Journal: Frontiers in Immunology

Article Title: Heterozygous premature termination in zinc-finger domain of Krüppel-like factor 2 gene associates with dysregulated immunity

doi: 10.3389/fimmu.2022.819929

Figure Lengend Snippet: (A) CD3 + CD4 + T cell subgroups in KLF2 c.951dup p.(Glu318Argfs*87) variant positive family members compared to healthy controls. Mutation carriers´ and controls´ unstimulated PBMCs were stained for regulatory T helper cell markers CD4, CD25, CD127 and FOXP3 and analyzed by flow cytometry. Th1 and Th 17 subpopulations were stained ex vivo (whole blood) with CD4, CXCR3, CCR6, CD45RA and CD10 antibodies and analyzed by flow cytometry. (B) Representative results of flow cytometry analysis of CD25 + CD127 low FOXP3 + regulatory T cells (Treg). The gating strategy is presented along with the data. Regulatory T cells of patient (III:2) and sex-matched controls´ were studied for the expression of CD45RA, CCR6, CCR7 and CXCR3 with flow cytometry. (C) The Treg populations I-V were studied for the expression of CCR6, CCR7 and CXCR3. Representative results of patient and control Treg populations are shown. The regulatory T cell populations I, II, III, IV and V are presented as the number of cells and percentages. The percentage of CXCR3, CCR6 and CCR7 positive cells.

Article Snippet: The antibodies against anti-human CD3 Alexa Fluor 488 (557694), anti-human CD4 PE Cy7 (557852) were purchased from BD Biosciences.

Techniques: Variant Assay, Mutagenesis, Staining, Flow Cytometry, Ex Vivo, Expressing

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

Journal: Frontiers in Immunology

Article Title: Immune regulatory adjuvant approach to mitigate subcutaneous immunogenicity of monoclonal antibodies

doi: 10.3389/fimmu.2024.1496169

Figure Lengend Snippet: Impact of OPLS on RTX- and ADM-mediated T-cell activation when administered subcutaneously in mice. CD4 + T cells in the draining lymph nodes (DLN) of mice at the study terminal were stained for the activation marker CD44 and classical immune checkpoints CLTA-4 and PD-1. Supplementary Figure 3 is the flow cytometry gating strategy. Frequency (%) of (A) CD44 ++ and (B) CTLA-4 + PD-1 + cells out of live CD3 + CD4 + T cells in DLN of mice administered RTX + rHuPH20 in vehicle, 25 mM OPLS, or 100 mM OPLS or (C) in DLN of mice administered ADM in vehicle, 25 mM OPLS, or 50 mM OPLS. Dots represent individual mice and bars are mean ± SD. Statistical significance was determined by Student’s unpaired t-test (one-tailed). *p<0.05, **p<0.01.

Article Snippet: Where indicated, splenocytes were surface stained with anti-mouse CD4 APC-Cy7 (GK1.5), CD3 FITC (17A2), LAG-3 APC (C9B7W), CD49 PE (HMA2), and LAP PerCP-Cy5.5 (TW7-16B4) and stained intracellularly with anti-mouse FoxP3 Alexa Fluor 700 (MF-14) using a fixation/permeabilization kit (Tonbo Biosciences).

Techniques: Activation Assay, Staining, Marker, Flow Cytometry, One-tailed Test

Journal: Frontiers in Immunology

Article Title: Immune regulatory adjuvant approach to mitigate subcutaneous immunogenicity of monoclonal antibodies

doi: 10.3389/fimmu.2024.1496169

Figure Lengend Snippet: Co-administration with OPLS promotes differentiation of regulatory T cells in vivo and in vitro . In the ADM study ( Figure 1 ), mouse splenocytes were collected at the terminal endpoint and analyzed for Tr1 phenotype. Supplementary Figure 4 is the flow cytometry gating strategy. (A) Frequency (%) of LAG-3 + CD49b + FoxP3 - CD4 + Tr1 cells in the spleen. Dots represent individual mice and bars are mean ± SD. Statistical significance was determined by Student’s unpaired t-test (one-tailed). *p<0.05. (B, C) Naïve immature SW mouse BMDCs were cultured for 24 h with media, VitD3/Dex, OVA (a model antigen), and OVA + 25 mM or 50 mM OPLS. Splenocytes from SW mice (n=2) subcutaneously immunized with OVA (2 μg/100 μL) once weekly were collected 4 days after the second dose. Isolated CD4 + T cells were co-cultured with treated BMDCs at a ratio of 1:5 BMDC, CD4 + T cell for 72 h. Frequency (%) (B) LAG-3 + CD49 + Tr1 and (C) LAP + of CD4 + FoxP3 - T cells. Error bars are mean ± SD of triplicate wells.

Article Snippet: Where indicated, splenocytes were surface stained with anti-mouse CD4 APC-Cy7 (GK1.5), CD3 FITC (17A2), LAG-3 APC (C9B7W), CD49 PE (HMA2), and LAP PerCP-Cy5.5 (TW7-16B4) and stained intracellularly with anti-mouse FoxP3 Alexa Fluor 700 (MF-14) using a fixation/permeabilization kit (Tonbo Biosciences).

Techniques: In Vivo, In Vitro, Flow Cytometry, One-tailed Test, Cell Culture, Isolation

Journal: Frontiers in Immunology

Article Title: Immune regulatory adjuvant approach to mitigate subcutaneous immunogenicity of monoclonal antibodies

doi: 10.3389/fimmu.2024.1496169

Figure Lengend Snippet: OPLS does not impact immunocompetence of NHP and mice. CD-1 mice received daily SC doses of vehicle (n=6), 20 mg/kg CYP (n=3), or OPLS (n=6/group) at 18-450 mM for 28 days. NHP (rhesus macaques) (n=3) received 21 daily SC doses of 54 mM OPLS. (A, B) Frequencies of lymphocyte populations—CD19 + B cells, CD3 + T cells, CD3 + CD4 + T cells, and CD3 + CD8 + T cells—in (A) spleens of mice on day 28 and (B) peripheral blood of NHP on day 0 (baseline), 8, 18, 22, and 35. Supplementary Figure 5 is the gating strategy. (C) Anti-KLH IgM and (D) IgG titers (log 2 ) in plasma of mice on day 28 following a single IV dose of KLH (2 mg) on day 15. (E) Anti-KLH IgM and IgG titers (log 2 ) in plasma of NHP on day 18 and 22, respectively, following a single IM dose of KLH (10 mg) on day 8. Each dot represents an individual animal and all bars are mean ± SD. (F-K) Microscopic images of H&E-stained spleens from mice treated with (F) vehicle or (G) 18 mM, (H) 45 mM, (I) 90 mM, (J) 225 mM, and (K) 450 mM OPLS. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. ****p<0.0001.

Article Snippet: Where indicated, splenocytes were surface stained with anti-mouse CD4 APC-Cy7 (GK1.5), CD3 FITC (17A2), LAG-3 APC (C9B7W), CD49 PE (HMA2), and LAP PerCP-Cy5.5 (TW7-16B4) and stained intracellularly with anti-mouse FoxP3 Alexa Fluor 700 (MF-14) using a fixation/permeabilization kit (Tonbo Biosciences).

Techniques: Clinical Proteomics, Staining