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Immunology - Lecture 3 Adaptive Immune System 2 Immunology - Lecture 3 Adaptive Immune System 2 Generation of Immune Diversity Lymphocyte Antigen Receptors - 8 Lymphocyte Development- 9 Lymphocyte Activation – 10 Lymphocyte Effector Functions – 11 Regulation of Adaptive Responses - 12 Innate versus Adaptive specificity? Epitopes recognized by innate receptors have been predetermined by evolution and germ-line encoded • Based on Ag that ancestors encountered • Limited range of view = TLR4 = LPS, 4 or 5 other things • View is looking back in time Epitopes recognized by Ig or TCR are determined before they ever encounter Ag • View is looking forward for thing you might see • Randomness of the future needs to be accounted for as well as the diverse Ag that will be accounted for 15 • It is estimated that each individual has the capacity to recognize 10 epitopes How do adaptive immune cells generate such diversity? Chromosome rearrangement Junctional diversity Allelic exclusion Isotype Switching Somatic Hypermutation (B cells) BCRs and TCRs are similarly organized. Each receptor is composed of two distinct subunit chains (BCR: light chain and heavy chain, TCR: α chain and β chain). The antigen binding surface is formed by the variable region of each chain, which is encoded by recombined V, J, and D (BCR heavy and TCRβ) gene segments. VDJ recombination or somatic recombination: The antigen receptors are encoded by roughly 300 different gene segments - but can be used to generate 1010-15 receptors DNA chromosomal rearrangement gene segments to produce this significant diversity Relies on Recombination -Activating Genes (RAG) RAG deficiency - results in severe combine immunodeficiency (SCID) Fig. 8.2 Some genes are inverted before being reannealing Terminal deoxynucleotide transferase (TdT) - adds or removes nucleotides to exposed ends of DNA before renealing to mediate junctional diversity http://www.youtube.com/watch?v=QTOBSFJ WogE Fig. 8.3 Steps in V(D)J recombination: Antigen receptor gene segments are flanked by a recombination signal sequence (RSS) In the first phase, recombination activating gene 1 (RAG1) and RAG2 proteins bind to the RSSs Capture of the second RSS (a process termed synapsis) results in the formation of the paired complex, within which the RAG proteins introduce double strand breaks between the gene segments and the RSSs. In the second phase, the RAG proteins cooperate with non-homologous end joining (NHEJ) DNA repair factors to rejoin the DNA ends. Gene segment ends typically undergo non-templated nucleotide addition by terminal deoxynucleotidyl transferase (TdT) and nucleotide loss before being joined to form the coding joint. RSS ends are typically joined without processing to form the signal joint. T Cell Receptor (TCR): Heterodimers composed of ab pairs - 1 x V and 1x C CD3 and CD247 for signaling TCR generation LC: α on chromosome 14 HC: β is on chromosome 7 Fig. 8.4 LC = VJC chains HC = VDJC chains Each developing T cell randomly produces a light- heavy chain combination with unique specificity due to the V region at the N-terminus of the protein V regions combined with limited set of D, J, C Random combinations of light and heavy chains genetic rearrangement and junctional diversity randomly create TCR chains that vary among, but not within, individual T cells The theoretical number of possible combinations produced within the body may be estimated to be the product of several possible light chains and several possible heavy chains Estimated that 1 to 5 million epitope-binding B-cell receptors or “Ig” are composed of: 2 identical LC (κ or λ) 2 identidal HC Associated Igα-Igβ for signaling (CD79/a/CD79b) which are required for signaling IgD>IgM>IgG can all be membrane bound monomers Gene clusters encoding BCR chains are found on multiple chromosomes HC V regions account for a significant level of diversity >9x106 Ag-Binding combinations Just like the TCR, the different combination of V domains on L and H chains to light and heavy chain results in significant diversity The choice to use which V, D, J genes is random for each cells In the end – 1 cells synthesizes an Ig of a single specificity made up of the VL and VH domain Isotype Switch: After contact with its specific epitope B cells cease production of lgD, and most of them differentiate into plasma cells that secrete lgM. Some B cells become memory B cells. Express only lgM on their surface. If reactivated by a new contact with their specific epitope and via interactions with T cells, they can complete rearrangements of their heavy chain genes at the DNA level to juxtapose their VDJ units with other constant genes. Whatever constant gene is brought adjacent to the VDJ determines what heavy chain gene will be produced. Some isotype switches may involve the deletion of large tracts of DNA to bring VDJ segments together with distant constant genes. Reactivations of memory B cells may result in a series of shorter deletions as lgM memory B cells may switch and become lgG memory B cells, then be reactivated again and switch to yet another isotype. Repeated or constant stimulation by the same epitope drives B cells from lgM expression to other isotypes The cell's epitope specificity is not altered by isotype switching. Lymphocyte Development - 9 The thymus is organized into outer or cortical and inner or medullary regions. Pro-thymocytes enter the thymus to under several maturational steps. B cell development: Takes place in the bone marrow: Contains HPSC, stomal cells for instruction, and vasculature enabling cells to come and go. First step is to express Iga and Igb (Signaling complex) Generate and test HC with SLC (pre-B cell receptor If non-functional –cell dies of apoptosis Generation of LC and IgM – tested for functionality Tested for self reactivity on stromal cells of the bone marrow Too strong – receptor editing (reactivates VDJ recombinases) If fails again – delete If successful a IgM positive B cells release to periphery T cell activation involves the formation of an immune synapse: Initiated by the TCR recognition of a Ag presented on MHC Weak interaction of TCR with MHC is stabilized by CD4 or CD8 Generates signal 1 through TCR/CD3 Necessary, but not sufficient to activate a naïve T cell (never activated before - versus a memory cell) Cell adhesion molecules (CAMs) expressed by the T cells (LFA-1) interact with ICAM-1 on APC form a ring with LFA on outside, then CD4/8 and TCR inside. This helps to initiate appropriate signaling. Second signal is received from an ACTIVATED APC Activated APC have recognized non-self and are “pro-inflammatory” CD86 hi CD40 hi Co-Stim CD80 hi MHC hi Secreting cytokines Without these APC derived signals - TCR stimulated T cells become, “anergic” or harder to stimulate into a productive immune response. CD4 Differentiation: Cytokines and local factors dictate the shape the CD4 responses takes after activation: IL-12 from activated DC- Type 1 helper cells (Th1) making IFNgamma and mediating cellular immunity. Helps CD8s and innate immune system remove pathogens IL-4 from innate cells and other T cells results in a T helper type 2 cells making more IL-4, IL-5, IL-13 which support humoral immunity, allergy, and tissue repair. Generation of T cell memory: Activated T cells complete effector responses and undergo activation induced cell death, or upregulate moleucles like CD152 which interact with co-stimulatory molecules to generate regulation. A small subset of Ag stimulated T cells will upregulate anti-apoptotic proteins such as Bcl2 and avoid death They will also decrease molecules that direct them into the lymphoid organs. Thus they sit in the blood and peripheral tissues where they can mount a rapid response to their recognized Ag. Memory cells take less stimulation to respond Went from a situation where you had a few naïve clones to an expand population of memory cells with the same Ag recognition ready for a rapid response BCR signaling: Initiated by clustered BCR - recognition of multiple epitopes Both T cells dependent and independent B cell activation T cell dependent is most common and relies on co- stimulation via CD154 on activate T cells and CD40 on B cells T cell independent involves mitogens - that bind structures other then the BCR Ex. LPS via TLR4 and BCR Innate APC, B cells, and T cells interact to shape and coordinate immune response to pathogens and after vaccination. Immunological Memory: Lymphocyte Effector Functions Upon proper activation via their Ag-receptor, adaptive immune cells undergo clonal expansion (multiple divisions of clone recognizing that Ag and expressing the EXACT same Ag-receptor) and while most will do their function and die, a small percentage will upregulate anti-apoptotic genes to survival. Become long lasting memory cell that express that Ag-receptor and need less stimulation to initiate immune response. Immune memory increases both the number of cells that can potentially recognize an antigen and also decreases the intensity of the stimuli need to activate them. Memory increases intensity, duration, and amplitude of adaptive immune response Memory is the reason vaccination is possible. Vaccines often use disabled “attenuated live” or killed microbes for vaccines. Still have Ag to stimulate adaptive response - but can not make you sick. Two effector arms of the adaptive immune system Humoral Immunity: involves fluids or “humors”; antibody and complement Ancient Greek and Romans; Humors = fluids of the body that define the makeup of the workings of the human body • Secreted antibodies that are targeted to specific epitopes on antigens via variable domain generated epitope binding sites and attached to heavy chains with FC regions that shape the function of bound antibodies • Once bound – work with complement and innate cells to clear Ag • There is a T cell-dependent component to humoral immunity, as T cell help (CD40 ligation via Th cell CD154 and Th cytokines) are required for an effective and diverse humoral response.
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