Extended Data Fig. 5: SATB1 expression is repressed by TGF-β and limits differentiation of short-lived effect CD8⁺ T cells.

(a) Histogram and quantification of SATB1 expression in wild-type CD8⁺ T cells that were stimulated in vitro in the presence or absence of titrated doses of TGF-β (n = 8). (b-d) Mixed bone marrow chimeric mice containing TGF-βR2 deficient and control CD8⁺ T cells were infected with LCMV-Docile. Spleens were analyzed on day 21 p.i. by flow cytometry. (b) Schematic of experimental set up. (c) SATB1 expression in TGF-βR2 deficient and control Gp33-specific CD8⁺ T cells (n = 10). (d) SATB1 expression in TGF-βR2 deficient and control Gp33-specific CD8⁺ T cell subsets (n = 10). (e,f) Naïve CD45.1⁺ P14 cells were adoptively transferred into naïve wildtype CD45.2⁺ mice and infected with LCMV-Armstrong. Spleens were harvested on days 7 or 28 p.i. and analyzed using flow cytometry. (e) Quantification showing the expression of SATB1 in MPC and SLEC cells compared to naïve CD8⁺ T cells and B cells (n = 10). (f) Histograms and quantification showing the expression of SATB1 in TCM and TEM cells compared to naïve CD8⁺ T cells and B cells (n = 9). (g-n) Naïve CD45.2⁺ SATB1-deficient P14 cells (Satb1^flex/flex/Cd8^Cre, SATB1-KO) or control cells (Satb1^flex/flex) P14 cells were adoptively transferred into naïve wildtype CD45.1⁺ mice and subsequently infected with LCMV-Armstrong. Mice were analyzed on day 7 p.i. (g) Schematic of experimental set up. (h,i) Flow cytometry plots (h) and quantification (i) of SATB1-KO and control P14 frequencies and numbers per spleen (n = 10). (j) Flow cytometry plots and quantification showing TIM-3 and PD-1 expression in SATB1-KO and control splenic P14 cells (n = 10). (k) Quantification of SATB1-KO and control SLEC and MPC numbers per spleen (n = 10). (l) Flow cytometry plots of IFN-γ and TNF production from SATB1-KO or control SLEC and MPC cells after incubation with Gp33 peptide (n = 10). (m) Quantification of frequencies of IFN-γ and TNF producing SATB1-KO and control SLEC and MPC (n = 10). (n) Quantification of IFN-γ and TNF expression in SATB1-KO and control SLEC and MPC (n = 10). Flow cytometry plots are representative. Dots in graphs represent biological replicates; horizontal lines and error bars of graphs indicate means and SD respectively (a). Data are pooled from at least two experiments. Dots in graphs represent individual mice (c-n); horizontal lines and error bars of bar graphs indicate means ± SEM, respectively. P values are from unpaired, two-way ANOVA (e,f), or two-tailed paired student’s t-tests (c,d). P values are from unpaired student’s t test (a,i-n).