Thrombospondin-1 is A Key Mediator of Mtor Inhibitor-Associated Induction of T and B Lymphocyte Regulatory Phenotype in Kidney Transplanted Patients.

Kidney transplantation (KT) tolerance is associated with increasing levels of circulating T (CD4+CD25+Foxp3+) and B (CD19+Tim-1+) regulatory cells (Tregs/Bregs): mTOR inhibitors (mTORi) increase Tregs in KT. Thrombospondin-1 (TSP-1) is a glycoprotein able to induce a Treg phenotype after CD47 binding. The aim of this study was to investigate the role of TSP-1 in mTORi-associated induction of Treg/Breg phenotype. We enrolled 60 KT patients with stable graft function: 20 in therapy with tacrolimus (TAC), 20 with sirolimus (SLR) and 20 with everolimus (EVE). We studied by FACS the percentage of circulating Tregs, Bregs, T memory (Tmem: CD4+CD45RO+) and B memory (Bmem: CD19+CD27+) cells. Plasma and urine ELISA for TSP-1 was performed. In vitro, TSP-1 mRNA/protein expression was evaluated in T/B cells or in human kidney tubular epithelial cells (TEC) incubated with therapeutic doses of TAC, SLR or EVE. KT patients treated with mTORi showed increased levels of circulating Tregs and Bregs in comparison to TAC (p<0.05). Higher levels of Tregs/Bregs were found in EVE patients. No differences in percentage of circulating Tmem and Bmem between mTORi and TAC groups were observed. Higher levels of plasma and urine TSP-1 were found in mTORi-treated patients in respect to TAC. TSP-1 levels correlated with percentage of circulating Tregs. In vitro, we found that SLR and EVE but not TAC induced an increased mRNA/protein expression of TSP-1 in T and B cells. In addition, TSP-1 induced a regulatory phenotype in both T and B cells without influencing Tmem and Bmem differentiation. TSP-1-mediated Treg/Breg induction was significantly decreased by using a CD47 blocking antibody. A similar effect was observed incubating T or B cells with supernatants released by TEC cultured with mTORi but not with TAC. mTORi-associated Treg/Breg induction was significantly decreased using supernatants of TEC previously engineered to knock-down TSP-1 by siRNA. TSP-1 produced by lymphocytes in an autocrine manner or released by intragraft resident cells such as TEC induce T and B cells to acquire a regulatory phenotype without affecting the percentage of memory cells. TSP-1 may represent a new biomarker and mediator of mTORi-associated Treg/Breg increase and tolerance in KT.