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Glucuronoxylomannan in the Cryptococcus species capsule as a target for CAR+ T-cell therapy

By Thiago Aparecido da Silva, Paul J. Hauser, Irfan Bandey, Tamara Laskowski, Qi Wang, Amer M. Najjar, Pappanaicken R. Kumaresan

Posted 26 Jul 2019
bioRxiv DOI: 10.1101/715045

The genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: C. gattii and C. neoformans. To establish invasive human disease, inhaled Cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infection's mortality rate ranges from 20% to 70%. HIV+/AIDS patients are highly susceptible to Cryptococcus infection. Therefore, we hypothesized that CD8+ T cells could be redirected to target glucuronoxylomannan (GXM), a sugar present in the Cryptococcus species capsule, via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR) for treatment of cryptococcosis. GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk, a CD28 transmembrane domain, and CD3-? and CD28 signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR+ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined using bright-field microscopy. Large clusters of proliferating GXMR-CAR+ T cells were observed, while no clusters were present in the control cells. Moreover, the interaction of GXM with GXMR-CAR+ T cells was detected via flow cytometry using a GXM-specific antibody. The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by fluorescent microscopy, but no binding was detected with NoDNA T cells. Furthermore, when GXMR-CAR+ T cells were administered to immunocompromised NSG mice infected with C. neoformans their C. neoformans burden was significantly lower than mock-transduced control T cell treated mice as shown via immunofluorescence using an anti-GXM antibody and Gomori methenamine-silver (GMS) staining of Titan cells in lung tissue. Thus, these findings demonstrated the effectiveness of GXMR-CAR+ T-cell therapy for cryptococcosis in a murine model.

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