Analysis of TCell Receptor V Region Gene Usage and Complementarity Determining Region 3 Sequences

A very straightforward approach to measuring antigen specific T-cell responses would be to use easily identified phenotypic markers for the response. Specimens of lymphocytes obtained before and after an immunization strategy could be tested using standard flow cytometric methods. By exploiting the ability to phenotype T cells by the variable chain usage of their TCRs, it is possible to follow the expansions of specific TCR gene subfamilies of T cells following vaccination. Initial attempts to directly visualize antigen specific T cells used combinations of antibodies that recognize different variable (V) region subfamily of the TCR alpha or beta chains. An increase in the number of cells expressing a particular V-a or V-P chain would denote development of oligoclonality, a possible sign of induction of a specific immune response (19). Unfortunately, this approach has limited value for a number of reasons. First, only a minority of T cells expressing a particular V-a or V-P combination will be specific for a particular antigen. Second, the response to most antigens is quite diverse and utilizes many different variable regions. Third, monospecific antibodies are not available for all V region gene subfamilies, especially the TCR a V region subfamilies, so this analysis is incomplete at best.

It has been suggested, for some antigens, that antigen-specific T cells have a restricted TCR repertoire (20) that can be detected by sequencing the third complementarity-determining region (CDR3) of the TCR. The CDR3 region encodes the highly polymorphic portion of the TCR responsible for recognizing peptide-MHC complexes. For the P chain, the CDR3 region encodes the Variable (V) region-Diversity (D) segment and Diversity segment-Joining (J) segment junctions, whereas for the alpha chain it encodes the V-J junction. Using V, D, or J-region subfamily specific polymerase chain reaction (PCR) primers, PCR may be performed to detect the development of restricted TCR gene usage (21,22). There is compelling evidence for restricted TCR V-region usage in the response to viral diseases (23,24). Some studies in melanoma patients (25,26) and renal cell carcinoma patients (27) have detected a restricted TCR gene usage. However, other studies in melanoma have found unrestricted TCR gene usage (28,29; Clay TM, unpublished observations). More studies are needed to determine the role of this new technology in monitoring immune responses in clinical trials. Advantages include the small amount of specimen required, the ability to perform the analysis from cells directly isolated from the blood to avoid introducing biases caused by ex vivo expansion, the reproducibility, and internal controls that permit analysis of samples collected at different times. Recently, a more automated and rapid fluorescence-based method for CDR3 length analysis of expressed TCR gene families that was able to distinguish between polyclonal, oligoclonal, and monoclonal CDR3 distributions has been developed (30).

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