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. 2019 Sep 10;216(11):2531–2545. doi: 10.1084/jem.20181868

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Figure 6. — TGFβ signaling promotes the LZ to DZ transition and antibody affinity maturation in nonmucosal GCs. (A) Confocal microscopy of pLNs of Tgfbr1^f/+ or Cr2-cre Tgfbr1^f/f animals that had been immunized s.c. with SRBCs stained for IgD (white), Bcl6 (green), and pSmad2 (red). Scale bar, 150 μm. (B) Percentage of IgD⁺ B cells in follicle or GC B cells in pLNs staining positive for pSmad2 quantified by histocytometry. Data in A and B are representative of or pooled from three to five GCs per mouse from two mice of each type from two independent experiments. Each dot represents one GC. (C) Confocal microscopy of pLNs from immunized Tgfbr1^f/+ or Cr2-cre Tgfbr1^f/f animals stained for IgD (white) and CD35 (red). Scale bars: 150 μm. Data are representative of two independent experiments with a total of four mice per group. (D–F) Ratio of LZ to DZ GCBs (D); percentages of CD45.2⁺ GCBs staining positive for IgA, IgG1, IgG2b, or IgM (E); or frequency of FoBs or GCBs derived from CD45.2 bone marrow (F) in SRBC-immunized pLNs from mixed bone marrow chimeras generated with a mixture of 70% WT (CD45.1/2) and 30% CD45.2 bone marrow that was Tgfbr1^f/+ or ^f/f or Cr2-cre Tgfbr1^f/f assessed by FACS. Data in D are pooled from three independent experiments with 15 and 14 total mice in each group. Data in E and F are pooled from two independent experiments with 10 total mice per group. (G and H) Ratio of high-affinity IgG1 binding to NP (NP₂) versus total IgG1 binding to NP (NP₃₀) in serum of littermate control or Cr2-cre Tgfbr1^f/f (G) or Aicda^cre/+ Tgfbr1^f/+ or Aicda^cre/+ Tgfbr1^f/f (H) animals that had been immunized 14 d prior with NP-CGG in alum by ELISA. Data in G are pooled from two independent experiments with 19 and 12 total mice per group. Data in H are pooled from three independent experiments with 12 and 14 total mice per group. (I and J) Frequency of K59R or Y99G mutations (I) or overall variable region mutation frequency (J) in V_H186.2 IgG1 reads from heavy-chain repertoire sequencing (see Materials and methods) of pLN GCBs from NP-CGG–immunized Aicda^cre/+ Tgfbr1^f/+ or Aicda^cre/+ Tgfbr1^f/f animals. Data are pooled from three mice per group from one experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test for data in A–H. ****, P < 0.0001; χ² test for I.