BET Bromodomain Inhibitors Which Permit Treg Function Enable a Combinatorial Strategy to Suppress GVHD in Pre-clinical Allogeneic HSCT

A recent approach for limiting production of pro-inflammatory cytokines has been to target bromodomain and extra-terminal (BET) proteins. These epigenetic readers of histone acetylation regulate transcription of genes involved in inflammation, cardiovascular disease, and cancer. Development of BET inhibitors (BETi) has generated enormous interest for their therapeutic potential. Because inflammatory signals and donor T cells promote graft-versus-host disease (GVHD), regulating both pathways could be effective to abrogate this disorder. The objective of the present study was to identify a BETi which did not interfere in vivo with CD4+FoxP3+ regulatory T cell (Treg) expansion and function to utilize together with Tregs following allogeneic hematopoietic stem cell transplantation (aHSCT) to ameliorate GVHD. We have reported that Tregs can be markedly expanded and selectively activated with increased functional capacity by targeting TNFRSF25 and CD25 with TL1A-Ig and low dose IL-2, respectively. Here, mice were treated over 7 days (TL1A-Ig + IL-2) together with BETi. We found that the BETi EP11313 did not decrease frequency/numbers or phenotype of expanded Tregs as well as effector molecules, such as IL-10 and TGF-β. However, BETi JQ1 interfered with Treg expansion and altered subset distribution and phenotype. Notably, in Treg expanded mice, EP11313 diminished tnfa and ifng but not il-2 RNA levels. Remarkably, Treg pSTAT5 expression was not affected by EP11313 supporting the notion that Treg IL-2 signaling remained intact. MHC-mismatched aHSCT (B6 → BALB/c) was performed using in vivo expanded donor Tregs with or without EP11313 short-term treatment in the recipient. Early post-transplant, improvement in the splenic and LN CD4/CD8 ratio along with fewer effector cells and high Treg levels in aHSCT recipients treated with expanded Tregs + EP11313 was detected. Interestingly, this group exhibited a significant diminution of GVHD clinical score with less skin and ocular involvement. Finally, using low numbers of highly purified expanded Tregs, improved clinical GVHD scores were observed in EP11313 treated recipients. In total, we conclude that use of this novel combinatorial strategy can suppress pre-clinical GVHD and posit, in vivo EP11313 treatment might be useful combined with Treg expansion therapy for treatment of diseases involving inflammatory responses

Improved NK Cell Recovery Following Use of PTCy or Treg Expanded Donors in Experimental MHC-Matched Allogeneic HSCT

•Post-transplantation cyclophosphamide (PTCy) and regulatory T cell (Treg) treatment for graft-versus-host disease prophylaxis improves natural killer (NK) cell reconstitution after MHC-matched allogeneic hematopoietic stem cell transplantation.•Effector function of the NK cell compartment is augmented after PTCy or Treg treatment. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is complicated by graft- versus-host disease (GVHD), which causes immune dysfunction and further delays immune reconstitution through its effects on primary and secondary lymphoid organs. Treatments to prevent GVHD and improve immune recovery following allo-HSCT are needed. Post-transplantation cyclophosphamide (PTCy) is a well-established and clinically widely used method for GVHD prophylaxis after HLA-matched as well as haploidentical allo-HSCT, as well as a promising strategy in the setting of mismatched unrelated donor allo-HSCT. Recently, regulatory T cells (Tregs), a critical subset for immune homeostasis and tolerance induction, have been evaluated for use as GVHD prophylaxis in experimental models and clinical trials. Natural killer (NK) cells are one of the first lymphoid populations to reconstitute following allo-HSCT and are important mediators of protective immunity against pathogens, and are also critical for limiting post-transplantation relapse of hematologic cancers. Several reports have noted that a delay in NK cell recovery may occur following experimental mouse allo-HSCT as well as after clinical allo-HSCT. Here we examined how 2 treatment strategies, PTCy and donor expanded Tregs (TrED), in experimental MHC-matched allo-HSCT affect NK recovery. Our experiments show that both strategies improved NK cell numbers, with PTCy slightly better than TrED, early after allo-HSCT (1 month) compared with untreated allo-HSCT recipients. Importantly, NK cell IFN-γ production and cytotoxic function, as reflected by CD107 expression as well as in vivo killing of NK-sensitive tumor cells, were improved using either PTCy or TrED versus control allo-HSCT recipients. In conclusion, both prophylactic treatments were found to be beneficial for NK recovery and NK cell function following MHC-matched minor antigen-mismatched experimental allo-HSCT. Improved NK recovery could help provide early immunity toward tumors and pathogens in these transplant recipients

Improved NK Cell Recovery Following Use of PTCy or Treg Expanded Donors in Experimental MHC-Matched Allogeneic HSCT

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is complicated by graft- versus-host disease (GVHD), which causes immune dysfunction and further delays immune reconstitution through its effects on primary and secondary lymphoid organs. Treatments to prevent GVHD and improve immune recovery following allo-HSCT are needed. Post-transplantation cyclophosphamide (PTCy) is a well-established and clinically widely used method for GVHD prophylaxis after HLA-matched as well as haploidentical allo-HSCT, as well as a promising strategy in the setting of mismatched unrelated donor allo-HSCT. Recently, regulatory T cells (Tregs), a critical subset for immune homeostasis and tolerance induction, have been evaluated for use as GVHD prophylaxis in experimental models and clinical trials. Natural killer (NK) cells are one of the first lymphoid populations to reconstitute following allo-HSCT and are important mediators of protective immunity against pathogens, and are also critical for limiting post-transplantation relapse of hematologic cancers. Several reports have noted that a delay in NK cell recovery may occur following experimental mouse allo-HSCT as well as after clinical allo-HSCT. Here we examined how 2 treatment strategies, PTCy and donor expanded Tregs (TrED), in experimental MHC-matched allo-HSCT affect NK recovery. Our experiments show that both strategies improved NK cell numbers, with PTCy slightly better than TrED, early after allo-HSCT (1 month) compared with untreated allo-HSCT recipients. Importantly, NK cell IFN-γ production and cytotoxic function, as reflected by CD107 expression as well as in vivo killing of NK-sensitive tumor cells, were improved using either PTCy or TrED versus control allo-HSCT recipients. In conclusion, both prophylactic treatments were found to be beneficial for NK recovery and NK cell function following MHC-matched minor antigen-mismatched experimental allo-HSCT. Improved NK recovery could help provide early immunity toward tumors and pathogens in these transplant recipients

Recipient Tregs: Can They Be Exploited for Successful Hematopoietic Stem Cell Transplant Outcomes?

Human and mouse CD4(+)FoxP3(+) T cells (Tregs) comprise non-redundant regulatory compartments which maintain self-tolerance and have been found to be of potential therapeutic usefulness in autoimmune disorders and transplants including allogeneic hematopoietic stem cell transplantation (allo-HSCT). There is substantial literature interrogating the application of donor derived Tregs for the prevention of graft versus host disease (GVHD). This Mini-Review will focus on the recipient's Tregs which persist post-transplant. Although treatment in patients with low dose IL-2 months post-HSCT are encouraging, manipulating Tregs in recipients early post-transplant is challenging, in part likely an indirect consequence of damage to the microenvironment required to support Treg expansion of which little is understood. This review will discuss the potential for manipulating recipient Tregs in vivo prior to and after HSCT (fusion proteins, mAbs). Strategies that would circumvent donor/recipient peripheral blood harvest, cell culture and ex-vivo Treg expansion will be considered for the translational application of Tregs to improve HSCT outcomes

TNFRSF25 and CD25 Stimulation Expands Tregs and ILC2s in the GI Tract: Recipient Modulation Pre-HSCT

Our lab has shown that using a 2-pathway strategy of stimulating TNFRSF25 and CD25 with a TL1A-Ig fusion protein (FP) and low dose IL-2, Treg frequency and numbers in the lymphohematopoietic (LHC) compartment can be markedly increased. Tregs and innate lymphoid cells (ILCs) are critical to maintaining immune homeostasis and generating tolerance in the GI tract. In addition to a highly diverse population of Tregs, the GI also contains various populations of ILCs, recently shown to modulate GVHD (Bruce, 2017). ILCs express CD25 as well as TNFRSF25 and respond individually to IL-2 and TL1A administration respectively. Here we asked how our 2-pathway (FP+IL-2) approach affected Tregs and ILCs in the GI of healthy mice. First, we examined Tregs from B6-Nur77GFP or B6-Nur77GFPFoxP3RFP mice to assess the Treg TcR signaling status in different tissues. Mice were administered FP, IL-2, or FP+IL-2 over 1 wk. The frequency of Treg/CD4 in the lamina propria (LP) of the large intestine (LI) reached levels >60% (1A). This elevation included FoxP3+RORyt− and FoxP3+RORyt+ Tregs. In contrast to our previous findings in the LHC, treatment with FP alone elevated levels to the same extent as the FP+IL-2 in the LI/LP, whereas IL-2 treatment alone had only a modest effect on elevating the frequency of Tregs (1A), suggesting that the activation status of Tregs differs based on compartmental location. The frequency of GFP+ Tregs and the GFP MFI was clearly highest (>45%) in the SI and LI LP vs. LHC (<35%) (1C). An independent experiment verified these findings and found that only conjunctival Tregs had a similar activation (GFP) phenotype (1D). Interestingly, without exogenous stimulation, Tregs exhibited higher baseline TcR activation levels vs. Tcon (1B). Next, we evaluated FP+IL-2 treated B6-Nur77GFP mice. Tregs - but not CD8 T-cells in the LHC had elevated GFP by frequency and MFI vs untreated mice (1E). Notably, we also found that FP+IL-2 treatment in vivo increases the relative frequency of both ILC2s and ILC3s (1F). Regarding ILC2s, FP+IL-2 increased their frequency greater than either TNFRSF25 or CD25 stimulation alone in the GI tract (1G). We then asked if our FP+IL-2 approach could be used to augment these populations as a potential strategy to protect HSCT recipients. FP+IL-2 stimulation prior to TBI in BALB/c mice significantly augmented Tregs and ILC2s in the GI tract for at least 1 wk post-irradiation (2A,2B). In total, these findings demonstrate 1) basal Treg activation status differs depending on the compartment and mucosal Tregs express highest TcR activation, 2) FP alone vs. FP+IL-2 stimulation results in equivalent expansion of GI Tregs, 3) only FP+IL-2 effectively expand ILC2s and 4) pre-TBI FP+IL-2 treatment results in elevated Treg and ILC2 levels post-irradiation. Ongoing experiments are examining GI GVHD and the role of Tregs/ILCs in TNFRSF25 +/- CD25 stimulated aHSCT recipients

The Innate Immune Sensor Sting Can Augment or Ameliorate Graft-Versus-Host Disease Dependent on the Genetic Disparity between Donors and Recipients

Abstract 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 Location of CD4+FoxP3+ Cells with Regard to CD25 and TNFRSF25 Receptor Signals Matters: Different Requirements for GI and Peripheral Tregs

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 (<35%) (1C). In an independent experiment, we verified these findings and learned that only conjunctival Tregs demonstrated similarly increased frequency of GFP+ Tregs with elevated GFP MFI (1D). We also found that TL1A-Ig + IL-2 treatment in vivo increases the relative frequency of both innate ILC2 and ILC3 lymphoid populations (2A). The combination also increased the frequency of ILC2 cells greater than either reagent alone in the GI tract (2B). 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

The Innate Immune Sensor Sting Regulates Intestinal Inflammation and GVHD after Allogeneic Hematopoietic Stem Cell Transplantation in Knock-out and Human Allele Knock-in Recipient Mice

Abstract 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