Journal: Frontiers in Immunology

Article Title: Characterisation of the bovine C-type lectin receptor Mincle and potential evidence for an endogenous ligand

doi: 10.3389/fimmu.2023.1189587

Figure Lengend Snippet: Identification of a potential boMincle ligand expressed on bovine CD4 + T cells. To identify potential self-derived boMincle ligands, CFSE-labelled CD4 + T cells were incubated with pUNO1-boMincle-transfected CHO cells. CHO cells were either untreated or pre-incubated with anti-boMincle antibody AbD31662. CHO cells transfected with empty pUNO1 and pUNO1-boDC-SIGN plasmid DNA served as negative and positive control, respectively. The number of CHO cell-bound CD4 + T cells was analysed by fluorescence microscopy. The results of two individual experiments, for which six randomly selected fields of view per condition were counted, are summarised as truncated violin plot. The median for each condition is shown as continuous line and the 75% and 25% quartiles by dotted lines (p = 0.0332 (*), p = 0.0021 (**), p = 0.0002 (***)).

Article Snippet: Bovine CD4 + T cells were isolated from PBMCs by magnetic-activated cell sorting (MACS) using a mouse anti-bovine CD4 antibody (clone CC8, Bio-Rad), anti-mouse IgG MicroBeads (Miltenyi Biotec), and LS columns (Miltenyi Biotec), according to the manufacturer’s instructions.

Techniques: Derivative Assay, Incubation, Transfection, Plasmid Preparation, Positive Control, Fluorescence, Microscopy

Journal: Frontiers in Immunology

Article Title: A Recombinant BCG Vaccine Is Safe and Immunogenic in Neonatal Calves and Reduces the Clinical Disease Caused by the Respiratory Syncytial Virus

doi: 10.3389/fimmu.2021.664212

Figure Lengend Snippet: Vaccination with rBCG-N-hRSV elicits antigen-specific CD4 and CD8 T cell proliferative responses. (A, C) Study 1 PBMCs and (B, D) Study 2 Tracheobronchial lymph node cells (TBLNs) were isolated on day 7 after infection, labeled with Cell Trace Violet, and restimulated in vitro with PPD-B, Ag85A/TB10.4, N-hRSV or bRSV strain 375. Mock stimulated cultures were used as negative controls. ConA stimulated cultures were used as positive controls (not shown). Six days later, (A, B) CD4 + T and (C, D) CD8 + T cell proliferation, as measured by dilution of the Cell Trace dye, was analyzed by flow cytometry. Data represented as mean ± SEM *p<0.05 **p<0.01 as determined by 2-way ANOVA and Sidak’s multiple comparisons test.

Article Snippet: PBMCs were suspended in FACS buffer (10% FBS and 0.02% NA-azide in PBS) and incubated 30 minutes at 4°C with 10 μg/mL of mouse anti-bovine CD4 and 10 μg/mL of mouse anti-bovine CD8α (clones ILA11A & BAQ111A respectively, both from Kingfisher Biotech, Inc).

Techniques: Isolation, Infection, Labeling, In Vitro, Flow Cytometry

Journal: Acta Veterinaria Scandinavica

Article Title: Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: Characterization of lung pathology, persistence of variable surface protein antigens and local immune response

doi: 10.1186/1751-0147-54-9

Figure Lengend Snippet: Antibodies used for immunohistochemistry on paraffin and/or frozen sections

Article Snippet: CC30 , 1:5 b , Mouse IgG1 , Bovine CD4 , w/o a , Biozol, Eching, Germany.

Techniques: Immunohistochemistry

Journal: Acta Veterinaria Scandinavica

Article Title: Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: Characterization of lung pathology, persistence of variable surface protein antigens and local immune response

doi: 10.1186/1751-0147-54-9

Figure Lengend Snippet: Total numbers of peribronchially located CD4 + and CD8 + T lymphocytes and numbers of immunoglobulin-positive plasma cells

Article Snippet: CC30 , 1:5 b , Mouse IgG1 , Bovine CD4 , w/o a , Biozol, Eching, Germany.

Techniques: Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Identification of a DRB3*011:01-restricted CD4 + T cell response against bovine respiratory syncytial virus fusion protein

doi: 10.3389/fimmu.2023.1040075

Figure Lengend Snippet: Stimulation of CD4 + T cells with synthetic, overlapping peptides from the BRSV-375 F protein. CD4 + T cells from dairy cows were positively selected and cultured with autologous antigen presenting cells loaded with synthetic peptide. T cell activation was assessed via IFN-γ secretion using ELISpot assays. Only cells from DRB3*011:01 positive animals responded to stimulation with five out of 88 peptides tested. T cell responses from animals 1141 (A) , 1310 (B) , 2938 (C) , and 2942 (D) displayed a measurable T cell response. Peptides sharing N-terminal amino acid with BRSV-375 F protein residue 253 generated a slight response while those with N-terminal amino acids matching residues 257-260 displayed a larger response. T cells were stimulated with PHA at a concentration of 5μg/mL as a positive control, and cRPMI media as a negative control. Data presented as mean ± SEM, p < 0.05 was considered significant and indicated by asterisk.

Article Snippet: Cell suspensions were labeled with the same mouse anti-bovine CD4 monoclonal antibody as above followed by goat anti-mouse IgG1-AlexaFluor 488 then analyzed on a FACS Symphony custom flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA).

Techniques: Cell Culture, Activation Assay, Enzyme-linked Immunospot, Generated, Concentration Assay, Positive Control, Negative Control

Journal: Frontiers in Immunology

Article Title: Identification of a DRB3*011:01-restricted CD4 + T cell response against bovine respiratory syncytial virus fusion protein

doi: 10.3389/fimmu.2023.1040075

Figure Lengend Snippet: Identification of the minimum peptide of a CD4 + T cell epitope in BRSV-375 F protein. C-terminal truncated peptides were used to stimulate CD4 + T cells in culture with autologous DRB3*011:01 antigen presenting cells. T cell stimulation was observed for peptides greater than 8 amino acids in length. The 9-mer minimum peptide sequence of INDMPITND was identified. T cells were stimulated with PHA at a concentration of 5μg/mL as a positive control, and cRPMI media as a negative control. Cows 1141 (A) and 2942 (D) had a significantly higher response to peptides containing the core INDMPITND sequence while T cells from 1310 (B) , 2938 (C) , 1503 (E) responded to these peptides, the results were not significant. T cells collected from cow 1613 (F) did not produce an observable response to peptide stimulation. Data presented as mean ± SEM, p < 0.05 was considered significant and indicated by asterisk.

Article Snippet: Cell suspensions were labeled with the same mouse anti-bovine CD4 monoclonal antibody as above followed by goat anti-mouse IgG1-AlexaFluor 488 then analyzed on a FACS Symphony custom flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA).

Techniques: Cell Stimulation, Sequencing, Concentration Assay, Positive Control, Negative Control

Journal: Frontiers in Immunology

Article Title: Identification of a DRB3*011:01-restricted CD4 + T cell response against bovine respiratory syncytial virus fusion protein

doi: 10.3389/fimmu.2023.1040075

Figure Lengend Snippet: Inhibition of BoLA DR, but not BoLA DQ, significantly reduces CD4 + T cell activation. Antigen presenting cells were incubated with antibodies against BoLA-DR or BoLA-DQ molecules prior to stimulation with synthetic overlapping peptides (A) or C-terminal truncated peptides (B) . Antibodies raised against MHC class II molecules were provided as mouse ascites fluid. A reduction of in T cell stimulation was observed only with following pre-treatment with anti-BoLA DR antibodies (A) . Antibodies were purified to ensure the reduction in T cell stimulation was due to antibody binding and not background from ascites fluid (B) . Anti-bovine IL-4 antibody was used as an isotype control. Cells were cultured in cRPMI as a media only control. Data is presented as mean ± SEM, p < 0.05 was considered significant and indicated by asterisk.

Article Snippet: Cell suspensions were labeled with the same mouse anti-bovine CD4 monoclonal antibody as above followed by goat anti-mouse IgG1-AlexaFluor 488 then analyzed on a FACS Symphony custom flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA).

Techniques: Inhibition, Activation Assay, Incubation, Cell Stimulation, Purification, Binding Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: Identification of a DRB3*011:01-restricted CD4 + T cell response against bovine respiratory syncytial virus fusion protein

doi: 10.3389/fimmu.2023.1040075

Figure Lengend Snippet: CD4 + T cell response to computationally predicted peptides presented by artificial antigen presenting cells (aAPC). Synthetic peptides computationally predicted to bind strongly to select BoLA-DRB3 alleles used to stimulate CD4 + T cells after loading to aAPCs expressing BoLA-DR and CD80 molecules. DRB3*011:01 predicted peptides, with a core sequence of INDMPITND, were observed to induce T cell activation (B) while those predicted to bind to *001:01 (A) , *012:01 (C) , and *014:01:01 (D) did not. Cells were stimulated with 5μg/mL PHA as a positive control. Data presented as mean ± SEM, p < 0.05 was considered significant and indicated by asterisk.

Article Snippet: Cell suspensions were labeled with the same mouse anti-bovine CD4 monoclonal antibody as above followed by goat anti-mouse IgG1-AlexaFluor 488 then analyzed on a FACS Symphony custom flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA).

Techniques: Expressing, Sequencing, Activation Assay, Positive Control