Fig. 3: Penk-deficient chimeric mice display virtually no functional T cell alteration.

The relative frequency of CD4⁺ T lymphocyte subsets in colonic lamina propria and spleen from penk^+/+ (white circles and white histogram) and penk^−/− (black circles and gray histogram) chimeric mice was estimated by cytofluorometry. a Gating strategy for flow cytometry data analysis. Living CD45-expressing cells gated on TCRβ chain⁺ CD4⁺ T lymphocytes were immunophenotyped as FoxP3⁺ regulatory T cells and FoxP3^- conventional T lymphocytes. Activated conventional CD4⁺ T lymphocytes were then distinguished based on the expression of CD44 and/or CD69 cell surface markers. b Frequency of conventional CD4⁺ T lymphocytes, activated conventional CD4⁺ T lymphocytes and regulatory T cells among living CD45⁺ TCRβ-expressing cells within the lamina propria (n = 7). c IFNγ (Th1), IL-17 (Th17), and IL-13 (Th2) cytokine expression levels expressed as geometric mean of fluorescence intensity (gMFI) in CD4⁺ T lymphocytes from colonic lamina propria (n = 6). d Frequency of CD4⁺ T lymphocytes, conventional CD4⁺ T lymphocytes, activated conventional CD4⁺ T lymphocytes and regulatory T cells among living CD45⁺ TCRβ-expressing cells within the spleen (n = 8). Each symbol represents one mouse. Statistical analysis was performed using Mann-Whitney U test. e Spleen cells from chimeric penk^+/+ and penk^−/− mice (n = 4) were isolated by cell-sorting as myeloid cells, B lymphocytes and CD4 and CD8 T lymphocytes based on the expression of the cell surface antigens CD45/CD11b, B220, TCRβ chain/CD4 and TCRβ chain/CD8 respectively (left panels). Penk mRNA expression was quantified by real-time PCR. mRNA content was normalized to the HPRT mRNA and quantified relative to standard mouse brain cDNA (n = 4). Each symbol represents one mouse. Statistical analysis was performed using one-way ANOVA followed by Sidak’s multiple comparison test; ***p < 0.001, ****p < 0.0001.