Vaccine Designs Utilizing Invariant NKT-Licensed Antigen-Presenting Cells Provide NKT or T Cell Help for B Cell Responses

Vaccines against a variety of infectious diseases have been developed and tested. Although there have been some notable successes, most are less than optimal or have failed outright. There has been discussion about whether either B cells or dendritic cells (DCs) could be useful for the development of antimicrobial vaccines with the production of high titers of antibodies. Invariant (i)NKT cells have direct antimicrobial effects as well as adjuvant activity, and iNKT-stimulated antigen-presenting cells (APCs) can determine the form of the ensuing humoral and cellular immune responses. In fact, upon activation by ligand, iNKT cells can stimulate both B cells and DCs as via either cognate or non-cognate help. iNKT-licensed DCs generate antigen-specific follicular helper CD4+ T cells, which in turn stimulate B cells, thus leading to long-term antigen-specific antibody production. Follicular helper iNKT cell-licensed B cells generally produce rapid, but short-term antibody. However, under some conditions in the presence of Th cells, the antibody production can be prolonged. With regards to humoral immunity, the quality and quantity of Ab produced depends on the APC type and the form of the vaccine. In terms of cellular immunity and, in particular, the induction of cytotoxic CD8+ T cells, iNKT-licensed DCs show prominent activity. In this review, we discuss differences in iNKT-stimulated APC types and the quality of the ensuing immune response, and also discuss their application in vaccine models to develop successful preventive immunotherapy against infectious diseases.

Vaccines against a variety of infectious diseases have been developed and tested. Although there have been some notable successes, most are less than optimal or have failed outright. There has been discussion about whether either B cells or dendritic cells (DCs) could be useful for the development of antimicrobial vaccines with the production of high titers of antibodies. Invariant (i)NKT cells have direct antimicrobial effects as well as adjuvant activity, and iNKT-stimulated antigen-presenting cells (APCs) can determine the form of the ensuing humoral and cellular immune responses. In fact, upon activation by ligand, iNKT cells can stimulate both B cells and DCs as via either cognate or non-cognate help. iNKT-licensed DCs generate antigen-specific follicular helper CD4 + T cells, which in turn stimulate B cells, thus leading to long-term antigen-specific antibody production. Follicular helper iNKT cell-licensed B cells generally produce rapid, but short-term antibody. However, under some conditions in the presence of Th cells, the antibody production can be prolonged. With regards to humoral immunity, the quality and quantity of Ab produced depends on the APC type and the form of the vaccine. In terms of cellular immunity and, in particular, the induction of cytotoxic CD8 + T cells, iNKT-licensed DCs show prominent activity. In this review, we discuss differences in iNKT-stimulated APC types and the quality of the ensuing immune response, and also discuss their application in vaccine models to develop successful preventive immunotherapy against infectious diseases.
Keywords: nKT, Tfh, nKTfh, dendritic cell, vaccines inTRODUCTiOn The success of vaccination strategies against viral infection or cancer depends on the efficient gene ration of appropriate antigenspecific T and B cell responses. Adequate antibody (Ab) responses of appropriate specificity elicited by vaccination are required to control and protect against many viral pathogens, such as influenza, human immunodeficiency virus, and human papilloma virus (1). Not only the suitable form of antigen, e.g., the commonly used inactivated virus, live attenuated virus, and recombinant viral protein, but also the optimal adjuvant are required for a successful vaccine. The development of ideal vaccine systems has been intensively explored to enhance the efficacy of weak antigens and broaden the immune response profile, leading to generation of high titer broadly neutralizing antiviral antibodies.
Vaccines targeting B cells are essentially, of two types, Tdependent and Tindependent, based on the requirement for Tcell help for Ab production (2). Tindependent B cell responses are usually elicited by nonprotein antigens that are unable to stimulate Th cells. Multimeric haptens or polysaccharides are typi cal Tindependent B cell antigens that are recognized via the B cell antigen receptor (BCR). Tindependent antigens generally induce robust and rapid B cell antibody responses, but with a low level of somatic hypermutation and thus affinity maturation, and limited isotype switching. Tdependent responses are typically induced by protein antigens and, as the term implies, there is cognate Tcell help for the antigenspecific B cells (3), which is provided by a specialized subset of CD4 + T cells called T follicular helper (Tfh) cells. When antigens contact B cells in the follicles of secondary lymphoid organs, the antigen is internalized by the B cells upon binding to antigenspecific BCRs. The antigen is then processed and antigenderived peptides are presented in the context of MHC class II (MHC II) molecules. Subsequently, the activated B cells are recruited to the border of the T cell and B cell zones, in which Tfh cells are generated following interacting with dendritic cells (DCs) presenting the same antigen. For the generation of Tfh cells, upregulation of the transcriptional rep ressor Bcl6, costimulation by CD28, and stimulation with IL21 have been reported as important factors (3). Also, by upregulating CXCR5, Tfh cells in turn localize to the boundary of the T and B cell zone (3), which is critical location for B cells to encounter Tfh cells.
Besides these classical Tdependent and Tindependent vac cines, NKT cellmediated vaccines have also been tested as a third vaccine candidate. NKT cells constitute approximately 0.05-0.2% of lymphocytes among human peripheral blood mononuclear cells and are classified into two groups: type I NKT cells express the invariant Vα14Jα18 TCRα chain paired with either Vβ2, Vβ7, or Vβ8 in mice and Vα24Jα18/Vβ11 in humans (4). The type I, invariant NKT cells (hereafter iNKT) recognize glycolip ids, such as αGalCer. By contrast, type II NKT cells display more diverse αβTCR pairings and respond to sulfatide, but do not to αGalCer (5). Several reports have shown that iNKT cells can deliver helper signals to B cells directly or indirectly. In infec tious diseases, neutralizing Ab production induced by vaccines represents a major protection mechanism against pathogens. Here, we review the features of iNKT cellmediated Ab produc tion, particularly by interacting directly or indirectly with B cells. We also discuss how these two pathways, i.e., vaccines utilizing iNKT cell help for B cells or iNKT cell help for DCs, augment efficient antigenspecific Ab production in the development of vaccination strategies against infectious diseases.

THe ROLe OF inKT CeLLS in inFeCTiOUS DiSeASeS
Realization of the importance of iNKT cells in protection from infectious diseases has largely been based studies of the res ponses of Jα18 or CD1ddeficient mice, both of which lack iNKT cells, to viruses, bacteria, and parasites (6,7). The outcome of most of these infectious models is worse in the iNKTdeficient animals. In studies of viral infections, iNKT cells play a protective role against influenza virus and cytomegalovirus (8,9), herpes simplex virus type 1, and hepatitis B virus (10). In bacterial infec tion models, iNKT cells have been shown to be important against Pseudomonas aeruginosa, Streptococcus pneumoniae, Mycobac terium tuberculosis (11), Chlamydia pneumoniae, Sphingomonas paucimobilis, and Staphylococcus aureus (12). The protective responses of iNKT cells during infections are mediated by two mechanisms. First is the direct activation by stimulation of the NKT TCR by iNKT cell ligands expressed on various pathogens. Second is indirect activation of iNKT cells is through other immune cells and is due to the cytokine milieu and tolllike receptors (TLRs) agonists. In the first type of response, iNKT cells directly recognize glycolipids and lipoproteins, highly abundant in cell walls of many pathogens. These include glycosphingo lipid in Gramnegative bacteria Sphingomonas, diacylglycerol in Borrelia burgdorferi, phosphatidylinositol mannoside in Mycobac terium tuberculosis, and glycosphingophospholipid in the proto zoa Leishmania donovani (13). In the second type of response, iNKT cells are activated through macrophages during microbial infections. When infected, antigenpresenting cells (APCs) recog nize bacterial signals via innate receptors, such as TLRs of mac rophages, e.g., TLR4, TLR7, and TLR9. These allow macrophages to produce inflammatory cytokines, e.g., IL12, which activate iNKT cells (14).

Two Types of inKT Based vaccines by Focusing on inKT-Licensed B Cells and inKT-Licensed DCs to induce Humoral immunity
B cell response is generally defined as being cognate helper T (Th), namely Tfhdependent (TD) or Tfhindependent (TI) (3). In terms of iNKTmediated vaccines directed toward B cell responses, there are two strategies using "iNKT mediatedDC therapy" or "iNKT mediatedB cell therapy" (Figure 1). For the induction of humoral responses, iNKT celllicensed DCs (noncognate help) can induce Tfh cells, resulting in longlasting antibody responses. On the other hand, iNKT celllicensed B cells (cognate help) generally result in rapid and robust, but shortterm responses. However, under some conditions, iNKT cell and B cell interactions seem to be more flexible and may be dependent on the experimental models. These are further discussed below.

Vaccines Using iNKT-Licensed B Cell Responses to Induce iNKTfh-Mediated Humoral Immunity
A lipid antigen component, e.g., a hapten or a lipid antigen con jugated to CD1dbinding glycolipids, can induce Tindependent Ab production (15,16). In addition to lipid antigens, a coad ministration of Tdependent protein antigen plus αGalCer, certain protein antigen conjugates, including protein-αGalCer conjugates or protein incorporated into αGalCerliposomes, have been introduced as vaccine formulations for cognate iNKT help (15).
When such immunogens are taken up via antigenspecific BCRs, they promote extensive BCR crosslinking and enhance BCR internalization. Simultaneously, the iNKT cell ligand from the immunogen is loaded on CD1d inside of B cells and then expressed on the cell surface, resulting in a strong cognate iNKT-B cell interaction (16)(17)(18)(19) (Figure 1A). Without incorporating the iNKT cell ligand, a Tindependent response usually elicits only shortlived IgM antibody without germinal center (GC) formation, affinity maturation, and class switch recombination (18). However, with an iNKT cell ligand, even as a Tindependent response, iNKT cells are converted to iNKTfh cells in a BCL6 dependent manner. iNKT cells help B cells to proliferate and dif ferentiate into extrafollicular plasma cells. They also robustly and rapidly induce GC formation, lowaffinity antibody maturation, secretion of high titers of specific IgM, and early classswitched antibodies. On the other hand, this humoral immunity is short lived and does not form a memory response (20)(21)(22).
In studies of the mechanism of iNKTlicensed B celldependent Ab production by lipid and/or proteinconjugated complexes, several factors have turned out to be essential. Since iNKT cells in steady state are usually located in the marginal zone in spleen or interfollicular or medulla areas in LNs (23,24), iNKT cells can more easily be activated by contacting marginal zone B cells than by follicular B cells after administration of αGalCer (16,21). The cognate help of iNKT cells for B cells presenting CD1dlipid is dependent on IFNγ, IL21, CD40/CD40L, and B71/2 (16,20,21). SAP expression in iNKT cells plays a key role in the cognate help for antigenspecific B cells, although this is not the case for non cognate helper functions (25).
In terms of vaccine strategy, several types of vaccines have been described including Tdependent protein antigens combined with αGalCer and these in nanoparticle liposome formulations (20)(21)(22). These showed the direct interaction between B cells and iNKT cells in response to Tdependent antigen, however, these strategies did not bring about high or long duration Ab produc tion (20)(21)(22). On the other hand, other investigators have used different conditions and could demonstrate longterm responses. Lang et al. showed that immunization with Tdependent protein antigen together with αGalCer in the presence of Th cells resulted in longlasting Ab titers (26)(27)(28). A model using coadministration of NPKLH and αGalCer elicits Ig classswitched Ab production and requires Th cells and CD1d on B cells, but not on DCs. Here, BAFF and APRILsecreting NKT cells play a role in plasma cell longevity (27,28). Also, liposomal nanoparticles displaying both lipid and polysaccharide antigens induce interactions with DCs for iNKT cell activation and then can elicit longterm B cell memory (29). Thus, the successful induction of iNKT celllicensed B cells in situ demands three signals (Figure 1), BCR crosslinking (signal 1), costimulatory molecules, such as CD28CD80/CD86, SLAMfamily receptor signaling, and CD40CD40L (signal 2), and inflammatory cytokines (IFNγ and IL21) (signal 3).

Vaccines Using iNKT-Licensed DC Responses to Induce Tfh-Mediated Humoral Immunity
We previously showed that coadministration of OVA protein antigen plus αGalCer elicited both CD4 + and CD8 + T cell responses via DC maturation (30). Similar responses have been demonstrated in some cancer and infectious disease models (31)(32)(33). We elucidated the mechanism of iNKTlicensed DCs for T cell response; activated iNKT cells promote DC maturation via CD40/40L signaling and cytokines (IFNγ and TNFα). DCs in situ capture the protein antigen and αGalCersimultaneously, then present αGalCer on CD1d to iNKT cells and the peptide on MHC class II to CD4 + T cells or class I to CD8 + T cells (34,35). The antigenspecific CD4 + Tfh cells that are derived from the helper CD4 + T cells stimulate antigenspecific B cells that had already taken up the protein via the BCR and presented peptide in MHC class II (18, 36) ( Figure 1B). Therefore, expression of both CD1d and MHC class II on DCs and MHC, II but not CD1d on B cells is essential for Ab production (37). Such a vaccine elicits a strong Ab response, i.e., characterized by GC formation, high affinity maturation, primary classswitched Ab, plasma cells, and memory B cells (37,38).
In the steady state, DCs express CD80/86 to some extent. After activation by iNKT cells, expression of costimulatory molecules, i.e., CD80/86 and CCR7, on DCs is promptly upregulated. The mature DCs are key players for priming CD4 + Tfh cells (3). When focused on DC subsets, we and others showed that the XCR1 − DC (CD8 − DC) subset is superior to the CD8 + DC subset for CD4 + Tfh cell priming (39). Shin et al. demonstrated this by using mAb to identify specific DC subsets, i.e., antiDEC205 for the CD8 + DC subset or antiDCIR2 antibodies for the CD8 − DC subset, and also showed that the CD8 − DCs subset is superior to the CD8 + DC sub set because of its dominant ICOSL and OX40L expression (40).
The location of DC and iNKT cells before and after iNKT cell activation has recently been clarified. As discussed previously, in the steady state, iNKT cells are localized in the marginal zone (MZ) of the spleen or medulla in LN (41), whereas XCR1 − DCs (CD8 − DC or 33D1 + DCs) are localized in the bridging channel (BC), a unique region of the spleen that spans the interface between the red pulp (RP) and white pulp (WP) and XCR1 + DCs reside in the MZ. After i.v. immunization with antigens and/or adjuvants, the majority of both DC subsets migrate into the WP, but XCR1 + DCs preferentially go to the CD8 + T cell area and XCR1 − DCs prefer to go to the CD4 + T cell area (42,43). After activation by αGalCer or ligandcontaining cells, activated iNKT cells accu mulate in the MZ or BC and can be in close contact with DCs that have already taken up antigen and αGalCer (44,45). iNKT licensed DCs then relocate to each T cell area.
Germinal center formation in secondary lymphoid organs is considered key for inducing better Ig class switching, somatic mutation, affinity maturation, and longlasting Ab responses (46). In addition to Bcl6 fl/fl CD4cre mice, LTα −/− mice and Lyn −/− mice are deficient in GC formation. When LTα −/− mice and Lyn −/− mice are immunized with antigen plus adjuvant, they respond with longlasting Ab production, probably due to the generation of longlived plasmablasts (19,(47)(48)(49)(50)(51). However, judging from the data using Bcl6 fl/fl CD4cre mice, the formation of GC is essential for iNKT cellmediated Ab production. When NKTfh and Tfh cells are compared, iNKTfh cells induce earlier GC formation, but it is more shortlived compared to GCs induced by Tfh cells (4). On the other hand, Tfh cells help in the induction of mature GCs better than iNKTfh cells (4). The expression of Bcl6 by Th cells is apparently crucial for efficient Ab production. Thus, iNKT cell licensed DCs induce antigenspecific CD4 + Tfh cells and drive them into the B cell zone (Figure 1B). Then, Tfh cells are engaged in cognate interactions with B cells, resulting in the formation of early GCs and also leading to the longlasting production of antigenspecific Abs.
The development of effective vaccines is a critical need. As discussed above, immunization by coadministration of protein antigen together with an iNKT cell ligand clearly generates potent Ab production. Traditional immunization protocols usually require a high dose of protein, e.g., 100-500 µg OVA protein per mouse is typically injected (15) yet, even so, Ab production is not impressively high (52). We have reported the artificial adjuvant vector cell (aAVC) system as an efficient vaccine strategy that can potently induce innate and adaptive immunity and this will be discussed in detail in the next section. But, to summarize this sec tion on the induction of antigenspecific Tfh cells and Ab, effec tive iNKT celllicensed DCs in situ are required (30,34,35,53). The DCs require three factors: (i) expression of the appropriate antigen peptide-MHC complex, (ii) upregulation of costimula tory and chemokine molecules, including CD80/CD86, ICOSL, and OX40L, and (iii) production of inflammatory cytokines and chemokines, such as IL6, IL12, and CCL17.

An Efficient Strategy Using iNKT-Licensed DCs to Induce Humoral Immunity
We have established the aAVC system, comprised of a CD1d + cellular vaccine incorporating foreign protein antigen plus an iNKT cell glycolipid antigen. We chose "adjuvant vector cell" as the name for this cellular vaccine to describe the fact that the "vector like cells" deliver the antigen as well as an iNKT cell adjuvant to host DCs. We used NIH3T3 cells for mouse and HEK293 cells for humans as vector cells (54,55). These cells are cotransfected with CD1d and antigen mRNA, and then loaded with αGalCer for use (54)(55)(56). The aAVC, therefore, express the αGalCer-CD1d complex on their surface and antigen protein intracellularly. The aAVCs directly activate iNKT cells via the αGalCer ligand, and iNKT cells producing IFNγ can then simultaneously activate NK cells. The combination of innate killer iNKT/NK cells capable of producing IFNγ then eliminates the adjuvant vector cells, which is not syngeneic with the recipient. Subsequently, the killed aAVC are taken up by DCs (CD8 + or XCR1 + DCs) in situ, thereby several immunogenic features of DCs are engaged. The aAVC captured by DCs in lung, liver, and spleen undergo maturation due to interaction with CD40L on iNKT cells and then produce inflammatory cytokines. The aAVC vaccine can efficiently generate antigenspecific CD8 + T cells and memory T cells (36). Interestingly, in aAVCvaccinated mice, antigenspecific CD4 + Tfh primed by XCR1 − DCs and GC were both generated, resulting in induction of longterm Ab produc tion (39). Killed aAVCs are phagocytosed, mainly by XCR1 + DC (CD8 + DC) cells and, presumably, the separated protein antigen and αGalCer are endocytosed simultaneously by the XCR1 − DC subset. MHC II −/− mice do not have CD4 + T cells but do have iNKT cells. Immunization with aAVCOVA elicited no detectable specific Ab response in these mice (39), indicating that iNKT cell licensed DC strategies require MHC II for CD4 + T cellmediated humoral immunity. Although both Tfh and iNKTfh coexist in the aAVCmouse models, Tfh cells are superior to iNKTfh cells for inducing Ab production (39).

APPLiCATiOn OF An inKT CeLL-TRiGGeReD DC-DeSiGneD vACCine "ARTiFiCiAL ADJUvAnT veCTOR CeLL" FOR inFLUenZA inFeCTiOn
Influenza virus is a member of the orthomyxoviridae family that contains a segmented negativesense singlestranded RNA gen ome (57). Influenza infection is a major global health prob lem, which is initially caused by viral infection of the respiratory tract. Upon viral infection, iNKT cells in the follicular areas of LNs facilitate IL4 signaling to B cells which triggers the seeding of GC cells and B cell immunity (23). However, to provide sufficient protection against influenza virus in humans, both adaptive T cell and Abproducing B cells need to be established and maintained as immunological memory.
Immunity against influenza virus is largely mediated by neutral izing antibodies that target the major surface glycoprotein hemag glutinin (HA) (58), in particular, the immunodominant head region of HA, or the viral neuraminidase (NA) (59). Preexisting neutral izing Ab, rather than the recall of virusspecific CTL, is thought to account for memory antiviral protection. Unfortunately, however, the antiviral antibodies generated by immunization or natural infection are only effective against a limited number of viral strains.
Several studies have addressed the possibility of using a combined vaccine approach, i.e., coadministration of inactivated influenza virus (IIV) together with αGalCer, to enhance pro tective efficacy via subcutaneous or intranasal administration (60)(61)(62). We established CD1d + HA mRNAtransfected cells loaded with αGalCer (aAVCHA) and demonstrated that this is a more efficient strategy for generating antigenspecific Ab production than coadministration of antigen plus αGalCer (Figure 2) (39). The efficacy of aAVC appears to depend on the GC and Tfh. We could easily modify the HA protein, depending on the circulating virus strain, and the manufacturing process for this vaccine has a much shorter timeframe than others, e.g., eggderived vaccines, which would be especially valuable during a flu pandemic. The aAVC vaccine thus holds great promise as a potential broad spectrum prophylactic or therapeutic agent and for the development of a universal influenza or other viral vaccine (39). As a future study, if the optimal stem region antigen were incorporated into an iNKT cellmediated vaccine, a universal HA stem antigenexpressing aAVC could be developed.

COnCLUSiOn
iNKT cells play an immunomodulatory role during immune responses. To utilize this capacity as an adjuvant in terms of novel vaccine strategies, it is essential to understand the interaction of iNKT cells with APCs in the host. We here summarized details of the relationship of iNKT celltriggered B cells and DCs with Ab production and compared them in terms of vaccine develop ment. Now we need to consider the future directions and challenges in translating these findings from experimental data obtained from mice to use in the clinic.

AUTHOR COnTRiBUTiOnS
SF and KS conceptualized, wrote, and edited the manuscript. SY and YS wrote and edited the manuscript.

ACKnOwLeDGMenTS
The authors are grateful to Dr. Peter Burrows for peerreviewing and helpful comments in the preparation of the manuscript. This work is supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to KS and SF, grant number 15K09590) and the Japan Agency for Medical Research and Development (translational research network program) (to SF, grant number 15lm0103002j0004).