A novel in vivo regulatory role of P-glycoprotein in alloimmunity

Abstract

P-glycoprotein (P-gp) is required for adaptive immunity through defined functions in T cell activation and antigen presenting cell (APC) maturation. The potential role of P-gp as an in vivo regulator of alloimmunity is currently unknown. Here we show that P-gp blockade prolongs graft survival in a murine heterotopic cardiac allotransplantation model through in vivo inhibition of the T helper 1 (Th1) cytokine IFN-gamma and the Th2 product IL-4, and via downregulation of the APC-expressed positive costimulatory molecule CD80. In vitro, the P-gp antagonist PSC833, a non-calcineurin-inhibitory cyclosporine A analogue, specifically inhibited cellular efflux of the P-gp substrate rhodamine-123 in wild-type CD3(+) T cells and MHC class II(+) APCs but not their P-gp knockout counterparts that lacked rhodamine-123 efflux capacity. Additionally, P-gp blockade significantly inhibited murine alloimmune T cell activation in a dose-dependent fashion. In vivo, P-gp blockade significantly prolonged graft survival in Balb/c recipients of C57BL/6 cardiac allografts from 8.5+/-0.5 to 11.7+/-0.5 days (P<0.01), similar in magnitude to the effects of monotherapy with cyclosporine A. Moreover, P-gp blockade, compared to controls, attenuated intragraft expression of CD3 and CD80, but not CD86, and inhibited IFN-gamma and IL-4 production (P<0.05). In the setting of systemic CD86 inhibition, P-gp blockade suppressed IFN-gamma and IL-4 production significantly further (to 98% and 89% inhibition, respectively) compared to either P-gp or anti-CD86 blockade alone, and markedly prolonged allograft survival compared to anti-CD86 blockade alone (40.5+/-4.6 versus 22.5+/-2.6 days, respectively, P<0.01). Our findings define a novel in vivo regulatory role of P-gp in alloimmunity and identify P-gp as a potential therapeutic target in allotransplantation.

Figure 1. P-gp gene expression and function in murine splenocyte subsets

(A) RT-PCR amplification of murine splenocyte P-gp cDNA. (B) Flow cytometry analysis of P-gp-mediated rhodamine-123 efflux transport (F11 fluorescence) in untreated, P-gp-inhibited (PSC833) or ATP hydrolysis-inhibited (ice) wild-type FVB strain splenocyte subsets (gated for PE-labeled CD3⁺, CD4⁺, CD8⁺ or MHC class II⁺ subsets at the Fl2 emission spectrum) as a time course (T=0 min and T=120 min upon rhodamine-123 loading). The percentages of rhodamine-123-effluxing cells are shown in the R1 gates. (C) Flow cytometry analysis of mdr1a/1b KO FVB strain splenocyte subsets studied and analyzed as in (B).

Figure 2. Effects of in vitro P-gp blockade on murine alloimmune T cell proliferation

(A) Top panel: MLR proliferation (³H-thymidine uptake) of wildtype (wt) versus mdr1a/1b knockout (KO) FVB splenocytes stimulated with allogeneic wt C57BL/6 splenocytes. Bottom panel: MLR proliferation (³H-thymidine uptake) of wt C57BL/6 splenocytes stimulated with allogeneic wt versus mdr1a/1b KO FVB splenocytes. Illustrated are mean values of n=16-32 wells±SEM representative of n=3-4 independent experiments, respectively. (B) Top panel: ³H-thymidine uptake of C57BL/6 donor strain (MLR)-stimulated Balb/c splenocyte cultures compared to untreated controls, plotted against PSC833 concentration (μm). Illustrated are mean values of quadruplicate wells±SEM. Bottom Panel: ³H-thymidine uptake of C57BL/6 donor strain (MLR)-stimulated Balb/c splenocyte cultures treated with PSC833, anti-CD86 mAb, anti-CD80, control Ig, or combinations thereof. Illustrated are means of quadruplicate wells±SEM. (C) Assessment of cell death by Annexin V-PE/7-AAD staining of untreated and PSC833-treated C57BL/6 donor strain (MLR)-stimulated Balb/c splenocyte cultures at 1 day (top panel) and 5 days (bottom panel) of incubation. Illustrated are mean values±SD of percentage changes in cell death (n=3 independent experiments).

Figure 3. Effects of in vivo P-gp blockade on murine cardiac allograft survival

(A) Effects of in vivo P-gp blockade on C57BL/6 cardiac allograft survival in Balb/c recipients. Kaplan-Meier analyses of the effects of PSC833, vehicle, or cyclosporine A monotherapy on cardiac allograft survival are illustrated. (B) Effects of concurrent P-gp and CD86 blockade on C57BL/6 cardiac allograft survival in Balb/c recipients. Kaplan-Meier analyses of the effects of PSC833+anti-CD86 compared to vehicle+anti-CD86, PSC833+control Ig, or vehicle+control Ig are illustrated.

Figure 4. Effects of in vivo P-gp blockade on graft inflammatory infiltration and T helper responses

(A) Depicted are H& E- or HRP-stained (isotype control, CD3, CD80, or CD86) representative axial ventricular tissue sections of cardiac allografts dissected 10 days post transplantation from vehicle- (top row) or PSC833-treated (bottom row) allograft recipients. Size bars: 50μm. (B) Quantitative analysis of intragraft CD3, CD80, and CD86 expression from vehicle- versus PSC833-treated allograft recipients. Mean percentages of positive cells±SEM are illustrated (*: P<0.05, NS: not significant). (C) ELISPOT analysis of INF-γ (left) and IL-4 (right panel) production by Balb/c recipient splenocytes isolated from vehicle- or PSC833-treated allograft recipients 10 days post transplantation. Naïve, irradiated C57BL/6 donor-strain splenocytes were used as stimulators. Spots per well (mean±SEM, n=6) are shown for each treatment group.