Evolution and the

Immune System
Sean D. Pitman M.D.

Everyone has experienced

sickness at one time or another in

his or her life. However, most

people get better and often do not

get that same “bug” again. Why is

this? There are literally millions of

"bugs" and other things in our

everyday environment that can make us sick. Why then do we generally remain

so healthy? The reason is because most of us have a highly effective immune

system. A healthy immune system is very good at searching out and killing

foreign invaders that can make the body sick. However, if the immune system is

not functioning well, there will certainly be a lot of problems.

As an example of this, consider the famous HIV virus (Human

Immunodeficiency Virus). This virus causes the disease known as AIDS

(Acquired Immune Deficiency Syndrome). A person with AIDS has a very

weakened immune system. Because of a lack of immunity, infections plague the

AIDS victim that do not normally infect humans. Interestingly enough, it is not the

HIV virus itself that makes these people sick. The HIV virus

specifically attacks immune cells called T-helper cells (click on the

video illustration - i.e., the black box). When these cells are lost in

high enough numbers, the immune system starts to shut down. The symptoms

of AIDS are the result of a loss of immunity. With the loss of immunity, a host of

infectious processes start to overwhelm the body. Eventually, these infections kill

their unfortunate host.

Now that we understand just how important it is to recognize bad "bugs", how

do our bodies become capable of recognizing the millions and billions and even

trillions of different arrangements of things that can make us sick?

Well, it is through a Darwinian-style selection process where the fittest survive

and the weakest die. In our bodies we have cells that are specialized immune

cells called T-cells. They go to "school" to learn the difference between "self"

and "non-self". Certainly one would not want his/her own immune system to

attack his/her own body! Sometimes this does happen and it is referred to as an

autoimmune disease. However, normally, the T-cells are educated in a very

tough school so that they do not attack one's own body or "self". But how exactly

are they trained to recognize the difference between self and non-self?
 Well, T-cells are

capable of being able to

tell the difference

between certain

molecules (antigens) that

are presented (by MHC

class I molecules) on the

surfaces of all "self" cells

in the body that they are

supposed to protect

compared to all foreign or

"abnormal" antigens that

are associated with outside invaders or internal disease processes (like cancer).

For example, when an outside invader, like a bacterium, enters the body, other

cells (macrophages) may catch this invader and "eat" it. The macrophage then

presents small parts of the invader on its own surface in association with a

certain molecule called MHC class II. If a cell within the body gets "sick" or

diseased (i.e., cancerous), certain molecules that are usually hidden from the

immune system may be produced. These are now presented on the surface of

this sick cell in association with MHC class I molecules. T-cells recognize these

new molecules as "non-self" and kill the sick or diseased cell. Once the T-

lymphocyte recognizes an infected cell, it produces a set of new proteins that it

places on the surface of the sick or diseased cell. Those proteins then bind to
receptors on the infected cell called "death domain receptors" (including the Fas

ligand and Trail receptors). This binding triggers a cascade of events in the

infected cell that leads to cell suicide, called apoptosis.

T-cells sense or "feel" these antigen molecules with their own fairly tall Y-

shaped molecules called T-cell receptors (like little hands on the ends of long

arms). These T-cell receptors (TCRs) are analogous to wider Y-shaped antibody

molecules (also known as "immunoglobulins") produce by B-cells (see

illustrations below). However, TCRs exist only in a membrane-bound form. They

are able to carry out their particular function without the need to leave the cell

surface. Their receptors are used for the detection of foreign antigens, and do

not directly mediate an effector response (except in the case of specialized

"cytotoxic" T-cells).4

Those T-cells that do not recognize self-antigens as being part of the body

are killed off before they get out of school (and this is a rather large majority of

the "students" that enter this school). It is a tough school indeed if the students

do not learn their lessons! Those cells that do recognize self-antigens as "self"

graduate to go and search the body for non-self antigens to attack and eliminate.

Each educated T-cell has only one type of receptor so only one specific

antigen can be recognized. But, how many possible antigens are there? "The

total number of possible epitopes is, therefore, 20B since there are 20 different

amino acids."5 Well, the typical length of an antigen epitope ("B" in the preceding

formula) is about 20 amino acid residues.5 So, the total number of possible
antigen epitopes is about 2020 or 104,857,600,000,000,000,000,000,000 or ~100

trillion trillion.

Since there are trillions of different possible antigen epitopes, how does one's

immune system cope with such a variety of potential enemies? Well, there are

many immune cells produced by the body. In humans, in particular, about 1012

lymphocytes are present at any given time.6

Not all

the T-

cells

have

different Y-shaped receptors, but many of them do. Chances are that if enough

non-self enemies get into the body at least one of the immune cells will recognize

the non-self marker sequences or "antigens" located on this invader as "foreign"

to at least some useful degree. The odds that a single T-cell will recognize a

random epitope to at least some useful degree is about 1 in 1012. So, does this

mean it would take a trillion different T-cells to cover all possible invaders? Well,

no. The reason is because an average cell or foreign invader "bug" has about

1012 different epitopes. So, on average, a single T-cell will recognize at least one

of the potential antigen epitopes of a foreign invader.5

When this happens this particular T-cell sounds the alarm that the body has

been invaded. Other immune cells, called B-cells, are also activated, but only
 those that

specifically produce

antibodies that have

a pretty good match

to the foreign

antigen epitope

expressed by the

invading organism. The invader, with its non-self antigens, is attacked.

However, if only a few immune cells recognize the invader upon initial exposure,

the initial attack might be rather weak. The resulting sickness may linger on for

some time before the body can kill off the offending invader. The good thing is

that the immune system remembers this particular invader for the future so it can

kill the invader more quickly if it ever

sees its particular antigen marker again. But how does this memory work?

The B-cell that recognized the foreign antigen clones itself to make many

nearly identical copies of itself - with slight variations. Now, there are many B-

cells that will recognize this particular foreign antigen. If infected again by an

invader with this particular antigen, the immune system is ready and produces

many more specific antibodies than before. This kills the invader much more

quickly - making the body "immune" to this particular bug.

This is how vaccines work. A vaccine presents the body with the antigens of

either a dead or a weakened bug. In this way the body can prepare to kill that
particular bug without first having to go through the sickness that the bug may

causes.

Programmed Variability

Many people, including scientists, claim that this process is evolution in

action.3,4,5 Is this actually true? After all, this system does use survival of the

fittest and a function-based selection process to create an incredible diversity of

immune cells. Is this not evolution in action? In thinking about this question,

remember that the immune system had no initial knowledge about all the evil

antigens in the world or just which ones it might have to combat. So, how did it

get its gigantic arsenal of options?

Well, the initial production of variety of T-cell receptors and B-cell produced

antibodies is done by purely random mutation without any function-based

selection. However, subsequent refinements of immune system function are

indeed based on both random mutations and function-based selection over

several generations of B-cells.

This may be a form of evolution in action, but it isn't quite what many

evolutionists think it is. Lets look into this process in a bit more detail.

Antibody Structure
 Antibodies are proteins and so they are coded for by DNA. Antibodies are Y-

shaped molecules with two different protein strands called heavy and light

chains. At the tips of the V-ends of the Y there are “variable regions” on both the

light and heavy chains that can be different from cell to cell. Also, within this

variable region are half a dozen or so "hypervariable regions". The rest of the

antibody does not vary in its protein sequencing from other antibodies within the

body. Each one of the two chains (heavy and light) are coded for by specific

sections of DNA. Each section of DNA can code for part of the final antibody for

that cell if it becomes an immune cell. Even though there are many options for

each section only one option will be chosen for a particular section. This choice

is determined by the random recombination of one option for one section with

one option for each one of the other sections (see illustration).
 For light chains there are about 250 V-segments (or separate options in the

original DNA), four J-segments, and three different ways that a V-segment can

join to a J-segment when the DNA is spliced. The final product for a light chain in

DNA is one V-segment followed by a single J-segment. This makes up the

“variable region” of the light chain. The constant region of DNA for the light chain

follows this region. The rest of the gene options are discarded for that cell.

For the heavy chains, there are about 250 V-segments, 15 D-segments, and

5 J-segments - followed by the constant region. Just like in the light chain DNA,

only one option from each segment type is chosen for the final splicing of DNA so

that just one V-option is followed by one D-option which is followed by one J-

option. This makes up the “variable region” of the heavy chain. The constant

region follows just like in the light chain.

 The large number of different options and their different possible

combinations give rise to the huge variety of antibody possibilities. This number

can be roughly calculated as follows: Light chains have 250 V-segments, 4 J-

segments, and 3 possible joining frames. This gives a total of 250 x 4 x 3 or

3000 different kinds of complete light chain possibilities. Heavy chains have 250

V-segments, 15 D-segments, 5 J-segments, and 3 different joining frames. This

gives a total of 250 x 15 x 5 x 3 or 56,250 different kinds of complete heavy chain

possibilities. Combining the chains gives around 1.7 x 108 (~170 million) different

possible antibody arrangements. Adding in the various hypervariable differences

within the variable regions gives around 1 x 1010 (~10 billion) different antibody

specificities.1,2

Discussion

Certainly, the potential for immune cell variety is great, but is the process that

produces this variety really an example of the Darwinian-style evolution at work?

3
According to the theory of evolution all living things evolved from a single

common ancestor over time. The differences between the resulting life forms

arose when random mutations happened upon some sort of new beneficial

function that was beneficial to a particular life form in a particular environment or

situation. More and more of these differences added up over time until it resulted
in the vast diversity of living things that we see today. That's the theory of

evolution in a nut shell. That's how it is supposed to have worked.

Now, in thinking about the immune system, is there a parallel with the theory

of evolution? What is evolving in the immune system? The overall structure and

function of the immune cells does not evolve over time and neither do the types

of antibodies that are produced. All the major types of immune cells and

antibodies are produced before any functional selection takes place. Only after

the T-cells are formed do they go to school to be "selected". Also, this selection

process only selects for a very limited ability - the ability to recognize and refrain

from attacking self antigens. This particular selection process is very specific

and it does not change over time to produce anything new. So, the major

differences in antibody specificity that are maintained were all made before any

selection process took place. As far as the immune system is concerned, the

initial action of "selection" and survival of the fittest only narrows the field. It

reduces the antibody diversity that was initially created before selection came

along.

This is different from Darwinian-style evolution where natural selection is

supposed to be able to create diversity. Selection, in the case of the immune

system, does not expand or create more diversity - at least not in the initial steps

of immune system education. Perhaps, then, later steps in the function of the

immune system show Darwinian-style evolution of truly novel functions?

 Real Evolution

As a review, remember that only a small fraction of the T-cells survive the

first selection step. When the body is exposed to a foreign invader with a specific

antigenic marker sequence, T-cells that match that marker stimulate B-cells that

also match to reproduce themselves - in a clonal fashion. If just any immune cell

was allowed to clone or reproduce itself, the immune system would not be nearly

as effective. However, mass reproduction of nearly identical copies of a B-cell

that did in fact recognize a specific type of foreign invader, is very helpful in

preventing future sickness by that particular invader should it ever invade the

body again.

Interestingly though, once a particular B-cell is selected for mass replication,

the offspring of that parent cell are not exactly the same. The antibodies that are

produced by the offspring B-cells are slightly different - usually in their

"hypervariable regions". These changes are indeed random changes that were

not present in the original pool of immune system options. In other words, they

are truly new sequences. When the same foreign antigen is encountered again,

those slightly different clones of the original B-cell that recognize the antigen

better will be preferentially selected, over the siblings that do not have as close a

match, to be cloned and produce the offspring of the next generation. In this

manner the specificity of immunity will indeed evolve in a stepwise truly

Darwinian-style fashion of improvement over time.

 "The B-cells expressing low affinity antibody on their surface become

progressively less able to bind and be stimulated by antigen; in the environment

of the germinal center, these poorly stimulated B cells are programmed to die by

a specific process known as "apoptosis" (Choe et al, J Immunol 157:1006,1996).

In contrast, the cells with high affinity antibody continue to bind antigen, and thus

continue be stimulated to proliferate and secrete antibody. As the antigen

concentration progressively falls while mutation and selection continue, the

intensity of the selective pressure for high affinity increases. Repeated cycles of

mutation and selection can lead to affinity levels 100-fold higher than that of the

original unmutated antibody. The 'competition' for efficient antigen binding has

been shown to be the selective force driving the rise in antibody affinity, since if

antigen is repeatedly administered to prevent the drop in antigen level and

thereby eliminate the selective pressure for efficient antigen binding, antibody

affinity does not rise (Eisen and Siskind, Biochemistry 3:996, 1964). Furthermore,

when selection pressure has been experimentally removed by engineering mice

with impaired capacity for programmed death by apoptosis, many B cells are

found that make mutated antibodies with low affinity (Takahashi et al. J Exp Med.

190:39, 1999).

Late in the course of an immune response, as antigen becomes completely

cleared from the bloodstream the amount of antibody secreted gradually falls and

the immune response ends; but a subset of the last group of highly efficient cells

persists as a quiescent population known as 'memory cells,' ready to respond

with rapid secretion of high affinity antibody should they ever be triggered by

another encounter with the same antigen in the future." 3

So, basically what we have here is random mutation combined with function-

based selection that makes improvements over time. By definition, this sounds

an awful lot like real evolution in action, but there is just one little catch.

One Little Catch

Imagine a sequence space of all possible antibody specificities that an

immune system can make. Such a space would contain literally billions upon

billions, and probably even trillions of potential antibody specificities - - right?

Now, from the perspective of a given antigen (actually an epitope or a small

portion of an antigen), there would be a continuum of reactivity if it were brought

in contact with each one of the potential antibodies in sequence space. The

continuum would range from an extremely poor fit to an extremely good fit.

Imagining this continuum as a line where, on every point of that line, there may

be a number of antibodies that would match our particular antigen to exactly the

same degree of selectability (i.e., they would activate the immune response to

the same selectable degree). Although there may be a few equivalent antibody

options at every point on our continuum, the odds are that the majority of

potential antibodies are fairly evenly spread out over the entire continuum.
 Given such a continuum as a true picture of reality, there are basically no

significant neutral gaps (with regard to the function of immunity) to cross in the

evolution of antibody specificity of a given antigen. In other words, the odds are

therefore very high that a give change/recombination/mutation to an antibody

sequence will actually be functionally different (either more or less binding affinity

for a specific antigen epitope) compared to what came before. Since a high ratio

of potential changes would be functionally different, evolution, even of highly

specific sequences, will occur rapidly. This explains the relatively rapid

refinement of antibody specificity when immune cells are repeatedly exposed to

the same antigen.

Another way to look at it is to think of the foreign antigen as a pre-formed

pattern, model, goal sequence, or type of template. The goal of creating random

antibody sequences is to change sequences that already match the antigen

template to see if random changes can keep what already matches and yet come

up with additional matches to create an even better fit. This system of evolution

works because the antigen is limited to a certain number of potential characters

at each position of its sequence, and the random antibody sequence generator is

programmed to cover all of these possibilities at each possible sequence

position.

Methinks it is Like a Weasel

 For example, say that the antigen target or "goal" reads like Dawkins's

famous, "Methinks it is like a weasel" illustration. Starting with a bunch of random

phrases (antibodies) and mutating them at random sequence positions in each

"generation" of phrases, all that has to be done is to find out which one matches

the goal phrase, "Methinks it is like a weasel" at the most positions and then use

that "best" phrase to populate the next generation. Obviously, if the goal is

already in place ahead of time, it is easy to evolve other phrases to match the

goal phrase in short order using random changes and sequence-based

comparison - as Dawkins's illustration clearly establishes.

Exactly the same thing happens with antibody evolution. It is already pre-

established that certain molecules will be attracted to each other. If more of these

molecules are lined up in the right places, there will be a stronger attraction. If the

immune system is programmed to recognize stronger binding as "beneficial",

stronger and stronger binding will quickly evolve with the random changes of the

sequences of each subsequent generation.

The Non-Beneficial Gap Problem

Of course, the problem for evolutionists is clearly illustrated by Behe and

many others in their devastating critique of Dawkins's "Methinks it is like a

weasel" scenario. If all potentially beneficial types of functions could be built by

matching sequences to other pre-established sequences, then Darwinian-style

evolution of all types of functional systems would be a piece of cake. Of course,

for most functions found in operation within living things, evolution is not thought

to have worked like this at all. There simply is no pre-established sequence or

"ideal" with which to compare the evolving sequence. For many types of complex

functions, like bacterial motility for example, there simply is no pathway of step-

by-step improvements for each and every single amino acid change/mutation.

The forces of Darwinian-style evolution run into the Non-Beneficial Gap Problem

when it comes to such higher-level functions.

Consider the function of bacterial motility more closely for comparison.

Bacterial motility will not be realized at all until a minimum number and specified

arrangement of amino acids are entirely in place. Before such assembly is

realized, there is no motility and there is no pre-established model sequence

available to guide the random changes via function-based selection to produce

such an integrated system one single step or single character change at a time.

Without such a model sequence acting as a function-based guide directing each

change toward continual improvement, the system of function-based selection

gets blinded pretty quickly. The exponentially expanding gaps of neutral and

detrimental sequences quickly separate the fewer and fewer potentially beneficial

sequences at higher and higher levels of functional/meaningful complexity.

It is the resulting random walk created by the huge ocean of functionally

neutral and detrimental sequences in sequence space at higher levels of

functional complexity that destroys the evolutionary mechanism of random

mutation and natural selection. The random walk is made possible by the
blindness of nature to functionally neutral sequence changes/mutations as well

as nature's aversion to functionally detrimental changes. Because of this

blindness to neutral differences and aversion to detrimental differences in

different sequences, a seemingly small neutral/detrimental gap between

potentially beneficial functions translates into a correspondingly enormous

random walk, which grows exponentially in length (by a factor of 2) with each

doubling of the average neutral gap. So, beyond the lowest levels of functional

complexity evolution simply stalls out this side of a practical eternity of time (i.e.,

trillions upon trillions upon "zillions" of years of time).

Low Level Evolution

So, we see that even though the immune system does undergo random

mutation with function-based selection and survival of the fittest with improved

fitness over time, this is not a very close parallel to Darwinian-style evolution

when it comes to explaining the evolution of many systems of function that exist

within all living things. Most of the great varieties within “kinds” in the animal and

plant kingdoms can easily be explained by “preprogrammed” or inherent genetic

abilities for variety (such as the phenomenon of genetic recombination as first

described by Gregor Mendel). There are certain limited situations were new

functions can and do evolve without pre-established templates, such as in the

cases of lactase and nylonase evolution as well as antibiotic resistance in

bacteria, but these examples of evolution in action have clear boundaries that

have never been crossed. However, even with their limitations, the evolution of

antibiotic resistance, lactase function, computer codes, and the like, are far better

examples of evolution creating novel sequences than is the process of directed

immune system variability which does nothing more "evolutionary" than evolve a

string of characters to match another pre-established string of characters. Such

template-matching evolution just doesn't solve the problem for the larger Theory

of Evolution.

1. Stryer, Lubert. Biochemistry, 3rd ed., 1988.

B-cell development and tumorigenesis
FROM THE FOLLOWING ARTICLE:
Multiple myeloma: evolving genetic events and host interactions

W. Michael Kuehl & P. Leif Bergsagel

Nature Reviews Cancer 2, 175-187 (March 2002)

doi:10.1038/nrc746

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Three B-cell-specific DNA remodelling mechanisms that modify immunoglobulin (Ig) genes are

involved at different stages of B-cell development135: VDJ recombination, somatic hypermutation

and Ig heavy chain (IgH) switch recombination. Sequential and regulated VDJ recombination

assembles combinations of the many Ig V, D and J segments in precursor bone-marrow B cells to

create Ig heavy chain (H)/Ig light chain (L) antigen receptors. A D segment combines with a J

segment and then a V segment combines with this DJ segment to form a unique IgH VDJ sequence

in pro-B cells; then, V and J segments combine to form a unique IgL VJ sequence in pre-B cells;

additional joining of V and J segments occurs as part of receptor editing in self-reactive immature B

cells. Immature B cells that express functional surface IgM exit the bone marrow and home to

secondary lymphoid tissues as mature B cells.

Productive interaction of mature B cells with antigen results in proliferation and differentiation. The

primary immune response generates pre-germinal-centre plasma cells that typically are short lived,

and usually secrete IgM but can secrete other Ig isotypes as a result of IgH switch recombination116,

. Germinal centres are generated during the primary immune response. Antigen-activated
117, 136

lymphoblasts that enter a germinal centre are subjected to multiple rounds of somatic

hypermutation of IgH and IgL V(D)J sequences and antigen selection. Cells that express high-

affinity antigen receptors are selected for survival, with subsequent differentiation to memory B

cells or post-germinal-centre plasma cells. Post-germinal-centre plasmablasts/plasma cells

(including those generated from memory B cells) that undergo IgH switch recombination typically
home to the bone marrow, where they reside as terminally differentiated, non-proliferating, long-

lived plasma cells for >30 days but sometimes years.

Nearly 80% of B-cell tumours arise from germinal-centre or post-germinal-centre B cells120,

indicating that the intrinsic genetic instability of B cells that is unleashed in the germinal centre is

important for their development. Although somatic hypermutation can cause mutations in some

non-Ig genes137, 138, it is unclear if the germinal-centre environment is responsible for any

oncogenetic changes other than primary Ig translocations. Multiple myeloma cells are the

transformed counterparts of post-germinal-centre bone-marrow plasmablasts/plasma cells

(reviewed in Refs 66,139).

Pathways regulating chondrocyte activation and cartilage

degradation in rheumatoid arthritis.
FROM THE FOLLOWING ARTICLE:
Cytokines in the pathogenesis of rheumatoid arthritis

Iain B. McInnes & Georg Schett

Nature Reviews Immunology 7, 429-442 (June 2007)

doi:10.1038/nri2094

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Cartilage degradation is a multistep process based on the release of matrix-degrading enzymes

such as aggrecanases (ADAMTS) and matrix metalloproteinases (MMPs). Cytokines such as

interleukin-1 (IL-1) and IL-17 induce a switch in the synthesis pattern of chondrocytes from matrix

molecules to matrix-degrading enzymes. In addition, synovial fibroblasts start producing matrix-

degrading enzymes and invade cartilage when activated by cytokines such as tumour-necrosis

factor (TNF) and IL-1. Chondrocyte death is another feature of cartilage damage, it leads to the

formation of empty lacunae and deprives cartilage from the ability to replenish matrix.
The production of inflammatory cytokines is a tightly
controlled process that involves both transcriptional and
posttranscriptional mechanisms. In stimulated cells,
maximal induction of cytokine expression requires
activation of the p38 MAPK/ MK2 stress kinase pathway.
Our group is focused on understanding how the p38/MK2
cell signaling pathway regulates inflammatory cytokine
expression and bone loss. Specifically we interested in
p38 MAPK signaling which regulates many cytokine
genes at the post transcriptional level. In one funded
study, we are identifying cis-elements of the IL-6 mRNA
which regulate IL-6 mRNA stability and how RNA-binding
proteins regulate IL-6 mRNA expression.

Inactivation of MAPKs is achieved mainly by a family of dual-specific MAPK phosphatases (MKPs) which target the
regulatory sites of these kinases. MKP-1 is the prototype of this class of phosphatases capable of negatively
regulating MAPK activity to attenuate the innate immune inflammatory cytokine response. Recent evidence from
acute inflammatory models suggests that MKP-1 contributes towards LPS tolerance and over-expression of MKP-1
accelerates p38 inactivation resulting in diminished proinflammatory cytokine production. In a related study, the
role of p38/MKP-1 signaling in chronic inflammation (i.e. periodontal diseases) has been initiated recently.

We have recently established small animal models of periodontal disease where periodontal pathogenic LPS can
induce significant bone loss. Using this model, we have evaluated the potential of p38 MAPK small molecule
inhibitors to prevent or attenuate LPS-induced periodontal bone loss.

Other areas of recent focus have recently been established in the field of oral cancer. Along with other
investigators, models of oral cancer in nude mice have been established to help us understand the role of tumor-
derived cytokine and matrix metalloproteinase expression which may be important for oral cancer local invasion
into bone.

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