Fig 1: Butyrate suppresses the GC response and autoantibody production in CIA mice(a, b) CII-specific IgG responses in CIA mice fed the control HAMS or HAMSB diet. The number of CII-specific IgG antibody-forming cells (AFC) cells per 5 × 105 DLN cells three weeks after booster immunization (a, each dot indicates data from 1 well, n= 36 from 6 mice). Statistical analysis was performed on the mean of each mouse. Serum levels of CII-specific IgG1 and IgG2a upon or three weeks after booster immunization (b, n= 5–10).(c–e) Colonic patches (CoPs) and solitary intestinal lymphoid tissues (SILTs) of CIA mice fed the control HAMS or HAMSB diet. Representative whole-mount B220 immunostaining (brown) of colon tissues (c), and the number of CoPs (d) and SILTs (e) in unimmunized DBA1/J mice and CIA mice one week after the initial immunization (n= 5 or 6).Dashed line, CoP, arrowheads, SILT. Scale bars, 5 mm.(f–h) GCB cells in CoPs from CIA mice fed the control HAMS or HAMSB diet. Representative flow cytometry contour plots of GL7 and intracellular Bcl-6 staining within CD19+ gate (f), and the frequency (g) and total number (h) of Bcl-6+GL7+ GCB cellstwo weeks after the initial immunization (n= 12).(i) GC area (mm2) of DLNs from CIA mice fed the control HAMS, or HAMSB diet Data were obtained two weeks after the initial immunization. Each dot indicates a single GC area of DLNs on cross-sections (n= 43, 30). Statistical analysis was performed on the mean of each mouse.(j–l) GCB cells in DLNs from CIA mice fed control HAMS or HAMSB diet. Representative flow cytometry contour plots of GL7 and intracellular Bcl-6 staining within CD19+ gate (j), and the frequency (k) and total number (l) of Bcl-6+GL7+ GCB cellstwo weeks after the initial immunization (n= 12).(m) Arthritis scores after antibody cocktail injection of collagen antibody-induced arthritis (CAIA) mice fed control HAMS or HAMSB diet (n= 3; means ± s.e.m.). AUC was calculated for each mice.Results show one representative experiment of at least two experiments. *P< 0.05, **P< 0.01, ***P< 0.001 (a, b, g–i, k, l, Welch's t-test or unpaired two-tailed Student's t-test; d, e, one-way ANOVA followed by Tukey's posthoc test; m, Log-rank test).
Fig 2: In vitro TFR (iTFR) cell differentiation(a) Schematic of iTFR and iTREG cell differentiation culture.(b) Relative mRNA expression of Pdcd1, Cxcr5, Bcl6, and Tcf7 in sort-purified TCRß+CD4+Foxp3-hCD2+ cells fro, iTFR or iTREG cell culture conditions (n= 5). Sort-purified naïve CD4+T cells from Foxp3hCD2reporter mice were used for the culture.(c, d) Expression of CD25 and Foxp3 by cells from iTFR or iTREG cultures. Representative flow cytometry contour plots of CD25 and Foxp3-hCD2 staining (c), and the frequency of CD25+Foxp3+ and CD25-Foxp3+ cells (d) within CD4+ TCRß+ gate (n= 5).(e, f) Expression of TFR cell phenotypic markers by cells from iTFR or iTREG cultures. Representative flow cytometry histograms of FSC, PD-1, CXCR5, Bcl-6, and TCF-1 expression (e), and the gMFI of PD-1, CXCR5, Bcl-6, and TCF-1 (f) within CD25+Foxp3+ and CD25-Foxp3+ gate (n= 5).(g, h) Expression of Bcl-6-tdTomato reporter by cells from iTFR or iTREG cultures. Sort-purified naïve CD4+T cells from Bcl-6-tdTomato Foxp3hCD2 double reporter mice or Foxp3hCD2reporter mice (described as control) were used for the culture. Representative flow cytometry histograms of Bcl-6-tdTomato reporter and control expression (g), and Bcl-6-tdTomato gMFI (h) within CD25+Foxp3+ and CD25-Foxp3+gate (n= 5).(i–l) B cell class switching to IgG1 and IgG2a in suppression assays. IgG-CD43-CD19+ resting B cells, Foxp3-hCD2-CXCR5+Bcl-6-Eyfp+ TFH cells, and CD25-Foxp3-hCD2+CXCR5+Bcl-6-Eyfp+ iTFR cells sort-purified from Bcl-6-Eyfp Foxp3hCD2 double reporter mice were used.The resting B cells from were co-cultured with TFH cells alone, TFH and TFR cells, or TFH and sort-purified CD25-Foxp3-hCD2+CXCR5+Bcl-6-Eyfp+ iTFR cells under the exsitance of 5 µg ml-1 anti-IgM and 2 µg ml-1 anti-CD3e Abs. Representative flow cytometry histograms of Fixable Viability Stain 780 (FVS780) staining (i), and the frequency of FVS780- live cells (j) within GL7+CD19+B cells (n= 3–5). Representative flow cytometry histograms of intracellular IgG1 and IgG2a staining (k), and the frequency of IgG1+ and IgG2a+ cells (l) within GL7+ CD19+B gate (n= 3–5).Results show one representative experiment of at least two experiments.***P< 0.001 (b, d, Welch's t-test or unpaired two-tailed Student's t-test; j, l, one-way ANOVA followed by Dunnett's post-hoc test; g, h, two-way ANOVA followed by Sidak's post-hoc test).
Fig 3: iTFR cells prevent autoimmune responses and CIA development(a, b) CII-specific IgG responses in CIA mice inoculated with Foxp3-hCD2+ cells cultured under iTFR-cell conditions in the absence or presence of 100 µM SB. Sort-purified 5 × 106 of Foxp3-hCD2+CD4+T cells were injected intravenously into DBA/1 J mice one week before the initial CII immunization.All mice were fed the control HAMS diet (n= 10, 11). The number of CII-specific IgG AFC cells per 1 × 105 DLN cells (a, each dot indicates data from 1 well, n= 44, 32, statistical analysis was performed on the mean of each mouse.), and serum levels of CII-specific IgG1 and IgG2a three weeks after booster immunization (b, n= 5, 6).(c) Arthritis scores after booster immunization of CIA mice inoculated with Foxp3-hCD2+ cells from iTFR cell culture (n= 10, 11). AUC was calculated in each mice.Results show one representative experiment of at least two experiments.**P< 0.01, ***P< 0.001 (a-c, Welch's t-test or unpaired two-tailed Student's t-test).
Fig 4: Butyrate increased TFR cells in CoPs(a, b) Follicular T cells in the DLNs and CoPs from CIA mice fed the control HAMS or HAMSB diet two weeks after the initial immunization. Representative flow cytometry contour plots of CXCR5 and intracellular Bcl-6 staining (a), and the frequency (b) of Bcl-6+CXCR5+ follicular T cells within CD4+ TCRß+ gate (n= 11, 10).(c–g) TFR and TFH cells in the DLNs and CoPs from CIA mice fed the control HAMS or HAMSB diet two weeks after the initial immunization. Representative flow cytometry contour plots of CD25 and intracellular Foxp3 staining (c), and the frequency of CD25-Foxp3+ TFR (d) and Foxp3- TFH (e) cells within Bcl-6+CXCR5+ follicular T cell gate and the total number of CD25-Foxp3+ TFR (f) and Foxp3- TFH (g) cells (n= 11, 10). Gating strategy is depicted in Figure S4.(h) TFR/TFH ratio calculated using the total number of CD25-Foxp3+ TFR and Foxp3- TFH cells in F and G.(i, j) B cell class switching to IgG1 by TFH cells. IgG-CD19+B cells sort-purified from the DLNs of CIA mice fed the control HAMS or HAMSB diet (described as HAMS-B or HAMSB-B respectively) two weeks after the initial immunization were cultured alone with 100 µg/ml type II collagen (CII),and IgG-CD19+B cells sort-purified from the DLNs of CIA mice fed the control HAMS diet were co-cultured with CXCR5+ICOS+CD4+ TFH cells sort-purified from DLNs of CIA mice fed the control HAMS or HAMSB diet (described as HAMS-TFH or HAMSB-TFH respectively) in the presence or absence of CII. Representative flow cytometry contour plots of GL7 and intracellular IgG1 staining six days after cultivation (i, n= 6), and the frequency of GL7+IgG1+B cells within CD19+ gate (j, n= 6).(k–n) Differentiation of TFR and TFH cells in CoPs of Tcrb-/-Tcrd-/- mice transferred with Foxp3-hCD2+iTREG cells. Sort-purified CD45.1+Foxp3-hCD2+T cells cultured for five days under iTREG conditions were injected intravenously with CD45.2+CD4+T cells into Tcrb-/-Tcrd-/- mice fed the control HAMS or HAMSB diet. Representative flow cytometry contour plots of hCD2 (Foxp3) and CD25 staining (k), and the frequency of Foxp3-, CD25+Foxp3+ and CD25-Foxp3+ cells (L) within CD45.1+CD4+ TCRß+ gate (n= 7). Representative flow cytometry contour plots of CXCR5 and PD-1 staining among Foxp3-, CD25+Foxp3+ and CD25-Foxp3+ gate (m), and the frequency of CXCR5+PD-1+ cells (N) among Foxp3- (TFH cells), CD25+Foxp3+ (CD25+TFR cells) and CD25-Foxp3+(CD25-TFR cells) gates (n, n= 7).Results show one representative experiment of at least two experiments. *P< 0.05, **P< 0.01, ***P< 0.001 (b, d–h, j, l, n, Welch's t-test or unpaired two-tailed Student's t-test).
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