Fundamentals of the Immune System

© Science photo/Shutterstock© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION CHAPTER 2 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORFundamentals SALE OR DISTRIBUTION of theNOT FOR SALE OR DISTRIBUTION

Barsha Dash, PhD; Brittney Dinkel, PhD; and Ian Clift, PhD, MLS(ASCP)CM

Alternative pathway Immune organs Neutrophils Basophils Lectin pathway Pattern recognition receptors Chemokines Lymphoid tissue inducer (PRR) © JonesClassical & Bartlett pathway Learning, LLC cells (LTi) © JonesPlasma & cells Bartlett Learning, LLC NOT FORClonal SALE expansion OR DISTRIBUTIONMacrophages NOT FORPrimary SALElymphoid OR tissues DISTRIBUTION Cytokines Major histocompatibility Secondary lymphoid tissues Dendritic cells (DCs) complex (MHC) Terminal pathway Hematopoietic stem cells (HSCs) Mast cells Histology Monocytes © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION LEARNING OBJECTIVES

Upon completion of this chapter, the reader should be able to: 1. Review© Jones the hist &ory Bartlett of immunology Learning, up to present LLC laboratory practices. © Jones & Bartlett Learning, LLC 2. UnderstandNOT FOR and differentiateSALE OR the DISTRIBUTION three lines of defense that make up the immuneNOT system. FOR SALE OR DISTRIBUTION 3. Specify the function of the major immune organs. 4. Outline the pathway of development for B and T cells via lineage and location. 5. Compare and contrast the pathophysiological role of the various complement pathways. 6. Identify and specify pattern recognition particles involved in innate immune response based on pathology. © Jones & 7.Bartlett Demonstrate Learning, understanding LLC of the cell types, function and form© Jones of MHC molecules. & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

© Jones & Bartlett Learning,© Jones LLC & Bartlett Learning LLC, an Ascend Learning© Jones Company. & NOT Bartlett FOR SALE Learning, OR DISTRIBUTION. LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

9781284173017_CH02_Clift.indd 15 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 16 Chapter 2 Fundamentals of the Immune System

▸▸ in cattle, and Koch experimentally demonstrated Introduction© Jones & Bartlett Learning,the LLC virulence of the bacteria by© firstJones isolating & Bartlett Learning, LLC The immune systemNOT hasFOR the SALE remarkable OR abilityDISTRIBUTION it, and then injecting healthy animalsNOT FOR with it.SALE OR DISTRIBUTION to discriminate between host and pathogens to Injected animals developed disease, definitively eliminate infections and preserve organismal demonstrating the bacteria to be the causative homeostasis. To this end, it relies on two key agent of the disease. Koch developed numer- elements—the innate and adaptive systems— ous techniques to isolate, grow, and inoculate © tJoneshat work & hand Bartlett in hand Learning, to eradicate LLC bacte- bacteria that are© Jonesstill in use & Bartletttoday. The Learning, works LLC NOTria, FORviruses, SALE and parasites.OR DISTRIBUTION Innate responses of Pasteur and NOTKoch elucidatedFOR SALE the importance OR DISTRIBUTION are driven by encoded pattern recognition of hypothesizing and developing experimental receptors (PRR) that detect a limited and con- tools to demonstrate the cause of an infectious served set of molecular patterns present on disease, enabling departure from early ideas of microbes and usually absent on mammalian spontaneous generation of disease. Koch went © Jones & Bartlettcells. Learning,However, adaptive LLC mechanisms depend © onJones to demonstrate & Bartlett that Learning,tuberculosis wasLLC caused NOT FOR SALEon OR many DISTRIBUTION genetic rearrangements, engender- NOTby aFOR bacterium SALE (Mycoba OR DISTRIBUTIONcterium tuberculosis). ing spectacular diversity in molecules that aid Pasteur also discovered that long-term culturing in recognition of pathogen-derived substances of bacteria attenuated the bacteria or made them called antigens. In addition, a salient feature of less virulent, generating the roots for attenuated the adaptive immune system is clonal expan- © Jones & Bartlett Learning,vaccine LLC applications, which involves© Jones adminis & -Bartlett Learning, LLC sion whereby adaptive cells that find their cog- tration of a weakened pathogen as vaccine to nate antigen undergo rapid expansion. These NOT FOR SALE OR DISTRIBUTIONcreate memory. Edward Jenner, anNOT 18th FOR century SALE OR DISTRIBUTION cells then partake in pathogen elimination and physician from England, had already shown that memory formation, which is essential for rapid inoculation with cowpox conferred protection elimination of pathogens upon reencounter. To against smallpox by immunizing a young boy summarize, collective specificities of the innate with bacteria from cowpox of a dairy farmer. © andJones adaptive & Bartlett responses Learning, allow for detection LLC and © Jones & Bartlett Learning, LLC NOTmounting FOR ofSALE potent OR defenses DISTRIBUTION against invading These observationsNOT FORand heroic SALE experimental OR DISTRIBUTION pathogens, while minimizing harm to the host. endeavors emphasized the importance of under- The specificity of the adaptive cell repertoire to standing biology, establishing methodologies minimize host damage is called immune toler- to find disease etiology and methods to exploit ance. Breakdown of tolerance can result in auto- immunological memory, although immunity © Jones & Bartlettimmune Learning, diseases that LLC entail immune-mediated © hadJones not yet & beenBartlett understood. Learning, LLC NOT FOR SALEdestruction OR DISTRIBUTION of organs. This chapter aims to pro- NOT FORIn the SALE 1800s, OR Paul DISTRIBUTION Erlich, a German vide an overview of immune cell development, physician-scientist, developed histologic meth- function, and immune organs at the anatomical ods to examine innate cells such as neutrophils, and molecular levels. basophils, and eosinophils. Histology involves staining of cells with different compounds to © Jones & Bartlett Learning,enable LLC visualization of cell morphology.© Jones Erlich’s & Bartlett Learning, LLC History NOT FOR SALE OR DISTRIBUTIONbackground in chemistry allowedNOT him FOR to put SALE OR DISTRIBUTION Nineteenth century paradigms created a fer- forth early theories of immune recognition of tile foundation for modern immunology. Louis microbes and microbe-derived substances. He Pasteur, a French scientist, and Robert Koch, a proposed that “antitoxins,” produced by certain German scientist, put forth the “germ theory”, cells of the host, bind via chemically specific © i.e.,Jones the postulation & Bartlett that Learning, microorganisms LLC cause interactions with© Jonestoxins, neutralizing & Bartlett their Learning, vir- LLC NOTdiseases. FOR Pasteur SALE hypothesized OR DISTRIBUTION that the bacte- ulence. He demonstratedNOT FOR this SALE by isolating OR serumDISTRIBUTION rium Bacillus anthracis caused Anthrax disease containing “antitoxin” and inoculating animals

9781284173017_CH02_Clift.indd 16 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Antimicrobial Small Molecules as the First Barrier of Protection 17

exposed to diphtheria toxin. Animals that were must cooperate to mount immune responses, given the© “antitoxin” Jones &survived. Bartlett Learning, LLCi.e., phagocytes express MHC© Jones molecules & that Bartlett dis- Learning, LLC Now,NOT it is wellFOR known SALE that OR B cells, DISTRIBUTION cells of play peptides derived fromNOT antigens FOR for SALE activa- OR DISTRIBUTION adaptive immunity, produce antibodies that tion of T cells. Currently, with the help of gene bind to toxins and neutralize their virulence, sequencing and protein structure determination, protecting the host. This was the first step it is well appreciated that MHC molecules are toward understanding humoral or B-cell medi- highly diverse (polymorphic) within the human © Jonesated & immunity. Bartlett AsLearning, new methodological LLC tools, population© andJones govern & susceptibility Bartlett Learning, to infectious LLC NOT FORenabling SALE protein OR separation DISTRIBUTION based on charge and diseases,NOT autoimmunity, FOR SALE and cancer. OR DISTRIBUTION size, enzymatic digestive methods for proteins, Elegant experiments of the past have built and protein structural analysis techniques were the foundations of modern immunology. Our developed, appreciation of the structure and understanding of immunology has led to devel- chemical nature of antibodies grew. In the 20th opment of diagnostic tools, which, in turn, have © Jones & Bartlett Learning,century, focus LLC shifted to an examination of© the Jones led & to Bartlett fine-tuning Learning, of our knowledge LLC about the NOT FOR SALE ORimmune DISTRIBUTION cells, laying down foundations forNOT cel- FORimmune SALE system, OR giving DISTRIBUTION birth to many avenues lular immunology. of immunology: developmental immunology, Frank Macfarlane Burnet, an Australian host-pathogen interaction, structural immu- immunologist, proposed the theory of clonal nology, cancer immunology, and auto immunity. expansion. This theory postulated that there These fundamental understandings underpin were many© immuneJones cells& Bartlett of varying Learning, specificities, LLCour clinical aptitude in immunodiagnostics© Jones & Bartlett and Learning, LLC and thatNOT upon exposureFOR SALE to a stimulant, OR DISTRIBUTION such as serology. NOT FOR SALE OR DISTRIBUTION a bacterium, the cell with highest recognition affinity would multiply, i.e., clonally expand. It is now known that both T and B cells of the adaptive arm of the immune system undergo ▸▸ Antimicrobial Small © Jonesclonal & Bartlett expansion Learning, upon antigen LLC encounter. © Jones & Bartlett Learning, LLC NOT FORAustralian SALE scientists OR DISTRIBUTION Peter Doherty and Rolf MoleculesNOT FOR asSALE the OR First DISTRIBUTION Zinkernagel discovered that T cells recognized Barrier of Protection antigens presented by molecules called major histocompatibility complex (MHC). This was Before antigens encounter immune cells, they a seminal finding and is called MHC-restriction are confronted by barrier immunity, a combi- © Jones & Bartlett Learning,and the fundamental LLC basis of T-cell immune© rec Jones- nation & Bartlett of physical Learning, and biochemical LLC protection1 NOT FOR SALE ORognition. DISTRIBUTION NOT FOR(FIGURE SALE 2–1 ).OR Physical DISTRIBUTION protection primarily With the advent of cloning techniques and consists of the skin and walls of the oral, intes- protein crystallization techniques, these ideas tinal, and respiratory systems. Biochemical pro- paved the way to greater structural knowledge tection consists of tears, mucus, antimicrobial of immune-cell receptors and proteins, ulti- peptides (APPs), and proteins such as defensins mately leading© Jones to discovery & Bartlett of the T-cellLearning, surface LLCand cathelicidins; and proteolytic© Jones enzymes & Bartlett such Learning, LLC receptor NOT(TCR) FOR by Mark SALE Davis, OR an DISTRIBUTION American as lysozyme secreted by NOTspecialized FOR epithelial SALE OR DISTRIBUTION immunologist; and to the structural character- cells of the skin and respiratory and digestive ization of a MHC molecule, by Pamela Bjork- tracts. APPs are primarily cationic molecules man, also an American immunologist. The TCR and prevent pathogen dissemination by disrupt- guides T-cell functions, including direct killing ing anionic bacterial cell walls through electro- © Jonesof &infected Bartlett cells Learning,(cytolytic function) LLC and helper static interactions.© Jones Physical & Bartlett mechanisms Learning, such as LLC NOT FORT-cell SALE functions OR that DISTRIBUTION allow for B-cell activation. It ciliary motionsNOT FOR and SALEperistalsis OR facilitate DISTRIBUTION the soon became clear that innate and adaptive cells expulsion of pathogens trapped in mucus. Thus,

9781284173017_CH02_Clift.indd 17 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 18 Chapter 2 Fundamentals of the Immune System

Barrier Innate Adaptive immunity © Jones & Bartlett Learning, LLC immunity immunity© Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

First line Second line Third line of defense of defense of defense © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Pathogen

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION FIGURE 2–1 The three major lines of defense against pathogens. Barrier immunity including skin and intact mucous membranes, innate immunity including in inflammation and phagocytosis, and adaptive immunity or the cellular response. Modified from Engelkirk, P., et al. Microbiology for the Health Sciences, 11e. Philadelphia, Pennsylvania: Lippincott Williams & Wilkins, 2018. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC biochemical barriersNOT FORaid in SALEpathogen OR destruc DISTRIBUTION- focus of infection, leading to theNOT recruitment FOR ofSALE OR DISTRIBUTION tion and prevent their dissemination. innate and adaptive cells, setting off more com- Only a few weeks before the birth of a plex molecular pathways, cellular crosstalk, and human baby, fetal skin produces copious quan- activity. © titiesJones of antimicrobial & Bartlett molecules Learning, in anticipation LLC © Jones & Bartlett Learning, LLC of exposure to microbes following birth, form- NOTing aFOR thick protectiveSALE OR layer DISTRIBUTION called vernix caseosa. NOT FOR SALE OR DISTRIBUTION Immediately after birth, the skin is still vulnera- ▸▸ Organs of the Immune ble to microbial breach as its most resilient layer is still in the making. Hence, a baby born before System: Sites of Immune the burst of microbial molecule secretion occurs © Jones & Bartlettis susceptible Learning, to fungal LLC and gram-negative bac- © JonesCell & Development Bartlett Learning, and LLC NOT FOR SALEterial OR infections. DISTRIBUTION With current knowledge about NOT FOR SALE OR DISTRIBUTION the importance of barrier functions and the Activity mechanisms of APP activity, research is being The immune system is highly compartmental- geared toward treatment of infants with anti- ized. The organs of the immune system provide microbial peptides that are effective in reducing niches for development of immune cells and the incidence of severe© Jones diseases & such Bartlett as meningitis Learning, LLC © Jones & Bartlett Learning, LLC and sepsis causedNOT by FOR bacteria. SALE An ORimportant DISTRIBUTION generation of immune responses viaNOT immune FOR cell SALE OR DISTRIBUTION 2 FIGURE 2–2 implication of using APPs in preterm infants activation ( ). Lymphocytes develop in is the reduction of antibiotic administration to the primary lymphoid tissues: bone marrow or combat opportunistic infections. In recent years, thymus. Secondary lymphoid tissues, such as numerous bacterial strains are gaining antibiotic lymph nodes and spleen, help maintain mature © resistance,Jones & and Bartlett it is an Learning,urgent necessity LLC to find lymphocytes until© Jonesan infection & Bartlettarises. Learning, LLC NOTalternative FOR SALEtreatment OR options. DISTRIBUTION During development,NOT FOR these SALE immune OR organs DISTRIBUTION Pathogens that evade microbicidal activities (including lymphoid organs) provide optimal of the skin and other barrier defenses create a niches replete with necessary developmental cues

9781284173017_CH02_Clift.indd 18 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Organs of the Immune System: Sites of Immune Cell Development and Activity 19

Axillary node Bronchial lymphoid patch Spleen Mucosa associated © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,lymphoid LLC tissue (MALT) NOT FOR SALE OR DISTRIBUTIONLymph NOT FOR SALE OR DISTRIBUTION vessels

Peyer’s lymphoid © Jones & Bartlett Learning, LLC patches of © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONintestinal mucosa NOT FOR SALE OR DISTRIBUTION Inguinal lymph nodes © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Bone marrow

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC FIGURE 2–2NOT Lymphoid FOR organs SALE of the OR human DISTRIBUTION body. NOT FOR SALE OR DISTRIBUTION Modified from McConnell, Thomas H. The nature of Disease: Pathology for the Health Professions. Lippincott Williams & Wilkins, 2013.

such as cytokines; soluble messengers that bind as foci for efficient interaction between innate receptors on cell surface and initiate gene expres- and adaptive cells, allowing for the concentration © Jonession & changes,Bartlett and Learning, chemokines LLC; soluble and of antigens© Jonesand an increasing & Bartlett chance Learning, for antigenic LLC NOT FORinsoluble SALE molecular OR DISTRIBUTION cues for migration. These encounterNOT with FOR T cells SALE and B cells OR possessing DISTRIBUTION the cues allow for cell-fate determination and matu- optimally specific receptors for the antigens. DCs ration. During immune cell activation, they serve initiate cell activation, functioning as a crucial

9781284173017_CH02_Clift.indd 19 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 20 Chapter 2 Fundamentals of the Immune System

link between innate and adaptive systems. Cel- (Immune dysregulation, polyendocrinopathy, lular interactions© Joneswithin lymphoid& Bartlett organs Learning, are enteropathy, LLC X-linked) in which© Jonesthe immune & Bartlett Learning, LLC mediated via receptorNOT FORengagement, SALE communica OR DISTRIBUTION- system attacks many different organsNOT FORin a sys SALE- OR DISTRIBUTION tion through secretion of cytokines, and chemo- temic manner.3 kines. Integrity and homeostasis of these organ systems are imperative for functional immu- Hematopoiesis © nityJones and are& Bartlettindicators Learning,of the general LLC health of Hematopoiesis ©is Jonesthe process & Bartlettof creating Learning, all of LLC the host. Examination of lymphoid organs may NOT FOR SALE OR DISTRIBUTION the blood cells NOTin the FORbody, i.e.,SALE red bloodOR DISTRIBUTION cells reveal vital information about the status of an and immune cells (innate and adaptive).4 Over individual’s health during the diagnosis of the the course of development of humans, hema- disease. For example, lymphadenopathy, or the enlargement of lymph nodes, indicates ongo- topoiesis takes place at three main locations. © Jones & Bartletting inflammation Learning, asLLC a result of an infection. © PrimitiveJones &hematopoiesis Bartlett Learning, begins in the LLC yolk sac and upon onset of blood circulation, shifts to the NOT FOR SALELymphadenopathy OR DISTRIBUTION in the absence of infection is NOT FOR SALE OR DISTRIBUTION often because of the breakdown of immune tol- liver, followed by a final shift to the bone mar- erance or autoimmune diseases such as Myas- row, the site of definitive hematopoiesis, sup- thenia Gravis, whereby the immune system porting the development of blood cells in young attacks the neuromuscular system, or IPEX, and adult humans (FIGURE 2–3). © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Fetal liver Bone marrow Blood AGM © Jones & Bartlett Learning,islands LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Embyronic PrimitiveDefinitive

17 27 30 After- 40 days Days

© FIGUREJones 2–3 &Sites Bartlett of hematopoiesis Learning, during humanLLC development. The major sites© ofJones hematopoiesis & Bartlett shift during Learning, LLC NOTembryonic FOR and SALE fetal development OR DISTRIBUTION with hematopoiesis starting in the yolk sac,NOT then shifting FOR to SALE the aorta-gonado- OR DISTRIBUTION mesonephros (AGM), then to the fetal liver, and then to the bone marrow. The arrows indicate the various sites of hematopoiesis based on the developmental stage. Modified from Orazi, Attilio, et al. Knowles Neoplastic Hematopathology. Lippincott Williams & Wilkins, 2013.

9781284173017_CH02_Clift.indd 20 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Organs of the Immune System: Sites of Immune Cell Development and Activity 21

Primary Lymphoid Organs: many other cells that regulate and support self- Bone Marrow© Jones & Bartlett Learning, LLCrenewal of HSCs and differentiation© Jones & of Bartlett HSCs Learning, LLC NOT FOR SALE OR DISTRIBUTIONinto myeloid and lymphoidNOT lineages. FOR The SALE marrow OR DISTRIBUTION The bone marrow is a spongy tissue in the central is abundantly vascularized, allowing for influx cavity of bone.5 It is the home to hematopoietic of oxygen and nutrients via arteries, trafficking stem cells and their renewal, the site of generations of precursors to distant locations and egress of of myeloid and lymphoid precursors (FIGURE 2–4), mature cells that have completed development via © Jonesand & the Bartlett primary Learning,organ of B-cell LLC development. venules (very© Jones small veins) & Bartlett called sinusoids. Learning, Migra- LLC NOT FORHematopoietic SALE OR stem DISTRIBUTION cells (HSCs) reside in the tory patternsNOT are FOR governed SALE by chemokines. OR DISTRIBUTION HSCs bone marrow. The two salient features of HSCs are are found close to these sinusoids surrounded by self-renewal and differentiation.4 HSCs are found nonhematopoietic stromal cells. along with nonhematopoietic cell types such as The knowledge of regenerative capacity endothelial cells, osteoblasts, chondrocytes, and of HSCs, i.e., self-renewal, has been critical for © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION LymphoidNOT FOR SALE OR DISTRIBUTION stem cells

Lymphocyte production

Lymphoid Lymphoid © Jones & Bartlett Learning, LLCstem cell stem cell © Jones & Bartlett Learning, LLC Thymosine NOT FOR SALE OR DISTRIBUTIONThymus NOTRed bone FOR SALE OR DISTRIBUTION marrow

Mature Mature © Jones & Bartlett Learning, LLC T- lymphocyte© JonesB-lymphocy & teBartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Stored in secondary lymphoid © Jones & Bartlett Learning,tissue LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Immune action Immune action

Cell-mediated Antibody-mediated © Jones & Bartlett Learning,immune LLC response immune response© Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION FIGURE 2–4 Lymphocytes are produced in the bone marrow. Lymphocytes emerge in the red bone marrow and either stay and mature into B lymphocytes or migrate to the thymus to become T lymphocytes. Modified from Archer, Pat, and Lisa A. Nelson. Applied Anatomy & Physiology for Manual Therapists. Lippincott Williams & Wilkins, 2012.

9781284173017_CH02_Clift.indd 21 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 22 Chapter 2 Fundamentals of the Immune System

modern medicine regenerative therapies such as culminating in a large repertoire of cells with bone marrow transplantation.© Jones &6 InBartlett addition, Learning,greater novel LLC specificities, which may include© Jones potentially & Bartlett Learning, LLC understanding ofNOT step-by-step FOR SALE hematopoiesis OR DISTRIBUTION and autoreactive TCRs, and nonfunctionalNOT FOR recep SALE- OR DISTRIBUTION markers to identify precursor cells and terminally tors. Thymic epithelial cells not only support the differentiated cells have been instrumental in development of cells but also aid in the selection characterizing hematopoietic failures in aplastic of cells that are functional and nonautoreactive. anemia, whereby Th1 cells mediate destruction In addition, the thymus harbors DCs, macro- © ofJones HSCs and& Bartlett chronic myeloid Learning, leukemia LLC (CML) phages, and B cells© Jones that facilitate & Bartlett T-cell selection. Learning, LLC NOTcharacterized FOR SALE by malignant OR DISTRIBUTION transformation of an Two major kindsNOT of T cellsFOR develop SALE in theOR thymus, DISTRIBUTION HSC that gives rise to myeloid progenitors with CD4 T cells and CD8 T cells.8 As mentioned ear- self-renewal capacity and superior proliferative lier, CD4 T cells, when activated, differentiate into capacity. Treatments for both of these conditions helper T cells, activating B cells and CD8 T cells; include bone marrow transplantation in combi- and CD8 T cells mediate direct killing of infected © Jones & Bartlettnation Learning, with other treatments. LLC © cells.Jones Following & Bartlett development, Learning, T cells egressLLC from NOT FOR SALE OR DISTRIBUTION NOTthe thymusFOR andSALE make OR their DISTRIBUTION way to secondary lymphoid organs, guided by chemokine molecules in Primary Lymphoid Organs: Thymus the blood. The fundamental role that the thymus The thymus consists of two main kinds of cells: plays in immunity is now well appreciated. epithelial cells that form the thymic architec- The human thymus is multilobed while ture, and thymocytes,© Jones which & Bartlett are lymphoid Learning, murine LLC thymus has two lobes. ©T-cell Jones develop & -Bartlett Learning, LLC cells.7,13 In adultNOT humans FOR and SALE mice, the OR thymus DISTRIBUTION ment takes places in two main locationsNOT FOR in both SALE OR DISTRIBUTION is located above the heart. During embryogene- human and mouse thymi, namely, the cortex and sis, as foregut development takes place, pharyn- the medulla. The migration of T cells occurs from geal pouches arise from the foregut. The thymus the cortex to the medulla as development pro- originates from the endoderm of a pharyngeal gresses and is dependent on chemokine cues. The © pouch,Jones which & Bartlett also gives Learning, rise to the parathyroid LLC medullary thymic© Jonesepithelial &cell Bartlett is a gene called Learning, an LLC autoimmune regulator (AIRE), which is critical NOTgland. FOR Thymus SALE development OR DISTRIBUTION happens in four NOT FOR SALE OR9 DISTRIBUTION major steps: formation of pharyngeal pouches, for elimination of autoimmune T cells. Congeni- tal defects in thymus development or loss of AIRE formation of the thymus from the pouch, protein function culminate in severe disorders detachment of the thymus from the endoderm, such as DiGeorge Syndrome and autoimmune and continued development followed by migra- polyendocrinopathy-candidiasis-ectodermal © Jones & Bartletttion toLearning, final location LLC at the pericardium by © Jones & Bartlett Learning, LLC dystrophy (APECED).10–13 DiGeorge syndrome NOT FOR SALE9 weeks OR DISTRIBUTION of gestation. The adult thymus consists NOT FOR SALE OR DISTRIBUTION results in thymic aplasia, creating reduced T-cell of a mesenchymal capsule covering the epi- development leading to recurrent infections. thelial framework. It is vascularized to aid the Current treatment for DiGeorge includes thymus influx of common lymphoid progenitors and the transplantation. APECED results in a severe mul- exit of thymocytes that have completed develop- tiorgan autoimmunity. The discovery of AIRE’s ment. Maintenance© Jones of thymus & Bartlettintegrity in Learning, adults LLC © Jones & Bartlett Learning, LLC importance has definitively established that cen- is dependent onNOT crosstalk FOR with SALE lymphocytes. OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION tral tolerance is enforced in the thymus. The thymus is the site of T-cell development from the site of T-cell development from common lymphoid progenitors (CLPs). Thymic epithelial Secondary Lymphoid Organs: cells orchestrate T-cell development by provid- © ingJones molecular & Bartlett cues for Learning,commitment ofLLC CLPs to Lymph Nodes© Jones & Bartlett Learning, LLC NOTthe T-cellFOR lineageSALE and OR production DISTRIBUTION of functional Lymph is fluid fromNOT the FOR tissue SALE that is drained OR DISTRIBUTION away T cells. Development of T cells involves genetic from tissues into lymph nodes (LN) (FIGURE 2–5) rearrangement at the T-cell receptor gene loci, for filtration and supplied back to the blood.14

9781284173017_CH02_Clift.indd 22 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Organs of the Immune System: Sites of Immune Cell Development and Activity 23

Outer cortex © Jones & Bartlett Learning,(B cells, macrophages, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONand dendritic cells) NOT FOR SALE OR DISTRIBUTION Afferent lymphatic vessels

Artery © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTIONVein

Germinal center

FIGURE 2–5 Anatomy of a Lymph Node. © Jones & BartlettData from Reisner, Learning, H. (2017). Crowley's an introduction LLC to human disease, 10e. Jones & Bartlett Learning. © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Lymph nodes develop from lymphoid tissue macrophages, awaiting antigen entry, and poised inducer cells (LTi).15–17 LTi cells arise at around to capture and present to B cells and T cells to 8 weeks of gestation from the fetal liver. Lymph trigger adaptive responses. Chemokines produced nodes form when LTi cells interact with mesen- by LN attract lymphocytes to enter the LN via © Jones & Bartlett Learning,chymal cells requiredLLC for organization of lymph© Jones the &high Bartlett endothelial Learning, venules. B LLCcells entering the NOT FOR SALE ORnodes DISTRIBUTION in the presence of appropriate molecularNOT FORLNs areSALE confined OR to DISTRIBUTION follicles called the B-cell zone, signals. These signals have not been fully dissected while T cells are found in paracortical areas called in humans. During inflammatory responses, T-cell zones where DCs are positioned to stimu- tertiary lymph nodes may arise from cells that late T cells. The positioning of these cells is depen- resemble LTi cells in their molecular signature. dent on chemokine gradients within the LN. The LNs are ©hubs Jones of interaction & Bartlett for innate Learning, cells and LLCencountering of B cells and© T cells Jones with & antigens Bartlett Learning, LLC adaptive NOTcells and FOR for theSALE concentration OR DISTRIBUTION of anti- begins a cascade of events thatNOT include FOR interaction SALE OR DISTRIBUTION gens for immune cell activation. of B cells with T cells, clonal expansion to create LNs are scattered throughout the body and effector cells that will actively fight infections, and capture pathogens that are not eliminated by long-lived memory cells for future responses. In initial innate responses by filtering lymph- and addition, LNs are important sites for tolerance © Joneschemokine-mediated & Bartlett Learning, recruitment LLC of activated enforcement.© Jones DCs from & tissuesBartlett continually Learning, present LLC NOT FORDCs SALEcarrying ORantigens. DISTRIBUTION Afferent lymphatic vessels self-derivedNOT peptides FOR to SALE T cells thatOR may DISTRIBUTION be poten- drain lymph into subcapsular sinuses (SCS). The tially autoreactive, rendering them anergic or in a subcapsular sinuses within the LN are lined by state of inactivity, a form of peripheral tolerance.

9781284173017_CH02_Clift.indd 23 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 24 Chapter 2 Fundamentals of the Immune System

Secondary Lymphoid Organs: whereby allergies and autoimmunity increase as exposure to infectious microbes decrease.20 Spleen © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONIn summary, the immune systemNOT is FOR a highly SALE OR DISTRIBUTION The spleen arises from the mesenchymal layer of compartmentalized system, and its components tissue during embryonic development. It is made are scattered throughout the body to capture and of two functionally and anatomically distinct eliminate pathogens and to direct the develop- regions: a large red pulp area where red blood cells ment of immune cells. This widespread distribu- © areJones broken & down Bartlett and discarded Learning, as well LLC as acting tion is important© for Jones local tissue & Bartlett surveillance. Learning,16 LLC NOTin iron FOR turnover, SALE and OR the smallerDISTRIBUTION white pulp area. NOT FOR SALE OR DISTRIBUTION Immune interactions happen primarily in the white pulp. Lymphocytes enter the white pulp via ▸ the highly branched trabecular artery. The white ▸ Innate and Adaptive Cells pulp is organized similarly to that in LN, consist- The cells of the immune system are what we are © Jones & Bartletting of B-cellLearning, and T-cell LLC zones. Just like the LNs, the © mostJones familiar & Bartlett with in the Learning, diagnostic LLClaboratory. NOT FOR SALEspleen OR hasDISTRIBUTION resident phagocytic cells that capture NOTThe variousFOR SALE myeloid ORand lymphoid DISTRIBUTION cells that make antigens to aid in pathogen clearance and T- and up the cellular immune system are a common B-cell activation essential for clonal expansion, source of information in the clinic. Along with the effector functions, and memory formation. lymphoid organs described above, these cells are fundamental to the processes of innate and adap- © Jones & Bartlett Learning,tive LLC immunity. The myeloid and ©mono Jonescytic cells& Bartlett Learning, LLC Secondary LymphoidNOT FOR Organs: SALE OR DISTRIBUTIONare described below, which haveNOT funda FORmental SALE OR DISTRIBUTION Mucosa-Associated Lymphoid Organs roles in both innate and adaptive systems. Absent Ingestion of food and colonization of the gut with from this discussion are the lymphocytes, in friendly but foreign bacteria requires constant which a more robust discussion is provide in a subsequent chapter. © immuneJones modulation& Bartlett at Learning,the interface ofLLC mucosal © Jones & Bartlett Learning, LLC surfaces that like the gut.17,18 Additionally, the air NOTthat FORwe breathe SALE via the OR nose DISTRIBUTION contains foreign par- NOT FOR SALE OR DISTRIBUTION ticles and pathogens, which must be tackled by Myeloid Cells: Granulocytes: the mucosa lining the nose and respiratory tract. Neutrophils, Basophils, The mucosal tissues that line these surfaces have many lymphoid structures specialized in antigen Eosinophils, and Mast Cells © Jones & Bartlettcapture Learning, and clearance. LLC The fundamental archi- © NeutrophilsJones & Bartlett are polymorphonuclear Learning, LLCleukocytes NOT FOR SALEtecture OR ofDISTRIBUTION these structures is akin to that of the NOTwith FORlobulated SALE nuclei OR21 (FIGURE DISTRIBUTION 2–6). Their cyto- LN and spleen, consisting of demarcated areas plasms are rich in granulocytic factors that develop for B cells and T cells and the presence of phago- in the bone marrow. During infections, neutrophil cytic cells for antigen capture and display. These production increases and neutrophils migrate to tissues are called Nasal Associated Lymphoid Tis- tissues for pathogen clearance. Neutrophils kill sue (NALTS), ©Bronchus Jones Associated & Bartlett Lymphoid Learning, pathogens LLC by phagocytosis followed© by Jones killing with & Bartlett Learning, LLC Tissue (BALTS)NOT and GutFOR Associated SALE ORLymphoid DISTRIBUTION the help of reactive oxygen species (ROS),NOT degranuFOR SALE- OR DISTRIBUTION Tissue (GALTS).19 These surfaces are colonized by lation of proteolytic enzymes, or NET (Neutrophils commensal bacteria, which are critical for over- Extracellular Traps) formation. NETs consist of all immune system maturation. Epidemiological DNA and protein traps to prevent dissemination of studies suggest an increased association of aber- pathogens. These are short-lived cells with a half- © rantJones immune & Bartlett function, suchLearning, as allergies LLC to innoc- life of 1.5 hours© in Jonesmice and &8 hours Bartlett in humans. Learning, LLC NOTuous FOR substances SALE because OR DISTRIBUTIONof lack of exposure to BasophilsNOT are also FOR granulocytes SALE that OR develop DISTRIBUTION commensal as well as infectious microbes. This in the bone marrow.22 They harbor granules con- observation has led to the hygiene hypothesis, taining histamines, proteoglycans, granzyme B, as

9781284173017_CH02_Clift.indd 24 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Complement Cascade 25

Granulocytes and DCs are phagocytic professional antigen-pre- © Jones & Bartlett Learning, LLCsenting cells (pAPCs). Macrophages© Jones & are Bartlett large Learning, LLC NOT FOR SALE OR DISTRIBUTIONmononuclear leukocytes thatNOT inhabit FOR lymphoid SALE and OR DISTRIBUTION nonlymphoid organs. They are important senti- nels of immune surveillance because of their supe- Nucleus rior phagocytic capacity. Phagocytosis allows them Erythrocyte to sample their environment for potential tissue © Jones & BartlettA Neutrophil Learning, LLC damage or© infection,Jones leading& Bartlett to initiation Learning, of mech- LLC NOT FOR SALE OR DISTRIBUTION anisms toNOT kill pathogens,FOR SALE mitigate OR damage,DISTRIBUTION and reestablish tissue homeostasis. Macrophages are a heterogeneous population of cells and are highly Granules plastic; their immediate environments shape their Nucleus properties. The different kind of macrop hages are Erythrocyte © Jones & Bartlett Learning, LLC © JonesKupffer & Bartlett cells in the Learning, spleen, Lang erhansLLC cells in the NOT FOR SALE OR DISTRIBUTIONB Eosinophil NOT FORskin, SALEand microglia OR DISTRIBUTIONin the brain. Dendritic cells (DCs) are phagocytic leukocytes that circulate in the blood or take up resi- Granules dence in peripheral tissues.25,27,28 They are essential Nucleus for immune surveillance and activation of adap- © Jones & Bartlett Learning, LLCtive immune cells. While there© Jones are subcategories & Bartlett of Learning, LLC NOTC Basophil FOR SALE OR DISTRIBUTIONDCs, all DCs are derived fromNOT common FOR SALEDC pro- OR DISTRIBUTION genitors (cDCPs).29 In the bone marrow, cDCPs FIGURE 2–6 Granulocytes images of N, E, B. differentiate into pre-DCs that migrate via blood Modified from Ruth McCall, Phlebotomy Exam Review, Jones & Bartlett Learning, 2015. to tissues to complete differentiation in lymphoid well as growth factors (Figure 2–6). Upon activa- and nonlymphoid organs. cDCPs are also precur- © Jonestion, & basophils Bartlett degranulate. Learning, Like LLC neutrophils, they sors to plasmacytoid© Jones &DCs Bartlett that aid Learning,in virus clear- LLC NOT FORalso haveSALE a short OR lifespan. DISTRIBUTION Eosinophils are granu- ance. DCsNOT sample FOR the environmentSALE OR for DISTRIBUTION pathogens locytes with bi-lobed nuclei and which contain using their PRRs and, upon antigen encounter, secretory granules.23 They leave the bone marrow they mature and migrate to lymph nodes to acti- fully mature and circulate in the blood. Eosino- vate T cells and secrete chemokines to shape the phils are known to modulate functions of other course of the adaptive immune responses. © Jones & Bartlett Learning, LLC © Jones &DC Bartlett macrophages Learning, are professional LLC antigen- immune cells by secretion of growth factors and 26 NOT FOR SALE ORmolecular DISTRIBUTION cues known as chemokines. Similarly,NOT FORpresen SALEting cells. OR DISTRIBUTIONThey engulf antigens and mast cells are granulated leukocytes that originate display them as small peptide fragments on their in the bone marrow.24 They circulate in the blood cell surface to activate T cells, setting off the adap- and take up residence in tissues where they mature tive responses in motion. Fragmented peptides further. Engagement of receptors on mast cells are displayed on protein molecules called major leads to degranulation,© Jones & causing Bartlett the releaseLearning, of his- LLChistocompatibility complex© (MHC). Jones T cells & Bartlett recog- Learning, LLC tamines, NOTproteases, FOR and SALEother molecules OR DISTRIBUTION that aid in nize peptide-MHC complexes;NOT this FOR recogni SALEtion is OR DISTRIBUTION pathogen clearance. a prerequisite for T-cell priming and activation for their adaptive immune functions. Mononuclear Phagocytes: © JonesMonocytes, & Bartlett Macrophages,Learning, LLC and DCs ▸▸ Complement© Jones & Bartlett Cascade Learning, LLC NOT FORMonocytes SALE areOR mononuclear DISTRIBUTION leukocytes that cir- In the lateNOT 19th FORcentury, SALE Hans Buchner, OR DISTRIBUTION a Germa n culate in the blood and differentiate into macro- bacteriologist, is credited with describing, for the phages and DCs (dendritic cells).25,26 Macrophages first time, protective substances, which he called

9781284173017_CH02_Clift.indd 25 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 26 Chapter 2 Fundamentals of the Immune System

alexins; alexins were capable of killing micro- components are recruited to pathogens or host- organisms. These© Joneshumoral &molecules Bartlett were Learning, later cell LLC membranes through the initial© Jones binding & of Bartlett Learning, LLC called “complement”NOT by FOR Paul SALEEhrlich, ORwhen DISTRIBUTION they immunoglobulins (Igs), both IgGNOT and IgM, FOR with SALE OR DISTRIBUTION were found to be important in both cellular and IgM being the most efficient activator. The first humoral responses. Unlike the antibody system, recruited element is named C1 and is broken into also described by Ehrlich, the complement system subunit C1q, C1r, and C1s. C1 has two substrates, is considered a nonadaptive piece of the humoral C4 and C2, the next two molecules activated in © response.Jones &Currently, Bartlett the Learning, term “complement” LLC the classical pathway.© Jones The complex & Bartlett of C4b2a Learning, stim- LLC NOTdescribes FOR 35 SALE proteins OR found DISTRIBUTION in plasma or on the ulates C3 to cleaveNOT and FOR form, SALE creating OR C4b2a3b DISTRIBUTION cell surface. that binds and cleaves the C5 molecule, leading The complement cascade may be mediated to the formation of the membrane attack complex by both innate and adaptive immune mecha- (MAC) and the terminal pathway. nisms (FIGURE 2–7). Specifically, the classical A second common pathway for complement © Jones & Bartlettpathway Learning, of complement LLC activation is through an © activationJones &is Bartlettcalled the Learning, alternative LLCpathway, NOT FOR SALEanti ORbody-dependent DISTRIBUTION trigger. The first complement NOTwhich FOR derived SALE from OR the DISTRIBUTIONantibody independent

Classical Pathway Alternative Pathway Antigen–antibody Microorganisms interaction triggers and/or their ©activation Jones of &initial Bartlett Learning, LLC components interact© Jones & Bartlett Learning, LLC complement components with factors NOTC1, C4,FOR and C2SALE OR DISTRIBUTION B, D, and P NOT FOR SALE OR DISTRIBUTION

C3 component activated © Jones & Bartlett Learning, LLCand activates other © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONcomplement components NOT FOR SALE OR DISTRIBUTION in sequence C3A C3B

© Jones & Bartlett Learning, LLC C3B© Jones & Bartlett Learning, LLC Produces mediators of C5 Enhances NOT FOR SALE OR DISTRIBUTIONinflammation, and NOT FOR SALE OR DISTRIBUTION phagocytosis attracts leukocytes C6 C7 C8 © Jones & Bartlett Learning,C9 LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Forms large complex (“attack complex”) that punches holes in cell membrane of © Jones & Bartlett Learning, LLCtarget cell or bacterium © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION FIGURE 2–7 Complement Pathways. Data from Reisner, H. (2017). Crowley's an introduction to human disease, 10e. Jones & Bartlett Learning.

9781284173017_CH02_Clift.indd 26 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Pattern Recognition Receptors 27

activation of complement from certain pathogens. which forms a 10 nm pore in the surface of the Current ©speculation Jones &exists Bartlett as to Learning,whether this LLCcell, releasing water and ions© fromJones the cell.& Bartlett Learning, LLC pathway NOTis completely FOR independentSALE OR of DISTRIBUTION the clas sical The complement cascadeNOT and FORMAC are SALE highly OR DISTRIBUTION pathway, or if it simply acts as an amplification effective at destroying and inactivating many bac- to that pathway. Nonetheless, it does require the terial pathogens, including Neisseria meningitides, recruitment of several components not found in Streptococcus pneumonia, and Haemophilus influ- the classical pathway, namely Factors B, D, and P, enzae. Complement is also consumed at a higher © Joneswhich & Bartlettcan bind the Learning, C3 component LLC and converge rate during© Jones certain &autoimmune Bartlett Learning,diseases such LLC NOT FORwith theSALE classical OR pathway DISTRIBUTION through the stimulation as systemicNOT lupus FOR erythematosus SALE OR (SLE). DISTRIBUTION Serum of C5b and the formation of the MAC. is collected for detection of the C3 and C4 com- A third complement pathway, known as the ponents via the immunoturbidometic method in lectin pathway is also antibody independent many labs as an assessment of a number of patho- and has similar conversion points to the prior physiologic conditions. © Jones & Bartlett Learning,two, but is distinctive LLC in its initiation through© the Jones & Bartlett Learning, LLC NOT FOR SALE ORmannan-binding DISTRIBUTION lectin (MBL), a lectin that sharesNOT FOR SALE OR DISTRIBUTION structural similarities with the C1 complex found ▸▸ Pattern Recognition in the classical pathway. Beyond this point, the pathway converges with the stimulation of C4 and Receptors C2 as before, and leads to the formation of the Pattern recognition receptors (PRRs) allow MAC and© the Jones terminal & pathway. Bartlett Learning, LLCdetection of pathogens by© innate Jones immune & Bartlett cells Learning, LLC The NOTterminal FOR pathway SALE, also OR referred DISTRIBUTION to as outside of the cell and withinNOT different FOR SALE cellular OR DISTRIBUTION the membrane attack pathway, begins with the compartments27,30 (TABLE 2–1). They recognize cleavage of the molecular C5 on the surface of common pathogen-associated molecular pat- the target cell. The C5b component is joined by terns, such as bacterial cell-wall molecules and molecules C6, C7, and C8, which, together, can viral RNA. Macrophages and DCs use PRRs to © Jonescause & membraneBartlett Learning,disruptions. However, LLC the MAC sense antigens© Jones in secondary & Bartlett lymphoid Learning, organs and LLC NOT FORis completed SALE byOR the DISTRIBUTIONrecruitment of multiple copies within tissues.NOT PRR FOR sensing SALE of pathogens OR DISTRIBUTION activates (estimated at 10) of complement molecular C9, macrophages and DCs, promoting their ability to

© Jones & Bartlett Learning,TABLE 2–1 LLC Pattern Recognition Receptor© Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Pattern Recognition Receptor Ligand Location Primary Outcome

Toll-like receptors Viral RNA, bacterial flagellin, Cell surface and Modulation of immune (TLRs) © Jones & Bartlettbacterial Learning,LPS LLC luminal space response© Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION RIG-I like receptors Microbial nucleic acids Cytosol Modulation of immune (RLRs) response

Dectins Microbial carbohydrates Cell surface Internalization of pathogen © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORLPS SALE receptor OR DISTRIBUTIONBind to bacterial Cell surfaceNOT FOR SALE Internalization OR ofDISTRIBUTION pathogen Lipopolysaccharide walls (continues)

9781284173017_CH02_Clift.indd 27 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 28 Chapter 2 Fundamentals of the Immune System

TABLE 2–1 ©Pattern Jones Recognition & Bartlett Receptor Learning, LLC © (JonesContinued &) Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Pattern Recognition Receptor Ligand Location Primary Outcome

Mannose receptors Mannose residues on Cell surface Internalization of pathogen © Jones & Bartlett Learning,bacterial and LLC viral proteins © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Complement receptors Bind to pathogens Cell surface Internalization of pathogen opsonized by complement

Fc receptors Bind to Fc portions of Cell surface Internalization of pathogen © Jones & Bartlett Learning, LLC antibodies that bind © Jones & Bartlett Learning, LLC pathogens NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

create an inflammatory milieu and to modulate both foreign antigen and self. For efficient immune responses by interaction with innate phagocytosis, particles derived from foreign and adaptive cells.© Jones Engagement & Bartlett of pathogens Learning, antig LLCens are coated by small molecules© Jones presen & tBartlett Learning, LLC with PRRs initiatesNOT signaling FOR cascadesSALE ORthat aidDISTRIBUTION in in the blood called opsonins, suchNOT as FORcomple SALE- OR DISTRIBUTION the production of inflammatory chemokines and ment proteins or lectins that facilitate uptake of enhance internalization of pathogen. these particles by phagocytic cells. Receptors such The production of various chemokines leads as dectins, Fc receptors, complement receptors, to recruitment of neutrophils, basophils, and scavenger receptors, and many others on phago- © eosinophilsJones & toBartlett the area ofLearning, inflammation. LLC In addi- cytic cells bind ©to opsonizedJones & fragments Bartlett and Learning, inter- LLC NOTtion, FOR macrophages SALE and OR DCs DISTRIBUTION produce cytokines to nalize them. BoundNOT antigensFOR SALE are taken OR up DISTRIBUTION into enhance activation of innate and adaptive cells. phagosomes, which undergo acidification and PRRs are strategically positioned in different sub- subsequent fusion with lysosomes that contain cellular compartments to maximize the chances digestive enzymes. Low pH of the acidified pha- of detecting a pathogen that is present extracel- gosome activates proteolytic enzymes allowing © Jones & Bartlettlularly Learning,or is phagocytosed LLC and is located within © forJones enzymatic & Bartlett degradation Learning, of antigens. LLC Pinocyto- NOT FOR SALEphag ORosomes DISTRIBUTION or the cytosol. For example, toll-like NOTsis refers FOR to SALEfluid-phase OR engulfment DISTRIBUTION of pathogens. receptors (TLRs) are found on the cell surface and Macrophages are more phagocytic and DCs are cell vacuoles, while nod-like receptors (NLRs), are less phagocytic, but are superior at pinocytosis found in the cytosol. and are thought to be better T-cell activators. Phagocytosis of antigens has a two-pronged © Jones & Bartlett Learning,effect: LLC it results in activation and maturation© Jones of &the Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONphagocytic cells, which allows themNOT to migrate FOR to the SALE OR DISTRIBUTION ▸▸ Mechanism of LN in search of the right T-cell clone and enhances their ability to activate T cells by upregulation of Internalization of MHC molecules and cytokine expression. Pathogens: Endocytosis The response of the innate immune sys- © Jones & Bartlett Learning, LLC tem provides mechanisms© Jones &to Bartlettkeep an infection Learning, LLC NOTEndocytosis FOR SALE or internalization OR DISTRIBUTION of extracellular under control, NOTsuch as FOR pathogen SALE recognition OR DISTRIBUTION via particles can occur in two main ways: Phagocy- various pathogen-associated molecular patterns tosis and pinocytosis.16,27,28,31–33 Phagocytosis refers (PAMPS), while the adaptive response has time to uptake of extracellular particles, which includes to form. The innate immune response also works

9781284173017_CH02_Clift.indd 28 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Mechanism of Internalization of Pathogens: Endocytosis 29

to prime the adaptive immune response by pro- express a T-cell receptor (TCR) that recognizes cessing the© Jonesantigen &for Bartlett presentation Learning, to T cells LLCantigen in the context of ©peptide Jones bound & Bartlett to the Learning, LLC 34 and releasingNOT proinflammatoryFOR SALE OR cytokines. DISTRIBUTION The Major HistocompatibilityNOT Complex FOR (MHC). SALE OR DISTRIBUTION adaptive immune response, which is composed of T cells play a key role in the immune response T and B cells, provides a specific targeted response by directing the response of other immune cells to antigens associated with the pathogens while through cytokines or by killing infected cells. also forming a memory response to provide pro- Each B-cell and T-cell has a unique BCR or © Jonestection & Bartlett against re-exposures Learning, to LLCthe same patho- TCR that© allows Jones it to & recognize Bartlett a specific Learning, patho- LLC NOT FORgen. ItSALE is the adaptive OR DISTRIBUTION response to disease that has gen. WhenNOT a B FOR or T-cell SALE recognizes OR DISTRIBUTION its antigen been exploited by immunodiagnostic manufac- through the binding of its receptor, it undergoes turers for the production of commercially avail- clonal expansion where every cell that is gen- able antibodies with specific targets. erated from that activated cell expresses the same The adaptive immune system has several clone of the receptor. © Jones & Bartlett Learning,unique features LLC that are not shared with the innate© Jones &Broadly, Bartlett two Learning, lineages of LLCcells carry out NOT FOR SALE ORimmune DISTRIBUTION system. The cells of the innate immuneNOT FORimmune SALE functions: OR DISTRIBUTIONthe myeloid and the lymphoid system have receptors on their surface that rec- lineages.4 Myeloid cells are innate cells, composed ognize pathogens that are coded for by germline of dendritic cells (DCs), macrophages, neutro- genes, i.e., genes that are found in all cells of the phils, eosinophils, and basophils. The lymphoid organism. The adaptive immune system requires lineage consists of T cells and B cells, which are receptor ©assembly Jones through & Bartlett gene rearrangement, Learning, LLCadaptive cells, and NK cells,© Joneswhich harbor & Bartlett both Learning, LLC i.e., genesNOT that areFOR specific SALE to the OR cells. DISTRIBUTION Adaptive innate and adaptive features.NOT A FORmore extensiveSALE OR DISTRIBUTION immune cells undergo a process of gene rearrange- examination of these two systems can be found ment that combines gene segments to produce in Chapter 5: Innate Immunity, and Chapter 6: their receptors. This receptor assembly provides Adaptive Immunity. a unique ability to express more than 1011 unique Immune cells develop in primary lymphoid © Jonesreceptors. & Bartlett The ability Learning, of the adaptiveLLC immune organs, namely,© Jones the bone & Bartlett marrow and Learning, the thymus, LLC NOT FORsystem SALE to create OR the DISTRIBUTION large repertoire of unique and undergoNOT activation FOR SALE in secondary OR DISTRIBUTION lymphoid receptors allows for greater protection against organs such as the spleen and the lymph nodes. more pathogens than a static germline system All immune cells develop from bone marrow would allow. Another unique feature to the adap- residing pluripotent hematopoietic stem cells tive immune system is memory. Once there has (HSCs).4 The two defining features of HSCs are © Jones & Bartlett Learning,been an adaptive LLC immune response to the patho© Jones- the & ability Bartlett to self-renew Learning, and to LLCdifferentiate into NOT FOR SALE ORgen, DISTRIBUTION memory toward that pathogen is generated,NOT FORprecursors SALE of ORimmune DISTRIBUTION cells. HSCs differentiate protecting against future infections. This memory into CLP, which give rise to the lymphoid lineages allows a quicker and more robust response to the and the common myeloid progenitors (CMP), pathogen upon re-exposure. which differentiate into the myeloid lineages. The adaptive immune system is composed of lymphocytes.© Jones Lymphocytes & Bartlett are composed Learning, of two LLCMajor Histocompatibility© Jones Complex & Bartlett Learning, LLC specific cellNOT types: FOR B cells SALE and T cells.OR BDISTRIBUTION cells, origi- NOT FOR SALE OR DISTRIBUTION nally isolated and named for the bursa of chickens, Major histocompatibility complex consists of a class have a unique B-cell receptor (BCR) expressed on of heterodimeric glycoprotein molecules, mainly the cell surface and develop in the bone marrow. important for antigen presentation to T cells and Each B-cell clone expresses a unique BCR that rec- for directing NK cell function.35,36 The two main © Jonesognizes & Bartlett a specific Learning,antigen. Once LLC the BCR binds to classes of© MHCJones proteins & Bartlett responsible Learning, for peptide LLC NOT FORits antigen, SALE it activatesOR DISTRIBUTION the B-cell to proliferate and presentationNOT to TFOR cells areSALE MHC-I OR and DISTRIBUTION MHC-II. differentiate into plasma cells. Plasma cells secrete Central to understanding the role of MHC in their BCRs as immunoglobulin, also known as immunity is the appreciation of structures of these antibodies. T cells develop in the thymus and molecules as well as the genetics that drive their

9781284173017_CH02_Clift.indd 29 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 30 Chapter 2 Fundamentals of the Immune System

expression.37 MHC molecules are polygenic, i.e., that bind MHC-I. Structural stability of MHC within one individual,© Jones multiple & Bartlett genes will Learning,encode molecules LLC is determined by the peptides© Jones loaded & Bartlett Learning, LLC MHC-I and II. NOTIn addition, FOR MHCSALE molec ORules DISTRIBUTION are onto these molecules. Peptides ensureNOT appropriFOR SALE- OR DISTRIBUTION highly polymorphic, which means that many ate folding of MHC molecules and their stability variants of a single gene exist in a population. on the cell’s surface. MHC-I molecules consist of two chains, the α and Peptide-MHC complexes serve as signals β domains. α chain comprises the larger part of of cellular health. While MHC-I genes are © theJones molecule, & Bartlett folding into Learning, three domains LLC α1, α2, expressed on all© nucleated Jones somatic & Bartlett cells, MHC-II Learning, LLC NOTand αFOR3. Folding SALE of theOR α 1DISTRIBUTION and α2 domains into is expressed onlyNOT on DCs, FOR macrophages, SALE OR and DISTRIBUTION thy- anti-parallel, β-pleated sheets gives rise to the mic epithelial cells. MHC molecules present on peptide-binding groove, while α3 is transmem- thymic epithelial cells are critical for instructing brane and pairs with β2-microglobulin for struc- developing T cells to recognize peptides in the tural stability. Amino acids that form the groove are context of MHC. This is called MHC restriction © Jones & Bartlettareas ofLearning, a high degree LLC of polymorphism, allowing © andJones allows & CD4+ Bartlett T cells Learning, to recognize peptidesLLC in NOT FOR SALEMHC OR proteins DISTRIBUTION to display a wide variety of peptides. NOTthe context FOR ofSALE MHC-II OR and DISTRIBUTION CD8+ T cells to recog- Amino-acid residues of the peptide that nize peptides in the context of MHC-I. Stimula- facilitate its binding to the MHC groove are tion of CD4+ T cells by antigenic peptides causes called anchor residues and are generally aro- them to differentiate into various helper lineages matic residues with the ability to have ionic that facilitate CD8+ T-cell and B-cell activation. interactions as ©well Jones as the ability & Bartlett to form Learning,hydro- CD8+ LLC T cells are direct killers of ©infected Jones cells. & Bartlett Learning, LLC gen bonds (BOX 2–1NOT). FOR MHC-II SALE molecules OR DISTRIBUTIONhave In addition to classical MHCNOT molecules FOR that SALE OR DISTRIBUTION two chains, α and β, which pair and contribute present peptides to T cells, nonclassical MHC- to the peptide-binding groove that is struc- like molecules (structurally similar to classical turally similar to MHC-I’s peptide binding MHC) play important roles in the modulation groove (FIGURE 2–8, BOX 2–2). Peptides that bind of immune responses. MHC studies have been © MHC-IIJones are& longerBartlett and moreLearning, variable LLCthan those pivotal in understanding© Jones &the Bartlett self-vs-nonself Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION A DEEPER LOOK BOX 2–1

Bonds are chemical interactions between atoms to create molecules (intramolecular) or an interaction © Jones & Bartlettbetween Learning, molecules (intermolecular)LLC to create macromolecular© Jones networks & Bartlett and structure. Learning, LLC NOT FOR SALE ORIntramolecular DISTRIBUTION forces are ionic or covalent bonds. IonicNOT bonds FOR are electrostatic SALE OR interactions DISTRIBUTION between atoms of opposite charges that occur as a result of donation and acceptance of electrons. For example, sodium (Na) donates an electron to chlorine (Cl) to form sodium chloride (NaCl). In the process, Na becomes positively charged and Cl becomes negatively charged. Covalent bonds involve the sharing of electrons between atoms. Carbon (C) bonding is covalent and is abundant in biological molecules. Carbon can make single, double, and triple bonds, allowing it to create a multitude of interactions. In a single water molecule, the interaction between one oxygen© Jones (O) molecule & Bartlett and two hydrogenLearning, (H) molecules LLC is a covalent bonds. © Jones & Bartlett Learning, LLC IntermolecularNOT forces FOR are weak SALE bonds. OR These DISTRIBUTION include Van der Waals forces and hydrogen bonds.NOT Van FORder SALE OR DISTRIBUTION Waals forces are distance dependent and exist between temporary dipoles. Hydrogen bonds are electrostatic interactions between electronegative (electron pulling) atoms. Although water molecules are a result of electron sharing, the sharing is unequal between O and H. O is electronegative and pulls electrons toward itself in a H–O–H molecule, becoming slightly negatively charged, and H atoms adopt a slightly positive © Jonescharge (dipole& Bartlett formation). Learning, The slightly negative LLC charge on O in a water molecule© Jones can attract & Bartletta slightly positive Learning, LLC NOTcharge FOR on SALEH on a neighboring OR DISTRIBUTION water molecule. Hydrogen bonding confers NOThigh surface FOR tension SALE to water. OR DISTRIBUTION Such interactions can lend stability to a molecule. For example, DNA consists of two strands held together by stacking hydrogen bond interactions.

9781284173017_CH02_Clift.indd 30 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Mechanism of Internalization of Pathogens: Endocytosis 31

Antigen-binding Antigen-binding region region © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

α2 α1 α1 β1

α3 β2 microglobulin α2 β2 © Jones & Bartlett Learning, LLCN © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

C © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

CC C

© Jones & Bartlett MHCLearning, IM LLC HC II © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION FIGURE 2–8 MHC I and MHC II. The major histocompatibility complex (MHC) I and II (also called Human Leukocyte Antigens; HLA in humans) are glycoproteins expressed on the surface of cells. MHC I is expressed in a variety of nucleated cells and is recognized by cytotoxic T cells prior to targeted destruction of the cell. MHC II, by contrast, is found only on specific antigen-presenting cells (APCs) and when bound with antigen, is recognized by helper T cells. © JonesModified & from Bartlett Michael H Ross, Wojciech Learning, Pawlina. Histology: A Text and LLCAtlas: With Correlated Cell and Molecular Biology, Lippincott Williams© & Wilkins, Jones 2015. & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION recognition capacity of the immune system. This in the context of self-MHC. If a T-cell encount ers understanding has been critical for appreciating a foreign MHC, it will perceive the MHC as the role of MHC in transplantation of organs. “nonself.” Hence, the MHC match between During T-cell development, T-cell repertoire of organ donors and recipients is critical to pre- © Jones & Bartlett Learning,an individual LLCis instructed to recognize peptides© Jones vent & immune-mediatedBartlett Learning, rejection LLC of grafts after NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION A DEEPER LOOK BOX 2–2 The Major Histocompatibility Complex (MHC)

The two classes of MHC molecules, class I and class II, are a reflection of the health of the cell that synthesized them (class I) or the local cellular environment (class II). In humans, the MHCs are typically referred to as HLA (for human© Jones leukocyte &antigen), Bartlett but have Learning, been named LLC variously in other organisms. Nomenclature© Jones for newly& Bartlett Learning, LLC found HLANOT antigens FOR are SALE governed OR by the DISTRIBUTION World Health Organization (WHO). NOT FOR SALE OR DISTRIBUTION In general, the class of glycoproteins called MHC are recognized to perform a range of immunological functions on the surface of somatic cells. In their prototypical form, they are known for presenting foreign and self-antigens to the surface of the cell, whereby they can be detected by the TCR on T cells or the NK cell receptors on NK cells. These antigens can be the debris from a pathophysiological condition underway within © Jones the& cell,Bartlett such as aLearning, viral infection, orLLC as a result of a cellular dysregulation© Jones as may occur & Bartlett in certain cancers Learning, or LLC NOT FORautoimmune SALE OR diseases. DISTRIBUTION HLA matching is crucially important for determiningNOT FORtransplantation SALE acceptance OR DISTRIBUTION or rejections. Furthermore, a large number of human diseases are affected by expression or polymorphisms of this molecule. Therefore, it is an important antigenic target in immunodiagnostic analysis.

9781284173017_CH02_Clift.indd 31 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 32 Chapter 2 Fundamentals of the Immune System

transplantation. MHC-related studies within presentation and T-cell activation. Activated CD4 human populations© Jones have revealed & Bartlett that these Learning, are T LLCcells facilitate the activation of© BJones cells, CD8 & Bartlett Learning, LLC important determinantsNOT FOR of cancer,SALE autoimmuOR DISTRIBUTION- T cells, (which deliver humoral NOTimmunity) FOR and SALE OR DISTRIBUTION nity, and susceptibility to infections. Diversity targeted cellular killing of infected cells, respec- of MHC is studied through sequencing of MHC tively. Because T cells offer help in activating other encoding genes and structural analysis through cells of the adaptive immune system, they are methods such as X-ray crystallography. known as helper T cells. Depending on the nature © Jones & Bartlett Learning, LLC of the PRR engaged© Jones by the &pathogen Bartlett on theLearning, DC, LLC NOT FOR SALE OR DISTRIBUTION the DC will produceNOT FORcytokines SALE to differen OR DISTRIBUTIONtiate The Adaptive Immune Response helper cells into specific fates, which need supe- Both the innate and adaptive cells play key roles rior capacity to fight specific pathogens. in determining pathogen clearance. DCs have the unique capacity to initiate T-cell responses, and © Jones & Bartlettmacrophages Learning, synergize LLC and amplify the process © KeyJones Tenets & Bartlett of Adaptive Learning, Immune LLC NOT FOR SALEof ORactivation DISTRIBUTION of T and B cells by capturing anti- NOTResponse FOR SALE OR DISTRIBUTION gens. Pathogens can either be transported by DC cells to the LN, or free pathogens can migrate to 1. The ability to discriminate between self and SLOs activating DCs. Activation of DCs results nonself in DC maturation, which involves increased 2. Protective memory formation expression of MHC© Jones molecules & Bartlettand CCR7, Learning, which 3. LLC Clonal expansion during activation© Jones & Bartlett Learning, LLC results in migrationNOT and FOR retention SALE of DCs OR in DISTRIBUTION LN. 4. Clonal deletion during developmentNOT FOR as basis SALE OR DISTRIBUTION Pathogen proteolytic degradation leads to peptide of central tolerance

© JonesCASE & STUDY Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Three Immune System Disorders Case Study 1 This case has been adapted from Markert et al, N Engl J Med, 1999.38 The DiGeorge syndrome is a heritable disorder that prevents normal thymus development leading to © Jones & Bartletta reduction Learning, of T-cell function.LLC Five infants, from age 1 to© 4 Jonesmonths with & profoundBartlett DiGeorge Learning, syndrome LLC were NOT FOR SALE treatedOR DISTRIBUTION with transplantation of postnatal thymus tissue.NOT Follow-up FOR tests SALE involved OR immune DISTRIBUTION phenotyping, proliferation assays of peripheral-blood mononuclear cells. Thymic production of new T cells was assessed in peripheral blood. Following transplantation, T-cell proliferative responses developed in four infants. Two patients survived and attained immune function. The remaining three patients died from infections or anomalies independent of transplantation. Biopsies of grafted thymus in the surviving patients showed normal morphologic features and active T-cell production. In one surviving patient, T-cell development occurred within© the Jones graft, leading & Bartlett to accumulation Learning, of recently LLC developed T cells in the periphery© andJones & Bartlett Learning, LLC acquisition of normalNOT T-cell FOR function. SALE In the OR second DISTRIBUTION patient, thymus function and T-cell developmentNOT FOR SALE OR DISTRIBUTION was observed for more than 5 years after transplantation. This study led to the conclusion that in some infants with severe DiGeorge syndrome, transplantation of the thymus can lead to restoration of the T-cell compartment and function. However, it is likely that early thymus transplantation is essential, prior to any infections, to increase chances of survival. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOTCritical FOR Thinking SALE ORQuestion DISTRIBUTION NOT FOR SALE OR DISTRIBUTION With crucial knowledge about DiGeorge syndrome in hand, you diagnose it in a newborn. Suppose you are a physician. What treatment would you administer to restore T-cell function in the infant?

9781284173017_CH02_Clift.indd 32 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Mechanism of Internalization of Pathogens: Endocytosis 33

Case Study© Jones 2 & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC This case was adapted from Gottleib et al., N Engl J Med, 1981.39 In NOT1981, the FOR first cases SALE of Acquired OR DISTRIBUTIONImmunodeficiency Syndrome (AIDS) arose aroundNOT the world. FOR The SALE OR DISTRIBUTION following is an (edited) abstract from a case study from 1981 published in the New England Journal of Medicine documenting initial observations of AIDS-positive patients. Four homosexual patients displayed opportunistic infections, loss of CD4 helper T cells, and telltale signs of inflammation such as lymphadenopathy. Soon a virus, Human Immunodeficiency Virus 1 (HIV1), was cloned and described as the causative © Jonesagent & Bartlett of AIDS. The Learning, key observation LLC of depletion of helper CD4 T cells© Jones led to the & discovery Bartlett of CD4 Learning, as a LLC NOT FORkey SALE receptor ORthat HIV1DISTRIBUTION used to bind to CD4 T cells and initiate its replicationNOT FOR cycle SALE within CD4+ OR T cells DISTRIBUTION that caused T-cell death. CD4 T-cell count is now an important criterion to monitor HIV status of patients. HIV struck at a time when clinicians and scientists were armed with tools such as monoclonal antibodies to immune phenotype cells, sequencing techniques to isolate the virus and monitor its evolution. Furthermore, general knowledge of the immune system, coupled with ideas about viral, bacterial, and © Jones & Bartlett Learning,fungal infections, LLC were already in place to recognize© Jones that patients & Bartlett were susceptible Learning, to opportunistic LLC infections that an immune-competent person could easily thwart. Although HIV remains a global NOT FOR SALE OR DISTRIBUTIONinfectious disease challenge with no effective NOTvaccine FOR in place, SALE powerful OR antiviral DISTRIBUTION drugs that halt viral replication and allow for CD4 T-cell persistence have enabled patients to lead longer and healthier lives. The case of HIV demonstrates an ongoing global collaboration between clinicians and scientists to overcome an enormous public health challenge with many successes and many failures and along the way. The following study was adapted from “Pneumocystis Carinii Pneumonia and Mucosal Candidiasis in Previously© JonesHealthy Homosexual & Bartlett Men. Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Evidence of a New Acquired Cellular Immunodeficiency Four previously healthy patients were hospitalized with fever, lethargy and lymphadenopathy, candidiasis infection, and respiratory infections. All patients had reduced T-cell numbers, as determined by monoclonal antibody function and virtual absence of T-cell proliferative response. Cytomegalovirus infection was determined by viral cultures performed in human-embryonic-lung fibroblasts. Antibodies to cytomegalovirus, © Jonesadenovirus, & Bartlett and herpesLearning, simplex were LLC found in the sera of the patients.© BronchialJones brushings& Bartlett showed Learning, evidence LLC NOT FORof PneumocystisSALE OR carinii DISTRIBUTION in all patients. One patient died, two patientsNOT recovered, FOR and SALE Kaposi’s sarcomaOR DISTRIBUTION developed in the remaining patient. These manifestations suggest severe immunosuppression. Critical Thinking Question Confronted with a conglomeration of symptoms that you have never seen before, how do you go about © Jones & Bartlett Learning,characterizing LLC a disease? © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONCase Study 3: RAG Deficiency LeadingNOT to Impaired FOR SALEAntibody OR Production DISTRIBUTION This case has been adapted from Geier et al, PLoS One, 2015.40 Patient A, a 27-year-old woman, was tested for a primary immunodeficiency. The woman’s family had no history of immunological defects. The woman suffered from reoccurring bacterial infections of the upper respiratory system starting at age 7. At the age of 8, she was hospitalized due to inflammation of the blood vessels ©of theJones skin of both& Bartlett legs, which Learning, reoccurred at ageLLC 13. When she was 10 years old,© she Jones was diagnosed & Bartlett Learning, LLC with chronicNOT interstitial FOR SALE pneumonia. OR The DISTRIBUTION chronic bacterial infections of the lungs led toNOT chronic FOR obstructive SALE OR DISTRIBUTION airway disease. At age 13, she was diagnosed with hypogammaglobulinemia, the lack of antibodies. She started intravenous (IV) IgG (IVIG) infusions at the age of 20. Prior to IgG infusions, she had a serum IgG level of 393 mg/dL and normal levels of IgA and IgM. Despite recurrent Streptococcus pneumoniae infections, the patient had very low levels of S. pneumoniae-specific IgG antibodies but high levels of pathogen-specific © Jones IgM& Bartlettantibodies. ContinuedLearning, susceptibility LLC to pulmonary and middle© ear Jones infections & required Bartlett regular Learning, IVIG LLC substitution therapy. Unfortunately, the chronic lung infections led to decreasing lung function, resulting in NOT FORthe SALE patient’s ORdeath DISTRIBUTION at age 48. NOT FOR SALE OR DISTRIBUTION (continues)

9781284173017_CH02_Clift.indd 33 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 34 Chapter 2 Fundamentals of the Immune System

CASE STUDY© Jones & Bartlett Learning, LLC ©(Continued Jones) & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Patient B is a 41-year-old woman who was tested for a primary immunodeficiency at the age of 35. The patient has two healthy children. She has a history of pneumonia and recurrent bacterial bronchitis. She presented with low levels of IgM, IgA, and IgG. She also lacked antibodies against common bacterial polysaccharides. The patient was successfully treated with IV infusions of IgG and sinus surgery. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Laboratory Diagnostics NOTThe FOR patients’ SALE blood samples OR DISTRIBUTION were evaluated for serum antibodies against commonNOT bacterialFOR SALE and viral OR DISTRIBUTION antigens. The first laboratory test was to determine the serum concentrations of the various antibody isotypes. Serum antibody concentrations are calculated by nephelometry. Nephelometry (discussed in more detail in Chapter 2: The Serological Specimen) measures the concentration of the antibodies in suspension via laser light. The more the light that is scattered, the higher the concentration of antibodies present. The antibody © Jones & Bartlettspecificity Learning, is determined LLC by ELISA. ELISA plates are coated© withJones the antigens & Bartlett of interest Learning, (tetanus, diphtheria LLC NOT FOR SALE tORoxin, HaemophilusDISTRIBUTION influenzae type b, or pneumococcal NOTcapsular FOR polysaccharide). SALE ORThe patient’s DISTRIBUTION serum is added to the wells and any antibodies specific to those antigens will bind. Noncognate antigens will be washed away. A secondary antibody that recognizes human IgG or IgM linked to an enzyme will be added to the wells to detect the antigen-specific antibodies. The enzymes linked to the end of the secondary antibody will interact with its substrate to produce fluorescence that can be quantified. These laboratory diagnostic tests revealed that the two patients© Jones had low & antibody Bartlett concentrations Learning, and lacked LLC antigen-specific antibodies. © Jones & Bartlett Learning, LLC Diagnostic tests were also performed to look at B-cell antibody secretion and proliferation. The patient PBMCs and controlNOT cells FOR were stimulated SALE ORwith Epstein-BarrDISTRIBUTION virus (EBV) and cultured for 8 days. TheNOT antibody FOR SALE OR DISTRIBUTION secretion was measured by nephelometry. Proliferation of the B cells was measured by 3H-Thymidin incorporation, which incorporates as the cells grow and divide. The patients’ B cells had decreased IgG production and proliferation. B-cell differentiation was also tested by flow cytometry. Patient A had normal levels of B cells in the periphery (CD19+ and CD20+) while patient B had decreased numbers. A decrease was © Jonesdetected & in Bartlettclass-switched Learning, B cells and memory LLC B cells. The patients also had© increased Jones CD21 & lowBartlett B cells, which Learning, LLC NOTare FOR characterized SALE by ORan exhausted DISTRIBUTION phenotype. Overall, the diagnostic tests revealedNOT FOR a defect SALE in B cells. OR DISTRIBUTION The patients also had their T cells tested. The patients had decreased CD3+, CD4+, and CD56+ lymphocytes and decreased Tregs. Further testing revealed decreased levels of naïve CD4+ T cells but normal levels of memory and effector cells. The CD8+ population was normal. A spectratyping analysis was done to examine the TCR Vβ repertoire of CD3+ T cells. Spectratyping analysis purifies RNA from the T cells and it is converted into cDNA. Polymerase chain reaction (PCR) amplifies the cDNA. During PCR, the complementary- © Jones & Bartlettdetermining Learning, region-3 LLC (CDR3) is fluorescently labeled with© variousJones primers. & Bartlett The cDNA isLearning, passed through LLC a laser NOT FOR SALE thatOR measures DISTRIBUTION the frequency of the different Vβ chains. NOTThe spectratyping FOR SALE of the patientOR DISTRIBUTION samples revealed normal diversity of the TCR Vβ chains. These tests revealed a defect in T cells as well as the B cells. The defect in T and B cells suggested a possible role for the endonucleases RAG1 and RAG2. RAG1 and RAG2 are required for T-cell receptor and B-cell receptor generation. DNA sequencing revealed mutations in the RAG1 and RAG2 loci. Patient A was heterozygous for a RAG1 mutation. Patient B was heterozygous for a RAG2 mutation.© Molecular Jones characterization & Bartlett of Learning, RAG expression LLC by Western Blot showed significantly© Jones decreased & Bartlett Learning, LLC levels of RAG inNOT these patients. FOR TheSALE mutation OR did DISTRIBUTION not completely delete RAG, resulting in an intermediateNOT FOR SALE OR DISTRIBUTION phenotype instead of severe combined immunodeficiency (SCID). The lower levels of RAG allowed some B cells and T cells to rearrange their receptors and successfully mature. The RAG defect did, however, decrease the number of T and B cells able to develop. The decrease in the number of CD4+ T cells would result in less T-cell help for the B cells, resulting in decreased class switching and affinity maturation. © JonesCritical & Thinking Bartlett Questions Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR1. Which SALE immunodiagnostic OR DISTRIBUTION tests were incorporated into this diagnosis?NOT FOR SALE OR DISTRIBUTION 2. How did the RAG1 and RAG2 deficiencies affect the patients’ antibody production?

9781284173017_CH02_Clift.indd 34 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION References 35

▸▸ References© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC 1. KollmannNOT TR, FORKampmann SALE B, Mazmanian OR DISTRIBUTION SK, et al. 20. Okada H, Kuhn C, Feillet H, etNOT al. The ‘hygieneFOR hypothesis’SALE OR DISTRIBUTION Protecting the newborn and young infant from infectious for autoimmune and allergic diseases: an update. Clin Exp diseases: lessons from immune ontogeny. Immunity. Immunol 2010;160(1):1–9. 2017;46(3):350–363. 21. Kolaczkowska E, Kubes P. Neutrophil recruitment and 2. Cyster JG. Lymphoid organ development and cell function in health and inflammation. Nat Rev Immunol. migration. Immunol Rev. 2003;195:5–14. 2013;13(3):159–175. © Jones 3. &Leite Bartlett MI, Jones M,Learning, Ströbel P, et al.LLC Myasthenia gravis 22. Chirumb©olo Jones S. State-of-the-art & Bartlett review Learning, about basophil LLC NOT FORthymus: SALE complement OR DISTRIBUTION vulnerability of epithelial and researchNOT in immunology FOR SALE and allergy: OR is DISTRIBUTIONthe time right myoid cell, complement attack on them, and correlations to treat these cells with the respect they deserve? Blood with autoantibody status. Am J Pathol. 2007;171(3): Transfus. 2012;10(2):148–164. 893–905. 23. Rosenberg HF, Dyer KD, Foster PS. Eosinophils: changing 4. Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm perspectives in health and disease. Nat Rev Immunol. for stem cell biology. Cell. 2008;132(4):631–644. 2013;13(1):9–22. © Jones & Bartlett Learning, 5. Morrison SJ, LLCScadden DT. The bone marrow niche© forJones 24. &Werner Bartlettsson S, Pejler Learning, G. Mast cell LLC secretory granules: haematopoietic stem cells. Nature. 2014;505(7483): NOT FOR SALE OR DISTRIBUTION NOT FORarmed SALE for battle. OR Nat DISTRIBUTION Rev Immunol. 2014;14(7):478–494. 327–334. 25. Auffray C, Sieweke MH, Geissmann F. Blood monocytes: 6. Gratwohl A, Baldomero H, Aljurf M, et al. Hematopoietic development, heterogeneity, and relationship with stem cell transplantation: a global perspective. JAMA. dendritic cells. Annu Rev Immunol. 2009;27(1):669–692. 2010;303(16):1617–1624. 26. Guilliams M, Ginhoux F, Jakubzick C, et al. Dendritic cells, 7. Rodewald HR. Thymus organogenesis. Annu Rev monocytes and macrophages: a unified nomenclature Immunol.© 2008;26:355–388.Jones & Bartlett Learning, LLC based on ontogeny. Nat Rev Immunol.© Jones 2014;14(8):571–578. & Bartlett Learning, LLC 8. Hogquist KA, Jameson SC. The self-obsession of T cells: 27. Pauwels AM, Trost M, Beyaert R, et al. Patterns, receptors, how TCRNOT signaling FOR thresholds SALE affect OR fate DISTRIBUTION‘decisions’ and NOT FOR SALE OR DISTRIBUTION and signals: regulation of phagosome maturation. Trends effector function. Nat Immunol. 2014;15(9):815–823. Immunol. 2017;38(6):407–422. 9. Bansal K, Yoshida H, Benoist C, et al. The transcriptional 28. Savina A, Amigorena S. Phagocytosis and antigen regulator Aire binds to and activates super-enhancers. Nat presentation in dendritic cells. Immunol Rev. 2007; Immunol. 2017;18(3):263–273. 219:143–156. 10. Baldini A. Dissecting contiguous gene defects: TBX1. Curr 29. Mildner A, Jung S. Development and function of dendritic © Jones &Opin Bartlett Genet Dev. 2005;15(3):279–284.Learning, LLC © Jones & Bartlett Learning, LLC cell subsets. Immunity. 2014;40(5):642–656. 11. Davies EG. Immunodeficiency in DiGeorge syndrome NOT FOR SALE OR DISTRIBUTION 30. TakeucNOThi O, Akira FOR S. Pattern SALE recognition OR DISTRIBUTION receptors and and options for treating cases with complete athymia. Front Immunol. 2013;4:322. inflammation. Cell. 2010;140(6):805–820. 12. Malchow S, Leventhal DS, Lee V, et al. Aire enforces 31. Henneke P, Golenbock DT. Phagocytosis, innate immune tolerance by directing autoreactive T cells into the immunity, and host-pathogen specificity. J Exp Med. regulatory T-cell lineage. Immunity. 2016;44(5):1102–1113. 2004;199(1):1–4. © Jones & Bartlett Learning,13. Pitkänen J, Peterson LLC P. Autoimmune regulator: from loss© Jonesof 32. &Iadecol Bartletta C, Anrather Learning, J, Nat, et LLCal. Mannose-binding function to autoimmunity. Genes Immun. 2003;4(1):12–21. lectin—the forgotten molecule? Nature Medicine. 2011; NOT FOR SALE OR14. DISTRIBUTION Ruddle NH, Akirav EM. Secondary lymphoid organs:NOT FOR17(26):796–808. SALE OR DISTRIBUTION responding to genetic and environmental cues in ontogeny 33. Jin J, He S. The complement system is also important and the immune response. J Immunol. 2009;183(4): in immunogenic cell death. Nat Rev Immunol. 2017; 2205–2212. 17(2):143. 15. Cupedo T. Human lymph node development: an 34. De Silva NS, Klein U. Dynamics of B cells in germinal inflammatory© Jones interaction. & BartlettImmunol Lett. Learning,2011;138:4–6. LLC centres. Nat Rev Immunol. 2015;15(3):137–148.© Jones & Bartlett Learning, LLC 16. Delves PJ, Roitt IM. The Immune system: first of two 35. Wieczorek M, Abualrous ET, Sticht J, et al. Major parts. NNOT Engl J Med. FOR 2000;343(1):37–49. SALE OR DISTRIBUTIONhistocompatibility complex NOT(MHC) FORclass I andSALE MHC OR DISTRIBUTION 17. van de Pavert SA, Mebius RE. New insights into the class II proteins: conformational plasticity in antigen development of lymphoid tissues. Nat Rev Immunol. presentation. Frontiers Immunol.2017;8:292. 2010;10(9):664–674. 36. Bjorkman PJ, Parham P. Structure, function, and diversity 18. Randall TD, Mebius RE. The development and function of class I major histocompatibility complex molecules. © Jones &of mucosalBartlett lymphoid Learning, tissues: a balancing LLC act with micro- 1990;59:253–288.© Jones & Bartlett Learning, LLC organisms. Mucosal Immunol. 2014;7(3):455–466. 37. Trowsdale J, Knight JC. Major histocompatibility complex NOT FOR19. Elmor SALEe SA. Enhanced OR DISTRIBUTION histopathology of mucosa-associated genomicsNOT and humanFOR disease. SALE Annu OR Rev DISTRIBUTIONGenomics Hum lymphoid tissue. Toxicol Pathol. 2006;34(5):687–696. Genetics. 2013;14(1):301–323.

9781284173017_CH02_Clift.indd 35 30/12/19 8:24 PM © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 36 Chapter 2 Fundamentals of the Immune System

38. Markert ML, Boeck A, Hale LP, et al. Transplantation of cellular immunodeficiency. N Engl J Med. 1981;305(24): thymus tissue in complete DiGeorge syndrome. N Engl 1425–1431. J Med. 1999;341(16):1180–1189.© Jones & Bartlett Learning,40. LLCGeier CB, Piller A, Linder A, et al. Leaky© Jones RAG deficiency & Bartlett Learning, LLC 39. Gottlieb MS, SchroffNOT R, SchankerFOR SALEHM, et al. PneumocystisOR DISTRIBUTION in adult patients with impaired antibodyNOT FORproduction SALE OR DISTRIBUTION carinii pneumonia and mucosal candidiasis in previously against bacterial polysaccharide antigens. PLoS One. healthy homosexual men: evidence of a new acquired 2015;10(7):e0133220.

9781284173017_CH02_Clift.indd 36 30/12/19 8:24 PM