Biology Dept
Kenyon College
Chapter 8 B.
Fall Section Spring Section 1 Spring Section 2
Genetics of the Immune System
Monoclonal Antibodies

Genetics of the Immune System

The purpose of the immune system is to attack potential pathogens that have invaded the body.  But the pathogens possess defenses, too; they recombine and mutate their own external proteins to thwart the host defenses.  Since pathogens reproduce far faster than multicellular hosts, how can host organisms use their own DNA to fight back?

The immune system poses special problems for genetic regulation:
(1) How to attack potential pathogens but avoid attacking one's own proteins?
(2) How to produce an infinite range of different antibodies that attack different specific pathogens?
(3) How to produce a specific antibody ONLY at the time when the pathogen is present?

We shall see how all levels of gene regulation are involved:
    DNA recombination
    RNA splicing
    Protein translation and processing

Many classes of cells are involved, including white blood cells and lympocytes.  Here we focus on only a tiny fraction of these classes: the B and T lymphocytes which produce antibodies  and antigen receptors.

Structure of Antibody Molecule
The structure of an antibody protein, or of an antigen receptor, looks like this.  Two light chains and two heavy chains are held together by disulfide bridges:   P-SH   HS-P  -->  P-S-S-P
Both light and heavy chains each contain a variable region and a constant regionThe variable region differs in amino acid sequence for different antibodies, which recognize different antigensThe constant region is the part that distinguishes different functional classes of antibody and antigen receptor.  For example, an antibody and an antigen receptor can recognize the same antibody if they share the same amino acids in their variable region; but the constant region of the antigen receptor keeps it bound to the membrane of its cell, whereas the constant region of the antibody permits secretion of the antibody into the blood.
The hypervariable regions are explained below.

Griffiths et al, AN INTRODUCTION TO GENETIC ANALYSIS, 6E, Freeman, 1996
Development of immune cells
To solve the problem of distinguishing self from non-self, the early stages of immune cell development and genetic regulation begins even before birth.  DNA rearrangement of antibody genes produces about 107 different clones of pre-antibody producer cells.  Then all antibody-producing cells which recognize antigens are eliminated--because any antigen they find must be "self."

Populations of immune cells recognizing external antigens slowly develop and mature.  The maturation of the immune system takes about 18 months after birth--one reason why breast feeding, which provides maternal antibodies, is so helpful during this period.

An external antigen stimulates the immune system by binding to the "variable" (i.e. clone-specific) portions of an antigen receptor on a precursor T cell, and on a precursor B cell.    Note that antigen receptors are similar to antibodies in structure.  The main difference lies in the constant (C) domain.  The C domain of antigen receptors keeps them plugged into the membrane, whereas the C domain of antibodies allows secretion.

The B cell then divides, proliferates, and differentiates into:

  • Circulating B secretor cells (or "terminal B cells") which produce antibodies in the blood
  • B memory cells, which persist for many years, expressing antigen receptors, ready to respond again
The T cell differentiates into:
  • T helper cells which are needed to activate and sustain the B cell response.  Defects in T cells result in immunodeficiency disease, such as HIV infection causing AIDS.
  • T suppressor cells which suppress anti-self reactions and turn off response when no longer needed.  Defects in T suppressor cells result in autoimmune disease such as lupus and scleroderma.
Antibody Diversity, through Intramolecular DNA recombination
Where do the ten million different clones come from?
The genes encoding antibody and antigen receptors are divided into sets of domains, for portions of a protein with different functions.
  • V domains -- Variable region of heavy or light chain.  The region of greatest diversity among the recombinant clones.
  • D domains -- Diverse regions, shorter than V domains.  Only on heavy chain.
  • J domains -- some diversity.  J segments connect V with C segments.
  • C domains -- constant for a given functional class.  All clones of a given class would share the same C domain.
For a given clone, a human chromosome can recombine together one each of the following:

Heavy chain: (300 V) (10 D) (4 J) = 12,000 different possible combinations (VERY approximate)
Light chain: (300 V) (4 J) = 1,200 different possible combinations
(12,000 heavy) (1,200 light) = 24,000,000 different clones

Note that any one cell will only recombine its DNA in one of these possible ways, and produce one possible antigen receptor or antibody.

Modified from Griffiths et al, AN INTRODUCTION TO GENETIC ANALYSIS, 6E, Freeman, 1996
Expression of the antigen receptor or the antibody
When the cell has been induced to mature and express its protein, the mRNA has to be spliced:
  • To link the J domain to the C.  All the intervening J genes are removed.
  • To link the leader peptide to the V region, enabling export of the antigen receptor to the membrane (or secretion of the antibody).  Note that this system provides one example of the usefulness of  intron splicing.
Hypermutation: Fine tuning
Where do the two-billion possible antibodies come from?
Each time the B and T cell clones are stimulated by an antigen, the clones divide and proliferate.  Their sub-clones inherit small mutations in the hypervariable regions (see above).  Those sub-clones whose mutations produce slightly stronger-binding antigen receptors will proliferate more than those whose mutations weaken the binding.  The product of the possible mutations with the ten-million-odd clones makes billions of possible antibody structures!

Monoclonal Antibody Technology
Modified from the MIT Hypertextbook, Excerpted from "What is Biotechnology?" Washington, D.C.: Biotechnology Industry Organization, 1989. Obtained from Genentech's Access Excellence

From the standpoint of gene technology, antibody molecules have two very useful characteristics:

  • Extreme specificity.  Each antibody binds to and attacks one particular antigen.
  • B memory cells provide future source of antibodies, indefinitely.
The B memory cells make it possible to develop vaccines. A vaccine is a preparation of killed or weakened bacteria or viruses that, when introduced into the body, stimulates the production of antibodies against the antigens it contains.

The specificity of antibodies  makes monoclonal antibody technology so valuable for biotechnology. Not only can antibodies be used therapeutically, to protect against disease; they can also help to diagnose a wide variety of illnesses, and can detect the presence of drugs, viral and bacterial products, and other unusual or abnormal substances in the blood.

Given such a diversity of uses for these disease-fighting substances, their production in pure quantities has long been the focus of scientific investigation. The conventional method was to inject a laboratory animal with an antigen and then, after antibodies had been formed, collect those antibodies from the blood serum (antibody-containing blood serum is called antiserum). There are two problems with conventional antibodies:

  • The antiserum  contains a poorly defined mixture of antibodies with diverse binding properties, as well as undesired substances
  • The B cells are mortal.  They produce only a very small amount of usable antibody, and they die in tissue culture.
Monoclonal antibody technology is based on a hybrid cell that combines the characteristic of "immortality" with the ability to produce the desired substance; in effect, a factory to produce antibodies that worked around the clock.  In monoclonal antibody technology, tumor cells that can replicate endlessly are fused with mammalian cells that produce an antibody. The result of this cell fusion is a "hybridoma," which will continually produce antibodies.

These antibodies are called monoclonal because they come from only one type of cell, the hybridoma cell; antibodies produced by conventional methods, on the other hand, are derived from preparations containing many kinds of cells, and hence are called polyclonal. An example of how monoclonal antibodies are derived is described below.

Monoclonal Antibody Production

A myeloma is a tumor of the bone marrow that can be adapted to grow permanently in cell culture. Myeloma cells can be fused with antibody-producing mammalian spleen cells, using a membrane fusion agent such as polyethylene glycol (PEG) or SV40 virus.  The nuclei fuse, and their chromosomes replicate and undergo mitosis together.   The resulting hybrid cells, or hybridomas, produce large amounts of monoclonal antibody. This product of cell fusion combines the desired qualities of the two different types of cells: the ability to grow continually, and the ability to produce large amounts of pure antibody.

Monoclonal antibodies have enormous clinical as well as research applications; for example, the Western Blot test for HIV.