The role of B lymphocyte stimulator in B cell biology: implications for the treatment of lupus.

B lymphocyte stimulator (BLyS; also known as B cell activating factor (BAFF)) plays a key role in peripheral B cell tolerance. Mounting evidence indicates that B cell tolerance can be either broken or modulated by deliberately manipulating BLyS levels, and belimumab, a BLyS-neutralizing antibody, was recently approved for the treatment of systemic lupus erythematosus (SLE). Thus, intense investigation has focused on understanding how therapeutics targeting BLyS may work, and accumulating evidence suggests multiple points of action. BLyS signaling, in conjunction with B cell receptor (BCR) signaling, determines the size and quality of the mature primary B cell compartment. Moreover, BLyS family members play roles in antigen-experienced B cell selection and differentiation. Together, these findings have implications for the continued development of novel therapeutics that target BLyS.

Cellular basis for neonatally induced T-suppressor activity. Primary B cell maturation is blocked by suppressor-helper interactions restricted by loci on chromosome 12.

The cellular mechanism and genetic restriction of neonatally induced HA-specific suppressor T (Ts) cells have been examined. The in vivo effect of these Ts cells on antibody production, primary B cell proliferation, B cell surface marker changes, and helper T (Th) cell priming during primary responses to HA have been determined. The results indicate that, although antigen-induced B cell proliferative responses and surface marker changes occur in the presence of Ts cells, differentiation to Ig secretion, and long-lived memory B cell production are prevented. Further, antigen-specific Th cell priming is completely ablated by Ts cells, suggesting that Ts act by preventing the delivery of Th signals required for both the later stages of primary B cell maturation, and the formation of memory B cell populations. Finally, in vivo cell mixing experiments using congenic mice indicate that this Ts-Th interaction is restricted by loci on mouse chromosome 12.

The diversity of the influenza-specific primary B-cell repertoire in BALB/c mice.

The primary immune response of BALB/c mice to influenza (PR8) hemagglutinin (HA), a complex protein antigen, has been examined by the splenic focus assay, and the resulting monoclonal anti-HA antibodies have been characterized by their reactivity with heterologous viruses. The analysis of the primary B-cell response to HA revealed marked differences from responses previously defined for haptenic determinants. There were following differences: (a) the frequency of HA-specific B cells in both conventional and germ-free BALB/c mice was 1 in 1.0-1.5 X 10(5) splenic B cells, which is substantially lower than the frequency of B cells responsive to various simple haptenic determinants; (b) monoclonal anti-HA antibodies were predominantly of the IgA or IgM isotypes instead of IgG, which dominates antihapten responses; and (c) after immunization, the frequency of anti-HA-specific B cells increases by 10- to 50-fold, which is much greater increase than that observed after immunization with haptenic determinants. Fine specificity analysis of primary monoclonal HA-specific antibodies revealed extensive diversity and a considerable overlap with the specificities obtained from immune mice. Given the low overall frequency of HA-specific B cells, it could be calculated that the representation of most HA-specific clonotypes within the B-cell repertoire could not exceed 1 in 10(7) B cells. These findings indicate that the primary B-cell clonotype repertoire is extremely diverse and largely antigen independent in its generation.

Restricted adult clonal profiles induced by neonatal immunization. Influence of suppressor T cells.

The effects of neonatal antigen exposure on the adult B cell repertoire have been examined by characterizing the influenza hemagglutinin (HA)-specific response of adult BALB/c mice given antigen soon after birth. Ligand exposure during early life exerts a profound and lasting effect upon the B cell repertoire, characterized by the expansion and preservation of particular antigen-reactive clones and the apparent loss of others. The precise subset of clonotypes selectively preserved depends upon the age at which antigen is first encountered; and is predictable given a knowledge of the emerging primary pool's dynamics and composition. The preserved (secondary) B cells differ from their unprimed precursors with respect to (a) expression of the surface marker detected by the monoclonal antibody J11d, and (b) susceptibility to T cell-mediated suppression. These studies thus demonstrate a strong relationship between the heritable dynamics of the emerging primary B cell repertoire and the effect of ligand-driven events upon repertoire phenotype. In addition, they provide a mechanistic model for certain forms of antigen-induced oligoclonal dominance, especially the phenomenon of original antigenic sin.

B cell repertoire diversity in athymic mice.

The extent of B cell repertoire diversity among nu/nu BALB/c mice has been assessed and compared with that of normal BALB/c mice. This was accomplished through the characterization of monoclonal, influenza hemagglutinin-specific antibodies by reactivity pattern analysis. The results indicate that the repertoire of athymic mice is equivalent in diversity to that of normal mice. Moreover, because these responses were obtained in recipients that were histocompatible but distinct at immunoglobulin allotype loci, these findings indicate that a very diverse array of B cell clonotypes may be stimulated in the absence of allotype-identical T cells.

Analysis of a novel VHS107 haplotype in CLA-2 and WSA mice. Evidence for gene conversion among IgVH genes in outbred populations.

Gene conversion has been suggested as the basis for many VH allelic differences, particularly in the murine VHS107 family. Whether conversion among IgVH genes is likely to have occurred in outbred populations has not been directly addressed. The CLA-2/Cn and WSA strains, which were recently and independently derived from a feral population exhibiting low responsiveness to PC, provide the opportunity to approach this question. In previous studies, the heavy chain cDNA sequence of a PC-specific hybridoma derived from CLA-2/Cn suggested gene conversion events within the VHS107 family. Accordingly, we have examined the germline VHS107 genes of CLA-2/Cn and WSA. The results indicate that: (a) The CLA-2 and WSA strains bear an identical but novel VHS107 family haplotype, which lacks a V3 element and contains a V1, a V13, and two V11 genes; (b) low PC responsiveness in these populations is unlikely due to an inability to express the V1 member of the VHS107 gene family; and (c) when compared with the other known VHS107 haplotypes, the proportion of differences consistent with gene conversion greatly exceeds that expected by random base substitution. Thus, gene conversion events appear to have occurred with considerable frequency in the evolution of the murine VHS107 family, especially among the V3, V13, and V11 members.

Individual antigen-specific T lymphocytes: helper function in enabling the expression of multiple antibody isotypes.

In recent years antigen-specific T cells have been shown to be capable of mediating a number of diverse functions in collaboration with B cells in humoral immune responses. One of the more intriguing roles attributed to helper T cells is the promotion of the synthesis of multiple immunoglobulin isotypes by B cells in T-dependent antibody responses. The experiments presented in this report were carried out to determine if an individual antigen-specific T lymphocyte has the capability to enable the production of antibodies of multiple immunoglobulin heavy chain isotypes. We describe an experimental system which allows for the isolation and antigenic stimulation of individual helper T cells in a splenic environment which provides an excess of primary B cells for collaboration with isolated T lymphocytes. Employing this system we have demonstrated that an individual antigen-specific T lymphocyte, specific for the PR8 strain of influenza virus, has the capacity to enable primary B-cell PR8-specific antibody responses of more than a single immunoglobulin isotype. The implications made by these studies regarding the problem of genetic restrictions regulating T-cell-B-cell interaction is discussed.

Differential expression of an equivalent clonotype among BALB/c and C57BL/6 mice.

The primary anti-phosphorylcholine (PC) response in BALB/c, C57BL/6, and congenic and recombinant inbred strains of these parental types has been examined in the splenic focus system. The frequencies of PC-specific precursors were shown to vary among these strains from 2 to 20 precursors per 10(6) splenic B cells. The distribution of these frequencies suggests that elements closely linked to or within the major histocompatibility complex may play a role in the determination of this parameter, although additional experiments are necessary to adequately assess this possibility. Moreover, all strains tested, regardless of immunoglobulin allotype, expressed monoclonal antibodies indistinguishable from the TEPC 15 myeloma protein (T15) clonotype. Further, the frequency of this clonotype in a given strain did not appear related to allotype, since both high and low T15 frequencies were found among strains of either the BALB/c (a(1)) or C57BL/6 (a(2)) allotype. The examination of normal serum for the T15 idiotype, however, revealed that only mice of the BALB/c allotype (a(1)) expressed the T15 idiotype in detectable quantities. After immunization with Diplococcus pneumoniae, sera from mice of the a(1) allotype consistently contained large quantities of the T15 idiotype, whereas sera from mice of the a(2) allotype exhibited various degrees of cross-reactivity with anti-T15 antibody. These results suggest that: (a) the allotype of an individual, although closely related to serum levels of an idiotype, is unrelated to the proportion of the precursor population which expresses that idiotype and; (b) the serum expression of a given idiotype may reflect regulatory processes, which act either during or before antigenic stimulation, rather than the actual clonotype representation in the repertoire. These findings indicate that distinctions must be made between the expression of idiotypic determinants within precursor B-cell populations and elements which regulate the subsequent appearance of those idiotypes in serum antibodies.

Competition for BLyS-mediated signaling through Bcmd/BR3 regulates peripheral B lymphocyte numbers.

Striking cell losses occur during late B lymphocyte maturation, reflecting BcR-mediated selection coupled with requisites for viability promoting signals. How selection and survival cues are integrated remains unclear, but a key role for B lymphocyte stimulator (BLyS(TM); trademark of Human Genome Sciences, Inc.) is suggested by its marked effects on B cell numbers and autoantibody formation as well as the B lineage-specific expression of BLyS receptors. Our analyses of the B cell-deficient A/WySnJ mouse have established Bcmd as a gene controlling follicular B cell life span, and recent reports show Bcmd encodes a novel BLyS receptor. Here we show that A/WySnJ B cells are unresponsive to BLyS, affording interrogation of how Bcmd influences B cell homeostasis. Mixed marrow chimeras indicate A/WySnJ peripheral B cells compete poorly for peripheral survival. Moreover, in vivo BrdU labeling shows that (A/WySnJ x BALB/c)F(1) B cells have an intermediate but uniform life span, indicating viability requires continuous signaling via this pathway. Together, these findings establish the BLyS/Bcmd pathway as a dominant mediator of B cell survival, suggesting competition for BLyS/Bcmd signals regulates follicular B cell numbers.

B cell production and turnover in CBA/Ca, CBA/N and CBA/N-bcl-2 transgenic mice: xid-mediated failure among pre B cells is unaltered by bcl-2 overexpression.

The CBA/N strain carries xid, a murine btk missense mutation that reduces peripheral B cell numbers. Using in vivo BrdU labeling and cytofluorimetry, we have compared the magnitude, production rates, and turnover rates of each B lineage subset in the marrow and periphery of CBA/Ca and CBA/N mice. Our results show the pro-B compartment is largely unaffected by xid. In contrast, the pre-B cell pool is markedly reduced, reflecting a diminished production rate and unaltered turnover time. Despite diminished pre-B cell formation, the size of the immature B cell pool is relatively normal in CBA/N mice, due to increased proportional survival of pre-B cells. In addition, we have assessed the marrow and peripheral B cell subsets of CBA/N mice transgenic for bcl-2. These results indicate that while the bcl-2 transgene promotes lengthened survival in most B cell subsets, the pro/pre-B cell losses mediated by xid are not abrogated by bcl-2 overexpression. Taken together, these findings suggest that the initial [not readable: see text] from the pro- to pre-B cell pools, and that anomalies in subsequent compartments likely reflects the action of homeostatic mechanisms compensating for compromised pre-B cell production.

B cell maturation and selection at the marrow-periphery interface.

More than 95% of newly formed B cells die in the short interval spanning sIgM acquisition in the bone marrow and entry into the long-lived pool, suggesting that selective events dictating B cell longevity occur at this stage. These likely include both ligand-induced deletion as well as discrete events that mediate recruitment to the long-lived recirculating pool. We are probing these events through the examination of normal B cell differentiation during this critical period: the characterization of a natural mutation that blocks late maturation, an irradiation/autoreconstitution model of marrow-derived B cell differentiation, and the identification of life span regulatory genes whose expression changes within this window.

The BLyS/BAFF family of ligands and receptors: key targets in the therapy and understanding of autoimmunity.

The B lymphocyte stimulator (BLyS; also termed BAFF) family of ligands and receptors plays a central role in B lymphocyte development, selection, and homoeostasis. Members of this family can independently influence different B cell subsets, because the interactions between the two ligands and three receptors vary, and the receptors themselves are differentially expressed among developing, naive, and antigen experienced B cell subsets. These properties prompt careful assessment of how ablative therapies may influence the behaviour of upstream or downstream B lineage populations, as well as how the implementation and expectations of therapeutics targeting BLyS family members must be guided by knowledge of the B cell subsets contributing to pathogenesis.

Multiple loci mediate B cell repertoire ontogeny: analysis of the hemagglutinin-specific repertoires in neonatal C.B20 and (C.B20 x B10.D2)F1 mice.

The HA-specific B cell repertoires of 12 to 14-day-old C.B20 and (C.B20 X B10.D2)F1 mice have been characterized and compared to those of BALB/c, B10.D2, and (BALB/c X B10.D2)F1 mice to assess the role of allotype-linked loci in the process of repertoire formation. Both C.B20 and (C.B20 X B10.D2)F1 neonates express repertoires similar to (BALB/c X B10.D2)F1s of the same age, and allotypic analysis indicates that nonparental clonotypes observed in (BALB/c X B10.D2)F1s at this developmental stage are derived predominantly from the B10.D2 parent. In conjunction, these findings indicate that: a) allotype-linked loci, although influential in repertoire formation, clearly interact with loci elsewhere in the genome to shape the expressed repertoire phenotype; b) developmental preference may exist among heterozygous individuals regarding the time at which particular elements of each parental VH haplotype are expressed; and c) the factors that control this preference are dictated by heritable elements unlinked to Ig allotype loci.

Probing the cellular basis for immunologic memory: approaching functional distinctions between primed and unprimed B-cell populations.

The physiologic distinctions between secondary and primary lymphoid populations remain largely conceptual. Altered activation, differentiation, and compartmentalization properties likely underlie these differences, but little precise knowledge of how these parameters are affected by antigen priming exists. Because lymphoid populations are dynamic entities and priming is a temporal process, lineage and life span analyses of definable subpopulations are required for the design and interpretation of experiments to probe functional distinctions between primed versus unprimed B-cell populations. Subsequent studies, which address differences in activation requirements and collaborative potential of primed versus unprimed B-cells are further required to evaluate the cellular basis of immunologic memory. Recent advances in the ability to dissect lymphoid differentiation subsets and lineages, coupled with a rapidly expanding knowledge of molecules which mediate B-lymphocyte growth and differentiation, render these possibilities amenable to experimental analysis.

B cell repertoire ontogeny: heritable but dissimilar development of parental and F1 repertoires.

This study examines the genetics of antibody repertoire ontogeny by the comparison of the repertoires in B10.D2, BALB/c, and (BALB X B10.D2)F1 mice at 12 to 14 days of age. This was accomplished by fine specificity analysis of clonotypes responsive to the influenza virus hemagglutinin in each strain. The results indicate that: a) neonatal antibody repertoires are in general less heterogeneous than their adult counterparts, which confirms and extends previous observations and reinforces the assertion that repertoire ontogeny is a heritable, patterned process; b) the BALB/c and B10.D2 neonatal repertoires differ from one another in clonotype compositions, facilitating genetic analyses of repertoire establishment; c) the BALB/c X B10.D2)F1 neonatal repertoire, although of restricted heterogeneity, is clearly dissimilar from either parental repertoire. These findings indicate that B cell repertoire ontogeny may be governed by heritable factors that interact with but are not necessarily themselves variable region structural genes. Furthermore, it appears that the expressed responsive B cell repertoire is a selected subset of the inherited potential repertoire.

Characterization of an inbred mouse exhibiting low responsiveness to phosphorylcholine. Antibodies from low-responder mice suggest gene conversion events within the S107VH gene family.

An inbred strain was derived from feral mice that respond poorly to phosphorylcholine (PC) but responded normally to a variety of other Ag. Low responsiveness is inherited as a single recessive autosomal trait, characterized by very low levels of both PC-binding serum antibody and PC-specific B cells. Analyses of PC-reactive clonotypes generated in limiting dilution culture, as well as analyses of PC-specific hybridomas, indicate that this strain expresses L and H chain V regions not previously associated with high affinity murine PC-binding antibodies. Further, sequence analyses suggest gene conversion events occurred within the S107VH gene family among the progenitors of this strain subsequent to their divergence from those of most common laboratory strains.

Long-lived B cells are distinguished by elevated expression of A1.

Only 5% of the 15 million B cells formed daily reach the long-lived peripheral B cell pool, presumably reflecting both negative and positive selection. These selective events occur primarily during late stages of differentiation in the marrow and periphery, when newly formed B cells bear surface IgM (sIgM), but differ from mature B cells in their expression of heat-stable Ag (CD24), B220 (CD45), and sIgD. Because genes of the Bcl-2 family influence longevity, we compared the expression of Bcl-2, Bax, and A1 among immature vs mature peripheral B cells using semiquantitative reverse-transcriptase PCR. While the levels of both Bcl-2 and Bax mRNA remain constant in these two populations, A1 expression is strikingly up-regulated among mature B cells. In addition, A1 expression is low among pro- and pre-B cells, as well as in immature (sIgM+) marrow B cells. Together, these data indicate that A1 mRNA expression is low at all stages of B cell development before final maturation in the periphery and, unlike other Bcl-2 family members whose expression changes little after marrow egress, A1 is up-regulated 10-fold as cells are recruited into the long-lived peripheral B cell pool.

Dynamics of B cell repertoire formation: normal patterns of clonal turnover are altered by ligand interaction.

The dynamics of B cell repertoire formation was examined by defining the kinetics and clonal composition of the influenza hemagglutinin- (HA) responsive BALB/c repertoire at 1 and 2 wk of age. Although the size and diversity of the HA-responsive repertoire remain constant during this period, the clonal composition changes significantly. These findings indicate a rapid and regular turnover of clonal specificities within the emerging primary repertoire. In addition, the effect of ligand exposure on this process was analyzed by characterizing the repertoire of 2-wk-old BALB/c mice that had been immunized with virus during their first week of life. This treatment markedly alters the normal kinetics and turnover of the emerging repertoire. First, many clonotypes that normally arise between 1 and 2 wk of age fail to be expressed in detectable numbers. Second, several clonotypes that are normally only transiently expressed at 1 wk of age are preferentially expanded and preserved within the responsive B cell pool. In conjunction, these results demonstrate that a) the primary repertoire is characterized by rapid and regular turnover in clonotype composition, b) antigenic exposure perturbs the normal kinetics and pattern of this turnover, and c) the exact effects of ligand exposure may depend on the developmental stage at which it occurs.

Patterns of Ig V region expression among neonatal hemagglutinin-responsive B cells. Evidence for non-random VH gene representation during later stages of development.

Hybridoma libraries were established whose specificities reflect those within the BALB/c hemagglutinin-responsive B cell repertoire at 1 or 2 wk of age. These libraries were generated through chronic immunization regimes that induce responses dominated by clonotypes available at the age of initial immunization. Dot blot analyses of cytoplasmic RNA from these hybridomas were performed to determine the Ig H chain V region (VH) families associated with the repertoire at each age. Although genes from most known VH families can generate hemagglutinin-specific antibodies, clonotypes prevalent during the first week of life disproportionately use VH7183 gene segments. In contrast, hybridomas representative of the repertoire in 2-wk-old individuals preferentially use VHS107, VH36-60, and VHX24 gene segments. These results demonstrate changes in VH gene family predominance that correlate with the age-related patterns of clonal emergence and turnover previously shown in the hemagglutinin-reactive B cell pool. Taken together, these findings suggest that the very early neonatal Ag-responsive B cell pool closely reflects preferential VH gene rearrangements within the pre-B cell compartment. Further, they suggest that either non-random strategies of VH gene expression, or selective clonal expansion strategies based on VH, operate even at later stages of development.