Engineering Mannan-specific Chimeric Antigen Receptor (m-car) T Cells With Distinct Costimulatory Domains to Improve Antifungal Activity Against Candida Spp.
Year: 2026
Type: article
Abstract: Invasive fungal infections (IFIs) are neglected diseases associated with high morbidity, mortality and costs worldwide. Invasive candidiasis (IC) is the most common health care associated IFIs. Candida albicans remains the predominant etiological agent, and non-albicans Candida species have emerged as a public health concern associated with antifungal resistance. Development of effector T cells play a critical role in the host immune response against IFIs. However, most patients with candidiasis exhibit a compromised T cell activity, which increases their susceptibility to IC. In this context, chimeric antigen receptor (CAR) technology has emerged as a promising strategy to redirect T cells to combat specifically fungal pathogens, as previously reported against Candida spp. M-CAR developed by our group recognizes α-1,6 mannose backbone localized in the Candida spp. cell wall, M-CAR induced Jurkat cell activation against multiple Candida species. M-CAR contains an N-terminal FLAG-tag followed by single-chain variable fragment (scFv) specific to mannan, the CD8 molecule as hinge/transmembrane, and CD3ζ as signaling domain. The original M-CAR construct contains CD137 (4-1BB) molecule as costimulatory domain, and the present study evaluated other costimulatory domains to enhance T-cell activation against Candida spp.. M-CAR variants incorporated either CD28 or iCOS as costimulatory domains followed by CD3ζ. The M-CAR constructs were generated using the Gibson Assembly. Lentiviral particles containing the coding sequence of the M-CAR constructs were produced via HEK 293T cell transfection and titrated by Jurkat cells transduction. Primary human T cells isolated from peripheral blood mononuclear cells (PBMCs) using Ficoll were modified with M-CAR variants. The engineered T cells were co-cultured with heat-killed C. albicans, C. tropicalis, C. parapsilosis, or C. glabrata in different morphological forms to evaluate the antigen-recognition capacity and subsequent M-CAR T cell activation. In addition, M-CAR T cells were incubated with live C. albicans yeast cells to assess their antifungal activity. Primary T cells were effectively transduced with all M-CAR variants. After 48 h of co-culture with distinct Candida species, M-CAR T cells exhibited high production of IFN-γ which was observed in response to heat-killed C. albicans and C. tropicalis hyphae, as well as C. parapsilosis pseudohyphae. Additionally, M-CAR variants mediated high IFN-γ production in response to live C. albicans yeasts, and M-CAR containing the iCOS as costimulatory domain induced the highest levels of IFN-γ production. Despite the enhanced M-CAR T cell activation, preliminary data of fungal burden measured by CFU revealed no significant reduction mediated by engineered T cells. Interestingly, the XTT assay demonstrated an increase of fungal damage mediated by M-CAR-iCOSζ T cells. These findings open perspectives to evaluate the fungicidal activity of human T cells expressing the M-CAR variants in response to Candida spp.
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: diamond
OpenAlex ID: W7223149823