BIOL 318 Immunology Lecture 1 PDF

BIOL 318 Immunology Lecture 1 Lecture by Andrea Verdugo Meza, PhD Candidate, MSc, BioEng Email: [email protected] September 3, 2024 Learning Objectives ✓To understand how we...

BIOL 318 Immunology Lecture 1 Lecture by Andrea Verdugo Meza, PhD Candidate, MSc, BioEng Email: [email protected] September 3, 2024 Learning Objectives ✓To understand how we generate several unique antibodies and TCRs ✓To overview the organization and expression of the lymphocyte receptor genes Antibodies and TCRs are the hallmark of the Adaptive Immune Response  B cell receptor is a future antibody Immune Receptors bear immunoglobulin domains Immunoglobulin lacking the carboxyl terminus transmembrane segment is secreted  B cell receptor is a future antibody (Ab) An antibody consists of two heavy chains and Fab = Variable regions determine which antigenic two light chains molecules the Ab recognizes formed at the “top Both type of chains have variable (V) and of the Y” of the antibody constant (C) regions Antigen specificity: the Ab molecule binds to its Held together by intra-/interchain disulfide specific antigen 2x covalent bonds  B cell receptor is a future antibody (Ab) Antiserum to the constant region of the heavy chain identifies five distinct classes of antibodies called isotypes IgA: alpha (α) heavy chain IgD: delta (δ) heavy chain IgE: epsilon (ε) heavy chain IgG: gamma (γ) heavy chain IgM: mu (μ) heavy chain Light chain isotypes are Kappa (κ) Lambda (λ)  B cell receptor is a future antibody (Ab) The heavy chain isotype determines the Ab-mediated effector functions 1. IgG: Main responder for infection; transferred to fetus 2. IgM: present early in infection: large size helps with complement activation leads to phagocytosis and/or death of the cell 3. sIgA: protection of mucosal surfaces 4. IgE: Parasitic response (allergies) 5. IgD: signal the B cells to be activated; binds to basophils and mast cells and activate these cells to produce antimicrobial factors to participate in respiratory immune defense in humans  Antibody Diversity Diversity (repertoire) is the total of all the Ab specificities that an organism is capable of expressing It’s estimated that humans can make a billion (107-9) or more different specific antibodies BUT we only have about 3 x 104 protein-producing genes How can we get so many different proteins from so few genes? How can an immunoglobulin heavy or light chain be identical in one part of its sequence but extraordinarily variable in another?  Origin of Antibody Diversity Germ-line theory: genes encoded by the genome of germ cells (egg and sperm) and passed to each generation; no genetic rearrangements Implies that antibody diversity generated in a species over evolutionary time Somatic mutation theory: germ line only contained a few H and L chain genes; during B cell maturation, different mutations in different B cells occurred so that new H and L chains were generated Implies that antibody diversity is generated by individuals during their lifetime Both ideas have proved to be partially correct (and also partially wrong): ✓ The germ line contains many Ab genes ✓ Genetic rearrangements occur in each lymphocyte ✓ Further diversity can be generated through mutations 10  The puzzle of immunoglobulin gene structure Antibodies can be produced for an enormous variety of different specific antigens 107–1011 different specificities But how can a limited amount of DNA produce such an extensive variety of possible antibodies? The mechanisms used by B cells to produce this vast repertoire were elucidated by very elegant experiments in the 1970s 11 Hozumi and Tonegawa in 1976 In embryonic liver cells, the DNA sequences encoding the variable and constant regions were located on different restriction endonuclease fragment The C region mRNA probe shows 1 band indicating that the cut is not in the middle of the light-chain C region but that the whole constant region is encoded on 1 fragment The light-chain sequence (V + C) probe yields 2 bands indicating that the cut site is somewhere between the V and C regions These two sequences were co-located on a single restriction fragment in the Ab- producing cells Both the liver-cell fragments disappeared indicating the DNA environment has changed C + V are on the same DNA location implies they moved together which was supported by DNA sequencing Recombination among the various V region gene segments generates a diverse repertoire of antibody combining sites Like shuffling a deck of cards, dealing out different hands Tightly regulated machinery controls the recombination processes  The puzzle of immunoglobulin gene structure There are variable (V), diversity (D), joining (J), and constant (C) region gene segments D segments are used in antibody heavy chains only  B cells use recombination of gene segments to create diff. possible antibodies  Gene segments are joined by the RAG1/2 recombinase RAG = recombination activating gene Both RAG1 and RAG2 proteins are needed for recombination Severe combined immunodeficiency (SCID) results from impaired receptor gene recombination Treatment: bone marrow transplantation  The puzzle of immunoglobulin gene structure There are variable (V), diversity (D), joining (J), and constant (C) region gene segments D segments are used in antibody heavy chains only  Recombination is directed by signal sequences Recombination signal sequences (RSSs) flank each antibody gene segment Each has a conserved nonamer and heptamer sequence V(D)J recombination results in a functional Ig variable region gene This figure presents a brief overview of the process RAG proteins bind to RSSs and cleave the DNA Other proteins process the hairpin loops that form after RAG has done its job Products include a recombined coding joint and a leftover signal joint that is later degraded See Figure 6.12 @Kuby for the complex and detailed steps 19  Mechanisms that generate antibody diversity in naïve B cells Recombination of multiple gene segments Combinatorial diversity: which H chain pair with which L chain Nucleotide addition: i.e. terminal deoxynucleotidyl-transferase (TdT) adds random nucleotides between joints Exonuclease trimming: sometimes occurs at junctions, losing nucleotides and changing reading frames 20 How many different H chains can a mouse generate? Assuming that the different segments can recombine randomly, if there are: 130 VH segments, 13 D segments and 4 J segments for 130 x 13 x 4 = 7,000 different H chain sequences. If there are 346 different light chain sequences 7,000 x 346 = 2 x 106 different Ig molecules. Combinatorial rearrangement of a relatively few gene segments can give rise to an enormous number of different antibody molecules. 21 Allelic exclusion ensures that each B cell synthesizes only one heavy and one light chain An individual B cell only produces one kind of Ig with one kind of light chain V rearrangement occurs during B cell maturation in the bone marrow Each B cell will have ONE functional variable region DNA sequence for each chain Only one allele (maternal or paternal) will be expressed in one B cell: allelic exclusion 22  Recombination of the B cell receptor is a very ordered process Heavy chains are recombined and expressed first Expression of a functional heavy chain shuts down recombination machinery temporarily The heavy chain is paired with a surrogate light chain (SLC) to form a pre-BCR If the SLC will pair with the heavy chain, the machinery is started up again Then Light-chain recombination takes place 23 Nonproductive arrangements lead to programmed cell death (apoptosis) during development 24  Receptor editing of autoreactive receptors occurs in light chains A functional Ab in an immature B cell may bind to self-antigens Recombination machinery can be turned back on to edit the original rearrangement to salvage the rearrangement or at least inactivate it 25  Mature B cells express both IgM and IgD antibodies An mRNA splicing mechanism is the cause Heavy chain transcripts have VDJ put together, but there is a spacer RNA sequence between the VDJ and C regions mRNA splicing removes the spacer, leaving VDJC mRNA ready to be translated Primary transcripts for IgM heavy chains may result from RNA polymerase transcribing through both the IgM and IgD constant regions Depending on which C segment becomes polyadenylated, IgM or IgD could end up being produced  B-cell receptor expression mRNA splicing mechanisms also control whether the cell produces membrane-bound or secreted IgM  T cell receptors (TCR) TCRs are not immunoglobulins but are structurally similar They have Ig domains They possess variable (V) domains and constant (C) domains, just as in Ab molecules Two TCR types, αβ and γδ, have diverse antigen-binding characteristics Each T cell has a TCR of only ONE specificity TCRs work as a part of a complex that includes CD3 CD3 necessary for cell surface expression of TCR Essential for transducing signal after Ag interaction with TCR  T-cell receptor genes and expression TCR genes undergo a process of rearrangement very similar to Ig genes Similar machinery for rearrangement TCR genes are located somewhat differently between mice and humans 30  Everything we just talked about occurs PRIOR to ever seeing a specific antigen Predetermined genetic One has all the antigen binding rearrangement receptors (either Antibody or TCR) before ever being exposed to antigens 31  Clonal selection hypothesis: AgR pre-formed on B and T cells and Ag selects the clones with the correct receptor  Class Switching Class switching to start expressing IgG, IgA or IgE requires exposure to antigen and to signalling molecules from T cells Cytokine exposure activates a new round of recombination that cuts out some of the CH genes and relocates the VDJ segments adjacent to a new CH region → Ex. IL-4 induces a switch to either IgG1 or IgE; interferon-γ causes a switch to IgG2a; transforming growth factor-β causes a switch to IgG2b or IgA. 33  Ab Changes during 1o and 2o Responses Total IgM IgG Ab Ab Ab Class switching – 1o - IgM Ab Titer 1o Ag 2o Ag – 2o - IgG, IgA or IgE Days After Immunization Summary The story of antibodies and T-cell receptor structures and gene arrangements was one of the largest advances in immunology ever Once gene arrangements were determined, discovering recombination mechanisms proved just as crucial By understanding the arrangement and rearrangement of these genes, we can understand much more about how B and T lymphocytes operate, both normally and abnormally 35