Objective:To determine whether Th17 cells induced after renal transplantation are viral antigen-specific or whether they are recruited to MCMV-infected allografts via antigen-independent, cytokine mediated pathways.

Methods:Murine renal transplantation was performed using Donor BALB/cJ and recipient C57BL/6J or IL-17A-GFP-reporter mice. Mice were infected with MCMVΔ157 at 10^6pfu intraperitoneally for D+R+ transplants. Uninfected mice were used for D-R- transplants. At post-transplant day 7, leucocytes from spleen and allografts were analyzed for polyfunctionality by ICS for T-helper(Th) cells expressing cytokines,TNF-α,IFN-γ and IL-17A. Intragraft level of Th cell associated cytokines were quantified using cytokine bead array.

Results:Compared to D-R-transplants, allografts from D+R+transplants had 1.75 fold higher frequency of total CD4+IL-17A+Th17 cells(1.88±0.40vs.3.29±1.17,p=0.0351) that expressed significantly higher frequencies of multifunctional cytokines including IL-17A+TNF-α+(1.16±0.22vs.2.18±0.88,p=0.0373), IL-17A+IFN-γ+(0.77±0.32vs.1.33±0.22,p=0.0136) and IL-17A+TNF-α+IFN-γ+(0.43±0.13vs.0.92±0.27,p=0.0064). D+R+ allografts had significantly higher quantities of IL-17A(9378±2630vs.5334±4403pg/g,p=0.0276) compared to D-R- allografts. In D+R+ transplants, allografts had significantly higher frequencies of CMV-specific Th17 cells compared to spleen(0.19±0.04%vs.0.002±0.002%,p=0.0002), as well as a higher ratio of CMV specific to total Th17 cells(graft,5.88±0.86%vs.Spleen,0.39±0.30,p<0.0001). The CMV specific Th17 cells from allografts predominantly expressed either IL17A alone or IL17A and IFN-γ combined, in contrast to the Th17 cells not responding to CMV peptides, which also expressed TNF-α with IL17A+/-IFN-γ. CMV specific Th17 cells in allografts more often co-expressed IFN-γ compared to those in spleens. Although not reaching statistical significance, the intragraft level of Th17 differentiating cytokines, IL-6 and IL-23 were higher in D+R+ as compared to D-R- allografts.

Conclusion:In this murine renal transplant model, Th17 cells are differentially recruited to MCMV-infected allografts compared to CMV-uninfected allografts, but CMV-specific Th17 cells only comprised a small percentage of total Th17 cells. CMV-specific Th17 cells differed from those directed against nonviral antigens, by lacking TNF-α co-expression. Further studies are needed to understand mechanisms by which CMV induces viral antigen-dependent and -independent Th17 cell recruitment to infected allografts.