Search

Gastrointestinal (GI) tract involvement is the major cause of morbidity and mortality in acute graft-versus-host disease (GVHD), and pathological damage is largely attributable to inflammatory cytokine production. Recently, granulocyte-macrophage colony stimulating factor (GM-CSF) has been identified as a cytokine that mediates inflammation in the GI tract, but the transcriptional program that governs GM-CSF production and the mechanism by which GM-CSF links adaptive to innate immunity within this tissue site have not been defined. In the current study, we identified Bhlhe40 as a key transcriptional regulator that governs GM-CSF production by CD4+T cells and mediates pathological damage in the GI tract during GVHD. In addition, we observed that GM-CSF was not regulated by either interleukin 6 (IL-6) or IL-23, which are both potent inducers of GVHD-induced colonic pathology, indicating that GM-CSF constitutes a nonredundant inflammatory pathway in the GI tract. Mechanistically, GM-CSF had no adverse effect on regulatory T-cell reconstitution, but linked adaptive to innate immunity by enhancing the activation of donor-derived dendritic cells in the colon and subsequent accumulation of these cells in the mLNs. In addition, GM-CSF promoted indirect alloantigen presentation, resulting in the accumulation of donor-derived T cells with a proinflammatory cytokine phenotype in the colon. Thus, Bhlhe40+GM-CSF+CD4+T cells constitute a colitogenic T-cell population that promotes indirect alloantigen presentation and pathological damage within the GI tract, positioning GM-CSF as a key regulator of GVHD in the colon and a potential therapeutic target for amelioration of this disease.

CD8+Foxp3+T cells (Tregs) are a potent regulatory population whose functional and ontological similarities to CD4+Fox3+T cells have not been well delineated. Using an experimental model of graft-versus-host disease (GVHD), we observed that CD8+Tregs were significantly less potent than CD4+Tregs for the suppression of GVHD. To define the mechanistic basis for this observation, we examined the T-cell repertoire and the transcriptional profile of in vivo-derived CD4+and CD8+Tregs that emerged early during this disease. Polyclonal and alloantigen-induced CD8+Tregs had repertoire diversity that was similar to that of conventional CD8+T cells, indicating that a restricted repertoire was not the proximate cause of decreased suppression. Transcriptional profiling revealed that CD8+Tregs possessed a canonical Treg transcriptional signature that was similar to that observed in CD4+Tregs, yet distinct from conventional CD8+T cells. Pathway analysis, however, demonstrated that CD8+Tregs had differential gene expression in pathways involved in cell death and survival. This was further confirmed by detailed mRNA sequence analysis and protein expression studies, which demonstrated that CD8+Tregs had increased expression of Bim and reduced expression of Mcl-1. Transplantation with CD8+Foxp3+Bim−/−Tregs resulted in prolonged Treg survival and reduced GVHD lethality compared with wild-type CD8+Tregs, providing functional confirmation that increased expression of Bim was responsible for reduced in vivo efficacy. Thus, Bim regulates the survival and suppressive capability of CD8+Tregs, which may have implications for their use in regulatory T-cell therapy.

Gastrointestinal (GI) tract involvement is a major determinant for subsequent morbidity and mortality arising during graft versus host disease (GVHD). CD4+T cells that produceGM-CSF have emerged as central mediators of inflammation in this tissue site as GM-CSF serves as a critical cytokine link between the adaptive and innate arms of the immune system.However, cellular heterogeneity within the CD4+GM-CSF+T cell population due to the concurrent production of other inflammatory cytokines has raised questions as to whether these cells have a common ontology or if there existsa uniqueCD4+GM-CSF+subset that differs from other defined T helper (TH) subtypes.Using single cell RNA sequencing analysis, we identified two CD4+GM-CSF+T cell populations that arose during GVHD and were distinguishable by the presence or absence of IFN-γ co-expression. CD4+GM-CSF+IFN-γ−T cellswhich emerged preferentially in the colonhad a distinct transcriptional profile, employed unique gene regulatory networks, and possessed a non-overlapping TCR repertoire when compared to CD4+GM-CSF+IFN-γ+T cellsas well asall other transcriptionally defined CD4+T cell populations in the colon.Functionally, thisCD4+GM-CSF+T cell populationcontributed topathological damagein the GI tract which was critically dependent upon signaling through the IL-7 receptor but was independent of type 1 interferon signaling.Thus, these studies help to unravel heterogeneity within CD4+GM-CSF+T cells that arise during GVHD and define a developmentally distinct colitogenic THGM-CSF+subset that mediates immunopathology.