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ANTIGEN PRESENTING CELL
- 1. Antigen presenting cells 1 Presented by Dr. Sreelakshmi. P 1ST YEAR MDS JKKNDCH
- 2. Content • Introduction • Antigen presenting cells • Various Terminologies • Requirement for Antigen presenting cells • Mechanism of action • Types of APC • Properties and functions of APC • Routes of antigen entry • Antigen recognition by T and B cell • Roles of APC • Antigen processing and presentation • Role of APC in periodontal disease • Conclusion • References 2
- 3. Introduction • A majority of the periodontal diseases are an immunoinflammatory response elicited by the microorganisms present in the microbial plaque. • The immune system of the body consists of organized network of different cells and molecules that work together in a systematic and orchestrated manner. • The immune system defends the body against the invading microorganisms through innate (natural) and adaptive (acquired) mechanisms. • The stable response is mediated by T helper cells 1 (Th1) and dominated by the presence of T cells, while the progressive lesion shows predominance of B cells through the T helper 2 (Th2) response. - Ohlrich EJ , 2009 3
- 4. Antigen presenting cells • The killing of microorganisms or neutralization of the toxins by T cells and B cells does not occur by direct stimulation of these cells by viral or bacterial antigens. • This lymphocyte stimulation is achieved via antigens displayed on the surface of specialized cells that are called antigen-presenting cells (APC). • APC :- A group of cells play important roles in the immune response which can uptake, process antigens and present peptide-MHC complexes to T cells. • Once initial lymphocyte response is generated following the initial processes of antigen presentation, a wide spectrum of cell types will present antigen to the activated cells. Azuma M.2006 4
- 5. • The immune system primarily contains three types of APCs namely, 1.Dendritic cells (DC) , 2.Macrophages and 3.B-lymphocytes. • These cells are specialized to initiate or promote the development of lymphocyte activation and are also termed as “Professional APC.” • They express major histocompatibility complex (MHC) molecules along with other mechanisms for effective antigens uptake and expression of costimulatory molecules that promote cellular interaction. - Cutler CW 2007 5
- 6. Various Terminologies • Endogenous Ags: antigens synthesized within cells, including self and unself protein----class Ⅰ MHC molecules. • Exogenous Ags: antigens comes outside the cells, including self and unself protein----class Ⅱ MHC molecules. • Antigen processing: the conversion of native proteins to peptides which can combine with MHC molecules. • Antigen presentation: the course of formation and display of peptide-MHC complexes on the surface of APCs and the course of peptide-MHC complexes recognition by T cells. • Ag capturing - Endocytosis (internalization) - Phagocytosis, Pinocytosis, Receptor-mediated endocytosis 6
- 7. Requirement for antigen presenting cells T cells cannot respond to the free antigen but only to the processed antigen by the APCs 7
- 8. Mechanism of action of APCs 8
- 9. Types of Antigen presenting cells Professional VS Non-Professional APCs 9
- 10. 10
- 11. Dendritic cell (DC) History: DCs were first found by Steinman in 1973，named for their special spinelike projections. DCs were cultured successfully in vitro in 1993 by Inaba. Characteristic: The most efficient APC, can present antigens to naive T cells to elicit primary immune response. Identification of DC : Typical morphology - spinelike projection & stimulate naïve T cells activation Surface markers : CD1a, CD11c, CD83(human)- high expression of classⅡMHC - co-stimulatory molecules-- CD80,CD86 Source of DC : Pluripotent hematopoietic stem cells myeloid DC myeloid progenitor lymphoid DC lymphoid progenitor ` GM-CSF, IL-4 11
- 12. Classification of DC DC in lymphoid tissue: Interdigitating DC (IDC) Folicular DC (FDC) DC in non lymphoid tissue: Langerhans cell (LC) DC in body fluid: Veiled cell, Blood DC 12
- 13. Pre-DC Blood Non-lymphoid tissue Differentiation ImmatureDC DistributeWidely distributed in the body Possess ability of Ag capture and process Cytokines and Ag DC mature and move into lymphoid tissue Ability of Ag capture and processing decreases while its ability of Ag presenting increases Development and Maturation of DC 13
- 14. Pre-DC stage 14
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- 16. Antigen Capture and Transport Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. Membrane Receptors (C-type lectins) Capture and Endocytose microbes or microbial products Process ingested proteins into peptides capable of binding to MHC Microbial products recognized by TLR Signals and Cytokines activate DC (TNF) Activated DC lose adhesiveness and migrate to lymph nodes 16
- 17. Antigen Capture and Transport Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. DC CCR7 Lymphatic Vessels T cell zones of Lymph Nodes CCL 19 CCL 21 Naïve T cell CCR7 “Colocalization” 17
- 18. Antigen Capture and Transport Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. DC Capture Antigen Present Antigen to Naïve T cells Activate Lymphocytes Express high levels of MHC Activated DC develop Into potent APCs 18
- 19. Role of Dentritic cells in Antigen Capture and Display Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. 19
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- 21. 2. Macrophage（ MФ） • Stem from monocytes in blood • Have strong phagocytosis (big phagocyte) • Can not stimulate naïve T cells • Capture antigens by phagocytosis, pinocytosis, receptor-mediated endocytosis 21
- 22. 22
- 23. • B cells function as APC in the process of expansion of the immune responses through their interaction with lymphocytes already responded to antigenic stimulus. • Once the response is established, B cells produce specific antibodies, and effector lymphocytes including cytotoxic T cells may be released and return to the site of infection. • The antigen presentation to B cells is encountered in secondary lymphoid organs. • B cells activation is initiated following engagement of the B-cell receptor (BCR) by a specific antigen. • B cells can recognize and respond to both soluble and membrane-associated antigen, although recent insights suggest that membrane-associated antigens are more important for B-cell activation - Carrasco YR, 2006 3. B - Cell 23
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- 26. Properties and functions of Apc 26
- 27. Functions of different Apc 27
- 28. Routes of antigen entry 28
- 29. Antigen recognition by T and B cells T and B cells exhibit fundamental differences in antigen recognition. • B cells recognize antigen free in solution (native antigen). • T cells recognize antigen after it has been phagocytosed, degraded and small pieces of the antigen have been bound by MHC molecules. 29
- 30. Role of antigen-presenting cells (Apc) Helper T cells: recognize antigen after processing and presentation by MHC-II on APC (dendritic cells, macrophages, B cells). Cytotoxic T cells: recognize antigen when it is presented on MHC-I. Dentritic cell : Always express high levels of MHC-II molecules and co-stimulatory activity (B7 molecule) Macrophages : requires activation to up-regulate MHC-II molecules and co-stimulatory molecules (B7 molecules) B cells : always express MHC-II molecules but needs to be activated to express co-stimulatory activity (B7 molecule) 30
- 31. Major Histocompatibility Complex (MHC) • The MHC loci encode two major classes of membrane-bound glycoproteins: class I and class II MHC molecules. • They function as antigen-recognition molecules. • TH cells recognize antigen combined with class II molecules, whereas TC cells generally recognize antigen combined with class I molecules. • MHC molecule can bind to a spectrum of antigenic peptides derived from the intracellular degradation of antigen molecules. • These variable regions form a cleft within which the antigenic peptide sits and is presented to T lymphocytes. 31
- 32. • Foreign protein antigen are degraded into small antigenic peptides that form complexes with class I or class II MHC molecules. • This conversion of proteins into MHC-associated peptide fragments is called antigen processing and presentation. • Whether a particular antigen will be processed and presented together with class I MHC or class II MHC molecules appears to be determined by the route that the antigen takes to enter a cell. 32
- 33. Antigen processing and presentation 33
- 34. 1. Location of pathogen - viruses in cytosol, MHC class I pathway, Tc response (Cytosolic pathway) - extracellular bacteria, MHC class II pathway, Th2 response Ab formation (Endocytic pathway) - intracellular bacteria, MHC class II pathway, Th1 response cellular response (Endocytic pathway) 2. Peptides derived from both self & non-self proteins can associate with MHC class I and class II molecules. 3. Chemical nature of MHC groove determines which peptides it will bind. 4. MHC-I and MHC-II associated with peptides processed in different intracellular compartments - Class I MHC binds peptides derived from endogenous antigens - Class I MHC binds peptides from antigens that have been processed via the cytosolic pathway (derived from the cytoplasm of the cell) - Class II MHC molecules bind peptides derived from exogenous antigens. These antigens were internalized by phagocytosis or endocytosis. 34
- 35. Processing of protein antigens Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. 35
- 36. CLASS I MHC PATHWAY Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. Injected via Bacterial secretory mechanisms Phagocytosed Escape Mechanism 36
- 37. Class I MHC Pathway Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. • Peptides are generated by proteasome degradation • Peptides are transported from cytosol to the RER • Peptides loading onto MHC-I is aided by Chaperones 37
- 38. Class I MHC Pathway Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. 38
- 39. TAP (Transporter associated with Antigenic Processing) • Peptides from proteasome degradation of cytoplasmic proteins are transported across the membrane of the rough endoplasmic reticulum by a heterodimeric protein designated as TAP. • TAP is composed of two subunits - TAP1 and TAP-2 • TAP-mediated transport is ATP-dependent • The genes for TAP1 and TAP2 are encoded within the MHC 39
- 40. Class I MHC Pathway Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. Membrane Chaperone: Calnexin Luminal Chaperone: Calreticulin 40
- 41. • 1. The class I alpha chain is stabilized by calnexin. • 2. When the alpha chain binds beta-2-microglobulin: - calnexin is lost - calreticulin and tapasin bind • 3. Tapasin & calreticulin brings the class I molecules into the vicinity of the TAP. • 4. A cytoplasmic peptide transported through the TAP is loaded onto the class I molecule. • 5. Class I MHC dissociates from calreticulin and tapasin. Class I MHC-peptide complex is transported to Golgi and to the cell surface. Assembly of the class I-peptide complex 41
- 42. Class I MHC Pathway Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. 42
- 43. CLASS I MHC PATHWAY 43
- 44. Class II Processing: • The endocytic antigen processing pathway – processing of externally-derived peptides • Antigen can be taken into cells by various means: phagocytosis, endocytosis, pinocytosis, receptormediated endocytosis • Antigen taken up in these ways passes through a series of intracellular compartments of increasing acidity - early endosome (pH 6.5-6.0), late endosome (pH 6.0-5.0), phagolysosome (pH <5.0). 44
- 45. • Three major events occur in the endosomal pathway: 1) Degradation of material that was taken in – endosome goes through acidification and fusion with lysosome which contain a wide array of degradative enzymes 2) Loading of peptides from this material on to class II MHC molecules 3) Transport of class II MHC – peptide complexes back to the cell surface. • The endosomal compartment is completely separate from the endoplasmic reticulum ---> So, externally derived peptides are usually not loaded on to MHC-I. 45
- 46. Class II MHC Pathway Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. Endosome-Lysosome Phagolysosomes Autophagosomes 46
- 47. Class II MHC is synthesized in the ER, but is not loaded with peptides there because it's peptide binding site is blocked by the invariant chain (Ii). STEP 1: Cathepsins degrade Invariant Chain CLIP STEP 2: HLA-DM removes CLIP 47
- 48. • Once class II MHC enters the endosomal compartments, the invariant chain is degraded, leaving a small fragment - CLIP - in the peptide binding site. CLIP is removed by HLA-DM, which loads a peptide on to class II MHC HLA-DO inhibits HLA-DM until the cell is Activated 48
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- 51. 51
- 52. Role of antigen-presenting cells in periodontal diseases • Various studies Konermann A 2012 in the past have suggested the role of APCs in the maintenance of oral health as well as pathogenesis of periodontal diseases. APCs have a very important role in inducing and maintaining mucosal immune homeostasis to harmless commensal bacteria and to self-antigens. • They migrate in the immature state to various lymphoid organs and induce the antigen-specific unresponsiveness or tolerance. Smits HH 2005 • In the process of transition from health to disease, the APCs induce a strong immune response under the influence of various pro- and anti-inflammatory cytokines. Pulendran B, 2001 52
- 53. • The chemokines and their receptors control these cell movements through their molecular signals. The expression patterns of the chemokines, and macrophage inflammatory protein 3 (MIP 3), and their respective receptors CCR6 and CCR7, suggests that these molecules mediate influx of APC, especially DCs into the tissues and efflux to T-cell rich areas in lymph nodes. • Failure of migrating DCs to mature en route to lymph nodes or the ability of the DCs to depolarize cytokine profiles toward a T helper type 2 (Th2) profile can lead to immune suppression. • Porphyromonas gingivalis lipopolysaccharide (LPS) has shown to suppress the immunity thereby inducing weak DC maturation and Th2 response. • It is also observed that the resolution of the inflammation is dominated by the migration of fully matured DCs from inflamed sites into lymph nodes leading to Th1 type cell-mediated immune response. 53
- 54. • After the DC migrates out of the periphery, monocytic DC precursors migrate in from the bloodstream and from the bone marrow. DC precursors are also recruited in elevated numbers to infected sites by different chemokines. These chemokines may have particular relevance to chronic periodontitis, which shows a dominant Th2 response. • Immature DCs that are activated at the site by pathogen-associated molecular patterns (PAMPs), in-turn secrete chemokines that attract other inflammatory cells, including activated T cells, natural killer cells, and additional DCs. • The recent studies show that the human gingiva is infiltrated with large numbers of TLR 2 positive and TLR 4 positive cells, and that their numbers increase significantly in chronic periodontitis. • The NACHT-LRR (domain present in NAIP, CIITA, HET-E, and TP1 with leucine-rich repeat) proteins or nucleotide-binding oligomerization domains (NODs) have been identified on DCs that, like TLRs, possess the capability to recognize microbes and convert this information to elicit appropriate downstream signaling events. 54
- 55. • When the transition from gingivitis to periodontitis occurs,it is accompanied by a shift in lymphocyte populations whereby T cells form the majority of lymphocytes in the inflammatory infiltrates in gingivitis tissues, while there is an increase in the numbers of B cells and plasma cells in periodontitis lesions. Seymour et al - 1979 • Different antigen-presenting cells (APCs) have been suggested to direct T cells to a Th1 or Th2 pathway, possibly via presentation of different antigenic epitopes involving different second signals resulting in the secretion of different cytokine patterns. Bloom et al - 1992 • Macrophage numbers do not increase and there is little evidence of macrophage activation in advanced periodontitis compared with minimally inflamed tissues Chapple - 1998 • Consistent with this, it has been demonstrated that, in the progression from gingivitis to periodontitis, there is a decrease in the macrophage/B-cell ratio Gemmel - 2000 55
- 56. 56
- 57. • Dendritic cells, Langerhans cells, and certain tissue macrophages are also equipped to capture microorganisms and self-antigens by the expression of distinct sets of C-type lectin receptors. • Many of the C-type lectin receptors are expressed in human gingiva, including the Langerhans cell-specific marker Langerin (CD207), macrophage mannose receptor (CD206), DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN; CD209), with Langerin decreasing in the gingival epithelium in chronic periodontitis, while the macrophage mannose receptor and DC-specific intercellular adhesion molecule-3 grabbing nonintegrin increase in the lamina propria in chronic periodontitis. • The active lesions of periodontal disease harbor both mature and immature DCs located in different compartments of the affected periodontal tissues surrounding bone. 57
- 58. • Moreover, DCs in the actively inflamed tissues of chronic periodontitis form aggregates with T cells, where the DC/ T-cell interactions through receptor activator of nuclear factor-B-ligand (RANK-RANKL) signaling take place. • The DCs are thought to indirectly influence the periodontal bone loss through mediation of inflammation, but recently it is seen that human peripheral blood monocyte-derived DCs can transdifferentiate into osteoclasts in the presence of macrophage colony-stimulating factor and RANKL in vitro, suggesting a direct involvement of DCs in osteoclastogenesis Theill LE 2002. 58
- 59. Conclusion • The APC form a very vital component of the human immune mechanism. • The role of the different APCs in the immune mechanism still needs more elaboration and research. • The DCs play a central role in the immune response and its interaction with cytokines and T cells is an important component of the immune cascade. • The detailed understanding of the APCs, especially DCs, will help in developing the therapeutic strategies for various inflammatory and autoimmune diseases. 59
- 60. References • Cellular and Molecular Immunology 8th Ed. (2015) by Abbas et al. – Chapter 6: MHC Molecules and Antigen Presentation to T Lymphocytes 60 • Antigen presenting cells in periodontal disease Neeraj Deshpande, Deepak Dave Indian Journal of Oral Sciences Vol. 3 Issue 2 May-Aug 2012 69. • Antigen Presentation and the MHC Herbert L. Mathews, Ph.D. Host Defense 2013 • Azuma M. Fundamental mechanisms of host immune responses to infection. J Periodontal Res 2006;41:361 73. • Cutler CW, Teng YT. Oral mucosal dendritic cells and periodontitis: Many sides of the same coin with new twists. Periodontol 2000 2007; 45:35 50. • Cutler CW, Jotwani R. Antigen-presentation and the role of dendritic cells in periodontitis. Periodontol 2000 2004 ; 35:135 57. • Cutler CW, Jotwani R, Palucka KA, Davoust J, Bell D, Banchereau J. Evidence and a novel hypothesis for the role of dendritic cells and Porphyromonas gingivalis in adult periodontitis. J Periodontal Res 1999; 34:406 12.
- 61. • Antigen presenting cells in periodontal disease Neeraj Deshpande, Deepak Dave Indian Journal of Oral Sciences Vol. 3 Issue 2 May-Aug 2012 69. • Antigen Presentation and the MHC Herbert L. Mathews, Ph.D. Host Defense 2013 • Azuma M. Fundamental mechanisms of host immune responses to infection. J Periodontal Res 2006;41:361 73. • Cutler CW, Teng YT. Oral mucosal dendritic cells and periodontitis: Many sides of the same coin with new twists. Periodontol 2000 2007; 45:35 50. • Cutler CW, Jotwani R. Antigen-presentation and the role of dendritic cells in periodontitis. Periodontol 2000 2004 ; 35:135 57. • Cutler CW, Jotwani R, Palucka KA, Davoust J, Bell D, Banchereau J. Evidence and a novel hypothesis for the role of dendritic cells and Porphyromonas gingivalis in adult periodontitis. J Periodontal Res 1999; 34:406 12. 61
Notas do Editor
- The innate immune response acts as the first line of defense mediated by release of inflammatory cytokines and chemokines by phagocytic cells. The adaptive immune response acts as the second line of defense and requires some time to respond. The latter is mediated by antigen‑specific response by T cells and B cells and can react to only those microbes that can generate this response. Azuma M.2006 The clinical expression of periodontal disease can be in the form of a relatively stable lesion or the progressive one
- Antigenic determinant – epitope present in antigen will bind to the paratope of antibody or the T cell receptors
- Professional APCs Efficient at phagocytosis and endocytosis Antigen presentation done by MHC class 2 molecules Expression of co-stimulatory molecules is the defining feature of the professional APCs. Some of the professional APCs are dendritic cells, macrophages, certain B cells,certain activated epethelial cells and others. II. Non professional APCs do not constitutively express the MHC class 2 during presentation. are responsible for little role of antigen presentation compared to professional APCs. include fibroblasts, thymic epethilial cells, thyroid epethelial cells, glial cells, pancreatic beta cells, vascular endothelial cells and others.
- IDC :-Express high level of classⅠ, Ⅱ MHC molecules and B7，lack of FcR and CR, can stimulate T cells. FDC :-Lie in follicle of LN, no expression of class Ⅱ MHC, high level of FcR and C3bR. LC Birbeck particle :- Lie in the epithelia of the skin, gastrointestinal and respiratory tracts, express FcR and C3bR. After uptaking antigens, migrating to draining LN and becoming IDC.
- Dendritic cells that are resident in epithelia and tissues capture protein antigens and transport the antigens to draining lymph nodes (Fig. 6-5). Resting tissue-resident dendritic cells (sometimes referred to as immature dendritic cells) express membrane receptors, such as C-type lectins, that bind microbes. Dendritic cells use these receptors to capture and endocytose microbes or microbial products and then process the ingested proteins into peptides capable of binding to MHC molecules. Apart from receptor-mediated endocytosis and phagocytosis, dendritic cells 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 dendritic cells. At the time that microbial antigens are being captured, microbial products are recognized by Toll-like receptors and other innate pattern recognition receptors in the dendritic cells and other cells, generating innate immune responses The dendritic cells are activated by these signals and by cytokines, such as tumor necrosis factor (TNF), produced in response to the microbes. The activated dendritic cells (also called mature dendritic cells) lose their adhesiveness for epithelia or tissues and migrate into lymph nodes.
- The dendritic cells also begin to express a chemokine receptor called CCR7 that is specific for two chemokines, CCL19 and CCL21, which are produced in lymphatic vessels and in the T cell zones of lymph nodes. These chemokines attract the dendritic cells bearing microbial antigens into draining lymphatics and ultimately 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 dendritic cells are concentrated (see Chapter 3). The colocalization of antigen-bearing activated dendritic cells and naive T cells maximizes the chance of T cells with receptors for the antigen finding that antigen.
- Activation also converts the dendritic cells from cells whose primary function is to capture antigen into cells that are able to present antigens to naive T cells and to activate the lymphocytes. Activated dendritic cells express high levels of MHC molecules with bound peptides as well as costimulators required for T cell activation. Thus, by the time these cells arrive in the 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.
- Role of Dendritic Cells in Antigen Capture and Display Microbes and protein antigens are transported after being collected from their portal of entry into the peripheral lymphoid organs with the use of the numerous lymphatic capillaries that drain into the regional lymph nodes. (Furthermore, if microbial antigens produced from any tissue colonized/infected by a microbe, ) Initiation of the primary responses of naïve T cells are done in these peripheral lymphoid organs Some antigens are transported in the lymph by APCs (primarily dendritic cells) that capture the antigen and enter lymphatic vessels, and other antigens enter the lymphatics in cell-free form. Thus, the lymph contains a sampling of all the soluble and cell-associated antigens present in tissues. The antigens become concentrated in lymph nodes, Antigens that enter the blood stream may be similarly sampled by the spleen. The cells that are best able to capture, transport, and present antigens to T cells are the dendritic cells. Now lets discuss dendritic cells
- PROCESSING OF PROTEIN ANTIGENS The pathways of antigen processing convert protein antigens present in the cytosol or internalized from the extracellular environment into peptides and load these peptides onto MHC molecules for display to T lymphocytes (Fig. 6-14). The mechanisms of antigen processing are designed to generate peptides that have the structural characteristics required for associating with MHC molecules, and to place these peptides in the same cellular location as newly formed MHC molecules with available peptide-binding clefts. Peptide binding to MHC molecules occurs before cell surface expression and is an integral component of the biosynthesis and assembly of MHC molecules. In fact, as mentioned earlier, peptide association is required for the stable assembly and surface expression of class I and class II MHC molecules. Protein antigens that are present in the cytosol (usually synthesized in the cell) generate class I–associated peptides that are recognized by CD8+ T cells, whereas antigens internalized from the extracellular environment into the vesicles of APCs usually generate peptides that are displayed by class II MHC molecules and recognized by CD4+ T cells. The different fates of cytosolic and vesicular antigens are due to the segregated pathways of biosynthesis and assembly of class I and class II MHC molecules (see Fig. 6-14 and Table 6-5).
- The Class I MHC Pathway for Processing and Presentation of Cytosolic Proteins Class I MHC–associated peptides are produced by the proteolytic degradation of mainly cytosolic proteins in proteasomes, and the generated peptides are transported into the endoplasmic reticulum (ER), where they bind to newly synthesized class I molecules. Sources of Cytosolic Protein Antigens Most cytosolic protein antigens are synthesized within cells, some are injected into the cytosol via bacterial secretory mechanisms, and others are phagocytosed and transported from vesicles into the cytosol. Foreign antigens in the cytosol may be the products of viruses, bacteria, 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 18). 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. AS LONG AS ANTIGEN IS IN THE CYTOSOL 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. Although microbial proteins that are presented on class I MHC molecules are typically cytosolic, proteins from other cellular compartments may also enter the class I MHC antigen processing pathway. 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. In addition, nuclear proteins may be processed by proteasomes in the nucleus and presented on class I MHC molecules.
- Digestion of Proteins in Proteasomes Ubiquitinated proteins, with chains of four or more ubiquitins, are recognized by the proteasomal cap and then are 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 them completely into single amino acids). There are mechanisms wherein IFN-γ can ENHANCE antigen presentation By increased expression of MHC molecules Increased transcription and synthesis of 3 novel catalytic subunits of proteosome: 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 such that the peptides produced usually contain carboxy-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. Thus, proteasomes are organelles whose basic cellular function has been adapted for a specialized role in antigen presentation.
- Transport of Peptides from the Cytosol to the Endoplasmic Reticulum Peptides generated in proteasomes are translocated by a specialized transporter into the ER, where newly synthesized class I MHC molecules are available to bind the peptides. This delivery is mediated by a dimeric protein called transporter associated with antigen processing (TAP), which is a member of the ABC transporter family of proteins, many of which 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 earlier, 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.
- 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 non-polymorphic regions of the class I molecule.
- The Class II MHC Pathway for Processing and Presentation of Vesicular Proteins The generation of class II MHC–associated peptides from endocytosed antigens involves the proteolytic degradation of internalized proteins in endocytic vesicles and the binding of peptides to class II MHC molecules in vesicles. Generation of Vesicular Proteins Most class II MHC–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 12). 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 denser 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. 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, cytosolic 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.
- Association of Processed Peptides with Class II MHC Molecules in Vesicles Within the endosomal vesicles, the invariant chain 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 invariant chain blocks access to the peptide-binding cleft of class II MHC molecules, it must be removed before complexes of peptide and class II MHC molecules can form. The same proteolytic enzymes that generate peptides from internalized proteins, such as cathepsins, also act on the invariant chain, 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 must 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 MHC molecules in the MIIC endosomal compartment. Unlike class II MHC molecules, HLA-DM molecules 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. If peptides with a higher affinity for the class II MHC cleft than CLIP are available in endosomes they will be able to displace CLIP because of the HLA-DM mediated exchange mechanism. If higher affinity peptides are not available CLIP will remain in the class II MHC cleft and these molecules will not undergo the presumed conformational change and stabilization required to efficiently travel to the cell surface. 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.
- Schematic illustration of T-cell differentiation. Early inflammatory signals from macrophages and peripheral dendritic cells such as interleukin-1 G3 (IL-1 j3) activate the CD4+ T-helper (Th) T cell. The Th cell then produces many cytokines, including IL-2, interferon-y (IFN 7 ) and IL-4, and is referred to as a ThO T cell. The ThO T cell further differentiates to form Thl or Th2 T cells, which regulate immune responses against intracellular and extracellular antigens, respectively. A key signal determining which pathway the T cell follows is the level of IL-12 provided by the antigen-presenting cell. High levels of IL-12 favor the Thl pathway, and low levels of IL-12 favor the Th2 pathway. Subsequently, the Thl phenotype is favored in the presence of Thl cytokines such as IFN ,1, which stimulates Thl differentiation and blocks Th2 differentiation. Similarly, the Th2 cytokines such as IL-4 block Thl differentiation while promoting Th2 differentiation. The Th3 T cell, which produces IgA and functions in mucosal defense, is not illustrated here. Ag-MHC, Antigen major histocompatibility complex; NK, natural killer; TNF-0, tumor necrosis factor beta.