Fig. 3: Level of CD28 expression impacts Tpex self-renewal and differentiation.

(A) Experimental layout: control Cd28^fl/fl CreERT2^neg, heterozygous Cd28^fl/wt CreERT2⁺ and homozygous Cd28^fl/fl CreERT2⁺ mice with established life-long LCMV chronic infection received tamoxifen treatment. 15–21 days post tamoxifen, CD28^hi (black), CD28^int (blue) and CD28^neg (red) CD45.2 Tpex were sorted, CellTrace Violet (CTV)-labeled and transferred into infection matched CD45.1 recipients. Analysis was performed 4–5 weeks post-transfer. (B) Representative flow cytometry plots showing the frequency of CD45.2⁺ donor cells among CD8 T cells in spleen (left) and lung (right) of CD45.1 recipient mice. (C) Total number of CD45.2⁺ donor CD8 T cells recovered in spleen and (D) lung. (E) Representative CD39 and TCF-1 expression on donor CD45.2⁺ cells in spleen. (F) Frequency and (G) total number of differentiated donor CD39⁺ Tex in spleen. (H-I) Frequency of divided cells (CTV^low) among TCF-1⁺ CD45.2⁺ T cells in spleen. (J) Total number of donor TCF-1⁺ Tpex in spleen. (K) Frequency of CD39⁺ differentiated cells among divided (CTV^low) transferred cells in spleen. Data in (B-K) are representative of 4 independent experiments with two to five mice per group. Data in (C-D, F-G and I-K) show combined data from all experiments. Symbols represent individual mice, bars show mean value of all animals analyzed and error bars indicate SEM. Significance was determined using (C-D, F-G and I-J) Kruskall-Wallis with Dunn’s correction for multiple comparisons, (K) Mann-Whitney. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.