Activation of natural killer T cells potentiates or prevents experimental autoimmune encephalomyelitis.

Natural killer (NK) T cells recognize lipid antigens in the context of the major histocompatibility complex (MHC) class 1-like molecule CD1 and rapidly secrete large amounts of the cytokines interferon (IFN)-gamma and interleukin (IL)-4 upon T cell receptor (TCR) engagement. We have asked whether NK T cell activation influences adaptive T cell responses to myelin antigens and their ability to cause experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. While simultaneous activation of NK T cells with the glycolipid alpha-galactosylceramide (alpha-GalCer) and myelin-reactive T cells potentiates EAE in B10.PL mice, prior activation of NK T cells protects against disease. Exacerbation of EAE is mediated by an enhanced T helper type 1 (Th1) response to myelin basic protein and is lost in mice deficient in IFN-gamma. Protection is mediated by immune deviation of the anti-myelin basic protein (MBP) response and is dependent upon the secretion of IL-4. The modulatory effect of alpha-GalCer requires the CD1d antigen presentation pathway and is dependent upon the nature of the NK T cell response in B10.PL or C57BL/6 mice. Because CD1 molecules are nonpolymorphic and remarkably conserved among different species, modulation of NK T cell activation represents a target for intervention in T cell-mediated autoimmune diseases.

Cancer vaccines based on dendritic cells loaded with tumor-associated antigens.

Dendritic cells (DCs) can be used as an antigen presentation platform for vaccination against cancer. In this approach, DCs are expanded in vitro from monocyte-derived progenitors, and subsequently loaded with well-characterized tumor-associated antigens (TAAs). TAAs can be incubated with DCs in various forms, including peptides, recombinant proteins, plasmid DNA, formulated RNA, or recombinant viruses. Advantages and limitations of DC-based cellular vaccines against cancers, as well as preliminary results of clinical studies already performed in humans, are discussed. Importantly, significant advances in our understanding of the biology of DCs can be used to support the design of new vaccines or adjuvants in order to elicit TH1 cellular immune responses.

Structural requirements for antigen presentation by mouse CD1.

The structural basis for the T cell response to glycolipid antigens (Ags) remains poorly understood. T lymphocytes autoreactive for mouse CD1 (mCD1.1) or reactive for the glycosphingolipid alphagalactosylceramide (alpha-GalCer) presented by mCD1.1 have been described previously. In this paper it is shown that mutations at the top of the alpha helices and in the bottom of the Ag-binding groove can disrupt both mCD1.1 autoreactivity and alpha-GalCer recognition. The locations of the positions that affect T cell responses indicate that recognition of mCD1.1 is not likely to be unconventional or superantigen-like. Furthermore, the effects of the bottom of the pocket mutation suggest that the autoreactive response could require an autologous ligand, and they indicate that alpha-GalCer binds to the groove of mCD1.1, most likely with the shorter 18-carbon hydrophobic chain in the A' pocket. Natural killer T cell hybridomas with identical T cell antigen receptor (TCR) alpha chains and different beta chains respond differently to alpha-GalCer presented by mCD1.1 mutants. This finding indicates a role for TCR beta in defining natural killer T cell specificity, despite the more restricted diversity of the alpha chains in these cells. Overall, the data are consistent with a mode of lipoglycan recognition similar to that proposed for glycopeptides, in which the TCR alpha and beta chains survey a surface composed of both mCD1.1 and the carbohydrate portion of alpha-GalCer.

Membrane lymphotoxin is required for the development of different subpopulations of NK T cells.

The development of lymphoid organs requires membrane-bound lymphotoxin (LT), a heterotrimer containing LTalpha and LTbeta, but the effects of LT on T cell function have not been characterized extensively. Upon TCR cross-linking in vitro, splenocytes from both LTalpha-/- and LTbeta-/- mice failed to produce IL-4 and IL-10 due to a reduction in NK T cells. Concordantly, LTalpha-/- and LTbeta-/- mice did not respond to the lipoglycan alpha-galactosylceramide, which is presented by mouse CD1 to Valpha14+ NK T cells. Interestingly, both populations of NK T cells, including those that are mouse CD1 dependent and alpha-galactosylceramide reactive and those that are not, were affected by disruption of the LTalpha and LTbeta genes. NK T cells were not affected, however, in transgenic mice in which LT signaling is blocked, beginning on day 3 after birth, by expression of a soluble decoy LTbeta receptor. This suggests that membrane-bound LT is critical for NK T cells early in ontogeny, but not for the homeostasis of mature cells.

Cutting edge: activation of NK T cells by CD1d and alpha-galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype.

NK T cells recognize glycolipid Ags such as alpha-galactosylceramide (alpha-GalCer) presented by the MHC class I-like molecule CD1d. In this paper we have studied the in vivo effects of alpha-GalCer on the generation of adaptive immune responses. Treatment of mice with alpha-GalCer resulted in rapid activation of NK T cells and production of the cytokines IL-4 and IFN-gamma. However, after this initial stimulation, NK T cells became polarized for the production of IL-4. Further, as soon as 6 days after alpha-GalCer injection, a marked increase in serum IgE levels was observed. Administration of alpha-GalCer at the time of priming of mice with protein Ag resulted in the generation of Ag-specific Th2 cells and a profound increase in the production of IgE. Collectively, these findings indicate that alpha-GalCer may be useful for modulating immune responses toward a Th2 phenotype during prophylaxis and therapy.

CD1-mediated immune responses to glycolipids.

Major advances have recently been accomplished in understanding the biochemistry of interactions between lipid antigens and CD1. The diversity of lipid antigens loaded onto CD1 molecules in normal cells, the ligand requirements for CD1 recognition by autoreactive T cells, including NK T cells, and the significance of CD1-mediated autoreactivity, as opposed to pathogen reactivity, remain controversial issues.

Immunization with alpha-galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis.

We have compared the immune responses of mice immunized either with alpha-galactosylceramide (alpha-GalCer), capable of eliciting a CD1-metiated stimulation of V alpha14+ NK T cells, or with lipoarabinomannan (LAM), a glycophospholipid derived from mycobacteria which is known to be presented by CD1b in humans. Within 24 h, alpha-GalCer induces a burst of IFN-gamma secretion in vivo, and recall with antigen in vitro leads to the synthesis of IL-4 and IL-10 in addition to IFN-gamma. Associated with this in vivo cytokine release is a polyclonal activation of splenic B and T cells. CD1-reactive NK T lymphocytes mediate these events, because none of them are observed in alpha-GalCer-immunized CD1-/- mice. LAM immunization fails to promote similar early responses in vivo. Repeated exposure of mice to alpha-GalCer induces splenic T cells to secrete IL-4 and IL-10 but dramatically reduced levels of IFN-gamma. Such a bias in the cytokine balance triggered by NK T cells stimulated with multiple doses of alpha-GalCer suggests that this compound might be useful in the induction of Th2 immune responses and the prevention of chronic inflammatory conditions mediated by Th1 cytokines.

A single intramuscular injection with an adenovirus-expressing IL-12 protects BALB/c mice against Leishmania major infection, while treatment with an IL-4-expressing vector increases disease susceptibility in B10.D2 mice.

Experimental infection of the susceptible BALB/c (H-2d) mouse with the intracellular parasite Leishmania major induces a predominant Th2-type T cell response that eventually leads to death. In contrast, the resistant B10.D2 (H-2d) strain develops Th1 cells that control parasite replication and disease. In this study, we tested the ability of a recombinant adenovirus vector-expressing IL-12 to skew the immune response in a Th1 direction and prevent leishmaniasis in susceptible mice. We report that BALB/c mice treated with the Ad5IL-12 vector on the same day as parasitic challenge are significantly protected against leishmaniasis and acquired long-lasting immunity, because upon rechallenge with L. major parasites they were resistant to disease. The vector-derived IL-12 expression was transient and highly localized to the tissue after i.m. injection; it caused an increase in the number of Ag-specific IFN-gamma-secreting lymphocytes and enhanced NK cell activity in the draining popliteal node. In contrast, resistant B10.D2 mice given i.m. injections with a recombinant adenovirus-expressing IL-4 displayed greater susceptibility to disease, and severe lesions were produced in some of the infected animals. These results suggest the potential use of recombinant adenoviruses expressing cytokines as potent immunomodulatory agents for the generation of protective immune responses against intracellular pathogens.

Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing.

Mouse CD1(mCD1) molecules have been reported to present two types of antigens: peptides or proteins and the glycolipid alpha-galactosylceramide. Here, we demonstrate that a protein antigen, chicken ovalbumin (Ova), must be processed to generate peptides presented by mCD1 to CD8(+) T cells. The processing and mCD1-mediated presentation of chicken Ova depend on endosomal localization because inhibitors of endosomal acidification and endosomal recycling pathways block T cell reactivity. Furthermore, a cytoplasmic tail mutant of mCD1, which disrupts endosomal localization, has a greatly reduced capacity to present Ova to mCD1 restricted cells. Newly synthesized mCD1 molecules, however, are not required for Ova presentation, suggesting that molecules recycling from the cell surface are needed. Because of these data showing that mCD1 trafficks to endosomes, where it can bind peptides derived from exogenous proteins, we conclude that peptide antigen presentation by mCD1 is likely to be a naturally occurring phenomenon. In competition assays, alpha-galactosylceramide did not inhibit Ova presentation, and presentation of the glycolipid was not inhibited by excess Ova or the peptide epitope derived from it. This suggests that, although both lipid and peptide presentation may occur naturally, mCD1 may interact differently with these two types of antigens.

Structural requirements for galactosylceramide recognition by CD1-restricted NK T cells.

The reactivity of a group of mouse Valpha14+ NK T cell hybridomas was tested with a panel of analogs of the glycolipid alpha-galactosylceramide (alpha-GalCer). Interestingly, the nearly complete truncation of the acyl chain from 24 to 2 carbons does not significantly affect the mouse NK T cell response to glycolipid presented by either mouse CD1 (mCD1) or its human homolog CD1d (hCD1d). Therefore, we propose that only one of the two hydrophobic pockets of the CD1 Ag-binding groove needs to be filled by Ag. In terms of the sphingosine base, the mCD1 binding groove has less-demanding structural requirements for presentation to NK T cells than hCDld. Tests of NK T cell reactivity to analogs presented by hCDld demonstrates that the invariant TCRs expressed by mouse and human NK T cells are surprisingly similar in their requirements for glycolipid recognition.

CD1d-mediated recognition of an alpha-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution.

Natural killer (NK) T cells are a lymphocyte subset with a distinct surface phenotype, an invariant T cell receptor (TCR), and reactivity to CD1. Here we show that mouse NK T cells can recognize human CD1d as well as mouse CD1, and human NK T cells also recognize both CD1 homologues. The unprecedented degree of conservation of this T cell recognition system suggests that it is fundamentally important. Mouse or human CD1 molecules can present the glycolipid alpha-galactosylceramide (alpha-GalCer) to NK T cells from either species. Human T cells, preselected for invariant Valpha24 TCR expression, uniformly recognize alpha-GalCer presented by either human CD1d or mouse CD1. In addition, culture of human peripheral blood cells with alpha-GalCer led to the dramatic expansion of NK T cells with an invariant (Valpha24(+)) TCR and the release of large amounts of cytokines. Because invariant Valpha14(+) and Valpha24(+) NK T cells have been implicated both in the control of autoimmune disease and the response to tumors, our data suggest that alpha-GalCer could be a useful agent for modulating human immune responses by activation of the highly conserved NK T cell subset.

Selective ability of mouse CD1 to present glycolipids: alpha-galactosylceramide specifically stimulates V alpha 14+ NK T lymphocytes.

Mouse CD1 (mCD1) glycoproteins are known to present peptides, while human CD1 molecules present glycolipids. In mice, mCD1-autoreactive NK T cells play critical roles in various immune responses, through the secretion of high amounts of cytokines. This study was initiated to determine whether glycolipids are involved in the autorecognition of mCD1 by NK T cells. Alpha-galactosylceramide (alpha-GalCer) was the only glycolipid tested capable of eliciting an mCD1-restricted response by splenic T cells. Moreover, splenic T cells derived from mCD1-deficient mice were not stimulated by alpha-GalCer, suggesting that the responsive T cells are selected by mCD1. Using cytoflow techniques, we confirmed that, in response to alpha-GalCer, IFN-gamma-secreting cells displayed an NK T cell phenotype. The predominance of IFN-gamma vs IL-4, however, is determined by the type of mCD1+ APC, suggesting the potential for APC regulation of cytokine production by NK T cells. Among a panel of 10 mCD1-autoreactive T cell hybridomas, only the ones that express the typical V alpha 14 J alpha 281 TCR rearrangement of NK T cells responded to alpha-GalCer. Fixation or treatment of mCD1+ APCs with an inhibitor of endosomal acidification and the use of mCD1 mutants unable to traffic through endosome still allowed alpha-GalCer to stimulate NK T cells. Thus, endosomal trafficking and Ag processing are not required for glycolipid recognition. In summary, alpha-GalCer might be the autologous ligand, or a mimic of a glycolipid ligand, involved in the mCD1-mediated stimulation of NK T cells.

Antigen-presenting function of mouse CD1: one molecule with two different kinds of antigenic ligands.

Mouse CD1 (mCD1) is an antigen-presenting molecule that is constitutively expressed by most bone marrow-derived cells. Peptides with a hydrophobic binding motif can bind to mCD1, and the peptide-CD1 complex is recognized by CD8+ cytolytic T cells. In contrast, NK1.1+ T cells, which are CD8-, are autoreactive for mCD1 molecules. This autoreactivity, along with the ability of NK T cells to rapidly produce large amounts of cytokine, has led to the suggestion that these cells may be immunoregulatory. We have shown that the mCD1-autoreactive T cells can distinguish between different cell types that express similar levels of mCD1, suggesting that mCD1-bound autologous ligands may be critical for T-cell stimulation. Consistent with this, some of these mCD1-restricted T cells can recognize the glycolipid alpha-galactosylceramide presented by mCD1, while others do not respond. The mCD1 crystal structure reveals a deep and narrow hydrophobic antigen-binding site which can more easily bind lipid antigens than the long hydrophobic peptides that we have defined as mCD1 antigens. The ability of mCD1 to bind and present two different types of ligands raises the question as to how mCD1 can accommodate both types of antigens.

Mouse CD1 is mainly expressed on hemopoietic-derived cells.

The mouse CD1 (mCD1) is a class I-like molecule that is encoded outside the MHC. Recent studies demonstrate that mCD1 presents hydrophobic peptides to CD8+ T cells and also that it is recognized by a population of NK1.1+ T cells that are thought to play an immunoregulatory role because of their ability to secrete IL-4. It has previously been reported that mCD1 is expressed predominantly by intestinal epithelial cells, although most NK1.1+ T cells are located elsewhere. We, therefore, have generated new mAbs to mCD1 to investigate its tissue distribution. The principal site of mCD1 expression in normal mice is on cells in the hemopoietic series, including constitutive expression on nearly all T and B cells, on macrophages, and on dendritic cells. Other than bone marrow-derived cells, mCD1 is not widely expressed and is not detectable on great majority of intestinal epithelial cells. The B cells, but not the T cells, from beta2m-deficient mice can be recognized by two mCD1 autoreactive T hybridomas. Therefore, although we could not detect a beta2m-independent form of mCD1 using these mAbs, mCD1 in a different conformation or a mCD1-related molecule is likely to be expressed in the absence of beta2m on some cell types. The pattern of expression of mCD1 correlates with the distribution of NK1 T cells and is consistent with an important Ag-presenting function for this molecule.

B-cell-derived IL-10: production and function.

In vitro and in vivo studies identify IL-10 as a key cytokine that inhibits cell-mediated immunity and inflammation while promoting humoral responses. The present review summarizes our studies regarding the ability of B cells to secrete IL-10. When established lines are considered, the production of human IL-10 is restricted to mature B-cell lines and correlates with Epstein-Barr Virus (EBV) expression. Accordingly, EBV infection induces purified tonsil B lym phocytes to produce high levels of human IL-10, whereas viral IL-10 (encoded by EBV) remains undetectable. Exogenous IL-10 enhances EBV-induced B-cell growth, while a neutralizing anti-IL-10 antibody inhibits it, suggesting that IL-10 acts as an autocrine growth factor for EBV-infected B lymphocytes. Normal B lymphocytes secrete lower levels of human IL-10 following activation through B-cell receptor or CD40 antigen. While addition of exogenous IL-4 diminishes CD40-induced secretion of IL-10, addition of Staphyloccocus aureus Cowan I (SAC) particles increases it. Neutralization of endogenous IL-10 does not alter growth of CD40-activated B cells but inhibits their IgG, IgA, and IgM secretion, especially when costimulated with SAC particles. Finally, the simul taneous blocking of both endogenous IL-10 and IL-6 inhibits two-thirds of the production of the three isotypes when B cells were triggered through CD40 in the presence of SAC particles, suggesting that IL-10 synergizes with IL-6 to sustain differentiation of CD40-activated B lymphocytes. Overexpression of B-cell-derived IL-10 is likely to contribute in vivo to different pathologies such as B-lymphoproliferative disorders and autoimmune diseases.

Inability to produce IL-6 is a functional feature of human germinal center B lymphocytes.

In response to Ag encounter, B lymphocytes undergo a complex maturation process yielding phenotypically distinct subpopulations that are located in highly organized compartments of secondary lymphoid organs. This study describes the patterns of cytokine secretion of naive, memory, and germinal center (GC) human tonsillar B lymphocytes, activated either through CD40 or B cell receptor or with Staphylococcus aureus Cowan I particles. The three B cell subpopulations produced comparable levels of IL-10 and TNF-alpha, regardless of the stimulation pathway. Interestingly, activated GC B lymphocytes fail to express IL-6, as determined both at mRNA and at protein levels, whereas both naive and memory B cells can be induced to secrete IL-6. Likewise, naive B lymphocytes undergoing dual ligation of CD40 and B cell receptor fail to express IL-6, since they acquire a GC-like phenotype. IL-6 receptors are up-regulated on both ex vivo-purified GC B lymphocytes and in vitro generated GC-like B cells, following CD40 activation. Consistent with this, addition of exogenous IL-6 sustains growth of CD40-stimulated GC B lymphocytes. Taken together, these results demonstrate that loss of IL-6 secretion is a functional characteristic of human GC B lymphocytes. The swap from an autocrine to a paracrine IL-6 response may permit a better control of B cell growth and differentiation during the germinal center reaction.

Negative selection of human germinal center B cells by prolonged BCR cross-linking.

The antigen receptors on T and B lymphocytes can transduce both agonist and antagonist signals leading either to activation/survival or anergy/death. The outcome of B lymphocyte antigen receptor (BCR) triggering depends upon multiple parameters which include (a) antigen concentration and valency, (b) duration of BCR occupancy, (c) receptor affinity, and (d) B cell differentiation stages. Herein, using anti-immunoglobulin kappa and lambda light chain antibodies, we analyzed the response of human naive, germinal center (GC) or memory B cells to BCR cross-linking regardless of heavy chain Ig isotype or intrinsic BCR specificity. We show that after CD40-activation, anti-BCR (kappa + gamma) can elicit an intracellular calcium flux on both GC and non-GC cells. However, prolonged BCR cross-linking induces death of CD40-activated GC B cells but enhances proliferation of naive or memory cells. Anti-kappa antibody only kills kappa + GC B cells without affecting surrounding gamma + GC B cells, thus demonstrating that BCR-mediated killing of GC B lymphocytes is a direct effect that does not involve a paracrine mechanism. BCR-mediated killing of CD40-activated GC B cells could be partially antagonized by the addition of IL-4. Moreover, in the presence of IL-4, prestimulation through CD40 could prevent subsequent anti-Ig-mediated cell death, suggesting a specific role of this combination in selection of GC B cells. This report provides evidence that in human, susceptibility to BCR killing is regulated along peripheral B cell differentiation pathway.

CD40 and B cell antigen receptor dual triggering of resting B lymphocytes turns on a partial germinal center phenotype.

Phenotypic alterations occur when resting human B lymphocytes become germinal center (GC) cells. These include the induction of surface CD38, CD95 (FAS/APO-1), and carboxy-peptidase-M (CPM), a recently described GC marker. However, the factors that govern the in vivo induction of these surface molecules on B cells remain unknown. Here, we purified resting (CD38-) human B lymphocytes from tonsils in an attempt to establish culture conditions resulting in the induction of these three GC markers. We show that interferon (IFN) alpha or IFN-gamma, as well as antibodies against the B cell antigen receptor (BCR), could induce CD38 on resting B lymphocytes, a phenomenon further enhanced by CD40 stimulation. Concomitantly, CD95 was upregulated by CD40 ligation and, to a lesser extent, by IFN-gamma. By contrast, CPM expression could be upregulated only through BCR triggering. This CPM induction was specifically enhanced by CD19 or CD40 ligation. CD40 + BCR stimulation of resting B cells with CD40 ligand-transfected fibroblastic cells in the presence of cross-linked anti-BCR monoclonal antibodies resulted in the coexpression of CD38, CD95, and CPM. As GC cells, these cells also expressed CD71, CD80 (B7.1), and CD86 (B7.2), but not CD24. However, CD10+ or CD44- B cells could not be detected in these culture conditions, suggesting that yet other signals are required for the induction of these GC markers. Consistent with a GC phenotype, CD40 + BCR-stimulated cells exhibited reduced viability when cultured for 20 h in the absence of stimulus. These results first demonstrate that cotriggering of resting B cells through BCR and CD40 induces both phenotypic and functional GC features. They also show that IFN and CD19 triggering of resting B cells specifically modulate the expression of GC markers.