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3 protocols using αcd28 37.51

1

Modulating T Cell Activation Pathways

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CD4+ T cells isolated from spleen and LN using CD4 microbeads
(Miltenyi). 1×106 T cells cultured with platebound
α-CD3 (2C11) and α-CD28 (37.51) from BioXcell at 1μg/mL
each for 48 or 66 hrs. Inhibitors purchased and used as follows: ZSTK474
(1μM, Sigma), Rapamycin (10nM, Selleckchem), NFAT inhibitor (10μM,
Tocoris), Mek inhibitor PD0325901 (100nM, Peprotech) and Erk inhibitor FR180204
(10μM, Tocoris). In vitro TR suppression
assays were performed as previous described (20 (link)). Chemotaxis assay performed as previously described (21 (link)).
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2

T Cell Isolation and Activation

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VAT was pooled from two or three mice of the same genotype to ensure adequate cell number for culture. Cells were isolated as described above, and CD4+ T cells were enriched by incubating cells with CD4 MicroBeads and positively selecting with MACS cell separation MS columns (Miltenyi Biotec). Purified cells were resuspended in RPMI-C with 500 U/ml recombinant IL-2 (eBioscience). Cells were cultured for 48 h in 96-well flat-bottom plates with plate-bound αCD3 (2C11) and αCD28 (37.51) from Bio X Cell at 1 µg/µl, with or without the addition of plate-bound αICOS (C398.4A; BioLegend) at 2 µg/µl. Expression of CCR3 was assessed by flow cytometry as described above.
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3

CFSE-Based T Cell Proliferation Assay

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Purified CD4+ T cells (CD4 T cells beads, Miltenyi or CD4 Dyna Beads, Invitrogen) were labeled with CFSE or CellTrace Volet (CTV) (Thermofisher) according to manufacturer’s protocol and cultured in RPMI 1640 complete medium supplemented with 10% fetal bovine serum (FBS, GIBCO Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine (all from GIBCO) together with DC and irradiated feeder cells (40 Gy) in 2:1:1 ratio for 72 or 96h. Polyclonal CD4+ T cells were stimulated with αCD3 (2C11) and αCD28 (37.51) (BioXcell); OT-II cells with OVA323-339 (ISQAVHAAHAEINEAGR) peptide (Pepscan) and Trp1 cells with Trp1106-130 (SGHNCGTCRPGWRGAACNQKILTVR) peptide (Pepscan) at concentration indicated in the Figure 2. Cells were additionally supplemented with IL-2, IL-15 or IL-7 (Peprotech) at a concentration indicated in the Figure 2. Mouse CD4+ T cells were cultured with αCD25 (PC61, BioXcell) and αIL-2 (JES6-1A12, BioXcell), added to the culture 24h post stimulation with αCD3 and αCD28 in a concentration of 5 μg/ml.
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