Fig. 5: P. anaerobius induces MDSC activation via the interaction between lytC_22 and Slamf4.
a, Levels of TLR and SLAMF mRNA expression in MDSCs treated with PA-CM or control broth (left); n = 2 biologically independent samples. Western blot showing the levels of Slamf4 protein in MDSCs treated with PA-CM or broth (right). b, Slamf4 mRNA in MDSCs after recombinant lytC_22 treatment; n = 3 biologically independent samples. c–e, MDSCs were pre-incubated with 20 μg ml−1 anti-Slamf4 (a-Slamf4), followed by lytC_22 treatment. c, MFI of Arg1 and iNOS in MDSCs; n = 5 biologically independent samples. d, IFN-γ and GzmB expression in CD4+ and CD8+ T cell populations after co-culture with MDSCs; n = 3 (lytC_22) and 4 (Ctrl, Ctrl + a-Slamf4 and lytC_22 + a-Slamf4) biologically independent samples. e, CD8+ T cell proliferation after co-culture with MDSCs at a 1:1 ratio; n = 6 biologically independent samples. Percentages in the flow plots are the percentages of subsequent cell divisions in total CD8+ cells. f, GST-tag pulldown assay showing the interaction between GST–lytC_22 and endogenous Slamf4 in MDSCs. g, GST-tag pulldown of GST–lytC_22 with recombinant hSLAMF4. f,g, n = 3 independent experiments, with similar results; a-GST, anti-GST and IP, immunoprecipitation. h, MST assay for direct binding between lytC_22 and hSLAMF4. The dissociation constant (Kd) is provided. The blue colour band indicates the time for the instrument to settle. The pink color indicates the MST on time chosen in MO.Affinity Analysis software. a–e,h, Data are the mean ± s.e.m. P values were calculated using an unpaired two-tailed Student’s t-test (a,b) or one-way ANOVA, followed by Tukey’s post-hoc test (c–e).
