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Ig-like transcript 4 (ILT4) (also known as leukocyte Ig-like receptor 2, CD85d, and LILRB2) is a cell surface receptor expressed mainly on myelomonocytic cells, whereas ILT2 (also known as leukocyte Ig-like receptor 1, CD85j, and LILRB1) is expressed on a wider range of immune cells including subsets of natural killer and T cells. Both ILTs contain immunoreceptor tyrosine-based inhibitory receptor motifs in their cytoplasmic tails that inhibit cellular responses by recruiting phosphatases such as SHP-1 (Src homology 2 domain containing tyrosine phosphatase 1). Although these ILTs have been shown to recognize a broad range of classical and nonclassical human MHC class I molecules (MHCIs), their precise binding properties remain controversial. We have used surface plasmon resonance to analyze the interaction of soluble forms of ILT4 and ILT2 with several MHCIs. Although the range of affinities measured was quite broad (Kd= 2–45 μM), some interesting differences were observed. ILT2 generally bound with a 2- to 3-fold higher affinity than ILT4 to the same MHCI. Furthermore, ILT2 and ILT4 bound to HLA-G with a 3- to 4-fold higher affinity than to classical MHCIs, suggesting that ILT/HLA-G recognition may play a dominant role in the regulation of natural killer, T, and myelomonocytic cell activation. Finally, we show that ILT2 and ILT4 effectively compete with CD8 for MHCI binding, raising the possibility that ILT2 modulates CD8+T cell activation by blocking the CD8 binding as well as by recruiting inhibitory molecules through its immunoreceptor tyrosine-based inhibitory receptor motif.

Subsets of dendritic cells (DCs) have been described according to their functions and anatomical locations. Conventional DC subsets are defined by reciprocal expression of CD11b and CD8α in lymphoid tissues (LT), and of CD11b and CD103 in non-LT (NLT). Spleen CD8α+ and dermal CD103+ DCs share a high efficiency for Ag cross-presentation and a developmental dependency on specific transcription factors. However, it is not known whether all NLT-derived CD103+ DCs and LT-resident CD8α+ DCs are similar despite their different anatomical locations. XCR1 was previously described as exclusively expressed on mouse spleen CD8α+ DCs and human blood BDCA3+ DCs. In this article, we showed that LT-resident CD8α+ DCs and NLT-derived CD103+ DCs specifically express XCR1 and are characterized by a unique transcriptional fingerprint, irrespective of their tissue of origin. Therefore, CD8α+ DCs and CD103+ DCs belong to a common DC subset which is unequivocally identified by XCR1 expression throughout the body.

Type II Toxoplasma gondii KU80 knockouts (Δ ku80 ) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δ ku80 Δ hxgprt strain measured at the orotate ( OPRT ) and the uracil ( UPRT ) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δ ku80 Δ hxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes ( GRA4 , GRA6 , ROP7 , and tgd057 ) that encode characterized CD8 + T cell epitopes that elicit corresponding antigen-specific CD8 + T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8 + T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δ gra4 and Δ gra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δ ku80 Δ hxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.

International audience; Memory CD8(+) T lymphocytes are critical effector cells of the adaptive immune system mediating long-lived pathogen-specific protective immunity. Three signals - antigen, costimulation and inflammation - orchestrate optimal CD8(+) T-cell priming and differentiation into effector and memory cells and shape T-cell functional fate and ability to protect against challenge infections. While among the conventional spleen DCs (cDCs), the CD8α(+) but not the CD8α(-) cDCs most efficiently mediate CD8(+) T-cell priming, it is unclear which subset, irrespective of their capacity to process MHC class I-associated antigens, is most efficient at inducing naïve CD8(+) T-cell differentiation into pathogen-specific protective memory cells in vivo. Moreover, the origin of the required signals is still unclear. Using mice infected with the intracellular bacterium Listeria monocytogenes, we show that splenic CD8α(+) cDCs become endowed with all functional features to optimally prime protective memory CD8(+) T cells in vivo within only a few hours post-immunization. Such programming requires both cytosolic signals resulting from bacterial invasion of the host cells and extracellular inflammatory mediators. Thus, these data designate these cells as the best candidates to facilitate the development of cell-based vaccine therapy.

International audience; BACKGROUND & AIMS: In the present work, we address the requirement for intestinal-specific homing molecules, the chemokine/chemokine receptor pair CCL25/CCR9 and beta7 integrin, in the pathogenesis of the CD8(+) T cell-dependent Tnf(DeltaARE) mouse model of Crohn's-like inflammatory bowel disease. METHODS: We investigated by flow cytometry lymphocyte recruitment in the intestinal epithelium and lamina propria (LP); cytokine production by intraepithelial and LP lymphocytes; and peripheral expression of CCR9, alpha4beta7, and alphaEbeta7 integrin. The functional significance of CCL25/CCR9 and beta7 integrin in inflammatory lymphocyte recruitment and intestinal disease development was assessed in Tnf(DeltaARE) mice genetically lacking these molecules. RESULTS: Intestinal inflammation in the Tnf(DeltaARE) mice is associated with early reduction of CD8alphaalpha-expressing intraepithelial lymphocytes, decreased T helper cell 1 and increased T helper cell 17 responses by LP CD4(+) lymphocytes, increased alphaEbeta7 integrin expression in peripheral activated/memory intestinal-homing CD8alphabeta lymphocytes, and predominance of tumor necrosis factor/interferon-gamma-producing CD8alphabeta lymphocytes in the epithelium. Although CCL25/CCR9 have been strongly implicated in T-lymphocyte recruitment to the small intestine, inflammatory pathology develops unperturbed in the genetic absence of CCL25/CCR9. Furthermore, CD8alphabeta lymphocyte recruitment in the intestinal epithelium and inflammatory infiltration in the LP are not impaired in CCR9- or CCL25-deficient Tnf(DeltaARE) mice. In contrast, genetic ablation of beta7 integrin results in complete amelioration of intestinal pathology. CONCLUSIONS: Our findings demonstrate that development of intestinal inflammation in the Tnf(DeltaARE) mice is critically dependent on beta7 integrin-mediated T-lymphocyte recruitment, whereas the function of the CCL25/CCR9 axis appears dispensable in this model.

International audience; Despite several decades of research, current antidepressant (AD) treatments remain of a limited efficacy justifying the need to find new drugs. These drugs have to be more efficacious, more rapid and display lesser side effects. Using rodent models, we recently identified spadin as a new antidepressant molecule that acts more quickly than classical ADs, working within 4 days to get same effects obtained with other ADs after 21 days. Spadin blocks TREK-1 K(2P) potassium channels that are considered as new targets for ADs. Deletion of the TREK-1 channel is known to increase sensitivity to pain, seizures and ischemia. Thus blocking these channels could result in deleterious side effects. In this study we showed that spadin did not interfere with other TREK-1 controlled functions such as pain, epilepsy and ischemia. We also demonstrated that spadin was unable to inhibit currents generated by TREK-2, TRAAK, TASK and TRESK four other K2P channels. More importantly, spadin did not induce cardiac dysfunctions, did not block I(Kr) and I(Ks) and did not modify the systolic pressure or cardiac pulses. After a three week treatment spadin remained an efficacious AD and did not modify the infarct size in brain following focal ischemia. Finally, we showed that kainate induced seizures and glycemia were not modified by spadin treatments. These data, together with those previously published reinforce the idea that spadin represents a good candidate for a new generation of ADs. This article is part of a Special Issue entitled 'Anxiety and Depression'.

International audience; The dendritic cells (DCs) present in lymphoid and non-lymphoid organs are generated from progenitors with myeloid-restricted potential. However, in the thymus a major subset of DCs expressing CD8α and langerin (CD207) appears to stand apart from all other DCs in that it is thought to derive from progenitors with lymphoid potential. Using mice expressing a fluorescent reporter and a diphtheria toxin receptor under the control of the cd207 gene, we demonstrated that CD207(+) CD8α(+) thymic DCs do not share a common origin with T cells but originate from intrathymic precursors that express markers that are normally present on all (CD11c(+) and MHCII molecules) or on some (CD207, CD135, CD8α, CX3CR1) DC subsets. Those intrathymic myeloid-type precursors correspond to CD44(+) CD25(-) double-negative 1c (DN1c) cells and are continuously renewed from bone marrow-derived canonical DC precursors. In conclusion, our results demonstrate that the earliest intrathymic precursors of CD8α(+) thymic DCs correspond to myeloid-type DN1c cells and support the view that under physiological conditions myeloid-restricted progenitors generate the whole constellation of DCs present in the body including the thymus.

Cross-presentation of cell-associated Ags by dendritic cells (DC) plays an important role in immunity. DC in lymphoid tissues are short lived, being continuously replaced by precursors that proliferate and differentiate locally. Paradoxically, although TLR ligands promote immune responses and stimulate DC replenishment, they impair the cross-priming capacity of terminally differentiated splenic CD8α+ DC, the major subset involved in cross-priming. In this study, we have investigated the cross-presentation capacity of newly generated murine DC and especially immediate precursors of CD8α+ DC. We show that these DC do not cross-present Ag from dead cells unless stimulated by TLR ligands before Ag capture. TLR ligand CpG induced the expression of costimulatory molecules required for CD8 T cell activation but also regulated the intracellular mechanisms of cross-presentation such as Ag degradation rates without regulating Ag uptake. GM-CSF, an inflammatory cytokine associated with infections, also promoted cross-presentation acquisition by pre-CD8α+ DC and synergized with TLR9 ligand. The concept that TLR ligands as well as inflammatory cytokines promote the acquisition of cross-presenting properties by pre-CD8α+ DC has important implications during immune responses and when considering the use of these cells for vaccination.

International audience; The mouse lymphoid organ-resident CD8alpha(+) dendritic cell (DC) subset is specialized in Ag presentation to CD8(+) T cells. Recent evidence shows that mouse nonlymphoid tissue CD103(+) DCs and human blood DC Ag 3(+) DCs share similarities with CD8alpha(+) DCs. We address here whether the organization of DC subsets is conserved across mammals in terms of gene expression signatures, phenotypic characteristics, and functional specialization, independently of the tissue of origin. We study the DC subsets that migrate from the skin in the ovine species that, like all domestic animals, belongs to the Laurasiatheria, a distinct phylogenetic clade from the supraprimates (human/mouse). We demonstrate that the minor sheep CD26(+) skin lymph DC subset shares significant transcriptomic similarities with mouse CD8alpha(+) and human blood DC Ag 3(+) DCs. This allowed the identification of a common set of phenotypic characteristics for CD8alpha-like DCs in the three mammalian species (i.e., SIRP(lo), CADM1(hi), CLEC9A(hi), CD205(hi), XCR1(hi)). Compared to CD26(-) DCs, the sheep CD26(+) DCs show 1) potent stimulation of allogeneic naive CD8(+) T cells with high selective induction of the Ifngamma and Il22 genes; 2) dominant efficacy in activating specific CD8(+) T cells against exogenous soluble Ag; and 3) selective expression of functional pathways associated with high capacity for Ag cross-presentation. Our results unravel a unifying definition of the CD8alpha(+)-like DCs across mammalian species and identify molecular candidates that could be used for the design of vaccines applying to mammals in general.

International audience; During evolution, vertebrates have developed an adaptive immune system able to cope with a variety of pathogens. Dendritic cells (DCs) are central to this process. DCs integrate information derived from pathogens or endogenous danger signals and convey them to T lymphocytes. Most of the present knowledge on DCs was generated in mice or by using human DCs differentiated in vitro from monocytes. In both species, several DC subsets have been identified in vivo based on differences in their phenotypes, anatomical locations or functions. In mice, protective immunity against intracellular pathogens or tumors can be induced most efficiently by targeting antigens to the CD8 alpha(+) DCs, a subset of DCs which resides in lymphoid tissues and is especially efficient at cross-presenting exogenous antigens to CD8(+) T lymphocytes. In contrary, harnessing human DC subsets for medical purposes is currently hampered by insufficient knowledge about these cells. To overcome this cognitive gap, we are using comparative genomics as a tool for designing hypotheses and experiments to further characterize DC subset functions and their molecular control, including the investigation of the functional equivalences that might exist between human and mouse DC subsets.

International audience; Intestinal intraepithelial lymphocytes carrying the γδ TCR (γδ iIEL) are involved in the maintenance of epithelial integrity. γδ iIEL have an activated phenotype, characterized by CD69 expression and increased cell size compared with systemic T lymphocytes. As an additional activation marker, the majority of γδ iIEL express the CD8αα homodimer. However, our knowledge about cognate ligands for most γδ TCR remains fragmentary and recent advances show that γδ T cells including iIEL may be directly activated by cytokines or through NK-receptors, TLR and other pattern recognition receptors. We therefore asked whether the TCR of γδ iIEL was functional beyond its role during thymic selection. Using TcrdH2BeGFP (Tcrd, T-cell receptor δ locus; H2B, histone 2B) reporter mice to identify γδ T cells, we measured their intracellular free calcium concentration in response to TCR-crosslinking. In contrast to systemic γδ T cells, CD8αα(+) γδ iIEL showed high basal calcium levels and were refractory to TCR-dependent calcium-flux induction; however, they readily produced CC chemokine ligand 4 (CCL4) and IFN-γ upon TCR triggering in vitro. Notably, in vivo blocking of the γδ TCR with specific mAb led to a decrease of basal calcium levels in CD8αα(+) γδ iIEL. This suggests that the γδ TCR of CD8αα(+) γδ iIEL is constantly being triggered and therefore functional in vivo.

International audience; Patients with mutations in the Artemis gene display a complete absence of T- and B lymphocytes, together with increased cellular radiosensitivity; this leads to a radiosensitive severe combined immunodeficiency (RS-SCID). Allogenic hematopoietic stem-cell (HSC) transplantation is only partially successful in the absence of an human leukocyte antigen-genoidentical donor, and this has prompted a search for alternative therapeutic approaches such as gene therapy. In this study, a self-inactivated lentiviral vector expressing Artemis was used to complement the Artemis knockout mouse (Art(-/-)). Transplantation of Artemis-transduced HSCs into irradiated Art(-/-) mice restored a stable (over a 15-month period of follow-up) and functional T- and cell repertoire that was comparable to that of control mice. The success of secondary transplantations demonstrated that the HSCs had been transduced. One of thirteen mice developed a thymoma 6 months after gene therapy. Although thymic cells were seen to be carrying two lentiviral integration sites, there was no evidence of lentivirus-driven oncogene activation. The Art(-/-) mice were found to be prone to develop T-cell lymphomas, either spontaneously or after irradiation. These data indicate that the observed lymphoproliferation was probably the consequence of the chromosomal instability associated with the Artemis-deficient background. As a whole, our work provides a basis for supporting the gene therapy approach in Artemis-deficient SCID.

International audience; Antigen recognition by T cell antigen receptors (TCRs) is thought to 'unmask' a proline-rich sequence (PRS) present in the CD3epsilon cytosolic segment, which allows it to trigger T cell activation. Using 'knock-in' mice with deletion of the PRS, we demonstrate here that elimination of the CD3epsilon PRS had no effect on mature T cell responsiveness. In contrast, in preselection CD4+CD8+ thymocytes, the CD3epsilon PRS acted together with the adaptor protein SLAP to promote CD3zeta degradation, thereby contributing to downregulation of TCR expression on the cell surface. In addition, analysis of CD4+CD8+ thymocytes of TCR-transgenic mice showed that the CD3epsilon PRS enhanced TCR sensitivity to weak ligands. Our results identify previously unknown functions for the evolutionarily conserved CD3epsilon PRS at the CD4+CD8+ developmental stage and suggest a rather limited function in mature T cells.

International audience; Background We have demonstrated previously that retroviral-mediated transfer of a suicide gene into bone marrow (BM) donor T cells allows an efficient control of graft-versus-host disease (GvHD) after allogeneic BM transplantation. However, the 12 days of ex vivo culture required for the production of gene-modified cells (GMC), including soluble CD3 monoclonal antibody (MAb)-mediated activation and expansion with interleukin (IL)-2, induced a decrease of GMC alloreactivity and a reversal of their CD4/CD8 ratio. Improving the culture protocol in order to maintain the highest alloreactivity is of critical importance in obtaining an optimal graft-versus-leukemia (GvL) effect. Methods Peripheral blood mononuclear cells were activated with soluble CD3 MAb or CD3 and CD28 MAb co-immobilized on beads and expanded for 12 days in the presence of IL-2, IL-7 or IL-15 before analysis of alloreactivity and phenotype. Results Replacing the CD3 MAb by CD3/CD28 beads led to similar in vitro alloreactivity but improved the expansion and in vivo alloreactivity of GMC. Replacing the IL-2 with IL-7, but not IL-15, or decreasing IL-2 or IL-7 concentrations, improved the in vitro alloreactivity of expanded cells but was associated with lower expansion. Indeed, the alloreactivity of expanded cells was negatively correlated with cell expansion and positively correlated with CD4/CD8 ratio and CD8 expression level. Discussion Quantitative (i.e. low CD4/CD8 ratio) and qualitative (e.g. low CD8 expression) defects may account for the decreased alloreactivity of GMC. Using CD3/CD28 beads and/or IL-7 is more beneficial than CD3 MAb and IL-2 for preventing perturbations of the alloreactivity and phenotype of GMC.

International audience; Studies of the quantitative and qualitative aspects of anti-microbial, anti-tumoral or autoreactive immune responses have been greatly facilitated by the possibility to stain antigen-specific CD8(+) T cells using fluorescently labeled multimeric major histocompatibility complex (MHC) class I/peptide complexes. So far, this technology has been developed for human and mouse, but not yet in the rat. Here, we describe the generation of the first rat MHC multimer. We produced a rat RT1(l) Pro5 MHC Pentamer combined with the immunodominant peptide for Borna disease virus (BDV), in order to study the characteristics of the antiviral CD8(+) T cell response. BDV is an RNA virus that can cause persistent infections of the central nervous system (CNS), often associated with prominent brain inflammation. In adult Lewis rats, of the RT1(l) MHC haplotype, BDV infection leads to severe immune-mediated neurological symptoms. The pathogenic role of the immune response is due primarily to antiviral CD8(+) T cells, many of them being specific for an immunodominant epitope located in the BDV nucleoprotein (N(230-238)). Ex vivo flow cytometry analyses revealed that 3 to 12% of CD8(+) T cells found in the brains of BDV-infected rats stained positively with the BDV-Pentamer. Interestingly, the frequency of Pentamer-positive cells increased up to 3.3 fold after a short resting period in culture. Virus-specific CD8(+) T cells were mainly detected in the brain and were virtually undetectable in peripheral lymphoid organs. This novel rat Pro5 MHC Pentamer represents an attractive tool for the detection, isolation and characterization of antigen-specific CD8(+) T cell responses in the rat.

Langerhans cells have been thought to play a major role as APCs for induction of specific immune responses to Leishmania major. Although their requirement for control of infection has been challenged recently, it remains unclear whether they can transport Ag to lymph nodes and promote initiation of T cell responses. Moreover, the role of dermal dendritic cells (DCs), another population of skin DCs, has so far not been addressed. We have investigated the origin and characterized the cell population responsible for initial activation of L. major-specific T cells in susceptible and resistant mice. We found that Ag presentation in draining lymph nodes peaks as early as 24 h after infection and is mainly mediated by a population of CD11chighCD11bhighGr-1−CD8−langerin− DCs residing in lymph nodes and acquiring soluble Ags possibly drained through the conduit network. In contrast, skin-derived DCs, including Langerhans cells and dermal DCs, migrated poorly to lymph nodes and played a minor role in early T cell activation. Furthermore, prevention of migration through early removal of the infection site did not affect Ag presentation by CD11chigh CD11bhigh DCs and activation of Leishmania major-specific naive CD4+ T cells in vivo.

The role of Notch signaling in T cell commitment during lymphoid development is well established. However, the identity of the ligand that triggers this critical signal in vivo is still unclear. By overexpressing Delta-1 and Delta-4 ligands in the hemopoietic cells of athymic nu/nu host mice, we demonstrate that, in vivo and in the absence of a thymus, Delta-1 or Delta-4 expression is sufficient to promote T cell development from the most immature progenitor stages to complete maturation of both CD8+ and CD4+ αβ T cells. The mature T cells developing in a Delta-1- or Delta-4-enriched environment express a diverse TCR repertoire, are able to proliferate upon in vitro TCR stimulation, but show different profiles of cytokine production after in vitro anti-CD3 stimulation.

Structural studies of cellular immune receptors such as MHC molecules, T cell receptors (TCR), and TCR/MHC complexes have been carried out with recombinant, soluble forms of the extracytoplasmic domain of these glycoproteins [1]. The important role of the membrane bilayer in T cell recognition and antigen presentation has become increasingly obvious with the description of lipid microdomains [2, 3]. These rafts appear to regulate recognition and signaling by clustering receptors and facilitating the formation of the immune synapse [4]. However, the interactions and orientation of these receptors at the lipid bilayer are unknown. We have used H-2K b , a major-histocompatibility (MHC) class I molecule, and tethered its soluble domain to a lipid bilayer via a surrogate connecting peptide to reveal the disposition of MHC molecule on the membrane surface. We demonstrate that the long axis of the MHC molecule is approximately parallel to the plane of the membrane with the peptide binding pocket close to the membrane surface. This result was determined by analyzing 4.5A resolution electron crystallographic projection data from frozen-hydrated 2-dimensional crystals. Ionic interactions between the lipid headgroup and the protein appear to be responsible for this orientation, which could establish a "fourth dimension" during MHC/T cell receptor interactions critical for activation.

Background Adoptive cell therapy (ACT) has emerged as an effective treatment for patients with metastatic melanoma. However, there are several logistical and safety concerns associated with large-scale ex vivo expansion of tumour-specific T lymphocytes for widespread availability of ACT for cancer patients. To address these problems we developed a specific compartmentalised bag allowing efficient expansion of tumour-specific T lymphocytes in an easy handling, closed system. Methods Starting from lymph nodes from eight melanoma patients, we performed a side-by-side comparison of Tumour-Infiltrating Lymphocytes (TIL) produced after expansion in the compartmentalised bag versus TIL produced using the standard process in plates. Proliferation yield, viability, phenotype and IFNγ secretion were comparatively studied. Results We found no differences in proliferation yield and cell viability between both TIL production systems. Moreover, each of the cell products complied with our defined release criteria before being administered to the patient. The phenotype analysis indicated that the compartmentalised bag favours the expansion of CD8+ cells. Finally, we found that TIL stimulated in bags were enriched in reactive CD8+ T cells when co-cultured with the autologous melanoma cell line. Conclusions The stimulation of TIL with feeder cells in the specifically designed compartmentalised bag can advantageously replace the conventional protocol using plates. In particular, the higher expansion rate of reactive CD8+ T cells could have a significant impact for ACT.