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4 protocols using αifn γ clone xmg1

1

Immune Checkpoint Modulation Protocols

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αGr-1 (clone RB-8C5), αLg6G (clone 1A8), αIFNAR-1 (clone MAR1-5A3), αIL-4 (clone 11B11), αCD8 (clone 2.43), αNK1.1 (clone PK136), αIL-21R (clone 4A9), and αIFNγ (clone XMG1.2) were purchased from BioXCell. Mouse cytokines IL-4, IL-6, IL-12, and human IL-4, TGFβ, IL-2 and IL-6 were purchased from R&D Systems. eATP signaling inhibitor Suramin (Catalog# sc-200833), CD39 inhibitor POM-1 (Catalog# sc-203205) and NF-κB inhibitor QNZ (Catalog# sc-200675) were purchased from Santa Cruz Biotechnology.
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2

Naïve SMARTA Cell Polarization Protocols

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Naïve SMARTA cells were negatively isolated by using CD4+ T cell isolation kit (Miltenyi or StemCell). 2 × 106 SMARTA cells were seeded on 24 well plates coated with 8 µg/mL αCD3 (clone 17A2, BioXcell) and αCD28 (clone 37.51, BioXcell). For TH1polarization, Smarta cells were given with 20 µg/mL of αIL-4 (clone 11B11, BioXcell) and αTGF-β (clone 1D11, BioXcell) and 20 ng/mL of rmIL-12 (Peprotech). For IL-6 condition, 10 µg/mL of αIFN-γ (clone XMG1.2, BioXcell) and αIL-12 (clone R1-5D9, BioXcell) and 20 ng/mL of rmIL-6 (Peprotech) were added in culture media.
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3

Murine Infection Models for Immunology

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The University of Iowa institutional animal care and use committee approved all experiments. C57BL/6 WT (JAX stock 002288), Ifngr1−/− (JAX stock 025545), and MD4 (JAX stock 002595) mice were purchased from The Jackson Laboratory. C57BL/6 Myd88−/− and Ticam1−/− were generous gifts from Michelle Callegan (University of Oklahoma Health Sciences Center, Oklahoma City, OK); Tim1−/− mice were a gift from Paul Rothman (Johns Hopkins, Baltimore, MD); and Axl−/− bone marrow was a generous gift from Carla Rothlin (Yale University, New Haven, CT). Infections in experimental mice were initiated by either serial transfer (i.v.) of 106Plasmodium yoelii (Py; American Type Culture Collection) parasitized RBCs (pRBCs) derived from a single donor C57BL/6 mouse, or i.p. injections of either 2.5 × 106 PFUs of LCMV-Arm or 5,000 Tb Antat1.1 parasites (Tb; a generous gift from Kent Hill, University of California, Los Angeles, Los Angeles, CA). In some experiments, Py-infected mice were treated with 500 µg of either α-IFN-γ (clone XMG1.2; BioXcell) or rat IgG1 isotype control or with 400 µg of either mouse chimeric PtS-blocking antibody clone mch1N11 or mouse IgG2a (clone 1.18.4; BioXcell) isotype control, as indicated in the figure legends. Parasitemia was measured using flow cytometry as described (Malleret et al., 2011 (link); Villarino et al., 2016 (link)).
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4

Naïve SMARTA Cell Polarization Protocols

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Naïve SMARTA cells were negatively isolated by using CD4+ T cell isolation kit (Miltenyi or StemCell). 2 × 106 SMARTA cells were seeded on 24 well plates coated with 8 µg/mL αCD3 (clone 17A2, BioXcell) and αCD28 (clone 37.51, BioXcell). For TH1polarization, Smarta cells were given with 20 µg/mL of αIL-4 (clone 11B11, BioXcell) and αTGF-β (clone 1D11, BioXcell) and 20 ng/mL of rmIL-12 (Peprotech). For IL-6 condition, 10 µg/mL of αIFN-γ (clone XMG1.2, BioXcell) and αIL-12 (clone R1-5D9, BioXcell) and 20 ng/mL of rmIL-6 (Peprotech) were added in culture media.
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