Question

1. Write about the following:

1. The major events that transform a hematopoietic stem cell into a mature and naïve T cell
2. The microenvironment of the thymus where each stage of T-cell development occurs.
3. The changes in expression of CD3, CD8 and TCR that occur during T-cell development
4. The basis for positive and negative selection; relationship between positive and negative selection and MHC restriction; and negative selection and central tolerance.
5. General functions of Treg cells

Answer 1

Question a. The major events that transform a hematopoietic stem cell into a mature and naïve T cell

Answer: All the cellular elements of blood, including the red blood cells that transport oxygen, the platelets that trigger blood clotting in damaged tissues, and the white blood cells of the immune system, derive ultimately from the same progenitor or precursor cells—the hematopoietic stem cells in the bone marrow. As these stem cells can give rise to all of the different types of blood cells, they are often known as pluripotent hematopoietic stem cells. Initially, they give rise to stem cells of more limited potential, which are the immediate progenitors of red blood cells, platelets, and the two main categories of white blood cells.

The myeloid progenitor is the precursor of the granulocytes, macrophages, dendritic cells, and mast cells of the immune system. Macrophages are one of the three types of phagocyte in the immune system and are distributed widely in the body tissues, where they play a critical part in innate immunity. They are the mature form of monocytes, which circulate in the blood and differentiate continuously into macrophages upon migration into the tissues. Dendritic cells are specialized to take up antigen and display it for recognition by lymphocytes. Immature dendritic cells migrate from the blood to reside in the tissues and are both phagocytic and macropinocytic, ingesting large amounts of the surrounding extracellular fluid. Upon encountering a pathogen, they rapidly mature and migrate to lymph nodes.

Mast cells, whose blood-borne precursors are not well defined, also differentiate in the tissues. They mainly reside near small blood vessels and, when activated, release substances that affect vascular permeability. Although best known for their role in orchestrating allergic responses, they are believed to play a part in protecting mucosal surfaces against pathogens.

The granulocytes are so called because they have densely staining granules in their cytoplasm; they are also sometimes called polymorphonuclear leukocytes because of their oddly shaped nuclei. There are three types of granulocyte, all of which are relatively short lived and are produced in increased numbers during immune responses, when they leave the blood to migrate to sites of infection or inflammation. Neutrophils, which are the third phagocytic cell of the immune system, are the most numerous and most important cellular component of the innate immune response: hereditary deficiencies in neutrophil function lead to overwhelming bacterial infection, which is fatal if untreated. Eosinophils are thought to be important chiefly in defense against parasitic infections, because their numbers increase during a parasitic infection.

The common lymphoid progenitor gives rise to the lymphocytes. There are two major types of lymphocyte: B lymphocytes or B cells, which when activated differentiate into plasma cells that secrete antibodies; and T lymphocytes or T cells, of which there are two main classes. One class differentiates on activation into cytotoxic T cells, which kill cells infected with viruses, whereas the second class of T cells differentiates into cells that activate other cells such as B cells and macrophages.

Lymphocytes are remarkable in being able to mount a specific immune response against virtually any foreign antigen. This is possible because each individual lymphocyte matures bearing a unique variant of a prototype antigen receptor, so that the population of T and B lymphocytes collectively bear a huge repertoire of receptors that are highly diverse in their antigen-binding sites. The B-cell antigen receptor (BCR) is a membrane-bound form of the antibody that the B cell will secrete after activation and differentiation to plasma cells. Antibody molecules as a class are known as immunoglobulins, usually shortened to Ig, and the antigen receptor of B lymphocytes is therefore also known as membrane immunoglobulin (mIg). The T-cell antigen receptor (TCR) is related to immunoglobulin but is quite distinct from it, as it is specially adapted to detect antigens derived from foreign proteins or pathogens that have entered into host cells. A third lineage of lymphoid cells, called natural killer cells, lack antigenspecific receptors and are part of the innate immune system. These cells circulate in the blood as large lymphocytes with distinctive cytotoxic granules. They are able to recognize and kill some abnormal cells, for example some tumor cells and virus-infected cells, and are thought to be important in the innate immune defense against intracellular pathogens.

The lymphoid organs are organized tissues containing large numbers of lymphocytes in a framework of nonlymphoid cells. In these organs, the interactions lymphocytes make with nonlymphoid cells are important either to lymphocyte development, to the initiation of adaptive immune responses, or to the sustenance of lymphocytes. Lymphoid organs can be divided broadly into central or primary lymphoid organs, where lymphocytes are generated, and peripheral or secondary lymphoid organs, where adaptive immune responses are initiated and where lymphocytes are maintained. The central lymphoid organs are the bone marrow and the thymus, a large organ in the upper chest; the location of the thymus, and of the other lymphoid organs.

Both B and T lymphocytes originate in the bone marrow but only B lymphocytes mature there; T lymphocytes migrate to the thymus to undergo their maturation. Thus B lymphocytes are so-called because they are bone marrow derived, and T lymphocytes because they are thymus derived. Once they have completed their maturation, both types of lymphocyte enter the bloodstream, from which they migrate to the peripheral lymphoid organs.

Question b. The microenvironment of the thymus where each stage of T-cell development occurs.

Answer: The thymus is the specialized central lymphoid organ where precursors of T cells undergo differentiation, selection and proliferation processes. The primary function of the thymus is to develop immature T cells into cells that will be able to carry out immune functions.

The organ, located in the anterior mediastinum, consists of two encapsulated lobes that are divided by numerous septa into multiple lobules. Each lobule presents two different regions, i.e., cortex and medulla. The outer cortical portion is densely populated by pre-T lymphocytes (or thymocytes while in the thymus), and the inner medullary portion contains few, but fully mature, T lymphocytes. Alongside the thymocytes, at different stages of maturation, the thymic environment is formed by epithelial cells, which form a meshwork to provide mechanical support and stimuli for the proliferation and development of thymocytes and by macrophages, dendritic cells, fibroblasts and matrix molecules. Also importantly, this cellular network involves blood vessels in the cortex and so effectively isolates developing lymphocytes to preclude the possibility of contaminating these cells with antigens (blood-thymus barrier).

T lymphocytes are generated from bone marrow-derived lymphocyte precursors that enter the thymic cortex through blood vessels. Currently, little is known of the mechanisms that attract precursors to the thymus or facilitate their migration through the surrounding tissues. Also, the phenotype of T-cell precursors remains unclear but some markers, such as c-Kit (CD117) and CD34 have been reported to be associated with them. More recently, signaling through the Notch receptors and the Notch ligands named Delta-like and Jagged have also been implicated during T-cell development because T cells fail to develop from Notch1-deficient bone marrow precursors.

The development of T cells within the thymus is a complex process that involves four main stages based on their expression of CD4 and CD8 co-receptors. At early stage of development, T-cell precursors have a CD34+CD4−CD8− “double negative” (DN) phenotype. This phase is also characterized by differential expression of the CD44 and CD25 molecules and these cells represent 5% of the total thymic lymphocytes. At the end of this phase, when the CD3 expression increases, the thymocytes present the CD3lowCD4−CD8− phenotype and a first step toward the expression of a functional T-cell receptor (TCR) takes place. Immature single-positive (ISP) cells (CD3low CD4+ CD8− or CD3low CD4− CD8+) start to rearrange the TCRB gene. Subsequently, the TCRβ chain becomes assembled into the pre-TCR complex with the invariant pre-Tα chain. Pre-TCR signaling confers survival and allows development to proceed through a CD4+CD8+TCRlow double-positive (DP) subset of thymocytes, which represent about 80% of the total cells in the organ. The thymocytes that were not able to generate a functional TCR die through apoptosis, whereas those expressing functional TCR will be exposed to endogenous peptides presented by self-major histocompatibility complex (MHC) molecules present on thymic microenvironmental cells. These interactions will determine the positive and negative selection events that will be decisive for the selection of a mature T-cell repertoire.

In the positive selection, double-positive thymocytes (CD4+CD8+) interact with self-major histocompatibility complex (MHC) molecules present on the cortical epithelium and this event leads cells to lose one of the co-receptor molecules (CD4 or CD8). Thus, a self MHC-restricted T-cell repertoire is generated. Then, the “single-positive” (SP) thymocytes (CD4+CD8− or CD4−CD8+) undergo negative selection in which those whose TCR recognizes self- peptides present in the thymic microenvironment are eliminated. Cells that fail the positive or negative selection die through apoptosis. On the other hand, the selected cells survive and migrate as mature T lymphocytes to the peripheral lymphoid tissues where they will mount and regulate cell-mediated immune responses.

Although it has long been known that interactions between the thymocytes and thymic environment are crucial during T-cell development, the molecular nature of such interactions that lead to positive and negative selection are yet unknown. The medullary thymic epithelial cells (mTEC), which correspond to the vast majority of cells in the thymus, and cells of the monocyte/dendritic cell lineage of the thymus are considered to play a major role in the establishment of self tolerance by eliminating auto-reactive T cells (negative selection) and/or by producing immune-regulatory T cells, which prevent CD4 T-cell mediated organ specific autoimmune diseases. mTECs express the autoimmune regulator (AIRE) gene that regulates the expression of tissuespecific antigens (TSAs) to the developing thymocytes in a dosage-dependent manner. So, a slight decrease in AIRE gene function can lead to a decrease in thymic protein expression, allowing the emigration of auto reactive T-cell clones to the periphery.

The successful development of mature T cells depends on the constant migration of the thymocytes through the thymic microenvironment. Such migration is essential for thymic stromal cells to provide signals to thymocytes that lead to proliferation, differentiation and generation of diversity. Although the mechanisms directing this migration are poorly understood, clear evidence has been obtained showing that the thymic microenvironment, collectively, influences the process of T-cell development through surface molecules and by secreting soluble polypeptides as cytokines, chemokines and hormones.

Lymphoid progenitors which have developed from hematopoietic stem cells in the bone marrow migrate to the thymus to complete their antigen-independent maturation into functional T cells . In the thymus, T cells develop their specific T cell markers, including TCR, CD3, CD4 or CD8, and CD2. T cells also undergo thymic education through positive and negative selection.

The thymus is a multi-lobed organ composed of cortical and medullary areas surrounded by a capsule. T cell precursors enter the subcapsular cortical areas, where they encounter networks of cortical epithelial calls (the thymic stroma) and undergo a period of proliferation. As they differentiate, they move from the cortex towards the medulla of the thymus; different microenvironments within the thymus direct T cell development. Most cells that enter the thymus die by apoptosis without successfully completing the steps required for becoming a mature naive T cell.

Question c.The changes in expression of CD3, CD8 and TCR that occur during T-cell development

Answer: When progenitor cells begin to express CD2 but have not yet rearranged their TCR genes (CD2+ CD3-), they are double negative for CD4 and CD8 ( CD4- CD8-), the markers for Th and Tc lineages. Of the double negative cells in the thymus, about 20% have rearranged gd TCR, about 20% have very homogenous ab TCR, and 60% are committed to becoming the majority of mature ab T cells. These cells next express the adhesion molecule CD44, then the a chain of the IL-2 receptor (CD25). CD44low CD25+ double negative T cells rearrange TCR b chain. b chain rearrangement begins with D-J joining, followed by V-DJ joining. The chances of successful b chain rearrangement are increased by the presence of two DJCb gene clusters. If rearrangement in the first cluster fails, rearrangement in the second can occur

Productive rearrangement of b chain is followed by its expression on the T cell membrane with CD3 and surrogate a chain, pTa (analogous to l5 in B cells). Signaling through the preT receptor causes the cells to stop rearranging b chain, undergo a period of proliferation, and begin to express both CD4 and CD8, becoming double positive T cells. Membrane CD25 is lost at this stage. Double positive cells re-express RAG-1 and RAG-2 to rearrange their a chain genes.a chain rearrangement can occur on both chromosomes and continue until the cell undergoes selection or dies, so T cells are not allelically excluded for a chain. However, even cells with two different TCR have only one which can bind self MHC with enough affinity to pass positive selection (one functional receptor specificity). Double positive ab T cells move into the cortico-medullary junction, where they undergo positive and negative selection and mature into Th and Tc cells.

T cell development is greatest during fetal development and before puberty. After puberty the thymus shrinks and T cell production declines; in adult humans, removal of the thymus does not compromise T cell function. Children born without a thymus because of an inability to form a proper third pharyngeal pouch during embryogenesis (DiGeorge Syndrome) were found to be deficient in T cells. Of several different T cell deficiencies that have been identified in mice, two complementary defects are found in SCID and nude mice. Nude mice, also called athymic, have a defective thymic epithelium and lack T cells. They are called nude because the defect also affects skin epithelium and results in lack of body hair as well as T cells. SCID mice (Severe Combined Immune Deficiency) have a thymus but cannot produce lymphocytes because of defects in enzymes (such as RAG and TdT) required for somatic recombination. Lymphoid progenitors from nude mice can develop normally when transferred into SCID mice with a normal thymus microenvironment.

Question d. The basis for positive and negative selection; relationship between positive and negative selection and MHC restriction; and negative selection and central tolerance.

Answer:

The basis for positive and negative selection.

Positive selection

To address the necessity that T cells be capable of binding MHC complexes, T cells undergo positive selection. In this process, cells in the thymus present short pieces of proteins, called peptides, on their own MHC class I and class II molecules, allowing immature T cells to bind. If TCRs are incapable of binding, the T cell will undergo a type of cell death celled apoptosis. If, however, a T cell’s TCR successfully binds to the MHC complexes on the thymic cells, they T cell receives survival signals and is thus positively selected. Further, this positive selection process also determines if a T cell will become a CD8+ T cell or a CD4+ T cell. Specifically, if a TCR complex binds strongly to MHC class II, the complex will send intracellular signals to induce the expression of a protein called ThPOK. This protein reduces the expression of another key protein, called Runx3, which is important in driving CD8 expression. If, however, a developing T cell does not bind strongly to MHC class II, ThPOK levels will be low and thus Runx3 levels will be high, pushing the T cell to differentiate into a CD8+ cell. In sum, the process of positive selection leads to the survival of mature CD8+ and CD4+ T cells capable of recognizing MHC complexes.

Negative selection

While the ability of T cells to recognizes antigen-MHC complex is vital for their ability to fight pathogens and other foreign cells, it is equally important that these T cells do not recognize and attack our own cells. This is where negative selection comes into play. As described above, developing T cells in the thymus are presented with peptides bound to MHC molecules, to which they may be able to bind. Importantly, while a moderate degree of binding leads to survival and positive selection, TCRs that bind too strongly to these MHC complexes are destined for the opposite fate. It is thought that, when TCRs bind too strongly to the MHC complexes in the thymus, the intracellular signaling is so strong that it actually leads to cell death, thereby eradicating immature T cell that have a high likelihood of being self-reactive and attacking our own cells.One of the most intriguing aspects of negative selection is that it primarily occurs in the thymus, which means that T cells rely solely on the cells in the thymus to present self-peptides on MHC molecules.

Negative selection, involves the depletion of all cell types except your cell type of interest. With our T cell isolation example, our negative selection kit would likely involve antibodies specific for B cells (CD19), monocytes (CD14), NK cells (CD56), and so on. With the depletion of these cell types we would only be left with our cells of interest, in this case T cells (CD3).

Relationship between positive and negative selection and MHC restriction

T cell antigen receptors (TCRs) recognize peptides of foreign protein antigens complexed with self-MHC (major histocompatibility complex) molecules. Indeed, the mature T cell repertoire is biased toward recognition of foreign peptides associated with self-MHC molecules, as opposed to those associated with nonself-MHC molecules. This characteristic is imposed by positive selection of developing T cells as they mature in the thymus. At the same time, a negative selection process purges the T cell repertoire of cells that are reactive with complexes of thymic self-peptides with self-MHC molecules.

positive selection favors immature CD4+CD8+ thymocytes that bind weakly or strongly to self-MHC/peptide complexes, whereas negative selection induces the death of thymocytes that bind strongly to self-MHC/peptide complexes . Operating together, positive and negative selection yield a repertoire of T cells that bind too weakly to self-MHC/self-peptide complexes for induction of immune responses. Binding contacts of the TCR with a foreign peptide in the groove of a self-MHC molecule may provide the added affinity necessary for triggering of a low but sufficient fraction of T cells, often estimated as 10−5–10−4 T cells per foreign peptide.

Negative selection and Central tolerance

Central tolerance, also known as negative selection, is the process of eliminating any developing T or B lymphocytes that are reactive to self. Through elimination of autoreactive lymphocytes, tolerance ensures that the immune system does not attack self peptides. Lymphocyte maturation (and central tolerance) occurs in primary lymphoid organs such as the bone marrow and the thymus. In mammals, B cells mature in the bone marrow and T cells mature in the thymus.

Central tolerance is not perfect, so peripheral tolerance exists as a secondary mechanism to ensure that T and B cells are not self-reactive once they leave primary lymphoid organs. Peripheral tolerance is distinct from central tolerance in that it occurs once developing immune cells exit primary lymphoid organs (the thymus and bone-marrow), prior to their export into the periphery.

Question e. General functions of Treg cells.

Answer:

1) Regulatory T cells (also called Tregs) are T cells which have a role in regulating or suppressing other cells in the immune system. Tregs control the immune response to self and foreign particles (antigens) and help prevent autoimmune disease. Tregs produced by a normal thymus are termed ‘natural’. Treg formed by differentiation of naïve T cells outside the thymus, i.e. the periphery, or in cell culture are called ‘adaptive’.

2) Natural Treg are characterised as expressing both the CD4 T cell co-receptor and CD25, which is a component of the IL-2 receptor. Treg are thus CD4+ CD25+. Expression of the nuclear transcription factor Forkhead box P3 (FoxP3) is the defining property which determines natural Treg development and function.

3) FoxP3 is crucial for maintaining suppression of the immune system. Naturally occurring mutations in the FOXP3 gene can result in self-reactive lymphocytes that cause a rare but severe disease IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked) in humans and scurfy in mice.

4) Tregs suppress activation, proliferation and cytokine production of CD4+ T cells and CD8+ T cells, and are thought to suppress B cells and dendritic cells. Tregs can produce soluble messengers which have a suppressive function, including TGF-beta, IL-10 and adenosine.