Fortifying B cells with CD154: an engaging tale of many hues

Although the original antibody to what is now defined as CD40 was raised against and shown to react with bladder carcinoma cells, the major insights into the functional attributes of this important receptor molecule – now known to be found on a diverse range of cell types – have arisen from studies on B lymphocytes. This 50 000 MW glycoprotein member of the tumour necrosis factor receptor (TNF-R) family, expressed constitutively at high density throughout B-cell differentiation, provides the focus for T-cell delivered cognate help to the B cell via the capacity to engage its inducible counterstructure – CD154; or ‘CD40 ligand’ (CD40L). 1–5 The aim of this article is to review the multiple stages at which the cognate CD40–CD40L pairing might serve to direct and modify the B-cell response with a focus on data generated from human in vitro studies set within an in vivo context gleaned primarily from observations in the mouse. The consequences of engaging CD40 on lymphoma B cells are discussed while, finally, we address how the quantity and quality of the CD154 being offered to its receptor may dictate the functional outcome of the cell in question. Absence, or blockade, of the CD40–CD40L interaction results in gross impairment of the physiological and molecular B-cell pathways that are unique to T-dependent (TD) antibody responses. Thus in patients with X-linked Hyper–immunoglobulin M (IgM) Syndrome (who carry a functionally crippled CD40L gene), 6 in genetically engineered CD40- or CD154- deficient mice,7,8 or in mice administered with antibodies that impede CD40–CD154 interaction, 9 there is a failure of germinal-centre (GC) formation accompanied by a marked reduction in immunoglobulin (Ig) isotype switching and a lack of affinity maturation that would normally arise from the processes of somatic hypermutation and selection that occur in GC responses. Central and exclusive to B cells responding to TD antigens is their entry, expansion, and differentiation that occur within the follicles of secondary lymphoid tissues. Prior to their follicular development, B cells bearing receptors specific for a challenge antigen need to engage TH cells primed to peptides from that same antigen within the context of interdigitating dendritic cells (IDC), the potent antigen-presenting cells (APC) located in the T-rich zones that surround the follicles. 10 Appropriate signalling among this cellular triumvirate will, independently of any follicular involvement, drive the extrafollicular (EF) expansion of the B cells and generate plasma cells producing some IgG but primarily IgM of low-to-moderate affinity for the antigen that initiated the response. While clearly not essential, it remains open as to whether CD40 engagement might modify the initial EF phase of B cells responding to TD antigen. This possibility was first highlighted by the study of Wheeler et al. where it was shown that co-ligating CD40 with IgM on the surface of resting (primarily EF) tonsillar B cells lowered the threshold requirement for signalling through the B-cell receptor (BCR) by approximately 1000-fold. 11 Unlike the well-established threshold lowering of the BCR signal seen on co-engaging the CD19/CD21 complex, 12 the CD40-dependent enhancement of proliferation that was observed with low levels of antigen surrogate (anti-Ig) was not a consequence of amplifying the BCR-dependent Ca2+ response (K. Wheeler & J. Gordon, unpublished obser-vations). Despite the dramatic outcome of CD40/BCR co-ligation in vitro, it was unclear as to when this combination of signals might be encountered physiologically: as na?ve B cells fail to turn on na?ve T cells13,14 it would be necessary to implicate the IDC – either directly or indirectly – in the provision of CD154 during an early phase of the response. Following their migration into 2° lymphoid tissue, Langerhans cells that have captured and processed antigen in the periphery turn off their antigen-processing machinery 15 and may thus hold residual antigen in a native configuration to be recognized by the B cell. This possibilty has recently been addressed by Wykes et al. 16 with in vivo studies in the rat. When spleen DC were transferred from rats immunized with haptenated keyhole limpet haemocyanin (KLH) to na?ve control animals, antibody responses to both hapten and carrier protein were observed in the recipients. 16 This strongly suggests that DC can retain intact antigen in vivo. Importantly, Wykes et al. were also able to conclude that such retention could reach 36 hr, a time-frame compatible with the reported kinetics for activation of both TH cells and B cells during primary antibody responses in vivo.16 Therefore, as long as it were still retained at the site of priming, a CD154-expressing TH cell would have the opportunity to signal – via CD40 engagement – an antigen-captured (and BCR-triggered) B cell ( Fig. 1). The triumverate model of B-cell activation. Having first taken up and processed antigen outside of secondary lymphoid tissue dendritic cells then migrate to the T cell-rich zones ready to present the foreign peptides to specific TH cells traversing these sites (1). Tissue-resident IDC constitutively express high-level major histocompatibilit complex (MHC) class II, CD40 and ligands for CD28. In this model, peptide/costimulatory molecule activation of the TH cell induces CD154 expression (2) that on docking with IDC-CD40, promotes expression of IDC-CD154 (3). Residual unprocessed antigen on the IDC allows receptor-specific B cells to be trapped and engaged (4) not only by BCR but concomitantly via CD40 engagement of the IDC-expressed CD154; CD154 can also be delivered to the B cell via the T-cell-expressed ligand. Thus an environment for BCR/CD40 co-ligation is established for efficient B-cell activation in response to a low concentration of captured native antigen. One difficulty with the concept of a physical ‘ménage-?-trois’ (as evoked in Fig. 1) is the sheer implausibility of the three cell types, each infrequent and motile, being together in the same place at the same time. A way out of this conundrum comes from the mounting evidence that implicates DC directly as providers of co-stimulatory signals to B cells, including that of CD40L. 17 Clark and co-workers have shown that ligating CD40 on human DC leads to an induction of both CD154 mRNA and functional protein: in this setting, CD40 and BCR could be simultaneously engaged by ligands presented at the DC surface. 18 This would closely mimic the in vitro system established by Wheeler and colleagues where B cells are activated by antibodies to CD40 and BCR presented on the surface of mouse fibroblasts. 11 Thus, the amount of native antigen needed to be held by a DC in order to stimulate a na?ve B cell into the proliferative cycle would be greatly diminished where CD40L had been induced: perhaps as a consequence of CD40–CD154 interactions generated during prior union of the DC with a specifically primed TH cell ( Fig. 2). This would be analogous to the model proposed by Matzinger and colleagues for the activation of cytotoxic T cells, whereby a CD40-positive DC is ‘licensed’ by a CD154-expressing TH cell to stimulate – at some later juncture – an antigen-specific TC cell. 19 Licensing of IDC for B-cell activation. (a) Foreign peptide-specific TH cells are primed by IDC as described in Figure 1. (b) Induction of CD154 on IDC (3) by the above interaction licenses the IDC to subsequently activate B cells bearing BCR specific for residual native antigen (4) even once TH cells have left the site of initial priming. Again, conditions for joint BCR/CD40 ligation are established, equipping the B cell with co-stimulatory molecules and processed peptide (5) for subsequent cognate engagement with primed TH cells. Cytokines released by the activated IDC, among which will be IL-12, contribute to ensuing B-cell development. Given the capacity of CD40L to stimulate na?ve B cells directly, the possibility then exists of bystander activation for any B lymphocyte that happened to be traversing sites of priming in an ongoing TD response. However, Nature seems to have tailored an elegant fail-safe mechanism to militate against such a potentially disastrous outcome. Garrone and colleagues first reported the rapid induction of Fas (CD95) that occurs following the ligation of CD40 on resting tonsillar B cells: the of Fas the B cell to as an of the CD40-dependent B-cell and differentiation that would in the of In an elegant to this and co-workers that engagement of BCR – not induction of Fas on either na?ve or B cells – from Thus, B cells engaging CD40L at sites of priming – either by or – would be for that had been into the response by of their antigen would be this cells are a of the with CD40 ligation an important signal for its has recently been and B cells TD signals – potentially at DC – to human B cells primed by CD40 and BCR was found to drive to a by that was from that with the The of were shown to be the induction of was of by but to the of The of in B cells primed with signals was left by the of was that the results a model for the and of B cells to during a TD response at the of the TH cell that of the This was with from the mouse that B cells in TD responses in vivo a or antigen challenge with the taken being the of the and colleagues have shown that B cells to conditions that normally drive T cells a to for this to the that DC not – by – an exclusive environment at sites of priming in T-dependent B-cell responses. a na?ve B cell being into a TD response a follicular or extrafollicular of it be as by na?ve B cells with to primary and is it a of the signals the CD40–CD40L interaction is known to the follicular of B cells in TD to model this in vitro to address such have the provision of a CD40 In reported on how EF B cells react to co-ligation of their CD40 and BCR by a with at of GC cells. Wheeler and found that signalling of EF B cells through CD40 and BCR and expression of a of and with the of a but of the of While these are compatible with a GC were Thus was on the cells the GC are are A outcome was by and colleagues not only the induction of as described but also reported the of the in EF B cells following BCR/CD40 Again, the failure to and an to highlighted the of EF B cells to a GC A that engagement of may contribute the of a GC recently from studies by et al. in which provision of a signal was reported to the of BCR/CD40 co-ligation in this that the of the CD40 signal might be a factor in whether a B cell the follicular or and colleagues established of EF tonsillar B cells on levels of this were able to – for the first – high-level induction of a antigen for B cells located within induction of to levels to found on GC B cells a high threshold expression of a reduction in the amount of CD40L by the was to capacity Induction was being as early as 12 While the physiological of remains the molecule was shown to be functional in that subsequent of cells to – a known for the – in their Importantly, the of et al. established that the engagement of an EF B cell with cell levels of CD40L was to into the cell a threshold of CD40 engagement needed to be it could the GC of is that such a would be only once a of T-cell priming had been Thus the of CD40L on the TH cell – or as discussed on the DC following its prior during T-cell priming – would dictate the to be taken by an B cell ( Fig. elegant study by and colleagues mice a CD154 to an by that CD40L was a factor in the of IgG antibodies GC in response to by TD above of CD40L were to drive in the quantity of and antibodies whether to or outside of the antigen-specific B cells and TH cells can now cognate interactions via peptide and that in CD154 on the TH cell to signalling through B-cell CD40 of the (3) will on the of the ensuing B-cell response. The quantity of CD154 (as on the TH cell but with a from the whether the subsequent taken by the B-cell remains extrafollicular leads to its the CD40 the B cell into a where it is by activated TH cells (5) to proliferation and to a low of CD40 an extrafollicular response in the of plasma cells producing antibody (primarily IgM but with some of affinity B cells that have either or high-level engagement are to expression but are from – either within or outside of the – by prior BCR Following a of expansion, B cells that have into this then and to the Fig. the for somatic hypermutation the from which antigen trapped on follicular dendritic cells the cells from the GC either to or to an of and in the and of the the within the where follicular dendritic cells are now also throughout the GC in to B foreign peptide-specific TH cells to the and within the that the follicular B cells which were the resting prior to its by lymphocytes. and from the within the with interactions (1). cells the for hypermutation (2) with bearing receptors with high affinity for the antigen that initiated the response – now trapped as on (3). to (and antigen to TH cells CD154 is for surface to B-cell CD40 high this provides a signal for to to the for of somatic A that has of engaging and antigen – on to cognate T-cell would be signals from TH cells with a in could serve to direct out of the plasma cells an of antibody with a for a and response to antigen The early that CD40 antibody delivered a potent signal to GC B cells was a physiological when CD40L was first and as an inducible surface molecule of TH cells. studies have subsequently the of the CD40–CD40L interaction both in GC responses and in the of into the 1–5 Fig. the studies with CD40 highlighted the of cells from the GC – compatible with differentiation – the possibility that CD40 signalling might also be in a was by studies of et al. 36 it was shown that GC B cells on from cells with CD40L not only to cycle but the of is a that among B cells is not only unique to within the GC but is also a necessary of their to be for into the to of the GC B-cell in vitro, it has been that CD40 signals might be into the to of and 36 GC B cells can be on a CD40 signal for a of 36 In the GC there would be the opportunity for to be encountered being delivered for or the TH cells located in the A study of that might the CD40-dependent of GC B cells in vitro of in this for GC B-cell was to and – an combination for CD40-dependent GC B-cell – had been reported by et al. as conditions for in human B-cell of the found to be in GC B cells in vitro have been at the mRNA in GC T cells. has been shown at both the mRNA and protein in tonsillar and colleagues have reported a requirement for in the of cells of dendritic were for the of in this et al. have described a unique of mRNA for and that this DC may also contribute to the of GC B cells in Although GC B cells could be for to with both a CD40 signal and the of from such retained of GC B cells with a being the of The being by the but could be for an normally by for et al. recently described a protein on that the CD40-dependent of GC B cells. A on CD40 signalling for has been for B cells from lymphoid follicles of In and colleagues have a requirement for CD154 in GC responses in vivo. that of to mice even as as into a TD response to an of the GC 9 to the system described it was whether – in combination with CD40 signals – could the that occurs within in vivo. GC B cells were by of during proliferation in with of the of levels of CD40-dependent and of from cells by that the combination was This was by which also that the generated were located in the and and with in hypermutation in vivo. This to the that somatic in the GC B-cell may be and not a consequence of The of CD40 signalling to the hypermutation is unclear the of the combination to CD40-dependent as GC B cells could not be in the of CD40 of the on CD154 in its capacity to out the possibility that – among the generated during the – some may now be receptors for CD154 exists located in the of TH cells within the and in the of GC are to as to of B 10 on this CD154 is to the cell surface. and colleagues have that GC B cells are of processed antigen to GC T cells while, it was shown that the T cells a with TD antigen are of the TH cells activated during the priming phase and thus for the processed that initiated the the above in a of can be established that – – would militate against the consequences of of high affinity into the with CD154 as the Thus, that have for foreign antigen held in the of on then present the peptides to GC T cells, the T-cell receptor engagement to the induction of CD40L that on docking with the B CD40 and differentiation any and antigen would be such with the T cells by to in the of engaging Again, to that such a fail-safe mechanism remains that it has been proposed that – as during the initial EF response – any could are as a consequence of interactions for which would now be Following selection in the what is the The possibility that some may to the to the machinery has been A of in vitro primarily on B-cell have that CD40L provides a potent signal for plasma cell have that the may be – that ligating CD40 B cells from cells. A study to address this in of the Thus, et al. that both in B-cell and GC B cells from CD154 antibody by the levels of but not could not be by or BCR on of the interaction could B cells be to was that the data for the observed lack of plasma cells within a for the selection of cells. The are with observations that CD40 ligation the of cells with a from GC are also by the lack of differentiation in CD40-dependent of GC B cells with among which and that are with antibody The focus of this review has been on CD154 as a delivered to the B cell via an T cell. to its in antigen-specific T-dependent B-cell responses is the that CD40L is both and during cognate first at the EF priming phase then during the selection in the However, the of CD154 has been and cells have also been described as providers of CD40L. long as CD40L expression is which it to its on these cell types is not but what B cells could express seems that – conditions – Induction of CD154 activation has now been described for both and B cells. The levels expressed to be Thus, in the first of CD40L expression on human B cells by et al. of were to a signal for CD154 low levels only of surface CD154 could be its being through by A study by the same has that B-cell expression of CD40L may be to stages of maturation such that the potentially that might arise via this would not be an of B cells. The of the is in from both human and mouse of the where a of B-cell CD154 has been A expression of CD40L has been for B-cell and that CD40–CD154 signalling might contribute to the or of the cells in such for a B CD40L can be a has established that lymphoma a of cells with GC B-cell as highlighted by the following and While in tumour cells and early from fail to express the in B-cell and in carry and established from these to the original even on the tumour is by a the and of that normally GC in order to the at these retain the in of cell not only are but cells can also be to by a of signals including that of BCR The of to GC is to the outcome of ligating CD40 on the tumour cells. Thus, CD40 or CD154 of 36 This to a of the potent by and for GC B cells cells with an signal to in a concomitantly a signal from these is to be through the expression of it has been shown that the of any high-level engagement of CD40 by CD154 can in the of this now out of a to CD40L is to for with CD40L a of for a this was the formation of together with gross in cell and a of and In some a and of was also was a the induction of protein and the of CD40 in cells to in response to CD154; the functional of CD40 is unclear it may serve to the of cells to CD154 signals as discussed the quantity and quality of the CD154 offered to a B cell to be important in the functional response that 1). One of the CD40-dependent described to is the of seen among both B cells and such formation both and of While B-cell can be initiated with low levels of either or CD40 its is Thus, only or the site on CD40 are in this fail to reported to occur in an is the of in human B cells: there is a that this outcome be on the of that is by the of on While – in resting B cells – of the cell cycle is there a requirement for of the receptor to the this can be by of and CD40 by with a secondary or by CD40 CD154 as a In marked receptor any need for for a CD40-dependent GC B cells can be from by the provision of CD40L CD40L has been in from activated B cells and activated TH cells, CD40L from the can on may be that within an in vivo B-cell of CD154 on traversing through the of a The for the of that can arise in B cells on their stages of and the way that CD40L is presented to is of the may be to the cells for the of The of CD40 of of but signalling pathways via a of known as tumour necrosis factor the – and – have been reported to directly to can and some can with each Given that can pathways to the and levels of could be in the of the response to a CD40L of expression among B cells has been described et The molecular configuration of CD40 within the may also be a factor the of engaging Thus, the of CD40 – as discussed for cells – could the way is to the of the in of the proposed need for in order to signalling via of the EF B cells CD40 in GC B cells from the same levels of & J. Gordon, unpublished has been shown to be an signalling through the CD40 with to be The of CD40 might thus serve as a for CD154 as observed with cells to CD40L in or as in GC B cells as a consequence of CD40 a in molecular could the of CD40 that occurs during the of B cells. but not of CD40 engagement be is could within the CD40 could in the of to is to whether there is a physiological to such A of a CD40 expressed by some B cells and from was recently as from an of the to its is that of CD40L specific to a Thus it was discussed how CD154 was at high on cell in order to on EF B cells. this not the of CD40 that would be by of the high-level of the receptor with in combination with a CD40 outside of the site could not for & reported that CD154 could in B cells, could remains in such is the from the cells, to the that The CD40–CD154 pairing has to be a molecular union in B-cell in that and are generated T-dependent antigen Despite its now the consequences of the of CD154 on B in both and of the quality and of the its and the of CD40-dependent have been The of CD40L its in the of B-cell and in an it is to that for B cells engaging at is in some cellular that with can also at J. J. Gordon, J. & The of the for such for The of the described in this was through from the and the is a would to and colleagues have to and presented – and are for their and of the