T Blankenstein

Retroviral interleukin-7 gene transfer into human dendritic cells enhances T cell activation

Tumor vaccination with dendritic cells (DC) presenting tumor antigens to T cells is a promising approach in immunotherapy. The aim of this study was to enhance T cell stimulatory ability of human DC by retroviral expression of the interleukin-7 (IL-7) gene. IL-7 has been shown to provide a potent costimulatory signal for the proliferation of T cells and the generation of cytotoxic T cells (CTL). DC were generated from human peripheral blood mononuclear cells (PBMC). DC were analyzed by light- and electron-microscopy, immunophenotype (CD1a+, CD14−, CD80+, CD86+, HLA-DR+) and functional assays. According to these criteria, 75–85% of the cells were DC. The cells did not produce measurable amounts of IL-7 spontaneously nor did they express the IL-7 receptor. A retroviral IL-7 expression vector was constructed. Retroviral infection was performed with either the LXSN-hIL-7 vector or its variant LXSN. Using the LXSN-hIL-7 vector, IL-7 production of 2296 pg/106 cells/24 h could be achieved on average. Transduction of DC was confirmed by RT-PCR in a CD1a-enriched cell fraction. Transduction efficiency by a control virus coding for β-galactosidase was about 30%. In autologous mixed lymphocyte reaction (MLR), IL-7 transduced DC augmented T cell proliferation by a factor of two compared with unmodified or mock-transfected DC, and in allogeneic MLR there was a 2.7-fold increase in T cell proliferation. The increase in T cell proliferation could be correlated to IL-7 secretion by DC. Dendritic cells that have been simultaneously peptide-loaded and gene-modified to secrete IL-7 are a potential tool to amplify activation of tumor-specific T cells.

Bcr/abl+ autologous dendritic cells for vaccination in chronic myeloid leukemia.

In chronic myeloid leukemia (CML) ex vivo generated DC are characterized by constitutive expression of bcr/abl and possibly other yet undefined leukemia-associated antigens, since these DC share a common progeny with leukemic cells. Induction of anti-leukemic T cell responses has been described in vitro. For a phase I vaccination study, autologous bcr/abl+ DC are generated under GMP conditions mainly from monocyte precursors in chronic phase CML patients. Lin−, CD80+, CD86+, CD83+, DR+ DC could be generated in sufficient numbers for s.c. vaccination with 1 × 106–5 × 107 DC. Using monocyte precursors, the yield of DC per seeded PBMC was in the range of 1–6%. Furthermore, we could demonstrate in vitrothat the T cell stimulatory ability of CD34+-derived DC can be augmented by a factor 2–3 by retroviral transduction with a gene coding for interleukin-7. DC-based vaccination strategies are a promising clinical approach, particularly as post-remission immunotherapy in the setting of autologous stem cell transplantation. Bone Marrow Transplantation (2000) 25 , Suppl. 2, S46–S49.

Flt-3 ligand as adjuvant for DNA vaccination augments immune responses but does not skew TH1/TH2 polarization.

Since transfection of dendritic cells (DC) plays a key role in DNA vaccination, in vivo expansion of DC might be a tool to increase vaccine efficacy. We asked whether Fms-like tyrosine kinase-3 ligand (Flt-3L), a growth factor for DC, can be used as an adjuvant for DNA vaccination. Beta-galactosidase (β-gal) was used as a model antigen in C57BL/6 mice. Mice were immunized i.m. with DNA coding for β-gal with or without additional injection of Flt-3L. In both cases, antigen-specific CD4+ and CD8+ T cells were detectable after vaccination. Compared with DNA alone, additional administration of Flt-3L led to a significant increase in the antigen-specific proliferative response. However, increased cytotoxicity by T cells was not observed. The cytokines secreted by splenocytes of immunized mice upon in vitro stimulation with antigen had a TH2 profile. Humoral responses against β-gal preferentially consisted of IgG1 antibodies. Analysis of DC from Flt-3L-treated mice revealed an immature phenotype with low or absent expression levels of CD80, CD86 and CD40. We conclude that Flt-3L does not generally skew immune responses towards a TH1 type. More likely, factors determined by the antigen and/or the vaccination procedure itself are crucial for the resulting type of immune response. Flt-3L – under circumstances such as the one we have investigated – can also lead to suppression of TH1 T cell immunity, possibly by expansion of immature/unactivated DC.

Allogeneic gene-modified tumor cells (RCC-26/IL-7/CD80) as a vaccine in patients with metastatic renal cell cancer: a clinical phase-I study

Despite novel targeted agents, prognosis of metastatic renal cell cancer (RCC) remains poor, and experimental therapeutic strategies are warranted. Transfection of tumor cells with co-stimulatory molecules and/or cytokines is able to increase immunogenicity. Therefore, in our clinical study, 10 human leukocyte antigen (HLA)-A*0201+ patients with histologically-confirmed progressive metastatic clear cell RCC were immunized repetitively over 22 weeks with 2.5–40 × 106 interleukin (IL)-7/CD80 cotransfected allogeneic HLA-A*0201+ tumor cells (RCC26/IL-7/CD80). Endpoints of the study were feasibility, safety, immunological and clinical responses. Vaccination was feasible and safe. In all, 50% of the patients showed stable disease throughout the study; the median time to progression was 18 weeks. However, vaccination with allogeneic RCC26/IL-7/CD80 tumor cells was not able to induce TH1-polarized immune responses. A TH2 cytokine profile with increasing amounts of antigen-specific IL-10 secretion was observed in most of the responding patients. Interferon-γ secretion by patient lymphocytes upon antigen-specific and non-specific stimulation was substantially impaired, both before and during vaccination, as compared with healthy controls. This is possibly due to profound tumor-induced immunosuppression, which may prevent induction of antitumor immune responses by the gene-modified vaccine. Vaccination in minimal residual disease with concurrent depletion of regulatory cells might be one strategy to overcome this limitation.

Adenoviral transduction of tumor cells induces apoptosis in co-cultured T lymphocytes

Adenoviral gene transfer of immunmodulatory molecules has been employed successfully in tumor vaccination studies to induce rejection of transplanted syngeneic tumors. In contrast, the response observed when treating chemically induced murine tumors is rather limited. The same applies for human malignancies. A number of reasons including poor transduction efficiency or insufficient T cell infiltration have been held accountable for this lack of efficacy. However, little attention has been given to effects of the adenoviral transduction itself on the T cell system. Here, we show that T cells are sensitized for activation-induced cell death after co-culture with adenovirally infected tumor cells. The levels of CD95/Fas ligand or TNF-α, both known mediators of activation induced cell death, however were not affected by the presence of adenovirus-infected target cells. Furthermore, supernatant transfer from adenovirally transduced or non-infected tumor cell cultures did not result in increased T cell apoptosis. This suggests that cell contact rather than a soluble factor is responsible for the induction of T cell apoptosis upon co-culture with adenovirally transduced tumor cells. Interestingly, and in line with our previous observations, activation-induced cell death was partially inhibited if T cells were co-cultured with tumor cells adenovirally transduced to express IL-7 and CD80, both molecules having the capacity to prevent T cell apoptosis.

Influence of local cytokines on tumor metastasis : Using cytokine gene-transfected tumor cells as experimental models

The molecular mechanisms and, in particular, the role of cytokines involved in the process of metastasis, though one of the most important aspects of cancer research, are largely unknown. In order to metastasize, a malignant cell has to detach from the primary tumor, invade the extracellular matrix and the blood or lymphatic vessels, arrest in the circulatory system, extravasate into target organs, and initiate tumor cell growth. This multistep process may involve interactions between metastatic tumor cells and the local environment, where cytokines, chemokines, angiogenetic factors, and other factors produced by tumor cells or tumor-infiltrating cells will influence or control the metastatic process in different ways.

In vivo splenic CD11c cells downregulate CD4 T-cell response thereby decreasing systemic immunity to gene-modified tumour cell vaccine

One of the factors influencing the efficacy of tumour cell vaccines is the site of immunization. We have shown previously that gene-modified vaccines delivered directly inside the spleen induced antigen cross-presentation by splenic antigen-presenting cells (not B cells). Here, we examined the interaction between splenic CD11c+ cells and antigen-specific CD4+ T cells. We used tumour cells expressing ovalbumin (OVA), a situation where CD4+ T-cell help is required for the generation of a cytotoxic T lymphocyte response. Using in vivo bioluminescence imaging of luciferase-expressing EL4-OVA cells, we could demonstrate that tumour cells were located exclusively inside the spleen following intrasplenic injection. We showed that after intrasplenic immunization with T/SA-OVA cells, splenic class I+ class II+ CD11c+ cells engulfed and presented in vivo the OVA class I-restricted peptide SIINFEKL. However, in vivo previously adoptively transferred 5,6-carboxy-succinimidyl-fluorescein-ester-labelled transgenic CD4+KJI-26+ cells specific for the class II OVA323–339 peptide underwent abortive proliferation in the spleen. These CD4+KJI-26+ cells were only transiently activated and produced IL-10 and IL-4 and not IFN-γ. It appears that splenic CD11c+ cells can downregulate splenic specific CD4+ T-cell response thereby leading to a decrease in antitumour systemic immunity.

34 Rejection of cytokine gene transfected mouse tumors: Therapeutical implications

Cytokines provided locally at the tumor site may initiate an effective anti-tumor immune response which leads to rejection of a tumor which otherwise grows progressively. Experimentally, this can be tested by gene transfer into cultured tumor cells followed by the analysis of the tumorigenicity of such genetically engineered cells. This approach allows to analyse the function of a given cytokine in vivo and to elucidate the therapeutical value of genetically engineered tumor cells as vaccines. Our experience includes experiments with about ten cytokines and the results can be summarized as follows: (1) some cytokines possess antitumor activity in this system, others do not; (2) a local and continuous cytokine supply seems to be essential for tumor rejection; (3) the tumor cell derived cytokines act in a dose-dependent manner and in the absence of systemic toxicity; (4) the immunological effector mechanisms induced by different cytokines are partly cytokine-specific, partly redundant (and usually involve T cell dependent and independent mechanisms; (5) tumor rejection and mechanism thereof may be different with different tumor cell lines transfected with the same cytokine gene. Cytokine gene modified tumor cells as vaccines are currently tested in first clinical trials. However, critical parameters such as vaccine potency of cytokine gene transfected tumor cells, optimal level of cytokine expression, reasons for varying vaccine effects in different tumor models, influence of irradiation of vaccine cells on their efficacy and attempts to improve vaccine efficacy (e.g. by coexpression of cytokines and T cell costimulatory molecules as B7) have to be further addressed in experimental tumor models.