Project: Cancer Immunotherapy

Analyzing change in metabolic homeostasis and cytokine signalling during immune-tumour cell interactions and generation of anti-tumour immune response using adjuvant coated therapy along with limiting the immune regressive signals

Introduction 


    Tumor cells evade immune response by taking advantage of peripheral tolerance. Peripheral tolerance is the downregulation of T-cell priming and getting rid of auto-reactive T-cells at the lymphatic system and tissue site. T-cell priming is the activation of T-cells in response to foreign antigens and to generate an immune response against it. In this process, the T-cell receptor (TCR) binds to the antigen displayed in MHC on the surface of antigen-presenting cells. Sufficient binding of co-stimulatory molecules such as CD80 and CD86 (B7 family) present on APC binds to CD28 on T-cells to upregulate proliferation and increase survival of T-cells (10). 


    Immune checkpoints such as CTLA-4 and PD-1 help tumour cells bypass immune response by limiting T- and B-cell activity. The role of CTLA-4 is to mediate early immune tolerance through suppression of immune priming of naive T-cells into T-cytotoxic cells and by regulating T-cell proliferation in lymph nodes and spleen. CTLA-4 is a CD28 homolog with much higher affinity and avidity for B7 family members than CD28. It is localised intracellularly but, upon co-stimulation, is exocytosed in vesicles as part of the -ve feedback loop system.


    This lowers the IL-2 production, limits the growth of T cytotoxic cells, and increases the survivability of T regulatory cells (Tregs) cells. CTLA-4 increases the cell survivability of Tregs by decreasing the B7 expression of APC (3, 4, 6, 7, 8). PD-1 mediates later immune tolerance by suppressing T-cytotoxic cells and B-cells and increasing the survival of Tregs while establishing tumour antigen-specific tolerance at the tumour-affected tissue site (1, 5). Breast cancer cell lines such as MDA-MB-231 and MCF-7 show comparatively higher levels of PD-1 on their surface than other tumour cell lines and imparts higher immune checkpoint mediated suppression on the T-cell proliferation while PD-1 silencing in these cells reduces the stemness property. Thus, solid tumor models would be right to study the immune-tumor cell interaction both in vitro and in vivo (11, 12).


    Metabolic homeostasis and tumour micro-environment in either T-cells or tumour cells highly regulate the proliferation of tumour cells and immune suppression. While both cancer and T-cells use anaerobic glycolysis for rapid proliferation, the difference is the production of lactic acid from pyruvate in cancer cells, which promotes T-cell exhaustion and reduced proliferation and cytokine production. Limited nutrients in the tumour microenvironment lead to competition between tumour cells and tumour-infiltrating lymphocytes for their survival and proliferation, which, most of the time, is the advantage for cancer cells (13, 14, 15, 16).



Objectives 


  • In-vitro analysis of metabolic rewiring, regulation of cytokine production in T cytotoxic cells via PD-1 mediated inhibition after co-culturing of T-cells and breast cancer cells (MDA-MB-231 and MCF-7). 

  • Analysis of differential regulation of apoptosis in both T regulatory and T cytotoxic cells through PD-1/CTLA-4 signalling by identifying different factors involved in downstream signalling (9). 

  • In-vivo analysis of anti-tumour immune response through combined interference of PD-1 and CTLA-4 expression along with drugs inhibiting downstream molecules responsible for maturation of Tregs such as FOXP3 and CD25. 

  • In-vivo analysis of T-cell priming and revival of T-cell function after their exhaustion using adjuvant coated tumour specific antigen with and without combined CTLA-4 and PD-1 silencing. 


Future Outcomes 

    Synergistic blockade of both immunosuppressants (CTLA-4 and PD-1) and increased priming of T-cells from outside the body against tumour cells could lead us to a successful therapeutic approach. Simultaneously PD-L1, PD-L1-based anti-cancer vaccines could provide help in combating immune tolerance at later stages of tumour growth (2). At this level of my education, my most important question in this field is how PD-1 differentially regulates the activity of different types of T cells. As in the case of cytotoxic T cells (CD8+), it represses the inflammatory activity while it reduces the apoptosis in regulatory T cells (anti-inflammatory cells). I want to know the differential apoptotic activity of PD-1 in CD8+ cells and Treg cells and the use of PD-1 inhibitors as immune checkpoint inhibitors against the anti-apoptotic activity of PD-1 in Treg cells while against the anti-inflammatory activity of PD-1 in CD8+ cells. Countering metabolic changes in immune cells mediated by tumour cells would be a more efficient way to upregulate anti-tumour immune response and proliferation of T cytotoxic cells.
    
    Priming the host adaptive immune response against heterogeneous tumour cell mass would eventually be more effective rather than selective chemotherapy targeting only one type of signalling cascade at once in tumour cells. Recent advances in pre-oncolytic viral therapy have been developed where it induces tumour cells to become susceptible to immunotherapy and other conventional therapies, aiding researchers in better understanding to counter tumour growth. Thus, a wide range of immunotherapy (or in combination with chemo- and radiotherapy) could be developed against the heterogeneous cancer cells that are repopulating in between the intervals of targeted therapies. 

References 


  1. Sotomayor, E. M., Borrello, I., Tubb, E., Allison, J. P., & Levitsky, H. I. (1999). In vivo, blockade of CTLA-4 enhances the priming of responsive T cells but fails to prevent the induction of tumour antigen-specific tolerance. Proceedings of the National Academy of Sciences, 96(20), 11476-11481. 

  2. Lin, Z., Zhang, Y., Cai, H., Zhou, F., Gao, H., Deng, L., & Li, R. (2019). A PD-L1-Based Cancer Vaccine Elicits Antitumor Immunity in a Mouse Melanoma Model. Molecular Therapy-Oncolytics, 14, 222-232. 

  3. Barber, D. L., Wherry, E. J., Masopust, D., Zhu, B., Allison, J. P., Sharpe, A. H., ... & Ahmed, R. (2006). Restoring function in exhausted CD8 T cells during chronic viral infection. Nature, 439(7077), 682-687. 

  4. Blank, C., Brown, I., Peterson, A. C., Spiotto, M., Iwai, Y., Honjo, T., & Gajewski, T. F. (2004). PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8+ T cells. Cancer research, 64(3), 1140-1145.

  5. Iwai, Y., Ishida, M., Tanaka, Y., Okazaki, T., Honjo, T., & Minato, N. (2002). Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proceedings of the National Academy of Sciences, 99(19), 12293-12297. 

  6. Walunas, T. L., Lenschow, D. J., Bakker, C. Y., Linsley, P. S., Freeman, G. J., Green, J. M., ... & Bluestone, J. A. (1994). CTLA-4 can function as a negative regulator of T cell activation. Immunity, 1(5), 405-413. 

  7. Walunas, T. L., Bakker, C. Y., & Bluestone, J. A. (1996). CTLA-4 ligation blocks CD28-dependent T cell activation. The Journal of experimental medicine, 183(6), 2541-2550. 

  8. Krummel, M. F., & Allison, J. P. (1995). CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. The Journal of experimental medicine, 182(2), 459-465.

  9. Riley, J. L. (2009). PD‐1 signaling in primary T cells. Immunological reviews, 229(1), 114-125. 

  10. Fife, B. T., & Bluestone, J. A. (2008). Control of peripheral T‐cell tolerance and autoimmunity via the CTLA‐4 and PD‐1 pathways. Immunological reviews, 224(1), 166-182.

  11. Mazel, M., Jacot, W., Pantel, K., Bartkowiak, K., Topart, D., Cayrefourcq, L., ... & Alix-Panabières, C. (2015). Frequent expression of PD-L1 on circulating breast cancer cells. Molecular oncology, 9(9), 1773-1782. 

  12. Gao, L., Guo, Q., Li, X., Yang, X., Ni, H., Wang, T., ... & Zheng, L. (2019). MiR-873/PD-L1 axis regulates the stemness of breast cancer cells. EBioMedicine, 41, 395-407. 

  13. Fox, C. J., Hammerman, P. S., & Thompson, C. B. (2005). Fuel feeds function: energy metabolism and the T-cell response. Nature Reviews Immunology, 5(11), 844-852.

  14. Jones, R. G., & Thompson, C. B. (2007). Revving the engine: signal transduction fuels T cell activation. Immunity, 27(2), 173-178. 

  15. Chang, C. H., Qiu, J., O’Sullivan, D., Buck, M. D., Noguchi, T., Curtis, J. D., ... & Tonc, E. (2015). Metabolic competition in the tumor microenvironment is a driver of cancer progression. Cell, 162(6), 1229-1241.

  16. Ho, P. C., Bihuniak, J. D., Macintyre, A. N., Staron, M., Liu, X., Amezquita, R., ... & Kleinstein, S. H. (2015). Phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cell responses. Cell, 162(6), 1217-1228.

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