title: Definition of an immunologic response using the major histocompatibility complex tetramer and enzyme-linked immunospot assays.
abstract: OBJECTIVE
  Define an immunologic response using the tetramer and enzyme-linked immunospot (ELISPOT) assays.
  
  
  METHODS
  Ten healthy subjects and 21 patients with melanoma (all HLA-A*0201) donated a total of 121 blood samples to determine the lower limit of detection (LLD), analytic coefficient of variation (aCV), and physiologic CV (pCV) of the tetramer and ELISPOT assays. The mean, SD, and reference change value (RCV) were calculated to define changes beyond the assay imprecision, and its application was tested in the monitoring of T-cell expansion after CTLA4 blockade with ticilimumab (CP-675,206).
  
  
  RESULTS
  The LLD for the tetramer assay was 0.038% CD8+ cells and seven spots per 10(5) peripheral blood mononuclear cells for the ELISPOT assay. The aCV of the tetramer assay was <10% and was higher for the ELISPOT (24.69-36.32%). There was marked between-subject variability on baseline homeostatic values, which was correlated to prior antigen exposure. An immunologic response was defined as an increase beyond the mean + 3 SD in antigen-specific cells for subjects with baseline levels below the LLD, or beyond the assay RCV for baseline levels above the LLD. In four patients receiving ticilimumab, expansions of antigen-specific T cells beyond the assay variability were noted for EBV and MART1 antigens.
  
  
  CONCLUSIONS
  A combined approach of change from negative (below the LLD) to positive (above the LLD) and a percentage change beyond the assay variability using the RCV score can be computed to define which change in circulating antigen-specific T cells represents a response to immunotherapy.
authors.affiliation: Department of Surgery, Division of Surgical, University of California at Los Angeles, Los Angeles, California, USA.
authors.forename: Begoña
authors.initials: B
authors.lastname: Comin-Anduix
authors.forename: Antonio
authors.initials: A
authors.lastname: Gualberto
authors.forename: John A
authors.initials: JA
authors.lastname: Glaspy
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Seja
authors.forename: Maribel
authors.initials: M
authors.lastname: Ontiveros
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Reardon
authors.forename: Roberto
authors.initials: R
authors.lastname: Renteria
authors.forename: Brigitte
authors.initials: B
authors.lastname: Englahner
authors.forename: James S
authors.initials: JS
authors.lastname: Economou
authors.forename: Jesus
authors.initials: J
authors.lastname: Gomez-Navarro
authors.forename: Antoni
authors.initials: A
authors.lastname: Ribas
mesh_terms: D000328:Adult; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007118:Immunoassay; D007167:Immunotherapy; D007371:Interferon-gamma / Q000096:biosynthesis / Q000276:immunology; D008285:Major Histocompatibility Complex / Q000276:immunology*; D008297:Male; D008545:Melanoma / Q000276:immunology* / Q000628:therapy; D008875:Middle Aged; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D007371:Interferon-gamma