Chapter 4: Adaptive Immune - Antibody & B-Cell Diversity Flashcards
Immunoglobulin
Cell-surface B-cell antigen receptors & secreted antibodies
5 isotypes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM
- constant regions vary
Antibodies
Secreted form of B-cell immunoglobulin
- in blood, lymph, and on mucosal surfaces
- specific (bind to one antigen)
Function of antibodies?
Bind specific antigen and deliver to other immune components
Antibody repertoire
Individual’s total # of specific abs – as high as 10^16, closer to 10^9
Plasma cells
Effector B lymphocytes that secrete abs
Secreted antibodies from plasma cells…
B-cells must come before!
Antigen
molecular fragment/toxin/etc from a pathogen
How many antibodies secreted at a time of infection?
Multiple
B cell to Plasma cell
- Resting B cell
- Encounter with antigen
- Stimulated B cell (clonal selection) gives rise to antibody-secreting plasma cells
Antibody: general structure
*Glycoproteins made of 4 polypep chains
- 2 identical heavy chains & 2 identical light chains
- Arm
- variable (V) region —– antigen binding site
- constant region
Arm
L chain covalently linked to amino-terminal part of H chain
- Disulfide bonds link H-L & H-H
Variable (V) region
N-terminal area of L and H chains – amino acid sequence varies between different abs
—Antigen binding site: V regions of 1H & 1L
Constant region
Amino acid sequence similar b/t abs of same isotype
- similar to TIR of TLRs
Hinge region
Allows flexibility
- can be cleaved by proteases to form:
— Fragment antigen binding (Fab) region
— Fragment crystallizable (Fc) region
Fragment crystallizable (Fc) region
Effector function - binds to serum protein and cell surface receptors
—imp for cell recognition & elimination of pathogen
Isotypes
- Ig isotype determined by C region of H chain
- Hinge region, H chain length, and carb binding varies between isotypes
- L chain isotypes: kappa or lambda
Immunoglobulin domain
Stable protein domain of ~100 AAs
- Variable domain
- Constant domain
Variable (V) domain
Vh and Vl
- Vh + Vl = antigen-binding site
Constant (C) domain
- One Cl per light chain
- 3-4 Ch per heavy chain
Hypervariable (HV) regions
Regions of V domains that have variable amino acid sequences
- aka complementary-determining regions (CDRs)
- 3 domain
Framework regions
Less variable regions of V domains
Epitope
Part of the antigen to which an antibody binds
- usually carbs or proteins
- multivalent
Multivalent
Antigen with more than one epitope or multiple copies of same epitope
Antigen-binding sites vary in?
Shape, size, and chemical properties to “fit” different epitopes
Epitopes can be…
- Linear: composed of AAs in sequence
- Discontinuous: brought together when protein is folded
Antigen/antibody interactions are?
Noncovalent
- Electrostatic forces, H bonds, van der Waals forces, hydrophobic interactions
Affinity
Binding strength of one antigen-binding site to its antigen
- Different abs may bind to same antigen with varying affinity
Avidity
Overall strength of binding to multiple epitopes of an antigen
Monoclonal antibodies
- Fusion of B cells from mouse immunized q antigen & myeloma cells
- Grow in drug-containing medium (only hybrid cells live)
- Select for antigen-specific hybridoma
- Clone the selected hybridoma cells
Gene segments
Fragments of genes across a chromosome that must come together to be expressed
- all cells have fragmented Ig heavy- and light-chain loci
— Germline form/config
— Only B cells can rearrange & assemble the functional Ig gene
——occurs during B cell development from B-cell precursors in bone marrow
Immunoglobulin genes
- Heavy-chain locus – chrom 14
- Kappa light-chain locus – chrom 2
- Lambda light-chain locus – chrom 22
— diff segments encode leader peptide (L), V region (V), and constant region (C)
Random recombination: L and C
Introns and exons, ready to be transcribed
Random recombination: V regions
Encoded by 2 Vl or 3 Vh gene segments - must be selected and rearranged to produce an exon.
Light chain V regon
1 variable (V) gene segment + 1 joining (J) gene segment
Heavy chain V region
1 variable (V) gene segment + 1 diversity (D) gene segment + 1 joining (J) gene segment
Somatic recombination
Process of bringing gene segments together during cell development
During B cell development, a single gene segment from each type brought together to form?
DNA sequence encoding V region of Ig chain
Somatic recombination: Light chain
single recombination b/t V (light) and J (light) segments
Somatic recombination: Heavy chain
two recombinations – D joined to J (heavy) then DJ to V (heavy) segment
V, D, and J gene segments joined together are randomly selected, resulting in
Diversity of Ig V regions
- 295 diff light chains possible
- 5520 diff heavy chains possible
- randomly combines = 1.6 million diff antibodies
—–abs repertoire = 10^9
Somatic
“Body”
Recombination signal sequences (RSSs)
Direct the recombination of the V, J, and D gene segments
- Flank 3’ side of V, both sides of D, and 5’ side of J
V(D)J recombinase
Set of enzymes that recombines V,D, and J gene segments
- “chopping up DNA”
- 2 of the components: recombination-activating genes (RAG) 1 and 2 – only made in developing lymphocytes
Recombination follows which rule?
12/23 rule
12/23 rule
RAG complex binds to an RSS and recruits the other RSS to the complex…RAG cleaves DNA…Coding joint or signal joint
- DNA is bent
- 23 always combines with 12
Coding joint
Ends of two gene segments joined
Signal joint
Ends of removed DNA joined
RAG has what type of activity?
Exonuclease
Junctional diversity
Contribution of P and N nucleotides to variation in coding joints
- increases diversity by 3 x 10 ^7
Initial DNA cleavage generates?
Hairpins that are opened by DNA repair enzymes
- nick in the hairpin occurs at random site
- P (palindromic) nucleotides generated
Ends of hairpins can be modified by
Terminal deoxynucleotidyl transferase (TdT)
TdT
Randomly adds nucleotides…until we get complementarity
- N nucleotides = not coded in germline
Exonucleases can remove what?
Nucleotides
Naive B cells
Circulating, haven’t encountered antigen (not stimulated)
- Express IgM and IgD on cell surface (don’t express other isotypes
What produces the different isotypes?
Alternative splicing of same mRNA transcript
Allelic exclusion
*Immunoglobulin diversity #4
Expression of only one heavy chain and one light chain by a single B cell
- ensures B cell produces IgM and IgD specific to a single antigen
Antigen-binding sites formed by?
1 H chain and 1 L chain
- random combinatorial association of one heavy chain with one light chain increases overall diversity of immunoglobulins
Heavy chains of IgM and IgD:
C-terminus hydrophobic sequence to associate with cell membranes, but very short cytoplasmic region
Ig alpha + Ig beta:
Travel with Ig from ER to cell surface; imp for signal transduction
B-cell receptor complex/B-cell receptor:
IgM or IgD + Ig alpha & Ig beta
Binding of antigen to Ig of a mature naive B cell…
Proliferation and differentiation, then antibody secretion
IgM produced in?
Large amounts
IgD produced in?
Respiratory tract
During differentiation, alternative RNA splicing results in?
Hydrophilic sequence in C-terminus of heavy chain
Somatic hypermutation
Single-nucleotide substitutions introduced randomly into V-domain coding sequence
- occurs after antigen binding
- Activation-induced cytidine deaminase (AID)
- may increase affinity for antigen
Activation-induced cytidine deaminase (AID)
Converts cytosine in single-stranded DNA to uracil
- produced only in proliferating B cells
- other enzymes convert U to one of four bases
Affinity maturation
Increase affinity for antigen
IgM is first class of antibody made in?
Primary immune response
Surface IgM is?
Monomeric
Secreted IgM is a?
Pentamer – bulky, restricted in effector mechanism recruitment
Isotype (aka class) switching
Further DNA recombination events allows V-region to combine with other heavy-chain C genes
- only in B cells responding to antigen
- previously expressed C gene is cleaved and new gene is transcribed
- patterns of isotype switching regulated by cytokines secreted by other immune cells
Antibodies clear pathogens by?
- Neutralization
- Opsonization
- Activating complement
Neutralization
Directly inactivating a pathogen/toxin and preventing it from interacting with human cells — *neutralizing antibodies
ex: Dimeric IgA = secreted in gut, milk, saliva, sweat, tears
Opsonization
Phagocytes have receptors for Fc regions of some abs
ex: Most abundant isotype in blood and lymph, very flexible
Activating complement
Direct lysis
IgE
Recruits effector functions of mast cells, basophils and eosinophils – expel and kills parasites; causes allergies/asthma
IgD
Recruits basophils in the respiratory tract
IgA: subclasses
IgA1 & IgA2
IgG
IgG1, IgG2, IgG3, and IgG4
- Different effector functions
ex: IgG3 best at activating complement
IgG4
Can exchange one H/L chain with another IgG4
- becomes monovalent
- anti-inflammatory.
