Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8(+) T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8(+) T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8(+) T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8(+) T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors.
Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471-1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis.
Live attenuated nonpathogenic Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediates long-lasting immune responses, has been safely administered as a tuberculosis vaccine to billions of humans, and is affordable to produce as a vaccine vector. These characteristics make it very attractive as a human immunodeficiency virus (HIV) vaccine vector candidate. Here, we assessed the immunogenicity of recombinant BCG (rBCG) constructs with different simian immunodeficiency virus (SIV)gag expression cassettes as priming agents followed by a recombinant replication-incompetent New York vaccinia virus (NYVAC) boost in rhesus macaques. Unmutated rBCG constructs were used in comparison to mutants with gene deletions identified in an in vitro screen for augmented immunogenicity. We demonstrated that BCG-SIVgag is able to elicit robust transgene-specific priming responses, resulting in strong SIV epitope-specific cellular immune responses. While enhanced immunogenicity was sustained at moderate levels for >1 year following the heterologous boost vaccination, we were unable to demonstrate a protective effect after repeated rectal mucosal challenges with pathogenic SIVmac251. Our findings highlight the potential for rBCG vaccines to stimulate effective cross-priming and enhanced major histocompatibility complex class I presentation, suggesting that combining this approach with other immunogens may contribute to the development of effective vaccine regimens against HIV.
NKT cells are a subpopulation of T lymphocytes with phenotypic properties of both T and NK cells and a wide range of immune effector properties. In particular, one subset of these cells, known as invariant NKT cells (iNKT cells), has attracted substantial attention because of their ability to be specifically activated by glycolipid antigens presented by a cell surface protein called CD1d. The development of synthetic ?-galactosylceramides as a family of powerful glycolipid agonists for iNKT cells has led to approaches for augmenting a wide variety of immune responses, including those involved in vaccination against infections and cancers. Here, we review basic, preclinical and clinical observations supporting approaches to improving immune responses through the use of iNKT cell-activating glycolipids. Results from preclinical animal studies and preliminary clinical studies in humans identify many promising applications for this approach in the development of vaccines and novel immunotherapies.
Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8?(+) DEC-205(+) dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of ?-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8?(+) dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.
Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid ?-galactosylceramide (?-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of ?-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an ?-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.
We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the ?MT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous ?MT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization.
CD1c is expressed with high density on human dendritic cells (DCs) and B cells, yet its antigen presentation functions are the least well understood among CD1 family members. Using a CD1c-reactive T cell line (DN6) to complete an organism-wide survey of M. tuberculosis lipids, we identified C32 phosphomycoketide (PM) as a previously unknown molecule and a CD1c-presented antigen. CD1c binding and presentation of mycoketide antigens absolutely required the unusual, mycobacteria-specific lipid branching patterns introduced by polyketide synthase 12 (pks12). Unexpectedly, one TCR responded to diversely glycosylated and unglycosylated forms of mycoketide when presented by DCs and B cells. Yet cell-free systems showed that recognition was mediated only by the deglycosylated phosphoantigen. These studies identify antigen processing of a natural bacterial antigen in the human CD1c system, indicating that cells act on glycolipids to generate a highly simplified neoepitope composed of a sugar-free phosphate anion. Using knowledge of this processed antigen, we generated human CD1c tetramers, and demonstrate that CD1c-PM complexes stain T cell receptors (TCRs), providing direct evidence for a ternary interaction among CD1c-lipid-TCR. Furthermore, PM-loaded CD1c tetramers detect fresh human T cells from peripheral blood, demonstrating a polyclonal response to PM antigens in humans ex vivo.
The critical role of peptide antigen-specific T cells in controlling mycobacterial infections is well documented in natural resistance and vaccine-induced immunity against Mycobacterium tuberculosis. However, many other populations of leukocytes contribute to innate and adaptive immunity against mycobacteria. Among these, non-conventional T cells recognizing lipid antigens presented by the CD1 antigen presentation system have attracted particular interest. In this chapter, we review the basic immunobiology and potential antimycobacterial properties of a subset of CD1-restricted T cells that have come to be known as Natural Killer T cells. This group of lipid reactive T cells is notable for its high level of conservation between humans and mice, thus enabling a wide range of highly informative studies in mouse models. As reviewed below, NKT cells appear to have subtle but potentially significant activities in the host response to mycobacteria. Importantly, they also provide a framework for investigations into other types of lipid antigen-specific T cells that may be more abundant in larger mammals such as humans.
Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by ?? T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and ??T cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.
Despite a high degree of conservation, subtle but important differences exist between the CD1d antigen presentation pathways of humans and mice. These differences may account for the minimal success of natural killer T (NKT) cell-based antitumor therapies in human clinical trials, which contrast strongly with the powerful antitumor effects in conventional mouse models. To develop an accurate model for in vivo human CD1d (hCD1d) antigen presentation, we have generated a hCD1d knock-in (hCD1d-KI) mouse. In these mice, hCD1d is expressed in a native tissue distribution pattern and supports NKT cell development. Reduced numbers of invariant NKT (iNKT) cells were observed, but at an abundance comparable to that in most normal humans. These iNKT cells predominantly expressed mouse V?8, the homolog of human V?11, and phenotypically resembled human iNKT cells in their reduced expression of CD4. Importantly, iNKT cells in hCD1d knock-in mice exert a potent antitumor function in a melanoma challenge model. Our results show that replacement of mCD1d by hCD1d can select a population of functional iNKT cells closely resembling human iNKT cells. These hCD1d knock-in mice will allow more accurate in vivo modeling of human iNKT cell responses and will facilitate the preclinical assessment of iNKT cell-targeted antitumor therapies.
The guinea pig has a storied history as a model in the study of infectious disease and immunology. Because of reproducibility of data and availability of various reagents, inbred mice have since supplanted the guinea pig as the animal model-of-choice in these fields. However, several clinically-significant microorganisms do not cause the same pathology in mice, or mice may not be susceptible to these infections. These demonstrate the utility of other animal models - either as the primary method to study a particular infection, or to confirm or refute findings in the mouse before translating basic science into clinical practice. The mononuclear phagocyte, or macrophage (M?), plays a key role in antigen presentation and the pathogenesis of intracellular bacteria, such as Mycobacterium tuberculosis and Legionella pneumophila. Because of variable yield and difficult extraction from tissue, the preferred method of producing M? for in vitro studies is to expand murine bone marrow (BM) precursors with mouse macrophage colony-stimulating factor (M-CSF). This has not been shown in the guinea pig. Here, we report the empiric observation that human M-CSF - but not mouse M-CSF, nor human granulocyte/macrophage colony-stimulating factor - can be used to induce BM precursor differentiation into bonafide M?. The differentiated cells appeared as enlarged adherent cells, capable of both pinocytosis and large particle phagocytosis. Furthermore, we showed that these guinea pig BM-derived M?, similar to human monocyte/M? lines but unlike most murine BM M?, support growth of wild type L. pneumophila. This method may prove useful for in vitro studies of M? in the guinea pig, as well as in the translation of results found using mouse BM-derived M? towards studies in human immunology and infectious disease.
Immunity conferred by antigen-specific CD4+ T cells is critical for controlling infection with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. However, despite research that spans more than a century, many of the characteristics of protective immune responses to Mtb remain elusive. Defining the repertoire of antigenic targets is central to understanding the immune response against this pathogen. Although traditional methods of antigen discovery have identified many immunodominant antigens, they afford limited proteome coverage. Recent advances in proteomic techniques that are based on peptide library and protein microarray technology have enabled interrogation of the entire proteome of Mtb for antigens. Though these techniques have limitations and are still evolving, early studies using these techniques provide an unbiased view of the immune response to Mtb. Here we review proteome-wide approaches to antigen discovery and summarize what these have revealed so far on the composition of the Mtb immunoproteome.
Natural killer T (NKT) cells express a semi-invariant V?14 T cell receptor (TCR) and recognize structurally diverse antigens presented by the antigen-presenting molecule CD1d that range from phosphoglycerolipids to ?- and ?-anomeric glycosphingolipids, as well as microbial ?-glycosyl diacylglycerolipids. Recently developed antibodies that are specific for the complex of the prototypical invariant NKT (iNKT) cell antigen ?GalCer (KRN7000) bound to mouse CD1d have become valuable tools in elucidating the mechanism of antigen loading and presentation. Here, we report the 3.1 ? resolution crystal structure of the Fab of one of these antibodies, L363, bound to mCD1d complexed with the ?GalCer analog C20:2, revealing that L363 is an iNKT TCR-like antibody that binds CD1d-presented ?GalCer in a manner similar to the TCR. The structure reveals that L363 depends on both the L and H chains for binding to the glycolipid-mCD1d complex, although only the L chain is involved in contacts with the glycolipid antigen. The H chain of L363 features residue Trp-104, which mimics the TCR CDR3? residue Leu-99, which is crucial for CD1d binding. We characterized the antigen-specificity of L363 toward several different glycolipids, demonstrating that whereas the TCR can induce structural changes in both antigen and CD1d to recognize disparate lipid antigens, the antibody L363 can only induce the F roof formation in CD1d but fails to reorient the glycolipid headgroup necessary for binding. In summary, L363 is a powerful tool to study mechanism of iNKT cell activation for structural analogs of KRN7000, and our study can aid in the design of antibodies with altered antigen specificity.
Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR ?-chain is invariant, NKT TCR V? exhibits greater diversity, with one (V?11) and three (V?8, V?7, and V?2) V? chains in humans and mice, respectively. With the exception of the V?2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2? that are critical for CD1d binding. Thus, how V?2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2?-encoded tyrosine residues, we show that the V?2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the V?8.2 and V?7 NKT TCRs. Accordingly, the germline-encoded regions of the TCR ?-chain do not exclusively dictate the innate NKT TCR-CD1d-Ag docking mode. Nevertheless, clear fine specificity differences for the CD1d-Ag existed between the V?2 NKT TCR and the V?8.2 and V?7 NKT TCRs, with the V?2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the V?2 NKT TCR-CD1d-?GalCer complex, the CDR2? loop mediated fewer contacts with CD1d, whereas the CDR1? and CDR3? loops contacted CD1d to a much greater extent compared with most V?11, V?8.2, and V?7 NKT TCRs. Accordingly, there is a greater interplay between the germline- and nongermline-encoded loops within the TCR ?-chain of the V?2 NKT TCR that enables CD1d-Ag ligation.
CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8?(+) and CD4(-)CD8?(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8?(+) and DN iNKT cells upon mitogen stimulation. CD8?(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-? and TNF-?) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8?>DN>CD4 pattern for Th1 and CD4>DN>CD8? for Th2. All iNKT subsets could simultaneously produce IFN-? and IL-4, but single-positivity for IFN-? or IL-4 was strikingly rare in CD4(+) and CD8?(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8?(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.
The mechanisms permitting nonpolymorphic CD1 molecules to present lipid antigens that differ considerably in polar head and aliphatic tails remain elusive. It is also unclear why hydrophobic motifs in the aliphatic tails of some antigens, which presumably embed inside CD1 pockets, contribute to determinants for T-cell recognition. The 1.9-Å crystal structure of an active complex of CD1b and a mycobacterial diacylsulfoglycolipid presented here provides some clues. Upon antigen binding, endogenous spacers of CD1b, which consist of a mixture of diradylglycerols, moved considerably within the lipid-binding groove. Spacer displacement was accompanied by F pocket closure and an extensive rearrangement of residues exposed to T-cell receptors. Such structural reorganization resulted in reduction of the A pocket capacity and led to incomplete embedding of the methyl-ramified portion of the phthioceranoyl chain of the antigen, explaining why such hydrophobic motifs are critical for T-cell receptor recognition. Mutagenesis experiments supported the functional importance of the observed structural alterations for T-cell stimulation. Overall, our data delineate a complex molecular mechanism combining spacer repositioning and ligand-induced conformational changes that, together with pocket intricacy, endows CD1b with the required molecular plasticity to present a broad range of structurally diverse antigens.
NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag ?-galactosylceramide (?-GalCer). A modified analog of ?-GalCer with a carbon-based glycosidic linkage (?-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to ?-C-GalCer, and related C-glycoside ligands, is weaker than that of ?-GalCer. Furthermore, the V?8.2 and V?7 NKT TCR affinity for CD1d-?-C-GalCer, and some related analogs, is ?10-fold lower than that for the NKT TCR-CD1d-?-GalCer interaction. Nevertheless, the crystal structure of the V?8.2 NKT TCR-CD1d-?-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-?-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-?-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.
CD1e is a member of the CD1 family that participates in lipid antigen presentation without interacting with the T-cell receptor. It binds lipids in lysosomes and facilitates processing of complex glycolipids, thus promoting editing of lipid antigens. We find that CD1e may positively or negatively affect lipid presentation by CD1b, CD1c, and CD1d. This effect is caused by the capacity of CD1e to facilitate rapid formation of CD1-lipid complexes, as shown for CD1d, and also to accelerate their turnover. Similar results were obtained with antigen-presenting cells from CD1e transgenic mice in which lipid complexes are assembled more efficiently and show faster turnover than in WT antigen-presenting cells. These effects maximize and temporally narrow CD1-restricted responses, as shown by reactivity to Sphingomonas paucimobilis-derived lipid antigens. CD1e is therefore an important modulator of both group 1 and group 2 CD1-restricted responses influencing the lipid antigen availability as well as the generation and persistence of CD1-lipid complexes.
Natural killer T (NKT) cells recognize glycolipids presented by CD1d. The first antigen described, ?-galactosyl ceramide (?GalCer), is a potential anticancer agent whose activity depends upon IFN-? secretion. We report two analogs of ?GalCer based on a naturally occurring glycosphingolipid, plakoside A. These compounds induce enhanced IFN-? that correlates with detergent-resistant binding to CD1d and an increased stability of the lipid-CD1d complexes on antigen-presenting cells. Structural analysis on one of the analogs indicates that it is more deeply bound inside the CD1d groove, suggesting tighter lipid-CD1d interactions. To our knowledge, this is the first example in which structural information provides an explanation for the increased lipid-CD1d stability, likely responsible for the Th1 bias. We provide insights into the mechanism of IFN-?-inducing compounds, and because our compounds activate human NKT cells, they could have therapeutic utility.
The varied rates of protection induced by Mycobacterium bovis BCG vaccine against tuberculosis has been attributed to many factors such as genetic variability among BCG strains, rapid clearance of BCG in some populations, and different levels of previous exposure of vaccinated populations to environmental mycobacteria. However, the methods and conditions employed to prepare this vaccine for human usage by various manufacturers have not been investigated as potential factors contributing to the variation in vaccine efficacy. A review of the literature indicates discrepancies between the approach for growing BCG vaccine in the laboratory to assess immune responses and protective ability in animal models, and that employed for production of the vaccine for administration to humans. One of the major differences is in the growth medium used for routine propagation in the laboratory and the one used for bulk vaccine production by manufacturers. Here we compared the immunogenicity of the BCG vaccine grown in Middlebrook 7H9 medium, the most commonly used medium in laboratory studies, against that grown in Sauton medium, which is used for growing BCG by most manufacturers. Our results showed clear differences in the behavior of BCG grown in these different culture media. Compared to BCG grown in Middlebrook 7H9 medium, BCG grown in Sauton media was more persistent inside macrophages, more effective at inhibiting apoptosis of infected cells, induced stronger inflammatory responses and stimulated less effective immunity against aerosol challenge with a virulent Mtb strain. These findings suggested that the growth medium used for producing BCG vaccine is an important factor that deserves increased scrutiny in ongoing efforts to produce more consistently effective vaccines against Mtb.
The most potent foreign antigens for natural killer T cells (NKT cells) are ?-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct ?-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated ?-linked agonists ?-galactosylceramide (?-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the ?-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the ?-linked self ligands to resemble the conformation of foreign ?-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to ?-linked antigens.
We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis ?esx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4(+) memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4(+) cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.
Structural variants of ?-galactosylceramide (?GC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct ?GC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-? secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with ?GC, we screened a library of ?200 synthetic ?GC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids.
Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(?)14-J(?)18) T cell antigen receptor (TCR) ?-chain and recognition of the glycolipid ?-galactosylceramide (?-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of ?-GalCer-reactive NKT cells that expressed a canonical V(?)10-J(?)50 TCR ?-chain, which showed a preference for ?-glucosylceramide (?-GlcCer) and bacterial ?-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCR? sequence identity, the V(?)10 TCR-CD1d-?-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-?-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.
Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.
Type 1 or invariant NKT (iNKT) cell agonists, epitomized by ?-galactosylceramide, protect against cancer largely by IFN-?-dependent mechanisms. Here we describe what we believe to be a novel IFN-?-independent mechanism induced by ?-mannosylceramide, which also defines a potentially new class of iNKT cell agonist, with an unusual ?-linked sugar. Like ?-galactosylceramide, ?-mannosylceramide directly activates iNKT cells from both mice and humans. In contrast to ?-galactosylceramide, protection by ?-mannosylceramide was completely dependent on NOS and TNF-?, neither of which was required to achieve protection with ?-galactosylceramide. Moreover, at doses too low for either alone to protect, ?-mannosylceramide synergized with ?-galactosylceramide to protect mice against tumors. These results suggest that treatment with ?-mannosylceramide provides a distinct mechanism of tumor protection that may allow efficacy where other agonists have failed. Furthermore, the ability of ?-mannosylceramide to synergize with ?-galactosylceramide suggests treatment with this class of iNKT agonist may provide protection against tumors in humans.
Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major global health problem, despite the widespread use of the M. bovis Bacille Calmette-Guerin (BCG) vaccine and the availability of drug therapies. In recent years, the high incidence of coinfection of M. tuberculosis and HIV, as well as escalating problems associated with drug resistance, has raised ominous concerns with regard to TB control. Vaccination with BCG has not proven highly effective in controlling TB, and also has been associated with increasing concerns about the potential for the vaccine to cause disseminated mycobacterial infection in HIV infected hosts. Thus, the development of an efficacious and safe TB vaccine is generally viewed as a critical to achieving control of the ongoing global TB pandemic. In the current study, we have analyzed the vaccine efficacy of an attenuated M. tuberculosis strain that combines a mutation that enhances T cell priming (?secA2) with a strongly attenuating lysine auxotrophy mutation (?lysA). The ?secA2 mutant was previously shown to be defective in the inhibition of apoptosis and markedly increased priming of antigen-specific CD8(+) T cells in vivo. Similarly, the ?secA2?lysA strain retained enhanced apoptosis and augmented CD8(+) T cell stimulatory effects, but with a noticeably improved safety profile in immunosuppressed mice. Thus, the M. tuberculosis ?secA2?lysA mutant represents a live attenuated TB vaccine strain with the potential to deliver increased protection and safety compared to standard BCG vaccination.
Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.
The programmed death-1 (PD-1) costimulatory receptor inhibits T and B cell responses and plays a crucial role in peripheral tolerance. PD-1 has recently been shown to inhibit T cell responses during chronic viral infections such as HIV. In this study, we examined the role of PD-1 in infection with Mycobacterium tuberculosis, a common co-infection with HIV. PD-1-deficient mice showed dramatically reduced survival compared with wild-type mice. The lungs of the PD-1-/- mice showed uncontrolled bacterial proliferation and focal necrotic areas with predominantly neutrophilic infiltrates, but a lower number of infiltrating T and B cells. Proinflammatory cytokines, such as TNF-alpha, IL-1, and especially IL-6 and IL-17 were significantly increased in the lung and sera of infected PD-1-/- mice, consistent with an aberrant inflammation. Microarray analysis of the lungs infected with M. tuberculosis showed dramatic differences between PD-1-/- and control mice. Using high-stringency analysis criteria (changes of twofold or greater), 367 transcripts of genes were differentially expressed between infected PD-1-/- and wild-type mice, resulting in profoundly altered inflammatory responses with implications for both innate and adaptive immunity. Overall, our studies show that the PD-1 pathway is required to control excessive inflammatory responses after M. tuberculosis infection in the lungs.
In this study, we investigated whether dyslipidemia-associated perturbed invariant natural killer T (iNKT) cell function is due to intrinsic changes in iNKT cells or defects in the ability of antigen-presenting cells to activate iNKT cells.
CD1d-restricted natural killer T cells with invariant T cell receptor ? chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of ?-galactosylceramide (?GalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-? (IFN?), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-? secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.
Several L-fucoglycolipids are associated with diseases such as cancer, cystic fibrosis and rheumatoid arthritis. Activation of iNKT cells is known to lead to the production of cytokines that can help alleviate or exacerbate these conditions. alpha-Galactosyl ceramide (alpha-GalCer) is a known agonist of iNKT cells and it is believed that its fucosyl counterpart might have similar immunogenic properties. We herein report the synthesis of alpha-L-fucosyl ceramide derivatives and describe their biological evaluation. The key challenge in the synthesis of the target molecules involved the stereoselective synthesis of the alpha-glycosidic linkage. Of the methods examined, the per-TMS-protected glycosyl iodide donor was completely alpha-selective, and could be scaled up to provide gram quantities of the azide precursor 11, from which a range of N-acylated alpha-L-fucosyl ceramides were readily obtained and evaluated for ex vivo expansion of human iNKT cells.
Alpha-glucosyl ceramides 4 and 5 have been synthesised and evaluated for their ability to stimulate the activation and expansion of human iNKT cells. The key challenge in the synthesis of both target molecules was the stereoselective synthesis of the alpha-glycosidic linkage. Of the methods examined, glycosylation using per-TMS-protected glucosyl iodide 16 was completely alpha-selective and provided gram quantities of amine 11, from which alpha-glucosyl ceramides 4 and 5 were obtained by N-acylation. alpha-GlcCer 4, containing a C24 saturated acyl chain, stimulated a marked proliferation and expansion of human circulating iNKT cells in short-term cultures. alpha-GlcCer 5, which contains a C20 11,14-cis-diene acyl chain (C20:2), induced extremely similar levels of iNKT cell activation and expansion.
The prevalence of asthma continues to increase in westernized countries, and optimal treatment remains a significant therapeutic challenge. Recently, CD1d-restricted invariant NKT (iNKT) cells were found to play a critical role in the induction of airway hyperreactivity (AHR) in animal models and are associated with asthma in humans. To test whether iNKT cell-targeted therapy could be used to treat allergen-induced airway disease, mice were sensitized with OVA and treated with di-palmitoyl-phosphatidyl-ethanolamine polyethylene glycol (DPPE-PEG), a CD1d-binding lipid antagonist. A single dose of DPPE-PEG prevented the development of AHR and pulmonary infiltration of lymphocytes upon OVA challenge, but had no effect on the development of OVA-specific Th2 responses. In addition, DPPE-PEG completely prevented the development of AHR after administration of alpha-galactosylceramide (alpha-GalCer) intranasally. Furthermore, we demonstrate that DPPE-PEG acts as antagonist to alpha-GalCer and competes with alpha-GalCer for binding to CD1d. Finally, we show that DPPE-PEG completely inhibits the alpha-GalCer-induced phosphorylation of ERK tyrosine kinase in iNKT cells, suggesting that DPPE-PEG specifically blocks TCR signaling and thus activation of iNKT cells. Because iNKT cells play a critical role in the development of AHR, the inhibition of iNKT activation by DPPE-PEG suggests a novel approach to treat iNKT cell-mediated diseases such as asthma.
KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can stimulate NKT cells to release a broad range of bioactive cytokines. In an effort to understand the structure-activity relationships, we have carried out syntheses of 26 new KRN7000 analogues incorporating aromatic residues in either or both side chains. Structural variations of the phytosphingosine moiety also include varying stereochemistry at C3 and C4, and 4-deoxy and 3,4-dideoxy versions. Their biological activities are described.
Certain glycolipid Ags for Valpha14i NKT cells can direct the overall cytokine balance of the immune response. Th2-biasing OCH has a lower TCR avidity than the most potent agonist known, alpha-galactosylceramide. Although the CD1d-exposed portions of OCH and alpha-galactosylceramide are identical, structural analysis indicates that there are subtle CD1d conformational differences due to differences in the buried lipid portion of these two Ags, likely accounting for the difference in antigenic potency. Th1-biasing C-glycoside/CD1d has even weaker TCR interactions than OCH/CD1d. Despite this, C-glycoside caused a greater downstream activation of NK cells to produce IFN-gamma, accounting for its promotion of Th1 responses. We found that this difference correlated with the finding that C-glycoside/CD1d complexes survive much longer in vivo. Therefore, we suggest that the pharmacokinetic properties of glycolipids are a major determinant of cytokine skewing, suggesting a pathway for designing therapeutic glycolipids for modulating invariant NKT cell responses.
The attenuated strain of Mycobacterium bovis known as bacille Calmette-Guérin (BCG) has been widely used as a vaccine for prevention of disease by Mycobacterium tuberculosis, but with relatively little evidence of success. Recent studies suggest that the failure of BCG may be due to its retention of immune evasion mechanisms that delay or prevent the priming of robust protective cell-mediated immunity. In this study, we describe an approach to enhance the immunogenicity of BCG by incorporating glycolipid activators of CD1d-restricted NKT cells, a conserved T cell subset with the potential to augment many types of immune responses. A method was developed for stably incorporating two forms of the NKT cell activator alpha-galactosylceramide into live BCG organisms, and the impact of this on stimulation of T cell responses and protective antimycobacterial immunity was evaluated. We found that live BCG containing relatively small amounts of incorporated alpha-galactosylceramide retained the ability to robustly activate NKT cells. Compared with immunization with unmodified BCG, the glycolipid-modified BCG stimulated increased maturation of dendritic cells and markedly augmented the priming of Ag-specific CD8(+) T cells responses. These effects were correlated with improved protective effects of vaccination in mice challenged with virulent M. tuberculosis. These results support the view that mycobacteria possess mechanisms to avoid stimulation of CD8(+) T cell responses and that such responses contribute significantly to protective immunity against these pathogens. Our findings raise the possibility of a simple modification of BCG that could yield a more effective vaccine for control of tuberculosis.
In spite of their relatively limited antigen receptor repertoire, CD1d-restricted NKT cells recognize a surprisingly diverse range of lipid and glycolipid antigens. Recent studies of natural and synthetic CD1d-presented antigens provide an increasingly detailed picture of how the specific structural features of these lipids and glycolipids influence their ability to be presented to NKT cells and stimulate their diverse immunologic functions. Particularly for synthetic analogues of alpha-galactosylceramides which have been the focus of intense recent investigation, it is becoming clear that the design of glycolipid antigens with the ability to precisely control the specific immunologic activities of NKT cells is likely to be feasible. The emerging details of the mechanisms underlying the structure-activity relationship of NKT cell antigens will assist greatly in the design and production of immunomodulatory agents for the precise manipulation of NKT cells and the many other components of the immune system that they influence.
The semi-invariant natural killer (NK) T-cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the alpha-chain of the NKTcr is invariant, the beta-chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an alpha-linked monosaccharide (alpha-galactosylceramide and alpha-galactosyldiacylglycerol) and the beta-linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their beta-chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including alpha-galactosylceramide, the structurally similar alpha-galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr beta-chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr beta-chain allows these cells to recognise unique aspects of structurally diverse CD1d-restricted ligands.
Short or polyunsaturated lipid variants of the NKT cell antigen alpha-galactosylceramide (alphaGC) exhibit decreased potency and a Th2 bias in vivo despite conserved TCR contact residues and stable binding to CD1d at neutral and acidic pH. Using reagents to directly visualize lipids in their free or CD1d-bound form, we determined that, contrary to predictions, these lipids reached the lysosome better than alphaGC. However, in contrast with alphaGC, they loaded CD1d at the cell surface and underwent immediate pH-dependent dissociation upon recycling to the lysosome. In cell-free assays, ultrafast dissociation of preformed complexes could be induced at acidic pH only when free competitor lipids were added, suggesting active lipid displacement. These findings provide a common cell biological explanation for the decreased stimulatory properties of short and polyunsaturated alphaGC variants. They also suggest that direct lipid displacement is a potent mechanism underlying highly dynamic lipid exchange reactions in the lysosomal compartment that shape the repertoire of lipids associated with CD1d.
The recent discovery of dideoxymycobactin (DDM) as a ligand for CD1a demonstrates how a nonribosomal lipopeptide antigen is presented to T cells. DDM contains an unusual acylation motif and a peptide sequence present only in mycobacteria, but its discovery raises the possibility that ribosomally produced viral or mammalian proteins that commonly undergo lipidation might also function as antigens. To test this, we measured T cell responses to synthetic acylpeptides that mimic lipoproteins produced by cells and viruses. CD1c presented an N-acyl glycine dodecamer peptide (lipo-12) to human T cells, and the response was specific for the acyl linkage as well as the peptide length and sequence. Thus, CD1c represents the second member of the CD1 family to present lipopeptides. lipo-12 was efficiently recognized when presented by intact cells, and unlike DDM, it was inactivated by proteases and augmented by protease inhibitors. Although lysosomes often promote antigen presentation by CD1, rerouting CD1c to lysosomes by mutating CD1 tail sequences caused reduction in lipo-12 presentation. Thus, although certain antigens require antigen processing in lysosomes, others are destroyed there, providing a hypothesis for the evolutionary conservation of large CD1 families containing isoforms that survey early endosomal pathways.
Invariant natural killer T cells (iNKT cells) have pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects. iNKT cells are activated through their T-cell receptors by glycolipid moieties (typically the alpha-galactosylceramide [alpha-GalCer] derivative KRN7000) presented within CD1d. We investigated the ability of modified alpha-GalCer molecules to differentially modulate alloreactivity and GVL. KRN7000 and the N-acyl variant, C20:2, were administered in multiple well-established murine models of allogeneic stem cell transplantation. The highly potent and specific activation of all type I NKT cells with C20:2 failed to exacerbate and in most settings inhibited GVHD late after transplantation, whereas effects on GVL were variable. In contrast, the administration of KRN7000 induced hyperacute GVHD and early mortality in all models tested. Administration of KRN7000, but not C20:2, was found to result in downstream interleukin (IL)-12 and dendritic cell (DC)-dependent natural killer (NK)- and conventional T-cell activation. Specific depletion of host DCs, IL-12, or donor NK cells prevented this pathogenic response and the induction of hyperacute GVHD. These data demonstrate the ability of profound iNKT activation to modulate both the innate and adaptive immune response via the DC-NK-cell interaction and raise concern for the use of alpha-GalCer therapeutically to modulate GVHD and GVL effects.
Invariant NKT cells (iNKT cells) recognize glycolipid Ags via an invariant TCR alpha-chain and play a central role in various immune responses. Although human CD4(+) and CD4(-) iNKT cell subsets both produce Th1 cytokines, the CD4(+) subset displays an enhanced ability to secrete Th2 cytokines and shows regulatory activity. We performed an ex vivo analysis of blood, liver, and tumor iNKT cells from patients with hepatocellular carcinoma and metastases from uveal melanoma or colon carcinoma. Frequencies of Valpha24/Vbeta11 iNKT cells were increased in tumors, especially in patients with hepatocellular carcinoma. The proportions of CD4(+), double negative, and CD8alpha(+) iNKT cell subsets in the blood of patients were similar to those of healthy donors. However, we consistently found that the proportion of CD4(+) iNKT cells increased gradually from blood to liver to tumor. Furthermore, CD4(+) iNKT cell clones generated from healthy donors were functionally distinct from their CD4(-) counterparts, exhibiting higher Th2 cytokine production and lower cytolytic activity. Thus, in the tumor microenvironment the iNKT cell repertoire is modified by the enrichment of CD4(+) iNKT cells, a subset able to generate Th2 cytokines that can inhibit the expansion of tumor Ag-specific CD8(+) T cells. Because CD4(+) iNKT cells appear inefficient in tumor defense and may even favor tumor growth and recurrence, novel iNKT-targeted therapies should restore CD4(-) iNKT cells at the tumor site and specifically induce Th1 cytokine production from all iNKT cell subsets.
The role of CD8 T cells in anti-tuberculosis immunity in humans remains unknown, and studies of CD8 T cell-mediated protection against tuberculosis in mice have yielded controversial results. Unlike mice, humans and nonhuman primates share a number of important features of the immune system that relate directly to the specificity and functions of CD8 T cells, such as the expression of group 1 CD1 proteins that are capable of presenting Mycobacterium tuberculosis lipids antigens and the cytotoxic/bactericidal protein granulysin. Employing a more relevant nonhuman primate model of human tuberculosis, we examined the contribution of BCG- or M. tuberculosis-elicited CD8 T cells to vaccine-induced immunity against tuberculosis. CD8 depletion compromised BCG vaccine-induced immune control of M. tuberculosis replication in the vaccinated rhesus macaques. Depletion of CD8 T cells in BCG-vaccinated rhesus macaques led to a significant decrease in the vaccine-induced immunity against tuberculosis. Consistently, depletion of CD8 T cells in rhesus macaques that had been previously infected with M. tuberculosis and cured by antibiotic therapy also resulted in a loss of anti-tuberculosis immunity upon M. tuberculosis re-infection. The current study demonstrates a major role for CD8 T cells in anti-tuberculosis immunity, and supports the view that CD8 T cells should be included in strategies for development of new tuberculosis vaccines and immunotherapeutics.
An alpha-galactosyl ceramide (alpha-GalCer) 2 was synthesized and evaluated for its ability to stimulate iNKT-cell proliferation and elicit T-helper cytokines, IL-4 and IFNgamma. Compound 2 combines the acyl chain of the potent, Th2 biasing alpha-GalCer 1 with a sphingoid base of the same length as that found in OCH, which also exhibits Th2 skewing, Such complementation may enhance cytokine bias, which is thought to be important for therapeutic applications of iNKT cell stimulation. Two related alpha-GalCers, 3 and 4, with saturated acyl chains were prepared for comparison.
CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.
Current influenza A virus vaccines do not generate significant immunity against serologically distinct influenza A virus subtypes and would thus be ineffective in the face of a pandemic caused by a novel variant emerging from, say, a wildlife reservoir. One possible solution would be to modify these vaccines so that they prime cross-reactive CD8(+) cytotoxic T lymphocytes (CTL) cell-mediated immunity directed at conserved viral epitopes. A further strategy is to use novel adjuvants, such as the immunomodulatory glycolipid alpha-galactosylceramide (alpha-GalCer). We show here that giving alpha-GalCer with an inactivated influenza A virus has the paradoxical effect of diminishing acute CTL immunity via natural killer T (NKT) cell-dependent expression of indoleamine 2,3-dioxygenase (IDO), an important mediator of immune suppression, while at the same time promoting the survival of long-lived memory CTL populations capable of boosting protection against heterologous influenza A virus challenge. This enhancement of memory was likely due to the alpha-GalCer-induced upregulation of prosurvival genes, such as bcl-2, and points to the potential of alpha-GalCer as an adjuvant for promoting optimal, vaccine-induced CD8(+) T cell memory.
In this study, the early pulmonary cytokine and chemokine responses in mice immunized with either BCG vaccine, a DeltasecA2 mutant of Mycobacterium tuberculosis, or a DNA vaccine expressing an ESAT6-antigen 85B fusion protein and then aerogenically challenged with a low dose of M. tuberculosis were evaluated by PCR array. The cellular immune responses at day 10 postchallenge were essentially equivalent in the lungs of mice immunized with either the highly immunogenic BCG vaccine or the DeltasecA2 M. tuberculosis mutant strain. Specifically, 12 immune biomolecules (including gamma interferon [IFN-gamma], interleukin-21 [IL-21], IL-27, IL-17f, CXCL9, CXCL10, and CXCL11) were differentially regulated, relative to the levels for naïve controls, in the lungs of vaccinated mice at this time point. Although the vaccine-related immune responses evoked in mice immunized with the DNA vaccine were relatively limited at 10 days postinfection, upregulation of IFN-gamma RNA synthesis as well as increased expression levels of CXCL9, CXCL10, and CXCL11 chemokines were detected.
Mycobacterium bovis BCG is an attractive vaccine vector against breast milk HIV transmission because it elicits Th1-type responses in newborns. However, BCG causes disease in HIV-infected infants. Genetically attenuated Mycobacterium tuberculosis (Mtb) mutants represent a safer alternative for immunocompromised populations. In the current study, we compared the immunogenicity in mice of three different recombinant attenuated Mtb strains expressing an HIV envelope (Env) antigen construct. Two of these strains (DeltalysA DeltapanCD Mtb and DeltaRD1 DeltapanCD Mtb) failed to induce significant levels of HIV Env-specific CD8(+) T cell responses. In striking contrast, an HIV-1 Env-expressing attenuated DeltalysA Mtb containing a deletion in secA2, which encodes a virulence-related secretion system involved in evading adaptive immunity, generated consistently measurable Env-specific CD8(+) T cell responses that were significantly greater than those observed after immunization with BCG expressing HIV Env. Similarly, another strain of DeltalysA DeltasecA2 Mtb expressing SIV Gag induced Gag- and Mtb-specific CD8(+) T cells producing perforin or IFNgamma, and Gag-specific CD4(+) T cells producing IFNgamma within 3 weeks after immunization in adult mice; in addition, IFNgamma-producing Gag-specific CD8(+) T cells and Mtb-specific CD4(+) T cells were observed in neonatal mice within 1 week of immunization. We conclude that DeltalysA DeltasecA2 Mtb is a promising vaccine platform to construct a safe combination HIV-TB vaccine for use in neonates.
Human and mouse type I natural killer T (NKT) cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognizes, to varying degrees of affinity, a range of Ags. Presently, it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of ?-galactosylceramide (?-GalCer) and have determined the structures of a human NKT TCR in complex with CD1d-4,4?-deoxy-?-GalCer and CD1d-?-GalCer with a shorter, di-unsaturated acyl chain (C20:2). Altered glycolipid ligands with acyl chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4-OH position in comparison with the 3-OH position of ?-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4-OH > 3-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity-determining region 1? loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important consideration for therapeutic development and NKT cell physiology.
Huisgen [3+2] dipolar cycloaddition of 6?-azido-6?-deoxy-?-galactosyl ceramide 11 with a range of alkynes (or a benzyne precursor) yielded a series of triazole-containing ?-galactosyl ceramide (?-GalCer) analogues in high yield. These ?-GalCer analogues and the precursor azide 11 were tested for their ability to activate iNKT cells and stimulate IL-2 cytokine secretion in vitro, and IFN-? and IL-4 cytokine secretion in vivo. Some of these analogues, specifically 11, 12b, 12f and 13, were more potent IL-2 stimulators than the prototypical CD1d agonist, ?-GalCer 1. In terms of any cytokine bias, most of the triazole-containing analogues exhibited a small Th2 cytokine-biasing response relative to that shown by ?-GalCer 1. In contrast, the cycloaddition precursor, namely azide 11, provided a small Th1 cytokine-biasing response.
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