Lymphoid Organs Introduction The slides for this lab are located in the “Lymphoid” folder on the Virtual Microscope. In this lab, you will learn about the lymphatic system and lymphoid organs that filter and clean the blood and contribute to immunity.

Lymphoid organs can be classified based on whether or not they have a connective tissue capsule. So-called encapsulated organs include the thymus, spleen and lymph nodes. While un- encapsulated organs include the appendix, ileum and tonsils, many of which you have encountered during your studies of histology so far.

Learning objectives and activities Using the Virtual Slidebox:

A Examine the thymus and identify the developing T-cells and epithelial-reticular cells of the thymic parenchyma

B Examine a lymph node and identify the cells that perform functions in generating immune responses.

C Examine the spleen and identify the cells that are involved in filtering the blood and reacting to blood-borne antigens.

D Differentiate between the MALT (Mucosa Associated Lymphoid Tissue) of the palatine, pharyngeal and lingual tonsils.

E Review the GALT (Gut Associated Lymphoid Tissue) in the appendix and ileum.

F Complete the self-quiz to test your understanding and master your learning Examine the thymus and identify the developing T-cells and epithelioreticular cells of the thymic parenchyma.

Examine Slide 1 and approximate a thymic lobule in the thymus by identifying the following:

The thymus is a bilobed lymphoepithelial organ located in the superior mediastinum. Multipotential lymphoid stem cells from the bone marrow are destined to develop into immunocompetent T-cells within the thymic parenchyma. The thymus is encapsulated by connective tissue which also divides the parenchyma into thymic lobules. i. General organization

a. Connective tissue capsule Identify the thymic A thin outer layer of dense irregular connective tissue. Contains capsule in blood vessels, efferent lymphatic vessels and nerves. Slide 1a

b. Trabeculae Extend into the parenchyma of the organ to establish domains Identify the within the thymus called thymic lobules. They also contain blood trabeculae in vessels, efferent lymphatic vessels and nerves. Slide 1b

c. Thymic lobule Identify a thymic Defined by connective tissue trabeculae it is composed of the lobule in cellular parenchyma organized into a cortex and medulla. Slide 1c

d. Thymic cortex The outer portion of parenchyma within a lobule. It is markedly Identify the thymic basophilic because it is packed full of T-lymphocytes and scattered cortex in epithelioreticular cells. Slide 1d

e. Thymic medulla The inner portion of the parenchyma within a lobule. It is less Identify the thymic basophilic as the T-lymphocytes here are more loosely packed and medulla in are larger. It also contains prominent epithelioreticular cells called Slide 1e “Hassall’s corpuscles”. ii. Parenchyma of the thymus

f. Thymocytes T-lymphocytes in the thymus. In the cortex they are small and Identify thymocytes tightly packed. In the medulla they are larger and more loose. in Slide 1f

g. Epithelioreticular cells There are six types of epithelioreticular cell in the thymus

Type I - separate connective tissue capsule/trabeculae/blood vessels Identify Type I from the parenchyma of the cortex. Epithelioreticular cells - adjacent reticular cells are connected by occluding junctions in Slide 1g - forms blood-thymus barrier to isolate T-cells from other tissues

Type II - are stellate cells within the cortex Identify Type II - have long cytoplasmic processes joined by desmosomes Epithelioreticular cells - compartmentalize the cortex into isolated areas of T-cells in Slide 1g - express MHCI/MHCII molecules at their surface - involved in thymic education

Type III and Type IV - are located at the cortico-medullary junction Identify Type III and IV - have long cytoplasmic processes joined by tight junctions Epithelioreticular cells - form a functional barrier between the cortex and medulla in Slide 1g - express MHCI /MHCII molecules at their surface - involved in thymic education

Type V Identify Type V - located throughout the medulla Epithelioreticular cells - have long cytoplasmic processes joined by desmosomes in Slide 1g - compartmentalizes the medulla into groups of T-cells

Type VI - form isolated masses of packed cells called Hassall’s corpuscles Identify Type VI - may contain keratin in the center Epithelioreticular cells - secrete IL-4/IL-7 that assists in differentiation/education of T- in Slide 1g cells

h. Macrophages Located in the thymic cortex but difficult to see in standard H&E Macrophages cannot preps. They phagocytose T-cells that do not fulfill thymic be seen in Slide 1 education requirements. 98% of T-cells will be phagocytosed. Examine a lymph node and identify the cells that perform functions in generating immune responses.

Examine the general organization of a lymph node in Slide 2 (H&E) and Slide 3 (Silver)

The role of the lymph node is to filter the lymph (extracellular fluid derived from blood). Lymph enters the node via numerous afferent vessels and leaves via a single efferent lymphatic vessel at the hilum. It is composed of a cortex, medulla and intervening sinuses through which lymphatic fluid flows. i. General organization

a. Connective tissue capsule Identify the capsule in A thin outer layer of dense irregular CT that surrounds the node Slide 2a and Slide 3a

b. Trabeculae Identify trabeculae in CT extending into the parenchyma forming a gross framework Slide 2b and Slide 3b c. Subcapsular sinus A lymphatic channel interposed between the CT capsule and the Identify subcapsular outer cortex. Afferent lymphatic vessels bring lymph into this sinus in sinus. Slide 2c and Slide 3c

d. Trabecular sinus Identify a trabecular Originates from the subcapsular sinus and carries lymph through sinus in the cortex into the medullary sinuses. Slide 2d and Slide 3d e. Reticular tissue You can only see Composed of reticular cells (fibroblast-like cells) and reticular reticular tissue in the fibers (Type II collagen) that forms a fine supporting meshwork silver stained throughout the organ. The fibers are best seen when stained with Slide 3e a silver stain.

f. Cortex The outer portion of parenchyma within a lymph node. It is Identify the cortex in composed mainly of reticular tissue and lymphocytes. It can be Slide 2f and Slide 3f sub-divided into outer/superficial and para/deep cortex and is home to lymphoid nodules.

g. Medulla The inner part of the lymph node. It contains medullary cords Identify the medulla in (composed of reticular tissue, lymphocytes (mostly B- Slide 2g and Slide 3g lymphocytes) and medullary sinuses through which lymph drains. ii. Cortex of the lymph node

h. Outer/Superficial/Nodular cortex The outermost region of the cortex that contains primary and Identify the superficial secondary lymphoid follicle cortex in Slide 2h and Slide 3h i. Inner/Deep/Para/Thymus-dependent cortex The portion of the cortex that separates the outer cortex from the Identify the deep medulla. It does not contain lymphoid nodules and is mainly cortex in composed of T-cells (hence thymus-dependent, perinatal Slide 2i and Slide 3i thymectomy results in an underdeveloped deep cortex). It is home to multiple high endothelial venules (HEV).

j. Primary nodule/follicle Located in the superficial cortex, nodules are considered ‘primary’ Identify a primary if they consist of small lymphocytes that have not been exposed nodule in to antigen and have not commenced differentiation. As a result Slide 3j and Slide 3j primary nodules appear as basophilic aggregates of lymphocytes.

k. Secondary nodule/follicle Located in the superficial cortex, nodules are considered Identify a secondary ‘secondary’ if the lymphocytes have been exposed to antigen and nodule in have commenced differentiation to form memory B-cells and Slide 2k and Slide 3k plasma cells. These cells predominate in a germinal center. Most nodules are secondary.

l. Germinal center The central pale region of a secondary nodule. Recall that a germinal center develops when a B-lymphocyte has recognized an Identify a germinal antigen and has interacted with a complimentary T-lymphocyte. center in This interaction between B-cell and T-cell triggers proliferation Slide 2l and Slide 3l and differentiation of the B-lymphocyte to form memory B-cells and plasma cells within the germinal center.

m. High endothelial venules (HEVs) Some lymphocytes enter lymph nodes through afferent lymphatic vessels (as a component of lymph) but most enter the node through the walls of HEVs located in the deep cortex. They are so Identify an HEV in named because they are lined with cuboidal endothelial cells (not Slide 2m and Slide 3m squamous). These special endothelial cells have receptors for antigen primed lymphocytes triggering them to migrate across the

endothelium into the lymph node. ii. Medulla of the lymph node

n. Medullary cords Cords of reticular fibers synthesized by surrounding reticular cells Identify medullary (fibroblasts) that serve as a framework for lymphocytes (mainly B- cords in cells), plasma cells from the cortex and macrophages that clean up Slide 2n and Slide 3n the lymph.

o. Medullary sinuses Identify the medullary A pathway for the flow of lymph that runs between medullary sinuses in cords. Sinuses converge at the hilum where they drain into Slide 2o and Slide 3o efferent lymphatic vessels.

trabecular sinus

trabecula

capsule

afferent lymphatics

efferent lymphatic vessel inner (para) cortex

medullary cord

outer cortex medullary sinus germinal center

secondary lymphatic subcapsular sinus nodules Examine the spleen and identify the cells that are involved in filtering the blood and reacting to blood-borne antigens.

Examine the general organization of the spleen in Slide 4

The spleen has a number of important roles. It acts as a store for platelets; reacts to blood borne antigens by producing antibodies (and is in fact the main source of circulating antibodies); it also removes defective red blood cells and platelets from the circulation. i. General organization

a. Connective tissue capsule Identify the capsule in A thin outer layer of dense irregular CT that surrounds the node Slide 4a

b. Trabeculae Identify trabeculae in CT extending into the parenchyma forming a gross framework Slide 4b c. White pulp Named after its appearance in fresh tissue white pulp forms Identify white pulp in circular or elongated areas scattered throughout the spleen. It Slide 4c consists of accumulations of lymphocytes that surround an artery.

d. Red pulp Identify red pulp in Named after its appearance in fresh tissue, red pulp contains large Slide 4d numbers of red blood cells that it filters and degrades. iii. Blood supply to the spleen Blood flows from the splenic artery into the following vessels before leaving the spleen via the splenic vein. Can you find evidence of these vessels in Slide 4?

e. Trabecular artery Identify a trabecular Branches of the splenic artery they run alongside the connective artery in Slide 4e tissue trabeculae in the spleen.

f. Central artery Identify a central Central arteries pass through splenic lymphoid nodules (white artery in Slide 4f pulp).

g. Marginal zone sinuses Identify marginal Blood flows from the central arteries into sinuses that surround sinuses in Slide 4g lymphoid nodules.

h. Penicillar (paintbrush) arteries Identify a penicillar Paintbrush shaped vessels that receive blood from the marginal artery in Slide 4h sinuses and the central artery.

i. Sinusoids Identify sinusoids in Large spaces filled with blood. They form a large portion of the red Slide 4i pulp

j. Trabecular vein Identify a trabecular Running alongside the trabecula. They drains the sinusoids and vein in Slide 4j transmit blood to the splenic vein.

splenic splenic artery vein

trabecular central marginal penicillar venous trabecular artery artery zone arteries sinusoids vein sinuses iv. Parenchyma of the spleen

White pulp

k. Lymphoid nodules A seemingly randomly distributed collections of lymphocytes Identify a lymphoid organized into round nodules. Each nodule has the five nodules in components listed below. Slide 4k

l. Central artery Identify a central A single distinctive arteriole located at the periphery of a artery in Slide 4l lymphoid nodule.

m. Periarteriolar lymphatic sheath (PALS) Identify PALS in A dense area of T-lymphocytes surrounding the central artery. Slide 4m n. Marginal zone - forms the margin of the lymphoid nodule. - separates white pulp from the surrounding red pulp. - is associated with blood-filled marginal zone sinuses Identify the marginal - antigen presenting cells and macrophages here collect blood- zone in borne antigens and destroy pathogens respectively. Slide 4n - is the site where lymphocytes in the bloodstream enter the lymphoid nodule and begin to percolate through the nodule being exposed to antigens associated with antigen presenting cells as they do so.

o. Peripheral white pulp Identify peripheral A population of B-lymphocytes surrounding the germinal center. white pulp in In some sections this region may appear paler than the germinal Slide 4o center.

p. Germinal center Recall that a germinal center develops when B-lymphocytes percolating through the white pulp recognize an antigen and Identify a germinal interact with a complimentary T-lymphocyte. These interactions center in Slide 4p trigger the proliferation and differentiation of B-lymphocytes to form memory B-lymphocytes and plasma cells within the germinal center. Red pulp

q. Venous sinusoids These are the termination of the penicillar arteries and are filled Identify venous with blood. Therefore they appear red (hence ‘red pulp’). sinusoids in Slide 4q r. Cords of Billroth Region of lymphoid tissue (reticular fibers, lymphocytes, Identify cords of macrophages, plasma cells, granulocytes) that occupy the space Billroth in between adjacent sinusoids. It is here that macrophages will Slide 4r phagocytose morphologically impaired/old erythrocytes.

white pulp (lymphoid nodule) red pulp

PALS marginal peripheral germinal venous Cord of zone pulp center sinusoid Bilroth

splenic splenic artery vein

trabecular central marginal penicillar venous trabecular artery artery zone arteries sinusoids vein sinuses Differentiate between the palatine, pharyngeal and lingual tonsils.

Examine the general organization of the lingual tonsil in Slide 5

The lingual tonsils are numerous small unencapsulated regions of lymphoid tissue located at the base of the tongue. Their location means they are covered in oral epithelium and are associated with salivary glands and the skeletal muscle of the tongue itself. i. General organization

a. Connective tissue capsule Identify the capsule in The surface of the lingual tonsil is not encapsulated but its base is Slide 5a surrounded by dense irregular connective tissue.

b. Crypts The deep fold above the tonsil is an epithelium lined crypt. It is Identify the crypt in often disrupted in the crypt due to many lymphocytes in the area. Slide 5b Lingual tonsils usually have crypts that are not branched.

c. Stratified squamous epithelium (non-keratinized) Identify oral A continuation of the oral epithelium. It covers the surface of the epithelium in tonsil in place of a capsule and descends into the crypt where it is Slide 5c more difficult to classify.

d. Secondary lymphoid nodule Identify a lymphoid Distinctive collections of lymphoid nodules with germinal centers. nodule in Slide 5d

e. Germinal center Recall that a germinal center develops when B-lymphocytes Identify a germinal percolating through the white pulp recognize an antigen and center in interact with a complimentary T-lymphocyte. These interactions Slide 5e trigger the proliferation and differentiation of B-lymphocytes to form memory B-lymphocytes and plasma cells within the germinal center.

f. Lingual salivary gland Identify the lingual salivary gland in Large mucous salivary glands. Slide 5f

g. Skeletal muscle of the tongue Identify skeletal muscle in Skeletal muscle fiber bundles. Slide 5g Examine the general organization of the palatine tonsil in Slide 6

The palatine tonsils are covered by oral epithelium and anchored by a capsule of dense irregular connective tissue that surrounds their base. Each tonsil contains 10-20 branching crypts invaginating from the surface. The space between the capsule and the epithelium is filled with lymphoid nodules. The interior of the crypts is often filled with desquamated epithelial cells, lymphocytes and micro-organisms that can become calcified. The crypts are also associated with palatine structures like the palatine salivary glands and skeletal muscle of the soft palate. i. General organization

a. Connective tissue capsule Identify the capsule in Dense irregular connective tissue forms large projections that Slide 6a separate the tonsillar tissue. There is no surface capsule.

b. Crypts Identify crypts in Multiple folds of the overlying epithelium that are often Slide 6b branching. They contain cellular and bacterial debris.

c. Stratified squamous epithelium (non-keratinized) A continuation of the oral epithelium of the soft palate. It covers Identify trabeculae in the surface of the tonsil in place of a capsule and descends into Slide 6c the crypts where it is more difficult to classify.

d. Secondary lymphoid nodule Identify a lymphoid Distinctive collections of lymphoid nodules with germinal centers. nodule in Slide 6d

e. Germinal center Recall that a germinal center develops when B-lymphocytes Identify a germinal percolating through the white pulp recognize an antigen and center in interact with a complimentary T-lymphocyte. These interactions Slide 6e trigger the proliferation and differentiation of B-lymphocytes to form memory B-lymphocytes and plasma cells within the germinal center.

f. Palatine salivary glands Identify palatine salivary glands in Small mucous salivary glands. Slide 6f Examine the general organization of the pharyngeal tonsil in Slide 7

The pharyngeal tonsil (adenoids) is covered by a disrupted respiratory epithelium. The tonsil does not have crypts like the other tonsils but instead has numerous surface folds (pleats) i. General organization

a. Connective tissue capsule Identify the capsule in Dense irregular connective tissue core. There is no surface Slide 7a capsule.

b. Pleats Identify trabeculae in The pharyngeal tonsil is folded rather than having deep crypts. Slide 7b c. Respiratory epithelium A continuation of the epithelium of the nasopharynx. It covers the surface of the tonsil in place of a capsule. It is often indistinct but Identify trabeculae in gives way to clear patches with distinct pseudostratification, cilia Slide 7c and goblet cells.

d. Secondary lymphoid nodules Identify white pulp in Distinctive collections of lymphoid nodules with germinal centers. Slide 7d

e. Germinal center Recall that a germinal center develops when B-lymphocytes percolating through the white pulp recognize an antigen and Identify red pulp in interact with a complimentary T-lymphocyte. These interactions Slide 7e trigger the proliferation and differentiation of B-lymphocytes to form memory B-lymphocytes and plasma cells within the germinal center. Review the lymphoid tissue in the intestine

Review the structure of the appendix and examine its lymphoid nodules in Slide 8

Lymph nodules surround the lumen of the appendix and extend from the mucosa into the submucosa. Their most luminal aspect is associated with the epithelium which contains specialized M-cells (microfold cells) that are involved in sampling antigen from the GI tract and handing it off to antigen presenting cells in this area in a controlled mechanism that assists in maintaining immunity. i. General organization

a. Review the organization of the wall of the appendix You should be able to distinguish the appendix from all other Review the appendix immune organs and name the layers of its wall and identify its in Slide 8a basic tissue components.

b. Lymphoid nodules Identify in Slide 8b c. Simple columnar epithelium with microvilli and goblet cells Identify gut epithelium The region covering the lymphoid nodules is where the M-cells in Slide 8c are located. These cells are not individually visible in histology. d. Germinal center Recall that a germinal center develops when B-lymphocytes Identify germinal percolating through the white pulp recognize an antigen and center in interact with a complimentary T-lymphocyte. These interactions Slide 8d trigger the proliferation and differentiation of B-lymphocytes to form memory B-lymphocytes and plasma cells within the germinal center. Review the structure of the ileum and examine its Peyer’s patches in Slide 9

Lymph nodules are located on one side of the ileum and are known as Peyer’s patches. They extend from the mucosa into the submucosa. Their most luminal aspect is associated with the epithelium which contains specialized M-cells (microfold cells) that are involved in sampling antigen from the GI tract and handing it off to antigen presenting cells in this area in a controlled mechanism that assists in maintaining immunity. i. General organization

a. Review the organization of the wall of the ileum You should be able to distinguish the appendix from all other Review the ileum in immune organs and name the layers of its wall and identify its Slide 9a basic tissue components.

b. Peyer’s patches Identify a Peyer’s A Peyer’s patch is named often its appearance as a whitish/grey patch in region visible in the wall of the gross ileum. Each patch is Slide 9b composed of a number of lymphoid nodules.

c. Lymphoid nodules Identify in Slide 9c d. Simple columnar epithelium with microvilli and goblet cells Identify gut epithelium The region covering the lymphoid nodules is where the M-cells in Slide 9d are located. These cells are not individually visible in histology. e. Germinal center Recall that a germinal center develops when B-lymphocytes Identify a germinal percolating through the white pulp recognize an antigen and center in interact with a complimentary T-lymphocyte. These interactions Slide 9e trigger the proliferation and differentiation of B-lymphocytes to form memory B-lymphocytes and plasma cells within the germinal center. In addition to all the basic tissues in slides and all previously covered items from MOHD1 that are relevant to interpreting tissue structure you must be able to identify the following structures and their features:

Thymus Spleen Capsule Capsule Trabecula(e) Trabecula(e) Thymic lobule Thymocytes Vasculature Type I epithelioreticular cells Trabecular artery Type II epithelioreticular cells Central artery/arteriole Type III/IV epithelioreticular cells Marginal zone sinuses Type V epithelioreticular cells Penicillar arteries/arterioles Type VI epithelioreticular cells Venous sinusoids Hassall’s corpuscles Trabecular vein

Lymph node White pulp Capsule Lymphoid nodule Trabecula(e) Periarteriolar Lymphatic Subcapsular sinus Sheath (PALS) Trabecular/intermediate sinus Peripheral white pulp Cortex Marginal zone Medulla Germinal center Outer/Superficial cortex Inner/Deep/Para Cortex Red pulp Lymphoid nodules/follicles Venous sinusoids (primary) Cords of Billroth Lymphoid nodules/follicles (secondary) Tonsils Germinal center Lingual tonsil High Endothelial Venule (HEV) Palatine tonsil Medullary cord Pharyngeal tonsil Medullary sinus All basic tissues Type III collagen Lymphoid nodule (reticular fibers) Germinal center

Appendix and Ileum All regions and basic tissues Peyer’s patch Lymphoid nodule Germinal center