Figure 5. Increasing basal TCR signaling in LLO118 cells inhibits Tfh development.

a, Outline of the Scn5a construct that was used to generate the F/S/F-Scn5a mouse, previously described²⁸, and the breeding scheme of the LLO118⁺Scn5a⁺Cd4-creErt2⁺ line. Upon tamoxifen treatment, ectopic Scn5a expression in peripheral CD4⁺ T cells can be detected by GFP expression. b, Representative flow plots depicting GFP detection in CD4⁺ T cells from LLO118⁺Scn5a⁺Cd4-creErt2⁺ mice 7 days post-tamoxifen treatment. Both the LLO118⁺Scn5a⁺Cd4-creErt2⁺ mouse and its littermate control were treated with tamoxifen. c-e, Flow cytometry analysis of CD5, Ly6C, and TCRβ MFI in LLO118 GFP⁺ and GFP⁻ T cells from LLO118⁺Scn5a⁺Cd4-creErt2⁺ mice 7 days post-tamoxifen treatment. Data points are the paired GFP⁺ and GFP⁻ populations from individually treated mice; three independent experiments (n=7). f, Analysis of LLO118⁺Scn5a⁺Cd4-creErt2⁺ GFP⁺ T cell expansion during a primary immune response. LLO118⁺Scn5a⁺Cd4-creErt2⁺ and LLO118⁺Scn5a⁻Cd4-creErt2⁺ mice were treated with tamoxifen and 7 days later CD4⁺ T cells were enriched and transferred into Tcra^−/− recipients. The following day, recipients were immunized with NP-LLO^LT-N. Graphs show the frequency of GFP⁺ cells in the LLO118 populations immediately prior to the transfer (unstimulated, left graph) and 7 days post-immunization (d7, right graph). Three independent experiments (n=7 for LLO118⁺Scn5a⁺Cd4-creErt2⁺ and n=9 for LLO118⁺Scn5a⁻Cd4-creErt2⁺). Mean ± SEM are shown. g, In the same experiments as (f), in vivo-activated LLO118⁺Scn5a⁺Cd4-creErt2⁺ cells were also analyzed at day 7 post-immunization for the frequency of Tfh cells within the GFP⁺ and GFP⁻ populations. ****p < 0.0001, **p < 0.01, *p < 0.05. Paired t test or Wilcoxon matched-pairs signed rank test for nonnormally distributed data (c-e, g). Unpaired t test or Mann-Whitney test for nonnormally distributed data (f).