Cyclosporin but not everolimus inhibits chemokine receptor expression on CD4+ T cell subsets circulating in the peripheral blood of renal transplant recipients

Abstract

The peripheral chemokine receptors chemokine receptor 3 (CXCR3) and CC chemokine receptor 5 (CCR5) have been reported to be associated with allograft rejection. The impact of the expression of immunosuppressive drugs on peripherally circulating CD4(+) T cell subsets after renal transplantation is unknown. Expression of CXCR3 and CCR5 was investigated by flow cytometry in 20 renal allograft recipients participating in a prospective, randomized trial (NCT00514514). Initial immunosuppression consisted of basiliximab, cyclosporin A (CsA), mycophenolate sodium and corticosteroids. After 3 months, patients were treated either with CsA, mycophenolate sodium (MPA) plus corticosteroids (n = 6), CsA and everolimus plus corticosteroids (n =8) or CsA-free (CsA(free)) receiving everolimus, MPA and corticosteroids (n = 6). After initial reduction of CD4(+) forkhead box protein 3 (FoxP3)(+) and CD4(+) CD25(hi) FoxP3(+) regulatory T cells (T(regs)) (P < 0.05; P < 0.01), 3-month post-transplant percentages of T(regs) were reconstituted in CsA(free) and CsA(lo) arms compared to CsA(reg) 12 months post transplant. Expression of CCR5 and CXCR3 on CD4(+) FoxP3(+) and CD4(+) FoxP3(-) T cells 12 months post transplant was increased in CsA(free) versus CsA(reg). Increase in CCR5(+) CXCR3(+) co-expressing CD4(+) FoxP3(-) cells between 3 and 12 months correlated negatively with the glomerular filtration rate (GFR) slope/year [modification of diet in renal disease (MDRD); r = -0.59, P < 0.01]. CsA, but not everolimus, inhibits both T(reg) development and expression of CXCR3 and CCR5 on CD4(+) T cell subsets. Increase in CCR5(+) CXCR3(+) co-expressing CD4(+) FoxP3(-) T cells is associated with early loss in allograft function.

Fig. 1

Study arms. The three study arms and their patient numbers are depicted. The orange-tinted parts of the time-line indicate time-periods in which samples were collected for fluorescence activated cell sorter (FACS) analysis and the block arrows the time-points at which expression data were used for statistical analysis. BSX, basiliximab; CsA, cyclosporin A; EVR, everolimus; MPA, mycophenolate sodium; PRED, prednisone; 144 × 123 mm (300 × 300 dots per inch).

Fig. 2

Longitudinal development of percentages of CD4⁺ regulatory T cells (T_regs) in the three treatment arms. The percentages of expression of forkhead box protein 3 (FoxP3) (a) and CD25^hiFoxP3 (b) are shown after gating on the CD4⁺ T cell subset and summarized as scatter-plots with mean. Dot-blots on the right depict representatives of each T_reg subset. The asterixes represent statistical significance (*P < 0·05; **P < 0·01); 123 × 88 mm (300 × 300 dots per inch).

Fig. 3

Cyclosporin inhibits CD4⁺ regulatory T cell (T_reg) development. Scatter-plot graphs in (a) show expression in percentage of forkhead box protein 3 (FoxP3) after gating in CD4⁺ T cells at 3 (pre-randomization) and 12 months after transplantation for each patient in each study arm. The scatter-plot graph with mean in (b) depicts the ratio of either FoxP3/CD45RO (left panel) or CD25^hiFoxP3/CD45RO (right panel) after gating on the CD4⁺ T cell subset in samples collected 3 (pre-randomization), 6 and 12 months after transplantation; 96 × 59 mm (300 × 300 dots per inch).

Fig. 4

Cyclosporin inhibits expression of CXCR3 and CC chemokine receptor 5 (CCR5) in CD4⁺ T cell subsets. Scatter-plot graphs show expression of CXCR3 (a) and CCR5 (b) for the three treatment arms after gating in CD4⁺ forkhead box protein 3 (FoxP3)⁺ T cells (left) and CD4⁺FoxP3^- T cells (right) at 3 (pre-randomization) and 12 months after transplantation; 157 × 126 mm (300 × 300 dots per inch).

Fig. 5

Correlation of CD4⁺ T cell subsets with allograft function during the first year after transplantation. The scatter-plot graph depicts the difference in percentage of CC chemokine receptor 5 (CCR5) and chemokine receptor 3 (CXCR3) co-expression (ΔCCR5⁺CXCR3⁺) of CD4⁺ forkhead box protein 3 (FoxP3)^- T cells (y-axis) over the slope of modified diet in renal disease (MDRD) (ml/min/year; x-axis) between 3 and 12 months post transplantation (n = 20); 117 × 84 mm (300 × 300 dots per inch).