Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Human Invariant Natural Killer T (iNKT) cells express cell-surface Immunoglobulin-like Transcript 2 (ILT2). ILT2 expression on CD3 + CD56 + NKT cells and CD3 + 6B11 + iNKT cells was evaluated with fresh PBMC by flow cytometry and was compared to that of CD4 + T, CD8 + T, and NK cells. (A) Gating strategy to identify CD4 + T, CD8 + T, NK, CD3 + CD56 + NKT, and iNKT cells from lymphocytes gated according to the FSC-SSC profile and to the CD3, CD4, CD8, CD56, and 6B11 expression. Percentage of parent population is indicated. (B) CD1d tetramer-αGC reactivity and CD56 expression of iNKT cells. Percentage of parent population is indicated. (C) ILT2 expression on different subsets of one representative blood donor. Percentage of ILT2 + cells is indicated. (D) Percentage of ILT2 + cells of each subpopulation from 14 healthy donors. Bars indicate mean ± SEM. (E) ILT2 expression on iNKT cells during expansion. iNKT cells at day 0 and 2 weeks after stimulation by CD1d-αGC and cytokines are shown. Percentage of ILT2 + cells is indicated.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Activation of human Invariant Natural Killer T (iNKT) cells is inhibited through the Human Leucocyte Antigen G (HLA-G):Immunoglobulin-like Transcript 2 (ILT2) interaction. A20CD1d or A20CD1d-HLA-G1-B2M cells were used as Antigen Presenting Cells (APC) and human iNKT cells were used as effector cells. Intracellular IFN-γ and IL-4 expression by effector NKT cells was evaluated by flow cytometry after a 4-h co-culture with APC loaded or not with αGC as indicated. Anti-HLA-G antibody 87G, or anti-ILT2 antibody GHI/75, were used to block the HLA-G/ILT2 interaction as indicated. (A) Before functional assay, expanded human iNKT cells were checked using 6B11 and CD1d-tetramer-αGC. Their surface ILT2 levels were measured. (B) Gating strategy to identify human iNKT cells after the coculture assay with stimulator cells, gated on lymphocytes according to the FSC-SSC profile and CD3+ and 6B11+ expression. Percentage of lymphocytes is indicated. (C) IFN-γ (upper panel) and IL-4 (lower panel) expression levels by effector cells of one representative experiment. The gate of cytokine expression is based on the no activation control in each experiment. (D) Two independent experiments were performed; bar indicates mean ± SEM.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Activation Assay, Expressing, Flow Cytometry, Co-Culture Assay, Blocking Assay, Functional Assay, Co-culture Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Human Invariant Natural Killer T (iNKT) cells express cell-surface Immunoglobulin-like Transcript 2 (ILT2). ILT2 expression on CD3 + CD56 + NKT cells and CD3 + 6B11 + iNKT cells was evaluated with fresh PBMC by flow cytometry and was compared to that of CD4 + T, CD8 + T, and NK cells. (A) Gating strategy to identify CD4 + T, CD8 + T, NK, CD3 + CD56 + NKT, and iNKT cells from lymphocytes gated according to the FSC-SSC profile and to the CD3, CD4, CD8, CD56, and 6B11 expression. Percentage of parent population is indicated. (B) CD1d tetramer-αGC reactivity and CD56 expression of iNKT cells. Percentage of parent population is indicated. (C) ILT2 expression on different subsets of one representative blood donor. Percentage of ILT2 + cells is indicated. (D) Percentage of ILT2 + cells of each subpopulation from 14 healthy donors. Bars indicate mean ± SEM. (E) ILT2 expression on iNKT cells during expansion. iNKT cells at day 0 and 2 weeks after stimulation by CD1d-αGC and cytokines are shown. Percentage of ILT2 + cells is indicated.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Activation of human Invariant Natural Killer T (iNKT) cells is inhibited through the Human Leucocyte Antigen G (HLA-G):Immunoglobulin-like Transcript 2 (ILT2) interaction. A20CD1d or A20CD1d-HLA-G1-B2M cells were used as Antigen Presenting Cells (APC) and human iNKT cells were used as effector cells. Intracellular IFN-γ and IL-4 expression by effector NKT cells was evaluated by flow cytometry after a 4-h co-culture with APC loaded or not with αGC as indicated. Anti-HLA-G antibody 87G, or anti-ILT2 antibody GHI/75, were used to block the HLA-G/ILT2 interaction as indicated. (A) Before functional assay, expanded human iNKT cells were checked using 6B11 and CD1d-tetramer-αGC. Their surface ILT2 levels were measured. (B) Gating strategy to identify human iNKT cells after the coculture assay with stimulator cells, gated on lymphocytes according to the FSC-SSC profile and CD3+ and 6B11+ expression. Percentage of lymphocytes is indicated. (C) IFN-γ (upper panel) and IL-4 (lower panel) expression levels by effector cells of one representative experiment. The gate of cytokine expression is based on the no activation control in each experiment. (D) Two independent experiments were performed; bar indicates mean ± SEM.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Activation Assay, Expressing, Flow Cytometry, Co-Culture Assay, Blocking Assay, Functional Assay, Co-culture Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Phenotypic characteristics of mature dendritic cell (mDC) and autologous DC-10 cells. Phenotype was validated based on the expression of CD14, CD1a, CD1d, HLA-DR, and Human Leucocyte Antigen G (HLA-G); numbers indicate the percentages of positive cells for CD1d and HLA-G.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Human Invariant Natural Killer T (iNKT) cells express cell-surface Immunoglobulin-like Transcript 2 (ILT2). ILT2 expression on CD3 + CD56 + NKT cells and CD3 + 6B11 + iNKT cells was evaluated with fresh PBMC by flow cytometry and was compared to that of CD4 + T, CD8 + T, and NK cells. (A) Gating strategy to identify CD4 + T, CD8 + T, NK, CD3 + CD56 + NKT, and iNKT cells from lymphocytes gated according to the FSC-SSC profile and to the CD3, CD4, CD8, CD56, and 6B11 expression. Percentage of parent population is indicated. (B) CD1d tetramer-αGC reactivity and CD56 expression of iNKT cells. Percentage of parent population is indicated. (C) ILT2 expression on different subsets of one representative blood donor. Percentage of ILT2 + cells is indicated. (D) Percentage of ILT2 + cells of each subpopulation from 14 healthy donors. Bars indicate mean ± SEM. (E) ILT2 expression on iNKT cells during expansion. iNKT cells at day 0 and 2 weeks after stimulation by CD1d-αGC and cytokines are shown. Percentage of ILT2 + cells is indicated.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Human Invariant Natural Killer T (iNKT) cells express cell-surface Immunoglobulin-like Transcript 2 (ILT2). ILT2 expression on CD3 + CD56 + NKT cells and CD3 + 6B11 + iNKT cells was evaluated with fresh PBMC by flow cytometry and was compared to that of CD4 + T, CD8 + T, and NK cells. (A) Gating strategy to identify CD4 + T, CD8 + T, NK, CD3 + CD56 + NKT, and iNKT cells from lymphocytes gated according to the FSC-SSC profile and to the CD3, CD4, CD8, CD56, and 6B11 expression. Percentage of parent population is indicated. (B) CD1d tetramer-αGC reactivity and CD56 expression of iNKT cells. Percentage of parent population is indicated. (C) ILT2 expression on different subsets of one representative blood donor. Percentage of ILT2 + cells is indicated. (D) Percentage of ILT2 + cells of each subpopulation from 14 healthy donors. Bars indicate mean ± SEM. (E) ILT2 expression on iNKT cells during expansion. iNKT cells at day 0 and 2 weeks after stimulation by CD1d-αGC and cytokines are shown. Percentage of ILT2 + cells is indicated.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Activation of human Invariant Natural Killer T (iNKT) cells is inhibited through the Human Leucocyte Antigen G (HLA-G):Immunoglobulin-like Transcript 2 (ILT2) interaction. A20CD1d or A20CD1d-HLA-G1-B2M cells were used as Antigen Presenting Cells (APC) and human iNKT cells were used as effector cells. Intracellular IFN-γ and IL-4 expression by effector NKT cells was evaluated by flow cytometry after a 4-h co-culture with APC loaded or not with αGC as indicated. Anti-HLA-G antibody 87G, or anti-ILT2 antibody GHI/75, were used to block the HLA-G/ILT2 interaction as indicated. (A) Before functional assay, expanded human iNKT cells were checked using 6B11 and CD1d-tetramer-αGC. Their surface ILT2 levels were measured. (B) Gating strategy to identify human iNKT cells after the coculture assay with stimulator cells, gated on lymphocytes according to the FSC-SSC profile and CD3+ and 6B11+ expression. Percentage of lymphocytes is indicated. (C) IFN-γ (upper panel) and IL-4 (lower panel) expression levels by effector cells of one representative experiment. The gate of cytokine expression is based on the no activation control in each experiment. (D) Two independent experiments were performed; bar indicates mean ± SEM.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Activation Assay, Expressing, Flow Cytometry, Co-Culture Assay, Blocking Assay, Functional Assay, Co-culture Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Phenotypic characteristics of mature dendritic cell (mDC) and autologous DC-10 cells. Phenotype was validated based on the expression of CD14, CD1a, CD1d, HLA-DR, and Human Leucocyte Antigen G (HLA-G); numbers indicate the percentages of positive cells for CD1d and HLA-G.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Human Invariant Natural Killer T (iNKT) cells express cell-surface Immunoglobulin-like Transcript 2 (ILT2). ILT2 expression on CD3 + CD56 + NKT cells and CD3 + 6B11 + iNKT cells was evaluated with fresh PBMC by flow cytometry and was compared to that of CD4 + T, CD8 + T, and NK cells. (A) Gating strategy to identify CD4 + T, CD8 + T, NK, CD3 + CD56 + NKT, and iNKT cells from lymphocytes gated according to the FSC-SSC profile and to the CD3, CD4, CD8, CD56, and 6B11 expression. Percentage of parent population is indicated. (B) CD1d tetramer-αGC reactivity and CD56 expression of iNKT cells. Percentage of parent population is indicated. (C) ILT2 expression on different subsets of one representative blood donor. Percentage of ILT2 + cells is indicated. (D) Percentage of ILT2 + cells of each subpopulation from 14 healthy donors. Bars indicate mean ± SEM. (E) ILT2 expression on iNKT cells during expansion. iNKT cells at day 0 and 2 weeks after stimulation by CD1d-αGC and cytokines are shown. Percentage of ILT2 + cells is indicated.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: NKT1.2-ILT2 cells are inhibited through the Human Leucocyte Antigen G (HLA-G)/Immunoglobulin-like Transcript 2 (ILT2) interaction. A20CD1d or A20CD1d-HLA-G1-hB2M cells were used as Antigen Presenting Cells (APC) and NKT1.2 or NKT1.2-ILT2 cells were used as effector cells. Intracellular IL-2 expression of effector NKT cells was evaluated by flow cytometry after 24 h of co-culture with APC loaded or not with αGC as indicated. Anti-HLA-G antibody (Ab), 87G, was used to block the HLA-G/ILT2 interaction when indicated. (A) Gating strategy to identify NKT1.2 cells after the coculture assay. FSC-SSC profile was used to identify living cells, then the NKT1.2 cells are identified as CD3+ singlet living cells. Percentage of lymphocytes is indicated. (B) Intracellular IL-2 expression in NKT1.2 cells of one representative experiment. The gate of IL-2 expression is based on the no activation control in each experiment. (C) Six independent experiments were performed. Each bar indicates mean ± SEM. Horizontal bars indicate statistical significance (One-way ANOVA and Bonferroni post hoc , *p ≤ 0.05; ***p ≤ 0.001).

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Expressing, Flow Cytometry, Co-Culture Assay, Blocking Assay, Co-culture Assay, Activation Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Activation of human Invariant Natural Killer T (iNKT) cells is inhibited through the Human Leucocyte Antigen G (HLA-G):Immunoglobulin-like Transcript 2 (ILT2) interaction. A20CD1d or A20CD1d-HLA-G1-B2M cells were used as Antigen Presenting Cells (APC) and human iNKT cells were used as effector cells. Intracellular IFN-γ and IL-4 expression by effector NKT cells was evaluated by flow cytometry after a 4-h co-culture with APC loaded or not with αGC as indicated. Anti-HLA-G antibody 87G, or anti-ILT2 antibody GHI/75, were used to block the HLA-G/ILT2 interaction as indicated. (A) Before functional assay, expanded human iNKT cells were checked using 6B11 and CD1d-tetramer-αGC. Their surface ILT2 levels were measured. (B) Gating strategy to identify human iNKT cells after the coculture assay with stimulator cells, gated on lymphocytes according to the FSC-SSC profile and CD3+ and 6B11+ expression. Percentage of lymphocytes is indicated. (C) IFN-γ (upper panel) and IL-4 (lower panel) expression levels by effector cells of one representative experiment. The gate of cytokine expression is based on the no activation control in each experiment. (D) Two independent experiments were performed; bar indicates mean ± SEM.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Activation Assay, Expressing, Flow Cytometry, Co-Culture Assay, Blocking Assay, Functional Assay, Co-culture Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC

doi: 10.3389/fimmu.2020.608614

Figure Lengend Snippet: Tolerogenic DC-10 cells do not support human Invariant Natural Killer T (iNKT) cell activation. Mature Dendritic Cells (mDC) or tolerogenic DC-10 cells were used as antigen-presenting cell (APC), and autologous human iNKT cells were used as effector cells. Intracellular IFN-γ and IL-4 expression of effector cells was evaluated by flow cytometry after 72 h of co-culture with APC loaded or not with αGC antigen as indicated. (A) Gating strategy to identify iNKT cells after the coculture assay with stimulator cells, gated on lymphocytes according to the FSC-SSC profile and CD3 + and CD161 + expression. Percentage of lymphocytes is indicated. (B) IFN-γ (upper panel) and IL-4 (lower panel) expression levels by effector cells of one representative experiment. The gate of cytokine expression is based on the no activation control in each experiment. (C) Two independent experiments were performed; bar indicates mean ± SEM.

Article Snippet: Antibodies used in flow cytometry analysis were: i) Anti-murine: CD1d-PE (clone 1B1, BD Phamingen), CD3-APC (clone REA606, Miltenyi Biotech), IL-2-FITC (clone JES6-5H4, eBioscience), and PIR-A/B-PE (clone 10-1-PIR, BD Phamingen); ii) Anti-human: CD1d-PE (clone 51.1, eBioscience), CD1d tetramer-APC (pre-loaded with and without αGC, ProImmune), CD3-eFluor450 (clone OKT3, eBioscience), CD11c-APC (clone 3.9, eBioscience), CD14-PerCP-Cy5.5 (clone 61D3, eBioscience), CD56-PE-Cy7 (clone CMSSB, eBioscience), CD86-PE-Cy7 (clone IT2.2, eBioscience), HLA-DR-FITC (clone MEM-12, Exbio), HLA-G (clone MEM-G/09, Exbio), IFNg-PerCP-Cy5.5 (clone 4S.B3, eBioscience), IL-4-APC (clone 8D4-8, eBioscience), ILT2-PE (clone HP-F1, eBioscience), CD161-APC (clone HP-3G10, eBioscience), and Va24Ja18 TCR-FITC (clone 6B11, eBioscience).

Techniques: Activation Assay, Expressing, Flow Cytometry, Co-Culture Assay, Co-culture Assay