Journal: Cell reports

Article Title: Sialyl-Lewis X Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy

doi: 10.1016/j.celrep.2020.107991

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Ulex Europaeus Agglutinin I (UEA I), Fluorescein , Vector Labs , cat# FL-1061-2.

Techniques: Isolation, Recombinant, Plasmid Preparation, Staining, Cell Culture, Activation Assay, Infection, Luciferase, Cell Isolation, Lysis, BIA-KA, Protease Inhibitor, Software

Journal: Cell reports

Article Title: Sialyl-Lewis X Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy

doi: 10.1016/j.celrep.2020.107991

Figure Lengend Snippet: (A–D) CD4 + T cells from HIV-infected ART-suppressed individuals were sorted based on levels of cell-surface fucosylation, SLe X , or CLA expression (lowest 5%, medium, and highest 5%); levels of HIV DNA and cell-associated HIV RNA (total elongated transcripts) were measured by qPCR. The ratio of cell-associated HIV RNA to HIV DNA was calculated. (A and B) Cells with high fucose, either total/core (A) or branched (B), exhibited higher levels of cell-associated HIV RNA compared with cells with low fucose, despite a less dramatic difference in levels of HIV DNA. (C and D) Cells with high levels of SLe X (C) or CLA (D) exhibited higher levels of cell-associated HIV RNA compared with cells with low levels of SLe X or CLA, despite similar levels of HIV DNA in vivo . Furthermore, cells with SLe X-Hi exhibited higher ratio of cell-associated HIV RNA to HIV DNA compared with cells with SLe X-Low (C). Lines and error bars represent the median and IQR. All statistical comparisons were performed using two-tailed non-parametric Wilcoxon rank tests. *p < 0.05. (E and F) Percentage of SLe X (E) and CLA (F) within total and memory CD4 + T cells of HIV-negative, HIV-infected ART-suppressed, and HIV-infected viremic individuals. Lines and error bars represent the median and IQR. Statistical comparisons were performed using two-tailed non-parametric Mann-Whitney U tests. n = 6 for HIV-negative controls; 7 for HIV-infected ART-suppressed individuals, and 8 for HIV-infected viremic individuals. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Cells were stained with AOL-biotin conjugated (TCI America, cat# A26591ML) followed with Streptavidin-APC (Biolegend, cat# 405207), UEA-I FITC (Vector labs, cat# FL-1061–2), SLe X (CSLEX1, AF647; BD biosciences, cat# 563526), or CLA (HECA-452, AF647; Biolegend, cat# 321309).

Techniques: Infection, Expressing, In Vivo, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: Sialyl-Lewis X Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy

doi: 10.1016/j.celrep.2020.107991

Figure Lengend Snippet: (A) Isolated CD4 + T cells from three HIV-negative donors were infected with either the HIV 89.6 virus (at a multiplicity of infection [MOI] of 6) or the HIV DH12 virus (at MOI of 10) by spinoculation in the presence of CD44 MicroBeads. Four days later, intracellular p24 expression and cell-surface expression of SLe X were determined by flow cytometry. (B) Isolated CD4 + T cells from three HIV-negative donors were depleted of CLA + cells. CLA-depleted cells were infected with either HIV 89.6 or HIV DH12 by spinoculation in the presence of CD44 MicroBeads. Four days later, intracellular p24 expression and cell-surface expression of CLA were determined by flow cytometry. (A and B) Top panels show the percentage of SLe X or CLA on total, p24+, and p24-negative cells, as well as controls (cells treated with CD44 MicroBeads alone), for each HIV strain. Bottom panels are representative flow cytometry plots of dually stained CD4 + T cells for HIV p24 protein and SLe X or CLA. Mean and standard error of the mean (SEM) are presented. Paired t tests were used for statistical analysis. (C) H9 cell line was infected with either HIV 89.6 (MOI of 6) or NL4–3 (MOI of 0.1) in the presence of CD44 MicroBeads. Protein expression of several glycosyltransferases involved in SLe X and CLA production (FUT6, FUT7, and B4GALT5), as well as HIV p24, was measured by western blotting. Diagrams show locations of glycosidic bonds catalyzed by indicated glycosyltransferases.

Article Snippet: Cells were stained with AOL-biotin conjugated (TCI America, cat# A26591ML) followed with Streptavidin-APC (Biolegend, cat# 405207), UEA-I FITC (Vector labs, cat# FL-1061–2), SLe X (CSLEX1, AF647; BD biosciences, cat# 563526), or CLA (HECA-452, AF647; Biolegend, cat# 321309).

Techniques: Isolation, Infection, Expressing, Flow Cytometry, Staining, Western Blot

Journal: Cell reports

Article Title: Sialyl-Lewis X Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy

doi: 10.1016/j.celrep.2020.107991

Figure Lengend Snippet: (A) Memory subset distribution of SLe X+ cells in HIV − , HIV + ART-suppressed, and HIV + viremic donors. (B) Frequency of SLe X+ cells within each memory CD4 + T cell subset in the analyzed groups. Subsets were defined as T N (naive; CD45RA + CD27 + CCR7 + CD95 − ), T SCM (stem cell memory; CD45RA + CD27 + CCR7 + CD95 + ), T EMRA (effector memory RA + ; CD45RA + CD27 − ), T EM (effector memory; CD45RA − CD27 − CCR7 − ), T CM (central memory; CD45RA − CD27 + CCR7 + ), and T TM (transitional memory; CD45RA − CD27 + CCR7 − ). Lines and error bars represent the median and IQR. All statistical comparisons were performed using two-tailed Wilcoxon rank tests. (C) Heatmaps showing the statistical difference in the frequency of all measured phenotypic markers in SLe X+ and SLe X− cells. All data were analyzed using a Friedman test (paired, non-parametric). n = 6 for HIV − , 7 for HIV + ART-suppressed individuals, and 8 for HIV + viremic individuals. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Cells were stained with AOL-biotin conjugated (TCI America, cat# A26591ML) followed with Streptavidin-APC (Biolegend, cat# 405207), UEA-I FITC (Vector labs, cat# FL-1061–2), SLe X (CSLEX1, AF647; BD biosciences, cat# 563526), or CLA (HECA-452, AF647; Biolegend, cat# 321309).

Techniques: Two Tailed Test

Journal: Cell reports

Article Title: Sialyl-Lewis X Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy

doi: 10.1016/j.celrep.2020.107991

Figure Lengend Snippet: CyTOF analysis of CD4 + T cells freshly isolated from PBMCs of nine HIV-infected ART-suppressed individuals. Cells were stained for SLe X , PD-1, TIGIT, CTLA-4, CD30, CCR6, or Survivin. (A) Percentage of SLe X+ and SLe X− CD4 + T cells expressing each of the indicated factors. (B) Mean signal intensity of SLe X on the surface of cells expressing or not each of the indicated factors. Median and IQR are presented, and p values were calculated using Wilcoxon signed-rank test. **p < 0.01.

Article Snippet: Cells were stained with AOL-biotin conjugated (TCI America, cat# A26591ML) followed with Streptavidin-APC (Biolegend, cat# 405207), UEA-I FITC (Vector labs, cat# FL-1061–2), SLe X (CSLEX1, AF647; BD biosciences, cat# 563526), or CLA (HECA-452, AF647; Biolegend, cat# 321309).

Techniques: Isolation, Infection, Staining, Expressing

Journal: Cell reports

Article Title: Sialyl-Lewis X Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy

doi: 10.1016/j.celrep.2020.107991

Figure Lengend Snippet: (A) Relative expression of GCNT1 and FUT7 genes in SLe X-Hi and SLe X-Low CD4 + T cells isolated from four HIV + ART-suppressed individuals. Mean and SEM are presented, and p values were calculated using paired t tests. FDR was calculated using the Benjamin-Hochberg procedure. (B and C) List of significantly enriched pathways (B) and functions (C) found by IPA among 1,086 genes significantly different (FDR < 5%, at least 2-fold) between SLe X-Hi and SLe X-Low CD4 + T cells. N, number of genes significantly changed in the pathway; % up, percentage of genes expressed at higher levels in SLe X-Hi compared with SLe X-Low CD4 + T cells; Z, activation Z score of the pathway or function predicted by IPA based on the direction of changes and contribution of membership genes; Positive, activated; negative, inhibited in SLe X-Hi . (D and E) Genes differentially expressed (FDR < 5%) in SLe X-Hi compared with SLe X-Low CD4 + T cells that belong to (D) leukocyte extravasation signaling or (E) NF-κB signaling. Red dots are genes expressed at higher levels in SLe X-Hi compared with SLe X-Low CD4 + T cells, whereas blue dots are genes expressed at lower levels in SLe X-Hi compared with SLe X-Low CD4 + T cells.

Article Snippet: Cells were stained with AOL-biotin conjugated (TCI America, cat# A26591ML) followed with Streptavidin-APC (Biolegend, cat# 405207), UEA-I FITC (Vector labs, cat# FL-1061–2), SLe X (CSLEX1, AF647; BD biosciences, cat# 563526), or CLA (HECA-452, AF647; Biolegend, cat# 321309).

Techniques: Expressing, Isolation, Activation Assay

Journal: Cell reports

Article Title: Sialyl-Lewis X Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy

doi: 10.1016/j.celrep.2020.107991

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were stained with AOL-biotin conjugated (TCI America, cat# A26591ML) followed with Streptavidin-APC (Biolegend, cat# 405207), UEA-I FITC (Vector labs, cat# FL-1061–2), SLe X (CSLEX1, AF647; BD biosciences, cat# 563526), or CLA (HECA-452, AF647; Biolegend, cat# 321309).

Techniques: Isolation, Recombinant, Plasmid Preparation, Staining, Cell Culture, Activation Assay, Infection, Luciferase, Cell Isolation, Lysis, BIA-KA, Protease Inhibitor, Software