Regulation of alloimmune Th1 responses by the cyclin-dependent kinase inhibitor p21 following transplantation

Abstract

Background: The cyclin-dependent kinase (cdk) inhibitor p21 inhibits cellular proliferation of many cell types, including T cells. Autoimmune models, however, have yielded conflicting results regarding the role of cdk inhibitors and T-cell function. The role of p21 in T-cell function after transplantation has not been investigated directly. We hypothesized that p21 plays an important role in alloantigen-driven responses in vitro in mixed lymphocyte cultures (MLC) and in vivo using the heterotopic murine cardiac allograft model.

Methods: Wild type (WT) and p21-deficient (p21-/-) mice were used as recipients, and the effects of p21 overexpression were assessed by transplanting p21 adenoviral-transfected cardiac allografts. Enzyme-linked immunospot (ELISPOT) and 3H-thymidine incorporation were used to evaluate for T-cell priming and proliferation in vitro, whereas graft histology was evaluated for rejection.

Results: When stimulated with alloantigens in vitro, splenocytes from p21-/- mice mounted enhanced proliferative responses and decreased Th2 responses relative to their WT counterparts. No differences in Th1 responses were noted when p21-/- cells were stimulated with alloantigens in vitro; however, after cardiac transplantation, Th1 responses were enhanced in p21-/- recipients relative to WT mice. This enhanced in vivo Th1 response was associated with exacerbated graft rejection in p21-/- recipients. Interestingly, p21 transfection of WT allografts inhibited graft rejection and Th1 priming.

Conclusions: p21 controls the intensity of the immune response posttransplantation, with overexpression inhibiting allograft rejection. Our data demonstrate that p21 controls T-cell priming and suggest that p21 and other cdk inhibitors may serve as potential targets for therapeutic manipulation of alloimmune responses.

Figure 1. Increased pathology of rejection in p21−/− cardiac allograft recipients

Representative H & E stained sections are depicted of rejecting BALB/c cardiac allografts in WT recipients on days 5 (Panel A) and 7 (Panel C) and in p21−/− recipients on day 5 (Panel B) and 7 (Panel D) post transplantation. Note early vascular involvement (white arrow) and hemorrhage (black arrows) on day 5 in p21−/− recipients (Panel B). Magnification = 200X.

Figure 2. Impact of p21 over-expression on allograft survival and donor-reactive Th1 responses

BALB/c cardiac allografts were transfected with 5 × 10⁸ pfu of Adβ-gal or Adp21 or left non-transfected prior to transplantation into WT C57BL/6 mice as described in the Materials and Methods section. Panel A: Graft function was monitored by daily palpation and rejection was recorded as the day on which the graft ceased to function (n = 6–8 transplants per group). p < 0.01 for the mean survival of Adp21 transfected allografts (18.6 days) compared to non-transfected (7.7 days) or Adβ-gal transfected allografts (7.5 days). Panel B: Splenocytes were harvested from recipients on the day of rejection and assessed for in vivo primed Th1 and Th2 responses by ELISPOT. Data represent the mean number of spots per 1 × 10⁶ cells +/− SEM. *p < 0.05 vs. Adβ-gal transfected and non-transfected groups. Panel C: RT-PCR was performed using primers specific for recombinant p21 or γ actin (see Materials and Methods) to verify appropriate expression of p21 following transfection with Ad21, but not Adβ-gal. Lane 1 – MW ladder. Lanes 2 and 3 – 293 cells were transfected with Adp21 or Adβ-gal, respectively, at an M.O.I. of 25 and RNA was isolated 24 hours post-transfection. Lanes 4-7 – Allografts were perfused with Adp21 or Adβ-gal and RNA was isolated from the transplanted hearts (H) and recipient spleens (S) on day 2 post-transplantation. Lanes 8 and 9 – Allografts were transfected with Adp21 or Adβ-gal, respectively, and RNA was harvested on day 7 post transplant. Results are representative of 3 transplanted mice per group.