/− mice, and P-selectin−/− mice which had received the allo-BMT conditioning regimen but not a donor graft, had similar cellularity in the majority of tissues examined as corresponding WT controls. This suggests that the differential cellularity of donor alloactivated T cells in WT and P-selectin−/− allo-BMT recipients with GVHD is probably largely dependent on trafficking and tissue infiltration during inflammation. Since P-selectin glycoprotein ligand 1 (PSGL1) is the best-described P-selectin ligand, and all leukocytes constitutively bear high levels of membrane PSGL1, we next hypothesized that PSGL1−/− donor alloreactive T cells would be defective in trafficking into GVHD target organs, and that PSGL1−/− donor T cells would cause decreased target organ damage, systemic GVHD, and mortality. However, allo-BMT recipients of WT and PSGL1−/− donor T cells had comparable survival and clinical GVHD scores, and further analyses on day 14 post-transplant revealed that recipients of WT and PSGL1−/− donor T cells also had similar numbers of donor alloactivated T cells in the spleen, liver, mesenteric and peripheral lymph nodes, and Peyer's Patches. Additionally, WT and PSGL1−/− donor T cells had comparable proliferation as measured by CFSE dilution, and comparable alloactivation in vivo as determined by levels of CD25, CD44, and CD62L, suggesting similar T cell function. As PSGL1−/− and WT donor T cells appeared to have equal functionality and accumulated in GVHD target tissues and lymphoid tissues in a similar fashion, we asked whether PSGL1−/− T cells might display other P-selectin ligands. Flow cytometric analyses of T cells from non-transplanted PSGL1−/− mice, and analyses of PSGL1−/− alloactivated T cells on day 14 after allo-BMT, revealed that these cells displayed substantial levels of cell-surface P-selectin ligands as defined by positive staining with recombinant P-selectin-IgG-Fc fusion protein at levels similar to those found on WT T cells, suggesting that although absence of P-selectin on host tissues may ameliorate GVHD, multiple donor leukocyte P-selectin ligands interact meaningfully with P-selectin. Our studies suggest that P-selectin may be required for trafficking into inflamed tissues but not SLO, and that donor T cells may utilize multiple P-selectin ligands apart from PSGL1 to interact with P-selectin and traffic into inflamed tissues during GVHD. We conclude that targeting P-selectin may be a viable target for GVHD prophylaxis or treatment. Disclosures No relevant conflicts of interest to declare.

Description: Background: Allogeneic hematopoietic cell transplantation is a potentially curative therapy for patients with hematological malignancies. However a fraction of patients will develop corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) which both cause a high mortality and impaired quality of life. Pre-clinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib by modification of T cells and dendritic cells. Methods: In this retrospective analysis, 19 stem cell transplant centers in Europe and the United States reported clinical outcome data from 95 patients who had received ruxolitinib as salvage-therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grade III or IV) or SR-cGvHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1-7) and SR-cGvHD (1-10). The median follow-up times were 26.5 (3-106) for SR-aGVHD and 22.4 (3-135) weeks for SR-cGVHD-patients. Results: The ORR was 81.5% (44/54) in SR-aGVHD including 25 CRs (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). The median time to response was 1.5 (1-11) and 3 (1-25) weeks after initiation of ruxolitinib treatment in SR-aGVHD and SR-cGVHD, respectively. Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3%-90.7%,95% CI) and 97.4% (92.3%-100%,95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and CMV reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Relapse of the underlying malignancy occurred in 9.3% (5/54) and 2.4% (1/41) of the patients with SR-aGVHD or SR-cGVHD, respectively. Conclusion: Ruxolitinib constitutes a promising new treatment option for SR-aGVHD and SR-cGVHD. Its activity in SR-aGVHD and SR-cGVHD should be validated in a prospective trials in both, SR-aGvHD and cGvHD. Disclosures Bertz: GILEAD Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Scheid:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding.

Description: Success of adoptive T cell therapy (ATT) is dependent on sufficient numbers of T cells and the characteristics of the final T cell product. In several studies, clinical grade CD19 CAR T cell products could not be generated from about 6-30% patients, particularly if they were isolated from older or heavily pretreated diffuse large B cell lymphoma (DLBCL) patients. In cyclophosphamide/fludarabine-lymphodepleted patients with persistent or progressive disease a sequential second dose of T cells has been shown to be effective resulting in tumor regression. Here we investigated to what extend T cell numbers could be increased via prolonged expansion with standard cytokines IL-7/IL-15 and how transcriptome and function of central memory T cells (Tcm) longitudinally change during culture. Method: Murine and human T cells were cultured with the cytokine combination IL-7/IL-15. Short-term expanded (ST, one week) and long-term expanded (LT) CD8+ (4 weeks) and CD4+ (3 weeks) T cells were compared for proliferation capacity (CFSE), extent of apoptosis (AnnexinV), up-regulation of T cell inhibitory receptors (TIRs) and cytokine expression pattern after in vitro re-stimulation upon anti-CD3/CD28 stimulation. Further, RNA sequencing of ST and LT expanded murine CD8+ and CD4+ Tcm followed by unsupervised hierarchical clustering, principal component analysis (PCA) and differential expression analysis was performed. In vivo mouse models were used to analyze engraftment, persistence and anti-tumor capacity applying our bioluminescent dual-luciferase reporter mouse (BLITC - bioluminescent imaging of T cells) allowing us to monitor migration, expansion (RLuc luciferase) and activation (NFAT-driven Click-beetle luciferase) of adoptively transferred T cells in vivo. Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors and DLBCL patients. Results: There was a 50-fold increase of T cells in LT vs. ST culture, the Tcmproportion was extended and stem cell markers were comparable or even higher expressed in LT expanded T cells. Differential analysis revealed 2786 (CD8) and 912 (CD4) with statistically significant expression alterations with generally only moderate effect size when comparing LT and ST expanded T cells. Interestingly, the dynamically modified genes largely overlapped for CD8 and CD4 T cells suggesting culture-associated changes. Comparable RLuc signals and T cells counts in peripheral lymph nodes (LN) and spleen indicate similar engraftment (4 weeks post ATT) and persistence capacities (up to 6 months post ATT) of transferred ST and LT T cells. SV40-TAg+ tumor bearing mice were treated with TCR-I retrovirally transduced CD8+ BLITC T cells, which were ST or LT expanded. The T cells infiltrated rapidly in the tumor where they got similarly activated resulting in a complete tumor rejection in all recipient mice. Finally, we analyzed the expansion and in vitro properties of T cells from healthy donors (n=3-5) and DLBCL patients (n=3) who were eligible for CAR T cell therapy. LT T cell expansion from healthy donors resulted in a 10.000-fold increase of CD8+CD45RO+CCR7+ T cells. In vitro assays showed comparable apoptosis and expression of TIRs between ST and LT CD8 T cells and stable expression of IFN-g and TNF-a within the first 3 weeks. The CD8+CD45RO+CCR7+ T cell expansion from DLBCL patients was weaker in comparison to healthy donors. The extent of cell death and up-regulation of TIRs after re-stimulation was comparable between ST and LT T cells, whereas cytokine expression varied individually. Conclusion: Our data suggest that it is feasible to expand CD8+ and CD4+ murine and human T cells up to a month, thereby increasing numbers of T cells with Tcm/Tscm properties and with sustained function for murine and human T cells from healthy donors, whereas there seems to be a high individual variance for DLBCL patients, which warrants further investigation in larger patient cohorts. Disclosures Bullinger: Bayer: Other: Financing of scientific research; Abbvie: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria.

Description: IL-21 is a proinflammatory cytokine produced by Th17 cells. Abrogation of IL-21 signaling has recently been shown to reduce GVHD while retaining graft-versus-leukemia/lymphoma (GVL) responses. However, the mechanisms by which IL-21 may lead to a separation of GVHD and GVL remain incompletely understood. In a murine MHC-mismatched BM transplantation model, we observed that IL-21 receptor knockout (IL-21R KO) donor T cells mediate decreased systemic and gastrointestinal GVHD in recipients of a transplant. This reduction in GVHD was associated with expansion of transplanted donor regulatory T cells and with tissue-specific modulation of Th-cell function. IL-21R KO and wild-type donor T cells showed equivalent alloactivation, but IL-21R KO T cells showed decreased infiltration and inflammatory cytokine production within the mesenteric lymph nodes. However, Th-cell cytokine production was maintained peripherally, and IL-21R KO T cells mediated equivalent immunity against A20 and P815 hematopoietic tumors. In summary, abrogation of IL-21 signaling in donor T cells leads to tissue-specific modulation of immunity, such that gastrointestinal GVHD is reduced, but peripheral T-cell function and GVL capacity are retained. IL-21 is thus an exciting target for therapeutic intervention and improvement of clinical transplantation outcomes.

Description: Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a ubiquitously expressed transcription factor that is well known for its role in regulating the cellular redox pathway. Although there is mounting evidence suggesting a critical role for Nrf2 in hematopoietic stem cells and innate leukocytes, little is known about its involvement in T-cell biology. In this study, we identified a novel role for Nrf2 in regulating alloreactive T-cell function during allogeneic hematopoietic cell transplantation (allo-HCT). We observed increased expression and nuclear translocation of Nrf2 upon T-cell activation in vitro, especially in CD4+ donor T cells after allo-HCT. Allo-HCT recipients of Nrf2−/− donor T cells had significantly less acute graft-versus-host disease (GVHD)-induced mortality, morbidity, and pathology. This reduction in GVHD was associated with the persistence of Helios+ donor regulatory T cells in the allograft, as well as defective upregulation of the gut-homing receptor LPAM-1 on alloreactive CD8+ T cells. Additionally, Nrf2−/− donor CD8+ T cells demonstrated intact cytotoxicity against allogeneic target cells. Tumor-bearing allo-HCT recipients of Nrf2−/− donor T cells had overall improved survival as a result of preserved graft-versus-tumor activity and reduced GVHD activity. Our findings characterized a previously unrecognized role for Nrf2 in T-cell function, as well as revealed a novel therapeutic target to improve the outcomes of allo-HCT.