8536d_ch08_185-199 8/22/02 11: 49 AM Page 185 mac100 mac 100: 12B8_tm: 8536d: Goldsby et al./Immunology 5e chapter8 Antigen processing g and presentation ECOGNITION OF FOREIGN PROTEIN ANTIGENS BY a T cell requires that peptides derived from the antigen be displayed within the cleft of an MHO molecule on the membrane of a cell. The formation of these peptide-MHC complexes requires that a protein antigen be degraded into peptides by a sequence of events called anti- gen processing. The degraded peptides then associate with Antigen Processing for Presentation by Class I MHC MHC molecules within the cell interior, and the peptide MHC complexes are transported to the membrane, where they are displayed (antigen presentation) Self-MHC Restriction of T Cells Class I and class II MHC molecules associate with pep- tides that have been processed in different intracellular com- Role of Antigen-Presenting Cells partments. Class I MHC molecules bind peptides derived from endogenous antigens that have been processed within a Evidence for Two Processing and Presentation Pathways the cytoplasm of the cell(e.g, normal cellular proteins,tu mor proteins, or viral and bacterial proteins produced a Endogenous Antigens: The Cytosolic Pathway within infected cells). Class II MHC molecules bind peptides a Exogenous Antigens: The Endocytic Pathway derived from exogenous antigens that are internalized by phagocytosis or endocytosis and processed within the endo- Presentation of Nonpeptide Antigens cytic pathway. This chapter examines in more detail the mechanism of antigen processing and the means by which processed antigen and MHC molecules are combined. In ad dition, a third pathway for the presentation of nonpeptide The results of these experiments, outlined in Figure8-1 showed that strain-2 antigen-pulsed macrophages activated strain-2 and FI T cells but not strain-13 T cells. Similarly, strain-13 antigen-pulsed macrophages activated strain-13 Self-MHC Restriction of t cells and Fi T cells but not strain-2 T cells. Subsequently, congenic and recombinant congenic strains of mice, which differed Both CD4 and CD8 T cells can recognize antigen only when from each other only in selected regions of the H-2 complex, it is presented by a self-MHC molecule, an attribute called self- were used as the source of macrophages and T cells. These ex- MHC restriction. Beginning in the mid-1970s, experiments periments confirmed that the CD4* TH cell is activated and conducted by a number of researchers demonstrated self- proliferates only in the presence of antigen-pulsed MHC restriction in T-cell recognition. A. Rosenthal and E rophages that share class II MHC alleles. Thus, Shevach, for example, showed that antigen-specific prolifera- recognition by the CD4 TH cell is class II MHC restricted tion of TH cells occurred only in response to antigen presented In 1974 R. Zinkernagel and P. Doherty demonstrated the y macrophages of the same MHC haplotype as the T cells In self-MHC restriction of CD8 T cells. In their experiments, their experimental system, guinea pig macrophages from mice were immunized with lymphocytic choriomeningitis strain 2 were initially incubated with an antigen. After the (LCM) virus; several days later, the animals'spleen cells, antigen-pulsed" macrophages had processed the antigen and which included Tc cells specific for the virus, were isolated presented it on their surface, they were mixed with T cells from and incubated with LCM-infected target cells of the same or the same strain(strain 2), a different strain(strain 13), or different haplotype( Figure 8-2). They found that the Tc cells (2 X 13)FI animals, and the magnitude of T-cell proliferation killed only syngeneic virus-infected target cells. Later studies in response to the antigen-pulsed macrophages was measured. with congenic and recombinant congenic strains showed
chapter 8 The results of these experiments, outlined in Figure 8-1, showed that strain-2 antigen-pulsed macrophages activated strain-2 and F1 T cells but not strain-13 T cells. Similarly, strain-13 antigen-pulsed macrophages activated strain-13 and F1 T cells but not strain-2 T cells. Subsequently, congenic and recombinant congenic strains of mice, which differed from each other only in selected regions of the H-2 complex, were used as the source of macrophages and T cells. These experiments confirmed that the CD4 TH cell is activated and proliferates only in the presence of antigen-pulsed macrophages that share class II MHC alleles. Thus, antigen recognition by the CD4 TH cell is class II MHC restricted. In 1974 R. Zinkernagel and P. Doherty demonstrated the self-MHC restriction of CD8 T cells. In their experiments, mice were immunized with lymphocytic choriomeningitis (LCM) virus; several days later, the animals’ spleen cells, which included TC cells specific for the virus, were isolated and incubated with LCM-infected target cells of the same or different haplotype (Figure 8-2). They found that the TC cells killed only syngeneic virus-infected target cells. Later studies with congenic and recombinant congenic strains showed ■ Self-MHC Restriction of T Cells ■ Role of Antigen-Presenting Cells ■ Evidence for Two Processing and Presentation Pathways ■ Endogenous Antigens: The Cytosolic Pathway ■ Exogenous Antigens: The Endocytic Pathway ■ Presentation of Nonpeptide Antigens Antigen Processing and Presentation R a T cell requires that peptides derived from the antigen be displayed within the cleft of an MHC molecule on the membrane of a cell. The formation of these peptide-MHC complexes requires that a protein antigen be degraded into peptides by a sequence of events called antigen processing. The degraded peptides then associate with MHC molecules within the cell interior, and the peptideMHC complexes are transported to the membrane, where they are displayed (antigen presentation). Class I and class II MHC molecules associate with peptides that have been processed in different intracellular compartments. Class I MHC molecules bind peptides derived from endogenous antigens that have been processed within the cytoplasm of the cell (e.g., normal cellular proteins, tumor proteins, or viral and bacterial proteins produced within infected cells). Class II MHC molecules bind peptides derived from exogenous antigens that are internalized by phagocytosis or endocytosis and processed within the endocytic pathway. This chapter examines in more detail the mechanism of antigen processing and the means by which processed antigen and MHC molecules are combined. In addition, a third pathway for the presentation of nonpeptide antigens derived from bacterial pathogens is described. Self-MHC Restriction of T Cells Both CD4 and CD8 T cells can recognize antigen only when it is presented by a self-MHC molecule, an attribute called selfMHC restriction. Beginning in the mid-1970s, experiments conducted by a number of researchers demonstrated selfMHC restriction in T-cell recognition. A. Rosenthal and E. Shevach, for example, showed that antigen-specific proliferation of TH cells occurred only in response to antigen presented by macrophages of the same MHC haplotype as the T cells. In their experimental system, guinea pig macrophages from strain 2 were initially incubated with an antigen. After the “antigen-pulsed” macrophages had processed the antigen and presented it on their surface, they were mixed with T cells from the same strain (strain 2), a different strain (strain 13), or (2 13) F1 animals, and the magnitude of T-cell proliferation in response to the antigen-pulsed macrophages was measured. Antigen Processing for Presentation by Class I MHC Molecules 8536d_ch08_185-199 8/22/02 11:49 AM Page 185 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-:
8536d_ch08_185-199 8/2/02 10:08 AM Page 186 mac79 Mac 79: 45_Bwppldsby et al./ Immunology Se 186 PART 11 Generation of B-Cell and T-Cell Response y LCM virus Strain 2 or 13 Strain 2 or 13 orC2×13)F1 orC2×13)F1 Peritoneal exudate cells Lymph node cells (containing Tc cells) (retains Peritoneal macrophages H-2k target cells H-2 LCM-infected H-2b LCM-infected target cells arget cells Antigen-pulsed Antigen-primed T-cells ⊙⊙⊙ B!:8 51Cr release +51Cr release 51 Cr release (no lysis) lysis) (no lysis) FICURE8-2 Classic experiment of Zinkernagel and Doherty demonstrating that antigen recognition by Tc cells exhibits MHC re- strictionH-2 mice were primed with the lymphocytic choriomeni gitis(LCM)virus to induce cytotoxic T lymphocytes(CTLs) specific Measure t-cell for the virus Spleen cells from this LCM-primed mouse were then added to target cells of different H-2 haplotypes that were intracellu- larly labeled withCr(black dots)and either infected or not with the LCM virus CTL-mediated killing of the target cells, as measured by Antigen-primed Antigen-pulsed macrophages the release of Cr into the culture supernatant, occurred only if the Strain 2 Strain 13(2x 13)F, target cells were infected with LCM and had the same MHC haplo- ype as the CTLs. ADapted from P C. Doherty and R. M. Zinkemagel, Strain 13 1975,.Exp.Med.141:502 (2×13)F1 restricted. In 1996, Doherty and Zinkernagel were awarded FIGURE8-1Experimental demonstration of self-MHC restriction of the Nobel prize for their major contribution to the under TH cells. Peritoneal exudate cells from strain 2, strain 13, or(2 X 13)F1 standing of cell-mediated immunity. guinea pigs were incubated in plastic Petri dishes, allowing enrichment of macrophages, which are adherent cells. The peritoneal macro- phages were then incubated with antigen. These "antigen-pulse macrophages were incubated in vitro with T cells from strain 2, strain Role of Antigen-Presenting Cells 13, or(2X 13)F1 guinea pigs, and the degree of T-cell proliferation As early as 1959, immunologists were confronted with data was assessed. The results indicated that TH cells could proliferate only suggesting that T cells and B cells recognized antigen by dif- in response to antigen presented by macrophages that shared MHC al- ferent mechanisms. The dogma of the time, which persisted leles.(Adapted from A Rosenthal and E Shevach, 1974, J. Exp. Med. until the 1980s, was that cells of the immune system recog- 138: 1194, by copyright permission of the Rockefeller University Press. I nize the entire protein in its native conformation. However, experiments by P G H. Gell and B Benacerraf demonstrated that, while a primary antibody response and cell-mediated that the Tc cell and the virus-infected target cell must share response were induced by a protein in its native conforma class I molecules encoded by the K or D regions of the MHC. tion, a secondary antibody response(mediated by B cells) Thus, antigen recognition by CD8 Tc cells is class I MHc could be induced only by native antigen, whereas a secondary
restricted. In 1996, Doherty and Zinkernagel were awarded the Nobel prize for their major contribution to the understanding of cell-mediated immunity. Role of Antigen-Presenting Cells As early as 1959, immunologists were confronted with data suggesting that T cells and B cells recognized antigen by different mechanisms. The dogma of the time, which persisted until the 1980s, was that cells of the immune system recognize the entire protein in its native conformation. However, experiments by P. G. H. Gell and B. Benacerraf demonstrated that, while a primary antibody response and cell-mediated response were induced by a protein in its native conformation, a secondary antibody response (mediated by B cells) could be induced only by native antigen, whereas a secondary 186 PART II Generation of B-Cell and T-Cell Responses Antigen-pulsed macrophages Antigen-primed T cell Strain 2 Strain 13 (2 × 13)F1 Strain 2 Strain 13 (2 × 13)F1 + + − + − + + + + Strain 2 or 13 or (2 × 13)F1 Strain 2 or 13 or (2 × 13)F1 Antigen Peritoneal exudate cells Peritoneal macrophages Adherent cells Antigen Antigen-pulsed macrophages Measure T-cell proliferation Lymph node cells Antigen-primed T-cells Adherence column (retains macrophages) 7 days FIGURE 8-1 Experimental demonstration of self-MHC restriction of TH cells. Peritoneal exudate cells from strain 2, strain 13, or (2 13) F1 guinea pigs were incubated in plastic Petri dishes, allowing enrichment of macrophages, which are adherent cells. The peritoneal macrophages were then incubated with antigen. These “antigen-pulsed” macrophages were incubated in vitro with T cells from strain 2, strain 13, or (2 13) F1 guinea pigs, and the degree of T-cell proliferation was assessed. The results indicated that TH cells could proliferate only in response to antigen presented by macrophages that shared MHC alleles. [Adapted from A. Rosenthal and E. Shevach, 1974, J. Exp. Med. 138:1194, by copyright permission of the Rockefeller University Press.] that the TC cell and the virus-infected target cell must share class I molecules encoded by the K or D regions of the MHC. Thus, antigen recognition by CD8 TC cells is class I MHC Spleen cells (containing Tc cells) H–2k target cells H–2k LCM-infected target cells H–2b LCM-infected target cells –51Cr release (no lysis) –51Cr release (no lysis) +51Cr release (lysis) H–2k LCM virus 51Cr FIGURE 8-2 Classic experiment of Zinkernagel and Doherty demonstrating that antigen recognition by TC cells exhibits MHC restriction. H-2k mice were primed with the lymphocytic choriomeningitis (LCM) virus to induce cytotoxic T lymphocytes (CTLs) specific for the virus. Spleen cells from this LCM-primed mouse were then added to target cells of different H-2 haplotypes that were intracellularly labeled with 51Cr (black dots) and either infected or not with the LCM virus. CTL-mediated killing of the target cells, as measured by the release of 51Cr into the culture supernatant, occurred only if the target cells were infected with LCM and had the same MHC haplotype as the CTLs. [Adapted from P. C. Doherty and R. M. Zinkernagel, 1975, J. Exp. Med. 141:502.] 8536d_ch08_185-199 8/2/02 10:08 AM Page 186 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch08_185-199 8/2/02 10:08 AM Page 187 mac79 Mac 79: 45_Bwppldsby et al./ Immunology Se Antigen Processing and Presentation CHAPTER 8 187 cell-mediated response could be induced by either the native 8-3a, b). During that interval of 1-3 h, the antigen-presenting or the denatured antigen(see Table 3-5). These findings were cells had processed the antigen and had displayed it on the viewed as an interesting enigma, but implications for antigen membrane in a form able to activate T presentation were completely overlooked until the early Subsequent experiments by R. P. Shimonkevitz showed that internalization and processing could be bypassed if anti- gen-presenting cells were exposed to peptide digests of an Processing of Antigen Is Required antigen instead of the native antigen(Figure 8-3c). In these for Recognition by T Cells experiments, antigen-presenting cells were treated with glu taraldehyde(this chemical, like paraformaldehyde, fixes the The results obtained by K. Ziegler and E. R. Unanue were cell, making the membrane impermeable)and then incu among those that contradicted the prevailing dogma that bated with native ovalbumin or with ovalbumin that had ntigen recognition by B and T cells was basically similar. been subjected to partial enzymatic digestion. The digested These researchers observed that TH-cell activation by bacter- ovalbumin was able to interact with the glutaraldehyde-fixed ial protein antigens was prevented by treating the antigen- antigen-presenting cells, thereby activating ovalbumin presenting cells with paraformaldehyde prior to antigen specific TH cells, whereas the native ovalbumin failed to do posure. However, if the antigen-presenting cells were first so. These results suggest that antigen processing involves the allowed to ingest the antigen and were fixed with paraform- digestion of the protein into peptides that are recognized by ldehyde 1-3 h later, TH-cell activation still occurred( Figure the ovalbumin-specific TH cell EXPERIMENTAL CONDITIONS T-CELL ACTIVA THell APO APC Fixation TH cell APC APC FIGURE8-3Experimental demonstration that antigen process. before antigen exposure and incubated with peptide digests of the ing is necessary for TH-cell activation.(a)When antigen-presenting antigen(rather than native antigen), they also can activate TH cells cells(APCs)are fixed before exposure to antigen, they are unable TH-cell activation is determined by measuring a specific TH-cell to activate TH cells.(b) In contrast, APCs fixed at least 1 h after response(e. g, cytokine secretion antigen exposure can activate TH cells. (c) When APCs are fixed
cell-mediated response could be induced by either the native or the denatured antigen (see Table 3-5). These findings were viewed as an interesting enigma, but implications for antigen presentation were completely overlooked until the early 1980s. Processing of Antigen Is Required for Recognition by T Cells The results obtained by K. Ziegler and E. R. Unanue were among those that contradicted the prevailing dogma that antigen recognition by B and T cells was basically similar. These researchers observed that TH-cell activation by bacterial protein antigens was prevented by treating the antigenpresenting cells with paraformaldehyde prior to antigen exposure. However, if the antigen-presenting cells were first allowed to ingest the antigen and were fixed with paraformaldehyde 1–3 h later, TH-cell activation still occurred (Figure 8-3a,b). During that interval of 1–3 h, the antigen-presenting cells had processed the antigen and had displayed it on the membrane in a form able to activate T cells. Subsequent experiments by R. P. Shimonkevitz showed that internalization and processing could be bypassed if antigen-presenting cells were exposed to peptide digests of an antigen instead of the native antigen (Figure 8-3c). In these experiments, antigen-presenting cells were treated with glutaraldehyde (this chemical, like paraformaldehyde, fixes the cell, making the membrane impermeable) and then incubated with native ovalbumin or with ovalbumin that had been subjected to partial enzymatic digestion. The digested ovalbumin was able to interact with the glutaraldehyde-fixed antigen-presenting cells, thereby activating ovalbuminspecific TH cells, whereas the native ovalbumin failed to do so. These results suggest that antigen processing involves the digestion of the protein into peptides that are recognized by the ovalbumin-specific TH cells. Antigen Processing and Presentation CHAPTER 8 187 FIGURE 8-3 Experimental demonstration that antigen processing is necessary for TH-cell activation. (a) When antigen-presenting cells (APCs) are fixed before exposure to antigen, they are unable to activate TH cells. (b) In contrast, APCs fixed at least 1 h after antigen exposure can activate TH cells. (c) When APCs are fixed before antigen exposure and incubated with peptide digests of the antigen (rather than native antigen), they also can activate TH cells. TH-cell activation is determined by measuring a specific TH-cell response (e.g., cytokine secretion). T-CELL ACTIVATION EXPERIMENTAL CONDITIONS + Antigen peptides Fixation APC Fixation – APC APC Antigen 1h Antigen 1h APC APC TH cell APC + Fixation APC TH cell (a) (b) (c) 8536d_ch08_185-199 8/2/02 10:08 AM Page 187 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch08_185-199 8/22/02 11: 49 AM Page 188 mac100 mac 100: 128_tm: 8536d: Goldsby et al./ Immunology 5e- 188 PART I1 Generation of B-Cell and T-Cell Response TABLE8-1 Antigen-presenting cells Professional antigen-presenting cells Nonprofessional antigen-presenting cells Dendritic cells(several types) Fibroblasts(skin) Thymic epithelial cells Glial cells(brain) Thyroid epithelial cells Pancreatic beta cells Vascular endothelial cells At about the same time, A. Townsend and his colleagues Macrophages must be activated by phagocytosis of began to identify the proteins of influenza virus that were particulate antigens before they express class II mHC ecognized by Tc cells. Contrary to their expectations, they molecules or the co-stimulatory B7 membrane found that internal proteins of the virus, such as matrix molecule and nucleocapsid proteins, were often recognized by Tc. b cells constitutively express class II MHC molecules but more Moreover, Townsends work revealed that Tc cells recog must be activated before they express the co-stimulatory B7 molecule nized short linear peptide sequences of the influenza pro- tein. In fact, when noninfected target cells were incubated Several other cell types, classified as nonprofessional in vitro with synthetic peptides corresponding to se- antigen-presenting cells, can be induced to express class ll quences of internal influenza proteins, these cells could be MHC molecules or a co-stimulatory signal (Table 8-1) cognized by Tc cells and subsequently lysed just as well Many of these cells function in antigen presentation only as target cells that had been infected with live influenza for short periods of time during a sustained inflammator virus. These findings along with those presented in Figure response 8-3 suggest that antigen processing is a metabolic process Because nearly all nucleated cells express class I MHC that digests proteins into peptides, which can then be dis- molecules, virtually any nucleated cell is able to function played on the cell membrane together with a class I or class target cell presenting endogenous antigens to Tc cells. Most II MHC molecule ften, target cells are cells that have been infected by a virus or some other intracellular microorganism. However, altered Most Cells Can Present Antigen with Class I self-cells such as cancer cells, aging body cells, or allogeneic MHC: Presentation with Class ll mhc cells from a graft can also serve as targets Is Restricted to apcs Since all cells expressing either class I or class lI MHC mole. Evidence for Two Processing les can present peptides to T cells, strictly speaking they all could be designated as antigen-presenting cells. However, by and Presentation Pathways convention,cells that display peptides associated with class I MHC molecules to CD8 Tc cells are referred to as target cells, The immune system uses two different pathways to eliminate cells that display peptides associated with class II MHC mole. intracellular and extracellular antigens. Endogenous anti- les to CD4* tH cells are called antigen-presenting cells gens(those generated within the cell) are processed in the cy- (APCs). This convention is followed throughout this text. rosolic pathway and presented on the membrane with class I A variety of cells can function as antigen-presenting cell MHC molecules; exogenous antigens( those taken up by en Their distinguishing feature is their ability to express class l sented on the membrane with class I mhc molecul MHC molecules and to deliver a co-stimulatory signal. Three cell types are classified as professional antigen-presenting (Figure 8-4) cells: dendritic cells, macrophages, and B lymphocytes. These Experiments carried out by L. A. Morrison and T. J. cells differ from each other in their mechanisms of antigen Braciale provided early evidence that the antigenic peptides uptake, in whether they constitutively express class II MHc presented by class I and class II MHC molecules are derived molecules, and in their co-stimulatory activity: from different processing pathways. These researchers based their experimental protocol on the properties of two clones a Dendritic cells are the most effective of the antigen- of Tc cells, one that recognized influenza hemagglutinin a high level of class II MHC molecules and co-k presenting cells. Because these cells constitutively (HA)associated with a class I MHC molecule, and an atypical Tc line that recognized the same antigen associated stimulatory activity, they can activate naive TH cells with a class II MHC molecule.(In this case, and in son
At about the same time, A. Townsend and his colleagues began to identify the proteins of influenza virus that were recognized by TC cells. Contrary to their expectations, they found that internal proteins of the virus, such as matrix and nucleocapsid proteins, were often recognized by TC cells better than the more exposed envelope proteins. Moreover, Townsend’s work revealed that TC cells recognized short linear peptide sequences of the influenza protein. In fact, when noninfected target cells were incubated in vitro with synthetic peptides corresponding to sequences of internal influenza proteins, these cells could be recognized by TC cells and subsequently lysed just as well as target cells that had been infected with live influenza virus. These findings along with those presented in Figure 8-3 suggest that antigen processing is a metabolic process that digests proteins into peptides, which can then be displayed on the cell membrane together with a class I or class II MHC molecule. Most Cells Can Present Antigen with Class I MHC; Presentation with Class II MHC Is Restricted to APCs Since all cells expressing either class I or class II MHC molecules can present peptides to T cells, strictly speaking they all could be designated as antigen-presenting cells. However, by convention, cells that display peptides associated with class I MHC molecules to CD8 TC cells are referred to as target cells; cells that display peptides associated with class II MHC molecules to CD4 TH cells are called antigen-presenting cells (APCs). This convention is followed throughout this text. A variety of cells can function as antigen-presenting cells. Their distinguishing feature is their ability to express class II MHC molecules and to deliver a co-stimulatory signal. Three cell types are classified as professional antigen-presenting cells: dendritic cells, macrophages, and B lymphocytes. These cells differ from each other in their mechanisms of antigen uptake, in whether they constitutively express class II MHC molecules, and in their co-stimulatory activity: ■ Dendritic cells are the most effective of the antigenpresenting cells. Because these cells constitutively express a high level of class II MHC molecules and costimulatory activity, they can activate naive TH cells. ■ Macrophages must be activated by phagocytosis of particulate antigens before they express class II MHC molecules or the co-stimulatory B7 membrane molecule. ■ B cells constitutively express class II MHC molecules but must be activated before they express the co-stimulatory B7 molecule. Several other cell types, classified as nonprofessional antigen-presenting cells, can be induced to express class II MHC molecules or a co-stimulatory signal (Table 8-1). Many of these cells function in antigen presentation only for short periods of time during a sustained inflammatory response. Because nearly all nucleated cells express class I MHC molecules, virtually any nucleated cell is able to function as a target cell presenting endogenous antigens to TC cells. Most often, target cells are cells that have been infected by a virus or some other intracellular microorganism. However, altered self-cells such as cancer cells, aging body cells, or allogeneic cells from a graft can also serve as targets. Evidence for Two Processing and Presentation Pathways The immune system uses two different pathways to eliminate intracellular and extracellular antigens. Endogenous antigens (those generated within the cell) are processed in the cytosolic pathway and presented on the membrane with class I MHC molecules; exogenous antigens (those taken up by endocytosis) are processed in the endocytic pathway and presented on the membrane with class II MHC molecules (Figure 8-4). Experiments carried out by L. A. Morrison and T. J. Braciale provided early evidence that the antigenic peptides presented by class I and class II MHC molecules are derived from different processing pathways. These researchers based their experimental protocol on the properties of two clones of TC cells, one that recognized influenza hemagglutinin (HA) associated with a class I MHC molecule, and an atypical TC line that recognized the same antigen associated with a class II MHC molecule. (In this case, and in some 188 PART II Generation of B-Cell and T-Cell Responses TABLE 8-1 Antigen-presenting cells Professional antigen-presenting cells Nonprofessional antigen-presenting cells Dendritic cells (several types) Fibroblasts (skin) Thymic epithelial cells Macrophages Glial cells (brain) Thyroid epithelial cells B cells Pancreatic beta cells Vascular endothelial cells 8536d_ch08_185-199 8/22/02 11:49 AM Page 188 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-:
8536d_ch08_185-199 8/2/02 10: 08 AM Page 189 mac79 Mac 79: 45_BWfpldsby et al./ Immunology 5e Antigen Processing and Presentation CHAPTER 8 189 CYTOSOLIC PATHWAY Endogcnous-ATP-Cytoplasmic--Peptides TAP Endoplasmic--Peptide-cassI proteasome reticulum MHC complex Exopeptidases、 Amino acids ENDOCYTIC PATHWAY Exogen Endocytic compartments Endow phagocytosis FIGURE 8-4 Overview of cytosolic and endocytic pathways for to the cell membrane. TAP(transporter of antigenic peptides) processing antigen. The proteasome complex contains enzymes transports the peptides to the endoplasmic reticulum. It should be that cleave peptide bonds, converting proteins into peptides. The noted that the ultimate fate of most peptides in the cell is neither antigenic peptides from proteasome cleavage and those from of these pathways, but rather to be degraded completely into endocytic compartments associate with class I or class ll MHc amino acids molecules, and the peptide- MHC complexes are then transported others as well, the association of T-cell function with MHc only to target cells treated with infectious virions. Similarly, restriction is not absolute). In one set of experiments, target target cells that had been treated with infectious influenza cells that expressed both class I and class lI MHC molecules irions in the presence of emetine, which inhibits viral pro were incubated with infectious influenza virus or with UV- tein synthesis, stimulated the class II-restricted Tc cells but inactivated influenza virus. (The inactivated virus retained not the class I-restricted Tc cells. Conversely, target cells that its antigenic properties but was no longer capable of replicat- had been treated with infectious virions in the presence of ing within the target cells. ) The target cells were then incu- chloroquine, a drug that blocks the endocytic processing bated with the class I-restricted or the atypical class II- pathway, stimulated class I- but not class II-restricted Tc restricted Tc cells and subsequent lysis of the target cells was cells determined. The results of their experiments, presented in These results support the distinction between the process- Table 8-2, show that the class Il-restricted Tc cells responded ing of exogenous and endogenous antigens, including the to target cells treated with either infectious or noninfectious preferential association of exogenous antigens with class Il influenza virions. The class I-restricted Tc cells responded MHC molecules and of endogenous antigens with class I TABLE Effect of antigen presentation on activation of class I and class ll MHC-restricted Tc cells CTL ACTIVITYt Class I restricted Class il restricte UV-inactivated virus(noninfectious) Infectious virus chloroquine Target cells, which expressed both class I and class ll MHC molecules, were treated with the indicated preparations of influenza virus and other agents. Emetine inhibits viral protein synthesis, and chloroquine inhibits the endocytic processing pathway. DEtermined by lysis(+)and no lysis(-)of the target cells. SOURCE: Adapted from T. ). Braciale et al, 1987, Immunol. Rev. 98: 95
others as well, the association of T-cell function with MHC restriction is not absolute). In one set of experiments, target cells that expressed both class I and class II MHC molecules were incubated with infectious influenza virus or with UVinactivated influenza virus. (The inactivated virus retained its antigenic properties but was no longer capable of replicating within the target cells.) The target cells were then incubated with the class I–restricted or the atypical class II– restricted TC cells and subsequent lysis of the target cells was determined. The results of their experiments, presented in Table 8-2, show that the class II–restricted TC cells responded to target cells treated with either infectious or noninfectious influenza virions. The class I–restricted TC cells responded only to target cells treated with infectious virions. Similarly, target cells that had been treated with infectious influenza virions in the presence of emetine, which inhibits viral protein synthesis, stimulated the class II–restricted TC cells but not the class I–restricted TC cells. Conversely, target cells that had been treated with infectious virions in the presence of chloroquine, a drug that blocks the endocytic processing pathway, stimulated class I– but not class II–restricted TC cells. These results support the distinction between the processing of exogenous and endogenous antigens, including the preferential association of exogenous antigens with class II MHC molecules and of endogenous antigens with class I Antigen Processing and Presentation CHAPTER 8 189 FIGURE 8-4 Overview of cytosolic and endocytic pathways for processing antigen. The proteasome complex contains enzymes that cleave peptide bonds, converting proteins into peptides. The antigenic peptides from proteasome cleavage and those from endocytic compartments associate with class I or class II MHC molecules, and the peptide-MHC complexes are then transported to the cell membrane. TAP (transporter of antigenic peptides) transports the peptides to the endoplasmic reticulum. It should be noted that the ultimate fate of most peptides in the cell is neither of these pathways, but rather to be degraded completely into amino acids. CYTOSOLIC PATHWAY ENDOCYTIC PATHWAY Endogenous antigens ± Ubiquitin ATP Exogenous antigens Cytoplasmic proteasome complex Peptides Peptides TAP Endoplasmic reticulum Peptide–class I MHC complex Peptide–class II MHC complex Exopeptidases Amino acids Endocytosis or phagocytosis Endocytic compartments TABLE 8-2 Effect of antigen presentation on activation of class I and class II MHC-restricted TC cells CTL ACTIVITY† Treatment of target cells* Class I restricted Class II restricted Infectious virus UV-inactivated virus (noninfectious) Infectious virus emetine Infectious virus chloroquine * Target cells, which expressed both class I and class II MHC molecules, were treated with the indicated preparations of influenza virus and other agents. Emetine inhibits viral protein synthesis, and chloroquine inhibits the endocytic processing pathway. † Determined by lysis () and no lysis () of the target cells. SOURCE: Adapted from T. J. Braciale et al., 1987, Immunol. Rev. 98:95. 8536d_ch08_185-199 8/2/02 10:08 AM Page 189 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch08_185-199 8/2/02 10: 08 AM Page 190 mac79 Mac 79: 45_BWFpldsby et al./Immunology 5e: 190 PART I1 Generation of B-Cell and T-Cell Response MHC molecules. Association of viral antigen with class I(a) MHC molecules required replication of the influenza virus E-amino group ol and viral protein synthesis within the target cells; association lysine side chain with class II did not. These findings suggested that the pep- tides presented by class I and class II MHC molecules are trafficked through separate intracellular compartments; class I MHC molecules interact with peptides derived from cy- NH2 tosolic degradation of endogenously synthesized proteins, ②=三② class II molecules with peptides derived from endocytic degradation of exogenous antigens. The next two sections examine these two pathways in detail enzyme subuni Endogenous Antigens The Cytosolic Pathway ② In eukaryotic cells, protein levels are carefully regulated Every protein is subject to continuous turnover and is de- Protein Proteasome graded at a rate that is generally expressed in terms of its ha life.Some proteins(e.g, transcription factors, cyclins, and FIGURE 8-5 Cytosolic proteolytic system for degradation of intra- metabolic enzymes)have very short half-lives; dena- cellular proteins. (a) Proteins to be degraded are often covalently tured, misfolded, or otherwise abnormal proteins also are de linked to a small protein called ubiquitin. In this reaction, which re- graded rapidly. The pathway by which endogenous antigens quires ATP, a ubiquinating enzyme complex links several ubiquitin are degraded for presentation with class I MHC molecules molecules to a lysine-amino group near the amino terminus of the utilizes the same pathways involved in the normal turnover protein. (6) Degradation of ubiquitin-protein complexes occurs of intracellular proteins peptides Proteasomes are large cylindrical particles whose subunits catalyze cleavage of peptide bo Peptides for Presentation Are Generated by Protease Complexes Called Proteasomes residues. As described in Chapter 7, peptides that bind to Intracellular proteins are degraded into short peptides by a cy- class I MHC molecules terminate almost exclusively with hy- tosolic proteolytic system present in all cells. Those proteins drophobic or basic residues targeted for proteolysis often have a small protein, called ubiquitin, attached to them(Figure 8-5a). Ubiquitin-protein mplex called a proteasome. Each proteasome is a large to the Rough Endoplasmic Reticulumo/ conjugates can be degraded by a multifunctional protease Peptides Are Transported from the Cytosol lindrical particle consisting of four rings of pro- tein subunits with a central channel of diameter 10-50 A. Insight into the role that peptide transport, the delivery of A proteasome can cleave peptide bonds between 2 or 3 peptides to the MHC molecule, plays in the cytosolic pro- different amino acid combinations in an ATP-dependent cessing pathway came from studies of cell lines with defects process(Figure 8-5b). Degradation of ubiquitin-protein in peptide presentation by class I MHC molecules. One such omplexes is thought to occur within the central hollow of mutant cell line, called RMA-S, expresses about 5% of the proteasome. normal levels of class i mhc molecules on its membrane. Al Experimental evidence indicates that the immune system though RMA-S cells synthesize normal levels of class I o tilizes this general pathway of protein degradation to chains and B2-microglobulin, neither molecule appears on produce small peptides for presentation with class I MHC the membrane. a clue to the mutation in the RMA-S cell line molecules. The proteasomes involved in antigen processing was the discovery by A. Townsend and his colleagues that include two subunits encoded within the MHC gene cluster,"feeding "these cells peptides restored their level of mem LMP2 and LMP7, and a third non-MHC protein, LMP10 brane-associated class I MHC molecules to normal. These (also called MECL-1) All three are induced by increased lev- investigators suggested that peptides might be required to els of the T-cell cytokine IFN-Y. The peptidase activities of stabilize the interaction between the class I a chain and proteasomes containing LMP2, LMP7, and LMP10 preferen- B2-microglobulin. The ability to restore expression of class ally generate peptides that bind to MHC class I molecules. I MHC molecules on the membrane by feeding the cells Such proteasomes, for example, show increased hydrolysis predigested peptides suggested that the RMA-S cell line of peptide bonds that follow basic and/or hydrophobic might have a defect in peptide transport
MHC molecules. Association of viral antigen with class I MHC molecules required replication of the influenza virus and viral protein synthesis within the target cells; association with class II did not. These findings suggested that the peptides presented by class I and class II MHC molecules are trafficked through separate intracellular compartments; class I MHC molecules interact with peptides derived from cytosolic degradation of endogenously synthesized proteins, class II molecules with peptides derived from endocytic degradation of exogenous antigens. The next two sections examine these two pathways in detail. Endogenous Antigens: The Cytosolic Pathway In eukaryotic cells, protein levels are carefully regulated. Every protein is subject to continuous turnover and is degraded at a rate that is generally expressed in terms of its halflife. Some proteins (e.g., transcription factors, cyclins, and key metabolic enzymes) have very short half-lives; denatured, misfolded, or otherwise abnormal proteins also are degraded rapidly. The pathway by which endogenous antigens are degraded for presentation with class I MHC molecules utilizes the same pathways involved in the normal turnover of intracellular proteins. Peptides for Presentation Are Generated by Protease Complexes Called Proteasomes Intracellular proteins are degraded into short peptides by a cytosolic proteolytic system present in all cells. Those proteins targeted for proteolysis often have a small protein, called ubiquitin, attached to them (Figure 8-5a). Ubiquitin-protein conjugates can be degraded by a multifunctional protease complex called a proteasome. Each proteasome is a large (26S), cylindrical particle consisting of four rings of protein subunits with a central channel of diameter 10–50 Å. A proteasome can cleave peptide bonds between 2 or 3 different amino acid combinations in an ATP-dependent process (Figure 8-5b). Degradation of ubiquitin-protein complexes is thought to occur within the central hollow of the proteasome. Experimental evidence indicates that the immune system utilizes this general pathway of protein degradation to produce small peptides for presentation with class I MHC molecules. The proteasomes involved in antigen processing include two subunits encoded within the MHC gene cluster, LMP2 and LMP7, and a third non-MHC protein, LMP10 (also called MECL-1). All three are induced by increased levels of the T-cell cytokine IFN-. The peptidase activities of proteasomes containing LMP2, LMP7, and LMP10 preferentially generate peptides that bind to MHC class I molecules. Such proteasomes, for example, show increased hydrolysis of peptide bonds that follow basic and/or hydrophobic residues. As described in Chapter 7, peptides that bind to class I MHC molecules terminate almost exclusively with hydrophobic or basic residues. Peptides Are Transported from the Cytosol to the Rough Endoplasmic Reticulum Insight into the role that peptide transport, the delivery of peptides to the MHC molecule, plays in the cytosolic processing pathway came from studies of cell lines with defects in peptide presentation by class I MHC molecules. One such mutant cell line, called RMA-S, expresses about 5% of the normal levels of class I MHC molecules on its membrane. Although RMA-S cells synthesize normal levels of class I chains and 2-microglobulin, neither molecule appears on the membrane. A clue to the mutation in the RMA-S cell line was the discovery by A. Townsend and his colleagues that “feeding” these cells peptides restored their level of membrane-associated class I MHC molecules to normal. These investigators suggested that peptides might be required to stabilize the interaction between the class I chain and 2-microglobulin. The ability to restore expression of class I MHC molecules on the membrane by feeding the cells predigested peptides suggested that the RMA-S cell line might have a defect in peptide transport. 190 PART II Generation of B-Cell and T-Cell Responses COOH H2N NH C O Ubiquitin (b) COOH NH2 (a) ε-amino group on lysine side chain COOH H2N NH C O Ubiquitin NH2 Ubiquinating enzyme complex + ubiquitin ATP AMP + PPi Protein Proteasome Peptides Proteolytic enzyme subunit FIGURE 8-5 Cytosolic proteolytic system for degradation of intracellular proteins. (a) Proteins to be degraded are often covalently linked to a small protein called ubiquitin. In this reaction, which requires ATP, a ubiquinating enzyme complex links several ubiquitin molecules to a lysine-amino group near the amino terminus of the protein. (b) Degradation of ubiquitin-protein complexes occurs within the central channel of proteasomes, generating a variety of peptides. Proteasomes are large cylindrical particles whose subunits catalyze cleavage of peptide bonds. 8536d_ch08_185-199 8/2/02 10:08 AM Page 190 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch08_185-199 8/2/02 10: 08 AM Page 191 mac79 Mac 79: 45_BWfpldsby et al./ Immunology 5e Antigen Processing and Presentation CHAPTER8 191 Subsequent experiments showed that the defect RMA-S cell line occurs in the protein that transports pep- (a) TAP TAP2 tides from the cytoplasm to the RER, where class I molecules are synthesized. When RMA-S cells were transfected with a (ATP) (ATP) functional gene encoding the transporter protein, the cells began to express class I molecules on the membrane. The transporter protein, designated TAP(for transporter asso ciated with antigen processing) is a membrane-spanning heterodimer consisting of two proteins: TAPI and TAP2 (Figure 8-6a) In addition to their multiple transmembrane RER membrane segments, the TAPI and TAP2 proteins each have a domain projecting into the lumen of the RER, and an ATP-binding lumen domain that projects into the cytosol. Both TAPI and TAP2 belong to the family of ATP-binding cassette proteins found in the membranes of many cells, including bacteria; these proteins mediate ATP-dependent transport of amino acid sugars,ions,and peptides. Amino acids Peptides generated in the cytosol by the proteasome ranslocated by TAP into the rer by a process that requires the hydrolysis of ATP(Figure 8-6b. TAP has the highest Peptides affinity for peptides containing 8-10 amino acids, which is the optimal peptide length for class I MHC binding In addi ATP tion, TAP appears to favor peptides with hydrophobic or ba ADP Pi sic carboxyl-terminal amino acids, the preferred anchor Class residues for class I MHC molecules. Thus, TAP is optimized I MH to transport peptides that will interact with class I MHC molecules Tapasin RER The TAPI and TAP2 genes map within the class II MHC region, adjacent to the LMP2 and LMP7 genes(see Figure Class I a chain 7-15). Both the transporter genes and these LMP genes ar Calnexin polymorphic; that is, different allelic forms of these genes exist within the population. Allelic differences in LMP-me diated proteolytic cleavage of protein antigens or in the transport of different peptides from the cytosol into the rer may contribute to the observed variation among individuals FIGURE 8-6 Generation of antigenic peptide-dlass I MHC com in their response to different endogenous antigens. TAP plexes in the cytosolic pathway. (a) Schematic diagram of TAP, a het deficiencies can lead to a disease syndrome that has aspects erodimer anchored in the membrane of the rough endoplasmic of both immunodeficiency and autoimmunity(see Clinical reticulum(RER). The two chains are encoded by TAPI and TAP2. The cy Focus) tosolic domain in each TAP subunit contains an ATP-binding site, and peptide transport depends on the hydrolysis of ATP.(b)In the cytosol, Peptides Assemble with Class I MHC Aided association of LMP2, LMP7, and LMP10(black spheres)with a protea- by Chaperone Molecules some changes its catalytic specificity to favor production of peptides that bind to class I MHC molecules Within the RER membrane, a newly Like other proteins, the a chain and B2-microglobulin thesized class I a chain associates with calnexin until Br-microglobulin components of the class I MHC molecule are synthesized binds to the a chain. The class I a chain/Bz-microglobulin heterodimer on polysomes along the rough endoplasmic reticulum. As- then binds to calreticulin and the TAP-associated protein tapasin. When sembly of these components into a stable class I MHC a peptide delivered by TAP is bound to the class I molecule, folding of molecular complex that can exit the RER requires the MHC class l is complete and it is released from the RER and transported presence of a peptide in the binding groove of the class I through the Golgi to the surface of the cell molecule. The assembly process involves several steps and includes the participation of molecular chaperones, which facilitate the folding of polypeptides. The first molecular chaperone involved in class I MHC assembly is calnexin, ates with the chaperone calreticulin and with tapasin resident membrane protein of the endoplasmic reticulum. Tapasin(TAP-associated protein) brings the TAP trans Calnexin associates with the free class I a chain and pro- porter into proximity with the class I molecule and motes its folding. When B2-microglobulin binds to the a allows it to acquire an antigenic peptide( figure 8-7). The hain, calnexin is released and the class I molecule associ- physical association of the a chain-B2-microglobulin
Antigen Processing and Presentation CHAPTER 8 191 (a) Amino acids Peptides Calreticulin Tapasin Class I α chain Calnexin (b) Cytosol TAP Protein RER lumen RER lumen TAP1 TAP2 Cytosol ATP ATP RER membrane ATP ADP + Pi Class I MHC FIGURE 8-6 Generation of antigenic peptide–class I MHC complexes in the cytosolic pathway. (a) Schematic diagram of TAP, a heterodimer anchored in the membrane of the rough endoplasmic reticulum (RER). The two chains are encoded by TAP1 and TAP2. The cytosolic domain in each TAP subunit contains an ATP-binding site, and peptide transport depends on the hydrolysis of ATP. (b) In the cytosol, association of LMP2, LMP7, and LMP10 (black spheres) with a proteasome changes its catalytic specificity to favor production of peptides that bind to class I MHC molecules. Within the RER membrane, a newly synthesized class I chain associates with calnexin until 2-microglobulin binds to the chain. The class I chain/2-microglobulin heterodimer then binds to calreticulin and the TAP-associated protein tapasin. When a peptide delivered by TAP is bound to the class I molecule, folding of MHC class I is complete and it is released from the RER and transported through the Golgi to the surface of the cell. Subsequent experiments showed that the defect in the RMA-S cell line occurs in the protein that transports peptides from the cytoplasm to the RER, where class I molecules are synthesized. When RMA-S cells were transfected with a functional gene encoding the transporter protein, the cells began to express class I molecules on the membrane. The transporter protein, designated TAP (for transporter associated with antigen processing) is a membrane-spanning heterodimer consisting of two proteins: TAP1 and TAP2 (Figure 8-6a). In addition to their multiple transmembrane segments, the TAP1 and TAP2 proteins each have a domain projecting into the lumen of the RER, and an ATP-binding domain that projects into the cytosol. Both TAP1 and TAP2 belong to the family of ATP-binding cassette proteins found in the membranes of many cells, including bacteria; these proteins mediate ATP-dependent transport of amino acids, sugars, ions, and peptides. Peptides generated in the cytosol by the proteasome are translocated by TAP into the RER by a process that requires the hydrolysis of ATP (Figure 8-6b). TAP has the highest affinity for peptides containing 8–10 amino acids, which is the optimal peptide length for class I MHC binding. In addition, TAP appears to favor peptides with hydrophobic or basic carboxyl-terminal amino acids, the preferred anchor residues for class I MHC molecules. Thus, TAP is optimized to transport peptides that will interact with class I MHC molecules. The TAP1 and TAP2 genes map within the class II MHC region, adjacent to the LMP2 and LMP7 genes (see Figure 7-15). Both the transporter genes and these LMP genes are polymorphic; that is, different allelic forms of these genes exist within the population. Allelic differences in LMP-mediated proteolytic cleavage of protein antigens or in the transport of different peptides from the cytosol into the RER may contribute to the observed variation among individuals in their response to different endogenous antigens. TAP deficiencies can lead to a disease syndrome that has aspects of both immunodeficiency and autoimmunity (see Clinical Focus). Peptides Assemble with Class I MHC Aided by Chaperone Molecules Like other proteins, the chain and 2-microglobulin components of the class I MHC molecule are synthesized on polysomes along the rough endoplasmic reticulum. Assembly of these components into a stable class I MHC molecular complex that can exit the RER requires the presence of a peptide in the binding groove of the class I molecule. The assembly process involves several steps and includes the participation of molecular chaperones, which facilitate the folding of polypeptides. The first molecular chaperone involved in class I MHC assembly is calnexin, a resident membrane protein of the endoplasmic reticulum. Calnexin associates with the free class I chain and promotes its folding. When 2-microglobulin binds to the chain, calnexin is released and the class I molecule associates with the chaperone calreticulin and with tapasin. Tapasin (TAP-associated protein) brings the TAP transporter into proximity with the class I molecule and allows it to acquire an antigenic peptide (Figure 8-7). The physical association of the chain–2-microglobulin 8536d_ch08_185-199 8/2/02 10:08 AM Page 191 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch08_185-199 8/22/02 11: 49 AM Page 192 mac100 mac 100: 128_tm: 8536d: Goldsby et al./ Immunology 5e- 192 PART II Generation of B-Cell and T-Cell Response CLINICAL FOCUS ells were isolated from biopsied skin Deficiency in Transporters from several patients, supporting this ssibility. Normally, the activity of NK Associated with Antigen cells is limited through the action of killer-cell-inhibitory receptors (KIRs) Presentation(TAP) Leads to a which deliver a negative signal to the NK Diverse Disease Spectrum cell following interaction with class I molecules(see Chapter 14). The defi. ciency of class I molecules in TAP-related blS patients explains the excessive activ- A relatively of the upper respiratory tract, and in the ity of the NK cells. Activation of NK cells econd decade begins to have chronic in. further explains the absence of severe dition known as bare lymphocyte syn. fection of the lungs. It is thought that virus infections, which are limited by NK drome,or BLS, has been recognized for post-nasal-drip syndrome common in and y8 cells more than 22 years. The lymphocytes in younger patients promotes the bacterial The best treatment for the character- BLS patients express MHC molecules at lung infections in later life. Noteworthy is istic lung infections appears to be antibi- below-normal levels and, in some cases, the absence of any severe viral infection, otics and intravenous immunoglobulin not at all. In type 1 BLS, a deficiency in which is common in immunodeficien. Attempts to limit the skin disease by im- MHC class I molecules exists; in type 2 cies with T-cell involvement(see Chapter munosup s. such BLS, expression of class ll molecules is 19). Bronchiectasis (dilation of the steroid treatment or cytotoxic agents, impaired. The pathogenesis of one type bronchial tubes )often occurs and red can lead to exacerbation of lesions and is of BLS underscores the importance of ring infections can lead to lung damage therefore contraindicated. Mutations in the class I family of MHC molecules in that may be fatal. The most characteristic the promoter region of TAP that preclude their dual roles of preventing autoim. mark of the deficiency is the occurrence expression of the gene were found for munity as well as defending against of necrotizing skin lesions on the extrem. several patients, suggesting the possibil- pathogens ities and the midface. These lesions ul- ity of gene therapy, but the cellular distri. Defects in promoter sequences that cerate and may cause disfigurement(see bution of class I is so widespread that it preclude MHc gene transcription were figure). The skin lesions are probably due is not clear what cells would need to be found for some type 2 BLS cases, but in to activated NK cells and y8 T cells; NK corrected to alleviate all symptoms many instances the nature of the under lying defect is not known. A recent stud has identified a group of patients with type 1 BLS due to defects in TAP1 TAP2 genes. Manifestations of the TAP deficiency were consistent in this patient group and define a unique disease. As teins are necessary for the loading of peptides onto class I molecules, a step that is essential for expression of class I MHC molecules on the cell surface. Lym- phocytes in individuals with TAP defi ency express levels of class I molecules an no Other cellular abnormalities include in. creased numbers of NK and y8 T cells and decreased levels of CD8* aB Tcells As we shall see. the disease manifesta Necrotizing granulomatous lesions in the midface of patient with TAP-deficiency syn- tions are reasonably well explained by drome. TaP deficiency leads to a condition with symptoms characteristic of autoimmu these deviations in the levels of certain uch as the skin lesions that on the extremities and the midfau ells involved in immune function immunodeficiency that causes chronic sinusitis, leading to recurrent lung infection g In early life the TAP-deficient individ- (From S.D. Gadola et al., 1999, Lancet 354: 1598, and 2000, Clinical and Experimental al suffers frequent bacterial infections Immunology 121: 173. 1
192 PART II Generation of B-Cell and T-Cell Responses of the upper respiratory tract, and in the second decade begins to have chronic infection of the lungs. It is thought that a post-nasal-drip syndrome common in younger patients promotes the bacterial lung infections in later life. Noteworthy is the absence of any severe viral infection, which is common in immunodeficiencies with T-cell involvement (see Chapter 19). Bronchiectasis (dilation of the bronchial tubes) often occurs and recurring infections can lead to lung damage that may be fatal. The most characteristic mark of the deficiency is the occurrence of necrotizing skin lesions on the extremities and the midface. These lesions ulcerate and may cause disfigurement (see figure). The skin lesions are probably due to activated NK cells and T cells; NK cells were isolated from biopsied skin from several patients, supporting this possibility. Normally, the activity of NK cells is limited through the action of killer-cell-inhibitory receptors (KIRs), which deliver a negative signal to the NK cell following interaction with class I molecules (see Chapter 14). The deficiency of class I molecules in TAP-related BLS patients explains the excessive activity of the NK cells. Activation of NK cells further explains the absence of severe virus infections, which are limited by NK and cells. The best treatment for the characteristic lung infections appears to be antibiotics and intravenous immunoglobulin. Attempts to limit the skin disease by immunosuppressive regimens, such as steroid treatment or cytotoxic agents, can lead to exacerbation of lesions and is therefore contraindicated. Mutations in the promoter region of TAP that preclude expression of the gene were found for several patients, suggesting the possibility of gene therapy, but the cellular distribution of class I is so widespread that it is not clear what cells would need to be corrected to alleviate all symptoms. A relativelyrare condition known as bare lymphocyte syndrome, or BLS, has been recognized for more than 22 years. The lymphocytes in BLS patients express MHC molecules at below-normal levels and, in some cases, not at all. In type 1 BLS, a deficiency in MHC class I molecules exists; in type 2 BLS, expression of class II molecules is impaired. The pathogenesis of one type of BLS underscores the importance of the class I family of MHC molecules in their dual roles of preventing autoimmunity as well as defending against pathogens. Defects in promoter sequences that preclude MHC gene transcription were found for some type 2 BLS cases, but in many instances the nature of the underlying defect is not known. A recent study has identified a group of patients with type 1 BLS due to defects in TAP1 or TAP2 genes. Manifestations of the TAP deficiency were consistent in this patient group and define a unique disease. As described earlier in this chapter, TAP proteins are necessary for the loading of peptides onto class I molecules, a step that is essential for expression of class I MHC molecules on the cell surface. Lymphocytes in individuals with TAP deficiency express levels of class I molecules significantly lower than normal controls. Other cellular abnormalities include increased numbers of NK and T cells, and decreased levels of CD8 T cells. As we shall see, the disease manifestations are reasonably well explained by these deviations in the levels of certain cells involved in immune function. In early life the TAP-deficient individual suffers frequent bacterial infections CLINICAL FOCUS Deficiency in Transporters Associated with Antigen Presentation (TAP) Leads to a Diverse Disease Spectrum Necrotizing granulomatous lesions in the midface of patient with TAP-deficiency syndrome. TAP deficiency leads to a condition with symptoms characteristic of autoimmunity, such as the skin lesions that appear on the extremities and the midface, as well as immunodeficiency that causes chronic sinusitis, leading to recurrent lung infection. [From S. D. Gadola et al., 1999, Lancet 354:1598, and 2000, Clinical and Experimental Immunology 121:173.] 8536d_ch08_185-199 8/22/02 11:49 AM Page 192 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-:
8536d_ch08_185-199 8/2/02 10: 08 AM Page 193 mac79 Mac 79: 45_ Bwppldsby et al./ Immunology 5e 193 B2 microglobulin → Exit RER 9 Class I mHC Calnexin CaInexin-associated Calreticulin-tapasin Class I mhc class I mHc a chain associated class I molecule MHC molecule Tapasin Calreticulin Tapasin Calreticulin IGURE 8-7 Assembly and stabilization of class I MHC mole- chaperonin calreticulin and to tapasin, which is associated with the cules. Newly formed class I a chains associate with calnexin, a peptide transporter TAP. This association promotes binding of an molecular chaperone, in the RER membrane. Subsequent binding antigenic peptide, which stabilizes the class I molecule-peptide to B2-microglobulin releases calnexin and allows binding to the complex, allowing its release from the reR heterodimer with the TAP protein(see Figure 8-6b)pro- the experiment shown in Figure 8-3 demonstrated, internal- motes peptide capture by the class I molecule before the pep- ized antigen takes 1-3 h to transverse the endocytic pathway tides are exposed to the luminal environment of the RER. and appear at the cell surface in the form of peptide-class Il Peptides not bound by class I molecules are rapidly degraded. MHC complexes. The ende ay appears to involve As a consequence of peptide binding, the class I molecule dis- three increasingly acidic compartments: early endosomes (pH plays increased stability and can dissociate from calreticulin 6.0-6.5); late endosomes, or endolysosomes(pH5.0-6.0); and and tapasin, exit from the RER, and proceed to the cell sur- lysosomes(pH 4.5-5.0). Internalized antigen moves from face via the Golgi. An additional chaperone protein, ERp57, early to late endosomes and finally to lysosomes, encountering has been observed in association with calnexin and calretic- hydrolytic enzymes and a lower pH in each compartment(Fig ulin complexes. The precise role of this resident endoplasmic ure 8-9). Lysosomes, for example, contain a unique collection reticulum protein in the class I peptide assembly and loading of more than 40 acid-dependent hydrolases, including pro- process has not yet been defined, but it is thought to con- teases, nucleases, glycosidases, lipases, phospholipases, and tribute to the formation of disulfide bonds during the matu- phosphatases. Within the compartments of the endocytic tion of class I chains. Because its role is not clearly defined, pathway, antigen is degraded into oligopeptides of about 13- ERp57 is not shown in Figures 8-6 and 8-7. 18 residues, which bind to class lI MHC molecules. Because the hydrolytic enzymes are optimally active under acidic condi tions (low pH), antigen processing can be inhibited by chem Exogenous Antigens: The Endocytic cal agents that increase the pH of the compartments (e.g chloroquine)as well as by protease inhibitors(e. g, leupeptin Pathway The mechanism by which internalized antigen moves Figure 8-8 recapitulates the endogenous pathway discussed from one endocytic compartment to the next has not bee previously (left side), and compares it with the separate exoge conclusively demonstrated. It has been suggested that early nous pathway (right), which we shall now consider Whether ndosomes from the periphery move inward to become late an antigenic peptide associates with class I or with class ll mol ndosomes and finally lysosomes. Alternatively, small trans- port vesicles may carry antigens from one compartment to ecules is dictated by the mode of entry into the cell, either ex- the next. Eventually the endocytic compartments,or por- ogenous or endogenous, and by the site of processing Antigen-presenting cells can internalize antigen by phago- ns of them, return to the cell periphery, where they fuse cytosis, endocytosis, or both. Macrophages internalize antigen th the plasma membrane. In this way, the surface receptor by both processes, whereas most other APCs are not phago- re recycled. cytic or are poorly phagocytic and therefore internalize exoge- nous antigen only by endocytosis(either receptor-mediated The Invariant Chain Guides Transport endocytosis or pinocytosis). B cells, for example, internalize of Class II MHC Molecules ntigen very effectively by receptor-mediated endocytosis u to Endocytic Vesicles ing antigen-specific membrane antibody as the receptor. Since antigen-presenting cells express both class I and class Il Peptides Are Generated from Internalized MHC molecules, some mechanism must exist to prevent Molecules in Endocytic Vesicle lass II MHC molecules from binding to the same set of anti- nic peptides as the class I molecules. When class II MHC Once an antigen is internalized, it is degraded into peptides molecule are synthesized within the RER, three pairs of class vithin compartments of the endocytic processing pathway. As II aB chains associate with a preassembled trimer of a
Antigen Processing and Presentation CHAPTER 8 193 heterodimer with the TAP protein (see Figure 8-6b) promotes peptide capture by the class I molecule before the peptides are exposed to the luminal environment of the RER. Peptides not bound by class I molecules are rapidly degraded. As a consequence of peptide binding, the class I molecule displays increased stability and can dissociate from calreticulin and tapasin, exit from the RER, and proceed to the cell surface via the Golgi. An additional chaperone protein, ERp57, has been observed in association with calnexin and calreticulin complexes. The precise role of this resident endoplasmic reticulum protein in the class I peptide assembly and loading process has not yet been defined, but it is thought to contribute to the formation of disulfide bonds during the maturation of class I chains. Because its role is not clearly defined, ERp57 is not shown in Figures 8-6 and 8-7. Exogenous Antigens: The Endocytic Pathway Figure 8-8 recapitulates the endogenous pathway discussed previously (left side), and compares it with the separate exogenous pathway (right), which we shall now consider. Whether an antigenic peptide associates with class I or with class II molecules is dictated by the mode of entry into the cell, either exogenous or endogenous, and by the site of processing. Antigen-presenting cells can internalize antigen by phagocytosis, endocytosis, or both. Macrophages internalize antigen by both processes, whereas most other APCs are not phagocytic or are poorly phagocytic and therefore internalize exogenous antigen only by endocytosis (either receptor-mediated endocytosis or pinocytosis). B cells, for example, internalize antigen very effectively by receptor-mediated endocytosis using antigen-specific membrane antibody as the receptor. Peptides Are Generated from Internalized Molecules in Endocytic Vesicles Once an antigen is internalized, it is degraded into peptides within compartments of the endocytic processing pathway. As the experiment shown in Figure 8-3 demonstrated, internalized antigen takes 1–3 h to transverse the endocytic pathway and appear at the cell surface in the form of peptide–class II MHC complexes. The endocytic pathway appears to involve three increasingly acidic compartments: early endosomes (pH 6.0–6.5); late endosomes, or endolysosomes (pH 5.0–6.0); and lysosomes (pH 4.5–5.0). Internalized antigen moves from early to late endosomes and finally to lysosomes, encountering hydrolytic enzymes and a lower pH in each compartment (Figure 8-9). Lysosomes, for example, contain a unique collection of more than 40 acid-dependent hydrolases, including proteases, nucleases, glycosidases, lipases, phospholipases, and phosphatases. Within the compartments of the endocytic pathway, antigen is degraded into oligopeptides of about 13– 18 residues,which bind to class II MHC molecules.Because the hydrolytic enzymes are optimally active under acidic conditions (low pH), antigen processing can be inhibited by chemical agents that increase the pH of the compartments (e.g., chloroquine) as well as by protease inhibitors (e.g., leupeptin). The mechanism by which internalized antigen moves from one endocytic compartment to the next has not been conclusively demonstrated. It has been suggested that early endosomes from the periphery move inward to become late endosomes and finally lysosomes. Alternatively, small transport vesicles may carry antigens from one compartment to the next. Eventually the endocytic compartments, or portions of them, return to the cell periphery, where they fuse with the plasma membrane. In this way, the surface receptors are recycled. The Invariant Chain Guides Transport of Class II MHC Molecules to Endocytic Vesicles Since antigen-presenting cells express both class I and class II MHC molecules, some mechanism must exist to prevent class II MHC molecules from binding to the same set of antigenic peptides as the class I molecules. When class II MHC molecule are synthesized within the RER, three pairs of class II chains associate with a preassembled trimer of a FIGURE 8-7 Assembly and stabilization of class I MHC molecules. Newly formed class I chains associate with calnexin, a molecular chaperone, in the RER membrane. Subsequent binding to 2-microglobulin releases calnexin and allows binding to the chaperonin calreticulin and to tapasin, which is associated with the peptide transporter TAP. This association promotes binding of an antigenic peptide, which stabilizes the class I molecule–peptide complex, allowing its release from the RER. + + Peptides Exit RER Calnexin Calnexin Class I MHC α chain Class I MHC molecule Calreticulin-tapasin– associated class I MHC molecule Calnexin-associated class I MHC α chain β2 microglobulin + + Tapasin Calreticulin Tapasin Calreticulin 8536d_ch08_185-199 8/2/02 10:08 AM Page 193 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch08_194 8/23/02 11: 59 AM Page 194 mac100 mac 100: 1268_*:8536d: Goldsby et al./Immunology se- 194 PART 11 Generation of B-Cell and T-Cell Response VISUALIZING CONCEPTS Endogenous pathway Exogenous pathway (class I MHC (class II MHC) Endogenous tigen Endogenous antigen is Proteasome Peptid reticulum (RER) RER via TAP Invariant T Class I MHC a and阝 nd invariant chain, TAP blocking binding of endogenous antigen. B2-microglobulin Class I Class ll MHC Calnexin MHC complex is routed Peptide hrough Golgi to Class I MHC o chain binds calnexin, then B2 microglobulin. Calnexin dissociates Calreticulin Golgi complex Invariant chain is and Tapasin bind MHO degraded, leaving CLIF fragment. chaperones dissociate. Digested Invariant④ Exogenous antigen is routed to endocytic pathway compartments Class I MHC-peptide is transported from RER to (③ COe- exogenous Golgi complex to plasma exchange of CLIP for antigenic peptide. Class lI MHC-peptide is Class I Class ll transported to plasma membrane MHC FIGURE 8-8 Separate antigen-presenting pathways are utilized termine whether antigenic peptides associate with class I MHC for endogenous (green )and exogenous(red)antigens. The mode molecules in the rough endoplasmic reticulum or with class ll of antigen entry into cells and the site of antigen processing de- molecules in endocytic compartments protein called invariant chain(li, CD74). This trimeric pro- from binding to the cleft while the class ll molecule is within tein interacts with the peptide-binding cleft of the class il the RER (see right side of Figure 8-8). The invariant chain molecules, preventing any endogenously derived peptides also appears to be involved in the folding of the class ll a and
194 PART II Generation of B-Cell and T-Cell Responses VISUALIZING CONCEPTS FIGURE 8-8 Separate antigen-presenting pathways are utilized for endogenous (green) and exogenous (red) antigens. The mode of antigen entry into cells and the site of antigen processing determine whether antigenic peptides associate with class I MHC molecules in the rough endoplasmic reticulum or with class II molecules in endocytic compartments. Endogenous pathway (class I MHC) Exogenous pathway (class II MHC) Peptide TAP Invariant chain Class II MHC Class I MHC Class I MHC Class II MHC Rough endoplasmic reticulum (RER) Proteasome Calreticulum Tapasin β2-microglobulin Golgi complex Digested invariant chain Exogenous antigen CLIP Calnexin Endogenous antigen Class II MHC α and β bind invariant chain, blocking binding of endogenous antigen. 1 Endogenous antigen is degraded by proteasome. 1 Peptide is transported to RER via TAP. 2 MHC complex is routed through Golgi to endocytic pathway compartments. 2 Class I MHC α chain binds calnexin, then β2 microglobulin. Calnexin dissociates, Calreticulin and Tapasin bind. MHC captures peptide, chaperones dissociate. 3 Invariant chain is degraded, leaving CLIP fragment. 3 Exogenous antigen is taken up, degraded, routed to endocytic pathway compartments. Class I MHC–peptide is transported from RER to Golgi complex to plasma membrane. 4 4 HLA-DM mediates exchange of CLIP for antigenic peptide. 5 Class II MHC–peptide is transported to plasma membrane. 6 protein called invariant chain (Ii, CD74). This trimeric protein interacts with the peptide-binding cleft of the class II molecules, preventing any endogenously derived peptides from binding to the cleft while the class II molecule is within the RER (see right side of Figure 8-8). The invariant chain also appears to be involved in the folding of the class II and 8536d_ch08_194 8/23/02 11:59 AM Page 194 mac100 mac 100: 1268_tm:8536d:Goldsby et al. / Immunology 5e-:
