*Purpose: Our group has shown that renal transplant patients with acute rejection have elevated blood mRNA RORγt: FOXP3 ratio compared to patients with stable graft function (2018 ATC abstract D186). We hypothesized that this blood RORγt: FOXP3 ratio may reflect an imbalance in T helper 17 (Th17) and regulatory T cells (Tregs) within acutely rejecting allografts. To study this, we utilized a rejecting murine renal transplant model to identify the Th17: Treg ratio within allografts and to characterize the phenotype of graft-infiltrating Th17 cells.

*Methods: Allogeneic (BALB/c to C57BL/6) and syngeneic (C57BL/6 to C57BL/6) murine renal transplantation was performed with cyclosporine immunosuppression (n=5-7 mice/group). Allografts and organs were analyzed at post-op days (POD) 7 and 14 for T helper cell frequencies and phenotypes by intracellular cytokine staining. T helper cell-associated cytokines and chemokines were quantitated in organs using cytokine bead array. Groups (mean±SD) were compared using Student’s t-test or ANOVA.

*Results: At POD7, allogeneic grafts had greater frequency of IL-17A+ CD4+ (Th17) cells compared to syngeneic grafts (0.59±0.25 vs. 0.04±0.10, p= 0.002). Compared to syngeneic grafts, Th17 cells from allografts expressed multiple inflammatory cytokines including IL-17A+/TNF-α+ (0.69±0.31 vs. 0.20±0.34, p= 0.049), IL-17A+/IFN-γ+ (0.29±0.14 vs. 0.00, p= 0.002) and IL-17A+/TNF-α+/IFN-γ+ (0.49±0.25 vs. 0.00, p= 0.002) cells, with sustained polyfunctional cytokine expression by intragraft Th17 cells observed at POD 14. In contrast, Th17 cells in spleens, lymph nodes, blood and livers of allogeneic transplant recipients had predominantly IL-17A+ or IL-17A+/TNF-α+ Th17 cells at the same timepoints. Allografts had significantly higher quantities of Th17 differentiating cytokines (IL-1β and IL-6) and Th17 cell recruiting chemokines (CCL4, CCL20 and CXCL10) compared to spleens. The ratio of Th17 cells to FOXP3+ Tregs was significantly higher in allografts compared to spleens at both 7 and 14 days (p=0.002 and p=0.001, respectively), as was the IL-17A: IL-10 cytokine ratio (2.1 ± 2.1 vs. 0.19 ± 0.11 p= 0.025).

*Conclusions: Acutely rejecting allografts had higher frequency of polyfunctional Th17 cells compared to syngeneic grafts, spleens and other organs, which persisted through post-transplant day 14 and were associated with Th17 cell-differentiating and -recruiting cytokines/chemokines. The elevated intragraft Th17: Treg and IL-17A: IL-10 cytokine ratios in allografts were consistent with our prior observations in the blood of kidney transplant patients. We conclude that polyfunctional inflammatory cytokine-expressing Th17 cells are present in rejecting allografts and may have a detectable signature in blood during clinical acute rejection.

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