Programmers of CD4 T Cell Responses

222 Infectious Disorders – Drug Targets, 2012, 12, 222-231 B Cells: Programmers of CD4 T Cell Responses

Tom A. Barr*, Mohini Gray and David Gray

Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3JT, UK

Abstract: B cells are once again gaining prominence as important programmers of CD4 T cell responses. With widespread use of B cell depletion therapy in the clinic, proving effective in treating diseases previously considered T cell- mediated, the time is right for a re-appraisal of the B cell. Though typically considered weak, Th2 driving APC, it is now clear that they are necessary for a robust and long-lived CD4 T cell response in many settings. The sphere of B cell influence extends well beyond that of simply antibody production; antigen presentation, cytokine secretion, costimulation and development of lymphoid tissue architecture are all critical aspects of B cell immunobiology, the absence of which has se- rious impacts for T cell priming and memory. The aim of this review is to look at non-antibody mediated B cell function and to ask how, where and when do B cells influence the CD4 T cell response? Keywords: B cell, T cell, antigen presentation, TLR, memory.

INTRODUCTION programme the CD4 response. Although B cells are not “professional” with regard to the diversity of antigens they The term “professional” antigen presenting cell (APC) can present, they are exquisite APCs for their specific anti- was first coined by Lassila, Vainio and Matzinger in 1988, gen. Thus DCs, though “professional”, can be viewed as a following the demonstration that dendritic cells (DCs) and jack of all trades, whereas the “amateur” B cell is the master macrophages are necessary and sufficient for T cell priming of one!. [1] Whilst the fact remains that initiation of the immune response is dependent upon the “professional” APC, a role for Increasing use of B cell depletion therapies (BCDT) in B cells in T cell priming and maintenance, though for years the clinic has lead to a revival in interest in B cells and is controversial, now seems irrefutable. forcing a re-appraisal of their role in orchestrating immunity; responses previously considered predominantly T cell medi- The potential of B cells to influence the CD4 T cell re- ated are proving highly responsive to B cell depletion ther- sponse was first demonstrated in 1982 by Ron et al who apy in a way not reconcilable by ablation of auto-antibody showed a failure of proliferative T cell responses to protein [16, 17]. Thus, factors other than antibody secretion by B antigens in B cell depleted mice [2]. Since these early ex- cells are also at play here. Ablation of B cells at defined periments, a requirement for B cells in robust T cell immu- points in the immune response also removes the complica- nity has been demonstrated in many models; including sal- tion of developmental issues seen in B cell deficient mice, monella [3, 4], malaria [5-7], coronavirus [8] and herpesvi- and thus impacts on T cell responses can be more directly rus [9] infections. These studies have not been without con- attributable to the transient depletion. troversy. Given gross abnormalities seen in genetically- modified B cell deficient mice, including aberrant lymphoid The questions of what aspects of B cell biology influence organogenesis [10], reduced T cell numbers [11] and loss of T cell responses and where and when these interactions oc- follicular DC [12], directly attributing the T cell defects in cur remain unclear. The aim of this review is to reconsider these studies to a lack of B functionality per se has been the B cell, to look beyond their role as antibody producers problematic. In particular, a role for B cells in T cell priming and ask how do they programme the CD4 T cell response?. is highly contentious, with demonstrations that the potent antigen presenting capacity of antigen-specific B cells is INNATE AND ADAPTIVE SIGNALS IN B CELL AC- reserved for re-activation of memory cells [1, 13, 14]. TIVATION Relegation of B cells to the “amateur” league has left Professional APC become activated by sensing patho- them as under-appreciated participants in the T cell response. gens and/or danger signals in the environment, through their In particular, the potency of an antigen specific B cell to pre- expression of a range of receptors for pathogen-associated sent antigen to primed or memory T cells is considerable molecular patterns (PAMPS), (e.g. TLRs, NLRs and RLRs) [15]. Given that memory is the hallmark of adaptive immu- [18, 19]. These receptors allow the APC to sense local dan- nity, the holy grail of vaccine design, and when directed ger cues and provide an immunological context in which to against self-antigens, the cause of chronic autoimmunity, it is appropriately respond. This activation signal, often called important that we do not lose sight of the B cells’ capacity to ‘signal 0’, represents the primary activation pathway for innate APC, driving maturation following antigen acquisition and their subsequent migration from the periphery to T cell *Address corresponding to this author at the Institute of Immunology and Infection Research, University of Edinburgh, Ashworth Laboratories, zones of secondary lymphoid structures. B cells on the other King’s Buildings, West Mains Road, Edinburgh, EH9 3JT, Tel: +44 131 hand, through their dual expression of hypervariable B cell 650 5488; Fax: +44 131 650 7322; E-mail: [email protected]

2212-3989/12 $58.00+.00 © 2012 Bentham Science Publishers B Cells: Programmers of CD4 T Cell Responses Infectious Disorders – Drug Targets, 2012, Vol. 12, No. 3 223 receptors (BCR) and germline encoded PAMP receptors, with appropriately armed T cells. For example, we have have two primary pathways for activation. TLR on B cells, shown that B cells stimulated with CpG rapidly shed as with professional APC, sense a broad range of pathogenic CD62L, which focuses them to the spleen during Salmonella patterns and cause direct activation, proliferation and matu- infection [33]. ration [20, 21]. The BCR however is highly specific, recog- One of the most important outcomes of TLR stimulation nising defined epitopes on antigens in their native conforma- of B cells is the induction of cytokine production [21]. In tion. This specificity lies at the heart of B cell clonal selec- particular IL-10 and IL-6 are produced in significant quanti- tion and expansion. Unlike innate APC, B cells must com- ties following TLR stimulation, pointing to potential regula- pete for the affections of T cells within the germinal centre, tory and pro-inflammatory roles for cytokine producing B with the fittest B cells profiting from T cell help and prolif- cells. Other cytokines are reported to be produced following erating as the immune response matures [22]. The influence TLR stimulation; for example IFN is secreted when B cells of B cells on T cell responses is generally attributed to these are stimulated with combinations of TLRs (eg. TLRs 2, 4 later stages of the response. However the sphere of influence and 9) [21]. Production of immunomodulatory cytokines is of B cells is now recognised to extend to the primary re- an area of great interest at the moment, and is dealt with in sponses through their expression of a diversity of innate re- more detail below. ceptors. ANTIGEN-SPECIFIC ACTIVATION OF B CELLS INNATE ACTIVATION OF B CELLS VIA TLRS - VIA BCR - SIGNAL 1 SIGNAL 0 The BCR is a unique receptor for cellular activation; it Both human and mouse B cells express many of the has dual functionality acting both as a mechanism to initiate TLRs. B cells respond rapidly and robustly to stimulation via signalling cascades and as a means to internalise antigen for these receptors (see [20, 23-25] for reviews). Of the TLRs processing and presentation. During B cell development the expressed by B cells, TLR4 and TLR9 (receptors for LPS BCR is pivotal to controlling B cell fate, with ligation to and CpG respectively) have been the most extensively stud- self-antigens leading to anergy or deletion. Once mature B ied. LPS has long been known to act as a B cell mitogen, cells enter the periphery, as transitional B cells, the BCR causing activation and polyclonal expansion of B cells irre- becomes the principal receptor for activation and prolifera- spective of BCR reactivity through complex binding to LPS- tion. Engagement of the BCR by antigen leads to phosphory- binding protein (LBP), CD14 and TLR4 [26]. B cells also lation of the intracellular ITAM tyrosines by LYN, SYK and express high levels of the TLR9 receptor. The outcomes of SH2 kinases resulting in the initiation of a variety of signal- TLR stimulation are diverse and there are several points at ling cascades, inducing activation and proliferation of the B which such innate activation of B cells can impact on the T cell (see [34] for review). cell response. Following BCR mediated activation B cells rapidly Perhaps the most obvious impact of TLR stimulation is upregulate the expression of numerous co-stimulatory mole- the influence on antibody secretion and this in turn influ- cules; key players in T:B interactions and the mediators of ences antigen opsonisation. Complexing of antigen with an- the cognate interactions known as T cell “help”. As with tibody has an adjuvant effect, enhancing uptake and the effi- innate activation, BCR ligation also influences the secretion ciency of processing and presentation by APC. The require- of cytokines. Though BCR ligation alone does not seem to ment for TLR ligation on B cells as a necessary prelude to a induce cytokines, this activation signal often strongly syner- class-switched T dependent antibody response has been con- gises with other signals such as TLR (e.g. TLR9) [35] and T troversial in recent years, with compelling data for the TLR cell help (e.g. CD40 + BCR IL-4 [36]). requirement [27] and also against [28]. Disparities in these results are striking and difficult to reconcile, but our own The second critical role of the BCR in T cell program- work on this problem shows the requirement for B cell- ming is its utility as a means of internalising and processing specific TLR stimulation is highly dependent on the nature antigen. Internalised protein antigens are processed in the of the immune challenge and the isotype of the antibody endosomal compartments and loaded onto class II molecules produced [29]. So during the response to Salmonella the iso- for transportation to the cell surface and presentation to T type that is most affected in chimeric mice with a B cell cells. This represents the first pivotal step in T:B interac- compartment that cannot signal via TLR (MyD88-deficient) tions. If this interaction is interrupted, either through specifi- is IgG2a/c that is dependent on IFN. cally inhibiting class II expression by B cells [37] or because B cells have a non-functional BCR (MD4), then the subse- TLR ligation on B cells causes upregulation of several quent T cell response often fails [4]. important cell surface molecules involved in T:B cell interactions, including MHC II for antigen presentation, CD40 , CD80 and CD86, ICOSL (B7h), BAFF and many others (see INTEGRATING SIGNALS: DUAL ENGAGEMENT OF TLR AND BCR [20, 24, 30-32] for reviews). These changes again impact on the effectiveness of B cells as APC and as potential pro- In most settings stimulation of TLRs and BCR are un- grammers of T cell differentiation. In addition, B cell ex- likely to occur independently, pathogen products often ac- pression of homing molecules and chemokine receptors is company, or actually are themselves, BCR antigens (e.g. differentially regulated upon exposure to TLR ligands, ena- flagellin [38]), thus it is important that we understand the bling B cells to traffic to distinct areas of lymphoid tissues, kinetics of TLR / BCR interplay if we are to gain insight into and thus improving the chances of meaningful interactions how these signals influence the T cell response. We have 224 Infectious Disorders – Drug Targets, 2012, Vo l. 12, No. 3 Barr et al. recently demonstrated that TLR and BCR signals to B cells yond the scope of this review. To highlight the reciprocal drive two functionally and temporally separate waves of in- nature of this dialogue, two examples are given below. fluence during bacterial infection; the early phase is largely A critical early costimulatory molecule is CD40. This TLR dependent and results in secretion of cytokines (e.g. IL- receptor is expressed by B cells and acts as the target ligand 6, IFN and IL-10) which are critical for optimal Th1 prim- for CD154, expressed by activated T cells. Ligation of CD40 ing. Conversely, the late phase is dependent upon BCR me- on B cells provides a potent activating signal, synergising diated uptake of antigen and subsequent presentation, as B with BCR signals to promote B cell survival and differentia- cells become “follow-on” APC necessary for memory T cell tion [42]. However, in addition to driving B cell activation, development [4]. Similar BCR/TLR co-ligation is also im- signals also occur in the reverse direction via CD154 leading portant in autoimmunity; antibody:auto-antigen complexes, to enhanced activation and proliferation of T cells [43, 44]. containing DNA, bypass the requirement for T cell help and TCR/CD154 stimulated T cells do not produce IL-2 and driving development of lupus [39] and recovery from EAE quickly succumb to apoptosis, thus it has been suggested that requires both TLR [40] and BCR [41] signalling in B cells. this pathway favours generation of short lived effector cells [43]. T:B COGNATE INTERACTIONS A second important costimulatory pathway is CD80/86 – In addition to recognition of pathogen associated signals CD28; activated B cells express the former and activated T via TLR (signal 0) and antigen via the BCR (signal 1), B cells the latter. Cognate interaction of CD80/86 with the cells also require additional signals derived from T cells CD28 receptor is essential for robust T cell activation and is (signal 2). These signals, termed T cell “help” enable B cells well characterised for innate APC such as DC [45]. The spe- to generate high-affinity class switched antibody. T cell cific requirement for B cell expression of B cell CD80/86 “help” occurs in two distinct phases; early events are medi- has been demonstrated by O’ Neill et al. In a mouse model ated by cell contact (e.g. CD154-CD40) and later events by of arthritis they showed that T cells failed to proliferate in soluble factors (e.g. cytokines). This interaction is viewed the absence of B cell CD80/86 expression [46]. largely as a uni-directional event, with T cells directing “help” at the antigen presenting B cell. However, our in- B cells are highly efficient at presenting antigen that is creased understanding of T:B interactions shows that help is bound by their BCR, facilitating activation and antigen up- a dialogue, with B cells giving as well as receiving informa- take [15]. As clonal expansion of antigen-specific B cells tion Fig. (1). T:B cell interactions are initiated following takes several days, the contribution of B cells is usually con- BCR binding to Ag and presentation of processed peptides sidered to happen in this late phase of the immune response on MHC II molecules as described above. In addition to pro- [47-50]. However evidence is accumulating that B cells exert viding the points of cognate interaction (i.e. TCR/MHC II their influence in T cell programming far earlier than previ- peptide complex), BCR binding to antigen also up-regulates ously anticipated. We have previously shown that antigen accessory molecules crucial to successful T cell help. Many presentation by B cells is necessary for optimum priming to molecules are involved in the cognate dialogue between B soluble antigen, as bone marrow chimeric mice in which B cells and T cells, and a thorough discussion of them is be- cells do not express MHC class II have impaired T cell activation very early after immunisations [37]. Recently we have

IL-12R Th1 IFN ‘Late’ co-stimuli IL-2 T-bet (CD80-CD28) LT (B7H-ICOS) IL-12 ‘Early’ co-stimuli IL-4R Th2 IL-4 IFN IL-5 (CD40-CD154) GATA-3 IL-2 IL-13 Antigen T IL-4 presentation T Signal 0 IILL-2121RR T IL-6 FH IL-21 (TLR) Bcl-6 B Later IL-6 TGF cytokines IL-23R Th17 IL-17 IL-21 TGF Signal 1 ROR T IL-22 (BCR) IL-2 IL-10 Treg TGF FoxP3 IL-10 IL-2R IL-35

Fig. (1). B cell dependent CD4 T cell polarization. Activation of B cells is dependent upon 3 distinct signals; signal 0 via innate receptors such as TLRs, signal 1 via BCR recognition of specific antigen and signal 2 in the form of T cell help. Though often considered as a one-way conversation, T cell help is actually a dialogue. B cells present antigen and secrete cytokines in response to activating signals, which in turn, drive T cell polarization. B Cells: Programmers of CD4 T Cell Responses Infectious Disorders – Drug Targets, 2012, Vol. 12, No. 3 225 used anti-CD20 depletion to remove B cells at defined time- seen as analogous to Th1 and Th2 T cells. Be1 cells secrete points during Salmonella infection; surprisingly T cell re- IFN, IL-6, IL-10 and IL-12, whereas Be2 cells secrete IL-2, sponses are impaired in mice depleted of B cells within the IL-4, IL-13 and IL-10. The interesting point in this study is first 10 days of infection, but not later in the response (TB, the requirement for pre-polarised T cells, that is the Be cells DG manuscript in preparation). Work from other groups seem to part of a positive feedback loop to increase T cell supports this idea of B cells as important primers of the T reactivity (e.g. Th1 drives Be1, which drives Th1). B cells cell response. Within hours of intradermal injection of anti- are not entirely dependent upon T cells for cytokine produc- gen, peptide:MHC II complexes are detected upon the sur- tion; antigen and innate signals also drive cytokine produc- face of B cells by acquiring soluble antigen from the subcap- tion, as discussed above, thus opening the possibility of T- sular sinus of draining lymph nodes in a DC independent independent Be generation and subsequent T cell polarisa- fashion [51, 52]. Similarly, B cells have been shown to ac- tion. quire particulate antigens and immune complexes from macrophages that capture such antigens in the subcapsular B CELLS AND EFFECTOR T CELLS sinus of lymph nodes [53, 54]. Thus B cells collect and present antigen within hours of the immune response and not Several studies have pointed to a key role for B cells in days or weeks as often suggested. The mechanisms em- the development of a robust inflammatory response in range ployed by B cells to capture antigen this early in the re- of viral and bacterial infections. B cell deficient mice in- sponse remain unclear. Antigen-specific B cell frequencies fected with lymphocytic choriomeningitis virus (LCMV) are too low to account for uptake, thus some other mecha- show impaired IFN production as early as day 8, a pheno- nism must be responsible; non-ag-specific uptake of soluble type which becomes even more pronounced when looking at antigen has been shown [55]. Complement and Fc receptors the memory response [60]. Though this group did not dem- can bind and internalise soluble antigen following opsonisa- onstrate directly that B cell-derived pro-inflammatory cyto- tion [56]. TLRs have been shown to bind and internalise kines were responsible, they did demonstrate that it was not PAMPs and associated peptide antigens in innate APCs [38], due to impaired cognate interactions. BCR transgenic mice providing potential antigen non-specific route. However (mIg-Tg mice which have no LCMV-specific antibody) CpG:Ag complexes have been shown to gain entry only to B mounted a normal memory Th1 response. A role for B cells cells carrying an appropriate Ag-specific BCR [35]. in driving the Th1 response has also been reported in HSV [61]. By depleting different populations of APC (DC, macro- Less controversial is the role of B cells as antigen pre- phage and B cell) authors of this paper showed that the senting cells late in a response, once clonal expansion has protective Th1 response was dependent on both B cells and occurred. Ag-specific B cells, through expression of a high- dendritic cells. Though again a direct role of pro- affinity BCR are excellent at capturing and presenting when inflammatory cytokines from B cells was not investigated. antigen levels are low [57, 58] and so at this stage of the Two models which have directly addressed the role of B immune response B cells likely become the predominant APC. Roles for B cell antigen presentation in CD4 T cell cell derived TNF and IFN are Toxoplasma gondii and responses are well documented. For example, in Salmonella Salmonella typhimurium respectively. Menard et al showed that B cells from T. gondii infected mice promote IFN pro- infection, we have demonstrated that an absence of B cell presentation, either through a lack of B cell MHC expres- duction in a TNF dependent manner. In the Salmonella sion, or through having a fixed BCR of irrelevant specificity, model, early studies by Mastroeni et al uncovered a critical results in failure of the memory CD4 response [4]. These role for B cells in the development of Th1 memory [3], and mice then rapidly succumb to secondary infection, dying if as discussed above, we have now demonstrated that antigen challenged with a virulent Salmonella strain. presentation by B cells is key here [4]. However, we have also shown that B cells produce IFN during Salmonella infection, and that in the absence of B cell-derived IFN, the B CELL-DERIVED CYTOKINES AND THE T CELL Th1 response is also impaired [4]. Similarly, we show the RESPONSE Th17 response seen during Salmonella infection is partially In addition to the ability of B cells to influence the T cell dependent upon B cell secretion of IL-6. The IL-17 response response through the cognate interactions of antigen presen- is clearly not critical in Salmonella infection as knock-out tation and co-stimulation, B cells also secrete soluble media- mice with no Th17 response still clear infection [4]. How- tors, such as cytokines. Despite demonstrations of the B ever, in EAE, a disease model involving Th17 cells [62] we cell’s capacity to secrete cytokines almost two decades ago have uncovered a critical role for B cell-derived IL-6. We [59], this aspect of their function is frequently overlooked, find that mice in which the B cells cannot make IL-6 show and is only recently gaining prominence as an important con- less severe disease, improved recovery and reduced IL-17 tributor to T cell function. Thus, as with other APC, B cells production when compared with WT controls (Barr, Filla- promote differentiation of CD4 T cells appropriate to the treau and Gray manuscript in preparation). We conclude that prevailing conditions and context of antigenic encounter; B cells are critical contributors to the cytokine environment driving Th1, Th2, Th17 and Treg responses through provi- that leads to primary T cell polarization, even under inflam- sion of appropriate cytokines. matory Th1 or Th17 conditions. An important study by Lund et al showed that, in much A role for B cells in Th2 responses has been proposed as the same way as T cells, B cells can become polarised and result of their inability to secrete IL-12 [63] but may also be express discrete profiles of cytokines [36]. These distinct B related to the strength of signal they can deliver as APC [64]. cell subsets, termed B effector 1 and 2 (Be1 and Be2), can be Their Th2 driving capacity has been shown in several mod- 226 Infectious Disorders – Drug Targets, 2012, Vo l. 12, No. 3 Barr et al. els. For example, in the mouse model of malaria (Plasmo- of AC to the cultures, except CpG, the TLR9 ligand. We dium chabaudi), immunity is characterised by an early Th1 have recently shown that the IL-10 enhancing effect of AC is spike, which switches to a Th2 response after 14-21 days [5]. lost if they are first DNase-treated or if B cell endosome In the absence of B cells, either through depletion [6] or acidification is prevented (necessary for TLR9 binding of through genetic deficiency [7], this shift to Th2 fails to oc- ligand). Furthermore the activity of AC in vivo to suppress cur. Clearly, in malaria, B cells are needed to bring about the collagen-induced arthritis is lost after DNase treatment and Th2 switch. Similarly, Schistosoma mansoni infected MT EAE, which is exacerbated in TLR9ko mice, cannot be alle- mice mount an aberrant T cell response, with reduced Th2 viated by injection of AC as it can in wild-type mice. Thus, it and enhanced Th1 cytokines [65, 66]. Though Jankovic et al, seems that DNA displayed at the surface of AC [77] is a state there is no change in the Th1/Th2 balance, IL-4 produc- physiological inducer of regulatory B cell function. Delivery tion by antigen restimulated mesenteric lymph nose cells is of the AC-displayed DNA to TLR9 in the endosome is likely greatly reduced. None of these investigations have directly to require recognition and uptake of the DNA-containing addressed a role of Be2 derived cytokines in driving Th2 complex on the AC surface by the B cell BCR, as B cells polarisation. This has recently been addressed by Lund and from mice which have a limited repertoire (BCR transgenics) colleagues. During infection with the Th2 driving intestinal show no response to AC. Interestingly the cells that show parasite, Heligomosomoides polygyrus, Be2 cells are critical most activity in response to AC are marginal zone and B1 B for the development of protective Th2 immunity, and that cells, both of which are known to contain cells with BCR this is dependent on their secretion of IL-2 [67]. reactivity (low affinity) to self-antigens [78]. This raises the spectre of self-reactive B cells that as a result of ubiquitous B CELLS AND REGULATION interactions with AC, have constitutive regulatory activity. These could act as a broad-based mechanism to prevent The production by B cells of IL-10 [68] and to a lesser breaking of tolerance and, in particular, to re-impose toler- extent TGF [69] is the basis of their regulatory function and ance and induce resolution during inflammatory lesions. has led to certain B cell subsets being termed regulatory B cells (Bregs). The first indication of a regulatory role came B CELL DEPLETION THERAPY (BCDT) from a number of studies in autoimmune disease models [41, 70] and more recently in infectious disease models [71], as Much of the renewed interest in the role of B cells stems discussed by Fillatreau et al in this issue. The answers to the from the increasingly widespread application of BCDT for following questions will greatly aid our understanding of B the treatment of a range of autoimmune conditions. Initially cell regulation: i) Is regulatory activity by B cells confined to proposed as a means of depleting autoantibody in autoim- certain subsets or are many types of B cells involved? Ii) mune settings, these treatments have proven effective even What are the physiological signals that induce IL-10 produc- when autoantibody is not depleted. Originally developed as a tion and regulatory finction? In answer to the first, it seems treatment for B cell lymphoma [79], BCDT has now been clear that some B cells when they are isolated have a greater studies extensively in both the clinic; Pemphigus vulgaris propensity to make IL-10 than others. Marginal zone and B1 [80}, rheumatoid arthritis [81], lupus [82] and multiple scle- B cells have long been shown to make most IL-10 [21]. rosis (MS) [83-85] and in animal models including lupus Mauri and colleagues have championed transitional B cells [86], diabetes [87], arthritis [88] and EAE [89]. A recent [72] as major producers, while Tedder’s group shows that a study by Tedder et al highlights an important consideration subset of CD1d expressing B cells in the spleen, termed B10 in BCDT; namely the timing of therapy in relation to disease cells [73] have most activity. It seems likely that all these progression [89]. Using the mouse model of MS and anti- cells for reasons related to their cell cycle/ activation status CD20 depletion, this group show that depletion prior to dis- are primed (ex vivo) to secrete IL-10. As some authors have ease induction increases severity, whereas ablation during shown, however, they also have a higher capacity to make disease progression ablates disease. other cytokines (eg. IL-6) [21]. In our experiments and those The studies above deal largely with complete ablation of of others [73], the suppressive IL-10 activity seems to be the B cell compartment. Though effective, future treatments dominant. Even if certain cells predominate directly ex vivo will almost certainly see refinements in this approach. Sev- it is also informative to ask what markers are expressed by eral more specific approaches are currently in development; IL-10 producers? The answer seems to be a much more di- in particular anti-CD79 [90] and anti-BAFF [82] treatments verse phenotype than predicted by the subset notion [74]. We have been used with some success. Clearly different targets believe that most B cells can be induced by the conditions have different potential therapeutic benefits. Different B cell and context of activation to become IL-10–producing regula- subsets, or their relative activation status, have been ascribed tory cells. different roles. This opens the opportunity to specific target- Many signals have been described that up-regulate B cell ing of distinct B cell effector subsets to treat specific aspects IL-10 production and regulatory activity. These include of a disease. For example MZ B cells are known to be a po- TLR2, 4, 9, BCR and anti-CD40 [21]. Given their role in the tent source of IL-6 [21]. They also have a unique phenotype resolution of autoimmune inflammation we wondered amongst B cells in expressing CD23, CD1d and CD9 [78] - whether an endogenous ligand would induce B cells to be- thus depletion therapy aimed at these receptors could prove come regulatory. We have found that apoptotic cells (AC) effective in specifically reducing B cell derived IL-6 and that have long been known to suppress inflammatory macro- associated Th17 responses, such as those seen in MS. Con- phage function [75] also induce a regulatory phenotype in B versely, we have shown that follicular B cells are the pri- cells, causing them to make enhanced levels of IL-10 [76]. mary source of B cell derived IFN [21]. Thus targeting fol- All IL-10 inducing stimuli could be augmented by addition licular B cells, for example by anti-CXCR5 therapy, may B Cells: Programmers of CD4 T Cell Responses Infectious Disorders – Drug Targets, 2012, Vol. 12, No. 3 227 prove to be an effective way of easing B cell mediated Th1 neity in the TFH compartment and we propose that the early inflammatory diseases. These approaches are very much in wave of TFH comprised of memory precursors. This hy- their infancy, but our increased understanding of the role of pothesis is supported by several arguments; first, Bcl-6, the B cells in controlling the T cell responses will enable the master transcription factor for TFH, is also necessary for development of specific B cell depletion approaches tailored CD4 [101] and CD8 [102] T cell memory differentiation and to treat specific aspects of disease. also memory B cell formation [103]. As Bcl-6/Blimp-1 balance represents the principal axis for effector cell versus B CELLS AND CONTROL OF T CELL MEMORY memory cell differentiation in both B cells and CD8 cells, it has been suggested that this may represent a universal path- A specialised role for B cells as APC only for memory T way for all lymphocytes [104]. A hypothetical mechanism is cells was proposed many years ago, following the demon- presented below in Fig. (2). Indeed, Bcl-6-deficient CD4+ T stration that B cells could activate primed, but not naive, T cells appear unable to generate a long-lived memory popula- cells. Certainly the data discussed in this review shows how tion [101]. A second corollary between TFH and memory important B cells are for a full, robust and long-lived re- CD4 T cells is the requirement for B cells [99] - MT mice, sponse, with T cell memory being particularly susceptible to which do not develop TFH do not develop memory in many perturbations in the B cell compartment. Though the specific models. Though the idea that B cells are in some way unique requirements for B cells in generating and/or maintaining T as APCs for TFH differentiation has recently been contested cell memory may vary from model to model, a growing by Deenick et al who show that though B cells typically act number of studies show that deficiencies in the B cell com- as APC for TFH, other cells can also induce TFH differentia- partment lead to specific failure of the memory response. It tion [105]. Another clue to the connection between TFH and is important to note that not all responses are dependent upon memory is gleaned from ICOS, a costimulatory molecule B cells for the generation of robust T cell memory. Normal, highly expressed by TFH. Patients with a deficiency in ICOS protective CD4 T responses have been demonstrated in B expression, also show impaired T cell memory [106]. Simi- cell deficient animals infected with Chlamydia [91], Listeria larly SLAM-associated protein (SAP) expression has also [92] and Francisella [93] to name but a few. Table 1 lists been shown to be necessary for both memory formation and some of the models, and outlines their dependence on the TFH differentiation [107-109]. presence of B cells for T cell memory. To further investigate the connection between CXCR5+ The possibility that B cells are “memory specific” APC is T cells homing to the follicle and subsequent memory, we an intriguing one, however a direct demonstration of this role have recently performed a series of experiments to preclude has been elusive. Could it be that B cell antigen presentation T cells from follicular entry. Thus by transferring CXCR5-/- is in some way specific in driving T cells down the memory T cells to Ly5 distinct recipients we show that only cells pathway? Or perhaps that they provide a specific niche, capable of entering the follicle differentiated to memory which favours memory T cell development or survival? Re- cells. We also show that ablation of B cells by anti-CD20 cent interest in the T-follicular helper (TFH) subset of T cells rapidly leads to reduction in TFH, and if ablated within the [94-97] could be shedding light on the role of B cells in first 7 days of an immune response, fail to generate a stable promoting and maintaining memory. TFH are a specialised CD4 memory population (Barr and Gray manuscript in subset of CD4 T cells which migrate to the B cell follicles preparation). This data is only correlative and not causal, but and deliver ‘help’ to B cells [98]. Entry to the follicle and strengthens the potential link between these two populations. thus, intimate interactions with B cells, is facilitated through We propose that migration of antigen-activated T cells the expression of the chemokine receptor CXCR5. Their through the follicle, by gaining a transient TFH-like pheno- development is controlled by the transcriptional repressor type, facilitates cognate B-T cell interactions, which pro- Bcl-6, which is antagonised by Blimp-1 [99]. motes B cell-driven expression of Bcl-6 and thus differentia- Clear correlative links exist between TFH cells and tion to memory cells. memory cells and the possibility that TFH are a heterogeneous population of T cells, which contain memory T cell pre- FUTURE PERSPECTIVES AND CONCLUSIONS cursors is a tantalising one. Recent work by Zaretsky et al The data accumulated over the last decade show that B has shown that CXCR5+ T cells which home to follicles during helminth infection display properties of both TFH cells are critical for the development of robust T cell immunity and that the mechanisms by which B cells program T (Bcl6 and IL-21 expression) and Th2 (GATA-3 and IL-4 cells are complex and varied. B cells exert their influence in expression) [100]. The development of these TFH cells was at least two temporal phases with TLR signalling important shown to be dependent on B cell cognate interactions via in early responses and antigen presentation critical in the late CD40/CD154. Similarly, our own recent studies of TFH dur- phase. In addition to the timing of the response and the na- ing Salmonella infection indicate that these cells are indeed a highly heterogeneous population. Though large numbers of ture of the activating signal, several groups have speculated that distinct subsets of B cells exist, with designated inflam- CXCR5+ T cells are found in the B cell follicles of infected matory and regulatory roles. Though robust data currently animals, they also possess Th1 characteristics (e.g. IFN) supports this idea, it seems likely there is subtlety to the role and, crucially, their arrival precedes germinal centre forma- of B cells as T cell programmers, which currently allude us. tion and T cell help by several weeks (TB, DG manuscript in For example CD1dhi marginal zone B cells are potent pro- preparation). Thus, it seems that follicular homing T cells go there to facilitate their entry into the memory pool, not sim- ducers of IL-10; thus it has been speculated that they are Bregs, however these very same cells also produce IL-6 and ply as designated helpers. This suggests functional heteroge- thus can act to promote inflammation. Thus any individual B 228 Infectious Disorders – Drug Targets, 2012, Vo l. 12, No. 3 Barr et al.

Table 1. Models of B Cell Dependent CD4 T Cell Memory

Infection/Antigen B Cell Deficiency(ies) T Cell Impairment(s) Reference(s)

Bacteria

MT; BCR null (MD4); MHC-IIko Th1 priming in MyD88ko chimera; Impaired Th1 mem- Salmonella enterica chimera; MyD88ko chimera; IL-6ko ory in MHC II chimera and BCR null; Impaired Th17 in [3, 4] chimera IL-6 chimera

Impaired IFN, IL-6 and IL-10 in lung; normal in geni- Chlamydia trachomatis MT [91, 110] tal tract

Listeria monocytogenes MT Reduced T cell proliferation and IFN [92]

Fungal

Pneumocystis carinii MT and CD40KO chimera Reduced activated T cells in lungs and dLNs [111]

Viral

IL-6 and IL-10 impaired in primary, elevated in mem- Influenza MT [50] ory; decreased CD62L expression

Lymphocytic choriomeningitis MT and Secretory Ig null (mIg-Tg) Impaired memory in MT; normal memory in mIg [60] virus (LCMV)

Impaired memory in MT; partially impaired in Herpes simples virus (HSV) MT and HELMT [9] HELMT

Coronavirus MT Impaired IFN in spleen and CNS [8]

Eukaryotic Parasites

MT; BCR null (MD4); MHC-IIko IL-4, IL-5, IL-10 and IL-13 impaired; dependent upon Heligomosomoides polygyrus chimera; IL-ko chimera; IL-2ko [67] BCR/MHC/ class II cytokines chimera; TNFko chimera

Plasmodium chabaudi B cell depleted; MT Reduced Th2; increased Th1 [5-7]

Schistosoma mansoni MT Impaired IL-4 and IFN [7, 66]

Soluble Protein

Keyhole limpet hemacyanin (KLH) MT; SCID+B cells Impaired memory precursor frequency and IL-2; [112, 113]

Primed early Short-lived effector(SLEC)

Naïve CD4 effector Th1 Th2 DC T Th17 B cell independent Blimp-1 TReg

DC priming ? Bcl-6

Bcl-6 B B cell dependent

Interactions with B Memory precursor Memory

cells effector cell (MPEC)

BCR? MHC? TFH-like cells?

CD40-CD154?

ICOS?

Fig. (2). B cell dependent CD4 memory differentiation. Priming of naïve CD4 T cells is dependent on presentation by professional APCs such as DC giving rise to early effector cells. These cells can they either differentiate to cytokine secreting effectors or the precursors of long- lived memory cells. We propose that upon interaction with B cells in the follicle, TFH-like memory pre-cursor cells differentiate to memory cells – a process antagonized by B-cell independent Blimp-1 driven effector cell differentiation. B Cells: Programmers of CD4 T Cell Responses Infectious Disorders – Drug Targets, 2012, Vol. 12, No. 3 229 cell may well have the capacity to act either to promote or [12] Fu, Y. X.; Huang, G.; Wang, Y.; Chaplin, D. D. B lymphocytes suppress a T cell response dependent upon the immunologi- induce the formation of follicular dendritic cell clusters in a lymphotoxin alpha-dependent fashion. J. Exp. Med., 1998, 187(7), cal “context” in which it finds itself. It is important to note 1009-1018. that the B cells influence is early and late. The early phases [13] Fuchs, E. J.; Matzinger, P. B cells turn off virgin but not memory T have been overlooked in the past, but a role for B cells in T cells. Science, 1992, 258(5085), 1156-1159. cell priming is now becoming clear. Unearthing the signals, [14] Epstein, M. M.; Di Rosa, F.; Jankovic, D.; Sher, A.; Matzinger, P. which control pathogenic versus regulatory function in B Successful T cell priming in B cell-deficient mice. J. Exp. Med., 1995, 182(4), 915-922. cells will almost certainly provide a rich area for the devel- [15] Sallusto, F.; Lanzavecchia, A. Efficient presentation of soluble opment of novel therapeutics. That B cells act as memory antigen by cultured human dendritic cells is maintained by specific APC is a tantalising idea; B cells are certainly nec- granulocyte/macrophage colony-stimulating factor plus interleukin essary for T cell memory. 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Received: January 18, 2012 Accepted: February 25, 2012