2B4 is an immunoglobulin superfamily member expressed constitutively on NK cells, and inducibly on some CD8+ T cells. We recently showed in a murine skin transplant model that treatment with selective CD28 blockade resulted in an upregulation of 2B4, which acted as a functionally important coinhibitor of T cell responses in the absence of CD28 costimulation. Here, we made the interesting observation that virtually all CD28 negative CD8+ T cells in humans express 2B4. The abundant expression of 2B4 specifically on CD28 negative CD8+ T cells suggests that this pathway may be therapeutically manipulated to better control this population following transplantation. Specifically, we hypothesized that increasing 2B4-mediated signals would diminish graft-specific CD8+ T cell responses following transplantation. To test this, we created 2B4 “retrogenic” donor-reactive TCR tg CD8+ OT-I T cells which are stably transduced with a retroviral vector constitutively expressing murine 2B4. Using this tool, we established a model wherein donor-reactive 2B4 retrogenic CD8+ T cells constitutively expressing high levels of 2B4 (or empty vector-transduced control cells) were adoptively transferred along with wild-type donor-reactive CD4+ OT-II T cells into naïve animals, which then received an OVA-expressing skin graft. Results indicated that overexpression of 2B4 reduced absolute numbers of donor-specific T cells in the spleen ten days following transplantation by over 10-fold, with spleens of WT-PMY mice containing an average of 6.51×106 OT-I cells (SEM +/- 2.81×106 cells) compared to the 2B4 RtTg mice, which averaged only 1.23×106 OT-I cells (SEM +/- 4.27×105 cells) (p = 0.0267), indicating that expression of 2B4 can independently attenuate donor-reactive CD8+ T cell responses. Interestingly, attenuation of CD8+ T cell responses by retrogenic 2B4 was dependent on the anatomical compartment, as the reduction of donor-specific CD8+ T cells was evident in the peripheral blood, bone marrow, and spleen, but not in the draining lymph nodes. Further analyses revealed that overexpression of 2B4 preferentially inhibited KLRG-1+ donor-reactive CD8+ cells, suggesting that the coinhibitory function of this molecule is differentiation-status specific. Taken together, these data suggest that manipulation of the 2B4 signaling pathway may be a novel therapeutic strategy to inhibit the donor-reactive effector memory T cell responses that underlie acute rejection following transplantation.

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