Sca-1-positive cells can reconstitute a 950 rad (9.5 Gy)-irradiated mouse instead of more than 10,000 unpurified BM cells.

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1 Immunology Dr. John J. Haddad Cells and Organs of the Immune System Chapter 2 Human hematopoiesis The process begins in yolk sac of embryo is 1 st week. In month 3, stem cells migrate to fetal liver and then spleen. During months 3-7, both organs play major role. By birth, BM is site of hematopoiesis. Most of the cells involved in the immune system are derived as part of hematopoiesis. The key cell in this process is the hematopoietic stem cell (HSC). Less than 1 HSC/5 x 10 4 bone marrow cells Sca-1-positive in mouse CD34-positive in humans Sca-1: This murine cell surface antigen is called also Ly-6A/E. It is the murine homologue of human CD34 expressed on immature hematopoietic progenitor cells Sca-1-positive cells can reconstitute a 950 rad (9.5 Gy)-irradiated mouse instead of more than 10,000 unpurified BM cells. Ability to purify HSCs is a major benefit in bone marrow transplantation. Purify stem cells away from cells that can give GVH (graft-versus-host) disease. It improves chance of success with unrelated donor. Also, possibility for gene therapy exists with biotechnology. HSCs are self-renewing, pluripotent cells. They give rise to progenitors that give rise to lymphoid and myeloid series. Figure 2.1. HSCs require stromal cells to grow. Stromal cells give rise to colonies in spleen of irradiated mouse or in culture with correct microenvironment. Stromal cells secrete necessary colony-stimulating factors. Steady state average human makes about 3.7 x cells/day by hematopoiesis. In a normal adult human s blood: Cell type cells/cubic mm % Total/adult Life RBCs 5.0 x 10 6 Platelets 2.5 x 10 5 Leukocytes (WBCs) 7.3 x 10 3 Neutrophils x day Lymphocytes Monocytes 1-6 Eosinophils 1-3 Basophils <1 Many growth factors exist. The direction of differentiation in any one area depends on: The factors that are being secreted by stroma The factors secreted by the cells themselves The receptors expressed on the cells Apoptotic signals 4

2 Apoptosis is one of two major forms of cell death. Necrosis resulting from cell injury involves: - Clumping of chromatin - Swollen organelles - Disintegration of cell and release of cell contents - Inflammation and cleanup by macrophages Apoptosis involves: - Chromosome compaction and nuclear fragmentation - DNA breakdown - Blebbing of membrane to give apoptotic bodies - Phagocytosis and destruction Apoptosis involves turning on genes that activate an internal program of active cell destruction. Other genes can be activated that oppose death by apoptosis. For example: Bcl-2, Bcl-X L oppose cell death Bax, BCL-X S promote cell death Which genes get turned on depend upon the cytokines in the area and the receptors expressed on the cells. Lets talk now about Lymphoid cells. Descend from lymphoid stem cell, the T or B lineage Resting cell about 6 microns in diameter. Mostly nucleus Blast cell (T or B) about 15 microns in diameter Similar size for plasma cell that makes antibodies Blasts have much more cytoplasm and synthetic activity Null cells are neither T nor B cells. Include NK cells Under microscope (even EM), T and B cells look alike, but they have very different functional capabilities and express very different functional molecules. Many of these are on the cell surface, and are referred to as CD (cluster of designation/differentiation) antigens. Cluster of differentiation is defined using antibodies produced by immunization with cells. A unique feature of the immune system can use it to generate reagents useful for studying the system itself. Examples are shown in book. There are probably > 200 by now. B lymphocytes the series of cells that will actually synthesize antibodies. They have a number of surface molecules that distinguish them, but the most important one is surface immunoglobulin, approximately 1.5 x 10 5 per resting B cell. As we shall see, B cells are generated in the bone marrow of an adult, and seed the rest of the body. T lymphocytes the series responsible for cellular immunity and also for providing the second opinion needed before B cells leave the resting state and become blasts, eventually to secrete antibody. They also have a characteristic set of surface molecules, most important of which is the αβ-t cell receptor (TCR). This determines what antigen they recognize. Particularly important additional surface molecules that distinguish mature T cells are CD8 (on cytotoxic T cells, CTLs) and CD4 (on helper T cells and cells that mediate delayed type hypersensitivity, DTH, e.g. poison ivy). 5

3 We ll have a lot more to say about these and other cell surface molecules later. Now let s talk about myeloid cells. These descend from the myeloid stem cell that gives rise to a number of progenitors. Monocytic cells Monocytes these circulate in the blood and give rise to macrophages when they leave blood stream and enter tissues. Differentiation to tissue macrophage involves a great increase in size and elaboration of factors that help them deal with organisms or other antigens that they phagocytose. Alveolar macrophages lung Histiocytes connective tissue Kupffer cells liver Mesangial cells kidney Microglial cells brain Osteoclasts bone These cells can move towards substances generated by tissues that are damaged or invaded or elaborated by other cells of the immune system chemotaxis. They surround an antigen (e.g. bacterium), take it into a phagosome, fuse lysosomes to it, and digest the bacterium to molecular pieces. Some of these they excrete others they keep to put on display for processes we will describe later under the guise of antigen processing and presentation. Macrophages and other cells of the myeloid series can destroy microbes by a variety of mechanisms that utilize oxygen-dependent killing mechanisms. Reactive oxygen intermediates/species (ROS) as well as reactive nitrogen intermediates. Radicals are generated by enzymatic machinery in the cells. Some microorganisms (e.g. Mycobacterium tuberculosis) are able to live inside the macrophages. They pose a special problem to the immune system one solved in part by delayed type hypersensitivity. Granulocytic cells These myeloid cells contain obvious staining granules that are mobilized to attack and destroy bacteria and foreign antigens. Neutrophils Their granules stain with both acid and basic dyes. They are generally the first cells to arrive at site of inflammation. They extravasate from the blood vessels in response to chemotactic factors. They phagocytose antigen, but instead of introducing lytic enzymes via lysosomes, they store them in granules. Also kill by reactive oxygen and nitrogen intermediates, and show a respiratory burst when activated. Eosinophils These are stained with the acid dye, eosin red. They also phagocytose main function appears to be against parasites. Basophils These stain with the basic dye, methylene blue. They do not phagocytose, and they function in immediate hypersensitivity response allergy. The granules contain histamine and other potent mediators. 6

4 Mast cells These are non-motile cells that populate various tissues of the body. Like basophils, they play a role in allergy by releasing histamine and other pharmacologically active compounds from granules. Dendritic cells Their name is reminiscent of dendrites of neurons long processes extending from cells. These are the best presenters of antigen of all. Langerhans cells epidermis and mucus membranes Interstitial dendritic cells heart, lungs, liver, kidney, GI tract Interdigitating dendritic cells thymus medulla, T cell areas of 2 o lymphoid The first two types of cells capture antigen and then migrate ( veiled dendrite-like processes disappear) via lymph and/or circulation to 2 o lymphoid tissue where adaptive immune response is initiated. They constitute 0.1% of blood WBCs. Some dendritic cells express one chain of the CD8 surface molecule that characterizes one class of T lymphocytes (CTLs). Others are CD8-negative. It had been thought that CD8-positive cells came from the lymphoid lineage and CD8-negative ones from the myeloid lineage. A recent paper from Irv Weissman s lab at Stanford (Science 290:2152-4, 2000) shows that both types can arise from a common myeloid progenitor. Different functions attributed to these phenotypically distinct dendritic cell subsets probably reflects the tissue microenvironments in which they matured. While they are critical participants in antigen stimulation of T cells, dendritic cells also appear to play a significant role in HIV-1 infection of T cells. (Geijtenbeek et al., Cell 100: , 2000) They have a unique surface molecule called DC-SIGN that helps support the primary immune response. This protein also binds gp120, the envelope protein of HIV-1. It appears that dendritic cells that encounter HIV-1 (e.g., in the mucosa), transport it to T cell-rich areas in 2 o lymphoid tissues. There, after interaction of CD40L on CD4-positive T cells with CD40 on the dendritic cell, HIV-1 undergoes one cycle of replication in the dendritic cell, followed by future replication cycles in the T cells. The virus is exploiting the intimate interaction of dendritic cells and T cells to gain access to the T cell compartment. Since dendritic cells are the best APCs that we know of, scientists are trying to exploit them to heighten immune responses against difficult-to-immunize antigens like cancer cells. Two approaches show hope: 1) TRANCE (for tumor necrosis factor-related activation-induced cytokine) is a newly discovered cytokine made by T cells that reacts with a receptor on dendritic cells and increases their longevity and abundance in areas of 2 o lymphoid tissue. Ex vivo treatment of DCs with TRANCE and antigen followed by delivery in vivo enhances responses. (Josien et al. J. Exp. Med. 191: (2000). 2) Dendritic cells can take up antigens from both apoptotic and necrotic tumor cells. They can present antigens from apoptotic cells well, but they require for maturation a signal from necrotic cells or supernatants from necrotic cells. This knowledge may be used to better use DCs to deliver antigens from tumor cells to stimulate immunity. (Sauter et al. J. Exp. Med. 191: , 2000) A final kind of dendritic cell is referred to as a follicular dendritic cell. These cells are located in the B-cell rich areas of the lymph nodes. They are MHC class II-positive (i.e., specialized for antigen presentation), and they have receptors for antibodies and complement components. They are thought to present antigen/antibody complexes over a very long period (years). Follicular dendritic cells have a beaded appearance, and it is thought that they may contribute to long term immunological memory by providing a stimulus long after the antigenic threat has disappeared. 7

5 Organs of the Immune System The primary lymphoid organs are the bone marrow and the thymus. The secondary lymphoid organs include: lymph nodes spleen mucosa-associated lymphoid tissue (MALT) Tertiary lymphoid tissue consists of areas that generally have few lymphoid cells but which can import them during a local inflammatory response for example, cutaneous-associated lymphoid tissue (CALT). Mature lymphocytes are generated in the primary lymphoid tissues, and they circulate in the blood and lymphatic system. The bone marrow is the major site of hematopoiesis in the adult. The B cells develop under the influence of stromal cells in the marrow. The progenitors of T cells arise in the bone marrow, and then migrate to the thymus where they undergo a complex differentiation program. They enter the thymus cortex, and complete their development in the thymic medulla. In the thymus the pro-t cells encounter at least two kinds of three-dimensional networks of thymic stromal cells that include: epithelial cells (nurse cells) interdigitating dendritic cells macrophages Factors secreted by the epithelial cells in the cortex induce appearance of characteristic T cell surface molecules (e.g., CD4, CD8). Cytokines like IL-7 stimulate growth and division of thymic lymphocytes (or thymocytes). Each cell is stimulated to express a T cell receptor (TCR), and through processes called positive and negative selection, the cells that will make up our immune systems library of T lymphocytes are selected (Chapter 10). The thymus is large in infants (about 70 gm) and decreases in size and activity with age (about 3 gm in an old person) (it involutes). However, it remains active in producing T cells throughout life. Patients who lack a thymus due to genetic mutations (DiGeorge s Syndrome in humans, nude mice) lack a T cell system and are severely immuno-compromised. Lymphatic system As the heart pumps blood around the body, some fluid leaves the vessels (between endothelial cells) and becomes interstitial fluid in the tissue spaces. Our normal muscular contraction causes this interstitial fluid and anything in it to move into a system of tiny open capillaries that lead to larger and larger lymphatic vessels. These have one-way valves to prevent back-flow. Afferent lymphatics carry things to lymph nodes or other 2 o lymphoid tissues such as lymph nodes. They act as filters for the lymph. Things that exit lymph nodes do so through efferent lymphatics. Gradually they reach larger and larger lymphatics that return the lymph and anything in it to the circulation via the right subclavian vein. 8

6 Lower body thoracic duct Upper body right lymphatic duct Antigens that penetrate the outer mechanical layers of the body and enter the tissues are brought via the lymph to 2 o lymphoid organs like the lymph nodes where they encounter T-cell and B-cell-rich areas. Organization of T- cell rich and B-cell rich areas and antigen-presenting cells is such that antigen has a good chance of encountering lymphocytes with receptors that recognize it. Immune response ensues. Lymphocytes from the blood enter lymph nodes through specialized post-capillary venules called high endothelial venules (HEV). Special receptors are involved a process to be discussed later. Products of immune response leave lymph node by way of efferent lymphatic. Antigens that get into the blood stream are brought to the spleen via the splenic artery, which acts a filter for the blood. The spleen has two major zones: red pulp, where defective red blood cells are destroyed, and white pulp, which surrounds branches of the splenic artery. This periarteriolar sheath (PALS) is rich in T cells. Antigens (and circulating lymphocytes) are deposited in the diffuse marginal zone, an area rich in B cells that separates red and white pulp, and they are brought to the PALS. Immune response ensues with development of primary and (with re-stimulation) secondary follicles in the marginal zone. Products of immune response leave spleen via the blood. Splenectomy in children can lead to increased incidence of bacterial sepsis. In adults, some increase in blood-borne bacterial infections. Mucosa-associated lymphoid tissues (MALT) Respiratory, gastrointestinal (GI) and urogenital tracts have mucosal lining. These are major sites for pathogen entry, and there is much 2 o lymphoid tissue associated with these sites. The GI tract is a major site with 3 regions: 1) Mucosal epithelial layer adjacent to lumen. Contains intraepithelial lymphocytes (IELs) which are poorly understood in that they have an alternative type of T cell receptor (γδtcr). 2) The lamina propria which contains many B cells, plasma cells, activated T H cells and macrophages in loose clusters 3) The submucosal layer which contains organized follicles called Peyer s patches. So-called M cells in the outer mucosal layer transport Ag from the lumen via endocytosis across the cell into a large basolateral pocket filled with B and T cells and macrophages. The M cells overlay organized lymphoid follicles in the lamina propria. Activated B cells mature into plasma cells and secrete a specialized type of antibody called IgA that is transported across the epithelial cells and is secreted into the lumen. Many grams are secreted each day. Some pathogens exploit the M cells to enter the body, since they cannot penetrate the physical barrier set by the tight junction between mucosal epithelial cells. Vibrio cholerae Some Salmonella species Polio virus 9

7 Cutaneous-associated lymphoid tissue (CALT) The outer epidermal layer is made up of epithelial cells called keratinocytes. They secrete cytokines in response to penetration that non-specifically can promote a local inflammatory response. They can also be induced to express class II, a MHC-encoded surface protein that, we will learn, is specific to antigen presenting cells. Thus keratinocytes may function as APCs. Langerhans cells (resident macrophages in epidermis) can pick up the antigen, become veiled (I.e., lose their processes), and transport it via lymphatic drainage to draining lymph nodes. There they present antigen to T cells. The epidermis also contains interepithelial lymphocytes that are mostly CD8-positive and have γδtcrs. Much as the case in the IELs in the gut mucosa, their function is poorly understood. They may be specialized receptors for pathogens that enter through these portals. In the dermis (deeper), there are macrophages and both CD4- and CD8-positive T cells in the dermis (deeper) that contain γδtcrs. These T cells have properties that suggest they were previously activated, and they may be memory cells. 10