Clinical significance of CD161+CD4+ T cells in the development of chronic antibody-mediated rejection in kidney transplant recipients

Abstract

In this study, we investigated whether CD161+CD4+ T cells can reflect the Th17 pathway in kidney transplant recipients (KTRs) and investigated the clinical significance of this cell type in chronic antibody-mediated rejection (cAMR) in KT. First, we investigated the relationship between CD161+CD4+ T and Th17 cells by flow cytometry and microarray analysis in an in vitro study. Second, we compared the proportion of T cell subsets including CD161+CD4+ T cells in cAMR (n = 18), long-term graft survival (LTGS) (n = 46), and interstitial fibrosis/tubular atrophy (IF/TA) (n = 22). We compared CD161+ cell infiltration between cAMR and IF/TA and also examined the effect of CD161+ T cells on human renal proximal tubular epithelial cells (HRPTEpiC). In flow cytometry, the proportion of CD161+CD4+ T cells showed a significant correlation with the proportion of Th17 cells. In microarray analysis, transcripts associated with the Th17 pathway such as IL18RAP, IL-18R1, IL23R, IL12RB2, RORC, TBX21, and EOMES were upregulated in CD161+ cells compared with CD161- cells. In an ex vivo study, only CD161+CD4+ T cells showed a significant increase in the cAMR group compared with IF/TA and LTGS groups. In allograft tissue, CD161+ cells showed a higher level of infiltration in the cAMR group than the IF/TA group. Lastly, CD161+ T cells increased the production of inflammatory cytokines from HRPTEpiC in a dose-dependent manner. This study suggests that monitoring of CD161+ T cells can be useful to detect the progression of cAMR.

Fig 1. Association between CD161⁺ T cells and Th17 cells.

PBMCs were stained with anti-CD4 PE-cy7, anti-CD161 APC, and anti-IL-17 PE antibodies. Lymphocytes were gated for further analysis. (A) Proportion (%) of CD161⁺/CD4⁺ IL-17⁺ T cells. (B) Proportion (%) of IL-17⁺/CD4⁺ CD161⁺ T cells in PBMCs. (C) PBMCs were cultured for 48 hours under Th0 differentiation conditions and the proportion (%) of CD161⁺/CD4⁺ IL-17⁺ T cells in PBMCs was analyzed. (D) PBMCs were cultured for 48 hours under Th17 differentiation conditions and the proportion (%) of CD161⁺/CD4⁺ IL-17⁺ T cells in PBMCs was analyzed. (E) The proportion (%) of CD161⁺/CD4⁺ T cells showed a significant positive correlation with the proportion (%) of IL-17⁺/CD4⁺ T cells (P = 0.02, r2 = 0.16). *p<0.05 for each comparison.

Fig 2. Gene expression in CD161⁺ T cells and CD161^- T cells using microarray.

(A) Hierarchical clustering of gene expression in CD161⁺ T cells and CD161^- T cells. Heatmap is showing 691 significantly (p<0.05) differentially expressed transcripts between CD161⁺ and CD161^- T cells in three donors. The 691 genes were selected for this analysis by the criteria described in ‘‘Materials and Methods”. Expression levels are normalized for each gene and shown by color, with yellow representing high expression and blue representing low expression. (B) Scatter plot of expression level between CD161⁺ and CD61^- T cells.

Fig 3. Comparison of CD4⁺ T cell subset among cAMR, IFTA, and LTGS groups.

(A) PBMCs were stained with anti-CD4 PE-cy7, anti-CD45RA–FITC, and anti-CCR7 APC antibodies. CD4⁺ T cells were gated for further analysis. (B-F) Proportion (%) of (B) CD4⁺ T cells/lymphocytes, (C) TCM/CD4⁺ T (CD45RA–CCR7⁺/CD4⁺ T cells), (D) T naïve/CD4⁺ T (CD45RA⁺CCR7⁺/CD4⁺ T cells), (E) TEM/CD4⁺ T (CD45RA^–CCR7^–/CD4⁺ T cells), (F) CD161⁺/CD4⁺ T cells in each patient group. *p<0.05 for LTGS, †p<0.05 for IF/TA. Abbreviations; cAMR, chronic antibody-mediated rejection; LTGS; long-term graft survival; IF/TA, interstitial fibrosis/tubular atrophy.

Fig 4. Comparison of CD161⁺ cell infiltration between cAMR group and IF/TA group.

Representative staining of CD161⁺ cells in renal allograft tissue of (A) cAMR and (B) IF/TA groups. CD161⁺ cells were found mostly within interstitial lymphocyte infiltration (A, B: Original magnification ×400). cAMR, chronic antibody-mediated rejection; IF/TA, interstitial fibrosis/tubular atrophy.

Fig 5. CD161⁺ T cells induced inflammation in human renal tubular epithelial cells.

(A) IL-6 and (B) IL-8 productions by HRPTEpiC that were co-cultured for 48 or 72 hours with sorted CD161⁺ T cells. Treatment with sorted CD161⁺T cells increased the production of IL-6 and IL-8 by HRPTEpiC. *p<0.05 vs. TEC. Values are expressed as the mean and SD of triplicate cultures. HRPTEpiC (TEC), human renal proximal tubular epithelial cells.