Fig. 4.

Butyrate increased T_FR 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⁺ TCRβ⁺ gate (n= 11, 10).

(c–g) T_FR and T_FH 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⁺ T_FR (d) and Foxp3⁻ T_FH (e) cells within Bcl-6⁺CXCR5⁺ follicular T cell gate and the total number of CD25⁻Foxp3⁺ T_FR (f) and Foxp3⁻ T_FH (g) cells (n= 11, 10). Gating strategy is depicted in Figure S4.

(h) T_FR/T_FH ratio calculated using the total number of CD25⁻Foxp3⁺ T_FR and Foxp3⁻ T_FH cells in F and G.

(i, j) B cell class switching to IgG₁ by T_FH 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⁺ T_FH cells sort-purified from DLNs of CIA mice fed the control HAMS or HAMSB diet (described as HAMS-T_FH or HAMSB-T_FH 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 T_FR and T_FH cells in CoPs of Tcrb^−/−Tcrd^−/− mice transferred with Foxp3-hCD2⁺iT_REG cells. Sort-purified CD45.1⁺Foxp3-hCD2⁺T cells cultured for five days under iT_REG 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⁺ TCRβ⁺ 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⁻ (T_FH cells), CD25⁺Foxp3⁺ (CD25⁺T_FR cells) and CD25⁻Foxp3⁺(CD25⁻T_FR 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).