HC5: B- and T-cell generation and diversity

Diverse repertoire

A diverse repertoire is necessary because humans are exposed to many different pathogens:

• Innate immunity can react to some constant properties of pathogens
• Adaptive immunity should respond to all possible pathogens, previously encountered and new
• Adaptive immunity shows a high degree of antigen specificity
• Adaptive immunity shows memory and is able to improve upon re-exposure

Antigen specificity:

B- and T-cells play a very important part in adaptive immunity. The difference between B-cells and T-cells is obvious:

• B-cells: host defense against extracellular pathogens
• T-cells: host defense against intracellular pathogens

Both cells are characterized by receptors on the surface which have specificity for one antigen → one cell has one specificity. This makes it possible for cells to respond do different pathogens.

B-cell receptors and secreted antibodies

Immunoglobulin:

Antibodies are also known as immunoglobulin (Ig). They are large, Y-shaped proteins produced by B-cells to neutralize pathogens. Antibodies recognize unique molecules of the pathogen → antigens. Antibodies can occur in 2 physical forms:

• Soluble form: has been secreted from the cell to be free in the blood plasma
• Membrane-bound form: the B-cell receptor

Every B-cell has a B-cell receptor → a surface immunoglobulin protein which is attached to the membrane of the B-cell. The B-cell receptor has a transmembrane region with which it signals to B-cells. When an antigen binds to the immunoglobulin, it will start producing soluble antibodies → the final antibodies present in circulation.

Clonal selection:

Clonal selection has 2 main functions:

• Generate a large variety of lymphocytes
• Select and expand the cell with wanted specificity

Clonal selection makes it possible for the immune system to respond to things it hasn't dealt with before.

During clonal selection, a large variety of B-cells is generated out of which the correct cell is selected:

1. A precursor cell makes many different cells which prepare the body for every threat that may be present
   • There are lymphocytes with receptors for many different antigens
2. During infection, lymphocytes with receptors that recognize the antigen give an activation signal
   • The receptors form crosslinks with the antigen
3. The lymphocytes with the correct receptor proliferate and differentiate
4. The lymphocytes give effector cells that terminate the infection

This is peculiar, because one cell has one specificity, and one gene is one protein. This would mean that an unlimited amount of genes is necessary to be able to have receptors for every antigen.

Antibody properties:

B-cells secrete antibodies of the same specificity as the membrane-bound immunoglobulin. The immunoglobulin/antibody is a central molecule of the B-cell. It consists of:

• 2 heavy chains and 2 light chains
• N-termini and C-termini
  • The C-termini attach the antibody to the plasma membrane
• Disulfide bonds → the heavy and light chains are covalently bound to each other
• A constant region and variable region
  • The constant region is the same for every immunoglobulin
  • The variable region is specific for every immunoglobulin → it contains the antigen binding site

The gene loci for Ig heavy chain and Ig light chain are composed of different building blocks:

1. V-genes: variable genes
2. D-genes: diversity genes
   • D-genes are only present in the heavy chain
3. J-genes: joining genes
4. The first chromosome

Antibody generation:

The diversity is created by there being more/less V-, D- and J-genes in different heavy- and light-chains. By combining different components of germline DNA, a variable region which gives high specificity for an antigen is made:

1. Germline DNA is DNA that is present in every cell
2. When a B-cell wants to make antibodies, it starts to recombine its DNA
3. 1 V-gene, 1 J-gene, 1 constant region and in case of a heavy chain 1 D-gene are joined together in a unique way for every B-cell
4. The DNA → RNA → protein route is followed
5. A polypeptide chain is created → the immunoglobulin molecule

The process makes it possible for the body to make a huge variety proteins.

Soluble antibodies:

Every antibody produced can be connected to the original B-cell. There is a sequence which can attach for example an IgM molecule to the plasma membrane. At mRNA level, the B-cell can make a soluble molecule when it lacks the tail with which it is made membrane-anchored → soluble antibodies are produced.

Selection points:

This process takes place in the bone marrow → both the precursor cells and B-cells are developing. To ensure that the correct cells are made, there are a few selection points during the process:

1. Repertoire assembly: generation of diverse and clonally expressed B-cell receptors in the bone marrow
2. Negative selection: alteration, elimination or inactivation of B-cell receptors that bind to components of the human body
3. Positive selection: promotion of a fraction of immature B-cells to become mature B-cells in the secondary lymphoid tissues → B-cells need to be structurally functional and have a correct antibody
4. Searching for infection: recirculation of mature B-cells between lymph, blood and secondary lymphoid tissues
5. Finding infection: activation and clonal expansion of B-cells by pathogen-derived antigens in secondary lymphoid tissues
6. Attacking infection: differentiation to antibody-secreting plasma cells in secondary lymphoid tissue

T-cell receptor

The T-cell receptor, like the B-cell receptor, is transmembrane-bound. It also has constant and variable regions and has an antigen binding site at the top of the molecule. The biggest difference between B-cell and T-cell receptors is the way they recognize antigens:

• B-cells are able to recognize everything that they encounter
• T-cells recognize certain peptide fragments produced after degradation of pathogens

MHC-molecules:

T-cell receptors recognize an MHC-molecule which is presenting a peptide:

1. Pathogens are degraded in the cell
2. An MHC-molecule presents a peptide
3. The T-cell receptor recognizes the antigen MHC-complex

There are 2 kinds MHC (major histocompatibility genes)/HLA (human leukocyte antigens) molecules. Both molecules have a groove in which the peptide is presented and are very polymorphic → there is a huge variation in MHC molecules:

• MHC class I
  • Made out of 1 chain with a common factor
  • Intracellular antigens
• MHC class II
  • Made out of 2 chains
  • Presents extracellular proteins

For there to be a response, the T-cell receptor has to recognize both the MHC-molecule and the peptide → the combination has to be correct for there to be proper recognition.

Generating diversity in T-cells and selection:

A T-cell receptor is formed out of an a- and b-chain:

• The a-chain is similar to the light-chain in immunoglobulins → only has V- and J-domains
  • Located on chromosome 14
• The b-chain is similar to the heavy-chain in immunoglobulins → has V- and J- and D-domains
  • Located on chromosome 7

The thymus is a small organ located near the heart. It is largest during infancy. Because a T-cell receptor only sees peptides in the context of MHC-molecules, it has to undergo a special process in the thymus before it is mature:

1. T-cell precursors travel from the bone marrow to develop in the thymus
2. The progenitor cells receive signals → proliferation
   • These cells are still precursor cells and are considered as "double-negative" → they lack 2 essential markers for selection
3. The precursor cells start rearranging the b-genes of their DNA
4. Check point: everything which is useless or doesn't make sense after the rearrangement is eliminated
5. Proliferation continues and the a-chain is rearranged
6. Check point: it is determined whether the cells are useful or not
7. Mature T-cells leave the thymus and travel to secondary lymphoid tissues

T-cell selection:

The thymus has specialized cells which can interact with developing T-cells → thymocytes. After the T-cells have developed, positive and subsequent negative selection of T-cells in the thymus occurs:

1. Positive selection: T-cells which have developed and have a receptor try to interact with epithelial cells in the thymus, which express MHC-molecules
   • If the T-cell is unable to recognize the MHC + peptide it will not get a signal → cell death
   • If the T-cell is able to recognize the MHC + peptide it will get a positive signal → continues to live
2. Negative selection: T-cells with a positive signal encounter different cells
   • If the binding is too strong, the cell is eliminated
   • If the binding is moderate, the cell continues living

Diversity in the B- and T-cell repertoire

T-cells and B-cells are very diverse:

• B-cells
  • More D-segments
  • Rarely have D-segments read in 3 frames
  • More junctional diversity
  • Total diversity: 5 x 10¹³
• T-cells
  • More V-segments
  • Often have D-segments read in 3 frames
  • More J-segments
  • More V-gene pairs
  • Total diversity: 10¹⁸

It may seem as if T-cells are much more diverse than B-cells, but this is not completely true. When B-cells are developed, are in secondary lymphoid organs and encounter an antigen, they have the capacity to change again → in the germinal centers, extra mutations are introduced in the V-regions to introduce extra affinity for antigens.

This is important while distinguishing immune responses → when exposed to a vaccine twice, the response will be much stronger and faster the second time.