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The rate of elimination of a pathogenic agent is of critical importance for the host and determines the extent and consequences of the infection. Antibody production, along with the activity of other cells of the immune system, plays an important role early and late in the response and contributes to all containment and elimination of the organism. B-cell clones reaching the mature long-lived pool are heterogeneous: some belong to the B1 B-cell subset, some are enriched in the CD21high compartment (mostly marginal zone (MZ)), whereas others recirculate primarily among the B-cell follicles (FO). This segregation is a T-independent, CD40L-independent but BCR/CD19-dependent process. Antigen encounter will recruit antigen-specific cells from the pool of mature B-lymphocytes and activate them to perform effector functions. CD21highCD23low B cells enriched in the MZ of the spleen initiate the early plasmablast wave during the first 3 days of an antibody response against particulate T-independent bacterial antigens. These findings indicate a functional heterogeneity within the mature B-lymphocyte population. MZ B cells and B1 B, in contrast to FO B cells, have the unique capacity to generate effector cells in early stages of the immune response against (particulate) antigens that are scavenged efficiently in these specialized anatomical sites.

In mice, the absence of terminal deoxynucleotidyl transferase (Tdt) expression during fetal and neonatal life provides a window in development where clones of lymphocytes are generated that provide protective immunity. Introducing premature Tdt activity interferes with the development of these clones and results in an impaired ability to make protective antibodies. Conversely, gene-targeted disruption of Tdt prevents N additions at all stages of T and B-lymphocyte development and promotes the development of fetal-like T and B-cell clones into adulthood, with accompanying alterations in repertoire. The alternative splice forms of Tdt may be necessary to provide regulatory mechanisms to restrict N addition to appropriate stages of the developmental pathways, the details of which are being revealed. The evidence continues to build that Tdt is a key player in influencing the outcome of V(D)J recombination during lymphocyte and repertoire development.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.

Collectively, these findings suggest that MZ B cells have unique signaling and subsequent differentiative capabilities that permit them to react much more vigorously than the majority of splenic B cells (FO) in the earliest stages of an in vivo immune response. This is particularly evident with limiting T cell help, low concentration of thymus-independent mitogens or low amounts of particulate blood-borne antigen in the spleen (Fig. 4). They are uniquely situated adjacent to the marginal sinuses and a rich array of antigen trapping macrophages. Because of this location the MZ B cells are ideally positioned for immediate exposure to blood-borne antigens. In contrast, the FO B cells are in juxtaposition to the PALS which may expedite the interactions of FO B cells with T cells and antigen presenting cells. Collectively these properties point to a role for FO B cells in antibody responses to T dependent antigens generated in germinal centers. These responses occur temporally later in immune responses and may be involved principally in the response to protein antigens. [figure: see text].

Ag encounter will recruit Ag-specific cells from the pool of mature B lymphocytes in the spleen and activate them to perform effector functions: generation of Ab-forming cells (plasma cells) and presentation of Ag to T cells. We have compared the ability of mature follicular and marginal zone cells to develop into effector B cells. The generation of marginal zone B cells and their localization in the marginal sinus area are T cell and CD40 ligand independent, suggesting that they do not represent a postgerminal center population. Compared with mature recirculating follicular B cells, they express several characteristics of previous antigenic experience, including higher levels of B7.1 (CD80) and B7.2 (CD86) when freshly isolated and following in vitro stimulation. After a brief 6- to 8-h in vitro stimulation with LPS or anti-CD40 Abs, marginal zone B cells become potent APCs. In addition, their ability to proliferate and differentiate into plasma cells in response to low doses of T-independent polyclonal stimuli (LPS) is far greater than that of follicular B cells. These findings indicate a functional heterogeneity within splenic mature B lymphocytes, with marginal zone B cells having the capacity to generate effector cells in early stages of the immune response against particulate Ags scavenged efficiently in this special anatomical site.

The generation of VH81X heavy chain lambda-light chain-expressing B cells (VH81X-lambda+ B cells) was studied in VH81X heavy chain transgenic mice as well as in VH81X JH (-/-) and VH81X JH (-/-) Ck (-/-) mice, in which competition resulting from expression of heavy and light chains from the endogenous heavy and kappa light chain loci was prevented. We show that although lambda light chain gene rearrangements occur normally and give rise to light chains that associate with the transgenic heavy chain to form surface and soluble IgM molecules, further B cell development is almost totally blocked. The few VH81X-lambda+ B cells that are generated progress into a mature compartment (expressing surface CD21, CD22, CD23, and low CD24 and having a relatively long life span) but they also have reduced levels of surface Ig receptor and express higher amounts of Fas Ag than VH81X-kappa+ B cells. These VH81X-lambda+ B cells reach the peripheral lymphoid organs and accumulate in the periarteriolar lymphoid sheath but are unable to generate primary B cell follicles. In other heavy chain transgenic mice (MD2, M167, and M54), lambda+ B cells are generated. However, they seem to be preferentially selected in the peripheral repertoire of some transgenic heavy chain mice (M54) but not in others (MD2, M167). These studies show that a crucial selection step is necessary for B cell survival and maintenance in which B cells, similar to T cells, receive signals depending on their clonal receptors.

The most D-proximal functional VH gene, VH81X, is preferentially expressed in the mouse fetal B cell repertoire; however, it is expressed in few B cells in the adult. To determine when VH81X gene expression affects size and phenotype of particular stages in B cell differentiation, transgenic mice have been developed expressing a germline fetal liver-derived VH81X-mu rearrangement. Comparative analysis of B lymphopoiesis reveals similarities and differences between fetal liver and adult bone marrow which pinpoint developmental stages in mice during which VH81X-expressing B cell progenitors expand or deplete compartment sizes. These include a similar reduction in c-kitR+ and establishment of a predominant CD43low/HSAhigh phenotype within the B220+ CD43+ compartment which is dependent on the association of the transgene with lambda 5. In contrast, the CD43- pre-B and immature B cell compartments are expanded in the fetus but not in the adult. In addition, there are other factors that later disfavor the survival of VH81X-expressing B1 and B2 cells. Thus the failure to detect VH81X-bearing B cells in the adult is the result of a multistep selection process occurring at all stages during B repertoire expansion.

Germinal center formation is the result of antigenic stimulation of B cells in a T cell-rich area. B cells cycle through the germinal centers, and a small percentage survive to become plasma cells or memory B cells. The transformation from a mature B cell into a germinal center B cell and finally into a terminally differentiated B cell is not well understood. Human CD38 is highly expressed on both germinal center B cells and plasma cells, and is useful in delineating these B cell subsets and in understanding the signaling events involved in the development of these B cells. To determine whether CD38 expression on activated germinal center B cells and postgerminal center B cells influences germinal center differentiation, we studied the expression of CD38 in the mouse. CD38 is expressed on follicular B cells in the Peyer's patches but is down-regulated on germinal center B cells located within the Peyer's patches. CD38dim/-B220+ germinal center B cells are also found in the spleens of immunized but not control mice, suggesting that Ag-stimulated germinal center formation is involved in the production of CD38dim/-B220+ B cells. Furthermore, mature plasma cells isolated from in vitro LPS cultures do not express CD38, but do contain high levels of cytoplasmic Ig. These results are in contrast to studies in humans in which CD38 is not found on follicular B cells but is highly expressed on germinal center B cells and plasma cells.

Human CD7 is a 40 kDa protein expressed on thymocytes, early T, B, NK and myeloid lineage cells in bone marrow, and on mature T and NK cells. Previous studies suggested human CD7 may be involved in T and NK cell activation and/or adhesion, and that CD7-mediated cell activation may be transduced via the lipid kinase phosphatidylinositol 3-kinase (Pi3-kinase), a heterodimeric cytosolic protein consisting of an 85 kDa adaptor subunit that is coupled to a 110 kDa catalytic subunit. It has recently been shown that a sequence motif present in the cytoplasmic tall of both human and mouse CD7 bound with high affinity to recombinant SH2 domains present in the p85 subunit of Pi3-kinase. In this work, we used co-precipitation with anti-CD7 mAb 3A1 and recombinant p85 SH2-GST fusion proteins and peptide competition analysis to demonstrate that the cytoplasmic tail of CD7 interacts with a functional Pi3-kinase via the pTyr-X-X-Met motif. Furthermore, we show that cross-linking of CD7 markedly increased the amount of Pi3-kinase activity associated with CD7. The interaction of CD7 with the Pi3-kinase signal transduction pathway provides a mechanism for the previously observed functional responses attributed to CD7-mediated T and NK cell activation.

Normal differentiation of B lineage cells has been the subject of intensive investigation over the past three decades. Current models of this process in humans are melded from the results of studies in a variety of organisms, including humans, mice and birds. Several recent developments have significantly reshaped and refined these models. The technique of homologous recombination in embryonic stem cells has allowed the production of mice with selectively disrupted genes that are important for B cell development in mice. At the same time, functional studies of human B cell differentiation, together with analysis of naturally occurring mutations that disrupt this process, have progressed rapidly. This has provided insight into the pathogenesis of lymphoproliferative and immunodeficiency diseases as well as a clearer view of normal developmental events. In this chapter we have reviewed human B cell differentiation with particular emphasis on newly emerging concepts. We also discussed CD5, a pan-T cell antigen that is expressed in low levels on a subpopulation of B cells implicated in the pathogenesis of chronic lymphocytic leukaemia (CLL). Finally, we discussed the issue of restricted variable region gene usage during B cell ontogeny and in CLL.

BALB/c mice were inoculated i.p. with a cross-reactive anti-Idiotypic mAb (designated FD5-1) in the absence of Ag or adjuvants. Injection with unmodified FD5-1 resulted in the induction of serum antibodies reactive with both FD5-1 (Ab3) and the hapten DNP (Ab1'). Endpoint titers of the Ab3 response showed an increase in serum IgM, which was dose-responsive to both the number of injections and the amount of FD5-1 antibody injected. The serum IgM Ab3 response was found to be thymus dependent and idiotypically specific for FD5-1. Athymic mice injected with FD5-1 were unable to produce a serum IgM Ab3 response, whereas their euthymic littermates produced strong Ab3 responses. Serum Ab3 responses and Ab1' were detectable only in the IgM isotype; no specific IgG responses were observed. Indeed, IgG recognized by FD5-1 appeared to be suppressed by FD5-1. Injection of mice with FD5-1 modulated serum IgM responses to DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one (OX), phosphorylcholine (PC), and alpha 1,3-dextran (DEX) in a thymus-dependent manner. FD5-1 injection induced IgM responses against DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one, and DEX but decreased IgM binding to PC. No detectable Ab1' responses to any of the aforementioned molecules were found when the same sera were probed for IgG. The specificity of serum Ab1' from FD5-1-injected mice was evaluated by antigenic inhibition. Binding of serum Ab1' to DNP-BSA was inhibitable by DNP-lysine, whereas equivalent concentrations of lysine alone had no inhibitory effect. The antigenic specificity of IgM from normal serum binding to PC-BSA was demonstrated by inhibition with free PC, and the binding of Ab1' from FD5-1-injected mice to DEX-coated plates was shown to be inhibitable by DEX. We have described in vivo network perturbation in adult BALB/c mice injected with anti-Id antibody in the absence of Ag or adjuvants. Our findings show that injection of the cross-reactive anti-Id FD5-1 can induce thymus-dependent Ag-specific responses. In two systems where FD5-1 functions as an anti-anti-anti-Id antibody (PC and DEX), thymus-dependent responses were also observed. FD5-1 injection suppressed antibodies binding to PC, whereas DEX-specific responses were induced.

In this study BALB/c B cell precursors responsive to the T-independent (TI) type 2 (TI-2) antigen, dextran B1355S (DEX), and the T-dependent (TD) derivative, dextran-Limulus hemocyanin (DEX-Hy) were examined for isotype and idiotope expression using the splenic focus assay. The predominant isotype detected in the TI assay was IgM, while IgA was the predominant isotype expressed in the TD assay. There was also a fourfold increase in the number of foci secreting more than one isotype in the TD assay vs. the TI assay without an overall change in anti-DEX precursor frequency, suggesting that carrier-primed T cells enhance the expression of non-IgM isotypes possibly by increasing the frequency of isotype switching by individual B cell precursors. A panel of distinct monoclonal antiidiotype antibodies (MAIDs) was then used to examine idiotope expression by antibodies secreted in splenic foci responding to DEX and DEX-Hy. This analysis revealed considerable diversity in the idiotope profiles expressed by all isotypes tested. There appeared to be no differences in idiotope diversity among the various isotypes. A similar diversity of idiotope profiles was obtained from both TI and TD splenic foci, indicating that a comparable degree of diversity was associated with the antibodies generated by TI and TD precursors. Idiotype analysis of IgM-IgA-secreting foci with a panel of monoclonal antiidiotope antibodies revealed slight idiotypic differences between the two isotypes secreted in the same focus in about half the cases. These results suggest that somatic variation occurs during the antigen-driven maturation of B cell precursors, within the 15-d time frame of the splenic focus assay, and may be associated with isotype switching.

We describe a surface determinant shared by human monocytes and cells of the megakaryocytic axis that has been identified using a mouse monoclonal antibody. This monocyte-platelet antigen (MPA) is expressed on all (greater than 99%) of peripheral blood monocytes, platelets, and megakaryocytes. It is also expressed weakly on the monocytic cell line U937 and the promyelocytic line HL60 and is present on cells from 3 of 4 AML patients examined. It is absent from polymorphonuclear leukocytes, T and B lymphocytes, erythrocytes, and a panel of hematopoietic cell lines. MPA is stripped from monocyte membranes with pronase and is reexpressed overnight. The determinant is carried on a noncovalently linked biomolecular complex with molecular weights of 93,000 and 135,000.

Determining the mechanisms by which B cells develop that culminates in the generation of a diverse repertoire is critical to our understanding of how the immune response is regulated. The B cell specificity repertoire appears to be developmentally acquired in a predetermined, temporally ordered fashion. Thus, in the Balb/c mouse, the ontogenetic appearance of functional B cells specific for various hapten probes occurs in the order of dinitrophenol (DNP), fluorescein (Fl), and phosphorylcholine (PC). In addition, when the influenza virus hemagglutinin molecule was used as an antigen probe, similar conclusions were drawn regarding a patterned acquisition of the specificity repertoire. More recently, we have used a fetal organ culture system to show that hapten-responsive B cells appear in the same predictable, temporal order in vitro. The importance of these studies was that the effects of environmental influences were minimized in the absence of circulation and cell migration, and therefore the results indicated that the patterned emergence of the specificity repertoire observed was largely the result of genetic regulatory processes. Recent molecular findings may relate to such genetic regulatory processes. The VH genes in Balb/c mice have been grouped into eight families based on sequence homology, and have been mapped relative to the constant region genes. Yancopoulos et al. and Perlmutter et al. have shown that the VH gene segments closest to the JH cluster, the VH1B 7183 family, are preferentially utilized in transformed fetal pre-B cell lines and in fetal B cell hybridomas. This led to the hypothesis that the developmental control of the expression of VH gene segments is related to chromosomal organization. A logical extension of these findings is that the programmed appearance of particular clonotypes in ontogeny may be explained, in part, by the preferential use of particular VH gene segments. However, to what extent the transformed B cell lines represent members of the functional expressed repertoire could not be evaluated. In the studies described herein, the fetal organ culture system was used to assess the early expressed repertoire at the clonotypic level using idiotypic analysis. Anti-DNP secreting clones were derived from fetal organ cultures and tested for the presence of two idiotypes, 36 and MOPC 460 (460). The 36 idiotype is a predominant DNP clonotype of the neonatal repertoire, while the 460 idiotype is a major cross-reactive idiotype of the adult DNP response.(ABSTRACT TRUNCATED AT 400 WORDS)

Paired immunofluorescent staining with antibodies specific for the major isotypes of mouse immunoglobulin was used to study the ontogenetic expression of diversity of cell surface immunoglobulin. The first B lymphocytes to emerge, derived from cytoplasmic IgM+ precursors, express sIgM exclusively. Between birth and 3 days of age separate populations of sIgM+ B lymphocyte acquire a second isotype: sIgD, one of the subclasses of sIgG, or sIgA. At 3 days, all splenic B lymphocytes that bear sIg or sIgA also express sIgM, but virtually none stain for sIgD. By 7 days, a substantial porportion of sIgG+ or sIgA+ lymphocytes in spleen and most of those in lymph node express both sIgM ans sIgD. Anti-mu antibody treatment from birth prevented development of B lymphocytes expressing any isotype. These observations suggest that the immature sIgM+ B lymphocyte is the pivotal cell in the generation of the different sublines of B cells and that sIgD ig or IgA. The frequency of lymphocytes bearing only sIgG or sIgA is higher in old than in young mice, suggesting that sIgD and sIgM may be lost after stimulation by antigens. The occurrence of a nearly identical distribution of sIg isotypes on B lymphocytes from athymic, pathogen-free mice suggests that primary expression of isotype diversity does not require T cells.

We have used two rat monoclonal antibodies (Mab) (Bet-1 and 331.12), a mouse Mab (AF6-78.25), and an alloantiserum (SJA anti-BAB/14) in two-color immunofluorescence analysis and enzyme-linked immunosorbent assays to examine the expression of IgM allotypes at various stages of mouse B cell development, especially at the pre-B cell stage. In agreement with findings previously reported by others, these antibodies displayed the patterns of allotypic reactivity with IgM+ B cells and plasma cells from the appropriate strains of mice: Bet-1 antibody is specific for a allotype, AF6-78.25 and SJA anti-BAB/14 for b allotype, and 331.12 for both a and b allotypes but not e allotype. These antibodies, however, did not react with isolated mu-heavy chains prepared from secreted molecules or synthesized in situ by normal pre-B cells or pre-B cell-derived hybridomas. The requirement of light chain participation in the expression of IgM allotypic determinant(s) was additionally suggested by analysis of a fetal liver-derived hybridoma (F1-26-11) that has been shown to secrete IgM heteromolecules composed of C57BL/6-derived mu-chain and BALB/c-derived kappa-chains. In contrast to the nonreactivity with C57BL/6 mu-only pre-B cell-derived hybridomas, all three antibodies with specificity for the b allotype (AF6-78.25, SJA anti-BAB/14, and 331.12) reacted with this particular hybridoma. After treating surface IgM molecules on B lymphocytes with papain to cleave Fab fragments, the b allotype reactivity of AF6-78.25 mouse Mab, but not 331.12 rat Mab, disappeared, whereas the a allotype reactivities of two rat Mab (Bet-1, 331.12) were not altered. These results suggest that the IgM allotypic epitopes defined by these antibodies appear to be sterically dependent on the assembly of the whole IgM molecules, and rat Mab 331.12 and mouse Mab AF6-78.25 define two separate b allotype specificities, one on the Fc portion and the other on the Fab portion of the IgM molecules, respectively.

Monoclonal anti-DNA antibodies from 13 cloned hybridoma cell lines have been used to analyze the clonal heterogeneity of autoimmune anti-DNA antibodies. The hybridomas were obtained by fusing spleen cells from a single, autoimmune (NZB X NZW)F1 mouse. With the use of the criteria of isotype, isoelectric focusing patterns, fine specificity for various nucleic acids, and idiotype, the monoclonal anti-DNA antibodies were found to be heterogeneous in that 12 different clonotypes were identified among the 13 monoclonal antibodies studied. However, eight out of the 13 monoclonal antibodies have similar antigen specificity and share a common idiotype. This suggests that although autoimmune anti-DNA antibodies within a single (NZB X NZW)F1 mouse are the products of a large number of different antibody-producing clones, many of the clones produce antibodies with similar antigen-binding regions. Furthermore, in the majority of (NZB X NZW)F1 mouse sera the appearance of detectable levels of the common idiotype coincides with the appearance of anti-DNA autoantibody.

C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed.

The potential light chain repertoires expressed by IgM antibodies of fetal liver and adult spleen cells were compared. B cells from these sources were stimulated by the polyclonal mitogen bacterial lipopolysaccharide and after 3 days were fused to construct hybridomas. The light chains synthesized by these hybridomas were isolated by electrophoresis and their heterogeneity was examined by isoelectric focusing. A total of 109 BALB/c and 77 CBA/J hybridoma-derived light chains obtained from fetal liver and adult spleen cells were analyzed in this way. Fifty-three and 45 unique light chains were obtained from BALB/c and C57BL/6 hybridomas, respectively. Some of these were repeated more frequently than others, but no significant differences were seen in the heterogeneity of the light chain spectrotypes of fetal and adult CBA/J and BALB/cJ mice. Furthermore, the kappa:lambda ratio of light chains were the same in fetal liver as adult-derived hybridomas. These results show that the heterogeneity of light chains as determined by IEF and the kappa:lambda ratio of IgM immunoglobulins is established very early in ontogeny.

Molecular relationships between beta2m and other cell surface antigens (H-2, Tla, Ia, and Thy-1) were studied with the double immunofluorescence method. Cells were incubated with an antiserum against one antigen capped, and then tested with an antiserum against a second antigen. Capping of beta2m on thymocytes led to simultaneous capping of H-2 and Tla but not Thy-1 antigens; capping of H-2 and TIa (but not Thy-1) antigens resulted in capping of all beta2m detectable by the immunofluorescence method. Similarly, capping of beta2m on B or T lymphocytes resulted in capping of H-2 and vice versa. Ia antigens on B lymphocytes were not capped after the redistribution of beta2m. We conclude from these data that, in the cell membrane of thymocytes, virtually all the beta2m molecules are associated with H-2 and Tla, but not with Thy-1, and that on the cell surface of T or B lymphocytes, virtually all beta2m is associated with H-2 but not with Ia. We found no evidence of any significant free beta2m on either thymocytes or splenocytes.

Monoclonal IgM antibodies with specificity for Moloney murine sarcoma virus (M-MuSV)-Moloney murine leukemia virus (M-MuLV) from two hybridoma clones have been isolated and characterized. The monoclonal antibodies have specificity for a cytoplasmic and cell surface Friend-Moloney-Rauscher group-specific antigen. Immunoelectron microscopy revealed antibody binding to the surface of virus-expressing cells but not to the budding virus particles. Treatment of M-MuSV-injected mice with monoclonal IgM anti-M-MuSV significantly inhibited tumor growth compared to virus-inoculated animals receiving either saline or MOPC 104E. Nude mice exhibited delayed tumor induction following treatment with the monoclonal antibodies but ultimately died from tumor growth. Virus-injected euthymic mice that were treated with monoclonal IgM anti-M-MuSV generated a potentiated spleen cell-mediated cytotoxicity against Moloney sarcoma cells compared to virus-infected treated with saline. This potentiation of cytotoxicity remained after trypsinization of the spleen cells and thus was probably not due to passively adsorbed monoclonal antibody. The antibodies alone or in the presence of complement did not neutralize M-MuLV. The IgM antibodies induced specific tumor cell cytotoxicity in vitro mediated by complement spleen cells, lymph node cells, or thymus cells. In conclusion, two monoclonal IgM anti-M-MuSV antibodies that bind to the tumor cell surface did not neutralize virus can inhibit primary M-MuSV-induced tumor growth in vivo. The regression event appeared to involve heterogeneous mechanisms. Complete regression remained thymus dependent even with passive antibody therapy, but significant tumor growth inhibition was produced independent of T-cells. In vitro these IgM antibodies induced complement and cell-mediated cytotoxicity.

Direct immunofluorescence was used to determine the classes of immunoglobulins expressed on the surface membrane and in the cytoplasm of newborn and adult B lymphocytes differentiating in response to LPS in vitro. In both newborn and adult spleen, a small proportion of IgM-bearing B lymphocytes also stained for IgG2; adult spleen contained an additional population of lymphocytes bearing IgG2 alone. Combined surface and cytoplasmic staining at intervals after culture initiation and demonstrated both IgM and IgG2 on the membranes of the earliest cells synthesizing cytoplasmic IgG2. At later stages the proportion of IgM-IgG2 surface doubles and of cells synthesizing cytoplasmic IgG2 which had surface IgM fell significantly. Detection of surface IgM on IgG2 precursors correlated with susceptibility of the IgG2 precursors to anti-mu-suppression over the first 3 days in cultures of newborn spleen cells. After 3 days when these cells no longer expressed surface IgM, 2gG2 responses were not suppressed although the IgM response was still inhibited. These results suggest that virgin IgG2 precursors may be B lymphocytes expressing both IgM and IgG2, and that "switching" involves the loss of IgM receptors as these cells proliferate and mature.

Monoclonal antibodies to idiotypic determinants are being used with increasing frequency for analysis and treatment of B cell malignancies. In the present study we have compared the idiotypic specificities of a panel of 39 mouse monoclonal anti-idiotype (anti-Id) antibodies developed against 16 monoclonal human immunoglobulins (Ig). The Id cross-reactivities of these antibodies with Ig products of normal and abnormal B cells were examined by immunofluorescence and immunochemical methods. The reactivity patterns of these anti-Id antibodies with a normal population of plasma cells were highly variable in the immunofluorescence assay. Six were reactive with 2 to 10% of normal plasma cells, 30 with 0.1 to 2% of plasma cells, and three with less than 0.1% of plasma cells from blood, bone marrow, spleen, or tonsils. These reactivity patterns were relatively consistent among samples from 23 Caucasian, black, and Oriental adults. Although the reactivities of most anti-Id antibodies in the panel were not restricted to a particular Ig isotype, several were preferentially reactive with a particular heavy or light chain isotype: one IgM-, two IgA-, two kappa-, and three lambda-restricted antibodies. The immunofluorescence data was confirmed by biosynthetic analysis of Id+ molecules produced by a normal plasma cell population. When the reactivity of this panel of anti-Id antibodies with nonhomologous B cell neoplasms was examined, seven of 30 myelomas or leukemia-derived products and one of nine B cell leukemias or lymphomas without paraproteins were found to be cross-reactive with one or two of the anti-Id antibodies. Although clearly significant, the cross-reactivity between the Id of these paraproteins appeared to be of lower affinity than the reactivity of the homologous Id with their respective anti-Id antibodies. The results reveal a remarkable diversity in the specificities of monoclonal antibodies classified by conventional criteria as anti-Id antibodies, and indicate the potential usefulness of a panel of antibodies for analyzing clonal diversity in normal and abnormal B cell development.

Monoclonal hybridoma antibodies to the human IgA subclasses were produced by immunizing mice with purified myeloma proteins. These antibodies were shown to be specific for the appropriate IgA subclass by enzyme-linked immunoabsorbant assay (ELISA) and by immunofluorescent staining of myeloma plasma cells and B cells from normal individuals. These antibodies were used to demonstrate age-related shifts in the proportions of IgA1- and IgA2-bearing B cells that could be correlated with 3 distinct staining patterns. In the newborn equal numbers of IgA1 and IgA2, B cells were found. These cells had only small amounts of surface IgA in a patchy distribution. They also expressed surface IgM. In the infant, large lymphoblastoid cells were observed that bore more IgA in a homogeneous pattern but did not express IgM. Of these cells, 98% were positive for IgA1. In the adult, 80% of the IgA B cells were positive for surface IgA1, and 20% were positive for IgA2. These were small lymphocytes brightly stained for IgA and negative for IgM. In culture, the adult B cells responding to pokeweed gave rise to roughly equal numbers of IgA1 and IgA2 plasma cells. These results suggest that there are equal numbers of precursor cells for IgA1 and IgA2 whose expansion, further differentiation, and migration are selectively affected by immunoregulatory controls.

Considerable heterogeneity in the amount of surface antigen can regularly be demonstrated by cytofluorometric analysis among genetically identical cells in a tumor clone. We have used monoclonal idiotype-specific antibodies to investigate the patterns of change in amounts of an idiotypic tumor antigen and how such changes affect the immune escape of malignant B cells expressing this antigen. By the use of fluorescence-activated cell sorting, we separated cells expressing either very large or very small amounts of the idiotypic target antigen and then analyzed these subpopulations of tumor cells at various times after isolation for expression of idiotype. We found that the differences in amount of antigen expression were not heritable, and that over a period of about 7 days of continuous growth in vitro, the fluorescence-activated cell sorted populations gradually came to express normal amounts of idiotype. The mechanisms regulating the quantity of surface idiotype were independent of those affecting the amounts of other membrane-associated molecules such as H-2 antigen. Furthermore, these nonheritable intraclonal differences in amounts of antigen expression were unrelated to stages of the cell cycle but clearly did affect the susceptibility of the cells from immune lysis. Thus, tumor cells expressing the lowest amount of surface idiotype were much more resistant to the lytic effects of antiidiotypic antibody and complement but had the same cell cycle distribution as did unseparated cells. These results demonstrate that nonheritable, non-cell cycle-related heterogeneity in amount of tumor antigen expression can significantly determine which cells in a cloned malignant cell population preferentially escape monoclonal tumor-specific antibody therapy.

The antibody response to alpha 1 leads to 3 dextran (DEX) in BALB/c mice consists of a family of closely related yet highly heterogeneous molecules. Although these antibodies have been previously characterized both idiotypically and structurally, detailed analysis of responding clones has not been possible using conventional anti-idiotype antibodies. Monoclonal syngeneic and allogeneic anti-idiotype antibodies (MAIDs) specific for anti-DEX antibodies were used in this study to dissect the serum antibody response to DEX in BALB/c mice. The constructed MAIDs showed considerable heterogeneity by isoelectric focusing and by their binding characteristics to a series of DEX specific myeloma and hybridoma proteins. The predominant heavy chain isotype of these MAIDs was gamma 1. These antibodies were used to identify individual idiotypic structures (IdI) on J558, or M104E as well as cross-reactive determinants common to both (IdX). Although both IdX and IdI MAIDs were obtained, IdI specific antibodies were obtained more frequently. BALB/c mice immunized with DEX produced antibodies expressing both IdI but in highly variable amounts. A large percentage of, but not all DEX specific antibody, could be accounted for by IdX bearing antibodies. Suppression of adult and neonatal mice by IdI specific MAIDs was effective with precise elimination of only those clones expressing IdI determinants leaving the total lambda bearing anti-DEX response intact. Suppression of adults and neonates by an IdX specific MAID resulted in a temporary and partial suppression of the total lambda bearing anti-DEX response along with total suppression of the IdX portion of the response. Unlike other systems these monoclonal antibodies produce only suppression, and under a variety of conditions enhancement of anti-DEX responses has not been observed.

Lipopolysaccharide-(LPS) induced differentiation of mouse B lymphocytes to cells synthesizing large amounts of cytoplasmic IgM and IgG2 could be suppressed by antibodies to mu-chains. Maximal inhibition of LPS-induced differentiation was associated with increased cellular proliferation as measured by incorporation of 3H-thymidine, whereas treatment with anti-mu alone over a wide dosage range did not stimulate cellular proliferation. Spleen cells from newborn mice were suppressed by concentrations of anti-mu several hundred-fold lower than required for adult spleen cells; the adult pattern of susceptibility to suppression was acquired by 1 week of age. No significant differences in susceptibility to anti-mu were found in comparisons of adult spleen, lymph node, bone marrow, and Peyer's patch lymphocytes.

Cultured cells from mouse thoracic duct, spleen, lymph node, Peyer's patches, and bone marrow gave rise to cells containing large amounts of cytoplasmic IgM, IgGl, and IgG2 when stimulated by bacterial lipopolysaccharide (LPS). Differntiation of IgA producers occurred in bone marrow only, and to a lesser extent than other classes. Significant IgM responses preceded development of cells containing other classes. The differentiation of IgG and IgA producers did not appear to depend on T cells, since cultures from nu/nu or thymectomized-irradiated, bone marrow-protected mice responded as well as normals. Cultures from mice rendered deficient in B cells by anti-mu treatment responded normally to T cell mitogens, but did not proliferate or give rise to immunoglobulin-secreting cells when stimulated with LPS. Bone marrow cultures gave relatively meager proliferative responses to LPS, but generated as many or more immunoglobulin-secreting cells as did other tissues.

Cultures of mouse fetal liver and spleen, stimulated by bacterial lipopolysaccharide, gave rise to plasma cells staining for IgM, IgG2, IgG1, and IgA. Cells containing Igm and IgG2 were found in cultures from 17-day fetuses, coincident with the appearance of B lymphocytes bearing cell-surface IgM. IgG1- and IgA-containing cells were induced in cultures from 19-day fetuses and 1-day-old mice. The capacity to give rise to immunoglobulin-secreting cells of all classes preceded the development of a significant proliferative response to LPS; the proportions of cells staining for each class reached adult values by 1 day of age whereas the proliferative response did not mature until 3 weeks.

Our findings indicate that a subset of VHIII antibodies, which we refer to as J606-GAC, contains a determinant(s) that is present on 5 to 15% of normal splenic B cells and plasma cells as detected by immunofluorescence. This subpopulation is detected by purified antibody, 0-1, which was prepared against a murine anti-group A carbohydrate (anti-GAC) hybridoma antibody. The J606-GAC subset includes the beta 2, 1 fructosan myelomas J606, EPC109, W3082, ABPC4, and UPC61, as well as 13 anti-GAC hybridomas. The 0-1 antiserum failed to react with hybridoma and myeloma immunoglobulins from murine VH groups I and II or other VHIII antibodies. By Western blot analysis, it was observed to react with isolated heavy, but not light, chains of J606-GAC-bearing antibodies. 0-1 failed to react with myelomas XRPC44 and J539, which have the same J region as J606 but a very different VH region. These observations indicate that 0-1 is detecting a VH region determinant. The J606-GAC marker recognized by 0-1 was expressed as early as 4 days after birth and was expressed at similar frequencies in germfree and conventional mice. Immunoprecipitation of both surface and biosynthetically labeled proteins from spleen cells or J606-GAC-positive hybridoma cell lines, respectively, confirmed that 0-1 was recognizing an immunoglobulin determinant.

By the analysis of hybridomas constructed from B cells early in development we have shown that: (i) the early neonatal B cell repertoire consists of a highly autoreactive set of B cells showing extensive multispecificity and interconnectivity; (ii) many of these antibodies express anti-idiotypic activity towards autologous germline-encoded idiotypic antibodies; (iii) the anti-idiotypic activities of such B cells and/or their antibody products play a major role in establishing the clonal dominance of certain well-characterized idiotypes in the responses to phosphorylcholine (PC) and alpha 1----3 dextran; and (iv) results obtained in comparisons between antibodies to the acetylcholine receptor and alpha 1----3 dextran in humans and mice showed extensive idiotypic connectivity. Some of the anti-idiotypic specificities involved were also apparent in the neonatally derived antibodies. These results suggest that there are extensive idiotype-directed interactions between B cells early in development which appear to be essential for establishing the adult B cell repertoire and the accompanying clonal dominance of appropriate idiotypes. Similar interactions may also play a role in the development of certain autoimmune disorders.

The clonal origin of an IgA1 kappa B cell leukemia in a 71-year-old man (WF) was examined using a monoclonal anti-Id antibody and a panel of monoclonal anti-VH antibodies. Immunofluorescent studies revealed that all surface IgA1 kappa + leukemic cells in WF's blood and 10% of the IgM+ B cells in his bone marrow expressed the WF Id. Three percent of the IgA1 kappa + leukemic cells in blood also expressed gamma-chains in their cytoplasm. Approximately 0.1%, 1%, and 10% of bone marrow mononuclear cells, respectively, expressed mu-chains, gamma-chains, and alpha-chains in their cytoplasm, but no detectable light chains or surface immunoglobulins. These mu, gamma, and alpha-positive cells had the convoluted nucleus and narrow cytoplasm characteristic of normal mu+ pre-B cells. Sequential isotype switching among this unusual pre-B population was indicated by co-expression of mu-chains and alpha-chains by 11% and 63%, respectively, of the gamma pre-B cells. These pre-B cells and the surface alpha-chains and cytoplasmic gamma-chains of the leukemic B cells were reactive with one of four monoclonal anti-VH antibodies. The data suggest malignant transformation of the clone before isotype switching, and also imply light chain precommitment at the pre-B cell level of differentiation.

By fusing BALB/c splenic lymphocytes from mice immunized with phosphorylcholine (PC) to an immunoglobulin nonproducing plasmacytoma cell line, a B cell hybridoma was isolated (MM-60) that has been shown by multiple criteria to produce a bona fide auto-anti-(anti-T15 idiotype) antibody. In vivo administration of MM-60 antibody suppressed T15+ anti-PC antibody production in an idiotope-specific manner by activation of an intervening set of anti-T15 B cells. These T15-specific B cells i) appeared to express germline-encoded variable region gene products, ii) developed in parallel to, but independent of, T15+ B cells, and iii) suppressed the anti-PC response in a T cell-independent fashion. Variants of T15+ anti-PC B cells possessing aberrant immunoglobulin heavy chain D region structure escaped from the suppression imposed by this anti-T15 B cell set, suggesting that a function of the heavy chain D region may be to contribute to the formation of molecular target sites for idiotype-directed regulatory cells and/or antibodies. The indigenous nature of these particular populations of anti-idiotypic and anti-(anti-idiotypic) B cells and the ability of their immunoglobulin products to regulate antigen-specific B cells in vivo provides strong supportive evidence for the significant role idiotype-directed network interactions play in regulating specific antibody production during a normal immune response.

We investigated the effect of three monoclonal anti-idiotype antibodies (anti-M104E) on various functions of MOPC 104E myeloma cells in vitro. The antibodies used were N-20-2 [immunoglobulin M (IgM), BALB/c], SJL18-1 [IgM, BALB/c X SJL F1], and CD3-2 [immunoglobulin G1 (IgG1), BALB/c X A/J F1]. The two IgM antibodies were very efficient in blocking surface M104E IgM as shown by rosette inhibition, whereas the IgG1 isotype was not very effective. The reexpression of surface M104E IgM was different from antibody to antibody. The secretion of M104E IgM by MOPC 104E cells was partially blocked by the two IgM antibodies, but the IgG1 antibody had no effect. All three anti-idiotype antibodies inhibited the stem cell renewal activity of MOPC 104E cells assayed by colony formation assay. On the other hand, in suspension culture, the two IgM antibodies inhibited the growth of MOPC 104E cells in the absence of complement or effector cells of antibody-dependent cellular cytotoxicity, but IgG1 antibody had no effect. The starting tumor inoculum size was critical in the observations of the effects seen on both the growth and the colony-forming activity of MOPC 104E cells. The results of this study show the functional differences between various monoclonal anti-idiotype antibodies and also indicate that some anti-idiotype antibodies can inhibit the growth of MOPC 104E myeloma cells directly without any help of complement or effector cells of antibody-dependent cellular cytotoxicity.

A large panel of enteric organisms was screened for agglutination with a panel of lambda monoclonal antibodies of different heavy chain isotypes specific for alpha 1-3 dextran (DEX). Two strains were initially isolated that were bound by most of the anti-DEX antibodies. One organism, Enterobacter cloacae strain MK7, which was characterized in detail, induced a typical lambda anti-DEX response in Igh-Ca mice that had a fine idiotope profile comparable with that induced by purified B1355S dextran containing alpha 1-3 glucosidic linkages (alpha 1-3-DEX). The determinant on the bacterial surface was shown by binding inhibition with nigerotriose to contain alpha 1-3 linkages. Hyperimmunization with these organisms of normal, athymic (nu/nu), or germ-free mice induced large amounts of IgM antibodies but very little IgG. This is the first description of an organism isolated from the normal gut flora of mice that can be shown directly to be bound by alpha 1-3-DEX antibodies and to induce the typical germ-line response of the DEX family of antibodies.

Chronic treatment of mice from birth with anti-mu antibodies aborts development of B lymphocytes and plasma cells. In these studies we show that bone marrow from anti-mu-treated mice contains a population of cells with cytoplasmic IgM, but which lack detectable cell-surface IgM. These cells are analogous to pre-B cells, defined in ontogenetic studies as the immediate precursors of B lymphocytes. Pre-B cells from bone marrow of anti-mu treated mice retain their functional integrity, as evidenced by their ability to give rise to sIgM+, LPS-responsive lymphocytes in culture. We also show that cyclophosphamide treatment destroys pre-B cells and that recovery of pre-B cells in bone marrow precedes the regeneration of sIgM+ B lymphocytes. Generation of B lymphocytes in adult mice apparently occurs exclusively in the bone marrow because induction of extramedullary hemopoiesis in spleen was not accompanied by the appearance of pre-B cells in that organ.