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.

1 . A method, comprising:
 generating a plurality of amplicons comprising rearranged TCR or IG CDR3 nucleic acid sequences from a first sample comprising nucleic acid molecules obtained from lymphocytes from a mammalian subject, using a plurality of V-segment primers and a plurality of J-segment primers in a single multiplex PCR, wherein each V-segment primer is complementary to one or more functional TCR or IG V-region gene segments, wherein each J-segment primer is complementary to one or more functional TCR or IG J-region gene segments, wherein said plurality of V-segment primers and said plurality of J-segment primers are capable of amplifying in said single multiplex PCR at least 10 4  distinct amplicons representing a diversity of rearranged TCR or IG CDR3 nucleic acid sequences present in said first sample;   sequencing said plurality of amplicons; and   determining a total diversity of rearranged TCR or IG CDR3 nucleic acid sequences from said plurality of sequenced amplicons of said first sample.   
     
     
         2 . The method of  claim 1 , wherein said determining comprises quantifying said plurality of sequenced amplicons generated in said single multiplex PCR. 
     
     
         3 . The method of  claim 2 , wherein said quantifying comprises quantifying the number of distinct rearranged TCR or IG CDR3 nucleic acid sequences from said first sample. 
     
     
         4 . The method of  claim 2 , wherein said determining comprises estimating a total diversity of rearranged TCR or IG CDR3 nucleic acid sequences. 
     
     
         5 . The method of  claim 1 , wherein said total diversity comprises at least 10 4  distinct rearranged TCR or IG CDR3 nucleic acid sequences. 
     
     
         6 . The method of  claim 1 , wherein said total diversity comprises at least 10 5  distinct rearranged TCR or IG CDR3 nucleic acid sequences. 
     
     
         7 . The method of  claim 1 , wherein said total diversity comprises at least 10 6  distinct rearranged TCR or IG CDR3 nucleic acid sequences. 
     
     
         8 . The method of  claim 1 , wherein said nucleic acid molecules comprise genomic DNA. 
     
     
         9 . The method of  claim 1 , wherein said nucleic acid molecules comprise cDNA. 
     
     
         10 . The method of  claim 1 , wherein said nucleic acid molecules comprise mRNA. 
     
     
         11 . The method of  claim 1 , wherein sequencing comprises high-throughput sequencing of said plurality of amplicons using a set of sequencing oligonucleotides that hybridize to a defined region in each of said plurality of amplicons. 
     
     
         12 . The method of  claim 1 , wherein said TCR V-region gene segment comprises a TCR Vδ segment and wherein said TCR J-region gene segment comprises a TCR Jδ segment. 
     
     
         13 . The method of  claim 1 , wherein said TCR V-region gene segment comprises a TCR Vγ segment and wherein said TCR J-region gene segment comprises a TCR Jγ segment. 
     
     
         14 . The method of  claim 1 , wherein said TCR V-region gene segment comprises a TCR Vα segment and wherein said TCR J-region gene segment comprises a TCR Jα segment. 
     
     
         15 . The method of  claim 1 , wherein said TCR V-region gene segment comprises a TCR Vβ segment and wherein said TCR J-region gene segment comprises a TCR Jβ segment. 
     
     
         16 . The method of  claim 1 , wherein said Ig V-region gene segment comprises an IGH V gene segment and wherein said Ig J-region gene segment comprises an IGH J gene segment. 
     
     
         17 . The method of  claim 1 , wherein said Ig V-region gene segment comprises an IGL V gene segment and wherein said Ig J-region gene segment comprises an IGL J gene segment. 
     
     
         18 . The method of  claim 1 , wherein said Ig V-region gene segment comprises an IGK V gene segment and wherein said Ig J-region gene segment comprises an IGK J gene segment. 
     
     
         19 . The method of  claim 1 , wherein each of said distinct rearranged TCR or IG CDR3 nucleic acid sequences spans a V-D-J or V-J junction. 
     
     
         20 . The method of  claim 1 , further comprising:
 generating a plurality of amplicons comprising rearranged TCR or IG CDR3 nucleic acid molecules from a second sample comprising nucleic acid molecules obtained from lymphocytes from a mammalian subject, using said plurality of V-segment primers and said plurality of J-segment primers in a single multiplex PCR;   sequencing said plurality of amplicons; and   determining a total diversity of rearranged TCR or IG CDR3 nucleic acid sequences from said plurality of sequenced amplicons of said second sample.   
     
     
         21 . The method of  claim 20 , wherein said first sample and said second sample are from the same mammalian subject. 
     
     
         22 . The method of  claim 21 , wherein said first sample is obtained from said mammalian subject before a treatment and said second sample is obtained from said mammalian subject after said treatment. 
     
     
         23 . The method of  claim 21 , further comprising comparing the total diversity of rearranged TCR or IG CDR3 nucleic acid sequences of said first sample with the total diversity of rearranged TCR or IG CDR3 nucleic acid sequences of said second sample. 
     
     
         24 . The method of  claim 23 , further comprising assessing an effect of a treatment based on differences in measurements of diversity between said first and second samples. 
     
     
         25 . The method of  claim 23 , further comprising assessing an immunocompetence of said subject based on differences in measurements of diversity between said first and second samples.