Introduction to Cellular Immunology Dr. Colin R.A. Hewitt crah1@le.ac.uk Movie credits: The movies of cells are used with the permission of Dr. James A. Sullivan of Cells Alive http://www.cellsalive.net/

The purpose of this preliminary lecture is to remind students of the immunology learnt in the second year, and introduce key concepts that are required for a full understanding of the later lectures To use the lecture, click on the projection screen icon below , then just click your way through the presentation. Don’t forget to try the online multiple choice questions at the end to find your strengths and weaknesses

What you should know by the end of this lecture  The basic terms used in immunology  The characteristics and interdependence of adaptive and innate immunity  The names and functions of cells in the immune system  The structure and function of peripheral lymphoid organs The purpose of lymphocyte recirculation  How cells communicate in the immune system and how this is tested  How the clonal distribution of antigen receptors in the immune system allows for diverse recognition, self tolerance and memory  That the compartments invaded by pathogens require different effector mechanisms of immunity.

History & impact of immunology on human health 1700 1900 1800 2000 Jenner Vaccination 1600 Jansen Microscope Müller Bacteria Koch’s Postulates Metchnikoff Phagocytosis Wright Antisera Kohler & Milstein Monoclonal Abs 1955 Miller T cells 1965 1970 1975 1980 Countries with more than one smallpox case per month 30 15 200 years after Jenner WHO announce smallpox eradicated

Why study immunology now? Infectious diseases Mechanisms of pathogenicity Vaccine development Diseases caused by a disturbed immune system ALLERGY: Immune responses to innocuous materials e.g. ASTHMA AUTOIMMUNITY: Anti-self immunity e.g. MULTIPLE SCLEROSIS GRAFT REJECTION: Immune responses to TRANSPLANTED TISSUE IMMUNODEFICIENCY: Defects in immune responses e.g. SCID Manipulation of immunity to treat disease IMMUNOSUPPRESSION: Treatment of immune diseases IMMUNOREGULATION: Immunotherapeutic interventions

Reminder of basic immunological terms ANTIGENS (Ag) are substances recognised by • ANTIBODIES (Immunoglobulin, Ig, Ab) and • T LYMPHOCYTES (T CELLS) Antibodies are made by B LYMPHOCYTES (B CELLS) T cells help B cells make antibodies: T HELPER (Th) cells T cells kill infected cells T CYTOTOXIC (CTL)

Cellular and humoral defences Immune responses Invasion & infection Barriers Innate immunity Adaptive immunity + Inflammation Skin & Mucous membranes rapidly regenerating surfaces, peristaltic movement, mucociliary escalator, vomiting, flow of urine/tears, coughing Cellular and humoral defences lysosyme, sebaceous/mucous secretions, stomach acid, commensal organisms,complement proteins, phagocytosis, NK cells Cellular and humoral defences Antibodies, cytokines, T helper cells, cytotoxic T cells

Immunity established to adapt to infection Adaptive immunity Immunity established to adapt to infection • Learnt by experience • Confers pathogen-specific immunity • Enhanced by second exposure • Has memory • Uses cellular and humoral components • Is poorly effective without innate immunity Antibodies reflect infections to which an individual has been exposed- diagnostic for infection

Innate immune response Inbuilt immunity to resist infection • Present from birth • Not antigen-specific • Not enhanced by second exposure • Has no memory • Uses cellular and humoral components • Is poorly effective without adaptive immunity Also involved in the triggering and amplification of adaptive immune responses

Leucocytes Adaptive and innate immunity depends upon LEUCOCYTES Innate immunity is mediated largely by GRANULOCYTES Adaptive immunity mediated by LYMPHOCYTES The growth, development and activities of granulocytes and lymphocytes are interconnected and often co-operative.

Cells Of The Immune System Lymphocyte Adaptive immunity Macrophage Monocyte Neutrophil PMN Eosinophil Basophil Mast cell Phagocytosis Ag presentation Phagocytic Anti-bacterial Anti-parasite immunity ?Protection of mucosal surfaces? Protection of mucosal surfaces Common lymphoid progenitor Myeloid Pluripotent haemopoietic stem cell

CLP T B Th CTL PC Lymphocyte subsets Common lymphoid precursor T CELLS B CELLS Activate B cells and macrophages T HELPER CELLS Th Kill virus- infected cells CYTOTOXIC T LYMPHOCYTES CTL Produce antibodies PLASMA CELLS PC

Cytotoxic T- lymphocyte killing target Look for some excellent low power images and electron micrographs of the cells at the following site: http://www-medlib.med.utah.edu/WebPath/webpath.html Resting Lymphocyte Activated Lymphocyte Plasma cell T and B cells are morphologically identical Movie: Cytotoxic T- lymphocyte killing target (click on this link)

Human macrophage ingesting Candida albicans Look for some excellent low power images and electron micrographs of the cells at the following site: http://www-medlib.med.utah.edu/WebPath/webpath.html Erythrocyte (Red blood cell) Blood monocyte Platelet (thrombocyte) Tissue macrophage Movie: Human macrophage ingesting Candida albicans (click on this link)

Chemotaxis of human neutrophils Look for some excellent low power images and electron micrographs of the cells at the following site: http://www-medlib.med.utah.edu/WebPath/webpath.html Neutrophil Movie: Chemotaxis of human neutrophils (click on this link)

Look for some excellent low power images and electron micrographs of the cells at the following site: http://www-medlib.med.utah.edu/WebPath/webpath.html Eosinophil Basophil Neutrophil Lymphocyte Monocyte

Lyc B T Lymphocyte antigen receptors Until the 1960’s, lymphocytes had no known function. T and B cells are essentially inactive until they encounter antigen. T and B cells express ANTIGEN RECEPTORS Lyc B The B cell antigen receptor is a membrane-bound antibody SURFACE IMMUNOGLOBULIN T The T cell antigen receptor IS NOT membrane bound antibody but a distinct molecule T CELL ANTIGEN RECEPTOR Each antigen receptor binds to a different antigen Each cell has only one antigen specificity

Lymphoid organs Organised tissue in which lymphocytes interact with non lymphoid cells Sites of maturation & initiation of adaptive immune responses CENTRAL LYMPHOID ORGANS PERIPHERAL LYMPHOID ORGANS Central lymphoid organs: THYMUS – T cell maturation BONE MARROW – B cell maturation Peripheral lymphoid organs: LYMPH NODES SPLEEN WHITE PULP MUCOSAL-ASSOCIATED LYMPHOID TISSUE T and B cell activation Antigen trapping

Lymph node 4. Germinal centre (site of intense B cell proliferation) 5. Medullary cords (Macrophage & plasma cell area) 3. Secondary lymphoid follicle 6. Efferent lymphatic vessel 2. Primary Lymphoid follicle (B cell area) Artery Paracortical (T cell) area Vein 1. Afferent lymphatic vessel. Lymph, cells & Ag drained from tissues enters here Medullary sinus

Look for an excellent image of a sectioned lymph node at the following site: http://www-medlib.med.utah.edu/WebPath/webpath.html

Look for an excellent image of a germinal centre at the following site: http://www-medlib.med.utah.edu/WebPath/webpath.html

Spleen white pulp Transverse section Marginal sinus B cell corona Red pulp Germinal centre Marginal zone Periarteriolar lymphocytic sheath (PALS) – T cell area Central arteriole

Look for an excellent image of a sectioned spleen at the following site: http://www-medlib.med.utah.edu/WebPath/webpath.html

Lymphocyte recirculation NAIVE LYMPHOCYTES enter blood, are seeded to the peripheral lymphoid organs and recirculate Cells & antigens from a site of infection are trapped in draining lymphoid tissue. Cells proliferate and re-enter the RECIRCULATING LYMPHOCYTE POOL to re-seed the peripheral lymphoid organs

Look for an excellent images of Wuchereria bancrofti and elephantiasis at the following site: http://www-medlib.med.utah.edu/WebPath/webpath.html

How immune cells communicate: SOLUBLE MEDIATORS Infection Phagocyte activation CYTOKINES & CHEMOKINES Diverse collection of soluble proteins made by cells that affect the behaviour of other cells. The balance & level of cytokines and chemokines secreted affects the outcome of the response INFLAMMATION Early events involve endothelial cells and result in the accumulation of fluid, plasma proteins & leucocytes. Later events involve the activation and maturation of lymphocytes and other granulocytes.

Bio-assay of cytokines in vitro Activation of M in vitro Test for effect on other cells Cytokine secretion +/- Remove cytokine containing supernatant Which cytokine?

Specificity of cytokine bioassays Test for a characteristic effect on other cells e.g. interleukin-1 Induces proliferation in thymocytes Include an antibody that blocks interleukin-1 + + IL-1 absent - IL-1 present

How immune cells communicate: CELL-CELL CONTACT Peripheral lymphoid tissues trap antigen-containing phagocytic cells and concentrate cells together to promote cell-cell contact. Cell-cell contact occurs at many stages of immune responses. T CTL Target cell Killing T Accessory cell activation Antigen presentation B Y Ab production

Cell surface molecules mediate cell-cell contact Resting cells Activated cells Expression and level of expression controls cell-cell adhesion Activation can induce expression. Cell adhesion, migration, antigen specificity, antigen presentation, costimulation, helper function, effector function. Cell surface molecules influenced by activation include cytokine receptors.

Bio-assay of cell cell contact requirements in vitro Physically separate cells with permeable membrane T Include a blocking anti-MHC molecule antibody + Due to cytokine or cell-cell contact? T T + MHC molecules not important T - Not due to a cytokine Which cell surface molecule? - MHC molecules important

Clonal nature of the adaptive immune response Each lymphocyte expresses a single antigen receptor specificity. There are millions of lymphocytes in the body, and thus millions of different antigen receptors. Each naive lymphocyte bearing a unique receptor is the progenitor of a genetically identical CLONE of daughter cells. PROBLEM: The CLONAL DISTRIBUTION of antigen receptors means that lymphocytes of a particular specificity will be too infrequent to mount an effective response. A process akin to natural selection, CLONAL SELECTION raises the clonal frequency of cells with a particular antigen specificity

Clonal selection theory: MacFarlane Burnet 1957 Daughter cells bear identical antigen specificity to the parent cell. Each lymphocyte bears a single type of receptor of unique specificity. Antigen interaction leads to lymphocyte activation.

Clonal selection induces proliferation and increases effector cell frequency No. of cell divisions No. of cells with useful specificity Threshold of protective effector function

Clonal nature of adaptive immune response allows for removal of harmful cells Opportunity to remove harmful specificity at an early stage of development IMMUNOLOGICAL TOLERANCE !!!!Cells specific for self antigen!!!! Antigen receptors recognising self antigens can be individually purged from the antigen receptor REPERTOIRE before clonal expansion

Clonal nature of adaptive immune response allows for immunological memory 4 16 12 8 20 64 68 72 Antibody titre Days 2° response to antigen A 1° response to antigen B Lymphocyte proliferation to Ag B Lymphocyte proliferation to Ag A 1° response to antigen A Lymphocyte apoptosis A A B

Immune effector mechanisms against extracellular pathogens & toxins NEUTRALISATION Y ` Y ` Y ` Y ` Adhesion to host cells blocked Prevents invasion Toxin release blocked Prevents toxicity NEUTRALISING ANTIBODIES

Effector mechanisms against extracellular pathogens OPSONISATION Bacteria in extracellular space Ab + Fc receptor Phagocytosis OPSONISATION binding

Effector mechanisms against extracellular pathogens COMPLEMENT Bacteria in plasma Lysis binding Ab & COMPLEMENT + Phagocytosis Complement & Fc receptor Opsonisation

Effector mechanisms against intracellular pathogens CYTOXICITY CTL CTL CTL Target cell death Viral infection Lethal hit

Th Th Effector mechanisms against intracellular bacteria MACROPHAGE ACTIVATION Inflammatory T cell Th Cytokines Th Activation of killing mechanisms Activated macrophage Resting Macrophage

NOW TRY THE MULTIPLE CHOICE QUESTIONS Summary:  Reminder of 2nd year immunology  Characteristics and components of adaptive and innate immunity  Peripheral lymphoid organs & lymphocyte recirculation  Intercellular communication by cytokines and cell-cell contact  Clonal selection: Ag recognition, self tolerance and memory Effector mechanisms NOW TRY THE MULTIPLE CHOICE QUESTIONS (click on this link)