Steven A. Porcelli

A subset of liver NK T cells is activated during Leishmania donovani infection by CD1d-bound lipophosphoglycan

Natural killer (NK) T cells are activated by synthetic or self-glycolipids and implicated in innate host resistance to a range of viral, bacterial, and protozoan pathogens. Despite the immunogenicity of microbial lipoglycans and their promiscuous binding to CD1d, no pathogen-derived glycolipid antigen presented by this pathway has been identified to date. In the current work, we show increased susceptibility of NK T cell–deficient CD1d−/− mice to Leishmania donovani infection and Leishmania-induced CD1d-dependent activation of NK T cells in wild-type animals. The elicited response was Th1 polarized, occurred as early as 2 h after infection, and was independent from IL-12. The Leishmania surface glycoconjugate lipophosphoglycan, as well as related glycoinositol phospholipids, bound with high affinity to CD1d and induced a CD1d-dependent IFNγ response in naive intrahepatic lymphocytes. Together, these data identify Leishmania surface glycoconjugates as potential glycolipid antigens and suggest an important role for the CD1d–NK T cell immune axis in the early response to visceral Leishmania infection.

Mycobacterium tuberculosis Lipomannan Induces Apoptosis and Interleukin-12 Production in Macrophages

ABSTRACT The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as a virulence factor of Mycobacterium tuberculosis, and modification of the terminal arabinan residues of this compound with mannose caps (producing mannosyl-capped LAM [ManLAM]) in M. tuberculosis or with phosphoinositol caps (producing phosphoinositol-capped LAM [PILAM]) in Mycobacterium smegmatis has been implicated in various functions associated with these lipoglycans. A structure-function analysis was performed by using LAMs and their biosynthetic precursor lipomannans (LMs) isolated from different mycobacterial species on the basis of their capacity to induce the production of interleukin-12 (IL-12) and/or apoptosis of macrophage cell lines. Independent of the mycobacterial species, ManLAMs did not induce IL-12 gene expression or apoptosis of macrophages, whereas PILAMs induced IL-12 secretion and apoptosis. Interestingly, uncapped LAM purified from Mycobacterium chelonae did not induce IL-12 secretion or apoptosis. Furthermore, LMs, independent of their mycobacterial origins, were potent inducers of IL-12 and apoptosis. The precursor of LM, phosphatidyl-myo-inositol dimannoside, had no activity, suggesting that the mannan core of LM was required for the activity of LM. The specific interaction of LM with Toll-like receptor 2 (TLR-2) but not with TLR-4 suggested that these responses were mediated via the TLR-2 signaling pathway. Our experiments revealed an important immunostimulatory activity of the biosynthetic LAM precursor LM. The ratio of LAM to LM in the cell wall of mycobacteria may be an important determinant of virulence, and enzymes that modify LM could provide targets for development of antituberculosis drugs and for derivation of attenuated strains of M. tuberculosis.

Improved Outcomes in NOD Mice Treated with a Novel Th2 Cytokine-Biasing NKT Cell Activator

Activation of CD1d-restricted invariant NKT (iNKT) cells by α-galactosylceramide (αGalCer) significantly suppresses development of diabetes in NOD mice. The mechanisms of this protective effect are complex, involving both Th1 and Th2 cytokines and a network of regulatory cells including tolerogenic dendritic cells. In the current study, we evaluated a newly described synthetic αGalCer analog (C20:2) that elicits a Th2-biased cytokine response for its impact on disease progression and immunopathology in NOD mice. Treatment of NOD mice with αGalCer C20:2 significantly delayed and reduced the incidence of diabetes. This was associated with significant suppression of the late progression of insulitis, reduced infiltration of islets by autoreactive CD8+ T cells, and prevention of progressive disease-related changes in relative proportions of different subsets of dendritic cells in the draining pancreatic lymph nodes. Multiple favorable effects observed with αGalCer C20:2 were significantly more pronounced than those seen in direct comparisons with a closely related analog of αGalCer that stimulated a more mixed pattern of Th1 and Th2 cytokine secretion. Unlike a previously reported Th2-skewing murine iNKT cell agonist, the αGalCer C20:2 analog was strongly stimulatory for human iNKT cells and thus warrants further examination as a potential immunomodulatory agent for human disease.

The Bovine CD1 Family Contains Group 1 CD1 Proteins, but No Functional CD1d

The CD1 family of proteins presents lipid Ags to T cells. Human CD1a, CD1b, and CD1c have been shown in humans to present mycobacterial lipid Ags. Cattle, like humans, are a natural host of several mycobacterial pathogens. In this study, we describe the CD1 family of genes in cattle (Bos taurus) and provide evidence that B. taurus expresses CD1a, CD1e, and multiple CD1b molecules, but no CD1c and CD1d molecules. In mice and humans, CD1d is known to present Ag to NKT cells, a T cell lineage that is characterized by a limited TCR repertoire, capable of rapidly secreting large amounts of IFN-γ and IL-4. In cattle, two CD1D pseudogenes were found and no intact CD1D genes. Consistent with this, we found complete lack of reactivity to a potent, cross-reactive Ag for NKT cells in mice and humans, α-galactosylceramide. Our data suggest the absence of NKT cells in cattle. It remains open whether other cells with the NKT-like phenotype and functions are present in this species. With its functional CD1A and CD1B genes, B. taurus is well equipped to present Ags to CD1-restricted T cells other than NKT cells. Cattle can be used as a model to study group 1 CD1-restricted T cell immunity, including its role in the defense against mycobacterial infections that occur naturally in this species.

Expansion and Hyperactivity of CD1d-Restricted NKT Cells during the Progression of Systemic Lupus Erythematosus in (New Zealand Black × New Zealand White)F1 Mice

CD1d-restricted NKT cells expressing invariant TCR α-chain rearrangements (iNKT cells) have been reported to be deficient in humans with a variety of autoimmune syndromes and in certain strains of autoimmune mice. In addition, injection of mice with α-galactosylceramide, a specific glycolipid agonist of iNKT cells, activates these T cells and ameliorates autoimmunity in several different disease models. Thus, deficiency and reduced function in iNKT cells are considered to be risk factors for the development of such diseases. In this study we report that the development of systemic lupus erythematosus in (New Zealand Black (NZB) × New Zealand White (NZW))F1 mice was paradoxically associated with an expansion and activation of iNKT cells. Although young (NZB × NZW)F1 mice had normal levels of iNKT cells, these expanded with age and became phenotypically and functionally hyperactive. Activation of iNKT cells in (NZB × NZW)F1 mice in vivo or in vitro with α-galactosylceramide indicated that the immunoregulatory role of iNKT cells varied over time, revealing a marked increase in their potential to contribute to production of IFN-γ with advancing age and disease progression. This evolution of iNKT cell function during the progression of autoimmunity may have important implications for the mechanism of disease in this model of systemic lupus erythematosus and for the development of therapies using iNKT cell agonists.

Enrichment of Human CD4+ Vα24/Vβ11 Invariant NKT Cells in Intrahepatic Malignant Tumors

Invariant NKT cells (iNKT cells) recognize glycolipid Ags via an invariant TCR α-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 Vα24/Vβ11 iNKT cells were increased in tumors, especially in patients with hepatocellular carcinoma. The proportions of CD4+, double negative, and CD8α+ 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.

Control of NKT Cell Differentiation by Tissue-Specific Microenvironments

CD1d-restricted Vα14 NKT cells play an important role in both Th1- and Th2-type immune responses. To determine whether NKT cells develop two functionally distinct subsets that provoke different types of responses, we examined the phenotypes and cellular functions of NK1.1+ and DX5+ T cells. We found that both NK1.1+ and DX5+ T cells are CD1d-restricted Vα14 T cells with identical Ag specificities, phenotypes, tissue locations, and functions. Similar to the NK1.1 marker, the DX5 marker (CD49b) is expressed on mature NKT cells in both NK1.1 allele-positive and allele-negative strains. However, when NK1.1+ and DX5+ NKT cells isolated from different tissues were compared, we found that thymic and splenic NKT cells differed not only in their cytokine profiles, but also in their phenotype and requirements for costimulatory signals. Thymic NKT cells displayed the phenotype of activated T cells and could be fully activated by TCR ligation. In contrast, splenic NKT cells displayed the phenotype of memory T cells and required a costimulatory signal for activation. Furthermore, the function and phenotype of thymic and splenic NKT cells were modulated by APCs from various tissues that expressed different levels of costimulatory molecules. Modulation of NKT cell function and differentiation may be mediated by synergic effects of costimulatory molecules on the surface of APCs. The results of the present study suggest that the costimulatory signals of tissue-specific APCs are key factors for NKT cell differentiation, and these signals cannot be replaced by anti-CD28 or anti-CD40 ligand Abs.

Alteration of the relative levels of iNKT cell subsets is associated with chronic mycobacterial infections.

CD1d-restricted invariant natural killer T cells (iNKT cells) have been identified as an important type of effector and regulatory T cell, but their roles in the chronic infectious diseases caused by Mycobacterium tuberculosis and Mycobacterium leprae remain poorly defined. Here, we studied circulating human iNKT cells in blood samples from tuberculosis (TB) and leprosy patients. We found that the percentages of iNKT cells among total circulating T cells in TB and leprosy patients were not significantly different from those in normal controls. However, both TB and leprosy patients showed a selective reduction of the proinflammatory CD4(-)CD8beta(-) (DN) iNKT cells with a proportionate increase in the CD4(+) iNKT cells. Similar phenotypic alterations in circulating iNKT cells were observed in a mouse model of M. tuberculosis infection. Taken together, these findings indicate that the selective reduction of circulating DN iNKT cells is associated with chronic infections caused by M. tuberculosis and M. leprae.

Mycobacterial Membrane Vesicles Administered Systemically in Mice Induce a Protective Immune Response to Surface Compartments of Mycobacterium tuberculosis

ABSTRACT 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. IMPORTANCE This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation. This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation.

Direct Measurement of Antigen Binding Properties of CD1 Proteins Using Fluorescent Lipid Probes

CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses. In the current study, we investigated ligand binding by CD1 proteins by developing a fluorescent probe binding approach using soluble recombinant human CD1 proteins. To increase stability and yield, soluble group 1 CD1 (CD1b and CD1c) and group 2 CD1 (CD1d) proteins were produced as single chain secreted CD1 proteins in which β2-microglobulin was fused to the N termini of the CD1 heavy chains by a flexible peptide linker sequence. Analysis of ligand binding properties of single chain secreted CD1 proteins by using fluorescent lipid probes indicated significant differences in ligand preference and in pH dependence of binding by group 1 versus group 2 CD1 proteins. Whereas group 1 CD1 isoforms (CD1b and CD1c) show stronger binding of nitrobenzoxadiazole (NBD)-labeled dialkyl-based ligands (phosphatidylcholine, sphingomyelin, and ceramide), group 2 CD1 (CD1d) proteins were stronger binders of small hydrophobic probes such as 1-anilinonaphthalene-8-sulfonic acid and 4,4′-dianilino-1,1′-naphthyl-5,5′-disulfonic acid. Competition studies indicated that binding of fluorescent lipid probes involved association of the probe with the hydrophobic ligand binding groove of CD1 proteins. Analysis of selected alanine substitution mutants of human CD1b known to inhibit antigen presentation showed that NBD-labeled lipid probe binding could be used to distinguish mutations that interfere with ligand binding from those that affect T cell receptor docking. Our findings provide further evidence for the functional specialization of different CD1 isoforms and demonstrate the value of the fluorescent lipid probe binding method for assisting structure-based studies of CD1 function.