Transcriptional programming by GATA3 regulates CD4+ T cell GM-CSF production in the gastrointestinal tract during graft versus host disease Free

Acute graft versus host disease (GVHD) of the gastrointestinal (GI) tract is a major complication of allogeneic hematopoietic stem cell marrow transplantation (HSCT). CD4⁺ T cells that produce GM-CSF have emerged as central mediators of inflammation in the GI tract as GM-CSF serves as a critical proinflammatory cytokine link between the adaptive and innate arms of the immune system. Notably, there exists cellular heterogeneity within the CD4⁺ GM-CSF⁺ T cell population due to the concurrent production of other inflammatory cytokines that has raised questions as to whether these cell populations are regulated by different transcription factors. To examine this question, we employed a major histocompatibility mismatched murine GVHD model (C57BL/6 [H-2b]→Balb/c [H-2d]) and performed single cell RNA sequencing analysis on T cells isolated from the GI tract which identified two CD4⁺ GM-CSF⁺ T cell populations that were distinguishable by the presence or absence of IFN-γ co-expression. Bioinformatic analysis of gene regulatory networks in CD4⁺ GM-CSF⁺ T cells revealed that CD4⁺ GM-CSF⁺ IFN-γ⁺ T cells had increased expression of Batf and Bhlhe40, whereas CD4⁺ GM-CSF⁺ IFN-γ^- T cells had preferential expression of Gata3. To determine the functional significance of these transcriptional factors in CD4⁺ GM-CSF⁺ T cell biology, we employed CRISPR Cas9-mediated gene editing to create a GM-CSF-Cre mouse that we crossed to Batf^fl/fl, Bhlhe40^fl/fl or Gata3^fl/fl animals so that each transcription factor could be individually deleted from GM-CSF expressing cells. We observed that irradiated Balb/c mice reconstituted with B6 bone marrow and splenocytes from either GM-CSF-Cre Batf^fl/fl or GM-CSF-Cre Bhlhe40^fl/fl animals had significantly less GVHD-induced mortality when compared to non-Cre floxed control mice. In addition, these cohorts had a significant reduction in both CD4⁺ GM-CSF⁺ IFN-γ^- and CD4⁺ GM-CSF⁺ IFN-γ⁺ T cells in the colon, demonstrating that Batf and Bhlhe40 regulated both CD4⁺ GM-CSF T cell populations. In contrast, there was significantly increased GVHD lethality in mice transplanted with marrow grafts from GM-CSF-Cre Gata3^fl/fl animals. Strikingly, this was accompanied by a marked reduction in CD4⁺ GM-CSF⁺ IFN-γ⁺ T cells and a near complete absence of CD4⁺ GM-CSF⁺ IFN-γ^- T cells, while there was a significant increase in CD4⁺ GM-CSF^- IFN-γ⁺ T cells in the colon. These data indicated that Gata3 was critical for expression of GM-CSF in CD4⁺ T cells and that absence of this transcription factor in CD4⁺ T cells poised to make GM-CSF markedly skewed these cells towards a T_H1 cytokine phenotype. In addition, we observed that there was a significant increase in serum IFN-γ and IL-17 levels and augmented pathological damage in the colon of these mice, demonstrating that deletion of Gata3 in poised CD4⁺ GM-CSF⁺ T cells augmented their pathogenicity. To verify that Cre excision of GATA3 occurred and was directly responsible for the absence of GM-CSF production in CD4⁺ T cells we crossed GM-CSF-Cre Gata3^fl/fl animals with mT/mG mice to create GM-CSF-Cre Gata3^fl/fl mT/mG double fluorescent animals. In these mice, Cre-mediated excision of Gata3 in CD4⁺ T cells poised to make GM-CSF results in GFP expression thereby validating effective excision in the CD4⁺ T cell population of interest. Analysis of CD4⁺ GFP⁺ T cells in the colon confirmed the near compete absence of GM-CSF production in Gata3-deleted CD4⁺ T cells. As further validation of a pivotal role for Gata3 in driving GM-CSF production in CD4⁺ T cells, we stimulated these cells in vitro with anti-CD3 and anti-CD28 antibodies along with culture in IL-7 and soluble anti-IFN-γ antibody to polarize these cells towards a CD4⁺ GM-CSF⁺ IFN-γ^- T cell population. Culture of naive CD4⁺ T cells derived from GM-CSF-Cre Gata3^fl/fl animals resulted in near complete elimination of CD4⁺ GM-CSF⁺ IFN-γ^- T cells with a corresponding increase in CD4⁺ GM-CSF^- IFN-γ⁺ T cells thereby confirming in vivo results. A requisite role for IL-7 as well was corroborated by transplantation of GM-CSF Cre Il7r^fl/fl marrow grafts into lethally irradiated recipients which phenocopied results observed with CD4⁺ T cells from GM-CSF-Cre Gata3^fl/fl animals. In summary, these studies identify a critical and nuanced role for Gata3 in driving GM-CSF production in CD4⁺ T cells while also preventing reversion of these cells to more proinflammatory T_H1 cytokine phenotype in the GI tract during GVHD.