CXCR5 PD1 non-T FH cells CXCR5 + PD1 + T FH cells were sorted from lung-draining lymph nodes and spleens in the WT and Trim37 ko mice infected with PR8 influenza virus. a Bcl6 protein expression was measured by Western blot analysis. b Bcl6 mRNA expression was measured by RT-PCR. c The WT and Trim37 ko CD4 + T cells were cultured under T FH-like conditions and stimulated with anti-CD3 and anti-ICOS for 4 h. Then, CHX was added to the medium for the indicated hours. Immunoblot analysis of Bcl6 in lysates of CD4 + T cells was performed. Residual Bcl6 protein was normalized to actin and presented relative to that before the addition of CHX (right). d Immunoblot analysis of lysates from the HEK293T cells transfected with HA-tagged Trim37 and Flag-tagged Bcl6 plasmids, followed by immunoprecipitation with anti-Flag affinity gel and immunoblot analysis with anti-HA or anti-Flag antibodies (Abs). e Confocal microscopy of the HEK293T cells transfected with HA-tagged Trim37 and Flag-tagged Bcl6 plasmids. Immunofluorescence was performed with anti-HA (green) and anti-Flag (red) Abs. Nuclei were stained with DAPI (blue). f The schematic design of Bcl6 TST mice (up). The WT and Bcl6 TST CD4 + T cells were cultured under T FH-like conditions and stimulated with anti-CD3 and anti-ICOS for 4 h, followed by affinity enrichment of TST with Strep-tactin and immunoblot analysis with anti-Bcl6, anti-Trim37 and anti-actin Abs (down). Data are representative of at least three independent experiments, and were analyzed by two-way ANOVA ( b ). Data are mean SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.
