An Essential Role for STAT1 Signaling in Regulation of Donor CD4⁺ T Cell Dependent Acute Graft Versus Host Disease (GVHD) Following Allogeneic Bone Marrow Transplantation (BMT).

Abstract 3551

Poster Board III-488

We have previously demonstrated that activation of STAT1 and STAT3 in target tissue and secondary lymphoid organs belong to the earliest events during induction of GVHD. Using STAT1-gene-deficient (STAT1KO) mice we tested the role of donor STAT1 in fully MHC-mismatched (129Sv[H2^b] to BALB/c [H2^d]) and MHC-matched minor histocompatibility antigen (mHA)-mismatched strain combinations (129Sv[H2^b] to B6[H2^b]). GVHD was induced lethal irradiation and transplantation of allogeneic donor bone marrow cells and whole spleen cells. GVHD in the MHC-mismatched model is primarily CD4 dependent. Induction of GVHD was associated with activation of STAT1 and significant expansion of activated STAT1 expressing CD4⁺ and CD8⁺ T cells as assessed by analysis of STAT1 Tyr701 phosphorylation using phosphoflow staining. Using STAT1KO whole splenocytes we were able to show that lack of STAT1 significantly inhibited development of GVHD in both major and mHA mismatched recipients with significantly extended median survival times (MST) and lower GVHD morbidity. Protection against GVHD in recipients of STAT1KO splenocytes was associated with significant contraction of CD8+ T cells, but expansion of CD4 T cells on days +3 and +6 post-BMT in the MHC-mismatched setting. Most importantly, we observed a significant expansion of CD4⁺CD25⁺ FOXP3⁺ T_reg cells in recipients of STAT1KO splenocytes. Lack of STAT1 in donor splenocytes resulted in a significantly attenuated and skewed systemic inflammatory response on day +6 post-BMT as demonstrated by significantly reduced IFN-g levels 508pg/ml vs 84.pg/ml (p<0.05), but significantly increased IL-4 (p=0.003), IL-5 (p=0.007) and IL-17 (p=0.03) levels. IL-6 levels were also increased with a trend towards statistical significance (p=0.08). In vitro studies demonstrated that STAT1KO CD8⁺ T cells produced much less IFN-g upon combined engagement of TCR and costimulation, but that this decrease in IFN-g secretion could be rescued if cells were simultaneously cultured under Th1 conditions (ie in the presence of IL-12 and anti-IL4 antibody). In contrast, lack of STAT1 completely inhibited the differentiation of naïve CD4⁺ T cells to IFN-g -producing cells upon TCR commitment and this capacity was also severely impaired under Th1 conditions. Furthermore, we observed a significantly reduced number of CXCR3⁻expressing CD4⁺ T cells in recipients of STAT1 KO splenocytes. In parallel to the afore-mentioned observations, tissue samples from BMT mice on day +3 and day +6 showed significantly less inflammation in liver and gut in recipients of STAT1 KO splenocytes compared to wild type cells. These data indicate that donor STAT1 is important for the induction of acute GVHD and that attenuation of GVHD in the absence of STAT1 involves expansion of Treg cells, perturbation of T cell polarization and subsequent reduced expression of the chemokine receptor CXCR3 on donor T cells leading to impaired target organ infiltration.