Combination of MHC-peptide multimer-based T cell sorting with the Immunoscope permits sensitive ex vivo quantitation and follow-up of human CD8+ T cell immune responses.
Identification of MHC-restricted antigens and progress in the induction and control of adaptive cytotoxic immune responses have led to renewed interest in immunotherapy as a treatment for severe pathologies such as cancer and autoimmune diseases. Reliable procedures for detecting and monitoring T cell responses induced by the treatment throughout a clinical trial are needed in order to design rational protocols with increased efficiency. We have attempted to develop such a procedure by combining T cell sorting using HLA-peptide complexes multimerized on magnetic beads together with the quantitative Immunoscope approach. Once a recruited patient has been typed for HLA and target antigens, relevant HLA--peptide multimers can be selected and used for sorting specific peripheral T cells prior to any treatment and at the peak of the expected response to treatment. Clonotypic primers specific for the TCR rearrangements of the specific T cell clones can then be designed and used for measuring the frequency of their TCR transcripts by quantitative PCR on blood samples or T cell subsets throughout the trial. In reconstruction experiments as well as in samples from one rheumatoid arthritis patient, we were readily able to detect and follow several T cell clones with a frequency as low as 10(-5) among CD8+ T cells. The main advantages of this procedure over other currently available assays are that it does not require any assumptions on the functional status of the specific T cells and it permits the monitoring of individual T cell clones whose phenotypic shift can thus be evaluated.
Lim
Annick
A
Unité de Biologie Moléculaire du Gène, INSERM U277, Institut Pasteur, Paris, France.
Baron
Véronique
V
Ferradini
Laurent
L
Bonneville
Marc
M
Kourilsky
Philippe
P
Pannetier
Christophe
C
D000951:Antigens, Neoplasm; D001327:Autoimmune Diseases / Q000276:immunology / Q000628:therapy; D001483:Base Sequence; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002985:Clinical Protocols; D018076:DNA, Complementary / Q000235:genetics; D015329:Gene Rearrangement, T-Lymphocyte; D015789:HLA-A2 Antigen; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000706:statistics & numerical data; D018189:Immunomagnetic Separation; D007167:Immunotherapy; D066298:In Vitro Techniques; D008545:Melanoma / Q000276:immunology; D009369:Neoplasms / Q000276:immunology / Q000628:therapy; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D000951:Antigens, Neoplasm; D018076:DNA, Complementary; D015789:HLA-A2 Antigen