Fig 2: Gating strategy for analysis of tumor-infiltrating CD8+ T cells(A) Single cell gating.(B) Forward and sideward scatter gating to identify lymphoid cells.(C) Live CD45+ cell gating.(D) CD8+ cell gating.(E) CD44+ cell gating.(F) PD-1+ cell gating.(G) Identification of SLAMF6+TIM3- stem-like and SLAMF6-TIM3+ terminally exhausted CD8+ T cells.(H) FMO SLAMF6 control.(I) FMO TIM3 control.

Fig 3: UTX demethylase activity is not required for effector CD8+ T cell function during chronic infectionWT, UTX-KI (catalytic dead knockin), and UTX-KO P14+CD8+ T cells were introduced into the same recipients, followed by CD4 depletion and infection with LCMV-A22. At day 8 post-infection, splenic donor P14T cells were analyzed for surface marker expression, transcription factor levels, and cytokine expression.(A) Illustration of the approach.(B) The three donor P14 T cell populations and host CD8+ T cells were identified by surface expression Ly5a, Ly5b, and Thy1.1.(C) Frequency of each donor P14 group among all donor CD8+Ly5a+ P14 T cells in spleen. Lines connect pairs of WT, UTX-KI, and UTX-KO P14T cells present in each recipient.(D) Histogram plots and gMFI for the transcription factors T-bet, Eomes, Tox, Tcf1, and Bcl2.(E and F) The fraction of donor P14 T cells producing granzyme (E) and the gMFI of granzyme expression among granzyme-positive cells (F).(G and H) The fraction of donor cells making IFN? (G) and the gMFI among IFN?-positive cells (H).(I) The distribution of IFN?+ (left) and IFN?- (right) donor cell populations after re-stimulation with GP33–41 peptide in an ICCS assay. The left graph shows the distribution of donor cells among the IFN?+ cells; the right graphs shows the distribution of donor cells that failed to make IFN?.(J) The histograms show several activation and inhibitory receptors expressed by IFN?+ donor cells.(K) The surface expression of Ly108 and CD69 on each donor cell population was used to identify Texprog1, Texprog2, Texint, and Texterm subsets.(L) Distribution of the four developmental stages of exhaustion for donor cell populations. Each circle represents 1% of the total population.Data show one experiment with five recipient mice. Samples from the five recipient mice were concatenated to generate histograms. Significance was determined by a paired Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).See also Figure S7.

Fig 4: Analysis of TCF-1 and TOX expression in tumor-infiltrating exhausted CD8+ T cells(A) Histograms showing TCF-1 expression in SLAMF6+TIM3- stem-like (SLAMF6+) and SLAMF6-TIM3+ terminally exhausted (TIM3+) CD8+ T cells.(B) Histograms showing TOX expression in SLAMF6+TIM3- stem-like (SLAMF6+) and SLAMF6-TIM3+ terminally exhausted (TIM3+) CD8+ T cells.

Fig 5: Impact of immune checkpoint blockade on tumor-infiltrating CD8+ T cells(A–H) Representative images of flow cytometry analysis of tumor-infiltrating CD8+ T cells (A and E), PD-1+ CD8+ T cells (B and F), SLAMF6+TIM3- stem-like and SLAMF6-TIM3+ terminally exhausted CD8+ T cells (C and G) and Ki67high SLAMF6+TIM3- stem-like CD8+ T cells (D and H) in the MCAprog tumor model following treatment with Isotype control (Iso) or combined anti-PD-1 and anti-CTLA-4 antibodies (ICB).