Journal: iScience

Article Title: A fully human pan VL9 HLA-E TCRm antibody enables functional dissection of HLA-E biology and checkpoint signaling

doi: 10.1016/j.isci.2026.115669

Figure Lengend Snippet: ABX002 exhibits peptide selectivity for fSP/HLA-E complexes (A) ELISA-based binding of ABX002 and 3D12 to peptide/HLA-E complexes. Left: 3D12; right: ABX002 binding to a panel of peptides, including fSPs and control peptide HLA-E complexes. Red and blue indicate fSP/HLA-E detection by 3D12 and ABX002, respectively; green denotes detection of non-canonical peptides or non-SPs presented by HLA-E; yellow indicates detection of fSP/HLA-E orthologs from mouse or non-human primates. (B) ELISA comparing ABX002 and 3D12 binding to HLA-E isoforms. Left: 3D12; right: ABX002 binding to SPs or control peptide presented by either HLA-E01:03 or HLA-E∗01:01. (C) Affinity measurements of ABX002 for all fSP/HLA-E complexes compared to NKG2A. Binding kinetics was assessed using ResoSens Ultra Mab Pro. (D) Cell-based analysis of ABX002 binding to peptide-loaded K562 cells expressing HLA-E (K562-E). K562-E cells were pulsed with fSPs or control peptides and stained with 3D12 (red) or ABX002 (blue). Orange indicates non-canonical peptides, and green represents pathogen-derived peptides. Dashed line represents the basal detection level of 3D12 in unpulsed cells or of ABX002 in the absence of pulsed peptide. (E) Alanine scanning of the L-G fSP in K562-E cells. ABX002 binding is quantified as gMFI of the indicated peptide normalized to gMFI of L-G × 100. Dashed line indicates ABX002 binding to cells pulsed with wild-type peptide, used as a reference for comparing other pulsed conditions. (F) Titration of ABX002 on K562-E cells loaded with fSPs or control peptides. (G) HLA-E tetramer staining of NKG2A + NK-92 cells. L-G/HLA-E tetramer staining was assessed in the presence or absence of HLA-E blockade using 3D12 or ABX002 to disrupt the NKG2A axis. HSP60/HLA-E tetramer used as control protein for this assay. Data are represented as mean ± SD of n = 3. See also .

Article Snippet: K562 cells were cultured in IMDM supplemented with 10% FBS (Cytiva), 2% penicillin/streptomycin (ATCC) and 2mM glutamine (ATCC).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Control, Expressing, Staining, Derivative Assay, Titration

Journal: iScience

Article Title: A fully human pan VL9 HLA-E TCRm antibody enables functional dissection of HLA-E biology and checkpoint signaling

doi: 10.1016/j.isci.2026.115669

Figure Lengend Snippet: ABX002 unleashes NK- and CTL-mediated cytotoxicity against target cells (A) NK cell mediated cytotoxicity using primary NK cells against IFN-γ treated JY cells pulsed with L-C3 or PODXL2 peptides in the presence of ABX002 (effector-silent format, ES), anti-NKG2A, or corresponding control antibodies. (B) Cytotoxic responses of primary NK cells against K562-E cells positively sorted for fSP/HLA-E complexes, assessed in the presence of ABX002 (ES), anti NKG2A, or control antibodies. (C) NK cell mediated cytotoxicity against RPMI-8226 tumor cells by primary NK cells in the presence of ABX002 (ES) or control antibody. (D) CTL-mediated cytotoxicity by NKG2A + and NKG2A − Flu/M1-specific CD8 + T cells against IFN-γ treated JY cells pulsed with M1 peptide, in the presence of ABX002, anti-NKG2A, or controls. Cytotoxicity was assessed at 1:1 (left) and 1:3 (right) target to effector (T:E) cell ratios. Data are represented as mean ± SD. Sample sizes: (A) n = 5, (B) n = 6, (C) n = 4, (D) n = 2. Statistical analysis was performed using one-way ANOVA (A, B, D), unpaired t test (C). Significance thresholds: p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗), p < 0.0001 (∗∗∗∗). Error bars represent mean ± SD. Sample sizes: (A) n = 5, (B) n = 6, (C) n = 4, (D) n = 2, and (E) n = 10. See also .

Article Snippet: K562 cells were cultured in IMDM supplemented with 10% FBS (Cytiva), 2% penicillin/streptomycin (ATCC) and 2mM glutamine (ATCC).

Techniques: Control

Journal: Nature Communications

Article Title: Whole-protein screening and multi-modal profiling of antigen-specific CD4 + T cells at single-cell resolution

doi: 10.1038/s41467-026-72396-7

Figure Lengend Snippet: a Genetic design encoding the class II SCT protein. The MHC α and β chains are connected to the antigen through flexible linkers (L1, L2, and a partial invariant chain pIi), followed by purification tags (AviTag/HisTag). A secretion signal is placed upstream of the α chain to facilitate protein export. Created in BioRender . b Streamlined workflow for high-throughput SCT plasmid construction and protein expression. High-throughput production of SCT libraries is enabled by automated primer design and an optimized pipeline for plasmid assembly and protein expression/purification. Created in BioRender . c Flow cytometric assay to assess binding of SCT tetramers of various common class II HLA alleles to their cognate TCRs ( n = 3 ). Previously reported TCR-antigen pairs: DRB1*01:01 (DR1) – mutTPI 23-37 :T28I, DRB1*11:01 (DR11) – HIV Gag 299-311 , DRB1*07:01 (DR7) – ADM2 51–65 , DPB1*04:01 (DP4) – mutPly 427–441 (Supplementary Data ). d Comparison of the occurrence of aromatic residues F/W/Y in 2-mers that are positively correlated with SCT expression versus those that are negatively associated. The p value was calculated using Fisher’s exact test on the full 2 × 2 contingency table comparing F/W/Y presence across groups. e A 23-Element DRB1*01:01 CEFT SCT library. SCT expression was evaluated through SDS-PAGE. L, molecular weight ladder; +, protein standard for quantification of expression level. Schematics created in BioRender . f Antigen-specific T cells were detected through dual tetramer staining to enhance the true positive rate. An HIV Gag 41–56 SCT tetramer was used to exclude non-specific binding cells. A representative flow cytometry plot is shown. g Tetramer binding validation of 16 identified CD4 TCRs, each reactive to one of the five CEFT antigens (A–E) ( n = 3 ). Error bars represent standard deviation. h Schematic of peptide-pulsed activation assay. TCR-transduced NFAT-GFP Jurkat cells were co-cultured overnight with peptides and DR1-K562 cells. Created in BioRender . i T cell activation measured through the percentage of GFP+ Jurkats ( n = 3 ). P < 0.01 for each group relative to the negative control peptide. Statistical significance was determined by a one-tailed independent t -test assuming equal variances. All replicates are biological replicates. All bar plots ( n = 3 ) are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: DR1+ K562 cells were established from WT K562 (ATCC, CCL-243), which lack functional expression of wild-type class II pMHC , by lentivirally transducing a plasmid encoding the DRA/DRB1*01:01 allele with the transmembrane and cytoplasmic tail domains.

Techniques: Purification, High Throughput Screening Assay, Plasmid Preparation, Expressing, Flow Cytometry, Binding Assay, Comparison, SDS Page, Molecular Weight, Staining, Biomarker Discovery, Standard Deviation, Activation Assay, Cell Culture, Negative Control, One-tailed Test

Journal: Nature Communications

Article Title: Whole-protein screening and multi-modal profiling of antigen-specific CD4 + T cells at single-cell resolution

doi: 10.1038/s41467-026-72396-7

Figure Lengend Snippet: a Design of an SCT library with overlapping 15-mer peptides spanning the entire RBD of the SARS-CoV-2 spike protein. b . Schematic overview for the experimental workflow of high-throughput screening. 50 PBMC samples from 22 HLA-DR1+ participants were barcoded by timepoint, pooled, enriched for CD4 + T cells, stained with the 64-element SCT-dextramer pool, FACS-sorted, and sequenced. Single-cell analysis included RNA expression profiling, TCRαβ sequencing, antigen-MHC pairing, and timepoint identification. Comparison of genetic variants between transcriptome and whole-genome sequencing (WGS) was used to demultiplex patient identity (Methods). Created in BioRender . c Correlation between SCT expression levels, quantity of antigen-specific cells captured, and predictions from class II antigen-presentation algorithms. SCT expression quantified prior to purification ( n = 2–4 biological replicates). Data were shown as mean ± SEM. %Rank_EL, percentile rank of eluted ligands. d Clonal and patient diversity in CD4 + T cell responses to each SARS-CoV-2 antigen. Antigens are color-coded based on their parent protein. e Tetramer binding validation of SARS-CoV-2-specific CD4 TCRs. A representative subset of SARS-CoV-2 TCRs ( n = 10) from Fig. 2d were selected and validated through SCT tetramer binding assays with biological triplicates. A DR1-restricted influenza A M1 17–31 SCT was used as the negative control. f Peptide-pulsed activation of SARS-CoV-2 CD4 TCRs in NFAT-GFP Jurkats ( n = 3). e , f P < 0.0001 for each group relative to the negative control peptide, determined by one-tailed independent t -test assuming equal variances. Exact P values are provided in the Source Data. All replicates are biological replicates. All bar plots ( n = 3) are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: DR1+ K562 cells were established from WT K562 (ATCC, CCL-243), which lack functional expression of wild-type class II pMHC , by lentivirally transducing a plasmid encoding the DRA/DRB1*01:01 allele with the transmembrane and cytoplasmic tail domains.

Techniques: High Throughput Screening Assay, Staining, Single-cell Analysis, RNA Expression, Sequencing, Comparison, Expressing, Immunopeptidomics, Purification, Binding Assay, Biomarker Discovery, Negative Control, Activation Assay, One-tailed Test

Journal: Nature Communications

Article Title: Whole-protein screening and multi-modal profiling of antigen-specific CD4 + T cells at single-cell resolution

doi: 10.1038/s41467-026-72396-7

Figure Lengend Snippet: a Antigen coverage across oncogenic proteins E6 and E7 in the SCT library for whole-protein screening. Left panel schematics created in BioRender . b Schematic overview of TCR repertoire profiling integrated with other key modalities. Created in BioRender . c Antigen specificity validation of HPV-specific CD4 TCRs (H1–H5) through tetramer binding and peptide-pulsed activation assays ( n = 3). **** P < 0.0001, * P < 0.05, ns P > 0.05 labeled for each group relative to the negative control peptide. d Polyfunctionality of TCR-transduced primary CD4 + T cells assessed by cytokine production (IFNγ, TNF, IL2, and GZMB) via ELISA assay following antigen stimulation ( n = 3). OD 450 values were normalized to TCR expression levels across each cytokine. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and unlabeled tiles indicate P ≥ 0.05, all relative to negative control peptides. e Cytotoxicity of TCR-transduced CD4 + T cells against DR1-K562 cells pulsed with cognate HPV-16 E6 peptides, monitored over 42 h ( n = 2). The heat map summarizes mean target cell killing at the 42-h time point. Non-cognate peptides served as negative controls for each TCR. f Cross-reactivity of TCR H2 and H5 against human self-antigens ( n = 2). H2- and H5-TCR-transduced T cells were co-cultured with LCLs pulsed with peptides identified from BLAST search and motif scans (Supplementary Data ). H2 TCR used a single 1 µM dose; H5-TCR was dose titrated at 1, 2, and 10 µM. g Alloreactivity screen. H2- and H5-TCR-transduced CD4 + T cells were tested against 41 LCL lines representing 92 distinct class II HLA alleles ( n = 2). DRB1*01:01-positive LCLs ( n = 4) pulsed with peptide L-2 (E6 91–107 ) or F-2 (E6 129–142 ) served as positive controls. Alloreactivity of H5 to HLA-DRB1*13:05-positive LCLs is marked with arrows. h Confirmation of H2 TCR reactivity to naturally processed E6 antigen. DRB1*01:01+ LCL (GM17281B) was titrated with full-length E6 protein (0–10 μg/mL), co-cultured with H2 TCR-transduced CD4 + T cells, and evaluated for IFNγ secretion ( n = 2). A DRB1*01:01- LCL (GM12244A) served as a negative control. Statistical significance was determined by a one-tailed independent t -test assuming equal variances. All replicates are biological replicates. All bar plots where n = 3 are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: DR1+ K562 cells were established from WT K562 (ATCC, CCL-243), which lack functional expression of wild-type class II pMHC , by lentivirally transducing a plasmid encoding the DRA/DRB1*01:01 allele with the transmembrane and cytoplasmic tail domains.

Techniques: Biomarker Discovery, Binding Assay, Activation Assay, Labeling, Negative Control, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, One-tailed Test

Journal: bioRxiv

Article Title: Compact adenine base editors to enable therapeutic rescue of Duchenne muscular dystrophy

doi: 10.64898/2026.05.08.723843

Figure Lengend Snippet: (A) Maximum-likelihood phylogenetic tree of MG102 Cas9d homologs generated using MAFFT-linsi multiple sequence alignment and RAxML. The reconstructed ancestral protein is indicated by the circle. Scale bar denotes substitutions per site. (B) Pairwise protein sequence alignment of MG102-2 and MG102-71. Conserved residues are shown in green and non-conserved residues are shown in white. (C) PAM SeqLogo of MG102-71 based on NGS of cleaved plasmids from a plasmid PAM library tested with MG102-2 guide scaffold. (D) Indel editing efficiencies of ancestral MG102 variants in K562 cells using the wild-type MG102 sgRNA scaffold across 8 spacers targeting AAVS1 loci adjacent to NRC PAMs. Values represent the mean of two biological replicates.

Article Snippet: Hepa1-6 (ATCC #CRL-1830) and K562 (ATCC #CCL-243) cells were cultured in IMDM + GlutaMAX (Corning) supplemented with 10% FBS (Corning) for 1–2 passages prior to nucleofection.

Techniques: Generated, Sequencing, Plasmid Preparation

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: (A) K562 cells were infected with the indicated Salmonella ( S .Tm) strain harboring the intracellular vacuolar p ssaG -GFP reporter for 30 min, followed by Gentamicin treatment and reporter maturation for an additional 3.5 h. Overlays of a single-plane image for the differential interference contrast (DIC) channel and maximum intensity projections for the GFP channel are shown. Scale bars: 10 µm. (B-D) K562 and HeLa cells were infected in parallel as in A and (B) analyzed by flow cytometry to determine (C) the percentage of infected ( ssaG -GFP-positive) cells. (D) Infection with a panel of Salmonella mutants reveals the T3SS-1 effector requirements for invasion of K562 and HeLa cells. (E) Intracellular Salmonella replication in K562 and HeLa cells was assessed by CFU (colony forming units) plating at 2 h and 18 h p.i. in a Gentamicin protection assay. Data is plotted as CFU/well, with a well harboring ∼750’000 cells. (F-G) Establishment of an early interaction assay with constitutively fluorescent Salmonella p rpsM -mCherry strains inoculated with K562 cells. (F) Fluorescence microscopy confirms specific detection of cell-associated bacteria without contamination by unbound bacteria. Scale bars: 10 µm. (G) Parallel infection of K562 cells with Salmonella wt, Δ4 and Δ invG indicates that both adhesion and early invasion are quantified, and reveals a role for T3SS-1 during stable binding. (H) Schematic of the experimental setup for early interaction and invasion assays in round-bottom 96-well plates compatible with flow cytometry. Data is plotted as mean + range of 3 replicates (D, E); or as mean + standard deviation (sd) of ≥ 4 replicates (C, G). Statistical analysis was performed by 2-way ANOVA (C-E, G) with Tukey’s honestly significant difference (TukeyHSD) post-hoc test (D, E, G). **, p < 0.01; ***, p < 0.001, MFI; median fluorescence intensity.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Infection, Flow Cytometry, Fluorescence, Microscopy, Bacteria, Binding Assay, Standard Deviation

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: (A) K562 infection with Salmonella ( S .Tm) wt p ssaG -GFP for 30 min in different formats yields comparable invasion efficiencies. (B) Infection in 96-well plates allows for a high throughput invasion assay, in which multiple strains and MOIs can be compared in a single experiment. (C) An invasion assay with the vacuolar p ssaG -GFP and cytosolic p uhpT -GFP reporters reveals a small cytosolic Salmonella subpopulation in K562 cells. (D) An adhesion assay using Cytochalasin-D (CytD) treatment to block uptake of Salmonella wt results in low numbers of cell-associated bacteria regardless of the media type. Different media were tested because the presence of serum might reduce bacterial association with host cells, and hence supplementation with BSA is preferable. 10% FCS: complete RPMI (RPMI/10% FBS/1 mM NaPyr; see ); 1% BSA: infection medium (RMPI/1% BSA; see ). (E) Effect of infection time on Salmonella association with K562 cells to establish an early interaction assay. Data is plotted as mean + range of 2 (D) or 3 (A) replicates, or as mean + sd of 6-10 replicates (B, C, E). Statistical analysis was performed by 2-way ANOVA (A-C, E) and TukeyHSD (A, B, E). Comparisons to wt are indicated in B, and comparisons to 30 min in E. ***, p < 0.001.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Infection, High Throughput Screening Assay, Invasion Assay, Cell Adhesion Assay, Blocking Assay, Bacteria

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: (A-B) Overnight (o/n) Taxol treatment arrests K562 cells in mitosis. (A) Untreated (top) or o/n Taxol-treated (bottom) K562 cells were permeabilized with 0.1% Triton-X-100 and stained with propidium iodide (PI) to distinguish between G1 and G2/M phase cells based on the amount of DNA per cell (left panels), with G2/M cells displaying twice the intensity of G1 cells. G2 and M cells can be distinguished based on their side scatter (SSC), as granularity decreases upon nuclear membrane degradation during mitosis. (B) The percentages of cells assigned to each cycle phase show an increase in the mitotic population for o/n Taxol-treated cells. Data is plotted as mean + sd of 4 replicates and statistical analysis was performed by 2-way ANOVA and TukeyHSD. (C) Filipin staining indicates an increase in cell surface cholesterol levels in mitotic-arrested cells. Data is plotted as mean + sd of 4 replicates and statistical analysis was performed by Kruskal-Wallis test with Dunn’s post-hoc test and Benjamini-Hochberg adjustment for multiple comparisons. (D) No preferential Salmonella targeting of mitotic-arrested cells was observed in an early interaction assay. Data is plotted as mean + sd of ≥ 11 replicates and statistical analysis was performed by 2-way ANOVA and TukeyHSD. (E) Subsequent Filipin staining of infected cells and calculation of the Filipin MFI ratio in Salmonella p rpsM -mCherry-positive over -negative cells reveals that cholesterol-enriched cells are preferentially targeted across all conditions. Pooled data for all MOIs >0 from 2 replicates is plotted. In boxplots, the height of the boxes represents the interquartile range (IQR), whereas the horizontal line depicts the median. Whiskers extend to the most extreme data point within 1.5x IQR. All data points are indicated as circles. (F-H) Methyl-β-cyclodextrin (MBCD) treatment for 30 min reduces (F) cell surface cholesterol levels as determined by Filipin staining, (G) Salmonella early interaction with and (H) invasion of K562 cells. Data is plotted as mean + sd of 6 (F, G) or 8 (H) replicates and statistical analysis was performed by Mann-Whitney U test (F) or 2-way ANOVA, whereby significance for the factor ‘treatment’ is indicated (G, H). (I-J) Simvastatin treatment for 48 h reduces (I) cholesterol levels and (J) Salmonella invasion. Data is plotted as mean + range or 3 replicates (I) or mean + sd of 9 replicates (J). Statistical analysis was performed by Mann-Whitney U test (I) or 2-way ANOVA, whereby significance for the factor ‘treatment’ is indicated (J). Dotted lines in C, F and I depict the MFI for unstained samples. *, p < 0.05; **, p < 0.01; ***, p < 0.001; MFI, median fluorescence intensity.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Staining, Membrane, Infection, MANN-WHITNEY, Fluorescence

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: Transcriptome and proteome analysis of K562 cells at steady-state and comparison to differentiated human 2D enteroid-derived IEC monolayers (data from ). (A, E) Transcripts with TPM (transcript per kilobase million) values >0 are plotted in a histogram, bins containing (A) well-known high abundance transcripts or (E) transcripts for inflammasome components and caspases are highlighted. (B, F) Lists of transcripts highlighted in A, E. For K562 cells, the mean of 4 replicates is depicted, while the mean of 3 replicates for independent enteroid lines derived from 2 patients is shown for IECs. (C, G) Identified proteins (≥1 unique + razor peptide) with TPA (total protein amount in fmol/µg total protein) values >0 are plotted in a histogram, bins containing (C) high abundance proteins or (G) inflammasome components or caspases are highlighted. (D, H) Lists proteins highlighted in C, G. The mean of 3 (IECs) or 4 (K562) replicates is depicted.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Comparison, Derivative Assay

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: (A-D) Transcriptome and proteome analysis of K562 cells at steady-state and comparison to differentiated human 2D enteroid-derived IEC monolayers (data from ). (A) Transcripts with TPM (transcript per kilobase million) values > 0 are plotted in a histogram, bins containing transcripts for HS or CS core proteins are highlighted. (B) List of TPM values for transcripts highlighted in A. The mean of 4 replicates is depicted for K562, while the mean of 3 replicates for independent human enteroid lines derived from 2 patients is shown for IECs. (C) Identified proteins (≥1 unique + razor peptide) with TPA (total protein amount in fmol/µg total protein) values > 0 are plotted in a histogram, bins containing HS or CS core proteins are highlighted. (D) List of proteins highlighted in C. The mean of 3 (IECs) or 4 (K562) replicates is depicted. Asterisks indicate identified proteins with <3 unique + razor peptides. (E-F) Analysis of (E) HS and CS disaccharides and (F) CS sulfation patterns plotted as mean + range of 6 replicates with 1-4x 10 7 cells per replicate. ND, not detected.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Comparison, Derivative Assay

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: (A-B) Neuraminidase (Neu) treatment of K562 cells for 1 h (A) reduces cell surface glycosylation as assessed by Wheat germ agglutinin (WGA) staining, and increases (B) Salmonella wt and Δ4 early interaction and (C) Salmonella wt invasion of host cells. Data is plotted as mean + sd of 4 replicates (A), ≥10 replicates (B), or 8 replicates (C). Statistical analysis was performed by Mann-Whitney U test (A) or 2-way ANOVA (B-C), whereby significance for the factors ‘strain’ and ‘treatment’ is indicated. (D) Deletion of the large SiiE adhesin encoded on SPI4, but not removal of the fimbrial tip protein FimH, increases Salmonella invasion of K562 cells. (E) Deletion of flagellar subunits (Δ fljBfliC ) increases Salmonella invasion efficiency in K562 cells compared to a non-motile, but still flagellated strain (Δ motA ). Additional removal of SiiE (Δ fljBfliC Δ SPI4 ) further facilitates invasion. Infection was allowed to proceed for 60 min to allow non-motile bacterial strains to reach the K562 cells in the absence of centrifugation. In D-E, data is plotted as mean + sd of ≥ 5 replicates and statistical analysis was performed by 2-way ANOVA and TukeyHSD. *, p < 0.05; ***, p < 0.001; MFI, median fluorescence intensity.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Glycoproteomics, Staining, MANN-WHITNEY, Infection, Centrifugation, Fluorescence

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: (A-D) Transcriptome (A-B) and proteome (C-D) analysis reveals that differentiated human IECs in 2D enteroid-derived monolayers express various transmembrane mucins and the epithelial marker EPCAM (data from ), whereas K562 cells display low to undetectable levels of mucin expression. (A) Transcripts with TPM > 0 are plotted in a histogram and bins containing transcripts for transmembrane mucins and other cell surface proteins are highlighted. (B) List of TPM values for transcripts highlighted in A. The mean of 4 replicates is depicted for K562, while the mean of 3 replicates for independent human enteroid lines derived from 2 patients is shown for IECs. (C) Identified proteins (≥1 unique + razor peptide) with TPA > 0 are plotted in a histogram, bins containing transmembrane mucins and other surface proteins are highlighted. (D) List of proteins highlighted in C. The mean of 3 (IECs) or 4 (K562) replicates is depicted. Asterisks indicate identified proteins with <3 unique + razor peptides. (E-F) Western blot analysis indicates successful ectopic expression and time-dependent glycosylation of GP2, MUC1 and MUC1ΔCT (lacking the cytoplasmic tail) in K562 cells. The expected sizes for non-glycosylated proteins, marked by black arrowheads, are ∼64 kDa for GP2 and ∼175 kDa for MUC1, whereas the glycosylated versions, marked by green arrowheads, are estimated to be ∼70-80kDa for GP2 and >250 kDa for MUC1. Time is indicated as hours post induction. (G) Ectopic expression of GP2 reduces invasion of Salmonella wt and Δ fimH . Data is plotted as mean + sd of 8 (wt) or 4 replicates (Δ fimH ), whereby one replicate corresponds to an individually transfected culture infected with 2 different inocula (mean for bacterial replicates). (H) Ectopic expression the larger surface glycoprotein MUC1ΔCT strongly reduces Salmonella invasion, indicating that the glycocalyx forms a size- dependent barrier for host cell targeting. Data is plotted as mean + sd of 12 (wt) or 4 replicates (Δ SPI4 ), with replicates defined as in G. Statistical analysis was performed by 2-way ANOVA (G-H) and TukeyHSD (H). ***, p < 0.001; EV, empty vector.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Derivative Assay, Marker, Expressing, Western Blot, Glycoproteomics, Transfection, Infection, Plasmid Preparation

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: (A-B) Viability as assessed by PI staining (A) and relative cell size measurements based on median FSC (B) following transfection with pMEP4-GP2. Data is plotted as mean + sd of 8 replicates. (C) Staining with respective primary and secondary antibodies listed in , followed by flow cytometry confirms successful expression of GP2. Data is plotted as mean + sd of 4 replicates. (D-E) Viability as assessed by PI staining (D) and relative cell size measurements based on median FSC (E) following transfection with pMEP4-MUC1ΔCT. Data is plotted as mean + sd of 12 replicates. Time is indicated as hours post induction. (F) Both expression of full-length MUC1 and a truncated version lacking the cytoplasmic tail (MUC1ΔCT) in K562 cells results in massive reduction of Salmonella wt and Δ SPI4 invasion. Data is plotted as mean + range of 3 replicates, except for the control (EV) infected with Δ SPI4 , where only 1 replicate is plotted. A replicate corresponds to an individually transfected culture infected with 2 different inocula (mean for bacterial inocula). (G-H) Viability as assessed by PI staining (G) and relative cell size measurements based on median FSC (H) upon treatment of MUC1-transfected K562 cells with the mucinase StcE for 2 h. Data is plotted as mean + sd of 4 replicates. (I-J) K562 cells ectopically expressing HiBiT-tagged MUC1 versions with varying numbers of VNTRs, resulting in different lengths, display comparable cell viability as assessed by PI staining (I) and relative cell size measurements based on median FSC (J). (K) WGA staining of K562 cells expressing MUC1 versions of different lengths reveals that glycosylation levels are dependent on the number of VNTRs. Data is plotted as mean + sd of 6 replicates. Statistical analysis in C, F and K was performed by 2-way ANOVA and TukeyHSD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; EV, empty vector; MFI, median fluorescence intensity.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Staining, Transfection, Flow Cytometry, Expressing, Control, Infection, Glycoproteomics, Plasmid Preparation, Fluorescence

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: (A-E) MUC1-transfected K562 cells were stained with (A) Jacalin, (B) PNA, (C) SNA-I, (F) UEA-I, or (G) WGA to assess potential increases in cell surface glycosylation and gain insight into glycosylation patterns. Jacalin (A), PNA (B) and WGA stainings (E) show a robust increase upon ectopic expression of MUC1. Data is plotted as mean + sd of 4 replicates. Dashed lines indicate the mean of all unstained samples. Statistical analysis was performed by Mann-Whitney U test. *, p < 0.05. (F-I) Transcriptome (F-G) and proteome (H-I) analysis of glycosyltransferases involved in the first 2 steps of mucin-type O-glycosylation towards core 1 structures in differentiated human IECs (data from ) and K562 cells. (F) Transcripts with TPM > 0 are plotted in a histogram and bins containing transcripts for selected glycosyltransferases are highlighted. (G) List of TPM values for transcripts highlighted in F. The mean of 4 replicates is depicted for K562, while the mean of 3 replicates for independent human enteroid lines derived from 2 patients is shown for IECs. (H) Identified proteins (≥1 unique + razor peptide) with TPA > 0 are plotted in a histogram, bins containing selected glycosyltransferases are highlighted. (I) List of proteins highlighted in H. The mean of 3 (IECs) or 4 (K562) replicates is depicted. MFI, median fluorescence intensity; EV, empty vector.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Transfection, Staining, Glycoproteomics, Expressing, MANN-WHITNEY, Derivative Assay, Fluorescence, Plasmid Preparation

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: Transcriptome data for glycosyltransferases obtained from K562 cells or differentiated human IECs (data from ) were employed to predict mucin-type O-glycan structures using the Human GlycoMaple tool available at the GlyCosmos portal ( https://glycosmos.org/glycomaple/Human ) . The predictions reveal a higher diversity of O-glycan structures in human IECs, but indicate that glycan structures generated by K562 cells are present also in IECs. Pathways including at least one step that is catalyzed by a glycosyltransferase expressed at < 1 TPM are considered to have a minor contribution in mucin-type O-glycosylation and are depicted with decreased opacity. A list of all glycosyltransferases considered in the analysis (numbered 1-22) and relevant lectin binding motifs (according to ) are included.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Glycoproteomics, Generated, Binding Assay

Journal: bioRxiv

Article Title: A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

doi: 10.64898/2026.05.11.724231

Figure Lengend Snippet: (A) WGA staining increases upon induced (18h) ectopic expression of MUC1. Data is plotted as mean + sd of 4 replicates. (B) A subpopulation of transfected cells does not successfully express MUC1 upon induction, as indicated by low WGA intensity comparable to background. (C-E) WGA staining following Salmonella infection of transfected and induced cells indicates that almost exclusively the non-expressing, WGA low subpopulation is targeted in MUC1ΔCT-transfected cells. (C) Virtually no ssaG -GFP+ WGA high double-positive cells (Q2) are observed in MUC1ΔCT-transfected cells infected with Salmonella wt. (D) WGA high cells are specifically detected in MUC1ΔCT-transfected samples. Pooled data for all MOIs is depicted. (E) Quantification of population sizes illustrated in C confirms that cells successfully expressing MUC1 largely remain uninvaded. Data for MOI 200 is depicted. In D- E, data is plotted as mean + sd of 4 replicates, whereby one replicate corresponds to an individually transfected culture infected with 2 different inocula (mean for bacterial replicates). (F-G) Treatment of MUC1-transfected K562 cells with the mucinase StcE (F) reverts increased cell surface glycosylation as assessed by WGA staining, and (G) restores Salmonella invasion (MOI 50). Data is plotted as mean + sd of 4 (F) or 7 (G) replicates. (H-J) K562 cells ectopically expressing HiBiT-tagged MUC1 versions with varying numbers of VNTRs, resulting in different lengths, were infected with Salmonella at MOI 50. (H) Luminesence-based detection of HiBiT-tagged MUC1 versions reveals robust and comparable expression of all constructs upon induction. (I) Early interaction and (J) invasion assays illustrate the size-dependent barrier effect of MUC1 towards Salmonella targeting of the K562 host cell surface. Data is plotted as mean + sd of 4 (H) or 6 (I-J) replicates, whereby one replicate is defined as described above. Statistical analysis was performed by 2-way ANOVA (A, D-J) and TukeyHSD (A, E-J). *, p < 0.05; **, p < 0.01; ***, p < 0.001; MFI, median fluorescence intensity; EV, empty vector; AU, arbitrary units.

Article Snippet: K562 CCL-243 cells (ATCC) were maintained in complete RPMI ( ) supplemented with 1x PenStrep (Gibco) at 37°C, 5% CO 2 in slightly tilted cell culture flasks at densities between 50’000 to 500’000 cells/mL.

Techniques: Staining, Expressing, Transfection, Infection, Glycoproteomics, Construct, Fluorescence, Plasmid Preparation