Increasing evidence suggests that elicitation of donor–reactive T-cell immunity is regulated by pro-inflammatory neuropeptides. Substance P (SP) is the prototypic pro-inflammatory neuropeptide released by leukocytes and nerves that promotes innate and adaptive immunity by binding the Neurokinin-1 Receptor (NK1R) expressed by dendritic cells (DC) and T cells. Aim: we investigated the mechanisms by which mice lacking the NK1R exhibit impaired allo-DTH responses and delayed allograft rejection. Methods: Allo-DTH was assessed in wt or NK1R^KO B6 mice sensitized (footpad) with BALB/c bone marrow (BM) DC. SP was detected by RT qPCR and fluorescence microscopy. The stimulatory capacity of NK1R^KO DC and T-cell responses were assessed (i) in vitro: in CFSE-MLC, and (ii) in vivo: in CFSE-TCR tg 1H3.1 CD4 T cells [specific for (BALB/c) IE^Α52-68 -(B6) IAb ] transferred into wt or NK1R^KO B6 mice skin-sensitized of grafted with BALB/c skin 1 d later. Detection of translocation of transcription factors was done by SDS-PAGE and Western blot. Results: The cutaneous allo-DTH response was abrogated in NK1R^KO mice, with minimal skin infiltration of macrophages and T cells, compared to wt controls. NK1R^KO B6 mice also rejected BALB/c skin grafts with delayed tempo. Traffic studies indicated that this was not due to inability of skin NK1R^KO DC to migrate to the draining lymph nodes. However, NKR1^KO DC secreted significantly lower levels of IL-12p70 and higher amounts of IL-10 and TGF-Β1 and therefore were deficient at polarizing type-1 T cells in vitro and in vivo, the latter assessed in adoptively transferred CFSE-labeled 1H3.1 CD4 T cells. On the other hand, NK1R^KO T cells proliferated as efficiently as wt cells, but died by apoptosis due to their low IL-2RΑ expression, IL-2 secretion and Bcl-2 content. Activated NK1R^KO T cells exhibited reduced nuclear translocation of the IL-2 transcription factors NFAT1c and NFAT2c. Conclusions: After allo-sensitization, NK1R-signling via pro-inflammatory neuropeptides is critical for generation of type-1 polarizing DC and for nuclear translocation of NFAT1c and NFAT2c in T cells, the latter necessary for T-cell survival.

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