Single-cell transcriptomics uncovers an instructive T-cell receptor role in adult γδ T-cell lineage commitment.

After entering the adult thymus, bipotent T-cell progenitors give rise to αß or γδ T cells. To determine whether the γδ T-cell receptor (TCR) has an instructive role in γδ T-cell lineage commitment or only "confirms" a pre-established γδ Τ-cell lineage state, we exploited mice lacking expression of LAT, an adaptor required for γδ TCR signaling. Although these mice showed a T-cell development block at the CD4- CD8- double-negative third (DN3) stage, 0.3% of their DN3 cells expressed intermediate levels of γδ TCR (further referred to as γδint ) at their surface. Single-cell transcriptomics of LAT-deficient DN3 γδint cells demonstrated no sign of commitment to the γδ T-cell lineage, apart from γδ TCR expression. Although the lack of LAT is thought to tightly block DN3 cell development, we unexpectedly found that 25% of LAT-deficient DN3 γδint cells were actively proliferating and progressed up to the DN4 stage. However, even those cells failed to turn on the transcriptional program associated with the γδ T-cell lineage. Therefore, the γδ TCR-LAT signaling axis builds upon a γδ T-cell uncommitted lineage state to fully instruct adult γδ T-cell lineage specification.

Diversity of innate immune cell subsets across spatial and temporal scales in an EAE mouse model.

In both multiple sclerosis and its model experimental autoimmune encephalomyelitis (EAE), the extent of resident microglia activation and infiltration of monocyte-derived cells to the CNS is positively correlated to tissue damage. To address the phenotype characterization of different cell subsets, their spatio-temporal distributions and contributions to disease development we induced EAE in Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined high content flow cytometry, immunofluorescence and two-photon imaging in live mice and identified a stepwise program of inflammatory cells accumulation. First on day 10 after induction, EGFP+ neutrophils and monocytes invade the spinal cord parenchyma through the meninges rather than by extravasion. This event occurs just before axonal losses in the white matter. Once in the parenchyma, monocytes mature into EGFP+/EYFP+ monocyte-derived dendritic cells (moDCs) whose density is maximal on day 17 when the axonal degradation and clinical signs stabilize. Meanwhile, microglia is progressively activated in the grey matter and subsequently recruited to plaques to phagocyte axon debris. LysM-EGFP//CD11c-EYFP mice appear as a powerful tool to differentiate moDCs from macrophages and to study the dynamics of immune cell maturation and phenotypic evolution in EAE.

Broad and Largely Concordant Molecular Changes Characterize Tolerogenic and Immunogenic Dendritic Cell Maturation in Thymus and Periphery.

Dendritic cells (DCs) are instrumental in the initiation of T cell responses, but how thymic and peripheral tolerogenic DCs differ globally from Toll-like receptor (TLR)-induced immunogenic DCs remains unclear. Here, we show that thymic XCR1(+) DCs undergo a high rate of maturation, accompanied by profound gene-expression changes that are essential for central tolerance and also happen in germ-free mice. Those changes largely overlap those occurring during tolerogenic and, more unexpectedly, TLR-induced maturation of peripheral XCR1(+) DCs, arguing against the commonly held view that tolerogenic DCs undergo incomplete maturation. Interferon-stimulated gene (ISG) expression was among the few discriminators of immunogenic and tolerogenic XCR1(+) DCs. Tolerogenic XCR1(+) thymic DCs were, however, unique in expressing ISGs known to restrain virus replication. Therefore, a broad functional convergence characterizes tolerogenic and immunogenic XCR1(+) DC maturation in the thymus and periphery, maximizing antigen presentation and signal delivery to developing and to conventional and regulatory mature T cells.

Cytomegalovirus Infection of the Rat Developing Brain In Utero Prominently Targets Immune Cells and Promotes Early Microglial Activation.

BACKGROUND: Congenital cytomegalovirus infections are a leading cause of neurodevelopmental disorders in human and represent a major health care and socio-economical burden. In contrast with this medical importance, the pathophysiological events remain poorly known. Murine models of brain cytomegalovirus infection, mostly neonatal, have brought recent insights into the possible pathogenesis, with convergent evidence for the alteration and possible involvement of brain immune cells. OBJECTIVES AND METHODS: In order to confirm and expand those findings, particularly concerning the early developmental stages following infection of the fetal brain, we have created a model of in utero cytomegalovirus infection in the developing rat brain. Rat cytomegalovirus was injected intraventricularly at embryonic day 15 (E15) and the brains analyzed at various stages until the first postnatal day, using a combination of gene expression analysis, immunohistochemistry and multicolor flow cytometry experiments. RESULTS: Rat cytomegalovirus infection was increasingly seen in various brain areas including the choroid plexi and the ventricular and subventricular areas and was prominently detected in CD45low/int, CD11b+ microglial cells, in CD45high, CD11b+ cells of the myeloid lineage including macrophages, and in CD45+, CD11b- lymphocytes and non-B non-T cells. In parallel, rat cytomegalovirus infection of the developing rat brain rapidly triggered a cascade of pathophysiological events comprising: chemokines upregulation, including CCL2-4, 7 and 12; infiltration by peripheral cells including B-cells and monocytes at E17 and P1, and T-cells at P1; and microglia activation at E17 and P1. CONCLUSION: In line with previous findings in neonatal murine models and in human specimen, our study further suggests that neuroimmune alterations might play critical roles in the early stages following cytomegalovirus infection of the brain in utero. Further studies are now needed to determine which role, whether favorable or detrimental, those putative double-edge swords events actually play.

Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice.

Inflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP(+) cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP(+) cells. Fluorescent invading cells were conventional XCR1(+) and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP(+)/EYFP(+) monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response.

Barcoding T cell calcium response diversity with methods for automated and accurate analysis of cell signals (MAAACS).

We introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells.

A hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia.

Using N-ethyl-N-nitrosourea-induced mutagenesis, we established a mouse model with a novel form of neutropenia resulting from a point mutation in the transcriptional repressor Growth Factor Independence 1 (Gfi1). These mice, called Genista, had normal viability and no weight loss, in contrast to mice expressing null alleles of the Gfi1 gene. Furthermore, the Genista mutation had a very limited impact on lymphopoiesis or on T- and B-cell function. Within the bone marrow (BM), the Genista mutation resulted in a slight increase of monopoiesis and in a block of terminal granulopoiesis. This block occurred just after the metamyelocytic stage and resulted in the generation of small numbers of atypical CD11b(+) Ly-6G(int) neutrophils, the nuclear morphology of which resembled that of mature WT neutrophils. Unexpectedly, once released from the BM, these atypical neutrophils contributed to induce mild forms of autoantibody-induced arthritis and of immune complex-mediated lung alveolitis. They additionally failed to provide resistance to acute bacterial infection. Our study demonstrates that a hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia characterized by a split pattern of functional responses, reflecting the distinct thresholds required for eliciting neutrophil-mediated inflammatory and anti-infectious responses.

Skin-draining lymph nodes contain dermis-derived CD103(-) dendritic cells that constitutively produce retinoic acid and induce Foxp3(+) regulatory T cells.

Small intestinal CD103(+) dendritic cells (DCs) have the selective ability to promote de novo generation of regulatory T cells via the production of retinoic acid (RA). Considering that aldehyde dehydrogenase (ALDH) activity controls the production of RA, we used a flow cytometry-based assay to measure ALDH activity at the single-cell level and to perform a comprehensive analysis of the RA-producing DC populations present in lymphoid and nonlymphoid mouse tissues. RA-producing DCs were primarily of the tissue-derived, migratory DC subtype and can be readily found in the skin and in the lungs as well as in their corresponding draining lymph nodes. The RA-producing skin-derived DCs were capable of triggering the generation of regulatory T cells, a finding demonstrating that the presence of RA-producing, tolerogenic DCs is not restricted to the intestinal tract as previously thought. Unexpectedly, the production of RA by skin DCs was restricted to CD103(-) DCs, indicating that CD103 expression does not constitute a "universal" marker for RA-producing mouse DCs. Finally, Toll-like receptor (TLR) triggering or the presence of a commensal microflora was not essential for the induction of ALDH activity in the discrete ALDH(+) DC subsets that characterize tissues constituting environmental interfaces.

Agricultural pesticide exposure and the molecular connection to lymphomagenesis.

The t(14;18) translocation constitutes the initiating event of a causative cascade leading to follicular lymphoma (FL). t(14;18) translocations are present in blood from healthy individuals, but there is a trend of increased prevalence in farmers exposed to pesticides, a group recently associated with higher risk of t(14;18)(+) non-Hodgkin's lymphoma development. A direct connection between agricultural pesticide use, t(14;18) in blood, and malignant progression, however, has not yet been demonstrated. We followed t(14;18) clonal evolution over 9 yr in a cohort of farmers exposed to pesticides. We show that exposed individuals bear particularly high t(14;18) frequencies in blood because of a dramatic clonal expansion of activated t(14;18)(+) B cells. We further demonstrate that such t(14;18)(+) clones recapitulate the hallmark features of developmentally blocked FL cells, with some displaying aberrant activation-induced cytidine deaminase activity linked to malignant progression. Collectively, our data establish that expanded t(14;18)(+) clones constitute bona fide precursors at various stages of FL development, and provide a molecular connection between agricultural pesticide exposure, t(14;18) frequency in blood, and clonal progression.

Follicular lymphoma-like B cells in healthy individuals: a novel intermediate step in early lymphomagenesis.

Follicular lymphoma is one of the most common adult lymphoma, and remains virtually incurable despite its relatively indolent nature. t(14;18)(q32;q21) translocation, the genetic hallmark and early initiating event of follicular lymphoma (FL) pathogenesis, is also present at low frequency in the peripheral blood of healthy individuals. It has long been assumed that in healthy individuals t(14;18) is carried by circulating quiescent naive B cells, where its oncogenic potential would be restrained. Here, we question this current view and demonstrate that in healthy individuals, t(14;18) is actually carried by an expanding population of atypical B cells issued from germinal centers, displaying genotypic and phenotypic features of FL, and prone to constitute potent premalignant FL niches. These findings strongly impact both on the current understanding of disease progression and on the proper handling of t(14;18) frequency in blood as a potential early biomarker for lymphoma.

IL-2 therapy and thymic production of naive CD4 T cells in HIV-infected patients with severe CD4 lymphopenia.

OBJECTIVES: IL-2 therapy increases memory and naive CD4 T cells in HIV-infected patients, but its effect on thymopoiesis is unknown. To investigate this effect, we quantified T-cell receptor rearrangement excision circles (TREC) in CD4 T cells from lymphopenic AIDS patients treated with highly active antiretroviral therapy and IL-2. METHODS: CD4 cell subsets were evaluated by flow cytometry using anti-CD45RO/RA, CD62L, Ki67 and CD95 monoclonal antibodies. The proportion of recent thymic emigrant had been quantified by a real-time polymerase chain reaction assay for signal joint TREC in peripheral blood mononuclear and purified CD4 T cells. RESULTS: At initiation of IL-2, TREC copies/microl of blood were correlated with naive T cell numbers and age. Both naive and TREC numbers/microl significantly increased over time in all patients, with a wide range of TREC increases. Higher percentages of CD4+CD45RO-negative cells positive for the Ki67 cell-cycle marker were found in patients with a low TREC increase, but remained stable under IL-2. TREC and naive cell recovery were correlated; they also correlated with the numbers of TREC and naive cells at the start of IL-2, and with age, suggesting a thymic origin for naive T-cell recovery. A mathematical model showing the linear recovery of naive cells and TREC under IL-2 also strongly suggested that a naive T-cell increase reflects thymic export and involves little net death and proliferation. CONCLUSION: Although we cannot rule out a mechanism of altered proliferation or death rate, the thymus plays an important role in the long-term recovery of naive T cells under IL-2 therapy.