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These notes were made by Hadley Wickham, and are licensed under the Creative
Commons   ...
Table of Contents
Overview of Immunity and Immune System.....................................................................
Overview of Immunity and Immune System
What is the Immune System?

 •   non-specific or innate immune mechanisms 1st line ...
•   permits Ag entering body to be brought rapidly into contact with effector mechanisms for disposal


•   cells i...
How Ag’s get to Meet Immune System
How Foreign Ag’s Meet Immune System

 •   response of immune system to foreign substanc...
Major Histocompatibility Complex (MHC)

 •   studies of grafts between different individuals led to identification of cell...
B Cells and Ab Responses

 •   2 heavy and 2 light chain, covalently-bonded structure with globular domains a...
•   IgG predominates in 20 responses although small IgM component generally still evident
•   term affinity refers general...
Cells and Cell-Mediated Immunity
Natural Killer (NK) Cells

 •   NK cells generally large granular leukocytes comprising 1...
•   immune responses occurring during graft rejection can be mimicked by mixing lymphocytes from different
Clonal Selection and T Cell Activation

 •   20 lymphoid organs and circulating lymphocyte pool contain repertoire of Ag-s...
T and B Cell Development and Thymus
T Cell Receptor Genes

 •   very similar to Ig genes and are considered part of Ig gen...
Ig                   TCR αβ               TCR γδ
                                       H                κ     α          ...
T Cell Ontogeny

 •   T cell development occurs in thymus (bilobed organ overlying heart, each lobe organized into lobules...
•   analysis of frequency of TCR gene usage shows variation from person to person at any one time
Cytokines, Lymphocyte Surface and Adhesion Molecules
T Cell Subsets And Cytokine Production

 •   TH major source of many ...
Class           Cytokines                                          Function
     Chemotactic        IL-8                  ...
Cell Adhesion Molecules

 •   fundamentally important to cell, determining whether it circulates or moves from blood vesse...
Rolling and Tethering
       •   selectin molecules bind mucin-like CHO ligands
       •   initially (within seconds) P-se...
Clinical Applications

•   current research on cell adhesion molecules extended into clinical field
•   monoclonal Ab’s to...
How Immune System Deals with Infections
Process of Infection

 •   often initial focus of infection does not stimulate imm...
•    however, if Ab’s not initially present, virus infection will often become well established in target tissues
•   Ab’s play important role in protection from many viral infections but it is are not only effector
          mechanism ...
Immunodeficiency (ID)

 •   immune system has many components: Ab’s, complement, B and T lymphocytes, cytok...
•   should suspect ID if patient has recurrent bacterial infections, or infections with unusual organisms
•   may be famil...
•   2:        Ab mediated
      •   3:        Immune complex mediated
      •   4:        Cell mediated (delayed type hype...
•   complexes deposit in skin (rash), joints (arthritis) and kidney (nephritis)
      •   formerly seen with horse Ig for ...
 •   mechanisms causing autoimmune disease are not fully understood

     Natural AutoAbs
       •   loss of self-...
•    lipopolysaccharide in G- bacteria is polyclonal B lymphocyte activator
        •    superAgs secreted by certain bact...
several mechanisms may account for this: peptide binding, molecular mimicry, receptor model
    Other genes             ge...
Immunology in Diagnostic and Clinical Medicine

 •   make use of exquisite specificity of Ag-binding part of Ab mo...
Name                            Differences                                                 Notes
     Western Blot       ...
Flow Cytometry
 •   cells in suspension incubated with fluorochrome-tagged Ab’s, then washed
       •      labelled cells ...
•   Ab and Ag diffusing towards each other from two small wells cut in agar gel will form visible line
       •   Ag embed...
What is the Immune System?.doc.doc
What is the Immune System?.doc.doc
What is the Immune System?.doc.doc
What is the Immune System?.doc.doc
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  1. 1. Immunology These notes were made by Hadley Wickham, and are licensed under the Creative Commons NonCommercial-ShareAlike License. To view a copy of this license, visit or send a letter to Creative Commons, 559 Nathan Abbott Way, Stanford, California 94305, USA.
  2. 2. Table of Contents Overview of Immunity and Immune System...........................................................................................3 How Ag’s get to Meet Immune System....................................................................................................5 B Cells and Ab Responses.........................................................................................................................7 Cells and Cell-Mediated Immunity..........................................................................................................9 T and B Cell Development and Thymus..................................................................................................12 Cytokines, Lymphocyte Surface and Adhesion Molecules.....................................................................16 How Immune System Deals with Infections..........................................................................................21 Immunodeficiency (ID)..........................................................................................................................24 Hypersensitivity......................................................................................................................................25 Autoimmunity.........................................................................................................................................27 Immunology in Diagnostic and Clinical Medicine..................................................................................31 Factors Influencing Immune Responsiveness.......................................................................................35
  3. 3. Overview of Immunity and Immune System What is the Immune System? • non-specific or innate immune mechanisms 1st line of defence against infectious agents • generally very effective at preventing invasion but if are breached, specific immune system called upon • both immune systems composed of variety of molecules and cells distributed throughout body • interdependent with considerable interaction between them Non-Specific Immune System Specific Immune System Repeated infection resistance not improved resistance improved Soluble factors Ab’s, complement, acute phase lysozyme, complement, proteins, interferons (IFNα and IFNβ), cytokines Cells phagocytes, natural killer (NK) cells lymphocytes, monocytes, APC Non-Specific Immune System • exterior (especially skin) presents effective barrier to most organisms • importance evident when individual suffers serious burns • most enter via epithelial surfaces (nasopharynx, gut, lungs or GU tract) • variety of physical and biochemical defences protect these areas from most infections • after penetration encounters phagocytic cells of reticulo-endothelial system • macrophages process Ag as well as engulfing them – vital in specific and non-specific immunity Acute Phase Proteins • acute phase proteins ↑↑ in response to early ‘alarm’ mediators (e.g. IL-1) released by tissue injury • many produced in liver, activate complement, ↑ phagocytosis by opsonization and ↓ bacterial enzymes Specific Immune System • 8 general properties which characterize immune system: • processing of molecular shape • can recognise diverse range of Ag’s • generates response specific to stimulus • responds to unexpected stimuli • effective discrimination between self and non-self Ag’s • adaptability to provide rapid, appropriate responses • specific immunological memory • regulation by complex 'internal' and 'external' networks • immune system consists of collection of lymphoid organs connected by circulatory and lymphatic system 10 lymphoid organs 20 lymphoid organs Role mature stem cells into Ag-sensitive T or B lymphocytes receive mature lymphocytes formed in primary organs Components bone marrow (source of stem cells and B lymphocytes) spleen, lymph nodes, gut-associated lymphoid tissue thymus (source of T lymphocytes) (GALT – tonsils, adenoids, Peyer’s patches) fetal liver (source of stem cells and B lymphocytes) • blood and lymphatic vessels connect organs to provide comprehensive network throughout body • enables lymphocytes and granulocytes to circulate freely and quickly reach important areas
  4. 4. • permits Ag entering body to be brought rapidly into contact with effector mechanisms for disposal Cells • cells include lymphocytes, macrophages, monocytes and APC (APC) • can be subdivided into number of subpopulations on basis of functions • effector’s responsible for responding to and disposing of Ag’s, regulator’s for control of effector cells Group Cell Description Surface Markers Effectors B Ab production CD20 CTL T cell-mediated cytotoxicity CD8 NK/K Natural killer cells (early defence in anti-viral and tumour immunity) K/Null Ab-dependent cell-mediated cytotoxicity (ADCC) CD16, CD56 Regulators TH Induction of effector responses CD4 (helper/inducer, recruit non-specific mechanisms. TH1 and TH2 subsets) TS Suppression of some effector responses CD8 (indistinguishable from those responsible to cytotoxicity)
  5. 5. How Ag’s get to Meet Immune System How Foreign Ag’s Meet Immune System • response of immune system to foreign substance depends on nature of substance and route of entry Route of Entry Response Skin much foreign matter dealt with at 10 site (local ingestion by macrophages) Agic fragments are transported in macrophage-like phagocytic cells to local lymph nodes (and spleen), or arrive by themselves at node and are collected by resident scavenger macrophages in node, Agic fragments passed to dendritic reticular cells (specialized APCs) which process fragments into smaller pieces (epitopes) and present them to Ag-sensitive T and B lymphocytes Bloodstream filtered out by macrophages in spleen (capable of mounting immune response), liver and lungs Upper respiratory, GI tract filtered through local lymph nodes and specialized lymphoid organs in gut Spleen • differentiated into two types of tissue: lymphoid white pulp and erythroid red pulp • white pulp, surrounds small splenic aa. from origins to termini (analogous to cortex of lymph node) • red pulp involved in scavenging old RBCs and serves as reserve site for haematopoiesis • within lymph nodes and spleen, T and B lymphocytes reside in specific areas Lymph node Spleen B lymphocytes follicles of cortical region less well-defined follicular areas of white pulp T lymphocytes paracortical areas, medullary cords peri-arteriolar cuffs • macrophages (especially dendritic reticular macrophages) closely associated with lymphocytes, play vital role in presenting foreign Ag’s to lymphoid cells Reticulo-Endothelial System • bone marrow-derived myeloid progenitors give rise to cells of mononuclear phagocytic system • two main functions, performed by two different types of cells • ‘professional’ phagocytic macrophages remove particulate Ag’s • APCs present Ag’s to specific Ag-sensitive lymphocytes • phagocytic tissue macrophages form network called reticulo-endothelial system (RES) • adherence and ingestion by cells of RES is promoted through opsonisation by complement and Ig • once ingested, degraded by cytoplasmic lysosomal granules (characteristic feature of phagocytes) • both respond to and secrete cytokines • phagocytosis and degradation important method of controlling foreign matter and dead/dying tissues, but process has additional importance as it usually first step in process of lymphocyte activation Tissue RES Cell Transport destination Skin Langerhans cells Regional node Lungs alveolar macrophages Blood blood monocytes, splenic macrophages Spleen or regional nodes Liver Kupffer cells Gut epithelial M cells Peyer’s patches Functions of APC • Ag collection, concentration, processing and presentation to lymphocytes • co-stimulation (accessory surface molecules, cytokines) • tolerance induction
  6. 6. Major Histocompatibility Complex (MHC) • studies of grafts between different individuals led to identification of cell surface structures associated with tissue compatibility (Ag’s which caused strong immune responses against grafted tissues) • gene complex responsible called major histocompatibility complex (MHC), located on chr 6 • aka human lymphocyte Ag’s (HLA) Class I and Class II MHC Gene Products • class I and II MHC genes code for surface proteins • class I found on virtually all nucleated cells in body, (A, B, C) • class II restricted to B lymphocytes, macrophages and other specialized APC, (DP, DQ, DR) • class I genes expressed as single glycoprotein (43kD) associated on cell surface with 12kD β2-microglobulin • class II gene products are expressed as heterodimers of α and β chains, encoded by genes of HLA-D locus • both have two other important properties: co-dominant and polymorphic • highly unlikely that two unrelated individuals will have exactly same set of HLA genes MHC Proteins and Ag Presentation • MHC proteins crucial structures for presenting Ag’s to T lymphocytes • both class I and II MHC have groove in top (peptide-binding groove) into which small pieces of foreign Ag can be fitted • some differences between class I and class II in sort of peptides presented • peptide-binding groove on class I restricted to peptides of 8-10 aa, while class II restricted to ~14 aa Pathways of Ag Processing • Ag processing within cells differs according to whether Ag’s are taken up by cell or originate from within cell • class I peptides usually derived from infectious process or from breakdown of normal cell products • class II peptides usually result from processing of Ag’s taken up and degraded by APCs • not all fragments resulting from Ag processing will be immunogenic epitopes • only those that can bind to peptide-binding groove can be presented to lymphocytes • different people have different MHC molecules and might present different epitopes to immune system • because class I MHC products expressed on virtually all cells, almost all cells can present Agic fragments derived from metabolic breakdown or from intracellular infectious processes • only B cells and certain specialized Ag-processing cells express class II MHC so only these cells can present Agic fragments from ingested material
  7. 7. B Cells and Ab Responses Summary Ab’s • 2 heavy and 2 light chain, covalently-bonded structure with globular domains and variable (Ag-binding) and constant (biologically-functional) regions • κ and λ light chain and µ, γ, α, ε and δ heavy chain classes • allotypic variation is intraspecies allelic variability • idiotypic variation refers to diversity at binding site (relates to hypervariable regions) • functional properties of Ab’s include: • direct neutralization • agglutination • opsonisation • ADCC • complement activation Class Properties Serum Ab IgM large pentamer, confined to bloodstream, does not cross placenta 10% produced early in 10 Ab responses - good defence against bacterial spread efficient agglutinator and complement activator IgG small monomer, diffuses easily out of blood into extravascular areas, crosses placenta 70-75% major class in secondary responses good complement activator, opsonin and Fc receptor-mediated effector mechanisms IgA predominant Ab class in sero-mucus secretions (dimer) 15-20% defence of exterior surfaces IgD highly sensitive to proteolysis trace receptor on virgin, Ag-sensitive B lymphocytes IgE high affinity Fcε receptor on mast cells and basophils trace involved in symptoms of allergy elevated levels in some helminthic parasite infections Complement cascade • classical pathway triggered by Ag-Ab complexes, involves formation of C3 convertase from activation of C1, C2 and C4 components • alternative pathway does not involve Ag-Ab complexes but cell wall components • alternative pathway C3 convertase requires factor B, factor D and C3B • cleavage of C3 into C3a and C3b is central event in complement activation • C3b binds to cell surfaces and serves as focus for activation of late components (C5 to C9) → cell lysis Generation Of Ab Responses • 10 Ab responses result from activation of naive, Ag-sensitive B cells, 20 (or anamnestic) responses result from activation of (long-lived) memory B cells • generation of Ab responses occurs in 20 lymphoid organs after Ag-sensitive B cells have bound sufficient Ag through their surface Ig (sIg) receptors and received activation signals from TH cells • activation of Ag-sensitive B cells results in proliferation and maturation to form population of plasma cells which secrete Ab’s of same Ag-binding specificity as was present on sIg of their precursor B cell, and population of memory B cells which can be restimulated subsequently to produce secondary responses • IgM 1st class of Ab’s produced in 10 response and then class switches to IgG
  8. 8. • IgG predominates in 20 responses although small IgM component generally still evident • term affinity refers generally to strength of binding between receptor and its ligand • In Ab response, there will be range of Ab affinities generated and average affinity of Ab population produced will depend on concentration of Ag and immunological history of individual Immunological Memory • during course of Ab response, new population of small B lymphocytes (memory cells) distinct from Ab- forming plasma cells are generated • histologically, memory B cells appear very similar to original small, Ag-sensitive B cells • role is 'super' Ag-sensitive cells • serve as replacements for their original Ag-sensitive precursors but have lower activation threshold so can be stimulated more easily • can mount more vigorous response than naive B cells so give higher concentrations of Ab’s (usually of different class) that persist for longer periods in serum Ab Class Switching • IgM 1st class to be synthesized in 10 Ab response but after time replaced by production of IgG • individual activated B cell clones begin by producing IgM but subsequently switch to IgG production • B cell remains true to Ag that originally stimulated it, although some mutation of Ag-binding region fine tuning specificity • IgG synthesis dramatically increased in 20 responses because memory B cells preferentially make IgG • IgM component of 20 response mostly due to activation of new naive Ag-sensitive B cells
  9. 9. Cells and Cell-Mediated Immunity Natural Killer (NK) Cells • NK cells generally large granular leukocytes comprising 1-5% mononuclear cells in blood • present in spleen and peritoneal exudate, absent from thymus and few in lymph nodes and bone marrow • can be identified by presence of Fcγ receptor (CD16) and CD56 surface marker • do not have to recognize target Ag’s in association with MHC determinant • do not require activation by Ag, although activity ↑ by certain cytokines (e.g. IL-2, IFN-γ) • limited repertoire of receptors directed at CHO-containing target structures • recognize targets by variety of mechanisms: • e.g. IgG Fc-γ receptors (CD16) to bind Ab’s bound to target cells and activate NK cell • even target cells resistant to NK cytotoxicity in absence of Ab can be killed by ADCC mechanism • another way NK cells might recognize target cells is through combinations of adhesion molecules such as various of integrin and cell adhesin molecules • very little known about origins or their lineage relationships, but thought to develop in bone marrow • mice with SCID genetic mutation don’t develop T and B cells but have normal NK cells, indicating that NK cell development diverges from B and T lymphocyte development relatively early Adoptive Transfer • possible to temporarily transfer immunity passively by injecting Ab’s from donor • provides short-term immunity, dependent on half-life of transferred Ab’s in recipient • situations where adoptive transfer of Ab’s does not protect against infection (e.g. intracellular parasites) • transfer of immunity can often be effected by injection of lymphocytes from immune donor • cytotoxic T-cells, rather than B-cell, important in this effect Cell-Mediated Immunity and Graft Rejection • cell-mediated immunity (CMI) = any response in which Ab’s play subordinate role • most responses involve both Ab and cell-mediated events, each influencing degree and magnitude of other • tissue transplanted from one animal to a genetically-dissimilar animal does not survive long • initially graft tissue begins to grow and be accepted but after days-weeks (depending on type of tissue and amount of genetic dissimilarity) graft begins to die • histologically, lymphocytes infiltrate graft and some appear to attack and kill cells of graft • by using adoptive transfer techniques, found that T-cells most important • if 2 graft from same donor placed on same recipient at later time, rejected much more rapidly, showing that nd immune responses capable of developing immunological memory • grafts between genetically-identical pairs not rejected because Ag’s not recognised as foreign • tissue from genetically-identical donor syngeneic, from genetically-unrelated donor allogeneic • immune responses that occur in graft rejection called alloreactive responses Mixed Lymphocyte Reaction • similar immunological rejection processes occur when blood is transplanted from one individual to another
  10. 10. • immune responses occurring during graft rejection can be mimicked by mixing lymphocytes from different individuals in culture and observing reaction over few days by measuring uptake of radioactive nucleotides • test called mixed lymphocyte reaction (MLR) and is still important way to test immunological compatibility Cells and MHC-Restricted Recognition of Ag’s • T cells and B cells do not respond to exactly same foreign determinants • T cells respond best to Ag’s associated with cell surface • B cells bind soluble Ag’s • proof: • infect mouse from one inbred strain (strain 1) with lymphocytic choriomeningitis virus (LCM virus) • short time later, when cytotoxic T cells specific for virus-infected target cells developed, remove spleen from mouse and study specificity • found that virus-specific TCT could effectively kill infected target cells derived from syngeneic mouse (i.e. strain 1) but would not kill infected target cells derived from allogeneic mouse (i.e. strain 2) • indicates that cytotoxic T cells not only recognizing Ag’s specified by LCM virus but also some normal component of infected target cells • so T cell receptor for Ag more complex than surface immunoglobulin receptor in that it recognizes foreign Ag’s in association with some normal 'self' Ag’s • shown to be MHC • MHC-restricted recognition of Ag general property of T lymphocytes, although different T cells recognize Ag’s associated with different MHC structures Ag-Recognition Surface Molecules on T-Cells • all T cells have complex of transmembrane proteins (CD3) on surface, known to be required for activation • TH cells also have CD4 molecules and TCT cells CD8 • both CD4 and CD8 are intimately associated with CD3 • T cells have two other transmembrane polypeptides involved in Ag recognition • called α and β chains, together known as T cell receptor (TCR) • striking similarities between structure of Ig and TCR: both chains have constant and variable domain, hydrophobic membrane-spanning region and short intra-cytoplasmic domain • genes that coding for TCR possess similar arrangement to that found in Ig genes • variable regions undergo rearrangement during T lymphocyte maturation in thymus Ag Recognition and Roles Of CD4, CD8 & CD3 • both CD4 and CD8 T lymphocytes use same genes to synthesize TCR • CD4 helper T lymphocytes: class II MHC-restricted recognition of Ag’s • CD8 cytotoxic T lymphocytes: class I MHC-restriction Ag • CD4 and CD8 bind to non-polymorphic regions of class II and class I respectively • TCT lymphocytes potentially able to recognize Ag’s expressed on any nucleated cell in body • TH lymphocytes have much more limited Agic universe • CD3 (intimately associated with TCR) consists of complex of 5 polypeptide chains • must be involved in activation, as monoclonal Ab’s against CD3 block induction and effector phases • other surface molecules involved in complex process of interaction, but TCR, CD4/CD8, and MHC contribute most to specificity
  11. 11. Clonal Selection and T Cell Activation • 20 lymphoid organs and circulating lymphocyte pool contain repertoire of Ag-sensitive T cells, each cell bearing set of TCRαβ receptors through which it can recognize outside world • foreign Ag’s come into contact with these T cells • those that can bind strongly to foreign Agic determinants presented in MHC are selected and activated • as well as requirement for binding to Ag presented by MHC activation requires and interaction between other surface molecules called co-stimulators • without co-stimulator activation does not occur and T cell can become anergized • include B7 expressed on activating APC and CD28 expressed on T cell • for cytotoxic T lymphocyte precursors (CTLP), Ag binding and other surface interactions induces cell to express cytokine-R, in particular IL-2-R (secreted by activated TH cells) • in response to Ag, IL-2 and other cytokines, CTLP can then proliferate and differentiate to form cytotoxic effector cells • cells in cytotoxic clone have same Ag-R and can recognize same foreign Ag presented in MHC • seek out and destroy any cells which bear foreign determinants that they can recognize • during processes of T-cell activation by Ag, memory cells also generated • similar to and have same specificity as precursors but more sensitive and can respond more vigorously • responsible for maintenance of immune state, causes rapid 2nd set rejection in transplant immunology Cytotoxic Mechanisms Used by CD8 T Cells • do not release effector molecules but require direct physical contact with targets to exert cytotoxic effect • kill targets by three mechanisms • insert complex of molecules called perforin into membrane which assembles into doughnut-shaped holes and compromise integrity of target cell’s membrane • release enzymes which digest target cell membrane • release cytokines that can bind to receptors on target cells and induce apoptosis
  12. 12. T and B Cell Development and Thymus T Cell Receptor Genes • very similar to Ig genes and are considered part of Ig gene superfamily • divided into arrays of interchangeable V, D and J exons scattered over large tracts of DNA • undergo rearrangement during T cell development in same way as Ig genes during B cell development, so that individual mature T cells end up with ability to produce only one type of receptor • separate gene complexes for α, β and γ chains and each contains V, J and D exons followed by C exons • δ chain unusual in that D, J and C exons are located between V and J exons of α chain gene complex • As result, α and δ chains use same V segments • C gene segments made up of 4 exons which include constant domain segment, short hinge-like region, transmembrane and intracytoplasmic parts • during T cell development in thymus, V, D and J segments are selected and recombined to form complete V- coding domain adjacent to particular C region Two Types of TCR • most T cells use α and β chains but small subset use γ and δ chains • represent lineage distinct from αβ T cell lineage • found in small numbers in peripheral blood and lymphoid organs (1-10%) • present in larger numbers in intestinal epithelium and represent bulk of dendritic T cells in skin • home to lungs, reproductive tract and spleen • in lymphoid tissue, γδ T cells reside in regions distinct from those occupied by αβ T cells; splenic γδ T cells predominate in sinusoids, αβ in follicular areas • majority have CD3 associated with their TCR but lack CD4 and CD8 (although small population of intestinal γδ also have CD8 on their surface) • cannot recognize Ag’s in same way as αβ T cells because do not have CD4 or CD8 molecules • physiological role uncertain, but may have role in control of infectious processes and autoimmunity • relative abundance epithelial surfaces suggests importance as early line of defence • proliferate in response HSPs T Cell Receptor Diversity • at least 3 highly diverse regions in both α and β chains (correspond to CDRs of Ig) • 2 encoded by V gene segments • 1 encoded by V and J gene segments (CDR3, in α chain) or V, D and J segments (in β chain) • many fewer V genes employed by TCR than by Ig, other ways are used by TCR to generate diversity - particularly within CDR3 region • include: • N-region diversity documented in all 4 TCR chains but only occurs in 1 Ig chain (heavy chain locus) • large number of TCR J region gene segments (cf. small number in Ig loci) • several Vαs, Vγs and Vδ s flexible with respect to position of their 3' joining points (not seen in Ig) • can use both D regions in δ chain and N-region addition occurs at 3 different positions • additionally, translation of D region sequences in all possible reading frames rare in Ig heavy chains but common in TCR β and δ chains, compensating for smaller number of TCR segments
  13. 13. Ig TCR αβ TCR γδ H κ α β γ δ Variable (V) segments ~100 ~70 50 57 8 3 Diversity (D) segments ~4 - - 2 0 3 Joining (J) segments 6 5 70 7 3 3 Combinatorial diversity ~10 6 ~10 6 ~10 3 N-region addition V-D None V-J V-D V-J V-D1 D-J D-J D1-D2 D2-J Ds read in all frames Rarely - - Often - Often Somatic mutation yes yes no no no no Total diversity ~1010 ~1010 ~106 by all mechanisms • concentration of diversity in V-J junction of TCRs may be reflection of known function of TCRs (recognize very diverse small molecules embedded in much less diverse and larger MHC molecules) • γδ T cells, in spite of great potential for diversity, show very limited repertoire for V, D and J gene usage • e.g. within given epithelium, γδ T cells show little or no diversity • however, certain receptor genes used appear to be specific for location where γδ cell found B And T Cell Ontogeny and Self-Tolerance • both B and T cell repertoires are designed for maximum recognition versatility • only cells that can recognize Ag through surface receptors activated to generate responses • because there does not have to be exact complementarity between sIg receptors and Ag, cells with range of affinities triggered in response to any particular Ag • high probability that there will always be some cells capable of responding to even most obscure Ag’s • but immune system must be able to discriminate between self and non-self and respond differently B Cell Ontogeny • lymphoid progenitor cells and early pro-B cells bind to VCAM-1 on bone marrow stromal cells through integrin VLA-4 and other adhesion molecules • promotes binding of surface c-Kit tyrosine kinase to stem-cell factor, inducing proliferation • late pro-B cells require both stem cells factor of stromal cell surface and IL-7 for growth and maturation • first type of cell that can be identified as being committed to B cell lineage is pre-B cell • can synthesize (but not secrete) μ heavy chains (can be observed in cytoplasm) • have undergone DNA rearrangements in heavy but not light chain genes • differentiate further and become fully committed by rearranging either their κ or λ light chain genes so that they acquire ability to make complete immunoglobulins • next, negative selection of self-reactive cells occurs • if immature B cell expresses s-IgM-R that recognizes self Ag in bone marrow it will bind to it • unlike mature B cells, any immature B cells that bind Ag become inactivated • never reach full maturity, and do not appear in Ag-sensitive B cell repertoire of 20 lymphoid organs • immature B cells that don’t recognize self Ag’s permitted to differentiate into fully mature B-cells and express s-IgD and IgM • s-IgD functions as B cell receptor delivering activation signal following Ag recognition, often referred to as triggering receptor
  14. 14. T Cell Ontogeny • T cell development occurs in thymus (bilobed organ overlying heart, each lobe organized into lobules) • in each lobule, bone marrow-derived lymphoid cells arranged in outer cortex of immature proliferating thymocytes and inner medulla of more mature cells • interdigitating bone marrow-derived APCs expressing MHC molecules • both thymic ep cells and interdigitating cells are involved in intrathymic ‘education’ of T lymphocytes • requirements to selection T cell lineage: • effective T cell receptor gene rearrangement • +ve selection for recognition of self MHC • -ve selection to eliminate strongly auto-reactive cells • differentiation of CD4 and CD8 subpopulations • development of αβ and δγ T cell subpopulations • during development, stem cells move from cortex to medulla, becoming progressively more mature • each thymocyte will rearrange either its TCRαβ or TCRδγ genes during sojourn in thymus • cortical thymocytes express CD4 and CD8 accessory molecules (double positive), subsequent maturation leads to these cells becoming ‘single positive’ medullary thymocytes • immature CD4/CD8 cells express only 1/5-1/10 TCR molecules of mature peripheral blood T cells • mature thymocytes develop ↑ TCR density ~20-40,000 per cell, similar to peripheral T cells Positive And Negative Selection In Thymus • of cells expressing TCRαβ are two types of selection that must occur before develop into fully mature T cells • must express TCR which recognizes MHC molecules with low affinity so that they will be able to recognize MHC in association with foreign epitopes with higher affinity (+ve) • must not recognize MHC + self peptide so strongly to be self-reactive in absence of foreign Ag (-ve) Positive Selection • recognize Ag exclusively in association with self-MHC • not property of all possible TCRαβ combinations • T cells unable to interact with self MHC die in thymus • only T cells with TCRαβ capable of interacting with self-MHC are positively selected for survival • self-MHC molecules responsible reside on thymic epithelial cells of cortex • probably occurs when thymocytes express both CD4 and CD8 accessory molecules Negative Selection • T cell repertoire arising from random rearrangement of TCRαβ genes would include combinations that would recognize self Ag’s plus self-MHC • strongly self-MHC autoreactive T-cells clonally deleted to maintain self tolerance • for clonal deletion to explain all tolerance every self-Ag in body must manifest itself in thymus • some researchers think idea credible because thymus crossroads for macrophages, other APC and soluble molecules circulating through body • other mechanisms, such as clonal anergy or suppression probably operate as well Mature Repertoire • as well as selection constraints, repertoire of mature T cells is dynamic and in constant process of flux, influenced by such factors as exposure during life to various microbial Ag’s
  15. 15. • analysis of frequency of TCR gene usage shows variation from person to person at any one time
  16. 16. Cytokines, Lymphocyte Surface and Adhesion Molecules T Cell Subsets And Cytokine Production • TH major source of many cytokines and very influential in modulating immune and inflammatory responses • TH can be divided into different populations on basis of different cytokines produced • TH0 thought to be common precursors Cytokines produced TH0 TH1 TH2 Cytoxic/Inflammatory    (IL-2, IFN-γ, lymphotoxin) IL-12    TNFα, GM-CSF, IL-3    Ab response    (IL-4, 5, 6, 10) • TH1 important in elimination of intracellular infections (e.g. Leishmania) • TH2 responses amplify humoral immune response against extracellular micro-organisms • TH1 and TH2 responses regulated themselves by cytokines produced • IL-4 produced by TH2 cells promotes further TH2 expansion and down-regulates TH1 response while IL-12 promotes TH1 responses and down-regulates TH2 cells Activated T cells and their Products CD8 T cells Cytotoxins perforin 1, granzymes Others Fas ligand, IFN-γ, lymphotoxin (TNF-α and TNF-β) TH1 cells Macrophage-activating cytokines IFN-γ, GM-CSF, TNF Others IL-3, lymphotoxin, IL-2 TH2 cells B cell-activating cytokines CD40 ligand, IL-4, IL-5, IL-6 Others IL-3, GM-CSF, IL-10, TGF-β Cytokines • glycoproteins that act as soluble messenger molecules between cells • initiate many different cellular events including production of further cytokines and other mediators of inflammation, expression of adhesion molecules, cell growth and differentiation • actions of some are inhibitory, playing important role in regulating immune response • number of cytokines termed interleukins (IL) meaning “between white cells” • most cytokines display pleiotropism (diverse actions), redundancy (many cytokines have similar functions), diverse origins, networking and high affinity binding Class Cytokines Function Multifunctional IL-1, TNFα, IL-6 T and B cell activation, hepatocyte acute phase response, fever induction, IL-1 and TNFα mediate fibroblast proliferation, PGE2 and collagenase synthesis, bone resorption T cell IL-2 activation and growth of T helper and T cytotoxic cells (IL-4, IL-6, IL-7) B cell IL-4, IL-5, B cell stimulation and growth IL-6 B cell differentiation to plasma cells IL-7 stimulation of pre-B cells Haematopoietic IL-3 proliferation of haematopoietic cells G-CSF, M-CSF granulocyte and macrophage growth /differentiation GM-CSF eosinophil growth factor IL-5, erythrocyte and megakaryocyte growth IL-9
  17. 17. Class Cytokines Function Chemotactic IL-8 neutrophil migration and degranulation Interferons IFNα, IFNβ, IFNγ anti-viral enhance HLA Class I & II expression activate NK cells, macrophages Growth factors TGFβ, PDGF fibroblast proliferation and matrix synthesis TGFα, EGF anchorage independent growth IGF, FGF wound healing Inhibitory IL-10 CSIF (cytokine synthesis inhibitory factor) TGFβ inhibits IL-1 and TNFα synthesis Properties • can be classified on basis of their functions, emphasizing role in regulating immune response Innate Immune Response important in viral and bacterial infections e.g. IFN-α & β play vital roles in viral infections, TNF-α, IL-1 & 6 important in bacterial infections TNF-α, IL-1 and IL-6 all ↑ inflammatory responses (“pro-inflammatory” cytokines) Adaptive Immune Response IL-1 (and to lesser extent TNF-α) secreted by APC following Ag presentation, and has been termed second messenger for T cell activation stimulates secretion of another cytokine, IL-2, by TH, which stimulates T cell proliferation link initial “innate” response with specific “adaptive” immune response driven by Ag IL-2 originally called T cell GF as its main action is ↑ T cell growth and clonal proliferation produced mainly by TH, has autocrine and paracrine effects IL-2 also important stimulator of NK cell activity and may help to stimulate B cell growth IFN-γ IL-4 performs important function in switching Ab production from IgM to other isotypes, particularly important in allergy where it promotes production of IgE IL-1,6 important in promoting B cell differentiation are IL-1 and IL-6 Chemotaxis IL-8 representative of large family of chemokines, small (8-10kD) chemotactic glycoproteins all contain 2 internal disulphide loops, structurally divided into 2 groups depending on whether cysteines are adjacent (C-C) or separated (C-X-C) IL-8 is C-X-C chemokine, is produced at sites of inflammation promoting chemotaxis of neutrophils binds to receptors on neutrophils coupled to GTP-binding proteins and have characteristic structure featuring 7 transmembrane domains important participants in leukocyte trafficking as they activate integrins, promoting firm adhesion of leucocytes to endothelium as well as directing movement of neutrophils may be thought of as 20 mediators of inflammation as their secretion is stimulated by pro- inflammatory cytokines C-C chemokines include monocyte chemotactic protein and macrophage inflammatory protein, play important role in allergic inflammation by stimulating basophils to release histamine Haematopoietic Growth Factors number of cytokines appear to act primarily to promote growth and differentiation of various lineages of haematopoietic cells Anti-Inflammatory Cytokines - Promotion Of Repair • TGFβ produced by certain tumours, allows normal cells to grow in soft agar • important immunoregulatory properties, secreted by Ag-activated T cells and macrophages • highly pleiotropic – can ↓ or ↑ growth of many cell types depending on experimental conditions • important ↓ actions on T cell proliferation and maturation of cytotoxic T cells • can ↓ synthesis of pro-inflammatory cytokines such and counteract effects of others • promotes wound healing by generation of connective tissue and new blood vessels • IL-10 (aka cytokine synthesis inhibitory factor, CSIF) important in regulating inflammatory response • FGF, PDGF, IGF all important growth factors participating in wound healing
  18. 18. Cell Adhesion Molecules • fundamentally important to cell, determining whether it circulates or moves from blood vessels • granulocytes and monocytes migrate through vessels in response to changes in adhesion molecules expressed by endothelial cells and home to target areas within tissue - do not recirculate • lymphocytes continually patrol body for foreign Ag by recirculating from blood stream, into tissue, through lymph nodes, into lymph and ultimately back to blood stream • also involved in growth and development of tissues, wound healing and tumour invasion • 4 major families of cell adhesion molecules which work in co-ordinated way to facilitate movement of leucocytes and their interaction with endothelial cells and ECM Family Molecule Notes Selectins mediate processes of tethering and rolling interactions have rapid association and dissociation constants and so adhesion is short-term and labile selectins bind to mucin-like ligands, which contain sialylated CHO residues which specifically bind to Ca2+-dependent lectin domain L-selectin expressed on all circulating leucocytes except for memory lymphocytes ligands: GlyCAM and CD34 produced by HEV P-selectin in α-granules of platelets and in pre-formed Weibel-Palade bodies of endothelial cells ligands: P-selectin glycoprotein ligand (PSLG-1) present on neutrophils E-selectin induced on vascular endothelial cells by cytokines such as IL-1 and TNF-α ligands: sialyl LewisX epitopes on leucocytes one of most versatile and diverse families of adhesion molecules, important in cell-cell and cell-ECM adhesion Integrins adhesiveness can be rapidly regulated by chemokines such as IL-8 composed of α and β subunit, subfamilies are classified according to β subunit β1 responsible for cell-ECM adhesion some recognize tripeptide sequence ‘RGD’ found on ligands (e.g. fibronectin) interaction between β1 integrin VLA-4 on monocytes, lymphocytes and eosinophils and VCAM-1 on endothelial cells provides important means of leucocyte-endothelium adhesion β2 found on surface of lymphoid and myeloid cells, mediate adhesion of these leucocytes to endothelium particularly important in neutrophil adhesion as these cells lack β1 integrins present constitutively on leucocytes and undergo conformational change upon activation to ↑ adhesiveness β3 found on platelets platelet adhesion to fibrinogen, fibronectin, von-Willebrand factor and vitronectin β7 found on lymphocytes and mediates homing of lymphocytes to Peyer’s patches in gut recognizes ligand on HEV of Peyer's patch called mucosal addressin cell adhesion molecule (MAdCAM-1) Cadherins Immunoglobulin Superfamily Molecules (IgSF) ICAM-1 expressed on inflamed endothelium binds to LFA-1 ICAM-2 constitutively expressed on endothelial cells binds to LFA-1 on leucocytes ICAM-3 expressed on leucocytes VCAM-1 expressed on activated endothelium binds VLA-4 MAdCAM expressed by HEV of Peyer’s patches MAdCAM-1 also contains mucin-like domain which can bind L-selectin and mediate lymphocyte rolling Ca2+-dependent adhesion molecules, mediate “homophilic” cell-cell binding N-cadherin expressed in embryonic ectoderm and influences separation of neural tube tissue from ectoderm (neural tube) Leucocytes in Inflammation • leucocytes circulating in bloodstream must be able to slow down and stop within blood vessels near site of inflammation, adhere strongly to endothelium, penetrate into underlying tissue and migrate through ECM
  19. 19. Rolling and Tethering • selectin molecules bind mucin-like CHO ligands • initially (within seconds) P-selectin expressed on surface of endothelial cells, hooks on to PSLG-1 on neutrophils causing them to roll along endothelial surface • L-selectin on leucocytes then interacts with CD34 on endothelial cells • E-selectin and sialyl-Lewis X interaction occurs later as E-selectin only presented 2-8 hours after stimulation with pro-inflammatory cytokines Triggering • chemokines act as leukocyte chemoattractants and are produced at sites of inflammation • diffuse outward producing concentration gradient down which leucocytes migrate • also produced by endothelial cells under influence of other cytokines (e.g. IL-1 and TNF-α) • platelet activating factor, CD31 and C5a other important chemoattractants • chemoattractants from endothelial cells attach to chemoattractant-R on leucocytes • ligand bound to receptor activates G protein cascade • G proteins transduce signals which activate integrin adhesiveness bringing Strong Adhesion • leukocyte integrins (e.g. LFA-1) express “activation epitopes” which ↑ affinity for ligands >200x • β2 integrin/ICAM interactions used by all leucocytes, while T lymphocytes also use VLA-4/VCAM • strong adhesion followed by decay of integrin function allowing cells to be released and extravasate • release phase also associated with shedding of L-selectin from leukocyte surface • migration of leucocytes within tissues associated with β1 integrin interactions as ligands for these adhesion molecules are found within ECM • leucocytes then migrate down chemoattractant concentration gradients to sites of inflammation Lymphocyte Trafficking • ~10% lymphocytes circulating at any time • re-circulating lymphocytes pass from blood through lymphoid system and back to blood • tend to recirculate preferentially to site where they have previously encountered Ag • naive T cells specifically migrate to lymph nodes and memory T cells to non-lymphoid tissue (homing) • specialized cuboidal endothelial cells in HEV express various adhesion molecules (e.g. GlyCAM-1 and CD34) to direct flow of lymphocytes into tissues • molecules which function to direct traffic of T cells through lymph node called addressins • suggested that lymphocyte homing process may also involve sequential steps including selectin-mucin interactions, followed by integrin activation and then coupling with IgSF molecules • interestingly, memory T cells lack L-selectin and therefore do not move through peripheral lymph nodes • long-lived lymphocytes (mostly T and memory cells), most mobile and participate in continual pattern of movement through lymph nodes, spleen, blood and lymphatic system T-Cell Activation • T cells also use adhesion molecules during adaptive immune response • activation occurs after encounter with Ag-presenting cell when Ag interacts with T cell receptor • adhesion molecules on both cell surfaces are required to stabilize this process • important molecules include β2 integrins, LFA-1, MAC-1 and CD2 • corresponding ligands on APC are ICAM-1 and LFA-3
  20. 20. Clinical Applications • current research on cell adhesion molecules extended into clinical field • monoclonal Ab’s to ICAM-1 used in transplantation research to block renal graft rejection • Ab’s to β2 integrins have been used experimentally to prevent cardiac re-perfusion injury by blocking migration of leucocytes to ischaemic tissue • Ab to β1 integrin has been shown to block experimental allergic encephalitis in rats (animal model for MS)
  21. 21. How Immune System Deals with Infections Process of Infection • often initial focus of infection does not stimulate immune enough to dispose of infectious agent completely • not until agent spread to 20 sites and replicated further that immune systems responds adequately • summary of events: (e.g. bacterial infection occurring at cut in skin) • initial lesion damages tissues and capillaries, provoking local inflammation • chemotactic factors released, attracting phagocytic cells – resulting neutrophil influx cleans up site by ingesting and digesting foreign matter and dead tissue • local mononuclear phagocytes as well as some monocytes also take up foreign matter, break it down into fragments and transport fragments to nearest lymph node through lymphatic vessels • bacteria that have entered lesion may begin multiplying at site • many bacteria will be ingested by local phagocytes, but some may escape and migrate through lymphatic and/or blood systems • other phagocytic cells reside in lymph nodes and spleen which serve to filter such bacteria Immune Responses to Infectious Agents • complex interactions among microbe, host factors, immunity, and pathogenesis occur during infections and degree and importance of each interaction varies depending on infectious agent • specific immune system can respond to infectious agents in variety of ways, in particular elaboration or inflammatory or inhibitory cytokines, cytotoxic T cell generation, and Ab formation • importance of each depends on nature of infectious agent • Ab’s can be generated against structural Ag’s of infectious agent, or against metabolic products • TCT generation is important in dealing with virus infected cells while of less value with extracellular pathogenic infections such as bacteria Factor Micro-organism Type of micro-organism (e.g.virus, bacterium, parasite) Dose (i.e. degree of exposure) Virulence of organism Route of entry Host Integrity of non-specific defences Competence of specific immune system Genetic capacity to respond normally to specific organism Evidence of previous exposure (natural or acquired) Existence of co-infection Immune Direct neutralization by Ab’s Opsonization and phagocytosis Complement-mediated effects MHC-restricted T cell-mediated cytotoxicity Inflammatory and immunoregulatory cytokines Anti-viral cytokines (interferons) Ab’s In Infection • specific Ab’s in high concentrations at time of infection may act to prevent viruses infecting target tissues
  22. 22. • however, if Ab’s not initially present, virus infection will often become well established in target tissues before production has been induced • other immune mechanisms, particularly TCT and TH-mediated effects will be more important • of value in bacterial and parasitic infections if they lead to inactivation or destruction of micro-organisms, but many bacteria have capsules or cell walls that are inherently resistant to complement-mediated damage • in situations where pathogen’s preferred site of infection is phagocytic cell, Ab’s may actually help • may occur in mycobacterial infections where mycobacteria grow inside macrophages and are fairly resistant to killing by Ab-mediated mechanisms Ab’s at Mucosal Surfaces • adherence to epithelial cells of mucous membranes often essential for viral and bacterial infection • IgA affords protection in external body fluids (i.e. tears, saliva, nose, intestines and lungs) • if infectious agent penetrates IgA barrier, comes up against IgE facet of secretory system • most serum IgE arises from plasma cells in mucosal tissues and in lymph nodes that drain them • although present in low concentration, IgE bound very firmly to Fc receptors of mast cells, and contact with Ag leads to mediator release Cytotoxic T Cells • because TCT cell activity restricted to recognition of peptides presented by class I MHC, not all Ag’s of pathogens will be accessible as targets of these effector cells • peptides presented by class I MHC arise from cytoplasmic processing of proteins and so only in pathogens that have intracellular cytoplasmic phase will T cell-mediated cytotoxicity play important role • TCT important in most viral infections but will be relatively unimportant in handling extracellular bacteria • recovery from viral infections strongly influenced by TCT activity and interferon rather than Ab’s Cytokines in Infection Secondary messengers important second messengers in cytotoxic T cell and B cell activation Pro-inflammatory TNFα TNFα mediates defence against bacteria (especially G-), release is triggered by LPS anti-TNFα results in widespread sepsis and sometimes death TNFα may enter bloodstream and act as hormone resulting in systemic host response to infection (e.g. fever, acute phase response, cachexia) IL-1 very similar actions to TNFα, both termed “endogenous pyrogens” primary source is activated macrophage but also produced by endothelium, ep cells and chondrocytes second messenger for T cell activation IL-6 shares many actions with IL-1 and TNF-α promotes “acute phase response”, acts upon hepatocytes to stimulate secretion elevation of fibrinogen is basis of clinical test which gives non-specific indication of degree of inflammation in body; erythrocyte sedimentation rate or ESR Chemotaxis e.g. IL-8 function to limit infectious processes by aiding chemotaxis and mobilization of effector cells as described Cytotoxicity IL-2, IFN-γ NK cell activity ↑ and Class I MHC expression ↑ NK and cytotoxic T cells lysis ↑ IFN-γ activates intracellular killing of bacteria by macrophages Interferon important antiviral proteins IFN-γ product of CD4 T cells, IFN–α and IFN-β transiently induced in most cells of body during viral infection act on uninfected cells to induce transient antiviral state by inducing of specific enzymes that cleave viral RNA Helper Cell Subsets in Infection • infectious disease immunology focused for many years on circulating Ab’s or capacity to generate rapid 2 0 Ab response as most effective means of protective immunity
  23. 23. • Ab’s play important role in protection from many viral infections but it is are not only effector mechanism nor are they protective in all infections • not only do TCT responses against virus-infected target cells constitute means of limiting viral infection but many bacterial and parasitic infections appears to depend more on TH than Ab’s • in some cases one class of T cells (e.g. TH1) associated with protection while another class (e.g. TH2) associated with exacerbation • protective immunity must be seen in context of each particular infection as relationship among B cells, TCT, TH and innate (e.g. NK cells) responses which may vary greatly depending on organism associated with infection • unstimulated macrophages do not deliver co-stimulatory signal to T cells recognizing non-bacterial Ag’s and consequently result in T cell anergy • bacteria stimulate macrophages to deliver co-stimulatory signal to T cells recognizing bacterial Ag resulting in proliferation and differentiation to specific T cell effectors • if non-bacterial Ag’s also presented by macrophages induced to express co-stimulatory signal then they too will lead to activation of specific T cells which recognize them Long-Lasting Immunity • generally speaking, vaccines designed to prevent viral infections do not give same degree of long-lived immunity as natural infection • one of factors contributing to this may be length of time Ag persists in infected or vaccinated individual • Ab-secreting B cells and effector T cells generally have short half-lives (days-weeks) • when Ag persists after infection, memory cells recruited over time and become Ab-secreting cells, thus providing for continual presence of Ab’s or at least allowing rapid production following re-exposure • injected Ag’s trapped in germinal centres of lymphoid tissues and undegraded Ag’s in form of Ag-Ab complexes can persist for long periods on follicular dendritic cells • in this form, Ag is highly immunogenic and can be endocytosed by B cells and presented to T cells, resulting in rapid B cell proliferation with formation of Ab-secreting cells in germinal centres Host Pathology in Infection • when large numbers of Ab’s produced with high concentrations of Ag’s, Ab’s will bind readily with Ag’s and immune complexes formed will tend to lodge in capillaries of kidney • activation of complement system can result in localized inflammatory reaction causing damage to surrounding tissues and compromising function of organs • when some Ag’s of infectious organism sufficiently similar to some host Ag’s, Ab’s generated against infectious Ag’s may cross-react and damage host tissues • when large numbers of infected cells in vital organ killed rapidly by TCT, organ may be unable to fulfil its functions properly • mechanism important in hepatitis B infections and in AIDS
  24. 24. Immunodeficiency (ID) Classification • immune system has many components: Ab’s, complement, B and T lymphocytes, cytokines and receptors, adhesion and other accessory molecules, HLA Ag’s, phagocytes etc • malfunction of any may result in state of immunodeficiency Classification congenital (due to genetic defect or in utero disease) acquired (in addition to HIV, acquired ID may be secondary to drug therapy or systemic disease) primary (inherited / genetic) secondary (due to some other factor, e.g. HIV) according to part of immune system affected (humeral, cellular, combined, complement, and phagocytic) • selective IgA deficiency is common (~1/500 people), but other primary ID are rare Primary ID Syndromes • very heterogenous range of syndromes • molecular basis of many ID is now known, and in future they will be classified by their cause • most due to autosomal or X-linked recessive gene defects Name Notes Adenosine Deaminase (ADA) build-up toxic byproducts of purine metabolism inside cells, T-cells especially affected deficiency first use of gene therapy, with limited success Bruton mutation in tyrosine kinase enzyme in B lymphocytes agammaglobulinaemia first ID described, X-linked, presents in childhood with recurrent pyogenic infections Severe Combined many potential causes, e.g. mutation in γ chain of IL-2-R critical cytokine, also part of Immunodeficiency (“SCID”) receptor for cytokines IL-4, -7, -9, and -15, so has widespread effects defects in both Ab and cellular immunity compatible bone marrow transplantation offers only chance of permanent cure Wiskott Aldrich syndrome defective gene codes for protein that regulates actin cytoskeleton platelet abnormalities as well as addition to B and T cell problems Leukocyte Adhesion defect in CD18 adhesion molecule, leukocytes can’t adhere properly, so can’t get to Deficiency infected and inflamed sites Hyper IgM Syndrome mutation in CD40 molecule so can’t make class switch high levels of IgM Ab’s but low IgG and IgA bare lymphocyte syndrome transcription of class II MHC genes abnormal, so can’t be expressed on surface MHC deficiency severely limits ability of T cells to respond to Ag’s Chronic Granulomatous defective enzymes in NADPH oxidase system needed to produce respiratory burst Disease (CGD) defective killing of phagocytosed bacteria Complement Deficiency may be asymptomatic or unusually prone to infection with certain bacteria Syndromes Hereditary angioneurotic deficiency of C1 inhibitor oedema Clinical Presentation • usually presents clinically with infection • type of infection is clue to underlying immune defect, but is usually not in itself diagnostic Organism Immunological Responses Ab T-cells Other Extracellular bacteria IgM, IgG complement, phagocytes Intracellular bacteria  macrophages Viruses IgG, IgA  complement, IFN Parasites IgE  eosinophils/mast cells Fungi IgA  neutrophils
  25. 25. • should suspect ID if patient has recurrent bacterial infections, or infections with unusual organisms • may be family history, many genetically are X-linked so will appear only in boys, most others autosomal recessive • 20 ID may be suspected: • in patients taking steroid or cytotoxic regimens for other disorders • with known major disease in other organ systems • with lifestyle risk factors for HIV • should always seek specialist advice before going beyond basic screening investigations • infections should be treated aggressively, and may require prolonged antibiotic therapy • except for Ig replacement therapy for Ab deficiency syndromes, no specific curative therapies for 10 ID • If ID is 20 to medication or disease, if possible underlying problem should be remedied Immunology Of HIV Infection • well established that HIV infects and depletes CD4 T-cells, molecular pathogenesis still poorly understood • co-factors (e.g. other infectious agents) may be involved • also infects macrophages, Ag-presenting dendrtitic cells, and some haemopoietic stem cells • virus’ gp120 molecule binds to CD4 on lymphocyte and low Mr cytokines receptors (chemokines-R) • some strains have preference for macrophages rather than T cells, and may reflect chemokine-R that predominate on different cell types • certain receptor polymorphisms seem to provide degree of protection, as does strong host chemokine response (competitive inhibition of binding to receptor?) • treatments based on chemokines being developed • infected cells may undergo cell death because of cell fusion/syncytia formation and can act as source of infection in cell to cell transmission • may also have more subtle functional effects on cells it infects, poorly understood • immune destruction results in loss of tumour surveillance, tumour cytotoxicity, and IFN-γ production • autoimmune problems can occur in AIDS, but not usually of clinical significance • result of losing T lymphocytes which normally suppress self-reactive immune responses? • due to excess of Th2 (B cell help) helper T lymphocytes? • vaccination against HIV possibility, and clinical trials with number of vaccines using gp120 are underway • produce successful Ab responses in host, crucial thing is probably to induce cytotoxic T lymphocytes Reasons for Poor Immune Response • progression associated with preferential loss of TH1 cells • virus mutates very rapidly, and immune system has trouble keeping up • good at “hiding” in dormant integrated state in lymphocytes resting in lymph nodes • current anti-retroviral therapies ↓ viral load and prolong survival but cannot eliminate latent virus Hypersensitivity • excessive immune response, so that host is damaged in some way • traditionally been divided into four types of reaction • 1: IgE mediated (anaphylactic, or “true” allergy)
  26. 26. • 2: Ab mediated • 3: Immune complex mediated • 4: Cell mediated (delayed type hypersensitivity, DTH) Type I Hypersensitivity : IgE mediated • occurs when divalent allergen cross-links 2 IgE molecules, previously passively bound to high affinity IgE Fc-R • mast cell mediators are released • granule-associated preformed mediators: • histamine, heparin • enzymes: tryptase, b-glucosaminidase • chemotactic and activating factors: eosinophil chemotactic factor (ECF), neutrophil chemotactic factor (NCF) and platelet activating factor (PAF) • newly formed mediators: • lipoxygenase products: SRS and leucotrienes • cyclooxygenase products: prostaglandins, thromboxanes • main effects are vasodilatation, vascular leakiness, pruritis, and smooth muscle contraction • may be localised, effects depend on which part of body is involved: Organ Symptoms Respiratory tract allergic rhinitis (hay fever, perennial rhinitis) sinusitis (NB ? secondary bacterial infection) asthma (allergic component VERY common) Eyes allergic conjunctivitis Skin urticaria (wheals) angioedema (deeper skin involvement) Gut food allergy (diarhoea, abdominal cramps, vomiting) Multiple organ anaphylaxis • generalised anaphylactic reactions can be fatal, as can severe asthma • patient’s with tendency to make IgE Ab’s to multiple allergens called atopic • runs in families, but basis poorly understood: one gene may be for variant of IgE Fc receptor • bronchial reactions to allergens show immediate and late phase reaction Type 2 Hypersensitivity: Ab mediated • complement fixation: (eg haemolytic disease of newborn, autoimmune haemolytic anaemia) • receptor-binding: blocking (anti-AChR in myasthenia gravis) or stimulating (anti-TSH receptor, Grave’s) • K/ADCC cells: receptors for Fc portion of Ig molecules, and lyse target cells when cross-linked • can be mediated by neutrophils, macrophages, eosinophils, and possibly other cell types • in vivo importance uncertain Type 3 Hypersensitivity: Immune complex mediated • binding of Ag and complementary Ag • conformational change in Ig Fc allows binding and activation of complement • direct action of complement split products • recruitment of other inflammatory cells by soluble mediators • systemic: classic example is serum sickness, caused by injection of foreign protein
  27. 27. • complexes deposit in skin (rash), joints (arthritis) and kidney (nephritis) • formerly seen with horse Ig for tetanus, problem with non-human monoclonal Ab’s used in therapy • postulated mechanism for vasculitis and renal disease seen in SLE • localised: occurs in tissues • farmer’s lung - extrinsic allergic alveolitis caused by inhaled actinomycete fungi which grow in hay, IgG Ab’s from circulation meet this Ag in alveoli • complement activation and recruitment of inflammatory cells • deposition of immune complexes in blood vessel walls Type 4 Hypersensitivity: Cell mediated (delayed type, DTH) • T cell mediated, takes 24- 48 hours • classic example: reaction to intradermal injection of purified protein derivative of Tuberculin, to test for cell mediated immunity against Mycobacteria • used to assess immunity (e.g. mumps, candida) as part of investigation of suspected immunodeficiency • also mechanism underlying contact sensitivity (e.g. to metals in jewellery) • initial phase involves uptake, processing, and presentation by dendritic cell in skin • presents Ag to T cells locally and in paracortical zones of nearby lymph nodes • T lymphocyte (mainly TH1) secretes cytokines, macrophages are recruited and activated • up-regulation of adhesion molecules and MHC expression on keratinocytes Autoimmunity • tolerance of “self” is central property of normal immune system • if discrimination broken, immune system may react to self structures and cause autoimmune diseases Mechanisms of Tolerance • Self-reactive lymphocytes are removed from immune repertoire when they are at immature stage of development • There are several possible mechanisms: • clonal deletion - complete removal of self-reacting cells • dominant mechanism for Ag’s expressed in primary lymphoid organs (thymus and bone marrow) • clonal anergy - self-reacting lymphocytes still exist, but are usually resistant to stimulation • important for Ag’s found only in periphery • immunological ignorance • self-reactive cells present, but do not mount pathological response, because Ag’s sequestered or lacking adequate T cell help • immune system needs to see Ag’s in context of certain “danger” signals before it responds • suppression • self-reacting lymphocytes present and potentially active, but are kept in check by “suppresser cells” • discrete suppressor population not found, now thought to be either TH2 or competing anergic cells
  28. 28. AutoAbs • mechanisms causing autoimmune disease are not fully understood Natural AutoAbs • loss of self-tolerance does not always lead to autoimmune disease • healthy immune repertoire has some B cells which have potential to produce autoAbs • usually IgM, low titre, and/or low affinity • directed against Ag’s that are not normally accessible in significant amounts (“sequestered” Ag’s) • referred to as “natural” autoAbs • postulated that they have regulatory role, or help dispose of breakdown products Why don’t they cause disease? • amounts or affinity are low • need “help” from corresponding self-reactive T helper lymphocyte to get stronger response going • Ab not pathogenic (many of Ab’s associated with connective tissue diseases don’t cause disease) • patient DOES have autoimmune disease, but this is still sub-clinical AUTOIMMUNITY IS COMMON, AUTOIMMUNE DISEASE IS RARE Sequestered Ag’s • tolerance not always established to Ag’s normally hidden from immune system (e.g. lens crystallin) • autoAbs can often be detected following trauma (allows contact of Ag with immune system) • may cause frank autoimmune disease (e.g. sympathetic ophthalmia, damaging good eye after trauma or surgery to other) Cross Reactions • tolerance can sometimes be by-passed by immunization with closely cross-reacting Ag • e.g. Ab’s against viral RNA or DNA may cross-react with self RNA or DNA, may help explain autoAbs to RNA or DNA in connective tissue disease • infection with certain bacteria (e.g. salmonella, shigella, chlamydia) can trigger reactive arthritis • rheumatic fever • similar cross-reactive theories put forward for infection triggering range of autoimmune diseases • alternative term for this theory of autoimmune disease is “molecular mimicry” Adjuvant Effects • adjuvants non-specifically enhance immune response • mixed with vaccines to improve efficacy and with Ag’s when experimental animals used to produce antisera • can help produce autoimmune disease when mixed with self or cross-reactive Ag’s • experimental examples: • collagen arthritis produced in rats and mice by immunising with bovine collagen in adjuvant, used as model (albeit not very convincing one) for human rheumatoid arthritis • thyroiditis can be induced in rabbits with thyroglobulin in adjuvant • experimental autoimmune encephalomyelitis produced in mice and rats using myelin basic protein, model for human multiple sclerosis • postulated that infections (both bacterial and viral) may have adjuvant-like effect, in particular:
  29. 29. • lipopolysaccharide in G- bacteria is polyclonal B lymphocyte activator • superAgs secreted by certain bacteria are polyclonal T lymphocyte activators Abnormal MHC Expression • viruses may have analogous adjuvant-like effect: • viral infection leads to release of interferons • IFN-γ causes up-regulation of Class II HLA Ag synthesis • most cells (except for immune and haemopoietic) don’t normally express Class II HLA • may present peptides derived from intracellular autoAgs • immune system isn’t normally exposed to these - intracellular version of sequestered Ag’s • T lymphocytes then respond to HLA+ sequestered-self • CD4+ T cells can now provide “help” for B lymphocytes that previously ignored intracellular Ag • examples of MHC Class II up-regulation include: • islet cells in type I diabetes mellitus • thyroid glandular cells in autoimmune thyroiditis • synovial endothelia and lining cells in rheumatoid arthritis • keratinocytes in psoriasis • unresolved issue is whether class II increase is cause or effect Regulatory Abnormalities • older theory for autoimmune disease was that regulatory suppressor T lymphocytes became defective, allowing emergence of autoimmune processes previously held in check • not widely believed, may regulate size of some immune responses rather than all-or-nothing tolerance effect Clinical Features Of Autoimmune Disease • vary widely, depending on organ and exactly what immune response is directed against • examples include: • thyrotoxicosis, caused by Ab’s against TSH receptor, mimic action of TSH and stimulate thyroid gland • pernicious anaemia, autoimmune destruction of gastric parietal cells or Ab’s directed against IF itself • idiopathic thrombocytopenic purpura, due to Ab’s against platelets • Addison’s disease, due to destruction of adrenal cortex • myasthenia gravis, due to autoAbs blocking acetylcholine receptor on NMJ • pemphigus, bullous inflammatory skin disease caused by autoAbs against cadherins Genetic Predisposition Factor Notes Immunoglobulin genes genes for some autoAbs encoded in germline somatic hypermutation may also be responsible for producing autoAbs T cell receptor (TCR) some TCR Vß genes are used preferentially by autoreactive T cells elimination of T cells which use specific Vß genes can prevent or control disease in animal models with Vß-specific anti-TCR Ab, T cells mediating experimental allergic encephalomyelitis can be eliminated, curing disease Complement human SLE is associated with deficiencies of complement components thought to be due to impaired clearance of immune complexes MHC many autoimmune disease patients have HLA-B8, -DR3 haplotype rheumatoid arthritis HLA-DR4, ankylosing spondylitis HLA-B27, IDDM aa substitution in β chain of HLA-DQ area of intensive research
  30. 30. several mechanisms may account for this: peptide binding, molecular mimicry, receptor model Other genes genome screens have been performed for several autoimmune diseases, and several candidates identified tend to code for proteins involved in regulating immune response New Treatment Approaches • cytokine inhibitors: antagonists of TNF-alpha and IL-1 are now in clinical use for rheumatoid arthritis • oral tolerance: works well in some animal models, e.g. multiple sclerosis and arthritis, but so far has had little success in human disease
  31. 31. Immunology in Diagnostic and Clinical Medicine Summary • make use of exquisite specificity of Ag-binding part of Ab molecule, and detection system attached to constant portion of molecule • can be used to detect either Ag’s, or Ab’s directed against them ELISA • enzyme-linked immunosorbent assay (ELISA) widely used technique Step Instructions 1 Ag attached to solid phase (often –ve plastic plate) best under alkaline conditions (pH 9-10), so Ag’s have net positive charge 2 plate then washed with saline solution (to remove any unattached Ag’s) wash solution contains weak detergent to help reduce non-specific binding 3 patient’s serum added, diluted in saline solution, incubated with Ag on plate specific Ab’s attach to Ag, while other Ab’s stay in solution plate is washed again, removing any unattached Ab 4 addition of “secondary” Ab” directed against Fc portion of patient’s Ab, chemically coupled to enzyme plate washed again 5 enzyme substrate then added (often horseradish peroxidase (HRP), in presence of H2O2 produces brown colour) measured by automated spectrophotometer Results amount of colour proportional to amount of HRP, in turn proportional to how much secondary Ab has bound, in turn proportional to amount of Ab in patient’s serum Sandwich ELISA Technique • simple variation on basic ELISA principle, and used to detect Ag’s • Ag “sandwiched” between two Ab’s • used to measure wide range of proteins (e.g. polypeptide hormones) • instructions: • coat 1st Ab onto solid phase • incubate patient’s serum, containing Ag, on plate • add Ab directed against same Ag (can coupled directly to enzyme detected by 3rd Ab) • wash plastic plates between successive incubations • colour developing when substrate added proportional to amount of Ag in patient’s serum Similar Techniques Name Differences Notes Radio-Immunoassay instead of enzyme, 20 Ab labelled with most assays are moving towards ELISA instead of RIA because of (RIA) radio-isotope safety concerns Micro Particle Enzyme solid phase made of numerous much greater surface area, so much more Ag can be used, making Immunoassay microscopic beads coated with Ag it more sensitive assay (MPEIA) many of serological assays performed in Auckland now performed by MPEIA (eg Hep B, Hep C, HIV 1 & 2) Radio Allergo-Sorbent 20 Ab directed against Fc portion of IgE performed to detect IgE Ab’s against specific Ag’s Assay only use Ag that might be causing patient’s allergy (e.g. house dust (RAST) mite) most RAST tests are now performed by ELISA or MPEIA, but old terminology still used allergy usually diagnosed on clinical grounds or with skin testing, and RAST tests are $$$ so only used in special circumstances
  32. 32. Name Differences Notes Western Blot Ag’s 1st dissociated with detergent, then can tell size of Ag electrophoresed (SDS-PAGE), important in diagnosing HIV infection: if patient’s serum tests +ve separates components according to Mr by MPEIA, Western blot is performed transferred to nitrocellulose membrane, ideally, Ab’s should bind to p24, p31, gp40, and either gp120 or incubated with patient’s serum, Ab bind gp160 of envelope for definite +ve to their respective Ag on membrane after washing, Ab’s detected using enzyme-coupled 20 Ab Immunofluorescence (IF) • fluorochromes (chemicals that emit light when stimulated) can be chemically coupled to Ab • fluorescein (FITC) emits green (520nm) colour, phycoerythrin (PE) emits orange (570nm) colour Direct IF • can be used to detect Ag’s found in tissue sections, or expressed on outsides of cells • tissue or cells incubated with fluorochrome-coupled Ab • after washing, specimen is examined under fluorescence microscope • areas with Ag show up as bright, coloured regions on dark background (band-pass filtering) • more than one Ag can be detected if Ab’s coupled to difference fluorochromes Confocal Microscopy • fluorescent images hard to examine at high magnification because of glare from slightly out-of-focus planes • confocal microscope focuses sharp laser beam on fine plane within tissue • photomultiplier tubes scans area, collects emitted light only from same plane, analysed digitally • result is high definition image that looks like very thin cross section of tissue • can image organelles within cells, and examine proteins secreted immediately surrounding cell Indirect IF • analogous to ELISA technique, often used to detect autoAbs ANA • anti-nuclear Ab’s (ANA) present in many patients with SLE, detection useful for diagnosis • assay starts with tissue section (or cultured cells) with prominent nuclei • attached to glass microscope slide, treated with chemical fixatives to stop Ag’s degrading and make membrane permeable (allows ANA to access nucleus) • patient’s serum added, and incubated with fixed tissue • after washing, FITC-coupled anti-Ig 20 Ab is added • after washing, anti-Ig will only remain if serum contained anti-nuclear Ab’s • quantitated by performing serial dilutions (1:10, 1:20, 1:40, 1:160, etc) of patient’s serum • result expressed as highest dilution that gave positive result • because some non-specific binding can still occur, ANA below 1:40 regarded as -ve • different nuclear Ag’s (e.g. nucleoli, centromere, DNA) distributed differently within nucleus ANCA • anti-neutrophil cytoplasmic Ab’s (ANCA) found in patients with vasculitis • assay performed as above, but using neutrophils • different patterns (perinuclear (pANCA) and cytoplasmic (cANCA)) associated with different types • Ag’s involved are enzymes myeloperoxidase and proteinase 3, respectively
  33. 33. Flow Cytometry • cells in suspension incubated with fluorochrome-tagged Ab’s, then washed • labelled cells pass through flow cytometer one by one • laser beam focused on each cell and fluorescence analysed, size and granularity of cell also measured • by using Ab’s against surface Ag’s, composition of cell population can be precisely determined • measurements are quantitative, so that estimate can be made of amount of Ag on particular cell type • possible to simultaneously examine up to 4 different Ag’s on each cell, but usually only 1 or 2 are • e.g. determination of ratio of CD4+ to CD8+ lymphocytes in AIDS patients: lymphocytes incubated with mixture of anti-CD4 (coupled to fluorescein) and anti-CD8 (coupled to phycoerythrin) • also used to examine white blood cells from patients suspected to have leukaemia: different types of leukemic cells have particular combinations of CD Ag’s on their surfaces • able to sort each cell into different receptacles • e.g. peripheral blood lymphocytes could be separated into CD4 and CD8 subpopulations • currently only done in research settings Immunocytochemistry • used to examine tissue under microscope for presence and distribution of particular Ag • enzyme-coupled Ab used, yielding insoluble coloured product • stays in tissue where Ab bound to Ag, and can be seen down microscope • disadvantages: lower definition but adequate for lower power microscopy • advantages: can be examined with ordinary microscope, colours also last long time • similar technique can be used with transmission electron microscopy (EM), but Ab’s coupled with gold This is Complement Fixation Tests (CFT) • often used to diagnose recent infections with organisms that are hard to culture • e.g. in diagnosis of recent infection with adenovirus, respiratory syncytial virus, influenza and B, parainfluenza 1- 3, Mycoplasma pneumoniae and Chlamydia psitticae • steps: • patient’s serum incubated with preparation of viral Ag’s • if patient’s serum contains Ab’s against virus these will bind virus to form immune complex • complement then added and immune complexes bind, then fix complement • sheep red blood cells (SRBC) that incubated with anti-SRBC added to mixture • if there complement left (i.e. no anti-viral Ab’s present), SRBC will be lysed • if no complement left (i.e. anti-viral Ab’s present), SRBC will not be lysed • made quantitative by using serial dilutions of serum • many patients will have Ab’s against common viruses because of prior infection, so to diagnose recent infection, need to show 4-fold rise in titre between acute and convalescent phases Complex Formation Immunodiffusion • when Ab’s combine with complementary Ag’s, insoluble complex forms and precipitates • can be visualised in several ways
  34. 34. • Ab and Ag diffusing towards each other from two small wells cut in agar gel will form visible line • Ag embedded in agar and serum put in well, visible ring will form as Ab slowly diffuses away from well • to assay for Ag, Ab embedded in gel, and serum added to wells • diameter of ring proportional to concentration of Ab • technique known as radial immunodiffusion • used to measure auto-Ab’s against certain of nuclear Ag’s (e.g. ANA SSA and SSB) Nephelometry • serum mixed with Ag in clear tube • light passing through tube is impeded by complexes, measured by spectrophotometer • light absorption proportional to amount of Ab present • used to quantitate Ab’s against DNA in SLE patients Agglutination • IgM Ab’s are much more effective at agglutination than IgG • classical application is blood grouping • Ag’s can also be artificially coated onto red blood cells • other particles (e.g. microscopic latex beads coated with Ag’s) can also be used HLA Tissue Typing • performed to achieve close match between recipient and donor before organ transplant Lymphocytotoxicity • traditional method uses cytotoxicity assay • peripheral lymphocytes incubated with wide range of Ab’s directed against all main HLA Ag’s • complement added, if lymphocytes have Ag, will be lysed by respective antisera • >100 different antisera are used to determine person’s HLA-A, B, and C Ag’s • anti-HLA antisera are scarce and expensive, so assays scaled down to microlitre proportions Polymerase Chain Reaction (PCR) • now use PCR to determine HLA types • match PCR primers (“sequence specific primer”, SSP-PCR) • probe PCR products with labelled nucleotides • directly determine DNA sequence of patient’s HLA genes • currently PCR mainly used for class II typing but may eventually replace all cytotoxicity assays Mixed Lymphocyte Reaction (MLR) • if lymphocytes from 2 patients mixed together, recognise other HLA Ag’s (mainly class II) as “foreign”, and begin to proliferate • in vitro equivalent to early stages of graft rejection