Journal: bioRxiv

Article Title: γδ T cells are effectors of immune checkpoint blockade in mismatch repair-deficient colon cancers with antigen presentation defects

doi: 10.1101/2021.10.14.464229

Figure Lengend Snippet: a. Flow cytometry gating strategy on PDTO cells for analysis of surface staining. Selected cells were gated on single, live cells before quantification of staining signal. b. Histogram representation and count for surface staining of MHC-I, PD-L1, and β2m expression on two PDTO lines B2MWT and B2MKO after IFNγ pre-stimulation. Staining with isotype antibodies for each fluorochrome (PE, APC and FITC) were included as negative control. c. Flow cytometry gating strategy on γδ T cell samples for analysis of intracellular staining to test antitumor reactivity upon PDTO stimulation. Lymphocyte population was further gated on single cells, live and CD3+ cells, γδ TCR+ cells and CD8+ as well as CD8–CD4– cells. Reactivity of the sample was based on IFNγ+ cells of the selected population. d. Histogram representation and count for surface staining of NKG2D ligands MICA/B, ULBP1, ULBP2/5/6, ULBP3, and ULBP4 on two PDTO lines B2MWT and B2MKO after IFNγ pre-stimulation. e. Flow cytometry gating strategy on γδ T cell samples for analysis of intracellular staining after stimulation with PDTOs in the presence of NKG2D ligand blocking. Lymphocyte population was further gated on single cells, live and CD3+ cells, followed by γδ TCR+ and CD8+ as well as CD8– cells. Reactivity of final population was based on IFNγ+ or CD107a+ cells.

Article Snippet: Briefly, cells were incubated with human Fc receptor block (BioLegend) and stained with the different cell surface antibodies (1:10 anti-CD112-PE [clone R2.525, BD Biosciences], 1:10 anti-CD155-PE [clone 300907, R&D Systems], 1:50 anti-CD277/BTN3A1-PE [clone BT3.1, Miltenyi], 1:100 anti-HLA-A,B,C-FITC [clone W6/32, eBioscience], 1:20 anti-HLA-E­BV421 [clone 3D12, BioLegend], 1:20 anti-HLA-G-APC [clone 87G, BioLegend], 1:300 anti-MICA/B-PE [clone 6D4, BioLegend], 1:10 anti-ULBP1-PE [clone 170818, R&D Systems], 1:20 anti-ULBP2/5/6-PE [clone 165903, R&D Systems], 1:20 anti-ULBP3-PE [clone 166510, R&D Systems], or 1:20 anti-ULBP4-PE [clone 709116, R&D Systems] for 45 min at 4°C.

Techniques: Flow Cytometry, Staining, Expressing, Negative Control, Blocking Assay

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

Article Title: Anti-leukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody-conjugate, in acute myeloid leukemia

doi: 10.1158/1078-0432.CCR-16-1904

Figure Lengend Snippet: (A) Percentages of CD123+ and CD131+ fractions within CD45dim blast gate on primary AML samples are shown. (B) Samples were treated with SL-101 at indicated doses for 48 hours. Normalized viable cell counts (left) were determined in a cohort of primary AML samples. (C) Gating scheme for the LSC population (CD45dimCD34+CD38−CD123+). Specific apoptosis was calculated based on percentage of Annexin-V+ cells using the following formula: percentage of specific apoptosis = 100 × (% apoptosis of treated cells − % apoptosis of control cells)/(100 − % apoptosis of control cells). Percentage of growth inhibition was calculated based on Annexin-V−/DAPI− viable cells using the following formula: 100 – 100 × % viable cells of treated cells/% of viable cells of control cells in both CD45dim blasts and LSC-enriched blasts. (D) Colony-forming cell assay was performed in primary AML samples and normal bone marrow (NBM) cells. Data were normalized to those from untreated control groups and represent the mean values of triplicate assays. The absolute colony numbers were shown in Supplementary Figure 1.

Article Snippet: MNCs isolated from primary AML specimens were stained with a cocktail of antibodies comprising CD34-FITC (Cat. 555821), CD38-PE-Cy7 (Cat. 335790), CD45-APC-Cy7 (Cat. 557833), CD123-PerCP-Cy5.5 (Cat. 558714; all from BD Biosciences), and Annexin-V–APC for 30 minutes at room temperature in dark.

Techniques: Inhibition

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

Article Title: Anti-leukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody-conjugate, in acute myeloid leukemia

doi: 10.1158/1078-0432.CCR-16-1904

Figure Lengend Snippet: The primary AML patient sample 11 (4030094) was left untreated or pre-treated with SL-101 (1.0 μg/mL) overnight prior to transplantation via intravenous injection into NSG mice (1×106 viable cells for each mouse). (A) Engraftment of human AML cells was measured by flow cytometry in blood by using anti-human CD45 antibody at week 5 after injection (left). CD123+ cells were gated on the human CD45+ fraction (right). (B) At week 6, all mice were sacrificed and engraftment of human AML was measured in both bone marrow (BM) and spleen. (C) Representative spleens from untreated (U) and SL-101-treated (S) mice are shown. (D) The gating scheme is shown for the CD45dimCD34+CD38−CD123+ LSC-enriched population in untreated and SL-101–treated mice. (E) Percentages of LSCe in human CD45+ cells were determined in both bone marrow and spleen by flow cytometry. AML11 cells (0.6×106) were injected into NSGS) mice that were treated intravenously with PBS or SL-101 at 0.1 mg/kg every other day for 6 doses, after confirming human CD45 engraftment. (F) Leukemia burden was measured after treatment (G) Body weight was monitored over treatment.

Article Snippet: MNCs isolated from primary AML specimens were stained with a cocktail of antibodies comprising CD34-FITC (Cat. 555821), CD38-PE-Cy7 (Cat. 335790), CD45-APC-Cy7 (Cat. 557833), CD123-PerCP-Cy5.5 (Cat. 558714; all from BD Biosciences), and Annexin-V–APC for 30 minutes at room temperature in dark.

Techniques: Transplantation Assay, Injection, Flow Cytometry

Journal: ImmunoHorizons

Article Title: Characterization of Canine Peyer’s Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing

doi: 10.4049/immunohorizons.2300091

Figure Lengend Snippet: Structural organization of canine Peyer’s patches, according to immunofluorescence data. ( A ) Sample collection and preparation. Intestinal tissues from two dogs were sampled, and regions of ∼2 cm in length were identified as Peyer’s patches (PPs). The PPs were isolated, sectioned on a cryostat, and prepared for microscopy, with the various structures within the intestine identified before DAPI staining. ( B ) Visualization of PPs with H&E show different staining intensities within the follicles. The staining with Ki67, for proliferating cells, and CD21, for B cells, allows the visualization of proliferating B cells within the GC. ( C ) Immunofluorescence staining of PPs. PPs were stained with DAPI in combination with Abs against CD8 and one of the following markers: CD21, CD4, or FOXP3. Epifluorescence microscopy is shown at original magnification ×10, visualizing specific structures, including the subepithelial dome (SED), T cell areas, and B cell follicles. ( D ) Myeloid cell staining. Immunostaining of the CD14 and CD11c markers, along with CD21, was used to identify dendritic cells within the SED. Different images from the lower and upper parts of the sample were captured to adequately show the CD11c staining in proximity to both the epithelium and the follicle.

Article Snippet: We used the following Abs: mouse anti-dog CD3 Alexa Fluor 700 (Bio-Rad, MCA1774A700), rat anti-dog CD4 PE-Cy7 (Bio-Rad, MCA1038PeCy7), rat anti-dog CD8 Pacific Blue (Bio-Rad, MCA1039PB), and mouse anti-dog CD21 Alexa Fluor 647 (Bio-Rad, MCA1781A647).

Techniques: Immunofluorescence, Isolation, Microscopy, Staining, Epifluorescence Microscopy, Immunostaining

Journal: ImmunoHorizons

Article Title: Characterization of Canine Peyer’s Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing

doi: 10.4049/immunohorizons.2300091

Figure Lengend Snippet: Characterization of T and B cell populations in canine Peyer’s patches by flow cytometry. ( A ) Peyer’s patches (PPs) from two dogs were dissociated, and single-cell suspensions were stained for flow cytometry in a Cytek Northern Lights flow cytometer. ( B ) Proportions of T and B cells. T cells accounted for 41.9% and B cells for 22.2% of the cell population analyzed. Within the B cell population, 11.6% of the cells were positive for Bcl-6. T cells were further separated into CD4 + , CD8 + , and CD4 + CD8 + subsets, revealing high levels of heterogeneity in the expression of the regulatory marker FOXP3. ( C ) FOXP3 expression in T cell subsets. The percentage of FOXP3 + cells was significantly higher among CD3 + CD4 + CD8 + cells than among CD3 + CD4 + or CD3 + CD8 + cells. Statistical analysis was performed by two-way ANOVA. * p ≤ 0.05, **** p ≤ 0.001. dp, double-positive.

Article Snippet: We used the following Abs: mouse anti-dog CD3 Alexa Fluor 700 (Bio-Rad, MCA1774A700), rat anti-dog CD4 PE-Cy7 (Bio-Rad, MCA1038PeCy7), rat anti-dog CD8 Pacific Blue (Bio-Rad, MCA1039PB), and mouse anti-dog CD21 Alexa Fluor 647 (Bio-Rad, MCA1781A647).

Techniques: Flow Cytometry, Staining, Northern Blot, Expressing, Marker

Journal: ImmunoHorizons

Article Title: Characterization of Canine Peyer’s Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing

doi: 10.4049/immunohorizons.2300091

Figure Lengend Snippet: Gene expression distribution, B and T cell subpopulations, and the Peyer’s patch atlas. ( A ) Marker expression. The expression of markers such as ICOS, TOP2A, JCHAIN, CCR10, and RORC was visualized across the clusters, facilitating cell subtype identification. ( B ) T cell subpopulations. The distribution of different T cell subpopulations is presented as a pie chart, with the corresponding percentages. ( C ) B cell subpopulations. The distribution of different B cell subpopulations is shown as a pie chart, with the corresponding percentages. ( D ) We propose an annotation of the immune cells identified within clusters, based on the specific expression patterns of marker genes. Bcl-6, B cell lymphoma 6; CD62L, CD62 L-selectin; DZ, dark zone; GC, germinal center; ILC3, innate lymphoid cells group 3; LZ, light zone; PD-1, programmed cell death protein 1; Tfh, T follicular helper cell.

Article Snippet: We used the following Abs: mouse anti-dog CD3 Alexa Fluor 700 (Bio-Rad, MCA1774A700), rat anti-dog CD4 PE-Cy7 (Bio-Rad, MCA1038PeCy7), rat anti-dog CD8 Pacific Blue (Bio-Rad, MCA1039PB), and mouse anti-dog CD21 Alexa Fluor 647 (Bio-Rad, MCA1781A647).

Techniques: Gene Expression, Marker, Expressing