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

Section II. Mechanisms of the Immune Response

Lecture 8. Costimulatory Molecules and Lymphokines

As discussed in the previous lecture, the activation of T AND B lymphocytes, which is crucial for The Development of the Immune Response, requires the interaction of the antigen-specific receptor with an antigen (soluble for B lymphocytes, and presented with an MHC molecule for T lymphocytes). The signal generated As a result of this interaction is transmitted to the Nucleus of the T or B lymphocyte, triggering the activation process. Signal propagation is facilitated by a coreceptor, which, firstly, enhances the avidity of the receptor-antigen interaction and, secondly, recruits Tyrosine kinase to the signaling complex. The intracellular signal branches, leading to the activation of several nuclear factors. The combined action of these factors can activate a specific Gene. Thus, Cell activation is triggered by the non-covalent interaction of the receptor with the Ligand, enhanced by additional coreceptor interactions. However, it turns out that a single signal passing through the receptor and coreceptor is not sufficient for full activation. The so-called costimulatory signal, which passes through specialized molecules on The Cell surface, is also necessary.

Based on the functional outcome of their interaction, costimulatory molecules are divided into:

- T lymphocyte activation molecules;

- B lymphocyte activation molecules;

- adhesion molecules.

Structurally, B7.1, B7.2, CD28, and CTLA-4 belong to the immunoglobulin superfamily (i.e., they are immunoglobulin-like molecules); CD40L and TNFa belong to the TNF (tumor necrosis factor) family, whose members can be either membrane-bound or soluble Proteins; TNF-R and CD40 belong to the type III cytokine receptor family, as will be discussed later.

In each interacting pair, except for adhesion molecules, one component is constitutively expressed on the cell (c), while the other is inducible (i). This means that via costimulatory molecules, cells can interact not at any given moment, but only upon reaching a specific state of readiness, i.e., in the presence of other activation signals. The expression of costimulatory molecules determines the maturity and readiness of a cell to engage in antigen-stimulated contact. The level of expression of costimulatory molecules is regulated by soluble factors known as cytokines.

Thus, the successful activation of lymphocytes, which is the key process of immune recognition, requires the presence of three activation signals:

1) a properly presented antigen for T cells, and a free antigen for B cells;

2) costimulatory molecules that determine cell maturity and responsiveness;

3) cytokines that stimulate the expression of costimulatory molecules.

Lymphocyte activation results in several distinct processes: the cells

begin to divide and differentiate, meaning they synthesize new products. This implies that various mechanisms must be engaged: the transition of the cell from a resting state (G_o phase) to the Cell Cycle progression (proliferation), the synthesis of new intracellular proteins altering cell function (differentiation), and the synthesis of export proteins such as Antibodies and cytokines. These mechanisms are triggered by various external signals, suggesting that the signals provided by the antigen, costimulatory molecules, and cytokines are functionally distinct.

The presence of all costimulatory signals in the absence of an antigen does not lead to cell activation and the initiation of an immune response. During accidental cell contacts, such as in the bloodstream, the avidity of their interaction is low, and they quickly part ways "having shaken hands." The antigen serves as the trigger for the entire system, significantly increasing interaction avidity and allowing the activation signal to pass. On the other hand, the system fires only if all its components are ready to respond upon the appearance of the antigen. If not (if any of the costimulatory signals are missing), several outcomes are possible:

1) activation does not occur, but the cells remain potentially capable of future activation;

2) cells enter a state of anergy and become incapable of further activity;

3) cells are eliminated as incompetent to perform their Functions, triggering the programmed cell death mechanism—apoptosis.

4) in the absence of a cytokine signal (at a certain level of costimulatory molecules), a partial activation decision may occur, for example, the cell secretes an activating cytokine but does not proliferate, or vice versa.

Therefore, the presence of costimulatory molecules also acts as a system reliability control, ensuring that the tasks of antigen recognition and potential destruction are performed only by experienced and qualified workers. Cells that fail to meet these requirements are excluded from further activity (anergy) or destroyed (apoptosis).

Interaction via costimulatory molecules increases the overall avidity of cell-cell interaction and serves as a source of additional activation signals. This leads to The formation of the so-called immune synapse (Fig. 17). This term, borrowed from neurobiology, emphasizes the presence of a localized, tight contact between two cells mediated by the interaction of multiple types of molecules, though it is not fully analogous to a neural synapse.

The Formation of the immune synapse fulfills another function whose importance has only recently become clear. This function is the close apposition of the presented antigen and the specific receptor. To ensure effective interaction between the T-cell receptor and the peptide-MHC complex, the interacting cells must approach each other to a distance of about 15 nm. However, both T Lymphocytes and antigen-presenting cells are densely covered by the glycocalyx—large molecules of heavily glycosylated, negatively charged proteins that prevent close cell approximation. Therefore, initial contacts between these cells occur via adhesion molecules LFA and ICAM, which can interact at a distance of about 40 nm. In the resting state, LFA is bound to the Cytoskeleton and cannot move within the membrane plane. It is released and can diffuse along the membrane under METABOLISM/18.html">The Influence of chemokines—a specific type of soluble factors (to be discussed in subsequent lectures). This enables the rearrangement of surface molecules on the interacting cells. During immune synapse formation, adhesion molecules remain at the periphery, while antigen-specific receptors, MHC molecules presenting the antigenic peptide, and

Costimulatory molecules become concentrated inside. CD28 and CTLA-4 molecules are also larger in size than the antigen-specific receptor, which is why they are believed to take the next step in bringing interacting cells closer together. Finally, if the T-cell receptor specifically interacts with the presented antigen, a whole cascade of activation events is triggered. Thus, the cell's first step toward activation occurs under the influence of chemokines, which essentially act as a fourth activation signal.

Interaction with the presented antigenic peptide is the natural pathway for immune cell activation. However, evolution first, and humans in experiments later, developed additional means of activation. One could say that the immune system possesses alternative, secondary pathways of activation that are utilized under certain specific conditions.

Nonspecific mitogens.

Nonspecific mitogens are substances of plant or bacterial origin that interact nonspecifically with Glycoproteins on the membranes of immune cells. These include plant Lectins (such as concanavalin A (Con A), phytohemagglutinin (PHA), and many others) and bacterial lipopolysaccharides (LPS). They are called mitogens because they induce the proliferation of T (Con A, PHA) or B (LPS) lymphocytes. Mitogens do not trigger a specific immune response, but they can serve as a source of additional signals, enhancing the overall activation state of lymphocytes and acting, to some extent, as substitutes for costimulatory and cytokine signals. It is believed that the response to LPS is one of the key factors in nonspecific defense against Bacteria.

Superantigens.

Superantigens include proteins from certain bacteria and Viruses, such as staphylococcal enterotoxins and the rabies virus nucleocapsid. These Antigens tightly bind MHC proteins On the surface of antigen-presenting cells and the T-cell receptor outside the binding sites for conventional antigens within these molecules (Fig. 13B). This means that all T lymphocytes carrying antigen-specific receptors of a specific family become activated. If the lymphocytes are mature and possess a sufficient number of costimulatory molecules, a very strong polyclonal response develops. Conversely, if superantigens bind resting T lymphocytes, the interaction leads to anergy in the absence of costimulatory molecules. Obviously, such a mechanism gives microorganisms an advantage in their confrontation with the immune system of higher animals.

It turned out that so-called endogenous superantigens, the information for which is stored in the host genome, operate on the very same principle. These are Mls and Mls-like antigens. Detailed study revealed that all of them are products of mouse mammary tumor retrovirus genes. Presumably, at some point, this virus integrated into the mouse genome and began encoding endogenous antigens. Its gene products bind in a superantigen-like manner to specific types of T-cell receptors and promote the elimination of the corresponding T cells during development (negative Selection). They are present in activated B cells, cytotoxic T lymphocytes, and thymic dendritic cells.

A signal somewhat resembling antigen binding is the binding of the corresponding receptor by specific antibodies, which typically leads to the clustering of these receptors. The consequence of such binding can be either full activation (for example, upon binding CD40 on B lymphocytes) or anergy, if the signal is interpreted as defective. For T cells, the cross-linking of the CD3 complex by antibodies serves as a proliferative signal. Antibodies are a convenient artificial tool that allows researchers to track how a cell responds to the binding of a particular surface receptor, thereby modeling, to some extent, the outcome of a receptor binding its specific ligand.

Cytokines and cytokine receptors.

Cytokines are a group of secreted proteins that cells use to communicate with one another. Cytokines influence the development and activation state of immune cells. Unlike Hormones, which affect target cells over considerable distances, most cytokines act locally via paracrine signaling—affecting cells in direct contact or even the secreting cell itself (autocrine action). Cytokines bind to specialized receptors, thereby triggering their Intracellular Signaling cascades. The half-life of cytokines in the bloodstream is measured in minutes, and their secretion is also a brief process, meaning cytokines act locally and transiently. As a rule, they are not stored in cells as precursors, but are synthesized de novo following gene activation (for example, after a signal passes through the T-cell receptor, as discussed in the previous lecture). In terms of their physicochemical properties, cytokines are glycoproteins with a Molecular Weight of 15–25 kDa. Each of them exhibits significant size heterogeneity due to varying degrees of carbohydrate attachment, post-translational modifications (N- and C-terminal Processing), and oligomerization.

Historically, the first cytokines discovered in the 1950s were interferons—antiviral substances produced by cells in response to a viral attack. The term lymphokines was proposed in 1969 to denote cell-mediated Immunity mediators of a non-antibody nature. Lymphokines were produced by activated lymphocytes, as opposed to monokines, which were produced by monocytes. When it became clear that there was no sharp distinction between them, the umbrella term cytokines was introduced.

It also became evident that the same cytokine can elicit different responses in various cells and regulate many other bodily functions beyond immune reactions. Among Blood Cells, the most active producers of cytokines are macrophages and activated T lymphocytes.

Until 1980, cytokines were studied simply as biological activities associated with specific cellular extracts. Over the past 20 years, the Amino acid sequences of more than 20 cytokines have been isolated, purified, and elucidated, and their genes have been cloned. Their receptors have also been extensively studied.

1. Cytokine nomenclature.

Initially, cytokines were named after their functions (biological effects): macrophage migration inhibitory factor (MIF), lymphocyte inhibitory factor (LIF), macrophage-activating factor (MAF), and so on. When it became clear that cytokines are pleiotropic, The Need for a universal Classification arose. They are now divided into four categories.

1) Hematopoietic factors—those that stimulate the growth and maturation of immature blood cells. These include colony-stimulating factors (CSF), interleukins 3 and 7, and Erythropoietin.

2) Regulators of innate immunity—those involved in the nonspecific defense of the Organism against bacterial and viral infections. These include interferons α and β, inflammatory cytokines such as interleukins 1 and 6 and tumor necrosis factor (TNF-α), as well as chemokines.

3) Cytokines regulating specific immune responses—those involved in the activation, growth, and differentiation of mature lymphocytes, such as interleukins 2 and 4.

4) Cytokines regulating inflammatory reactions that develop during a specific immune response. These include, for example, interferon γ, lymphotoxin, and interleukins 5 and 10.

Such categorization is somewhat arbitrary because, as already noted, a single cytokine can affect different cells, mediating various effects, and conversely, a single function may be regulated by more than one cytokine. Immune system cytokines form a cascade. An antigen initially stimulates the synthesis of «first-wave» cytokines—pro-inflammatory cytokines such as interleukins 1 and 6 and TNF-α—which then induce The Biosynthesis of «second-wave» cytokines, including interleukins 2, 3, 4, 5, etc., which in turn modulate the synthesis of early cytokines. This principle allows the Immune Response to be regulated and amplified, recruiting progressively more cells into the process. We will examine the functions of some of these that are relevant to immune cell activity.

2. Functions of cytokines.

Interferons

Interferons α and β are produced by virus-infected cells. They exert antiviral effects and also induce enhanced MHC class I expression on target cells (for better antigen presentation), B lymphocyte proliferation, and enhanced natural killer cell activity.

Interferon γ is produced by T lymphocytes. It also exhibits antiviral activity and activates macrophages, T lymphocytes, B lymphocytes, and natural killer cells, induces MHC class I and II expression, and enhances antibody secretion.

Interleukins (IL)

The term "interleukins" refers to soluble factors that mediate communication between leukocytes. Historically, it was introduced in an attempt to unify the designations of many known cytokines. The conference where this term was adopted took place in the Swiss town of Interlaken ("situated between lakes"), making the term doubly symbolic.

More than a dozen and a half interleukins are currently known. The main ones are described below.

IL-1: produced by activated macrophages and B lymphocytes. It induces T cell proliferation, pre-B Cell Differentiation, and B cell proliferation and antibody secretion. It also stimulates fibroblast and endothelial cell proliferation, TNF secretion by endothelial cells, and enhanced production of acute-phase proteins. It is considered the major inflammatory cytokine and also plays a role in wound healing and bone calcium metabolism.

IL-2: produced by T lymphocytes. It is considered the main regulatory cytokine. In fact, along with γ-interferon, it acts as an activator of the cytotoxic branch of the immune response. It induces the proliferation and enhanced cytotoxic activity of activated T lymphocytes, upregulation of the IL-2 receptor, production of other cytokines by T cells, and B lymphocyte proliferation.

IL-3: produced by activated T lymphocytes, mast cells, and basophils. It stimulates the growth and differentiation of stem cells, promotes the release of basophil mediators, and enhances the phagocytosis and functions of eosinophils.

IL-4: produced by stimulated T lymphocytes, Bone Marrow stromal cells, and mast cells. It induces the proliferation of activated B lymphocytes and immunoglobulin isotype switching from IgM to IgG1 and IgE. To some extent, IL-4 acts as an antagonist to IL-2, enhancing phagocytosis, antigen presentation, and The production of IL-1, IL-6, and TNFα (inflammatory cytokines) in monocytes.

IL-5: produced by T lymphocytes and mast cells. It controls the differentiation of B cells into antibody-secreting cells. It also induces a recombinase component that acts together with IL-4 in switching from Cμ to Cγ1. It stimulates the production of IgA and IgM by activated B lymphocytes, hematopoietic cell differentiation, and eosinophil differentiation.

IL-6: produced by T lymphocytes, monocytes, fibroblasts, and endothelial cells. It stimulates the growth and antibody production of B cells, enhances the synthesis of acute-phase proteins, the expression of IL-2 and its receptor, and the differentiation of CTLs. IL-6 receptors are also found on Nerve Cells, where this cytokine exerts its neurotrophic effect.

IL-7: produced by bone marrow and Thymus stromal cells. It is essential for the development of immune cells and induces the proliferation of early pro-B, pre-B, and T lymphocyte precursors.

IL-8: produced by monocytes, lymphocytes, as well as fibroblasts, Skin cells, and endothelial cells. It serves as a chemoattractant for neutrophils, basophils, and T lymphocytes.

IL-9: produced by stimulated T helper cells. It induces the proliferation of T lymphocytes and mast cells.

IL-10: produced by T lymphocytes and monocytes stimulated by lipopolysaccharide. It acts as an antagonist to γ-interferon and IL-2, suppresses the EFFECTOR FUNCTIONS OF macrophages, and decreases the surface expression of MHC class II antigens. It induces the proliferation of B cells, thymocytes, and mast cells, and the differentiation of B lymphocytes into IgG4-secreting cells.

IL-11: produced by bone marrow stromal cells and fibroblasts. It enhances antibody synthesis by B cells and stimulates blood cell development.

IL-12: produced by activated B lymphocytes and monocytes. It is one of the most potent stimulators of natural killer cells and also affects the activation of cytotoxic T lymphocytes.

IL-13: produced by activated T lymphocytes. It inhibits the production of inflammatory cytokines by monocytes and stimulates B lymphocyte proliferation and antibody secretion.

IL-14: produced by T lymphocytes. It enhances the proliferation of activated B lymphocytes and suppresses immunoglobulin biosynthesis.

IL-15: produced by many cell types; it shares many properties with IL-12, inducing the proliferation of cytotoxic T lymphocytes and natural killer cells.

Cytokines are not exclusively elements of the immune system; they regulate many other processes, such as hematopoiesis, inflammation, tumor and general tissue growth, embryonic development, and neuroendocrine processes.

As noted previously, cytokines are characterized by two important features:

1) functional redundancy, which means that a given biological function is typically regulated by more than one cytokine;

2) pleiotropy (multifunctionality) of each cytokine.

These two features bear a certain analogy to the polyspecificity and heterogeneity of IMMUNOGLOBULINS. They imply that the reliability of the system as a whole is an integral value dependent on the cumulative action of multiple factors that can substitute for one another to a certain extent. This has been clearly demonstrated using IL-2 gene-knockout mice. Despite the key role of this cytokine in Immune cell development and activation, mice lacking the IL-2 gene proved to be completely normal, as other cytokines took over the function of IL-2. However, when mice deficient in the IL-2 receptor gene were generated, they exhibited severe immune system dysfunctions: T cells were rendered anergic, and B cells underwent rapid differentiation in the absence of signals restraining their maturation. These experiments demonstrated that the receptor itself plays a crucial role in regulating immune processes by transmitting the appropriate signals into the cell, whereas the ligand (cytokine) can be replaced.

3. Cytokine receptors.

Each cytokine has its own specific receptor, typically of high affinity. The number of receptors per cell ranges from 10² to 10⁴ and increases upon cell activation.

Based on their Structure, cytokine receptors are divided into four types. What unites them is that most are oligomeric—meaning they consist of multiple subunits—and the same subunits are frequently shared among several receptors. For instance, the IL-2 receptor consists of α, β, and γ subunits; the γ chain is also part of the IL-15 receptor, while the γ chain is shared by the receptors for IL-4, IL-7, and IL-9. Thus, the multifunctionality of cytokines can be partially explained by the presence of shared subunits in their respective receptors.

Type I receptors are receptors for IL-2, 3, 4, 5, 7, and 9. Structurally, they resemble Fibronectin, an Extracellular matrix adhesive glycoprotein. They are characterized by the presence of two extracellular domains: the first is stabilized by Disulfide Bonds, while the second features a juxtamembrane motif containing Tryptophan and Serine residues. IL-6 and IL-12 receptors possess an additional N-terminal domain. Despite the general similarity of their extracellular regions, these receptors feature completely distinct intracellular domains and, accordingly, mediate separate signaling pathways. Their sole shared function is mitogenic.

Type II receptors include those for y-interferon and IL-10. Their extracellular region also consists of two domains, each cross-linked by two disulfide bonds.

Type III receptors are Cysteine-rich receptors for TNFα and β, NGF (nerve growth factor), CD40L, and Fas (the apoptosis receptor).

Type IV receptors are proteins belonging to the immunoglobulin superfamily. They contain three supramembranous immunoglobulin-like domains. This type includes receptors for IL-1 (α, β), as well as membrane-bound forms of IL-6 and IL-7.

It should be noted that just as cytokines can exist in both soluble and membrane-bound forms, their receptors also have soluble counterparts. For example, IL-2 binds to a high-affinity receptor composed of two subunits, 75 and 55 kDa. Following T-cell activation via the IL-2 receptor, a fraction of the 55 kDa subunits is cleaved from the membrane, becoming a soluble IL-2 receptor. Little is known about the BIOLOGICAL FUNCTIONS OF soluble receptors; however, it is believed that they may bind excess circulating cytokine.

Cytokine receptors exhibit Homology with certain oncogenes. For instance, the c-kit proto-oncogene encodes the stem cell factor receptor. Furthermore, a single Arginine-to-serine amino acid substitution in the erythropoietin receptor leads to uncontrolled activation of the respective cell and, consequently, malignancy. This serves as another example that malignant transformation stems from impaired Regulation of Cell growth and proliferation.

The multifunctional nature of these receptors is explained by the fact that a single receptor can interact with Different types of tyrosine Kinases. Signal Specificity is controlled at multiple levels:

1) The structure of the intracellular domains of the receptor determines its potential kinase interaction partners;

2) the specific type of kinases and the signaling cascades they trigger depend on receptor density on the cell surface (low receptor density engages one pathway, whereas high density engages another).

Cytokine receptors are associated with the third family of tyrosine kinases—Janus kinases (Jak). Jak kinases phosphorylate cytoplasmic Stat proteins; Stat proteins then form homo- or heterodimers and migrate to The Nucleus, where they act as Transcription factors. Different Stat proteins lie downstream of different receptors, even though the latter may be associated with identical Jak kinases. Thus, much like the two-faced Roman god, Janus kinases are dual-natured: the same Jak phosphorylates different Stats depending on the receptor context. For example, IL-2 and IL-4 receptors signal through identical Jak 1 and Jak 3 kinases but different Stats; conversely, activation of IL-2 and prolactin receptors both result in Stat 5 phosphorylation, but this is mediated by different Jak kinases.

Summary.

Lymphocyte activation requires multiple activation signals, the combined effect of which brings cells into close proximity and enhances the avidity of their interaction. Chemokines induce alterations in adhesion molecule properties and the rearrangement of Membrane Proteins. Cytokines promote the surface expression of costimulatory molecules. These costimulatory molecules bridge the antigen-specific T-cell receptor and the MHC molecule on the antigen-presenting cell. Upon

complementarity between the presented antigenic peptide and the T-cell receptor, an immunological synapse is formed, facilitating signal Transduction into the interacting cells. The absence of even one activation signal can lead to cellular anergy or apoptosis.

Cytokines are crucial factors in the development and activation of immune cells. They act over a distance, yet typically exert their effects on cells brought into proximity by other interactions. Depending on their function, cytokines are classified into several categories, although the effects of different cytokines may overlap. They bind to specific receptors intracellularly linked to Janus tyrosine kinases and Stat transcription factors. Cytokines serve as a vital link connecting the immune system with other body systems, primarily the nervous and endocrine systems. For example, the action of the cytokine IL-1 on specific receptors located in Brain cells elicits symptoms characteristic of inflammation, such as fever and headache.