M4HS2, a new human T-cell line for the efficient generation of human T-cell hybrids.

We have developed and characterised a variant of the human T-cell tumour, MOLT-4F, which is resistant to both 8-azaguanine and 6-thioguanine. This new cell line, M4HS2, is suitable for the efficient production of lymphokine-secreting human T-cell hybrids and is available for distribution.

Identification of the major Ascaris allergen and its purification to homogeneity by high-performance liquid chromatography.

The major allergen from the body fluid of adult Ascaris suum (ABF) has been identified and purified to homogeneity using gel permeation high-performance liquid chromatography (HPLC). The purity of the Ascaris body fluid allergen (ABA-1) was confirmed by HPLC and SDS-PAGE. ABA-1 appears to be a 25-kDa dimer in its native form, which runs as a 10-kDa monomer on SDS-PAGE under reducing conditions. The allergenicity of the HPLC-purified protein was confirmed using isolated in vivo-sensitised mast cells from infected rats in an in vitro histamine release assay. ABA-1 is responsible for less than 80% of the allergenicity of ABF in this sensitive and specific system. Amino acid composition analysis and N-terminal amino acid sequencing were performed on ABA-1. Comparisons are made between ABA-1 and some of the heterogeneous Ascaris allergen preparations previously published. It is suggested that Asc-1 and allergen A both contain ABA-1 in large quantities and that discrepancies in the literature result from contaminating proteins in these preparations and technical differences in characterization of the predominant molecules present in the preparations. Compositional data suggests that the ABA-1 monomer is a molecule of 94 amino acids (based on a molecular weight estimate of 10 kDa) with a composition resembling that previously published for allergen A. The first 10 amino acids are identical to those of a protein affinity purified from the body fluid of Ascaris suum at the Wellcome Laboratories for Experimental Parasitology (WLEP-14K). The similarity between ABA-1 and WLEP-14k is also apparent on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity amongst infected children in IgE antibody repertoire to the antigens of the parasitic nematode Ascaris.

Plasmas from a sample of 6-year-old children chronically infected with Ascaris lumbricoides were examined by immunoblotting for IgE antibody to the pseudocoelomic fluid of adult parasites. This revealed a high degree of heterogeneity in the antibody repertoire to putative allergens of the parasite, such that none of those screened had identical recognition patterns. This heterogeneity occurred despite the fact that infection with A. lumbricoides involves both tissue-invasive and intestinal phases of the infection, which should result in close contact between the immune system and potentially allergenic components of the parasite.

MHC class II (I-A) region control of the IgE antibody repertoire to the ABA-1 allergen of the nematode Ascaris.

ABA-1 is an approximately 14,000 molecular weight (MW) allergen which is among the most abundant proteins synthesized by the nematode parasite Ascaris. IgG and IgE responses to it are major histocompatibility complex (MHC)-restricted in rodents and have only been found to occur in rats of the RT1u haplotype and mice of the H-2s haplotype. Humans infected with the parasite vary substantially in their immune response to the allergen, but the genetic basis for this unknown. H-2 recombinant mice were used to identify the region within the MHC controlling antibody responses to the allergen. IgG antibody to immunoaffinity purified ABA-1 was assayed by radio-immunoassay and IgE by passive cutaneous anaphylaxis. This showed that the restriction element is the I-A molecule and that there was some evidence for I-E modulation of the level of response.

The human bladder carcinoma line T-24 secretes a human B-cell differentiation factor.

The effect of a B-cell differentiation factor (BCDF) found in the supernatant of the human bladder carcinoma cell line T-24 (T-24.BCDF) was assessed using the human lymphoblastoid line CESS (Muraguchi et al., 1981) and TPA-stimulated human B-CLL B cells. This T-24.BCDF was shown to cause both these cell types to secrete immunoglobulin, and therefore indicates that culture supernatants from this bladder carcinoma line contain a potent differentiation factor for human B cells.

Ability of alpha (but not beta) form interleukin-1 to support DNA synthesis in human tonsillar B cells.

The ability of the two forms of recombinant human interleukin-1 (alpha and beta) to support DNA synthesis in human tonsillar B cells was assessed. The results suggested that in two out of three cases the alpha form was able to support DNA synthesis when the beta form was not. These results support the hypothesis that each form of human interleukin-1 has a different role in supporting the growth of human B cells.

The ABA-1 allergen of the nematode Ascaris suum: epitope stability, mass spectrometry, and N-terminal sequence comparison with its homologue in Toxocara canis.

ABA-1 is a major allergen of nematode parasites of the genus Ascaris which includes the large roundworms of humans and pigs, A. lumbricoides and A. suum, respectively. The allergen was purified from A. suum by immunoaffinity chromatography for immunochemical examination. The IgE antibody repertoire is under MHC control in infected rodents and the IgE-binding epitopes were robust to treatment with heat or periodate, and electroblotting on nitrocellulose. This implies that the IgE epitopes comprise primary peptide sequence or an unusually stable secondary or tertiary structure. The molecular mass of ABA-1 is controversial, but mass spectrometry analysis indicated that there were five components of similar size, with the major species being 14,643.2 +/- 1.4 D. Finally, N-terminal sequence analysis of ABA-1 and TBA-1 (the homologue in the canine nematode infective to humans, Toxocara canis) revealed a high degree of similarity, and we have previous evidence that ABA-1 homologues are widespread amongst ascaridid parasites of humans. ABA-1 and its homologues might, therefore, be important to the immunopathology of many infections with nematode parasites, upon which the genetic constitution of the hosts will also have a bearing.

Comparison between the MHC-restricted antibody repertoire to Ascaris antigens in adjuvant-assisted immunization or infection.

Genetic restrictions to the immune repertoire will be an important consideration in the development of anti-nematode vaccines. It has already been established that the major histocompatibility complex (MHC) limits responsiveness to nematode antigens in infection, but little is known of whether this also applies under other routes of sensitization, such as with adjuvants. The specificity of the antibody response was, therefore, compared in infection and adjuvant-assisted immunization using secreted and somatic antigens of Ascaris suum as a model system in mice and rats. The findings were, first, that the lack of responsiveness to certain antigens in infection was not circumvented by Freund's adjuvant-based immunization, despite the fact that the latter generally elicited higher levels of response. Secondly, that adjuvant-assisted immunization could elicit responses to parasite products which were not detectable in the context of infection. Conversely, some specificities were detectable in infection but absent under adjuvant immunization. Finally, immunization with a defined parasite allergen (ABA-1) in Freund's adjuvant did not provoke an IgE response which would be anticipated if the molecule were to have an intrinsic allergenic property. These results are likely to be of general importance to the application of subunit or recombinant vaccines against nematodiases and to the hypersensitivity reactions which vaccination might engender or recall.

The specificity of the antibody response to internal antigens of Ascaris: heterogeneity in infected humans, and MHC (H-2) control of the repertoire in mice.

Children from an area of Africa endemic for the large roundworm of humans, Ascaris lumbricoides, were found to vary considerably in the specificity of their serum IgG response to the internal antigens of the parasite. This was particularly noticeable for responses to a 14-kD protein (ABA-1) of the parasite that has previously been shown to be the subject of a strong IgE antibody response in infected animals. The possibility that this heterogeneity in immune repertoire has a genetic basis was explored in inbred mice infected with Ascaris suum. This showed that no strain responded to all the potential antigens, that the recognition profiles of strains bearing independent haplotypes were unique, and only H-2-identical strains had responses of similar specificities. Major histocompatability complex (MHC) restriction was confirmed using H-2-congenic animals on BALB and B10 backgrounds, which responded according to their H-2 haplotype. It is likely, therefore, that it is the MHC which controls the repertoire to Ascaris antigens in infected people. If this is so, then there will be implications for immunopathology associated with ascariasis, and possibly also for resistance and susceptibility to infection.

T-24.B-cell differentiation factor induces immunoglobulin secretion in human B cells without prior cell replication.

Stimulation of B lymphocytes from B-cell chronic lymphocytic leukaemia (B-CLL) with 12-0-tetradecanoylphorbol-13-acetate (TPA) has shown that these cells are capable of differentiation (Totterman, Nilsson & Sundstrom, 1980). Increases in the expression of different class II MHC antigens (Guy et al., 1983, 1986) and responsiveness to growth factors (Kabelitz et al., 1985; Suzuki, Butler & Cooper, 1985) have been studied. Supernatant from the human bladder carcinoma line T-24 contains a B-cell differentiation factor (BCDF) able to induce immunoglobulin secretion from CESS cells. We investigated the induction of proliferation and immunoglobulin secretion in human B cells by studying the effects of this factor on B-CLL cells, in both the presence and absence of TPA. We report here that this material (termed T-24.BCDF) causes immunoglobulin secretion to be initiated in these cells, and that this is not accompanied by detectable DNA synthesis. These observations were extended to normal human B cells and demonstrate that human B cells can secrete immunoglobulin in the absence of clonal expansion.

N-terminal amino acid sequence identity between a major allergen of Ascaris lumbricoides and Ascaris suum, and MHC-restricted IgE responses to it.

A protein allergen of the parasitic nematode Ascaris has been purified to homogeneity by immunoaffinity chromatography. It is the most abundant protein species in the parasite's body fluid and has been named ABA-1. The allergen's molecular weight (MW) has been previously estimated at 14,000, but this sizing is currently under re-evaluation. The immunological activity of the protein was intact after purification, as attested by immunoprecipitation and passive cutaneous anaphylaxis. The IgE response to ABA-1 was under major histocompatibility complex (MHC) restriction in the rat, in which only RT1u strains were found to respond following infection with the parasite. The tissue-invasive and intestinal stages of both Ascaris lumbricoides (of humans) and Ascaris suum (of pigs) have an antigen of similar MW to ABA-1 in their secretions or among their somatic antigens. These are antigenically indistinguishable; they were found to have similar amino acid compositions, and their N-terminal amino acid sequences were identical to 41 residues. Finally, the apparent MW, amino acid composition and isoelectric point of ABA-1 all argue for close similarity to the previously described Allergen A of the parasite.

Responses of human B-cells to recombinant lymphokines.

The effects of recombinant or purified cytokines on the growth and differentiation of human B-cells from a variety of sources were evaluated. The results suggest that IL-1, BCGF and BSF-2 have either "growth-factor" or "differentiation-factor" activity and that only IL-2 has both these functions. In addition, evidence is presented that the two forms of human IL-1 affect B-cell growth differently. Finally, the separation of human normal and leukaemic B-cells into subsets defined by their buoyant cell density, is described.

MHC restriction of the antibody repertoire to secretory antigens, and a major allergen, of the nematode parasite Ascaris.

Humans vary considerably in the antigen specificity of their immune responses to parasitic nematodes, and in the infection loads of individuals living in the same environment. The possibility that the former has a genetic basis operating through repertoire control of the immune system was investigated using infection of mice with the nematode Ascaris. The specificity of the antibody response was examined using excretory/secretory (ES) materials of the parasite as target Ag. No strain of mouse was found to recognize all of the potentially antigenic components of ES, and the Ag recognition patterns varied considerably from strain to strain. Using H-2 congenic mice on both the BALB and B10 backgrounds, it was established that the antigen recognition patterns were MHC-determined. Focusing on one particular component of ES, of Mr 14,000, only H-2s strains responded in IgG. This MHC restriction of the repertoire was confined to infection, and broke down under adjuvant-assisted immunization with the purified protein. The Mr 14,000 molecule was also found to be a potent allergen in a passive cutaneous anaphylaxis assay, and the IgE response to it was also restricted to H-2s. This haplotype was, however, a low IgE responder on the SJL background. There is, therefore, MHC control of the specificity of the immune response to this molecule, but non-MHC control of the amplitude of the IgE antibody response to it. Hybrids between responder and nonresponder strains (BALB/c x SJL)F1, responded to the Mr 14,000, but their responses to other ES components could not be predicted from the response patterns of parental strains. For example, the BALB/c parent responded to a 118-kDa component, but the SJL parent and the F1 progeny did not. Moreover, the response to a 41-kDa Ag was substantially down-regulated in the F1, whereas both parental strains responded vigorously. This new model system, therefore, has implications for MHC control of responses to the allergens of pathogens, and for the complex immunoregulation in heterozygotes in the context of infection.