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Somatic mutation of immunoglobulin (Ig) genes plays an important role in generating antibody diversity. The frequency of somatic mutation appears to vary throughout life. However, this process has been difficult to study in vivo because the DNA in and around rearranged V genes undergoes random mutation, causing silent or replacement mutations. Therefore, we have developed a transgenic mouse model for studying the frequency of B cells exhibiting mutation in young and old mice. The system is based on a reporter transgene (HuG-X) that encodes a chimeric Ig heavy chain composed of a murine VDJ segment and a human IgG1 constant region. The VDJ has been mutated to contain a TAG stop codon in the D segment. Therefore, the transgene is transcribed but not translated. Point mutation of the stop codon results in expression of the chimeric H chain, which is readily detected as human IgG1 expression. In vivo, we found that the transgene undergoes spontaneous reverse somatic mutation at a low frequency. Treatment of HuG-X mice with anti-IgD greatly increases the frequency of somatic mutation. The observed mutation frequency in anti-IgD-treated mice increases with age until adulthood, then plateaux and finally declines in aged mice. The mutations in the stop codon were associated with increased double-stranded DNA breaks (DSB) within and around the TAG site. Our results demonstrate that the rate of frequency of spontaneous reverse mutation is very low in vivo, yet it is significantly increased after stimulation with anti-IgD antibodies. The frequency of point mutation is age dependent and correlates with increased DSB.

Neonates display lower immune responsiveness and higher susceptibility for high-dose tolerance. Quantitative as well as functional differences between the neonatal and adult lymphocytes or antigen presenting cells (APC) respectively, explain the particular immune responsiveness during the early stages of the postnatal development. Reduced numbers of lymphocytes and APCs as well as a modified responsiveness of T cells in neonates, are the main factors that account for the low threshold of tolerance in newborns. Taking into account these particularities, the design of effective vaccines for neonates poses significant difficulties. We hypothesized that a continuous exposure to low doses of antigens may avoid high-zone tolerance and may lead instead, to effective expansion of effector and memory cells. Indeed, inoculation of newborn mice with plasmids encoding nucleoprotein (NP) or hemagglutinin (HA) of influenza virus, led to the priming of specific cytotoxic (CTL), helper (Th) and B cells, rather than induction of unresponsiveness. Mice immunized as neonates with naked DNA and challenged later with lethal doses of influenza virus, displayed significant protection. Thus, DNA immunization may be a promising strategy for vaccination against serious infectious diseases of infants and children.

Motheaten mice homozygous for the recessive mev mutation develop a fatal immunodeficiency syndrome associated with hypergammaglobulinemia, thymic aplasia, production of autoantibodies and development of a severe lupus like systemic autoimmune disease. Most B lymphocytes in this mutant strain belong to B-1 subset. We have addressed the question if differences existed in the V-gene repertoire of autoimmune mev/mev mice as compared to phenotypically normal mev/+ and C57BL/6 background strain by examining the VH and V kappa gene family expression as well as the association of VH and V kappa gene families among B lymphocyte clones. The data outlined here demonstrate that both the expression of VH and V kappa gene families and their association is skewed in mev/mev mice, suffering from systemic autoimmune disease, and differs significantly from phenotypically normal mev/+ litter mates as well as the C57BL/6 background strain. In addition, VH+V kappa gene family pairs in phenotypically normal mev/+ differed from normal C57BL/6 mice suggesting that motheaten mutation, whether homozygous or heterozygous, alters the development of the B lymphocyte repertoire. These observations suggest positive selection of B-1 lymphocytes in autoimmune motheaten mice either as a result of selective processes, via receptor-ligand interactions, operating on the development of the primary antibody repertoire or defective B lymphocyte haematopoiesis due to the deficiency of haematopoietic cell phosphatase involved in determining the threshold by which B cells respond to self antigen(s).

The contribution of VH11 gene family to the development of the primary B cell repertoire has been studied by analyzing 1.8 x 10(4) mitogen induced B lymphocyte colonies. The data demonstrate that VH11 family is predominantly expressed among neonatal splenic as well as adult peritoneal B cell colonies, both rich in Ly-1+ B cells. VH11 gene family expression among B splenocytes decreases during ontogeny and VH11 family pairs stochastically with different V kappa families among mitogen-activated neonatal B cell colonies, which are representative of an antigen unselected B cell repertoire. Thus, an increased VH11 expression among peritoneal and neonatal B cells points towards its biased expression among Ly-1+ B lymphocytes. The restricted V gene rearrangements and VH11-V kappa 9 pairing observed among anti-bromelain-treated mouse red blood cells autoantibodies are likely to be an outcome of both intrinsic gene recombination processes per se as well as selection by an autoantigen and/or local selective environmental factors.

Malignant CD5 B cells obtained from patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) were analyzed for immunoglobulin variable gene usage, CD5 gene expression and autoantibody production. A statistically significant biased usage of the VH5, VH6 and VKIII immunoglobulin variable gene families was observed. It is important to point out that both VH5 and VH6 are extremely small families which are located at the 3' extremity of the immunoglobulin variable gene locus. We determined that the transcription of the CD5 gene in T cell malignancies, CLL, SLL and a selected group of EBV transformed lines was identical. Autoantibody production was studied in a panel of heterohybridomas obtained by the fusion of CLL cells with mouse myeloma line SP2/0. A large fraction of these heterohybridomas secrete autoantibodies; some were monospecific, some bispecific and some polyspecific.

Frequencies of 25 immunoglobulin heavy-chain and kappa light-chain variable (VH + V kappa) gene-family pairings expressed in splenic B-cell populations were determined by hybridization of VH- and V kappa-family-specific DNA probes to mitogen-induced B-cell colonies from C57BL/6 mice or hybridomas derived from BALB/c and NZB mice. Both analyses support the conclusion that VH and V kappa gene families pair without bias; as would be expected for random association, the frequencies of specific VH + V kappa pairs may be estimated by the product of the independent VH and V kappa frequencies. Based upon the frequencies at which 9 VH and 12 V kappa gene families are expressed, we calculated the expected usage for approximately 100 VH + V kappa family pairings in neonatal and adult C57BL/6 mice. Variability in the expression of such VH + V kappa pairings is considerable; pairs representing greater than 10% to less than 0.01% of the splenic B-cell population occur. This variability is most pronounced in the neonate, where 6 VH + V kappa family pairs account for nearly 40% of all mitogen-reactive B cells. As the neonate matures, the distribution of frequencies for VH + V kappa pairings becomes more nearly uniform. This process may underlie the patterned acquisition of humoral immune responsiveness.

CBA/N female mice, which express an X-linked defect in B-lymphocyte function, were mated with C3H/HeJ male mice, which are unresponsive to lipopolysaccharide (LPS). The resulting F1 hybrid females were mated to C3H/HeJ males. Approximately one-half of the backcross (BC.1) males obtained from this mating expressed a more profound immunologic defect than either of the parental strains. Spleen cells from these mice were unresponsive to a series of B-cell mitogens including LPS prepared from Escherichia coli K235 and from E. coli 0111:B4, lipoprotein mitogen from E. coli, and Nocardia water-soluble mitogen (NWSM). They failed to give in vitro antibody responses to the thymus-independent type 2 (TI-2) antigen trinophenylated Ficoll and most were unresponsive to the TI-1 antigens trinitrophenylated Brucella abortus, trinitrophenylated LPS, and trinitrophenylated NWSM. This synergistic defect in B-lymphocyte function depended on the presence of the CBA/N xid gene but the critical gene(s) from the C3H strain was not the defective Lps gene (Lpsd). These mice should provide a valuable tool for the elucidation of B-lymphocyte ontogeny, heterogeneity, and function.

Secific stimulation of T-cells by PPD was inhibited by their autologous B cells. This inhibition was obtained with B cells separated either by depletion of E-RFC or by elution, with human IgG of lymphocytes bound to Sephadex beads coated with rabbit antibodies anti-human Fab fragments. The suppression was proportional to the number of B cells added to 10(6) T cells incubated with PPD and as previously reported was more marked in the case of B or T cells from BCG-vaccinated subjects with negative skin tests. The suppressive phenomenon required viable B cells and was inhibited by cycloheximide but was not altered by pretreatment of suppressor cells with actinomycin D or colchicine. It seems that B-suppressor cells interfere with recognition of PPD by T cells rather than with the proliferative phase of the specific blast response. Using various surface markers (i.e. Ig, C3 and Fc receptors) it was shown that the suppressor cells represent a subset of Ig-bearing B cells which do not carry Fc receptors.

Nocardia water soluble mitogen (NWSM), a mitogen specific for mouse and rabbit B cells, activates blood, spleen, and tonsil lymphocytes from all the humans tested. Evidence is presented that it acts selectively on human B lymphocytes. B-enriched cells were recovered from anti-Ig-coated Sephadex columns or after elimination of E rosette-forming cells: they were able to incorporate tritiated thymidine when they were cultivated in the presence of NWSM whereas they were not activated by PWM. Conversely T-enriched suspensions did not respond to NWSM although they were able to be stimulated by PHA and PWM. Moreover, the response to NWSM remained unchanged after T cells had been killed by anti-HTLA serum and complement and disappeared after B cells had been killed by anti-HBLMA serum. After stimulation by NWSM, the number of direct plaque-forming cells to HRBC, LPS, and TNP was significantly increased, showing a polyclonal activation of human B lymphocytes. Since no evidence of T cell help for B cell activation has been found, NWSM thus appears to be a T-independent human B lymphocyte mitogen.

Autoantibodies play an important role in the pathogenesis of type II collagen-induced arthritis in mice. We have earlier reported a high frequency of cells producing anti-CII autoantibodies and a low frequency of cells producing multispecific antibodies, in regional lymph nodes 9 to 11 days after primary immunization with CII. It is shown here that anti-CII antibodies produced during primary immune response are IgG-antibodies mainly of IgG2a, IgG1 and IgG2b subclasses while IgM antibodies dominate primary responses elicited by OVA and denatured CII as analyzed with a large panel of hybridomas. Anti-CII antibodies generated during the primary response recognize at least five different epitopes on the CII molecule. The specificities of these antibodies for various epitopes result from combinational association of products encoded by genes derived from various VH and VK families and/or by the occurrence of somatic mutations. It is suggested that the primary anti-CII autoantibody response involves activation of memory B cells and is in this aspect different from the origin of "natural" autoantibodies.

We investigated the proliferative responses and immunoglobulin production of highly purified E-rosette negative largely leukemic B cells from patients with CLL to Staphylococcus aureus Cowan I (SAC) or to SAC in combination with anti-mu or conditioned medium (CM). The latter was derived by stimulating human peripheral blood mononuclear leukocytes with PHA. We observed: (1) that purified E-rosette negative largely leukemic B cells from 25% (five out of 20) of the patients exhibited proliferative responses to SAC; (2) inhibition of SAC-induced proliferation by anti-mu in certain patients, whereas synergism between SAC and anti-mu in inducing proliferative responses in others; (3) the lack of synergism between SAC and CM in inducing proliferative responses, which is in contrast to the strong synergism that was observed between anti-mu and CM in inducing proliferation; and (4) induction or enhancement by SAC alone of Ig production by largely leukemic B-cell populations from few patients with CLL and purified tonsillar B lymphocytes, but not peripheral blood B cells from normal donors. These results suggest that SAC and anti-mu induce proliferation of B cells by different mechanisms and that B-cell proliferation and differentiation is dependent not only on the mitogen but also on the activation state of the cells.

Several idiotypic (Id) specificities have been identified by immunofluorescence on the membranes of B cells in the spleens of nonimmune mice. These determinants are displayed at frequencies varying from 0.22 to 1.83% of B cells and are entirely immunoglobulin (Ig) in nature. No Id determinants were observed on the membranes of four Slg- Abelson virus-transformed pre-B cell lines that are sensitive to LPS. Under our experimental conditions, we did not observe a significant increase in 3H-thymidine incorporation subsequent to the in vitro incubation of splenic lymphocytes from normal mice with various amounts of anti-Id antibodies specific for several cross-reactive (IdX) or individual Id. Similarly, in utero exposure to anti-Id antibodies against myeloma proteins specific for T-independent antigens displaying B cell mitogenic properties did not alter the proliferation of lymphocytes induced by these mitogens. In contrast, exposure to anti-Id antibodies in vitro as well as in utero had a profound and specific effect on the corresponding antibody responses and maturation of small lymphocytes into plasma cells. When normal B cells were cultured with the B cell mitogen LPS in the presence of anti-Id antibodies directed against the J558IdX, specific suppression of the maturation of IdX+ plasma cells was observed. In mice exposed to maternal anti-Id in utero, we observed a severe inability to mount an immune response to any compound that elicited antibody molecules bearing the Id that was complementary to the maternal anti-Id. These same maternally Id-suppressed mice, however, gave a normal response when the same compound was presented as a mitogen. Our results reinforce the concept that Id on membranes of B lymphocytes are associated only with Ig receptors and do not support the possibility of mitogen-like poly-clonal stimulatory properties of anti-Id antibodies.

Introduction of the CBA/N X-linked gene into C3H mice has resulted in the establishment of a new strain of mice that has profound immunologic defects. B cells from these mice show significantly impaired in vitro immune responses to the T cell-independent type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA) as well as markedly reduced proliferative responses to a number of B cell mitogens when compared with the responses of the parental control mice. The in vivo response of such mice to TNP-BA is, however, comparable to that of CBA/N mice. Furthermore, B cells from C3.CBA/N mice are unresponsive to the plaque-forming cell enhancing effects induced by EL4-derived supernatant in the presence of TNP-BA, unlike B cells obtained from CBA/N or C3H/Hen mice whose responsiveness to TNP-BA can be significantly enhanced in the presence of EL4-derived supernatant. The model we have presented to best explain these results suggests that B cells from C3.CBA/N mice can be stimulated only under conditions in which they can interact with carrier-specific T cell help and not under conditions where factor-dependent responses are dominant.

The B-cell mitogens lipopolysaccharide (LPS), Nocardia water-soluble mitogen (NWSM), and dextran sulfate (DxS) react with different subpopulations of B lymphocytes. Selective in vitro killing of cells responding to either LPS or NWSM has little effect on the in vitro response to the other mitogen, although the response to DxS is reduced in both cases. If, after selective in vitro killing, cells are injected into irradiated mice for 2-3 wk before measuring their in vitro mitogen responses, the same specificity pattern is seen. Thus, one is dealing with different B-cell subpopulations rather than different stages of maturation of a single population. Treatment with various alloantisera and complement before measuring the mitogen response to LPS and NWSM shows that (a) whereas all LPS response cells carry surface Ig, a subpopulation of NWSM responsive cells does not; (b) both LPS- and NWSM-responsive cells carry I-A antigens but might not I-E or I-J antigens; (c) all LPS-responsive cells carry I-C antigens, whereas approximately 25% of NWSM responsive cells do not: (d) there is a subpopulation of NWSM-responsive cells carrying neither surface Ig nor I-C antigens and resistant to anti-theta treatment.

Specific stimulation of thymus-derived cells by purified protein derivative or tetanus toxoid was inhibited by autologous lymphocytes. The lymphocytes, which were collected from healthy humans who had been vaccinated with bacille Calmette-Guérin were purified on nylon columns after removal of cells that form rosettes with sheep erythrocytes. The degree of purification of the lymphocytes was controlled by appropriate techniques.

Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.

Nocardia water soluble mitogen (NWSM) is known to stimulate mouse and rabbit lymphoid cells and to act selectively on murine B-derived lymphocytes. In this paper, evidence is presented that NWSM is also a mitogen for rabbit bursal equivalent cells and does not bring about blast transformation of thymus-derived rabbit cells. Pretreatment of rabbit spleen lymphocytes with antibody directed against rabbit thymus lymphocyte antigen (anti RTLA serum) and with complement did not affect the strong increase of thymidine incorporation which follows stimulation with NWSM. The mitogen-induced polyclonal activation of antibody-forming cells and resulted in the presence of 30% of cells with the ultrastructural characteristics of plasmocytes. These observations led to the conclusion that NWSM is a mitogen for a B-derived lymphocyte of the rabbit.

We have dissected the human influenza-specific B cell repertoire by performing Epstein-Barr virus (EBV) limiting dilution analysis of lymphocytes obtained from donors before and after immunization with a commercially available influenza vaccine. In addition to an analysis of precursor frequency and light chain diversity, we studied sera and culture supernatants containing human anti-influenza antibodies with a panel of murine monoclonal antibodies specific for idiotopes identified on murine anti-PR8 and anti-X-31 antibodies. An idiotypic specificity present on the X-31-specific murine monoclonal PY206 has previously been shown to be shared by murine antibodies specific for PR8, X-31, and other influenza viruses. We observed little correlation among the following parameters: anti-viral titer, serum idiotope content, precursor frequency and immune status. More interestingly, there was a striking predominance of human influenza-specific antibodies that utilized lambda light chains. In addition, 12 of 26 human anti-influenza monoclonals strongly inhibited the binding of one of the murine anti-idiotopes to the labeled murine antibody, PY206. This is the same idiotope that is shared among murine antiinfluenza antibodies and all six individuals studied contained clones reactive with this anti-idiotope. Seven of these 12 idiotope-positive human antibodies gave partial cross-reactivity in a second anti-idiotypic system. These observations imply that a significant level of homology exists between the binding sites of human and murine influenza-specific antibodies and suggest that idiotypic manipulation of the human immune response to influenza virus may have important therapeutic implications.