References

Benschop, R. J., and J. C. Cambier. 1999. B-cell development: signal transduction by antigen receptors and their surrogates. Curr. Opin. Immunol. 11:143.

Berek, C. 1999. Affinity Maturation. In Fundamental Immunology, 4th ed., edited by W. E. Paul. Lippincott-Raven, Philadelphia and New York.

Go to www.whfreeman.com/immunology Self-Test

Review and quiz of key terms

Berland, R. and H. H. Wortis. 2002. Origins and functions of B-1 cells with notes on the role of cd5. Annu. Rev. Immunol. 20:253.

Bruton, O. C. 1952. Agammaglobulinemia. Pediatrics 9:722.

Hardy, R. R., and K. Hayakawa. 2001. B-cell development pathways. Annu. Rev. Immunol. 19:595.

Jacob, J., G. Kelsoe, K. Rajewsky, and U. Weiss. 1991. Intraclonal generation of antibody mutants in germinal centres. Nature 354:389.

Manis, J. P., M. Tian, and F. W. Alt. 2002. Mechanism and control of class-switch recombination. Trends Immunol. 23:31.

Matsuuchi, L., and M. R. Gold. 2001. New views of BCR structure and organization. Curr. Opin. Immunol. 13:270.

Melchers, F., and A. Rolink. 1999. B-lymphocyte development and biology. In Fundamental Immunology, 4th ed., edited by W. E. Paul. Lippincott-Raven, Philadelphia and New York.

Meffre, E., R. Casellas, and M. C. Nussenzweig. 2000. Antibody regulation of B-cell development. Nature Immunology 1:379.

Papavasiliou, F. N., and D. G. Schatz. 2002. Somatic hypermutation of immunoglobulin genes. Merging mechanisms for genetic diversity. Cell 109:S35.

SEFUL WEB SITES " *

http://www.ncbi.nlm.nih.gov/Omim/>

http://www.ncbi.nlm.nih.gov/htbinpost/Omim/getmim

The Online Mendelian Inheritance in Man Web site contains a subsite that lists more than a dozen different inherited diseases associated with B-cell defects.

http://www.bioscience.org/knockout/knochome.htm>

The Frontiers in Bioscience Database of Gene Knockouts features information on the effects of knockouts of many genes important to the development and function of B cells.

Study Questions

Clinical Focus Question Patients with X-linked agammaglobu-linemia are subject to infection by a broad variety of pathogens. Suppose you have three sources of highly purified human immunoglobulin (HuIg) for the treatment of patients with X-linked agammaglobulinemia. The human Ig from all three sources is equally free of disease-causing agents and is equally well tolerated by recipients, but the number of donors whose blood was pooled for the preparation of each source differs widely: 100 individuals for source A, 1000 for source B, and 60,000 for source C. Which would you choose and what is the basis of your choice?

1. Indicate whether each of the following statements concerning B-cell maturation is true or false. If you think a statement is false, explain why.

a. Heavy chain VH-DH-JH rearrangement begins in the pre-B-cell stage.

b. Immature B cells express membrane IgM and IgD.

c. The enzyme terminal deoxyribonucleotidyl transferase (TdT) is active in the pre-B-cell stage.

d. The surrogate light chain is expressed by pre-B cells.

e. Self-reactive B cells can be rescued from negative selection by the expression of a different light chain.

f. In order to develop into immature B cells, pre-B cells must interact directly with bone-marrow stromal cells.

g. Most of the B cells generated every day never leave the bone marrow as mature B cells.

2. You have fluorescein (Fl)-labeled antibody to the heavy chain and a rhodamine (Rh)-labeled antibody to the 8 heavy chain. Describe the fluorescent-antibody staining pattern of the following B-cell maturational stages assuming that you can visualize both membrane and cytoplasmic staining: (a) progenitor B cell (pro-B cell); (b) precursor B cell (pre-B cell); (c) immature B cell; (d) mature B cell; and (e) plasma cell before any class switching has occurred.

3. Describe the general structure and probable function of the B-cell-coreceptor complex.

4. In the Goodnow experiment demonstrating clonal anergy of B cells, two types of transgenic mice were compared: single transgenics carrying a transgene-encoded antibody against hen egg-white lysozyme (HEL) and double transgenics carrying the anti-HEL transgene and a HEL transgene linked to the zinc-activated metallothionine promoter.

a. In both the single and double transgenics, 60%-90% of the B cells expressed anti-HEL membrane-bound antibody. Explain why.

b. How could you show that the membrane antibody on these B cells is specific for HEL and how could you determine its isotype?

c. Why was the metallothionine promoter used in constructing the HEL transgene?

d. Design an experiment to prove that the B cells, not the TH cells, from the double transgenics were anergic.

5. Discuss the origin of the competence and progression signals required for activation and proliferation of B cells induced by (a) soluble protein antigens and (b) bacterial lipopolysaccharide (LPS).

6. Fill in the blank(s) in each statement below (a-i) with the most appropriate term (s) from the following list. Terms may be used more than once or not at all.

dark zone light zone paracortex cortex centroblasts centrocytes follicular dendritic cells medulla memory B cells plasmablasts TH cells a. Most centrocytes die by apoptosis in the .

b. Initial activation of naive B cells induced by thymus-dependent antigens occurs within the of lymph nodes.

c. are rapidly dividing B cells located in the _of germinal centers.

d. _expressing high-affinity mIg interact with antigen captured by_in the light zone.

e. Class switching occurs in the and requires direct contact between B cells and .

f. Centrocytes expressing mIg specific for a self-antigen present in the bone marrow are subjected to negative selection in the .

g. Within lymph nodes, plasma cells are found primarily in the_of secondary follicles.

h. Generation of_in the _ of germinal centers is induced by interaction of centrocytes with IL-1 and CD3.

i. Somatic hypermutation, which occurs in proliferating

, is critical to affinity maturation.

7. Activation and differentiation of B cells in response to thymus-dependent (TD) antigens requires TH cells, whereas the B-cell response to thymus-independent (TI) antigens does not.

a. Discuss the differences in the structure of TD, TI-1, and TI-2 antigens and the characteristics of the humoral responses induced by them.

b. Binding of which classes of antigen to mIg provides an effective competence signal for B-cell activation?

8. B-cell-activating signals must be transduced to the cell interior to influence developmental processes. Yet the cytoplas-mic tails of all isotypes of mIg on B cells are too short to function in signal transduction.

a. How do naive B cells transduce the signal induced by crosslinkage of mIg by antigen?

b. Describe the general result of signal transduction in B cells during antigen-induced activation and differentiation.

9. In some of their experiments, Nemazee and Burki mated mice carrying a transgene encoding Kb, a class I MHC molecule, linked to a liver-specific promoter with mice carrying a transgene encoding antibody against Kb. In the resulting double transgenics, Kb-binding B cells were found in the bone marrow but not in lymph nodes. In contrast, the anti-Kb single transgenics had Kb-binding B cells in both the bone marrow and lymph nodes.

a. Was the haplotype of the mice that received the transgenes H-2b or some other haplotype?

b. Why was the Kb transgene linked to a liver-specific promoter in these experiments?

c. What do these results suggest about the induction of B-cell tolerance to self-antigens?

10. Indicate whether each of the following statements is true or false. If you believe a statement is false, explain why.

a. Cytokines can regulate which branch of the immune system is activated.

b. Immunization with a hapten-carrier conjugate results in production of antibodies to both hapten and carrier epitopes.

c. All the antibodies secreted by a single plasma cell have the same idiotype and isotype.

d. If mice are immunized with HRBCs and then are immunized a day later with SRBCs, the antibody response to the SRBCs will be much higher than that achieved in control mice immunized only with SRBCs.

11. Four mice are immunized with antigen under the conditions listed below (a-d). In each case, indicate whether the induced serum antibodies will have high affinity or low affinity and whether they will be largely IgM or IgG.

a. A primary response to a low antigen dose b. A secondary response to a low antigen dose c. A primary response to a high antigen dose d. A secondary response to a high antigen dose

12. DNA was isolated from three sources: liver cells, pre-B lymphoma cells, and IgM-secreting myeloma cells. Each DNA sample was digested separately with the restriction enzymes BamHI and £coRI, which cleave germ-line heavy-chain and k light-chain DNA as indicated in part (a) of the figure below. The digested samples were analyzed by Southern blotting using a radiolabeled C^1 probe with the BamHI digests (blot #1) and a radiolabeled Ck probe with the £coRI digests (blot #2). The blot patterns are illustrated in part (b) of the figure. Based on this information, which DNA sample (designated A, B, or C) was isolated from the (a) liver cells, (b) pre-B lymphoma cells, and (c) IgM-secreting plasma cells? Explain your assignments.

For use with Question 12 (a)

LVh1 LVHn Dh1 Dh13

Bam HI

Bam HI

Eco RI

L VK1 L Vn

EcoR I

Jk1 Jk5 Ck

Blot #1

Blot #2

Cytokines

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