09.Part I: Immunology Overview
10.Fig. 1.1: Bridging of innate and adaptive immune responses. (a) Dendritic cells (DCs) are major sentinel cells responsible for detecting and responding to molecules released from damaged and dying cells and from pathogen invasion of tissues. Immature DCs (iDCs) expressing pattern recognition receptors (PRRs) such as TLRs, CLRs, NLRs, RLRs, and SRs on the cell surface and within the cytoplasm bind to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) from damaged tissue and initiate innate immune responses. Signals received by iDCs may lead to T lymphocyte activation for the production of effector T cells or signals may induce anergy or tolerance. (b) Cytokines released by iDCs polarize T helper subsets (TH1, TH2, TH9, TH17) and enhance T cell recognition of antigen and effector cell and memory cell development. Cytokines secreted enhance B and T cell cooperation in adaptive immune responses. Cross presentation of antigens to cytotoxic T cells (Tc) also leads to activation and induction of apoptosis in targets cells. (c) Inhibitory signals and lack of coreceptor expression or engagement can lead to anergy and tolerance. The Tyro-3, Ax1 and Mer (TAM) family receptor tyrosine kinases become activated, inhibiting DC activation and maturation. [Republished with permission of SpringerNature publishing from Irvine et al. 2013. Figure 1. Conveyed through STM permission guidelines 2014]
11.2. Components of Immunity
12.Cluster of Differentiation (CD) Antigens
13.4. Transcription Factors Important in Hematopoiesis and Immune Cell Function
14.Fig. 5.3: Neutrophil extracellular traps (NETs) are formed in response to binding of PAMPs to PRRs on neutrophils and phagocytosis of pathogens. Degranulation and release of granule contents and release of enzymes and chromatin result in the formation of NETs. Extracellular DNA fibers are released following nuclear condensation and rupture. NETs trap and immobilize pathogens. Antimicrobial peptides (AMPs), histones, NE, MPO, HMGB1, PAD4 and ANCA are released and together other activities in the cell such as reactive oxygen species and recruitment of platelets, can kill trapped pathogens. Abbreviations: ANCA, Anti neutrophil cytoplasmic antibody; cfDNA, cell free DNA; HMGB1, high-mobility group box 1; MPO, myeloperoxidase; NE, neutrophil elastase; PAD4, peptidyl arginine deiminase 4
15.6. Innate Lymphoid Cells
16.Fig. 7.1: Immune response in the oral cavity during periodontal disease. Influx of cells of the innate and adaptive immune response. Neutrophils are recruited into the gingiva, junctional epithelium and periodontal pocket in response to bacterial products and antigens like LPS. Immune cells produce inflammatory mediators. Abbreviations: ILCs, innate lymphoid cells; DCs dendritic cells; M1, macrophage subset 1; M2, macrophage subset 2; LPS, lipopolysaccharide
17.Fig. 8.1: Comparison of gut immune homeostasis and dysbiosis leading to inflammation. Immune homeostasis of the GI tract is maintained by cells within the epithelium including enterocytes, M cells, Goblet cells, Paneth cells, Tuft cells, intraepithelial cells and no breach of the epithelium by bacteria. Antimicrobial peptides (AMPs), sIgA secreting plasma cells, thick mucus layer, SCFA, Tregs and cryptopatches characterize a healthy gut barrier. A dysfunctional barrier is characterized by bacteria gaining access to the lamina propria and the presence of APCs to take up bacteria for processing and presentation to T cells. With dysbiosis epithelial integrity is lost and bacteria have breached the epithelium, inflammatory reactions, and recruitment of cells like sIgA secreting plasma cells and dendritic cells to the submucosa can be seen
18.Fig. 9.6: Peyer’s Patch from rat (31-day-old male Sprague-Dawley rat) small intestine. (1) Follicle (F) with germinal center (GC) surrounded by mantle zone or corona (C) is shown. The GC is flanked by interfollicular regions (IFR). The corona is surrounded by the subepithelial dome (SED) and follicle-associated epithelium (FAE). (5) High magnification of high endothelial venule (HEV) showing lymphocytes on endothelial cell surface. [Republished with permission of SAGE publishing from Cesta 2006b, Figures 1 and 5. Conveyed through STM permission guidelines 2014]
19.Fig. 10.2: Toll-like receptor (TLR) signaling showing some of the pathways activated following PAMP binding to PRR. Binding of a ligand to the membrane-bound TLR1/TLR2 dimerized receptor activates cytoplasmic signaling through the MyD88 adaptor protein. IRAK1 is recruited to the MyD88 and becomes activated by phosphorylation. TRAF 6 is recruited along with TAB1 and TAB2. Activation of TAK1 results in the activation of the MAP kinase pathway leading to production of the transcription factor AP-1. Activation of the IKK complex results in the production of the transcription factor NFκB. Transcription factors influence gene expression of various molecules including cytokines. Interferon regulatory factors (IRFs), IL-1 receptor-associated kinase (IRAK), Myeloid differentiation factor 88 (MyD88), NFκB essential modifier (NEMO), Toll/IL-1 receptor (TIR), Transforming growth factor β-activated kinase (TAK1), TAK 1-binding protein 1 and 2 (TAB1 and TAB2), Tumor necrosis factor receptor-associated factor 6 (TRAF6)
20.Fig. 11.1: Assembly and activation of the NLRP3 inflammasome platform. Cytosolic pattern recognition receptor (PRR) sensors sense and ligate pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). The adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) polymerizes in the cytoplasm and binds to the sensor protein NLP3, triggering activation of the canonical inflammasome pathway. Procaspase 1 is recruited along with the adaptor protein ASC to the NLRP3 subunits. Activation of procaspase 1 to caspase 1 leads to the cleavage of GSDMD to release a cytotoxic amino-terminal fragment-GSDMDN. The N-terminal fragment of gasdermin D associates with the plasma membrane leading to pore formation, intracellular K+ efflux, and lysis of the cell. Caspase 1 also cleaves Pro-IL-1β and Pro-IL-18, precursors for IL-1β and IL-18, respectively, which become secreted from the cell. Abbreviations: NLRP3, nucleotide-binding domain leucine-rich repeat containing protein family 3; ASC, apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain); GSDMD, gasdermin D; N-GSDMD, amino-terminal fragment of gasdermin D
21.Fig. 12.2: Trained innate immunity and adaptive immune memory development. (a) First exposure of innate immune cells to antigen (PAMPs) activates naïve innate immune cells resulting in a primary response. Upon re-exposure to the same or heterologous antigen triggers a secondary amplified response regulated by epigenetic modifications and metabolic changes. Epigenetic reprogramming of innate cells occurs between the first and second exposures (blue shaded arrow) leading to trained innate immune responses. (b) Comparison of memory cell development in adaptive immunity following B and MHC-restricted T cell recognition of antigen using antigen-specific BCR and TCR, activation and proliferation, respectively. Specific antigen recognition is based on V(D)J recombination of receptor genes and recognition of diverse antigens, somatic hypermutation generating high affinity, antibodies, isotype switching, and cytokine involvement of B and T cell response to antigen. Trained immunity is shown alongside development of primary and secondary adaptive immune responses
24.Fig. 15.3: Mechanisms contributing to evasion of apoptosis and carcinogenesis. (Republished with permission of SpringerNature publishing from Wong (2011), Figure 2. Conveyed through STM permission guidelines 2014)
25.Fungal Antigens and Immunity
26.Fig. 17.2: Antibody structure consisting of 2 heavy (H) chains (blue/green) and 2 light chains (orange/yellow). Both H and L chains contain variable (V) regions in the amino terminus and constant (C) regions in the carboxyl terminus of the antibody molecule. Carbohydrate molecules are associated with the H chain constant region (not shown) at the second C domain (CH2) of the H chain. The antigen binding domain of the antibody molecule is composed of the VL and VH regions known as the Fab fragment. The constant region of the H chain is known as the Fc region and is important for binding to Fc receptors on cells expressing the Fc receptor
27.Fig. 18.2: Germline organization and rearrangement of the antibody L chain gene. From multiple VJ genes, recombination and rearrangements take place in L chain gene to generate the variable region for antigen binding. The V regions are combined with constant region genes to encode the full L chain. Antibody molecules can have either a κ or λ L chain combined with the H chain
28.Fig. 19.2: Antigen processing and presentation. Endogenous antigens produced by intracellular pathogens or through changes in protein expression due to neoplastic growth are processed in immunoproteasomes. Peptides generated are transported to the ER where they interact with MHC class I proteins. MHC class I bound proteins are transported to the host cell surface where they are recognized by CD8+ cytotoxic T cells. Exogenous antigens are phagocytosed or endocytosed by dendritic cells or macrophages. Phagocytic vesicles fuse with lysosomes to form phagolysosomes. MHC class II proteins synthesized in the ER associate with the invariant chain protein; the invariant proteins become cleaved leaving CLIP in the antigen binding site and become transported into the endosome where antigenic peptides are located. CLIP is exchanged for antigenic peptide and MHC class II bound to antigen peptide is transported to the surface of the antigen presenting cell for recognition by CD4+ T helper cells
29.Fig. 20.2: T cell activation. Antigen binding to the T cell receptor (TCR) leads to the phosphorylation of the tyrosine kinases Lck and ZAP-70, leading to the phosphorylation of tyrosines on the ITAMS of the TCR coreceptor molecule CD3 and the adaptor proteins LAT and SLP-76. Docking sites exposed following phosphorylation leads to recruitment of signaling molecules possessing SH2 domains, such as Grb2 and phospholipase Cγ (PLCγ). Phosphorylation and activation of PLCγ leads to the cleavage of PIP2 and the release of IP3 and DAG. The second messenger IP3 stimulates the release of calcium from calcium stores, which binds calmodulin. The phosphatase calcineurin becomes activated by dephosphorylation allowing calcineurin to activate the transcription factor NFAT. DAG binds to protein kinase C theta (PKCθ). The transcription factor NFκB becomes activated by phosphorylation. The adaptor molecule Grb2binds to LAT and recruits members of the Ras pathway activating the MAP kinase (MAPK) pathway. MAP kinases such Erk activates the transcription factor AP-1. The transcription factors translocate into the nucleus and influence the expression of cytokine and cytokine receptor genes
30.Fig. 21.1: Mechanisms of costimulatory and inhibitory receptor signaling in T cells. Costimulatory receptors CD226, ICOS, CD28, 4-1BB, CD40L, and OX40, bind to the ligands CD155, ICOSL, CD80/86, 4-1BBL, CD40 and OX40L, respectively. Following the ligation of TCR to peptide presented on MHC class II proteins, signals transduced by binding of coreceptors to their ligand provides signals for T cell activation, cell proliferation, differentiation and cell survival. Inhibitory receptors PD1, TGIT, TIM-3, CTLA-4, and LAG3 bind to their ligands PD-L1/PD-L2. CD155, CEACAM, CD80/86, and MHC class II, respectively. Inhibitor receptor-ligand interaction results in checkpoint inhibition of T cell activation which suppresses T cell activity. CTLA-4 and PD1 binding to their ligands inhibits TCR/CD3 signaling and CD28 signaling which interferes with the co-stimulatory signals that enhance T cell activation. PD1 ligation to PD-L1 and PD-L2 inhibit the phosphorylation of ZAP70 and CD3 resulting in the inhibition of downstream signaling to production of NFAT. LAG3 disrupts the interaction with CD4 or CD8 binding to Lck and interacts with MHC class II proteins which interferes with TCR signaling. LAG3 also binds other ligands like Gal-3 and LSECtin. Competitive binding of the TIM-3 ligands Fyn and BAT3 can lead to T cell anergy. Phosphorylation of Lck in the inhibitory residue inhibits TCR/CD3 signaling. TIGIT competes with CD226 for binding to CD155, inhibiting the costimulatory effect of CD226 for T cell activation. Ligation of TGIT to CD155, phosphorylates the ITIM and ITT motifs on the cytoplasmic tail of CD155 which transduces inhibitory signals to the APC. Recruitment of Grb2, b-arrestin and the phosphatase SHIP1, inhibits TCR/CD3 signaling
31.Fig. 22.2: Five generations of chimeric antigen receptors for expression on T cells. Each generation consists of a modular design that includes an extracellular ScFv domain with a linker and co-stimulatory cytoplasmic domains. The first-generation CAR consists of ScFv, transmembrane CD8α and intracellular CD3ζ domain. The second and third-generation CARs include CD28 and 4-1BB cytoplasmic signaling domains, and OX40 and 4-1BB domains, respectively. The fourth-generation CAR includes a cytokine inducer domain to allow for cytokine expression, and the fifth-generation CAR includes the IL-2 Rβ (IL-2 receptor β chain) for JAK/STAT and CD28 signaling to enhance T cell activation
32.Cytokine Receptor Families
33.Fig. 24.2: The illustration depicts the general mechanisms of signal transduction of most class 1 and 2 cytokine receptor family members. Binding of the cytokine to the heterodimeric receptor molecule in the cell membrane leads to receptor dimerization and activation of cytoplasmic domains in the receptor by phosphorylation and recruitment of Janus kinases (JAK) and signal transducer and activation of transcription (STAT). JAK kinases phosphorylate the STATs which dimerize and translocate into the nucleus where they influence gene expression
34.Fig. 25.1: Illustration of the three pathways of complement fixation. The classical, lectin, and alternative pathways are shown. All pathways utilize complement component C3 which is cleaved to generate C3a, a soluble fragment, and C3b, a tissue bound fragment. After cleavage of complement component C5 to generate C5a and C5b, assembly of the membrane attack complex (MAC) is initiated by C5b to assemble, C6, C7, C8, and multiple components of C9 to form the pore which is inserted into the cell membrane. (Republished with permission of Elsevier publishing from Thurman (2015), Fig. 1. Conveyed through STM permission guidelines 2020)
35.Other Cytotoxicity Reactions
38.Bacterial Evasion of Immunity
39.Viral Evasion of Immunity
41.Fig. 32.1: Proposed mechanisms of synthesis, delivery, and action of the mRNA vaccine against SARS-CoV-2. The PCR template DNA or linearized plasmid DNA containing the designed vaccine sequences is transcribed in vitro in a media containing RNA polymerase and nucleotide phosphates. A mixture of dsRNAs and other aberrant products is obtained. Chromatographic purification such as FPLC is performed to obtain the mRNA with desired content and length. After vector-mediated delivery into the body, the mRNA transits into the cytosol. In the cytosol, the cellular translation machinery synthesizes proteins which undergo posttranslational modifications, resulting in properly folded, fully functional proteins. The secretory signal and MITD sequences direct the peptides to specific compartments of the endoplasmic reticulum and Golgi body for efficient secretion (linear B lymphocytes) and presentation by class I MHC for cytotoxic T lymphocytes and class II MHC for helper T cells. [Republished with permission of Elsevier publishing from Ahammad and Sultana Lira (2020). Figure 7. Conveyed through STM permission guidelines 2020]
42.Graft-Versus-Host Disease
43.Experimental Animals Used
44.Secondary Immunodeficiencies
46.Laboratory Report Topics
47.Fig. 37.1: Compound microscope showing labeled parts
48.Table 38.1: Pipet settings for measured volumes
49.Table 39.2: Typical profile of a differential blood cell count from a peripheral human blood smear
50.Table 40.2: Average cell count and viability determination obtained from a typical experiment where the cell suspension was diluted 1:20 (experiment 1) and 1:50 (experiment 2)
51.Fig. 41.1: Phagocytosis of bacterial cells by mouse macrophages isolated from the peritoneal cavity and macrophage cell line suspension. Cells are centrifuged, resuspended in medium, and incubated with bacteria
52.Fig. 42.2: Clearance of bacteria from blood by the reticuloendothelial system. Tissues (liver, spleen, lungs, thymus, and kidneys) collected from a mouse after bacterial injection and blood collection are ground in a mortar containing saline. The saline suspension is diluted tenfold and plated onto agar plates using the spread plate method
53.Fig. 43.1: Hemolytic plaque assay. Spleen cells obtained from a mouse immunized with sheep red blood cells (SRBCs) after 4 days are incubated with SRBCs and Guinea pig complement and poured on an agar plate. The formation of plaques indicates positive IgM antibody production. No plaques are formed in the negative control
54.Collection of Preimmune Serum
55.Fig. 45.3: Determination of hemolysin titer in SRBC agglutination. Positive agglutination is shown in wells 1–7 for a titer of 1:128
56.Fig. 46.1: Widal Test for identification Salmonella typhi antigens in patient sera
57.Fig. 47.1: ADCI assay performed to determine growth inhibition of P. falciparum
58.Fig. 48.3: Illustration of electrophoresis of antigens and precipitation by antibodies. (a) Immunoelectrophoresis of antigens placed in wells alongside a trough that is filled with antibody after antigens are electrophoresed. Precipitin arcs form after antibody diffuses and combines with antigen. (b) Rocket immunoelectrophoresis of antigens placed in wells and electrophoresed in agar mixed with antibody. Incubation of agar results in “rockets” showing antigen reactivity with the antibody
59.Fig. 49.1: Protein-A affinity purification of rabbit IgG. Rabbit antiserum is incubated with protein-A beads, followed by washes and elution of bound IgG with 0.58% acetic acid or 0.2 M glycine, pH 2
60.Table 50.1: Components required for preparing, separating, and stacking acrylamide gel mixtures
61.Fig. 51.2: Schematic illustration of SDS-PAGE and western plot procedure showing antibody identification of a single protein band from a heterogeneous mixture of proteins
63.Fig. 53.1: Indirect and capture ELISA. In indirect ELISA, antigen is coated on the wells of a microtiter plate, the wells are blocked, and the primary antibody is added to wells. Secondary antibody conjugated to HRP is added, and following the addition of substrate, reactive wells are identified. In capture ELISA, the capture antibody is coated to wells of a 96-well plate, the wells are blocked, antigen is added, followed by detection antibody. Biotinylated secondary antibody is added, followed by streptavidin-HRP. After substrate addition, wells are developed to detect the antigen
64.Fig. 54.1: ELISPOT Assay. Antibody or cytokine-secreting cells will be identified as dark spots on the nitrocellulose membrane
65.Fig. 55.2: Immunoelectron microscopy of Plasmodium chabaudi rhoptries
66.Fig. 56.1: Immunocytochemistry and immunohistochemistry assays performed to identify antigens in cells and tissue sections using antibodies specific to the antigens in cultured cells of cells obtained from patients or antigens in biopsied tissue or prepared tissue from experimental animal models
67.Fig. 57.2: (a) Illustration of side scatter and forward scatter plot of peripheral white blood cells (WBCs). (b) Different WBCs can be gated for further analysis. (c) The distribution of CD4+ and CD8+ T lymphocytes can be investigated using specific antibodies labeled with different colored fluorophores
68.Fig. 58.1: Complement fixation. Indicator system determines the presence of specific antibody in patient serum. If specific antibody is absent in patient serum, complement will not be fixed and addition of SRBC and anti-SRBC will result in hemolysis of SRBC
69.Procedure for Using Tissue Culture Hood
70.Fig. 60.1: Mitogen-induced response of B and T lymphocytes. Mononuclear cells obtained from spleen cell suspension is treated with mitogens to induce cell proliferation. The lectins concanavalin A (Con A), wheat germ agglutinin (WGA), and the molecule lipopolysaccharide (LPS)
71.Fig. 61.1: Induction of IL-2 secretion from mouse spleen cells and lymphoma cells. Spleen cells were incubated with concanavalin A (Con A) and lipopolysaccharide (LPS) and lymphoma cells were incubated with phorbol-12-myristate-13-acetate (PMA). Culture supernatants are tested for the secretion of the cytokine IL-2
73.Fig. 63.1: Immune system reconstitution of irradiated mouse to demonstrate the requirement of B and T lymphocyte cooperation in immune response to antigen
74.Fig. 64.1: Macrophage inhibitory factor (MIF) assay to demonstrate macrophage migration in response in absence of MIF and inhibition of macrophage migration in the presence of MIF
76.Fig. 66.1: Illustration of procedure for producing monoclonal antibodies. Spleen cells obtained from immunized mouse are fused with myeloma cells using polyethylene glycol (PEG). Hybridoma supernatants are screened for antibody production by ELISA, IFA or dot blots. Antibody positive hybridomas are cloned by limiting dilution for injection into mice to collect ascites fluid or grown in culture flasks to collect antibody in culture supernatant
77.Polymerase Chain Reaction (PCR)
78.Immunization of Mice for Challenge Infection
