Kamel Benlagha

Distinct Functional Lineages of Human Vα24 Natural Killer T Cells

CD1d-restricted autoreactive natural killer (NK)T cells have been reported to regulate a range of disease conditions, including type I diabetes and immune rejection of cancer, through the secretion of either T helper (Th)2 or Th1 cytokines. However, mechanisms underlying Th2 versus Th1 cytokine secretion by these cells are not well understood. Since most healthy subjects express <1 NKT cell per 1,000 peripheral blood lymphocytes (PBLs), we devised a new method based on the combined used of T cell receptor (TCR)-specific reagents α-galactosylceramide (αGalCer) loaded CD1d-tetramers and anti-Vα24 monoclonal antibody, to specifically identify and characterize these rare cells in fresh PBLs. We report here that CD4+ and CD4−CD8− (double negative [DN]) NKT cell subsets represent functionally distinct lineages with marked differences in their profile of cytokine secretion and pattern of expression of chemokine receptors, integrins, and NK receptors. CD4+ NKT cells were the exclusive producers of interleukin (IL)-4 and IL-13 upon primary stimulation, whereas DN NKT cells had a strict Th1 profile and prominently expressed several NK lineage receptors. These findings may explain how NKT cells could promote Th2 responses in some conditions and Th1 in others, and should be taken into consideration for intervention in relevant diseases.

Testing the NKT cell hypothesis of human IDDM pathogenesis

Defects in IL-4-producing CD1d-autoreactive NKT cells have been implicated in numerous Th1-mediated autoimmune diseases, including diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and systemic sclerosis. Particular attention has been focused on autoimmune insulin-dependent diabetes mellitus (IDDM) because nonobese diabetic (NOD) mice and humans with IDDM are both reported to express severe deficiencies in the frequency and Th2 functions of NKT cells. Furthermore, experimental manipulations of the NKT defect in the NOD mouse induced corresponding changes in disease. Taken together, these converging studies suggested a general role of NKT cells in natural protection against destructive autoimmunity. However, in previous reports the identification of NKT cells was based on indirect methods. We have now devised a direct, highly specific CD1d tetramer-based methodology to test whether humans with IDDM have associated NKT cell defects. Surprisingly, although we find marked and stable differences in NKT cells between individuals, our study of IDDM patients and healthy controls, including discordant twin pairs, demonstrates that NKT cell frequency and IL-4 production are conserved during the course of IDDM. These results contradict previous conclusions and refute the hypothesis that NKT cell defects underlie most autoimmune diseases.

Contribution of IL-17–producing γδ T cells to the efficacy of anticancer chemotherapy

IL-17 production by γδ T cells is required for tumor cell infiltration by IFN-γ–producing CD8+ T cells and inhibition of tumor growth in response to anthracyclines.

Characterization of the early stages of thymic NKT cell development

Upon reaching the mature heat stable antigen (HSA)low thymic developmental stage, CD1d-restricted Vα14-Jα18 thymocytes undergo a well-characterized sequence of expansion and differentiation steps that lead to the peripheral interleukin-4/interferon-γ–producing NKT phenotype. However, their more immature HSAhigh precursors have remained elusive, and it has been difficult to determine unambiguously whether NKT cells originate from a CD4+CD8+ double-positive (DP) stage, and when the CD4+ and CD4−CD8− double-negative (DN) NKT subsets are formed. Here, we have used a CD1d tetramer-based enrichment strategy to physically identify HSAhigh precursors in thymuses of newborn mice, including an elusive DPlow stage and a CD4+ stage, which were present at a frequency of ∼10−6. These HSAhigh DP and CD4+ stages appeared to be nondividing, and already exhibited the same Vβ8 bias that characterizes mature NKT cells. This implied that the massive expansion of NKT cells is separated temporally from positive selection, but faithfully amplifies the selected TCR repertoire. Furthermore, we found that, unlike the DN γδ T cells, the DN NKT cells did not originate from a pTα-independent pathway bypassing the DP stage, but instead were produced during a short window of time from the conversion of a fraction of HSAlow NK1.1neg CD4 cells. These findings identify the HSAhigh CD4+ stage as a potential branchpoint between NKT and conventional T lineages and between the CD4 and DN NKT sublineages.

CD1d Endosomal Trafficking Is Independently Regulated by an Intrinsic CD1d-Encoded Tyrosine Motif and by the Invariant Chain

Endosomal trafficking is an essential component of the CD1 pathway of lipid antigen presentation to T cells. We demonstrate that CD1d access to endosomal compartments is under dual regulation by an intrinsic tyrosine-based motif, which governs intense recycling between the plasma membrane and the endosome, and by the invariant chain, with which CD1d associates in the endoplasmic reticulum. Both pathways independently enhance antigen presentation to V(alpha)14(+) NKT cells, the main subset of CD1d-restricted T cells. These results reveal the complexity of CD1d trafficking and suggest that the invariant chain was a component of ancestral antigen presentation pathways prior to the evolution of MHC and CD1.

Multiple defects in antigen presentation and T cell development by mice expressing cytoplasmic tail–truncated CD1d

For members of the CD1 family of β2-microglobulin–associated lipid-presenting molecules, tyrosine-based motifs in the cytoplasmic tail and invariant chain (Ii) govern glycoprotein trafficking through endosomal compartments. Little is known about the intracellular pathways of CD1 trafficking and antigen presentation. However, in vitro studies with cells transfected with mutant CD1 that had a truncated cytoplasmic tail have suggested a role for these tyrosine motifs in some, but not all, antigenic systems. By introducing a deletion of the tyrosine motif into the germ line, and through homologous recombination in embryonic stem cells, we now describe knock-in mice with the CD1d cytoplasmic tail deleted. Despite adequate surface CD1d expression and the presence of Ii, these mutant mice showed multiple and selective abnormalities in intracellular trafficking, antigen presentation and T cell development, demonstrating the critical functions of the CD1d cytoplasmic tail motif in vivo.

Skin and peripheral lymph node invariant NKT cells are mainly retinoic acid receptor-related orphan receptor (gamma)t+ and respond preferentially under inflammatory conditions.

Lymph nodes (LNs) have been long considered as comprising few invariant NKT (iNKT) cells, and these cells have not been studied extensively. In this study, we unravel the existence of stable rather than transitional LN-resident NK1.1− iNKT cell populations. We found the one resident in peripheral LNs (PLNs) to comprise a major IL-17-producing population and to express the retinoic acid receptor-related orphan receptor γt (RORγt). These cells respond to their ligand α-galactosylceramide (α-GalCer) in vivo by expanding dramatically in the presence of LPS, providing insight into how this rare population could have an impact in immune responses to infection. PLN-resident RORγt+ NK1.1− iNKT cells express concomitantly CCR6, the integrin α-chain αE (CD103), and IL-1R type I (CD121a), indicating that they might play a role in inflamed epithelia. Accordingly, skin epithelia comprise a major RORγt+ CCR6+CD103+CD121a+ NK1.1− cell population, reflecting iNKT cell composition in PLNs. Importantly, both skin and draining PLN RORγt+ iNKT cells respond preferentially to inflammatory signals and independently of IL-6, indicating that they could play a nonredundant role during inflammation. Overall, our study indicates that RORγt+ iNKT cells could play a major role in the skin during immune responses to infection and autoimmunity.

Thymocyte expression of cathepsin L is essential for NKT cell development

CD1d antigen presentation to natural killer T (NKT) cells expressing the semi-invariant T cell receptor Vα14Jα18 requires CD1d trafficking through endosomal compartments; however, the endosomal events remain undefined. We show that mice lacking the endosomal protease cathepsin L (catL) have greatly reduced numbers of Vα14+NK1.1+ T cells. In addition, catL expression in thymocytes is critical not only for selection of these cells in vivo but also for stimulation of Vα14+NK1.1+ T cells in vitro. CD1d cell-surface expression and intracellular localization appear normal in catL-deficient thymocytes, as does the lysosomal morphology; this implies a specific role for catL in regulating presentation of natural CD1d ligands mediating Vα14+NK1.1+ T cell selection. These data implicate lysosomal proteases as key regulators of not only classical major histocompatibility complex class II antigen presentation but also nonclassical CD1d presentation.

Unaltered phenotype, tissue distribution and function of Vα14+ NKT cells in germ‐free mice

The expression pattern of mouse CD1d and the tissue distribution of CD1d‐restricted Vα14‐Jα281 NKT cells suggest that the liver and the marginal zone of the spleen might be preferred sites of activation of this potent innate pathway of early cytokine secretion. Because these tissues are particularly involved with the filtration of blood‐borne pathogens, and because NKT cells with an activated / memory phenotype accumulate over the first weeks of life and their CD1 ligands bind microbial glycolipids, it has been hypothesized that expansion of the NKT cell subset may be driven by exposure to the microbial environment. To test this hypothesis, we analyzed the frequency, surface phenotype and functional properties of NKT cells in normal and in germ‐free C57BL / 6 mice. Surprisingly, we found that the NKT cell subset develops in the presence or absence of a microbial environment. Although these results do not rule out the possibility that NKT cells exert a protective function against some microbial agents, they demonstrate that non microbial ligands, possibly self‐antigens are sufficient for the generation, maturation and peripheral accumulation of NKT cells.

iNKT cell development is orchestrated by different branches of TGF-beta signaling.

Invariant natural killer T (iNKT) cells constitute a distinct subset of T lymphocytes exhibiting important immune-regulatory functions. Although various steps of their differentiation have been well characterized, the factors controlling their development remain poorly documented. Here, we show that TGF-β controls the differentiation program of iNKT cells. We demonstrate that TGF-β signaling carefully and specifically orchestrates several steps of iNKT cell development. In vivo, this multifaceted role of TGF-β involves the concerted action of different pathways of TGF-β signaling. Whereas the Tif-1γ branch controls lineage expansion, the Smad4 branch maintains the maturation stage that is initially repressed by a Tif-1γ/Smad4-independent branch. Thus, these three different branches of TGF-β signaling function in concert as complementary effectors, allowing TGF-β to fine tune the iNKT cell differentiation program.