Overview of immunology

Introduction Overview of immunology Masayuki Miyasaka, MD, PhD Interdisciplinary Program for Biomedical Sciences WPI Immunology Frontier Research Center (IFReC) Osaka University Inside our body, we have an amazing protection mechanism called the immune system, which is designed to defend us against millions of bacteria, viruses, fungi, toxins and parasites. However, this system is not perfect, and sometimes it fails. 1

What is immunity? Immunity Immunis in Latin exemption from military service, tax payments, etc. protection from disease Immunity is defined as resistance to disease, specifically infectious diseases. Why is the immune system so important? Defense against infections Defense against newly arising transformed cells (tumor cells) Elimination of non-self (ex. newly introduced substances or molecules, such as tissue grafts) Induction of tissue injury/inflammation 2

Vaccination is very effective for certain infectious diseases Measles Rubella Mumps Polio 900,000/1940 s 60,000/1940 s Almost none 150,000/1960 s 20,000/1950 s Almost none among vaccinated children 0 200,000 400,000 600,000 800,000 1,000,000 cases/year How do we recognize invaders? 3

Mechanism of non-self recognition Immune cells bear sensors against these determinants Antigen: any substance that evokes an immune response Epitope: part of an antigen that is recognized by the immune system = antigenic determinant Innate immunity acts first, and adaptive immunity follows Microbial invasion Responses by the innate immune system Non-specific Innate to the host Cellular components Macrophages, Neutrophils, Dendritic cells, Natural killer cells Responses by the adaptive immune system Highly specific Acquired Cellular components Lymphocytes T cells, B cells, 4

Innate immunity provides the initial defense against infections, and adaptive immunity follows next to provide more specific and long-lasting protection Innate immunity Microbes Complement Epithelial barriers Phagocytes (macrophages, neutrophils, dendritic cells) NK cells Time after infection HOURS 6 12 Innate immunity provides the initial defense against infections, and adaptive immunity follows next to provide more specific and long-lasting protection Adaptive immunity B lymphocytes Antibodies T lymphocytes Effector T-Cells DAYS 1 3 5 Time after infection 5

Cells and molecules involved in innate immunity cells molecules Epithelial cells Defensins, IL-25, -33, -TSLP, etc Macrophages G-CSF, M-CSF, IL-1, -6, -8, -10, -12, etc Neutrophil phagocytes G-CSF, IL-1, -6, TNF, CCL3, CCL4 etc Dendritic cells GM-CSF, IFN, IL-1, -6, -10, -12, etc NK cells Granzymes, Perforin, IL-17A, -22, etc NKT cells IL-4, -17, IFN, etc Innate lymphoid cells (ILCs) Group 1 ILCs - tissue-resident immune cells IFN, TNF Group 2 ILCs IL-4, -5, -9, -33 Group 3 ILCs IL-17A, -22 Liver cells Complement components The complement system: consists of over 25 proteins and protein fragments, which are generally synthesized by the liver. They normally circulate as inactive precursors in the blood. When stimulated by one of several triggers, such as microbial products, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end-result of this activation cascade is massive amplification of the response and activation of the cellkilling MAC (membrane attack complex). Killing of microbes 6

Complement activation cascades Classical Pathway Alternative Pathway Antigen-antibody Complex Activates C1 C2 C4 Factor B Factor D Factor P(properdin) Microbe C1 C3 C4b C2a Polysaccharides on surface of microbe Antigen Antibody C3b C5 C4b C2a C3a C5b, C6, C7, C8, C9 Form MAC, which causes cytolysis of microbe C3a, C4a, C5a Blood vessel dilatation Histamine release from mast cells Recruitment of phagocytes C5a C5b C6 C5b C6 C7 C8 C3b Promotion of microbial phagocytosis C7 Membrane attack complex (MAC) C8 C9 Microbial membrane There are three pathways of complement activation. The consequences of complement activation are the recruitment of inflammatory cells, the opsonization of pathogens, and the direct killing of pathogens CLASSICAL PATHWAY Antigen: Antibody Complex Recruitment of inflammatory and immunocompetent cells ALTERNATIVE PATHWAY Pathogen surfaces Complement activation Promotion of phagocytosis LECTIN PATHWAY Lectin binding to pathogen surfaces Killing of pathogens 7

How is the innate immune system activated? Sensors of the innate immune system = Innate immunity receptors Innate immunity receptors are mainly activated by two types of molecular patterns; pattern recognition receptors PAMPs (pathogen-associated molecular patterns): microbial molecules that stimulate innate immunity, such as microbial DNAs, RNAs, and cell wall components DAMPs (damage/danger-associated molecular patterns): molecules that are released from damaged or necrotic cells, such as HMGB1, S100 proteins, uric acid, and purine metabolites. 8

Innate immunity receptors are expressed ubiquitously Bacterial cell Extracellular TLRs Microbial polysaccharide Lectin receptors Plasma membrane Endosome Cytosolic NOD-like receptors Nucleic acids of ingested microbes TLRs Bacterial Peptidoglycans Products of damaged cells RIG-like receptors Endosomal membrane Viral RNA TLRs respond to various PAMPs and DAMPs Bacterial peptidoglycan Bacterial Lipopeptiodes LPS Bacterial Flagellin TLR-1:TLR-2 TLR-2 TLR-4 TLR-5 Bacterial lipopetides TLR-2:TLR-6 Different TLRs respond to many different, structurally diverse products of microbes. Plasma membrane MD2 Endosome Endosomal TLRs respond to only nucleic acids. TLR-3 dsrna TLR-7 ssrna TLR-8 ssrna All TLRs contain a ligand-binding domain composed of leucine-rich motifs and a cytoplasmic signaling domain (TIR domain). TLR-9 CpG DNA 9

TLR engagement leads to activation of NF-kB and IRFs, resulting in induction of anti-viral states, acute inflammation, and stimulation of adaptive immunity Leucine-rich repeats TLR engagement by bacterial or viral molecules Toll-IL-1 receptor (TIR) signaling domain Recruitment of adaptor proteins Nucleus NF-КB Activation of transcription factors IRFs (interferon regulatory factors) Increased expression of: cytokines, adhesion molecules, costimulatory molecules Acute inflammation Stimulation of adaptive immunity Production of type 1 interferon (IFNαβ) Antiviral state Pathogenic bacteria Extracellular ATP NLRP-3 (sensor) Adaptor Caspase-1 (inactive) Innate signals (e.g.,tlrs) + + K + K + Plasma membrane Bacterial products Crystals K + efflux Reactive oxygen species NLRP-3 inflammasome Caspase-1(active) Engagement of NOD-like receptors stimulates the inflammasome complex that proteolytically generates the active form of IL-1 from an inactive precursor Upon sensing of PAMPs/DAMPs, NLRP-3 oligomerizes with an adaptor protein and an inactive form of caspase-1. Once recruited, caspase-1 is activated, and cleaves pro-il-1 to generate biologically active IL-1. Pro-IL1β gene transcription Pro-IL 1β IL-1β Secreted IL-1β Acute inflammation IL-1 induces acute inflammation and causes fever. Proinflammatory cytokines: IL-1, IL-18, TNF, IL-6.. Nucleus 10

Innate immunity vs. adaptive immunity Specificity Receptors Nature of receptors Self/non-self discrimination Innate immunity Broad; recognizes molecular patters (PAMPs and DAMPs) TLRs, NLRs, RLR, lectins, Limited diversity Encoded in germline Non-clonal: all cells have identical receptors Yes Adaptive immunity Fine; recognizes antigens or structural detail of microbial or non-microbial molecules BCR (immunoglobulins) & TCRs Greater diversity Encoded by genes that undergo gene rearrangements and somatic mutations Clonal: each lymphocyte clone expresses a unique receptor Yes (due to positive and negative selection in the primary lymphoid tissues) Cells and molecules involved in adaptive immunity Cells Receptors Triggering molecules Secreted molecules Th1 TCR stimulation + IL-12 IL-2, IFN etc Th2 TCR stimulation + IL-4 IL-4, -5, -6, -10, -13, etc T cells Th17 TCR TCR stimulation + TGF, IL-1b, -6, -23 IL-17A, -17F, -21, -22, etc Treg TCR stimulation + IL-12, TGFβ, etc IL-10, -35, TGF, etc Tc TCR stimulation + IL-12 perforin,granzymes,etc B1 T-independent antigen IgM (natural antibodies) B cells B2 BCR antigen + 2 nd signal from dendritic cells IgM, IgG, IgA, IgE, etc 11

Lymphocytes express antigen-specific receptors αβ heterodimer T cells T cell receptor (TCR) (recognizes peptide-mhc complex presented by dendritic cells) B cells 2H+2L tetramer B cell receptor (BCR) (recognizes antigen (proteins, sugars) in the absence of dendritic cells) Two arms of the adaptive immune system: Humoral immunity vs. cell-mediated immunity Humoral immunity Extracellular microbes B lymphocyte Selected antibody Block infections and eliminate extracellular microbes Microbe Responding lymphocytes Effector mechanism Functions 12

Two arms of the adaptive immune system: Humoral immunity vs. cell-mediated immunity Cell-mediated immunity phagocytosed microbes in dendritic cell Helper T lymphocyte Activate dendritic cells to kill phagocytosed microbes Intracellular microbes (e.g., viruses) replicating within infected cell Cytotoxic T lymphocyte Kill infected cells and eliminate reservoirs of infection Microbe Responding lymphocytes Effector mechanism Functions Properties of adaptive immune responses Feature Specificity Adaptive immunity Highly specific; clonal responses Diversity Memory Duration Non-reactivity to self Successful response to a large variety of antigens More rapid, effective response to a repeated exposure to an antigen compared with the primary response Regulated response with limited duration No concomitant tissue injury; self-reactivity is normally down-regulated 13

Each lymphocyte bears a single type of receptor with a unique specificity, and antigen binding is required for cell activation Antigen X Anti-X antibody Lymphocyte precursor Mature Lymphocyte clones Antigen Y Anti-Y antibody Lymphocytes respond to antigen at the clonal level Clone 1 Ag receptor Clone 2 Clone 3 Clone 4 Antigen Membrane-bound antigen receptors are secreted as antibody molecules from plasma cells Clone X A specific clone is selected for proliferation by antigen = clonal selection theory 14

The secondary immune response occurs more rapidly and more vigorously than the primary immune response Antigen X Antigen X + Antigen Y Activated B cells Serum antibody titer Naïve B cells Primary anti-x response Activated B cells Naïve Memory B cells B cells Secondary anti-x response Primary anti-y response Activated B cells 2 4 6 8 10 12 Weeks Lymphocytes are produced in primary lymphoid organs. After maturation, lymphocytes enter the peripheral lymphoid organs, where they may encounter Ag. They subsequently recirculate in the blood and lymph Primary lymphoid organs B Bone Iymphocyte marrow lineage stem cell Bone marrow Mature B lymphocytes Secondary lymphoid organs Blood Recirculation Lymph nodes Spleen T Iymphocyte lineage Thymus Mature T lymphocytes Blood, lymph Recirculation Peripheral Lymphoid tissues 15

Microbe Antigens that entered lymphatic vessels are transported to lymph nodes, whereas those that entered blood vessels are transported to the spleen Free Antigen in Tissue Connective tissue Lymphatic vessel To lymph node antigen-loaded dendritic cells Blood vessel To circulation and spleen Epithelium Antigen that enters bloodstream Microbial antigens are displayed in peripheral lymphoid tissues for recognition by T cells Lymph node Lymph node captures antigen from epithelium and connective tissue Blood-borne antigen are captured by antigenpresenting cells in the spleen Upon encounter with antigen, naïve cells differentiate into effector cells and memory cells Cell Type Naϊve cells Stage Effector cells Memory cells B lymphocytes Antigen recognition Proliferation Differentiation Helper T lymphocytes Antigen recognition Proliferation Differentiation 16

Properties of naïve, effector and memory cells Naïve cells Effector cells Memory cells Cell cycle quiescent cycling quiescent Life span weeks-months usually short (days) long (years) Effector function none B cells: antibody secretion Th cells: cytokine secretion Tc cells: cell killing none Migration to lymph nodes to peripheral tissues (sites of infection) to lymph nodes, bone marrow, How do T cells recognize antigen? 17

Dendritic cells play an essential role in T cell activation pathogen Dendritic cell MHC TCR peptide T cell Costimulatory molecules Antigen uptake and degradation Antigen presentation Antigen recognition by T cells T cell activation requires not only TCR ligation with antigen (MHC-peptide complex) but also costimulation Antigen recognition T cell response Resting APC (lacking in costimulatory molecule expression) CD28 Naïve T cell Activation of APCs by microbes, innate immune response B7 CD28 Cytokines (e.g.,il-12) IL-2 No response Effector T cells Activated APC: Increased expression of costimulatory molecules, Increased secretion of cytokines T cell proliferation and differentiation 18

T cell activation requires not only TCR ligation with antigen (MHC-peptide complex) but also costimulation Antigen recognition T cell reponse Resting (contimulatordeficient)apc Activated APC: increased expression of costimulators, secretion of cytokines B7 CD28 Activation of APCs by microbes, innate immune response Native T cell TCR-[MHC+peptide] interactions provide signal 1, whereas interactions among costimulatory molecules No provide response signal 2 to T cells Cytokines (e.g.,il-12) IL-2 Effector T cells T cell activation requires signal 1 (Ag specific) and signal 2 (Ag nonspecific). T cell proliferation and differentiation MHCs are the markers of self Every cell in our body carries the same set of distinctive surface proteins that distinguish us as self. This set of markers on our cells is called the major histocompatibility complex (MHC). There two classes; MHC Class I proteins, which are on all cells, and MHC Class II proteins, which are only on certain specialized cells ( =antigen presenting cells). 19

MHC genes are highly polymorphic Chromosome 6 Human: HLA Class II MHC locus DP DQ DR Class III MHC locus Class I MHC locus B C A DM Proteasome Components; TAP1, TAP2 Complement proteins: C4, factor B, C2 Cytokines: LTβ, TNF-α, LT >150 alleles >200 >1,000 >2,000 >1,000 >1,500 MHC genes are co-dominantly expressed = the alleles inherited from both parents are expressed equally Identical twin brothers express identical MHCs Nobu Masa rheumatologist immunologist 20

MHC molecules are membrane proteins on antigen-presenting cells that display peptide antigens for recognition by T lymphocytes MHC Class I Peptide-binding cleft Peptides MHC Class II Peptide-binding cleft Peptides α1 α 2 α1 α 2 α1 β 1 α1 β 1 N N α3 α 3 α 2 N N β 2 β 2 - microglobulin C Transmembrane region C β2m Disulfide bond Ig domain expressed by all nucleated cells Transmembrane region α 2 β 2 C C expressed by DCs, macrophages, B cells Binding of peptides to MHC molecules MHC class I molecule Each MHC molecule can present only one peptide at a time, but is capable of presenting many different peptides α1 MHC class molecule α2 9 peptides Class I MHC molecules acquire peptides from cytosolic proteins α1 β1 15 peptides (10 30) Class II MHC molecules acquire peptides from proteins in intracellular vesicles (internalized proteins) Pockets in floor of peptide binding groove of MHC class II molecule Peptide Anchor residues 21

The MHC class I pathway vs. MHC the class II pathway Class I MHC pathway Cytosolic microbe Peptides in cytosol CD8 + CTL Mcrobial protein Unfolded protein Class I MHC ER CD8 T cells recognize antigen presented in the cleft of MHC class I proteins Class II MHC pathway CD4 + T cell Endocytosis of extracellular microbe Endocytic vesicle Class II MHC Invariant chain (I i ) ER CD4 T cells recognize antigen presented in the cleft of MHC class II proteins Antigen uptake Antigen processing MHC biosynthesis Peptide-MHC association Ag presentation to T cells How do B cells recognize antigen? 22

Lymphocytes respond to antigen at the clonal level Clone 1 Ag receptor Clone 2 Clone 3 Clone 4 Antigen Membrane-bound antigen receptors are secreted as antibody molecules from plasma cells Clone X A specific clone is selected for proliferation by antigen, and eventually produces antibody molecules Antibody molecules exist as either membrane-bound form (=Ag receptor) or secreted form Antigenbinding site N N Membrane IgM C L C H 2 C H 3 V L C H 1 Antigenbinding site N N V H H: heavy chain L: light chain V: variable region C: constant region Secreted IgG Antigenbinding site Heavy N N chain Fc receptor/ Complement binding site Light chain Hinge V L C L C H 1 C C C H 2 C H 3 Antigenbinding site N N V H Fab region Fc region Plasma Membrane of B cells C C C H 4 S-S Disulfide bond Ig domain Tail piece C C Abbas and Lichtman: Basic Immunology, 3 rd Ed. 23

There are at least 5 different Ig isotypes Upon antigen stimulation, IgM is produced first IgG Major Ig against invading pathogens IgE Binds to allergens, triggers histamine release IgD Bound to membrane together with IgM IgA Found in mucosa and prevents colonization by pathogens IgM Early stage protection Subsequent to antigen-induced proliferation, B cells undergo isotype switching to produce antibodies of different isotypes Isotype switching Plasma cells IgM Principal effector functions Complement activation (classical pathway) B cell Helper T cells; CD40L, cytokines IFN-γ IL-4 Cytokines produced in mucosal tissues, e.g., TGF-β, BAFF, others IgM IgG subclasses IgE IgA IgG subclasses (IgG1,IgG3) IgE IgA Fc receptor-dependent phagocyte responses; complement activation; neonatal immunity (placental transfer) Immunity against helminths Mast cell degranulation (Immediate hypersensitivity) Mucosal immunity (transport of IgA through epithelia) neutralization of microbes and toxins 24

Antigen receptor diversity is generated by the use of different combinations of V, D, J gene segments (combinatorial diversity) Germline DNA Somatic recombination (D-J joining) Somatic recombination (V-DJ joining) D1-Dn J1-6 V1 Vn Cμ 5 3 >100 ~30 6 D1J1 V1 Vn Cμ 5 3 V1D1J1 Cμ 5 3 Heavy chain diversity = > 100 x 30 x 6 = > 10,000 combinations Transcription RNA processing (splicing) V1D1J1 Cμ 5 3 V1D1J1 Cμ AAA Somatic recombination of different gene segments Translation V CDR3 Cμ Antigen receptor diversity is initiated by recombination of gene segments that code for the variable regions of the receptors, and diversity is generated during this process The VDJ recombinase is expressed in lymphocytes Number of V gene segments Number of diversity(d) gene segments Number of joining(j) gene segments Combinatorial diversity: Immunoglobulin Heavy chain Light chain κ T cell receptor ~100 35 54 67 27 0 0 2 6 5 61 4 Mechanism specifically V1 D1J1 C Vn D2J2 C α β The total potential repertoire of immunoglobulin is the order of 10 11, and this is even larger with TCR Number of possible V-(D)-J combinations Junctional diversity: Total potential repertoire with junctional diversity Ig:~10 6 TCR:~3 10 6 V1 D1J1 V1 D1J1 C V1 D1 J1 C Removal of nucleotides Ig:~10 11 TCR:~10 16 C Addition of nucleotides (N region orp nucleotides) 25

Antigen receptors are non-covalently attached to other invariant molecules whose function is to deliver to the inside of the cell the activation signals that are triggered by antigen recognition Antibody (Immunoglobulin) T cell receptor (TCR) Membrane Ig TCR Antigen MHC Igα Signal transduction Igβ Antigen Signal transduction CD3 Antigen-presenting cell (APC) Innate immunity provides the initial defense against infections, and adaptive immunity follows next to provide more specific and long-lasting protection Innate immunity Microbes Complement Epithelial barriers Phagocytes NK cells Time after infection HOURS 6 12 26

Innate immunity provides the initial defense against infections, and adaptive immunity follows next to provide more specific and long-lasting protection Adaptive immunity B lymphocytes Antibodies T lymphocytes Effector T-Cells DAYS 1 3 5 Time after infection Introduction Overview of immunology Masayuki Miyasaka, MD, PhD Interdisciplinary Program for Biomedical Sciences WPI Immunology Frontier Research Center (IFReC) Osaka University 27