CD28-/CD8 Subset of Lymphocytes May Escape Co-Stimulation Blockage With Belatacept to Mediate Acute Cellular Rejection in Allograft Kidneys.

Abstract

Purpose. Belatacept (Bela), a CD28/B7 co-stimulation pathway inhibitor, used as an alternative to calcineurin inhibitor (CNI)-based immunosuppression after renal transplantation, is associated with a higher incidence of acute cellular rejection (ACR). Recent experimental data indicate that ACR in patients treated with Bela might be mediated by alloreactive CD8 memory effector T cells with low CD28 expression. Our study tested the hypotheses that in renal transplant biopsies with ACR in patients on Bela the proportions of CD4 and CD8 lymphocytes co-expressing CD28 are different from those treated with CNI. Methods. Frozen kidney biopsies with type 1 ACR from patients on Bela (n=14) or CNI (n=11) were co-stained with CD4/CD28 and CD8/CD28 double immunofluorescence (IF). To assess CD4/CD28 and CD8/CD28 co-expression, M1 and M2 Manders' Coefficients, which represent the fraction of CD4 or CD8 positivity overlapping with CD28 positivity (M1) and the fraction of CD28 positivity overlapping with CD4 or CD8 positivity (M2), were calculated from multiple representative 40x IF digital images taken from the renal cortex. The M1 and M2 Manders' Coefficients were compared between the Bela and CNI groups using Student's T-test. Results. The M1 Coefficient for CD4/CD28 was significantly higher (p=0.007) in the Bela group (0.55) than in the CNI group (0.3), consistent with a higher proportion of the CD28+/CD4 cells in the Bela group. Although the M2 Coefficient for CD8/CD28 was significantly higher (p=0.01) in the CNI group (0.55 in CNI vs. 0.3 in Bela), suggestive of a higher proportion of the CD28-/CD8 cell subset in the Bela group, the differences were statistically insignificant (p=0.09) for the M1 Coefficient between the two groups (0.22 in Bela vs. 0.08 in CNI). Conclusion. Differences in CD28 expression of graft infiltrating lymphocytes between the Bela and CNI groups might indicate pathogenic heterogeneity for ACR in the two groups. Bela-associated ACR might be mediated, at least in some cases, by CD28- memory effector T cells escaping co-stimulation blockage. Substantial differences in the CD28 expression between individual cases in both groups raise the possibility of CD28 as a candidate marker to identify various subsets of ACR.