Journal: Frontiers in Veterinary Science

Article Title: Canine Transforming Growth Factor-β Receptor 2-Ig: A Potential Candidate Biologic for Melanoma Treatment That Reverses Transforming Growth Factor-β1 Immunosuppression

doi: 10.3389/fvets.2021.656715

Figure Lengend Snippet: Effect of TGF-β1 on Th1 cytokine production and Tregs differentiation in PBMC cultures. (A) IL-2, (B) IFN-γ, and (C) TNF-α concentrations in cell culture supernatant were measured by ELISA. (D) The percentage of CD25 + Foxp3 + cells among CD4 + lymphocytes was measured by flow cytometry. The Wilcoxon signed-rank sum test was used for statistical analysis ( n = 6, * p <0.05).

Article Snippet: Subsequently, 1 μg/mL of anti-canine CD4 mouse monoclonal antibody (R&D systems) and 1 μg/mL phycoerythrin (PE)-conjugated anti-canine CD25 mouse monoclonal antibody (Thermo Fisher Scientific) were added and incubated at 25°C for 30 min.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Frontiers in Veterinary Science

Article Title: Canine Transforming Growth Factor-β Receptor 2-Ig: A Potential Candidate Biologic for Melanoma Treatment That Reverses Transforming Growth Factor-β1 Immunosuppression

doi: 10.3389/fvets.2021.656715

Figure Lengend Snippet: Effect of TGF-βRII-Ig on Th1 cytokine production and Tregs differentiation of in PBMC cultures in the presence of TGF-β1. (A) IL-2, (B) IFN-γ, and (C) TNF-α in cell culture supernatants were measured by ELISA. (D) The percentage of CD25 + Foxp3 + cells among CD4 + lymphocytes was calculated by flow cytometry. The Wilcoxon signed-rank sum test was used for statistical analysis ( n = 6, * p <0.05).

Article Snippet: Subsequently, 1 μg/mL of anti-canine CD4 mouse monoclonal antibody (R&D systems) and 1 μg/mL phycoerythrin (PE)-conjugated anti-canine CD25 mouse monoclonal antibody (Thermo Fisher Scientific) were added and incubated at 25°C for 30 min.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: European journal of immunology

Article Title: Sub-optimal CD4+ T-cell activation triggers autonomous TGF-β-dependent conversion to Foxp3+ regulatory T cells.

doi: 10.1002/eji.201040896

Figure Lengend Snippet: Figure 1. Non-depleting anti-CD4 MAbs induce low-frequency Treg conversion in vivo. OT-II.RAG/ mice were transplanted with skin grafts from OVA-transgenic mice in the presence of anti-CD4 MAb. At day 30, the frequency of Foxp31 T cells was evaluated in axillary LN and spleens of mice that rejected the skin grafts (control) or that were rendered tolerant (anti-CD4). Bar graph and representative dot plots from one of two independent experiments (n 5 5; bars show mean7SD; po0.05; po0.01, two-tailed non-parametric Mann–Whitney test).

Article Snippet: Inhouse-produced anti-CD4 (IgG2a, YTS177), the control anti-canine CD4 (IgG2a, YKIX302), neutralizing anti-TGF-b (1D11), anti-CD3 (145-2C11, eBiosciences), and recombinant TGF-b (R&D Systems) were used as indicated in the text.

Techniques: In Vivo, Transgenic Assay, Control, Two Tailed Test, MANN-WHITNEY

Journal: European journal of immunology

Article Title: Sub-optimal CD4+ T-cell activation triggers autonomous TGF-β-dependent conversion to Foxp3+ regulatory T cells.

doi: 10.1002/eji.201040896

Figure Lengend Snippet: Figure 2. Suboptimal T-cell stimulation drives Foxp3 expression by T cells. (A and B) OVA-specific TCR-transgenic T cells were sorted from DO11.10.RAG/ mice and cultured for 3 days with bone marrow DCs loaded with 0.1 mM OVA in the presence of different concentrations of anti- CD4 MAb. In some wells, 5 ng/mL TGF-b or neutralizing anti-TGF-b (100 mg/mL) were added to the medium. (A) Bar graph and (B) representative dot plots from one out of three independent experiments are shown (mean1SD). (C and D) OVA-specific T cells, prepared as in A, were cultured for 3 days with bone marrow DCs and different concentrations of agonist peptide (mean7SD, representative of three independent experiments). (E) Sorted OT-II.RAG/ T cells were cultured for 3 days in plates coated with 3 mg/mL of a mixture of anti-CD3 MAb and a control MAb (YKIX302), where the anti-CD3 MAb was 100, 50, 10, 1 or 0% of the MAb in the mixture. In some wells, 5 ng/mL TGF-b was added to the medium (mean1SD, representative of three independent experiments). (F and G) CD25 expression by Foxp31 and Foxp3 T cells at the end of cultures described in (E). (H) Proliferation of effector T cells in the presence of equal number of in vitro induced or natural Foxp31 Tregs (mean1SD, n 5 5, po0.05, two-tailed non-parametric Mann–Whitney test). (I) Addition of neutralizing anti-TGF-b MAb to the culture conditions described in (E) in the absence of exogenous TGF-b (mean1SD, representative of three independent experiments).

Article Snippet: Inhouse-produced anti-CD4 (IgG2a, YTS177), the control anti-canine CD4 (IgG2a, YKIX302), neutralizing anti-TGF-b (1D11), anti-CD3 (145-2C11, eBiosciences), and recombinant TGF-b (R&D Systems) were used as indicated in the text.

Techniques: Cell Stimulation, Expressing, Transgenic Assay, Cell Culture, Control, In Vitro, Two Tailed Test, MANN-WHITNEY

Journal: Blood

Article Title: Stable long-term mixed chimerism achieved in a canine model of allogeneic in utero hematopoietic cell transplantation.

doi: 10.1182/blood-2013-11-537571

Figure Lengend Snippet: Figure 1. Development of immune profile in fetal thymus by immunohistochemistry. (A,C,E; 320, H&E) Shows the evolution in tissue architecture, as well as the appearance and expansion of CD4/8 DP cells (B,D,F; 320, CD41 [blue], CD81 [red], CD4/8 DP [purple]). At 33 days (A-B), the thymus is relatively devoid of T cells or their precursors. By 39 days (C-D), DP cells begin to appear and undergo significant expansion by 46 days (E-F), when the tissue architecture takes on a relatively normal postnatal appearance. Scale bars represent 50 mm. Flow cytometry (G) confirms rapid proliferation of DP thymocytes between 39 and 42 days, with even more significant increase by 46 days.

Article Snippet: Primary antibody was applied (mouse anti-canine CD45 [AbD Serotec], goat anti-canine CD4 [R&D Systems, Minneapolis, MN], or mouse anti-canine CD8 [Lifespan, Seattle,WA]) and incubated overnight at 4°C.

Techniques: Immunohistochemistry, Flow Cytometry

Journal: Inflammation

Article Title: CXCL9/Mig Mediates T cells Recruitment to Valvular Tissue Lesions of Chronic Rheumatic Heart Disease Patients

doi: 10.1007/s10753-013-9606-2

Figure Lengend Snippet: In situ expression of chemokine and chemokine receptors. Cardiac tissue sections from RHD patients were stained with primary antibodies against CD4-Alexa Fluor 488, CD8-Alexa Fluor 488, CCL3, CXCL9, CCR5, and CXCR3 followed by incubation with Alexa Fluor 633-conjugated secondary antibodies. DAPI was used for nuclear staining. Fluorescent images were obtained using an LSM/Meta 510 Zeiss microscope and analysis was performed using LSM Image Examiner software (Zeiss). The figure depicts some examples of positive staining from different patients. a , b CD4 + and CD8 + staining ( green ) in the mitral valve of patient 15; c , d CCR5 and CCL3/MIP1α staining (pink and red, respectively) in the myocardium of patient 22; e , f CXCL9/Mig and CXCR3 staining ( red ) in the mitral valve of patient 16.

Article Snippet: The following primary antibodies were used: CCL1 (clone 35305), CCL3 (clone 93321), CXCL9 (clone 49106), CCR5 (clone 45523), CXCR3 (clone 49801), FITC-conjugated CD14 (clone TuK4), Alexa Fluor 488-conjugated CD4 (clone S3.5), and Alexa Fluor 488-conjugated CD8 (clone 3B5; all unconjugated primary antibodies were purchased from R&D Systems Inc., Minneapolis, MN, and all conjugated primary antibodies were purchased from Invitrogen Corporation Carlsbad, CA).

Techniques: In Situ, Expressing, Staining, Incubation, Microscopy, Software

Journal: Inflammation

Article Title: CXCL9/Mig Mediates T cells Recruitment to Valvular Tissue Lesions of Chronic Rheumatic Heart Disease Patients

doi: 10.1007/s10753-013-9606-2

Figure Lengend Snippet: Phenotypic Characterization of Valvular Tissue-Derived T Cells that Migrate upon Specific Chemokine Gradient

Article Snippet: The following primary antibodies were used: CCL1 (clone 35305), CCL3 (clone 93321), CXCL9 (clone 49106), CCR5 (clone 45523), CXCR3 (clone 49801), FITC-conjugated CD14 (clone TuK4), Alexa Fluor 488-conjugated CD4 (clone S3.5), and Alexa Fluor 488-conjugated CD8 (clone 3B5; all unconjugated primary antibodies were purchased from R&D Systems Inc., Minneapolis, MN, and all conjugated primary antibodies were purchased from Invitrogen Corporation Carlsbad, CA).

Techniques:

Journal: Inflammation

Article Title: CXCL9/Mig Mediates T cells Recruitment to Valvular Tissue Lesions of Chronic Rheumatic Heart Disease Patients

doi: 10.1007/s10753-013-9606-2

Figure Lengend Snippet: Heart-infiltrating T cells migrate towards specific chemokines gradient. a Absolute numbers of heart infiltrating T cells that migrated toward CCL1/I-309, CCL3/MIP1α, CXCL9/Mig, and CCL17/TARC gradient compared with untreated cells (controls). Data represent mean ± SD of three to four valvular tissue-derived T cell lines from different RHD patients that transmigrated toward chemokine gradient. Statistical analysis was performed using nonparametric unpaired t test. P values are depicted in the figure. b The phenotype of heart tissue-infiltrating T cells that migrated toward CCL1/I-309 gradient (250 nM). The percentage of cells was determined by flow cytometric analysis (FACS) and the results of patient 19 are depicted. Pre- and post-migration CD4 + CD45RO + and CD8 + CD45RO + subpopulations are shown.

Article Snippet: The following primary antibodies were used: CCL1 (clone 35305), CCL3 (clone 93321), CXCL9 (clone 49106), CCR5 (clone 45523), CXCR3 (clone 49801), FITC-conjugated CD14 (clone TuK4), Alexa Fluor 488-conjugated CD4 (clone S3.5), and Alexa Fluor 488-conjugated CD8 (clone 3B5; all unconjugated primary antibodies were purchased from R&D Systems Inc., Minneapolis, MN, and all conjugated primary antibodies were purchased from Invitrogen Corporation Carlsbad, CA).

Techniques: Derivative Assay, Migration