Legends %e2%80%93 Immunology Diagrams

Legends %e2%80%93 Immunology Diagrams - Legends –...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: Legends – Immunology Diagrams Imm. – 1. Development of Immune Cells (hematopoiesis). The pluripotent hematopoietic stem cells can give rise to all cells in the blood. The pluripotent hematopoietic stem cell is selfrenewing and some stem cells differentiate into precursor cells that can give rise to a more limited set of cell types. The lymphoid precursor cell gives rise to T and B cells and probably NK cells. The myeloid precursor cell gives rise to monocytes, neutrophils, basophils, mast cells and eosinophils. When monocytes enter tissues, they differentiate into macrophages. The precursor cells that give rise to platelets and red blood cells are also derived from the myeloid precursor cell. Only the immune cells that will be discussed in MICB202 are depicted here. Imm. – 2. Pattern Recognition Receptors. These are molecules that are either cell membrane associated or secreted and recognize distinct molecular patterns found on pathogens. Shown in the figure is an endocytic receptor and a Toll-like receptor. The recognition of a PAMP by a Tolllike receptor results in a signaling cascade and the activation of T cells to initiate an adaptive immune response. Imm. – 3. The Inflammatory Response. Penetration of the body’s first line of defense, the skin, by bacteria elicits inflammation. Cells at the sites of tissue damage release compounds that increase blood flow to the area and recruit phagocytes (neutrophils and macrophages). These phagocytic cells engulf the bacteria and damaged cellular material. Phagocytic cells leave the blood vessels and enter the tissue by squeezing between the endothelial cells that line the blood vessel wall, and migrate towards the bacteria. In most cases, this eliminates the infection. However, if bacteria at the wound site succeed in invading lymphatic vessels or blood vessels, a more general bacterial invasion occurs and an adaptive immune response must be mounted. Imm. – 4. Phagocytosis. Bacteria are engulfed by the cell and taken into a special organelle called a phagosome. The phagosome fuses with lysosomes to form a phagolysosome. In the phagolysosome, low pH, hydrogen peroxide, lysozyme, and proteases kill the bacteria and degrade it into small fragments. Large fragments of dead bacteria are released from the cell by a process called exocytosis while smaller fragments such as amino acids are recycled in the cell. Imm. – 5. The Complement System. There are three pathways of complement activation; only two of the pathways are discussed in MICB202. The classical pathway is triggered by the binding of complement component C1q to antibodies that have already bound to the pathogen surface. The alternative pathway is triggered by the spontaneous breakdown of complement component C3 and the binding of C3b to the pathogen surface. Both pathways lead to the formation of the C3 convertase which is a critical step for the generation of the effector molecules. The three main consequences of complement activation are opsonization of pathogens, recruitment of inflammatory cells and the direct killing of pathogens. Imm. – 6. MHC Class I Proteins and Antigen (Ag) Processing and Presentation. MHC class I molecules are found on most nucleated cells. MHC class I molecules consist of a membranebound a chain which is non-covalently associated with β2-microglobulin. In the endogenous pathway (bottom left), the peptides are derived from cytoplasmic proteins or proteins synthesized during a viral replication that are processed by the proteasome. The peptides are transported into the endoplasmic reticulum by the TAP transporter proteins. In the ER, the peptides bind to MHC class I proteins and the peptide/MHC class I complexes pass through the Golgi apparatus and are brought to the cell surface in vesicles. The peptide/MHC class I complexes are then displayed on the cell surface and the peptides are "presented" to T cells. In the cross-presentation pathway that has been described in dendritic cells, the peptides are derived from antigen taken up by the cell in an endosome (e.g., virus particle), and the contents of the endosome are diverted to the proteasome. This pathway allows a dendritic cell to present antigenic peptides on MHC class I molecules to activated p-CTLs. Imm. – 7. MHC Class II Proteins and Antigen (Ag) Processing and Presentation. MHC class II molecules are only found on B cells, dendritic cells and activated macrophages. b) Antigens taken up from the medium are degraded into peptides in endosomes or phagolysosomes. MHC class II proteins travel from the ER to these sites of antigen processing. The peptide-binding site of the MHC class II proteins coming from the ER is blocked by a polypeptide called the invariant chain (Ii). In the Golgi complex, Ii is partially degraded, resulting in a small peptide called CLIP blocking the peptide binding site of the MHC class II molecule. The vesicle containing the peptide fragments fuses with a vesicle containing the MHC class II-CLIP complex, and CLIP is released and the MHC class II proteins bind peptides. The peptide/MHC II complexes are transported to the cell surface in vesicles. The peptide/MHC II complexes are then displayed on the cell surface and presented to T cells. Imm. – 8. The T Cell Receptor (TCR) Complex. The TCR complex of a mature T cell consists of an antigen-binding portion that is comprised of TCRα and TCRβ polypeptides, associated signaling subunit (CD3 complex) and accessory molecules (CD4 or CD8, but not both). The TCR antigen-binding subunit recognizes peptides that are bound to MHC proteins. It does not recognize native, intact antigens. The signaling subunit (CD3) of the TCR sends signals to the inside of the T cell that activates it and causes it to divide. The co-receptors CD4 and CD8 define the effector function – CD4 is typically found on T helper cells whereas CD8 is typically found on cytotoxic T cells. CD4 and CD8 molecules bind to the MHC proteins of the APC, but at a different site to the TCR (i.e., not the site involved in peptide binding). This increases the affinity of interaction between the T cell and the APC. Imm. – 9. Positive and Negative Selection of T cells. During T cell development in the thymus, thymocytes (immature T cells) start to express the TCR and both CD4 and CD8 co-receptors. Thymocytes that have TCRs that can interact with MHC class I or MHC class II molecules are positively selected by thymic epithelial cells and receive a “survival signal”; those that TCRs that cannot interact with MHC, do not receive a survival signal and die of neglect. After positive selection, the thymocytes begin to express only the CD4 co-receptor or the CD8 co-receptor. The thymocytes are then subjected to negative selection by dendritic cells and macrophages; those that have TCRs that bind MHC and self-peptide with high affinity receive a signal to undergo apoptosis. Only thymocytes that have TCR that bind MHC with weak affinity are permitted to mature into functional T cells and leave the thymus. Imm. – 10. The "Two Signal" Model for the Activation of T Cells. "Professional" APCs such as dendritic provide the co-stimulatory signal ("signal 2") required (CD28/B7 interaction) for T cell activation. "Non-professional" APCs such as endothelial cells can provide "signal 1", but are less efficient at providing "signal 2". If a T cell receives "signal 1" but does not receive "signal 2", it will enter a state of non-responsiveness (anergy). Imm. – 11. Cytokine Mediated Generation of TH Subsets. Naive CD4 T cell proliferating in an IL-12 dominated environment will develop into TH1-type helper T cells. Naive CD4 T cell proliferating in an IL-4 dominated environment will develop into TH2-type helper T cells. The cytokines produced by one subset of T helper cell positively regulates the subset that produces it, and negatively regulates the other subset. Imm. – 12. Activation of Cytotoxic T Cells. After the TCR has interacted with the peptide-MHC I complex on the antigen-presenting cell (APC) and received the co-stimulatory signal, the CTL-P expresses IL-2 receptors. The proliferation and activation of the CTL-Ps requires additional IL-2 secreted by TH1 cells (which had been activated by interaction with the antigen-presenting cell). The T cell receptor of the CTL recognizes the viral peptide complexed with MHC I proteins. This interaction is enhanced by the binding of CD8 to the MHC I protein. The activated CTL then destroys the target cell by the targeted release of perforin to lyse the infected cell and granzyme to induce apoptosis in the infected cell. Imm. – 13. B Cell Antigen Receptor (BCR) Complex. The BCR is a modular receptor. One subunit of the receptor, the membrane-bound immunoglobulin (mIg, either IgM or IgD; IgM is shown here) is responsible for binding antigen. The other subunit, Ig-α/Igβ, is responsible for sending a signal to the inside of the cell once an antigen has bound to the BCR. This signal activates the B cell. When the B cell receives additional signals from activated helper T cells, it will proliferate. Some of the progeny cells will differentiate into antibody-secreting plasma cells while others differentiate into memory cells. Imm. – 14. Activation of B cells. The cross-linking of membrane bound Ig by antigen results in the internalization of the antigen, with its subsequent processing and presentation on MHC class II molecules. The B cell interacts with a TH2 cell (that had been previously activated by a dendritic cell) and receives cytokines from the TH2 cell, resulting in the complete activation of the B cell. The B cell is then able to proliferate and differentiate into antibody-secreting plasma cells. Imm. – 15. Antibody Structure. The structures of an IgM (top) and IgG (bottom) antibodies are shown. For each antibody, the 2 heavy (H) and 2 light (L) chains are held together by disulfide bonds. The N-terminal portions of the H and L chains are the variable regions (VH, VL) and vary from one antibody to another. The rest of the H and L chains are the constant region which are identical for all chains of that type (e.g., all µ or γ chains for the heavy chain). The hypervariable (HV) regions of the VH and VL regions make up the antibody binding sites of the antibody. The amino acids that make up these HV regions determine the shape of the side chains of the HV region amino acids and bind to the antibody with high affinity. The ability of an antibody to bind an antigenic determinant (or epitope) depends on the ability to form close contacts between the surface of the antigenic determinant and the surface of the antigen-binding site of the antibody. The constant region of the heavy chain determines the functionality of the antibody in the immune system. Both IgM and IgG are capable of activating complement, but only IgG binds to receptors on phagocytic cells. Imm. – 16. Primary and Secondary Responses. In a primary response, there is a delay of about 5 – 7 days after immunization with the antigen before antibody can be detected. During this time, the T helper cells and B cells are being activated, proliferating and undergoing differentiation. As plasma cells develop and start to secrete antibody, the concentration of antibody in the blood serum gradually increases (this demonstrates the “inducibility” of the adaptive immune system). The antibody is typically IgM and will last for a few weeks. If re-immunized with the same antigen, the adaptive response occurs much quicker and much more vigourously – antibody can be detected within 2 days. The memory T helper cells and memory B cells are re-activated, and the B cells quickly respond by differentiating into plasma cells (this demonstrates the “memory” of the adaptive immune system). Because of the proliferation of the T helper cells and B cells during the primary response, there are more cells that recognize this specific antigen. Thus, when the plasma cells develop, there is a larger pool of plasma cells to secrete antibody. The antibody is typically IgG, and will last for many months or years. The specificity of the immune response can be demonstrated by immunizing with a second, different antigen – the immune system will develop a separate, primary response to this antigen (this demonstrates the “specificity” of the adaptive immune system). Imm. – 17. Activation of Macrophages by TH1 Cells. TH1 cells provide the macrophages with cytokines so that they can more efficiently kill intracellular bacteria. Activated macrophages fuse their lysosomes more efficiently to the phagosomes and kill intracellular bacteria and recently ingested pathogens and synthesize toxic reactive nitrogen metabolite nitric oxide (NO), oxygen radicals and proteases. Imm. – 18. IgE and Allergies. Sequences in the constant region of the ε heavy chain allow IgE to bind to specific receptors (Fcε receptors) on mast cells. When antigen binds to these IgE molecules, they trigger the mast cell to degranulate and release large quantities of histamine that cause allergic responses. Imm. – 19. Transgenic and Gene Knock-out Mice. a) DNA containing the transgene is injected into the male pronucleus of a mouse embryo that is then transferred to a pseudo-pregnant mouse. Offspring are then screened for the presence of the transgene. b) DNA containing the disrupted gene is injected into an embryonic stem (ES) cell. Drug selection is used to isolate the ES cells in which the DNA has undergone homologous recombination. The ES cells are injected into a mouse blastocyst that is then implanted into the uterus of a pseudo-pregnant mouse. Offspring are screened for the presence of the disrupted gene. Source: Chen, J., and Roop, D.R., J. Investig. Dermatol. Symp. Proc., 10:37 – 46, 2005. Imm. – 20. Hybridomas and Monoclonal Antibodies. A mouse is repeatedly immunized with an antigen so that a strong antibody response is generated. The spleen is removed and the B cells are then fused to myeloma cells. Several possible fusions may occur: B cell – B cell, myeloma – myeloma, and B cell – myeloma. The B cell – myeloma fusion is called a hybridoma, and can be selected by the use of special drugs. The hybridoma has characteristics of both the B cell (i.e., the ability to make antibody and the ability to grow in the presence of the special drugs), and the myeloma (i.e., immortality). The hybridoma can be cloned, and then grown in large-scale culture. The antibody secreted into the culture medium can be collected and purified. The concentration of the monoclonal antibody can be determined by ELISA. Imm. – 21. Indirect ELISAs for Detecting the Presence of Antibodies. One way to use ELISA would be for example, the screening a patient’s serum for antibodies against a particular pathogen. The same protocol would be used for screening hybridomas for the production of antibodies against any antigen. The antigen (i.e., protein molecule, carbohydrate, fragments from the pathogen) is coated in the wells of a microtitre plate. The primary antibody (from the patient’s serum or the hybridoma culture supernatant) is added and incubated for a period of time to allow the antibodies (if present) to bind to the antigen. The unbound material is then washed away, and a secondary antibody is added to the wells. The secondary antibody is from a different species than the primary antibody, and would recognize and bind to the constant region of the heavy chain of the primary antibody. The secondary antibody also has an enzyme conjugated to it. After a period of time, the plate is washed again, and unbound secondary antibody is removed. If there were primary antibodies bound to the antigen, there will be some secondary antibody bound. If there were no primary antibodies bound to the antigen, none of the secondary antibody will remain (it does not bind to the antigen). A colourless substrate is added, and if the secondary antibodies are present, the enzyme converts the substrate into a coloured product. The concentration of the coloured product can be determined, and the concentration of the primary antibody could be determined (if a standard curve had been constructed). Imm. – 22. Direct ELISA to Determine the Concentration of Soluble Antigens. In this particular ELISA, one is determining the presence and/or concentration of a soluble antigen (i.e., a hormone). Paired sets of antibodies are used – both will bind to the antigen, but will bind to different parts (or epitopes) of the antigen. The primary antibody is unlabelled and is used to coat the wells of the microtitre plate. The sample is added, and incubated to allow the antigen (if present) to bind to the antibody. After the plate is washed, the secondary antibody is added – this antibody has an enzyme conjugated to it. After a period of time, the plate is washed again, and unbound secondary antibody is removed. If there were antigens present in the sample, it would have bound to the primary antibodies, and the secondary antibodies would have then bound to the antigen. If there were no antigens presents in the sample, the secondary antibodies would have nothing to bind to, and would be washed away (the secondary antibody does not bind to the primary antibody). A colourless substrate is added, and if the secondary antibodies are present, the enzyme converts the substrate into a coloured product. The concentration of the coloured product can be determined, and the concentration of the antigen could be determined (if a standard curve had been constructed). Imm. – 23. Immunofluorescence. Antibodies labeled with a fluorescent dye such as fluorescein (FITC) are used to reveal the presence of antigens on cells or tissues. Antibodies linked to FITC will bind to cells that have the relevant antigen on their surface. When exposed to light of the correct wavelength, the FITC will emit light. Special filters on a fluorescence microscope can be used so that only the light emitted by the FITC is detected. In this way, the presence of cells expressing certain antigens can be detected. Imm. – 24. Analyzing Cells by FACS. The FACS can determine the number of cells of a particular type in a mixed population. A fluorescent antibody to a cell surface antigen is added to the mixed population of cells. The fluorescent antibody will only bind to those cells which have that surface antigen. When the cells are analyzed by the FACS, it counts the total number of cells as well as the number of cells that fluoresce due to the fluorescent antibody bound to them. Imm. – 1. Development of Immune Cells (hematopoiesis). Bone marrow pluripotent hematopoietic stem cell lymphoid stem cell T cell precursor B cell precursor myeloid stem cell Thymocyte (TCR– CD4– CD8–) Re- arrangement of TCR genes, expression of CD4 and CD8 (TCR+ CD4 + CD8+ “double positive”) Ig gene rearrangements, expression of membrane bound IgM (immature B cell) neutrophil monocyte Bone marrow elimination of self-reactive B cells by bone marrow stromal cells expression of both membranebound IgM and IgD on cell surface mature, naïve B cell Bone marrow mast cell precursor (?) immature dendritic cell other granulocytes Thymus positive selection by cortical epithelial cells, or death by neglect immature T cell (TCR+ CD4+ CD8– , or TCR+ CD4– CD8+ “single positive”) negative selection by thymic macrophages and dendritic cell mature, naïve T cell mature CD4+ T cell mature CD8 + T cell mature B cell neutrophil monocyte immature dendritic cell mast cell precursor ( ?) other granulocytes Blood Lymph node Function of cells involved in the immune response to pathogens Macrophage Dendritic cell Neutrophil Mast cell B cell T helper cells (Th1) T helper cells (Th2) Cytotoxic T cells Phagocytosis and activation of bacteriocidal mechanisms, antigen presentation Antigen uptake in peripheral sites, antigen presentation in lymph nodes Phagocytosis and activation of bacteriocidal mechanisms Release of granules containing histamine and active agents On activation by antigen, differentiate into antibody producing plasma cells Provide cytokines to immune responses that mainly depend on antibodies Provide cytokines to immune responses that mainly depend on cell mediated responses (i.e., activation of cytotoxic T cells, activation of macrophages) Mediates lysis of target cells that display antigenic peptides complexed with MHC proteins Tissue s T cell B cell mature dendritic cell immature dendritic cell mast cell macrophage Imm. – 2. Pattern Recognition Receptors. Pathogen Toll- like recep tor Endocytic PRR PAMP or pathogen B7 co-stimulatory molecule MHC class II with peptide derived from pathogen Antigen-presenting cell (e.g., dendritic cell) Imm. – 3. The Inflammatory Response. monocyte neutrophil red blood cells endothelial cells vein resident mast cell resident macrophage tissue endothelial cells vein histamine act on bacteria act on TNF-α IL-1 IL-6 resident mast cell resident macrophage tissue vein neutrophils start to phagocytose the bacteria bacteria neutrophil squeezing between the endothelial cells lining the blood vessel endothelial cells – express adhesion molecules, increase permeability between cells tissue Imm. – 4. Phagocytosis. A bacterium binds to the phagocyte (a cell capable of phagocytosis) by an endocytic PRR endocytic PRR phagolysosome phagosome lysosome Imm. – 5. The Complement System. CLASSICAL PATHWAY Ab from previous adaptive immune response bind to pathogen surface C1q binds to Ab-Ag complex additional complement components bind to pathogen surface ALTERNATIVE PATHWAY Spontaneous breakdown of C3 to C3a + C3b in serum C3b binds to pathogen surface additional complement components bind to pathogen surface C4b2a (classical C3 convertase) C3 C3bBbP (alternative C3 convertase) formation of the C3 convertase on pathogen surface act on blood vessels to increase permeability and express adhesion molecules, stimulates macrophages to release additional TNF-α binds to pathogen surface to function as an opsonin C3a + C3b C3b binds to pathogen surface additional complement components bind to pathogen surface C5 formation of the C5 convertase on pathogen surface C5a + C5b C5b binds to pathogen surface additional complement components bind to pathogen surface act on blood vessels to increase permeability and express adhesion molecules, stimulates mast cells to release additional histamine, chemoattractant for neutrophils and monocytes formation of the MAC in the pathogen cytoplasmic membrane death of the pathogen resulting from membrane disruption Imm. – 6. MHC Class I Proteins and Antigen (Ag) Processing and Presentation. antigenic peptide; 8 – 10 amino acids long, bound to the MHC by the ends and within the α chain peptide-binding cleft α chain β2-microglobulin; noncovalently associated with the α chain Exogenous antigen is digested normal cytoplasmic protein or viral protein synthesized during a viral infection Fragments are diverted to the proteasome proteasome peptides Golgi complex Golgi complex TAP proteasome peptides TAP ER ER Imm. – 7. MHC Class II Proteins and Antigen (Ag) Processing and Presentation. antigenic peptide; at least 13 amino acids long, not bound to the MHC by the ends, and between the α and β chains peptide-binding cleft β chain α chain Exogenous antigen is digested An antigenic peptide is exchanged for CLIP peptides Invariant chain is digested, leaving CLIP Golgi complex Invariant chain ER Imm. – 8. The T Cell Receptor (TCR) Complex. T helper cells TCR αβ CD4 recognizes peptide that is complexed with MHC class II co-receptor – b inds to MHC class II of APC to strengthen interaction between T cell and APC γε εδ ζζ -S–S-S–S- extracellular environment plasma membrane cytoplasm CD3 p56lck Cytotoxic T cells TCR αβ CD8 recognizes peptide that is complexed with MHC class I αβ γε εδ ζζ -S–S-S–S- co-receptor – b inds to MHC class I of APC to strengthen interaction between T cell and APC extracellular environment plasma membrane cytoplasm CD3 p56lck signaling Imm. – 9. Positive and Negative Selection of T cells. Immature “double-positive” thymocyte (expresses the TCR, and both CD4 and CD8 co-receptors, i.e., CD4 +CD8+) Thymocytes that have a TCR that cannot interact with MHC do not receive a “survival signal” and die of neglect. Positive selection of thymocytes that have TCR that can bind to MHC class I or MHC class II molecules. Negative selection and death (by apoptosis) of thymocytes that have TCR that binds to MHC class I or MHC class II with high affinity. Mature CD4+ T cells Mature CD8+ T cells Imm. – 10. The "Two Signal" Model for the Activation of T Cells. Professional antigen-presenting cell – constitutive or induced expression of B7 – e.g., dendritic cell, macrophage Naïve CD4 T cell (Helper T cell) CD4 TCR MHC class II SIGNAL 1 SIGNAL 2 CD3 CD28 B7 T cell activation Non-professional antigen-presenting cell – no expression of B7 – e.g., endothelial cell CD4 Naïve CD4 T cell (Helper T cell) TCR MHC class II SIGNAL 1 CD3 CD28 T cell anergy Imm. – 11. Cytokine Mediated Generation of TH Subsets. Professional antigen-presenting cell –e.g., dendritic cell, macrophage CD4 CD28 TCR CD3 T when infected with viruses or intracellular bacteria CC SIGNAL 2 R IGNAL 1 SD 4 B 7 Naïve CD4 T cell (TH0 cell) IL-12 + IL- 18 IL-2 IL-4 INF-γ Activated TH1 cell IL-2 IL-4 Activated TH2 cell IL-2 Imm. – 12. Activation of Cytotoxic T Cells. IL-2R (IL-2 receptor) TH0 cell TH1 cell IL-2 IL-12 + IL-18 proliferation TH1 cell IL-2 CTL-P cell activation Ag activated CTL-P proliferation differentiation CTL recognition of target cell death of target cell Target cell CTL perforin and granzyme Imm. – 13. B Cell Antigen Receptor (BCR) Complex. Ag recognition and binding N-terminus of the H chain Ag recognition and binding Variable region N-terminus of the L chain L chain Constant region C-terminus of the L chain H chain Ig β Ig α extracellular environment plasma membrane cytoplasm C-terminus of the H chain signaling Imm. – 14. Activation of B cells. signal #1 antigen-presenting cell e.g., dendritic cell MHC class II with peptide derived from the antigen B7 B cell antigen B cell CD28 TCR complex CD4 T helper cell proliferation differentiation B cell effector TH2 cell MHC class II with peptide derived from the antigen memory TH2 cell B cell B cell CD40L CD40 signal #2 proliferation differentiation effector TH2 cell (CD28 molecule not shown) memory B cell plasma B cell secreted antibodies Imm. – 15. Antibody Structure. IgM Variable region of the heavy chain, VH Ag binding site Hypervariable region (HV) – within the V H and VL region Ag recognition and binding Constant region of the heavy chain, CH Variable region Variable region of the light chain, VL L chain Constant region of the light chain, CL hinge region H chain di-sulphide bonds Constant region Fc region Complement activation a “domain|” of the CH chain (domains are also present in L chains) IgG Variable region of the heavy chain, VH Ag binding site Hypervariable region (HV) – within the V H and VL region Ag recognition and binding Constant region of the heavy chain, CH Variable region Variable region of the light chain, VL L chain Constant region of the light chain, CL Complement activation H chain di-sulphide bonds Constant region Fc region a “domain|” of the CH chain (domains are also present in L chains) Macrophage binding Imm. – 16. Primary and Secondary Responses. Antibody concentration in serum Events during the “lag” of primary response: • Innate responses occur • Dendritic cells engulf antigen, process it, migrate to lymph node to activate T helper cells • T helper cells become TH2 type, proliferate, some differentiate to memory TH2 cells • Ag carried to lymph nodes, activate B cells • B cell receive cytokine help from TH2 cells • B cells proliferate, some differentiate to memory B cells, others differentiate to plasma cells and start to secrete antibody Secondary response to antigen “A” During the secondary response: • Memory TH2 cells activated, memory B cells activated and undergo class-switching (?) • TH2 and B cells proliferate • B cells differentiate into plasma cells quickly and start to secrete antibody “lag” Primary response to antigen “A” “lag” Primary response to antigen “B” 4 8 12 16 20 64 68 72 Days after immunization Immunization with antigen“A” Re-immunization with antigen“A”, immunization with antigen “B” For the primary response: • Antibody can be detected in the serum after ~5 – 7 days • Concentration of antibody increases slowly with time • A small amount of antibody is made, most IgM • A lot of antigen was required to induce the antibody response • The antibody remains in the blood circulation for a few weeks For the secondary response: • Antibody can be detected in the serum after ~1 – 2 days • Concentration of antibody increases very rapidly • 10 – 100X more antibody is made, most IgG • Very little antigen was required to induce the antibody response • The antibody remains in the blood circulation for a many months - years Imm. – 17. Activation of Macrophages by TH1 Cells. Professional antigen-presenting cell – e.g., dendritic cell, macrophage Naïve CD4 T cell (TH0 cell) Endocytic PRR bacterium TLR B7 CD4 TCR CD3 CD28 SIGNAL 1 SIGNAL 2 R IL-12 + IL- 18 polarization to TH1-type of T helper cell TH1 cell IL-2 IL-2R (IL-2 receptor) proliferation differentiation effector TH1 cell memory TH1 cell macrophag e with intracellular bacterial infection secreted IFN-γ MHC class II TCR CD3 CD40L membrane TNF-β TNF-β receptor TDTH cell TNF receptor CD40 activated macrophage Imm. – 18. IgE and Allergies. cross-linking of two adjacent IgE molecules by the allergen (antigen) IgE FcεRI allergen (antigen) signal to degranulate granule containing histamine Fc receptor for IgE type antibodies, FcεRI Imm. – 19. Transgenic and Gene Knock-out Mice. Imm. – 20. Hybridomas and Monoclonal Antibodies. Immunize mouse with antigen “X” Check for presence of antibodies to antigen “X” by ELISA If no antibodies can be detected, or the response is weak, re- immunize mouse with antigen X If positive, remove spleen – source of antibody producing B cells Myeloma, a B cell tumour: • immortal • no longer secretes antibody • drug sensitive (drugS) • B cells: • mortal • secrete antibody • drug resistance (drugR) fuse together Grow cells in an in vitro culture system, use drug selection to select cells that have specific properties of both the B cell and myeloma, clone individual hybridomas in microtitre plates Hybridoma • immortal • secrete antibody • drug resistance (drugR) • Hybridoma that produces antibody of the desired specificity – grow in cell culture Remaining hybridomas produce antibody, but not of the desired specificity Imm. – 21. Indirect ELISAs for Detecting the Presence of Antibodies. 1 2 3 4 a colourless substrate is added and is converted to a coloured product by the enzyme on the secondary antibody – the concentration of this product can be measured S S E P P the sample is added to the well, if antibody is present, it binds to the antigen – this is the primary antibody – the excess antibody is washed away E E E antigen is coated to the well of a microtitre plate – unbound antigen is washed away the secondary antibody (with an enzyme covalently bonded to it) is added – the secondary antibody is from a different species than the primary antibody, and binds to the Fc region of the heavy chain of the primary antibody – excess antibody is washed away 1 2 the sample is added to the well, since there is no antibody, nothing binds to the antigen that is coating the plate 3 4 a colourless substrate is added – since the secondary antibody did not bind, there is no enzyme to convert the substrate into a coloured product E S S E antigen is coated to the well of a microtitre plate – unbound antigen is washed away the secondary antibody (with an enzyme covalently bonded to it) is added – since the sample did not have primary antibody to bind to the antigen, the secondary antibody has nothing to bind to and is washed away– the secondary antibody does not bind to the antigen! Imm. – 22. Direct ELISA to Determine the Concentration of Soluble Antigens. 1 2 3 4 a colourless substrate is added and is converted to a coloured product by the enzyme on the secondary antibody – the concentration of this product can be measured S S E E P P the sample is added to the well, if antigen is present, it binds to the primary antibody – the excess antigen is washed away E E antibody specific for the antigen is coated to the well of a microtitre plate – this is the primary antibody – excess antibody is washed away the secondary antibody (with an enzyme covalently bonded to it) is added – the secondary antibody recognizes a different epitope on the antigen than the primary antibody (and does not bind the primary antibody) – excess antibody is washed away 1 2 3 4 E the sample is added to the well, since there is no antigen present, nothing binds to the primary antibody E a colourless substrate is added – since there secondary antibody did not bind, there is no enzyme to convert the substrate into a coloured product S S antibody specific for the antigen is coated to the well of a microtitre plate – this is the primary antibody – excess antibody is washed away the secondary antibody (with an enzyme covalently bonded to it) is added – since there is no antigen for it to bind to, it is washed away Imm. – 23. Immunofluorescence. cell surface proteins recognized by an antibody tagged with a fluorochrome incubate with antibody tagged with a fluorochrome, wash away unbound excess antibody UV light source expose to UV light of the correct wavelength to excite the fluorochrome, detect emitted light with microscope exciting light emitted light Imm. – 24. Analyzing Cells by FACS. vibrating nozzle – droplets leaving the nozzle contain a single cell detectors laser + – ++++ Cell suspension – cells labeled with either “anti-A” antibody or “anti-B” antibody – the antibodies are tagged with different fluorochromes charger + Relative number of cells Data for a FACS experiment using one fluorescent-labeled antibody A – deflection plates – – – – + – – + + A + + – – + + + + Anti-A antibody fluorescence Data for a FACS experiment using two fluorescent-labeled antibodies AB Anti- B antibody fluorescence –+ AB ++ AB –– AB +– cells without bound antibody cells with “anti-A” antibody bound to the surface cells with “anti-B” antibody bound to the surface Anti-A antibody fluorescence ...
View Full Document

Ask a homework question - tutors are online