Immunology Level 2 Notes PDF

IMMUNOLOGY LEVEL 2 NOTES Compiled by H.S.R Table of contents Topic Lecturer Page 1.Introduction to Immunology Dr Barasa 4 2.Cells and Organs of the Immune System Dr Rashmi 56 3.Innate and Adaptive Immun...

IMMUNOLOGY LEVEL 2 NOTES Compiled by H.S.R Table of contents Topic Lecturer Page 1.Introduction to Immunology Dr Barasa 4 2.Cells and Organs of the Immune System Dr Rashmi 56 3.Innate and Adaptive Immunity Dr Njagi 78 4.Antigenicity and Immunogenicity Prof Nyagol 117 5.T-Cell Ontogenesis Dr Oyaro 139 6.Antigen and Antibodies Dr Gontier 165 7.Complement System Dr Njagi 210 8.Major Histocompatibility(MHC) Prof Nyagol 215 9.Antigen Processing,Presentation and Recognition by Dr Oyaro 269 T-Cells 10.B Cell Ontogenesis Dr Barasa 287 11.Cytokines Dr Barasa 307 12.Phagocytosis Dr Gontier 344 13.Mechanisms of Humoral Immunity Dr Njagi 360 14.Cell mediated Immunity Prof Nyagol 390 15.Regulation of the Immune Response Dr Oyaro 405 16.Immunology of Inflammation Dr Barasa 436 17.Immunological Tolerance Dr Gontier 489 18.Immunity to Bacterial/viral infections Dr Njagi 519 19.Immune Responses To Parasites and Fungi Prof Nyagol 543 20.Microbial Immune Evasion Mechanisms Dr Oyaro 579 21.Immunization Dr Barasa 612 22.Primary Immunodeficiency Dr Gontier 649 23.Secondary Immunodeficiency Dr Njagi 696 24.Type I &II Hypersensitivity Reactions Dr Nyagol 729 25.Type III&IV Hypersensitivity Reactions Dr Oyaro 744 26.Introduction to Transplantation Immunology Dr Barasa 763 27.Autoimmunity and Autoimmune Disease Dr Njagi 790 Introduction to Immunology History and Overview Dr. Barasa AK H.S.R  Outline History of Immunology Concepts of the Immune Response Failure/Challenges to the development of healthy immune responses H.S.R  Overview of the Immune System Protection of multicellular organisms from pathogens Diverse potential pathogens – tiny intracellular viruses to large parasitic worms Requires a range of recognition & destruction mechanisms Complicated & dynamic network of cells, molecules & pathways H.S.R Historical Perspectives of Immunology H.S.R  Concept of Immunity Observation that individuals who had recovered from certain infectious diseases were thereafter protected from the disease Latin term immunis meaning “exempt” Immunity – a state of protection from infectious disease H.S.R Early Vaccination Studies Deliberate induction of immunity 15th Century – Chinese &Turks Attempts to prevent smallpox Dried crusts from smallpox pustules inhaled or inserted into small cuts in the skin (variolation) H.S.R Kuby Immunology, 7e  Early Vaccination Studies 1718; Lady Mary Montagu (wife of British ambassador to Constantinople) Observed the positive effects of variolation Had it performed on her children H.S.R  Early Vaccination Studies 1798; English physician, Edward Jenner Milkmaids who had contracted cowpox were subsequently immune to the more severe smallpox Reasoned that inoculating fluid from a cowpox pustule into people would be protective Inoculated an 8-yr old boy & later infected him with smallpox Technique spread throughout Europe H.S.R  Early Vaccination Studies Louis Pasteur Grew the bacterium that causes fowl cholera in culture Confirmed this by injecting it into chicken, that then developed fatal cholera Reinjection with an old bacterial culture – the chickens became sick but recovered Grew a fresh culture & injected a mix of previously injected chicken and unexposed birds H.S.R  Early Vaccination Studies Only the fresh chickens died Aging had weakened the virulence of the pathogen A weakened/attenuated strain could be administered to provide immunity against a disease He called this attenuated strain a vaccine (Latin vacca = cow) H.S.R  Early Vaccination Studies 1881; Pasteur Vaccinated a group of sheep with Bacillus anthracis that were attenuated by heating Challenged vaccinated & unvaccinated sheep with a virulent culture of the bacteria Vaccinated sheep lived and unvaccinated ones died H.S.R  Early Vaccination Studies 1885; Pasteur administered his 1st vaccine to a young boy who had been repeatedly bitten by a rabid dog Series of attenuated rabies virus preparations The boy lived; later became a caretaker at the Pasteur Institute (1887) H.S.R  Vaccination The study of immunology is linked to the discovery of vaccines Vaccination has aided in improving mortality rates worldwide, esp among young children Measles, Mumps, Rubella, Polio, Tetanus….. Still a major challenge for some pathogens – malaria, AIDS 1977 - last known case of naturally acquired smallpox (Somalia) H.S.R  Vaccination Herd immunity – as a critical mass of people acquire protective immunity through vaccination or infection, this decreases the number of individuals who can harbor or spread an infectious agent Significantly reduces the chances that a susceptible person will become infected H.S.R Immunology is about more than just vaccines and infectious disease Immune response can be manipulated to boost, inhibit or redirect the specific efforts of immune cells, in Rx of autoimmune disease, allergy and other disorders H.S.R  Mechanisms of Immunity Pasteur showed that vaccination worked, but did not understand the mechanisms 1883; Elie Metchnikoff demonstrated that cells contribute to immunity Observed that certain wbc (phagocytes) ingested microorganisms & other foreign material 1890; Emil von Behring & Shibasaburo Kitasato, 1890 Demonstrated that serum from animals previously immunized with diphtheria could transfer the immune state to unimmunized animals H.S.R  Humoral Immunity Soluble mediators of immunity Various researchers in the early 1900s helped characterize the active immune component in serum Could neutralize or precipitate toxins; could agglutinate bacteria The component was named antitoxin, precipitin, agglutinin 1930s; Elin Kabat - a fraction of serum, gamma globulin (now immunoglobulin) was responsible for all these activities (antibodies) Body fluids were then known as humors H.S.R  Humoral Immunity Antiserum – the antibody-containing serum fraction from a pathogen-exposed individual (horses) was given to patients suffering from diphtheria and tetanus Current therapies that rely on transfer of immunoglobulins to protect susceptible individuals: Snake antivenom (immune serum containing Abs against snake venom) to treat bite victims H.S.R  Humoral Immunity Passive immunity Short-lived and limited – cells that produce the Abs are not transferred Active immunity - administration of a vaccine or natural infection (production of one’s own immunity) H.S.R  Cell-Mediated Immunity 1940s; Merrill Chase (Rockefeller Institute) - conferred immunity against TB by transferring wbc between guinea pigs 1950s - lymphocyte was identified as the cell type responsible for both cellular & humoral immunity Bruce Glick, (Mississippi State University), experiments in chicken – 2 types of lymphocytes: T lymphocytes (T cells), derived from the thymus, & B lymphocytes (B cells), derived from the bursa of Fabricious in birds H.S.R Cellular immunity is imparted by T cells Antibodies produced by B cells confer humoral immunity Both are needed for a complete response against most pathogens H.S.R The Immune System Recognizes Foreign Substances Antigen – substance that elicits a specific response by B or T cells 1900s; Jules Bordet (Pasteur Institute) – nonpathogenic substances (rbc from other species) can serve as antigens Serum from an animas that had been inoculated with noninfectious but otherwise foreign (nonself) material would react with the injected material in a specific manner Karl Landsteiner et al – injecting an animal with almost any nonself organic chemical could induce production of Abs that would bind specifically to the chemical H.S.R Outline for the humoral and cellular branches of the immune system H.S.R Kuby Immunology, 7e Concepts of the Mammalian Immune Response H.S.R  Pathogens Disease-causing organisms Pathogenesis – the process by which they induce illness in the host Viruses, fungi, parasites, bacteria H.S.R Major categories of microorganisms causing human disease H.S.R Kuby Immunology, 7e  Pathogens The immune system exploits some shared characteristics that are common to groups of pathogens, but not to the host, for recognition & destruction H.S.R  Pathogens The same pathogen can be treated differently depending on the context/microenvironment in which it is encountered Some areas of the body e.g. CNS are “off limits” for the immune system (the immune response can cause more harm than the pathogen) Some foreign compounds that enter via the GIT e.g. commensal microbes, are tolerated by the immune system; however if they enter the bloodstream, they are treated aggressively H.S.R  Pathogens Immune pathways do not become engaged until the foreign organisms first breach the physical barriers of the body – skin, mucous membranes Other barriers – acidity of stomach, vagina, perspiration (some microorganisms cannot survive in the low pH) H.S.R  The Immune Response The cells and molecules that become activated in a given immune response depend on The chemical structures present on the pathogen Whether it resides inside or outside of host cells The location of the response H.S.R  The Immune Response Pathogen recognition involves an interaction between the foreign organism & a recognition molecule or molecules expressed by host cells (membrane-bound) Can also be soluble receptors or secreted recognition molecules H.S.R  The Immune Response Ligands for recognition molecules include whole pathogens, antigenic fragments of pathogens, or products secreted by pathogens Outcome of binding is an intracellular or extracellular cascade of events that leads to labeling and destruction of the pathogen – The Immune Response H.S.R  The Immune Response E.g. Viruses cause intracellular infection CTLs must be able to detect changes that occur in a host cell after it becomes infected by virus Recognition molecules inside the cells bind to viral proteins present in the cytosol & initiate events that alert the CTLs to the presence of an invader The virally-infected cells must be killed so as to eradicate the pathogen Can cause disruption to normal function; e.g. in HIV H.S.R  Pathogen Recognition Molecules Pathogen-associated molecular patterns (PAMPs) – chemical structures that characterize whole groups of pathogens Not typically found in mammals E.g. polysaccharide coat on encapsulated bacteria Recognized by the immune system – Pattern recognition receptors (PRRs) on wbc Recognize sugar residues and other foreign structures A cascade of events then labels the target pathogen for destruction H.S.R  Pathogen Recognition Molecules PRRs are conserved, germline-encoded recognition molecules; first line of defense Pathogens evolve to express unique structures that avoid host detection H.S.R  Pathogen Recognition Molecules Generation of diversity – each B and T cell expresses many copies of one unique recognition molecule Resulting in a population with the potential to respond to virtually any antigen Occurs by rearrangement and editing of the genomic DNA that encodes the antigen receptors expressed by B and T cells H.S.R  Generation of diversity and clonal selection Kuby Immunology, 7e H.S.R  Tolerance The immune system must avoid accidentally recognizing and destroying host tissues Principle that relies on self/nonself discrimination To establish tolerance, the antigen receptors on developing B and T cells must first pass a test of unresponsiveness against host structures H.S.R  Tolerance Downside of robust self-tolerance is that the immune system frequently ignores cancerous cells that arise in the body, as long as these cells continue to express self structures that that immune system has been trained to ignore Dysfunctional tolerance – autoimmune diseases H.S.R  Innate Immunity In-built cellular and molecular mechanisms encoded in the germline Primitive Aimed at preventing infection or quickly eliminating common invaders Physical and chemical barriers PRRs Complement – serum proteins that bind common pathogen- associated structures and initiate a cascade of labeling and destruction events H.S.R  Adaptive Immunity B and T lymphocytes Takes some time to be activated (5-6 days) Antigen-specific Evolves in real time in response to infection, and adapts to better recognize, eliminate and remember the invading pathogen Second line of defense H.S.R  Adaptive Immunity Goal of vaccination against infectious disease is to elicit the development of specific & long-lived adaptive responses So that the vaccinated individual will be protected in future, when they encounter the actual pathogen H.S.R  Innate and adaptive immunity work together The 2 systems must be able to communicate with one another Achieved by cell-cell contact and soluble messengers – Cytokines Cytokines bind to receptors on responding cells & signal them to perform new functions, e.g. synthesis of other soluble factors, or differentiation to a new cell type Chemokines – cytokines that have chemotactic activity (recruit specific cells to a site) H.S.R Immunologic memory – ability of the immune system to respond more swiftly and with greater efficiency during a second exposure to the same pathogen 1st exposure with foreign Ag – adaptive immunity undergoes a primary immune response Key lymphocytes that will be used to eradicate the pathogen are clonally selected Subsequent exposure with the same Ag - secondary response Memory cells (kin of B and T cells trained during the primary response) are re-enlisted to fight again H.S.R Comparison of innate and adaptive immunity Kuby Immunology, H.S.R 7e Kuby Immunology, H.S.R 7e The Good, Bad and Ugly of the Immune system Failures and challenges to the development of healthy immune responses H.S.R  Dysfunctional (Inappropriate) Immune Responses The healthy immune response involves a balance between immune aggression & immune suppression pathways Improper regulation of the immune response: Attacks something it shouldn’t or fails to attack something it should H.S.R  Dysfunctional (Inappropriate) Immune Responses Hypersensitivity – exaggerated/overzealous attacks on common benign but foreign antigens; e.g. allergy Autoimmune disease – erroneous targeting of self- proteins or tissues by immune cells Immune deficiency – insufficiency of the immune response to protect against infectious agents H.S.R  Challenges of Transplantation The immune system will attack and reject a transplanted organ (or cells, or tissue) that is nonself or not a genetic macth Even when it is a lifesaving Rx Transplant rejection responses can be suppressed by immune inhibitory drugs; Which also suppress general immune function, leaving a host susceptible to infections H.S.R  Cancer (Malignancy) and the Immune Response Occurs in host cells, when they begin to divide out of control Self-tolerance mechanisms can inhibit the development of an immune response, making detection and eradication of cancerous cells challenging H.S.R  Cancer (Malignancy) and the Immune Response Many tumour cells express unique or developmentally inappropriate proteins This makes them potential targets for immune cell recognition and elimination As well as targets for therapeutic intervention The immune system actively participates in the detection and control of cancer in the body H.S.R CELLS AND ORGANS OF THE IMMUNE SYSTEM Rashmi Mali National Institute of Virology H.S.R  Components of the Immune System Cells originate in the bone marrow. Arise from pluripotent hematopoietic stem cells. (HSCs) HSCs give rise to precursor cells which are myeloid progenitors and common lymphoid progenitors. Myeloid progenitors give rise to granulocytes, macrophages, dendritic cells and mast cells. Common lymphoid progenitors give rise to lymphocytes, dendritic cells and NK cells. There are more myeloid progenitors in the bone marrow than lymphoid progenitors. H.S.R H.S.R Granulocytes or Polymorphonuclear (PMN) Leukocytes  A group of white blood cells is collectively referred to as granulocytes or polymorphonuclear leukocytes (PMNs).  Granulocytes are composed of three cell types as Neutrophils Eosinophils Basophils  These cells are important in the removal of bacteria and parasites from the body.  They engulf these foreign bodies and degrade them using their powerful enzymes. H.S.R H.S.R Neutrophils.  Neutrophils constitutes 50-70% of the circulating WBC’s.  Circulate for 7-10 h prior to migration to tissue; live 2-4 days in tissue.  They are first cell to arrive to the infection site.  It plays important role in inflammatory response.  Phagocytic and bactericidal.  Leukocytosis  Extravasation  Chemotactic factors H.S.R Eosinophils  Eosinophils constitutes 1-3 % of the circulating WBC’s.  Important in anti-parasite defenses.  It presents Ag to T cell in body.  Release of contents in eosinophilic granules can damage the parasite membrane.  Eosinophils are associated with allergic diseases.  The inappropriate release of their granule content can cause host cell damage leading to airway remodeling (fibrosis) in many cases. H.S.R BASOPHILS MAST CELLS H.S.R  BASOPHIL  Only present in the bloodstream, and represent 1% at a single genetic locus ina population of individuals MHC genes are the most polymorphic known The type and variant MHC molecules do not vary in the lifetime of the individual The diversity in MHC molecules exists at the population level This sharply contrast diversity in T and B cell antigen receptors which exists within the individual 53 H.S.R  Summary of Aspects of MHC MHC molecules are membrane-bound. Recognition by T cells requires cell-cell contact. Peptide from cytosol associates with class | MHC and is recognized by Tc cells. Peptide from vesicles associates with class Il MHC and is recognized by Th cells. 54 H.S.R  Antigen Processing, presentation & recognition by T-cells By Dr Oyaro H.S.R  Learning objectives Identify and explain the locations and functions of MHC molecules. Identify and explain antigen processing and presentation. Identify and explain antigen recognition and T- cell selection. Identify the characteristics of T-dependent and T- independent antigens (including superantigens). Identify the similarities and differences of T- dependent and T-independent antigens. H.S.R  Antigen Processing and Presentation Ag recog by T cells REQUIRES presentation by MHC on a cell membrane (MHC restriction) Pathways for Ag presentation: a) Class I MHC assoc. with peptides from endogenous Ag’s b) Class II MHC assoc with peptides from exogenous Ag’s H.S.R “self-MHC II” restriction in (CD4) TH cells shown by Rosenthal & Shevach (1974) Ag-specific stim. of TH cells occurs only in response to Ag presented by APC’s with the same MHC haplotype as the T cells H.S.R “self-MHC I” restriction of (CD8) TC cells shown by Doherty & Zinkernagel (1974) TC cells kill only syngeneic virally-infected target cells Both the TC cell and the infected cell must share the same set of MHC genes H.S.R  Distinctions between MHC I and MHC II MHC I MHC II Most cells (target cells) can Only APC’s can present Ag w/ present Ag w/ MHC I to TC’s MHC II to TH’s Nearly all nucleated cells APC’s are of 2 categories: infected by MO/virus, or abnormal proteins prod by – Professional APC’s cancer cells, aging cells, or by – Non-professional APC’s allogeneic cells from transplants Assoc w/ MHC II does not Assoc w/ MHC I requires require replication of entity w/i replication of foreign entity target cells (i.e., abnormal protein synth) Phagocytosis is important in within the target cell Ag-processing H.S.R Ag is processed thru 2 separate pathways: *MHC I interacts w/ peptides from cytosolic degradation *MHC II interacts w/ peptides from endocytic degradation H.S.R  Processing Endogenous Ag: the Cytosolic pathway Processing of endogenous Ag involves 3 activities: Peptide generation from proteolysis Transport to ER Peptide binding to MHC I H.S.R Endogenous Ag processing & peptide generation Proteins targeted for lysis combine w/ a small protein  ubiquitin Ubiquitin-protein complex is degraded by a proteosome Specific proteosomes generate peptides which can bind to MHC I H.S.R Endogenous Ag processing transport to ER Peptides from proteolysis bind to a “transporter protein assoc w/ Ag processing” (TAP) TAP is a heterodimer which uses ATP to help transport peptides (8-10 aa’s) to lumen of ER Usually basic aa’s @ COOH end of peptide H.S.R chain Endogenous Ag processing peptide binding to MHC I MHC I assembly occurs w/ the aid of chaperone proteins to promote folding (calnexin + MHC I α chain) Tapasin + calreticulin brings TAP/ peptide close to MHC assembly Allows MHC I to bind to peptides MHC I-Ag exits ER to Golgi to plasma membrane H.S.R Assembly and stabilization of MHC I – Ag complex H.S.R  Processing of Exogenous Ag’s: the Endocytic pathway Exogenous Ag’s are typically phagocytized/ endocytized by MØ and APC’s – Foreign Ag is degraded w/i endocytic vacuole of endocytic pathway. The pathway includes: – Early endosomes (pH 6-6.5) – Late endosomes or endolysosome (pH 5-6) – Lysomes (pH 4.5 – 5) Ag is degraded into 13-18 aa polypeptides which bind to MHC II Eventually endocytic vacuole returns to PM  recycling surface receptors H.S.R Processing of Exogenous Ag’s: the Endocytic pathway H.S.R  Processing of Exogenous Ag’s: manufacture of MHC II w/i ER, α and β chains of MHC II combine w/ a protein – “the invariant chain” (Ii, CD74) the IC binds to MHC @peptide binding cleft + then exits the ER to Golgi apparatus as proteolytic activity continues, the IC is degraded to a small fragment (CLIP*) another MHC II (HLA-DM (found in endosomes)) substitutes Ag for CLIP w/i lysosome MHC II – Ag complex is transported to the PM *CLIP = class II associated invariant chain peptide H.S.R Comparison of Ag-processing pathways H.S.R  What Does the αβ T Cell Receptor (TCR) Recognize?-(T-cell activation) 1. Only fragments of proteins (peptides) associated with MHC molecules on surface of cells Helper T cells (Th) recognize peptide associated with MHC class II molecules Cytotoxic T cells (Tc) recognize peptide associated with MHC class I molecules H.S.R  Superantigens Proteins produced by pathogens Not processed by antigen presenting cells Intact protein binds to variable region of β chain on TCR of T cells and to MHC class II on antigen presenting cells (APC) Large numbers of activated T cells release cytokines having pathological effects H.S.R Ontogenesis of B lymphocytes Dr. Barasa AK 16012023  Learning Objectives Outline the origin and development of B lymphocytes from a primitive lymphoid progenitor to a mature cell  Introduction The process of B-cell development & differentiation is defined by the regulated expression of specific sets of transcription factors, Ig and cell surface molecules Leads to formation of a functional BCR which consists of membrane-bound Ig B cells produced throughout life  Introduction BM stromal cells provide contact-dependent signals and growth factors for developing lymphocytes B cells that express a diverse repertoire of BCRs are continually generated in the BM Undergo negative selection to remove self-reactive Ag receptor  Introduction B cells that survive negative selection disperse to peripheral lymphoid organs Activated when they encounter foreign Ag they have specificity for Terminally differentiate into plasma cells B cells that do not encounter appropriate Ag die over a few weeks  Multipotent haematopoietic stem cells Lymphoid-primed multipotent progenitors (LMPPs) Common myeloid Common lymphoid progenitor progenitor T-cells, B-cells, NK-cells Early B Lymphocyte Development: Stem Cell to Immature B cell Developmental stages defined by: Rearrangement status of Ig heavy & light chain genes Expression of differentiation- specific molecules on the cell surface Helbert M. Immunology for Medical Students, 3e Structure of Immunoglobulin  B-cell Receptor Development 2 heavy (H) chains (50kd) 2 light (L) chains (50kd) L & H chain loci composed of V (variable) gene elements D (diversity) segments (H chain) J (joining segments) C (constant region) exons Genes of 9 different H chain types on chromosome 14 IgM, IgD, IgG1-4, IgA1-2, IgE Early B Lymphocyte Development: Stem Cell to Immature B cell Pro-B cell Earliest B-lineage cell Recognized by appearance of surface markers characteristic of the B lineage e.g. CD19 RAGs are active; induce rearrangements of Ig heavy chain diversity (DH), joining (JH) & variable region (VH) gene segments, to produce a heavy chain TdT is also active; increases junctional diversity (by adding nucleotides) Early B Lymphocyte Development: Stem Cell to Immature B cell Pre-B cell Surface expression of µ heavy chain, constituting a pre-BCR Pre-BCR plays a role in transducing signals that lead to proliferation of the pre-B cells, facilitating their further development V(D)J recombinase initiates light chain gene rearrangement Cell first attempts to combine a κ light chain then a 𝜆 light chain if the former is unsuccessful Early B Lymphocyte Development: Stem Cell to Immature B cell Immature B cell Appearance of paired light & heavy chain polypeptides on the cell surface constituting a complete IgM molecule, the BCR Transcription of RAG & TdT genes is switched off for the remainder of the cell’s life Early B Lymphocyte Development: Stem Cell to Immature B cell Recombination that does not result in successful expression of a pre-BCR or BCR is fatal for cells Only B cells with intact BCRs survive the maturation process Recombination gives rise to extensive receptor diversity May generate some Ag receptors that possess self-reactivity  Tolerance Induction of Immature B cells Clonal deletion Self Ags that cross-link immature BCRs (multivalent Ags) induce apoptosis (Ags that bind withhigh affinity) – negative selection Self Ag in this context are macromolecules present in the BM that could be bound by the BCR E.g. molecules on surface of healthy cells, or present in solution in the ECM  Tolerance Induction of Immature B cells Anergy Small soluble proteins/Ags (cannot crosslink BCR) in high doses lead to downregulation of IgM expression These cells are rendered incapable of becoming activated upon subsequent antigenic challenge  Tolerance Induction of Immature B cells Receptor editing IgM antibodies undergo repeated rounds of light chain rearrangement to lessen the self specificity of the antibody  Tolerance Induction of Immature B cells B cells that survive negative selection express increased IgM levels and also begin to express membrane-bound IgD Expressed after alternative splicing of heavy chain transcripts  Mature B cell Mature B cells express surface IgM and surface IgD Exit the BM and migrate to peripheral lymphoid organs – naïve B cells Have fully rearranged genes but have not encountered non self antigen Continuously recirculate through blood to follicles in the secondary lymphoid organs until they encounter their specific Ag  Cytokines MBChB 2 Immunology Lecture Dr. A. Barasa 16012023  Learning Objectives Describe the general properties of cytokines Describe the role of cytokines in the immune system Development of immune cells Innate immune responses Adaptive immune responses Outline some cytokine-related disease processes Outline the clinical utility of cytokines  Introduction Small protein molecules used by immune cells to communicate with each other Soluble messenger molecules secreted by cells of the immune system (and some non-immune cells) Some are bound to the cell surface e.g. CD40L, FasL Involved in: Development and regulation of the immune system Innate and adaptive immune responses  Properties of Cytokines Small molecules Short half-lives Very potent effects; expression is tightly regulated Bind to receptors on target cells and alter gene expression Through intracellular signaling, e.g. JAK/STAT pathway Outcome of cytokine stimulation depends on the cytokine & the target cells Cell suppression or activation Cell proliferation Cell differentiation Cell migration Kuby Immunology, 7e  Properties of Cytokines Modes of actions: Cytokines produced at site of infection (e.g., TNF) can travel to the brain (hypothalamus) to cause an increase in body temperature. This inhibits growth of pathogens & increases effectiveness of the immune response E.g. Macrophage activation by IFN-γ produced by T cells E.g. activated T cell produces IL-2 which binds to IL-2R on same cell to cause proliferation/clonal expansion Kuby Immunology, 7e  Properties of Cytokines Redundancy – different cytokines can induce similar effects Act in synergy Clinical attempts to block effects of cytokines may not guarantee clinical outcomes; e.g. anti-TNF mAbs used to prevent joint damage in RA do not completely prevent disease as they have no effect on IL-1 Kuby Immunology, 7e  Properties of Cytokines Pleiotropism - many cytokines act on different cell types Side effects of cytokine therapy e.g. IFN-α used to treat HBV makes patients feel unwell as it induces an acute-phase response Kuby Immunology, 7e  Properties of Cytokines Cytokines can inhibit each other Kuby Immunology, 7e  Nomenclature of Cytokines ~300 cytokines described No standard naming system that includes all of them Interleukins Largest group Named so as they were thought to act between leucocytes; have effects on many cell types Numbered according to order of discovery Interferons Named after presumed function; interfere with viral replication Are also potent activators of the immune system  Nomenclature of Cytokines TNF Can induce necrosis in cancers when injected into animals at high concentrations In vivo effects are more subtle Colony stimulating factors Chemokines Stimulate cell migration Named based on number & spacing of the specific cysteine residue e.g. CCL2, CXCL8  Classification of Cytokines Based on structure: Based on function: 1. Haemopoietin family (e.g. IL-2, IL-4) 1. Growth factors 2. Interferon family (e.g. IFN-α, β, 𝛾) 2. Pro- & anti-inflammatory agents 3. Chemokine family 3. Polarizing agents (cause cell specialization) 4. Tumor necrosis family Kuby Immunology, 7e  Cytokine Secretion Secreted by various cells types Most secreted only when cells become activated, as part of the response to infection Some are constitutively secreted (constantly, at low levels)  Cytokine Secretion Secreted at variable levels Adaptive immune system cytokines secreted at very low levels; have paracrine & autocrine effects Maintains specificity of the adaptive immune response e.g. IL-2 by activated T cells Low levels; may be impossible to detect in fresh blood samples Innate immune system cytokines often secreted at low levels, over a short range e.g. chemokines directed at neutrophils during inflammation Can be secreted at high levels & act in endocrine fashion; measurable in blood samples; e.g. IL-1, IL-6 & TNF secreted during the acute-phase response  Cytokine Secretion Secretion is usually transient – in response to infection; secretion stops once infection is resolved Inhibitory cytokines (IL-10, TGF-β) may be produced towards the end of an immune response Cytokine receptors also usually expressed transiently by activated T cells Prevents inappropriate activation of the immune system Kuby Immunology, 7e  Cytokine Receptors Based on structural homology, there are 6 major cytokine receptor families: Ig superfamily receptors Interferon receptors TNF receptor superfamily Chemokine receptors TGF receptor family Haemopoietin receptors (cytokine receptor superfamily) Kuby Immunology, 7e  Cytokine Receptors and Signaling Molecules Most cytokine receptors not expressed in high numbers on resting cells Upregulated after cell activation e.g. after a T cell is activated through its TCR After upregulation, the receptors are spread across the surface of the cells Cytokine binding to its receptor causes aggregation of the receptors at the cell surface  Cytokine Receptors and Signaling Molecules Each family of receptors is linked to different signal transduction molecules Signal transduction molecules convey signals from the receptor to the inside of the cell Cytokine binding causes changes in gene expression of the target cell General Model of Signal Transduction Kuby Immunology, 7e  Roles of Cytokines in the Immune Response Initiation of inflammation Communication between innate and adaptive immune systems T cell priming Development of T cell specialization Winding down of the immune response  Cytokines in the Innate Immune Response Resident tissue macrophages recognize pathogens & secrete pro-inflammatory cytokines (IL-1, TNF, IL-6) & chemokines to recruit neutrophils & other immune cells to a site of infection – Acute inflammation Type I IFNs secreted early in the innate immune response Cause virally infected cells to become more susceptible to killing Increase resistance of unaffected cells to infection by inducing synthesis of cellular proteins that interfere with viral replication  Cytokines in T cell Priming Ag presentation, in presence of costimulation (CD40, CD80, ICAM), activates Th cells Costimulatory cytokines secreted by APCs also help initiate T cell responses - IL-1 & type I IFNs Activated Th cells upregulate the IL-2R & begin to secrete IL-2, which has an autocrine effect (or paracrine); leading to expansion of T cell clones Subsequent events depend on type of pathogens triggering the response & the site of the response  Cytokines in the Development of Specialized T cell Responses Th1 responses Intracellular pathogens stimulate APCs to secrete IL-12 & type I IFNs These induce T cells to secrete IFN-𝛾, and acquire a Th1 phenotype Th1 cells activate macrophages to kill intracellular pathogens; and favour production of IgG by B cells Additional IFN-𝛾 and TNF secretion when pathogens cannot be cleared leads to granuloma formation  Cytokines in the Development of Specialized T cell Responses Th2 responses Helminths favour Th2 responses (likely due to the T cell transcription factor GATA3 induction, as opposed to T-bet in Th1 responses) Lead to IL-4 production, which favours B cell production of IgE Th2 cells also secrete IL-5 & eotaxin (chemokine), which stimulate maturation of mast cells & eosinophils  Cytokines in the Development of Specialized T cell Responses Th17 responses Extracellular pathogens (bacteria, fungi) stimulate innate immune system to produce IL-23 This stimulates generation of Th17 cells These secrete IL-17 that attracts N⏀ to the site of infection  Cytokines in the Development of Specialized T cell Responses Gut immunity Gut derived T cells secrete TGF-β This induces Ig class switch from IgM to IgA (mucosal immunity) Also has anti-inflammatory effects; can induce T cells to become Tregs  Cytokines in the Development of Specialized T cell Responses Tregs Peripheral tolerance; prevent responses to self antigen Secrete IL-10 and TGF-β; which inhibit responses to self antigens Roles of Cytokines in Ending the Immune Response On clearance of the pathogens, levels of innate immune cytokines fall Less antigen is available for presentation T cell stimulation declines Lower levels of cytokine production & receptor expression IL-10, TGF-β, & IL-35 are inhibitory cytokines secreted by regulatory T cells & other cell types Limit effector T cell proliferation; play a role in peripheral tolerance Tumours can also secrete suppressive cytokines to protect themselves from the immune response  Cytokine-Related Diseases Cytokines may cause injury to host tissues Exposure to certain bacterial toxins (e.g. TSS toxin, staphylococcus enterotoxin) made during an infection can activate large numbers of T cells, which produce large quantities of IFN-𝛾 This activates macrophages to release large amounts of pro- inflammatory cytokines – IL-1, IL-6, TNF, resulting in a “cytokine storm” Can cause sepsis with shock, DIC & multi-organ failure Some forms of immunotherapy can also cause a similar situation (often milder) – cytokine release syndrome  Cytokine-Related Diseases Loss of Function of Cytokines or their Receptors Mutation of IL-2𝛾R chain Causes immune deficiency The mutation also affects receptors for IL-4, IL-7, IL-9, IL-15 (share a common gamma chain with IL-2) Results in a decrease in NK & T cells and non-functional B cells due to the need of some of these cytokines for lymphocyte development  Cytokine-Related Diseases Loss of Function of Cytokines or their Receptors CD40 Ligand Deficiency CD40L interaction with CD40 to activate B lymphocytes is critical for immunoglobulin (antibody) class switching Patients lacking a functional CD40L (also called CD154) in X- linked hyper IgM syndrome present with high IgM and almost no IgG, IgE, or IgA These individuals have a poor response to extracellular bacteria  Cytokine-Related Diseases Loss of Function of Cytokines or their Receptors Defects in Fas ligand (CD178) FasL/Fas interaction is important in removing lymphocytes after an immune response and during development (e.g., elimination of autoreactive lymphocytes) The secondary lymphoid organs of these patients are filled with these lymphocytes which should have undergone apoptosis Autoimmune Lymphoproliferative Syndrome (ALPS)  Cytokine-Related Diseases Cytokines and Infectious Disease Chemokine receptors CXCR4 & CCR5 serve as coreceptors for HIV infection Genetic polymorphism of CCR5 (∆32 del) prevents expression of the receptors on the cell surface Confers resistance to HIV infection  Cytokines Assays Identification & quantification of cytokine profiles in biopsies, isolated cells or in body fluids Blood levels of cytokines are not measured in routine clinical practice Can be measurable in blood in cases of very severe infection e.g toxic shock (high levels of TNF or IL-6 in blood) Flow cytometry, ELISA, Luminex bead assays, ELISPOT assay, gene expression assays  Cytokine Therapy Stimulate/enhance immune responses (immunotherapy) E.g. in cancer patients: IL-2 (to increase T cell proliferation), IL-4 (to increase B activation), IFN-𝛾 (to increase antigen presentation), IL-12 (to activate NK cells) G-CSF in neutropaenia; or post HSCT Suppress immune responses (cytokine blockade) E.g. in transplant recipients: anti-CD25 (inhibit IL-2R) or calcineurin inhibitors (prevent T cell activation & IL-2 synthesis) Autoimmune disease e.g. RA: anti-TNF Alter the cytokine balance e.g. in patients with autoimmune disease Kuby Immunology, 7e Phagocytosis and Intracellular Killing Defination  Phagocytosis: An Evolutionarily Conserved Mechanism to Remove Apoptotic Bodies and Microbial Pathogens Phagocytes Cells that protect the body by ingesting harmful foreign particles- bacteria, and dead or dying cells  Neutrophils  Monocytes  Macrophages  Dendritic cells  Mast cells Professional Phagocytes location Variety of phenotypes Blood neutrophils, monocytes Bone marrow macrophages, monocytes, sinusoidal cells, lining cells Bone tissue osteoclasts Gut and intestinal macrophages Peyer's patches Connective tissue histiocytes, macrophages, monocytes, dendritic cells Liver Kupffer cells, monocytes Lung self-replicating macrophages, monocytes, mast cells, dendritic cells Lymphoid tissue free and fixed macrophages and monocytes, dendritic cells Nervous tissue microglial cells (CD4+) Spleen free and fixed macrophages, monocytes, sinusoidal cells Thymus free and fixed macrophages and monocytes Skin resident Langerhans cells, other dendritic cells, conventional macrophages, mast cells Phagocytes - Neutrophils (PNMs) Characteristic nucleus, cytoplasm Granules CD 66 membrane marker Characteristics of Neutrophil Granules primary granules secondary granules azurophilic; specific for mature neutrophils characteristic of young neutrophils; contain cationic proteins, contain lysozyme, NADPH lysozyme, defensins, oxidase components, elastase and lactoferrin and B12-binding myeloperoxidase protein Phagocytes - Macrophages Characteristic nucleus Lysosomes CD14 membrane marker Phagocyte Response to Infection The SOS Signals N-formyl methionine- containing peptides Clotting system peptides Complement products Cytokines released by tissue macrophages Phagocyte response Vascular adherence Diapedesis Chemotaxis Activation Phagocytosis and killing Initiation of Phagocytosis Attachment via Receptors: IgG FcR Complement R ScavengerR Toll-like R Cellular Barriers Phagocytosis  carried out by 2 types of leukocytes in the blood: neutrophils, a type of granulocyte (60-70%) monocytes, a type of agranulocyte (3-8%)  phagocytes in the body can be divided into 2 types: microphages (e.g., neutrophils) - phagocytic against bacteria in the early phases of infection macrophages - fixed in tissues (reticuloendothelial system); active against microbes, dead and dying microphages, dead tissue  Phagocytosis Phagocyte Bacterium or Particulate Matter Lysosome Fusion Binding P rimary Lysosome Endocytosis Hydrolysis Endosome Secondary Lysosome Fusion Excretion  Post-phagocytic Events Pathogen Macrophage 2O2  2O2- + H+ NADPH oxidase 2O2- + NO ONOO- Peroxynitrite H2O2 + Cl-  HOCl + OH Myeloperoxidase O2- + O2- + 2H+  H2O2 + O2 Superoxide dismutase 1. Phagosome-Oxidase Fusion 2. Generation of H2O2 3. Myeloperoxidase Activity 4. Peroxynitrite Production Mechanisms to avoid destruction by phagocytes  kill the phagocyte  inhibit binding or endocytosis  overwhelm phagocytes  resistance to digestion in the phagolysosome,e.g., TB, leprosy, leishmaniasis  Immunological Consequences of Phagocytosis Clearance of pathogens Death of pathogenic microbe Persistence of pathogenic microbe Resolution of infection Failure of resolution of infection Clearance of apoptotic corpses Suppression of inflammation Inappropriate inflammation Tolerance Break in tolerance Bacterial Virulence Factors Subvert Host Defenses Modification of phagocytic receptors (P. aeruginosa) Escape from phagosome into cytosol (Listeria, Shigella) Ingestion phase impaired (Yersinia) Phagosome maturation stalled Resistance to (M. tuberculosis; Legionella) lysosomal degradation (Salmonella) Functions of Phagocytosis  clear the body of debris  secrete defense proteins, e.g., interferon  ingest and kill bacteria  inflammation - synthesize endogenous pyrogen (IL-1)  antigen processing Mechanisms of Humoral Immunity Ephantus Njagi H.S.R Background  Mechanisms of humoral immunity are accomplished after interaction of:  B cells  Antibody production  antigen binding H.S.R Interaction of antigen with B cells  Each antibody binds to a specific antigen similar to a lock and key through:  Noncovalent bonds or Intermolecular forces.  Hydrogen bonds  Electrostatic bonds  Van der Waal forces  Hydrophobic bonds  An immune complex is formed from the integral binding of an antibody to a soluble antigen.  The bound antigen acting as a specific epitope, bound to an antibody is referred to as a singular immune complex. H.S.R B-cell Activation and Differentiation H.S.R Clonal expansion and Differentiation H.S.R B cells mediated immune response Humoral immunity(HI) or antibody mediated immunity: The total immunological reaction that B cells recognize antigen, then activate, proliferate, differentiate into plasma cells and produce Ab.  B2 cells mediated immune response to TD-Ag  B1 cells mediated immune response to TI-Ag H.S.R PartⅠ: Immune response of B2 cell to TD-Ag Characteristics of TD-Ag:  PossessT cell epitope and B cell epitope  Need Th cells participation  Both CMI and HI  Produce several types of antibodies: IgG  Produce immune memory H.S.R  1. B cells recognize antigen  BCR directly recognize the conformational determinant, capture Ag and present Ag signal to Th cells  No APC , no MHC restriction  Specificity H.S.R linear conformational H.S.R  Clonal Selection Antigens Only B-cells that bind antigen are selected B-cells Antibody on Surface H.S.R  Clonal Expansion B-cells Antibody on Surface Proliferation of selected B-cell H.S.R  Somatic Mutation Somatic Mutation B-cells Antibody on Surface Improved Antibody Binding By Genetic Rearrangement H.S.R  2. B cells activation, proliferation and differentiation (1) B cell activation: double signals  First signal : antigen signal BCR--conformational determinant on the surface of Ag Igα/Igβtransduct first signal CD19/CD21 (co-receptor) binds to C3d on Ag  Second signal: co-stimulatory signal The CD40 on B cells binds to CD40L on activated T cells H.S.R  Antigen CD40 T helper TCR B cell cell B MHC II cell 1. Antigen presentation to Th cell B7 CD28 2. B7 expressed Cytokine Immunoglobulin receptor 3. Th cell is receptor activated and expresses CD40 ligand, Cytokines CD40 secreted ligand B B B B T helper cell cell cell cell cell 5. B cell activated Cytokine 6. B cells proliferate, differentiate, secrete Ig H.S.R Interaction between Th cell and B cell  B cells act on T helper cells:  B cells present Ag to Th cells  B cells provide B7 for Th cells  T helper cells act on B cells:  Activated Th cells provide co-stimulatory molecule for B cells: CD40L-D40  Activated Th produce Cytokines to help B cells proliferate and differentiate  Activated B cells express receptors of cytokines  Activated Th2 secrete cytokines IL-4, IL-5, IL-6 to enhance proliferation and differentiation of B cells  B cells differentiate into plasma cells (antibody forming cells)- produce Ab, some activated B cells become memory B cells H.S.R  B cell Th cell Th cell Ag processing activation B cell activation Interactions between B cell and Th cell H.S.R Cytokines affect proliferation and Class switch of B cells H.S.R H.S.R H.S.R 3. Stage of effect----the function of Ab 1. Neutralization-Ab covers up sites on toxic molecule/virus. 2. Opsonization-Ab-mediated phagocytosis. 3. Complement fixation-cascade reacts to ag-ab complexes. 4. Agglutination/precipitation-Cross-link ags into complexes-aids phagocytosis. 5. Immobilization-Abs bind to flagella etc. & prevent escape macrophage death. 6. ADCC-NK, macrophage 7. Participate in hypersensitivity-I,II,III H.S.R 1. Neutralization: * to neutralize microbial toxins and animal venoms * to prevent viruses and bacteria from infecting cells H.S.R 2. ADCC—NK Antibody-coated target cells can be killed by natural killer cells(NK cell) in antibody-dependent cell-mediated cytotoxicity, that is called ADCC. H.S.R 3.Opsonization – macrophage Fc receptors on phagocytes trigger the uptake of antibody-coated bacteria. As a result, this action enhance phagocytosis of the bacterium. H.S.R 4.Activation of complement Complement can be activated to directly lyse bacteria by the presence of antibodies bound to the bacteria. Complement proteins bind to antibodies. H.S.R 5. Participate in hypersensitivity IgE binds to high-affinity Fc receptors on mast cells and basophils, and leads to the rapid release of granules containing inflammatory mediators into surrounding tissue, cause hypersensitivity H.S.R H.S.R H.S.R H.S.R H.S.R Primary and secondary antibody responses to protein antigens differ qualitatively and quantitatively H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R Regulation of the immune response Dr Oyaro H.S.R  POINTS TO BE DISCUSSED ✓ Regulation by antigen and antigen-presenting cell ✓ Regulation by antibody ✓ The role of T cells (Treg) and NK T cells ✓ The role of telomeres ✓ Idiotypes and idiotypic network ✓ Neuroendocrine mechanisms ✓ Genetic aspects in immune regulation ✓ Immune regulation vs immune modulation - vaccines H.S.R  REGULATION BY ANTIGEN Chemical nature of Ag-polysaccharides vs.proteins Soluble vs.intracellular Ags Large doses vs. small doses of Ag Competition between antigens and peptides The route of administration of Ag The role of adjuvants H.S.R  THE SIGNIFICANCE OF ANTIGEN-PRESENTING CELL Professional vs. non-professional APC CD40L on T cell- CD40 on APC interaction CD28 |CTLA4 | vs. CD80 and CD86 (B7-1 & B7-2) on APC The level of expression of MHC on APC H.S.R  THE SIGNIFICANCE OF ANTIGEN-PRESENTING CELL 2 The extent of cytokines secreted by APC Cross-presentation (cross-priming) of viral Ags by APC to Tc (CD8+) cells via MHC-I APC killing by cytotoxic T cells H.S.R  IMMUNE RESPONSE: STIMULATORY AND INHIBITORY CYTOKINES Interleukin-2 (IL-2) IL-4 IL-1 IL-10 IL-4 IL-11 IL-5 IL-6 IL-13 IL-12 Transforming growth IL-18 factor beta (TGF-) Interferon gamma IFN-/ (IFN- ) IFN- H.S.R  REGULATION BY T LYMPHOCYTES CTLA-4, instead of CD28, on T cells, binds B7 (on APC) – inhibition of activation Fratricide – mutual killing of T cells by Fas- FasL system Prevention of induction of autoimmunity by CD4+ CD25+Treg cells H.S.R  FEATURES OF Treg CELLS Quantity: 5-10 % of CD4+ T cells in the blood Surface markers: CD25, CD103, Foxp3 GITR,(glucocorticoid-induced TNF receptor) Cytokine expression/secretion:IL-10, IFN-, TGF- Suppression mechanism: contact with activated target CD8+ / CD4+ T cells, secretion of cytoki- nes (IL-10, TGF-) and non-specific inhibition („bystander effect”) H.S.R  Regulation of cell senescence by telomere shortening Telomeres – repeats of the DNA sequence (GGGTTA) and protein located at the end of chromosomes – up to 2000 copies per cell Provide stabilization and protect chromosomal ends from damage; regulate cell replication At every cell division they shorten by 50-100 bp H.S.R  Regulation of cell senescence by telomere shortening -2 When telomeres become too short, chromosome gets unstable and DNA damage can occur. To prevent damaged cells being replicated such cells: - die by apoptosis, - Enter cell arrest, known as cellular senescence. T cells in elderly people have significantly shorter telomeres than young ones, People with some premature ageing syndromes have short telomeres and usually have low life expectancy H.S.R IDIOTYPIC REGULATION OF IMMUNE RESPONSES (IR) Idiotype = the sum of idiotopes, variable determinants in a given antibody molecule or TCR There are public and private idiotopes: - public: those found on other cells private: unique for given cell or cell clone H.S.R IDIOTYPIC REGULATION OF IMMUNE RESPONSES (IR) Anti-idiotypic antibodies may block antigen binding and thus regulate immune response These antibodies are present in small amounts within immunoglobulin pool of all humans and participate in normal both, humoral and cellular IR H.S.R  JERNE’S IDIOTYPIC NETWORK (NOBEL PRIZE IN 1984) Idiotypic determinants are immunogenic Anti – idiotypic antibodies are formed following formation anti - antigen antibody Anti-idiotypic Ab induce anti-anti-idiotypic response This leads to gradual fading of immune response against given antigen H.S.R  B cells, Tcells and NK cells express receptors that contain immuno-receptor tyrosine based inhibitory motifs (ITIMs), apart from ITAMs (activating ones). H.S.R NEUROENDOCRINE MODULATION OF IMMUNE RESPONSES Most lymphoid tissues possess sympathetic innervation Lymphocytes express receptors for a variety of hormones, neurotransmitters and neuropeptides H.S.R NEUROENDOCRINE MODULATION OF IMMUNE RESPONSES -2 Examples include steroids, catecholamines, enkephalins, endorphins and others When released in vivo during stress, most of them are immunosuppressive H.S.R NEUROENDOCRINE MODULATION OF IMMUNE RESPONSES - 3 On the other hand, IL-1 and IL-6 cytokines act as stimulants of adrenal corticosteroid production Both IL-1 and IL-6 are synthesized by neurons and glial cells and, in addition by cells in pituitary and adrenal glands H.S.R  GENETIC CONTROL OF IMMUNE RESPONSES Strains of mice with different MHC haplotypes vary in their ability to mount immune response to given antigens Peptide-binding groove of APC is formed by the most polymorphic residues in MHC molecules (encoded by different alleles) Thus, MHC dependent aminoacid sequences of the groove determine the accuracy of peptide binding and in turn, antigen presentation H.S.R CLINICAL IMPLICATIONS OF GENETIC CONTROL OF IR MHC-linked genes control the response to several infections Certain HLA haplotypes confer protection from malaria (Plasmodium falciparum) Susceptibility to autoimmune diseases is influenced by MHC-linked genes H.S.R CLINICAL IMPLICATIONS OF GENETIC CONTROL OF IR (2) Linkage disequilibrium denotes grouping of some genes that increase risk of particular disease(example:HLA-DR3/DR4 -diabetes) Non-MHC-linked genes also affect susceptibility to several diseases H.S.R IMMUNE MODULATION – MANIPULATION OF THE IMMUNE RESPONSE Vaccination – passive and active Application of cytokines Application of monoclonal antibodies Suppression by glucocorticoids and other immunosuppressive drugs Infusion of immune cells Gene therapy related to the immune system H.S.R  FEATURES OF GOOD VACCINE 1. Safe to use (live vaccines bear potential risk) 1. Induce the right type of immunity 1. Is affordable by the population concerned 1. Easy to produce and store H.S.R ADVANTAGES OF LIVE ATTENUATED VACCINES 1. Preserve immunogenicity of virulent agent, especially conformational antigens involved in antibody production 1. Mimic natural infection better than inactive vaccines 1. Usually stimulate multiple components of the immune system including T cell and mucosal immunity mediated by IgA 1. Herd immunity H.S.R  DISADVANTAGES OF LIVE ATTENUATED VACCINES May contain adventitious agents Can revert to virulence by mutation or interserotypic recombination Can cause serious ilness in immunosupressed individuals Stringent storage instructions for vaccine efficacy and safety H.S.R  THERAPEUTIC MODULATION OF IMMUNE RESPONSES Non-specific immunization (BCG, bacterial lysates, cytokines) Passive immunization: direct infusion of antibodies Active immunization = vaccination: – live attenuated organisms(measles,mumps) – non-living organisms or subcellular fragments(pertussis, polio) – recombinant DNA-based (hepatitis B) – Edible transgenic plants (HBs in lettuce) H.S.R  NEW APPROACHES FOR BETTER VACCINES Inactivated vaccines Recombinant proteins produced in yeast, bacteria, cell culture or plants Synthetic peptides Anti-idiotypic vaccines Nucleic acid vaccines Novel adjuvants Novel carriers H.S.R  ADJUVANTS, IMMUNOSTIMULANTS, PROBIOTICS Adjuvants – organic and inorganic compounds enhancing immune response while applied together with an antigen (Freunds a. ) Immunostimulants – microbial compounds that enhance general immune response(ribomunyl) Probiotics – living microorganisms exerting favourable effect on patient’s health (Lactobacillus) H.S.R  Immunomodulation by monoclonal antibodies Advantages structural stability unlimited supply of reagent, high specificity Disadvantages: risk of sensitization for foreign protein (murine or rat Ig), potential hazard of anafilactic shock, difficult accesibility, high cost H.S.R  Modifications of monoclonal antibodies Mabs Chimeric Mabs –-Variable parts from mice, constant regions (C) human (75% human sequences) Humanized Mabs – hypervariable regions from mice, C regions from man (95% human sequences) Human Mabs – human Ig gene expresion in various biological carriers (bacteriofages, transgenic animals, bacteria and even plants – „plantibodies”) Minibodies – miniaturised Mabs (have better penetration) Bispecific Mabs – specificity for 2 antigens H.S.R  Examples of Mabs currently used in therapy Infliximab /anti TNF/ rheumatoid arthritis |RA|, Crohn disease Rytuksymab /Anti CD20/ RA, B cell leukemias /lymphomas Efalizumab /anti CD11a/ psoriasis Trastuzumab /HER 2/neu/ breast carcinoma Cetuksymab /EGFR/ large bowel carcinoma H.S.R  Importance of immune regulation Prevents inappropriate immune response to self- antigens (self-tolerance) Prevents immune response against harmless environmental antigens (commensal microbes) To avoid excessive lymphocyte activation and tissue damage during normal protective responses against infections Failure of control mechanism is the underlying cause of immune-mediated inflammatory diseases H.S.R Immunology of Inflammation Dr. Barasa H.S.R  Learning Objectives By the end of this lecture, you should be able to: – Describe the processes of initiation and resolution of inflammation – Describe the phases of inflammation – Outline the mediators of inflammation – Describe the types of inflammation H.S.R  Inflammation Reaction of living tissue to injury or infection – Protective host response to “danger” signals – infective, traumatic, ischaemic, physical, chemical Exposure to PAMPs and DAMPs leads to activation of cells of the monocyte- macrophage lineage Results in expression of pro-inflammatory and suppression of anti-inflammatory genes H.S.R  Inflammation Inflammatory reaction consists mainly of responses of blood vessels and leukocytes Cytokines, chemokines and other chemicals cause cellular recruitment and vascular changes Principal defenders against foreign invaders are plasma proteins, circulating leukocytes & tissue phagocytes H.S.R H.S.R  Inflammation The vascular and cellular reactions amplify the inflammatory response & determine its pattern, severity, & clinical & pathologic manifestations Inflammation can be Acute or Chronic, depending on depending on – Nature of the stimulus – Effectiveness of the initial reaction in eliminating the stimulus or damaged tissues H.S.R  Inflammation Acute inflammation Rapid in onset (typically minutes) Short duration (hours or few days) Main characteristics are – Exudation of fluid & plasma proteins (oedema) – Emigration of leukocytes, predominantly neutrophils H.S.R  Inflammation When acute inflammation is successful in eliminating the offenders the reaction subsides If the response fails to clear the invaders it can progress to a chronic phase H.S.R  Inflammation Chronic inflammation May follow acute inflammation or be insidious in onset Longer duration Associated with presence of lymphocytes & macrophages, proliferation of blood vessels, fibrosis, & tissue destruction H.S.R  Inflammation Terminated when the offending agent is eliminated The reaction resolves rapidly because – Mediators are broken down and dissipated – Leukocytes have short life spans in tissues – Anti-inflammatory mechanisms are activated Control the response & prevent it from causing excessive damage to the host H.S.R  Inflammation Without inflammation, – Infections would go unchecked – Wounds would never heal – Injured tissues might remain permanent festering sores H.S.R  Inflammation The ideal inflammatory response is rapid and destructive (when necessary), yet specific and self limiting H.S.R  Cardinal Signs of Inflammation Rubor (redness) Tumor (swelling) Calor (heat) Dolor (pain) Functio laesa (loss of function) H.S.R  Roles of Inflammation in Combating Infection Delivery of additional effector molecules & cells to the site of infection to augment the killing of invading microorganisms by the front line macrophages Provision of a physical barrier in the form of microvascular coagulation to prevent the spread of infection upstream Promotion of repair of injured tissue H.S.R  Phases of Inflammation I. Recognition of infection/offending agent II. Recruitment of inflammatory cells III. Elimination of the microbe/offending agent IV. Resolution of inflammation H.S.R  Recognition of Infection Macrophages and DCs engulf microbes, which they detect via PRRs that are expressed on their cell surface Net result of pathogen recognition is activation of resident phagocytes & release of pro-inflammatory cytokines & preformed mediators H.S.R  Recruitment of Inflammatory Cells Cytokines induce changes in local blood vessels that promote conversion of infected tissue to an inflamed state Leucocytes travel by laminar flow, contacting endothelium intermittently, rolling for some distance, then detaching if no further adherent stimuli are encountered If the rolling leucocyte encounters inflamed endothelium expressing activated integrin adhesion molecules, H.S.R tight binding ensues & the leucocyte can then migrate through the endothelium (diapedesis)  Elimination of the Microbe Phagocytosis & killing by neutrophils If N⏀ detect TNF-α but don’t directly encounter microbial particles after entering tissue, they release their granules into the extracellular space in an effort to create an inhospitable environment for nearby pathogens Granule contents are not uniquely toxic for microbial organisms; generate significant damage to host tissues and cells – E.g. elastase, cathepsin G, Proteinase 3 are serine proteases that can break down components of extra cellular matrix H.S.R  Resolution of Inflammation If the inflammatory response is able to contain the microbial infection, the overall response is shifted toward anti-inflammatory signals & resolution of inflammation H.S.R  Mediators of Inflammation Soluble diffusible molecules that act locally at the site of tissue damage or infection, and at more distant sites Produced in response to various stimuli – Microbial products – Substances released from necrotic cells – Proteins of the complement, kinin, & coagulation systems (are also activated by microbes & damaged tissues) H.S.R  Mediators of Inflammation Exogenous mediators – Bacterial products and toxins e.g. endotoxin (LPS) Endogenous mediators – Generated either from cells or from plasma proteins H.S.R Cell-derived Mediators of Inflammation Normally sequestered in intracellular granules Can be rapidly secreted by granule exocytosis (e.g., histamine in mast cell granules) Or are synthesized de novo (e.g., prostaglandins, cytokines) in response to a stimulus Cells: Platelets, neutrophils, monocytes/macrophages, mast cells, mesenchymal cells (endothelium, smooth muscle, fibroblasts), epithelia H.S.R  Plasma-derived Mediators of Inflammation Mainly produced in the liver Present in the circulation as inactive precursors that must be activated, (proteolytic cleavage), to acquire their biologic properties E.g., complement proteins, kinins H.S.R Mediators of Inflammation H.S.R Basis of Disease, 8e Robbins & Cotran Pathologic Role of Mediators in Different Reactions in Inflammation Robbins & Cotran Pathologic Basis of H.S.R Disease, 8e Inter-relationships between the four plasma mediator systems triggered by activation of factor XII Thrombin induces inflammation by binding to protease-activated receptors in platelets, endothelium & sm cells H.S.R  Mediators of Inflammation One mediator can stimulate the release of other mediators – E.g. TNF acts on endothelial cells to stimulate production of IL-1 Secondary mediators may have the same actions as the initial mediators but may also have different and even opposing activities Such cascades provide mechanisms for amplifying—or, in certain instances, counteracting—the initial action of a mediator H.S.R  Mediators of Inflammation Most are short-lived – They decay quickly (e.g., arachidonic acid metabolites) – Or are inactivated by enzymes (e.g., kininase inactivates bradykinin) – Or are scavenged (e.g., antioxidants scavenge toxic oxygen metabolites) – Or inhibited (e.g., complement regulatory proteins break up and degrade activated complement components) There is thus a system of checks and balances that regulates mediator actions H.S.R Generation of Arachidonic Acid Metabolites and their Roles in Inflammation Robbins & Cotran Pathologic H.S.RBasis of Disease, Robbins & Cotran Pathologic 8th edition Basis of Disease, 8e  Cytokines in Inflammation TNF and IL-1 have autocrine and paracrine effects leading to local activation of macrophages and neutrophils When releasedin large amounts, they can exert endocrine effects Induction of acute phase proteins in the liver, platelet activation, fever, fatigue, anorexia H.S.R Basis of Disease, 8e Robbins & Cotran Pathologic  Resolution of Inflammation Mechanisms that shut down the inflammatory response and mediate homeostasis Promote resolution and repair H.S.R  Anti-inflammatory Mediators Lipoxins – Stop the influx of neutrophils – Promote uptake of apoptotic neutrophils – Recruit additional monocytes Secretory leukocyte protease inhibitor (SLPI) – Produced by macrophages, neutrophils and epithelial cells – Inactivates the proteases released from neutrophil granules H.S.R  Anti-inflammatory Mediators Anti-inflammatory cytokines (IL-10, TGF-b) IL-10 produced by Treg cells; TGF-b by monocytes and platelets Glucocorticoids – production induced by systemic elevation of inflammatory cytokines (esp. IL-1) Cleaved extracellular domains of cytokine receptors (e.g. soluble TNFR, IL-1R) serve as decoy receptors; limit inflammation by binding and neutralizing their respective cytokines H.S.R  Anti-inflammatory Mediators Complement inhibitors Prostaglandins and lipid mediators e.g. resolvins exert negative feedback loops by suppressing transcription and release of cytokines Acute phase proteins e.g. α-1 antitrypsin Stress hormones e.g. corticosteroids and catecholamines are negative regulators of TLR signaling H.S.R  Acute Inflammation Rapid host response that serves to deliver leukocytes & plasma proteins (antibodies), to sites of infection or tissue injury H.S.R  Major Components of Acute Inflammation Vascular dilatation & increased blood flow (causing erythema & warmth) Extravasation & extravascular deposition of plasma fluid & proteins (oedema) Leucocytes emigration & accumulation in the site of injury Robbins & Cotran Pathologic Basis of Disease, 8e H.S.R  Stimuli for Acute Inflammation Infections Tissue necrosis (ischemia, trauma, chemical injury, thermal injury, irradiation) Foreign bodies (splinters, dirt, sutures) Immune reactions/hypersensitivity reactions in which the normally protective immune system damages the individual's own tissues H.S.R  Reactions of Blood Vessels in Acute Inflammation Undergo changes designed to maximize movement of plasma proteins & circulating cells out of the circulation, into site of infection or injury Exudation - escape of fluid, proteins, & blood cells from the vascular system into the interstitial tissue or body cavities H.S.R Ultrafiltrate of plasma Low protein content (mostly albumin; little or no cellular material; low specific gravity) High protein concentration (contains cellular debris; high SG) Increase in the normal permeability of small blood vessels in an area of injury (inflammatory reaction) H.S.R Robbins & Cotran Pathologic Basis of Disease, 8e Mechanisms of Increased Vascular Permeability H.S.R Robbins & Cotran Pathologic Basis of Disease, 8e  Reactions of Blood Vessels in Acute Inflammation Oedema - excess of fluid in the interstitial tissue or serous cavities; it can be an exudate or a transudate Pus – A purulent exudate – Inflammatory exudate rich in leukocytes (mostly neutrophils), the debris of dead cells and microbes H.S.R  Reactions of Leucocytes in Inflammation Leucocyte adhesion to endothelium Leucocyte migration through endothelium Chemotaxis of leucocytes Recognition of microbe and dead tissues Phagocytosis and Removal of the offending agents H.S.R H.S.R  Termination of the Acute Inflammatory Response Inflammation has an inherent capacity to cause tissue damage; needs tight controls to minimize the damage In part, inflammation declines because the mediators – Are produced in rapid bursts, only as long as the stimulus persists – Have short half-lives, & are degraded after their release Neutrophils also have short half-lives in tissues & die by apoptosis within a few hours after leaving the blood H.S.R  Termination of the Acute Inflammatory Response Inflammation also triggers a variety of stop signals that serve to actively terminate the reaction: Switch in the type of AA metabolite produced, from pro- inflammatory LTs to anti-inflammatory lipoxins Liberation of anti-inflammatory cytokines, e.g. TGF-β and IL- 10, from macrophages and other cells Production of anti-inflammatory lipid mediators (resolvins & protectins), derived from polyunsaturated fatty acids Neural impulses (cholinergic discharge) that inhibit the production of TNF in macrophages H.S.R Outcomes of Acute Inflammation Limited or short- lived injury/little tissue destruction Substantial tissue Persistence of injurious destruction/tissues agent/interference with incapable of normal process of regeneration; abundant healing fibrin exudation H.S.R Robbins & Cotran Pathologic Basis of Disease, 8e  Chronic Inflammation Prolonged duration (weeks or months) Tissue injury, and attempts at repair coexist, in varying combinations May follow acute inflammation Or may begin insidiously, as a low-grade, smouldering response without any manifestations of an acute reaction *Cause of tissue damage in some of the most common & disabling human diseases – RA, atherosclerosis, TB, pulmonary fibrosis etc H.S.R  Causes of Chronic Inflammation Persistent infections by microorganisms that are difficult to eradicate e.g. mycobacteria Immune-mediated inflammatory diseases Prolonged exposure to potentially toxic agents, either exogenous or endogenous H.S.R  Features of Chronic Inflammation Infiltration with mononuclear cells (macrophages, lymphocytes, plasma cells) Tissue destruction, induced by the persistent offending agent or by the inflammatory cells Attempts at healing by connective tissue replacement of damaged tissue, accomplished by proliferation of small blood vessels (angiogenesis) and fibrosis H.S.R  H.S.R Basis of Disease, 8e Robbins & Cotran Pathologic  Granulomatous Inflammation Distinctive pattern of chronic inflammation encountered in a limited number of infectious and some non-infectious conditions Activation of T lymphocytes leading to production of IFN that activates macrophages Macrophages differentiate into epithelioid cells with enhanced secretory function, and diminished phagocytic capacity H.S.R  Granulomatous Inflammation Tuberculosis is the prototype of the granulomatous diseases Recognition of the granulomatous pattern in a biopsy specimen is important because of the limited number of possible conditions that cause it and the significance of the diagnoses associated with the lesions H.S.R  Granuloma Cellular attempt to contain an offending agent that is difficult to eradicate Focus of chronic inflammation consisting of a microscopic aggregation of macrophages that are transformed into epithelium-like cells, surrounded by a collar of mononuclear leukocytes, principally lymphocytes and occasionally plasma cells Frequently, epithelioid cells fuse to form giant cells in the periphery or sometimes in the center of granulomas H.S.R Basis of Disease, 8e Robbins & Cotran Pathologic H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R H.S.R IMMUNE RESPONSES TO PARASITIC AND FUNGAL INFECTIONS. 1 H.S.R Objectives and learning outcomes. Learn immune responses to parasites and fungal infections. Immunopathology of parasitic and fungal infections. Know range of strategies used by parasites and fungi to evade hosts’ immune mechanisms. Be able to give specific examples of parasites and fungi, & their immune evasion strategies. 2 H.S.R  Introduction. Parasitology – Protozoa Intracellular – Red blood cells – macrophage extracellular – Helminth 3 H.S.R  Parasite immunology Exposure – Host - susceptible - parasite survives. – Host - insusceptible - parasite killed by innate immunity. E.g. Humans insusceptible to larval stages of bird schistosomes (e.g. Trichobilharzia). But get cercarial dermatitis (‘swimmers itch’). 4 H.S.R  Immunoparasitology. o Spontaneous-cure - parasite establishes but eventually expelled, e.g., Nippostrongylus brasiliensis. o Adult Nippostrongylus, releases protective antigens - not stage specific. o Hence, antibodies produced recognise targets on both adult worm & migrating infective larvae. 5 H.S.R  Immunoparasitology. Parasites successfully adapt to innate & acquired immune responses of host. Many factors involved in host susceptibility – e.g. genetic background, age, nutritional & hormonal status of individual. 6 H.S.R  Immunoparasitology. § Immune response mounted to protozoal & helminth infections. § Evidence- 1. Prevalence of infection declines with age. 2. Immunodepressed individuals quickly succumb. 3. Acquired immunity in laboratory models. 7 H.S.R  Parasites damage host by: Competing for nutrients (e.g. tapeworms). Disrupting tissues (e.g. Hydatid disease). Destroying cells (e.g. malaria, hookworm, schistosomiasis). Mechanical blockage (e.g. Ascaris). Severe disease often has immune / inflammatory component. 8 H.S.R Immunopathology - examples. Cerebral malaria - TNF, IFN & other proinflammatory cytokines in brain. Hepatosplenic schistosomiasis - anti-egg immune responses – granuloma & fibrosis. Onchocerciasis - anti-microfilarial responses in eye = blindness. Anaphylactic shock – e.g. rupture of hydatid cyst. Immediate hypersensitivity by parasite antigens. Nephropathy - immune complexes in kidney (e.g. malaria, schistosomiasis). 9 H.S.R  Immune responses to Protozoan parasites. 1. Innate immune responses. Extracellular protozoa eliminated -phagocytosis & complement activation 1. Acquired immune responses 1. T cell responses. - Extracellular protozoa - TH2 cytokines - ab production. - Intracellular protozoa – TC (cytotoxic lymphocytes) kill infected cells. - TH1 cytokines activate macrophages & TC. 2. Antibody + Complement, e.g. lysis of blood dwelling trypanosomes. 10 H.S.R Note Activated macrophages effective against intracellular protozoa, e.g. Leishmania, Toxoplasma, Trypanosoma cruzi. CD8+ cytotoxic T cells kill parasite infected host cells, e.g. Plasmodium infected liver cell. 11 H.S.R  Acquired immune responses. 1. Antibody responses. a) Extracellular protozoa 1. opsonization, 2. complement activation 3. Antibody Dependent Cellular Cytotoxicity (ADCC). b) Intracellular protozoa 1. Neutralisation: e.g. neutralising ab prevents malaria sporozoites entering liver cells. 12 H.S.R  Immune responses to helminth infections. Most of the helminths are extracellular organisms. Size: too large for phagocytosis. – Some gastrointestinal nematodes - host develops inflammation & hypersensitivity. – Eosinophils & IgE initiate inflammatory response in intestine / lungs. – Histamine elicited - similar to allergic reactions. 13 H.S.R Immune responses to helminth infections. 1. Acute response IgE & eosinophil mediated systemic inflammation = worm expulsion. 2. Chronic exposure = chronic inflammation: a) DTH, Th1 / activated macrophages - granulomas. a) Th2 / B cell responses increase IgE, mast cells & eosinophils = inflammation. 14 H.S.R  Immune responses to helminth infections. Helminths induce Th2 responses - IL-4, IL-5, IL- 6, IL-9, IL-13 & eosinophils & ab (IgE). Characteristic ADCC reactions, i.e. killer cells directed against parasite by specific ab. – E.g. Eosinophil killing of parasite larvae by IgE. 15 H.S.R  Parasite Immune Evasion –Evasion strategies. 1. Parasites need time in host - development, reproduce & ensure vector transmission. 2. Chronic infections normal. 3. Parasites evolved variety immune evasion strategies. 16 H.S.R  Protozoan immune evasion strategies. 1. Anatomical seclusion in vertebrate host. Parasites may live intracellularly - avoid host immune response. E.g. Plasmodium inside RBC’s - when infected not recognised by TC & NK cells. Other stages Plasmodium inside liver cells. Leishmania parasites & Trypanosoma cruzi inside macrophages. 17 H.S.R  2. Antigenic variation In Plasmodium, different stages of life cycle express different antigens. Antigenic variation also in extracellular protozoan, Giardia lamblia. African trypanosomes -1 surface glycoprotein that covers parasite = variant surface glycoprotein (VSG). – Immunodominant for antibody responses. – Trypanosomes have “gene cassettes” of VSG’s allowing regular switching to different VSG. – Host mounts immune response to current VSG but parasite already switching VSG to another type. – Parasite expressing new VSG escapes ab detection, replicates & continue infection. – Allows parasite survival - months / years. – Up to 2000 genes involved. 18 H.S.R  Host mounts immune response to current VSG Parasitaemia fluctuates. After Ross, P. (1910), Proc. Royal Soc. London, B82, 411 After each peak, tryp population antigenically different from that earlier / later peaks. 19 H.S.R 3. Shedding / replacement surface e.g. Entamoeba histolytica. 4. Immunosupression – manipulation host immune response e.g. Plasmodium. 5. Anti-immune mechanisms - Leishmania - anti-oxidases to counter macrophage oxidative burst. 20 H.S.R  Helminth immune evasion strategies 1. Large size - difficult to eliminate. 1. Primary response – inflammation. 2. Often worms not eliminated. 21 H.S.R  Helminth immune evasion strategies. 2. Coating with host proteins. Tegument cestodes & trematodes adsorb host components, e.g. RBC ags. a) Immunological appearance of host tissue. 1. E.g. Schistosomes - host blood proteins, (blood group ags & MHC class I & II). 2. worms seen as “self”. 22 H.S.R  Helminth immune evasion strategies 3. Molecular mimicry. Parasite mimics host structure / function. E.g. schistosomes have E-selectin - adhesion / invasion. 4. Anatomical seclusion - nematode larva does this -Trichinella spiralis inside mammalian muscle cells. 5. Shedding / replacement surface e.g. trematodes, hookworms. 23 H.S.R Helminth immune evasion strategies 6. Immunosupression – manipulation of the immune response. High nematode burdens - apparently asymptomatic. Parasite may secrete anti-inflammatory agents - suppress recruitment & activation effector leukocytes or block chemokine-receptor interactions. E.g. hookwor