Distinct phenotypic expression of immunogenicity and immunosensitivity in sublines of a tumor

Continuous in vitro or in vivo passage of a BALB/c leukemia has resulted in generation of 2 immunologically distinct sublines. The subline maintained by in vitro passage failed to stimulate an allogeneic response but was susceptible to lysis by alloreactive cytotoxic cells. Conversely, the subline maintained by in vivo passage induced an allogeneic response but was resistant to lysis by cytotoxic T lymphocytes (CTL) reactive to H-2d antigens. Resistance to lysis occurred despite expression of H-2d antigens in a form recognizable by differentiated alloreactive CTL, as determined by cold-target inhibition experiments. Moreover, resistance was immunologically specific, in that the subline was susceptible to immune lysis mediated through recognition of other determinants. The results imply that the display and/or orientation of antigen in the cell membrane of these sublines that is required for a lytic event is distinct from the antigen expression necessary for immunologic recognition.     

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/10J) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.     

The expression of Ia antigens on immunocompetent cells in the quinea pig. I the differential expression of Ia antigens on T cell subpopulations.

We have examined the effect of negative selection with anti-Ia serum and C on a number of T cell functions and have clearly defined two subpopulations of guinea pig T lymphocytes. One subpopulation is susceptible to the lytic effects on anti-Ia serum and C and includes the majority of the primed T cells which proliferate and which produce migration inhibition factor in response to specific antigen stimulation in vitro. The lytic effects of anti-Ia serum were directed against the antigen-specific T cell and not an accessory cell such as a macrophage or nonantigen-specific T cell. No evidence for allelic exclusion of the Ia antigens of the antigen-responsive cell could be demonstrated. The susceptibility of the mitogen-responsive T cell to lysis by anti-Ia serum and C varied with the mitogen used, anatomic origin of the T cell, and the strain of animals studied. A second subpopulation of T cells is completely resistant to the lytic effects of anti-Ia serum and C and includes the primed T helper cell and the T cell that proliferates in response to alloantigenic stimulation in the MLR.     

Relationship between trinitrophenyl and H-2 antigens on trinitrophenyl-modified spleen cells. I. H-2 antigens on cells treated with trinitrobenzene sulfonic acid are derivatized.

Spleen cells were treated with TNBS in order to determine if cell surface H-2 antigens are derivatized with TNP. By labeling the cell membrane of the TNP-modified cells with 125I, followed by detergent lysis and immune precipitation with anti-TNP, it was determined that no H-2 antigenic activity remained in the supernatant. Further, by the use of an antibody-induced antigen redistribution assay it was found that previous exposure to TNP-modified cells to anti-TNP in the absence of complement rendered these cells resistant to lysis by anti-H-2 in the presence of complement. Together these data indicate that at the concentration of TNBS used for modification, H-2 antigens are derivatized with TNP. However, in addition to H-2, other proteins including immunoglobulin were also derivatized with TNP. Anti-TNP cytotoxic effector cells were blocked from their cytotoxic activity by anti-TNP antiserum. These data indicate that TNP directly couples to H-2 antigens on the cell surface of TNP-modified cells and that TNP is associated with the antigenic determinant that the cytotoxic T cell recognizes.     

Multiple splenic lymphoid cell subpopulations regulate H-2 antigen expression on teratocarcinoma cells in vivo

Undifferentiated murine 402AX teratocarcinoma cells do not express MHC antigens when passaged in vitro or in vivo in genetically susceptible host mice. When passaged in vivo in genetically resistant mice, however, the tumor cells become H-2b antigen positive regardless of the H-2 haplotype of the resistant host mouse. The present studies use monoclonal anti-H-2b antibodies to corroborate these earlier findings, which were performed with conventional antisera. Previous studies have established that host bone marrow plus lymphoid cells from resistant primed donors regulate tumor cell H-2b antigen expression. Using bone marrow and mature lymphoid cell reconstitution techniques, the present studies indicate that splenic Ig- cells from genetically resistant host mice are the most efficient lymphoid cell subpopulation in tumor cell H-2b antigen induction. Ig+ spleen cells also reconstitute the capacity to induce teratocarcinoma cell H-2 antigens but are less effective than Ig- spleen cells. Tumor cell H-2 antigen induction in C57BL/6 beige mice is impaired compared to C57BL/6 hosts, which suggests that host NK cells may also be involved in tumor cell H-2 antigen induction. Reconstitution of lethally irradiated resistant hosts for teratocarcinoma cell H-2 antigen expression requires bone marrow plus resistant primed lymphoid cell subpopulations; bone marrow alone is insufficient. These results indicate that multiple splenic lymphoid cell subpopulations requiring a radiosensitive host environment and/or factor for differentiation regulate teratocarcinoma 402AX H-2b antigen expression in vivo in genetically resistant mice.     

Murine trophoblast resists cell-mediated lysis. I. Resistance to allospecific cytotoxic T lymphocytes

Research on the mechanisms of nonrejection of the fetoplacental allograft has focused on the tissues composing the fetomaternal interface, of which the placental trophoblast, the tissue directly confronting the maternal environment, is considered a prime candidate responsible for the survival of the fetus. We recently developed a method for isolating murine trophoblast, and found that a proportion of trophoblast cells from mature placentas, cultured for 2 days, express class I antigens on their surface, and this expression can be enhanced in vitro by interferon-alpha/beta (IFN-alpha/beta). In the present study, it was determined that cultured trophoblast cells from day 14 placentas were resistant to allospecific cytotoxic T lymphocytes (allo-CTL), while being susceptible to alloantibody and complement-mediated lysis. The trophoblast cells remained resistant to allo-CTL-mediated lysis despite IFN-mediated enhanced expression of class I H-2 antigens on their surface and the addition of phytohemagglutinin into the assay. Inhibition of protein synthesis also had no effect. In contrast, fetal fibroblasts, isolated from the same conceptuses, were readily susceptible to allo-CTL-mediated lysis. That the trophoblast cells do interact with the effector cells was shown by their ability to specifically inhibit the lysis of tumor target cells in a dose-dependent manner. Additionally, trophoblast culture supernatants did not inhibit the lytic activity of allo-CTL, even when concentrated 10- to 25-fold, indicating that a soluble suppressor factor was not inactivating the effector cells. These results suggest that trophoblast cells have a protein synthesis-independent mechanism of resistance to lysis by allo-CTL, which could play an important role in protecting the fetoplacental allograft from maternal immune rejection.     

TPA induction of EL4 resistance to macrophage-released TNF: role of ADP-ribosylation in tumoricidal activities of TNF and other factors

Activated macrophages synthesize and release numerous tumoricidal soluble factors that can be divided into receptor- or nonreceptor-dependent agents. Tumor necrosis factor (TNF) would be an example of the former. In our experimental model the killing of EL4 thymoma cells by syngeneic activated macrophages involves, but not exclusively, TNF. Our results show that approximately 50% of the anti-EL4 activity expressed by macrophages can be specifically inhibited with rabbit anti-mouse TNF antibody. EL4 variants resistant to the lytic activity of TNF were still susceptible to macrophage-mediated lysis. A tumor-promoting phorbol ester, TPA, rendered TNF-sensitive and -insensitive EL4 cells resistant to M phi-mediated lysis. However, TPA down-regulated TNF-specific binding sites on both TNF-sensitive and -resistant cell surface membranes, suggesting that resistance to TNF involves postligand:receptor events. Tumor cell G-protein involvement (ADP-ribosylation), as a result of TNF-TNF receptor interactions, was investigated. The results showed that pertussis toxin was cytotoxic against TNF-sensitive and -resistant EL4 cells but not against TPA-treated target cells. Inhibitors of ADP-ribosyltransferase inhibited pertussis toxin cytotoxicity and macrophage-mediated lysis but did not interfere with recombinant TNF lytic activity.     

Target cell expression of MHC antigens is not (always) a turn-off signal to natural killer cells

Recent evidence has demonstrated that the lytic function of natural killer cells might be regulated by potential target cells through the target cells' expression of cell surface components that are able to inhibit the lytic process. Specifically, it has been shown in many target cell systems that the expression of class I MHC proteins by target cells is inversely proportional to their susceptibility to lysis by NK cells. It has been suggested, therefore, that MHC proteins may act as important negative regulatory elements in the ongoing control of NK cell function. Herein, we examined two closely related murine lymphoma cells (ASL1 and ASL1w), both in terms of their susceptibility to lysis by NK cells as well as their expression of both H-2K and H-2D class I MHC proteins. The results of these studies showed that whereas ASL1 and ASL1w cells differed greatly in their susceptibility to NK cell lysis (ASL1 was much more NK resistant than ASL1w), both expressed high levels of H-2K and D proteins. In contrast to what might have been predicted base on reports from other target cell systems, the more NK susceptible ASL1w cells expressed somewhat higher levels of H-2K Ag than did ASL1 cells. These results indicate that expression of H-2 class I proteins by target cells, in and of itself, is not sufficient to inhibit the lytic activity of murine NK cells.     

Differential ability to resist to complement lysis and invade host cells mediated by MBL in R4 and 860 strains of Trypanosoma cruzi

To produce an infection Trypanosoma cruzi must evade lysis by the complement system. During early stages of infection, the lectin pathway plays an important role in host defense and can be activated by binding of mannan-binding lectin (MBL) to carbohydrates on the surface of pathogens. We hypothesized that MBL has a dual role during parasite-host cell interaction as lectin complement pathway activator and as binding molecule to invade the host cell. We used two polarized strains of T. cruzi, R4 (susceptible) and 860 (resistant) strains, to investigate the role of MBL in complement-mediated lysis. Interestingly R4, but not 860 metacyclic strain, markedly increases the invasion of host cells, suggesting that MBL drives the invasion process while the parasite deactivates the Lectin complement pathway.     

402AX teratocarcinoma MHC class I antigen expression is regulated in vivo by Lyt 1, Lyt 2, and L3T4 expressing splenic T cells

The murine 402AX teratocarcinoma is a MHC class I antigen negative tumor of 129 strain origin. Host resistance to the 402AX tumor is genetically controlled. When passed intraperitoneally in genetically resistant mice, the tumor cells are induced to express MHC Class I antigens of the 129 genotype. When passed in genetically susceptible mice, the tumor cells remain MHC class I antigen negative. Earlier studies have demonstrated that resistance to the tumor and regulation of tumor cell MHC class I antigen expression are under the control of the host's immune system. The present studies indicate that splenic Lyt 1-, Lyt 2-, and L3T4-expressing cells regulate tumor cell MHC class I antigen expression, and that these cells require a genetically resistant host environment in which to differentiate. Splenic T cells primed to the 402AX tumor and transferred into genetically susceptible 129 mice give rise to GVHD, suggesting that immunity to the tumor involves reactivity to 129 minor histocompatibility antigens.     

Newly isolated Nodularia phage influences cyanobacterial community dynamics

Cyanophages, that is, viruses infecting cyanobacteria, are a key component driving cyanobacterial community dynamics both ecologically and evolutionarily. In addition to reducing biomass and influencing the genetic diversity of their host populations, they can also have a wider community‐level impact due to the release of nutrients by phage‐induced cell lysis. In this study, we isolated and characterized a new cyanophage, a siphophage designated as vB_NpeS‐2AV2, capable of infecting the filamentous nitrogen fixing cyanobacterium Nodularia sp. AV2 with a lytic cycle between 12 and 18 hours. The role of the phage in the ecology of its host Nodularia and competitor Synechococcus was investigated in a set of microcosm experiments. Initially, phage‐induced cell lysis decreased the number of Nodularia cells in the cultures. However, around 18%–27% of the population was resistant against the phage infection. Nitrogen was released from the Nodularia cells as a consequence of phage activity, resulting in a seven‐fold increase in Synechococcus cell density. In conclusion, the presence of the cyanophage vB_NpeS‐2AV2 altered the ecological dynamics in the cyanobacterial community and induced evolutionary changes in the Nodularia population, causing the evolution from a population dominated by susceptible cells to a population dominated by resistant ones.     

Protective antigens of bloodstage Plasmodium knowlesi parasites

The simian malaria Plasmodium knowlesi provides many favourable features as an experimental model; it can be grown in vivo or in vitro. Parasites of defined variant specificity and stage of development are readily obtained and both the natural host and a highly susceptible host are available for experimental infection and vaccination trials. Proteins synthesized by erythrocytic P. knowlesi parasites are characteristic of the developmental stage, as are the alterations that the parasite induces in the red cell surface. Erythrocytic merozoites are anatomically and biochemically complex, their surface alone is covered by at least eight distinct polypeptides. Immune serum from merozoite-immunized rhesus recognizes many parasite components, especially those synthesized by schizonts. All of the merozoite surface components and some of the schizont-infected red cell surface antigens are recognized by such immune sera. Rhesus monkeys rendered immune by repeated infection may by contrast recognize comparatively few antigens; a positive correlation was established for these 'naturally' immunized monkeys between protection and antibody directed against a 74 000 molecular mass antigen. Immunization with this purified antigen confers partial protection. Other putative protective antigens have been identified by monoclonal antibodies that inhibit merozoite invasion of red cells in vitro. The antigens recognized by inhibitory monoclonal antibodies are synthesized exclusively by schizonts and are processed, at the time of schizont rupture and merozoite release, to smaller molecules that are present on the merozoite surface. The multiplicity of protective antigens is clearly demonstrated by the fact that seven distinct merozoite surface antigens are recognized by three different inhibitory monoclonals. None of the protective antigens identified are variant or strain specific.     

Functional properties of Ly 11.2 lymphocytes: a role for these cells in leukemia?

Functional studies of lymphocyte subpopulations reveal that Ly 11.2, a newly defined T cell surface antigen, is present on prothymocytes and natural killer cells, but not on suppressor T cells for antigen-specific IgE antibody responses, Ly 1+, 2-, 3- helper T cells nor on tumor-specific cytotoxic effector cells. Changes in the expression of Ly 11.2 regularly accompany leukemogenesis and are quite distinct from changes of other cell surface antigens thus far observed. After intrathymic inoculation of radiation leukemia virus (RadLV), many more Ly 11.2-positive cells are found expressing viral antigens than cells expressing other cell surface phenotypes. In addition, after RadLV inoculation, significantly more Ly 11.2-positive cells can be found in the thymus of susceptible mice than in the thymus of resistant mice. The greater availability of permissive (Ly 11.2-positive) cells in susceptible vs resistant hosts at the time when infectious virus is present may account for the shorter latency period and high leukemia incidence of susceptible vs resistant mice.     

The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules

Human herpesviruses utilize an impressive range of strategies to evade the immune system during their lytic replicative cycle, including reducing the expression of cell surface major histocompatibility complex (MHC) and immunostimulatory molecules required for recognition and lysis by virus-specific cytotoxic T cells. Study of possible immune evasion strategies by Epstein-Barr virus (EBV) in lytically infected cells has been hampered by the lack of an appropriate permissive culture model. Using two-color immunofluorescence staining of cell surface antigens and EBV-encoded lytic cycle antigens, we examined EBV-transformed B-cell lines in which a small subpopulation of cells had spontaneously entered the lytic cycle. Cells in the lytic cycle showed a four- to fivefold decrease in cell surface expression of MHC class I molecules relative to that in latently infected cells. Expression of MHC class II molecules, CD40, and CD54 was reduced by 40 to 50% on cells in the lytic cycle, while no decrease was observed in cell surface expression of CD19, CD80, and CD86. Downregulation of MHC class I expression was found to be an early-lytic-cycle event, since it was observed when progress through late lytic cycle was blocked by treatment with acyclovir. The immediate-early transactivator of the EBV lytic cycle, BZLF1, did not directly affect expression of MHC class I molecules. However, BZLF1 completely inhibited the upregulation of MHC class I expression mediated by the EBV cell-transforming protein, LMP1. This novel function of BZLF1 elucidates the paradox of how MHC class I expression can be downregulated when LMP1, which upregulates MHC class I expression in latent infection, remains expressed in the lytic cycle.     

Distinct effects of interferon-gamma and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.     

Clonal analysis of cytolytic T lymphocyte-mediated lysis of target cells with inducible antigen expression: correlation between antigen density and requirement for Lyt-2/3 function

The lysis by allospecific cytolytic T lymphocytes (CTL) of the BALB/c lymphoma ST-4.5, a cell line that can be induced by interferon-gamma (IFN-gamma) to express increased amounts of major histocompatibility complex (MHC) class I antigens, was investigated. Culture of ST-4.5 in IFN-gamma increased the surface expression of Kd molecules from originally low levels and Dd from undetectable amounts by approximately fivefold as determined by fluorescence-activated cell sorter (FACS) analysis, whereas the levels of several other antigens (Ld, I-Ad, Thy-1, Lyt-2, L3T4, and LFA-1) were not affected. The lysis of ST-4.5 by Dd- and Ld-specific CTL clones correlated with the expression of those antigens on target cells as determined by both FACS and biochemical analysis. Lysis of ST-4.5 by CTL clones specific for Kd antigen fell into two distinct groups: those that could lyse targets cultured either normally or in IFN-gamma, and those that could only lyse targets that had been precultured in IFN-gamma. The apparent sensitivity to antigen exhibited by the Kd-specific CTL clones predicted their sensitivity to inhibition of target lysis by anti-Lyt-2/3 antibody. Those CTL clones that were only active against ST-4.5 expressing higher amounts of surface antigen (resulting from IFN-gamma preculture) were readily inhibited by anti-Lyt-2/3 antibody, whereas those CTL capable of lysing normally cultured targets having lower amounts of surface antigen were heterogeneous in their sensitivity to anti-Lyt-2/3; some were inhibitable, whereas others were resistant. In addition, another CTL clone that was resistant to inhibition by anti-Lyt-2/3 alone was readily inhibited by a synergistic combination of anti-Lyt-2/3 plus anti-Kd (but not anti-Dd or Ld) antibodies. These results indicate that CTL antigen receptor sensitivity to (or affinity for) antigen and the level of specific antigen expression by the target cell may both be important criteria in assessing Lyt-2/3 molecule function in CTL-mediated cytolysis. The function of recognition-associated molecules such as Lyt-2/3 may be to strengthen and increase the number of receptor-ligand binding events that facilitate CTL-target membrane interactions that lead to the lysis of the target cell.     

Activation of the Epstein-Barr virus replicative cycle by human herpesvirus 6.

One common attribute of herpesviruses is the ability to establish latent, life-long infections. The role of virus-virus interaction in viral reactivation between or among herpesviruses has not been studied. Preliminary experiments in our laboratory had indicated that infection of Epstein-Barr virus (EBV) genome-positive human lymphoid cell lines with human herpesvirus 6 (HHV-6) results in EBV reactivation in these cells. To further our knowledge of this complex phenomenon, we investigated the effect of HHV-6 infection on expression of the viral lytic cycle proteins of EBV. Our results indicate that HHV-6 upregulates, by up to 10-fold, expression of the immediate-early Zebra antigen and the diffuse and restricted (85 kDa) early antigens (EA-D and EA-R, respectively) in both EBV producer and nonproducer cell lines (i.e., P3HR1, Akata, and Raji). Maximal EA-D induction was observed at 72 h post-HHV-6 infection. Furthermore, expression of late EBV gene products, namely, the viral capsid antigen (125 kDa) and viral membrane glycoprotein gp350, was also increased in EBV producer cells (P3HR1 and Akata) following infection by HHV-6. By using dual-color membrane immunofluorescence, it was found that most of the cells expressing viral membrane glycoprotein gp350 were also positive for HHV-6 antigens, suggesting a direct effect of HHV-6 replication on induction of the EBV replicative cycle. No expression of late EBV antigens was observed in Raji cells following infection by HHV-6, implying a lack of functional complementation between the deleted form of EBV found in Raji cells and the superinfecting HHV-6. The susceptibility of the cell lines to infection by HHV-6 correlated with increased expression of various EBV proteins in that B95-8 cells, which are not susceptible to HHV-6 infection, did not show an increase in expression of EBV antigens following treatment with HHV-6. Moreover, UV light-irradiated or heat-inactivated HHV-6 had no upregulating effect on the Zebra antigen or EA-D in Raji cells, indicating that infectious virus is required for the observed effects of HHV-6 on these EBV products. These results show that HHV-6, another lymphotropic human herpesvirus, can activate EBV replication and may thus contribute to the pathogenesis of EBV-associated diseases.     

Selective MHC expression in tumours modulates adaptive and innate antitumour responses

Progress towards developing vaccines that can stimulate an immune response against growing tumours has involved the identification of the protein antigens associated with a given tumour type. Epitope mapping of tumour antigens for HLA class I- and class II-restricted binding motifs followed by immunization with these peptides has induced protective immunity in murine models against cancers expressing the antigen. MHC class I molecules presenting the appropriate peptides are necessary to provide the specific signals for recognition and killing by cytotoxic T cells (CTL). The principle mechanism of tumour escape is the loss, downregulation or alteration of HLA profiles that may render the target cell resistant to CTL lysis, even if the cell expresses the appropriate tumour antigen. In human tumours HLA loss may be as high as 50%, inferring that a reduction in protein levels might offer a survival advantage to the tumour cells. Alternatively, MHC loss may render tumour cells susceptible to natural killer cell-mediated lysis because they are known to act as ligands for killer inhibitory receptors (KIRs). We review the molecular features of MHC class I and class II antigens and discuss how surface MHC expression may be regulated upon cellular transformation. In addition, selective loss of MHC molecules may alter target tumour cell susceptibility to lymphocyte killing. The development of clinical immunotherapy will need to consider not only the expression of relevant CTL target MHC proteins, but also HLA inhibitory to NK and T cells. Received: 20 March 1999 / Accepted: 3 May 1999     

The role of interferon in the regulation of virus infections by cytotoxic lymphocytes

Interferon (IFN) induced during a virus infection mediates antiviral effects both by direct inhibition of virus replication and by influencing the proliferation, differentiation, and chemotaxis of cyto-toxic lymphocytes which control the infection. Cells from tissue taken from virus-infected mice are conditioned by IFN to resist lysis by natural killer (NK) cells, while they become increasingly susceptible to lysis by cytotoxic Tlymphocytes (CTL). This is due to marked IFN-induced biochemical changes, including an up-regulation of major histocompatibility antigens, which are targets for CTL. Cytopathic viruses, which inhibit cellular RNA and protein synthesis, render target cells refractory to IFN-mediated protection against NK cells, thereby providing a mechanism for NK cells to mediate antiviral effect by preferentially lysing virus-infected but not uninfected cells.