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Antigen
This module will help you

• recognize antigens and factors that contribute to their immunogenicity.

• learn the kinds of immune responses made to different types of antigens.
• understand how molecules on our own cells (CD antigens) can be used to
identify and isolate functional cell types.
• test your knowledge and immunology problem solving skills.

Foreign (Pathogen) Antigen

CD Antigen Markers
Key Concepts

Foreign (Pathogen) Antigen

Antigens were originally defined as non-self molecules which bound specifically

to antibodies. In practice, the term antigen is used to mean any molecule
recognized by the immune system.

Antigens which induce adaptive immunity are called immunogens. All

immunogens are antigens, and are usually called antigens unless their ability to
induce an immune response is being discussed. Some antigens, called haptens,
are not immunogenic unless they are covalently linked to immunogenic carriers
(usually proteins). Haptens can bind antibodies once the antibodies are
produced, but haptens will not induce antibody synthesis on their own. Small
non-protein organic molecules, for example the antibiotic penicillin, are haptens.

Immunogenicity is influenced by

• the chemical nature of the antigen.

• the antigen's size.
 • the antigen's usual presence in the body.
• antigen dose and route and timing of administration.
• whether the antigen is easily phagocytosed.
• whether antigen is efficiently presented to T cells on MHC.
• the maturity of the immune system and specific lymphocytes.

Note that immunogenicity depends on the immune system as well as on the

antigen.

Immunogenicity is generally higher for proteins than for other organic molecules.
Protein antigens, which are the predominant sort that activate helper T cells,
consequently induce more B cell antibody production, synthesis of IgG or IgA,
and generation of memory B and T cells. T-independent antigens, which
activate only B cells, induce IgM synthesis but not other adaptive immune
responses.

Immunogenicity increases with molecular size and complexity. Haptens are

generally non-proteins or they are too small to be immunogenic unless they are
covalently attached to a carrier protein molecule. Some carbohydrate antigens
can stimulate B cells to secrete IgM in the absence of T cell help but do not elicit
IgG or memory cells. Human infants make poor responses to carbohydrate
antigens, so early vaccinations with bacterial polysaccharide antigens employ
protein carriers. Aggregated proteins are easily phagocytosed and more
immunogenic than soluble proteins. Adjuvants, including bacterial products,
alum, or oil emulsification of antigen, attract and activate antigen-presenting cells
(dendritic cells and macrophages) and slow antigen release to prolong exposure
and improve immunogenicity.

Each part of the antigen bound by a unique antibody is called an epitope. Most
proteins have several epitopes that are recognized by different B cells and
induce a polyclonal antibody response. In a polyclonal response, several
clones of B cells each make different antibodies, all able to bind to the same
antigen but at different epitopes. Epitopes may be shared by closely related
antigens (cross-reactivity), so that antibody made to tetanus toxoid binds
tetanus toxin. A protein epitope may be a linear sequence of amino acids or it
may be assembled by protein folding. Epitopes with definite three-dimensional
shapes and charged amino acids are particularly well recognized by antibodies.
External membrane and cell wall molecules, often present in many copies on the
pathogen, are common B cell antigens. A small peptide of 4-6 amino acids could
fit into an antibody binding site, but larger proteins have more extended epitopes
across their surfaces.

Active immunity is induced by exposure to antigen. Antigen dose and the route
and timing of antigen contact, as well as immunogenicity, influence the
magnitude and nature of the immune response. Very high or very low doses of
antigen induce tolerance, the inability to respond to that antigen, while
intermediate doses induce immunity. We are generally tolerant to cell-bound and
soluble antigens present in our own bodies. Oral tolerance to foods is common,
although some foods induce allergic reactions.

Antigens encountered in body tissues through subcutaneous or intramuscular

injection are carried by lymph and macrophages into the lymph nodes, where
they usually elicit formation of serum IgG antibody. Antigens encountered by
mucosal routes (orally or in the respiratory or urogenital tracts) are taken up by M
cells and induce secretory IgA production. IgE is often made to worm parasites
and to protein allergens such as pollen that are encountered at low doses across
mucus membranes. Introducing antigens into the blood stream requires larger
doses for inducing immunity, as the majority of antigen is quickly removed and
destroyed in the spleen and liver reticuloendothelial systems (RES).

Exogenous (extracellular) pathogens and their toxins, and endogenous

(intracellular) pathogens encountered when they are extracellular, induce
formation of antibodies by B cells with the help of Th2 cells. Intracellular
pathogens elicit formation of cytotoxic T cells (Tc) to kill virus-infected cells or of
inflammatory cytokine-producing Th1 cells to promote macrophage destruction of
engulfed pathogens. A virus must be capable of infecting and replicating in a host
cell to induce formation of cytotoxic T cells, but both live and killed viruses induce
antibody formation. Mycobacteria that survive inside macrophage phagosomes
induce Th1 activation.

Upon initial (primary) contact with an antigen, a lag of several days occurs before
increased antibody production or cellular immunity can be detected. During this
time the innate immune response is occurring. Macrophages and neutrophils
engulf and destroy the pathogens; complement is activated to stimulate
inflammation and pathogen lysis; NK cells kill virus-infected host cells; and
cytokine production results in inflammation, increased body temperature (fever),
and increased hematopoiesis.

If antigen is not removed by innate immune mechanisms, adaptive immune

effectors are activated. Antigen in the tissues is carried by macrophages, PMNs,
and dendritic cells to nearby secondary lymphoid organs where it stimulates
lymphocytes to become immune effector cells. Measures of adaptive immune
effector functions such as plasma antibody, increase for about 10-14 days after
antigen contact, plateau for several days, and, when antigen has been
successfully eliminated, drop but remain higher than in unimmunized individuals.
The predominant antibody made during the primary immune response is IgM.
Upon secondary antigen contact, the lag period is shorter, the peak response
higher, and increased antibody levels may persist for weeks or months. IgG (or
IgA for mucosal antigens) is the predominant antibody made during secondary
immune responses.
B cell responses are usually measured by assays of secreted (circulating)
antibodies. Blood is removed from the individual whose immunity is being tested
and allowed to clot; antibodies are present in the unclotted liquid serum. The
presence of specific antibody, its isotype and concentration, its affinity (tightness
of binding) for antigen, its neutralizing ability, and changes in these properties
with time give information about the humoral immune response. The presence of
specific antibody is usually detected by its binding to antigen using enzyme
activity, radioactivity, or antigen agglutination to detect the binding (see ToolBox).

T cell responses are usually detected by cell division, cytokine secretion, or

cytotoxicity. Circulating T cells must be separated from erythrocytes and other
leukocytes and challenged with antigen presented on self Class I or Class II
MHC in order to detect T cell activation. Proliferation or target cell lysis are
detected by incorporation of 3H-thymidine or release of 51Cr, respectively.
Secreted cytokines are detected by binding them to their specific antibodies or
measuring their effects on other cell populations(see ToolBox).

CD Antigen Markers

The identification of membrane "markers" on immune system cells has been

instrumental in the identification and characterization of functional cell types. B
cells, helper T cells, and cytotoxic T cells, as well as some of their immature
precursors, look identical under the light microscope but have very different
functions. Plasma membrane molecules unique to distinct functional cell types
were identified by making antibodies to intact cells and studying the ability of
those antibodies to bind to or inhibit the activities of different cells. An
international CD (Cluster of Differentiation) classification system insures that
every antibody identified as anti-CD4, for example, binds the same molecule (not
necessarily the same epitope).

Although we call the membrane CD antigens "markers" because they help us

identify cells, they are on the membrane to serve as receptors for antigens or
cytokines or binding molecules on other cells. CD antigens have been identified
on many different immune cells and on cells outside the immune system. Some
common cell surface markers, for example BCR (membrane Ig) and TCR, have
not been given CD designations, while other markers (B220 on B cells) have
both a common name and a CD designation. A list of CD designations can be
found at http://www.ebioscience.com/ebioscience/whatsnew/humancdchart.htm
Many are listed in Janeway et al. Immunobiology. The Immune System in
Health and Disease Appendix 1. A few key cell markers are shown in the table
below.

Selected Leukocyte Membrane Markers

Cell Type Marker Function
B cells mIgM and mIgD Binds antigen
 Igα Igβ (CD79 α
Transduces binding signal
β)
CD 19, CD 21,
Co-receptors to enhance signaling
CD 81
Co-stimulator for T cell activation,
activated B cells, dendritic B7.1 (CD 80), B
binds T cell CD28 (binds CTLA-4
cells, macrophages 7.2 (CD86)
for inactivation of T cells)
activated B cells, dendritic Co-stimulator for APC activation,
CD40
cells, macrophages binds T cell CD40L (CD154)
CD209 (DC-
dendritic cells Binds ICAM-3 on naïve T cells
SIGN)
TCR Binds antigen peptide on MHC
T cells
CD3 Transduces binding signal
Binds CD40 to communicate with
CD40L (CD154)
APC
activated T cells Binds B cell B 7.1 and B7.2 to
CD28
activate T cell
Binds APC B 7.1, 7.2 to inactivate
CTLA-4
T cells
Adhesion molecule, binds CD58
T & NK cells CD2
(LFA-3)
Binds class II MHC, co-receptor to
Th CD4
enhance signaling
Binds class I MHC, co-receptor to
Tc CD8
enhance signaling
NK cells CD56 Adhesion molecule
Monocyte/macrophages,
CD64 (FcγRI) Binds antigen-bound IgG
neutrophils
Monocyte/macrophages, Binds antigen-bound IgG; low-
CD16 (FcγRIII)
neutrophils, NK cells affinity Fcγ Receptor
Granulocytes and
CD 114 Binds G-CSF growth factor
monocytes
CD45 (CLA,
All hematopoietic cells Augments signaling
B220, T200)
Antigen presenting cells:
B cells, Macrophages, Class II MHC Antigen presentation to Th cells
and dendritic cells
All nucleated cells Class I MHC Antigen presentation to Tc cells
Antibodies to unique CD antigens can be used to purify particular cell
populations. Some of the commonly used techniques involve linking the
antibodies to plastic dishes, beads, or paramagnetic particles. The desired cells
bind to the specific antibodies, and other cell types can be washed away. The
use of fluorescent antibodies to CD antigens in flow cytometry allows for the
quantification of different cell types (and the amount of marker on each cell) as
well as their purification. Additional information about these important techniques
can be found in the ToolBox.

Key Concepts

Practice Quiz

Pick the one BEST answer for each question by clicking on the letter of the
correct choice.

1. The ability of an antigen to induce an immune response does NOT depend on

the antigen's

a. ability to enter the thyroid.

b. degree of aggregation.
c. dose.
d. size.
e. usual presence in the body.

2. Alum is an effective adjuvant because it

a. disaggregates the antigen.

b. is immunogenic for stem cells
c. is immunogenic for T cells.
d. slows the release of antigen.
e. transports antigen into the cytoplasm of antigen-presenting cells.

3. Antibody cross-reactivity is demonstrated by antibody binding to

a. a cell surface marker.

b. a hapten.
c. a hapten-carrier complex.
d. an antigen that is structurally similar to the immunogen
e. the immunogen.

4. The antibiotic penicillin is a small molecule that does not induce antibody
formation. However, penicillin binds to serum proteins and forms a complex that
in some people induces antibody formation resulting in an allergic reaction.
Penicillin is therefore

a. an antigen.
b. a hapten.
c. an immunogen.
d. both an antigen and a hapten.
e. both an antigen and an immunogen.

5. Antigen entering the body in a subcutaneous injection activates its specific

lymphocytes in the

a. blood circulation.
b. draining lymph nodes.
c. MALT.
d. skin.
e. spleen.

6. To detect a humoral immune response to influenza virus, you would measure

a. cytotoxicity of virus-infected cells in the lung.

b. cytotoxicity of virus-infected cells in tissue culture.
c. dividing T cells in the draining lymph nodes.
d. plasma cytokine levels.
e. serum antibody titer.

7. During the lag period between antigen contact and detection of adaptive
immunity,

a. antigen is hidden from the immune system in macrophages.

b. cellular immunity can be detected but antibodies cannot.
c. innate immune effectors are eliminating antigen.
d. innate immunity blocks the activation of adaptive immune effector cells.
e. new B and T cells with the appropriate antigen specificity must be produced in
the bone marrow.

8. To elicit the best antibodies to mouse MHC I, you should inject it into

a. a goat.
b. a mouse of the same genetic background (strain).
c. a mouse of a different strain.
d. a rat.
e. the mouse you isolated it from.

9. For specific antigen recognition by T cells,

a. antigen is bound by a T cell membrane antibody.
b. denaturation of antigen does not reduce epitope recognition.
c. MHC molecules are not required.
d. soluble antigen is bound directly without processing.
e. antigen exposure during T cell maturation is required.

10. The immune response to a booster vaccine is called a(n)

a. cellular response.
b. humoral response.
c. innate response.
d. primary response.
e. secondary response.

11. Immunogenicity

a. depends on the ability of the native antigen to be presented by MHC.

b. is usually a property of "self" antigens such as eye tissue.
c. is not a property of antibodies.
d. is not a property of haptens.
e. only applies to antigens that are composed of proteins.

12. Lymphocytes are activated by antigen in the

a. blood stream.
b. bone marrow.
c. liver.
d. lymph nodes.
e. skin.

13. A molecule that can be covalently linked to a non-immunogenic antigen to

make it an immunogen is called a(n)

a. adjuvant.
b. carrier.
c. hapten.
d. mitogen.
e. superantigen.

14. A polyclonal antibody response

a. is not antigen-specific.
b. is produced only in response to polymeric antigens.
c. is produced by several B cells recognizing different epitopes on the same
antigen.
d. occurs during the lag phase of the immune response.
e. violates clonal selection.

15. Very low doses of antigen may induce

a. a secondary response.
b. hypersensitivity.
c. immunological ignorance.
d. low zone tolerance.
e. low zone immunity.

16. A virus vaccine that can activate cytotoxic T cells MUST contain

a. a high dose of virus particles.

b. an adjuvant to stimulate T cell division.
c. foreign MHC.
d. live virus.
e. virus peptides.

17. Which statement about antigen epitopes is FALSE?

a. An epitope may be shared by two different antigens.

b. A protein molecule usually contains multiple epitopes.
c. B cells bind only processed antigen epitopes.
d. Epitopes may be linear or assembled.
e. Some epitopes are more immunogenic than others

18. CD antigens

a. allow leukocytes to recognize antigen.

b. are each expressed on only one cell type.
c. are expressed on immune cells by immunologists to "mark" them for
separation.
d. are found only on leukocytes.
e. function as receptors for cytokine and CAMs.

19. A patient desperately needs a bone marrow transplant, and a perfect match
cannot be found. The rejection response in unmatched marrow is primarily due to
the presence of mature T cells that recognize the recipient's cells as foreign. To
minimize this rejection response, the marrow can be treated before transfusion
into the recipient with complement plus antibody to human

a. CD3.
b. CD4.
c. CD8.
d. CD28.
e. CD154.

20. Antibody to membrane receptors sometimes inhibits receptor function and

sometimes mimics the action of the normal receptor ligand. (For example, some
antibodies to insulin receptor block the action of insulin and some mimic the
action of insulin.) An antibody which should NOT either block or stimulate B cell
function would be anti-

a. CD21.
b. CD56.
c. CD80.
d. Igα .
e. µ chain.

Problems

1. For your independent study, your mentor has asked you to test the specificity
of several antibodies: anti-mouse CD3, anti-mouse CD4, anti-mouse µ chain,
and anti-mouse CD79α . The anti-CD3 and anti-CD79α are labeled with FITC
(fluorescein isothiocyanate, which fluoresces green). The anti CD4 and anti-µ
chain are tagged with PE (phycoerythrin, which fluoresces red), and you have a
flow cytometer at your disposal.

a) Which cells will you stain to test for specificity and what results do you expect.
HINT: think about which cells should be stained by each antibody and where they
will be found in the mouse. (Erythrocytes would always be removed before
staining.) Draw a flow diagram showing log fluorescence intensity (horizontal
axis) vs. number of cells (vertical axis) for a positive and negative sample.

b) What will you use as your controls? (Some background fluorescence is usually
seen even with nonspecific antibodies and must be checked.)

c) Because you have two fluorochromes, you can do two color fluorescence by
staining one population with two antibodies at once. Draw the flow diagram for
spleen cells (erythrocytes removed) stained with both FITC-anti-CD3 and PE
anti-µ , where log FITC intensity is plotted on the vertical axis and log PE
intensity is plotted on the horizontal axis. HINT: Draw a square and divide it into
four equal boxes.
Write anti-CD3 on the vertical axis and anti-µ on the horizontal axis. Cells
positive for CD3 but not µ go in the top left box. Cells positive for µ but not
CD3 go in the bottom right box. Cells positive for both CD3 and µ (if any) go in
the top right box, and cells negative for both (if any) go in the bottom left box.

d) Now do the same kind of plot for anti-CD3 vs. anti-CD4 and for anti-µ vs.
anti-CD79α . Which cells in the spleen are stained for one (single positive),
both (double positive), or neither (double negative) marker? [If you can't draw
the diagrams, just list the cells that will stain with each marker).

e) Would the results differ if you used cells from the blood? the thymus? The
bone marrow?

References

Website listing antibodies available to animal CD antigens:

http://www.serotec.com/asp/veterinarymarkers.htm

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http://microvet.arizona.edu/Courses/MIC419/Tutorials/antigen.html
Written by Janet M. Decker, PhD jdecker@u.arizona.edu
Last modified February 15, 2007