INHIBITION OF HUMAN SKIN ALLOGRAFT REJECTION BY BLOCKADE OF CD28-B7 AND CD40-CD154 COSTIMULATION IN THE HUMANIZED SCID MOUSE (HuSCID).

Abstract

438 We have developed a HuSCID model of rejection to examine the effect of CD28-B7 and CD40-CD154 on the development of alloreactive human mononuclear cells (MNCs) in vivo. In this study, human foreskins were transplanted onto SCID mice and were allowed to engraft for 4-6 weeks. Following successful engraftment, 3 × 108 human MNCs allogeneic to the skin were adoptively transferred into the mice by IP injection. In untreated animals, skin grafts reject in association with CD3, CD4, CD8, CD25 and CD68 +ve MNCs which infiltrate from day 7, and peak at 14-21 days post transfer. There were 4 experimental groups: 1) Rejection control: received control fusion Ig 200μg IP on day 0, 2, 4, 6 or no reagent 2) CD154 blockade: anti-human CD154 mAb 250 μg given on days 0, 2, 4, 6 3) B7 blockade: Human CTLA4-Ig 200μg IP given on days 0, 2, 4, 6 and 4) B7 and CD154 blockade: both given on days 0, 2, 4, 6. Humanization of animals was confirmed by detection of human Ig in mouse sera by ELISA. Graft tissue was harvested at day 14 or day 28. CD3+ T cell infiltrates were assessed by immunohistochemistry and counted over 7 areas of each specimen using the calibrated grid method. In specimens harvested at either day 14 (Table) or day 28 (n = 5/5), marked CD3+ infiltration seen in rejection controls was inhibited in animals who received CTLA4-Ig alone or in combination with anti CD154 mAb. Inhibition of rejection closely correlated with preservation of tissue architecture. Quantitative RT-PCR (fg/pg GAPDH) was also performed on day 14 samples to evaluate the expression of perforin, granzyme B and Fas ligand, reported to be associated with human rejection. Lack of rejection was also associated with a reduced expression of all three genes, as shown below.When HuSCID were humanized at the same time as skin grafting, thereby ensuring circulating MNCs at the time of grafting, skins failed to engraft and were rejected prior to 28 days (n = 4) or had marked infiltrates at 28 days (n= 2). CTLA4-Ig combined with anti CD154 mAb also prevented rejection in this model, with graft survival in 4/4 animals at 28 days. These new findings suggest that simultaneous blockade of both CD28-B7 and CD40-CD154 interactions inhibits the recruitment of alloreactive MNCs and rejection in a“human” transplant model.