*Purpose: Cytomegalovirus infection in transplant recipients has been associated with renal allograft rejection and graft failure. We have previously shown that murine CMV (MCMV) infected allografts have greater infiltration of T helper 17(Th17) cells compared to uninfected transplants and are associated with more severe allograft injury. We hypothesized that only a fraction of these Th17 cells are CMV specific and are phenotypically different than the rest of the Th17 cells that infiltrate the allograft via alternate antigen independent mechanism.

*Methods: Murine renal transplantation was performed between Donor BALB/cJ and recipient C57BL/6J/ OT-II/ IL-17A-GFP reporter mice (N=3-5 mice/group). The mice were infected with MCMVΔ157 at 10⁶ pfu intraperitoneally for D+R+ transplants. At post-transplant day 7, leucocytes from allografts were stimulated with PMA-Ionomycin or MCMV peptide pool and were analyzed for polyfunctionality for CD4+ cells expressing cytokines (TNFα, IFNγ and IL17A) and chemokine receptors (CCR6, CCR4 and CXCR3). Intragraft level of Th cell associated cytokines were quantified using cytokine bead array.

*Results: Compared to D-R-, allografts from D+R+ transplants had 1.75-fold higher frequency of CD4⁺IL17A⁺Th17 cells that co-expressed higher frequencies of IL17A⁺IFNγ⁺(p=0.0166) and IL17A⁺TNFα⁺IFNγ⁺(p=0.0075). CMV specific Th17 cells in the D+R+ allografts constituted an average of 5.47±0.96% of the total Th17 cells and predominantly expressed IL17A⁺IFNγ⁺. The CMV infected allografts also had 18.9-fold higher infiltration of antigen non-specific (OVA tetramer+) CD4+cells and Th17 cells (p=0.0009) that more often co-expressed IL17A and TNFα. The intragraft level of Th17 differentiating cytokines, IL6, IL1β and IL23 showed a higher trend in D+R+ group, compared to D-R- group. D+R+ allografts had greater proportion of Th17 cells expressing CCR6 (p=0.0235) or CXCR3 (p= 0.0235) alone or in combination (p= 0.0235), compared with D-R- allografts.

*Conclusions: Cytomegalovirus infection induces the infiltration of CMV specific IL17-A⁺IFN-γ⁺ as well as antigen non-specific IL17-A⁺TNF-α⁺ polyfunctional Th17 cells into the allografts. The allograft microenvironment favors the cytokine mediated differentiation of naïve T cells to polyfunctional Th17 cells. Additionally, recruitment of Th17 cells in CMV infected allografts are mediated by CCR6 and CXCR3 during allograft rejection. These data suggest that multifunctional inflammatory cytokine expressing Th17 cells infiltrate MCMV infected allografts by both antigen- dependent and -independent mechanisms in CMV infected allografts. Therefore, blockage of Th17 recruitment into the allograft can be good target to prevent Th17 mediated allograft injury in clinical solid organ transplantation.

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