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. 2021 Jun 24;12:650977. doi: 10.3389/fimmu.2021.650977

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Copyright © 2021 Klein, Witalisz-Siepracka, Gotthardt, Agerer, Locker, Grausenburger, Knab, Bergthaler and Sexl

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Cdk6^fl/fl CD4-Cre mice do not show a defect in thymic T cell development and splenic CD8+ T cell numbers, while in vitro expansion of CD8+ T cells is impaired. (A) Thymocytes were lysed from Cdk6 ^fl/fl and Cdk6 ^fl/fl CD4-Cre mice for western blot analysis of CDK6 protein levels. HSC70 served as a loading control. (B) Absolute numbers of thymocytes were analyzed in Cdk6 ^fl/fl and Cdk6 ^fl/fl CD4-Cre mice (n=4-7). (C) The frequencies of CD4/CD8 double-negative (DN), CD4/CD8 double-positive (DP), CD4 single-positive (CD4 SP) and CD8 single-positive (CD8 SP) thymocytes were analyzed by flow cytometry in Cdk6 ^fl/fl CD4-Cre mice and Cdk6 ^fl/fl controls (n=4-7). Representative dot plots are shown. Figure S2A shows the corresponding bar graph with mean ± SEM values. (D) Splenic CD8+ T cells from Cdk6 ^fl/fl and Cdk6 ^fl/fl CD4-Cre mice were cultured for six days and lysed for western blot analysis of CDK6 protein levels. HSC70 served as a loading control. (E, F) Percentage (E) and total numbers (F) of splenic CD3+CD8+ T cells were analyzed by flow cytometry in Cdk6 ^fl/fl and Cdk6 ^fl/fl CD4-Cre mice (n=7-9). (G) Splenic CD8+ T cells were isolated from Cdk6 ^fl/fl and Cdk6 ^fl/fl CD4-Cre mice, activated with αCD3/αCD28 for 48h and cultured in the presence of IL-2. Numbers of CD8+ T cells were analyzed every two days during the culture (n=4). (B, E, F) Bar graphs represent mean ± SEM, pooled from 2-3 independent experiments. (G) Symbols in growth curves represent mean ± SEM, pooled from two independent experiments; ***p < 0.001 (for comparison on d6), two-way ANOVA. ns, not significant.