Felicia M. Rosenthal

Lack of Effector Cell Function and Altered Tetramer Binding of Tumor-Infiltrating Lymphocytes

Tumor-specific CD8 T cell responses to MCA102 fibrosarcoma cells expressing the cytotoxic T cell epitope gp33 from lymphocytic choriomeningitis virus were studied. MCA102gp33 tumors grew progressively in C57BL/6 mice, despite induction of peripheral gp33-tetramer+ T cells that were capable of mediating antiviral protection, specific cell rejection, and concomitant tumor immunity. MCA102gp33 tumors were infiltrated with a high number (∼20%) of CD11b+CD11c− macrophage-phenotype cells that were able to cross-present the gp33 epitope to T cells. Tumor-infiltrating CD8 T cells exhibited a highly activated phenotype but lacked effector cell function. Strikingly, a significant portion of tumor-infiltrating lymphocytes expressed TCRs specific for gp33 but bound MHC tetramers only after cell purification and a 24-h resting period in vitro. The phenomenon of “tetramer-negative T cells” was not restricted to tumor-infiltrating lymphocytes from MCA102gp33 tumors, but was also observed when Ag-specific T cells derived from an environment with high Ag load were analyzed ex vivo. Thus, using a novel tumor model, allowing us to trace tumor-specific T cells at the single cell level in vivo, we demonstrate that the tumor microenvironment is able to alter the functional activity of T cells infiltrating the tumor mass.

Crucial Role of TNF-α in CD8 T Cell-Mediated Elimination of 3LL-A9 Lewis Lung Carcinoma Cells In Vivo

The role of perforin, IFN-γ, and TNF-α in anti-tumor CD8 T cell immunity was examined in a new tumor model using a CD8 T cell epitope (GP33) derived from lymphocytic choriomeningitis virus as a tumor-associated Ag. In contrast with parental 3LL-A9 (A9) Lewis lung carcinoma cells that progressively grow in C57BL/6 mice, s.c. injection of GP33-transfected A9GP33 tumor cells induced a protective GP33-specific CD8 T cell response that led to complete tumor cell elimination. Tumor regression was dependent on perforin, IFN-γ, or TNF-α, because A9GP33 tumors developed in mice deficient in one of these genes. A9GP33 tumors arising in perforin- and IFN-γ-deficient mice represented GP33 Ag-loss variants, demonstrating that GP33-specific CD8 T cells from these mice were able to exert an Ag selection pressure. In contrast, tumor cells growing in TNF-α knock-out mice still expressed the tumor-associated GP33 peptide despite the presence of activated GP33-specific CD8 T cells. These findings provide evidence for a crucial role of TNF-α in A9 tumor cell elimination by CD8 T cells in vivo.

Human tumor vaccines and genetic engineering of tumors with cytokine and histocompatibility genes to enhance immunogenicity.

Genetically engineered tumor cells can be used as vaccines in order to stimulate an immune response. To date, tumor cells have been modified in vitro so that they secrete cytokines or express histocompatibility molecules that they naturally fail to express. These tumor cells differ in the types of immune responses they induce and in whether the responses have local or systemic efficacy. Many questions have been raised during the past year, including whether allogeneic or autologous tumor cells should be employed and whether there may be a risk of inducing autoimmune disease along with the antitumor response. Nevertheless, because of the paucity of available therapies for patients with advanced cancer, investigators must attempt to refine the approaches used in order to minimize patient risk while maximizing tumor cell destruction.

Cloning of idiotype immunoglobulin genes in B cell lymphomas by anchored PCR and production of individual recombinant idiotype vaccines in Escherichia coli.

Abstract:  Objectives: Individual immunoglobulins expressed by B‐cell lymphomas represent tumor‐specific antigens (‘idiotypes’). Immunization with idiotype in follicular lymphoma patients may induce specific immune responses, sustained progression‐free survival, and disappearance of minimal residual disease. Manufacturing of idiotype vaccines has mostly relied on heterohybridomas established from viable lymphoma cells. This paper describes the feasibility of production of GMP‐grade idiotype vaccines as recombinant Fab fragments in Escherichia coli.

Production of stem-cell transplants according to good manufacturing practice

Abstract Peripheral blood stem cells (PBSCs) are used for transplantation to reconstitute the hematopoietic system after high-dose chemotherapy. They are harvested from peripheral blood after mobilization by cytokines and/or chemotherapy. Further ex vivo manipulation steps (e.g., selection of CD34+ PBSCs, purging, expansion, and differentiation or gene transfer) can be performed. In 1997, more than 12,000 PBSC preparations were transplanted in Europe and the total number is steadily increasing [1]. To ensure quality and safety of the final cell products intended for clinical use, national and international guidelines and regulations have been issued. The implementation of a quality assurance (QA) program including the principles of good manufacturing practice (GMP) and a quality control system is a major requirement. GMP regulations apply to all phases of cell collection, processing, and storage, and to documentation, training of personnel, and equipment of the cell processing laboratory. They have to be followed by pharmaceutical companies and medical doctors who are involved in PBSC processing at academic institutions. The complicated regulatory network for the manufacturing of cell products will help to standardize these procedures and ensure consistent quality and safety in the long term. This will be in the interest of patients and reduce risks of application of individual cell preparations.

Opinion paper on the current status of the regulation of gene therapy in europe

THE POTENTIAL APPLICATIONS of gene therapy are many, extending from monogenic hereditary diseases to acquired and multifactorial disorders. Therapeutic gene transfer addressing such a variety of conditions is currently being investigated, creating therapeutic options for diseases where none had previously been available. The field of gene therapy represents one of the most challenging therapeutics for the new millennium. As such the concept might have been oversold ahead of its time. Nevertheless, in some instances, potential clinical efficacy is currently being found with reports of significant successes, for example, in inherited severe combined immunodeficiency disorders (SCID), hemophilia, arteritis obliterans, and even cancer (CavazzanaCalvo et al., 2000; Cohen, 2000; Heise et al., 2000; Kay et al., 2000). The development of recombinant DNA technology has caused fear in the public and speculation regarding its potential risks. Public reaction to the implementation of gene therapy is at best ambivalent, ranging from enthusiasm about its formidable therapeutic promises to apprehension concerning its putative harmful consequences. Indeed, the report of accidents in the United States resulted in a broad debate on the subject of gene therapy regulation brought to the attention of the U.S. Senate and Government (Barinaga, 2000; Commander, 2000; Hollon, 2000a,b; Marshall, 2000; Renault, 2000). In addition, the recent occurrence of a malignant proliferation of the hematopoietic system in one gene therapy-treated SCID patient has been reported (www.astg.org). (Check and Schiermeier, 2002) with a monoclonal insertion of the retrovirus vector at a sensitive locus (LMO2).

Tumor-bearing animals contain suppressed antitumor effectors the function of which can be unmasked by IL-2.

CMS5 fibrosarcoma cells were infected with retroviral constructs containing interleukin-2 (IL-2) cDNA and selected in G418. Parental tumor cells and those that produced IL-2 were injected in vivo. Whereas injection of parental tumor cells resulted in progressive tumor growth, those secreting high levels of IL-2 were rejected. Furthermore, the immunosuppression associated with inoculation of parental tumor cells was not seen. To understand the failure of mice to reject non-IL-2-secreting tumor cells, functional responses of spleen cells from immune and tumor-bearing mice were studied in vitro. As expected, immune spleen cells proliferated under a variety of conditions but were inhibited in the presence of parental tumor cells. Even spleen cells from tumor-bearing animals responded well in the absence of parental tumor cells or in the presence of parental tumor cells, if supplied with adequate levels of IL-2. These results suggest that both tumor-bearing and immune mice generate antitumor effectors but that the cells might be functionally suppressed because of their inability to secrete IL-2 after contact with parental tumor cells.

Cytokine Gene Transfer into Tumor Cells and its Application to Human Cancer

The success of conventional tumor therapy depends on the elimination of all tumor cells. Toxicity, inaccessibility or resistance to chemotherapy, however causes residual malignant cells to survive and ultimately to kill the host. Several new strategies attempt to induce host anti-tumor responses. Theoretically two approaches seem promising: 1. Activation of the afferent arm of the immune system by enhancing immunogenicity of tumor cells, e.g. by enhancing expression of MHC molecules, adhesion molecules or by transfection of tumor-specific antigens. 2. Activation of effector cells of the immune system known to be capable of killing tumor cells in vitro.