Fig 2: Loss of TET-function impairs GC maintenance and CSR upon immunization. (A) qRT-PCR analysis for TET2 and TET3 mRNA expression in FACS-sorted splenic GC B cells of the indicated genotypes 5 and 10 days post immunization with NP-CGG (n = 3 for Cg1-Cre and n = 3 for Cg1-Cre;Tet2 F/F;Tet3 F/F). (B) Mice of the indicated genotypes were immunized at the age of 8–10 weeks with NP-CGG and analysed 14 days later. Flow cytometry analysis of splenic single-cell suspensions identified the fractions of total B cells (B220+ CD19+), GC B cells (CD19+B220+ CD138- CD95hi CD38lo/-) and NP +IgG1+ GC B cells within total splenocytes. Total cell numbers for the respective cell populations are shown (n = 5 for Cg1-Cre and n = 5 for Cg1-Cre;Tet2 F/F;Tet3 F/F). (C) Representative flow cytometry plots (left) and bar graph (right; n = 5 for Cg1-Cre and n = 5 for Cg1-Cre;Tet2 F/F;Tet3 F/F) depict fractions of B cells, GC B cells and IgG1+ NP + GC B cells shown in (B). Arrows indicate gating strategy. (D) Spleen sections from the indicated genotypes 14 days post immunization with NP-CGG were analysed for Ki67 (proliferating cells) and for the ability of cells to bind PNA (GC B cells). The brown precipitate indicates positive staining. Sections were counterstained with haematoxylin. One representative mouse out of three per group is shown. Scale bars: 200 µm. (E) Representative flow cytometry plots (left) depict fractions of DZ and LZ GC B cells within total GC B cells. Scatter graph (right; n = 5 for Cg1-Cre and n = 5 for Cg1-Cre;Tet2 F/F;Tet3 F/F) shows the ratio of DZ and LZ GC B cells. (F) Scatter graph depicts the ratio of IgM+ and IgG1+ cells within GC B cells (n = 5 for Cg1-Cre and n = 5 for Cg1-Cre;Tet2 F/F;Tet3 F/F). (G) qRT-PCR analysis for Ki67 and AID mRNA in FACS-sorted naïve Cg1-cre B cells (n = 3) and GC splenic B cells (n = 3 for Cg1-Cre and n = 3 for Cg1-Cre;Tet2 F/F;Tet3 F/F) 14 days post NP-CGG immunization. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 vs Cg1-Cre control (Student's t-test).

Fig 3: Flow cytometric analysis of antigen-matured engineered B cells.Host mice receiving LPS activated only (Mock), H + ?, or H -targeted B cells were analyzed 14 d after the first boost (w8). n = 4 mice in each group. a–c, Germinal center (GC) B cells. a Representative flow cytometry and statistical analysis of total GC B cells pre-gated as live, CD19+ singlets. b CD45.1+ (donor) GC B cells. c Fraction of cells gated in (b) which were class-switched IgG1+ and GT8++KO11- (engineered VRC01-expressing). d–e Plasma cells (PCs). Frequency of class-switched and antigen-specific PCs in the spleen (d) and bone marrow (e) as a fraction of total donor PCs. GT8++KO11–CD45.1+CD45.2-IgG+ PCs were analyzed by intracellular staining after the permeabilization of surfaced-stained cells. Plasma cells are gated as F4/80-IgD-TCRß-CD138+Sca-1+. f Memory cells (MCs). Enumeration of IgG1+GT8++KO11-, engineered MCs (sIgD-CD45.1+CD45.2-CD38+GL7-). g Vaccine-induced expansion of engineered cells. GT8++KO11-CD45.1+CD45.2- cell expansion at w8 compared with w0 as a percentage of the total donor cells. h Engineered memory cells have become germinal center dependent (CD73+) after vaccination. GT8+ or GT8- donor memory cells were compared with host naïve and memory B cell controls for expression of the indicated memory cell surface markers. i Quantification of B cells memory subtypes gated as in (h). a–i Bars represent mean ± SD for all data points in each group. *P < 0.05; **P < 0.01; ns, not significant; unpaired 2-tailed T test.

Fig 4: IgMhi B-1a cells in CKO mice internalize surface IgM and start expressing the pre-GC APC phenotype.a Live myeloidneg CD3neg CD19+ splenic B cells in control or CKO mice were gated to show IgM and IgD expression. IgMhi IgDlo/neg, IgMint IgDneg, and IgMneg IgDneg B cells in CKO mice were gated to reveal IgMhi, IgMint, and IgMneg B-1a cells, based on CD5 and CD43 expression. Two FMO stainings were used to define IgM and CD5 positive cells. FACS histogram on the left shows the intracellular IgM level in IgMneg B-1a cells (red line) and IgM-secreting PCs (IgDneg CD138+) (gray line). Blue line shows the surface IgM staining level in IgMneg B-1a cells that were stained with anti-IgM antibody before fixation and permibilization. b FACS plots shows cells expressing the differentiated CD95+ CD38lo GL7+ CD150+ phenotype in IgMhi, IgMint, and IgMneg B-1a cells from CKO mice. c Data summarizing the percentage of CD95+ CD38lo GL7+ CD150+ cells in IgMhi, IgMint and IgMneg B-1a cells from control and CKO mice is shown. Each dot represents data for an individual mouse, n = 7–10 mice per subset, *p < 0.001, **p < 0.002, ns, not significant (p < 0.2), nonparametric Wilcoxon one-way test. d IgMhi B-1a and IgMneg B-1a cells from CKO mouse were sorted and then sequenced to obtain their IgH transcripts. Data summarizing the frequencies of IgH sequences containing >5 nucleotide changes and the mutation rate of each sample is shown, n = 7 samples per group with each sample from an individual mouse. Data for IgMhi B-1a and IgMneg B-1a cells from the same mouse are connected. *p < 0.001, nonparametric Wilcoxon one-way test. All mice are 2 months old. Box plots in c, d: box draws 75% (upper), 50% (center line), and 25% (down) quartile, the maxima and minima outliers are shown as top and bottom line, respectively.

Fig 5: (A) Representative flow cytometry analyses and bar charts denote the frequency of CD138+ cells in splenocytes before and after splenocytes were CpGC stimulated for 24 h. (B) Representative flow cytometry analyses and bar charts denote the frequency of CD138+ T cells in CD3+ T cells before and after splenocytes were CpGC stimulated for 24 h. (C) Representative flow cytometry analyses and bar charts denote the frequency of CD138+ cells in CD4+ T cells before and after 24 h of CpGC stimulation. All data are presented as mean ± SD; n=4 per group from two independent experiments; #P<0.05, ##P<0.01 by Mann-Whitney U or one-way analysis of variance test. PNS, prednisone.