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Monolayer and suspension cell cultures prepared from Hodgkin's disease tumors in the spleen were examined microscopically and by cytogenetics, tested for lymphocyte and monocyte cell surface properties, and assayed for enzymes by histochemical and spectrophotometric techniques. Hodgkin's disease monolayer cultures were composed of rapidly proliferating round and polygonal cells that were capable of propagation in vitro for an indefinite period of time. Abnormal aneuploid chromosomes were found in short-term Hodgkin's disease monolayers that had been passaged 16-20 times, and in established cell lines carried in culture longer than 3 yr and passaged more than 200 times. Cells fromHodgkin's disease monolayers contained lysozyme (muramidase), fluoride-resistant alpha naphthol acetate esterase, acid and alkaline phosphatase, and chymotrypsin-like activity. The monolayers did not exhibit specific cell surface markers or phagocytosis. Suspension cultures derived from Hodgkin's disease monolayers were composed of cells with aneuploid karyotypes and similar enzymes. The Hodgkin's disease suspension culture cells had surface receptors for complement and IgGFc, lacked surface or cytoplasmic immunoglobulin, and did not form Erosettes, react with an antithymocyte serum, nor exhibit phagocytosis. Normal monolayer culture cells, derived from adult spleen and human fetal spleen and thymus, were composed of spindle cells with a diploid number of chromosomes that could be carried for only a finite period of time in vitro. Normal cultured cells contained similar esterases and phosphatases, but were devoid of lysozyme and chymotrypsin-like activity. The morphologic, cytogenetic, cell surface, and enzymatic findings indicate that our Hodgkin's disease monolayer and suspension cultures are composed of cells with many properties suggesting an origin from monocytes (macrophages) rather than lymphocytes or fibroblasts. The presence of aneuploid karyotypes is consistent with a neoplastic origin and derivation from a malignant cell of Hodgkin's disease.

Long-term monolayer cell cultures have been prepared from tumor nodules in spleens removed from 28 patients with Hodgkin's disease and from 84 spleens that did not have tumors from Hodgkin's disease patients, normal adult spleens, and human fetal spleens and thymuses. After 5 to 20 serial passages in culture, cells from nine of the Hodgkin's disease monolayers underwent morphologic change in vitro with transition from a spindle and reticular pattern of replication to polygonal and round cells that propagated in mosaic arrays. Four of such Hodgkin's disease monolayer cell lines were injected subcutaneously into 43 nude, athymic mice. In 36 animals (84%), neoplasms developed at the inoculation site that werel ocally destructive, capable of pulmonary metastasis, and eventually fatal to the recipients. Transplanted tumors were not observed in 18 athymic mice injected with cultures prepared from normal human adult spleen and fetal spleen and thymus, nor were tumors seen in 16 similar animals that received fresh, noncultured Hodgkin's disease tumor tissue. On microscopic examination, xenografts derived from Hodgkin's disease cultures were pleomorphic malignant neoplasms composed of large, undifferentiated cells, resembling reticulum cell sarcoma. These neoplasms did not involve the lymphoreticular organs of mice. Chromosome studies indicated that the transplanted neoplasms were composed of human cells with an aneuploid karyotype and that monolayer cultures prepared from the heterotransplants contained a karyotype similar to that of the cultured cells prior to passage in mice. The ability of these Hodgkin's disease cell lines to produce invasive tumors with human karyotypes in nude mice is evidence of the neoplastic nature of the monolayer cells and their relationship to the malignant cell of the human disorder.

We examined 90 serums from patients with Hodgkin's disease for immune complexes and for reactivity with established monolayer tissue cultures prepared from the tumor. All 23 serums with immune complex levels greater than 20 microgram per milliliter were found to react with cultured cells of patients with Hodgkin's disease when tested with antiserums against immunoglobulin heavy and light chains and the C3 component of complement. Five of 11 serums with borderline elevations of immune complexes (10 to 20 microgram per milliliter) and only four of 56 with levels less than 10 microgram per milliliter reacted. Absorption of patients' serums with cultured cells removed immune complexes and eliminated binding to monolayers. Immune-complex-containing serums from 19 control patients did react with cultured cells of patients with Hodgkin's disease; none of serums reacted with normal cultured spleen. Antibodies within complement-containing immune complexes in serums of patients with Hodgkin's disease react with an antigen on the surface of cultured cells of such patients.

Answers are beginning to emerge to the questions posed in the introduction to the preceding section. In vitro techniques that allow characterization of malignant cells have particular relevance when, as in Hodgkin's disease, the precise identity of the cells remains in doubt. Monolayer tissue cultures derived from Hodgkin's disease tumours and maintained as established cell lines have proven amenable to a variety of cytogenetic, immunological, enzymatic, and ultrastructural studies. Tissue culture experiemnts, in conjunction with meticulous immunological studies of individual Reed-Sternberg cells from non-cultured tumours, suggest that neoplastic cells of Hodgkin's disease are related to, and possibly derived from, cells of the monocyte-macrophage system. The lymphocytes that comprise an integral part of the cellular proliferation and form the basis for histological subclassification of the tumour could be a manifestation of cell-mediated immunity against this non-lymphoid malignant cell. The immunodeficiency of patients with untreated Hodgkin's disease of limited anatomical extent is not the primary event of the disorder and probably not related to the site at which the aetiological agent acts. The deficit does not result solely from impaired T-cell function and appears to arise as a consequence of excessive suppressor cell activity. Inhibitory monocyte-lymphocyte interactions may be one of the causes of defective cell-mediated immunity in Hodgkin's disease. The possible significance of elevated levels of circulating immune complexes in the serum of patients with Hodgkin's disease is indicated by the finding that such complexes react with cells of long-term monolayer tissue cultures derived from the tumour. Circulating immune complexes may be one source for intracellular immunoglobulin in non-cultured Hodgkin's disease cells. The presence of polyclonal immunoglobulin G on the membrane and within the cytoplasm of Reed-Sternberg cells could be due to in vivo binding and ingestion of immune complexes by such cells. The specificity of the interaction between soluble complement-containing immune complexes and neoplastic cells of Hodgkin's disease depends on the nature of the complexed antigen. The complexes could non-specifically attach via an Fc receptor or, if the complexed antigen is identical to a tumour cell antigen, the binding could be specific. If the immune complexes are tumour specific they could provide a source for isolation and identification of tumour-associated antigens. However, the aetiological significance of antigens and putative oncogenic viruses thus far identified in association with Hodgkin's disease remains to be clarified.

The surface of lymphocytes obtained from fresh biopsy specimens from 41 patients with malignant lymphoma and from 30 normal subjects or patients with non-neoplastic lymphadenopathy were investigated. Immunoglobulin on the cell surface was used to identify B cells, whereas T cells were recognized by their reactivity with an antithymocyte antiserum and their ability to form rosettes with sheep erythrocytes. Normal and inflammatory lymph nodes were composed predominantly of T lymphocytes, as were nodes from 14 patients with Hodgkin's disease. Two thymomas were T cell proliferations, whereas a node from a patient with ataxia-telangiectasia was devoid of T lymphocytes. The presence of immunoglobulin on the cell surface indicated that 19 of 21 lymphocytic lymphomas were B cell proliferations, whereas the cells from 3 histiocytic lymphomas (reticulum cell sarcomas) and 1 mixed histiocytic and lymphocytic lymphoma were devoid of surface immunoglobulin. In immunoglobulin-positive tumors, one predominant heavy chain and one predominant light chain could usually be identified, thus establishing the clonal character of the neoplastic proliferation. Ten of 11 diffuse poorly differentiated lymphocytic lymphomas were composed of cells with large amounts of surface immunoglobulin, whereas only 1 of 5 diffuse well differentiated lymphocytic tumors contained such abundant surface immunoglobulin. The surface immunoglobulin data indicate the existence of at least two subspecies of B cell neoplasms. A small lymphocyte with sparse surface immunoglobulin proliferates as diffuse well differentiated lymphocytic lymphoma and chronic lymphocytic leukemia, whereas a larger lymphocyte with abundant surface immunoglobulin proliferates as diffuse poorly differentiated lymphocytic lymphoma and lymphosarcoma cell leukemia.

Rabbits were immunized with an antigen of specific gravity, 1.15-1.21 isolated by density gradient sedimentation of the centrifuged medium of long-term monolayer cultures derived from spleens involved by Hodgkin's disease. The globulin fraction of the antiserum was absorbed to reduce reactivity with normal cellular antigens and tissue culture components, and was tested by the indirect fluorescent antibody technique with cells from 18 different Hodgkin's disease cultures, and 16 normal cultures derived from adult spleen and fetal spleen and thymus. With anti-Hodgkin's disease globulin diluted 1:40 and 1:80, positive surface staining was observed in 48% and 41%, respectively, of viable cells from Hodgkin's disease cultures, and in less than 5% of cells cells from normal cultures. Fluorescent staining of the cytoplasm without nuclear staining was observed in 51% of acetone-fixed cells from the Hodgkin's disease cultures and in 4-8% of cells from normal cultures. Reactivity of the antiserum with Hodgkin's disease target cells could be removed by absorption of the antibody with additional antigen of density 1.15-1.21 obtained from other Hodgkin's disease cultures. Antisera to fractionated medium from a normal spleen culture and to noncultured Hodgkin's disease tumor tissue were used as controls: 2-10% of viable and acetone-fixed target cells reacted and no difference was observed between Hodgkin's disease and normal cell cultures. In vitro propagation of tumor cells from patients with Hodgkin's disease is needed for detection of the Hodgkin's disease tissue culture antigen; the antigen could not be demonstrated in noncultured Hodgkin's disease tissue.

Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells.

An antigen in tissue cultures derived from Hodgkin's disease tumors was investigated by polyacrylamide gel electrophoresis, column chromatography, and isotopic antibody techniques. Fourteen long-term, serially passaged monolayer cultures prepared from tumor nodules of Hodgkin's disease in the spleen were studied; 11 monolayers derived from normal adult spleen and human fetal spleen and thymus were used as controls. Cell-free medium from Hodgkin's disease and normal cultures were centrifuged, and the pellet fractions were sedimented in a discontinuous sucrose gradient, solubilized with dodium dodecyl sulfate, and labeled with radioiodine. Gel filtration and electrophoresis revealed a component in samples prepared from medium of Hodgkin's disease cultures that was not observed in medium from normal cultures. An antiserum made in rabbits against this component reacted by radioiodine-labeled antibody assay with an antigen on the surface on cells from Hodgkin's disease cultures that was present in very small amounts, or in a cryptic state, on normal cultured cells. This antigen, intimately associated with propagation of cells obtained from the tumor in vitro, was not demonstrable in noncultured Hodgkin's disease tissue...

An antiserum was prepared in rabbits against an antigen obtained by density gradient sedimentation of centrifuged medium from monolayer cultures of spleens involved by Hodgkin's disease. The antiserum was tested by isotopic antibody techniques with cells from each of eight cultures derived from spleens involved by Hodgkin's disease, four cultures derived from normal adult spleen, and one culture each of fetal spleen and thymus. By an indirect radioiodine-labeled antibody assay, anti-Hodgkin's disease globulin reacted with an antigen on the surface of cells from the Hodgkin's disease cultures, the quantity of which was related to the number of target cells and the amount of antibody used. This Hodgkin's disease tissue-culture antigen did not react with a rabbit antiserum against fractionated medium from a normal spleen culture, nor against noncultured Hodgkin's disease tumor tissue. The tumor specificity of the Hodgkin's disease tissue-culture antigen was assessed by a direct technique using (125)I-labeled anti-Hodgkin's disease globulin absorbed with either cultured Hodgkin's disease cells or with cultured normal cells. By this method the quantity of antigen on cells from Hodgkin's disease cultures was 15- to 30-fold greater than that on cells from normal cultures. The Hodgkin's disease tissue-culture antigen is intimately associated with the propagation of the tumor in monolayer cultures, but its identity has not been established: it could be a viral component, a tumor or fetal antigen, or a normal tissue constituent.

Cells from 9 monolayer tissue cultures prepared from Hodgkin's disease tumors in the spleen were examined in the electron microscope. Three established culture lines (carried in vitro for greater than 3 years and passaged greater than 200 times) that contained aneuploid karyotypes were composed of oval cells with numerous interdigitating surface microvilli. The nuclei were complex and convoluted with multiple large nucleoli and dispersed chromatin. The cytoplasm contained lysosomes, microfilaments, a complex Golgi apparatus, nondilated rough endoplasmic reticulum, polyribosomes, fat, and glycogen. One Hodgkin's disease monolayer with aneuploid chromosomes examined from the 4th to 48th passage in culture was composed of larger cells with fewer microvilli and numerous multinuclear giant cells. Two monolayers derived from transplanted tumors in nude mice inoculated with Hodgkin's disease cultured cells were similar to the original cell lines. The ultrastructural features of these 6 cultures with aneuploid karyotypes differed from those of 3 monolayers which, although prepared from Hodgkin's disease splenic tumors, were composed of fibroblastic cells with diploid chromosomes. The aneuploid Hodgkin's disease cultures did not resemble 6 normal spleen, thymus, or lung monolayers, Raji lymphoblastoid suspension cultures, or Hela cells. Our electron microscopic studies indicate that adherent cells which replicate in some monolayer tissue cultures derived from Hodgkin's disease tumors are related to and possibly derived from neoplastic macrophages.

Levels of circulating immune complexes (CIC) in the serum of patients with Hodgkin's disease were measured by the Raji cell radioimmunoassay. Elevated levels of immune complexes (mean value of 49 microgram/ml +/- 21 SE) were detected in 20 of 40 (50 per cent) untreated patients. After treatment, the level of CIC was normal (less than 15 microgram/ml) in 39 of 41 patients. Recurrent disease developed in two of the 39 patients with normal post-treatment levels of CIC and in one of the two patients with elevated post-treatment levels during the follow-up period of six months to six years. Elevated levels of CIC were detected in patients with Hodgkin's disease in stages I, II and III but not in stage IV. No significant correlations were found in the frequency of elevated levels of CIC or the values observed, and the presence or absence of symptoms (fever, sweats, weight loss) or the histologic subtype of the tumor. Our data indicate that the measurement of CIC by the sensitive and specific raji cell assay may prove useful in the management of patients with Hodgkin's disease. In particular, serial measurement of the level of CIC could be employed to monitor the response to treatment and to detect recurrent diseases.

We examined the binding of soluble immune complexes in sera from patients with Hodgkin's disease to established tissue cultures derived from the tumor. Circulating immune complex levels were determined by the Raji cell assay, and the reaction of serum with cultured cells was examined with a radioimmune assay and by immunoferritin electron microscopy. Serum with elevated immune complexes was found to react with cells of Hodgkin's disease monolayers when tested with radioiodine-labeled antisera against human IgG heavy and light chains and the complement 3 (C3) component. When examined with the electron microscope, monolayers incubated with Hodgkin's disease serum containing immune complex and labeled with ferritin-conjugated antiserum to C3 contained surface-bound ferritin particles with a uniform but discontinuous pattern. Absorption of Hodgkin's disease serum with monolayer cells reduced immune complexes and decreased reactivity of the sample with cultured cells by radioimmune assay. Sera of patients with other disorders and aggregated gamma-globulin with complement, despite markedly elevated immune complex levels, did not react positively with monolayers derived from Hodgkin's disease tumors, and none of the sera reacted with normal cultured spleen. The approximate size of serum components reacting with Hodgkin's disease monolayers was estimated by sucrose density gradient centrifugation. Sedimentation fractions in the 19S region reacted with monolayer cells when tested with 125I-labeled antisera to both IgG and C3 and contained immunoglobulin-complement complexes by gel diffusion and immunoabsorption. A component sedimenting at 7-9S contained immunoglobulin not complexed with complement; this component reacted with monolayer cells when tested with anti-IgG antiserum but did not react when tested with antibody to C3. The reaction of Hodgkin's disease monolayers with serum containing immune complexes differed from that of two suspension culture lines composed of cells with surface complement and IgG Fc receptors. Inasmuch as cells of our long-term Hodgkin's disease monolayers do not contain these surface receptors, possibly the antibody component of the immune complex reacts with antigens on the surface of cultured cells.

Pellets obtained from supernatant fluids of monolayer cultures of cells from patients with Hodgkin's disease were fractionated by isopycnic density sedimentation. Material in a peak of specific gravity 1.15-1.21 g/ml from two Hodgkin's disease cultures was used to immunize rabbits, and the antisera obtained in this manner were reacted by agar-gel diffusion and immunoelectrophoresis with antigens from the purified peaks and the unfractionated pellets of centrifuged culture medium from all the cultures. The antisera reacted with material from 9 of 10 lines derived from spleens of patients with Hodgkin's disease, 2 of 8 cell lines from histologically negative spleens from patients with Hodgkin's disease and with 3 of 6 lymphoma cell lines not diagnosed as Hodgkin's disease. The antisera did not react with 12 cell cultures prepared from normal adult and fetal spleen and thymus. The antigen from cultures from patients with Hodgkin's disease was not found in material sedimenting at lower specific gravities; it resisted Tweenether solubilization, and migrated as a single band by immunoelectrophoresis. The antigen was not found in disrupted, noncultured tumor cells from patients with Hodgkin's disease, and an antiserum against noncultured, minced tumor tissue did not react with the Hodgkin's disease tissue-culture material. No immunological relationship was found between the tissue culture antigen and Epstein-Barr, RD-114, or Rauscher murine leukemia viruses. The Hodgkin's disease antigen may be a tumorrelated antigen or a component of an oncogenic virus.