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. Author manuscript; available in PMC: 2020 Jul 15.
Published in final edited form as: Methods Enzymol. 2019 Oct 22;631:43–59. doi: 10.1016/bs.mie.2019.08.012

Fig. 3.

Fig. 3

Effects of GITR agonism in Treg suppression assay. Donor-derived CFSE-labeled Teff were cultured alone, or with an equal amount of CTV-labeled Teff or Tregs and stimulated for 4 days with anti-CD3 and anti-CD28 in the presence or the absence of rhGITRL. (A) Representative gating strategy of proliferating CFSE-labeled and CTV-labeled CD4+Teff. CFSE-labeled and CTV-labeled cells are gated among live single CD4+ T cells. Proliferating CFSElowand CTVlow cells are then gated among the CFSE+ and CTV+ CD4+ cell subsets in the respective histogram plot. (B) Representative FACS analysis of proliferation (CFSE dilution) and activation (CD25 up-regulation) in target Teff cultured alone or with an equal amount of CTV-labeled Teff or Tregs and stimulated with (+rhGITRL) or without (−rhGITRL) rhGITRL. (C) Quantification of the indicated cytokines in the supernatants from Teff:Teff and Tregs:Teff co-cultures as shown in (B).