Journal: World Journal of Gastroenterology : WJG

Article Title: Investigation on correlation between expression of CD58 molecule and severity of hepatitis B

doi: 10.3748/wjg.v12.i26.4237

Figure Lengend Snippet: Content of CD58 in serum.

Article Snippet: Detection of membrane CD58 in PBMC One ml of PBMC sample was mixed with 10 μL of mouse anti-human CD58 antibody marked by fluorescence FITC (American Southern Biotech), incubated in the dark at room temperature, washed twice with PBS, and centrifuged at 2500 r/min before abandoning supernatant.

Techniques:

Journal: World Journal of Gastroenterology : WJG

Article Title: Investigation on correlation between expression of CD58 molecule and severity of hepatitis B

doi: 10.3748/wjg.v12.i26.4237

Figure Lengend Snippet: Content of membrane CD58 molecule in mild chronic hepatitis B.

Article Snippet: Detection of membrane CD58 in PBMC One ml of PBMC sample was mixed with 10 μL of mouse anti-human CD58 antibody marked by fluorescence FITC (American Southern Biotech), incubated in the dark at room temperature, washed twice with PBS, and centrifuged at 2500 r/min before abandoning supernatant.

Techniques: Membrane

Journal: World Journal of Gastroenterology : WJG

Article Title: Investigation on correlation between expression of CD58 molecule and severity of hepatitis B

doi: 10.3748/wjg.v12.i26.4237

Figure Lengend Snippet: Content of membrane CD58 molecule in severe hepatitis B.

Article Snippet: Detection of membrane CD58 in PBMC One ml of PBMC sample was mixed with 10 μL of mouse anti-human CD58 antibody marked by fluorescence FITC (American Southern Biotech), incubated in the dark at room temperature, washed twice with PBS, and centrifuged at 2500 r/min before abandoning supernatant.

Techniques: Membrane

Journal: Cancer cell

Article Title: CMTM6 shapes antitumor T cell response through modulating protein expression of CD58 and PD-L1

doi: 10.1016/j.ccell.2023.08.008

Figure Lengend Snippet: (A) Quantitative proteomics analysis of CMTM6-proficient (WT) and -deficient (KO) 8505C cells. Fold changes of significantly differentially expressed proteins (two-sided Student’s t test, p < 0.05) are depicted. (B) List of top 10 downregulated proteins in CMTM6-deficient 8505C cells. (C) Western blot analysis of CMTM6, CD58, and PD-L1 expression in parental 8505C cells (WT) and independent CMTM6-knockout clonal cells (CMTM6 KO). HSP90 served as a control. (D) Schematic illustration of the flow cytometry-based haploid genetic screen for modulators of CD58 expression. (E) Identification of modulators of CD58 expression by the haploid genetic screen depicted in (D). Each dot represents an individual gene, with the x axis indicating the number of disruptive insertions in each gene, and the y axis showing the fold changes in the frequency of unique insertions in the CD58 high population compared to the CD58 low population. Genes with a significant enrichment of insertions (two-sided Fisher’s exact test, FDR-corrected p < 0.05) in either the CD58 high or CD58 low populations are represented by light orange and blue dots, respectively. (F and G) CD58 expression levels in parental HAP1 cells (WT) and independent CMTM6-knockout clonal cells (CMTM6 KO). Levels of CD58 expression were determined by flow cytometry (F) and CMTM6 expression was analyzed by Western blot (G). HSP90 served as a control in the Western blot analysis. (H and I) Flow cytometry analysis of CD58 and PD-L1 expression in wild-type (WT), CMTM6-knockout (CMTM6 KO), CMTM6-overexpressing (WT + CMTM6 OE), and CMTM6-reconstituted (CMTM6 KO + CMTM6 OE) 8505C (H) and A375 cells with or without IFNγ exposure (I). Data represent the mean ± standard deviation of triplicates and were analyzed using a two-way ANOVA test (with Tukey’s multiple comparisons test). A p value greater than 0.05 indicates non-significance (ns), while a p value less than 0.0001 is denoted as **.

Article Snippet: Mouse anti-human CD58 BUV395 (Clone L306.4) , BD Biosciences , Cat# 745571; RRID: AB_2743091.

Techniques: Western Blot, Expressing, Knock-Out, Flow Cytometry, Standard Deviation

Journal: Cancer cell

Article Title: CMTM6 shapes antitumor T cell response through modulating protein expression of CD58 and PD-L1

doi: 10.1016/j.ccell.2023.08.008

Figure Lengend Snippet: (A) qPCR analysis of mRNA levels of CD58 in CMTM6-deficient and -proficient A375, 8505C, and RKO cells. (B) Stability of cell surface-expressed CD58, PD-L1, and MHC class I in parental (WT) and CMTM6-deficient (CMTM6 KO#6, CMTM6 KO#12) A375 cells. A375 cells were treated with IFNγ for 24 h and then individually incubated with APC-conjugated antibodies specific for CD58, PD-L1, or MHC class I at 4°C. After removing unbound antibodies, the cells were further incubated at 37°C for the indicated time periods, and the APC signal was measured by flow cytometry. The percentage of signal remaining at the indicated time points relative to time 0 is shown. (C) Stability of cell surface-expressed CD58 in the parental (WT) and CMTM6-deficient (CMTM6 KO#6, CMTM6 KO#12) A375 cells in the presence of the proteasome inhibitor bortezomib (Bor.) or the lysosome inhibitor concanamycin A (ConA). The untreated samples presented in (B) served as the control. Data acquisition and presentation were performed as described in (B). (D) Western blot analysis of cell lysates and indicated immunoprecipitates from A375 cells. HSP90 served as a control. (E) Western blot analysis of cell lysates and indicated immunoprecipitates by cell surface immunoprecipitation from A375 cells. For the cell surface immunoprecipitation, live cells were incubated with antibodies that recognize the extracellular domains of CD58 or PD-L1. After removal of unbound antibodies, the cells were lysed for (co)immunoprecipitation. HSP90 served as a control. The triangles indicate the position of background bands, which are present when the anti-CD58 antibody (R&D Cat# AF1689) was used for detection, whereas the anti-CD58 antibody (BioLegend, Cat# 330924) does not produce such background signal. Data represent mean ± standard deviation of at least triplicates (A–C) and were analyzed using unpaired Student’s t test. Statistical significance is indicated by *p < 0.05.

Article Snippet: Mouse anti-human CD58 BUV395 (Clone L306.4) , BD Biosciences , Cat# 745571; RRID: AB_2743091.

Techniques: Incubation, Flow Cytometry, Western Blot, Immunoprecipitation, Standard Deviation

Journal: Cancer cell

Article Title: CMTM6 shapes antitumor T cell response through modulating protein expression of CD58 and PD-L1

doi: 10.1016/j.ccell.2023.08.008

Figure Lengend Snippet: (A) Schematic illustration of the antigen-specific T cell-tumor cell coculture system. Primary T cells were isolated from human PBMCs, activated, and transduced with a MART-1-specific TCR. HLA-A2-positive tumor cells were loaded with MART-1 peptide and incubated with the TCR-transduced T cells. (B) Effect of CMTM6 loss on T cell activation. MART-1 TCR-transduced T cells were cocultured with MART-1 peptide-loaded CMTM6 wild-type (WT), CMTM6-knockout (CMTM6 KO), or CMTM6-reconstituted (CMTM6 KO + OE) A375 cells. After 24 h, expression of indicated T cell activation markers and cytokines in CD8 + and CD8 − T cells was determined by flow cytometry. To allow detection of TNFα and IL-2, brefeldin A was added 4 h before T cell harvesting. (C–E) Effect of PD-L1 blockade, CD58 blockade, and CMTM6 deletion on T cell activation and tumor cell viability. MART-1 TCR-transduced T cells were co-cultured with MART-1 peptide-loaded tumor cells in the presence of PD-L1-blocking antibody (atezolizumab, aPD-L1), CD58-blocking antibody (aCD58), or their combination. The coculture without antibody treatment (untreated) served as control. After 18–60 h of coculture, T cell activation and tumor cell viability were analyzed. (C) Flow cytometric analysis of T cells that were cocultured for 18 h with A375 cells in the absence or presence of the indicated blocking antibodies. Expression of CD137 was analyzed within CD3 + CD8 + cell populations stratified by CD2 expression (CD2 high : top 33%, CD2 inter : middle 33%, CD2 low : bottom 33%), as well as PD-1 expression (PD-1 + or PD-1 − ). Representative contour plots are presented. (D) Flow cytometric analysis of T cells that were cocultured with CMTM6 wild-type and -knockout A375 cells for 24 h. Percentages of CD137 + , and IL-2 + cells within the CD8 + CD3 + and CD8 − CD3 + cell populations are depicted. (E) Viability of CMTM6 wild-type and -knockout A375 cells after coculture with T cells for 60 h was determined by CellTiter-Blue Cell Viability Assay. The data presented depict relative tumor cell viability (relative to tumor cells that were cultured in the absence of T cells). (F and G) Viability of A375 cells, genetically modified as indicated, was assessed using the CellTiter-Blue Cell Viability Assay after 60 h of coculture with T cells. A375 cells with wild-type CMTM6 (Ctrl.) or CMTM6 knockout (CMTM6 KO), with or without additional CD58 knockout (CD58 KO) (F), or CD58 overexpression (CD58 OE) (G), were examined separately. Cells were either treated with a PD-L1-blocking antibody or a control IgG antibody, as specified. The data presented depict tumor cell viability relative to tumor cells that were cultured in the absence of T cells. Data represent mean ± standard deviation of at least triplicates (B) (D–G) and were analyzed using two-way ANOVA (Tukey’s multiple comparisons test). The statistical significance levels are indicated as follows: ns (not significant; p ≥ 0.05), * (p < 0.05), and ** (p < 0.0001).

Article Snippet: Mouse anti-human CD58 BUV395 (Clone L306.4) , BD Biosciences , Cat# 745571; RRID: AB_2743091.

Techniques: Isolation, Transduction, Incubation, Activation Assay, Knock-Out, Expressing, Flow Cytometry, Cell Harvesting, Cell Culture, Blocking Assay, Viability Assay, Genetically Modified, Over Expression, Standard Deviation

Journal: Cancer cell

Article Title: CMTM6 shapes antitumor T cell response through modulating protein expression of CD58 and PD-L1

doi: 10.1016/j.ccell.2023.08.008

Figure Lengend Snippet: (A–D) Tumor biopsies from 88 melanoma and 102 colon cancer patients were analyzed for CMTM6 and CD58 expression levels using immunohistochemistry. (A, C) Spearman correlation analysis was performed to evaluate the correlation between CMTM6 and CD58 expression levels in tumor cells. (B and D) The association between CMTM6 and CD58 expression levels in tumor cells was analyzed using chi-squared tests. The samples were divided into CMTM6-high, CMTM6-low, CD58-high, and CD58-low groups based on the median expression values, and the results were presented as contingency tables. (E) The association between CMTM6 and CD58 expression in tumor cells and the response to ICB therapies in the melanoma cohort. The expression levels of CMTM6 and CD58 were individually plotted against the patient’s response to therapy, classified as clinical benefit (Yes) or no clinical benefit (No). Patients with complete response (CR), partial response (PR), and stable disease (SD) lasting for 6 months or more were classified as having clinical benefit (Yes, n = 49), while patients with progressive disease or with an SD for less than 6 months were categorized as having no clinical benefit (No, n = 39). Comparisons were made between patients that showed clinical benefit (Yes) and those that showed no clinical benefit (No) using the Wilcoxon rank-sum test. Statistical significance is indicated by *p < 0.05.

Article Snippet: Mouse anti-human CD58 BUV395 (Clone L306.4) , BD Biosciences , Cat# 745571; RRID: AB_2743091.

Techniques: Expressing, Immunohistochemistry

Journal: Cancer cell

Article Title: CMTM6 shapes antitumor T cell response through modulating protein expression of CD58 and PD-L1

doi: 10.1016/j.ccell.2023.08.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-human CD58 BUV395 (Clone L306.4) , BD Biosciences , Cat# 745571; RRID: AB_2743091.

Techniques: Generated, Conjugation Assay, Virus, Recombinant, Mutagenesis, Staining, Plasmid Preparation, Sequencing, CRISPR, Knock-Out, shRNA, Software

Journal: bioRxiv

Article Title: Perfusion bioreactor culture of adipose-derived stromal cells on decellularized adipose tissue scaffolds enhances in vivo regeneration

doi: 10.1101/2020.04.10.036194

Figure Lengend Snippet: A) Representative immunostaining showing Ku80 labelled cells (red) counterstained with DAPI (blue). Scale bars represent 100 µm. B) Bar graph showing the density of Ku80 + DAPI + ASCs in the implants at 4 and 8 weeks. Data was compiled from 6 implants per group (N=6), with 3 cross-sections per scaffold taken from varying depths and 10 random fields analyzed per cross-section. Error bars represent standard deviation.

Article Snippet: To detect the human ASCs within the DAT implants, cross-sections were stained for human-specific Ku80 using a rabbit anti-Ku80 antibody (Cedarlane, Burlington, Canada; 1:100 dilution) followed by Alexa Fluor ® 594 conjugated goat anti-rabbit (1:200 dilution) secondary antibody as described above.

Techniques: Immunostaining, Standard Deviation

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: Screening for CD14 incorporation by lab-adapted HIV-1 isolates. ( A ) Schematic describing the virion capture assay used throughout. Briefly, antibodies or LPS (not depicted) were adsorbed onto 96-well plates and blocked before adding virus samples to allow precipitation of virus particles bearing the antigen of interest. Unbound virions were washed away and captured virions were lysed for quantitation of HIV-1 by Gag p24 ELISA. ( B ) Virion capture assay was performed on four HIV-1 isolates (IIIB, NL4-3, BaL, and SF162) produced by infected PBMCs using anti-CD14 (orange) and anti-gp120 (blue). Bars are isotype control-subtracted mean ± SD Gag p24 of duplicate assays and are representative of two independent experiments. ( C ) Western blot corroborating the incorporation of CD14. HIV-1 grown in monocyte-derived macrophages (MDMs) (BaL and SF162, two donors each) and PBMCs (IIIB, two donors; NL4-3, four donors) were concentrated, lysed, and resolved by SDS-PAGE before blotting for p24 (loading control), gp120 (positive control for viral surface protein), and CD14. Data are representative of three independent blots.

Article Snippet: Primary antibodies were diluted in TBST and added overnight at 4°C: human anti-HIV gp120 (mAb 39F, 0.15 μg/mL, NIH ARP #11437), mouse anti-HIV gp120 (mAb B23, 1:5, gifted from G. Lewis), rabbit anti-human CD14 (mAb ARC54507, 0.2 μg/mL, ABclonal #A21256), and mouse anti-HIV p24 (mAb AG3.0, 1:1,000, NIH ARP #4121).

Techniques: Virus, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Produced, Infection, Control, Western Blot, Derivative Assay, SDS Page, Positive Control

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: HIV-1 pseudovirus reliably models virion incorporation of CD14. ( A ) Schematic depicting the process of transfecting 293T cells with plasmids encoding the HIV-1 backbone (pSG3ΔEnv) and CD14 to produce PV bearing CD14 on the surface. ( B ) Confirming the transfection efficiency of 293T cells ectopically expressing CD14. Cells were transfected with backbone only (black histogram) or backbone plus CD14 (orange histogram) to produce WT or CD14 + PVs, respectively. Cells were collected and stained with PE-conjugated anti-CD14 to confirm cell-surface expression of CD14 available for incorporation. ( C ) Gating strategy to identify PV populations by FVM. Phosphate-buffered saline (PBS) or WT virions were acquired, and data are displayed on pseudocolored dot plots. Fluorescence data (y-axis) were calibrated to produce quantitative units of MESF (molecules of equivalent soluble fluorophore), which describe the number of fluorophores and, indirectly, the number of antigens detected. Side scatter data (x-axis) were calibrated to produce arbitrary units of scattering cross section (nm 2 ), which relates to the size and refractive index of particles. Gates to the far left (PBS and WT PV) represent the instrument’s optical noise and are present of every FVM plot. Virions are identified as the dense, monodispersed population (right gate) adjacent to the optical noise. ( D ) WT (top row) and CD14 + (bottom row) virions were stained with PE-conjugated isotype control (left column) or anti-CD14 (right column) (0.4 µg/mL) and analyzed by FVM. Gates were positioned above the unstained PV populations, identifying PE + events, and were set respective to each isotype control stain. PE MESF values are enumerated where PE + events are present (mean ± SD, n = 3 independent stains), describing the number of fluorophores detected in each PE + gate. Virus stocks used in C and D had a titer of 187 ng/mL (WT) and 114 ng/mL (CD14 + ) of p24 as measured by ELISA. ( E ) Virion capture assay was performed on WT (teal) and CD14 + (orange) PVs to corroborate CD14 incorporation. “Nil” represents PBS buffer, “mIgG2a” is an isotype-matched control, and “αCD14” is anti-CD14 mAb M5E2. Bars are mean ± SD p24 of four independently produced virus stocks and are representative of at least two independent assays.

Article Snippet: Primary antibodies were diluted in TBST and added overnight at 4°C: human anti-HIV gp120 (mAb 39F, 0.15 μg/mL, NIH ARP #11437), mouse anti-HIV gp120 (mAb B23, 1:5, gifted from G. Lewis), rabbit anti-human CD14 (mAb ARC54507, 0.2 μg/mL, ABclonal #A21256), and mouse anti-HIV p24 (mAb AG3.0, 1:1,000, NIH ARP #4121).

Techniques: Transfection, Expressing, Staining, Saline, Fluorescence, Refractive Index, Control, Virus, Enzyme-linked Immunosorbent Assay, Produced

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: CD14 remains functional in the viral envelope and binds to LPS. ( A ) FVM was used to read out a checkboard titration of LPS-bio and PE-SA on CD14 + virions to identify optimal staining concentrations. LPS-bio was incubated overnight at 4°C with virus samples and PE-SA was added for 4 h at 4°C the next day before acquisition. Gates to the left identify optical noise used for ( C ), and gates to the right identify PE + events that were set respective to each negative control PE-SA stain (top row). CD14 + virus stock shown in FVM plots had a titer of 57 ng/mL p24 as measured by ELISA. ( B ) The number of particles in the PE + gates of ( A ) were tabulated and heat mapped with blue, yellow, and red identifying low, intermediate, and high values, respectively. Higher particle counts identify LPS-bio and PE-SA stains that support CD14 + virions binding. ( C ) SI was calculated and plotted for each PE-SA concentration using the PE MESF measured in the PE + gates of ( A ). Peak SI identifies optimal staining pairs (orange stars and blue inverted triangles). ( D ) Incubation times for optimal PE-SA pairs from ( C ) were evaluated over a 16-h time course. Particle counts in PE + gates (right gates) are enumerated on each plot. CD14 + virus stock shown in FVM plots had a titer of 73 ng/mL p24 as measured by ELISA. ( E ) SI of PE MESF measured in ( D ) were plotted over time. All titration data were reproducible across two independent experiments using two independently produced virus stocks. All LPS-bio and PE-SA concentrations in A–E are in units of ng/mL and µg/mL, respectively.

Article Snippet: Primary antibodies were diluted in TBST and added overnight at 4°C: human anti-HIV gp120 (mAb 39F, 0.15 μg/mL, NIH ARP #11437), mouse anti-HIV gp120 (mAb B23, 1:5, gifted from G. Lewis), rabbit anti-human CD14 (mAb ARC54507, 0.2 μg/mL, ABclonal #A21256), and mouse anti-HIV p24 (mAb AG3.0, 1:1,000, NIH ARP #4121).

Techniques: Functional Assay, Titration, Staining, Incubation, Virus, Negative Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Produced

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: LPS binds to HIV-1 in a CD14-specific manner and does not passively associate with the lipid envelope. ( A ) Optimized LPS-bio (300 ng/mL, overnight, 4°C) and PE-SA (0.2 µg/mL, 4 h, 4°C) staining on CD14 + virions. Particle counts in PE + gates are enumerated on each plot. ( B ) Virion capture assay using immobilized untagged LPS. Ninety-six-well plates were coated with buffer (PBS) or LPS and virions with (CD14 + , orange bars) or without CD14 (WT, teal bars) were added. Captured virions were quantified by Gag p24 ELISA. Bars are mean ± SD of duplicate capture assays and are representative of three independent experiments. ( C ) FVM evaluating the contribution of recombinant LPS-binding protein (LBP) on LPS-bio binding to CD14 + virions. Varying amounts of recombinant LBP (rLBP) were mixed with LPS-bio prior to incubating with WT (top row) and CD14 + (bottom row) virus samples and staining with PE-SA. Gates define PE + events based on negative control (PE-SA only). WT and CD14 + virus stocks shown in FVM plots for ( A ) and ( C ) had a respective titer of 76 and 57 ng/mL p24 as measured by ELISA. ( D ) Particle counts in PE + gates from ( C ) were plotted as a function of PE-SA concentrations for WT (teal triangles) and CD14 + virions (orange circles). Data are mean ± SD of two independent virus stocks. ( E ) FVM measuring neutralization of LPS-bio binding to virions. Anti-CD14 (M5E2) was added to WT (top row) and CD14 + (bottom row) virions before incubation with LPS-bio and PE-SA. Gates are as described for ( C ). WT and CD14 + virus stocks shown in FVM plots had a respective titer of 368 and 141 ng/mL p24 as measured by ELISA. ( F ) Particle counts in PE + gates from ( E ) were plotted over the range of anti-CD14 concentrations tested for WT (teal triangles) and CD14 + (orange circles) virions. Data are representative of two independent virus stocks.

Article Snippet: Primary antibodies were diluted in TBST and added overnight at 4°C: human anti-HIV gp120 (mAb 39F, 0.15 μg/mL, NIH ARP #11437), mouse anti-HIV gp120 (mAb B23, 1:5, gifted from G. Lewis), rabbit anti-human CD14 (mAb ARC54507, 0.2 μg/mL, ABclonal #A21256), and mouse anti-HIV p24 (mAb AG3.0, 1:1,000, NIH ARP #4121).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Virus, Negative Control, Neutralization, Incubation