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[Preprint]. 2024 Mar 12:2024.02.26.582122. Originally published 2024 Mar 1. [Version 2] doi: 10.1101/2024.02.26.582122

Figure 3. Rag-GTPases are critically required for GC formation and antibody production independent of mTORC1.

Figure 3.

(A-E) Tamoxifen was administered to animals by oral gavage daily for 4 consecutive days. Mice were immunized intraperitoneally (100 μg NP-OVA/alum) 7 days after the last injection. (A) Representative flow plots of GL-7 and Fas expression in splenic B cells from μMT:CreER control (WT, n = 7), μMT:CreERRragafl/flRragbfl/fl (n = 9), and μMT:CreERRptorfl/fl (n = 7) mice. Right, summary of the percentages and numbers of GC (GL-7+Fas+) B cells. (B) Representative flow plots of CXCR4 and CD86 expression in GC B cells. Right, summary of the ratio between dark zone (DZ) (CXCR4+CD86−) and light zone (LZ) (CXCR4−CD86+) cells. (C) Representative flow plots of CD138 and B220 expression in spleens of WT (n = 7), μMT:CreERRragafl/flRragbfl/fl (n = 9), and μMT:CreERRptorfl/fl (n = 7) mice. Right, summary of the percentages and numbers of CD138+ cells. (D) Total NP-specific antibodies of all classes were measured using the sera of immunized WT (n = 7), μMT:CreERRragafl/flRragbfl/fl (n = 8), and μMT:CreERRptorfl/fl (n = 7) mice. (E) high-affinity NP-specific antibodies of all classes were measured using the sera of immunized WT (n = 5), μMT:CreERRragafl/flRragbfl/fl (n = 5), and μMT:CreERRptorfl/fl (n = 7) mice. (F) Representative flow plots of p-S6 and p-4EBP1 expression in fresh or overnight anti-IgM/anti-CD40/IL-4 stimulated splenic B cells from immunized WT (n = 3), μMT:CreERRragafl/flRragbfl/fl (n = 3), and μMT:CreERRptorfl/fl (n = 2) mice. (G) Heatmap of the mRNA expression of indicated genes on mouse follicular (FO) B cells, marginal zone (MZ) B cells and germinal center (GC) B cells. (H-N) NP-OVA was administered to WT (n = 11), AicdaCreRragafl/flRragbfl/fl (n = 10), and AicdaCreRptorfl/fl (n = 6) mice. Animals were analyzed 9 days after immunization. (H) Representative flow plots of GL-7 and Fas expression in splenic B cells. Right, summary of the GC B cell percentages. (I) Representative flow cytometry plots of NP+IgG1+ GC B cells in splenic B cells. Right, summary of the percentages of NP+IgG1+ GC B cells. (J) Representative flow plots of CXCR4 and CD86 expression in GC B cells. Right, summary of the DZ/LZ ratio. (K) Representative flow plots of CD138 and B220 expression in splenic lymphocytes. Right, summary of the percentages of CD138+ cells. (L) Representative flow plots of CD138 and IgG1 expression in plasmablasts. Right, summary of the percentages of IgG1+ plasmablasts. (M-N) The anti-NP2 (M) and anti-NP23 (N) titers of different immunoglobulin isotypes in the sera of immunized mice were measured by ELISA. Data in graphs represent mean ± SEM. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA (A, B, C, H, I, J, K and L), two-way ANOVA (D, E, F, M and N).