Fundamentals of Immunology - Lecture Course by M. V. Skok - Kyiv 2002

Chapter III. Immune Processes at the Organism Level

Lecture 12. Anatomy of the Immune System, Lymphocyte Migration

1. Structure of Lymphoid Organs.

Immune Cells are generated and undergo "education" in Primary lymphoid organs: the Bone Marrow and the Thymus. This is where their set of surface markers—which determine functional differentiation—and their repertoire of specificities are established. From here, lymphocytes migrate to Secondary Lymphoid Organs, where they reside to carry out their functions. Primary and secondary lymphoid organs are not merely accumulations of lymphoid cells; rather, they possess a specific, highly ordered architecture.

The thymus consists of several lobes, each divided into cortical and medullary zones (compartments). Their framework is formed by epithelial cells that synthesize thymic Hormones—growth factors essential for T lymphocyte differentiation and maturation. The cortex contains so-called "nurse cells," which are large epithelial cells with numerous processes expressing MHC Class II molecules. They form a network through which T lymphocytes migrate during their maturation. A layer of macrophages is located at the cortico-medullary junction. The medulla contains dendritic and epithelial cells expressing both MHC class I and MHC class II. T lymphocytes from the bone marrow enter the thymic cortex, where The First stage of positive Selection takes place. Cells that fail to pass these initial checkpoints undergo apoptosis and are cleared by the macrophage layer. Final maturation of T lymphocytes occurs in the medullary zone, from which they emerge as mature cells into the periphery.

Cortical lymphocytes are cortisol-sensitive, meaning that adrenal Steroid Hormones induce their apoptosis. Cortisol is one of the factors promoting apoptosis in cells eliminated during positive selection. Medullary T lymphocytes are resistant to corticosteroids, firstly due to the presence of the 20-α-hydroxysteroid dehydrogenase enzyme, and secondly due to upregulated expression of the bcl-2 Gene. This was demonstrated in experiments using bcl-2 and bax knockout Transgenic Mice. These mice developed normally for up to two weeks after birth, after which all T AND B lymphocytes disappeared (underwent apoptosis). During this period, mice begin to synthesize the glucocorticoids adrenaline and noradrenaline in response to stress, and these levels are sufficient to destroy all lymphocytes unless protected by the bcl-2 gene product.

Lymph Nodes are likewise divided into cortical, paracortical, and medullary zones and are enclosed by a capsule. Lymphocytes enter the lymph nodes through reticular Cell sinuses and move directionally from the cortex to the medulla, exiting on the other side. Within the lymph node, B AND T cells occupy separate compartments. B cells are predominantly located in the cortex, where they form primary and secondary follicles (the latter developing following antigenic stimulation). It is here that germinal centers form, serving as sites for somatic hypermutation, selection, and the generation of memory cells. T lymphocytes reside in the paracortical zone. Plasma cells, generated through B Cell Differentiation and maturation, exit the lymph nodes via the medullary sinuses.

The Spleen is also encapsulated and comprises red and white pulp. The red pulp consists of erythrocytes and macrophages. Interspersed within the red pulp are regions of white pulp, which is composed of lymphoid cells. Each area of white pulp features a marginal zone—housing B cells and follicles where germinal centers develop—and a periarteriolar lymphoid sheath containing T lymphocytes. Plasma cells are found in the marginal zone at the interface between the red and white pulp.

Mucosa-associated lymphoid tissue (MALT) comprises diffuse clusters of cells—such as lymphocytes and macrophages—located in the Lungs, intestines, Appendix, Peyer's patches, and Tonsils. The total mass of this tissue is substantial: for instance, a two-meter stretch of the intestine contains as many lymphocytes as the bone marrow, spleen, and lymph nodes combined. They are arranged in an orderly fashion, forming distinct B and T cell zones. Peyer's patches even contain specialized compartments for γδ-T lymphocytes. Mucosal lymphocytes predominantly produce IgA and IgE.

2. Lymphocyte Migration. Adhesion Molecules.

The developmental pathway of lymphocytes and their response to foreign Antigens are inextricably linked to their migration throughout the human or animal body. Lymphocyte production begins in the bone marrow, specifically on the inner surface of long bones, where common progenitors diverge into lymphoid and myeloid lineages. A fraction of the lymphoid precursors differentiates into immature IgM⁺ B lymphocytes. These leave the bone marrow via venous Blood, travel to The Heart, and enter the arterial Circulation. T lymphocyte precursors migrate to the thymus, where they differentiate into CD4⁺ or CD8⁺ T lymphocytes. They likewise exit the thymus via the Venous system, pass through the heart, and enter arterial blood. Through the arterial circulation, T and B cells reach secondary lymphoid organs and peripheral Tissues. Exchange between Blood Vessels and lymphoid tissues occurs at the level of lymph nodes. Lymphocytes enter lymph nodes from the blood via high endothelial venules (HEVs) and exit via the lymph through medullary sinuses. Lymphatic vessels eventually merge into the Thoracic duct (ductus thoracicus), which empties into the subclavian vein (vena subclavia). Thus, lymphocytes re-enter the venous blood, completing the circulatory loop.

En route from the bone marrow and thymus, lymphocytes colonize secondary lymphoid organs. Immature B lymphocytes enter the spleen via central arterioles and marginal sinuses, accumulating in the white pulp surrounding the arteriole. There, negative selection against autoantigens not present in the bone marrow takes place. B lymphocytes mature into IgM⁺IgD⁺ cells and form primary follicles. T lymphocytes similarly enter the spleen through marginal sinuses and accumulate in the periarteriolar lymphoid sheaths. In lymph nodes, B and T cells segregate into the cortical and paracortical zones, respectively. The majority of lymphocytes reside within secondary lymphoid organs prior to an Immune Response.

Antigen-presenting cell (APC) precursors migrate into tissues, where they differentiate into macrophages and dendritic cells. Consequently, prior to the onset of an immune response, lymphocytes and antigen-presenting cells are segregated: lymphocytes reside mainly in secondary lymphoid organs, whereas APCs are located in peripheral tissues. Upon antigen invasion, dendritic cells transport the antigen to the nearest lymphoid organ, where T and B lymphocyte activation and cognate interaction take place. Activated effector cells (cytotoxic T lymphocytes [CTLs], helper T cells [Th]) migrate to the site of inflammation or infection. Finally, the newly generated memory cells become distributed throughout the entire immune system.

Thus, all processes encompassing both the pre-immune development of lymphocytes and their Participation in the immune response rely on migration from one organ of the body to another. This cellular movement is far from random; it is guided by specific "homing signals" and "address codes" that dictate the precise organ a given cell must target. For instance, activated lymphocytes, if extracted from the body and re-introduced elsewhere, retain The ability to home back to the specific site where their activation occurred.

Cellular migration within the Organism is directed by adhesion molecules and chemokines.

Two MAIN TYPES OF adhesion molecules participate in lymphocyte migration.

1. Selectins are Glycoproteins whose N-terminal domain is homologous to Ca²⁺-dependent Lectins. They are expressed exclusively on leukocytes and vascular endothelial cells, making them specific to interactions between these particular cell types.

L-selectin is constitutively expressed on lymphocytes, monocytes, and dendritic cells, but is absent on memory cells. Its affinity increases following lymphocyte activation.

P-selectin is expressed on endothelial cells and platelets, rapidly appearing on The Cell surface via exocytosis from intracellular granules in response to TNF-α, histamine, Thrombin, and Complement components. E-selectin is likewise found on vascular endothelial cells, and its synthesis is stimulated by TNF-α and IL-1.

The ligands for selectins are CARBOHYDRATES linked to mucins—cell Membrane Proteins rich in Serine and Threonine. All of them contain sialic acid and fucose, and L-selectin ligands are additionally sulfated. The interaction between selectins and their ligands is characterized by low affinity and high reversibility.

2. Integrins are heterodimeric proteins composed of non-covalently linked α and β subunits featuring characteristic Structural motifs. At least 12 types of integrins are known to participate in lymphocyte adhesion.

The ligands for integrins include membrane proteins belonging to the immunoglobulin superfamily (e.g., ICAM [intercellular adhesion molecule], found on many cell types including lymphocytes; VCAM [vascular Cell Adhesion molecule], expressed on vascular endothelial cells; and MAdCAM [mucosal addressin cell adhesion molecule], found in mucosal tissues), as well as Extracellular matrix proteins (Collagen, Laminin, Fibronectin) and Components of the coagulation and complement systems. Integrin-Ligand interactions are generally of higher affinity.

Chemokines and their Receptors.

The phenomenon of chemotaxis is widely recognized in cell biology as the directed movement of cells along a concentration gradient of an attractant. For instance, the C5a complement component serves as a chemoattractant for monocytes, neutrophils, and eosinophils, while Leukotrienes act as attractants for monocytes and neutrophils. In 1991–1992, a distinct class of molecules termed chemokines was discovered. These are small Polypeptides comprising 70–80 amino acid residues, produced by various body tissues and exhibiting Specificity toward distinct lymphocyte subpopulations. Chemokines play a critical role in directing and regulating the efficiency of lymphocyte migration throughout the organism.

To date, more than 50 different chemokines are known. They are classified into four families based on The structure of the Cysteine-rich region at the N-terminus: C, CC, CXC, and CX3C. Depending on their physiological functions, chemokines are divided into homeostatic (or constitutive) and inflammatory (or inducible). Homeostatic chemokines are constantly produced in the Skin and mucous membranes, directing cell movement under normal conditions: the homing of secondary lymphoid organs during lymphocyte development, antigen transport, and immune surveillance by memory cells. Inflammatory chemokines start to be produced by cells in affected tissues under METABOLISM/18.html">The Influence of inflammatory cytokines (IL-1β, TNFα). They recruit effector T lymphocytes, as well as monocytes and granulocytes, to the site of inflammation. This Classification is not absolute, as some chemokines perform both homeostatic and inflammatory functions.

Lymphocytes can sense a chemokine gradient as shallow as 1% across their diameter and migrate toward the highest concentration.

Chemokine receptors are transmembrane proteins that span the membrane seven times, which is why they are referred to as serpentine (or G-protein-coupled) receptors. They are linked to a G protein that activates phospholipase Cβ2. Corresponding to the chemokine families, their receptors are also divided into the CXCR, CCR, and CX3CR families. Currently, 18 chemokine receptors are known. Different cell types (monocytes, dendritic cells, lymphocytes) and various lymphocyte subpopulations express receptors with different specificities. The interaction between constitutive chemokines and their receptors is highly specific (meaning one chemokine interacts with only a specific receptor), which determines the precise homing address for lymphocyte migration. For example, B-lymphocyte precursors are retained in the bone marrow under the Influence of the SDF-1 (CXC) chemokine, which is produced by the bone marrow and embryonic Liver. T-lymphocyte precursors HEAD toward the thymus directed by the TECK chemokine, produced by thymic dendritic cells. In contrast, the interaction of inducible chemokines with receptors is somewhat degenerate, meaning that each receptor can bind several chemokines, and a single chemokine can interact with several receptors. Such degeneracy facilitates the mass migration of effector cells into the focus of inflammation.

Cell migration in the body occurs through the participation and interplay of adhesion molecules and chemokines. This has been demonstrated using the example of diapedesis—the migration of cells through the blood vessel wall. This is how lymphocytes enter lymphoid organs and body tissues. Diapedesis takes place in so-called post-capillary venules, a part of the venous system that begins immediately after the arterial capillaries. The walls of venules consist of a single layer of endothelial cells that express adhesion molecules and are sensitive to inflammatory mediators.

A multi-step model of leukocyte movement has been proposed (Fig. 20). According to this model, a cell moving with the bloodstream does not normally contact the vessel wall. As a result of injury, stress, or inflammation, endothelial cells begin to express P-selectin. This happens very rapidly, within minutes, because P-selectin is stored in intracellular granules and exocytosed to the membrane. Later, newly synthesized E-selectin, integrin ligands, ICAM-1, and VCAM-1 appear. The primary contact with the vessel wall occurs through the interaction of P- and E-selectins on endothelial cells with their corresponding ligands on lymphocytes, as well as lymphocyte L-selectins with ligands on endothelial cells. This interaction is low-affinity and short-lived, causing the cell to "roll" along the vessel wall.

A chemokine produced, for example, by cells of a lymphoid organ or at an inflammatory site, directs the cell's movement in the right direction (along a concentration gradient). In the process, the lymphocyte becomes polarized and reorganizes its Cytoskeleton. The action of the chemokine also alters the adhesive properties of integrins on the lymphocyte surface: conformational changes occur, resulting in a 200-fold increase in binding affinity for their corresponding ligands on endothelial cells. Assisted by integrins, leukocytes arrest and firmly attach to the vessel wall. The exact mechanism of diapedesis remains not fully understood. It has been shown that the attachment of the cell to the vessel wall leads to the dissociation of adhesion molecules known as cadherins, which link endothelial cells together, thereby facilitating the "squeezing" of the lymphocyte through the endothelial cell layer. It is unknown how the lymphocyte crosses the vessel's basement membrane: whether it mechanically disrupts the collagen and laminin layer or enzymatically cleaves them.

Thus, The process of lymphocyte migration from the vessel into the tissue consists of the following stages:

- tethering to the vessel (selectins);

- rolling along the wall (selectins);

- activation — expression of additional receptors, integrin conformation change (chemokine);

- arrest and strengthening of the bond with the vessel (integrins);

- diapedesis.

The vascular endothelium in different organs and tissues features a distinct set of adhesion molecules. Accordingly, lymphocyte subpopulations and cells at various stages of activation express different adhesion molecules. Since there are quite a few varieties of selectins, chemoattractants, and integrins, a unique "postal code" — an address code — can be determined for each cell type and each migration pathway, where the first digit represents the selectin type, the second the chemoattractant type, and the third the integrin type.

It is believed that there are separate streams of lymphocytes: those circulating through the skin, gastrointestinal tract, and lungs, and those permanently residing in lymphoid tissues. However, circulating lymphocytes periodically pass through lymph nodes, the spleen, and mucosal lymphoid tissue.

Let us now examine how cell migration occurs during an immune response. Precursors of antigen-presenting cells migrate from the bone marrow into tissues, where they differentiate into macrophages and dendritic cells. Thus, prior to the immune response, lymphocytes and antigen-presenting cells are segregated: lymphocytes reside mostly in secondary lymphoid organs, whereas antigen-presenting cells reside in tissues. T and B lymphocytes are also segregated in specialized zones of lymphoid organs. For an Immune Response to develop, they must be brought together in one place. An antigen enters the body through external barriers — the skin and mucous membranes — where it first contacts dendritic cells. For example, upon skin injury, the antigen is engulfed by so-called Langerhans cells. Under normal conditions, Langerhans cells are tightly connected to skin keratinocytes via homophilic E-cadherin adhesion. Inflammatory cytokines accompanying skin damage, particularly TNFα, downregulate cadherin expression on Langerhans cells and alter their chemokine receptor profile, notably inducing the expression of the CCR7 receptor. Such cells can migrate to the nearest lymph node and enter it through high endothelial venules (HEVs). This is a specialized type of endothelium that expresses L-selectin ligands GlyCAM-1 and CD34, the integrin ligand ICAM-1, as well as the chemokine SLC, which binds to CCR7. Dendritic cells express MHC class II and also secrete the chemokines MDC and TARC, which attract T lymphocytes to the paracortical area. If a T-lymphocyte receptor matches the antigenic peptide, the dendritic cell expresses additional adhesion molecules, and the T lymphocyte becomes activated and proliferates. This process takes 2–4 days. In the cortical zone of the lymph node, the antigen is presented by another type of dendritic cell — follicular dendritic cells (FDCs). Unlike the dendritic Cells of the paracortical area, follicular dendritic cells do not process the antigen; instead, they present it in its intact form as immune complexes with Antibodies or complement. The antigen can persist on follicular dendritic cells for years (!) and serve as an activator for corresponding B lymphocytes. Interaction with native antigen stimulates the primary activation of B lymphocytes. Now, the pre-primed T and B lymphocytes must meet. Activated T lymphocytes migrate to the B zone (cortex) and activate B lymphocytes, which, in turn, migrate to primary follicles, proliferate, and form germinal centers. They are attracted by the BLC chemokine, produced by follicular dendritic cells, which interacts with the CXCR5 receptor on B lymphocytes. The chemokines MDC and TARC stimulate the interaction of activated Th cells with pre-activated B lymphocytes. Thus, migration not only brings together all participants of the immune response but also differentiates activated lymphocytes from naive ones. The antigen presented by follicular dendritic cells serves as a selection factor for B lymphocytes whose genes undergo somatic Mutations. As a result, the selected cells proliferate, undergo immunoglobulin class switching, and generate plasma cells and memory cells that migrate to the medullary cords of the lymph node and enter the circulation via the medullary sinuses. It should be noted that naive and memory lymphocytes enter lymph nodes via different routes: memory cells through tissue endothelium, whereas naive cells enter through high endothelial venules. Naive and memory cells differ in their expression of adhesion molecules, and memory cells circulating in different PARTS OF THE body also differ from one another.

The spleen lacks high endothelial venules, and lymphocytes migrate into it from the capillaries of the so-called marginal zone. From there, they move into the white pulp and segregate into B and T compartments. Marginal zone B lymphocytes and follicular B lymphocytes of the white pulp differ in their properties. The maturation of the immune response in the splenic follicles proceeds somewhat analogously to that in lymph nodes. The resulting plasma cells migrate to the red pulp and remain there.

Each organ of the body is characterized by its own type of endothelium with a unique set of adhesion molecules and chemokines. For instance, entry into Peyer's patches through high endothelium is permitted in the presence of L-selectin, α4β7, and α4β2 integrins on the lymphocyte, and MAdCAM-1 and ICAM-1 molecules on the endothelium. The address for the skin comprises a set of P- and E-selectins, the chemokine MCP-1, and the integrin ligands VCAM-1, ICAM-1, and ICAM-2; lymphocytes, in turn, must carry α4β1 and α4β2 integrins along with the specific selectin ligand CLA (Cutaneous Lymphocyte-Associated Antigen).

During lymphocyte development, adhesion molecules change. For example, embryonic T lymphocytes express α4β7 and αEβ7 integrins, whereas a week after birth they begin to express L-selectin. THE SPECTRUM OF adhesion molecules also changes following activation. Activated T lymphocytes upregulate the expression of CD44, CD2, LFA-1, ICAM-2, and α₁β₁, α₂β₁, α₃β₁, α₅β₁, α₆β₁ integrins. T lymphocytes destined to act in mucosal tissues lose L-selectin (which is specific for lymph node ligands) but retain the α4β7 integrin required for mucosal entry. This means that throughout life, the spectrum of a lymphocyte's "vital interests" shifts, which is reflected in the types of adhesion molecules that determine both the ability of lymphocytes to interact with each other and their migration pathways.

Activated effector lymphocytes and memory cells migrate to the site of antigen invasion. Corresponding chemokines are produced as a result of a non-specific response, namely inflammation. Inflammation also enhances lymphocyte migration into lymphoid organs: blood flow increases, and The entry of memory cells into lymph nodes is facilitated.

Antigens entering the body via various routes induce immune responses in different lymphoid organs:

- food antigens are recognized by lymphocytes of Peyer's patches and the appendix; activated lymphocytes migrate via mesenteric lymph nodes into the thoracic duct and blood;

- antigens entering through the respiratory tract (respiratory infections) are recognized by the lingual, palatine, and pharyngeal tonsils (tonsils and adenoids), submandibular lymph nodes, and lung lymphoid tissue;

- antigens entering through the skin are directed to the draining lymph nodes;

- the spleen is responsible for clearing generalized bloodborne infections.

Summary.

Lymphocytes produced in the bone marrow migrate to the thymus and secondary lymphoid organs, and also patrol the entire body by moving through Blood and Lymphatic vessels. Lymphocyte migration is ordered and driven by weak interactions between surface adhesion molecules. Several types of such molecules are known, and their combinations form distinct "address codes" for various organs and tissues. The type of adhesion molecules changes over the course of life and upon lymphocyte activation. There are distinct pools (streams) of lymphocytes that monitor different parts of the body and respond to antigens entering the body via various pathways.