Fig 1: Importance of Early IL-4 Production on B Cell Immunity, Related to Figure 7(A) Representative confocal microscopy images of mediastinal lymph node sections from mice that were treated as in Figure 7E. Sections were stained with antibodies to the B lymphocyte marker, IgD (green) and the germinal center marker, GL7 (magenta). Scale bar, 450 µm. Quantification of the germinal center area in confocal images by ImageJ is shown in the right chart.(B) Flow cytometry analysis at day 8 of mediastinal lymph nodes from wild type mice that were treated with PBS or anti-IL4 blocking antibody on day -3, and infected with 200 PFU of influenza virus at day 0. Contour plots show Fas+GL7+ germinal center cells. The responses are quantified in right charts.(C) Experimental scheme showing the strategy for the generation of antigen-specific TfH cell precursors.(D–F) Flow cytometry analysis of mediastinal lymph node cells from mice treated as in (C). Representative contour plots display (D) the gating strategy used to differentiate endogenous CD4+ T cells from adoptively transferred OT-II cells, (E) the percentage of endogenous and exogenous cells bearing the TfH cell markers PD-1 and CXCR5 and (F) the percentage of B cells that acquire GL7 marker. Quantifications are shown on the right charts.(G and H) OCR at baseline and after sequential treatment with oligomycin, FCCP and Rotenone of B cells that were (G) stimulated ex vivo with anti-IgM and/or IL-4 or (H) isolated from lymph node cells of influenza-infected mice (day 3). Bar charts show the respiratory capacity obtained as (OCR after FCCP)- (basal OCR). Each dot represents an individual measurement.(I) Experimental scheme showing Zika virus infection strategy and time points at which lymph nodes and serum were collected from macaques.(J) Linear regression model of gene signatures for NKT cells, TfH cells, B cells, IL-4, STAT6 and IL-18 from harvested lymph nodes at day 14 with neutralizing antibodies measured in plasma at day 17. Red squares represent positive correlation, blue squares represent negative correlation and white squares represent no significant correlation.(K) Gene interacting network inference using GeneMANIA software representing interconnectivity of NKT, TfH cells, B cells, IL-4, STAT6 and IL-18 pathways and the co-expression of their leading genes.Unless stated, each dot represents a single mouse. Horizontal bars – mean; error bars – SEM. Data are representative of two independent experiments. Statistical analysis, two tailed Student’s t test, *p < 0.05, **p < 0.01 and ****p < 0.0001.
Fig 2: Expression Analysis of TfH Markers by NKT Cells and Strategy for the Generation of CD1d-Floxed ESCs, Related to Figure 2(A) Flow cytometry gating strategy for the analysis of NKT cells (TCRß+ CD1d tetramer+), CD4+ T cells (TCRß+ CD1d tetramer- CD4+) and TfH cells (TCRß+ CD1d tetramer- CD4+ CXCR5+ PD-1+) in mediastinal lymph nodes at day 9 of influenza infection.(B) RT-qPCR analysis of IL-21 expression at day 3 and 9 of influenza infection by sorted NKT and TfH cells compared to CD4+ cells.(C and D) Representative contour plots show the expression of (C) Bcl-6 and (D) SLAM by NKT, TfH and CD4+ T cells at day 3 and 9 of infection. Quantification on the right charts shows the mean fluorescence intensity for (C) Bcl-6 and (D) SLAM.(E) Scheme showing the wild type and targeted CD1d locus containing two loxP sites flanking the exon 3 of CD1d as well as two FRT sites for the removal of the Neomycin resistance cassette.(F) Specific fragments amplified by long-range PCR for the 5' end (7.1kb) and 3' end (6.6kb) of the clones that correctly performed homologous recombination.(G) DNA from three positive clones was digested with SacI restriction enzyme and Southern blot performed using a labeled Southern probe. The upper band corresponds to the wild type locus (10.6kb) and the lower band to the targeted locus (7.3kb).(H) Southern blot of the three positive clones performed with a labeled Southern probe against the Neomycin cassette. Orange arrow indicates the single 5.2kb band recognized by this probe, indicating single insertion of the construct. The clone 2, in red, was chosen for injections.In all quantification charts, each dot represents one mouse. Data are representative from three experiments. Statistical analysis two-tailed Student’s t test; **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Fig 3: NKT Cells Deliver Non-cognate Help to B Cells during Viral Infection(A and B) Flow cytometry analysis of wild-type mice infected with influenza virus.(A) Gating strategy for NKT and CD4+ T cells. B220+ cells were excluded from the analysis.(B) Analysis of the percentage of NKT (blue) and CD4+ T (red) cells that acquire CXCR5 and PD-1 markers after infection.(C) Confocal microscopy analysis of lymph nodes on day 9 of influenza infection incubated with CD1d tetramer (magenta) and GL7 (green). Charts show the number and density of NKT cells inside and outside of germinal centers. Each dot represents a single germinal center. Scale bar, 60 µm.(D) Schematic showing the CD1d locus targeted for deletion with two loxP sites flanking exon 3 of CD1d.(E) Representative contour plot shows the gating strategy for B cells.(F and G) Flow cytometry analysis of CD1d expression in B cells from (F) CD1dflox/floxCD19-Cre or (G) CD1dflox/floxMb1-Cre mice: Cre- (blue) and Cre+ (red).(H–K) Flow cytometry analysis of germinal center and TfH cell formation in (H and I) CD1dflox/floxCD19-Cre or (J and K) CD1dflox/floxMb1-Cre mice on day 9 of influenza infection: Cre- (blue dots) and Cre+ (red dots).All data are representative of 3 independent experiments. Unless specified, each dot represents one mouse. Horizontal bars, mean; error bars, SEM; statistical analysis, two tailed Student’s t test, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig 4: NKT Cells Generate a Pre-TfH Cell Wave of IL-4(A) Flow cytometry analysis of CD69 levels in the TCRß+ CD1d tetramer+ population after 0–6 days of influenza infection. MFI, mean fluorescence intensity.(B) Representative plots showing the percentage of NKT cells producing IFN-? (blue) and IL-4 (red) after 3 days of influenza infection.(C) Flow cytometry analysis of IFN-? (blue) and IL-4 (red) production on day 3 of influenza infection. Gates were drawn on IFN-?+ and IL-4+ lymph node cells to analyze the percentage of NKT cells inside each cytokine+ population.(D–F) Flow cytometry analysis of NKT (blue) and TfH (red) cells from IL-4 GFP mice infected with influenza virus.(D) Numbers of NKT and TfH cells at different stages of influenza infection.(E) GFP expression in NKT and TfH cells at different times of infection.(F) Percentage of CD1d-tetramer+ NKT cells and CXCR5+ TfH cells inside the IL-4 GFP+ lymph node cell population at different stages of influenza infection.In all charts, each dot represents one mouse. In graphs showing statistical analysis, data are representative of three independent experiments. Horizontal bars, mean; error bars, SEM. Student’s t test, ****p < 0.0001.
Fig 5: NKT Cell-Deficient Mice Display Early Impairment in B Cell Immunity, Related to Figure 1(A–D) Flow cytometry analysis of mediastinal lymph node cells from wild type or CD1d-/- mice that were intranasally infected with 200 PFU of influenza virus for (A-B) 7 or (C-D) 21 days. Representative contour plots display the percentage of (A-C) B220+ cells bearing biomarkers of germinal cell activity, Fas+GL7+, and (B–D) CD4+ T cells bearing biomarkers of TfH cells, CXCR5+PD-1+. Dot plots show quantification of (A-C) germinal center and (B-D) TfH cells.(E and F) Flow cytometry analysis of mediastinal lymph nodes harvested at day 10 from wild type mice that were intranasally challenged with (E) 10 µg of HA trimmer or (F) PBS in the presence or absence of poly I:C or a-GalCer. Representative contour plots display the percentage of HA-specific B220+ cells differentiated into Fas+GL7+ germinal center cells.(G and I) ELISPOT analysis of IgG1 HA-specific ASCs in mediastinal lymph nodes from wild type and CD1d-/- mice treated as in figure S1E.(H and J) Flow cytometry analysis of HA-specific germinal center cells in mediastinal lymph nodes from wild type and CD1d-/- mice treated as in figure S1E.In all quantification charts, each dot represents one mouse. Data are representative from two experiments. Statistical analysis two-tailed Student’s t test; *p < 0.05.
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