CD8+hla-DR+CD27+ t cells define a population of naturally occurring regulatory precursors in humans

The biology of CD8 regulatory T cells (Tregs) under steady-state conditions remains poorly understood due to their phenotypic heterogeneity and context-dependency. In our previous study, single-cell RNA sequencing (scRNA-seq) identified a distinct population of CD8⁺ regulatory precursors (CD8⁺ Trps), enriched in patients who successfully restored immune homeostasis following allo-HSCT. To further characterize the immunophenotype of CD8⁺ Trps, we analyzed peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 10) and allo-HSCT recipients with restored homeostasis (n = 21). A 16-color flow cytometry panel was designed based on scRNA-seq markers, revealing that CD8⁺ HLA-DR⁺ CD27⁺ cells are predominantly enriched within the effector-memory compartment and exhibit high expression of both co-stimulatory (CD28, ICOS) and co-inhibitory (PD-1, TIGIT) receptors. Unsupervised clustering showed a distinct population co-expressing MHC class II molecules (HLA-DR, HLA-DQ) and inhibitory receptors (PD-1, TIGIT), consistent with the CD8⁺ Trp phenotype identified from our scRNA-seq. These cells also expressed CD27 and lacked CD45RA, confirming their identity as non-naïve, non-terminally differentiated memory T cells. We next investigated the transcriptomic and epigenetic profiles of sorted CD8⁺ HLA-DR⁺ CD27⁺ cells. RNA-seq analysis revealed strong similarities to CD4⁺ Tregs, including elevated expression of co-stimulatory receptors (CD28, ICOS), co-inhibitory receptors (TIGIT, PDCD1), and the transcription factor TCF-1 under steady state. ATAC-seq analysis showed enhanced chromatin accessibility at key regulatory loci (CD28, ICOS, TIGIT, PDCD1, NR4A2). Moreover, integration of scRNA-seq with bulk RNA-seq confirmed that the CD8⁺ Trp signature was most highly enriched in this subset, further validating that CD8⁺ HLA-DR⁺ CD27⁺ cells faithfully recapitulate the CD8 Trp cluster. Functionally, in vitro co-culture of CD8⁺ Trps with PBMCs significantly suppressed PBMC proliferation. In a mixed lymphocyte reaction (MLR) model using MHC-knockout NSG mice (PrkdcscidIL2rg-/-H2-K1-/-H2-Ab1-/-H2-D1-/-), PBMCs from donor A (HLA-A02:01–negative) were infused on day 0; on day 14, mice were challenged with MMC-treated PBMCs from donor B (HLA-A02:01–positive), co-infused with either CD8⁺ Trps or CD4⁺ Tregs from donor A. CD8⁺ Trps significantly suppressed human CD45⁺ expansion and reduced IL-2 and TNF-α production by both CD4⁺ and CD8⁺ T cells. In a xenogeneic graft-versus-host disease (x-GVHD) model, co-infusion with CD8⁺ Trps mitigated graft-versus-host disease (GVHD) damage and prolonged NSG mouse survival. Importantly, when AML cells (THP-1) were co-transferred with PBMCs and CD8⁺ Trps into NSG mice, GVHD was alleviated without compromising anti-leukemia effects, thereby prolonging survival. Mechanistically, CD8⁺ Trps expanded CD4⁺ Tregs and upregulated FOXP3 expression in themselves. In both the MLR and x-GVHD models, we observed increased frequencies of CD4⁺ FOXP3⁺ and CD8⁺ FOXP3⁺ cells in the CD8⁺ Trp groups. Co-culture assays revealed that CD8⁺ Trps enhanced FOXP3 expression in CD4⁺ Tregs and promoted CD4⁺ conventional T cells (CD4 Tconvs) into CD4⁺ FOXP3⁺ regulatory T cells. Meanwhile, CD8⁺ Trps also served as precursors to CD8⁺ FOXP3⁺ cells, with FOXP3 expression increasing in vivo x-GVHD model and following in vitro TGF-β stimulation. Moreover, long-term in vitro culture of CD8⁺ Trps induced progressive upregulation and eventual stabilization of CD4 Treg signature proteins (FOXP3, CD25, CTLA-4, HELIOS) in CD8⁺ Trps. Finally, the frequency of HLA-DR⁺ CD27⁺ cells was significantly elevated in CD8⁺ T cells from allo-HSCT recipients with restored homeostasis compared to healthy donors or chronic GVHD patients, suggesting that CD8⁺ Trps contribute to the remodeling of immune homeostasis following allo-HSCT. In summary, our study identifies a naturally occurring subset of CD8⁺ regulatory precursors with the immunophenotype CD8⁺ HLA-DR⁺ CD27⁺, which exhibit potent immunosuppressive activity through dual mechanisms—promoting CD4⁺ Treg expansion and differentiating into CD8⁺ FOXP3⁺ cells. These findings highlight the therapeutic potential of CD8⁺ Trps in transplantation and autoimmunity by promoting immune homeostasis.