• How do the rare lymphocytes specific for any microbial antigen find that microbe, especially considering that microbes may enter virtually anywhere in the body?

The immune system has developed a highly specialized system for capturing and displaying antigens to lymphocytes. In this course, we have previously discussed how protein antigens are captured, broken down, and displayed by major histocompatibility complex (MHC) molecules for recognition by T lymphocytes. Here we will focus on how antigens and naïve T cells are brought together.

Protein antigens of microbes that enter the body are captured by professional antigen-presenting cells (APCs) and concentrated in the peripheral lymphoid organs where immune responses are initiated. Microbes enter the body mainly through three portals — the skin (by contact), the gastro-intestinal tract (by ingestion), and the respiratory tract (by inhalation). (Some insect-borne microbes may be injected into the bloodstream as a result of insect bites.) All the interfaces between the body and the external environment are lined by continuous epithelia, whose principal function is to provide a physical barrier to infection. The epithelia contain a population of professional APCs that belong to the lineage of dendritic cells. The term "professional"APC refers to the ability of these cells to both display antigens for T cells and provide the additional signals needed to activate naïve T cells. In the skin, where they have been studied most thoroughly, the epidermal dendritic cells are called Langerhans cells. These epithelial dendritic cells are said to be "immature," because they are inefficient at stimulating T lymphocytes. Their function is to capture the antigens of microbes that enter the epithelium, by the processes of phagocytosis (for particulate antigens) and pinocytosis (for soluble antigens). Immature dendritic cells may express receptors that enable them to bind microbes. One such receptor recognizes terminal mannose residues on glycoproteins, a typical feature of microbial but not mammalian glycoproteins. When epithelial cells, macrophages and lymphocytes that reside in epithelia encounter microbes, these cells respond by producing cytokines, such as tumor necrosis factor (TNF) and interleukin-1 (IL-1). The production of these cytokines is part of the innate immune response to microbes. TNF and IL-1 act on the epithelial dendritic cells that have captured microbial antigens, and cause the dendritic cells to round up and lose their adhesiveness for the epithelium. Now the dendritic cells are ready to leave the epithelium with their cargo of antigen.

Immature dendritic cells also express surface receptors for a group of chemo-attracting cytokines (chemokines) that are normally produced in the T cell-richareas of lymph nodes. These chemokines stimulate the dendritic cells that have exited the epithelium to migrate into the lymph nodes draining that epithelium. During this process of migration, the dendritic cells mature into APCs capable of stimulating T lymphocytes. This maturation is reflected in increased synthesis and stable expression of MHC molecules, whose function is to display antigen to T cells, and of other molecules, called costimulators, that are required for full T cell responses. The net result of this sequence of events is that the protein antigens of microbes that enter through the common portals are transported to and concentrated in lymph nodes in the regions where they are most likely to encounter T lymphocytes. Naive T lymphocytes continuously recirculate through lymph nodes, and it is estimated that every naive T cell in the body may cycle through some lymph nodes at least once a day. Therefore, professional APCs bearing captured antigen and naive T cells poised to recognize antigens come together in lymph nodes. This process is very efficient; it is estimated that if microbial antigens are introduced at any site in the body, a T cell response to these antigens will begin in the lymph nodes draining that site within 12-18 hours.

If the microbe breaches the epithelium and enters connective tissues, the bloodstream, or parenchymal organs, it may be captured by dendritic cells that live in these tissues and in the spleen. Other APCs in these sites may also capture and display antigens. Perhaps the most important of these other APCs are macrophages, which are abundant in all tissues. B lymphocytes also ingest protein antigens and display them to helper T cells; this process is important for the development of humoral immune responses.

The sequence of events described above is best understood for the presentation of antigens of extracellular microbes to CD4⁺ T lymphocytes. But some microbes, such as viruses, rapidly infect host cells and can only be eradicated by CTLs destroying the infected cells. The immune system, and especially CD8⁺ CTLs, must be able to recognize and respond to the antigens of these intracellular microbes. A likely mechanism of recognition of intracellular antigens is that professional APCs ingest infected cells, and display the antigens present in the infected cells for recognition by CD8⁺ T lymphocytes (which then differentiate into CTLs). This process is called cross-presentation (or cross-priming), to indicate that one cell type, the professional APCs, can present the antigens of other cells, the infected cells, and prime (or activate) naïve T lymphocytes specific for these antigens. The professional APCs that ingest infected cells may also present microbial antigens to CD4⁺ helper T lymphocytes. Thus, both classes of T lymphocytes, CD4⁺ and CD8⁺ cells, specific for the same microbe are activated close to one another. This process may be important for the antigen-stimulated differentiation of naïve CD8⁺ T cells to effector CTLs, a process that often requires help from CD4⁺ cells.

Cell-mediated immunity (CMI) is the effector function of T lymphocytes, and it serves as the defense mechanism against microbes that survive within phagocytes or infect non-phagocytic cells. Historically, adaptive immunity has been divided into humoral immunity, which can be adoptively transferred from an immunized donor to a naive host by antibodies in the absence of cells, and cell-mediated immunity, which can be adoptively transferred only by viable T lymphocytes. The effector phase of humoral immunity is triggered by the recognition of antigen by secreted antibodies; therefore, humoral immunity neutralizes and eliminates extracellular microbes and toxins that are accessible to antibodies, but it is not effective against microbes that survive and replicate inside infected cells. In contrast, in cell-mediated immunity, the effector phase is initiated by the recognition of antigens by T cells. T lymphocytes recognize protein antigens of intracellular microbes that are displayed on the surfaces of infected cells as peptides bound to self major histocompatibility complex (MHC) molecules. This ensures that T cells recognize and respond only to cell-associated antigens, such as the antigens of intracellular microbes. Defects in CMI result in increased susceptibility to infections by viruses and intracellular bacteria. Cell-mediated immune reactions are also important for elimination of cells that express foreign MHC molecules, as in an allograft, or express cells that tumor-specific antigens, as in a malignant tumor. In this part of the lecture, we will discuss the effector mechanisms of cell-mediated immune reactions.

T cell-mediated macrophage activation

Elimination of microbes by activated macrophages

Role of T_H2 cells in cell-mediated immunity

There are two types of cell-mediated immune reactions designed to eliminate different types of intracellular microbes: CD4⁺ T cells activate phagocytes to destroy microbes residing in the vesicles of these phagocytes, and CD8⁺ T cells kill cells containing cytoplasmic microbes, thus eliminating the reservoir of infection. This separation of the effector functions of T lymphocytes is not absolute. Some CD4⁺ T cells are capable of killing infected macrophages, and CD8⁺ T cells activate macrophages to eliminate phagocytosed microbes. Nevertheless, phagocyte activation, largely the function of CD4⁺ T cells, and killing of infected cells, mediated by CD8⁺ T cells, are fundamentally different immune reactions, and we will describe them separately.

Microbial infections may occur anywhere in the body, and some infectious pathogens are able to infect and live within host cells. Pathogenic microbes that infect and survive inside host cells include many bacteria and some protozoa that are ingested by phagocytes but resist the killing mechanisms of these phagocytes, and viruses that infect phagocytic and non-phagocytic cells and live in the cytoplasm of these cells. Effector T cells whose function is to eradicate these microbes are generated in lymph nodes and spleen, where naïve T cells are stimulated by microbial antigens. The differentiated effector T cells than migrate to the site of infection. Phagocytes at these sites ingest the microbes into intracellular vesicles, and generate peptide fragments of microbial proteins that are displayed by class II MHC molecules for recognition by effector T cells of the CD4⁺ subset. Peptide antigens derived from microbes, such as viruses, living in the cytoplasm of infected cells are displayed by class I MHC molecules for recognition by CD8⁺ effector T cells. Antigen recognition by the effector T cells then activates these cells to perform their task of eliminating the infectious pathogens. Thus, in cell-mediated immunity, T cells recognize protein antigens at two stages — naive T cells recognize antigens in lymphoid tissues and respond by proliferating and by differentiating into effector cells, and effector T cells recognize the same antigens anywhere in the body and respond by eliminating these microbes.

We will next describe how differentiated effector T cells locate microbes in tissues, and then how CD4⁺ and CD8⁺ cells eliminate these microbes.

At the same time as effector T lymphocytes are being arrested on the endothelium, macrophages and endothelial cells respond to the infectious microbes by producing another set of cytokines, the chemokines. The principal function of chemokines is to attract and stimulate the motility of leukocytes. Chemokines are often displayed on endothelial cells bound to cell surface proteoglycans, thus providing a high local concentration near the site of infection. Chemokinesare also produced at the extravascular infection site by leukocytes that are reacting to the infectious microbe, and this creates a concentration gradient of chemokines towards the infection. The endothelial cell-associated chemokines act on loosely adherent T cells to increase the affinity of their integrins for endothelial ligands. The chemokines also act on firmly adherent T cells and stimulate the motility of these cells, and the concentration gradient draws the T cells through the vessel wall into the site of infection. Thus, circulating effector T lymphocytes migrate, or "home," to sites of infection and become concentrated at these sites.

The homing of effector T cells to a site of infection is independent of antigen recognition, but lymphocytes that recognize microbial antigens are preferentially retained at the site. Because the homing of effector T cells to sites of infection is dependent on adhesion molecules and chemokines, and not on antigen recognition, all effector T cells present in the blood that were generated in response to different microbial infections can enter the site of any infection. This non-selective migration presumably maximizes the ability of effector lymphocytes to search out the microbes they are designed to eliminate. However, the same lack of selectivity creates a problem — how are lymphocytes specific for a microbe focused on to that microbe for long enough to perform their function? The answer is that if an effector T lymphocyte that has left the circulation and entered a tissue specifically recognizes microbial antigen, the cell is again activated. One consequence of activation is an increase in the expression and binding affinity of VLA integrins on the T cells. Some of these integrins specifically bind to molecules present in the extracellular matrix, such as hyaluronic acid and fibronectin. Therefore, the antigen-stimulated lymphocytes adhere firmly to the tissue near the antigen, and the cells stay long enough to respond to the microbe and eradicate the infection. Lymphocytes that enter the tissue but do not recognize an antigen are not activated to adhere. They enter lymphatic vessels draining the tissue and return to the circulation, prepared to home to another site of infection in search of the microbial antigen for which they are specific.

The net result of this sequence of cell migration and retention is that effector T lymphocytes, which were produced in the peripheral lymphoid organs in response to an infection, are able to locate that infectious microbe at any site in the body. These effector lymphocytes are activated by the microbe and respond in ways that eliminate the microbe. In contrast to the activation of naive T cells, which requires antigen presentation and costimulation by professional APCs, differentiated effector cells are activated by antigen recognition, and appear to be less dependent on costimulation than are naïve cells. Because of this difference, the proliferation and differentiation of naive T cells are confined to lymphoid organs where professional APCs display antigens, but the functions of effector T cells may be directed at any host cell displaying microbial antigens, not just professional APCs.

CD4⁺ helper T lymphocytes and CD8⁺ CTLs eliminate infections by distinct mechanisms. Therefore, we will discuss the effector mechanisms of these lymphocyte classes individually, and conclude by describing how the two classes of lymphocytes may cooperate to get rid of intracellular microbes.

Cell-mediated immunity was discovered as a form of immunity to an intracellular bacterial infection that could be transferred from immune animals to naive animals by cells (now known to be T lymphocytes) but not by serum antibodies. It was known from the earliest studies that the specificity of cell-mediated immunity against different microbes was a function of the lymphocytes, but the elimination of the microbes was a function of activated macrophages. The roles of T lymphocytes and phagocytes in cell-mediated immunity are now well understood.

Essentially the same reaction may be elicited by injecting a microbial protein into the skin of an individual who has been immunized against the microbe by prior infection or vaccination. This reaction is called delayed-type hypersensitivity (DTH), because it occurs 24-48 hours after an immunized individual is challenged with a microbial protein (i.e. the reaction is delayed) and because it reflects an increased sensitivity to antigen challenge. The delay occurs because it takes 24-48 hours for circulating effector T lymphocytes to home to the site of antigen challenge and to respond to the antigen at this site. DTH reactions are manifested by infiltrates of T cells and monocytes into the tissues, edema and fibrin deposition caused by increased vascular permeability in response to cytokines produced by CD4⁺ T cells, and tissue damage induced by the products of macrophages activated by T cells. DTH reactions are often used to determine if individuals have been previously exposed to and have responded to an antigen. For instance, a DTH reaction to a mycobacterial antigen (called PPD, for purified protein derivative) is an indicator of a T cell response to the mycobacteria. This is the basis for the PPD skin test, which is frequently used to detect past or active mycobacterial infection.

In the following section we will describe how T lymphocytes activate macrophages and how the macrophages eliminate phagocytosed microbes.

T CELL-MEDIATED MACROPHAGE ACTIVATION

CD4⁺ T lymphocytes perform functions in addition to macrophage activation in cell-mediated immune reactions. Antigen-stimulated CD4⁺ T cells secrete cytokines such as TNF, which act on vascular endothelium to increase the expression of adhesion molecules and production of chemokines. As a result, more T cells and other leukocytes, including blood neutrophils and monocytes, are recruited into the site of infection. Thus, the T cell response is amplified, and additional phagocytes are called in to assist in eradicating the infection. This T cell-stimulated cellular infiltration, and an accompanying vascular reaction, are typical of inflammation. Inflammation is a component of T cell-mediated reactions, such as DTH, and is also seen in innate immune reactions to microbes. In addition to their role in eradicating phagocytosed microbes, CD4⁺ T cells help CD8⁺ T cells to differentiate into active CTLs, and help B lymphocytes to differentiate into antibody-producing cells.

ELIMINATION OF MICROBES BY ACTIVATED MACROPHAGES

Macrophage activation leads to the expression of enzymes that catalyze the production of microbicidal substances in phagosomes and phagolysosomes. There are several major microbicidal mechanisms of activated phagocytes. Microbiocidal substances produced in the lysosomes of macrophages are reactive oxygen intermediates, nitric oxide, and proteolytic enzymes. These mechanisms are activated in innate immunity when macrophages encounter microbes. As described above, effector T_H1 cells are potent activators of the same microbicidal mechanisms in cell-mediated immunity. These mechanisms are critical for host defense in two situations — when macrophages are not activated by the microbes themselves, i.e. when innate immunity is ineffective, and when pathogenic microbes have evolved to resist the defense mechanisms of innate immunity. In these situations, the additional macrophage activation by T cells changes the balance between microbes and host defense in favor of the macrophages, thus serving to eradicate intracellular infections. The substances that are toxic to microbes may injure normal tissues if they are released into the extracellular milieu, because they do not distinguish between microbes and host cells. This is the reason for tissue injury (a reflection of "hypersensitivity") in delayed-type hypersensitivity (DTH) reactions, which often accompany protective cell-mediated immunity. It is also the reason why prolonged macrophage activation in chronic cell-mediated immune reactions is associated with considerable injury to adjacent normal tissues. For instance, in mycobacterial infections, which are difficult to eradicate, much of the pathology is caused by a sustained T cell and macrophages response to the mycobacterial antigens. The chronic cell-mediated immune response in such persistent infections may lead to the formation of granulomas, which are collections of activated lymphocytes and macrophages often with fibrosis and tissue necrosis, around the microbe.

Activated macrophages perform several functions, in addition to killing microbes, that are important in cell-mediated immunity. Activated macrophages secrete cytokines, including TNF, IL-1, and chemokines, which stimulate the recruitment of neutrophils, monocytes, and effector T lymphocytes to the site of infection. As a result, cell-mediated immunity is usually accompanied by inflammation. Macrophages produce other cytokines, such as platelet-derived growth factor, that stimulate the growth and activities of fibroblasts and endothelial cells, helping to repair tissue after the infection is cleared. Macrophage activation also leads to the increased expression of class II MHC molecules and costimulators on these cells, thus enhancing their antigen presenting function, which promotes T cell activation and amplifies the cell-mediated immune reaction.

ROLE OF T_H2 CELLS IN CELL-MEDIATED IMMUNITY

The T_H2 subset of CD4⁺ T lymphocytes stimulates eosinophil-rich inflammation and also functions to limit the injurious consequences of macrophage activation. When differentiated T_H2 cells recognize antigens, the cells produce the cytokines IL-4 and IL-5 (and also IL-10, which is produced by many other cell populations). IL-4 stimulates the production of IgE antibody, and IL-5 activates eosinophils. This reaction is important for defense against helminthic infections, because eosinophils bind to IgE-coated helminths and the helminths are killed by the granule proteins of eosinophils. The cytokines produced by T_H2 cells, including IL-10 and IL-4, also inhibit macrophage activation. Because of this action, T_H2 cells may serve to terminate DTH reactions and thus limit the tissue injury that often accompanies T_H1 cell-mediated protective immunity.

The relative activation of T_H1 and T_H2 cells in response to an infectious microbe may determine the outcome of the infection. For instance, the protozoan parasite Leishmania major lives inside macrophages and its elimination requires the activation of the macrophages by L. major-specific T_H1 cells. Most inbred strains of mice make an effective T_H1 response to the parasite, and are thus able to eradicate the infection. In some inbred mouse strains the response to L. major is dominated by T_H2 cells, and these mice succumb to the infection. Mycobacterium leprae, the bacterium that causes leprosy, is a pathogen for humans that also lives inside macrophages and may be eliminated by cell-mediated immunity. Some individuals infected with M. leprae are unable to eradicate the infection and develop destructive lesions, called lepromatous leprosy. In contrast, other patients develop strong cell-mediated immunity with activated T cells and macrophages around the infection, and few surviving bacteria; this form of less destructive disease is called tuberculoid leprosy. Some studies have shown that the tuberculoid form is associated with the activation of M. leprae-specific T_H1 cells, whereas the destructive lepromatous form is associated with a defect in T_H1 cell activation and a dominant T_H2 response. The same principle, that the T cell cytokine response to an infectious pathogen is an important determinant of the outcome of the infection, may be true for many other infectious diseases.

As we mentioned earlier, activated macrophages are best at killing microbes that are confined to vesicles, and microbes that directly enter the cytoplasm (e.g. viruses) or escape from phagosomes into the cytoplasm (e.g. some phagocytosed bacteria) are relatively resistant to the microbicidal mechanisms of phagocytes. Eradication of such pathogens requires the second major effector mechanism of cell-mediated immunity, namely cytolytic T lymphocytes (CTLs).

CD8⁺ CTLs recognize class I MHC-associated peptides on infected cells and kill these cells, thus eliminating the reservoir of infection. The sources of class I-associated peptides are protein antigens synthesized in the cytoplasm, and protein antigens of phagocytosed microbes that are transported or escape from phagocytic vesicles into the cytoplasm. Differentiated CD8⁺ CTLs recognize class I MHC-peptide complexes on the surface of infected cells by their TCR and by the CD8 co-receptor. The adhesion of CTLs to infected cells is stabilized by integrins on the CTLs binding to their ligands on the infected cells. (These infected cells are also called "targets" of CTLs, because they are destined to be killed by the CTLs.) The antigen receptors and co-receptors of the CTL cluster at the site of contact with the target cell. The CTLs are activated by antigen recognition; at this stage in their lives, the CTLs do not require costimulation or T cell help for activation. Therefore, differentiated CTLs are able to kill any infected target, even though the development of effector CTLs from naive CD8⁺ T lymphocytes requires costimulation by professional APCs and, in many cases, cytokines produced by helper T cells. Antigen recognition by the CTLs results in the activation of signal transduction pathways that lead to the exocytosis of the contents of the CTL’s granules to the region of contact with the targets.

CTLs kill target cells mainly as a result of granule contents creating pores in target cell membranes and introducing into the target cells substances that induce DNA fragmentation and apoptosis. The pore-forming protein of CTL granules is called perforin. When perforin is secreted from CTLs, it inserts into the target cell membrane and is induced to polymerize by the high concentration of Ca⁺⁺ ions present in the extracellular environment. Polymerized perforin forms a pore in the target cell membrane. At the same time the CTLs secrete granule enzymes called granzymes, which enter target cells through the perforin pores. Granzymes cleave and thereby activate enzymes called caspases that are present in the cytoplasm of the target cells and the active caspases induce apoptosis. (Caspases are so named because they cleave proteins C-terminal of aspartic acid residues; their major function is to induce apoptosis.) Activated CTLs also express a membrane protein called Fas ligand, which binds to a death-inducing receptor, called Fas (CD95), on target cells. Engagement of Fas activates caspases and induces target cell apoptosis; this pathway of CTL killing does not require granule exocytosis and is probably a minor pathway. The net result of these effector mechanisms of CTLs is that the infected cells are killed. Cells that have undergone apoptosis are rapidly phagocytosed and eliminated. The mechanisms that induce fragmentation of target cell DNA, which is the hallmark of apoptosis, may also break down the DNA of microbes living inside the infected cells. Each CTL can kill a target cell, detach, and go on to kill additional targets.

Recognition of Infected Cells by NK Cells

The activating receptors used by NK cells to recognize infected cells in the absence of antibody are not known with certainty. Several potential candidates have been identified by in vitro studies, including CD2 and integrins (which are also present in T cells), and various NK cell-specific molecules. The killer inhibitory receptor family, which is described below, may contain some members that associate with signaling subunits that contain ITAMs (rather than the inhibitory motifs typical of most of the inhibitory receptors). This subset of receptors may recognize class I MHC molecules on target cells and mediate NK cell activation. It is also possible that merely the stable binding of NK cells to their targets, which may be mediated by various adhesion molecules such as integrins, may be sufficient to activate NK cells, especially if inhibitory receptors are not engaged (see below).

NK cells express inhibitory receptors that recognize class I MHC molecules, and therefore NK cells are inhibited by class I-expressing cells and activated by target cells lacking class I molecules. The physiologic importance of this unusual specificity of NK cells is that it prevents the killing of normal host cells by NK cells, because all nucleated cells normally express class I MHC molecules. Many viruses have evolved to inhibit the expression of class I molecules in infected cells, and thus to evade lysis by virus-specific CD8⁺ CTLs. NK cells are an adaptation that allows the host to eliminate cells infected by such viruses. Two types of inhibitory receptors have been identified in humans. Some killer inhibitory receptors (KIRs) are members of the Ig superfamily and contain in their cytoplasmic domains immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which are distinct from the ITAM motifs we have mentioned earlier. Clustering of these receptors leads to phosphorylation of tyrosine residues within the ITIMs and recruitment and activation of protein tyrosine phosphatases. These phosphatases remove phosphate groups from the tyrosine residues of various substrates, and thus antagonize the activating kinases that are recruited to the ITAMs of activating receptors and block the activation of the NK cells. A second class of inhibitory receptors on human NK cells consists of heterodimers containing an invariant protein, called CD94, and a variable lectin subunit, called NKG-2. Like KIRs, NKG-2 molecules contain ITIMs, and are believed to function by recruiting tyrosine phosphatases. A third type of lectin inhibitory receptor, called Ly49A, is expressed on rodent but not human NK cells. As mentioned earlier, a subset of KIRs associates with an ITAM-containing chain and functions to activate NK cells.

The ligands for the inhibitory receptors of NK cells are particular alleles of class I MHC molecules. In humans, many of the inhibitory receptors recognize class I alleles encoded by the HLA-B or C locus. Some recognize less polymorphic class I-like molecules, also called class Ib molecules, such as HLA-E and HLA-G. Different NK cells express inhibitory receptors that are specific for different class I MHC alleles. Target cells that express the appropriate MHC allele are able to signal the NK cells that express the corresponding KIR or inhibitory lectin, and thus prevent NK cell activation. When a virus-infected cell loses expression of class I MHC molecules, it is unable to inhibit some NK cells in the host. This allows the activating receptors to function unopposed, resulting in NK cell-mediated lysis of the infected cells.

Effector Functions of NK Cells

One approach for tilting the balance between the host and microbes in favor of protective immunity is to vaccinate individuals to enhance immune responses.