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.

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).

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 β)	Transduces binding signal
	CD 19, CD 21, CD 81	Co-receptors to enhance signaling
activated B cells, dendritic cells, macrophages	B7.1 (CD 80), B 7.2 (CD86)	Co-stimulator for T cell activation, binds T cell CD28 (binds CTLA-4 for inactivation of T cells)
activated B cells, dendritic cells, macrophages	CD40	Co-stimulator for APC activation, binds T cell CD40L (CD154)
dendritic cells	CD209 (DC-SIGN)	Binds ICAM-3 on naïve T cells
T cells	TCR	Binds antigen peptide on MHC
T cells	CD3	Transduces binding signal
activated T cells	CD40L (CD154)	Binds CD40 to communicate with APC
	CD28	Binds B cell B 7.1 and B7.2 to activate T cell
	CTLA-4	Binds APC B 7.1, 7.2 to inactivate T cells
T & NK cells	CD2	Adhesion molecule, binds CD58 (LFA-3)
Th	CD4	Binds class II MHC, co-receptor to enhance signaling
Tc	CD8	Binds class I MHC, co-receptor to enhance signaling
NK cells	CD56	Adhesion molecule
Monocyte/macrophages, neutrophils	CD64 (FcγRI)	Binds antigen-bound IgG
Monocyte/macrophages, neutrophils, NK cells	CD16 (FcγRIII)	Binds antigen-bound IgG; low-affinity Fcγ Receptor
Granulocytes and monocytes	CD 114	Binds G-CSF growth factor
All hematopoietic cells	CD45 (CLA, B220, T200)	Augments signaling
Antigen presenting cells: B cells, Macrophages, and dendritic cells	Class II MHC	Antigen presentation to Th 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.

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

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

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

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

13. A molecule that can be covalently linked to a non-immunogenic antigen to make it an immunogen is called a(n)

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

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-

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 m chain, and anti-mouse CD79a. The anti-CD3 and anti-CD79a are labeled with FITC (fluorescein isothiocyanate, which fluoresces green). The anti CD4 and anti-m 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-m, 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-m on the horizontal axis. Cells positive for CD3 but not m go in the top left box. Cells positive for m but not CD3 go in the bottom right box. Cells positive for both CD3 and m (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-m vs. anti-CD79a. 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?

Tutorials

Antigen