8/9/01

Lecture #17 - Immune Diversity

Main question: How does the immune system specifically respond to a wide range of invaders?

Antigen

anything that leads to an immune response
foreign proteins that aren't encoded by the host

Antibody

proteins made by animals that can specifically interact with the antigen
also called immunoglobins
the most common antibody is IgG, which is a circulating antibody that accounts for 20% of the proteins in blood
when antibodies encounter antigens, antibodies stimulate the immune system
the binding of the antibody to the antigen leads to the inactivation or degredation of the antigen.

White blood cells are called lymphocytes - B cells and T cells are major classes of lymphocytes

B cells

B cells are derived from cells in the bone marrow.
In chemotherapy, B cell precursors are killed (B cell precursors replaced in bone marrow transplants)
B cells make antibodies involved in recognition of the antigen.
The humoral response is mediated by B cells.
While the immune system as a whole can produce many different antibodies, a single B cell produces only 1 type of antibody molecule.

T cells

the T cell is derived from cells in the thymus
antigen recognition is mediated by T cell receptors
T cells cause a cell mediate response
T cell receptors recognize antigens on the surface of other cells. Cells infected with a virus produce viral proteins on their cell surfaces. T cells recognize these foreign proteins and kills those cells.
A single T cell produces only 1 type of T-cell receptor (which recognizes the antigen)
When the antigen binds to the T-cell receptor, the immune system is stimulated and the cell bearing the antigen is killed.
T cell receptors are generated in a similar way as antibodies.

Distinguishing between self vs. non-self

We don't want to unleash the immune system against our own proteins.
Immune cells are "tolerant", meaning they don't attack self-antigens
Tolerance works by clonal deletion
B cells and T cells that produce antibodies that (T cell receptors) recognize self antigens are deleted from the repertoire.
When clonal deletion fails, autoimmune diseases occur:
- In lupus, antibodies attack U1 snRNA and other snRNPs in the splicing machinery. Lupus can be deadly if the response gets out of control.
- In myasthemia gravis, the immune responds against acetylcholine receptors involved in nerve transmission. This can also be deadly.
The immune system usually does a good job of distinguishing between self vs. non-self.

Immune memory

Concentration of Antibodies vs. Time graph. After initial immunization with antigen #1 at t=0, the animal mounts an immune response and the [antibodies] reaches a low peak after about 20 days and then [antibodies] falls until t=40 days where the [antibodies] is very low. If you reimmunize (administer a booster) at approximately t=40 days with antigen #1, the [antibodies] reaches a high peak and then take a long time to fall back to low levels. At t=40 days, if you immunize with antigen #2, the [antibodies] for antigen #2 reaches the same low peak as was observed for the initial immunization of antigen #1. After 10, 20, or 30 years, if you reimmunize with antigen #1, then the [antibodies] reaches the same high peak as was observed for the first booster for antigen #1. The immune system somehow remembers after long periods of time to which antigens it mounted an immune response against.

Epitopes = antigenic determinants

Epitopes are a regions of the antigen that ellicit an immune response such as the part of the antigen. There are 2 types of epitopes:

"local" epitopes are a primary amino acid sequence of several adjacent amino acids. Different antibodies can recognize different epitopes of the antigen.
"global" epitopes are secondary and tertiary structures of the antigen proteins. These epiopes contain amino acids that are NOT adjacent to each other.

Polyclonal response

Polyclonal immune response is one where the animal produces different antibodies which are directed against a particular antigen. The response is directed against different epitopes of the antigen. When an animal is immunized with a foreign protein, the animal ALWAYS has a polyclonal response.

Monoclonal response

Artificial situation that is important for research and medicine.
These are a result of lab technique and the animals normally do NOT do this.
In a monoclonal response, a single antibody is produced and this single antibody is directed against a single epitope of the antigen.
This cell line makes a single type of antibody.

How to make monoclonal antibodies

Inject antigen into mouse and you get a polyclonal response.
You want to isolate B cells that make an immune response so you remove the spleen to isolate B cells.
In the spleen, you have plasma cells which are short lived and secret antibodies (IgG) extracellularly.
Myeloma cells are cancerous B-lymphocytes which are long-lived and don't produce antibodies.
Myeloma and plasma cells are fused to yield hybridomas that are long lived, producing and secreting antibodies.
Monoclonal cell lines make a single antibody directed against a single epitope.
Monoclonal cell lines and are used widely in research and medicine.

Antibody structure

consist of 2 heavy chains and 2 light chains
the 2 heavy chains are identical to each other
the 2 light chains are identical to each other
the immunoglobin tetramer has 2 light chains and 2 heavy chains with the following structure:
the variable domain is generated by association between the variable regions of the light and heavy chain. The variable domains are identical (they are on the arms of the Y shaped antigen and each arm has a copy of the V domain) to each other on a single antibody, and they recognize the antigen.
the first C (constant) domain is generated from the association of the constant region of the light chain with part of the constant region of the heavy chain. Disulfide bonds link the constant regions of the light and heavy chains. The second C domain is from the association between C regions of the heavy chains, and they are linked together by disulfide bonds.
the heavy chain constant region is called the effector and determines the class of the antibody

Figure 24.17 &endash; classes of antibodies made by animals

IgG is secreted into blood stream (20% of blood protein) and has a particular heavy chain constant region
IgM is not secreted extracellularly but instead appears on cell surfaces and has a different heavy chain constant region from IgG.

B cell lineage

derived from bone marrow cells
pre-B-cells are present in the bone marrow and are called stem cells
pre-B stem cells can differentiate into different cell types and they do not make antibodies
pre-B-cells stem cells are pluripotent, meaning they are somewhat limited in the cell types which they can become
totipotent stem cells can differentiate into anything and embryonic stem cells are totipotent
pre-B-cell proliferate and differentiate into many different cells that have different IgM molecules on their cell surfaces. If you expose B cells with IgM on their cell surface to the antigen, then IgM antibodies can bind to the antigen and stimulate that cell to proliferate and differentiate into:
- 1) Memory cells that persist for a long time
- 2) Plasma cells that are short lived and secrete antibodies (IgG). Secretion of IgG by these plasma cells is the major component of the primary immune response.

Specificity and diversity of the immune system

1965, several theories to explain how animals generate specific and diverse immune response

1) Instructive Theory

a limited # of antibodies are made
the antigen causes the antibody to fold into a certain structure allowing the antibody to bind to the antigen with high affinity
problem = how could cell remember the folded up structure over a long period of time (i.e. how is immune system memory explained?)

2) Clonal selection

a large # of different antibodies are produced in animals
in response to antigen, a small subset of antibodies were stimulated to proliferate
problem = where does information come from to allow for so many different antibodies to be encoded? The immune system can respond to 10⁷ antigens. If each gene has 10³ b.p. and 1 gene makes 1 antibody, then 10⁷ x 10³ = 10¹⁰b.p. needed for all the possible antigens. BUT, the human genome is only 3 x 10⁹ b.p.
Post-translational modification by glycosylation or phosphorylation could increase the number of different molecules produece from a given number of genes. As could alternative splicing (pre-mRNA splicing not discovered until 1977).

In actuality, a little of both instinctive theory and clonal selection mechanisms contribute to diversity and specificity of the immune response

mostly clonal selection is responsible
in the end, the genome does contain the capacity to code for large numbers of different antibodies.
but the antigen is instructive and does have an affect on specificity of antibody produced.

3 ways that diversity and specificity are generated

1. Somatic Recombination
2. Joining
3. Somatic Mutation

Somatic Recombination

DNA in B cells rearranges
DNA arrangement in B cells is different from the DNA arrangement in other cells of the body.
Site-specific recombination
NOT alternative splicing! Remember, DNA rearranges in B cells!
DNA of both heavy and light chains encode multiple variable region gene segments and so they are adjacent to each other in the chromosome.
They recombine in different combinations
DNA rearrangement occurs in both heavy and light chains and produces the variable antigen-binding regions.

2 light chain loci in man and mouse

Kappa light chain locus
l light chain locus

Kappa light chain rearrangement:

V1 V2 V3 V4Š..V45Š..V75 V76 J1 J2 J3 J4 J5 C

Above is shown the germ-line pre-DNA arrangement. A DNA recombination event brings 1 V and 1 J segment together. This is a B cell specific event. We will analyze what happens when V45 and J4 are recombined together.

The region between V45 and J4 is lost when V45 and J4 are put together, yielding:

V1 V2 V3 V4 Š..V45 J4 J5 C

The above is B cell DNA after rearrangement. After transcription, the following pre-mRNA is yielded:

V45 J4 J5 C

splicing occurs and introns removed yielding

V45 J4 C

Figure 24.6 depicts Kappa light chain DNA rearrangement

note that the mRNA has a leader, intron, J2 and J3
the purple J region is also variable

Figure 24.9 shows that there are 76 variable region gene segments, 5 J regions, and 1 C

Figure 24.8 - l light chain

Heavy chains have an additional segment

in light chains, V and J recombine
in heavy chains, V, D (diversity), and J recombine
you have 2 recombination events for heavy chains where the first recombination involves D and J recombining into DJ.
the second recombination brings V and DJ together
then transcription and splicing occurs.

see Figure 24.7

How much diversity?

Heavy chain
- 300 V, 20 D, 6 J
- 300 x 20 x 6 = 36,000 different combinations
Light chain (l)
- 300V x 6J-C = 1800
- 80V x 5J = 400
- 1800 + 400 = 2200
TOTAL different combinations = 8 x 10⁷

SUMMARY: Somatic Recombination

DNA rearrangement
yields approximately 10⁸ combinations.

Joining

diversity is introduced during site specific recombination
site specific recombination is where extra nucleotides are added in randomly at recombination joints
diversity is introduced in conserved sequences that flank the gene segments to be recombined

These are 2 different consensus sequences at the mouse Ig loci:

conserved heptamer (7bp) ---------- variable (12bp) --------------- conserved nonamer (9bp)

nonamer-----------23bp spacer-----------heptamer

Rule: consensus sequence with 1 type of spacing can be recombined only to a consensus sequence with the other kind of spacing.

see Figure 24.12

At the Kappa locus:

3' to ALL 76 Variable gene segments the consensus sequence heptamer-12bp-nonamer appears
5' to ALL J segments the consensus sequence nonamer-23bp-heptamer appears

At the l locus:

The V and J consensus sequences are inverted in orientation.
The different spacing in V segments vs. J segments serves to prevent one V gene segment from one combining with another, or one J segment from recombining with another. Thus, the rule about spacing above means that different V's and J's will directly combine.

In heavy chains, the mechanism for assembly is the same except there is a different arrangement of consensus sequences. 5' and 3' to each D you have recombination segments with 23bp spacers 5' to each J and 3' to each V, there are 12 bp spacers

Figure 24.14

V gene segment ends at the green arrow -- heptamer &endash; 23 bp &endash; nonamer
5' to J segment &endash; heptamer &endash; 23bp - nonamer
nicks are made at the junction only at one strand, where RAG1,2 nick only 1 phosphodiester bond
the cut is at the end of V and the beginning of J
hairpin forms via free 3'-OH attack phosphorus on other strand. Hairpin forms on both right and left sides
2nd cleavage is variable within a few nucleotides at the ends
cuts lead to variable overhangs that are relatively short
exonuclease activity causes synthesis of random nucleotides where nucleotides can be lost or added
ends are filled in with random nucleotides
ends ligated together with somewhat random nucleotides inserted during ligation

implications

1/3 of time get productive rearrangement
2/3 of the time, you ends up with a V and J out of frame which is bad and leads to a nonproductive antibody. BUT this is the price paid to increase diversity of the antibodies produces

SUMMARY: Joining

Light chain &endash; assume 2 amino acids randomized &endash; 20² = 400 different combinations
Heavy chain &endash; assume 2 amino acids randomized at each join so 400 x 400
6 x 10⁷ different additional combinations of light and heavy chains

Somatic Muations &endash; bizarre!

entails random mutagenesis of VH
this was first noticed when people started sequencing VH from B cells that underwent rearrangement because this DNA of VH sequence was different from any VH sequence in germline.
Rules:
- 1) variation &endash; mutagenesis confined to VH
- 2) 1-4% of amino acids appeared to be different in VH &endash; this was definitely pronounced
- 3) appeared to be random
  - 3rd base silent mutation had no effect
  - stop codons
- 4) somatic mutation appeared to occur after DNA rearrangement and took place after initial exposure to the antigen.
Idea: somatic mutations increase affinity of antibody for antigen. A small % of these mutations lead to increased affinity of antibody to antigen but MOST lead to a decreased affinity. Apparently, the immune system considers it worthwhile.
5 amino acids randomized, 20⁵ = 3.2 x 10⁶ different combinations

Figure 24.20

shows B cell lineage with steps of diversity
pre-B cells make no antibodies
DNA recombination takes place initially
IgM present on B cell surface
There are many B cells and the ability to produce different combinations exceeds the # of lymphocytes in body.

Total diversity

(somatic recombination) X (joining) X (somatic mutation)
(8 x 10⁷) X (6 x 10⁷) X (3.2 x 10⁶) = ~ 10²¹ !!!!
lymphocytes = 2 x 10¹²
Ability to generate diversity exceeds the number of cells in the immune system.

Back to B cell linage

most B cells circulate with membrane bound antigens
primary response involves isotype switching to IgG producing B cells (plasma cells) and somatic mutation leads to B memory cells that are proliferated and mutated (somatic mutation), presumably to produce antibody molecules that bind with higher affinity to the antigen.
secondary response is greater because there are more B cells that can recognize the antigen (because these B memory cells are pre-selected for the antigen and have proliferated in the primary response) and the antibodies have greater specificity for the antigen (because of somatic mutation).