Extended Data Fig. 9. Attenuated activation of NFATc2 and NFATc1 in exhausted CD8 T cells.

a,b Flow cytometry analysis of sorted PD1⁺Tim3⁺ exhausted (T_EX) and PD1⁻Tim3⁻ progenitor (T_Pro) CD8 TILs (a) and the expression of TCF1 (b) in PD1⁺Tim3⁺ exhausted (T_EX) and PD1⁻Tim3⁻ progenitor (T_Pro) CD8 cells derived from day 20tumor of B16-OVA tumor-bearing wildtype (WT) or Dapl1 KO (KO) mice. c-e, Flow cytometry analysis of nuclear NFATc2 (c) and NFATc1 (d,e) in wildtype and Dapl1 KO T_Pro CD8 TILs (c), wildtype T_EX and T_Pro CD8 TILs (d), or wildtype and Dapl1 KO T_EX CD8 TILs (e), either untreated (NT) or ionomycin-stimulated (Iono, 30 min). n =5 per genotype. f,g, Flow cytometry analysis of sorted PD1⁺Tim3⁺ exhausted (T_EX) and PD1⁻Tim3⁻ progenitor (T_Pro) CD8 T cells (f) and the expression of TCF1 (g) in PD1⁺Tim3⁺ exhausted (T_EX) and PD1⁻Tim3⁻ progenitor (T_Pro) CD8 cells from LCMV clone 13-infected wildtype or Dapl1 KO mice. h-j, Flow cytometryanalysis of nuclear NFATc2 (h) and NFATc1 (i,j) in wildtype and Dapl1 KO progenitor (T_Pro) CD8 T cells(h), wildtype T_EX and T_Pro cells (i), and wildtype and Dapl1 KO T_EX cells (j) from the spleen of LCMV clone 13-infected mice, either not treated (NT) or stimulated with ionomycin (Iono) for 30 min. n =5 per genotype. k,l, Gating strategies for sorting PD1⁺Tim3⁺ exhausted (T_EX) and PD1⁻Tim3⁻ progenitor (T_Pro) Pmel1 CD8 TILs from the tumor (k) of B16F10-implanted B6.SJL mice (CD45.1⁺) adoptively transferred with Dapl1 KO Pmel1 CD8 T cells (CD45.2⁺) transduced with either an empty vector or HA-Dapl1 expression vector, and flow cytometry analysis of nuclear NFATc1 in untreated (NT) or ionomycin-stimulated (Iono, 30 min) PD1⁺Tim3⁺ exhausted (T_EX) and PD1⁻Tim3⁻ progenitor (T_Pro) Pmel1 CD8 TILs (l). n =5 per genotype. m, Flow cytometry analysis of nuclear NFATc2 in in vitro generated exhausted human CD8 T cells transduced with a non-silencing control shRNA (Ctrl shRNA) or a Dapl1-specific shRNA, either not treated (NT) or stimulated for 30 min with ionomycin. n=5 biologically independent repeats. n-p, Flow cytometry analysis of nuclear NFATc2 (n) and nuclear NFATc1 (o,p) in PD1⁺Tim3⁺ CD8 T cells in the tumor of B16-OVA-bearing wildtype or Dapl1 KO mice injected with anti-Tim3 or IgG isotype control on days 7, 9, and 11. n=5 per genotype. Summary data are shown as the mean±s.d. with P values determined using a two-tailed unpaired Student’s t-test (c-e, h-j, l, m, o). ns, not significant.