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Stimulator of interferon genes (STING) is an innate immune sensor of cytoplasmic dsDNA originating from microorganisms and host cells. STING plays an important role in the regulation of murine graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and may be similarly activated during other transplantation modalities. In this review, we discuss STING in allo-HSCT and its prospective involvement in autologous HSCT (auto-HSCT) and solid organ transplantation (SOT), highlighting its unique role in nonhematopoietic, hematopoietic, and malignant cell types.

Mobilized hematopoietic stem / progenitor cells have become the primary option used for adult allogeneic hematopoietic stem cell transplants (aHSCTs) although GVHD remains a major immunologic complication (CIBMTR, 2017). The present studies are directed to developing a translational model for the application of donor regulatory T cells (Tregs) together with the use of mobilized peripheral blood (mPB) for more widespread application of these aHSCTs. We have previously reported that two-pathway Treg expansion strategy markedly elevates Treg numbers and function (Wolf, 2017; Copsel 2018). Additionally, this approach could be effectively applied in a concomitant strategy to amplify Treg suppressor activity in mPB to diminish pre-clinical GVHD (Barreras H, ASTCT Meeting S292 ,2019). Here, we present new findings using mPB transplants examining GVL reactivity in an MHC-matched minor antigen mismatched pre-clinical model and test the notion that human donor Tregs can be employed with mobilized healthy donor PB to effectively regulate xenogeneic GVHD. Donor C3H.SW (H2b) mice were treated with filgrastim for 4 days prior to use for transplants and PB analysis demonstrated significant elevations in c-kit + cells. Some PB donors were concurrently administered the two-pathway Treg expansion strategy (D-6 through D-1) consisting of TL1A-Ig, targeting TNFRSF25, and low dose huIL-2. A significant increase (up to 40%) in the frequency of CD4 +FoxP3 + Tregs occurred during the mobilization process. Pooled C3H.SW PB was collected from both mobilized and Treg unexpanded ("TrUM") or mobilized and Treg expanded ("TrEM") donors and transplanted into lethally irradiated, MHC-matched B6 (H2b) recipients. Recipients of TrEM donors exhibited significantly reduced weight loss and clinical GVHD scores compared to recipients of TrUM (Fig.1A). GVL responses were tested in animals administered B6-MLL-AF9 GFP leukemia cells administered at the time of transplant. Notably, recipients of TrEM exhibited comparable GVL activity to TrUM recipients evidenced by B6-MLL-AF9 GFP levels in bone marrow and spleen (Fig.1B). To begin to translate these findings, we tested the use of ex-vivo expanded human donor Tregs (huTregs) to ameliorate xenogeneic GVHD (xGVHD). First, sorted huTregs (CD4 +CD25 +CD127 lo) from a healthy donor were expanded ex-vivo using anti-CD3/anti-CD28 beads 1 week prior to transplant (Fig 2A). next, these expanded huTregs were combined with human mPB from the same healthy donor (6x10 6 PBMC) and transplanted into NSG/ NOD-scid IL2Rgamma null mice. We found that treatment with ex-vivo expanded huTregs resulted in significant reduction of mortality rate and clinical xGVHD (Fig 2B,C). Notably, 1 week post-transplant PB huTregs levels were still elevated and frequency of huCD4 +Tconv cells was diminished supporting xGVHD outcomes (Fig 2D). In total, these findings demonstrated that the use of mPB containing elevated Treg levels significantly reduced pre-clinical GVHD without loss of GVL activity in an MHC-matched allogeneic model. Moreover, utilizing ex-vivo expanded huTregs from a mPB donor and added back to the same donor at the time of transplant ameliorated xGVHD. The observations indicate that during the donor stem / progenitor cell mobilization process, manipulation of donor Tregs using our two-pathway strategy can be successfully accomplished in PB resulting in an effective and translational approach to ameliorate GVHD following aHSCT. In total, the present studies support the notion that in vivo or ex-vivo manipulation of donor tregs together with mobilized peripheral blood could provide therapeutic approaches to improve aHSCT outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Aberrant transcriptional programs are cardinal features of Acute Myeloid Leukemia (AML). Recently, it has been shown that specific distal cis-regulatory elements called enhancers communicate with promoters through 3-D DNA looping to regulate tissue-specific gene expression. Recurrent mutations in epigenetic regulators that modify enhancers, transcription factors that bind enhancers and the structural proteins that promote DNA looping, such as the Cohesin complex and its major binding partner CTCF have been demonstrated in AML. However, how these mutations regulate chromatin and alter 3D-DNA topology and communication between enhancers and promoters to generate leukemia-specific transcriptional programs remains poorly understood. In addition, many AML cases lack mutations in epigenetic regulators, transcription factors or DNA structural proteins, yet still demonstrate aberrant transcription, suggesting indirect effects of other mutations on enhancer function and the epigenetic landscape. To address these questions, we have utilized an allelic series of mice carrying the most common mutations in AML, namely Flt3-ITD and Npm1c (co-mutated in ~15% of all AMLs). These model different "transition states" (normal: wild type (WT), Pre-Malignant: single mutant (SM) with either Flt3-ITD or Npm1c mutations and Malignant: double mutant (DM)) during AML induction. Moreover, our design allows analysis of the SM mice to deconvolute the contribution of individual mutations to altered chromatin regulation. We have analyzed hematopoietic stem and progenitor cells (HSPCs) from WT and mutant mice for gene expression (RNA-seq), chromatin activation states (ChIP-seq for H3K4me1, H3K4me3, H3K27ac and H3K27me3), chromatin accessibility (ATAC-seq), and promoter-anchored 3-D chromatin interaction (promoter capture HiC, pCHiC)(Figure 1) and have integrated these analyses to determine the transcriptional, epigenetic and DNA-topological evolution of AML. Through pairwise comparisons between mutant and WT HSPCs, our data demonstrated that SM cells, with either Flt3-ITDor Npm1c mutations, alter gene expression only very modestly. However, when both mutations are present in DM cells, much larger gene programs that drive leukemia are both up- and downregulated. To examine the epigenetic regulation of these programs, we next built an enhancer compendium across all 4 allelic states using the H3K4me1 mark. Layering on H3K27ac activation, our data demonstrated that, in contrast to gene expression, significant alterations in enhancer specification and activation occur in advance of gene expression changes, to "prime" critical genes in Flt3-ITD, but not in Npm1c HSPCs. By contrast, Flt3-ITD and Npm1c mutations both altered global chromatin accessibility, with losses and gains evident at multiple critical genes. Similarly, our pCHiC data demonstrated significant alterations in DNA topology in mutant HSPCs that culminate in alterations in DNA "compartments" in DM HSPC. Moreover, they identified "hardwired" and "rewired" interactions between promoters and enhancers important for expression of critical leukemia programs. Analyses of all of these separate layers demonstrated a uniform pattern; progressive alterations in the transition from SM to DM HSPCs. Integrating these layers of analysis clearly demonstrated synergy between the mutations and a correlation between gene expression changes and chromatin dynamics in mutant cells. Furthermore, performing de novo motif analysis suggested a signal-specific transcription factor (TF) network downstream of Flt3-ITD that was amplified in the DM HSPC and that was corroborated by GSEA analysis. Our data had identified long-range regulatory control regions at the Spi1/PU.1 and Hoxa cluster loci amongst many others, and motif analysis had suggested Hox and Pu.1 to be important TFs in our malignant networks. Using these as examplars, we then perturbed the genes and regulatory elements at these loci by shRNA knockdown and CRISPR-mediated excision and could abrogate leukemic growth, validating the importance of our proposed networks. Taken together, these integrated analyses demonstrate a highly dynamic and coordinated process, where the effects of individual mutations synergize to remodel the chromatin landscape and 3D-DNA topology to generate networks that initiate and maintain AML transcriptional programs. Figure Disclosures Vassiliou: Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding.

Regulatory T cells (Tregs) are critical to maintaining immune homeostasis and generating tolerance in the gastrointestinal (GI) tract. GI complications play a prominent role following allogeneic HSCT (aHSCT) and we are interested in manipulating GI Tregs to regulate GVHD. Previously our lab has shown that using a two-pathway strategy of stimulating the TNFRSF25 and CD25 receptors with a TL1A-Ig fusion protein (FP) and IL-2low dose, Treg frequency and numbers in the lymphohematopoietic (LHC) compartment can be markedly increased (Wolf, BBMT 2017; Copsel, BBMT 2018). As a consequence of microbes and food antigens, Treg populations in the GI have a relative highly diverse composition compared to the lymphohematopoietic Treg compartment. This includes, but is not limited to, the presence of a stable and suppressive FoxP3+RORyt+ Treg population. Additionally, the GI tract contains various populations of innate lymphoid cells (ILCs) that interact with Tregs. ILCs express CD25 as well as TNFRSF25, and respond individually to IL-2 and TL1A administration respectively (Danny, JCI 2017; Verneris, Blood 2013). Based on the extensive differences between the lymphohematopoietic and GI Treg compartments, we evaluated how our two-pathway strategy may be differentially affecting the levels and activation status of Tregs in the GI tract. To address this question, we initially utilized B6-Nur77GFP mice, where GFP expression occurs with activation of the Nur77 promoter. However, since Nur77 is only produced following TcR engagement and not inflammatory signals, the strength of TcR stimulation in all T cell populations can be monitored by GFP levels. We first examined Tregs from 2-pathway treated B6-Nur77GFP mice and subsequently generated B6-Nur77GFPFoxP3RFP mice to readily assess the TcR signaling status of Tregs in different compartments. Mice were systemically administered FP, IL-2, or FP + IL-2 over a 1 wk period. The frequency of Tregs (FoxP3+CD4+) / Tcon (CD4+FoxP3-) in the lamina propria (LP) of the large intestine (LI) reached levels >60%. (1A). This elevation of Treg / Tcon frequency included FoxP3+RORyt- Tregs as well as FoxP3+RORyt+ double positive Treg populations. In contrast to our previous findings in the LHC, treatment with TL1A-Ig FP alone elevated levels to the same extent as the combination of FP + IL-2 (1A). Importantly, IL-2 treatment alone - as reported in the LHC - again had only a modest effect on elevating the frequency of Tregs in the LI/LP (1A). These observations suggest that the activation status of Tregs may differ based on the compartmental location. To asses activation status, we evaluated 2-pathway treated B6-Nur77GFP mice. Tregs in the LN/spleen had elevated frequency of GFP+ Tregs and higher GFP MFI than untreated mice. This elevated TcR stimulation was present in peripheral Tregs - but not CD8 - T cells (1B). Without exogenous stimulation, Tregs exhibited higher baseline TcR activation levels vs. Tcon cells (1B). The frequency of GFP+ Tregs and the GFP MFI was clearly highest (>45%) in the SI and LI LP vs. LN/spleen ( In total, our results objectively validate selective Treg vs. Tcon targeting via TNFRSF25. These findings also demonstrate that basal Treg activation status differs depending on the compartment. Notably, Tregs in mucosal vs. LHC tissue expressed higher TcR activation levels. Such Tregs have the potential for co-stimulation via TNFRSF25. Moreover, since IL-2 is required to maintain Tregs under both basal and activated conditions, our findings suggest a local source of IL-2 is present to maintain GI Tregs. ILC3 cells are recently reported to generate IL-2 (Zhou L, Nature 2019). We hypothesize that TNFRSF25 stimulation in ILC3 cells results in a local source of IL-2 which can account for our finding that TL1A-Ig alone vs. TL1A-Ig + IL-2 stimulation results in equivalent levels of GI Tregs. Ongoing experiments are examining how GI GVHD in aHSCT recipients are affected by TNFRSF25 +/- CD25 Treg/ILC stimulation. Disclosures Levy: Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells, leading to damage of particular host tissues characteristic of GVHD. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to pro-inflammatory cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) aHSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of MUD C3H.SW bone marrow (BM) + T cells into irradiated B6 wildtype (WT) or STING-/- recipients. Colon tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs WT. MUD STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 wks post-HSCT vs WT. Double chimerism studies showed that the absence of STING in non-hematopoietic cells was responsible for GVHD amelioration. Conversely, a single dose of the highly specific STING agonist DMXAA given in vivo increased IFNβ, TNFα and IL-6 mRNA expression in WT, but not STING-/-, colon tissue 48 hrs after transplant and increased GVHD scores and lethality post-HSCT. Post-transplant cytoxan treatment abolished the ability of DMXAA to augment GVHD, supporting the notion that STING signaling increases donor T cell activation during aHSCT. To evaluate the potential impact of STING in the clinical setting, we transplanted C3H.SW BM + T cells into mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) and found that these mice also exhibited diminished GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD aHSCT contrasts to reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) aHSCT (Fischer J, et al, Sci. Transl. Med. 2017). Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we hypothesized that STING's effect on the predominant T cell subset in each model may explain these seemingly paradoxical results in STING-/- vs WT recipients. Therefore, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING-/- mice and found that - in contrast to MMUD recipients of combined CD4+ and CD8+ T cells - STING-/- recipients developed lower GVHD clinical scores, reduced skin pathology and had lower frequencies of activated T cells 8 wks post-HSCT vs WT, supporting a role for STING in the promotion of CD8+ T cell-mediated GVHD. Next, we investigated if recipient APCs played a role in STING's enhancement of CD8+ T cell-mediatedGVHD. We found that STING-/- mice had greater frequencies and numbers of recipient splenic CD11b+CD11c+ APCs 1 day after MMUD B6 into BALB/c aHSCT (Fig. A). BALB/c-STING-/- APCs also expressed reduced MHC class I protein levels (Fig. B). Moreover, STING-/- recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C). These data support the hypothesis that STING contributes to early activation of donor CD8+ T cells and elimination of recipient APCs. Next, to identify if the loss of host MHC II+ APCs affected subsequent donor CD4+ T cell activation, B6-Nur77GFP transgenic donor T cells were used to explicitly monitor T cell receptor signaling. Consistent with increased numbers of host MHC II+ APCs in the spleens of STING-/- recipients 1 day post-aHSCT, we found greater frequencies and numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ in STING-/- spleens 6 days after transplant (Fig. D). In summary, our studies demonstrate that STING plays an important role in regulating aHSCT and provide one potential mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Overall, our studies suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Blazar: BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding. Levy:Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

Background:The PML;RARA fusion protein is the hallmark driver of Acute Promyelocytic Leukemia (APL). The fusion disrupts retinoic acid signalling, leading to the proliferation of myeloid precursors halted at the promyelocyte stage of maturation. Chromatin conformation plays a fundamental role in controlling regulatory networks driving cell differentiation, however the genome wide organisational changes that occur during the PML;RARA differentiation block remains to be elucidated. Aims: We have aimed to characterise three hallmarks of PML;RARA driven transcriptional mis-regulation: transcription factor binding, epigenetic remodelling and higher order chromatin organisation. Methods: To a PML;RARA inducible U937-PR9 cell line model (Figure 1a), we have applied 1) Promoter Capture Hi-C (PCHi-C) to characterise long-range regulatory interactions, 2) Cut&Run, a newly described chromatin profiling technique, and 3) RNA-seq. All datasets are paired and have been modelled as induced vs non-induced. Results: PML;RARA induction resulted in 855 significantly differentially expressed genes (DEGs, adjusted pvalue < 0.01). Up regulated genes (457) were enriched for genes involved in proliferative and oncogenic pathways. Down regulated genes (398) were enriched for drivers of cell differentiation, including the master regulators of myeloid differentiation: CEBPE, CEBPB and CEBPA. We identified 15,412 PML;RARA binding sites using Cut&Run (n=2). These sites encompassed 95% of previously uncovered PML;RARA binding sites, in addition to ~12,000 novel sites. 53% of these regions were distributed at gene promoters, 23% were intergenic and 24% at gene bodies. We observed a global decrease in H3k27ac after PML;RARA induction (86% regional loss), a significant proportion of these regions coincide with PML;RARA binding (Fisher's test 2.2-16). 1,900 PML;RARA peaks were associated with 66% DEGs. Interestingly, a significant proportion of PML;RARA peaks were not associated with DEGs. Applying PCHi-C (n=3) we identified >60,000 consistent differential interactions (DIs, ihw < 0.01), with a mean interaction distance of 100kb. 30,039 interactions increased and 30,403 interactions decreased after PML;RARA induction. Over half of DIs directly involved 8,066 PML;RARA binding sites (Fisher's test 2.2-16). We observed that genes losing long-range interactions were more likely to have decreased expression (59%) and this observation increased if a gene is also bound by PML;RARA (65%). SPI1, ID2, CEBPA, CEBPB, CEBPE, EGR1, and TFEB, key regulators of myeloid differentiation, display this pattern of PML;RARA driven negative regulation. We also observed that genes with increased expression were more likely to gain long-range interactions (60%). The top ranked gene across all datasets was the prostaglandin receptor 4 (PTGER4), a G-Protein coupled receptor highly expressed in AML and upregulated in our datasets (3.1 fold) (figure 1b). PTGER4 gained long-range interactions with an intergenic region ~300kb downstream of its promoter. This intergenic region is highly enriched for both H3k27ac and PML;RARA. This suggests that PML;RARA facilitates the long-range activation of PTGER4. We see a similar pattern to PTGER4 in 15% of DEGs where PML;RARA is anchored to both ends of the DI. Summary/Conclusion: Applying novel NGS techniques to a simple model, we have highlighted that specific chromosome conformations are pivotal to the transcriptional mis-regulation driven by PML;RARA. We show PML;RARA may be directly involved in the re-organisation of the genome and this 3D architecture is pivotal in driving the Leukemia differentiation block. Disclosures Dillon: Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; TEVA: Consultancy, Honoraria.

BACKGROUND: Regulatory T cells (Tregs) are non-redundant mediators of immunity and tolerance. Adoptive transfer of CD4+Foxp3+ Tregs has emerged as a promising therapy for graft-versus-host disease (GVHD) following allogeneic HSCT (aHSCT), solid organ transplantation and autoimmune diseases (Hanash AM, Blood 2005; Copsel S, Wolf D, Haematologica 2019; Marek-Trzonkowska N, Diabetes Care 2012; Tang Q, J Mol Cell Biol 2012). Our prior work was the first to demonstrate a two-pathway in vivo strategy targeting TNFRSF25 (with TL1A-Ig fusion protein) and CD25 (with low dose IL-2, IL-2LD) receptors can elicit a rapid and strong increase in Treg numbers and function (Wolf D, BBMT 2017). In fact, very low numbers of these in vivo expanded donor Tregs exhibited effective GVHD suppression in recipients following aHSCT (Copsel S, BBMT 2018). Based on these findings and the success of IL-2LD in pre-clinical and clinical studies, we asked: are there phenotypic/functional differences between Tregs expanded via IL-2LD, TL1A-Ig or their combined application (TL1A-Ig+IL-2LD)? METHODS: Mice were administered IL-2LD, TL1A-Ig or TL1A-Ig+IL-2LD over 6 days. Splenic and lymph node (LN) Treg phenotype was determined by flow cytometry. Treg functionality was assessed with sorted populations using an in vivo MHC-mismatched aHSCT. RESULTS: Treatment of C57BL/6-FoxP3RFP mice with TL1A-Ig+IL-2 LD versus IL-2LD only treatment resulted in significantly higher levels of activation/differentiation and functional markers on Tregs including KLRG1, CD103, Nrp1, ICOS (Fig. 1A). Ki67 expression was higher in two-pathway versus IL-2LD stimulation alone (Fig.1B). These data suggested a key role for TNFRSF25 stimulation. Notably, Treg stimulation with TL1A-Ig alone drove the above phenotype indicating a pathway difference between the TNFRSF25 and IL-2 receptors. This difference was further apparent as high affinity IL-2 receptor (CD25) expression was reduced after TL1A-Ig +/- IL-2LD -mediated expansion compared with IL-2 LD alone stimulated Tregs (Fig. 1A). Results were corroborated using a second independent mouse strain, BALB/c, following use of these protocols. To begin addressing if the observed phenotypic differences between CD25 vs. TNFRSF25 + CD25 expanded Tregs could be related to a more potent Treg in vivo suppressive activity, an initial MHC-mismatched aHSCT (donor/recipient = C57BL/6-BALB/c) was performed. We employed 200,000 sorted Tregs (>98% purity by CD4+FoxP3+ selection from C57BL/6-FoxP3RFP reporter mice) from donor unexpanded, IL-2LD, or TL1A-Ig+IL-2LD treated mice combined with 1.0 x106 T cells. As anticipated, transfer 200,000 TL1A-Ig+IL-2LD stimulated Tregs ameliorated acute GVHD (Fig. 1C). Remarkably, lower GVHD clinical scores were obtained using the same number of IL-2LD only expanded Tregs compared with TL1A-Ig+IL-2LD stimulated Tregs (Fig. 1C). Moreover, early post-transplant, higher LN and splenic CD4/CD8 ratios were detected in aHSCT recipients treated with IL-2LD expanded Tregs vs. TL1A-Ig+IL-2LD (Fig. 1D). CONCLUSION: Our donor TL1A-Ig+IL-2LD Treg expansion protocol promotes a more activated/differentiated and proliferative phenotype versus IL-2LD stimulation alone. This finding may have accounted for their initial effectiveness - but less efficient long-term GVHD amelioration compared to IL-2LD only stimulated Tregs. Multiple variables are associated with the application of Tregs for therapy including numbers, persistence, and suppressive capacity. Our findings suggest a rationale that one-pathway and / or two-pathway stimulated Tregs may be beneficial for use in aHSCT recipients dependent on whether there is a perceived need for prolonged Treg presence and the stage of GVHD. Disclosures Levy: Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (allo-HSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells. Damage to host tissues initiates a cytokine storm, promoting activation and expansion of donor anti-host alloreactive T cells. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched (MUD) allo-HSCT models. Our studies show that the STING pathway rapidly regulates cytokine production in the intestinal tract and non-hematopoietic cells can contribute to these responses. Using mice expressing a human STING allele associated with decreased STING activity (Patel S, et al, J Immunol. 2016), we demonstrate its potential clinical importance. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of C3H.SW bone marrow (BM) + T cells into irradiated B6-WT or STING-/- recipients. Colonic tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs. WT. On day 10 post-transplant, colons from STING-/- recipients exhibited reduced inflammation and overall pathology scores than WT. MHC-matched STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 weeks post-HSCT vs. WT. Chimeric studies demonstrated that the absence of STING in non-hematopoietic cells was responsible for the amelioration of GVHD. Therefore, to test STING signaling in non-hematopoietic intestinal cells, we generated intestinal organoid cultures. Intestinal organoids upregulated IFNβ, TNFα, IL-6 and CXCL10 mRNA 6hrs after stimulation with the highly specific STING agonist DMXAA, supporting the notion that STING in intestinal tissues can contribute to inflammation in vivo. Interestingly, expression of these cytokines returned to baseline levels 24 hrs after stimulation (Fig. 1A). Next, we posited that if the absence of the STING pathway in recipients ameliorated GVHD after MHC-matched HSCT, pathway stimulation would exacerbate GVHD. B6-WT mice were injected with DMXAA immediately prior to HSCT with donor C3H.SW BM + T cells. Administration of a single dose of DMXAA increased expression of IFNβ, TNFα and IL-6 mRNA in colon tissue 48 hrs after transplant (Fig. 1B). Importantly, DMXAA treatment of WT - but not STING-/- - recipients significantly increased GVHD scores and lethality post-HSCT. To evaluate the potential impact of STING in the clinical setting, we evaluated recipients after transplant of C3H.SW BM + T cells into mice homozygous for a human allele associated with diminished STING activity (HAQ-MPYS knock-in mice, termed B6N-STINGHAQ/HAQ here) and found that STINGHAQ/HAQ mice contained a lower frequency of donor T cells expressing an activated phenotype (CD44hiCD62Llo) vs. WT recipients and the former also exhibited diminished GVHD (Fig. 1C,D). In contrast to STING knock-out recipients completely lacking protein, these results indicate that reduced STING activity can also affect GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MHC-matched allo-HSCT contrast reported observations that STING activation can exacerbate GVHD after MHC-mismatched HSCT (Fischer J, et al, Sci. Transl. Med. 2017). We are currently investigating how the STING pathway regulates CD4+ and CD8+ T cell mediated GVHD and initial findings may provide insight into understanding the pathway's involvement in MHC-matched vs. mismatched allo-HSCT. In total, our studies demonstrate that STING plays an important role in regulating allo-HSCT and suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Levy: Allergan: Consultancy; Capricor Therapeutics: Consultancy; HEAT Biologics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pelican Therapeutics: Consultancy; OccuRx: Research Funding; Shire: Research Funding.

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (allo-HSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration which results in the death of rapidly dividing cells and the initiation of a cytokine storm, promoting activation and expansion of donor anti-host alloreactive T cells in HSCT recipients. However, the precise mechanism of cytokine production remains unclear. Cell death following pre-transplant conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to cytokine production. One such sensor is Stimulator of Interferon Genes (STING) which following activation induces phosphorylation of IRF3 and IκBα. Although STING activation was recently reported to worsen GVHD after MHC-mismatched allo-HSCT (Fischer J, et al, Sci. Transl. Med. 2017), STING involvement in MHC-matched allo-HSCT has not yet been thoroughly evaluated. Here, using B6-STING knock-out recipients and either MHC-mismatched or matched ("MUD") donor strains we corroborate that STING deficiency worsens - but in contrast - ameliorates GVHD in the former and latter pre-clinical mouse models, respectively. To evaluate the effect of STING deficiency on GVHD after MHC-mismatched allo-HSCT, BALB/c BM + T cells were transplanted into 11.5Gy irradiated B6-WT and B6-STING-/- recipients. Consistent with previously reported results using B6-STINGgt/gt mice, STING-/- recipients experienced increased weight loss and GVHD clinical scores post-HSCT as well as decreased survival relative to WT recipients (Fig. 1A). Notably, when irradiation was given on day 0 instead of day -1 using the same donor/recipient combination no significant difference in GVHD was observed between WT and STING-/- recipients (Fig. 1B & 1E). We next investigated the role of STING after clinically relevant models of MHC-matched allo-HSCT. To assess for a role by STING immediately after HSCT, we examined cytokine mRNA expression 48 hrs. after transplant of LP/J BM + T cells → 8.5Gy irradiated B6-WT and B6-STING-/- recipients. Colonic tissue from STING-/- recipients had reproducibly >2x reduction in IFNβ, TNFα and IL-6 mRNA compared to WT (Fig. 1C). Importantly, IFNβ is one of the major downstream effector molecules produced as a result of IRF3 phosphorylation, consistent with the notion that STING activation occurs shortly after conditioning and/or allo-HSCT. Notably, LN examination at this time demonstrated fewer donor CD8+ T cells and CD8+CD44hiCD62Llo cells in STING-/- vs WT recipients. In contrast to STING-/- recipients of MHC-mismatched HSCT, MHC-matched STING-/- HSCT recipients experienced decreased weight loss and GVHD clinical scores vs. WT mice. STING-/- recipients contained 20-50% fewer donor lymphocytes and a significantly reduced frequency of activated donor CD4+ and CD8+ T cells in lymphoid tissues on D7. Analysis of peripheral LN and spleen 1-3 mo. post-HSCT revealed a similar profile and a higher frequency of naïve T cells - consistent with their decreased clinical signs of disease. Histological examination of recipient skin also showed higher pathology scores in WT recipients relative to STING-/- recipients. These findings were corroborated using a second MUD model, i.e. C3H.SW → B6 (Fig. 1D). Furthermore, measurement of serum cytokines 6 wks. post-transplant showed that B6-STING-/- recipients had higher levels of the immunosuppressive cytokine IL-10. The effect of donor T cell dose was also examined. Increased donor T cell numbers resulted in enhanced GVHD clinical scores in STING-/- recipients, however, the GVHD remained less severe than that observed in WT. Notably, HSCT studies using fully reconstituted chimeric B6-CD45.1↔B6-STING-/- indicated that STING expression in non-hematopoietic tissues is important for the development of GVHD. In total, this is the first report we are aware of in which the same pathway appears to differentially impact the outcome of allogeneic HSCT based upon the genetic disparity across the transplant. These findings reveal that STING's contribution to the development of GVHD apparently differs depending on the presence or absence of an MHC-disparity between donors and recipients as well as the timing of the pathway's activation and HSCT. Disclosures Levy: Capricor Therapeutics: Consultancy; OccuRx: Research Funding; Shire: Research Funding; Allergan: Consultancy.

The 7.2 kilobase (kb) Corfu δβ thalassemia mutation is the smallest known deletion encompassing a region upstream of the human δ gene that has been suggested to account for the vastly different phenotypes in hereditary persistence of fetal hemoglobin (HPFH) versus β thalassemia. Fetal hemoglobin (HbF) expression in Corfu heterozygotes and homozygotes is paradoxically dissimilar, suggesting conflicting theories as to the function of the region on globin gene regulation. Here, we measure γ- and β-globin gene transcription, steady-state mRNA, and hemoglobin expression levels in primary erythroid cells cultured from several patients with Corfu δβ thalassemia. We show through RNA fluorescence in situ hybridization that the Corfu deletion results in high-level transcription of the fetal γ genes in cis with a concomitant reduction in transcription of the downstream β gene. Surprisingly, we find that elevated γ gene transcription does not always result in a corresponding accumulation of γ mRNA or fetal hemoglobin, indicating a post-transcriptional regulation of γ gene expression. The data suggest that efficient γ mRNA accumulation and HbF expression are blocked until β mRNA levels fall below a critical threshold. These results explain the Corfu paradox and show that the deleted region harbors a critical element that functions in the developmentally regulated transcription of the β-globin genes.

Regulatory T cells (Treg) are critical for the maintenance of self-tolerance and Treg expansion is being investigated as a promising therapy for graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. Because the numbers of donor graft Tregs necessary for maximal efficacy are significant, ex vivo expansion presents both practical and scientific challenges. We and others have demonstrated that in situ expansion of CD4+ FoxP3+ Tregs using IL-2 may be used to inhibit alloreactive T cells that mediate GVHD. Recently, we developed a novel combination approach to expand donor Tregs, wherein we administered a fusion protein (TL1A-Ig) and IL-2, which target, respectively, TNF receptor super family 25 (TNFRSF25) and CD25, the high affinity IL-2 receptor. Following a 6-day sequence of Tl1A-Ig and IL-2, we observed markedly greater peripheral Treg expansion (5-8 fold) vs. either reagent alone (2-3 fold). The resulting Treg expansion in healthy animals is transient, peaking by 7-8 days, and results in no long-term complications, as assessed by changes in blood counts, immune phenotype / function and tissue pathology 6 months following cessation of treatment. Analyses of animals 1 week following sequential therapy revealed Treg expansion in blood, spleen, lymph nodes, and the colon but not bone marrow. Expanded Tregs were highly activated (Ki67+) with an elevated KLRG1+ fraction. High functional activity was also reflected in their hypomethylated FoxP3 promoter region, elevated pSTAT5 expression and the ability of sorted Tregs to mediate functional suppression of lymphocyte proliferation. Notably, assessment of the splenic vs. LN Treg compartment indicated that there were consistently higher Treg frequencies an d greater levels of Treg effector proteins (Gzmb/TNFa/IFNg/IL-10) in the splenic Treg compartment. To assess the functional significance of this difference, we stimulated splenocytes and lymph node cells from TL1A-Ig/IL-2 treated animals with anti-CD3 mAb and LPS, and observed significantly greater suppression in the spleen vs LN cultures. Importantly, even when cultures of LN cells contained the same or greater numbers of Tregs compared with the spleen, greater suppression was detected in the splenic cultures. Based on the heightened suppressive environment in spleens post-Treg expansion, we performed HSCT across a complete MHC mismatch using the B6àBALB/c donor/recipient combination. Groups of 8.5 Gy TBI-conditioned mice received T cell depleted B6 bone marrow and either spleen or LN cells from TL1A-Ig/IL-2 treated or untreated B6 mice. While GVHD was reduced in recipients of either spleen or lymph node T cells, relative to recipients of control (non-expanded) donors, suppression of GVHD was significantly greater in recipients of spleen cells (by weight loss, clinical score and survival). In summary, we have identified a novel strategy to rapidly and transiently expand donor Treg cell numbers in situ. The splenic compartment contained increased numbers and more functionally active Tregs compared to lymph nodes. Overall, TL1A-Ig/IL-2 expanded Tregs demonstrated excellent suppressive function both ex vivo and in vivo, as assessed by GVHD incidence and severity following allogeneic transplantation. This approach may be a promising strategy for GVHD prevention or therapy in the clinical setting. Figure 1. Figure 1. Disclosures Podack: Heat Biologics: Equity Ownership, Patents & Royalties: Dr. Podack is an inventor of patents used in the study and stands to gain royalties from future commercialization of "immunomodulating tumor necrosis factor receptor 25 (TNFR25) agonists, antagonists and immunotoxins, Research Funding. Levy:Allergan: Consultancy.

The placenta was recently unveiled as an important hematopoietic organ, harboring a large pool of HSCs during midgestation. Yet, it has not been defined whether the placenta can generate HSCs de novo. By using the Runx1-LacZ and Ncx1 knockout mouse models we show that the placenta is a site of HSC generation and identify the cellular niches in which placental HSCs reside. Runx1 is essential for the emergence of definitive HSCs and remains expressed in HSCs throughout fetal development and adult life. Analysis of Runx1LacZ/+ and Runx1LacZ/LacZ placental sections nominated the large vessels of the placenta and the chorioallantoic mesenchyme as putative sites of HSC origin. Once formed, LacZ+ candidate HSCs convened in the labyrinth vessels. Co-staining of Runx1LacZ/+ placentas with an antibody specific for phosphorylated Ser 10 at histone 3, a marker of mitosis, showed mitotically active definitive hematopoietic cells in the labyrinth vessels, suggesting that the labyrinth is a microenvironmental niche capable of stimulating HSC expansion. In wild-type placentas, CD41+ nascent hematopoietic cells were found in the same vascular sites as in the Runx1-LacZ placentas but never in the mesenchyme. Instead, placental stroma was populated by F4/80+CD45+/−CD41- macrophages, suggesting that the placenta harbors two distinct hematopoietic lineages that are supported by different microenvironments. To verify that the CD41+ nascent HSCs were generated de novo in the placenta, we analyzed Ncx1−/− embryos, which lack heartbeat due to lack of the sodium-calcium exchange pump 1. In the absence of circulation, trafficking of hematopoietic cells between tissues is abolished. Strikingly, CD41+ HSCs emerge in the large vessels of the placenta in Ncx1−/− mutants. In some sections CD41+ cells formed clusters that were still connected to the vessels of the placenta and umbilical cord. These findings imply that formation of HSCs extends to a much larger anatomical area than was previously thought, including the placenta. Importantly, the placentas in both Ncx1−/− and control embryos (E8.5–9.5) generated mixed hematopoietic outgrowth including definitive progenitors in OP-9 co-culture, as verified by expression of c-kit, CD41 and CD45. When the differentiation of the definitive progenitors was assessed on methylcellulose, Ncx1−/− tissues demonstrated similar potential as Ncx1+/− hematopoietic organs (yolk sac, aorta gonad mesonephros (AGM) and placenta), yielding erythroid, myeloid and mixed colonies and B220+ lymphoid cells. These studies reveal that definitive hematopoietic cells with both myeloerythroid and lymphoid potential are generated de novo within the placental vasculature. Furthermore, the placental labyrinth provides a unique hematopoietic niche that is conducive for proliferation of hematopoietic cells and, unlike the AGM or the yolk sac, serves as a supportive niche for a large pool of HSCs prior to liver colonization.