4: Antigen Recognition in the Adaptive Immune System

Specific antigen reception is whose task?

Specific antigen recognition is the task of cell surface proteins of lymphocytes

What form of antigens do B cell receptors recognize?

Conformational and linear epitopes

• Macromolecules (proteins, polysaccharides, lipids, and nucleic acids)

• Small chemicals

What form of antigens do T cell receptors recognize?

Linear epitopes

• Mainly peptides displayed by MHC molecules on antigen-presenting cells

Epitopes

An epitope is the specific part of a foreign substance or antigen that an antibody or immune cell recognizes and binds to.

Diversity of B cell receptors:

Each clone has a unique specificity

• potential for >10^9 distinct specificities

Diversity of T cell receptors:

Each clone has a unique specificity

• potential for >10^11 distinct specificities

What mediates antigen recognition in B cell receptors?

Variable (V) regions of heavy and light chains of membrane Ig

What mediates antigen recognition in B cell receptors?

Variable regions of alpha and beta chains of the TCR

What is the variable region?

The variable region of a cell receptor is the part that differs between receptors and determines which specific antigen or epitope it can recognize and bind to.

What mediates signaling functions in B cell receptors?

Proteins (Igα and lgβ) associated with membrane lg

What mediates signaling functions in T cell receptors?

Proteins (CD3 and ζ) associated with the TCR

What mediates the effector functions of B cell receptors?

Constant (C) regions of secreted lg

What mediates the effector functions of T cell receptors?

TCR does not perform effector functions!

What are effector functions?

An effector function is the action a cell or immune molecule performs after recognizing a target, helping the body respond to and eliminate a threat.

Secreted lgG

An antibody released by a B cell into the blood or tissues to help fight pathogens

Membrane IgM

An antibody attached to the surface of a B cell that acts as a receptor to recognize antigens

What regions does IgG contain?

• 2 heavy chains + 2 light chains.

• Heavy chain: VH + CH1 + CH2 + CH3.

• Light chain: VL + CL.

• 1 variable region + 3 constant regions per heavy chain.

• Has a hinge region.

• Fab region: antigen binding.

• Fc region: binds Fc receptors and complement.

• Secreted IgG is released into blood and tissues.

What regions does IgM contain?

• 2 heavy chains + 2 light chains in each antibody unit.

• Heavy chain: VH + CH1 + CH2 + CH3 + CH4.

• Light chain: VL + CL.

• 1 variable region + 4 constant regions per heavy chain.

• No conventional hinge region.

• Fab region: antigen binding.

• Fc region: CH2 + CH3 + CH4.

• Membrane IgM is attached to the B-cell membrane and acts as a B-cell receptor.

• Secreted IgM is usually a pentamer (5 antibody units joined together).

How many heavy and light chains are in IgG?

2 heavy and 2 light

What do the heavy and light chains of IgG make up?

These chains are held together by disulfide bonds and make up an antigen-binding site. The antigen-binding site is the part of the antibody that recognizes and attaches to a specific epitope on an antigen.

Fab region in IgG:

Fragment antigen-binding

Functions:

• Recognize the antigen

• Bind to the antigen through the antigen-binding site

What kind of regions does the Fab region contain?

The Fab region contains both the variable and constant portions of the heavy and light chains.

Where is the Fc region?

The Fc region is located on the lower stem of the Y

What is the Fc region and what is its function?

Fragment crystallizable

There are two regions of Fc binding: receptor and complement

Receptor Binding:

• It docks onto specialized Fc receptors located on the surface of immune cells like macrophages, natural killer (NK) cells, and neutrophils

Complement Binding:

• The Fc region can bind to complement proteins to jump-start the complement pathway

ALLOWS AN ANTIBODY THAT HAS BOUND TO AN ANTIGEN TO RECRUIT OTHER IMMUNE DEFENSES

Where is the hinge region located?

The hinge is the flexible region between the Fab arms and the Fc stem

Complementarity-determining regions (CDRs)

CDRs are small, highly variable parts of an antibody’s variable regions that directly contact and bind to a specific epitope of an antigen

• The antibody’s variable region contains CDRs, which determine which epitope an antibody can bind to

Bivalent binding of an antibody

An antibody has bivalent binding. This means that one antibody can bind to two antigen molecules because it has two Fab regions (antigen-binding sites)

What is hybridoma technology?

Hybridoma technology is a technique used to produce large amounts of one specific antibody by fusing an antibody-producing B cell with an immortal myeloma cell.

Process of hybridoma technology:

1. A mouse is injected with antigen X

2. The mouse’s immune system responds by producing B cells that make antibodies against that antigen

3. Scientists remove the mouse’s spleen and isolate the spleen cells, including B cells that produce the X antibodies

4. The antibody-producing B cells are fused with immortal myeloma cells

5. The cells are cultured, and only the successfully fused hybridoma cells survive and grow

6. Scientists isolate clones derived from the single hybridoma cells, so their descendants produce the same antibody

7. Scientists test to make sure that each clone is producing an anti-X antibody

8. Those producing the correct antigen are selected and grown

Why do we fuse B cells and myeloma cells in hybridoma technology?

The purpose of hybridoma technology is to produce a large amount of one specific antibody. B cells produce antibodies, but normal antibody-producing B cells have a limited lifespan and cannot divide indefinitely. On the other hand, cancer cells can divide indefinitely, but they do not produce the desired antibodies.

By fusing the two, we get:

• The antibody-producing ability of the B cell.

• The indefinite growth ability of the myeloma cell.

What do T cell antigen receptors consist of?

• α (alpha) chain

• β (beta) chain

These two chains are connected by a disulfide bond

What are the regions of a TCR (T Cell Receptor)

• Variable region (Vα or Vβ): Helps determine which antigen the TCR recognizes.

• Constant region (Cα or Cβ): Provides structural support.

• Transmembrane region: Anchors the receptor in the T-cell membrane.

• Short cytoplasmic tail: Extends inside the cell.

What forms the antigen-binding portion of the TCR?

the V and Vß domains

What are amino-terminal and carboxy-terminal ends of the polypeptides

1. Amino-terminal end (N-terminus)

• The end of the polypeptide with a free amino group (–NH₂).

• It is the beginning of the protein chain.

2. Carboxy-terminal end (C-terminus)

• The end of the polypeptide with a free carboxyl group (–COOH).

• It is the end of the protein chain.

What is immunoglobulin?

Immunoglobulin (Ig) is another name for an antibody, a protein made by B cells that recognizes and binds to specific antigens.

What antigen-binding chains make up Ig?

Made up of three CDRs in VH and three CDRs in VL

How many antigen-binding segments make up TCR?

Made up of three CDRs in Vα and three CDRs in Vβ

What changes are there in constant regions of Ig?

Heavy-chain class switching and change from membrane to secretory Ig

What changes are there in constant regions of TCR?

No changes

What is the affinity of antigen binding in Ig?

The average affinity of Ig increases during immune responses to protein antigens

What is the affinity of antigen binding in TCR?

There is no change during immune responses

On-rate and off-rate

These terms describe how quickly an antibody (Ig) or T-cell receptor (TCR) binds to and releases an antigen.

On-rate & off-rate of Ig:

Rapid on-rate, variable off-rate

On-rate & off-rate of TCR

Slow on-rate, slow off-rate

V(D)J recombination

is the process that rearranges DNA segments in immature B and T cells to create unique antigen receptors in mature cell DNA

• Mixing and matching DNA segments to create unique antigen receptors

What does V(D)J stand for?

• V = Variable

• D = Diversity

• J = Joining

These are gene segments that get cut and joined together in different combinations.

What is the purpose of V(D)J recombination?

Create millions of different B-cell receptors (BCRs) and T-cell receptors (TCRs) so they can recognize different antigens.

Steps in the maturation of lymphocytes:

1. It starts with a common lymphoid progenitor from the bone marrow

2. The common lymphoid progenitor develops into pro-B/T cells

   1. Cells that fail to express pre-antigen receptors undergo cell death

3. These cells then go on to produce one chain of their antigen receptors, making them Pre- B/T cells

   1. Those that do not produce one of their chains undergo cell death

4. To keep the one receptor chain stable on the cell’s surface, a surrogate chain is formed while the second chain is waiting to develop

5. The Pre-B/T cells then go on to produce the 2nd chain of the antigen receptors, making them immature B/T cells

What is a pro-B/T cell?

It is an early developing B cell or T cell that has started rearranging its antigen receptor genes but has not yet made a complete functional receptor.

What are Pre-B/T cells?

Pre-B/T cells are developing B or T cells that have successfully produced one chain of their antigen receptor and are testing whether it works.

What happens to immature B/T cells with weak antigen recognition?

They undergo positive selection and develop into mature B/T Cells!

• The body hopes that these receptors are random enough to recognize foreign antigens, but so weak that they dont recognize self antigens found in high concentration.

What happens to immature B/T cells with strong antigen recognition?

Negative selection! They undergo cell death

• Strong affinity means that they will be attracted to our self-antigens, which is not good! We do not want to kill our body’s antigens!

Germline organization

Germline organization means the arrangement of the gene segments in the inherited DNA before the cell rearranges them.

What are the different types of gene segments?

• Variable

• Diversity

• Joining

• Constant

• Leader

Variable gene segment

Part of the variable region that helps determine what antigen the receptor recognizes.

Diversity gene segment

Adds additional variation to the variable region

Joining gene segment

Joins gene segments together to help form a variable region

Constant gene segment

Encodes the constant region of the receptor, which contributes to its structure and functions

Leader gene segment

A short sequence that helps direct the newly made protein into the endoplasmic reticulum; cleaved from mature proteins

What happens to people who do not have RAD recombinase?

Their B and T cells won’t have receptors, so they will be deleted.

• NO B & T CELLS!

• NO ADAPTIVE IMMUNITY

CDR3

Complementarity-determining region 3

• It is one of the most variable parts of the antigen-binding region.

Process: Recombination and expression of Ig genes

1. There is germline DNA at the Ig Heavy locus

2. RAG-1 and RAG-2 initiate recombination by cutting the DNA at appropriate gene segments

3. One D segment joins one J segment

   1. The DNA that was between the D and J is removed or excluded from the rearranged gene

4. One V segment joins the DJ segment

   1. The DNA that was between the V and DJ is removed or excluded from the rearranged gene

5. The rearranged DNA is transcribed into RNA

6. The RNA is processed (introns are removed and exons are joined together)

7. The mRNA is translated into the μ heavy-chain protein

8. The heavy chain can pair with a light chain to form an antibody

VDJ Segment

Encodes the variable region of the heavy chain

• This is good because different cells can produce different VDJ segments, which contributes to each B cell having a unique antigen receptor

Ligase

Acts as a "molecular glue" to join DNA ends together

• When RAG-1 and RAG-2 help cut the DNA, the DNA must be repaired and joined back together.

What kind of chain are κ and λ?

(kappa and lambda) light chain

Combinatorial diversity

Combinatorial diversity means that a developing B cell or T cell randomly selects different V, D, and J gene segments and combines them to make its antigen receptor gene.

Junctional diversity

Junctional diversity is the variation created by adding or removing nucleotides at the places where V, D, and J gene segments are joined.

Terminal deoxynucleotidyl transferase

TdT

TdT adds nucleotides to the DNA ends during V(D)J recombination.

N-region nucleotides

Additional, non-templated nucleotides are added at the junctions of gene segments

P-nucleotides

Short nucleotide sequences that can be added during the DNA repair process

Selection in MHC-restricted T lymphocytes

Weak recognition of class II MHC + peptide

• They will undergo positive selection

Weak recognition of class I MHC + peptide

• Positive selection

No recognition of MHC + peptide

• Failure of positive selection (death by neglect)

Strong recognition of either class I or class II MHC + peptide

• Negative selection