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

International audienceIn 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

Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species.

Dendritic cells (DCs) are professional antigen-presenting cells that hold great therapeutic potential. Multiple DC subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. Here, we provide and validate a universal toolbox for the automated identification of DCs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. The use of a minimal set of lineage-imprinted markers was sufficient to subdivide DCs into conventional type 1 (cDC1s), conventional type 2 (cDC2s), and plasmacytoid DCs (pDCs) across tissues and species. This way, a large number of additional markers can still be used to further characterize the heterogeneity of DCs across tissues and during inflammation. This framework represents the way forward to a universal, high-throughput, and standardized analysis of DC populations from mutant mice and human patients

Towards the identification of the thymus seeding progenitor: "fate mapping" of early T-cell stages using gene "knock-in" strategies

The pTa gene encodes the pre-T cell receptor alpha chain, an essential component of the pre-TCR complex. Using pTa expression as a specific marker for early T lineage cells, we developed a lineage-tracing strategy to visualize T lineage-committed precursor cells and their progeny. A cDNA encoding an improved version of the Cre recombinase (iCre) was targeted into the pre-T cell receptor alpha-chain (pTa) gene locus. In parallel, we introduced a Cre-inducible expression cassette encoding for a very bright variant of red fluorescent protein (tdRFP) into the ubiquitously expressed mouse locus ROSA26. We generated pTa-iCre deleter and ROSA26-tdRFP indicator mouse strains, respectively. In cells bearing both "knock-in" alleles, pTa-driven iCre recombinase expression leads to permanent activation of the ROSA26-tdRFP reporter locus. As a result, pTa-expressing cells and their progeny exhibited persistent red fluorescence. Red fluorescence was detected in cells considered to belong developmentally to the T-lineage (gd, ab, natural killer-T cells, but not in other hematopoietic cells, demonstrating the exquisite specificity of pTa expression. Exclusive reporter activation in T but not B-cells indicated that onset of pTa-iCre expression occurs after the T/B lineage decision. Over 95% of gd-T cells were tdRFP-positive, demonstrating for the first time that almost all gd-T cells develop - like TCRab lymphocytes - from a pTa-expressing precursor. The extent of T-cell labeling in athymic pTa-iCre x ROSA26-RFP nude mice indicate that pTa is also expressed during extrathymic T-cell development. In the thymus, onset of reporter activation was found at the TN2 to TN3 stage but not in the most immature population (ETP). Finally, red fluorescence was detected in a minute subset of Lin- bone marrow cells. pTa-iCre deleter mice should be useful for both efficient T-lineage-specific genome manipulations and identification of pTa-expressing T precursor cells in extra-thymic tissues

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

International audienceAfter entering the adult thymus, bipotent T-cell progenitors give rise to ab or cd T cells. To determine whether the cd T-cell receptor (TCR) has an instructive role in cd T-cell lineage commitment or only "confirms" a pre-established cd Τ-cell lineage state, we exploited mice lacking expression of LAT, an adaptor required for cd TCR signaling. Although these mice showed a T-cell development block at the CD4 À CD8 À double-negative third (DN3) stage, 0.3% of their DN3 cells expressed intermediate levels of cd TCR (further referred to as cd int) at their surface. Single-cell transcriptomics of LAT-deficient DN3 cd int cells demonstrated no sign of commitment to the cd T-cell lineage, apart from cd TCR expression. Although the lack of LAT is thought to tightly block DN3 cell development, we unexpectedly found that 25% of LAT-deficient DN3 cd int 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 cd T-cell lineage. Therefore, the cd TCR-LAT signaling axis builds upon a cd T-cell uncommitted lineage state to fully instruct adult cd T-cell lineage specification

The earliest intrathymic precursors of CD8α(+) thymic dendritic cells correspond to myeloid-type double-negative 1c cells.

International audienceThe 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

CD64 expression distinguishes monocyte-derived and conventional dendritic cells and reveals their distinct role during intramuscular immunization.

International audienceAlthough most vaccines are administered i.m., little is known about the dendritic cells (DCs) that are present within skeletal muscles. In this article, we show that expression of CD64, the high-affinity IgG receptor FcγRI, distinguishes conventional DCs from monocyte-derived DCs (Mo-DCs). By using such a discriminatory marker, we defined the distinct DC subsets that reside in skeletal muscles and identified their migratory counterparts in draining lymph nodes (LNs). We further used this capability to analyze the functional specialization that exists among muscle DCs. After i.m. administration of Ag adsorbed to alum, we showed that alum-injected muscles contained large numbers of conventional DCs that belong to the CD8α(+)- and CD11b(+)-type DCs. Both conventional DC types were capable of capturing Ag and of migrating to draining LNs, where they efficiently activated naive T cells. In alum-injected muscles, Mo-DCs were as numerous as conventional DCs, but only a small fraction migrated to draining LNs. Therefore, alum by itself poorly induces Mo-DCs to migrate to draining LNs. We showed that addition of small amounts of LPS to alum enhanced Mo-DC migration. Considering that migratory Mo-DCs had, on a per cell basis, a higher capacity to induce IFN-γ-producing T cells than conventional DCs, the addition of LPS to alum enhanced the overall immunogenicity of Ags presented by muscle-derived DCs. Therefore, a full understanding of the role of adjuvants during i.m. vaccination needs to take into account the heterogeneous migratory and functional behavior of muscle DCs and Mo-DCs revealed in this study

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

International audienceInflammatory 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

Immunology: Egocentric pre-T-cell receptors.

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