Fig 2: Increased germinal center B cell expansion in Dnmt3-deficient mice. a Germinal centers identified using Fas and GL7 expression on inguinal and periaortic lymph node (LN) B220+ B cells in naive mice (day 0) and PE-CFA immunized mice (day 30) in Cd19cre/+Dnmt3afl/flDnmt3bfl/fl mice (KO) and littermate controls (C). b Immunofluorescence of lymph nodes 30 days after PE-CFA immunization showing T cells (blue; Thy1.2), IgD B cells (red; IgD), and activated B cells (green; GL7). The germinal center area is outlined in white and a 100 µM scale is shown (bottom left). Quantitation of germinal center area is shown (right). c B220 expression and PE-binding of LN lymphocytes from naive mice (day 0) and PE-CFA immunized mice (day 30). Flow cytometry data are lymphocyte size gated and CD11b-. d Serum PE-specific antibodies for C and KO mice 30 days post-immunization. Mean and standard deviation shown on beeswarm plots and P values are calculated using a two-sided t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Data are from either two, day 0 experiments with eight mice each and two, day 30 experiments with 9 mice and 17 mice (a, c) or from one, day 30 experiment with 10 mice (b, d), where open circles represent females and closed circles males and no sex differences were observed in multivariate analysis

Fig 3: Differentiation of icCD3e+ cells in the small intestine(A) Lin-CD127+ cells from WT small intestine were first divided into CD45lo and CD45hi. icCD3e levels were determined for each subset. Transcription factor levels in CD45hi KLRG1- cells were further analyzed and the correlation between the percentages of icCD3e+ cells and the levels of GATA3 or ROR?t are plotted on the right.(B) MLN cells from mice with or without Nb infection for 6 days were analyzed. Lin-CD127+ cells were separated into Thy1+icCD3e- and Thy1+icCD3e+ subsets, which were then scored for IL-5 and IL-13 expression. Numbers of cytokine+ cells were calculated. One-way ANOVA was used for determining the statistical significance. *p<0.05. Data are represented as mean ± SD.

Fig 4: Cellular response of splenocytes in mice immunized with different vaccines. (A,C) The percentage of GCB cells was analyzed by means of flow cytometry in mice immunized with the HB02 + Delta, HB02 + Beta + Delta, and HB02 vaccines or the control group after 42 days (n = 5–6), with GCB cells gated in live CD45 + B220 +; (B,D) The percentage of IFN-?-expressing T cells was analyzed by means of flow cytometry in mice immunized with HB02 + Delta, HB02 + Beta + Delta, and HB02 vaccines or the control group after 42 days (n = 5–6), with IFN-?-expressing T cell gated in live CD45 + CD90 +. Error bars represent SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 ((A–D), Mann–Whitney test). See also Figure S1.

Fig 5: Age-dependent output of blood td-ILCs and their differentiation(A) Peripheral blood of indicated strains of mice were analyzed. The frequencies of Lin-Thy1+CD127+ and subsequent CD62L+icCD3e+ subsets are quantified.(B) The average frequency of blood CD62L+icCD3e+ cells of the Lin-Thy1+CD127+ compartment in WT mice of indicated ages. Data are represented as mean ± SD. One-way ANOVA was used for determining the statistical significance of data shown in (A) and (B). *p<0.05, **<0.01, ***p<0.001, ****p<0.0001, ns, not significant.(C) In vitro differentiation on OP9-DL stroma of the indicated cell populations (800 cells per well) in 30 ng/mL of each IL-2, IL-7, and stem cell factor. Cells were analyzed on day 9 of the culture and the frequencies of indicated subsets are shown in the graphs. Data shown are representative of three independent experiments.