Endogenous antigens

• Antigen presentation stimulates T cells to become either

"cytotoxic" CD8+ cells or "helper" CD4+ cells.
• Endogenous antigens are produced by intracellular
bacteria and viruses replicating within a host cell.
• The host cell uses enzymes to digest virally associated
proteins, and displays these pieces on its surface to T-cells
by coupling them to MHC.
• Endogenous antigens are typically displayed on MHC class
I molecules, and activate CD8+ cytotoxic T-cells.
• With the exception of non-nucleated cells (including
erythrocytes), MHC class I is expressed by all host cells.
 CD8+ T lymphocytes and cytotoxicity
• Cytotoxic T cells (also known as TC, killer T cell, or
cytotoxic T-lymphocyte (CTL)) are a sub-group of T cells
that induce the death of cells that are infected with
viruses (and other pathogens), or are otherwise damaged
or dysfunctional.
• Naive cytotoxic T cells are activated when their T-cell
receptor (TCR) strongly interacts with a peptide-bound
MHC class I molecule.
• This affinity depends on the type and orientation of the
antigen/MHC complex, and is what keeps the CTL and
infected cell bound together
• Once activated, the CTL undergoes a process called clonal
selection, in which it gains functions and divides rapidly to
produce an army of “armed” effector cells.
• Activated CTL then travels throughout the body searching
for cells that bear that unique MHC Class I + peptide.
• When exposed to these infected or dysfunctional somatic
cells, effector CTL release perforin and granulysin:
cytotoxins that form pores in the target cell's plasma
membrane, allowing ions and water to flow into the
infected cell, and causing it to burst or lyse.
• CTL release granzyme, a serine protease that enters cells
via pores to induce apoptosis (cell death).
Class I MHC proteins and cytotoxic T cells
(Tc)
The cell-cell interaction between
the infected cell and the Tc
cell is mediated by the
MHC/antigen complex and TCR

The Tc cell produces cytotoxic proteins

perforins—produce holes or pores in the
target cell and granzymes enter the
virus infected cell causing apoptosis or
programmed cell death

The cytotoxic proteins only affect those

cells to which the Tc cell has specifically
interacted
 Class I MHC proteins and
cytotoxic T cells (Tc)

Class I pathway is useful in destroying

cells that have been infected by viruses or
have been transformed by tumors

1. Protein antigens manufactured in the cell

by viruses or tumors are degraded in the
cytoplasm and transported to the
endoplasmic reticulum
2. The processed antigens bind to Class I
MHCs and are transported to the cell
surface
3. Together this complex interacts with the
TCR of a Tc cell, the binding of the
complex with the TCR is strengthened
by a CD8 co-receptor
• To limit extensive tissue damage during an infection, CTL
activation is tightly controlled and in general requires a
very strong MHC/antigen activation signal, or additional
activation signals provided by "helper" T-cells .
• On resolution of the infection, most effector cells die and
phagocytes clear them away—but a few of these cells
remain as memory cells.
• On a later encounter with the same antigen, these
memory cells quickly differentiate into effector cells,
dramatically shortening the time required to mount an
effective response.
 Exogenous antigens

• Dendritic cells engulf exogenous pathogens, such

as bacteria, parasites or toxins in the tissues and
then migrate, via chemotactic signals, to the T
cell-enriched lymph nodes.
• During migration, dendritic cells undergo a
process of maturation in which they lose most of
their ability to engulf other pathogens, and
develop an ability to communicate with T-cells.
• The dendritic cell uses enzymes to chop the pathogen into
smaller pieces, called antigens.
• In the lymph node, the dendritic cell displays these non-
self antigens on its surface by coupling them to a receptor
called the major histocompatibility complex, or MHC (also
known in humans as human leukocyte antigen (HLA).
• This MHC: antigen complex is recognized by T-cells passing
through the lymph node.
• Exogenous antigens are usually displayed on MHC class II
molecules, which activate CD4+T helper cells.
 Helper T-cells
• CD4+ lymphocytes, also called "helper" or
"regulatory" T cells, are immune response
mediators, and play an important role in
establishing and maximizing the capabilities of the
adaptive immune response.
• These cells have no cytotoxic or phagocytic
activity; and cannot kill infected cells or clear
pathogens, but, in essence "manage" the immune
response, by directing other cells to perform
these tasks.
• Helper T cells express T cell receptors (TCR) that
recognize antigen bound to Class II MHC
molecules.
• The activation of a naive helper T-cell causes it to
release cytokines, which influences the activity of
many cell types, including the APC (Antigen-
Presenting Cell) that activated it.
• Helper T-cells require a much milder activation
stimulus than cytotoxic T cells.
• Helper T cells can provide extra signals that "help"
activate cytotoxic cells.
 Class II MHC proteins
and helper T cells (TH)
The Class II proteins and antigen
are expressed on B cells, APCs
and macrophages

1. The APC takes up an external foreign

protein via phagocytosis or endocytosis
2. Class II proteins are produced in the
endoplasmic reticulum and assembled
with a blocking protein (Ii) or invarient
chain
3. The Class II proteins enter the
phagolysosome where the Ii is degraded
and the partially processed antigen
binds to the class II molecule
4. The complex is translocated to the
surface of the APC where it interacts
with the TCR of a T helper cell
Class II MHC proteins and helper T cells (TH)
Specialized TH cell involved in
the inflammatory response

Cell-cell interaction mediated

by the TCR and the class II
MHC-antigen complex activates
The TH cell which produces
cytokines
TNF-alpha (tumor necrosis factor)
IFN-gamma (interferon)
GM-CSF (granulocyte-monocyte
colony stimulating factor)

These cytokines further stimulate

macrophages to increase phagocytic
activity and to in turn produce cytokines
that promote inflammation
B lymphocytes and Humoural Immunity (antibody production)

• B cells function to protect the host by producing

antibodies that identify and neutralize foreign objects like
bacteria and viruses.
• B Cells are the major cells involved in the creation of
antibodies that circulate in blood plasma and lymph,
known as humoral immunity. Antibodies (also known as
immunoglobulin, Ig), are large Y-shaped proteins used by
the immune system to identify and neutralize foreign
objects.
• In mammals, there are five types of antibody: IgA, IgD,
IgE, IgG, and IgM, differing in biological properties; each
has evolved to handle different kinds of antigens.
• Upon activation, B cells produce antibodies, each of which
recognize a unique antigen, and neutralizing specific
pathogens.
• Antigen and antibody binding would cause five different protective
mechanisms:
• 1. Agglutination: Reduces number of infectious units to be dealt with
• 2. Activation of complement: Cause inflammation and cell lysis
• 3. Opsonization: Coating antigen with antibody enhances
phagocytosis
• 4. Antibody-dependent cell-mediate cytotoxicity: Antibodies attached
to target cell cause destruction by macrophages, eosinophils, and NK
cells
• 5. Neutralization: Blocks adhesion of bacteria and viruses to mucosa
• Like the T cell, B cells express a unique B cell receptor
(BCR), in this case, a membrane-bound antibody molecule.
• All the BCR of any one clone of B cells recognizes and
binds to only one particular antigen.
• A critical difference between B cells and T cells is how
each cell "sees" an antigen. T cells recognize their cognate
antigen in a processed form – as a peptide in the context
of an MHC molecule, whereas B cells recognize antigens in
their native form.
• Once a B cell encounters its cognate (or specific) antigen
(and receives additional signals from a helper T cell, it
further differentiates into an effector cell, known as a
plasma cell.
 B cells are coated with
antibodies that react with
Class II MHC proteins, specific antigens
helper T cells that When the antigen binds to the
stimulate antibody antibody, the B cell first acts
producing cells—the B cells as an APC.
The bound antigen is endo
cytosed and complexed with
MHC II and then surface
expressed

The surface
expressed complex
interacts with and
activates TH cells that produce
the cytokines interleukin 4 & 5
IL4 and 5 stimulates the B cells to produce
identical memory B cells and antibody
secreting plasma cells that secrete the
same antibody
• Plasma cells are short-lived cells (2–3 days) that secrete
antibodies.
• These antibodies bind to antigens, making them easier
targets for phagocytes, and trigger the complement
cascade.
• About 10% of plasma cells survive to become long-lived
antigen-specific memory B cells.
• Already primed to produce specific antibodies, these cells
can be called upon to respond quickly if the same
pathogen re-infects the host, while the host experiences
few, if any, symptoms.
 Immunological memory
• When B cells and T cells are activated some become memory B cells,
and some memory T cells.
• Throughout the lifetime of an animal these memory cells form a
database of effective B and T lymphocytes.
• Upon interaction with a previously encountered antigen, the
appropriate memory cells are selected and activated. In this manner,
the second and subsequent exposures to an antigen produce a
stronger and faster immune response.
• This is "adaptive" because the body's immune system prepares itself
for future challenges, but is "maladaptive" of course if the receptors
are autoimmune.
• Immunological memory can be in the form of either passive short-
term memory or active long-term memory.
 Passive memory

• Passive memory is usually short-term, lasting between a few days and

several months.
• Newborn infants have had no prior exposure to microbes and are
particularly vulnerable to infection.
• Several layers of passive protection are provided by the mother.
• In uterus, maternal IgG is transported directly across the placenta, so that,
at birth, human babies have high levels of antibodies, with the same range
of antigen specificities as their mother.
• Breast milk contains antibodies (mainly IgA) that are transferred to the gut
of the infant, protecting against bacterial infections, until the newborn can
synthesize its own antibodies.
• This is passive immunity because the fetus does not
actually make any memory cells or antibodies: It only
borrows them.
• Short-term passive immunity can also be transferred
artificially from one individual to another via antibody-rich
serum.
 Active memory

• In general, active immunity is long-term and can be

acquired by infection followed by B cells and T cells
activation, or artificially acquired by vaccines, in a
process called immunization.
 Hypersensitivity and Allergic Reactions
Hypersensitivity (hypersensitivity reaction) refers to
undesirable immune reactions produced by the normal
immune system.

• Allergies are hypersensitivities

• It is overreactions of the immune system to substances
that do not cause reactions in most people.
• Allergen (Substances that often cause reactions)also
called antigen that causes allergy.
 Types of Allergens
• Types of Allergens
• There are many different types of allergens that could
trigger an allergic
• Pollen
• Animals
• Dust Mites
• Foods (Egg, Milk, Peanut, Shellfish, Soy, Tree nut,
Wheat, Banana and strawberries)
• Insect Stings
• Latex
• Mold
Allergens
Sulfonamide Penicillins
drugs

salicylate
• Hypersensitivities are grouped into four types, I
through IV. Based on what parts of the immune
system are activated and how long it takes for a
reaction to occur.
• Atopy is the inherited propensity to respond
immunologically to such common naturally
occurring allergens with continuous production
of IgE antibodies.
nd Coombs classification of hypersensitiviti

Type I Type II Type III Type IV

IgE Mediated IgG/IgM IgG Mediated T cell

Mediated

Classic Allergy Immune Delayed

rbc lysis complex Type
Disease Hypersensitivity
 Types of Hypersensitibities

• two types of hypersensitivities commonly

associated with allergies: Type 1 and type 4

• type I (immediate hypersensitivities) in which

antigens (allergens) combine with specific IgE
(immunoglobulin E) antibodies to cause local and
sometimes systemic reactions – usually within
minutes
 Type I
• The first time a predisposed person (FH) is
exposed to a potential allergen, they will not have
a major reaction; instead, they will create a specific
IgE antibody and become “sensitized.

• ” The IgE antibody produced then attaches itself to

mast cells, in the tissues, and basophils in the
blood stream.
 Mechanism of type I hypersensitivity
Primary Generation
Allergen Individual IgE
Adhesion
Secondary
IgE binds to the FceRI on mast cell and basophil

Allergen binds to the IgE on primed target cell

Crosslikage of FceRI

Degranulate and release the biological mediators

Preformed granule mediators New generated mediators

Histamine BradykininLeukotrienes PAFProstaglandin D2

llaries,increase permeability, increase mucus secretion, contract sm

Systemic Skin Respiratory tract Degist tract

anaphylaxis
 Type IV
• type IV (delayed hypersensitivities) reactions caused by
the interactions of antigens with specific sensitized T
lymphocytes instead of antibodies.

• Type IV delayed hypersensitivity reactions are most often

skin reactions. Common examples include reactions to
metal and jewelry.
 Type IV
• They occur when an antigen interacts with specific
sensitized T lymphocytes. The lymphocytes release
inflammatory and toxic substances, which attract
other white blood cells to the exposure site,
resulting in tissue injury.

• Type IV hypersensitivity is usually a reaction

(redness, swelling, hardening of the skin, rash,
dermatitis) observed at the exposure site hours to
days after exposure
 Treatment of Allergy
• The three most effective ways to treat
allergies are
– 1- Avoidance
– 2- Immunotherapy
– 3- Medication.
 Immunologic tests: Antigen-Antibody interactions

1. Precipitation Rxns:
-Ab’s and Ag’s in aqueous soln’s form a lattice => Precipitin Lattice formation
requires: 1) polyvalent Ab’s
2) Ag must be bivalent, polyvalent

Precipitation rxns, once popular, have been replaced by faster, more sensitive tests
 Immunologic tests:

Precipitation rxns in gels

Immunologic tests:

2. Immunoelectrophoresis: Incorp electrophoresis

w/ double
diffusion
• An Ag mixture is 1st separated by charge
• Then, “troughs” are cut ∥to direction of electric
field and antisera is added to trough
• Ag’s and Ab’s diffuse towards each other to
produce precipitin bands
• Used to detect: a)presence/absence of
specific proteins or Ig
classes
b) immunodeficiency or immunoproliferative disorder
Immunologic tests:

Immunoelectrophoresis:
Immunologic tests:

3) Agglutination reactions – simple,

inexpensive, but
sensitive!
Several types exist:
a) Hemagglutination of RBC’s
b) Bacterial Agglutination
c) Passive Agglutination
d) Agglutination Inhibition
Immunologic tests:
4) Radioimmunoassay (RIA)– very sensitive test; used
for measuring hormones, serum proteins, drugs, etc.
at low [C]’s (≤ 0.001ug/ml)
measures “competitive binding” of radiolabelled Ag +
unlabelled (test) Ag to high affinity Ab
Immunologic tests:
5. ELISA tests: dep on enzyme conugated to 2 Ab reacting with a specific
substrate to produce a color rxn. Most sensitive of tests for Ag/Ab!!
Variations of ELISA’s:
Allows for qualitative or quantitative testing.
Each one can be used for qualitative detection of Ag or Ab
Also, a std curve based on known [C]’s of Ag/Ab can be prepped and an
unknown [C} can be ascertained
a. Indirect ELISA
b. Sandwich ELISA
c. Competitive ELISA
Immunologic tests: Types of ELISA’s…
Immunologic tests:
6) Western Blot

• Used to id specific
proteins in mixtures
• Proteins are separated on
SDS-PAGE
• Proteins then transferred
to membrane
• Membrane flooded w/
radio-labelled or enz-
linked poly/monoclonal
Ab’s specific for protein
Immunologic tests: 7) Immunoprecipitation

• Provides a quick and

sensitive test for finding
proteins/Ag’s
– Especially in low [C]’s
• Binds Ab to synthetic
bead support 
centrifuged
• Or 2° Ab w/ bead or
magnetic bead -> collect
by magnetism
Immunologic tests: 8) Immunofluorescence

• Provides a quick method for the id of

pathogens and lymphocytes
– Ab’s are conjugated with a fluorescent dye
(fluorescein, rhodamine, phycoerythrin)
– If Ab’s bind to specific Ag’s, they can be illum
w/ UV light and emit bright colors
– There are currently 2 methods employed:
• Direct staining
• Indirect staining
Direct and indirect Immunofluorescence
THANK
YOU