Fig 2: 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 3: T cells acquire CD80 and CD86 from neighboring cells.a CD80- and CD86-surface staining of Cd80-/-Cd86-/- P14 CD8+ T cells co-cultured with wild-type (WT) or Cd80-/-Cd86-/- antigen-loaded DCs. Gating: Thy1.2+ CD44+ T cells. b CD80 and CD86 staining of WT and Cd80-/-Cd86-/- P14 T cells on day 12 post LCMV-infection. Left: representative histograms of transferred T cells. Right: Proportion of CD80+ or CD86+ cells. LN denotes lymph nodes. c Z-projection (left) and focal plane (right) of membrane-Tomato P14 CD8+ T cells (blue) co-cultured for 48 h with CD80-TagGFP transgenic, antigen-loaded MEFs (yellow). Bottom right image solely depicts CD80 co-localizing with T cell membrane. Scale bar 10 µm. d Acquisition kinetics of CD80 and CD86 by P14 CD8+ T cells upon co-culture with CD80- or CD86-mScarlet-transgenic, antigen-expressing MEFs or their respective supernatants (SN). e Proportions of P14 CD8+ T cells acquiring CD80 or CD86 co-cultured with MEFs expressing native CD80- or CD86-mScarlet or mutants thereof (CD80 Y201A, CD86 Q35A) with or without antigen-loading. f Blockade of intercellular transfer of CD80 and CD86 upon addition of CTLA4-Fc to CD8+ T cells co-cultured with CD80- or CD86-mScarlet expressing MEFs. See also Figure S1 and Movie S1. Statistics: b: Pooled data from 4 independent experiments. Two-sided Wilcoxon matched-pairs signed rank test. p-values: CD80 spleen 0.0001 (n = 14 animals); CD80 iLN 0.0001 (n = 14 animals); CD80 lung 0.0020 (n = 10 animals); CD80 liver 0.0020 (n = 10 animals); CD80 blood 0.0078 (n = 8 animals); CD86 spleen 0.0001 (n = 14 animals); CD86 iLN 0.0001 (n = 14 animals); CD86 lung 0.0020 (n = 10 animals); CD86 liver 0.0020 (n = 10 animals); CD86 blood 0.0078 (n = 8 animals). e: Representative data from 1 of 3 independent experiments. Brown-Forsythe/Welch’s ANOVA with Dunnett’s T3 correction for multiple comparison. p-value: CD86 vs. gp33 + CD86: 3 × 10-5 (n = 3 biologically independent samples). f: Pooled data from 3 independent experiments. Two-sided Mann–Whitney test. p-values: CD80: 7 × 10-7 CD86: 1 × 10-11 (n = 12 biologically independent samples). Source data are provided as a Source data file.

Fig 4: Enforced CD38 expression did not impact the exhaustion differentiation of adoptively transferred OT-1 cellsThy1.1-Vector or Thy1.1-CD38 were ectopically expressed in Cd38-/- OT-1 cells by retrovirus infection.(A) Histogram of CD38 expression in Thy1.1+ subsets of indicated two groups.(B) Representative FACS plots of CD62L and CD44 expression in Vector and CD38 expressed Cd38-/- OT-1 cells (left). Percentage of CD62L+CD44+ Thy1.1+ cells in two groups (right). Mean ± SD (n = 3), Student’s t test, ns: not significant.(C) Flow cytometry analysis of ?-H2AX expression in indicated two groups.(D and E) Intracellular cytokines staining for TNFa, IFN?, and IL-2 in Vector or CD38-overexpressed OT-1 cells at day 3 postinfection (left). Values indicate the TNFa+IFN?+ or IL-2+ frequencies of two groups (right). Mean ± SD (n = 3), Student’s t test, ns: not significant.(F and G) The three groups of WT OT-1 cells with Thy1.1-Vector expression, Cd38-/- OT-1 cells with Thy1.1-Vector or Thy1.1-CD38 expression were adoptive transferred in B16-OVA tumor-bearing mice, respectively. Summary of CD62L+CD44+ Thy1.1+, or TNFa+IFN?+ Thy1.1+ frequencies of indicated transferred OT-1 cells in recipient mice spleen. Data are shown as Mean ± SD (n = 5), Student’s t test, ns: not significant.(H) Flow cytometry analysis of CD38 expression in indicated Thy1.1+ OT-1 TILs.(I) Representative FACS plots of PD-1 and TIM-3 expression in indicated OT-1 TILs (left). Values indicate the frequency of PD-1+TIM-3+ in OT-1 TILs (right). Data are shown as Mean ± SD (n = 5), Student’s t test, ns: not significant.(J) Left: representative contour plots of indicated OT-1 TILs producing cytokines TNFa, IFN?. Right: summary of the frequencies of indicated Thy1.1+ OT-1 TILs producing cytokines. Data are shown as Mean ± SD (n = 3), Student’s t test, ns: not significant.