Functional activity of T cells modified with GXMR-CAR expressing the intracellular domain of CD28 or CD137, to control of experimental cryptococcosis

Abstract

Among neglected diseases, invasive fungal infections (IFIs) have a higher rate of mortality and morbidity. Moreover, there is a great difference of investment in the treatment of IFIs compared to malaria and tuberculosis, that have mortality rates similar to those reported in IFIs. The fungus genus Cryptococcus spp. comprises the main causes of IFIs, affecting about 1 million individuals/year and its mortality rates varies between 20 to 70% in HIV+ immunocompromised patients. Specifically, C. gattii has a greater predilection for lung tissue and its infection can occur in both immunosuppressed and immunocompetent individuals. The capsule of C. gatti is its major virulence factor and is composed primarily of a sugar moiety glucuronoxylomannan (GXM). The capsule provide protection to the fungus preventing recognition by the host immune system, thus compromising the differentiation and action of CD4+ T cells ( Th1 and Th17) and CD8+ T cells (Tc1 and Tc17). To circumvent this process, the current project proposes to redirect T cells for the recognition of C. gattii via interaction with polysaccharide-GXM present in the capsule, through chimeric antigenic receptors (CAR), which in this case is characterized as GXMR-CAR. Therefore, we propose a functional comparative analysis of T cells GXMR-CAR expressing the intracellular portion of CD28 (GXMR-CD28 CAR) or CD137 (GXMR-CD137 CAR), to treat experimental infections caused by C. gattii. The GXMR-CAR contains a hinge and transmembrane region from the CD8 molecule, and in the intracellular region the activation motifs of the CD28 or CD137 molecules, together with CD3z motifs, will be responsible for the activation of T cells after interaction with C. gattii. Initially, the Jurkat cell line will be modified with GXMR-CD28 CAR or GXMR-CD137 CAR, by lentiviral transduction, to confirm expression and integrity of these receptors and the ability to interact with C. gattii followed by cell activation signal. In a second step, transduction of PBMC with GXMR-CD28 CAR or GXMR-CD137 CAR will allow to quantify the secretion of pro-inflammatory mediators and cytotoxic granules by the modified T cells incubated with C. gattii. Thus, the ability of the modified T cells to inhibit in vitro growth of C. gattii will be investigated. Finally, the effect of infusion of T cells modified with GXMR-CD28 CAR or GXMR-CD137 CAR in NSG mice infected with C. gattii will be evaluated through a survival curve and pulmonary fungal load. (AU)