Clinical significance of CCR7+CD8+ T cells in kidney transplant recipients with allograft rejection

Abstract

The regulatory function of CCR7⁺CD8⁺ T cells against effector T-cells involved in T-cell mediated rejection (TCMR) in kidney transplant recipients was investigated. In vitro experiments explored the ability of CCR7⁺CD8⁺ T cells to suppress T-cell proliferation under T-cell activation conditions or during coculture with human renal proximal tubular epithelial cells (HRPTEpiC). In an ex vivo experiment, the proportion of CCR7⁺/CD8⁺, FOXP3⁺/CCR7⁺CD8⁺ T and effector T-cell subsets were compared between the normal biopsy control (NC, n = 17) and TCMR group (n = 17). The CCR7⁺CD8⁺ T cells significantly suppressed the proliferation of CD4⁺ T cells and significantly decreased the proportion of IFN-γ⁺ and IL-17⁺/CD4⁺ T cells and inflammatory cytokine levels (all p < 0.05). After coculturing with HRPTEpiC, CCR7⁺CD8⁺ T cells also suppressed T-cell differentiation into IL-2⁺, IFN-γ⁺, and IL-17⁺/CD4⁺ T cells (all p < 0.05). The TCMR group had significantly fewer CCR7⁺/CD8⁺ and FOXP3⁺/CCR7⁺CD8⁺ T in comparison with the NC group, but the proportions of all three effector T-cell subsets were increased in the TCMR group (all p < 0.05). The proportion of CCR7⁺/CD8⁺ T was inversely correlated with those of effector T-cell subsets. The results indicate that CCR7⁺CD8⁺ T cells may regulate effector T-cells involved in TCMR in an in vitro and in an ex vivo transplant model.

Figure 1

Induction and expansion of CCR7⁺CD8⁺ T cells. PBMCs (n = 5) were collected from healthy individuals, plated at 2 × 10⁵ cells per well and stimulated with anti-CD3 Abs (0.1 μg/ml), recombinant IL-15 (20 ng/ml), IL-2 (20 ng/ml), and retinoic acid (1 μg/ml). On day 3, cells were harvested, stained with antibodies specific to CD8, CCR7, and Foxp3, and analyzed by flow cytometry. The percentage of CCR7⁺ cells was determined using cells gated for CD8⁺ (a,b). The percentage of the Foxp3⁺ and Foxp3⁺ isotype was determined using cells gated for CD8⁺CCR7⁺ (c,d). (e) T-bet and Eomes mRNA expression was by real-time PCR. Bars represent the median with range. ^*p < 0.05, ^**p < 0.01 vs. Nil.

Figure 2

CCR7⁺CD8⁺ T cell-mediated suppression of activation of isolated CD4⁺ T cells from healthy donors. They were then cultured under CD4⁺ T-cell activation conditions with anti-CD3 and anti-CD28 for 72 hours (n = 6). (a) CCR7⁺CD8⁺ T cell-mediated suppression of the proliferation of T cells within the PBMC population isolated from healthy donors was measured using a ³H-thymidine incorporation assay. The cells were cultured for 3 days. (b) PBMCs were stained with PE-cy7-CD4, FITC-IFN-γ, PE-IL-17, and APC-IL-10. The proportion of (c) IFN-γ⁺ in the CD4⁺ T cells (%), (d) IL-17⁺ in the CD4⁺ T cells (%), and (e) IL-10⁺ in the CD4⁺ T cells (%) was performed using flow cytometry. ^*p < 0.05, ^**p < 0.01 vs. unstimulated CD4⁺ T cells, ^#p < 0.05, ^##p < 0.01 vs. T cell activation (Th0 condition).

Figure 3

Effect of CCR7⁺CD8⁺ T cell-mediated suppression on activated CD4⁺ T-cell cytokine levels. To induce CD4⁺ T cell activation, CD4⁺ T cells (5 × 10⁵) (n = 4) were incubated for 72 h with anti-CD3 and anti-CD28. To investigate the suppressive effects of CCR7⁺CD8⁺ T cells, stimulated CD4⁺ T cells were cultured with CCR7⁺CD8⁺ T cells. The secretion of (a) IL-2, (b) IL-17, and (c) IL-10 by stimulated CD4⁺ T cells was performed by ELISA of the culture supernatant. Note that addition of CCR7⁺CD8⁺ T cells significantly decrease IL-2, IL-17, and IL-10 levels that were increased by stimulated CD4⁺ T cells. ^**p < 0.01, ^***p < 0.001 vs. unstimulated CD4⁺ T cells and ^###p < 0.001 vs. stimulated CD4⁺T. Values are the median with range of triplicate cultures.

Figure 4

Effect of CCR7⁺CD8⁺ T cell-mediated suppression on T-cell proliferation by contact with HRPTEpiC. (a) The protocol for T-cell and HRPTEpiC coculturing. (b) The percentages of IFN-γ, IL-17, and IL-10⁺ in the CD4⁺T PKH⁻ cells were obtained by flow cytometry. Activated CD4⁺ T cells were cultured with HRPTEpiC for 72 h, and the percentage of (c) CD4⁺PKH⁻, (d) IFN-γ⁺/CD4⁺PKH⁻, (e) IL-17⁺/CD4⁺PKH⁻, and (f) IL-10⁺/CD4⁺PKH⁻ was performed by flow cytometry (n = 6). Note that addition of CCR7⁺CD8⁺ T cells significantly decreased CD4⁺PKH⁻, IFN-γ⁺/CD4⁺PKH⁻, and IL-17⁺/CD4⁺PKH⁻ levels, which was further increased with HRPTEpiC. ^*p < 0.05, ^**p < 0.01 vs. stimulated CD4⁺ T cells and ^#p < 0.05, ^##p < 0.01 vs. stimulated CD4⁺ T cells + HRPTEpiC. Values are the median with range of triplicate cultures.

Figure 5

Effect of CCR7⁺CD8⁺ T cell-mediated suppression on inflammatory cytokines production by stimulated CD4⁺ T cells cocultured with HRPTEpiC. (a) IL-2, (b) IFN-γ, (c) IL-17, and (d) IL-10 secretion by stimulated CD4⁺ T cells cocultured with/without CCR7⁺CD8⁺ T cells (as indicated) cocultured for 72 hours with HRPTEpiC (n = 6). Note that CCR7⁺CD8⁺ T cells suppressed the CD4⁺ T-cell production of IL-2, IFN-γ, and IL-17. ^*p < 0.05, ^**p < 0.01 vs. stimulated CD4⁺ T cells and ^#p < 0.05, ^##p < 0.01 vs. stimulated CD4⁺ T-cell + HRPTEpiC. Values are expressed as the median with range of triplicate cultures.

Figure 6

Comparison of CCR7⁺CD8⁺ T and effector T cells in PBMCs from kidney transplant recipients with or without TCMR. (a) The representative figure for the flow cytometric analysis of CCR7⁺ CD8⁺, Foxp3⁺/CCR7⁺CD8⁺, CD28^nullCD57⁺, CD45RA⁺CCR7⁻/CD8⁺, and CCR4⁺CCR6⁺/CD4⁺ T cells. Panels (b–f) show the comparison of the distribution of (b) CCR7⁺/CD8⁺ (c) Foxp3⁺/CCR7⁺CD8⁺, (d) CD28^nullCD57⁺/CD8⁺, (e) CD45RA⁺CCR7⁻/CD8⁺ T (CD8⁺TEMRA), and (f) CCR4⁺CCR6⁺/CD4⁺ T within the peripheral blood mononuclear cells (PBMCs) between the NC (n = 17) and TCMR group (n = 17). (g–i) shows the correlation curve between CCR7⁺/CD8⁺ T with (g) CD28^nullCD57⁺/CD8⁺, (h) CD45RA⁺CCR7⁻/CD8⁺, and (i) CCR4⁺CCR6⁺/CD4⁺ T cells. ^*p < 0.05 between NC and TCMR.