Humoral Immunity

This module will help you

• see how B cells are activated to synthesize antibody.
• learn the effector functions of antibodies.
• test your knowledge and immunology problem-solving skills.

Antibody Production
Isotype Distribution and Functions
FcR⁺ Accessory Cells

Antibody Production

Humoral immunity refers to antibody production, and all the accessory processes that accompany it: Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibody, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

B cells need two signals to initiate activation. Most antigens are T-dependent, meaning T cell help is required for maximal antibody production. With a T-dependent antigen, the first signal comes from antigen cross linking BCR and the second from the Th2 cell. T dependent antigens contain protein so that peptides can be presented on B cell Class II MHC to Th2 cells, which then provide co-stimulation to trigger B cell proliferation and differentiation into plasma cells. Isotype switching to IgG, IgA, and IgE and memory cell generation occur in response to T-dependent antigens.

Some antigens are T-independent, meaning they can deliver both the antigen and the second signal to the B cell. Mice without a thymus (nude or athymic mice) can respond to T-independent antigens. Many bacteria have repeating carbohydrate epitopes that stimulate B cells to respond with IgM synthesis in the absence of T cell help.

T-dependent responses require that B cells and their Th2 cells respond to epitopes on the same antigen. T and B cell epitopes are not necessarily identical; for example, T cells respond well to internal viral proteins while B cells produce neutralizing antibodies to viral coat proteins. (Once virus-infected cells have been killed and unassembled virus proteins released, B cells specific for internal proteins can also be activated to make opsonizing antibodies to those proteins.) Attaching a carbohydrate to a protein can convert the carbohydrate into a T-dependent antigen; the carbohydrate-specific B cell internalizes the complex and presents peptides to Th2 cells, which in turn activate the B cell to make antibodies specific for the carbohydrate. This linked recognition of T and B cells was first discovered with work on haptens; see Problem 2 below.

Only about 1 in 10,000 to 1 in a million lymphocytes is specific for a particular antigen. The chances that these cells will find each other in the circulation are extremely small. However, lymphocyte recirculation ensures that T cells, B cells, and dendritic cells carrying antigen leave the circulation at the HEV and migrate into the T cell zone of the lymph nodes. If the lymphocytes bind their specific antigen there, they are retained (probably by increased expression of certain CAMs) to increase their chances of interaction and activation. Early in a primary response, naïve Th2 are activated by dendritic cells, since antigen-specific B cells are rare. Armed effector Th2 then activate antigen-binding B cells as they arrive in the lymph nodes.

B cell activation is initiated by the cross-linking of BCR. Antigen must have at least two identical epitopes situated so that they can cross-link B cell surface Ig to activate B cells. Aggregation of BCR-IgaIgb complexes promotes phosphorylation of ITAMs by cytoplasmic tyrosine kinases Fyn, Blk, and Lyn, to initiate a second messenger cascade. Once antigen is bound, it is internalized with its BCR; antigen can then be degraded (processed), combined with Class II MHC, and presented to effector Th2 cells, which must bind specific antigen in order to provide help.

Effector Th2 cells bind B cells via CAMs and then specific peptide on Class II MHC with TCR. T cell CD40 ligand (CD40L) binds B cell CD40 and sends the co-stimulatory signal for B cell activation. The Th2 cell reorganizes its membrane molecules and golgi to concentrate CD40L binding and cytokine secretion towards the specific B cell to maintain the antigen-specificity of the response. Cytokines, predominantly IL-4, IL-5, and IL-6, signal the B cell to divide and differentiate into antibody-secreting plasma cells. In a primary immune response, IgM is secreted before isotype switching occurs and is the predominant isotype produced.

Th cytokines direct isotype switching in activated B cells. Although Th1 cells promote cellular immunity and do not initiate antibody formation, they can induce isotype switching to certain isotypes. Depending on which lymphokines are in highest concentration near a given B cell, it can follow several differentiation pathways (these studies have been done in the mouse). Th1 cells secrete mainly IL-2 and IFNg, while Th2 produce mainly IL-4, IL-5, and IL-6. IL-2 stimulates production of J chain. IFNg stimulates switching to IgG_2a. IL-4 stimulates switching to IgE or IgG₁, while IL-5 stimulates switching to IgA. IL-6 promotes antibody secretion. Th cells are thought to produce certain cytokines in response to their location and antigen density on the APC. For example, Th2 cells in mucosal tissues produce cytokines that promote isotype switching to a chain. Each antibody isotype has specific effector functions in humoral immunity.

Isotype switching requires DNA recombination. It increases the functional diversity of Ig molecules but does not affect their antigen-binding specificities. Human gene segments for C_H are arranged linearly in the order Cm, Cd, Cg3, Cg1, yCe, Ca1, Cg2, Cg4, Ce, and Ca2. Each C gene segment, except Cd, is preceded by an intron containing a switch region sequence. This sequence is different from the recombination signal sequences found flanking the V region segments, and the recombinases mediating isotype switching are not encoded by RAG-1 and RAG-2. Rearranged VDJ_H is always expressed first with membrane Cm in the developing B cell, with both membrane Cm and Cd in the mature B cell, and with secreted Cm as the B cell begins responding to antigen. When the B cell receives the proper signals from antigen and cytokines to switch to IgG₁ production, for example, recombination occurs between the switch regions Sm and Sg1, looping out the intervening DNA, including the coding sequences for m, d and g3 heavy chains. All isotype switching events are productive, since DNA splicing occurs within introns.

A few activated B cells differentiate into short-lived plasma cells that migrate directly to the medullary cords and begin secreting IgM and later IgG. Most activated B cells go to the B cell areas of the lymph node, called primary follicles, which are present in unstimulated peripheral lymphoid tissues, in the fetus, and in germ free animals. They contain mainly follicular dendritic cells (FDC) and IgM⁺IgD⁺ B cells (naïve resting B cells). B cell blasts (dividing cells) form secondary follicles (germinal centers) within a few days of antigen stimulation. Germinal centers are sites of intensive B cell proliferation and are surrounded by the Th2 cells that activated the B cells and migrated with them. By a week after initial antigen contact, many germinal centers are seen. Athymic mice have few germinal centers following immunization.

Isotype switching, affinity maturation, and generation of memory B cells occur late in a primary immune response. Antigen-binding specificity can be altered by somatic hypermutation, which occurs in rapidly dividing B cells in the germinal centers. Mutation occurs preferentially in the CDR (hypervariable regions) of H and L chains at a rate high enough that about half of B cells undergo mutation of their Ig antigen-binding regions. B cells which remain specific for the stimulating antigen are selected by binding antigen held on FcR and complement receptors on follicular dendritic cells (FDC), which do not express Class II MHC or act as APC for T cells. Only B cells which bind antigen with high enough affinity continue to secrete antibody and differentiate into plasma cells; low affinity B cells undergo apoptosis.

The B cell co-receptor complex of CD21 (CR2), CD19, and TAPA 1 responds to complement or FDC CD23 binding by initiating a phosphorylation cascade that synergizes with BCR signaling, increasing the sensitivity of B cells to antigen, and with IL-1 promotes B cell differentiation into plasma cells. Affinity maturation occurs by clonal selection as antigen levels drop due to antigen clearance, resulting in survival of B cells with the highest antigen-binding affinity. Further Th2 cell interaction with these B cells signals them to become plasma cells or memory cells.

Plasma cells are antibody-producing cells that no longer divide or respond to antigen. They are larger than B cells and have more ribosomes, endoplasmic reticulum, and golgi, but no membrane Ig. Some survive only a few weeks, while others continue to produce antibody for longer periods, providing rapid protection against re-infection. Other B cells become memory cells, which can be rapidly re-stimulated by antigen and are present in higher frequency than the naïve resting B cells (more of the latter continue to be produced by the bone marrow). Memory B cells are functionally and physically distinguishable from naïve B cells. They often have membrane IgG, IgA, or IgE and higher levels of ICAM-1 and CR then naïve B cells, and are thought to live longer. Low levels of antigen may remain on FDC for years, so that B cells may be continually activated at low levels to replenish memory cell populations.

Some T-independent antigens (TI-1 antigens) include polyclonal B cell activators (mitogens), which are often microbial surface molecules like LPS or carbohydrates that bind B cell membrane receptors and provide co-stimulatory signals. At high concentrations, TI-1 antigens stimulate B cells of many specificities to divide and secrete antibody (polyclonal activation). At lower concentration, they stimulate only B cells whose BCR specifically bind them to divide and secrete specific IgM; this is classic clonal selection. Responses to polyclonal B cell activators can be observed in the absence of T cells and occur more quickly than T-dependent responses because no T cell activation is required.

TI-2 antigens have multiple repeating determinants; for example, bacterial polysaccharide capsules are TI-2 antigens. TI-2 antigens extensively cross-link BCR on specific B cells, stimulating them to produce IgM. Antibody responses to polysaccharides are not made in infants, because their less mature B cells are inactivated by BCR cross-linking. Responses to polysaccharides occur in mice lacking a thymus but not in the total absence of T cells. This suggests that T cells which develop outside the thymus may be activated by TI-2 antigens, possibly presented on non-classical MHC-like molecules, to provide some help to responding B cells. Another possibility is that TI-2 antigens act as polyclonal activators of T cells through membrane molecules other than TCR. Because T cells seem to be involved in responses to TI-2 antigens, IgG as well as IgM can be produced. IgG is especially important in responses to bacteria with polysaccharide capsules, which are difficult for macrophages to phagocytose. Opsonizing IgG bound to FcR on macrophages promotes phagocytosis of encapsulated bacteria.

Isotype Distribution and Function

Microbes usually enter the body through the epithelial cells of the respiratory, digestive, or genital tracts or through skin broken by a scrape, cut, insect bite or hypodermic needle. Once inside, the microbe may begin replicating locally or get into the circulation and move throughout the body. Bacterial toxins also travel from the initial infection site. The Fc regions of the Ig isotypes allow them to bind Fc receptors and cross tissue barriers to reach pathogens throughout the body.

IgM is secreted first in a primary response. No somatic hypermutation has yet occurred, so it is low affinity antibody. IgM avidity is high, however, because it is a pentamer, and IgM fixes complement very efficiently to promote inflammation and pathogen lysis. Because IgM is so large, it cannot enter the tissues very efficiently; but it is effective in controlling pathogens in the circulation.

Once isotype switching occurs, IgG predominates in serum and in tissues. IgG both neutralizes pathogens and their toxins and opsonizes them for phagocytosis by neutrophils and macrophages. IgG can also activate complement on the pathogen surface once concentrations are high enough for two IgG molecules to bind nearby epitopes.

IgA is the predominant antibody that is secreted across epithelial cells of the respiratory, digestive and genital tracts to block pathogen entry into the body. IgE binds FceR on mast cells lining the blood vessels throughout the body. When pathogen binds to the mast cell IgE, the mast cells immediately release inflammatory mediators that trigger coughing, sneezing or vomiting to expel pathogens from the body.

The selective transport of various Ig isotypes to particular regions of the body occurs because of isotype-specific Fc receptors on different tissues. Dimeric Ig A (and, to a lesser extent, pentameric IgM) bind to the poly Ig receptor on the body side of epitheilial cells in the intestines, respiratory tract, tear and salivary glands, and lactating mammary gland. The antibody-poly Ig receptor complex is endocytosed into the epithelial cell and travels in an endocytic vesicle across the cytoplasm (transcytosis) to be secreted on the outer surface of the epithelium (into the intestine or lung surface or tears, saliva, or milk). Maternal IgA in milk can neutralize pathogen in the infant's digestive tract until the infant's immune system is mature enough to take over that task. IgG which has crossed the placenta into the fetal circulation offers additional protection during the first few months of life. An FcRn has been identified on placental cells for transport of IgG across the placenta, and a similar molecule has been identified on intestinal cells of some mammals that may allow uptake of IgG in colostrum, the first fluid secreted by the mammary gland after birth.

Numerous bacteria cause disease by releasing toxins that damage cells. Like viruses, bacterial toxins must enter cells via specific receptors in order to damage the cells. Neutralizing antibodies, usually IgG, block binding to the target cells. Toxins may kill us before we can produce neutralizing antibodies, so they are a natural target for vaccination. We are immunized as infants with inactivated diphtheria and tetanus toxins (toxoids); we may become infected with Corynebacterium diphtheriae or Clostridium tetani, but any toxin they produce will be neutralized before it can harm our cells. For snake venoms, passive immunization with antitoxins produced in horses can neutralize the venom.

Adapted from Leffell, M. S., A. D. Donnenberg, and N. R. Rose. Handbook of Human Immunology. CRC Press, Boca Raton, 1997.

Viruses are vulnerable to neutralizing antibody, which can block their infection of host cells. The Salk (killed) polio vaccine is very effective in inducing protective immunity against polio even without activating CTL because it induces strong neutralizing antibodies. Humoral immunity offers protection against many virus infections. High affinity IgG and IgA are also important for blocking bacterial adherence to host cells. Without adherence, bacteria often fail to cause an infection.

FcR⁺ accessory cells

In order to act as opsonins or to activate cells, antigen-bound antibodies bind to Fc receptors. Several different FcR have been identified on the membranes of granulocytes, dendritic cells, B cells, NK cells, and mast cells. Some are there constitutively, while expression of others is induced during an immune response. The isotype specificity of each FcR, its binding affinity, and its structure all influence its function. Free antibody (unbound to antigen) binds to FcR with a very low affinity except in the case of IgE. Antibody aggregated by antigen, with several nearby Fc regions, binds much more tightly to FcR. Binding of ligand to FcR acts through signal transduction complexes to alter gene expression.

FcgRI on macrophages, dendritic cells, neutrophils and eosinophils has a high binding affinity for IgG, which means the cells bind antibody-coated antigen even at low concentration (as soon as IgG is available). In addition to promoting antigen uptake, FcgRI have a cytoplasmic region with ITAMs which can signal the FcR+ cells to activate killing mechanisms. FcgRI binds IgG₁ and IgG₃ best, IgG₄ and IgG₂ less well, and other isotypes not at all. Macrophages and dendritic cells constitutively express FcgRI; neutrophils and eosinophils can be stimulated to express them. Macrophages and neutrophils also have an FcR for alpha chain (FcaRI). Opsonization is especially important for the elimination of bacteria that resist phagocytosis, for example because they have polysaccharide capsules.

Bactericidal agents released in response to FcR binding include oxygen radicals and peroxides, nitric oxide, defensins, and lysozyme. Respiratory burst is the process by which phagocytes generate the toxic oxygen compounds that inactivate key microbial enzymes and structural proteins by oxidizing them. Phagocytes also acidify their phagocytic vesicles to activate degradative enzymes and release molecules such as lactoferrin and vitamin B12-binding protein that compete with microbes for essential nutrients. For antibody-coated microbes which are too large to phagocytose, phagocytes excrete these bactericidal agents into the extracellular space. Helminth parasites generally induce secretion of IgE, which on eosinophil FceR1 signals eosinophils to kill the parasite .

Cells infected with enveloped viruses express the envelope proteins on their membranes before the viruses take pieces of membrane for their envelope as they but from the cell. Antibodies to those viral proteins can bind to FcgRIII (CD16) on NK cells and activate them to kill the virus-infected cell, a process called ADCC (Antibody-Dependent Cell-mediated Cytotoxicity). NK cells use perforin and granzymes, present in their granules (remember NK cells are also called Large Granular Lymphocytes), to kill their target cells.

In response to some antigen challenges, especially helminth parasites and allergens, the body responds by producing IgE. Mast cells with their membrane FceR1 are found just below the skin and respiratory and digestive epithelia. Mast cell cytoplasm is packed with granules containing histamine and other mediators of inflammation. Unlike other FcR, FceR1 binds free IgE (without bound antigen), so most IgE in the body is found on mast cell surfaces and on circulating basophils. When antigen enters the body, it cross-links the IgE on mast cells and ITAMs signal the mast cells to immediately secrete their granule contents. Mast cells also secrete cytokines, including IL-4 that stimulates IgE synthesis by B cells. Eosinophils express FceR1 when activated at an infection site. Experiments with mice deficient in mast cells show that these mice have difficulty eliminating helminth parasites compared to their normal counterparts. Mast cells, IgE, basophils and eosinophils have been implicated in resistance of mice to certain bloodsucking ticks, such as those that transmit Lyme disease.

FcgRII-B1 (CD32) on B cells and mast cells has cytoplasmic ITIMS instead of ITAMS, and inhibits cell function. On B cells, CD32 binds Ig with a lower affinity than FcgRI, so high amounts of IgG antibody inhibit activation of naïve B cells (late in immune responses) and mast cells.

Practice Quiz

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

1. T independent antigens do NOT

a. bind to BCR.
b. get presented on MHC Class II.
c. have repeating epitopes.
d. induce B cell proliferation.
e. provide co-stimulatory signals to B cells

2. The humoral immune response to T-independent antigens includes production of

a. IgA.
b. IgM.
c. memory B cells.
d. memory T cells.
e. all of the above.

3. T-independent antigens are often

a. components of self cell membranes.
b. polyclonal B cell activators.
c. repeating peptide epitopes
d. too small to be phagocytosed and presented.
e. none of the above

4. Before a B cell can receive T cell help, the B cell must

a. express membrane B7.
b. express membrane CD40L.
c. express membrane IFNg receptors.
d. go to the site of infection.
e. process and present peptide on Class II MHC.

5. Antigen-binding B cells entering the secondary lymphoid organs initially go to the

a. B cell areas where they can bind antigen presented by follicular dendritic cells.
b. B cell areas where they can process and present antigen to T cells.
c. plasma cell areas where they can secrete antibody.
d. T cell areas where they can be the predominant activator of naïve T cells.
e. T cell areas where they can find specific helper T cells.

6. Germinal centers are

a. areas of lymphocyte generation in the bone marrow.
b. common in unimmunized mice.
c. sites of rapid antigen-induced B cell division in the lymph nodes and spleen.
d. surrounded by naïve T cells waiting to be activated.
e. None of the above is true

7. In order for T cells to provide help to B cells, T cell and B cell epitopes must be

a. covalently linked.
b. identical.
c. non-identical.
d. peptides.
e. T-dependent.

8. B cell isotype switching is important for

a. increasing the avidity of the antibody.
b. providing antibodies of many different antigen specificities.
c. providing antibodies which can perform different effector functions.
d. signaling the B cells to become activated by follicular dendritic cells
e. signaling B cells to leave the lymph nodes and secrete antibody at the site of infection.

9. Affinity maturation of the humoral immune response is due to

a. continued stimulation of B cells by high levels of antigen on the FDC.
b. DNA recombination by products of the RAG genes.
c. isotype switching.
d. negative selection of T cells with the lowest helper potential.
e. positive selection of B cells with the highest affinity for antigen.

10. Isotype switching by B cells occurs in response to T cell

a. IFNg.
b. IL-4.
c. IL-5.
d. IL-6.
e. all of the above.

11. Isotype switching resembles somatic recombination because both processes

a. are catalyzed by the products of RAG-1 and RAG-2.
b. are regulated by helper T cell cytokines.
c. can result in stop codons in coding sequences.
d. occur in developing B cells in the bone marrow.
e. result in the irreversible loss of DNA from the B cell.

12. Somatic hypermutation results in

a. antibody with different CDR than the membrane Ig which originally bound antigen.
b. B cell apoptosis.
c. B cells with higher affinity for the stimulating antigen.
d. B cells which can no longer bind the stimulating antigen.
e. All of the above result from somatic hypermutation

13. Plasma cells

a. are all very long-lived.
b. divide and differentiate into memory B cells.
c. produce most of their antibody at the site of infection.
d. secrete antibodies as long as antigen binds their membrane Ig receptors.
e. None of the above are true.

14. Which of the following is NOT a similarity between the cellular and humoral immune responses?

a. Antigen-specific lymphocytes undergo clonal selection and expansion.
b. Cytokine signals promote effector cell differentiation.
c. Memory cells are generated.
d. Macrophage cytotoxicity is increased.
e. Receptor isotype switching occurs.

15. Humoral immunity involves all of the following EXCEPT

a. antibody-dependent cell-mediated cytotoxicity.
b. antibody secretion by plasma cells.
c. B cell activation by antigen plus cytokines.
d. immunoglobulin isotype switching.
e. macrophage activation by Th1 cells

16. In the problem below dealing with linked T and B cell immune responses to haptens, the investigators chose to look at production of IgG rather than IgM because

a. IgG antibodies are easier to detect than IgM.
b. IgG is made before IgM in an immune response.
c. no IgM antibody can be produced to a hapten.
d. production of IgG requires T cell help but production of IgM does not.
e. None of the above is true.

17. In the practice problem below dealing with linked T and B cell immune responses to haptens, IgG is not produced in experiment #4 because

a. HEL is not an effective carrier for PC.
b. PC cannot be presented to B cells.
c. T and B cell epitopes need to be identical in order for the T cells to provide help.
d. T and B cell epitopes need to be physically linked in order for the T cell to provide help.
e. the same antigen epitopes were not given in both primary and secondary exposures.

18. Epithelial cells which secrete IgA to block pathogen entry are NOT found in the

a. digestive tract.
b. mammary glands.
c. respiratory tract.
d. salivary glands.
e. skin.

19. Different Ig isotypes are found in different body locations because they

a. are secreted in different tissues.
b. bind to different FcR that allow them to cross tissue barriers.
c. have different addressins.
d. have different affinities for antigen.
e. None of the above is true.

20. Neutralizing antibody provides effective immunity to

a. bacterial infection.
b. bacterial toxins.
c. virus infection.
d. Both a and b are correct.
e. All of the above can be blocked by neutralizing antibodies.

21. An inactivated toxin used in a vaccine is called a(n)

a. adjuvant.
b. attenuated vaccine.
c. hemagglutinin.
d. neutralizing antigen.
e. toxoid.

22. FcgRI on binds with highest affinity to

a. antigen-bound IgG₁ and IgG₃.
b. complement-activated IgG₁ and IgG₃.
c. free IgE.
d. free IgG₁ and IgG₃.
e. None of the above.

23. Binding of ligand to FcgRI on macrophages and neutrophils does NOT signal the cells to

a. acidify their phagocytic vesicles.
b. engulf antigen.
c. release their histamine-containing granules to initiate inflammation.
d. produce lactoferrin to compete with microbes for iron.
e. undergo oxidative burst.

24. Successful immune responses to bacteria which resist phagocytosis because of a polysaccharide capsule depends on the production of

a. active complement anaphylatoxins.
b. armed effector CTL.
c. neutralizing antibodies.
d. opsonizing antibodies.
e. oxidative burst.

25. Successful immune responses to bacteria which adhere to mucosal surfaces in order to initiate infection depends on the production of

a. active complement.
b. armed effector CTL.
c. neutralizing antibodies.
d. opsonizing antibodies.
e. oxidative burst.

26. Successful immune responses to bacterial toxins depend on the production of

a. active complement.
b. armed effector CTL.
c. neutralizing antibodies.
d. opsonizing antibodies.
e. oxidative burst.

27. In order for NK cells to do ADCC, they must bind

a. antibodies to virus proteins expressed on infected cell membranes
b. B7.
c. CD16.
d. toxic oxygen radicals.
e. virus peptides expressed on infected cell membrane MHC.

28. NK cells kill their targets in ADCC using

a. complement-mediated lysis.
b. Fas and FasL.
c. oxidative burst.
d. perforins and granzymes.
e. all of the above.

29. Mast cells release their granule contents to stimulate inflammation in response to

a. antigen binding to IgE on mast cell FceR.
b. antigen binding to IgG on mast cell FcgR.
c. antigen binding to mast cell antigen receptors.
d. antigen binding to mast cell FceR.
e. IgE-coated antigen binding to mast cell FceR.

30. Successful immune responses to helminth parasites depends on the production of

a. active complement.
b. armed effector CTL.
c. neutralizing IgA.
d. opsonizing IgE.
e. opsonizing IgG.

Problems

1. Haptens helped immunologists understand antigen recognition and the T cell-B cell cooperation required for IgG synthesis. In the experiment shown below, mice were injected with the small non-protein hapten phosphorylcholine, either unlinked (PC) or chemically linked to a protein carrier, hen egg lysozyme (PC-HEL) or human serum albumin (PC-HSA). IgG antibody to PC was measured after the second injection. Explain how the data shown below supports the requirement for Th and B cells to recognize epitopes on the same T-dependent antigen.

HSA, human serum albumin; HEL, hen egg lysozyme; PC, phosphorylcholine (a hapten)