Natural Abs specific for the carbohydrate Ag Galα1–3Galβ1–4GlcNAc-R (αGal) play an important role in providing protective host immunity to various pathogens; yet little is known about how production of these or other anti-carbohydrate natural Abs is regulated. In this study, we describe the generation of Ig knock-in mice carrying functionally rearranged H chain and L chain variable region genes isolated from a B cell hybridoma producing αGal-specific IgM Ab that make it possible to examine the development of B cells producing anti-carbohydrate natural Abs in the presence or absence of αGal as a self-Ag. Knock-in mice on a αGal-deficient background spontaneously developed αGal-specific IgM Abs of a sufficiently high titer to mediate rejection of αGal expressing cardiac transplants. In the spleen of these mice, B cells expressing αGal-specific IgM are located in the marginal zone. In knock-in mice that express αGal, B cells expressing the knocked in BCR undergo negative selection via receptor editing. Interestingly, production of low affinity αGal-specific Ab was observed in mice that express αGal that carry two copies of the knocked in H chain. We suggest that in these mice, receptor editing functioned to lower the affinity for self-Ag below a threshold that would result in overt pathology, while allowing development of low affinity anti-self Abs.
Natural Abs, those produced without intentional immunization, play a major role in providing protective host immunity (1, 2, 3). However, the study of natural Abs has been difficult because in many cases their Ag specificity is unknown. The specificity of many natural Abs remains undefined; however, a significant portion are specific for carbohydrates such as blood group Ags. In addition to anti-ABO blood group Abs, natural Abs specific for a carbohydrate Ag Galα1–3Galβ1–4GlcNAc-R (αGal), represent a significant population of natural Abs (4, 5, 6, 7, 8, 9, 10, 11). The αGal Ag is synthesized by the glucosyltransferase UDP galactose:β-d-galactosyl-1,4-N-acetyl-d-glucosaminide α(1, 2, 3) galactosyltransferase (Enzyme classification E.C. 22.214.171.124), or αGT. All placental mammals except humans, apes and Old World monkeys express a functional αGT enzyme and αGal epitopes on most tissues (12), and are consequently tolerant to αGal because it is recognized as part of self. In contrast, humans, apes and Old World primates carry a nonfunctional αGT gene whose function appears to have been lost ∼30 million years ago (5). Because these species do not recognize αGal structures as self, they consequently produce αGal-specific Abs.
αGal-specific natural Abs are estimated to comprise 1–8% of circulating Ig in humans, and ∼1% of EBV-transformed peripheral blood B cells make Abs that bind αGal (6, 13). In humans, αGal-specific Abs are encoded for by a restricted set of Ig Vh genes from the VH3 family (14). Production of Abs specific for αGal is believed to be elicited in response to normal bacterial flora that colonize the human gastrointestinal tract (11, 15). The presence of anti-αGal Ab in serum and secretory fluids, such as colostrum and saliva, suggests that these Abs have evolved to play a protective role in primate immunity. Viruses produced in αGT-expressing cells that display αGal-modified glycoproteins within their envelop, such as lymphocytic choriomeningitis virus, Newcastle disease virus and vesicular stomatitis virus, as well as C-type retroviruses, have all been shown to be susceptible to inactivation by serum anti-αGal Abs (16, 17). αGal-specific Abs are therefore believed to play an important role in preventing cross-species infection by pathogens. αGal-specific Abs have also been shown to play an important role in rejection of xenogeneic tissue when transplanted into non-human primates (18, 19, 20, 21, 22). Despite the importance of αGal-reactive Abs to host immunity little is known about how development of B cells producing αGal-reactive or other anti-carbohydrate Abs is regulated.
αGal-deficient mutant mice lacking a functional αGT gene (GT0/0 mice) generated by gene targeting in embryonic stem cells lack expression of αGal epitopes and consequently develop αGal-specific natural Abs, the majority of which are IgM (23, 24, 25). The serum titer of αGal-specific Abs in GT0/0 mice increases in an age-dependent fashion. αGal-specific Abs in GT0/0 mice share many features with human αGal-specific Abs, including usage of related V genes (26, 27, 28, 29, 30, 31). These mice therefore represent a small animal model in which αGal-reactive Abs can be studied. However, the frequency of B cells that produce αGal-specific Abs in these mice is low, making direct analysis of B cells producing αGal-specific Abs difficult. We and others have generated Ig transgenic mice to study regulation of B cells producing αGal-specific Abs (32, 33). However, the use of these mice to address several aspect of B cell development is limited because the Ig transgenes are randomly integrated. They are not transcriptionally regulated in an identical fashion to endogenous Ig genes, and in the case of Ig L chain transgenes, unlike endogenous self-reactive rearranged κ L chain genes, are not subject to deletion during receptor editing. Therefore, to develop a mouse model in which regulation of αGal-specific Ab production could be studied, we used gene targeting in embryonic stem cells to construct Ig gene knock-in mice. To this end, the rearranged VH and VL regions encoding specificity for αGal were isolated from M86, a hybridoma (IgM, κ L chain), derived from GT0/0 mice (34). M86VHVL knock-in mice were generated on either an Ag-deficient GT0/0 or Ag-sufficient GT+/+ or GT+/− background to address fundamental issues in regulation of anti-carbohydrate natural Abs. Using these mice, we examined the source of B cells producing αGal-specific Abs and mechanisms leading to negative selection of B cells producing anti-carbohydrate Abs. Our data indicate that restricting the ability of B cells producing self-reactive anti-carbohydrate Ags to undergo receptor editing significantly affects B cell development and allows for the production of low affinity anti-self Abs.
Materials and Methods
C57BL/6 GT0/0 mice and derivation of the colony used in these studies have been described in (35). C57BL/6 and BALB/c mice were used as controls and were obtained from The Jackson Laboratory. All mice were housed in viral Ab-free microisolator conditions. All animal experiments were conducted in accordance with Institutional guidelines.
Generation of M86VHVL knock-in mice
The functionally rearranged M86 VDJH and VJκ regions were isolated using standard genomic cloning techniques. To construct Ig H chain and L chain targeting vectors the rearranged M86VH and VL gene segments were then cloned using standard techniques into the plVhL2Neo (36) and pVKRNeo (37) H and L chains targeting vectors kindly provided by Dr. K. Rajewsky (Immune Disease Institute, Harvard Medical School, Boston, MA). Vector integrity was confirmed by restriction mapping. Gene targeting and generation of chimeric mice was performed essentially as described (38). Briefly, each targeting vector was linearized and transfected separately into ES-J1 embryonic stem cells. Transfected cells were selected in presence of G418 (300 mg/ml) and gancyclovir (2 mM). DNA prepared from double drug-resistant colonies was then screened for the presence of homologous recombination by Southern blotting. ES clones containing either the targeted M86VH or VL regions were injected into C57BL/6 blastocysts and then transferred into (BALB/c × C57BL/6) F1 foster mothers. Blastocyst injection, and embryo transfer, was performed by the Massachusetts General Hospital Microinjection Core Facility (Boston, MA). Chimeric mice were mated to C57BL/6 mice. DNA prepared from tail biopsy samples of resulting offspring was analyzed by Southern blotting to confirm germline transmission. Resulting knock-in mice were then crossed with cre mice (provided by the Massachusetts General Hospital Core Facility) to delete the Neor gene from targeted H chain and L chain loci. Resulting knock-in mice were then crossed to GT0/0 mice on the C57BL/6 background to generate M86VHGT+/0 and M86VLGT+/0 mice. These mice were then bred to generate αGal expressing M86VHVLGT+/0 and αGal-deficient M86VHVLGT0/0 mice.
Flow cytometry and Abs
Single cell suspensions were prepared from blood or lymphoid tissues and then stained and analyzed by flow cytometry as described (35). αGal epitopes were detected using the Gal-specific IB4 lectin (Sigma-Aldrich) from Bandeiraea simplicifolia (BS-I isolectin B4) (39). The following Abs used in this study were purchased from BD Pharmingen: RA3-6B2 (anti-CD45R/B220), 187.1 (anti-mouse Igκ), R26-46 (anti-mouse λ1, λ2, and λ3), 11-26c.2a (anti-IgD FITC), R6-60.2 (anti-IgM), 7G6 (anti-CD21), S7 (anti-CD43), and anti-Mac-1 (anti-CD11b). Goat anti-mouse IgM was purchased from Jackson ImmunoResearch Laboratories. RS3.1 (anti-Igh-6a (40)) and MB86 (anti-Igh-6b (41)) were provided by Dr. H. Wortis (Tufts University Sackler School of Biomedical Sciences, Boston MA). B cells capable of binding αGal were detected by staining with FITC- or biotin-conjugated Gal-BSA (V-Labs).
ELISAs were conducted as previously described (35). Briefly, ELISA plates (Corning) were coated overnight at 4°C with either αGal conjugated to BSA (Gal-BSA) or lactosamine conjugated to BSA (Lac-BSA; V-Labs) in carbonate buffer (pH 9.5), and then washed with PBS containing 0.05% Tween 20 (PBS-Tween 20). Lac-BSA shares all determinants with Gal-BSA, except for the terminal galactose structure, and serves as a specificity control. The wells were blocked with 1% BSA in PBS-Tween for 1 h at room temperature and then washed. Serum samples were serially diluted in PBS-Tween 20, added to the plates, and incubated for 1 h at 37°C. The plates were then washed extensively with PBS-Tween 20, and bound Abs were detected using HRP-conjugated goat anti-mouse IgM (1/4000; Jackson ImmunoResearch Laboratories). To determine the relative contribution of transgene-encoded vs endogenously encoded anti-αGal, bound Abs were detected with purified biotinylated RS3.1 or MB86, followed by HRP-conjugated streptavidin (1/800; Amersham Biosciences). The plates were incubated for 1 h at 37°C and then washed five times with PBS-Tween 20. A total of 0.01 mg/ml o-phenylenediamine dihydrochloride (Sigma-Aldrich) in substrate buffer was then added for 20 min at room temperature to develop the assays. The reaction was terminated by adding sulfuric acid to each well, and absorbency was read at 492 nm. Background values obtained from Lac-BSA-coated plates were subtracted from those obtained using Gal-BSA-coated plates. Assays were performed in duplicate. In some instances, serum from immunized mice was used. Mice were immunized i.p. with 107 irradiated (3000 rad) pig PBMC as described (42).
Anti-αGal B cell ELISPOT assay
Multiscreen-HA plates (Millipore, Bedford, MA) were coated with 10 μg/ml of either Gal-BSA or Lac-BSA in PBS at 4°C overnight. The plates were then washed three times with PBS, allowing the plates to soak for 5 min between each wash. The plates were blocked with IMDM containing 0.4% BSA and penicillin and streptomycin for 2 h at 37°C. The blocking medium was then removed and 10-fold serial dilutions (starting at 1 × 106 cells per well) of spleen cells prepared in blocking IMDM were added to the wells. The cells were incubated at 37°C in 5% CO2 for 24, 48, or 72 h in the presence or absence of LPS (0.5 μg/well). After culture, the plates were washed three times in PBS, followed by three additional washes in PBS-Tween 20. HRP-conjugated goat anti-mouse IgM was then added to each well and incubated for 2 h at 4°C. The plates were washed three more times with PBS-Tween 20, followed by PBS, at which point the assays were developed by adding filtered chromogen substrate (3-amino-9-ethyl-carbazole) in acetate buffer (pH 5.0). Plates were incubated in the presence of chromogen substrate at room temperature for 5 min and the reaction terminated by washing the plate with water. Spots were enumerated using an automated ELISPOT reader (ImmunoSpot; Cellular Technology). In all assays, the number of background spots obtained on Lac-BSA-coated plates was subtracted from the number obtained on corresponding Gal-BSA-coated plates. All samples were plated in duplicate.
B cell precursors from M86VHVL-GT0/0 mice were grown in vitro as previously described (43, 44). Briefly, bone marrow cells were depleted of erythrocytes and were cultured in BMB220 medium (IMDM supplemented with 10% FCS, 2-ME, l-glutamine, penicillin/streptomycin, and 50–100 U/ml recombinant murine IL-7; R&D Systems) at a concentration of 2 × 106 cells/ml for 5 days. Washed cells were then cultured for 2–42 h in the absence of IL-7 in wells with irradiated (2000 rad) confluent primary adherent bone marrow-derived stroma from Ag-negative GT0/0 or Ag-bearing wild-type C57BL/6 mice. After culturing the cells on the stroma, nonadherant pre-B cells were washed and frozen at −80°C until RNA was extracted. Stromal cultures were initiated by plating erythrocyte-depleted bone marrow cells to confluence in stromal medium (RPMI 1640, 5% FCS, 2-ME, l-glutamine, penicillin/streptomycin, sodium pyruvate, and nonessential amino acids) for 3 days, then washing away nonadherent cells and allowing the adherent stroma to grow for at least 2 wk before use. Stromal cell lines were tested for the expression of αGal by staining with αGal-specific IB4 lectin.
Detection of RAG2 expression
RNA was prepared from cells using an RNeasy mini kit (Qiagen). Complementary DNA was prepared from DNaseI-treated (Invitrogen Life Technologies) RNA with oligo(dT) primers with the Superscript first-strand synthesis kit (Invitrogen Life Technologies). Primer sequences used are as follows: RAG2 forward primer, 5′-CACATCCACAAGCAGGAAGTACAC-3′; RAG2 reverse primer, 5′-GGTTCAGGGACATCTCCTACTAAG-3′; β-actin forward primer, 5′-ACCCCAAGGCCAACCGCGAGAAGATGACC-3′; β-actin reverse primer, 5′-GGTGATGACCTGGCCGTCAGGCAGCTCGTA-3′. PCR were performed in a final volume of 50 μl using 3–5 μl of cDNA and 2 U Taq polymerase (Fischer Scientific) on a GeneAmp PCR 2400 thermal cycler (PerkinElmer). Except for the first cycle, which had a 2 min 94°C denaturation step, each cycle consisted of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C, with a 7 min 72°C extension cycle to end the PCR. For PCR analysis, 35 cycles was used. The PCR product was then fractionated by agarose gel electrophoresis, transferred to Hybond- N nylon membranes (Amersham Biosciences) and RAG2 amplicons detected by hybridization to 32P-labeled RAG2 probes as described (45).
RAG2 expression was also analyzed by quantitative real-time PCR. One microgram of total RNA was reverse transcribed using the SuperScript III First-Strand Synthesis SuperMix for quantitative real-time PCR kit (Invitrogen Life Technologies) and used to quantitate relative levels of RAG2 expression using the TaqMan predeveloped assay Mm00501300_m1 for RAG2, and 4352932E for GADPH (Applied Biosystems). The assays were performed in triplicates following standard protocols. The values shown are presented as the difference in cycle threshold (Ct) values normalized to GADPH for each sample (RQ).
Data analysis and reproducibility
Data throughout the study are representative of experiments containing at least three age- and sex-matched mice per group. All experiments have been shown to be reproducible by multiple individuals and have been conducted and confirmed on multiple occasions.
Construction of M86VHVL Ig knock-in mice
The low frequency of B cells that produce αGal-specific Abs in GT0/0 mice (25, 46, 47) limits the use of these mice to study the regulation of αGal-specific Ab production. To develop a mouse model in which B cells producing αGal-specific Abs could be directly tracked during their development, we generated Ig knock-in mice that express an αGal-specific BCR. To this end, the rearranged Ig H chain and L chain variable region segments were cloned from the hybridoma M86. M86 was derived from GT0 mice and produces an αGal-specific IgM Ab that uses a κ L chain (26, 32, 34). The H chain (rearranged to Jh4) and L chain (rearranged to Jκ1) variable region gene segments were then cloned into the plVhL2Neo (36) and pVKRNeo (37) H and L chains Ig targeting vectors. Each targeting vector was then electroporated separately into 129/Sv embryonic stem cells (ES-J1) as described (38). ES clones containing either the targeted M86VH or M86VL region were then injected separately into C57BL/6 blastocysts that were then transferred into (BALB/c × C57BL/6) F1 foster mothers to generate chimeric mice as described (38). Offspring carrying a knocked in M86VH or M86VL allele were then crossed with Cre recombinase transgenic mice to delete the neomycin resistance gene from targeted H chain and L chain loci. Resulting knock-in mice were then crossed to GT0/0 mice on the C57BL/6 background to generate M86VHGT+/0 and M86VLGT+/0 mice (Fig. 1 A) that were then bred to generate αGal-expressing M86VHVLGT+/0 mice and αGal-deficient M86VHVLGT0/0 mice.
Characterization of αGal-specific Ab production in M86VHVL knock-in mice
Analysis of natural serum Abs revealed that M86VHVLGT0/0 mice contain high titers of αGal-specific Ab in their serum (Fig. 1,B). The level of αGal-specific Abs in M86VHVLGT0/0 mice was significantly higher than that observed in GT0/0 controls at all ages examined (Fig. 1,B). Immunization of M86VHVLGT0/0 mice with αGal-expressing pig cells led to an increase in the titer of αGal-specific Abs (Fig. 1,B). In contrast, we were unable to detect production of αGal-specific Abs in the serum of M86VHVLGT+/0 or M86VHVLGT+/+ mice even after immunization (Fig. 1,B). In M86VHVL mice the endogenous H chain locus is IgH-6b (IgMb,μb), whereas the knocked in H chain locus is IgH-6a (IgMa,μa), which can be detected using the anti-allotypic mAbs MB86 (41) and RS3.1 (40), respectively. αGal-specific Abs in M86VHVLGT0/0 mice were encoded for by the knocked in IgMa allotype, rather than the endogenous IgMb allotype (Fig. 1,C). B cells spontaneously producing αGal-specific Abs were detected in the spleen, lymph node, and bone marrow but not the peritoneum of M86VHVLGT0/0 mice (Fig. 1,D). Immunization with pig cells did not result in the detection of αGal-producing B cells in the peritoneum of immunized M86VHVLGT0/0 mice (data not shown). The frequency of αGal-producing B cells in the spleen, bone marrow, and lymph nodes of M86VHVLGT0/0 mice was at least 10-fold higher than in GT0/0 controls (Fig. 1,D). We were unable to detect B cells producing αGal-specific Abs in lymphoid tissues from M86VHVLGT+/0 or M86VHVLGT+/+ mice (Fig. 1 D).
We next examined whether the titer of αGal-specific Abs in M86VHVLGT0/0 mice was sufficient to induce Ab-mediated transplant rejection. MHC-matched hearts from littermate M86VHVLGT+ or C57BL6 mice were heterotopically transplanted into the abdomen of M86VHVLGT0/0 mice. Hearts from M86VHVLGT+ and C57BL/6 mice were uniformly rejected within 24–72 h. In contrast, hearts from M86VHVLGT0/0 mice were accepted long-term (Fig. 1 E). These data suggest that αGal-specific Abs in M86VHVLGT0/0 mice are functional and of sufficient titer to induce Ab-mediated rejection.
Characterization of early lymphocyte development in M86VHVL knock-in mice
M86VHVLGT0/0 and M86VHVLGT0/+ mice were sacrificed and lymphoid tissues analyzed to examine lymphocyte development. αβ T cell development in the thymus was essentially normal when compared with GT0/0 or GT+ controls (data not shown). Similarly, T cell development in the spleen and lymph nodes was normal (data not shown). Characterization of B cell development in the bone marrow of M86VHVLGT0/0 or M86VHVLGT+/+ knock-in mice demonstrated that pro-B cells (B220+,CD43+) and immature (B220+,CD43−) B cells, as defined in (48, 49), were present in similar proportions to those observed in normal mice, although the frequency of each of these fractions was reduced when compared with normal controls (Fig. 2 A), as observed in other Ig knock-in mice (32, 33). The frequency of pre-B cells was similar in M86VHVLGT0/0 or M86VHVLGT+/+ knock-in mice (data not shown).
The majority of B220+ B cells in the bone marrow of M86VHVLGT+ and M86VHVLGT0/0 mice expressed the knocked in H chain allele (B220+, sIgMa+) (40.4 ± 9.3% and 46.6 ± 10.1%, respectively) rather than endogenously encoded (B220+, sIgMb+) allele reflecting exclusion by the rearranged M86VH gene (Fig. 2,B). Essentially all of the B220+, sIgMa+ B cells in the bone marrow of M86VHVLGT0/0 mice were able to bind αGal as determined by staining with fluorescently labeled BSA conjugated to αGal (αGal-BSA) (Fig. 2,C). B220+, sIgMb+ B cells able to bind αGal-BSA were not detected in M86VHVLGT+ or M86VHVLGT0/0 mice (Fig. 2,C). Staining of M86VHVLGT0/0 bone marrow cells for expression of B220 and CD43 revealed that B cells capable of binding αGal-BSA were CD43−, pre-B or newly formed B cells (Fig. 2,D). B cells able to bind αGal were not detected in the bone marrow of M86VHVLGT+ mice, suggesting that B cells expressing the knocked in transgene are tolerized during their development (Fig. 2 C).
Receptor editing prevents the development of B cells producing self-reactive Abs
Because the frequency of B cells expressing surface IgMa (sIgMa)3 in the bone marrow of M86VHVLGT+ is relatively high (Fig. 2,B) even though we were unable to detect B cells that bind αGal in the bone marrow of M86VHVLGT+ mice (Fig. 2,C) we reasoned that tolerance to αGal in M86VHVLGT+ mice was not deletional. We therefore examined whether tolerance was the result of receptor editing (45, 50, 51, 52). To this end, IL-7 driven pre-B cells from the bone marrow of M86VHVLGT0/0 mice were cultured on irradiated bone marrow stromal cells from GT+ and GT0/0 mice and the levels of RAG2 expression assayed by real-time PCR. Expression of RAG2 was up-regulated in M86VHVLGT0/0 pre-B cells cultured on stromal cells from GT+ cells when compared with pre-B cells from the same mice cultured on GT0/0 stromal cells (Fig. 3,A). Analysis of RAG2 expression by quantitative real-time PCR confirmed this finding (Fig. 3 B). These data suggest that pre-B cells producing self-reactive anti-carbohydrate Abs undergo tolerance through a mechanism that involves receptor editing.
Peripheral B cell development in M86VHVL knock-in mice
We next characterized B cell development in the periphery of M86VHVL mice. In the spleen of M86VHVLGT0/0 and M86VHVLGT+/0 mice the majority of B cells were B220+, sIgMa+ (51.8 ± 8.3% and 69.4 ± 7.6%, respectively) with the frequency of B220+, sIgMa+ B cells being slightly higher in M86VHVLGT+/0 mice (Fig. 4,A). B220+, sIgMb+ B cells were detected in both types of mice (Fig. 4,A). When compared with bone marrow (Fig. 2,B), the proportion of B220+, sIgMb+ B cells observed was higher in the spleen of both M86VHVLGT0/0 and M86VHVLGT+/0 mice (Fig. 4,A). B cells expressing both sIgMa and sIgMb were not detected in either M86VHVLGT0/0 or M86VHVLGT+/0 mice, suggesting allelic exclusion by the knocked in H chain (Fig. 4,B). As observed in the bone marrow, B cells capable of binding αGal were detected in the spleen of M86VHVLGT0/0 but not M86VHVLGT+/0 mice (Fig. 4,C). Although we were unable to detect B cells that spontaneously secrete αGal-specific Abs in the peritoneum of M86VHVLGT0/0 mice (Fig. 1,D), B220+, CD11b+, CD5− B cells in the peritoneum were capable of binding αGal in M86VHVLGT0/0 mice (Fig. 4,D). In both the spleen and peritoneum, B cells able to bind αGal were B220+, sIgMa+. However, B cells in the peritoneum did not produce αGal-specific Abs even after stimulation with LPS (Fig. 4,E). Consistent with data in Fig. 1 E, B cells secreting αGal-specific Abs were only observed in the spleen, bone marrow, and lymph nodes.
Immature B cells in the bone marrow first emigrate to the red pulp in the spleen where they can be detected as sIgMhigh, sIgDlow, CD21low, CD23− newly formed transitional or T1 cells. This emigration to the spleen is an Ag-regulated process (reviewed in Ref. 53). T1 cells then colonize the lymphoid follicles present in the spleen, and up-regulate IgD to become sIgMhigh, sIgDhigh, CD21int follicular B cell precursors. These cells give rise to sIgMlow, sIgDhigh, CD21int, CD23+ naive follicular B cells, and after additional Ag stimulation, sIgMhigh, sIgDhigh mature follicular B cells. There is also a subpopulation of B cells that express high levels of CD21 and CD1d, and are sIgMhigh, sIgDlow, CD21high, CD23−, CD1dhigh marginal zone (MZ) B cells, which exist outside the follicle (54, 55). Analysis of the spleen of M86VHVLGT0/0 mice by cell surface staining with Abs specific for IgM, IgD, and CD21 and with FITC-labeled αGal-BSA revealed that within the spleen B cells capable of binding αGal were sIgMhigh, IgDlow, CD21high MZ B cells (60 ± 22% MZ B cells are αGal specific, Fig. 5,A). We were unable to detect sIgMlow, IgDhigh, CD21int follicular B cells capable of binding αGal-BSA (Fig. 5,A). Analysis of tissue sections from the spleens of M86VHVLGT0/0 mice revealed that essentially all B cells capable of binding αGal-BSA reside in the MZ and were excluded from the follicle (Fig. 5 B). These data suggest that in M86VHVLGT0/0 mice αGal-binding B cells are committed to a MZ fate.
Increasing M86VHVL copy number alters the fate of αGal-specific B cells when self-Ag is encountered
While breeding M86VHVL knock-in mice, we also generated mice carrying two copies of the knocked in M86VH region and either one or two copies of knocked in M86VL region (M86VH2VL or M86VH2VL2 mice) on a GT0/0 or GT+ background. Additionally, we generated mice carrying two copies of the knocked in M86VL region (M86VHVL2 mice) on a GT0/0 or GT+ background. In blood, the frequency of B cells in M86VH2VL, M86VH2VL2, and M86VHVL2 mice on the GT0/0 background was similar to the frequency observed in M86VHVLGT0/0 mice (Fig. 6). However, the frequency of B220+ B cells in the periphery of M86VH2VLGT+ (18.7 ± 2.3%) mice was significantly reduced when compared with the frequency in M86VHVLGT+ mice (24.7 ± 2.9%; p < 0.05, Student’s t test). M86VHVL2GT+ and M86VH2VL2GT+ mice exhibited an even greater reduction in B220+ cells (10.1 ± 0.3% and 6.6 ± 0.6%, p < 0.05) when compared with the frequency in M86VHVLGT+ mice (Fig. 6). These data suggest that the presence of multiple knocked in H chain or L chain alleles affects B cell development. Interestingly, the reduction in B cell numbers is observed only in mice that express αGal as a self-Ag, suggesting that the effect observed may be related to alterations in negative selection.
Production of autoreactive αGal-specific Abs in M86VH2VL2GT+ knock-in mice
Our data indicate that B cells producing αGal-specific Abs were efficiently tolerized in M86VHVLGT+; however, we consistently observed the presence of αGal-specific serum IgM Abs in M86VH2VL2GT+ mice (Fig. 7,A). These Abs did not bind the control Ag Lac-BSA which shares all determinants with αGal-BSA except for the terminal galactose residue, indicating the binding observed was specific for αGal. Essentially all αGal-specific Abs in these mice were of the M86VH-encoded IgMa allotype (data not shown). The titer of anti-αGal Abs in these mice was consistently lower than that observed in littermate M86VH2VLGT0/0 and M86VH2VL2GT0/0 mice, but was similar to the titers observed in GT0/0 control mice (Fig. 7,A). Furthermore, the titers of these Abs could be boosted by pig cell immunization (Fig. 7,A). To confirm these results, spleen cells from pig cell immunized M86VH2VL2GT+/0, C57BL/6 GT+ controls and GT0/0 mice were analyzed for their ability to produce αGal-specific Ab in Ig ELISPOT assays using αGal-BSA- or Lac-BSA-coated plates. We detected B cells producing αGal-specific IgM Abs in the spleens of M86VH2VL2GT+ mice, at a frequency similar to that observed in immunized GT0/0 controls (Fig. 7 B). Similar results were observed in M86VH2VLGT+ mice (data not shown). These data suggest that B cells producing self-reactive anti-αGal Abs are able to develop in M86VH2VL2GT+ mice.
The development of B cells producing αGal-specific Ab in M86VH2VL2GT+ mice may reflect lowered affinity for Ag
The ability to detect anti-αGal Abs in M86VH2VL2GT+ mice was unexpected. To begin to characterize these Abs, we analyzed the capacity of αGal-specific Abs from these mice to bind BSA conjugated to different numbers of αGal epitopes. We were readily able to detect binding of αGal-specific IgM from these mice when using ELISA plates coated with αGal-BSA conjugates in which the molar ratio of αGal to BSA molecules was high (15:1) (Fig. 7,C). However, the ability to detect binding of αGal in these mice was significantly reduced when ELISA plates were coated with αGal-BSA conjugates in which the molar ratio of αGal to BSA was lower (10:1) (Fig. 7,C). Importantly, serum IgM from M86VHVLGT0/0 (Fig. 7 C) and M86VH2VL2GT0/0 (data not shown) was able to bind to both αGal-BSA preparations to a similar degree. These data suggested that αGal-specific Abs produced in M86VH2VL2GT+ mice exhibit different binding characteristics than those of littermate mice on the GT0/0 background. We suggest that B cells making self-reactive αGal-specific Abs in M86VH2VL2GT+ and M86VH2VLGT+ mice (data not shown) may develop because the Ab they produce exhibits a lowered affinity for αGal.
Anti-carbohydrate natural Abs are thought to play a key role in providing protective host immunity and have been well described as being important in mediating transplantation rejection across blood group disparities as well as discordant species. Yet little is known about B cells that produce anti-carbohydrate Abs, in part due to a lack of small animal models in which the development and regulation of B cells producing anti-carbohydrate Abs can be studied in a physiologically relevant manner. The development of GT0/0 mice has made it possible to begin to address some of these issues. However, the frequency of B cells producing αGal specific Abs in these mice is too low to allow for their direct analysis. We and others have previously attempted to overcome this difficulty by generating Ig transgenic mice that express transgenes encoding αGal-specific Abs (32, 33). However, these models are limited because the expressed transgenes were not subject to regulation by elements within the endogenous Ig loci, making it difficult to assess physiological relevance. To overcome these issues we used gene targeting in embryonic stem cells to construct Ig knock-in mice that carry rearranged H chain and L chain V regions that encode specificity for αGal that are under regulatory control of the endogenous Ig H chain and κ L chain loci. Breeding M86VHVL mice to GT0/0 mice offered us the opportunity to directly examine the development of B cells producing αGal-specific Abs in the presence or absence of αGal as a self-Ag.
M86VHVLGT0 mice show high serum levels of αGal-specific Abs produced by the knocked in allele. The titer of these Abs was sufficient to mediate rejection of H-2-matched heart transplants from GT+ donors. The titer of αGal-specific Abs in M86VHVLGT0 mice could be increased by immunization. αGal-specific Abs were not detected in the serum of M86VHVLGT+ mice, indicating that expression of αGal in these mice prevented development of αGal-specific Abs. However, we were still able to detect sIgMa+ B cells in the spleens of M86VHVLGT+ mice, suggesting that B cells expressing the knocked in M86VH region were not deleted during their development. Because the frequency of B cells expressing surface IgMa in the bone marrow of M86VHVLGT+ and M86VHVLGT0/0 knock-in mice was similar even though we were unable to detect B cells that bind αGal in the bone marrow of M86VHVLGT+ mice, we reasoned that tolerance to αGal in M86VHVLGT+ mice was most likely the result of receptor editing rather than deletion. Analysis of IL-7 driven pre-B cells from the bone marrow of M86VHVLGT0/0 mice revealed that exposure to αGal as a self-Ag led to an up-regulation of RAG2 expression in M86VHVLGT0/0 pre-B cells. These data are consistent with the idea that pre-B cells producing self-reactive anti-carbohydrate Abs undergo tolerance induction via receptor editing in the bone marrow. Interestingly, although receptor editing has been suggested to lead to an increase in the frequency of immature B cells expressing λ light chains (37, 45, 56), we did not detect an increase in the frequency of B cells expressing a λ L chain in the bone marrow or periphery of M86VHVLGT+ mice (data not shown). Therefore, in this model, receptor editing most likely occurs preferentially on the κ L chain. At least three distinct mechanisms that shape the naive B cell repertoire have been described, including clonal deletion (57, 58, 59, 60, 61); anergy (62, 63); and receptor editing (45, 50, 51, 64). The mechanism by which B cells producing Abs to self-carbohydrate Ags are tolerized has been studied in relatively little detail. Although receptor editing has been described as a major mechanism of B cell tolerance, to our knowledge this is the first description suggesting that receptor editing is the mechanism of tolerance for B cells producing anti-carbohydrate Abs.
Our analysis of B cell development in M86 knock-in mice shows that anti-αGal B cells undergo editing at the H chain locus even in the absence of α-Gal, resulting in the development of sIgMb+ B cells. When comparing the frequency of sIgMb+ B cells in bone marrow and spleen, it is apparent that the relative frequency of such cells is increased in spleen (see Figs. 2 and 4), suggesting a selection of B cells expressing sIgMb+ in the periphery. One possibility is that the knocked in H chain does not exclude well and that a selection process in GT+ mice ensures that only B cells expressing the endogenous H chain differentiate into peripheral mature lymphocytes. However, based on the analysis shown in Fig. 4 B in which we were unable to detect B cells producing both sIgMa and IgMb, we would suggest that apparently allelic exclusion in this system is not very leaky. Rather, there seems to be a selection process by which B cells expressing endogenous sIgMb are selected for in the periphery. It seems reasonable to suggest that this selection would be important to allow for an increase in the diversity of the Ig repertoire.
There is an unresolved issue regarding which B cell subsets produce anti-carbohydrate Abs. Using Ig transgenic mice, it has been suggested previously that B cells producing αGal-specific Abs are skewed to a MZ B cell phenotype (33). However, the use of Ig transgenic mice has made it difficult to determine whether this observation is of physiological relevance. Analysis of spontaneous αGal Ab production in lymphoid tissue of M86VHVLGT0/0 mice revealed the presence αGal-specific Ab production in the spleen, bone marrow, and lymph nodes, but not the peritoneum. In the spleen of M86VHVLGT0/0 mice, B cells capable of binding αGal were sIgMhigh, IgDlow, CD21high MZ B cells. We were unable to detect sIgMlow, IgDhigh, CD21int follicular B cells capable of binding αGal-BSA. These data together with analysis of αGal-binding B cells in tissue sections suggest that B cells producing αGal anti-carbohydrate Abs are committed to a MZ B cell fate.
Analysis of mice carrying two copies of the knocked in M86VH region and either one or two copies of the knocked in M86VL region revealed that copy number can significantly impact B cell development in mice expressing αGal as a self-Ag. Although the frequency of B cells in M86VH2VL2, M86VH2VL, and M86VHVL2 mice on the GT0/0 background was similar to the frequency in M86VHVLGT0 mice, the frequency of B cells in M86VH2VLGT+ mice was significantly reduced. M86VHVL2GT+ and M86VH2VL2GT+ mice exhibited an even greater reduction in B cells when compared with M86VHVL animals. Interestingly, the greatest affect on B cell numbers was observed in M86VHVL2GT+ and M86VH2VL2GT+ mice that carry two copies of the knocked in L chain allele. Because the effect of copy number on B cell number was seen only in mice expressing αGal as a self-Ag, we suggest that the effect observed is related to alterations in negative selection and that increasing the copy number of knocked in alleles may alter mechanisms of negative selection by restricting receptor editing, thereby skewing tolerance toward a deletional mechanism. This would support the idea that B cells primarily use editing to eliminate self-reactive B cells, and that negative selection via deletion is secondary to receptor editing.
Although B cells producing αGal-specific Abs were efficiently tolerized in M86VHVL mice on the GT+ background, we consistently observed the presence of αGal-specific serum IgM Abs in M86VH2VL2GT+ mice. This unanticipated finding prompted us to examine the binding characteristics of αGal specific Abs in these animals. Interestingly, although we were readily able to detect binding of αGal-specific IgM from these mice when using ELISA plates coated with αGal-BSA conjugates in which the molar ratio of αGal to BSA molecules was high, the ability to detect binding of αGal in these mice was significantly reduced when ELISA plates were coated with αGal-BSA conjugates in which the molar ratio of αGal to BSA was relatively low. These data suggest that αGal-specific Abs in M86VH2VL2GT+ mice have a lower affinity for αGal than that observed in GT0 mice. Such low affinity Abs appear to require an increase in binding avidity to be detected, which can be achieved by using BSA molecules that are highly substituted with αGal epitopes. Why then would such Abs develop in mice that express αGal? Although this issue is under study, it is important to point out that production of αGal-specific Abs are produced only in mice containing two copies of the knocked in H chain allele and either one or two copies of the knocked in L chain allele. In M86 mice, the knocked in H chain allele is rearranged to Jh4, which prevents receptor editing but not V gene replacement. We suggest that in the context of multiple copies of the knocked in H chain, V gene replacement via homologous recombination may select for VH genes of similar sequences to the M86VH region, which can encode H chain variable regions that bind αGal with an affinity that is sufficiently low to allow for their development in GT+ mice. We are currently evaluating this hypothesis.
The development of M86 Ig knock-in mice provides us with a unique model to study the development of B cells producing anti-carbohydrate Abs. The initial description of these mice has provided a novel insight into mechanisms of tolerance for B cell producing anti-carbohydrate Abs. Importantly, unanticipated findings resulting from this work may aid our understanding of self-non-self recognition. Although M86VH2VL2GT+ mice producing αGal-specific Abs appear to be grossly healthy, the presence of αGal-specific Abs in these mice may provide us with a model to examine how autoimmunity can be precipitated and lead to pathology.
We thank Uri Galili, (University of Massachusetts) for providing the M86 hybridoma, members of the Dr. Iacomini laboratory for helpful discussions, and John Kearney (University of Alabama, Birmingham) for instruction in the staining of spleen sections.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by Grants R01AI044268-09 and R01 AI050602-06 from the National Institutes of Health (to J.I.).
Abbreviations used in this paper: sIgM, surface IgM; MZ, marginal zone.