Germinal centre cell kinetics

, . 185: 229–233 (1998)

REVIEW ARTICLE

GERMINAL CENTRE CELL KINETICS

1* . 2

1Department of Cellular Pathology, Level 1, John Radcliffe Hospital, Headley Way, Headington, Oxford, OX3 9DU, U.K.2Department of Pathology, Raigmore Hospital NHS Trust, Inverness, U.K.

SUMMARY

The germinal centre is a fundamental component of the humoral immune response, representing a unique microenvironment whereantigen-activated B lymphocytes undergo clonal expansion, mutate their immunoglobulin, and are subject to a stringent selection processbased on their antigen affinity. This review highlights recent advances in the understanding of the cell kinetic process of activation,proliferation, differentiation, and death of germinal centre cells, which are beginning to provide important insights into the regulation ofthis highly complex reaction. Their definition may have considerable pathological import given the involvement of the germinal centrein non-Hodgkin’s lymphomas and recent evidence suggesting that abnormal germinal centre reactions may be involved in thepathogenesis of Hodgkin’s disease and some autoimmune diseases. ? 1998 John Wiley & Sons, Ltd.

J. Pathol. 185: 229–233, 1998.

KEY WORDS—germinal centre; B lymphocyte; lymphoma; Hodgkin’s disease; proliferation; apoptosis

INTRODUCTION

Germinal centres (GCs) play a critical role in thegeneration of high-affinity humoral immune re-sponses.1,2 Developing within primary B-cell folliclesfollowing antigen (Ag) stimulation, they display a char-acteristic temporal pattern of morphological changeswhich has been termed the germinal centre reaction(GCR).3 Whereas many of the morphological andfunctional aspects of the GCR have been defined, itsregulatory mechanisms are less well delineated.1–5

Fundamentally, it represents a complex interaction ofthe basic cell kinetic processes of activation, prolifer-ation, differentiation, and death of B lymphocytes.6,7

Investigation of these events is beginning to providegreater understanding of the nature of the mechanismscontrolling the GCR. Their definition is not only ofbasic immunological importance, but is also begin-ning to impinge on our understanding of lymphomasinvolving the GC and of autoimmune disease.8,9

ACTIVATION AND PROLIFERATION

Primary (1)) and secondary (2)) GCRs possess distinctkinetic and functional properties, a prototypical GCRprobably only occurring following primary immuniz-ation.1,5,6 The reaction begins with activation (cells inG0 progress to the G1 phase of the cell cycle and becomeresponsive to cell cycle progression factors) of smallrecirculating B cells. Activation occurs in extrafollicularsites, at least in 1) responses.1,2

*Correspondence to: Kevin Hollowood, Department of CellularPathology, Level 1, John Radcliffe Hospital, Headley Way,Headington, Oxford OX3 9DU, U.K.

CCC 0022–3417/98/070229–05 $17.50? 1998 John Wiley & Sons, Ltd.

The activated B cells migrate to 1) follicles, appearingas Ag-specific B blasts within the follicular dendritic cell(FDC) network 2–4 days after 1) immunization.5 Initialexperiments suggested that these blast cell foci werederived oligoclonally from one to three precursors5,10

but immunoglobulin (Ig) gene analysis of microdissectedsingle GC B cells has shown that the initial follicularcolonization is polyclonal, the reaction later becomingoligoclonal due to selection pressures.11 These blast cellsshow massive proliferative activity, reflecting a veryrapid cell cycle time of 6–7 h and a high growth fractionapproaching unity, which produces a peak cell birth rateon day 4 of a 1) response and day 2 of a 2) response.5–7

Four to 6 days after 1) immunization, the developingGCs undergo profound morphological changes, withcompartmentalization of the blast cell population (cen-troblasts) and their progeny (centrocytes) into darkand light zones.1,5 Centroblasts continue to proliferaterapidly, reflected by the maintenance of a high absoluteGC cell birth rate, producing a continuous output ofcentrocytes (many of which are no longer in the cellcycle) for 7–14 days.5–7 Some Ag-selected centrocytesare re-routed to the proliferative compartment.2,12,13

Following this plateau phase, overall GC proliferativeactivity diminishes quite rapidly.6 Interestingly, overallGC proliferation is considerably less in 2) responses.5,6

Although the GC has been long thought of as a B-cellproliferative and differentiation compartment, recentmurine experiments have demonstrated that some ofthe GC proliferative activity represents intrafollicu-lar Ag-specific T-cell proliferation,14,15 the functionalaspects of which, currently, are unclear.

Activation of the GC precursor cells is strictlyAg-dependent with a typical GCR probably onlyproduced by T-dependent Ag.5,16 The importance of

Received 9 July 1997Accepted 17 December 1997

230 K. HOLLOWOOD AND J. R. GOODLAD

cognate T–B cell interaction involving CD40 and the B7family of receptors on B cells and their respective ligandsCD40L and CD28/CTLA4 on T helper cells is supportedby several lines of evidence. In particular, GCs areabsent in patients with the hyper-IgM immunodeficiencysyndrome (due to CD40L mutation), in gene-targetedCD40 and CD40L-deficient mice, and following disrup-tion of CD40–CD40L and B7–CD28/CTLA4 signallingby administration of anti-CD40L or constitutive pro-duction of CTLA4–Ig constructs in transgenic mice.17–21

The majority of data regarding the control of GCB-cell proliferation has been provided by in vitro culturestudies; GC B-cell proliferation is stimulated by Ag,cognate FDC–B cell interaction, FDC-derived solublefactors, cognate T–B cell interaction particularly involv-ing CD40–CD40L signalling, and cytokines includinginterleukin-2 (IL2), IL4, IL5, and IL10.1–3,22–27 How-ever, the relative importance, precise timing of action,and interaction of these factors in vivo remain unclear.

The morphological differences between the earlyexpansive and established zonal phases of the GCR,together with the differential disruption of proliferationin the two stages by cyclosporin A, suggest that differentmechanisms of proliferation control are predominant indifferent phases of the GCR.7 For example, the paucityof GC T cells in the early blast cell stage suggests thatthe combination of complement-containing immunecomplexes, FDC–B cell cognate interaction, and FDC-derived soluble factors are predominant. In contrast,interference with T–B cell interaction during the estab-lished phase diminishes centroblast proliferation, imply-ing an important role for GC T cells probably, giventheir mainly light zone location, in reactivating andre-routing centrocytes to the dark zone.13 Nonetheless, iflymphocyte cell cycle studies showing that Ag stimula-tion is required for each cell cycle28 are applicable to GCB cells, FDCs and Ag in the dark zone would continueto provide a vital proliferative stimulus.

The apparent paradox that there is very much lessproliferative activity in the FDC–Ag-dense light zonemight be explained by functional FDC heterogeneity. Insome in vitro conditions, FDCs can diminish rather thanaugment B-cell proliferation.29

DIFFERENTIATION AND DEATH

The established concept of GC differentiation anddeath holds that centrocytes expressing mutatedIg—hypermutation of the Ag-binding regions of theIg genes being initiated in centroblasts—undergo aDarwinian positive selection process based on theiraffinity for Ag held on FDCs in the light zone.1,30 Highaffinity mutants survive, whereas low affinity ones can-not engage Ag and die by apoptosis.30,31 However, it isevident that the mechanisms of GC cell death andsurvival are more complex and other selection pro-cedures, together with factors such as unrepaired DNAdamage, contribute significantly to GC cell death.

Positive GC B-cell selection in fact begins before theonset of somatic hypermutation.11,32 Ig variable regiongene analysis demonstrates a considerable reduction in

? 1998 John Wiley & Sons, Ltd.

GC clonal diversity, with preservation of B cells express-ing higher affinity but as yet unmutated Ig.32 The deathof unselected, low affinity B cells produces a massiveincrease in apoptosis by day 7 of a 1) response.33

Maintenance of this high level of apoptosis for at least7 days together with the intra-GC accumulation ofcentrocytes expressing high affinity mutated Ig attests tothe veracity of the original concept of positive selectionand affinity maturation in the GC.1,2,33,34 The level ofapoptosis is considerably less during a 2) GCR, suggest-ing that somatic hypermutation is much reduced in 2)responses.33

There is increasing evidence that negative selection,whereby cells binding Ag undergo apoptosis insteadof surviving/proliferating (akin to elimination of self-reactive T and B cells in 1) lymphopoietic organs), alsooccurs in the GC, eliminating centrocytes expressingautoreactive mutated Ig and thereby preserving self-tolerance.9 Undoubtedly, self-reactive clones can begenerated by somatic hypermutation in experimentalautoimmune disease models such as MRL-lpr/lpr mice.9GC B cells are particularly sensitive to tolerance induc-tion; administration of soluble Ag—which acts as asurrogate self-Ag—during a GCR produces a suddenmassive increase in GC apoptosis.9,12,35 Consequently,negative B-cell selection seems to contribute to the highlevel of GC apoptosis during the established phase of theGCR. Regression of the GCR is associated with declin-ing apoptosis, implying that the reaction is curtailed bydiminishing proliferation and continuing emigrationrather than massive cell death.6,33 Interestingly, emerg-ing evidence suggests that intrafollicular T cells arealso subject to positive and negative selection withinGCs and, therefore, also contribute to overall GCapoptosis.15

Regulation of GC B-cell death and differentiationappears to be as complex as that of proliferation. Itseems likely that, in common with other tissues, survivalor death is determined by a complex interplay of positiveand negative, external and internal signals integratedinto a final common pathway involving Ced-3-likecysteine proteases.36 In the GCR, the external control isbiased towards Ag as evidenced by the dominanceof high affinity mutants and the in vitro demonstrationthat cross-linked Ag is required for survival of GC Bcells.30,34

Even so, Ag only provides a short-lived survivalsignal, and other factors are of importance. CognateFDC–B cell interaction, involving ICAM-1, VCAM-1,and CD23 on FDCs and their respective ligands LFA-1,VLA-4, and CD21 on GC B cells, supplies a very strongsurvival signal to GC B cells in vitro.37,38 A role forGC T cells—which can rapidly express CD40L39—issuggested by their spatial distribution and by in vitroexperiments documenting improved survival of CD40L-stimulated GC B cells,30,40–43 although the importanceof CD40–CD40L signalling in vivo remains uncertain,given the recent observation that administration ofanti-CD40L during the established phase of an in vivoGCR produced only slightly increased GC apoptosis.12

Activation of the apoptosis promoter Fas byFas-ligand on T cells mediates apoptosis in some

, . 185: 229–233 (1998)

231GERMINAL CENTRE CELL KINETICS

lymphocytes but its role in the GCR is unresolved.Although Fas is highly expressed on GC B cells and theyare susceptible to Fas-induced apoptosis in vitro, thereare conflicting data on the effects of its interaction withGC survival factors41–43 and Fas-deficient lpr micemaintained normal positive and negative selectionin vivo.12

Similarly, the intracellular regulation of GC B-cellsurvival remains contentious. The anti-apoptotic mol-ecule bcl-2 has been implicated by its association withreduced apoptosis in neoplastic GC B cells31,44 and itsinduction in vitro by most GC B-cell survival factors.45

However, few centrocytes express bcl-2 in vivo; its con-stituent expression does not disturb affinity matu-ration;46 and its expression following CD40 ligation isdelayed.40 These observations could be reconciled ifbcl-2 expression is induced just prior to or duringmigration from the GC but, in normal circumstances, itdoes not mediate intra-GC survival. In contrast, otheranti-apoptotic members of the bcl-2 family, bcl-xL andmcl-l, are expressed by GC B cells47–49 and bcl-xL israpidly induced by CD40 ligation.50 Currently, there areno data as to how these molecules interact in GC B cellswith the apoptotic promoters bax, c-myc, and p53,which are also present in high levels.48

The absence of appropriate FDC and T-cell derivedsignals in the presence of B-cell receptor signalling isthought to account for the death of autoreactive mutantcentrocytes which encounter non-FDC-bound Ag withinthe GC.9


Surviving centrocytes differentiate into B memorycells, plasma cell precursors (particularly in 2) re-sponses), or re-enter the dark zone. There is in vivo andin vitro evidence that the pathway is determined by thedifferential effects of CD40L, CTLA4, and cytokinestimulation. For example, disruption of CTLA4 signal-ling in vivo diminishes memory B-cell production13 and,in vitro, CD40L together with IL2 and IL10 inducesmemory cell differentiation.51 In contrast, cytokinecombinations—IL4 and IL10 or CD23 and IL1-á—promote plasma cell differentiation.1,51

GERMINAL CENTRE CELL KINETICS ANDDISEASE

There is increasing evidence that abnormal GC cellkinetics, particularly GC B-cell death and differen-tiation, are involved in the pathogenesis of follicu-lar lymphoma, some autoimmune disorders, andintriguingly, Hodgkin’s disease.

Although direct experimental proof is lacking, there isstrong circumstantial evidence that diminished apop-tosis plays a significant role in the development of fol-licular lymphoma. In particular, malignant folliclesdemonstrate a much lower apoptotic index than GCs,31

follicular lymphoma cells survive longer than GC B cellsin culture;52 and, in most follicular lymphomas, there isaberrant follicular expression of bcl-2 secondary to thet14,18 chromosomal translocation (which appears to

Fig. 1—Schematic diagram illustrating the cell kinetic events occurring in a germinal centre during the early (A) and established (B) phases ofa primary germinal centre reaction. cc=centrocyte; FDC=follicular dendritic cell; LZ=light zone; DZ=dark zone

, . 185: 229–233 (1998)

232 K. HOLLOWOOD AND J. R. GOODLAD

occur at the pre-B-cell stage of B-cell development).44 Itis suggested that prolonged centrocyte survival (perhapswith intervening episodes of peripheral circulation)coupled with the activity of their hypermutationmechanism, predisposes to the introduction of furthertransforming genetic mutations and consequentdevelopment of the malignant phenotype.53

Doubts about the importance of bcl-2 in follicularlymphomagenesis might be raised, given that bcl-2 maynot normally mediate intra-GC survival (see above), andthat other apoptosis-modulating molecules includingmcl-l and bcl-x are expressed by follicular lymphomas.54

However, in support of its importance, abnormal exper-imental constitutive expression of bcl-2 is associatedwith follicular expansion55 and can interfere, at leastpartially, with some intra-GC selection processes.35,56

Interestingly, cell death is not completely deranged infollicular lymphoma, since Ig heavy chain variableregion gene analysis demonstrates persistence of anAg-driven selection process—and presumably, therefore,death of unselected mutants—in follicular lymphoma.53

Perhaps the most exciting recent development isthe proposal that nodular lymphocyte predominantHodgkin’s disease (NLPHD) and at least some cases ofclassical Hodgkin’s disease arise in transformed GCcells. Microdissection and PCR-based gene sequencingstrategies have shown that both L and H and Hodgkinand Reed–Sternberg (H–R–S) cells possess hypermu-tated clonal Ig genes.57,58 The degree of variable regiongene mutation in L and H cells of 7–27 per cent isusually associated with diminished Ag affinity, andmany of the H–R–S clones possessed crippling hyper-mutations (such that viable Ig would not be expressed).This implies that not only have these cells arisen in theGC, but that they have evaded the normal positiveselection procedure and survive despite being unable tocompete effectively for Ag.

Kanzler et al.58 have hypothesized that their survivalmay be related to upregulation of bcl-2 by Epstein–Barrvirus (EBV). However, the observation that bcl-2expression is lower in EBV-positive than in EBV-negative cases casts doubt on this mechanism.59 Analternative candidate might be bcl-x, which is highlyexpressed by H–R–S cells and shows a close correlationwith EBV status (it should be noted that this study couldnot discriminate between bcl-xL and bcl-xS).60 Regard-less of the precise molecular mechanisms, the demon-stration of crippling or reduced-affinity mutationsprovides compelling evidence that abnormal GCapoptosis is critically involved in the pathogenesis ofHodgkin’s disease.

The third possible pathological consequence ofderanged GC B-cell death is that of autoimmune dis-ease. The recent observation that autoreactive antibodyspecificity is generated by somatic hypermutation duringnormal immune reactions to foreign antigens,61 togetherwith the presence of hypermutated antibodies in exper-imental systems and human disease, implies that GCcells with autoreactive mutant Ig may evade the normalnegative selection process in GCs.9 Several groups haverecently suggested that such a mechanism may occurin thymic GCs in myasthenia gravis, although obser-


vations of aberrant GC bcl-2 and CD23 expression areconflicting.62,63

FUTURE PROSPECTS

Unsurprisingly, given the highly complex nature ofthe GCR, an overall integrated picture of its cell kineticsand regulation remains elusive. Nevertheless, increas-ingly sophisticated GC cell separation techniques,improved in vitro models, and, crucially, the use ofstrategies which specifically disturb putative regulatorypathways during an in vivo GCR provide encourage-ment that the goal is attainable. Its impact on the basicunderstanding and treatment of diseases involving theGC could be considerable.

REFERENCES

1. MacLennan ICM. Germinal centers. Annu Rev Immunol 1994; 12: 117–139.2. Kelsoe G. In situ studies of the germinal center reaction. Adv Immunol 1995;

60: 267–288.3. Nieuwenhuis P, Opstelten D. Functional anatomy of germinal centers. Am

J Anat 1984; 170: 420–435.4. Thorbecke GJ, Amm AR, Tsiagbe VK. Biology of germinal centers in

lymphoid tissue. FASEB J 1994; 8: 832–840.5. Liu Y-J, Zhang J, Lane PJL, Chan EY-T, MacLennan ICM. Sites of specific

B cell activation in primary and secondary responses to T cell-dependentand T cell-independent antigens. Eur J Immunol 1991; 21: 2951–2962.

6. Hollowood K, Macartney JC. Cell kinetics of a germinal center reaction—astathmokinetic study. Eur J Immunol 1992; 22: 261–266.

7. Goodlad JR, Macartney JC. Regulation of murine germinal centrecell proliferation in vivo: a stathmokinetic study examining the effect ofdifferently timed doses of cyclosporin A. J Pathol 1995; 176: 87–97.

8. Isaacson PG. Malignant lymphomas with a follicular growth pattern.Histopathology 1996; 28: 487–495.

9. Pulendran B, van Driel R, Nossal GJV. Immunological tolerance ingerminal centres. Immunol Today 1997; 18: 27–32.

10. Kroese FGM, Wubbena AS, Seijen HG, Nieuwenhuis P. Germinal centersdevelop oligoclonally. Eur J Immunol 1987; 17: 1069–1072.

11. Kuppers R, Zhao M, Hansmann ML, Rajewsky K. Tracing B cell develop-ment in human germinal centres by molecular analysis of single cells pickedfrom histological sections. EMBO J 1993; 12: 4955–4967.

12. Han S, Zheng B, Dal Porto J, Kelsoe G. In situ studies of the primaryimmune response to (4-hydroxy-3-nitrophenyl) acetyl. IV. Affinity-driven Bcell apoptosis in germinal centers as a mechanism for maintaining selftolerance. J Exp Med 1995; 182: 1635–1644.

13. Han S, Hitchcock K, Zheng B, Kepler TB, Hodes R, Kelsoe G. Cellularreactions in germinal centers. Roles of CD40 ligand and B7-2 in establishedgerminal centers. J Immunol 1995; 155: 556–567.

14. Gulbranson-Judge A, MacLennan ICM. Sequential antigen-specific growthof T cells in the T zones and follicles in response to pigeon cytochrome c.Eur J Immunol 1996; 26: 1830–1837.

15. Zheng B, Han S, Zhu Q, Goldsby R, Kelsoe G. Alternative pathways for theselection of antigen-specific peripheral T cells. Nature 1996; 384: 263–266.

16. Goodlad JR, Macartney JC. Germinal center cell proliferation in responseto T-independent antigens: a stathmokinetic, morphometric and immuno-histochemical study in vivo. Eur J Immunol 1995; 25: 1918–1926.

17. Callard RE, Armitage RJ, Fanslow WC, Spriggs MK. CD40 ligand and itsrole in X-linked hyper-IgM syndrome. Immunol Today 1993; 14: 559–564.

18. Kawabe T, Naka T, Yoshida K, et al. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinalcenter formation. Immunity 1994; 1: 167–178.

19. Xu J, Foy TM, Laman JD, et al. Mice deficient for the CD40 ligand.Immunity 1994; 1: 423–431.

20. Foy TM, Laman JD, Ledbetter JA, Aruffo A, Claessen E, Noelle RJ.gp39–CD40 interactions are essential for germinal center formation and thedevelopment of B cell memory. J Exp Med 1994; 180: 157–163.

21. Lane P, Burdet C, Hubele S, et al. B cell function in mice transgenic formCTLA4-Hã1: lack of germinal centers correlated with poor affinitymaturation and class switching despite normal priming of CD4+ T cells.J Exp Med 1994; 179: 819–830.

22. Kosco MH, Pflugfelder E, Gray D. Follicular dendritic cell dependentadhesion and proliferation of B cells in vitro. J Immunol 1992; 148:2331–2339.

23. Burton GF, Conrad DH, Szakal AK, Tew JG. Follicular dendritic cells andB cell costimulation. J Immunol 1993; 150: 31–38.

, . 185: 229–233 (1998)

233GERMINAL CENTRE CELL KINETICS

24. Kim HS, Zhang X, Klyushnenkova E, Choi YS. Stimulation of germinalcenter B lymphocyte proliferation by an FDC-like cell line, HK. J Immunol1995; 155: 1101–1109.

25. Butch AW, Nahm MH. Functional properties of human germinal center Bcells. Cell Immunol 1992; 140: 331–344.

26. Grouard G, Bouteiller O, Banchereau J, Liu YJ. Human follicular dendriticcells enhance cytokine-dependent growth and differentiation of CD40-activated B cells. J Immunol 1995; 155: 3345–3352.

27. Rabinowitz JL, Tsiagbe VK, Nicknam MH, Thorbecke GJ. Germinalcenter cells are a major IL-5 responsive B cell population in peripherallymph nodes engaged in the immune response. J Immunol 1990; 145:2440–2447.

28. Lernhardt W, Karasuyama H, Rolink A, Melchers F. Control of the cellcycle of murine B lymphocytes: the nature of á- and â-B-cell growth factorsand of B-cell maturation factors. Immunol Rev 1987; 99: 241–262.

29. Freedman AS, Munro JM, Rhynhart K, et al. Follicular dendritic cellsinhibit human B-lymphocyte proliferation. Blood 1992; 80: 1284–1288.

30. Liu Y-J, Joshua DE, Williams GT, Smith CA, Gordon J, MacLennan ICM.Mechanism of antigen-driven selection in germinal centres. Nature 1989;342: 929–931.

31. Hollowood K, Macartney JC. Reduced apoptotic cell death in follicularlymphoma. J Pathol 1991; 163: 337–342.

32. McHeyzer-Williams MG, McLean MJ, Lalor PA, Nossal GJV. Antigendriven B cell differentiation in vivo. J Exp Med 1993; 178: 295–307.

33. Goodlad JR, Hollowood K. Temporal changes in germinal centre apoptosisduring an experimental immune response and its alteration by cyclosporinA. J Pathol 1995; 176(Suppl): 34A.

34. Berek C, Ziegner M. The maturation of the immune response. ImmunolToday 1993; 14: 400–404.

35. Pulendran B, Kannourakis G, Nouri S, Smith KGC, Nossal GJV. Solubleantigen can cause enhanced apoptosis of germinal-centre B cells. Nature1995; 375: 331–334.

36. Wyllie AH. Apoptosis. In: Anthony PP, MacSween RNM, Lowe DG, eds.Recent Advances in Histopathology. Edinburgh: Churchill Livingstone,1997; 1–14.

37. Koopman G, Keehnen RMJ, Lindhout E, et al. Adhesion through theLFA-1 (CD11a/CD18)–ICAM-1(CD54) and the VLA-4 (CD49d)–VCAM-1(CD106) pathways prevents apoptosis of germinal center B cells. J Immunol1994; 152: 3760–3767.

38. Bonnefoy J-Y, Henchoz S, Hardie D, Holder MJ, Gordon J. A subset ofanti-CD21 antibodies promotes the rescue of germinal center B cells fromapoptosis. Eur J Immunol 1993; 23: 969–972.

39. Casamayor-Palleja M, Khan M, MacLennan ICM. A subset of CD4+

memory T cells contains preformed CD40 ligand that is rapidly buttransiently expressed on their surface after activation through the T cellreceptor complex. J Exp Med 1995; 181: 1293–1301.

40. Holder MJ, Wang H, Milner AE, et al. Suppression of apoptosis in normaland neoplastic human B lymphocytes by CD40 ligand is independent ofbcl-2 induction. Eur J Immunol 1993; 23: 2368–2371.

41. Cleary AM, Fortune SM, Yellin MJ, Chess L, Lederman S. Opposing rolesof CD95Fas/APO-1) and CD40 in the death and rescue of human lowdensity tonsillar B cells. J Immunol 1995; 155: 3329–3337.

42. Koopman G, Keehnen RHN, Lindhout E, Zhou DFH, de Groot C, PalsST. Germinal center B cells rescued from apoptosis by CD40 ligation orattachment to follicular dendritic cells but not by engagement of surfaceimmunoglobulin or adhesion receptors become resistant to CD95-inducedapoptosis. Eur J Immunol 1997; 27: 1–7.

43. Choe J, Kim HS, Zhang X, Armitage RJ, Choi YS. Cellular and molecularfactors that regulate differentiation and apoptosis of germinal center B cells.Anti-Ig down-regulates Fas expression on CD40 ligand stimulated germinalcenter B cells and inhibits Fas-mediated apoptosis. J Immunol 1996; 157:1006–1016.


44. Tsujimoto Y, Cossman J, Jaffe E, Croce CM. Involvement of the bcl-2human follicular lymphoma. Science 1985; 228: 1440–1443.

45. Liu Y-J, Mason DY, Johnson JD, et al. Germinal center cells express bcl-2protein after activation by signals which prevent their entry into apoptosis.Eur J Immunol 1991; 21: 1905–1910.

46. Smith KGC, Weiss U, Rajewsky K, Nossal GJV, Tarlinton DM. Bcl-2increases memory B cell recruitment but does not perturb selection ingerminal centers. Immunity 1994; 1: 803–813.

47. Krajewski S, Bodrug S, Krajewska M, et al. Immunohistochemical analysisof mcl-1 protein in human tissues. Differential regulation of mcl-1 andbcl-2 protein production suggests a unique role for mcl-1 in control ofprogrammed cell death in vivo. Am J Pathol 1995; 146: 1309–1319.

48. Martinez-Valdez H, Guret C, de Bouteiller O, Fugier I, Banchereau J,Liu YJ. Human germinal center B cells express the apoptosis-inducing genesFas, c-myc, p53, and bax but not the survival gene bcl-2. J Exp Med 1996;183: 971–977.

49. Tuscano JM, Druey KM, Riva A, Pena J, Thompson CB, Kehrl JH. Bcl-xrather than bcl-2 mediates CD40-dependent centrocyte survival in thegerminal center. Blood 1996; 88: 1359–1364.

50. Zhang X, Li L, Choe J, et al. Up-regulation of bcl-xL expression protectsCD40-activated human B cells from Fas-mediated apoptosis. Cell Immunol1996; 173: 149–154.

51. Arpin J, Dechanet J, van Kooten C, et al. Generation of memory B cells andplasma cells in vitro. Science 1995; 268: 720–722.

52. Johnson PWM, Watt SM, Betts DR, et al. Isolated follicular lymphomacells are resistant to apoptosis and can be grown in vitro in the CD40/stromal cell system. Blood 1993; 82: 1848–1857.

53. Zelenetz AD, Chen TT, Levy R. Clonal expansion in follicular lymphomaoccurs subsequent to antigenic selection. J Exp Med 1992; 176: 1137–1148.

54. Schlaifer D, Krajewski S, Galoin S, et al. Immunodetection of apoptosis-regulating proteins in lymphomas from patients with and without humanimmunodeficiency virus infection. Am J Pathol 1996; 149: 177–185.

55. McDonnell TJ, Deane N, Platt FM, et al. bcl-2-immunoglobulin transgenicmice demonstrate extended B cell survival and follicular lymphoprolifer-ation. Cell 1989; 57: 79–88.

56. Shokat KM, Goodnow CC. Antigen-induced B cell death and eliminationduring germinal-centre immune responses. Nature 1995; 375: 334–338.

57. Marafioti T, Hummel M, Anagnostopoulos I, et al. Origin of nodularlymphocyte-predominant Hodgkin’s disease from clonal expansion ofhighly mutated germinal-center B cells. N Engl J Med 1997; 337: 453–458.

58. Kanzler H, Kuppers R, Hansmann ML, Rajewsky K. Hodgkin andReed–Sternberg cells in Hodgkin’s disease represent the outgrowth of adominant clone derived from (crippled) germinal center B cells. J Exp Med1996; 184: 1495–1505.

59. Jiwa NM, Oudejans JJ, Bai MC, et al. Expression of bcl-2 protein andtranscription of the Epstein–Barr virus homologue BHRF-1 in Hodgkin’sdisease: implications for different pathogenic mechanisms. Histopathology1995; 26: 547–553.

60. Schlaifer D, March M, Krajewski S, et al. High expression of the bcl-x genein Reed–Sternberg cells of Hodgkin’s disease. Blood 1995; 85: 2671–2674.

61. Ray SK, Putterman C, Diamond B. Pathogenic autoantibodies are routinelygenerated during the response to foreign antigen: a paradigm for auto-immune disease. Proc Natl Acad Sci USA 1996; 93: 2019–2024.

62. Shiono H, Fujii Y, Okumara M, Takeuchi Y, Inoue M, Matsuda H.Failure to down-regulate bcl-2 protein in thymic germinal center B cells inmyasthenia gravis. Eur J Immunol 1997; 27: 805–809.

63. Murai H, Hara H, Hatae T, Kobayashi T, Watanabe T. Expression ofCD23 in the germinal center of thymus from myasthenia gravis patients.J Neuroimmunol 1997; 76: 61–69.

, . 185: 229–233 (1998)

Documents

Germinal centre cell kinetics