RT Journal Article SR Electronic T1 Glucuronoxylomannan in the Cryptococcus species capsule as a target for CAR+ T-cell therapy JF bioRxiv FD Cold Spring Harbor Laboratory SP 715045 DO 10.1101/715045 A1 da Silva, Thiago Aparecido A1 Hauser, Paul J. A1 Bandey, Irfan A1 Laskowski, Tamara A1 Wang, Qi A1 Najjar, Amer M. A1 Kumaresan, Pappanaicken R. YR 2019 UL http://biorxiv.org/content/early/2019/07/26/715045.abstract AB 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.