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Brief Definitive Report |
nussen{at}mail.rockefeller.edu
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) repertoire expressed by naive and antigen-selected memory B cells in humans. We found that the Ig repertoire differs between naive and memory B cells and that this shift in the repertoire does not occur in the absence of somatic hypermutation in patients lacking activation-induced cytidine deaminase (AID). Our work suggests that somatic hypermutation makes a significant contribution to shaping the antigen-selected antibody repertoire in humans.
Key Words: immunoglobulin repertoire • activation-induced cytidine deaminase • somatic hypermutation • memory B cell • affinity maturation
Activation-induced cytidine deaminase (AID) is a germinal center B cell–restricted molecule that carries cytidine deaminase activity and is required for switch recombination and somatic hypermutation in mice and humans 161718. In the absence of AID, B cells are unable to undergo somatic hypermutation or produce secondary antibodies despite germinal center formation 1718. Here we report on the Ig
Reverse Transcription PCR, Cloning, and Sequencing.
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The affinity of antibodies for their cognate antigens increases during immune responses 1. In depth analysis of hybridoma antibodies specific for influenza hemagglutinin or for small chemical haptens such as 2-phenyl-5-oxaz-olone or 4-hydroxy-3-nitrophenyl acetyl (NP) revealed that somatic hypermutation is one of the mechanisms that produce this increased affinity 23456789. For example, the VH186–2 + Ig antibodies dominate the initial antibody response to NP, and mutation from TrpH33 to LeuH33 brings about a 10-fold increase in affinity of these VH186–2 + Ig antibodies 810111213. Increased affinity is also accompanied by a shift in the antibody repertoire, and secondary high-affinity responses to NP are dominated by Ig
antibodies and not Ig, suggesting that repertoire shifts contribute to affinity maturation 3101415. Little is known about this shift in the repertoire and how it relates to somatic hypermutation. antibody repertoire in humans deficient in AID. We find that AID is essential for the shift in repertoire between naive and antigen-selected memory B cells.
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Patient Samples and Cell Preparation.
AID-deficient patients and AID mutations have been described 18. Patients P1, P13, P14, P17, and P18 were 10, 11, 4, 14, and 2 yr old, respectively at the time of blood donation, and they did not suffer from chronic infections. They were treated with intravenous Ig supplementation. Control donors C1, C2, C3, C4, C5, C6, and C7 were healthy and 32, 11, 35, 2, 28, 41, and 33 yr old, respectively when blood samples were obtained. Blood mononuclear cells were isolated on Ficoll gradients. Control CD19+ B cells were fractionated into naive CD19+IgM+CD27– and memory CD19+IgM+CD27+ B cells by cell sorting on FACS VantageTM. Due to absence of secondary isotypes in AID-deficient patients, AID B cells are all IgM+ and were therefore sorted into naive CD19+CD27– and memory CD19+CD27+ B cells without IgM staining. Antibodies used for staining were FITC–anti-CD19, PE–anti-CD27 (Immunotech/Beckman Coulter), and biotin-conjugated anti-IgM mAb (PharMingen), which was visualized with Streptavidin Red 670 (GIBCO BRL).
Total RNA was extracted from 104–105 purified cells using TRIzol Reagent (GIBCO BRL) and reverse transcribed in a 10-µl reaction with Superscript II (GIBCO BRL). For reverse transcription (RT)-PCR reactions, 1 µl of cDNA was amplified for 30–35 cycles of 30 s at 94°C, 30 s at 58°C (VH1-Cµ) or at 55°C (V-C) and 30 s at 72°C with a final 10-min extension at 72°C using HotStarTaqTM DNA polymerase (QIAGEN) and the following primers: V1–8 family consensus sense, 5'-GGG(G/A)TC(T/C)CTGA(C/T/G)CG(A/C/G)TTCTCTGG(C/G)TCC-3'; V9 sense, 5'-ATCCCTGATCGCTTCTCAGTCTTG-3'; V10 sense, 5'-GATCTCAGAGAGATTATCTGCATCC-3'; and C antisense, 5'-CACAC(T/C)AGTGTGGCCTTGTTGGCTTG-3'. Sense FR1 VH1 and antisense Cµ primers were as described previously 1920. RT-PCR products were run on 2% agarose gels, and PCR products were gel purified (QiaquickTM; QIAGEN) and cloned into TA vectors (Invitrogen). Double-stranded DNA sequences were obtained using antisense Cµ or C primers and Dye Terminator Cycle Sequencing (PE Applied Biosystems). Sequences were analyzed using Ig BLAST®. When two or more identical sequences were found, they were counted as a single clone. Sequences were considered mutated when they displayed two or more nucleotide differences from their germline counterparts. Differences in gene distribution between naive and memory B cells were analyzed with chi-square tests (Cochran-Mantel-Haenszel test) adjusted by the Bonferroni method for multiple testing and they were considered significant when P values were equal to or less than 0.05.
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To determine whether there is a shift in repertoire between naive and antigen-selected B cell compartments in humans, we compared the unmutated germline Ig sequences to mutated Ig sequences obtained from CD19+ peripheral B cells from four control donors. In humans, Ig light chains are found in 30–40% of all antibodies, and among the 10 V gene families three (V1, V2 and V3) represent >80–90% of all V genes 2122. We found that the distribution of V1 and V2, two of the most frequently used human V families, differs between germline-encoded and mutated antibodies (Fig. 1 A; total of 239 individual sequences): V1 is decreased and V2 increased among mutated Igs, and this difference is independent of the age of the donors (Fig. 1a and Fig. b).
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repertoire between naive and memory B cells, we fractionated peripheral B cells using CD27 memory marker and isolated naive (CD19+IgM+CD27–) and memory (CD19+IgM+CD27+) B cells from control donors 2324. The difference in V distribution was also found when comparing naive and memory B cell compartments (Fig. 2; total of 262 sequences). Antibodies cloned from memory B cells were predominantly mutated and showed decreased V1 and increased V2 gene usage (Fig. 2a and Fig. b). We conclude that there is a shift in the Ig repertoire between the naive and antigen-selected memory B cell compartments in humans.
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repertoire between the naive and memory compartments is related to somatic hypermutation, we analyzed the Ig genes expressed in naive and memory B cells from patients lacking activation-induced deaminase (AID) 1718. AID has been shown to be essential for both hypermutation and switch recombination but does not appear to be necessary for normal B cell development in mice and humans 1718. Patients with AID deficiency showed no secondary antibodies and no somatic mutation; nevertheless, these individuals displayed enlarged tonsils with germinal centers and showed normal numbers of CD19+CD27+ B cells 18. The CD27+IgM+ B cells found in AID-deficient patients resembled authentic CD27+IgM+ memory B cells in that they showed normal selection against VH1–69, a VH gene that is frequently found in B lymphoid chronic lymphocytic leukemias producing autoreactive antibodies (Fig. 3) 192526. However, the antibodies expressed in antigen-selected memory B cells in five AID-deficient patients differed from the three controls in that they showed no mutations, and there was no shift in the V repertoire between naive B cells and antigen-selected memory B cells (compare Fig. 2 and Fig. 4; total of 330 sequences). In particular, there was no increase in V2 gene expression and no relative decrease in V1 (Fig. 4). In addition, VH5–51 gene usage was favored in the memory CD27+ B cells from AID-deficient patients but not in normal controls (Fig. 3).
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antibody repertoire between naive and memory B cells from normal donors. This shift in repertoire is associated with somatic hypermutation and is AID dependent. We conclude that AID and hypermutation make a significant contribution to shaping the antigen-selected memory B cell repertoire in humans.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health to M.C. Nussenzweig and from Institut National de la Santé et de la Recherche Médicale. M.C. Nussenzweig is an investigator in the Howard Hughes Medical Institute.
Submitted: 1 May 2001
Revised: 13 June 2001
Accepted: 19 June 2001
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References |
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