Identification of CAR with high specificity to Cryptococcus gattii by Jurkat cell line

• Grant number: 21/07174-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2021
• End date: July 31, 2022
• Field of knowledge: Biological Sciences - Immunology - Applied Immunology
• Principal Investigator: Thiago Aparecido da Silva
• Grantee: Camilly Melo Garcia Ferreira
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 18/18538-0 - Bioengineered of T- and NK-cells by CAR against invasive fungal infections, AP.JP

Abstract

Cryptococcus gattii is an enveloped yeast that belongs to the genus Cryptococcus remains one of the leading causes of criptococose in humans by inhalation of the propagules. The infection is persistent on respiratory tissues of immunocompromised individuals, but can also progress to meningoencephalitis and disseminate to other tissues. The C. gattii is an endemic pathogen classically located in tropical, subtropical, and transition zones. The polysaccharide capsule on Cryptococcus ssp is associated with the fungi membrane and provides greater potential for increased virulence, allowing the organism to bypass the host innate immune response due to components like glucuronoxylomannan (GXM) and, at a low rate, galactoxylomannan (GalXM) and mannoproteins. The immune response evasion occurs directly with T-cells function interference, furthermore dysregulation of cytokines and macrophage proliferation, stimulation of apoptosis, moreover effects like pathogen adhesion to lung epithelial cells. To perform antifungal protecting response, T CD4+ cells with T-helper (Th) profile, as Th1 and Th17, are important to create and maintain the microenvironment at the infection site. The cell's profiles Tc1 e Tc17, by the differentiation of T CD8+, release cytotoxic granules and pró-inflammatory mediators important at effective immune response at the fungal invasive infection site. *In brief, the objective of this current proposal is to redirect functional cytotoxic T-cells to Cryptococcus gattii by cell surface components of the yeast. The specificity redirection of T-cells will be promoted by chimeric antigen receptors (CAR), GMXR-CAR, which are recombined on a recognized region with GXM, from scFv monoclonal antibody anti-GXM. Therefore, several GXMR-CAR constructs and auxiliar plasmids will be transfected to HEK-293FT cells, with the goal to produce lentiviral particles containing GXMR-CAR constructs. Those lentiviral particles will be presented to the Junkart cell line transfection to express GXMR-CAR. Following, transformed Junkart cells with different CAR constructs will be co-cultivated with C. gattii to evaluate the production of pró-inflammatory mediators. These specific objectives will allow differentiating the capacity of several GXMR-CAR constructs to induce modified T-cells activation face to C. gattii. (AU)