- Email: [email protected]
B-cell differentiation in vivo
104
mechanism of selection theory. eagerly awaited.
cytotoxic lymphocytes are expressed on normal and transformed lymphoid cells, fibroblasts and peritoneal exudate cells. In a complementary paper, in the same issue, Risser and Crunwald describe the production of cytotoxic anti-self FI-2 antibodies by Fl mice hyper-immunized with a parental Abelson virus-induced lymphoma. Since most of the mice repeatedly challenged with the tumour do not survive, it is difficult to assess whether the production of such anti-self antibodies is associated with the development of an autoimmune or other pathological lesions. Nevertheless, there is a clear demonstration in these two papers of the production of cytotoxic T lymphocytes and cytotoxic antibodies by Fl hybrid animals against their own self antigens. The mechanism by which a vertebrate animal produces antibody diversity’” and distinguishes between self and non-self, especially in an Fl hybrid, is at present unknown. The results discussed here, taken with those in previous reports, present a serious challenge to the
self tolerance proposed in the New paradigms in immunology
clonal are
References 1 Kuhn, T. S. (1962) ‘7% Slrurlw/, uj S~1(‘7112/i( KPnolullonl University of Chicago Press. 2 Nakano, K., Nakamura, I. and Cudkowicz, G. (1981) jV&rr (Imdm) 289, 559-563 3 Risscr, R. and Grunwald, D. J. (1981) .Natzrre (l,wu/onj 289, 563-568 4 Burnet, F. M. (1959) T/u, (:lorinl Srlrition Thmry o/ Arqud Immunz~y Cambridge University Press, London 5 Snell, C. D. (195S)J. .N&/. (:nnc~r In.\l., 20, 787-824 6 Cudkowicz, G. and Stimpfling, J. H. (1964) ~V&rw (Lon&l 204,450-453 7 Adler, W. H., Takiguchi, ‘I‘., Marsh, B. and Smith, R. T. (1970) J. Zrrimitnol., 105, 984-l 000 8 Subramaniam, R. and Ebringer, A. (1978) Hzoriwm. G. Tmniac., 6, 1079-1081 9 Ebringer, A., Deacon, N. ~J. and Young, C. R. (1976) j’. Imrruini~grnul. 3, 401-409 10 Ebringer, A. (1975) .7. 7hmr. Hu~i., 5 1, 293-302
mpii--)
B-cell differentiation
in uiuo
Paul Nieuwenhuis Department
of Histology,
Kijksuniversiteit
Groningen,
The
immunologically competent cell Only twenty five years ago, in 1956, a lymphocyte was defined as ‘. a somewhat inconspicuous cell with no particularly striking functional or morphological characteristics of its own’; ‘... it has been and still is the cell around which a violent haematological controversy has been waged”. In his Croonian Lecture, in 1958, Medawars introduced the term ‘Immunologically competent cell, that is, a cell which is fully qualified to undertake an immunological response’ whereas, in 1963, at a Ciba Foundation Study group he added: ‘I think we can
This review draws heavily on three papers (Kefs 11, 19, 23) presented by D. G. Osmond (section 3), G.C.B. Klaus (section 5) and the author (section 6) at the CIBA Foundation Symposium no. 84 on ‘Micro-environments in haemopoietic and lymphoid differentiation’. Proceedings of this symposium will shortly be published by Pitman Medical Ltd, London. No attempt has been made to fully cover the increasingly cornplex field of B cell differentiation, of which excellent reviews have appeared in previous issues of 1rruruu/o/r~~1~ 7urlo~ (J. M. Adams (1980) 1, 10 and J. W. Schrader (1981) 2, 7).
9713
EZ Groningen,
‘l’he Netherlands
now say there is such a thing as an immunologically competent cell and that it is a lymphocyte.. .“‘. In subsequent years it became evident that not every lymphocyte could perform in the various types of immune responses and by 1970 it was clear that at least two distinct subpopulations appeared to exist: thymus-derived T lymphocytes and non-thymus derived B lymphocytes”. T cells, although originating in bone marrow, undergo a process of proliferation and differentiation in the thymus, a well structured organ, possibly providing the essential micro-environment to permit or induce the maturation process. B cells seem to lack a comparably structured micro-environment, at least in mammals, an absence that has not facilitated the study of B-cell differentiation in oinr~. Origin of B cells During ontogeny poiesis are found cytoplasmic IgM IgM(sIgM) - and
early signs of H lymphocytothe liver’ where first cells with (cIgM) and later surface C3 - receptor positive cells can be
in
IO5
detected. Recent observations by Melchers” indicate that in the placenta B lymphocytopoiesis takes place even earlier. In postnatal life the majority of B lymphocytes should derive from the bone marrow @Ml. Chromosome marker studies, e.g. by Abramson et al.‘, have shown that - like other blood cells - all lymphoid cells derive from pluripotent haemopoietic stem cells and that for the T-cell lineage a committed T-cell precursor may exist. No such cell has been identified for the B-cell lineage, suggesting a direct descendencc from the stem cell pool. One might speculate that this relates to the need for pre-T cells to migrate to the thymus for further processing, whereas early phases of Bcell differentiation may occur locally in the BM microenvironment. Whether the BM microenvironment is essential for B lymphocytopoiesis is uncertain: experiments by Rozing et al. 8 indicate that bone-marrow function in this respect can be taken over by the spleen. In the chicken, B lymphocytopoiesis has been postulated to be dependent upon the presence of the Bursa of Fabricius”. Althoug!i it is quite clear that at a certain stage during ontogeny removal of the Bursa of Fabricius can lead to the development of a completely B-cell-less chicken, it is equally clear that when removal of the bursa after the 7th day post hatching is followed by near lethal whole-body X irradiation, a normal B-cell system will regenerate in the absence of the bursa of Fabricius”‘. Whether and how, in the chicken, the bone marrow contributes to the B lymphocyte pool is beyond the scope of this review. Suffice it to say, that the remarks above at least illustrate the rather erratic pattern of B lymphocytopoiesis during ontogeny and the apparent absence of a fixed relation to a specific micro-environment. B lymphocytopoiesis
in bone
marrow
From experiments by Osmond and co-workers (for references see Ref.ll), performed in both mice and guinea pigs, there is evidence that B cells are not only BM-derived, but that at least the earlier differentiation stages actually take place in the marrow cavity (+ stroma) itself. The first sign of B-lymphocyte differentiation is :he appearance of cytoplasmic IgM, which is detected in about 30% of all small lymphocytes present in BM. Another 50% express readily detectable surface IgM molecules. By these criteria, some 80% of the marrow small lymphocytes are of the B-cell lineage. [3H]thymidine labelling experiments have shown that the vast majority (over 90%) of marrow B lymphocytes are rapidly renewed indigenous cells. Eventually these still immature B cells leave the BM to migrate to the spleen and other sites. At the same time surface IgM increases in density and other markers such as Fc receptors and Ia antigens appear, followed by C3 receptors and a low to medium density of IgD (see Fig. 1). These later maturation stages seem
BONE Stem a
Commited
cells
MARROW Droaenltors _
LYMPH010 1
Moturlno
CELLS cells -
Functlonol
cells
I Large
lymphold Cytoplosmlc
cells p
Small
lymphocytes’
EZZI Surface IgM [ +IgM+IgD Fc 8 C3 receptors] 10 ontlqens
B Lymphocyte
0 -----* c
-f\ ’ 12 \’ ,’
Slowly
Fig. 1. Schdme of B lymphocytopoiesis in marrow. With slight modifications from Ref. 12 by permission Press Inc.
renewed
mouse
bone
of Academic
to be independent of micro-environmental influences of either marrow or spleen because they can also proceed in z&o. In addition to the population of small lymphocytes, there are rapidly dividing large lymphoid cells in the marrow, of which 6OY0 are cIgM posit& and thus preB cells. Large lymphoid cells have never been found to sIg. Attempts are being made, using express monoclonal antibodies, to identify intermediate stages between the pluripotent stem cell and the pre-B cells (see Refs 13 and 14). Micro-environmental
aspects
Microscopically, in transverse sections, the marrow parenchyma is arranged between blood vessels, which radiate from longitudinally running central arterioles to an elaborate subendosteal capillary plexus. Venous sinuses converge to the central venous sinus. After continuous [3H]-thymidine infusion for varying periods of time the highest incidence of labelled nucleated cells was found in the immediately subendosteal region (47% after 1 day; 71% after 4 days) decreasing towards the centre of the marrow (20.5% after one day; 54.7% after 4 days). In contrast, when frozen sections of marrow were studied to detect sIgM positive cells, the highest incidence of small lymphoid cells showing a membrane ring fluorescence was found towards the marrow centre. These and other data (not presented here) ‘are consistent with a centripetal movement of maturing B lymphocytes from peri-
106
pherally situated progenitors and their release into adjacent venous sinuses at various stages en route” ‘. Local concentrations of fluorescing cells were not detected. This might indicate that local microenvironmental factors - if operative - act at earlier stages of differentiation, perhaps in the immediate subendosteal region, possibly by influencing the localisation of (pre-B-)precursor cells. Whether this micro-environmental influence is associated with the cortical bone or its associated stromal cells is uncertain and perhaps even unlikely, because Rozing et ~1.~ have shown that, by eliminating haemopoiesis from the BM with “‘Sr, normal follicular structures develop in the spleen of mice after lethal irradiation and reconstitution with BM cells. Regulation of BM lymphocyte production Apart from local micro-environmental factors, other - exogenous - regulatory factors might affect marrow B lymphocytopoiesis: e.g. thymus hormones, peripheral B-lymphocyte pool size and antigen stimulation. In neonatally thymectomized as well as congenitally athymic nude mice, BM lymphocyte production shows virtually the same characteristics (turnover, proportion of IgM-bearing cells) as in normal controls, indicating the independence of BM B lymphocytopoiesis from either thymic hormones or (levels of) recirculating T cells. Depletion of the circulating Blymphocyte pool also had no marked effect on the various stages of B lymphocytopoiesis occurring in BM. For example, repeated treatment of mice with anti-IgM serum after birth led to a severe depletion of circulating B cells as well as BM sIg-positive cells, but the population of cIgM-positive cells did not seem to be affected. Depletion of the circulating B-lymphocyte pool by partial body irradiation and shielding of the marrow in two hind limbs (guinea pigs), did not induce any change in the number of small lymphocytes in the shielded marrow. In contrast, the granulocyte precursor population reacted with a burst of expansion. From these data, Osmond et al. concluded ‘Apparently the production of marrow small lymphocytes is independent of feedback control from the B Regulation in these experiments lymphocyte pool”‘. thus seems to result from local microenvironmental influences. In mice, raised under germfree conditions from birth, the proportion of small lymphocytes and IgMbearing cells was normal. However, the rate at which these cells are produced is strikingly reduced, with prolongation of the half turnover time from 32 to 125 h. On the other hand, after iv. injection of sheep red cells into conventionally reared animals marrow lymphocyte production increases. In conclusion, it seems fair to say that BM B lymphocytopoiesis ‘represents the summation of two steps: (1) a basal level regulated
immunolu,~y 1uday,Junr
11)x1
by local microenvironmental factors; (2) an amplification resulting from extrinsic stimuli”‘. B lymphodytopoiesis in peripheral lymphoid tissues Following the i.v. or S.C. administration of an antigen (e.g. bacterial vaccine, foreign protein) three types of immune reaction, involving bothTand B lymphocytes, occur in spleen and draining lymph nodes. As a consequence both cellular and humoral immunity usually develop, as well as immunological memory. With or without T-cell help, antigen-reactive B cells respond by transforming to plasmablasts which proliferate and eventually mature into antibodysecreting plasma cells. Histologically, these plasmablasts - within 24-48 h after antigenic stimulation develop in a rather diffuse way in primary nodules or at the periphery of secondary follicles or - in the case of T-cell dependent antigens - at the boundary between follicles and the adjoking T-cell areals. In contrast, from 3 days %nwards, centrally in follicular structures, an accumulation of large pyroninophylic cells gives rise to typical germinal centres by proliferation and probably the recruitment of more B lymphocytes. After several days the progeny of the rapidly dividing blast cells find their way to the periphery of the germinal centre to migrate through the lymphocyte corona to the marginal zone, from where the now medium-sized or small germinal-centrederived lymphocytes reach the blood or lymph circulation. Eventually these cells may home to follicular structures elsewhere’“. Thus, along with the B lymphocytopoiesis in the BM, B lymphocytes are generated in peripheral lymphoid tissues and more precisely in the germinal centres of the follicular B-cell areas. The remainder of this review will deal with this - essentially antigen-dependent - production of B lymphocytes outside the BM. Germinal centres: sites for B memory cell production There are several excellent reviews on the possible role of germinal centres in the generation of B memory cells (e.g. Refs 17 and 18). Here attention will be focused on recent data from Klaus and Kunkl’” on the special role of antigen-antibody complex localization in follicular structures and the micro-environmental implications in relation to germinal-centre formation and B memory-cell production. After administration of, for example, a soluble protein antigen, some antigen is retained in peripheral lymphoid tissue for a long period of time (several weeks) at a rather specific site, i.e. at the surface of the follicular dendritic cells in the thinly populated area of a germinal centre. By means of radioautography or immunofluorescence or other suitable techniques the antigen can be detected in that area in a lacey pattern or as a crescentic cap just underneath the lymphocyte corona (see e.g. Ref. 20).
107
Questions such as how the antigen is transported to that particular area and in what form the antigen is retained are still matters of discussion. The phenomenon is usually described as ‘antigen trapping’ and experimental data indicate that what is ‘trapped’ is not the antigen as such but antigen-antibody complexes. When antigen-antibody complexes are, for example injected iv., trapping in the spleen occurs within a matter of hours, in contrast when antigen fier se is injected, follicular localisation usually takes at least 5 or 6 days, presumably only after some antibody has been produced. Role of complement memory-cell formation
in antigen
trapping
and
B-
From experiments by Klaus and Humphrey2’ it has become clear that the presence of C3 is essential for the development of B-cell memory. When mice were adult thymectomized, lethally irradiated and reconstituted with fetal liver (T- mice) and then chronically depleted of C3 by serial injections of cobra venom factor (CVF), a potent C3 activator, primary immunisation with DNP-KLH did not result in B-memory formation. (Relative) T-cell deficiency could not explain this observation, because non CVF-treated T- mice showed excellent memory generation. Relatively T-cell-deficient mice were essential in these studies to facilitate chronic decomplementation.
When T- mice were injected i.p. with DNP-KLHr251, follicular localisation of the radio-labelled antigen occurred only after active primary immunisation ten days previously or after the administration of passive anti-DNP-KLH antibody. Chronic decomplementation of T- mice could totally abrogate follicular localisation of DNP-KLH-lZSI in both actively and passively immunised mice. These results show that both antibody and C3 are essential for follicular localisation and suggest that Bmeory-cell formation may be impaired after chronic decomplementation because of the prevention of follicular localisation of antigen-antibody-(complement) complexes. Role of antigen-antibody memory-cell formation
in B-
The above model, if true, would predict that ‘immunisation with preformed immune complexes should be an efficient method for priming B cells”li. To test this prediction syngeneic lethally irradiated mice were reconstituted with (i) spleen cells from mice primed with immune precipitates of DNP-KLH and anti-DNP antibodies, and (ii) spleen cells from mice primed with ovalbumin (OVA). Animals thus reconstituted were challenged with DNP-OVA. Results showed that complexes at equivalence, or in antigen excess were at least 100-fold more immunogenic for priming B cells than were equivalent doses of
7
70-*--Ag-Ab -=-APAg ---&lubleAg NothIng
6
5 67 -1 ‘0‘;
1‘‘k. ‘. +-1‘N \.
:
6
T
I
i i ;i
4
./’ L ./
-i
Days after Primary Immunization Fig. 2. Kinetics Groups of DNP-KLH-mouse together with figure shows methyl green
.I’
‘G 0 8
0 ./’
60-
-.AyAb ---SolubleAg -=-A.PAg -Nothing
50-
./’
73
./
3
From
complexes
5
7
Days after Primary
10
14
Immunization
of B-cell priming and germinal-centre formation in mice immunmized with different forms of antigen. mice were given Opg DNP-KLH Pither as soluble antigen or as an alum precipitate plus Z~o~~/~/~//n /wr/u\\i\ (A, P.Ag), or as anti-IINP antibody complexes (Ag-Ab). In the left panel spleen cells from these mice were transferred 3-14 days later spleen cells from mice primed with (A.P.) ovalbumin and 2Opg soluble DNP-ovalbumin to irradiated (650r) recipients. The the splenir IgG (IPFC) anti-IINP transfer. In the right panel spleens from similarly primed mice were lixed, stained with pyronin, and the average number of germinal centres/sections were counted.
Ref. 19 by permission
of Pitman
Medical
Limited.
soluble DNP-KLH’“. Moreover, not only the number of transferable B memory cells was greatly increased after immunisation with antigen-antibody complexes, but also the appearance of these cells was significantly accelerated: maximal priming was already induced by day 5 (see Fig. 2). This earlier appearance of transferable memory B cells coincided with earlier appearance of germinal centres, which were also strikingly increased in number (also see Fig. 2). These results clearly support the ‘hypothesis that giving antigen-antibody complexes immediately concentrates the antigen into follicles, and thereby initiates germinal centre formation and memory cell formation much more rapidly than conventional immunisation.‘“. From the above data it is clear that a particular micro-environmental characteristic of germinal centres - i.e. the capacity to ‘trap’ antigenantibody-(complement) complexes - greatly influences, if not determines B lymphocytopoiesis in these locations. It is tempting to speculate that the intrafollicularly retained antigen-antibody complexes have a local immunoregulatory function e.g. in in-situ differentiation towards B memory cells capable of high affinity antibody production upon re-exposure to antigen. Further experiments, however, are needed to elucidate this point. The role of T cells in the induction and continuation of germinal centre activity is beyond the scope of the present review. Germinal amplification
centres:
sites
of virginal
B-cell
From experiments in the rabbit on the contribution of the gut-associated lymphoid tissue, and in particular the appendix, to the post-irradiation regeneration of B follicular structures, we obtained evidence that, as well as having a postulated role in B memory-cell production, germinal centres may also function as sites of virginal B-cell amplification’“. Appendectomy before irradiation (450 rads, whole body) not only delayed follicular regeneration, for example in spleen and lymph nodes, but also delayed regeneration of primary IgM-antibody-forming potential against, for example, Hagellar (H) antigens of a Sulmon~llnj~va bacterial vaccine. Conversely, shielding the appendix during the irradiation led to accelerated follicular regeneration. It could bc shown that these effects were related to the activity of the germinal ccntre compartment of appendix follicular structures. From these and related experimcnts’“J2 we concluded that (appendix) germinal centres add to the B-cell population by producing among other cells virginal B cells capable of primary (IgM) antibody formation to antigens unrelated to the antigen that originally induced the germinal centre. How
many
From
B-~11
.~1~l3~o~)~tlnt~on.s.~
the discussion
above
it seems that ‘germinal
900 rad
24 hr b
BM
+
108
SRBC
14days
WI’=” histology
7days
sp’een histology
109
5
900 rad
24 hr
TDL
108
controls
: no
+
SRBC log
SRBC
Fig. 3. Experimental design to test cell suspensions TDL) for presence of germinal centre-precursor cells minal centre formation assay). Sections of spleens were screened for germinal centres. From Ref. 26 by permission of Springer Verlag.
(BM, (ger-
centres can produce at least two types of H cells: (i) B memory cells with specificity related to the antigen inducing the germinal centre, and (ii) virginal B cells with specificities unrelated to the inducing antigen. Next one might ask the question what types of B cells are, on the afferent side, involved in the two types of immune reactions into which K cells can be recruited, namely the plasma cell reaction and the germinal-centre reaction? Why, or how, does antigenic stimulation of a H ccl1 in one instance lead to terminal differentiation into antibody-forming plasma cells and in another instance result in the formation of a germinal ccntre? Could it be that these B cells have differing potentials or, alternatively, are B cells bipotential and is their ultimate fate after antigenic stimulation dependent on differences in the local micro-environment (e.g. way of antigen presentation, regulation by different T cell subsets)? Is there a difference in receptors (IgD?) or is there a difference in signals (or perhaps both)? In short, is an antibodyforming-cell precursor B cell (AFCP) different from a germinal-ccntre-precursor B cell (GCPC) or are the two types of reactions just two types of phenotypic expressions of a single but bi-potential type of B cell?
An attempt to answer these questions was made using two different experimental models, one in the rabbit, the other in the ratz3. The reader may have noticed that in the section above on regeneration of primary antibody forming potential after X-irradiation no mention was made of an experiment in which the appendix was shielded during irradiation rather than being surgically removed prior to irradiation. The reason is that results from that type of experiment - much to our surprise showed that appendix shielding did not result in accelerated regeneration of primary antibody-forming
potential but that the cells derived from the appendix could respond to primary antigenic challenge only after a 7-day delayz3J1. From this observation it was tentatively concluded that prospective antibody forming cell precursor cells recently derived from the appendix (germinal centre) were not yet fully mature when leaving the appendix and needed extra time (and micro-environment?) for further maturation. Virtually the same results were obtained using appendix germinal-centre cell suspensions to reconstitute appendectomized and irradiated recipients (autologous transfer). Moreover, although incapable of an immediate antibody response, appendix (germinal centre) derived cells were fully qualified to form new germinal centres, e.g. in the spleen, and to respond to antigenic challenge by memory-cell production23,24. In another approach the migration pattern of appendix germinal-centre cells was compared to that of ‘normal’ B cells obtained from other sources23,25. Using [3H] leucine labelled appendix germinal-centre cell suspensions transferred i.v. to normal recipients it was found that, in contrast to ‘normal’ B cells which tend to localize in the lymphocyte corona of follicular structures, appendix germinal centre cells (or at least
GERMINAL
a subpopulation thereof) showed central localisation in both primary and secondary follicles. It is tempting to speculate that these follicle-centre-seeking or germinal-centre-seeking cells are actually germinalcentre precursor cells, apparently differing from other 8 cells migrating to the lymphocyte corona. In the rat, lethally irradiated recipients were reconstituted with either BM cells or thoracic duct lymphocytes (TDL) and challenged with sheep red cells iv.. After 7 or 14 days spleen samples were studied for the presence of germinal centres (germinal centre formation assay) (see Fig. 3). Once more to our surprise BM-reconstituted recipients - at least over the observation period - did not show germinal-centre formation, in contrast to 1’DL-reconstituted animals, where germinal centres were a prominent feature23J”. Further analysis by separating TDL into T and B cell subpopulations indicated that in TDL the germinal centre precursor cell was among the B-cell fraction but needed T cells for expression of its germinal-centre forming capacity. At present we are trying to characterize further the germinal centre precursor cell present in TDL, its surface markers such as sIg, C3-receptors, its nylon wool adherence, and Ia antigens etc
CENTRE anti
antigen (
stem
cell
A
A
primary response)
pre-B
cell
immature
B cell
memory
Bcell
antigen
A
mature
B cell
antigen
A
plasmacell
(secondary
response)
plasmacell
(primary
Fig. 4. Schematic representation of possible role of germinal centres in B cell differentiation. A-Z: B cells with specificities ranging from A-Z. Afferent side of germinal centre: A: germinal centre precursor antigen A; B-Z: germinal centre precursor cells with specificity unrelated to antigen A. Y: immunoglobulin secreted). From Ref. 23 by permission of Pitman Medical Ltd.
response)
cells with specificity (surface, cytoplasmic
for or
B-cell
differentiation
in &JO:
a synopsis
In Fig. 4 an attempt is made to give a synoptic view of B-cell differentiation in uivo compatible with the data as described in the three main sections above. Early phases of B-cell differentiation and B lymphocytopoiesis take place in the BM micro-environment and to a certain extent appear to be antigen independent. Without antigenic stimulation BMderived B cells will mature further to immunocompetent but still virginal (antigen inexperienced) primary antibody-forming cell precursors (A-Z) (base line sequence in Fig. 4). Upon antigenic stimulation if necessary with the appropriate help of 1’ cells - these AFCPs will eventually differentiate into antibody forming plasma cells. Superimposed upon this basic pattern of antigen independent B lymphocytopoiesis, germinal centre activity seems to add to the population of immunologically competent B cells in two ways: (i) by virtue of B-memory-cell production (more A) and (ii) by nonspecific amplification of other B cells (more B-Z) thereby adding to the pool of primary AFCP (A-Z). A working hypothesis is that germinal centre formation might result from the specific interaction of antigen with a germinal-centre precursor cell (A), possibly an as yet not fully mature B cell, perhaps differing in surface receptors (qualitatively or quantitatively) from AFCP, and that the interaction results in specifi? amplification and differentiation of these cells to antigen (A)-specilic B memory cells. As a corollary to this process germinal centre precursor cells with specificities (B-Z) other than fitting the antigen that induced the germinal centre, nevertheless as ‘innocent bystanders’ may become involved in the amplification process by virtue of a surface receptor for (a) mitogenic factor(s) present in the germinal-centre micro-environment. The result of this non-specific amplification process will be the production of more immature (still antigen inexperienced) B cells (more B-Z), which subsequently may either (i) be specifically recruited into another germinal centre or (ii) upon maturation (like other antigen inexperienced immature B cells) become part of the pool of primary AFCP. ‘It is clear, that in this way germinal centres contribute to the expansion of the pool of B cells, which may well be of advantage to an organism that has suffered from an antigenic attack, preparing it not only in a specific way - by the formation of memory cells - for a second attack by the same antigen, but also for any other antigenic determinant that the organism may subsequently have to cope with’23.
Acknowledgements
I extend my sincerest thanks to Prof. 11. G. Osmond and Dr G. G. B. Klaus for allowing me to draw heavily on the
manuscripts of their presentations at the CIBA Foundation Symposium no. 84 on ‘Micro-environments in haemopoietic and lymphoid differentiation’. I wish to stipulate, however, that views expressed in the present paper are my sole responsibility. My studies and those of my associates were supported by the Foundation for Medical Research (FUNGO), which is subsidized by the Netherlands Organisation for the Advancement of Pure Research (ZWO) (grants nos. 13-27-17 and 13-27-39).
References
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4 5 6 7
P. B. (1963)
‘Thr Immunolr~~dly
C,im$rtunt
ad
Cell, CIBA
Foundation Study Group no 16,4 Roitt, J. M., Grcaves, M. F., Torrigiani, G., Brostoff, J. and Playfair,J. H. L. (1969) Thr Lanrct, ii, 367 Owen, J. J. T., Wright, D. E., Habu, S., Raff, M. C. and Cooper, M. D. (1977) j’. Irr,munol.118, 2067 M&hers, F. (personal communication) Abramson, S., Miller, R. G. and Phillips, R. A. (1977)J. &/I. Med. 145, 1567
8
Razing, J., Brons, N. H. C., Ewijk, W. van and Benner, R.
9
Cooper, M. D., Lawton, A. R. and Kincade, P. W. (1972)
(1978)
Ml
T~\\NP Krs. 189, 19
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33
10 Nieuwenhuis, P. (1976) /Ido. E.xP./Mpd.Bid. 66, 25 11 Osmond, D. G., Fahlman, M. T. E. F., Fulop, G. IM. and Rahal, D. M. (1981) MzcmPnuimnmr,nt.~ zn hurmopozrtir and lymhhozridzfirwntintion, CIBA Foundation Svmposium no 84, I . %nan Medical, London (in press) 12 Osmond,
D.
C.
(1976)
Strm cellr
I!/ renewing
cdl
popnlntzons
(Lairnie, A. K., Lala, P. K. and Osmond, D. G. Eds.) 195, Academic Press 13 Coffman, R. L. and Weissman, I. L. (1981) ,7. hp. Md. 153, 169 14
Paige, C. J., Kincade, P. W., Shinefield, L. A. and Sam, V. L.
15
Veldman, J. E., Keuning,
(1981)J. Vurhows
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153, 154
F. J. and Molenaar,
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16 Nieuwenhuis, P. and Keuning, F. J. (1974) Immwdqy 26, 509 17 Thorbecke, C. J., Romano, T. J. and Lerman, S. J. (1974) I’mg. Immunr~l. II, 3, 25
18 Klaus, G. G. B., Humphrey, 1. H., Kunkl, A. and DonEworth, ,, D. W. (1980) Immrrnoi:ItrJ: 53;3 19 Klaus. G. G. B. and Kunkl. A. (1981) /Mz~rornolronmrntl zn hnPm&xrt~c rind lymphoid cl;llrmtzhtum, CIBA Foundation Symposium no. 84, Pitman Medical, London (in press) 20 Rooijen, N. van (1980) Phylogeny q/zmmtlnol~~,~zrnl memory(Manning, M. J. ed.) 281 21 Klaus, G. G. B. and Humphrey, J. H. (1977) Ivummolr~gy 33, 31 22 Nieuwenhuis, P., Nouhuys, C. E. van, Eggens, J. II. and Keuning, F. J. (1974) Immwwlogy 26, 497 23 Neuwenhuis, P., Gastkemper, N. A. and Opstelten, I). (1981) Micm~nu~mrrmenl.,~t~ m hnpmopozrtzr
rind lymphod
rl$/evntzntmn,
CIBA
Foundation Symposium no. 84, Pitman Medical London (in press) 24 Opstelten, D., Stikker, R., IHeiiden, D. H. van der. and N&venhuis, P. (1980) V&ozer irh. B. 34, 53 25 Opstelten, I).. Stikker, R.. Deenen. G. I., Bos, I,. and Nieuwenhuis, P. (1981) &ll 7i.u~ Rr.r. (in press) 26 Gastkemper, N. A., Wubbena, A. S., GimbrPre, F. ,J. H., Graaf, A. de, and Nieuwenhuis, I’. (1981) Cpll 7i.1~ &I. (in press)
mechanism of selection theory. eagerly awaited.
cytotoxic lymphocytes are expressed on normal and transformed lymphoid cells, fibroblasts and peritoneal exudate cells. In a complementary paper, in the same issue, Risser and Crunwald describe the production of cytotoxic anti-self FI-2 antibodies by Fl mice hyper-immunized with a parental Abelson virus-induced lymphoma. Since most of the mice repeatedly challenged with the tumour do not survive, it is difficult to assess whether the production of such anti-self antibodies is associated with the development of an autoimmune or other pathological lesions. Nevertheless, there is a clear demonstration in these two papers of the production of cytotoxic T lymphocytes and cytotoxic antibodies by Fl hybrid animals against their own self antigens. The mechanism by which a vertebrate animal produces antibody diversity’” and distinguishes between self and non-self, especially in an Fl hybrid, is at present unknown. The results discussed here, taken with those in previous reports, present a serious challenge to the
self tolerance proposed in the New paradigms in immunology
clonal are
References 1 Kuhn, T. S. (1962) ‘7% Slrurlw/, uj S~1(‘7112/i( KPnolullonl University of Chicago Press. 2 Nakano, K., Nakamura, I. and Cudkowicz, G. (1981) jV&rr (Imdm) 289, 559-563 3 Risscr, R. and Grunwald, D. J. (1981) .Natzrre (l,wu/onj 289, 563-568 4 Burnet, F. M. (1959) T/u, (:lorinl Srlrition Thmry o/ Arqud Immunz~y Cambridge University Press, London 5 Snell, C. D. (195S)J. .N&/. (:nnc~r In.\l., 20, 787-824 6 Cudkowicz, G. and Stimpfling, J. H. (1964) ~V&rw (Lon&l 204,450-453 7 Adler, W. H., Takiguchi, ‘I‘., Marsh, B. and Smith, R. T. (1970) J. Zrrimitnol., 105, 984-l 000 8 Subramaniam, R. and Ebringer, A. (1978) Hzoriwm. G. Tmniac., 6, 1079-1081 9 Ebringer, A., Deacon, N. ~J. and Young, C. R. (1976) j’. Imrruini~grnul. 3, 401-409 10 Ebringer, A. (1975) .7. 7hmr. Hu~i., 5 1, 293-302
mpii--)
B-cell differentiation
in uiuo
Paul Nieuwenhuis Department
of Histology,
Kijksuniversiteit
Groningen,
The
immunologically competent cell Only twenty five years ago, in 1956, a lymphocyte was defined as ‘. a somewhat inconspicuous cell with no particularly striking functional or morphological characteristics of its own’; ‘... it has been and still is the cell around which a violent haematological controversy has been waged”. In his Croonian Lecture, in 1958, Medawars introduced the term ‘Immunologically competent cell, that is, a cell which is fully qualified to undertake an immunological response’ whereas, in 1963, at a Ciba Foundation Study group he added: ‘I think we can
This review draws heavily on three papers (Kefs 11, 19, 23) presented by D. G. Osmond (section 3), G.C.B. Klaus (section 5) and the author (section 6) at the CIBA Foundation Symposium no. 84 on ‘Micro-environments in haemopoietic and lymphoid differentiation’. Proceedings of this symposium will shortly be published by Pitman Medical Ltd, London. No attempt has been made to fully cover the increasingly cornplex field of B cell differentiation, of which excellent reviews have appeared in previous issues of 1rruruu/o/r~~1~ 7urlo~ (J. M. Adams (1980) 1, 10 and J. W. Schrader (1981) 2, 7).
9713
EZ Groningen,
‘l’he Netherlands
now say there is such a thing as an immunologically competent cell and that it is a lymphocyte.. .“‘. In subsequent years it became evident that not every lymphocyte could perform in the various types of immune responses and by 1970 it was clear that at least two distinct subpopulations appeared to exist: thymus-derived T lymphocytes and non-thymus derived B lymphocytes”. T cells, although originating in bone marrow, undergo a process of proliferation and differentiation in the thymus, a well structured organ, possibly providing the essential micro-environment to permit or induce the maturation process. B cells seem to lack a comparably structured micro-environment, at least in mammals, an absence that has not facilitated the study of B-cell differentiation in oinr~. Origin of B cells During ontogeny poiesis are found cytoplasmic IgM IgM(sIgM) - and
early signs of H lymphocytothe liver’ where first cells with (cIgM) and later surface C3 - receptor positive cells can be
in
IO5
detected. Recent observations by Melchers” indicate that in the placenta B lymphocytopoiesis takes place even earlier. In postnatal life the majority of B lymphocytes should derive from the bone marrow @Ml. Chromosome marker studies, e.g. by Abramson et al.‘, have shown that - like other blood cells - all lymphoid cells derive from pluripotent haemopoietic stem cells and that for the T-cell lineage a committed T-cell precursor may exist. No such cell has been identified for the B-cell lineage, suggesting a direct descendencc from the stem cell pool. One might speculate that this relates to the need for pre-T cells to migrate to the thymus for further processing, whereas early phases of Bcell differentiation may occur locally in the BM microenvironment. Whether the BM microenvironment is essential for B lymphocytopoiesis is uncertain: experiments by Rozing et al. 8 indicate that bone-marrow function in this respect can be taken over by the spleen. In the chicken, B lymphocytopoiesis has been postulated to be dependent upon the presence of the Bursa of Fabricius”. Althoug!i it is quite clear that at a certain stage during ontogeny removal of the Bursa of Fabricius can lead to the development of a completely B-cell-less chicken, it is equally clear that when removal of the bursa after the 7th day post hatching is followed by near lethal whole-body X irradiation, a normal B-cell system will regenerate in the absence of the bursa of Fabricius”‘. Whether and how, in the chicken, the bone marrow contributes to the B lymphocyte pool is beyond the scope of this review. Suffice it to say, that the remarks above at least illustrate the rather erratic pattern of B lymphocytopoiesis during ontogeny and the apparent absence of a fixed relation to a specific micro-environment. B lymphocytopoiesis
in bone
marrow
From experiments by Osmond and co-workers (for references see Ref.ll), performed in both mice and guinea pigs, there is evidence that B cells are not only BM-derived, but that at least the earlier differentiation stages actually take place in the marrow cavity (+ stroma) itself. The first sign of B-lymphocyte differentiation is :he appearance of cytoplasmic IgM, which is detected in about 30% of all small lymphocytes present in BM. Another 50% express readily detectable surface IgM molecules. By these criteria, some 80% of the marrow small lymphocytes are of the B-cell lineage. [3H]thymidine labelling experiments have shown that the vast majority (over 90%) of marrow B lymphocytes are rapidly renewed indigenous cells. Eventually these still immature B cells leave the BM to migrate to the spleen and other sites. At the same time surface IgM increases in density and other markers such as Fc receptors and Ia antigens appear, followed by C3 receptors and a low to medium density of IgD (see Fig. 1). These later maturation stages seem
BONE Stem a
Commited
cells
MARROW Droaenltors _
LYMPH010 1
Moturlno
CELLS cells -
Functlonol
cells
I Large
lymphold Cytoplosmlc
cells p
Small
lymphocytes’
EZZI Surface IgM [ +IgM+IgD Fc 8 C3 receptors] 10 ontlqens
B Lymphocyte
0 -----* c
-f\ ’ 12 \’ ,’
Slowly
Fig. 1. Schdme of B lymphocytopoiesis in marrow. With slight modifications from Ref. 12 by permission Press Inc.
renewed
mouse
bone
of Academic
to be independent of micro-environmental influences of either marrow or spleen because they can also proceed in z&o. In addition to the population of small lymphocytes, there are rapidly dividing large lymphoid cells in the marrow, of which 6OY0 are cIgM posit& and thus preB cells. Large lymphoid cells have never been found to sIg. Attempts are being made, using express monoclonal antibodies, to identify intermediate stages between the pluripotent stem cell and the pre-B cells (see Refs 13 and 14). Micro-environmental
aspects
Microscopically, in transverse sections, the marrow parenchyma is arranged between blood vessels, which radiate from longitudinally running central arterioles to an elaborate subendosteal capillary plexus. Venous sinuses converge to the central venous sinus. After continuous [3H]-thymidine infusion for varying periods of time the highest incidence of labelled nucleated cells was found in the immediately subendosteal region (47% after 1 day; 71% after 4 days) decreasing towards the centre of the marrow (20.5% after one day; 54.7% after 4 days). In contrast, when frozen sections of marrow were studied to detect sIgM positive cells, the highest incidence of small lymphoid cells showing a membrane ring fluorescence was found towards the marrow centre. These and other data (not presented here) ‘are consistent with a centripetal movement of maturing B lymphocytes from peri-
106
pherally situated progenitors and their release into adjacent venous sinuses at various stages en route” ‘. Local concentrations of fluorescing cells were not detected. This might indicate that local microenvironmental factors - if operative - act at earlier stages of differentiation, perhaps in the immediate subendosteal region, possibly by influencing the localisation of (pre-B-)precursor cells. Whether this micro-environmental influence is associated with the cortical bone or its associated stromal cells is uncertain and perhaps even unlikely, because Rozing et ~1.~ have shown that, by eliminating haemopoiesis from the BM with “‘Sr, normal follicular structures develop in the spleen of mice after lethal irradiation and reconstitution with BM cells. Regulation of BM lymphocyte production Apart from local micro-environmental factors, other - exogenous - regulatory factors might affect marrow B lymphocytopoiesis: e.g. thymus hormones, peripheral B-lymphocyte pool size and antigen stimulation. In neonatally thymectomized as well as congenitally athymic nude mice, BM lymphocyte production shows virtually the same characteristics (turnover, proportion of IgM-bearing cells) as in normal controls, indicating the independence of BM B lymphocytopoiesis from either thymic hormones or (levels of) recirculating T cells. Depletion of the circulating Blymphocyte pool also had no marked effect on the various stages of B lymphocytopoiesis occurring in BM. For example, repeated treatment of mice with anti-IgM serum after birth led to a severe depletion of circulating B cells as well as BM sIg-positive cells, but the population of cIgM-positive cells did not seem to be affected. Depletion of the circulating B-lymphocyte pool by partial body irradiation and shielding of the marrow in two hind limbs (guinea pigs), did not induce any change in the number of small lymphocytes in the shielded marrow. In contrast, the granulocyte precursor population reacted with a burst of expansion. From these data, Osmond et al. concluded ‘Apparently the production of marrow small lymphocytes is independent of feedback control from the B Regulation in these experiments lymphocyte pool”‘. thus seems to result from local microenvironmental influences. In mice, raised under germfree conditions from birth, the proportion of small lymphocytes and IgMbearing cells was normal. However, the rate at which these cells are produced is strikingly reduced, with prolongation of the half turnover time from 32 to 125 h. On the other hand, after iv. injection of sheep red cells into conventionally reared animals marrow lymphocyte production increases. In conclusion, it seems fair to say that BM B lymphocytopoiesis ‘represents the summation of two steps: (1) a basal level regulated
immunolu,~y 1uday,Junr
11)x1
by local microenvironmental factors; (2) an amplification resulting from extrinsic stimuli”‘. B lymphodytopoiesis in peripheral lymphoid tissues Following the i.v. or S.C. administration of an antigen (e.g. bacterial vaccine, foreign protein) three types of immune reaction, involving bothTand B lymphocytes, occur in spleen and draining lymph nodes. As a consequence both cellular and humoral immunity usually develop, as well as immunological memory. With or without T-cell help, antigen-reactive B cells respond by transforming to plasmablasts which proliferate and eventually mature into antibodysecreting plasma cells. Histologically, these plasmablasts - within 24-48 h after antigenic stimulation develop in a rather diffuse way in primary nodules or at the periphery of secondary follicles or - in the case of T-cell dependent antigens - at the boundary between follicles and the adjoking T-cell areals. In contrast, from 3 days %nwards, centrally in follicular structures, an accumulation of large pyroninophylic cells gives rise to typical germinal centres by proliferation and probably the recruitment of more B lymphocytes. After several days the progeny of the rapidly dividing blast cells find their way to the periphery of the germinal centre to migrate through the lymphocyte corona to the marginal zone, from where the now medium-sized or small germinal-centrederived lymphocytes reach the blood or lymph circulation. Eventually these cells may home to follicular structures elsewhere’“. Thus, along with the B lymphocytopoiesis in the BM, B lymphocytes are generated in peripheral lymphoid tissues and more precisely in the germinal centres of the follicular B-cell areas. The remainder of this review will deal with this - essentially antigen-dependent - production of B lymphocytes outside the BM. Germinal centres: sites for B memory cell production There are several excellent reviews on the possible role of germinal centres in the generation of B memory cells (e.g. Refs 17 and 18). Here attention will be focused on recent data from Klaus and Kunkl’” on the special role of antigen-antibody complex localization in follicular structures and the micro-environmental implications in relation to germinal-centre formation and B memory-cell production. After administration of, for example, a soluble protein antigen, some antigen is retained in peripheral lymphoid tissue for a long period of time (several weeks) at a rather specific site, i.e. at the surface of the follicular dendritic cells in the thinly populated area of a germinal centre. By means of radioautography or immunofluorescence or other suitable techniques the antigen can be detected in that area in a lacey pattern or as a crescentic cap just underneath the lymphocyte corona (see e.g. Ref. 20).
107
Questions such as how the antigen is transported to that particular area and in what form the antigen is retained are still matters of discussion. The phenomenon is usually described as ‘antigen trapping’ and experimental data indicate that what is ‘trapped’ is not the antigen as such but antigen-antibody complexes. When antigen-antibody complexes are, for example injected iv., trapping in the spleen occurs within a matter of hours, in contrast when antigen fier se is injected, follicular localisation usually takes at least 5 or 6 days, presumably only after some antibody has been produced. Role of complement memory-cell formation
in antigen
trapping
and
B-
From experiments by Klaus and Humphrey2’ it has become clear that the presence of C3 is essential for the development of B-cell memory. When mice were adult thymectomized, lethally irradiated and reconstituted with fetal liver (T- mice) and then chronically depleted of C3 by serial injections of cobra venom factor (CVF), a potent C3 activator, primary immunisation with DNP-KLH did not result in B-memory formation. (Relative) T-cell deficiency could not explain this observation, because non CVF-treated T- mice showed excellent memory generation. Relatively T-cell-deficient mice were essential in these studies to facilitate chronic decomplementation.
When T- mice were injected i.p. with DNP-KLHr251, follicular localisation of the radio-labelled antigen occurred only after active primary immunisation ten days previously or after the administration of passive anti-DNP-KLH antibody. Chronic decomplementation of T- mice could totally abrogate follicular localisation of DNP-KLH-lZSI in both actively and passively immunised mice. These results show that both antibody and C3 are essential for follicular localisation and suggest that Bmeory-cell formation may be impaired after chronic decomplementation because of the prevention of follicular localisation of antigen-antibody-(complement) complexes. Role of antigen-antibody memory-cell formation
in B-
The above model, if true, would predict that ‘immunisation with preformed immune complexes should be an efficient method for priming B cells”li. To test this prediction syngeneic lethally irradiated mice were reconstituted with (i) spleen cells from mice primed with immune precipitates of DNP-KLH and anti-DNP antibodies, and (ii) spleen cells from mice primed with ovalbumin (OVA). Animals thus reconstituted were challenged with DNP-OVA. Results showed that complexes at equivalence, or in antigen excess were at least 100-fold more immunogenic for priming B cells than were equivalent doses of
7
70-*--Ag-Ab -=-APAg ---&lubleAg NothIng
6
5 67 -1 ‘0‘;
1‘‘k. ‘. +-1‘N \.
:
6
T
I
i i ;i
4
./’ L ./
-i
Days after Primary Immunization Fig. 2. Kinetics Groups of DNP-KLH-mouse together with figure shows methyl green
.I’
‘G 0 8
0 ./’
60-
-.AyAb ---SolubleAg -=-A.PAg -Nothing
50-
./’
73
./
3
From
complexes
5
7
Days after Primary
10
14
Immunization
of B-cell priming and germinal-centre formation in mice immunmized with different forms of antigen. mice were given Opg DNP-KLH Pither as soluble antigen or as an alum precipitate plus Z~o~~/~/~//n /wr/u\\i\ (A, P.Ag), or as anti-IINP antibody complexes (Ag-Ab). In the left panel spleen cells from these mice were transferred 3-14 days later spleen cells from mice primed with (A.P.) ovalbumin and 2Opg soluble DNP-ovalbumin to irradiated (650r) recipients. The the splenir IgG (IPFC) anti-IINP transfer. In the right panel spleens from similarly primed mice were lixed, stained with pyronin, and the average number of germinal centres/sections were counted.
Ref. 19 by permission
of Pitman
Medical
Limited.
soluble DNP-KLH’“. Moreover, not only the number of transferable B memory cells was greatly increased after immunisation with antigen-antibody complexes, but also the appearance of these cells was significantly accelerated: maximal priming was already induced by day 5 (see Fig. 2). This earlier appearance of transferable memory B cells coincided with earlier appearance of germinal centres, which were also strikingly increased in number (also see Fig. 2). These results clearly support the ‘hypothesis that giving antigen-antibody complexes immediately concentrates the antigen into follicles, and thereby initiates germinal centre formation and memory cell formation much more rapidly than conventional immunisation.‘“. From the above data it is clear that a particular micro-environmental characteristic of germinal centres - i.e. the capacity to ‘trap’ antigenantibody-(complement) complexes - greatly influences, if not determines B lymphocytopoiesis in these locations. It is tempting to speculate that the intrafollicularly retained antigen-antibody complexes have a local immunoregulatory function e.g. in in-situ differentiation towards B memory cells capable of high affinity antibody production upon re-exposure to antigen. Further experiments, however, are needed to elucidate this point. The role of T cells in the induction and continuation of germinal centre activity is beyond the scope of the present review. Germinal amplification
centres:
sites
of virginal
B-cell
From experiments in the rabbit on the contribution of the gut-associated lymphoid tissue, and in particular the appendix, to the post-irradiation regeneration of B follicular structures, we obtained evidence that, as well as having a postulated role in B memory-cell production, germinal centres may also function as sites of virginal B-cell amplification’“. Appendectomy before irradiation (450 rads, whole body) not only delayed follicular regeneration, for example in spleen and lymph nodes, but also delayed regeneration of primary IgM-antibody-forming potential against, for example, Hagellar (H) antigens of a Sulmon~llnj~va bacterial vaccine. Conversely, shielding the appendix during the irradiation led to accelerated follicular regeneration. It could bc shown that these effects were related to the activity of the germinal ccntre compartment of appendix follicular structures. From these and related experimcnts’“J2 we concluded that (appendix) germinal centres add to the B-cell population by producing among other cells virginal B cells capable of primary (IgM) antibody formation to antigens unrelated to the antigen that originally induced the germinal centre. How
many
From
B-~11
.~1~l3~o~)~tlnt~on.s.~
the discussion
above
it seems that ‘germinal
900 rad
24 hr b
BM
+
108
SRBC
14days
WI’=” histology
7days
sp’een histology
109
5
900 rad
24 hr
TDL
108
controls
: no
+
SRBC log
SRBC
Fig. 3. Experimental design to test cell suspensions TDL) for presence of germinal centre-precursor cells minal centre formation assay). Sections of spleens were screened for germinal centres. From Ref. 26 by permission of Springer Verlag.
(BM, (ger-
centres can produce at least two types of H cells: (i) B memory cells with specificity related to the antigen inducing the germinal centre, and (ii) virginal B cells with specificities unrelated to the inducing antigen. Next one might ask the question what types of B cells are, on the afferent side, involved in the two types of immune reactions into which K cells can be recruited, namely the plasma cell reaction and the germinal-centre reaction? Why, or how, does antigenic stimulation of a H ccl1 in one instance lead to terminal differentiation into antibody-forming plasma cells and in another instance result in the formation of a germinal ccntre? Could it be that these B cells have differing potentials or, alternatively, are B cells bipotential and is their ultimate fate after antigenic stimulation dependent on differences in the local micro-environment (e.g. way of antigen presentation, regulation by different T cell subsets)? Is there a difference in receptors (IgD?) or is there a difference in signals (or perhaps both)? In short, is an antibodyforming-cell precursor B cell (AFCP) different from a germinal-ccntre-precursor B cell (GCPC) or are the two types of reactions just two types of phenotypic expressions of a single but bi-potential type of B cell?
An attempt to answer these questions was made using two different experimental models, one in the rabbit, the other in the ratz3. The reader may have noticed that in the section above on regeneration of primary antibody forming potential after X-irradiation no mention was made of an experiment in which the appendix was shielded during irradiation rather than being surgically removed prior to irradiation. The reason is that results from that type of experiment - much to our surprise showed that appendix shielding did not result in accelerated regeneration of primary antibody-forming
potential but that the cells derived from the appendix could respond to primary antigenic challenge only after a 7-day delayz3J1. From this observation it was tentatively concluded that prospective antibody forming cell precursor cells recently derived from the appendix (germinal centre) were not yet fully mature when leaving the appendix and needed extra time (and micro-environment?) for further maturation. Virtually the same results were obtained using appendix germinal-centre cell suspensions to reconstitute appendectomized and irradiated recipients (autologous transfer). Moreover, although incapable of an immediate antibody response, appendix (germinal centre) derived cells were fully qualified to form new germinal centres, e.g. in the spleen, and to respond to antigenic challenge by memory-cell production23,24. In another approach the migration pattern of appendix germinal-centre cells was compared to that of ‘normal’ B cells obtained from other sources23,25. Using [3H] leucine labelled appendix germinal-centre cell suspensions transferred i.v. to normal recipients it was found that, in contrast to ‘normal’ B cells which tend to localize in the lymphocyte corona of follicular structures, appendix germinal centre cells (or at least
GERMINAL
a subpopulation thereof) showed central localisation in both primary and secondary follicles. It is tempting to speculate that these follicle-centre-seeking or germinal-centre-seeking cells are actually germinalcentre precursor cells, apparently differing from other 8 cells migrating to the lymphocyte corona. In the rat, lethally irradiated recipients were reconstituted with either BM cells or thoracic duct lymphocytes (TDL) and challenged with sheep red cells iv.. After 7 or 14 days spleen samples were studied for the presence of germinal centres (germinal centre formation assay) (see Fig. 3). Once more to our surprise BM-reconstituted recipients - at least over the observation period - did not show germinal-centre formation, in contrast to 1’DL-reconstituted animals, where germinal centres were a prominent feature23J”. Further analysis by separating TDL into T and B cell subpopulations indicated that in TDL the germinal centre precursor cell was among the B-cell fraction but needed T cells for expression of its germinal-centre forming capacity. At present we are trying to characterize further the germinal centre precursor cell present in TDL, its surface markers such as sIg, C3-receptors, its nylon wool adherence, and Ia antigens etc
CENTRE anti
antigen (
stem
cell
A
A
primary response)
pre-B
cell
immature
B cell
memory
Bcell
antigen
A
mature
B cell
antigen
A
plasmacell
(secondary
response)
plasmacell
(primary
Fig. 4. Schematic representation of possible role of germinal centres in B cell differentiation. A-Z: B cells with specificities ranging from A-Z. Afferent side of germinal centre: A: germinal centre precursor antigen A; B-Z: germinal centre precursor cells with specificity unrelated to antigen A. Y: immunoglobulin secreted). From Ref. 23 by permission of Pitman Medical Ltd.
response)
cells with specificity (surface, cytoplasmic
for or
B-cell
differentiation
in &JO:
a synopsis
In Fig. 4 an attempt is made to give a synoptic view of B-cell differentiation in uivo compatible with the data as described in the three main sections above. Early phases of B-cell differentiation and B lymphocytopoiesis take place in the BM micro-environment and to a certain extent appear to be antigen independent. Without antigenic stimulation BMderived B cells will mature further to immunocompetent but still virginal (antigen inexperienced) primary antibody-forming cell precursors (A-Z) (base line sequence in Fig. 4). Upon antigenic stimulation if necessary with the appropriate help of 1’ cells - these AFCPs will eventually differentiate into antibody forming plasma cells. Superimposed upon this basic pattern of antigen independent B lymphocytopoiesis, germinal centre activity seems to add to the population of immunologically competent B cells in two ways: (i) by virtue of B-memory-cell production (more A) and (ii) by nonspecific amplification of other B cells (more B-Z) thereby adding to the pool of primary AFCP (A-Z). A working hypothesis is that germinal centre formation might result from the specific interaction of antigen with a germinal-centre precursor cell (A), possibly an as yet not fully mature B cell, perhaps differing in surface receptors (qualitatively or quantitatively) from AFCP, and that the interaction results in specifi? amplification and differentiation of these cells to antigen (A)-specilic B memory cells. As a corollary to this process germinal centre precursor cells with specificities (B-Z) other than fitting the antigen that induced the germinal centre, nevertheless as ‘innocent bystanders’ may become involved in the amplification process by virtue of a surface receptor for (a) mitogenic factor(s) present in the germinal-centre micro-environment. The result of this non-specific amplification process will be the production of more immature (still antigen inexperienced) B cells (more B-Z), which subsequently may either (i) be specifically recruited into another germinal centre or (ii) upon maturation (like other antigen inexperienced immature B cells) become part of the pool of primary AFCP. ‘It is clear, that in this way germinal centres contribute to the expansion of the pool of B cells, which may well be of advantage to an organism that has suffered from an antigenic attack, preparing it not only in a specific way - by the formation of memory cells - for a second attack by the same antigen, but also for any other antigenic determinant that the organism may subsequently have to cope with’23.
Acknowledgements
I extend my sincerest thanks to Prof. 11. G. Osmond and Dr G. G. B. Klaus for allowing me to draw heavily on the
manuscripts of their presentations at the CIBA Foundation Symposium no. 84 on ‘Micro-environments in haemopoietic and lymphoid differentiation’. I wish to stipulate, however, that views expressed in the present paper are my sole responsibility. My studies and those of my associates were supported by the Foundation for Medical Research (FUNGO), which is subsidized by the Netherlands Organisation for the Advancement of Pure Research (ZWO) (grants nos. 13-27-17 and 13-27-39).
References
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4 5 6 7
P. B. (1963)
‘Thr Immunolr~~dly
C,im$rtunt
ad
Cell, CIBA
Foundation Study Group no 16,4 Roitt, J. M., Grcaves, M. F., Torrigiani, G., Brostoff, J. and Playfair,J. H. L. (1969) Thr Lanrct, ii, 367 Owen, J. J. T., Wright, D. E., Habu, S., Raff, M. C. and Cooper, M. D. (1977) j’. Irr,munol.118, 2067 M&hers, F. (personal communication) Abramson, S., Miller, R. G. and Phillips, R. A. (1977)J. &/I. Med. 145, 1567
8
Razing, J., Brons, N. H. C., Ewijk, W. van and Benner, R.
9
Cooper, M. D., Lawton, A. R. and Kincade, P. W. (1972)
(1978)
Ml
T~\\NP Krs. 189, 19
ContPmp. Top. Immlmobiol.,
33
10 Nieuwenhuis, P. (1976) /Ido. E.xP./Mpd.Bid. 66, 25 11 Osmond, D. G., Fahlman, M. T. E. F., Fulop, G. IM. and Rahal, D. M. (1981) MzcmPnuimnmr,nt.~ zn hurmopozrtir and lymhhozridzfirwntintion, CIBA Foundation Svmposium no 84, I . %nan Medical, London (in press) 12 Osmond,
D.
C.
(1976)
Strm cellr
I!/ renewing
cdl
popnlntzons
(Lairnie, A. K., Lala, P. K. and Osmond, D. G. Eds.) 195, Academic Press 13 Coffman, R. L. and Weissman, I. L. (1981) ,7. hp. Md. 153, 169 14
Paige, C. J., Kincade, P. W., Shinefield, L. A. and Sam, V. L.
15
Veldman, J. E., Keuning,
(1981)J. Vurhows
Exp. Mrd. Arch.
153, 154
F. J. and Molenaar,
I. (1978)
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