Polyreactive antigen-binding B cells in the peripheral circulation are IgD+ and B7−

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen-binding B cells further. A panel of fluorescein isothiocyanate-labeled antigens (insulin, IgG Fc fragment or β-galactosidase) served as probes to select polyreactive antigen-binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen-binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7⁻ cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen-binding B cells have properties that are consistent with the ability to induce immunological tolerance.     

CD5+ B lymphocytes, polyreactive antibodies and the human B-cell repertoire

The lock and key model of antigen-antibody reaction has traditionally been used to explain the specificity of antibodies and the need for antibody diversity. Recently it has become clear that certain antibodies are polyreactive and recognize a variety of self- and foreign antigens. It is now clear that these antibodies are made by a novel subset of B cells that bear the surface CD5 marker. Careful analysis has shown that about 20% of peripheral blood B lymphocytes in adults are CD5+ and, therefore, represent a major component of the normal human B-cell repertoire. The precise role of the antibodies produced by these cells is still not clear, but because of their polyreactivity they might function in clearing autoantigens from the circulation and/or as a rapid first line of defense against foreign invaders, such as bacteria and viruses. Sequence analysis showed that these antibodies use gene segments in germ-line configuration for their antigen-binding portion. In this article, Paolo Casali and Abner Notkins propose that polyreactive antibodies are what, for years, have been referred to as the 'natural antibodies' of serum and that under certain circumstances they may contribute to the pathogenesis of autoimmune diseases.     

ANTIGEN BINDING TO LYMPHOID CELLS OF UNIMMUNIZED MICE

In order to determine the cell type responsible for the antigen-binding reaction in the bone marrow and spleen of mice, cells derived from pure in vitro derived colonies of neutrophils, eosinophils, macrophage-megakaryocytes and B lymphocytes were tested for their ability to bind fluorescent protein antigens. Only B lymphocytes bound antigen. An unexpectedly high percentage of bone marrow B lymphocytes (20%) bound a given antigen. This frequency was considerably higher than that found for spleen cells. As might be expected from such high binding frequencies, some cells bound two fluorchromated antigens when these are added together. As a direct test of the clonality of antigen binding to bone marrow B lymphocytes, whole colonies of B cells were tested for antigen binding of two non-cross-reacting protein antigens. The frequency of antigen-binding clones, including double antigen-binding clones, reflects exactly the frequencies observed for dispersed colony B cells and for in vivo derived Ig-bearing bone marrow B cells. The frequency of double antigen-binding colonies was equal to the product of the frequencies of the colonies binding each of the two antigens alone. No ‘mixed’ colonies containing single binding cells for each antigen were found. Thus, the ability to bind any two given antigens is a clonally distributed property of the bone marrow B lymphocyte population. Heterogenous receptors for multiple antigen binding on each cell are either randomly distributed among the B cell population, or homogenous antigen-binding receptors on each cell have a random chance of cross-reaction with the two antigens tested.     

CD23/CD21 interaction is required for presentation of soluble protein antigen by lymphoblastoid B cell lines to specific CD4+ T cell clones

Previous studies have documented a role for membrane-bound CD23 (the low affinity FcεRII) in presentation of alloantigens by B cells. The aim of the present study was to examine the involvement of cell surface CD23 in presentation of more conventional soluble protein antigens to T cells. We show that antibodies to CD23 and to its lymphocyte-associated second ligand, CD21, inhibit presentation of the cow's milk allergen casein, by autologous CD23⁺CD21⁺ B-EBV cell lines to casein-specific HLA-DP-restricted CD4⁺ T cell clones obtained from patients with either reaginic or enterophatic forms of cow's milk protein intolerance. Maximal inhibition was achieved when the antibodies were added at the initiation of the culture. The absence of specific inhibition by an anti-DRα monoclonal antibody (mAb) argues against a steric hindrance phenomenon impeding access of the T cell receptor to major histocompatibility complex class II molecules. Rather, anti-CD23 and anti-CD21 mAb-induced inhibition of antigen presentation seems to affect at least partly, heterotypic conjugate formation through CD23/CD21 interaction. Double immunofluorescence labeling of the T cell clones and antibody inhibition of T/B conjugate formation shows that functional CD23 and CD21 molecules are induced on T cells following contact with B-EBV cell lines. Taken together, these data indicate that CD23/CD21 interactions between T and B cells are required for presentation of soluble protein antigens by B-EBV cell lines to specific CD4⁺ T cells. The potential implications of these findings for allergen-specific T cell activation are discussed.     

The Antibody Repertoire of Early Human B Cells I. High Frequency of Autoreactivity and Polyreactivity

Cord blood and fetal liver B cells were immortalized using Epstein-Barr virus, and IgM antibodies from the resulting lines and clones were examined for their binding to a variety of auto-antigens and micro-organisms by ELISA and fluorescence assays. Auto-antigens tested included Fc of IgG, ssDNA and dsDNA, cardiolipin, histones 1-4, collagens type I and II, thyroglobulin, cytoskeletal components, and a tissue section screen. Of 71 cell lines tested, all but 19 showed some autoreactivity. All 32 fetal liver lines reacted to some self-antigens. In cord blood clones, 16 out of 26 bound to auto-antigens. Many of the clones reacted with more than one auto-antigen and were 'polyreactive'. Some of the cord blood clones bound to extracts of micro-organisms, showing specificity for both endogenous and exogenous antigens. The high frequency of CD5+ B cells in the cord blood (greater than 50%) and fetal liver (greater than 70%) argues for many of these clones being derived from this subset. Therefore, our data support the concept that many 'early' B cells produce polyreactive IgM which can bind to a variety of different auto-antigens and micro-organisms. These IgM antibodies are similar to those described by others as 'natural antibodies'.     

Scarcity of autoreactive human blood IgA+ memory B cells

Class‐switched memory B cells are key components of the “reactive” humoral immunity, which ensures a fast and massive secretion of high‐affinity antigen‐specific antibodies upon antigenic challenge. In humans, IgA class‐switched (IgA⁺) memory B cells and IgA antibodies are abundant in the blood. Although circulating IgA⁺ memory B cells and their corresponding secreted immunoglobulins likely possess major protective and/or regulatory immune roles, little is known about their specificity and function. Here, we show that IgA⁺ and IgG⁺ memory B‐cell antibodies cloned from the same healthy humans share common immunoglobulin gene features. IgA and IgG memory antibodies have comparable lack of reactivity to vaccines, common mucosa‐tropic viruses and commensal bacteria. However, the IgA⁺ memory B‐cell compartment contains fewer polyreactive clones and importantly, only rare self‐reactive clones compared to IgG⁺ memory B cells. Self‐reactivity of IgAs is acquired following B‐cell affinity maturation but not antibody class switching. Together, our data suggest the existence of different regulatory mechanisms for removing autoreactive clones from the IgG⁺ and IgA⁺ memory B‐cell repertoires, and/or different maturation pathways potentially reflecting the distinct nature and localization of the cognate antigens recognized by individual B‐cell populations.     

Presence of Activated B-1 Cells in Chronic Inflamed Gingival Tissue

B-1 cells are physically and functionally unique B cells that produce polyreactive natural antibody. This study examined the activation of B-1 cells in inflamed gingival tissue. Serum IgG antibodies to phosphorylcholine, E. coli LPS, DNA, and some commensal bacteria were examined in adult periodontitis patients and healthy subjects. In addition, the proportion of B-1a (CD20⁺ CD5⁺) cells and the amount of IL-6 and IL-10 in the inflamed gingival tissues were examined. The serum levels of IgG antibodies to phosphorylcholine, E. coli LPS, and commensal bacteria were significantly higher in the adult periodontitis patients than the healthy subjects. The proportion of B-1a cells and the amount of IL-6 and IL-10 were significantly higher in the inflamed gingival tissues than in peripheral blood from the healthy subjects. These results suggest the activation of B-1 cells in the inflamed gingival tissue of adult periodontitis patients, and that B-1 cells may serve as the first line of defense by producing polyreactive antibodies to phosphorylcholine, LPS, and commensal bacteria.     

Antibody specificity and immunoglobulin VH utilization of human monoclonal CD5+ B cell lines

Human B lymphocytes that bear the CD5 antigen are relatively abundant in early ontogeny and comprise a small fraction of the B cell population in adults. The CD5 B cell subset has attracted much attention because of its possible involvement in autoimmune disease and certain B cell malignancies. To begin to understand the role of CD5 B cells in disease processes, we have generated a panel of ten human monoclonal B cell lines selected for expression of the CD5 antigen. These cell lines were obtained by Epstein-Barr virus transformation of B lymphocytes isolated from the spleen, liver and bone marrow of a 19-week-old fetus, from cord blood and from peripheral blood of healthy volunteers. In addition, one cell line was isolated from the spleen of a patient with chronic lymphocytic leukemia. Here, we describe the antibody and immunoglobulin V_H gene repertoire of this panel of CD5 B cell lines. The results of these experiments show that (a) some but not all CD5 B cell lines secrete polyreactive antibodies that bind to a variety of self- and xenoantigens and (b) members of the small V_H4, V_H5 and V_H6 gene families are overrepresented in this panel of cell lines. Nucleotide sequence analysis revealed the expression of V_H gene elements that have been previously reported in the preimmune B cell repertoire, in CD5 B cell tumors and in polyreactive antibodies.     

Vaccine molecules targeting Xcr1 on cross‐presenting DCs induce protective CD8+ T‐cell responses against influenza virus

Targeting antigens to cross‐presenting dendritic cells (DCs) is a promising method for enhancing CD8⁺ T‐cell responses. However, expression patterns of surface receptors often vary between species, making it difficult to relate observations in mice to other animals. Recent studies have indicated that the chemokine receptor Xcr1 is selectively expressed on cross‐presenting murine CD8α⁺ DCs, and that the expression is conserved on homologous DC subsets in humans (CD141⁺ DCs), sheep (CD26⁺ DCs), and macaques (CADM1⁺ DCs). We therefore tested if targeting antigens to Xcr1 on cross‐presenting DCs using antigen fused to Xcl1, the only known ligand for Xcr1, could enhance immune responses. Bivalent Xcl1 fused to model antigens specifically bound CD8α⁺ DCs and increased proliferation of antigen‐specific T cells. DNA vaccines encoding dimeric Xcl1‐hemagglutinin (HA) fusion proteins induced cytotoxic CD8⁺ T‐cell responses, and mediated full protection against a lethal challenge with influenza A virus. In addition to enhanced CD8⁺ T‐cell responses, targeting of antigen to Xcr1 induced CD4⁺ Th1 responses and highly selective production of IgG2a antibodies. In conclusion, targeting of dimeric fusion vaccine molecules to CD8α⁺ DCs using Xcl1 represents a novel and promising method for induction of protective CD8⁺ T‐cell responses.     

Cell contact interaction between adipose‐derived stromal cells and allo‐activated T lymphocytes

Mesenchymal stromal cells regulate immune cell function via the secretion of soluble factors. Cell membrane interactions between these cell types may play an additional role. Here, we demonstrate that subpopulations of allo‐activated T cells are capable of binding to human adipose‐derived stromal cells (ASC). The bound T‐cell population contained CD8⁺ T cells and was enriched for CD4^?CD8^? T cells, whereas the proportion of CD4⁺ T cells was decreased compared with the non‐bound T‐cell population. Bound CD4⁺ T cells had high proliferative activity and increased CD25 and FoxP3 expression. However, they also expressed CD127, excluding regulatory T‐cell function. In CD8⁺ T cells, IL‐2 sensitivity, as determined by the analysis of phosphorylated STAT5, was lower in the presence of ASC and even lower in bound cells. In contrast, IL‐2‐induced phosphorylated STAT5 levels were higher in bound CD4⁺ T cells than in non‐bound CD4⁺ T cells. Additionally, pro‐proliferative TGF‐β signalling via endoglin and SMAD1/5/8 phosphorylation was detected in bound CD4⁺ T cells. Even after prolonged co‐culture with ASC, the activated phenotype of bound CD4⁺ T cells persisted. In conclusion, these results demonstrate that the binding of lymphocytes to ASC represents an immunomodulatory mechanism in which CD8⁺ T cells are inhibited in their responsiveness to pro‐inflammatory stimuli and reactive CD4⁺ T cells are depleted from the immune response.     

Co-expression of CD173 (H2) and CD174 (Lewis Y) with CD44 suggests that fucosylated histo-blood group antigens are markers of breast cancer-initiating cells

Histo-blood group antigens CD173 (H2) and CD174 (Lewis Y) are known to be developmentally regulated carbohydrate antigens which are expressed to a varying degree on many human carcinomas. We hypothesized that they might represent markers of cancer-initiating cells (or cancer stem cells, CSC). In order to test this hypothesis, we examined the co-expression of CD173 and CD174 with stem cell markers CD44 and CD133 by flow cytometry analysis, immunocytochemistry, and immunohistochemistry on cell lines and tissue sections from breast cancer. In three breast cancer cell lines, the percentage of CD173⁺/CD44⁺ cells ranged from 17% to >60% and of CD174⁺/CD44⁺ from 21% to 57%. In breast cancer tissue sections from 15 patients, up to 50% of tumor cells simultaneously expressed CD173, CD174, and CD44 antigens. Co-expression of CD173 and CD174 with CD133 was also observed, but to a lesser percentage. Co-immunoprecipitation and sandwich ELISA experiments on breast cancer cell lines suggested that CD173 and CD174 are carried on the CD44 molecule. The results show that in these tissues CD173 (H2) and CD174 (LeY) are associated with CD44 expression, suggesting that these carbohydrate antigens are markers of cancer-initiating cells or of early progenitors of breast carcinomas.     

CD8+ T‐cell responses to Theileria parva are preferentially directed to a single dominant antigen: Implications for parasite strain‐specific immunity

Although immunodominance of CD8⁺ T‐cell responses is a well‐recognised feature of viral infections, its role in responses to more antigenically complex pathogens is less clear. In previous studies we have observed that CD8⁺ T‐cell responses to Theileria parva exhibit different patterns of parasite strain specificity in cattle of different MHC genotypes. In the current study, we demonstrated that animals homozygous for the A10 and A18 MHC haplotypes have detectable responses to only one of 5 T. parva antigens. Over 60% of the responding T cells from the A18⁺ and A10⁺ animals recognised defined epitopes in the Tp1 and Tp2 antigens, respectively. Comparison of T‐cell receptor β chain expression profiles of CD8⁺ T‐cell lines and CD8⁺ T cells harvested ex vivo confirmed that the composition of the T‐cell lines was representative of the in vivo memory CD8⁺ T‐cell populations. Analysis of the Tp1 and Tp2 antigens revealed sequence polymorphism, which was reflected by differential recognition by T‐cell lines. In conclusion, we have demonstrated a profound immunodominance in the CD8⁺ T‐cell response to T. parva, which we propose is a major determinant of the parasite strain specificity of the response and hence immune protection.     

Monoreactive and Polyreactive Rheumatoid Factors Produced by in Vitro Epstein-Barr Virus-Transformed Peripheral Blood and Synovial B Lymphocytes from Rheumatoid Arthritis Patients

The CD5 membrane molecule, initially identified as an exclusive T-cell marker, also defines a phenotypically and functionally distinct B-lymphocyte population. In normal individuals, CD5+ B cells are committed to secrete 'natural' polyreactive (auto)antibodies, that is antibodies, mainly IgM, endowed with multiple antigen-binding capabilities, including rheumatoid factor (RF) activity. At variance with this, in rheumatoid arthritis (RA) as well as in other autoimmune conditions, monoreactive autoantibodies binding with high affinity and specificity to a given self antigen have been isolated and the cells from which they originate differently related to the CD5+ B-lymphocyte subset. Here, we studied the proportions of CD5+ B cells and the characteristics, in terms of polyreactivity and monoreactivity, of RF produced by B lymphocytes in RA patients with classified disease activity. Our results suggest that patients with a more severe disease activity have higher proportions of CD5+ B cells and higher frequencies of B lymphocytes committed to secrete RF, with the characteristics of polyreactive antibodies. On the other hand, we did not find a significant difference between the proportions of peripheral B cells producing monoreactive RF in patients with high- versus patients with low-activity RA. However, in two highly active RA patients, we found that synovial fluid, compared with peripheral blood, was significantly enriched for (IgG and IgA) monoreactive RF-producing B cells.     

Poor memory B cell generation contributes to non-protective responses to DTaP vaccine antigens in otitis-prone children

We recently identified a cohort of children with recurrent episodes of acute otitis media (AOM) who fail to generate protective antibody titres to otopathogens and several vaccine antigens. In this study we determined the antibody levels against DTaP vaccine antigens, diphtheria toxoid (DT), tetanus toxoid (TT) and acellular pertussis toxoid (PT) in sera from 15 stringently defined otitis-prone (sOP) children and 20 non-otitis-prone (NOP) children. We found significantly lower concentrations of immunoglobulin (Ig)G antibodies against vaccine antigens in the serum of sOP children compared to age-matched NOP children. To elucidate immunological cellular responses to the vaccines in these children, we investigated memory B cell responses to DTaP vaccination. We used fluorescently conjugated vaccine antigens to label antigen receptors on the surface of memory B cells and examined the frequency of antigen-specific CD19⁺ CD27⁺ memory B cells in the peripheral blood. sOP children showed a significantly lower percentage of antigen-specific CD19⁺ CD27⁺ memory B cells than NOP children. We also found a linear correlation between the frequencies of memory B cells and circulating IgG titres for DT, TT and PT proteins. To our knowledge, this is the first study to show significant differences in memory B cell responses to DTaP vaccine antigens and their correlation with the circulating antibodies in young children with recurrent AOM.     

Monoclonal antibody (B27M2) subdividing HLA-B27

Because HLA-B27 shows strong association with ankylosing spondylitis, it was of interest to produce murine monoclonal antibodies to study this antigen in detail. The first such anti-B27 antibody, anti-B27M1, reacted with 100% of B27 + cells and cross-reacted with homozygous B7 cells. In the present report a second monoclonal antibody, anti-B27M2, is described which subdivides HLA-B27 into two variants. Among healthy B27⁺ individuals, 87% were B27M1⁺ and B27M2⁺, and 13% were B27M1⁺ but B27M2⁺ by standard lymphocytotoxicity assays. The specificity of B27M2 antibody for the HLA-B27 molecule was confirmed with blocking studies using F(ab′)₂ fragments of HLA alloantibodies. Both the B27M2⁺ and B27M2 variants of HLA-B27 bred true in family studies. Unlike B27M1, B27M2 antibody did not react with B7 but did react with the rare Bw47 allele.For antibody binding studies Epstein-Barr virus-transformed B lymphoblastoid cell lines were derived from normal donors KCA (by lymphocytotoxicity shown to be B27⁺, B27M2⁺. VC (B27⁺, B27M1⁺, B27M2^?), and WH (B27^?, B27M1^?, B27M2^?). Each cell line bound equivalent amounts of W6/32 (monoclonal anti-HLA-ABC): KCA and VC bound similar amounts of anti-B27M1, but only KCA bound substantial anti-B27M2 antibody. These data are consistent with a model in which all B27 antigens possess a B27M1 epitope: whereas most, but not all, possess an additional and distinct epitope, B27M2. Although the relation of these genetic variants to disease susceptibility remains to be determined, the availability of epitope-specific monoclonal antibodies should help to refine our understanding of the structure and function of HLA molecules.     

Characterizing CEACAM5 interaction with CD8α and CD1d in intestinal homeostasis

Normal intestinal epithelial cells (IECs) could act as non-professional antigen-presenting cells, selectively activating CD8⁺-suppressor T cells. An epithelial cell surface glycoprotein, gp180, recognized by monoclonal antibodies B9 and L12 was determined to be critical in this process. Purification and sequence analysis of mAb B9 reactive material revealed amino-acid sequence homology with CEACAM5. We demonstrate that CEACAM5 has properties attributed to gp180, such as CD8α binding and activation of CD8-associated Lck. CEACAM5 is the only CEACAM member interacting with CD1d through the B3 domain. Its N domain (recognized by B9) is required for CD8α binding. Removal of the N-domain glycosylated residues reduces B9 recognition, CD8α binding affinity, and activation of LcK. Therefore, conformational changes in CEACAM5 glycosylation site are critical for its interaction with CD8α. CEACAM5-activated CD8⁺ T cells acquire the ability to suppress the proliferation of CD4⁺ T cells in vitro in the presence of interleukin (IL)-15 or IL-7. We provide new insights into the role of CEACAM5 and define its specific immunoregulatory properties among the CEACAMs expressed on IECs. We suggest that unique set of interactions between CEACAM5, CD1d, and CD8 render CD1d more class I-like molecule, facilitating antigen presentation and activation of CD8⁺-suppressor regulatory T cells.     

Different epitopes of the CD31 antigen identified by monoclonal antibodies: cell type-specific patterns of expression

3 mAb-5A2.G5, B2B1 and 2BD4-all of IgG1 isotype were identified as belonging to the CD31 cluster by their binding to transfected murine cell lines expressing the CD31 antigen and by sequential immunoprecipitation experiments. Competitive binding experiments were carried out using the human myelomonocytic cell line RC-2A. mAb B2B1 and 2BD4 did not cross block. mAb 5A2.G5 partly inhibited binding of 2BD4 but not B2B1. Thus the epitopes identified by 5A2.G5 and 2BD4 appear to overlap but to be quite separate from that recognized by B2B1. All 3 antibodies bound to a 130 kDa species on Western blots after nonreducing polyacrylamide gel electrophoresis of platelet lysates. Culture of RC-2A cells in the presence of tunicamycin (after removal of surface antigens by pronase) blocked re-expression of the epitopes recognised by all 3 mAb suggesting that they involve N-linked glycosylation. Furthermore, treatment of platelet lysates with Endoglycosidase F prior to electrophoresis and Western blotting abolished the binding of the mAb but not a rabbit polyclonal antiserum to CD31. Nevertheless, neuraminidase treatment of RC-2A cells failed to affect mAb binding. The antibodies displayed typical properties of CD31 antibodies in that all 3 precipitated at 130 kDa cell surface protein and bound strongly to platelets, monocytes, neutrophils and vascular endothelium. All 3 antibodies were positive on hemopoietic progenitor cells which give rise to colonies in the CFU-C assay. However, differences in binding to peripheral blood lymphocytes and to certain human leukemic cell lines were noted. In particular, mAb 5A2.G5 bound weakly to lymphocytes and to the lymphoid cell line HSB-2 compared with the other 2 antibodies.     

Anti-Pneumococcal Capsular Polysaccharide Antibody Response and CD5 B Lymphocyte Subsets

The role of CD19⁺ CD5⁺ and CD19⁺ CD5⁻ B cell subpopulations in the antibody response to pneumococcal capsular polysaccharides (caps-PSs) is controversial. In the present study, we evaluated the role of human CD19⁺ CD5⁺ and CD19⁺ CD5⁻ cell populations in the serotype-specific antibody response to caps-PS. After vaccination of 5 healthy human adults with Pneumovax (23-valent pneumococcal polysaccharide vaccine [PPV23]), IgG anti-caps-PS serotype 4 antibody-producing cells resided mainly in the CD19⁺ CD5⁻ B cell subset, as assessed by enzyme-linked immunosorbent spot (ELISpot) analysis. Moreover, in a humanized SCID mouse model, CD19⁺ CD5⁻ B cells were more effective than CD19⁺ CD5⁺ cells in producing IgG anti-cap-PS antibodies. Finally, an association was found between the level of IgG anti-caps-PS antibodies and the number of CD19⁺ CD5⁻ B cells in 33 humans vaccinated with PPV23. Taken together, our data suggest that CD5 defines a functionally distinct population of B cells in humans in the anti-caps-PS immune response.     

Effect of age on the expressed B cell repertoire: role of B cell subsets

Aged humans and experimental animals are impaired in their responsesto most foreign antigens although they produce greater amountsof autoantibodies. We have examined the effect of age on theproduction of antibodies to a prototypic foreign antigen, sheeperythrocytes (SRBC), and to a prototypic autoantigen, bromelain-treatedmouse erythrocytes (BrMRBC), in young and old ice before andafter immunization with SRBC. Old mice express more anti-BrMRBCplaqueforming cell (PFC) antibodies before and an even greaternumber after immunization with SRBC than young mice. Conversely,old mice produce far fewer anti-SRBC PFC than young mice followingimmunization with SRBC. We hypothesized that the differencesin the responses of old mice to BrMRBC and SRBC reflects differencesin the activity of CD5⁺ and CD5^– B cells. To test thishypothesis we immunized young and old mice with foreign antigensreported (and confirmed in our studies) to stimulate CD5⁺ Bcells [TNP-ficoll and phosphorylcholine - keyhole limpet hemocyanln(KLH)] or CD5^– B cells (SRBC and TNP-KLH). We found thatthe PFC response of old mice to antigens mediated by CD5⁺ Bcells was equal to or greater than that of young mice. In contrastthe PFC response of old mice induced by antigens mediated byCD5⁺ B cells was only 10% that of young mice. Thus it appearsthat the immune response of old mice is well maintained forantigens which elicit a CD5⁺ B cell response but not for thosewhich elicit a CD5^– B cell response. This conclusion issupported by our finding that a higher percentage of splenicB cells from old compared to young mice express that V_H11 lghgene family. This V_H gene family is preferentially expressedby CD5⁺ B cells.     

Cell surface expression of surrogate light chain (ΨL) in the absence of μ on human pro-B cell lines and normal pro-B cells

Surrogate light chains (ΨL) encoded by λ-like (λ5) and VpreB genes play a critical role in controlling the early steps of B cell differentiation. We prepared new anti-VpreB monoclonal antibodies (mAb) (3C7/6F6) which preferentially recognize the VpreB epitope at the cell surface of human cell lines that do not express the μ chain. These mAb provide the first characterization of human pro-B cell lines expressing surface ΨL. We demonstrate that surface ΨL expression is considerably enhanced upon interleukin-7 stimulation and that the ΨL complex is formed independently of the Igα/Igβ heterodimer. Finally, using these antibodies, we confirm the existence of a normal pro-B cell population in human adult bone marrow. These cells are CD34⁺ CD38⁺ ΨL⁺, do or do not express CD19, CD10, or both epitopes, and may represent the earliest cell population committed to B cell differentiation.