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
Authors: Bader Cameron S. , Barreras Henry , Lightbourn Casey O. , Copsel Sabrina , Ahn Jeonghyun , Barber Glen N. , Jin Lei , Levy Robert B. 
Published: October 2019

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.