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 obtained from a mammalian subject with a composition, said sample comprising rearranged nucleic acid molecules of a T-cell receptor (TCR) or Immunoglobulin (IG) complementarity determining region 3 (CDR3) region obtained from lymphocytes of said subject, said composition comprising:
 (a) a plurality of V-segment primers, and 
 (b) a plurality of J-segment primers, 
 each of said plurality of V-segment primers and said plurality of J-segment primers consisting of 15 to 50 nucleotides, 
 wherein each of said V-segment primers comprises a first sequence and a second sequence, wherein said first sequence is complementary to a portion of a first region of at least one V-segment of said TCR or IG CDR3 region, said first region located immediately 5′ to a second region of said V-segment where untemplated deletions occur during TCR or IG gene rearrangement, wherein said second region of said V-segment is adjacent to and 5′ to a V-recombination signal sequence (V-RSS) of said V-segment, wherein said first sequence is located 3′ to said second sequence on said V-segment primer, 
 wherein each of said J-segment primers has a first sequence and a second sequence, wherein said first sequence is complementary to a portion of a first region of at least one J-segment of said TCR or IG CDR3 region, said first region located immediately 3′ to a second region of said J-segment where untemplated deletions occur during TCR or IG gene rearrangement, wherein said second region of said J-segment is adjacent to and 3′ to a J-recombination signal sequence (J-RSS) of said J-segment, wherein said first sequence is located 3′ to said second sequence on said J-segment primer, wherein each of said plurality of J-segment primers is not specific to all J-segment genes; and 
   amplifying said rearranged nucleic acid molecules with said plurality of V-segment primers and said plurality of J-segment primers in a single multiplex polymerase chain reaction (PCR) amplification to produce at least 10 5  distinct amplicons representing a diversity of rearranged TCR or IG nucleic acid molecules present in said sample.   
     
     
         2 . The method of  claim 1 , wherein said second sequences of said plurality of V-segment primers and said plurality of J-segment primers each comprise a universal sequence complementary to a sequencing oligonucleotide. 
     
     
         3 . The method of  claim 1 , wherein said second sequence of said plurality of V-segment primers is not complementary to a V-segment of said TCR or IG CDR3 region and wherein said second sequence of said plurality of J-segment primers is not complementary to a J-segment of said TCR or IG CDR3 region. 
     
     
         4 . The method of  claim 1 , further comprising sequencing said at least 10 5  distinct amplicons using a set of sequencing oligonucleotides that hybridize to a region of said at least 10 5  distinct amplicons. 
     
     
         5 . The method of  claim 4 , wherein said set of sequencing oligonucleotides comprise sequences of SEQ ID NOs. 470-482. 
     
     
         6 . The method of  claim 1 , wherein a 3′-end of said first region of said V-segment is at least 4 base pairs upstream from a 5′-end of said V-RSS. 
     
     
         7 . The method of  claim 1 , wherein said second region of said V-segment is at least 4 base pairs in length. 
     
     
         8 . The method of  claim 1 , wherein a 5′-end of said first region of said J-segment is at least 4 base pairs away from a 3′end of said J-RSS. 
     
     
         9 . The method of  claim 1 , wherein said second region of said J-segment is at least 4 base pairs in length. 
     
     
         10 . The method of  claim 1 , wherein said plurality of V-segment primers have a consistent annealing temperature. 
     
     
         11 . The method of  claim 1 , wherein said plurality of J-segment primers have a consistent annealing temperature. 
     
     
         12 . The method of  claim 1 , further comprising quantifying a total diversity of rearranged TCR or IG nucleic acid molecules in said sample. 
     
     
         13 . The method of  claim 1 , wherein said total diversity is greater than 10 6  rearranged TCR or IG nucleic acid molecules. 
     
     
         14 . The method of  claim 1 , wherein said plurality of J-segment primers comprises IGH J-segment primers and each of said plurality of J-segment primers is not specific to all IGH J-segment genes. 
     
     
         15 . The method of  claim 1 , wherein said rearranged nucleic acid molecules comprise genomic DNA. 
     
     
         16 . The method of  claim 1 , wherein said rearranged nucleic acid molecules comprise mRNA or cDNA. 
     
     
         17 . The method of  claim 1 , wherein said V-segment of said TCR or IG CDR3 region comprises a TCR Vδgene segment, a TCR Vγ gene segment, a TCR Vα gene segment, a TCR Vβ segment, an IGH V gene segment, an IGL V gene segment, or an IGK V gene segment. 
     
     
         18 . The method of  claim 1 , wherein said J-segment of said TCR or IG CDR3 region comprises a TCR Jδ gene segment, a TCR Jγ gene segment, a TCR Jα gene segment, a TCR Jβ gene segment, an IGH J gene segment, an IGL J gene segment, or an IGK J gene segment. 
     
     
         19 . The method of  claim 1 , wherein each of said at least 10 5  distinct amplicons is approximately 200 bases in length. 
     
     
         20 . A method of diagnosing immunodeficiency in a human subject, comprising:
 (i) measuring a diversity of at least 10 5  rearranged TCR or IG CDR3 sequences of the subject, wherein measuring said diversity of at least 10 5  rearranged TCR or IG CDR3 sequences comprises the steps of:
 (a) contacting a plurality of V-segment primers and a plurality of J-segment primers with a sample of genomic DNA comprising rearranged nucleic acid molecules of a TCR or IG CDR3 region from lymphocytes obtained from the subject, said plurality of V-segment primers and a plurality of J-segment primers each consisting of 15 to 50 nucleotides,
 wherein each of said V-segment primers comprises a first sequence and a second sequence, wherein said first sequence is complementary to a portion of a first region of at least one V-segment of said TCR or IG CDR3 region, said first region located immediately 5′ to a second region of said V-segment where untemplated deletions occur during TCR or IG gene rearrangement, wherein said second region of said V-segment is adjacent to and 5′ to a V-recombination signal sequence (V-RSS) of said V-segment, wherein said first sequence is located 3′ to said second sequence on said V-segment primer, 
 wherein each of said J-segment primers has a first sequence and a second sequence, wherein said first sequence is complementary to a portion of a first region of at least one J-segment of said TCR or IG CDR3 region, said first region located immediately 3′ to a second region of said J-segment where untemplated deletions occur during TCR or IG gene rearrangement, wherein said second region of said J-segment is adjacent to and 3′ to a J-recombination signal sequence (J-RSS) of said J-segment, wherein said first sequence is located 3′ to said second sequence on said J-segment primer, wherein each of said plurality of J-segment primers is not specific to all J-segment genes; 
 
 (b) amplifying said rearranged nucleic acid molecules from said sample using said V-segment and J-segment primers, thereby producing at least 10 5  distinct amplicons representing a diversity of TCR CDR3 genes in said sample; 
 (c) sequencing said at least 10 5  distinct amplicons; 
 (d) determining a total diversity of TCR or IG CDR3 sequences among said at least 10 5  distinct amplicons for said subject; and 
   (ii) comparing said total diversity of TCR or IG CDR3 sequences of said subject to a total diversity of TCR or IG CDR3 sequences obtained from said subject at an earlier time point or from a control subject.