Immunology Exam 2

Antigen-binding site

The region of an antibody capable of interacting with antigens to initiate an immune response. Also known as complementarity determining region.

Pepsin

Proteolytic enzyme whose cleavage of immunoglobulin releases two F(ab')2 fragments and degrades the Fc region

IgM and mu heavy chains

An antibody class that serves as a receptor on naive B cells. Alse the first class of antibody to be secreted during the course of the immune response. When secreted, exists primarily in pentameric form.

Mature B cell

A B cell expressing both IgM and IgD

Heterokaryon or hybrid cell

A cell containing two or more genetically different nuclei; in immunology, hybridomas are used to produce monoclonal antibodies

Follicular dendritic cell

A cell with extensive dendritic extensions that is found in the follicles of lymph nodes. Although they do no express class II MHC molecules, they are richly endowed with receptors for complement and Fc receptors for antibody. they are of a lineage that is distinct from class II MHC-bearing dendritic cells.

Activated B cell

A mature B cell whose receptors have attached to an antigen and will respond to it

Frameshift mutation in Ig genes

A mutation that alters the reading frame of an Ig gene in some way; can be either productive or non-productive (the latter is more common). Examples include addition and deletion.

Open reading frame (ORF)

A part of a reading frame containing no stop codons

J-chain

A polypeptide that links the heavy chains of monomeric units of polymeric IgM and di-or trimeric IgA. The linkage is by disulfide bonds between the J chain and the carboxyl-terminal cysteines of IgM or IgA heavy chains.

Allelic exclusion

A process that permits expression of only one of the allelic forms of a gene

Papain

A proteolytic enzyme derived from papyas and pineapples. Papain cleavage of immunoglobulin releases two Fab and one Fc fragment per IgG

Framework regions of V region

A relatively conserved sequence of amino acids located on either side of the hypervariable regions in the variable domains of immunoglobulin heavy and light chains

Leader sequence on a nascent protein

A short hydrophobic sequence of amino acids at the N-terminus of newly synthesized immunoglobulins; it inserts into the lipid bilayer of the vesicles that transport Ig to the cell surface. The leader is removed from the ends of mature antibody molecules by proteolysis

Idiotypic determinants

A single antigenic determinant in the variable domains of an antibody or T-cell receptor; also call an idiotype. These are generated by the unique amino acid sequence specific for each antigen.

Ig superfamily

Know examples - any class of immunoglobulings, MHC cl I/II, CD4, CD8, FcR, poly IgR, TCR, CAMs Many are membrane bound

Cross-reactivity

Ability of a particular antibody to react with two or more antigens that possess a common epitope; used in research (Western blot, ELISA), but may cause false positives in clinical application

AID

Activation-induced cytidine deaminase; an enzyme that removes an amino group from deoxycytidine, forming deoxyuridine This is the first step in the process of both somatic hypermutation and class switch recombination.

Indirect ELISA

Adding antibody to well lined with antigen to measure level of antibody

DSBR enzymes used during DNA rearrangement

Aids in reforming double stranded DNA after rearrangement

Primary structure of a protein

Amino acid sequence

FcεR

An FcR specific for the Fc region of epsilon Ig molecules; present on cells responsible for releasing histamine and inducing an allergic response

Cysteine

An amino acid capable of binding to other molecules of the same type and forming strong disulfide bonds

Isotype

An antibody class that is determined by the constant-region sequence of the heavy chain. Five human isotypes are IgA, IgD, IgG, IgE, and IgM

Link between cross-reactivity and autoimmunity

An antibody that is able to recognize epitopes common to multiple proteins is more likely to recognize an epitope on a self protein that one that is specific to only one kind of protein

Allotypic determinants

An antigenic determinant that varies among members of a species or between different inbred strains of animals. The constant regions of antibodies possess allotypic determinants

Isotypic determinants

An antigenic determinant within the immunoglobulin constant regions that is characteristic of a species

Flow cytometry

Analysis of immune cells and their molecular markers based on presence/absence of those markers, which have been tagged with antibody

Anti-allotypic antibodies

Antibodies directed towards allotypic determinants

Anti-idiotypic antibodies

Antibodies directed towards antigenic determinants located in the antigen binding site of other antibodies

Anti-isotypic antibodies

Antibodies directed towards antigenic determinants located in the constant regions of antibodies that are shared among all members of a species

IgE and epsilon heavy chains

Antibodies responsible for the allergic response (formerly parasites, but because they are rare now, function against allergens)

Antigen (Ag)

Any substance (usually foreign) that binds specifically to an antibody or a T-cell receptor; often used as a synonym for immunogen

S-exon on Ig HC gene

Located to splice out different C isotype regions to alter expression of C segment

B cells versus activated B cells versus plasma cells

B cells - express immunoglobulin to detect immunogens Activated B cells - B cells that have been stimulated by an antigen Plasma cells - high affinity B cells that secrete different isotypes of antibodies during infection

Promoters and enhancers in Ig genes

Brought closer together by splicing and recombination to ensure expression only when rearrangement is completed

Antigenic specificity

Capacity or antibody and T-cell receptor to recognize and interact with a single, unique antigenic determinant or epitope

FcR

Cell-surface receptor specific for the Fc portion of certain classes of immunoglobulin. It is present on lymphocytes, mast cells, macrophages, and other accessory cells.

Ig-fold

Characteristic structure in immunoglobulins that consists of a domain of 100 to 110 amino acids folded into two beta-pleated sheet, each containing three or four antiparallel beta strands and stabilized by an intrachain disulfide bond

Ab affinity

Combined strength of all non-covalent interactions between a single antigen binding site on an antibody and a single B-cell epitope on the antigen

CTL

Cytotoxic T lymphocytes; an effector T cell that can mediate the lysis of target cells bearing antigenic peptides complesed with a class I MHC molecule

Humanized monoclonal antibodies for clinical applications

Increases the half life of Ab because the humanized portion of the heavy chain is less likely to be degraded; the entire process of hybridoma formation cannot be done with humans because we require our spleens to be intact

Booster shot (immunization)

Inoculation given to stimulate and strengthen an immunological memory response

Pro-B cell

Earliest distinct cell of the B-cell lineage

Conformational determinants

Epitopes of a protein that are composed of amino acids that are close together in the three-dimensional structure of the protein but may not be near each other in the amino acid sequence

Immature B cell

Express a fully-formed IgM receptor on their cell surface. Contact with antigen at this stage of B cell development results in tolerance induction rather than activation. Express lower levels of IgD and higher levels of IgM than mature B cells. Also have lower levels of anti-apoptotic molecules and higher levels of Fas than mature B cells, reflective of their short-half lives.

IgD and delta heavy chains

Expressed in mature, naive B cells

FACS-analysis and flow cytometry

Fluorescence activated cell sorter; used to analyze and sort individual ceells from complex cell suspension. Each cell is analyzed based on presence/absence of molecular markers, which have been tagged with antibody

Germ-line DNA versus rearranged DNA at Ig-loci

Germ line DNA contains all of the possibilities of Ig proteins; rearranged DNA has been spliced to only produce one specific molecule

Coding segments (V, D, J)

Heavy chain - V, D, J Light chain - V, J DNA segments that are recombined to make contact with each other and be expressed together; this recombination serves to increase diversity within B cells and antibodies

Role of disulfide bonds in formation of Ig molecule

Helps to stabilize and strengthen bonds

Recombination signal sequences (RSS)

Highly conserved heptamer and nonamer nucleotide sequences that serve as signals for the gene rearrangement process and flank each germ line V, D, and J segment

Cell-mediated immunity

Host defenses that are medicated by antigen-specific T cells. It protects against intracellular bacteria, viruses, and cancer and is responsible for graft rejection. Transfer of primed T cells confers this type of immunity of the recipient.

in-frame and out-of-frame joining of segments

In-frame - maintains proper reading frame Out-of-frame - introduces early stop codon to prevent complete function of protein

Ig classes and subclasses (isotypes)

IgM IgG - IgG1, IgG2, IgG3, IgG4 IgA - IgA1, IgA2 IgE IgD

One turn/two turn RSS

Immunoglobulin gene-recombination signal sequences are separated by an intervening sequence of 12 base pairs

Light chains

Immunoglobulin polypeptides of the lambda or kappa type that join with heavy chain polypeptides to form the antibody heterodimer

Antibody (Ab)

Immunoglobulin proteins consisting of two identical heavy chains and two identical light chains, that recognize a particular epitope on an antigen and facilitates clearance of that antigen. Membrane-bound Ab is expressed by B cells that have not encountered antigen; secreted Ab is produced by plasma cells. Some Abs are multiples of the basic four-chain structure.

Monoclonal versus polyclonal antibodies

Monoclonal - deriving from a single clone of dividing cells (only one type of antibody) Polyclonal - a mixture of antibodies produces by a variety of B-cell clones that have recognized the same antigen, although all of the antibodies react with the immunizing antigen, they differ from each other in amino acid sequence

IgG and gamma heavy chains

Most important antibody for immunization; 2nd most important for immune response/memory; 4 subclasses; can cross placenta

Productive versus non-productive rearrangements

Non-productive - introduce stop codon early in nucleotide sequence and prevent continued expression Productive - introduce diversity into genome without introducing new stop codon

Immunoglobulin protein fragments formed by pepsin versus papain

Pepsin - F(ab')2 (cuts below disulfide bond on heavy chain Papain - Fab (cuts Ag above disulfide bond)

Example 2 of alternative RNA splicing in Ig genes

Polyadenylation RNA splicing

Primary versus secondary antibodies and use in Western blot and ELISA

Primary antibodies - specific to the antigen of interest Secondary antibodies - directed against isotypic determinants on primary antibody Use of secondary antibodies - amplification

Cell differentiation

Process by which unspecialized or less specialized cells become more specialized

RAG-1 and RAG-2

Recombinases; only expressed when needed for rearrangement; only in B and T lymphocytes

Effect of chemical reduction on the structure of Ig molecule (eg. beta-mercaptoethanol)

Reduction of disulfide bonds can disconnect heavy and light chains of Ig and creates single strands of amino acids

Immunodominant epitope

Referring to antigenic determinants that produce a more pronounced immune response than others under the same condition

Fab fragment

Region at the N-terminus of the antibody molecule that interact with antigen. This antibody fragment, consisting of one light chain and part of one heavy chain, linked by an interchain disulfide bond, is obtained by brief papain digestion

Fc fragment

Regions at the C terminus of the antibody molecule that interacts with Fc receptors on other cells and with components of the complement system. This crystallizable antibody fragment consists of the carboxyl-terminal portions of both heavy chains and is obtained by brief papain digestion.

M1 and M2 exons on Ig-gene

Required for membrane binding of Ig molecules

ELISA

Sandwich - Measures the level of an antigen/protein using Ab coated well Indirect - Measures level of an antibody using Ag coated well

Example 1 of alternative RNA splicing in Ig genes

Secreted versus nonsecreted (membrane) Ig

IgA and alpha heavy chains

Seen in areas in close contact with the outside world; exists as monomer, dimer, or tetramer; j-chain holds multiple IgA molecules together; contains secretory component which allows transportation of IgA from site of formation to lumen where it will function

Germ-line model

States that all Ig molecule segments are encoded by all different genes; this would take up 15% of the genome, which is highly unlikely

Immunofluorescence

Technique of staining cells or tissues with fluorescent antibody and visualizing them under a fluorescent microscope.

C regions: CH and CL

The 3' coding of a rearranged immunoglobulin gene. There are multiple C gene segments in germ-line DNA, but as a result of gene rearrangement and, in some cases, RNA processing, only one segment is expressed in a given protein. CH- on the heavy chain; CL - on the light chain

Agglutination

The aggregation or clumping of cells or particles; in particular, a hemagglutinin (HA) causes clumping of red blood cells

DNA-rearrangement or recombination

The alteration of germ line DNA to permanently alter its structure and ensure the expression of a single protein type; ex. the rearrangement of the VDJ region of a heavy chain

Plasma cell

The antibody-secreting effector cell of the B lineage

Class switching

The generation of antibody genes for heavy chain isotypes other than mu or delta by DNA recombination

Affinity maturation

The increase in average antibody affinity for an antigen that occurs during the course of an immune response or in subsequent exposures to the same antigen

Heavy chains

The larger polypeptide of an antibody molecule; it is composed of one variable domain VH and three or four constant domains (CH1, CH2, etc.). there are five major classes of heavy chains in humans, which determine the isotype of an antibody.

Constant region domains

The nearly invariant portion of the immunoglobulin molecule that does not contain antigen-binding domains. The sequence of amino acids in the constant region determines that isotype of heavy chains and the type of light chains

Coding joint

The nucleotide sequences at the point of union of coding sequences during V(D)J rearrangement to form rearranged antibody or T-cell receptor genes

B cell epitopes

The portion of an antigen that is bound by an antibody; also known as an antigenic determinant

Epitopes or antigenic determinants

The portion of an antigen that is recognized and bound by an antibody or TCR-MHC combination

Complementarity determining region (CDR) or hypervariable region (HVR)

The same thing! Those parts of the variable regions of the BCR and TCR that exhibit the most sequence variability and interact with the antigen.

Pre-B cell

The stage of B-cell development that follows the pro-B stage. Produce cytoplasmic mu heavy chains and most display the pre-B cell receptor

Alternative RNA splicing in Ig genes

The variable splicing of mRNA transcripts from DNA to create variation in Ig gene products

Gene-segment model

Theory stating that Ig genes are encoded by multiple gene segments and vary due to random rearrangement at the genomic level

HVR1, 2, and 3

Those parts of the variable regions of the BCR and TCR that exhibit the most sequence variability and interact with the antigen.

Ig domains - structure and function

Three dimensional structures characteristic of immunoglobulin and related proteins including T cell receptors, MHC proteins, and adhesion molecules. Consists of a domain of 100 - 110 amino acids folded into two beta-pleated sheets, each containing three or four antiparallel beta strands and stabilized by an intrachain disulfide bond.

F(ab')2 fragment

Two Fab units linked by disulfide bridges between fragments of the heavy chain. They are obtained by digestion of antibody with pepsin.

HAT selection

Unmerged myeloma cells that do not form hybridomas with lymphocytes cannot survive in HAT medium, which results in their death

One-turn-two-turn joining rule

Upon rearrangement, one-turn RSS's can only join with 2-turn RSS's

Hapten-carrier conjugate

Use in hybridomas; used to select for multiple different antibodies specific for different B cell epitopes

ELISPOT

Used to detect percent of secreting cells in a heterogeneous cell/tissue sample

Ig gene segments (V, D, J)

V - variable region of the gene D - diversity region; only in heavy chain J - joining segment

Hybridomas

What are they used for? Production of monoclonal antibodies How are they generated? Fusion of myeloma cells with lymphocytes (from mice/other animal)