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Definition of an Immunologic Response Using the Major Histocompatibility Complex Tetramer and Enzyme-Linked Immunospot Assays

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Definition of an Immunologic Response Using the Major Histocompatibility Complex Tetramer and Enzyme-Linked Immunospot Assays Imaging, Diagnosis, Prognosis DefinitionofanImmunologicResponseUsingtheMajor Histocompatibility Complex Tetramer and Enzyme-Linked Immunospot Assays Begon‹ aComin-Anduix,1 Antonio Gualberto,4 John A. Glaspy,2,3 Elisabeth Seja,2 Maribel Ontiveros,2 Deborah L. Reardon,4 Roberto Renteria,5 Brigitte Englahner,4 James S. Economou,1, 3 Jesus Gomez-Navarro,4 and Antoni Ribas1,2,3 Abstract Purpose: Define an immunologic response using the tetramer and enzyme-linked immunospot (ELISPOT) assays. Experimental Design: Ten healthy subjects and 21patients with melanoma (all HLA-A*0201) donated a total of 121blood samples to determine the lower limit of detection (LLD), analytic coefficient of variation (aCV), and physiologic CV (pCV) of the tetramer and ELISPOTassays. The mean, SD, and reference change value (RCV) were calculated to define changes beyond the assay imprecision, andits application was testedin the monitoring ofT-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 105 peripheral blood mononuclear cells for the ELISPOTassay. 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. Infour patients receiving ticilimumab, expansions of antigen-specificTcells beyond the assay variability were noted for EBVand MART1antigens. 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-specificTcells represents a response to immunotherapy. Modern cellular immunologic assays, like the enzyme-linked rapidly replaced older assays requiring extensive ex vivo immunospot (ELISPOT; refs. 1–4), the MHC tetramer binding manipulations, like the limiting dilution microcytotoxicity assay (5, 6), and the intracellular cytokine flow cytometry assay and the lymphoproliferative assay (10). However, few assay (7, 8), allow for the enumeration of antigen-specific studies had been conducted to define key methodologic T lymphocytes at a single cell level (9–11). These assays have variables, like accuracy, precision, and reproducibility (10). These variables are critical to determine which level of T-cell expansion is beyond the baseline variability of the assay and therefore would represent a positive immune response. Authors’ Affiliations: 1Department of Surgery, Division of Surgical; 2Department of Medicine, Division of Hematology/Oncology; 3Jonsson Comprehensive Cancer The most commonly used approach to define a positive Center, Oncology, University of California at Los Angeles, Los Angeles, California; immune response is the detection of circulating antigen-specific 4Pfizer Global Research and Development, Groton-New London, Connecticut; and T cells after immunotherapy beyond the mean and 2 or 3 SD 5Beckman Coulter, Inc., San Diego, California from negative values, using results from a negative control Received 1/18/05; revised 9/11/05; accepted 10/10/05. antigen or a positive antigen in a negative population (10, 12). Grant support: Flow cytometry and enzyme-linked immunospot assays were done in the University of California at Los Angeles Jonsson Comprehensive Cancer This approach is logical for values that are below a certain Center and Center for AIDS Research Flow Cytometry Core Facility that is threshold of negativity at baseline, which would reflect the assay supported by NIH grants CA-16042 and AI-28697, Jonsson Comprehensive background. However, the baseline values of circulating antigen- Cancer Center, University of California at Los Angeles AIDS Institute, and David specific T cells for many infectious disease and tumor antigens Geffen School of Medicine at University of California at Los Angeles. can be positive at baseline for a significant subset of patients The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance (12, 13). In this case, the definition based on a fixed value with 18 U.S.C. Section 1734 solely to indicate this fact. (an increase beyond the mean + SD from negatives) would not Requests for reprints: Antoni Ribas, University of California at Los Angeles be easily applicable. This approach could only be used if several Medical Center, 11-954 Factor Building, 10833 Le Conte Avenue, Los Angeles, CA baseline values were collected for each patient, allowing defining 90095. Phone: 310-206-3928; Fax: 310-206-0914; E-mail: aribas@mednet. ucla.edu. a mean and SD for their individual baseline values of circulating F 2006 American Association for Cancer Research. antigen-specific T cells, and then comparing it to its change after doi:10.1158/1078-0432.CCR-05-0136 immunotherapy. This is not feasible in routine practice. www.aacrjournals.org 107 Clin Cancer Res 2006;12(1) January 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Imaging, Diagnosis, Prognosis The calculation of the reference change value (RCV) may immunologic studies at redosing at 3 or 6 mg/kg under a separate allow a more reliable definition of an immune response if redosing study (IRB 03-01-014). Two were redosed after being enrolled baseline values are above the assay lower limit of detection at the initial low dose cohorts, and two were enrolled after having (LLD). The RCV allows determining the change that must clinical benefit to the initial dose. Peptides and tetramers. The following peptides were used: AFP occur in an individual’s serial test results before the change is 325-332 (GLSPNLNRFL), CMVpp65 (NLVPMVATV), EBV BMLF1 considered significant by taking into account the main 495-503 259-267 (GLCTLVAML), MART-126-35 (ELAGIGILTV), MART-127-35 (AAGIGILTV), components of assay variability and a desired statistical and a HLA-A*0201-binding nonrelevant peptide (ref. 21; referred to as significance. Its calculation has the goal to define the the negative peptide from here on). All tetramers were purchased from magnitude of change in results that would be beyond the Beckman Coulter, Inc. (San Diego, CA), except a PE-labeled HLA-A*0201 assay imprecision. Since its original description by Harris and MART127-35 tetramer, which was obtained from the National Institute of Brown in 1979 (14), different versions of the formula have Allergy and Infectious Disease (Emory University, Atlanta, GA). been proposed (15–19). We reasoned that when applied to Tetramer-binding assay. Cryopreserved PBMC aliquots were thawed immunologic monitoring, it would allow us to define positive and diluted with RPMI supplemented with 10% human AB serum, 1% and negative changes in circulating antigen-specific T cells after penicillin, streptomycin, amphotericine (PSA, OmegaSci), and DNase j an immunotherapy intervention for subjects with baseline (0.002%, Sigma) for an hour at 37 C. PBMCs were then washed and resuspended at 1 to 3 Â 106/100 AL in 100% adult bovine serum measurable values (above the assay LLD), thereby representing (OmegaSci). Only samples with viability of >90% by trypan blue a true positive or negative immune response and not the assay exclusion were used. PBMCs were stained for 30 minutes at room noise. temperature in the dark with 8 AL MHC tetramer, 8 AL FITC-conjugate anti-CD8 (SFI21Thy2D3), and 8 AL iMASC antibodies (a pre-prepared Materials and Methods mixture with PC5-conjugated CD4 À13B8.2À, CD13 ÀIMMU103.44À, and CD19 ÀJ4.119À, all from Beckman Coulter), which were used to Study eligibility and screening procedures for the methodology study. gate out CD8À lymphocytes. Cells were washed in 3 mL of PBS and, HLA-A*0201-positive healthy subjects and HLA-A*0201-positive immediately before flow cytometric analysis, 10 AL of 7-amino- patients with melanoma were eligible, provided that they were not actinomycin D was added to gate out dead cells. PBMCs were analyzed receiving active therapy or having an intercurrent event (like an on a FACSCalibur (BD Biosciences, San Jose, CA) within 1 hour of influenza infection or symptoms of common cold) for a minimum staining, without adding any fixative. Stained cells were kept constantly period of 30 days before the blood draws and during the blood draws. at 4jC. A minimum of 30,000 CD8+ T cells were acquired, and up to Eligible patients with melanoma included stage II to IV melanoma, a 200,000 were preferred. Analysis was done both with CellQuest Karnofsky performance status of >70%, and adequate hematopoietic, (Beckman Coulter) and FCS Express (DeNovo Software, Thornhill, hepatic, and renal function. To be eligible, tumors were required to Ontario, Canada) software. In addition to the routine daily FACSCa- express Melan-A/MART-1 (MART-1) by immunohistochemistry or libur calibration, FLOW-SET Fluorospheres (Beckman Coulter) were reverse transcription-PCR. Exclusion criteria included prior therapy < used to set peak channels before every tetramer flow cytometry 4 weeks before study entry, untreated central nervous system metastasis, experiment. pregnancy, or concurrent immunosuppressive conditions or therapy. For tetramer spiking
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