Family of disulphide-linked dimers containing the zeta and eta chains of the T-cell receptor and the gamma chain of Fc receptors.

Stimulation of T cells by antigen activates many signalling pathways. The capacity for this range of biochemical responses may reside in the complex structure of the seven-chain T-cell antigen receptor (TCR). In addition to the complexity shared by all TCRs, coexpression of zeta (zeta) and the distinct but related eta (eta) chains creates structural diversity among the TCR complexes expressed on a given cell. In most murine T cells that we have studied, about 90% of the heptameric receptor complexes contain a zeta zeta disulphide homodimer, whereas 10% contain a zeta eta disulphide heterodimer. Recent studies suggest that zeta has a critical role in allowing antigen to activate the cell, whereas eta expression has been correlated with the capacity for antigen-induced phosphoinositide turnover. A third zeta-related protein, the gamma (gamma) chain of the Fc epsilon and some Fc gamma receptors, exists as a disulphide homodimer in those complexes. The structural relatedness of zeta and gamma is emphasized by the recent demonstration of zeta zeta in association with CD16 in TCR-negative natural killer cells. Here we identify T cells lacking Fc receptors but coexpressing zeta, gamma, and eta, document the formation of novel heterodimers between zeta and gamma and between eta and gamma and show their association with the TCR. A greater range of homologous coupling structures than previously thought may be one way of achieving the variety of TCR-mediated (and possibly Fc receptor-mediated) biochemical responses and effector functions.

Monoclonal antibodies against the human interferon-gamma receptor(s).

Enriched human interferon-gamma (HuIFN-gamma) receptor preparations were obtained by affinity chromatography of non-ionic detergent solubilized COLO 205 cell membranes on immobilized recombinant HuIFN-gamma. The active fractions, identified by a competition ELISA, were used as the immunogen in a BALB/c mouse. Fusion of its splenocytes with myeloma cells yielded several hybrids secreting antibodies that inhibit the antiviral activity of HuIFN-gamma; the two most active ones were selected for further characterization. This blocking activity was restricted to both the human species and the gamma type of IFN. Affinity purification of cell membrane extracts on the immobilized monoclonal antibodies resulted in the visualization of a major protein band with an Mr of 90,000, which is in good agreement with the results obtained by other authors [Aguet M. and Merlin G. (1987) J. exp. Med. 165, 988-999; Novick D., Orchansky P., Revel M. and Rubinstein M. (1987) J. biol. Chem. 262, 8483-8487; Sheehan K. C. F., Calderon J. and Schreiber R. D. (1988) J. Immun. 140, 4231-4237].

Rat (and mouse) monoclonal antibodies. VIII. ELISA measurement of Ig production in mouse hybridoma culture supernatants.

We describe a rat monoclonal antibody (MAb)-based solid-phase immunoenzymometric assay for the quantification of mouse hybridoma culture supernatant immunoglobulins (Igs). This assay involves the use of two rat MAbs, LO-MK-1 and LO-MK-2, which bind distinct mouse kappa light chain epitopes expressed by all murine kappa Igs. The assay permits reliable measurement of all murine kappa IgG subclasses in the 2-120 ng/ml range and murine kappa IgM class in the 2-30 ng/ml range. The intra- and interassay coefficients of variation in the measurement of Ig in mouse hybridoma culture supernatants averaged 7.5% and 5% respectively. The assay is simple, reproducible, rapid and does not require specific equipment. It is of potential value to all laboratories engaged in hybridoma technology.

Purification of soluble cytokine receptors from normal human urine by ligand-affinity and immunoaffinity chromatography.

Affinity chromatography of crude human urinary proteins on either human recombinant interleukin-6 (rIL-6) or human recombinant interferon-gamma (rIFN-gamma) or anti IFN-gamma receptor (IFN-gamma-R) monoclonal antibodies (McAb) yielded the two respective soluble receptors in significant amounts. A single sequence of 30 amino acid residues was obtained by N-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reversed-phase high-performance liquid chromatography. This sequence was identical with the predicted N-terminal sequence of IL-6-R as previously reported. The purified IL-6-R retained its biological activity. It was used for the preparation of specific anti IL-6-R monoclonal antibodies. Analysis of the eluted proteins from both IFN-gamma and anti IFN-gamma-R columns by inhibition of solid-phase radioimmunoassay, enzyme-linked immunosorbent assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting proved the existence of soluble IFN-gamma-R in normal urine. This finding together with the already known presence of soluble TNF receptors and a soluble IL-2 receptor found both in plasma and in urine indicates that release of soluble cytokine receptors into body fluids is a general phenomenon which occurs under normal physiological conditions.

Lack of somatic mutation in a kappa light chain transgene.

Analysis of mice transgenic for immunoglobulin genes should allow definition of the cis-acting DNA sequences required to target somatic mutation to antibody V genes. We have looked for mutations in a chimeric kappa transgene encoding a V region specific for the hapten 2-phenyloxazolone (phOx) linked to a rat C kappa gene. Two independent lines of transgenic mice were hyperimmunized with phOx and splenic hybridomas established. In B cells that had been selected by antigen and which used mouse anti-phOx genes, the endogenous sequences were found to be mutated whereas the transgene remained unchanged. These results suggest either that (a) if the transgene is a "passenger" gene expressed at a low level, transgene mutation is a rare event, or that (b) sequences far from the kappa coding region are necessary to direct somatic mutation.

Products of cells from gliomas: VIII. Multiple-well immunoperoxidase assay of immunoreactivity of primary hybridoma supernatants with human glioma and brain tissue and cultured glioma cells.

To test the feasibility of primary screening of hybridoma supernatants against human glioma tissue, over 5000 combinations of hybridoma supernatants with glioma tissue, cultured glioma cells, and normal central neural tissue were screened with a new multiple-well (M-well) screening system. This is an immunoperoxidase assay system with visual endpoints for screening 20-30 hybridoma supernatants per single microscope slide. There were extensive differences between specificities to tissue and to cultured glioma cells when both were screened with M-wells and when cultured cells were screened with standard semi-automated fluorescence. Primary M-well screening with glioma tissue detected seven hybridoma supernatants that specifically identified parenchymal cells of glioma tissue and that were not detected with cultured cells. Immunoreactivities of individual supernatants for vascular components (nine supernatants), necrosis (five supernatants), and nuclei (three supernatants) were detected. Other supernatants bound multiple sites on glioma tissue and/or subpopulations of neurons and glia of normal tissue. The results show that primary screening with glioma tissue detects a number of different specificities of hybridoma supernatants to gliomas not detected by conventional screening with cultured cells. These are potentially applicable to diagnosis and therapy.

Flow sorting of hybrid hybridomas using the DNA stain Hoechst 33342.

A two-step sorting procedure with the fluorescence-activated cell sorter (FACS) is described for the selection of hybrid hybridomas producing bispecific monoclonal antibodies. Parental hybridoma cells were first labelled before fusion with fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC); heterofluorescent cells were recovered after fusion. After a period of growth in culture, the cells were then stained with the DNA-specific dye bis-benzimidazole Hoechst 33342 and sorted on the basis of their DNA content. The staining conditions (10 micrograms/ml of Hoechst 33342, 90 min incubation time at 37 degrees C) were found to be optimal for obtaining a well resolved DNA histogram with minimal effect on the growth properties of cells from different mouse hybridoma lines. Employing this method we have isolated hybrid hybridomas synthesizing bispecific monoclonal antibodies reacting with human low density lipoprotein and alkaline phosphatase from calf intestine.

[The detection of lipofuscin in a culture of hybridoma cells]. / Obnaruzhenie lipofustsina v kul'ture gibridomnykh kletok.

Lipofuscin granules (LG) are found in the cultured hybridoma cells producing monoclonal antibodies to phage. LG have been studied using light and electron microscopy. Luminescent spectra of LG clusters in hybridoma cells are presented. The increase of own luminescence intensity of LG in the course of excitation by ultraviolet (365/nm) is shown. The advantages of hybridoma cells culture for investigation of LG on the cell level are discussed.

Transcriptional down-regulation of the rearranged C-myc expression in murine cell hybrids between a plasmacytoma and a T-cell lymphoma.

Regulation of the rearranged and non-rearranged c-myc expression was studied in murine cell hybrids (SBWI and SBWII) between plasmacytoma (S194) and T-cell lymphoma (BW5147) cells. Expression of the rearranged c-myc of heterogeneous mRNA sizes (1.8 approximately 2.4 kb) was markedly down-regulated in these hybrids regardless of retention of the gene. On the other hand, expression of the non-rearranged c-myc (2.4 kb) was not significantly affected in these hybrids. Treatment of SBWI hybrid cells with cycloheximide enhanced the non-rearranged c-myc 2- to 4-fold but did not release the down-regulation of the rearranged c-myc at all, suggesting that the down-regulation of the rearranged c-myc in the hybrid cells was mainly at a transcriptional rather than a post-transcriptional level. This was supported by the results of nuclear run-on assay: the high level of run-on transcripts in S194 cells declined in SBWI hybrid cells comparable to the level in BW5147 cells. The rearranged c-myc was hemi-methylated in S194 cells and the pattern was the same in SBWI hybrid cells. Furthermore, down-regulation of the rearranged c-myc in the hybrid was also not restored by treatment with 5-azacytidine (5-AzaC), 12-O-tetradecanoylphorbol-13-acetate (TPA) or forskolin, suggesting no causative involvement of DNA methylation or protein phosphorylation in down-regulation. Higher DNase I sensitivity of the rearranged c-myc in S194 cells decreased to a similar extent to that of the non-rearranged c-myc after cell fusion with BW5147 cells. These results suggest that expression of the rearranged c-myc is down-regulated at the level of transcription in murine cell hybrids between a plasmacytoma and a T-cell lymphoma, probably by changing chromatin configuration around the gene from the open to the closed state.

Evaluation of immunocytochemical detection methods of incorporated bromodeoxyuridine in hybridomas.

Bivariate distributions obtained from nominal acid hydrolysis or thermal treatment methods used in the cell cycle analysis of incorporated bromodeoxyuridine were shown to be unacceptable with hybridomas. Four different cell treatment and staining methods were compared. These methods are acid hydrolysis, thermal denaturation, nuclei extraction with pepsin digestion, and simultaneous pepsin digestion and acid hydrolysis. The nuclei extraction method was determined to be the most appropriate for the immunocytochemical staining of incorporated bromodeoxyuridine in hybridomas. The resulting bivariate distribution provides a clear distinction between labelled and unlabelled cell fractions. The method based on nuclei extraction with pepsin digestion was optimized for a hybridoma line used in this study.

Molecular cloning of a human immunoglobulin heavy chain variable (VH) region with anti-myelin-associated glycoprotein activity.

A cDNA clone that encodes the heavy chain variable region (VH) of an IgM M-protein with anti-myelin-associated glycoprotein (MAG) activity secreted by chronic lymphocytic leukemia cells (B-C11) from a patient with peripheral neuropathy was cloned and sequenced. The JH region was identical to the germline JH4 sequence except for deletion of a thymidine residue at the site of D-JH recombination, and the D region showed greatest homology to DM2. Sequence analysis of the VH region revealed greatest homology to VH26, a member of the VH3 gene family, but homology was only 83.7% over 326 bases, suggesting that it was derived from as yet an unidentified member of the VH3 gene family.

Diversity in the available repertoire of murine antibodies reactive with bromelain-treated isologous erythrocytes.

Antibodies specific for bromelain-treated mouse RBC (BrMRBC) are of interest as models of "natural autoantibodies" and because of their primary source is Ly-1+ (CD5+) B cells. In earlier work by others, anti-BrMRBC hybridomas prepared by using CBA or NZB "spontaneously activating" peritoneal B cells were all found to produce mAb with a single common H chain V region sequence, by using a novel gene (VH11p), and a single common L chain V region sequence, a member of the Vk9 group (VkBrMp). We prepared anti-BrMRBC hybridomas by using LPS-activated B10.A splenic B cells in order to reveal the maximum available diversity in this repertoire. Data based on binding studies, Northern blot analyses with V region-specific probes, and mRNA nucleotide sequence analysis indicated that there is combining-site diversity in the repertoire of anti-BrMRBC hybridomas. There was considerable variation in trimethylammonium (a constituent of phosphatidyl choline) binding efficiency, and one of the anti-BrMRBC mAb showed no detectable binding. Northern blot analyses indicated 6 of 11 mAb to be of the VH11p/VkBrMp type, including one dual reactive anti-[BrMRBC + SRBC] mAb. Sequence analyses of the H chain V regions of four of the non-VH11p mAb revealed utilization of four distinct VH, three of which are very similar to the VH expressed by Ly-1+ B cell clones or lymphomas, as reported by others. However, because the VH11p/VkBrMp-type mAb were all relatively efficient at lysing BrMRBC and binding trimethylammonium, we suggest that affinity considerations may determine the selective predominance of B cells with this V region configuration from an available repertoire of considerable diversity.

Sequence and fine specificity analysis of primary 511 anti-phosphorylcholine antibodies.

The primary antibody response of mice to phosphorylcholine (PC) is dominated by antibodies using the T15 L chain. Anti-PC antibodies using the 511 L chain are prominent only in secondary responses to PC coupled to proteins, are somatically mutated, and all have an extra amino acid at the Vh-D junction, compared with T15 antibodies. The aim of the experiments reported here was to determine if the extra junctional amino acid alone was sufficient to generate a 511 PC-binding antibody, or if somatic mutation or other junctional changes were also necessary. We also wished to determine if unmutated 511 antibodies had sufficient affinity for PC to appear in the primary response. To increase the frequency of primary 511 antibodies, we generated a series of hybridomas from M167 L chain transgenic mice immunized 4 days earlier with either Streptococcus pneumonia R36a or PC-keyhole limpet hemocyanin (KLH). We determined the relative affinity of the antibodies, and sequenced their H chain V regions. The results showed that: 1) somatic mutations are not required for 511 antibodies to bind PC; 2) primary 511 antibodies all had lower relative affinities for PC than T15 while having similar affinities to T15 for TNP-aminophenyl PC, and higher affinities for the PC analogs nitrophenyl PC and choline; 3) all antibodies had the 511-specific insertion of an extra amino acid, usually Ala, at the VhD junction, compared with T15; 4) immunization with R36a, but not PC-hemocyanin, elicited antibodies with a specific Tyr----Asp substitution in the D region, indicating Ag selection based on fine specificity differences; 5) the total length of CDR3 was conserved in most anti-PC-hemocyanin antibodies, whereas the anti-R36a antibodies predominantly had longer CDR3 sequences; and 6) there were unique substitutions in most antibodies, including significant sequence heterogeneity in the D-Jh junction. We conclude that Ag selection on the basis of affinity for PC biases the primary anti-PC response in favor of T15, and that 511 precursors with their alternative fine specificities contribute the precursors that are expanded in the secondary anti-PC-KLH responses.

Clonal diversity, somatic mutation, and immune memory to phosphocholine-keyhole limpet hemocyanin.

Group II antibodies to phosphocholine (PC)-keyhole limpet hemocyanin in BALB/c mice are genetically diverse and of a defined binding phenotype which recognizes the hapten, phenyl-PC, and PC coupled to protein but not free PC. We sequenced the V regions of 14 kappa and lambda-bearing group II antibodies. Both types show extensive somatic mutations. The pattern of the mutations differs between kappa and lambda antibodies. The nature of the somatic mutation in lambda chains suggests strong Ag selection on the L chain but not the H chain of the lambda-bearing antibodies. The reverse pattern of selection was observed among kappa-containing antibodies wherein the accumulation of replacement mutations in the CDR of the H chain appears to result from selection while changes in the L chain appear unselected. From these findings it appears that somatic mutation plays a major role in anti-PC-keyhole limpet hemocyanin memory development because all 14 antibodies displayed changes from germ-line sequences.

Characterization of syngeneic antiidiotypic monoclonal antibodies to murine anti-human high molecular weight melanoma-associated antigen monoclonal antibodies.

Hybridization with murine myeloma cells P3-X63-Ag8.653 of splenocytes from BALB/c mice immunized with syngeneic anti-human high molecular weight melanoma-associated Ag (HMW-MAA) mAb 149.53, 225.28, 763.74, and TP41.2 has resulted in the formation of antiidiotypic antibody-secreting hybridomas with a frequency ranging between 1.2% and 5.2%. No marked difference was detected in the frequency of antibody secreting hybridomas in the fusions generated from mice immunized with the four anti-HMW-MAA mAb, suggesting that the idiotopes expressed by each of them display similar immunogenicity in a syngeneic combination. The number of antiidiotypic mAb that did not inhibit the binding of immunizing mAb to melanoma cells was higher than that of those that died, suggesting that idiotopes not associated with the Ag-combining site are more immunogenic than those that are. The idiotopes recognized by mAb were not detected on a large panel of anti-HLA Class I mAb, anti-HLA Class II mAb, and anti-human melanoma-associated Ag mAb. The latter included also mAb that cross-inhibit the immunizing anti-HMW-MAA mAb. The idiotopes recognized by mAb were not detected on the isolated H and L chain of the immunizing anti-HMW-MAA mAb. Cross-blocking experiments with a selected number of antiidiotypic mAb identified three distinct idiotopes on mAb 149.53, 225.28, and TP41.2 and two on mAb 763.74. Three, 5, 2, and 5 antiidiotypic mAb to idiotopes within the Ag-combining site of mAb 149.53, 225.28, 763.74, and TP41.2, respectively, were tested for their ability to induce anti-HMW-MAA antibodies. Serological and immunochemical assays detected anti-HMW-MAA antibodies only in sera from BALB/c mice immunized with mAb MK2-23. Therefore, mAb MK2-23 can be classified as beta, while the remaining 14 can be classified as gamma.

Production of hapten-specific T cell hybridomas and their use to study the effect of ultraviolet B irradiation on the development of contact hypersensitivity.

We produced a series of T cell hybridomas that produce IL-2 when cultured with syngeneic APC coupled to FITC or TNP. These hybridomas are hapten specific and Ia restricted. The hybridomas were used to detect hapten-bearing APC in draining lymph nodes of mice sensitized with trinitrochlorobenzene or FITC in vivo. Hapten-bearing APC capable of stimulating the hybridomas were detectable in draining lymph nodes of hapten-painted mice within 3 h after sensitization. The ability of lymph node APC to stimulate the hybridomas peaked at 24 h and declined by 48 h. The dendritic cell subpopulation was the subpopulation of cells that were found in the regional lymph nodes of hapten-painted animals that were capable of stimulating the hybridomas to produce IL-2. Prior treatment of the skin with low dose UVB irradiation before epicutaneous application of contact sensitizers significantly reduced the capacity of hapten-bearing APC to stimulate the hybridomas. This observation was corroborated by results obtained from flow microfluorometry analysis of lymph node cells from FITC-sensitized mice. Lymph node dendritic cells obtained from FITC-painted mice contain a brightly staining group of cells by flow microfluorometry analysis. Lymph node dendritic cells from FITC-painted, UVB-irradiated mice did not contain this brightly staining population. These results indicate that low dose, local UVB irradiation may affect APC migration and/or function. We believe that these hybridomas will prove to be useful tools in the study of the development and regulation of contact hypersensitivity.

Idiotypic replica of an anti-human tumor-associated antigen monoclonal antibody. Analysis of monoclonal Ab1 and Ab3 fine specificity.

CaMBr1 is a tissue-specific and tumor-associated saccharidic epitope, defined by mAb MBr1 (Ab1), expressed on glycoconjugates of the human mammary carcinoma cell line MCF-7 and of normal and neoplastic mammary epithelial cells. An anti-anti-idiotypic monoclonal Ab3, 2G-3, identifying a human breast tumor associated antigen, was raised by using as immunogen a mouse anti-idiotypic monoclonal Ab2, A3B10, which behaves as the internal image of CaMBr1. mAb 2G-3, as well as MBr1, defines a saccharidic epitope on glycoconjugates extracted from MCF-7 cells and shows MBr1-like reactivity on normal and neoplastic-tissues. Experimental evidence, however, suggests that the fine immunoreactivity of the two antibodies is not identical, because MBr1 has a preferential reactivity with glycolipids and 2G-3 with glycoproteins. We suggest that a possible biologic explanation for our findings could reside in the nature of the immunogens used to raise the two mAb (glycolipid vs protein "internal image").

My 43, a monoclonal antibody that reacts with human myeloid cells inhibits monocyte IgA binding and triggers function.

A mAb My 43 of the IgM isotype was obtained from a fusion of spleen cells immunized against human monocytes. This mAb inhibited monocyte binding of both soluble FITC-labeled IgA and IgA-coated E, whereas it did not inhibit IgG binding. The Ag recognized by My 43 was induced on HL-60 cells in parallel with IgA binding ability by 1-25 dihydroxy-vitamin D3 treatment. Phagocytosis of IgA-coated E by monocytes and 1-25 dihydroxyvitamin D3-treated HL-60 cells was inhibited by My 43. Furthermore, a heteroantibody of My 43 x F(ab)'2 anti-E promoted phagocytic uptake of E by monocytes. Production of superoxide anion by IFN-gamma treated U-937 cells was stimulated by My 43 but not by other IgM mAb recognizing myeloid cells. By these criteria My 43 recognized a molecule capable of triggering function. Moreover, its binding reactivity, ability to block binding of IgA and IgA-complexes, and its ability to induce activation of IgA receptor bearing myeloid cells, are consistent with the possibility that My 43 reacts with the IgA receptor on these cells.

Identification of distinct T cell receptor (TCR)-gamma delta heterodimers using an anti-TCR-gamma variable region serum.

T cell hybridomas were generated from CD3+, CD4-, CD8- splenocytes and fetal thymocytes. V gamma 1-expressing proteins present on these murine TCR-gamma delta hybridomas were identified by using an anti-TCR V gamma 1 peptide serum. This antiserum specifically immunoprecipitated 41-kDa TCR V gamma-C gamma 4 chains and 31-kDa TCR V gamma-C gamma 1/2 chains from distinct heterodimers expressed on the TCR-gamma delta T cell hybridomas. The RNA from a hybridoma with a 31-kDa TCR-gamma chain hybridized with a V gamma 1 probe but failed to hybridize with a V gamma 2 probe. In contrast, the RNA from a hybridoma with a 32-kDa TCR-gamma chain hybridized with a V gamma 2 probe. This 32-kDa TCR-gamma chain was not immunoprecipitated by the anti-V gamma 1 serum. These data were consistent with the conclusion that the 31-kDa protein was the product of a V gamma 1 to C gamma 2 rearrangement, whereas the 32-kDa protein was the product of a V gamma 2 to C gamma 1 rearrangement. Furthermore, Southern analyses confirmed that the 32-kDa protein was the product of a V gamma 1.2-J gamma 2 rearrangement, and all three of the 41-kDa TCR-gamma chains were the results of V gamma 1.1-J gamma 4 rearrangements. This was the first demonstration at the clonal level of TCR-gamma proteins which use members of the V gamma 1 gene family, as well as the C gamma 2 constant region. Additional biochemical analyses of the TCR-gamma and -delta proteins from three independently derived C gamma 4-bearing T cell hybridomas suggested that most of the molecular mass diversity observed in the bulk subpopulation of peripheral C gamma 4-containing heterodimers may be contributed by the TCR-delta chains.

Molecular analysis of clonal stability and longevity in B cell memory.

We used the antiphosphocholine response induced by Proteus morganii and an adoptive transfer protocol to study the contribution of individual clones to B cell memory. Spleen cells from donor mice immunized with P. morganii were injected into irradiated hosts. These recipients were then immunized and their spleen cells fused 12 to 14 wk thereafter. The sequences of hybridoma VH and VL were obtained and DNA rearrangements at both V region loci were studied to ascertain clonal relationships. In all three adoptive transfer experiments, each mouse of a pair receiving cells from the same donor contained hybridomas which were clonally related to each other. In two of these experiments paired recipients possessed cells that had identically mutated V genes. These results lead us to conclude that once a B cell clone(s) dominates a response, progeny of that clone form the memory cell population for many months. Moreover, stability appears to be generated in some memory B cells through inactivation of the hypermutation mechanism.