Fig. 7: Plekho1 plays a stage-specific role in regulating the competitive fitness of GC-Tfh cells. | Nature Communications

Fig. 7: Plekho1 plays a stage-specific role in regulating the competitive fitness of GC-Tfh cells.

From: Spatiotemporal resolution of germinal center Tfh cell differentiation and divergence from central memory CD4+ T cell fate

Fig. 7

a Purified OT-II cells were transferred into CD45.1+ SMARTA recipient mice followed by intranasal infection with PR8-OVA. Indicated donor cell populations at day 14 p.i. in the medLN were analyzed by real time RT-PCR for Plekho1 mRNA levels (n = 3). b ATAC-seq read density at the Plekho1 gene locus of naïve and indicated donor cell populations as in Fig. 3d. c Purified CD4+ T cells from C57BL/6 mice were activated in vitro and infected with control retrovirus (RV-Ctrl) or retrovirus expressing Maf (RV-c-Maf) under Th0 and Tfh-like culture conditions. On day 4, retrovirally infected cells were sorted and analyzed by real time RT-PCR for Plekho1 mRNA levels (n = 3). d–h Purified OT-II cells from CD45.1+CD45.2+ and CD45.2+ OT-IITg mice were activated in vitro and infected with retrovirus expressing shRNAmir against CD19 (shCD19, CD45.1+CD45.2+) and Plekho1 (shPlekho1, CD45.2+). Retrovirally infected OT-II cells were co-transferred into CD45.1+ SMARTA recipient mice followed by intranasal infection with PR8-OVA two days after cell transfer. d Donor OT-II cells at days 14 and 21 p.i. in the medLN were analyzed for PD-1 and CXCR5 staining (day 14, n = 4; day 21, n = 8). e–g Donor OT-II cells at day 14 p.i. in the medLN were analyzed by RNA-seq. e Clustered heatmap of shCD19 and shPlekho1 GC-Tfh cells. f Numbers and percentages of potential Foxo1 targets in Cluster i and Cluster ii genes from Fig. 7e. g Enrichr enrichment analysis of transcription factors associated with DEGs in Fig. 7e. h ImageStream analysis of Foxo1 sublocation in GC-Tfh cells 30 min after stimulation with anti-CD3/ICOS antibodies (n = 7). Scale bars, 12 μm. Data in a, c, d, h are representative (or pooled) results of two independent experiments. Bars represent average ±SD. The P-values were determined by a two-tailed paired t-test (d, h), a one-way ANOVA with Tukey’s multiple comparisons test (a), or a two-way ANOVA with Sidak’s multiple comparisons test (c). RNA-seq and ATAC-seq samples are independent biological replicates. Source data are provided as a Source Data file.

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