Immunogens and Antigens

Chapter 3
 Introduction
Antigen: any agent capable of binding specifically to
components of the immune system (eg. BCR on B cells)
Substances that can mobilize adaptive defenses and provoke an
immune response
Targets of all adaptive immune responses
Immunogen: any agent capable of inducing an immune
response immunogenic
All immunogens are antigens but not all antigens are
immunogens (eg. low molecular weight compounds such
as many antibiotics and drugs)
By themselves, these compounds (low MW hapten) are incapable
of inducing an immune response but when coupled with a larger
entity (carrier protein), the conjugate induces a response
Complete Antigens
Important functional properties
Immunogenicity: ability to stimulate
proliferation of specific lymphocytes
Reactivity: ability to react with activated
lymphocytes and antibodies released by
immunogenic reactions
Examples: foreign protein,
polysaccharides, lipids, and nucleic
acids
Requirements for Immunogenicity
Foreignness vs. self
we will not make antibody to human albumin but
will make antibody to rabbit albumin; the more
foreign the compound, the more immunogenic; autoimmunity is an
exception!!!
High molecular weight: small substances have immunogenicity
while large substances have immunogenicity

compounds less than 1000 daltons are not

immunogenic, i.e.., penicillin, aspirin,
progesterone
compounds greater than 6000 daltons are
immunogenic, i.e.., albumin, tetanus toxoid
150 kDa
66 kDa
 Chemical Complexity
homopolymers of amino acids such as a
polymer of lysine (30,000 Da) is not
immunogenic: not chemically complex enough
homopolymer of glutamic acid that makes up
the capsule of Bacillus anthracis (50 kDa) is not
immunogenic
Copolymers (coupled) of several amino acids
are highly immunogenic such as
polyglutamine, alanine and lysine
In general, increased chemical complexity means increased
immunogenicity!!!
Degradability
Many antigens are proteins and they
must be degraded by APC (antigen
processing: enzymatic degradation of antigen) and
presented on their surface for Th cells
The antigen must be susceptible to
enzymatic processing
Polypeptides of D-amino acids are not
immunogenic. Why? They are resistant to
enzymatic degradation (we do not have enzymes to cut them
into small pieces!!!)
Haptens
They are incomplete antigens
Haptens fail to induce an immunogenic
response
E.g., peptides, nucleotides, some
hormones
They are low molecular weight
compounds with chemical simplicity
When conjugated (chemical bound) to
high-molecular weight, complex
carriers, they are immunogenic
Drug Allergies
97% of drug
reaction allergies
that result in death
each year are due to
penicillin. (313-334)
1-5% of people
receiving penicillin
will develop some
form of allergy
Other Requirements for
Immunogenicity
Dosage and route of administration of
antigens are important
Number of doses administered affects
outcome
If dose (insufficient doses of antigen??) fails to activate
lymphocytes, no immune response, may require
repeated administration of antigen for a strong immune response

Route of administration can alter response

IV injection goes to spleen, subcutaneous
goes to local lymph nodes and Langerhans
 Orally administered antigens (GI route)
elicit local Ab response within intestine
Intranasal route can elicit allergic
responses
Seeding Secondary Lymphoid
Organs and Circulation
Immunocompetent B and T cells not yet
exposed to antigen called naive
Exported from primary lymphoid organs
(bone marrow and thymus) to "seed"
secondary lymphoid organs (lymph
nodes, spleen, etc.)
Increases chance of encounter with
antigen
B lymphocytes
Do not activate naive T cells
Present antigens to helper T cell to
assist own activation
Activation and
Differentiation of B Cells
B cell activated when antigens bind to
its surface receptors and cross-link
them
Receptor-mediated endocytosis of
cross-linked antigen-receptor
complexes (clonal selection)
Proliferation and differentiation into
effector cells
Fate of the Clones
Most clone cells become plasma cells
Secrete specific antibodies at rate of 2000
molecules per second for four to five days,
then die
Antibodies circulate in blood or lymph
Bind to free antigens and mark for destruction
by innate or adaptive mechanisms
Clone cells that do not become plasma
cells become memory cells
Antigen Encounter and
Activation
Clonal selection
Naive lymphocyte's first encounter with
antigen selected for further development
If correct signals present, lymphocyte will
complete its differentiation
Proliferation and
Differentiation
Activated lymphocyte proliferates
exact clones
Most clones effector cells that fight
infections
Few remain as memory cells
Able to respond to same antigen more
quickly second time
B and T memory cells and effector T
cells circulate continuously
Antigen Receptor Diversity
Genes, not antigens, determine which
foreign substances immune system will
recognize
Immune cell receptors result of acquired
knowledge of microbes likely in
environment
Lymphocytes make up to billion
different types of antigen receptors
Coded for by ~25,000 genes
Gene segments are shuffled by somatic
recombination
Table 21.3 Overview of B and T Lymphocytes
Cellular Immune Response
T cells provide defense against
intracellular antigens
Some T cells directly kill cells; others
release chemicals that regulate immune
response
Cellular Immune Response
Two populations of T cells based on
which glycoprotein surface receptors
displayed
CD4 cells usually become helper T cells
(TH); activate B cells, other T cells,
macrophages, and direct adaptive immune
response
Some become regulatory T cells which
moderate immune response
Can also become memory T cells
Cellular Immune Response
CD8 cells become cytotoxic T cells (TC)
Destroy cells harboring foreign antigens
Also become memory T cells
Helper, cytotoxic, and regulatory T cells are
activated T cells
Naive T cells simply termed CD4 or CD8
cells
Figure 21.16 Major types of T cells.
Adaptive defenses Cellular immunity

Immature
lymphocyte
Red bone marrow

T cell T cell
receptor receptor
Maturation

Class II MHC CD8 Class I MHC

protein displaying CD4 protein displaying
cell cell
antigen Thymus antigen

Activation Activation

APC
(dendritic cell) Memory APC
cells (dendritic cell)

CD4 CD8

CD4 cells become Lymphoid CD8 cells become

either helper T tissues and
cells or cytotoxic T
organs cells
regulatory T cells

Effector
cells

Blood plasma
 Primary and Secondary
Responses
First exposure to antigen is the primary
immunization
The first measurable response is the
primary response
The second exposure results in a stronger
secondary response
quick onset and higher magnitude
also called memory (anamnestic) response by
memory B & T cells
Memory T cells: central memory Tcm & effector memory Tem expressing for
different chemokine receptors
More on memory T cells
Recent studies suggest that Tcm are mostly
found in lymph nodes and have limited
effector function but can become effector
cells with subsequent stimulation
Intended for long term protection?
By contrast, Tem subpopulation has a strong
presence in peripheral tissues which can
rapidly produce cytokines (IFN) with antigenic
encounter but are limited in proliferative
capacity
Short term or immediate protection?
Antigenic Determinants
Only certain parts (antigenic
determinants) of entire antigen are
immunogenic
Antibodies and lymphocyte receptors
bind to them as enzyme binds substrate
Antigenic Determinants
Most naturally occurring antigens have
numerous antigenic determinants that
Mobilize several different lymphocyte
populations
Form different kinds of antibodies against
them
Large, chemically simple molecules
(e.g., plastics) have little or no
immunogenicity
Figure 21.7 Most antigens have several different antigenic determinants.

Antigen- Antigenic determinants

binding
Antibody A sites

Antigen

Antibody B
Antibody C
 Major Classes of Antigens
Carbohydrates (polysaccharides)
may be part of cell-surface polysaccharides
Lipids (may be regarded as haptens)
are rarely immunogenic unless conjugated to
carrier proteins
Nucleic acids
poor immunogens by themselves but are very
good when bound to carrier proteins
Proteins (due to size and complexity)
best immunogens, multideterminant antigens
Summary of Antigens
Self-antigens: MHC Proteins
Protein molecules (self-antigens) on
surface of cells not antigenic to self but
antigenic to others in transfusions or
grafts
Example: MHC glycoproteins
Coded by genes of major histocompatibility
complex (MHC) and unique to individual
Have groove holding self- or foreign
antigen
T lymphocytes can only recognize antigens that
are presented on MHC proteins
MHC Proteins and Antigen
Presentation
T cells respond only to processed
fragments of antigens displayed on
surfaces of cells
Antigen presentation vital for activation
of naive T cells and normal functioning
of effector T cells
MHC Proteins
Two types of MHC proteins important to
T cell activation
Class I MHC proteins displayed by all
cells except RBCs
Class II MHC proteins displayed by APCs
(dendritic cells, macrophages, and B cells)
Both types are synthesized at ER and
bind to peptide fragments
Class I MHC Proteins
Bind with fragment of protein synthesized in
the cell (endogenous antigen)
Endogenous antigen is self-antigen in normal
cell; a nonself antigen in infected or abnormal
cell
Crucial for CD8 cell activation
Inform cytotoxic T cells of microorganisms
hiding in cells (cytotoxic T cells ignore
displayed self-antigens)
Act as antigen holders; form "self" part that T
cells recognize
Class II MHC Proteins
Bind with fragments of exogenous
antigens that have been engulfed and
broken down in a phagolysosome
Recognized by helper T cells
Signal CD4 cells that help is required
What do T and B Cells See in
an Antigen?
T cells, unlike B cells, are not able to
bind soluble antigen
TCRs require processing of antigen and
association with MHC I (Tc) or MHC II (Th)
Generally T cells recognize internal,
denatured protein segments
Polysaccharides do not yield internal
epitopes and therefore do not activate T
cells
What do T Cells See in an
Antigen?
What do T and B Cells See in an Antigen?
B cells recognize 5-7 amino acids, the size that
binds to the IgD antibody
Th and Tc cells recognize 8-15 amino acids that
binds to the TCR
a few more amino acids are needed to bind with the
MHC II (Th) or MHC I (Tc) on APC
B cells have membrane-bound antibody that
binds free antigen in solution
the antigen will be on the outside of the molecule and
accessible (for interaction with B cell receptor)
antigen may be sequential or nonsequential amino
acids
 Protein
Structure
Sequential Epitopes

Sperm whale
myoglobin
showing
location of five
sequential
epitopes
Nonsequential Epitopes
Chicken egg
lysozyme showing
location of
nonsequential
epitopes
residues in red contact
antibody heavy chain

residues in blue contact

antibody light chain

residues in white contact

both antibody heavy and
light chains
Interaction between influenza virus and
antibody paratope on light & heavy chains
T cell Activation
T cell activation two-step process
Antigen binding
Co-stimulation
Both occur on surface of same APC
Both required for clonal selection
T cell Activation: Antigen
Binding
T cell antigen receptors (TCRs) bind to
antigen-MHC complex on APC surface
TCR that recognizes the nonself-self complex
linked to multiple intracellular signaling
pathways
Other T cell surface proteins involved in T cell
activation (e.g., CD4 and CD8 help maintain
coupling during antigen recognition)
T cell Activation: Co-
stimulation
Requires T cell binding to other surface
receptors on an APC co-stimulatory
signals
Dendritic cells and macrophages produce
surface B7 proteins when innate defenses
mobilized
B7 binding crucial co-stimulatory signal
Cytokines (interleukin 1 and 2 from
APCs or T cells) trigger proliferation and
differentiation of activated T cell
T cell Activation: Co-
stimulation
Without co-stimulation, anergy occurs
T cells
Become tolerant to that antigen
Are unable to divide
Do not secrete cytokines
T cell Activation: Proliferation
and Differentiation
T cells that are activated
Enlarge and proliferate in response to
cytokines
Differentiate and perform functions
according to their T cell class
Figure 21.17 Clonal selection of T cells involves simultaneous recognition of self and nonself. Slide 1
Adaptive defenses Cellular immunity

Bacterial antigen 1 Antigen

presentation
Class lI MHC Dendritic cell engulfs
protein an exogenous
displaying antigen, processes it,
processed and displays its
bacterial antigen fragments on class II
Dendritic Co-stimulatory MHC protein.
cell molecule

CD4 protein 2 Double recognition

T cell 2a CD4 T cell
receptor recognizes antigen-
(TCR) MHC complex. Both
TCR and CD4 proteins
bind to antigen-MHC
Co-stimulatory CD4 T cell complex.
molecules
2b Co-stimulatory
Clone molecules bind
formation together.

3 Clone
formation Activated
CD4 T cells proliferate
(clone), and become
memory and effector
cells.
Memory
CD4 T cell Helper
T cells
T cell Activation: Proliferation
and Differentiation
Primary T cell response peaks within a week
T cell apoptosis occurs between days 7 and
30
Benefit of apoptosis: activated T cells are a hazard
produce large amount inflammatory cytokines
hyperplasia, cancer
Effector activity wanes as amount of antigen
declines
Memory T cells remain and mediate
secondary responses
Roles of Helper T (TH) cells
Play central role in adaptive immune
response
Activate both humoral and cellular arms
Once primed by APC presentation of
antigen, they
Help activate T and B cells
Induce T and B cell proliferation
Their cytokines recruit other immune cells
Without TH, there is no immune
response
Helper T cells: Activation of
B cells
Interact directly with B cells displaying antigen
fragments bound to MHC II receptors
Stimulate B cells to divide more rapidly and
begin antibody formation
B cells may be activated without TH cells by
binding to T cellindependent antigens
Response weak and short-lived
Most antigens require TH co-stimulation to
activate B cells: T celldependent antigens
Figure 21.18a The central role of helper T cells in mobilizing both humoral and cellular immunity. Slide 1

Helper T cells help in humoral immunity

Helper T cell
1 TH cell binds
T cell receptor (TCR) with the self-nonself
complexes of a B cell
that has encountered
Helper T cell
its antigen and is
CD4 protein displaying it on
MHC II on its surface.
MHC II protein
of B cell displaying
processed antigen 2 TH cell releases
interleukins as co-
IL-4 and other stimulatory signals to
cytokines complete B cell
activation.

B cell (being activated)

Helper T cells: Activation of
CD8 cells
CD8 cells require TH cell activation into
destructive cytotoxic T cells
Cause dendritic cells to express co-
stimulatory molecules required for CD8
cell activation
Figure 21.18b The central role of helper T cells in mobilizing both humoral and cellular immunity. Slide 1

Helper T cells help in cellular immunity

CD4 protein Helper T cell

1 TH cell binds
Class II MHC
protein dendritic cell.
APC (dendritic cell)
2 TH cell
IL-2 stimulates dendritic
cell to express
co-stimulatory
molecules.

3 Dendritic cell
can now activate
Class I CD8 CD8 cell with the
MHC protein protein help of interleukin 2
CD8 T cell secreted by TH cell.
(becomes TC cell
after activation)
Helper T cells: Amplification of
Innate Defenses

Amplify responses of innate immune

system
Activate macrophages more potent
killers
Mobilize lymphocytes and
macrophages and attract other types of
WBCs
Helper T cells: Subsets of
TH cells
TH1 mediate most aspects of cellular
immunity
TH2 defend against parasitic worms;
mobilize eosinophils; promote allergies
TH17 link adaptive and innate
immunity by releasing IL-17; may play
role in autoimmune disease
Cytotoxic T (TC) cells
Directly attack and kill other cells
Activated TC cells circulate in blood and
lymph and lymphoid organs in search of
body cells displaying antigen they
recognize
Roles of Cytotoxic T (TC)
cells
Targets
Virus-infected cells
Cells with intracellular bacteria or parasites
Cancer cells
Foreign cells (transfusions or transplants)
Cytotoxic T cells
Bind to a self-nonself complex
Can destroy all infected or abnormal
cells
Cytotoxic T cells
Lethal hit two methods:
TC cell releases perforins and granzymes
by exocytosis
Perforins create pores through which
granzymes enter target cell
Granzymes stimulate apoptosis
TC cell binds specific membrane receptor
on target cell, and stimulates apoptosis
 Figure 21.19 Cytotoxic T cells attack infected and cancerous cells.

Adaptive defenses Cellular immunity

Cytotoxic 1 2 TC releases 3 Perforin molecules insert into

T cell (TC) TC identifies
perforin and the target cell membrane,
foreign antigens granzyme polymerize, and form transmembrane
on MHC I proteins molecules from itspores (cylindrical holes) similar to
and binds tightly granules by those produced by complement
to target cell. exocytosis. activation.

Granule
Perforin

TC cell
membrane Cytotoxic
T cell

Target
cell
membrane
Target Cancer cell
cell Perforin
pore
Granzymes

4 Granzymes enter the

5 The TC detaches target cell via the pores.
and searches for Once inside, granzymes
another prey. activate enzymes that
trigger apoptosis.
A mechanism of target cell killing by TC cells. Scanning electron micrograph of a
TC cell killing a cancer cell (2100x).
Regulatory T (TReg) cells
Dampen immune response by direct
contact or by inhibitory cytokines such
as IL-10 and TGF-
Important in preventing autoimmune
reactions
Suppress self-reactive lymphocytes in
periphery (outside lymphoid organs)
Research into using them to induce
tolerance to transplanted tissue
Figure 21.20 Simplified summary of the primary immune response.
Cellular Humoral Antigen (Ag) intruder
immunity immunity

Inhibits Inhibits
Triggers

Adaptive defenses Innate defenses

Surface Internal
barriers defenses

Free Ags
may directly
activate B cell
Ag-infected
body cell engulfed
by dendritic cell Antigen-
activated
Becomes B cells

Clone and

Present Ag to activated helper T cells

Co-stimulate and release cytokines
give rise to
Ag-presenting cell
(APC) presents
self-Ag complex

Activates Activates Memory

B cells
Naive Naive
CD8 CD4
T cells T cells

Activated to clone Activated to clone

and give rise to Induce and give rise to Plasma cells
Memory Memory
co-stimulation CD4 (effector B cells)
CD8
T cells T cells

Secrete
Cytotoxic Helper
T cells T cells

Cytokines stimulate

Nonspecific killers
(macrophages and Antibodies (Igs)
Together the nonspecific killers
and cytotoxic T cells mount a NK cells of innate Circulating lgs along with complement
physical attack on the Ag immunity) mount a chemical attack on the Ag
 Cross-Reactivity
A toxoid cross-reacts with its toxin
10-12 grams tetanus toxin kills a mouse while
10-6 grams required for immunization
Toxoid that is modified still acts as an
immunogen that cross-reacts with toxin
Toxoid still shares epitopes with toxin
Human blood group A reacts with antiserum
raised against Streptococcus capsule or
influenza virus
Human blood group B reacts to serum raised
to E. coli
Immunologic Adjuvants
To enhance the immune response to a
given immunogen, additives are added
Adjuvant (Latin, to help) enhances the
immune response
aluminum potassium sulfate (alum) is
the only adjuvant used in the United
States
causes immunogen to precipitate resulting
in time-release (released more slowly) & increased
precipitated antigen size is more likely to be phagocytized
 Immunologic Adjuvants
In Europe, they use BCG adjuvant (bacille
Calmette-Guerin): most widely used vaccination in the world
composed of attenuated Mycobacterium bovis,
Corynebacterium, and Bordetella pertussis
also acts as TB vaccine
very good at activating macrophages to
phagocytize and present to T cells
In animals, use Freunds complete adjuvant
that contains killed Mycobacterium
tuberculosis