Disparate Activation And Function of B Cells in Benign And Malignant Tumors of the Skin: Implications for Cancer Immunotherapy
Open Access
- Author:
- Podolsky, Michael Adam
- Graduate Program:
- Immunology and Infectious Diseases
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 23, 2017
- Committee Members:
- Adam Glick, Dissertation Advisor/Co-Advisor
Adam Glick, Committee Chair/Co-Chair
Na Xiong, Committee Member
Andrea Mastro, Committee Member
Connie Rogers, Outside Member - Keywords:
- Squamous Cell Carcinoma
B-Cell
CD40
T-Cell
Ras
Immunotherapy - Abstract:
- The generation and progression of both benign and malignant cutaneous tumors is dependent upon initiating mutations in transformed keratinocytes, as well as supporting factors from stromal cells, blood vessels, and lymphatics. Steady-state immunity is tumor-suppressive through immunosurveillance, and recently developed immune checkpoint inhibitors have shown promise in treating various solid tumors by blocking inhibitory signals. Chronic inflammation however remains an important prognostic factor for the development of many cancers, highlighting an important dichotomy with regards to the relationship between inflammation and cancer. To elucidate the specific roles of inflammation in cutaneous tumor establishment and progression, we used two murine models of epidermally targeted Harvey-RasG12V, specifically expressed in the basal/stem cell layer with a Keratin14 driver gene (K14Ras) under control of a reverse tetracycline transactivator, or the suprabasal/differentiated layers with an Involucrin driver gene (InvRas) under control of a tetracycline transactivator. To induce H-RasG12V expression in these mice, doxycycline was added to or removed from drinking water respectively. Specifically targeting this proto-oncogene to the basal and suprabasal layers caused generation of malignant squamous cell carcinomas (SCC) or benign papillomas respectively. H-Ras induced tumors caused initial pro-inflammatory responses in both models, but in malignant SCCs, inflammation shifted towards an immunosuppressive response. In later stages, K14Ras mice displayed a Treg/Breg skewed adaptive immune response, while InvRas mice displayed a Th2/APC+ B cell phenotype. To specifically examine the roles of lymphocytes in these cutaneous tumor models, we crossed both lines onto a Recombination-Activating Gene-1 knockout (Rag1-/-) background, and found that K14RasRag1-/- mice had significantly greater tumor numbers and increased morbidity, while InvRasRag1-/- tumors regressed following 15 days of Ras-induction, indicating tumor suppressive and tumor maintaining roles for lymphocytes respectively. Depletion of CD4, CD8, and B cells was accomplished with intraperitoneal injection with specific monoclonal antibodies (mAbs) against CD4, CD8, and CD20 respectively, and revealed the importance of CD4 T cells and B cells in protective immunity in K14Ras mice and tumor-promoting inflammation in InvRas mice. Adoptive transfer of CD4 T cells, B cells, or CD4 T cells with B cells highlighted that the tumor-suppressive anti-inflammatory roles in K14Ras and tumor-promoting inflammation in InvRas was a product of collaboration between these cells, as neither lymphocyte was capable of perpetuating these responses alone. Flow cytometric analysis of skin draining lymph nodes (SDLN) and tumor tissue indicated that these interactions occur within the tumor microenvironment rather than the secondary lymphoid tissue, and this was confirmed in vitro where only B cells from the tumor microenvironment were able to specifically polarize naïve CD4 T cells, dependent upon direct cell-cell contact. Finally, we show the essential role that CD40/CD40L interactions have during the priming of both K14Ras and InvRas tumor-associated immune responses, as both in vitro and in vivo blockade of CD40L inhibited polarization of CD4 T cells, and altered tumor growth in manners that resembles Rag1-/- mice of each model. Artificial stimulation of CD40 using a CD40 agonist antibody in vivo enhanced the Rag1+/+ phenotypes of both K14Ras and InvRas mice, reducing tumor growth and inflammation in the former, and enhancing tumor growth and inflammation in the latter. The results of these studies strongly implicate CD4 T cell/B cell interactions in the establishment of the tumor-associated immune response, and that specific targeting with immunotherapeutics can tailor these responses with notable influences on tumor growth. With recent advances in immunotherapeutics as cancer treatments, our studies highlight the importance of specifically tailoring these therapies to the local immune microenvironment, as to both maximize clinical benefit as well as minimize non-responders and unintended exacerbation of a tumor-promoting immune response.