CD101 genetic variants modify regulatory and conventional T cell phenotypes and functions

Abstract

We recently reported that the risk of sexually acquired HIV-1 infection is increased significantly by variants in the gene encoding CD101, a protein thought to modify inflammatory responses. Using blood samples from individuals with and without these variants, we demonstrate that CD101 variants modify the prevalence of circulating inflammatory cell types and show that CD101 variants are associated with increased proinflammatory cytokine production by circulating T cells. One category of CD101 variants is associated with a reduced capacity of regulatory T cells to suppress T cell cytokine production, resulting in a reduction in the baseline level of immune quiescence. These data are supported by transcriptomics data revealing alterations in the intrinsic regulation of antiviral pathways and HIV resistance genes in individuals with CD101 variants. Our data support the hypothesis that CD101 contributes to homeostatic regulation of bystander inflammation, with CD101 variants altering heterosexual HIV-1 acquisition by facilitating increased prevalence and altered function of T cell subsets.

Keywords: CD101; HIV acquisition; T cell; host genetic variation; immune quiescence; inflammation; inflammatory homeostasis.

Graphical abstract

Figure 1

CD101⁺ immune cell frequency and phenotype vary based on the presence of genetic variants in the Ig-like region of CD101 Circulating PMBCs from study participants possessing Ig-like variants in CD101, including rs12093834, rs17235773, and rs3754112 (N = 85), versus no functional variant (N = 117) were assessed by high-parameter flow cytometry for expression of various subset-specific and activation markers on CD101⁺ cells. (A–C) The mean and SD of the frequency of activation and subset-specific markers expressed on CD101⁺CD4 T cells (A), CD101⁺CD8 T cells (B), and CD101⁺ Treg cells (C) for Ig-like variants versus controls. (D) CD101⁺ DCs are broken into CD1c⁺ DCs and CD141⁺ DCs alongside various activation markers within those subsets (D). (E) The total frequencies of classic (CD14⁺⁺CD16⁻), nonclassic (CD14⁺CD16⁺⁺), and intermediate (CD14⁺⁺CD16⁺) monocytes expressing CD101 alongside the frequencies of various activation markers within each indicated subset. (F) CD101⁺ B cells and expression of CD80 and CD40 among CD101⁺ B cells. Data points represent the mean and SD of biologic replicates as indicated by N per group. Two-sample t tests were performed with Bonferroni correction for 100 comparisons to determine significance. ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 2

Circulating CD101⁺ immune cell frequency and phenotype vary minimally based on the presence of genetic variants in the cytoplasmic domain of CD101 Circulating PMBCs from study participants possessing cytoplasmic variants in CD101, including rs34248572 (N = 20) and rs150494742 (N = 13), versus no functional variant (N = 117) were assessed by high-parameter flow cytometry for expression of various subset-specific and activation markers on CD101⁺ cells. (A–C) The mean and SD of the frequency of activation and subset-specific markers expressed on CD101⁺CD4 T cells (A), CD101⁺CD8 T cells (B), and CD101⁺ Treg cells (C) for cytoplasmic variants versus controls. (D) CD101⁺ DCs are broken into CD1c⁺ DCs and CD141⁺ DCs, alongside various activation markers within those subsets. (E) The total frequencies of classic (CD14⁺⁺CD16⁻), nonclassic (CD14⁺CD16⁺⁺), and intermediate (CD14⁺⁺CD16⁺) monocytes expressing CD101 alongside the frequencies of various activation markers within each indicated subset. (F) CD101⁺ B cells and expression of CD80 and CD40 among CD101⁺ B cells. Data points represent the mean and SD of biological replicates as indicated by N per group. Two-sample t tests were performed, comparing each variant to samples with no functional variants, with Bonferroni correction for 100 comparisons to determine significance. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001.

Figure 3

T cells from individuals with variants in CD101 have increased potential to express cytokines (A–D) Cytokine-producing CD8⁺ or CD4⁺ T cells from EBV-stimulated (A and C) or αCD3/αCD28-stimulated (B and D) whole PBMCs for CD101 Ig-like (N = 42) and cytoplasmic variants (N = 18) compared with no functional CD101 variants (missense, 3′ or 5′ untranslated region, splice site) (N = 40). Live-sorted PBMCs, including lymphocytes and APCs, were recovered and stimulated with EBV lysate or glycine control (A and C) or αCD3/αCD28 or medium control (B and D) for 6 h prior to staining for intracellular cytokine production. The frequencies of CD101⁺CD8⁺ T cells producing IFN-γ after subtracting background values were affected by the presence of Ig-like and cytoplasmic-CD101 variants (A and B). Similarly, production of IFN-γ by CD101⁺CD4⁺ T cells after EBV stimulation was increased significantly for individuals carrying an Ig-like variant, whereas possessing a cytoplasmic variant did not have a significant effect (C). CD8⁺ T cells co-producing IFN-γ and TNF-α after αCD3/αCD28 stimulation also trended toward an increased frequency in individuals with the cytoplasmic variant (D). Four participants were homozygous for Ig-like variant rs12093834; they are denoted by orange symbols. (E–H) Live-sorted PBMCs, including lymphocytes and APCs, were recovered and stimulated with EBV lysate or glycine control (E and G) or αCD3/αCD28 or medium control (F and H) for 6 h prior to staining for intracellular cytokine production. Total CD4⁺CD3⁺ or CD8⁺CD3⁺ T cells were gated by CD101 positivity and then assessed for their ability to co-produce IFN-γ and TNF-α. EBV lysate stimulation of PBMCs isolated from individuals with cytoplasmic variants resulted in an increased frequency of IFN-γ⁺TNF-α⁺CD8⁺ (E) and CD4⁺ (G) T cells, whereas having an Ig-like variant did not produce a significant effect. Stimulation with αCD3/αCD28 antibodies elicited an increased frequency of IFN-γ⁺TNF-α⁺ CD8⁺ (F) and CD4⁺ (H) T cells in individuals with both Ig-like and cytoplasmic-variants. Matched background control values were subtracted for all participants. Four participants were homozygous for Ig-like variant rs12093834; they are denoted by orange symbols. Each data point represents one individual. Adjusted p values were calculated as described in STAR Methods.

Figure 4

CD101 variation diminishes Treg cell-mediated restraint of effector T cells (A) Schematic of the PBMC sorting and stimulation protocol (created with BioRender). PBMCs (N = 100) were sorted into (1) whole live PBMCs and (2) Treg cell-depleted or (3) purified CD3⁺ T cell fractions. Cells were stimulated with EBV lysate or control or αCD3/αCD28 or control for 6 h. Cells were then analyzed for their expression of cytokines by intracellular cytokine staining (ICS). (B) The frequency of CD4⁺ and CD8 T cells producing proinflammatory cytokines in response to EBV among Treg-cell-depleted PBMCs was analyzed per case or control. (C and D) Individual trajectory plots (C) and summary results (D) of Δ% IL-2⁺CD4⁺ T cells from Treg cell-depleted and whole PBMCs for CD101 cytoplasmic (N = 18) and Ig-like variants (N = 42) compared with no functional variants (N = 40). Live-sorted “whole” PBMCs, including lymphocytes and APCs, or PBMCs sorted to deplete Treg cells were recovered and stimulated with EBV lysate or glycine control for 6 h prior to staining for intracellular cytokine production. In (C), the frequencies of IL-2-producing CD4⁺ T cells recovered from the “whole” PBMC fraction and the Treg cell-depleted fraction are plotted as a trajectory plot, and in (D), the difference between the percentage of IL-2⁺ CD4⁺ T cells in Treg cell-depleted compared with whole PBMCs is plotted. Results are stratified by the presence of an Ig-like or cytoplasmic variant. Each data point represents one individual. Adjusted p values were calculated as described in STAR Methods.

Figure 5

CD101 variation is associated with transcriptional changes in circulating CD4⁺ and CD8⁺ T cells CD4⁺ conventional or CD8⁺ T cells were sorted from PBMCs sampled from 3 individuals with no functional variants in CD101 or from 3 individuals with an Ig-like variant (rs12093834) or 3 with a cytoplasmic variant (rs34248572) in CD101. (A) The gating strategy for sorting included gates for lymphocytes and singlets, and CD8⁺ T cells were sorted as CD3⁺CD8⁺, whereas conventional CD4⁺ T cells were sorted as CD3⁺CD4⁺ and, to exclude Treg cells, were further gated as CD25⁻. (B) Volcano plots showing genes that are differentially expressed between the indicated groups. Genes were considered differentially expressed when false discovery rate (FDR) values were less than 0.05. (C) Heatmaps showing the top 10 differentially upregulated genes and top 10 differentially downregulated genes for each genotype category (Ig-like or cytoplasmic) versus no variant, with samples ordered by genotype.