Determining the replicative history of lymphocytes

Abstract

The invention relates to the field of immunology and immunodiagnostics. Provided is a method for determining the replicative history of a lymphocyte, preferably a B cell, the method comprising detecting a signal joint nucleotide sequence on an extrachromosomal circular excision product in the lymphocyte, wherein the excision product is deleted from a chromosome to give a chromosomal-coding joint nucleotide sequence, wherein the coding joint is retained in the chromosome, and detecting the coding joint nucleotide sequence in the lymphocyte. Also provided are primers, probes and a control cell for use in a method of the invention. A method provided herein is among others advantageously used to assess recovery of the precursor B-cell compartment, for example, in a patient following bone marrow transplantation

1 . A method for determining the replicative history of a lymphocyte, the method comprising:
 detecting a signal joint nucleotide sequence on an extrachromosomal circular excision product in said lymphocyte, wherein said excision product is deleted from a chromosome to give a chromosomal coding joint nucleotide sequence that is retained in the chromosome, and   detecting said coding joint nucleotide sequence in said lymphocyte.   
     
     
         2 . The method according to  claim 1 , further comprising:
 calculating the ratio between said chromosomal coding joint nucleotide sequence and said extrachromosomal signal joint nucleotide sequence in said lymphocyte.   
     
     
         3 . The method according to  claim 1 , wherein said lymphocyte is a B cell. 
     
     
         4 . The method according to  claim 3 , wherein said circular excision product is a kappa-deleting element rearrangement excision circle (KREC). 
     
     
         5 . The method according to  claim 3 , wherein the signal joint nucleotide sequence comprises the intron recombination signal sequence (intronRSS), located between the J and C gene segments of the IGK locus. 
     
     
         6 . The method according to  claim 1 , wherein detecting comprises polymerase chain reaction (PCR) analysis or real-time quantitative (RQ) PCR analysis using SYBR Green I Dye, hydrolysis probes or hybridization probes. 
     
     
         7 . A nucleic acid amplification primer selected from the group of oligonucleotides consisting of: 
       
         
           
                 
                 
               
                     
                   (Vk3-20 Up) 
                 
                     
                   5′-TCTCACCATCAGCAGACTGGAG-3′, 
                 
                     
                     
                 
                     
                   (Intron Up1) 
                 
                     
                   5′-CCGATTAATGCTGCCGTAGC-3′, 
                 
                     
                     
                 
                     
                   (Intron Up2) 
                 
                     
                   5′-CCCGATTAATGCTGCCGTAG-3′, 
                 
                     
                     
                 
                     
                   (Intron Up3) 
                 
                     
                   5′-GGCACCGCGAGCTGTAGAC-3′, 
                 
                     
                     
                 
                     
                   (Kde Down2) 
                 
                     
                   5′-CCTAGGGAGCAGGGAGGCTT-3′, 
                 
                     
                     
                 
                     
                   (Kde Down3) 
                 
                     
                   5′-CCTCAGAGGTCAGAGCAGGTTGTCCTA-3′, 
                 
                     
                     
                 
                     
                   (Kde Down4) 
                 
                     
                   5′-TACAGACAGGTCCTCAGAGGTCAG-3′, 
                 
                     
                     
                 
                     
                   (Vk3-20 Down) 
                 
                     
                   5′-CTATCTGTAAAGGAAGCAGCTGGTA-3′, 
                 
                     
                     
                 
                     
                   (Kde-germline Up) 
                 
                     
                   5′-CTTACCCTAGAGTTTCTGCACGG-3′, 
                 
                     
                     
                 
                     
                   (Int-Kde BREC F) 
                 
                     
                   5′-TCAGCGCCCATTACGTTTCT-3′, 
                 
                     
                     
                 
                     
                   (Int-Kde BREC R) 
                 
                     
                   5′-GTGAGGGACACGCAGCC-3, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       and 
       (see Table I), a variant of any thereof. 
     
     
         8 . A set of at least two pairs of nucleic acid amplification primers comprising:
 at least a first pair of primers for detecting a signal joint nucleotide sequence on an extrachromosomal circular excision product in a lymphocyte and   a second pair of primers for detecting a chromosomal coding joint nucleotide sequence in a lymphocyte or a B cell.   
     
     
         9 . The set of  claim 8 , wherein said excision product is a kappa-deleting element rearrangement excision circle (KREC) and/or wherein said coding joint is derived from a Kde rearrangement or an intronRSS-Kde rearrangement. 
     
     
         10 . The set of  claim 8 , wherein at least one of the primers is a nucleic acid amplification primer selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   (Vk3-20 Up) 
                 
                     
                   5′-TCTCACCATCAGCAGACTGGAG-3′, 
                 
                     
                     
                 
                     
                   (Intron Up1) 
                 
                     
                   5′-CCGATTAATGCTGCCGTAGC-3′, 
                 
                     
                     
                 
                     
                   (Intron Up2) 
                 
                     
                   5′-CCCGATTAATGCTGCCGTAG-3′, 
                 
                     
                     
                 
                     
                   (Intron Up3) 
                 
                     
                   5′-GGCACCGCGAGCTGTAGAC-3′, 
                 
                     
                     
                 
                     
                   (Kde Down2) 
                 
                     
                   5′-CCTAGGGAGCAGGGAGGCTT-3′, 
                 
                     
                     
                 
                     
                   (Kde Down3) 
                 
                     
                   5′-CCTCAGAGGTCAGAGCAGGTTGTCCTA-3′, 
                 
                     
                     
                 
                     
                   (Kde Down4) 
                 
                     
                   5′-TACAGACAGGTCCTCAGAGGTCAG-3′, 
                 
                     
                     
                 
                     
                   (Vk3-20 Down) 
                 
                     
                   5′-CTATCTGTAAAGGAAGCAGCTGGTA-3′, 
                 
                     
                     
                 
                     
                   (Kde-germline Up) 
                 
                     
                   5′-CTTACCCTAGAGTTTCTGCACGG-3′, 
                 
                     
                     
                 
                     
                   (Int-Kde BREC F) 
                 
                     
                   5′-TCAGCGCCCATTACGTTTCT-3′, 
                 
                     
                     
                 
                     
                   (Int-Kde BREC R) 
                 
                     
                   5′-GTGAGGGACACGCAGCC-3′, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       and
 (see Table 1), a variant of any thereof. 
 
     
     
         11 . A nucleic acid amplification assay comprising the set of primers of  claim 8 . 
     
     
         12 . An oligonucleotide probe homologous to an internal sequence of an amplified nucleic acid sequence produced in the nucleic acid amplification assay of  claim 11 . 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . A cell comprising a modification selected from the group consisting of:
 a recombinant nucleic acid sequence that is homologous to a signal joint nucleotide sequence derived from an extrachromosomal circular excision product in a lymphocyte or a B cell, preferably a signal joint nucleotide sequence derived from a kappa-deleting element rearrangement excision circle (KREC);   on one IGK allele with a nucleic acid sequence that is homologous to a coding joint nucleotide sequence, preferably wherein said coding joint sequence comprises the result of an intronRSS-Kde rearrangement;   a recombinant nucleic acid sequence that is homologous to a signal joint nucleotide sequence derived from an extrachromosomal circular excision product in a B cell and with a nucleic acid sequence that is homologous to a coding joint nucleotide sequence in a B cell.   
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . The cell of  claim 15 , wherein the cell is provided with a recombinant nucleic acid sequence that is homologous to a signal joint nucleotide sequence derived from an extrachromosomal circular excision product in a B cell and with a nucleic acid sequence that is homologous to a coding joint nucleotide sequence in a B cell, and wherein said cell is provided with a kappa-deleting element rearrangement excision circle (KREC). 
     
     
         19 . The cell of  claim 18 , wherein said signal joint and coding joint sequences are the result of an intronRSS-Kde rearrangement. 
     
     
         20 . A diagnostic kit comprising a diagnostic component selected from the group consisting of
 a nucleic acid amplification primer selected from the group of oligonucleotides consisting of:   
       
         
           
                 
                 
               
                     
                   (Vk3-20 Up) 
                 
                     
                   5′-TCTCACCATCAGCAGACTGGAG-3′, 
                 
                     
                     
                 
                     
                   (Intron Up1) 
                 
                     
                   5′-CCGATTAATGCTGCCGTAGC-3′, 
                 
                     
                     
                 
                     
                   (Intron Up2) 
                 
                     
                   5′-CCCGATTAATGCTGCCGTAG-3′, 
                 
                     
                     
                 
                     
                   (Intron Up3) 
                 
                     
                   5′-GGCACCGCGAGCTGTAGAC-3′, 
                 
                     
                     
                 
                     
                   (Kde Down2) 
                 
                     
                   5′-CCTAGGGAGCAGGGAGGCTT-3′, 
                 
                     
                     
                 
                     
                   (Kde Down3) 
                 
                     
                   5′-CCTCAGAGGTCAGAGCAGGTTGTCCTA-3′, 
                 
                     
                     
                 
                     
                   (Kde Down4) 
                 
                     
                   5′-TACAGACAGGTCCTCAGAGGTCAG-3′, 
                 
                     
                     
                 
                     
                   (Vk3-20 Down) 
                 
                     
                   5′-CTATCTGTAAAGGAAGCAGCTGGTA-3′, 
                 
                     
                     
                 
                     
                   (Kde-germline Up) 
                 
                     
                   5′-CTTACCCTAGAGTTTCTGCACGG-3′, 
                 
                     
                     
                 
                     
                   (Int-Kde BREC F) 
                 
                     
                   5′-TCAGCGCCCATTACGTTTCT-3′, 
                 
                     
                     
                 
                     
                   (Int-Kde BREC R) 
                 
                     
                   5′-GTGAGGGACACGCAGCC-3′, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       and 
       (see Table I), a variant of any thereof;
 an oligonucleotide probe, 
 the cell of  claim 15 , and any combination of any thereof. 
 
     
     
         21 . The method according to  claim 1  wherein the lymphocyte is a normal or a diseased lymphocyte selected from the group consisting of a B cell, bone marrow precursor-B cell, plasma cell, neonatal cord blood B cell, childhood peripheral blood B cell, adult peripheral blood B cell, tonsil B cell, virgin subset B cell, and memory subset B cell. 
     
     
         22 . The method according to  claim 1  further comprising assessing normal and aberrant lymphocyte development. 
     
     
         23 . The method according to  claim 1  further comprising:
 determining the antigen response by the lymphocyte. 
 
     
     
         24 . The oligonucleotide probe of  claim 12 , wherein said oligonucleotide probe is a TaqMan probe.