Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein

The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of alpha-galactosylceramide (alphaGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-gamma production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by alphaGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of alphaGalCer, mice were given alphaGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-gamma producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, alphaGalCer, NKT, or NK cells. Therefore a single dose of alphaGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein.     

The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation

Dendritic cell (DC) maturation is an innate response that leads to adaptive immunity to coadministered proteins. To begin to identify underlying mechanisms in intact lymphoid tissues, we studied alpha-galactosylceramide. This glycolipid activates innate Valpha14(+) natural killer T cell (NKT) lymphocytes, which drive DC maturation and T cell responses to ovalbumin antigen. Hours after giving glycolipid i.v., tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were released primarily by DCs. These cytokines induced rapid surface remodeling of DCs, including increased CD80/86 costimulatory molecules. Surprisingly, DCs from CD40(-/-) and CD40L(-/-) mice did not elicit CD4(+) and CD8(+) T cell immunity, even though the DCs exhibited presented ovalbumin on major histocompatibility complex class I and II products and expressed high levels of CD80/86. Likewise, an injection of TNF-alpha up-regulated CD80/86 on DCs, but CD40 was required for immunity. CD40 was needed for DC interleukin (IL)-12 production, but IL-12p40(-/-) mice generated normal ovalbumin-specific responses. Therefore, the link between innate and adaptive immunity via splenic DCs and innate NKT cells has several components under distinct controls: antigen presentation in the steady state, increases in costimulatory molecules dependent on inflammatory cytokines, and a distinct CD40/CD40L signal that functions together with antigen presentation ("signal one") and costimulation ("signal two") to generate functioning CD4(+) T helper cell 1 and CD8(+) cytolytic T lymphocytes.     

Cross-presentation of glycolipid from tumor cells loaded with alpha-galactosylceramide leads to potent and long-lived T cell mediated immunity via dendritic cells

We report a mechanism to induce combined and long-lived CD4⁺ and CD8⁺ T cell immunity to several mouse tumors. Surprisingly, the initial source of antigen is a single low dose of tumor cells loaded with α-galactosylceramide (α-GalCer) glycolipid (tumor/Gal) but lacking co-stimulatory molecules. After tumor/Gal injection intravenously (i.v.), innate NKT and NK cells reject the tumor cells, some of which are taken up by dendritic cells (DCs). The DCs in turn cross-present glycolipid on CD1d molecules to NKT cells and undergo maturation. For B16 melanoma cells loaded with α-GalCer (B16/Gal), interferon γ–producing CD8⁺ T cells develop toward several melanoma peptides, again after a single low i.v. dose of B16/Gal. In all four poorly immunogenic tumors tested, a single dose of tumor/Gal i.v. allows mice to become resistant to tumors given subcutaneously. Resistance requires CD4⁺ and CD8⁺ cells, as well as DCs, and persists for 6–12 mo. Therefore, several immunogenic features of DCs are engaged by the CD1d-mediated cross-presentation of glycolipid-loaded tumor cells, leading to particularly strong and long-lived adaptive immunity.     

OX40 ligand expressed by DCs costimulates NKT and CD4+ Th cell antitumor immunity in mice

The exceptional immunostimulatory capacity of DCs makes them potential targets for investigation of cancer immunotherapeutics. We show here in mice that TNF-alpha-stimulated DC maturation was accompanied by increased expression of OX40 ligand (OX40L), the lack of which resulted in an inability of mature DCs to generate cellular antitumor immunity. Furthermore, intratumoral administration of DCs modified to express OX40L suppressed tumor growth through the generation of tumor-specific cytolytic T cell responses, which were mediated by CD4+ T cells and NKT cells. In the tumors treated with OX40L-expressing DCs, the NKT cell population significantly increased and exhibited a substantial level of IFN-gamma production essential for antitumor immunity. Additional studies evaluating NKT cell activation status, in terms of IFN-gamma production and CD69 expression, indicated that NKT cell activation by DCs presenting alpha-galactosylceramide in the context of CD1d was potentiated by OX40 expression on NKT cells. These results show a critical role for OX40L on DCs, via binding to OX40 on NKT cells and CD4+ T cells, in the induction of antitumor immunity in tumor-bearing mice.     

Natural killer T cell ligand alpha-galactosylceramide enhances protective immunity induced by malaria vaccines

The important role played by CD8(+) T lymphocytes in the control of parasitic and viral infections, as well as tumor development, has raised the need for the development of adjuvants capable of enhancing cell-mediated immunity. It is well established that protective immunity against liver stages of malaria parasites is primarily mediated by CD8(+) T cells in mice. Activation of natural killer T (NKT) cells by the glycolipid ligand, alpha-galactosylceramide (alpha-GalCer), causes bystander activation of NK, B, CD4(+), and CD8(+) T cells. Our study shows that coadministration of alpha-GalCer with suboptimal doses of irradiated sporozoites or recombinant viruses expressing a malaria antigen greatly enhances the level of protective anti-malaria immunity in mice. We also show that coadministration of alpha-GalCer with various different immunogens strongly enhances antigen-specific CD8(+) T cell responses, and to a lesser degree, Th1-type responses. The adjuvant effects of alpha-GalCer require CD1d molecules, Valpha14 NKT cells, and interferon gamma. As alpha-GalCer stimulates both human and murine NKT cells, these findings should contribute to the design of more effective vaccines against malaria and other intracellular pathogens, as well as tumors.     

Sustained expansion of NKT cells and antigen-specific T cells after injection of alpha-galactosyl-ceramide loaded mature dendritic cells in cancer patients

Natural killer T (NKT) cells are distinct glycolipid reactive innate lymphocytes that are implicated in the resistance to pathogens and tumors. Earlier attempts to mobilize NKT cells, specifically, in vivo in humans met with limited success. Here, we evaluated intravenous injection of monocyte-derived mature DCs that were loaded with a synthetic NKT cell ligand, alpha-galactosyl-ceramide (alpha-GalCer; KRN-7000) in five patients who had advanced cancer. Injection of alpha-GalCer-pulsed, but not unpulsed, dendritic cells (DCs) led to >100-fold expansion of several subsets of NKT cells in all patients; these could be detected for up to 6 mo after vaccination. NKT activation was associated with an increase in serum levels of interleukin-12 p40 and IFN-gamma inducible protein-10. In addition, there was an increase in memory CD8+ T cells specific for cytomegalovirus in vivo in response to alpha-GalCer-loaded DCs, but not unpulsed DCs. These data demonstrate the feasibility of sustained expansion of NKT cells in vivo in humans, including patients who have advanced cancer, and suggest that NKT activation might help to boost adaptive T cell immunity in vivo.     

A nonclassical non-Valpha14Jalpha18 CD1d-restricted (type II) NKT cell is sufficient for down-regulation of tumor immunosurveillance

The importance of immunoregulatory T cells has become increasingly apparent. Both CD4+CD25+ T cells and CD1d-restricted NKT cells have been reported to down-regulate tumor immunity in mouse tumor models. However, the relative roles of both T cell populations have rarely been clearly distinguished in the same tumor models. In addition, CD1d-restricted NKT cells have been reported to play a critical role not only in the down-regulation of tumor immunity but also in the promotion of the immunity. However, the explanation for these apparently opposite roles in different tumor models remains unclear. We show that in four mouse tumor models in which CD1d-restricted NKT cells play a role in suppression of tumor immunity, depletion of CD4+CD25+ T cells did not induce enhancement of immunosurveillance. Surprisingly, among the two subpopulations of CD1d-restricted NKT cells, Valpha14Jalpha18+ (type I) and Valpha14Jalpha18- (type II) NKT cells, type I NKT cells were not necessary for the immune suppression. These unexpected results may now resolve the paradox in the role of CD1d-restricted NKT cells in the regulation of tumor immunity, in that type II NKT cells may be sufficient for negative regulation, whereas protection has been found to be mediated by alpha-galactosylceramide-responsive type I NKT cells.     

NKT cell-dependent leukemia eradication following stem cell mobilization with potent G-CSF analogs

NKT cells have pivotal roles in immune regulation and tumor immunosurveillance. We report that the G-CSF and FMS-like tyrosine kinase 3 ligand (Flt-3L) chimeric cytokine, progenipoietin-1, markedly expands the splenic and hepatic NKT cell population and enhances functional responses to alpha-galactosylceramide. In a murine model of allogeneic stem cell transplantation, donor NKT cells promoted host DC activation and enhanced perforin-restricted CD8+ T cell cytotoxicity against host-type antigens. Following leukemic challenge, donor treatment with progenipoietin-1 significantly improved overall survival when compared with G-CSF or control, attributable to reduced graft-versus-host disease mortality and paradoxical augmentation of graft-versus-leukemia (GVL) effects. Enhanced cellular cytotoxicity was dependent on donor NKT cells, and leukemia clearance was profoundly impaired in recipients of NKT cell-deficient grafts. Enhanced cytotoxicity and GVL effects were not associated with Flt-3L signaling or effects on DCs but were reproduced by prolonged G-CSF receptor engagement with pegylated G-CSF. Thus, modified G-CSF signaling during stem cell mobilization augments NKT cell-dependent CD8+ cytotoxicity, effectively separating graft-versus-host disease and GVL and greatly expanding the potential applicability of allogeneic stem cell transplantation for the therapy of malignant disease.     

Route of immunization with peptide-pulsed dendritic cells controls the distribution of memory and effector T cells in lymphoid tissues and determines the pattern of regional tumor control

We have established that the route of immunization with peptide-pulsed, activated DC leads to memory CD8+ T cells with distinct distributions in lymphoid tissues, which determines the ability to control tumors growing in different body sites. Both intravenous (i.v.) and subcutaneous (s.c.) immunization induced memory T cells in spleen and control of metastatic-like lung tumors. s.c. immunization also induced memory T cells in lymph nodes (LNs), imparting protection against subcutaneously growing tumors. In contrast, i.v. immunization-induced memory was restricted to spleen and failed to impart protective immunity against subcutaneously growing tumors. Memory cell distribution and tumor control were both linked to injection route-dependent localization of DCs in lymphoid compartments. Using peripheral LN-ablated mice, these LNs were shown to be essential for control of subcutaneously growing tumors but not lung metastases; in contrast, using immunized asplenic mice, we found that the spleen is necessary and sufficient for control of lung tumors, but unnecessary for control of subcutaneously growing tumors. These data demonstrate the existence of a previously undescribed population of splenic-resident memory CD8 T cells that are essential for the control of lung metastases. Thus, regional immunity based on memory T cell residence patterns is an important factor in DC-based tumor immunotherapy.     

Equipotent generation of protective antitumor immunity by various methods of dendritic cell loading with whole cell tumor antigens

Multiple clinically applicable methods have been used to induce dendritic cells (DCs) to express whole cell tumor antigens, including pulsing DCs with tumor lysate, and mixing DCs with apoptotic or live tumor cells. Herein we demonstrate, using two different tumor systems, that these methods are equipotent inducers of systemic antitumor immunity. Furthermore, tumor lysate pulsed DC vaccines generate more potent antitumor immunity than immunization with irradiated tumor cells plus the classic adjuvant, Corynebacterium parvum.     

Murine induced pluripotent stem cells can be derived from and differentiate into natural killer T cells

NKT cells demonstrate antitumor activity when activated to produce Th1 cytokines by DCs loaded with α-galactosylceramide, the prototypic NKT cell–activating glycolipid antigen. However, most patients do not have sufficient numbers of NKT cells to induce an effective immune response in this context, indicating a need for a source of NKT cells that could be used to supplement the endogenous cell population. Induced pluripotent stem cells (iPSCs) hold tremendous potential for cell-replacement therapy, but whether it is possible to generate functionally competent NKT cells from iPSCs has not been rigorously assessed. In this study, we successfully derived iPSCs both from embryonic fibroblasts from mice harboring functional NKT cell–specific rearranged T cell receptor loci in the germline and from splenic NKT cells from WT adult mice. These iPSCs could be differentiated into NKT cells in vitro and secreted large amounts of the Th1 cytokine IFN-γ. Importantly, iPSC-derived NKT cells recapitulated the known adjuvant effects of natural NKT cells and suppressed tumor growth in vivo. These studies demonstrate the feasibility of expanding functionally competent NKT cells via an iPSC phase, an approach that may be adapted for NKT cell–targeted therapy in humans.     

Utilizing the adjuvant properties of CD1d-dependent NK T cells in T cell-mediated immunotherapy

Activation of invariant CD1d-dependent NK T cells (iNKT cells) in vivo through administration of the glycolipid ligand alpha-galactosylceramide (alpha-GalCer) or the sphingosine-truncated alpha-GalCer analog OCH leads to CD40 signaling as well as the release of soluble molecules including type 1 and gamma interferons that contribute to DC maturation. This process enhances T cell immunity to antigens presented by the DC. The adjuvant activity is further amplified if APCs are stimulated through Toll-like receptor 4, suggesting that iNKT cell signals can amplify maturation induced by microbial stimuli. The adjuvant activity of alpha-GalCer enhances both priming and boosting of CD8(+) T cells to coadministered peptide or protein antigens, including a peptide encoding the clinically relevant, HLA-A2-restricted epitope of the human tumor antigen NY-ESO-1. Importantly, alpha-GalCer was used to induce CD8(+) T cells to antigens delivered orally, despite the fact that this route of administration is normally associated with blunted responses. Only T cell responses induced in the presence of iNKT cell stimulation, whether by the i.v. or oral route, were capable of eradicating established tumors. Together these data highlight the therapeutic potential of iNKT cell ligands in vaccination strategies, particularly "heterologous prime-boost" strategies against tumors, and provide evidence that iNKT cell stimulation may be exploited in the development of oral vaccines.     

Fatty acid amide hydrolase shapes NKT cell responses by influencing the serum transport of lipid antigen in mice

The potent regulatory properties of NKT cells render this subset of lipid-specific T cells a promising target for immunotherapeutic interventions. The marine sponge glycolipid α-galactosylceramide (αGalCer) is the proto­typic NKT cell agonist, which elicits this function when bound to CD1d. However, our understanding of the in vivo properties of NKT cell agonists and the host factors that control their bioactivity remains very limited. In this report, we isolated the enzyme fatty acid amide hydrolase (FAAH) from mouse serum as an αGalCer-binding protein that modulates the induction of key effector functions of NKT cells in vivo. FAAH bound αGalCer in vivo and in vitro and was required for the efficient targeting of lipid antigens for CD1d presentation. Immunization of Faah-deficient mice with αGalCer resulted in a reduced systemic cytokine production, but enhanced expansion of splenic NKT cells. This distinct NKT response conferred a drastically increased adjuvant effect and strongly promoted protective CTL responses. Thus, our findings identify not only the presence of FAAH in normal mouse serum, but also its critical role in the tuning of immune responses to lipid antigens by orchestrating their transport and targeting for NKT cell activation. Our results suggest that the serum transport of lipid antigens directly shapes the quality of NKT cell responses, which could potentially be modulated in support of novel vaccination strategies.     

Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance

To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal alphaDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c- cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When alphaDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4-48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of alphaDEC-205:OVA to DCs in the steady state initially induced 4-7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with alphaDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic alphaCD40 antibody produced large amounts of interleukin 2 and interferon gamma, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation.     

Monoclonal antibodies targeted against melanoma and ovarian tumors enhance dendritic cell-mediated cross-presentation of tumor-associated antigens and efficiently cross-prime CD8+ T cells

Dendritic cells (DCs) constitute very attractive vectors for cancer immunotherapy due to their ability to efficiently capture and present tumor antigens, which initiates tumor-directed T-cell responses. Because the initiation of cytotoxic anti-tumor immune responses requires the cross-presentation mechanism, antigen targeting to DCs represents a very important step in the chain of events that constitutes the cross-priming immune process. In the current study, we explored the ability of DCs loaded with antibody-coated melanoma and ovarian carcinoma tumor cells to cross-present tumor antigens to CD8+ T cells and elicit in vitro anti-tumor immune responses. Coating melanoma and ovarian cancer cells with monoclonal antibodies against different surface antigens (CD44, ME491, LFA-3, and CD24) expressed by the tumor cells promoted the cross-presentation of the tumor-associated antigens as MART-1, gp100, tyrosinase, and NY-ESO-1 by DCs to CD8+ T. These tumor antigen-specific CD8+ T-cell populations resulting from the DC-mediated cross-priming process were identified using specific immune tetramers and were a few fold larger than the ones generated using peptide-pulsed or apoptotic tumor cell-loaded DCs. The CD8+ T cells generated by DCs loaded with monoclonal antibody-coated tumor cells were cytotoxic against the primary melanoma and ovarian carcinoma cells. Thus, targeting monoclonal antibody-coated tumor cells to DCs is a novel method that opens new perspectives for immunotherapy strategies.     

Mechanisms involved in radiation enhancement of intratumoral dendritic cell therapy

We have previously reported that local tumor irradiation, without inducing cell death, can augment the therapeutic efficacy of intratumoral (IT) dendritic cell (DC) vaccination. This study examined potential mechanisms underlying radiation enhancement of IT DC therapy in this setting. Even though ionizing radiation did not mediate tumor cell killing, bone marrow-derived DCs acquired in vitro tumor antigens from irradiated D5 murine melanoma cells more efficiently than from untreated cells. This radiation-enhanced loading of DCs did not induce DC maturation, but was associated with improved cross-priming of T cells both in vitro and in vivo. Furthermore, in vivo pulsing of DCs with irradiated versus untreated tumor cells resulted in superior presentation of tumor antigens to T cells. In addition, tumor irradiation facilitated homing of IT administered DCs to the draining lymph node, possibly by down-regulating CCL21 expression within the tumor mass. Studies of the tumor microenvironment in irradiated versus untreated tumors did not reveal significant inflammatory changes. Moreover, radiation did not promote accumulation of CD4 or CD8 effector T cells within solid tumors. Our results indicate that, without inducing cytotoxicity, tumor irradiation can enhance the ability of DCs to capture tumor antigens, migrate to the draining lymph node, and present processed antigens to T cells. These findings may prove useful in designing future strategies for human cancer immunotherapy.     

New ways to turn on NKT cells

Natural killer T (NKT) cells are CD1d-restricted, lipid antigen-reactive T cells with powerful immunoregulatory potential. The prototypic antigen for NKT cells is a marine sponge-derived glycolipid, α-galactosylceramide (α-GalCer), but this is not normally encountered in the mammalian environment. Thus, there is great interest in the identification of more physiological stimuli for NKT cells, and numerous studies have shown that NKT cells are capable of responding to a range of microbial lipid-based antigens. Two new studies expand our understanding of environmental NKT cell stimuli, with one showing that CD1d-restricted NKT cell antigens are present within common house dust extract (HDE), whereas the other shows that NKT cells can respond to innate stimuli irrespective of the presence of foreign microbial antigens. Collectively, these two investigations indicate that NKT cells are far more likely to encounter foreign antigens, or innate activating signals, than previously recognized, suggesting a more central role for these cells in the immune system.     

Dendritic cells are responsible for the capacity of CpG oligodeoxynucleotides to act as an adjuvant for protective vaccine immunity against Leishmania major in mice

Vaccination with leishmanial Ag and CpG oligodeoxynucleotides (ODN) confers sustained cellular immunity and protection to infectious challenge up to 6 mo after immunization. To define the cellular mechanism by which CpG ODN mediate their adjuvant effects in vivo, the functional capacity of distinct dendritic cell (DC) subsets was assessed in the lymph nodes (LNs) of BALB/c mice, 36 h after immunization with the leishmanial antigen (LACK) and CpG ODN. After this immunization, there was a striking decrease in the frequency of the CD11c+B220+ plasmacytoid DCs with a proportionate increase in CD11c+CD8-B220- cells. CD11c+CD8+B220- cells were the most potent producers of interleukin (IL)-12 p70 and interferon (IFN)-gamma, while plasmacytoid DCs were the only subset capable of secreting IFN-alpha. In terms of antigen presenting capacity, plasmacytoid DCs were far less efficient compared with the other DC subsets. To certify that DCs were responsible for effective vaccination, we isolated CD11c+ and CD11c- cells 36 h after immunization and used such cells to elicit protective immunity after adoptive transfer in naive, Leishmania major susceptible BALB/c mice. CD11c+ cells but not 10-fold higher numbers of CD11c- cells from such immunized mice mediated protection. Therefore, the combination of LACK antigen and CpG ODN adjuvant leads to the presence of CD11c+ DCs in the draining LN that are capable of vaccinating naive mice in the absence of further antigen or adjuvant.     

The adaptor protein AP-3 is required for CD1d-mediated antigen presentation of glycosphingolipids and development of Valpha14i NKT cells

Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 molecules. Here we show that the adaptor protein complex 3 (AP-3) is required for the efficient presentation of glycolipid antigens that require internalization and processing. AP-3 interacts with mouse CD1d, and cells from mice deficient for AP-3 have increased cell surface levels of CD1d and decreased expression in late endosomes. Spleen cells from AP-3-deficient mice have a reduced ability to present glycolipids to natural killer T (NKT) cells. Furthermore, AP-3-deficient mice have a significantly reduced NKT cell population, although this is not caused by self-tolerance that might result from increased CD1d surface levels. These data suggest that the generation of the endogenous ligand that selects NKT cells may also be AP-3 dependent. However, the function of MHC class II-reactive CD4+ T lymphocytes is not altered by AP-3 deficiency. Consistent with this divergence from the class II pathway, NKT cell development and antigen presentation by CD1d are not reduced by invariant chain deficiency. These data demonstrate that the AP-3 requirement is a particular attribute of the CD1d pathway in mice and that, although MHC class II molecules and CD1d are both found in late endosomes or lysosomes, different pathways mediate their intracellular trafficking.