VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites.

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.

Azithromycin promotes relapse by disrupting immune and metabolic networks after allogeneic stem cell transplantation.

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.

Human inflammatory dendritic cells induce Th17 cell differentiation.

Dendritic 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.

X-linked primary immunodeficiency associated with hemizygous mutations in the moesin (MSN) gene.

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.

Human Primary Immune Cells Exhibit Distinct Mechanical Properties that Are Modified by Inflammation.

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.

MAIT cells detect and efficiently lyse bacterially-infected epithelial cells.

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.

Cytokine secretion by CD4+ T cells at the immunological synapse requires Cdc42-dependent local actin remodeling but not microtubule organizing center polarity.

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.

SUV39H1 Ablation Enhances Long-term CAR T Function in Solid Tumors.

Failure of adoptive T-cell therapies in patients with cancer is linked to limited T-cell expansion and persistence, even in memory-prone 41BB-(BBz)-based chimeric antigen receptor (CAR) T cells. We show here that BBz-CAR T-cell stem/memory differentiation and persistence can be enhanced through epigenetic manipulation of the histone 3 lysine 9 trimethylation (H3K9me3) pathway. Inactivation of the H3K9 trimethyltransferase SUV39H1 enhances BBz-CAR T cell long-term persistence, protecting mice against tumor relapses and rechallenges in lung and disseminated solid tumor models up to several months after CAR T-cell infusion. Single-cell transcriptomic (single-cell RNA sequencing) and chromatin opening (single-cell assay for transposase accessible chromatin) analyses of tumor-infiltrating CAR T cells show early reprogramming into self-renewing, stemlike populations with decreased expression of dysfunction genes in all T-cell subpopulations. Therefore, epigenetic manipulation of H3K9 methylation by SUV39H1 optimizes the long-term functional persistence of BBz-CAR T cells, limiting relapses, and providing protection against tumor rechallenges. SIGNIFICANCE: Limited CAR T-cell expansion and persistence hinders therapeutic responses in solid cancer patients. We show that targeting SUV39H1 histone methyltransferase enhances 41BB-based CAR T-cell long-term protection against tumor relapses and rechallenges by increasing stemness/memory differentiation. This opens a safe path to enhancing adoptive cell therapies for solid tumors. See related article by Jain et al., p. 142. This article is featured in Selected Articles from This Issue, p. 5.

Human MAIT cells inhibit alloreactive T cell responses and protect against acute graft-versus-host disease.

Adoptive transfer of immunoregulatory cells can prevent or ameliorate graft-versus-host disease (GVHD), which remains the main cause of nonrelapse mortality after allogeneic hematopoietic stem cell transplantation. Mucosal-associated invariant T (MAIT) cells were recently associated with tissue repair capacities and with lower rates of GVHD in humans. Here, we analyzed the immunosuppressive effect of MAIT cells in an in vitro model of alloreactivity and explored their adoptive transfer in a preclinical xenogeneic GVHD model. We found that MAIT cells, whether freshly purified or short-term expanded, dose-dependently inhibited proliferation and activation of alloreactive T cells. In immunodeficient mice injected with human PBMCs, MAIT cells greatly delayed GVHD onset and decreased severity when transferred early after PBMC injection but could also control ongoing GVHD when transferred at delayed time points. This effect was associated with decreased proliferation and effector function of human T cells infiltrating tissues of diseased mice and was correlated with lower circulating IFN-γ and TNF-α levels and increased IL-10 levels. MAIT cells acted partly in a contact-dependent manner, which likely required direct interaction of their T cell receptor with MHC class I-related molecule (MR1) induced on host-reactive T cells. These results support the setup of clinical trials using MAIT cells as universal therapeutic tools to control severe GVHD or mucosal inflammatory disorders.

Crosstalk between T lymphocytes and dendritic cells.

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.

Integration of intermittent calcium signals in T cells revealed by temporally patterned optogenetics.

T cells become activated following one or multiple contacts with antigen-presenting cells. Calcium influx is a key signaling event elicited during these cellular interactions; however, it is unclear whether T cells recall and integrate calcium signals elicited during temporally separated contacts. To study the integration of calcium signals, we designed a programmable, multiplex illumination strategy for temporally patterned optogenetics (TEMPO). We found that a single round of calcium elevation was insufficient to promote nuclear factor of activated T cells (NFAT) activity and cytokine production in a T cell line. However, robust responses were detected after a second identical stimulation even when signals were separated by several hours. Our results suggest the existence of a biochemical memory of calcium signals in T cells that favors signal integration during temporally separated contacts and promote cytokine production. As illustrated here, TEMPO is a versatile approach for dissecting temporal integration in defined signaling pathways.

T cell polarity at the immunological synapse is required for CD154-dependent IL-12 secretion by dendritic cells.

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.

Mucosal-associated invariant T (MAIT) cells, a new source of universal immune cells for chimeric antigen receptor (CAR)-cell therapy.

Treatment of hematological malignancies by autologous T cells expressing a chimeric antigen receptor (CAR) is a breakthrough in the field of cancer immunotherapy. As CAR-T cells are entering advanced phases of clinical development, there is a need to develop universal, ready-to-use products using immune cells from healthy donors, to reduce time to treatment, improve response rate and finally reduce the cost of production. Mucosal-associated invariant T cells (MAIT) are unconventional T cells which recognize microbial-derived riboflavin derivatives presented by the conserved MR1 molecule and are endowed with potent effector functions. Because they are not selected by classical MHC/peptide complexes and express a semi-invariant T cell receptor, MAIT cells do not mediate alloreactivity, prompting their use as a new source of universal effector cells for allogeneic CAR-T cell therapy without the need to inactivate their endogenous TCR. We produced CD19-CAR MAIT cells as proof-of-concept allowing subsequent head-to-head comparison with currently used CD19-CAR T cells. We demonstrated their anti-tumor efficacy in vitro and their capacity to engraft without mediating GVHD in preclinical immunodeficient mouse models. Universal, off-the-shelf CAR-MAIT cells could provide a suitable alternative to current autologous CAR-T cells to treat patients regardless of HLA disparity, without production delay, enabling a cost-effective manufacturing model for large-scale clinical application.

Human MAIT cells are devoid of alloreactive potential: prompting their use as universal cells for adoptive immune therapy.

BACKGROUND: Mucosal-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 antimicrobial defense. MAIT cells are very few at birth and accumulate gradually over a period of about 6 years during the 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. METHODS: We used ex vivo, in vitro and in vivo models to determine if human MAIT cells contribute to allogeneic responses. RESULTS: We show that recovery of MAIT cells after allogeneic hematopoietic stem cell transplantation recapitulates their slow physiological expansion in early childhood, independent of recovery of non-MAIT 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 in immunodeficient mice. CONCLUSIONS: Altogether, 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. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number NCT02403089.

Optogenetic manipulation of calcium signals in single T cells in vivo.

By offering the possibility to manipulate cellular functions with spatiotemporal control, optogenetics represents an attractive tool for dissecting immune responses. However, applying these approaches to single cells in vivo remains particularly challenging for immune cells that are typically located in scattering tissues. Here, we introduce an improved calcium actuator with sensitivity allowing for two-photon photoactivation. Furthermore, we identify an actuator/reporter combination that permits the simultaneous manipulation and visualization of calcium signals in individual T cells in vivo. With this strategy, we document the consequences of defined patterns of calcium signals on T cell migration, adhesion, and chemokine release. Manipulation of individual immune cells in vivo should open new avenues for establishing the functional contribution of single immune cells engaged in complex reactions.

Termination of T cell priming relies on a phase of unresponsiveness promoting disengagement from APCs and T cell division.

T cells are primed in secondary lymphoid organs by establishing stable interactions with antigen-presenting cells (APCs). However, the cellular mechanisms underlying the termination of T cell priming and the initiation of clonal expansion remain largely unknown. Using intravital imaging, we observed that T cells typically divide without being associated to APCs. Supporting these findings, we demonstrate that recently activated T cells have an intrinsic defect in establishing stable contacts with APCs, a feature that was reflected by a blunted capacity to stop upon T cell receptor (TCR) engagement. T cell unresponsiveness was caused, in part, by a general block in extracellular calcium entry. Forcing TCR signals in activated T cells antagonized cell division, suggesting that T cell hyporesponsiveness acts as a safeguard mechanism against signals detrimental to mitosis. We propose that transient unresponsiveness represents an essential phase of T cell priming that promotes T cell disengagement from APCs and favors effective clonal expansion.

T-lymphocyte passive deformation is controlled by unfolding of membrane surface 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.

Force generation upon T cell receptor engagement.

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.