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The production of heparanase, an endoglycosidase capable of degrading heparan sulfate from the subendothelial extracellular matrix (ECM), was investigated in various murine B-lymphoid tumors representing distinct maturation stages of the B-cell lineage. We found that heparanase is produced and released by 3 out of 4 pre-B lymphomas and by 4 B lymphomas examined. In contrast, 5 plasmacytomas and resting normal B lymphocytes, expressed little, if any, heparanase activity. Treatment with LPS resulted in high expression of the enzyme by normal B-lymphocytes, but there was no effect on the constitutive production of heparanase by myeloma or B-lymphoma cells. Our results indicate that heparanase is produced by B cells during discrete stages of their maturation. We suggest that heparanase may play a role in B-cell migration by enabling pre-B and B lymphocytes to leave the bone-marrow compartment and recirculate among peripheral lymphoid organs.

Human B cell lines were tested for the capacity to convert C3 to C3b/iC3b through the alternative C pathway (ACP) and to fix the generated C3 fragments. The panel of lines included four Epstein-Barr virus (EBV)-negative Burkitt's lymphomas and their sublines converted with two EBV strains: the prototype B958 (B virus) and the nontransforming P3HR-1 (P virus). Comparison of these groups of parallel lines (derived from a single patient) showed that the EBV-carrying cells were more efficient in activation and fixation of C3. All cell lines activated C3 with higher efficiency when treated with interferon gamma or tumor necrosis factor alpha. After treatment, the comparative relationships between the sublines changed. The treated EBV-negative and the P virus converted sublines showed similar levels of ACP activation, while the B virus carrying sublines were more efficient. The amounts of bound C3 fragments on the activating cells correlated with the ACP efficiencies. In accordance with the elevated ACP activation capacity of the cells after cytokine treatment, the amounts of C3 fragments detected on their surfaces were higher. Among the lines tested, only two (BL28 and Ramos) were sensitive to C-mediated lysis. The lytic sensitivity of these two lines increased after treatment with the cytokines. The results indicate, therefore, that the cytokine-induced increase in ACP activation by Burkitt's lymphoma line is not sufficient to overcome resistance to C lysis.

C57BL mice inoculated with radiation leukemia virus (RadLV) develop preleukemic cells long before the onset of leukemia. These cells are potentially immunogenic but fail to elicit an immune response in the host because of the appearance of virus-specific suppressor T cells. We have studied the effect of polysaccharide K (PSK) on the generation of RadLV-specific cell-mediated immune responses in vitro. Long-term exposure to PSK in culture potentiated the ability of immunized T cells to respond to a RadLV-induced lymphoma. It also abrogated the suppressive activity of suppressor T cells and simultaneously boosted the ability of reactive T cells to respond. The dual immunostimulating activity of PSK resulted in the generation of T cytotoxic lymphocytes that could lyse lymphoma cells in vitro. The results suggest that PSK could be used as a prophylactic immune response modifier in preleukemia.

The complement receptor CR3 molecule functions in direct intercellular contacts mediated by its beta chain, CD18. Similarly to the Fc receptor (CD16), CR3 is a marker of human natural killer cells. We have shown that opsonization of NK targets with iC3b leads to their increased lytic sensitivity. Opsonization could be achieved by incubating certain B and T cell lines in human serum. The expression of CR2 was a prerequisite for C3 fragment fixation. The CR2 negative cell line, P3HR1 could be opsonized by incubation in human serum when induced to express the EBV envelope glycoprotein gp350. C3b or iC3b could also be deposited artificially on cell surfaces by chemical coupling to surface reactive antibodies. Similarly to the function of macrophages and monocytes, contact with opsonized targets exclusively through the iC3b binding site of CR3 did not seem to trigger NK function. We attempted to clarify the functional role of other CR3 ligands. The beta chain of the molecule, CD18, was essential to the NK effect. The NK targets did not seem to interact with the beta-glucan binding epitope on the alpha chain of CR3, CD11b. On the other hand, the cytolytic function could be enhanced through this epitope with the appropriate ligand.

BALB/c mice immunized with keyhole limpet hemocyanin (KLH) develop Thy-1+ and CD8+, KLH-specific cytotoxic T cells (Tc). Such Tc cells can lyse TNP-specific B cells activated with TNP-KLH, but not with TNP-ovalbumin. Cytotoxicity was inhibited by anti-H-2K/D antibodies, but not by anti-Ia antibodies, suggesting a major histocompatibility complex class I-restricted killing. Selective enrichment for virgin and memory TNP-specific B cells revealed that the latter cells were relatively resistant to lysis by KLH-specific Tc cells. Depletion of CD8+ T cells from cultures of TNP-specific virgin B cells activated with TNP-KLH and KLH-primed T cells, increased the titer of anti-TNP antibodies in the culture supernatants. This increase was reduced if KLH-primed CD8+ T cells were added to the culture 1 day before its termination. Anti-TNP antibody secretion by memory B cells activated in the same manner was not affected by depletion or addition of CD8+ cells. These results suggest that Tc cells are generated following immunization with a soluble antigen which may participate in the down-regulation of primary B cell responses.

Raji cells activate the alternative complement pathway (ACP) and fix C3 fragments when incubated in human serum (HS). Earlier experiments have shown that CR2 molecules are involved in this phenomenon and the opsonized cells have elevated sensitivity to the lytic effect of CR3-bearing NK cells. We show here that Raji cells treated with CR2 site-specific ligands, (C3d, OKB-7 and HB-5 mAbs, and a synthetic peptide which binds to CR2) generated and bound C3 fragments after exposure to HS. The elevated lytic sensitivity of HS-treated cells was not altered by the presence of the various CR2 ligands. Thus, the membrane-bound C3 fragments are not fixed at the C3dg receptor binding site.

The cell line designated PIR-2 is a nonimmunogenic X-ray-induced thymoma of C57BL/6 origin that is unable to induce antitumor immunity in syngeneic lymphocytes in vitro and in mice in vivo. Fusion of PIR-2 with an allogeneic "universal fuser" A9HT (clone 3c) resulted in the establishment of a somatic cell hybrid designated A9/PIR. C57BL/6 lymphocytes sensitized in vitro with A9/PIR could lyse parental PIR-2 cells, as well as other syngeneic tumors. However, immunization of mice with the hybrid significantly enhanced PIR-2 tumor takes while it partially protected the animals against a challenge with unrelated syngeneic tumors. The results imply that somatic cell hybridization can increase the immunogenicity of an otherwise nonimmunogenic tumor. However, in view of the enhancing effects of hybrid preimmunization on parental tumor cell growth, the possible application of this approach for immunotherapy is questionable.

Adult C57BL/6 (B6) mice exposed to fractionated irradiation or inoculated with a highly leukemogenic RadLV (A-RadLV) develop high incidence of lymphatic leukemias after a latency of 3-5 months. Inoculation of a low leukemogenic RadLV variant (D-RadLV) does not result in lymphoma development unless the animals receive a single, sublethal dose of irradiation. Whereas virus-induced primary lymphomas produce virus, express gp70 and are all immunogenic, those induced by X-rays are devoid of virus expression and are not immunogenic. A-RadLV inoculation into B6 mice is associated with early induction of virus specific, cyclophosphamide sensitive suppressor T cells which are capable of abrogating a potential antitumor immune responsiveness. D-RadLV, on the other hand, induces reactive T lymphocytes that arrest leukemogenesis unless their function is impaired by irradiation. In (B6 X BALB/c)F1 mice both A-RadLV and D-RadLV leukemogenesis depend on sublethal irradiation of the host, and both induce reactive T cells. These results suggest that (a) RadLV and X-ray induced leukemogeneses involve different etiologies. (b) Different leukemogenic treatments evoke different host responses early in latency which can either sustain or interfere with lymphoma progression.

Virus production, EBV (P3HR-1 substrain) superinfectability, IdUrd inducibility, EBV receptor and complement (C3) receptor expression were assessed in two independently maintained jijoye lines, the derived P3HR-1 clone that releases a growth inhibitory and cytopathic, non-transforming viral mutant, and in non-producer sublines derived from the P3HR-1 line by the spontaneous cessation of virus production. Both jijoye lines were superinfectable, inducible, and carried EBV and C3 receptors. Virus-producing P3HR-1 cells and recently derived non-producer sublines lacked EBV-receptors and C3 receptors, could not be superinfected, but were IdUrd inducible. Two long-passaged, non-producer sublines of P3HR-1 reexpressed EBV and C3 receptors to an equal degree (different in the two sublines). EBV-superinfectability became partially reestablished in the subline with the higher expression of EBV and C3 receptors. These findings support the hypothesis that the EBV-receptor/C3 receptor negativity of the producer P3HR-1 sublines and their recent non-producer derivatives is due to negative selection by the growth-inhibitory, cytopathic P3HR-1 virus variant. The closely linked disappearance and reappearance of EBV-receptors and complement receptors gives further support to the idea that these two receptors are either identical or closely linked constituents of the cell membrane.

Pretreatment with the P3HR-1 substrain of Epstein-Barr virus (EBV) inhibited approximately 85% of the DNA stimulation induced by Staphylococcus aureus in human B lymphocytes. In parallel experiments, the DNA stimulation induced by the transforming B95-8 substrain of EBV was almost completely inhibited by prior exposure to P3HR-1 virus. Phytohemagglutinin stimulation was only slightly inhibited, whereas the DNA synthesis of continuously growing Raji cells was inhibited to a considerable extent. The degree of DNA inhibition was correlated with the induction of the EBV-determined antigens (membrane, early, and nuclear antigens) in sensitive indicator cells.

Raji and Daudi cells were opsonized with C3b, iC3b, and C3d fragments by using purified complement components. The sensitivity of C3-opsonized cells to lysis mediated by low density blood lymphocytes was studied. Raji and Daudi cells carrying C3b or C3d fragments were lysed with similar efficiencies as the nonopsonized cells. The presence of iC3b on the target surface imposed elevated NK sensitivity. The iC3b-mediated enhancement of NK lysis was inhibited when iC3b fragments or rabbit anti-human C3 antibodies were included into the lytic assays. These results indicate that the iC3b fragments fixed on the targets bind to the CR3 on the lymphocytes. Results obtained in immobilized conjugate-lytic assays showed that iC3b-opsonized targets interact more readily with the lymphocytes. This was reflected by the elevated proportion of lymphocytes that were bound to the iC3b-carrying targets. The proportions of conjugates in which target damage occurred were similar with the control and with the iC3b-carrying cells. It seems therefore that opsonization of targets with iC3b leads to recruitment of effector lymphocytes due to contact with their CR3. However, once the effector-target contact is established, the triggering of lytic function does not seem to be influenced by the iC3b/CR3 bridge.

The majority of cell lines derived from Burkitt lymphomas carry CR2 on their plasma membrane cell lines of haematopoietic origin can activate C3 present in human serum through the alternative pathway. However, only the lines that carry CR2 were shown to bind C3 fragments. This bond can be either fixation to acceptor sites or attachment to the CR. Our studies with Raji cells showed that when the possibility for the covalent acceptor bond was eliminated by using methylamine (MA)- or zymosan-treated serum, considerably lower amounts of C3 were bound. In the zymosan-treated serum C3 fragments are present that can bind to receptors but their capacity for acceptor bond is absent. These results indicate that when Raji cell are incubated in human serum some of the generated C3 fragments are bound to acceptors and a lower proportion through the specific interaction with complement receptors. Pretreatment of the CR2 carrying cell lines with human serum elevated their sensitivity to the lytic effect of human blood lymphocytes. We showed in this work that MA-treated serum did not induce this elevation. Zymosan-treated serum under conditions that excluded activation of the residual native C3 molecules, i.e., in the presence of EDTA, did not have the enhancing effect either. These results suggest that the increased lytic efficiency imposed by human serum was due to cleavage of C3 molecules by Raji and fixation of the C3 fragments by acceptor sites. Natural killer cells carry CR3; therefore it is likely that the attached C3 fragments bind also to the effector cells. The C3 molecules could elevate thereby the avidity between the target and the lytic lymphocytes. The observation that C3 fragments are not bound to the surface of CR2 negative lines in spite of their capacity to activate C3 suggests that the receptor molecule is either involved in the activation and/or serves also as an acceptor.

Complement (C3) receptors, EBV receptors, Fc receptors, membrane IgM and beta2microglobulin (beta 2m) were individually stripped from the surface of human B lymphoma lines. The cells were subsequently tested for their ability to absorb infectious EB virus. Stripping of Fc receptors, IgM and beta2m did not reduce EBV absorption. Stripping of either EBV receptors or C3 receptors eliminated or drastically reduced EBV-absorptive capacity. The results confirm the distinctive association between EBV receptors and C3 receptors on human lymphoma cells.

C57BL/6 (B6) mice inoculated with the highly leukemogenic variant of the radiation leukemia virus (A-RadLV) develop suppressor cells capable of abrogating potential anti-tumor immunity in vitro and in vivo. Inoculation of B6 animals with the low-leukemogenic D-RadLV variant does not result in suppressor cell generation but induces antitumor reactive lymphocytes. A-RadLV and D-RadLV are not leukemogenic in BALB/c or (B6 X BALB/c)F1(F1) mice, and reactive but not suppressor lymphocytes could be demonstrated in F1 animals inoculated with either virus. Infectivity assays and fingerprint analysis revealed that A-RadLV and D-RadLV contain viruses with N and B tropism. In addition, thymoma cells induced by A-RadLV produced another virus with a fingerprint pattern containing X-MuLV elements. The possible implications of the different virus types on the immunogenic and leukemogenic properties of the RadLV variants are discussed.

The cytotoxic activity of human blood lymphocytes toward Raji cells was strongly elevated when human serum (HS) was included in the cytotoxicity assay. This phenomenon also occurred when the effector cells were activated by interferon (IFN). Hypogammaglobulinemic serum (HyS) and heat-inactivated serum could also augment cytotoxicity, but C3-depleted serum was inefficient. IFN treatment of Raji cells decreased their sensitivity to lysis and this effect was counteracted by addition of HS to the system. It is likely that C3 activation by, and deposition on, Raji cells when used as targets for cytotoxicity facilitate their recognition and lysis by lymphocytes. These events may represent one mechanism operating in the natural killing phenomenon.

Lysis of Raji cells by human lymphocytes was found to be enhanced if human serum was added to the assay. This was not due to the contribution of antibodies because hypogammaglobulinemic serum also augmented cytotoxicity. The results suggest that the mechanism of the enhancement was due to activation of C3 by the Raji cells. We assume that the cleavage products are deposited on cell surfaces in such a way that they contribute to contact between effector and target. Previous reports in two antibody-dependent cellular cytotoxicity systems provided other examples for the existence of this phenomenon.

A thymoma cell line (PXT), originally induced by X-irradiation in a C57Bl/6 mouse, was found capable of generating cytotoxic T lymphocytes (CTL) in a syngeneic mixed lymphocyte tumour culture (MLTC), but failed to stimulate allogeneic lymphocytes. Serologically defined H-2 antigens could readily be detected on PXT cells, which were also susceptible to lysis by alloreactive CTL generated against C57Bl/6 lymphoblasts or other thymomas of C57Bl/6 origin. These observations suggest that the PXT line lacks lymphocyte-activating determinants (LAD) essential for allosensitization but possesses other determinants enabling stimulation of syngeneic CTL, and that the cellular events leading to generation of anti-H-2 and anti-'modified self' CTL proceed along distinct T-cell differentiation pathways.

Mouse mastocytoma cells (P 815) formed rosettes with normal mouse spleen lymphocytes which had been coated with uncleaved human C 3; this interaction was clearly dependent on the amount of C 3. Lymphocytes treated with C 3 b or buffer alone were ineffective. Formation of cell contact could be inhibited by the presence of protease inhibitors such as diisopropyl fluorophosphate, phenyl methyl sulfonyl fluoride and tosyllysyl chloromethyl ketone. Seve n out of 13 different cell lines behaved like P 815 cells. The results strongly suggested that a proteolytic activity on mouse tumor cells led to a cooperation with uncleaved C 3 on a carrier cell to connect these two cells. We interpreted these data in analogy to the complement-dependent bridge formation mechanism (M. P. Dierich and B. Landen, J. Exp. Med. 1977. 146: 1484): uncleaved C 3, attached to mouse spleen lymphocytes as carriers, becomes cleaved by enzymes associated with the tumor cells tested; by this cleavage, the labile binding site is released on C 3 (nascent C 3 b) and anchors the C 3-carrying cell to the protease-carrying cell; since this labile binding site is short-lived, this process can be induced by membrane-associated proteases only. The nature of the proteases and the biological implications of this process are as yet uncertain.

Suppressor cells capable of enhancing tumor growth in vivo and of abrogating a potential anti-tumor immunity in vitro are generated in C57BL/6 mice inoculated with the high-leukemogenic A-RadLV. Mice inoculated with low-leukemogenic D-RadLV do not develop suppressor cells but contain anti-tumor reactive lymphocytes that can inhibit in vivo tumor growth. Cyclophosphamide (CyF) treatment of mice inoculated with A-RadLV hampered suppressor cell function and rendered the animals' lymphocytes responsive to A-RadLV induced tumor cells in vitro. Administration of CyF also reduced leukemia incidence in mice inoculated with A-RadLV, but had no effect on leukemia induction by D-RadLV in irradiated mice. It is suggested that the high leukemogenic activity of A-RadLV depends on the virus' ability to recruit CyF-sensitive suppressor cells early in latency and that tumor progression in mice inoculated with D-RadLV is arrested due to the host immune response.

Adult C57BL/6 mice inoculated intrathymically (i.t.) with the highly leukemogenic variant of the radiation leukemia virus (A-RadLV) develop suppressor cells capable of specifically abrogating a potential anti-tumor cytotoxic response in vitro. Suppressor cells were generated directly by the virus, independently of an initiation of a leukemogenic process. Inoculation of C57BL/6 animals with the low leukemogenic D-RadLV variant did not result in suppressor cell generation. It is proposed that induction of tumor-specific immune suppression by A-RadLV is essential for tumor progression, and the leukemogenic activity of D-RadLV is attributed to its inability to recruit suppressor cells.

The relationship between MCSA and the viral structural proteins in YAC Moloney lymphoma cells was further investigated by using membrane immunofluorescence and immunoadsorbent columns. As previously observed, MCSA showed only minimal capping and thereby differed in behaviour from gp70, p30 and p12 virion antigens. Whereas antibody-induced capping of gp70 did not change the membrane distribution of MCSA, co-capping was observed between MCSA and p30 and p12 gag protein antigens. This indicated that, whereas MCSA is distinct from gp70, it is linked to p30 and p12 on the membrane of living cells. It was then attempted to isolate MCSA on anti-p30 and anti-p15 immunoadsorbent columns from solubilized YAC cells. Contrary to what was expected, no MCSA was found to the anti-p30 column. On the other hand, a part (10-15%) of the MCSA was bound to the anti-p15 column, indicating that MCSA is linked to the gag p15.

A murine carcinoma cell line was grown in a microcarrier culture and was used for immunization of allogeneic mice. It was found that inoculation of cells attached to microcarrier beads resulted in heightened serum titers of cytotoxic antibodies and in a stronger cell-mediated cytotoxic reactivity in the spleen compared to cells detached from the substrate. It is proposed that immunization of animals with anchorage-dependent cells should be carried out while the cells are still adherent to the culture microcarriers.

In vitro converted, Epstein-Barr (EBV) genome-carrying cell lines showed a reduced ability to shed surface-bound antibodies, compared to their EBV-free parental B lymphoma lines. On the other hand, antibody degradation was higher with EBV-positive cells. Analysis of antibody-coated single cells revealed that cells which failed to redistribute or cap their ligand-bound surface components at 37 degrees C, were also capable of shedding bound antibody. This suggests that capping and shedding are essentially independent phenomena, although both may be influenced by the same virally induced change in the lateral mobility of membrane constituents.

Clones of N-, B- and NB-fibrotropic viruses were isolated from weakly (D-RadLV) and strongly (A-RadLV) leukomogenic RadLV preparations. A highly leukemogenic, thymotropic virus (TV) was isolated by ex-vivo infection of thymocytes with A-RadLV. This virus could not be isolated from D-RadLV. Two-dimensional fingerprint analysis suggested that TV recombines unique RNA sequences with RNA genomic material derived from a B-tropic endogenous virus. C57BL/6 (B6) mice injected with B- or NB-fibrotropic clones, but not with TV or N-tropic viral clones, developed reactive T lymphocytes (Tr), capable of differentiating into anti-tumor cytotoxic cells. The N-tropic virus isolates were non-immunogenic in B6 mice whereas the TV isolate induced suppressor T lymphocytes (Ts) that abrogated a potential Tr response. These results suggest that emergence of highly leukemogenic RadLV involves activation of endogenous fibrotropic virus which is immunogenic in its natural host strain (B6). This virus can further recombine with other retroviral genetic sequences, resulting in a suppressogenic and thymotropic, highly leukemogenic virus.

Clonality of radiation leukemia virus (RadLV)-induced thymic lymphomas was determined by detection of rearrangements in the genetic locus coding for the beta chain of the T-cell receptor (T beta). Unique T beta rearrangements were detected in four of six lymphomas. Two of the T beta-rearranged thymic lymphomas and two in which such a rearrangement was not detected had a biallelic deletion of C beta 1. With an anti-RadLV monoclonal antibody it was found that 1-2 days after virus inoculation more than one-third of the cells in the thymus were infected by the virus. The frequency of virus-positive cells gradually declined and persisted at 1-2% until the appearance of a clonal lymphoma at which time virtually all the cells in the thymus were virus positive. Transfer of thymocytes from a single, preleukemic mouse 21 days post-virus inoculation into several adoptive recipients resulted in donor-type thymic lymphomas in the majority of the mice. T beta rearrangement analysis revealed that these lymphomas were clonal and derived from different potentially leukemic (preleukemic) cells in the thymus of the donor mouse. Eleven of 15 lymphomas had a biallelic deletion of C beta 1. These results suggest that clonal, RadLV-induced thymomas are selected from an oligoclonal, RadLV-infected preleukemic T-cell population.

Haemolysis of sensitized A1 erythrocytes by blood monocytes or complement was measured under conditions where antibody, complement, target and effector concentrations varied relatively to each other. When sensitization was limiting, cell-mediated lysis remained constant with increasing red cell number. When antibody was in excess and complement was limiting, complement-mediated lysis remained constant with increasing red cell number. These effects could be explained if a non-homogeneous expression of A1 antigens on individual erythrocytes is assumed. Considering that the capacity of antibody to bind to the antigen is random and uniform, at high dilution of antibody only those erythrocytes with sufficiently high antigen density will be appropriately sensitized for cell-mediated lysis. At antibody excess binding sites are saturated, and therefore the number and distribution of cell bound antibodies reflect the number and distribution of antigens. The fact that under such conditions increasing target concentrations did not reduce lysis induced by a suboptimal complement concentration also argues for an uneven antigen distribution among erythrocytes.

The thymus is populated by various Ia+ cell populations, including epithelial cells, macrophages and dendritic cells. Thymic cell suspensions were stained with an anti-Ia antibody and shown by cytofluorometry to contain a small number of strongly Ia+ cells characterized by a large diameter. The cell population was separated with the aid of the fluorescence-activated cell sorter (FACS) and characterized. They were shown to express high levels of membranal Ia antigens; they demonstrated ATPase activity and displayed the ultrastructural features characteristic of the previously described thymic interdigitating cells. C57BL/6 mice were submitted to various regimens of X-irradiation. Whereas exposure to a single dose of X-irradiation was followed by an increase in the percentage of strongly Ia+ cells, exposure to a leukemogenic regimen of fractionated X-irradiation led to a decrease in the percentage and absolute numbers of these cells in the thymus. Of the C57BL/6 mice that were irradiated with fractionated X-irradiation, 77% developed leukemia. Intravenous injection of syngeneic bone marrow one day following the last irradiation or protection of the femur during irradiation prevented both the appearance of leukemia and the disappearance of interdigitating cells. Therefore an inverse correlation between the presence of thymic dendritic cells and the incidence of leukemia in C57BL/6 mice could be demonstrated. These findings are discussed in relation to the putative role of dendritic cells in the thymus.

Receptors for the third component of complement (C3) were demonstrated on the surface of established human lymphoid cell lines by a membrane fluorescence test with FITC- or TRITC-conjugated antibodies against human C3. Two-color fluorescence staining of EBV receptors and C3 receptors showed complete overlapping of green and red fluorescence. Capping of the EBV receptor induced co-capping of the C3 receptor and vice versa. There was neither overlapping nor co-capping when EBV or C3 receptors were examined in relation to Fc receptors, surface IgM or beta2 microglobulin. The kinetic pattern of EBV receptor capping was identical with the pattern of C3 receptor capping but differed from the pattern of IgM capping. These results suggest a close association between EBV and C3 receptors on the human B-lymphocyte.