Concepts in immunity


ANTIBODY-ANTIGEN REACTIONS



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ANTIBODY-ANTIGEN REACTIONS

  • Basics

    • Interaction is non-covalent: H-bonding, electrostatic, Van Der Waals, hydrophobic

    • Better fit  tighter bonding

    • Interaction is reversible

      • Mass action: K = [Ab—Ag]/[Ab][Ag]

  • Types of antigen-Ig reactions

    • Agglutination: antigen particle plus specific Ig  aggregation of particle

    • Precipitation: soluble antigen plus specific Ig  insoluble lattice formation

    • Complement activation: antigen in solution/on cell plus specific IgG or IgM  complement inactivation

    • Cytolysis: cell plus anti-cell Ig plus complement  lysis of cell

    • Opsonization: antigenic particle plus Ig plus complement  enhanced phag

    • Neutralization: toxins/viruses/hormones plus specific Ig  inactivation

  • Antigens

    • Microbes have lots of surface molecules each with many antigenic determinants

      • Some elicit stronger responses than others (based on health, age, genetics)

    • Haptens (e.g. Penicillin): small molecules that can’t elicit Ig response unless attached to larger molecules (carriers)

  • Cross-reactivity

    • Similar/identical antigenic determinant on multiple molecules or cells (shared epitope)

    • Cross-reactive antigen may not bind to Ig quite as well

    • E.g. people have Ig’s to blood type antigens other than their own bc they’re similar to carb antigens on some microbes

    • May explain natural/innate Ig’s to a variety of molecules

    • Autoimmune (e.g. group A strep infection  rheumatic fever; strep antigens similar to heart muscle)

  • Precipitation: antigenic particles initially soluble

    • Anti-hapten Ig plus hapten  soluble complex

    • Anti-hapten Ig plus protein with many haptens attached  insoluble complex

    • Lattice formation: if protein has several hapten determinants, Ig can bind to more than one antigen (hapten) at a time

      • Leads to precipitation

      • Adding hapten to anti-hapten Ig before adding hapten-protein conjugate inhibits precipitation

    • Level of precipitation

      • Antibody excess zone: lattice doesn’t develop, soluble

      • Equivalence zone: maximum precipitation; no antigen or Ig detected in supernatant

      • Antigen excess zone: lattice doesn’t develop, soluble

    • Protein antigens don’t usually have repeating sequences  1 determinant of a particular kind per protein antigen

      • So, there are usually multiple Ig’s formed for each protein antigen (specific to its diff determinants)

      • So, lattice formation doesn’t occur for most soluble proteins (unless it has repeats)

  • Agglutination: linking insoluble antigenic particles

    • Interaction of surface antigens on insoluble particles (e.g. cells) with antigen-specific Ig

    • Need much less Ig than for precipitation

      • Clinical uses:

        • Blood type

        • If bacteria is present in blood

    • IgM is more efficient at it than IgG bc it has 10 binding sites

    • Sometimes Ig binding to cell doesn’t cause agglutination

      • Use second Ig reactive to first Ig

      • Coombs test

        • Identifying patients with hemolytic anemia

        • Use Ig reactive to human Ig  causes agglutination of RBCs if human Ig is bound to RBCs

        • Or indirectly: add patient serum to cells and then add mouse or rabbit anti-human Ig to detect circulating Ig reactive to cell surface Ig

    • Results in bigger circle on plates

  • Monoclonal antibodies

    • Making monoclonal antibodies (mAbs)

      • Fuse immortal cell (myeloma tumor cell) with specific Ig-producing B cell from immunized animal/human  hybridoma cell

      • Hybridoma cell is immortal and makes specific mAbs

      • Used for clinical applications

    • Fv libraries to make mAbs

      • Get mRNA for Vh and Vl regions from lots of B cells

      • Use the mRNA to make cDNA for each H chain V region and randomly join it with cDNA for each L chain V region  produces genes with lots of diff antigen combining sites (Fv regions)

      • Clone the Fv’s into cells  source for specific mAbs

  • Antigen-Antibody assays used clinically

    • Background

      • Antigen on cell can be detected via labeled Ig

        • Radioisotopes, fluorescein, enzymes

    • Radioimmunoassay (RIA)

      • Measure serum level of Ig to a specific antigen

      • Add antigen to wells  wash  add test Ig  wash  detect with radiolabeled ligand that binds to Ig

    • Enzyme-linked immunoabsorbent assays (ELISA)

      • Like RIA, but ligand is coupled to enzyme (e.g. peroxidase)

      • Bind ligand to test Ig  wash  detect by adding substrate that reacts with enzyme  color

    • RadioAllergoSorbent Test (RAST)

      • Like RIA, but measures antigen-specific IgE with radiolabeled anti-IgE

    • Antigen detection

      • Competitive assay: Ig on plate  add test antigen (serum) and labeled antigen  test antigen will out-compete labeled if it’s present

      • Two-site capture assay (more common): capturing Ig on plate  add test antigen (serum)  binds to capturing Ig  add labeled Ig  binds to other site on test antigen

    • ELISPOT: for cells that produce cytokines

      • Cytokine-specific Ig on plate  add activated T cells  T cells secrete cytokines  cytokines bind to Ig  add second cytokine-specific Ig that’s labeled with enzyme  colored precipitate

    • Mistakes during antigen detection

      • Caused by heterophillic Ig (HA) – anti-animal human Ig

      • False positives: HA bridges test Ig and labeled Ig via Fc or Fb regions

      • False negatives:

        • Anti-idiotype HA steric hindrance: HA blocks test-Ig binding sites by binding to Fab

        • Anti-isotype HA steric hindrance: HA blocks test-Ig binding sites by binding to Fc

        • Antigen binding HA steric hindrance: HA binds to antigen so it can’t bind to test Ig

    • Flow cytometry

      • Bind labeled Ig to cells in suspension  run through flow cytometer  cytometer measures fluorescence of each cell

      • If labeled Ig cells are run through fluorescence-activated cells sorter (FACS), cells get separated into diff tubes based on their brightness

      • Use to test for cancer types with blood cancer

    • Immunohistochemistry (IHC)

      • Detect antigens on histological specimens

      • Bind labeled Ig to tissue section  wash  detect expression via fluorescence or enzymes

      • Advantage: detect antigen within complex tissue

        • Counterstain all/adjacent cells and tissues to get location of antigen

      • Use to test for cancer types with solid tumors

  • What to know

    • Understand the basic nature of antigen-antibody reactions

    • Know the difference between a hapten and an immunogen

    • Understand cross-reactivity and the consequences of it

    • Know requirements for lattice formation and precipitation

    • Know what a Coombs test is

    • Know what monoclonal antibodies are and their uses

    • Be familiar with the basic concepts involved in antigen-antibody assays, including RIA, RAST, ELISA, and flow cytometry



ANTIGEN PROCESSING AND PRESENTATION

  • Immune system must monitor for pathogens inside and outside cells

    • Outside: bacteria, protozoa, worms, fungi

    • Inside: viruses, bacteria, protozoa, tumors

    • Monitor outside via Ig

    • Hard to get at the inside ones  small pieces of proteins inside cells (peptides) are presented on surface via MHC

    • MHC-peptide complex scanned by TcR

    • If TcR binds MHC-peptide complex AND gets costimulatory signals, T cell responds  proliferation, cytokine production, differentiation

  • MHC Class I pathway: presentation to CD8 T cells (CTL) – kill virus-infected cells ----

    • Occurs in all nucleated cells

    • Proteosome: protein complex that degrades defective cellular proteins in cytoplasm for reuse

      • During immune response, proteosome changes shape better  makes peptides that have better fit with MHC I

    • Transporters associated with antigen processing (TAP): Brings peptides into ER

    • In the ER: peptides associate with MHC I exported to cell surface

    • Cell surface: MHC-peptide complex recognized by CD8 T cells

  • MHC Class II pathway: present to CD4 helper T cells – interact with DC, macro, B cell

    • Occurs in antigen presenting cells (APC) – macrophage, B cell, dendritic cell

    • APC phagocytoses extracellular proteins

      • Macrophage and DC take up lots of antigens via nonspecific receptors

      • B cell takes up specific antigen via specific Ig  B cell activation

    • Endocytic/lysosomal vesicles: pathogen proteins broken into peptides

    • MHC II fuses with endocytic vesicle and gets loaded with peptide

      • MHC II is assembled in ER but doesn’t take up peptides there (MHC I does)

      • Invariant chain blocks binding pocket of MHC II when in ER

      • Invariant chain degraded in endosome, leaving CLIP peptide

      • HLA-DM removes CLIP, so MHC II can bind to peptides in endosome

    • MHC-peptide complex goes to cell surface  recognition by CD4

      • TcR binds to specific MHCII (recognizes antigen)

      • CD4 binds to invariant region of MHC II

  • Features of MHCs

    • One peptide displayed at a time  each T cell responds to single MHC-peptide

    • Peptides acquired during MHC complex assembly intracellularly (endosome for MHC II, ER for MHC I)

    • Low affinity, broad specificity  different peptides can bind to the same MHC

    • Slow off-rate  peptide displayed long enough to get specific T cell

    • Stable expression requires peptide  empty MHCs recycled

    • MHCs only bind peptides  MHC-restricted T cells only respond to protein antigens (not carb, lipid, na!)

  • Cross-presentation

    • Costimulation is only provided well by APCs (especially DCs)

    • So, CD8 cells need a way to respond to virus-infected cells, which don’t provide costimulation

    • DCs ingest virus-infected cells and present viral peptide on MHC I

      • = cross-presentation

      • antigens move from endosome to cytoplasm!

      • Then they follow MHC I pathway and are presented to CD8

      • They’re also present to CD4 via MHC II like usual

  • MHC molecules and peptide binding

    • MHC’s have polymorphic peptide binding domains

    • Peptides that bind are 9-12 aa

      • Have anchor residues (Y) which bind to MHC

    • Diff MHC’s bind to diff peptides from same antigen bc they have diff aa’s

    • MHC must be able to bind to peptides of an antigen to have T cell response to that antigen

  • MHC structure

    • MHC I and II genes linked on chromosome 6

    • MHC I

      • HLA- A, B, C genes

      • 10-70 alleles for each

      • Complex: MHC I alpha chain, beta-2-microglobulin, peptide

        • CD8 binds to alpha 3 domain

    • MHC II

      • HLA- DR, DQ, DP genes

      • 7-70 alleles for each

      • Complex: MHC II alpha chain, MHC II beta chain, peptide

        • Alpha and beta are polymorphic, mostly in alpha 1 and beta 1 domains, which interact with TcR

        • CD4 binds to beta 2 domain

    • Codominant expression; complete set of genes inherited from each parent  increases diversity of MHC

    • Polymorphic genes  diff individuals present and respond to diff microbial peptides


Examples:

1. influenza virus

2. mycobacterium tuberculosis



3. strept pneumoniae



  • Tissue distribution of MHC I and II antigens

    • B cells, DC, macrophages express lots of MHC II  proficient at presenting to CD4 T cells

    • All nucleated cells express MHC I  proficient at presenting to CD8 T cells

  • Non-classical MHC molecules

    • HLA-G

      • MHC I

      • Nonpolymorphic, no HLA-A, B, or C

      • In extravillous trophoectoderm (fetal tissue contacting maternal circulation)

      • May prevent maternal immune response (T and NK cells) against paternal antigens in the fetus

    • CD1d

      • Nonpolymorphic

      • Not encoded in MHC region

      • On B cells, DC, and some non-APCs

      • Presents non-peptide antigens (lipid and glycolipid) to T cells

      • Restricts T cell response to foreign microbial antigens

  • Uptake and presentation of foreign antigens (MHC II, or MHC I via cross-presentation)

    • Immature DCs take in foreign antigens at epithelium/CT

    • DCs get signals that there’s a pathogen

      • Pathogen-associated molecular signals (lipopolysaccharide, double stranded RNA)

      • Inflammatory cytokines

    • DCs migrate to lymph nodes via lymphatics to present to T lymphocytes

      • Migrate to T cell areas using cytokine gradients

      • DCs mature and upregulate expression of costimulatory molecules  activate antigen-specific T cells by presenting antigen and giving costimulation

    • Antigens that enter blood stream can be captured by APCs in spleen

  • What to know

    • Understand MHC class I pathway

    • Understand MHC class II pathway

    • Know the fundamental differences in the Class I and II MHC processing pathways, and with which subsets of T cells they interact.

    • Know the general properties of class I and class II MHC molecules

    • Know the restrictions for peptide binding to MHC molecules.

    • Know the significance of MHC gene polymorphisms

    • Understand the role of dendritic cells in antigen presentation to naïve T cells



T CELL RECOGNITION OF ANTIGEN

  • Combatting intracellular infections is primary role of T lymphocyte

    • Microbes in phagosome can resist proteolysis

    • Viruses in cytoplasm

  • T lymphocytes

    • Scan for intracellular pathogens via TcR

    • TcR recognizes antigenic peptides on MHC

    • Once recognized, T cells become effectors  attack, recruit other cells, increase killing efficiency

    • CD8 cells (CTL)

    • CD4 cells (Th)

      • Can differentiate to effectors, secrete cytokines

      • Target of AIDS virus

    • Induce adaptive immune response and immunological memory

  • Antigen recognition

    • DO NOT recognize native antigen

      • Recognize peptides from antigens (bacteria, virus) on MHC

    • TcR --------------------------------------------------------------------------------

      • Alpha and beta chains created from gene recombination

      • Each T cell expresses TcR with diff sequence that recognizes unique peptide-MHC complex

      • Rearrangement/expression in thymus during T cell devel.

      • TcR complex




  • Coreceptors needed for T cell activation

    • CD4: binds to MHC II invariant residues in beta 2 domain

    • CD8: binds to MHC I invariant residues in alpha 3 domain

  • Immunological synapse

    • Naïve T cells circulate through nodes and spleen looking for APCs with MHC-peptide that matches their TcRs

    • T cell stimulation requires:

      • TcR binding to MHC-peptide

      • CD4/8 coreceptor activation

      • CD3 signal conduction

      • Adhesion molecule (integrin) engagement

        • ICAM-1 on T cell, LFA-1 on APC

        • Surround TcR-MHC proteins and hold cells together

          • = immunological synapse

        • Chemokines and antigen-TcR binding make integrins become high-affinity  strong adhesion between cells  T cell response

      • Costimulation (see below)

  • Costimulation

    • Activates specific pathways, ESSENTIAL for naïve T cell activation

    • CD80 (aka B7): on APCs, binds to CD28 on T cells

    • CD86: on APCs

    • Expression on APC increases in response to microbe  signal 2

    • Only on DC, mac, B cell  ONLY THES PROFESSIONAL APCs can activate naïve T cells!!

    • Two-signal model to prevent response to self antigens

      • Signal 1: TcR binds to MHC-peptide

        • If CD8 T cell only gets signal 1  tolerance (T cell is unresponsive/inactived – anergy)

      • Signal 2: CD28 binds to costimulator, which upregulates at APC surface in presence of microbe

        • Signal 1 and 2  growth and cytokine production of T cell

    • Cross-presentation of extracellular peptides by MHC I is needed so that CD8 T cells can get activated!

      • ??????

  • Early T cell activation, growth, cytokine production

    • Clonal expansion

      • Activated T cell begins to divide A LOT

      • Daughter cells (clones) have same TcR and same MHC-peptide specificity

    • T cells make growth factors to support their growth

      • Interleukin 2 (IL-2)

        • Interleukin/lymphokine: soluble proteins make by immune cells

        • Production stimulated by TcR stim

      • T cell increases high-affinity Il-2 receptor as well

        •  autocrine signaling loop

      • Also paracrine function of IL-2 on other cells

        • B cell differentiation

        • Growth factor for NK cells

  • Turning off immune response

    • Activated T cells begin expressing CTLA-4

    • CTLA-4 binds strongly to CD80 and 86, replacing CD28  inhibitory signal

    • Lack of CTLA-4 function can cause inflammatory disorders, tissue injury

  • Superantigens

    • Some bacteria/viruses make proteins that are superantigens bc they stimulate lost of diff T cells

    • Bind to sides of MHC II and Vb region of TcR, gluing APC and TcR together

    • Tons of cytokine production  vascular leakage, shock

    • Staphylococcal enterotoxins (food poisoning, toxic shock syndrome)

  • What to know

    • How the TCR recognizes antigen

    • Coreceptors involved in T cell activation

    • What is costimulation?

    • Clonal expansion of antigen specific T cells

    • Role of cytokines in T cell activation

    • Inhibition of T cell activation by CTLA-4

    • What are superantigens?



T CELL RESPONSE AND T CELL MEDIATED IMMUNITY

  • T cell selection

    • T cell precursors migrate to thymus (progenitor cells committed to T cell lineage)

    • Most die within thymus

    • T cells undergo gene rearrangement in thymus  TcR expressed on thymocytes

      • Have CD4 and CD8 = double positive thymocytes

      • Many can’t express usable TcR

    • Positive selection: cells with TcR that weakly binds to MHC I or II survive

      • Thymic epithelial cells in thymic cortex are the presenting cells, present lots of peptides that would normally be expressed elsewhere

      • Then express only CD4 or 8 depending on if they bind MHC I or II

    • Negative selection: cells with TcR that strongly binds to MHC I or II die

      • Autoreactive, so may cause problems in periphery

      • DC, mac, medullary epithelium are the presenting cells

    • Select T cells that recognize self MHC and foreign peptide, not self peptide!

    • Regulatory T cells: possibly come from cells with very high affinity for self

      • CD4 cells that suppress immune responses when exported to periphery

      • Inhibit via cytokine production (active) and interaction with other cells (passive)

  • Lymphocyte trafficking and recirculation: speed dating btwn lymphocytes and DC’s

    • Naïve B and T cells (have not found their antigen yet) recirculate through secondary lymph organs

    • Adhesion molecules on surface let them attach to endothelial cells to exit blood stream via HEV

    • Homing adhesion molecules attach to specific molecules on endothelial cells (addressins) in certain sites ---------------------

      • Allows lymphocytes to target mucosal or peripheral lymph node areas

      • MALT: lymphocytes stimulated in one MALT site will migrate to other MALT sites via homing

    • Once activated, lymphocytes alter chemokine receptors so they can go into blood stream and infection

  • Tolerance

    • Antigen-specific T or B cells reactive to self tissues may be:

      • Clonally deleted

      • Made functionally unresponsive

      • Prevented from responding (suppressed)

      • Lacking antigen-specific receptor or MHC element (CD4/8)

    • Tolerance is specific

      • Can still respond to other antigens

      • May be partial (e.g. weak/altered immune response)

      • Can be humoral or cellular

    • Central tolerance

      • Clonally deleting T and B self-reactives in the thymus and marrow (i.e. negative selection)

      • Antigen exposure during development

        • Virus in neonate may be seen as self  deletion of lymphocytes responsive to it

    • Peripheral tolerance

      • Some autoreactive T cells make it to the periphery

      • If self-antigen is on an APC when the APC also happens to have antigen particles, autoreactive T cell can get costimulation and respond; otherwise, they won’t respond  anergy (expression of proapopotic proteins or of death receptor and death receptor ligands Fas/Fas-L)

      • CD4 T regulatory cells can keep T cells from responding

  • Effector cells in T cell immunity -----------------------------------------------

    • Activation requires:

      • Specific signal via TcR

      • General costimulatory signal

    • Activation  autocrine growth factors (IL-2), alters cells surface molecules (for trafficking), inc membrane proteins to trigger other cells (e.g. CD154), production of cytokines

  • CD8 cytotoxic T cells (CTL): recognize MHC I

    • Host resistance to pathogens that live in cytosol (e.g. viruses)

    • Recognize pathogens via MHC I

    • Killing mechanisms

      • Lyse cells they bind to with perforin and granzymes  pores in cell membrane  apoptosis

      • Release factors (TNF-alpha, IFN-gamma) onto cells they bind to or express molecules on surface (Fas-L) that trigger apoptosis

  • CD4 helper T cells (Th): recognize MHC II

    • Early mature cells: Th0

      • Broad spectrum of cytokines

    • Later mature cells: Th1, Th2, Th17

      • Th1: inflammatory responses (activate mac and CTL) via cytokines IFN-gamma, IL-2, TNF-alpha

        • Intracellular microbes – help macrophages become completely activated (IFN, TNF)

        • Makes cytokines that recruit macrophages and inc release from marrow (GM-CSF, IL-3)

        • TNF promotes adhesion of macrophages to endothelial cells at infection site

        • Induce B cells to make IgG

        • IFN-gamma: prevents induction of Th2; increase production of cytokines that inc Th1 differentiation (pos feedback)

      • Th2: help B cells grow/differentiate via cytokines IL-4, IL-5, IL-13

        • Induce B cells to make Ig, isotype switching, affinity maturation!! Via:

        • Cytokines

          • IL-4  IgE  IgE binds to mast cells  mast cells make IL-4  Th2 induction (pos fdbck)

          • Allergic rxns

        • Surface molecules that engage B cells

        • IL-5  eosinophil production in marrow (worms, large parasites)

      • Th17: inflammation, autoimmunity, inflammatory disorders via IL-17

  • CD4 regulatory T cells (Treg)

    • Usually derived from strongly self-reacting CD4 cells in the thymus; become T reg with IL-2 and Foxp3

    • Suppress T cell immune responses in thymus (inhibit activation) and periphery (inhibit effector function)

    • Maintain tolerance to self-proteins

    • Contact-dependent mechanism that reduces inflammatory response via IL-10, TGF-beta

  • Cytokines

    • Act like hormones and NTs for cellular communication

    • Proteins, peptides, or glycoproteins

    • Interleukins, interferons, etc

    • Have lots of functions; related to allergies, cancer, inflammatory disease, immune reconstitution

    • Cytokines enhance or suppress cell-mediated immunity  treatment of autoimmunity, cancer, etc.

  • What to know

    • Thymic structure

    • T cell development in the thymus

    • What is MHC restriction? Why is this important?

    • Mechanisms of central vs peripheral tolerance

    • T cell trafficking in the periphery and into tissue

    • Role of cytokines in T cell effector cell development

    • Mechanisms of CD8+ T cell function

    • CD4+ T cell subsets and their function in immunity





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