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The Journal of Immunology, 1998, 161: 5720-5732.
Copyright © 1998 by The American Association of Immunologists

The Dual Phases of the Response to Neonatal Exposure to a VH Family-Restricted Staphylococcal B Cell Superantigen1

Gregg J. Silverman2, Jayakar V. Nayak3, Klaus Warnatz4, Fred F. Hajjar, Stephen Cary, Helen Tighe and Virginia E. Curtiss

The Sam and Rose Stein Institute for Research on Aging and the Theodore Gildred Cancer Center, Department of Medicine, University of California at San Diego, La Jolla, CA 92093


   Abstract
Top
 Abstract
Introduction
 Methods and Materials
 Results
 Discussion
 References
 
In vitro studies of several naturally occurring proteins have characterized VH family-specific B lymphocyte binding and stimulatory properties that appear analogous to those of T cell superantigens. To examine the in vivo consequences of exposure to a putative B cell superantigen, we treated neonatal BALB/c mice with a form of staphylococcal protein A (MS) devoid of Fc{gamma} binding activity, which retains the clan VHIII Fab binding specificity. In naive adults, about 5% of peripheral B cells and >13% of splenic IgM-secreting cells display MS binding activity, in association with high IgM and low IgG circulating anti-MS Ab titers. Neonatal exposure to MS elicited two distinct temporal phases of immune responsiveness. The early phase, representing the first approximately 5 wk of life, was associated with MS-specific B cell and T cell tolerance. Microfluorometric assays revealed that exposure caused a dramatic MS-specific B cell clonal loss in bone marrow and spleen, but levels normalized by about 3 wk of life. The late phase (>6 wk of age) was associated with spontaneous priming for MS-specific T cell responses and production of MS-specific IgG1 Abs despite long term persistently depressed in vivo and in vitro MS-specific IgM responses. In vivo challenge during the late phase induced high frequencies of MS-specific IgG-secreting cells, indicating recruitment of highly focused Ab responses that were predominantly encoded by rearrangements of the S107 family, a member of the VHIII clan. These studies document the immunodominance of the VH-restricted Fab binding site on staphylococcal protein A and demonstrate the diverse effects of a B cell superantigen on the emerging peripheral B cell compartment.


   Introduction
Top
 Abstract
 Introduction
Methods and Materials
 Results
 Discussion
 References
 
The fate of a maturing B cell is largely determined by the signals it receives through its Ag receptor (BCR)5 complex. The nature of the encountered Ag, the maturational stage of the B cell, the anatomic and lymphoid context of the encounter, the availability of costimulatory signals, and the relative fitness of each clone all contribute to whether BCR-mediated interactions result in positive (survival and proliferation) or negative (death or anergy) clonal selection (reviewed in 1 . These principles have been confirmed in Ig transgenic mouse models, but due to the relative infrequency of clones of a defined conventional antigenic specificity, the full implications for naturally developing polyclonal B cell populations have been difficult to assess.

In contrast to conventional protein Ags, which are generally recognized by <0.1% of lymphocytes in a naive mature B cell pool, recent reports have described a group of natural proteins that can be bound by the BCR of up to 50% of human peripheral B cells (2). They have been termed B cell superantigens because they display properties comparable to those of bacterial and viral proteins with special stimulatory properties for T cells. We have hypothesized that these exogenous (and perhaps endogenous (3, 4, 5)) proteins may be capable of influencing large scale shifts in B cell clonal representation (reviewed in 6 . B cell superantigens may contribute to the abnormal B cell and Ab patterns detected in HIV infection (7, 8, 9, 10) as well as to the clonal selection of autoreactive B cells in certain autoimmune diseases (reviewed in 11 . In addition, analysis of Ab gene expression in single human B cells has provided evidence of positive selection of VH3 family-expressing clones in healthy adults (12). The in vivo clonal response to experimental challenge with a B cell superantigen has not been previously investigated.

Of putative B cell superantigens, the bacterial membrane protein, staphylococcal protein A (SpA), is the best characterized. This 42-kDa membrane protein, composed of five homologous 58–61 amino acid Ig binding domains and a transmembrane domain (13, 14), is produced by virtually every clinical isolate of Staphylococcus aureus. Experimental systems indicate that SpA is a virulence factor (15, 16), but the mechanism of its effect has not been defined. The immunologic properties of SpA are linked to two distinct types of Ig binding sites (i.e., an Fc{gamma} binding site and a VH-restricted Fab binding site) that are separate and functionally noncompetitive (17, 18).

In the human system, Fab-mediated SpA binding is restricted to the products of the human VH3 gene family, and at least 16 of the approximately 22 potentially functional VH3 gene segments can encode for an SpA binding site (19, 20, 21, 22). Moreover, 85% of adult VH3 IgM and about 65% of VH3 IgG have Fab-mediated SpA binding activity (19, 20, 23) (G. J. Silverman, unpublished observations), while every unmutated VH3 Ab from fetal liver B cells evaluated was found to bind SpA (22). VH3 IgM Abs can bind SpA with a Kd of <10-8 M, which is comparable to conventional Ab responses (24). Chain recombination studies demonstrate that L chains are relatively passive in binding (20). In vitro, SpA selectively binds to VH3-expressing B cells (2), and in the correct cytokine milieu selectively induces the differentiation of VH3 Ig-producing cells (25, 26). Fab-mediated interactions between human VH3 Ig and SpA were recently shown to induce an in vivo immune complex-mediated inflammatory response in a rabbit model (27).

SpA binding capacity correlates with conserved clan VHIII-specific sequences that include the VH framework 1 and 3 subdomains, and these Abs display a hierarchy of affinities that appears to be linked to specific VH gene segment usage (20, 24). Almost every mammalian species uses clan VHIII-related genes, and their Ab responses include this germline-linked SpA binding activity (28). In the mouse, Abs from the clan VHIII, J606, S107 (29), and large 7183 family (G. J. Silverman, manuscript in preparation) commonly display Fab-mediated binding capacity, but other related murine families may also be associated with this activity.

The current studies were initiated to investigate the outcome of immune exposure to SpA during the neonatal period. For more than 35 years, neonatal injection has been known to cause Ag-specific neonatal unresponsiveness or tolerance in which re-exposure fails to elicit a detectable Ab response (30, 31, 32). It is now believed that T cell tolerance is not an intrinsic property of the newborn immune system but is the nature of the APCs prevalent in the neonate (33, 34, 35). Immune tolerance can be the result of Th cell immune deviation (36, 37, 38, 39, 40, 41) or the induction of suppressor T cell clones (42, 43). In the B cell compartment, neonatal exposure to BCR-selective ligands can also result in profound changes in the relative representation of Ag-specific precursors (44, 45, 46, 47). The current studies examine the immediate and long term immunologic consequences of neonatal exposure of polyclonal BALB/c populations to a B cell superantigen. The results indicate that exposure elicits two distinct temporal phases in which superantigen-specific tolerance dominates the early phase while the late phase is associated with T cell priming and mixed B cell responsiveness. These studies begin to elucidate the predominant immunologic features of B cell superantigen-induced responses.


   Methods and Materials
Top
 Abstract
 Introduction
 Methods and Materials
Results
 Discussion
 References
 
Immunogens

As previously described (2), endotoxin-free recombinant SpA (Repligen, Cambridge MA) was chemically modified to create a form of SpA, termed modified SpA (MS), that retains VHIII Fab binding specificity but is devoid of Fc binding activity. For certain studies, MS was biotinylated. OVA (Sigma, St. Louis, MO), BSA (Sigma), recombinant ß-galactosidase (ß-gal; Sigma), and phycoerythrin (PE) (Molecular Probes, Eugene OR) were used as control protein immunogens.

Mice and immunizations

BALB/cJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were bred at University of California-San Diego under specific pathogen-free conditions. Applying a protocol previously reported for neonatal treatment with a bacterial T cell superantigen (48), neonatal mice, beginning within 24 h of birth, were injected with 100 µg of protein in PBS i.p. every other day for the first 2 wk of life (eight times, 100 µg protein). Certain mice were challenged by s.c. injection with 200 µg of protein emulsified in an equal volume of CFA (Difco, Detroit, MI), then sacrificed 9 or 10 days later (41), and the immune response was examined. Blood was collected retro-orbitally in heparinized tubes, and cells were removed by centrifugation before storage of the plasma at -80°C. Animals were maintained in accordance with the guidelines of the University of California-San Diego Office of Animal Resources. In all experiments, mice were matched for age and sex.

T cell proliferation

Assays for Ag-induced proliferation used draining lymph node suspensions and splenocytes, which can be more reflective of general immune responsiveness (40). Mononuclear cells were cultured at 5 x 105/well in triplicate in sterile 96-well plates (catalogue no. 25860, Corning Costar, Cambridge, MA) in 0.2 ml of serum-free medium (HL-1, BioWhittaker, Walkersville, MD) supplemented with 2 mM L-glutamine without Ag or with MS, control proteins, Con A at 1 µg/ml (Sigma), or purified protein derivative (PPD) of Mycobacterium tuberculosis at 700 U/ml (gift from W. O. Weigle) for 5 days. Proliferation was assessed by the incorporation of 1 µCi of [3H]thymidine during the last 18 h of culture, and the incorporation of label was measured by liquid scintillation counting. To evaluate Ag-specific T cell anergy, certain cultures included murine rIL-2 at 50 U/ml (PharMingen, La Jolla CA) (49, 50).

Enzyme-linked assays of Ab response

A standard ELISA was used to quantify the Ab responses to MS and control Ags. Briefly, microtiter plates (catalogue no. 3690, Corning Costar) were coated overnight with protein at 5 µg/ml in PBS. After blocking with 2% BSA-PBS, serum samples diluted in block were incubated for 4 h at room temperature. The amount of bound Ab was determined by incubation with horseradish peroxidase-labeled affinity-purified goat F(ab')2 anti-mouse IgM or IgG (Jackson ImmunoResearch, West Grove, PA), with values obtained after incubation of substrate for 15 min. Calibration studies with the OVA-specific mAb, clone OVA-14 (Sigma), and the MS-specific mAb, J606, demonstrated >40-fold greater relative sensitivity for the detection of OVA binding. To compare Ab titers, values from different groups of mice were compared at sample dilutions at which the lower mean signal provided an OD of approximately 0.2. A mouse serum pool was used as a standard. For competition studies, sera were titrated to a linear portion of the binding curve (i.e., 1/2,500 for IgM anti-MS activity in naive mice and 1/12,500 for IgG anti-MS activity in postchallenge mice). The relative inhibition capacity of a competitor was determined by comparison to dilutions of the same samples without inhibitor.

Enzyme-linked immunospot (ELISPOT) assays

To quantitate the frequency of Ig- and specific Ab-secreting splenocytes, wells were precoated overnight at 4°C with 5 µg/ml of affinity-purified goat anti-mouse IgM, anti-IgG (Southern Biotechnology Associates, Birmingham, AL), MS, OVA, or 10% FBS in PBS. Plates were washed once with 0.05% Tween-20/PBS and blocked for 1 h at 37°C with HL-1 medium containing 10% FBS that was previously passed over a SpA-Sepharose column (Pharmacia, Piscataway, NJ). Freshly isolated splenocytes in 100 µl of supplemented HL-1 medium were added to triplicate wells with fourfold dilutions starting at 100,000 cells/well and incubated in 5% CO2 at 37°C for 4 h. Adapting a reported protocol (51), in certain studies 6 x 106 fresh splenocytes were first cultured in 5 ml of supplemented HL-1 medium in a 25-ml flask without and with Salmonella minnesota LPS (Sigma) and incubated for 72 h before examination of Ig secretion by ELISPOT assay. To identify the isotype or subclass of specific Ig/Ab secretors, plates were vigorously washed to remove cells, and then alkaline phosphatase-conjugated anti-mouse IgM or IgG (Jackson ImmunoResearch), anti-IgG1, or anti-IgG2a (Southern Biotechnology Associates) was incubated overnight at 4°C. Plates were washed six times before adding 100 µl of 0.6% agarose containing the substrate 5-bromo-4-chloro-3-indoyl phosphate (Sigma) to the well and were developed overnight at room temperature. When possible, values were based on the mean of replicate wells containing >10 but <100 spots/well.

To evaluate the availability of Ag-inducible Th cells, IL-2-producing cells were quantitated in a capture ELISA format employing purified paired mAb, as previously described (52). Microtiter wells were precoated overnight at 4°C with rat anti-mouse IL-2 (PharMingen), then washed and blocked with 10% FBS (SpA column depleted) in HL-1 at 37°C for at least 1 h. Freshly isolated splenocytes in supplemented HL-1 medium were incubated at varying dilutions starting at 200,000 cells/well without or with proteins at 10 µg/ml. After incubation in 5% CO2 at 37°C for 48 h, cells were removed, and plates were washed vigorously, then biotin-conjugated rat anti-mouse IL-2 (PharMingen) was added, and incubated at 4°C overnight. After washing, plates were incubated with streptavidin-alkaline phosphatase (Kirkegaard & Perry, Gaithersburg MD) at 37°C for 30 min, then plates developed and read as described above.

Flow cytometric analysis

Adapting previously reported methods (2), staining of mononuclear cells was performed in PBS containing 1% FBS that had been passed over an SpA column (Pharmacia). In multiparameter studies, Fab-mediated SpA binding was detected with biotin- or PE-labeled MS and compared with that of FITC-labeled anti-mouse IgM (clone 11/41), APC-labeled anti-B220 (clone RA3–6B2), and biotin-labeled anti-mouse IgD (clone AMS 9.1), used with streptavidin-labeled Cychrome (PharMingen). Data were acquired using a FACSCaliber (Becton Dickinson, Sunnyvale, CA) and were analyzed with CellQuest software (Becton Dickinson). Dead cells were excluded based on light scatter and propidium iodide uptake.

RT-PCR assays

Total RNA from control and MS-treated mice was isolated from 40 x 106 fresh splenic cells using Trizol reagent (Life Technologies, Grand Island, NY). After chloroform extraction and isopropanol precipitation, all RNA samples were individually stored in diethylpyrocarbonate-treated H2O at -80°C and quantitated on a GeneQuant Spectrophotometer (Pharmacia). Briefly, from each sample, 3 ng of RNA was reverse transcribed with oligo(dT) using the 1st Strand Synthesis Kit (Life Technologies) to make a 100-µl stock of cDNA. For the amplification of murine genes, a previously calibrated amount of cDNA was added to a reaction mixture containing 5 µl of 10x PCR buffer (Boehringer Mannheim, Indianapolis, IN), 2.5 µl of 10 mM dNTP (Pharmacia), and 1.5 µl of each 20 mM primer solution (Operon, Alameda, CA). Nuclease-free H2O was added to a final reaction volume of 50 µl. Thermocycling conditions included a hot start of 95°C for 3 min with the addition of 2 U of Taq polymerase (Boehringer Mannheim), then 30 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 1 min, followed by a final 72°C for 7 min. Products were individually stored at -20°C. Aliquots from each PCR sample were separated on a 2% agarose gel containing 1% ethidium bromide. Under UV light, gel images were directly digitized through a high resolution digital camera (UVP, San Gabriel, CA) and then quantitated using ImageQuant according to the manufacturer’s protocol (Molecular Dynamics, Sunnyvale, CA). The signal strength of each sample was determined by interpolation from each standard curve of a pooled splenocyte cDNA sample. The content of ß-actin cDNA was used to equalize the amount of cDNA used in VH-{gamma} PCR amplifications (53) and to normalize the quantitated values. Each of the seven VH family-specific separate reactions employed the same antisense {gamma}1 CH1-derived oligonucleotide primer (5'-ggc tta caa tca caa tcc ctg g-3') and a different sense FR1-derived oligonucleotide (CLIII-FR1A (7183), 5'-gtg gag tct ggg gga ggc tta-3' (54); CLIII-FR1B (X24), 5'-gga ggt gac ctg gtg cag cct gga-3'; CLIII-FR1C (J606), 5'-gga gga tgc ttg gtg caa cct gga-3'; CLIII-FR1D (S107), 5'-gga gga agc ttg gta cag cct ggg-3' (55); CLII-FR1A (Q52), 5'-gga cct gac ctg gtg cag ccc tca-3'; CLI-FR1A (J558), 5'-ggt gaa gct tgg ggc ttc agt ga-3' (54); CLI-FR1B (Vgam3), 5'-cag atc cag ttg gtg cag tc-3'). Specificity was confirmed by sequencing and hybridization studies (54, 55) (F. Hajjar and G. Silverman, manuscript in preparation).

Statistical analysis

Comparisons between MS-treated and control groups used the rank sum Wilcoxon and Mann-Whitney one-tailed U tests.


   Results
Top
 Abstract
 Introduction
 Methods and Materials
 Results
Discussion
 References
 
Ab responses in naive and neonatally treated mice

In naive mice, Fab-mediated MS binding activity is high in circulating IgM, while a much lower activity is associated with the IgG isotype (Figs. 1Go and 2). With maturation, the spontaneous levels of anti-MS Abs in naive mice gradually increase, achieving a steady level at 7–12 wk of age. Because this age-dependent change parallels the natural increase in total Ig levels, it does not appear to represent a preferential induction of anti-MS Abs. Presumably, this unconventional germline-associated binding specificity is prevalent among IgM Abs, reflecting the representation of clan VHIII-related families. The lower activity among IgG Abs is a carryover in clones selected by other Ags and probably reflects the lower avidity of IgG binding interactions and the effects of hypermutation (56).



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  		FIGURE 1. Long-term Ab changes induced by neonatal treatment with MS. After neonatal treatment with MS or a control protein Ag, mice were bled periodically, and MS-specific IgM (A) or MS-specific IgG (B) activities were evaluated by ELISA. IgM anti-MS activity was always lower in MS-treated mice than in age-matched control mice. Two distinct temporal phases in levels of IgG anti-MS activity were detected in mice that received neonatal MS treatment. Lower IgG anti-MS levels were induced during the early phase and higher levels during the late phase of the response compared with those in age-matched controls. Quantitation of anti-MS activity in individual serum was performed by comparison with a pooled mouse serum standard, and data represent the arithmetic mean for relative units ± SEM from groups of five.

 
In comparison to age-matched control mice, the neonatal treatment regimen with MS resulted in two distinct temporal phases of altered serum levels of IgG anti-MS Abs. During the first 5 wk of life, here termed the early phase, IgG anti-MS Ab levels are lower than those in age-matched naive mice. Beginning at 6 to 7 wk of life, the late phase of the response to neonatal treatment is associated with serum titers of IgG anti-MS Abs that increase spontaneously to exceed those in age-matched controls and reach a stable elevated level after 7 wk of age (Fig. 1Go). The early phase following neonatal MS treatment is associated with IgM anti-MS levels that are lower than those in naive age-matched controls (p < 0.0001), and these levels also remain lower in the late phase (Figs. 1Go and 2Go). MS binding activity in the serum of naive mice is indistinguishable from the response in PE- or OVA-treated age-matched control mice (not shown).



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  		FIGURE 2. IgM and IgG Ab responses to MS following different treatment regimens. The Ab responses of different groups of mice at 30 days of life (A–D; early phase) and at 9 wk of life (E–H; late phase) were evaluated. A, B, E, and F represent ELISA results using wells precoated with MS, while C, D, G, and H depict studies with OVA precoats. For each group, treatment regimens are indicated in which the Ag used for neonatal treatment is first, followed by challenge, as appropriate (e.g., MS/none, mice that received neonatal treatment without challenge; MS/MS, mice neonatally conditioned with MS that received challenge with MS). The responses of additional treatment groups that did not receive neonatal treatments but subsequently were challenged are also displayed (i.e., None/MS and None/OVA). Challenge immunizations included either MS in CFA or OVA in CFA, and sera were collected 9 or 10 days later. Results represent the arithmetic mean ± SEM of values obtained by ELISA from age-matched groups of five mice.

 
Significantly, in competition binding studies a human VH3-encoded IgA at 20 µg/ml inhibited 96% of IgM anti-MS activity from naive 10-wk-old mice and 83% of IgG anti-MS activity from 10-wk-old mice following priming and in vivo challenge, while non-VH3 IgA had no significant effect (<10%; data not shown). These results are comparable to inhibition studies with human Ig and B cells (2, 20), suggesting that the clan VHIII-restricted Fab binding site of SpA is immunodominant for recognition by both natural nonimmune and postimmune exposure Abs.

To determine whether the two distinct temporal phases of altered IgG MS binding levels reflect different intrinsic levels of immune responsiveness, mice neonatally treated with MS were subsequently challenged with the immunogen. During the early phase, challenge with MS did not affect circulating IgM anti-MS levels in either naive or MS neonatally conditioned mice (Fig. 2GoA). Early phase challenge of neonatally MS-conditioned mice induced only a small IgG response that was blunted compared with the induced IgG response in naive age-matched controls (Fig. 2GoB).

During the late phase, adult MS challenge induced only modest increases in IgM anti-MS activity in mice that had been neonatally treated with MS or in age-matched naive mice (Fig. 2GoE). Significantly, during the late phase, postchallenge IgG anti-MS responses of MS neonatally conditioned mice greatly exceeded the postchallenge responses of naive controls (p < 0.001; Fig. 2GoF), thus confirming recent reports that neonatal treatment generally results in priming for Ab responses to adult challenge (40, 41).

To compare these findings to the responses induced by a conventional soluble protein Ag, groups of neonatal mice received equivalent treatment with OVA (Fig. 2Go, C, D, G, and H). In age-matched naive mice, the titers of IgM anti-OVA activity were 30- to 100-fold lower than those of IgM anti-MS activity (p < 0.0001; Fig. 2Go, C and G). In these control groups, neonatal conditioning with OVA did not affect IgM anti-OVA activity during the early phase (Fig. 2GoC), while it resulted in lower IgM anti-OVA activity during the late phase (Fig. 2GoG) compared with that in age-matched naive mice. With OVA challenge at 3 wk of age, about half the OVA-conditioned mice displayed OVA-specific IgM and IgG Ab responses (Fig. 2Go, C and D), proliferation, and induction cytokine production (not shown). The remainder of this group displayed OVA-specific tolerance (see below). Hence, OVA was less effective than MS at inducing and maintaining Ag-specific tolerance in the early phase. During the late phase, all OVA-conditioned mice displayed induced vigorous IgM and IgG anti-OVA responses following adult challenge. However, after challenge during the late phase, the OVA-specific Ab activity in neonatally OVA-primed mice was significantly lower than the postchallenge anti-MS Ab response in equivalently MS-treated mice (p = 0.001; Fig. 2Go, G andH).

Neonatal treatment transiently decreases the frequency of MS-specific B cells

To determine whether the two different phases of immune responsiveness reflect changes in the representation of MS-specific B cells, cytofluorometric studies were performed (Fig. 3Go). Consistent with specificity studies of human mononuclear cells (2), MS binding activity was restricted to a subset of murine immature and mature B cells (Fig. 3Go). In naive adult mice, about 5% of sIgM+ splenocytes displayed MS binding activity (57). The proportion of splenic sIgM+ B cells with MS binding activity varied little among individuals of the same age and was greatest among sIgD+ peripheral B cells (not shown), consistent with findings in the human immune system (2, 11). Binding was detected in both Ig {kappa}- and {lambda}-expressing populations in a normal distribution (not shown).



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  		FIGURE 3. Transient deletion in the peripheral and central compartments of MS-binding B cells following neonatal exposure. A, Multiparameter flow cytometric studies were performed on splenocyte suspensions from 15-day-old mice following neonatal treatment with MS or a control Ag, OVA. In the control Ag-treated mice, 5.1% of B220+ cells display MS binding activity, while after MS treatment <0.3% of the B220+ cells display MS binding activity, indicating a selective and near complete loss of detectable MS-binding B cells. B, Comparable studies from bone marrow suspensions from 15-day-old mice following neonatal treatment with MS or a control Ag, OVA, are presented. C, To ensure that post-treatment changes did not arise from surface modulation of sIgM, findings for the proportion of MS-binding cells among sIgM+ cells are depicted. Following neonatal treatment, the selective loss of MS-binding splenic B cells in MS-treated mice (filled circle) was most pronounced at 15 days of age, and by 21 days of age was undetectable. Control Ag-treated mice were unaffected (open square). Results are representative of four independent experiments using 25 neonatally treated mice, including those depicted in A and B.

 
After neonatal MS treatment, on day 15 of life there was a near complete loss of detectable MS-binding splenic B cells (Fig. 3Go). Similar losses of MS-binding mature and immature B cell subsets were detected in the bone marrow. At this time, neonatal MS treatment also caused modest decreases in the representation of B220+ mononuclear cells in the spleen (Fig. 3GoA), suggesting that this represented MS-induced B cell clonal deletion and not modulation of sIgM affecting the detection of MS binders.

To evaluate whether residual in vivo MS might be blocking the binding of labeled MS during these in vitro staining studies, certain mice were neonatally treated with biotinylated MS. However, in vitro staining of mononuclear cells with fluorochrome-labeled streptavidin failed to detect B cell-associated biotinylated MS (not shown), suggesting that residual MS from the treatments did not significantly contribute to the observed decrease in the frequency of MS-binding B cells.

As the MS-treated animals aged, the proportion of B cells with MS binding activity in the central and peripheral compartments reverted to the levels present in naive mice. Within 1 wk after the last MS treatment, or at 3 wk of age, levels of splenic MS binders were completely normalized (Fig. 3GoC). These ephemeral changes resemble the central clonal deletion described following Ag treatment of Ag-specific monoclonal Ig transgenic mice (58, 59). However, in our studies of the polyclonal populations of BALB/cJ mice, it remains a distinct possibility that with the observed normalization of the overall frequency of MS-binding B cells masks a subtle but persistent change in specific BCR VH region expression by the B cell repertoire.

Neonatal treatment causes a persistent decrease in the frequency of MS-specific IgM-secreting cells

The functional capacity of the B cell compartment was assessed using ELISPOT studies that detect in vivo activated Ig-secreting cells. Consistent with reports that this compartment of activated IgM-secreting B cells is homeostatically autonomous (60, 61), we found comparable frequencies of splenic IgM-secreting cells (1300–9000/106 splenocytes) in 4- to 35-wk-old naive mice raised under specific pathogen-free conditions (Figs. 4Go and 5). In naive mice at the early and late phase ages, B cells secreting MS-binding IgM represented 15.9 ± 1.7 and 13.1 ± 0.8% (mean ± SEM) of all IgM-secreting B cells, respectively. The demonstration that this higher proportion of IgM-secreting B cells in naive mice displays MS specificity is consistent with a greater detection sensitivity for binding than can be attained in cytofluorometric studies (Fig. 3Go).



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  		FIGURE 4. During the early phase, neonatal treatment induces a selective loss of MS-specific IgM-secreting cells and a tolerance to MS challenge. Each panel displays results from a treatment group of 30-day-old mice in immunospot assays of spontaneous in vitro splenic IgM-secreting cells, and the frequencies for individual mice for total IgM-secreting cells, MS-binding IgM-secreting cells, OVA-binding IgM-secreting cells, and FBS-binding IgM secreting cells (background) are independently displayed. Arithmetic mean values for MS-specific Ig secretors are indicated by a short horizontal line and are compared with the values from naive age-matched control mice (broken line). Statistically significant differences between groups are indicated, as determined by a one-tailed Mann-Whitney U test. Results are compiled from four multigroup experiments in which certain mice were challenged 9 or 10 days before harvest of splenocytes. The numbers of IgM-secreting cells are expressed per 1 x 106 splenocytes. For each group, ELISPOTs of cultured splenic cells for more than five mice are displayed.

 
When mice were evaluated within 1 day of the last neonatal MS treatment (15 days of age), MS-specific IgM-secreting cells were undetectable (not shown). Persistent MS-specific functional impairment was demonstrated at 4 wk of life (2 wk after the last MS exposure) when there was still an 84% reduction in the mean frequency of MS-specific IgM-secreting cells compared with that in naive age-matched controls (Fig. 4Go). Notably, in all studies mice that received neonatal treatment with MS had a lower frequency of spontaneous IgM anti-MS secretors than naive mice (Figs. 4Go and 5Go) even after prolonged periods (i.e., 72 wk of age; p < 0.004). Hence, this post-treatment decrease in MS-specific IgM-secreting cells continued throughout the late phase despite the development of MS-specific T cell responsiveness (see below). In mice neonatally treated with conventional protein Ags, OVA (Fig. 4Go) or ß-gal (not shown), there was no affect on the frequencies of MS-specific IgM secretors. Using these same methods, conventional Ag-specific IgM-secreting cells in naive and neonatally conditioned mice could not be detected (Figs. 4Go and 5Go). Significantly, in vitro LPS stimulation of splenic B cells caused a three- to fivefold relative increase in the frequency of IgM secretors without affecting the proportion of cells secreting MS-specific IgM (not shown). Hence, despite normal frequencies of MS-binding splenic B cells, mice that received neonatal MS treatment displayed the same reduction in MS-specific IgM-secreting cells after in vitro LPS stimulation, a pattern of in vitro nonresponsiveness associated with anergic B cells (51, 62, 63, 64, 65).



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  		FIGURE 5. During the late phase, neonatal treatment primes for the induction of Ag-specific IgG1-secreting cells despite a selective and persistent loss of MS-specific IgM-secreting cells. The frequencies of splenic IgM- and IgG1-secreting cells in individual mice were enumerated after different treatment regimens. In A, arithmetic mean values for MS-specific IgM secretors are indicated by a short horizontal line; the value from naive age-matched control mice (broken line) is compared with those from other groups. In B, arithmetic mean values for total IgG1 secretors and Ag-specific IgG1 secretors are indicated by a short horizontal line. The broken line compares the mean values of MS-specific and OVA-specific IgG1 secretors in age-matched groups that were neonatally primed and adult challenged with their respective immunogens. Results were obtained from four multigroup experiments using 9- to 14-wk-old mice, in which certain mice were challenged 9 or 10 days before harvest of splenocytes. The numbers of Ig-secreting cells are expressed per 1 x 106 splenocytes. Statistically significant differences between groups are indicated, as determined by a one-tailed Mann-Whitney U test. For each group, ELISPOTs of cultured splenic cells for more than five mice are displayed.

 
To determine the stability of the MS-specific tolerance induced by neonatal treatment, mice were challenged in vivo with MS emulsified in CFA. Following challenge of neonatally conditioned mice during the early phase (at 21 days of life), there was still a >80% decrement in the frequency of anti-MS IgM-secreting cells compared with that in control mice (p = 0.0002; Fig. 4Go). During the early phase, MS challenge also failed to induce levels of anti-MS IgG-secreting cells above the low background level (170 ± 35/106 splenocytes; not shown).

During the late phase, adult challenge with MS did induce an increase in total IgM-secreting cells that was associated with a modest increment in IgM anti-MS-secreting cells compared with that in nonchallenged MS-conditioned mice (Fig. 5Go). However, the ratios of MS-specific IgM-secreting cells/total IgM-secreting cells remained depressed in these late phase neonatally conditioned mice (p = 0.0003). Challenge with CFA alone of adult MS-conditioned mice or naive age-matched controls also did not affect the frequency of total IgM-secreting cells or of MS-specific IgM-secreting cells (not shown).

Significantly, during this late phase, challenge of MS-conditioned mice resulted in a significant induction of IgG1-secreting B cells and a mean proportion of 91% of these cells secreting MS-specific IgG1 Ab (Fig. 5GoB). By comparison, the induction of IgG2a-secreting cells was modest (200–400/106 splenocytes), and MS-specific IgG2a-secreting B cells were not detected (not shown).

In mice neonatally conditioned with OVA, adult challenge with OVA/CFA resulted in a significant induction of IgG1 anti-OVA-secreting cells (Fig. 5GoB), while few (<200/106 splenocytes) IgG2a-secreting cells were induced (not shown). Significantly, in these OVA-conditioned mice only a mean of 7% of induced IgG1-secreting cells was specific for the OVA immunogen (Fig. 5GoB). Hence, there was a significant difference in the mean frequency of postimmunization OVA-specific IgG-secreting cells, which was about 30-fold lower than that induced by equivalent treatment with MS (p < 0.0004). These findings are consistent with past reports that the secondary immune response to the conventional protein Ags in adjuvant are highly degenerate (66, 67). In contrast, the same treatment regimen with the prototype B cell superantigen, MS, recruits essentially only MS-specific Ab-forming cells. In these studies there were no detectable age- or sex-associated differences in the response patterns of mice between 9 and 30 wk of age.

T cell response to neonatal treatment with MS

To assess T cell recognition of the immunogen, Ag-specific proliferation assays were performed on neonatally treated mice after in vivo challenge. Splenocytes (Fig. 6GoA) and lymph node cells (not shown) of 30-day-old mice displayed MS-specific nonresponsiveness, the hallmark of tolerance (68, 69), while there was a vigorous response to PPD, a component of the adjuvant used in the challenge immunizations. Addition of IL-2 to cultures did not result in a proliferative response to MS (not shown), suggesting that MS nonresponsiveness was not due to Ag-specific T cell anergy (49, 50). Notably, in control studies splenocytes (Fig. 6Go) or purified B cells (not shown) from naive animals did not proliferate in the presence of MS, suggesting that under these conditions MS is not a direct B cell mitogen. To evaluate the T cell responsiveness during the late phase, neonatally treated mice were challenged at 6 wk of age or older (Fig. 6GoB). Splenocytes from these mice displayed an Ag-specific proliferative response (e.g., p = 0.014 at 10 µg/ml MS), indicating spontaneous in vivo priming during the late phase.



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  		FIGURE 6. Early phase MS-specific proliferative tolerance and late phase immune responsiveness following neonatal exposure to MS. The in vitro responses of splenocytes following culture without or with different proteins are displayed, and in vitro concentrations of MS and OVA (micrograms per milliliter) are indicated. During the early phase (A), neonatal treatment with MS induced MS-specific nonresponsiveness, while stimulation with PPD, a component in the challenge, was vigorous. During the late phase (B), neonatally MS-conditioned mice demonstrated a primed response to MS, while splenocytes from naive mice were nonresponsive (p = 0.014 at 10 µg/ml MS). The data represent mean scintillation count values from triplicate wells from individual MS-treated mice (filled circles) or naive control mice (open squares), and the arithmetic means for groups of four MS-treated (+) and naive control (X) mice are displayed. The dashed line indicates 3 times the counts per minute value for the control group samples. For mice neonatally treated with MS, splenocytes were harvested 9 or 10 days after in vivo challenge with MS emulsified in CFA. Cultured cells from more than five mice are shown. The data are representative of results from four experiments.

 
To enumerate the frequency of MS-responsive effector Th cells, cytokine-specific ELISPOT assays were performed. In vitro culture of splenocytes from 30-day-old neonatally conditioned mice (early phase) did not reveal an increased frequency of MS-induced IL-2-secreting cells (not shown). These findings along with the lack of induction of IgG anti-MS secretors and the evidence of nonresponsiveness in the proliferation assays are consistent with previous descriptions of active tolerance affecting Th effector cells (42, 43, 70).

Neonatally conditioned mice were also evaluated for the frequency of splenic cytokine-secreting cells during the late phase of the response. Even without subsequent in vivo exposure, the splenocytes of about half the group of mice neonatally treated with MS had significantly increased levels of MS-responsive IL-2 secretors, while all OVA-conditioned mice contained increased levels of OVA-responsive IL-2 secretors (Fig. 7Go). Importantly, after equivalent neonatal priming and adult challenge with their respective immunogens in CFA, based on cytokine secretion we found comparable frequencies of MS-specific Th cells in MS-treated mice and of OVA-specific Th cells in OVA-treated mice (Fig. 7Go). This suggests that a greater availability of T cell help (i.e., clonal size of Ag-specific T effector cells) was not responsible for the higher frequency of inducible immunogen-specific IgG-secreting cells detected during late phase-associated secondary anti-MS responses.



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  		FIGURE 7. Induction of IL-2-secreting cells by in vitro challenge during the late phase. Following neonatal conditioning, 9-wk-old mice were challenged in vivo, and IL-2 production by splenocytes was measured following in vitro culture. In each panel the response was measured for splenocytes from different treatment regimens following culture without (-) or with MS, OVA, or ß-gal at 10 µg/ml or with PPD at 700 U/ml, as indicated. Mice neonatally treated and later challenged with MS (MS/MS) showed MS-specific induction of IL-2-secreting cells. A comparable frequency of IL-2-secreting cells was induced in mice receiving equivalent treatment with OVA (OVA/OVA) or ß-gal (b-gal/b-gal). Values represent arithmetic mean ± SEM for groups of four mice. The data are representative of three experiments.

 
VH gene usage in MS-induced Ab responses

To further characterize the B cell response to MS challenge, based on the documented IgG1 bias of the induced response we evaluated the expressed {gamma}1 gene rearrangements for evidence of VH-mediated clonal restriction. For these studies, we developed an RT-PCR based system to compare splenocyte samples for their relative content of mRNA from each of seven different VH families that are representative of the three clans of murine VH genes. These gene families are cumulatively the source of >85% of the expressed Ig repertoire of BALB/c mice (71, 72, 73, 74).

In general, for any sample the cumulative level of VH-{gamma}1 gene expression paralleled the relative frequencies of IgG1-secreting cells in the splenocyte samples (Figs. 8Go and 9). As expected, splenic samples from naive mice contained very low levels of VH-{gamma}1 mRNA. Mice that did not receive both a priming and an adult challenge of Ag also displayed very low levels of detectable {gamma}1 rearrangement mRNA. By comparison to naive mice, in each sample from a mouse neonatally conditioned with OVA and then challenged as an adult (n = 4), there was at least a 10-fold mean increase in the expression of all seven VH families, indicating a non-VH-selective stimulation. Significantly, the VH gene expression of mice that were MS conditioned and challenged (n = 6) uniformly demonstrated much greater levels of expression of the S107 family (VHIII clan), representing 40-fold mean higher levels than those in the OVA-treated and challenged mice (p < 0.005) and >400-fold mean higher levels than those in naive mice. In comparisons of these MS-treated and the OVA-treated mice, the MS responders also displayed lower expression levels of the six other VH families, which were statistically significant for the Q52 (p < 0.005), 7183 (p < 0.005), and X24 families (p < 0.005), indicating selective B cell recruitment in the anti-MS response. These data document the dominance of a clan VHIII-restricted component in the cellular recruitment into the secondary immune response to a bacterial B cell superantigen.



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  		FIGURE 8. Expression of VH gene families in {gamma}1 rearrangements. A semiquantitative RT-PCR method was used to evaluate the splenic expression of major murine VH gene families after different treatments. For each sample, actin gene expression was quantitated by comparison of the intensity of the specific 623-bp band product to a standard curve. Actin content was then used to normalize template in each amplification, and values from repeat actin reamplifications were used as a denominator to standardize VH gene cDNA quantitation. At the left, a standard curve for each specific amplification is shown. At the top right, the neonatal treatment/adult challenge regimens is indicated above the results for each individual splenic sample. In each panel at the right, the approximately 630- to 660-bp products from representative splenic samples are shown for each of the VH-{gamma}1 reactions. Values were derived from separate VH-specific reactions by comparison to standard curves for each amplification type. For naive mice (none/none) and mice that received a challenge with CFA alone, very low VH-{gamma}1 gene expression levels were detected. In contrast, after neonatal treatment with OVA and adult challenge (OVA/OVA-CFA), greatly increased levels of expression of all seven VH families were demonstrated. Neonatal treatment followed by adult MS challenge (MS/MS-CFA) resulted in predominant expression of VH S107 gene rearrangements. Relative unit values were not comparable between different amplification types. These data and the results from two other sets of experiments are compiled in Fig. 9Go.

 

   Discussion
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
References
 
MS induces a B cell superantigen response

Within the current investigations of SpA, an experimental Ag studied for decades, we provide the first characterization of the clonal response following in vivo challenge with this B cell superantigen. Both cytofluorometric studies and serologic assays document that the frequency of B cells in the naive repertoire capable of Fab-mediated SpA interactions is orders of magnitude greater than that for conventional protein Ags. The specific IgG Ab response after challenge of primed adults is also of higher magnitude. To independently survey the functional capacity of the B cell compartment, we quantitated spontaneous Ig/Ab-secreting cells. We found that a mean of 13% of the IgM-secreting cells in naive animals displays Fab-mediated SpA binding activity, presumably reflecting the composition of the primary B cell repertoire (60, 61). Notably, during secondary immune responses to MS >90% of IgG1-secreting cells are MS specific, while in naive mice IgG-secreting cells are infrequent and MS-specific IgG-secreting cells are undetectable under the same conditions.

The IgG response to MS contrasts with results for OVA, which appears to induce a highly degenerate response, containing only a small proportion of Ag-specific IgG-responding B cells, confirming earlier reports for other soluble homogeneous conventional protein Ags (66, 67). Calibration experiments of the immunospot assay demonstrated that it is unlikely that sensitivity thresholds (e.g., the epitopic heterogeneity or precoating density) artifactually limited the detection of OVA-specific Ig secretors. Similarly, we found that after equivalent neonatal priming and adult challenge with another conventional protein immunogen, ß-gal, <20% of the IgG secretors are Ag specific (not shown). Therefore, in studies designed to juxtapose MS-induced findings with responses to conventional homogeneous soluble protein Ags, we believe that the results fairly highlight the potent ability of SpA to induce highly focused Ab responses.

The current studies address the fundamental question of the role of the clan VHIII-restricted Fab binding site in active immune responses to SpA. To sample the specific repertoire we could have evaluated panels of MS-specific hybridomas, but there was concern in light of recent reports that this method can rescue B cells that are anergic in vivo, which are therefore not representative of the active in vivo response (65). In support of our more direct approach, we found that >90% of IgG1-forming cells induced by secondary immunization recognize SpA, and >80% of these MS-specific IgG Abs can be blocked by the binding of a VHIII Ig from a naive host. Hence, most of this MS-induced response appears to be directed at the site responsible for binding of VHIII-encoded Abs. Therefore, we surveyed for VH-{gamma}1 expression and discovered that the secondary responses to MS recruited an overwhelming predominance of S107 rearrangements, which have been shown to generally encode for SpA binding activity (29). These data document that the clan VHIII-restricted Fab binding site of SpA is dominant during active immune responses.

Epitope dominance resulting in the recruitment of a highly restricted Ab response has been described in several systems, but most often this pattern has been associated with structurally simpler Ags (e.g., carbohydrates, haptens, or peptides). In the few reports of highly restricted responses to protein Ags, the dominant B cell epitopes are generally redundant determinants in functionally multivalent proteins (75, 76). Hence, while the focused B cell response elicited by SpA can be considered as another type of epitope dominance, the clan VHIII restriction of binders and the high frequency of potential responders are very different from those of other characterized Ags. Taken together, these immunologic properties of a very high binder frequency in the naive repertoire, immunodominance of the Fab binding site, and recruitment of a VH-restricted B cell responders that dominates the immune response embody the hallmarks of a B cell superantigen.

MS is a superantigen, but a B cell is still a B cell

Aside from the unconventional structural correlates (VH dependence) of binding that are responsible for the high frequency of potential responders, in most important respects the cellular correlates for immune responsiveness to this B cell superantigen do not appear to be distinct from those of conventional Ags (i.e., SpA binders display a range of affinities, and T lymphocytes play a central role in determining the outcome of exposure). Preliminary findings in S107-{gamma}1 libraries suggest that secondary anti-MS responses are associated with the expression of clonally restricted gene sets representing independent S107 gene rearrangements, which presumably were selected based on their relative affinity within the accessible B cell pool (G. J. Silverman, manuscript in preparation).

The current studies also suggest that this immunogen can induce a range of Ag-specific B cell clonal fates among the specific B cells in polyclonal lymphocyte populations, findings that previously could only be considered in transgenic monoclonal Ig systems. Hence, our studies begin to illustrate how a B cell superantigen can be exploited for the dissection of the events that contribute to active immune responses and stage-dependent influences on the formation of the mature B cell repertoire. Offering another attraction for the future development of model systems, SpA can be manipulated to alter intrinsic Fab binding affinity and effective valency.

Neonatal treatment with a B cell superantigen causes shifts in tolerance and immunologic memory

During the early phase following neonatal treatment we found an absence of proliferative, cytokine, or Ab responses to Ag re-exposure. In other reports it has been shown that Ag-specific tolerance can affect all types of effector Th cells (42, 43, 70). The inducible tolerance associated with the neonatal period has been ascribed to an age-associated immune defect in which Ag is presented in the context of inadequate costimulatory signals (33, 34, 35, 39, 77). Ag-specific tolerance can be mediated by dendritic cells (78) or by resting B cells (79, 80, 81), which are especially susceptible to tolerization or negative selection during early development (82) and which clearly display MS binding capacity at exceptionally high frequency (Fig. 3Go).

Consistent with earlier (30, 31) and more recent reports (40, 41), our studies demonstrate that the eventual outcome of neonatal Ag conditioning is an enhanced adult capacity for Ag-specific Ab and proliferative responses, indicating in vivo priming and immunologic memory. We found that adult challenge induces an MS-specific IgG1 Ab response, suggesting that neonatal exposure to a B cell superantigen, such as conventional self and non-self Ags (36, 37, 38, 40, 41), leads to a Th2-biased response. Notably, the duration of complete tolerance to MS could also be prolonged by weekly i.p. instillations of 100 µg of MS in alum (not shown) or by the substitution of native SpA that has higher intrinsic Fab binding affinity (G. J. Silverman, unpublished observations), which is predictable based on classical models of neonatal tolerance (30, 83).

Our studies of the late phase of the response demonstrate that despite the same high frequency of peripheral MS-binding B cells as that detected in naive mice, neonatal MS treatment results in lower IgM anti-MS circulating Ab levels and the persistent loss of in vitro spontaneous anti-MS IgM secretion. In transgenic Ig systems, this loss of spontaneous Ig-secreting cells of a defined Ag specificity has been used as a marker of clonal functional inactivation (i.e., anergy) (63, 64, 65), although in the MS neonatal treatment system other regulatory or trafficking-related processes have not been ruled out.

The late phase of the MS response is also associated with spontaneous priming of MS-specific T cell responses and production of IgG anti-MS Abs. Presumably, these divergent effects on the IgM and IgG responses, demonstrated to persist for more than a year after the last infusion, reflect the continued influence of an Ag that may be retained in lymphoid tissue for years (84). While partial tolerance has been previously reported following neonatal exposure to mixed immunogens, this was interpreted as representing nonresponsiveness to certain antigenic determinants at the same time as other determinants are the targets of active immune responses (31). In contrast, our studies characterize the response to a homogeneous Ag that is recognized by B cells at very high frequency via a VHIII-restricted Fab binding site.

Based on the transgenic anti-HEL/HEL tolerance model, MS-specific partial anergy would be predicted to involve B cell clones expressing BCR with higher binding affinity (85). Functional inactivation of MS-specific clones could occur during the continuous generation of immature/transitional B cells in the bone marrow, which are highly sensitive to BCR-mediated tolerance induction and negative selection (86, 87), or their newly emergent counterparts in the periphery that have similar sensitivity (58, 82, 88). A possibly related process is responsible for the sensitivity to tolerization reportedly associated with memory B cell differentiation (89, 90, 91, 92, 93, 94). Hence, we question whether the active anti-MS response truly reflects the products of B lymphocytes expressing BCR with the highest binding affinity. Significantly, recent rapid amplification of cDNA ends-PCR studies have demonstrated that neonatal MS treatment results in a persistent loss of VH 7183 family expression in the primary B cell pool (J. V. Nayak and G. J. Silverman, manuscript in preparation), possibly due to MS-induced clonal loss. The further dissection of these phenomena will therefore include a more rigorous definition of the relative affinities of the superantigen binding activities conveyed by different murine Ab genes.

B cell superantigens and repertoire development

During the neonatal stage of development, BCR-mediated signals apparently affect the rate at which the maturing B lymphocytes fill the periphery. In a recent report maternal IgG was shown to act as a nonselective BCR-interacting agent on the emerging B cell compartment, resulting in enhanced cellular expansion in the periphery. Concurrently, this influence delayed the spontaneous differentiation of evolving B cell clones to the Ig-secreting stage (95). In a more clonally restricted manner, specific encounters with cross-reacting Ag or their anti-idiotypic surrogates during early immune development have been reported to shift the Ab V gene/clonal representation in subsequent Ag-specific responses (44, 45, 46, 47). Our studies suggest that neonatal exposure to a bacterial product conveys a VH-selective stimulation/differentiation of certain superantigen-specific B cell clones, concurrent with the functional inhibition or down-regulation of others. As discussed, Ag persistence is almost certainly responsible for the observed prolonged effects on the B cell compartment, while the range of BCR affinities for the B cell superantigen probably contributes to the different clonal outcomes, including the continual or recurrent "tickling" of certain clones (96, 97). Hence, we wonder whether the current results predict that neonatal B cell superantigen exposure can bias the accessible pool of B cell responders for subsequent exposures to conventional ligands, and whether an equivalent natural process may be responsible for evidence in humans and mice of unconventional VH family-directed selection (12, 98). Recent reports of endogenous proteins with B cell superantigen-like properties (3, 4, 5) warrant further investigation of these postulated processes.



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  		FIGURE 9. The VH-restricted response to MS. Each panel represents compiled relative values obtained from analyses as depicted in Fig. 8Go. These data include results from control samples that did not include cDNA (-) and those from representative age-matched adult mice that received neither neonatal treatment nor challenge (Naive), that received no neonatal treatment but were given an adult challenge of CFA alone (None/CFA), that received neonatal MS treatment followed by adult challenge with CFA alone (MS/CFA), or that received no neonatal treatment but were adult challenged with MS in CFA (None/MS). The number of individual samples evaluated is indicated at the bottom (N). For the mice neonatally conditioned with OVA and later adult challenged with OVA in CFA (OVA/OVA), results from four mice are presented. For the group that were neonatally conditioned with MS and adult challenged with MS in CFA (MS/MS), results from six mice are compared. Compared with the nonselective induction of VH family expression in the comparable OVA/OVA-responding mice, MS/MS treatment uniformly elicited a response dominated by VH S107 gene family expression, representing a mean 40-fold increase compared with that in the OVA responders (p < 0.005) and a >400-fold increase compared with that in naive age-matched adults. In the MS/MS samples, there were uniform decreases in the expression of the other VH families. *, Significant differences between the OVA/OVA- and MS/MS-treated groups (p < 0.005).

 

   Acknowledgments
 
We thank Heather Morrell, Raymond Wagenknecht, and Laura Sandoval for their expert technical assistance and Dr. Mark Shlomchik for advice and criticism throughout the project. We also acknowledge the useful discussions provided by Drs. Ann Feeney, Roy Riblet, Helen Quill, Susan Wormsley, John Monroe, Garnet Kelsoe, Dan Gold, and William O. Weigle.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants R29-AI34001, R01-AI40305, and 5P60-AR40770; in part by Training Grant AR07567 (to V.E.C.); by a Career Development Award (K02-AI01378) from the National Institute of Allergy and Infectious Diseases (to G.J.S.); by biomedical sciences awards from the Arthritis Foundation; and by a research grant award from the Crohn’s and Colitis Foundation of America. Back

2 Address correspondence and reprint requests to Dr. Gregg J. Silverman, Department of Medicine-0663, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0663. E-mail address: Back

3 Current address: University of Pittsburgh School of Medicine, Pittsburgh Cancer Institute, BST 1013E, 211 Lothrop St., Pittsburgh, PA 15213. Back

4 Current address: Klinische Forschergruppe fur Rheumatologie, Universitat Freiburg, Breisacher Str. 64, 79106 Freiburg i. Breisgau, Germany. Back

5 Abbreviations used in this paper: BCR, B cell receptor for Ag; SpA, staphylococcal protein A; MS, chemically modified staphylococcal protein A that retains Fab binding activity; ß-gal, ß-galactosidase; PE, phycoerythrin; PPD, purified protein derivative; ELISPOT, enzyme-linked immunospot; sIgM, surface IgM. Back

Received for publication May 8, 1998. Accepted for publication July 8, 1998.


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 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
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