Anti cd4 clone gk1

Single-cell suspensions from spleen of mice were stained with anti-CD4 (clone GK1.5; BioLegend), anti-CD25 (clone 3C7; BioLegend), and anti-FoxP3 (clone MF-14; BioLegend). In the case of intracellular cytokine staining, splenocytes were cultured in RPMI 1640 medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin in presence of phorbol-12-myristate-13-acetate (PMA) (50 ng/ml; Sigma), ionomycin (1 μg/ml; Sigma), and monensin (BioLegend) for 5 h. Cells were washed, fixed, permeabilized, and stained with anti-IL-17 (clone TC11-18H10.1; BioLegend). Viable single cells were analyzed based on forward and side light scatter properties with a CyFlow Cube 6 flow cytometer (Sysmex) or MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec).

An intraperitoneal injection with 500 µg of anti-PDCA1 (clone 927, BioLegend) or intravenous injection with 100 µg of anti-CD4 (clone GK1.5, BioLegend) was performed to deplete pDCs or CD4⁺ cells 1 day before immunization with the vaccine. To deplete CD8⁺ cells, an intraperitoneal injection with 100 µg of anti-CD8α (clone 2.43, Bio X cell, Lebanon, NH) was performed at 15, 17, 19, and 21 days after the inoculation of tumor. To deplete macrophages, 200 µL of clophosome-A (FormuMax Scientific, Inc., Sunnyvale, CA) was intravenously administered on days 1 and 6 before immunization with the vaccine.

Atm-deficient mice received 0.125 mg/mL anti CD4 (clone GK1.5, BioLegend Cat# 100435, RRID:AB_2075571) and 0.125 mg/mL anti CD8 (clone 53–6.7, BioLegend Cat# 100735, RRID:AB_2075237) monoclonal antibody, 7 days before bone marrow transplantation (BMT) and a second dose of each antibody in combination with cyclophosphamide (CP, 80 ng/mL, Endoxan, Baxter, Unterschleißheim, Germany) 1 day before BMT as non-myeloablative conditioning. 5 × 10⁶ bone marrow donor cells (BMDCs) were sterilely harvested from 129S6/SvEv GFP-transfected wild-type mice and were then injected intravenously into conditioned recipients (22 (link), 24 (link)). Grafting of ATM-competent GFP⁺ bone marrow-derived donor cells was traced in the peripheral blood of Atm-deficient mice over the time, 6, 12, and 24 weeks post-transplantation.

To assess the role of IL-33 in pulmonary responses to O₃, WT and db/db mice were treated with an antibody directed against the extracellular domain of recombinant murine ST2 (10 mg/kg, i.p.) or with isotype (IgG1) antibodies. At this dose, anti-ST2 blocks responses to exogenous IL-33 in mice (Palmer et al. 2009 (link)). Mice were exposed to O₃ 24 hr later, and evaluated 24 hr after exposure. Evaluation included measurement of airway responsiveness, BAL, and lung tissue and blood harvest.
To evaluate the role of CD4 cells in O₃-induced changes in type 2 cytokines, we depleted CD4 cells. Db/db mice were injected once with anti-CD4 (clone: GK1.5, Biolegend) (8 mg/kg) or isotype antibody (Rice and Bucy 1995 (link)). Mice were exposed to O₃ 6 days later and evaluated as described above. Confirmation of CD4 depletion in lung tissue was assessed by flow cytometry.
To examine the role of γδ T cells in obese mice, WT and TCRδ^–/– mice were fed either an HFD or normal chow, and then exposed to air or to O₃, and evaluated as described above.
Methods for BAL, measurement of cytokines and chemokines, RNA extraction and RT-qPCR, and flow cytometry were as previously described (Krishnamoorthy et al. 2015 (link); Williams et al. 2013 (link)) and are found in an online supplement (see “Supplemental Methods” in the Supplemental Material).