Clonal Expansion and Differentiation of Stem-like Memory T Cells to Tissue-Resident Memory T Cells Perpetuates Pathogenesis of Chronic Graft-Versus-Host Disease

We and others have shown that tissue-resident memory CD4 ⁺ T cells play a critical role in maintaining chronic GVHD pathogenesis. However, the cellular and molecular mechanisms remain largely unknown. Ly108 and (TCF1) expression have been shown to reflect the stemness of memory CD8 ⁺ T cells. In the current studies, using the markers Ly108 (TCF1) and CD69, we identified four subsets of memory CD4 ⁺ T (Tm) cells in the GVHD target tissues, including Ly108 ⁺ CD69 ^- and Ly108 ⁺CD69 ⁺ stem-like Tm cells, as well as Ly108 ^- CD69 ^- and Ly108 ^- CD69 ⁺ differentiatedTm cells. Compared to other Tm subsets, the Ly108 ^- CD69 ⁺ subset expressed the highest levels of IFN-γ and GM-CSF without upregulating anergy/exhaustion markers, indicating that they represent a terminally differentiated, tissue resident, pathogenic CD4 ⁺ memory T (Trm) subset. The Ly108 ⁺ Tm subsets showed self-renewal capacity and differentiation into Ly108 ^- Tm subsets, as demonstrated by adoptive transfer experiments. Using single-cell RNA sequencing (scRNA-seq) in conjunction with scTCR-seq analysis, we observed that Ly108 ⁺CD69 ^- Tm subset was clonally related to the expanded Ly108 ^- CD69 ^- and Ly108 ^- CD69 ⁺ Tm subsets, suggesting a clonal expansion and differentiation of Ly108 ⁺ CD69 ^- stem-like memory T (Tsm) cells. In addition, we found that IFN-γ primed donor-type antigen-presenting cells (APCs) play an essential role in optimizing the transition from CD4 ⁺ Tsm cells to CD4 ⁺ Trm cells in STAT3- and BCL6-dependent manner, as indicated by ATAC-Seq analysis. Our results have elucidated a novel pathway of clonal expansion and differentiation of Tsm cells to Trm cells in the GVHD target tissues. These observations provide cellular and molecular mechanisms that explain how chronic GVHD is perpetuated by memory T cells in local tissues.

XK and BW contributed equally.