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GXMR-CAR containing distinct GXM-specific single-chain variable fragment (scFv) mediated the cell activation against Cryptococcus spp. And had difference in the strength of tonic signaling

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Machado, Michele Procopio ; dos Santos, Matheus Henrique ; Guimaraes, Julia Garcia ; de Campos, Gabriela Yamazaki ; Oliveira Brito, Patricia Kellen Martins ; Ferreira, Camilly Melo Garcia ; Rezende, Caroline Patini ; Frota, Natalia Fernandes ; Soares, Sandro Gomes ; Kumaresan, Pappanaicken R. ; Lourenzoni, Marcos Roberto ; da Silva, Thiago Aparecido
Número total de Autores: 12
Tipo de documento: Artigo Científico
Fonte: BIOENGINEERED; v. 14, n. 1, p. 26-pg., 2023-12-31.
Resumo

Cryptococcus spp. has a polysaccharide capsule composed of glucuronoxylomannan-GXM, a major virulence factor that can prevent the recognition of fungi by immune cells. Chimeric Antigen Receptor (CAR) redirects T cells to target Cryptococcus spp. as previously demonstrated by a CAR specific to GXM, GXMR-CAR. The current study evaluated the strength of the signal transduction triggered by GXMR-CAR, composed of a distinct antigen-binding domain sourced from a single-chain variable fragment (scFv). GXM-specific scFv derived from mAbs 2H1 and 18B7, 2H1-GXMR-CAR and 18B7-GXMR-CAR, respectively, were designed to express CD8 molecule as hinge/transmembrane, and the costimulatory molecule CD137 (4-1BB) coupled to CD3 zeta. The 2H1-GXMR-CAR or 18B7-GXMR-CAR Jurkat cells recognized soluble GXM from C. gattii and C. neoformans, and the levels of IL-2 released by the modified cells did not differ between the GXMR-CAR constructs after exposure to Cryptococcus spp. 18B7-GXMR-CAR triggered tonic signaling was more pronounced in modified Jurkat cells, and a protein kinase inhibitor of the Src family (dasatinib) significantly reduced GXMR-CAR tonic signaling and inhibited cell activation against ligands. 18B7 scFv showed a structural modification of the variable heavy (VH) chain that clarified the difference in the strength of tonic signaling and the level of cell activation between 2H1-GXMR-CAR and 18B7-GXMR-CAR. GXMR-CAR constructs induced T-cell activation against clinical isolates of Cryptococcus spp. and serum from patients with cryptococcosis induced high levels of IL-2, mainly in cells modified with 18B7-GXMR-CAR. Thus, 18B7-GXMR-CAR and 2H1-GXMR-CAR mediated T cell activation against Cryptococcus spp. and 18B7 and 2H1 scFv influenced the strength of tonic signaling. HIGHLIGHTS center dot 2H1-GXMR-CAR and 18B7-GXMR-CAR are efficiently expressed on the cell surface; center dot 2H1-GXMR-CAR and 18B7-GXMR-CAR redirected T cells toward the ligands; center dot 18B7-GXMR-CAR provided highest levels of tonic signaling; center dot Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR; center dot Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR; [GRAPHICS] . (AU)

Processo FAPESP: 18/18538-0 - Bioengenharia de células T e NK através de receptores CAR contra infecções fúngicas invasivas
Beneficiário:Thiago Aparecido da Silva
Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
Processo FAPESP: 21/02758-4 - Construção de receptores CAR para redirecionar células T e NK no controle da infecção invasiva por Candida albicans
Beneficiário:Gabriela Yamazaki de Campos
Modalidade de apoio: Bolsas no Brasil - Doutorado
Processo FAPESP: 20/16738-2 - Redirecionamento de células T através de receptor CAR para controlar a infecção por Candida albicans
Beneficiário:Júlia Garcia Guimarães
Modalidade de apoio: Bolsas no Brasil - Mestrado
Processo FAPESP: 19/26074-7 - Bioengenharia de células T e NK através de receptores CAR contra infecções fúngicas invasivas
Beneficiário:Thiago Aparecido da Silva
Modalidade de apoio: Bolsas no Brasil - Jovens Pesquisadores
Processo FAPESP: 20/11307-3 - Impacto de distintos scFv em GXMR-CAR expresso por linfócitos T na etapa de ativação celular frente a Cryptococcus spp
Beneficiário:Michele Procópio Machado
Modalidade de apoio: Bolsas no Brasil - Mestrado