T cells lose costimulation dependence following primary activation and many phenotypes of terminally differentiated cells have been identified as contributing to costimulation blockade (CoB)-resistant rejection (RR). In particular, CD57+PD1- CD4 T cells are significantly increased in patients with renal failure, resistant to immunosuppression with belatacept, but remain susceptible to inhibition with tacrolimus. Study of these cells has been hampered by limited understanding of their growth requirements and the biology leading to their formation. We therefore sought to investigate signaling pathways that may contribute to their differentiation and expansion. Genomic analysis of sorted CD57+ compared to CD57- CD4 T cells revealed increased expression of genes highly associated with inflammation and allograft rejection signaling, and was used as a guide for subsequent in vitro manipulations. To determine what signals CD57+ CD4 T cells require for activation, peripheral blood was collected from patients with renal failure consented at Duke Transplant Center. Isolated PBMCs were labeled with VPD450 and sorted into populations based on expression of CD57 and PD1, then cultured in non-stimulated, anti-CD3, or anti-CD3 and anti-CD28 conditions in the presence of no drug, tacrolimus or belatacept for 96 hours as sorted cells alone, or with unlabeled bulk PBMCs. Cell culture supernatant was collected from each condition to analyze cytokine and chemokine production with a Human 25-Plex Luminex Panel, and cells were collected to assess proliferation and phenotypic changes by flow. When cultured with bulk PBMCs, CD57+PD1- CD4 T cells were CoB-resistant but controlled by tacrolimus. However, proliferative capacity was lost in the absence of PBMCs. Candidate factors contributed by PBMCs were investigated and inflammatory cytokines IL-1b, IL-6, IL-17, IFNg and TNFa, pro-survival and proliferation cytokines IL-2, IL-7 and IL-15, and chemokines CCL2, CCL5 and CXCL10, which recruit leukocytes to sites of inflammation, were increased in cell culture supernatants in the presence of belatacept, but not tacrolimus. Despite lack of proliferative capacity, polyfunctionality (dual IFNg and TNFa secretion) of CD57+PD1- CD4 T cells was maintained in the absence of bulk PBMCs. Taken together, these data suggest preformed CoB-resistant cells are dependent on interactions with less differentiated cells, particularly for their expansion, and provide additional insight into the aggressive but transient nature of CoBRR.

CITATION INFORMATION: Espinosa J., Adams B., Kearns J., Shaw F., Osborne R., Woody T., Ellis M., Cheeseman J., Kirk A. Activation Requirements of Costimulation Blockade-Resistant CD4 T Cells Am J Transplant. 2017;17 (suppl 3).

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