Journal: Cell Reports Medicine

Article Title: Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells

doi: 10.1016/j.xcrm.2024.101483

Figure Lengend Snippet: Characterization of CD32-driven trogocytosis (A) 293T cells transiently expressing C-terminal GFP fusion proteins of FcγRs CD32A, CD32B, or CD32C or, as a control, the nucleocytoplasmic dNTPase SAMHD1 served as donors in co-cultures with CellTrace dye-stained SupT1 T target cells. All culture media contained IgG-depleted FCS. Shown are representative flow cytometry dot plots and the percentages of CD32 + and GFP + target T cells. One experiment out of two is shown. (B) Schematic of topology determination of transferred CD32-GFP (top). Bottom: SupT1 T cells were co-cultured as described in (A) and stained with either an anti-GFP mAb or an isotype control antibody, both conjugated to Alexa 647, with or without prior cell permeabilization. One representative experiment is shown (n = 3). The illustration was created with BioRender.com . (C) 293T cells were co-transfected with plasmids encoding C-terminal GFP fusion proteins of CD32A, CD32B, or CD32C or, as a control, histone H2B-GFP, together with a plasmid encoding CCR5. After 2 days, cells were either left untreated or pre-treated with an anti-CD32 Ab or an isotype control Ab prior to co-cultivation with SupT1 T cells. One day later, the expression of GFP and CCR5 on the target T cells was determined by flow cytometry. Mean ± SEM are shown (n = 3). Asterisks indicate statistical significance by two-way ANOVA. p values were corrected for multiple comparison (Tukey). (D) Half-life of CD32 and CCR5 surface expression on SupT1 target cells following co-culture as in (A). Following 1 day of co-culture, SupT1 T cells positive for CD32-GFP were sorted by flow cytometry and kept in culture for an additional 9 days. The expression of CD32 (top) or CCR5 (bottom) on sorted cells was determined for up to 192 h of cultivation. One representative experiment is shown (n = 2). (E) Schematic of CD32B with important amino acids and motifs indicated. (F) Transfer of the indicated CD32B mutants, CD32A WT, CD32C WT, or H2B (GFP fusion proteins), assessed as in (A) (mean ± SEM; n = 4). Asterisks indicate statistical significance by one-way ANOVA. p values were corrected for multiple comparison (Dunnett). (G) Visualization of the material transfer from CD32B-GFP expressing 293T cells to LifeAct-mCherry-expressing SupT1 using live-cell imaging. 293T cells transiently expressing CD32B-GFP (green) were co-cultured with LifeAct-mCherry-expressing SupT1 cells (magenta), cultivated in IgG-depleted FCS and boosted with PGT151 antibody, and imaged using spinning disc microscopy for 4 h. The left panel shows the beginning of co-culture. (a) Labels the area with the first transfer event (middle panel). (b) Labels the area of the second transfer event (right panel). Dashed white box marks the area that is zoomed and depicted with individual time points before and after the transfer event (shown below). The time stamp (upper right corner, relative to the time frame which shows the transfer event (time 00:00) in zoom-ins). Scale bar, 10 μm. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001.

Article Snippet: Human T cell line SupT1 (DSMZ, ACC 140) was cultivated in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% (v/v) FBS and Penicillin-Streptomycin (100 IU/mL).

Techniques: Expressing, Staining, Flow Cytometry, Cell Culture, Transfection, Plasmid Preparation, Comparison, Co-Culture Assay, Live Cell Imaging, Microscopy

Journal: Cell Reports Medicine

Article Title: Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells

doi: 10.1016/j.xcrm.2024.101483

Figure Lengend Snippet: CD32-driven trogocytosis is boosted by T cell-autoreactive antibodies associated with chronic HIV-1 infection (A) CD32 expression on CD4 T cells from peripheral blood of healthy donors (HD) (n = 23) and chronic HIV-1 infected patients (CHI) (n = 39). Median with 95% CI are shown. Asterisks indicate statistical significance by Mann-Whitney test. (B) 293T cells transiently co-expressing CD32B-GFP and CCR5 were pre-treated with the indicated patient sera before 1 day of co-culture with SupT1 T cells. Shown are the percentage of CD32B-GFP + and CCR5 + target cells (median with 95% CI, each dot represents a different patient; see also Figure S7 C). CHI, chronic HIV-1 infection; ART, anti-retroviral therapy; AHI, acute HIV-1 infection. Fiebig stages II-III of acute HIV-1 infection ; HIV-2, HIV type 2; HTLV-1, human T cell lymphotropic virus type 1; HCV, hepatitis C virus; DENV, dengue virus; YFV, yellow fever virus-vaccinated; SARS-CoV-2, severe acute respiratory syndrome coronavirus type 2; EC, Echinococcus multilocularis ; SCH, Schistosoma spp.; TB, Mycobacterium tuberculosis ; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; CG, cryoglobulinemia. Asterisks indicate statistical significance by Mann-Whitney test. (C) Percentage of GFP + target cells after 1 day of co-culture with 293T cells as in (B). IgG was depleted from the sera of two healthy donor (HD) and two HIV-1 patient (CHI) samples from (B, pink and red) and input (original sera), flowthrough and eluate of the IgG depletion were used for pre-treatment of cells prior to co-culture. Mean of two donors from each category is shown. (D) Correlation of antibody binding to SupT1 T cells and CD32B-GFP trogocytosis as in (B), with sera from HIV-1 patients. P, Pearson correlation coefficient. (E) Binding of sera with high or low trogocytotic activity (pink and red dots in B) to primary CD4 T cells as detected with fluorochrome-coupled anti-human IgG Ab (median with 95% CI, CD4 T cells; n = 3). Kruskal-Wallis test with Dunn’s multiple-testing correction. (F) A panel of bNAbs was analyzed for binding to uninfected resting CD4 T cells (top) or activated CD4 T cells (bottom). Mean ± SEM; n = 3. Asterisks indicate statistical significance by one-way ANOVA (top) or three-way ANOVA (bottom). p values were corrected for multiple comparison (Dunnett). (G) Purified, CMV-encoded, soluble Fc-binding proteins gp34 and gp68, or control proteins gp34 non-binding mutant (mtrp; W65F) and soluble ICOSL (inducible T cell co-stimulator ligand) were added to 293T donor cells as in (A), in the presence of PGT151 Ab, and subsequently co-cultured with SupT1 T cells. CD32 transfer was evaluated as in (B). Asterisks indicate statistical significance by two-way ANOVA. p values were corrected for multiple comparison (Tukey). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; n.s., not significant. (H) Schematic of the determinants of antibodies for trogocytosis enhancement.

Article Snippet: Human T cell line SupT1 (DSMZ, ACC 140) was cultivated in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% (v/v) FBS and Penicillin-Streptomycin (100 IU/mL).

Techniques: Infection, Expressing, MANN-WHITNEY, Co-Culture Assay, Virus, Binding Assay, Activity Assay, Comparison, Purification, Mutagenesis, Cell Culture

Journal: Cell Reports Medicine

Article Title: Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells

doi: 10.1016/j.xcrm.2024.101483

Figure Lengend Snippet:

Article Snippet: Human T cell line SupT1 (DSMZ, ACC 140) was cultivated in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% (v/v) FBS and Penicillin-Streptomycin (100 IU/mL).

Techniques: Purification, Virus, Recombinant, Staining, Western Blot, Cell Isolation, Isolation, Marker, BIA-KA, Software, Membrane, Cell Culture

Journal: Scientific Reports

Article Title: Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

doi: 10.1038/s41598-017-16451-w

Figure Lengend Snippet: HIV-1 infection of αRep4E3- and αRep9A8-expressing SupT1 cells. ( a ) Kinetics of GFP expression . Control cells (unmodified SupT1 and SupT1/αRep9C2-GFP) and αRep-GFP-expressing SupT1 cells (SupT1/αRep4E3-GFP and SupT1/αRep9A8-GFP) were infected with HIV-1 at MOI 1, and monitored by flow cytometry from day 3 (D3) to D21 pi. Data presented are mean ± SD of triplicate experiments. ( b ) Kinetics of e xtracellular release of viral particles . Samples from culture media were collected at different time points pi, as indicated, and the levels of CAp24 antigen determined by ELISA. (c) Kinetics of e xtracellular release of viral genomes . Viral RNA yields were determined at D7, D14, and D21 pi in samples of culture media, and results expressed as copy number per mL.

Article Snippet: Human T cells lymphoblastic lymphoma cells SupT1 and Jurkat LTR-GFP cells (JLTRG; obtained from the NIH AIDS Reagent Program) were grown in RPMI-1640 medium containing penicillin (100 U/mL), streptomycin (100 µg/mL), 2mM L-glutamine, and 10% FBS (HyClone).

Techniques: Infection, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

doi: 10.1038/s41598-017-16451-w

Figure Lengend Snippet: Cytopathogenicity of HIV-1 in αRep-expressing cells. HIV-1-infected cells were maintained in culture for several weeks. Control cell cultures consisted of unmodified SupT1 cells, and of SupT1/αRep9C2-GFP cells expressing an irrelevant αRep serving as negative control for αRep4E3 and αRep9A8. At different time intervals, as indicated, samples were collected and monitored for cell morphology, concentration, and viability. ( a ) Cell morphology, analyzed by phase contrast microscopy. ( b ) Total cell number per mL. ( c ) Percentage of viable cells. Bars represent the mean ± SD ( n = 3).

Article Snippet: Human T cells lymphoblastic lymphoma cells SupT1 and Jurkat LTR-GFP cells (JLTRG; obtained from the NIH AIDS Reagent Program) were grown in RPMI-1640 medium containing penicillin (100 U/mL), streptomycin (100 µg/mL), 2mM L-glutamine, and 10% FBS (HyClone).

Techniques: Expressing, Infection, Negative Control, Concentration Assay, Microscopy

Journal: Scientific Reports

Article Title: Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

doi: 10.1038/s41598-017-16451-w

Figure Lengend Snippet: Antiviral activity of αRep4E3 and αRep9A8. ( a ), PR-mediated maturation cleavage of Pr55Gag . Control cells (unmodified SupT1 and SupT1/αRep9C2-GFP), and αRep-expressing cells (SupT1/αRep4E3-GFP and SupT1/αRep9A8-GFP) were infected with HIV-1 at the same MOI, and extracellular media collected. After normalization to the CAp24 level (80 µg per sample), the degree of proteolytic cleavage of Pr55Gag at the MA-CA junction by the viral protease (PR) was evaluated using an ELISA-based maturation assay. The bar graph represents the percentage of cleaved Pr55Gag (mean ± SD; n = 3), estimated from the degree of accessibility and reactivity of a Gag-embedded MA epitope towards its specific monoclonal antibody, in competition with a free synthetic peptide reproducing its sequence (refer to Materials & Methods ). (b) , Viral genome packaging . Bar graph representing the concentrations of HIV-1 genomes in culture media after normalization to CAp24 levels (genome copy number per 25 µg CAp24; mean ± SD; n = 3 ), and the fold changes in culture media of SupT1/αRep4E3-GFP and SupT1/αRep9A8-GFP cells, compared to control cell lines SupT1 and SupT1/αRep9C2-GFP. (**), P < 0.01; (*), P < 0.05; ns, not significant.

Article Snippet: Human T cells lymphoblastic lymphoma cells SupT1 and Jurkat LTR-GFP cells (JLTRG; obtained from the NIH AIDS Reagent Program) were grown in RPMI-1640 medium containing penicillin (100 U/mL), streptomycin (100 µg/mL), 2mM L-glutamine, and 10% FBS (HyClone).

Techniques: Activity Assay, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: Scientific Reports

Article Title: Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

doi: 10.1038/s41598-017-16451-w

Figure Lengend Snippet: Infectivity of virus progeny released by HIV-1-infected, αRep-expressing SupT1 cells. Control cells (unmodified SupT1 and SupT1/αRep9C2-GFP), and αRep-expressing cells (SupT1/αRep4E3-GFP and SupT1/αRep9A8-GFP) were infected with HIV-1 at the same MOI. Samples from culture media were collected at D14 pi, and re-inoculated to Tat-dependent, indicator T-cells (Jurkat-GFP cells) after adjustment of the CAp24 levels to 15 µg per sample. Jurkat-GFP cells were harvested at D10 and D14 post-reinfection, and GFP-positive cells determined by flow cytometry. The results presented in the bar graph (mean ± SD; n = 3 ) are the percentages of GFP-positive, fluorescent cells. (**), P < 0.01; ns, not significant.

Article Snippet: Human T cells lymphoblastic lymphoma cells SupT1 and Jurkat LTR-GFP cells (JLTRG; obtained from the NIH AIDS Reagent Program) were grown in RPMI-1640 medium containing penicillin (100 U/mL), streptomycin (100 µg/mL), 2mM L-glutamine, and 10% FBS (HyClone).

Techniques: Infection, Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

doi: 10.1038/s41598-017-16451-w

Figure Lengend Snippet: Resistance of SupT1/αRep9A8 cells to HIV-1 infection. ( a ), HIV-1-infected SupT1/αRep9A8-GFP cells were harvested at D38 pi, and cell morphology analyzed by light and fluorescence microscopy (magnification 100X). ( b – d) , Samples from the cell cuture medium collected at D38 pi were assayed for ( b ) CAp24 levels, (c) viral RNA concentration, and (d) infectivity expressed as the percentage of GFP-positive Jurkat-GFP reporter cells. Data (mean ± SD; n = 3 ) were compared with those obtained at D14 pi.

Article Snippet: Human T cells lymphoblastic lymphoma cells SupT1 and Jurkat LTR-GFP cells (JLTRG; obtained from the NIH AIDS Reagent Program) were grown in RPMI-1640 medium containing penicillin (100 U/mL), streptomycin (100 µg/mL), 2mM L-glutamine, and 10% FBS (HyClone).

Techniques: Infection, Fluorescence, Microscopy, Concentration Assay

Journal: Scientific Reports

Article Title: Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

doi: 10.1038/s41598-017-16451-w

Figure Lengend Snippet: Schematic representation of the distinct antiviral effects of αRep4E3 and αRep9A8 in HIV-1-infected SupT1 cell. The normal pathway of HIV-1 assembly and egress is represented in (1); the antiviral effects of the αRep4E3 and αRep9A8 proteins are displayed in pathways (2) and (3). αRep4E3 (blue symbol) negatively interferes with Gag-RNA interaction and genome packaging; αRep9A8 (pink symbol) acts as a virus maturation inhibitor. Of note, the model presented in this figure was built by the first author, using a combination of elements available from the Servier Medical Art site http://smart.servier.com/ . Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License ( https://creativecommons.org/licenses/by/3.0/ ). As specified, users are free to share, copy and redistribute the material in any medium or format, and to adapt, remix, transform, and build upon the material for any purpose, even commercially.

Article Snippet: Human T cells lymphoblastic lymphoma cells SupT1 and Jurkat LTR-GFP cells (JLTRG; obtained from the NIH AIDS Reagent Program) were grown in RPMI-1640 medium containing penicillin (100 U/mL), streptomycin (100 µg/mL), 2mM L-glutamine, and 10% FBS (HyClone).

Techniques: Infection