Human intestinal acute graft-versus-host disease is governed by a subset of transition-like tissue-resident memory CD8+ T cells Free

Background:

Tissue-resident memory T cells(TRM) have recently emerged as crucial cellular players for murine acute graft-versus-host disease(aGVHD) development. However, the role of distinct subsets of CD8⁺TRM cells in human intestinal aGVHD is not well known yet.Herein, we combined multiplex-immunohistochemical staining with single-cell RNA sequencing to elucidate the lineage commitment, clonal expansion, differentiation trajectory, and functional properties of distinct CD8⁺TRM cell subsets in human intestinal aGVHD.

Method：

Intestinal biopsy samples were obtained from 50 patients with intestinal acute graft-versus-host disease (aGVHD) , 15 patients with no aGVHD and 12 healthy controls. Clinical symptoms of the patients were evaluated according to the Glucksberg criteria. 9 samples were subjected to Single-cell RNA-seq and V(D)J-seq after CD8⁺ magnetic bead sorting.All samples were detected by multiplex immunohistochemistry(mIHC).

Results:

We performed further analyses of CD8⁺ TRM cells from 9 intestinal biopsies samples after transplantation (6 intestinal acute GVHD and 3 no intestinal acute GVHD) to characterize the intrinsic structure and potential functional subsets of CD8⁺ TRM cells. We identified 7 distinct clusters according to the gene expression profile and paired VDJ profile.Compared with the TCF1⁺CD8⁺TRM_recir-stem (recirculating stem-like CD8⁺TRM), CX3CR1⁺CD8⁺TRM_eff (effector-like CD8⁺TRM) and TIGIT⁺CD8⁺TRM_exh(exhausted-like CD8⁺TRM) subsets, the GZMK⁺CD8⁺TRM_trans (transition-like CD8⁺TRM) subset was the highest proportion subset, and specifically expressed higher levels of GZMK and ITGA1. Furthermore,the GZMK⁺CD8⁺TRM_trans subset was dominant in the expanded TCR clonotypes among CD8⁺TRM subsets. We observed two distinct CD8⁺ TRM subset pseudotime trajectories based on shared TCR clonotypes. Based on the marker genes among the CD8⁺ TRM subsets, we found that compared with those in the HCs and patients without intestinal acute GVHD, the mIHC findings in intestinal acute GVHD patients revealed greater numbers and ratios of GZMK⁺CD8⁺TRM_transsubset infiltration (P<0.01 and P<0.001). In addition, there was no evidence of significant differences in other CD8⁺ TRM subsets. GSEA analysis revealed that the upregulated DEGs in the GZMK⁺CD8⁺TRM_transsubset were associated with GVHD, the IFN-γ response and antigen processing/presentation signaling pathways.pSTAT1, IRF1, and ISG20 were significantly upregulated in the GZMK⁺CD8⁺TRM_transsubset of intestinal acute GVHD patients by performing mIHC (P<0.05).GZMK⁺CD8⁺TRM_transsubset from acute GVHD patients secreted more IFN-γ (P<0.05).And we found that the GZMK⁺CD8⁺TRM_transsubset in acute GVHD patients exhibited a greater proximity with the CX3CR1⁺CD8⁺TRM_eff compared to the TIGIT⁺CD8⁺TRM_exh subset.Moreover, increased GZMK⁺CD8⁺TRM_trans infiltration was associated with the severity of intestinal acute GVHD(severe aGVHD: clinical grade 3 to 4,mild/moderate aGVHD: clinical grade 1 to 2), and high GZMK⁺CD8⁺TRM_trans infiltration correlated with worse overall survival and disease-free survival outcomes (P<0.01 and P<0.05).

Conclusion:

In conclusion,we have investigated, using scRNA-seq, scTCR-seq and multiplex immunofluorescence, that the lineage commitment, clonal expansion，differentiation trajectory, and functional properties of distinct CD8⁺ TRM cell subsets in human intestinal acute GVHD. First, we provide a framework for better understanding the CD8⁺ TRM subset landscape in human intestinal acute GVHD. We revealed seven functionally distinct CD8⁺ TRM subsets and uncovered a significant CD8⁺ TRM subset, known as GZMK-expressing transition-like CD8⁺ TRM cells subset, characterized by the GZMK and CD49a markers. Second, we provided novel insights into the functions of GZMK⁺CD8⁺TRM_trans subset, which exhibited enhanced clonal expansion and lineage bifurcation differentiation properties, differentiating into either effector-like CD8⁺TRM cells or exhausted-like CD8⁺TRM cells. Third, GZMK⁺CD8⁺TRM_trans subset upregulated IFN signaling pathway-associated proinflammatory signatures and differentiated more into CX3CR1⁺CD8⁺TRM_effsubset in intestinal acute GVHD. Fourth, high GZMK⁺CD8⁺TRM_trans subset infiltration is correlated with human intestinal acute GVHD severity and poor prognosis. Therefore, we demonstrated that human intestinal acute graft-versus-host disease is in good part mediated by pivotal GZMK⁺CD8⁺TRM_trans subset.