Chapter 1 Document PDF

Immune Mechanisms Section 1. Basic Science 1 ANTIGENS An antigen – a contraction of “antibody generator” – is any substance that gives rise to a specific immune response or is recognized by lymphocytes or antibodies. Definitions • Antigen is a molecule or substance that is recognized by the imm...

Immune Mechanisms Section 1. Basic Science 1 ANTIGENS An antigen – a contraction of “antibody generator” – is any substance that gives rise to a specific immune response or is recognized by lymphocytes or antibodies. Definitions • Antigen is a molecule or substance that is recognized by the immune system • Immunogen is a molecule that induces an immune response, used interchangeably wit\ h antigen • Hapten is a small-molecule antigen that, when bound to a larger carrier such as a protein, is capable of eliciting the production of antibodies; hapten-specific B cells interact with carrier-specific T cells to generate these antibodies; once an antibody to a hapten is generated, the carrier is no longer needed to stimulate an immune response • Epitope is the specific portion of a macromolecular antigen to which an antibody or T cell receptor binds Key Fact Haptens require a carrier protein to stimulate an initial antibody respo\ nse. • Carrie r is a macromolecular substance to which a hapten is coupled in order to produce an immune response against the hapten • Adjuvants are molecules given in vaccines to enhance the body’s immune response to an antigen; typically this involves activating the innate immune system, which leads to costimulatory expression, and cytokine production, which stimulates an adaptive immune response; examples: ° Alum – Used most commonly in humans ° AS01B – monophosphoryl lipid A (MPL), isolated from the surface of bacteria, and QS-21, a natural compound extracted from the Chilean soapbark tree (Quillaja saponaria M\ olina) 2 / CHAPTER 1 Key Fact Adjuvants are necessary to achieve maximal adaptive immune response to a\ vaccine. • Superantigen: ° Antigens (Table 1-1) that activate a large number of polyclonal T lymphocytes by directly binding to the Vβ domain of the T cell receptor and the external surface of the MHC molecule (Figure 1-1\ ) ° Cause massive cytokine release Table 1-1. Superantigens Source ToxinDisease Staphylococcus aureus SEBFood poisoning SEC2 Food poisoning TSST Toxic shock syndrome Streptococcus pyogenes SPE-CStreptococcal toxic shock syndrome Abbreviations: SEB, staphylococcal enterotoxin B; SEC, staphylococcal enterotoxin C; SPE-C, streptococcal pyrogenic exotoxins C; TSST, toxic shock syndrome toxin. Superantigens stimulate all T cells in an individual that express a particular family of V β chain (variable portion of the β chain) of the T-cell receptor (TCR). They bind to the V β chain of the TCRs and class II MHC molecules outside of the peptide binding cleft, so are capable of activating a much larger polyclonal T-cell response than a typical antigen (Figure 1-1). Unchecked stimulation of T cells by superantigens can lead to “cytokine storm.” Key Fact Superantigens bind the V β region of TCRs that are outside of the peptide-binding groove on the MHC molecule.\ Figure 1-1. Superantigen. IMMUNE MECHANISMS / 3 • Cytokine Release Syndrome (also known as Cytokine Storm) is an acute multi-organ inflammatory syndrome in response to infection, immune therapy ° Results in increased levels of inflammatory cytokines (particularly IL-6, TNF-α, and IL-10) and activation of T lymphocytes, macrophages, and endothelial cells ° Results in complement activation, vascular leakage, activation of coagulation cascade, cardiomyopathy, DIC Types of Antigens Table 1-2 summarizes the types of antigens. Table 1-2. Types of Antigens Antigen Immune Cell Involved Surface Molecule Involved B-Cell Response Vaccines Protein Follicular B cells T cells “T-cell dependent” MHC class I MHC class II Isotype switch Affinity maturation Induced memory response Diphtheria Tetanus Polysaccharide Marginal zone B cells B1 cells Macrophages “T-cell independent” No isotype switch Predominantly IgM No affinity maturation Limited memory 23-valent Pneumococcal vaccine Typhoid vaccine Nucleic acids B cells CTL and DCs MHC class I MHC class II TLRs DNA vaccines in clinical trials Lipids NKT cells γδ T cells MHC-like CD1 (NKT) Abbreviations: CTL, cytotoxic T lymphocyte; DCs, dendritic cells; MHC, major histocompatibility complex; NKT, natural killer T cell; TLR, Toll-like receptor. Antigen Structure Epitope (Antigenic Determinant) – Antigenic component identified by a unique antibody. A single antigen may have many different epitopes. Recognized by B lymphocyte: • Linear determinants or tertiary structure in “native” conformation • Carbohydrates, amino acids (4 to 8 residues), nucleic acids, and phospholipids Recognized by T lymphocyte: • Linear determinants of amino acids only • Length limited by MHC binding cleft (MHC class I 8–11 aa and MHC class II 10–30 aa) An understanding of antigen composition and factors influencing immunogenicity and tolerogenicity is critical to immunization development and the assessment of response to i\ mmunizations (Table 1-3). 4 / CHAPTER 1 Table 1-3. Factors That Determine the Immunogenicity and Tolerogenicity of Protein Antigens Favor Stimulation of Immune ResponsesFavor Tolerance Persistence Short-lived (eliminated by immune response) Prolonged, leading to persistent antigen receptor engagement Portal of entry; location Subcutaneous, intradermal; absence from generative organs Intravenous, mucosal; presence in generative organs Presence of adjuvants Antigens with adjuvants: stimulate helper T cells Antigens without adjuvants: absence of costimulation Properties of APCs Mature dendritic cells: High levels of costimulators Immature (resting) dendritic cells: Low levels of costimulators and cytokines Key Fact Conjugated vaccines are T-independent antigens linked to a carrier protein, which can trigger a T-dependent response and memory. Examples of conjugated vaccines include the 13-valent pneumococcal vac\ cine (Prevnar 13 ®), Hemophilus influenzae type B (Hib) vaccine, and meningococcal vaccines\ (MCV4-Menactra ® and Menveo ®). MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) MHC molecules, also known as human leukocyte antigens (HLA), are located on the short arm of chromosome 6. Molecules encoded in this region are involved in antigen presentation, inflammation regulation, complement system, and the innate and adaptive immune respon\ ses. Key Fact MHC molecules are expressed co-dominantly, which means an individual expresses one haplotype from each parent. MHC inherited from both parents is expressed on cell surfaces. Polymorph\ isms in the MHC locus influence many biological traits and individuals’ susceptibility to autoimmune and infectious diseases. Shared feature of MHC molecules: • Single binding site and cell-membrane bound • Interaction with T lymphocyte requires direct contact (via TCR and the immune synapse) IMMUNE MECHANISMS / 5 Structure Table 1-4 summarizes differences between MHC class I and MHC class II molecules. Table 1-4. Structure of MHC Class I and Class II Molecules MHC Class IMHC Class II GenesHLA-A, -B, and -C HLA-DP, -DQ, and -DR Polypeptide chains (domains) α chain (α 1, α 2, α 3) β 2-microglobulin α chain (α 1, α 2) β chain (β 1, β 2) Restriction CD8 CD4 Binding site for TCR (nonpolymorphic) α 3 binds CD8 β 2 binds CD4 Binding site for peptide (polymorphic) α 1 and α 2 α1 and β 1 Peptide-binding cleft Peptides 8–11 amino acids Peptides 10–30 amino acids Antigenic sampling Intracellular Extracellular Location Nucleated cells Antigen presenting cells MHC Distribution Key Fact CD8 only recognizes MHC class I molecules. CD4 only recognizes MHC class\ II molecules. This restricts which type of T cell may be activated by different APCs. Key Fact T cells only recognize antigens that are presented as part of the MHC com\ plex (“MHC restriction”), which is restricted to peptide. Lipids, nucleic acids, and polysaccharides are not presented by\ MHC molecules. Features of MHC expression are reviewed in Table 1-5. MHC expression is induced by cytokines produced by innate and/or adaptive immune responses. 6 / CHAPTER 1 Table 1-5. Features of MHC Class I and MHC Class II Expression MHC Class IMHC Class II Constitutive Most nucleated cells APC (dendritic cells, macrophages, and B lymphocytes), thymic epithelia, and activated T lymphocytes Molecules HLA-A, HLA-B, HLA-C HLA-DP, HLA-DQ, HLA-DR Cytokine Interferon IFNα, IFNβ, and IFNγ IFNγ Abbreviation: APC, antigen-presenting cell; IFN, interferon. MHC Genome • MHC molecules are encoded on the short arm of chromosome 6 (Figure 1-2) • The β 2-microglobulin chain is encoded on chromosome 15 (outside of the MHC) • MHC molecules are the most polymorphic of any in the human genome Figure 1-2. Map of the human MHC genome. Key Fact On chromosome 6, there are three regions that encode MHC molecules (in \ order): class II, class III, and class I. In addition to encoding the MHC polypeptides, the MHC genome includes genes whose products are involved with antigen processing. Other class II-like proteins are highly conserved: • HLA-E: NK cell recognition • HLA-F: Localized to endoplasmic reticulum and Golgi apparatus ° HLA-F is expressed primarily in trophoblasts in concert with HLA-E and HLA-G and plays a role in immune defense of the fetus • HLA-G: On fetal-derived placental cells ° Peptide-bound HLA-G-B2M complex acts as a ligand for inhibitory/activating KIR2DL4, LILRB1 and LILRB2 receptors on uterine immune cells to promote fetal development while maintaining maternal-fetal tolerance • HLA-H: Involved in iron metabolism IMMUNE MECHANISMS / 7 The class III region encodes: • Proteins of the complement system: Factor B, C4, and C2 • Heat shock proteins • Cytokines: Tumor necrosis factor TNFα and lymphotoxins α and β Antigen Processing and MHC Presentation Intracellular and extracellular proteins are processed by specific pathways and presented in associa\ tion with MHC class I (Figure 1-3) or MHC class II (Figure 1-4) molecules, res\ pectively. Figure 1-3. MHC class I antigen-processing pathway. MHC I Pathway 1) Newly synthesized MHC class I polypeptides remain sequestered in the endoplasmic reticulum (ER) by interacting with calnexin, calreticulin, Erp57, and tapasin (“chaperone” proteins) 2) Cytoplasmic proteins (that enter) are degraded to antigenic peptides by the proteasome a) The proteasome is a multi-subunit proteinase; four rings of 7-subunits some of which rings have catalytic activity b) Low-molecular-mass polypeptide (LMP)7 and LMP2 subunits (encoded by class II locus) 3) Antigenic peptides are transported into the ER by transporter of antigenic-processing ( TA P) proteins a) Energy-dependent transport of peptides b) TAP1 and TAP2 subunits (encoded in class II locus); both must be present for function c) TAP protein associated with tapasin which brings the TAP transporter into a complex with the MHC Class I molecules 4) Antigenic peptides are loaded onto newly synthesized MHC class I polypeptides 5) MHC class I and peptide are transported to cell surface 8 / CHAPTER 1 Key Fact Viruses develop strategies to evade MHC class I presentation. Herpes simp\ lex virus (HSV) can block TAP transportation, and cytomegalovirus (CMV) can remove MHC class I molec\ ule from ER, inhibit proteasomal activity and/or block MHC synthesis or retention in the ER. MHC II Pathway 1) Extracellular antigen is endocytosed and compartmentalized in cytosolic phagosomes 2) Phagosomes fuse with lysosomes; phagolysosome degrades the microbe into antigenic peptides by proteases (cathepsins) 3) MHC class II is synthesized in the ER and transported to the phagolysosome, forming the MHC class II vesicle 4) The MHC class II-binding cleft is occupied by the invariant chain (Ii) prior to peptide loading 5) In the vesicle, Ii is degraded by proteolytic enzymes, leaving behind a short peptide named class II associated invariant chain peptide (CLIP) 6) HLA-DM removes CLIP and allows antigenic peptides to be loaded in the MHC-binding cleft 7) MHC class II and peptide are transported to cell surface Key Fact HLA-DM is an intracellular protein involved in MHC class II antigen proc\ essing and does not present antigenic peptides nor is it a component of MHC class II. Figure 1-4. MHC class II antigen-processing pathway. IMMUNE MECHANISMS / 9 Cross Presentation Cross presentation is the process by which an APC can present antigens from another cell and activate (prime) T lymphocytes specific for that antigen. • DCs present antigens via MHC Class II antigen processing pathway to CD4+ T lymphocytes • Contemporaneously, the same DCs ingest virus- infected cells or tumor cells • These ingested antigens are transported from vesicles to the cytosol • Antigen peptides are processed via the MHC Class I pathway • Cross presentation occurs when the DC presents these antigens to specific CD8+ T lymphocytes • CD8+ T lymphocytes are primed (activated) • Cross presentation is important for CD8+ T cell responses to virus, tumors, and cytosolic microbes Defect in MHC Expression and Disease Bare Lymphocyte Syndromes (MHC Class I and MHC Class II Deficiencies) The bare lymphocyte syndromes are primary immune deficiencies due to a lack of MHC expression (Table 1-6). Table 1-6. Bare Lymphocyte (MHC Deficiency) Syndromes MHC Class I Deficiency MHC Class II Deficiency Mutation Genes encoding for TAP or tapasin Genes encoding for several transcription factors required for MHC class II expression: MHC2TA, RFX5, FRXAP, FRXANK Inheritance Autosomal recessive Autosomal recessive Clinical features Bacterial sinopulmonary infections Necrotizing granulomatous skin lesions Necrobiosis lipoidica Present with viral, bacterial, fungal and/or protozoal infections Pneumonia/pneumonitis, bronchitis, gastroenteritis, sepsis Infections usually start in 1st year of life Associated with failure to thrive, diarrhea, malabsorption Diarrhea, hepatosplenomegaly, transaminitis, sclerosing cholangitis Laboratory CD8 lymphopenia CD4 lymphopenia Absent germinal centers, hypogammaglobulinemia Treatment Monitor for infection Treat with antibiotics Use aggressive pulmonary toileting and chest PT Hematopoietic stem cell transplantation (HSCT) IVIG Antibiotics Abbreviations: CF, cystic fibrosis; DTH, delayed-type hypersensitivity; IVIG, Intravenous immunoglobulin; MHC, major histocompatibility complex; PBMC, peripheral blood mononuclear cells; PT, physiotherapy; RSV, respiratory syncytial virus; TAP, transporter-associated with antigen presentation. 10 / CHAPTER 1 IMMUNOLOGIC TOLERANCE Definitions • Tolerance : unresponsiveness to antigen ° Self-antigens (ie self-tolerance) or to foreign antigens ° Self-tolerance is part of educating the immune system to not react to se\ lf ° Failure of self-tolerance promotes autoimmunity • Tolerogens: antigens that induce tolerance ° A foreign tolerogen is conditional ° May only induce tolerance under certain conditions (age, antigen concen\ tration of (very low or high)) • Anergy: state of unresponsiveness to antigenic stimulation ° Antigen is recognized but weak signaling, due to a lack of costimulation\ , leads to anergy ° Other factors include antigen type and dose Central Tolerance Occurs in the primary lymph organs (bone marrow and thymus). Central T-Lymphocyte Tolerance • A developing T-lymphocyte is exposed to self-antigen in the thymus • If the T lymphocyte reacts to a self-antigen, there are 2 outcomes: ° (1) apoptosis, which is also known as negative selection (strong binding affinity) ° (2) regulatory T (Treg) cell development (intermediary binding) ° The main factors determining tolerance or negative selection are antigen concentration and affinity to the TCR. High concentration and high affinity promote negative selection The autoimmune re gulatory (AIRE) gene is expressed by medullary thymic epithelial cells (MTECs) . • Promotes expression of nonthymic tissue antigens (neuroendocrine self-peptides) • Self-antigens are presented in the context of MHC class I and II by medullary dendritic cells and macrophages Key Fact Mutation in the AIRE gene leads to autoimmune polyglandular syndrome (APS-1) also know\ n as APECED. Lymphocytes are not deleted or tolerized to endocrine self-antigens durin\ g selection in the thymus. The endocrine organs are attacked by autoreactive T lymphocytes and autoantibodies in the parathyroid glands, adrenal gland\ s, and pancreatic islets. IMMUNE MECHANISMS / 11 Central B-Lymphocyte Tolerance • A precursor B lymphocyte is exposed to self-antigen in the bone marrow during development • If the B lymphocyte reacts to self-antigen, there are 3 outcomes: ° Receptor editing ° Apoptosis (negative selection) ° Anergy Receptor editing involves reactivation of RAG1/2 when a self-antigen is recognized by a B-cell receptor (BCR) • RAG1/2 deletes the previously rearranged VκJκ exon and gives the BCR a new kappa light chain • If unsuccessful, a lambda light chain will be used • If both recombinations recognize self-antigen (failure of receptor editing), the B lymphocyte will be deleted by apoptosis • If there is low antigen concentration, the B lymphocyte may become anergic Key Fact The κ light chains are rearranged first. If receptor editing is needed, a λ light chain will be used. The kappa:lambda light chain ratio can be used to detect plasma cell dis\ orders (ie multiple myeloma). Peripheral Tolerance Occurs in peripheral lymph tissues when a naïve mature B or T lymphocyte encounters self-antigen • T lymphocyte: (1) apoptosis, (2) suppression via T regulatory cells , (3) anergy • B lymphocyte: (1) apoptosis or (2) anergy Peripheral T-Lymphocyte Tolerance • Peripheral tolerance outcomes: Anergy, apoptosis (deletion), or regulation • Lack of a second signal or costimulation produces anergy of peripheral T lymphocytes • Anergy is maintained by blockade of TCR signaling, ubiquitin ligases (which target proteins for degradation), and inhibitor costimulatory molecules (eg, CTLA-4 and PD\ -1) Key Fact Lack of costimulation, or lack of an innate immune system response to th\ e antigen, blunts the required upregulation to produce costimulators (ie, a second signal). T lymphocytes recognize the antigen but receive no support to activate. After repeated recognition without costimulation, the lymphocyte becomes\ unresponsive to that antigen (ie, anergic). Once a cell is anergic, costimulation will not restore activation. 12 / CHAPTER 1 Maintenance of Tolerance Resting (not activated) or immature dendritic cells (DCs) that are p\ resent self-antigen on their surfaces • These DCs do not express receptors • Thus antigen presented to T lymphocytes will not have a second signal, resulting in tolerance • This DC antigen presentation is ongoing and reminds cells to not be self-reactive (Table 1-7) Key Fact Regulatory T cells (Tregs) play a critical role in maintaining normal immune function. Tregs express CD4, CD25, and FoxP3 (a transcription factor). FoxP3 mutation causes immune dysregulation, polyendocrinopathy, and enteropathy X-linked (IPEX) syndrome, which is a fatal autoimmune disorder: triad o\ f watery diarrhea, eczema, and endocrinopathy. (Reminder: E in IPEX is for enteropathy). Table 1-7. Tolerance to Self-Protein Antigen Determinant Immunogenicity Tolerance Exposure Not displayed in central organs, novel protein in MHC (ie abacavir and HLA-B* 5701) Short lived Displayed in central organ (ie endocrine peptides expressed by AIRE in thymus) Prolonged exposure Delivery route Cutaneous and intradermal Oral Adjuvant Antigen with adjuvant Without an adjuvant APCs Mature, activated and costimulators Inactivated (resting) and naïve Abbreviation: APCs, antigen-presenting cells. • Regulatory T cells (Treg) are involved in suppressing the immune response of other cells Treg development depends on the binding affinity between the TCR and self-peptide/MHC II complex ° T cells with strong binding undergo negative selection (apoptosis) ° T cells with weak binding undergo positive selection (effector cells) ° T cells with intermediate binding become Treg cells • Despite being self-reactive, Tregs escape the thymus and help maintain self-tolerance • Tregs express CD4, CD25 (interleukin [IL-2R] α chain), and FoxP3 (Forkhead box P3, a master transcription factor of Treg cells) • Treg survival depends on IL-2 and transforming growth factor beta (TGFβ) • Tolerance or regulation is maintained by secretion of IL-10 and TGF • IL-10 targets macrophages and dendritic cells, and TGFβ inhibits lymphocytes and macrophages Apoptosis is a key regulator of self-reacting T lymphocytes: Intrinsic pathway: Self-antigens repeatedly recognized by a T lymphocyte without costimulation can activate Bim, which is a proapoptotic member of the Bcl-2 protein family. Activated Bim leads to cell apoptosis by binding pro-apoptotic effector proteins (Bax and Bak) which oligomerize and insert into the outer mitochondrial membrane, leading to increased permeability and cell death (mitochondrial pathway). IMMUNE MECHANISMS / 13 Extrinsic pathway: Fas ligand (FasL) (CD95L) is upregulated on repeatedly activated T lymphocytes. FasL can interact with Fas (CD95) on a cell presenting self-antigen or nearby cells, either deleting a self-reactive T lymphocyte or causing the death of an activated cell, thereby downregulating the immune response. The Fas:FasL interaction signals through the caspase system. Key Fact Mutations in Fas or caspase 10 manifest as autoimmune lymphoproliferativ\ e syndrome (ALPS). The lymphocytes do not know when to die and accumulate in the peripheral lymph organs (\ dramatic lymphadenopathy, splenomegaly, lymphoma). A lack of tolerance produces autoimmune features (ie cytopenias). Peripheral B-Lymphocyte Tolerance Because antigens cannot cross-link the BCR on their own, B cells cannot be activated if there is no help from T cells. B cells will then become anergic or be induced to undergo apoptosis. • Self-reactive B cells can become anergic after repeated stimulation by self-antigen • These anergic cells cannot compete with normal naïve B cells for the growth factor BAFF and are thus eliminated • Chronic antigen recognition downregulates CXCR5, inhibiting B-lymphocyte homing and interaction with T lymphocytes, which yields cell death • Self-reactive B cells may also undergo apoptosis via the mitochondrial pathway Central and peripheral tolerance of T and B lymphocytes are summarized in Table 1-8. Table 1-8. Summary of T- and B-Lymphocyte Tolerance T Lymphocytes B Lymphocytes Central Peripheral CentralPeripheral Location of tolerance Thymus Bone marrow Educational phenotype CD4+CD8+ (double positive) CD4+ or CD8+ (single positive) IgM+, IgD+ (immature) Basis for developing tolerance High avidity No costimulators No inflammation (innate) Repeated stimulation Multivalent antigen No T lymphocyte help Chronic stimulation No costimulation Fate of self-recognition Apoptosis (negative selection/deletion) T regulatory cell Anergy (no signal) Apoptosis (deletion) T regulatory cell Receptor editing Anergy Apoptosis (negative selection/deletion) Anergy (no signal) Death (apoptosis) Follicular exclusion 14 / CHAPTER 1 IMMUNOGENETICS DNA • Composed of subunits (or bases) called nucleotides, which include adenine (A), guanine (G), thymine (T), and cytosine (C) • A and G are purines, and T and C are pyrimidines • Organized into a double helix (the Watson-Crick model), in which A forms a base pair with T and G forms a base pair with C RNA • It contains the pyrimidine uracil (U), instead of T • During protein synthesis, mRNA is copied from DNA and travels to ribosome. tRNA transports amino acids to ribosome. rRNA and protein combine to make ribosomes Transcription is the synthesis of mRNA from DNA. Translation is the synthesis of proteins from mRNA. Genetic Mutations • Mutations result from changes in the nucleotide sequence of genes (Table 1-9). These mutations can occur at the genomic or mitochondrial or RNA level (Table 1-10) • Germ-line mutations can be passed down via reproductive cells • Somatic mutations involve cells outside the reproductive system and generally do not get passed to subsequent generations. A mutation early in embryogenesis can affect a large number of cells, but not all; this can lead to “mosaicism” where only a portion of a person’s cells have a given genetic mutation, but it is enough to cause disease expression Table 1-9. Types of Mutations Mutation Symbol Consequence DNA Code Protein Peptides Frameshift fsInsertion "ins" or deletion "del" causes a shift in the translational reading frame; more dramatic effect on peptide sequence UUU UAC AAA UUU ACA AAG Phe Tyr Lys Phe Thr Lys Missense > (general symbol for substitution) Single-nucleotide substitution causes the translation of a different amino acid UUU UAC AAA UUG UAC AAA Phe Tyr Lys Leu Tyr Lys Nonsense X or *Single-nucleotide substitution causes an early stop (or termination) codon UUU UAC AAA UUU UAA AAA Phe Tyr Lys Phe STOP Silent >Single-nucleotide substitution does not cause a change in amino acid sequence UUU UAC AAA UUU UAC AAG Phe Tyr Lys Phe Tyr Lys Neutral >Single-nucleotide substitution causes a different but similar amino acid to be translated UUU UAC AAA UAU UAC AAA Phe Tyr Lys Ty r Tyr Lys IMMUNE MECHANISMS / 15 Table 1-10. Understanding Genetic Readouts ExpressionMeaning g.38 G>C g refers to genomic DNA c.38 G>C c refers to cDNA m.38 G>C m refers to mitochondrial sequence r.38 G>C r refers to RNA sequence p.R38 G>C p refers to protein sequence Communicating About Mutations • Gain of function (GOF) mutation – refers to mutations that lead to overexpression or overactivation of a given gene. Example: Activated PIK3 Syndrome • Loss of function (LOF) mutation – refers to mutations that silence or dramatically reduce expression of a specific gene. Example: Wiskott-Aldrich Syndrome, vast majority of primary immunodeficiencies • Hypomorphic mutation – refers to mutations that allow for partial expression and function of a specific gene, such that the phenotype is different compared to that of a complete LOF or GOF mutation of the same gene. Example: Omenn syndrome • Dominant negative mutation – refers to a mutation when expressed leads to product that interferes with the activity of wild-type allele. Example: BCL11B SCID • Haploinsufficiency – when one allele is deleted or inactivated, and the remaining allele is not sufficient to preserve normal function. Example: CTLA4 haploinsufficiency Genome Wide Association Study (GWAS) • A tool that is implemented when genes involved in disease are unknown. DNA is obtained from as many patients with the disease as possible and as many controls as possible. Then each individual’s genome is examined for single-nucleotide polymorphism (SNP) – a variation in DNA sequence that occurs when a single nucleotide in a gene of an individual is different from that of other individuals • Goal is to identify genes associated with the disease • Several SNPs in specific genes have been identified to relate to allergic disease ° FLG (filaggrin) gene – atopic dermatitis and asthma ° ADAM33 (ADAM Metallopeptidase Domain 33) gene – increased risk of asthma and bronchial hyperresponsiveness ° ADRB2 (Adrenoreceptor Beta 2) gene – Arg/Arg phenotype with decreased albuterol response compared with the Gly/Gly phenotype at residue 16 Key Fact Histone acetylation opens the chromatin to allow transcription. Histone \ deacetylation represses gene expression and is reduced in chronic obstructive pulmonary disease (COPD). 16 / CHAPTER 1 Epigenetics Epigenetics refers to modification of the expression or repression of a gene rather than the DNA code. Examples include histone acetylation (see Key Fact) and DNA methylation, which typically represses gene expression. IMMUNOGLOBULINS (Ig) Igs are glycoprotein molecules produced by B cells and plasma cells in response to an immunogen. Ig is the key component of humoral immunity. The earliest cell in B cell lineage that produces Ig is the pre-B cell. An adult human produces approximately 2–3 g of Ig every day. Ig Structure The Ig molecule is a polypeptide heterodimer composed of two identical light chains (LCs) and two identical heavy chains (HCs) connected by disulfide bonds (Figure 1-5). Each chain consists of two or more Ig\ domains, which are compact globular structures of approximately 110 amino acids containing intra-chain disulfide bonds (Figure 1-5). Each HC and LC has constant and variable\ regions. Heavy chains are designated by letters of the Greek alphabet (ie, γ, α, µ, ε, δ) for Ig classes: G, A, M, E, and D, respectively. Human IgG consists of four isotypes: IgG1, IgG2, IgG3, and IgG4. For example, IgG1 contains Cγ1 as its heavy chain. The constant (C) regions of IgG, IgA, and IgD consist of only three C H domains. In IgM and IgE, the C regions consist of four C H domains. Heavy chain locus is on chromosome 14. Key Fact Omalizumab binds to C ε3. Light chains κ and λ are identified by their C regions: κ is encoded on chromosome 2 and λ is encoded on chromosome 22. They lack transmembrane domains and connect to HC’s by disulfide bond. They do not contribute to effector function. An Ig molecule has either κκ (60%) or λλ (40%) but never 1 of each. An individual B cell will produce only κ or λ chains but never both. Changes in this ratio occur in B cell tumors. Key Fact The ratio of κ-bearing lymphocytes to λ-bearing lymphocytes can be used as an indication of clonality and is, therefore, useful in diagnosing and typing B cell lymphomas and multiple\ myelomas. Hinge regions are proline-rich and provide Ig flexibility (allow a single immunoglobulin molecule to bind to 2 sites on one antigen). Interchain disulfide bonds exist between the heavy-heavy and heavy-light chains. Only IgG, IgA and IgD have distinct hinge regions. IMMUNE MECHANISMS / 17 Ig fragments are produced from enzymatic cleavage of the Ig molecule. Papain cleaves Ig above the hinge at the amino terminal side and results in two Fab (each antigen-binding) fragments and 1 Fc (crystallizable) fragment. Pepsin cleaves Ig below the hinge at multiple sites and produces a single F(ab′)2, which contains interchain disulfide bonds and exhibits two antigen-binding sites. F(ab) can bind but not cross-link; and F(ab′)2 both binds and cross-links. Neither F(ab) nor F(ab′)2 will fix complement or bind to the Fc receptor on the cell surface. Fc portion is usually digested into several smaller peptides by pepsin (pFc′). Key Fact The most variable part of the Ig molecule is CDR3. Variable regions V L and adjoining V H form the antigen-binding sites that consist of complementarity- determining regions (CDRs) of about 10 amino acids and account for antibody diversity. The three CDRs are loops that protrude from the surface of the two Ig V domains to form 2 antigen binding sites per molecule; CDR3 of both Vh and Vl is the most variable and, typically, has the most extensive contact with the antigen. Figure 1-5. Immunoglobulin structure. Constant regions C H and C L are located at C-terminals of the Ig molecule. Only C H mediates effector functions by binding to Fc receptors or binding complement. Glycosylation of Igs is important in maintaining their structural stability and effector functions. Human IgG has one conserved glycosylation site in the Cγ2 domain (asparagine-297). De-glycosylated IgG cannot bind FcγRs and C1q effectively and therefore is unable to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and complement activation. Decreased glycosylation has been associated with many inflammatory and infectious diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Crohn’s disease, and tuberculosis (TB). Sialic acid enrichment in intravenous immunoglobulin (IVIG) preparation significantly increases its anti-inflammatory activity. 18 / CHAPTER 1 Ig Forms Two forms of Ig exist that differ in the amino acid sequence of the C-terminal end of the C H. Membrane-bound Ig (or surface Ig) is attached to the B cell surface by its transmembrane region. Once Ig molecules bind to antigens and are cross-linked, they serve as B cell antigen receptors that mediate B cell activation. The membrane-bound form contains a hydrophobic α-helical transmembrane region and an intracellular juxtamembrane positively charged portion helping to anchor into the plasma membrane. In IgM and IgD, the cytoplasmic portion of the heavy chain is short (only three amino acid residues in length); in IgG and IgE molecules, it is longer (up to 30 amino acid residues in\ length). Secreted Ig molecules lack transmembrane regions and circulate in the plasma, mucosal sites, and interstitial fluids. Secreted Ig can be in the form of monomers (all Ig), dimers (IgA), or pentamers (IgM). Dimers or pentamers are formed by tailpieces connected by disulfide bonds to joining (J) chain. These tailpieces are hydrophilic. The secreted form is found in blood, mucosal surfaces, and extracellular\ fluid. Key Fact The shortest half-life of all IgG subclasses is IgG3. IgM fixes complement most efficiently of any Ig isotype. Rheumatoid factor (RF) is an antibody against the Fc portion of IgG. R\ F is most commonly IgM but can also be any other isotype. Ig Characteristics Affinity is the strength of the binding between each molecule of Ig and a single antigen epitopes; it is indicated by K d. A numerically lower K d indicates higher affinity. Avidity is determined by the net effect of affinity and valence. It is an estimate of the overall strength of the binding between Ig and antigen. Increases in avidity in an immune response are result of somatic hypermutation and selection of high-affinity B cells in germinal centers. A low-affinity IgM can produce a high-avidity interaction by simultaneous binding to multiple antigen epitopes through 10 contact sites on each IgM molecule. Ig Superfamily The Ig superfamily is a group of proteins that share similar structure to Ig by having one or more domains of two adjacent beta-pleated sheets composed of 70–110 amino acids, most typically containing an intrachain disulfide bridge. Examples of Ig superfamily members are TCR, MHC class I, CD4, CD8, CD19, B7-1, B7-2, Fc receptors, killer cell immunoglobulin-like receptor (KIR), and vascular cell adhesion molecule 1 (VCAM-1). IMMUNE MECHANISMS / 19 Antigen Recognition Ig can recognize highly diverse antigens through linear and conformational determinants found in various macromolecules (ie, proteins, polysaccharides, and lipids). TCRs only recognize linear determinants of peptides presented by MHC molecules. Key Fact The only Ig to cross the placenta is IgG. The Ig class with highest plasma concentration is IgG. The Ig class with highest total body concentration and daily production is IgA. The poly-Ig receptor is synthesized by mucosal epithelial cells and expr\ essed on their basolateral surfaces. Once inside the epithelial cell, IgA bound to the poly-Ig receptor is actively transported in vesicles to th\ e luminal surface. Ig Production • IgM is the first Ig produced after birth, the first to reach adult level, and the first to be synthesized following antigenic stimulation; transmembrane monomeric IgM is the earliest secreted by developing B cells; IgM is secreted as a pentamer with J chain, found largely in serum; IgM pentamer is the largest Ig molecule • Only IgG crosses the placenta; it does this by binding to the neonatal Fc receptor (FcRn), which allows IgG to be endocytosed and, instead of targeted to lysosomes, to be recycled to the cell surface; FcRn is also found on most endothelial cells and macrophages; this accounts for the long half-life of IgG since it is protected from degradation; IgG level reaches a nadir around 4–6 months after birth due to decline in passively transferred maternal IgG • IgA is produced in the highest quantity daily and is found in higher concentrations in the respiratory and GI mucosal surfaces; it may take several years for IgA to reach adult levels; in the gastrointestinal tract, IgA is produced by plasma cells in the lamina propria and is transported ac\ ross the mucosal epithelium by poly-Ig receptor (transcytosis). The cleaved part of the poly-Ig receptor, called the secretory component, remains associated with the dimeric IgA in the lumen • Smaller quantities of IgG and IgM are secreted into the gut lumen and bind to microbes and toxins and neutralize them. This is called secretory immunity • IgA is produced in lymphoid tissue in mammary gland and is secreted into the breast milk. Some IgG and IgM are also secreted into the breast milk (Passive Immunity) • IgE binds receptors with the highest affinity of all Ig isotypes Ig Isotypes There are five Ig heavy chain isotypes. The isotypes differ in their biological properties, functional locations, and interactions with different antigens, as depicted in Table 1-11. 20 / CHAPTER 1 Table 1-11. Immunoglobulin Isotypes IsotypeIgGIgAIgM IgEIgD Subclasses IgG1IgG2IgG3IgG4IgA1IgA2 none nonenone Heavy chain γ1γ2γ3γ4α1α2 µ εδ # Heavy chains (C H) 3 3443 Secreted form (MW in kDa) Monomer Monomer dimer Monomer Pentamer Monomer Monomer Serum level in adult (mg/dL) 430–1050 100–300 30–9015–6090– 325 80– 290 50–250 0.0015–0.2 0.3–30 Total: 700–1500 Total: 60–450 Half-life (days) 2323 82366 5 23 Cytokines inducing class switch IFNγ, IL-4 IFNγ, TGFβ IFNγ IL-4, IL-13 TGFβ, IL-5 N/AIL-4, IL-13 N/A Complement fixation ++ +++ ––+++ –– Placental transport ++ +++ ++ –––– Properties Th1 response, opsoniza - tion Best for ADCC Responses to protein antigens Antipoly - saccharide - Ab Latest to reach adult level of all IgG sub- classes Shortest half-life in IgG sub- classes Opsoniza - tion Antipoly - saccharide Ab Elevated in immuno - therapy Mucosal immunity IgA1 in serum and respiratory tract IgA2 in lower GI tract Primary re- sponse (eg, isohemag - glutinin and rheumatoid factor) Neutralizes antigens Allergic reaction The only Ig to bind to mast cells Binds receptors with highest affinity of all Ig isotypes Do not require isotype switching Exact function unknown B cell maturation marker Abbreviations: Ab, antibody; ADCC, antibody-dependent cell-mediated cytotoxicity; kDa, kilodaltons; MW, molecular weight. General Functions of Ig Antigen recognition by Ig initiates a humoral immune response. Ig selectively captures antigens and microbial pathogens, including bacteria and viruses, through noncovalent, reversible binding through the Ig V regions. Ig-mediated effector functions include neutralization of microbes or toxins, opsonization, ADCC, and immediate hypersensitivity (IgE). These effector mechanisms often require interaction of Ig with complement proteins or other immune cells, such as phagocytes, eosinophils, and mast cells, through Fc receptors. Fc receptors recognize and bind to the constant region of the antibody. The functional features of Ig are summarized in Table 1-12. Some of these characteristics are also shared by TCR. 1. Neutralization of antigen or toxin by formation of soluble complexes prevents further binding o\ f that antigen to other cells. 2. Promote phagocytosis of antigens through complement cascade activation o\ r interactions with FcRs. 3. Antibodies bound to FcRs on immune cells cross link leading to ADCC. IMMUNE MECHANISMS / 21 Key Fact Somatic hypermutation leads to changes in the V, not the C regions. Diversifies BCR’s used to recognize antigen. Class switch recombination changes the C but not the V regions. Does not\ change antigen specificity. Alternative splicing changes Ig from transmembrane to secretory form. Only IgM, IgG1, and IgG3 efficiently activate complement. Table 1-12. Functional Features of Ig Feature Description Results Specificity Ability of Ig and TCR to distinguish subtle differences in molecular structures of antigen Distinct antigens elicit specific responses Diversity Variability in the structures of the antigen-binding site of Ig and TCR Large structurally distinct antibody and TCR repertoires Germ-line variation Variation in inherited (germ-line) V, D, and J elements in Ig and TCR Inherited structural differences create different basic structural frameworks Combinatorial diversity (somatic recombination) Result of different V, D, and J segment rearrangement in developing B and T cells Moderate levels of immune receptor diversity Junctional diversity Random (nontemplated) addition or removal of nucleotide sequences at junctions between V, D, and J regions Extensive somatic variability in immune receptors Somatic hypermutation Point mutations in V regions of Ig in rapidly dividing B cells; diversifies BCR’s Selected increase (or decrease) in antibody affinity Receptor editing Changes in Ig specificity that express self-reactive antibody achieved through secondary rearrangements Elimination of self-reactive Ig Class switch recombination (Isotype switching) Change in heavy-chain C regions, with same V region at the gene locus; T cell dependent; however BCR signaling with TLR signaling can induce without T cell help in response to microbial pathogens Switch in Ig isotype from IgM or IgD to IgG, IgA, or IgE; no change in antigen specificity Affinity maturation Process of somatic hypermutation and selective survival of B cells that produce high-affinity antibodies ↑Ig affinity Alternative splicing Splicing at different locations in the 3' of C region exons Production of membrane-bound or secreted Ig forms Splicing at different location of the C-terminal of the IgM gene Production of IgD from the same RNA transcript with IgM. Not conventional class switch Fc Receptors (FcRs) FcRs are members of the Ig superfamily. Each Fc receptor functions as a receptor specific for the C H region of the Ig molecule (Table 1-13). FcRs contain domains for Ig-binding and signaling component\ s. 22 / CHAPTER 1 Table 1-13. Fc Receptors on Leukocytes FcRCD Marker Affinity for Ig Cell Distribution Function For IgG FcγRI CD64 HighMacrophages, neutrophils, monocytes, DCs Phagocytosis FcγRIIA CD32 LowMacrophages, neutrophils, eosinophils, platelets Phagocytosis (poor), cell activation FcγRIIB1/2 CD32 LowB cells, macrophages, dendritic cells, others Feedback inhibition of various cellular responses FcγRIIC CD32 LowMacrophages, neutrophils, NK cells Phagocytosis, cell activation FcγRIIIA CD16 LowNK cells, macrophages ADCC FcγRIIIB CD16 LowNeutrophils, macrophages, eosinophils Phagocytosis (poor) For IgE FcεRI HighMast cells, basophils, eosinophils Degranulation, ADCC FcεRII CD23 (not 32!) LowMonocytes, B cells, DCs Prevent IgE binding to FCER1, inhibit excess IgE levels For IgA FCαR CD89 LowNeutrophils, eosinophils, monocytes Cell activation? Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; NK, natural killer. Antibody-Mediated Immune Regulation IgA and Mucosal Immunity: Gut/Oral – The gastrointestinal and pulmonary tracts are the most common entry point for microbes. IgA acts as the major defense against microbes along gastrointestinal and respiratory mucosal surfaces. • IgA binds microbes and toxins in the gut and respiratory lumen, neutralizing them and preventing infection • Isotype switching to IgA occurs most efficiently in the mucosal lymphoid tissue and is stimulated by transforming growth factor ( TGFβ) and IL-5 • Secreted IgA is a dimer that is transported into lumen via an IgA-specific receptor called the poly-Ig receptor (Figure 1-6) ° The poly-Ig receptor is responsible for secreting IgA into milk, bile, saliva, and sweat ° Poly-Ig receptor can also transport IgM into intestinal secretions; thus, the use of the prefix “poly” in its name Figure 1-6. IgA dimer. 1 = H chain; 2 = L chain; 3 = J chain; 4 = secretory component. Adapted, with permission, from Wikimedia Commons. IMMUNE MECHANISMS / 23 Key Fact Although it constitutes less than one quarter of antibody in plasma, the\ amount of IgA produced in the body is greater than that of any other antibody isotype (60–70% of total antibody ou\ tput), with most of the antibody located in or near the mucosal tissues. IgG and Neonatal Immunity – Neonates are not capable of producing an effective immune response and, for the first several months after birth, they are protected passiv\ ely by maternal antibodies. • IgG is transported across the placenta from mother to infant • IgG is transported across the placenta and infant gut lumen by an IgG-specific Fc receptor called neonatal Fc receptor (FcRn), which resembles the major histocompatibility complex (MHC) class I molecule • Maternal IgA more than IgG is ingested by the infant from the breast milk • In adults FcRn resides on phagocyte cellular membranes, binds to pinocytosed IgG molecules, protects them from lysosomal degradation, and then recycles IgG to the cell surface Key Fact FcRn is also responsible for the remarkably long half-life of IgG. FcRn-medi\ ated cellular recycling saves IgG from intracellular degradation. T-CELL RECEPTORS AND SIGNALING The T-cell receptor (TCR) comes in two forms: αβ and γδ. αβ TCR Structure – It is a heterodimer of an α and β chain, each with two Ig-like domains. Each α and β chain consists of a variable (V) domain, a constant (C) domain, a transmembrane hydrophobic region, and a short cytoplasmic tail with no signaling molecules. The particular variable β or Vβ region of the TCR is the binding site for superantigen. Key Fact αβ TCRs recognize a single antigen only in the context of an HLA molecule. The receptor has no signaling ability and requires accessory molecules for signal transduction. The V regions contain short stretches of amino acids, which form the hypervariable or complementarity- determining regions (CDR); the CDRs of both chains are the sites of recognition of the peptide-HLA complex. The C regions of both the α and β chains contribute to a disulfide bond linking the two chains. 24 / CHAPTER 1 αβ TCR affinity is less than that of antibodies; this, and the lack of signaling motifs on the αβ receptor, sets the stage for the necessity for accessory molecules and adhesion molecul\ es to and from the TCR complex. Key Fact VDJ recombination creates TCR diversity. Unlike Ig, TCR genes do not undergo class switch or somatic mutation. TCR Complex – TCR complex consists of the TCR, CD3, and two zeta (ξ) chains (Figure 1-7). CD4 recognizes antigens presented in the context of HLA class II, and CD8 (not shown in Figure 1-7) recognizes antigens presented in the context of HLA class I antigens. In order for antigen-signaling transduction to be initiated, the entire TCR complex is required to be expressed. The accessory molecules contain immunoreceptor tyrosine-based activation motifs (ITAMs), which are required for signal transduction. Antigen recognition is connected to activation through the TCR complex. Key Fact The combination of the chains and their Ig-like domains are analogous to\ the immunoglobulin Fab fragment. These molecules undergo recombination for their diversity. Both chains have complementarity-determining regions (CDRs), and, like in an immunoglobulin, the αβ CDR3 region of the TCR imparts the most significant sequence variability. Figure 1-7. TCR complex with CD4. γδ T Cells γδ T cells are similar in structure and association to αβ T cells. γδ T cells require CD3 and the ζ chain for signal transduction. Most do not have CD4 or CD8. They are not HLA-restricted. Some antigens do not require processing. Some antigens are presented in MHC-like class I molecules. They are thought to be a bridge between innate and acquired immunity. IMMUNE MECHANISMS / 25 Natural Killer T (NKT) Cells These cells express NK cell and T cell markers. Key Fact NKT cells recognize lipids in the context of CD1. Key Fact CD40 ligand – CD40 interaction stimulates activation-induced cytosine\ deaminase (AID), which is crucial for somatic mutation and isotype switching. CD40 ligand mutations occur in X-linked \ Hyper IgM syndrome. T Cell Activation, Proliferation, and Differentiation Three signals involved in T cell activation, proliferation, and differentiation: • TCR binding with antigen in MHC • Co-stimulation • Cytokine production Once the TCR-antigen HLA complex is formed, then activation, proliferation and differentiation can occur as follows: • Costimulatory pathways in T cell activation: T cell receptor CD28, which binds B7-1 (CD80) and B7-2 (CD86) on activated APCs • Intracellular signaling pathways can trigger calcium signaling and amplify the activation of NFKB • The most important cytokine produced during activation is the T cell survival signal, provided by the interaction between IL-2 and its receptor CD25 Proliferation is clonal. • It is stimulated by IL-2 • Clonal expansion preserves the specificity of the T cell for its particular antigen Immunologic Synapse: A specialized cell-to-cell interaction where proteins are sorted into distinct regions known as supramolecular activations clusters (SMACs) • This requires the actin cytoskeleton to organize the synapse • This molecular interface between lymphocytes and antigen presenting cells (APCs) leads to activation of T cells • Clinical correlation: Patients with WAS cannot organize the immunologic synapse and thus cannot polarize T cells Of note, activation depends on strength and duration of binding to MHC. Full activation requires prolonged interaction between T cell and APC with adhesion molecules (LFA1 on T cell/ICAM on APC). 26 / CHAPTER 1 Once T cells are activated, they become either effector or memory T cells (Table 1-14). • Effector cells function to react to and eliminate antigen; with the decline in antigen stimulation, there is a decline in T cells activity and achievement of homeostasis (CD45RA) • In the CD4+ lineage, they induce differentiation of the T cell response, Th1, Th2, Treg, and Th17 • In the CD8+ population, the cells become cytolytic • Memory cells are a subset of the clonally expanded population • These cells are long-lived and functionally quiet • They provide a rapid secondary response (CD45RO) Table 1-14. T-Cell Differentiation Antigen Recognition Cell ActivationClonal ExpansionFunctional Differentiation Naïve CD4+ T cells Dendritic cells Naïve CD8+ T cells IL-2 production Upregulation IL-2 receptor Proliferation of cell with same antigen recognition Effector CD4+ T cells (helper) Memory CD4+ T cells Effector CD8+ T cells (killer) Memory CD8+ T cells Costimulation Between Antigen-Presenting Cells and T Cells The costimulatory molecules are on the APCs. Their ligands are on the T cells. There is no T cell response without costimulation. The T cell that recognizes the antigen, but is not costimulated, is said to be in a state of anergy (nonresponsiveness). Costimulation may produce activation or induce negative regulation of th\ e immune response (Table 1-15). Key Fact CD3 deficiency produces severe combined immunodeficiency (SCID). IMMUNE MECHANISMS / 27 Table 1-15. Costimulator Expression and Function APCDC/Mø/B cells DC/Mø/B cellsDC/Mø/B cellsDC/Mø/B cellsDC/Mø/B cells Costimulator on APC B7-1 (CD80), B7-2 (CD86) B7-1 (CD80), B7-2 (CD86) ICOS-L CD40PD-L1 (B7-H1, CD274) / PD-L2 (B7-DC, CD273) APC T cells Receptor on T cells CD28 CTLA4 (CD152) (Inhibitory through ITIM) ICOS (CD278) CD40LPD-1 (Inhibitory through ITIM) Expression Constitutive Inducible InducibleInducibleT, B, myeloid cells/ inducible Effect Activation of naïve cells Induction of CD40L, OX40, CXR5, ICOS, CTLA-4 T-lymphocyte tolerance Th1 development Costimulation of effector T lymphocytes, implicated in Ab class switching APC activation, germinal center development, class switching Negative regulation, cell death Abbreviations: APC, antigen-presenting cells; CTLA, cytotoxic T lymphocyte antigen; DC, dendritic cell; ICOS, inducible costimu- lator; ITIM, immunoreceptor tyrosine-inhibitory motifs; Mø, macrophage; PD-1, programmed death. TCR-Signaling Pathways Role of CD4 and CD8 Molecules – These molecules dictate HLA restriction. CD4 binds to HLA II molecules at a nonpolymorphic site. CD8 binds to HLA I molecules at a nonpolymorphic site. The class II- binding site is the β 2 region and the class I-binding site is the α 3 region; both contain Ig-like domains. These molecules stabilize the immune synapse. The cytoplasmic tails of CD4 and CD8 are also involved in signaling through a Src fam\ ily tyrosine kinase called lck, which is required for T cell activation and maturation (Figure 1-8). Costimulation – The interaction between CD28 and both CD80 and 86, CD2, and CD58, and a signaling lymphocytic activation molecule (SLAM) provides signals of costimulation for activation, survival, and stability of the immune synapse. SLAM (signaling lymphocytic activation molecule), a subgroup of CD2 family of proteins, has an immunoreceptor tyrosine-based switch motif (ITSM) that binds to SLAM-associated prote\ in (SAP), which links SLAM to Fyn that is physically linked to CD3 proteins in T cells. Mutations in SAP cause X-linked lymphoproliferative (XLP) syndrome. 28 / CHAPTER 1 Key Fact ZAP-70 deficiency is a form of SCID with no CD8 cells and no T cell function, but normal B cells and NK cells. Key Fact ZAP-70 deficiency is an SCID with no CD8 cells and no T-lymphocyte function, but normal B lymphocytes and NK cells. Figure 1-8. T-cell receptor signaling. ITAM vs. ITIM: • Phosphorylation of ITAM allows for recruitment of kinases/adaptor proteins that mediate activating response • Phosphorylation of ITIM leads to recruitment of one or other inhibitory \ phosphatases SHp1, SHp2, and SHIP • Antigen binds TCR (via MHC) • Lck (Src family Kinase, non-covalently associated with CD4 and CD8) phosphorylates the ITAMs in CD3 and zeta proteins facilitating the recruitment and activation of ZAP70 • ZAP70 (Syk family kinase, noncovalently associated with CD3 zeta chain) contains two SH domains which bind the ITAM phosphotyrosines and then itself phosphorylates LAT • LAT (an adaptor protein with no kinetic activity) becomes a docking site for PLC1 (a cytosolic enzyme; in B cells its PLC2) and Grp2 • When attached to LAT, Grb-2 recruits Ras GTP/GDP exchange factor SOS which catalyzes GTP for GDP • PLC hydrolyzes lipid PIP and generates DAG/IP3 (1, 4, 5-triphosphate), which activate two distinct downstream pathways • DAG activates PKC (serine/threonine kinase), which leads to activation and nucleolar transcription of NFKB (a transcription factor) • IP3 increases leads to increased intracellular calcium through the CRAC (Calcium release-activated calcium channel) on the cell membrane IMMUNE MECHANISMS / 29 • Increased intracellular calcium activates Calcineurin, activates NFAT by dephosphorylation, enabling it to move to the nucleus and turn on transcription of cytokines. Of note, calmodulin is a ubiquitous calcium dependent regulatory protein that binds calcium and interacts with calci\ neurin • Ras is bound to GDP in inactivate form. When replaced with GTP, Ras undergoes a conformational change and can recruit enzymes, most importantly c-Raf (a kinase). Raf phosphorylates and activates MEK-1, which phosphorylates ERK • The activated ERK translocates to the nucleus and phosphorylates a protein Elk, which stimulates transcription of C-Fos (a component AP-1) PI3K (not shown) is an enzyme recruited to adaptor proteins and phosphorylates PIP2 to generate PIP3. This activates PDK1 and signals downstream. PLC (not shown) can also be phosphorylated by other kinases like Tec family kinase ITK. Table 1-16. Transcription Factors Transcription Factor Function N FAT In inactive form in cytoplasm of resting T cells Encodes for cytokines IL-2, IL-4, TNF etc. Tacrolimus blocks T cell cytokine gene transcription AP-1 (FOS + JUN) Associated with other TF’s in the nucleus, works best with NFAT FOS: downstream from Ras, ERK JUN B: downstream from Rac and JNK NFKB Essential for cytokine synthesis and plays and important role in lymphoc\ yte development, malignant neoplasms, and formation of secondary lymphoid organs STAT (STATs 1-4, 5a, 5b and 6) Activate gene transcription Table 1-17. Kinases/Phosphatases Tyrosine KinasesSrc (Lyn (B cell), Lck (T cell), Fyn (B cell), Blk (T cell) Syk (Syk (B cell), Zap70 (T cell) Tec (BTK (B cell), ITK (T cell)) ZAP70 phosphorylates LAT and SLP-76 ZAP70 binds to the P on the ξ chain Fyn is non-covalently associated with CD3 Serine/Threonine Kinase PKC, MAPK, low molecular weight G protein PKC is θ isoform in T Cells; β form in B cells Lipid Kinase PI3K; phospholipase C PLC in T cells is γ1 isoform; Isotype in B cells is γ2 isoform MAP Kinases ERK MEK-1 Non-receptor Tyrosine Kinase JAK (JAK1-3 and TYK2) Paired with STAT Tyrosine Phosphatases CD45 SHP1/2 CD45 facilitates lymphocyte activation/ dephosphorylates inhibitory tyrosine residues in Src family (Lck, Flyn) SHP1/2: NK cells, B and T cells Serine/Threonine Phosphatase Calcineurin Intracellular calcium regulation Lipid Phosphatase SHIP Removes phosphate from PIP3. Inhibits PI3 kinase activity in lymphocytes, NK cells and innate immune cells Adaptor proteins: Zap70, SLP-76, GADS, GRB-2. In a parallel GTP/GDP exchange protein called Vav (similar to SoS) that acts on Rac (similar to Ras). This cascade results in activation of a distinct MAP kinase c-Jun N (JNK). Activated JNK phosphorylates c-Jun (a second component of the AP-1 transcription factor). 30 / CHAPTER 1 B-CELL RECEPTOR SIGNALING Cytoplasmic tails of transmembrane Ig HC isotypes lack signaling motifs (due to short length) and thus associate with polypeptides Igα and Igβ. B-lymphocyte-receptor complex (BCR) – Made up of the surface immunoglobulin and the associated Igα and Igβ chains. Includes Igα/Igβ heterodimer, CD19, CR2 (CD21) and CD81 (TAPA-1). Cross-linking – Recognition of the antigen by at least two receptors. Activation will not occur without receptor cross-linking. Igα and Igβ chains – Similar to the CD3 molecules and the ξ chain of the TCR; they contain the ITAMs, are noncovalently associated, and are required for signal transduction. Lipid rafts – Where many adaptor proteins and signaling molecules are concentrated. They bring the BCR and the Src family of kinases in close proximity. Figure 1-9.