βGL1-22/LGL1–specific type II murine NKT cells display NKT_FH phenotype and provide B-cell help with the induction of GC B cells and IgG and lipid-reactive antibodies in C57BL/6 and Jα18^−/− mice. (A-B) Spleen cells from WT (A, C57BL/6) mice or (B, Jα18^−/− mice) obtained 7 days after immunization with α-GalCer, βGL1-22, LGL1, or vehicle alone were stained to assess CXCR5, PD-1, ICOS, Bcl-6, and IL-21 expression among CD4⁺ tetrameter-positive NKT cells. Numbers in plots indicate percentage of the cells included in the gates. The histogram on the right shows differences in Bcl-6, Foxp3, and ICOS expression as reflected by MFI between follicular helper (PD-1⁺CXCR5⁺) T cells and nonfollicular helper (PD-1⁻CXCR5⁻) cells (top) as control. Panel on extreme right shows staining for IL-21. Data for MFI on isotype controls are on pooled cells. (C-D) Percentages of CD4⁺ CD1d tetrameter-positive cells (C) and CXCR5^hi PD-1^hi NKT-T_FH cells (D) in spleens of WT and Jα18^−/− mice after immunizations with vehicle or α-GalCer, βGL1-22, or LGL1. The results represent 1 of 3 comparable experiments, each performed with at least 3 mice per group is shown. § represents comparison of α-GalCer tetrameter-positive cells between WT mice injected with vehicle and βGL1-22 (P < .0001); ∞ represents comparison of βGL1-22 tetrameter-positive cells between vehicle and βGL1-22 immunized mice (WT and Jα18^−/−); # represents comparison of LGL1 tetrameter-positive cells between vehicle and LGL1-immunized mice (WT and Jα18^−/−); ¶ represents comparison of α-GalCer tetrameter-positive cells between WT mice injected with vehicle and α-GalCer. ***P < .0001; **P < .001; *P < .01. (E) Representative contour plots showing the percentage of FAS⁺GL-7⁺ GC B cells among total B cells (CD19⁺B220⁺) in WT, CD1d^−/−, and Jα18^−/− mice splenocytes 7 days after α-GalCer, βGL1-22, or LGL1 immunization. (F) Complied results showing the frequency of GC B cells in WT, CD1d^−/−, and Jα18^−/− mice splenocytes 7 days after α-GalCer, βGL1-22, or LGL1 immunization. Data are presented as mean ≥ SEM (n = 6). (G) Serum from WT, CD1d^−/−, and Jα18^−/− mice immunized with α-GalCer, βGL1-22, or LGL1 was collected, and total immunoglobulin of the indicated isotypes and IgM were measured by ELISA. Data are presented as mean ± SEM (n = 4). (H) Stacked bar graph showing the presence of anti- βGL1-22 or LGL1 antibodies in the sera of WT, CD1d^−/−, and Jα18^−/− mice immunized with α-GalCer, βGL1-22, or LGL1. ***P < .0001; **P < .001; *P < .01. ns, nonsignificant; PBS, phosphate-buffered saline.