Impact of different scFv in GXMR-CAR expressed by T lymphocytes during T-cell activation against Cryptococcus spp

Abstract

Cryptococcosis is an opportunistic fungal infection caused by inhalation of propagules or spores of the etiologic agent Cryptococcus gattii or Cryptococcus neoformans. The C. gattii species affects mainly immunocompetent individuals, while C. neoformans, the immunocompromised ones, being responsible for fungal meningitis in a high number of adults with HIV. The pathogens C. neoformans and C. gattii present as the main virulence factor the polysaccharide capsule associated with the fungal cell wall. The capsule contains mainly glucuronoxylomannan (GXM) and smaller amounts of glucuronoxylomannogalactan (GXMGal) and mannoproteins that represent less than 1% of the capsular weight. The GXM polysaccharide is able to inhibit T lymphocyte activation and reduce macrophage survival. In cryptocose, high serum GXM levels are found and immunotherapy with GXM-specific monoclonals is effective in reducing serum GXM levels and increasing the survival of animals affected by C. neoformans. Among these anti-GXM monoclonal antibodies, clones 2H1 and 18B7 are properly characterized in the context of the antigen-antibody complex. In view of the detailed knowledge of the interaction region of clone 18B7, this information was adopted in the construction of a chimeric antigenic receptor (CAR) to redirect cytotoxic T cells against Cryptococcus spp, this receptor is called GXMR-CAR. The use of this cell therapy in cryptococcosis was formulated by the person responsible for the current proposal, and this approach extension is contemplated by the Young Researcher Aid (2018 / 18538-0).The bioengineering of T lymphocytes through CAR technology allows T cells to recognize targets on the surface of fungal cells and, with this, be activated independently of MHC-TCR fusion. In this way, the current proposal will compare the effect of GXMR-CAR containing different scFv, recognition portion for GXM, from clones 2H1 and 18B7, during the activation of modified T cells and the cytotoxic reflex against Cryptococcus spp yeasts. The evaluation of different scFv in the CAR structure is important in the level of activation and survival of the modified T cells, this will reflect on the cytotoxic effect on the desired target. Thus, the current proposal will proceed with the following specific objectives: (i) production of lentiviral particles containing the coding sequence of the forms 2H1-GXMR-CAR and 18B7-GXMR-CAR; (ii) generation of stable Jurkat cells for these receptors; (iii) evaluate the interaction of these Jurkat cells with C. gattii and C. neoformans yeasts and the effect on cell activation; (iv) transduction of PBMC with the constructs of the 2H1-GXMR-CAR or 18B7-GXMR-CAR forms to quantify the production of pro-inflammatory mediators, cytotoxic granules and cell proliferation after incubation with yeasts; (v) evaluate in vitro the ability of T cells modified with 2H1-GXMR-CAR or 18B7-GXMR-CAR to reduce yeast growth and alter the susceptibility of Cryptococcus spp to antifungal drugs. (AU)