Immunology

Study
St d off a host’s
h t’ reactions
ti when
h foreign
f i substances
bt
are introduced into the body. A foreign substance
that induces such an immune response is called an
antigen
antigen.
g

Immunity (resistance) is the sum of all

naturally occurring defense mechanisms that protect
h
humans f
from i f i
infectious di
disease
1500s- Chinese developed a practice of inhaling powder made
1500s
from smallpox scabs in order to produce protection against
this dreaded disease. This practice of deliberately exposing an
individual to material from smallpox lesions was known as
variolation.
1700s- an English
1700s E li h country doctor
d b the
by h name off Edward
Ed d Jenner
J
discovered a remarkable relationship between exposure to cowpox and
immunity to smallpox.
smallpox After observing the fact that milkmaids who
were exposed to cowpox had apparent immunity to smallpox, he
deliberately
y injected
j individuals with material from a cowpox
p lesion
and then exposed them to smallpox. He thus proved that immunity to
cowpox, a very mild disease, provided protection against smallpox.
Thi procedure
This d off injecting
i j i cellular
ll l material
i l became
b k
known as
vaccination, from vacca, the Latin word for “cow.”
Louis Pasteur,
Pasteur a key figure in the development of both
microbiologygy and immunology, gy, accidentallyy found that old
cultures would not cause disease in chickens
Pasteur applied principle of attenuation to the prevention of
rabies
In 1885,, when a boy y who was severely y bitten by
y a rabid dog
g
was brought to his home, Pasteur tried out his new procedure.
The boy y received a series of 12 injections
j beginning
g g with
material from the least infectious cords and progressing to the
fresher,, more infectious material. Miraculously, y, the boyy
survived, and a modification of this procedure is the standard
treatment for rabies today
y
The phenomenon in which exposure to one agent produces
protection
t ti against
i t another
th agentt is
i known
k as
cross--immunity
cross
Natural, or innate, immunity

is the ability
y of the individual to resist infection by
y
means of normally present body functions. These are
considered non
non-adaptive
adaptive or nonspecific and are the
same for all pathogens or foreign substances to which
one is exposed.
exposed No prior exposure is required,
required and the
response does not change with subsequent exposures.
Many of these mechanisms are subject to influence by
such factors as nutrition, age,
g fatigue,
g stress, and
genetic determinants.
Acquired immunity (Specific),
is a type of resistance that is characterized by
specificity
ifi i for
f eachh individual
i di id l pathogen,
h or microbial
i bi l
agent, and the ability to remember a prior exposure,
which results in an increased response upon repeated
e posu e.
exposure.
Naturally acquired Placental transfer of antibody
(Passive)
Recovery from disease (Active)
Artificially acquired Administration of antitoxin
(Passive)
Vaccination
Vacc a o ((Active)
c ve)
Protection Against Invading Pathogens
1. First
Fi Line
Li off Defense:
Defense
D f : Non
N -specific
Non- ifi naturall
barriers which restrict entry of pathogen.
Examples:: Skin and mucous membranes.
Examples
2. Second Line of Defense:
Defense: Innate non
non--specific
p
immune defenses provide rapid local response to
pathogen
p g after it has entered host.
Examples:: Fever, phagocytes (macrophages and
Examples
neutrophils),
neutrophils ), inflammation
inflammation,, and interferon.
3. Third line of defense: Antigen-
Antigen-specific immune
responses, specifically
ifi ll target
t t andd attack
tt k invaders
i d
that get past first two lines of defense.
Examples:: Antibodies and lymphocytes.
Examples
Th
Three Lines
Li off Defense
D f Against
A i t Infection
I f ti
First Line of Defense:
Ski and
Skin dMMucous M
Membranes
b

I. Mechanical Defenses
1. Skin has two Layers:
A E
A. Epidermis
Epidermis:
id i : Thin
Thi outert layer
l off epithelial
ith li l tissue.
ti
Contains Langerhans cells, dead cells, and keratin
(waterproof).
B. Dermis:
Dermis: Thick inner layery of connective tissue.
Infections are rare in intact skin. Exceptions:
) Hookworms can penetrate intact skin
) Dermatophytes
Dermatophytes:: ““Skin
Skin loving” fungi
Intact Skin is an Effective Barrier
A i t Most
Against M t Pathogens
P th
I. Mechanical Defenses
2. Mucous Membranes
Membranes:: Line gastrointestinal,
genitourinary, and respiratory tracts.
‹ Two layers: Outer epithelial and inner connective layer.
‹ Epithelial
p layer
y secretes mucus which maintains moist
surfaces.
‹ Although they inhibit microbial entry, they offer less
protection than skin.
‹ Severalmicroorganisms are capable of penetrating
mucous membranes:
) Papillomavirus
p
) Treponema pallidum
) Enteroinvasive E. coli

) Entamoeba histolytica
I. Mechanical Defenses
3. Lacrimal
L i l apparatus:
apparatus
t : Continual
C ti l washing
hi and d
blinking prevents microbes from settling on the
eye surface.
surface
4. Saliva
Saliva:: Washes microbes from teeth and mouth
mucous membranes.
b
5. Mucus
Mucus:: Thick secretion that traps many
microbes.
6. Nose Hair
Hair:: Coated with mucus filter dust, pollen,
p
and microbes.
7. Ciliary
y Escalator:
Escalator: Cilia on mucous membranes of
lower respiratory tract move upwards towards
throat at 1-3 cm/hour.
I. Mechanical Defenses
8. Coughing
C hi and
d sneezing:
sneezing
i : Expel
E l foreign
f i objects.
bj
9. Epiglottis
Epiglottis:
pg : Covers larynx
y during
g swallowing.
g
10.. Urination
10 Urination:: Cleanses urethra.
11. Vaginal Secretions
11. Secretions:: Remove microbes from
genital tract.
Epiglottis Protects Respiratory System from
I f ti During
Infection D i Swallowing
S ll i
B. Chemical Defenses:
‹ Sebum
Sebum:: Oily substance produced by
sebaceous glands that forms a protective layer
over skin. Contains unsaturated fatty acids
which inhibit growth of certain pathogenic
bacteria and fungi.
‹ pH
pH::
Low, skin pH usually between 3 and 5.
Caused by lactic acid and fatty acids.
‹ Perspiration
P
Perspiration:
i ti : P d db
Produced by sweatt glands.
l d
Contains lysozyme and acids.
‹ Lysozyme
Lysozyme:: Enzyme that breaks down gram-
gram-
positive cell walls. Found in nasal secretions,
saliva, and tears.
B. Chemical Defenses (Continued)
‹ Gastric
G t i Juice:
Juice
J i : Mixture
Mi t off hydrochloric
h d hl i acid, id
enzymes, and mucus. pH between 1.2 to 3
kills many microbes and destroys most toxins.
toxins
Many enteric bacteria are protected by food
particles.
) Helicobacter pylori neutralizes stomach acid and
can grow in the stomach, causing gastritis and
ulcers.
‹ Transferrins
Transferrins:: Iron--binding proteins in blood
Iron
which inhibit bacterial growth by reducing
available iron.
Composition of Human Blood
II. Second Line of Defense
1. Phagocytosis
Phagocytosis::
‹ Derived from the Greek words “Eat and cell”.
cell”
‹ Phagocytosis is carried out by white blood cells:
macrophages,
h neutrophils
neutrophils,
t hil , and
d occasionally
i ll
eosinophils..
eosinophils
‹ Neutrophils predominate early in infection.
‹ Wandering g macrophages:
macrophages
p g : Originate
g from
monocytes that leave blood and enter infected
tissue,, and develop
p into p
phagocytic
g y cells.
‹ Fixed Macrophages ((Histiocytes
Histiocytes):
): Located in
liver, nervous system, lungs, lymph nodes, bone
marrow, and several other tissues.
Phagocytic Cells: Macrophages (Monocytes),
Neutrophils and Eosinophils
Neutrophils,

(Macrophages)
Stages of Phagocytosis
1. Chemotaxis
Chemotaxis:: Phagocytes are chemically
attracted to site of infection.
2. Adherence: Phagocyte plasma membrane
attaches to surface of ppathogen
g or foreign
g
material.
) Adherence can be inhibited by capsules (S.
pneumoniae) or M protein (S. pyogenes).
) Opsonization: Coating process with opsonins that
Opsonization:
facilitates attachment.
• Opsonins
O i include
i l d antibodies
tib di and
d complement
l t proteins.
t i
Phagocytes
Ph t are Att
Attracted
t d tto Sit
Site off
Infection by
y Chemotaxis
Stages of Phagocytosis (Continued)
3. Ingestion:
Ingestion: Plasma membrane of phagocytes
phagoc tes
extends projections (pseudopods) which engulf the
microbe Microbe is enclosed in a sac called
microbe.
phagosome..
phagosome
4. Digestion
Digestion:: Inside the cell, phagosome fuses with
lysosome to form a phagolysosome
phagolysosome..
Lysosomal enzymes kill most bacteria within 30
minutes and include:
) Lysozyme: Destroys cell wall peptidoglycan
) Lipases and Proteases

) RNAses and DNAses

After digestion, residual body with undigestable

material is discharged.
Process of Phagocytosis
g y
Inflammation
Triggered by
b tissue
tiss e damage due
d e to infection,
infection heat,
heat
wound, etc.
Four Major Symptoms of Inflammation:
1. Redness
2. Pain
3. Heat
4. Swelling
May also observe:
5. Loss of function
 Functions
u c o s oof Inflammation
o
1. Destroy and remove pathogens

2. If destruction
d t ti is i nott possible,
ibl tot limit
li it
effects by confining the pathogen and its
products.

3. Repair
p and replace
p tissue damaged
g byy
pathogen and its products.
Stages
g of Inflammation
1. Vasodilation:
Vasodilation: Increase in diameter of blood
vessels.
Triggered by chemicals released by damaged cells:
histamine kinins
histamine, kinins,, prostaglandins,
prostaglandins and
leukotrienes..
leukotrienes
2. Phagocyte
Ph t Mi
Migration
ti and d Margination
M i ti :
Margination:
Margination is the process in which phagocytes
stick
ti k tto li
lining
i off blood
bl d vessels.
l
Diapedesis (Emigration):
(Emigration): Phagocytes squeeze
between endothelial cells of blood vessels and
enter surrounding tissue.
Process of Inflammation
Stages
g of Inflammation ((Continued))
Phagocytes are attracted to site of infection
through chemotaxis.
Phagocytes destroy microbes, as well as dead and
damaged host cells
cells.
3. Tissue Repair
Repair:: Dead and damaged cells are
replaced.
l d
 Immunogens 
and 
d 
Immunization
IImmunogenicity
Immunogenicity:i it : Is
I the
th ability
bilit to t induce
i d a humoral
h l
(by induction of B lymphocytes to produce
i
immunoglobulins)
l b li ) and/or
d/ cell-mediated
ll di t d (by
(b activation
ti ti
of T lymphocytes to enter their specific responses
l di
leading t the
to th secretionti off lymphokines)
l h ki ) immune
i
response.
Antigenicity:: Is the ability to combine specifically
Antigenicity
with the final products of the [immune response] (i.e.
secretedd antibodies
ib di and/or d/ surfacef receptors on T-
cells). Although all molecules that have the property of
i
immunogenicity
i i alsol have
h the
h property off antigenicity,
i i i
the reverse is not true.
¾Antigen is a molecule recognized by the immune system.
O i i ll the
Originally th term
t came from
f antibody
tib d generatort and d was a
molecule that binds specifically to an antibody, but the term now also
refers to any
y molecule or molecular fragment
g that can be bound byy
a major histocompatibility complex (MHC) and presented to a T-cell
receptor. "Self" antigens are usually tolerated by the immune system;
whereas "Non-self"
"Non self" antigens are identified as intruders and attacked
by the immune system. Autoimmune disorders arise from the immune
system reacting to its own antigens.
¾Immunogen is a specific type of antigen. An immunogen is
defined as a substance that is able to provoke an adaptive
immune response if injected on its own. Said another way, an
immunogen is able to induce an immune response, while an antigen
is able to combine with the products of an immune response once
they are made. The overlapping concepts of
immunogenicity and antigenicity are thereby subtly different.
¾ Degree
D off “foreignness”‐Based
“f i ” B d on genetic i relatedness.
l d
¾ Chemical composition (complexity).
‰ proteins are the most potent immunogens‐ Primary, 
secondary, tertiary and quaternary structure.
‰Most polysaccharides are weak antigens.
‰Nucleic acids in their pure form are nonimmunogenic.
p g
¾Antigen size molecular size‐usually MW >100,000; <10,000 
non immunogenic; 10,000‐100,000 MW is imunogenetically 
g ; , , g y
variable.
¾Degradability : 
g y Polystyrene or asbestosis are nonimmunogenic 
y y g
cause they cannot processed by phagocyte cells.  
¾ Route of immunization : subcutaneous or intramuscular
route of antigen are best for inducing an antibody response.
While
Whil intravenous
i injection
i j i thwart
h or minimize
i i i theh immune
i
response.

¾Nature of the host .

¾ Dose of route of antigen presentation.

‰Immunological tolerance.

‰Vaccines
¾Autoantigens‐”self”
¾Autoantigens self
¾Alloantigens‐”same species”
¾Heteroantigens‐”different
g species”
p
¾Heterophile antigens‐”occur when an antibody to one
antigen reacts unexpectedly with unrelated antigen when
i fact
in f t both
b th contain
t i the
th same shared
h d epitops”.
it ”
¾T‐cell dependent antigens‐Requires T cell involvement;
proteins.
¾T‐cell independent antigens‐Does not require T cell
involvement; polysaccharides.
¾Superantigen ”enterotoxin by S. aureus induce release of
large quantities of cytokines (interlukin‐1 and tumour
necrosis factor from T cells ) ”
 Antigens

Epitope “ Antigenic determinant”

¾ Small
ll part off an antigen that
h interacts with
h
an antibody and T cells receptors.
receptors.
¾ Usually between one and six sugars or amino
acids on the surface of the antigen
¾ Any given antigen may have several epitopes.
epitopes.
¾ Each epitope is recognized by a different
antibody..
antibody
¾HAPTEN is i capablebl off binding
bi di t an antibody
to tib d but
b t
unlike an immunogen, is NOT capable of evoking an
i
immune response such h as the
th production
d ti off antibodies.
tib di
¾HAPTEN is small non –immunogenic molecule and is
the equivalent of an epitope and can bind to a single
antigen binding site on an antibody molecule.
¾ Haptens include low molecular weight chemicals (e.g.
benzene, dinitrophenol (DNP), drugs (e.g. aspirin), and
antibiotics (e.g. penicillin).
Adjuvant
j is a substance administered with an
immunogen that increases the immune response.
It acts byy p
producingg a local inflammatoryy response
p
that attracts a large number of immune system
cells to the injection
j site.
Types of adjuvants:
¾Aluminum
¾ l salts.
l
¾Complete Freund’s adjuvant(CFA)‐” mineral oil,
emulsifier, and killed mycobacteria”. Increase the
cell –mediated response .
¾Incomplete Freund
Freund’ss adjuvant (IFA)‐” water in oil
emulsion with antigen in water phase ”. Increase
the humoral immune response ‘100 100 folds
folds”.
¾Bacille Calmette Gurein “BCG” has also been
used
d as an immunotherapeutic
i th ti agentt to
t boost
b t the
th
immune system in patients with special types of
cancer(e.g.,
( superficial
fi i l bladder
bl dd cancer). )
¾Mitogenic substances: endotoxins and
phtohemaglutinin
h h l ”
”PHA” ” increasing the h clonal
l l
expansion of B and T cells .
•Active Immunity occurs when one
makes
k hi
his/her
/h own antibodies.
tib di Thi
This ttype
of immunity is long term.

•Gettingg the disease : If y

you g
get an
infectious disease (like Chicken
Pox), often times, that stimulates the
production of MEMORY cells which
are then stored to prevent the
infection in the future.
Vaccination: A vaccination is an injection 
of a weakened form of the actual antigen 
that causes the disease. The injection is too 
j
weak to make you sick, but your B 
lymphocytes will recognize the antigen and 
react as if it were the "real thing". Thus, you 
produce MEMORY cells for long term 
d  MEMORY  ll  f  l  t  
immunity. 
Passive Immunity occurs when the
antibodies come from some other source.
This type of immunity is short term.

Breast milk : Milk from

a mother's breast
contains antibodies
antibodies.
The baby is acquiring
passive immunity
immunity.
These antibodies will
only last several weeks.
Gamma Globulin: A Gamma Globulin shot is purely an
i j ti
injection off antibodies
tib di t provide
to id temporary
t i
immunity.
it
You might receive an Gamma Globulin shot if you travel
outside of the country.
country
 ‰Active Immunization 

¾The immunized individuals own cells contribute

to their immunity.
¾I
¾Immunity it is
i long
l l ti and
lasting d easily
il reactivated
ti t d by
b
booster injections of antigen.
¾ Free dangers as comapered with passive
immunity .
¾Both B‐cell and T‐cell are activated .
¾Prophylactic
op y act c immunization
u at o .
 ‰Passive Immunization 

¾This provides humoral (antibody ‐based)

immunity but not T
T‐cell
cell based immunity .
¾Immunity is short lasting ,which is approximately
3 weeks.
weeks
¾ There is danger of causing anaphylaxis or serum
sickness.
i k
¾Prophylactic and/or protective immunization .
¾ INJECTION by b intramuscular
i l or subcutaneous
b f
for
both active and passive immunization. Intravenous
injection used for immunoglobulins.
immunoglobulins
¾ ORAL administration ”oral polio vaccine “.
¾INTRANASAL immunization for f airborne
b pathogen.
h
¾HERD immunity
CELLS AND TISSUES OF THE IMMUNE SYSTEM

Ghassan Mohammad Sulaiman , Ph.D.

Assist.
A it P Prof.
f
Department of applied science, School of biotechnology
University of Technology
White Blood Cells
 OVERVIEW OF THE IMMUNE SYSTEM

Cells: lymphocytes, macrophages & monocytes, dendritic

cells, granulocytes. All arise from pluripotent
hematopoietic stem cells in bone marrow.

Organs: lymph nodes (found in various locations)

locations), thymus
thymus,
spleen - these constitute the lymphoid organs

Thymus and bursa (bone marrow) are called central

lymphoid organs

Peripheral Lymphoid Organs: lymph nodes,

spleen and tonsils,
spleen, tonsils liver,
liver intestine and skin are also
are also important parts of the immune system.
 MONOCYTES AND MACROPHAGES

Monocytes are immature macrophages; monos circulate in blood & accum-

ulate at sites of inflammation. Macrophages may differentiate in tissue in
absence of antigen (Kupffer cells in liver
liver, e
e.g.)
g ) or differentiate in response to
inflammation. They are Ag-presenting cells (APC). Also phagocytose
microbes; contain bacteriocidal mechanisms.

mono in blood smear

Wright-Giemsa
 DENDRITIC CELLS
•DCs are the most effect antigen presenting cells.

•2 sites of origin, plasmacytoid DCs (periphery & spleen) and myeloid DCs (bone
marrow).)

•Myeloid dendritic cells (mDC): Produce IL-12, express TLR2,4. Effective in antigen
presentation

•Plasmacytoid dendritic cells (pDC) (lymhoid origin?): interferon-alpha, express

TLR7 9
TLR7,9.

•Some DCs can be immunosuppressive.

LPS-activated DCs
 Polymorphonuclear leukocytes (PMNs)

Neutrophils:
N hil Predominant
P d i type off white
hi blood
bl d cell.
ll RRapidly
idl
migrate to sites of infection or inflammation. Phagocytic, they
have special enzymatic pathways for enhanced bacteriocidal
action.

Mono PMN

PMN, note tri-lobed nucleus Wright-Giemsa,

 BASOPHILS AND MAST CELLS

Have basophilic granules, which contain allergic mediators.

Basophils circulate, mast cells found in tissue.
b
basophil
hil

degranulating
mast cell

Basophilic granules of mast cells

intact
mast cell
 EOSINOPHILS

Have granules that stain red with eosin Y. Mediate late phase of allergic
response,
p , active in immune response
p to p
parasites & tumors ((antibody-
y
dependent cell-mediated cytotoxicity).

eosinophil

PMNs
comparison of PMNs to eosinophil
 Lymphocytes

•Major subtypes are T and B lymphocytes (or cells).

•Responsible
Responsible for immunological memory
memory.
•T cells mature in thymus; B cells in avian bursa of Fabricius,
mammalian fetal liver & bone marrow.
• T cells participate in cell
cell-mediated
mediated immunity & immunoregulation
• B cells synthesize antibodies.
• NK cells are morphologically similar to T & B cells
are cytotoxic.
y
Origin of B cells

LOCATION OF THE
BURSA OF FABRICIUS

In 1956,
1956 Bruce Glick and Timothy Chang reported:
the bursa of Fabricius plays an important role
in antibody production.
 ORGANS OF THE IMMUNE SYSTEM
PRIMARY LYMPHOID ORGANS

Primary lymphoid organs are where lymphocytes arise and mature in the
absence of antigenic stimuli. They are the bone marrow and thymus.

Bone marrow: Source of all hematopoietic progenitor (stem) cells,

cells site
of B cell maturation post-birth in mammals.

About 1 in every 10,000 to 15,000 bone marrow cells

is thought to be a stem cell.
In the blood stream 1 in 100,000 blood cells.
 PRIMARY LYMPHOID ORGANS: THYMUS

Thymic epithelial cells are derived from the third pharyngeal pouch.

The thymus is the site where T cells develop.

It gradually enlarges during childhood but after puberty it undergoes a process of involution.

The thymus
Th th iis arranged
d iinto
t an outer
t cortex
t and d an iinner medulla.
d ll IImmature
t llymphoid
h id cells
ll iin
the cortex. Mature T cells are in the medulla from where mature T lymphocytes enter the
circulation.
 SECONDARY LYMPHOID ORGANS

Peripheral lymphoid organs: lymph

nodes, spleen, tonsils, adenoids,
and
d llymphoid
h id ti
tissue associated
i t d with
ith
other organ systems (gut, skin,
mucosa).

LYMPH NODES: filter lymphatic fluid;

sites of Ag presentation & cell traffic
 LYMPH NODES

Lymph nodes have a fibrous capsule from which trabeculae extend towards the
center, forming a framework for the lymphatic sinuses, blood vessels, and
parenchyma (cortex,
(cortex paracortex,
paracortex and medulla).
medulla)

1 - cortex (B Cells) 2 - paracortical zone (T Cells)

3 – medulla(T,( , B,, Mac)) 4 - medullaryy cords
5 - lymphoid follicle of the cortex
6 - capsule 7 - subcapsular sinus
8 - cortical sinus 9 - medullary sinus
 LYMPH NODES, CONTINUED
Functions of structural elements of lymph nodes

Lymph Æ afferent lymphatics,

lymphatics
Æsinuses lined with macrophages
Æ efferent lymphatic (ultimately Subcapsular
all drain into the portal vein). Sinus

Lymphocytes enter the node

primarily from the blood via HEV
Æ efferent lymphatics.
lymphatics

DCs migrating from tissue enter

the node into the T cell areas.

B cells entering nodes from blood

must cross the T rich area in
transit to the B cell rich areas thus
optimizing T-B cooperation.
SPLEEN
 LYMPH NODES, CONTINUED

SPLEEN
Stained with haematoxylin and eosin
1 - lymphoid follicle (white pulp)
2 - redd pulp
l
3 - capsule
4 - trabeculae (connective tissue)

The spleen serves two major functions:

f
*It is responsible for the destruction of old red blood cells (RBCs) -
this occurs in the red pulp;
*It
It is a major site for mounting the immune response - the white pulp
pulp.
The spleen behaves like a lymph node, but instead of filtering lymph,
it filters blood.
Has the hematopoiesis function in mice.
MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT)

Lymphoid tissue found at the gastrointestinal tract, respiratory tract and

urogenital tract.

MALT consists of aggregates of lymphocytes, macrophages, DCs, and

other accessory cells.
 GUT-ASSOCIATED LYMPHOID TISSUE (GALT)

This is comprised of:

* tonsils, adenoids (Waldeyer's ring)
* Peyer
Peyer'ss patches
* lymphoid aggregates in the appendix and large intestine
* lymphoid tissue accumulating with age in the stomach
* diffusely
y distributed lymphoid
y cells and plasma cells in the lamina propria of the g
gut
 SKIN-ASSOCIATED
SKIN ASSOCIATED LYMPHOID TISSUE (SALT)

Skin is an active participant in host defense

defense. It
has the capability to generate and support local
immune and inflammatory responses to foreign
Ags that enter the body via the skin.

Cells of SALT include keratinocytes, Langerhans

cells (immature DCs found in skin),
intraepiethelial T cells,
cells and melanocytes
melanocytes.

Langerhans cells form a continuous epidermal

meshwork: they capture Ag, then migrate to
d i i llymph
draining h nodes,
d where
h they
h act as A
Ag-
presenting cells.
 Origin of T Cells

Jacques Miller found that removal of thymus (thymectomy)

from neonatal mice resulted in fewer lymphocytes
y p y and no
antibody to sheep red blood cells (1962). Later on, in
thymectomized animals, the ability to reject allografts and to
mount delayed hypersensitivity responses was drastically
reduced.

By the mid-1960s
mid-1960s, immunologists were convinced that there
were indeed two separate arms of the immune system: one
dealing exclusively with the production of circulating
antibodies (humoral immunity),
immunity) and another that is involved
in the delayed hypersensitivity-type reactions and graft
rejections (cell-mediated immunity). To have a good
i
immune response, bboth
hhhave to exist.
i
A T Cell ?
A B Cell?
 T Cell Subsets

T –cell subtype Symbol Identifying surface MHC Target cell Function

antigen restriction

Cytotoxic Tc CD8 Class I Tumours and Kills foreign

virally infected cell cells
Allografts
Helper Th CD4 Class II B cell and Tc cell Interleukin
secretion
Inducer Th CD4 Class II B cell and Tc cell Interleukin
secretion

Suppressor Ts CD8 Class I B cell ,Th and Down –

Tc cell regulates cell
growth
th

Delayed-type Tdth CD4 Class II Macrophage, Tc Release

hypersensitivity cell lymphokines
Memmory Tm CD8;CD4 Both B and T cell Anamnesis

CD Cluster of differentiation : Antigenic features of leukocytes that are identified by

groups off monoclonal
l l antibody
tib d expressing
i common or overlapping
l i activity.
ti it
Class I MHC expression – On all cells
Class II MHC expression - On B cells, monos/macs & DCs and activated human T cells
 Immunoglobulins
Immunoglobulins:
g :
Structure and Function
Immunoglobulins:
g Structure and Function
„ Definition: Glycoprotein molecules that are produced
Definition:
b plasma
by l cells
ll in
i response to an immunogen
i and
d
which function as antibodies
 Antibodies
‹ Proteins that recognize and bind to a particular
antigen with very high specificity.
‹ Made in response to exposure to the antigen.
‹ One virus or microbe may have several
antigenic determinant sites, to which different
antibodies may bind.
‹ Each antibody has at least two identical sites
that bind antigen: Antigen binding sites.
‹ Valence of an antibody:
antibody: Number of antigen
binding sites. Most are bivalent
bivalent..
‹ Belong to a group of serum proteins called
immunoglobulins (IgsIgs).
).
 Antibody
y Structure
‹ Monomer
Monomer:: A flexible Y-
Y-shaped molecule with
four protein chains:
‹2 identical light chains…..
chains…..25000
25000 Dalton
‹ 2 identical heavy chains………
chains
chains………50000
50000-77000 Dalton
50000-
‹ Variable Regions:
Regions: Two sections at the end of
Y’ arms. C
Y’s Contain
t i th
the antigen
ti bi
binding
di sites
it
(Fab
Fab)). Identical on the same antibody, but
vary from
f one antibody
tib d tto another.
th
‹ Constant Regions:
Regions: Stem of monomer and lower
parts of Y arms.
‹ Fc region:
region
g : Stem of monomer only. y Important
p
because they can bind to complement or cells.
Antibody Structure
 Antibody Structure
Fab
F b fragment
f t
antigen binding

Fc fragment
crystalizable
li bl
Human Immunoglobulin
g Light
g Chain Types
yp

„ Kappa
„ Lambda
 Human Immunoglobulin
g Classes

„ IgG – Gamma ((γγ) heavy chains

„ IgM – Mu (µ) heavy chains
„ IgA – Alpha (α) heavy chains
„ IgD – Delta (δ) heavy chains
„ IgE
g – Epsilon
p (ε) heavy
y chains
Different Immunoglobulins
 Immunoglobulin
g Classes
I. IgG (subclasses IgG
IgG11 , 2, 3, 4)
‹ Structure: Monomer
‹ Percentage g serum antibodies: 80
80%
%
‹ Location: Blood, lymph, intestine
‹ Half
Half--life in serum: 23 days
‹ Complement Fixation: Yes
‹ Placental Transfer: Yes
‹ Known Functions: Enhances phagocytosis,
phagocytosis,
neutralizes toxins and viruses, protects fetus and
newborn.
 Immunoglobulin
g Classes
II. IgM
‹ Structure: Pentamer
‹ Percentage g serum antibodies: 5-10%
10%
‹ Location: Blood, lymph, B cell surface (monomer)
‹ Half
Half--life in serum: 5 days
‹ Complement Fixation: Yes
‹ Placental Transfer: No
‹ Known Functions: First antibodies produced
during an infection. Effective against microbes and
agglutinating antigens.
 Immunoglobulin
g Classes
III. IgA (subclasses IgA 1, 2)
‹ Structure: Dimer
‹ Percentage g serum antibodies: 10 10--15%
15%
‹ Location: Secretions (tears, saliva, intestine, milk), blood
and lymph.
‹ Half
Half--life in serum: 6 days
‹ Complement
C l t Fixation:
Fi ti N
No
Secretory J
‹ Placental Transfer: No Piece Chain

‹ Known Functions: Localized protection of mucosal

surfaces. Provides immunity to infant digestive tract.
 Immunoglobulin
g Classes
IV. IgD
‹ Structure: Monomer
‹ Percentage g serum antibodies: 0.2%
‹ Location: B-cell surface, blood, and lymph
‹ Half
Half--life in serum: 3 days
‹ Complement Fixation: No
‹ Placental Transfer: No
‹ Known Functions: In serum function is unknown.
On B cell surface, initiate immune response.
 Immunoglobulin
g Classes
V. IgE
‹ Structure: Monomer
‹ Percentage g serum antibodies: 0.002%
002%
‹ Location: Bound to mast cells and basophils
throughout body. Blood.
‹ Half
Half--life in serum: 2 days
‹ Complement
C l t Fixation:
Fi ti N
No
‹ Placental Transfer: No
‹ Known Functions: Allergic reactions. Possibly
lysis of worms.
 Properties of Immunoglobulins
Property IgG IgA IgM IgE IgD
Heavy chain γ α µ ε δ
symbol
Molecular 150 170-400
170- 900 190 180
weight KDa KDa KDa KDa KDa

Percentage 75--80 %
75 10
10--15 % 5-10 % 0.002 % % 0.2
in serum
Complement Yes No Yes No No
fixation
Transplacental Yes No No No No
passage
Opsonization Yes No No No No
Antibody Response After Exposure to Antigen
Primary and Secondary antibody response

Primary antibody response Secondary antibody response

* first exposure to antigen * Subsequent exposure

* lag period: days or weeks * Lag period: hours

(slow onset) (rapid onset)

* Small amount immunogl

immunogl.. * large amount immunogl
immunogl..
low Ab level with gradual increase high Ab with rapid increase

• Ab Persist
P i for f short
h duration
d i * Persist
P i for f long
l periods
i d
• Weeks then decline rapidly (monthes
(monthes or years)

* Antibody is IgM * Antibody is IgG

Antigen-Antibody
Antigen-
I t
Interactions:
ti
Characterized as:
• Non-covalent interaction (similar to “lock and key” fit
of enzyme-substrate)
• Does not lead to irreversible alteration of Ag or Ab
• This exact and specific interaction has led to many
immunological assays used to:
» detect Ag or Ab
» diagnose disease
» measure magnitude of humoral IR
» identify molecules of bio and med interest
 Nature of Ag/Ab Reactions

- Four types of non-covalent forces operates over a very short distance

( generally 1 angstrom )
Structure of an antibody
 Affinityy
• Strength of the reaction between a single antigenic
d
determinant
i andd a single
i l Abb combining
bi i site
i

High Affinity Low Affinity

Ab Ab

Ag Ag

Affinity = ( attractive and repulsive forces

 Avidityy
• The overall strength of binding between an Ag
with
ith many determinants
d t i t andd multivalent
lti l t Abs
Ab

Keq = 104 106 1010

Affinity
y Avidity
y Avidity
y
 S
Specificity
ifi i
• The ability of an individual antibody combining site
to react with only one antigenic determinant.

• The physical state of the antigen is responsible

for the naming of antibodies:
• Agglutinins
gg :antibodies that clump
p or aggregate
gg g cellular antigen.
g
• Lysins : antibodies that cause dissolution of cell membrane.
• Hemolysins: antibodies lyse erythrocytes with the help of complement
proteins.
t i
• Precipitins : antibodies that form insoluble complexes with soluble
antigens.
g
 Cross Reactivity
• The ability of an individual Ab combining site to
react with
i h more than
h one antigenic
i i determinant.
d i
• The ability of a population of Ab molecules to
react with more than one Ag

C
Cross reactions
ti

Anti-A Anti-A Anti-A

Ab Ab Ab

Ag A Ag B Ag C

Shared epitope Similar epitope

 Factors Affecting Measurement of
Ag/Ab Reactions

• Affinity
Affi i
• Avidity Ab excess Ag excess

• Ag:Ab ratio
• Physical form of Ag

Equivalence – Lattice formation

 P i it ti reactions
Precipitation ti

• Immunodiffusion
• Mancini
• Oudin
• Ouchterlony
• Immunoelectrophoresis
 Precipitation Reactions

( no precipitate is formed
( Lattices or if an Ag contains only a
large aggregates ) single
i l copy off each h epitope
i )

FIGURE 6
6-4
4
Precipitation reactions in fluids yield a precipitin curve.
 Precipitation Curve

• Prozone – antibody excess, many

g sites- results
antibodies coat all antigen
in false negative
• Postzone – antigen excess,
excess antibody
coats antigen but cannot get lattice
f
formation, results in false
f negative
• Zone of Equivalence – antigen and
antibody present in optimal proportions
to bind and give visible reaction
Precipitation
ec p o reaction
e c o
 Radial Immunodiffusion (Mancini)
( )

A iin gel
Ab
• Method
– Ab in gel Ag Ag Ag Ag

– Ag in a well
• Interpretation
p
– Diameter of ring is
proportional to the

Diametter2
concentration
t ti
• Quantitative
– Ig
I levels
l l
Ag Concentration
 in the Ab-containing semisolid medium

-> The area is proportional to

the conc. of Ag.
The region
g of equivalence
q

FIGURE 6-5
Diagrammatic representation of radial & double immunodiffusion.
immunodiffusion
: precipitation reactions in gels yield visible precipitin lines; no visible
precipitate forms in regions of Ab or Ag excess.
Immunodiffusion
 Oudin Precipitation
• Solution
S l i off antibody
ib d isi carefully
f ll llayered
d on top off a solution
l i off
antigen, such that there is no mixing between the two.
• With time at the interface where the two layers
y meet,, antigen-
g
antibody complexes form a visible precipitate. The other two
tubes are negative controls, containing only antibody or only
antigen plus an irrelevant protein in the second layer
layer.
Ouchterlony Immunodiffusion

• Holes punched in agar.

• Known antibody or antigen added to
center well.
• Known
K sample
l added
dd d tto outer
t well.
ll
• U
Unknown
o sa
sample
p e added to oute
outer well
e
next to unknown sample.
• Wait for bands to form
form.
Ouchterlony Immunodiffusion
 Ouchterlony - Identity
• The precipitation appears as a continuous line in the
form of an angle between those two wells and the C
well There are no spurs at the angle and this type of
well.
reaction is termed a band of identity.
 Ouchterlony – Partial Identity
• FIGURE 2:
If a solution with antigens X and Y is placed in well 1, a solution with
antigen X only is placed in well 2, and antiserum containing antibodies
specific for both X and Y is placed in well 3, a reaction similar to that
appearing in FigFig. 2 will
ill occ
occur.
r Notice that there is a sp
spurr reaction
towards the XY well. This indicates that the two antigenic materials in
wells 1 and 2 are related, but that the material in well 1 possesses an
antigenic specificity not possessed by the material in well 2. Such a
reaction
ti with
ith spur fformation
ti indicates
i di t partial
ti l identity
id tit
 Ouchterlony – Non
Non-Identity
Identity
• If the material in wells 1 and 2 do not possess
common antigens and the antiserum in well 3
possesses specificities for both materials
materials, the
reaction will appear as two crossed lines as in Fig. 3
 Ouchterlony Interpret
Ouchterlony-Interpret
• Determine which interpretation fits for samples 1, 2
and 3.
Immunoelectrophoresis
 Precipitation Reactions(immunoelectrophorosis)

FIGURE 6-6 (a)

Immunoelectrophoresis.
- an antigen mixture is first electrophoresed to separate its components by charge
- diffusion & producing lines of precipitation.
 Countercurrent electrophoresis
p
• Method
– Ag
A and d Ab migrate
i t ttoward
d each
h other
th b by
electrophoresis
– Used
U d onlyl when
h A Ag and
d Ab h
have opposite
it
charges
- +
Ag Ab

• Qualitative
–Rapid
R id
Rocket Immunoelectrophoresis
„ Antigen
A ti iis electrophoresed
l t h d into
i t gell containing
t i i
antibody. The distance from the starting well to
the front of the rocket shaped arc is related to
antigen concentration.
 Agglutination reactions
• Direct agglutination
• Indirect agglutination
• Hemagglutination
 Direct agglutination
• Cross – linking and lattice formation
• Antibodies react with particulate
antigens (red blood cells
cells, bacteria
bacteria, fungi)
• Visible clumps
• Estimate amount of antibody
• Antigen found naturally on particle
particle.
• Blood Grouping is an example, antigen
on cell
 Indirect agglutination
• Soluble antigen is coated onto particles
(red bood cells, latex beads; polystyrene
latex,, bentonite or charcoal))
• Allow for visible clumps (agglutination)
 Agglutination/Hemagglutination
gg gg
• Definition - tests that have as their endpoint
the agglutination of a particulate antigen
– Agglutinin/hemagglutinin
• Qualitative agglutination test
– Ag or Ab

+ Þ
 Agglutination
gg Reactions

-visible clumping
p g by
y interaction between Ab & a p
particulate antigen
g such as RBC,
latex beads.
-routinely performed to type RBCs for blood transfusion.

+ + + (control)

FIGURE 6-7
67
Demonstration of hemagglutination using Ab against sheep
red blood cells (SRBCs).
 A ti
Antigen – antibody
tib d assays

• Radioimmunoassay (RIA)
• Enzyme – linked immunosorbant assay
(ELISA)
• Western blot
 R di i
Radioimmunoassay (RIA)

• Competitive inhibition assay

• Measure antigen or antibody
• Ex.
Ex Measure small amounts of hormones
or drugs in a clinical sample
• Ex. Measure small amounts of IgE
antibody (radioallergosorbent test)
RIA
 RIA

• From these data, a

standard binding
curve, like the one
shown in red, can be
drawn.
 RIA
• Radioimmunoassay is widely-used
because of its ggreat sensitivity.
y
• Using antibodies of high affinity, it is
possible to detect a few picograms (10−12 12

g) of antigen in the tube.

• The greater the specificity of the
antiserum, the greater the specificity of the
assay
 Enzyme – linked
immunosorbant assay (ELISA)
• Color reaction assay
• Indirect ELISA
• Direct ELISA
 to detect Ab (HIV, HCV) ELISA

to detect Ag

to detect Ag

FIGURE 6-10
6 10
Variations in the enzyme-linked immunosorbent assay (ELISA) technique, similar to
RIA except using an Enzyme (alkaline ⓟ, horseradish peroxidase, & β-galactosidase)
: safer & less costly.
 Western blotting

: separates the components according to

their molecular weight.

: the proteins in the gel are transferred to the

sheet of nitrocellulose or nylon by the passage
off an electric
l t i current.t

: probed with Ab & then radiolabeled or enzyme-linked

2nd Ab.

: a position is visualized by means of an ELISA reaction.

FIGURE 6-12
Western blot
 I
Immunofluorescence
fl tests
t t

• Direct fluorescent antibody test

• Indirect fluorescent antibody test
 Immunofluorescence

Fluorochromes
-Fluorescein
Fluorescein (490→517nm)
-Rhodamine (515→546nm)
-Phycoerythrin
: absorb light of one wavelength & emit
fluorescence at a longer wavelength than
fluorescein.
FIGURE 6-14
6 14
mIgM-producing B cells indirectly stained with rhodamine-conjurated
secondary Ab under a fluorescence microscope.
 Complement fixation test
• Measures the binding of complement by
an antigen – antibody interaction
• Indicator system determine positive or
negative reactions
Complement
fixation test
 Neutralization test
• Antibody bind to specific antigen (virus,
toxin)
• Antibody – antigen complex prevents
antigen from binding (neutralization)
 Cellular immunology test
• Identification of subsets of lymphocytes
• Lymphocyte response to mitogens
• Cytoxic T – cell function
• Cell – mediated immunity to infectious
agents
Monoclonal Antibodies
T-Cell Maturation and
the Thymus
The Normal Rat Thymus
 Nude mouse

DiGeorge’s syndrome
 Origin, generation and differentiation of T cells

„ T cell progenitors migrate from bone marrow and seed

thymus.. T cell progenitors undergo differentiation to CD
thymus CD44
and CD8
CD8 cells in thymus
thymus.. Mature CD4
CD4 and CD8
CD8 T cells
circulate between blood and lymphoid tissues until they
meet antigens presented on dendritic cells in lymphoid
tissues.. T cells further undergo maturation to become
tissues
functional memory or effector T cells in LT
TCR : T cell receptor; Heterodimer consisting of
two non-identical polypeptide chains which are
linked together by disulfide bonds. Two different
TCR molecules TCR1 and TCR2 are known.
known
‰ More than 95% of T cell express the αβ
heterdimer (TCR2), which is the member of
immunoglubulin superfamily of molecules.

‰TCR1 is composed of γ and δ chains.

CD3 : is a protein complex and is composed of five

distinct chains (γ, δ, ε, ζ,η)that closely
associated with TCR.

CD4

CD8
 TCR genes undergo DNA rearrangement in thymus

*No Ds in Vα gene; DJ first then VDJ in β gene rearrangement

Un-rearranged

rearranged

expression

rearranged

Un-rearranged
 The Thymus
stromal cells:
maturatiion
 Positive
P iti and
d
Negative
Selection of
Thymocytes
in the Thymus
Two-step Selection Process of Thymocytes

Positive Selection: permits the survival of only

those T cells whose TCRs recognize self-MHC
molecules.

Negative Selection: eliminates T cells that react

too strongly with self-MHC or with self-MHC
plus
l self-peptides.
lf tid

Thymic stromal cells

cells, which express high levels
of class I and class II MHC molecules, play a role
in this process.
p
- Any developing thymocytes that are unable to
recognize self-MHC molecules or that do have a high
affinity for self-Ag plus self-MHC (or self-MHC alone) are
eliminated by y programmed
p g cell death.

- Thus, only those cells whose receptors recognize a self-

MHC molecules
l l plus
l fforeign
i A
Ag are allowed
ll d tto mature.
t

- An estimated 95%
95%--99%
99% of all thymocyte progeny
undergo programmed cell death within the thymus
without ever maturing.
g
Positive
P iti selection
l ti selects
l t T cells
ll
that recognize peptides on self
MHC
This is to assure that mature T cells can respond
to antigen-presented on self MHC.

-Self MHC I and II harboring self peptides on

thymic epithelial cells recognize and activate
TCRs on some DP thymocytes.

-DP thymocytes should receive this signal within

3-4 days to survive.Otherwise they undergo
apoptosis.
apoptosis
Negative selection eliminates T cells with
TCRs that bind too strongly or too week to
self antigen/MHC complex.
This is to assure that T cells don’t react against self
antigens In other words,
antigens. words autoreactive cells are
removed by this process.

Dendritic cells and macrophages in cortico-medullary

junction mediate it.

Negative selection cannot eliminate T cells whose

receptors are specific for self peptides that are
expressed outside of thymus (These cells enter
circulation, but soon to be rendered anergicg or
unresponsive by other mechanims).
 C t ki
Cytokines
and
d
Ch
Chemokines
ki
 Cytokines
Low molecular weight soluble proteins
(polypeptides) produced in response to
microbes and other antigens

They act via cell surface receptors to mediate

and regulate the amplitude and duration of
the immune-
immune-inflammatory responses
responses,,
through activation of macrophages,
controlling growth and differentiation of T
and B cells
 C t ki N
Cytokine Names
„Interleukins - produced exclusively by
leukocytes
„Lymphokines - produced by lymphocytes
„Monokines - produced exclusively by
monocytes
y
„Interferons - involved in antiviral responses
„Colony Stimulating Factors - support the
growth of cells in semisolid medias
„Chemokines
C - promote chemotaxis.
chemotaxis.
 Functional Categories of Cytokines
Cytokines
C t ki classified
l ifi d according
di to
t their
th i biologic
bi l i actions
ti
into three groups:

1) Mediators and regulators of innate immunity

- Produced by activated microphages and NK cells

in response
p to microbial infection

- they act mainly on endothelial cells and

leukocytes to stimulate the early inflammatory
response to microbes
 Functional Categories of Cytokines
2) Mediators
M di t and
d regulators
l t off acquired
i d immunity
i it
- Produced mainly by T lymphocytes in response to
specific recognition of foreign antigens
- They include IL
IL--2, IL-
IL-4, IL-
IL-5,, IL-
IL-13
13,, IFN,
Transforming growth factor-
factor-β (TGF
(TGF--β) and
lymphotoxin (TNF
(TNF-- β)

3) Stimulators of haematopoiesis
- Produced byy bon marrow,, stormal cells,, leukocytes
y
- Stimulate growth and differentiation of leukocytes
- Stem cell factor, IL
IL--3, IL-
IL-7, GM-
GM-CSF
 Interferons (IFNs)
* Interferons (IFNs): are proteins secreted in response to
viral infections or other stimuli

* They
y include:

- INF
INF--α produced by leucocytes
induced by virus infected cells
- INF
INF--β produced by fibroblasts

- INF
INF--γ p
produced by
y NK cells,TH1
cells,TH1 cells, CD8
CD8 T-cells
 Interferons (IFNs)
Action of INF-
INF-α and IFN-
IFN-β :

- Prevent viral replication

- Increase MHC-
MHC-I expression on viral infected cells
helping their recognition by CD8 T-cells

- Increase cytotoxic action of Nk cells

- Inhibit cell p
proliferation and tumor g
growth
 Interferons (IFNs)
Action of IFN-
IFN-γ :

- Activate Macrophages

- Increase expression of MHC-

MHC-I and II on APCs

- Enhance cytotoxic actions of Nk cells

- Promote production of TH1

TH1 and inhibits
proliferation of TH
p TH22
 Therapeutic
p Uses of Cytokines
y
1) Interferon in treatment of viral diseases, cancer

2) Several cytokines are used to enhance T- T-cell activation

in immunofideficincy
imm nofideficinc diseases
diseases,, e.g.
e g IL
IL--2, IFN-
IFN-γ,TNF
,TNF-
TNF-α

3) IL-
IL-2 and lymphokine activating killer cells (LAK) in
treatment of cancer

4) GM
GM--CSF induces increase in white cell count,
count, it is used:
a- To restore leukocytic count after cytotoxic
chemotherapy induced neutropenia
b- After bon marrow transplantation
C- To correct AIDS
AIDS--associated leukopenia
 Therapeutic Uses of Cytokines
5) Anti-
Anti-cytokines antibodies in management of
autoimmune diseases and transplant rejection:
rejection:
a- Anti
A
Anti-
i-TNF ini treatment rheumatoid
h id arthritis
h ii
b- Anti
Anti--IL
IL22 to reduce graft rejection

6) Anti
Anti--TNF antibodies in treating septic shock

7) Anti
Anti--IL
IL--2 in treating adult T-
T-cell leukemia

8) Anti
Anti--IL
IL--4 is under trial for treatment of allergies
Monoclonal Antibodies
T-Cell Maturation and
the Thymus
The Normal Rat Thymus
 Nude mouse

DiGeorge’s syndrome
 Origin, generation and differentiation of T cells

„ T cell progenitors migrate from bone marrow and seed

thymus.. T cell progenitors undergo differentiation to CD
thymus CD44
and CD8
CD8 cells in thymus
thymus.. Mature CD4
CD4 and CD8
CD8 T cells
circulate between blood and lymphoid tissues until they
meet antigens presented on dendritic cells in lymphoid
tissues.. T cells further undergo maturation to become
tissues
functional memory or effector T cells in LT
TCR : T cell receptor; Heterodimer consisting of
two non-identical polypeptide chains which are
linked together by disulfide bonds. Two different
TCR molecules TCR1 and TCR2 are known.
known
‰ More than 95% of T cell express the αβ
heterdimer (TCR2), which is the member of
immunoglubulin superfamily of molecules.

‰TCR1 is composed of γ and δ chains.

CD3 : is a protein complex and is composed of five

distinct chains (γ, δ, ε, ζ,η)that closely
associated with TCR.

CD4

CD8
 TCR genes undergo DNA rearrangement in thymus

*No Ds in Vα gene; DJ first then VDJ in β gene rearrangement

Un-rearranged

rearranged

expression

rearranged

Un-rearranged
 The Thymus
stromal cells:
maturatiion
 Positive
P iti and
d
Negative
Selection of
Thymocytes
in the Thymus
Two-step Selection Process of Thymocytes

Positive Selection: permits the survival of only

those T cells whose TCRs recognize self-MHC
molecules.

Negative Selection: eliminates T cells that react

too strongly with self-MHC or with self-MHC
plus
l self-peptides.
lf tid

Thymic stromal cells

cells, which express high levels
of class I and class II MHC molecules, play a role
in this process.
p
- Any developing thymocytes that are unable to
recognize self-MHC molecules or that do have a high
affinity for self-Ag plus self-MHC (or self-MHC alone) are
eliminated by y programmed
p g cell death.

- Thus, only those cells whose receptors recognize a self-

MHC molecules
l l plus
l fforeign
i A
Ag are allowed
ll d tto mature.
t

- An estimated 95%
95%--99%
99% of all thymocyte progeny
undergo programmed cell death within the thymus
without ever maturing.
g
Positive
P iti selection
l ti selects
l t T cells
ll
that recognize peptides on self
MHC
This is to assure that mature T cells can respond
to antigen-presented on self MHC.

-Self MHC I and II harboring self peptides on

thymic epithelial cells recognize and activate
TCRs on some DP thymocytes.

-DP thymocytes should receive this signal within

3-4 days to survive.Otherwise they undergo
apoptosis.
apoptosis
Negative selection eliminates T cells with
TCRs that bind too strongly or too week to
self antigen/MHC complex.
This is to assure that T cells don’t react against self
antigens In other words,
antigens. words autoreactive cells are
removed by this process.

Dendritic cells and macrophages in cortico-medullary

junction mediate it.

Negative selection cannot eliminate T cells whose

receptors are specific for self peptides that are
expressed outside of thymus (These cells enter
circulation, but soon to be rendered anergicg or
unresponsive by other mechanims).
 C t ki
Cytokines
and
d
Ch
Chemokines
ki
 Cytokines
Low molecular weight soluble proteins
(polypeptides) produced in response to
microbes and other antigens

They act via cell surface receptors to mediate

and regulate the amplitude and duration of
the immune-
immune-inflammatory responses
responses,,
through activation of macrophages,
controlling growth and differentiation of T
and B cells
 C t ki N
Cytokine Names
„Interleukins - produced exclusively by
leukocytes
„Lymphokines - produced by lymphocytes
„Monokines - produced exclusively by
monocytes
y
„Interferons - involved in antiviral responses
„Colony Stimulating Factors - support the
growth of cells in semisolid medias
„Chemokines
C - promote chemotaxis.
chemotaxis.
 Functional Categories of Cytokines
Cytokines
C t ki classified
l ifi d according
di to
t their
th i biologic
bi l i actions
ti
into three groups:

1) Mediators and regulators of innate immunity

- Produced by activated microphages and NK cells

in response
p to microbial infection

- they act mainly on endothelial cells and

leukocytes to stimulate the early inflammatory
response to microbes
 Functional Categories of Cytokines
2) Mediators
M di t and
d regulators
l t off acquired
i d immunity
i it
- Produced mainly by T lymphocytes in response to
specific recognition of foreign antigens
- They include IL
IL--2, IL-
IL-4, IL-
IL-5,, IL-
IL-13
13,, IFN,
Transforming growth factor-
factor-β (TGF
(TGF--β) and
lymphotoxin (TNF
(TNF-- β)

3) Stimulators of haematopoiesis
- Produced byy bon marrow,, stormal cells,, leukocytes
y
- Stimulate growth and differentiation of leukocytes
- Stem cell factor, IL
IL--3, IL-
IL-7, GM-
GM-CSF
 Interferons (IFNs)
* Interferons (IFNs): are proteins secreted in response to
viral infections or other stimuli

* They
y include:

- INF
INF--α produced by leucocytes
induced by virus infected cells
- INF
INF--β produced by fibroblasts

- INF
INF--γ p
produced by
y NK cells,TH1
cells,TH1 cells, CD8
CD8 T-cells
 Interferons (IFNs)
Action of INF-
INF-α and IFN-
IFN-β :

- Prevent viral replication

- Increase MHC-
MHC-I expression on viral infected cells
helping their recognition by CD8 T-cells

- Increase cytotoxic action of Nk cells

- Inhibit cell p
proliferation and tumor g
growth
 Interferons (IFNs)
Action of IFN-
IFN-γ :

- Activate Macrophages

- Increase expression of MHC-

MHC-I and II on APCs

- Enhance cytotoxic actions of Nk cells

- Promote production of TH1

TH1 and inhibits
proliferation of TH
p TH22
 Therapeutic
p Uses of Cytokines
y
1) Interferon in treatment of viral diseases, cancer

2) Several cytokines are used to enhance T- T-cell activation

in immunofideficincy
imm nofideficinc diseases
diseases,, e.g.
e g IL
IL--2, IFN-
IFN-γ,TNF
,TNF-
TNF-α

3) IL-
IL-2 and lymphokine activating killer cells (LAK) in
treatment of cancer

4) GM
GM--CSF induces increase in white cell count,
count, it is used:
a- To restore leukocytic count after cytotoxic
chemotherapy induced neutropenia
b- After bon marrow transplantation
C- To correct AIDS
AIDS--associated leukopenia
 Therapeutic Uses of Cytokines
5) Anti-
Anti-cytokines antibodies in management of
autoimmune diseases and transplant rejection:
rejection:
a- Anti
A
Anti-
i-TNF ini treatment rheumatoid
h id arthritis
h ii
b- Anti
Anti--IL
IL22 to reduce graft rejection

6) Anti
Anti--TNF antibodies in treating septic shock

7) Anti
Anti--IL
IL--2 in treating adult T-
T-cell leukemia

8) Anti
Anti--IL
IL--4 is under trial for treatment of allergies
Hypersensitivity
H iti it
Reactions
Allergies
 Hypersensitivity Reactions
„ Allergies Greek = altered
reactivity
„ 1906 – von Pirquet coined
term: hypersensitivity
te ype se s t ty
 Definition
‰ Refers to damaging immunologic reactions that
result from the in vivo interaction of the antigen and
th immune
the i response the
th individual
i di id l makesk t that
to th t
antigen (usually called an allergen in this context).
context).
‰ The persons first contact with the allergen induces
an immune response.
response
p . Subsequent
q exposure
p produces a
p
reaction that leads to tissue damage.
damage.
‰ These reactions are called hypersensitivity reactions
because they are exaggerated responses (or
sensitivities ) to materials that are usually innocuous
when first encountered.
encountered.
 Hypersensitivity Reactions

„ (In 1968)
1968) Gell and Coombs classification :
• Type I – IgE mediated (allergy)
• Type
yp II – Antibody-
Antibody
y-mediated cytotoxic
y
• Type III – Immune Complex mediated
• Type IV – Delayed
Delayed--Type Hypersensitivity
(DTH)
„ T
Types II, II and
d III are “immediate”
“i di t ”
„ Type
ype IV iss delayed
de ayed
 …Types

„ Immediate Hypersensitivity
„ initiated by antibody or antigen
antigen--antibody complexes
„ Symptoms appear within minutes

„ Delayed Type Hypersensitivity

„ Due to cytokines produced by activated TH1 and cytotoxic T cells
„ Symptoms take days to appear
 Hypersensitivity reaction depends on:

‰ Chemical nature of allergen.

‰ Route involved in sensitization i.e.
i h l ti ingestion,
inhalation, i ti injection…
i j ti
‰ Physiological state of individual / genetic
p
potential
 Type I hypersensitivity
(Immediate
I di t hypersensitivity
h iti it reactions)
ti )

‰ Initiated by antigens reacting with cell

bound immunoglobulin E (IgE IgE)) antibody
antibody..
‰ Release of pharmacologically active
mediators from mast cells or basophils .
‰ This release is triggered by the cross
cross--linking
of cell bounds
bo nds IgE molecules
molec les by
b antigens
antigens..
 Cross linking of FcεRI to
Cross-linking
IgE bounded to Ag

Degranulation of Mediators
e.g. Histamin , Leukotrienes
and Prostaglandins
 List of Allergens
„ Pollens
„ W d
Weeds
„ Foods--
Foods -->Crab
>Crab shrimp,
>Crab, shrimp potato,
potato tuna etc
etc.
„ Epidermals--
Epidermals -->
> dog, cat, mouse
„ Insect bites (bee, wasp )
„ House dust
„ Drugs
 Diseases

② Asthma ③ Hay fever

① Urticaria

④Insect allergy
allergy:: serious or fatal
anaphylaxis may follow.
follow.
Edema of larynx, with airway
obstruction may occur
occur..
 Type I Hypersensitivity

„ Skin testing
• Potential allergens
g
are injected or
scratched into the
skin
• If the patient is
allergic a wheal &
flare response
occurs
 Laboratory tests for allergy

Total IgE
IgE::
Competitive solid phase
EIA
Or allergen specific IgE
Using wells coated with
the allergen
 Type II Hypersensitivity
(Cytotoxic reactions)
‰ Initiated by antibody
antibody--either IgG or IgM with
cell--bound antigen
cell antigen..
‰ Complement--mediated cytolysis
Complement cytolysis..
‰ Antibody--dependent cell
Antibody cell--mediated cytotoxicity
(ADCC).
(ADCC)
‰ Anti--receptor
Anti p antibodies.
‰ Complement
C
Complement-
l t-dependent
d d t reactions
ti that
th t lead
l d
to lysis
y of cells or render them susceptible
p to
phagocytosis.. e.g. Transfusion reactions
phagocytosis
‰ IgG
IIgG-
G-coatedd target cells
ll are killed
kill d by
b cells
ll that
h
bear Fc receptors
p for IgG
g (e.g.,
g , NK cells,,
macrophages). e.g. Graft rejection
‰ In this example, acetylcholine receptor
antibodies impair neuromuscular transmission
in myasthenia gravis.
 yp Ⅲ Hypersensitivity
3. Type yp y
(Immune complex-
complex-mediated)

„ Induced by antigen
antigen--antibody
complexes that produce tissue damage
as a result of their capacity to activate
complement
p system
y
 3. Type Ⅲ Hypersensitivity
(
(Immune complex-mediated))
complex-
„ Antibody produced in response to exposure to antigen, forms
immune complexes
p of antigen
g and antibody y which mayy circulate
circulate..
„ Complexes cause no symptoms, quickly disappear from the
circulation..
circulation
„ In some individuals the immune complexes persist in circulation
causing clinical symptoms, some of them serious.
serious.
„ Size of complexes
p produced seems important
p p in determining g
whether they will be eliminated quickly from the body or retained
long enough to cause damage
damage..
„ Classical clinical symptoms of immune complex disease are due
to blood vessel involvement, i.e., vasculitis
vasculitis..
„ Blood vessels of joints and the kidney are most frequently
affected
affected, giving rise to symptoms of arthritis and
glomerulonephritis..
glomerulonephritis
 yp Ⅲ Hypersensitivity
3. Type yp y
(Immune complex-
complex-mediated)
„ Mechanisms are as follows:
• Soluble immune complexes which contain a greater proportion of antigen
than antibody
y ppenetrate blood vessels and lodge
g on the basement
membrane
• At the basement membrane site, these complexes activate the complement
cascade.
• During complement activation, certain products of the cascade are
produced,`attract neutrophils to the area. Such substances are known as
chemotactic substances.
• Once
O th
the polymorphs
l h reachh the
th basement
b t membrane
b th
they release
l their
th i
granules, which contain lysosomal enzymes which are damaging to the
blood vessel.
• This total process leads to the condition recognized histologically as
vasculitis..
vasculitis
• When it occurs locally (in the skin) it is known as an Arthus Reaction,
when it occurs systemically as a result of circulating immune complexes it
is know as serum sickness.
Schematic illustration of the three sequential phases in the induction of
systemic type Ⅲ (immune complex) hypersensitivity.
Serum Sickness
 ..types off antigens
i
„ Exogenous antigens :
„ Bacteria :streptococci causing Glom.Nephritis,
Glom.Nephritis,
Infective endocarditis etc
„ Virus : Hepatitis B
„ Fungus : Actinomyces (Farmers lung)
„ Parasite : malarial parasites
„ D
Drugs : fforeign
i serum ((serum sickness),
i k )
quinidine (haemolytic anaemia
anaemia))
 ..types off antigens
i
„ Endogenous antigens : antibodies are produced
against self components
„ Nuclear antigens
g
„ Systemic Lupus Erythematosus (SLE)
„ Immunoglobulins
„ Rheumatoid Arthritis (RA)
„ Tumour antigens
„ Glomerulonephritis
p
 Systemic Diseases
„ Chronic immune complex diseases are
naturally occurring diseases caused by
deposits of immune complex and
complement
l in
i the
h tissues.
i
• SLE
• Acute g
glomerulonephritis
p
• Rheumatic fever
• Rheumatoid arthritis
 Type IV or Delayed
Hypersensitivity
„ First observed by Robert Koch in 1890
1890..
„ The reaction elicited by antigen occurs relatively slowly
(hence the name "delayed hypersensitivity"). Usually
takes 24 – 28 hours
„ The hypersensitivity is mediated via T-
T-cells and
macrophages.
„ The hypersensitivity illustrates both antigen-
antigen-specific (T-
(T-
cell) and antigen non-
non-specific (macrophage)
characteristics.
„ No histamine or chemically related substances are
released from cells
Type IV or Delayed
H
Hypersensitivity
iti it
 ..some of the causative agents (Intracellular
pathogens
th & contact
t t antigens)
ti )

„ Intracellular pathogens like : M.tuberculosis

M.tuberculosis,,
M.Leprae , Brucella etc..
„ Intracellular Fungi like :Candida albicans,
albicans,
Histoplasma,, Cryptococci etc
Histoplasma
„ Intracellular parasites like :Leishmania
:Leishmania species
etc..
t
„ Intracellular viruses like :Herpes
p simplex,
p
smallpox (variola
(variola),
), measles etc..
Contact antigens like : Hair dyes , poison ivy,
poison oak, nickel salts etc.
Tuberculin (TB) Test
 Diseases
Di

① Chronic active hepatitis

②Some
o autoimmune
o disease
③ Contact dermatitis
④ Graft rejection
⑤ Inflammatory bowel disease.
disease.
Blistering skin lesions
on hand
h d off patient
ti t
with poison
p ivy
y
contact dermatitis
Summary
 Tissue
Transplantation
 Types of Grafts
‰ Sy
Syngrafts,
g s, oor isografts
sog s , involve
vo ve thee transfer
se o of normal
o tissue
ssue
between identical (syngeneic) individuals (i.e., identical twins or the
same inbred line).

‰Autografts are grafts removed and placed in the same individual

(e g skin graft from a person’s leg to his face).
(e.g., face)

‰ Allografts
g or homografts
g , involve the transfer of normal tissue
between allogeneic individuals (i.e., genetically different individuals
of the same species).

‰Heterografts, or xenografts (also called xenogenec grafts), are

tissues transferred between animals of two different species (e.g.,
(e g
baboon liver transplanted into human)
 Rejection of transplanted tissue:

Graft versus Host reaction

‰ Fever
‰ Skin rashes
‰ Inflammation surrounding
g transplanted
p tissue
 Successful tissue transplantation
Histocompatibility
p y antigens
g or transplantation
p antigens
g =
tissue compatibility
compatibility..
Antigens expressed on cell surfaces that determine the compatibility
or incompatibility of transplanted tissue ,these antigens induce the
immune response in the host that may cause rejection of transplanted
tissue
i .
Main factors in successful tissue transplantation?
‰ ABO
A O antigens
i off red
d blood
bl d cell
ll system alsol act as strong
transplantation antigens.
antigens.
‰ Minor histocompatibility antigens,
antigens such as those coded for the Y
chromosome can also contribute to graft rejection.
rejection.
‰ Major histocompatibility antigens (MHC) (MHC)..These antigens are
termed Human Leukocyte Antigens (HLA) because they are found in
high concentrations on lymphocytes and other white blood cells .
HLAs also occur on the nucleated cells in the body like macrophage
and hepatocytes
hepatocytes..
 HLA
„ Found on the short arm of chromosome 6.
„ Also called H-
H-2 (histocompatibility group 2) in mice
found on chromosome 17 17..
HLA Components
Class I - HLA
HLA--A , HLA-
HLA-B, HLA
HLA--C: ~ 2000 bp
Class II - HLA
HLA--DR , HLA
HLA--DQ,
DQ HLA-
HLA-DP : ~ 1000
bp
Cl
Class III - C2 , C4
C4, TNF,
TNF 21
21--Hydroxulase....etc
H d l t :
~ 1000 bp
Genomic Structure of MHC Region
g
„ Class I antigens occur on all nucleated human cells (except
sperm ) and d on platelets
platelets.
l t l t . In
I the
th mouse, however,
h th
the
corresponding H-2 MHC antigens also present on
erythrocytes
erythrocytes.
th t .
„ Class II antigens are found chiefly on the surface of
immunocompetent cells, including dendritic cells,
macrophage, monocytes,
monocytes, resting T cells (in low amounts),
activated T cells, and B cells.
cells.
„ Class III antigens do not participate in MHC restriction or
graft rejection, are associated with certain complement
components (C2 (C2, C4,and factor B of the alternative
pathway),with tumor necrosis factors α and β (TNF (TNF--α and
TNF--β) ,and with two hydroxylase enzymes
TNF enzymes.. These
molecules are known as class III MHC antigens, although
they are not involved in histocompatibility
histocompatibility..
 Class I antigens
„ MHC-I consists of 2 glycopeptide chains
MHC-
„ Alpha (α) chain is 338 amino acids long
„ ~ 281 aa extracellular
„ 3 domains α1
α1, α2
α2, α3
α3 about 90aa
90aa each
„ α1-α2 regions – hypervariable – binding site
„ ~ 25 transmembrane
„ ~30 intracellular tail (heavily phosphorylated)
phosphorylated)
 Class I antigens
„ Beta (ß) chain is invariant - same in all individuals
„ Gene on chromosome #15 #15
„ Non--covalently bound to α chain between α2
Non α2 and α3
d
domains
i
„ 44 kDa
„ Not transmembrane
 Class II antigens
„ MHC-II consists of 2 glycopeptide chains
MHC-
„ Both transmembrane
„ Alpha (α)
(α) chain 33-
33-35 kDa
„ Beta (ß) chain 26-
26-28 kDa
„ 10--20 copies of MHC-
10 MHC-II per cell membrane
„ Increased with immune activation
 Class II antigens
„ MHC-II chains non-
MHC- non-covalently attached to each
other
„ α1 & α2 domains
„ ß1 & ß2 domains
d i
 HLA and diseases
„ HLA complex has been associated with over a 100 diseases,
many y of them autoimmune diseases
„ includes diseases such as asthma, type I diabetes, rheumatoid
arthritis,, p
psoriasis..
psoriasis
„ HLA--B27 linked to Ankylosing Spondylitis
HLA
„ 87%
87 % of patients are B27B27+
+ (males)
„ Reiter’s Syndrome (arthritis) Risk Factor 37x
37x
„ DR2
DR 2
„ Multiple Sclerosis 4x
„ Disseminated Leprosy 5x
„ Goodpasture’s Syndrome 16x 16x
„ Allele is one of two or more forms of the DNA sequence of a
particular gene
 HLA and diseases
„ HLA-DR3
HLA- DR3
„ Addison’s Disease – 6x
„ HLA--DR
HLA DR44
„ Rheumatoid Arthritis – 5x
„ HLA--B5 - low response to Tetanus Toxoid in
HLA
vaccine (& toxin)
toxin)..
„ HLA--B7 or HLA-
HLA HLA-B8 higher incidence of cat
allergies
„ HLA--A2 or HLA
HLA HLA--A28 high incidence of ragweed
allergies
 Roles of HLA
„ Utilization in medicine.
medicine
„ Transplant
p tissue typing.
yp g
„ Transfusion therapy.
„ Paternity testing.
„ Disease predictions.
predictions
„ Preventative measures can be implemented.
„ Insurance Ratings or Treatment Protocols.
„ Ethical Issues!!!
The Complement System

DR GHASSAN MOHAMMAD SULAIMAN

DR.
DEPARTMENT OF APPLIED SCIENCE
SCHOOL OF BIOTECHNOLOGY
• Complement – A series of serum and membrane
expressed proteins involved in the effector role of the
immune response to pathogens
• Made up of approximately 30 circulating and membrane-
membrane
bound proteins (10% of the total serum proteins).
• Synthesized
y in the liver and byy cells involved in the
inflammatory response.
• All Complement
p proteins
p are zymogens
y g :
A zymogen (or proenzyme) is an
inactive enzyme precursor. A zymogen requires
a biochemical
bi h i l changeh (
(suchh as a hydrolysis
h d l i reaction
ti
revealing the active site, or changing the configuration to
reveal the active site) for it to become an active enzyme.
enzyme
• Activation occurs by three mechanisms.
 Complement pathway
A) Classical pathway:

- Complement is activated by antigen –antibody

complex (IgM or IgG)

- Fc portion of the antibody form a binding site for C1q

- The numerical sequence of the complement factors in the

classic pathway is:
C1q,r,s , C4, C2, C3, C5, C6, C7, C8, C9
Component Protein Complex
C1
 A)) Classical Pathwayy
The reaction sequence divided into three stages:
1) Recognition
R iti stage:
t
- C1q act as the recognition element
- It
I binds
bi d to Fc
F portion
i off IgM
I M or IgG
I G
- The activated C1 molecule can cleave many C4 molec.

2) Activation stage:
The complement components C4, C2, C3, C5, C6, C7,
C8, C9 participate in that order

3) Membrane attack stage:

Complement components C5, C6, C7, C8, C9 participate where
cell membrane damage and cell lysis occur
 Recognition Unit C1qrs
C4,
C2 C4a, Anaphylatoxin
C2b Kinin-like activity
C4b2a
C3
Anaphylatoxin
C3a

Activation Unit C4b2a3b

C5
Anaphylatoxin
C5a Chemotoxin
Membrane Attack Complex C5b

C5b-6-7
Anaphylatoxin activity : triggering
degranulation of mast cells as well as
increasing vascular permeability and smooth
5b-6-7-8 muscle contraction

5b-6-7-8-9 Lysis
Classical pathway of complement
activation
Membrane attack complex
 Complement Classical pathway
• Classical pathway: is triggered by activation of the C1-complex (C1q,
two molecules of C1r,, and two molecules of C1s thus forming g
C1qr2s2)
• Activation of the C1-complex
p occurs when C1q q binds to IgM
g or IgG
g
complexed with antigens or when C1q binds directly to the surface of
the pathogen
• A single IgM can initiate the pathway, while multiple IgGs are
needed)
• Such bindings lead to conformational changes in the C1q molecule,
which leads to the activation of two C1r (a serine protease) molecules
 Complement Classical pathway

• They then cleave C1s (another serine protease)

• The C1r2s2 component now splits C4 and then C2, producing
C4a,C4b,C2a,and C2b
• C4b and C2a bind to form the classical pathway C3-convertase
(C4bC2a complex)
• C3-convertase (C4b2a complex) promotes cleavage of C3 into C3a
and C3b
• C3b later jjoins with C4b2a ((the C3 convertase)) to make C5
convertase (C4bC2aC3b complex).
• C3b binds to the surface of ppathogens
g leadingg to greater
g
internalization by phagocytic cells by opsonization
 Complement

• In all three pathways,

pathways a C3-convertase cleaves and activates
component C3, creating C3a and C3b and causing a cascade of
further cleavage and activation events
• C5b initiates the membrane attack pathway, which results in the
membrane attack complexp ((MAC), ), consistingg of C5b,, C6,, C7,, C8,,
and polymeric C9
• MAC is the cytolytic endproduct of the complement cascade; it
forms a transmembrane channel, which causes osmotic lysis of the
target cell.
• C3b binds to the surface of pathogens leading to greater
internalization by phagocytic cells by opsonization
 B)) Alternative pathway
p y
This pathway is initiated by:
* Bacterial endotoxin,
endotoxin polysaccharide capsule,
capsule
aggregates of IgE and properdin

* It starts at C3 then C5, C6, C7, C8, C9

* The complement components C1, C4, C2 are by-passed

* Antibodies are not required to initiate activation of

this
hi pathway
h

* This pathway provides a means of non-specific

resistance
 Complement
p Alternative p
pathway
y
• Alternative complement pathway: is triggered by spontaneous C3
hydrolysis directly due to the breakdown of the thioester bond via
condensation reaction (C3 is mildly unstable in aqueous
environment) to form C3a and C3b
• C3b is then capable of covalently binding to a pathogenic membrane
surface if it is near enough
• If there is no pathogen in the blood, the C3a and C3b protein
fragments will be deactivated by rejoining with each other.
• Upon
p binding g with a cellular membrane, C3b is bound by
y factor B to
form C3bB
• This complex
p in ppresence of factor D will be cleaved into Ba and Bb
Complement
p Alternative p
pathway
y

• Bb will remain covalently bonded to C3b to form C3bBb which

is the alternative pathway C3-convertase.
• The C3bBb complex, which is "hooked" onto the surface of the
pathogen, will then act like a "chain saw," catalyzing the
hydrolysis of C3 in the blood into C3a and C3b
• This positivelyy affects the number of C3bBb hooked onto a
pathogen .
• After hydrolysis
y y of C3,, C3b complexes
p to become C3bBb,,
which cleaves C5 into C5a and C5b.
 Complement
p Alternative p
pathway
y

• After hydrolysis of C3, C3b complexes to become C3bBb,

which
hich clea
cleaves
es C5 into C5a and C5bC5b withith C6,
C6 C7,
C7 C8,
C8 and
C9 (C5b6789) complex to form the membrane attack
complex
• The membrane attack complex, also known as MAC, which
is inserted into the cell membrane, "punches a hole," and
initiates cells lysis.
y
• C5a and C3a are known to trigger mast cell degranulation
• IgA is associated with activating the alternative path
 C) Complement Lectin pathway
• Lectin pathway (MBL - MASP): Mannose
Mannose-binding
binding lectin pathway
• The lectin pathway is homologous to the classical pathway, but
with the opsonin, mannose
mannose-binding
binding lectin (MBL), and ficolins,
instead of C1q
• Ficolins are homologous to MBL and function via MASP
(Mannan-binding lectin-Associated Serine Protease) in a similar
way In vertebrates without an adaptive immune system,
way. system ficolins
are expanded and their binding specificities diversified to
compensate for the lack of pathogen-specific
pathogen specific recognition
molecules.
• This pathway is activated by binding mannose-binding
mannose binding lectin to
mannose residues on the pathogen surface
 Complement Lectin pathway

• This activates the MBL-associated serine proteases, MASP-1, and

MASP-2 (very similar to C1r and C1s, respectively),which can
then split C4 into C4a and C4b and C2 into C2a and C2b
• C4b and C2a then bind together to form the C3-convertase, as in
the classical pathway
Classical C3 convertases
C1q/C1r/C1s
C1 /C1 /C1 C5 convertases
t
C4 C4b+C2 C4b2a
C4a C4b2a3b
C3 C3a C5 C5b
Lectin C3bBbC3b C5a C6
MBP/MASP-1 C5b6
/MASP2 C7
C3
C3a C3bBb C5b67
C3 C3b P,B,D C8
C3(H2O)+B
C3(H2O) B C3(H2O)Bb C5b678
D C9
Alternative Ba C5b6789

Complement pathway
 Classic And Alterenative pathways

Classic Pathway Alternative pathway

* Specific acquired immunity * Non-specific innate immunity

* Initiated by antibody * Bacterial endotoxin, capsule

* Interaction
I t ti off allll components
t * C1,
C1 C4,
C4 C2 are by-passed
b d

* Properdin system not involved * Properdin system is involved

 Biological Effects of Complement

Beneficial effects:
1) Cytolysis:

- activated complement proteins polymerize on cell

surfaces of bacteria or erythrocyte to form pores

in its membrane (killing by osmotic lysis)

 Biological Effects of Complement

2) Opsonization:

- binding of complement proteins opsonin (C3b)

to surfaces of foreign
g organisms
g or p
particles

- Phagocytic
g y cells express
p specific
p receptors
p for
opsonins, so promote phagocytosis
 Biological Effects of Complement
3) Inflammatory response :
S ll ffragments
Small t released
l dd during
i complement
l t
activation have several inflammatory actions:

a) C5a is chemotactic and attract neutrophiles and

macrophages

b) C3,C4 and C5 are anaphylatoxins

Cause degranulation of mast cells and release of
histamine and other inflammatory mediators
 Biological Effects of Complement

4) Immune complex clearance:

- C3b facilitate binding of immune complex to several
surfaces (erythrocytes) and enhance removal by liver
and spleen

- binds erythrocytes to blood vessels , make them as

easy
y prey
p y for p
phagocytosis
g y

- C3 deficiency
y associated with Immunocomplex p
disease and susceptibility to recurrent infections
 Biological Effects of Complement

5 Enhancement of antibody production:

5-Enhancement

- Binding
Bi di off C3b to
t its
it receptors
t on activated
ti t d B cells
ll
(CR2) greatly enhances antibody production

- Patient who are deficient in C3b produce

muchh lless antibody
ib d than
h normall iindividuals
di id l and
d
more susceptible to pyogenic infection
 Complement
p Deficiencies

• SLE – C1, C4, or C2 deficiencies

• Membranoproliferative
p Glomerulonephritis
p –
C3 deficiency.

• Properdin and factor B and D defects

 Complement deficiency and
disease

• C3 deficiency – severe,
severe recurrent life-
life
threatening infections with encapsulated
microbes.

• C1 inhibitor deficiency – hereditary

angioneurotic edema.

• C1, C2, C4 deficiency – autoimmune disease.

‫بسم اللة الرحمن الرحيم‬
 Tolerance
And
Autoimmune Diseases
 Tolerence
* It is a specific immunologic unresponsiveness

i.e. the absence of specific immunoresponses

to a particular
i l antigen
i in
i a fully
f ll
immunocomptent person

* Unresponsiveness to self antigens is known as

auto
t tolerance
t l

* Both B-cells and T

T--cells participate in tolerence

* But T-cells play the primary role

 Central Tolerance
Clonal–
Clonal–deletion:

* The process by which T-cells acquire the

ability to distinguish self from non self,
self,
in fetal thymus

* This involves the killing of T-

T-cells that
react against antigens present in the
fetus at that time
 Peripheral Tolerance
* T-cell tolerance (clonal anergy
anergy)):
a lack
ac oof reaction
eact o by tthe
e body
body's
s de
defense
e se mechanisms
ec a s s to
foreign substances, and consists of a direct induction of
peripheral lymphocyte tolerance

* Some self
self--reactive T cells are not killed
in thymus

* Functional inactivation of surviving

g self
self--
reactive T cells
 Tolerance
* B-
B-cells become tolerant to self by two mechanisms:

1) Clonal deletion
Probably while B-
B-cell precursors are in bon marrow

2) Clonal anergy
B cells in the periphery

* Tolerance in B-
B-cells is less complete than in T-
T-cells

* The most autoimmune diseases are mediated by

y
antibodies
 Factors Influencing
g The Induction Tolerance

1) Immunologic maturity of the host:

Neonates are immunologically immature and well
acceptt allograft
ll ft th
thatt would
ld be
b rejected
j t d by
b mature
t host
h t

2) Structure
St t and
d dose
d off antigen:
ti
a- Simple molecules induce tolerance more readily than
complex
l ones

b- Very high and very low doses of antigen may result

in tolerance
Factors Influencing The Induction Tolerance

3) T-cells become tolerant more readily and remain

tolerant longer than B-cells

4) The continuous presence of antigen helps to

maintain tolerance

5) Administration of immunosuppressive drugs

enhances tolerance as in transplantation
 Clinical Importance of Tolerance
1) Organ transplantation:
Introduction of tolerance may help in prevention of
rejection
j ti

2) Tumor
T d
development:
l t
Tolerance to tumor antigen results in growth of the
tumor without
ih b
being
i d
detected
dbby the
h iimmune
mechanisms

3) Autoimmune disorders:
Di t b
Disturbance off self-
self
lf-tolerance
t l results
lt iin autoimmune
t i
disease
Autoimmune Diseases
 Autoimmune Diseases
* Autoimmune diseases occur due to
breakdown of the mechanisms that
maintain auto tolerance

* Auto-
Auto-antibodies and self reactive T-
T-cells
are produced, resulting in tissue
damage by several mechanisms
 Etiology Of Autoimmune Diseases
1) Genetic predisposition:
- Familial incidence of autoimmune diseases
- Most of them appear to be associated with certain
MHC genes,
genes, specially MHC II genes

e.g. Rheumatoid arthritis is associated with DR4

DR4
Thyroditis with DR5
DR5
Multiple sclerosis with DR2
DR2
SLE with
ith DR2
DR2/DR
/DR33
Type I diabetes with DR3
DR3/DR4
/DR4
Ankylosing spondylitis with B27
B27
Rheumatoid Arthritis
 Etiology Of Autoimmune Diseases
2) Exposure to infectious antigens “Molecular mimicry”
mimicry”
that cross react with self antigens

- An immune response to these antigen will result

in immune attack against self antigens

e.g.
g Antibodies against
g M protein
p of Streptococcus
p
pyogens may react with heart valves and cause
Rheumatic fever
3) Alteration of self antigens or the appearance of new
antigens
g under the effect of drugs,
g , chemicals,, or viral
infections

4) Hormonal influences play a role e.g.

e g SLE affects
women 10 times more than men
 5) Cryptic Antigen

„ Ag sequestered from immune system

„ Damage & release of antigen. (injury or
surgery)
¾ e.g. Eye → sympathetic ophthalmia
¾ Testes → anti
anti--sperm & orchitis
Antibodies in blood can attack Myelin
y Basic
Protein if Blood-
Blood-Brain barrier is breached.
 Mechanisms Of Disease Production

* The disease may be organ specific

e.g Hashimoto thyroditis

* The disease may be systemic

e g SLE or rheumatoid arthritis
e.g.

1) Binding
Bi di off an autoantibody
ib d to host
h cells
ll result l
in complement fixation and tissue destruction
e.g. Haemolytic
H l i anemiai (Type II hypersensitivity)
 Mechanisms Of Disease Production

2) Formation of immune complexes and their

deposition in tissues, joints, kidney and skin
The immune complexes fix complement resulting in
tissue damage
e.g. SLE and rheumatoid arthritis (Type III hyper.)

3) DTH reactions (Type IV)) due to auto reactive T- T-cells

e.g. Ulcerative colitis, multiple sclerosis and
type I diabetes
 Laboratory
y Diagnosis
g
1- There is elevated serum immunoglobulins

2- Complement levels may be decreased

3- Immune complexe detected in serum or organ biopsy

4- Auto antibodies can be detected in serum

e.g. anti
anti-
ti-nuclear,
l anti
anti-
ti-smooth
th muscles,
l Rh ffactor
t
and anti-
anti-mitochondrial Ab

5- Testing for antibodies specific to particular Ag,

Ag,
involved in organ specific diseases (anti
(anti--thyroid Ab)
‫الرحيم‬
‫يم‬ ‫ن ر‬ ‫الرحمن‬
‫ببسمم ﷲ ر‬
Tumor immunology
 Tumor immunology
gy
* Pathological cell masses derived by abnormal and
uncontrollable clonal expansion of single cell

* Transformation of normal cells to malignant cells by:

a- Spontaneous mutation during daily cell division

chemical carcinogens
g
b- It may be induced by physical carcinogens
viruses

* Cells become antigenically different from normal cells

* They are recognized and destroyed by immune system

 Etiology Of Tumor
1) Inherited :
Expression of inherited oncogene
e.g.
g viral g
gene incorporated
p into host g
gene

2) Viral:
- Human
H papilloma
papilloma,
ill , herpes
h type 2, hepatitis
h i i B HBV
HBV,
Epstein - Barr Virus EBV (DNA)
- Human T T--cell leuckemia virus (RNA)

3) Chemical:
- Poly cyclic hydrocarbons cause sarcomas
- Aromatic amines cause mammary carcinoma
- Alkyl nitroso amines cause hepatoma

4) Radiological: Ultraviolet & ionizing irradiation

5) Spontaneous: failure in the cellular growth control

 Tumor Associated Antigens
1) Viral Antigen :
a- Viral p
proteins and g
glycoproteins
y p
b- New antigens produced by virally infected host
cells under control of viral nucleic acid

2) Tumor specific antigens :

- Tumor cells develop new antigen specific to
their carcinogens

3) Tumor specific transplantation antigens :

- Tumor cells express new MHC antigens due to
alteration of normally present MHC antigens
 Tumor Associated Antigens
g
4) Oncofetal antigens:

a- Carcino
Carcino--embryonic antigens (CEA)
- Normally expressed during fetal life on fetal gut
- Reappearance in adult life:
Gastrointestinal tract (GIT), pancreas, biliary
system and cancer breast

b- Alpha fetoprotein:
- Normally expressed in fetal life
- Reappearance in adult life; hepatoma
 Immune Surveillance System

* During neoplastic transformation, new antigen develop

* The host recognize them as nonself antigens

* Cell mediated immune reactions attack these

nonself tumor cells

* Immune response act as surveillance system

to detect and eliminate newly arising neoplastic cells
 Immune Surveillance System
y
This system include :
1) Natural
N l killer
kill (NK) cells
ll
They kill directly tumor cells,helped by interferon, IL
IL--2

2) Cytotoxic T-cells
They also kill directly tumor cells

3) C
Cell
ll mediated
di t d T
T--cells
ll ((effector
effector
ff t T-cells)
ll )
They produce and release a variety of lymphokines :
a-Macrophage activation factor that activate macrophag
b-Gamma interferon and interleukin-
interleukin-2 that activate NK
c-Tumor necrosis factor (cachectine
(cachectine))
 Immune Surveillance System
4) B
B--cells
ll :
- Tumor associated antigens stimulate production of
specific
p antibodies by
y host B-
B-cells

- These specific antibodies bind together on tumor cell surface

leading to destruction of tumor through:

a- Antibody mediated
mediated--cytotoxicity :
kill
Cytotoxic T-
T-cells IgG
IgG--coated tumor cells

b- Activation of macrophages
release
Sensitized T-
T-cells macrophage activating factor
IgG--coated tumor cells
IgG macrophages activate

c- Activation of classical pathway of complement leading to

Lysis of tumor cells
 Tumor Escape
p
Mechanisms by which tumor escape immune defenses:

1) Reduced levels or absence of MHCI molecule on

tumor so that they can not be recognized by CTLs

2) Some tumors stop expressing the antigens

These tumors are called “antigen loss variants”

3) Production of immunosuppressive factors by tumor

e g transforming growth factor (TGF
e.g. (TGF--β)

4) Tumor antigens may induce specific immunologic

tolerance
 Tumor Escape
p
5) Tumor cells have an inherent defect in antigen
processing and presentation

6) Blocking of receptors on T-
T-cells by specific antigen
antibodies complex (after
(after shedding of tumor Ag)
prevents them from recognizing and attacking
tumor cells

7) Antigens on the surface of tumors may be masked

by sialic acid-
acid-containing mucopolysaccharides

8) Immune suppression
pp of the host as in transplant
p
patients who show a higher incidence of malignancy
 Tumor Markers
* Tumor markers :

Tumor antigens
* They are either or
Tumor products
(enzymes and hormones)

* Tumor products are released in the serum of patients

* They are used to confirm diagnosis and follow up the

response to therapy
 Tumor Antigens
g
1) Alpha fetoprotein antigen (AFP) in cases of hepatoma

2) Carcinoembryoinic antigen (CEA) in gastrointestinal

tumors tumors of biliary system and cancer breast
tumors,

3) Cancer
C antigen
ti 125 (CA 125
125)) in
i ovarian
i carcinoma
i

4) Cancer
C antigen
ti 15--3 (CA
15 (CA15
15-3) in
15- i bbreastt cancer

5) C
Cancer antigen 19-
19
9-9 in colon
l and
d pancreatic tumor

6) Prostatic specific antigen (PSA) in prostatic tumors

 Tumor Products
a) Hormones :
- Human chorionic gonadotrophins (HCG) are secreted
in cases of choriocarcinoma
- Thyroxin (T3
(T3 & T4
T4) is secreted in cases of cancer
of thyroid gland

b) Enzymes :
- Acid phosphatase enzymes in cases of cancer
prostate

- Alkaline phosphatese,
phosphatese, lipase and amylase enzymes in
cases of cancer pancreas
 Immunology and Hematology

1. Clinical Immunology and Serology a Laboratory Prospective.

2010. Christine Dorresteyn Stevens, F.A. Davis Company,
USA.
2. HARRISON’S Hematology and Oncology. 2010. Dan L. Longo,
USA.
3. Advances in Medical Immunology. 2002. Fifth Edition Revised
and Expanded. Gabriel Virella Marcel Dekker, Inc. USA.
4. Basic Immunlogy Functions and Disorders of the Immune
System. 2004. Abdul K. Abbas and Andrew H. Lichtman,
USA.
 
 ‫الموضوع‪ :‬المناعة‬ ‫الجامعة التكنولوجية‬
‫المرحلة‪ :‬الثالثة‬ ‫قسم العلوم التطبيقية‬
‫الزمن‪ :‬ثالث ساعات‬ ‫فرع التقنيات االحيائية‬
‫التدريسي‪ :‬د‪ .‬غسان محمد سليمان‬ ‫األمتحانالنھائې‪/‬‬
‫‪2011-2010‬‬

‫‪Solutions.‬‬

‫)‪Q1: Draw with labeling the following? (Choose 2 only; 10 Marks‬‬

‫‪1- Digestion of IgG immunoglobulin‬‬

‫‪2- Phagocyte of microbe‬‬

3- The complement of classical pathway

Q2: Answer the following? (Choose 2 only; 10 Marks)

1- Precipitation reactions and give the principle assay for one test.
• Immunodiffusion
• Mancini
• Oudin
• Ouchterlony
• Immunoelectrophoresis

2- Types of allergens that associated with type I hypersensitivity.

Pollens
Weeds
Foods-->Crab, shrimp, potato, tuna etc.
Epidermals--> dog, cat, mouse
Insect bites (bee, wasp )
House dust
Drugs

3- Types of antigens that associated with detection of tumors.

1) Alpha fetoprotein antigen (AFP) in cases of hepatoma
2) Carcinoembryoinic antigen (CEA) in gastrointestinal tumors, tumors of biliary system
and cancer breast
3) Cancer antigen 125 (CA 125) in ovarian carcinoma
4) Cancer antigen 15-3 (CA15-3) in breast cancer
 5) Cancer antigen 19-9 in colon and pancreatic tumor
6) Prostatic specific antigen (PSA) in prostatic tumors

Q3: Compare between the following? (Choose 2 only; 10 Marks)

1-Positive selection and negative selection.
Positive Selection: permits the survival of only those T cells whose
TCRs recognize self-MHC molecules. Self MHC I and II harboring
self peptides on thymic epithelial cells recognize and activate TCRs
on some DP thymocytes.
Negative Selection: eliminates T cells that react too strongly with
self-MHC or with self-MHC plus self-peptides. Dendritic cells and
macrophages in cortico-medullary junction mediate it. Negative
selection cannot eliminate T cells whose receptors are specific for
self peptides that are expressed outside of thymus (These cells enter
circulation, but soon to be rendered anergic or unresponsive by other
mechanims).

2-Innate immunity and acquired immunity.

Components of the immune system

Innate immune system Adaptive immune system

Response is non-specific Pathogen and antigen specific response

Exposure leads to immediate maximal response Lag time between exposure and maximal response

Cell-mediated and humoral components Cell-mediated and humoral components

No immunological memory Exposure leads to immunological memory

Found in nearly all forms of life Found only in jawed vertebrates

 3-Class I MHC antigen and Class II MHC antigen.
„ Class I antigens:
MHC-I consists of 2 glycopeptide chains
Alpha (α) chain is 338 amino acids long
~ 281 aa extracellular
3 domains α1, α2, α3 about 90aa each
α1-α2 regions – hypervariable – binding site
~ 25 transmembrane
~30 intracellular tail (heavily phosphorylated)
Beta (ß) chain is invariant - same in all individuals
Gene on chromosome #15
Non-covalently bound to α chain between α2 and α3 domains
44 kDa
Not transmembrane

„ Class II antigens:
MHC-II consists of 2 glycopeptide chains
Both transmembrane
Alpha (α) chain 33-35 kDa
Beta (ß) chain 26-28 kDa
10-20 copies of MHC-II per cell membrane
Increased with immune activation
MHC-II chains non-covalently attached to each other
α1 & α2 domains
ß1 & ß2 domains
Q4: Define the following items? (Choose 5 only; 10 Marks)
(1) Antigenicity: Is the ability to combine specifically with the final
products of the [immune response] (i.e. secreted antibodies and/or
surface receptors on T-cells). Although all molecules that have the
property of immunogenicity also have the property of antigenicity, the
reverse is not true.
(2) Paratope: The paratope is the part of an antibody which recognises
an antigen, the antigen-binding site of an antibody. It is a small region
(of 15–22 amino acids) of the antibody's Fv region and contains parts
of the antibody's heavy and light chains.
(3)  
Adjuvant: is a substance administered with an immunogen that
increases the immune response. It acts by producing a local
inflammatory response that attracts a large number of immune system
cells to the injection site.
(4) C5 convertase: C5-convertase is an enzyme involved in
the complement system. Its primary function is to cleave C5 protein
to C5a and C5b. C5a is a smaller product and diffuses into the plasma,
whereas C5b remains and initiates the formation of membrane attack
complex (MAC).
(5) Molecular mimicry: Exposure to infectious antigens “Molecular
mimicry” that cross react with self antigens - An immune response to
these antigen will result in immune attack against self antigens e.g.
Antibodies against M protein of Streptococcus pyogens may react
with heart valves and cause Rheumatic fever.
(6) Clonal anergy: a lack of reaction by the body's defense
mechanisms to foreign substances, and consists of a direct induction
of peripheral lymphocyte tolerance.

Q5: Give the full name and the function of the following abbreviations?
(Choose 5 only; 10 Marks)).
(1) CA15-3 Cancer antigen 15-3 (CA15-3) in breast cancer.
(2) CFA: Complete Freund’s adjuvant (CFA)-” mineral oil,
emulsifier, and killed mycobacteria”. Increase the cell –mediated
response.
(3) RIA: Radioimmunoassay is widely-used because of its great
sensitivity. Using antibodies of high affinity, it is possible to detect a
few picograms (10−12 g) of antigen in the tube. The greater the
specificity of the antiserum, the greater the specificity of the assay.
 (4) CD: Cluster of differentiation : Antigenic features of leukocytes
that are identified by groups of monoclonal antibody expressing
common or overlapping activity.

(5) GM-CSF: Granulocyte-Macrophage Colony Stimulating Factor

induces increase in white cell count, it is used:
a- To restore leukocytic count after cytotoxic chemotherapy induced
neutropenia.
b- After bone marrow transplantation.
C- To correct AIDS-associated leukopenia

(6) GALT: Gut-Associated Lymphoid Tissue. This is comprised of:

Tonsils, adenoids (Waldeyer's ring).
Peyer's patches.
Lymphoid aggregates in the appendix and large intestine.
Lymphoid tissue accumulating with age in the stomach.
Diffusely distributed lymphoid cells and plasma cells in the lamina
propria of the gut.

Q6: Discrimination between T - Cell subsets using the following items:

(1) symbol (2) Surface antigen (3) MHC restriction (4) Target cell (5)
Function. (10 Marks)

T–cell Symbol Identifying MHC Target cell Function

subtype surface restriction
antigen

Cytotoxic Tc CD8 Class I Tumours and Kills foreign

virally cells
infected
cell
Allografts

Helper Th CD4 Class II B cell and Tc Interleukin

cell secretion

Inducer Th CD4 Class II B cell and Tc Interleukin

cell secretion

Suppressor Ts CD8 Class I B cell ,Th and Down –

Tc cell regulat
es cell
growth
Delayed-type Tdth CD4 Class II Macrophage, Release
hypersensi Tc cell lympho
tivity kines
Memmory Tm CD8;CD4 Both B and T cell Anamnesis
Q7: What is immunologic tolerance? What are its influencing factors,
and why is it important?
(10 Marks)

Immunologic tolerance: It is a specific immunologic unresponsiveness

1- Immunologic maturity of the host.
2- Structure and dose of antigen.
3- T-cells become tolerant more readily and remain tolerant longer
than B-cells
4- The continuous presence of antigen helps to maintain tolerance
5- Administration of immunosuppressive drugs enhances tolerance as
in transplantation.
Clinical Importance of Tolerance
1) Organ transplantation: Introduction of tolerance may help in
prevention of rejection.
2) Tumor development: Tolerance to tumor antigen results in growth
of the tumor without being detected by the immune mechanisms
3) Autoimmune disorders: Disturbance of self-tolerance results in
autoimmune disease.
 ‫ المناعة‬:‫الموضوع‬ ‫الجامعة التكنولوجية‬
‫ الثالثة‬:‫المرحلة‬ ‫قسم العلوم التطبيقية‬
‫ ثالث ساعات‬:‫الزمن‬ ‫فرع التقنيات االحيائية‬
‫ غسان محمد سليمان‬.‫ د‬:‫التدريسي‬ /‫األمتحانالنھائې‬
2011-2010

Solutions

Q1: A- Which of MHC genes groups include alleles closely associated

with the susceptibility to develop the following diseases?
(Choose 5 only; 5 Marks)
1- Ankylosing spondylitis (HLA-B27) 2-  Goodpasture’s Syndrome
(HLA- DR2) 3-  Ragweed allergies (HLA-A2 or HLA-A28)
4-  Rheumatoid Arthritis (HLA-DR4) 5-  Addison’s Disease
(HLA-DR3) 6-Tetanus (HLA-B5)
B- What is the phenomenon of negative selection, and what is its
importance? (5 Marks)
Negative Selection: eliminates T cells that react too strongly with self-MHC or
too weak with self-MHC plus self-peptides. Thymic stromal cells, which
express high levels of class I and class II MHC molecules, play a role in
this process. Dendritic cells and macrophages in cortico-medullary
junction mediate it.
Negative selection cannot eliminate T cells whose receptors are specific for self
peptides that are expressed outside of thymus (These cells enter circulation,
but soon to be rendered anergic or unresponsive by other mechanims).
Importance: This is to assure that T cells don’t react against self antigens. In
other words, autoreactive cells are removed by this process.

Q2: When antigens enter through the skin, in what organs are they
concentrated? What cell type (s) plays important roles in this process
of antigen capture? (10 Marks)
Skin is an active participant in host defense. It has the capability to
generate and support local immune and inflammatory responses to foreign Ags
that enter the body via the skin. Cells of SALT include keratinocytes,
Langerhans cells (immature DCs found in skin), intraepiethelial T cells, and
melanocytes. Langerhans cells form a continuous epidermal meshwork: they
capture Ag, then migrate to draining lymph nodes, where they act as Ag-
presenting cells.
Q3: Discrimination between Gell and Coombs reactions using the
following items:
(1) Common name (2) Principle of reaction (3) One example of
disease. (10 Marks)

Q4: Give the full name and the function of the following abbreviations?
(Choose 5 only; 10 Marks)

(1) LAK--- lymphokine activating killer cells (LAK) used in treatment

of cancer.
(2) AFP ----Alpha fetoprotein antigen (AFP) in cases of hepatoma.
(3) BCG ---- Bacille Calmette Gurein (BCG) has also been used as an
immunotherapeutic agent to boost the immune system in patients with
special types of cancer (e.g., superficial bladder cancer).
(4) PHA----Type of adjuvant phtohemaglutinin (PHA) increasing the
clonal expansion of B and T cells.
(5) PMN ---- Polymorphonuclear leukocyte (PMN) Neutrophil:
Predominant type of white blood cell. Rapidly migrate to sites of
infection or inflammation. Phagocytic, they have special enzymatic
pathways for enhanced bacteriocidal action.
(6) MALT ----- Mucosa – Associated Lymphoid Tissue (MALT)
Lymphoid tissue found at the gastrointestinal tract, respiratory tract and
urogenital tract. MALT consists of aggregates of lymphocytes,
macrophages, DCs, and other accessory cells.
Q5: Define the following items? (Choose 5 only; 10 Marks)
 
(1) Epitope: or Antigenic determinant Small part of an antigen that
interacts with an antibody and T cells receptors. Usually between one
and six sugars or amino acids on the surface of the antigen. Any given
antigen may have several epitopes. Each epitope is recognized by a
different antibody. 
(2) Hapten: is capable of binding to an antibody but unlike an
immunogen, is NOT capable of evoking an immune response such as
the production of antibodies. Is small non –immunogenic molecule
and is the equivalent of an epitope and can bind to a single antigen
binding site on an antibody molecule. Haptens include low molecular
weight chemicals (e.g. benzene, dinitrophenol (DNP), drugs (e.g.
aspirin), and antibiotics (e.g. penicillin).
(3) C4b2a: C3-convertase cleaves and activates component C3,
creating C3a and C3b and causing a cascade of further cleavage and
activation events. 
(4) Clonal selection:  The ability of the immune system to respond to
an antigen exists before it ever encounters that antigen.The immune
system relies on the prior formation of an incredibly diverse
population of: B cells (B lymphocytes) each with its surface covered
with thousands of identical copies of a receptor for antigen(the B-cell
receptor for antigen = BCR).T cells (T lymphocytes) each with its
surface covered with thousands of identical copies of a T-cell receptor
for antigen (TCR).
(5) CD3: is a protein complex and is composed of five distinct
chains (γ, δ, ε, ζ,η)that closely associated with TCR.

Q6: Compare between the following?

(Choose 2 only; 10 Marks)
1-Classical pathway and Alternative pathway.
Classic Pathway Alternative pathway
* Specific acquired immunity * Non-specific innate immunity
* Initiated by antibody * Bacterial endotoxin, capsule
* Interaction of all components * C1, C4, C2 are by-passed
* Properdin system not involved * Properdin system is involved
 2-Primary immune response and secondary immune response.

Primary antibody response Secondary antibody response

first exposure to antigen
lag period: days or weeks (slow onset)
Small amount immunogl.
low Ab level with gradual increase
Ab Persist for short duration Weeks then
decline rapidly
Antibody is IgM
Subsequent exposure
Lag period: hours (rapid onset)
large amount immunogl.
high Ab with rapid increase
Persist for long periods (monthes
or years)
Antibody is IgG

3-Passive immunization and active immunization.

Active Immunization Passive Immunization

The immunized individuals own cells This provides humoral (antibody

contribute to their immunity -based) immunity but not T-cell
based immunity.

Immunity is long lasting and easily Immunity is short lasting ,which

reactivated by booster injections of is approximately 3 weeks.
antigen.
Free dangers as comapered with passive There is danger of causing
immunity . anaphylaxis or serum sickness.
Both B-cell and T-cell are activated. B-cell

Prophylactic immunization Prophylactic and/or protective

immunization .
Q7: Draw with labeling the following? (Choose 2 only; 10 Marks)
1- Typical immunoglobulin.

2- Production of monoclonal antibodies.

3- Inflammatory reaction.
 ‫الموضوع‪ :‬المناعة‬ ‫الجامعة التكنولوجية‬
‫المرحلة‪ :‬الثالثة‬ ‫قسم العلوم التطبيقية‬
‫الزمن‪ :‬ثالث ساعات‬ ‫فرع التقنيات االحيائية‬
‫التدريسي‪ :‬د‪ .‬غسان محمد سليمان‬ ‫األمتحانالنھائې‪/‬‬
‫‪2011-2010‬‬

‫‪Solutions.‬‬

‫‪Q1: How do phagocytes ingest and kill microbes? Explain your answer‬‬
‫‪with drawing and methods of microbes killing mechanisms.‬‬
‫)‪(10 Marks‬‬
Q2: What are the functions of the complement system, and what
components of complement mediate these functions?
(10 Marks)

Beneficial effects:
1) Cytolysis:
- activated complement proteins polymerize on cell surfaces of
bacteria or erythrocyte to form pores in its membrane (killing by
osmotic lysis)
2) Opsonization:
- binding of complement proteins opsonin (C3b) to surfaces of
foreign organisms or particles - Phagocytic cells express specific
receptors for opsonins, so promote phagocytosis.
3) Inflammatory response:
Small fragments released during complement activation have several
inflammatory actions:
a) C5a is chemotactic and attract neutrophiles and
macrophages
b) C3,C4 and C5 are anaphylatoxins Cause degranulation of mast
cells and release of histamine and other inflammatory mediators
4) Immune complex clearance:
- C3b facilitates binding of immune complex to several surfaces
(erythrocytes) and enhances removal by liver and spleen
- binds erythrocytes to blood vessels , make them as easy prey for
phagocytosis
- C3 deficiency associated with Immunocomplex disease and
susceptibility to recurrent infections
5-Enhancement of antibody production:
- Binding of C3b to its receptors on activated B cells (CR2) greatly
enhances antibody production.
- Patient who are deficient in C3b produce much less antibody than
normal individuals and more susceptible to pyogenic infection.
Q3: Compare between the following? (Choose
2 only; 10 Marks)
1-Positive selection and negative selection.

Positive Selection: permits the survival of only those T cells whose

TCRs recognize self-MHC molecules. Self MHC I and II harboring
self peptides on thymic epithelial cells recognize and activate TCRs
on some DP thymocytes.
Negative Selection: eliminates T cells that react too strongly with
self-MHC or with self-MHC plus self-peptides. Dendritic cells and
macrophages in cortico-medullary junction mediate it. Negative
selection cannot eliminate T cells whose receptors are specific for
self peptides that are expressed outside of thymus (These cells enter
circulation, but soon to be rendered anergic or unresponsive by other
mechanims).

2-Innate immunity and acquired immunity.

Components of the immune system

Innate immune system Adaptive immune system

Response is non-specific Pathogen and antigen specific response

Exposure leads to immediate maximal response Lag time between exposure and maximal response

Cell-mediated and humoral components Cell-mediated and humoral components

No immunological memory Exposure leads to immunological memory

Found in nearly all forms of life Found only in jawed vertebrates

 3-Class I MHC antigen and Class II MHC antigen.

„ Class I antigens:
MHC-I consists of 2 glycopeptide chains
Alpha (α) chain is 338 amino acids long
~ 281 aa extracellular
3 domains α1, α2, α3 about 90aa each
α1-α2 regions – hypervariable – binding site
~ 25 transmembrane
~30 intracellular tail (heavily phosphorylated)
Beta (ß) chain is invariant - same in all individuals
Gene on chromosome #15
Non-covalently bound to α chain between α2 and α3 domains
44 kDa
Not transmembrane

„ Class II antigens:
MHC-II consists of 2 glycopeptide chains
Both transmembrane
Alpha (α) chain 33-35 kDa
Beta (ß) chain 26-28 kDa
10-20 copies of MHC-II per cell membrane
Increased with immune activation
MHC-II chains non-covalently attached to each other
α1 & α2 domains
ß1 & ß2 domains
Q4: Define the following items?
(Choose 5 only; 10 Marks)
(1) Crypt antigen:
Ag sequestered from immune system Damage & release of antigen.
(injury or surgery)
e.g. Eye → sympathetic ophthalmia
Testes → anti-sperm & orchitis . Antibodies in blood can attack
Myelin Basic Protein if Blood-Brain barrier is breached.
(2) Paratope: The paratope is the part of an antibody which
recognises an antigen, the antigen-binding site of an antibody. It is a
small region (of 15–22 amino acids) of the antibody's Fv region and
contains parts of the antibody's heavy and light chains.
(3)  
Adjuvant: is a substance administered with an immunogen that
increases the immune response. It acts by producing a local
inflammatory response that attracts a large number of immune system
cells to the injection site.
(4) C5 convertase: C5-convertase is an enzyme involved in
the complement system. Its primary function is to cleave C5 protein
to C5a and C5b. C5a is a smaller product and diffuses into the plasma,
whereas C5b remains and initiates the formation of membrane attack
complex (MAC).
(5) Molecular mimicry: Exposure to infectious antigens “Molecular
mimicry” that cross react with self antigens - An immune response to
these antigen will result in immune attack against self antigens e.g.
Antibodies against M protein of Streptococcus pyogens may react
with heart valves and cause Rheumatic fever.
(6) Clonal anergy: a lack of reaction by the body's defense
mechanisms to foreign substances, and consists of a direct induction
of peripheral lymphocyte tolerance.
 
Q5: Give the full name and the function of the following abbreviations?
(Choose 5 only) (10 Marks)
(1) CA125
Cancer antigen 125 (CA 125) in ovarian carcinoma
 (2) Fab: Fragment Antigen Binding
(3) ELISA: Enzyme – linked immunosorbant assay to detct Ag or
Ab .
(4) IFN-γ: Inerferon gama
Action of IFN-γ:
- Activate Macrophages
- Increase expression of MHC-I and II on APCs
- Enhance cytotoxic actions of Nk cells
- Promote production of TH1 and inhibits
proliferation of TH2

(5) GM-CSF
Granulocyte-Macrophage Colony Stimulating Factor induces
increase in white cell count, it is used:
a- To restore leukocytic count after cytotoxic chemotherapy induced
neutropenia.
b- After bone marrow transplantation.
C- To correct AIDS-associated leukopenia

(7) SALT: Skin Associated Lymphoid Tissue:

Skin is an active participant in host defense. It has the capability to
generate and support local immune and inflammatory responses to foreign
Ags that enter the body via the skin. Cells of SALT include keratinocytes,
Langerhans cells (immature DCs found in skin), intraepiethelial T cells,
and melanocytes. Langerhans cells form a continuous epidermal
meshwork: they capture Ag, then migrate to draining lymph nodes, where
they act as Ag-presenting cells

Q6: Discrimination between Igs types using the following items (1)
Heavy chain symbol (2) Complement fixation (3) Opsonization.
(10 Marks)

Property IgG IgA IgM IgE IgD

Heavy chain γ α µ ε δ
symbol
Complement Yes No Yes No No
fixation
Opsonization Yes No No No No
Q7: Answer the following? (Choose 2 only; 10 Marks)
1- Cytokine names and their functions.

Interleukins - produced exclusively by leukocytes

Lymphokines - produced by lymphocytes
Monokines - produced exclusively by monocytes
Interferons - involved in antiviral responses
Colony Stimulating Factors - support the growth of cells in semisolid
medias Chemokines - promote chemotaxis

Function:
1) Interferon in treatment of viral diseases, cancer
2) Several cytokines are used to enhance T-cell activation in
immunofideficincy diseases, e.g. IL-2, IFN-γ,TNF-α
3) IL-2 and lymphokine activating killer cells (LAK) in treatment of
cancer
4) GM-CSF induces increase in white cell count, it is used:
a- To restore leukocytic count after cytotoxic
chemotherapy induced neutropenia
b- After bon marrow transplantation
C- To correct AIDS-associated leukopenia
5) Anti-cytokines antibodies in management of
autoimmune diseases and transplant rejection:
a- Anti-TNF in treatment rheumatoid arthritis
b- Anti-IL2 to reduce graft rejection
6) Anti-TNF antibodies in treating septic shock
7) Anti-IL-2 in treating adult T-cell leukemia
8) Anti-IL-4 is under trial for treatment of allergies

2- Properties of immunogenicity, type of immunization and route

of administrations.
Degree of “foreignness”-Based on genetic relatedness.
Chemical composition (complexity).
proteins are the most potent immunogens- Primary, secondary, tertiary and
quaternary structure.
Most polysaccharides are weak antigens.
Nucleic acids in their pure form are nonimmunogenic.
Antigen size molecular size-usually MW >100,000; <10,000 non immunogenic;
10,000-100,000 MW is imunogenetically variable.
Degradability : Polystyrene or asbestosis are nonimmunogenic cause they cannot
processed by phagocyte cells.
 Type of immunization
1‐ Active Immunization 
2‐ Passive Immunization  

Route of administrations  
1- INJECTION by intramuscular or subcutaneous for both active and
passive immunization. Intravenous injection used for
immunoglobulins.
2- ORAL administration ”oral polio vaccine “.
3- INTRANASAL immunization for airborne pathogen.
4- HERD immunity

3- Characterization of Ag-Ab interactions and forces of binding.

1- Non-covalent interaction (similar to “lock and key” fit of enzyme-
substrate).
2- Does not lead to irreversible alteration of Ag or Ab.
3- This exact and specific interaction has led to many immunological
assays used to:
a. detect Ag or Ab
b. diagnose disease
c. measure magnitude of humoral IR
d. identify molecules of bio and med interest
Forces of binding:
1- Hydrogen Bond
2- Ionic Bond
3- Hydrophobic interactions
4- Van der Waals Interactions