Self-reactivity profiles of antibodies from IgM⁺ memory B cells

The self-reactivity profiles of IgM⁺ memory and naive B cell antibodies from the same donors were compared in ELISAs using lysates of the human cell line HEp-2 (Fig. 2 A and reference 2 for GO and JB). On average, 19.7% of the antibodies from mature naive B cells from three donors JH (17.6%; Fig. 2 A), GO (22.8% and reference 2), and JB (16.7% and reference 2) were self-reactive (Fig. 2 A and Tables S4–S6, available at http://www.jem.org/cgi/content/full/jem.20052033/DC1). In contrast, only 2.1% of antibodies expressed by IgM⁺ memory B cells from the same individuals were self-reactive (0% for JH and JB, 4.8% for GO; Fig. 2 A and Tables S1–S3). This result was confirmed by indirect immunofluorescence assay (IFA) with HEp-2 cell–coated slides (unpublished data). Thus, the frequency of self-reactive antibodies expressed by IgM⁺ memory B cells was significantly lower than that measured in mature naive B cells (Fig. 2 A; P < 0.0001 for all donors combined; P = 0.028 for JH, P = 0.033 for JB, and P = 0.021 for GO). We conclude that B cells expressing self-reactive antibodies are efficiently excluded from the IgM⁺ memory B cell pool.

Open in a separate window

Figure 2.

Polyreactive and self-reactive antibodies are excluded from the IgM⁺ memory B cell compartment. (A) Antibodies from mature naive (donor JH) and IgM⁺ memory B cells (donors JH, GO, JB) were tested for self-reactivity by HEp-2 cell ELISA. HEp-2 ELISA results for antibodies from mature naive B cells of donors GO and JB have been published previously (reference 2). Dotted line shows ED38 positive control antibody (references 2, 43). Horizontal line shows cut-off OD₄₀₅ for positive reactivity as determined by comparison to low positive control serum (red line) and negative control serum (green line) from patients and healthy individuals, respectively. Pie charts summarize the frequency of HEp-2 self-reactive and nonself-reactive clones for all three donors. The number of tested antibodies is indicated in the pie chart center (reference 2). (B) Antibodies from mature naive (donor JH) and IgM⁺ memory B cells (donors JH, GO, JB) were tested for polyreactivity by ELISA with ssDNA, dsDNA, LPS, and insulin. Dotted lines represent the high positive control antibody ED38 (references 2, 43). Horizontal lines show cut-off OD₄₀₅ for positive reactivity as determined by comparison to the previously published control antibodies mGO53 (negative control; green line) and eiJB40 (low positive control; red line) (reference 2). The frequency of polyreactive antibodies in mature naive B cells from donors GO and JB has been published (reference 2). Pie charts show the frequency of polyreactive clones in mature naive and IgM⁺ memory B cell antibodies from all three donors with the number of tested antibodies indicated in the center (reference 2).

To measure the frequency of polyreactive antibodies in mature naive and IgM⁺ memory B cells, we performed ELISAs on ss/dsDNA, LPS, or insulin (Fig. 2 B). As previously reported, 5.4% (5/93) of the antibodies cloned from mature naive B cells from donors GO and JB showed low levels of reactivity with at least two of these antigens (2). Antibodies cloned from mature naive B cells of JH were not significantly different from the previously published result showing 8.3% polyreactivity (3/36, P = 0.685; Table S6). On average, 6.2% of mature naive B cell antibodies were polyreactive (8/129; Fig. 2 B and reference 2). In contrast, only 1% of antibodies cloned from IgM⁺ memory B cells were polyreactive (1/105, P = 0.044; Fig. 2 B). In summary, polyreactive and self-reactive antibodies comprising 5% and 20% of the mature naive repertoire, respectively, are rarely found in the IgM⁺ memory antibody pool.

Antibody selection

Our experiments indicate that the transition to the IgM⁺ memory B cell compartment is associated with selection against self-reactivity. To determine whether selection occurs before or after somatic hypermutation, we reverted the somatic mutations of 27 IgM⁺ memory B cell antibodies to their unmutated germline form and tested the recombinant antibodies for self-reactivity and polyreactivity (Fig. 3, Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20052033/DC1, and Table S7, available at. http://www.jem.org/cgi/content/full/jem.20052033/DC1). Although an average of 19.7% of all antibodies produced by naive B cells were self-reactive (Fig. 2 A and reference 2), none of the 27 reverted antibodies displayed self-reactivity or polyreactivity, including the two self-reactive and one polyreactive IgM⁺ memory B cell antibodies (Fig. 3 and Table S7). We conclude that naive B cells expressing self-reactive antibodies do not contribute to the IgM⁺ memory B cell compartment. Furthermore, the rare IgM⁺ memory B cells that produce antibodies with low levels of self-reactivity acquire this reactivity by somatic hypermutation.

Open in a separate window

Figure 3.

Self-reactivity and polyreactivity profiles of mutated IgM⁺ memory B cell antibodies in comparison with their unmutated counterparts. IgH and IgL chains from IgM⁺ memory B cell antibodies were reverted into their germline counterparts by PCR. (A) Recombinant antibodies were tested for self-reactivity by HEp-2 ELISA. Dotted line shows ED38 positive control antibody (references 2, 43). Horizontal line shows cut-off OD₄₀₅ for positive reactivity as determined by comparison to low positive control serum (red line) and negative control serum (green line) from patients and healthy individuals, respectively. (B) Recombinant antibodies were tested for polyreactivity by ELISA with ss/dsDNA, insulin and LPS. Dotted lines represent the high positive control antibody ED38 (references 2, 43). Horizontal lines show cut-off OD₄₀₅ for positive reactivity as determined by comparison to the previously published control antibodies mGO53 (negative control; green line) and eiJB40 (low positive control; red line) (reference 2).

Antibody reactivity with bacteria and bacterial polysaccharide antigens

To determine whether antibodies cloned from IgM⁺ memory B cells react with bacteria or T-I bacterial antigens, we performed ELISAs using Streptococcus pyogenes, a mixture of 10 Streptococcus pneumoniae serotypes (1, 3–6, 9V, 14, 18, 19, 23F); Enterococcus faecalis; Escherichia coli, a protein A mutant (SpA-) strain of Staphylococcus aureus (Fig. 4 A); or bacterial polysaccharide antigens in human vaccines PNEUMOVAX 23 (Streptococcus pneumoniae), PedvaxHIB (Haemophilus influenzae), and Menomune (Neisseria meningitidis) (Fig. 4 B).

Open in a separate window

Figure 4.

Whole bacteria and bacterial polysaccharide ELISAs. (A) Non-polyreactive antibodies from mature naive (n = 79) and IgM⁺ memory B cells (n = 101) from three healthy donors were tested for reactivity with whole cell bacteria (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, Esherichia coli) or (B) bacterial polysaccharide vaccines (PNEUMOVAX 23, PedvaxHIB, Menomune) by ELISA. Dotted line shows the positive control antibody ED38 (references 2, 43). Horizontal line shows cut-off OD₄₀₅ for positive reactivity as determined by comparison to the negative control antibody mGO53 (green line) and the low positive control antibody eiJB40 (red line) (reference 2). (C) Pie charts summarize the frequency of polyreactive antibodies that recognize ssDNA, dsDNA, LPS, insulin, bacteria and bacterial polysaccharides (black), the frequency of antibodies polyreactive with bacteria and bacterial polysaccharides (gray), and the frequency of non-polyreactive antibodies (white) in mature naive and IgM⁺ memory B cells in all three donors. The number of tested antibodies is indicated in the pie chart centers.

As expected, polyreactive antibodies from mature naive (4.3%, 4/91) and IgM⁺ memory B cells (1%, 1/105) showed high levels of reactivity with whole bacteria and bacterial antigens (Fig. 4 and Tables S1–S6). In addition, we found a new group of antibodies that showed broad reactivity with bacteria or bacterial polysaccharide antigens, but not with DNA, LPS, or insulin (Fig. 4 and Tables S1–S6). These antibodies comprised 8.8% (8/91) of the mature naive but only 2.9% (3/105) of the IgM⁺ memory B cell repertoire (Fig. 4 C). When combined, the broadly bacterially reactive antibodies comprised 13.1% of the mature naive and 3.9% of the IgM⁺ memory B cell compartment; overall, IgM⁺ memory B cells show a lower frequency of broadly bacterially reactive antibodies than mature naive B cells (P = 0.0195).

Among 91 antibodies from naive B cells and 105 antibodies from memory B cells tested, we found only two antibodies with high levels of specific reactivity against bacterial antigens. Both antibodies were cloned from IgM⁺ memory B cells from donor JB (2/27, 7.4%) who had previously been vaccinated and both recognized polysaccharides from the serotype 11A vaccine strain of Streptococcus pneumoniae contained in PNEUMOVAX 23 (Fig. 4 B, top right; Tables S1–S3, and Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20052033/DC1).

Ig gene RT-PCR amplification and expression vector cloning.

Antibodies were cloned as described previously (2, 42). In brief, single cell cDNA was synthesized in the original sort plates in a total volume of 14.5 μl (4 μl sort lysis solution + 10.5 μl RT-PCR reaction mix containing 150 ng random hexamer primer [pd{N}₆]; GE Healthcare), 0.5% vol/vol Igepal CA-630 (NP40; Sigma-Aldrich), 7 mM DTT, 4 U RNAsin (Promega), 7.5 U Primer RNase Inhibitor (Eppendorf), 1.2 mM each dNTP, and 50 U Superscript II RT (Invitrogen). cDNA was synthesized at 42°C for 55 min. Individual IgH (μ) and IgL chain (κ or λ) gene rearrangements were amplified in two successive rounds of PCR (50 cycles each) using primers as described previously (2, 42). Ig genes were amplified from 3.5 μl cDNA or first PCR product in 40 μl of total reaction volume with 0.2 μM 5′ and 3′ primers or primer mixes, 312.5 μM each dNTP and 1 U Hotstar Taq DNA polymerase (QIAGEN). All PCR products were purified (Qiaquick; QIAGEN), sequenced, and analyzed by Ig BLAST comparison with GenBank. Digested PCR products were purified and cloned into human IgG1, Igκ, or Igλ expression vectors (2, 42). Plasmids were sequenced to select clones with inserts identical to the original PCR product.

Antibody production and purification.

Recombinant antibodies were produced as described previously (2, 42). In brief, 293A human embryonic kidney cells were cultured in DMEM supplemented with 10% ultra-low IgG FCS (Invitrogen) and cotransfected with 12.5 μg of IgH and IgL chain encoding plasmid DNA by calcium phosphate precipitation. Cells were washed with serum-free DMEM for 8-12 h after transfection and thereafter cultured in DMEM supplemented with 1% Nutridoma SP (Roche). Supernatants were collected after 7 d of culture. For self-reactive HEp-2 ELISAs, antibodies were purified with protein G–Sepharose (GE Healthcare).

Bacteria strains and media.

Streptococcus pneumoniae strains DCC1355 (serotype 19), DCC1335 (9V), DCC1420 (23F), DCC1490 (14), DCC1714 (3), DCC1850 (6), AR314 (5), AR620 (1), GB2017 (18), GB2092 (4), and Enterococcus faecalis V12 were grown in Brain Heart Infusion broth (Sigma-Aldrich). Streptococcus pyogenes D471 was grown in Todd-Hewitt broth (Sigma-Aldrich). Staphylococcus aureus protein A-negative Foster strain 8325–4 spa::Ebr was grown in super broth (10 g/liter MOPS, 30 g/liter tryptone, 20 g/liter yeast extract). Escherichia coli was grown in Luria-Bertani medium. Cell counts from log-phase bacteria cultures were determined before treatment with 50 μg/ml mitomycin-C for 1 h at 37°C. Whole bacteria were suspended at a concentration of 10⁸/ml PBS for ELISA.

ELISA.

To measure reactivity of antibodies with individual antigens, microtiter plates (COSTAR 9018) were coated with 50 μl/well of 10 μg/ml of ssDNA, dsDNA, LPS (Sigma-Aldrich), or 5 μg/ml of recombinant human insulin (Sigma-Aldrich) or 10 μg/ml of human bacterial polysaccharide vaccines PNEUMOVAX 23 (Merck & Co.), PedvaxHIB (Merck & Co.), or Menomune (Aventis) or 10 μg/ml of modified protein A. To measure antibody reactivity with specific bacteria, microtiter plates were coated with 100 μl/well of 100 μg/ml poly-l-lysine, 25 μl/well of bacteria solution, and 25 μl/well of 0.2% gluteraldehyde were added at room temperature for 20 min. Plates were centrifuged at 1,500 g for 20 min and washed with PBS. To block aldehydes, 50 μl/well of 0.1 M l- lysine in PBS was added and wells were washed with PBS.

Antibody concentrations in supernatants and after purification were determined by anti–human IgG1 ELISA using human monoclonal IgG1 as standard (Sigma-Aldrich). For polyreactivity and bacterial ELISAs, supernatants were adjusted to a starting antibody concentration of 1 μg/ml and with three subsequent 1:4 dilutions in PBS. Samples were considered negative if the OD₄₀₅ did not exceed a threshold value at any of the four dilutions in at least two independent experiments as indicated in each graph. In all assays, threshold values were set using the negative control antibodies mGO53, low positive control eiJB40, and high polyreactive antibody ED38 (2, 43). HEp-2 cell self-reactivity ELISAs used QUANTA Lite ANA ELISA plates (INOVA Diagnostics) and purified antibodies at a concentration of 10 μg/ml with three subsequent 1:4 dilutions in PBS. Positive and negative controls for HEp-2 ELISAs included control sera from patients and healthy individuals (INOVA Diagnostics) as well as ED38 and were included in every experiment. Consistent with our previously published results, the negative and low positive serum was used to determine the cut-off OD₄₀₅ for reactivity in each experiment (2). Cut-off OD₄₀₅ levels vary among experiments depending on the absolute OD₄₀₅ values and antibody concentrations, but allow us to compare the data among individual experiments. Antibodies were considered reactive if they showed reactivity levels above the cut-off OD₄₀₅ in at least two independent experiments and if the results could be confirmed by IFA on HEp-2 cell–coated slides (unpublished data). All ELISAs were developed with horseradish peroxidase–labeled goat anti–human IgG Fc antibodies (Jackson ImmunoResearch Laboratories) and horseradish peroxidase substrates (Bio-Rad Laboratories). OD₄₀₅ was measured with a microplate reader (Molecular Devices).

Reversion of somatic hypermutations into germline sequences.

Antibodies for reversion were chosen randomly and are listed in Table S7. Mutated IgH and IgL chain genes were reverted to their germline sequence by two separate PCR reactions for the V gene and the (D)J gene followed by a third overlap PCR to fuse the two PCR products (44). Germline V genes were amplified from previously cloned Ig genes from unmutated naive B cell templates using FWR1 sense and FWR3 antisense primers. Germline (D)J sequences were generated with sense primers spanning the 3′ end of the V gene and the CDR3 region and antisense primers in the J gene. A second 20-cycle PCR reaction fused the two overlapping V and (D)J PCR fragments to generate unmutated germline antibody sequences (Fig. S2). All unmutated IgH and IgL chain PCR products were sequenced before and after cloning into the corresponding expression vectors to confirm the lack of mutations. For all clones' mutated and unmutated IgH and IgL chain CDR3 regions, Ig gene repertoire and reactivity profiles are summarized in Table S7.

Statistics.

p-values for Ig gene repertoire analysis, analysis of positive charges in IgH CDR3, and antibody reactive were calculated by 2 × 2 of 2 × 5 Fisher's Exact test or Chi-square test. p-values for IgH CDR3 length were calculated by two-tailed Student's t test.

We thank Dr. G.J. Silverman, Dr. C.S. Goodyear, Dr. V.A. Fischetti, Dr. J.M. Loeffler, Dr. D. Ho, and Dr. Y. Huang for reagents and advice, and all members of the Nussenzweig laboratory and Dr. E. Besmer for discussion and critical reading of the manuscript.

This work was supported by grants from the National Institutes of Health (to M.C. Nussenzweig and S. Yurasov), the Dana Foundation (to H. Wardemann), and the Naito Foundation (to M. Tsuiji). M.C. Nussenzweig is a Howard Hughes Medical Investigator and S. Yurasov is a New York Chapter Arthritis Foundation Young Scholar.

The authors have no conflicting financial interests.