Host-derived Spi6 provides granzyme B-independent protection of intestinal epithelial cells in murine GVHD

Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic cell transplantation (allo-HCT) that limits the therapeutic potential of this treatment. Host antigen-presenting cells (APCs) play a vital role in activating donor T cells which subsequently use granzyme B (GzmB) and other cytotoxic molecules to damage host normal tissues. Serine protease inhibitor 6 (Spi6), known as the sole endogenous inhibitor of GzmB, has been implicated in protecting T cells and APCs against GzmB-inflicted damage. In this study, we used murine models to examine the previously unknown role of host-derived Spi6 in GVHD pathogenesis. Our results indicated that host Spi6 deficiency exacerbated GVHD as evidenced by significantly increased lethality, clinical and histopathological scores. Using bone marrow chimera system, we found that Spi6 in non-hematopoietic tissue played a dominant role in protecting against GVHD and was significantly upregulated in intestinal epithelial cells after allo-HCT, while Spi6 in hematopoietic APCs surprisingly suppressed alloreactive T cell response. Interestingly, the protective effect of Spi6 and its expression in intestinal epithelial cells appeared to be independent of donor-derived GzmB. We employed in silico modeling to explore potential targets of Spi6. Interaction tested in that could inhibit caspase-3 caspase-8 same functional loop an in vitro we T cell-derived inducing Spi6 expression in an intestinal epithelial cell line. our data indicate that host Spi6 plays a novel, GzmB-independent role in regulating alloreactive T cell response and protecting intestinal epithelial cells. Therefore, enhancing host-derived Spi6 function has the potential to reduce GVHD. in GVHD models, nor is known whether Spi6 can protect GVHD target cells such as intestinal epithelial cells. In this study, we have used several clinically relevant GVHD models to investigate the roles of Spi6 in host APCs and non-hematopoietic tissue cells. We have found that Spi6 deficiency in the host exacerbated GVHD. We demonstrate here that Spi6 plays important roles in host APC function and GVHD target organ protection in GzmB- independent manner. Using in silico methods we showed that Spi6 can inhibit caspase-3 and caspase-8 but with lower affinity compared to GzmB-Spi6 interaction. We confirmed that Spi6 expression and function in intestinal epithelial cells is dependent on donor T cell-derived IFN- γ . Altogether, our data suggest that Spi6 could have broader regulatory effect on inflammatory T cell response associated with GVHD that is beyond GzmB inhibition. provided by Dr. Dirk (Technische Universität München). This cell line was derived from C3H/He mice exhibits similar morphological and phenotypic characteristics of normal enterocytes as well as expressing MHC class II molecules and TLR9 23 . The cells were maintained at 37°C and 5% CO2 in DMEM high glucose supplemented with L-glutamine and 10% heat inactivated FBS. CD4+ and CD8+ cells were harvested from C57BL/6 mice by using EasySep mouse CD4+ isolation kit and EasySep CD8+ mouse isolation kit respectively (Stem Cells Technologies). MODE-K cells were cultured in 24-well plate. After 80% confluency, MODE-K cells were co-cultured with 2x10 5 either CD8+ T cells or CD4 + T cells in RPMI1640 culture media supplemented with L-glutamine, penicillin-streptomycin and 10% heat inactivated FBS. After 48 hours, cells were washed and MODE-K cells were detached using Trypsin and stained with live/dead, CD3 and Spi6 antibody. Spi6 protein was stained using rabbit anti mouse Spi6 antibody (Hycultbiotech) and Alexa-flour 488 donkey to rabbit IgG. CD3 and CD28 antibody (both form Bioxcell) were added in co-culture groups at 13μ/ml. Anti-mouse IFN- γ (clone H22; 53μ/ml) and purified Armenian hamster IgG isotype control (Clone HTK88; 53μ/ml) were used to block IFN- γ . To detect cytokine production by T cells in co-culture, we added Brefeldin A (Invitrogen) 4 hours before T cells staining. other criteria such as activity, severity of diarrhea and posture changes after consulting with veterinarian from animal facility. Statistical significances for Kaplan-Meier survival curves are evaluated by Log-rank (Mantel-Cox) tests and body weight loss, and clinical GVHD scores are evaluated