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Germinal centres in T-cell-dependent antibody responses
REVIEW
Germinal centres in T-cell-dependent antibody responses Yong-Jun Liu, Gerald D. Johnson, John Gordon and Ian C.M. MacLennan For more than a century follicles have been recognized as a site of intense cell proliferation and cell death. At last the significance of this activity is beginning to emerge: antigen-driven B-cell proliferation, somatic mutation, positive and negative selection, and memory and plasma cell development all appear to take place within the follicle. B-cell follicles are found in secondary lymphoid organs throughout the body, including the spleen, lymph nodes, Peyer's patches, the appendix, tonsils and other mucosaassociated lymphoid tissues. Follicles in which no antigen-driven processes are taking place consist of a network of follicular dendritic cells (FDCs), the spaces of which are filled mainly with recirculating surface immunoglobulin M + (slgM +) slgD + small B cells t,-'. These primary follicles, unlike secondary follicles that contain activated B cells, are already present in lymph nodes early in the second trimester of human foetal lifeL Primary follicles are characteristic of germ-free rodents 4 and are frequently found in normal spleen, reflecting relative protection of this lymphoid organ from exogenous antigen. This allows the spleen to be used to study the sequence of follicular responses to injected antigens. The formation of germinal centres during the first three weeks of a T-cell-dependent response can be clearly distinguished, using this system, from the relatively less dramatic level of B-cell activation that is seen during thc subsequent months of the response 5. Secondary follicles, found in tissues continuously exposed to antigen, such as the tonsils, are characterized by the presence of germinal centres. The relative, but not absolute, lack of secondary follicles in congenitally athymic rodents ~ reflects the T-cell dependence of most follicular responses. It is central to the function of follicles that antigen is taken up by FDCs in the form of immune complexes and can be held in a nondegraded form for many months-. The mechanism of antigen uptake and storage in a nonprocessed form has been investigated in detail s but will not be considered further here. It has been proposed that this stored antigen is important in maintaining T-celldependent responses: successful transfer of established (late secondary) antibody responses to syngeneic, naive animals depends on the recipient being given both cells and antigen ~n°. A further dimension to the significance of this antigen storage system is the recent demonstration that follicular B cells can take up antigen from FDCs and process this into a form that will activate CD4 + T cells LI.
Germinal centre development The sequence of follicular responses has been studied by many groups (reviewed in Refs 8,12-14). The location of antigen-specific B cells during follicular re-
sponses can bc studicd inlmunohistologicallv using techniques that identify antigen-specific B cells and plasma cells. The use ot two haptens, one conjugated to horseradish peroxidasc and the other to alkaline phosphatasel,.i,, allows two anti-hapten responscs to bc studled in the same tissue section. The initial stages of germinal centre formation are associatcd with the exponential growth of B-cell blasts within follicles. Kroesc et al.I- studied germinal centre reactions in radiation chimaeras and noted that thc rcsponscs in individual follicles were oligoclonal. This has been confirmed in more physiological svstems, using techniques to identify antigen-specific B cells ~s,~'. When two simultaneous primary anti-haptcn responses are studied using thc immunohistological techniques outlined above, the oligochmality of the germinal centre reaction is clearly seen I'. This has been performed in rats primed with spider crab haemocvanm (,k'ISH) and rcdmmunized with a mixture of 2,4-dinitrophenyI-MSH ',I)NI~-NISHI and phenyloxazalone-NISH (Ox-N1SH). In such cxpurimcnts 6-31",'o of the follicles on day three after immunization were found to contain blastsof one specificity {I)NP or Ox) while the remainder of the follicles wcrc of mixed specificity. Statistically, 12.5%, of follicles would be cxpected to bc monospecific if each follicle is colonized t~x three hapten-spccific blasts. As thc number of blasts in follicles three days after immunization is of the order ot 1-1.5 × 10 4, it tollows that the cell cycle time of these primary B blasts is about 6 h. This agrees closely with the cell cycle time for mouse germinal centrc blasts csnmated by stathmokinetic studies-'~L During the cxponcntial growth of 13blast cells seen during germinal centrc formation, the small recirculating B cells arc excluded from the follicle centre and form the familiar follicular mantle that surrounds the germinal centre, q-here is no clear explanation for the number of blasts that give risu to germinal centres being so restricted. Given the number of haptenspecific blasts seen in T-cell zones m the first few days of primary antibodv responses to haptcn protein and iheir polychmality, it appears not to bc duc to lack ot precursor cells *s. Experiments comparing thc rcsponsiveness of transferred memory and virgin B cells to antigen alrcad~ localized on the FDCs of recipient rats indicate that nlelnor~' cells respond rapidly and well while virgin cells
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;
N,,. ;
REVIEW (a)
(b)
FDC network
Zone
Follicle
Fine processes
Follicular mantle
Loose network
Outer zone
Dense network
Apical light zone
Dense network
Basal light zone
Fine processes
Dark zone
Fig. 1. (a) Photomicrograph of a secondary follicle with a well-developed germinal centre, from a normal human tonsil. Tbe zonal pattern of the tonsil is apparent. Green immunofluorescence identifies weak CD23 expression by B cells in the follicular mantle and strong CI)23 expression by follicular dendritic cells in the apical light zone. Red immunofluorescence shows the heavy concentration of cells in cell cycle in the clark zone identified by the Ki6 7 monoclonal antibody. The unstained area between the follicular mantle and apical light zone is the outer zone and that between the apical ligh} zone and the dark zone is the basal light zone. Both the outer zone and basal light zone contain occasional Ki67 + cells. (b) These regions are shown with a colour key.
respond slowly, if at all, even when T-cell help is provided I-3,21,22. The failure of the virgin B cells in these transfer experiments to respond to antigen on FDCs is not due to lack of antigen-specific B cells among the donor cells, since transferred virgin cells do give a full and rapid response if the recipient is immunized with soluble antigen after transfer, and if T-cell help is available 1-~,21,22. Excellent germinal centres develop during primary antibody responses, particularly when T-cell help is not limiting. As virgin B cells do not seem to respond to antigen prelocalized on FDCs it seems that they are recruited to form germinal centres by activation by antigen outside follicles 1-~,21-23. This may involve interaction among helper T cells, B cells and interdigitating cells in the T-cell zones, where antigen-specific B cells are known to proliferate during the first few days of primary T-cell-dependent antibody responses 5,1~.l~. Further investigation, comparing the proliferation of
hnmunology Today
memory cells in follicles with the germinal centre reaction seen when virgin B cells are induced to colonize follicles, is required. Centroblasts and centrocytes The first stage of germinal centre formation ends when the B-cell blasts, which are in exponential growth, have filled the FDC network. Over the next few hours blasts are lost from the FDC network and the classical polarization of the germinal centre develops (Fig. 1). The primary B-cell blasts move to one edge of the FDC network, known as the dark zone, where the dendritic processes are widely spaced. The blasts, now termed centroblasts, have an altered phenotype, most strikingly in having lost their slg (Table 1). Centroblasts, like their progenitors the B-cell blasts, are in rapid cell cycle, but, unlike the B-cell blasts, they do not increase in number. Rather, they continually give rise to progeny, centrocytes that are
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Vol. 13 No. 1 1992
REVIEW Table 1. Phenotype of FDCs and B cells in follicles with germinal centres Zone
Outer zone
FD(2 (11)54
slg
B cells (11")39 CD38 t)cl-2
Ki67 :'
CD75
++
++
--
++
--
+
+
++
_
++
_b
+
+++
+
+++
++
_
++
b
±
+
+++
+++
++
--
++
--
+
+
++
--
+++
+
CD21
CD23
+
-
)
++
±
+
+++
+++
+++
±
+
.
.
.
BUIO
.
.
•'Ki67 is a monoclonal antibody that stains cells in active cell cycle, bOccasional B cells it1 the light zone arc t~cl-2 ~. slg: surface immunogh~hulin. nondividing and express surface immunoglobulin. These centrocytes enter the dense FDC network of the follicle centre, known as the light zone of the germinal centre. Centroblasts and centrocytes have not been shown formally to originate from primary follicular B blasts, but this seems likely, for two reasons: first, the follicles remain oligoclonal as assessed by the double hapten system described above, and second, there is no other obvious source of antigen-specific precursor cells. DNA-labelling studies ~',e4 indicate that centroc)tes are a labile population of cells that is continually replenished from the proliferating pool of centroblasts. Local macrophages contain nuclear debris (tingible bodies) derived from cells that have recently been in cell cyclez~, indicating that there is a high death rate among germinal centre cells. The location of these macrophages suggests that both centroblasts and centrocytes are dying but that cell death is most marked among the cells in that part of the light zone adjacent to the dark zone.
Hypermutation and selection The recruitment of virgin B cells into T-cell-dependent antibody responses is confined to the first few days after exposure to antigen; the response is subsequently maintained by memory B-cell clonesa% Somatic mutation in Ig V region genes, in responses to Ox protein, in B cells in mouse spleen does not start until after the onset of antibody production z-,-'s. Together, these two sets of observations suggest that the lg V-region-directed mutagenic process is induced in cells that have already been activated by antigen. Furthermore, the peak period of accumulation of mutations in Ig V region genes coincides with the time when the germinal centre reaction is taking place-'7-:~L Based on these data, it was proposed that the mutational process occurs in centroblasts and that centrocytes are selected on the basis of their capacity for activation by antigen held on FDCs (Ref. 30 and Fig. 2). Direct evidence that somatic hypermutation is activated in germinal centres is still fragmentary, particularly in relation to the exact stage or stages when the process is activated. Recent evidence suggests that it may be switched on during the phase of exponential growth of B blasts in follicles and before the appearance of centroblasts and centrocytes ~. An indication of the way in which selection after lg V region mutation takes place has been provided by studies of germinal centre cells isolated from the human tonsil 32.
I~1ll lllnolog 3' l'~dax'
This isolation was based on the finding that most germinal centre B cells fail to penetrate 60% Percoll and lack surface expression of IgD and CD39 (Ref. 33). Provided they are isolated at 4°C these cells remain viable ~2,~4but, on culture at 37°C, a high proportion of the cells dies by apoptosis ~ within a few hours ~2,~4. Few non-germinalcentre tonsillar B cells undergo apoptosis on culture. The tendency of germinal centre cells to enter apoptosis can be delayed by one or two days if their antigen-binding receptors are cross-linked m t,itro using anti-immunoglobulin immobilized on a solid surface~-'. These findings suggest that germinal centre B cells with low affinity for antigen held on FDCs are lost by apoptosis while highaffinity cells survive. Study of genetic defects associated with tumours of germinal centre cells has provided some insight into the molecular control of germinal centre cell survival during this selection process. A translocation between a site on chromosome 18 adjacent to the t?cl-2 gene and the lg H genes on chromosome 14 is frequently found in centroblastic/centrocvtic lymphomas ~'. Bcl-2 encodes a 26 kDa protein that is located mainly in the mitochondria ~7,~sand cvtosol ~s. While this protein is not expressed in appreciable amounts in germinal centre cells, it is found in most, if not all, small lymphocytes and extrafollicular B-cell blasts. The anti-Ig signal tllat delays thc onset of apoptosis in germinal centre cells induces the expression of the bcl-2-encoded protein within four hours <~. Expression of bcl-2 has been implicated in ceil survival: first, the transfection of productively-expressed bcl-2 into interleukin 3 (IL-3)-dependent haemopoietic cell lines confers protection from apoptosis when IL-3 is withdrawn ~-41 and second, Burkitt's lymphoma cell lines, which have many of the features of centroblasts, enter apoptosis if cultured at low serum concentration but become serum-independent after transfection of bcl-2 (Ref. 42). As the neoplastic germinal centre cells with the t14,18 translocation express the bcl-2 gene constitutively, it may be that these cells survive, without the need for interaction with antigen on F1)(is.
Differentiation signals Three types of physiological signal can extend the survival of germinal centre cells and result in their differentiation. First, activation with the anti-CD40 antibody, G28-5 (Ref. 43), induces cells to leave cell cycle, many acquiring the phenotype of small lymphocytes ~4. This i's
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LSt
er e
Fig. 2. Hypothetical pathways of selection and differentiation in the germinal centres, slg centroblasts proliferating in the dark zone undergo somatic mutation in their immunoglobulin V region genes. These give rise to centrocytes which re-express slg and enter the basal light zone. If centrocytes interact with antigen held on the FDCs in the basal light zone, they progress to the apical light zone. Centrocvtes failing to interact with antigen die by apoptosis. Centrocytes entering the apical light zone receive differentiation signals indicated to become memory B cells or plasmablasts (or possibh, centroblasts).
consistent with the production of memory B cells, which are known to be produced by germinal centres 44,45. The CD40-derived signal also induces centrocytes to express bcl-2 protein ,;~. Cells rescued from apoptosis in this way can be maintained in cell cycle by the addition of recombinant IL-4 (rlL-4) 46. These cycling ceils, unlike centroblasts, express surface immunoglobulin. They might be representative of the secondary follicular B-cell blasts that are characteristic of the established phase of T-celldependent antibody responses. Second, a combination of the recombinant (25kDa) fragment of the CD23 molecule plus rlL-lu also prevents germinal centre cells from entering apoptosis. This combination of signals induces some of the features of plasmablasts, including the expression of cytoplasmic Ig and bcl-2 and the development of the endoplasmic reticulum ~4. Third, it recently became clear that a proportion of germinal centre cells responds to riL-2 to remain in cell cycle46. Preliminary phenotypic analysis suggests that the blasts resulting from this stimulus also resemble plasmablasts but differ from those induced with CD23 plus IL-lc~ in that they express little extranuclear protein encoded by the bcl-2 gene. Further work is required to determine the significance of these three groups of differentiation signals. Germinal centre microenvironments
Detailed immunohistological analysis indicates that the light zone of germinal centres in human tonsil is
Immunology Today
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subdivided into two distinct areas (Refs 47,48 and Figs 1 and 3): the apical zone, where the FDCs express high levels of CD23, and the basal zone, where the FDCs are essentially CD23 but strongly express CD54 (intercellular cell adhesion molecule (1CAM-I)). In both zones there is a dense FDC network that holds antigen in the form of immune complexes. Both the FDCs and the centrocytes in the basal light zone have relatively large amounts of mRNA in their cytoplasm, which is not associated with endoplasmic reticulum. The centrocytes in this zone express CDlla/CD18 (lymphocyte function-associated molecule 1 (LFA-1)), the natural ligand for CD54, on their surface. There are many cells that show features of apoptosis in this basal light zone. All these features are consistent with the suggestion that the basal light zone is the area where centrocytes are selected, on the basis of their capacity to be activated by antigen held on FDCs. The role of the many helper T cells in the light zone 4~ in these selection and differentiation events remains to be determined. In the apical light zone, the presence of CD23 on FDCs suggests that cells that have successfully survived transit through the basal light zone receive the paracrine signals delivered through soluble CD23 plus IL-h~. It may be that they also interact with the natural ligand for CD40 in this area but so far the nature and location of this ligand have remained elusive. The finding that anti-Ig, CD23 plus IL-lu and CD40 antibody alone or in combination, induce the expression of bcl-2 implies that cells receiving these signals in vivo soon leave the germinal centre: only a small proportion of centrocytes express bcl-2 and that expression is weak. Figure 2 depicts the possible processes that occur in germinal centres, based on these findings and interpretations. A further narrow zone is found between the CD23-rich apical light zone and the follicular mantle. This zone extends to surround the dark zone. The cells in this area express CDw75 particularly strongly. The significance of this outer zone and its cellular content remains obscure, but we suggest that it might provide a path by which a proportion of centrocytes return to the dark zone to go through a further cycle of proliferation and V region gene mutation 4s. Late follicular responses
In secondary lymphoid tissues, which have low background germinal centre activity, germinal centre reactions last about three weeks after antigen administration. When centroblasts and centrocytes are no longer present in the follicle, small numbers of B-cell blasts can still be found in the FDC network 5,19. As recirculating memory B ceils can respond to antigen held on FDCs 13,21-23,it seems likely that there is an equilibrium between these late follicular B-cell blasts and memory B cells. It is also possible that these follicular B-cell blasts produce plasmablasts that migrate to the bone marrow 5° or lamina propria of the gut 51, depending on the site of the follicle. The estimated life span of these plasma cells, about four weekss2, is too short to maintain secondary antibody responses which may continue for more than a year. Antigen has been shown to be held on FDCs for extended periods v.8 and presumably is involved in maintaining these late follicular B-cell blasts in cell cycle. ,
Vol 13 No. I 1992
REVIEW
Coherent concepts about the nature and functions of germinal centres are now being developed SL While many of these deserve rigorous evaluation, it seems likely that follicles are the principal sites where T-cell-dependent antibody responses are maintained and that germinal centres are intimately involved in the affinity maturation of these responses.
Yong-]un Liu, Gerald D..Johnson, John Gordon and Ian C.M. MacLennan are at the Dept of Immunology, University of Birmingham Medical School, Birmingham B 15 2 TT, UK. References 1 Nieuwenhuis, P. and Ford, W i . (1976) Cell. lmmunol. 23, 254-267 2 Gray, D., Mackennan, I.C.M., Bazin H. and Khan, M. (1982) Eur. 1. lmmunol. 12, 564-569 3 Namikawa, R., Mizuno, T., Matsuoka, H. et al. 11986) Immunology 57, 61-69 4 Thorbecke, G.J. 11959) Ann. NYAcad. Sci. 78, 237-246 5 MacLennan, I.C.M., Liu, Y-J. and Ling, N.R. 11988) Curr. Top. Microbiol. Immunol. 141, 138-148 6 Jacobson, E.B., Caporale, l.H. and Thorbecke, G.J. (19"4) Cell. hnmunol. 13, 416-431) 7 Tew, J.G. and Mandel, T.E. (1979) hnmunology 37, 69-76 8 Szakal, A.K., Kosco, M.H. and Tew, J.G. 11989) Annu. Rev. hnmunol. 7, 91-109 9 Gray, D. and Skarvall, H. (1988) Nature 336, 70-73 10 Askonas, B.A. and Williamson, A.R. (1972) Eur. J. lmmtmol. 2, 487-493 11 Gray, D., Kosco, M. and Stockinger, B. (1991) Int. hnmunol. 3, 141-148 12 Nieuwenhuis, P. and Opstelten, D. (1984) Ant..l. Anat. 170, 421-435 13 MacLennan, I.C.M., Liu, Y-J., Oldfield, S. et al. (1990) Curt. Top. Microbiol. hnmunol. 159, 37-63 14 Kroese, F.G.M., Timens, W. and Nieuwenhuis, P. (1990) Curr. "D~p. Pathol. 84, 103-148 15 Van Rooijen, N., Classen, E. and Eikelenboom, P. (1986) Immunol. Today 7, 193-196 16 Liu, Y-J., Oldfield, S. and MacLennan, I.C.M. (1988) Eur. J. bnntunol. 18,355-362 17 Kroese, F.G.M., Wubenna, A.S., Seijen, H.G. and Nieuwenhuis, P. (1987) Eur. J. Immunol. 17, 1069-1072 18 Jacob, J., Kassir, R. and Kelsoe, G. 11991)J. Exp. Med. 173, 1165-1176 19 Liu, Y-J., Zhang, J., Chan, E. et al. Eur. J. lmmunol. (in press 20 Zhang, J., MacLennan, I.C.M., Liu, Y-J. and Lane, P.J.L. (1988) hnmunol. Lett. 18,297-299 21 Gray, D. (1988) Immunology 65, 73-80 22 Vonderhei& R.H. and Hunt, S.V. (1990) Eur. J. Immunol. 20, 79-86 23 Kroese, F.G.M., Wubbena, A.S. and Nieuwenhuis, P. (1986) hnmunology 57, 99-104 24 Fliedner, T.M., Kress, M., Cronkite, E.P. and Robertson, J.S. 11964) Ann. N Y Acad. Sci. 113,578-594 25 Fliedner, T.M. 11967) in Germinal Centres in bnmune Responses (Cottier, H. et al., eds), pp. 218-224, SpringerVerlag 26 Gray, D., MacLennan, I.C.M. and Lane, P.J.L. (1986) Eur. J. hnmunol. 16, 641-648 27 Griffiths, G.M., Berek, C., Kaartinen, M. and Milstein, C. (1984) Nature 312, 271-275 28 Berek, C., Griffiths, G.M. and Milstein, C. (1985) Nature 316, 412--418
Immunology Tod,n'
29 XXeiss, 11 and Raiewsky, K. (1990)]. t(xp. Med. 172, 1681-1689 30 Macl.cnnan, I.C.M. and Gray, D. 11986) hnmunol. Rev. 91, 61-85 31 Gray, D. (1991) Res. Immunol. 142,237-242 32 Liu, Y-J., Joshua, D.E., Williams, G.T. et al. 11989) Nature 342, 929-93 I 33 Ling, N.R., MacLennan, I.C.M. and Mason, I).Y. 11987) m Leukocyte Typing Ill (McMichael, A. et al., eds), pp. 302-335, ()xford University Press 34 Liu, Y-J, Cairns, J.A., Abbot, S.l). et al. ( 1991) k[ur. I. hnmunol. 21, 1107-1114 35 Wyllie, A.H., Morris, R.G., Smith, A.L. and l)unh)p, 1). (1984i ]. Pathol. 142, 67-77 36 Tsujimoto, Y., Cossman, J., Jaffe, E. and Croce, C. (1985) Science 228, 1440-1443 37 Hockenbery, D., Nunez, G., Millm3an, C. et al. 11990) Nature 348, 334- ~36 38 kin, Y-J., Mason, D.Y, Johnson, G.I). et al. 199 I) Eur. J. lnzmtmol. 21, 1')05-1910 39 Vaux, D.L., C.~ry, S. and Adams, J.M. (1988) Nature 335,440-442 40 Nuncz, G., London, L., Hockenbcry, D. el al. (1990) J. lmmunol. 144, 3602-3610 41 Williams, G.T., Smith, C.A., Spooncer, E. etal. 11990) Nature 343, -76-7~} 42 Henderson, S., Rowe, M., Gregory, C. et ,I1. (;ell (in press) 43 ('lark, E.A. and Ledbetter, ].A. (1986) l>roc. Natl Acad. Sci. USA 8~, 4495-4498 44 Coico, R.F., Bhogal, B.S. and Tlmrbccke, G.J. (198,:;) J. lntmunol. 131, 2254-2257 45 Klaus, G.G.B., Humphrey, J.H., Kunkle, A. and Dongworth, I).W. (1980) hnmunol. Ret,. 53, 3-28 46 Holder, M., Liu, Y-J., de Prance, T. et ~d. Int. hnmunol. (in press) 47 Johnson, G.1)., Macl,emlan, I.C.M., Khan, M. et al. 11989'. m Leukocyte l),ping IV (Knapp, W. et ,tl., eds), pp. 183-184, Oxfords University Press 48 MacLennan, I.C.M., Johnson, G.I)., Liu, Y-J. and Gordon, J. ( 1991 ) Res. hnmunol. 142, 253-257 49 Stein, H., Gerdes, J. and Mason, I).Y. 1982 Clm. Haematol. 11, 531-559 50 Benner, R., Hiimans, W. and Haaiimau, J.J. 198 I) (;list. Exp. lmmunol. 46, 1-8 51 Gowans, J i . and Knight, E.J. (1964) l'roc. R. ),'oc. London Ser. B. 159,257-282 52 Ho, F.C.S., Lortan, J.L., Khan, M. and Macl~ennan, I.C.M. (1986) Eur..]. lmmunol. 12, 1297-1301 53 Kosco, M. (19~)1) Res. lmmunol. 142, 219-224
Erratum The cover illustration of the N o v e m b e r issue of IT contains a t a x o n o m i c error. There are no such things as ' N e w World Apes' and 'Old World Apes'. In Primata there is a suborder of new world monkeys (Simiae Platyrrhinae) and a suborder of old world monkeys (Simiae Catarrhinae). Many thanks to Professor Edward A. Clark for pointing out and correcting our mistake.
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v , , i ~ No. i 1992
Germinal centres in T-cell-dependent antibody responses Yong-Jun Liu, Gerald D. Johnson, John Gordon and Ian C.M. MacLennan For more than a century follicles have been recognized as a site of intense cell proliferation and cell death. At last the significance of this activity is beginning to emerge: antigen-driven B-cell proliferation, somatic mutation, positive and negative selection, and memory and plasma cell development all appear to take place within the follicle. B-cell follicles are found in secondary lymphoid organs throughout the body, including the spleen, lymph nodes, Peyer's patches, the appendix, tonsils and other mucosaassociated lymphoid tissues. Follicles in which no antigen-driven processes are taking place consist of a network of follicular dendritic cells (FDCs), the spaces of which are filled mainly with recirculating surface immunoglobulin M + (slgM +) slgD + small B cells t,-'. These primary follicles, unlike secondary follicles that contain activated B cells, are already present in lymph nodes early in the second trimester of human foetal lifeL Primary follicles are characteristic of germ-free rodents 4 and are frequently found in normal spleen, reflecting relative protection of this lymphoid organ from exogenous antigen. This allows the spleen to be used to study the sequence of follicular responses to injected antigens. The formation of germinal centres during the first three weeks of a T-cell-dependent response can be clearly distinguished, using this system, from the relatively less dramatic level of B-cell activation that is seen during thc subsequent months of the response 5. Secondary follicles, found in tissues continuously exposed to antigen, such as the tonsils, are characterized by the presence of germinal centres. The relative, but not absolute, lack of secondary follicles in congenitally athymic rodents ~ reflects the T-cell dependence of most follicular responses. It is central to the function of follicles that antigen is taken up by FDCs in the form of immune complexes and can be held in a nondegraded form for many months-. The mechanism of antigen uptake and storage in a nonprocessed form has been investigated in detail s but will not be considered further here. It has been proposed that this stored antigen is important in maintaining T-celldependent responses: successful transfer of established (late secondary) antibody responses to syngeneic, naive animals depends on the recipient being given both cells and antigen ~n°. A further dimension to the significance of this antigen storage system is the recent demonstration that follicular B cells can take up antigen from FDCs and process this into a form that will activate CD4 + T cells LI.
Germinal centre development The sequence of follicular responses has been studied by many groups (reviewed in Refs 8,12-14). The location of antigen-specific B cells during follicular re-
sponses can bc studicd inlmunohistologicallv using techniques that identify antigen-specific B cells and plasma cells. The use ot two haptens, one conjugated to horseradish peroxidasc and the other to alkaline phosphatasel,.i,, allows two anti-hapten responscs to bc studled in the same tissue section. The initial stages of germinal centre formation are associatcd with the exponential growth of B-cell blasts within follicles. Kroesc et al.I- studied germinal centre reactions in radiation chimaeras and noted that thc rcsponscs in individual follicles were oligoclonal. This has been confirmed in more physiological svstems, using techniques to identify antigen-specific B cells ~s,~'. When two simultaneous primary anti-haptcn responses are studied using thc immunohistological techniques outlined above, the oligochmality of the germinal centre reaction is clearly seen I'. This has been performed in rats primed with spider crab haemocvanm (,k'ISH) and rcdmmunized with a mixture of 2,4-dinitrophenyI-MSH ',I)NI~-NISHI and phenyloxazalone-NISH (Ox-N1SH). In such cxpurimcnts 6-31",'o of the follicles on day three after immunization were found to contain blastsof one specificity {I)NP or Ox) while the remainder of the follicles wcrc of mixed specificity. Statistically, 12.5%, of follicles would be cxpected to bc monospecific if each follicle is colonized t~x three hapten-spccific blasts. As thc number of blasts in follicles three days after immunization is of the order ot 1-1.5 × 10 4, it tollows that the cell cycle time of these primary B blasts is about 6 h. This agrees closely with the cell cycle time for mouse germinal centrc blasts csnmated by stathmokinetic studies-'~L During the cxponcntial growth of 13blast cells seen during germinal centrc formation, the small recirculating B cells arc excluded from the follicle centre and form the familiar follicular mantle that surrounds the germinal centre, q-here is no clear explanation for the number of blasts that give risu to germinal centres being so restricted. Given the number of haptenspecific blasts seen in T-cell zones m the first few days of primary antibodv responses to haptcn protein and iheir polychmality, it appears not to bc duc to lack ot precursor cells *s. Experiments comparing thc rcsponsiveness of transferred memory and virgin B cells to antigen alrcad~ localized on the FDCs of recipient rats indicate that nlelnor~' cells respond rapidly and well while virgin cells
1~"2, ll,~xw~ %i
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;
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REVIEW (a)
(b)
FDC network
Zone
Follicle
Fine processes
Follicular mantle
Loose network
Outer zone
Dense network
Apical light zone
Dense network
Basal light zone
Fine processes
Dark zone
Fig. 1. (a) Photomicrograph of a secondary follicle with a well-developed germinal centre, from a normal human tonsil. Tbe zonal pattern of the tonsil is apparent. Green immunofluorescence identifies weak CD23 expression by B cells in the follicular mantle and strong CI)23 expression by follicular dendritic cells in the apical light zone. Red immunofluorescence shows the heavy concentration of cells in cell cycle in the clark zone identified by the Ki6 7 monoclonal antibody. The unstained area between the follicular mantle and apical light zone is the outer zone and that between the apical ligh} zone and the dark zone is the basal light zone. Both the outer zone and basal light zone contain occasional Ki67 + cells. (b) These regions are shown with a colour key.
respond slowly, if at all, even when T-cell help is provided I-3,21,22. The failure of the virgin B cells in these transfer experiments to respond to antigen on FDCs is not due to lack of antigen-specific B cells among the donor cells, since transferred virgin cells do give a full and rapid response if the recipient is immunized with soluble antigen after transfer, and if T-cell help is available 1-~,21,22. Excellent germinal centres develop during primary antibody responses, particularly when T-cell help is not limiting. As virgin B cells do not seem to respond to antigen prelocalized on FDCs it seems that they are recruited to form germinal centres by activation by antigen outside follicles 1-~,21-23. This may involve interaction among helper T cells, B cells and interdigitating cells in the T-cell zones, where antigen-specific B cells are known to proliferate during the first few days of primary T-cell-dependent antibody responses 5,1~.l~. Further investigation, comparing the proliferation of
hnmunology Today
memory cells in follicles with the germinal centre reaction seen when virgin B cells are induced to colonize follicles, is required. Centroblasts and centrocytes The first stage of germinal centre formation ends when the B-cell blasts, which are in exponential growth, have filled the FDC network. Over the next few hours blasts are lost from the FDC network and the classical polarization of the germinal centre develops (Fig. 1). The primary B-cell blasts move to one edge of the FDC network, known as the dark zone, where the dendritic processes are widely spaced. The blasts, now termed centroblasts, have an altered phenotype, most strikingly in having lost their slg (Table 1). Centroblasts, like their progenitors the B-cell blasts, are in rapid cell cycle, but, unlike the B-cell blasts, they do not increase in number. Rather, they continually give rise to progeny, centrocytes that are
18
Vol. 13 No. 1 1992
REVIEW Table 1. Phenotype of FDCs and B cells in follicles with germinal centres Zone
Outer zone
FD(2 (11)54
slg
B cells (11")39 CD38 t)cl-2
Ki67 :'
CD75
++
++
--
++
--
+
+
++
_
++
_b
+
+++
+
+++
++
_
++
b
±
+
+++
+++
++
--
++
--
+
+
++
--
+++
+
CD21
CD23
+
-
)
++
±
+
+++
+++
+++
±
+
.
.
.
BUIO
.
.
•'Ki67 is a monoclonal antibody that stains cells in active cell cycle, bOccasional B cells it1 the light zone arc t~cl-2 ~. slg: surface immunogh~hulin. nondividing and express surface immunoglobulin. These centrocytes enter the dense FDC network of the follicle centre, known as the light zone of the germinal centre. Centroblasts and centrocytes have not been shown formally to originate from primary follicular B blasts, but this seems likely, for two reasons: first, the follicles remain oligoclonal as assessed by the double hapten system described above, and second, there is no other obvious source of antigen-specific precursor cells. DNA-labelling studies ~',e4 indicate that centroc)tes are a labile population of cells that is continually replenished from the proliferating pool of centroblasts. Local macrophages contain nuclear debris (tingible bodies) derived from cells that have recently been in cell cyclez~, indicating that there is a high death rate among germinal centre cells. The location of these macrophages suggests that both centroblasts and centrocytes are dying but that cell death is most marked among the cells in that part of the light zone adjacent to the dark zone.
Hypermutation and selection The recruitment of virgin B cells into T-cell-dependent antibody responses is confined to the first few days after exposure to antigen; the response is subsequently maintained by memory B-cell clonesa% Somatic mutation in Ig V region genes, in responses to Ox protein, in B cells in mouse spleen does not start until after the onset of antibody production z-,-'s. Together, these two sets of observations suggest that the lg V-region-directed mutagenic process is induced in cells that have already been activated by antigen. Furthermore, the peak period of accumulation of mutations in Ig V region genes coincides with the time when the germinal centre reaction is taking place-'7-:~L Based on these data, it was proposed that the mutational process occurs in centroblasts and that centrocytes are selected on the basis of their capacity for activation by antigen held on FDCs (Ref. 30 and Fig. 2). Direct evidence that somatic hypermutation is activated in germinal centres is still fragmentary, particularly in relation to the exact stage or stages when the process is activated. Recent evidence suggests that it may be switched on during the phase of exponential growth of B blasts in follicles and before the appearance of centroblasts and centrocytes ~. An indication of the way in which selection after lg V region mutation takes place has been provided by studies of germinal centre cells isolated from the human tonsil 32.
I~1ll lllnolog 3' l'~dax'
This isolation was based on the finding that most germinal centre B cells fail to penetrate 60% Percoll and lack surface expression of IgD and CD39 (Ref. 33). Provided they are isolated at 4°C these cells remain viable ~2,~4but, on culture at 37°C, a high proportion of the cells dies by apoptosis ~ within a few hours ~2,~4. Few non-germinalcentre tonsillar B cells undergo apoptosis on culture. The tendency of germinal centre cells to enter apoptosis can be delayed by one or two days if their antigen-binding receptors are cross-linked m t,itro using anti-immunoglobulin immobilized on a solid surface~-'. These findings suggest that germinal centre B cells with low affinity for antigen held on FDCs are lost by apoptosis while highaffinity cells survive. Study of genetic defects associated with tumours of germinal centre cells has provided some insight into the molecular control of germinal centre cell survival during this selection process. A translocation between a site on chromosome 18 adjacent to the t?cl-2 gene and the lg H genes on chromosome 14 is frequently found in centroblastic/centrocvtic lymphomas ~'. Bcl-2 encodes a 26 kDa protein that is located mainly in the mitochondria ~7,~sand cvtosol ~s. While this protein is not expressed in appreciable amounts in germinal centre cells, it is found in most, if not all, small lymphocytes and extrafollicular B-cell blasts. The anti-Ig signal tllat delays thc onset of apoptosis in germinal centre cells induces the expression of the bcl-2-encoded protein within four hours <~. Expression of bcl-2 has been implicated in ceil survival: first, the transfection of productively-expressed bcl-2 into interleukin 3 (IL-3)-dependent haemopoietic cell lines confers protection from apoptosis when IL-3 is withdrawn ~-41 and second, Burkitt's lymphoma cell lines, which have many of the features of centroblasts, enter apoptosis if cultured at low serum concentration but become serum-independent after transfection of bcl-2 (Ref. 42). As the neoplastic germinal centre cells with the t14,18 translocation express the bcl-2 gene constitutively, it may be that these cells survive, without the need for interaction with antigen on F1)(is.
Differentiation signals Three types of physiological signal can extend the survival of germinal centre cells and result in their differentiation. First, activation with the anti-CD40 antibody, G28-5 (Ref. 43), induces cells to leave cell cycle, many acquiring the phenotype of small lymphocytes ~4. This i's
19
Vc~I. I ~ %'~. I 1~)'~2
REVIEW ~A~rnnrv r~AII
LSt
er e
Fig. 2. Hypothetical pathways of selection and differentiation in the germinal centres, slg centroblasts proliferating in the dark zone undergo somatic mutation in their immunoglobulin V region genes. These give rise to centrocytes which re-express slg and enter the basal light zone. If centrocytes interact with antigen held on the FDCs in the basal light zone, they progress to the apical light zone. Centrocvtes failing to interact with antigen die by apoptosis. Centrocytes entering the apical light zone receive differentiation signals indicated to become memory B cells or plasmablasts (or possibh, centroblasts).
consistent with the production of memory B cells, which are known to be produced by germinal centres 44,45. The CD40-derived signal also induces centrocytes to express bcl-2 protein ,;~. Cells rescued from apoptosis in this way can be maintained in cell cycle by the addition of recombinant IL-4 (rlL-4) 46. These cycling ceils, unlike centroblasts, express surface immunoglobulin. They might be representative of the secondary follicular B-cell blasts that are characteristic of the established phase of T-celldependent antibody responses. Second, a combination of the recombinant (25kDa) fragment of the CD23 molecule plus rlL-lu also prevents germinal centre cells from entering apoptosis. This combination of signals induces some of the features of plasmablasts, including the expression of cytoplasmic Ig and bcl-2 and the development of the endoplasmic reticulum ~4. Third, it recently became clear that a proportion of germinal centre cells responds to riL-2 to remain in cell cycle46. Preliminary phenotypic analysis suggests that the blasts resulting from this stimulus also resemble plasmablasts but differ from those induced with CD23 plus IL-lc~ in that they express little extranuclear protein encoded by the bcl-2 gene. Further work is required to determine the significance of these three groups of differentiation signals. Germinal centre microenvironments
Detailed immunohistological analysis indicates that the light zone of germinal centres in human tonsil is
Immunology Today
20
subdivided into two distinct areas (Refs 47,48 and Figs 1 and 3): the apical zone, where the FDCs express high levels of CD23, and the basal zone, where the FDCs are essentially CD23 but strongly express CD54 (intercellular cell adhesion molecule (1CAM-I)). In both zones there is a dense FDC network that holds antigen in the form of immune complexes. Both the FDCs and the centrocytes in the basal light zone have relatively large amounts of mRNA in their cytoplasm, which is not associated with endoplasmic reticulum. The centrocytes in this zone express CDlla/CD18 (lymphocyte function-associated molecule 1 (LFA-1)), the natural ligand for CD54, on their surface. There are many cells that show features of apoptosis in this basal light zone. All these features are consistent with the suggestion that the basal light zone is the area where centrocytes are selected, on the basis of their capacity to be activated by antigen held on FDCs. The role of the many helper T cells in the light zone 4~ in these selection and differentiation events remains to be determined. In the apical light zone, the presence of CD23 on FDCs suggests that cells that have successfully survived transit through the basal light zone receive the paracrine signals delivered through soluble CD23 plus IL-h~. It may be that they also interact with the natural ligand for CD40 in this area but so far the nature and location of this ligand have remained elusive. The finding that anti-Ig, CD23 plus IL-lu and CD40 antibody alone or in combination, induce the expression of bcl-2 implies that cells receiving these signals in vivo soon leave the germinal centre: only a small proportion of centrocytes express bcl-2 and that expression is weak. Figure 2 depicts the possible processes that occur in germinal centres, based on these findings and interpretations. A further narrow zone is found between the CD23-rich apical light zone and the follicular mantle. This zone extends to surround the dark zone. The cells in this area express CDw75 particularly strongly. The significance of this outer zone and its cellular content remains obscure, but we suggest that it might provide a path by which a proportion of centrocytes return to the dark zone to go through a further cycle of proliferation and V region gene mutation 4s. Late follicular responses
In secondary lymphoid tissues, which have low background germinal centre activity, germinal centre reactions last about three weeks after antigen administration. When centroblasts and centrocytes are no longer present in the follicle, small numbers of B-cell blasts can still be found in the FDC network 5,19. As recirculating memory B ceils can respond to antigen held on FDCs 13,21-23,it seems likely that there is an equilibrium between these late follicular B-cell blasts and memory B cells. It is also possible that these follicular B-cell blasts produce plasmablasts that migrate to the bone marrow 5° or lamina propria of the gut 51, depending on the site of the follicle. The estimated life span of these plasma cells, about four weekss2, is too short to maintain secondary antibody responses which may continue for more than a year. Antigen has been shown to be held on FDCs for extended periods v.8 and presumably is involved in maintaining these late follicular B-cell blasts in cell cycle. ,
Vol 13 No. I 1992
REVIEW
Coherent concepts about the nature and functions of germinal centres are now being developed SL While many of these deserve rigorous evaluation, it seems likely that follicles are the principal sites where T-cell-dependent antibody responses are maintained and that germinal centres are intimately involved in the affinity maturation of these responses.
Yong-]un Liu, Gerald D..Johnson, John Gordon and Ian C.M. MacLennan are at the Dept of Immunology, University of Birmingham Medical School, Birmingham B 15 2 TT, UK. References 1 Nieuwenhuis, P. and Ford, W i . (1976) Cell. lmmunol. 23, 254-267 2 Gray, D., Mackennan, I.C.M., Bazin H. and Khan, M. (1982) Eur. 1. lmmunol. 12, 564-569 3 Namikawa, R., Mizuno, T., Matsuoka, H. et al. 11986) Immunology 57, 61-69 4 Thorbecke, G.J. 11959) Ann. NYAcad. Sci. 78, 237-246 5 MacLennan, I.C.M., Liu, Y-J. and Ling, N.R. 11988) Curr. Top. Microbiol. Immunol. 141, 138-148 6 Jacobson, E.B., Caporale, l.H. and Thorbecke, G.J. (19"4) Cell. hnmunol. 13, 416-431) 7 Tew, J.G. and Mandel, T.E. (1979) hnmunology 37, 69-76 8 Szakal, A.K., Kosco, M.H. and Tew, J.G. 11989) Annu. Rev. hnmunol. 7, 91-109 9 Gray, D. and Skarvall, H. (1988) Nature 336, 70-73 10 Askonas, B.A. and Williamson, A.R. (1972) Eur. J. lmmtmol. 2, 487-493 11 Gray, D., Kosco, M. and Stockinger, B. (1991) Int. hnmunol. 3, 141-148 12 Nieuwenhuis, P. and Opstelten, D. (1984) Ant..l. Anat. 170, 421-435 13 MacLennan, I.C.M., Liu, Y-J., Oldfield, S. et al. (1990) Curt. Top. Microbiol. hnmunol. 159, 37-63 14 Kroese, F.G.M., Timens, W. and Nieuwenhuis, P. (1990) Curr. "D~p. Pathol. 84, 103-148 15 Van Rooijen, N., Classen, E. and Eikelenboom, P. (1986) Immunol. Today 7, 193-196 16 Liu, Y-J., Oldfield, S. and MacLennan, I.C.M. (1988) Eur. J. bnntunol. 18,355-362 17 Kroese, F.G.M., Wubenna, A.S., Seijen, H.G. and Nieuwenhuis, P. (1987) Eur. J. Immunol. 17, 1069-1072 18 Jacob, J., Kassir, R. and Kelsoe, G. 11991)J. Exp. Med. 173, 1165-1176 19 Liu, Y-J., Zhang, J., Chan, E. et al. Eur. J. lmmunol. (in press 20 Zhang, J., MacLennan, I.C.M., Liu, Y-J. and Lane, P.J.L. (1988) hnmunol. Lett. 18,297-299 21 Gray, D. (1988) Immunology 65, 73-80 22 Vonderhei& R.H. and Hunt, S.V. (1990) Eur. J. Immunol. 20, 79-86 23 Kroese, F.G.M., Wubbena, A.S. and Nieuwenhuis, P. (1986) hnmunology 57, 99-104 24 Fliedner, T.M., Kress, M., Cronkite, E.P. and Robertson, J.S. 11964) Ann. N Y Acad. Sci. 113,578-594 25 Fliedner, T.M. 11967) in Germinal Centres in bnmune Responses (Cottier, H. et al., eds), pp. 218-224, SpringerVerlag 26 Gray, D., MacLennan, I.C.M. and Lane, P.J.L. (1986) Eur. J. hnmunol. 16, 641-648 27 Griffiths, G.M., Berek, C., Kaartinen, M. and Milstein, C. (1984) Nature 312, 271-275 28 Berek, C., Griffiths, G.M. and Milstein, C. (1985) Nature 316, 412--418
Immunology Tod,n'
29 XXeiss, 11 and Raiewsky, K. (1990)]. t(xp. Med. 172, 1681-1689 30 Macl.cnnan, I.C.M. and Gray, D. 11986) hnmunol. Rev. 91, 61-85 31 Gray, D. (1991) Res. Immunol. 142,237-242 32 Liu, Y-J., Joshua, D.E., Williams, G.T. et al. 11989) Nature 342, 929-93 I 33 Ling, N.R., MacLennan, I.C.M. and Mason, I).Y. 11987) m Leukocyte Typing Ill (McMichael, A. et al., eds), pp. 302-335, ()xford University Press 34 Liu, Y-J, Cairns, J.A., Abbot, S.l). et al. ( 1991) k[ur. I. hnmunol. 21, 1107-1114 35 Wyllie, A.H., Morris, R.G., Smith, A.L. and l)unh)p, 1). (1984i ]. Pathol. 142, 67-77 36 Tsujimoto, Y., Cossman, J., Jaffe, E. and Croce, C. (1985) Science 228, 1440-1443 37 Hockenbery, D., Nunez, G., Millm3an, C. et al. 11990) Nature 348, 334- ~36 38 kin, Y-J., Mason, D.Y, Johnson, G.I). et al. 199 I) Eur. J. lnzmtmol. 21, 1')05-1910 39 Vaux, D.L., C.~ry, S. and Adams, J.M. (1988) Nature 335,440-442 40 Nuncz, G., London, L., Hockenbcry, D. el al. (1990) J. lmmunol. 144, 3602-3610 41 Williams, G.T., Smith, C.A., Spooncer, E. etal. 11990) Nature 343, -76-7~} 42 Henderson, S., Rowe, M., Gregory, C. et ,I1. (;ell (in press) 43 ('lark, E.A. and Ledbetter, ].A. (1986) l>roc. Natl Acad. Sci. USA 8~, 4495-4498 44 Coico, R.F., Bhogal, B.S. and Tlmrbccke, G.J. (198,:;) J. lntmunol. 131, 2254-2257 45 Klaus, G.G.B., Humphrey, J.H., Kunkle, A. and Dongworth, I).W. (1980) hnmunol. Ret,. 53, 3-28 46 Holder, M., Liu, Y-J., de Prance, T. et ~d. Int. hnmunol. (in press) 47 Johnson, G.1)., Macl,emlan, I.C.M., Khan, M. et al. 11989'. m Leukocyte l),ping IV (Knapp, W. et ,tl., eds), pp. 183-184, Oxfords University Press 48 MacLennan, I.C.M., Johnson, G.I)., Liu, Y-J. and Gordon, J. ( 1991 ) Res. hnmunol. 142, 253-257 49 Stein, H., Gerdes, J. and Mason, I).Y. 1982 Clm. Haematol. 11, 531-559 50 Benner, R., Hiimans, W. and Haaiimau, J.J. 198 I) (;list. Exp. lmmunol. 46, 1-8 51 Gowans, J i . and Knight, E.J. (1964) l'roc. R. ),'oc. London Ser. B. 159,257-282 52 Ho, F.C.S., Lortan, J.L., Khan, M. and Macl~ennan, I.C.M. (1986) Eur..]. lmmunol. 12, 1297-1301 53 Kosco, M. (19~)1) Res. lmmunol. 142, 219-224
Erratum The cover illustration of the N o v e m b e r issue of IT contains a t a x o n o m i c error. There are no such things as ' N e w World Apes' and 'Old World Apes'. In Primata there is a suborder of new world monkeys (Simiae Platyrrhinae) and a suborder of old world monkeys (Simiae Catarrhinae). Many thanks to Professor Edward A. Clark for pointing out and correcting our mistake.
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v , , i ~ No. i 1992