Mannan-specific chimeric antigen receptor mediated the T cell activation against Candida spp.

Invasive fungal infections (IFI) are responsible for high morbidity and mortality rates, causing approximately 1.5 million deaths annually worldwide. Candida albicans has been among the main causative agents of IFIs in recent decades, while non-albicans Candida species have emerged as a growing global public health concern. In addition, COVID-19 has been associated with an increased incidence of IFIs, such as mucormycosis, which has Rhizopus oryzae as the most prevalent causative agent. A host effector immune response against IFIs depends on the activity of T cells that are susceptible to the regulatory effects triggered by fungal virulence factors, and this condition favors the establishment and progression of the infection. The fungal cell wall is an important virulence factor by acting as an important barrier against cytotoxic agents present in the environment, and also has the ability to regulate the expression and distribution of several molecules on the cell surface, which provides a subversion of the immune response of the host. This dynamic of the fungal cell wall aids in the establishment of the infection and often masks several immunogenic components that are amenable to recognition by host immune cells. A way of redirecting T cells to recognize the fungus through targets present in the cell wall and enabling the direct action of cytotoxic T cells against the pathogen is mediated by the application of chimeric antigen receptor (CAR) technology. This receptor induces cell activation immediately after interaction with the target. In this context, the present dissertation generated a specific CAR for mannan, called M-CAR, which is a component of the cell wall of several fungi, and the ability of M-CAR to induce the activation of T cells against the morphological forms of Candida spp.. Jurkat cells expressing M-CAR after transduction with a multiplicity of infection (MOI) of 1, 3, 5 or 10 were able to recognize different Candida spp. species. Furthermore, cells modified with M-CAR produced high levels of IL -2 and had a high expression of CD69 against the co-culture with C. albicans hypha. M-CAR also induced high levels of IL-2 in the presence of C. tropicalis hypha, C. parapsilosis pseudohypha, C. glabrata yeast, and R. oryzae spores. The ability of M-CAR to mediate T cell activation against Candida spp. promoted an increase in the expression of cell exhaustion markers and induction of apoptosis in the period of co-culture with C. albicans. In addition, the M-CAR-modified cells were responsive after incubation with the soluble forms of mannan, obtained from S. cerevisae, and β-glucan peptide (BGP); and the interaction of M-CAR and fluorophore-conjugated mannan demonstrated a distribution of M-CAR on the cell surface in the form of clusters by fluorescence microscopy. Expression of M-CAR cells in NK-92 cells was feasible and enabled the establishment of a stable population for M-CAR expression. Furthermore, modification of PBMC with M-CAR allowed the expansion of an M-CAR T cell population capable of recognizing C. albicans and inducing IFN-γ production. These findings together showed the ability of M-CAR to mediate T cell activation against Candida spp., opening new perspectives to evaluate the fungicidal activity of human T and NK cells after modification with M-CAR. (AU)