Down-Regulation of Surface CD28 under Belatacept Treatment: An Escape Mechanism for Antigen-Reactive T-Cells

BACKGROUND: The co-stimulatory inhibitor of the CD28-CD80/86-pathway, belatacept, allows calcineurin-inhibitor-free immunosuppression in kidney transplantation. However, aggressive T-cell mediated allogeneic responses have been observed in belatacept-treated patients, which could be explained by effector-memory T-cells that lack membrane expression of CD28, i.e. CD28-negative (CD28(NULL)) T-cells. CD28-positive (CD28(POS)) T-cells that down regulate their surface CD28 after allogeneic stimulation could also pose a threat against the renal graft. The aim of this study was to investigate this potential escape mechanism for CD28(POS) T-cells under belatacept treatment. MATERIALS & METHODS: PBMCs, isolated T-cell memory subsets and isolated CD28(POS) T-cells were obtained from end-stage renal disease (ESRD) patients and co-cultured with allo-antigen in the presence of belatacept to mimic allogeneic reactions in kidney-transplant patients under belatacept treatment. As a control, IgG was used in the absence of belatacept. RESULTS: Despite high in vitro belatacept concentrations, a residual T-cell growth of ±30% was observed compared to the IgG control after allogeneic stimulation. Of the alloreactive T-cells, the majority expressed an effector-memory phenotype. This predominance for effector-memory T-cells within the proliferated cells was even larger when a higher dose of belatacept was added. Contrary to isolated naïve and central-memory T cells, isolated effector-memory T cells could not be inhibited by belatacept in differentiation or allogeneic IFNγ production. The proportion of CD28-positive T cells was lower within the proliferated T cell population, but was still substantial. A fair number of the isolated initially CD28(POS) T-cells differentiated into CD28(NULL) T-cells, which made them not targetable by belatacept. These induced CD28(NULL) T-cells were not anergic as they produced high amounts of IFNγ upon allogeneic stimulation. The majority of the proliferated isolated originally CD28(POS) T-cells, however, still expressed CD28 and also expressed IFNγ. CONCLUSION: This study provides evidence that, apart from CD28(NULL) T-cells, also CD28(POS), mostly effector-memory T-cells can mediate allogeneic responses despite belatacept treatment.