title: Mechanisms of T-cell protection from death by IRX-2: a new immunotherapeutic.
abstract: OBJECTIVE
  IRX-2 is a novel immunotherapeutic containing physiologic quantities of several cytokines which protects human T lymphocytes from tumor-induced or drug-induced apoptosis. Here, we investigate the mechanisms responsible for IRX-2-mediated protection of T lymphocytes exposed to tumor-derived microvesicles (TMV).
  
  
  METHODS
  Jurkat cells or primary human T cells ± IRX-2 were co-incubated with TMV and then examined by flow cytometry or Western blots for expression of molecules regulating cell survival (FLIP, Bcl-2, Bcl-xL, Mcl-1) or death (Fas, caspase 8, caspase 9, Bax, Bid). ANX V binding, caspase activation or cytochrome c release were also measured ± cycloheximide (CHX) or ± the Akt-specific inhibitor. Jurkat cells transfected with the cFLIP gene were used to evaluate the role of cFLIP in IRX-2-mediated protection. Effects of CHX on IRX-2-mediated protection and activation of NF-κB upon the TMV/IRX-2 treatment were also measured.
  
  
  RESULTS
  IRX-2 protected T cells from apoptosis by preventing Fas overexpression induced by TMV and blocking caspase 8 activation by up-regulating cFLIP. Jurkat cells overexpressing cFLIP were more resistant to TMV-induced apoptosis than the mock-transfected cells (p < 0.02). Signaling via the PI3K/Akt pathway, IRX-2 corrected the imbalance of pro- versus anti-apoptotic proteins induced by TMV and promoted NF-κB translocation to the nucleus. CHX abolished IRX-2-mediated protection in T cells, suggesting that IRX-2 induces de novo synthesis of one or more proteins that are required for protection.
  
  
  CONCLUSIONS
  This biologic may be therapeutically useful for protection of activated T cells from tumor-induced immune suppression and death.
authors.affiliation: Research Pavilion at Hillman Cancer Center, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Suite 1.27, Pittsburgh, PA 15213-1863, USA.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Czystowska
authors.forename: Miroslaw J
authors.initials: MJ
authors.lastname: Szczepanski
authors.forename: Marta
authors.initials: M
authors.lastname: Szajnik
authors.forename: Karen
authors.initials: K
authors.lastname: Quadrini
authors.forename: Harvey
authors.initials: H
authors.lastname: Brandwein
authors.forename: John W
authors.initials: JW
authors.lastname: Hadden
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Whiteside
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002469:Cell Separation; D016207:Cytokines / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D019169:Jurkat Cells; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects*
chemical_list: D016207:Cytokines; C114857:IRX 2