Brief Definitive Report |
Address correspondence to Mark J. Shlomchik, Department of Laboratory Medicine and Section of Immunobiology, Yale University School of Medicine, 330 Cedar St. Rm CB465, New Haven, CT 06510. Phone: 203-688-2089; Fax: 203-688-2748; E-mail: mark.shlomchik{at}yale.edu; or Garnett Kelsoe, Department of Immunology, Duke University Medical Center, 117 Jones Bldg., DUMC 3010, Research Dr., Durham, NC 27710. Phone: 919-613-7815; Fax: 919-613-7878; E-mail: ghkelsoe{at}duke.edu
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Abstract |
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Key Words: mutation antibody affinity plasma cell variable region gene 1 immunoglobulin
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Introduction |
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To distinguish these models for the antigen-driven selection of B cells, we generated the following two lines of transgenic (Tg) mice expressing Ig H chain Tgs that encode extremely low affinity anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies when associated with the 1 L chain: 1.2 x 105 M-1 for H50Gµa and
3.0 x 104 M-1 for T1(V23)µa.
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Materials and Methods |
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Creation of H50Gma and T1(V23)ma Tg Mice.
The VH186.2/DFL16.1 rearrangement, containing a single mutation at codon 50, designated H50G and V23 analogue VH rearrangement from an unmutated VDJ rearrangement of the V23 gene taken from a C57BL/6 mouse 10 d after immunization with NP, were subcloned into a vector that contains the 11.6-kb Cµ fragment that includes the membrane and secretory exons (14). The H50Gµa lineage had two copies of the Tg, whereas two separate T1(V23)µa lineages with similar phenotypes were used with two to three copies or a single copy of the Tg. Tg mice were backcrossed with C.B-17 mice for five or more generations.
Flow Cytometry.
The following antibodies were used: phycoerythrin-conjugated anti-B220 (CD45RA-PE; BD PharMingen); fluorescein-conjugated antimouse IgMa (RS3.1-FITC); biotinylated AF6-7.8 (anti-IgMb); fluorescein-conjugated or biotinylated Ls136 (anti-1); and biotinylated goat antimouse
L chainspecific and biotinylated goat antimouse Ig (Southern Biotechnology Associates, Inc.). Red 670conjugated streptavidin (GIBCO BRL) was used to reveal biotin-coupled Ab staining.
Antigens and Immunizations.
Mice were immunized intraperitoneally with 50 µg of NP16-chicken -globulin (CG), (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)12-CG, or CG precipitated in alum. For secondary/memory responses and challenge of repopulated SCID mice, 20 µg of soluble NP16-CG in PBS was administered intravenously on the indicated days.
Immunohistology.
Immunohistology was performed as previously described (3, 15). GCs were enumerated as peanut agglutinin (PNA)+ areas within lymphoid follicles (± SD from three separate sections). Endogenous Igexpressing B cells in the GC of H50Gµa or T1(V23)µa mice were enumerated using both AF6-7.8-biotin and biotinylated goat antimouse IgG1, followed by streptavidin-alkaline phosphatase. To detect apoptotic cells, TUNEL assays were performed using in situ apoptosis detection kits (Oncor).
Microdissection of Cells, DNA Amplification, and Sequencing.
Microdissection of cells, DNA amplification, and sequencing was performed as previously described (14, 16). Cellular material (constituting 1020 cells) was microdissected from individual GCs (3, 15, 17). In H50Gµa mice, GCs sampled were also IgMa+ and devoid of endogenous Igexpressing (IgMb+ or IgG1+) cells. DNA was amplified by Pfu polymerase (Stratagene) using nested primers specific for V1 and J
1 elements (14).
Quantitation of Serum Ab.
Ab titers were determined by endpoint dilution in ELISA using plates coated with NP21-BSA, NIP19-BSA, or CG (7). The endpoint was the last serial dilution that demonstrated a signal greater than twofold above the background. NP-specific ELISAs using differentially haptenated substrates were used to quantitate high and low affinity Ab (3).
Reduction of Serum Ab by 2-Mercaptoethanol (2-ME).
Serum was assessed for NP reactivity by ELISA after treatment with 2-ME (3). For calculations of the percent of the IgMa titer resistant to 2-ME treatment, mock and 2-ME ELISA results were converted to relative Ab concentrations by comparison to a standard purified H50Gµa/1 transfectoma Ab.
Adoptive Transfers of Splenocytes.
5 x 106 splenocytes recovered from H50Gµa and C.B-17 mice 30 d after immunization with 50 µg of NP12-CG precipitated in alum (18) were injected intravenously into recipient SCID mice. Recipients were challenged with 20 µg of NP12-CG in PBS or PBS alone 24 h later. 9 d after immunization the recipient sera were collected.
Online Supplemental Material.
The supplementary material shows original V sequence data as well as summary tables that analyze the distribution of mutations in the sequences. Fig. S1 shows all codons that contained mutations in V
1 sequences derived from either C.B-17 or H50Gµa mice, as described in the Materials and Methods and Results sections. Table S1 is a comparison of the two datasets, showing the frequency of overall mutations as well as replacement and silent mutations. Table S2 shows the ratios of replacement and silent ratios in each subregion of V
. Online supplemental material available at http://www.jem.org/cgi/content/full/jem.20011550/CD1.
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Results |
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Humoral Immune Responses in H50Gµa Tg Mice.
Naive H50Gµa Tg mice had low titers (1:60) of NP-specific serum Ab that sharply increased after immunization with NP-CG (Fig. 1
A). At day 12, the titers of NP-binding Tg (IgMa) Ab increased 1,000-fold in H50Gµa mice. Only in 3 out of 31 H50Gµa mice were substantial levels of IgG1 anti-NP detected (Fig. 1 A), indicating that typically most anti-NP was Tg derived. Levels for anti-NP
1 and total Ig at day four were similar for Tg and control animals. However, C.B-17 mice later achieved 510-fold higher titers. To confirm that H50Gµa mice expressed only low affinity Tg-encoded antibodies in response to immunization, sera of H50Gµa and C.B-17 mice were mildly reduced with 2-ME (3). The binding of very low affinity IgM Ab is avidity dependent and lost upon 2-ME reduction (5), whereas higher affinity IgM or IgG Abs are unaffected. The reduction of Tg sera led to a near complete loss (
97%) of NP reactivity (Fig. 1 B). This loss was comparable to that observed for purified H50Gµa/
1 transfectoma Ab (3). In contrast, C.B-17 serum titers were unaffected.
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T1(V23)µa mice had 810-fold increases in hapten-specific IgMa Ab between 4 and 16 d after immunization with either immunogen, with some mice showing 2030-fold increases in Ab titer (Fig. 1, C and D; ,
). IgMa responses by T1(V23)µa mice to NP-CG or NIP-CG were lower (510-fold) than those observed in H50Gµa mice for the same immunogen (Fig. 1, C and D;
,
vs.
). IgG1 responses, presumably encoded by the endogenous Igh loci, were higher in T1(V23)µa mice compared with H50Gµa mice. The higher levels of endogenously derived anti-NP antibodies in T1(V23)µa mice may reflect the extraordinarily poor affinity of these Tg B cells, which confer little advantage in competition with rare B cells that express endogenous VH genes. B cells expressing endogenously encoded VH genes comprise 13% of the initial repertoire, of which only a small fraction of B cells would have NP/NIP specificity. Thus, the emergence of these B cells in response to NP/NIP reflects the competition between rare, presumably higher affinity B cells and the much more common low affinity Tg B cells.
At the height of the response, H50Gµa mice challenged with NIP had a 100-fold increase in hapten-binding IgMa Ab titers compared with controls (Fig. 1 D), which is 210-fold lower than those after challenge with NP-CG (Fig. 1, C and D). This observation establishes a relationship between B cell antigen receptor (BCR) affinity/avidity and the magnitude of the Ab response in a single Tg mouse line.
Low Affinity Tg B Cells Can Initiate Primary GCs.
PNA+ GCs in H50Gµa mice 12 d after immunization (Fig. 2 A) were observed in numbers (7080 per section) equivalent to normal mice (6, 15). 65 ± 5.8% of the GCs in H50Gµa mice were stained with both anti-IgMa and anti-
1 Ab, indicating that they contained NP-specific B cells expressing the H50Gµa Tg (Fig. 2, A and C). The percentage of
1+ GCs was only slightly higher than that seen in non-Tg C.B-17 mice for the same day, which was 54 ± 3.4%. Many B cells in the splenic red pulp exhibited strong cytoplasmic staining with
1- and IgMa-specific Ab (unpublished data), the characteristic phenotype of plasmablasts and antibody-forming cell (AFC) (18).
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Late GCs of H50Gµa Mice Show Evidence of Increased Apoptosis.
12 d after immunization with NP-CG, B cells bearing BCRs encoded by the endogenous Ighb loci are infrequent in the GC of H50Gµa Tg mice (Fig. 3
A). However, at day 16 of the primary response, endogenous Igexpressing B cells (IgMb+ or IgG1+) begin to appear in the GCs, increasing to 510% of all PNA+ GC cells by day 20 (Fig. 3, B and C). Late GCs (1620 d after immunization) of H50Gµa mice had two- to threefold more apoptotic B cells compared with C.B-17 controls, as indicated by TUNEL labeling (Fig. 3, D and E). GCs (n = 30) sampled from six H50Gµa mice at 16 or 20 d after immunization had an average 32% (±10%) TUNEL+/PNA+/IgD- cells. This was in contrast to GCs (n = 20) from four C.B-17 mice at similar days, which contained only 12% (±7%) TUNEL+ GC cells.
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The distribution and type of somatic mutations observed in V1 genes present in Tg and control GC B cells is summarized in Tables SI and SII. 57 of 68 H50Gµa V
1 mutations encoded amino acid replacements. The 68 unique mutations were a striking contrast to the 11 unique mutations found in C.B-17 GCs. Mutations in the H50Gµa V
1/J
1 rearrangements had an overall replacement/silent (R/S) ratio three times greater than C.B-17 rearrangements. In addition, the R/S ratio of mutations in CDR1 and CDR2 of H50Gµa V
1 genes was 10- and 6-fold higher, respectively, than the predicted ratios based on codon usage. These data demonstrate enhanced mutation and selection in the endogenous V
1 gene rearrangements of H50Gµa GC B cells.
Low Affinity Primary and Memory Responses in H50Gµa Mice.
To determine the degree of affinity maturation in the primary and memory responses of H50Gµa mice, Tg and C.B-17 mice were immunized with NP-CG and 60 d later were rechallenged intravenously with soluble NP-CG. Fig. 4
shows the change in the NP-specific IgMa-Tg Ab titers. Upon secondary immunization, a prompt and significant increase in NP-specific IgMa Ab was observed. By day 9 of the secondary response, Tg-IgMa endpoint titers increased from 1:4,500 to 1:72,000 (Fig. 4, ). No significant Ab response (>1 out of 200) was observed in unprimed H50Gµa mice given the same soluble antigen (Fig. 4,
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To exclude that T cell priming alone was responsible for the memory response of H50Gµa mice, Tg and control mice were primed with the carrier protein CG, and 60 d later challenged intravenously with soluble NP-CG. Average NP-specific 1+ Ab titers from CG-primed H50Gµa mice were 15-fold lower than cohorts primed with NP-CG (Table I; 1:2,200 vs. 1:33,400). On challenge with soluble antigen, NP-CGprimed H50Gµa mice exhibited a 38% absolute increase in the serum Ab titer capable of binding NP5-BSA, whereas there was no appreciable increase in the NP5-BSAbinding titer in CG-primed H50Gµa mice. Thus, T cell priming alone cannot account for the extent of low affinity Ab elicited in the memory responses of H50Gµa mice.
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Discussion |
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Although competition is reduced, low numbers of B cells expressing endogenous VH gene segments provide detectable interclonal competition. Endogenously derived cells are rare in H50Gµa Tg mice, especially early in the response. However, many B cells expressing non-Tg BCR emerge in immunized T1(V23)µa mice, and to a lesser extent H50Gµa mice, as demonstrated by the presence of non-Tg Ab (Fig. 1) and histology (Fig. 3). Presumably, the extremely low affinity of T1(V23)µa B cells for the NP and NIP haptens allows them to be overwhelmed by rare B lymphocytes that escape allelic exclusion and respond to NP/NIP, as indicated by their frequent expression of VH186.2, a hallmark of high affinity NP Ab. Thus, higher affinity B cells expressing endogenous genes outcompete a numerous but very low affinity Tg population.
As V(D)J hypermutation creates L chain diversity in GCs (Figure S1), intraclonal competition also exists. R/S ratios in V CDR1 and V
CDR2 suggest strong selection for GC B cell mutants. Although V
mutations can improve BCR affinity in H50Gµa mice, the low affinity of secondary Ab in these mice (Fig. 4) suggests that this pathway for affinity maturation is limited. Nonetheless, the high frequency (1.4%) of point mutations in the V
1 rearrangements of H50Gµa GC B cells is almost twice that found in a similar mouse with a germline affinity, VH 186.2 Tg (14), and much higher than wild-type mice. The high frequency and intense selection of V
1 mutations in H50Gµa mice could reflect the difficulty in improving BCR affinity through L chain mutations alone. However, mutation rates may be intrinsically higher in low affinity B cells.
Our data do not rule out the role(s) for differential signaling governed by affinity/avidity in determining B cell fates (11, 12). Aside from memory development, other aspects of B cell differentiation like isotype switching could be governed by BCR signal strength.
The ability of very low affinity B cells to establish GCs and humoral memory has implications for understanding natural B cell responses. Low affinity BCRs can acquire higher affinities with a single point mutation (19, 21), and the many low affinity B cells that reach GCs are the potential precursors of effective memory B cells. This view is also consistent with direct estimates of clonal diversity in nascent GCs (6) and computer models, which suggest that GCs are seeded by 4050 precursors (22). Finally, the latent genetic potential of low affinity clones could underlie "clonal shifts" in secondary responses whereby dominant, primary clonotypes are replaced by mutated, dissimilar clones (2, 23).
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Acknowledgments |
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Submitted: September 10, 2001
Revised: March 8, 2002
Accepted: March 13, 2002
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Footnotes |
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G. Kelsoe and M.J. Shlomchik contributed equally to this work.
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References |
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