Human iNKT cells as key modulators of macrophage survival and phenotype

• Autor(a) principal: Cruz, Mariana Santos
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/33028
• topic: Macrophages; iNKT cells; Cell viability; CD1d; Colorectal cancer; Cell invasion

Invariant natural killer T (iNKT) cells are innate-like cells that express a semi-invariant TCR. iNKT cells recognize lipid antigens loaded in CD1d molecules, which are expressed at the surface of antigen-presenting cells (APCs), such as macrophages. Upon activation, iNKT cells display crucial immune-stimulatory or regulatory roles in autoimmunity, infection, and anti-tumour immunity. Macrophages are professional phagocytes which are commonly found in the tumour microenvironment (TME). Macrophages may polarize into pro-inflammatory M1-like or anti-inflammatory M2-oriented profiles in response to different signaling cues. As they exhibit a broad spectrum of effector phenotypes, macrophages display bimodal roles in tumour progression. In the specific case of colorectal cancer (CRC), although both macrophage populations can be detected in the TME, higher infiltration of M2-like tumour-associated macrophages (TAMs) is suggested to be associated with poor prognosis. Although iNKT cells were shown to induce restriction of M2-like TAMs in a murine model of prostate cancer while protecting the M1-like population, preliminary results obtained in our group indicate that human iNKT cells kill human M1-like macrophages to a higher extent that their M2-like counterparts. Hence, this work aimed to clarify how human iNKT cells modulate the survival and activation of differently polarized macrophages, which molecular mechanisms support this effect and in what way features of the macrophage-iNKT cell crosstalk may translate into CRC. To accomplish these aims, human monocyte-derived macrophages were polarized into the pro- and anti-inflammatory phenotypes by stimulation with LPS+IFN-γ and IL-10, respectively. The roles of iNKT cells on macrophage viability and polarization were investigated by culturing macrophages with and without iNKT cells in the absence or presence of the lipid antigen α-Galactosylceramide (α-GalCer). Our results indicate that α-GalCer-activated iNKT cells are cytotoxic towards all macrophage subsets. Activation of iNKT cells was confirmed by detectable CD25 expression upon stimulation with α-GalCer. Importantly, no differences were observed in the ratios of iNKT-induced cell death across macrophage subpopulations, contrary to what has been previously pinpointed in human, in preliminary results obtained in our group, and in mice. Moreover, macrophages that survived iNKT cell cytotoxicity were activated and exhibited an M1-skewed phenotype, as evidenced by increased expression of CD86 and CD40, which are pro-inflammatory and activation markers, and decreased expression of the M2 marker CD163. Considering this, we investigated whether CD1d-TCR and CD40-CD40L axes played a role in the iNKT-mediated effect on macrophages. Co-cultures performed in the presence of CD1d and CD40L blocking antibodies highlighted that CD1d-TCR interactions contribute to the induction of macrophage death by iNKT cells but CD40-CD40L engagement did not seem essential to this effect. However, CD40L blocking attenuated the effect of iNKT cells on the activation and M1-like polarization of macrophages. A similar tendency was also observed upon CD1d blocking, although it was less pronounced. In addition, the putative relevance of the macrophage-iNKT cell crosstalk in CRC was also explored. Data obtained from Matrigel invasion assays suggested that iNKT cells were capable of mitigating macrophage-stimulated invasion of human colorectal cancer cells. Taking this into account, this thesis helped to characterize the human macrophage-iNKT cell crosstalk and its putative role in modulating CRC invasion.