Fig 2: Lymph nodes draining the ear skin of naïve C57BL/6 and BALB/c mice contain only a small population of ILC2. Flow cytometry of lymphomononuclear cells isolated from C57BL/6 (A) and BALB/c 4get mice (B). Single cells were pre-gated on viability and CD45 expression; eosinophils staining positively for SiglecF were excluded. ILC2 were defined as lineage marker-negative, CD90.2+ cells. For further characterization, the surface markers CD127, Sca-1, ICOS (CD278) and PD1 were used. Data are representative of 3 to 4 experiments with 3 to 5 mice pooled per group. Lineage-mix included antibodies directed against CD2, CD3, CD5, CD11b, CD11c, CD19, FceR1a, Ly6G, Nkp46 and Ter119. The numbers within the histograms indicate the mean fluorescence intensity of the respective surface markers (A,B).

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: Non-classical CD8+ T Cells Participate during Acute MCMV Infection in KbDb-/- Mice(A) Representative staining of CD8+ T cells in the spleen and liver of KbDb-/- mice on day 0 and day 7 post-MCMV infection.(B) Frequency (black) and number (gray) of CD8+ T cells in the spleen and liver of KbDb-/- mice on indicated days post-MCMV infection (n = 9). Numbers indicate fold change of cell number compared to day zero.(C) Frequency of CD8+ TEFF cells (KLRG1+CD127-) in the spleen (—) and liver (- -) of KbDb-/- mice on indicated days post-MCMV infection (n = 9). Data are pooled from three independent experiments and represent mean ± SEM.See also Figures S1 and S2.

Fig 5: Detection of ILC2 in the ear skin of naïve C57BL/6 and BALB/c mice. Flow cytometry of ear skin cells from naïve 4get-C57BL/6 (A) and 4get-BALB/c mice (B). Lymphomononuclear cells were isolated from ear dermis and epidermis by enzymatic and mechanical dissociation. Single cells were pre-gated on viability and CD45 expression; eosinophils staining positively for SiglecF were excluded. ILC2 were defined as lineage marker-negative, CD90.2+ cells. For further characterization, the surface markers CD127, Sca-1, ICOS (CD278), PD1 and ST2 were used. Data are representative of 3 to 4 experiments (A,B) or 2 experiments (C) with 3 to 5 mice pooled per group. The lineage marker-mix included antibodies directed against CD2, CD3, CD5, CD11b, CD11c, CD19, FceR1a, Ly6G, NKp46 and Ter119. The numbers within the histograms indicate the mean fluorescence intensity of the respective surface markers (A–C).