Antibodies and B cell development- Unit 2 - 541

What type of molecule is an antibody?

Generally a protein or carbohydrate, can also be a lipid.

What is the main function of an antibody?

Activates an adaptive immune response.

How heavy are antibody heavy chains?

50 kD each.

How heavy are antibody light chains?

25 kD each.

What does Fc stand for in antibodies?

Fragment crystallizable.

What is the function of the Fc fragment?

It is the constant fragment of the antibody.

What does Fab stand for in antibodies?

Fragment of antigen binding.

Where is the constant region located?

At the C-terminus.

What is the variation level of the constant region?

Limited variation.

Where is the variable region located?

At the N-terminus.

What does the variable region contain?

The antigen-binding site.

What is the main source of antibody diversity?

The variable region.

What is the hinge domain of an antibody?

The flexible branch of the Y-shaped antibody.

Name all five classes of immunoglobulins.

IgG, IgM, IgD, IgA, IgE.

Which antibody is the main circulating type in infection/vaccination?

IgG.

How many IgG subclasses exist?

Four (1-4).

Which antibody is first produced in any infection?

IgM

Where is IgD found?

In immature B cells.

Where is IgA secreted?

At mucosal surfaces.

How many IgA subclasses exist?

Two (1-2).

What does IgE do?

Binds mast cells and leads to degranulation.

How many total IgE types/subtypes exist?

Nine.

What are the Greek symbols for IgG, IgM, IgD, IgE?

IgG = Gamma, IgM = Miu, IgD = Delta, IgE = Epsilon.

Which part of the antibody do these classes refer to?

The heavy chain.

What is an antigen?

A molecule that forms antibody-binding sites; can have multiple epitopes.

What is an epitope?

A small part of an antigen recognized by antibodies.

What are the two types of epitopes?

Linear and discontinuous.

What is a linear epitope?

Sequential amino acids.

What is a discontinuous epitope?

Non-sequential amino acids close in tertiary structure.

Can antibodies recognize the same epitope differently?

Yes, with different affinities (binding strength).

What is somatic recombination?

Rearrangement of antibody gene segments (V, D, J) to form a functional Ig gene.

Why is somatic recombination necessary?

To generate antibody diversity and produce functional antibodies from fragmented germline genes.

Where does somatic recombination occur?

In developing B cells in the bone marrow.

What is the germline configuration?

The inherited, unarranged form of Ig genes found in non-B cells.

On which chromosome is the heavy chain gene located?

Chromosome 14.

On which chromosomes are the light chain genes located?

Kappa (κ): Chromosome 2; Lambda (λ): Chromosome 22.

What are the main parts of each Ig gene locus?

Leader (L), Variable (V), and Constant (C) regions.

What does the leader (L) sequence do?

Directs the antibody protein into the secretory pathway.

What do V region genes encode?

The variable part of the antibody that binds to antigens.

What do C region genes encode?

The constant region that determines antibody class and effector function.

Which gene segments make up the light chain variable region?

V and J segments.

Which gene segments make up the heavy chain variable region?

V, D, and J segments.

How many recombination events occur for the light chain?

One (V + J).

How many recombination events occur for the heavy chain?

Two (D + J, then V + DJ).

How many possible recombinations exist for each chain type?

κ: 35 V × 5 J = 175; λ: 30 V × 4 J = 120; Heavy: 40 V × 23 D × 6 J = 552

What is the total possible antibody combination count?

1,628,400 combinations.

What are RSSs?

Recombination Signal Sequences: Conserved DNA sequences guiding recombination.

What are the components of an RSS?

Conserved heptamer, spacer (12 or 23 bp), and nonamer sequence.

Where are RSSs found in the light chain locus?

3′ end of V segment and 5′ end of J segment.

Where are RSSs found in the heavy chain locus?

3′ end of V, both ends of D, and 5′ end of J.

What does the 12/23 rule ensure?

A one-turn (12 bp) sequence can only join a two-turn (23 bp) sequence.

Which enzymes perform somatic recombination?

RAG-1 and RAG-2 (Recombination-Activating Genes).

Which cells express RAG enzymes?

Lymphocytes only.

What is the first step of recombination by RAG?

RAG binds to RSSs and brings two gene segments together.

What happens next in RAG-mediated recombination?

A DNA hairpin loop forms between the segments.

What is a signal joint?

The DNA loop excised by RAG and discarded.

How does recombination add antibody diversity?

By adding random nucleotides to coding ends before joining.

Are recombination changes reversible?

No, they are permanent DNA changes.

What happens in the early pro-B cell stage?

D and J segments of the heavy chain are joined.

What happens in the late pro-B cell stage?

V segment joins to DJ, forming a complete VDJ exon.

Which antibody class is first made?

IgM.

What happens in the large pre-B cell stage?

Heavy chain is made; light chain rearrangement not yet started.

What happens in the small pre-B cell stage?

Light chain rearrangement begins — κ first, then λ if κ fails.

What is allelic exclusion?

Each B cell expresses antibody genes from only one chromosome pair.

Why is allelic exclusion important?

Ensures every B cell makes one antibody specificity.

What is a productive rearrangement?

Rearrangement that maintains the reading frame → functional Ig chain.

What is a nonproductive rearrangement?

Rearrangement causing frame-shift → nonfunctional protein.

What happens after a productive heavy chain rearrangement?

Recombination machinery shuts off for the other allele.

What happens if both heavy chain rearrangements fail?

The B cell dies by apoptosis.

What are polyclonal antibodies?

Mixture of antibodies from multiple B cell clones recognizing different epitopes.

How are polyclonal antibodies produced?

Inject animal with antigen → collect serum.

One advantage of polyclonal antibodies?

Can detect proteins even when sequence is unknown.

One disadvantage of polyclonal antibodies?

May bind to unintended proteins (less specific).

What are monoclonal antibodies?

Antibodies from one B cell clone recognizing a single epitope.

How are monoclonal antibodies made?

B cells fused with tumor cells → hybridomas → select desired antibody.

One advantage of monoclonal antibodies?

Highly specific and consistent.

One disadvantage of monoclonal antibodies?

May not cross-react with other species.

What are mouse monoclonal antibodies?

Fully mouse antibodies; humans mount immune response after one use.

What are chimeric antibodies?

Mouse V region + human C region (e.g., Rituximab).

What are humanized antibodies?

Mostly human; only binding site (CDR) is mouse (e.g., Omalizumab).

What are human monoclonal antibodies?

Fully human; made using genetically engineered mice.

Where does B cell education occur?

In the bone marrow.

What is the purpose of B cell education?

Eliminate self-reactive B cells to ensure self-tolerance.

Which cells test immature B cells for self-reactivity?

Bone marrow stromal and hematopoietic cells.

What happens to self-reactive B cells?

They are retained for receptor editing or undergo apoptosis.

What is clonal deletion?

Death of self-reactive B cells that fail editing.

What is anergy?

State where self-reactive B cells survive but remain inactive.

What percent of immature B cells are self-reactive?

About 75%.

How do B cells exit the bone marrow?

Enter the bloodstream as immature B cells.

How do they enter lymph nodes?

Through high endothelial venules (HEV).

What chemokine draws B cells into lymph nodes?

CCL21.

What receptor do B cells use to respond to CCL21?

CCR7 receptor.

Where do B cells go after entering lymph nodes?

Into primary lymphoid follicles.

Which cells support B cell survival in follicles?

Follicular dendritic cells (FDCs).

What happens if a B cell is not stimulated by antigen?

Leaves lymph node as a naive mature B cell.

What happens if a B cell encounters its antigen?

Becomes activated, differentiating into plasma and memory cells.

What do plasma cells produce?

Soluble antibodies.

How do antibodies become soluble?

Alternative RNA splicing removes membrane-binding sequence.

What is somatic hypermutation?

Single-base substitutions in the V region after activation.

What is the effect of somatic hypermutation?

Alters affinity of antibodies; leads to affinity maturation.