Figure 5:

Cytokine production by CCR6+CD4+ Th17 cells highlighting their pro-inflammatory phenotype in severe refractory allograft rejection. Transcriptome activation of cytokine, chemokine, and transcription factor Th17 related genes in rejection patients at the time of rejection diagnosis as defined by RT-PCR. A) LP CD4+ T cells isolated from non-rejector control and non-responder rejection graft biopsy samples during active rejection on treatment, stimulated with PMA/ionomycin and subsequently analyzed for intracellular IL-17, TNF-α, and CCR6 expression. Representative flow plots showing CD4+CD3+ gated populations exhibited intracellular cytokines IL-17 and TNF-α predominantly in the CCR6+ T cell populations; followed by violin plots of individual values representing comparative analysis of cytokine-expressing CCR6+CD4+ T cell subpopulations versus total cytokine-expressing CD4+ T cells in non-rejector control versus non-responder rejection patients. Statistics performed using Wilcoxon rank testing. Sample size for IL-17 producing CCR6+CD4+ group is control n=10, rejection n=11; for IL-17 CD4+ group control n=10, rejection n=11; for TNF-α CCR6+CD4+ control n=7, rejection n=11; TNF-α CD4+ control n=7, rejection n=11. B and C) Gene expression analysis on intestinal graft biopsies from non-rejector controls versus pre-rejection controls versus rejection patients at rejection diagnosis. Heat map visualization of pathway-focused panel for Human Th17 Response depicting normalized fold change 2^−ΔΔCt of genes exhibiting significant difference in expression on a magnitude of log₂ scale. All cycle threshold values normalized to GAPDH values and log transformed. Sample size for heatmap is control n=13, pre-rejection control n=13, rejection n=13.