Mutation in the Aire gene induced by the CRISPR-Cas9 system in an in vitro murine model (thymic medullary epithelial cells): impact on intercellular adhesion and antigen presentation

Author(s)

Ana Carolina Monteleone Cassiano

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2024-04-19

Examining board members

Eduardo Antonio Donadi; Daniella Arêas Mendes da Cruz; Maria Danielma dos Santos dos Reis

• Advisor: Eduardo Antonio Donadi

Abstract

In addition to controlling the expression of peripheral tissue antigens, the autoimmune regulatory gene (Aire) controls the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction. For this process to occur, it is important that the three-dimensional (3D) architecture of the thymus preserves the medullary layer. Aire knockout mice have disorganized thymic medulla, and there are no studies on the mTEC-mTEC interaction. In this study, we evaluated mTEC-mTEC adhesion in a 3D thymus culture model and the antigen presentation process of wild-type (WT) mTEC and Aire (E6) knockout mTEC at the NLS domain. E6 cells were obtained using CRISPR-Cas9. mTEC-mTEC adhesion was evaluated in an in vitro model for the formation of 3D spheroids. For the antigen presentation assay, we used WT and E6 cells stimulated with IFNγ in coculture with naïve CD4+ splenic T cells from OT II animals presenting specific receptor for the OVA323-339 peptide, in the presence or absence of dendritic cells. The absence of Aire: i) disorganizes the 3D structure of mTEC spheroids, ii) increases the proportion of cells in the G0/G1 phase of the cell cycle, iii) increases the rate of mTEC apoptosis, iv) decreases mTEC-mTEC adhesion strength, v) promotes differential regulation of classical mTEC surface markers, vi) modulates genes that encode adhesion molecules, vii) modulates genes involved in the antigen presentation pathway after stimulation with IFNγ (MHC II, CIITA, CD74), and viii) does not alter antigen processing pathways in the lysosomal stage. In contrast, transduction (overexpression) of the Aire gene inhibits responses induced by IFNγ. In conclusion, E6 mTECs show decreased mTEC-mTEC adhesion, making this the first study to address the topic. Although E6 mTECs present antigens in a similar way to WT, only some molecules related to presentation (MHC II and transactivators of MHC II molecules) and antigen processing (CD74) appear to be under the influence of the Aire gene, in the model studied. (AU)

• FAPESP's process: 19/23448-3 - Effect of Aire gene mutations (APS1 Syndrome) induced by CRISPR-Cas9 in protein conformation, transcriptome of mTEC cells and its interaction with thymocytes
• Grantee: Ana Carolina Monteleone Cassiano
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)