Immunotherapy for Cutaneous Malignancies: Rationale for a Basal Cell Carcinoma Vaccine and Modulation of PD-1 in Tissue by Local Growth Factors
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CitationHaitz, Karyn. 2017. Immunotherapy for Cutaneous Malignancies: Rationale for a Basal Cell Carcinoma Vaccine and Modulation of PD-1 in Tissue by Local Growth Factors. Doctoral dissertation, Harvard Medical School.
AbstractPart 1 Purpose: Basal cell carcinoma (BCC) vaccination could reduce treatment cost and improve quality of life. Hedgehog interacting protein 1 (HHIP1) is overexpressed in BCC and DEC205 is a dendritic cell (DC) antigen uptake receptor abundant on skin resident DCs. The first objective is to generate an anti-DEC205 and HHIP1 fusion construct for use as a BCC vaccine.
Methods: The immunogenic pPT27 domain of HHIP1 was cloned into anti-DEC205 heavy chain or IgG control, expressed by transient transfection in HEK293F cells, and purified on protein G columns. Protein characterization and functional testing were performed using Western blot and fluorescent-activated cell sorting (FACS) staining. Prime-boost immunization in wildtype mice was analyzed with CFSE Dilution Assay.
Results: Purified anti-DEC205-pPT27 was characterized by Western blot at ~230kD as expected using goat anti mouse IgG(H+L)-AP. FACS verified specific binding on CHO cells stably transfected with DEC205 using goat anti mouse IgG-FITC. Wildtype prime-boost immunization showed non-significant immune response.
Conclusions: These data show anti-DEC205-pPT27 can be generated, has adequate solubility for mammalian cell secretion, and functionally binds DEC205 in vitro. Further research should determine efficacy in a BCC mouse model.
Part 2 Purpose: PD-1 inhibitors are well-established melanoma treatment with unknown mechanism and variable outcomes. Our unpublished data show increased PD-1 expression in epidermal tissue resident memory cells (Trm), and TGF-beta is known to be important for Trm formation. We hypothesized TGF-beta may be a conditioning cue for epidermal PD-1 expression that determines who benefits from PD-1 inhibitors. Therefore, my second objective was to assess TGF-beta effect on epidermal PD-1.
Methods: In examining the role of PD-1 in Trm, normal human skin was digested and PD-1 expression analyzed by FACS. TGF-beta and anti-TGF-beta culture was performed with human epidermis and mouse splenic T-cells and in vivo by anti-TGF-beta injection in wildtype mice. FACS analysis of PD-1 expression on T-cells with Trm markers (CD69, CD103) was performed for all experiments.
Results: PD-1 is elevated on Trm-like cells in human epidermis, but not human dermis. TGF-beta culture increases initial cell division in human models and increases CD103 expression on T-cells in mouse models, but does not affect PD-1 expression.
Conclusions: These results confirm PD-1 elevation on epidermal Trm. TGF-beta may affect cell division and CD103 expression, but does not affect epidermal PD-1 expression and thus does not appear to be relevant in the immune checkpoint pathway for melanoma. Future studies should aim to identify alternative conditioning cues for epidermal PD-1 expression.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:40621377