Fig. 3. Dapl1 regulates the functionality of exhausted CD8 T cells.

a,b, Flow cytometry analysis of the frequency of Granzyme B- or IFN-γ-producing cells (a) and Ki67⁺ cells (b), gated on CD8⁺PD1⁺Tim3⁺ T cells in the tumor of B16-OVA-bearing wildtype (WT) or Dapl1 KO mice. n =5 per genotype. c,d, Flow cytometry analysis of the frequency of H2Db/GP_33–41 Tetramer⁺ CD8 T cells (c) and LCMV viral load (d) in the spleen of LCMV clone 13-infected wildtype and Dapl1 KO mice. n=5(c) or n=10 (d) per genotype. e,f, Flow cytometry analysis of the frequency of Ki67⁺ cells in gated H2Db/GP_33–41 tetramer⁺PD1⁺Tim3⁺ CD8 T cell population (e) and ICS detecting IFN-γ-, IL-2- or granzyme B-producing cells in the gated PD1⁺Tim3⁺ CD8 T cell population (f) derived from the spleen of wildtype or Dapl1 KO mice infected with LCMV clone 13 for 30 days. n =5 per genotype. g, Flow cytometry analysis of CFSE dilution to measure the proliferation of H2Db/GP_33–41 Tetramer⁺PD1⁺ CD8 T cells, stimulated in vitro with anti-CD3 plus anti-CD28 for 3 days. h,i, Tumor growth curve (h) and survival plot (i) of B16-OVA-bearing B6.SJL mice (CD45.1⁺) adoptively transferred (on day 7 after tumor injection) with in vitro exhausted wildtype or Dapl1 KO OT-I (CD45.2⁺) CD8 T cells (5×10⁵ cells/mouse). n =5 per genotype. j-m, Flow cytometry analysis of the frequency of donor OT-I CD8 T cells (CD45.2⁺) and host CD8 T cells (CD45.1⁺) (j) as well as donor (CD45.2⁺) PD1⁺Tim3⁺ cells (k), Ki67⁺ cells (l), and IFN-γ- or granzyme B-producing OT-I CD8 T cells (m) in gated tumor-infiltrating CD45.2⁺CD8⁺ T cells of the mice from h. n =5 per genotype. n,o, ICS analysis of IL-2-producing (n) or IFN-γ-producing (o) cells in human CD8 T cells transduced with a control (Ctrl) shRNA or a Dapl1-specific shRNA and subsequently cultured under exhaustion or unexhaustion conditions. Data are representative of three independent experiments. Summary data are shown as the mean ± s.d. with P values determined using a two-tailed unpaired Student’s t-test (a-f, j, l-o), two-sided log-rank Mantel–Cox tests (i) and two-way ANOVA with Bonferroni correction (h).