METHOD OF MEASURING ADAPTIVE IMMUNITY

摘要

A method of measuring immunocompetence is described. This method provides a means for assessing the effects of diseases or conditions that compromise the immune system and of therapies aimed to reconstitute it. This method is based on quantifying T-cell diversity by calculating the number of diverse T-cell receptor (TCR) beta chain variable regions from blood cells.

主权项

1. A method, comprising:
contacting a sample comprising genomic DNA from mammalian lymphoid cells with a plurality of distinct V-segment primers and at least 3 distinct J-segment primers,
wherein said plurality of distinct V-segment primers are each capable of specifically hybridizing to at least one immunoglobulin (Ig) V-region gene segment, wherein each V-segment primer hybridizes to a V-region gene sequence that is outside a first region where untemplated deletions occur during Ig gene rearrangement, wherein said first region of said V-region gene segment is adjacent to and 5′ to a V-recombination signal sequence (V-RSS) of said V-region gene segment, wherein each V-segment primer hybridizes with the 3′ end of said V-segment primer closer to the RSS than the 5′ end of said V-segment primer and is positioned with the 3′ end of said V-segment primer at least 10 nucleotides upstream from said V-RSS;wherein said at least 3 distinct J-segment primers are each capable of specifically hybridizing to at least one Ig J-region gene segment, wherein each J-segment primer hybridizes to a J-region gene sequence that is outside a second region where untemplated deletions occur during Ig gene rearrangement, wherein said second region of said J-region gene segment is adjacent to and 3′ to a J-recombination signal sequence (J-RSS) of said J-region gene segment, wherein said J-segment primer hybridizes with the 3′ end of said J-region primer closer to said J-RSS than the 5′ end of said J-segment primer and is positioned with the 3′ end of the J-segment primer at least 10 nucleotides downstream from the J-RSS; amplifying in a single multiplex PCR reaction said genomic DNA with said plurality of distinct V-segment primers and said at least 3 distinct J-segment primers to produce at least 104 distinct rearranged DNA amplicons representing the diversity of Ig genes in said sample; and sequencing each of said at least 104 distinct rearranged DNA amplicons using high-throughput sequencing.