Joachim Fensterle

Regulatory CD4+CD25+ T Cells Restrict Memory CD8+ T Cell Responses

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.

Immunogenicity of Constitutively Active V599EBRaf

Activating BRAF somatic missense mutations within the kinase domain are present in 60–66% of melanomas. The vast majority of these represent a single substitution of glutamate for valine (V599E). Here, we demonstrate spontaneous HLA-B*2705-restricted cytotoxic T-cell responses against an epitope derived from V599EBRaf. These T-cell responses were mutation specific as the corresponding epitope derived from wild-type BRaf was not recognized. The loss of the V599EBRAF genotype during progression from primary to metastatic melanoma in patients with V599EBRaf specific T-cell responses suggests an active immune selection of nonmutated melanoma clones by the tumor-bearing host.

Cell-Mediated Immunity Induced by Recombinant Mycobacterium bovis Bacille Calmette-Guérin Strains Against an Intracellular Bacterial Pathogen: Importance of Antigen Secretion or Membrane-Targeted Antigen Display as Lipoprotein for Vaccine Efficacy

Live recombinant vaccines expressing defined pathogen-derived Ags represent powerful candidates for future vaccination strategies. In this study, we report on the differential induction of protective cell-mediated immunity elicited by different recombinant Mycobacterium bovis Bacille Calmette-Guérin (BCG) strains displaying p60 Ag of Listeria monocytogenes in secreted, cytosolic, or membrane-attached form for T cell recognition. Anti-listerial protection evoked by the membrane-linked p60 lipoprotein of rBCG Mp60 and that of the p60 derivative secreted by rBCG Sp60-40 were nearly equal, whereas cytosolic p60 displayed by rBCG Np60 failed to protect mice from listeriosis. In vivo depletion of CD4 or CD8 T cell subpopulations in rBCG Mp60-vaccinated mice before listerial challenge revealed interactions of both T cell subsets in anti-listerial protection. In rBCG Sp60-40-vaccinated animals, CD4 T cells predominantly contributed to anti-listerial control as shown by the failure of anti-CD8 mAb treatment to impair the outcome of listeriosis in rBCG Sp60-40-vaccinated mice after L. monocytogenes challenge. Hence, differential Ag display by rBCG influences cell-mediated immunity, which in turn may impact vaccine efficacy due to the different requirements of CD4 or CD8 T cells for pathogen elimination.

Cancer immunotherapy based on recombinant Salmonella enterica serovar Typhimurium aroA strains secreting prostate-specific antigen and cholera toxin subunit B

Prostate cancer is the most common malignant tumor in men and is normally associated with increased serum levels of prostate-specific antigen (PSA). Therefore, PSA is one potential target for a prostate cancer vaccine. In this study we analyzed the functionality of new bacterial PSA vaccines, expressed and secreted via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of Type I secretion systems (T1SS) using an attenuated Salmonella enterica serovar Typhimurium aroA strain as carrier. The data demonstrate that a bacterial live vaccine encompassing T1SS in combination with cholera toxin subunit B can be successfully used for delivery of PSA to induce cytotoxic CD8+ T-cell responses resulting in an efficient prevention of tumor growth in mice.

Use of a recombinant Salmonella enterica serovar Typhimurium strain expressing C-Raf for protection against C-Raf induced lung adenoma in mice

BackgroundSerine-threonine kinases of the Raf family (A-Raf, B-Raf, C-Raf) are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models.MethodsThe antigen C-Raf has been fused to the C-terminal secretion signal of Escherichia coli α-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the α-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays.ResultsC-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas.ConclusionsThe combination of the C-Raf antigen, hemolysin secretion system and Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies.

B-Raf specific antibody responses in melanoma patients

BackgroundMutations of the BRAF gene are the most common genetic alteration in melanoma. Moreover, BRAF mutations are already present in benign nevi. Being overexpressed and mutated, B-Raf is a potential target for the immune system and as this mutation seems to be an early event, a humoral immune response against this antigen might serve as a diagnostic tool for detection of high risk patients.Methods372 sera of 148 stage IV melanoma patients and 119 sera of non-melanoma patients were screened for B-Raf, B-Raf V599E and C-Raf specific antibodies by an ELISA assay. Sera were screened for specific total Ig and for IgG. Serum titers were compared with a two tailed Mann-Whitney U test. Sera with titers of 1:300 or higher were termed positive and groups were compared with a two tailed Fishers exact test.ResultsB-Raf specific antibodies recognizing both B-Raf and B-Raf V599E were detected in 8.9% of the sera of melanoma patients and in 2,5% of the control group. Raf specific IgG was detected in some patients at very low levels. B-Raf specific antibody responses did not correlate with clinical parameters but in some cases, B-Raf antibodies emerged during disease progression.ConclusionThese findings imply that B-Raf is immunogenic in melanoma patients and that it might serve as a potential target for immunotherapy. However, B-Raf specific antibodies emerge at rather late stages of melanoma progression and are present only with a low frequency indicating that spontaneous B-Raf specific antibodies are not an early marker for melanoma, but rather may serve as a therapeutic target.

Shigella Mediated Depletion of Macrophages in a Murine Breast Cancer Model Is Associated with Tumor Regression

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TΔaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TΔaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression. These data support TAMs as useful targets for antitumor therapy and highlight attenuated bacterial pathogens as potential tools.

Vivotif® – A ‘Magic Shield’ for Protection against Typhoid Fever and Delivery of Heterologous Antigens

The attenuated Salmonella typhi strain Ty21a is the main constituent of Vivotif®, the only attenuated live oral vaccine against typhoid fever. In comparison with antibiotics, the ‘magic bullets’ which Paul Ehrlich was striving for to treat infectious diseases, this vaccine should be viewed as a ‘magic shield’, because rather than treating typhoid fever after the infection has started, immunisation with this vaccine strain prevents infection and disease by the induction of specific immune responses. Ty21a is also an attractive carrier for the delivery of heterologous antigens. Recently, we successfully used Ty21a for antigen delivery via the haemolysin secretion system of Escherichia coli, which allows efficient protein secretion from the carrier bacteria.

Reactive antibodies against bacillus Calmette-Guerin heat-shock protein-65 potentially predict the outcome of immunotherapy for high-grade transitional cell carcinoma of the bladder.

Intravesical immunotherapy with Mycobacterium bovis (M. bovis) bacillus Calmette‐Guerin (BCG) is the current standard of care against superficial, high‐grade transitional cell carcinoma (TCC) of the urinary bladder (carcinoma in situ and pathologic T1, grade 3 disease). However, individual patient outcome is barely predictable because of the lack of serum markers. Consequently, progression to muscle‐invasive bladder cancer and critical delay of treatments (such as neoadjuvant combination chemotherapy and/or radical cystectomy) often occur. The objectives of this study were to identify a marker for measuring the BCG‐induced immune response and to predict the outcomes and potential improvements of BCG immunotherapy.

Raf kinases in lung tumor development.

The specific response of a cell to an extracellular signal is mediated by signaling cascades that relay the excitation of one or more receptor(s) to the co-ordinated activity of a set of effector molecules which regulate cell division and growth and prevent an intrinsic cell death program. The small G protein Ras is an essential component in the transduction of extracellular signals. Raf is a serine/threonine protein kinase and a direct downstream effector of Ras, which is activated to its GTP-bound state in response to various ligands binding to their cognate receptors. Raf kinases are located at the head of one of the best characterized signaling cascades, the Raf-MEK-ERK kinase cascade which is involved in many signaling processes (Fig. 1). In multicellular organisms, the family of Raf kinases consists of three members, ARaf, BRaf and CRaf. Although all of these Raf kinases phosphorylate MEK, they differ in the way they are activated (Hagemann and Rapp, 1999). For example, cAMP potentiates BRaf kinase activity but inhibits CRaf (Erhardt et al., 1995; Cook and McCormick, 1993; Dhillon et al., 2002). Genetic and biochemical data also indicate that isoform specific functions exist for Raf kinases which correlate with specific protein interactions (Hagemann and Rapp, 1999). Recent findings regarding interaction of Raf with lipid membrane microdomains (Hekman et al., 2002; Raabe and Rapp, 2002) and the discovery of Rab binding scaffold proteins that may be regulated by MAP kinases in their ability to activate actin comet formation for vesicle transport (Kerkhoff et al., 2001; Eden et al., 2002) have suggested to us a revised model for membrane translocation of Raf (Fig. 2). The model incorporates suggestions on the role of rafts in protein sorting (Simons and Ikonen, 1997) and anticipates a self organizing element in the maturation of signaling complexes. As pictured in Fig. 2, joint sorting of Raf, Ras and upstream signal regulators, e.g., growth factor receptors together with elements of the Ras cycle as well as effectors of Ras/Raf signaling such as MEK and ERK (perhaps