title: Murine CD4 T cells produce a new form of TGF-β as measured by a newly developed TGF-β bioassay.
abstract: BACKGROUND
  It is generally assumed that T cells do not produce active TGF-β since active TGF-β as measured in supernatants by ELISA without acidification is usually not detectable. However, it is possible that active TGF-β from T cells may take a special form which is not detectable by ELISA.
  
  
  RESULTS
  We constructed a TGF-β bioassay which can detect both soluble and membrane-bound forms of TGF-β from T cells. For this bioassay, 293T cells were transduced with (caga)(12) Smad binding element-luciferase along with CD32 (Fc receptor) and CD86. The resulting cells act as artificial antigen presenting cells in the presence of anti-CD3 and produce luciferase in response to biologically active TGF-β. We co-cultured pre-activated murine CD4(+)CD25(-) T cells or CD4(+)CD25(+) T cells with the 293T-caga-Luc-CD32-CD86 reporter cells in the presence of anti-CD3 and IL-2. CD4(+)CD25(-) T cells induced higher luciferase in the reporter cells than CD4(+)CD25(+) T cells. This T cell-produced TGF-β is in a soluble form since T cell culture supernatants contained the TGF-β activity. The TGF-β activity was neutralized with an anti-mouse LAP mAb or an anti-latent TGF-β/pro-TGF-β mAb, but not with anti-active TGF-β Abs. An anti-mouse LAP mAb removed virtually all acid activatable latent TGF-β from the T cell culture supernatant, but not the ability to induce TGF-β signaling in the reporter cells. The induction of TGF-β signaling by T cell culture supernatants was cell type-specific.
  
  
  CONCLUSIONS
  A newly developed 293T-caga-Luc-CD32-CD86 reporter cell bioassay demonstrated that murine CD4 T cells produce an unconventional form of TGF-β which can induce TGF-β signaling. This new form of TGF-β contains LAP as a component. Our finding of a new form of T cell-produced TGF-β and the newly developed TGF-β bioassay system will provide a new avenue to investigate T cell function of the immune system.
authors.affiliation: Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Takatoku
authors.initials: T
authors.lastname: Oida
authors.forename: Howard L
authors.initials: HL
authors.lastname: Weiner
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D051940:B7-2 Antigen / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D002460:Cell Line; D018920:Coculture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D009500:Neutralization Tests; D009928:Organ Specificity; D010766:Phosphorylation; D017452:Receptors, IgG / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D051899:Smad2 Protein / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D051940:B7-2 Antigen; D017452:Receptors, IgG; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D016212:Transforming Growth Factor beta