Journal: Scientific Reports

Article Title: PGE2 displays immunosuppressive effects during human active tuberculosis

doi: 10.1038/s41598-021-92667-1

Figure Lengend Snippet: PGE2 regulates the expression of surface receptors on PBMC and neutrophils from HD and TB patients. ( A – E ) PBMC from HD (n = 7) and TB patients (n = 11) were stimulated with Mtb -Ag (10 µg/ml) in the presence or absence of PGE2 (2 µM) for 5 days. Afterwards, the surface expression of ( A ) SLAMF1 and ( B ) CD31 on CD3 + T cells and ( C ) CD80, ( D ) MHC-I and ( E ) MHC-II on CD14 + mononuclear phagocytes were determined by flow cytometry. Each bar represents the mean percentage of ( A – C ) CD3 + or ( D,E ) CD14 + Receptor + cells ± SEM or relative Mean Fluorescence Intensity (rMFI) ± SEM. ( F – H ) Human purified neutrophils from HD (n = 7) and TB patients (n = 6) were stimulated with Mtb -Ag (10 µg/ml) in the presence or absence of PGE2 (2 µM) for 2 h. Then, ( F ) SLAMF1, ( G ) PD-L1 and ( H ) PD-L2 expressions were evaluated by flow cytometry. Bars represent the mean values of the percentage of Receptor + neutrophils ± SEM. Statistical differences were calculated using one-way ANOVA and post hoc Dunnett’s multiple comparison test. * p < 0.05. # p < 0.05; Mann–Whitney nonparametric test for unpaired samples.

Article Snippet: To determine the expression of immune receptors on lymphocytes, mononuclear phagocytes and neutrophils, cells stimulated with Mtb -Ag (10 μg/mL) treated or not with PGE2 (2 µM) were blocked in PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 )-FBS 5% for 15 min and then stained for surface expression with fluorophore-marked antibodies against SLAMF1 (BD, 559572), CD31 (BD, 560984), CD80 (Biolegend, 305207), Major Histocompatibility Complex (MHC) -I (Biolegend, HLA-A2, 343306), MHC-II (Biolegend, HLA-DQ, 318106), PD-L1 (eBioscience, MIH1), PD-L2 (BD, MIH18), CD3 (Biolegend, 300306) and CD14 (Biolegend, 367116).

Techniques: Expressing, Flow Cytometry, Fluorescence, Purification, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mannose-Capped Lipoarabinomannan (LAM) from Mycobacterium tuberculosis Induces CD4 + T cell Anergy via GRAIL

doi: 10.4049/jimmunol.1500710

Figure Lengend Snippet: LAM does not affect receptor expression on LAM-anergized P25 TCR-Tg CD4+ T cells. (A) 1×106 CD4+ T cells were pre-treated with either LAM or left untreated (none) and co-cultured with Ag85B-pulsed APCs. After 48 h, cells were collected, washed and labeled with anti-PD1, anti-Lag3 or anti-Tim 3, anti-CD28, anti-CD40L mAbs. Intracellular staining was performed for CTLA4. Gating was performed on live CD4+ T cells. Alternatively, after 48 h cells were collected, washed and cultured in IL-7 for 5 d. Cells were then stained for surface expression of TCR and CD3. Gating was performed on live CD4+ T cells. Representative histograms of at least three independent experiments are shown. (B) The mean fluorescence intensity (MFI) of PD-1 staining in LAM-treated (LAM) and untreated (none) T cells was quantified after 48 h of primary stimulation. Data are means +/− SEM of three independent experiments.

Article Snippet: The following mAbs and isotype controls were purchased for analysis of receptor expression: anti-CD3-PE, anti-CD4-APC, anti-CD28-APC, anti-Tim3-PE, anti-CTLA4-PE, anti-CD25-alexa Fluor-488, anti-FoxP3-PE, anti-Annexin V-Alexa Fluor 450, anti-cholera toxin subunit B-Alexa Fluor 647, anti-CD3-Alexa Fluor 647, anti-mouse IgG-Alexa Fluor 488, and LIVE/DEAD violet and yellow cell stain (all from ebioscience); anti-PD1-PE (BD Pharmingen); anti-Lag3-APC (Biolegend); anti-TCR-Vβ-PE and anti-CD40L-PE (Miltenyi Biotech).

Techniques: Expressing, Cell Culture, Labeling, Staining, Fluorescence

Journal: ESMO Open

Article Title: Impact of the combined timing of PD-1/PD-L1 inhibitors and chemotherapy on the outcomes in patients with refractory lung cancer

doi: 10.1016/j.esmoop.2021.100094

Figure Lengend Snippet: Association between the activation states and cytotoxic effects of dicycloplatin (DCP) in peripheral blood mononuclear cells (PBMCs) stimulated by the anti-CD3 antibody (anti-CD3 Ab). (A, B) Proliferation of anti-CD3 Ab-stimulated PBMCs. Proliferation rate (day n ) = MFI (day n )/MFI (day n −1). (C, D) PD-1 expression in anti-CD3 Ab-stimulated PBMCs. Dynamic changes in (C) the positive percent of PD-1 expression compared to the isotype control, and (D) the MFI of PD-1 expression on CD3 + T cells after stimulation were detected by flow cytometry. (E, F) Cytotoxic effect of DCP in anti-CD3 Ab-stimulated PBMCs. PBMCs stimulated with the anti-CD3 Ab for different lengths of time were used to represent different activation states, and the cells were exposed to a full range of concentrations of DCP for 72 h before detection. (F) The 50% inhibitory concentration (IC 50 ) values of DCP in PBMCs stimulated for 2-6 days were compared with those in PBMCs stimulated for 1 day. The data are presented as mean ± SD of three independent experiments. ∗ P < 0.05, statistically significant. MFI, mean fluorescence intensity.

Article Snippet: The anti-PD-1-PE and PD-1 isotype antibodies were purchased from BD Pharmingen (San Diego, CA, USA).

Techniques: Activation Assay, Expressing, Flow Cytometry, Concentration Assay, Fluorescence

Journal: ESMO Open

Article Title: Impact of the combined timing of PD-1/PD-L1 inhibitors and chemotherapy on the outcomes in patients with refractory lung cancer

doi: 10.1016/j.esmoop.2021.100094

Figure Lengend Snippet: Impacts of the anti-PD-1 antibody (Ab) on cell proliferation and the cytotoxic effect of dicycloplatin (DCP) in peripheral blood mononuclear cells (PBMCs). (A) Effect of the anti-PD-1 Ab on anti-CD3 Ab-stimulated PBMCs. (B) Effect of the recombinant PD-L1 protein on anti-CD3 Ab-stimulated PBMCs. (C) Effect of the anti-PD-1 Ab on PD-L1-inhibited PBMCs. The recombinant PD-L1 protein and anti-PD-1 Ab were used at 2 μg/ml and 10 μg/ml, respectively, and cells were incubated for 6 days before detection. The graph shows a representative case from six sets of PBMCs, four of which demonstrated recovery from PD-L1-induced proliferation inhibition. (D) Effect of the anti-PD-1 Ab on the cytotoxicity of DCP in PD-L1-inhibited PBMCs. PBMCs pretreated with the PD-L1 protein (and anti-PD-1 Ab for the DCP + aPD-1 group) for 6 days were incubated with DCP for 72 h before detection. The graph shows the relative cytotoxicity of DCP at 1 μM. (E, F) Effect of the combined timing of anti-PD-1 Ab on the cytotoxicity of DCP in PD-L1-inhibited PBMCs. The cytotoxicity of DCP was measured by a (E) luminescent cell viability assay and (F) lactate dehydrogenase (LDH) assay. The relative cytotoxicity of DCP = [MFI (aPD-1) − MFI (DCP + aPD-1)]/MFI (aPD-1) for the cell viability assay. Each column indicates the mean ± SD of triplicate tests. ns, not statistically significant. ∗ P < 0.05, ∗∗ P < 0.01, statistically significant. ATP, adenosine triphosphate.

Article Snippet: The anti-PD-1-PE and PD-1 isotype antibodies were purchased from BD Pharmingen (San Diego, CA, USA).

Techniques: Recombinant, Incubation, Inhibition, Cell Viability Assay, Lactate Dehydrogenase Assay, Viability Assay

Journal: ESMO Open

Article Title: Impact of the combined timing of PD-1/PD-L1 inhibitors and chemotherapy on the outcomes in patients with refractory lung cancer

doi: 10.1016/j.esmoop.2021.100094

Figure Lengend Snippet: Survival analyses. (A, B) Receiver operating characteristic (ROC) curve analyses of (A) overall survival and (B) progression-free survival for the optimal cut-off value of the combined timing of PD-1/PD-L1 inhibitors and chemotherapy. (C-F) Patient survival. Kaplan–Meier survival curves comparing overall survival and progression-free survival between different combined-timing groups. CI, confidence interval; HR, hazard ratio; mOS, median overall survival; mPFS, median progression-free survival.

Article Snippet: The anti-PD-1-PE and PD-1 isotype antibodies were purchased from BD Pharmingen (San Diego, CA, USA).

Techniques:

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: A Purified tonsil CD4 + T cells (~91% purity) or CD4 + CD25 + CD127 low T reg cells (>88% FOXP3 + ) were TCR activated and infected with HIV as described in “Methods.” GFP was assessed in FOXP3 + (left) or FOXP3 − (right; gated on Foxp3 neg cells) fractions 48 h post-infection. Representative flow cytometric data (left) and statistical analyses from three independent tonsil donors (right) are shown. Mean +/− SEM are shown; * P < 0.05; **<0.005, ****<0.00005; two-tailed; Mann–Whitney test. B – F TCR activated whole human tonsil cultures (HTC) were infected with HIV and allowed to expand with IL-2 for 6 days. Flow cytometric analyses of CD3 + FOXP3 + cells pre-gated on CD8 − cells ( B ), viability of CD3 + CD8 − FOXP3 + cells ( C ), PD-1 and IFN-γ expression in viable CD3 + FOXP3 + CD8 − cells ( D ), with respective statistical analyses (right) are shown. B – D n = 5; * P < 0.05; ** P = 0.007; two-tailed; Mann–Whitney test. E , F Flow cytometric plots showing the expression of the indicated proteins in PD-1 high and PD-1 low populations gated in D in HIV-infected HTC. B – F Five independent experiments showed similar results.

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Purification, Infection, Two Tailed Test, MANN-WHITNEY, Expressing

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: Whole HTC was activated with TCR stimulation, infected with HIV, and allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days. Viral inhibitor Efavirenz (50 nM) or cell death/pan-caspase inhibitor z-VAD (10 μM) was added 28 h post-infection as described in “Methods.” Flow cytometry acquisition was done with constant time for all the samples. Percentage of CD3 + FOXP3 + cells pre-gated on CD8 − cells ( A ), viability of FOXP3 + cells pre-gated on CD3 + CD8 − cells ( B ), PD-1 and IFN-γ expression in viable CD3 + CD8 − FOXP3 + cells ( C ), Ki-67 expression in viable PD-1 high and PD-1 low FOXP3 + populations ( D ) are shown. A – D Representative contour plots (left), statistical analyses of proportions of the cells (middle), and statistical analyses of the absolute cell counts (right) are shown (ordinary one-way ANOVA alpha = 0.05* and multiple comparison t tests; * P < 0.05; **<0.005, ***<0.0005). Results are derived from three independent experiments ( n = 3) and are presented as mean value +/− SEM.

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Infection, Flow Cytometry, Expressing, Derivative Assay

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: Purified tonsil CD4 + T cells (~93% purity) were TCR activated, infected with HIV, and allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days post-infection unless otherwise noted. Efavirenz (50 nM), LPS (10 μg/ml), PG-LPS (5 μg/ml), HKGT (10 6 /ml), IL-1β (20 ng/ml), IL-33 (20 ng/ml), Anakinra (10 μg/ml), LY294002 (10 μM), and Nigericin (10 nM) were added as indicated, 36 h post infection. A PD-1 and IFN-γ (left) and AREG (right) expression in FOXP3 + cells. B ELISA quantification of IL-1β (left) and AREG (right) in cell culture supernatants collected on day 3 post infection. p-Akt ( C ) and AREG ( D ) expression in FOXP3 + cells. E Percentage and absolute cell numbers of PD-1 hi IFN-γ + FOXP3 + cells in CD4 + population. F AREG expression in FOXP3 + cells (left) and ELISA quantification of AREG (right), 6 days post infection. A – F Data are representative of three independent experiments and are presented as mean value +/− SEM. (* P < 0.05; **<0.005, ***<0.0005, ****<0.00005; unpaired t tests). Source data are provided as a Source data file.

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Purification, Infection, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: CD4 + T cells were stimulated as in Fig. . A , B AEP, pAEP, FOXP3, BCL-2, and Ki-67 staining in PD-1 high FOXP3 + (blue) and PD-1 low FOXP3 + cells 6 days post-infection. Some CD4 + T cells stimulated and infected as above were moved to a plate coated with recombinant human PD-L1/B7-H1 Fc chimera (5 μg/ml) or treated with AEP inhibitor (10 μM) 36 h after infection. Percentage of FOXP3 + cells in CD4 + population and FOXP3 MFI on FOXP3 + gated cells ( C ), percentage and absolute cell numbers of PD-1 hi IFN-γ + cells in FOXP3 + population ( D ), and AREG expression in FOXP3 + cells ( E ), as determined by flow cytometric analyses. Results represent triplicate experiments with similar results and are presented as mean value +/− SEM (ordinary one-way ANOVA and multiple t tests were conducted; * P < 0.05; **<0.005, ***<0.0005).

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Staining, Infection, Recombinant, Expressing

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: Purified CD4 + T cells and T regs were stimulated and infected as in “Methods.” A CD25 and FOXP3 expression in all cells in the cultures (above) and PD-1 and IFN-γ expression in CD25 + FOXP3 + cells (below) at 96 h post infection. B Statistical analyses of PD-1 hi IFN-γ + cells in FOXP3 + population from these two cultures. C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1. As controls, T resp cells were cultured alone or co-cultured with purified naive CD127 low CD25 + T regs that were sequentially sorted to remove CD45RO + CD4 + cells using human CD45RO kit (Miltenyi) and purify CD25 high T reg cells using STEMCELL technology CD25 + T reg isolation kits. These control T regs were also previously stimulated and infected the same manner (purple) before co-culture with T resp . T resp proliferation was determined by cell-trace dye dilution in PD-1 hi CD25 + co-culture (blue), control T reg co-cultures (purple), or those cultured alone (green). D Statistical analyses of % T resp suppression mean values from three independent experiments showing similar results. B , D Mean +/− SEM are shown; * P < 0.05; **<0.005, ****<0.00005; two-tailed; Mann–Whitney test).

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Purification, Infection, Expressing, Isolation, Labeling, Cell Culture, Co-Culture Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: HOILs from gingival mucosa from healthy controls and HIV + patients on cART were processed for flow cytometry ex vivo. A FOXP3 expression in CD3 + CD4 + gated HOIL cells. PD-1 and IFN-γ ( B ), IL-10 ( C ), AREG ( D ), and BCL-2 ( E ) expression in FOXP3 + population. Statistical analyses and comparison between the groups for % PD-1 hi IFN-γ + cells ( n = 20; ** P = 0.0025) ( F ), % PD-1 hi cells ( n = 35; ** P = 0.005) ( G ), % IL-10 + cells ( n = 22; * P = 0.024) ( H ), AREG expression ( n = 22; *** P = 0.0002) ( I ), and BCL-2 expression ( n = 12; ** P = 0.0022) ( J ) in FOXP3 + population. K ELISA quantification of AREG levels in saliva ( n = 78; * P < 0.03). L , M Correlation of % PD-1 hi CD25 + cells in FOXP3 + population ( L ) and salivary AREG ( M ), with effector CD4 hyperactivation (% CD38 + HLADR + in FOXP3 neg CD4 + T cells in gingival mucosa; Fig. ; n = 20). F – K P values two-tailed; Mann–Whitney test; each data point represents a study participant, and the data are presented as mean value +/− SEM. Source data are provided as a Source data file.

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Flow Cytometry, Ex Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Combination immune checkpoint blockade to reverse HIV latency

doi: 10.4049/jimmunol.1901191

Figure Lengend Snippet: A: Schematic representation of the experimental set-up. Resting CD4+ T cells, isolated from PBMC of HIV-uninfected individuals, were co-cultured with syngeneic monocytes in the presence of SEB and infected with full length nef competent CCR5-tropic EGFP-reporter HIV. B: Cells were harvested for flow cytometry analysis and single cells were selected by assessment of forward scatter (FSC) and side scatter (SSC). T cells were identified by gating for CD3+HLA-DR− cells, and then gated for EGFP−, eFluor670HI (non-proliferating) and eFluor670LO (proliferating) T cells. Representative dot-plots showing gating strategy for C: PD-1, D: TIM-3, E: CTLA-4, F: BTLA and G: TIGIT. All dot-plots shown were from 3 days post-infection. H: Percentage of cells expressing PD-1 (n=4), TIM-3 (n=8), CTLA-4 (n=3), BTLA (n=5) and TIGIT (n=9) on EGFP− non-proliferating (left) and proliferating (right) T cells cultured alone (black circles) or with syngeneic monocytes (grey squares) was assessed by flow cytometry. Data points represent mean values ±SEM. Open circles indicate that fewer donors were used for the analysis of IC marker expression on proliferating T cells, as some donors did not have sufficient (>50,000 cells) proliferating T cells at that time point for analysis. I: The percentage of cells expressing PD-1, TIM-3, CTLA-4, BTLA and TIGIT 3 days post infection, on T cells cultured alone (grey bars) or co-cultured with syngeneic monocytes (blue bars). Data points represent mean ±SEM. Individual donor results are shown as black symbols. *p<0.05, **p<0.01, as determined by Student’s T test (n≤5) or Wilcoxon matched pairs signed rank test (n>5).

Article Snippet: Single IC expression on T cells was determined by gating on CD3 + HLA-DR − EGFP − cells, which were subsequently separated into eFluor670 HI (non-proliferating) and eFluor670 LO (proliferating) populations using m-a-hCD3-PB (UCHT1, BD), m-a-hHLA-DR-PE-TexR (TU36, BD), m-a-hCD279/PD-1-PE (EH12.1, BD), m-a-hTIM-3-PE (F38–2E2, BioLegend), m-a-hCD152/CTLA-4-PE (BNI3, BD), m-a-hCD272/BTLA-PE (MIH26, BioLegend), m-a-hTIGIT-eFluor710 (MBSA43, eBioscience), mIgG2a-PE isotype control (MOPC-173, BioLegend) or mIgG1-eFluor710 isotype control ( {"type":"entrez-protein","attrs":{"text":"P36281","term_id":"549203","term_text":"P36281"}} P36281 , eBiosciences).

Techniques: Isolation, Cell Culture, Infection, Flow Cytometry, Expressing, Marker

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Combination immune checkpoint blockade to reverse HIV latency

doi: 10.4049/jimmunol.1901191

Figure Lengend Snippet: A: Resting CD4+ T cells were cultured with syngeneic monocytes in the presence of SEB and infected with EGFP-reporter virus. At day 5 post-infection CD3+HLA-DR− EGFP− (not productively infected), eFluor670HI (non-proliferating) and eFluor670LO (proliferating) T cells were further sorted based on IC molecule expression into low/negative (−) and high (+) expressing populations compared to FMO controls (see also Figure S1) or the expression of PD-1+CTLA-4+TIM-3+BTLA (multiple ICM) or none of these molecules. Sorted cells were cultured in the presence of the integrase inhibitor raltegravir (RAL) with and without anti-CD3/CD28+IL-7+IL-2. B: On day 8, EGFP+ cells were measured by flow cytometry and latent infection was calculated by subtracting the number of EGFP+ cells in the untreated culture from the number of EGFP+ cells in the stimulated culture. Red lines indicate median values and dots represent individual donors (n=4–6). *p<0.05, **p<0.01, as determined by Student’s T test (n≤5) or Wilcoxon matched pairs signed rank test (n>5). MFC denotes the median fold change between the samples with and without IC expression.

Article Snippet: Single IC expression on T cells was determined by gating on CD3 + HLA-DR − EGFP − cells, which were subsequently separated into eFluor670 HI (non-proliferating) and eFluor670 LO (proliferating) populations using m-a-hCD3-PB (UCHT1, BD), m-a-hHLA-DR-PE-TexR (TU36, BD), m-a-hCD279/PD-1-PE (EH12.1, BD), m-a-hTIM-3-PE (F38–2E2, BioLegend), m-a-hCD152/CTLA-4-PE (BNI3, BD), m-a-hCD272/BTLA-PE (MIH26, BioLegend), m-a-hTIGIT-eFluor710 (MBSA43, eBioscience), mIgG2a-PE isotype control (MOPC-173, BioLegend) or mIgG1-eFluor710 isotype control ( {"type":"entrez-protein","attrs":{"text":"P36281","term_id":"549203","term_text":"P36281"}} P36281 , eBiosciences).

Techniques: Cell Culture, Infection, Expressing, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Combination immune checkpoint blockade to reverse HIV latency

doi: 10.4049/jimmunol.1901191

Figure Lengend Snippet: A: Co-expression of IC molecules PD-1, TIM-3 and TIGIT on T cells was determined in CD3+HLA-DR−EGFP− non-proliferating (left) and proliferating (right) T cells cultured with syngeneic monocytes by flow cytometry at the day of infection (d0), and on each day post infection (d1-d4), compared to isotype control. The cells are grouped to show the distribution of cells expressing none IC (grey), one IC (yellow), any combination of two IC (blue) or all three IC (red). Data points represent the mean of 4 donors ±SEM. B: Pie chart showing the distribution of IC molecule expression on non-proliferating (top) and proliferating (bottom) T cells on day 3 post infection. Co-expression of IC molecules PD-1, CTLA-4, TIM-3 and TIGIT on T cells was determined in CD3+HLA-DR−EGFP− non-proliferating (C) and proliferating (D) T cells cultured with syngeneic monocytes by flow cytometry at day 5 post infection, compared to isotype control. Black lines indicate mean values of 6 donors ±SEM and equal colours represent equal donors across the panels. E: Pie chart showing the distribution of IC expression on non-proliferating (left) and proliferating (right) T cells day 5 post infection. The cells are grouped to show the distribution of cells expressing none IC (grey), one IC (yellow), any combination of two IC (blue), any combination of three IC (red) or four IC (green).

Article Snippet: Single IC expression on T cells was determined by gating on CD3 + HLA-DR − EGFP − cells, which were subsequently separated into eFluor670 HI (non-proliferating) and eFluor670 LO (proliferating) populations using m-a-hCD3-PB (UCHT1, BD), m-a-hHLA-DR-PE-TexR (TU36, BD), m-a-hCD279/PD-1-PE (EH12.1, BD), m-a-hTIM-3-PE (F38–2E2, BioLegend), m-a-hCD152/CTLA-4-PE (BNI3, BD), m-a-hCD272/BTLA-PE (MIH26, BioLegend), m-a-hTIGIT-eFluor710 (MBSA43, eBioscience), mIgG2a-PE isotype control (MOPC-173, BioLegend) or mIgG1-eFluor710 isotype control ( {"type":"entrez-protein","attrs":{"text":"P36281","term_id":"549203","term_text":"P36281"}} P36281 , eBiosciences).

Techniques: Expressing, Cell Culture, Flow Cytometry, Infection