Journal: Immunity

Article Title: Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function

doi: 10.1016/j.immuni.2020.07.006

Figure Lengend Snippet:

Article Snippet: Mouse anti-CD16 APC Cy7 Clone B73.1 , BD Biosciences , Cat No. 561306; RRID: AB_10643005.

Techniques: Recombinant, Molecular Cloning, Blocking Assay, Conjugation Assay, Staining, Western Blot, Lysis, Mutagenesis, Isolation, Luminex, Transfection, Amplification, Software, Variant Assay

Journal: iScience

Article Title: Peripheral immunophenotyping of AITD subjects reveals alterations in immune cells in pediatric vs adult-onset AITD

doi: 10.1016/j.isci.2021.103626

Figure Lengend Snippet: Manual gating analysis of myeloid and NK cell subsets reveals pediatric AITD patients show a tendency of reduced frequencies of total NK and the cytotoxic CD56 lo NK subset compared to pediatric controls (A) Frequencies of dendritic cell (DC) subsets are similar among AITD and control subjects. Conventional type 1 DCs (cDC1) were gated as CD3-CD19-HLA-DR + CD14-CD16-CD11c + CD1c-, conventional type 2 DCs (cDC2) were gated as CD3-CD19-HLA-DR + CD14-CD16-CD11c + CD1c+, and plasmacytoid DC (pDC) were gated as CD3-CD19-HLA-DR + CD45RA + CD123 + CD11c-. (B) The frequency of classical monocytes (CD3-CD19-HLA-DR + CD14+) is increased in pediatric AITD subjects compared to pediatric controls, whereas the frequency of intermediate monocytes (CD3-CD19-HLA-DR + CD14 + CD16+) and non-classical monocytes (CD3-CD19-HLA-DR + CD14-CD16+) are similar among AITD and control subjects. (C) Frequencies of cytokine producing monocytes at baseline demonstrates adult AITD patients have reduced frequencies of IL-23p19+ and IL-6+ monocytes compared to adult controls. (D) The frequency of natural killer (NK; CD3-CD19-HLA-DR-CD16+) cells is reduced, but not significantly, in pediatric AITD and adult AITD patients compared to controls. The reduced frequency of NK cells appears relegated to the cytotoxic CD56 lo NK cell subset, as opposed to the regulatory CD56 hi NK subset. PA: pediatric AITD, PH: pediatric healthy control, AA: adult AITD, AH: adult healthy control. Data are represented as mean ± SEM. Samples were processed as depicted in Figure S1 . Frequency of myeloid and NK cell subsets, and cytokine production were determined by manual gating, as depicted in and . Statistical significance determined by One-way ANOVA with Sidak multiple comparisons post-test. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Mouse monoclonal anti-human CD16 (clone B73.1) , eBioscience , Cat#16-0167-025 RRID: AB_2865639.

Techniques: Control

Journal: iScience

Article Title: Peripheral immunophenotyping of AITD subjects reveals alterations in immune cells in pediatric vs adult-onset AITD

doi: 10.1016/j.isci.2021.103626

Figure Lengend Snippet: Unsupervised clustering and dimension reduction analysis of myeloid and NK cells reveals similar frequencies of subpopulations in AITD patients and controls (A) Non-T/non-B cells which encompass myeloid and natural killer (NK) cells were manually gated as CD45 + CD3-CD19-and then analyzed using the unsupervised clustering and dimension reduction programs PhenoGraph and X-shift to generate distinct clusters based on cell surface marker expression (CD15, CD1c, CD11b, HLA-DR, CD56, CD11c, CD14, and CD16). A UMAP plot was created using total non-B/non-T cells from concatenated adult AITD, adult healthy control, pediatric AITD, and pediatric healthy control samples with a minimum of 17,000 cell events. Seven unique cell subpopulations were identified. UMAP plots of each surface marker are displayed to demonstrate varying expression levels among the myeloid and NK cell subpopulations. (B) Heatmap of cell surface marker expression for identification of the 5 myeloid and 2 NK cell subsets identified using PhenoGraph/X-Shift. cDC1: conventional type 1 dendritic cell; cDC2: conventional type 2 dendritic cell. (C) Frequencies of myeloid and NK cell subsets in adult AITD (AA), adult healthy control (AH), pediatric AITD (PA), and pediatric healthy control (PH) subjects demonstrate normal frequencies in AITD patients compared to controls. Data are represented as mean ± SEM. Samples were processed as depicted in Figure S1 . Statistical significance determined by Mann-Whitney non-parametric unpaired Student's t tests.

Article Snippet: Mouse monoclonal anti-human CD16 (clone B73.1) , eBioscience , Cat#16-0167-025 RRID: AB_2865639.

Techniques: Marker, Expressing, Control, MANN-WHITNEY

Journal: iScience

Article Title: Peripheral immunophenotyping of AITD subjects reveals alterations in immune cells in pediatric vs adult-onset AITD

doi: 10.1016/j.isci.2021.103626

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-human CD16 (clone B73.1) , eBioscience , Cat#16-0167-025 RRID: AB_2865639.

Techniques: Recombinant, Antibody Labeling, Immunopeptidomics, Mass Cytometry, Software

Journal: Frontiers in Microbiology

Article Title: HTLV-1 HBZ Protein Resides Exclusively in the Cytoplasm of Infected Cells in Asymptomatic Carriers and HAM/TSP Patients

doi: 10.3389/fmicb.2019.00819

Figure Lengend Snippet: Expression of cells surface markers in PBMCs of AC PH1339, HAM/TSP patient PH1509, and healthy control. The expression of HLA class I, HLA class II DR, CD3, CD4, CD8, CD25, CD19, and CD16 surface molecules on PBMCs from AC PH1339, HAM/TSP patient PH1509, and a healthy control was assessed by immunofluorescence and flow cytometry with antibodies specific for the various markers. Results are expressed as relative number of cells (ordinate) versus the mean intensity of fluorescence in arbitrary units (abscissa). In each histogram, negative controls, obtained by staining the cells with an appropriate isotype-matched antibody, are depicted as a dashed line.

Article Snippet: For flow cytometry, the following reagents were used: mouse anti-human HLA class I (clone B9.12); mouse anti-human HLA class II DR (clone D1.12), both revealed by FITC-labeled rabbit anti-mouse IgG F(ab’)2 antiserum (Sigma, Milan, Italy); FITC mouse anti-human CD3 (clone (UCHT1, BD Pharmingen); FITC mouse anti-human CD4 (clone RPA-T4, BD Pharmingen); PE-Cy5 mouse anti-human CD8a (clone RPA-T8; eBioscience, Milan, Italy); PE mouse anti-human CD16 (clone B73.1, eBioscience, Milan, Italy); FITC mouse anti-human CD19 (clone HIB19, BD Pharmingen); and phycoerythrin (PE) mouse anti-human CD25 (clone M-A251, BD Pharmingen).

Techniques: Expressing, Immunofluorescence, Flow Cytometry, Fluorescence, Staining

Journal: Frontiers in Microbiology

Article Title: HTLV-1 HBZ Protein Resides Exclusively in the Cytoplasm of Infected Cells in Asymptomatic Carriers and HAM/TSP Patients

doi: 10.3389/fmicb.2019.00819

Figure Lengend Snippet: Expression of cell surface markers in the CB-CD4/HTLV-1 cell line. The expression of HLA class I, HLA class II DR, CD3, CD4, CD8, CD25, CD19, and CD16 surface molecules on CB-CD4 HAM/TSP cells and healthy control was assessed by immunofluorescence and flow cytometry with antibodies specific for the markers indicated on the top of the panels. Results are expressed as relative number of cells (ordinate) versus the mean intensity of fluorescence in arbitrary units (abscissa). In each histogram, negative controls, obtained by staining the cells with an appropriate isotype-matched antibody, are depicted as a dashed line.

Article Snippet: For flow cytometry, the following reagents were used: mouse anti-human HLA class I (clone B9.12); mouse anti-human HLA class II DR (clone D1.12), both revealed by FITC-labeled rabbit anti-mouse IgG F(ab’)2 antiserum (Sigma, Milan, Italy); FITC mouse anti-human CD3 (clone (UCHT1, BD Pharmingen); FITC mouse anti-human CD4 (clone RPA-T4, BD Pharmingen); PE-Cy5 mouse anti-human CD8a (clone RPA-T8; eBioscience, Milan, Italy); PE mouse anti-human CD16 (clone B73.1, eBioscience, Milan, Italy); FITC mouse anti-human CD19 (clone HIB19, BD Pharmingen); and phycoerythrin (PE) mouse anti-human CD25 (clone M-A251, BD Pharmingen).

Techniques: Expressing, Immunofluorescence, Flow Cytometry, Fluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Long-Term Clinical and Immunological Profile of Kidney Transplant Patients Given Mesenchymal Stromal Cell Immunotherapy

doi: 10.3389/fimmu.2018.01359

Figure Lengend Snippet: Profile of circulating NK cell subsets. (A) Profile of NK cell counts in mesenchymal stromal cell (MSC)-treated patients ( n = 4), and kidney transplant patients undergoing induction therapy with bas/low dose rabbit anti-thymocyte globulin (RATG) ( n = 6) or with low-dose RATG alone ( n = 5) from 1 to 5 years posttransplant. Data are mean ± SEM. P = NS. (B) Gating strategy for identification of natural killer cell subsets and natural killer T cells. In the CD45 + live singlet cells, the population of CD3 − lymphocytes was gated on the morphology based on SSC and plotted for CD16 and CD56 expression. CD16 + CD56 dim cells positive for CD11b expression and negative for CD27 were identified as cytotoxic NK cells. The CD56 bright CD16 −/+ NK cells were divided into CD27 + and CD27 − subpopulations. NKT cells were defined as CD3 + CD56 + cells. The frequency (% on the gated CD3 − population) of CD56 dim NK cells, CD56 bright NK cells negative or positive for CD27 expression, and of NKT cells (% on CD3 + cells) in patients given MSC infusion and in kidney transplant patients undergoing induction therapy with bas/low dose RATG or with low-dose RATG alone from 1 to 5 years posttransplant are reported in panels (C–F) , respectively. Data are mean ± SEM, * P < 0.05 vs MSC-treated patients and control patients given low-dose RATG at the same time point.

Article Snippet: The mix antibodies contained BUV395 mouse anti-human CD45 (clone HI30), BV510 mouse anti-human CD3 (clone HIT3a), APC-R700 mouse anti-human CD27 (clone M-T271), BB700 mouse anti-human CD16 (clone B73.1), BV650 mouse anti-human CD56 (clone B159), BV711 mouse anti-human CD11b (clone D12), BUV737 mouse anti-human CD14 (clone M5E2), PE-CF594 mouse anti-human CD64 (clone 10.1), BV605 mouse anti-human HLA-DR (clone G46-6), and PE-Cy5 mouse anti-human CD33 (clone WM-53), all antibodies were purchased from BD Bioscience.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Long-Term Clinical and Immunological Profile of Kidney Transplant Patients Given Mesenchymal Stromal Cell Immunotherapy

doi: 10.3389/fimmu.2018.01359

Figure Lengend Snippet: Profile of circulating monocytes. (A) Gating strategy for identification of classical, non-classical, and intermediate monocytes in peripheral blood mononuclear cells. In the CD45 + live singlet cells, the population of CD3 − leukocytes was gated on the morphology based on SSC and plotted for CD14 expression. Classical monocytes were identified as CD64 + cells on gated CD33 + HLADR + cells in the CD14 + CD16 − cell population, intermediate monocytes as CD64 + cells on gated CD33 + HLADR + in the CD14 + CD16 + cell population, and non-classical monocytes as CD64 − cells on gated CD33 + HLADR + cells in the CD14 − CD16 + cell population. Percentages of classical monocytes (% on the gated CD3 − population) and their HLA-DR expression in patients given mesenchymal stromal cell (MSC) infusion ( n = 4) and in kidney transplant patients undergoing induction therapy with bas/low dose rabbit anti-thymocyte globulin (RATG) ( n = 6) or with low-dose RATG alone ( n = 5) from 1 to 5 years posttransplant are reported in panels (B,C) , respectively. Data are mean ± SEM, P = NS. Table in panel (D) reported mean ± SEM of intermediate and non classical monocytes; P = NS.

Article Snippet: The mix antibodies contained BUV395 mouse anti-human CD45 (clone HI30), BV510 mouse anti-human CD3 (clone HIT3a), APC-R700 mouse anti-human CD27 (clone M-T271), BB700 mouse anti-human CD16 (clone B73.1), BV650 mouse anti-human CD56 (clone B159), BV711 mouse anti-human CD11b (clone D12), BUV737 mouse anti-human CD14 (clone M5E2), PE-CF594 mouse anti-human CD64 (clone 10.1), BV605 mouse anti-human HLA-DR (clone G46-6), and PE-Cy5 mouse anti-human CD33 (clone WM-53), all antibodies were purchased from BD Bioscience.

Techniques: Expressing

Journal: PLoS Pathogens

Article Title: Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

doi: 10.1371/journal.ppat.1002814

Figure Lengend Snippet: A) Peripheral blood mononuclear cells (PBMC), purified CD3 + T-cells (CD3 + CD14 − ) or co-cultures of purified CD3 + T-cells with 10% purified CD14 + monocytes (CD3+ CD14+) were mock-infected (MOI = 0, white bars) or challenged with D39 Streptococcus pneumoniae (MOI = 50, black bars) for 16 h and accumulation of hypodiploid DNA (% Sub G0/1) measured in CD3 + T-cells, n = 4. B) caspase 3 activation in CD3 + T-cells under the same condition as A), n = 4. C) Representative western blot probed for Bid, Bim and actin from CD3 + T-cells isolated from PBMC or CD3 enriched cultures following bacterial challenge as in A), D) densitometry summarizes the fold change in Bid compared to the mock infected (MI), derived from three separate experiments. E) % Sub G0/1 purified CD3 + T-cells in co-cultures containing 10% or 20% of purified CD14 + monocytes under the same conditions as A), n = 4 or in F) co-cultures of CD3 + T-cells and purified CD14 + monocytes with (CD14 + /CD16 − ) or without (CD14 + /CD16 + ) CD16 + monocyte depletion, under the same conditions as A), n = 4, ns (not significant) * p<0.05, ** p<0.01, *** p<0.001; statistical analysis by ANOVA.

Article Snippet: To determine percentages of monocyte sub-populations after each isolation procedure we used 1 µg/ml mouse anti-human anti-CD14 (TuK4) pacific blue (Invitrogen) and anti-CD16 (B73.1) PE (eBioscience) or appropriate isotype controls.

Techniques: Purification, Infection, Activation Assay, Western Blot, Isolation, Derivative Assay