T LYMPHOCYTES • T cells constitute 70–80% of blood lymphocytes. • Unlike B cells, they do not have microvilli on their surface. • Bear specialized surface receptors called T cell receptors (TCR). T cell receptor • Main function - antigen recognition. • Unlike B cell receptor which binds to antigen directly, TCR does not recognize antigen by itself. • Can only respond to an antigen which is processed and presented by the antigen presenting cells such as macrophages. TCR-CD3 complex • Most T cell receptors (95%) comprise of two chains (α & β) which in turn have three regions: o Extracellular domain o Transmembrane domain o Cytoplasmic tail. • Extracellular domain of each polypeptide chain has 2 regions (variable and constant region). • TCR is active only when both the chains (α & β) complexes with CD3 molecule. T Cell Development • Major events of T cell maturation take place in thymus. • Progenitor T cells are originated from the fetal liver or in adults from the bone marrow and seed the thymus through blood stream. T cell development Types of T cells -Effector T Cells • Two types of effector T cells: o CD4+ helper T cells o CD8+ cytotoxic T cell. Helper T cells (TH) • Possess CD4 molecules as surface receptors. • Recognize the antigenic peptides that are processed by antigen presenting cells and presented along with MHC-II molecules. • Following antigenic stimulus, the helper T cells differentiate into either of the two types of cells- TH1 and TH2 subset. • Each secrete specific cytokines which modulate the cellular and humoral immune responses respectively. Helper T cells (TH) • TH17 cells: o Recently a third subset of T helper cells. o Produces IL-17 & IL-22 and is primarily involved in recruiting neutrophils which in turn kill the microbes as well as induce inflammation. o Contribute to the pathogenesis of many autoimmune inflammatory diseases such as psoriasis, inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis. Cytotoxic T cells (TC) • Possess CD8 molecules • Recognize the intracellular antigens processed by any nucleated cells and presented along with MHC-I. • TC cells are involved in destruction of virus infected cells and tumor cells. Rare subtypes of T cellsRegulatory T cells (TREG cells) • Subpopulation of T cells which regulate the immune system. • Provide tolerance to self-antigens (known as peripheral tolerance), and prevent the development of autoimmune disease. • Surface markers -TREG cells possess surface markers such as CD4, CD25 and Foxp3 (a forkhead family transcription factor) • Deficiency of Foxp3 receptors leads to a severe form of autoimmune disease known as Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome. Rare subtypes of T cellsγδ T cells • Small subset of T cells (5%) that possess a distinct TCR composed of γ & δ chains; instead of α/β chains. • Lack both CD4 and CD8 molecules. • Do not require antigen processing and MHC presentation of peptides. Rare subtypes of T cellsγδ T cells • Part of innate immunity. • Found in the gut mucosa, within a population of lymphocytes known as intraepithelial lymphocytes (IELs). • Encounter the lipid antigens that enter through the intestinal mucosa. B Lymphocytes • Mediators of humoral immunity; constitutes 10-15% of blood lymphocytes. • Named after their site of maturation (bursa of Fabricius in birds and bone marrow in human and other mammals). Development of B Lymphocytes B Lymphocytes • Development in peripheral lymphoid organs: o Immature B cells migrate from bone marrow to peripheral lymphoid organs (lymph node and spleen). o Transform into mature B cells following contact with appropriate antigen. Mature or naive B cells • Most mature B cells (95%) belong to the follicular B cell type and produce surface receptor IgD in addition to IgM. • Play an important role in humoral immune response. • Following antigenic stimulus, the mature B cells transform into activated B cells (lymphoblasts). • Which further differentiate into either effector B cells i.e. plasma cells (majority) or memory B cells. Mature or naive B cells • Plasma cells (antibody secreting cells): o Oval, large (15µm size), with an eccentrically oval nucleus containing large blocks of peripheral chromatin (cartwheel appearance) o Cytoplasm contains abundant organelles. o Short life span of two or three days. Rare mature B cell types • B-1 cells: o Found mostly in the peritoneal cavity. o Coated by surface markers IgM (natural antibodies) and CD5 molecules, but lack IgD. • Marginal-zone B cells: o Present at the edges of lymphoid follicles of spleen. o Produced in response to the polysaccharide antigens. Differences between T cell and B cell Property T-cell Origin Maturation Peripheral blood Bone marrow Thymus 70-80% of total lymphocytes Bone marrow Bone marrow 10-15% of total lymphocytes Antigen recognition receptors T cell receptors complexed with CD3 B cell receptor-Surface IgM or IgD complexed with Igα/Igβ CD markers Thymus specific Ag CD 3,4,8 Present CD19, 21, 24 Absent Microvilli on the surface Absent B-cell Present Natural killer (NK) cells • • • • Large granular lymphocytes. Constitute 10-15% of peripheral blood lymphocytes. Derived from a separate lymphoid lineage. Similar to cytotoxic T cells, NK cells also are involved in destruction of virus infected cells and tumor cells. Macrophage • First described - Russian scientist Metchnikoff (1883) • Monocytes/macrophages originate from bone marrow, from a separate lineage; i.e. from the granulocyte-monocyte progenitor cells. • Macrophages are motile, travel by amoeboid movement throughout the tissues and are called as free or wandering macrophages. • While, some reside in particular tissue, become non-motile and are called fixed macrophages. Macrophage • Monocytes: o Present in blood. o Largest blood cells measuring 12-20µm size. o Do not divide. o Average transit time -8 hours in blood; then they migrate to tissues. Macrophage • Macrophages: o When monocytes migrate to tissues, they transform into macrophages o Macrophages differ from monocytes in the following: 5-10 folds larger than monocytes Contain more lysozymes and cell organelles Produce more lytic enzymes and cytokines Possess greater phagocytic activity Have a longer life in tissues (months to years) Types macrophages Body sites Peripheral blood Tissues Liver Brain Kidney Lungs Bone Inflammation site Connective tissues Placenta Lymphoid follicle Macrophage designation Monocytes Macrophages Kupffer cells Microglial cells Mesangial cells Alveolar macrophages Osteoclasts Epithelioid cells Multinucleated cell (Langhans giant cells) Histiocytes Hofbauer cell Tingible body macrophage Secretory Products of Macrophages Enzymes Lysozyme, acid hydrolases, elastases, Phosphatases, lipases, collagenases Free radicals Reactive oxygen intermediates • Superoxide anion (O•2-) • Hydroxyl radicals (OH•) • Hydrogen peroxide (H2O2) • Hypochlorite anion(ClO-) Reactive nitrogen intermediates• Nitric oxide (NO) • Nitrogen dioxide (NO2) • HNO2(nitrous acid) Cytokines Coagulation factors Interferon α,β Interleukins (IL-1, IL-6, IL-8, IL-12) Tumor necrosis factor- α (TNF- α) Colony stimulating factors (CSF) Platelet derived growth factor (PDGF) Platelet activating factor (PAF) Transforming growth factor β (TGF-β) Factor V,VII,IX,X Prothrombin Complement factors C5, C8 properdin, Factor B, D, I Growth factors Functions of macrophage • Phagocytosis: o Macrophages are the principle cells involved in phagocytosis. o Steps: Functions of macrophage • Antigen presentation: o Promote adaptive immunity, by acting as APCs. Macrophages capture the antigen Process into smaller antigenic peptides Present the antigenic peptides along with the MHC class II molecules to the helper T cells Facilitates helper T cell activation. Functions of macrophage • Activated macrophages: o On exposure to cytokines such as interferon-γ, macrophages become activated  greater phagocytic ability  produce many cytokines  act against intracellular bacteria, virus infected cells and tumor cells. o Also express higher level of MHC class II, hence can act as efficient APCs. Functions of macrophage • Secretory products of macrophages have various biological functionso Interleukin 1 (IL-1):Promotes inflammatory responses, fever and activate helper T cells. o IL-6 & TNF-α:Promote innate immunity, (inflammation & fever) and eliminate the pathogens. o Interferon α & β -have antiviral activity. o TNF-α: Lyse the tumor cells (anti-tumor activity) o Growth factors such as CSF (colony stimulating factor): promote hematopoiesis. o Following tissue injury, various mediators are secreted from macrophage; which help in tissue repair and scar formation. Dendritic cells • Possess long membranous cytoplasmic extensions resembling dendrites of neurons - hence named as dendritic cells. • Originate from bone marrow, but the pathway is uncertain. • Develop as a separate lineage from stem cells or may originate from the macrophage lineage. Types of dendritic cells Types of dendritic cells Site Function -Antigen presentation, Langerhans cells Skin and mucosa Interstitial dendritic cells Organs (lungs, liver, spleen -Express high MHC-II and B7 molecules etc) Thymus Interdigitating dendritic cells Circulating dendritic cells Follicular dendritic cells Blood & lymph Lymph nodes -B cells maturation, & differentiation. -MHC-II and B7 molecules absent -Coated with Ag-Ab complex Functions of dendritic cells • Non-phagocytic in nature. • Most efficient APCs. • Main function is to capture, process and present the antigenic peptides on their cell surface to the helper T cells. • Carry high level of MHC class II and co-stimulatory B7 molecules. • Act as messengers between the innate and the adaptive immune systems. Follicular dendritic cells • Present in lymphoid follicles are an exception in the sense that they recognize antigen-antibody complex rather than antigen alone. • Do not act as APCs • Do not express MHC class II or B7 molecules. Granulocytic Cells • Granulocytes are a category of white blood cells characterized by the presence of granules in their cytoplasm. • E.g. neutrophils, eosinophils and basophils. • Differ from each other by cell morphology and cytoplasmic staining and function. Neutrophils • Possess multilobed nucleus and a granulated cytoplasm that stains with both acid and basic dyes. • Cytoplasm - heavily granular; contains several granules such as myeloperoxidase, lysozyme, defensins, elastase, gelatinase, etc. • Constitute 50%–70% of the circulating WBCs. Neutrophils • Level is greatly increased in presence of infection under the influence of certain cytokines such as IL-8. • Principal phagocytes of innate immunity. • Mechanism of microbial killing: o Both by oxygen dependent and independent mechanisms. Eosinophils • Bilobed nucleus and a granular cytoplasm that stains red with the acid dye eosin. • Phagocytic. • Constitute only 1-3% of total leukocytes. • Number is greatly increased in certain allergic conditions and helminthic infections. • Interleukin-5 is believed to be the eosinophil chemotactic factor. Basophils • Non-phagocytic granulocytes. • Function – secretes contents of granules. • Lobed nucleus and heavily granulated cytoplasm that stains with the basic dye methylene blue. • Resemble mast cells in their function. • Granules are rich in histamine & other mediators that play a major role in certain allergic responses. Mast cells • Present in various body sites such as skin, connective tissues of various organs, and mucosa (respiratory and intestinal). • Contain cytoplasmic granules rich in histamine and other active substances. • Play an important role in the development of certain allergic (type I hypersensitivity) reactions. MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) • Complex is a group of genes. • Coding for a set of host cell surface molecules that bind to peptide fragments derived from pathogens • Display them on the host cell surface for recognition by the appropriate T-cells. • Present in almost all the human cells, but first discovered on the surface of leukocytes; hence in humans, the MHC coded proteins are also called as human leukocyte antigens (HLA). MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) • Serve as a unique identification marker for every individual. • Following transplantation of a graft, the recipient mounts an immune response against the graft’s MHC molecule and vice versa. • The acceptance or rejection of the graft is directly dependent on the MHC molecules of the graft and the recipient. • As the MHC molecules determine the compatibility between the graft and host tissues, they are named as histocompatibility antigens. MHC genes (HLA complex) and their products • Located in short arm of chromosome-6. • HLA complex extends over 4000kbp length covering >100 genes. • Genes are clustered in three regions named as MHC region-I, II and III. Structure of human MHC complex Structure of MHC Class I molecule Structure of MHC Class I molecule • Association of β2 microglobulin with α chain is necessary for the expression of MHC I molecules on to the cell surface. • In Daudi cells (a type of human B cell tumor cell which are not able to produce β2 microglobulin) - observed that they synthesize MHC-I but do not express them on cell surface. Role of MHC class I molecules • Antigen peptide groove of class I MHC molecule (i.e. the site, where the antigen peptide binds) is formed by the cleft between α1 and α2 domains. • α3 domain binds to CD8 molecule present on cytotoxic T cells during antigen presentation. Structure of MHC molecule Differences between MHC class I and MHC class II molecules Present on MHC class I MHC class II All nucleated cells (except sperms) Antigen presenting cells and platelets (APCs) presented to CD8 T cells Nature of peptide antigen Endogenous or intracellular (viral / tumor antigen) Peptide antigen (size) 8-10 amino acid long Peptide antigen is Antigen presentation pathways Peptide-binding site CD4 or CD8 binding site presented to CD4 T cells Exogenous 13-18 amino acid long Cytosolic pathway Endocytic pathway α1/α2 groove α1/β1 groove α3 binds to CD8 molecules on TC cells β2 binds to CD4 on TH cells MHC polymorphism • Each MHC molecule specifically presents a certain antigenic peptide to the T cells. • Since there is a wide spectrum of different antigenic peptides (with specific sequences) derived from various antigens; there is a likewise need of different MHC molecules capable of recognizing these peptides. • Made possible by the polymorphic feature of MHC genome; by which it is capable of producing a wide array of MHC molecules with vast antigenic specificities. Mechanisms by which MHC molecules show such a high level of pleomorphism • Multiple gene loci o Both MHC class I & II molecules are coded by multiple genes. Gene of each locus codes for a similar but not identical chain. For example, MHC I molecules (α chain) are coded by any of the three loci of class I region i.e. HLA-A or B or C loci and MHC II molecules (α & β chains) are coded by any of the three loci of class II region i.e. DP or DQ or DR loci. Mechanisms by which MHC molecules show such a high level of pleomorphism • Multiple allele for each locus: o In a given species, extraordinarily large numbers of different alleles are known to exist for each locus. o MHC genes are one of the most polymorphic genes known. o For example: For class I MHC region in humans, there are 240 alleles for HLA-A, 470 alleles for HLA-B & 110 alleles for HLA-C. The MHC class I region of any individual would have one of the allele from each HLA-A, B and C allele bank. Total 240x470x110 number of theoretical combinations possible for class I MHC region. Mechanisms by which MHC molecules show such a high level of pleomorphism • Co-dominant expression: o MHC genes are expressed in codominant fashion, i.e. the alleles inherited from parents (one from father & one from mother) are simultaneously and equally expressed. Regulation of MHC Expression • Transcription factors-MHC genes have promoter sequences at their 5’ end which are regulated by certain transcription factors such as CIITA, and RFX (both bind to MHC II promoter genes and increase their transcription). Defects in CIITA, and RFX cause one of the form of bare lymphocyte syndrome. • Cytokines also influence MHC expression. o Interferon γ activates both MHC-I & II promoter genes. o IL-4 increases expression of class II MHC molecules on resting B cells. Regulation of MHC Expression • Corticosteroid and prostaglandins decrease the expression of MHC II molecules. • In many viral infection, the viral antigens inhibit various components of MHC-I (e.g., adenovirus proteins inhibit TAP, cytomegalovirus proteins inhibit β2 microglobulin). As a result, MHC-I expression is suppressed. Diseases associated with certain HLA alleles HLA allele HLA B27 DR-2 Associated disease Ankylosing spondylitis, Reactive arthritis (Yersinia, Salmonella, Gonococcus) and Reiter’s syndrome Multiple sclerosis, Goodpasture’s syndrome, Narcolepsy DR-3 Myasthenia gravis, systemic lupus erythematosus (SLE) DR-3/DR-4 DR-4 A3/B14 Insulin-dependent diabetes mellitus Rheumatoid arthritis Hereditary hemochromatosis CYTOKINES • Definition - Cytokines are chemical substances which serve as messengers, mediating interaction and communication between the various cells of immune system. • Major classes of Cytokines o Lymphokines- produced by lymphocytes o Monokines- produced by monocytes & macrophages o Interleukins- produced by WBCs and acting on the same or different WBCs o Chemokines- involved in chemotaxis and other leukocyte behavior Properties of Cytokines • Growth factors are produced constitutively while cytokines are inducible i.e. produced only after the activation of their cells of origin. • Cytokines have broad range of effects; which include: o Autocrine effect- Act on the same cell o Paracrine effect- Act on the adjacent cell o Endocrine effect- Act on a cell present at a distant site. Properties of Cytokines • Various types of interactions occurring between cytokines o Pleiotropy and redundancy effect:  Pleiotropy - Same cytokine having different actions on different target cells Redundancy - Different cytokines producing the same effect on the same target cell. Properties of Cytokines o Synergy and antagonism effect- Two cytokines may augment each other’s action producing a larger effect (synergism) or may oppose each other’s action (antagonism). o Cascade effect - series of effects mediated by different cytokines. One cytokine acts on a target cell to produce another cytokine which in turn acts on another target cell and so on. Structure of cytokines • Cytokines are glycoproteins with molecular weight less than 30kDa. • Most cytokines display high degree of α-helix structure but no βstructure. • Cytokines characterized so far belong to one of the four groups: the hematopoietin family, the interferon family, the chemokine family, or the tumor necrosis factor family. Sources and functions of cytokines Cytokine Cytokine secreting cells Interleukins (IL) Produced by all nucleated cells, but IL-1 principal sources are APCs such asMacrophages, monocytes dendritic cell, B cells & endothelial cell IL-2 TH 1 cells IL-3 TH cell, NK cell, Mast cell IL-4 TH 2 cells Target cells & Functions 1. TH cells - IL1 produced by APCs stimulates TH cells activation & proliferation • Promotes IL2 secretion by TH cells • Induces IL-2 receptor expression on TH cells • Induces ↑MHC-II expression on APCs 1. B cell -Promotes B cell development & maturation 2. Liver- Induces synthesis of acute phase reactant proteins 3. Hypothalamus- induction of fever 4. Macrophage & neutrophil activation-↑ expression of ICAM Induces proliferation activated TH cells, TC cells and some NK cells (Previously called as T cell growth factor) 1. Stimulates hematopoiesis (acts as multi-CSF) 2. Mast cell degranulation-↑ histamine secretion 1. TH cells -Promote TH 2 cell activity and inhibit TH 1 cell 2. B cell- Promote B cells activation & proliferation and induce B cell class switch over to produce IgE, IgG4, IgG1;previously called as B cell growth factor Sources and functions of cytokines Cytokine Cytokine secreting cells Interleukins (IL) IL-5 TH 2 cells IL-6 TH 2 cells, macrophages IL-7 IL-8 IL-9 IL-10 IL-11 IL-12 IL-13 IL-17 Bone marrow/thymic stromal cells Macrophages, endothelial cells TH cells TH 2 cells Bone marrow stromal cells Target cells & Functions Promote eosinophil growth and differentiation IL-1 & TNF like effects (synergistic effect) Promotes B cell proliferation and antibody production Serves as a growth factor for T cell and B cell precursors. Attracts neutrophils, NK cells, eosinophils and basophils. Hematopoietic & thymopoietic effects Reduces cytokine production by TH1 cell. Hematopoietic effect ( B cell and platelet development) Liver- Induce synthesis of acute phase reactant protein Promote TH 1 cell induction and inhibit TH 2 activity; promotes CMI Macrophages responses NK cell stimulatory factor Mimic IL-4 function TH 2 cells + CD4 activated memory TH cell Initiates and maintains inflammation Sources and functions of cytokines Cytokine Cytokine secreting cells Target cells & Functions Interferons (IFN)* IFN-α Leukocytes Antiviral activity IFN-β Fibroblasts Antiviral activity IFN-γ TH &TC cells, NK cells 1. 2. 3. 4. Macrophage -Activates the resting macrophages into activated macrophage B cells -Activate B cells to produce IgG Promotes inflammation of delayed type of hypersensitivity (along with TNFβ) TH 2 cell - Inhibits TH 2 cell proliferation Sources and functions of cytokines Cytokine Cytokine secreting cells Target cells & Functions Tumor necrosis factors (TNF) TNF-α Macrophage 1. 2. 3. 4. TNF- β TH 1 cell and TC cell Tumor cells- Similar effect like TNF-α Macrophage-Enhance phagocytic activity IL-1 like effect Tumor cells- Promote vascular thrombosis and tumor necrosis Inflammatory cells- Induce cytokine secretion Induces lipolysis, causes extensive weight loss associated with chronic inflammation Sources and functions of cytokines Cytokine Cytokine secreting cells Target cells & Functions Colony stimulating factor(CSF) GM-CSF G-CSF M-CSF Others TGF- β Fibroblasts, endothelium, T cells, macrophages Bone marrow stromal cells, macrophages Fibroblasts, endothelium Macrophage and granulocyte growth stimulation Macrophages, mast cells T and B cells, platelet 1. 2. 3. Granulocyte growth stimulation Macrophage growth stimulation Inhibit T and B cell proliferation and hematopoiesis: Promote wound healing Promotes class switching of B cells to the IgA class Essentials of Medical Microbiology © 2018, Jaypee Brothers Medical Publishers