title: Development of assay platforms for in vitro screening of Treg modulating potential of pharmacological compounds.
abstract: CD4 + CD25+ regulatory T cells (Tregs) are believed to be pivotal in controlling chronic inflammation as well as in opposing the effect of cancer immunotherapy. Therefore, identification of novel drug compounds that interfere with Treg function is of high priority together with research that investigates Treg modulation by current drugs. For such research as well as for novel cell based therapies based on Treg infusions, rapid in vitro assays as well as functional assays based on inhibitory capacity of Tregs are required. Here, we report on such assays using highly pure fluorescence-activated cell sorting (FACS) sorted CD4 + CD25(high)CD127(dim/-)CD45RA+ naïve Treg cells followed by in vitro expansion. We report on the use of these cells in a short-term assay based on Treg mediated inhibition of the early effector T cell activation markers CD69 and CD154. Additionally, we investigate the use of highly pure Tregs in a functional assay based on Treg mediated inhibition of effector T cell proliferation. We report highly reproducible Treg function in assays that test the effect of well-known model compounds such as CpG-A, anti-IL-6R (tocilizumab), anti-TNF-α (adalimumab) or a combination of IL-6 and TNF-α. In conclusion, these assays have the potential for use in pharmacological screening and discovery in relation to drug development in immunology.
authors.affiliation: Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark and.
authors.forename: Anders Elm
authors.initials: AE
authors.lastname: Pedersen
authors.forename: Kim
authors.initials: K
authors.lastname: Holmstrøm
authors.forename: Flemming
authors.initials: F
authors.lastname: Jørgensen
authors.forename: Simon S
authors.initials: SS
authors.lastname: Jensen
authors.forename: Monika
authors.initials: M
authors.lastname: Gad
mesh_terms: D000293:Adolescent; D000328:Adult; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology*; D015415:Biomarkers / Q000032:analysis; D015704:CD4 Antigens / Q000276:immunology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D055808:Drug Discovery / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology; D053699:Interleukin-7 Receptor alpha Subunit / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D008875:Middle Aged; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D055815:Young Adult
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; D015415:Biomarkers; D015704:CD4 Antigens; C508017:IL2RA protein, human; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit; D017493:Leukocyte Common Antigens
keywords: Drug screening; in vitro assay; pharmacology; regulatory T cells