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Blood, Vol. 114, Issue 2, 346-356, July 9, 2009
IL-10 is up-regulated in multiple cell types during viremic HIV infection and reversibly inhibits virus-specific T cells
Blood Brockman et al.
114: 346
Supplemental materials for: Brockman et al
Isolation of PBMC subsets with Dynabeads
PBMC subsets were initially isolated using paramagnetic Dynabeads Pan Mouse IgG (Dynal Biotech) conjugated with individual mouse monoclonal antibodies recognizing CD3 (clone UCHT1), CD11b (ICRF44), CD11c (B-Ly6), CD14 (M5E2), CD16 (3G8), and CD19 (HIB19) (all from BD Biosciences). Beads were incubated with 1 µg/mL antibody for 30 minutes at RT, washed 3 times with sterile PBS and stored at 4°C until use. One million PBMC were mixed with antibody-conjugated beads at a 1:1 ratio on ice for 30 minutes, captured on a magnet, washed twice with ice-cold PBS, and resuspended in RLT lysis buffer for RNA extraction using the RNeasy mini kit (Qiagen).
Files in this Data Supplement:
- Table S1. List of the HLA Class I −HIV epitope tetrameric and pentameric complexes used to quantify CTL responses (PDF, 88 KB)
- Figure S1. Impact of IL-10Rα blockade on CMV-specific CD4 cell responses correlates neither with HIV viral load nor with HIV-specific responses (JPG, 181 KB)
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Impact of IL-10Rα blockade on CMV-specific and HIV-specific CD4 T-cell responses was compared in a group of 30 HIV-infected individuals. Summary data of proliferative responses to (A) recombinant HIV p24 antigen and (B) CMV lysate in the presence and absence of anti–IL-10Rα antibody. The vertical axis (IL-10Rα proliferation index) corresponds to the ratio of the fraction of proliferating (%CD3+CD4+CFSElow) cells in the presence of IL-10Rα blocking antibody versus isotype control. Consistent with data on the entire cohort (Fig. 1), increase in HIV p24-specific CD4 T-cell proliferation is significantly greater in viremic than aviremic individuals. In contrast, an increase in CMV-specific CD4 T-cell responses upon IL-10Rα blockade is observed in some individuals but this effect does not correlate with HIV status. Statistical comparisons were made using the Mann-Whitney test. (C) There is no correlation between HIV viral load and impact of IL-10Rα blockade on CMV-specific proliferative responses (Spearman) and (D) no correlation between effect of IL-10Rα blockade on HIV-specific and CMV-specific proliferative responses (Spearman).
- Figure S2. Detection of IL-10 mRNA in multiple magnetic bead-isolated cell populations in HIV-infected persons (JPG, 259 KB)
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Compared to uninfected controls, investigation of HIV-infected individuals shows that IL-10 mRNA is upregulated in multiple cell subsets that were isolated from PBMC with antibody-conjugated magnetic beads. The vertical axis corresponds to the ratio of IL-10 copies to copies of the control gene human hypoxanthine guanine phosphoribosyltransferase (HPRT) for each cell sample, relative to the median of healthy controls. Statistical comparisons were made with the Mann-Whitney test and showed statistical significance for the CD3, CD11b, CD11c and CD14 subpopulations, as indicated on the figures.
- Figure S3. FACS cell-sorting strategy for PBMC subsets (JPG, 714 KB)
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Two panels of antibodies were used to isolate live cells on the basis of combinations of surface markers. (A) In the first panel, PBMC were stained with antibodies CD3-PE-Cy7, CD4-PerCp-Cy5.5, CD8-FITC, CD14-Pac Blue, CD19-APC-Cy7, and CD56−APC (BD Biosciences). In the live cell gate, doublet exclusion was sequentially performed for FSC (FSC-W vs FSC-H plot) and SSC (SSC-W vs SSC-H plot). Subsequent gates were designed to isolate CD4 T cells (CD3+CD4+), CD8 T cells (CD3+CD8+) CD14+ monocytes (CD14+, CD3−), B cells (CD14− CD3− CD19+) and NK cells ((CD14− CD3− CD56+). (B) Verification of the purity of the cell populations isolated. Immediately after sorting using the strategy described in A), cell subsets were acquired on the FACS Aria. Numbers indicate the percent cells located in each gate. (C) A second panel was used to isolate live dendritic cell (DC) subsets. PBMC were stained with a lineage exclusion antibody cocktail–FITC, CD11c-PE, CD123-PE Cy5, and HLA-DR-APC Cy7 (BD Biosciences). In the live cell gate, doublet exclusion was sequentially performed for FSC (FSC-W vs FSC-H plot) and SSC (SSC-W vs SSC-H plot). Subsequent gates were designed to exclude unwanted cell lineage (gating on lineage-negative PBMC), select the HLA-DR+ cells and, within this subset, identify the plasmocytoid (CD123+) and myeloid (CD11c+) DCs. (D) Verification of the purity of the cell populations isolated. Immediately after sorting using the strategy described in C), cell subsets were acquired on the FACS Aria. Numbers indicate the percent cells located in each gate.
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