iNKT cell help to B cells: a cooperative job between innate and adaptive immune responses
Keywords: CD1d, B cell help, vaccines, T follicular helper cells
Paolo Dellabona1*, Sergio Abrignani2*, Giulia Casorati1* 1 Experimental Immunology Unit, Division of Immunology Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy 2. Istituto Nazionale di Genetica Molecolare, Milano, Italy *corresponding authors
Contact author: Paolo Dellabona, Experimental Immunology Unit, Division of Immunology Transplantation and Infectious Diseases, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milano, Italy. E-mail: [email protected]; phone: +39-02.26434727
Received: 24-Feb-2014; Revised: 21-Apr-2014; Accepted: 25-Apr-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/eji.201344399. This article is protected by copyright. All rights reserved.
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Abstract T-cell help to B lymphocytes is one of the most important events in adaptive immune responses in health and disease. It is generally delivered by cognate CD4+ T follicular helper (TFH) cells via both cell-to-cell contacts and soluble mediators, and it is essential for both the clonal expansion of antibody-secreting B cells and memory B-cell formation. CD1d-restricted invariant natural killer T (iNKT) cells are a subset of innate-like T lymphocytes that rapidly respond to stimulation with specific lipid antigens that are derived from infectious pathogens or stressed host cells. Activated iNKT cells produce a wide range of cytokines and upregulate costimulatory molecules that can promote activation of dendritic cells (DCs), natural killer (NK) cells and T cells. A decade ago we discovered that iNKT cells can help B cells to proliferate and to produce IgG antibodies in vitro and in vivo. This adjuvant-like function of antigen-activated iNKT cells provides a flexible set of helper mechanisms that expand the current paradigm of T cell–B cell interaction and highlights the potential of iNKT-cell targeting vaccine formulations.
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Introduction The production of neutralizing antibodies by B cells represents a major parameter of host defense against infectious pathogens and is a key component of protective immune responses elicited by vaccines (reviewed in [1]). The innate and adaptive arms of the immune system are functionally separated, but seem to be highly coordinated to ensure an optimal outcome of immune responses. Although innate and adaptive immune cells are endowed with very distinct functions and timing of response, there are specialized lymphocyte subsets that straddle the two programs, — and these cells have been defined as innate-like lymphocytes (reviewed in [2]). CD1d-rectricted invariant V14 natural killer T (iNKT) cells are one of the best characterized types of innate-like T lymphocytes, which display multiple effector functions upon activation (reviewed in [3]). Here we review the mechanisms through which iNKT cells help B cells to promote antibody production and memory formation.
The T-D and T-I paradigm of the B-cell response B-cell responses are generally described as T-dependent (TD) or T-independent (TI), based on the requirement for T-cell help for antibody production. TD responses are induced by protein antigens that are recognized by specific B cells in the presence of cognate T-cell help, which is provided by a specialized subset of CD4+ T helper cells called T follicular helper (TFH) cells (reviewed in [4]). Antigens reach the follicles, the B-cell zone of secondary lymphoid organs, and activate B cells upon binding to the specific B-cell receptors (BCRs) (reviewed in [5]). This leads to antigen internalization, presentation of the processed antigen peptides into MHC class II (MHC II) molecules and migration of the activated B cells to the border of the T-B cell
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zone [4]. TFH cells are induced in the T-cell zone by dendritic cells (DCs) that have internalized and present peptides of the same antigens (reviewed in [6]). TFH-cell differentiation requires upregulation of the transcriptional repressor B cell lymphoma6 (BCL-6) and costimulation with CD28 and interleukin-21 (IL-21) [6], and results in the upregulation of CXC chemokine receptor 5 (CXCR5), which in turn drives TFHcell relocalization to the boundary of the T-B cell zone, where TFH cells encounter activated B cells [6]. Upon recognition of cognate peptide–MHC II complexes on B cells, TFH cells upregulate CD40L and secrete cytokines (such as IL-21, interferon- (IFN- and IL-4). This results in the provision of TFH cell-help to B cells, which initiates the Ig class switch recombination [7] and either induces B-cell differentiation into short-lived plasma cells that form extrafollicular foci, or support B cells to reenter the follicles and form germinal centers (GCs). GC formation is the hallmark of TD responses [4, 6]. In GCs, with further help by highly activated TFH cells (recognized as CXCR5hiPD1hiICOShiBCL-6hi cells), B cells undergo class switch recombination and somatic hypermutation and affinity maturation, which all result in the differentiation of long-lived plasma cells that produce high-affinity antibodies and memory B cells [6, 8]. TD responses are typically adaptive: antibody titers develop slowly (≥ 10 days) and result in the formation of memory responses characterized by a rapid boost in antibody production upon antigen recall (reviewed in [9]). In contrast, TI responses are elicited by non-protein antigens that are unable to stimulate T helper cells and are recognized by innate-like B-cell subsets (such as marginal zone B cells and B1 cells) that do not reside in follicles (reviewed in [10]). TI type 1 (TI 1) antigens are essentially mitogenic stimuli that elicit polyclonal B-cell activation via Toll-like receptors [10]. TI type 2 (TI 2) antigens are multimeric haptens
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or polysaccharides that strongly engage the BCR and induce brisk antigen-specific B-cell responses, stimulating the appearance of extrafollicular foci of plasma blasts, small abortive GCs, low levels of somatic hypermutation and limited isotype switching (IgA, IgG3) [10]. This generates robust primary antibody responses that develop more rapidly that the TD responses (3-5 days). TI responses therefore account for host’s protection in the early phase of infection but do not convert into typical recall responses [11]. iNKT cells Invariant NKT (iNKT) cells are a unique T-cell subset characterized by the expression of a conserved invariant T-cell receptor (TCR) V chain (V14J18 in mice, V24J18 in humans), paired with a restricted repertoire of diverse TCR V chains (V8.2, V7 or V2 in mice, V11 in humans) [12, 13]. The semi-invariant TCR recognizes CD1d [14], a member of the CD1 family of non-polymorphic, MHC Ilike lipid-antigen presenting molecules that is expressed on monocytes, DCs, B cells and some non-hematopoietic cells
(reviewed in [15]). Essentially all iNKT cells
recognize the synthetic glycosphingolipid antigen galactosyl ceramide (GalCer) presented by CD1d [3], and also respond variably to other glycolipids from various infectious pathogens, such as Sphingomonas; B. burgdorferi, S. pneumoniae, A. fumigatus [16-18]. In addition, iNKT cells are strongly autoreactive against endogenous glycosphingolipids and lysophospholipids that are upregulated in cells under stress conditions [19]. Unlike conventional MHC-restricted T cells, iNKT cells display innate effector functions even without sensitization by antigen [3], and are regarded as sentinels surveying cell stress and tissue integrity via the detection of ‘unphysiological’ lipid structures.
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iNKT cells can be divided into phenotypic subsets in mice and humans depending on their specific cytokine patterns and tissue homing [3, 20]. Administration of synthetic CD1d-restricted lipid agonists (typically GalCer) in vivo has been shown to rapidly activate iNKT cells, as a result of lipid-CD1d complex presentation by CD8+DCs [21, 22]. Activated iNKT cells upregulate co-stimulatory molecules, including CD40L, and within a few hours release considerable quantities of different type 1 and type 2 cytokines that, in turn, activate NK cells and induce DC maturation [21, 23, 24]. This adjuvant-like effect of GalCer has been successfully exploited to enhance T-cell responses against a variety of co-administered nominal antigens, such as the surrogate tumor antigen ovalbumin or the infectious pathogen-derived plasmodium circumsporozoite protein [24-26].
The role of iNKT cells in B-cell responses The observation that most mouse and human B cells express CD1d [27, 28] has led immunologists to suggest that B cells may receive cognate help by lipid antigenspecifc iNKT cells. Early studies showed that injection of GalCer alone into mice induces bystander B-cell activation and increases the levels of total (antigen nonspecific) serum IgE in a manner that depends on IL-4 secretion by the activated iNKT cells. [29]. However, attempts to demonstrate that iNKT cells help cognate B cells to mount a response against malaria sporozoite lipids in vivo have produced conflicting results [30, 31]. Galli et al were the first to demonstrate that iNKT cells provide cognate help to B cells [32]. The study showed that human iNKT cells induce the expansion of purified naïve and memory B cells and drive the secretion of IgM and IgG in vitro in a CD1d-
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dependent manner [32]. iNKT-cell help to B cells under these conditions was most pronounced in the presence of GalCer, though it also occurred at a lower level in the absence of GalCer, suggesting that endogenous B-cell lipids can also be recognized by iNKT cells [32]. Stimulation by both IL-4 and IL-13 was also required for iNKT-cell-derived B-cell help, whereas CD40L–CD40 interactions were only partially involved in these responses [32]. Accordingly, the TH0 CD4+ iNKT-cell subset was markedly more efficient than the T H1 CD4- iNKT-cell subset in providing B-cell help [32]. Subsequent studies demonstrated that immunization of mice with GalCer along with protein or hapten–carrier antigens elicits high titers of antigen-specific, classswitched antibodies [33-35], significantly enhancing features of the antibody response in vivo, such as protection from infections [33, 35] and long lasting B-cell memory [35]. The adjuvant effect of GalCer was comparable to that of benchmark adjuvants that are being utilized for human vaccination [35] and was independent of the route of administration [35]. B-cell help provided by GalCer-activated iNKT cells was found to critically depend on the expression of CD40L [35]. This implied that CD40 expression is required, but it was unclear whether CD40 should be expressed on B cells or other immune cells [35]. The T helper cytokines IFN- and IL-4 were shown to be dispensable for the iNKT-cell helper function, although they directed the IgG2a and IGg1 class switch, respectively [35]. The outcome of mouse immunization with protein antigens mixed with GalCer was optimal when CD4+ T cells were also present [34, 35]. However, unlike other classic adjuvants, GalCer was shown to enhance antibody production against both TD and TI antigens also in mice lacking CD4+ Th cells [34, 35], suggesting the possible provision of CD1-dependent B-cell
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help by iNKT cells in vivo. Immunization of mice lacking iNKT cells revealed that iNKT cells also display a spontaneous (non-GalCer induced) role in inducing and/or maintaining the circulating Ab levels through the control of plasma cell homeostasis [34, 36], which was found to depend on the iNKT-cell expression of B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) [37].
Mechanisms of iNKT cell-mediated B-cell help Studies gaining mechanistic insights into the provision of B-cell help by iNKT cells in vivo have uncovered two clearly distinct mechanisms. These mechanisms are dictated by the vaccine formulation’s capacity to recruit T helper cells and differentially rely on CD1d-cognate interactions between iNKT and B cells.
Non-cognate help by iNKT cells Immunization of mice with protein antigens admixed with GalCer has been shown to elicit strong antibody responses that, somewhat unexpectedly, do not require CD1d expression on B cells but, instead, depends on the co-expression of CD1d and MHC II on DCs [38]. Coordinate recognition of antigens in complex with CD1d and MHC II on the surface of DCs by iNKT cells and T helper cells has been shown to result in an enhanced induction of the T helper cell response and enhanced delivery of classic T-cell help [38, 39]. In addition, help by non-cognate iNKT cells has been shown to require CD40 expression on B cells [38] — but not the expression of the TFH cell-related molecule SLAM-associated protein (SAP) by iNKT cells [40] — and was reported to occur also in splenectomized mice, ruling out the need for innate-like B cells, which reside in the spleen [38]. Overall, the GalCer-induced, non-cognate
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help by iNKT cells recapitulates the functions of adjuvants that activate the innate immune system to support adaptive immune responses. The available topographic data allow inferring a model of non-cognate B-cell help by iNKT cells (Figure 1A-C). At the steady state, iNKT cells are mainly located in the marginal zone of the spleen, strategically pre-positioned in close contact with macrophages and DCs that capture blood-borne antigens flowing through the red pulp, with a scattered distribution in the red pulp and white pulp [41-43]. Upon GalCer immunization, marginal zone DCs uptake and present GalCer, and subsequently iNKT cells concentrate in the marginal zone and in bridging channels, where they rapidly produce cytokines [42, 43]. This promotes the maturation of DCs, which upregulate expression of CC chemokine receptor 7 (CCR7) and relocate into the T-cell zone of the white pulp [43]. In the white pulp, DCs can enhance the induction of TFH cells, which then provide help to cognate B cells. In popliteal lymph nodes, endogenous iNKT cells localize in the interfollicular region and in the medulla, but not in the T cell-rich paracortex [44], whereas adoptively transferred iNKT cells are found in the paracortex [45]. Nevertheless, in both locations iNKT cells can be activated upon contacting CD169+ subcapsular sinus resident macrophages, which express CD1d and can present lymph-borne soluble antigens [44, 45], possibly leading to the subsequent activation of TFH cells and B-cell responses. Pre-clinical results highlight the efficacy and attractiveness of the use of vaccine containing GalCer simply mixed with protein antigens derived from a variety of infectious pathogens, to activate non-cognate B-cell help by iNKT cells and induce efficient protective humoral responses and long-lasting memory through mucosal and parenteral administration routes [43, 46-49].
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Cognate help by iNKT cells CD1d- and lipid-antigen-dependent cognate interactions between iNKT and B cells have also been shown to elicit antibody responses in in vitro and in vivo studies. These studies used complex or particulated immunogens composed of proteins or hapten antigens that are either physically associated to GalCer [50, 51] or entrapped in bacterial fragments together with glycolipid analogs [52] (Figure 1 D-F). The linkage between protein antigens and GalCer is essential for promoting efficient contact between cognate iNKT cells and B cells. The specific BCRs bind the protein or hapten moiety of the complex and, provided sufficient receptor crosslinking is attained [51], are internalized together with the linked GalCer. Internalized GalCer can then be presented in complex with CD1d by B cells to activate cognate iNKT cells [50, 51]. In the case of protein antigens mixed with GalCer, B cells can internalize the free lipid only through its binding to the low-density lipoprotein receptor (LDL-R) [53]. As GalCer binding to LDL-R and protein antigen binding to the BCR are two independent events, the likelihood that a single B cell binds and internalizes both structures at the same time is low. Immunization of mice with protein complexed with GalCer in silica microspheres, or with haptenated-GalCer, elicits long-lasting iNKT cell–B cell interactions in the draining lymph nodes [54], rapid extrafollicular responses in the spleen [51] and swift elevation in the titers of protein- or hapten-specific IgM and IgG[50, 51]. These effects require the presence of iNKT cells, CD1d expression by B cells and the provision of CD40–CD40L signalling, CD80–CD86 co-stimulation and IFN-, but not of IL-4 [50]. Formulations involving proteins such as HEL in complex with GalCer, or haptenated-GalCer molecules, cannot generate MHC II-restricted epitopes, and
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this observation confirms the fact that cognate iNKT-cell-mediated B-cell help can elicit antibody production and class switch independently of T helper cell recruitment [50, 51]. Cognate iNKT cell-mediated B-cell help has also been described to occur spontaneously in a model of infection with the hepatotropic spingomonas N. aromaticivorans, which contains glycosphingolipid analogs (-glycurunilceramides) that activate iNKT cells and the B-cell antigen enzyme pyruvate dehydrogenases complex E2 (PDC-E2) [52]. Infection of mice with this bacterium elicits rapid iNKTcell dependent production of high-affinity and class-switched antibodies against bacterial PDC-E2, which also cross react with the homologous mammalian mitochondrial enzyme that is expressed in small bile ducts, thereby initiating organ damage that resembles human primary biliary cirrhosis [52]. In this model, CD1d expression on B cells and iNKT-cell activation are required for the induction, but not the maintenance of the autoantibody response, which instead relies on Th cells that sustain the late chronic phase [52]. Consistent with their ability to provide help to cognate B cells, iNKT cells have recently been found to adopt the TFH-cell phenotype [54, 55] and localize within GCs [54] in mice immunized with particulated or even with soluble GalCer. In the spleen and lymph nodes of immunized mice, the frequency of iNKTFH cells has been shown to be comparable with that of TFH cells induced by TD antigens [54, 55]. Furthermore, iNKTFH cells have also been detected in human tonsils, and this finding implied that iNKTFH cells are spontaneously induced by environmental antigens in humans [54]. The differentiation of iNKTFH and TFH cells follows the same requirements [54], with the notable exception of the fact that IL-21 is not required for iNKTFH-cell formation,
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although it is critical for their cognate helper functions [54, 56]. Cognate help from iNKTFH cells can induce the formation of extrafollicular plasmablasts, Ig class switch and moderate affinity maturation [54, 56]. Moreover, iNKTFH cells promote considerably rapid GC formation (within three days post immunization) as compared with TFH cells, which promote GC formation ten days post immunization with protein antigens. However, the GCs that are induced by iNKTFH-cell help are short-lived and do not produce long-lived plasma cells and memory responses [54-56]. Thus, the specific characteristics of humoral responses that are elicited with the help of cognate iNKTFH cells are distinct from the characteristics of previously described TI and TD B-cell responses and have prompted the new definition of TD type 2 (TD 2) responses [56] (Figure 1). The induction of iNKTFH cells and TD 2 humoral responses has also been shown to occur in mice lacking T helper cells upon immunization with protein antigens mixed with GalCer [55], a formulation that in the presence of T helper cells would instead activate non-cognate iNKT-cell help, leading to long-lasting antibody titers and B-cell memory are generated [38, 56]. In CD4+ T-cell-deficient mice, iNKTFH cells expanded more than in T helper-cell sufficient mice [55], suggesting that in physiological conditions the expansion of iNKTFH cells may be limited by the competition with TFH cells and/or by the suppression exerted by a specialized subset of Treg cells that have been found infiltrating GCs [57, 58]. It has been observed, however, that immunization of wild-type mice with the haptenated protein NP-KLH mixed with GalCer elicits class switched antibody responses that require both CD1d expression on B cells and the presence of T helper cells [34, 59]. At variance with the described examples of cognate and non-
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cognate iNKT-cell help [38, 39, 54], the induction of B-cell responses by this particular antigen model does not require CD40L and CD1d expression on iNKT cells and DCs, respectively [60, 61]. These results, which highlight the flexibility of the iNKT cell-helper functions, might be related to experimental differences between models, which might include a particular immunogenicity of NP-KLH–aGalCer mixture compared with the other protein–GalCer mixtures, or with the proteinGalCer conjugates utilized by the other studies. The above results have suggested that iNKTFH cells are less efficient than TFH cells in helping the induction of mature GCs and memory B-cell responses. This could be owed to a reduced persistence of the B-cell antigens and GalCer that are administered without adjuvant, and/or to quantitative or as yet unknown molecular differences between the two follicular helper cell types [62]. Recent data challenge this view by showing that the injection into mice of liposomal nanoparticles incorporating both S. pneumoniae capsular polysaccharide (PS) and GalCer activates long-lasting and class-switched antibody titers with features of memory response upon antigen recall [63]. Although this antigen formulation does not promote iNKTFH cell differentiation, it induces extrafollicular B-cell responses that require the expression of CD1d on DCs and B cells, which activate iNKT cells and support the subsequent cognate iNKT–B cell interactions, respectively[63]. The superior immunogenicity displayed by the PS–GalCer liposomes may be due to its increased capacity to cross-link the BCR of PS-specific B cells, delivering a stronger activation signal for B cells compared with the previously tested antigen-GalCer complexes. Although there is no evidence that this PS–GalCer vaccine can induce protection against S. pneumonia challenge [63], this formulation has the potential to
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induce efficient antibody responses against any “help-less” non-protein antigen (carbohydrates, lipids, haptens) that cannot induce the differentiation of peptidespecific TFH cells. It is unclear whether iNKT cells provide cognate help to innate-like or adaptive-like B cells. In principle, both subsets could be recruited in the extrafollicular responses induced by this form of help [64]. Marginal zone B cells express high CD1d levels and are more efficient than follicular B cells both in capturing haptenated-GalCer and in activating iNKT cells in vitro [50], while they may contact iNKT cells in vivo [42]. However, GalCer-dependent activation of iNKT cells in vivo has been reported to result in a contraction of marginal zone B cells [65]. Moreover, iNKTFH cells have been detected in splenic GCs following GalCer immunization, suggesting that iNKTFH cell-follicular B-cell interactions might also occur [54]. Hence, the crucial mechanistic details of the iNKT-cell cognate help to B cells remain to be defined.
Conclusions The results outlined above indicate that iNKT cells provide both non-cognate and cognate help to B cells through distinct mechanisms that depend on the ability of TFH cells to recognize immunization antigens. Non-cognate iNKT-cell help (as it is the case of immunization with protein antigens) does not require a physical link between the B-cell epitopes and GalCer, i.e., it is a classical inter-molecular help. In contrast, cognate iNKT-cell help (as in the case of immunization with oligosaccharide antigens) requires that B-cell epitopes and GalCer are linked to form a single molecule, i.e., it is an intra-molecular help. Further studies are needed to unravel the spontaneous role for iNKT-cell-derived B-cell help in the pathophysiology of B-cell
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responses during natural infections, autoimmunity and cancer, whereas the results reviewed here have immediate implications for the development of new NKT-cellstimulating adjuvants. In this respect, data obtained in clinical trials for cancer immunotherapy show that administration of GalCer in humans is safe and does not cause notable side effects [3]. Moreover, the data obtained in cancer patients who received GalCer-containing vaccines show clear expansion and functional activation of iNKT cells, correlating with the clinical responses, indicating a remarkable responsiveness of these cells to the immunogen in spite of their relatively low frequency in the human immune system [66, 67]. Thus, the possibility to exploit the flexible helper functions of iNKT cells opens the way to the design of a new generation of vaccines.
Acknowledgements The authors acknowledge the support of the Italian Ministry of Health, Fondazione Cariplo (Vaccine Program to PD and SA), Associazione Italiana Ricerca Cancro (AIRC IG-11523 to GC; AIRC Special Program Molecular Clinical Oncology 5 per mille n. 9965 to PD) and an ERC Advanced Grant to SA (no. 269022).
Conflict of interest The authors declare no financial or commercial conflict of interest.
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Figure 1. iNKT cells provide help for B-cell responses: non-cognate and cognate mechanisms. (A) Upon entering secondary lymphoid organs, protein– galactosylceramide (GalCer) mixtures or complexes of GalCer with proteins or haptens are non-selectively uptaken by CD8+ DCs, which activate iNKT cells by presenting GalCer-CD1d complexes. This DC–iNKT cell interaction results in CD40L–CD40 co-stimulation and licensing of antigen-presenting functions in DCs, which leads to enhanced priming of T helper cells that are specific for the coadministered protein antigen. (B) Activated T helper cells acquire TFH-cell functions and migrate to the border of the T–B zone, where they provide efficient help to cognate B cells. This occurs independently of CD1d expression on B cells. (C) TFH cell–B cell interactions induce a typical TD response, with formation of GCs, Ig affinity maturation and class switch, and generation of long-lived PCs and memory B cells. Depicted is a model of reactive spleen. PALS, periarteriolar lymphoid sheath; GC, germinal center. (D) Immunization of wild-type mice with complexes of GalCer with antigens (proteins, sugars, lipid, haptens) that cannot be recognized by T helper cells, or immunization of T helper cell-deficient mice with mixtures of GalCer and protein antigens both lead to GalCer presentation by DCs and iNKT-cell activation. These iNKT cells co-opt the follicular helper pathway. (E) iNKTFH cells are able to recognize B cells that have internalized Ag–GalCer complexes via the BCR, or free GalCer via Low Density Lipoprotein Receptor (LDL-R), and present GalCer on CD1d molecules. This unleashes the provision of CD1d-, CD40L- and IL-21dependent help by iNKT cells. (F) Help by cognate iNKT cells results in a newlydefined TD type 2 B-cell response, which is characterized by extrafollicular B-cell
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proliferation and poor GC formation, Ig class switch and modest levels of affinity maturation, sustained primary response but lack of memory generation. Depicted is a model of reactive spleen.
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Keywords: CD1d, B cell help, vaccines, T follicular helper cells
Paolo Dellabona1*, Sergio Abrignani2*, Giulia Casorati1* 1 Experimental Immunology Unit, Division of Immunology Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy 2. Istituto Nazionale di Genetica Molecolare, Milano, Italy *corresponding authors
Contact author: Paolo Dellabona, Experimental Immunology Unit, Division of Immunology Transplantation and Infectious Diseases, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milano, Italy. E-mail: [email protected]; phone: +39-02.26434727
Received: 24-Feb-2014; Revised: 21-Apr-2014; Accepted: 25-Apr-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/eji.201344399. This article is protected by copyright. All rights reserved.
1
Abstract T-cell help to B lymphocytes is one of the most important events in adaptive immune responses in health and disease. It is generally delivered by cognate CD4+ T follicular helper (TFH) cells via both cell-to-cell contacts and soluble mediators, and it is essential for both the clonal expansion of antibody-secreting B cells and memory B-cell formation. CD1d-restricted invariant natural killer T (iNKT) cells are a subset of innate-like T lymphocytes that rapidly respond to stimulation with specific lipid antigens that are derived from infectious pathogens or stressed host cells. Activated iNKT cells produce a wide range of cytokines and upregulate costimulatory molecules that can promote activation of dendritic cells (DCs), natural killer (NK) cells and T cells. A decade ago we discovered that iNKT cells can help B cells to proliferate and to produce IgG antibodies in vitro and in vivo. This adjuvant-like function of antigen-activated iNKT cells provides a flexible set of helper mechanisms that expand the current paradigm of T cell–B cell interaction and highlights the potential of iNKT-cell targeting vaccine formulations.
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Introduction The production of neutralizing antibodies by B cells represents a major parameter of host defense against infectious pathogens and is a key component of protective immune responses elicited by vaccines (reviewed in [1]). The innate and adaptive arms of the immune system are functionally separated, but seem to be highly coordinated to ensure an optimal outcome of immune responses. Although innate and adaptive immune cells are endowed with very distinct functions and timing of response, there are specialized lymphocyte subsets that straddle the two programs, — and these cells have been defined as innate-like lymphocytes (reviewed in [2]). CD1d-rectricted invariant V14 natural killer T (iNKT) cells are one of the best characterized types of innate-like T lymphocytes, which display multiple effector functions upon activation (reviewed in [3]). Here we review the mechanisms through which iNKT cells help B cells to promote antibody production and memory formation.
The T-D and T-I paradigm of the B-cell response B-cell responses are generally described as T-dependent (TD) or T-independent (TI), based on the requirement for T-cell help for antibody production. TD responses are induced by protein antigens that are recognized by specific B cells in the presence of cognate T-cell help, which is provided by a specialized subset of CD4+ T helper cells called T follicular helper (TFH) cells (reviewed in [4]). Antigens reach the follicles, the B-cell zone of secondary lymphoid organs, and activate B cells upon binding to the specific B-cell receptors (BCRs) (reviewed in [5]). This leads to antigen internalization, presentation of the processed antigen peptides into MHC class II (MHC II) molecules and migration of the activated B cells to the border of the T-B cell
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zone [4]. TFH cells are induced in the T-cell zone by dendritic cells (DCs) that have internalized and present peptides of the same antigens (reviewed in [6]). TFH-cell differentiation requires upregulation of the transcriptional repressor B cell lymphoma6 (BCL-6) and costimulation with CD28 and interleukin-21 (IL-21) [6], and results in the upregulation of CXC chemokine receptor 5 (CXCR5), which in turn drives TFHcell relocalization to the boundary of the T-B cell zone, where TFH cells encounter activated B cells [6]. Upon recognition of cognate peptide–MHC II complexes on B cells, TFH cells upregulate CD40L and secrete cytokines (such as IL-21, interferon- (IFN- and IL-4). This results in the provision of TFH cell-help to B cells, which initiates the Ig class switch recombination [7] and either induces B-cell differentiation into short-lived plasma cells that form extrafollicular foci, or support B cells to reenter the follicles and form germinal centers (GCs). GC formation is the hallmark of TD responses [4, 6]. In GCs, with further help by highly activated TFH cells (recognized as CXCR5hiPD1hiICOShiBCL-6hi cells), B cells undergo class switch recombination and somatic hypermutation and affinity maturation, which all result in the differentiation of long-lived plasma cells that produce high-affinity antibodies and memory B cells [6, 8]. TD responses are typically adaptive: antibody titers develop slowly (≥ 10 days) and result in the formation of memory responses characterized by a rapid boost in antibody production upon antigen recall (reviewed in [9]). In contrast, TI responses are elicited by non-protein antigens that are unable to stimulate T helper cells and are recognized by innate-like B-cell subsets (such as marginal zone B cells and B1 cells) that do not reside in follicles (reviewed in [10]). TI type 1 (TI 1) antigens are essentially mitogenic stimuli that elicit polyclonal B-cell activation via Toll-like receptors [10]. TI type 2 (TI 2) antigens are multimeric haptens
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or polysaccharides that strongly engage the BCR and induce brisk antigen-specific B-cell responses, stimulating the appearance of extrafollicular foci of plasma blasts, small abortive GCs, low levels of somatic hypermutation and limited isotype switching (IgA, IgG3) [10]. This generates robust primary antibody responses that develop more rapidly that the TD responses (3-5 days). TI responses therefore account for host’s protection in the early phase of infection but do not convert into typical recall responses [11]. iNKT cells Invariant NKT (iNKT) cells are a unique T-cell subset characterized by the expression of a conserved invariant T-cell receptor (TCR) V chain (V14J18 in mice, V24J18 in humans), paired with a restricted repertoire of diverse TCR V chains (V8.2, V7 or V2 in mice, V11 in humans) [12, 13]. The semi-invariant TCR recognizes CD1d [14], a member of the CD1 family of non-polymorphic, MHC Ilike lipid-antigen presenting molecules that is expressed on monocytes, DCs, B cells and some non-hematopoietic cells
(reviewed in [15]). Essentially all iNKT cells
recognize the synthetic glycosphingolipid antigen galactosyl ceramide (GalCer) presented by CD1d [3], and also respond variably to other glycolipids from various infectious pathogens, such as Sphingomonas; B. burgdorferi, S. pneumoniae, A. fumigatus [16-18]. In addition, iNKT cells are strongly autoreactive against endogenous glycosphingolipids and lysophospholipids that are upregulated in cells under stress conditions [19]. Unlike conventional MHC-restricted T cells, iNKT cells display innate effector functions even without sensitization by antigen [3], and are regarded as sentinels surveying cell stress and tissue integrity via the detection of ‘unphysiological’ lipid structures.
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iNKT cells can be divided into phenotypic subsets in mice and humans depending on their specific cytokine patterns and tissue homing [3, 20]. Administration of synthetic CD1d-restricted lipid agonists (typically GalCer) in vivo has been shown to rapidly activate iNKT cells, as a result of lipid-CD1d complex presentation by CD8+DCs [21, 22]. Activated iNKT cells upregulate co-stimulatory molecules, including CD40L, and within a few hours release considerable quantities of different type 1 and type 2 cytokines that, in turn, activate NK cells and induce DC maturation [21, 23, 24]. This adjuvant-like effect of GalCer has been successfully exploited to enhance T-cell responses against a variety of co-administered nominal antigens, such as the surrogate tumor antigen ovalbumin or the infectious pathogen-derived plasmodium circumsporozoite protein [24-26].
The role of iNKT cells in B-cell responses The observation that most mouse and human B cells express CD1d [27, 28] has led immunologists to suggest that B cells may receive cognate help by lipid antigenspecifc iNKT cells. Early studies showed that injection of GalCer alone into mice induces bystander B-cell activation and increases the levels of total (antigen nonspecific) serum IgE in a manner that depends on IL-4 secretion by the activated iNKT cells. [29]. However, attempts to demonstrate that iNKT cells help cognate B cells to mount a response against malaria sporozoite lipids in vivo have produced conflicting results [30, 31]. Galli et al were the first to demonstrate that iNKT cells provide cognate help to B cells [32]. The study showed that human iNKT cells induce the expansion of purified naïve and memory B cells and drive the secretion of IgM and IgG in vitro in a CD1d-
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dependent manner [32]. iNKT-cell help to B cells under these conditions was most pronounced in the presence of GalCer, though it also occurred at a lower level in the absence of GalCer, suggesting that endogenous B-cell lipids can also be recognized by iNKT cells [32]. Stimulation by both IL-4 and IL-13 was also required for iNKT-cell-derived B-cell help, whereas CD40L–CD40 interactions were only partially involved in these responses [32]. Accordingly, the TH0 CD4+ iNKT-cell subset was markedly more efficient than the T H1 CD4- iNKT-cell subset in providing B-cell help [32]. Subsequent studies demonstrated that immunization of mice with GalCer along with protein or hapten–carrier antigens elicits high titers of antigen-specific, classswitched antibodies [33-35], significantly enhancing features of the antibody response in vivo, such as protection from infections [33, 35] and long lasting B-cell memory [35]. The adjuvant effect of GalCer was comparable to that of benchmark adjuvants that are being utilized for human vaccination [35] and was independent of the route of administration [35]. B-cell help provided by GalCer-activated iNKT cells was found to critically depend on the expression of CD40L [35]. This implied that CD40 expression is required, but it was unclear whether CD40 should be expressed on B cells or other immune cells [35]. The T helper cytokines IFN- and IL-4 were shown to be dispensable for the iNKT-cell helper function, although they directed the IgG2a and IGg1 class switch, respectively [35]. The outcome of mouse immunization with protein antigens mixed with GalCer was optimal when CD4+ T cells were also present [34, 35]. However, unlike other classic adjuvants, GalCer was shown to enhance antibody production against both TD and TI antigens also in mice lacking CD4+ Th cells [34, 35], suggesting the possible provision of CD1-dependent B-cell
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help by iNKT cells in vivo. Immunization of mice lacking iNKT cells revealed that iNKT cells also display a spontaneous (non-GalCer induced) role in inducing and/or maintaining the circulating Ab levels through the control of plasma cell homeostasis [34, 36], which was found to depend on the iNKT-cell expression of B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) [37].
Mechanisms of iNKT cell-mediated B-cell help Studies gaining mechanistic insights into the provision of B-cell help by iNKT cells in vivo have uncovered two clearly distinct mechanisms. These mechanisms are dictated by the vaccine formulation’s capacity to recruit T helper cells and differentially rely on CD1d-cognate interactions between iNKT and B cells.
Non-cognate help by iNKT cells Immunization of mice with protein antigens admixed with GalCer has been shown to elicit strong antibody responses that, somewhat unexpectedly, do not require CD1d expression on B cells but, instead, depends on the co-expression of CD1d and MHC II on DCs [38]. Coordinate recognition of antigens in complex with CD1d and MHC II on the surface of DCs by iNKT cells and T helper cells has been shown to result in an enhanced induction of the T helper cell response and enhanced delivery of classic T-cell help [38, 39]. In addition, help by non-cognate iNKT cells has been shown to require CD40 expression on B cells [38] — but not the expression of the TFH cell-related molecule SLAM-associated protein (SAP) by iNKT cells [40] — and was reported to occur also in splenectomized mice, ruling out the need for innate-like B cells, which reside in the spleen [38]. Overall, the GalCer-induced, non-cognate
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help by iNKT cells recapitulates the functions of adjuvants that activate the innate immune system to support adaptive immune responses. The available topographic data allow inferring a model of non-cognate B-cell help by iNKT cells (Figure 1A-C). At the steady state, iNKT cells are mainly located in the marginal zone of the spleen, strategically pre-positioned in close contact with macrophages and DCs that capture blood-borne antigens flowing through the red pulp, with a scattered distribution in the red pulp and white pulp [41-43]. Upon GalCer immunization, marginal zone DCs uptake and present GalCer, and subsequently iNKT cells concentrate in the marginal zone and in bridging channels, where they rapidly produce cytokines [42, 43]. This promotes the maturation of DCs, which upregulate expression of CC chemokine receptor 7 (CCR7) and relocate into the T-cell zone of the white pulp [43]. In the white pulp, DCs can enhance the induction of TFH cells, which then provide help to cognate B cells. In popliteal lymph nodes, endogenous iNKT cells localize in the interfollicular region and in the medulla, but not in the T cell-rich paracortex [44], whereas adoptively transferred iNKT cells are found in the paracortex [45]. Nevertheless, in both locations iNKT cells can be activated upon contacting CD169+ subcapsular sinus resident macrophages, which express CD1d and can present lymph-borne soluble antigens [44, 45], possibly leading to the subsequent activation of TFH cells and B-cell responses. Pre-clinical results highlight the efficacy and attractiveness of the use of vaccine containing GalCer simply mixed with protein antigens derived from a variety of infectious pathogens, to activate non-cognate B-cell help by iNKT cells and induce efficient protective humoral responses and long-lasting memory through mucosal and parenteral administration routes [43, 46-49].
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Cognate help by iNKT cells CD1d- and lipid-antigen-dependent cognate interactions between iNKT and B cells have also been shown to elicit antibody responses in in vitro and in vivo studies. These studies used complex or particulated immunogens composed of proteins or hapten antigens that are either physically associated to GalCer [50, 51] or entrapped in bacterial fragments together with glycolipid analogs [52] (Figure 1 D-F). The linkage between protein antigens and GalCer is essential for promoting efficient contact between cognate iNKT cells and B cells. The specific BCRs bind the protein or hapten moiety of the complex and, provided sufficient receptor crosslinking is attained [51], are internalized together with the linked GalCer. Internalized GalCer can then be presented in complex with CD1d by B cells to activate cognate iNKT cells [50, 51]. In the case of protein antigens mixed with GalCer, B cells can internalize the free lipid only through its binding to the low-density lipoprotein receptor (LDL-R) [53]. As GalCer binding to LDL-R and protein antigen binding to the BCR are two independent events, the likelihood that a single B cell binds and internalizes both structures at the same time is low. Immunization of mice with protein complexed with GalCer in silica microspheres, or with haptenated-GalCer, elicits long-lasting iNKT cell–B cell interactions in the draining lymph nodes [54], rapid extrafollicular responses in the spleen [51] and swift elevation in the titers of protein- or hapten-specific IgM and IgG[50, 51]. These effects require the presence of iNKT cells, CD1d expression by B cells and the provision of CD40–CD40L signalling, CD80–CD86 co-stimulation and IFN-, but not of IL-4 [50]. Formulations involving proteins such as HEL in complex with GalCer, or haptenated-GalCer molecules, cannot generate MHC II-restricted epitopes, and
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this observation confirms the fact that cognate iNKT-cell-mediated B-cell help can elicit antibody production and class switch independently of T helper cell recruitment [50, 51]. Cognate iNKT cell-mediated B-cell help has also been described to occur spontaneously in a model of infection with the hepatotropic spingomonas N. aromaticivorans, which contains glycosphingolipid analogs (-glycurunilceramides) that activate iNKT cells and the B-cell antigen enzyme pyruvate dehydrogenases complex E2 (PDC-E2) [52]. Infection of mice with this bacterium elicits rapid iNKTcell dependent production of high-affinity and class-switched antibodies against bacterial PDC-E2, which also cross react with the homologous mammalian mitochondrial enzyme that is expressed in small bile ducts, thereby initiating organ damage that resembles human primary biliary cirrhosis [52]. In this model, CD1d expression on B cells and iNKT-cell activation are required for the induction, but not the maintenance of the autoantibody response, which instead relies on Th cells that sustain the late chronic phase [52]. Consistent with their ability to provide help to cognate B cells, iNKT cells have recently been found to adopt the TFH-cell phenotype [54, 55] and localize within GCs [54] in mice immunized with particulated or even with soluble GalCer. In the spleen and lymph nodes of immunized mice, the frequency of iNKTFH cells has been shown to be comparable with that of TFH cells induced by TD antigens [54, 55]. Furthermore, iNKTFH cells have also been detected in human tonsils, and this finding implied that iNKTFH cells are spontaneously induced by environmental antigens in humans [54]. The differentiation of iNKTFH and TFH cells follows the same requirements [54], with the notable exception of the fact that IL-21 is not required for iNKTFH-cell formation,
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although it is critical for their cognate helper functions [54, 56]. Cognate help from iNKTFH cells can induce the formation of extrafollicular plasmablasts, Ig class switch and moderate affinity maturation [54, 56]. Moreover, iNKTFH cells promote considerably rapid GC formation (within three days post immunization) as compared with TFH cells, which promote GC formation ten days post immunization with protein antigens. However, the GCs that are induced by iNKTFH-cell help are short-lived and do not produce long-lived plasma cells and memory responses [54-56]. Thus, the specific characteristics of humoral responses that are elicited with the help of cognate iNKTFH cells are distinct from the characteristics of previously described TI and TD B-cell responses and have prompted the new definition of TD type 2 (TD 2) responses [56] (Figure 1). The induction of iNKTFH cells and TD 2 humoral responses has also been shown to occur in mice lacking T helper cells upon immunization with protein antigens mixed with GalCer [55], a formulation that in the presence of T helper cells would instead activate non-cognate iNKT-cell help, leading to long-lasting antibody titers and B-cell memory are generated [38, 56]. In CD4+ T-cell-deficient mice, iNKTFH cells expanded more than in T helper-cell sufficient mice [55], suggesting that in physiological conditions the expansion of iNKTFH cells may be limited by the competition with TFH cells and/or by the suppression exerted by a specialized subset of Treg cells that have been found infiltrating GCs [57, 58]. It has been observed, however, that immunization of wild-type mice with the haptenated protein NP-KLH mixed with GalCer elicits class switched antibody responses that require both CD1d expression on B cells and the presence of T helper cells [34, 59]. At variance with the described examples of cognate and non-
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cognate iNKT-cell help [38, 39, 54], the induction of B-cell responses by this particular antigen model does not require CD40L and CD1d expression on iNKT cells and DCs, respectively [60, 61]. These results, which highlight the flexibility of the iNKT cell-helper functions, might be related to experimental differences between models, which might include a particular immunogenicity of NP-KLH–aGalCer mixture compared with the other protein–GalCer mixtures, or with the proteinGalCer conjugates utilized by the other studies. The above results have suggested that iNKTFH cells are less efficient than TFH cells in helping the induction of mature GCs and memory B-cell responses. This could be owed to a reduced persistence of the B-cell antigens and GalCer that are administered without adjuvant, and/or to quantitative or as yet unknown molecular differences between the two follicular helper cell types [62]. Recent data challenge this view by showing that the injection into mice of liposomal nanoparticles incorporating both S. pneumoniae capsular polysaccharide (PS) and GalCer activates long-lasting and class-switched antibody titers with features of memory response upon antigen recall [63]. Although this antigen formulation does not promote iNKTFH cell differentiation, it induces extrafollicular B-cell responses that require the expression of CD1d on DCs and B cells, which activate iNKT cells and support the subsequent cognate iNKT–B cell interactions, respectively[63]. The superior immunogenicity displayed by the PS–GalCer liposomes may be due to its increased capacity to cross-link the BCR of PS-specific B cells, delivering a stronger activation signal for B cells compared with the previously tested antigen-GalCer complexes. Although there is no evidence that this PS–GalCer vaccine can induce protection against S. pneumonia challenge [63], this formulation has the potential to
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induce efficient antibody responses against any “help-less” non-protein antigen (carbohydrates, lipids, haptens) that cannot induce the differentiation of peptidespecific TFH cells. It is unclear whether iNKT cells provide cognate help to innate-like or adaptive-like B cells. In principle, both subsets could be recruited in the extrafollicular responses induced by this form of help [64]. Marginal zone B cells express high CD1d levels and are more efficient than follicular B cells both in capturing haptenated-GalCer and in activating iNKT cells in vitro [50], while they may contact iNKT cells in vivo [42]. However, GalCer-dependent activation of iNKT cells in vivo has been reported to result in a contraction of marginal zone B cells [65]. Moreover, iNKTFH cells have been detected in splenic GCs following GalCer immunization, suggesting that iNKTFH cell-follicular B-cell interactions might also occur [54]. Hence, the crucial mechanistic details of the iNKT-cell cognate help to B cells remain to be defined.
Conclusions The results outlined above indicate that iNKT cells provide both non-cognate and cognate help to B cells through distinct mechanisms that depend on the ability of TFH cells to recognize immunization antigens. Non-cognate iNKT-cell help (as it is the case of immunization with protein antigens) does not require a physical link between the B-cell epitopes and GalCer, i.e., it is a classical inter-molecular help. In contrast, cognate iNKT-cell help (as in the case of immunization with oligosaccharide antigens) requires that B-cell epitopes and GalCer are linked to form a single molecule, i.e., it is an intra-molecular help. Further studies are needed to unravel the spontaneous role for iNKT-cell-derived B-cell help in the pathophysiology of B-cell
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responses during natural infections, autoimmunity and cancer, whereas the results reviewed here have immediate implications for the development of new NKT-cellstimulating adjuvants. In this respect, data obtained in clinical trials for cancer immunotherapy show that administration of GalCer in humans is safe and does not cause notable side effects [3]. Moreover, the data obtained in cancer patients who received GalCer-containing vaccines show clear expansion and functional activation of iNKT cells, correlating with the clinical responses, indicating a remarkable responsiveness of these cells to the immunogen in spite of their relatively low frequency in the human immune system [66, 67]. Thus, the possibility to exploit the flexible helper functions of iNKT cells opens the way to the design of a new generation of vaccines.
Acknowledgements The authors acknowledge the support of the Italian Ministry of Health, Fondazione Cariplo (Vaccine Program to PD and SA), Associazione Italiana Ricerca Cancro (AIRC IG-11523 to GC; AIRC Special Program Molecular Clinical Oncology 5 per mille n. 9965 to PD) and an ERC Advanced Grant to SA (no. 269022).
Conflict of interest The authors declare no financial or commercial conflict of interest.
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Figure 1. iNKT cells provide help for B-cell responses: non-cognate and cognate mechanisms. (A) Upon entering secondary lymphoid organs, protein– galactosylceramide (GalCer) mixtures or complexes of GalCer with proteins or haptens are non-selectively uptaken by CD8+ DCs, which activate iNKT cells by presenting GalCer-CD1d complexes. This DC–iNKT cell interaction results in CD40L–CD40 co-stimulation and licensing of antigen-presenting functions in DCs, which leads to enhanced priming of T helper cells that are specific for the coadministered protein antigen. (B) Activated T helper cells acquire TFH-cell functions and migrate to the border of the T–B zone, where they provide efficient help to cognate B cells. This occurs independently of CD1d expression on B cells. (C) TFH cell–B cell interactions induce a typical TD response, with formation of GCs, Ig affinity maturation and class switch, and generation of long-lived PCs and memory B cells. Depicted is a model of reactive spleen. PALS, periarteriolar lymphoid sheath; GC, germinal center. (D) Immunization of wild-type mice with complexes of GalCer with antigens (proteins, sugars, lipid, haptens) that cannot be recognized by T helper cells, or immunization of T helper cell-deficient mice with mixtures of GalCer and protein antigens both lead to GalCer presentation by DCs and iNKT-cell activation. These iNKT cells co-opt the follicular helper pathway. (E) iNKTFH cells are able to recognize B cells that have internalized Ag–GalCer complexes via the BCR, or free GalCer via Low Density Lipoprotein Receptor (LDL-R), and present GalCer on CD1d molecules. This unleashes the provision of CD1d-, CD40L- and IL-21dependent help by iNKT cells. (F) Help by cognate iNKT cells results in a newlydefined TD type 2 B-cell response, which is characterized by extrafollicular B-cell
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proliferation and poor GC formation, Ig class switch and modest levels of affinity maturation, sustained primary response but lack of memory generation. Depicted is a model of reactive spleen.
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