Figure 5. miR-21 contributes to Tfh cell differentiation in vitro.

(A) Healthy naive CD4⁺ T cells were cultured under Tfh cell–polarized conditions for 1, 3, and 5 days. Representative flow cytometry and quantification of CD4⁺CXCR5⁺PD-1⁺ Tfh cells are shown (n = 3). (B) qPCR of miR-21 during the differentiation process of Tfh cells in A. (C–E) Healthy naive CD4⁺ T cells were transfected with Agomir-NC or Agomir-21 and cultured under Tfh cell–polarized conditions for 3 days (n = 5). After 3 days of polarization, (C) the expression level of miR-21, (D) flow cytometry and quantification of CD4⁺CXCR5⁺PD-1⁺ Tfh cells, and (E) mRNA expression of CXCR5, PDCD1, IL21, and BCL6 were analyzed. (F–H) Healthy naive CD4⁺ T cells were transfected with Antagomir-NC or Antagomir-21 and cultured under Tfh cell–polarized conditions for 3 days (n = 5). After 3 days of polarization, (F) the expression of miR-21, (G) flow cytometry and quantification of CD4⁺CXCR5⁺PD-1⁺ Tfh cells, and (H) mRNA expression of CXCR5, PDCD1, IL21, and BCL6 were analyzed. (I) Representative flow cytometry and quantification of CD4⁺CXCR5⁺PD-1⁺ Tfh cells transfected with Agomir-NC, Agomir-21, and Agomir-21 plus 2,5-DHBA (n = 3). (J) Representative flow cytometry and quantification of CD4⁺CXCR5⁺PD-1⁺ Tfh cells transfected with Antagomir-NC, Antagomir-21, or Antagomir-21 plus iron dextran (n = 3). Data are shown as mean ± SEM. Data are representative of at least 2 independent experiments with 3–5 donors. *P < 0.05, **P < 0.01, ****P < 0.0001 (1-way ANOVA with Tukey’s multiple-comparisons test for A, B, I, and J and 2-tailed Student’s t test for C–H).