1st practice: Introduction, structure of lymphoid organs

Basic Immunology

University of Pécs, Clinical Center Department of Immunology and Biotechnology Introducing the subject 1.

• Quick tests at the beginning of each practice based on the LECTURES OF THE PREVIOUS WEEK (for 2 points), 10 tests for a maximum of 20 points. • In case of absence one catch-up test is allowed at the end of the semester. (But already written tests cannot be rewritten) • In order to accept the semester you must have at least 10 points and maximum 3 absences! These terms are non-negotiable. • You may only join another group in case of absence once in the semester. • Final grade is based on a written exam in the exam period. (for 100 points) • All points above 10 acquired from practice tests will be added to your final result. If you also have half a point by the end of the semester it will be rounded to an additional point. (e.g. If you have 14.5 points by the end of the year and get 64 points for the written exam your final result will be 69 points) • The Immunology competition is held annually in April where you can get additional extra points or even a final grade of 5 (excellent). • Laboratory coats are necessary for some of the practices, your teacher will advise you the previous week to bring them with you. • All the educational material will be available to download on our website. (www.immbio.hu) Introducing the subject 2. NEW: From this semester we introduce an interactive quiz during the lessons for the better follow-up and understanding. We will ask 1-2 questions from the given part of the practices during the slide presentation. Technically, the questions will appear on-line at www.medtraining.eu with randomized answer choices for every individual user. Registration is required for the use of the website: see next slides. For participating in the quiz, we recommend the use of mobile devices (smartphones, tablets, laptops), but it is also possible to hand in the answers on paper. Since the actual question will only be active for a limited time it is recommended that you follow the slides of the presentations during the lessons because you can only pick the correct answer this way. You can answer the questions only once and it is not possible to correct later or return to previous question(s).

Registration 1. Registration 2.

Username: NEPTUN code

A regisztráció kitöltése után jóváhagyásra kerül és pár percen belül használható a weboldal. Introducing the subject 2.

• Our official books you can learn from:

Abul K. Abbas, Andrew H.H. Lichtman, Abul K. Abbas, Andrew H.H. Shiv Pillai: Cellular and Molecular Lichtman, Shiv Pillai: Basic Immunology, 8th edition, 2015. Immunology, 4th edition, 2012.

• Attention! Our department has never published or lectored any notes for students, therefore we recommend you to be cautious in case you decide to study from them. Introducing the subject 3.

• What makes immunology worth studying? – The is involved one way or another in almost all human pathological conditions. – Many of the laboratory diagnostics are based on immunological methods. (see later) – More and more diseases get treatable by manipulating the immune system. (see later) – Autoimmune diseases affect 7-8% of the population, they are chronic and generally incurable, yet many can be treated effectively. (see later) – The number of immunocompromised patients increased recently. (Due to therapeutic immunosuppression and HIV, see later) – Laypeople also seem to have strong opinions regarding immunology. → Media tends to mix medical facts with quackery and pseudoscience.

A news report from June of 2015.

Main tasks of the immune system

Preserving the integrity of an organism

Defense against external pathogens Elimination of one’s pathologically (e.g. viruses, bacteria, parasites) altered cells (e.g. virally infected cells, cancer cells)

Altered foreign structures must be recognized and distinguished from the organism’s own healthy cells.

IMMUNE RESPONSE (either an aggressive response or immunological tolerance) Components of the immune system

• The components of immune system can be classified into subsystems (See the lectures for more details): – Innate immunity (e.g. granulocytes, macrophages, NK cells, complement system) – Natural immunity (e.g. B1 B cells, γδ T cells) – Adaptive immunity (e.g. αβ T cells, B2 cells, antibodies) • The distinction above is artificial, in the organisms these work hand in hand. • You will mostly learn about the adaptive immunity throughout the semester.

Antibody = Immunoglobulin Innate vs adaptive immunity

Innate Adaptive Recognition Pattern-based (not Antigen-specific antigen-specific) Kinetics Quick (minutes, hours) Slow (days, weeks) Amplification of response Linear Exponential Immunological memory No Yes

Antigen: Substance recognized by T and receptors (TCR and BCR) which induces either an immune response or immunological tolerance. Difference between pattern-recognition and antigen-recognition:

Innate: „The cell surface is Adaptive: „This is the 45- full of carbohydrates 60 amino acid segment of usually found on bacteria - E. coli flagellin.” it must be some kind of bacteria.” E. coli Quiz follows Organs of the immune system

• The immune system is organized into a network of cells and organs. (the entire body must be protected from pathogens) • Lymphoid organs: – Primary (production of immune cells) • , , embryonic liver (+bursa of Fabricius in birds [nomenclature: „B” lymphocytes originating from the bursa and „T” cells from the thymus[1.]]) – Secondary (site of antigen recognition, immune response) • Lymph nodes, , MALT (mucosa-associated lymphatic tissue), SALT (skin-associated lymphatic tissue) – Tertiary (pathological, usually due to chronic inflammation) • E.g. ectopic (=at an abnormal site) lymphoid follicles Lymph nodes Lymphatic vessels

Thymus Lymph nodes

Spleen

Peyer’s patches

Appendix

Bone Lymph nodes marrow Lymphatic vessels Bone marrow (medulla ossium)

• Spongiform tissue found within bones which constitutes 4-5% of the total body weight in adults. (≈2,6 kg)[2.] • Red bone marrow (medulla ossium rubra): – Found in short and flat bones (sternum, ribs, clavicle, scapula, pelvis, vertebrae, skull) and the epiphysis of long bones (e.g. femur) – Role: Producing blood cells (hematopoiesis) → 1011 new cells daily of neutrophils alone[3.] (the human body is made of approx. 3,7x1013 cells)[4.] • Yellow bone marrow (medulla ossium flava): – Found in the diaphysis of long bones – Mainly composed of adipocytes, can turn into red bone marrow when needed (e.g. after blood loss) Structure of the red bone marrow

Outer surface of bone

Red bone marrow Yellow bone marrow

• Spongy bone tissue with sinusoids, spaces are filled with Epiphysis cells of various lineages undergoing hematopoiesis (see later), stromal cells and adipocytes.[2.] • Mature and naive B cells leave the bone marrow, whereas Diaphysis T cells produced by the bone marrow are still immature and must undergo further maturation in the thymus. • Mature: capable of recognizing an antigen • Naive: haven’t yet encountered an antigen Clinical significance of the bone marrow

• Bone marrow biopsy or aspiration for histological or cytological assessment in case of hematological diseases (e.g. leukemias, aplastic anemia, etc.) – Performed from: iliac crest or sternum[5.]

• Collecting hematopoietic stem cells (HSC) to perform bone marrow transplantation – Usually gathered from the peripheral blood after cell mobilization[6.] Thymus

• A lobulated organ located in the superior , it is the primary site of maturation. • Consists of 2 lobes further divided into lobules separated by connective tissue septa. The inner layer of the lobules is called medulla, the peripheral layer is the cortex.

Thyroid gland

Windpipe Right lobe Left lobe

Thymus Septa

Lobules Thymus (H&E staining): the peripheral, basophilic layer is the cortex. The inner medulla seems more eosinophilic because it contains less cell nuclei. Hall of shame Histology of the thymus

Dead Capsule Dividing Trabecula thymocytes Thymocytes Nurse cells thymocytes Cortical epithelial cell Cortex Maturing T cell precursors Cortex

Medulla Mature T cells Medulla Interdigitating Blood vessel dendritic cell Medullary Hassal’s Macrophage epithelial cell Hassal’s body corpuscles • T cell precursors (=immature cells) produced by the bone marrow enter the thymus through blood vessels → MATURATION (see later) → Mature and naive T cells leave the thymus and enter circulation • Main cellular components of the thymus: T cells (thymocytes), thymus epithelial cells, dendritic cells, macrophages, epithelioreticular cells[7.] Involution of the thymus

Age-related composition of the thymus Thymic lobes Weight (g)

Fat

Cortex

Medulla Cross-section Prenatal months Age (years)

Newborn 7 years 17 years 30 years Clinical significance of the thymus

• Congenital abnormalities (e.g. ectopic thymus or thymic aplasia [=absence of thymus] for instance in DiGeorge syndrome → immunodeficiency)

• Tumors (, thymus carcinoma)[8.] – May be associated with autoimmune disorders such as myasthenia gravis (see later) – Might compress nearby structures (e.g. superior vena cava syndrome, dysphagia, see later in the clinical phase of your studies) Thoracic CT angiography (dye seen in blood vessels): The red line marks a thoracic mass later confirmed to be a thymoma by histological evaluation. Quiz follows (nodus lymphaticus) • They act as filters of the lymph: lymph reaching the node through the afferent lymphatic vessels is filtered for pathogens and cancer cells. (one of the organs where the adaptive immune cells can meet with antigens the first time) • This is the place where the antigens that entered the will be recognized by the adaptive immune cells followed by cell proliferation and differentiation. • Tremendous clinical significance: Infectious agents and cancer cells may spread via the lymphatic system.

Retroperitoneal lymphadenomegaly (=enlarged lymph nodes) seen on a CT scan image. Arrows mark enlarged lymph nodes. Structure of lymph nodes 1.

• Have outer fibrous capsule from which trabeculae radiate towards the inner part of the organ. • Layers from outermost to innermost: cortex, paracortex and the medulla. • Afferent lymphatic vessels enter through the convex surface; the efferent lymphatic vessels and blood vessels (artery and venule) are located at the hilum. • Reticular connective tissue forms the frameworks of the lymph nodes. • Sites where immune cells enter: – From the bloodstream: (HEV) – From the lymphatic system: afferent lymphatic vessels • Cellular zones:[9.] – Cortex: B cells organized into follicles, cells that recognized an antigen proliferate and form germinal centers – Paracortex: T cells and dendritic cells diffusely – Medulla: mainly antibody-producing plasma cells Structure of lymph nodes 2. Route of lymph Afferent Lymph flow Primary follicle (covered with Secondary endothelial cells): follicle Capsule 1. Afferent Subcapsular Germinal lymphatic vessel sinus center 2. Subcapsular sinus

3. Cortical sinus

4. Paracortical sinus Medullary Cortex sinus (B cell zone) 5. Medullary sinus

Paracortex Senescent (T cell zone) Medulla 6. Efferent Efferent lympahtic vessel lymphatic vessel Blood vessels Structure of lymph nodes 3.

B cell-specific chemokine Chemokine attracting T cells and denditic cells The cellular organization is Dendritic cell controlled by chemokines. (see Naive later in lectures) B cell

Afferent lymphatic vessel

Naive T cell B cell zone T cell zone T cell zone Artery (paracortex) HEV Naive B cell B cell zone (follicle) Naive T cell Immunofluorescence microscopy (see later) Lymphoid follicle (folliculus lymphaticus) Capsule Afferent vessel Main cellular components: B cells, macrophages, follicular helper T cells, follicular dendritic cells Secondary follicle, germinal center Follicle (B cells) (FDC)

Primary follicle 1. Primary follicle: Naive B cells that haven’t yet Paracortical zone (T cells) met with an antigen

Medulla 2. Secondary follicle (germinal center):[9.] § Dark zone: centroblasts (proliferating B cells) Mantle § Light zone: centrocytes (B zone cells undergoing antigen- Light zone dependent maturation, see later) Dark § Mantle zone: transient B cells zone (=passing through) High endothelial venules (HEV) T cells binding to the luminal surface of a HEV HEVs in a lymph node HEVs (electron microscopy image)

L-selectin ligand on T cells binding to HEV endothelial cells (IHC) (frozen section assay)

• Lymphocytes use HEVs to enter lymphoid organs. (through L-selectin, see later) • Found in all secondary lymphoid organs (e.g. lymph nodes, tonsils, T cells HEV Peyer’s patches), EXCEPT THE SPLEEN[10.] Filtration of lymph by nodes 1.

Lymph node Naive without antigen T cell Activated Blood vessels HEV T cell 1. Infection on the periphery 2. The same antigen may enter the Site of infection lymphatic vessels in different (peripheral tissue) Microbes forms: • Native bound antigen (e.g. Efferent lymphatic Afferent vessel lymphatic vessel living bacteria) Lymph node with antigen • Native soluble form (e.g. proteins derived from dead HEV Lymphatic Blood bacteria) vessel vessel • Processed form: dendritic cells phagocytose the antigen and present it as a Efferent lymphatic vessel Thoracic duct peptide to helper T cells

Vena cava (see later) 3. Lymphocytes enter lymph nodes either through afferent lymph vessels or HEVs and meet with the antigens (see the lectures for more details) Lymph node Dendritic cell ANTIGEN PRESENTATION AND T CELL RESPONSE Cervical lymph nodes

Intercostal lymphatic vessels

Axillary lymph nodes DRAINING LYMPH NODE Cisterna chyli Paraaortic lymph nodes

Lymphatic vessels from the gut Dendritic cell ANTIGEN CAPTURE with the antigen AND TRANSPORT Inguinal INFECTION Free lymph nodes antigen

Lymphatic vessel

Connective tissue Quiz follows Spleen (lien or splen)

• Located in the left hypochondriac region of the abdomen, weighs approx. 150-200 grams. • Functions: – Immunological: filtering the blood for pathogens – Hemoglobin metabolism: elimination of aged red Heart blood cells by the reticuloendothelial cells→ formation of bilirubin Spleen

– Site of hematopoiesis in the embryo as in the Pancreas liver (can produce red blood cells in pathological Kidney conditions even in adults) – Acts as a storage of red blood cells and platelets (less significant in humans) Structure of the spleen 1.

• Has a fibrous capsule and trabeculae. • THERE ARE NO afferent lymphatic vessels and HEVs. • Tissue architecture:[11.] – Red pulp: sinusoids with an open circulation filled with blood: has a reticular framework populated mainly by red blood cells, macrophages, plasma cells and reticular fibrocytes. – White pulp: lymphoid tissue • PALS (periarteriolar lymphatic sheath): T cells, dendritic cells • Follicles (Malpighian follicles): B cells and follicular dendritic cells (FDC) • : special, marginal zone B cells (MZB, see later) and MZ macrophages Structure of the spleen 2.

Capsule Trabecula

Sinusoids Follicle Central arteriole PALS (T cell Marginal zone) White zone PALS pulp

Marginal zone B cell zone Germinal center Trabecular artery Follicle Red pulp Splenic Splenic vein artery

B cell zone (follicle)

T cell zone Germinal center (PALS) (secondary follicle also called Malpighian follicle in the spleen) PALS (periarteriolar lymphatic sheath) White pulp with immunohistochemistry

follicle

PALS: T cells artery

Labeling T cells in a murine spleen Chromogen used: AEC, substrate: H2O2 Clinical significance of the spleen

• (=enlarged spleen): Can have several causes such as hematological malignancies, hypersplenism (e.g. hemolytic anemia), increased pressure in the portal veins (cirrhosis), infections (mononucleosis, malaria), storage diseases[12.] • Splenic rupture (ruptura lienis): Caused by trauma or an underlying pathological condition, high risk of intra-abdominal hemorrhage • Splenectomy (=surgical removal of the spleen): Leads to increased vulnerability to polysaccharide encapsulated bacteria (see later)[13.] CT scan of a patient with chronic lymphocytic leukemia (CLL) showing massive splenomegaly. Quiz follows MALT (mucosa-associated lymphoid tissue)

• Mucosa = enormous surface for the pathogens to enter the body! • MALT = The biggest lymphoid tissue. • MALT: can be further classified based on location:[14.] – GALT (gut-associated lymphoid tissue) – BALT (bronchus-associated lymphoid tissue) – NALT (nasopharynx-associated lymphoid tissue) • Organized MALT (site of antigen recognition): – Lymphoid cells form organized structures such as follicles (e.g. tonsils of the Waldeyer-ring, Peyer’s patches, cryptopatches, isolated follicles, see in the lectures) • Diffuse MALT (has effector functions): – Lymphocytes diffusely scattered in the epithelial layer and lamina propria of mucosal surfaces (IEL=intraepithelial lymphocyte) Organized MALT

Waldeyer-ring (tonsils): Peyer’s patches in the ileum (H&E, cross-section): Tubal Pharyngeal tonsil

Palatine tonsil

Lingual tonsil

Peyer’s patch Intestinal villi Lumen

Both tonsils and Peyer’s patches have tissue architecture similar to that of lymph nodes (follicles with B cells, separated T cell zones, HEVs), but unlike lymph nodes they do not have fibrous capsules. SALT (skin-associated lymphoid tissue)

Langerhans cells capture the antigen in the epidermis, then Langerhans cell process it and move to the Epidermis (DC in the skin) draining lymph node through Keratinocytes lymphatic vessels. In the lymph Intraepithelial lymphocytes node they present the Lymphocytes processed antigen to helper T DC cells.[15.] Dermis Several cell types participate in Afferent lymphatic the immunological defense of Tissue vessel macrophage the skin. (e.g. keratinocytes, macrophages, γδ T cells, see later) Lymph node

Interdigitating dendritic cell Quiz follows Example for tertiary lymphatic tissue

IT IS PATHOLOGICAL!

Healthy tissue Ectopic lymphoid follicles in the thyroid (medium magnification) gland in Hashimoto’s thyroiditis (small magnification) Thank you for your attention!

• Feedback both from students and teachers is highly appreciated, you may send your remarks via email to: [email protected] References 1.

1. Davison TF: The immunologists' debt to the chicken. Br Poult Sci. 2003 Mar;44(1):6-21. 2. Travlos GS: Normal structure, function, and histology of the bone marrow. Toxicol Pathol. 2006;34(5):548- 65. 3. Blood Journal: Of mice and men … and elephants (http://www.bloodjournal.org/content/100/13/4679?sso-checked=true) 4. Bianconi E, et al.: An estimation of the number of cells in the human body. Ann Hum Biol. 2013 Nov- Dec;40(6):463-71 5. Riley RS, et al.: A pathologist's perspective on bone marrow aspiration and biopsy: I. Performing a bone marrow examination. J Clin Lab Anal. 2004;18(2):70-90. 6. Levesque JP, Winkler IG: Mobilization of hematopoietic stem cells: state of the art. Curr Opin Organ Transplant. 2008 Feb;13(1):53-8. 7. Pearse G: Normal structure, function and histology of the thymus. Toxicol Pathol. 2006;34(5):504-14. 8. Tomaszek S, et al.: : review of current clinical practice. Ann Thorac Surg. 2009 Jun;87(6):1973- 80. 9. Willard-Mack CL: Normal structure, function, and histology of lymph nodes. Toxicol Pathol. 2006;34(5):409-24. 10. Umemoto E, et al.: Novel regulators of lymphocyte trafficking across high endothelial venules. Crit Rev Immunol. 2011;31(2):147-69. References 2.

11. Cesta MF: Normal structure, function, and histology of the spleen. Toxicol Pathol. 2006;34(5):455-65. 12. Mayo clinic: Enlarged spleen (splenomegaly) (http://www.mayoclinic.org/diseases-conditions/enlarged- spleen/basics/causes/con-20029324) 13. Weledji EP: Benefits and risks of splenectomy. Int J Surg. 2014;12(2):113-9. 14. Cesta MF: Normal structure, function, and histology of mucosa-associated lymphoid tissue. Toxicol Pathol. 2006;34(5):599-608. 15. Tay SS, et al.: The Skin-Resident Immune Network. Curr Dermatol Rep. 2013 Nov 28;3:13-22. eCollection 2014.