Phenotypic and genotypic characterization of BCL2/IGH junctions in isolated B cell subsets from both exposed and control HI
| B cell subsets | PCRa | RAG-mediated activity | AID-mediated activity | GC transitb | |||||||
| IgDc | CD10 | CD27d | Nb clones | BCL2-mbr (5′ to 3′)e | N nucleotides (5′ to 3′) | JH | CSR | SHMf | Rate (%) | ||
| Exposedg | |||||||||||
| 78 | + | + | − | SR (1) | CAGTGGTGCTTACGCTCCACCA | CCCCTGAGGCAGAT | J6 | n.a. | n.a. | – | – |
| n.d. | + | + | SR(3)/LR(1) | AAGAAAGCAGGAAe | GAACGTTACCTCAGGGGACTTCCCCGCGCCCCCCG | J6 | γ1 | 9/821 | 10.9 | yes | |
| + | + | − | SR (1) | CAGACCTCCCCCGGC | ∅︀ | J6 | n.a. | n.a. | – | ||
| + | − | + | LR (1) | CAGTGGTGCTTACGCTCC | TTCGGGTGTGATCGGTCGG | J6 | μ | 3/1,262 | 2.4 | yes | |
| + | − | + | LR (1) | AAGAAAGCAGGAAe | GAACGTTACCTCAGGGGACTTCCCCGCGCCCCCCG | J6 | γ1 | 8/821 | 9.8 | yes | |
| + | − | + | LR (1) | CAGTGGTG | TTGTAGTAGGAAA | J6 | μ | 1/1,262 | 0.8 | yes | |
| + | − | + | LR (1) | CAGTGGTG | TTGTAGTAGGAAA | J6 | γ1 | 1/474 | 2.1 | yes | |
| 138 | + | + | − | SR (1) | CAGTGGTGCTTACGCT | ATCGCGACGGGCTA | J3-6 | n.a. | n.a. | ||
| + | − | − | LR (1) | ACAAGTCCTTTA (*ICR) | GATCGGGGTCCCCCCGGGA | J6b | γ2 | 4/900 | 4.4 | yes | |
| +/−h | − | + | LR (3) | CAGACCTC | TTGTGAGGGGCCATGGTTATCCCAGGACGTCGTCGCCGA | J6b | γ1 | 44/4,176 | 10.5 | yes | |
| 326 | + | + | − | SR (1) | CAGTGGe | ATACTTGGGACAG | J4 | n.a. | n.a. | – | |
| + | + | − | SR (1) | CAGTGGTGCTTA | TCAGGTAGGGGG | J6 | n.a. | n.a. | – | ||
| − | − | + | LR (1) | CAGTGGe | ATACTTGGGACAG | J4 | γ1 | 1/687 | 1.5 | yes | |
| + | − | + | LR (2) | CCTCCTGCCCTCC | GGTAGACTAGACCC | J4 | μ | 6/2,524 | 2.4 | yes | |
| 431 | + | + | − | SR(7)/LR(2) | CAGTGGTGCTTAe | AATTTAGTCT | J6 | μ | 6/2,524 | 2.4 | yes |
| + | − | + | LR (2) | CAGTGGTGCTTAe | AATTTAGTCT | J6 | μ | 3/2,276 | 1.3 | yes | |
| 433 | + | + | − | SR (1) | CAGACCTCCCCCGGCGG | TTTTTCGAAGACGATATCAGGGT | J6 | n.a. | n.a. | – | |
| + | − | + | LR (1) | CAGACCTCCCCC | CCGAGCG | J4 | μ | 10/753 | 13.5 | yes | |
| 576 | +/− | − | + | LR (3) | CCTCCTGCCCTC | GTACGATTTTTGGAGTGGTCT | J6 | μ | 4/2,046 | 1.9 | yes |
| 605 | + | + | − | SR (1) | CCTCCTGCCCTCCTTCe | GGGTTTACTCCCCGGGTTTT | J5 | n.a. | n.a. | – | |
| +/− | + | + | SR(1)/LR(1) | TGCTTTACGT | TGCTC | J6 | μ | 1/1,262 | 0.8 | yes | |
| + | + | + | SR (1) | AAGAAAGCAGGAe | CCCCCACGGAG | J6 | n.a. | n.a. | yes | ||
| + | + | − | LR (1) | TCAGGGAACAGAA | CCCCTAGATAGTGGGAGCTCCAT | J6b | μ | 0/1,262 | 0 | – | |
| − | − | + | LR (1) | CAGTGGTGCTTA | AGGAAGAAGTCG | J4 | μ | 2/1,262 | 1.6 | yes | |
| + | − | + | LR (1) | AAGAAAGCAGGAe | CCCCCACGGAG | J6 | μ | n.d. | n.d. | yes | |
| + | − | + | LR (1) | CCTCCTGCCCTCCTTCCe | TCGAGAGAAG | J5 | μ | 5/1,262 | 4 | yes | |
| 611 | + | + | − | LR (3) | CAGTGGTGCe | CGAATGG | J6 | μ | 20/3,500 | 5.7 | yes |
| n.d. | + | + | SR (3) | CAGACCTCCCCCGGe | TTAGAGTTTGTGGGGCCCGTTGGGACG | J6 | n.a. | n.a. | yes | ||
| n.d. | + | + | SR (1) | CAGTGGTGCTTACG | TGCCGACCAAA | J3 | n.a. | n.a. | yes | ||
| + | + | − | SR (2) | AAGAAAGCAG | AGGGCCCCACCCTG | J6 | n.a. | n.a. | – | ||
| n.d. | + | + | SR (4) | AAGAAAGCA | AGGCAGGGCTTCGAGGG | J6 | n.a. | n.a. | yes | ||
| +/− | − | + | LR (4) | CAGTGGTGCe | CGAATGG | J6 | μ | 21/5,048 | 4.2 | yes | |
| +/− | − | + | LR (3) | CAGTGGTGCe | CGAATGG | J6 | γ1 | 2/95 | –i | yes | |
| − | − | + | LR (1) | CAGACCTCCCCCGGe | TTAGAGTTTGTGGGGCCCGTTGGGACG | J6 | γ3 | 23/1,109 | 20.7 | yes | |
| − | − | + | LR (1) | CAGACCTCCC | GAGATAGGGTTCAGGGGC | J6 | γ3 | 11/667 | 16.5 | yes | |
| + | − | + | LR (1) | CAGTGGTGCTTAC | CGCCCTAGCTGC | J6 | γ1 | 7/857 | 8.1 | yes | |
| Anonymous blood donorsf | |||||||||||
| 33 | n.d. | + | n.d. | LR (1) | CTTTACGTGGCCTe | TAGTACCAGCTGCCCCCTA | J6 | μ | n.d. | n.d. | – |
| n.d. | + | n.d. | SR (3) | CAGTGGTGCe | CAGCTGCCCCCCCCCGCCTGGCGGTGGGTTGTGCGGCCCC | J1 | n.a. | n.a. | – | ||
| n.d. | + | n.d. | LR (1) | CAGTGGTGC | CCTCGCCATATGTTCGTAACCCCGTCACCACC | J6 | γ3 | 2/521 | 3.8 | yes | |
| n.d. | − | + | SR (1) | CTTTACGTGGCCTe | TAGTACCAGCTGCCCCCTA | J6 | n.a. | n.a. | yes | ||
| n.d. | − | + | SR (7) | CAGTGGTGCe | CAGCTGCCCCCCCCCGCCTGGCGGTGGGTTGTGCGGCCCC | J1 | n.a. | n.a. | yes | ||
| n.d. | − | + | SR(13)/LR(3) | CAGTGG | GGGT | J6 | μ | 5/1,262 | 4 | yes | |
| 37 | n.d. | + | n.d. | LR (1) | CCTCCTGCCCTCCTT | AAACCA | J4 | μ | 13/1,282 | 11.1 | yes |
| n.d. | + | n.d. | SR(5)/LR (1) | CCTCCTGCCCTCCTTCCe | CCTTGGGGGGCT | J6 | μ | 3/1,448 | 2 | yes | |
| n.d. | + | n.d. | SR (1) | CAGTGGTGCTTe | GATTAGAGG | J5 | n.a. | n.a. | – | ||
| n.d. | + | n.d. | LR (1) | CAGACCTCCCCGe | AGAACCCCGCATAAA | J6 | μ | 1/1,526 | 0.7 | – | |
| n.d. | − | + | SR(4)/LR (1) | CCTCCTGCCCTCCTTCCe | CCTTGGGGGGCT | J6 | μ | 8/2,793 | 2.9 | yes | |
| n.d. | − | + | SR(1)/LR (1) | CAGTGGTGCTTe | GATTAGAGG | J5 | μ | 2/2,966 | 0.7 | yes | |
| n.d. | − | + | SR (4) | CAGACCTCCCCGe | AGAACCCCGCATAAA | J6 | n.a. | n.a. | yes | ||
| Controlsf | |||||||||||
| 118 | + | n.d. | + | LR (1) | CTCCTTCCGCGGG | A | J4 | μ | 5/2,620 | 1.9 | yes |
| + | n.d. | + | LR (1) | CAGTGGTGCTTA | GTGAATAGCGGGGCTC | J3 | μ | 3/1,262 | 2.4 | yes | |
| 92 | + | n.d. | + | LR (1) | GCTTTCTCATGG | TTCTTTTTCGGTAGGGTTGAGCACG | J5 | μ | 0/1,262 | 0 | yes |
| LR (1) | CAGTGGTGCTTAC | TGACCTCGGTCGA | J4 | μ | 2/1,262 | 1.6 | |||||
| 111 | + | n.d. | + | LR (2) | CAGTGGT | TTCTTTCGGGG-(DH3-9/DH3-10)-GTTAGG | J5 | μ | 17/2,524 | 6.8 | yes |
| 100 | + | n.d. | + | LR (1) | CAGTGGTG | GATATTTTCGGGGGCAACGACCGGGAGGATTGTCTCAAAA | J4 | μ | 1/1,262 | 0.8 | yes |
| 103 | + | n.d. | + | LR (1) | CAGACCTCCCCGGC | CCCCCCCCAGAA | J6 | μ | 2/1,262 | 1.5 | yes |
| LR (4) | CAGACCTCCCCGGC | CCCCCCCCAGAA | J6 | γ1 | 7/5,048 | 1.4 | yes | ||||
| 102 | + | n.d. | + | LR (4) | CCTCCTGCCCTCCTTCCG | TTCCTGTCCAAAAAG | J6 | μ | 5/5,048 | 0.9 | yes |
| + | n.d. | + | LR (1) | CAGACCTCCCCGGC | TGGGTTAGG | J5 | γ1 | 3/645 | 4.6 | yes | |
| − | n.d. | + | LR (1) | CCTCCTGCCCTCCTTCCGCGG | CCGTTGTGGGGACTCA | J6 | μ | 5/1,262 | 4 | yes | |
| 127 | + | n.d. | + | LR (2) | CAGACCTCCCCGG | TATCGCTAAACACGAT | J6 | γ2 | 20/1,932 | 10.3 | yes |
| − | n.d. | + | LR (3) | CAGACCTCCCCGG | TATCGCTAAACACGAT | J6 | γ2 | 110/7,329 | 15 | yes | |
| − | n.d. | + | LR (3) | CAGTGGTGCTTA | AGGTGGTC | J6 | γ1 | n.d. | n.d. | yes | |
| B cell subsets | PCRa | RAG-mediated activity | AID-mediated activity | GC transitb | |||||||
| IgDc | CD10 | CD27d | Nb clones | BCL2-mbr (5′ to 3′)e | N nucleotides (5′ to 3′) | JH | CSR | SHMf | Rate (%) | ||
| Exposedg | |||||||||||
| 78 | + | + | − | SR (1) | CAGTGGTGCTTACGCTCCACCA | CCCCTGAGGCAGAT | J6 | n.a. | n.a. | – | – |
| n.d. | + | + | SR(3)/LR(1) | AAGAAAGCAGGAAe | GAACGTTACCTCAGGGGACTTCCCCGCGCCCCCCG | J6 | γ1 | 9/821 | 10.9 | yes | |
| + | + | − | SR (1) | CAGACCTCCCCCGGC | ∅︀ | J6 | n.a. | n.a. | – | ||
| + | − | + | LR (1) | CAGTGGTGCTTACGCTCC | TTCGGGTGTGATCGGTCGG | J6 | μ | 3/1,262 | 2.4 | yes | |
| + | − | + | LR (1) | AAGAAAGCAGGAAe | GAACGTTACCTCAGGGGACTTCCCCGCGCCCCCCG | J6 | γ1 | 8/821 | 9.8 | yes | |
| + | − | + | LR (1) | CAGTGGTG | TTGTAGTAGGAAA | J6 | μ | 1/1,262 | 0.8 | yes | |
| + | − | + | LR (1) | CAGTGGTG | TTGTAGTAGGAAA | J6 | γ1 | 1/474 | 2.1 | yes | |
| 138 | + | + | − | SR (1) | CAGTGGTGCTTACGCT | ATCGCGACGGGCTA | J3-6 | n.a. | n.a. | ||
| + | − | − | LR (1) | ACAAGTCCTTTA (*ICR) | GATCGGGGTCCCCCCGGGA | J6b | γ2 | 4/900 | 4.4 | yes | |
| +/−h | − | + | LR (3) | CAGACCTC | TTGTGAGGGGCCATGGTTATCCCAGGACGTCGTCGCCGA | J6b | γ1 | 44/4,176 | 10.5 | yes | |
| 326 | + | + | − | SR (1) | CAGTGGe | ATACTTGGGACAG | J4 | n.a. | n.a. | – | |
| + | + | − | SR (1) | CAGTGGTGCTTA | TCAGGTAGGGGG | J6 | n.a. | n.a. | – | ||
| − | − | + | LR (1) | CAGTGGe | ATACTTGGGACAG | J4 | γ1 | 1/687 | 1.5 | yes | |
| + | − | + | LR (2) | CCTCCTGCCCTCC | GGTAGACTAGACCC | J4 | μ | 6/2,524 | 2.4 | yes | |
| 431 | + | + | − | SR(7)/LR(2) | CAGTGGTGCTTAe | AATTTAGTCT | J6 | μ | 6/2,524 | 2.4 | yes |
| + | − | + | LR (2) | CAGTGGTGCTTAe | AATTTAGTCT | J6 | μ | 3/2,276 | 1.3 | yes | |
| 433 | + | + | − | SR (1) | CAGACCTCCCCCGGCGG | TTTTTCGAAGACGATATCAGGGT | J6 | n.a. | n.a. | – | |
| + | − | + | LR (1) | CAGACCTCCCCC | CCGAGCG | J4 | μ | 10/753 | 13.5 | yes | |
| 576 | +/− | − | + | LR (3) | CCTCCTGCCCTC | GTACGATTTTTGGAGTGGTCT | J6 | μ | 4/2,046 | 1.9 | yes |
| 605 | + | + | − | SR (1) | CCTCCTGCCCTCCTTCe | GGGTTTACTCCCCGGGTTTT | J5 | n.a. | n.a. | – | |
| +/− | + | + | SR(1)/LR(1) | TGCTTTACGT | TGCTC | J6 | μ | 1/1,262 | 0.8 | yes | |
| + | + | + | SR (1) | AAGAAAGCAGGAe | CCCCCACGGAG | J6 | n.a. | n.a. | yes | ||
| + | + | − | LR (1) | TCAGGGAACAGAA | CCCCTAGATAGTGGGAGCTCCAT | J6b | μ | 0/1,262 | 0 | – | |
| − | − | + | LR (1) | CAGTGGTGCTTA | AGGAAGAAGTCG | J4 | μ | 2/1,262 | 1.6 | yes | |
| + | − | + | LR (1) | AAGAAAGCAGGAe | CCCCCACGGAG | J6 | μ | n.d. | n.d. | yes | |
| + | − | + | LR (1) | CCTCCTGCCCTCCTTCCe | TCGAGAGAAG | J5 | μ | 5/1,262 | 4 | yes | |
| 611 | + | + | − | LR (3) | CAGTGGTGCe | CGAATGG | J6 | μ | 20/3,500 | 5.7 | yes |
| n.d. | + | + | SR (3) | CAGACCTCCCCCGGe | TTAGAGTTTGTGGGGCCCGTTGGGACG | J6 | n.a. | n.a. | yes | ||
| n.d. | + | + | SR (1) | CAGTGGTGCTTACG | TGCCGACCAAA | J3 | n.a. | n.a. | yes | ||
| + | + | − | SR (2) | AAGAAAGCAG | AGGGCCCCACCCTG | J6 | n.a. | n.a. | – | ||
| n.d. | + | + | SR (4) | AAGAAAGCA | AGGCAGGGCTTCGAGGG | J6 | n.a. | n.a. | yes | ||
| +/− | − | + | LR (4) | CAGTGGTGCe | CGAATGG | J6 | μ | 21/5,048 | 4.2 | yes | |
| +/− | − | + | LR (3) | CAGTGGTGCe | CGAATGG | J6 | γ1 | 2/95 | –i | yes | |
| − | − | + | LR (1) | CAGACCTCCCCCGGe | TTAGAGTTTGTGGGGCCCGTTGGGACG | J6 | γ3 | 23/1,109 | 20.7 | yes | |
| − | − | + | LR (1) | CAGACCTCCC | GAGATAGGGTTCAGGGGC | J6 | γ3 | 11/667 | 16.5 | yes | |
| + | − | + | LR (1) | CAGTGGTGCTTAC | CGCCCTAGCTGC | J6 | γ1 | 7/857 | 8.1 | yes | |
| Anonymous blood donorsf | |||||||||||
| 33 | n.d. | + | n.d. | LR (1) | CTTTACGTGGCCTe | TAGTACCAGCTGCCCCCTA | J6 | μ | n.d. | n.d. | – |
| n.d. | + | n.d. | SR (3) | CAGTGGTGCe | CAGCTGCCCCCCCCCGCCTGGCGGTGGGTTGTGCGGCCCC | J1 | n.a. | n.a. | – | ||
| n.d. | + | n.d. | LR (1) | CAGTGGTGC | CCTCGCCATATGTTCGTAACCCCGTCACCACC | J6 | γ3 | 2/521 | 3.8 | yes | |
| n.d. | − | + | SR (1) | CTTTACGTGGCCTe | TAGTACCAGCTGCCCCCTA | J6 | n.a. | n.a. | yes | ||
| n.d. | − | + | SR (7) | CAGTGGTGCe | CAGCTGCCCCCCCCCGCCTGGCGGTGGGTTGTGCGGCCCC | J1 | n.a. | n.a. | yes | ||
| n.d. | − | + | SR(13)/LR(3) | CAGTGG | GGGT | J6 | μ | 5/1,262 | 4 | yes | |
| 37 | n.d. | + | n.d. | LR (1) | CCTCCTGCCCTCCTT | AAACCA | J4 | μ | 13/1,282 | 11.1 | yes |
| n.d. | + | n.d. | SR(5)/LR (1) | CCTCCTGCCCTCCTTCCe | CCTTGGGGGGCT | J6 | μ | 3/1,448 | 2 | yes | |
| n.d. | + | n.d. | SR (1) | CAGTGGTGCTTe | GATTAGAGG | J5 | n.a. | n.a. | – | ||
| n.d. | + | n.d. | LR (1) | CAGACCTCCCCGe | AGAACCCCGCATAAA | J6 | μ | 1/1,526 | 0.7 | – | |
| n.d. | − | + | SR(4)/LR (1) | CCTCCTGCCCTCCTTCCe | CCTTGGGGGGCT | J6 | μ | 8/2,793 | 2.9 | yes | |
| n.d. | − | + | SR(1)/LR (1) | CAGTGGTGCTTe | GATTAGAGG | J5 | μ | 2/2,966 | 0.7 | yes | |
| n.d. | − | + | SR (4) | CAGACCTCCCCGe | AGAACCCCGCATAAA | J6 | n.a. | n.a. | yes | ||
| Controlsf | |||||||||||
| 118 | + | n.d. | + | LR (1) | CTCCTTCCGCGGG | A | J4 | μ | 5/2,620 | 1.9 | yes |
| + | n.d. | + | LR (1) | CAGTGGTGCTTA | GTGAATAGCGGGGCTC | J3 | μ | 3/1,262 | 2.4 | yes | |
| 92 | + | n.d. | + | LR (1) | GCTTTCTCATGG | TTCTTTTTCGGTAGGGTTGAGCACG | J5 | μ | 0/1,262 | 0 | yes |
| LR (1) | CAGTGGTGCTTAC | TGACCTCGGTCGA | J4 | μ | 2/1,262 | 1.6 | |||||
| 111 | + | n.d. | + | LR (2) | CAGTGGT | TTCTTTCGGGG-(DH3-9/DH3-10)-GTTAGG | J5 | μ | 17/2,524 | 6.8 | yes |
| 100 | + | n.d. | + | LR (1) | CAGTGGTG | GATATTTTCGGGGGCAACGACCGGGAGGATTGTCTCAAAA | J4 | μ | 1/1,262 | 0.8 | yes |
| 103 | + | n.d. | + | LR (1) | CAGACCTCCCCGGC | CCCCCCCCAGAA | J6 | μ | 2/1,262 | 1.5 | yes |
| LR (4) | CAGACCTCCCCGGC | CCCCCCCCAGAA | J6 | γ1 | 7/5,048 | 1.4 | yes | ||||
| 102 | + | n.d. | + | LR (4) | CCTCCTGCCCTCCTTCCG | TTCCTGTCCAAAAAG | J6 | μ | 5/5,048 | 0.9 | yes |
| + | n.d. | + | LR (1) | CAGACCTCCCCGGC | TGGGTTAGG | J5 | γ1 | 3/645 | 4.6 | yes | |
| − | n.d. | + | LR (1) | CCTCCTGCCCTCCTTCCGCGG | CCGTTGTGGGGACTCA | J6 | μ | 5/1,262 | 4 | yes | |
| 127 | + | n.d. | + | LR (2) | CAGACCTCCCCGG | TATCGCTAAACACGAT | J6 | γ2 | 20/1,932 | 10.3 | yes |
| − | n.d. | + | LR (3) | CAGACCTCCCCGG | TATCGCTAAACACGAT | J6 | γ2 | 110/7,329 | 15 | yes | |
| − | n.d. | + | LR (3) | CAGTGGTGCTTA | AGGTGGTC | J6 | γ1 | n.d. | n.d. | yes | |
n.a., not applicable; n.d., not determined.
Type of PCR assay in which the fragment was obtained (number of positive amplicons of the given immunogeno-/phenotype; Fig. S6 B).
Based on CD27+ and/or CSR+ and/or SHM+.
See Fig. S3 for evaluation of the IgM status.
CD27 is a pertinent GC marker only when present, as the loss/absence of CD27 after GC transit has been shown to occur in recently identified memory B cell subsets (Ehrhardt et al., 2005; Wirths and Lanzavecchia, 2005; Tangye and Good, 2007) and is variably expressed on FL cells.
BCL2/JH junctions detected in both CD10+ and CD10− fractions.
SHM corresponds to the number of somatic mutations/total length of Sµ analyzed (bp). Background mutation levels were calculated to be 0.55‰ (Fig. S2 A).
Sorted according to CD10, CD27, and IgD for exposed samples (Fig. S6 A, top); according to CD10 and CD27 into CD10+ and CD10−CD27+ for anonymous blood donors (Fig. S6 A, bottom); and according to CD27 and IgD for controls (only the CD27+ is shown). Eight additional anonymous blood donors were screened and excluded from the analysis due to the absence of the t(14;18) detection.
+/− indicates PCR fragments detected in both positive and negative sorted B cell fractions.
The mutation rate was not estimated due to the partial deletion of the Sµ region.