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. 2023 Oct 3;220(12):e20230263. doi: 10.1084/jem.20230263

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© 2023 Tilstra et al.

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Figure 1. — Tamoxifen-induced deletion of Myd88 in B cells results in suppressed MyD88 activity. (A) Schematic of experimental design; Tam-hCD20-Cre;-Myd88^fl MRL.Fas^lpr or Cre negative controls were aged up to 12 wk of age, an early disease time point. Mice were then treated 2× per week with tamoxifen 1 mg in corn oil for 8 wk, after which mice were euthanized and assessed for disease endpoints. (B) Tam-hCD20-Cre;-Myd88^fl MRL.Fas^lpr (TamCreB ΔMyd88) (n = 6) or Cre negative controls (Ctrl) (n = 2) were treated for 3 wk with tamoxifen given by oral gavage. Splenocytes were then sorted and analyzed for Myd88 allelic presence by qPCR in the cell populations as indicated: B cells (CD19⁺, CD90⁻ cells), T cells (CD90⁺, CD19⁻), cDC (CD19⁻, CD11c⁺), and PBs (CD90⁻, CD138⁺, Kappa^high). (C) Splenocytes were isolated from Tam-hCD20-Cre;-Myd88^fl MRL.Fas^lpr (n = 10) or Cre-negative controls (n = 12) and stimulated with CpG DNA (10 μg/ml) for 2 h. Nuclear translocation of NF-κB, a downstream signaling component of the MyD88 pathway, was assessed by Amnis imaging cytometer. In the left panel, representative images of NF-κB and nuclear stain (DAPI) in CD19⁺ B cells are shown along with representative histograms on the right outlining the NF-κB location relative to the nuclear stain, with colocalization of DAPI and NF-κB signifying activation (represented by a shift of the orange/stimulated histogram to a higher similarity score, representing the overlap between NF-κB and DAPI) with the maintenance of NF-κB in the cytoplasm representing lack of activation (represented by overlap of black (unstimulated) and orange (stimulated) similarity scores on the associated histogram). (D) The median similarity score represents NF-κB nuclear translocation and was graphed for a subset of treated and control mice. Scatter plots display data from individual mice with black lines showing median values. ***P < 0.001, ****P < 0.0001, two-tailed Mann–Whitney U test. Data shown are representative of two independent experiments with n indicated for each experiment shown.