BASIC IMMUNOLOGY

Natural Defenses against Disease

• Variola diffusion in
the troops
• Auto-vaccine
• Variola auto-
innoculation
• Cells and Molecules
of the immune
system
• Memory Cells
 Defense Systems
• Humans defend themselves
against pathogens by both
nonspecific (innate) and
specific means.
• Innate defense: skin, a
super efficient barrier against
pathogens
• Specific defense: proteins
(antibodies) able to
recognize, link and destroy a
pathogen
• Humans nonspecific defenses include physical barriers, competing resident
microorganisms, and local agents, such as secretions that contain an antibacterial enzyme.
• The inflammation response uses several cells and proteins. Activated mast cells release
histamine, which causes blood capillaries to leak and inflame. Complement proteins attract
macrophages to the site, where they engulf bacteria and dead cells
A cell signaling
pathway
involved the toll
receptor
stimulates the
defense response
Many of our
defenses are
implemented
by cells and
proteins
carried in the
bloodstream
and in the
lymphatic
system.
• White blood cells, including lymphocytes (B and T cells) and phagocytes
(such as neutrophils and macrophages), play many defensive roles
The Immune • Four features
Response characterize the immune
response: specificity,
the ability to respond to
an enormous diversity of
antigens, the ability to
distinguish self from
nonself, and memory.
• The immune response is
directed against
antigens that evade the
nonspecific defenses.
• Each antibody or T cell
is directed against a
particular antigenic
determinant.
• There are two interactive immune responses: the humoral
immune response and the cellular immune response.
• The humoral immune response employs antibodies
secreted by B cells to target antigens in body fluids.
• The cellular immune response employs T cells to attack
body cells that have been altered by viral infection or
mutation or to target antigens that have invaded the body's
cells.
Clonal selection
accounts for the
rapidity,
specificity,
and diversity of
the immune
response as well
as
immunological
memory and
tolerance to self.
• Immunological memory plays roles in
both natural immunity and artificial
immunity based on vaccination.
 The Humoral
Immune Response
• Activated B cells form
plasma cells, which
synthesize and secrete
specific antibodies.
• The basic unit of an
antibody, or
immunoglobulin, is a
tetramer of four
polypeptides: two identical
light chains and two
identical heavy chains,
each consisting of a
constant and a variable
region.
• The variable regions of the light and heavy chains collaborate to form the
antigen-binding sites of a specific antibody.
• Each antigen usually has several different antigenic determinants (binding
sites for specific antibodies).
• The variable regions determine each antibody's specificity for a
determinant; the constant region determines the destination and
function of the antibody.
• There are five immunoglobulin classes. IgM, formed first, is a
membrane receptor on B cells, as is IgD. IgG is the most abundant
antibody class and performs several defensive functions. IgE
takes part in inflammation and allergic reactions. IgA is present in
various body secretions.
Monoclonal
antibodies consist
of identical
immunoglobulin
molecules
directed against a
single antigenic
determinant.
The Cellular Immune Response
• Receptor of a T cell
• The cellular immune response is
directed against altered or
infected cells of the body.
• TC cells attack virus-infected or
tumor cells, causing them to lyse.
• TH cells activate B cells and
influence the development of
other T cells and macrophages.
• T cell receptors in the cellular
immune response are analogous
to immunoglobulins in the
humoral immune response.
• The major
histocompatibility
complex (MHC)
encodes many
membrane
proteins. MHC
molecules in
macrophages, B
cells, or body cells
bind processed
antigen and
present it to T
cells.
• In the cellular immune
response, class I MHC
molecules, TC cells, CD8,
and cytokines collaborate
to activate TC cells with the
appropriate specificity.
• Developing T cells
undergo two tests: They
must be able to recognize
self MHC molecules, and
they must not bind to
both self MHC and any of
the body's own antigens.
T cells that fail either of
these tests die.
• The rejection of organ
transplants results from
the genetic diversity of
MHC molecules.
The presence of high affinity PROTECTIVE HLA alleles
allow NEGATIVE SELECTION
The presence of low affinity HLA PREDISPOSING
ALLELES determines the lack of negative selection of auto-
reactive cells.
 CELLULAR
HUMORAL
RESPONSE
RESPONSE
The antibody, T cell receptor and MHC loci

• According to the germ line theory each antibody is encoded by an

inherited gene, it is not modified during somatic development and
thus a very high numbe of antibody genes must exist in the germ
line. According to the somatic theory there exist only a small
number of antibody genes but these become greatly diversified in
somatic cells through mutation and/or recombination.
• We now know the number of antibody gene
segments in humans, we know in
substantial detail how these segments are
somatically modified by recombination and
mutation and what is the contribution of the
gene pool and the somatic processes to the
specificity and affinity of the antibody
response.
• We also know that certain strategies apply
both to antibody and T cell receptor genes
(gene recombination), others apply to
antibody but not T cell receptor genes
(gene hypermutation) and that the
strategy underlying the ability of MHC
proteins to interact with different antigens is
gene polymorphism. Thus the genetics of
the immune system has been largely
unravelled in the last few years.
 Evidence for somatic recombination (so-called
rearrangement) of antibody and TCR genes
• The hypothesis that antibody genes resulted from the
"fusion" of two different genes (a V gene and a C
gene) was put forward in 1965 by W Dreyer and J
Bennett. It provided an explanation for the protein
sequence data that was accumulating at the time and
for serological and genetic observations indicating that
the same idiotype could be found in association with
different isotypes.
• The "two genes - one polypeptide" hypothesis was
proven correct in the mid seventies by N Hozumi who
demonstrated that probes for the C or a V+C light
chain mRNA hybridized with different restriction
fragments of embryo genomic DNA but hybridized
with the same restriction fragment in the case of
DNA from the myeloma line. This experiment
confirmed that a process of somatic rearrangement
had occured in the antibody-producing cell.
 Somatic hypermutation
• Experimental evidence supports the view that the mechanism of SHM involves
deamination of cytosine to uracil in DNA by an enzyme called Activation-
Induced (Cytidine) Deaminase, or AID. A cytosine:guanine pair is thus directly
mutated to a uracil:guanine mismatch. Uracil residues are not normally found in
DNA, therefore, to maintain the integrity of the genome, most of these mutations
must be repaired by high-fidelity Base excision repair enzymes. The uracil bases
are removed by the repair enzyme, uracil-DNA glycosylase.Error-prone DNA
polymerases are then recruited to fill in the gap and create mutations.
 Somatic hypermutation
• The synthesis of this new DNA involves error-
prone DNA polymerases, which often
introduce mutations at the position of the
deaminated cytosine itself or neighboring base
pairs.
• During B cell division the immunoglobulin
variable region DNA is transcribed and
translated. The introduction of mutations in the
rapidly proliferating population of B cells
ultimately culminates in the production of
thousands of B cells, possessing slightly
different receptors and varying specificity for
the antigen, from which the B cell with highest
affinities for the antigen can be selected.
• The B cells with the greatest affinity will then
be selected to differentiate into plasma cells
producing antibody and long-lived memory B
cells contributing to enhanced immune
responses upon reinfection.
The location of the antibody, T cell receptor and MHC
loci in humans is shown in the Table below.
The antibody heavy chain, k light chain
and l light chain loci

• The human VH locus is located at the

telomeric end (i.e. within few kb of the
telomere) of the long arm of chromosome14
(14q32-33). The position has been confirmed
by a variety of cytogenetic and hybridization
techniques. The locus comprises 123-129 V
segments, the variation being due to
insertional polymorphism. The locus spans >
1Mb.
• The human Vk locus is located near the centromere on
the short arm of chromosome 2 (2p12). It comprises 76
Vk segments, 5 Jk segments and a single Ck segment.
The locus spans ~ 1.8 Mb and a large portion (440 kb)
is duplicated.
• The duplication is a recent event as it is >96%
identical at the sequence level. 40 genes reside in
the proximal half, 36 reside in the distal (duplicated)
half. 34 genes are functional, 16 have minor defects (one
or two nucleotide changes) and 25 are clearly
pseudogenes.
• Six duplicate genes yield the same transcription product
and two other yield the same translation product.
• The transcriptional orientation differ among different
groups of genes. Approximately half the genes rearrange
by a deletion mechanism. The other half by a
mechanism involving inversions of Mb size DNA.
 The T cell receptor (TCR) a/d, b and g loci
• Although there are four types of T cell receptor chains (a, b, g and d), there are
only three loci because the d locus is interdispersed in the a locus. The human a-d
locus is complex and includes 54 Va gene segments of which 45 are functional. The
locus also contains a remarkable number (61) of Ja segments, the majority of which is
functional. The human b locus was the first TCR locus to be fully mapped and
sequenced. It spans ~0.6 Mb and contains 62-65 V genes of which 39-41 are
functional. The human g locus instead is much simpler: it spans ~ 0.1 Mb and contains
12-15 V genes of which 4 -6 are functional.
 The MHC locus

• The human MHC locus spans approximately 4 Mb on the short arm

of chromosome 6 (6p21). The locus comprises three regions: a
group of around 20 class I genes (telomeric), a group of around 15
class II genes (centromeric) and a heterogeneous group of around
30 class III genes located between the class I and class II clusters.
• MHC plays an essential role in the immune answer: allows T
Lymphocytes to recognize the antigen.
• MCH works togheter with TCR and influences T antigen repertoire
able to recognize the antigen.
• This is reason why MHC complex plays a fundamemntal role in the
susceptibility to several complex diseases and autoimmunity.
 MHC I structure
• Expressed on cell surfaces
• Recognized by TCR of T CD8
(cytotoxic) lymphocytes
• CD8 links the comples peptide-
MHC I
• MHC I are requested for
endogenous antigen recognition
(citosolic way).
• Constituted by two chains: the
alpha encoded by MHC genes and
the microglobulin beta, encoded by
a gene external to the cluster MHC
• The alpha chain has 3 external
domains, 1 transmembrane domain
that, togheter with alfa 3 link the
molecule to the membrane and a
cytoplasmatic tail.
 MHC II structure
• MHC II are heterodimer molecules
costituted by two alpha eand two beta
chains, both encoded by genes of the
MHC cluster
• Both chains present two extra-cellular
domains, the transmembrane domain
and the citoplasmatic tail.
 MHC III structure
• The proteins encoded by MHC III are not membrane receptors and
are not involved in the antigen presentation
• It has been hypothesized that alterations of MHC III proteins are
involved in autoimmune diseases.

• MCH III proteins have different function and sequences: some of

them are complement proteins, others are enzymes (i.e. 21-
hydrolasis), inflammatory cytockines, tumor necrosis factors and
heat shock proteins.
 MHC LOCUS MAIN FEATURES

• MULTIGENIC: several genes encodes for different class I and II

molecules, each of them with different specificity for the various
peptides
• ALLELIC POLYMORPHISM: a great number of alleles are present
for each gene
• CODOMINANT: products of both alleles of each gene are
produced
• There are also three
polymorphic products of class
II genes (HLA-DR, HLA-DQ
and HLA-DP) and several class
II pseudogenes (for example
the DPA2 and DPB2 genes).
• The molecular basis of class I and class II gene polymorphism is
at the level of amino acid substitutions and most alleles are
present at significant frequencies in the populations. This argues
against neutral mutation and suggests overdominant selection
(heterozygote advantage) or frequency-dependent selection.
• The suggestion made by H Zinkernagel and P Doherty that the
polymorphism is maintained by viruses is a plausible one.
Recombination within locus occurs at preferred sites.
• This leads to linkage disequilibrium (i.e. the occurrence of alleles
of two genes together at frequencies higher than expected from
the frequencies of the single alleles; the loci are then said to be in
linkage disequilibrium).
The Genetic Basis of Antibody Diversity

• Immunoglobulin heavy-chain supergenes are

constructed from one each of numerous V, D, J, and C
segments. The V, D, and J segments combine by DNA
rearrangement, and transcription yields an RNA
molecule that is spliced to form a translatable mRNA.
Other gene families give rise to the light chains.
• As a result of these DNA rearrangements, there are millions of possible
antibodies as a result of these DNA combinations. Imprecise DNA
rearrangements, mutations, and random addition of bases to the ends of the
DNAs before they are joined contribute even more diversity.
• Class switching
after initial
immunoglobulin
production results
in antibodies with
the same antigen
specificity but a
different function.
It is accomplished
by cutting and
rejoining of the
genes encoding
the constant
region.
Disorders of the Immune System
• Allergies result from an
overreaction of the
immune system to an
antigen
• Autoimmune diseases
result from a failure in
the immune recognition
of self, with the
appearance of antiself B
and T cells that attack
the body's own cells.
• Immune deficiency disorders result from failures of one or
another part of the immune system. AIDS is an immune deficiency
disorder arising from depletion of the body's TH cells as a result
of infection with HIV. Depletion of the TH cells weakens and
eventually destroys the immune system, leaving the host
defenseless against "opportunistic" infections.
• HIV inserts a copy of its genome into a chromosome of a
macrophage or TH cell, where it may lie dormant for years. When
the viral genome is transcribed and translated, new viruses form.
• Currently the most effective drugs to treat HIV are those directed
against reverse transcriptase and protease. ･ Some treatments may
provide a dramatic reduction in HIV levels, but there is as yet no
indication that we can prevent infection with HIV, as by
vaccination. The only strategy currently available is for people to
avoid behaviors that place them at risk.
 Overview of Celiac Disease
• Celiac disease is a hereditary
intolerance to gluten, a protein found
in wheats and, to a lesser extent in
barley, rye, and oats. While gluten
intolerance is permanent, symptoms
can be alleviated by avoiding all
gluten from one’s diet. Inflammation
occurs when gliadin, a peptide
derivative of gluten, found in gluten-
containing foods is ingested and
presented to T cells. Inflammation
causes damage to mucosal tissue of
the small intestine, especially the villi
that absorb nutrients, which results in
malabsoption of food.
• Celiac disease affects as many as 1 in 300
people in Italy and southwestern Ireland, but is
extremely rare in Africa, Japan, and China
( Not et al., 1988). According to a multicenter
study in 2003, there is a 1 in 133 chance that
people with no risk factors or family history in
the U.S. have celiac disease. Additionally, a
person ﾕ s risk increases to a 1 in 22 chance if
they have a first-degree relative with celiac
disease and a 1 in 39 chance if they have a
second-degree relative (Fasano, 2002).
Around 60,000 Americans are diagnosed with
celiac disease annually and a total of over 2
million have the disease, making it perhaps
the most common genetic disorder in the
United States (Westerberg, et al., 2006).
Celiac disease can occur at any age, and
females are more commonly affected than
males. Of females presenting during their
fertile years, the male to female ratio is almost
3 to 1 (Feighery et al., 1998).
 Genetic Risk Factors
• Celiac disease is strongly
associated with the human
leukocyte antigen (HLA) DQ2 and
DQ8 haplotypes. HLA genes are
part of the major histocompatibility
complex (MHC).
• The function of MHC molecules is
to bind peptide fragments derived
from pathogens and display them on
the cell surface for recognition by T
cells. Many proteins involved in
antigen processing and presentation
are encoded by genes within the
MHC (Janeway, 2005).
• Symptoms of celiac disease are
caused by a glutamine and proline
rich 33-mer peptide found in
gluten that initiates the
inflammatory response when
bound to these HLA haplotypes.
• The primary HLA association in
most patients with celiac disease
is with DQ2 (DQA*05/DQB1*02)
and in a minority of patients with
DQ8 (DQA1*03/DQB1*0302).
Approximately 97% of individuals
with celiac disease have the HLA-
D2Q or HLA-DQ8, compared to
40% of the general population
(NIH, 2006).
• The HLA association in celiac
disease can be explained by a
superior ability of DQ2 to bind the
repertoire of proline-rich gluten
peptides that have survived
gastrointestinal digestion and that
have been deaminated by tissue
transglutaminase
• Celiac disease is diagnosed in about 10% of first degree relatives of an
individual with celiac disease (Logan, 1992). Although hereditary factors
play a significant role, genetic factors alone do not explain the
development of the disease because the disease is concordant in only
60% to 70% of identical twins. All people with HLA DQ2 and DQ8 do not
develop into disease, and others without those alleles can develop celiac
disease, so more genes may be involved. Additional factors such as
hormones and infectious agents may also be involved in linking the
ingestion of gluten with a chronic inflammatory reaction in the intestine in
genetically predisposed individuals (Fasano,1996).