Costimulatory function and expression of CD40 ligand, CD80, and CD86 in vascularized murine cardiac allograft rejection

Abstract

Recent data implicates a role for the CD40-CD40 ligand (CD40L) pathway in graft rejection. One potential mechanism is direct costimulation of T cells through CD40L. Alternatively, the ability of CD40 stimulation to induce CD80 (B7-1) and CD86 (B7-2) expression on antigen-presenting cells (APCs) has led to the hypothesis that the role of CD40-CD40L interactions in transplant rejection might be indirect, i.e., to promote the costimulatory capacity of APCs. Here, we have used a murine vascularized cardiac allograft model to test this hypothesis. Treatment of the recipients with donor splenocytes and a single dose of anti-CD40L mAb induces long-term graft survival (> 100 days) in all animals. This is associated with marked inhibition of intragraft Th1 cytokine [interferon gamma and interleukin (IL) 2] and IL-12 expression with reciprocal up-regulation of Th2 cytokines (IL-4 and IL-10). In untreated allograft recipients, CD86 is strongly expressed on endothelial cells and infiltrating mononuclear cells of the graft within 24 hr. In contrast, CD80 expression is not seen until 72 hr after engraftment. Anti-CD40L mAb has no detectable effect on CD86 up-regulation, but almost completely abolishes induction of CD80. However, animals treated with anti-CD80 mAb or with a mutated form of CTLA4Ig (which does not bind to CD86) rejected their cardiac allografts, indicating that blockade of CD80 alone does not mediate the graft-prolonging effects of anti-CD40L mAb. These data support the notion that the role of CD40-CD40L in transplant rejection is not solely to promote CD80 or CD86 expression, but rather that this pathway can directly and independently costimulate T cells. These data also suggest that long-term graft survival can be achieved without blockade of either T cell receptor-mediated signals or CD28-CD86 engagement.

Figure 1

Effect of anti-CD40L mAb on cardiac allograft survival. BALB/c hearts were transplanted into C57BL/6 recipients. Allograft recipients were treated with either a control Ig or anti-CD40L mAb (200 μg by intravenous injection) at the time of transplantation. Selected recipients also received a transfusion of 5 × 10⁶ donor lymphocytes (DST) at the time of transplantation.

Figure 2

Effect of anti-CD40L mAb on intragraft cytokine gene expression. C57BL/6 recipients of BALB/c cardiac allografts were treated with either DST plus 200 μg of control Ig (Upper) or DST plus 200 μg of anti-CD40L mAb (Lower) at the time of transplantation. The animals were killed after 3 days and the hearts were stained for expression of IL-2, IFN-γ, IL-4, and IL-10 as described. Arrowheads (a and c) indicate the typical mononuclear cell labeling for IL-2 and IFN-γ confined to control grafts, whereas grafts from anti-CD40L mAb-treated recipients showed dense mononuclear and some adjacent endothelial cell labeling for IL-4 (f) and IL-10 (h). Cryostat sections, hematoxylin counterstain. (Bar = 50 microns.)

Figure 3

Serial changes in B7-1 and B7-2 expression within murine cardiac allografts. BALB/c hearts transplanted into C57BL/6 recipients were harvested after 1 or 3 days and sections stained for the expression of CD80 (B7-1) or CD86 (B7-2). Day 0 panels represent a harvested donor heart immediately before transplantation. An isograft control (BALB/c donor into BALB/c recipient) was harvested on day 1. Arrowheads in a and c indicate the weak labeling for B7-1 that is restricted to interstitial dendritic cells in the first day posttransplantation, whereas by day 3, dense mononuclear and some capillary endothelial cell labeling is seen (g). In contrast, B7-2 is expressed by occasional graft endothelial cells (arrowheads in b) but is rapidly and densely up-regulated in allografts within 24 hr (d) but not in a day 1 isograft (f). Where indicated, the recipients were treated with DST plus 200 μg of anti-CD40L mAb at the time of transplantation (i and j). DST plus CD40L mAb administration blocked up-regulation of B7-1, without affecting graft cellularity (compare g and i). The dense endothelial expression of B7-2 was not modulated by DST plus anti-CD40L mAb (compare h and j). DST plus control Ig did not affect the marked increase in intragraft B7-1 seen at day 3 in control allografts (data not shown). Cryostat sections, hematoxylin counterstain. (Bar = 50 microns.)