Tertiary Lymphoid Tissue in Colorectal Cancer: A Key Player in the Tumour Immune Microenvironment

Tumour infiltrating lymphocytes influence colorectal cancer (CRC) progression. However, lymphocyte infiltration comes in different flavours and evidence has been provided that the spatial distribution of immune cells within the tumour tissue is an important immunological feature. The aim of this thesis was to investigate how the dual localization of tumour infiltrating lymphocytes (TILs) can affect their function in the tumour microenvironment. The project started with the analysis of the CD3 compartment, as CD3+ T cell infiltration (CD3-TILs) is a recognized positive prognostic factor for CRC patients. Results here presented show that CD3+ tumour-infiltrating lymphocytes are present both interspersed in the tumour tissue or scattered throughout the stroma (CD3-TILs) and also aggregated in lymphoid structures showing features of tertiary lymphoid tissue (CD3-TLT). Tumour-associated TLT had a peculiar compartmentalization, with CD3+ T cells and CD20+ B lymphocytes holding complementary positions and with distinct types of dendritic cell populations among them. The presence of HEVs (High Endothelial Venules) suggests a role for TLT in T cell recruitment at the tumour site. To test this hypothesis in human cancer, I performed a whole tissue analysis of the CD3+ infiltrate on CRC sections and found a positive correlation between CD3-TIL and CD3-TLT densities. I further confirmed the hypothesis in vivo in a murine model of colitis-associated cancer (AOM/DSS). AOM/DSS treated mice had expanded TLT compared to control mice. Intravenously injected GFP+ splenocytes localised in TLT of tumour-bearing mice more than in control mice. I then investigated the clinical significance of CD3-TLT in relationship with CD3-TILs in a cohort of 351 CRC patients. In patients with node-negative (without lymph node metastasis, stage II) CRC, a high density of CD3-TLT and CD3-TILs associated to a better prognosis, while in patients with node-positive (presence of lymph node metastasis, stage III) CRC, TLT and TIL density were irrelevant in predicting patient prognosis, thus behaving as biomarkers only for early stage CRC patients. In the second part of my thesis, I analysed the distribution of B cells in colorectal cancer and their possible contribution to disease progression. Despite still controversial, increasing evidence that B cells play a role in cancer progression has been provided, bringing up the hypothesis that also B-cell responses should be considered as targets of immunotherapeutic approaches. Similarly to CD3+ cells, I showed that, both in human and in preclinical models of CRC, B cells display a dual geographical distribution, either within tertiary lymphoid tissue (CD20-TLT) or dispersed at the tumour invasive margin (CD20-TILs). Therefore, I evaluated the role of B cells according to their localization in the microenvironment. I found that CD20-TLT associated to better prognosis, while CD20-TILs did not. Interestingly, CD20-TLT correlated with CD20-TILs only among patients who experienced cancer recurrence. This result suggests that, when located within a lymphoid site, B cells might have a protective anti-tumour function, participating in an antitumour immune response. Conversely, the distribution of B cells scattered in the microenvironment is likely to reflect a non-specific pro-tumour inflammatory reaction. To confirm the hypothesis in vivo and attempt to dissect the dual function of B cells, I took advantage of two CRC preclinical models in which B cells present a distinct geographical distribution in the tumour microenvironment. In the first model, B cells mainly localise within TLT, while in the second one B cells diffusely infiltrate the mucosa, without forming aggregates. I found that in a model in which B cells localize primarily within TLT, the genetic deficiency of B cells significantly increased tumour formation, suggesting that B cells within TLT might exert an important anti-tumour function. In contrast, in a model in which B cells only localize within the tissue, genetic deficiency of B cells reduces tumour growth, suggesting that infiltrating B-TILs might have a pro-tumour role. Therefore, the occurrence of TLT is associated with lymphocyte infiltration in CRC, contributing to recruitment of CD3-TILs. TLT and TILs work together to set up an anti-tumour immune response in patients with low-risk early-stage colorectal cancer. Thus, TLT represents a novel prognostic biomarker for CRC. As to the B cell compartment, their differential distribution in the tumour site corresponds to distinct prognostic functions. This evidence suggests that the design of novel immunotherapeutic drugs depleting B cells should take into account their ability to selectively targeting CD20-TILs but not C20-TLT