01.Immunology: Overview and Laboratory Manual
05.Part I: Immunology Overview
07.Organization of the Immune System
08.2. Components of Immunity
10.Cell Lineage Development from HSCs
12.Cluster of Differentiation (CD) Antigens
13.4. Transcription Factors Important in Hematopoiesis and Immune Cell Function
14.5. The Immune System: Pathogen Sensing and Detection
15.Cells of the Immune System
16.Categories of Immune Responses
19.6. Innate Lymphoid Cells
20.7. Immunity of the Oral Cavity
22.Antimicrobial Function of Saliva
23.Role of Gingival Crevicular Fluid (GCF) in Oral Immunity
24.Innate and Adaptive Immunity in the Oral Cavity
25.8. Skin and Mucosal Immunity: Microbiome and Immune Homeostasis
27.Skin Microbiome and Immunity: Epidermis
28.Role of Langerhans Cells (LCs) in Skin Immunity
29.Role of Programmed Cell Death (PCD) Pathways to Maintain and Regulate Skin Immunity
30.Programmed Cell Death Pathways
33.Short Chain Fatty Acids and Gut Immune Homeostasis
34.Functions of SCFAs in Immune Homeostasis
38.Gut Lymphoid Organs, Cells, and Immune Function
40.Lymphoid Organs and Tissue
42.Organization of Thymus
43.Organization of the Spleen
44.Direction of Blood Flow Entering and Exiting the Spleen
45.10. Innate and Adaptive Immunity
46.Neuroendocrine Effects on the Immune System
49.Z-DNA Binding Protein 1 (ZBP1) PANoptosome
50.Absent in Melanoma 2 (AIM2) PANoptosome
52.Canonical and Noncanonical Inflammasomes
54.Innate and Adaptive Immunity
55.Mechanisms of Trained Immunity
56.Role of Noncoding RNAs in Trained Immunity
57.Metabolic Changes in Trained Immune Cells
58.Changes in Glycolysis and Krebs Cycle
59.13. Epigenetic Regulation of Trained Immunity
60.The Epigenetic Code and Immunity
61.Long Noncoding RNAs and Epigenetic Regulation
62.Cell Metabolism and the Epigenetic Code
64.14. Leukocyte Homing, Migration, and Recirculation
65.Cell Adhesion Molecules
68.Characteristics of Apoptotic Cells
69.Intrinsic Pathway (Mitochondrial Pathway)
72.Antigen Characteristics
73.Bacterial Antigens and Immunity
74.Virus Antigens and Immunity
75.Fungal Antigens and Immunity
76.17. B Cell Development, Activation, and Immunoglobulin Structure
81.18. Immunoglobulin and T Cell Receptor Gene Rearrangements
82.General Mechanisms of Rearrangements in B and T Cell Antigen Receptor Genes
83.Generating Antibody Diversity.
84.Features Contributing to Antibody Diversity
85.Germinal Center Formation
87.19. Major Histocompatibility Complex Genes
88.Structure of Class I MHC Proteins
89.Structure of Class II MHC Proteins
90.Functional Assays for Class I MHC Proteins
91.Cell-Mediated Lympholysis (CML) (51Chromium Release Assay)
92.Functional Assays for Class I and II MHC Proteins
93.Mixed Lymphocyte Response (MLR)
94.Two-Way MLR (e.g., Typing Tissue for Transplantation)
97.Relative Risk Calculations
98.Antigen Presenting Cells
103.Cross-Presentation of Antigenic Peptides by Antigen Presenting Cells
104.20. T Cell Development and T Cell Receptor Structure
106.Positive and Negative Selection of T Cells
111.T Cell Activation Signals
112.B Cell Activation Signals
115.Accessory Molecules for T Cell Activation
116.Examples of Accessory Molecules
125.21. Immune Checkpoint Inhibitors
126.Historical Uses of Immune Therapy
127.Immune Checkpoint Molecules
134.GITR (Glucocorticoid-Induced TNFR-Related Protein)
141.TIGIT (T Cell Immunoreceptor with Ig and ITIM Domains)
143.Other Activators and Repressors of T Cell Activation and Innate Immune Checkpoint
144.22. Chimeric Antigen Receptor (CAR) T Cells
145.Chimeric Antigen Receptors (CARs)
146.T Cell Receptor-CD3 Complex
147.Chimeric Antigen Receptor Design
149.CAR T Cell Culture and Infusion
150.Side Effects of CAR T Cell Therapy
155.Cytokine Receptor Families
156.24. Lymphocyte Signals
157.Signal Transduction in Lymphocytes
158.T Cell Receptor/CD3 Complex
160.B Cell Receptor Complex
161.Cytokine/Chemokine Receptor
162.FcεRI Receptor on Mast Cells
163.25. Complement Fixation
164.Regulation of the Complement Pathway
165.Examples of Regulatory Proteins
166.26. Cell-Mediated Immunity
167.Cell-Mediated Immunity (T Cell Immunity)
168.Other Cytotoxicity Reactions
169.27. Hypersensitivities
171.28. Parasite Immunity
173.Malaria and Leishmaniasis
175.Giardiasis and Amebiasis
177.29. Immunity to Bacteria
178.Bacterial Evasion of Immunity
179.30. Immunity to Viruses
182.Viral Evasion of Immunity
183.31. Immunity to Fungi
187.Live Attenuated Vaccines
188.Inactivated (Killed) Vaccines
189.Protein-Based Vaccines
191.Polysaccharide Vaccines
192.Nucleic Acid Vaccines
193.Recombinant Vector Vaccines
196.Graft-Versus-Host Disease
197.34. Immune Regulation and Autoimmunity
199.Mechanisms Leading to the Development of Autoimmunity
200.Examples of Autoimmune Diseases
201.Myasthenia Gravis (Blocking Autoantibodies)
202.Graves’ Disease (Stimulating Antibodies)
203.Systemic Lupus (Red Wolf) Erythematosus (SLE)
204.Rheumatoid Arthritis, RA (IC-Mediated Disease)
205.Antibody and T Cell-Mediated Autoimmune Disease
206.Hashimotos’s Thyroiditis
207.Multiple Sclerosis (T Cell-Mediated Autoimmune Disease)
209.Experimental Animals Used
210.35. Immunodeficiencies
211.Immunodeficiency Diseases
212.Primary Immunodeficiencies
213.Adaptive Immunity (B and T Cells)
217.Innate and Adaptive Immunity
219.Secondary Immunodeficiencies
221.Classification of Cancers
231.Study Guide Immunology Examination 1
233.Vocabulary: Define or Explain the Following Terms
235.Multiple Choice Questions
243.Study Guide Immunology Examination 2
245.Multiple Choice Questions
246.Study Guide for Final Exam
247.Quizzes Covering Chapter Material
260.Immunology Laboratory Manual
261.Immunology Laboratory Policies
262.Laboratory Safety Guidelines
263.Instructions for Notebook Entries
264.Instructions for Writing Laboratory Reports
265.Points: 100 Points for Each Report
267.Abstract: (On a Separate Page; About ½ Page)
268.Introduction: (Approx. 1 Page)
270.Methods: (1–3 Pages Depending on the Exercise(s))
271.Results: (1–4 Pages Depending on the Exercise(s))
272.Discussion: (1–3 Pages Depending on the Exercise(s))
273.Conclusions: (1–2 Paragraphs)
276.Laboratory Report Topics
277.37. Exercise 1: Using a Compound Microscope
278.Materials and Reagents (Per Pair)
279.Examination of Mouse Blood Using Giemsa-Stained Slides with the Oil Immersion Lens
281.38. Exercise 2: Pipetting and Dilution Techniques, Identification of Mouse Lymphoid Organs
287.Materials and Reagents (per Pair)
290.Materials and Reagents (per Pair)
295.39. Exercise 3: Blood Cell Preparation, Leukocyte Differentiation, and ABO Blood Typing
298.Cells of the Immune System
299.Neutrophils (Polymorphonuclear Leukocytes, PMNs)
303.Materials and Reagents (per Pair)
305.40. Exercise 4: Single Suspension of Mouse Spleen Cells, Cell Viability Assays, and Identification of Specific Cells Using Cell Surface Antigens
307.Total Leukocyte Count (Mononuclear Cell Count) and Trypan Blue Viability Determination
308.Materials and Reagents (per Pair)
311.Collection of Lymphoid Cells
313.Determination of Cell Viability by Trypan Blue Exclusion
315.Giemsa Stain of Lymphoid Cells
318.41. Exercise 5: Isolation of Mouse Peritoneal Macrophages: In Vitro
320.Materials and Reagents (per Group)
321.Procedure (Fig. 41.1)
322.42. Exercise 6: Clearance of Bacteria from Mouse Blood by the Reticuloendothelial System (RES)
324.Materials and Reagents (per Group)
326.43. Exercise 7: Hemolytic Plaque Assay (The Jerne Plaque Assay)
328.Materials and Reagents (per Group)
332.44. Exercise 8: Rabbit and Mouse Immunizations: Preparation of Polyclonal Antibodies and Screening Assays
334.Handling of Experimental Animals
335.Blood Collection and Immunization
336.Materials (per Group)
338.Collection of Preimmune Serum
339.45. Exercise 9: Determination of Antibody Titer and Screening Techniques
342.Materials and Reagents (per Pair)
347.Materials and Reagents (per Pair)
351.Materials and Reagents (per Pair)
353.Tanning and Coating Sheep Red Blood Cells
354.Tanning Cells with Tannic Acid
355.Coating Tanned Cells with Bovine Serum Albumin (BSA)
357.Materials and Reagents (per Pair)
359.46. Exercise 10: Widal Test
360.Materials and Reagents
361.Procedure (Fig. 46.1)
362.47. Exercise 11: Antibody-Dependent Cellular Inhibition (ADCI)
363.Materials and Reagents (Per Group)
365.Procedure (Fig. 47.1)
366.48. Exercise 12: Immunodiffusion and Immunoelectrophoresis
368.Immunoelectrophoresis (IEP)
369.Ouchterlony Double Diffusion
370.Materials and Reagents (per Pair)
372.Immunoelectrophoresis
373.Materials and Reagents (per Pair)
375.Rocket Immunoelectrophoresis
376.Materials and Reagents (per Pair)
378.49. Exercise 13: Protein-A Affinity Purification of Rabbit IgG
380.Materials and Reagents (per Pair)
384.Digestion of Rabbit IgG with Pepsin for Generation of F(ab′)2 Fragments
386.50. Exercise 14: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
388.Define or Explain the Following Terms
389.Materials and Reagents (per Pair)
390.Assembling SDS-PAGE Gels
393.51. Exercise 15: Immunoblotting (Western Blotting) and Dot Blotting
395.Materials and Reagents (per Pair)
398.Materials and Reagents
400.52. Exercise 16: Protein Assay
402.Materials and Reagents (Per Group)
404.53. Exercise 17: Enzyme-Linked Immunosorbent Assay (ELISA)
406.Materials and Reagents (Per Pair)
407.Materials and Reagents (Per Pair)
408.Starting with Precoated Plates
409.Detection of LPS in Extracts of E. coli Lysates
411.Direct ELISA Procedure
412.Capture (Sandwich) ELISA
413.54. Exercise 18: Enzyme-Linked Immunospot Assay (ELISPOT)
415.Materials and Reagents (Per Pair)
417.55. Exercise 19: Immunofluorescence Assay (IFA)
419.Materials and Reagents (Per Pair)
421.56. Exercise 20: Immunocytochemistry and Immunohistochemistry
426.57. Exercise 21: Flow Cytometry
428.Materials and Reagents (Per Pair)
430.58. Exercise 22: Complement Fixation Assays
432.Materials and Reagents (Per Pair)
437.59. Tissue Culture Techniques: Introduction
438.Types of Cell Culture
439.Use of Pipets (See Exercise 2)
440.Procedure for Using Tissue Culture Hood
441.60. Exercise 23: Mitogen-Induced Response of Lymphocytes: Detection of B and T Lymphocytes Using Immunofluorescence
443.Materials and Reagents (Per Group)
445.61. Exercise 24: Induction of IL-2 Secretion from Mouse Lymphoma Cells (EL4.IL-2)
447.Materials and Reagents (Per Group)
449.Screening for IL-2 Production by Elisa Using Anti-IL-2 Antisera
450.62. Exercise 25: Intracellular Staining for Cytokine Detection
452.Materials and Reagents (Per Group)
454.63. Exercise 26: Adoptive Transfer of Lymphocytes: B and T Lymphocyte Cooperation for Antibody Production
456.Materials and Reagents (Per Pair)
458.64. Exercise 27: Macrophage Migration Inhibition Test
460.Materials and Reagents (Per Group)
462.65. Exercise 28: Griess Assay for Detection of Nitric Oxide (NO)
464.Materials and Reagents (Per Pair)
466.66. Exercise 29: Monoclonal Antibody Production
468.Materials and Reagents (Per Pair)
471.Cloning by Limiting Dilution
472.67. Exercise 30: Genomic DNA Isolation from Plasmodium falciparum and Colpodella sp. for Polymerase Chain Reaction
474.Materials and Reagents (Per Pair)
476.Polymerase Chain Reaction (PCR)
477.68. Exercise 31: Immunization of Mice with a Recombinant Protein
479.Materials (Per Group)
481.Immunization of Mice for Antibody Production
482.Immunization of Mice for Challenge Infection
483.69. Exercise 32: Polarization of T Helper Cells into TH1 Cells
487.70. Exercise 33: Immunoprecipitation
489.Materials (Per Group)
491.71. Exercise 34: Chromatin Immunoprecipitation (ChIP)
492.Materials and Reagents (Per Group)
494.72. Exercise 35: Antibody Immobilized Gold Electrodes for Antigen Detection
496.Materials and Reagents
498.73. Exercise 36: Induction of Apoptosis in T Lymphocytes and Detection of Apoptotic Markers
502.Annexin V Labeling Procedure
505.74. Exercise 37: Cloning and Sequencing cDNAs of Mouse Variable Regions of H and L Chains of Monoclonal Antibodies
512.Vendors Where Supplies, Reagents, Equipment, and Materials for Immunology Exercises Can Be Purchased
513.Helpful Immunology Websites and Sites That Have Supply Reagents Useful for Immunology Experiments
514.Buffers and Solutions
515.Sample Practice Questions
543.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]
544.Fig. 5.1: Dendritic cells cultured from bone marrow precursors with GM-CSF (a) cultures at 24 h, (b, c) cultures at 6 days, red arrows (b) showing long and thin extensions (dendrites). (d) Positive immunofluorescent label of MHC class II on iDC at 6 day culture. (e) Positive immunofluorescent label of MHC class II on iDC after 2 h culture with LPS. (e) Toluidine blue-stained DC showing extensions (dendrites). [Republished with permission of IntechOpen publishing from Castell-Rodriguez et al. 2017, Figure 2. Conveyed through STM permission guidelines 2014]
545.Fig. 5.2: Gut mucosal barrier. The illustration depicts two mucus layers overlaying the gut epithelium (left) and domain structures of secretory (MUC2) and membrane-bound (MUC3) mucins on the right. MUC3 and MUC17 are part of the glycocalyx, which makes up membrane-bound mucins covering the epithelium and loosely adherent mucin layer composed of MUC2 secreted by goblet cells, paneth cells, and enterocytes. Microbes are associated with the loose mucin layer. The domain structure of MUC2 monomer shows central tandem repeat (TR) regions rich in proline, threonine, and serine (PTS domain) to which many oligosaccharide side chains (O-linked glycan) are linked, and four von Willebrand factor D domains flanking the tandem repeat (PTS) domains and C-terminal knot (CK) domain, which is involved in initial MUC2 dimerization. The domain structure of MUC3 mucin shows that it consists of two subunits, one extracellular and one membrane-bound. The extracellular subunit consists of a glycosylated tandem repeat (PTS) domain and two epidermal growth factor (EGF)-like domains separated by sperm protein, enterokinase, and agrin (SEA) motif (a proteolytic cleavage site during biosynthesis), a membrane-bound subunit that consists of membrane-spanning domain and a cytoplasmic tail with potential phosphorylation (P) sites. [Republished with permission of SpringerNature publishing from Kim and Ho 2010, Figure 1. Conveyed through STM permission guidelines 2014]
546.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
547.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
548.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
549.Fig. 9.1: Mouse (6-month-old 129 mouse) bone marrow showing hematopoiesis. M Myeloid, E Erythroid regions and megakaryocytes (arrows). [Republished with permission of SAGE publishing from Travlos 2006, Figure 10. Conveyed through STM permission guidelines 2014] Lymphoid Organs and Tissue
550.Fig. 9.2: Bone marrow smear from a normal rat showing cells of different lineages during hematopoiesis. BN Band neutrophil, L Lymphocyte, MF Mitotic figure, MM Metamyelocyte, MR Metarubricyte, R Rubricyte, RM Ring form myelocyte, SN Segmented neutrophil. [Republished with permission of SAGE publishing from Travlos 2006, Figure 12. Conveyed through STM permission guidelines 2014]
551.Fig. 9.3: Rat and mouse thymus. (a) Rat (3-month-old female Wistar rat) thymus showing cortex and medulla and thymic lobules. (b) Higher magnification of (a). (c) Mouse (3-month-old B6C3F1 mouse) thymus showing absence of thymic lobules. (d) Higher magnification of C. [Republished with permission of SAGE publishing from Pearse 2006, Figure 8. Conveyed through STM permission guidelines 2014]
552.Fig. 9.4: Illustration of a single lymph node lobule showing afferent and efferent lymphatic vessels, subcapsular sinus, follicles, medullary sinuses, medullary cord, and deep cortical unit (DCU) containing paracortex. [Republished with permission of SAGE publishing from Willard-Mack 2006, Figure 1. Conveyed through STM permission guidelines 2014]
553.Fig. 9.5: Mouse (20-week-old female B6C3F1 mouse) spleen, (left) and rat (12-week-old male F344/N rat) (right). A Central artery, F Follicle, H Hilus, MS Marginal sinus region, MZ Marginal zone, P Periarteriolar lymphoid sheath, RP Red pulp. [Republished with permission of SAGE publishing from Cesta 2006a, Figure 3. Conveyed through STM permission guidelines 2014]
554.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]
555.Fig. 10.1: Pattern recognition receptor family members NLR and ALR from canonical inflammasomes. NLR or ALR sensors detect and bind to viral and bacterial PAMPs. NLRC4 is activated by bacterial flagellin and T3SS components, NLRP1b is activated by anthrax lethal toxin, and AIM2 is activated by cytosolic dsDNA. NLRP3 is activated by a wide variety of signals including pore-forming cytotoxins, ATP, uric acid, and alum. Once activated, the receptors form an inflammasome complex with or without the adaptor, ASC, and recruit procaspase-1, which is subsequently cleaved into caspase-1. Caspase-1 cleaves precursors of IL-1β and IL_18 into their active forms as well as induces cell death. (Republished with permission of Elsevier publishing from Vanaja et al. (2015), Figure 1. Conveyed through STM permission guidelines 2014)
556.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)
557.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
558.Fig. 12.1: Cells of the innate and adaptive immunity. Binding of PAMPs to PRRs on APCs like macrophages results in signaling that produces downstream expression of proinflammatory cytokines like IL-6 and TNFα. NK cells, mast cells, ILCs, monocytes, and neutrophils respond to cytokines and chemokines that promote migration. Processed antigen associates with MHC proteins for presentation to naïve T cells. Activated Th cells cooperate with B cells which become activated to secrete antibodies in the adaptive immune response
559.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
560.Fig. 15.1: Apoptosis induced by cytotoxic T lymphocytes (CTL) can occur through the Fas or perforin granzyme pathways. In the Fas pathway, Fas on the target host cell binds to Fas ligand (FasL) on the CTL. The adapter protein FADD binds to Fas; inactive caspase 8 is recruited to the adapter resulting in activation of caspase 8 and events leading to apoptosis within the target cell. Perforin and granzyme are released from granules in the CTL and taken up by the target cell. Perforin forms pores in the endocytic vesicle and releases granzyme which activates Bid, a proapoptotic protein and procaspase 3. Bid induces cytochrome c release from the mitochondria. Cytochrome c, Apaf-1, and procaspase 9 assemble to form the apoptosome, where caspase 9 is activated and also cleaves procaspase 3. Active caspase 3 along with cytochrome c initiates apoptosis in the target cell
561.Fig. 15.2: Detection of mitochondrial outer membrane (OM) permeabilization in H1975 cancer cells treated with 1 μM staurosporine (STS) for 12 h followed by immunofluorescence staining. Antibodies specific for Cyt c and active caspase-3 (Casp-3), as well counterstaining with Hoechst (which marks chromatin), were used to stain cells. In control cells (upper panels), Cyt c is detected in the intermembrane space (IMS). A “tubular” pattern of fluorescence was observed and caspase-3 was detected. STS-induced mitochondrial membrane permeabilization (lower panels) shows Cyt c released into the cytosol and observed as diffuse staining, where it leads to the activation of caspase-3. The pyknotic nucleus detected is characteristic of cells undergoing apoptosis. White scale bars represent 10 μm. (Republished with permission of SpringerNature publishing from Galluzi et al. (2007), Figure 1. Conveyed through STM permission guidelines 2014)
562.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)
563.Fig. 17.1: B lymphocyte activation. Antigen binding to membrane immunoglobulin (Ig) leads to phosphorylation of Igα/Igβ and CD19 by Lyn, followed by recruitment and activation of Syk and Btk (signalosome) through phosphorylation. Following phosphorylation of the adaptor proteins BLNK and BCAP, phosphatidylinositol 3-kinase is recruited to BCAP and Vav to BLNK resulting in PIP3 formation and MAP kinase activation, respectively. The guanine nucleotide exchange factor Vav mediates cytoskeletal reorganization. PLCγ2 recruited to BLNK is activated by Syk-mediated phosphorylation leading to the cleavage of phosphatidylinositol bisphosphate (PIP2) and generation of diacylglycerol (DAG) and inositol triphosphate (IP3). The MAP kinase pathway becomes activated once DAG binds to the GTP exchange factor RasGRP and PKC enhanced by IP3-mediated release of calcium from calcium stores. The transition factors cyclic-AMP responsive element binding protein (CREB), Jun, Ets-like-1 (Elk1), early growth response 1 (Egr1), and nuclear factor of activated T cells (NFAT) become activated and translocate into the nucleus. Elk1 and Egr1 are activated downstream of extracellular signal-regulated kinase1/2. PDK1 and Akt localize to the membrane, activated Akt phosphorylates and inactivates Bax and Bad resulting in inhibition of apoptosis and enhancement of cell survival
564.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
565.Fig. 18.1: Germline organization and rearrangement of the antibody H chain gene. From multiple V (D) J genes, recombination and rearrangements take place in H chain gene to generate the variable region for antigen binding. The V regions are combined with constant region genes to encode the full H chain
566.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
567.Fig. 19.1: Structure of the class I and class II MHC proteins. Class I MHC consists of a membrane protein α chain organized in three domains and associated with β2-microglobulin. The α1 and α2 domains make up the closed antigen binding pocket that fits peptides of 8–10 amino acid residues, with nine residues being the optimum length. A longer peptide would buckle to fit the binding pocket. The “hamburger in the bun” analogy used here shows the peptide confined within the binding groove or pocket. Class II MHC consists of α β membrane proteins associated as a heterodimer. The antigen binding pocket is made up of α1 and β1 domains. Longer antigen peptides can fit into the pocket with the ends of the peptides protruding out of the pocket in a “hot dog in a bun” analogy
568.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
569.Fig. 20.1: Germline organization and rearrangement of αβ T cell receptor genes. From multiple V (D) J genes, recombination and rearrangements take place in the α and β genes to generate the variable region for antigen binding. The V regions are combined with constant region genes to encode the full α and β chains
570.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
571.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
572.Fig. 22.1: Production and transfusion of autologous chimeric antigen receptor (CAR) T cells. Peripheral blood mononuclear cells (PBMCs) are collected from a patient by leukapheresis. T cells are selected, activated and engineered by transduction with a virus vector encoding the CAR transgene. The T cell population is expanded in culture after delivery and expression of the CAR gene. The CAR-T cells are adoptively transferred back to the patient by infusion after lymphodepletion therapy to permit CAR-T cell engraftment, establishment and targeting of tumor cells for destruction
573.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
574.Fig. 24.1: Lymphocyte signaling is initiated by ligand binding to membrane receptors, many of which are associated with co-receptor molecules that may possess long cytoplasmic tail containing ITAMs suitable for phosphorylation and activation to induce cytoplasmic signal transduction. Receptor binding leads to clustering of signaling molecules in the membrane to form lipid rafts. Easy access and exposure of activated domains following phosphorylation by serine-tyrosine kinases leads to recruitment of adaptor molecules, which are also activated and serve as platforms for the binding of other signaling molecules. Some common themes include the cleavage of PIP2 by PLCγ and the generation of the second messengers, DAG and IP3. Calcium release by IP3 leads to binding to calmodulin, which binds to the phosphatase calcineurin. Calcineurin dephosphorylates NFAT, a transcription factor that translocates into the nucleus. Diacylglycerol (DAG) activates PKC leading to events that activate the cytosolic molecule NFκB allowing its translocation into the nucleus. Recruitment of the guanosine exchange factors RasGRP and SOS leads to the activation of Ras which activates the MAP kinase pathway and the generation of the transcription AP-1. Entry of the transcription factors into the nucleus influences gene expression of genes encoding cytokines, cytokine receptors, and antibodies in B cells
575.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
576.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)
577.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]
578.Fig. 37.1: Compound microscope showing labeled parts
579.Fig. 38.1: Pipet pump with serological pipet, pipettor, and transfer pipet (squeezer)
580.Fig. 38.2: Serial tenfold (a) and twofold (b) dilution scheme in 1 mL volumes. Tenfold dilutions are performed using red blood cells, and twofold dilutions are performed using methylene blue to demonstrate the importance of dilutions in the immunology lab
581.Fig. 38.3: Identification of mouse thymus and spleen, primary and secondary lymphoid organs, respectively
582.Fig. 38.4: Preparing a mouse spleen cell suspension using the plunger from a 10 mL syringe. Following splenectomy, the spleen is placed on a wire mesh contained in a petri dish with tissue culture medium. Using the plunger, spleen cells are gently teased into the medium
583.Fig. 39.1: Leukocytes (white blood cells) and platelets (thrombocytes)
584.Fig. 39.2: ABO and + – Rhesus (D) blood typing. Individuals with type A blood have the A antigen, A lack anti-A antibodies but have anti-B antibodies. Individuals with type B blood have the B antigen, lack anti-B antibodies but B have anti-A antibodies. Individuals with type AB blood have A and B antigens and lack anti-A and anti-B antibodies. AB Individuals with type O blood have anti-A and anti-B antibodies and lack A and B antigens. The O presence of the D antigen on red cells will result in binding to anti-D antibodies which is a D positive test
585.Fig. 40.1: Hemocytometer showing the duplicate counting chambers with coverslips. Cells are added to the “V”-shaped groove. Capillary action moves the cells into the chamber. An enlarged counting chamber shows four large squares for counting white blood cells (WBCs). The center grid is used for counting red blood cells (RBCs)
586.Fig. 40.2: Separation of blood cells on Histopaque. Following centrifugation, the mononuclear cell fraction is located at the interface of Histopaque, and the medium and the red blood cells are found in the pellet fraction
587.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
588.Fig. 42.1: Clearance of bacteria from blood by the reticuloendothelial system. Blood collected after injection of bacteria is diluted tenfold, then plated onto agar using the spread plate method
589.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
590.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
591.Fig. 45.1: Precipitation curve. Immune complexes form, leading to precipitation at the zone of equivalence. In the presence of excess antibody (prezone) or excess antigen (postzone), precipitate will not form
592.Fig. 45.2: Dilution scheme for SRBC agglutination in round-bottomed 96-well microtiter plates. Anti-SRBC (hemolysin) serially diluted in a twofold dilution scheme is incubated with SRBC. Control wells contain PBS or saline and normal rabbit serum (NRS)
593.Fig. 45.3: Determination of hemolysin titer in SRBC agglutination. Positive agglutination is shown in wells 1–7 for a titer of 1:128
594.Fig. 46.1: Widal Test for identification Salmonella typhi antigens in patient sera
595.Fig. 47.1: ADCI assay performed to determine growth inhibition of P. falciparum
596.Fig. 48.1: Illustration of double diffusion immunoprecipitation (Ouchterlony) of antibody and antigens. Antibody in the lower well and antigens in the top two wells. Patterns of identity, partial identity, and nonidentity are shown
597.Fig. 48.2: Well template for Ouchterlony immunoprecipitation. In a petri dish containing agarose, punch holes as shown in the template and, using a Pasteur pipet with a rubber bulb or an aspirator, remove the agarose plugs. Fill the wells with the antibodies and antigens shown. Antigens: BSA, bovine serum albumin; FBS, fetal bovine serum; OVA, ovalbumin; HS, human serum; DS, Donkey serum; CHS, chicken serum; GS, goat serum; RPL, rabbit plasma; NRS, normal rabbit serum. Antibodies: anti-bovine serum albumin and anti-goat serum
598.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
599.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
600.Fig. 50.1: SDS-PAGE gel preparation. A mixture of acrylamide and bis-acrylamide prepared in Tris-glycine-SDS buffer is poured into the front and back plates assembled with spacers, first the separating or running gel, followed by the stacking gel. A comb used to form the wells is inserted into the stacking gel before the gel polymerizes (top left). The comb and bottom spacer are removed before the gel is placed in a gel chamber with buffer. Proteins loaded in the gel are electrophoresed
601.Fig. 50.2: Gel loading template for SDS-PAGE gel
602.Fig. 51.1: Western blot sandwich assembly showing SDS-PAGE gel “sandwiched” between filter papers and cassettes. Locked cassette is placed vertically in a wet transfer buffer system contained in an electrophoresis chamber
603.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
604.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
605.Fig. 54.1: ELISPOT Assay. Antibody or cytokine-secreting cells will be identified as dark spots on the nitrocellulose membrane
606.Fig. 55.1: Indirect immunofluorescent staining of Plasmodium falciparum merozoite rhoptries
607.Fig. 55.2: Immunoelectron microscopy of Plasmodium chabaudi rhoptries
608.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
609.Fig. 57.1: Flow cytometry. Cells in suspension are passed through laser light source. Forward and side scattered light is detected. Fluorescent light is also detected as cells are sorted
610.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
611.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
612.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)
613.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
614.Fig. 63.1: Immune system reconstitution of irradiated mouse to demonstrate the requirement of B and T lymphocyte cooperation in immune response to antigen
615.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
616.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
617.Table 38.1: Pipet settings for measured volumes
618.Table 39.1: ABO blood antigens
619.Table 39.2: Typical profile of a differential blood cell count from a peripheral human blood smear
620.Table 40.1: Summary of some frequently encountered types and sources of CD antigens
621.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)
622.Table 50.1: Components required for preparing, separating, and stacking acrylamide gel mixtures
