Figure 5.

Various immunostimulatory signals induce similar CV and R47H hTREM2 cleavage from the cell surface. (A) Representative flow cytometry plots demonstrating that both CV and R47H hTREM2 are lost from the cell surface of transduced RAW264.7 cells upon treatment with LPS, TNFα, or IFNγ. (B) Quantification of gMFI of LPS, TNFα, and IFNγ treated cells relative to untreated cells. Shown is mean ± SEM for three independent experiments. (C) Adam17 was knocked out in CV- and R47H-expressing RAW264.7 cells using lentivirally delivered Cas9 and guide RNA. LPS-stimulated cells were stained for hTREM2. Nontransduced cells (ctrl; gray shaded) uniformly lost hTREM2 surface expression. Transduced cells (solid red line) had two populations, one that retained hTREM2 surface expression (+) and one that lost hTREM2 surface expression (−), and these two populations were sorted by FACS. (D) Immunoblot for Adam17 demonstrates that the CV and R47H (+) populations have highly efficient knockout of Adam17 protein expression relative to either ctrl or (−) populations. (E) Experiments conducted as in A and B using Adam17 KO cells show that the effects of LPS, TNFα, and IFNγ on hTREM2 surface expression are ablated in Adam17-deficient cells. Shown is mean ± SEM for three independent experiments. **, P < 0.01; ****, P < 0.0001 by one-way ANOVA with Holm-Sidak multiple comparisons testing.