Fig 2. Perforin deficiency in CD8⁺ T cells from sentinel nodes.

(A) The expression of granzyme B and perforin in CD8⁺ T cells of different tissues were phenotyped by flow cytometry. The co-expression pattern in CD8⁺ T cells was shown in dot plots and gated for distinguishing between double and single expression of granzyme B and perforin. The gate was based on isotype control and the frequency of granzyme B and perforin expression was counted out of CD8⁺ T cells. Dot plots showed a representative data from a patient underwent transurethral resection of the bladder (TUR-B) and cystectomy. (B) The frequency of granzyme B⁺/perforin⁺ CD8⁺ T cells from PBMC, SN, and tumor tissues was shown in graphs and was calculated out of CD8⁺ T cells (n = 27). (C) Same as in (B) but the analysis was done on granzyme B⁺/perforin^- CD8⁺ T cells. The data are means with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test. (D) The expression of gene responsible in encoding granzyme B (GZMB) and perforin (PRF1) in CD8⁺ T cells isolated from PBMC, SN, and tumor (n = 6). RT-qPCR was done to analyze the gene expression followed by quantification using 2^-ΔΔCt method. The fold change was calculated in regards of PBMC as control, with RPII gene used as the housekeeping gene. The data are the means of Log₂ of fold change (2^-ΔΔCt) with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test on each gene. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.