Characterization of a human B cell-specific antigen (B2) distinct from B1

A human B lymphocyte-specific antigen (B2) was identified and characterized by the use of a monoclonal antibody. By indirect immunofluorescence and quantitative absorption, B2 was shown to be expressed exclusively on Ig+ B cells isolated from peripheral blood and lymphoid tissues. In contrast, B2 was not found on monocytes, resting and activated T cells, Null cells, or granulocytes, nor was it found on cell lines or tumor cells of T cell or myeloid origin. Functional studies demonstrated that only B2 antigen-positive splenocytes could be induced to differentiate into plasma cells under the stimulus of pokeweed mitogen, further confirming the B cell specificity of B2. It was then demonstrated that the B2 antigen was distinct from the previously described B cell-surface determinants including surface immunoglobulin, Ia-like antigens, and Fc and C3 receptors. More importantly, the B2 antigen has been clearly shown to be distinct from the previously described B cell-specific antigen, B1, by its m.w. and expression on normal and malignant B lymphocytes. The distinct distribution of B2 on normal and malignant lymphocytes supports the notion of B cell heterogeneity and provides further evidence for existence of subpopulations of human B lymphocytes.     

Isolation and characterization of a rabbit T lymphocyte-specific antigen.

Rabbit T lymphocytes may be differentiated from B lymphocytes by the presence of a T lymphocyte-specific surface antigen. This unique antigen has been extracted from the plasma membrane of rabbit thymocytes by 3 M KCl. The presence of the antigen in the membrane extract was demonstrated by inhibition of cytotoxicity with goat anti-rabbit T cell serum (ATS). The crude membrane extract was fractionated by gel electrophoresis and the fractions containing the T cell antigen identified by inhibition of cytotoxic ATS and by passive hemagglutination. The purified T cell antigen was found to have a m.w. of approximately 12,000 and contained approximately 2.5% carbohydrate. Evidence was also obtained to suggest that the rabbit T cell antigen exists in multiple forms, each having the same m.w. but exhibiting different electrophoretic characteristics.     

Characterization of the gene for prostate-specific antigen, a human glandular kallikrein

The gene for the human glandular kallikrein, prostate-specific antigen, has been cloned. The sequence of 7130 nucleotides encompassing the gene and 633 bp of 5' and 639 bp of 3' flanking DNA has been determined. The translation initiation site was slightly heterogeneous, yielding 5' non-translated leader sequences of 41 and 35 bp. The gene is divided into five exons, with introns located at positions identical with those found in other glandular kallikrein genes. The nucleotide sequence is very similar to that of the human kallikrein gene hGK-1, with 76 to 93% of the nucleotides being identical in the exons and 76 to 87% in the introns. The similarity also extends approximately 200 bp into the sequence flanking the 5' end of hGK-1 and several other, both human and rodent, glandular kallikrein genes.     

Characterization of antigen processing and presentation by resting B lymphocytes

The production of antibody to a thymus-dependent Ag requires cooperation between the B cell and an Ag-specific Th cell. MHC restriction of this interaction implies that the Th cell recognizes Ag on the B cell surface in the context of MHC molecules and that the Ag-specific B cell gets help by acting as an APC for the Th cell. However, a number of studies have suggested that normal resting B cells are ineffective as APC, implying that the B cell must leave the resting state before it can interact specifically with a Th cell. Other studies, including our own with rabbit globulin-specific mouse T cell lines and hybridomas, show that certain T cell lines can be efficiently stimulated by normal resting B cells. One possible explanation for the above contradiction is that our B cells have become activated before presentation. Here we show that presentation by size-selected small B cells is not the result of nonspecific activation signals generated by the T cells or components of the medium. Also, although LPS activation does increase the efficiency of presentation by small B cells, use of large cells in place of small cells or preincubation of resting B cells with mitogenic doses of anti-Ig does not. Another possibility that we considered was that small B cells are unable to process Ag and that we had selected T cell lines that were capable of recognizing native Ag on the B cell surface. In the majority of cases, experiments with B cell lines and macrophages have shown that Ag presentation requires Ag processing, a sequence of events that includes internalization of Ag into an acid compartment, denaturation or digestion of Ag into fragments, and its return to the cell surface in the context of class II MHC molecules. The experiments reported here show that our T cell lines require an Ag processing step and that small resting B cells, like other APC, process Ag before presenting it to T cells. Specifically, we show that an incubation of 2 to 4 h is required after the Ag pulse before Ag presentation becomes resistant to irradiation. Shortly after the pulse, the Ag enters a pronase-resistant compartment. Although efficient Ag presentation requires initial binding to membrane Ig, Ag is no longer associated with membrane Ig at the time of presentation and is not presented in its intact form, because removal of membrane Ig by goat anti-Ig blocks presentation before but not after the Ag pulse.     

Characterization of the human newborn response to herpesvirus antigen

An investigation was made into the human newborn cellular response to herpes simplex virus type 1 (HSV), cytomegalovirus (CMV), and varicella zoster virus (VZV) to understand more about the nature of the neonate's susceptibility to overwhelming infection by these viruses. Newborn mononuclear cells sustained the proliferation in culture of maternal (i.e., haplotype-matched) T cell blasts with specificity for HSV, CMV, or VZV (p less than 0.05). This is evidence for intact antigen-processing capability by newborn monocytes. The response of the maternal T cell blasts appeared to be HLA-haplotype-restricted as suggested by experiments in which maternal T cell blasts were limited in number. Our culture conditions elicited responses predominantly from the T4+ lymphocyte subset. A low frequency of herpesvirus-specific T4+ lymphocytes in newborn blood might contribute to deficient viral immunity, so we evaluated the virus-specific T cell responding frequency in human newborns in limiting dilution cultures. We were unable to find a herpesvirus-specific responder cell frequency greater than 1:1,400,000 in nonimmune newborns. Three of seven adults who had no serum antibody to CMV had a CMV responder cell frequency (RCF) of 1:100,000 to 1:200,000. The RCF to HSV in immune children, ages 18 mo to 12 yr, and adults, ages 13 to 80 yr, ranged from 1:14,000 to 1:18,000. We conclude that newborn monocyte processing of herpesvirus antigen is intact, that T cell RCF is low in neonates, and that immunity to HSV after infection outside the newborn period results in comparable RCF between adults and children.     

Characterization of a recombinant vaccinia virus expressing human melanoma-associated antigen p97.

p97 is a cell surface glycoprotein expressed at high levels in most human melanomas but present only in trace amounts in normal adult tissues. We are interested in exploring the possibility of using recombinant vaccinia virus to express a specific tumor-associated antigen as a vaccine against human cancer. To this end, we constructed a recombinant virus, v-p97NY, which contains the entire coding sequence for p97 under the control of the vaccinia virus 7.5K promoter. Upon infection of tissue culture cells, v-p97NY expressed high levels of a membrane-bound glycoprotein immunoreactive with a p97-specific monoclonal antibody. Immunization of mice with this recombinant elicited high-titered antibodies against p97. Spleen cells isolated from these mice proliferated in vitro when stimulated either with purified p97 protein or with syngeneic cells expressing p97 antigen. Delayed-type hypersensitivity was also observed in immunized mice after challenge with p97-expressing cells. These findings indicate the potential usefulness of v-p97NY and similar recombinants in tumor immunotherapy.     

Characterization of a human cervical carcinoma-associated antigen by lectin binding and immuno-electron microscopy

Summary The specific binding and nature of the epitope recognized by monoclonal antibody (Mab) 1H10, which binds an antigen expressed on human cervical tumors, was characterized by enzyme digestion, lectin competition assay and immuno-electron microscopy. Membrane homogenates of CaSki cervical carcinoma cells were digested with various enzymes, then analysed by SDS-PAGE and immunoblotting. Cells grown on coverslips were treated with various enzymes and in situ binding of Mab 1H10 to cells was analysed by electron microscopy. The ability of lectin-conjugates to block Mab 1H10 binding to CaSki cells was also examined. Treatment of samples with sodium periodate abrogated antigen recognition by Mab 1H10. Neuraminidase and hyaluronidase digestion decreased but did not eliminate Mab 1H10 binding to cells in situ. Chondroitinase ABC digestion, in contrast, removed Mab 1H10 binding sites both in vitro and in situ. Trypsin and chymotrypsin digestion of cell membrane homogenates decreased the molecular weight of the Mab 1H10 antigen but did not decrease the binding intensity. Wheat germ agglutinin (WGA) strongly bound to CaSki cells and partially blocked Mab 1H10 binding, indicating that the antigen contains N-acetyl-galactosamine residues at or near the epitope recognized by Mab 1H10. Ricinus communis agglutinin (RCA) exhibited a similar binding pattern to WGA. However, concanavalin A bound only weakly to CaSki cells and was ineffective at blocking Mab 1H10 binding. The tumor-associated antigen recognized by Mab 1H10 is concluded to be a chondroitin sulphate glycoprotein or proteoglycan rather than a mucopolysaccharide or lipoprotein.     

Characterization by human autoantibody of a nuclear antigen related to the cell cycle

Using a serum from a patient with an autoimmune disease, we have recently described a novel 55 000-dalton antigen (p55) in the nucleus of several animal cells including human ones. This antigen, designated PSL, was not related to the previously defined antigens recognized by sera from patients with systemic rheumatic diseases (Sm, n-RNP, SS-B, Scl-70). We have now found that p55 is associated with chromatin structures as it is released from the nucleus of mink cell fibroblasts by saline + DNase treatments. Analysis by sucrose gradient centrifugation of the nuclear material released in these conditions indicated that p55 co-migrated with core histones. Meanwhile, p55 was absent from the residual nuclear matrices (achromatinic nuclei). Localization of p55 in synchronized cells was performed by indirect immunofluorescence and immunoprecipitation. P55 appeared to accumulate in the nucleus during the S phase. Finally, it was not recognized by an anti-SV40 tumor serum that specifically precipitated the protein p53, which has been recently related to cell proliferation. Thus, PSL an p53, although apparently not antigenically related, appear to be implicated in the same step of the cell cycle.     

Characterization of a human synovial cell antigen: VCAM-1 and inflammatory arthritis

The contribution of synovial cells to the pathogenesis of rheumatoid arthritis (RA) is only partly understood. Monoclonal antibody (mAb) 1D5 is one of very few mAb ever raised against RA synovial cells in order to study the biology of these cells. Studies on the expression pattern and structural features of the 1D5 Ag suggest that 1D5 recognizes human vascular cell adhesion molecule-1 (VCAM-1), which is an intercellular adhesion molecule. Vascular cell adhesion molecule-1 may be involved in a number of crucial intercellular interactions in RA.     

Identification of a novel human B cell activation antigen involved in B cell growth factor-dependent proliferation

The B6.4 mAb we present here identifies a novel activation Ag of B cell lineage. The B6.4 mAb was generated by immunizing mice with B cell growth factor (BCGF)-responding BA3 cells, and selected by its capability to block BCGF-induced proliferation of BA3 cells. The inhibitory effect of this antibody on BCGF-dependent proliferation was further confirmed by using normal activated B cells in the presence of exogenous BCGF derived from T cells or B cells. Furthermore, it did not affect IL-2-dependent proliferation of B cells. The expression of the B6.4 Ag, as analyzed by FACS, is restricted to in vitro activated B cells, and remains undetectable on resting B or T cells, PHA-activated T cells, and monocytes. The B6.4 Ag is also expressed on some lymphoblastoid B cell lines and most of the lymphomas and CLL of B cell origin; however, it did not express on pre-B cell ALL and plasma cell leukemias. The B6.4 Ag has a Mr of 35,000 Da, as determined by SDS-PAGE of radiolabeled immunoprecipitates from activated B cells. The B6.4 Ag is detectable on B cells as early as 8 h after activation, and precedes that of the IL-2R or transferrin R. All of these results suggest that the B6.4 Ag is an early activation Ag of B cells and is functionally related to a receptor of BCGF, but not IL-2.     

Characterization of a unique glycoprotein antigen expressed on the surface of human neuroblastoma cells

In order to develop a molecular probe to delineate chemical and biological characteristics of human neuroblastoma cells, a murine monoclonal antibody (Mab 5G3) was produced that is directed to a glycoprotein, preferentially expressed on the surface of such cells. This antibody is of IgG2a isotype, has an association constant of 8 X 10(9) M-1, and reacts preferentially with human neuroblastoma cell lines and fresh frozen tissue sections in enzyme-linked immunosorbent assay and immunoperoxidase assays, respectively. Minimal reactivity is observed with a variety of lymphoblastoid cell lines and normal fetal and adult tissues. Mab 5G3 specifically recognizes a neuroblastoma target glycoprotein antigen of 215 kDa that is derived from a 200-kDa precursor, as evident from pulse-chase biosynthetic studies. Treatment with tunicamycin revealed that both molecules contain N-asparagine-linked oligosaccharides; however, only the 215-kDa species is resistant to treatment with endo-beta-N-acetylglucosaminidase H and sensitive to neuraminidase, indicating that it contains trimmed and terminally sialylated oligosaccharides of the "complex" type. In contrast, the 200-kDa precursor is sensitive to endo-beta-N-acetylglucosaminidase H and resistant to neuraminidase treatment indicating that it contains high-mannose non-processed oligosaccharides. The 215-kDa molecule is sulfated, phosphorylated at serine residues, and expressed on the cell surface. A molecule of 200 kDa is detected by Mab 5G3 in spent culture medium of human neuroblastoma cells which is neither sulfated nor phosphorylated.     

The human B cell-associated antigen CD24 is a single chain sialoglycoprotein

The CD24 human B cell-associated antigen was originally characterized in this laboratory with the use of monoclonal antibody BA-1 combined with a standard radioimmuneprecipitation technique, and was reported to be a three chain glycoprotein complex of 45, 55, and 65 kilodaltons. We have compared our standard radioimmuneprecipitation technique (BA-1 ascites followed by rabbit anti-mouse IgM-coated protein A-Sepharose) to BA-1 conjugated directly to CNBr-Sepharose (BA-1-Sepharose) and report striking differences in the electrophoretic profile of CD24 immuneprecipitates. The CD24 immuneprecipitate obtained with BA-1-Sepharose showed a single broad band with a relative mobility of 42 kilodaltons. A series of experiments performed with the two immuneprecipitation techniques, reducing or nonreducing electrophoretic conditions, and addition of preformed mock BA-1 immuneprecipitate to BA-1-Sepharose immuneprecipitates convincingly demonstrated that the previously described 55 and 65 kilodalton components were artifacts caused by co-migration of CD24 with IgG and IgM heavy chains, respectively. Because of the consistent association and co-migration of CD24 with IgG heavy chains, we investigated the possibility that CD24 might be related to the 45 kilodalton Fc-gamma receptor expressed on B cells and eosinophils. We found, however, no evidence for such a relationship by cross adsorption analysis with BA-1-Sepharose and IgG-Sepharose.     

Characterization of a membrane antigen of Leishmania amazonensis that stimulates human immune responses.

To investigate human immune responses to defined leishmania Ag we have begun to characterize biochemically and immunologically, an abundant 42-kDa surface Ag of Leishmania amazonensis, a causative agent of human leishmaniasis. We have shown that this Ag, La gp42, is expressed on the surface of L. amazonensis promastigotes, being anchored to the membrane by a glycosyl-phosphatidylinositol moiety. As demonstrated by lectin blotting studies, La gp42 is glycosylated, binding both Con A and wheat germ agglutinin. Immunologically, La gp42 is strongly recognized by sera from patients with different forms of leishmaniasis as well as by patients with Chagas' disease. In addition, we show that purified La gp42 stimulates the proliferation of human T lymphocytes obtained from several leishmaniasis patients. Finally, the N-terminal sequence of La gp42 was obtained and a serologically cross-reactive 42-kDa protein with a homologous sequence was identified in Leishmania major.     

Binding of human lymphocyte-specific monoclonal antibodies to common marmoset lymphoid cells

Commercially available monoclonal antibodies which bind to human lymphocyte subsets were screened for their ability to bind to lymphoid cells from the common marmoset Callithrix jacchus. Anti-Leu-5 and T11 were the only pan T-cell antibodies which reacted strongly. None of the antibodies which bind human lymphocytes of the helper/inducer subpopulation reacted with C. jacchus cells and only one antibody, T8, specific for the cytotoxic/suppressor subset, bound to the marmoset cells. The two antibodies tested which bind human B cells, B1 and anti-HLA-DR, were also reactive with marmoset cells. The cellular specificity of the T11, T8, and B1 antibodies was determined by dual binding studies on the fluorescence-activated cell sorter. The B1 antibody bound only Ig+ cells and all Ig+ cells were B1+. The T11 and T8 antibodies bound only to Ig- marmoset lymphoid cells and, as in the human, all T8+ marmoset cells were also T11+. Thus, using these monoclonal antibodies in the common marmoset one can identify three populations of lymphoid cells: (1) T11+, T8+ cells; (2) T11+, T8- cells; (3) B1+ cells.     

Characterization of an antigen expressed by human natural killer cells

A monoclonal antibody, anti-N901, was produced by fusing NS-1 myeloma cells with spleen cells of a mouse immunized with human CML cells. This antibody was reactive with a subpopulation of peripheral blood LGL, including the natural killer cells. Monocytes, granulocytes, B cells, T cells (T3+ cells), erythrocytes, and platelets were nonreactive. The N901-positive cells in the peripheral blood were heterogeneous with respect to expression of other cell surface antigens. The majority of N901+ cells co-expressed T11, Mo1, and HNK-1, whereas a smaller percentage expressed T8. Ia, T3, T4, Mo2, or B1 antigens were very uncommon on N901+ cells. The heterogeneity of the N901+ LGL was further investigated by examining the expression of N901 antigen on a series of cloned normal human NK cell lines. N901 antigen was expressed by each of the NK cell lines tested, and by a minority of cloned T cell lines without NK activity. Anti-N901 does not block NK activity and can be used to rapidly purify functional NK cells for further study.     

Characterization of a monoclonal antibody-defined human melanoma-associated antigen susceptible to induction by immune interferon

To develop monoclonal antibodies (mAb) recognizing human melanoma-associated antigens (MAA) susceptible to modulation by immune interferon (IFN-gamma), hybridomas were constructed with splenocytes from a BALB/c mouse immunized with IFN-gamma-treated melanoma cells Colo 38. Screening of supernatants with control and IFN-gamma-treated melanoma cells showed that the mAb CL203 and CL207 display preferential reactivity with IFN-gamma-treated melanoma cells. The two mAb recognize the same (or spatially close) determinant on a 96,000 MAA which has a density of 0.36 X 10(6) antigenic sites/cell on untreated melanoma cells Colo 38 and of 1.39 X 10(6) and 1.54 X 10(6) on melanoma cells Colo 38 treated with IFN-gamma (final concentration, 200 U/ml) for 24 and 48 hr, respectively. The effect of IFN-gamma on the 96,000 MAA is dose- and time-dependent, reversible, and blocked by inhibitors of RNA and protein synthesis. Furthermore, the effect of IFN-gamma on the induction of the 96,000 MAA appears to be specific, inasmuch as IFN-alpha and IFN-beta do not induce the expression of the 96,000 MAA. The latter is also induced by IFN-gamma in a variety of carcinoma cell lines, but its level is markedly lower than on melanoma cells. Furthermore, the apparent m.w. of the antigen synthesized by the carcinoma cell lines in the presence of IFN-gamma ranges between 93,000 and 96,000. This molecular heterogeneity appears to reflect differences in the degree of glycosylation of the polypeptide moiety because the antigen synthesized by a variety of cell lines in the presence of tunicamycin has an apparent m.w. of 51,000.