To investigate regulation of human immunoglobulin heavy chain expression, we have cloned DNA downstream from the two human Cα genes, corresponding to the position in the mouse IgH cluster of a locus control region (LCR) that includes an enhancer which regulates isotype switching. Within 25 kb downstream of both the human immunoglobulin Cα1 and Cα2 genes we identified several segments of DNA which display B lymphoid–specific DNase I hypersensitivity as well as enhancer activity in transient transfections. The corresponding sequences downstream from each of the two human Cα genes are nearly identical to each other. These enhancers are also homologous to three regions which lie in similar positions downstream from the murine Cα gene and form the murine LCR. The strongest enhancers in both mouse and human have been designated HS12. Within a 135-bp core homology region, the human HS12 enhancers are ∼90% identical to the murine homolog and include several motifs previously demonstrated to be important for function of the murine enhancer; additional segments of high sequence conservation suggest the possibility of previously unrecognized functional motifs. On the other hand, certain functional elements in the murine enhancer, including a B cell–specific activator protein site, do not appear to be conserved in human HS12. The human homologs of the murine enhancers designated HS3 and HS4 show lower overall sequence conservation, but for at least two of the functional motifs in the murine HS4 (a κB site and an octamer motif  ) the human HS4 homologs are exactly conserved. An additional hypersensitivity site between human HS3 and HS12 in each human locus displays no enhancer activity on its own, but includes a region of high sequence conservation with mouse, suggesting the possibility of another novel functional element.

SummaryUpon activation, B lymphocytes can change the isotype of the antibody they express by immunoglobulin (Ig) isotype switch recombination. In previous studies on the regulation of human IgG expression, we demonstrated that interleukin 10 (IL-10) could stimulate IgG1 and IgG3 secretion by human CD40-activated naive (slgD +) tonsillar B cells. To assess whether IL-10 actually promotes the DNA recombination underlying switching to these isotypes, we examined the effect of IL-10 on the generation of reciprocal products that form DNA circles as byproducts of switch recombination. The content of reciprocal products characteristic of p.-~/recombination was elevated after culture of CD40-activated tonsillar slgD + B cells with either IL-4 or IL-10, although high levels of IgG secretion were observed only with IL-10. Unlike IL-4, IL-10 did not induce reciprocal products of p~-e and ~/-e switch recombination. These results demonstrate that IL-10 promotes both switching to ",/and IgG secretion.

We have characterized the VH and VL genes of three low-affinity polyreactive and two high-affinity monoreactive IgM and IgA1 rheumatoid factor (RF) mAb generated using circulating CD5+ B cells from a single rheumatoid arthritis patient. We found that four and one RF mAb utilized genes of the VHIV and VHIII families, respectively. The VHIV gene usage by these RF mAb differs from the preferential VHIII, VHI, and, to a lesser extent, VHII gene usage by the IgM with RF activity found in patients with mixed cryoglobulinemia, Waldenstrom's macroglobulinemia, and other monoclonal gammopathies. In addition, in contrast to the preponderant kappa L chain usage by the RF in these patients, a lambda L chain was utilized by all RF mAb from our rheumatoid arthritis patient. Two RF mAbs utilized V lambda I, two V lambda IV, and one V lambda III L chains. The VH genes of the two low-affinity polyreactive IgM RF mAb were in germline configuration. When compared with the deduced amino acid sequence of the putatively corresponding genomic segment, the VH gene of the high-affinity monoreactive IgM RF mAb displayed five amino acid differences, all of which are in the complementarity determining regions (CDR), possibly the result of a process of somatic point mutation and clonal selection driven by Ag. The unavailability of the corresponding genomic VH segment sequences made it impossible to infer whether the VH genes utilized by the two IgA1 RF were in a germline or somatically mutated configuration. Sequencing of the genes encoding the H chain CDR3 (D segments) revealed that all three low-affinity polyreactive RF mAb displayed a much longer D segment (36-45 bases) than their high-affinity monoreactive counterparts (15-24 bases), raising the possibility that a long D segment may be one of the factors involved in antibody polyreactivity.

The maturation of the specific antibody response to foreign antigens (Ags)' has been thoroughly investigated in experimental animals (1-8). A first in vivo exposure to Ag induces a "primary" antibody response constituted mainly of IgM with relatively low affinity for the inducing Ag. A second and any further exposure to the same Ag evoke "secondaries" or "memory" responses that involve mainly IgG with a higher affinity for the Ag. Although study ofthe human antibody response to some Ags, e.g., tetanus toxoid (TT) and keyhole limpet hemocyanin, has been attempted (9-13), the enormous difficulty in generating human mAbs (14, 15) has hindered the definition of the clonal basis of such responses.The recent progress made in the generation of human mAb-producing cell lines (14-21) and in the characterization of novel B cell subsets (22, 23) allowed us to investigate the human antibody response to self and exogenous Ags at the clonal level (16)(17)(18)(19). In the present studies, we quantitated the circulating B cells committed to the production of antibodies to rabies virus and determined their phenotype in healthy humans before and after multiple administrations of inactivated rabies virus vaccine. Moreover, using EBV transformation and somatic cell hybridization techniques, we constructed 10 cell hybrids secreting IgM, IgG, and IgA mAbs to the virus. We found that in the preimmune B cell repertoire, circulating lymphocytes committed to the production ofvirus-binding IgM, but not IgG or IgA antibodies, are present in high number. These cells are surface CD5+ and the antibodies they produce are polyreactive and low affinity. After vaccination with inactivated rabies virus, B lymphocytes producing monoreactive high affinity IgG and IgA antibodies to the virus consistently appear in the circulating. Most ofthese cells are surface CD5-and account for >10% of the total IgG-and IgA-producing cell precursors, respectively.

A novel method for the deprotection of oligodeoxyribonucleotides has been developed. Gaseous amines such as ammonia or methylamine were employed under pressure to achieve mild and rapid deprotection conditions. For example, oligodeoxyribonucleotides having a (tert-butyl)phenoxyacetyl group for the protection of the exocyclic amino function of cytosine, adenine and guanine were released from controlled-pore glass supports and fully deprotected by ammonia or methylamine under gas phase conditions, at room temperature, within 35 or 2 min respectively.

Monoclonal polyreactive antibodies bind to a variety of self and foreign antigens. In contrast, monoclonal monoreactive antibodies bind to a single or restricted number of known antigens. The rate at which polyreactive antibodies are removed from the circulation compared to monoreactive antibodies has not been determined. In the present experiments, human monoclonal polyreactive and monoreactive antibodies of different isotypes were injected intravenously into mice and the clearance from the circulation was determined. The half-life of polyreactive IgM, IgA, and IgG antibodies was 8.0, 8.2, and 9.8 hr, respectively, compared to 35.4, 26.6, and 280 hr for monoreactive IgM, IgA, and IgG antibodies, respectively. Examination of tissue sections from animals given intravenous antibody showed substantial deposition of polyreactive, but not monoreactive, antibodies in several organs, the liver being the principal site of deposition. It is concluded that polyreactive antibodies are cleared from the circulation substantially faster than monoreactive antibodies.

Suppression of high M r tropomyosins (TMs) is a common feature of transformed cells. Previous work from this laboratory has demonstrated that the isoform 1 of TM, TM1, acts as an anti-oncogene in rastransformed murine ®broblasts. In this study, we have investigated whether TM1 is a ras-speci®c suppressor, or a general suppressor protein of the cellular transformation. V-src transformed ®broblasts, which express decreased TM1, were transduced with a full-length cDNA to overexpress TM1. Both the control and the transduced cells expressed v-src kinase at comparable levels. TM1 expressing (src-T1) cells grew at a lower rate in monolayer, exhibited well spread,¯at morphology than the control cells. Enhanced expression of TM1 resulted in improved micro®lamental architecture. More signi®cantly, src-T1 cells completely failed to grow under anchorage independent conditions. These data demonstrate that TM1 is as an anti-oncogene of functionally diverse oncogenes, and it is a class II tumor suppressor protein.

B lymphocytes committed to the production of antibodies binding to antigens on pathogenic bacteria and viruses (natural antibodies) are common components of the normal human B cell repertoire. A major proportion of natural antibodies is capable of binding multiple antigens (polyreactive antibodies). Using B cells from three HIV-1 seronegative healthy subjects, and purified HIV-1 and beta-galactosidase from Escherichia coli as selecting antigen, we generated three natural IgM mAb to HIV-1 and a natural IgM mAb to beta-galactosidase. The three HIV-1-selected antibodies (mAb102, mAb103, and mAb104) were polyreactive. They bound with different affinities (Kd = 10(-6) to 10(-8) M) to the HIV-1 envelope gp160, the p24 core protein, and the p66 reverse transcriptase, but not to the 120 glycosylated env protein. They also bound to beta-galactosidase (Kd approximately 10(-7) M), tetanus toxoid, and various various self antigens. In contrast, the natural mAb selected for binding to beta-galactosidase (mAb207.F1) was monoreactive, in that it bound with a high affinity (Kd < 10(-8) M) to this antigen, but to none of the other antigens tested, including HIV-1. Structural analysis of the VH and VL segments revealed that the natural mAb utilized three segments of the VHIV gene family and one of the VHIII family, in conjunction with VL segments of the V lambda I, V lambda II, V lambda III, or V kappa IV subgroups. In addition, the natural mAb VH and VL segments were in unmutated or virtually unmutated (germline) configuration, including those of the monoreactive mAb207.F1 to beta-galactosidase, and were identical or closely related to those utilized by specific autoantibodies or specific antibodies to viral and/or bacterial pathogens. Thus, the present data show that both polyreactive and monoreactive natural antibodies to foreign antigen can be isolated from the normal human B cell repertoire. They also suggest that the VH and VL segments of not only polyreactive but also monoreactive natural antibodies can be encoded in unmutated or minimally mutated genes, and possibly provide the templates for the specific high affinity antibodies elicited by self or foreign antigens.