Thomas Blankenstein

Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay

Successful adoptive T-cell therapy has been demonstrated in viral disease and selected forms of cancer. However, it is limited by the difficulty to efficiently isolate and amplify autologous tumor-reactive T-cell clones. Tetramers of major histocompatibility complex (MHC) class I and peptide have facilitated the characterization of CD8+ T cells specific for tumor-associated antigens. However, for adoptive T-cell therapy, MHC-tetramers have limitations: they require knowledge of tumor antigens, which is often not available; they select T cells with a single specificity, thereby posing risk for selection of tumor escape variants; they do not select for function, so that T cells may be anergic when isolated from cancer patients; and they do not allow the isolation of CD4+ T cells that can be essential for tumor rejection. Because interferon (IFN)-γ is essential for tumor rejection, we isolated live T cells based on their IFN-γ production. IFN-γ secreted by previously activated T cells is retained on the cell surface, allowing their specific isolation and expansion. We show here that IFN-γ+ but not IFN-γ− T cells from tumor-immunized mice are cytolytic and mediate tumor rejection upon adoptive transfer. Importantly, tumor-specific T cells can be enriched from lymphocytes infiltrating human renal cell carcinoma by the IFN-γ capture assay.

The role of IFN-γ in tumor transplantation immunity and inhibition of chemical carcinogenesis

IFN-gamma contributes to the rejection of transplantable tumors and the inhibition of methylcholanthrene (MCA)-induced carcinogenesis by different mechanisms. In most tumor transplantation models, tumor rejection requires IFN-gamma receptor expression by host cells, but not by tumor cells. IFN-gamma produced by either CD4+ or CD8+ T cells acts on non-hematopoietic tumor stroma cells and, either directly or indirectly, induces angiostasis. This prevents rapid tumor burden and allows residual tumor cells to be eliminated. In some models, IFN-gamma also contributes to the destruction of existing tumor blood vessels. During MCA-induced tumorigenesis IFN-gamma is involved in the inhibition of MCA diffusion by encapsulation and reduction of DNA damage. This mechanism may primarily protect tissue from damage and simultaneously inhibit tumor development.

Cutting Edge: CD8+ Effector T Cells Reject Tumors by Direct Antigen Recognition but Indirect Action on Host Cells

CD8+ effector T cells recognize malignant cells by monitoring their surface for the presence of tumor-derived peptides bound to MHC class I molecules. In addition, tumor-derived Ags can be cross-presented to CD8+ effector T cells by APCs. IFN-γ production by CD8+ T cells is often critical for tumor rejection. However, it remained unclear whether 1) CD8+ T cells secrete IFN-γ in response to Ag recognition on tumor cells or APCs and 2) whether IFN-γ mediates its antitumor effect by acting on host or tumor cells. We show in this study that CD8+ effector T cells can reject tumors in bone marrow-chimeric mice incapable of cross-presenting Ag by bone marrow-derived APCs and that tumor rejection required host cells to express IFN-γR. Together, CD8+ effector T cells recognize Ag directly on tumor cells, and this recognition is sufficient to reject tumors by IFN-γ acting on host cells.

CD4+ T cell-mediated HER-2/neu-specific tumor rejection in the absence of B cells

HER‐2/neu (HER‐2) is a cell surface proto‐oncogene that is often overexpressed in carcinomas. Passive administration of anti‐HER‐2 antibodies in breast cancer patients has achieved promising results, but less is known about the role of antibodies in active immunization. We asked whether B cells/antibodies are needed for tumor immunity induced by plasmid (HER‐2 and GM‐CSF) immunization. HER‐2 specific tumor immunity relied completely on both CD4+ and CD8+ T cells. IFN‐γ, and to a lesser extent IL‐4, seemed to be crucial cytokines during tumor rejection. Protection was associated with production of anti‐HER‐2 IgG antibodies in B cell competent mice. After immunization, however, B cell‐deficient mice rejected HER‐2‐expressing tumors as efficiently as control littermates. We conclude that T cells are the main effector cells in DNA vaccine induced immunity against HER‐2 and that anti HER‐2 antibodies are not necessary to elicit a protective anti tumor immune response in this model.

Cytokines and cancer: experimental systems.

The transfer of certain cytokine genes into cancer cells can provide very powerful suppression of tumor growth in the absence of any toxic side effects. Some of these cytokines, such as interleukin-4, granulocyte colony-stimulating factor and tumor necrosis factor, can mediate powerful immune suppression even in T-cell-deficient animals and appear to be effective for poorly or non-antigenic tumors. However, approaches must be found to induce or deliver cytokines locally at the tumor site.

Lack of tumorigenicity of interleukin 4 autocrine growing cells seems related to the anti-tumor function of interleukin 4

Recently, the failure of interleukin 4 (IL4) autocrine growing CT4S cells to grow in vivo has been demonstrated. Because it could not be excluded that the cells produce insufficient amounts of IL4 to support their growth in vivo, subclones were established which are unresponsive to exogenous IL4 and therefore have acquired full growth autonomy. From the fact that the subclones likewise did not give rise to tumors when injected into nude mice, one may conclude that the IL4 production of autocrine growing CT4S prevents their growth in vivo. To test this hypothesis, a retroviral vector containing the IL4 gene under the control of the immunoglobulin heavy chain (Igh) enhancer/promoter was constructed and used to infect the myeloma cell line J558L. An IL4 producing clone was established (J558L-XEPIL4) and the tumor progression in comparison to the parental clone J558L was monitored in nude mice. The IL4 production significantly delayed the growth of J558L-XEPIL4 in vivo. Tumor suppression was much more evident when J558L-XEPIL4 cells were injected into syngeneic BALB/c mice. These results may explain why autocrine growing CT4S do not grow in vivo and suggest the involvement of functional T lymphocytes in the effectiveness of the host dependent anti-tumor action of IL4.

Expression of B7.1 (CD80) in a renal cell carcinoma line allows expansion of tumor-associated cytotoxic T lymphocytes in the presence of an alloresponse

We have selected a well-characterized human renal cell carcinoma (RCC) line as the basis for development of a genetically engineered tumor cell vaccine to be applied in an allogeneic setting. This cell line was genetically modified by retroviral transduction to express B7.1 costimulatory molecules. The unmodified tumor cells and B7.1-expressing tumor cells were compared for their ability to induce tumor-associated responses in allogeneic peripheral blood mononuclear cells (PBMC) of two normal control donors having single MHC class I allele matches with the tumor cells. PBMC primed using B7.1-modified tumor cells showed a preponderance of CD3+CD8+ cytotoxic T lymphocytes (CTL) that proliferated over extended periods of time in mixed lymphocyte tumor cell (MLTC) cultures. Strong cytolytic activity developed in the primed populations and included allospecific CTL with specificity for mismatched HLA-A, -B and -C molecules. Nevertheless, it was possible to isolate CTL clones that were able to lyse tumor cells but not lymphoblastoid cells that expressed all the corresponding allospecificities. Thus, induction of complex allospecific responses did not hinder the development of tumor-associated CTL in vitro. These results support the use of this genetically modified allogeneic tumor cell line for vaccination of partial-MHC matched RCC patients.

Dual T cell receptor T cells with two defined specificities mediate tumor suppression via both receptors

Grafting T cells with new antigen specificity by T cell receptor (TCR) gene transfer could greatly facilitate adoptive T cell immunotherapy. Little is known about how two TCR on one T cell influence each other. Among other reasons, this is often due to the fact that only one TCR specificity is known. We have genetically generated murine dual TCR T cells (OT‐I/P14), specific for ovalbumin(ova257) and lymphocyte choriomeningitis virus glycoprotein (gp33). These cells retain both specificities and can be stimulated by either antigenic peptide to proliferate and produce IFN‐γ. Even though one TCR (P14) is expressed at reduced levels on dual TCR T cells, the peptide sensitivity of these cells is similar to that of single TCR T cells of the same specificity. TCR down‐modulation on dual TCR T cells depends primarily on binding of the specific ligand. Adoptively transferred dual TCR T cells suppress the growth of both B16‐ova and B16‐gp33 melanoma cells, regardless of the peptide used for in vitro activation. Taken together, despite a certain dominance of expression between two TCR on the same T cell, this need not necessarily have functional consequences.

Cross-priming versus cross-tolerance: are two signals enough?

Depending on their state of maturation, dendritic cells (DCs) can cross-prime or cross-tolerize T cells. Mature DCs provide T-cell-receptor ligands (signal 1) and costimulatory molecules (signal 2), and activate T cells. Immature DCs provide signal 1, but not signal 2, and tolerize T cells. However, new data show that it is not the expression of costimulatory molecules alone that determines whether DCs induce immunity or tolerance, because mature DCs can induce tolerance also.

The rat interleukin-5 gene: Characterization and expression by retroviral gene transfer and polymerase chain reaction

The rat interleukin-5 (IL-5) gene was isolated from a genomic lambda phage library and a fragment containing all four exons was inserted into the retroviral vector pXT1, resulting in pXTRIL5. Upon retroviral gene transfer into two IL-5-dependent mouse cell lines, B13 and T88M, autonomously growing cells were established and B-cell growth factor activity was detected in the supernatants of the infected cells. cDNA versions of the rat IL-5 gene were rescued by the polymerase chain reaction (PCR) with primers specific for the flanking regions of the cloning site in pXT1. Restriction or DNA sequence analysis of five different clones revealed precise splicing in two cases, while three of the clones had retained the first intron. In addition, in two of these about 400 bp of rat IL-5 5 flanking regions were deleted. The sequence comparison of rat, mouse, and human IL-5 genes revealed a high degree of conservation (e.g., mouse and rat were 92% homologous at the amino acid level). The combination of retroviral gene transfer and PCR may offer an alternative, efficient method for the cloning of cDNAs.