Fig. 5.

In vitro T_FR (iT_FR) cell differentiation

(a) Schematic of iT_FR and iT_REG cell differentiation culture.

(b) Relative mRNA expression of Pdcd1, Cxcr5, Bcl6, and Tcf7 in sort-purified TCRβ⁺CD4⁺Foxp3-hCD2⁺ cells fro, iT_FR or iT_REG cell culture conditions (n= 5). Sort-purified naïve CD4⁺T cells from Foxp3^hCD2reporter mice were used for the culture.

(c, d) Expression of CD25 and Foxp3 by cells from iT_FR or iT_REG 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⁺ TCRβ⁺ gate (n= 5).

(e, f) Expression of T_FR cell phenotypic markers by cells from iT_FR or iT_REG 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 iT_FR or iT_REG cultures. Sort-purified naïve CD4⁺T cells from Bcl-6-tdTomato Foxp3^hCD2 double reporter mice or Foxp3^hCD2reporter 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 IgG_2a in suppression assays. IgG⁻CD43⁻CD19⁺ resting B cells, Foxp3-hCD2⁻CXCR5⁺Bcl-6-Eyfp⁺ T_FH cells, and CD25⁻Foxp3-hCD2⁺CXCR5⁺Bcl-6-Eyfp⁺ iT_FR cells sort-purified from Bcl-6-Eyfp Foxp3^hCD2 double reporter mice were used.The resting B cells from were co-cultured with T_FH cells alone, T_FH and T_FR cells, or T_FH and sort-purified CD25⁻Foxp3-hCD2⁺CXCR5⁺Bcl-6-Eyfp⁺ iT_FR cells under the exsitance of 5 μg ml⁻¹ anti-IgM and 2 μg ml⁻1 anti-CD3ε 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 IgG_2a staining (k), and the frequency of IgG₁⁺ and IgG_2a⁺ 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).