Fig. 6

Aberrantly upregulated p19Arf antagonizes T_FH differentiation and survival. a Diagram showing the structure of WT and mutant forms of p19Arf, with their capacity of interacting with Mdm2 and Bcl6 summarized. b p19Arf and Bcl6 interaction capacity. WT or mutant p19Arf was co-transfected with WT Bcl6 expression vector into 293T cells. Cell lysates were immunoprecipitated with anti-Bcl6 and then immunoblotted with anti-p19Arf. Data are representative of two experiments. c Impact of p19Arf on T_FH differentiation and survival. In vivo primed WT Smarta CD4⁺ T cells were infected with EV-GFP retrovirus or that expressing WT or mutant p19Arf, followed by adoptive-transfer and LCMV-Arm infection. On 6 dpi (corresponding to day 5 after initial priming), CD45.2⁺CD4⁺ T cells were detected for GFP expression (top panels), and GFP⁺ cells were analyzed for AnnexinV positivity (middle) or CXCR5⁺SLAM^lo T_FH cells (bottom panels). d Interplay of p19Arf and Bcl6 in T_FH differentiation. WT Smarta CD4⁺ T cells were transduced with EV-GFP or ArfΔ14 retrovirus in combination with EV-mCherry or Bcl6 retrovirus, followed by adoptive-transfer and LCMV-Arm infection. On 6 dpi, GFP⁺mCherry⁺CD45.2⁺ CD4⁺ T cells were analyzed for frequency of CXCR5⁺SLAM^lo T_FH cells. e–f Effect of genetically ablating p19Arf on T_FH and B cell responses to protein immunization. WT, Ezh2^–/–, or Ezh2^–/–Arf^–/– CD45.2⁺ Smarta CD4⁺ T cells were adoptively transferred into CD45.1⁺Bcl6^–/– recipients followed by GP61-KLH immunization. On day 5 post-immunization, CXCR5⁺ T_FH cells were detected in the draining LNs (e), and on day 8, KLH-specific IgG was detected in the sera by ELISA (f). Data are in (c–f) are means ± s.d. from ≥2 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 for indicated pairwise comparison (c–e) or comparison with recipients of Ezh2^–/– Smarta CD4⁺ T cells (f) by Student’s t-test, coupled with one-way ANOVA for multi-group comparisons