Human antibody responses to bacterial antigens: studies of a model conventional antigen and a proposed model B cell superantigen.

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Intern. Rev. Immunol. Vol. 9, 1992, pp. 57-78 Reprints available directly from the publisher Photocopying permitted by license only O 1 9 9 2 Harwood Academic Publishers GmbH Printed in the United States of America

Human Antibody Responses to Bacterial Antigens: Studies of a Model Conventional Antigen and a Proposed Model B Cell Superantigen GREGG J. SILVERMAN Department of Medicine and the Sam and Rose Stein Institute for Research o n Aging, University of California, San Diego, La Jolla, California 92093-0663, USA

We have investigated the human antibody repertoires that bind to two different classes of bacterial antigens. Immunization with the conventional antigen, type b capsular polysaccharide of Haemophilus influenzae Hib PS, uniformly induces IgA and IgG responses dominated by clones that use heavy chains structurally related to two subsets of V 3 enes, while in a minority of subjects antibodies from the VH1 or VH4families are co-induced. In contrast, th,“ ‘kternative binding site” of Staphylococcal Protein A (SPA) represents an unconventional determinant, because; (i) SPA is bound by a large proportion of non-immune IgM, IgA and IgG F(ab’)2, (ii) SPA is bound only to Fab from the VH3 family, which can be encoded by at least four different germline genes, (iii) SPA binding is independent of V, usage, (iv) by flow cytometry SPA is bound by >15% of tonsilar B cells, but not to T cells. (v) In virro stimulation with an SPA containing mitogen induces the preferentially production of Ig bearing a VH3 marker. Taken together, these studies characterize a VH family restricted binding interaction that is distinct from the properties associated with conventional antigens such as Hib PS. Based on these data we propose that SPA represents a prototype for a B cell superantigen.

KEYWORDS: reperioire, superaniigen, protein A , Haemophilus influenzae, antibody, variable region

INTRODUCTION Defining the clonal and structural heterogeneity of antigen-specific antibody responses has been a central focus of contemporary immunology. Extensive investigations in inbred animal models have suggested that antibody responses to specific antigens are often dominated by a limited set of clonotypes that are the same in genetically identical individuals. Comparable studies have not been generally possible in humans, partly due to ethical considerations that limit the immunogens available for study, and also due to the relatively poor cloning efficiency of human B cells. Therefore, characterization of the clonal diversity of human antibody responses, and the determination of whether particular human antigen-specific responses are associated with biased variable region gene usage, has required the development of alternative approaches. We have focussed on two very different prototypic antigen binding activities. For a model conventional antigen binding system, we have evaluated the human antibody response to the capsular polysaccharide (PS) of the Haemophilus injuenzue type b (Hib), because of its clinical relevance as the immunodominant target for the immune defense from a common 57


58

G . J. SILVERMAN

childhood pathogen. We have also investigated the diversity of Ig that interact with the “alternative binding site” of the staphylococcal protein A (SPA), which is representative of a unique type of unconventional antigenic determinant. This site specifically interacts with a subset of human immunoglobulin Fab that is present at a very high frequency on circulating Ig and B cells, which are completely restricted in their VH gene family usage. Therefore, we have recently proposed that this activity is best described as a “Ig superantigen” for the human immune response [I]. The purpose of this review is to describe our findings regarding the functional utility of certain human variable regions in antibacterial human responses, and to discuss the implications for clonal selection and B cell repertoire formation.

EVALUATION OF VARIABLE REGION DIVERSITY IN CIRCULATING ANTIBODIES Conventional anti-idiotypes that recognize conserved structural motifs in an antibody variable (V) region can be used to demonstrate the heterogeneity within specific immune responses. In particular, if an idiotype represents a marker for a V gene segment, D segment or other germline element, it can be used to determine if antibody responses in different individuals all arise from the same set of genes. While most idiotypic systems have been found to be expressed only in inbred animal strains, several anti-idiotypes have been described that identify large sets of monoclonal immunoglobulin that are produced during human lymphoproliferative syndromes. Studies with these anti-idiotypes have led to the hypothesis that disorders such as chronic lymphocytic leukemia, mixed cryoglobulinemia and chronic cold agglutinin disease are each associated with the preferential expression of a limited set of immunoglobulin genes [2-51. While these investigations have documented that disease associated antibodies in unrelated patients can have a common origin, whether physiologic antigen-driven responses are also structurally and genetically restricted has only recently been addressed. As a complement to available conventional idiotype systems, we have developed a panel of serologic markers to distinguish the Ig products of the known human Variable (V) region gene families [6-lo]. This method was based on earlier reports which demonstrated that a synthetic peptide with the exact sequence present in a hypervariable region can be used to generate an antiserum that identifies a “primary-sequence dependent” idiotype [II, 121. These antibodies act as markers for particular V region gene segments and, at times, as clonotypic markers. We initially generated antisera that can distinguish the products of the V kappa gene families, and these serologic reagents each identifies a sequence in the first framework region portion that is highly conserved by members of a particular V, family, but differs from the homologous framework sequences form the other V, families [6]. These reagents have been demonstrated to identify the vast majority of kappa light chains into non-overlapping subsets, probably because replacement mutations are less common in this portion of the framework region as substitutions might impair basic Ig folding [13]. Reagents specific for the products of the six known human VH gene families have also been generated. Putnam and coworkers originally divided the available V, sequences into three Subgroups based on available protein sequences [141, but subsequently these V, Subgroups have been found to be encoded by V, genes that are arranged into at least six distinct families on the basis of DNA sequence homology [15]. The polypeptides belonging to the protein VHI Subgroup are encoded by the vH1 and vH5 families, the protein VHII

CONVENTIONAL AND UNCONVENTIONAL B CELL ANTIGENS

59


Subgroup consists of the products of the vH2, vH4 and V,6 families and the protein VHIII Subgroup derive from the V,3 family. By definition, the families within each subgroup are more similar to each other than to members of other Subgroups, as presumably the gene members of a Subgroup share a common primordial ancestor. In Table I are compiled the framework sequences which have been found to be representative of the vast majority of germline V, and VH genes. Extensive experience has now documented that these specific anti-peptide reagents can identify the VH and V, family origins of more than 95% of Ig from cell lines and lymphoproliferative syndromes (>120 monoclonal samples) (see Fig. 1) [6-101 and G. Silverman unpublished observations).

VARIABLE REGION RESTRICTION IN HUMAN ANTIBODY RESPONSES TO THE CAPSULAR POLYSACCHARIDE OF HAEMOPHILUS INFLUENZAE, TYPE B Due to its clinical relevance, the antibody response against the capsular PS of Haemophilus injuenzae,type b (Hib), has been the best structurally characterized of all human antigenspecific responses. Hib is a leading cause of meningitis in infants, and it is also responsible for other forms of invasive disease [16]. Capsular polysaccharides (PS) are known virulence factors for these pathogenic bacteria, and antibodies against these immunodominant structures are usually protective [17,18]. PS vaccines have been shown to generally provide broad clinical efficacy [ 191, however many individuals, especially certain ethnic groups, such as native Americans, are unable to mount adequate responses to PS antigens and are at increased for these infections [17, 201. In most populations the peak incidence of Hib meningitis and bacteremia is between 6 to 15 months of age, and susceptibility correlates with a period of anergy to Hib PS and low or absent circulating levels of protective antibodies [ 181. Insel et al. documented the restricted heterogeneity of antibodies induced by Hib PS by

TABLE I Human Variable Region Specific Peptide Sequences Peptide name VH sequences VHI-FRI VH2-FR 1 VH3-FR 1 VH3-FR 1a VH4-FR I VH4-FR 1a VH5-FR3 VH6-FR 1 VK sequences Vkl -FRI Vk2-FRI Vk3-FRI Vk4-FRI

Specificity

Site

Amino acid sequence

First framework region First framework region First framework region First framework region First framework region First framework region Third framework region First framework region

AEVKKPGASVKVSC PALVKATHTLTC GGLVQPGGSLRLSC GGLVKPGGSLRLSC PGLVKSETLSLTC AGLLKPSETLSLTC acetyl-STAYLQWSSLKASDC VKPSQTLSLTC

First First First First

SLSASVGDRVTITC TQSPLSLPVTTGGC SLSLSPGERATLSC SLAVSLGERATINC

framework region framework region framework region framework region

All sequences have been derived from variable region protein sequences, with carboxy terminal cysteines added in certain instances, to aid coupling to a carrier. Except for the VH5-FR3 reagent, the sequences and the reactivities of the anti-peptide sera have previously been reported [S-7, 9, 291.


G. J. SILVERMAN

wi VHS m w w w Vw I

I

n

n

VH3

PM

I U

m

VH1 VH5 VHZ vH( VH4 VHI YHB VH3 QM I

I

"

I

U

U

Iu

FIGURE 1 Binding specificities of antisera to V, family framework regions. Proteins are identified by V, family from comparison of H chain sequence, and are clustered into related protein V, Subgroups. Replicate immunoblots of the seven monoclonal IgM proteins and one monoclonal IgG protein, CESS, and pooled polyclonal IgM from normal adult donors. About one microgram of protein was loaded per lane and H and L chains separated under reducing conditions. V, family specific antisera are directed against first framework (FR1) sequences, and subsets within these families are identified based on identification by anti-peptide antibodies to second hypervariable region sequences (HV2). Reactivity was detected by incubation with 12sI-proteinA, with subsequent exposure of autoradiograms overnight at -70°C. In other studies the primary amino acid sequence dependence of binding specificity of these anti-peptide antibodies has been demonstrated, based on blocking of immunoblot reactivity after a serum is preincubated with immunizing peptide, but not after preincubation with peptides taken from other V gene families (not shown). Certain V,4 and V,6 chains share FR sequences, but can be distinguished with hypervariable region directed reagents.

isoelectric focusing studies [21,22]. Later, Lucas characterized a cross-reactive idiotype on anti-Hib PS antibodies, which provided the first evidence that antigen-specific antibodies from outbred human populations might share V region structural features [23]. To investigate the diversity within these antibodies, Nahm and coworkers have purified circulating IgG anti-Hib PS antibodies to monoclonality and determined the protein sequences of the V regions [24-271, and more recently Carroll and coworkers have determined the V region sequences expressed in a panel of anti-Hib heterohybridomas [28] (reviewed in other articles in this issue). We sought to extend these findings, by using a different approach to examine the genetic and structurally origins of the range of distinct clones that are concurrently expressed in a circulating response, in order to make comparisons between different time points and between different individuals.

V, USAGE IN ANTI-HIB PS ANTIBODIES In collaboration with Dr. A. Lucas (Oakland Children's Hospital, Oakland CA) we have recently described the pre- and post-immunization responses of 10 normal adult volunteers who were immunized with 25 mcg of purified Hib PS [29]. Aliquots of post-immunization sera were filtered and incubated with 2 mcg of Hib PS, which uniformly caused the



61

precipitation of Ig, but there was little or no Ig precipitated from pre-immunization samples. Radioimmunoassay of anti-Hib PS antibody content of serum samples was performed before and after PS precipitation, and the amount of antibody depleted by the precipitation correlated with signal intensity of the immunoblotted anti-Hib PS antibodies. Furthermore, prior incubation of post-immunization samples with capsular PS from other bacteria did not interfere with subsequent precipitation with Hib PS. The anti-Hib PS antibodies from ten adult volunteers were characterized with the panel of anti-peptide antibodies, and all subjects were found to have a major induction of V, derived L chains in their anti-Hib PS responses (Fig. 2), while only 3/10 individuals had significant populations of A isotype L chains. Corroborating earlier studies [25, 261, the induced responses from all ten individuals also included VK2derived L chains. In addition, based on the reactivity with an anti-peptide antibody that recognized the exact sequence in the second hypervariable region encoded by the VK2 germline gene, A2, the majority (6/10) of individuals were found to use L chains related to the A2 germline gene in their anti-Hib PS responses. All post-immunization responses were also shown to include VK3derived L chains, and 7/10 anti-Hib PS responses also had a minor subset of V,1 derived antibodies. Taken together, our analyses reiterated past reports of V, dominance in anti-Hib PS responses in adults [30] and we also confirmed that VK2gene, A2, is routinely used in these responses. Moreover, these data also indicated that within each anti-Hib PS response, clones with L chains from at least three V, families were concurrently expressed.

V, USAGE IN ANTI-HIB PS ANTIBODIES VHusage was found to be highly restricted in anti-Hib PS responses. Immunization induced IgM, IgA and IgG anti-Hib PS antibodies in all ten subjects, but the dominant responses were all derived from the V,3 family. In fact, in four subjects the anti-Hib PS response contained only V,3 derived H chains. However, we also found that the anti-Hib PS antibodies from several individuals included minor subpopulations of H chains derived from an additional V, family, either the vH1 and V,4 families. This pattern of VH region usage was novel, and has not previously been reported in a human anti-PS response. Strikingly, using the same analytic technique anti-tetanus antibodies from three normal adult volunteers were found to utilize L chains from V, and the four V, families, in association with H chains from at least the three major V, families (VHl, vH3, and V d ) [G. Silverman, unpublished observation]. Therefore, in contrast to the V region restriction common to anti-Hib PS antibodies there is no evidence of restriction in anti-tetanus responses, which have the same V region distribution as unfractionated Ig. And, the V, family usage in anti-tetanus responses was roughly equivalent to the distribution of V gene rearrangements detected by in situ hybridization studies of unfractionated adult peripheral B cells-the vast majority utilize vH1, vH3 and vH4 genes, while vH2, vH5 and V,6 genes each represent very small subsets of unselected peripheral blood mononuclear cells or are undetectable [31, 321. Therefore, in contrast to adult anti-tetanus toxoid responses, which are polyclonal and without V, or V, family restriction, anti-Hib PS responses are oligoclonal and associated with a VHrestriction that is greater than V, restriction, in which there is a preferential VH3 usage. Speculatively, these different patterns may be due to molecular selection of certain restricted sets of Fab by Hib PS and/or due to the limited number of epitopes on Hib PS.

G. J. SILVERMAN

62


JN AW SS CA I g G ~ l + I I - + I I - ~ +

I

g

JN

G

U AW

u SS

U

CA

CS

DTNFPTHFDF + + + +

UU DT NF P i HF DF CS

FIGURE 2 Light (L) chain variable region diversity in circulating anti-Hib PS antibodies. Replicate blots are displayed with the L chain bands from the blots used in Figure 2. The a n t i c , and anti-C, reagents identify sequences in the constant regions of kappa and lambda L chains, respectively. V, family specific reagents identify diagnostic sequences in the first framework regions (FRl), and the anti-VK2-HV2a identifies a diagnostic sequence in the second hypervariable region of the V,2 gene, A2. As in Figure 2, only two patients have detectable anti-Hib PS antibodies prior to immunization (-). For SS, these L chains are predominantly VK3-derived,and for CS they are VJ-derived. Of the ten individuals, six have L chains identified by the anti-VK2-HV2a reagent that was derived from the second hypervariable region of the V,2 gene, A2 gene. This antibody does not react with unfractionated IgG, or IgA and IgM (not shown). Anti-Hib PS antibodies derive from the V,1, V,2, and V,3 families, but not V,4 (not shown). Three individuals also have a major population of anti-Hib PS antibodies with A L chains (AW, CS and DF). (taken from [29], with permission).

HUMAN ANTIBODIES TO THE CAPSULAR PS OF HAEMUPHILUS INFLUENZAE, TYPE B PREFERENTIALLY USE TWO TYPES OF VH3 H CHAINS In a series of complementary studies we also found that anti-Hib PS antibodies were derived from limited V,3 subsets. A panel of reagents were generated against second hypervariable region sequences from a highly restricted repertoire of V, rearrangements isolated from a



63

130 day fetal liver cDNA library [33], and reagents derived from rearrangements of the germline vH3 gene, 9-1 gene [15], were shown to identify the alpha and/or gamma H chains of the anti-Hib PS antibodies from 4/10 individuals (Fig. 3). In addition, the antibody specific for the second hypervariable region of the products of the germline VH26 gene, a vH3 gene rearranged in this library, acted with H chains in the anti-Hib PS responses from all 10 individuals studied. In contrast, the anti-Hib PS antibodies were recognized neither by the serologic reagents against the hypervariable region sequences from two other V,3 genes, 56P1 and 38P1, nor from any of the other members of this V, fetal repertoire. These data suggest that immunization non-stochastically induces restricted IgG responses with dominant clonotypes that are structurally related to the V,9-1 and V,26 genes. The notion that these genes commonly encode for dominant clonotypes has been strengthened by the recent report that genes highly homologous to the 9-1 germline vH3 gene encode the V, region of three IgA and one IgG anti-Hib PS B cell heterohybridoma lines derived from three normal adult subjects, while the VH26gene had the closest homology to an additional IgM cell line from a four year child [28]. Comparison of the V, region diversity of anti-PS antibodies of different H chain isotypes has also been informative. We found that within a response both the IgG responses and the IgM responses were predominantly or completely from the vH3 family. In contrast, in two

JN

I

AW g

SS

CA

CS G

DTNFPTHFDF + +~ + + +

~ l

JN g

AW G ~

SS ~

CA C ~ 7

CS l

DTNFPTHFDF

+~

+ +~ + +

antiVH3-FRia VH3-FR1

peptide antiVH3-HV2a VH3-HV2b

peptide antiVH3-FR3a anti VH3-FR1 VH3-FRia

peplide anriVH3 MV2b VH3-HV2c

peplide

FIGURE 3 Heavy chain variable region diversity in circulatiing human antibodies to Huemophilus Injluenzue type b (Hib) polysaccharide (PS). Replicate blots included controls of polyclonal IgG (shown) and control monoclonal IgMs (not shown). Of five preimmunization samples (-), precipitating anti-Hib PS antibodies were detected in two individuals (SS and CS), but not in the other three. VH3-derived H chains were the major populations in all post-immunization (+) precipitating antibodies. Vd-derived antibodies were detected in four samples, but were smaller sub-populations. Patient SS had VH4-derived antibodies prior to immunization, but VH3-derived antibodies were later induced. VHI-derived antibodies were detected in three cases, but represented significant populations in only two samples (CS and DF). VH2, VH5 or VH6-derived H chains were not detected (not shown). Many VH3-derivedH chains were also identified by the VH9-1derived antibodies (VH3-FRla. VH3HV2a and VH3-FR3a) and the VH26-derived antibody (VH3-HV2h). In addition, anti-peptides to three VH1,two VH2,two VH3,six VH4, one VH5and one VH6hypervariable region sequences were also non-reactive with any of the anti-Hib PS antibodies (not shown). Anti-Hib PS antibodies were purified by incubation of 2 mcg of purified Hib PS for 1 hr at 37°C then overnight at 4°C with 1 ml of serum that had been filtered, and centrifuged to remove aggregates. Complexes were then washed twice with 1% bovine serum albumin 1% tween 20 in PBS, then dissolved in 8M urea 2% SDS, prior to polyacrylamide electrophoresis and immunoblotting (taken from [29], with permission).


64

G . J. SILVERMAN

instances in which subsets of gamma H chains from the vH1 and V& families were detected, concordant IgM subset from these families were not detected. We also found that IgG and IgA clones that are coexpressed in the same response may have different clonal origins. In one of the sera (AW), based on serologic markers the predominant clone in the IgA response derived from a gene related to the V,9-1 subfamily. However, the IgG antibodies in this specimen were identified only by the antibodies specific for the VH26 related subfamily. These data are consistent with a model of human anti-PS responses, in which several founder clones with independent V, rearrangements can contribute to an antigen-induced response. In one subject (SS) the pre-immunization level of anti-Hib PS antibodies was adequate to allow analysis of V region diversity. Significantly, the antibodies from this serum contained only VH4-derived antibodies, while immunization induced the new appearance of V,3derived anti-Hib PS antibodies. Additionally, the pre-immunization antibodies had a significant population of VK3-derivedantibodies, but immunization predominantly induced VK1and V,2 antibodies. As immunization induced a proportionately greater expansion and/ or differentiation of V&v,1 and V,3-VK2 expressing anti-Hib PS clones than the V&VK3 anti-Hib PS antibody producing clone(s), this observation may be reflective of differences either in intrinsic affinity or specificity which affect in vivo clonal competition. Our findings in anti-Hib responses are consistent with animal models of B cell responses to conventional antigens, but they are distinct from those associated with the alternative binding site of staphylococcal protein A.

STAPHYLOCOCCAL PROTEIN APROTOTYPE FOR A HUMAN B CELL SUPERANTIGEN Staphylococcal protein A (SPA) is a bacterial membrane protein, which possesses, in addition to its well described Fcy-binding sites, sites that are bound by the Fab portions of some IgM, IgA and IgG and IgE [34-361. Early studies provided evidence that these “alternative binding sites” are structurally and functionally distinct from the Fcy binding site, because chemical modification of SPA by iodination can selectively eliminate the Fc binding activity without altering the specificity for Fab [l, 371. In addition, Fc fragments from human IgG cannot inhibit IgM binding, while this activity can be inhibited by F(ab’)* from IgG [37]. Vidal and Conde demonstrated that this activity was associated with the V region of the H chain of a human monoclonal IgM [38], and SPA also binds Ig from several other mammalian species [39]. The structural basis of the SPA alternative binding site has been investigated by Drs. E. Sasso, M. Mannik and colleagues (University of Washington, Seattle). They demonstrated that 4 of 12 human monoclonal IgM rheumatoid factors (RF) (anti-IgG autoantibodies) bound to SPA [40]. In collaboration with this group, by radioimmunoassay we subsequently demonstrated that 6/16 purified monoclonal IgM RF bound SPA by radioimmunoassay, while 4/8 monoclonal IgM without RF activity also bound SPA [41]. Therefore, SPA binding was not exclusive to IgM with conventional RF binding activity. To determine whether there was a correlation between V gene family usage and SPA binding activity, we applied the previously described VHand V, family specific anti-peptide antibodies. Significantly, we found that all ten monoclonal IgM with SPA binding activity contained vH3 heavy chains, while the non-binders included seven V,1 IgM, six V& IgM and one other V,3 IgM. However, in contrast to the association of V,3 usage, SPA binding



65

was demonstrated to be independent from V, family expression, because SPA binders used L chains from at least four V, families. The V, composition of SPA binding and nonbinding fraction of polyclonal Ig from normal adult donors was next assessed. Polyclonal IgM, IgA or IgG F(ab’)2 were each separated into binding and non-binding fractions by passage over SPA-affinity columns [ l , 411, and the SPA binding fractions were all found to be greatly enriched in Ig reactive with the VH3 family specific reagent, and depleted in V,1 and V,4 Ig (Fig. 4 and Fig. 5). Therefore, these studies suggest that the association of SPA binding with vH3 usage was not limited to the Ig repertoire expressed in lymphoproliferative syndromes, but that it is common to the physiologic adult repertoire. In corroboration, Shokri et al. have subsequently reported that lymphoblastoid B cell lines that express a VH3 associated marker preferentially bind SPA [42], In more recent work, we have extended our studies of SPA binding to include monoclonal IgM and IgG proteins that are representative of all known V, families. In these studies we have also compared the binding activity of purified SPA, with SPA that has been modified by hyperiodination to destroy Fc binding activity [41]. To detect binding, both forms of SPA were biotinylated for use in an enzyme-linked immunoassay, and these new data are displayed in Figure 6, and are compiled with earlier data in Table 11. Among the monoclonal IgM we found that modified SPA (without Fc binding activity) was bound by 15/16 vH3, but none of the 7 V,1, 7 vH4, 1 vH5 and 1 V,6 proteins tested [41]. And, among monoclonal IgG we found that modified SPA was bound by 216 V,3, but none of the 4 VH1,l V,2, and 1

A

B

C

FIGURE 4 Immunoblots of electrophoretically separated H chains from pooled polyclonal IgM from normal adult donors. Total IgM (A) was passed over a sepharose-protein A (SPA) column, and divided into binding (B) and non-binding fractions (C). Replicate blots are displayed in which serial dilutions are displayed, in which the first lane represents 2.5 mcg, then 1.25 mcg, then 0.625 mcg, then 0.312 mcg. Blots are reacted with peptide induced serum to a sequence specific for the )L contant region (Anti-mu), or for antiserum specific for first framework regions of the largest families in the three protein VH subgroups; anti-V,I Subgroup (anti-VHI-FRI), anti-V,II Subgroup (anti-VH4-FRl), and anti-V,III Subgroup (anti-VH3-FRI). The IgM which was bound to protein A-sepharose column is shown to be greatly enriched in H chains that are identified by the V,3 family specific reagent. (taken from [41], with permission).

G. J. SILVERMAN

66

Total I9A

Flow Through

Eluant


Eluant

1 2 3

1 2 3

Flow Through

---

~~

1 2 3

Total I9G F(abh

1

2

3

1

2

3

1 2 3

FIGURE 5 Immunoblot of electrophoretically separated H chains from pooled polyclonal IgA (left) and polyclonal IgG F(ab’), (right) from normal adult donors. Comparable to Figure 2, the left three lanes of each panel show unfractionated Ig in serial dilutions, with the first lane containing 2.5 mcg, then 1.25 mcg, then 0.625 mcg Ig. The middle lanes contain the same amounts of Ig that bound to the protein A column (eluant), while the right lanes contain Ig that did not bind to the column (flow through). Replicate panels were reacted with anti-peptide to constant region sequences (anti-a or anti-y), or antisera to first framework, family specific sequences for the VH1 family (anti-VHI-FRl), VH4(anti-VH4-FRl), and VH3(anti-VH3-FR1). Both of the IgA and IgG F(ab’), fractions which bound to the SPA columns are highly enriched in VH3 H chains (taken from [I], with permission).

VH4 proteins tested. As expected, non-modified SPA was bound by all IgG proteins, however after hyperiodination, modified SPA was bound only by certain monoclonal IgM or IgG from the VH3 family. These data demonstrate that examples of Ig from the V families, V,1, vH2, vH4, vH5 and V,6, did not express SPA binding capacity, because SPA binding was limited to certain Fab that contain vH3 H chains ( ~ 2 P , < 0.001). Moreover, SPA binding was present in Ig from at least four V, families, including both kappa and lambda L chains. In addition, SPA binding activity was not restricted to antibodies with a single conventional antigen binding activity. Significantly, a much larger proportion of the vH3 IgM (15/16) than vH3 IgG (216) bound the modified SPA ( ~ 2 P, < 0.005), which may be due to differences in VH region expression in these IgG, and possibly because replacement mutations in IgG V, regions interfere with SPA binding. In Table 111 are compiled all available V, sequence data from monoclonal Ig that bind to

-

IgG Fine* Heb* Hou* Long* Ger*

-

+ +

-

+ + + + + + + + + + + + + + +

SPA

NT

A

K1

A

K3

NT

K4

A

K4

K1

K3 K3 K4

K1

K3 K3 K3 K3

K3

K1

K1

K1

V,

Cia*

Cor Far Gre Les Odo* Ore Ple

-

-

-

-

K1

K3

K3 K3 K3 K3

A

-

K3

-

V,

-

SPA

vH4+

A224* -

SPA

'H5

X

V, L16*

-

SPA

VH6

A

V,

Data compiled from a previously reported study, in which SPA reactivity was demonstrated by radioimmunoassay [l], and later studies, in which SPA binding was performed by immunoassay (see Figure 7). Variable region family was determined either by sequence analysis, or based on reactivity with a panel of V family specific anti-peptide antibodies, +Certain 1gM proteins initially identified as members of protein Subgroup 11, have subsequently been demonstrated to derive from the V d family [9]. The VH region sequences of several VH3 proteins are displayed in Table III. The panel is biased toward V,3 proteins, because many of the Ig are rheumatoid factors derived from patients with mixed essential cryoglobulmemia. NT,not tested, The idiotypic reactivities and source of many of these proteins have previously been reported [7,9].

A

Cal* Do* Fitz* Mag*

A

K1

-

Joh

4B4* 591*

Buc Cha Chr Dau* Erik Glo Hea Kim 4.6* Lay Pom Riv Sim 18/2*

SFL*

CeSS*

V,

V"3

Structural and Function Properties of Monoclonal Ig Proteins

A

K2

K3

-

-

-

-

K3 K3 K4 K3 K3 K3

-

IgM And Bor Gil Jan Kas Pal Sie

SPA

SPA

V,

VH2

VH1

TABLE I1



68 G . J. SILVERMAN


CONVENTIONAL AND UNCONVENTIONAL B CELL ANTIGENS 69

18/2

IgGFc IgGFc IgGFc IpGFC

DNA 9-1 sm 9-111 DNA

V826

++

+++

++

++++ ++++

V"1 vg2

SIE CE-1 LES

Vn5 V H ~ Poly

IgGFc Poly

[Ink

IgGFc IgGFc IgGFc

-

-

-

-

CDRl

cDR3

The amino acid sequences of heavy chains reactivc with modified-SPA by immunoassay arc displayed. The IgM proteins 484. IR/Z, and KIM4.6 dcrivc from B cell lines. and thc gcrmlinc VH Kgments from which they derive. arc displayed f67.68.69). The IgM proteins. LAY. POM. RIV. BOR. KAS AND SIE.arc from the scrum of patients with monoclonal cryoglobulincmia 170.71). and Ihe lgG S R was purified from the circulation of a . patient w i t h hypcrgammaglobulinemia. The amino acid scqucnccs of heavy chains cvaluntcd for binding to modified-SPA by immunoassay arc displaycd. The single letter code for aminu acid rcsidues is used, and U represents a pyrrolidonccarborylic acid rcsidd. S IS undetermined. and is a space. V H regions ~ are grouped above. and all other families arc listcd helow. The IgM proteins 464, 18/2. K1M4.6 and L16 directly derivc from the gcrmlinc configuration of VH gene segments. and these genes are listed under GL V H - . The conventional antigen binding activity of VH3 antibodics is listed under A S + . Rclntivc rcnctivity with modified-SPA is dcplctcd as OD405 0.100-0.400 +. 0.4014.800 ++. 0.801-1.200 +++. > I 2 0 0 ++++. NT. not tcstcd.

L16

A224

VHl

XAS

Vg4

Vgl

BOR

Non V H ~heavy chains

RIV SFL

POI4

LAY

KIM4.'

484

++ +

liudA!A

v ~ heavy 3 chains

%^&+

SPA Reactivity with Human VH Regions

SPA Reactivity with Human VH3 Proteins

TABLE 111




71

modified SPA, to determine whether there are conserved structural motifs. Sequence analysis confirms that the V, regions of SPA reactive Ig share considerable homology in the framework regions, as is expected in vH3 H chains. However, the composition of the hypervariable regions vary greatly, and these H chains express V, regions that represent rearrangements of at least four different vH3 germline genes. Therefore, unlike antibodies with a conventional antigen binding activity, which often share hypervariable region sequence motifs, these limited data did not detect shared motifs in the hypervariable region that correlate with SPA binding, and documented that many human vH3 genes can encode for SPA binding. The use of SPA as a B lymphocyte surface marker has also been examined. In a representative study displayed in Figure 7 , we found that biotinylated modified SPA (without Fc binding activity) bound to a subset of the tonsilar mononuclear cells that expressed the B cell, CD19 marker, but did not bind to cells identified by the T cell, CD3 marker. We also found that a significant proportion of B cells were identified by the SPA reagent whether they expressed IgM, IgD or IgG on the membrane surface. In subsequent studies we have found that a greater proportion of IgM than IgG bearing B cells bind modified SPA (that is devoid of Fcy binding activity) (G. Silverman and S. Wormsley, unpublished observation). Therefore, based on the identification of large subsets of both IgM and IgG cells, these studies demonstrate that modified SPA (without Fc binding activity) is an effective B cell phenotypic marker, presumably due to the recognition of surface vH3 Ig. To determine whether SPA may induce VH restricted lymphocyte mitogenesis, in our laboratory Dr. J. Crowley compared the effect of stimulation of purified neonatal B cells with either Epstein Barr virus or an extract of Staphylococcal aureus, cowan strain (SAC), which contains SPA (previously unpublished). The Ig composition of these supernatants was then assessed by measurement of the levels of two well characterized V, associated idiotypes-a subset of VH1 products that is identified by the murine monoclonal antiidiotype, G6 [7], and a subset of V,3 products that is recognized by the murine monoclonal anti-idiotype, B6 [43]. These studies revealed that these “polyclonal B cell activators” stimulate different B cell populations, and that the supernatants of SPNSAC stimulated B cells are comparatively enriched in Ig that bear a vH3 associated idiotype (Table IV). However, selective B cell stimulation required the use of crude SAC extract that includes SPA because purified recombinant SPA alone does not induce in vitro B cell proliferation [44,45]. This observation is consistent with the two signal model of B cell activation [46], in which SPA induces the preferential stimulation and secretion of V, restricted B cells, presumedly due to selective cross-linlung of surface Ig, while other factors in SAC, such as T cell mitogens, likely provide the second signal for in vitro B cell stimulation [44].

DISCUSSION The human antibody response to the carbohydrate determinant, Hib PS, is dominated by a preferential usage of H chains from a limited subset of the large vH3 family in association with L chains from diverse V, families. The diversity of antibody clones in the circulating responses was assessed using antibodies isolated by direct precipitation with Hib PS antigen, which makes it likely that this pattern of restriction is also representative of physiologic interactions responsible for clearance of encapsulated bacteria from the bloodstream. Although these immunochemical determinations could not identify the exact VH

G. J. SILVERMAN


12

FIGURE 7 Microfluorimetry of tonsilar mononuclear cells with biotinylated modified SPA. Lower left panel displays the relative proportion of B cells and T cells, identified by the CD19 and CD3 markers, respectively (Becton-Dickinson, Mountain View CA). Mononuclear cells were dissociated from human tonsilar lymphoid tissue, washed in RPMI 1640, separated on Ficoll gradient, then washed and frozen in DMSO/fetal bovine serum in liquid nitrogen until used. Studies were performed on a FACSCAN with lymphocyte gating. Quadrants were set using either tagged isotype controls (Becton-Dickinson), or tagged avidin (Southern Biotechnology, Birmingham AL). For double staining studies, samples were first incubated with 0.25 mcg/ml of biotinylated modified SPN106 cells, then washed and incubated with directed labelled CD antibody or anti-human IgG or IgD diluted in 1% human IgM, then washed and incubated with labelled avidin. For double staining using direct labelled anti-human IgM, a diluent of 1% human IgG (Calbiochem) was used.


13

TABLE IV V, Subset Expression after in v i m Stimulation SPA

EBV

% B6-IgM % G6-IgM

30.0 2 8.4 1.4 2 0.6

10.6 ? 4.0 5 . 5 2 2.1

Ratio (%B6-IgM/%G6-IgM)

21.4

1.9


Mononuclearumbilical cord supernatants were harvested after 8 days of SPNSAC stimulation and 28 days of EBV stimulation. The G6 and B6 idiotypes were measured by immunoassay using standard curves with the monoclonal IgM, Sie and Glo, respectively [7,43]. All values determined in mcg/ml +/- SEM. Expression of the idiotypes on IgM after SAC/SPA and EBV stimulation are significantly different ( p < 0.05).

genes encoding these antibodies, based on the primary amino acid sequence dependence of the analytic reagents these studies did demonstrate that dominant antibody clones were derived from genes that are highly homologous to the germline VH9-1 and VH26genes. In support, five anti-Hib PS B cell lines have recently been demonstrated to use V, genes that are highly homologous to these germline vH3 genes [28]. Significantly, available data suggest that the composition of the induced repertoire is not greatly affected by the use of different forms of the Hib PS immunogen. Lucas et al. demonstrated that both purified Hib PS and the diphtheria protein conjugate induced comparable expression of the V, associated Hib-id1 cross-reactive idiotype [23, 471, and rearrangements of the V,9-1 related gene segments have been reported in B cell lines isolated from adults immunized with either of these vaccines (281. The mechanisms responsible for the preferential usage of gene segments from the VH26 and V,9-1 subfamilies in anti-Hib PS antibodies requires further elucidation. This preferential usage may reflect a greater representation of B cells with these rearrangements in the preimmunization repertoire. Alternatively, certain B cells with rearrangements of these V gene segments may have an enhanced Hib PS binding capacity that leads to a clonal advantage during antigenic selection. By comparison the alternative binding site of SPA represents a very different type of antigen binding function for the human immune system. In contrast to the anti-Hib PS response which is antigen-induced and non-exclusively associated only with certain VH3H . chains, a large proportion of polyclonal Ig can bind SPA, independent of prior antigenic exposure. While V, sequence analysis has suggested that antibodies related to the VH9-1 and V,26 subfamilies dominate anti-Hib PS responses, many germline vH3 genes can encode for SPA binding activity. Furthermore, unlike antibodies to a conventional antigen, initial studies suggest that Ig that bind SPA may not share conserved hypervariable region sequences, and this activity exclusively correlates with Ig with vH3 family specific sequences. Significantly, sequences in the first and third framework regions of members of the V,3 family are extremely conserved within and between species [48-511, and framework residues have been shown to be critical for the reactivity of a Mls superantigen with Vp8.2 T cell receptors [52]. These framework regions are interposed and highly solvent exposed (Fig. 8; Color Plate I) [53], and because they correlate with SPA binding we have proposed that portions of these framework regions may have a direct role in SPA binding [l]. The specific interaction of SPA with a large proportion of human B cells evokes the general concept of a superantigen, and by analogy to the documented properties of Vp restricted T cell superantigens we have formulated criteria for the identification of B cell superantigens: 1) Superantigen binding should be expressed by a large proportion of nonimmune Ig, which are restricted by their V, usage. ii) Superantigen binding should be

G.J. SILVERMAN


74

FIGURE 8 The Fab region of V,3 KOL is displayed with the regions of interest differentially colored: the hypervariable regions of the heavy and light chains are in white: the heavy chain is in blue, the light chain is violet: FRl residues (6-24) are in yellow and FR3 (67-85) are in red. Note that the FRI and FR3 residues are solvent exposed and form a bridge between the hypervariable region and CHI domain of the heavy chain (kindly provided by Drs. P: M. Kirkham and H. W. Schroeder, Jr.) (adapted from [%I). (See Color Plate I)



75

present on antibodies that also express diverse conventional antigen binding activities. iii) Superantigens should bind both isolated Ig, and Ig on the surface of B cells. iv) Superantigen binding should be useful for the selection and stimulation of V, family restricted B cell populations. The notion that SPA may possess B cell superantigenic properties is also supported by a report that the V, sequences of murine B cell lines that bound SPA were derived from several members of the 5606 and S107 families, which share considerable framework homologies with the members of the human vH3 family 1541. In addition, based on descriptions of “non-immune” interactions with Ig Fab regions [55-57], we speculate that other self and microbial products are also candidate B cell superantigens, but their further classification will require molecular characterization of the binding Eg. The appreciation that B cell superantigens are likely ubiquitous within the environment has important implications for our understanding of the development of human immune response. The restriction of the V, repertoire during early ontogeny has been suggested to be due to anti-self and idiotype-anti-idiotypic interactions, and preferential rearrangements [58-61], but following birth these mechanisms are likely less important than the influence of external ligands. Bos et al. have demonstrated the impact of bacterial colonization on immune development, as germfree mice, fed filtered diets from birth, have only a fraction of the circulating IgG levels of their non-germfree littermates [62]. Because exposure to Staphylococcus aureus is universal, we postulate that unconventional antigens, such as SPA, influence the expressed B cell repertoire. In particular, SPA (or equivalent B cell superantigens) may contribute to the dominance of B cells with V,3 rearrangements within the human immune response, independent of clonal selection by conventional antigens. This proposed mechanism may also present an evolutionary advantage, because vH3 related genes in both human and murine systems are preferentially used in conventional anti-bacterial antibodies [28, 61-66]. Following stimulation and clonal expansion of B cell clones by a VH specific B cell superantigen a range of potential fates may hypothetically occur (illustrated in Fig. 9). If superantigenic influences are part of normal B cell physiology (and pathophysiology), these agents may contribute to the acquisition of protective immune responses, B cell memory or perhaps to the development of neoplastic or autoimmune processes. However, it remains to be defined whether these potential effects are completely analogous to those of T cell superantigens. Of particular interest will be the determination of whether clonal deletion may occur under the influence of an endogenous B cell superantigen, or from chronic exposure to an exogenous B cell superantigen. In summary, recent analyses of the human IgG antibody repertoire to the bacterial capsular polysaccharide, Haemophilus injuenzae, type b, have documented that these responses are structurally restricted to the common usage of a very small number of VH3 germline genes. These data suggest that exposure to Hib PS induces the production of antibodies with similar V, rearrangements throughout outbred human populations, while many different types of V, regions are expressed with these H chains. Related genes appear to also be used in certain anti-pneumococcal responses [65,66]. Therefore, we hypothesize that genes from these two V,3 subfamilies may have been selected for, during evolution, due to their utility in protective anti-bacterial responses. In contrast, the VHrestriction in Ig that bind the unconventional antigen, SPA, does not appear to be limited to a small number of the gene elements within the large V,3 family. Taken together, data from a variety of binding and stimulation studies suggest that the unconventional V, binding activity of SPA may represent a prototypic example of a B cell superantigen. Furthermore, based on these findings we suggest that the influence of B cell


76

G. J. SILVERMAN

FIGURE 9 Model of clonal fates of B cells expanded by exposure to a VH3 superantigen. Exposure to an exogenous superantigen stimulates and expands most B cell clones that express surface Ig with rearrangements of members of the VH3 family. Subsequently, these clones numerically dominate the B cell compartment. Hypothetically, B cells from clones with intrinsic affinity for exogenous antigen (VH3A),or self ligand (VH3B)may then be stimulated and selected by exposure to these conventional antigens. Due to clonal expansion there is a greater chance that a mutational event may contribute to autonomous growth of certain B cells (VH3C), with the subsequent outgrowth of a neoplastic population. Alternative fates may include the development into memory cells, or perpetuation by a sequestered carbohydrate antigen, or in the absence of continued stimulation clonal deletion due to apoptosis may occur.

superantigens on antibody repertoire development should be further investigated, due to the possible implications for in the creation of therapeutic modalities to modulate and augment the human immune response.

Acknowledgments I appreciate the assistance, advice and encouragement of my collaborators, E. H. Sasso, M. Mannik, A. H. Lucas, and colleagues,!I M. Kirkham and H. W. Schroeder, Jr. I am particularly indebted for the continued support of Dr. D. A . Carson. Expert technical assistance was contributed by E. Rapaport, and flow cytometry support was performed by K. Cox at the GCRC Core Lab of Scripps Clinic. I am the recipient of a NIAID-Physician Scientist Award, and this work was supported in part by NIH AI00866, AR25443 and RR00833.


77


References 1. Sasso, E. H., Silverman, G. J., and Mannik, M., J. Immunol. 147, 1877-1883, 1991. 2. Kipps, T. J., Fong, S., Tomhave, E., Chen, €? I?, Goldfien, R. D., and Carson, D. A,, Proc. Natl. Acad. Sci. USA 84, 2916-2920, 1987. 3. Humphries, C. G., Shen, A,, Kuziel, W. A., Capra, J. D., Blattner, E R., and Tucker, €? W., Nature 331,446449, 1988. 4. Chen, P. I?, Albrandt, K., Orida, N. K., Radoux, V., Chen, E.Y., Schrantz, R., Liu, E-T., and Carson, D. A,, Proc. Null. Acad. Sci. USA 83, 8318-8322, 1986. 5 . Silberstein, L. E., Jefferies, L. C., Goldman, J., Friedman, D., Moore, J. S., Nowell, €? C., Roelcke, D., Pruzanski. W., Roudier, J., and Silverman, G. J., Blood 78, 2372-2386, 1992. 6. Silverman, G. J., Carson, D. A., Solomon, A., and Fong, S., J. Immunol. Methods 95, 249-257, 1986. 7. Silverman, G. J., Goldfien, R. D., Chen, €?,Mageed, R. A., Jefferis, R., Goni, E, Frangione, B., Fong, S., and Carson, D. A., J. Clin. Invest. 82, 469-475, 1988. 8. Silverman, G . J., Goni, E, Chen, €? I?, Fernandez, J., Frangione, B., and Carson, D. A., J. Exp. Med. 168, 2361-2366, 1988. 9. Silverman, G. J., Schrohenloher, R. E., Accavitti, M. A., Koopman, W. J., and Carson, D. A,, Arthritis Rheum. 33, 1347-1360, 1990. 10. Silverman, G. J. and Carson, D. A., Eur. J. Immunol. 20, 351-356, 1990. 11. Seiden, M. V., Heuckeroth, R., Clevinger, B., McMillan, S., Lerner, R., and Davie, J. M., J. Immunol. 136, 582-587, 1986. 12. Chen, I? €?,Fong, S., Normansell, D., Houghten, R. A,, Karras, 1. G., Vaughan, J. H., and Carson, D. A,, J. Exp. Med. 159, 1502-1511, 1984. 13. Shlomchik, M. J., Litwin, S., and Weigert, M., The Influence of Somatic Mutation on Clonal Expansion. In:

Progress in Immunology, Edited by E Melchers, E. D. Albert, H. von Boehmer, M. F! Dierich, L. Du Pasquier, K. Eichmann, D. Gemsa, 0. Gotze, J. R. Kalden, S. H. E. Kaufmann, H. Kirchner, K. Resch, G. Rietmuller, A. Schimpl, C. Sorg, M. Steinmetz, H. Wagner and H. G. Zachau, Berlin: Springer-Verlag, 1989, pp. 415-423. 14. Kohler, H., Shimizu, A., Paul, C., Moore, V., and Putnam, E W., Nature 227, 1318-1320, 1970. 15. Berman, J. E., Mellis, S. J., Pollock, R., Smith, C. L., Suh, H., Heinke, B., Kowal, C., Surti, U., Chess, L., Cantor, C. R., and Alt, E W., EMBO J. 7, 727-738, 1988. 16. Sell, S. H. and Wright, €? E., Haemophilus influenzne. Epidemiology, Immunology, and Prevention of Disease. New York: Elsevier Science Publishing Co., 1982. 17. Siber, G. R., Santosham, M., Reid, G. R., Thompson, C., Almeido-Hill, J., Morell, A,, DeLange, G., Ketcham, J. K., and Callahan, E. H., N. Engl. J. Med. 323, 1387-1392, 1990. 18. Fothergill, L. D. and Wright, J., J. Immunol. 24, 273-284, 1933. 19. Peltola, H,. Kayhty, H., Sivonen, A., and Makela, I? H., Pediatrics 60, 730, 1977. 20. Ward, J., Brenneman, G., Letson, G. W., Heyward, W. L., and The Alaska H., influenzae Vaccine Study Group, N . Engl. J. Med. 323, 1393-1401, 1990. 21. Insel, R. A. and Anderson, I? W., J. Exp. Med. 163, 262-269, 1986. 22. Insel, R. A., Kittelberger, A., and Anderson, I?, J. Immunol. 135, 2810-2816, 1985. 23. Lucas, A. H., J. Clin. Invest. 81, 480-486, 1988. 24. Tarrand, J. J., Scott, M. G., Takes, I? A., and Nahm, M. H., J. Immunol. 142, 2519-2526, 1989. 25. Scott, M. G., Crimmins, D. L., McCourt, D. W., Zocher, I., Thiebe, R., Zachau, H. G., and Nahm, M. H., J. Irnmunol. 143, 4110-4116, 1989. 26. Scott, M. G., Tarrand, J. J., Crimmins, D. L., McCourt, D. W., Siegel, N. R., Smith, C. E., and Nahm, M. H., J. Immunol. 143, 293-298, 1989. 27. Scott, M. G., Crimmins, D. L., McCourt, S. W., Chung, G., Schable, K. E, Thiebe, R., Quenzel., E. M., Zachau, H. G., and Nahm, M. H., J. Immunol. 147, 4007-4013, 1991. 28. Adderson, E. E., Shackelford, €? G., Quinn, A., and Carroll, W. L., J. Immunol. 147, 1667-1674, 1991. 29. Silverman, G. J. and Lucas, A. H., J. Clin. Invest. 88, 911-920, 1991. 30. Ambrosino, D. M.. Greif, W.. Thompson, C., and Siber, G. R., J. Infect. Dis. 161, 922-925, 1990. 3 1. Guigou, V., Cuisinier, A.-M., Tonnelle, C., Moinier, D., Fougereau, M., and Fumoux, E, Mol. Immunol. 27, 935-940, 1990. 32. Zouali, M. and These, J., J. Immunol. 146, 2855-2864, 1991. 33. Schroeder, H. W., Jr., Hillson, J. L., and Perlmutter, R. M., Science 238, 791-793, 1987. 34. Langone, J. J., Adv. Irnmunol. 32, 157, 1982. 35. Harboe, M. and Folling, I., J. Immunol. 3, 471, 1974. 36. Inganas, M., Scand. J. Immunol. 13, 343-352, 1981. 37. Ramagnani, S., Guidizi, M. G., del Prete, G., Maggi, E., Biagiotti, R., Almerigogna, E, and Ricci, M., J. Immunol. 129, 596, 1982. 38. Vidal, M. A. and Conde, E I?, J. Immunol. 135, 1232, 1985.

G . J. SILVERMAN

78

Inganas, M., Johansson, G. 0.. and Bennich, H. H., Scand. J. Intmunol. 12, 23-31, 1980. Sasso, E. H., Barber, C. V,Nardella, A., Yount, W. J., and Mannik, M., J. Immunol. 140, 3098-3107, 1988. Sasso, E. H., Silverman, G . J., and Mannik, M., J. Immunol. 142, 2778-2783, 1989. Shokri, E, Mageed, R. A., Maziak, B. R., and Jefferis, R., J. Immunol. 146, 936-940, 1991. Crowley, J. J., Mageed, R. A., Silverman, G. J., Chen, F! F?, Kozin, E, Erger, R. A,, Jefferis, R., and Carson, D. A,, Mol. Immunol. 27, 87-94, 1990. 44. Schrezenmeier, H. and Fleischer, B., J. Immunol. Merhods 105, 133-137, 1987. 45. Person, U., Inganas, M., Smith, C. I. E., Hammarstrom, I., and Johansson, S. G. O., Scand. J. Immunol. 29, 39. 40. 41. 42. 43.


151-158, 1989.

Falkoff, R. J. M., Zhu, L. I?, and Fauci, A. S., J. Immunol. 129, 97-102, 1982. Lucas, A. H. and Granoff, D. M., J. Clin. Invest. 85, 1158-1166, 1990. Tutter, A. and Riblet, R., Proc. Natl. Acud. Sci. USA 86, 7460-7464, 1989. lbtter, A. and Riblet, R., Imrnunoglob. Superfurn. Genes 137, 106-115, 1988. Schroeder, H. W., Jr., Hillson, J. L., and Perlmutter, R. M., Inr. Immunol. 2 , 41-50, 1990. Kirkham, F? M., Mortari, E , Newton, J. A., and Schroeder, H. W., Jr., EMBO J. 11, 603-609, 1992. Pullen, A. M., Wade, T., Marrack, F?, and Kappler, J. W., Cell 6, 1365-1374, 1990. Satow, Y.. Cohen, G. H., Padlan, E. A., and Davies. D. R., J. Mol. Biol. 190, 593-604, 1986. Seppala, I., Kaartinen, M., Ibrahim, S., and Makela, O., J. Immunol. 145, 2989-2993, 1990. Bouvet, J.-I?, Pires, R., Charlemagne, J., Pillot, J., and Iscaki, S., Scand. J. Immunol. 34, 491-496, 1991. Eliasson, M., Andersson, R., Nygren, P-A., and Uhlen, M., Mol. Immunol. 28, 1055-1061, 1991. Erntell, M., Myhre, E. B., and Kronvall, G., Scand. J. Immunol. 17, 201-209, 1983. Vakil, M. and Kearney, J. E , Eur. J. Immunol. 16, 1151-1158, 1986. Vakil, M., Sauter, H., Paige, C., and Kearney, J. E , Eur. J. Itnmunol. 16, 1159-1165, 1986. Holmberg, D., Andersson, A., Carlsson, L., and Forsgren, S., Immunol. Rev. 110, 89-103, 1989. Coutinho, A,, Immunol. Rev. 110, 63-87, 1989. Bos, N. A., Kimura, H., Meeuwsen, C. G., de Visser, H., Hazenberg, M. F?, Wostmann, B. S., Pleasants, J. R., Benner, R., and Marcus, D. M., Eur. J. Immunol. 12, 2335-2339, 1989. 63. Briles, D. E., Forman, C., Hudak, S., and Claflin, J. L., J. Exp. Med. 156, 1177-1185, 1982. 64. Boswell, C. M., Irwin, D. C., Goodnight, J., and Stein, K. E., J. Immunol. 148, 3864-3872, 1992. 65. Schroeder, H. W., Jr., Stuber, E S., Kirkham, F? M., Gray, B. M., Tzeng, J.-J., and Shaw, D. R., FASEB J. 6, 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.

A1223, 1992. 66. 67. 68. 69.

Silverman, G. J. and Rapaport, E., FASEB J. 6, A1223, 1992 (Abstract). Sanz, I. H., Dang, H., Takei, M., Talal, N. et al., J. Immunol. 142, 883, 1989. Dersimonian, H., Schwartz, R. S., Barrett, K. J., and Stollar, B. D., J. Imrnunol. 139, 2496-2501, 1987. Cairns, E., Kwong, F? C., Misener, V., Ip, F?, Bell, D. A,, and Siminovitch, K. A,, J. Immunol. 143,685-691,

1989. 70. Klapper, D. G . and Capra, J. D., Ann. Immunol. (Paris) 127C, 261-271, 1976. 71. Newkirk, M. M. and Capra, J. D., Monogr. Allergy 22, 1-11, 1987.

Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model.

Virus-specific antibody secreting cell, memory B-cell, and sero-antibody responses in the human influenza challenge model.

Presentation of superantigen by human T cell clones: a model of T-T cell interaction.

The Mycoplasma arthritidis T-cell mitogen, MAM: a model superantigen.

Keyhole limpet haemocyanin - a model antigen for human immunotoxicological studies.

Conventional antigen and superantigen may be coupled to distinct and cooperative T-cell activation pathways.

Lactobacilli and Bifidobacteria enhance mucosal B cell responses and differentially modulate systemic antibody responses to an oral human rotavirus vaccine in a neonatal gnotobiotic pig disease model.

Human metapneumovirus virus-like particles induce protective B and T cell responses in a mouse model.

A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells.

Differentiation human leukocyte antigens: a proposed nomenclature.

Listeria monocytogenes: a model pathogen to study antigen-specific memory CD8 T cell responses.

Corrigendum: A generalized quantitative antibody homeostasis model: antigen saturation, natural antibodies and a quantitative antibody network.

Accessory function of human mononuclear phagocytes for lymphocyte responses to the superantigen staphylococcal enterotoxin B.

Hypersensitivity to bacteria in eczema. III. Arthus-like responses to bacterial antigens without specific antibody.

Antibody and B cell responses to Plasmodium sporozoites.

Antigen-conjugated human IgE induces antigen-specific T cell tolerance in a humanized mouse model.

Commentary on: Antibody and B Cell Responses to Plasmodium Sporozoites.

A topological model for hepatitis B surface antigen.

Human combinatorial antibody libraries to hepatitis B surface antigen.

Complementarity determining region-independent recognition of a superantigen by B-cell antigen receptors of mantle cell lymphoma.

Depressed antibody responses to a thymus-dependent antigen in toxoplasmosis.

A novel subset of antigenic cells triggers B-cell responses to MHC antigens.

Monoclonal antibody directed to a B-cell antigen present in rats, mice, and humans.

Human B-cell cancer cell lines as a preclinical model for studies of drug effect in diffuse large B-cell lymphoma and multiple myeloma.