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Immunological tolerance in germinal centres
IMMUNOLOGY
TODAY
Immunological tolerance in germinal centres Bali Pulendran, Rosemary
van
Driel and G.J.V. Hossal
The abiliOl to avoht autoimmunity is a cardinal feature of the immmw I
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work of follicular dendritic cells (FDCs) and T cells. FDCs can harbour unprocessed antithis is achieved by several of mature lymphocytes in the gen in the form of immune complexes on mechanisms that exclude self. . . . . . . . . :immune system. One serves their surface for several months24.~. The :o exclude those lymphocytes with noncentrocytes with high affinity for the antireactive h.lmphoqttes f r o m mature productive antigen-receptor rearrangements gen on the FDCs are presumed to be posihdmphocytc n,pertoires. Here, md the other serves to extinguish those tively selected2".a7, and can then differenBali Puh,ndran, Rosemary van DrM [ymphocytes that have acquired self-reactive tiate into memory B cells or into ~ntigen receptors. Evolution seems to place antibody-forming plasma cells-~7-3~,dependand G.I.V. Nossal rt'z,it~t, recent a great premium o~ avoiding the horror ing on the costimulatory signals they restudies indicatiKq that several ceive from the FDCs. Alternatively, the cen~utotoxicus envisaged by EhrliclP in 1901, trocytes are thought to return to the dark and has ingeniously devised a number mechanisms of self-tolerance operate zone and undergo further proliferation and of different contrivances to maintain selfit! gerttlitta| centres. mutation. Those centmcytes that are not tolerance2--q.Many of the negative selection selected by the FDCs are believed to die steps in the generation of B- and T-ceU repertoires occur early in the ontogeny of fl~ese cells in the bone through apoplosislx2~'.-'7.It appears that centrocytes cultured in the marrow and thymus, respectively. However, several additional presence of an antibody against CD40 and IL-4 (Refs 30, 31) or mechanisms of self-tolerance in the periphery have evolved to CD40-1igand, IL-2 and IL-10 (Ref. 29) can survive and acquire the silence mature B cells2"~ and T cells~.~ that have escaped central phenotype of memory B cells. Thus the two processes of mutation tolerance. Recently, there has bi.,enmuch interest in the possibility and selection acting in a coordinated and iterative manner are of there being self-tolerance mechanisms within germinal centres. It thought to give rise to the phenomenon of affinity maturation. has h.,en proposed that such mechanisms exist in order to prevent the emergence of autoreactive cells through somatic hypermutation 3,~'7, Recent studies by various groups indicate that several mechanisms Emergence of autoreactive B cells within germinal eentres opt,rate ill germinal centres to maintain self-toleranee~q-', Given the intense proliferation of the centroblasts, ancl the rapid rate of mutation in the V genes, and considering the large number of self-anUgens in the body, it is likely that the germinal centre Germinal centre development Germinal ~,ntres (Fig. I) are dynamk" mlcroenvlronments uf B-cell differentlathm which arise transiently in tile primary B.cell fullteles of ~ m d a r y lymphoid organs during immune responses~,". The B cells that form germinal centres are initially activated outside the primary follicles: [or example, in the T.cell-rich zones of the perlarlerlo|ar lymphatic sheath (PALS) in the splenic white pulp, in association with T-helper (Thl cells and interdigitating dendritic cells~.1". After this initial activation, the [I cells can either differentiate into anllbt~ty-forming cells of the foci, or migrate into the primary fnllicles and undergo further cloaal expansion and differentiation, giving rise to germinal centres. Once the activated B cells emer the primary follicles, they downregulate their immunoglobnlin fig) receptors and undergo a phase of massive clonal expansion during which they divide every 6--7 hours ~zls. In addition, they activate a site-specific hypermutation mechanism, which rapidly introduces random point m~tations Fig, I. Photomicrographof a section of spleen dmuqug Huegerminal cen-
system. In healthy individuals,
development of the repertoire
into their Ig variable region genes (V genes) w--'~(Fig. 2). These dividing eentroblasts form the 'dark zone' of the germinal centre and continually give rise to nondivlding centrocytes, which upregulate their lg receptors and migrate to the apical region of the germinal centre (the light zone), where they interact with an extensive netPll *aO167,~b99(96)lOObB
slaim~d with pemmt aggh,tinin (PNA) fblue) mid ml iuterw,ning T-cell zone staim'd with anti-CD4 (lavwn). Note that thereare rca.qanable mmlbers of CD4 ~ cells scattered throughout the germinal eentres. Note fio'ther that PNA stains vascular endothelimn and the marginal shins this ca, be useful in ~rientating seatfoas that at,, not comflerstahwd. trex
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Fig. 2. The germinal centre reaction showbzg the hnportance of T-cell-Bcell interaction, B-celI-FDC-interaction and apoptosis of B cells in the comph'x cascade of eveuts withbz the gennittal centre. Abbreviations: FDCo follicular dendritic cells; lg, immmzoglobulin; Th, T hellx, r cell, reaction often results in self-reactive B cells. Indeed, several obse-rations illustrate this possibility. A recent study has shmvn that pathogenic autoantibodies that bind doubh.~-stranded (ds) DNA are generated at a high frequency during the immune response to foreign antigen. The autoantlbodies, which were crossreactive with the immunizing antigen, were found to be encoded by somatically mutated V genes and did not arise until day l0 or ll after immunization. These observations suggest that the autoreactive specificity is generated by somatic mutation~-'. Furthermore, the analysis of autoreactive, anti-ds-DNA-binding hybridomas in the autoimmune MgL.Ipr/Ipr or NZB/NZW mice shmvs anti-ds-DNA B cells to be the pn~ducts of extensive somatic mutation and clonal selection. This suggests that, in these shains of mice, such cells persist as a result of a defect in their elimination~ . In a self, rate study it has been shown that healtl~y individuals, unlike subjects with rheumatoid arthritis (RA), do not produce high-affinity rheumatoid factors (RFs) after immunization3~. Moreover, there appears to be a strong selection against replacement of amino acids in the hypervarlable or complementarity.determining region of RFs in healthy individuals. These observations suggest thatRFs from R A patientsescape a tolerance mechanism which norma~y operates in healthy Individuals. 1~L~e and several other experiments are a fo~afful reminder of the possibility of autoreactive B cells arising in the germinal centres. Are there special mechanisms of self-tolerance which prevent this?
Negative selection of B cells w i t h i n germinaJ centres? Several experimenls indicate that negative selection steps occur ~ t h i n germinal centres, and that B cells can be eliminated by anti-
gen even during immune responses. Linton el al. showed that B cells typical of the secondary B-cell lineage iwhich express low levels of Jlld (heat-stable antigen) (ReL 40) and which have the capacity to seed into primary follicles and form germinal centres41] displayed an exquisite sensitivity to tolerization in vitro 6. This was interpreted to mean that B cells unde-rwent a 'second window" of tolerance during their differentiation into pre-memory B cells, presumably within germinal centres, in a separate study, by Dintzis et al. 42, it was shown that when mice undergoing a T-cell dependent immune response to fluoresce.in isothiocyanate (FlTC)-ovalbumin-haptencarrier were injected with FITC coupled with a nonimmunogeneic carrier, the response was suppressed such that much fewer antiFITC antibody-secreting cells were present in the spleens after confinued exposure to this tolerogen. Prompted by these and other observations~, we used the capacity of deaggregated forms of soluble antigen to induce immunologic tolerance in adult mice~.45 to try and explore tolerance within germinal centres. Initial experiments demonstrated that when deaggregated human serum albumin (HSA) was administered as a surrogate se.lf-antigen seven days prior to immunization with HSA, there was a marked reduction in the number of clonable anti-HSAspecific B cells with sufficient affinity to bind to HSA as a bivalent lgG1 dimer, rather than as a decavalent lgM (Ref. 46). Adoptive transfer experiments suggested that both the B- and particularly the T.-cell compartments were afro-ted47. The molecular and histological analysis of this tolerance were facilitated by the use of a hapten-carrier system, in which (4-hydroxy3-nitrophenyi)acetyl (NP) coupled to HSA (NP-HSA) was used as the antigen. The immune response to NP-pmtein conjugates has been well characterized in the C57BL/6 strain of miceI~,t~-'l'~.4~-~. Injections of deaggregated NP-HSA 4-7 days prior to immunization with the alum-precipitated, more highly haptenated form of the same antigen resulted in a profound and persistent state of unresponsivenessSL Hapten-carrier studies again showed a major eb feet in the T-cell compartment, thus confirming the earlier adoptive transfer studiesSL The NP-specific B cells in the spleens of the Immune and tolerant mice were directly identified using a multiparameter flow cytometry system designed to detect the ran, and heterogeneous population of isotype-switched, N[~.spceific cellss2. Such analysis revealed a significant reduction in the NP-bindlng cells of germinal centre origin in the tolerant mice~, again showing that the tolerance resided in B and T cells. This was corroborated by immunohistologic studies in which splenic sections were stained for germinal centers comprising cellq expressing the lg ~ light chain, which are characteristic of the anti-NP r,~spimse in C57BL/6 mice~ ' ~ - ~ . Tliese r~ults were interpreK,d to mean that the tolerance resulted in an impairment of antigen-specific gem~inal centre development both through a T-cell-dependent mechanisms~ and through a direct effect on B cells.
of soluble ont/gcn on an established immune response Interesting|); it was shmvn that a similiar effect could be elicited by the soluble NP-HSA, even when it was given as late as six days
IMMUNOLOGY
after inununization"~L In this case, there was a profound reduction in the high affinity, NP-specific B ceils that could be enumerated at day 14 of the response, in limiting dilution cultures. By contrast, there was only a modest reduction in the number of cells secreting low-affinity antibody. This suggested that soluble antigen iniected six days after immunization had selectively interfered with the development of high-affinity anti-NP cells. This tolerance appeared to be mediated through a direct effect on the B cells and indirectly through Th cells, as a partial reduction could be obtained by injecting soluble HSA, lacking the NP hapten. Further cellular and histological analysis confirmed this, as mice treated with soluble NP-HSA 6 days after immunization developed far fewer NPspecific, germinal centre cells and lg ~.~ germinal centres in their spleens compared with control miceu. However, the tolerant mice did contain large, Ig h - germinal centres, perhaps containing B cells specific for other antigens. In contrast to its effect on NP-specific, germinal centre B cells, the soluble antigen induced the B cells outside the follicles to expand and differentiate into antibody-secreting cells. This was consistent with the much larger lgG1 ÷ loci observed in splenic sections of the tolerant mice. Thus, despite an impairment of NP-specific germinal centre B cells, soluble antigen given after immunization appears to cause F"lyclonal activation of other B cells. This polyclonal activation coulo be mediated through immune complexes operating thwugh the Fc3, receptors on B cells - it is clear that immune complexes can exert stimulatory effects on B cells when formed in antigen-excess~-~, Therefore, soluble antigen given six days after immunization seems to act in two distinct ways: (1) it cau,~es a polyclonal activation of B cells both in rite germinal centre and the antibody-forming arms of the immune response and (2) it causes impairment of antigen-specific, germinal centre B cells.
C l o n a l d e l e t i o n o f B cells in 8 e r m l n a l ¢entres
TODAY
Fig. 3. (a) 0.45 Izm section of a Rerminal ccnhv firm manse ~ ~lee, 4 II after solubh' anti~e,t challc,ge, stained with Toluidine Blue m,~ A=m" il. The cluster of dead cells within the square is shown in (b). ~ 7hmsmisshllt eh,ctn), mictvgraph montage of m~'a selected firm (ok ~howill,~. a cluster of moribund cells partially eaveh)ped by a ma,~ophage. Within this cluster art' profiles of cells in various sta~es of apol,~,sis and prefiles of type B dark cells. (c) "133picaltype B dark cell, showin~: conden. sation of chwmatiu fwithout the 'ereseent' formath)ll seen in ,!~Jptasis), swollen mitochomlria altd endoplassnic reticulum, and cell ,./winka,~e without amnding: ,ore tm~ccsst~ I~'lu~'en ~d/awnt cells, This cell i~ ~hreeceU I~lx~filesaway fn,,i m0,tagt' ([p}.fd) l~lpicat early at,)l~lOsts: hi,~l, ma.~nifioltiott of con ill h)ll,er box/11(ll)l Ilotc holllO,~,t'llOUSa.ldet~sathm of c']lro. matin, and ~;m.~Mtcell tin,file,/e) High ma,~laificatton of area e.~losed by Uplwr box in (b), showi.g a t,lpe I] dark cell Imlfik (ri,~hl) adi.~.e.t to a partially digestedat~oplollcI.~dy (h,fl), All barscorrespondto 2~'~,I~m.
What is the medlanlsm of Impairment of germinal centre cells? Does the soluble anllt4en kill the B cells or does It make them anergic? D ~ it mediate its effect(s) directly on the B cells or through some other mechanism Ittvolving Tit cells? To explore these questions and to examine the sensitivity of germinal centre II cells to tl~is spike in the number of apuptotlc cel]s was antlgen-sI, cific, as apuptosls we used the TUNEL technique~L C57BL/6 mice were im- it coukl not be induced by injecting an irrelevant protei. ~uc~ as munized with NP-HSA and 14 days later, at the height of the im- transferrln. Further studies on germinal centre apoptosis w~re pernmlse n.,sDOllbe(whvll the germinal centre reaction was at its peak), formed using soluble carrier alone and the NP hapten attached to the mice were injected with 5 mg of soluble NI)~-HSA. Tile aim was the self-antigen, mouse serum albumin (MSA). These supported the to confrullt the germinal centre cent~t~cytes with ,~luble antigen be- view that the dominant effect was a direct killing of the haptenlure they could gain access [o the antigen displayed on the surface specific B cells. Furtbermore, this apoptosis was unique to germiof FDCs. At various inlervals after this injection (ranging from nal centres, as no other areas in the spleens (such as loci) e×hibited 10 rain to 24 h) tile mice were killed and the spleens removed and any noticeable increase in apoptosts'j. When tbe experiments were performed in bcl-2 transgenic mice~, analysed for apoptosis using tile TUNEL assay. The soluble antigen induced a rapid and dramatic increase in the number of TUNEL' which constitutively expressed tile anti-apoptotic bcl-2 transgene in cells within germinal centres, which peaked at 4--6 h, before declin- all B cells, there was only a partial inhibition of the soluble antigening to base-line levels by 24 h (Ref. 9). One hour after injection there induced apoptosls'j. By contrast, apoptosis which spontaneously ocwas already a significant increa~ in apoptotic cells. After 12 h, ex- curs within germinal centres during a normal immune respn,se (and tensive al~ptotic debris could be seen within macrophages, some which presumably reflects the death of centrocytes thai fail to be seof which appeared to be migrating out to the red pulp. Moreover, lected by FDC-held antigen 27) was effectively inhibited by bcl-2.
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This is in agreement with experimentss~that demonstrate a significant increase in the spontaneous apoptesis of mature B celts in bcl2-deficient mice. Thus, it appears that there is a fundamental difference in the mechanism between the two forms of apoptosis: (D the spontaneous, "background' apoptosis (which presumably reflects the rather passive 'death by neglect' in normal germinal centre functioning) being effectively inhibited by bcl~; and (2) the active process that might be at work in soluble antigen-induced apoptosis being only partially inhibited by bcl-2. Ultrastructural analysis of soluble-antigen-induced apoptosis revealed clusters of apoptotie ceils throughout germinal centres (Figs 3a and b). Such cells displayed the morphologically distinctive features of apeptosis°°, such as the striking reduction in cell and nuclear volume (Figs 3b and dl which accompanies the margination of the chromatin to form dense granular caps under the nuclear membrane. In contrast to the cytoplasmic organelles of necrotic cells, those of apoptotic cells appeared to be weU preserved, apart from the dilation of the endoplasmic reticulum. Cells with surface blebbing and nuclear disintegration (which typify the advanced stages of apopinsis) were also apparent (Figs 3b and el.
Type B dark cells In addition to classically apoptotic cells, we observed cells that were clearly moribund and yet mnrphologically quite distinctive from either apoptotic or nt.x-roticceils (Figs 3b, t. and O. These possess the characteristic features of 'type B dark cells "l'°-~ The condensed chvamatin in such cells does not have the smoothly contoured inner edt~es characteristic of apoptosis, and there is no radical redistribution of the chix~matin. Although there is overall cytoplasmic coltdeusation, tile mitochondria and endop]asmic reticulum tend to be swollen (unlike those in the type A dark cells)~. In addition, the cytoplasm is stretched out in the form of attenuated processes Ix~ tween adj,~cent cells, a feature that is quite unlike the budding to produce round and oval bodies, which o.-curs during apoptosls. Although dark cells have been implicated in ,,'-elldeath, to the Ix,st of our knowh,,dge there have been no studies desigmM to determine whether DNA is cleaved during their formatimz, and if ~ whether cleavage is random or selective. "Pype B dark cells have b~en observed in tomours where the turnout cells were most-ck~ely packed together, suggesting thnt cron,ding contributes to their development"~, It is tentptin~3to specu!ate whe!l'er their (.vo'urrence in germinal centrps could [~.,attributed to this reason. Similiar data have been obtaizu.~db~ Shoktt and Goodnow~, who develol:,.~t a system to track hen egg lysozyme (HELl.specific B tells fp_~mtrm~,enic mk'e, after seedtng them into the developing germinal centres hi: nnatmnsgenic mice immunized with duck egg ly,~zyme (DELl, The ,,~xted B ceils were identified by a unique LySa allutypic m;irker, attd were ~,en to multiply rapidly over the next five days, accuunting for m ~ t of the |g-k,aring B cells within the germinal centres. To model the fate of serf-reactive variants that ari~ in germinal cenm.,s, 5 mg of ,~oluble HEL 'was in~:ttM into the mice, and the sph.,ens analysed for apeptosis nsing rite TUNEL as~y. Therefor~ in rids model the germinal centre B cells were con................ .........................................................
fronted witll an antigen that lacked T-cell help (since DEL and HEL do not crossreact at the Th-cell level). Within 4 h, large numbers of apoptotic cells were seen within germinal centres, and the HELspecific B cells disappeared from the light zones of the germinal centres. Surprisingly, it was observed that some HEL-specific B cells relocated themselves to the T-cell zone, persisted for a while and then died eight b,ours later. Bd-2 could not protect against apoptosis within the germinal centre.s, but could prevent the death in the T-cell zone, suggesting a mechanistic difference between the two forms of apoptosis. Further supporting data have been obtained by Hart et al. I(~,who injected protein conjugates of (4-hydroxy-B-iodo-3-nitrophenyl}acetyl (NIPI into mice previously immunized with NP-.-chicken gamma-globulin (CGG). As in the previous hvo studies, a single injection of soluble antigen induced a large increase in apoptosis in germinal centres without significantly decreasing their numbers or average size. However, repeated injections over an 18 h period resulted in substantially smaller germinal centres; the germinal centre cells that survived long exposures to this antigen expressed c~,pical V(DlJ-gene rearrangements that were unlikely to encode hi#Paffinity antibodies. Recent studies by Galibert el a l l 2 demonstrate that CD.10activated germinal centre B cells front human tonsils can be killed by prolonged crossllnking of the light chains of their lg receptors. By contrast, naive or memory B cells were indueod to proliferate by such treatment, indicating that human germinal centre B cells are uniquely sensitive to anligen-mediated apoptosis. Taken together, the~' experiments suggest a novel censoring step v¢ithin germinal centres, whiclt acts by deleting B ceils that bind soluble antigen.
The germinal centre: a primary lymphoid organ? Since Flemming's original dl,~,~criptionof these structnres mort' than 100 years ago~'~. it has k~come incn,,asingly evident that germinM centres an., unique microenvimnments in tile periphery wizen., I,~tsl. tire and negative seh,,ction of B cells can occur. Kel~e el clip ~ have |~tinled out the striking analngy tirol ~.ems to Ix, enzerging I_~tweeu germinal centres alto primary lymphoid organs. EfMrIs aimed at dettnipg the molecules that mediate celluktr interacthuts within ger~ minM centres should eventually provide us with the molecular framework necessary to understand the cmnplex inttnunoregul0f dry loops involvt~| in the fomtation of tile sc~mdary II.cel) rvtx,rloire. It will be important to understand nnt only tile molecular bid. logical rules governing sumatic Ig V-gene hypermutation but al,~ tbe t.xlmplexcell-migrah~, eve, Is inv~tlvvd in the ~lect:ve survival of high-affinity mutants. Given that the memory B cells that nit. mately emerge from the germinal cent~, conshtute a quite dil|erent repertoire from tho,~,, originally ,~.~,ied from the bone marrow, to consider the gemtlnal centres as a primary, lymphoid organ is not at all unreachable. Further questMns about the "choices' that soh:cted germinal centlx, B eells must make after positive ,~lection by FDC.bound antigen ~,main unanswen.xl, 11tereare three aliematiw:s: (I) return to small lymphocyte morpbology and reMin the tx~citx-ulathlg p~.~l as a
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memory B cell; (2) m o v e out of the germinal centre to the splenic red pulp, the bone m a r r o w or some other location for the purposes o1: antibody formation; or (3) return to the d a r k zone so that the whole mutation-selection process can start again. We are only just beginning to face this additional level of complexity.
(1993) l- Exp. Mt'd. 178, 295-307 24 NossaI, G.l.V. and Ada, G.L. (1971) Antigens, Lymphoid Cclls, and the hmmme Response, Academic Press 25 Mandel, T.E., Phipps, R,E, Abbot, A. et al. (1080) ImmumJL ReP. 53, 29-h0 26 MacLennan, LC.M. and Gray, D. 0986) hmmmol. Rei,, 91, 61-85 27 Liu,J.l., Joshua, D.E., Williams, G.T. et al. (19891 Natun. 342, 929-931 Many of the experiments reviewed were periormed at the Hall hstitute. We 28 Liu, YJ., Cairns, J.A., Holder, M.J. et aL (tq~D Eur. I. ballttlnol. 21, thank Prof. J, Kerr for his invaluable insights into the morphology of apop- 1107-1114 relic cells and type B dark cells, and acknowk'dge the critical input of our 29 Arpil~.C., Dechanct, I., Van Kooten, C. el al. (lq95~ Sciesee 26tt,720.-722 colleagues at both the Hall Institute and Immunex. Original work summarized 30 Holder, MJ., Wang, H., Milner, A.E. et al. (lt}93) Elir. [. hmmmal. 23, in this article was supported by the National Health and Mt~iical Research 2~68--Y-~7{ Council, Canberra; by the National Institutt~ el Allergy and Infectious 31 Holder, M.I., Liu. Y'I., Defrance, T., Flares-Rome, L, MacLennan, I,C. Diseases, United States Public Health Service (grant AI-039581; and by the and Gordon, I. (11}91)Int. hnnllmtll.3, 124.'3--1251 32 Ray, K.R., Putterman, C. and Diamtmd, B. (1q9(*) Pn,c. NAtl. Acad. SoL Human Frontier Science Program, principal investigator, PrnL D. Mathis. U. S. A. 03, 2019-2024 33 Shlomchik, M.~., Aucx~in, A.H, Pisetsky, D.S. et aL (1987) Pnlc. NatL Bali Pulendran (l~ptllendrall@iltlillnlll,a',conl) is ill lilinillilex, 5 | Ullit~erAcad, Set. tl, S. A. 84, 9150--9154 sity Street, Seattle. W A 98101, USA; Rasmnary v a n Dr(el is at the Dept 34 Shlomchik, MJ., Marshak-Rothstein, A., Wolfowicz, C.B. et al. (10871 of Pathology aad bnullalohigy, Moaash Medil.al Sdlaol, Alfred I hispihll, Natmt, 328, 805-811 Cilnlnlell'ial Road,/lrahrall, Victoria 3181, AIislralill; G.].V. Nossal is aI 35 Shlomddk, M., Mascelli, M,, Sban, 1t. el al. (lqqO) I. Exp. Mt,d. 171, Tilt' Ikt~lltt,l' illitt Eli.-,1 Hall Ins/i/lib,. P O Rollal Melbollrlle Hospihll, 2/~5-297 li~elbollrllt., VIC 3l)50, Australia. 36 Tilhnan, D.M., lou, N-T,, Itill, R.I. and Marion. T.N. Ilqt)2) I. Exp. Med. 17fi, 7bl-779 37 Diamond. B,. Katz, I,B., Panl, E, Arannlt; C., Lustgarten, D. and References Sdlarlt, M D. (It)t)2) Atom. Rev,Imm,m)L IO, 731-757 1 Ehrlieh. RandMorg, enmth, l.(Iq57)inTheG~lhvtaiPap..rsofPaulEhrlh'h 38 Radic, M.Z. and Weigerl. M. (1q941 Atom. Rel.. hltmitnoL 12, 487-521! (Vol, 21 (Himmelweit, F., t~l.) pp. 246-255. Pergamon 39 Borretzen, M., Randen, I., Zdarsky, E., Forte, O., Natvig, l.B. and 2 Nt~ssaI, G.l.V.(Iq831Annu, Reo, hauumol 1.334,2 Thnmpt~m, K,M. Ilqq41 Pwr, Nail, Aold. Sci. U, 5. A. '~1, 12917-121121 3 Gtn~lnott; C,C., Cyster, I.G.. Harlley, S.B. el al. (lqt~5) A,h.. hlnllllll0l. 59, 40 Linlon, PJ., Decker, D.l. alad Klinman, N.R. (IqStJl G.Il StJ, lO.ltj-10,59 279-368 41 Llnton, P.l., Lo, D,, Lai, L., Thurbecku, G.i. and Kltnman, N.R. (lqq2) 4 Hammerling. G,I,. ,~hr,~lrich. G., Mombnrg. E el al. (lqql) hlntntllal, Rel.. Ela', I. hamulwl 22. 12113-12117 12", 47~,7 42 Dlnt/Is, H.M. and Dintl.is, R.Z (lt)tJ2) Pn~c. NaIL At'ad. Sci. LI, S. A. 89, $ Miller, l.EA.P, andMorahan, G.(l~)2)Amm, Rcv. hmmmnl, lll, 51-~tl 113-117 6 Llnton. P,l.,Rndle, A, andKlinlnan, N.R,(Iqgl)l, humllmll. 140,411qt)-4104 43 Galelli, A, and Charlol, ll. (l~lqtll l. hmmmol, 1,15,23q7-2,105 7 NossaI, G,l.V.(1992)Adl,.hm.mml, 52,283-311 44 Dresser. D,W, (19ti2) hm.lololQ,,y 5, I01-1014 8 Shnkat, K,M, alld Gt~tll'a~; C.C, ll°/)5) Nahln, 375, 334-33~ 43 Chiller, I,M., Hablcld, G.S, antt Weigle, W.O, 11970) Prtw, Nail, Aead. SoL 9 Pulendran, ll., Kanotmrakis, G,. Nouri. S, el at. (Iq~k~iNalmv 375, 331-334 il, S. ,'1. ~5, 551-5~ 10 Ilan0 S., Zhellg, I1.0Dal Porto, l, ¢l at, IIt)llS) ]. I~.,~ll,Ml',l, 1142,ltl.~3-lt~44 46 Nossal, GJ.V. and Karvela,, M. (It)till}Prec. Nail. At:ad. Sct. LI, S, A. 1~7, I I I~tliendralh I!,, Slnilh, K,G,C, alltt Nossal, G,l.V, ( I qtiSi/, hunnlliiil, 15.~, Itll 5-1(~lt,I 1141-11.N! 47 Karvelas. M, and Nossal, G,IN. (Iqtl2~Pr0c. Natl. Acad. SeL LI, S, A, 149, I1 (ialll~t, il, I=,,llurdill, N,, Ilarihelciny, C,¢laL (Iqql,)I. I;xp, Med. 11'13, 3150-3154 21175:2085 48 lack, R.S., ImanlshI.Kari, T, and Ralewsky, K. (Iq77) Em; l. hmmmoL T, 13 MacLenn,nL I.C.M. (It~4) Am,i. ReP. hmnmlal. 12,117-13t) 14 I,Itt, ¥,l. lohn~on. G,D, Gnrdnn. l, and MacLt, lumn, I.C.M. ( IOqZ) 4~ Reth, M., Hammerling, G.I. and Ralewsky, K, (ItJ77) Eur, l, lm,mm#, fl, IIIlllnlnlll. 'llkhl!l 13, 17-21 3ll3-.,I(1(I 1~ laeol% l., Kasslr, R, and Kelsnt,, G, IIt~t)l) I, I!sp, Med, 173, 11f~5-1175 50 Allen, D., Cnnlano, A., Dtldrop, I,L et al, (Iq1"17)hnmmwl. Reo. t)o, 5-22 16 L!u, Y,l,,Zhalltb l., Lane, l~.l.l..el al, (19tlll l'.ur, l, hllmllnal, 21, 51 Nnssal, G.I.V, Knrvelas, M. and Pulendran, It, (lt)t)31 Prac, Nail, Acad. 2@51-2962 ScL 1.1,S, A, ~)0. 3{IblPI-3{lt12 l? Hanna, MG. (l~)b41 I~lb. Im,e~t. 13, qS-104 52 Mcl-leyzer-Williams, M.G.. Nossal, GJ.V. and Lalor, I;A. (Iq911 Nah.'e ill Zhang, I.. MacLt,nnan, I,C.M., Lin, YI. ¢1 al. (lq['lS)hmmmat. Letl. 18, 3511,5112-505 297-2oAI 53 Pulendran, B., Karvelas, M, and NnssaI, G.I.V. l199,11Proc. Nall, Ao~d. 19 lacoh, I., I
JANUARY
1997
.i!.
I M M U N O L O G Y TODAY
58 Strasser,A., Whittingham, S., Vaux, D. etal. (1991) Prec. Na#L Acad. Sd. U, S. A. 88, 8661--8665 59 Nakayama, K., Nakayama, K.. Dust,n, L.B.and Loh. D.V. (1995)#. Ex'p. Med. 182, 1101-1110 60 Kerr,JER., Wyllie.A.H. arid Cure,e. A.R. (1972) Br. I. Cancer 26, 239--257
61 62 63 64 65
Johannisson, E. (1968)Acta EndocrinaL Copellhagen (Suppl. 130)58, ;-107 Harmon, B,V.(1987)[. Pathol. 153,245-355 Bud(z,EE. and Spies, I. (1989)Ceil Tissue/h.s. 256, 17">-486 Flemming, IN. (1885) Arch. Mikrosk. Anat. 24, 50--99 Kelsoe, G. (1996) lmm,nity 4,107-tll
Cytokines, tumour-cell death and immunogenicity: a question of choice Piero Musiani, Andrea Plodesti, Mirella Giovarelli, Federica C~v~llo, Plario P. Colombo, Pier Luigi Lollini and Guido Forni H o w do cytokines released by engmecred tumour cells provake
L't.lour rl'juction dtld all ;.lllllllltll" he way in which tumourence of small amounts of specific exogenous associated antigens (TAAs) cytokines. lift'//fliRt7 IS Ua£cJllatilllt wiHt enter the immune system detlllllOItr ceils t/t•/lauc bel'n termines the features of the ensuing immune response t. Whether or not a engim'ered to secn'h" Cl#hlkillC~ f/ Cy~okine-activated t~mour significant T-cell response will be el,died inhibition t,i~bh, thl'rapeulic tu'r.,;tll'l-lil,e? depends on the level of expression of major Numerous studies in mice have shmvn that Pil'l'll MIl~illlti alld COl/Clll%qlt'~ ]llqi~l" hislocempatibility complex (MHC) glycorepeated ink,ctions of low doses of 11.-1.IL-2, proteins, the availability of the accessory IL4 and interferov ,/(IFN-~) in the turnout Sillt q/It I?ll answi'r hi tltl'sC qlteStiOlt~ and adhesion molecules known to hcilitate I growth area a¢livate a strong antitumour Ill/ [rdlt.~!(,clilt~. till" Slllltl" ltllllOltr costimulationz-~, and the ability of lumour reaction" I~. Complele or partial clinical n.~. with thp ,TcHi's Of UllriOll~ t'l/tOkilli's cells to reach lymphoid tissues~. The feamissions have also been observed in hutures of the reaction an., also controlled by a mans". Similar strong reactions are actb Illid i'htcilDltillq the ~'lllitl('s (if the repertoire of helots and cytokines secreted rated when IL-2, IL-4 and IFN-~ ate tl'lll'tiOlls Iqit'ih'd. by each turnout'; these can modify the rerewatedly inlectod locally or r~lea.sed by sponses of potentially reactive leukocytes cyloktne-gene-engineered (cytoklne-engiand modulate the activity of locally present endothelial and stromal neered) tumour cells ".'~, However, tile histotypo and intrinsic ImcellsL A turnout can also forestall Ihe immune system by mouniing mmlogentcity of a turnout, the kind of extracellular matrix it proa suppression scenario through the secretion of prosiaglandin E: dutx.'s and the el,perle,re of cytokines it ¢lmslituttvely n, lea~,s (Rel. 8), transforming growth factor 13(TGF-I~I", interleukin 10 (IL- markedly modify the local effects of a givelt cylokine ~ "'. As a n.~ult, l0) "~and many olher factors". The cllaracteristics of the immune ix,- dissimilar and varionsly efficacious reaction nlechanisnls that am sponse elicited by the tumour art, a[~ influenced by the local pres- hard in deterlvme are activated when the ,~lne cytnkine Is released
U
Fig, I. Reco,str,ctioll of the cell els,,ts that taAv l,lace at the site of the challen,ge with TS.|-I~ and TSA calls engineered with eytvkiue genes 0is indioited) ,is ,teterlllilled by seqne, tial histological, i,mliolocytLw2w, lical and uttrastruetural oI,sertwtions, Gtouils Of thl~' nlit'~, tl~'rt, chasca randomly and ,~lerifict~l 1.2, 3, 4, 5, 7, 10. 13. 15, 20, 23, 25 and 28 days after the challeu.~e. Speckh/nt~ls. TSA o'lls; sl~rkled ot~ls R,ith black slnils, damaged TSA cells; enrage cells with a n'd auch.us, fibwbhists: n'd cn,'~euts anmnd n~! Slants, t~-g,qqsand n~t calls; n'd cn,~'ents awund a red circle, tlman[glsed ves.~,ls;rcd cres'ceutsaround an ¢mply st~lce,dillliaqei~ Pi.'s.,;els" Ry([cr~*scciltS allillih| r ~ s]tOts and a sla'c/¢h~fel,el ilL.6 patuq), ves.~.ls with a ,wlaslati:i,g tmno,r eel|: llatchcd areas lllarked with a while arrow (IL-IO m,d IFN-a pamqs), marked im~tuclion ig colla,~en fibres. The mellunls used ~lr theg, oba,nsaions hm¢ I,eea dL~crilwd im.viousl9 m detail :~,:::~x; ~:. The amounts of l~tokine pnntuced by | × 10~elkqineered TSA cells cultun.d ~lr 48 h hi I nd oJ mat,me evaluat:d I~y ELISA ilL.6. IL.IO):° or bi(~ls.~y (IL-2, IL.4, IL.7, IFN-a. IFN-I,, TNF.a) as &~crih'd Im, viously ":: ucw: TSA-IL-2 500 U; TSA.IL-4, 40 U; TSA-IL.5. fal U; TSA-IL.6,1250 I,R; TSA.ILL 30 U; TSA.IL-IO, 620 U: TSA-IFN-a, 200 U; TSA-IFN- 7. f~90U: TSA-TNF-,. 10 [.I, Abbreviations: e. ¢osinophil (enrage ci~h,): IFN-ct. illter[ewn ,; IL.2. interleukin 2: L, lyml~ha~, te ¢vhllet circle): M. ,uwnlpha,~e (bhw cell): n, ,e,ln)phil (Sree, cin'h,): uk, nahmtl kilkr cell (mamv owlet; TNF.a. tmnaur rex'rests ~rctor or; TSA-tr, TSA.l~m,ntal cell.
TODAY
Immunological tolerance in germinal centres Bali Pulendran, Rosemary
van
Driel and G.J.V. Hossal
The abiliOl to avoht autoimmunity is a cardinal feature of the immmw I
i,
work of follicular dendritic cells (FDCs) and T cells. FDCs can harbour unprocessed antithis is achieved by several of mature lymphocytes in the gen in the form of immune complexes on mechanisms that exclude self. . . . . . . . . :immune system. One serves their surface for several months24.~. The :o exclude those lymphocytes with noncentrocytes with high affinity for the antireactive h.lmphoqttes f r o m mature productive antigen-receptor rearrangements gen on the FDCs are presumed to be posihdmphocytc n,pertoires. Here, md the other serves to extinguish those tively selected2".a7, and can then differenBali Puh,ndran, Rosemary van DrM [ymphocytes that have acquired self-reactive tiate into memory B cells or into ~ntigen receptors. Evolution seems to place antibody-forming plasma cells-~7-3~,dependand G.I.V. Nossal rt'z,it~t, recent a great premium o~ avoiding the horror ing on the costimulatory signals they restudies indicatiKq that several ceive from the FDCs. Alternatively, the cen~utotoxicus envisaged by EhrliclP in 1901, trocytes are thought to return to the dark and has ingeniously devised a number mechanisms of self-tolerance operate zone and undergo further proliferation and of different contrivances to maintain selfit! gerttlitta| centres. mutation. Those centmcytes that are not tolerance2--q.Many of the negative selection selected by the FDCs are believed to die steps in the generation of B- and T-ceU repertoires occur early in the ontogeny of fl~ese cells in the bone through apoplosislx2~'.-'7.It appears that centrocytes cultured in the marrow and thymus, respectively. However, several additional presence of an antibody against CD40 and IL-4 (Refs 30, 31) or mechanisms of self-tolerance in the periphery have evolved to CD40-1igand, IL-2 and IL-10 (Ref. 29) can survive and acquire the silence mature B cells2"~ and T cells~.~ that have escaped central phenotype of memory B cells. Thus the two processes of mutation tolerance. Recently, there has bi.,enmuch interest in the possibility and selection acting in a coordinated and iterative manner are of there being self-tolerance mechanisms within germinal centres. It thought to give rise to the phenomenon of affinity maturation. has h.,en proposed that such mechanisms exist in order to prevent the emergence of autoreactive cells through somatic hypermutation 3,~'7, Recent studies by various groups indicate that several mechanisms Emergence of autoreactive B cells within germinal eentres opt,rate ill germinal centres to maintain self-toleranee~q-', Given the intense proliferation of the centroblasts, ancl the rapid rate of mutation in the V genes, and considering the large number of self-anUgens in the body, it is likely that the germinal centre Germinal centre development Germinal ~,ntres (Fig. I) are dynamk" mlcroenvlronments uf B-cell differentlathm which arise transiently in tile primary B.cell fullteles of ~ m d a r y lymphoid organs during immune responses~,". The B cells that form germinal centres are initially activated outside the primary follicles: [or example, in the T.cell-rich zones of the perlarlerlo|ar lymphatic sheath (PALS) in the splenic white pulp, in association with T-helper (Thl cells and interdigitating dendritic cells~.1". After this initial activation, the [I cells can either differentiate into anllbt~ty-forming cells of the foci, or migrate into the primary fnllicles and undergo further cloaal expansion and differentiation, giving rise to germinal centres. Once the activated B cells emer the primary follicles, they downregulate their immunoglobnlin fig) receptors and undergo a phase of massive clonal expansion during which they divide every 6--7 hours ~zls. In addition, they activate a site-specific hypermutation mechanism, which rapidly introduces random point m~tations Fig, I. Photomicrographof a section of spleen dmuqug Huegerminal cen-
system. In healthy individuals,
development of the repertoire
into their Ig variable region genes (V genes) w--'~(Fig. 2). These dividing eentroblasts form the 'dark zone' of the germinal centre and continually give rise to nondivlding centrocytes, which upregulate their lg receptors and migrate to the apical region of the germinal centre (the light zone), where they interact with an extensive netPll *aO167,~b99(96)lOObB
slaim~d with pemmt aggh,tinin (PNA) fblue) mid ml iuterw,ning T-cell zone staim'd with anti-CD4 (lavwn). Note that thereare rca.qanable mmlbers of CD4 ~ cells scattered throughout the germinal eentres. Note fio'ther that PNA stains vascular endothelimn and the marginal shins this ca, be useful in ~rientating seatfoas that at,, not comflerstahwd. trex
Copln~hl ~ 1997 [l~ev,cr S¢,erK¢ Lid All ~,C,hl~ reserve0 01hi ~699/9h$~ t'OU
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Fig. 2. The germinal centre reaction showbzg the hnportance of T-cell-Bcell interaction, B-celI-FDC-interaction and apoptosis of B cells in the comph'x cascade of eveuts withbz the gennittal centre. Abbreviations: FDCo follicular dendritic cells; lg, immmzoglobulin; Th, T hellx, r cell, reaction often results in self-reactive B cells. Indeed, several obse-rations illustrate this possibility. A recent study has shmvn that pathogenic autoantibodies that bind doubh.~-stranded (ds) DNA are generated at a high frequency during the immune response to foreign antigen. The autoantlbodies, which were crossreactive with the immunizing antigen, were found to be encoded by somatically mutated V genes and did not arise until day l0 or ll after immunization. These observations suggest that the autoreactive specificity is generated by somatic mutation~-'. Furthermore, the analysis of autoreactive, anti-ds-DNA-binding hybridomas in the autoimmune MgL.Ipr/Ipr or NZB/NZW mice shmvs anti-ds-DNA B cells to be the pn~ducts of extensive somatic mutation and clonal selection. This suggests that, in these shains of mice, such cells persist as a result of a defect in their elimination~ . In a self, rate study it has been shown that healtl~y individuals, unlike subjects with rheumatoid arthritis (RA), do not produce high-affinity rheumatoid factors (RFs) after immunization3~. Moreover, there appears to be a strong selection against replacement of amino acids in the hypervarlable or complementarity.determining region of RFs in healthy individuals. These observations suggest thatRFs from R A patientsescape a tolerance mechanism which norma~y operates in healthy Individuals. 1~L~e and several other experiments are a fo~afful reminder of the possibility of autoreactive B cells arising in the germinal centres. Are there special mechanisms of self-tolerance which prevent this?
Negative selection of B cells w i t h i n germinaJ centres? Several experimenls indicate that negative selection steps occur ~ t h i n germinal centres, and that B cells can be eliminated by anti-
gen even during immune responses. Linton el al. showed that B cells typical of the secondary B-cell lineage iwhich express low levels of Jlld (heat-stable antigen) (ReL 40) and which have the capacity to seed into primary follicles and form germinal centres41] displayed an exquisite sensitivity to tolerization in vitro 6. This was interpreted to mean that B cells unde-rwent a 'second window" of tolerance during their differentiation into pre-memory B cells, presumably within germinal centres, in a separate study, by Dintzis et al. 42, it was shown that when mice undergoing a T-cell dependent immune response to fluoresce.in isothiocyanate (FlTC)-ovalbumin-haptencarrier were injected with FITC coupled with a nonimmunogeneic carrier, the response was suppressed such that much fewer antiFITC antibody-secreting cells were present in the spleens after confinued exposure to this tolerogen. Prompted by these and other observations~, we used the capacity of deaggregated forms of soluble antigen to induce immunologic tolerance in adult mice~.45 to try and explore tolerance within germinal centres. Initial experiments demonstrated that when deaggregated human serum albumin (HSA) was administered as a surrogate se.lf-antigen seven days prior to immunization with HSA, there was a marked reduction in the number of clonable anti-HSAspecific B cells with sufficient affinity to bind to HSA as a bivalent lgG1 dimer, rather than as a decavalent lgM (Ref. 46). Adoptive transfer experiments suggested that both the B- and particularly the T.-cell compartments were afro-ted47. The molecular and histological analysis of this tolerance were facilitated by the use of a hapten-carrier system, in which (4-hydroxy3-nitrophenyi)acetyl (NP) coupled to HSA (NP-HSA) was used as the antigen. The immune response to NP-pmtein conjugates has been well characterized in the C57BL/6 strain of miceI~,t~-'l'~.4~-~. Injections of deaggregated NP-HSA 4-7 days prior to immunization with the alum-precipitated, more highly haptenated form of the same antigen resulted in a profound and persistent state of unresponsivenessSL Hapten-carrier studies again showed a major eb feet in the T-cell compartment, thus confirming the earlier adoptive transfer studiesSL The NP-specific B cells in the spleens of the Immune and tolerant mice were directly identified using a multiparameter flow cytometry system designed to detect the ran, and heterogeneous population of isotype-switched, N[~.spceific cellss2. Such analysis revealed a significant reduction in the NP-bindlng cells of germinal centre origin in the tolerant mice~, again showing that the tolerance resided in B and T cells. This was corroborated by immunohistologic studies in which splenic sections were stained for germinal centers comprising cellq expressing the lg ~ light chain, which are characteristic of the anti-NP r,~spimse in C57BL/6 mice~ ' ~ - ~ . Tliese r~ults were interpreK,d to mean that the tolerance resulted in an impairment of antigen-specific gem~inal centre development both through a T-cell-dependent mechanisms~ and through a direct effect on B cells.
of soluble ont/gcn on an established immune response Interesting|); it was shmvn that a similiar effect could be elicited by the soluble NP-HSA, even when it was given as late as six days
IMMUNOLOGY
after inununization"~L In this case, there was a profound reduction in the high affinity, NP-specific B ceils that could be enumerated at day 14 of the response, in limiting dilution cultures. By contrast, there was only a modest reduction in the number of cells secreting low-affinity antibody. This suggested that soluble antigen iniected six days after immunization had selectively interfered with the development of high-affinity anti-NP cells. This tolerance appeared to be mediated through a direct effect on the B cells and indirectly through Th cells, as a partial reduction could be obtained by injecting soluble HSA, lacking the NP hapten. Further cellular and histological analysis confirmed this, as mice treated with soluble NP-HSA 6 days after immunization developed far fewer NPspecific, germinal centre cells and lg ~.~ germinal centres in their spleens compared with control miceu. However, the tolerant mice did contain large, Ig h - germinal centres, perhaps containing B cells specific for other antigens. In contrast to its effect on NP-specific, germinal centre B cells, the soluble antigen induced the B cells outside the follicles to expand and differentiate into antibody-secreting cells. This was consistent with the much larger lgG1 ÷ loci observed in splenic sections of the tolerant mice. Thus, despite an impairment of NP-specific germinal centre B cells, soluble antigen given after immunization appears to cause F"lyclonal activation of other B cells. This polyclonal activation coulo be mediated through immune complexes operating thwugh the Fc3, receptors on B cells - it is clear that immune complexes can exert stimulatory effects on B cells when formed in antigen-excess~-~, Therefore, soluble antigen given six days after immunization seems to act in two distinct ways: (1) it cau,~es a polyclonal activation of B cells both in rite germinal centre and the antibody-forming arms of the immune response and (2) it causes impairment of antigen-specific, germinal centre B cells.
C l o n a l d e l e t i o n o f B cells in 8 e r m l n a l ¢entres
TODAY
Fig. 3. (a) 0.45 Izm section of a Rerminal ccnhv firm manse ~ ~lee, 4 II after solubh' anti~e,t challc,ge, stained with Toluidine Blue m,~ A=m" il. The cluster of dead cells within the square is shown in (b). ~ 7hmsmisshllt eh,ctn), mictvgraph montage of m~'a selected firm (ok ~howill,~. a cluster of moribund cells partially eaveh)ped by a ma,~ophage. Within this cluster art' profiles of cells in various sta~es of apol,~,sis and prefiles of type B dark cells. (c) "133picaltype B dark cell, showin~: conden. sation of chwmatiu fwithout the 'ereseent' formath)ll seen in ,!~Jptasis), swollen mitochomlria altd endoplassnic reticulum, and cell ,./winka,~e without amnding: ,ore tm~ccsst~ I~'lu~'en ~d/awnt cells, This cell i~ ~hreeceU I~lx~filesaway fn,,i m0,tagt' ([p}.fd) l~lpicat early at,)l~lOsts: hi,~l, ma.~nifioltiott of con ill h)ll,er box/11(ll)l Ilotc holllO,~,t'llOUSa.ldet~sathm of c']lro. matin, and ~;m.~Mtcell tin,file,/e) High ma,~laificatton of area e.~losed by Uplwr box in (b), showi.g a t,lpe I] dark cell Imlfik (ri,~hl) adi.~.e.t to a partially digestedat~oplollcI.~dy (h,fl), All barscorrespondto 2~'~,I~m.
What is the medlanlsm of Impairment of germinal centre cells? Does the soluble anllt4en kill the B cells or does It make them anergic? D ~ it mediate its effect(s) directly on the B cells or through some other mechanism Ittvolving Tit cells? To explore these questions and to examine the sensitivity of germinal centre II cells to tl~is spike in the number of apuptotlc cel]s was antlgen-sI, cific, as apuptosls we used the TUNEL technique~L C57BL/6 mice were im- it coukl not be induced by injecting an irrelevant protei. ~uc~ as munized with NP-HSA and 14 days later, at the height of the im- transferrln. Further studies on germinal centre apoptosis w~re pernmlse n.,sDOllbe(whvll the germinal centre reaction was at its peak), formed using soluble carrier alone and the NP hapten attached to the mice were injected with 5 mg of soluble NI)~-HSA. Tile aim was the self-antigen, mouse serum albumin (MSA). These supported the to confrullt the germinal centre cent~t~cytes with ,~luble antigen be- view that the dominant effect was a direct killing of the haptenlure they could gain access [o the antigen displayed on the surface specific B cells. Furtbermore, this apoptosis was unique to germiof FDCs. At various inlervals after this injection (ranging from nal centres, as no other areas in the spleens (such as loci) e×hibited 10 rain to 24 h) tile mice were killed and the spleens removed and any noticeable increase in apoptosts'j. When tbe experiments were performed in bcl-2 transgenic mice~, analysed for apoptosis using tile TUNEL assay. The soluble antigen induced a rapid and dramatic increase in the number of TUNEL' which constitutively expressed tile anti-apoptotic bcl-2 transgene in cells within germinal centres, which peaked at 4--6 h, before declin- all B cells, there was only a partial inhibition of the soluble antigening to base-line levels by 24 h (Ref. 9). One hour after injection there induced apoptosls'j. By contrast, apoptosis which spontaneously ocwas already a significant increa~ in apoptotic cells. After 12 h, ex- curs within germinal centres during a normal immune respn,se (and tensive al~ptotic debris could be seen within macrophages, some which presumably reflects the death of centrocytes thai fail to be seof which appeared to be migrating out to the red pulp. Moreover, lected by FDC-held antigen 27) was effectively inhibited by bcl-2.
!
This is in agreement with experimentss~that demonstrate a significant increase in the spontaneous apoptesis of mature B celts in bcl2-deficient mice. Thus, it appears that there is a fundamental difference in the mechanism between the two forms of apoptosis: (D the spontaneous, "background' apoptosis (which presumably reflects the rather passive 'death by neglect' in normal germinal centre functioning) being effectively inhibited by bcl~; and (2) the active process that might be at work in soluble antigen-induced apoptosis being only partially inhibited by bcl-2. Ultrastructural analysis of soluble-antigen-induced apoptosis revealed clusters of apoptotie ceils throughout germinal centres (Figs 3a and b). Such cells displayed the morphologically distinctive features of apeptosis°°, such as the striking reduction in cell and nuclear volume (Figs 3b and dl which accompanies the margination of the chromatin to form dense granular caps under the nuclear membrane. In contrast to the cytoplasmic organelles of necrotic cells, those of apoptotic cells appeared to be weU preserved, apart from the dilation of the endoplasmic reticulum. Cells with surface blebbing and nuclear disintegration (which typify the advanced stages of apopinsis) were also apparent (Figs 3b and el.
Type B dark cells In addition to classically apoptotic cells, we observed cells that were clearly moribund and yet mnrphologically quite distinctive from either apoptotic or nt.x-roticceils (Figs 3b, t. and O. These possess the characteristic features of 'type B dark cells "l'°-~ The condensed chvamatin in such cells does not have the smoothly contoured inner edt~es characteristic of apoptosis, and there is no radical redistribution of the chix~matin. Although there is overall cytoplasmic coltdeusation, tile mitochondria and endop]asmic reticulum tend to be swollen (unlike those in the type A dark cells)~. In addition, the cytoplasm is stretched out in the form of attenuated processes Ix~ tween adj,~cent cells, a feature that is quite unlike the budding to produce round and oval bodies, which o.-curs during apoptosls. Although dark cells have been implicated in ,,'-elldeath, to the Ix,st of our knowh,,dge there have been no studies desigmM to determine whether DNA is cleaved during their formatimz, and if ~ whether cleavage is random or selective. "Pype B dark cells have b~en observed in tomours where the turnout cells were most-ck~ely packed together, suggesting thnt cron,ding contributes to their development"~, It is tentptin~3to specu!ate whe!l'er their (.vo'urrence in germinal centrps could [~.,attributed to this reason. Similiar data have been obtaizu.~db~ Shoktt and Goodnow~, who develol:,.~t a system to track hen egg lysozyme (HELl.specific B tells fp_~mtrm~,enic mk'e, after seedtng them into the developing germinal centres hi: nnatmnsgenic mice immunized with duck egg ly,~zyme (DELl, The ,,~xted B ceils were identified by a unique LySa allutypic m;irker, attd were ~,en to multiply rapidly over the next five days, accuunting for m ~ t of the |g-k,aring B cells within the germinal centres. To model the fate of serf-reactive variants that ari~ in germinal cenm.,s, 5 mg of ,~oluble HEL 'was in~:ttM into the mice, and the sph.,ens analysed for apeptosis nsing rite TUNEL as~y. Therefor~ in rids model the germinal centre B cells were con................ .........................................................
fronted witll an antigen that lacked T-cell help (since DEL and HEL do not crossreact at the Th-cell level). Within 4 h, large numbers of apoptotic cells were seen within germinal centres, and the HELspecific B cells disappeared from the light zones of the germinal centres. Surprisingly, it was observed that some HEL-specific B cells relocated themselves to the T-cell zone, persisted for a while and then died eight b,ours later. Bd-2 could not protect against apoptosis within the germinal centre.s, but could prevent the death in the T-cell zone, suggesting a mechanistic difference between the two forms of apoptosis. Further supporting data have been obtained by Hart et al. I(~,who injected protein conjugates of (4-hydroxy-B-iodo-3-nitrophenyl}acetyl (NIPI into mice previously immunized with NP-.-chicken gamma-globulin (CGG). As in the previous hvo studies, a single injection of soluble antigen induced a large increase in apoptosis in germinal centres without significantly decreasing their numbers or average size. However, repeated injections over an 18 h period resulted in substantially smaller germinal centres; the germinal centre cells that survived long exposures to this antigen expressed c~,pical V(DlJ-gene rearrangements that were unlikely to encode hi#Paffinity antibodies. Recent studies by Galibert el a l l 2 demonstrate that CD.10activated germinal centre B cells front human tonsils can be killed by prolonged crossllnking of the light chains of their lg receptors. By contrast, naive or memory B cells were indueod to proliferate by such treatment, indicating that human germinal centre B cells are uniquely sensitive to anligen-mediated apoptosis. Taken together, the~' experiments suggest a novel censoring step v¢ithin germinal centres, whiclt acts by deleting B ceils that bind soluble antigen.
The germinal centre: a primary lymphoid organ? Since Flemming's original dl,~,~criptionof these structnres mort' than 100 years ago~'~. it has k~come incn,,asingly evident that germinM centres an., unique microenvimnments in tile periphery wizen., I,~tsl. tire and negative seh,,ction of B cells can occur. Kel~e el clip ~ have |~tinled out the striking analngy tirol ~.ems to Ix, enzerging I_~tweeu germinal centres alto primary lymphoid organs. EfMrIs aimed at dettnipg the molecules that mediate celluktr interacthuts within ger~ minM centres should eventually provide us with the molecular framework necessary to understand the cmnplex inttnunoregul0f dry loops involvt~| in the fomtation of tile sc~mdary II.cel) rvtx,rloire. It will be important to understand nnt only tile molecular bid. logical rules governing sumatic Ig V-gene hypermutation but al,~ tbe t.xlmplexcell-migrah~, eve, Is inv~tlvvd in the ~lect:ve survival of high-affinity mutants. Given that the memory B cells that nit. mately emerge from the germinal cent~, conshtute a quite dil|erent repertoire from tho,~,, originally ,~.~,ied from the bone marrow, to consider the gemtlnal centres as a primary, lymphoid organ is not at all unreachable. Further questMns about the "choices' that soh:cted germinal centlx, B eells must make after positive ,~lection by FDC.bound antigen ~,main unanswen.xl, 11tereare three aliematiw:s: (I) return to small lymphocyte morpbology and reMin the tx~citx-ulathlg p~.~l as a
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memory B cell; (2) m o v e out of the germinal centre to the splenic red pulp, the bone m a r r o w or some other location for the purposes o1: antibody formation; or (3) return to the d a r k zone so that the whole mutation-selection process can start again. We are only just beginning to face this additional level of complexity.
(1993) l- Exp. Mt'd. 178, 295-307 24 NossaI, G.l.V. and Ada, G.L. (1971) Antigens, Lymphoid Cclls, and the hmmme Response, Academic Press 25 Mandel, T.E., Phipps, R,E, Abbot, A. et al. (1080) ImmumJL ReP. 53, 29-h0 26 MacLennan, LC.M. and Gray, D. 0986) hmmmol. Rei,, 91, 61-85 27 Liu,J.l., Joshua, D.E., Williams, G.T. et al. (19891 Natun. 342, 929-931 Many of the experiments reviewed were periormed at the Hall hstitute. We 28 Liu, YJ., Cairns, J.A., Holder, M.J. et aL (tq~D Eur. I. ballttlnol. 21, thank Prof. J, Kerr for his invaluable insights into the morphology of apop- 1107-1114 relic cells and type B dark cells, and acknowk'dge the critical input of our 29 Arpil~.C., Dechanct, I., Van Kooten, C. el al. (lq95~ Sciesee 26tt,720.-722 colleagues at both the Hall Institute and Immunex. Original work summarized 30 Holder, MJ., Wang, H., Milner, A.E. et al. (lt}93) Elir. [. hmmmal. 23, in this article was supported by the National Health and Mt~iical Research 2~68--Y-~7{ Council, Canberra; by the National Institutt~ el Allergy and Infectious 31 Holder, M.I., Liu. Y'I., Defrance, T., Flares-Rome, L, MacLennan, I,C. Diseases, United States Public Health Service (grant AI-039581; and by the and Gordon, I. (11}91)Int. hnnllmtll.3, 124.'3--1251 32 Ray, K.R., Putterman, C. and Diamtmd, B. (1q9(*) Pn,c. NAtl. Acad. SoL Human Frontier Science Program, principal investigator, PrnL D. Mathis. U. S. A. 03, 2019-2024 33 Shlomchik, M.~., Aucx~in, A.H, Pisetsky, D.S. et aL (1987) Pnlc. NatL Bali Pulendran (l~ptllendrall@iltlillnlll,a',conl) is ill lilinillilex, 5 | Ullit~erAcad, Set. tl, S. A. 84, 9150--9154 sity Street, Seattle. W A 98101, USA; Rasmnary v a n Dr(el is at the Dept 34 Shlomchik, MJ., Marshak-Rothstein, A., Wolfowicz, C.B. et al. (10871 of Pathology aad bnullalohigy, Moaash Medil.al Sdlaol, Alfred I hispihll, Natmt, 328, 805-811 Cilnlnlell'ial Road,/lrahrall, Victoria 3181, AIislralill; G.].V. Nossal is aI 35 Shlomddk, M., Mascelli, M,, Sban, 1t. el al. (lqqO) I. Exp. Mt,d. 171, Tilt' Ikt~lltt,l' illitt Eli.-,1 Hall Ins/i/lib,. P O Rollal Melbollrlle Hospihll, 2/~5-297 li~elbollrllt., VIC 3l)50, Australia. 36 Tilhnan, D.M., lou, N-T,, Itill, R.I. and Marion. T.N. Ilqt)2) I. Exp. Med. 17fi, 7bl-779 37 Diamond. B,. Katz, I,B., Panl, E, Arannlt; C., Lustgarten, D. and References Sdlarlt, M D. (It)t)2) Atom. Rev,Imm,m)L IO, 731-757 1 Ehrlieh. RandMorg, enmth, l.(Iq57)inTheG~lhvtaiPap..rsofPaulEhrlh'h 38 Radic, M.Z. and Weigerl. M. (1q941 Atom. Rel.. hltmitnoL 12, 487-521! (Vol, 21 (Himmelweit, F., t~l.) pp. 246-255. Pergamon 39 Borretzen, M., Randen, I., Zdarsky, E., Forte, O., Natvig, l.B. and 2 Nt~ssaI, G.l.V.(Iq831Annu, Reo, hauumol 1.334,2 Thnmpt~m, K,M. Ilqq41 Pwr, Nail, Aold. Sci. U, 5. A. '~1, 12917-121121 3 Gtn~lnott; C,C., Cyster, I.G.. Harlley, S.B. el al. (lqt~5) A,h.. hlnllllll0l. 59, 40 Linlon, PJ., Decker, D.l. alad Klinman, N.R. (IqStJl G.Il StJ, lO.ltj-10,59 279-368 41 Llnton, P.l., Lo, D,, Lai, L., Thurbecku, G.i. and Kltnman, N.R. (lqq2) 4 Hammerling. G,I,. ,~hr,~lrich. G., Mombnrg. E el al. (lqql) hlntntllal, Rel.. Ela', I. hamulwl 22. 12113-12117 12", 47~,7 42 Dlnt/Is, H.M. and Dintl.is, R.Z (lt)tJ2) Pn~c. NaIL At'ad. Sci. LI, S. A. 89, $ Miller, l.EA.P, andMorahan, G.(l~)2)Amm, Rcv. hmmmnl, lll, 51-~tl 113-117 6 Llnton. P,l.,Rndle, A, andKlinlnan, N.R,(Iqgl)l, humllmll. 140,411qt)-4104 43 Galelli, A, and Charlol, ll. (l~lqtll l. hmmmol, 1,15,23q7-2,105 7 NossaI, G,l.V.(1992)Adl,.hm.mml, 52,283-311 44 Dresser. D,W, (19ti2) hm.lololQ,,y 5, I01-1014 8 Shnkat, K,M, alld Gt~tll'a~; C.C, ll°/)5) Nahln, 375, 334-33~ 43 Chiller, I,M., Hablcld, G.S, antt Weigle, W.O, 11970) Prtw, Nail, Aead. SoL 9 Pulendran, ll., Kanotmrakis, G,. Nouri. S, el at. (Iq~k~iNalmv 375, 331-334 il, S. ,'1. ~5, 551-5~ 10 Ilan0 S., Zhellg, I1.0Dal Porto, l, ¢l at, IIt)llS) ]. I~.,~ll,Ml',l, 1142,ltl.~3-lt~44 46 Nossal, GJ.V. and Karvela,, M. (It)till}Prec. Nail. At:ad. Sct. LI, S, A. 1~7, I I I~tliendralh I!,, Slnilh, K,G,C, alltt Nossal, G,l.V, ( I qtiSi/, hunnlliiil, 15.~, Itll 5-1(~lt,I 1141-11.N! 47 Karvelas. M, and Nossal, G,IN. (Iqtl2~Pr0c. Natl. Acad. SeL LI, S, A, 149, I1 (ialll~t, il, I=,,llurdill, N,, Ilarihelciny, C,¢laL (Iqql,)I. I;xp, Med. 11'13, 3150-3154 21175:2085 48 lack, R.S., ImanlshI.Kari, T, and Ralewsky, K. (Iq77) Em; l. hmmmoL T, 13 MacLenn,nL I.C.M. (It~4) Am,i. ReP. hmnmlal. 12,117-13t) 14 I,Itt, ¥,l. lohn~on. G,D, Gnrdnn. l, and MacLt, lumn, I.C.M. ( IOqZ) 4~ Reth, M., Hammerling, G.I. and Ralewsky, K, (ItJ77) Eur, l, lm,mm#, fl, IIIlllnlnlll. 'llkhl!l 13, 17-21 3ll3-.,I(1(I 1~ laeol% l., Kasslr, R, and Kelsnt,, G, IIt~t)l) I, I!sp, Med, 173, 11f~5-1175 50 Allen, D., Cnnlano, A., Dtldrop, I,L et al, (Iq1"17)hnmmwl. Reo. t)o, 5-22 16 L!u, Y,l,,Zhalltb l., Lane, l~.l.l..el al, (19tlll l'.ur, l, hllmllnal, 21, 51 Nnssal, G.I.V, Knrvelas, M. and Pulendran, It, (lt)t)31 Prac, Nail, Acad. 2@51-2962 ScL 1.1,S, A, ~)0. 3{IblPI-3{lt12 l? Hanna, MG. (l~)b41 I~lb. Im,e~t. 13, qS-104 52 Mcl-leyzer-Williams, M.G.. Nossal, GJ.V. and Lalor, I;A. (Iq911 Nah.'e ill Zhang, I.. MacLt,nnan, I,C.M., Lin, YI. ¢1 al. (lq['lS)hmmmat. Letl. 18, 3511,5112-505 297-2oAI 53 Pulendran, B., Karvelas, M, and NnssaI, G.I.V. l199,11Proc. Nall, Ao~d. 19 lacoh, I., I
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58 Strasser,A., Whittingham, S., Vaux, D. etal. (1991) Prec. Na#L Acad. Sd. U, S. A. 88, 8661--8665 59 Nakayama, K., Nakayama, K.. Dust,n, L.B.and Loh. D.V. (1995)#. Ex'p. Med. 182, 1101-1110 60 Kerr,JER., Wyllie.A.H. arid Cure,e. A.R. (1972) Br. I. Cancer 26, 239--257
61 62 63 64 65
Johannisson, E. (1968)Acta EndocrinaL Copellhagen (Suppl. 130)58, ;-107 Harmon, B,V.(1987)[. Pathol. 153,245-355 Bud(z,EE. and Spies, I. (1989)Ceil Tissue/h.s. 256, 17">-486 Flemming, IN. (1885) Arch. Mikrosk. Anat. 24, 50--99 Kelsoe, G. (1996) lmm,nity 4,107-tll
Cytokines, tumour-cell death and immunogenicity: a question of choice Piero Musiani, Andrea Plodesti, Mirella Giovarelli, Federica C~v~llo, Plario P. Colombo, Pier Luigi Lollini and Guido Forni H o w do cytokines released by engmecred tumour cells provake
L't.lour rl'juction dtld all ;.lllllllltll" he way in which tumourence of small amounts of specific exogenous associated antigens (TAAs) cytokines. lift'//fliRt7 IS Ua£cJllatilllt wiHt enter the immune system detlllllOItr ceils t/t•/lauc bel'n termines the features of the ensuing immune response t. Whether or not a engim'ered to secn'h" Cl#hlkillC~ f/ Cy~okine-activated t~mour significant T-cell response will be el,died inhibition t,i~bh, thl'rapeulic tu'r.,;tll'l-lil,e? depends on the level of expression of major Numerous studies in mice have shmvn that Pil'l'll MIl~illlti alld COl/Clll%qlt'~ ]llqi~l" hislocempatibility complex (MHC) glycorepeated ink,ctions of low doses of 11.-1.IL-2, proteins, the availability of the accessory IL4 and interferov ,/(IFN-~) in the turnout Sillt q/It I?ll answi'r hi tltl'sC qlteStiOlt~ and adhesion molecules known to hcilitate I growth area a¢livate a strong antitumour Ill/ [rdlt.~!(,clilt~. till" Slllltl" ltllllOltr costimulationz-~, and the ability of lumour reaction" I~. Complele or partial clinical n.~. with thp ,TcHi's Of UllriOll~ t'l/tOkilli's cells to reach lymphoid tissues~. The feamissions have also been observed in hutures of the reaction an., also controlled by a mans". Similar strong reactions are actb Illid i'htcilDltillq the ~'lllitl('s (if the repertoire of helots and cytokines secreted rated when IL-2, IL-4 and IFN-~ ate tl'lll'tiOlls Iqit'ih'd. by each turnout'; these can modify the rerewatedly inlectod locally or r~lea.sed by sponses of potentially reactive leukocytes cyloktne-gene-engineered (cytoklne-engiand modulate the activity of locally present endothelial and stromal neered) tumour cells ".'~, However, tile histotypo and intrinsic ImcellsL A turnout can also forestall Ihe immune system by mouniing mmlogentcity of a turnout, the kind of extracellular matrix it proa suppression scenario through the secretion of prosiaglandin E: dutx.'s and the el,perle,re of cytokines it ¢lmslituttvely n, lea~,s (Rel. 8), transforming growth factor 13(TGF-I~I", interleukin 10 (IL- markedly modify the local effects of a givelt cylokine ~ "'. As a n.~ult, l0) "~and many olher factors". The cllaracteristics of the immune ix,- dissimilar and varionsly efficacious reaction nlechanisnls that am sponse elicited by the tumour art, a[~ influenced by the local pres- hard in deterlvme are activated when the ,~lne cytnkine Is released
U
Fig, I. Reco,str,ctioll of the cell els,,ts that taAv l,lace at the site of the challen,ge with TS.|-I~ and TSA calls engineered with eytvkiue genes 0is indioited) ,is ,teterlllilled by seqne, tial histological, i,mliolocytLw2w, lical and uttrastruetural oI,sertwtions, Gtouils Of thl~' nlit'~, tl~'rt, chasca randomly and ,~lerifict~l 1.2, 3, 4, 5, 7, 10. 13. 15, 20, 23, 25 and 28 days after the challeu.~e. Speckh/nt~ls. TSA o'lls; sl~rkled ot~ls R,ith black slnils, damaged TSA cells; enrage cells with a n'd auch.us, fibwbhists: n'd cn,'~euts anmnd n~! Slants, t~-g,qqsand n~t calls; n'd cn,~'ents awund a red circle, tlman[glsed ves.~,ls;rcd cres'ceutsaround an ¢mply st~lce,dillliaqei~ Pi.'s.,;els" Ry([cr~*scciltS allillih| r ~ s]tOts and a sla'c/¢h~fel,el ilL.6 patuq), ves.~.ls with a ,wlaslati:i,g tmno,r eel|: llatchcd areas lllarked with a while arrow (IL-IO m,d IFN-a pamqs), marked im~tuclion ig colla,~en fibres. The mellunls used ~lr theg, oba,nsaions hm¢ I,eea dL~crilwd im.viousl9 m detail :~,:::~x; ~:. The amounts of l~tokine pnntuced by | × 10~elkqineered TSA cells cultun.d ~lr 48 h hi I nd oJ mat,me evaluat:d I~y ELISA ilL.6. IL.IO):° or bi(~ls.~y (IL-2, IL.4, IL.7, IFN-a. IFN-I,, TNF.a) as &~crih'd Im, viously ":: ucw: TSA-IL-2 500 U; TSA.IL-4, 40 U; TSA-IL.5. fal U; TSA-IL.6,1250 I,R; TSA.ILL 30 U; TSA.IL-IO, 620 U: TSA-IFN-a, 200 U; TSA-IFN- 7. f~90U: TSA-TNF-,. 10 [.I, Abbreviations: e. ¢osinophil (enrage ci~h,): IFN-ct. illter[ewn ,; IL.2. interleukin 2: L, lyml~ha~, te ¢vhllet circle): M. ,uwnlpha,~e (bhw cell): n, ,e,ln)phil (Sree, cin'h,): uk, nahmtl kilkr cell (mamv owlet; TNF.a. tmnaur rex'rests ~rctor or; TSA-tr, TSA.l~m,ntal cell.