ALLAH does not want from you to be the BEST ALLAH wants from you to do your BEST

1. بسم الله الرحمن الرحيم AND ALLAHdoes not want from you to be theBEST ALLAHwants from you to do yourBEST ALLAHwill take care of theREST

2. Lymphoid System Hazem M. Abu-Eisha Ph.D , MD Assistant Professor of Immunology Pathology Department King Saud University King Khaled University Hospital E - mail : hmt149@yahoo.com

3. Outlines General considerations about Haematopoiesis Organs , cells & tissues of the lymphoid system Development of T & B lymphocytes T & B cell receptors ( TCR & BCR ) T & B lymphocytes subsets Locations of T & B lymphocytes in secondary lymphoid organs & their interactions

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5. Haematopoiesis • All blood cells including lymphocytes arise from one cell type called : Haematopoietic Stem Cell ( HSC )that originate in the bone marrow ( BM ) & gives rise to two cell lineages [ Common Myeloid Progenitor ( CMP ) & Common Lymphoid Progenitor ( CLP ) ] • In comparison to unipotent cell which can differentiate into a single cell type, HSC is a pluripotent stem cell [ i.e. it has the ability to differentiate into various types of cells including { Erythrocytes , granulocytes , monocytes , mast cells , lymphocytes & megakaryocytes } ] • HSCs are few in number [ There is one HSC for every 5 x 104 cells in the bone marrow ]

6. All cells shown here arise from the hematopoietic stem cell. Platelets produced by megakaryocytes are released into the circulation. Polymorphonuclear granulocytes and monocytes pass from the circulation into the tissues. Mast cells are identifiable in all tissues. B cells mature in the fetal liver and bone marrow in mammals, whereas T cells mature in the thymus. The origin of the large granular lymphocytes with natural killer (NK) activity is probably the bone marrow. Lymphocytes recirculate through secondary lymphoid tissues. Interdigitating cells and dendritic cells act as antigen-presenting cells (APCs) in secondary lymphoid tissues.

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9. Primary Lymphoid Organs 1. Thymus • It is the main site for T - cell maturation • Flat , bilobed organ • Each lobe is surrounded by a capsule & is divided into lobules • Each lobule is organized into two compartments : Outer called Cortex ; densely packed with immature T - cells called Thymocytes & Inner called : Medulla ; sparsely populated with thymocytes

10. Role Of Thymus In The Immune Function Experimental studies in mice with neonatal thymectomy & DiGeorge’s syndrome ( Congenital thymic hypoplasia )show : • Dramatic in circulating lymphocytes • Absence of cell - mediated immunity

11. Primary Lymphoid Organs 2. Bone Marrow • The primary site for B - cell maturation • The bone marrow contains connective tissue, blood vessels, fat, and cells • Among these structures are the hematopoietic stem cells which are capable of giving rise to the stem cells of the myeloid, granuloid, erythroid, and lymphoid cells • Unlike lymphoid cells that are destined to differentiate into T - cells, those committed to the B - cell lineage remain within the bone marrow for further development & maturation

12. Secondary Lymphoid Organs 1. Spleen • Large , ovoid organ • Its main function is : Filtering blood and trapping blood-borne antigens • Blood borne antigens & lymphocytes are carried out to it through splenic artery • It is formed of compartmentalized structure which consists mainly of two pulps separated by diffuse marginal zone : • The splenic red pulp • The splenic white pulp

13. The red pulp consisted of sinusoids populated with macrophages & red blood cells ( RBCs ) where old & defective RBCs are destroyed and removed • The white pulp surrounds the branches of splenic artery forming a periarteriolar lymphoid sheath ( PALS ) populated mainly by T - lymphocytes • The marginal zone is located peripheral to the PALS , it is rich in B - lymphocytes • In the marginal zone , antigens & lymphocytes are trapped by DCs that carry it to PALS

14. Initial activation of T & B lymphocytes occur in PALS where DCs capture Ags & present it in combination with MHC class II to TH cells • Activated T- cells activate B - cells • Activated B - cells with some activated TH cells migrate to the primary follicles in the marginal zone • Antigenic challenge Primary follicles develop to secondary follicles where rapidly dividing B - cells and plasma cells are formed [ Antibody - forming cells ( AFCs ) ] • Effect of splenectomy : Depends on the age • In children : incidence of bacterial sepsis • In adults : Less serious but leads to blood-borne bacterial infection

15. Secondary Lymphoid Organs 2. Lymph Node • Histologically , LN is divided into 3 regions: Cortex , Paracortex & Medulla • Cortex : Contains B - lymphocytes , macrophages & follicular DCs arranged in primary follicles which enlarge after antigenic challenge to become secondary follicles each contain a germinal center ( GC ) • Paracortex : is populated mainly by T - lymphocytes & DCs which express high level of MHC class II needed for antigen presentation for TH cells • Medulla : is populated mainly with plasma cells that are actively secreting ABs

16. Lymphoid Follicle • Aggregates of lymphoid & non-lymphoid cells surrounded by a network of draining lymphatic capillaries • Until it is activated by antigen , lymphoid follicle is called PRIMARY FOLLICLEthat contains mainly small resting B - cells and DCs • After antigenic challenge , it enlarges to become a SECONDARY FOLLICLEwhich contains a ringed concentrically packed B - cells surrounding a center [ GERMINAL CENTER (GC) ] i.e., focus of proliferating B - cells with area of non-dividing B - cells & some TH cells , macrophages & follicular DCs

17. Secondary Lymphoid Organs 3. Mucosal-associated lymphoid tissues ( MALT ) Mucosal-associated lymphoid tissues ( MALT ): Are anatomical sites placed at strategic areas of potential microbial entry • Tonsils : In the nasopharynx at the portal entry of respiratory & gastrointestinal systems • Peyer’s patches : Lymphoid accumulations lying underneath the villi of the small intestine that protect the bowel from microbial invasion

18. Cells Of The Lymphoid System 1. T - cell lineage • αβ T cells • γδ T cells • NKT cells 2. B - cell lineage • B cell

19. Lymphatic Circulatory System Lymphatics, lymphoid organs, and tissues. The lymphatics serve as a drainage system to remove cellular debris and microbes from the body's tissues to the lymph nodes. Lymphatic trunk vessels join to form the thoracic duct, which returns fluid (lymph) to the cardiovascular circulation. Leukocytes and their products use two circulatory systems : • One, is the cardiovascular system which is responsible for the circulation of blood (both its soluble and cellular components) throughout the body • The other system, is the lymphatic circulatory system. It is an extensive capillary network that collects lymph, a watery clear fluid containing leukocytes and cellular debris, from various organs and tissues Lymphatic vessels within small intestine villi, contain a milk-white fluid, chyle, produced by digestion. The lymphatic capillaries drain into large lymphatic vessels that drain into lymph nodes for filtration Ultimately, the lymphatic trunk vessels join to form the thoracic duct that conveys lymph into the subclavian vein

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21. T - Lymphocytes • They arise in the bone marrow , but migrate to the thymus for complete maturation • During T - cell maturation , they express a unique antigen - binding molecule called : T - cell receptor ( TCR ) • B - cells recognize antigen alone while T - cells recognize antigens bound to cell membrane proteins called : MAJOR HISTOCOMPATABILITY COMPLEX( MHC ) • When a naϊve T- cell encounters antigen combined with MHC , T - cell proliferate & differentiate into : • Effector T - cells [ T - helper ( TH ) & Cytotoxic T - cells ( TC ) ] • Memory T - cells

22. T cells can be differentiated by their antigen receptors ( TCR ) • T - cell either expresses : Two - disulfide heterodimers linked polypeptides ( α & β ) OR Structurally similar heterodimer consisting of ( γ & δ polypeptides ) • About 90 - 95 % of blood T - cells are α β & the remaining 5 - 10 % are γδ T - cells

24. Development of αβ T - cells Cortical thymocytes are called double negative (DN) cells They soon begin to generate and express αβ TCRs, associated CD3 complex, and both CD4 and CD8 molecules & these immature thymocytes are called double positive (DP) cells In a process known as positive selection, DP thymocytes die within three to four days unless they recognize and bind to major histocompatability complex (MHC) molecules expressed by cortical epithelial cells. This process eliminates thymocytes that are incapable of recognizing self MHC Cells that pass the positive selection test are allowed to enter the medulla; those that fail the test die. DP cells whose CD8 molecules have engaged (p) MHC I then stop the expression of CD4 molecules and become single positive (SP) CD8+ cells. Likewise, those that are bound to (p)MHC II stop expression of CD8, becoming SP CD4+ cells Survivors of positive selection then run a second test called negative selection when they arrive at the corticomedullary junction. There, they meet and interact with a second set of epithelial reticular cells (antigen presenting cells such as dendritic cells and macrophages). Those that efficiently bind to self peptides of the pMHC I or pMHC II on these APCs are potentially autoreactive and undergo apoptotic death. Thymocytes that pass both positive and negative selection tests “graduate” from the thymus, entering the circulation through the medullary postcapillary venules as mature T- cells

25. Development of γδ T - cells The thymus is also the differentiation site for thymocytes that express γδ TCRs and CD3 complex molecules. Many of these cells fail to express CD4 and/or CD8. Consequently, they do not undergo the same positive and negative selective processes as αβ TCR-bearing thymocytes γδ cells are thought to be a transitional cell type that may represent a bridge between the innate and adaptive immune systems. γδ T cells develop early in embryogenesis before many αβ T cells and migrate preferentially to the respiratory organs, the skin, and the peritoneal cavity. They use a very limited in their recognition repertoire than are αβ T cells. They respond more quickly than do αβ T cells, but they do so without generating memory.

26. Natural Killer T (NKT) cells • Natural killer T (NKT) cells are a distinctive subset of T - cells that share some characteristics with NK cells • They express several surface markers and receptors found on natural killer ( NK ) cells [ CD16 & CD56 ] , but unlike NK cells, they undergo some development in the thymus • NKT cells express TCRs that are extremely limited in repertoire and are predominantly specific for lipids, glycolipids, and a few specialized types of peptides • Their TCRs have an unusual restriction pattern. Although they may be either CD4+ or CD4+CD8+, they specifically recognize epitopes presented by a “nonclassical” MHC class I molecule called CD1

27. Terminal deoxynucleotidyl transferase (TdT) is an enzyme in thymic stem cells. It decreases in stage II and is lost altogether in the medulla. Several surface glycoproteins appear during differentiation. CD1 is present on stage II cortical thymocytes and is lost in the medulla. CD2 and CD7 (the pan-T marker) appear very early in differentiation and are maintained through to the mature T cell stage. CD5 appears at an early stage and persists on mature T cells. CD3 is expressed first in the cytoplasm in stage I cells (cyto), and then on the surface simultaneously with the T cell receptor (TCR). In most stage II cells, both surface CD3 and the αβ TCR are expressed at low density, but these markers are present at high density on stage III cells. CD4 and CD8 are co-expressed on stage II cells (double positives). One of these molecules is lost during differentiation into mature stage III cells (single positives).

28. B - Lymphocytes • They originate from HSC & mature in the BM • They express a unique antigen binding receptor on its membrane therefore , B - cell receptor is a membrane - bound antibody molecule • When a naïve B - cell encounters the antigen for the first time , this binding leads to rapid division of naïve B - cell & their progeny will differentiate into : 1. Effector B - cell ( PLASMA CELLS ) which produce secreted Abs { Effector molecules of HUMORAL IMMUNITY } 2. Memory B - cells { They have a longer life span than naïve cells & continue to express same membrane - bound antibody }

29. B - Cell Surface Markers A. Immunoglobulin ( Chiefly IgM ) • It is the primary surface marker on the B - cell membrane • Surface IgM (sIgM) functions as antigen recognition site that binds specific epitopes • Binding initiates activation , proliferation & differentiation into plasma cells B. Other markers • CD antigens ( CD10, 19, 20, 21, 22 & 23 ) • Complement components ( C3b & C3D ) • MHC class I & II molecules

30. Values of CD molecules Allow lymphocytes to : • Communicate with their environment ( Cell Trafficking , Adhesion & Activation ) • Isolation from each other that allow them to be identified using fluorescent antibodies as probes ( Flow cytometry )

32. Development of B - cells • In humans, progenitors of immunoglobulin-producing cells are found in the yolk sac by the third week, in the fetal liver by the eighth week, and in the bone marrow by approximately the twelfth week of gestation. • B - cells are defined as cells that synthesize immunoglobulin ( antibodies ) and display it on their cell surfaces as B - cell receptors BCRs. • These cells are called bone marrow-derived lymphocytes or B - cells

33. Pro - B lymphocyte represents the early stage of B - cell development in which V , D & J gene segments combine to form an immunoglobulin μ heavy chain Maturation to pre - B cell starts with cytoplasmic expression of a surrogate light chain ( early stage ) followed by surface expression of both μ and surrogate light chain Immature B - cells starts to express IgMκ or IgMλ on their surfaces Mature B - cells express both IgM & IgD on their cell surfaces Plasma cells (AFCs ) represent the end stage of differentiation of B - cell lineage

34. B cell development reflects the stages of immunoglobulin heavy and light chain rearrangement and surface expression • Arising from a common lymphoid progenitor (CLP), the earliest identifiable cell committed to the B cell lineage is the pre-pro-B cell (Fraction A), within which the cell begins to express Igα and Igβ BCR accessory molecules • Immunoglobulin DJ gene joining and cytoplasmic expression of surrogate light chain (SLC) occurs at the early pro-B cell (Fraction B) stage followed by VDJ gene joining and cytoplasmic SLC expression at the late pro-B cell (Fraction C) stage • The early pre-B cell (Fraction C-prime or C') stage is characterized by the surface expression of pseudo-IgM (rearranged µ heavy chains plus SLC) and is accompanied by a burst of cellular proliferation • In the late pre-B cell (Fraction D) stage, immunoglobulin light chain kappa (κ) or lambda (λ) genes rearrange, and their products (κ or λ light chains) replace the SLCs • Immature B cells (Fraction E) express µ heavy chains plus κ or λ light chains on their cell surfaces • Mature B cells (Fraction F) coexpress IgM and IgD on their cell surfaces • B cell progenitors, like thymocytes, express molecules and receptors necessary for migration and interaction with other cells • Some attributes (e.g., DNA recombinase expression) are lost by the time cells reach the immature B cell (Fraction E) stage. If the IgM on the developing cells should bind to epitopes they encounter in the bone marrow, such cells undergo apoptotic death to prevent production of autoreactive B cells

36. Generation , Maturation & Differentiation of B - cells • Generation of mature, immunocopmpetent B - cells ( Maturation ) • Activation of mature B - cells • Differentiation of activated B - cells into plasma cells & memory cells • Generation of B - cells occurs in BM in absence of Ag ( Antigen - independent phase of B - cell development ) • B - cell activation & differentiation is Ag dependent ( Antigen - dependent phase of B - cell development )

38. 1 Antigen independent Bone Marrow s 2 Antigen dependent Peripheral Lymphoid Organs

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41. T - cell receptors ( TCR ) T - cell receptors are located on the surface of T - cells & play a critical role in the adaptive immune response T - cells recognize fragments of degraded proteins ( peptides ) that should be bound to antigen - presenting molecules called MAJOR HISTOCOMPATABILITY COMPLEX ( MHC ) molecules

42. TCR are of two varieties termed αβ & γδ Both molecules : • Resemble immunoglobulins & made up of heterodimers ( i.e. Either α & β or γ & δ subunits ) • Integral membrane proteins with large extracellular domains ( Responsible for antigen - recognition ) and short cytoplasmic tails ( Responsible signal transduction )

43. T - cell Receptor Complex : TCR - CD3 • The αβ & γδ heterodimers must associate with a series of polypeptide chains termed CD3 complex for antigen - binding domains of TCR to form a functional receptor stably expressed at the cell surface capable of transmitting a signal upon binding to the antigen • The TCR - CD3 complex is formed of four dimers : 1. The αβ or γδ TCR heterodimer AND 2. The CD3 dimers ( γε, δε & ζζ or ζη ) required for signal transduction

44. B - cell Receptor ( BCR ) • How membrane immunoglobulin ( mIg ) mediates an activating signal after contact with antigen ? AS IT HAS A SHORT CYTOPLASMIC TAIL ? • The answer is : mIg does not constitute the entire antigen - binding receptor on B - cells • B cell receptor ( BCR ) is a transmembrane protein complex composed of mIg and disulfide - linked heterodimers called Ig-α/Ig-β • The long cytoplasmic tails of both Ig-α/Ig-β enable them to interact with intracellular signaling molecules

45. ITAMs : Immunoreceptor Tyrosine - Based Activating Motifs

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47. Major subpopulation of T - cells • About 90 - 95 % of T - cell are αβ which are divided into two distinct populations 1.One carries CD4 marker ( CD4+ T helper - cells ) & mainly help or induce the immune response & recognize antigens with MHC class II molecules . CD4 TH - cells can be subdivided into two subsets depending on cytokines profiles they produce : CD4 TH1 - cells : Secrete IL-2 & IFN-γ CD4 TH2 - cells : Secrete IL-4, IL-5, IL-6 & IL-10 2. The other set carries CD8 marker (CD8+ T cytotoxic - cells ) & mainly cytotoxic that recognize antigens with MHC class I molecules • A small percentage of αβ T - cells neither express CD4 nor CD8 molecules ( DOUBLE NEGATIVE T - cells ) that may have a regulatory function ( Regulatory T - cells & AUTOIMMUNE DISEASES ) • In comparison , most circulating γδ are DOBLE NEGATIVE & most γδ T - cells in tissues express CD8 marker

48. B - cell subsets • There are 2 subsets of B cells : B - 1 B & B - 2 B cells ( Major group of B cells in Human & Mice ). • B - 1 B cells appear during fetal life , express IgM with little or no IgD & display CD5. • B - 1 B cells are minor population especially in secondary lymphoid organs but it is the major population in peritoneum. • Up till now , there is no definite function for B - 1 B cells but they may respond poorly to protein antigens but much better to CHO Ags. • Mostly are IgM bearing cells with less class switching than B - 2 B cells.

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