Method of measuring adaptive immunity

Abstract

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.

What is claimed: 
     
         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 T cell receptor (TCR) 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 TCR 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 TCR 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 TCR 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 10 4  distinct rearranged DNA amplicons representing the diversity of TCR genes in said sample; and   sequencing each of said at least 10 4  distinct rearranged DNA amplicons using high-throughput sequencing.   
     
     
         2 . The method of  claim 1 , further comprising quantifying the full diversity of rearranged TCR genes in said sample using sequencing data of said at least 10 4  distinct rearranged DNA amplicons. 
     
     
         3 . The method of  claim 1 , wherein amplifying produces at least 10 5  distinct rearranged DNA amplicons. 
     
     
         4 . The method of  claim 1 , wherein amplifying produces at least 10 6  distinct rearranged DNA amplicons. 
     
     
         5 . The method of  claim 1 , wherein sequencing comprises using a plurality of sequencing oligonucleotides that hybridize to a defined region of said distinct rearranged DNA amplicons. 
     
     
         6 . The method of  claim 5 , wherein said sequencing oligonucleotides are selected from the group consisting of SEQ ID NOs: 470-482. 
     
     
         7 . The method of  claim 5 , wherein said sequencing oligonucleotides hybridize adjacent to a unique n-base tag within a J gene segment of said distinct rearranged DNA amplicons. 
     
     
         8 . The method of  claim 7 , wherein n is four and wherein said sequencing oligonucleotides hybridize at positions +11 through +14 downstream of the J-RSS. 
     
     
         9 . The method of  claim 1 , wherein said plurality of V-segment primers have similar annealing temperatures. 
     
     
         10 . The method of  claim 1 , wherein said at least 3 distinct J-segment primers have similar annealing temperatures. 
     
     
         11 . The method of  claim 1 , wherein said each of said distinct rearranged DNA amplicons spans a V-D-J or V-J junction. 
     
     
         12 . The method of  claim 1 , wherein said TCR V-region gene segment comprises a TCR Vδ segment, a TCR Vγ segment, a TCR Vα segment, or a TCR Vβ segment. 
     
     
         13 . The method of  claim 1 , wherein said TCR J-region gene segment comprises a TCR Jδ segment, a TCR Jγ segment, a TCR Jα segment, or a TCR Jβ segment. 
     
     
         14 . The method of  claim 1 , further comprising contacting said sample with 14 to 45 distinct V-segment primers. 
     
     
         15 . The method of  claim 1 , further comprising contacting said sample with 3 to 13 distinct J-segment primers. 
     
     
         16 . The method of  claim 1 , wherein each of said plurality of distinct V-segment primers do not cross an intron/exon boundary. 
     
     
         17 . The method of  claim 1 , wherein each of said at least 3 distinct J-segment primers do not cross an intron/exon boundary. 
     
     
         18 . The method of  claim 1 , wherein each of said at least 3 distinct J segment primers hybridize to a conserved element of the J-region gene segment. 
     
     
         19 . The method of  claim 1 , wherein each of said at least 3 distinct J-segment primers specifically hybridizes to a different TCR J-region gene segment.