Single-cell multi-omics analysis presents the landscape of peripheral blood T-cell subsets in human chronic prostatitis/chronic pelvic pain syndrome

Abstract

Cumulative evidence suggests that abnormal differentiation of T lymphocytes influences the pathogenesis of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). Thus, understanding the immune activation landscape of CP/CPPS would be helpful for improving therapeutic strategies. Here, we utilized BD™ AbSeq to digitally quantify both the protein and mRNA expression levels in single peripheral blood T cells from two CP/CPPS patients and two healthy controls. We utilized an integrated strategy based on canonical correlation analysis of 10 000+ AbSeq profiles and identified fifteen unique T-cell subpopulations. Notably, we found that the proportion of cluster 0 in the CP/CPPS group (30.35%) was significantly increased compared with the proportion in the healthy control group (9.38%); cluster 0 was defined as effector T cells based on differentially expressed genes/proteins. Flow cytometry assays confirmed that the proportions of effector T-cell subpopulations, particularly central memory T cells, T helper (Th)1, Th17 and Th22 cells, in the peripheral blood mononuclear cell populations of patients with CP/CPPS were significantly increased compared with those of healthy controls (P < 0.05), further confirming that aberration of effector T cells possibly leads to or intensifies CP/CPPS. Our results provide novel insights into the underlying mechanisms of CP/CPPS, which will be beneficial for its treatment.

Keywords: T regular cell; chronic prostatitis/chronic pelvic pain syndrome; effector T cell; single-cell multi-omics.

FIGURE 1

Single‐cell isolation, library preparation and bioinformatic analyses. A, The CD3+ T‐cell enrichment by CD3+ T‐cell enrichment kit, followed by single‐cell isolation and library construction; B, the flow showed reverse transcription and library construction details; and C. bioinformatic analyses procedure

FIGURE 2

Single‐cell multi‐omics analysis revealed 15 distinct T‐cell subsets. A, UMAP plot showing dimensional reduction of the distribution of 10 000+ individual T cells extracted from two CP/CPPS cases and two healthy controls; B, the proportion variations of the T‐cell subsets between CP/CPPS cases and healthy controls (*P < 0.05; **P < 0.01); C, heatmap plot showing clustering with top 5 highly expressed genes/proteins within each transgenic line. Representative genes/proteins found within each specific cell lineage are denoted to the left. The cells were defined based on the marker genes/proteins identified and labelled on the right of the plot. D, Violin plots comparing the expression of key differentially expressed genes/proteins in different clusters

FIGURE 3

Analyses of the differentially expressed genes/proteins between CP/CPPS cases and healthy controls and their corresponded pathways. A, The differentially expressed genes between CP/CPPS cases and healthy controls; B, the pathway enrichment of the differentially expressed genes/proteins between CP/CPPS cases and healthy controls. CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome

FIGURE 4

Secondary UMAP analysis of cluster 0. A, UMAP analysis and cluster allocation for the single cells in cluster 0; B, heatmap plot showed the marker genes/proteins in each newly defined cluster; and C‐G. the identified marker genes/proteins in cluster 0 to 5. UMAP, Uniform Manifold Approximation and Projection

FIGURE 5

Flow cytometry revealed central memory T cell, Th1, Th17, Th22 and Treg proportions increased in PBMCs derived from CP/CPPS patients than healthy controls. PBMCs were incubated with various fluorescein‐labelled antigens for surface staining. PerCP/Cyanine5.5‐conjugated CD3 and FITC‐conjugated CD4 were used for the central memory T cell, Th1, Th17 and Th22 cell staining, and PE‐conjugated CD25 and FITC‐conjugated CD4 were incubated for staining Treg cells. After fixing and permeabilizing with cell fixation/permeabilization kit, (A) for samples staining central memory T cells were incubated with PE‐conjugated CD45RA and APC‐conjugated CD62L; (C) for samples staining, Th1 cells were incubated with PE‐conjugated IFN‐γ; (E) for samples staining, Treg cells were incubated with eFluor 660‐conjugated FoxP3; (G) for samples staining, Th17 cells were incubated with PE‐conjugated IL‐17A; and (I) for samples staining, Th22 cells were incubated with PE‐conjugated IL‐17A and APC‐conjugated IL‐22. The quantification data were presented in B, D, F, H and J. *P < 0.05; PBMC, peripheral blood mononuclear cell