preparation-single-cell-suspension-mouse-thymic-epithelial-cells

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the ...

National Institutes of Health

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)-related diseases. Three patients presented with EBV-associated Hodgkin's lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8 T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro-generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8 T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70-CD27 interactions therefore play a nonredundant role in T and B cell-mediated immunity, especially for protection against EBV and humoral immunity.

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency.

Mg is required at micromolar concentrations as a cofactor for ATP, enzymatic reactions, and other biological processes. We show that decreased extracellular Mg reduced intracellular Mg levels and impaired the Ca flux, activation marker up-regulation, and proliferation after T cell receptor (TCR) stimulation. Reduced Mg specifically impairs TCR signal transduction by IL-2-inducible T cell kinase (ITK) due to a requirement for a regulatory Mg in the catalytic pocket of ITK. We also show that altered catalytic efficiency by millimolar changes in free basal Mg is an unrecognized but conserved feature of other serine/threonine and tyrosine kinases, suggesting a Mg regulatory paradigm of kinase function. Finally, a reduced serum Mg concentration in mice causes an impaired CD8 T cell response to influenza A virus infection, reduces T cell activation, and exacerbates morbidity. Thus, Mg directly regulates the active site of specific kinases during T cell responses, and maintaining a high serum Mg concentration is important for antiviral immunity in otherwise healthy animals.

Mg regulation of kinase signaling and immune function.

Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches. In humans, loss-of-function mutations in the  gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasia (XMEN), a disease that has a broad range of clinical and immunological consequences. We have previously shown that EBV susceptibility in XMEN is associated with defective expression of the antiviral natural-killer group 2 member D (NKG2D) protein and abnormal Mg transport. New evidence suggests that MAGT1 is the human homolog of the yeast OST3/OST6 proteins that form an integral part of the -linked glycosylation complex, although the exact contributions of these perturbations in the glycosylation pathway to disease pathogenesis are still unknown. Using MS-based glycoproteomics, along with CRISPR/Cas9-KO cell lines, natural killer cell-killing assays, and RNA-Seq experiments, we now demonstrate that humans lacking functional MAGT1 have a selective deficiency in both immune and nonimmune glycoproteins, and we identified several critical glycosylation defects in important immune-response proteins and in the expression of genes involved in immunity, particularly CD28. We show that MAGT1 function is partly interchangeable with that of the paralog protein tumor-suppressor candidate 3 (TUSC3) but that each protein has a different tissue distribution in humans. We observed that MAGT1-dependent glycosylation is sensitive to Mg levels and that reduced Mg impairs immune-cell function via the loss of specific glycoproteins. Our findings reveal that defects in protein glycosylation and gene expression underlie immune defects in an inherited disease due to MAGT1 deficiency.

Magnesium transporter 1 (MAGT1) deficiency causes selective defects in linked glycosylation and expression of immune-response genes.

Major histocompatibility complex (MHC)-associated peptides generated and displayed by antigen-presenting cells in the thymus are essential for the generation of functional and self-tolerant T cells that protect our body from various pathogens. The peptides displayed by cortical thymic epithelial cells (cTECs) are generated by unique enzymatic machineries including the thymoproteasomes, and are involved in the positive selection of self-protective T cells. On the other hand, the peptides displayed by medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs) are involved in further selection to establish self-tolerance in T cells. Although the biochemical nature of the peptide repertoire displayed in the thymus remains unclear, many studies have suggested a thymus-specific mechanism for the generation of MHC-associated peptides in the thymus. In this review, we summarize basic knowledge and recent advances in MHC-associated thymic peptides, focusing on the generation and function of thymoproteasome-dependent peptides specifically displayed by cTECs.

Peptides for T cell selection in the thymus.

Thymoproteasomes and immunoproteasomes are two types of tissue-specific proteasomes, which contribute to the production of major histocompatibility complex (MHC) class I (MHC-I)-associated peptides that are important for the development and function of CD8 cytotoxic T cells. Thymoproteasomes are specifically expressed by cortical thymic epithelial cells and are important for MHC-I-dependent positive selection of developing thymocytes, whereas immunoproteasomes are abundant in many other cells, including hematopoietic cells and medullary thymic epithelial cells. Here we summarize the role of these two tissue-specific proteasomes, focusing on their functions in the development of CD8 T cells in the thymus.

Tissue-specific proteasomes in generation of MHC class I peptides and CD8 T cells.

The thymus is compartmentalized into the cortex and the medulla. Cortical and medullary thymic epithelial cells (TECs) characterize T cell-producing and T cell-selecting functions of cortical and medullary microenvironments in the thymus. Enzymatic digestion of the thymus and flow cytometric isolation of TECs and their subpopulations are useful for molecular and cellular characterization of TECs. However, the cellularity of cTECs and mTECs isolated from mouse thymus is limited. In this chapter, we describe the method for isolation of a large number of TECs using enlarged mouse thymus, which enables biochemical and proteomic analysis of TEC subpopulations.

Large-Scale Isolation of Mouse Thymic Epithelial Cells.

Medullary thymic epithelial cells (mTECs) are essential for the establishment of self-tolerance in T cells. Promiscuous gene expression by a subpopulation of mTECs regulated by the nuclear protein Aire contributes to the display of self-genomic products to newly generated T cells. Recent reports have highlighted additional self-antigen-displaying mTEC subpopulations, namely Fezf2-expressing mTECs and a mosaic of self-mimetic mTECs including thymic tuft cells. In addition, a functionally different subset of mTECs produces chemokine CCL21, which attracts developing thymocytes to the medullary region. Here, we report that CCL21 mTECs and Aire mTECs non-redundantly cooperate to direct self-tolerance to prevent autoimmune pathology by optimizing the deletion of self-reactive T cells and the generation of regulatory T cells. We also detect cooperation for self-tolerance between Aire and Fezf2, the latter of which unexpectedly regulates thymic tuft cells. Our results indicate an indispensable interplay among functionally diverse mTECs for the establishment of central self-tolerance.

Article	Year
Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency. The Journal of experimental medicine\| PubMed ID: 28011864	2017
Mg regulation of kinase signaling and immune function. The Journal of experimental medicine\| PubMed ID: 31196981	2019
Magnesium transporter 1 (MAGT1) deficiency causes selective defects in linked glycosylation and expression of immune-response genes. The Journal of biological chemistry\| PubMed ID: 31337704	2019
Peptides for T cell selection in the thymus. Peptides\| PubMed ID: 34624431	2021
Tissue-specific proteasomes in generation of MHC class I peptides and CD8 T cells. Current opinion in immunology\| PubMed ID: 35689940	2022
Large-Scale Isolation of Mouse Thymic Epithelial Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 36374458	2023
Functionally diverse thymic medullary epithelial cells interplay to direct central tolerance. Cell reports\| PubMed ID: 38581680	2024

Mami Matsuda-Lennikov

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