Numerous studies have implicated donor-reactive memory T cells in mediating costimulation blockade resistant rejection (CoBRR) following treatment with belatacept. In humans, these memory T cells contain a subset of CD28^null cells, which may be indifferent to CD28 costimulatory signals. Previous reports have purported a role for CD28^null cells in mediating CoBRR; however frequency of CD28^null cells has not consistently borne out as being positively associated with CoBRR. Here, we show that CD28 expression is dynamically regulated following T cell stimulation. CD28 was initially downregulated at 1, 2 and 4 hours ex vivo post-stimulation with PMA and ionomycin (P/I), and expression was restored by 24 and 48 hours post-stimulation. These data suggest that CD28 expression can change rapidly in the setting of immune activation. We hypothesized that the degree of CD28 downregulation following ex vivo restimulation may correlate with risk of rejection on belatacept. To test this, we isolated pre-transplant PBMC from 10 renal allograft recipients receiving belatacept. Within 7 months, 5 experienced acute cellular rejection (ACR) and 5 remained rejection-free. PBMC were stimulated with P/I or left unstimulated, and were analyzed 4 hours later for CD28 expression. In line with previous reports, the frequency of CD28^null cells directly ex vivo did not correlate with risk of rejection in this cohort. However, stimulation with P/I resulted in a larger increase in the frequency of CD4⁺ CD28^null cells in patients that experienced CoBRR as compared to patients that remained rejection-free (3.2-fold vs 1.3 fold increase, respectively). Similarly, analysis of the CD8+ compartment following ex vivo restimulation revealed a significant decrease in CD28 MFI within the CD45RA^–CCR7^– T_EM population in patients that went on to reject on belatacept, but not in patients that remained rejection-free. In contrast, preliminary analyses of tacrolimus-treated patients failed to identify a differential effect of stimulation-induced CD28 downregulation in patients that went on to reject vs. those that remained stable. In summary, these data suggest that the propensity for CD28 downregulation in the setting of immune activation following transplantation may identify a population of T cells more resistant to belatacept-resistant rejection, and raise the possibility that degree of CD28 downregulation post-ex vivo restimulation could be used as a biomarker to predict risk of rejection on belatacept.

CITATION INFORMATION: Cortes M, Laurie S, Sayed B, Ford M. Rapid Emergence of CD28^null Cells Following Ex Vivo Restimulation Is Associated with Belatacept Resistant Rejection. Am J Transplant. 2016;16 (suppl 3).

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