Constitutive ablation of dendritic cells breaks self-tolerance of CD4 T cells and results in spontaneous fatal autoimmunity.

Lack of immunological tolerance against self-antigens results in autoimmune disorders. During onset of autoimmunity, dendritic cells (DCs) are thought to be critical for priming of self-reactive T cells that have escaped tolerance induction. However, because DCs can also induce T cell tolerance, it remains unclear whether DCs are required under steady-state conditions to prevent autoimmunity. To address this question, we crossed CD11c-Cre mice with mice that express diphtheria toxin A (DTA) under the control of a loxP-flanked neomycin resistance (neo(R)) cassette from the ROSA26 locus. Cre-mediated removal of the neo(R) cassette leads to DTA expression and constitutive loss of conventional DCs, plasmacytoid DCs, and Langerhans cells. These DC-depleted (DeltaDC) mice showed increased frequencies of CD4 single-positive thymocytes and infiltration of CD4 T cells into peripheral tissues. They developed spontaneous autoimmunity characterized by reduced body weight, splenomegaly, autoantibody formation, neutrophilia, high numbers of Th1 and Th17 cells, and inflammatory bowel disease. Pathology could be induced by reconstitution of wild-type (WT) mice with bone marrow (BM) from DeltaDC mice, whereas mixed BM chimeras that received BM from DeltaDC and WT mice remained healthy. This demonstrates that DCs play an essential role to protect against fatal autoimmunity under steady-state conditions.

Accumulation of effector CD4 T cells during type 2 immune responses is negatively regulated by Stat6.

Th2 cells are important effector cells during allergic disorders and parasite infections. Efficient differentiation of Th2 cells requires signaling via the IL-4R and the transcription factor Stat6. Stat6 is further implicated in Th2 cell recruitment to the lung and might be required for the survival of memory Th2 cells. We analyzed the role of Stat6 in T cell expansion, survival, and recruitment to the lung using competitive adoptive transfer experiments and infection with the helminth parasite Nippostrongylus brasiliensis. Stat6 was not required in T cells or other cell types for recruitment of in vivo-generated Th2 cells to the lung. Functional analysis of Th2 memory cells revealed that Stat6 signaling in CD4 T cells was dispensable for memory cell generation, expansion, and cytokine secretion. However, Stat6-deficient T cells survived better than wild-type T cells, resulting in higher accumulation in the bronchoalveolar lavage, lung, and lymph nodes. This demonstrates that effector T cell expansion is negatively controlled by a novel Stat6-dependent mechanism which probably serves to limit the number of effector T cells during the acute phase of the immune response and thereby lowers the risk of bystander toxicity against healthy tissues.

Gene transfer preferentially selects MHC class I positive tumour cells and enhances tumour immunogenicity.

The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.

Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency.

BACKGROUND: Gene therapy is an attractive new approach for the treatment of cancer. Therefore, the development of efficient vector systems is of crucial importance in this field. Different adeno-associated virus (AAV) serotypes have been characterized so far, which show considerable differences in tissue tropism. Consequently, we aimed to characterize the most efficient serotype for this application. METHODS: To exclude all influences other than those provided by the capsid, all serotypes contained the same transgene cassette flanked by the AAV2 inverted terminal repeats. We systematically compared these vectors for efficiency in human cancer cell directed gene transfer. In order to identify limiting steps, the influence of second-strand synthesis and proteasomal degradation of AAV in a poorly transducible cell line were examined. RESULTS: AAV2 was the most efficient serotype in all solid tumor cells and primary melanoma cells with transduction rates up to 98 +/- 0.3%. Transduction above 70% could be reached with serotypes 1 (in cervical and prostate carcinoma) and 3 (in cervical, breast, prostate and colon carcinoma) using 1000 genomic particles per cell. In the colon carcinoma cell line HT-29 proteasomal degradation limited AAV1-AAV4-mediated gene transfer. Moreover, inefficient second-strand synthesis prevents AAV2-mediated transgene expression in this cell line. CONCLUSIONS: Recent advances in AAV-vector technology suggest that AAV-based vectors can be used for cancer gene therapy. Our comparative analysis revealed that, although AAV2 is the most promising candidate for such an application, serotypes 1 and 3 are valid alternatives. Furthermore, the use of self-complementary AAV vectors and proteasome inhibitors significantly improves cancer cell transduction.