Fig 2: Rescue of GPI-anchored protein expression and TLR4 signaling by PIGH re-expression. (a) Western blot analysis of GPI-negative (GPI-) BLaER1 clones that were either transduced with a lentivirus encoding for GFP, PIGH-Flag, or PIGA-Flag, or not transduced (/) as a negative control. One representative blot of three is shown. Full-length blots are presented in Supplementary Figure S9. (b) GPI-negative BLaER1 cells stably expressing GFP, PIGH-Flag, or PIGA-Flag were analyzed by flow cytometry. The percentage of CD48-expressing cells among GFP-positive cells is shown as the mean + SD from n = 3 independent measurements. For comparison, CD48 surface expression of a GPI-positive (GPI +) BLaER1 monoclonal cell line is shown on the right-hand side. Two-way ANOVA followed by Dunnett’s multiple comparisons test was used to determine statistical significance. Results from comparing cells transduced with either GFP, PIGH-Flag, or PIGA-Flag with the negative control (not transduced, /) are shown (ns – not significant, ****p ≤ 0.0001). (c) GPI-negative BLaER1 cells stably expressing GFP or PIGH-Flag were transdifferentiated for 5 days and then analyzed by flow cytometry. The percentage of CD14-expressing cells among GFP-positive cells is shown as the mean + SD from n = 3 independent measurements. (d) IL-6 and IL-1β in the supernatant were determined by ELISA 16 h after the stimulation of BLaER1 monoclonal cell lines, stably expressing GFP or PIGH-Flag, with LPS or medium control (0). Values correspond to the mean + SD of n = 3 independent experiments. (e–g) BLaER1 clones stably expressing GFP or PIGH-Flag were stimulated for 16 h with four different TLR7/TLR8 ligands and the supernatant was used to determine IL-6 (e) and IL-1β (g) concentrations by ELISA, or type I IFN concentration by HEK blue assay (f). Data for stimulation with R848 40 ng/ml and 200 ng/ml, TL8-506 20 ng/ml, and CL264 200 ng/ml are depicted as mean + SD of n = 2 independent experiments. For all other stimuli and concentrations, the mean + SD of n = 3 independent experiments is shown. (c–g) BLaER1 cells stably expressing PIGH-Flag were compared to GFP-expressing control cells using unpaired t tests. Statistical significance is depicted as follows: ns—not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig 3: Loss of CD14 on the cell surface in BLaER1 monocytes is the result of a general GPI anchor defect. (a) CD14 mRNA expression of BLaER1 clones, quantified by qRT-PCR. Shown is the mean ± SD of n = 3 independent measurements, normalized to GAPDH expression. Dots represent individual values. (b) Western blot analysis of CD14 expression. One representative blot of two is shown. Full-length blots are presented in Supplementary Figure S9. (c) Quantification of the CD14 protein in (b). CD14 expression was normalized to the housekeeping protein β-actin. The mean ± SD of pooled data from three different monoclonal cell lines from n = 2 independent measurements is shown. Dots represent the individual values. (d–f) Surface expression of the GPI-anchored proteins CD48 and CD55 (d,f) and the transmembrane proteins CD45 and CD64 (e) was measured by flow cytometry in transdifferentiated (d,e) or undifferentiated (f) BLaER1 monoclonal cell lines. Shown is the percentage of cells expressing the protein of interest among all GFP-positive cells from n = 2 independent measurements (mean ± SD). Individual values are depicted as dots. (a,c–f) Monoclonal cell lines with higher responsiveness to LPS (dark blue) were compared to cell lines with lower responsiveness to LPS (light blue) by Mann–Whitney test (ns—not significant, **p ≤ 0.01).

Fig 4: F. nucleatum subsp. nucleatum confers Siglec-7-dependent protection against NK cell-mediated cancer cell killing(A) Schematic representation of target cell killing by YTS EV or YTS Siglec-7 cells. YTS empty vector (EV) or YTS Siglec-7 cells are activated almost exclusively by CD48-expressing target cells via their activating receptor 2B4. In the presence of bacteria, killing of target cells is inhibited via Siglec-7.(B) Staining of YTS EV or YTS Siglec-7 cells with Siglec-7 (upper histograms) and 2B4 (lower histograms) antibodies. Filled gray histograms represent staining with isotype control and secondary antibody only, respectively.(C) Staining of 721.221 or (D) BCBL-1 cells for Siglec-7 ligands with Siglec-7-Ig or of CD48 expression using anti-CD48 antibodies. (E) YTS EV or YTS Siglec-7 cells were precincubated or not with F. nucleatum subsp. nucleatum ATCC 23726 (Fnn 23726) at a ratio of 1:20 for 30 min at 37°C. The cells were then incubated with Calcein AM-labeled 721.221 or (F) BCBL-1 cells at an E:T ratio of 10:1 for 4 h at 37°C and the Calcein release into the supernatant was quantified. Each graph shows data from three independent experiments and each independent experiment is colored differently (white, blue, and black). Each experiment was performed in triplicates. Significance was tested using mixed-effects analysis with the Geisser-Greenhouse correction and Sidak’s multiple comparisons test (∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001).

Fig 5: KDM6A regulates CD48 expression in MM cells.a Normalized lysis percentage of KDM6A WT or KO H929 cells after co-culture with primary NK cells at different E:T ratios with IL-2 for 6 hours (mean ± SEM, n = 3 biologically independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided student’s t test). b Intracellular IFN-γ staining of primary NK cells co-cultured with KDM6A WT or KO H929 cells with IL-2 for 6 hours (mean ± SEM, n = 3 biologically independent experiments). **p < 0.01 (two-sided student’s t test). c Volcano plot of differentially expressed genes in KDM6A KO H929 cells compared with WT cells assessed by RNA-seq. d Venn diagram showing the overlapped enriched genes between the CRISPR screen and RNA-seq. e, Representative flow cytometry analysis of CD48 expression in H929 transfected with indicated sgRNAs. f q-RT-PCR for CD48 mRNA in H929 cells transfected with indicated sgRNAs. Data were normalized against GAPDH (mean ± SEM, n = 3 biologically independent experiments). ****p < 0.0001 (two-sided student’s t test). g, ChIP-seq density profiles for H3K27me3 on the CD48 gene in KDM6A WT and KO H929 cells. h, H3K27me3 ChIP-qPCR analysis at the CD48 gene in KDM6A WT and KO H929 cells (mean ± SEM, n = 3 biologically independent experiments). ***p < 0.001 (two-sided student’s t test). i, ATAC-seq density profiles at the CD48 promoter region in KDM6A WT and KO H929 cells. j q-RT-PCR for CD48 mRNA in newly diagnosed (N/D) (n = 7) and Dara-resistant (R/R) (n = 12) patient samples. Data were normalized against GAPDH (mean ± SEM). *p < 0.05 (two-sided student’s t test). Source data are provided as a Source Data file.