To B, or not to B: that is the question Richard B. Gallagher and Dennis G. Osmond
Three major episodes of selection stand out as milestones in the generation and differentiation of B cells. These occur first during the primary B-cell genesis that occurs in the bone marrow of humans and mice (Dennis Osmond, Montreal), the bursa of Fabricius in birds (Olli Lassila, Turku) and the Peyer's patches (PP) in ruminants (Bruce Motyka, Calgary), second during the selection of the peripheral B-cell pool and third during the generation of B-cell memory within the germinal centres. A feature of these episodes is massive overproduction and culling of the population. The decisions of life and death that are taken by, or imposed upon, individual cells are based not only on the ability of the cells productively to rearrange their immunoglobulin genes but also on positive and negative selection, dependent on the specificity of the expressed Ig.
Primary genesis There is no exclusive B-cell domain in the bone marrow - in vivo immunolabelling of the B220 glycoprotein reveals B lineage cells essentially everywhere - but there is an identifiable migration from the edge of the marrow, where precursors are formed, towards the centre, where mature B cells congregate (Osmond) (Fig. 1). Each single progenitor at the stage of Ig gene rearrangement may give rise to as many as 64 progeny. This precociousness is closely associated with intimate cell contact between the precursor B cells and the long dendritic processes extended by the stromal reticular cells. Adhesion molecules and the production of short range growth factors, for example interleukin 7 (IL-7), by the stromal cells may also be important in B-cell regulation. It is not clear whether positive selection, negative selection or both are occurring during this stage of B lymphopoiesis. It was suggested (Klaus Rajewsky, Cologne) that the B-cell-stromal-cell interactions may
Irrespective of the prevailing fashion, Germinal Centre Conferences (GCC) have treated the immune system in a determinedly holistic manner. At the recent lOth GCC* the advantages of Selection and stability of the this approach were demonstrated in peripheral B-cell pool The second round of discrimielegant new schema for lymphocyte nation during B-cell development development, accessory cell function, occurs during the selection of the tolerance and autoimmunity, and for peripheral B-cell pool. The vast mathe organization and function of jority of the virgin B cells exported mucosal defence. This report concen- from the bone marrow have a very trates on only one aspect of the meet- short life span, most of these cells ing- the development ofan integrated being exported to the spleen. Negaview of the natural history of B cells. tive selection of autoreactive cells both before and after export from the bone marrow may play a major role in the deletion of this population. What of the cells that do become incorporated into the recirculating pool? Based on estimates of marrow production and experiments examining reconstitution following B-cell depletion, some previous calculations of the average turnover time of the periphera! B-cell pool were of the order of 4-5 days. More recent studies have either contradicted or supported this estimate. In rats 1 and mice (Irmgard Forster, Cologne) bromodeoxyuridine (BrdU) labelling techniques indicate that many B cells can survive for much longer periods. Forster demonstrated that while the majority of splenic B cells rapidly turn over during the first 4-6 weeks of life, once the peripheral lymphocyte pool has been built up in adult mice most of the peripheral B cells form a stable, long-lived population and the mean life span of splenic ~+8 + cells exceeds six weeks under nearphysiological conditions. Teleologically this makes sense, given that negative selection on the basis of anti-self reactivity appears to have already taken place. This suggestion is supported by the biased usage of certain VH gene families in peripheral ~+8+ B cells, compared with the broad distribution of VH genes expressed in the pre-B-cell pool (Forster). A recent publication 2 has indicated that one-third of the *The 10th International Conference splenic B cells in adult mice still conon Lymphatic Tissues and Germinal stitute a short-lived population, Centres in Immune Reactionswas held in which is renewed every 2-3 days as a result of the continuing influx of Compiegne, France on 1-5 July 1990.
mediate a form of positive selection, the rescue of a minority of cells from programmed cell death, namely those cells that successfully rearrange their Ig genes. Alternatively, the loss of cells may be a result of negative selection: ligation of newly expressed Ig molecules on the surface of the developing B cells may be the stimulus for this via a process analogous to deletion of self-reactive T cells in the thymus. These mechanisms of positive and negative selection are not mutually exclusive. In any event, of the pre-B cells generated in the marrow around 75% are culled (Osmond). Contact between B220 + cells and macrophages is a prelude to rapid phagocytosis of abnormal looking (and often frankly apoptotic) cells, a process accentuated in both severe combined immunodeficient (scid) and anti-IgM-treated mice in which all cells are deleted at early and late stages of B-cell development, respectively. In sheep PP, over 95% of the B cells produced are destroyed and show DNA fragmentation indicative of apoptotic programmed cell death (Motyka). Those cells in the bone marrow that escape deletion home to, and accumulate in, sinusoidal spaces near the central sinus, from which they are released en masse into the periphery in response to unknown signals (Fig. 1).
© 1991, Elsevier Science Publishers Ltd, UK. 0167--4919/91/$02.00
Immunology Today
1
Vol. 12, No. 1 1991
-___/
Fig. 1. Scheme of organization of B-cell genesis in the bone marrow, seen in a cross section of mouse femur (reproduced from Ref. 5 with permission). Precursor B cells are proposed to follow the numbered sequence while proliferating and differentiating from the stage of heavy chain gene rearrangement to the mature B cell. RC: reticular cell; ARC: adventitial reticular cell; Mac: macrophage; End: endosteum; Art: artery; Cap: capillary.
migrant plasma cells. Cells that fail to interact with the FDC die by apoptosis and are eliminated by macrophages. Somatic mutation and selection
newly-formed virgin B cells from the bone marrow.
Antigenic recruitment The third bout of B-cell selection occurs as a result of encounter with antigen, mainly in the lymph nodes and spleen. Again great diversity is generated, this time via somatic hypermutation and again selection, this time of antigen-specific Ig, takes place. Germinal centre formation
Earlier histological studies3 showed that plasmablasts develop within 24-48 h of T-cell independent and T-cell dependent antigenic challenge, at the periphery of secondary follicles or at the boundary of the follicles and adjoining T-cell areas, respectively. By 72h, however, proliferating blasts also appear centrally in the follicles, increasing rapidly in number accompanied by the appearance of macrophages containing tingible bodies (five days), and differentiating by seven days into typical germinal centres (GC) with a densely populated dark zone of rapidly dividing centroblasts and a thinly populated light zone of nondividing centrocytes (Fig. 2). After maintaining a kinetic steady state for 2-3 weeks, mitotic activity slowly subsides leaving quiescent secondary follicles. The clonality of germinal centres was studied by Mirjam Hermans (Groningen) and co-workers using rat RT7 (CD45) chimeras constructed by RT7.2 foetal liver cell
injection into non-irradiated newborn RT7.1 rats. In response to challenge with sheep red blood cells, entirely RT7.2, entirely non-RT7.2, and mixed GC could be detected, suggesting that germinal centres arise from oligoclonal proliferation. Ian MacLennan (Birmingham, UK) gave an overview of a proposed sequence of maturation of B cells in GC. He divided the sequence into four stages based on immunohistochemical studies of the follicular response to two hapten-protein conjugates in carrier-primed rats. First, follicles are colonized by fewer than five primary B-cell blasts which proliferate rapidly to fill the spaces in the follicular dendritic cell (FDC) network, to total about 104 cells within three days. Second, by around day four after antigenic challenge, the B-cell blasts metamorphose into centroblasts (which lack surface immunoglobulin and express CD77) and migrate to one pole of the FDC network, forming the dark zone; the centroblasts within it are a self-renewing but nonexpanding population that give rise to centrocytes. The third stage of maturation is the re-expression of surface Ig, migration into the light zone by the centrocytes and interaction with the FDC. This is the last judgement of B cells. Cells that interact with antigen expressed on the surface of the FDC are selected into the fourth stage of development. In this stage secondary follicular blasts are formed within the FDC network, giving rise to both memory cells and Immunology Today
2
Vol 12, No. 1 1991
Hypermutation of Ig V region genes is a feature of B-cell development, particularly during primary responses, which expands the antigen specificity repertoire and leads to the selection of memory B cells with enhanced antigen-binding affinity. Two promising approaches to the study of the process, and the controversial question of whether it occurs before or after isotype switching, were described. First, Michael Apel and colleagues (Cologne) produced hybridomas using PNA hi (i.e. GC) cells and PNA l° cells 10 days after primary immunization with phenyloxazolone. Many PNA cells had already undergone isotype switching; thus it occurs early during the primary response. Sequence analysis showed mutations, mainly silent, located apparently at random across light and heavy chain genes: hybridoma lines that had, through somatic mutation, lost the ability to bind antigen were detectable but characteristic key mutations known to enhance affinity for the antigen were absent. The GC cells had apparently begun to undergo somatic mutation but had yet to undergo positive affinity selection. Later mutations (14 days) became concentrated in certain VH gene regions. Second, the potential role of polymerase chain reaction (PCR) in studying the patterns and kinetics of B-cell hypermutation using RNA from PNA* spleen cells was highlighted by Tim Manser and colleagues (Princeton). Plasma cell or memory B cell?
The final question asked of antigen-selected B cells is whether
iiiiiii!iiiiiii!iiiitii!iiiiii!!!!ii!ii!iiiiiitiiiiiiiii!iiiiiii!tiiiii Memory B cells ~ they will terminally differentiate into antibody-secreting plasma cells or undergo phenotypic maturation into memory cells. This deliberation is still shrouded in mystery but an intriguing possibility is suggested by the work of Y-J. Liu (Birmingham, UK). Purified GC B cells can be rescued from apoptosis in vitro (1) by a combination of antigen plus antibody against CD40 and (2) by high levels of recombinant soluble CD23 plus IL-lc~. Under the former circumstances the rescued cells are morphologically small resting lymphocytes while the latter treatment promotes B-cell enlargement and the development of rough endoplasmic reticulum. Based on these findings Liu proposed the following sequence of differentiation: centrocytes are rescued from apoptosis by interaction with antigen immobilized on the surface of FDC. In the presence of CD23 (a molecule expressed by FDC) and IL-lo~, the cells are directed to become plasmablasts while rescue via ligation of CD40 results in memory cell generation. Fine tuning by the microenvironment Lymphoid follicles can be found in the lymphoid organs and in mucosaassociated lymphoid tissue along the digestive, respiratory and urinary tracts. At the latter sites the isotype produced is generally IgA, as opposed to IgM or lgG. What factors contribute to this site-specific variation? It has previously been suggested that chronic antigenic challenge, as occurs in the PP, is the stimulus for switching to IgA isotype. John Cebra and Peter Weinstein (Philadelphia) suggest otherwise. In germ-flee mice exposed to a single oral inoculum of reovirus, they found that mainly IgAproducing cells, but no IgG1 ÷ cells, develop in PP follicles and GC. Thus the regional GC microenvironment, rather than chronicity of antigenic stimulation, appears to be determinative in isotype switching. T cells may play a major role in microenvironmental control. That they are necessary for the development of GC is shown by the studies of H.G. Seijen and colleagues (Groningen). In experiments reconstituting lethally X-irradiated rats with thoracic duct lymphocytes depleted of different cell populations, the
Plasma cells
need for helper T cells in GC formaMantle ~ , F~ tion was clearly demonstrated. It rezone / ..----47---.' mains to be seen whether differences Apical / ~- Seco'rldary"'\ in PP and spleen T-cell populations, light----/--f-- B blasts \ for example in lymphokine secretion profiles, might play a role in differential antibody isotype expression. One way to examine this question l i g h t ~ .... I" ~-- // ] may be by in vitro reconstitution z°°e / of GC (Marie Kosco, Basel). The removal of lymph nodes from an D a r k e ~ / immune animal followed by enzymatic treatment, low density cell separation and enrichment for poorly adherent cells yields a mixed PrimaryB blasts population of B cells (85-90%), FDC (3-10%) and T cells (1-5%). Fig. 2. Schematic plan of a germinal centre. In culture, clusters of 20-50 B cells form in which cells of GC B-cell phenotype continue to proliferate Endpiece for several days, a process dependent After several years in the shadow on both FDC and T cells. Cluster of molecular biology a new era formation together with B-cell pro- of immunophysiology is dawning. liferation requires cell-cell contact Some of the major questions that and is antigen-specific. These fea- must be addressed in this new era tures may make this a useful ma- were articulated at the GCC. What nipulable model of the GC in vivo. are the selecting elements in B-cell development? How does stromal cell The maintenance of memory interaction regulate B-cell genesis? Once the primary response has What controls the release of B cells been established and memory B cells from the marrow? When does a B produced, what are the mechanisms lineage cell become committed to of memory retention? Multicolour either plasma cell or memory cell immunofluorescence has been used development? What controls this to study the turnover of antigen- decision? How is Ig gene hyperspecific memory B cells (Birgit mutation initiated and controlled? Shittek, Cologne). BrdU labelling of How does the microenvironment DNA-synthesizing cells after prim- influence the isotype of the B-cell ing mice with phycoerythrin in vivo response? Can phenotypic and revealed that soon after priming functional FDC subsets be identnearly all PE-binding memory cells ified? How is B-cell memory mainare newly formed from dividing tained ? A full understanding of what cells. Later, at 10-28 weeks, persist- makes B cells tick in vivo will require ing PE-binding cells remain un- a synthesis of the answers to these labelled by BrdU, even if given for fascinating questions. many (5-7) weeks, indicating that the PE-specific memory cells have Dennis Osmond is at the Dept of Anatbecome a long-lived population, omy, McGill University, 3640 Univerrather than being maintained by epi- sity Street, Montreal, PQ, Canada and sodes of cell activation and prolifer- Richard Gallagher is Editor of Immuation. This finding has implications nology Today. for, but does not solve, the longstanding question of the necessity for References antigen for B-cell memory. It has 1 Gray,D. (1988) J. Exp. Med. ]67, been reported that memory is lost if 805-816 antigen-specific B cells are trans- 2 Rocha, B., Penit, C., Baron, C. et al. ferred into unprimed hosts4. The (1990) Eur. J. Immunol. 20, well-demonstrated long-term reten- 1697-1708 3 Nieuwenhuis,P. (1981) Immunol. tion of antigen in the form of im- Today 2, 104-110 mune complexes on the surface of 4 Gray, D. and Skarvall, H. (1988) FDCs may be the key to the selection Nature 336, 70-73 of cells with high-affinity receptors 5 Jacobsen, K. and Osmond, D.G. Eur. to maintenance of B-cell memory. J. Immunol. (in press) Immunology Today
3
Vol. 12, No. 1 1991
Three major episodes of selection stand out as milestones in the generation and differentiation of B cells. These occur first during the primary B-cell genesis that occurs in the bone marrow of humans and mice (Dennis Osmond, Montreal), the bursa of Fabricius in birds (Olli Lassila, Turku) and the Peyer's patches (PP) in ruminants (Bruce Motyka, Calgary), second during the selection of the peripheral B-cell pool and third during the generation of B-cell memory within the germinal centres. A feature of these episodes is massive overproduction and culling of the population. The decisions of life and death that are taken by, or imposed upon, individual cells are based not only on the ability of the cells productively to rearrange their immunoglobulin genes but also on positive and negative selection, dependent on the specificity of the expressed Ig.
Primary genesis There is no exclusive B-cell domain in the bone marrow - in vivo immunolabelling of the B220 glycoprotein reveals B lineage cells essentially everywhere - but there is an identifiable migration from the edge of the marrow, where precursors are formed, towards the centre, where mature B cells congregate (Osmond) (Fig. 1). Each single progenitor at the stage of Ig gene rearrangement may give rise to as many as 64 progeny. This precociousness is closely associated with intimate cell contact between the precursor B cells and the long dendritic processes extended by the stromal reticular cells. Adhesion molecules and the production of short range growth factors, for example interleukin 7 (IL-7), by the stromal cells may also be important in B-cell regulation. It is not clear whether positive selection, negative selection or both are occurring during this stage of B lymphopoiesis. It was suggested (Klaus Rajewsky, Cologne) that the B-cell-stromal-cell interactions may
Irrespective of the prevailing fashion, Germinal Centre Conferences (GCC) have treated the immune system in a determinedly holistic manner. At the recent lOth GCC* the advantages of Selection and stability of the this approach were demonstrated in peripheral B-cell pool The second round of discrimielegant new schema for lymphocyte nation during B-cell development development, accessory cell function, occurs during the selection of the tolerance and autoimmunity, and for peripheral B-cell pool. The vast mathe organization and function of jority of the virgin B cells exported mucosal defence. This report concen- from the bone marrow have a very trates on only one aspect of the meet- short life span, most of these cells ing- the development ofan integrated being exported to the spleen. Negaview of the natural history of B cells. tive selection of autoreactive cells both before and after export from the bone marrow may play a major role in the deletion of this population. What of the cells that do become incorporated into the recirculating pool? Based on estimates of marrow production and experiments examining reconstitution following B-cell depletion, some previous calculations of the average turnover time of the periphera! B-cell pool were of the order of 4-5 days. More recent studies have either contradicted or supported this estimate. In rats 1 and mice (Irmgard Forster, Cologne) bromodeoxyuridine (BrdU) labelling techniques indicate that many B cells can survive for much longer periods. Forster demonstrated that while the majority of splenic B cells rapidly turn over during the first 4-6 weeks of life, once the peripheral lymphocyte pool has been built up in adult mice most of the peripheral B cells form a stable, long-lived population and the mean life span of splenic ~+8 + cells exceeds six weeks under nearphysiological conditions. Teleologically this makes sense, given that negative selection on the basis of anti-self reactivity appears to have already taken place. This suggestion is supported by the biased usage of certain VH gene families in peripheral ~+8+ B cells, compared with the broad distribution of VH genes expressed in the pre-B-cell pool (Forster). A recent publication 2 has indicated that one-third of the *The 10th International Conference splenic B cells in adult mice still conon Lymphatic Tissues and Germinal stitute a short-lived population, Centres in Immune Reactionswas held in which is renewed every 2-3 days as a result of the continuing influx of Compiegne, France on 1-5 July 1990.
mediate a form of positive selection, the rescue of a minority of cells from programmed cell death, namely those cells that successfully rearrange their Ig genes. Alternatively, the loss of cells may be a result of negative selection: ligation of newly expressed Ig molecules on the surface of the developing B cells may be the stimulus for this via a process analogous to deletion of self-reactive T cells in the thymus. These mechanisms of positive and negative selection are not mutually exclusive. In any event, of the pre-B cells generated in the marrow around 75% are culled (Osmond). Contact between B220 + cells and macrophages is a prelude to rapid phagocytosis of abnormal looking (and often frankly apoptotic) cells, a process accentuated in both severe combined immunodeficient (scid) and anti-IgM-treated mice in which all cells are deleted at early and late stages of B-cell development, respectively. In sheep PP, over 95% of the B cells produced are destroyed and show DNA fragmentation indicative of apoptotic programmed cell death (Motyka). Those cells in the bone marrow that escape deletion home to, and accumulate in, sinusoidal spaces near the central sinus, from which they are released en masse into the periphery in response to unknown signals (Fig. 1).
© 1991, Elsevier Science Publishers Ltd, UK. 0167--4919/91/$02.00
Immunology Today
1
Vol. 12, No. 1 1991
-___/
Fig. 1. Scheme of organization of B-cell genesis in the bone marrow, seen in a cross section of mouse femur (reproduced from Ref. 5 with permission). Precursor B cells are proposed to follow the numbered sequence while proliferating and differentiating from the stage of heavy chain gene rearrangement to the mature B cell. RC: reticular cell; ARC: adventitial reticular cell; Mac: macrophage; End: endosteum; Art: artery; Cap: capillary.
migrant plasma cells. Cells that fail to interact with the FDC die by apoptosis and are eliminated by macrophages. Somatic mutation and selection
newly-formed virgin B cells from the bone marrow.
Antigenic recruitment The third bout of B-cell selection occurs as a result of encounter with antigen, mainly in the lymph nodes and spleen. Again great diversity is generated, this time via somatic hypermutation and again selection, this time of antigen-specific Ig, takes place. Germinal centre formation
Earlier histological studies3 showed that plasmablasts develop within 24-48 h of T-cell independent and T-cell dependent antigenic challenge, at the periphery of secondary follicles or at the boundary of the follicles and adjoining T-cell areas, respectively. By 72h, however, proliferating blasts also appear centrally in the follicles, increasing rapidly in number accompanied by the appearance of macrophages containing tingible bodies (five days), and differentiating by seven days into typical germinal centres (GC) with a densely populated dark zone of rapidly dividing centroblasts and a thinly populated light zone of nondividing centrocytes (Fig. 2). After maintaining a kinetic steady state for 2-3 weeks, mitotic activity slowly subsides leaving quiescent secondary follicles. The clonality of germinal centres was studied by Mirjam Hermans (Groningen) and co-workers using rat RT7 (CD45) chimeras constructed by RT7.2 foetal liver cell
injection into non-irradiated newborn RT7.1 rats. In response to challenge with sheep red blood cells, entirely RT7.2, entirely non-RT7.2, and mixed GC could be detected, suggesting that germinal centres arise from oligoclonal proliferation. Ian MacLennan (Birmingham, UK) gave an overview of a proposed sequence of maturation of B cells in GC. He divided the sequence into four stages based on immunohistochemical studies of the follicular response to two hapten-protein conjugates in carrier-primed rats. First, follicles are colonized by fewer than five primary B-cell blasts which proliferate rapidly to fill the spaces in the follicular dendritic cell (FDC) network, to total about 104 cells within three days. Second, by around day four after antigenic challenge, the B-cell blasts metamorphose into centroblasts (which lack surface immunoglobulin and express CD77) and migrate to one pole of the FDC network, forming the dark zone; the centroblasts within it are a self-renewing but nonexpanding population that give rise to centrocytes. The third stage of maturation is the re-expression of surface Ig, migration into the light zone by the centrocytes and interaction with the FDC. This is the last judgement of B cells. Cells that interact with antigen expressed on the surface of the FDC are selected into the fourth stage of development. In this stage secondary follicular blasts are formed within the FDC network, giving rise to both memory cells and Immunology Today
2
Vol 12, No. 1 1991
Hypermutation of Ig V region genes is a feature of B-cell development, particularly during primary responses, which expands the antigen specificity repertoire and leads to the selection of memory B cells with enhanced antigen-binding affinity. Two promising approaches to the study of the process, and the controversial question of whether it occurs before or after isotype switching, were described. First, Michael Apel and colleagues (Cologne) produced hybridomas using PNA hi (i.e. GC) cells and PNA l° cells 10 days after primary immunization with phenyloxazolone. Many PNA cells had already undergone isotype switching; thus it occurs early during the primary response. Sequence analysis showed mutations, mainly silent, located apparently at random across light and heavy chain genes: hybridoma lines that had, through somatic mutation, lost the ability to bind antigen were detectable but characteristic key mutations known to enhance affinity for the antigen were absent. The GC cells had apparently begun to undergo somatic mutation but had yet to undergo positive affinity selection. Later mutations (14 days) became concentrated in certain VH gene regions. Second, the potential role of polymerase chain reaction (PCR) in studying the patterns and kinetics of B-cell hypermutation using RNA from PNA* spleen cells was highlighted by Tim Manser and colleagues (Princeton). Plasma cell or memory B cell?
The final question asked of antigen-selected B cells is whether
iiiiiii!iiiiiii!iiiitii!iiiiii!!!!ii!ii!iiiiiitiiiiiiiii!iiiiiii!tiiiii Memory B cells ~ they will terminally differentiate into antibody-secreting plasma cells or undergo phenotypic maturation into memory cells. This deliberation is still shrouded in mystery but an intriguing possibility is suggested by the work of Y-J. Liu (Birmingham, UK). Purified GC B cells can be rescued from apoptosis in vitro (1) by a combination of antigen plus antibody against CD40 and (2) by high levels of recombinant soluble CD23 plus IL-lc~. Under the former circumstances the rescued cells are morphologically small resting lymphocytes while the latter treatment promotes B-cell enlargement and the development of rough endoplasmic reticulum. Based on these findings Liu proposed the following sequence of differentiation: centrocytes are rescued from apoptosis by interaction with antigen immobilized on the surface of FDC. In the presence of CD23 (a molecule expressed by FDC) and IL-lo~, the cells are directed to become plasmablasts while rescue via ligation of CD40 results in memory cell generation. Fine tuning by the microenvironment Lymphoid follicles can be found in the lymphoid organs and in mucosaassociated lymphoid tissue along the digestive, respiratory and urinary tracts. At the latter sites the isotype produced is generally IgA, as opposed to IgM or lgG. What factors contribute to this site-specific variation? It has previously been suggested that chronic antigenic challenge, as occurs in the PP, is the stimulus for switching to IgA isotype. John Cebra and Peter Weinstein (Philadelphia) suggest otherwise. In germ-flee mice exposed to a single oral inoculum of reovirus, they found that mainly IgAproducing cells, but no IgG1 ÷ cells, develop in PP follicles and GC. Thus the regional GC microenvironment, rather than chronicity of antigenic stimulation, appears to be determinative in isotype switching. T cells may play a major role in microenvironmental control. That they are necessary for the development of GC is shown by the studies of H.G. Seijen and colleagues (Groningen). In experiments reconstituting lethally X-irradiated rats with thoracic duct lymphocytes depleted of different cell populations, the
Plasma cells
need for helper T cells in GC formaMantle ~ , F~ tion was clearly demonstrated. It rezone / ..----47---.' mains to be seen whether differences Apical / ~- Seco'rldary"'\ in PP and spleen T-cell populations, light----/--f-- B blasts \ for example in lymphokine secretion profiles, might play a role in differential antibody isotype expression. One way to examine this question l i g h t ~ .... I" ~-- // ] may be by in vitro reconstitution z°°e / of GC (Marie Kosco, Basel). The removal of lymph nodes from an D a r k e ~ / immune animal followed by enzymatic treatment, low density cell separation and enrichment for poorly adherent cells yields a mixed PrimaryB blasts population of B cells (85-90%), FDC (3-10%) and T cells (1-5%). Fig. 2. Schematic plan of a germinal centre. In culture, clusters of 20-50 B cells form in which cells of GC B-cell phenotype continue to proliferate Endpiece for several days, a process dependent After several years in the shadow on both FDC and T cells. Cluster of molecular biology a new era formation together with B-cell pro- of immunophysiology is dawning. liferation requires cell-cell contact Some of the major questions that and is antigen-specific. These fea- must be addressed in this new era tures may make this a useful ma- were articulated at the GCC. What nipulable model of the GC in vivo. are the selecting elements in B-cell development? How does stromal cell The maintenance of memory interaction regulate B-cell genesis? Once the primary response has What controls the release of B cells been established and memory B cells from the marrow? When does a B produced, what are the mechanisms lineage cell become committed to of memory retention? Multicolour either plasma cell or memory cell immunofluorescence has been used development? What controls this to study the turnover of antigen- decision? How is Ig gene hyperspecific memory B cells (Birgit mutation initiated and controlled? Shittek, Cologne). BrdU labelling of How does the microenvironment DNA-synthesizing cells after prim- influence the isotype of the B-cell ing mice with phycoerythrin in vivo response? Can phenotypic and revealed that soon after priming functional FDC subsets be identnearly all PE-binding memory cells ified? How is B-cell memory mainare newly formed from dividing tained ? A full understanding of what cells. Later, at 10-28 weeks, persist- makes B cells tick in vivo will require ing PE-binding cells remain un- a synthesis of the answers to these labelled by BrdU, even if given for fascinating questions. many (5-7) weeks, indicating that the PE-specific memory cells have Dennis Osmond is at the Dept of Anatbecome a long-lived population, omy, McGill University, 3640 Univerrather than being maintained by epi- sity Street, Montreal, PQ, Canada and sodes of cell activation and prolifer- Richard Gallagher is Editor of Immuation. This finding has implications nology Today. for, but does not solve, the longstanding question of the necessity for References antigen for B-cell memory. It has 1 Gray,D. (1988) J. Exp. Med. ]67, been reported that memory is lost if 805-816 antigen-specific B cells are trans- 2 Rocha, B., Penit, C., Baron, C. et al. ferred into unprimed hosts4. The (1990) Eur. J. Immunol. 20, well-demonstrated long-term reten- 1697-1708 3 Nieuwenhuis,P. (1981) Immunol. tion of antigen in the form of im- Today 2, 104-110 mune complexes on the surface of 4 Gray, D. and Skarvall, H. (1988) FDCs may be the key to the selection Nature 336, 70-73 of cells with high-affinity receptors 5 Jacobsen, K. and Osmond, D.G. Eur. to maintenance of B-cell memory. J. Immunol. (in press) Immunology Today
3
Vol. 12, No. 1 1991
