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

Abstract

Regulatory T_reg cells are essential for immune homeostasis. While CD4 T_reg cells are well characterized, CD8 T_reg cells remain less understood and are primarily observed in pathological or experimental contexts. Here, we identify a naturally occurring CD8 regulatory precursor T_rp cell at the steady state, defined by a CD8⁺HLA-DR⁺CD27⁺ phenotype and a transcriptome resembling CD4 T_reg cells. Multiomics analyses reveal activation of TCF7 and costimulatory and co-inhibitory molecules in CD8 T_rp cells. CD8 T_rp cells suppress T cell expansion in vitro and in vivo. In a humanized xenogeneic graft-versus-host disease (GVHD) model, they dampen T cell activation, alleviate GVHD pathology, and prolong survival without impairing antileukemia activity. Mechanistically, CD8 T_rp cells promote immune regulation by inducing FOXP3 expression in both CD4 T_reg cells and themselves. Their expansion also correlates with immune homeostasis restoration post-allogeneic stem cell transplantation. These findings establish CD8 T_rp cells as a naturally occurring regulatory precursor population that promotes transplantation tolerance.

Fig. 1.. Immunophenotype characterization of CD8⁺ regulatory precursor cells in the homeostatic state.

(A) Schematic illustration of the experimental workflow. Comprehensive immunological analysis is performed on PBMCs obtained from healthy individuals (n = 10) and homeostatic recipients following Haplo-SCT (n = 12) or MSDT (n = 9). (B) Flow cytometry analysis of CD8⁺ T cell subsets including T_naive (T_n), T_cm, T_em, and T_emra cell distribution within the CD8⁺HLA-DR⁺ and CD8⁺HLA-DR⁻ populations from the healthy individual group, Haplo-SCT recipient group, and MSDT recipient group. (C) Flow cytometry analysis of CD8⁺ T cell subsets including T_naive, T_cm, T_em, and T_emra cell distribution within the CD8⁺CD27⁺ and CD8⁺CD27⁻ populations from the healthy individual group, Haplo-SCT recipient group, and MSDT recipient group. (D) Distribution of CD8⁺ T cell subsets including T_naive, T_cm, T_em, and T_emra cells within the four quadrants extinguished by HLA-DR and CD27 in CD8⁺ T cells. Representative graph from the Haplo-SCT recipient group. (E) UMAP visualization of spectrum flow cytometry–detected CD3⁺ T cells from the healthy individual group, Haplo-SCT recipient group, and MSDT recipient group. (F) UMAP visualization of spectrum flow cytometry–detected CD8⁺ T cells from the healthy individual group, Haplo-SCT recipient group, and MSDT recipient group. (G and H) Heatmaps of the expression level of indicated markers in CD8⁺ T cells from the healthy individual group, Haplo-SCT recipient group, and MSDT recipient group. Unpaired t test for comparison between two groups. Error bars represent the means ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.. Protein and transcriptomic expression patterns of CD8⁺HLA-DR⁺CD27⁺ T cells.

(A and B) Representative histogram (A) and summary of median fluorescence intensity (MFI) (B) showing protein expression levels for the indicated markers of HLA-DR⁺CD27⁺, HLA-DR⁻CD27⁺, HLA-DR⁻CD27⁻, and HLA-DR⁺CD27⁻ within CD8⁺ T cells from the healthy individual group (n = 10), Haplo-SCT recipient group (n = 12), and MSDT recipient group (n = 9). (C) Heatmap of protein levels of CD8⁺HLA-DR⁺CD27⁺ and CD8⁺HLA-DR⁻CD27⁻ T cells from the indicated groups. (D) Principal components analysis (PCA) of classic CD8⁺ T cell subsets, including T_naive, T_cm, T_em, and T_emra cells, along with the CD8⁺HLA-DR⁺CD27⁺ and CD8⁺HLA-DR⁻CD27⁻ subsets. (E) Volcano plot showing the differentially expressed genes (DEGs) between CD8⁺HLA-DR⁺CD27⁺ T cells and CD8 T_naive cells. (F) The top 50 CD8 T_rp cell signature genes, identified from single-cell data in our previous study, were extracted, and their enrichment scores across different groups were calculated using the GSVA package. (G) Heatmap showing the expression levels of selected genes across different groups. (H) Gene enrichment in CD8⁺HLA-DR⁺CD27⁺ T cells and CD4 T_reg cells relative to CD8 and CD4 T_naive cells, respectively. (I) Transcripts per million (TPM) of the indicated genes across different groups. CD8 T_rp, CD8⁺HLA-DR⁺CD27⁺; CD8 T_naive, CD8⁺CCR7⁺CD45RA⁺; CD8 T_cm, CD8⁺CCR7⁺CD45RA⁻; CD8 T_em, CD8⁺CCR7⁻CD45RA⁻; CD8 T_emra, CD8⁺CCR7⁻CD45RA⁺; CD4 T_reg, CD4⁺CD25⁺CD127^low; CD4 T_naive, CD4⁺CD25^lowCCR7⁺CD45RA⁺. A one-way ANOVA was applied for comparisons among four groups. For the RNA-seq data of sorted T cell subsets from healthy individuals, classical subsets were sorted from four donors, and CD8⁺HLA-DR⁺CD27⁺ and CD8⁺HLA-DR⁻CD27⁻ subsets were sorted from five donors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.. CD8 T_rp cells suppress T cell proliferation and activation in vitro and in vivo.

(A) Proliferation analysis of PBMCs with or without coculture with CD8⁺HLA-DR⁺CD27⁺ T cells (n = 8). Combined data from two independent experiments. (B) Schematic illustration of the experimental workflow for in vivo MLR. T_regs, T_reg cells; T_rps, T_rp cells. (C) Representative flow cytometry graph and statistical analysis of hCD45⁺ cell engraftment in humanized mice from the peripheral blood (PB) and spleen (SP). (D) Absolute number of hCD45⁺ cells in the spleen of humanized mice. (E) Representative flow cytometry graph showing FOXP3 expression in CD4⁺ T cells and statistical analysis of the proportion and absolute number of CD4 T_reg cells in the spleen of humanized mice from the indicated groups. (F) Representative flow cytometry graph and statistical analysis of cytokine secretion levels in CD4⁺ or CD8⁺ T cells of humanized mice from the indicated groups. A paired t test was applied for comparison between two groups, and a one-way ANOVA was applied for comparisons among four groups. Each group included five mice. The data shown represent one experiment of three independent experiments. Symbols indicate individual mice, and error bars represent the means ± SEM. *P < 0.05, **P < 0.01.

Fig. 4.. CD8 T_rp cells inhibit GVHD damage in humanized xenogeneic GVHD models.

(A) Schematic illustration of the experimental workflow for the humanized xenogeneic GVHD (x-GVHD) model. (B) Survival curve, weight changes, and GVHD scores of humanized x-GVHD models. Twenty-two mice for group 1, 22 mice for group 2, and 19 mice for group 3. Median survival: group 1, 21 days; group 2, 30 days; group 3, 31 days. Combined data from three independent experiments. (C) Representative flow cytometry graph and statistical analysis of hCD45⁺ cell engraftment in x-GVHD mice from the peripheral blood (PB) and spleen (SP). (D) Representative flow cytometry graph and statistical analysis of CD4 T_reg cells in x-GVHD mice from the indicated groups. (E) Representative flow cytometry graph and statistical analysis of activation markers expression levels in CD4⁺ and CD8⁺ T cells in x-GVHD mice from the indicated groups. (F) Representative flow cytometry graph and statistical analysis of cytokine secretion levels in CD4⁺ and CD8⁺ T cells in x-GVHD mice from the indicated groups. (G) and (H) Tumor growth was monitored using bioluminescence imaging (BLI) on the dates indicated. (I) Survival curve of humanized x-GVHD and GVL models. Group 1: THP-1 alone, median survival of 41 days; group 2: PBMCs + THP-1, median survival of 33 days; group 3: PBMCs + THP-1 + CD4 T_reg cells, median survival of 39 days; group 4: PBMCs + THP-1 + CD8 T_rp cells, median survival of 42 days. Each group included five mice. Unless otherwise indicated, the data shown are representative of one of three independent experiments. For the comparison of recipient survival among groups, the log-rank test was used to determine statistical significance. A one-way ANOVA was applied for comparisons among three groups. Symbols indicate individual mice, and error bars represent the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.. CD8 T_rp cells enrich precursors of CD8⁺FOXP3⁺ T cells.

(A) Representative flow cytometry graph and statistical analysis of FOXP3 expression levels in CD4⁺ and CD8⁺ T cells from x-GVHD mice across the indicated groups. Thirteen mice for group 1, 14 mice for group 2, and 15 mice for group 3. Combined data from three independent experiments. (B) FOXP3 expression levels in CD4 T_reg cells with or without coculture with CD8 T_rp cells (n = 4). (C) FOXP3 expression levels in CD4 T_con cells stimulated with either IgG or HLA-DR agonist antibody (n = 5, P = 0.0099, paired t test). (D) Representative flow cytometry graph and statistical analysis of FOXP3 expression levels in the specific groups under the steady state and TGF-β stimulation. (E) Proliferation analysis of HLA-A02:01–positive PBMCs with or without coculture with HLA-A02:01–negative CD8⁺HLA-DR⁺CD27⁺ T cells (n = 8). Combined data from two independent experiments. (F) Representative flow cytometry graph of FOXP3 expression levels in the indicated groups. (G) Statistical analysis of FOXP3 expression levels in the indicated groups (n = 4). (H) Statistical analysis of FOXP3 expression levels in CD8 T_rp cells before and after coculture with PBMCs (n = 4). (I) Expression levels of CD4 T_reg cell signature proteins, including FOXP3, CD25, CTLA-4, and HELIOS, in long-term in vitro cultures of CD4 T_reg cells and CD8 T_rp cells (n = 5). d, days. (J) TGF-β levels in supernatants from the indicated groups during long-term in vitro cultures (n = 5). (K) Relative levels of 5-methylcytosine (5mC) DNA methylation measured by qMSP (n = 3). A paired/unpaired t test for comparison between two groups, and a one-way ANOVA was applied for comparisons among three groups. Symbols indicate individual samples, and error bars represent the means ± SEM. Unless otherwise noted, the data shown represent one of two or three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.. Epigenomic signatures of CD8 T_rp cells.

(A) Volcano plots showing genes proximal to differential accessible regions in CD8 T_rp cells compared to CD8 DN T cells. (B) Volcano plots showing genes proximal to differential accessible regions in CD4 T_reg cells compared to CD4 T_con cells. (C) Overlap between DEGs and different accessibility-proximal genes in CD8 T_rp cells compared to CD8 DN T cells. (D) Integrated analysis of RNA-seq and ATAC-seq data. DEGs and DA peak-proximal genes in CD8 T_rp cells compared to CD8 DN T cells. (E) Enriched transcription factor motifs in the differential accessible peaks of CD8 T_rp cells compared to CD8 DN T cells are shown. (F) WashU browser views show the gene expression level (RNA-seq) and chromatin accessibility (ATAC-seq) of specific genes in the indicated groups. n = 5. CD8 T_rp, CD8⁺HLA-DR⁺CD27⁺; CD8 DN, CD8⁺HLA-DR⁻CD27⁻; CD8 T_naive, CD8⁺CCR7⁺CD45RA⁺; CD8 T_cm, CD8⁺CCR7⁺CD45RA⁻; CD8 T_em, CD8⁺CCR7⁻CD45RA⁻; CD8 T_emra, CD8⁺CCR7⁻CD45RA⁺.

Fig. 7.. Proportion and signature of CD8 T_rp cells in healthy individuals, homeostatic recipients, and cGVHD recipients following allo-HSCT.

(A) Representative flow cytometry graph of CD8 T_rp cells in healthy individuals, homeostatic recipients, and cGVHD recipients following allo-HSCT. (B) Statistical analysis of the proportion of HLA-DR⁺CD27⁺ in CD8⁺ T cells from healthy individuals (n = 10), homeostatic recipients (n = 21), and cGVHD recipients (n = 12) following allo-HSCT. (C) Distribution of classical T cell subsets in CD8⁺HLA-DR⁺CD27⁺ T cells across different groups. (D) Expression level of signature genes in CD8⁺HLA-DR⁺CD27⁺ T cells across different groups. CD8 T_naive, CD8⁺CCR7⁺CD45RA⁺; CD8 T_cm, CD8⁺CCR7⁺CD45RA⁻; CD8 T_em, CD8⁺CCR7⁻CD45RA⁻; CD8 T_emra, CD8⁺CCR7⁻CD45RA⁺. A one-way ANOVA was applied for comparisons among three groups. Symbols indicate individual participants, and error bars represent the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.