Table I.
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 | |
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.