Neuropilin-1 is Required for the Suppressive Function of Human Regulatory T Cells

Regulatory T cells (Tregs), lymphocytes that suppress immunological reactions, are of great interest for our comprehension of homeostasis and regulation in the immune system and as a therapeutic target in the treatment of both immune-mediated pathologies and reactivation of the immune response in patients with cancer. Understanding the molecular mechanisms by which these cells are regulated in respone to their environment will help to inform clinical strategies targeting Tregs. We hypothesised that Neuropilin-1, a transmembrane co-receptor for ligands of the semaphorin and growth factor families, promotes the suppressive function of human Tregs. Utilising in vitro lentivirus-mediated transduction with shRNA to knock down neuropilin-1 in primary human Tregs, we demonstrated that neuropilin-1 knockdown Tregs were severely impaired in their capacity to suppress cell proliferation in vitro and in their ability to prolong allograft survival in a humanised mouse model of transplantation. While neuropilin-1-KD Tregs exhibited no defects in survival, proliferation and activation upon stimulation in vitro, we hypothesised that loss of NRP1 expression would alter the global gene expression profile of human Tregs, revealing a NRP1-dependent Treg-associated transcriptional signature. All samples are human T CD4+ T lymphocytes (FACS-sorted from peripheral blood), transduced with lentivirus and expanded in vitro for 16days. Either regulatory T cells (Tregs) (CD4+CD25+CD127lo FACS-sorted or effector T cells (CD4+CD25-) Either experimental (transduced with shRNA to NRP1 (neuropilin-1 gene) to yield NRP1 knockdown) or control (transduced with scrambled control (pLKO1)) Either stimulated (with anti-CD3 anti-CD28-coated beads for 72hrs) or unstimulated (cultured for 72hrs without beads) Groups: A. human Tregs, unstimulated, control vector pLKO1 (N=5 donors) - control for (B) B. human Tregs, unstimulated, NRP1-KD (N= 5 donors) C. human Tregs, stimulated, control vector pLKO1 (N=4 donors) = control for (C) D. human Tregs, stimulated, NRP1-KD (N= 4 donors) E. human Teffs, unstimulated, control vector pLKO1 (N=3 donors) = control for (F) F. human Teffs, unstimulated, NRP1-KD (N=3 donors) Samples derived from the same blood donor are paired between groups A and B, C and D, and E and F