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B-cell differentiation
Immunology Today, vol. 7, No. 11, 1986
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dence has already been set with IL-3, in that the murine entity has been characterized but the human molecule has not been isolated or identified. Although the opinions of those present were somewhat mixed on this point, the subcommittee recommends to the parent committee that cytokine activities be given an interleukin number only when the entity has been characterized from human origin. Accordingly, a particular cytokine from another species would carry an appropriate prefix (eg. murine IL-1), whereas the same molecule of human origin would not: rather, it is understood that an entity without a prefix is of human origin."
References 1 Noma, Y., P. Sideras,T. Naito, S. Bergstedt-Linquist, C. Azuma, E. Severinson,T. Tanabe, T. Kinashi, F. Matsuda, Y. Yaoita and T. Honjo (1986) Cloning of cDNA encoding the murine IgG1 induction factor by a novel strategy using SP6 promoter. Nature 319, 640 2 Lee, F., T. Yokota, T. Otsuka, P. Meyerson, D. Viltaret, R. Coffman, T. Mosmann, D. Rennick, N. Roehm, C. Smith, A. Zlotnik and K. Arai (1986) Isolation and characterization of a mouse interleukin cDNA clone that expressesB-cell stimulatory factor 1 activities and T-cell- and mast-celtstimulating activities. Proc. Natl Acad. Sci. USA 83, 2061 3 Yokota, T., T. Otsuka, T. Mosmann, J.
As lymphoid precursors in the bone marrow differentiate, they undergo an orderly series of gene rearrangements at the immunoglobulin (Ig) heavy and light chain loci which culminate in the synthesis of the Ig molecule. The genes involved in this process have been defined and their chromosomal location is known. It has also been possible to describe various cellular stages of B lymphopoiesis, owing to the availability of antibodies specific for Ig and various non-lg determinants. For example, B lineage cells that have not yet synthesized Ig can be recognized with antibodies directed against the B220 family of molecules. The recently described antibody to the BP-1 antigen, a 140 000 Mr molecule, should also prove useful in identifying early Bcell precursors. Subsequently, a population of pre-B cells expresses heavy chains of the I~ class in their cytoplasm. As light chain proteins are synthesized, the Ig molecule consisting of the I~ heavy chain and kappa or lambda light chains is assembled and expressed on the surface of newly produced B cells. This brief and obviously incomplete synopsis of B-cell development summarizes several years of work by many laboratories. Nevertheless, symposia, workshops, and informal discussions at the Congress defined several issues that remain to be resolved before the understanding of this differentiative pathway is complete.
B-cell differentiation
Identification of stem cellsand B lymphocyte precursors The pluripotent hemopoietic stem
Banchereau, T. DeFrance, D.
Blanchard, J.E. De Vries, F. Lee and K, Arai (1986) Isolationand characterization of a human interleukin cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expressesB-cell-and T-cellstimulating activities. Proc. NatlAcad. Sci. USA 83, 1
Readerswho wish to commenton these recommendationsshouldwrite to: ProfessorK.A. Smith Departmentof Medicine, Dartmouth MedicalSchool, Hanover, New Hampshire03756, USA
KennethDorshkind cell can generate B cells as well as myeloid progeny, but steady-state B-cell production may not depend on this population. A number of cells could sustain B-cell production: these include a common lymphoid stem cell, the existence of which is largely speculative, or a B restricted precursor. It would be particularly helpful if early hemopoietic precursors could be isolated and their differentiative potential directly analysed. The observation by R. Mueller, J. Whitlock, and I. Weissman of Stanford University that multipotent hemopoietic precursors can be purified from the marrow because they express low levels of Thy-1 antigen should facilitate the characterization of primitive hemopoietic cells with B-lymphocyte differentiative potential and the parameters that regulate their development. To establish the full differentiative potential of a particular precursor cell, it is necessary to give the cell an optimal environment and to develop ways of identifying its progeny unambiguously. The resolution of lineage relationships has been improved by the introduction of selectable genes into primitive stem cells with retroviral vectors. These integrate randomly in the genome of each infected cell, creating unique restriction fragment length polymorphisms. The integration site is transmitted during cell division and
can thus be used as a clonal marker of the progeny of a single precursor. With this technique, R. Phillips and colleagues (Toronto) have demonstrated the existence of pluripotent stem cells, myeloid restricted stem cells, and, provisionally, a common lymphoid precursor. To obtain the optimal conditions for cellular differentiation the selected hemopoietic population is often transplanted into mice. A recently described mutant mouse with severe combined immunodeficiency disease (SCID) has been particularly valuable for studies on lymphopoiesis. These mice have no functional B or T cells but these populations can be reconstituted with grafts of normal stem cells. Although no pretransplant conditioning is needed, G. Fulop and R. Phillips (Toronto) showed that prior irradiation facilitates engraftment. These mutant mice are proving valuable in testing the lymphoid differentiative potential of selected cell populations. For example, K. Dorshkind, K. Denis, and O. Witte (Riverside and Los Angeles) have demonstrated that cells from Whitlock-Witte cultures are fully functional following transplantation into SCID mice. SCID bone marrow cells can be transformed with Abelson murine leukemia virus, the preferential target of which is B lineage cells, and these transformants have Ig gene rearrangements. There have been sug-
~) 1986, Elsevier Science Publishers B.V., Amsterdam
0167
4919/86/$02.00
Immunology Today, vol. 7, No. 11, 1986
gestions that the SCID defect is in the Ig gene assembly.
Regulation of Ig gene expression Cycling precursors in the bone marrow eventually generate surface IgM + B cells that can respond to foreign agents. The Ig repertoire of specificities is apparently not acquired randomly because responsiveness to various antigens appears in an ordered sequence during ontogeny. How this process occurs is not completely understood. The development of clonal assay systems that allow B lineage cells of different maturational states to be grown would facilitate study of Ig gene use in those populations and permit the effects of genetic background and environment on that process to be analysed. Recent work by C. Paige and his colleagues (Basel) has permitted the analysis of heavy chain V region gene expression in early fetal mouse liver B cell precursors and has demonstrated that the expression of VH gene fragments varies according to strain and stage of development. Studies on Ig H chain gene expression suggest that the assembly of V region genes in early B cell precursors is a result of the accessibility of those areas to transcription. Once a I~ chain protein is synthesized, it may exert a feedback signal which stops all additional H chain assembly. This negative feedback would result in a cell with one productive V-D-J rearrangement and, for example, a frozen D-J complex on the second allele. This mechanism could explain allelic exclusion, the phenomenon in which there is phenotypic expression of only one of the two Ig alleles in a single B cell. The mechanisms by which I• proteins exert their effects is unknown. The synthesis of the I~ protein may serve to signal the synthesis of light chain assembly, a process that is also highly regulated. The combined light chain-heavy chain protein then signals cessation of light chain gene assembly (F. AIt and colleagues, New York). Transgenic mice are proving to be a valuable tool in these analyses. Such mice are produced by microinjecting a Cloned gene into fertilized
mouse eggs and implanting these into pseudopregnant females. Progeny that express the particular gene are then selected at birth. The expression of the introduced gene in various tissues can be studied and its effects and that of its products on endogenous Ig gene expression analysed. Microenvironmentsfor E-cell
differentiation This regulation at a molecular level must be seen in the context of the tissue-specific regulatory signals encountered by developing B cells in the bone marrow. For example, various hormone-like molecules analogous to those which act on other hemopoietic lineages could provide the signal that triggers events at the DNA level. This suggestion is plausible in light of recent reports that factors such as those described by K. Landreth (Morgantown) and his colleagues in the urine of cyclic neutropenic patients or by P. Kincade (Oklahoma City) in the serum of NZB mice affect B-cell differentiation. The source of these molecules and their characterization has developed into an active area of investigation. Particular emphasis has focused on marrow stromal cells as providing the microenvironment necessary for Bcell differentiation. Stromal cells form the supporting framework in the spaces between the sinusoids of the medullary cavity. D. Osmond and his colleagues (Montreal) have demonstrated that surface IgM positive cells are associated with stromal cell processes in vivo. The work of M. Cooper and his associates (Birmingham) further suggests that microenvironments for Bcell differentiation may exist in early postnatal liver. It is unclear if the stromal cells which form B-cell microenvironments are the same or different from those which support myelopoiesis and how they mediate their effects. A valuable model system with which these questions are being approached is the long-term bone marrow culture system. Different types of these cultures exist, but the common feature is the establish-
ment of an adherent layer of stromal cells in culture upon which longterm hemopoiesis depends. The system described by Dexter is optimal for myelopoiesis; no B cells are detected in these cultures but an early B-cell precursor is present. The Whitlock-Witte system produces B cells and their precursors but not myeloid cells. Although the adherent layers in the two cultures appears different, the stromal cells that comprise them are not restricted to the support of myelopoiesis or B lymphopoiesis. Under appropriate conditions, Dexter stroma supports B lymphopoiesis while Whitlock-Witte stroma supports myelopoiesis. This may explain in part the finding that B lymphopoiesis can be induced in the Dexter system by switching established cultures to the WhitlockWitte conditions. Several groups have developed cell lines from the adherent layers of the various long-term cultures with the goal of identifying the stromal cell(s) which support B lymphopoiesis. The B-cell supporting capacity of these cells has been tested by seeding them with cells from primary Whitlock-Witte cultures, fresh bone marrow, or the Thy-1 positive stem cell previously described. It is now clear that several groups have cell lines that support B lymphopoiesis. These appear to mediate their effects, at least in part, via the secretion of regulatory molecules that act on B-cell precursors. It will be of interest to determine how the lines and factors isolated by different groups compare with one another. The model systems used to study primary B-cell differentiation are not restricted to those mentioned in this report. For example, analyses in nonmammalian systems such as the chicken bursa are providing complementary information. In view of the variety of approaches being taken, it is not too optimistic to expect that the questions noted here will be resolved in the near future. Division of BiomedicalSciences, Universityof California, Riverside, Cafifomia 92521-0121, USA
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P.
322
dence has already been set with IL-3, in that the murine entity has been characterized but the human molecule has not been isolated or identified. Although the opinions of those present were somewhat mixed on this point, the subcommittee recommends to the parent committee that cytokine activities be given an interleukin number only when the entity has been characterized from human origin. Accordingly, a particular cytokine from another species would carry an appropriate prefix (eg. murine IL-1), whereas the same molecule of human origin would not: rather, it is understood that an entity without a prefix is of human origin."
References 1 Noma, Y., P. Sideras,T. Naito, S. Bergstedt-Linquist, C. Azuma, E. Severinson,T. Tanabe, T. Kinashi, F. Matsuda, Y. Yaoita and T. Honjo (1986) Cloning of cDNA encoding the murine IgG1 induction factor by a novel strategy using SP6 promoter. Nature 319, 640 2 Lee, F., T. Yokota, T. Otsuka, P. Meyerson, D. Viltaret, R. Coffman, T. Mosmann, D. Rennick, N. Roehm, C. Smith, A. Zlotnik and K. Arai (1986) Isolation and characterization of a mouse interleukin cDNA clone that expressesB-cell stimulatory factor 1 activities and T-cell- and mast-celtstimulating activities. Proc. Natl Acad. Sci. USA 83, 2061 3 Yokota, T., T. Otsuka, T. Mosmann, J.
As lymphoid precursors in the bone marrow differentiate, they undergo an orderly series of gene rearrangements at the immunoglobulin (Ig) heavy and light chain loci which culminate in the synthesis of the Ig molecule. The genes involved in this process have been defined and their chromosomal location is known. It has also been possible to describe various cellular stages of B lymphopoiesis, owing to the availability of antibodies specific for Ig and various non-lg determinants. For example, B lineage cells that have not yet synthesized Ig can be recognized with antibodies directed against the B220 family of molecules. The recently described antibody to the BP-1 antigen, a 140 000 Mr molecule, should also prove useful in identifying early Bcell precursors. Subsequently, a population of pre-B cells expresses heavy chains of the I~ class in their cytoplasm. As light chain proteins are synthesized, the Ig molecule consisting of the I~ heavy chain and kappa or lambda light chains is assembled and expressed on the surface of newly produced B cells. This brief and obviously incomplete synopsis of B-cell development summarizes several years of work by many laboratories. Nevertheless, symposia, workshops, and informal discussions at the Congress defined several issues that remain to be resolved before the understanding of this differentiative pathway is complete.
B-cell differentiation
Identification of stem cellsand B lymphocyte precursors The pluripotent hemopoietic stem
Banchereau, T. DeFrance, D.
Blanchard, J.E. De Vries, F. Lee and K, Arai (1986) Isolationand characterization of a human interleukin cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expressesB-cell-and T-cellstimulating activities. Proc. NatlAcad. Sci. USA 83, 1
Readerswho wish to commenton these recommendationsshouldwrite to: ProfessorK.A. Smith Departmentof Medicine, Dartmouth MedicalSchool, Hanover, New Hampshire03756, USA
KennethDorshkind cell can generate B cells as well as myeloid progeny, but steady-state B-cell production may not depend on this population. A number of cells could sustain B-cell production: these include a common lymphoid stem cell, the existence of which is largely speculative, or a B restricted precursor. It would be particularly helpful if early hemopoietic precursors could be isolated and their differentiative potential directly analysed. The observation by R. Mueller, J. Whitlock, and I. Weissman of Stanford University that multipotent hemopoietic precursors can be purified from the marrow because they express low levels of Thy-1 antigen should facilitate the characterization of primitive hemopoietic cells with B-lymphocyte differentiative potential and the parameters that regulate their development. To establish the full differentiative potential of a particular precursor cell, it is necessary to give the cell an optimal environment and to develop ways of identifying its progeny unambiguously. The resolution of lineage relationships has been improved by the introduction of selectable genes into primitive stem cells with retroviral vectors. These integrate randomly in the genome of each infected cell, creating unique restriction fragment length polymorphisms. The integration site is transmitted during cell division and
can thus be used as a clonal marker of the progeny of a single precursor. With this technique, R. Phillips and colleagues (Toronto) have demonstrated the existence of pluripotent stem cells, myeloid restricted stem cells, and, provisionally, a common lymphoid precursor. To obtain the optimal conditions for cellular differentiation the selected hemopoietic population is often transplanted into mice. A recently described mutant mouse with severe combined immunodeficiency disease (SCID) has been particularly valuable for studies on lymphopoiesis. These mice have no functional B or T cells but these populations can be reconstituted with grafts of normal stem cells. Although no pretransplant conditioning is needed, G. Fulop and R. Phillips (Toronto) showed that prior irradiation facilitates engraftment. These mutant mice are proving valuable in testing the lymphoid differentiative potential of selected cell populations. For example, K. Dorshkind, K. Denis, and O. Witte (Riverside and Los Angeles) have demonstrated that cells from Whitlock-Witte cultures are fully functional following transplantation into SCID mice. SCID bone marrow cells can be transformed with Abelson murine leukemia virus, the preferential target of which is B lineage cells, and these transformants have Ig gene rearrangements. There have been sug-
~) 1986, Elsevier Science Publishers B.V., Amsterdam
0167
4919/86/$02.00
Immunology Today, vol. 7, No. 11, 1986
gestions that the SCID defect is in the Ig gene assembly.
Regulation of Ig gene expression Cycling precursors in the bone marrow eventually generate surface IgM + B cells that can respond to foreign agents. The Ig repertoire of specificities is apparently not acquired randomly because responsiveness to various antigens appears in an ordered sequence during ontogeny. How this process occurs is not completely understood. The development of clonal assay systems that allow B lineage cells of different maturational states to be grown would facilitate study of Ig gene use in those populations and permit the effects of genetic background and environment on that process to be analysed. Recent work by C. Paige and his colleagues (Basel) has permitted the analysis of heavy chain V region gene expression in early fetal mouse liver B cell precursors and has demonstrated that the expression of VH gene fragments varies according to strain and stage of development. Studies on Ig H chain gene expression suggest that the assembly of V region genes in early B cell precursors is a result of the accessibility of those areas to transcription. Once a I~ chain protein is synthesized, it may exert a feedback signal which stops all additional H chain assembly. This negative feedback would result in a cell with one productive V-D-J rearrangement and, for example, a frozen D-J complex on the second allele. This mechanism could explain allelic exclusion, the phenomenon in which there is phenotypic expression of only one of the two Ig alleles in a single B cell. The mechanisms by which I• proteins exert their effects is unknown. The synthesis of the I~ protein may serve to signal the synthesis of light chain assembly, a process that is also highly regulated. The combined light chain-heavy chain protein then signals cessation of light chain gene assembly (F. AIt and colleagues, New York). Transgenic mice are proving to be a valuable tool in these analyses. Such mice are produced by microinjecting a Cloned gene into fertilized
mouse eggs and implanting these into pseudopregnant females. Progeny that express the particular gene are then selected at birth. The expression of the introduced gene in various tissues can be studied and its effects and that of its products on endogenous Ig gene expression analysed. Microenvironmentsfor E-cell
differentiation This regulation at a molecular level must be seen in the context of the tissue-specific regulatory signals encountered by developing B cells in the bone marrow. For example, various hormone-like molecules analogous to those which act on other hemopoietic lineages could provide the signal that triggers events at the DNA level. This suggestion is plausible in light of recent reports that factors such as those described by K. Landreth (Morgantown) and his colleagues in the urine of cyclic neutropenic patients or by P. Kincade (Oklahoma City) in the serum of NZB mice affect B-cell differentiation. The source of these molecules and their characterization has developed into an active area of investigation. Particular emphasis has focused on marrow stromal cells as providing the microenvironment necessary for Bcell differentiation. Stromal cells form the supporting framework in the spaces between the sinusoids of the medullary cavity. D. Osmond and his colleagues (Montreal) have demonstrated that surface IgM positive cells are associated with stromal cell processes in vivo. The work of M. Cooper and his associates (Birmingham) further suggests that microenvironments for Bcell differentiation may exist in early postnatal liver. It is unclear if the stromal cells which form B-cell microenvironments are the same or different from those which support myelopoiesis and how they mediate their effects. A valuable model system with which these questions are being approached is the long-term bone marrow culture system. Different types of these cultures exist, but the common feature is the establish-
ment of an adherent layer of stromal cells in culture upon which longterm hemopoiesis depends. The system described by Dexter is optimal for myelopoiesis; no B cells are detected in these cultures but an early B-cell precursor is present. The Whitlock-Witte system produces B cells and their precursors but not myeloid cells. Although the adherent layers in the two cultures appears different, the stromal cells that comprise them are not restricted to the support of myelopoiesis or B lymphopoiesis. Under appropriate conditions, Dexter stroma supports B lymphopoiesis while Whitlock-Witte stroma supports myelopoiesis. This may explain in part the finding that B lymphopoiesis can be induced in the Dexter system by switching established cultures to the WhitlockWitte conditions. Several groups have developed cell lines from the adherent layers of the various long-term cultures with the goal of identifying the stromal cell(s) which support B lymphopoiesis. The B-cell supporting capacity of these cells has been tested by seeding them with cells from primary Whitlock-Witte cultures, fresh bone marrow, or the Thy-1 positive stem cell previously described. It is now clear that several groups have cell lines that support B lymphopoiesis. These appear to mediate their effects, at least in part, via the secretion of regulatory molecules that act on B-cell precursors. It will be of interest to determine how the lines and factors isolated by different groups compare with one another. The model systems used to study primary B-cell differentiation are not restricted to those mentioned in this report. For example, analyses in nonmammalian systems such as the chicken bursa are providing complementary information. In view of the variety of approaches being taken, it is not too optimistic to expect that the questions noted here will be resolved in the near future. Division of BiomedicalSciences, Universityof California, Riverside, Cafifomia 92521-0121, USA
323