SOPHOMORE DENTAL/OPTOMETRY MICROBIOLOGY SECTION: IMMUNOLOGY CELLS AND ORGANS OF THE IMMUNE SYSTEM

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1 SOPHOMORE DENTAL/OPTOMETRY MICROBIOLOGY SECTION: IMMUNOLOGY CELLS AND ORGANS OF THE IMMUNE SYSTEM Lecturer: Dr. John F Kearney jfk@uab.edu Objectives: To learn: 1) the basic cell types that make up the immune system; 2) the important surface markers used to distinguish these cell types; and 3) their general function in an immune response. To become familiar with the organs of the immune system and their role in immune cell development and the immune response. Reading Assignment: Immunology 6 th Edition (Goldsby, Kindt, Osborne and Kuby) Chapter 2 - Cells and Organs of the Immune System I. HEMATOPOIESIS Hematopoietic Stem Cells (HSC): self-renewing stable numbers are maintained throughout adult life pluripotent: able to differentiate into red and white blood cells including erythrocytes, granulocytes, monocytes, lymphocytes and megakaryocytes Hematopoiesis: begins in embryonic yolk sac during first week at 3 months yolk-sac stem cells migrate to fetal liver and then to spleen 3-7 months hematopoiesis occurs in fetal liver and spleen >7 months stem cells migrate to bone marrow and this serves as the primary site of hematopoiesis. at birth little or no hematopoiesis occurs in liver or spleen Generation of Immune Cells from Pluripotent HSC: The Pluripotent HSC differentiates into either a Myeloid or a Lymphoid Lineage Progenitor cell. These progenitor cells have lost the capacity for self-renewal.

2 The common Myeloid Progenitor differentiates into erythrocyte precursors and platelets, as well as and white blood cells including neutrophils, eosinophils, basophils, and monocytes. The common Lymphoid Progenitor differentiates into B cells, T cells, and NK cells. Progenitor Commitment depends on: Acquisition of responsiveness to Cytokines and/or Growth Factors Stromal Cells which provide a Hematopoieticinducing Microenvironment consisting of cellular matrix and either membrane-bound or diffusible growth factors Hematopoiesis is Regulated: Hematopoiesis maintains a steady state in which the production of mature blood cells = their loss (primarily due to aging) Maintenance of steady state requires production of 3.7 x white blood cells per day Factors that Control Hematopoiesis: Control of the levels and types of cytokines produced by bone-marrow stromal cells The production of cytokines with hematopoietic activity by other cell types, such as activated T cells or macrophages The regulation of the expression of receptors for hematopoietically active cytokines in stem cells and progenitor cells The removal of some cells by the controlled induction of cell death II. CELLS OF THE IMMUNE SYSTEM Cluster of Differentiation (CD): Different lineages or maturational stages of lymphocytes (and other white blood cells) can be distinguished by the expression of membrane molecules that are recognized by monoclonal antibodies (mab). All of the mabs that react with a particular membrane molecule are grouped together as a cluster of differentiation (CD). New mabs are analyzed to determine if they fall into existing CDs and if not they are assigned a new CD designation.

3 Granulocytic Cells Granulocytes are classified as neutrophils, eosinophils or basophils based on cellular morphology and cytoplasmic staining characteristics Neutrophils: Multilobed nucleus and a granulated cytoplasm that stains with both acidic and basic dyes often called a polymorphonuclear leukocyte (PMN) for its multilobed nucleus Produced by hematopoiesis in the bone marrow and are released into the blood where they circulate briefly before migrating into tissues where they survive for a few days Constitute 50-70% of circulating white blood cells Generally first cells to accumulate at sites of inflammation in response to chemotactic factors including: complement components components of blood-clotting system cytokines secreted by T cells or macrophages Neutrophils are active phagocytic cells that contain primary and secondary granules that mediate elimination of ingested microbes Neutrophils also generate anti-microbial substances Eosinophils: Bilobed nucleus and a granulated cytoplasm that stains with the acid dye eosin red Constitute 1-3% of circulating white blood cells Motile phagocytic cells that migrate from the blood to tissue spaces Thought to play a role in the defense against parasitic infection Basophils: Lobed nucleus and heavily granulated cytoplasm that stains with the basic dye methylene blue Constitute <1% of circulating white blood cells Nonphagocytic granulocytes that function by releasing pharmacologically active substances from cytoplasmic granules Basophils express both high affinity (FcεRI) and low affinity (FcεRII, CD23) Fc receptors for IgE Cross-linking of IgE Fc receptors on basophils promotes degranulation of basophils and the release of mediators involved in Type I hypersensitivity

4 Mast Cells Formed in the bone marrow by hematopoiesis and released into the blood as undifferentiated cells after which they enter tissues and differentiate Found in wide range of tissues Contain cytoplasmic granules like basophils that contain histamine and other pharmacologically active substances Mast cells express both high affinity (FcεRI) and low affinity (FcεRII, CD23) Fc receptors for IgE Cross-linking of IgE Fc receptors on mast cells promotes degranulation and the release of mediators involved in Type I hypersensitivity Mononuclear Cells Mononuclear phagocytic system consists of monocytes and macrophages In the bone marrow granulocyte-monocyte progenitor cells differentiate into promonocytes that enter the blood where they further differentiate into monocytes Monocytes circulate in the blood and then migrate into different tissues where they differentiate into tissue-specific macrophages (Mφ) Aveolar Mφ in the lung Histiocytes in connective tissue Kupffer cells in liver Mesangial cells in the kidney Microglial cells in the brain Osteoclasts in bone Differentiation into tissue macrophages is associated with: cell enlargement increased number and complexity of organelles increased phagocytic activity production of hydrolytic enzymes secretion of soluble factors Macrophages can be activated by inflammatory and T cell-derived cytokines (e.g. IFN-γ) Activated macrophages are more effective than resting cells at eliminating pathogens because they exhibit increased: phagocytic activity ability to kill ingested microbes Oxygen-dependent killing RO intermediates, RN intermediates Oxygen-independent killing Defensins, Lysozyme, Hydrolytic enzymes

5 secretion of inflammatory mediators IL-1, TNFα, IL-6, Complement components ability to activate T helper cells through the presentation of antigen in the context of MHC Class II molcules Natural Killer Cells (NK or Null Cells): Surface Markers - CD2: receptor that binds the adhesion molecule LFA-3 CD16: the FcγIII receptor that recognizes the Fc portion of IgG and is involved in mediating Antibody Dependent Cell-Mediated Cytotoxicity (ADCC) CD28: receptor for the co-stimulatory B7 family molecules expressed on antigen-presenting cells NKR-P1: Activating receptor known as a C-type lectin that binds to carbohydrate NKp30, 44, 46: Activating receptors that promote NK cell killing by binding to ligands on target cells may be pattern recognition receptors CD94/NKG2: C-type lectin inhibitory receptor that binds to HLA-E, which is only expressed when bound to a peptide derived from HLA-A, B, or C KIR: Killer inhibitory receptors bind to MHC class I molecules Immune Function Cells derived from bone marrow that share a common lineage with T cells but do not express TCR or CD3 on their surface NK cells kill tumor cells and virus-infected cells by a process similar to that used by CTL important first line of defense against virus-infected cells prior to generation of CTL s NK cells are constitutively cytotoxic and possess large granules in their cytoplasm that contain granzymes and perforin NK cells can also mediate killing of antibody-coated target cells via CD16 in a process referred to as ADCC NK cell-mediated killing is regulated by the balance of signaling via activating (NKR-P1, NKp30, 44, 46, CD16, CD2) and inhibitory (CD94/NKG2, KIRs) receptors. In general, NK cells kill host cells that lack MHC class I due to viral infection of transformation. Lymphoid Cells % of white blood cells

6 99% of cells in the lymph Approximately 1 x white cells in human body Continually migrate through the blood and lymph and have the ability to migrate into tissue spaces and lymphoid organs Divided into three populations T lymphocytes, B lymphocytes and Natural Killer cells (NK cells) Each T and B cell expresses a unique receptor on its surface that is specific for antigen on the T cell this is referred to as the T cell antigen receptor or TCR, and on the B cell the BCR or membrane immunoglobulin. Resting B and T cells - have not encountered antigen via their respective antigen receptor, i.e. naïve cells small, motile, nonphagocytic cells that can not be distinguished cells are in G 0 phase of the cell cycle ~6 µm; cytoplasmic rim barely visible; densely packed chromatin, few mitochondria short life span Activated B and T cells have encountered antigen and cytokines enter cell cycle and begin to proliferate form lymphoblast cells, ~15 µm; higher cytoplasm:nucleus ratio; more organellar complexity Lymphoblasts differentiate into Effector Cells or Memory Cells Plasma Cells = effector cell of the B lymphocyte these cells secrete soluble antibody and mediate the humoral immune response Effector cells of the T lymphocyte include cytokine-secreting T helper cells (T H cell) and the cytotoxic T cell (CTL) which is important for cell-mediated immunity. B Lymphocytes: Surface Markers Membrane Immunoglobulin (BCR): 2 heavy and 2 light chain polypeptides that are disufide bonded to one another to form an intact antigen receptor. B220: a transmembrane protein tyrosine phosphatase that regulates signal transduction via the BCR MHC Class II Molecules: involved in presentation of antigen to T cells CR1 (CD35 and CR2 (CD21): receptors for certain complement products FcγRII (CD32): a receptor for Fc portion of IgG, an isotype of antibody CD22: a B cell receptor involved in regulation of BCR signaling

7 B7-1 (CD80) and B7-2 (CD86): molecules that interact with CD28 and CTLA-4, important regulatory molecules on the surface of T cells, including TH cells CD40: a molecule that interacts with CD40 ligand on helper T cells this interaction is critical for the survival of antigen-stimulated B cells and for development into antibody-secreting plasma cells or memory B cells Immune Function B cells specifically recognize antigen by virtue of expressing unique antigen receptors on their surface membrane immunoglobulin (i.e. the BCR) Binding of antigen to the BCR leads to cell activation and entry into the cell cycle Additional help from T cells in the form of soluble cytokines and cognate cell:cell contact promote clonal expansion of antigen-specific B cells and differentiation into antibody-secreting plasma cells T Lymphocytes: Surface Markers T Cell Antigen Receptor (TCR): comprised of a and b chain polypeptides that are disulfide-linked to form an intact antigen binding receptor that recognizes peptides in the context of either MHC Class I or MHC Class II CD3: a complex of polypeptides associated with the TCR that transduce signals upon TCR cross-linking CD4: a co-receptor on MHC class II-restricted T cells that binds to nonpolymorphic regions of MHC Class II CD8: a co-receptor on MHC Class I-restricted T cells that binds to nonpolymorphic regions of MHC Class I CD45: a transmembrane protein tyrosine phosphatase that is required for signal transduction via the TCR CD28: a receptor for the co-stimulatory B7 family of molecules present on B cells and other antigen-presenting cells Immune Function The TCR recognizes antigen that is bound to either MHC Class I or II molecules depending on whether the T cell expresses CD8 or CD4, respectively CD4+ T cells generally function as helper T cells whereas CD8+ cells generally

8 function as cytotoxic T cells the ratio of helper T to cytotoxic T cells can be evaluated by examining the ratio of CD4 versus CD8 expression Activation of helper T cells in response to antigen leads to proliferation and the secretion of cytokines that promote activation of B cells and other T cells Helper T cells can be classified as TH1 cells that produce cytokines, which support inflammation or TH2 cells that produce cytokines, which promote B cell activation and the development of immune responses that depend on antibody production Activation of cytotoxic T cells leads to proliferation and differentiation into cells that exhibit cytolytic activity Cytotoxic T cells acquire the ability to recognize and destroy cells that exhibit altered self antigens Dendritic Cells Arise from HSC through the myeloid lineage Dendritic cells are found in numerous tissues and the blood: Langerhans cells: found in the epidermis and mucous membranes Interstitial dendritic cells: populate most organs Interdigitating dendritic cells: present in T-cell areas of secondary lymphoid organs and the thymic medulla Circulating dendritic cells: constitute 0.1% of blood leukocytes Process and present antigen to T cells Constitutively express MHC Class I and II antigens as well as B7 family members (CD80/CD86) therefore dendritic cells are extremely important in antigen presentation to and activation of naïve T cells III. ORGANS OF THE IMMUNE SYSTEM The thymus and bone marrow are the primary (or central) lymphoid organs where maturation of lymphocytes takes place The lymph nodes, spleen, and various mucosal-associated lymphoid tissues (MALT) such as gut-associated lymphoid tissue (GALT) are the secondary (or peripheral) lymphoid organs, which trap antigen and provide sites for mature lymphocytes to interact with antigen Once lymphocytes have been generated in the primary lymphoid organs they circulate in the blood and lymphatic system a network of vessels that collect fluid that has escaped from tissues and capillaries.

9 Primary Lymphoid Organs Thymus: Site of maturation for T cells Bilobed organ separated into lobules by trabeculae each lobule is further organized into two compartments: the outer compartment or cortex which is densely packed with immature T cells called thymocytes the inner compartment or medulla which is sparsely populated with thymocytes that have undergone selection and maturation Cortex and medulla are criss-crossed by three-dimensional stromal-cell network composed of epithelial cells, interdigitating dendritic cells and macrophages these contribute to growth and maturation of thymocytes Thymocyte Development: Thymocytes enter the thymus and multiply rapidly in the cortex Thymocytes rearrange genes that encode TCR and express mature TCR on their surface Expression of TCR leads to two-step selection process in which thymocytes that react with self antigen or fail to see MHC are deleted via programmed cell death Thymocytes that recognize self MHC with foreign antigen are positively selected and allowed to mature Maturation of thymocytes is accompanied by migration from the cortex to the medulla Thymocyte development is also associated with phenotypic changes CD4-CD8- to CD4+CD8+ to CD4+CD8- or CD4-CD8+ Bone Marrow: Location where B cell maturation occurs Cavity within bones contains network of spongy bone partitions called trabeculae that are filled with bone marrow Inner surfaces of bone are lined with cell layer called the endosteum Bone marrow consists of a collagenous framework produced by highly branched stromal cells that support developing blood cells Interconnected blood vessels called sinuses cross bone marrow and empty into central sinus B Cell Development:

10 Stages of B cell development can be distinguished by rearrangement and expression of membrane immunoglobulin genes B cell progenitor cells differentiate into pro-b cells that exhibit rearrangement of heavy chain genes Pro-B cells differentiate into pre-b cells pre-b cells express heavy chain in conjunction with VpreB and λ5 (pre-bcr) and begin to rearrange light chain genes Pre-B cell differentiates into immature B cell that expresses intact BCR consisting of heavy and light chain polypeptides Immature B cells undergo negative selection to eliminate self-reactive B cells B cells that are specific for foreign antigen leave the bone marrow and differentiate into mature B cells that express IgM and IgD Lymphatic System Circulation of blood under pressure causes plasma to seep through the walls of capillaries into surrounding tissue interstitial fluid the fluid that does not return to the blood through the capillaries is considered to be lymph Lymph flows from the connective tissue into a network of lymphatic capillaries and then to a series of progressively larger vessels called lymphatic vessels The largest vessel is the thoracic duct, which empties into the left subclavian vein near the heart Lymph flows in only one direction due to one way valves in the vessels Foreign antigen in the tissue is picked up by the lymph and is carried to organized lymphoid tissues called lymphnodes where it is detected by the immune system Secondary Lymphoid Organs Lymphoid tissue can consist of diffuse collections of lymphocytes and macrophages (e.g. in lung or lamina propria of intestinal wall) other tissue is organized into structures called lymphoid follicles, which consist of aggregates of lymphoid and non-lymphoid cells surrounded by a network of draining lymphatic capillaries Lymph Nodes: Encapsulated bean-shaped structures containing reticular network packed with lymphocytes Located at junctions of lymphatic vessels and are the first organized structures to encounter foreign antigen Antigen in lymph node is detected by phagocytic cells and dendritic cells, it is

11 processed and then presented to T cells Structure: the lymph node is divided into three distinct areas: The Cortex outermost layer contains lymphocytes (mostly B cells), macrophages and folicular dendritic cells in primary follicles (unstimulated) After antigenic challenge the primary follicles enlarge and become secondary follicles each with a germinal center The Paracortex is populated largely by T cells and also contains interdigitating dendritic cells, which express high levels of MHC Class II necessary for presenting antigen to T cells The Medulla is the innermost region and is sparsely populated with lymphoidlineage cells; of those present many are plasma cells actively secreting antibody Transport of antigen: Afferent lymphatic vessels pierce the capsule of lymphnodes and empty lymph into the subcapsular sinus Lymph with antigen percolates from the cortex to medulla so that macrophages and dendritic cells can process and present it to T cells Lymph leaving the node via the efferent vessels is enriched with antibody specific for the antigen and with lymphocytes that have specificity for the antigen Increased numbers of lymphocytes are due to proliferation within the LN and migration of lymphocytes from the blood into the LN Spleen: Large ovoid organ located high in the left abdominal cavity LN are specialized for trapping antigen from the lymph whereas the spleen is important for filtering blood and trapping blood-borne antigens (i.e. systemic versus local infection) Spleen is not supplied by lymphatic vessels so antigens are carried into the spleen via the splenic artery more circulating lymphocytes pass through the spleen daily than all the LNs combined Structure: spleen is surrounded by capsule with extensions (trabeculae) that form compartments the compartment consist of two types: Red pulp consists of a network of sinusoids populated by macrophages and red blood cells White pulp surrounds the branches of the splenic artery forming a periarteriolar lymphoid sheath (PALS) populated mostly by T cells. The marginal zone located peripheral to the PALS is rich in B cells organized into

12 primary follicles Transport of antigen: Blood-borne antigen and lymphocytes enter the spleen via the splenic artery which empties into the marginal zone In the marginal zone antigen is trapped by interdigitating dendritic cells, which carry it to the PALS Initial activation of B and T cells occurs in PALS where IDC present antigen to T helper cells which then activate B cells Activated B cells migrate to primary follicles in marginal zone, which develop into secondary follicles with germinal centers Mucosal-Associated Lymphoid Tissue: Mucous membranes are defended by organized lymphoid tissues called MALT Structurally they range from loosely organized cluster of lymphoid cells to well organized structures such as tonsils, or Peyer s patches in intestinal lining Importance of MALT is attested to by the large number of antibody-secreting plasma cells more than in all other lymphoid organs combined