Therapy of human tumors in NOD/SCID mice with patient-derived reactivated memory T cells from bone marrow.

In an analysis of 84 primary-operated breast cancer patients and 11 healthy donors, we found that the bone marrow of most patients contained memory T cells with specificity for tumor-associated antigens. Patients' bone marrow and peripheral blood contained CD8+ T cells that specifically bound HLA/peptide tetramers. In short-term culture with autologous dendritic cells pre-pulsed with tumor lysates, patients' memory T cells from bone marrow (but not peripheral blood) could be specifically reactivated to interferon-gamma-producing and cytotoxic effector cells. A single transfer of restimulated bone-marrow T cells into NOD/SCID mice caused regression of autologous tumor xenotransplants associated with infiltration by human T cells and tumor-cell apoptosis and necrosis. T cells from peripheral blood showed much lower anti-tumor reactivity. Our findings reveal an innate, specific recognition of breast cancer antigens and point to a possible novel cancer therapy using patients' bone-marrow-derived memory T cells.

Escape of metastasizing clonal tumor cell variants from tumor-specific cytolytic T lymphocytes.

A metastasizing variant of a chemically induced lymphoma from a DBA/2 mouse is shown to carry a distinct tumor-associated transplantation antigen (TATA), which can be recognized by syngeneic secondary anti-tumor cytolytic T lymphocytes (CTL). During metastasis of twice-cloned cell lines of this tumor, variants develop that are specifically immunoresistant to lysis by anti-tumor CTL. The variants are detected in the spleen of normal syngeneic mice. They remain stable over long-term passage in tissue culture. The high frequency with which these immunoresistant metastatic variants develop was found to explain the relative ineffectiveness of specific immunization against this metastatic tumor. Compared with organ-selective metastatic variants, the immunoresistant tumor variants seem to arise with a much higher frequency. The change in TATA expression described here differs from antibody-induced antigenic modulation in that it is more stable and genetically transmitted.

Determination of antibody class in a system of cooperating antigenic determinants.

19S and 7S memory is analyzed in a system of cooperating antigenic determinants. Cooperation occurs in the induction of both 19S and 7S secondary antibodies, and for both responses carrier specificity can be entirely accounted for by presensitization of the animal to carrier determinants. The class distribution of secondary anti-hapten antibody depends on the dose of the hapten primary-carrier conjugate used for priming, and on the time interval between priming with the hapten primary-carrier conjugate and secondary injection. The conditions of priming with the secondary carrier influence the extent of the secondary response but not the class distribution of secondary antibody. The data confirm the cooperation hypothesis of antibody induction. Specifically, we interpret them to mean that in hapten-carrier cooperation, the hapten-specific memory cells are predetermined for the class of the emerging antibodies. Together with the hapten-specific memory cells, the carrier-specific helpers are responsible for the extent of the secondary response.

Immune responses against native and chemically modified albumins in mice. I. Analysis of non-thymus-processed (B) and thymus-processed (T) cell responses against methylated bovine serum albumin.

Immune cells induced by bovine serum albumin (BSA) and its methylated derivative (MBSA) have been compared in a cooperative cell transfer system for their content of BSA-specific antibody-forming cell precursors (AFCP, B) and BSA-specific helper (T) cells. When MBSA immune cells were transferred together with hapten-primed cells into recipient mice which were stimulated by a hapten-BSA conjugate, their cooperative secondary anti-hapten response was as good as in case of transferred BSA immune cells. Their secondary anti-BSA response, however, was markedly reduced (reduction factor > 30). Hapten-MBSA conjugates had the same capacity to react with BSA-specific helper cells in the cooperative secondary anti-hapten response as hapten-BSA conjugates but had a reduced ability to react with BSA-specific AFCP cells. In spite of the pronounced reduction of the B cell response, MBSA had the same threshold dose as BSA for activating BSA-specific T cells. These data suggest that B and T cells recognize different epitopes on the BSA molecule, only those recognized by B cells being affected by the methylation procedure.

Metastatic potential severely altered by changes in tumor cell adhesiveness and cell-surface sialylation.

A plastic adherent variant line (ESb-M) of a highly invasive and metastatic murine T cell lymphoma (ESb) was found to have lost its metastatic potential while still being tumorigenic in normal syngeneic hosts. The variant retained most of its ESb-derived antigenic and biochemical characteristics but differed at binding sites for certain lectins with specificity for terminal N-acetylgalactosamine residues. Whereas such sites were masked by sialic acid on metastatic ESb cells, they became unmasked on the adherent variant line. Metastatic revertants of ESb-M cells did not express the respective lectin receptor sites because these were again masked by sialic acid. It is suggested that the masking of specific lectin receptors sites on the tumor cell surface is of crucial importance for metastatis. If freely exposed, these sites may change adherence characteristics of the cells possibly not only in vitro (to plastic) but also in vivo.

Hepatocyte-tumor cell interaction in vitro. I. Conditions for rosette formation and inhibition by anti-H-2 antibody.

Murine hepatocytes, isolated by an in situ collagenase-perfusion technique and cultured in Petri dishes, were shown to form rosettes with liver-metastasizing syngeneic tumor cells. Pretreatment of the tumor cells with neuraminidase generally increased the binding, whereas pretreatment of the liver cells with neuraminidase abolished the binding completely. The tumor-cell binding may be mediated by the previously described lectin-like receptor of hepatocytes that also was sensitive to neuraminidase treatment and that bound desialylated cells better than normal cells. Anti-H-2 sera could efficiently inhibit the rosette formation of metastatic tumor cells with the hepatocytes, which points to a possible role of H-2 molecules in this interaction of neoplastic and normal cells.

Integration of a virus membrane protein into the lipid bilayer of target cells as a prerequisite for immune cytolysis. Specific cytolysis after virosome-target cell fusion.

Structural requirements for membrane antigens on target cells to mediate immune cytolysis were studied in a model system with purified membrane proteins from Semliki Forest virus (SFV). These SFV spike proteins were isolated in the form of detergent- and lipid-free protein micelles (29S complexes) or, after reconstitution into lipid vesicles, in the form of virosomes. Both the 29S complexes and the virosomes were found to bind well to murine tumor cells (P815 or Eb). When these cells, however, were used as target cells in complement-dependent lysis or in antibody-dependent cell- mediated cytotoxicity assays in the presence of anti-SFV serum, they were not lysed, although they effectively bound the antibody and consumed complement. The same tumor cells infected with SFV served as positive controls in both assays. Different results were obtained when inactivated Sendai virus was added as a fusion reagent to the cells coated with either virosomes or 29S complexes. Under these conditions the virosome-coated cells became susceptible to SFV- specific lysis, whereas the 29S complex-coated cells remained resistant. Evidence that the susceptibility to lysis ofvirosome-coated cells was dependent on active fusion and, therefore, integration of the viral antigens into the lipid bilayer of the target cells was derived from control experiments with enzyme-treated Sendai virus preparations. The 29S complexes and the virosomes partially and selectively blocked the target cell lysis by anti-H-2 sera but not by anti-non-H-2 sera confirming our previous finding that major histocompatibility antigens serve as receptors for SFV. The general significance of these findings for mechanisms of immune cytolysis is dicussed.

Interactions of Fc receptors with antibodies against Ia antigens and other cell surface components.

Two Fc receptor-dependent tests were investigated to study the question of a relationship between Fc receptors and known cell surface antigens, in particular I region-associated (Ia) antigens: (a) a rosette assay with antibody-coated erythrocytes (EA) as indicator cells and normal mouse lymphoid cells as source of rosette-forming cells, and (b) a cytotoxicity test with antibody-coated erythrocytes as target cells and normal mouse spleen cells as a source of cytotoxic cells (K cells). EA rosettes were specifically inhibited by antibodies reacting with Ia antigens. Various other antisera reacting with antigens on B lymphocytes, like anti-Ly 4.2 (raised in H-2 identical mice), rabbit antimouse B-cell serum, or rabbit antimouse immunoglobulin, also specifically inhibited the rosettes. No inhibition occurred in the presence of allogeneic or xenogeneic antisera reacting with T lymphocytes. K-cell cytotoxicity was specifically inhibited by each of the antisera (reacting with either B cells or T cells). F(ab')2 fragments of anti-Ia antibodies could still specifically inhibit EA rosettes but they could not inhibit K-cell cytotoxicity. Similar results were obtained with F(ab')2 fragments of anti-immunoglobulin antibodies. These results indicate that the mechanism of inhibition of Fc receptors in the two tests was different. In neither of the tests could we find any evidence for a unique association between the Fc receptors and Ia antigens. The Fc receptors on K cells did not seem to be associated at all with Ia antigens.

A new sensitive assay for antibody against cell surface antigens based on inhibition of cell-dependent antibody-mediated cytotoxicity. I. Specificity and sensitivity.

Inhibition of cell-dependent antibody-mediated cytotoxicity has been investigated as a new assay for antibody against cell surface antigens. The cytotoxicity system consisted of effector cells (normal mouse spleen cells), target cells ((61)Cr-labeled chicken erythrocytes), and antitarget cell antibody. Addition of antibody against cell surface antigens in the effector cell population regularly inhibited the cytotoxicity measured in this system. This cytotoxicity inhibition assay (CIA) detected antibody with a variety of specificities: anti-H-2, anti-Thy 1.2, anti-immunoglobulin, and antimouse bone marrow-derived lymphocyte antigen. When the inhibition by anti-H-2 sera was analyzed using effector cells from congenic mice, the activity was found to be directed against specificities mapping in the H-2K, H-2D, and I regions of the H-2 complex, correlating well with the specificities characterized by complement-dependent assays. A comparison between the sensitivity of the CIA and complement-dependent lysis revealed that the CIA was 2-11 times more sensitive for anti-H-2 antisera and 20-780 times more sensitive for certain antisera against subpopulations of the spleen cells (i.e., T cells or B cells). The CIA proved to be precise, sensitive, and reliable. It may become a very useful antibody assay in various species including man.

Acquisition of high metastatic capacity after in vitro fusion of a nonmetastatic tumor line with a bone marrow-derived macrophage.

A low metastatic, thioguanine-resistant murine T lymphoma line (EbTGR) was hybridized in vitro, with the help of polyethylene glycol, with syngeneic bone marrow-derived macrophages. Two HAT-resistant hybrid lines (Eb-F1 and Eb-F2) were obtained from independent fusion cultures. A cytogenetic analysis revealed that most of the macrophage chromosomes except No. 12 had segregated or become rearranged 60 d after fusion, a time at which the cell lines had become stabilized in culture. Syngeneic mice inoculated subcutaneously with the tumor macrophage hybrid lines developed, very quickly, visceral metastases and died after less than 2 wk, while those inoculated with the parental line lived for greater than 6 wk and developed only localized, large primary tumors. The metastatic hybridomas expressed a similar tumor antigen as a spontaneous, in vivo derived, high metastatic variant (ESb) of the same tumor. This suggests that ESb cells might have arisen from a spontaneous fusion with a host macrophage.

Cytotoxic immune cells with specificity for defined soluble antigens. IV. Antibody as mediator of specific cytotoxicity.

Spleen cells from mice immunized against ovalbumin (OA) or dinitrophenylated mouse serum albumin (DM) were found to be specifically cytotoxic in vitro towards target cells (chicken red blood cells) coated with these antigens. Inhibition of specific cytotoxicity was observed when free soluble antigen was added to the incubation mixtures. DM-immune cell cytotoxicity could be specifically and completely inhibited by DNP-lysine and was thus shown to be hapten specific. Complete and specific inhibition was also observed for OA-immune cell cytotoxicity using OA as inhibitor, but compared with the inhibition curves obtained with DNP-lysine, the OA cytotoxicity inhibition curves were shifted by a factor of about one hundred towards lower molar inhibitor concentrations. Very similar results were observed when the serum antibodies of DM- and OA-immune animals were analyzed by passive hemagglutination inhibition. With increasing time after immunization, both cytotoxicity inhibition curves and agglutination inhibition curves, shifted to lower antigen or hapten concentrations. Specific cytotoxicity against antigen-coated target cells was induced in nonimmune spleen cells (a) by serum from immune animals, and (b) by supernatants from in vitro immune cell cultures. In both instances, the factor which induced antigen-specific cytotoxic activity could be absorbed on anti-mouse Ig columns, thus demonstrating its immunoglobulin nature. The ability of target cell bound antibodies to induce cytotoxicity in nonimmune spleen cells was restricted to the 7S antibody class.

The requirement of more than one antigenic determinant for immunogenicity.

Rabbits primarily stimulated with a BSA (bovine serum albumin)-sulfanilic acid complex will produce a good secondary response to the sulfanilic acid hapten if the carrier used in the secondary stimulus is again BSA, and not if the secondary carrier is HGG (human gamma globulin). In the latter situation, a good secondary response is obtained, however, if the rabbits are pretreated a few weeks earlier with free HGG. We conclude that the immune stimulus involves the recognition of carrier determinants unrelated to the hapten. As the receptors for recognition of unrelated determinants are probably situated on different cells, we suggest that the immune stimulus leading to antibody formation requires the interaction of two antigen-bridged cells.

Anti-CD2 antibodies induce T cell unresponsiveness in vivo.

The CD2 receptor functions as an adhesion and signal molecule in T cell recognition. Multimeric binding of CD2 on T cells to its physiologic ligand LFA-3 on cognate partner cells in vitro efficiently augments the antigen-specific T cell signal delivered by the T cell receptor/CD3 complex. The precise contribution of the antigen-nonspecific CD2-LFA-3 interactions to T cell immune responses in vivo, however, has been difficult to assess. Here we analyzed the role of CD2 in the murine immune response using a nondepleting anti-CD2 monoclonal antibody that induces a marked, reversible modulation of CD2 expression on murine T and B cells in situ. This modulation is dose and time dependent, specific for CD2, and does not require the Fc portion of the antibody. Anti-CD2 antibodies [rat IgG1 or F(ab')2] significantly inhibit the CD4+ T cell-mediated response to hen egg lysozyme and the cytotoxic CD8+ T cell response to a syngeneic tumor cell line. In both cases, anti-CD2 antibodies are only effective when administered before or within 24 h after antigen priming. The suppression of the antitumor response corresponds to a sixfold reduction of specific cytotoxic T lymphocyte precursor cells and results in the abrogation of protective antitumor immunity. Anti-CD2 antibodies also affect the humoral immune response to oxazolone: the isotype switch from specific IgM to IgG1 antibodies is delayed, whereas the IgM response is unaltered. In addition, a single antibody injection results in sustained polyclonal unresponsiveness of T cells irrespective of antigen priming and CD2 modulation. These results document that CD2-mediated signals induce a state of T cell unresponsiveness in vivo.

Efficiency of adjuvant active specific immunization with Newcastle disease virus modified tumor cells in colorectal cancer patients following resection of liver metastases: results of a prospective randomized trial.

PURPOSE: Metastatic disease is a major cause of mortality in colorectal cancer patients. Even after complete resection of isolated liver metastases, recurrence develops in the majority of patients. Therefore, development of strategies to prevent recurrent liver metastases is of major clinical importance. The present prospectively randomised phase III trial investigates the efficiency of active specific immunotherapy (ASI) after liver resection for hepatic metastases of colorectal cancer. METHODS: Patients with histologically confirmed liver metastases from colorectal cancer were randomised to the vaccination or control group. After complete resection of liver metastases, patients randomised to the vaccination group received six doses of Newcastle disease virus (NDV) infected autologous tumour cell vaccine (ATV-NDV). The primary end-point was overall survival, secondary end-points were disease-free survival and metastases-free survival. RESULTS: Fifty-one patients were enrolled in the study with 50 patients available for analysis. The follow-up period was 116.1 +/- 23.8 month in the vaccination arm and 112.4 +/- 18.5 month in the control group. In the total patient group, no differences in the primary and secondary end-points were detected. Most interestingly, subgroup analysis revealed a significant advantage for vaccinated colon cancer patients with respect to overall survival [hazard ratio: 3.3; 95%, confidence interval (CI): 1.0-10.4; P = 0.042] and metastases-free survival (hazard ratio: 2.7; 95%, CI: 1.0-7.4; P = 0.047) in the intention-to-treat analysis. CONCLUSION: Active specific immunotherapy in unselected colorectal cancer patients was not effective for prevention of recurrent metastatic disease. However, in colon cancer patients, ASI with ATV-NDV appears to be beneficial prolonging overall and metastases-free survival.

Identification of asparagine-linked oligosaccharides involved in tumor cell adhesion to laminin and type IV collagen.

MDW4, a wheat germ agglutinin-resistant nonmetastatic mutant of the highly metastatic murine tumor cell line called MDAY-D2 has previously been shown to attach to fibronectin and type IV collagen, whereas MDAY-D2 and phenotypic revertants of MDW4 attached poorly to these substrates. The increased adhesiveness of the mutant cells appeared to be closely related to a lesion in cell surface carbohydrate structures. In an effort to identify the carbohydrates involved in cell attachment, glycopeptides isolated from mutant and wild-type cells as well as from purified glycoproteins were tested for their ability to inhibit the attachment of MDW4 cells to plastic surfaces coated with fibronectin, laminin, or type IV collagen. The addition of mannose-terminating glycopeptide to the adhesion assay inhibited MDW4 cell attachment to type IV collagen. In contrast, a sialylated poly N-acetyllactosamine-containing glycopeptide, isolated from wheat germ agglutinin-sensitive MDAY-D2 cells but absent in MDW4 cells, inhibited MDW4 attachment to laminin. None of the glycopeptides used in this study inhibited attachment of MDW4 cells to fibronectin-coated plastic. Peptide N-glycosidase treatment of the cells to remove surface asparagine-linked oligosaccharides inhibited MDW4 adhesion to type IV collagen, but not to laminin, and the same treatment of the wheat germ agglutinin-sensitive cells enhanced attachment to laminin. Tumor cell attachment to, and detachment from, the sublaminal matrix protein laminin and type IV collagen are thought to be important events in the metastatic process. Our results indicate that tumor cell attachment to these proteins may be partially modulated by the expression of specific oligosaccharide structures associated with the cell surface.

Polarization of human monocyte-derived dendritic cells to DC1 by in vitro stimulation with Newcastle Disease Virus.

PURPOSE: Dendritic cell (DC)-based tumor vaccines have been tested extensively to treat cancer patients. However, the results of several DC-based clinical trials have been disappointing. Amelioration of such a modality for cancer treatment seems warranted, i.e. by improving DC immunogenicity and polarization. The goal of our study was to evaluate the potential for immune activation of human DCs by incubating them in vitro with the Newcastle Disease Virus (NDV), a paramyxovirus with strong immunostimulatory properties. RESULTS: In vitro infection with NDV of human monocyte-derived DCs--generated from peripheral monocytes cultured with IL-4 and GM-CSF--induces the generation of viral M gene transcripts and RIG-I expression within DCs. Expression of both genes was increased upon co-stimulation with LPS. Surprisingly, LPS and NDV had opposite effects on induction of interferon (IFN)-alpha. Furthermore, NDV induced DC maturation (as measured by TNF-alpha production and CD80 cell surface expression) only in the presence of LPS. Most interestingly, an optimal combination of NDV and LPS caused polarization of the DCs to Th1 type cytokines with a high ratio of interleukin (IL)-12 to IL-10. CONCLUSION: These in vitro results provide a means and protocol for maturation and activation of DCs with enhanced and sustained T helper type 1-polarizing capacity. Such pretreated DCs may significantly improve DC-based cancer immunotherapy. The data encourage the use of RNA-based viral vectors as potential novel and powerful gene transfer modality for cytoplasmic gene expression in professional antigen-presenting cells (APC) to overcome immunosuppression in cancer patients.

p53 increases experimental metastatic capacity of murine carcinoma cells.

Transfection of a cloned p53 gene into a murine bladder carcinoma cell with a low metastatic capacity led to elevated levels of p53 protein in clonal transfectants. After intravenous inoculation into syngeneic mice, p53-transfected clones showed significantly increased metastatic potential in comparison with control transfectants. The observed change did not seem to be due to a change in growth potential per se since the cell lines showed similar growth properties in vitro.

Specific immune recognition of pancreatic carcinoma by patient-derived CD4 and CD8 T cells and its improvement by interferon-gamma.

Pancreatic carcinoma is a very aggressive disease and little is known about its immunobiology. We here describe the presence in pancreatic cancer patients of spontaneously induced functional CD4 and CD8 memory/effector T cells reactive to autologous tumor cells or to the pancreatic cancer associated antigen, MUC-1. Such specific cells were present in the bone marrow or peripheral blood of most of the 23 tested patients. Low dose stimulation of primary cultures of pancreatic cancer cells with 500 IU/ml IFN-gamma for 72 h enhanced HLA-I expression and induced the de novo expression of HLA-II molecules. This led to a much better immune recognition by autologous HLA-I restricted and purified CD8 T cells and allowed tumor cell recognition by HLA-II restricted purified CD4 T-helper cells. Thus, interferon-gamma appears to be a useful adjuvant cytokine to enhance the immunogenicity of a patients' tumor cells and their recognition by tumor reactive immune cells.

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. VII. Interaction of metastasizing and nonmetastasizing tumors with normal tissue in vitro.

Three mouse tumors known to metastasize in vivo and 3 nonmetastasizing mouse tumors that grow only locally in vivo were examined for their ability to adhere to and invade normal syngeneic lung organ cultures in vitro. All 3 metastasizing tumors adhered to and invaded the normal lung cultures. In contrast, tumors that grow only locally in vivo neither adhered to nor invaded the normal lung tissue. The described system is ideally suited to correlate the in vivo invasiveness of a given tumor with its potential to metastasize in vivo and to study in vitro how to influence the interaction of metastasizing tumor cells with normal tissue.

Inheritance of immunogenicity and metastatic potential in murine cell hybrids from the T-lymphoma ESb08 and normal spleen lymphocytes.

T-lymphoma cells were fused with normal lymphoid cells to examine the segregation of tumorigenicity and metastatic capacity in the hybrids. In independent fusions the immunogenic ESb08 T-lymphoma line fused successfully with normal syngeneic spleen cells (from DBA/2 and CD1 mice) enriched either with T-cells or B-cells. Ten times fewer hybrids were obtained with B-cells compared to the number obtained with T-cells, and marker assays showed that both types of fusions preferentially generated T-T hybridomas. Some of the hybrids resembled their tumor parent in their ability to form primary and secondary tumors only in irradiated DBA/2 mice, whereas other hybrids lost the high ESb08 immunogenicity, were equally tumorigenic, and in some cases metastatic, in nonirradiated mice. DNA distributions of the original hybrid lines ranged from a hexaploid DNA content (expected for complete hybrids derived from a tetraploid line and normal diploid cells) to a tetraploid DNA content, confirming the reported chromosome instability of T-T hybrids. No correlation was noted between the initial DNA content and tumorigenicity, but in the case of complete hybrids, reduction in the ploidy levels always was observed in the cells of primary and metastatic lesions. One chromosomally stable and highly malignant hybrid (C2), which was analyzed for segregation of chromosomes and for drug-resistance markers, showed preferential loss of chromosomes from the normal T-cell fusion partner. The decreased immunogenicity of this hybrid could not be related to any detectable loss of chromosomes from the ESb08 tumor parent.