Class II major histocompatibility complex gene sequences in rheumatoid arthritis. The third diversity regions of both DR beta 1 genes in two DR1, DRw10-positive individuals specify the same inferred amino acid sequence as the DR beta 1 and DR beta 2 genes of a DR4 (Dw14) haplotype.

The DR1 and DRw10 beta 1 chain genes were isolated from each of 2 individuals with rheumatoid arthritis who were heterozygous for these class II major histocompatibility complex specificities. The sequences of the DR1 beta 1 chains from both patients were identical, differing from previously reported DR beta 1 chains of individuals without RA by 2 amino acid substitutions, at positions 85 (Val-Ala) and 86 (Gly-Val), and by a silent mutation at the last nucleotide of codon 78 (C-T), resulting in the loss of a Pst I restriction endonuclease site. Identical DRw10 beta 1 chain genes were found in both patients. These were shown to encode the epitope recognized by monoclonal antibody 109d6. This antibody also recognizes an epitope on the DRw53 beta 2 chain of the DR4 haplotype. The third diversity regions of the DR1 beta (amino acids 67-74) and the DRw10 beta 1 chains (amino acids 67-73) were identical, respectively, with those of the DR4 (Dw14) beta 1 and beta 2 chains, raising the possibility that in these patients, the third diversity regions of the two DR beta 1 chain genes present in trans are conformationally equivalent to the cis-encoded third diversity regions of the DR4 (Dw14), DR beta 1, and beta 2 chains. The nucleotide sequences of the DQ beta complementary DNA clones were identical to that of the DQw1 beta chain, and no DR beta 2 complementary DNA clones were identified.

Nucleotide sequence of a DRw10 beta chain cDNA clone. Identity of the third D region with that of the DRw53 allele of the beta 2 locus and as the probable site encoding a polymorphic MHC class II epitope.

The nucleotide and inferred amino acid sequence of a DRw10 beta chain was obtained from cDNA clones isolated from a DR1, DRw10 heterozygous cell line. The sequence of this beta chain gene was distinctive, differing from those of all other defined DR types. The DRw10 beta chain gene was shown by transfection experiments to encode a polymorphic epitope recognized by mAb 109d6 that is also encoded by the DRw53 beta 2 chain gene. Comparison of the nucleotide sequence of both genes revealed that their third D regions (amino acids 67 to 73) were identical. This suggested first that the 109d6 epitope could be encoded by residues of this region, and second, that a putative gene conversion event transferred this sequence along with the information encoding the 109d6 epitope from a donor gene such as DRw53 beta 2. The sequence of the DRw10 beta chain gene was observed to be identical to that of clone pII beta 4 derived from the non-DR3 haplotype in the Raji cell line, which was also demonstrated to express the determinant recognized by antibody 109d6, suggesting that the typing of this cell line is HLA-DR3/DRw10. No evidence was found for the existence of a DR beta 2 chain gene product encoded by the DRw10 haplotype. The DRw10 haplotype was of particular interest because it was present along with a DR1 haplotype in the propositus who had rheumatoid arthritis, and was shared by the DR4-positive son of the propositus, who also had rheumatoid arthritis. This raised the possibility that the DRw10 haplotype, and most probably one or more specific conformations encoded by the DR beta chain, are involved in the definition of the disease susceptibility phenotype.

Paraproteinemias in homosexual men with HIV infection. Lack of association with abnormal clinical or immunologic findings.

During a prospective immunologic study of 130 homosexual men, the authors looked for the presence of paraprotein bands in serum by electrophoresis. Antibody to the human immunodeficiency virus (HIV) was present in 65 of the 130 men, the lymphadenopathy syndrome (LAS) in 26, and the acquired immune deficiency syndrome (AIDS) in 3. Abnormal bands were detected in the serum of six men, as single paraproteins in four and as oligoclonal bands in two. All six were seropositive for anti-HIV; one has LAS, two had persistent but minor lymphadenopathy, and three were apparently normal. There was no significant difference between the T-cell subsets or ratios between those seropositive men with or without paraproteins. This high incidence of paraproteins is another accompaniment of B-cell hyperactivation in persons infected with HIV.

Acute scleroderma in stable mixed connective tissue disease: treatment by plasmapheresis.

This report describes a case of stable mixed connective tissue disease (MCTD) with development of acute scleroderma with hypertension, oliguric renal failure, microangiopathic hemolytic anemia, and pulmonary infiltrates. The renal histology in the acute episode was that of scleroderma with intimal sclerosis and 'onion skinning' of vessels and glomerular ischemic injury but with no evidence of damage by immune complexes either histologically or by immunofluorescence. improvement occurred after treatment with plasmapheresis, cyclophosphamide, and captopril with return of near normal renal function.

Familial hairy cell leukemia.

A mother and son are reported who both developed hairy cell leukemia. The mother aged 74 presented with pancytopenia and responded well to splenectomy. Four years later her son aged 48 presented with pancytopenia; splenectomy was less effective but he improved after treatment with interferon-alpha. Histological examination of the splenic tissue in both cases showed changes characteristic of hairy cell leukemia. This is the third report of this rare disease occurring in family members.

Immunological abnormalities in asymptomatic homosexual men: correlation with antibody to HTLV-III and sequential changes over two years.

A prospective study on 100 homosexual male volunteers was designed to examine immunological function in relation to sexual activity and infection with the human T cell lymphotropic virus Type III (HTLV-III). Complete data were available for 71 men. In a comparison with 100 age-matched heterosexual men, the study group of 100 men had a significantly higher mean serum IgG level (12.1 +/- SD 2.7 g/l vs. 10.9 +/- 2.4 g/l, p less than 0.01) and a significantly lower mean number of CD4 (T4) cells (845 +/- 310 X 10(-6)/l vs. 1128 +/- 375; p less than 0.01). For the study group, seropositivity for anti-HTLV-III was present initially in 22 per cent and was associated with a higher mean level of serum IgG and lower mean number of CD4 cells. Among seropositive homosexual men a low CD4/8 ratio was attributable to low numbers of CD4 cells in those without lymphadenopathy and to high numbers of CD8 cells in those with lymphadenopathy. For the seronegative homosexual men, a low CD4/8 ratio as a result of an increased CD8 cell count was present in 12 of 60, and was associated with numerous sexual partners and semen culture positive for cytomegalovirus. In two seropositive subjects a low CD4/8 ratio due to a decrease in the CD4 cell count was predictive of the development of AIDS by some two years. For the 71 men with complete data over two years, indices of cell-mediated immunity, including mean counts of CD4 cells, the CD4/8 ratio, and score for recall of cutaneous delayed type hypersensitivity increased during the first year but not during the second year in both seropositive and seronegative subjects. These increases occurred in association with changes in sexual practices and activity, but could not be attributed to any one particular factor.

Association between anorectal dysplasia, human papillomavirus, and human immunodeficiency virus infection in homosexual men.

Cells from the anorectal mucosa of 61 homosexual men were examined microscopically for evidence of papillomavirus infection and dysplastic changes. There was cytological evidence of dysplasia with concomitant features of human papillomavirus (HPV) infection on at least one occasion in 24 men and of papillomavirus infection without dysplasia on at least one occasion in a further 26: dysplasia was present for over one year in 9 of 14 men who were re-examined. Dysplasia was associated with a history of anal warts, frequent receptive anal intercourse, presence of serum antibody to human immunodeficiency virus (HIV), and immune dysfunction as judged by a low CD4/CD8 ratio, but not with the lifetime number of sexual partners. The association of longlasting dysplasia with anti-HIV was independent of the association with immune dysfunction. Thus infection of anorectal mucosal cells with papillomavirus seems to be frequent among homosexual men and may predispose to dysplasia.

Evidence for the in vivo production and release into the serum of a T-cell lymphokine, persisting-cell stimulating factor (PSF), during graft-versus-host reactions.

The T-cell lymphokine, persisting-cell stimulating factor (PSF or interleukin-3), was detected in the serum of mice undergoing graft-versus-host reactions (GVHR). Gel filtration under non-dissociating conditions indicated that the PSF in the serum had an apparent molecular weight of 34,000, a figure identical with that of PSF generated from activated T cells in vitro, indicating that PSF was not bound by serum proteins. The GVHR was accompanied by increases in the numbers in the bone marrow and spleen of precursors of PSF-dependent mast cells, and increases in the numbers of mast cells, megakaryocytes and immature and mature neutrophils in the spleen. These effects of GVHR on haemopoietic cells paralleled those seen when haemopoietic tissues were stimulated with pure PSF in vitro and closely resembled those induced in previous studies by the presence of a tumour that secreted PSF alone. These studies are the first to show that PSF can enter the circulation during immune reactions in vivo and suggest that much of the stimulation of haemopoietic cells seen in GVHR, can be accounted for by the release of PSF from activated T cells.

'Acute' autoimmune hepatitis.

A previously well young woman presented with an acute hepatitis resembling viral hepatitis and a liver biopsy after 5 weeks showed features of acute hepatitis. Infection with identifiable viruses or other organisms known to cause hepatitis was excluded. Evidence for autoimmune chronic active hepatitis ab initio included prolonged fever, lymphadenopathy, urticaria, arthralgia, Coombs' positive hemolytic anemia, lymphopenia, a markedly raised level of immunoglobulin G and a positive antinuclear antibody test. Liver biopsies after 4 and 28 months showed typical histologic features of autoimmune chronic active hepatitis and the subsequent clinical course was typical, being marked by relapses and remissions responsive to prednisolone. Thus, described here is a woman in whom an acute onset of autoimmune chronic active hepatitis was clinically and histologically identified.

Stimulation of bone marrow-derived and peritoneal macrophages by a T lymphocyte-derived hemopoietic growth factor, persisting cell-stimulating factor.

Several lines of evidence indicated that P cell-stimulating factor (PSF), a T lymphocyte-derived lymphokine known to stimulate the growth of hemopoietic stem and progenitor cells, also acted on macrophages. PSF was absorbed from medium that had been mixed for two hours at 0 degrees C with either resident or thioglycollate-elicited peritoneal cells, suggesting the presence of receptors for PSF on cells in the population. The addition of pure PSF to populations highly enriched in either resident or elicited adherent peritoneal macrophages resulted in stimulation of macrophages with morphological changes, including increases in size, spreading, vacuolation, and the number of cytoplasmic processes, together with stimulation of proliferation and the phagocytosis of opsonized yeast. PSF also stimulated the incorporation of [3H]thymidine by bone marrow-derived adherent macrophages. Addition of pure PSF to cultures that contained only a single macrophage resulted in enhanced survival and proliferation of these isolated cells, demonstrating that the effect of PSF on macrophages was direct. These results indicate that PSF can stimulate well-differentiated functional macrophages and raise the possibility that the effects of PSF on macrophages may play a regulatory role in immune responses.

Analysis of the binding of a hemopoietic growth factor, P-cell-stimulating factor, to a cell surface receptor using quantitative absorption of bioactivity.

Quantitative absorption of biological activity was used to study the interaction of persisting (P)-cell-stimulating factor (PSF), a T-cell-derived lymphokine, with PSF-dependent lines of hemopoietic cells (P cells). It was shown that suspension of P cells in medium containing PSF at 4 degrees C resulted in a diminution of PSF activity in the medium. Similar results were obtained with homogeneous, pure PSF or crude supernatants from cells secreting PSF. This diminution was specific and involved saturable, reversible binding of PSF to the cells rather than degradation of PSF or the release of an inhibitor. Calculations based on the measurement of PSF activity remaining after absorption and estimates of the specific activity of PSF indicated that there were approximately 1000 receptors/cell and that PSF bound with an equilibrium dissociation constant (Kd) of 5 X 10(-12) M. Increased amounts of PSF were absorbed at 37 degrees C; however, in the presence of metabolic inhibitors, the amount of PSF activity removed was equivalent to that seen at 4 degrees C.

Antibody stimulation of hemopoietic progenitor cells.

Antisera were raised by immunizing rabbits with cloned lines of murine hemopoietic progenitor cells (P cells) that depended on the presence of a specific hemopoietic growth factor, persisting cell-stimulating factor (PSF), for their growth and survival. The unabsorbed antiserum was inhibitory, but after absorption with murine spleen cells and the mastocytoma, P815, significant stimulation of both P cell growth and thymidine incorporation was evident. IgG antibodies isolated from the antiserum by staphylococcal protein A chromatography or further purified by diethylaminoethyl anion exchange chromatography, ammonium sulphate precipitation, and gel filtration using Sephacryl S-300 were responsible for the stimulation. The absorbed antiserum promoted the survival of normal murine bone marrow cells in liquid culture over a four-day period, and the inclusion of IgG antibodies in agar cultures of normal bone marrow promoted the in vitro survival, over a 48-hour period, of cells capable of subsequently generating, in the presence of a source of PSF, colonies of neutrophils, macrophages, and megakaryocytes. It is postulated that the antibodies act by stimulating the PSF receptor on both the factor-dependent cell lines and normal myeloid progenitor cells.

In vivo transfer of persisting (P) cells; further evidence for their identity with T-dependent mast cells.

Previously we described the persistent in vitro growth of lines of cells (persisting [P] cells) that shared many cytochemical, biochemical, and functional characteristics with mast cells and depended for their survival and growth on a specific T cell-derived factor, P cell-stimulating factor (PSF). Here we present further evidence for their identity with the T-dependent or atypical subset of mast cells and show that they retain characteristics of T-dependent mast cells when transferred in vivo. One week after the injection of P cells into the dermis of mutant Wf/Wf mice, which have a genetically determined deficiency in mast cells, large numbers of mast cells were present at the injection site, although by 2 wk or later these had disappeared. These mast cells resembled T-dependent mast cells rather than connective tissue mast cells in terms of their size and staining characteristics. Further evidence that these mast cells belonged to the T-dependent subset was that they retained their sensitivity to PSF. Thus, if P cells were injected into the dermis of Wf/Wf mice that bore in one groin a subcutaneous tumor (WEHI-3B) that produced PSF, increased numbers of mast cells were still evident at the injection site 4 wk later; this was not the case in mice bearing a non-PSF-producing variant of the same tumor. Experiments with cloned P cells generated from mice bearing the beige (bgJ/bgJ) mutation and with the giant granules of cells of this genotype used as a marker showed conclusively that the mast cells at the injection sites were derived from the injected P cells. P cells sensitized in vitro with monoclonal antigen-specific IgE or IgG1 antibodies and then injected intracutaneously into W/Wv mice transferred local cutaneous anaphylactic responses. P cells sensitized with IgG1 transferred local cutaneous anaphylactic responses to rats. These results support the view that P cell lines are cognate with the atypical or T-dependent subset of mast cells and that these cells retain their functional capabilities when injected in vivo.

The in vivo functions and properties of persisting cell-stimulating factor.

We present evidence that persisting (P) cell-stimulating factor (PSF), a T cell lymphokine, is produced and active in vivo. Mice injected in one footpad with keyhole limpet haemocyanin or intravenously with sheep erythrocytes had substantial increases in numbers of splenic P cell precursors; the increase following the sheep erythrocytes did not occur in athymic mice implying a dependence on T lymphocytes. The increase in P cell precursors correlated with the local release of PSF; thus cells from the ipsilateral draining lymph node of mice injected in one footpad with KLH, but not cells from the contralateral node, showed both increased numbers of P cell precursors and the production of PSF. PSF could, in other situations, enter the circulation and exert effects distal to its release. Mice bearing a localized tumour that produced PSF (WEHI-3B), but not those bearing a non-producing subline, showed both a significant increase in P cell precursors in the spleen and bone marrow, and a marked increase in the numbers of mast cells, megakaryocytes, metamyelocytes and polymorphs in the spleen. PSF was detected in the serum of the mice bearing the PSF-producing tumour. Following intravenous injection of PSF into normal mice there was a rapid initial clearance (t 1/2 4 min), followed after 10 mins by a phase of slower clearance (t 1/2 40 min). This was due to removal of PSF rather than inhibition or destruction by serum factors, as when PSF was mixed in vitro with mouse serum for 24 hr at 37 degrees, no activity was lost.

Activated lymphocyte killer cells derived from melanoma tissue or peripheral blood.

Lymphoid cells infiltrating metastatic melanomas were grown directly from cell suspensions of tumour tissue by the addition of T cell growth factor. Lymphoid cells grew out at the expense of tumour cells in six of seven freshly excised tumours, and cells from two cultures were expanded for in vitro testing of cytolytic function against different target cells. Early in culture the tumour derived lymphocytes killed fresh autologous melanoma cells and, particularly later in culture, were highly and non-specifically cytolytic for cultured melanoma and non-melanoma cells. Cultured peripheral blood lymphocytes from patients with melanoma, and from normal subjects, were cytolytic to the same degree as tumour derived lymphocytes, and also resembled cells grown from tumour tissue in possessing acid phosphatase activity which was resistant to tartrate. Cultured lymphoblasts from both tumour and peripheral blood had a T cell phenotype when analysed with monoclonal antibodies. An in vitro co-culture system was employed to study the kinetics and the precursors of these non-specific killer cells among blood mononuclear cells. Blood mononuclear cells cultured with irradiated B lymphoblasts led to the generation of non-specific cytolytic cells, referred to as activated lymphocyte killer (ALK) cells, after 7-10 days of culture and the progenitors of these ALK cells were demonstrated to be distinct from those of specific cytolytic T cells.

In vitro approaches to lymphopoiesis, hemopoiesis, and oncogenesis.

Lymphopoiesis still remains a black box. Much remains to be learned about the identification of the cellular stages and the factors that regulate the rate of production of lymphocytes. Modifications of the Dexter system should assist with answers to these questions. Our work on PSF and that of other groups who have studied what is almost certainly the same factor under names such as hemopoietic growth factor, IL-3, BPA or multi-CSF suggests that this factor may provide an alternative means of generating cells for transplantation and replacement therapy. At present the only established sources of PSF are the activated T cell or tumors in which we think the gene has been anomalously activated, such as WEHI-3B; the long-term bone marrow culture system, however, seems to be defining a factor or influence that may be identical with, or is able to replace, the T cell factor. Finally, our experiments with the heterogeneous P cell lines and the initiation of oncogenesis by activation of the PSF gene raise some caveats about the use of cultured cells for human therapy. In the mouse system the production of immortalized factor-dependent lines appears to be more frequent in cells taken from long-term bone marrow cultures rather than normal bone marrow. Obviously, further information on the mechanism of immortalization and on the frequency and mechanism of activation of PSF genes in such lines will be of great importance in guiding the practical use of in vitro-derived cells and in the understanding of leukemogenesis.

Autogenous production of a hemopoietic growth factor, persisting-cell-stimulating factor, as a mechanism for transformation of bone marrow-derived cells.

Lines of hemopoietic progenitor cells can be grown for prolonged or indefinite periods in vitro in the presence of a specific growth factor produced by activated T lymphocytes, persisting (P)-cell-stimulating factor (PSF). From such a PSF-dependent line, we report the emergence of variants that had concomitantly acquired both the capacity for autonomous growth in the absence of exogenous PSF and the capacity for the autogenous production of PSF. The link between these two new properties was strengthened by the demonstration that the variant lines absorbed PSF and, in some culture conditions, responded to exogenous PSF. Thus when variant cells were plated at low density in 1-ml agar cultures, cloning efficiency and colony size were enhanced by supplementation with sources of PSF, including medium conditioned by concanavalin A-stimulated normal spleen cells, T-cell tumors and T-cell hybridomas, and, importantly, medium conditioned by the autonomous P-cell lines themselves. In contrast to the parental line, the autonomous clones tested formed progressively growing tumors in vivo. It is proposed that the myelomonocytic leukemia WEHI-3B that produces PSF arose from a neutrophil-macrophage progenitor through acquisition of the capacity for the autogenous production of PSF and that the autogenous production of PSF may play a similar role in a range of proliferative disorders both of the blood and of other tissues containing components derived from the bone marrow.