title: Comparative dose-responses of recombinant human IL-2 and IL-7 on STAT5 phosphorylation in CD4+FOXP3- cells versus regulatory T cells: a whole blood perspective.
abstract: Interleukin(IL)-2 and IL-7 are cytokines with important functions related to CD4(+) lymphocyte proliferation, differentiation and survival. Depending on doses, they theoretically activate regulatory (Treg) and/or effector T cells (Teff) and thus may be indicated with different therapeutic objectives. In this study we assessed ex vivo the differential dose-responses of CD4(+) T cell subsets (Treg versus CD4(+)FOXP3(-) cells) to recombinant human (rh) IL-2 and rhIL-7. Fresh whole blood from healthy donors was stimulated with increasing doses of cytokines. By using a novel flow cytometry procedure of intracellular signaling pathway staining (e.g., detection of STAT5 phosphorylation; a pivotal marker of cytokine-induced activation; in combination with intracellular FOXP3 staining), we were able to specifically measure Treg and CD4(+)FOXP3(-) cell responses in the same tube. Half maximal effective concentrations (EC50) were calculated. We observed a dose-response effect on Treg and CD4(+)FOXP3(-) cells for both cytokines. Interestingly, low doses of hIL-2 preferentially activated Treg (EC50 Treg = 0.15 pg/ml versus CD4(+)FOXP3(-) cells = 750 pg/ml - p < 0.0001) whereas low doses of rhIL-7 preferentially induced CD4(+)FOXP3(-) cell activation (EC50 Treg = 25 pg/ml and CD4(+)FOXP3(-) cells = 2.5 pg/ml - p < 0.0001). To our knowledge, this work is the first to show differential dose-response effects on CD4(+)FOXP3(-) cells versus Treg of rhIL-7 and rhIL-2 in one ex vivo whole blood single tube assay including two intracellular stainings (i.e., pSTAT5 and FOXP3). Beyond the confirmation of the dose-dependent differential effects of IL-2 versus IL-7 on CD4(+)FOXP3(-) cells/Treg, our results illustrate the value of this approach for monitoring drugs' activities by flow cytometry in daily clinical practice.
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Laboratoire d'Immunologie, Lyon F-69003, France; Département d'anesthésie réanimation, Centre hospitalier universitaire de Saint-Etienne, Saint-Etienne F-42055, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Dupont
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Laboratoire d'Immunologie, Lyon F-69003, France; Université Claude Bernard Lyon 1, EAM 4174, Lyon F-69008, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Demaret
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Laboratoire d'Immunologie, Lyon F-69003, France; Université Claude Bernard Lyon 1, EAM 4174, Lyon F-69008, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Venet
authors.affiliation: Beckman Coulter Immunotech, Life Sciences Global Assay and Applications Development, Marseille, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Malergue
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Laboratoire d'Immunologie, Lyon F-69003, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Malcus
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Laboratoire d'Immunologie, Lyon F-69003, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Poitevin-Later
authors.affiliation: Département d'anesthésie réanimation, Centre hospitalier universitaire de Saint-Etienne, Saint-Etienne F-42055, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Morel
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Laboratoire d'Immunologie, Lyon F-69003, France; Université Claude Bernard Lyon 1, EAM 4174, Lyon F-69008, France. Electronic address: guillaume.monneret@chu-lyon.fr.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Monneret
mesh_terms: D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D015851:Interleukin-7 / Q000494:pharmacology*; D008213:Lymphocyte Activation / Q000187:drug effects*; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000494:pharmacology; D050799:STAT5 Transcription Factor / Q000378:metabolism*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; C508594:IL2 protein, human; C508605:IL7 protein, human; D007376:Interleukin-2; D015851:Interleukin-7; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; D025521:Tumor Suppressor Proteins
keywords: FOXP3; Flow cytometry; Interleukin-2; Interleukin-7; STAT5