Effect of hinge-transmembrane and signal transduction domains of GXMR-CAR specific to Cryptococcus spp. in T and NK cell activation

Author(s)

Matheus Henrique dos Santos

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-09-02

Examining board members

Thiago Aparecido da Silva; Allan Jefferson Guimarães

• Advisor: Thiago Aparecido da Silva

Abstract

Invasive fungal infections (IFIs) represent a great challenge to public health. Among neglected diseases, IFIs are responsible to cause as many deaths as malaria or tuberculosis. The clinal manifestation of Cryptococcus spp. infection is the cryptococcosis, and is responsible to cause around 180.000 deaths annually. The main risk factor to the development of cryptococcosis is the impairment of immune system, and the species Cryptococcus gattii and Cryptococcus neoformans represent greater clinical relevance, accountable for cause disease whether in immunocompromised or immunocompetent individuals. These pathogens display a diverse arsenal of virulence factors and physiological mechanisms that allow evasion from the host immune response, evading phagocytosis and modulating the T cell differentiation. To overcome these characteristic of Cryptococcus spp, this work evaluated the redirection of T cells and natural killer cells (NK) to recognize Cryptococcus spp. through the interaction with the polysaccharide glucuronoxilomanana (GXM) in the Cryptococcus capsule. For this, the chimeric antigen receptor (CAR) specific for this structure, GXMR-CAR, was expressed in T and NK cells modified with lentiviral transduction. Different constructs of GXMRCAR were considered to evaluate the functional properties against yeasts of Cryptococcus spp., and it is based in the following structural modifications: the IgG4 as hinge and CD28 as transmembrane compose the construct GXMR-IgG4-28ζ; CD8α as hingetransmembrane is present in the GXMR-8-BBζ and GXMR-8-28ζ; the co-stimulatory molecule is represented by 4-1BB (GXMR-8-BBζ) or CD28 (GXMR-IgG4-28ζ and GXMR-8-28ζ). Jurkat cells were considered as expression plataform to evaluate GXMRCAR against the reference species C. gattii R265 and C. neoformans H99 or clinical isolates of Cryptococcus spp. The cell linage NK-92 was used to perform in vitro studies evaluating the control of Cryptococcus spp. infection. The GXMR-CAR carrying CD8α as hinge-transmembrane demonstrated greater expression in Jurkat cells and the cells modified with GXMR-8-BBζ or GXMR-8-28ζ showed greater cell proliferation compared with GXMR-IgG4-28ζ during culture. Jurkat cells expressing GXMR-CAR with CD8α demonstrated tonic singnaling, and the co-stimulatory molecule 4-1BB was responsible to induce more levels of IL-2 compared with CD28. Besides the tonic signaling, the IL-2 production by the cells modified with GXMR-8-BBζ or GXMR-8-28ζ was increased upon incubation with soluble GXM or inactivated yeast of Cryptococcus spp, whereas the Jurkat cells expressing GXMR-IgG4-28ζ did not trigger cell activation. When the GXMR-CAR signaling cascade is inhibited by the use of dasatinib, a Scr tyrosine kinase inhibitor, the tonic signaling is attenuated. Moreover, the cell activation induced by interaction of GMXR-CAR and yeast is also attenuated by dasatinib and the proliferation of the cells treated is ameliorated when in presence of yeast. In addition, Jurkat cells modified with GXMR-8-BBζ or GXMR-8-28ζ are able to recognize different clinical isolates serotypes of C. gattii and C. neoformans, resulting in cell activation. The expression of GXMR-CAR in NK-92 cells demonstrated greater rates of expression of the CD8α-containing constructs, and the modified population remained stable for longer periods of culture. The fungicide capacity of NK-92 expressing GXMR-8-BBζ was evaluated against viable yeasts of Cryptococcus spp. The co-culture of cells and yeast resulted in NK activation, with increasing in INF-γ production, and we verified that GXMR-8-BBζ mediated tonic signaling in this cell line. The modified cells were able to increase the susceptibility of Cryptococcus spp. to amphotericin B, whereas the fungicide and fungistatic effect were not observed. In conclusion, the functional characterization of distinct GXMR-CAR in different cell lines and against a set of ligands, indicated the importance of CAR domains that resulted in the best options of GXMR-CAR against Cryptococcus spp. (AU)

• FAPESP's process: 20/09113-6 - Functional activity of T cells modified with GXMR-CAR expressing the intracellular domain of CD28 or CD137, to control of experimental cryptococcosis
• Grantee: Matheus Henrique dos Santos
• Support Opportunities: Scholarships in Brazil - Master