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. 2017 Sep 4;214(9):2795–2810. doi: 10.1084/jem.20161955

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Figure 1. — Nonredundant requirements for MHCII on DCs and B cells in GC response. (A) DC-specific MHCII cKO (MHCII^flox/flox × CD11c-Cre, termed DC-MHCII cKO) mice were immunized with NP-KLH/Alum. At day 7 after immunization, NP-specific GC B cell numbers were analyzed in the spleen. Each strain, n = 3. Data are representative of three independent experiments. (B) DC-MHCII cKO mice were immunized with NP-KLH/Alum. 3 wk after immunization, anti-NP IgG1 titers were analyzed by ELISA. Dashed line indicates background OD value in empty wells. MHCII^flox/flox, n = 4; DC-MHCII cKO, n = 4; and MHCII KO, n = 2. Data are representative of two independent experiments. (C and D) OT-II T cells (CD45.1) were transferred to the indicated recipient mice (CD45.2) followed by NP-OVA/Alum immunization, and transferred OT-II cells were analyzed at day 7. (C) Recovered total OT-II cell number (top), frequency of Tfh cells among OT-II (middle), and total OT-II Tfh cell number (bottom) from the spleen are shown. (D) Representative flow cytometry profile of splenic OT-II cells (CD4⁺ B220⁻ CD45.1⁺ Vα2⁺) analyzed for the Tfh CXCR5^high PD-1^high phenotype. Unimmunized, n = 4; MHCII^flox/flox, n = 5; and DC-MHCII cKO, n = 6. Data shown are the combined result of two independent experiments. (E) Cell-intrinsic requirement of MHCII on B cells for antigen-specific GC development. MHCII WT (CD45.1) + MHCII KO (CD45.2) mixed BM chimeric mice and control MHCII WT (CD45.1) + MHCII WT (CD45.2) BM chimeric mice were prepared and immunized with NP-KLH. At day 7 after immunization, chimerism of total B cells (B220⁺; left) and NP-binding GC B cells from the spleen (B220⁺ GL7⁺ Fas⁺; right) was analyzed. Each group, n = 4. Data are representative of two independent experiments. (F) Cell-intrinsic requirement of MHCII on B cells for antigen-specific IgG1 production. MHCII WT (Igh^a) and MHCII KO (Igh^b) mixed BM chimera mice and control MHCII WT (Igh^a) and MHCII WT (Igh^b) BM chimera mice were prepared and immunized with NP-KLH/Alum. At 3 wk after immunization, anti–NP-IgG1 titers were analyzed by ELISA. Each group, n = 3. Data are representative of two independent experiments. (G and H) BM chimeras were reconstituted by a mixture of B cell KO (μMT) + MHCII KO BM (MHCII^−/−+ μMT; Fig. S2 E), resulting in the selective absence of MHCII on all B cells. Control chimeras were reconstituted by a mixture of B cell KO (μMT) + MHCII WT BM (MHCII^+/+ + µMT) BM. OT-II T cells (CD45.1) were transferred to recipient BM chimera mice (CD45.2) followed by NP-OVA/Alum immunization 1 d later, and splenic OT-II cells were analyzed at day 8 after immunization. (G) Recovered total OT-II cell number (top), frequencies of OT-II Tfh cells (middle), and number of total OT-II Tfh cells were analyzed (bottom). ns, not significant. (H) Representative flow cytometry plot of OT-II cells (CD4⁺ B220⁻ CD45.1⁺ Vα2⁺) analyzed for the Tfh CXCR5^high PD-1^high phenotype. Data presented are the combined result of three independent experiments. The total numbers of mice in the three combined experiments are unimmunized, n = 4; (MHCII^+/+ + µMT) BM chimera, n = 3; and (MHCII^−/− + μMT) BM chimera, n = 5. Statistical significance was evaluated by Student’s t test. All error bars represent mean ± SEM.