Glucuronoxylomannan in the Cryptococcus species capsule as a target for Chimeric Antigen Receptor T-cell therapy

Author(s)

Silva, Thiago Aparecido da ^{[1, 2]} ; Hauser, Paul J. ^[1] ; Bandey, Irfan ^[1] ; Laskowski, Tamara ^[1] ; Wang, Qi ^[3] ; Najjar, Amer M. ^[1] ; Kumaresan, Pappanaicken R. ^[4]

Affiliation

^[1] Univ Texas MD Anderson Canc Ctr, Deparment Pediat Res, Houston, TX 77030 - USA

^[2] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Cellular & Mol Biol & Pathogen Bioagents, Sao Paulo - Brazil

^[3] Univ Texas MD Anderson Canc Ctr, Dept Bioinformat & Computat Biol, Houston, TX 77030 - USA

^[4] Univ Texas MD Anderson Canc Ctr, Dept Lymphoma & Myeloma, 7455 Fannin St, Unit 903, Houston, TX 77054 - USA

• Document type: Journal article
• Source: CYTOTHERAPY; v. 23, n. 2, p. 119-130, FEB 2021.
• Web of Science Citations: 0

Abstract

Background aims: The genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: Cryptococcus gattii and Cryptococcus 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%. Many HIV+/AIDS patients are affected by Cryptococcus infections, with 220,000 cases of cryptococcal meningitis reported worldwide in this population every year (C. neoformans infection statistics, via the Centers for Disease Control and Prevention, https://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/statistics .html). To escape from host immune cell attack, Cryptococcus covers itself in a sugar-based capsule composed primarily of glucuronoxylomannan (GXM). To evade phagocytosis, yeast cells increase to a >45-mu m perimeter and become titan, or giant, cells. Cryptococci virulence is directly proportional to the percentage of titan/giant cells present during Cryptococcus infection. To combat cryptococcosis, the authors propose the redirection of CD8(+) T cells to target the GXM in the capsule via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR). Results: GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk in the extracellular domain, a CD28 transmembrane domain and CD28 and CD3-sigma 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 with the use of brightfield microscopy. Large clusters of proliferating GXMR-CAR(+) T cells were observed in GXM-treated cells, whereas no clusters were observed in control cells. Moreover, the interaction of GXM with GXMR-CAR(+) T cells was detected via flow cytometry by using a GXM- specific antibody, and the recognition of GXM by GXMR-CAR T cells triggered the secretion of granzyme and interferon gamma (IFN-gamma). 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 in mock-transduced control T cells (NoDNA T cells). Moreover, lung tissue sections were stained with Gomori Methenamine Silver and evaluated by NanoZoomer (Hamamatsu), revealing a significantly lower number of titan cells, with perimeters ranging from 50 to 130 mu m and giant cells >130 mu m in the CAR T-cell treated group when compared with other groups. Therefore, the authors validated the study's hypothesis by the redirection of GXMR-CAR(+) T cells to target GXM, which induces the secretion of cytotoxic granules and IFN-gamma that will aid in the control of cryptococcosis Conclusions: Thus, these findings reveal that GXMR-CAR(+) T cells can target C neoformans. Future studies will be focused on determining the therapeutic efficacy of GXMR-CAR(+) T cells in an animal model of cryptococcosis. Published by Elsevier Inc. on behalf of International Society for Cell \& Gene Therapy. (AU)

• FAPESP's process: 16/04877-2 - Design of new therapeutic strategies, based on the carbohydrate recognition, against cryptococcosis
• Grantee: Thiago Aparecido da Silva
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 18/18538-0 - Bioengineered of T- and NK-cells by CAR against invasive fungal infections
• Grantee: Thiago Aparecido da Silva
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 16/23044-1 - Engineered T cells expressing carbohydrate recognition domain of ArtinM lectin to activate macrophages and treat cryptococcosis
• Grantee: Thiago Aparecido da Silva
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor