Fig. 4.
Provision of B cell help by αV-deficient Tfh. (A and B) Antigen-dependent T:B conjugation of WT OVA-loaded LPS-stimulated B cells with IL-2 cultured WT (αV+/+ CD4-Cre+) or αV-CD4 cKO OTII CD4+ T cells. (A) Representative FACS plots (numbers, percent of CD4 cells in conjugate with CD19 B cells). (B) Antigen-dose response. (C) B cell activation by Tfh. Resting B cells from naïve animals cultured with in vivo Tfh from day-10 OVA/CFA-immunized WT and αV-CD4 cKO mice ± anti-CD3 and anti-IgM Abs. Percent CD19+ CD4− GL7+ B cells after 6 d of coculture. (D) IgG in supernatant of day-11 Tfh and B cell cocultures. (A and B) One of three independent experiments. (C and D) Mean of two to four individual experiments. Statistics by two-tailed t test; *P < 0.05. (E–G) In vivo B cell help assay: MDA transfer ± OT-II (C57BL/6) or OT-II αV-CD4 cKO into 2D2-recipient mice followed by immunization with HEL-OVA/CFA. Analysis of MD4 expansion (F) and Bcl6 induction (E and G) day 5 postimmunization. (E) Representative FACS plots (numbers, percentage of MD4 cells IgDlow, Bcl6+). Two independent experiments, five mice per group. Statistics by Mann–Whitney U test; **P < 0.01, ***P < 0.001. ns, not statistically significant.