Search

Administration of azithromycin after allogeneic hematopoietic stem cell transplantation for hematologic malignancies has been associated with relapse in a randomized phase 3 controlled clinical trial. Studying 240 samples from patients randomized in this trial is a unique opportunity to better understand the mechanisms underlying relapse, the first cause of mortality after transplantation. We used multi-omics on patients’ samples to decipher immune alterations associated with azithromycin intake and post-transplantation relapsed malignancies. Azithromycin was associated with a network of altered energy metabolism pathways and immune subsets, including T cells biased toward immunomodulatory and exhausted profiles. In vitro, azithromycin exposure inhibited T-cell cytotoxicity against tumor cells and impaired T-cell metabolism through glycolysis inhibition, down-regulation of mitochondrial genes, and up-regulation of immunomodulatory genes, notably SOCS1. These results highlight that azithromycin directly affects immune cells that favor relapse, which raises caution about long-term use of azithromycin treatment in patients at high risk of malignancies. The ALLOZITHRO trial was registered at www.clinicaltrials.gov as #NCT01959100.

BackgroundMucosal associated invariant T (MAIT) cells are semi-invariant T cells that recognize microbial antigens presented by the highly conserved MR1 molecule. MAIT cells are predominantly localized in the liver and barrier tissues and are potent effectors of anti - microbial defense. MAIT cells are very few at birth and accumulate gradually over a period of about 6 years during infancy. The cytotoxic potential of MAIT cells, as well as their newly described regulatory and tissue repair functions, open the possibility of exploiting their properties in adoptive therapy. A prerequisite for their use as “universal” cells would be a lack of alloreactive potential, which remains to be demonstrated.MethodsWe used ex vivo, in vitro and in vivo models to determine if human MAIT cells contribute to allogeneic responses.ResultsWe show that recovery of MAIT cells after allogeneic hematopoietic stem cell transplantation recapitulates their slow physiological expansion in early childhood, independent of recovery of conventional T cells. In vitro, signals provided by allogeneic cells and cytokines do not induce sustained MAIT cell proliferation. In vivo, human MAIT cells do not expand nor accumulate in tissues in a model of T-cell mediated xenogeneic graft-versus-host disease (GVHD) in immunodeficient mice.ConclusionsAltogether, these results provide evidence that MAIT cells are devoid of alloreactive potential and pave the way for harnessing their translational potential in universal adoptive therapy overcoming barriers of HLA disparity.

T-lymphocyte passive deformation when squeezing through narrow capillaries is limited by the excess membrane contained in microvilli and membrane folds. During active processes such as transendothelial migration, larger deformations are made possible by an increase in membrane area, possibly through recruitment of internal membrane reservoirs., T-lymphocytes in the human body routinely undergo large deformations, both passively, when going through narrow capillaries, and actively, when transmigrating across endothelial cells or squeezing through tissue. We investigate physical factors that enable and limit such deformations and explore how passive and active deformations may differ. Employing micropipette aspiration to mimic squeezing through narrow capillaries, we find that T-lymphocytes maintain a constant volume while they increase their apparent membrane surface area upon aspiration. Human resting T-lymphocytes, T-lymphoblasts, and the leukemic Jurkat T-cells all exhibit membrane rupture above a critical membrane area expansion that is independent of either micropipette size or aspiration pressure. The unfolded membrane matches the excess membrane contained in microvilli and membrane folds, as determined using scanning electron microscopy. In contrast, during transendothelial migration, a form of active deformation, we find that the membrane surface exceeds by a factor of two the amount of membrane stored in microvilli and folds. These results suggest that internal membrane reservoirs need to be recruited, possibly through exocytosis, for large active deformations to occur.

Cytokine secretion by T lymphocytes plays a central role in mounting adaptive immune responses. However, little is known about how newly synthesized cytokines, once produced, are routed within T cells and about the mechanisms involved in regulating their secretions. In this study, we investigated the role of cytoskeleton remodeling at the immunological synapse (IS) in cytokine secretion. We show that a key regulator of cytoskeleton remodeling, the Rho GTPase Cdc42, controls IFN-γ secretion by primary human CD4+ T lymphocytes. Surprisingly, microtubule organizing center polarity at the IS, which does not depend on Cdc42, is not required for cytokine secretion by T lymphocytes, whereas microtubule polymerization is required. In contrast, actin remodeling at the IS, which depends on Cdc42, controls the formation of the polymerized actin ring at the IS, the dynamic concentration of IFN-γ–containing vesicles inside this ring, and the secretion of these vesicles. These results reveal a previously unidentified role of Cdc42-dependent actin remodeling in cytokine exocytosis at the IS.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) with the unique property of inducing priming and differentiation of naïve CD4+ and CD8+ T cells into helper and cytotoxic effectors. Their efficiency is due to their unique ability to process antigen, express costimulatory molecules, secrete cytokines, and migrate to tissues or lymphoid organs to prime T cells. DCs also play an important role in T-cell peripheral tolerance. There is ample evidence that the DC ability to present antigens is regulated by CD4+ helper T cells. Indeed, interactions between surface receptors and ligands expressed respectively by T cells and DCs, as well as T-cell-derived cytokines modify DC functions. This T-cell-induced modification of DCs has been called "education" or "licensing." This intimate crosstalk between DCs and T lymphocytes is key in establishing appropriate adaptive immune responses. It requires cognate interactions between T lymphocytes and DCs, which are organized in time and space by structures called immunological synapses. Here we discuss the particular aspects of immunological synapses formed between T cells and DCs and the role these organized interactions have in T-cell-DC crosstalk.

Ag-specific interaction between T lymphocytes and dendritic cells (DCs) leads to both T cell and DC activation. CD154 (CD40 ligand)/CD40 interactions have been shown to play a major, although not exclusive, role in this functional cross-talk. Interactions between T cells and DCs are structured by an immunological synapse (IS), characterized by polarization of the T cell microtubule cytoskeleton toward the interacting DCs. Yet the role T cell polarization may play in T cell-induced DC activation is mostly unknown. In this study, we address the role of T cell polarity in CD154-dependent activation of DCs in a human model, using two different tools to block T cell polarity (i.e., a microtubule depolymerizing drug and an inhibitor of atypical protein kinase C). We show that CD154 is recruited and concentrated at the IS formed between human primary T cells and autologous DCs and that this recruitment requires T cell polarity at the IS. Moreover, we show that T cell polarization at the IS controls T cell-dependent CD154–CD40 signaling in DCs as well as CD154-dependent IL-12 secretion by DCs. This study shows that T cell polarity at the IS plays a key role in CD154/CD40-dependent cross-talk between CD4+ T cells and DCs.

International audience; BACKGROUND: We investigated 7 male patients (from 5 different families) presenting with profound lymphopenia, hypogammaglobulinemia, fluctuating monocytopenia and neutropenia, a poor immune response to vaccine antigens, and increased susceptibility to bacterial and varicella zoster virus infections. OBJECTIVE: We sought to characterize the genetic defect involved in a new form of X-linked immunodeficiency. METHODS: We performed genetic analyses and an exhaustive phenotypic and functional characterization of the lymphocyte compartment. RESULTS: We observed hemizygous mutations in the moesin (MSN) gene (located on the X chromosome and coding for MSN) in all 7 patients. Six of the latter had the same missense mutation, which led to an amino acid substitution (R171W) in the MSN four-point-one, ezrin, radixin, moesin domain. The seventh patient had a nonsense mutation leading to a premature stop codon mutation (R533X). The naive T-cell counts were particularly low for age, and most CD8(+) T cells expressed the senescence marker CD57. This phenotype was associated with impaired T-cell proliferation, which was rescued by expression of wild-type MSN. MSN-deficient T cells also displayed poor chemokine receptor expression, increased adhesion molecule expression, and altered migration and adhesion capacities. CONCLUSION: Our observations establish a causal link between an ezrin-radixin-moesin protein mutation and a primary immunodeficiency that could be referred to as X-linked moesin-associated immunodeficiency.

The mechanisms by which Lat (a key adaptor in the T cell antigen receptor (TCR) signaling pathway) and the TCR come together after TCR triggering are not well understood. We investigate here the role of SNARE proteins, which are part of protein complexes involved in the docking, priming and fusion of vesicles with opposing membranes, in this process. Here we found, by silencing approaches and genetically modified mice, that the vesicular SNARE VAMP7 was required for the recruitment of Lat-containing vesicles to TCR-activation sites. Our results indicated that this did not involve fusion of Lat-containing vesicles with the plasma membrane. VAMP7, which localized together with Lat on the subsynaptic vesicles, controlled the phosphorylation of Lat, formation of the TCR-Lat-signaling complex and, ultimately, activation of T cells. Our findings suggest that the transport and docking of Lat-containing vesicles with target membranes containing TCRs regulates TCR-induced signaling.

SummaryDendritic cells (DCs) are critical regulators of immune responses. Under noninflammatory conditions, several human DC subsets have been identified. Little is known, however, about the human DC compartment under inflammatory conditions. Here, we characterize a DC population found in human inflammatory fluids that displayed a phenotype distinct from macrophages from the same fluids and from steady-state lymphoid organ and blood DCs. Transcriptome analysis showed that they correspond to a distinct DC subset and share gene signatures with in vitro monocyte-derived DCs. Moreover, human inflammatory DCs, but not inflammatory macrophages, stimulated autologous memory CD4+ T cells to produce interleukin-17 and induce T helper 17 (Th17) cell differentiation from naive CD4+ T cells through the selective secretion of Th17 cell-polarizing cytokines. We conclude that inflammatory DCs represent a distinct human DC subset and propose that they are derived from monocytes and are involved in the induction and maintenance of Th17 cell responses.

Mucosal associated invariant T cells (MAIT) are innate T lymphocytes that detect a large variety of bacteria and yeasts. This recognition depends on the detection of microbial compounds presented by the evolutionarily conserved major-histocompatibility-complex (MHC) class I molecule, MR1. Here we show that MAIT cells display cytotoxic activity towards MR1 overexpressing non-hematopoietic cells cocultured with bacteria. The NK receptor, CD161, highly expressed by MAIT cells, modulated the cytokine but not the cytotoxic response triggered by bacteria infected cells. MAIT cells are also activated by and kill epithelial cells expressing endogenous levels of MRI after infection with the invasive bacteria Shigella flexneri. In contrast, MAIT cells were not activated by epithelial cells infected by Salmonella enterica Typhimurium. Finally, MAIT cells are activated in human volunteers receiving an attenuated strain of Shigella dysenteriae-1 tested as a potential vaccine. Thus, in humans, MAIT cells are the most abundant T cell subset able to detect and kill bacteria infected cells., Author Summary Human Mucosa-Associated Invariant T cells (MAIT) detect microbe-derived compounds presented by the MHC-like molecule, MR1. These foreign antigens are produced by a wide variety of microbes, including commensal and pathogenic bacteria or yeasts. MAIT cells expend shortly after birth and constitute the major antibacterial T cell subset described and, hence, could play important roles in infectious diseases. Here we show that MAIT cells recognize epithelial cells infected by the intestinal pathogen Shigella flexneri in a process requiring endogenous MR1, while the closely related bacterium Salmonella Tyhpimurium is not. Upon recognition, infected epithelial cells are efficiently lysed by MAIT cells. We also show that the triggering of CD161, a natural killer receptor highly expressed by MAIT cells, can modulate the cytokine but not the cytotoxic function of these cells. Finally, we provide evidence that MAIT cells are activated during the course of an experimental enteric infection in humans. Our study provides important insight on the antibacterial function of MAIT cells and their interaction with pathogenic bacterial species.

International audience; T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca 2+ concentration ([Ca 2+ ] i) was monitored simultaneously to verify the cell commitment in the activation process. [Ca 2+ ] i increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically-regulated activation process.

T lymphocytes are key modulators of the immune response. Their activation requires cell-cell interaction with different myeloid cell populations of the immune system called antigen-presenting cells (APCs). Although T lymphocytes have recently been shown to respond to mechanical cues, in particular to the stiffness of their environment, little is known about the rigidity of APCs. In this study, single-cell microplate assays were performed to measure the viscoelastic moduli of different human myeloid primary APCs, i.e., monocytes (Ms, storage modulus of 520 +90/−80 Pa), dendritic cells (DCs, 440 +110/−90 Pa), and macrophages (MPHs, 900 +110/−100 Pa). Inflammatory conditions modulated these properties, with storage moduli ranging from 190 Pa to 1450 Pa. The effect of inflammation on the mechanical properties was independent of the induction of expression of commonly used APC maturation markers, making myeloid APC rigidity an additional feature of inflammation. In addition, the rigidity of human T lymphocytes was lower than that of all myeloid cells tested and among the lowest reported (Young’s modulus of 85 ± 5 Pa). Finally, the viscoelastic properties of myeloid cells were dependent on both their filamentous actin content and myosin IIA activity, although the relative contribution of these parameters varied within cell types. These results indicate that T lymphocytes face different cell rigidities when interacting with myeloid APCs in vivo and that this mechanical landscape changes under inflammation.