Studies in both murine and NHP models have shown that CTLA-4Ig-resistant rejection is mediated by CD8+ T cells, particularly memory CD8+ T cells. Because CTLA-4 Ig binds to CD80/86, both CD28 costimulatory and CTLA-4 coinhibitory signals are inhibited. We hypothesized that blocking CTLA-4 mediated coinhibitory signals may detract from the overall goal of attenuating alloreactive T cell responses. To test this hypothesis we made use of novel recombinant domain antibodies specific for CD28 (CD28dAbs), which selectively block CD28 and leave CTLA-4 signals intact. B6 recipients of BALB/c skin grafts were treated with CTLA-4 Ig or CD28dAbs. Results indicated that both graft rejection and CD8+ alloreactive T cell responses as measured by ICCS were attenuated in CD28dAb treated recipients as compared to CTLA-4 Ig treated controls (MST > 60 d vs 29d). To elucidate the mechanism by which selective CD28 blockade better attenuates allospecific CD8+ T cell responses, we examined the phenotype of donor-reactive CD8+ T cells under both treatment conditions. Strikingly, CD8+ T cells from mice treated with CD28dAbs exhibited a significant increase in the expression of the coinhibitory receptor 2B4 (SLAMf4, CD244). We next sought to determine whether 2B4 expression is induced by selective CD28 blockade on alloreactive memory CD8+ T cells. We employed an adoptive transfer approach which CD45.2+ B6 animals were pre-sensitized with a BALB/c skin graft. CD44hi CD8+ CD45.2+ T cells were purified at day 30 post transplant and transferred into naÏve CD45.1+ animals, which then received a BALB/c skin graft in the presence or absence of CD28 dAbs. Results revealed that CD28dAbs significantly attenuated and induced a striking upregulation of 2B4 on polyclonal alloreactive CD8+ memory T cells. Next, we interrogated whether 2B4 plays a causal role in attenuating donor-reactive memory T cell responses following transplantation. We utilized a similar experimental approach, but this time compared T cell responses from WT and 2B4-/- recipients. Results showed that splenocytes from 2B4-/- recipients exhibited enhanced alloreactive CD8+ T cell responses upon secondary restimulation and exhibited a more activated phenotype as measured by CD69 expression. Thus, these data suggest that the 2B4 coinhibitor plays a functional role in the attenuation of polyclonal donor-reactive CD8+ memory T cell responses in transplantation.

Suchard, S.: Employee, Bristol-Myers Squibb. Nadler, S.: Employee, Bristol-Myers Squibb. Ford, M.: Grant/Research Support, Bristol-Myers Squibb.

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