title: Preculture of PBMCs at high cell density increases sensitivity of T-cell responses, revealing cytokine release by CD28 superagonist TGN1412.
abstract: Human volunteers receiving TGN1412, a humanized CD28-specific monoclonal antibody, experienced a life-threatening cytokine release syndrome during a recent trial. Preclinical tests using human PBMCs had failed to announce the rapid release of TNF, IFN-γ, and other toxic cytokines in response to this CD28 "superagonist" (CD28SA). CD28SA activate T-lymphocytes by ligating CD28 without overt engagement of the TCR. They do, however, depend on "tonic" TCR signals, which they amplify. Here we show that short-term preculture of PBMCs at high, but not at low, cell density results in massive cytokine release during subsequent stimulation with soluble TGN1412. Restoration of reactivity was cell-contact dependent, involved functional maturation of both monocytes and T cells, was sensitive to blockade by HLA-specific mAb, and was associated with TCR polarization and tyrosine phosphorylation. CD4 effector memory T cells were identified as the main source of proinflammatory cytokines. Importantly, responses to other T-cell activating agents, including microbial antigens, were also enhanced if PBMCs were first allowed to interact under tissue-like conditions. We provide a protocol, which strongly improves reactivity of circulating T cells to soluble stimulants, thereby allowing for more reliable preclinical testing of both activating and inhibitory immunomodulatory drugs.
authors.affiliation: Institute for Virology and Immunobiology, University of Würzburg, Germany.
authors.forename: Paula S
authors.initials: PS
authors.lastname: Römer
authors.forename: Susanne
authors.initials: S
authors.lastname: Berr
authors.forename: Elita
authors.initials: E
authors.lastname: Avota
authors.forename: Shin-Young
authors.initials: SY
authors.lastname: Na
authors.forename: Manuela
authors.initials: M
authors.lastname: Battaglia
authors.forename: Ineke
authors.initials: I
authors.lastname: ten Berge
authors.forename: Hermann
authors.initials: H
authors.lastname: Einsele
authors.forename: Thomas
authors.initials: T
authors.lastname: Hünig
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology* / Q000494:pharmacology; D018106:CD28 Antigens / Q000819:agonists / Q000276:immunology*; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D007376:Interleukin-2 / Q000276:immunology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D008198:Lymph Nodes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018613:Microscopy, Confocal; D016853:Muromonab-CD3 / Q000276:immunology / Q000494:pharmacology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D018106:CD28 Antigens; D016207:Cytokines; D007376:Interleukin-2; D016853:Muromonab-CD3; D011948:Receptors, Antigen, T-Cell; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; C509303:TGN-1412