Antigens and Immunogens

• An antigen is any compound that can bind to adaptive immune receptors and antibodies.
• Immunogens are antigens that trigger an immune response.
• Haptens are antigens too small to provoke immune responses and must attach to carrier molecules.
• Good immunogens are foreign, large, chemically complex, and degradable (for T cell antigens).
• Self-antigens are usually not immunogenic.
• A size of 6 kDa is likely immunogenic.
• Homopolymers (only one type of monomer) are weak immunogens.
• High molecular diversity leads to higher immunogenicity.
• Antigens must be digested by APC or in a proteosome prior to presentation for T cells.

Epitopes and Paratopes

• An epitope is the specific part of an antigen that antibodies or receptors bind to.
• Epitopes are also called antigenic determinants.
• Molecules that share epitopes are cross-reactive, such as with the tuberculosis (BCG), attenuated flu, smallpox, and tetanus vaccines.
• The paratope is the complementary binding site on the antibody/receptor.
• Nucleic acids are highly immunogenic, while lipids are weakly immunogenic.

Epitope Size and Macromolecule Type

• B cells need receptor-antigen interaction only.
• T cells need receptor-antigen AND MHC-antigen interaction.
• MHC processing ONLY happens to proteins.
• Carbohydrate antigens are humoral antigens only.
• T cells only get antigen from phagocytic processes.
• B cells can take up free-floating antigen from the environment.

Antigen Presentation

• T-cell antigen is processed before being presented.
• B cells take up whatever is available.
• B cell epitopes vs T cell epitopes differ.
• Binding is noncovalent.
• Each chain has multiple intrachain bonds that cause folding at regular intervals (~100 aa) within globular domains.
• Light chains have 2 domains.
• Heavy chains have 4-5 domains.

Hinge Region

• The hinge region is proline/cysteine rich.
• Proline allows flexibility in the distance between the top arms.
• Cysteine allows for one or more disulfide bonds.
• The hinge region is easily cleaved by papain.
• Two Fab regions can bind to antigens.
• One Fc region can be crystallized and does not bind to antigens.

Variable Domain

• The variable domain is designated VL or VH based on which chain it's on (Fab).
• Antigenic specificity comes from the variable domain, which has three hypervariable regions per chain.
• The framework region of the variable domain is constant.

Heavy and Light Chain Genes

• Heavy chain genes include IgM (mu), IgD (delta), IgG (gamma), IgA (alpha), and IgE (epsilon).
• Each isotype has subclasses with subtle differences; humans have 4 subclasses of IgG and 2 subclasses of IgA.
• Light chain genes are lambda and kappa.
• IgG, IgD, and IgE exist as monomers.
• IgA usually exists as a dimer.

IgG Characteristics

• IgG is the most common isotype in serum and has a half-life of ~23 days (except IgG3).
• IgG can be reused via a specialized IgG receptor (FcRp).
• Agglutination is the clumping of multiple antigens, which allows for phagocytosis/neutralization.
• Opsonization coats antigens to make them easier for phagocytes to ingest.
• ADCC (Antibody-Dependent Cell-mediated Cytotoxicity) involves an effector cell (NK cells for IgG) killing a target cell.
• Multiple IgGs must bind to activate the complement system.
• IgG neutralizes toxins and viruses, and its long half-life makes it ideal for antitoxins/antivenom.
• IgG binding to flagella and clumping immobilize/restrict bacterial movement.
• IgG can cross the placenta.

IgM Characteristics

• IgM is the first antibody on the scene and is secreted as a pentamer.
• IgM has one extra CH domain and a half-life of ~5 days.
• The primary response is the host's first encounter with an antigen, lasting roughly 7-14 days in a latent period.
• During the steady state, production/degradation is balanced, resulting in a generally flat amount.
• The declining phase involves the shutdown of antibody production or isotype switching to IgG production.
• The secondary response is any follow-up response with the antigen.
• Isohemagglutinins are naturally occurring anti-A/B antibodies, mostly IgM.

IgA Characteristics

• IgA is commonly found in external secretions as a dimer.
• IgA has two subclasses and is linked by the same J chain found in IgM.
• The secretory component is found in secretions but is not produced by plasma cells.

IgD Characteristics

• IgD almost entirely membrane-bound and co-expressed with IgM, acting as a B-cell receptor.

IgE Characteristics

• IgE is expressed as a monomer, has one extra CH domain, and has a half-life of only 2 days.
• The main function of IgE is to kill helminths and cause allergies.
• The eosinophil/basophil response can be associated with fighting off helminth infections.

Agglutination Reactions

• Agglutination reactions include direct typing and back typing.
• Direct typing applies anti-A and anti-B antibodies (usually IgM) to drops of blood, and clumping is observed.
• Back typing applies known blood type A or B cells, and the sera response is observed.
• Precipitation reactions work with soluble antigens instead of particulate antigens.
• Too little antigen causes competition to bind twice.
• Too much antigen yields not enough antibodies.
• A perfect balance yields enough antibodies to bind to the same antigen, but not so much that it outcompetes.
• Common precipitation experiments include Gel diffusion assays and Western Blots.

Immunoassays

• ELISA is an easy method to measure the quantity of antibody in serum or the amount of soluble marker in the solution.
• Flow Cytometry is a specialized technique that allows for rapid phenotyping of immune cells.
• Clusters of Differentiation are surface proteins that distinguish different cell types.
• In Flow Cytometry, cells flow through a chamber and fluid dynamics limits them to 1 cell at a time (usually).
• As it passes through the chamber, fluorescent antibodies are excited by a laser.
• Cells that fluoresce will give off light that a detector identifies.
• Forward Scatter measures the size of the cell, with larger cells bending the light more.
• Side Scatter measures the granularity of the cell.
• Granulocytes have high side scatter, while monocytes tend to be the largest.
• The flow graph represents Red > Yellow > Green > Blue.
• B cells are CD19+ CD3-, while T cells are CD19- CD3+.
• FACS (Fluorescence Activated Cell Sorting) electronically charges a cell of interest to allow isolation from other cells.
• DUMP is a common strategy when you want to avoid certain cell types.

Light Chain Genes

• There are four possible light chain genes: Kappa- 2 on chromosome 2 and Lambda- 2 on chromosome 22.
• In the Kappa Model (Gene 1), Kappa has about 40 V segments and 5 J segments.
• Gene 2, the C gene (constant) Kappa, has 1.
• The light chain V segment codes for the first 95 amino acids of the variable region.
• The light chain J segment codes for the remaining 13.
• The B cell splices its own genome, selecting one V segment and one J segment and splicing out everything in between them.
• V(D)J recombination is a mechanism of random assembly of gene segments that generates the great diversity of unique receptors on B cells and T cells during their early development in the bone marrow.
• V(D)J recombinase is the set of enzymes needed to recombine V, D, and J segments.
• Kappa is preferred.
• Rearrangement will be attempted on one kappa segment and if that fails, it moves to the homologous gene, then the lambda gene if that fails.
• Lambda genes have 30 V segments and 4 J segments that are linked to a unique C gene.
• The H-chain locus is on chromosome 14.
• The heavy variable domain is like the light variable domain.
• The heavy chain region has V 50 segments and J 6 segments.
• Heavy Chain rearrangement involves splicing between D and J first, then V and D, ending with a single locus with V:D:J.

Recombination

• Each gene segment has a Recombination Recognition Sequence (RSS) that is recognized by a complex of two Recombination Activating Genes (RAG-1 and RAG-2).
• Rag-1/2 binds with itself causing the DNA to loop back onto itself then cleaves the loop.
• The loop is a circle.
• Sticky ends of the loop are joined together with no further known role for the loop.
• Repair enzymes fuse the two ends back together to fix the genome
• This results in a completed V-J fusion.

Alternative Splicing

• Mature B cells have both receptors due to alternative splicing, where two splice variants form, allowing for IgM and IgD coexpression.
• Activation-induced cytidine deaminase (AID) is involved in somatic hypermutation; it is an enzyme that converts cytosine to uracil, which are then excised and replaced with non-template nucleotides.
• Cytidine deaminase converts cytidine to uridine.
• NHEJ (non-homologous end joining) typically results in excision of the middle genes.
• CD19 is a B cell marker used in humans.
• Pro-B cells express CD34+, CD19+, and CD10+ on their surface.
• Pre-B cells Express Cytoplasmic μ and pre-B receptor-associated μ (surrogate light chain).
• Ig alpha and Ig beta are expressed on the surface of naive B lymphocytes.
• Immature B cells express IgM on the surface with CD20.
• Mature B cells express IgM and IgD on the surface.
• Memory B cells are CD27+ and respond to an antigen more strongly when the body is reinfected with an antigen than it does during its first encounter with the antigen.

B Cell Development

• Central Tolerance involves the elimination of cells that bind to "self" cells in bone marrow.
• Deletion is triggered apoptosis.
• Receptor editing is the process by which B cells that bind self are edited; RAG gene expression allows light chain editing.
• Activation of B cells leads to either of two fates: plasma cell and memory cell.
• T cell dependent activation requires a B cell to phagocytose and present an antigen to a CD4+ T cell; MHC II complex is used.
• The germinal center is the area within the lymph node that produces lymphocytes.
• Somatic hypermutation occurs in B cells in lymph nodules, which rapidly mutate, creating new sequences.
• High affinity selection involves a limited population of follicular dendritic cells holding the antigen on their cell surface for long periods of time.
• Plasma cells can be short-lived or long-lived, and most migrate back to the bone marrow.
• Memory cells develop in the germinal centers of lymph nodes and other lymphoid organs.
• Marginal-Zone B cells are found in the spleen marginal zones; they do not circulate and are distinguished from conventional B cells by a distinct set of surface proteins.
• B-1 cells are a class of atypical, self-renewing B cells (also known as CD5 B cells) found mainly in the peritoneal and pleural cavities in adults. They have a much less diverse antigen-receptor repertoire than conventional B cells.
• Common surface molecules of the B cell include BCR, B-cell co-receptor, MHC-II, CD40, cytokine receptors, chemokine receptors, and integrins.
• The co-receptor doubles-binding the co-receptor and the BCR to a pathogen enhances the signal
• CD10 is present on Pro-B and Pre-B cells.
• CD19 is present on All B cells but plasma cells.
• CD20 is present on all B cells except pro-B and pre-B cells.
• CD27 is present on Memory B cells and plasma cells.