331

Development of B-cell memory and effector function Peter Lane

The past year has seen significant advances in our

understanding of molecules that both positively and negatively regulate B- and T-cell responses. Of particular interest is the lethal phenotype of CTLA-4-deficient mice, which has illuminated the importance of downregulation of T-cell responses and the increasingly complicated role of CD40 and its ligand in directing both T- and B-cell priming.

Address Base1 Institute for Immunology, Grenzscherstraese 487, Base1 CH-4005, Switzerland; e-mail: lane@bii.ch

Current Opinion in Immunology 1996, 8:331-335

0 Current Biology Ltd ISSN 0952-7915

Abbreviattons APC antigen-presenting cell FDC follicular dendritic cell L ligand TCR T-ceil receptor Th2 T-helper type 2

Introduction The observation that unmethylated dinucleotides from bacteria activate murine B cells was the most unexpected observation of the year and an acute reminder that Darwinian selection ensures that all is possible [l’]. This review pretends that the B cell immune system is organized in a logical way. Some of the questions that are still poorly understood are laid out below and I have concentrated on the recent developments in these areas of T cell dependent B-cell activation. The following have been addressed: regulation of T cell priming; factors that specifically direct T-cell help for B cells; signals which control B-cell proliferation and differentiation; signals to generate germinal centers and switch on somatic mutation; tolerance in the peripheral B-cell pool; generation of memory B cells and their characteristics; and follicular dendritic cells.

Regulation of T-cell priming We have been reminded that successful priming of CD4+ T cells does not require B cells [Z]. Nor is it as dependent on costimulation through CD28 as was once thought [3]. There are most likely other costimulatory molecules that can act as substitutes, such as the recently described SLAM molecule [4].

Perhaps the most exciting observation has been that mice deficient in CTLA-4 die from an aggressive lym- phoproliferative disorder of greater severity than that associated with Fas ligand (L) deficient T-cell regulation [5**,6**]. CTLA-4, a molecule homologous to CD28 but only expressed by activated T cells, appears to switch

off uncontrolled proliferation of T cells. This is similar to the excessive priming of CD4 cells, which was seen in mice that overexpressed a soluble form of CTLA4 [7]. It therefore appears that CTLA4 and Fas represent distinct mechansims for regulating T cells. Contrary to the FasL-dependent deletion seen following immunization with the superantigen staphylococcal enterotoxin B, we have found that the induction of ‘anergy’ is blocked in CTLA4 transgenic mice (P Lane, unpublished data). This is consistent with the idea that signalling through CTLA4 induces proliferative unresponsiveness in T cells without prejudicing T-cell capacity to deliver help for other effector cells. An example of this might occur in locations such as germinal centers, where T cells help antigen-selected B cells, but do not usually proliferate themselves.

The crucial role of CD40 in T-cell priming has been demonstrated using several different approaches. Firstly, blocking the binding of CD40 ligand with monoclonal antibodies (mAbs) promotes the induction of tolerance by allogeneic B cells [SO]. Secondly, using mice deficient in CD40L, Flavell and colleagues found that T-cell priming was profoundly impaired, although not absent [9*]. Fur- thermore, although CD40L-deficient transgenic T cells failed to proliferate in U&J in response to antigen, they were able to respond in vitro. The interpretation of these experiments is that the effects of CD40L are indirect, and that there is no intrinsic signal through CD40L required to prime T cells. CD40L probably exerts its effect by promoting the differentiation of immature CD40-bearing dendritic cells to effective antigen-presenting cells (APCs) by upregulating the expression of costimulatory molecules and lymphokines, particularly IL-12 (A Lanzavecchia, personal communication). Significantly, T cells are not rendered tolerant by antigen in the experiments described, which shows that blocking CD4OL is permissive for an- other as yet unidentified tolerogenic signal from allogeneic B cells.

The third approach, which has given slightly different results, has involved the use of mice deficient in CD40. The numbers of T cells primed in CD40-deficient mice were normal [lo’], but T-cell help for B cells was impaired. It is not clear what accounts for the discrepancies with mice deficient in CD40 ligand, but one possibility is that there are alternative ligands in the CD40 system.

Factors which specifically direct T cells to help B cells Primed CD4+ T cells provide different kinds of help and an unanswered question is what specifically directs them to help B cells. B cell antigen presentation promotes the differentiation of T-helper type 2 (Th2) cells [l 1.1

332 Lymphocyte activation and effector functions

as was originally suggested using mice in which the B cells were suppressed [12]. One interesting idea is that B-helper function in T cells is induced by engagement of CD40L on T cells by B cells expressing CD40 [lo’]. Macrophages, however, which evoke the opposite T-cell response, also express CD40. An alternative possibility is that signalling of B cells through CD40 induces specific B cell accessory molecules, which evoke the appropriate T-cell help. Candidate molecules include other members of the CD40L family of receptors, CD27L [13] and OX4OL [14], which are induced on B cells activated by CD40. In particular, CD27 ligand (CD70) is expressed on early germinal center B cells at a time when T cells are expanding at this location so would be a good candidate for evoking specific T-cell help. The differential expression of B7-1 and B7-2 could also account for this [15*,16*].

Regulation of B-cell differentiation and proliferation at primary activation sites T-cell dependent B-cell responses are initiated in the T-cell zone [17,18] to which antigen-activated B cells specifically migrate [19*], an appropriate response for B cells looking for T-cell help. Activated B cells have one of three fates: they may differentiate into plasma cells; proliferate and form germinal centers; or if they receive the ‘wrong’ kind of T-cell help, they die.

Using a system with transgenic B cells and T cells, Goodnow and colleagues have shown that antigen-acti- vated ‘anergic’ B cells are eliminated by antigen-specific T cells expressing FasL [ZO*]. In some TCR transgenic mice, immunization favors the development of Thl cells, which fail to help B cells [ll*]. Both of these experiments illustrate the importance of B cells receiving the appropriate type of T-cell help. The importance of FasL in regulating B-cell responses is seen in deficient mice where the gross overproduction of autoantibody is associated with massive proliferation of plasmablasts in the T-cell zones [21*]. All of these experiments point to the notion that, under normal circumstances, the activation of B cells by Th2 cells within the T-cell zones is balanced by negative signals from Thl cells, which prevent uncontrolled overexpansion.

B cells that do get positive signals from T cells may either differentiate or proliferate. What determines this dichotomy? Klinman and co-workers [ZZ] think there are distinct B-cell precursors, although this has been disputed [ 181. The differences in capacity to form germinal centers could also reflect different stages of B-cell maturity: it has been suggested previously that newly formed B cells are the precursors of germinal centers [17]. The difference in fate could again be determined by T cells. Activated memory T cells in the outer T-cell zone are heterogeneous with regard to their expression of CD40L [23*]. As T cells deficient in CD40 ligand can stimulate the B cells to differentiate into plasma cells without significant B-cell proliferation [24], one might speculate that this forms the

basis for two kinds of help for B cells: CD40L pushing B cells down the proliferative pathway; and CD40L independent help promoting antibody formation.

Molecular signals that induce the formation of germinal centers Because germinal centers are absent in both CD40-de- ficient [25] and CD40L-deficient mice [26], it has been widely assumed that signalling through CD40 is required for germinal center formation. Recent experiments by Gray and colleagues [lo*] suggest that the situation may be more complex. Injection of soluble CD40 into CD40-deficient mice that had been immunized with protein antigens, partially restored germinal center formation although there was no class switch or memory antibody formation. This suggests that there may be some signalling through CD40L on T cells to induce ‘germinal center factor’.

Intriguingly, CD40L has been reported to be expressed on activated B cells [27] and this has been speculated to play a role in B-cell expansion in germinal centers. This seems unusually altruistic given that B cells expressing CD40L would help B cells other than themselves in the highly competitive environment of the germinal center, unless of course only members of a clone expanded. It seems unlikely that this could operate in mutating B-cell populations, as then B cells with ‘bad’ specificities might be rescued.

Two groups [28*,29*] have reported that CD19-deficient mice lack germinal centers; however, the molecular mech- anism accounting for this is still not clear. Most plausibly, one would expect signalling through the CDZl-CD19 complex to be impaired [30] although CD19 expressing follicular dendritic cells (FDCs) might be absent too. The phenotype is reminiscent of complement-depleted animals.

Somatic mutation The signals that regulate the onset of somatic mutation in germinal center B cells are poorly understood, although the mutating B-cell population can be clearly identified in humans [31]. Although primary B cells activated through CD40 do proliferate, they do not undergo somatic hypermutation of their immunoglobulin variable regions, so some additional signals must be required to switch on the mutator apparatus.

Recent extensive experiments have dissociated somatic mutation from germinal center formation. Immunization with phosphor+ choline conjugates, in contrast to the haptens, oxazolone and NP (nitrophenyl), poorly induce mutation although all three antigens induce histologically indistinguishable PNA+ germinal centers [32-l. These authors correlated mutation with quantitative T-cell help. In additional experiments, mutation was found to be much reduced in old mice [33’] and this was associated

Development of B-cell memory and effector function Lane 333

with deficient expression of B7-2 on germinal center B cells. In support of this notion anti-B72 .monoclonal antibodies reduced the mutation rate in normal mice [34]. Both CD40L and anti-immunoglobulin signals upregulate expression of B7-2. One could speculate that the early phase of clonal expansion without mutation is driven by T cells expressing CD40L within follicles. The trigger for mutation might be through immunoglobulin derived signals from immune complexes on follicular dendritic cells.

Generation of memory 6 cells The progeny of centroblasts differentiate into surface immunoglobulin-positive centrocytes, which have gener- ally switched the class of their immunoglobulin isotypes [35]. Most centrocytes die through lack of selection. Rescued centrocytes are induced to become plasma cells if CD40 signalling is blocked, or to become memory B cells if CD40 signalling is present [24,36*]. CD40 signalling appears to be crucial for memory B-cell development although it is not clear whether the signals that determine re-entry into the centroblast pathway are qualitatively or quantitatively different from those determining memory B-cell formation.

Deletion of germinal center and other autoreactive B cells The sensitivity of immature B cells to deletion or anergy by antigen is well known. Two recent publications [37*,38*] hypothesize that deletion of germinal center B cells by soluble antigen, may represent a normal mechanism for the deletion of mutated B cells with acquired self reactivity. The injection of soluble antigen, into animals with preformed antibody and germinal centers, led to the deletion of germinal center B cells accompanied by significant migration of antigen-specific B cells towards T-cell populated areas. This was associated with plasma-cell differentiation [39]. An alternative expla- nation is that immune complexes, perhaps via the indirect release of lymphokines, are toxic for germinal center B cells, which like thymocytes are sensitive to stress. Specificity for these experiments would be enhanced if irrelevant germinal centers were spared.

Location and properties of memory 6 cells Selected B cells that leave the germinal center environ- ment can either differentiate or become memory B cells. Two areas are selectively colonized by memory B cells: the marginal zone of spleen [40] and the subepithelial regions of lymph nodes [41]. Direct confirmation that memory B cells enter the marginal zone is provided by the existence of memory B cells with mutated immunoglobulin variable regions [42]. Reactivation of memory B cells leads to more rapid upregulation of costimulatory molecules compared with unprimed cells [41].

Follicular dendritic cells The elusive cell which is much ignored in the studies of T-cell dependent B-cell activation, due to difficulty in isolation, is the FDC. Unfortunately, many FDC-like cell lines do not behave like the real thing. The production of a reliable source of FDCs in vim is still a sisyphean goal. On balance, the importance of FDCs still seems to be, providing a framework of accessory molecules and immune complexes that form a scaffold for T cells and proliferating B lymphocytes. (For recent publications in this area, see [43-45].)

The significant message of the year is the importance of Conclusions

negative regulation of both B- and T-cell responses as demonstrated by experiments on mice deficient in FasL and CTLA4 signal transduction. It now seems that B cells are responsible for evoking the appropriate help from T cells although it is not clear what the precise molecular signals are. Signalling through CD40 or its ligand are implicated as T-cell priming is diminished in CD40L deficient mice, probably via indirect effects on ARCS. There is also new light shed on somatic mutation although it is still not known what induces this phenomenon in germinal center centroblasts. Finally, the molecular signals that regulate differentiation as distinct from proliferation in B cells certainly involve CD40 signalling but the role of other ligand pairs is still poorly understood.

Acknowledgements The Base1 Institute was founded and is supported by F Hoffman La-Roche Ltd. Co., Hasei, Switzerland. I would like to thank Fiona McConnell. Ton Roiink and Antonio i,anzavecchia for reading this manuscript and making many useful suggestions.

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