HC7: Effector mechanisms of antibodies

B-cells

B-cells form the basis of humoral immunity by producing antibodies. They are part of the adaptive immune system. They possess antigen specificity and capacity to form memory upon clonal selection. During their final developmental stage, they form plasma cells.

Adaptive immunity is divided into 2 categories:

• Humoral immune response: antibody-mediated, B-cells
• Cell-mediated immune response: T-cells

An antigen is engulfed by an antigen-presenting cell and simultaneously presented to a helper T-cell and B-cell. To be presented to a T-cell, it's chopped into little pieces and attached to an MHC molecule. To be presented to a B-cell, it remains in its 3D-structure. Only when there's B-cell activation and sufficient T-cell help, there'll be formation of memory B-cells and plasma cells. Plasma cells produce the secreted antibody. Memory B-cells will remain in the body to protect the body from future attacks from the same antigen.

B-cell receptors:

On the surface of B-cells, there are antibodies → membrane bound immunoglobulins which serve as B-cell receptors:

• B-cells are characterized by the presence of surface immunoglobulins: the B-cell receptor (BCR)
• Each B-cell has multiple copies of the B-cell receptor
• Each B-cell generates BCR's with a single specificity
• The repertoire of BCR's is capable of recognizing millions of antigens

B-cell activation:

Globally, B-cell activation goes as follows:

1. A resting B-cell has a membrane bound Ig → the B-cell receptor
2. The membrane bound Ig encounters an antigen
3. T-helper cells stimulate the B-cell to give rise to antibody-secreting plasma cells

B-cell stages:

Plasma cells may derive from B-cells, but they have different properties:

• Resting B-cell
  • Surface Ig → only resting B-cells have receptors
  • Surface MHC class II → present peptides to T-cells
  • Growth
  • Somatic hypermutation
  • Isotype switch
• Plasma cell
  • High-rate Ig secretion → produce and secrete antibodies

Antibody structures

An antibody comprises 2 light chains and 2 heavy chains. These chains come from different genes, meet together in the B-cell and are connected via disulfide bonds. The left-hand side of the antibody is exactly the same as the right-hand side.

Antibodies have 2 regions:

• Constant region: genetically the same in every antibody in every human
• Variable region: forms the antigen binding sites → explains why one antibody binds to a specific antigen

Antibodies have different domains and nomenclature:

• Fab domain: mediate the binding to the antigen → mediate antigen neutralization
• Fc domain: complement activation and triggering of Fc receptors
  • Fc receptors are cellular receptors

Antibody isotypes:

Different isotypes have different immunological properties. The beginning of an antibody response starts with an IgM molecule that can be T-cell independent or T-cell dependent. Later on, it will switch to different isotypes. However, the basic structure of the antibodies remains the same. The differences are in the number of domains. When an antibody is switching isotypes, it's switching the constant domain → it's not necessarily switching the variable domain. Processes like affinity maturation and additional mutation will make the antibody bind stronger → the variable domain does change here.

There are 5 antibody isotypes:

• IgM
  • Activation of the complement system
  • IgM isn't a monomere in the circulation, it's a pentamere
    • This is caused by a J chain that locks 5 fragments together
    • This is necessary because in the beginning the affinity is very low → IgM needs many arms to increase the affinity
• IgD
• IgG1, IgG2, IgG3, IgG4
  • Neutralization
  • Opsonization (IgG1 and IgG3)
  • Activation of the complement system (IgG1, IgG3)
• IgA
  • IgA is largely dimeric in the circulation
    • This is caused by a J-chain
    • This makes it possible for IgA to be transported to epithelial surfaces
• IgE
  • Sensitization of mast cells

Antibody functions

Antibodies have many functions:

• Neutralization
• Opsonization
• Complementing dependent cytotoxicity

Neutralization:

An example of neutralization is when bacterial toxins are released from bacteria:

• Normally the toxins interact with receptors on epithelial cells → the epithelial cells go "crazy"
  • This can cause diarrhea
• If antibodies are present, they bind the toxins → the toxins can't bind to the epithelial cells anymore → the complexes are internalized and degraded

Opsonization:

In case of opsonization, a bacterium is present in the extracellular space:

1. Antibodies bind to the outside of the bacteria
2. Phagocytes have Fc receptors for the constant domain of the antibodies
3. The bacterium is ingested by the phagocyte and destroyed

Complementing dependent cytotoxicity:

In parallel to opsonization, there are antibodies that not only bind to the bacteria but also induce complement activation. Several complement-molecules are deposited on the outside of the bacteria, which are recognized by phagocytes that have receptors for these molecules.

The complement system can be activated either by IgM or IgG. Complementing dependent cytotoxicity activated by IgM has the following steps:

1. A pentameric IgM molecule binds to antigens on bacterial surface and adopts a "staple" form
2. C1q binds to a single IgM molecule
3. The complement system is activated

Complementing dependent cytotoxicity activated by IgG has the following steps:

1.  IgG molecules bind to antigens on bacterial surface
2. C1q binds to 2 or more IgG molecules and initiates complement activation
3. Activated C1s and C1r start to cleave complement proteins

C1q never binds to IgM or IgG in the circulation → if it would, a human would die immediately. After the complement system is activated, multiple processes take place:

• Recruitment of inflammatory cells
• Opsonization of pathogens → facilitates uptake and killing by phagocytes
• Perforation of pathogen cell membranes

In detail, the following happens:

1. C3 cleaves C4 to C4a and C4b
   • Some C4b binds covalently to the microbial surface
2. Activated C1s also cleaves C2 to C2a and C2b
3. C2a binds to surface C4b → the classical C3 convertase, C4b2a, is formed
4. C4b2a binds C3 and cleaves it to C3a and C3b → C3b binds covalently to the microbial surface
5. C5 convertase activates C5
   • This is complement mediated target lysis
6. Multiple processes take place → C5 mediates C6, C7, C8 and C9
7. When there are multiple copies of C9, a hole is made in the membrane
8. The pathogen dies

The complement system is very important in cases of encapsulated bacteria → encapsulated bacteria cannot be recognized and engulfed by neutrophils:

1. An antibody bound to bacteria activates the complement system and the bonding of C3b to the bacteria
   • The neutrophil can now recognize the bacteria
2. Fc receptors and complement receptors mediate the engulfment of bacteria by neutrophils
3. Granules fuse with phagosomes → toxic oxygen metabolites that kill the bacteria are released

Cellular Fc-receptors

Not every cell type has the same receptor → every cell type responds in a different way. There are multiple kinds of cellular Fc-receptors. The receptors also have different binding strengths:

• FCgRII-B1 binds very strongly to IgG1
• FCgRIII, will only bind to IgG1 when there are a lot of antibodies together → the bond is weak
• The binding of FceRI to IgE is super strong → all IgE receptors will be occupied

The functional consequence of binding to an Fc receptor can be very different:

• Binding of IgG1 to FCgRII-B2 will lead to inhibition of the cells → macrophages will be inhibited in their functional properties
• Binding of IgG1 to FCgRII-B1 will stimulate the cells → the antigen is killed and phagocytosis occurs
• Binding of IgE to FceRI will lead to the secretion of granules
• Binding of IgA FcaRI will uptake the induction of killing

NK-cells:

NK-cells have Fc receptors which can bind to antibodies:

1. An antibody binds antigens on the surface of a target cell
2. Fc receptors on an NK-cell recognize the bound antibody
   • The antibodies have the capacity to bind very strongly to FCgRIII, receptors on NK-cells
3. The NK-cell gets a very strong activation signal → it releases mediators from its intracellular space
4. The target cell dies by apoptosis

Mast cells:

The concept of the mast cell is a unique process in the immune response. They generate an allergic response and can fight parasites. Mast cells are full of granules with active components like histamine and other mediators. On the outside, a mast cell is decorated by Fcereceptors which binds to IgE with a very high affinity → all IgE is already bound to the mast cell, waiting for an antigen to come by. If an antigen comes by, this results in an immediate release of all the granules → all the content is released into the surrounding tissue, leaving a very deformed mast cell. The antigen has to be cross-linked to multiple receptors → binding to just one receptor doesn't generate a strong enough response.

Distribution of antibodies throughout the body

As a consequence of there being different antibody isotypes and properties, they are distributed differently throughout the body:

• Most of the body is filled with IgG and monomeric IgA
  • IgG1 and IgG3 can be transported across the placenta
    • If IgM would've been transported to the fetus, the consequences would be deadly if the fetus doesn't have the same AB0-system as the mother
  • IgG1 has the highest concentration in the circulation
• IgE is mostly located on lining epithelia of the gastro-intestinal tract and outside surface of the body
  • There is barely any IgE in the circulation
• IgM is mainly located by the heart
• Dimeric IgA is mainly located inside the gastro-intestinal tract
  • Because it exists in dimer form, only IgA can be transported across epithelium
  • Only dimeric IgA can be transported to the mother milk → the mother protects the intestines of the child from bacteria in the environment
• IgD is only present on the cell surface of early B-cells
  • It cannot be transported

Brain:

In principle, there are no antibodies in the brain, because immune responses in the brain can be deadly. If one can fight an immune response from outside the brain, it usually isn't necessary to do it inside the brain.

Mother and child:

Before birth, a child doesn't produce any IgG because it receives passively transferred maternal IgG. After birth, the child will slowly start to produce its own IgG. The child won't synthesize much IgM in the first year, because it receives maternal IgM through the breast milk. The concept of making new IgA will start even later. IgA levels aren't visible in the blood, because it will only be present in the intestines.

Antibodies in the lab

There are roughly 2 types of antibodies:

• Polyclonal: antibodies recognizing many different epitopes form 1 protein
  • Antibodies derived from larger animals
• Monoclonal: antibodies derived from 1 B-cell, recognizing only 1 antigen
  • Antibodies derived from smaller animals
    • Up to this era, mainly derived from mice and rats

Polyclonal and especially monoclonal antibodies are used in research, diagnostics and clinic.

Monoclonal antibodies:

Monoclonal antibodies can be made as follows:

1. B-cells from a mouse immunized with antigens are fused with myeloma cells
   • This is necessary to make the cells live forever
2. The cells are grown in a drug-containing medium
3. Cells are selected for antigen-specific hybridoma
   • These are the cells that make the right antibody
4. The selected hybridoma cells are cloned

These cells are used for many different purposes:

• They can be conjugated with different colors
• They can be used for tissue staining
• They can be coupled to a radioactive tracer, to locate tumors in cancer patients
• They can be conjugated to a special toxin

Monoclonal antibodies can be used in diagnostics and research:

• Flow cytometry: antibodies with different colors are used to bind to different cell types
  • 1 antibody for T-cells, 1 antibody for B-cells, etc.
• Analysis of cells stained with labeled antibodies
• Dot plot

Monoclonal antibodies can also be used as therapeutics, for instance in tumor-cell killing. There are 4 types of therapeutic monoclonal antibody:

• Mouse: mouse antibodies
  • These antibodies are foreign to the human body and will trigger an immune response → not very effective
• Chimeric: part mouse, part human antibodies
• Humanized: even more human antibodies are present
• Human: fully human antibodies

Polyclonal antibodies:

Polyclonal antibodies can also be used in therapeutics. This mainly happened in the past, because they are less effective than monoclonal antibodies.

Rheumatoid arthritis:

In rheumatoid arthritis the cytokine TNF plays a key role. Nowadays there are many different antibodies that target TNF and neutralize its function, which is a huge help to TNF patients.

Development of Chimeric Antigen Receptors (CAR) T-cells:

T-cells have a fantastic capacity to kill virus-infected cells, but cannot recognize tumor cells very well. Currently, T-cells with chimeric antigen receptors are being developed. These receptors are meant to recognize tumor cells:

1. T-cells are isolated from the blood of the patient
2. A gene is inserted into the T-cells
3. The T-cells start to express the gene → the chimeric antigen T-cell
4. The T-cells proliferate in the lab
5. The T-cells are given back to the patient