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, we and others identified GM-CSF as a cytokine that is produced primarily by donor-derived CD4+ T cells and mediates inflammation in the GI tract. However, the precise mechanism by which GM-CSF induces pathological damage and the transcriptional profile of this novel colitogenic CD4+ GM-CSF+ T cell population have not been defined. To address these questions, we employed a well-defined murine model of GVHD [C57BL/6 (H-2b)→Balb/c (H-2d)] and demonstrated that GM-CSF induces inflammation by enhancing the activation of donor-derived dendritic cells in the colon as evidenced by increased expression of costimulatory molecules (i.e. CD80 and CD86) and the production of IL-23. In addition, GM-CSF linked adaptive to innate immunity by promoting indirect alloantigen presentation in the mesenteric lymph nodes which was IL-23 dependent and characterized by an increased number of CD103+ CD11b+ dendritic cells and donor CD4+ T cells with a proinflammatory cytokine phenotype. Unexpectedly, we observed two distinct CD4+ GM-CSF+ populations in the GI tract that were distinguishable by the presence or absence of IFN-γ production by intracellular cytokine staining (i.e. CD4+ GM-CSF+ IFN-γ+ and CD4+ GM-CSF+ IFN-γ-). Notably, CD4+ GM-CSF+ IFN-γ- cells were largely absent from other target organs (e.g. liver, lung), suggesting that this population had a unique role in the biology of GVHD in the GI tract. To determine whether these two populations represented transcriptionally distinct lineages or reflected TH1-biased lineage plasticity, we performed single cell RNA sequencing and immunological profiling on donor-derived sort-purified T cells from the colons of GVHD mice one and three weeks post-transplant using the 10X Genomics platform. After selecting only high quality reads, we recovered 6315 unique barcodes corresponding to individual cells and identified several transcriptionally distinct cell clusters that spatially segregated following Seurat and UMAP analysis. Colonic T cells obtained on days 7 and 21 post transplantation completely separated, indicating that the transcriptional profile of these cells changes dramatically between early and later time points. Detectable transcription of GM-CSF was observed in two distinct populations of CD4+ T cells only at the 21-day timepoint. Notably, only one of these clusters co-expressed IFN-γ, confirming our flow-based results, and indicating that CD4+ GM-CSF+ IFN-γ+ and GM-CSF+ IFN-γ- T cells represented distinct populations. Further analysis revealed that CD4+ GM-CSF+ IFN-γ+ T cells were T-bet+ and differentially expressed high levels of costimulatory molecules (CD137, OX40, and CD81) and PD-1, indicative of an activated T cell phenotype. In contrast, CD4+ GM-CSF+ IFN-γ- T cells were distinguishable by the co-expression of T-bet and Gata-3, which is a TH2-defining transcription factor, as well as by the IL-7R and a series of interferon stimulated genes (IFITM1, IFITM2, and IFITM3), supporting the premise that these cells constitute a discrete TH cell lineage. To further characterize these CD4+ T cell populations, we examined the T cell repertoire (TCR) using a targeted sequencing analysis approach of our barcoded cDNA library. We identified 444 unique clonotypes among CD4+ GM-CSF+ T cells based on sequencing of CDR3 regions of TCR alpha and beta chains. Notably, only 5 clonotypes were shared between CD4+ GM-CSF+ IFN-γ+ and CD4+ GM-CSF+ IFN-γ-T cells, representing 58 of 1154 (~5%) of the total cells in both clusters. Thus, this minimal overlap suggested that these T cells were responding to non-overlapping antigens within the GI tract. Analysis of V beta TCR gene usage revealed that CD4+ GM-CSF+ IFN-γ- cells had a highly-biased repertoire with approximately half of cells utilizing a single V beta gene, Vbeta3. In contrast, CD4+ GM-CSF+ IFN-γ+ T cells had a much more evenly distributed Vbeta receptor profile with no predominant Vbeta usage. Collectively, these studies demonstrate the existence of two transcriptionally distinct CD4+ GM-CSF+ T cell populations that accumulate within the GI tract, possess non-overlapping T cell repertoires, promote indirect alloantigen presentation, and mediate pathological damage during GVHD.
Disclosures
No relevant conflicts of interest to declare.
Single cell immune profiling reveals a developmentally distinct CD4+ GM-CSF+ T cell lineage that induces GI tract GVHD
