Supplementary Figure 3: iNKT cell and T cell phenotypes, plus leukocyte repopulation following neutrophil depletion during inflammation.

(a) Flow cytometry measure of percentage of CD69⁺ CD1d-tet⁺ iNKT cells (left panel) and MFI level on CD1d-tet⁺ iNKT cells (right panel) 4 and 12 hrs after IL-18 or vehicle administration to wild type (C57BL/6) mice in vivo. (b) Frequency of splenic γδT cells in vehicle or IL-18 injected mice at day 12 as assessed by flow cytometry. (c) Flow cytometry plots displaying representative neutrophil populations in mouse spleen (top panels) or blood (bottom panels) on day 12 following treatments indicated. Depletion antibody (anti-Ly6G- clone 1A8) administered on days 0 and 4. (d) Summary of flow cytometry data shown in (c), displaying kinetics of neutrophil numbers in spleen (upper panel) and blood (lower panel) in mice depleted at days 0 and 4. (e) Flow cytometry determination of number of splenic iNKT cells following treatment as indicated (depletion as in c). (f) ELISA measure of serum BAFF in mice treated as indicated. Data expressed as % of BAFF detected in vehicle treated mice. (g) Cytometric bead array kinetic measure of serum concentrations of IFN–γ from IL-18 or vehicle treated wild-type (C57BL/6) mice in the presence or absence of neutrophil depleting antibody (1A8) given days 0 and 4. (h) Intracellular flow cytometry data displaying MFI values for T-bet expression by iNKT cells in vehicle or IL-18 injected mice on day 12. (i) Intracellular flow cytometry data displaying MFI values for CD4⁺ and CD8⁺ T cell expression of the nuclear transcription factor GATA3 on day 12 after injection with IL-18 or vehicle. (j) Flow cytometry measure of FasL expression for conventional CD1d-tet^– T cells after IL-18 injection, normalized to levels in cells from vehicle treated mice. *P < 0.05 (two-tailed unpaired Student’s t-test [(a, n = 3 per group), (b, d; n = 4 per group), (e; n = 8 mice per group), (h; n = 5 mice per group), (i; n = 5 mice per group), (j; 14 mice per group)] or [1way ANOVA with Tukey correction (f; n = 8 per group), (g; n = 5 per group)]. Data are representative of one (a, g), two (h, i), or pooled from two (b, e, j), three (f), or four experiments (d). Box height or symbol center indicates group mean + s.e.m.