ALBERT

Description: 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.

Description: 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 (

Description: 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.

Description: 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.