Regulation of cytolytic cell populations from human peripheral blood by B cell stimulatory factor 1 (interleukin 4).

The effects of B cell stimulatory factor 1 (BSF-1) on the generation of human CTL and lymphokine-activated killer (LAK) cells in vitro were investigated. Both L-2 and BSF-1 were potent helper factors for the generation of antigen-specific CTL in MLC; detection of optimal BSF-1-induced CTL activity in this system occurred when BSF-1 was added to cultures after an initial period of activation during which exogenous BSF-1 was not present. In contrast to IL-2, BSF-1 failed to induce an LAK cell population, as detected with Daudi tumor targets, in cultures that had not been allosensitized. Furthermore, when both lymphokines were added together at culture initiation, BSF-1 inhibited the IL-2-driven generation of cytolytic cells. The differential ability of BSF-1 to promote the generation of CTL but not LAK could have important implications for lymphokine-mediated immunotherapy.

Production of IL-1 alpha and IL-1 beta by clones of EBV transformed, human B cells.

An EBV transformed B-cell line was derived from peripheral blood cells from a single donor. B-cell clones were isolated from that line and analyzed for transcription of IL-1 genes, production of biologically detectable IL-1 and antigen presenting capabilities. One clone was found to secrete IL-1 activity into tissue culture medium, and to express the gene for IL-1 alpha. Three other clones were positive for cell surface IL-1 activity but no activity could be detected in culture supernatants. Those clones express the gene for IL-1 beta but not IL-1 alpha. All clones were approximately equal in their ability to present antigen to resting and primed T cells.

Targeted macrophage cytotoxicity using a nonreplicative live vector expressing a tumor-specific single-chain variable region fragment.

Antigen-specific recognition and subsequent destruction of tumor cells is the goal of vaccine-based immunotherapy of cancer. Often, however, tumor antigen-specific cytotoxic T lymphocytes (CTLs) are either not available or in a state of anergy. In addition, MHCI expression on tumor cells is often downregulated. Either or both of these situations can allow tumor growth to proceed unchecked by CTL control. We have shown previously that tumor antigen-specific monoclonal antibodies can be expressed in vaccinia virus and that activated macrophages infected with this virus acquire the ability to kill tumor cells expressing that antigen. Here we show that a membrane-anchored form of the scFv portion of the MUC1 tumor antigen-specific monoclonal antibody, SM3, can be expressed on activated macrophages with the highly attenuated poxvirus, modified vaccinia Ankara (MVA), as a gene transfer vector. Cells infected with the MVA-scFv construct were shown to express the membrane-bound scFv by Western blot and FACS analysis. That cells expressing the membrane-anchored scFv specifically bind antigen was shown by FACS and by BIAcore analysis. GM-CSF-activated macrophages were infected with the construct and shown to recognize specifically MUC1-expressing tumor cells as measured by IL-12 release. Furthermore, activated macrophages expressing the membrane-bound scFv specifically lyse target cells expressing the MUC1 antigen but not cells that do not express MUC1.

Redirected cellular cytotoxicity by infection of effector cells with a recombinant vaccinia virus encoding a tumor-specific monoclonal antibody.

Cytotoxicity is an important function of the immune system that results in the destruction of cellular targets by humoral and/or cellular mechanisms. We wanted to assess the possibility of targeting the lytic function of immune cells toward cancer cells, which express the gene coding for a known tumor antigen (Ag) (GA733-2/epithelial cell adhesion molecule), using a viral vector encoding a monoclonal antibody (mAb) specific for said tumor Ag (CO17-1A). To this end, we have constructed recombinant vaccinia viruses expressing the sequences corresponding to mAb CO17-1A, which recognizes a specific Ag (GA733-2) that is present on the surface of most gastrointestinal carcinomas. The recombinant vectors encoding either a secreted or membrane-anchored form of CO17-1A mAb were used to infect effector cells, which were subsequently assessed for their cytotoxic activity. The recombinant viruses were able to infect both granulocyte-macrophage colony-stimulating factor-activated human macrophages and Ag-stimulated murine cytotoxic T lymphocytes. Infected granulocyte-macrophage colony-stimulating factor-activated macrophages were found to be able to kill GA733-2-expressing tumor cells. Likewise, infected cytotoxic T lymphocytes, although conserving their original alloreactivity, gained the capability of killing GA733-2-expressing cancer cells.

Differential MUC 1 expression in normal and neoplastic human pancreatic tissue. An immunohistochemical study of 60 samples.

Neoplastic transformation of epithelial cell sis commonly associated with altered synthesis of mucin glycoproteins. Few studies have been performed on the correlation between MUC 1 expression and pancreatic carcinoma using immunohistochemical methods. We compared the patterns of MUC 1 expression in normal pancreatic tissue, in pancreatic carcinoma, and in chronic pancreatitis. Immunohistochemical studies were performed using 3 monoclonal anti-MUC 1 antibodies (12C10, 1G5, and H23) on surgical specimens and on fine-needle aspiration biopsy specimens. In the neoplastic cells from adenocarcinomas, high levels of cytoplasmic MUC 1 expression were observed, with some membrane staining. No such cytoplasmic expression was observed in normal tissue, tissue from chronic pancreatitis, or benign neoplastic tissue. These data show conspicuous quantitative and qualitative differences between the patterns of MUC 1 expression observed in nonmalignant vs malignant pancreatic tissue and may be useful in the histologic diagnosis of adenocarcinoma in biopsy samples.

IL 1 expression in a clone of human T cells.

Three human T cell clones, all of which are T3+, T4+, T8-, and T11+, were examined for IL 1 production. Two clones were found to express readily detectable, membrane-bound IL 1 activity upon stimulation with OKT3 antibody, rIL 2, and PMA. Northern blot analysis of RNA from one of the clones shows that cells can be induced to express the genes for both IL 1 alpha and IL 1 beta. Furthermore, the pattern of expression in response to different stimuli suggests that the genes for IL 1 alpha and IL 1 beta are regulated independently.

Effects of platelet-derived growth factor and epidermal growth factor on antigen-induced proliferation of human T-cell lines.

When the serum content of tissue culture medium is reduced from 10% to 1%, the capacity of T cells to proliferate in response to antigen within that medium is dramatically reduced. Physiological concentrations of platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) are able to partially replace the requirement for serum, in that they are able to increase antigen-driven T-cell proliferation at a serum concentration of 1%. Neither growth factor is mitogenic for T cells in the absence of antigen, and neither is able to act synergistically with T-cell growth factor (TCGF) or IL-2) in the absence of antigen. Antigen-presenting cells (APC) pulsed with antigen in the presence of PDGF or EGF are able to stimulate antigen-specific T-cell proliferation to a greater extent than antigen-presenting cells pulsed in the absence of exogenous PDGF or EGF. Both growth factors increase the expression of MHC Class II antigens on antigen-presenting cells.

Vaccinia virus MUC1 immunization of mice: immune response and protection against the growth of murine tumors bearing the MUC1 antigen.

MUC1 is a mucin found on the apical surfaces of some normal mammalian mucin-secreting cells. It is characterized by heavy glycosylation and a 20-amino-acid tandem repeat segment. In most cases of human breast adenocarcinoma, this antigen is overexpressed. Moreover, abnormal glycosylation exposes a novel peptide epitope within the tandem repeat, such that antibodies to this epitope can distinguish normal from malignant adenocarcinomatous breast tissue. We have constructed a vaccinia virus (VV) that carries the cDNA for the MUC1 antigen. Murine and human cells infected with this virus express the MUC1 molecule, with three to four tandem repeats per molecule and with the tumor-associated epitopes exposed. Mice immunized with this virus produce antibodies that recognize MUC1 outside the tandem repeat, within the tandem repeat, and within the tumor-associated protein core epitope. Tumorigenic P815 (DBA) and 3T3 (BALB/c) cells have been transfected with MUC1. Thirty percent of DBA mice immunized with VV-MUC1 are protected from growth of P815-MUC1 tumors when implanted with 10(5) cells. Immunized BALB/c mice show a late development of transfected 3T3 tumor cells. Immunized mice show a moderate MUC1-specific IgG titer, but it cannot be correlated with subsequent tumor rejection. No evidence for a MUC1-specific cytotoxic T lymphocyte response has been found after immunization with VV-MUC1.

Rapid phosphorylation and modulation of the T4 antigen on cloned helper T cells induced by phorbol myristate acetate or antigen.

A cell surface protein known as T4 (CD4, Leu3), Mr = 55,000, is expressed on the subset of human T lymphocytes which provides helper function for B cell and cytotoxic T cell activities. We show that T4 is constitutively phosphorylated and that phorbol myristate acetate (PMA) induces a rapid serine phosphorylation which is followed by a fast dephosphorylation. Within 5 min after PMA treatment, there is a 24% reduction of T4 on the cell surface, by 4 h the loss is nearly complete, and by 20 h T4 is re-expressed. Addition of antigen to a T4+ antigen-reactive T cell clone induces both phosphorylation and dephosphorylation with kinetics similar to that described for PMA. Antigen also causes reduction of cell surface T4, although to a lesser degree than stimulation with PMA. The rapid phosphorylation/dephosphorylation of T4 as well as its movement from the cell surface suggest that T4 functions as a receptor for an unknown ligand.

Phosphorylation of the CD8 antigen on cytotoxic human T cells in response to phorbol myristate acetate or antigen-presenting B cells.

We have used the monoclonal antibody OKT8 to demonstrate that the cluster designation (CD)8 antigen on cytotoxic human T cells undergoes a previously unreported protein modification. Immunoprecipitation of CD8 with OKT8 from a CD8+ and CD4+ T cell line prelabeled with [32P]phosphate demonstrates that CD8 can be constitutively labeled with phosphate. CD8 undergoes rapid and intense phosphorylation in serine upon addition of phorbol myristate acetate to the cells. CD8 phosphorylation is induced upon addition of heterologous, Epstein-Barr virus-transformed B cells, which cause proliferation and are target cells for a cytotoxic CD8+CD4+ T cell line and a CD8+CD4- T cell clone. Phosphorylation induced by targets is dose-dependent, rapid, and followed by a fast dephosphorylation. Epstein-Barr virus-transformed B cells that do not induce proliferation of and are not targets for the CD8+CD4+ line and the CD8+CD4- clone do not induce CD8 phosphorylation. Cloned CD8+CD4- cells that proliferate in response to target cells, but lyse them only in the presence of lectin do not undergo target cell-induced CD8 phosphorylation. These data suggest that induction of CD8 phosphorylation is antigen-specific and is coincident with the cytotoxic response. Finally, preincubation of effector and target cells with an antibody to a monomorphic determinant of major histocompatibility complex class I antigens reduces target-induced CD8 phosphorylation to a greater extent than antibody to a major histocompatibility complex class II subregion (DR) monomorphic determinant, reinforcing the notion that major histocompatibility complex class I antigens interact with CD8+ cells.

Lack of evidence for an immunosuppressive role for MUC1.

The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1-and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells.

Cloning the interleukin 1 receptor from human T cells.

cDNA clones of the interleukin 1 (IL-1) receptor expressed in a human T-cell clone have been isolated by using a murine IL-1 receptor cDNA as a probe. The human and mouse receptors show a high degree of sequence conservation. Both are integral membrane proteins possessing a single membrane-spanning segment. Similar to the mouse receptor, the human IL-1 receptor contains a large cytoplasmic region and an extracellular, IL-1 binding portion composed of three immunoglobulin-like domains. When transfected into COS cells, the human IL-1 receptor cDNA clone leads to expression of two different affinity classes of receptors, with Ka values indistinguishable from those determined for IL-1 receptors in the original T-cell clone. An IL-1 receptor expressed in human dermal fibroblasts has also been cloned and sequenced and found to be identical to the IL-1 receptor expressed in T cells.

Recombinant MUC 1 vaccinia virus: a potential vector for immunotherapy of breast cancer.

Breast cancer is considered as the major cause of mortality by cancer for women. Even if chemotherapy, radiotherapy and surgery have improved the life expectancy of patients bearing tumours, breast cancer is responsible for the death of 42,000 women per year in USA and 25,000 women in France. In this context, cancer vaccines may add an attractive alternative therapeutic strategy to the current existing treatments. We describe here the construction of recombinant vaccinia viruses co-expressing a tumour associated antigen (MUC 1) and an "adjuvant" cytokine, which have potential applications in the active immunotherapy of breast cancer. Indeed, recombinant vaccinia viruses have been extensively used during the past decade to induce a protective response against a whole variety of pathogens, and has proven to be of great value in the elicitation of a cellular immune response leading to the rejection of tumour grafts in mouse models.