Fig 2: 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).Source data

Fig 3: P. anaerobius activates MDSCs activation via lytC_22/Slamf4 interaction.a. The 50–100 kDa fraction from Pa-CM was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. b. Representative images of MDSCs migrated to the lower chamber and quantification of MDSC migration towards different concentration of lytC_22 (n = 4 biologically independent samples). Scale bar: 100 μm. c. tSNE plots showing expression levels of Cd244a in MDSCs clusters from tumours treated with P. anaerobius or not (Ctrl, control). d. Percentage of CD244+ MDSCs in MDSCs after LytC_22 treatment was determined by FACS analysis. e. MDSCs were pretreated with lytC_22 and lytC_22 mutant protein. CD8+ T cell proliferation after co-culture with MDSCs. c-d n = 3 biologically independent sample. Data are presented as mean ± SEM. P values were calculated by one-way ANOVA followed by Tukey’s post-hoc test (b, e) and unpaired two-tailed Student’s t-test (d). Source data

Fig 4: Blocking the lytC_22–Slamf4 interaction can reverse the effect of lytC_22 and P. anaerobius on MDSC modulation.a, Schematic of the lytC_22–Slamf4 binding sites and their corresponding prediction result (pink, lytC_22; cyan, Slamf4 and yellow dash, hydrogen bond). LytC_22 binds to Slamf4 protein with a binding energy of −416.43 kcal mol−1. LytC_22 is attached to Slamf4 with seven key residues: Glu570, Asp653, Asp725, Tyr661, Asp712, Arg660 and Lys669. b, LytC_22 mutant protein was purified following substitution of key binding residues with alanine. GST-tag pulldown of the GST–lytC_22 mutant with recombinant hSLAMF4. c, MDSCs were treated with lytC_22 or its mutant. Flow cytometry was used to determine the MFI of Arg1 and iNOS in MDSCs; n = 3 biologically independent samples. d, Following MDSC pre-treatment with lytC_22 or lytC_22 mutant protein, the expression of IFN-γ and GzmB in CD4+ and CD8+ T cells was assessed after co-culture with MDSCs; n = 4 biologically independent samples. e–g, Anti-Slamf4 reversed lytC_22-induced anti-PD1 resistance in the MC38 allograft mouse model; n = 6 mice per group. e, Tumour growth (left) and tumour weight (left). f, Percentage of MDSCs (left) as well as Arg1+ (middle) and iNOS+(right) MDSCs in tumours from mice in the different treatment groups. g, Percentage of IFN-γ+CD8+ T cells in the tumours. c–g, Data are the mean ± s.e.m. P values were calculated using a one-way ANOVA, followed by Tukey’s post-hoc test (c,d) or a one-way ANOVA, followed by Fisher’s least significant difference test (e–g).Source data

Fig 5: Schematic model of P. anaerobius-driven anti-PD1 resistance in CRC and potential therapeutic approaches.P. anaerobius engaged with CRC cells via integrin α2/β1 receptor to promote nuclear translocation of NF-κB p65, which in turn stimulates release of CXCL1 to recruit CXCR2+ MDSCs to tumours. In addition, lytC_22 protein secreted from P. anaerobius directly activates Slamf4 receptor on MDSCs, leading to elevated arginase 1 (Arg1) and iNOS expression. P. anaerobius infection thus increased MDSCs accumulation and activation, leading to suppression of CD8+ T cells and non-response to anti-PD1 therapy. Blockade of integrin α2/β1 or Slamf4 could interrupt this process to impair MDSCs in tumour microenvironment and reactivate IFNγ expressing CD8+ T cells to overcome P. anaerobius-driven anti-PD1 resistance.