Morphology and Populations of Dendritic Cells

Dendritic cells (DCs) are present in lymphoid organs, in the epithelia of the skin and gastrointestinal and respiratory tracts, and in the interstitium of most parenchymal organs. These cells (introduced in Chapter 2) are identified morphologically by their membranous or spine-like projections (Fig. 6-4). All DCs are thought to arise from bone marrow precursors, and most are related in lineage to mononuclear phagocytes (see Fig. 2-2). Several subsets of DCs have been identified that may be distinguished by the expression of various cell surface markers and may play different roles in immune responses. The two main types are called conventional DCs and plasmacytoid DCs (Table 6-3).

FIGURE 6–4 Dendritic cells.

A, Light micrograph of cultured dendritic cells derived from bone marrow precursors. B, A scanning electron micrograph of a dendritic cell showing the extensive membrane projections. C, D, Dendritic cells in the skin, illustrated schematically (C) and in a section of the skin (D) stained with an antibody specific for Langerhans cells (which appear blue in this immunoenzyme stain). E, F, Dendritic cells in a lymph node, illustrated schematically (E) and in a section of a mouse lymph node (F) stained with fluorescently labeled antibodies against B cells in follicles (green) and dendritic cells in the T cell zone (red).

(A, B, and D courtesy of Dr. Y-J Liu, M.D. Anderson Cancer Center, Houston, Texas; F courtesy of Drs. Kathryn Pape and Jennifer Walter, University of Minnesota School of Medicine, Minneapolis.)

TABLE 6–3 The Major Subpopulations of Dendritic Cells

Other subsets of dendritic cells have been described on the basis of the expression of various surface markers (such as CD4, CD8, and CD11b) or migration from tissue sites (Langerhans-type dendritic cells from epithelia and interstitial dendritic cells from tissues). Note that all DCs express class II MHC molecules. Some authorities also refer to monocyte-derived dendritic cells, which can be generated from blood monocytes cultured with various cytokines and may develop in vivo during inflammatory reactions.

DCs that migrate from tissue sites to lymph nodes can also be characterized as immature or mature. Many DCs normally residing in lymphoid organs and in nonlymphoid tissues, including epithelia, in the absence of infection or inflammation, appear to be in an immature state, that is, able to capture antigens but unable to activate T cells. These DCs may function to present self antigens to self-reactive T cells and thereby cause inactivation or death of the T cells or generate regulatory T cells. These mechanisms are important for maintaining self-tolerance and preventing autoimmunity (see Chapter 14). As discussed below, DCs that have encountered microbes undergo maturation and function to present the antigens to T cells and to activate the T cells.

Antigen Capture and Transport by Dendritic Cells

DCs that are resident in epithelia and tissues capture protein antigens and transport the antigens to draining lymph nodes (Fig. 6-5). Resting (immature) DCs express membrane receptors, such as C-type lectins, that bind microbes. DCs use these receptors to capture and endocytose microbes and their antigens and then process the ingested proteins into peptides capable of binding to MHC molecules. Apart from receptor-mediated endocytosis and phagocytosis, DCs can ingest antigens by micropinocytosis and macropinocytosis, processes that do not involve specific recognition receptors but capture whatever might be in the fluid phase in the vicinity of the DCs. At the same time, an innate immune response develops during which microbial products are recognized by Toll-like receptors and other microbial sensors in the DCs and other cells. The DCs are activated by these signals and by cytokines, such as tumor necrosis factor (TNF), produced in response to the microbes. The activated DCs (also called mature DCs) lose their adhesiveness for epithelia or tissues and migrate into lymph nodes. The DCs also begin to express a chemokine receptor called CCR7 that is specific for two chemokines, CCL19 and CCL21, that are produced in the T cell zones of lymph nodes. These chemokines attract the DCs bearing microbial antigens into the T cell zones of the regional lymph nodes. Naive T cells also express CCR7, and this is why naive T cells migrate to the same regions of lymph nodes where antigen-bearing DCs are concentrated (see Chapter 3). The colocalization of antigen-bearing DCs and naive T cells maximizes the chance of T cells with receptors for the antigen finding that antigen. Maturation also converts the DCs from cells whose function is to capture antigen into cells that are able to present antigens to naive T cells and to activate the lymphocytes. Mature DCs express high levels of MHC molecules with bound peptides as well as costimulators required for T cell activation. Thus, by the time these cells become resident in lymph nodes, they have developed into potent APCs with the ability to activate T lymphocytes. Naive T cells that recirculate through lymph nodes encounter these APCs, and the T cells that are specific for the displayed peptide-MHC complexes are activated. This is the initial step in the induction of T cell responses to protein antigens.

FIGURE 6–5 Role of dendritic cells in antigen capture and presentation.

Immature dendritic cells in the skin (Langerhans cells) or dermis (dermal DCs) capture antigens that enter through the epidermis and transport the antigens to regional lymph nodes. During this migration, the dendritic cells mature and become efficient APCs. The table summarizes some of the changes during dendritic cell maturation that are important in the functions of these cells.

Antigens may also be transported to lymphoid organs in soluble form. Resident DCs in the lymph nodes and spleen may capture lymph- and blood-borne antigens, respectively, and also be driven to mature by microbial products. When lymph enters a lymph node through an afferent lymphatic vessel, it drains into the subcapsular sinus, and some of the lymph enters fibroblast reticular cell conduits that originate from the sinus and traverse the cortex (see Chapter 2). Once in the conduits, low-molecular-weight antigens can be extracted by DCs whose processes interdigitate between the reticular cells. Other antigens in the subcapsular sinus are taken up by macrophages and DCs, which carry the antigens into the cortex. B cells in the node may also recognize and internalize soluble antigens. DCs, macrophages, and B cells that have taken up protein antigens can then process and present these antigens to naive T cells and to effector T cells that have been generated by previous antigen stimulation.

The collection and concentration of foreign antigens in lymph nodes are supplemented by two other anatomic adaptations that serve similar functions. First, the mucosal surfaces of the gastrointestinal and respiratory systems, in addition to being drained by lymphatic capillaries, contain specialized collections of secondary lymphoid tissue that can directly sample the luminal contents of these organs for the presence of antigenic material. The best characterized of these mucosal lymphoid organs are Peyer’s patches of the ileum and the pharyngeal tonsils (see Chapter 13). Second, the blood stream is monitored by APCs in the spleen for any antigens that reach the circulation. Such antigens may reach the blood either directly from the tissues or by way of the lymph from the thoracic duct.

Antigen-Presenting Function of Dendritic Cells

Many studies done in vitro and in vivo have established that the induction of primary T cell–dependent immune responses to protein antigens requires the presence of DCs to capture and to present the antigens to the T cells. This was first shown for CD4⁺ T cell responses but is now known to be true for CD8⁺ T cells as well.

Several properties of DCs make them the most efficient APCs for initiation of primary T cell responses.

DCs can ingest infected cells and present antigens from these cells to CD8⁺ T lymphocytes. DCs are the best APCs to induce the primary responses of CD8⁺ T cells, but this poses a special problem because the antigens these lymphocytes recognize may be produced in any cell type infected by a virus, not necessarily DCs. Some specialized DCs have the ability to ingest virus-infected cells or cellular fragments and present antigens from these cells to CD8⁺ T lymphocytes. This process is called cross-presentation, or cross-priming, and is described later in the chapter.

The Mouse MHC (H-2 Complex)

The MHC was discovered from studies of tissue transplantation, well before the structure and function of MHC molecules were elucidated. It was known that tissues, such as skin, exchanged between nonidentical animals are rejected, whereas the same grafts between identical twins are accepted. This result showed that inherited genes must be involved in the process of tissue rejection. In the 1940s, to analyze the genetic basis of graft rejection, George Snell and colleagues produced inbred mouse strains by repetitive mating of siblings. Inbred mice are homozygous at every genetic locus (i.e., they express only one allele of every gene, even the polymorphic genes), and every mouse of an inbred strain is genetically identical (syngeneic) to every other mouse of the same strain (i.e., they all express the same alleles). Different strains may express different alleles and are said to be allogeneic to one another. By breeding congenic strains of mice that rejected grafts from other strains but were identical for all other genes, these investigators showed that a single genetic region is primarily responsible for rapid rejection of tissue grafts, and this region was called the major histocompatibility locus (histo, tissue). The particular locus that was identified in mice by Snell’s group was linked to a gene on chromosome 17 encoding a blood group antigen called antigen II, and therefore this region was named histocompatibility-2, or simply H-2. Initially, this locus was thought to contain a single gene that controlled tissue compatibility. However, occasional recombination events occurred within the H-2 locus during interbreeding of different strains, indicating that it actually contained several different but closely linked genes, many of which were involved in graft rejection. The genetic region that controlled graft rejection and contained several linked genes was named the major histocompatibility complex. Although not known at the time of Snell’s experiments, transplant rejection is in large part a T cell–mediated process (see Chapter 16), and therefore it is not surprising that there is a relationship between MHC genes, which encode the peptide-binding MHC molecules that T cells recognize, and graft rejection.

The Human MHC (HLA)

The human MHC was discovered by searching for cell surface molecules in one individual that would be recognized as foreign by another individual. This task became feasible when Jean Dausset, Jan van Rood, and their colleagues discovered that individuals who had received multiple blood transfusions and patients who had received kidney transplants contained antibodies that recognized cells from the blood or kidney donors and multiparous women had circulating antibodies that recognized paternal cells. The proteins recognized by these antibodies were called human leukocyte antigens (HLA) (leukocyte because the antibodies were tested by binding to the leukocytes of other individuals, and antigens because the molecules were recognized by antibodies). Subsequent analyses have shown that as in mice, the inheritance of particular HLA alleles is a major determinant of graft acceptance or rejection (see Chapter 16). Biochemical studies gave the satisfying result that the mouse H-2 proteins and the HLA proteins had essentially identical structures. From these results came the conclusion that genes that determine the fate of grafted tissues are present in all mammalian species and are homologous to the H-2 genes first identified in mice; these are called MHC genes. Other polymorphic genes that contribute to graft rejection to a lesser degree are called minor histocompatibility genes; we will return to these in Chapter 16, when we discuss transplantation immunology.

Immune Response Genes

For almost 20 years after the MHC was discovered, its only documented role was in graft rejection. This was a puzzle to immunologists because transplantation is not a natural phenomenon, and there was no reason that a set of genes should be preserved through evolution if the only function of the genes was to control the rejection of foreign tissue grafts. In the 1960s and 1970s, it was discovered that MHC genes are of fundamental importance for all immune responses to protein antigens. Baruj Benacerraf, Hugh McDevitt, and their colleagues found that inbred strains of guinea pigs and mice differed in their ability to make antibodies against some simple synthetic polypeptides, and responsiveness was inherited as a dominant mendelian trait. The relevant genes were called immune response (Ir) genes, and they were all found to map to the MHC. We now know that Ir genes are, in fact, MHC genes that encode MHC molecules that differ in their ability to bind and display peptides derived from various protein antigens. Responder strains, which can mount immune responses to a particular polypeptide antigen, inherit MHC alleles whose products can bind peptides derived from these antigens, forming peptide-MHC complexes that can be recognized by helper T cells. These T cells then help B cells to produce antibodies. Nonresponder strains express MHC molecules that are not capable of binding peptides derived from the polypeptide antigen, and therefore these strains cannot generate helper T cells or antibodies specific for the antigen. It was also later found that many autoimmune diseases were associated with the inheritance of particular MHC alleles, firmly placing these genes at the center of the mechanisms that control immune responses. Such studies provided the impetus for more detailed analyses of MHC genes and proteins.

The Phenomenon of MHC Restriction

The formal proof that the MHC is involved in antigen recognition by T cells came from the experimental demonstration of MHC restriction by Rolf Zinkernagel and Peter Doherty. In their classic study, reported in 1974, these investigators examined the recognition of virus-infected cells by virus-specific CTLs in inbred mice. If a mouse is infected with a virus, CD8⁺ CTLs specific for the virus develop in the animal. These CTLs recognize and kill virus-infected cells only if the infected cells express alleles of MHC molecules that are expressed in the animal in which the CTLs were generated (Fig. 6-6). By use of MHC congenic strains of mice (mice that were identical at every genetic locus except the MHC), it was shown that the CTLs and the infected target cell must be derived from mice that share a class I MHC allele. Thus, the recognition of antigens by CD8⁺ CTLs is restricted by self class I MHC alleles. Subsequent experiments demonstrated that responses of CD4⁺ helper T lymphocytes to antigens are self class II MHC restricted.

FIGURE 6–6 Experimental demonstration of the phenomenon of MHC restriction of T lymphocytes.

Virus-specific cytotoxic T lymphocytes (CTLs) generated from virus-infected strain A mice kill only syngeneic (strain A) target cells infected with that virus. The CTLs do not kill uninfected strain A targets (which express self peptides but not viral peptides) or infected strain B targets (which express different MHC alleles than does strain A). By use of congenic mouse strains that differ only at class I MHC loci, it has been proved that recognition of antigen by CD8⁺ CTLs is self class I MHC restricted.

We continue our discussion of the MHC by describing the properties of the genes and then the proteins, and we conclude by describing how these proteins bind and display foreign antigens.

Human and Mouse MHC Loci

In humans, the MHC is located on the short arm of chromosome 6 and occupies a large segment of DNA, extending about 3500 kilobases (kb). (For comparison, a large human gene may extend up to 50 to 100 kb, and the size of the entire genome of the bacterium Escherichia coli is approximately 4500 kb.) In classical genetic terms, the MHC locus extends about 4 centimorgans, meaning that crossovers within the MHC occur with a frequency of about 4% at each meiosis. A molecular map of the human MHC is shown in Figure 6-8.

FIGURE 6–8 Map of the human MHC.

The genes located within the human MHC locus are illustrated. In addition to the class I and class II MHC genes, HLA-E, HLA-F, and HLA-G and the MIC genes encode class I–like molecules, many of which are recognized by NK cells; C4, C2, and factor B genes encode complement proteins; tapasin, DM, DO, TAP, and proteasome encode proteins involved in antigen processing; LTα, LTβ, and TNF encode cytokines. Many pseudogenes and genes whose roles in immune responses are not established are located in the HLA complex but are not shown to simplify the map.

The human class I HLA genes were first defined by serologic approaches (antibody binding). There are three class I MHC genes called HLA-A, HLA-B, and HLA-C, which encode three class I MHC molecules with the same names. Class II MHC genes were first identified by use of assays in which T cells from one individual would be activated by cells of another individual (called the mixed lymphocyte reaction; see Chapter 16).There are three class II HLA gene loci called HLA-DP, HLA-DQ, and HLA-DR. Each class II MHC molecule is composed of a heterodimer of α and β polypeptides, and the DP, DQ, and DR loci each contain separate genes designated A or B, encoding α and β chains, respectively. More recently, DNA sequencing methods have been used to more precisely define MHC genes and their differences among individuals. The nomenclature of the HLA locus takes into account the enormous polymorphism (variation among individuals) identified by serologic and molecular methods. Thus, based on modern molecular typing, individual alleles may be called HLA-A*0201, referring to the 01 subtype of HLA-A2, or HLA-DRB1*0401, referring to the 01 subtype of the DR4 allele in the B1 gene, and so on.

The mouse MHC, located on chromosome 17, occupies about 2000 kb of DNA, and the genes are organized in an order slightly different from the human MHC gene. One of the mouse class I genes (H-2K) is centromeric to the class II region, but the other class I genes are telomeric to the class II region. There are three mouse class I MHC genes called H-2K, H-2D, and H-2L, encoding three different class I MHC proteins, K, D, and L. These genes are homologous to the human HLA-A, B, and C genes. The MHC alleles of particular inbred strains of mice are designated by lowercase letters (e.g., a, b), named for the whole set of MHC genes of the mouse strain in which they were first identified. In the parlance of mouse geneticists, the allele of the H-2K gene in a strain with the k-type MHC is called K^k (pronounced K of k), whereas the allele of the H-2K gene in a strain with d-type MHC is called K^d (K of d). Similar terminology is used for H-2D and H-2L alleles. Mice have two class II MHC loci called I-A and I-E, which encode the I-A and I-E molecules, respectively. These are located in the A and E subregions of the Ir region of the MHC and were discovered to be the Ir genes discussed earlier. The mouse class II genes are homologous to human HLA-DP, DQ, and DR genes. The I-A allele found in the inbred mouse strain with the K^k and D^k alleles is called I-A^k (pronounced I A of k). Similar terminology is used for the I-E allele. As in humans, there are actually two different genes, designated A and B, in the I-A and I-E loci that encode the α and β chains of each class II MHC molecule.

The set of MHC alleles present on each chromosome is called an MHC haplotype. For instance, an HLA haplotype of an individual could be HLA-A2, HLA-B5, HLA-DR3, and so on. All heterozygous individuals, of course, have two HLA haplotypes. Inbred mice, being homozygous, have a single haplotype. Thus, the haplotype of an H-2^d mouse is H-2K^d I-A^d I-E^d D^d L^d.

Expression of MHC Molecules

Because MHC molecules are required to present antigens to T lymphocytes, the expression of these proteins in a cell determines whether foreign (e.g., microbial) antigens in that cell will be recognized by T cells. There are several important features of the expression of MHC molecules that contribute to their role in protecting individuals from diverse microbial infections.

Class I molecules are constitutively expressed on virtually all nucleated cells, whereas class II molecules are expressed only on dendritic cells, B lymphocytes, macrophages, and a few other cell types. This pattern of MHC expression is linked to the functions of class I–restricted and class II–restricted T cells. The effector function of class I–restricted CD8⁺ CTLs is to kill cells infected with intracellular microbes, such as viruses, as well as tumors that express tumor antigens. The expression of class I MHC molecules on nucleated cells serves provides a display system for viral and tumor antigens. In contrast, class II–restricted CD4⁺ helper T lymphocytes have a set of functions that require recognizing antigen presented by a more limited number of cell types. In particular, naive CD4⁺ T cells need to recognize antigens that are captured and presented by dendritic cells in lymphoid organs. Differentiated CD4⁺ helper T lymphocytes function mainly to activate (or help) macrophages to eliminate extracellular microbes that have been phagocytosed and to activate B lymphocytes to make antibodies that also eliminate extracellular microbes. Class II molecules are expressed mainly on these cell types and provide a system for display of peptides derived from extracellular microbes and proteins.

The expression of MHC molecules is increased by cytokines produced during both innate and adaptive immune responses (Fig. 6-9). On most cell types, the interferons IFN-α, IFN-β, and IFN-γ increase the level of expression of class I molecules. The interferons are cytokines produced during the early innate immune response to many viruses (see Chapter 4). Thus, innate immune responses to viruses increase the expression of the MHC molecules that display viral antigens to virus-specific T cells. This is one of the mechanisms by which innate immunity stimulates adaptive immune responses.

FIGURE 6–9 Enhancement of class II MHC expression by IFN-γ.

IFN-γ, produced by NK cells and other cell types during innate immune reactions to microbes or by T cells during adaptive immune reactions, stimulates class II MHC expression on APCs and thus enhances the activation of CD4⁺ T cells. IFN-γ and type I interferons have a similar effect on the expression of class I MHC molecules and the activation of CD8⁺ T cells.

The expression of class II molecules is also regulated by cytokines and other signals in different cells. IFN-γ is the principal cytokine involved in stimulating expression of class II molecules in APCs such as dendritic cells and macrophages (see Fig. 6-9). IFN-γ may be produced by NK cells during innate immune reactions and by antigen-activated T cells during adaptive immune reactions. The ability of IFN-γ to increase class II MHC expression earlier than APCs is an amplification mechanism in adaptive immunity. As mentioned earlier, the expression of class II molecules also increases in response to signals from Toll-like receptors responding to microbial components, thus promoting the display of microbial antigens. B lymphocytes constitutively express class II molecules and can increase expression in response to antigen recognition and cytokines produced by helper T cells, thus enhancing antigen presentation to helper cells (see Chapter 11). IFN-γ can also increase the expression of MHC molecules on vascular endothelial cells and other nonimmune cell types; the role of these cells in antigen presentation to T lymphocytes is unclear. Some cells, such as neurons, never appear to express class II molecules. Activated human but not mouse T cells express class II molecules after activation; however, no cytokine has been identified in this response, and its functional significance is unknown.

The rate of transcription is the major determinant of the level of MHC molecule synthesis and expression on the cell surface. Cytokines enhance MHC expression by stimulating the transcription of class I and class II genes in a wide variety of cell types. These effects are mediated by the binding of cytokine-activated transcription factors to DNA sequences in the promoter regions of MHC genes. Several transcription factors may be assembled and bind a protein called the class II transcription activator (CIITA), and the entire complex binds to the class II promoter and promotes efficient transcription. By keeping the complex of transcription factors together, CIITA functions as a master regulator of class II gene expression. CIITA is synthesized in response to IFN-γ, explaining how this cytokine increases expression of class II MHC molecules. Mutations in several of these transcription factors have been identified as the cause of human immunodeficiency diseases associated with defective expression of MHC molecules. The best studied of these disorders is the bare lymphocyte syndrome (see Chapter 20). Knockout mice lacking CIITA also show reduced or absent class II expression on dendritic cells and B lymphocytes and an inability of IFN-γ to induce class II on all cell types.

The expression of many of the proteins involved in antigen processing and presentation is coordinately regulated. For instance, IFN-γ increases the transcription not only of class I and class II genes but also of several genes whose products are required for class I MHC assembly and peptide display, such as genes encoding the TAP transporter and some of the subunits of proteasomes, discussed later in the chapter.

General Properties of MHC Molecules

All MHC molecules share certain structural characteristics that are critical for their role in peptide display and antigen recognition by T lymphocytes.

Class I MHC Molecules

Class I molecules consist of two noncovalently linked polypeptide chains, an MHC-encoded 44- to 47-kD α chain (or heavy chain) and a non–MHC-encoded 12-kD subunit called β₂-microglobulin (Fig. 6-10). Each α chain is oriented so that about three quarters of the complete polypeptide extends into the extracellular milieu, a short hydrophobic segment spans the cell membrane, and the carboxyl-terminal residues are located in the cytoplasm. The amino-terminal (N-terminal) α1 and α2 segments of the α chain, each approximately 90 residues long, interact to form a platform of an eight-stranded, antiparallel β-pleated sheet supporting two parallel strands of α helix. This forms the peptide-binding cleft of class I molecules. Its size is large enough (~25 Å × 10 Å × 11 Å) to bind peptides of 8 to 11 amino acids in a flexible, extended conformation. The ends of the class I peptide-binding cleft are closed so that larger peptides cannot be accommodated. Therefore, native globular proteins have to be “processed” to generate fragments that are small enough to bind to MHC molecules and to be recognized by T cells (described later). The polymorphic residues of class I molecules are confined to the α1 and α2 domains, where they contribute to variations among different class I alleles in peptide binding and T cell recognition (Fig. 6-11). The α3 segment of the α chain folds into an Ig domain whose amino acid sequence is conserved among all class I molecules. This segment contains the binding site for CD8. At the carboxyl-terminal end of the α3 segment is a stretch of approximately 25 hydrophobic amino acids that traverses the lipid bilayer of the plasma membrane. Immediately following this are approximately 30 residues located in the cytoplasm, which include a cluster of basic amino acids that interact with phospholipid head groups of the inner leaflet of the lipid bilayer and anchor the MHC molecule in the plasma membrane.

FIGURE 6–10 Structure of a class I MHC molecule.

The schematic diagram (left) illustrates the different regions of the MHC molecule (not drawn to scale). Class I molecules are composed of a polymorphic α chain noncovalently attached to the nonpolymorphic β₂-microglobulin (β₂m). The α chain is glycosylated; carbohydrate residues are not shown. The ribbon diagram (right) shows the structure of the extracellular portion of the HLA-B27 molecule with a bound peptide, resolved by x-ray crystallography.

(Courtesy of Dr. P. Bjorkman, California Institute of Technology, Pasadena.)

FIGURE 6–11 Polymorphic residues of MHC molecules.

The polymorphic residues of class I and class II MHC molecules are located in the peptide-binding clefts and the α helices around the clefts. The regions of greatest variability among different HLA alleles are indicated in red, of intermediate variability in green, and of the lowest variability in blue.

(Reproduced with permission from Margulies DH, K Natarajan, J Rossjohn, and J McCluskey. Major histocompatibility complex [MHC] molecules: structure, function, and genetics. In WE Paul [ed]: Fundamental Immunology, 6th ed. Lippincott Williams & Wilkins, Philadelphia, 2008.)

β₂-Microglobulin, the light chain of class I molecules, is encoded by a gene outside the MHC and is named for its electrophoretic mobility (β₂), size (micro), and solubility (globulin). β₂-Microglobulin interacts noncovalently with the α3 domain of the α chain. Like the α3 segment, β₂-microglobulin is structurally homologous to an Ig domain and is invariant among all class I molecules.

The fully assembled class I molecule is a heterotrimer consisting of an α chain, β₂-microglobulin, and a bound antigenic peptide, and stable expression of class I molecules on cell surfaces requires the presence of all three components of the heterotrimer. The reason for this is that the interaction of the α chain with β₂-microglobulin is stabilized by binding of peptide antigens to the cleft formed by the α1 and α2 segments, and conversely, the binding of peptide is strengthened by the interaction of β₂-microglobulin with the α chain. Because antigenic peptides are needed to stabilize the MHC molecules, only potentially useful peptide-loaded MHC molecules are expressed on cell surfaces.

Most individuals are heterozygous for MHC genes and therefore express six different class I molecules on every cell, containing α chains encoded by the two inherited alleles of HLA-A, HLA-B, and HLA-C genes.

Class II MHC Molecules

Class II MHC molecules are composed of two noncovalently associated polypeptide chains, a 32- to 34-kD α chain and a 29- to 32-kD β chain (Fig. 6-12). Unlike class I molecules, the genes encoding both chains of class II molecules are polymorphic and present in the MHC locus.

FIGURE 6–12 Structure of a class II MHC molecule.

The schematic diagram (left) illustrates the different regions of the MHC molecule (not drawn to scale). Class II molecules are composed of a polymorphic α chain noncovalently attached to a polymorphic β chain. Both chains are glycosylated; carbohydrate residues are not shown. The ribbon diagram (right) shows the structure of the extracellular portion of the HLA-DR1 molecule with a bound peptide, resolved by x-ray crystallography.

(Courtesy of Dr. P. Bjorkman, California Institute of Technology, Pasadena.)

The amino-terminal α1 and β1 segments of the class II chains interact to form the peptide-binding cleft, which is structurally similar to the cleft of class I molecules. Four strands of the floor of the cleft and one of the α-helical walls are formed by the α1 segment, and the other four strands of the floor and the second wall are formed by the β1 segment. The polymorphic residues are located in the α1 and β1 segments, in and around the peptide-binding cleft, as in class I molecules (see Fig. 6-11). In human class II molecules, most of the polymorphism is in the β chain. In class II molecules, the ends of the peptide-binding cleft are open, so that peptides of 30 residues or more can fit.

The α2 and β2 segments of class II molecules, like class I α3 and β₂-microglobulin, are folded into Ig domains and are nonpolymorphic, that is, they do not vary among alleles of a particular class II gene. The β2 segment of class II molecules contains the binding site for CD4, similar to the binding site for CD8 in the α3 segment of the class I heavy chain. In general, α chains of one class II MHC locus (e.g., DR) most often pair with β chains of the same locus and less commonly with β chains of other loci (e.g., DQ, DP). The carboxyl-terminal ends of the α2 and β2 segments continue into short connecting regions followed by approximately 25–amino acid stretches of hydrophobic transmembrane residues. In both chains, the transmembrane regions end with clusters of basic amino acid residues, followed by short, hydrophilic cytoplasmic tails.

The fully assembled class II molecule is a heterotrimer consisting of an α chain, a β chain, and a bound antigenic peptide, and stable expression of class II molecules on cell surfaces requires the presence of all three components of the heterotrimer. As in class I molecules, this ensures that the MHC molecules that end up on the cell surface are the molecules that are serving their normal function of peptide display.

Humans inherit, from each parent, one DPA1 and one DPB1 gene encoding, respectively, the α and β chains of an HLA-DP molecule; one DQA1 and one DQB1 gene; and one DRA1 gene, a DRB1 gene, and a separate duplicated DRB gene that may encode the alleles DRB3, 4, or 5. Thus, each heterozygous individual inherits six or eight class II MHC alleles, three or four from each parent (one set each of DP and DQ, and one or two of DR). Typically, there is not much recombination between genes of different loci (i.e., DRα with DQβ, and so on), and each haplotype tends to be inherited as a single unit. However, because some haplotypes contain extra DRB loci that produce β chains that assemble with DRα, and some DQα molecules encoded on one chromosome can associate with DQβ molecules produced from the other chromosome, the total number of expressed class II molecules may be considerably more than six.

Characteristics of Peptide-MHC Interactions

MHC molecules show a broad specificity for peptide binding, in contrast to the fine specificity of antigen recognition of the antigen receptors of lymphocytes. There are several important features of the interactions of MHC molecules and antigenic peptides.

Structural Basis of Peptide Binding to MHC Molecules

The binding of peptides to MHC molecules is a noncovalent interaction mediated by residues both in the peptides and in the clefts of the MHC molecules. As we shall see later, protein antigens are proteolytically cleaved in APCs to generate the peptides that will be bound and displayed by MHC molecules. These peptides bind to the clefts of MHC molecules in an extended conformation. Once bound, the peptides and their associated water molecules fill the clefts, making extensive contacts with the amino acid residues that form the β strands of the floor and the α helices of the walls of the cleft (Fig. 6-13). In the case of class I MHC molecules, association of a peptide with the MHC groove depends on the binding of the positively charged N terminus and the negatively charged C terminus of the peptide to the MHC molecule by electrostatic interactions. In most MHC molecules, the β strands in the floor of the cleft contain “pockets.” Many class I molecules have a hydrophobic pocket that recognizes one of the following hydrophobic amino acids—valine, isoleucine, leucine, or methionine—at the C-terminal end of the peptide. Some class I molecules have a predilection for a basic residue (lysine or arginine) at the C terminus. In addition, other amino acid residues of a peptide may contain side chains that fit into specific pockets and bind to complementary amino acids in the MHC molecule through electrostatic interactions (charge-based salt bridges), hydrogen bonding, or van der Waals interactions. Such residues of the peptide are called anchor residues because they contribute most of the favorable interactions of the binding (i.e., they anchor the peptide in the cleft of the MHC molecule). Each MHC-binding peptide usually contains only one or two anchor residues, and this presumably allows greater variability in the other residues of the peptide, which are the residues that are recognized by specific T cells. In the case of some peptides binding to MHC molecules, especially class II molecules, specific interactions of peptides with the α-helical sides of the MHC cleft also contribute to peptide binding by forming hydrogen bonds or charge interactions. Class II MHC molecules accommodate larger peptides than class I MHC molecules. These longer peptides extend at either end beyond the floor of the groove.

FIGURE 6–13 Peptide binding to MHC molecules.

A, These top views of the crystal structures of MHC molecules show how peptides lie in the peptide-binding clefts. The class I molecule shown is HLA-A2, and the class II molecule is HLA-DR1. The cleft of the class I molecule is closed, whereas that of the class II molecule is open. As a result, class II molecules accommodate longer peptides than do class I molecules.

(Reprinted with permission of Macmillan Publishers Ltd. from Bjorkman PJ, MA Saper, B Samraoui, WS Bennett, JL Strominger, and DC Wiley. Structure of the human class I histocompatibility antigen HLA-A2. Nature 329:506-512, 1987; and Brown J, TS Jardetzky, JC Gorga, LJ Stern, RG Urban, JL Strominger, and DC Wiley. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 364:33-39, 1993.)

B, The side view of a cutout of a peptide bound to a class II MHC molecule shows how anchor residues of the peptide hold it in the pockets in the cleft of the MHC molecule.

(From Scott CA, PA Peterson, L Teyton, and IA Wilson. Crystal structures of two I-A^d–peptide complexes reveal that high affinity can be achieved without large anchor residues. Immunity 8:319-329, 1998. Copyright 1998, with permission from Elsevier Science.)

Because many of the residues in and around the peptide-binding cleft of MHC molecules are polymorphic (i.e., they differ among various MHC alleles), different alleles favor the binding of different peptides. This is the structural basis for the function of MHC genes as “immune response genes”; only animals that express MHC alleles that can bind a particular peptide and display it to T cells can respond to that peptide.

The antigen receptors of T cells recognize both the antigenic peptide and the MHC molecules, with the peptide being responsible for the fine specificity of antigen recognition and the MHC residues accounting for the MHC restriction of the T cells. A portion of the bound peptide is exposed from the open top of the cleft of the MHC molecule, and the amino acid side chains of this portion of the peptide are recognized by the antigen receptors of specific T cells. The same T cell receptor also interacts with polymorphic residues of the α helices of the MHC molecule itself (see Fig. 6-1). Predictably, variations in either the peptide antigen or the peptide-binding cleft of the MHC molecule will alter presentation of that peptide or its recognition by T cells. In fact, one can enhance the immunogenicity of a peptide by incorporating into it a residue that strengthens its binding to commonly inherited MHC molecules in a population.

Because MHC molecules can bind only peptides but most antigens are large proteins, there must be ways by which these proteins are converted into peptides. The conversion is called antigen processing and is the focus of the remainder of the chapter.

Sources of Cytosolic Protein Antigens

Most cytosolic protein antigens are synthesized within cells, and some are phagocytosed and transported into the cytosol. Foreign antigens in the cytosol may be the products of viruses or other intracellular microbes that infect such cells. In tumor cells, various mutated or overexpressed genes may produce protein antigens that are recognized by class I–restricted CTLs (see Chapter 17). Peptides that are presented in association with class I molecules may also be derived from microbes and other particulate antigens that are internalized into phagosomes but escape into the cytosol. Some microbes are able to damage phagosome membranes and create pores through which the microbes and their antigens enter the cytosol. For instance, pathogenic strains of Listeria monocytogenes produce a protein, called listeriolysin, that enables bacteria to escape from vesicles into the cytosol. (This escape is a mechanism that the bacteria may have evolved to resist killing by the microbicidal mechanisms of phagocytes, most of which are concentrated in phagolysosomes.) Once the antigens of the phagocytosed microbes are in the cytosol, they are processed like other cytosolic antigens. In dendritic cells, some antigens that are ingested into vesicles enter the cytosolic class I pathway, in the process called cross-presentation that is described later. Other important sources of peptides in the cytosol are misfolded proteins in the ER that are translocated into the cytosol and degraded like other cytosolic proteins; this process is called ER-associated degradation.

Proteolytic Digestion of Cytosolic Proteins

The major mechanism for the generation of peptides from cytosolic protein antigens is proteolysis by the proteasome. Proteasomes are large multiprotein enzyme complexes with a broad range of proteolytic activity that are found in the cytoplasm and nuclei of most cells. The proteasome appears as a cylinder composed of a stacked array of two inner β rings and two outer α rings, each ring being composed of seven subunits, with a cap-like structure at either end of the cylinder. The proteins in the outer α rings are structural and lack proteolytic activity; in the inner β rings, three of the seven subunits (β1, β2, and β5) are the catalytic sites for proteolysis.

The proteasome performs a basic housekeeping function in cells by degrading many damaged or improperly folded proteins. Protein synthesis normally occurs at a rapid rate, about six to eight amino acid residues being incorporated into elongating chains every second. The process is error prone, and it is estimated that approximately 20% of newly synthesized proteins are misfolded. These defective ribosomal products as well as older effete proteins are targeted for proteasomal degradation by covalent linkage of several copies of a small polypeptide called ubiquitin. Ubiquitinated proteins, with chains of four or more ubiquitins, are recognized by the proteasomal cap and are then unfolded, the ubiquitin is removed, and the proteins are “threaded” through proteasomes, where they are degraded into peptides. The proteasome has broad substrate specificity and can generate a wide variety of peptides from cytosolic proteins (but usually does not degrade proteins completely into single amino acids). Interestingly, in cells treated with the cytokine IFN-γ, there is increased transcription and synthesis of three novel catalytic subunits of the proteasome known as β1i, β2i, and β5i, which replace the three catalytic subunits of the β ring of the proteasome. This results in a change in the substrate specificity of the proteasome so that the peptides produced usually contain carboxyl-terminal hydrophobic amino acids such as leucine, valine, isoleucine, and methionine or basic residues such as lysine or arginine. These kinds of C termini are typical of peptides that are transported into the class I pathway and bind to class I molecules. This is one mechanism by which IFN-γ enhances antigen presentation, another mechanism being increased expression of MHC molecules (see Fig. 6-9). Thus, proteasomes are excellent examples of organelles whose basic cellular function has been adapted for a specialized role in antigen presentation.

Some protein antigens apparently do not require ubiquitination or proteasomes to be presented by the class I MHC pathway and are presumably degraded by cytosolic proteases. In addition, the signal sequences of membrane and secreted proteins are usually cleaved by signal peptidase and degraded proteolytically soon after synthesis and translocation into the ER. This ER processing generates class I–binding peptides without a need for proteolysis in the cytosol.

Transport of Peptides from the Cytosol to the Endoplasmic Reticulum

Peptides generated in the cytosol are translocated by a specialized transporter into the ER, where newly synthesized class I MHC molecules are available to bind the peptides. Because antigenic peptides for the class I pathway are generated in the cytosol but class I MHC molecules are synthesized in the ER, a mechanism is needed to deliver cytosolic peptides into the ER. This transport is mediated by a dimeric protein called transporter associated with antigen processing (TAP), which is homologous to the ABC transporter family of proteins that mediate ATP-dependent transport of low-molecular-weight compounds across cellular membranes. The TAP protein is located in the ER membrane, where it mediates the active, ATP-dependent transport of peptides from the cytosol into the ER lumen. Although the TAP heterodimer has a broad range of specificities, it optimally transports peptides ranging from 8 to 16 amino acids in length and containing carboxyl termini that are basic (in humans) or hydrophobic (in humans and mice). As mentioned before, these are the characteristics of the peptides that are generated in the proteasome and are able to bind to class I MHC molecules.

On the luminal side of the ER membrane, the TAP protein associates with a protein called tapasin, which also has an affinity for newly synthesized empty class I MHC molecules. Tapasin thus brings the TAP transporter into a complex with the class I MHC molecules that are awaiting the arrival of peptides.

Assembly of Peptide–Class I MHC Complexes in the Endoplasmic Reticulum

Peptides translocated into the ER bind to class I MHC molecules that are associated with the TAP dimer through tapasin. The synthesis and assembly of class I molecules involve a multistep process in which peptide binding plays a key role. Class I α chains and β₂-microglobulin are synthesized in the ER. Appropriate folding of the nascent α chains is assisted by chaperone proteins, such as the membrane chaperone calnexin and the luminal chaperone calreticulin. Within the ER, the newly formed empty class I dimers remain linked to the TAP complex. Empty class I MHC molecules, tapasin, and TAP are part of a larger peptide-loading complex in the ER that also includes calnexin, calreticulin, and the oxidoreductase Erp57, all of which contribute to class I assembly and loading. Peptides that enter the ER through TAP and peptides produced in the ER, such as signal peptides, are often trimmed to the appropriate size for MHC binding by the ER-resident aminopeptidase ERAP. The peptide is then able to bind to the cleft of the adjacent class I molecule. Once class I MHC molecules are loaded with peptide, they no longer have an affinity for tapasin, so the peptide–class I complex is released from tapasin, and it is able to exit the ER and be transported to the cell surface. In the absence of bound peptide, many of the newly formed α chain–β₂-microglobulin dimers are unstable and cannot be transported efficiently from the ER to the Golgi. These misfolded empty class I MHC complexes are transported into the cytosol and are degraded in proteasomes.

Peptides transported into the ER preferentially bind to class I but not class II MHC molecules, for two reasons. First, newly synthesized class I molecules are attached to the luminal aspect of the TAP complex, and they capture peptides rapidly as the peptides are transported into the ER by the TAP. Second, as discussed later, in the ER, the peptide-binding clefts of newly synthesized class II molecules are blocked by the associated I_i.

Surface Expression of Peptide–Class I MHC Complexes

Class I MHC molecules with bound peptides are structurally stable and are expressed on the cell surface. Stable peptide–class I MHC complexes that were produced in the ER move through the Golgi complex and are transported to the cell surface by exocytic vesicles. Once expressed on the cell surface, the peptide–class I complexes may be recognized by peptide antigen–specific CD8⁺ T cells, with the CD8 coreceptor playing an essential role by binding to nonpolymorphic regions of the class I molecule. In later chapters, we will return to a discussion of the role of class I–restricted CTLs in protective immunity. Several viruses have evolved mechanisms that interfere with class I assembly and peptide loading, emphasizing the importance of this pathway for antiviral immunity (see Chapter 15).

Generation of Vesicular Proteins

Most class II–associated peptides are derived from protein antigens that are captured from the extracellular environment and internalized into endosomes by specialized APCs. The initial steps in the presentation of an extracellular protein antigen are the binding of the native antigen to an APC and the internalization of the antigen. Different APCs can bind protein antigens in several ways and with varying efficiencies and specificities. Dendritic cells and macrophages express a variety of surface receptors that recognize structures shared by many microbes (see Chapter 4). These APCs use the receptors to bind and internalize microbes efficiently. Macrophages also express receptors for the Fc portions of antibodies and receptors for the complement protein C3b, which bind antigens with attached antibodies or complement proteins and enhance their internalization. Another example of specific receptors on APCs is the surface immunoglobulin on B cells, which, because of its high affinity for antigens, can effectively mediate the internalization of proteins present at very low concentrations in the extracellular fluid (see Chapter 11).

After their internalization, protein antigens become localized in intracellular membrane-bound vesicles called endosomes. The endosomal pathway of intracellular protein traffic communicates with lysosomes, which are more dense membrane-bound enzyme-containing vesicles. A subset of class II MHC–rich late endosomes plays a special role in antigen processing and presentation by the class II pathway; this is described later. Particulate microbes are internalized into vesicles called phagosomes, which may fuse with lysosomes, producing vesicles called phagolysosomes or secondary lysosomes. Some microbes, such as mycobacteria and Leishmania, may survive and even replicate within phagosomes or endosomes, providing a persistent source of antigens in vesicular compartments.

Proteins other than those ingested from the extracellular milieu can also enter the class II MHC pathway. Some protein molecules destined for secretion may end up in the same vesicles as class II MHC molecules and may be processed instead of being secreted. Less often, cytoplasmic and membrane proteins may be processed and displayed by class II molecules. In some cases, this may result from the enzymatic digestion of cytoplasmic contents, referred to as autophagy. In this pathway, cytoplasmic proteins are trapped within membrane-bound vesicles called autophagosomes; these vesicles fuse with lysosomes, and the cytoplasmic proteins are proteolytically degraded. The peptides generated by this route may be delivered to the same class II–bearing vesicular compartment as are peptides derived from ingested antigens. Autophagy is primarily a mechanism for degrading cellular proteins and recycling their products as sources of nutrients during times of stress. It also participates in the destruction of intracellular microbes, which are enclosed in vesicles and delivered to lysosomes. It is therefore predictable that peptides generated by autophagy will be displayed for T cell recognition. Some peptides that associate with class II molecules are derived from membrane proteins, which may be recycled into the same endocytic pathway as are extracellular proteins. Thus, even viruses, which replicate in the cytoplasm of infected cells, may produce proteins that are degraded into peptides that enter the class II MHC pathway of antigen presentation. This may be a mechanism for the activation of viral antigen–specific CD4⁺ helper T cells.

Proteolytic Digestion of Proteins in Vesicles

Internalized proteins are degraded enzymatically in late endosomes and lysosomes to generate peptides that are able to bind to the peptide-binding clefts of class II MHC molecules. The degradation of protein antigens in vesicles is an active process mediated by proteases that have acidic pH optima. The most abundant proteases of late endosomes are cathepsins, which are thiol and aspartyl proteases with broad substrate specificities. Several cathepsins contribute to the generation of peptides for the class II pathway. Partially degraded or cleaved proteins bind to the open-ended clefts of class II MHC molecules and are then trimmed enzymatically to their final size. Immunoelectron microscopy and subcellular fractionation studies have defined a class II–rich subset of late endosomes that plays an important role in antigen presentation (Fig. 6-18). In macrophages and human B cells, it is called the MHC class II compartment, or MIIC. (In some mouse B cells, a similar organelle containing class II molecules has been identified and named the class II vesicle.) The MIIC has a characteristic multilamellar appearance by electron microscopy. Importantly, it contains all the components required for peptide–class II association, including the enzymes that degrade protein antigens, the class II molecules, and two molecules involved in peptide loading of class II molecules, the invariant chain and HLA-DM, whose functions are described later.

FIGURE 6–18 Morphology of class II MHC–rich endosomal vesicles.

A, Immunoelectron micrograph of a B lymphocyte that has internalized bovine serum albumin into early endosomes (labeled with 5-nm gold particles, arrow) and contains class II MHC molecules (labeled with 10-nm gold particles, arrowheads) in MIICs. The internalized albumin will reach the MIICs ultimately.

(From Kleijmeer MJ, S Morkowski, JM Griffith, AY Rudensky, and HJ Geuze. Major histocompatibility complex class II compartments in human and mouse B lymphoblasts represent conventional endocytic compartments. Reproduced from The Journal of Cell Biology 139:639-649, 1997, by copyright permission of The Rockefeller University Press.)

B, Immunoelectron micrograph of a B cell showing location of class II MHC molecules and DM in MIICs (stars) and invariant chain concentrated in the Golgi (G) complex. In this example, there is virtually no invariant chain detected in the MIIC, presumably because it has been cleaved to generate CLIP.

(Courtesy of Drs. H. J. Geuze and M. Kleijmeer, Department of Cell Biology, Utrecht University, The Netherlands.)

Biosynthesis and Transport of Class II MHC Molecules to Endosomes

Class II MHC molecules are synthesized in the ER and transported to endosomes with an associated protein, the invariant chain (I_i), which occupies the peptide-binding clefts of the newly synthesized class II molecules (Fig. 6-19). The α and β chains of class II MHC molecules are coordinately synthesized and associate with each other in the ER. Nascent class II dimers are structurally unstable, and their folding and assembly are aided by ER-resident chaperones, such as calnexin. A protein called the invariant chain (I_i) promotes folding and assembly of class II molecules and directs newly formed class II molecules to the late endosomes and lysosomes where internalized proteins have been proteolytically degraded into peptides. The I_i is a trimer composed of three 30-kD subunits, three of which bind one newly synthesized class II αβ heterodimer in a way that blocks the peptide-binding cleft and prevents it from accepting peptides. As a result, class II MHC molecules cannot bind and present peptides they encounter in the ER, leaving such peptides to associate with class I molecules (described before). The class II MHC molecules are transported in exocytic vesicles toward the cell surface. During this passage, the vesicles taking class II molecules out of the ER meet and fuse with the endocytic vesicles containing internalized and processed antigens. Thus, class II molecules encounter antigenic peptides that have been generated by proteolysis of endocytosed proteins, and the peptide-MHC association occurs in the vesicles.

FIGURE 6–19 The functions of class II MHC–associated invariant chain and HLA-DM.

Class II molecules with bound invariant chain, or CLIP, are transported into vesicles, where the I_i is degraded and the remaining CLIP is removed by the action of DM. Antigenic peptides generated in the vesicles are then able to bind to the class II molecules. Another class II–like protein, called HLA-DO, may regulate the DM-catalyzed removal of CLIP. CIIV, class II vesicle.

Association of Processed Peptides with Class II MHC Molecules in Vesicles

Within the endosomal vesicles, the I_i dissociates from class II MHC molecules by the combined action of proteolytic enzymes and the HLA-DM molecule, and antigenic peptides are then able to bind to the available peptide-binding clefts of the class II molecules (see Fig. 6-19). Because the I_i blocks access to the peptide-binding cleft of a class II MHC molecule, it must be removed before complexes of peptide and class II molecules can form. The same proteolytic enzymes, such as cathepsins, that generate peptides from internalized proteins also act on the I_i, degrading it and leaving only a 24–amino acid remnant called class II–associated invariant chain peptide (CLIP), which sits in the peptide-binding cleft in the same way that other peptides bind to class II MHC molecules. Next, CLIP has to be removed so that the cleft becomes accessible to antigenic peptides produced from extracellular proteins. This removal is accomplished by the action of a molecule called HLA-DM (or H-2M in the mouse), which is encoded within the MHC, has a structure similar to that of class II MHC molecules, and colocalizes with class II molecules in the MIIC endosomal compartment. HLA-DM molecules differ from class II MHC molecules in several respects; they are not polymorphic, and they are not expressed on the cell surface. HLA-DM acts as a peptide exchanger, facilitating the removal of CLIP and the addition of other peptides to class II MHC molecules.

Once CLIP is removed, peptides generated by proteolysis of internalized protein antigens are able to bind to class II MHC molecules. The HLA-DM molecule may accelerate the rate of peptide binding to class II molecules. Because the ends of the class II MHC peptide-binding cleft are open, large peptides may bind and are then “trimmed” by proteolytic enzymes to the appropriate size for T cell recognition. As a result, the peptides that are actually presented attached to cell surface class II MHC molecules are usually 10 to 30 amino acids long and typically have been generated by this trimming step.

Expression of Peptide–Class II MHC Complexes on the Cell Surface

Class II MHC molecules are stabilized by the bound peptides, and the stable peptide–class II complexes are delivered to the surface of the APC, where they are displayed for recognition by CD4⁺ T cells. The transport of class II MHC–peptide complexes to the cell surface is believed to occur by fusion of vesiculotubular extensions from the lysosome with the plasma membrane, resulting in delivery of the loaded class II MHC complexes to the cell surface. Once expressed on the APC surface, the peptide–class II complexes are recognized by peptide antigen–specific CD4⁺ T cells, with the CD4 coreceptor playing an essential role by binding to nonpolymorphic regions of the class II molecule. Interestingly, whereas peptide-loaded class II molecules traffic from the late endosomes and lysosomes to the cell surface, other molecules involved in antigen presentation, such as DM, stay in the vesicles and are not expressed in the plasma membrane. The mechanism of this selective traffic is unknown.

Nature of T Cell Responses

The presentation of cytosolic versus vesicular proteins by the class I or class II MHC pathways, respectively, determines which subsets of T cells will respond to antigens found in these two pools of proteins and is intimately linked to the functions of these T cells (Fig. 6-21). Endogenously synthesized antigens, such as viral and tumor proteins, are located in the cytoplasm and are recognized by class I–restricted CD8⁺ CTLs, which kill the cells producing the intracellular antigens. Conversely, extracellular antigens usually end up in endosomal vesicles and activate class II–restricted CD4⁺ T cells because vesicular proteins are processed into class II–binding peptides. CD4⁺ T cells function as helpers to stimulate B cells to produce antibodies and macrophages to enhance their phagocytic activity, both mechanisms that serve to eliminate extracellular antigens. Thus, antigens from microbes that reside in different cellular locations selectively stimulate the T cell responses that are most effective at eliminating that type of microbe. This is especially important because the antigen receptors of CTLs and helper T cells cannot distinguish between extracellular and intracellular microbes. By segregating peptides derived from these types of microbes, the MHC molecules guide CD4⁺ and CD8⁺ subsets of T cells to respond to the microbes that each subset can best combat.

FIGURE 6–21 Presentation of extracellular and cytosolic antigens to different subsets of T cells.

A, Cytosolic antigens are presented by nucleated cells to CD8⁺ CTLs, which kill (lyse) the antigen-expressing cells. B, Extracellular antigens are presented by macrophages or B lymphocytes to CD4⁺ helper T lymphocytes, which activate the macrophages or B cells and eliminate the extracellular antigens.

Immunogenicity of Protein Antigens

MHC molecules determine the immunogenicity of protein antigens in two related ways.

FIGURE 6–22 Immunodominance of peptides.

Protein antigens are processed to generate multiple peptides; immunodominant peptides are the ones that bind best to the available class I and class II MHC molecules. The illustration shows an extracellular antigen generating a class II–binding peptide, but this also applies to peptides of cytosolic antigens that are presented by class I MHC molecules.