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Invariant natural killer T (iNKT) cells are a T-cell subset with potent immunomodulatory properties. Experimental evidence in mice and observational studies in humans indicate that iNKT cells have antitumor potential as well as the ability to suppress acute and chronic graft-versus-host-disease (GVHD). Murine iNKT cells differentiate during thymic development into iNKT1, iNKT2, and iNKT17 sublineages, which differ transcriptomically and epigenomically and have subset-specific developmental requirements. Whether distinct iNKT sublineages also differ in their antitumor effect and their ability to suppress GVHD is currently unknown. In this work, we generated highly purified murine iNKT sublineages, characterized their transcriptomic and epigenomic landscape, and assessed specific functions. We show that iNKT2 and iNKT17, but not iNKT1, cells efficiently suppress T-cell activation in vitro and mitigate murine acute GVHD in vivo. Conversely, we show that iNKT1 cells display the highest antitumor activity against murine B-cell lymphoma cells both in vitro and in vivo. Thus, we report for the first time that iNKT sublineages have distinct and different functions, with iNKT1 cells having the highest antitumor activity and iNKT2 and iNKT17 cells having immune-regulatory properties. These results have important implications for the translation of iNKT cell therapies to the clinic for cancer immunotherapy as well as for the prevention and treatment of GVHD.

Many studies have shown the important role that invariant natural killer T (iNKT) cells play in suppression of graft-versus-host disease (GVHD). Murine studies have also demonstrated multiple subsets of iNKT cells, including those with pro-inflammatory and immunosuppressive properties. The prevention of murine acute GVHD by the adoptive transfer of immunomodulatory iNKT cells suggests that this approach would be ideal for clinical translation. However, the heterogeneity of human iNKT cells remains poorly understood, and the uncertain ability to identify immunosuppressive human iNKT cells and expand them to clinically relevant numbers remains a limitation in clinical translation. Therefore, engineering bulk human iNKT cells to enhance immunosuppressive capacity represents a novel approach to ensuring sufficient cell numbers of immunoregulatory cells for adoptive transfer. Herein, we describe the lentiviral transduction of human iNKT cells to overexpress immunomodulatory cytokines. We focused on IL-4, because murine iNKT cell-mediated GVHD suppression is IL-4 dependent, and IL-10, because an immunosuppressive regulatory T cell-like subset of murine iNKT cells highly produces IL-10. Human iNKT cells were transduced with a lentiviral construct expressing IL-4 or IL-10 (or green fluorescent protein (GFP) as a control) and an inert truncated nerve growth factor receptor (deltaNGFR) to facilitate sorting of transduced cells. Enzyme linked immunosorbent assay revealed significantly increased expression of IL-4 or IL-10 by cells transduced with the corresponding lentivirus, as compared to untransduced, mock transduced, or GFP transduced cells, replicated with 3 iNKT donors (p Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Regulatory T cells are a critical population for the maintenance of self-tolerance and the use of these cells for therapeutic purpose in transplantation has emerged based on their capacity to control immune activation, exhibiting positive therapeutic potential to ameliorate acute graft-versus-host disease (GvHD), one of the major complications after allogeneic bone marrow transplantation (allo-BMT). One of the remaining technical challenges in the clinical practice of Treg administration is the number of Treg required to effectively prevent GvHD. In this study, we engineered murine Treg cells to express an altered IL-2 receptor β subunit that selectively interacts with an orthogonal mutant IL-2 (oIL2) without interacting with the natural cytokine or receptor counterparts, thus preventing acute -GvHD. For investigation of the suppressive function of Treg orthogonal IL-2/IL-2 receptor system in a murine major mismatch of acute GvHD model, the BALB/c mice were lethally irradiated (8.8 Gy) and transplanted with 5x10 6 T cell-depleted bone marrow cells (TCD-BM) from C57Bl/6 mice alone or together with fresh 1x10 6 C57Bl/6 FoxP3 DRT-GFP-luc+ Treg cells (fTreg) or with 0.2x10 6 and 1x10 6 oTreg (transduced with the construct for murine oIL-2 receptor β chain) on day 0. On day 2, C57Bl/6 Tcon cells (1x10 6) were injected to induce GvHD. Recipients were treated with PBS or oIL-2 (25K IU/daily) for 14 days and then 3 times a week for another 14 days. Using bioluminescence imaging, we observed that ortho IL2Rβ +Treg not only maintains migration ability to the gut, mLN and LN but also exhibited significant expansion ability treated with oIL2 at day 7 post-transplant. The expansion of oTreg with PBS and fTreg was observed on day 14 after allo-BMT. The administration of oTreg-oIL2 and fTreg (1:1 Tcon:Treg ratio) significantly increased the survival of the mice compared to Tcon group (P oIL-2 protein injection was able to selectively expand the oTreg in vivo, with a significantly increased percentage of Foxp3 GFP+ in CD4 + T cells (PBS;14.96±2.4%, oIL-2; 27.76±23.6%, P=0.02) in the peripheral blood (PB) and spleen after 7 days post-transplant. We also observed a significant decrease in the percentage of effector T cells in the LN, mLN and spleen of the mice transplanted with oTreg and oIL-2 administration after 7 days post transplantation compared with GvHD group. We further demonstrated that ortho-IL2 administration-maintained graft-versus-leukemia (GvL) responses against lymphoma cells (A20). Engineered orthogonal cytokine receptor improves the suppressing abilities of Treg potential in a murine model of acute GvHD with the ability to maintain GvL, representing a novel approach to the utilization of Treg in Allogeneic Hematopoietic Stem Cell Transplant. Disclosures Garcia: Synthekine: Membership on an entity's Board of Directors or advisory committees. Blazar: Tmunity Therapeutics: Other: Co-founder; Equilibre Pharmaceuticals Corp: Research Funding; Carisma Therapeutics, Inc: Research Funding; Rheos Medicines: Research Funding; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Death receptor 3 (DR3) is a tumor necrosis factor receptor superfamily member (TNFRSF25), which is minimally expressed on resting conventional T cells (though readily inducible upon cell activation), yet highly expressed on resting FoxP3+ regulatory T cells (Treg). We recently demonstrated that activation of DR3 with an agonistic antibody (4C12) leads to selective expansion and activation of Treg in healthy mice and suppression of graft-versus-host disease (GVHD) in recipient mice when donor mice are treated. However, given the long antibody half-life and concomitant safety concerns, along with the lack of a humanized agonistic antibody to DR3, both human and murine fusion proteins incorporating the natural DR3 ligand TL1A (TL1A-Ig) have been developed. Herein, we show that DR3 activation with 4C12 or with TL1A-Ig, with or without the addition of low dose IL-2 to the treatment regimen, led to a significant expansion of murine Treg in spleen, lymph nodes, and peripheral blood. Bioluminescent imaging revealed peak Treg expansion around day 7-8, with return to near baseline after 2-3 weeks. In addition to expansion, all DR3 agonist treatment regimens led to increased activation of Tregs, with significant upregulation of the activation markers ICOS, KLRG-1, PD-1, and CD103, and the proliferation marker Ki-67. The near absence of activated Treg populations in control treated spleens was also detected on tSNE analysis of flow cytometry data. Subtly different patterns of splenic Treg activation by the different DR3 agonists were noted in both tSNE analysis of flow cytometry data and RNA-sequencing analysis. However, upregulation of gene transcripts which play important roles in cell proliferation, trafficking, activation, and effector function were observed regardless of the DR3 agonist treatment regimen used. In the major MHC-mismatch model of hematopoietic cell transplantation, DR3 agonist-mediated expansion and activation of Tregs in donor mice led to a significant improvement in GVHD in recipient mice. These data provide important preclinical information regarding the outcome of DR3 activation with an agonistic antibody or natural ligand and provide insight into the therapeutic use of this approach to reduce GVHD in recipients and improve outcomes of hematopoietic cell transplantation.

Successful hematopoietic stem cell transplantation (HSCT) requires vacating recipient hematopoietic stem cell (HSC) niches in the bone marrow to permit donor HSC engraftment that can provide life-long hematopoietic and immune function. Currently, HSCT in SCID relies on DNA damaging chemotherapy to eliminate recipient HSC and achieve niche clearance. We have pursued a non-toxic approach to target and deplete HSC using a humanized monoclonal antibody, JSP191, that binds human CD117 (c-Kit). We previously showed the safety and successful HSC engraftment in a Phase 1 trial of the first 6 patients with severe combined immunodeficiency (SCID), who underwent a second transplant because of HSC engraftment failure and poor immunity after their first transplantation. In these re-transplant patients even a low level of stringently measured myeloid chimerism resulted in significant and sustained generation of naive T cells and clinical improvement. Based on these results, the study of JSP191 (NCT#02963064)has opened a cohort of newly diagnosed infants with SCID. Here we report data from the first patient in this cohort, a SCIDX1 patient who received a primary HSCT with haploidentical CD34+ cells after conditioning with JSP 191. The patient had a c.270-15A>G variant in the IL2RG gene, which is predicted to cause a null phenotype. Besides a T- B+ NK- phenotype typical of SCIDX1 including dysfunctional B cells, the patient had anemia and intermittent neutropenia and thrombocytopenia. Despite evidence of maternal T cell engraftment, the patient had no clinical graft-versus-host disease (GVHD). The patient was initially enrolled in a trial of lentiviral gene therapy, but harvested bone marrow cells died in vitro during transduction and culture. The patient also mobilized poorly with G-CSF/Plerixafor. Further investigation revealed heterozygosity for loss-of-function mutations in two genes involved in DNA repair, BRCA1 and RAD51; Diepoxybutane (DEB) breakage study showed greater than normal pathologic chromosomal breaks, but less than that seen in Fanconi anemia. Because of concern for possible hypersensitivity to alkylating agent-based conditioning, the patient was referred for transplant with JSP191 conditioning. The patient received a CD34+ peripheral blood HSCT from his father after conditioning with 0.3 mg/kg of JSP 191 antibody intravenously over an hour on Day -8 and rATG (Thymoglobulin) on Day -5, -4, -3 and -2 (3.5 mg/kg total) to prevent rejection by the maternal T cells. The cryopreserved donor CD34+ cells were administered after sufficient clearance of the JSP191 serum level. The antibody infusion was well tolerated without toxicity, and the post-transplant course was uneventful without acute toxicities or GVHD. As a surrogate marker for HSC engraftment, CD15+ myeloid cells from peripheral blood were stringently sorted by flow cytometry and donor levels were quantified by short-tandem repeat (STR) analysis. Progressive levels of myeloid engraftment were observed beginning at Week 4. The level of donor chimerism at 12 weeks was 8% in the sorted CD15+ blood cells, and a marrow aspirate showed 25% donor CD34+ cells. By 3 months pre-existing abnormal CD19-CD20+ host B lymphocytes were significantly reduced, and CD19+ donor-derived B lymphocytes were emerging. At 2 months, CD4+ recent thymic emigrant and naïve T lymphocytes were observed, and by 3 months, overall T and NK lymphocyte numbers were 390/uL and 117/uL, respectively. Normal blastogenic responses to the T cell mitogen PHA were observed at 3 months. These first-in-class results provide proof of concept of the safety and efficacy of the use of JSP191 antibody to clear host marrow niche space to enable sufficient donor HSC engraftment and immune reconstitution as primary therapy of SCID. Non-genotoxic conditioning with JSP191 may replace conventional conditioning for newly diagnosed infants with SCID, thereby avoiding toxicities of chemotherapy. Disclosures Kohn: Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. De Oliveira:Orchard Therapeutics: Research Funding; bluebird bio, Inc.: Research Funding. Czechowicz:Rocket Pharmaceuticals, Inc.: Research Funding. Brown:Merck: Membership on an entity's Board of Directors or advisory committees; Ansun: Membership on an entity's Board of Directors or advisory committees; Cidara: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Cellerant Therapeutics: Membership on an entity's Board of Directors or advisory committees. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

Patients who fail to adequately reconstitute the donor-derived immune system after allogeneic hematopoietic stem cell transplantation (HSCT) are at increased risk for infections and leukemia relapse. In the past, pan T-cell depleted haploidentical grafts were associated with delayed immune reconstitution (IR). Recently, the majority of patients receiving αβ T-cell/B-cell depleted haploidentical HSCT (αβhaplo-HSCT) reach a threshold of 200 CD3+ T cells/mcl by 100 days after HSCT (Bertaina A et al. Blood 2014 Jul 31;124(5):822-6). However, a proportion of patients experience a slower IR with consequent higher morbidity and mortality. Early prediction of delayed IR may permit prompt clinical intervention such as infusion of donor lymphocytes or of virus-specific cytotoxic T cells. Flow cytometry, the most widely applied approach for IR analysis, suffers from intrinsic limitations, such as high lymphocyte number requirement, degradation of samples, and insufficient standardization due to technical and operator variability. To overcome these limitations, we used a DNA methylation-based quantitative PCR that detects the epigenetic signature of different peripheral blood immune cell subsets (epigenetic quantification). This technique provides relative and absolute immune cell counts applicable to fresh, frozen, or paper-spotted dried blood. Epigenetic measurements are based on a per cell DNA copy number and provide a clear positive or negative signal rather than arbitrarily defined thresholds for "positivity" as in flow cytometry. We hypothesize that epigenetic quantification at day 15 after αβhaplo-HSCT could predict flow-based IR at day 100. Patients were consented at Lucile Packard Children's Hospital (Stanford, CA). Blood was collected between days 10-17 for epigenetic quantification and days 82-124 for flow cytometry. Bisulfite treated DNA underwent qPCR quantification of cell type-specific DNA regions of de-methylation (Baron U et al. Sci Transl Med 2018 Aug 1;10(452):eaan3508). Flow cytometry was performed using directly conjugated antibodies. Absolute cell counts were determined, plotted, and then analyzed using a linear regression model. In the first 5 αβhaplo-HSCT patients evaluated, we found a direct correlation between the epigenetic quantification at day 15 and flow cytometry at day 100 for CD4+ T cells (P=0.01), while the early epigenetic quantification of CD3+ and CD8+ T cells was not informative (Fig. 1). Preliminary data suggest that the use of epigenetic quantification early after αβhaplo-HSCT can predict the IR of CD4+ T cells at day 100 in αβhaplo-HSCT recipients. Ongoing analysis on a larger cohort of both αβhaplo-HSCT and unmanipulated HSCT recipients, will confirm if epigenetic quantification results obtained early post-HSCT can be used as a clinical biomarker of delayed IR and guide physicians in the use of post-HSCT adoptive immunotherapy.

Natural killer (NK) cells are a population of cytotoxic innate lymphocytes that evolved prior to their adaptive counterparts and constitute one of the first lines of defense against infected/mutated cells. Several studies have shown that in patients with acute leukemia given haploidentical hematopoietic stem cell transplantation, donor-derived NK cells play a key role in the eradication of cancer cells. The antileukemic effect is mostly related to the presence of “alloreactive” NK cells, that is, mature KIR+ NK cells that express inhibitory KIR mismatched with HLA class I (KIR-L) of the patient. A genotypic analysis detecting KIR B haplotype and the relative B content is an additional donor selection criterion. These data provided the rationale for implementing phase I/II clinical trials of adoptive infusion of either selected or ex vivo-activated NK cells, often from an HLA-mismatched donor. In this review, we provide a historical perspective on the role played by NK cells in patients with acute leukemia, focusing also on the various approaches to adoptive NK cell therapy and the unresolved issues therein. In addition, we outline new methods to enhance NK activity, including anti-KIR monoclonal antibody, bi-/trispecific antibodies linking NK cells to cytokines and/or target antigens, and CAR-engineered NK cells.

Invariant natural killer T (iNKT) cells serve as a bridge between innate and adaptive immunity and have been shown to play an important role in immune regulation, defense against pathogens, and cancer immunity. Recent data also suggest that this compartment of the immune system plays a significant role in reducing graft-versus-host disease (GVHD) in the setting of allogeneic hematopoietic stem cell transplantation. Murine studies have shown that boosting iNKT numbers through certain conditioning regimens or adoptive transfer leads to suppression of acute or chronic GVHD. Preclinical work reveals that iNKT cells exert their suppressive function by expanding regulatory T cells in vivo, though the exact mechanism by which this occurs has yet to be fully elucidated. Human studies have demonstrated that a higher number of iNKT cells in the graft or in the peripheral blood of the recipient post-transplantation are associated with a reduction in GVHD risk, importantly without a loss of graft-versus-tumor effect. In two separate analyses of many immune cell subsets in allogeneic grafts, iNKT cell dose was the only parameter associated with a significant improvement in GVHD or in GVHD-free progression-free survival. Failure to reconstitute iNKT cells following allogeneic transplantation has also been associated with an increased risk of relapse. These data demonstrate that iNKT cells hold promise for future clinical application in the prevention of GVHD in allogeneic stem cell transplantation and warrant further study of the immunoregulatory functions of iNKT cells in this setting.

CD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system and enhance immune tolerance after transplantation. Donor-derived Treg prevent the development of lethal acute graft-versus-host disease (GVHD) in murine models of allogeneic hematopoietic stem cell transplantation. We recently demonstrated that a single treatment of the agonistic antibody to DR3 (death receptor 3, αDR3) to donor mice resulted in the expansion of donor-derived Treg and prevented acute GVHD, although the precise role of DR3 signaling in GVHD has not been elucidated. In this study, we comprehensively analyzed the immunophenotype of Treg after DR3 signal activation, demonstrating that DR3-activated Treg (DR3-Treg) had an activated/mature phenotype. Furthermore, the CD25+Foxp3+ subpopulation in DR3-Treg showed stronger suppressive effects in vivo. Prophylactic treatment of αDR3 to recipient mice expanded recipient-derived Treg and reduced the severity of GVHD, whereas DR3 activation in mice with ongoing GVHD further promoted donor T-cell activation/proliferation. These data suggest that the function of DR3 signaling was highly dependent on the activation status of the T cells. In conclusion, our data demonstrated that DR3 signaling affects the function of Treg and T-cell activation after alloantigen exposure in a time-dependent manner. These observations provide important information for future clinical testing using human DR3 signal modulation and highlight the critical effect of the state of T-cell activation on clinical outcomes after activation of DR3.

Cellular therapy with regulatory T cells (Tregs) has shown promising results for suppressing graft versus host diseases (GvHD) in animal models and phase I/II clinical trials. However, a paucity of Tregs in the peripheral blood makes it difficult to acquire sufficient numbers of cells and hampers further clinical applications. Invariant natural killer T (iNKT) cells are another compartment of regulatory cells that ameliorate GvHD by interacting with Tregs upon either transplantation with activation with α-galactosylceramide (αGalCer) or adoptive transfer of iNKT cells. The aim of this study was to explore the proliferation and anti-GvHD effect of Treg following stimulation of iNKT cells with αGalCer.We utilized a liposomal formulation of αGalCer that was found to have the potential of preventing GvHD in a recently published phase I/IIa clinical trial. Lethally (8.8Gy) irradiated BALB/c recipient mice were transferred with 5 x 106T cell depleted bone marrow cells and 1 x 106conventional CD4+and CD8+T cells (Tcons) from WT C57BL/6 donor mice, with or without 1 x 105CD4+CD8-CD25hiFoxp3-GFP+cells (Tregs) that were freshly isolated from Foxp3-GFP+C57BL/6 mice. Immediately after cell injection, αGalCer-loaded or unloaded (Ctrl) liposomes were injected IV at 10 μg/kg. We utilized a subtherapeutic dose of Treg (1 x 105) that did not provide survival benefit compared to those without Treg transfer. αGalCer injection alone did not result in improved survival, suggested that a single injection of αGalCer is not sufficiently potent to prevent GvHD. Mice transferred with a subtherapeutic dose of Treg and liposomal αGalCer injection showed significant improvement in survival.Tissue damage examined 10 days after BMT was less severe in the Treg plus αGalCer group compared to those of the other groups. We analyzed the Treg population among CD4+T cells in the spleen, superficial/mesenteric lymph nodes, and liver on day 10 following transplant. As αGalCer stimulation promoted both Tcon-derived (CD45.1+Foxp3+) and transferred (CD45.2+Foxp3-GFP+) Treg, the proportion of Treg in the mice treated with the combination of Treg plus αGalCer stimulation significantly increased in all organs compared to those of the other groups. We evaluated the transferred Tregs in vivo by bioluminescent imaging. Treg-signals in αGalCer treated mice did not show significant difference compared to those in Ctrl mice until day 7 and increased thereafter especially in the gastrointestinal tract and became significantly higher 2-3 weeks after BMT. Host iNKT cells rather than donor iNKT cells contributed more to GvHD suppression because survival benefit of αGalCer stimulation was not shown in the transplantation of WT C57BL/6 mice into Jα18-/- BALB/c mice, whereas it was in that from Jα18-/-C57BL/6 mice into WT BALB/c mice. In conclusion, liposomal αGalCer injection enhances Treg function through activation of host derived iNKT cells and could make Treg cell therapy more feasible and safer by reducing the requirement of Treg number. Disclosures No relevant conflicts of interest to declare.

Invariant natural killer T (iNKT) cells are an interesting subpopulation of T cells that can potently inhibit graft-versus-host-disease (GVHD) through the production of Interleukin 4 (IL-4), while also carrying anti-tumor potential (Leveson-Gower et al. Blood. 2011;117:3220-9; Schneidawind et al. Blood. 2014;124:3320-8). Murine iNKT cells differentiate during thymic development into three distinct sublineages, named according to the classification of conventional T cells: Th1-like iNKT (iNKT1) cells, Th2-like iNKT (iNKT2) cells, and Th-17 like iNKT (iNKT17) cells (Brennan et al. Nat Rev Immunol. 2013; 13:101-1). In this study we investigated the immune regulatory and anti-tumor potential of iNKT1, iNKT2 and iNKT17 cell subsets. Thymic iNKT1 cells, iNKT2 cells and iNKT17 cells were isolated from 8-10 week-old FVB/NJ mice by flow cytometry based on PBS-57-CD1d-Tetramer and a combination of cell surface molecules (iNKT1: ICOS- PD1- CD27+ CD24-; iNKT2: ICOS+ PD1+ CD27+ CD4+ CD24-; iNKT17: ICOS+ PD1+ CD4- CD27- CD24-) and purity was confirmed by intra-nuclear staining for the transcription factors PLZF and RORγT. RNA sequencing analysis determined that iNKT1 cells were the main subset expressing proinflammatory and cytotoxic genes, such as Interferon gamma (IFN-γ), Fas Ligand (FasL), Perforin, and Granzyme B (Gzmb), whereas IL-4 was expressed by iNKT2 cells and, at a lesser extent, by iNKT17 cells. To assess the immuno-regulatory potential of the three iNKT sublineages, we employed a murine major histocompatibility complex (MHC)-mismatched bone marrow transplantation model. BALB/c (H-2Kd) recipients were lethally irradiated with 8.8 Gy; on the same day, 4 x 106 TCD-BM cells and 1 x 106 conventional CD4 and CD8 T cells (Tcon) from FVB/NJ (H-2kq) mice were injected intravenously. Additionally, 5 x 104 purified iNKT1, iNKT2 or iNKT17 cell subsets from FVB donors were injected. A significant survival benefit was observed when iNKT2 (p=0.017) and iNKT17 (p=0.033) cells were adoptively transferred compared to mice that only received TCD-BM and Tcon, whereas there was no survival benefit in the group that received iNKT1 cells. In addition, body weight was improved in mice that received iNKT2 (day +41: p=0.009, day +49: p=0.005 and day +59: p= 0.005) or iNKT17 (day +59: p= 0.006) compared to mice that received iNKT1 cells. Clinical GVHD scores were also improved in mice that received iNKT2 (day +41: p= 0.012, day +51: p= 0.005) or iNKT17 (day +28: p=0.05, day +51: p=0.007) compared to mice that received iNKT1 cells. Interestingly, we found that even 1 x104 iNKT2 (p= 0.008) and iNKT17 (p= 0.04) significantly suppressed GVHD. As iNKT1, iNKT2 and iNKT17 have a very different gene expression profile, we tested the ability of the sublineages to kill a B-cell lymphoma cell line transduced to express high levels of CD1d (A20-CD1d) in vitro and found that iNKT1 cells killed A20-CD1d cells significantly better than iNKT2 (p=0.006) or iNKT17 (p=0.0001) cells. These findings are in line with the sequencing data mentioned above, showing that iNKT1 cells express a more inflammatory phenotype. In summary, we demonstrate here that only iNKT2 and iNKT17 cells protect from GVHD, whereas iNKT1 cells have cytotoxic function. To our knowledge, this is the first study to show functional differences between the iNKT sublineages, suggesting that iNKT1, iNKT2 and iNKT17 cells have diverse functions. Therefore, these data provide new biological insights, which will be useful for developing iNKT cell-based cell therapy. Disclosures Chang: Spring Discovery: Membership on an entity's Board of Directors or advisory committees; Epinomics and Accent Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Objective Rheumatoid arthritis (RA) is a destructive autoimmune disease characterized by an increased inflammation in the joint. Therapies which activate the apoptotic cascade may have potential as a future therapy for RA, however few therapeutics fit this category. Recently, therapies that mimic the action of Bcl-2 homology 3 (BH3) domain-only proteins such as Bim have shown success in preclinical studies of cancer but their potential in autoimmune disease is unknown.

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21((WAF1/CIP1)), an established suppressor of cell cycle progression, is a inhibitor of IL-1beta synthesis in macrophages. Mice deficient in p21 (p21(-/-)) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL-1beta. Administration of IL-1 receptor antagonist reduces LPS-induced lethality in p21(-/-) mice. Analysis of isolated macrophages, which are one of the central producers of IL-1beta, reveals that deficiency for p21 led to more IL-1beta mRNA and pro-protein synthesis following TLR ligation. The increase in IL-1beta pro-protein is associated with elevated secretion of active IL-1beta by p21(-/-) macrophages. siRNA-mediated knockdown of p21 in human macrophages results in increased IL-1beta secretion as well. A peptide mapping strategy shows that the cyclin-dependent-kinase (CDK)-binding domain of p21 is sufficient to reduce the secretion of IL-1beta by p21(-/-) macrophages. These data suggest a novel role for p21 and specifically for the CDK-binding domain of p21((WAF1/CIP1)) in inhibiting inflammation.

Background : CD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system and enhance immune tolerance after transplantation. Donor-derived Treg prevent the development of lethal acute graft versus host disease (GVHD) in murine models of allogeneic hematopoietic cell transplantation (HCT). We recently demonstrated that a single treatment of the agonistic antibody to DR3 (Death receptor 3, aDR3) to donor mice resulted in the expansion/activation of donor derived Treg and prevented acute GVHD (Blood 126:546, 2015), although the precise role of DR3 signaling in GVHD has not been elucidated. In this study, we investigated the efficacy of αDR3 treatment to recipient mice in model of murine GVHD. Methods To analyze the DR3 expression in immune cells with or without TCR stimulation, we comprehensively analyzed the cells with multicolor cytometry using viSNE (visualization of stochastic neighbor embedding algorithm). In transplantation experiments, 5x10e6 T cell depleted bone marrow (from WT C57BL/6 mice, H2kb) and 1x10e6 T cells (C57BL/6-Luciferase mice, H2kb) were injected intravenously into lethally irradiated (8Gy in total) BALB/c recipient mice (H2kd). aDR3 was intraperitonealy injected at different time point after transplantation. The transplanted mice were monitored by clinical GVHD score, weight, bioluminescence imaging (BLI) for donor T cell trafficking, and survival time. To investigate the role of donor or recipient derived Treg in this model, in vivo Treg depletion using B6-Foxp3DTR mice was also performed. Results viSNE analysis demonstrated that DR3 was preferentially expressed on resting-Treg (79%), although a subpopulation of CD4+Foxp3-T cells (59%), CD8+T cells (24%), and NK1.1+TCRb+NKT celsl (42%) also expressed DR3. However, DR3 expressions in CD4+Foxp3-T cells and CD8+T cells were elevated after TCR stimulation in vitro (p However, the prophylactic treatment of animals with aDR3 (day 0 αDR3 and day 2 allogeneic T cells) resulted in the expansion of recipient derived Treg (H2kd+CD4+Foxp3+ cells, p Conclusion In conclusion, we demonstrated that activation through DR3 signaling not only expands and activates Treg, but also further activates conventional alloreactive T cells and has very different clinical impact depending upon the timing of administration. These data provide important information for future clinical translation using modification of DR3 signaling. Disclosures Negrin: Stanford University: Patents & Royalties.

OBJECTIVE: The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin-dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis. METHODS: Experimental arthritis was induced in wild-type or p21-/- (C57BL/6) mice, using the K/BxN serum-transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide-induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex-based assay, flow cytometry, or enzyme-linked immunosorbent assay. RESULTS: Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21-/- mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21-intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages. CONCLUSION: These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA., The authors are grateful for the assistance of staff of the Washington University School of Medicine Center for Musculoskeletal Biology and Medicine and the Northwestern University Feinberg School of Medicine Cell Imaging Core Facility and the Methodology and Data Management Core.

The incidence of basal cell carcinoma (BCC), the most frequent human tumor, is rising. BCC incidence and aggressiveness are greatly increased in immunocompromised patients, indicating a role for the immune response in their prevention, formation, and growth. Human biopsies have exhibited a peritumoral infiltrate of lymphocytes with low numbers within the tumor itself. The Ptc1 mouse, a phenocopy of nevoid BCC syndrome, in which patients may acquire thousands of BCCs, develops tumors ~12 months after radiation exposure. To further study the immunobiology of BCC, a more practical tumor model was needed. We evaluated the use of ASZ001, a murine BCC cell line isolated and cultured from a Ptc mouse. Injection of 2.5x106 cells s.c. into Ptc1 mice elicited rapid tumor development with an average tumor volume of 58mm3 on day 2. Microscopic analysis showed an aggressive phenotype and a paucity of intratumoral T cells, similar to BCCs in the Ptc1 mouse and human biopsies. Thus, injection of ASZ001 cells into Ptc1 mice is a rapid tumor model that will be used to study the immune response to BCC. This work was supported by the Saint Louis University Cancer Center and Department of Pediatrics.