Current thinking holds that costimulation-blockade resistant graft rejection may be mediated by CD28^null T cells. To further investigate this possibility, we performed a retrospective immunophenotypic analysis of adult renal transplant recipients who experienced acute rejection on belatacept treatment compared to those that did not. We identified a subset of CD28^null CD4⁺ T_EM that are elevated at baseline in patients that did not go on to experience acute rejection (p<0.0001). Our data demonstrate that this CD28^null CD4⁺ T_EM population contained fewer IL-2 producing cells and showed increased expression of the coinhibitor 2B4 compared to rejecting patients (p=0.05). To investigate whether 2B4 could confer exhaustion in transplantation, we retrogenically expressed 2B4 on murine CD8⁺ OT-I T cells (2B4rg) and transferred these cells into naïve recipients prior to giving an OVA-expressing skin graft. We found that 2B4 expression results in significantly reduced accumulation of antigen-specific CD8⁺ T cells in the spleen 10 days post-transplantation (p=0.03). We observed a marked reduction in the proliferation of CD8⁺ Thy1.1⁺ 2B4rg cells when compared to controls as measured by CellTrace Violet (CTV) analysis (p=0.002). The 2B4rg population contained a higher frequency of CTV^hi undivided cells compared to the controls, indicating that expression of 2B4 on donor-reactive CD8⁺ T cells results in reduced recruitment into the response. To investigate the mechanisms by which 2B4 expression results in decreased proliferation, we interrogated differences in the metabolism of 2B4-deficient (2B4KO) T cells. We used the Seahorse extracellular flux assay to determine that 2B4KO cells activated in vitro exhibited increased glycolytic capacity compared to WT controls (p<0.0001). In vivo analysis of 2B4KO vs. WT T cells following infection with Listeria monocytogenes revealed that that 2B4KO T cells show a trend towards increased Glut-1 expression and significantly enhanced uptake of the glucose analog 2-NBDG ex vivo (p=0.02). Thus, we conclude that 2B4 signals serve to inhibit glycolysis, which limits the proliferation and accumulation of alloaggressive CD8⁺ T cells following transplantation. These data suggest that 2B4 signaling on CD8⁺ T cells may promote the metabolic shift from resting to effector cell, and raise the possibility that 2B4-mediated manipulation of metabolism may be a novel therapeutic strategy to target the donor-reactive memory T cell responses following transplantation.

CITATION INFORMATION: Laurie S, Liu D, Cortes M, Wagener M, Ford M. 2B4-Mediated Inhibition of Proliferation and Glycolytic Function Attenuates T Cell Alloreactivity. Am J Transplant. 2017;17 (suppl 3).

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