The CD38-CD20 cells generated in our culture system have all the features of memory B cells: phenotype, ability to re- spond to proliferative signals, and low levels of intracytoplasmic and secreted lg. Al- though most memory B cells isolated from human tonsils are medium-size resting B cells (7), the memory B cells generated here are large cells. Large memory B blasts have been identified in vivo in the B cell follicles, where they have been construed as long- term memory B cells undergoing chronic stimulation by immune complexes on follic- ular dendritic cells (1), and among the recir- culating thoracic duct lymphocytes, where they have been construed as memory B cells recently generated from GCs (15). The gen- eration of CD38-CD20 memory B blasts from GC B cells described here will provide a model to identify the signals that allow such blasts to revert to small resting cells. Our results show that interruption of the CD40 signal after 3 days of primary culture results in the terminal differentiation of proliferating B blasts into plasma cells. They are characterized by typical pheno- type and morphology, large amounts of in- tracellular Ig and secretion of large amounts of Ig, and the inability to undergo further proliferation. During humoral immune re- sponses, responding B cells differentiate into either memory B cells or plasma cells. These two facets of the response must be tightly integrated to ensure adequate amounts of antibody production and the generation of memory B cells. CD40L plays a critical role at two stages of memory B cell generation: (i) in the induction phase of GC reaction, as demonstrated by the lack of GCs in hyper-IgM patients (16) and in mouse models where CD40-CD4OL interac- tions were interrupted (17, 18), and (ii) in the differentiation phase of high-affinity GC B cells toward memory B cells, as dem- onstrated here. This second phase (CD4OL- dependent GC B cell differentiation) was also suggested by an interesting in vivo observation: Mice receiving soluble CD40- IgG chimeric molecules have normal GCs but no memory B cell generation. Because soluble CD40-IgG chimeric molecules have a lower affinity for CD40L than do antibod- ies to CD4OL, this observation suggests that the GC differentiation phase is more easily blocked than the GC induction phase (19). The recent identification of T cells that express CD40L in the light zone of GCs from human tonsils (9) supports the hy- pothesis that the GC differentiation phase occurs here (20). After somatic mutation and positive selection, high-affinity GC B cells pick up antigen from follicular dendrit- ic cells and present it to GC T cells (20). During this cognate T-B cell interaction, T cells may be induced to secrete cytokines and to express CD4OL (21), resulting in the 722 generation of CD38-CD20 memory B blasts. Because CD40L expression on T cells can be rapidly down-regulated by CD40 antigen on B cells (11), a proportion of proliferating B blasts will differentiate into CD38+CD20- plasma cells in the ab- sence of CD4OL signaling. REFERENCES AND NOTES 1. Y.-J. Liu, J. Zhang, P. J. L. Lane, E. Y.-T. Chan, I. C. M. MacLennan, Eur. J. Immunol. 21, 2951 (1991); J. Jacob and G. Kelsoe, J. Exp. Med. 176, 679 (1992). 2. C. Berek, A. Berger, M. Apel, Cell 67, 1121 (1991); J. Jacob, G. Kelsoe, K. Rajewsky, U. Weiss, Nature 354, 389 (1991); R. Kuppers, M. Zhao, M.-L. Hans- mann, K. Rajewsky, EMBO J. 12, 4955 (1993); M. G. MacHeyzer-Williams, M. J. MacLean, P. A. La- lor, G. J. V. Nossal, J. Exp. Med. 178, 295 (1993). 3. R. F. Coico, B. S. Bhogal, G. J. Thorbecke, J. lmmu- nol. 131, 2254 (1983); G. G. B. Klaus, J. H. Hum- phrey, A. Kunki, D. W. Dongworth, Immunol. Rev. 53, 3 (1980). 4. M. H. Kosco, G. F. Burton, Z. F. Kapasi, A. K. Szakal, J. G. Tew, Immunology 68, 312 (1989). 5. Y.-J. 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Xu et al., ibid., p. 423. 18. T. M. Foy et al., J. Exp. Med. 180,157 (1994). 19. D. Gray, P. Dullforce, S. Jainandunsing, ibid., p. 141. 20. Y.-J. Liu, G. D. Johnson, J. Gordon, l. C. M. MacLen- nan, Immunol. Today 13,17 (1992); I. C. M. MacLen- nan, Annu. Rev. Immunol. 12,117 (1994); G. J. Thor- becke, A. R. Amin, V. K. Tsiagbe, FASEB J. 8, 832 (1994). 21. A. W. Butch, G.-H. Chung, J. W. Hoffmann, M. H. Nahm, J. Immunol. 150, 39 (1993); J. Andersson et al., Immunology 83,16 (1994). 22. Y. -J. Liu et al., Immunity, in press. 23. GC B cells were isolated from total tonsillar B cells as described (6, 22). The purity of isolated GC B cells (Fig. 1A) was greater than 98% in all experi- ments. Their GC nature was confirmed by their mutated immunoglobulin V genes (7). For the pri- mary culture, cells (106/ml) were cultured with IL- 10 (100 ng/ml) and IL-2 (10 U/mI) on CD40L-trans- fected murine fibroblasts (2 x 105 fibroblasts per milliliter) irradiated with 75 Gy (where 1 Gy equals 100 rads) in Iscove medium containing 5% fetal calf serum for 3 days (10). For the secondary culture, the cells were washed and recultured for 4 days under three conditions, as described in the text. The antibody to CD4OL used in the third condition was LL2 (C. Van Kooten, in preparation). 24. We thank 1. Durand for FACS sorting, N. Courbi6re and M. Vatan for editorial help, l. C. M. MacLennan for communicating the manuscript in press, and J. Chiller for support. CA., J.D., P.M., and G.G. re- ceived fellowships from the Fondation Merieux. 30 November 1994; accepted 6 February 1995 Immune System Impairment and Hepatic Fibrosis in Mice Lacking the Dioxin-Binding Ah Receptor Pedro Fernandez-Salguero, Thierry Pineau, David M. Hilbert, Timothy McPhail, Susanna S. T. Lee, Shioko Kimura, Daniel W. Nebert, Stuart Rudikoff, Jerrold M. Ward, Frank J. Gonzalez* The aryl hydrocarbon (Ah) receptor (AHR) mediates many carcinogenic and teratogenic effects of environmentally toxic chemicals such as dioxin. An AHR-deficient (Ahr-'-) mouse line was constructed by homologous recombination in embryonic stem cells. Almost half of the mice died shortly after birth, whereas survivors reached maturity and were fertile. The Ahr-'- mice showed decreased accumulation of lymphocytes in the spleen and lymph nodes, but not in the thymus. The livers of Ahr-'- mice were reduced in size by 50 percent and showed bile duct fibrosis. Ahr-'- mice were also nonresponsive with regard to dioxin-mediated induction of genes encoding enzymes that catalyze the metabolism of foreign compounds. Thus, the AHR plays an important role in the devel- opment of the liver and the immune system. The AHR is a ligand-activated transcrip- tion factor that is distinct from members of the steroid receptor superfamily (1). It is a member of the basic helix-loop-helix (bHLH) superfamily of DNA binding pro- teins and is activated by ligand binding and by dimerization with the AHR nuclear trans- locator (Amt) (1, 2). A functional AHR is required in laboratory animals to mediate the harmful effects of toxic environmental chemicals such as dioxin (2,3,7,8-tetra- chlorodibenzo-p-dioxin, or TCDD), benzo- [alpyrene in cigarette smoke and the prod- ucts of other combustion processes, poly- SCIENCE * VOL. 268 * 5 MAY 1995 INIMIM111111110 Ill 1111m on February 1, 2015 www.sciencemag.org Downloaded from on February 1, 2015 www.sciencemag.org Downloaded from on February 1, 2015 www.sciencemag.org Downloaded from on February 1, 2015 www.sciencemag.org Downloaded from on February 1, 2015 www.sciencemag.org Downloaded from