Role of p21 in CD4+ T cells in the antitumor response against colorectal cancer

Abstract

Cancer is the second deadliest disease in the world. Both environmental and genetic factors contribute to its onset, leading to an uncontrolled cell growth of malignant cells. Colorectal cancer (CRC) is one of the most diagnosed types of cancer worldwide, with an increasing incidence each year. CRC progression is shaped by the interplay between cancer cells and extrinsic factors such as immune system and microbiota. Immune cells, for example, can both promote and inhibit tumor development. Amongst all, CD4+ T cells are key players in cancer, by promoting CD8+ T cell cytotoxic function and contributing to tumor cell killing themselves. Unfortunately, increased exposure to persistent antigens gives rise to dysfunctional T cells, which are unable to control cancer growth. To protect against further damage, mechanisms such as p21 activation regulate CD4+ T cell division. Until now, the functional role of p21 activation in CD4+ T cells during colorectal cancer development has not been investigated. This thesis aimed to understand p21’s function in CD4+ T cells in their antitumor response. For this, mice with a deletion of p21 in all host cells (Cdkn1a-/-, also known as p21CIP/WAF1 or p21-/-) were evaluated. First, changes in their immune system under steady state conditions were evaluated. Further, Cdkn1a-/- mice along with control C57Bl6/J (or B6/J) mice were used as donor animals for in vitro models and the reconstitution of the immune system in Rag1-/- mice. These Rag1-/- mice were then further subjected to MC38 orthotopic and AOM/DSS CRC models. Cdkn1a-/- mice showed a different immune phenotype in comparison to control mice under steady state conditions. Even though they develop normally, decreased relative numbers of CD4+ and CD8a+ T cells were observed, accompanied with a loss of CD27/CD28 co-stimulatory molecules. The maturation status of Cdkn1a-/- T cells was also predominantly changed, leading to an accumulation of effector / effector memory cells. When Rag1-/- mice were reconstituted with Cdkn1a-/- CD4+ T cells and subjected to MC38 orthotopic and AOM/DSS tumor models, an increased tumor growth was observed in comparison to Rag1-/- mice injected with control CD4+ T cells. Further analysis of the tumor microenvironment revealed a decrease in the relative number of Tbet+ T cells in Rag1-/- mice injected with p21-deficient CD4+ T cells. These cells showed higher frequencies of the effector / effector memory cells and an accumulation of CD27-CD28- Tbet+ T cells. A similar phenotype was observed in in vitro experiments, where polarized Cdkn1a-/- Th1 cells produced less IFNγ compared to control Th1 cells. p21 loss also led to a significant increase in relative numbers of effector / effector memory T cells and a decrease in the CD28 expression in vitro. Interestingly, in vitro polarized p21-/- Th1 cells showed increased expression of exhaustion markers such as BRCA1 and CTLA4 and affected the relative cell numbers in G0/G1 phase, in comparison to B6/J Th1 cells. Last, this phenotype could be restored by using Palbociclib, a CDK4/6 inhibitor during cell cycle. This resulted in increased IFNγ production in Palbociclib-treated p21-/- Th1 cells compared to the untreated Cdkn1a-/- and B6/J Th1 cells. In conclusion, this thesis proves the importance of p21 in CD4+ T cells in controlling cell cycle progression during cell differentiation and effector function in CRC development. Overall, p21 loss led to an exhausted-like phenotype in CD4+ T cells, which impacted tumor growth. The addition of Palbociclib restored Cdkn1a-/- Th1 cell function in vitro, which might be promising in further in vivo CRC studies, such as CAR-T cell therapy. Show more