Basic Research
Anti-Tumor T-Cell Response
Inducing Anti-Tumor T-Cell Response
The schematic diagram above shows the potential mechanism for the induction of an anti-tumor T-cell response. Antigen(s) derived from tumor cells are taken up by dendritic cells (DC).
DCs present these peptide epitopes via major histocompatibility complexes (MHC) class I or class II to CD8+ T-cells or CD4+ T-cells, respectively.
Signalling through costimulatory molecules expressed on CD4+ T-cells leads to the activation and maturation of DCs. Mature DCs can then up-regulate receptors necessary for the activation of antigen-specific CD8+ T-cells.
The cytokine milieu at the tumor microenvironment is also determining the outcome of the T-cell response (Th1 vs Th2).
Lack of proper costimulation or the exposure to inhibitory cytokines release by the tumor (e.g. TGF-ß) can lead to anergy or ignorance of antigen-specific T-cells.
Once CD8+ T-cells are fully activated, they are able to lyse tumor cells via perforin release or through Fas/ FasL interaction.
Our research efforts focus on the manipulation of these cell-cell interactions through delivery of costimulatory molecules or antigen via recombinant Vaccinia virus, or through attraction of various cell types, e.g. DCs or T-cells, to the tumor environment via chemokines expressed by recombinant Vaccinia virus.
Chemokines
Figure: Migration of enriched T-cells following exposure to supernatants from rV-SLC infected BSC-1 cells, as compared to rV-LacZ infected cells, or effect being neutralized by anti-SLCmAb |
Construction of recombinant vaccinia virus encoding chemokines e.g. Secondary lymphoid chemokine is chemoattractant for mature (antigen presenting) dendritic cells, and naïve T cells.
Characterization of vector construct and assessment of biological activity e.g. by transmigration assays (see figure)Application of rV-mSLC in animal tumor studiesImproved anti-tumor response through enhanced antigen-presentation and cross-priming due to presence of antigen-presenting cells and naïve T-cells at the local tumor site.
Costimulatory Molecules
Construction of recombinant vaccinia virus encoding costimulatory molecules
e.g. CD40L Characterization of vector construct and assessment of biological
activity e.g. testing cell surface expression of CD40L by flow cytometry
(see figure) Application of rV-mSLC in animal tumor studies Improved antigen
presentation through co-expression of antigen with costimulatory molecules
Improved TAA cross-presentation through co-expression of different combinations of costimulatory molecules
Figure: FACS analysis of rV-B7.1 and rV-CD40L infected BSC1 cells stained with a direct PE-labeled anti-B7.1 antibody or primary hamster anti-mouse or CD40L monoclonal antibody and a secondary rabbit anti-hamster FITC polyclonal antibody.
The shaded region shows rVCD40L (a) and rV-B7.1 (b) infected cells.
No staining was seen with vaccinia-LacZ infected BSC1 cells (not shown) or with isotype control antibody (solid lines).
Figure: FACS analysis of rV-B7.1 and rV-CD40L infected BSC1 cells stained with a direct PE-labeled anti-B7.1 antibody or primary hamster anti-mouse or CD40L monoclonal antibody and a secondary rabbit anti-hamster FITC polyclonal antibody.
The shaded region shows VCD40L(a) and rV-B7.1 (b) infected cells.
No staining was seen with vaccinia-LacZ infected BSC1 cells (not shown) or with isotype control antibody (solid lines). |
Apc1638N/CEA Mouse Model
Figure:Immunohistochemical staining with an anti-CEA mAb of small intestinal polyp in an 8-month-old mouse. |
Hybrid transgenic mice were generated by crossing human carcinoembryonic antigen (CEA) mice with adenomatous polyposis coli (Apc1638N) knock-out mice (both H-2b).
Mice developed gastrointestinal polyps in 6-8 months that progress to invasive carcinomas with a similar pattern of dysplasia and CEA expression as observed in human colorectal cancer (Hörig et al., Cancer Res. 2001, 61:8520-6).
Delayed growth to a CEA-expressing tumor indicated incomplete or partial tolerance to CEA, as compared to CEA transgenic mice displaying no growth inhibition.
Optimization of prime-boost protocols for preventive/ therapeutic vaccination against colorectal cancer in Apc/CEA mouse model Obtaining and characterization of antigen-specific CTLs from immunized animals - flow cytometry analysis (tetramer staining, intracellular cytokines, perforin), chromium releasing assay Future studies are assessing the efficacy of therapeutic vaccine strategies for colorectal cancer in this model.
This model allows the evaluation of antigen-specific tumor immunity against spontaneous tumors arising in an orthotopic location.
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