Hervé Luche

Intestinal Lamina Propria Dendritic Cell Subsets Have Different Origin and Functions

The intestinal immune system discriminates between tolerance toward the commensal microflora and robust responses to pathogens. Maintenance of this critical balance is attributed to mucosal dendritic cells (DCs) residing in organized lymphoid tissue and dispersed in the subepithelial lamina propria. In situ parameters of lamina propria DCs (lpDCs) remain poorly understood. Here, we combined conditional cell ablation and precursor-mediated in vivo reconstitution to establish that lpDC subsets have distinct origins and functions. CD103(+) CX(3)CR1(-) lpDCs arose from macrophage-DC precursors (MDPs) via DC-committed intermediates (pre-cDCs) through a Flt3L growth-factor-mediated pathway. CD11b(+) CD14(+) CX(3)CR1(+) lpDCs were derived from grafted Ly6C(hi) but not Ly6C(lo) monocytes under the control of GM-CSF. Mice reconstituted exclusively with CX(3)CR1(+) lpDCs when challenged in an innate colitis model developed severe intestinal inflammation that was driven by graft-derived TNF-alpha-secreting CX(3)CR1(+) lpDCs. Our results highlight the critical importance of the lpDC subset balance for robust gut homeostasis.

Faithful activation of an extra-bright red fluorescent protein in "knock-in" Cre-reporter mice ideally suited for lineage tracing studies.

The considerable potential of Cre recombinase as a tool for in vivo fate‐mapping studies depends on the availability of reliable reporter mice. By targeting a tandem‐dimer red fluorescent protein (tdRFP) with advanced spectral and biological properties into the ubiquitously expressed ROSA26 locus of C57BL/6‐ES cells, we have generated a novel inbred Cre‐reporter mouse with several unique characteristics. We directly demonstrate the usefulness of our reporter strain in inter‐crosses with a “universal Cre‐deleter” strain and with mice expressing Cre recombinase in a T lineage‐specific manner. Cytofluorometric and histological analyses illustrate: (i) non‐toxicity and extraordinary brightness of the fluorescent reporter, allowing quantitative detection and purification of labeled cells with highest accuracy, (ii) reliable Cre‐mediated activation of tdRFP from an antisense orientation relative to ROSA26 transcription, effectively excluding “leaky” reporter expression, (iii) absence of gene expression variegation effects, (iv) quantitative detection of tdRFP‐expressing cells even in paraformaldehyde‐fixed tissue sections, and (v) full compatibility with GFP/YFP‐based fluorescent markers in multicolor experiments. Taken together, the data show that our C57BL/6‐inbred reporter mice are ideally suited for sophisticated lineage‐tracing experiments requiring sensitive and quantitative detection/purification of live Cre‐expressing cells and their progeny.

Identification and targeting of the ROSA26 locus in human embryonic stem cells.

The derivation of human embryonic stem (hES) cells has opened new avenues for studies on human development and provided a potential source of cells for replacement therapy. To reveal the full potential of hES cells, it would be advantageous to be able to genetically alter them as is routinely done with mouse ES cells through homologous recombination. The mouse Rosa26 locus is particularly useful for genetic modification as it can be targeted with high efficiency and is expressed in most cell types tested. Here we report the identification of the human homolog of the mouse Rosa26 locus. We demonstrate targeting of a red-fluorescent protein (tdRFP) cDNA to this locus through homologous recombination and expression of this targeted reporter in multiple hES cell–derived lineages. Through recombinase-mediated cassette exchange, we show replacement of the tdRFP cDNA with other cDNAs, providing a cell line in which transgenes can be readily introduced into a broadly expressed locus.

Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor.

NKp46 is a cell surface receptor expressed on natural killer (NK) cells, on a minute subset of T cells, and on a population of innate lymphoid cells that produce IL-22 and express the transcription factor retinoid-related orphan receptor (ROR)-γt, referred to as NK cell receptor (NKR)+ROR-γt+ cells. Here we describe Nkp46iCre knock-in mice in which the gene encoding the improved Cre (iCre) recombinase was inserted into the Nkp46 locus. This mouse was used to noninvasively trace cells expressing NKp46 in vivo. Fate mapping experiments demonstrated the stable expression of NKp46 on NK cells and allowed a reappraisal of the sequential steps of NK cell maturation. NKp46 genetic tracing also showed that gut NKR+ROR-γt+ and NK cells represent two distinct lineages. In addition, the genetic heterogeneity of liver NK cells was evidenced. Finally, Nkp46iCre mice also represent a unique mouse model of conditional mutagenesis specifically in NKp46+ cells, paving the way for further developments in the biology of NKp46+ NK, T, and NKR+ROR-γt+ cells.

CD64 Expression Distinguishes Monocyte-Derived and Conventional Dendritic Cells and Reveals Their Distinct Role during Intramuscular Immunization

Although 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.

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

Summary 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.

Deletion of Notch1 Converts Pro-T Cells to Dendritic Cells and Promotes Thymic B Cells by Cell-Extrinsic and Cell-Intrinsic Mechanisms

Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

Cutting Edge: Expression of XCR1 Defines Mouse Lymphoid-Tissue Resident and Migratory Dendritic Cells of the CD8α+ Type

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.

Preferential localization of IgG memory B cells adjacent to contracted germinal centers

It has long been presumed that after leaving the germinal centers (GCs), memory B cells colonize the marginal zone or join the recirculating pool. Here we demonstrate the preferential localization of nitrophenol-chicken γ-globulin-induced CD38+IgG1+ memory B cells adjacent to contracted GCs in the spleen. The memory B cells in this region proliferated after secondary immunization, a response that was abolished by depletion of CD4+ T cells. We also found that these IgG1+ memory B cells could present antigen on their surface, and that this activity was required for their activation. These results implicate this peri-GC region as an important site for survival and reactivation of memory B cells.

Multicolor fate mapping of Langerhans cell homeostasis

The adult epidermal Langerhans cell network is formed by adjacent proliferative units composed of dividing cells and their terminally differentiated daughter cells.