JIA SOON LEN
Immune cells are important cells that play many functions in the body: defending against pathogens and repairing tissue after injury. Perhaps the most well-known function of the immune cells is its protection against pathogens. Each day, we breathe in about a million microorganisms into our lungs, including fungi, bacteria, and viruses, and this number can be much higher depending on the environment we are in. As such, immune cells are vital in promoting rapid clearance and removal of these pathogens before they can cause an infection.
There are two types of immune defense mechanisms in the body: innate immunity and adaptive immunity. In innate immunity, immune responses against foreign particles are elicited minutes or hours upon exposure since it is the body’s initial fighting response. Compared to innate immunity, adaptive immune responses are elicited days or weeks after exposure but can efficiently eliminate infectious pathogens by recognizing specific antigens. They take days or weeks to build specific defenses to these pathogens and target them more efficiently. However, have you ever wondered how immune cells in adaptative immunity distinguish foreign particles (such as pathogens) from self? An important cell involved in adaptive immunity would be T cells, named because the development and maturation of functional T cells lie within a primary lymphoid organ called the thymus.
Role of T cells in the body
T cells are responsible for mediating various functions such as inflammation, cytotoxicity, and regulation of immune responses. There are many T cell subsets, each performing a specific function. Depending on the specific proteins displayed on the cell surface, cells perform different functions. The two main cell surface proteins expressed by T cells are CD4 and CD8. These cell surface proteins are called co-receptors. They are expressed alongside the T cell receptor and help recognize peptides loaded on a specific major histocompatibility complex (MHC) class I or II. MHC are glycoproteins expressed on a cell surface, and they function to present antigens for recognition by T cells. Glycoproteins are proteins that have sugar residues attached to their amino acid side chain. For CD4 T cells, they recognize antigens loaded on MHC class II, while CD8 T cells recognize antigens loaded on MHC class I. An antigen is any substance that is capable of stimulating an immune response.
Intracellular parasites such as Mycobacterium tuberculosis are usually engulfed by another type of immune cell called macrophages. However, since these intracellular parasites have evolved mechanisms to evade killing by macrophages, they can survive intracellularly and cause infection of the cell.
An effector subset of CD4 T cells, TH1 cells, recognizes the antigen presented by MHC class II on infected macrophages and produces a molecule called interferon-γ (IFN-γ) to promote the efficient killing of these intracellular pathogens. CD8 T cells, on the other hand, are called cytotoxic T cells. They release granzymes and perforins upon recognizing the antigen presented on MHC class I infected cells that eventually cause infected cells to be killed. Perforins are proteins that cause pores to form on the target cell membrane, resulting in direct cellular damage. Perforins allow granzymes to enter the target cell. Granzymes are proteases (enzymes that cleave proteins) that are activated intracellularly. In the cell, granzymes cleave proteins to initiate signaling cascades (series of changes in the cell that signal the cell to perform specific tasks), leading to programmed cell death known as apoptosis.
T cells and immunological tolerance
Given the ability of T cells to exert their cytotoxic activity, it is essential to have regulatory mechanisms, termed immunological tolerance, in place to prevent the destruction of normal cells/tissues by T cells. The T cell development occurs after multipotent hematopoietic stem cells in the bone marrow migrate to the thymus, where they receive signals to proliferate and become committed to a specific T cell lineage as characterized by the T-cell receptor chains they have. Amongst these T cell lineages, α:β T cells will later develop to give rise to CD4 and CD8 T cells. In T cell development, the desired outcome is one where T cells are functional (able to recognize antigens on either MHC class I or II) while not reacting strongly to self-antigens. This is achieved through positive selection and negative selection, respectively. In positive selection, only T cells that can recognize self-peptides presented on either MHC class I or II will survive. Positive selection ensures the production of functional T cells since recognition of the antigen presented by MHC class I or II is essential for it to become activated to perform its effector functions. However, if T cells react too strongly to the self-peptide presented on the MHC, they will either be eliminated via apoptosis or develop into T regulatory cells in a process called negative selection. Negative selection ensures the T cells generated will not target our own cells or tissues, which will otherwise give rise to autoimmune diseases such as type I diabetes and multiple sclerosis. This is because, for T cells to become activated, they will need to bind strongly to the antigen presented on the MHC class I or MHC class II.
Generation of thymic cells from induced pluripotent stem cells (iPSCs)
Although we have gained a considerable amount of knowledge on T cell development over the years, many questions remain unanswered. In an effort to drive the field forward, in a paper published in the Journal of Allergy and Clinical Immunology, Ramas and colleagues describe a protocol for generating functional thymic cells from iPSCs. In the paper, Ramas and colleagues sought to explore the possibility of using patient-derived induced pluripotent stem cells (iPSC) to generate functional thymic cells. iPSCs are cells that have been reprogrammed from other differentiated cell types to become pluripotent stem cells. Pluripotency refers to the ability of cells to differentiate and give rise to any cell type in the body. Cell differentiation refers to the process whereby progenitor cells gradually lose their ability to give rise to other cell types. These cells will become committed to giving rise to a limited range of cell types and perform specific functions. Conversely, de-differentiation is a process that is the opposite of differentiation.
Figure 1. (A) PBMC (obtained from human volunteers and cord blood) and human neonatal dermal fibroblast are reprogrammed and de-differentiated to induced pluripotent stem cells (iPSCs). These iPSCs are then differentiated to thymic epithelial progenitor (TEP) cells.
(B) TEP cells generated from iPSC are then transplanted into athymic nude mouse (mouse lacking functional thymus) to assess their functionality (ability to differentiate into thymic epithelial cells (TECs)). As T cells migrate to secondary lymphoid organs such as spleen after maturation, the spleen of TEP-transplanted athymic nude mice is harvested and analysed. If the spleen of athymic node mice contains mature, functional T cells, this would imply the transplanted TEP is able to perform its function of supporting T cell development. The cell populations in TEP graft is analysed and compared with human primary neonatal thymus tissue. The results indicated that the cell populations in TEO graft and human primary neonatal thymus tissue are very similar.
(C) The main findings by Ramas and colleagues are shown.
To generate iPSCs, the authors obtained peripheral blood mononuclear cells (PBMCs) from human volunteers. As de-differentiation is a sequential process, the authors first converted PBMC to erythroid progenitor cells. Following that, erythroid progenitor cells are de-differentiated to become iPSCs by introducing several transcriptional factors, collectively known as Okita factors. To prove the versatility of their approach, the authors generated iPSC from multiple cell types (refer to Figure 1A). The authors tested six cell differentiation conditions individually to differentiate iPSCs into thymic epithelial progenitor (TEP) cells. They subsequently found one of them to be most optimal in differentiating iPSCs to TEP cells. The differentiation of iPSC into TEP is a three-stage process whereby iPSC is first converted to definitive endoderm (DE) cells, followed by differentiation to third pharyngeal pouch cells, and finally to TEP cells. As with any other cell type, these cells are characterized by a distinct set of cell markers. For instance, DE cells have cell markers such as FOXA2 and SOX17, while TPP cells have a different set of cell markers: EYA1 and HOXA3. By looking at the protein markers a cell has, researchers will be able to identify its identity. Identification of cell identity will enable subsequent analysis or experiments.
Although TEPs can be obtained from iPSCs, it is imperative to test their functionality. To assess the functionality of the TEPs obtained, Ramas and coworkers transplanted them into athymic nude mice (mice lacking normal, functional thymus) and subsequently checked if these TEP cells could differentiate into thymic epithelial cells (TEC) in-vivo (Figure 1B). Through quantitative polymerase chain reaction (qPCR) and immunostaining for TEC cell markers, researchers confirmed that iPSC-derived TEP is functionally capable of differentiating into TEC.
Given that TEC functions in the selection and maturation of T cells, the author harvested the spleen from TEP-transplanted and control mice (without TEP transplantation). As T cells migrate to secondary lymphoid organs such as the spleen after undergoing positive and negative selection and maturation in the thymus, analysis of cells in these peripheral lymphoid organs will indicate if these TEC can perform their function in T cell development. Flow cytometry (a technique used to analyze cell characteristics) and in-vitro T cell activation tests showed the spleen from TEP-transplanted mice contain mature and functional T cells. Not only that, through single-cell RNA sequencing, the TEC-derived from iPSC was found to be indistinguishable from human primary neonatal TECs (Figure 1C).
These results highlight the therapeutic potential of using iPSC cells for treating patients with thymic disorders. Furthermore, the ability to generate iPSC from different cell types and the differentiation of iPSC to TEP cells, as described by Ramas et al., will promote the ease of study for other researchers and enable more breakthroughs in the T-cell development research field.
References
Gusareva, E. S. et al. (2019) 'Microbial communities in the tropical air ecosystem follow a precise diel cycle', Proceedings of the National Academy of Sciences 116, 23299-23308, doi:10.1073/pnas.1908493116
Ramos, S. A. et al. (2022) 'Generation of functional human thymic cells from induced pluripotent stem cells', Journal of Allergy and Clinical Immunology 149(2), 767-781, doi:10.1016/j.jaci.2021.07.021 (2022)