Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Myeloid Cells Obtained from the Blood but Not from the Tumor Can Suppress T-cell Proliferation in Patients with Melanoma

doi: 10.1158/1078-0432.CCR-12-1108

Figure Lengend Snippet: Characterization of myeloid cells in peripheral blood of healthy donors and melanoma patients. A, flow cytometry evaluation of expression of Lin (CD3/CD19/CD20/CD56/CD57), CD11b, CD14, and CD15 in whole blood. An example of representative dot plots after excluding aggregates, dead cells, and RBCs is shown. Gates were set based on isotype controls. Numbers represent the percentages from the parental populations gated. Names above FACS plots indicate the population gated that was analyzed. Markers analyzed are indicated in the axis of each FACS plot. The gating strategy used to analyze the samples is illustrated. B, frequency of the different phenotypes of myeloid cells in peripheral blood of healthy donors (HD; n = 10) and patients with melanoma (Mel; n = 20) within all the live cells. Each dot represents one patient. C, frequency of CD14+ HLA-DR− cells within CD14+ cells (left) and frequency of CD14+ HLA-DR− within all live cells (right) in peripheral blood.

Article Snippet: Intracellular staining for the quantification of T-regulatory cells was conducted as previously described ( 33 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Variable/trait Total (%) Total no. patients 32 (100) Sex Male 22 (69) Female 10 (31) Age 11–20 1 (3) 21–30 5 (16) 31–40 3 (9) 41–50 12 (37) 51–60 7 (22) 61–70 4 (13) Prior treatment Surgery 28 (88) Chemotherapy 9 (28) Radiotherapy 2 (6) Immunotherapy 19 (59) Any 2 or more 18 (56) Any 3 or more 13 (41) Open in a separate window Patient characteristics Antibodies and reagents The following monoclonal antibodies (mAb) specific for human antigens and their corresponding isotype controls were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (SK7), anti-CD19 (HIB19), anti-CD20 (2H7), anti-CD56 (NCAM 16.2), anti-CD57 (NK-1), phycoerythrin (PE)-Cy7–conjugated anti-HLA-DR L243 (G46–6), allophycocyanin cell (APC)-conjugated anti-CD15 (H198), Alexa-700–conjugated anti-CD14 (M5E2), and APC-Cy7–conjugated anti-CD11b (ICRF44).

Techniques: Flow Cytometry, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Myeloid Cells Obtained from the Blood but Not from the Tumor Can Suppress T-cell Proliferation in Patients with Melanoma

doi: 10.1158/1078-0432.CCR-12-1108

Figure Lengend Snippet: Frequency and absolute numbers of myeloid subsets in peripheral blood of healthy donors and melanoma patients

Article Snippet: Intracellular staining for the quantification of T-regulatory cells was conducted as previously described ( 33 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Variable/trait Total (%) Total no. patients 32 (100) Sex Male 22 (69) Female 10 (31) Age 11–20 1 (3) 21–30 5 (16) 31–40 3 (9) 41–50 12 (37) 51–60 7 (22) 61–70 4 (13) Prior treatment Surgery 28 (88) Chemotherapy 9 (28) Radiotherapy 2 (6) Immunotherapy 19 (59) Any 2 or more 18 (56) Any 3 or more 13 (41) Open in a separate window Patient characteristics Antibodies and reagents The following monoclonal antibodies (mAb) specific for human antigens and their corresponding isotype controls were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (SK7), anti-CD19 (HIB19), anti-CD20 (2H7), anti-CD56 (NCAM 16.2), anti-CD57 (NK-1), phycoerythrin (PE)-Cy7–conjugated anti-HLA-DR L243 (G46–6), allophycocyanin cell (APC)-conjugated anti-CD15 (H198), Alexa-700–conjugated anti-CD14 (M5E2), and APC-Cy7–conjugated anti-CD11b (ICRF44).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Myeloid Cells Obtained from the Blood but Not from the Tumor Can Suppress T-cell Proliferation in Patients with Melanoma

doi: 10.1158/1078-0432.CCR-12-1108

Figure Lengend Snippet: Relative frequency of myeloid subsets in peripheral blood and tumors

Article Snippet: Intracellular staining for the quantification of T-regulatory cells was conducted as previously described ( 33 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Variable/trait Total (%) Total no. patients 32 (100) Sex Male 22 (69) Female 10 (31) Age 11–20 1 (3) 21–30 5 (16) 31–40 3 (9) 41–50 12 (37) 51–60 7 (22) 61–70 4 (13) Prior treatment Surgery 28 (88) Chemotherapy 9 (28) Radiotherapy 2 (6) Immunotherapy 19 (59) Any 2 or more 18 (56) Any 3 or more 13 (41) Open in a separate window Patient characteristics Antibodies and reagents The following monoclonal antibodies (mAb) specific for human antigens and their corresponding isotype controls were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (SK7), anti-CD19 (HIB19), anti-CD20 (2H7), anti-CD56 (NCAM 16.2), anti-CD57 (NK-1), phycoerythrin (PE)-Cy7–conjugated anti-HLA-DR L243 (G46–6), allophycocyanin cell (APC)-conjugated anti-CD15 (H198), Alexa-700–conjugated anti-CD14 (M5E2), and APC-Cy7–conjugated anti-CD11b (ICRF44).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Myeloid Cells Obtained from the Blood but Not from the Tumor Can Suppress T-cell Proliferation in Patients with Melanoma

doi: 10.1158/1078-0432.CCR-12-1108

Figure Lengend Snippet: Morphologic traits of myeloid cells in peripheral blood and tumors of patients with melanoma. Myeloid cells were stained with PI and cell surface antibodies for Lin (CD3/CD56/CD57/CD19/CD20), CD11b, CD14, and CD15, and PI-negative cells (live cells) were sorted according to cell surface expression of Lin, CD11b, CD14, and CD15 and cytospins and Wright-Giemsa stains were conducted. Representative pictures of the populations sorted from patient #2 are shown. Cytospins of myeloid cells sorted from peripheral blood (A) and from melanoma single cells suspension (B).

Article Snippet: Intracellular staining for the quantification of T-regulatory cells was conducted as previously described ( 33 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Variable/trait Total (%) Total no. patients 32 (100) Sex Male 22 (69) Female 10 (31) Age 11–20 1 (3) 21–30 5 (16) 31–40 3 (9) 41–50 12 (37) 51–60 7 (22) 61–70 4 (13) Prior treatment Surgery 28 (88) Chemotherapy 9 (28) Radiotherapy 2 (6) Immunotherapy 19 (59) Any 2 or more 18 (56) Any 3 or more 13 (41) Open in a separate window Patient characteristics Antibodies and reagents The following monoclonal antibodies (mAb) specific for human antigens and their corresponding isotype controls were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (SK7), anti-CD19 (HIB19), anti-CD20 (2H7), anti-CD56 (NCAM 16.2), anti-CD57 (NK-1), phycoerythrin (PE)-Cy7–conjugated anti-HLA-DR L243 (G46–6), allophycocyanin cell (APC)-conjugated anti-CD15 (H198), Alexa-700–conjugated anti-CD14 (M5E2), and APC-Cy7–conjugated anti-CD11b (ICRF44).

Techniques: Staining, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Myeloid Cells Obtained from the Blood but Not from the Tumor Can Suppress T-cell Proliferation in Patients with Melanoma

doi: 10.1158/1078-0432.CCR-12-1108

Figure Lengend Snippet: Characterization of myeloid cells infiltrating fresh melanoma tumors. A, flow cytometry evaluation of expression of Lin (CD3/CD19/CD20/CD56/ CD57), CD11b, CD14, and CD15 in melanoma tumor cell digests. An example of representative dot plots after excluding aggregates, dead cells, and RBCs is shown. Gates were set based on isotype controls. B, frequencies of myeloid cells and subpopulations of myeloid cells infiltrating melanoma tumors (n = 26) represented as a percentage of all the live cells in the tumor (after excluding aggregates, dead cells, and RBCs). Each dot represents one melanoma sample. C, differences in the relative frequency of the myeloid subpopulations present in blood and tumors from patients with melanoma (Blood n = 20; Tumor n = 26). D, correlation of tumor-infiltrating myeloid cells and T-regulatory cells. For 11 melanoma tumors, both T-regulatory cell frequency (CD4+ CD25 FOXP3+) and frequency of myeloid cells as well as frequency of CD14+ and CD14- CD15+ within myeloid population were determined and correlation was assessed using a Spearman test.

Article Snippet: Intracellular staining for the quantification of T-regulatory cells was conducted as previously described ( 33 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Variable/trait Total (%) Total no. patients 32 (100) Sex Male 22 (69) Female 10 (31) Age 11–20 1 (3) 21–30 5 (16) 31–40 3 (9) 41–50 12 (37) 51–60 7 (22) 61–70 4 (13) Prior treatment Surgery 28 (88) Chemotherapy 9 (28) Radiotherapy 2 (6) Immunotherapy 19 (59) Any 2 or more 18 (56) Any 3 or more 13 (41) Open in a separate window Patient characteristics Antibodies and reagents The following monoclonal antibodies (mAb) specific for human antigens and their corresponding isotype controls were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (SK7), anti-CD19 (HIB19), anti-CD20 (2H7), anti-CD56 (NCAM 16.2), anti-CD57 (NK-1), phycoerythrin (PE)-Cy7–conjugated anti-HLA-DR L243 (G46–6), allophycocyanin cell (APC)-conjugated anti-CD15 (H198), Alexa-700–conjugated anti-CD14 (M5E2), and APC-Cy7–conjugated anti-CD11b (ICRF44).

Techniques: Flow Cytometry, Expressing

Journal: Aging (Albany NY)

Article Title: Dectin-1 stimulation promotes a distinct inflammatory signature in the setting of HIV-infection and aging

doi: 10.18632/aging.204927

Figure Lengend Snippet: Effects of age and HIV-infection on Dectin-1 induced cytokine production in monocytes and dendritic cells. HIV-negative young adults (Young) (n=21), HIV-negative older adults (Older) (n= 24), HIV-positive young adults (HIV-Young) (n =14), and HIV-positive older adults (HIV-Older) (n = 22). Dot plots showing percent change in production of interleukin IL-10, IL-12 (p70 isoform), IL-6, and tumor necrosis factor-α (TNF-α) compared to baseline after stimulation with whole glucan particles (WGP or 1,3/1,6-β-glucan). The following comparisons indicated by asterisks were statistically significant using a Wilcoxon two-sample test with t approximation, which were then adjusted with a false discover rate (FDR) calculation for multiple comparisons. ( A ) Total cytokine production in CD11b+ activated monocytes. Young vs. Older: IL-12 (p= 0.00065), IL-6 (p= 0.006), TNF-α (p= 0.026), Young vs. HIV-Young: IL-12 (p=0.011), TNF-α (p=0.016), Older vs. HIV-Older: IL-12 (p=0.01), TNF-α (0.0057). ( B ) Total cytokine production in Inflammatory (CD14+CD16+) monocytes. Young vs. HIV-Young: IL-12 (p=0.0199), TNF-α (p=0.024), Older vs. HIV-Older: IL-10 (p= 0.011), TNF-α (0.032). ( C ) Total cytokine production in myeloid dendritic cells. HIV-negative young adults (n=21), HIV-negative older adults (n= 18), HIV-positive young adults (n =10), and HIV-positive older adults (n = 17). Dot plots showing percent change in production of interleukin IFN-α, TNF-α, IL-12, and IL-6 compared to baseline after stimulation with whole glucan particles (WGP or 1,3/1,6-β-glucan) (labeled as percent positive). The following comparisons indicated by asterisks were statistically significant using a Wilcoxon two-sample test with t approximation, which were then adjusted with a false discovery rate (FDR) calculation for multiple comparisons. i) IFN-α, Young vs. Older (p=0.0373), Young vs. HIV-Young (p=0.0004), Older vs. HIV-Older (p=0.011). ii) TNF-α, Young vs. Older (p=0.023), iii) IL-12, Young vs. HIV-Young (p=0.0001), Older vs. HIV-Older (p=0.001) iiii) IL-6, Older vs. HIV-Older (p=0.035). ( D ) Total cytokine production in plasmacytoid dendritic cells. HIV-negative young adults (n=21), HIV-negative older adults (n= 18), HIV-positive young adults (n =10), and HIV-positive older adults (n = 17). Dot plots showing percent change in production of interleukin IFN-α, TNF-α, IL-12, and IL-6 compared to baseline after stimulation with whole glucan particles (WGP or 1,3/1,6-β-glucan) (labeled as percent positive). The following comparisons indicated by asterisks were statistically significant using a Wilcoxon two-sample test with t approximation, which were then adjusted with a false discovery rate (FDR) calculation for multiple comparisons. i) IFN-α, Young vs. HIV-Young (p=0.0003), Older vs. HIV-Older (p=0.011). ii) TNF-α, Older vs. HIV-Young (p=0.0085), iii) IL-12, Older vs. HIV-Older (p=0.0011). iiii) IL-6, Older vs. HIV-Older (p=0.035). All other comparisons were not significant.

Article Snippet: Cells were then surface stained with anti-CD14-PE-CF594 (3G8, BD-Biosciences), anti-CD16-PE-Cy7 (3G8, BD-Biosciences), and anti-CD11b-APC-Cy7 (ICRF44, BD-Biosciences) to identify monocytes and separate them into activate, inflammatory, classical and non-classical subsets.

Techniques: Infection, Labeling

Journal: Aging (Albany NY)

Article Title: Dectin-1 stimulation promotes a distinct inflammatory signature in the setting of HIV-infection and aging

doi: 10.18632/aging.204927

Figure Lengend Snippet: Dectin-1 surface expression in monocytes and dendritic cells. The cohort consists of the following: Monocytes: [HIV-negative young adults (Young) (n=21), HIV-negative older adults (Older) (n= 24), HIV-positive young adults (HIV-young) (n =14), and HIV-positive older adults (HIV-Older) (n = 22)]. Dendritic cells: [Young (n=21), Older (n=18), HIV-Young (n=10), HIV-Older (n= 17)]. Dot plots showing the Median of the Mean Fluorescence Intensity (MFI) of Dectin-1 surface expression. The following comparisons indicated by asterisks were statistically significant using a Wilcoxon two-sample test with t approximation, which were then adjusted with a false discover rate (FDR) calculation for multiple comparisons. ( A ) Activated Monocytes (CD11b+ CD14+). Young vs. Older (p= 0.026), Young vs. HIV-Young (p=0.001), Older vs. HIV-Older, (p=0.005). ( B ) Inflammatory Monocytes (CD14+CD16+). Young vs. Older (p= 0.003), Young vs. HIV-Young (p=0.001). ( C ) Classical Monocytes (CD14+CD16lo). Young vs. Older (p= 0.0039), Young vs. HIV-Young (p=0.0026). ( D ) Non-classical monocytes (CD14lo, CD16H). Young vs. HIV-Young (p=0.011), Older vs. HIV-Older, (p=0.048). ( E ) Dectin-1 Surface Expression in Myeloid Dendritic cells (mDC) and Plasmacytoid Dendritic Cells (pDC). i). mDC, Young vs. HIV-Young (p=0.0002), Older vs. HIV-Older (p=0.0018). ii). pDC, Young vs. HIV-Young (p=0.012), Older vs. HIV-Older (p=0.0085).

Article Snippet: Cells were then surface stained with anti-CD14-PE-CF594 (3G8, BD-Biosciences), anti-CD16-PE-Cy7 (3G8, BD-Biosciences), and anti-CD11b-APC-Cy7 (ICRF44, BD-Biosciences) to identify monocytes and separate them into activate, inflammatory, classical and non-classical subsets.

Techniques: Expressing, Fluorescence

Journal: Aging (Albany NY)

Article Title: Dectin-1 stimulation promotes a distinct inflammatory signature in the setting of HIV-infection and aging

doi: 10.18632/aging.204927

Figure Lengend Snippet: ( A ) Principal component analysis highlights transcriptional differences between the HIV-positive older adults and all other cohorts. Principal component analysis was performed to identify age, HIV, and Dectin-1 stimulation associated differences among Inflammatory (CD14+CD16+) monocytes. Dectin-1 stimulated vs. unstimulated Inflammatory monocytes were separated with 16% variance on PC2, and monocytes from HIV older adults vs all other cohorts are separated by 53%. [HIV-Older n= 4, HIV-Young n =4, Young = 3, Young* = 4, Older = 3]. Please note that the asterisk (*) indicates the presence of co-morbidities. None = Unstimulated, WGP= Dectin-1 stimulation. ( B ) Heatmap of top 100 variable genes. A total of 314 (Young (Y)), 541 (Young * (Y*)), 402 (Older (O)), 860 (HIV-Young (HIV-Y)), and 156 (HIV Older (HIV-O)) unique DEG (differentially expressed genes) were noted for each of the cohorts respectively. The genes with a fold change of (FC) 1.2 and FDR < 0.1 were defined as differentially expressed genes (DEGs). This heat map notes differences in HIV Older vs. all other cohorts–the effect of stimulation is seen in the second row (Pink column). The heatmap was constructed using Pheatmap. The transcripts were normalized using variance stabilizing transformation function. The color represents relative expression of transcripts that covary across cohorts and condition.

Article Snippet: Cells were then surface stained with anti-CD14-PE-CF594 (3G8, BD-Biosciences), anti-CD16-PE-Cy7 (3G8, BD-Biosciences), and anti-CD11b-APC-Cy7 (ICRF44, BD-Biosciences) to identify monocytes and separate them into activate, inflammatory, classical and non-classical subsets.

Techniques: Construct, Transformation Assay, Expressing