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B cell differentiation in bone marrow
CLINICAL IMMUNOLOGYAND [MMUNOPATHOLOGY Vol. 76, No. 3, September, pp. S188-S191, 1995
B Cell Differentiation in Bone Marrow 1 FRITZ M E L C H E R S
Basel Institute for Immunology, CH-4005 Basel, Switzerland
Lymphocytes of mouse and humans are generated throughout life in the primary lymphoid organs. The similarities of the cellularsity, the composition of subcompartments, the modes of successive antigen receptor gene rearrangements, the turnover, and the rates of selection into the peripheral, mature pool for T lineage cells in the thyn/us and for B lineage cells in bone marrow are striking. This presentation summarizes our knowledge of B cell differentiation in bone marrow. For the early stages of development, our knowledge relies solely" on results obtained with mouse bone marrow, while the development of later stages can be discussed with data obtained in mouse and humans. To conclude, B cell development in bone marrow is compared with T cell development in the thymus. Progenitors which are derived from pluripontent hemotopoietic stem cells are induced in bone marrow to be committed to the B lineage pathway of differentiation. This pathway is highlighted by the expression of specific intracellular and surface-bound marker molecules, some with known functions in this development, and by immunoglobulin (Ig) heavy (H) and light (L) chain gene rearrangements. Rearrangements are ordered in time and space during lymphocyte development. DH to JH rearrangements in IgH chain gene loci precede V s to DHJ H rearrangements, which are followed by V K to JK, and finally V~ to J~, rearrangements (1). Order in the rearrangements is imposed by molecular and cellular selection mechanisms. Molecular selections are thought to involve the opening of a given Ig gene locus, its sterile transcription, and the recognition of specific heptamer/nonamer palindromic sequences by the rearrangement enzyme complex. Cells with productively rearranged gene loci, hence with Ig chains expressed in them and on their surface, are selected by either suppression or enhancement of proliferation of a given stage of precursor cell. Other stages are selected by the suppression or induction of the transit of a cell from one compartment to the next by differentiation without proliferation (2). Nevertheless differentiation to more mature stages 1 Presented as part of the fifth Jeffrey Modell Immunodeficiency Symposium titled "Advances in Primary Immunodeficiency Disease," October 10-11, 1994, Paris, France.
of precursor cells can occur without any Ig gene rearrangements, although the selection of cells to proliferate or their suppression to do so, does not take place in cells which do not express Ig chains. Hence the number of cells in bone marrow remains very low in m u t a n t mice (and humans) and leads to severe combined immunodeficiency states (SCID) in the B cell compartments. DIFFERENTIATION OF PRECURSOR B LYMPHOCYTES W I T H O U T Ig G E N E R E A R R A N G E M E N T S
The roles of Ig H and L chains in these cellular selections becomes most evident in m u t a n t mice, such as SCID, RAG-1T, or RAG-2T (3-5). "T" stands for targeted integration of a defective gene by homologous recombination into the germline, leading to mouse strains which are defective for the targeted (T) gene (6, 7). In SCID mice, Ig gene r e a r r a n g e m e n t s are attempted but never productive; in RAG-1T or RAG-2T mice, they are not even attempted. In recent experiments from our laboratory it has been demonstrated that a cellular program of differentiation of B lineage cells from the earliest progenitor to the stage of an immature B cell can occur without productive, or even without any, Ig gene rearrangements (8). Differentiation can be monitored by changes in expression of preB cell-specific markers, by changes of in vitro growth properties, by the induction of apoptosis, and by the expression of sterile transcripts of the IgH and KL chain gene loci. Stromal cell/IL-7-reactive B220 + B cell precursors are found in fetal liver and bone marrow of SCID, SCID/bcl-2 transgenic, and RAG-2T mice in frequencies comparable to normal mice. Like cells from normal mice, they proliferate normally on stromal cells in the presence of IL-7 and differentiate in vitro upon IL-7 deprivation from c-kit +, CD43 +, s u r r o g a t e L chain +, RAG-1 +, RAG-2 +, and CD25- clonable preB cells into c-kit-, CD43-, surrogate L chain-, RAG-1 +, RAG-2 +, and CD25 + immature B cells which are no longer clonable on stromal cells and IL-7. Concomitantly, they enter a program of apoptosis, unless a bcl-2 transgene is expressed. While long-term proliferating precursor B cells of RAG-2T express sterile transcripts of the ~H, but little of the KL, chain gene locus, in vitro differentiation to later stages of development
$188 0090-1229/95 $12.00 Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
B CELL DIFFERENTIATION IN BONE MARROW induces sterile transcription of the KL chain gene locus. These findings suggest that B cell differentiation to the stage of immature B cells in vivo might be controlled by cell-to-cell contacts and by cytokines provided by the microenvironment in the bone marrow, but not by Ig gene rearrangements or Ig proteins expressed from them. While differentiation of cells without Ig gene expression appears to be possible, the development of normal numbers of precursors in bone marrow and thymus is severely impaired in rearrangement-defective mice (and in similar h u m a n SCID patients) (9). They have at best 1% of the normal number of cells which express m a r k e r s characteristic of the ~H chain-expressing preB-II cells or of the ~H chain/L chain-expressing immature B cells in bone marrow. Hence, expansion of precursor cells to fill the compartments in bone marrow and thymus with normal numbers of cells is impaired and mature, peripheral B cells are missing. Precursor thymocyte development occurs at very similar stages of development in the absence of TCR gene rearrangements, i.e., in RAG-1T and RAG-2T mice. Again, the expansion of precursors in the thymus and, hence, the generation of normal numbers of peripheral, mature T cells is impaired. T H E P R E - B CELL R E C E P T O R OF S U R R O G A T E L CHAIN AND ~H CHAIN POSITIVELY SELECTS P R O D U C T I V E L Y VHDHJH-REARRANGED B CELL P R E C U R S O R S BY I N D U C T I O N OF PROLIFERATION
One of the ways by which productive rearrangements of the H chain gene locus could be monitored by a precursor B cell would be to deposit DHJHC~ proteins and ~H chains in association with the surrogate L chain, encoded by the preB cell-specific genes h a and VpreB, into the surface membrane (10). This view of B cell differentiation is supported by the analysis of mice carrying a targeted deletion of the exon coding for the t r a n s m e m b r a n e portion of ~H chain (~MT mice) (11) or a targeted deletion of the entire JH gene cluster of the IgH gene locus (JHT mice) (12, 13). Both m u t a n t mouse strains are devoid of peripheral, mature B cells and are blocked in the development of B cell precursors at the transition for DHJ Hrearranged preB-I to VHDHJH-rearranged preB-II cells (for nomenclature used in this study, see 2). Mice with a targeted disruption of the ha gene (h a T mice) (14) are blocked at the same stage of B cell differentiation; however, they can generate B cells from the smaller pool of early preB-1 cells, so that peripheral B cells are produced, although at a slower rate (15). The analysis of B cell generation in haT mice shows that the surrogate L chain is not mandatory, but helpful, for B'cell development. It allows the expansion of precursor B cells with productive VHDHJH rearrangements and thereby the generation of a 20- to 100-fold larger preB-II cell compartment, which is large enough to secure the generation of sufficient numbers of sIg ÷ B cells.
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In support of this scenario, it has been shown by Decker et al. (16) that once a V H gene segment has been rearranged to DHJ H gene segments yielding a productive VHDHJ H rearrangement, an estimated five to six divisions may occur. This would allow the estimated 2-5 x 106 pro/preB-I cells to expand to 30-50 x 106 cytoplasmic ~H chain-positive preB-II cells in vivo. This preB-II compartment has been found to express CD25 (17). T cell development could be guided by very similar p r o l i f e r a t i v e e x p a n s i o n of p r o d u c t i v e l y V~D~J~rearranged CD4- CD8- precursor thymocytes which have recently been shown to express a glycoprotein of 33 kDa together with the TcR/3 chain on their surface (18). This 33-kDa protein might well serve the role of a "surrogate a chain" in T cell development. ALLELIC EXCLUSION One B cell makes only one H chain, and one T cell only one/3 chain. This phenomenon, called allelic exclusion, can be explained by a number of events contributing to the final representation of cells expressing only one of the two possible H and /3 chains, respectively. First, when the first productive .VHDHJH or V~D~J~ rearrangement has been made in a preB-II cell or in a double-negative thymocyte, while the second allele may still be only either DHJ H or D~J~ rearranged, or m a y already have made a VHDHJ H or V~D~J~ rearrangement, although nonproductive, the expression of the ~H chain/surrogate L chain preB cell receptor would lead to a 100-fold expansion by proliferation of this cell with only one productive rearrangement. Furthermore, however, it is possible that the preB cell receptor and, respectively, the preT cell receptor signal not only proliferative expansion but also inhibition of the rearrangement machinery and the transcriptional activity at the second allele. N E G A T I V E A N D P O S I T I V E SELECTIONS OF IMMATURE L Y M P H O C Y T E S E X P R E S S I N G ~ T c R or ~H/L C H A I N Ig
MOLECULES ON THEIR SURFACE During the transit into the mature, peripheral compartments of the immune system, immature TcR ÷ and sIg ÷ cells are negatively and positively selected (2, 19). Negative selection deletes in primary lymphoid organs and anergizes in the periphery those cells which are autoreactive with high avidity for self-antigens. Immature lymphocytes differ from mature, peripheral cells by their continued expression of the recombination-active genes RAG-1 and RAG-2 (20, 21). Immature sIgM ÷ sIgD- B cells generated from precursors "in vitro" therefore continue to rearrange K and h L chain gene loci. M a t u r e B cells no longer express RAG-1 and RAG-2, no longer rearrange ~ and h L chain gene loci, express sIgM and sIgD, and have change~l their life expectancy. ~n the primary lymphoid organs practically all lymphdid precursors, i.e., all immature " i n - f r a m e " - r e a r r a n g e d cells, such as TcR or Ig-
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FRITZ MELCHERS
expressing cells, or out-of-frame-rearranged cells, such as TcR or Ig-negative cells, have a short life, around 3 days. Each day a small proportion (between 0.1 and 3%) of these immature cells transit from the primary to the secondary lymphoid organs. In the secondary organs only TcR + and Ig + cells, but not the TcR- and Igcells, are found, and they now are much longer lived. Their half-lives have been measured to be at least 6 weeks and sometimes longer (22). In the transition from immature to mature B cells, surface-bound Ig molecules signal the downregulation of RAG-1 and RAG-2 expression, the upregnlation of surface IgD expression, and a change in the migratory patterns and life expectancy of these cells. It is not clear whether this transition from immature to mature cells is, again, accompanied by proliferative expansion. Positive selection is well understood for T cells (19). Immature thymocytes expressing a TcR with intermediate avidity for self-MHC class I or II molecules and self-peptides bound into them are positively selected to fill the pool of peripheral, mature T cells in which all those with nonfitting, useless TcR are discarded. Positive ~election of fitting T cells probably prolongs their life expectancy, while nonfitting T cells die within 3 days, as rapidly as those which never made a TcR due to out-of-frame rearrangements. As a result of this positive selection, a repertoire of self-MHC-restricted T cells is generated for the periphery. A foreign peptide, presented on the self-MHC molecules upon uptake and processing of the corresponding foreign antigen by antigen-presenting cells, will elicit the response o f a T cell to proliferate and mature to effector functions whenever it manages to increase the avidity of a TcR on a peripheral mature T cell for the presenting complex of foreign peptide and self-MHC molecule. Positive selection also occurs during transit from immature sIg ÷ B cells in primary lymphoid organs to mature sIg ÷ B cells in the periphery (2). It is not yet clear whether self-antigens can positively select, and if so, which can. The repertoires or peripheral, mature B cells do not give any evidence of dominant selecting self-antigens such as the MHC molecules for T cells. Positive selection of B cells certainly occurs when they are stimulated by foreign antigens (23). This can occur with and without the help of T cells. Helper T cell-independent antigens stimulate B cells mainly to proliferate and maturate into clones of IgM-secreting cells. No memory develops. Helper T cell-dependent antigens, on the other hand, stimulate not only proliferation and maturation to IgM secretion, but also induce switching of IgH class expression and somatic hypermutations of variable (V) regions of Ig H and L chain genes. This occurs in germinal centers of secondary lymphoid organs. Memory of the antigenic experience develops. Some memory cells can be accounted for by an increase in antigen-specific T and B cells, by changes in the expression p a t t e r n s of lymphokine genes in T H cells, by changes in the class and the avid-
ity of the Ig molecules specific for the antigen made by the B cells, and by different migratory patterns and increased life expectancies of the experienced antigenspecific T and B cells. ACKNOWLEDGMENT The Basel Institute for Immunology was founded and is supported by F. Hoffman La Roche, Ltd., Basel, Switzerland. REFERENCES 1. A]t, F.W., Blackwell, T. K., Depinho, R.A., Reth, M. G., and Yancopoulos, G. D., Regulation ofgenome rearrangement events during lymphocyte differentiation. Immunol. Rev. 89, 5-30, 1986. 2. Rolink, A., and Melchers, F., Generation and regeneration of cells of the B-lymphocyte lineage. Curr. Opin. Immunol. 5, 207217, 1993. 3. Bosma, G. C., Custer, R. P., and Bosma, M. J., A severe combined immunodeficiency mutation in the mouse. Nature 301, 527-530, 1983. 4. Mombaerts, P., Iacomini, J., Johnson, R. S., Herrup, K., Tonegawa, S., and Papaioannou, V. E., RAG-l-deficient mice have no mature B and T lymphocytes. Cell 68, 869-877, 1992. 5. Shinkai, Y., Rathbun, G., Lam, K. P., Oltz, E. M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A. M., and Alt, F. W., RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855867, 1992. 6. Gu, H., Zou, Y.-R., and Rajewsky, K., Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-medicated gene targeting. Cell 73, 1155-1164, 1993. 7. Chen, J., Gorman, J. R., Stewart, V., Williams, B., Jacks, T., and Alt, F. W., Generation of normal lymphocyte populations by Rbdeficient embryonic stem cells. Curr. Biol. 3, 405, 1993. 8. Melchers, F., Haasner, D., Grawunder, U., Kalberer, C., Karasuyama, H., Wink]er, T., and Rolink, A. G., Roles of IgH and L chains and of surrogate H and L chains in the development of cells of the B lymphocyte lineage. Annu. Rev. Immunol. 12, 209225, 1994. 9. Rolink, A., Karasuyama, H., Haasner, D., Grawunder, U., Martensson, I. L., Kudo, A., and Melchers, F., Two pathways of B lymphocyte development in mouse bone marrow and the roles of surrogate L chain in this development. Immunol. Rev. 137, 185201, 1994. 10. Melchers, F., Karasuyama, H., Haasner, D., Bauer, S., Kudo, A., Sakaguchi, N., Jameson, B., and Rolink, A., The surrogate light chain in B-cell development. Immunol. Today 14, 60-68, 1993. 11. Kitamura, D., Roes, J., Kfihn, R., and Rajewsky, K., A B celldeficient mouse by targeted disruption of the membrane exon of the immunoglobulin Ix chain gene. Nature 350, 423-426, 1991. 12. Chen, J., Trounstine, M., Alt, F. W., Young, F., Kurahara, C., Loring, J. F., and Huszar, D., Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus. Int. Immunol. 5, 647-656, 1993. 13. Ehlich, A., Schaal, S., Gu, H., Kitamura, D., Mtiller, W., and Rajewsky, K., Immunoglobulin heavy and light chain genes rearrange independently at early stages orB cell development. Cell 72, 695-704, 1993. 14. Kitamura, D., Kudo, A., Schaal, S., Mfiller, W., Melchers, F., and Rajewsky, K., A critical role of As in B cell development. Cell 69, 823-831, 1992.
B CELL DIFFERENTIATION IN BONE MARROW 15. Rolink, A., Karasuyama, H., Grawunder, U., Haasner, D., Kudo, A., and Melchers, F., B cell development in mice with a defective A5 gene. Eur. J. Immunol. 23, 1284-1288, 1993. 16. Decker, D. J., Boyle, N. E., Koziol, J. A., and Klinman, N. R., The expression of the Ig H chain repertoire in developing bone marrow B lineage cells. J. Immunol. 146, 350-361, 1991. 17. Rolink, A., Grawunder, U., Winkler, T. H., Karasuyama, H., and F., M., IL-2 receptor a chain (CD25, TAC) expression defines a crucial stage in preB cell development. Int. Immunol. 6, 12571264, 1994. 18. Groettrup, M., and yon Boehmer, H., A role for a preT-cell receptor in T-cell development. Immunol. Today 14, 610-614, 1993. Received March 16, 1995; accepted May 12, 1995
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19. yon Boehmer, H., Thymic selection: A matter of life and death. Immunol. Today 13, 454-458, 1992. 20. Rolink, A., Grawunder, U., Haasner, D., Strasser, A., and Melchers, F., Immature surface Ig÷ B cells can continue to rearrange ~ and A L chain gene loci. J. Exp. Med. 178, 1263-1270, 1993. 21. Borgulya, P., Kishi, H., Uematsu, Y., and yon Boehmer, H., Exclusion and inclusion of ~ and/3 T cell receptor alleles. Cell 69, 529-537, 1992. 22. Rajewsky, K., B-cell lifespans in the mouse--Why to debate what? Immunol. Today 14, 40-41, 1993. 23. Clark, E. A., and Ledbetter, J. A., How B and T cells talk to each other. Nature 367, 425-428, 1994.
B Cell Differentiation in Bone Marrow 1 FRITZ M E L C H E R S
Basel Institute for Immunology, CH-4005 Basel, Switzerland
Lymphocytes of mouse and humans are generated throughout life in the primary lymphoid organs. The similarities of the cellularsity, the composition of subcompartments, the modes of successive antigen receptor gene rearrangements, the turnover, and the rates of selection into the peripheral, mature pool for T lineage cells in the thyn/us and for B lineage cells in bone marrow are striking. This presentation summarizes our knowledge of B cell differentiation in bone marrow. For the early stages of development, our knowledge relies solely" on results obtained with mouse bone marrow, while the development of later stages can be discussed with data obtained in mouse and humans. To conclude, B cell development in bone marrow is compared with T cell development in the thymus. Progenitors which are derived from pluripontent hemotopoietic stem cells are induced in bone marrow to be committed to the B lineage pathway of differentiation. This pathway is highlighted by the expression of specific intracellular and surface-bound marker molecules, some with known functions in this development, and by immunoglobulin (Ig) heavy (H) and light (L) chain gene rearrangements. Rearrangements are ordered in time and space during lymphocyte development. DH to JH rearrangements in IgH chain gene loci precede V s to DHJ H rearrangements, which are followed by V K to JK, and finally V~ to J~, rearrangements (1). Order in the rearrangements is imposed by molecular and cellular selection mechanisms. Molecular selections are thought to involve the opening of a given Ig gene locus, its sterile transcription, and the recognition of specific heptamer/nonamer palindromic sequences by the rearrangement enzyme complex. Cells with productively rearranged gene loci, hence with Ig chains expressed in them and on their surface, are selected by either suppression or enhancement of proliferation of a given stage of precursor cell. Other stages are selected by the suppression or induction of the transit of a cell from one compartment to the next by differentiation without proliferation (2). Nevertheless differentiation to more mature stages 1 Presented as part of the fifth Jeffrey Modell Immunodeficiency Symposium titled "Advances in Primary Immunodeficiency Disease," October 10-11, 1994, Paris, France.
of precursor cells can occur without any Ig gene rearrangements, although the selection of cells to proliferate or their suppression to do so, does not take place in cells which do not express Ig chains. Hence the number of cells in bone marrow remains very low in m u t a n t mice (and humans) and leads to severe combined immunodeficiency states (SCID) in the B cell compartments. DIFFERENTIATION OF PRECURSOR B LYMPHOCYTES W I T H O U T Ig G E N E R E A R R A N G E M E N T S
The roles of Ig H and L chains in these cellular selections becomes most evident in m u t a n t mice, such as SCID, RAG-1T, or RAG-2T (3-5). "T" stands for targeted integration of a defective gene by homologous recombination into the germline, leading to mouse strains which are defective for the targeted (T) gene (6, 7). In SCID mice, Ig gene r e a r r a n g e m e n t s are attempted but never productive; in RAG-1T or RAG-2T mice, they are not even attempted. In recent experiments from our laboratory it has been demonstrated that a cellular program of differentiation of B lineage cells from the earliest progenitor to the stage of an immature B cell can occur without productive, or even without any, Ig gene rearrangements (8). Differentiation can be monitored by changes in expression of preB cell-specific markers, by changes of in vitro growth properties, by the induction of apoptosis, and by the expression of sterile transcripts of the IgH and KL chain gene loci. Stromal cell/IL-7-reactive B220 + B cell precursors are found in fetal liver and bone marrow of SCID, SCID/bcl-2 transgenic, and RAG-2T mice in frequencies comparable to normal mice. Like cells from normal mice, they proliferate normally on stromal cells in the presence of IL-7 and differentiate in vitro upon IL-7 deprivation from c-kit +, CD43 +, s u r r o g a t e L chain +, RAG-1 +, RAG-2 +, and CD25- clonable preB cells into c-kit-, CD43-, surrogate L chain-, RAG-1 +, RAG-2 +, and CD25 + immature B cells which are no longer clonable on stromal cells and IL-7. Concomitantly, they enter a program of apoptosis, unless a bcl-2 transgene is expressed. While long-term proliferating precursor B cells of RAG-2T express sterile transcripts of the ~H, but little of the KL, chain gene locus, in vitro differentiation to later stages of development
$188 0090-1229/95 $12.00 Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
B CELL DIFFERENTIATION IN BONE MARROW induces sterile transcription of the KL chain gene locus. These findings suggest that B cell differentiation to the stage of immature B cells in vivo might be controlled by cell-to-cell contacts and by cytokines provided by the microenvironment in the bone marrow, but not by Ig gene rearrangements or Ig proteins expressed from them. While differentiation of cells without Ig gene expression appears to be possible, the development of normal numbers of precursors in bone marrow and thymus is severely impaired in rearrangement-defective mice (and in similar h u m a n SCID patients) (9). They have at best 1% of the normal number of cells which express m a r k e r s characteristic of the ~H chain-expressing preB-II cells or of the ~H chain/L chain-expressing immature B cells in bone marrow. Hence, expansion of precursor cells to fill the compartments in bone marrow and thymus with normal numbers of cells is impaired and mature, peripheral B cells are missing. Precursor thymocyte development occurs at very similar stages of development in the absence of TCR gene rearrangements, i.e., in RAG-1T and RAG-2T mice. Again, the expansion of precursors in the thymus and, hence, the generation of normal numbers of peripheral, mature T cells is impaired. T H E P R E - B CELL R E C E P T O R OF S U R R O G A T E L CHAIN AND ~H CHAIN POSITIVELY SELECTS P R O D U C T I V E L Y VHDHJH-REARRANGED B CELL P R E C U R S O R S BY I N D U C T I O N OF PROLIFERATION
One of the ways by which productive rearrangements of the H chain gene locus could be monitored by a precursor B cell would be to deposit DHJHC~ proteins and ~H chains in association with the surrogate L chain, encoded by the preB cell-specific genes h a and VpreB, into the surface membrane (10). This view of B cell differentiation is supported by the analysis of mice carrying a targeted deletion of the exon coding for the t r a n s m e m b r a n e portion of ~H chain (~MT mice) (11) or a targeted deletion of the entire JH gene cluster of the IgH gene locus (JHT mice) (12, 13). Both m u t a n t mouse strains are devoid of peripheral, mature B cells and are blocked in the development of B cell precursors at the transition for DHJ Hrearranged preB-I to VHDHJH-rearranged preB-II cells (for nomenclature used in this study, see 2). Mice with a targeted disruption of the ha gene (h a T mice) (14) are blocked at the same stage of B cell differentiation; however, they can generate B cells from the smaller pool of early preB-1 cells, so that peripheral B cells are produced, although at a slower rate (15). The analysis of B cell generation in haT mice shows that the surrogate L chain is not mandatory, but helpful, for B'cell development. It allows the expansion of precursor B cells with productive VHDHJH rearrangements and thereby the generation of a 20- to 100-fold larger preB-II cell compartment, which is large enough to secure the generation of sufficient numbers of sIg ÷ B cells.
S189
In support of this scenario, it has been shown by Decker et al. (16) that once a V H gene segment has been rearranged to DHJ H gene segments yielding a productive VHDHJ H rearrangement, an estimated five to six divisions may occur. This would allow the estimated 2-5 x 106 pro/preB-I cells to expand to 30-50 x 106 cytoplasmic ~H chain-positive preB-II cells in vivo. This preB-II compartment has been found to express CD25 (17). T cell development could be guided by very similar p r o l i f e r a t i v e e x p a n s i o n of p r o d u c t i v e l y V~D~J~rearranged CD4- CD8- precursor thymocytes which have recently been shown to express a glycoprotein of 33 kDa together with the TcR/3 chain on their surface (18). This 33-kDa protein might well serve the role of a "surrogate a chain" in T cell development. ALLELIC EXCLUSION One B cell makes only one H chain, and one T cell only one/3 chain. This phenomenon, called allelic exclusion, can be explained by a number of events contributing to the final representation of cells expressing only one of the two possible H and /3 chains, respectively. First, when the first productive .VHDHJH or V~D~J~ rearrangement has been made in a preB-II cell or in a double-negative thymocyte, while the second allele may still be only either DHJ H or D~J~ rearranged, or m a y already have made a VHDHJ H or V~D~J~ rearrangement, although nonproductive, the expression of the ~H chain/surrogate L chain preB cell receptor would lead to a 100-fold expansion by proliferation of this cell with only one productive rearrangement. Furthermore, however, it is possible that the preB cell receptor and, respectively, the preT cell receptor signal not only proliferative expansion but also inhibition of the rearrangement machinery and the transcriptional activity at the second allele. N E G A T I V E A N D P O S I T I V E SELECTIONS OF IMMATURE L Y M P H O C Y T E S E X P R E S S I N G ~ T c R or ~H/L C H A I N Ig
MOLECULES ON THEIR SURFACE During the transit into the mature, peripheral compartments of the immune system, immature TcR ÷ and sIg ÷ cells are negatively and positively selected (2, 19). Negative selection deletes in primary lymphoid organs and anergizes in the periphery those cells which are autoreactive with high avidity for self-antigens. Immature lymphocytes differ from mature, peripheral cells by their continued expression of the recombination-active genes RAG-1 and RAG-2 (20, 21). Immature sIgM ÷ sIgD- B cells generated from precursors "in vitro" therefore continue to rearrange K and h L chain gene loci. M a t u r e B cells no longer express RAG-1 and RAG-2, no longer rearrange ~ and h L chain gene loci, express sIgM and sIgD, and have change~l their life expectancy. ~n the primary lymphoid organs practically all lymphdid precursors, i.e., all immature " i n - f r a m e " - r e a r r a n g e d cells, such as TcR or Ig-
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FRITZ MELCHERS
expressing cells, or out-of-frame-rearranged cells, such as TcR or Ig-negative cells, have a short life, around 3 days. Each day a small proportion (between 0.1 and 3%) of these immature cells transit from the primary to the secondary lymphoid organs. In the secondary organs only TcR + and Ig + cells, but not the TcR- and Igcells, are found, and they now are much longer lived. Their half-lives have been measured to be at least 6 weeks and sometimes longer (22). In the transition from immature to mature B cells, surface-bound Ig molecules signal the downregulation of RAG-1 and RAG-2 expression, the upregnlation of surface IgD expression, and a change in the migratory patterns and life expectancy of these cells. It is not clear whether this transition from immature to mature cells is, again, accompanied by proliferative expansion. Positive selection is well understood for T cells (19). Immature thymocytes expressing a TcR with intermediate avidity for self-MHC class I or II molecules and self-peptides bound into them are positively selected to fill the pool of peripheral, mature T cells in which all those with nonfitting, useless TcR are discarded. Positive ~election of fitting T cells probably prolongs their life expectancy, while nonfitting T cells die within 3 days, as rapidly as those which never made a TcR due to out-of-frame rearrangements. As a result of this positive selection, a repertoire of self-MHC-restricted T cells is generated for the periphery. A foreign peptide, presented on the self-MHC molecules upon uptake and processing of the corresponding foreign antigen by antigen-presenting cells, will elicit the response o f a T cell to proliferate and mature to effector functions whenever it manages to increase the avidity of a TcR on a peripheral mature T cell for the presenting complex of foreign peptide and self-MHC molecule. Positive selection also occurs during transit from immature sIg ÷ B cells in primary lymphoid organs to mature sIg ÷ B cells in the periphery (2). It is not yet clear whether self-antigens can positively select, and if so, which can. The repertoires or peripheral, mature B cells do not give any evidence of dominant selecting self-antigens such as the MHC molecules for T cells. Positive selection of B cells certainly occurs when they are stimulated by foreign antigens (23). This can occur with and without the help of T cells. Helper T cell-independent antigens stimulate B cells mainly to proliferate and maturate into clones of IgM-secreting cells. No memory develops. Helper T cell-dependent antigens, on the other hand, stimulate not only proliferation and maturation to IgM secretion, but also induce switching of IgH class expression and somatic hypermutations of variable (V) regions of Ig H and L chain genes. This occurs in germinal centers of secondary lymphoid organs. Memory of the antigenic experience develops. Some memory cells can be accounted for by an increase in antigen-specific T and B cells, by changes in the expression p a t t e r n s of lymphokine genes in T H cells, by changes in the class and the avid-
ity of the Ig molecules specific for the antigen made by the B cells, and by different migratory patterns and increased life expectancies of the experienced antigenspecific T and B cells. ACKNOWLEDGMENT The Basel Institute for Immunology was founded and is supported by F. Hoffman La Roche, Ltd., Basel, Switzerland. REFERENCES 1. A]t, F.W., Blackwell, T. K., Depinho, R.A., Reth, M. G., and Yancopoulos, G. D., Regulation ofgenome rearrangement events during lymphocyte differentiation. Immunol. Rev. 89, 5-30, 1986. 2. Rolink, A., and Melchers, F., Generation and regeneration of cells of the B-lymphocyte lineage. Curr. Opin. Immunol. 5, 207217, 1993. 3. Bosma, G. C., Custer, R. P., and Bosma, M. J., A severe combined immunodeficiency mutation in the mouse. Nature 301, 527-530, 1983. 4. Mombaerts, P., Iacomini, J., Johnson, R. S., Herrup, K., Tonegawa, S., and Papaioannou, V. E., RAG-l-deficient mice have no mature B and T lymphocytes. Cell 68, 869-877, 1992. 5. Shinkai, Y., Rathbun, G., Lam, K. P., Oltz, E. M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A. M., and Alt, F. W., RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855867, 1992. 6. Gu, H., Zou, Y.-R., and Rajewsky, K., Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-medicated gene targeting. Cell 73, 1155-1164, 1993. 7. Chen, J., Gorman, J. R., Stewart, V., Williams, B., Jacks, T., and Alt, F. W., Generation of normal lymphocyte populations by Rbdeficient embryonic stem cells. Curr. Biol. 3, 405, 1993. 8. Melchers, F., Haasner, D., Grawunder, U., Kalberer, C., Karasuyama, H., Wink]er, T., and Rolink, A. G., Roles of IgH and L chains and of surrogate H and L chains in the development of cells of the B lymphocyte lineage. Annu. Rev. Immunol. 12, 209225, 1994. 9. Rolink, A., Karasuyama, H., Haasner, D., Grawunder, U., Martensson, I. L., Kudo, A., and Melchers, F., Two pathways of B lymphocyte development in mouse bone marrow and the roles of surrogate L chain in this development. Immunol. Rev. 137, 185201, 1994. 10. Melchers, F., Karasuyama, H., Haasner, D., Bauer, S., Kudo, A., Sakaguchi, N., Jameson, B., and Rolink, A., The surrogate light chain in B-cell development. Immunol. Today 14, 60-68, 1993. 11. Kitamura, D., Roes, J., Kfihn, R., and Rajewsky, K., A B celldeficient mouse by targeted disruption of the membrane exon of the immunoglobulin Ix chain gene. Nature 350, 423-426, 1991. 12. Chen, J., Trounstine, M., Alt, F. W., Young, F., Kurahara, C., Loring, J. F., and Huszar, D., Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus. Int. Immunol. 5, 647-656, 1993. 13. Ehlich, A., Schaal, S., Gu, H., Kitamura, D., Mtiller, W., and Rajewsky, K., Immunoglobulin heavy and light chain genes rearrange independently at early stages orB cell development. Cell 72, 695-704, 1993. 14. Kitamura, D., Kudo, A., Schaal, S., Mfiller, W., Melchers, F., and Rajewsky, K., A critical role of As in B cell development. Cell 69, 823-831, 1992.
B CELL DIFFERENTIATION IN BONE MARROW 15. Rolink, A., Karasuyama, H., Grawunder, U., Haasner, D., Kudo, A., and Melchers, F., B cell development in mice with a defective A5 gene. Eur. J. Immunol. 23, 1284-1288, 1993. 16. Decker, D. J., Boyle, N. E., Koziol, J. A., and Klinman, N. R., The expression of the Ig H chain repertoire in developing bone marrow B lineage cells. J. Immunol. 146, 350-361, 1991. 17. Rolink, A., Grawunder, U., Winkler, T. H., Karasuyama, H., and F., M., IL-2 receptor a chain (CD25, TAC) expression defines a crucial stage in preB cell development. Int. Immunol. 6, 12571264, 1994. 18. Groettrup, M., and yon Boehmer, H., A role for a preT-cell receptor in T-cell development. Immunol. Today 14, 610-614, 1993. Received March 16, 1995; accepted May 12, 1995
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19. yon Boehmer, H., Thymic selection: A matter of life and death. Immunol. Today 13, 454-458, 1992. 20. Rolink, A., Grawunder, U., Haasner, D., Strasser, A., and Melchers, F., Immature surface Ig÷ B cells can continue to rearrange ~ and A L chain gene loci. J. Exp. Med. 178, 1263-1270, 1993. 21. Borgulya, P., Kishi, H., Uematsu, Y., and yon Boehmer, H., Exclusion and inclusion of ~ and/3 T cell receptor alleles. Cell 69, 529-537, 1992. 22. Rajewsky, K., B-cell lifespans in the mouse--Why to debate what? Immunol. Today 14, 40-41, 1993. 23. Clark, E. A., and Ledbetter, J. A., How B and T cells talk to each other. Nature 367, 425-428, 1994.