Antigens on human plasma cells identified by monoclonal antibodies

Two monoclonal antibodies that define distinct plasma cell-associated antigens, termed PCA-1 and PCA-2, were developed against human plasma cell leukemia cells. These antigens are strongly expressed on human myelomas, plasma cell leukemia, and plasmacytoma tumor cells, but are not detected on other lymphoid malignancies of B, T, null, or myeloid origin. PCA-1 and PCA-2 are not expressed on either normal T or B lymphocytes, but are weakly expressed on granulocytes and monocytes. When pokeweed mitogen is used to induce human B lymphocyte differentiation, PCA-1 is expressed when other B cell determinants are lost and plasmacytoid morphology, intracytoplasmic immunoglobulins, and surface T10 staining characteristic of plasma cells appear. In contrast, PCA-2 cannot be induced and may therefore appear later in the B cell differentiation scheme. These antigens may be of utility for the study and regulation of normal and abnormal plasma cell growth, traffic, and tissue distribution and may aid in understanding heterogeneity within plasma cell dyscrasias.     

Separation and isolation of rat natural killer (NK) cells from T cells with monoclonal antibodies

It has been difficult to clearly differentiate rat NK cells from cytotoxic T cells. In this study we have shown that rat NK cells do not express the T cell protein defined by the OX 19 antibody. By using the FACS we have isolated the OX 19- OX 8+ lymphocyte subset that contains virtually all the NK activity. The simultaneous use of the OX 19 and OX 8 antibodies allows the separation of NK cells from T cells.     

Regulation of natural killer cell activity by anti-I-region monoclonal antibodies

The effects of a monoclonal antibody directed against immune response gene products on mouse NK activity were examined. In vivo administration of an anti-I-A^k antibody to C3H/He (H-2^k) mice modulated their peritoneal cell (PC) and spleen cell (SC) natural killer (NK) activity against YAC-1 lymphoma target cells in vitro. No such effect was observed when BALB/c (H-2^d) mice were treated with this antibody. Administration of anti-I-A^k antibody to mice before and after infection with Toxoplasma or treatment with poly(I:C) leads to suppression of NK activity in comparison to NK activity of mice infected with Toxoplasma or injected with poly(I:C) alone. A similar treatment regimen with M5/114 antibody which reacts with I-A^b, I-A^d, I-E^d, and I-E^k molecules resulted in decreased NK activity in B10.D2 (H-2^d) but not in B10.BR (H-2^k) mice. Serum and cell culture supernatant interferon (IFN) concentrations were not altered as a result of anti-I-A^k treatment. Removal of adherent cells did not restore NK activity of anti-I-A^k-treated Toxoplasma-infected mice to levels obtained with mice infected with Toxoplasma. In contrast, depletion of Ly 2.1⁺ cells from nylon-wool nonadherent SC of mice treated with anti-I-A^k antibody, before and after infection with Toxoplasma, resulted in restoration of NK activity to the same level as that observed in Toxoptasma-infected mice.     

Antigens on human monocytes identified by monoclonal antibodies

Two antigens (Mo1 and Mo2) present on human peripheral blood monocytes have been defined by lytic IgM monoclonal antibodies. Both antigens are present on greater than 70% of adherent mononuclear cells (predominantly monocytes). Mo1 is expressed by monocytes, granulocytes, and Null cells, but is absent from T and B lymphocytes. Mo2, on the other hand, appears specific for peripheral blood monocytes. Neither antigen is present on Ia-positive B cell lines or on tumor cells from patients with B cell lymphoproliferative malignancies, further excluding the possibility that Mo1 and Mo2 are Ia antigens. Mo1 and Mo2 are, however, present on a significant number of blast cells from patients with monocytic leukemia (both myelomonocytic and pure monocytic variants), but relatively infrequently expressed by cells from patients with acute granulocytic leukemia. These results indicate that Mo1 and Mo2 are unique antigens that may represent distinct stages of late monocyte-granulocyte differentiation.     

Diurnal variation of lymphocyte subsets identified by monoclonal antibodies.

Monoclonal antibodies specific for lymphocyte subsets were used to examine circulating lymphocytes obtained at frequent intervals from healthy subjects. A diurnal rhythm was found in the total numbers of lymphocytes, T cells, inducer/helper cells, suppressor/cytotoxic cells, Ia positive cells, and B cells. The lowest levels of all subsets were seen at 0900 hours and the highest levels at 2100. In some subjects the ratio of helper to suppressor cells varied considerably during the sample period, though the ratio was relatively constant for the group as a whole.     

Cell surface glycoproteins of hepatocytes and hepatoma cells identified by monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MABs) directed to cell surface components of rat hepatocytes were isolated. The antigens of seven MABs were identified as glycosylated plasma membrane proteins. The presence of these glycoproteins on normal hepatocytes and hepatocellular carcinoma cells was analyzed. A semi-quantitative enzyme-linked immunosorbent assay revealed that only two MABs (Be 8.7, Ne 11.3) recognized proteins which were expressed not only in normal liver but also in chemically induced transplantable Morris hepatomas and hepatoma-derived cell lines. The expression of six antigens was found to be sensitive to transformation. The domain specificity of the MABs was determined by indirect immunofluorescence on sections of liver tissue containing neoplastic nodules. Three MABs (Be 8.4, Ne 11.1, Ne 11.3) specifically bound to the sinusoidal domain and two MABs (Be 9.2, De 13.4) to the bile canalicular domain. These five antigens were transformation-sensitive except for the glycoprotein recognized by the MAB Ne 11.3. Three MABs (Be 8.7, Be 9.1, De 13.2) also showed intracellular immunofluorescence. Two of the antigens (Be 9.1, De 13.2) were not present in hepatomas. The relative molar masses (Mr) of the glycoproteins were determined after protein immunoblotting and immunoprecipitation. Four MABs (Be 8.7, Be 9.1, Be 9.2, De 13.4) recognized antigens with a Mr of 110 000 but did not mutually cross-react. The antigen recognized by MAB De 13.4 was identified as the ectoenzyme dipeptidyl peptidase IV (EC 3.4.14.-).     

Tissue-specific mammary gland antigen identified by monoclonal antibodies

A new antigen of mammary tumor cells has been revealed using monoclonal antibodies. A similar antigen was detected also in certain types of secretory epithelia of oesophagus, stomach, salivary gland, and embryonic large intestine. Upon malignization, antigen persists in invasive ductal cancer of mammary gland and is expressed in cancers of ovary and rectum. The molecular weight of the antigen is greater than 2,000 kD.     

Generation of anti-idiotypic monoclonal antibodies recognizing vasopressin receptors in cultured cells and kidney sections.

To produce anti-idiotypic antibodies against receptors for the neurohypophyseal hormone vasopressin, an anti-vasopressin monoclonal antibody with a ligand specificity similar to that of vasopressin receptors was employed for immunization. Three anti-idiotypic monoclonal antibodies were obtained which induced, like vasopressin, plasminogen activator production in the renal epithelial cell line LLC-PK1 (expressing V2-receptors). Induction of plasminogen activator synthesis by the anti-idiotypic antibodies could be inhibited by coincubation with a vasopressin antagonist. In a fashion similar to that of vasopressin itself, the anti-idiotypic antibodies induced receptor down-regulation. The anti-idiotypic antibodies were employed to visualize vasopressin receptors on LLC-PK1 and A7r5 (V1-receptor-expressing) smooth muscle cells by immunofluorescence. Antibody-mediated fluorescence was not observed in receptor-deficient mutant cell lines or vasopressin-receptor-down-regulated cells. Furthermore, these antibodies were used for immunohistochemical localization of vasopressin receptors in rat and bovine kidney preparations. In accordance with earlier physiological and biochemical observations, vasopressin receptors were detected predominantly in collecting ducts in cortex and medulla. On the cellular level, a differential staining pattern was observed.     

Novel allosteric sites on human erythrocyte acetylcholinesterase identified by two monoclonal antibodies.

Monoclonal antibodies against human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) have been examined for inhibition of enzyme activity. Of sixteen antibodies analyzed, only one (C1B7) inhibited enzyme activity, indicating selection of an unusual susceptible site. The inhibitory activity of C1B7 was characterized and compared to another inhibitory antibody, AE-2, previously described by Fambrough et al. (Proc. Natl. Acad. Sci. USA 79, 1078, 1982). Maximal demonstrated inhibition was 84% for C1B7 and 72% for AE-2 and antibody inhibition of enzyme activity was equivalent for the reduced and alkylated acetylcholinesterase monomer and the intact dimer. The Ki (stoichiometry of the enzyme-antibody reaction estimated from enzyme kinetics) was 1.0 for C1B7 and 4.8 molecules of antibody per monomer of acetylcholinesterase for AE-2. The antibodies did not compete with one another for binding to acetylcholinesterase, indicating that they have different target epitopes on the enzyme. Antibody binding to the enzyme was not specifically affected by any of the anticholinesterase agents tested: (a) the irreversible esteratic site-directed inhibitor diisopropylfluorophosphate; (b) the reversible active site-directed inhibitors edrophonium, neostigmine, BW284c51, and carbachol; and (c) allosteric site-directed compounds propidium and gallamine. Kinetic analysis of their effects provide evidence that both antibodies decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.     

A conserved coronavirus epitope, critical in virus neutralization, mimicked by internal-image monoclonal anti-idiotypic antibodies.

Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.     

Detection of function-associated molecules on rat leukemic NK cells: activation by monoclonal antibody or phorbol ester

In the present study a rat leukemia NK cell line designated CRC- (derived from RNK-16 cells) was shown to spontaneously transform into a noncytolytic (NL) line referred to as CRC-/NL cells. CRC- and CRC-/NL cells were utilized to study pathways of NK activation by phorbol esters, calcium ionophore (A23187), and monoclonal antibody (mAb). 10(-6)-10(-7) M phorbol myristate acetate (PMA) but not phorbol didecanoate or 4-beta-phorbol activated CRC-/NL to lyse YAC-1 targets. Activated CRC-/NL cells produced 20-90% specific cytotoxicity compared to 0-5% for nonactivated cells. 10(-7) M PMA inhibited normal CRC- cytotoxicity. The optimum concentration of PMA for activation was 10(-6)-10(-7) M and 3-6 h treatment time. Augmentation of cytotoxicity by PMA occurred at different E:T ratios. The time required to reverse the PMA activation of CRC-/NL cells was approximately 9-10 h posttreatment. In an effort to attempt to differentiate pathways which initiated activation, CRC-/NL cells were treated with FAM binding mAb, or with combinations of mAb and ionophore, mAb and PMA, or PMA and A23187. mAb singly or in combination with 10(-7) M PMA increased cytotoxicity. However, A23187 either singly or when combined with PMA or mAb did not produce an augmented lysis of YAC-1 target cells. Additional experiments were conducted to determine if PMA activation was associated with FAM binding. This was accomplished by analyzing redirected killing of various FAM mAb-producing myeloma cells in the presence of 10(-7) M PMA. PMA treatment of the CRC-/NL cells caused a significant increase in the lysis of myeloma/mAb-producing cells compared to control cells. Further evidence that FAM binding was associated with cytotoxicity was presented by demonstrating specific inhibition of redirected lysis by homologous mAb. Phenotype analysis of CRC- and CRC-/NL cells demonstrated that OX-7 and OX-1 expression on CRC-/NL cells was increased by 71.8 and 86.8% respectively compared to CRC-. FAM expression (78-83% positives) by CRC- and CRC-/NL cells was not different. These experiments indicated at the functional level that rat NK cells can be activated for increased cytotoxicity by FAM-specific mAb binding and/or by treatment with the diacylglycerol analogue PMA. This implies that protein kinase C mobilization either singly or in concert with inositol-1,4,5-trisphosphate activation following FAM mAb binding may play important roles in NK cell cytotoxicity.     

Human granulocyte surface molecules identified by murine monoclonal antibodies

We have investigated the nature of the antigens recognized by four classes of mouse anti-human monoclonal antibodies that characteristically reacted with neutrophilic granulocytes and their precursor cells, but not with monocytes or other normal hemopoietic cells. The antigenic targets of the majority (9/12) of the independently isolated monoclonal antibodies were present on two surface glycoproteins (Mr 145,000 and 105,000) and glycolipids. This antigen(s) was also detected on granulocyte precursor cells, including the bone marrow granulocyte/monocyte progenitor cells (CFU-GM). The same antigen(s) detected by these monoclonal antibodies was also present in non-hemopoietic cell lines (colon carcinoma and neuroblastoma). Three other antigens, defined by monoclonal antibodies AHN-8, L12.2, and L13.1 and present on granulocytes and their mid-late precursor cells, could not be identified as proteins but were detected in a protein-free glycolipid extract of these cells. The diversity of the antigens was confirmed by cross-competition experiments and by the identification of their different patterns of reactivity with cell lines and bone marrow cells.     

Serological study of yeast killer toxins by monoclonal antibodies

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.     

Identification of a human natural killer cell target cell antigen with monoclonal antibodies

mAb have been derived against NK cell-sensitive target cells in an effort to identify the target cell structure involved in Ag recognition by NK cells. Several mAb were selected for further study based on their preliminary target cell binding characteristics. Flow cytometry demonstrated that each of these mAb bound to a series of NK-sensitive target cells of various origins (e.g., K562 and Molt-4) while having little or no reactivity with several NK-resistant target cell lines (e.g., SB and Daudi). Functional studies revealed that two of the mAb were able to inhibit the lysis of NK-sensitive K562 target cells by freshly isolated, endogenous NK cells in a dose-dependent fashion. Further, these mAb also could inhibit the killing of K562 target cells by both activated NK cells and cultured lymphokine-activated killer cells, as well as the cytolysis of other NK-sensitive target cells by each of these effector cell populations. Control experiments with another mAb which bound to the target cells but did not inhibit lysis implied that the effects of these mAb on NK cell function was not the result of steric hindrance. Single cell conjugate assays demonstrated that the mAb inhibited NK cell lysis via the inhibition of binding (recognition). Biochemical analysis of this target cell structure revealed that it was a molecule of approximately 42 kDa which may exist as a homodimer in its native state. Thus, it appears that the mAbs identify an unique Ag on the surface of NK cell-sensitive target cells which is involved in NK cell Ag recognition.     

Human newborn natural killer cell responses to activation by monoclonal antibodies. Effect of culture with herpes simplex virus

Deficient responses by NK cells may contribute to the susceptibility of human newborns to severe HSV infections. Furthermore, HSV disseminates widely during neonatal infection and may also infect and interfere with the function of blood mononuclear cells (MNC). To determine whether the function of newborn NK cells would be affected by contact with HSV, and whether newborn NK cells might be permissive for HSV replication, newborn MNC were cultured with HSV in vitro for 3 days. Before culture, the intracellular calcium mobilization in newborn NK cells induced by mAb to CD2 and CD16 did not differ from that of adult NK cells. This result argues against immaturity of an early NK cell activation event in human newborns. After culture with HSV the Ca2+ flux response was unaffected by lysis of K562 targets by newborn NK cells and NK-dependent suppression of HSV replication in fibroblasts were preserved or increased. Incorporation of thymidine by NKH-1 cells following stimulation with PHA and IL-2 was not suppressed. NK cells recovered from these cultures did not contain infectious HSV and synthesis of HSV-specific proteins was not detected by immunoprecipitation although HSV genome was detected by DNA hybridization. Our results extend the in vitro model stimulation systems in which newborn NK cells respond positively to include triggering through the CD2 Ag, cross-linking of cell surface CD 16 Ag and response to a pathogen, HSV.     

Characterization and utilization of a monoclonal antibody inhibiting porcine natural killer cell activity for isolation of natural killer and killer cells

A mAb, porcine NK-inhibitory mAb (PNK-I) that inhibits porcine NK activity without affecting antibody-dependent cellular cytotoxicity (ADCC) has been developed. PNK-I acts at the level of the effector cell and inhibition of NK activity is independent of complement. Inhibitory effects are seen against various human and murine NK-susceptible targets. Addition of PNK-I antibody up to 60 min after assay initiation was effective at inhibiting NK activity. Furthermore PNK-I does not inhibit E:T conjugation and inhibits during the Ca2(+)-dependent phase of NK cytolysis. PNK-I Ag is present on virtually all PBL showing a bimodal distribution with 74% "dim" and 15% "bright" by flow cytometry. Monocytes and granulocytes stain with an intermediate intensity with greater than 90% and 95% staining positively, respectively. F(ab')2 fragments of PNK-I antibody show identical staining and functional activity as the whole molecule indicating that PNK-I acts independently of FcR. PNK-I immunoprecipitates molecules of molecular mass of 166, 155, 95 kDa under reducing and nonreducing conditions. PNK-I appears to be recognizing an epitope on a CD18 molecule. The CD18 molecule (beta-chain of CD11a,b,c) is ubiquitous on the surface of leukocytes and is implicated in a variety of cellular functions. Dim and bright populations were sorted and assessed functionally for NK and ADCC activity. It is demonstrated that PNK-I+ bright lymphocytes contain all detectable NK and ADCC activity in porcine PBL. Furthermore PNK-I+ bright lymphocytes contain the cytokine responsive NK cells capable of stimulation by IL-2, porcine NK-activating factor, and porcine natural killer-enhancing mAb. PNK-I+ dim cells were devoid of all baseline as well as inducible NK and ADCC activity. Giemsa stain of sorted populations show PNK-I+ bright cells containing the large granular lymphocytes whereas dim are devoid of these. Two color analysis show that PT4+ cells are PNK-I+ dim whereas PT8+ lymphocytes are divided between PNK-I+ bright and dim populations. Our results indicate that we are able to isolate all active as well as inducible NK and ADCC effector cells from porcine PBL based on relative Ag expression of CD18. Therefore quantitative as well as qualitative antigen expression is important in NK/ADCC-mediated cytotoxicity.     

Distinct epitopes on the T cell antigen receptor of HPB-ALL tumor cells identified by monoclonal antibodies

The T cell antigen receptor is a approximately 90,000 dalton disulfide linked heterodimer that is non-covalently associated with the CD3 complex. Prior studies have demonstrated that anti-CD3 or -Ti antibodies can mimic antigen and induce cellular proliferation and the secretion of lymphokines. An early event in activation via CD3/Ti is a rapid increase in concentration of intracellular Ca2+ levels. In the present studies, we have produced a panel of monoclonal antibodies (MAb) against the Ti expressed on HPB-ALL tumor cells. All MAb immunoprecipitate a approximately 90,000 dalton disulfide linked heterodimer and induced co-modulation of Ti and CD3. On the basis of competitive binding studies, four distinct epitopes on the Ti of HPB-ALL were identified with MAb L38, L39, L41, and L42. These epitopes were additionally discriminated on the basis of reactivity with normal polyclonal T cell populations and functional effects on HPB-ALL. L39 reacted with a monomorphic epitope present on approximately 2 to 5% of peripheral blood T lymphocytes from all donors examined and was specifically mitogenic for peripheral blood T cells expressing this epitope. L39+ T cells in blood included both CD4+ and CD8+ lymphocytes. In contrast, L38, L41, and L42 failed to react with peripheral blood T cells and were not mitogenic for peripheral blood lymphocytes. Anti-Leu-4, L38, L39, and L41 MAb all induced a rapid increase in (Ca2+)i in HPB-ALL tumor cells, similar to previous findings with anti-CD3 and anti-Ti MAb against various tumor cells and peripheral blood T cells. In contrast, L42 MAb did not induce a substantial increase in (Ca2+)i. Failure of L42 to induce a substantial increased (Ca2+)i could not be attributed to the apparent titer, avidity, or isotype of the antibody. These findings suggest that induction of increased (Ca2+)i upon binding of Ti is epitope dependent. Furthermore, these data demonstrate that several distinct public and private epitopes can be identified on the T cell antigen receptor.     

Human monoclonal anti-idiotypic antibodies. I. Establishment of immortalized cell lines from a tumor patient treated with mouse monoclonal antibodies

Patients who undergo immunotherapy with a murine anti-colon carcinoma mAb (mAb17-1A) generate high titers of anti-idiotype and anti-isotype antibodies. Specifically selected anti-idiotypic antibodies that elicit in vivo a humoral and a cellular immune response against the nominal Ag can be used as surrogate Ag for immunization. We established from the B lymphocytes of a treated patient a series of EBV-transformed cell lines. Three weeks after immortalization, the cells were selected for production of antibodies (Ab2) against the Fab fragment of the murine mAb17-1A. The selected cells were cloned and screened by ELISA for specific anti-mAb17-1A idiotypic antibodies. Thirty-six out of 89 clones were anti-idiotypes. Cell culture supernatants and the purified Ig derived from 10 clones completely inhibited the specific binding of radiolabeled mAb17-1A to HT-29 colon carcinoma cells thus resembling Ab2-gamma anti-idiotypes. These cell lines which grow now in culture for 18 mo, continuously secrete IgG,K anti-Ab1-idiotype mAb. Human anti-idiotypic mAb might be candidates for vaccines when the nominal Ag itself is not available or cannot be used as such.