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

Abstract

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-ς 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. Author summary Cryptococcus gattii infects both immunocompetent and immunodeficient patients such as those with HIV/AIDS, while C. neoformans usually infects only immunocompromised patients. Every year, almost one million HIV/AIDS patients suffer from Cryptococcus fungal co-infection. At present, no curative treatment is available to treat cryptococcosis in chronic HIV/AIDS patients. The objective of this research was to develop novel “Bioengineered” Cryptococcus specific chimeric antigen receptor (CAR) CD8+ T-cells to target and kill Cryptococcus. By using a culture model, we demonstrated that the Cryptococcus specific CAR T cells were able to bind to the yeast form of C. neoformans. Using a mouse model of Cryptococcus, the Cryptococcus specific CAR treated group showed a significant reduction of fungal burden in lung tissue when compared to the control group. This gives new hope to HIV/AIDS patients suffering from cryptococcal infections.