ANALYSIS OF THE ROLE OF NEGATIVE T CELL COSTIMULATORY PATHWAYS IN CD4 AND CD8 MEDIATED ALLOIMMUNE RESPONSES IN VIVO

Abstract

O42 Aims: Negative signals through cell surface molecules such as CTLA-4 and PD-1 have been shown to play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unknown. This study examines the effects of these inhibitory pathways on alloreactive CD4 and CD8 T cells in a vascularized heart transplant model. Furthermore, we investigate the interplay between CD28 costimulatory signals and these inhibitory pathways in vivo. Methods: C57BL/6 wild-type, CD8, CD4, CD28, CD28/CD8 and CD28/CD4 deficient mice on the B6 background are used as recipients of BALB/c vascularized heart grafts. Recipients are treated with a blocking anti-CTLA-4, anti-PD-1, anti-PD-L1 or anti-PD-L2 mAb (0.5 mg i.p. on day 0 and 0.25 mg i.p. on day 2,4,6,8 and 10 post transplant). Results: CTLA-4 blockade accelerates graft rejection in wild-type recipients (MST 6.3±0.5 days, n=6 compared to untreated group MST 8.9±1.6 days, n=7, p=0.001) and a proportion of CD4−/− recipients (rejection in 38% of treated mice within 40 days, n=6 versus untreated mice MST>130, n=5, p=0.04), but not in CD8−/− recipients. The same treatment leads to prompt rejection of heart allografts in CD28−/− (MST 11.1±4.4, n=8 vs. MST 17.7±3.1 days, n=6 in untreated group, p=0.023) and a proportion of CD28−/−CD4−/− mice (33% of mice within first 40 days, n=6 vs. MST>65 days, n=4 in control group, p=N.S.) without any effect on allograft survival in CD28−/−CD8−/− recipients. These results indicate that the B7-CTLA-4 pathway provides a critical negative signal to CD8+ T cells, particularly in the presence of CD28. In contrast, PD-1 blockade only leads to accelerated rejection of heart allografts in CD28−/− (MST 11.1±3.6 days, n=9, p=0.017) and CD28−/−CD8−/− recipients (MST 14.2±3, n=6, p=0.0015 compared to MST 122, n=8 in controls) with no apparent effect in all other recipients. Finally, PD-L1 blockade leads not only to accelerated rejection in CD28−/− (MST 7.2±0.8, n=6, p=0.0008) and CD28−/−CD8−/− recipients (MST 9.3±0.7, n=6, p<0.001), but also in wild-type mice (MST 6.5±0.5, n=6, p=0.0017) and in CD28−/−CD4−/− recipients (MST 20±2.8, n=6, p=0.003). These data indicate that PD-1:PDL-signaling mediates negative regulation of alloimmune responses affecting mainly CD4 T cells, especially in the absence of positive CD28 costimulation. Conclusions: Understanding the intricate balance of positive and negative T cell costimulatory signals in determining the fate of the alloimmune response will allow development of new strategies for therapeutic intervention in transplantation. We hypothesize that induction of reproducible tolerance will require blockade of positive and enhancement of negative costimulatory signals in vivo.