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Recently, single-cell PCR studies have demonstrated that Hodgkin and Reed-Sternberg (HRS) cells are clonally related in many cases of Hodgkin disease. To investigate the lineage and clonality of neoplastic cells in local environments in nodular sclerosis Hodgkin disease (NSHD), we microdissected multiple distinct nodules from patients with NSHD and analyzed them for IgH gene rearrangement by PCR. These results were correlated with immunophenotype, Epstein-Barr-encoded RNA (EBER) expression, and clinical outcome. Forty individual nodules from 10 patients with NSHD (11 specimens) were microdissected from formalin-fixed paraffin-embedded tissue. DNA extracts were analyzed for IgH gene rearrangement by using PCR with FRIIIa and JHa primers. Cases were immunophenotyped in paraffin sections with antibodies to CD20(L26), CD79a(HM57), CD45RO(A6), CD15 (Leu-M1), and CD30(Ber-H2). Infection of HRS cells by Epstein-Barr virus was evaluated by using EBER in situ hybridization (EBER-ISH). DNA extracts from 12 of 40 microdissected nodules from 8 of 10 patients demonstrated a monoclonal pattern by IgH-PCR. Three patients demonstrated 2 individual nodules with different monoclonal patterns. One patient demonstrated 2 nodules with bands that appeared similar in size but were found to be different from one another upon further testing. All 28 remaining nodules demonstrated a polyclonal pattern. Six of 10 patients were positive for the Epstein-Barr virus genome by EBER-ISH. No correlation was found between IgH monoclonality, immunophenotypic features, Epstein-Barr virus infection, or clinical outcome. It was concluded that a subset of NSHD cases contain detectable monoclonality within individual nodules by IgH-PCR, suggesting that HRS cells are clonally related within local microenvironments.

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Using Southern blot and fluorescence in situ hybridization analyses, we have shown that the novel human gene EPAG, expressed in association with the mitogenic activation of T and B cells in peripheral blood, maps to chromosome region Xq21-->q22. These data establish EPAG as a candidate gene in X-linked disorders.

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We have isolated a novel 667-bp cDNA clone, designated epag, from a Hodgkin's-disease cell line-derived library that is expressed in association with T cell activation and which is not related to any known gene family. By using reverse transcription/PCR, we have demonstrated that epag mRNA is expressed as early as 1 h after stimulation of normal PBMCs with anti-CD3. The levels of mRNA peaked by 4 h, and no expression was detectable by 12 h postactivation or in resting cells incubated in culture without activation. Expression of epag was also detected in PMA- and PHA-stimulated, but not in nonstimulated Jurkat cells, and overall its expression in transformed cell lines of hemopoietic origin is highly restricted. Sequence analysis of multiple independent cDNA clones showed that epag expressed in the Hodgkin's-disease cell line L428 is identical to the gene expressed in normal activated PBMC. Epag expression was detected by reverse transcription/PCR in RNA preparations made from various normal nonlymphoid tissues. Computer analysis of the sequence identified an open reading frame encoding a putative protein of 13.2 kDa initiating at a CUG translational codon. In vitro translation and Western blot analysis with anti-peptide serum supported this analysis. We hypothesize that epag functions as an early signal that helps mediate the activation of T cells.

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Patients with Hodgkin's disease, at presentation or in remission, exhibit a persistent defect in cellular immunity. Natural killer cell mediated cytotoxicity is depressed in untreated patients. Humoral immune function is transiently reduced following treatment. The cellular immune defect appears to be the result of enhanced sensitivity to suppressor monocytes and T-suppressor cells, in addition to abnormal interleukin-2 production. Patients with advanced disease have an inherent T-lymphocyte defect. Reed-Sternberg cells function as antigen-presenting cells for mitogen-induced and mixed lymphocyte T-cell proliferation.

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Patients with Hodgkin's disease, either untreated or in remission, exhibit a persistent defect in cellular immunity. This cellular immune defect appears to be the result of increased sensitivity to suppressor monocytes and T-suppressor cells, in addition to abnormal Interleukin-2 production. T-lymphocyte function is abnormal in patients with advanced disease. The precise origin of Reed-Sternberg and Hodgkin's cells is unknown. Reed-Sternberg cells function as antigen-presenting cells and as accessory cells in mitogen-induced T-cell proliferation. They have properties in common with dendritic cells and activated lymphocytes. L428 cells express a transformation-associated phosphorylated transmembrane protein, with properties of a growth factor receptor, that may play a role in tumorigenic transformation.

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The HeFi-1 mAb recognizes a membrane protein on Hodgkin's disease cells and on a limited number of other human cells that are either tumorigenically transformed or virally activated. Herein biochemical and structural analyses of the HeFi-1 reactive membrane protein (HRMP) were done to identify its potential importance in cellular transformation in the Hodgkin's disease cell line L428, in the T cell lymphoma line HuT 78, and in several EBV-transformed lymphoblastoid cell lines. Immunoprecipitation studies demonstrated that the mature form of the HRMP had an apparent Mr of 120 kDa in tumor cells and 116 kDa in the EBV-transformed cell lines and that it was phosphorylated at both serine and tyrosine residues in all cell lines tested. The precursor to the HRMP is an 86-kDa core protein that, after processing by high mannose N-linked glycosylation, migrates with an apparent Mr of 90 kDa. This protein is then further processed to the mature 120-kDa HRMP in part by O-linked glycosylation, the addition of sialic acid residues, and by the conversion of N-linked oligosaccharides from the high mannose to the complex type. Detectable amounts of the 90-kDa molecule can be found in the membrane and, although this protein can be phosphorylated in vitro, it is not phosphorylated in intact cells. The combined results of this study suggest that the HRMP is involved in cellular metabolism and show that an unusual amount of post-translational processing of the 90-kDa precursor results in the formation, and perhaps phosphorylation, of the mature 120-kDa HRMP.

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With the use of cells doubly labeled with 51Cr and 125I, an analysis was made of the distribution and lysis in normal and immune syngeneic C58 mice of weakly immunogenic malignant lymphocytes (Ib) and a highly immunogenic variant (IbN) derived from them. The results showed that 51Cr release was not a valid measure of tumor cell lysis in vivo. Prior exposure to 55 degrees C for 30 minutes caused Ib to lyse immediately after iv injection. Prior X-irradiation (10,000 R) enhanced the lysis of Ib in vivo but had only minor effects on IbN. In normal mice 125I-labeled Ib were entrapped in the lungs (approximately to 95%) immediately after iv injection, released in a diphasic manner, and then accumulated in the liver, spleen, bone marrow, and lymph nodes. In immune mice initial entrapment of Ib in the lungs was only about 42%, and label did not accumulate in the described organs. Heat-inactivated Ib were not retained to a significant degree in any of the organs of normal or immune mice. IbN were retained initially (2 hr) at values lower than Ib in the whole body, lungs, and livers of normal mice. 125I-labeled Ib were used to analyze how they were distributed and lysed in normal and immune mice during the first 6 hours after iv injection. To account for the loss of 125I from a tissue because of cell lysis (as distinct from cell redistribution), the amount of 125I associated with cells and blood plasma, and lost from the whole body, was quantified. A significant loss of 125I from the whole body began at 2 hours and continued at a linear rate thereafter. Non-cell-associated 125I occurred in the blood plasma at 1-2 hours and increased at a linear rate thereafter. These results made it possible to distinguish between loss of 125I from a tissue because of cell lysis as distinct from the redistribution of intact labeled cells.

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The phosphorylation of a 34,000-molecular-weight (34K) cell protein, purported to be a substrate of the avian retrovirus pp60src-associated protein kinase activity, was compared in three types of Rous sarcoma virus-infected vole cells: fully transformed cells, partial revertants which are morphologically normal in appearance but retain their tumorigenic potential, and full revertants which are similar to normal vole cells in all parameters including a lack of tumorigenicity. Although similar amounts of 34K protein are present in all three cell types, phosphorylation of the 34K protein was significantly reduced in the full revertant cell type. The reduced phosphorylation occurred at the tyrosine residue.

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A computer model was constructed to simulate the lymphocyte-mediated destruction of line Ib malignant lymphoid cells (Ib cells) as they circulated through the major tissue compartments of immune syngeneic C58 mice. The technique of discrete-event simulation was used to account for the arterial and venous circulation of blood-borne Ib cells through the lung, spleen, liver, and carcass. Simulation was carried out by means of IBM computer program 360, using the technique of General Purpose System Simulation. The parameters analysed were the mean residence times of viable and killed Ib cells in each tissue compartment, the rate or proliferation of Ib cells, the rate of generation of cytotoxic splenic lymphocytes, the rate of lysis of 51Cr labelled Ib cells, and the organ-specific rate constants for target cell kill. Direct laboratory measurements of these parameters validated the model and made it possible to calibrate computer simulations by the technique of best-fit analysis. The computer modelling technique accurately simulated the growth of viable Ib cells in vivo and the retention times of 51Cr in the spleen, lung, liver and carcass when viable or heat-killed Ib cells were inoculated intravenously (i.v.) into normal and immune mice. Computer simulations quantitatively defined the mean residence times of viable and heat-killed Ib cells in the major tissue compartments and the mean rate constants for target cell lysis in such compartments. The applicability of modelling approach to an analysis of immunological phenomena is discussed.

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RNA extracted from the spleens of tumour-bearing (TLRNA) and tumour-immune (ILRNA) mice was shown to transfer to normal lymphocytes (NL) the ability to produce factors that blocked specific tumour-cell cytotoxicity and mediated specific antibody-dependent cell cytotoxicity (ADCC). Aliquots of normal C3H mouse lymphocytes were treated with TLRNA or ILRNA and cultured in vitro in the absence of tumour antigen. Supernatants were collected at 24h intervals and tested in a microcytotoxicity assay for blocking and ADCC activities. Factors that inhibited tumour destruction by specifically sensitized lymphocytes at the level of both the tumour cells and effector cells were demonstrable in culture supernatants of NL pretreated with TLRNA (50 or 100 microgram/4 X 10(6) cells) but not ILRNA. However, treatment of NL with either RNA resulted in the production factors that mediated tumour-specific ADCC. Cytotoxicity testing and absorption studies of the tumour cell and a control cell (LM) indicated that factors mediating ADCC and blocking at the target-cell level were specific for the tumour. Suppressor activity at the effector-cell level was not absorbed by tumour cells and represents a separate and distinct mechanism of immunosuppression. These data indicate that RNA faithfully transfers "suppressive" as well as "positive" types of immune responses that have been reported previously for lymphocytes obtained directly from tumour-bearing and tumour-immune animals.

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