Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell responses in humans. To identify novel antigenic lipids and increase our understanding of lipid-mediated immune responses, we established a panel of T cell clones with different lipid specificities. Using this approach we characterized a novel lipid antigen belonging to the group of diacylated sulfoglycolipids purified from Mycobacterium tuberculosis. The structure of this sulfoglycolipid was identified as 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2′-sulfate-α-α′-d-trehalose (Ac2SGL). Its immunogenicity is dependent on the presence of the sulfate group and of the two fatty acids. Ac2SGL is mainly presented by CD1b molecules after internalization in a cellular compartment with low pH. Ac2SGL-specific T cells release interferon γ, efficiently recognize M. tuberculosis–infected cells, and kill intracellular bacteria. The presence of Ac2SGL-responsive T cells in vivo is strictly dependent on previous contact with M. tuberculosis, but independent from the development of clinically overt disease. These properties identify Ac2SGL as a promising candidate to be tested in novel vaccines against tuberculosis.

Complexes between CD1 molecules and self or microbial glycolipids represent important immunogenic ligands for specific subsets of T cells. However, the function of one of the CD1 family members, CD1e, has yet to be determined. Here, we show that the mycobacterial antigens hexamannosylated phosphatidyl-myo-inositols (PIM6) stimulate CD1b-restricted T cells only after partial digestion of the oligomannose moiety by lysosomal alpha-mannosidase and that soluble CD1e is required for this processing. Furthermore, recombinant CD1e was able to bind glycolipids and assist in the digestion of PIM6. We propose that, through this form of glycolipid editing, CD1e helps expand the repertoire of glycolipidic T cell antigens to optimize antimicrobial immune responses.

Liposomes are closed bilayer structures spontaneously formed by hydrated phospholipids that are widely used as efficient delivery systems for drugs or antigens, due to their capability to encapsulate bioactive hydrophilic, amphipathic, and lipophilic molecules into inner water phase or within lipid leaflets. The efficacy of liposomes as drug or antigen carriers has been improved in the last years to ameliorate pharmacokinetics and capacity to release their cargo in selected target organs or cells. Moreover, different formulations and variations in liposome composition have been often proposed to include immunostimulatory molecules, ligands for specific receptors, or stimuli responsive compounds. Intriguingly, independent research has unveiled the capacity of several phospholipids to play critical roles as intracellular messengers in modulating both innate and adaptive immune responses through various mechanisms, including (i) activation of different antimicrobial enzymatic pathways, (ii) driving the fusion–fission events between endosomes with direct consequences to phagosome maturation and/or to antigen presentation pathway, and (iii) modulation of the inflammatory response. These features can be exploited by including selected bioactive phospholipids in the bilayer scaffold of liposomes. This would represent an important step forward since drug or antigen carrying liposomes could be engineered to simultaneously activate different signal transduction pathways and target specific cells or tissues to induce antigen-specific T and/or B cell response. This lipid-based host-directed strategy can provide a focused antimicrobial innate and adaptive immune response against specific pathogens and offer a novel prophylactic or therapeutic option against chronic, recurrent, or drug-resistant infections.

CD1-restricted lipid-specific T lymphocytes are primed during infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. Here we describe the antigenicity of glycerol monomycolate (GroMM), which stimulates CD1b-restricted CD4(+) T cell clones. Chemical characterization of this antigen showed that it exists as two stereoisomers, one synthetic isomer being more stimulatory than the other. The hydroxyl groups of glycerol and the mycolic acid length are critical for triggering the T cell responses. GroMM was presented by M. tuberculosis-infected dendritic cells, demonstrating that the antigen is available for presentation during natural infection. Ex vivo experiments showed that GroMM stimulated T cells from vaccinated or latently infected healthy donors but not cells from patients with active tuberculosis, suggesting that GroMM-specific T cells are primed during infection and their detection correlates with lack of clinical active disease.

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SummaryWe previously described an escape mechanism exploited by Mycobacterium tuberculosis (Mtb) to prevent the generation of fully competent dendritic cells (DC). We have now tested the effect of isolated mycobacterial components on human monocyte differentiation into DC and demonstrated that cell wall (CW)-associated alpha-glucan induces monocytes to differentiate into DC (Glu-MoDC) with the same altered phenotype and functional behaviour of DC derived from Mtb-infected monocytes (Mt-MoDC). In fact, GluMoDC lack CD1 molecule expression, fail to upregulate CD80 and produce IL-10 but not IL-12. We also showed that Glu-MoDC are not able to prime effector T cells or present lipid antigens to CD1-restricted T-cell clones. Thus, we propose a mechanism of Mtbmonocyte interaction mediated by CW-associated alpha-glucan, which allows the bacterium to evade both innate and acquired immune responses.

Effective control of tuberculosis (TB) includes discrimination of subjects with active TB from individuals with latent TB infection (LTBI). As distinct interferon (IFN)-gamma and interleukin (IL)-2 profiles of antigen-specific T-cells have been associated with different clinical stages and antigen loads in several viral and bacterial diseases, we analysed these cytokines in TB using a modified QuantiFERON-TB Gold In Tube test. Detection of IL-2 in addition to IFN-gamma distinguishes not only Mycobacterium tuberculosis-infected subjects from healthy controls, but also individuals with LTBI from active TB patients. This may help to improve diagnostic tests for TB.

Cellular immune responses are crucial for the control of EBV-associated lymphoproliferative diseases. To induce an anti-EBV cell-mediated immunity, we have used dendritic cells (DCs) generated by a 3-day culture of human CD14+ monocytes in the presence of GM-CSF and type I IFN (IFN-DCs) and pulsed with peptides corresponding to CTL EBV epitopes. The functional activity of IFN-DCs was compared with that of APCs differentiated by culturing monocytes for 3 days with GM-CSF and IL-4 and indicated as IL-4-DCs. Stimulation of PBLs from EBV-seropositive donors with EBV peptide-pulsed autologous IFN-DCs resulted in a stronger expansion of specific T lymphocytes producing IFN-γ with respect to stimulation with peptide-loaded IL-4-DCs, as assessed by ELISPOT assays. When purified CD8+ T cells were cocultured with EBV peptide-pulsed IFN-DCs or IL-4-DCs, significantly higher levels of specific cytotoxic activity were observed in CD8+ T cell cultures stimulated with IFN-DCs. Injection of peptide-pulsed IFN-DCs into SCID mice transplanted with autologous PBLs led to the recovery of a significantly greater number of EBV-specific human CD8+ T cells from the spleen and the peritoneal cavity with respect to that recovered from mice injected with peptide-pulsed IL-4-DCs. Moreover, a significant delay in lymphoma development was observed when peptide-pulsed IFN-DCs were injected into SCID mice reconstituted with PBMCs endowed with a high capability of lymphoma induction, whereas injection of unpulsed IFN-DCs was ineffective. Our results indicate that IFN-DCs efficiently promote in vitro and in vivo the expansion of CD8+ T lymphocytes acting as cytotoxic effectors against EBV-transformed cells.