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

<div><p>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 <i>in vitro</i> 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 <i>IL18RAP</i>, <i>IL-18R1</i>, <i>IL23R</i>, <i>IL12RB2</i>, <i>RORC</i>, <i>TBX21</i>, and <i>EOMES</i> were upregulated in CD161⁺ cells compared with CD161^- cells. In an <i>ex vivo</i> 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.</p></div

Baseline characteristics of the patient populations.

<p>Baseline characteristics of the patient populations.</p

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

<p>(A) Hierarchical clustering of gene expression in CD161⁺ T cells and CD161^- T cells. Heatmap is showing 691 significantly (<i>p</i><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.</p

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

<p>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.</p

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

<p>(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. *<i>p</i><0.05 vs. TEC. Values are expressed as the mean and SD of triplicate cultures. HRPTEpiC (TEC), human renal proximal tubular epithelial cells.</p

Association between CD161⁺ T cells and Th17 cells.

<p>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). *<i>p</i><0.05 for each comparison.</p

A flow diagram of the effect of gemigliptin against high-phosphate-induced vascular calcification.

<p>Gemigliptin attenuated vascular calcification and osteogenic trans-differentiation in VSMCs via multiple steps including downregulation of PiT-l expression, and suppression of ROS generation, phosphor-PI3K/AKT, and Wnt signaling pathway.</p

Gemigliptin attenuates the expression of sodium phosphate co-transporter PiT-1.

<p>The expression of PiT-1 was evaluated by qRT-PCR and western blot after treatment with 3 mM phosphate and/or 50 μM gemigliptin for 2 days (A) or 4 days (B). GAPDH was used as the loading control. The results are expressed as percentage of control. Results are presented as mean ± SEM (n = 5–6 in each group). Con, control; Gemi50, 50 μM gemigliptin; Pi, 3mM phosphate; PG50, 3mM phosphate and 50 μM gemigliptin. *P<0.5, **P<0.01, ***P<0.001 compared with control and #P<0.5, ##P<0.01, ###P<0.001 compared with phosphate group.</p

Gemigliptin attenuates high phosphate-induced Wnt signaling.

<p>The expression of <i>FDZ3</i> and <i>DKK-</i>1 was evaluated by qRT-PCR (A and B) and western blot (C and D) in VSMCs after incubating with 3 mM phosphate and/or 50 μM gemigliptin for 7 days (A and C) and 14 days (B and D). The results are expressed as percentage of control. Results are presented as mean ± SEM (n = 5–6 in each group). FDZ3, frizzled-3; DKK-1, dickkopf-1; Con, control; Gemi50, 50 μM gemigliptin; Pi, 3mM phosphate; PG50, 3mM phosphate and 50 μM gemigliptin. *P<0.5, **P<0.01, ***P<0.001 compared with control and #P<0.5, ##P<0.01, ###P<0.001 compared with phosphate group.</p

Gemigliptin attenuates phosphate-induced phospho-PI3K-AKT signaling pathway.

<p>(A) VSMCs were treated with 3 mM phosphate and/or 50 μM gemigliptin for 24 h. Representative immunoblot images are shown. Graph represents quantitative data as (B) ratio of phospho-AKT/total-AKT or (C) phospho-PI3K/total PI3K. The results are expressed as percentage of control. Results are presented as mean ± SEM (n = 4–5 in each group). Con, control; Gemi50, 50 μM gemigliptin; Pi, 3 mM phosphate; PG50, 3 mM phosphate and 50 μM gemigliptin. *P<0.5, **P<0.01, ***P<0.001 compared with control, #P<0.5, ##P<0.01, ###P<0.001 compared with phosphate group.</p