Fig 2: Cardiolipin Is Necessary for In Vivo and In Vitro CD8+ T Cell Responses(A) Schematic of the cardiolipin synthesis pathway.(B) PTPMT1 expression in CD8+ T cells from PTPMT1 WT (Ptpmt1flox/floxCD4-Cre-/-) and PTPMT1-?T (Ptpmt1flox/floxCD4-Cre+/-) mice.(C) Cardiolipin content in freshly isolated Ptpmt1 WT and Ptpmt1-/- CD8+ T cells.(D) Percentage of CD8+ T cells in spleens from PTPMT1 WT and PTPMT1-?T mice. Dots represent individual mice.(E) PTPMT1 WT and PTPMT1-?T mice infected i.v. with 1 × 106 colony-forming units (CFU) LmOVA ?Acta and challenged i.v. with 5 × 107 CFU LmOVA 21 days after primary infection. Blood analyzed at indicated time.(F) Percentage of CD8+ T cells in PTPMT1 WT and PTPMT1-?T mice infected as in (E). Dots represent individual mice.(G) Percentage of CD8+ Tet+ T cells in PTPMT1 WT and PTPMT1-?T mice infected as in (E). Dots represent individual mice.(H) Bacterial burden shown as CFU per µg of liver of PTPMT1 WT and PTPMT1-?T mice 7 days post-LmOVA ?Acta challenge. Dots represent individual mice.(I) TN (CD62LhiCD44lo), TE (CD62LloCD44hi), and TM (CD62LhiCD44hi) CD8+ T cells from PTPMT1 WT and PTPMT1-?T mice 7 days after primary infection. Dots represent individual mice.(J) EEC, SLEC, MPEC, and DPEC CD8+ T cells from PTPMT1 WT and PTPMT1-?T mice 7 days after primary infection according to KLRG1 and CD127 expression. Dots represent individual mice.(K) OCR/ECAR ratio of WT and Ptpmt1-/- CD8+ T cells analyzed 7 days after secondary infection. Dots represent individual mice.(L) Ptpmt1 WT and Ptpmt1-/- CD8+ T cells stimulated with PMA/Iono for 4 h. Also shown is the percentage of CD8+ T cells producing IFN-? and IFN-? MFI of cytokine-producing cells.(M) Cytoplasmic calcium levels at baseline and after aCD3 crosslinking via IgG, tapsigargin (TG), and ionomycin (iono) in Ptpmt1 WT and Ptpmt1-/- CD8+ T cells. Representative of 3 experiments.(N) CD5, CD44, CD25, and CD69 expression in Ptpmt1 WT and Ptpmt1-/- CD8+ T cells 24 h after activation with aCD3/aCD28 + IL-2.(O) Survival of Ptpmt1 WT and Ptpmt1-/- CD8+ T cells 24 h after activation.(P) Cell cycle analysis assessed by FxCycle + Ki67 staining of Ptpmt1 WT and Ptpmt1-/- CD8+ T cells 24 h and 48 h after activation with aCD3/aCD28 + IL-2.(Q) Active proliferation assessed by Ki67 expression in Ptpmt1 WT and Ptpmt1-/- CD8+ T cells 24 h and 48 h after activation with aCD3/aCD28 + IL-2.(R) Cell proliferation assessed by CTV dilution of Ptpmt1 WT and Ptpmt1-/- CD8+ T cells 72 h after activation with aCD3/aCD28 + IL-2. Histogram representative of 4 independent experiments.Data shown as mean ± SEM of =3 independent experiments unless indicated. Statistical comparisons for 2 groups calculated by unpaired two-tailed Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001.See also Figure S2.

Fig 3: Phenotype of artARENA-induced CTLs and their dependence on IL-33-ST2 alarmin signaling(A–F) We immunized BALB/c mice with artPICV-P1A and artLCMV-P1A in homologous or heterologous prime-boost vaccination i.v. on day 0 and day 27. On day 34, we analyzed P1A-Tet-binding and CD62L expression by splenic CD8+ T cells (A; gated on CD8+B220– lymphocytes). Unimmunized control mice are shown for comparison in (A) only. Numbers in (A) indicate the percentage of cells in the respective quadrant. Total P1A-Tet+ CTLs (B), P1A-specific effector/effector memory CTLs (CD62Llo; C), and P1A-specific central memory CTLs (CD62Lhi; D) were enumerated in the spleen on day 34. In both subsets of P1A-specific CTLs, CD62Lhi and CD62Llo, we determined the surface expression of KLRG1, CX3CR1, CD27, CD43, and CD127 as well as the master transcription factors Tcf-1, Tbet, and Eomes (E). Total numbers of marker-expressing P1A-specific CTLs were enumerated in (F). (A) shows representative FACS plots from individual mice. Symbols in (B)–(D) and (F) represent individual mice, and bars in (B)–(D) and (F) indicate the mean ± SD. Numbers in (A) and (E) indicate the percentage of gated cells (mean ± SD) or the mean fluorescence intensity (MFI ± SD). Means were calculated from six mice per immunization group (A–F) or from three unimmunized controls (A). N = 2. **p < 0.01 by unpaired two-tailed Student’s t test.(G and H) We immunized ST2-/- and WT mice with artLCMV-E7E6, artPICV-E7E6 or arCAND-E7E6 i.v. Controls were left unimmunized (“no vaccine” in G). E7-tetramer-binding cells in blood were determined on day 7. Representative FACS plots are shown in (G); values indicate E7-Tet+CD8+ T cells as a percentage of lymphocytes. Symbols in (H) represent individual mice (n = 5 per group) with mean ± SD. N = 2. **p < 0.01 by two-way ANOVA with Sidak’s post-test.See also Figures S5 and S6.

Fig 4: ST2 suppression in the peritoneal lavage and lung during Heligmosomoides polygyrus infection.Mice were infected with 200 H. polygyrus L3 larvae, and at day seven post-infection peritoneal lavage (PL) and lung cells were stained for flow cytometry of ST2 on peritoneal lavage KLRG1+CD25+lineage–CD45+ ILC2s (A, B), lung ICOS+lineage–CD45+ ILC2s (C, D), or peritoneal lavage SSChilineage–CD45+ cells (E, F). Geometric mean fluorescence intensity of ST2 is shown in A, C and E, representative ST2 histograms on gated cells is shown in B, D and F. Data from a single experiment with five mice per group. Error bars show SEM.