Novel mouse models based on intersectional genetics to identify and characterize plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are the main source of type I interferon (IFN-I) during viral infections. Their other functions are debated, due to a lack of tools to identify and target them in vivo without affecting pDC-like cells and transitional DCs (tDCs), which harbor overlapping phenotypes and transcriptomes but a higher efficacy for T cell activation. In the present report, we present a reporter mouse, pDC-Tom, designed through intersectional genetics based on unique Siglech and Pacsin1 coexpression in pDCs. The pDC-Tom mice specifically tagged pDCs and, on breeding with Zbtb46GFP mice, enabled transcriptomic profiling of all splenic DC types, unraveling diverging activation of pDC-like cells versus tDCs during a viral infection. The pDC-Tom mice also revealed initially similar but later divergent microanatomical relocation of splenic IFN+ versus IFN- pDCs during infection. The mouse models and specific gene modules we report here will be useful to delineate the physiological functions of pDCs versus other DC types.

The activation trajectory of plasmacytoid dendritic cells in vivo during a viral infection.

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN-I). What other functions pDCs exert in vivo during viral infections is controversial, and more studies are needed to understand their orchestration. In the present study, we characterize in depth and link pDC activation states in animals infected by mouse cytomegalovirus by combining Ifnb1 reporter mice with flow cytometry, single-cell RNA sequencing, confocal microscopy and a cognate CD4 T cell activation assay. We show that IFN-I production and T cell activation were performed by the same pDC, but these occurred sequentially in time and in different micro-anatomical locations. In addition, we show that pDC commitment to IFN-I production was marked early on by their downregulation of leukemia inhibitory factor receptor and was promoted by cell-intrinsic tumor necrosis factor signaling. We propose a new model for how individual pDCs are endowed to exert different functions in vivo during a viral infection, in a manner tightly orchestrated in time and space.

Viral infection engenders bona fide and bystander subsets of lung-resident memory B cells through a permissive mechanism.

Lung-resident memory B cells (MBCs) provide localized protection against reinfection in respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. We found that two main transcriptionally distinct subsets of MBCs colonized the lung peribronchial niche after infection. These subsets arose from different progenitors and were both class switched, somatically mutated, and intrinsically biased in their differentiation fate toward plasma cells. Combined analysis of antigen specificity and B cell receptor repertoire segregated these subsets into "bona fide" virus-specific MBCs and "bystander" MBCs with no apparent specificity for eliciting viruses generated through an alternative permissive process. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.

Human germinal center transcriptional programs are de-synchronized in B cell lymphoma.

Most adult B cell lymphomas originate from germinal center (GC) B cells, but it is unclear to what extent B cells in overt lymphoma retain the functional dynamics of GC B cells or are blocked at a particular stage of the GC reaction. Here we used integrative single-cell analysis of phenotype, gene expression and variable-region sequence of the immunoglobulin heavy-chain locus to track the characteristic human GC B cell program in follicular lymphoma B cells. By modeling the cyclic continuum of GC B cell transitional states, we identified characteristic patterns of synchronously expressed gene clusters. GC-specific gene-expression synchrony was lost in single lymphoma B cells. However, distinct follicular lymphoma-specific cell states co-existed within single patient biopsies. Our data show that lymphoma B cells are not blocked in a GC B cell state but might adopt new dynamic modes of functional diversity, which opens the possibility of novel definitions of lymphoma identity.

Class-switched memory B cells remodel BCRs within secondary germinal centers.

Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive rediversification of B cell receptors (BCRs), but the underlying mechanisms remain unresolved. Here, the integrated specificity and function of individual memory B cell progeny revealed ongoing evolution of polyclonal antibody specificities through germinal center (GC)-specific transcriptional activity. At the clonal and subclonal levels, single-cell expression of the genes encoding the costimulatory molecule CD83 and the DNA polymerase Polη segregated the secondary GC transcriptional program into four stages that regulated divergent mechanisms of memory BCR evolution. Our studies demonstrate that vaccine boosts reactivate a cyclic program of GC function in class-switched memory B cells to remodel existing antibody specificities and enhance durable immunological protection.

High-Dimensional Single-Cell Analysis Identifies Organ-Specific Signatures and Conserved NK Cell Subsets in Humans and Mice.

Natural killer (NK) cells are innate lymphoid cells (ILCs) involved in antimicrobial and antitumoral responses. Several NK cell subsets have been reported in humans and mice, but their heterogeneity across organs and species remains poorly characterized. We assessed the diversity of human and mouse NK cells by single-cell RNA sequencing on thousands of individual cells isolated from spleen and blood. Unbiased transcriptional clustering revealed two distinct signatures differentiating between splenic and blood NK cells. This analysis at single-cell resolution identified three subpopulations in mouse spleen and four in human spleen, and two subsets each in mouse and human blood. A comparison of transcriptomic profiles within and between species highlighted the similarity of the two major subsets, NK1 and NK2, across organs and species. This unbiased approach provides insight into the biology of NK cells and establishes a rationale for the translation of mouse studies to human physiology and disease.

Integrative single-cell chromatin and transcriptome analysis of human plasma cell differentiation.

Plasma cells (PC) are highly specialized cells representing the end stage of B cell differentiation. We have shown that PC differentiation can be reproduced in vitro using elaborate culture systems. The molecular changes occurring during PC differentiation are recapitulated in this in vitro differentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drives the early stages of PC differentiation. We combined single cell (sc) RNA-seq and single cell ATAC-seq to decipher the trajectories involved in PC differentiation. ScRNA-seq experiments revealed a strong heterogeneity of the preplasmablastic and plasmablastic stages. Among genes that were commonly identified using scATAC-seq and scRNA-seq, we identified several transcription factors with significant stage specific potential importance in PC differentiation. Interestingly, differentially accessible peaks characterizing the preplasmablastic stage were enriched in motifs of BATF3, FOS and BATF, belonging to the AP-1 transcription factor family, that may represent key transcriptional nodes involved in PCD. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of preplasmablasts already undergone epigenetic remodeling related to PC profile together with UPR activation and are committed to differentiate in PC. These results and the supporting data generated with our in vitro PC differentiation model provide a unique resource for the identification of molecular circuits that are crucial for early and mature plasma cell maturation and biological functions. These data thus provide critical insights into epigenetic- and transcriptional-mediated reprogramming events that sustain PC differentiation.

Heterogeneity of germinal center B cells: New insights from single-cell studies.

Upon antigen exposure, activated B cells in antigen-draining lymphoid organs form microanatomical structures, called germinal centers (GCs), where affinity maturation occurs. Within the GC microenvironment, GC B cells undergo proliferation and B cell receptor (BCR) genes somatic hypermutation in the dark zone (DZ), and affinity-based selection in the light zone (LZ). In the current paradigm of GC dynamics, high-affinity LZ B cells may be selected by cognate T- follicular helper cells to either differentiate into plasma cells or memory B cells, or re-enter the DZ and initiate a new round of proliferation and BCR diversification, before migrating back to the LZ. Given the diversity of cell states and potential cell fates that GC B cells may adopt, the two-state DZ-LZ paradigm has been challenged by studies that explored GC B-cell heterogeneity with a variety of single-cell technologies. Here, we review studies and single-cell technologies which have allowed to refine the working model of GC B-cell cellular and molecular heterogeneity during affinity maturation. This review also covers the use of single-cell quantitative data for mathematical modeling of GC reactions, and the application of single-cell genomics to the study of GC-derived malignancies.

TLR-9 agonist and CD40-targeting vaccination induces HIV-1 envelope-specific B cells with a diversified immunoglobulin repertoire in humanized mice.

The development of HIV-1 vaccines is challenged by the lack of relevant models to accurately induce human B- and T-cell responses in lymphoid organs. In humanized mice reconstituted with human hematopoietic stem cells (hu-mice), human B cell-development and function are impaired and cells fail to efficiently transition from IgM B cells to IgG B cells. Here, we found that CD40-targeted vaccination combined with CpG-B adjuvant overcomes the usual defect of human B-cell switch and maturation in hu-mice. We further dissected hu-B cell responses directed against the HIV-1 Env protein elicited by targeting Env gp140 clade C to the CD40 receptor of antigen-presenting cells. The anti-CD40.Env gp140 vaccine was injected with CpG-B in a homologous prime/boost regimen or as a boost of a NYVAC-KC pox vector encoding Env gp140 clade C. Both regimens elicited Env-specific IgG-switched memory hu-B cells at a greater magnitude in hu-mice primed with NYVAC-KC. Single-cell RNA-seq analysis showed gp140-specific hu-B cells to express polyclonal IgG1 and IgG3 isotypes and a broad Ig VH/VL repertoire, with predominant VH3 family gene usage. These cells exhibited a higher rate of somatic hypermutation than the non-specific IgG+ hu-B-cell counterpart. Both vaccine regimens induced splenic GC-like structures containing hu-B and hu-Tfh-like cells expressing PD-1 and BCL-6. We confirmed in this model that circulating ICOS+ memory hu-Tfh cells correlated with the magnitude of gp140-specific B-cell responses. Finally, the NYVAC-KC heterologous prime led to a more diverse clonal expansion of specific hu-B cells. Thus, this study shows that CD40-targeted vaccination induces human IgG production in hu-mice and provides insights for the development of a CD40-targeting vaccine to prevent HIV-1 infection in humans.

Single-cell RNA sequencing unveils the shared and the distinct cytotoxic hallmarks of human TCRVδ1 and TCRVδ2 γδ T lymphocytes.

γδ T lymphocytes represent ∼1% of human peripheral blood mononuclear cells and even more cells in most tissues of vertebrates. Although they have important anticancer functions, most current single-cell RNA sequencing (scRNA-seq) studies do not identify γδ T lymphocytes because their transcriptomes at the single-cell level are unknown. Here we show that high-resolution clustering of large scRNA-seq datasets and a combination of gene signatures allow the specific detection of human γδ T lymphocytes and identification of their T cell receptor (TCR)Vδ1 and TCRVδ2 subsets in large datasets from complex cell mixtures. In t-distributed stochastic neighbor embedding plots from blood and tumor samples, the few γδ T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVδ1 and TCRVδ2 subsets form close yet distinct subclusters, respectively neighboring NK and CD8 T cells because of expression of shared and distinct cytotoxic maturation genes. Similar pseudotime maturation trajectories of TCRVδ1 and TCRVδ2 γδ T lymphocytes were discovered, unveiling in both subsets an unattended pool of terminally differentiated effector memory cells with preserved proliferative capacity, a finding confirmed by in vitro proliferation assays. Overall, the single-cell transcriptomes of thousands of individual γδ T lymphocytes from different CMV+ and CMV- donors reflect cytotoxic maturation stages driven by the immunological history of donors. This landmark study establishes the rationale for identification, subtyping, and deep characterization of human γδ T lymphocytes in further scRNA-seq studies of complex tissues in physiological and disease conditions.

Integrative molecular profiling of autoreactive CD4 T cells in autoimmune hepatitis.

BACKGROUND & AIMS: In most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-soluble liver antigen (SLA) autoantibodies is associated with reduced overall survival, but the associated autoreactive CD4 T cells have not yet been characterised. Herein, we isolated and deeply characterised SLA-specific CD4 T cells in patients with AIH. METHODS: We used brief ex vivo restimulation with overlapping SLA peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterise their transcriptome and T-cell receptor (TCR) repertoire. Autoreactive TCRs were cloned and used to identify dominant SLA-derived epitopes. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing to identify their phenotypic niche. We further characterised disease-associated peripheral blood T cells by high-content flow cytometry in 42 patients with AIH and 17 controls with non-alcoholic steatohepatitis. RESULTS: Autoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1+CXCR5-CCR6-CD27+ phenotype. ScRNA-seq revealed their pro-inflammatory/B-helper profile. SLA81-100 and SLA177-204 contain dominant T-cell epitopes. Autoreactive TCR clonotypes were predominantly found in the memory PD-1+CXCR5-CD4 T cells, which were significantly increased in the blood of patients with AIH and supported B-cell differentiation through IL-21. Finally, we identified specific T-cell phenotypes linked to disease activity and IgG level during AIH. CONCLUSIONS: We provide a deep characterisation of rare circulating autoreactive CD4 T cells and identify their peripheral reservoir in AIH. We also propose a specific phenotype of autoreactive T cells related to AIH disease activity, which will be essential to track, delineate, and potentially target these pathogenic cells. LAY SUMMARY: One principal characteristic of autoimmune hepatitis (AIH), like for many other autoimmune diseases, is the accumulation of autoantibodies produced by B lymphocytes following their interaction with autoreactive CD4 T lymphocytes. In this study, we identified and characterised with high resolution these CD4 T cells. This will be essential to track, delineate, and potentially target them during AIH.

Premalignant cell dynamics in indolent B-cell malignancies.

PURPOSE OF REVIEW: Follicular lymphoma and chronic lymphocytic leukemia (CLL) are indolent B-cell malignancies characterized by a long preclinical phase and frequent relapses once treatment is initiated. The present review gathers recent findings on the occurrence, relevance, and dynamics of premalignant cells in the development of follicular lymphoma and CLL. RECENT FINDINGS: The frequency of circulating cells bearing the follicular lymphoma hallmark translocation t(14;18) in healthy persons is correlated to the risk of developing follicular lymphoma later in life. Chronic B-cell receptor stimulation induces cyclic re-entries of BCL2 B cells into germinal centers that propagate clonal evolution and early follicular lymphoma progression. The lymph node microenvironment is a key activation/proliferation niche for malignant cells in CLL, also active in its preclinical antecedent monoclonal B-cell lymphocytosis. SUMMARY: Considering recent studies of premalignant cells in both diseases and of their putative normal cell counterparts, we propose different models of premalignant evolution for the two pathologies. Before overt follicular lymphoma, t(14;18) B cells exploit the dynamics of memory B cells to re-enter multiple times into local or distant germinal centers, gather activation/proliferation signals, and gain additional mutations to progress to malignant lymphoma. In monoclonal B-cell lymphocytosis, CLL-like activated/memory B cells follow cycles of germinal center-independent activation/proliferation in lymph node. Finally, we discuss the next level genetic and functional analyses that should result in a better understanding of the origins and mechanisms of frequent relapses in follicular lymphoma and CLL.

Early posttransplantation donor-derived invariant natural killer T-cell recovery predicts the occurrence of acute graft-versus-host disease and overall survival.

Invariant natural killer T (iNKT) cells can experimentally dissociate GVL from graft-versus-host-disease (GVHD). Their role in human conventional allogeneic hematopoietic stem cell transplantation (HSCT) is unknown. Here, we analyzed the post-HSCT recovery of iNKT cells in 71 adult allografted patients. Results were compared with conventional T- and NK-cell recovery and correlated to the occurrence of GVHD, relapse, and survival. We observed that posttransplantation iNKT cells, likely of donor origin, recovered independently of T and NK cells in the first 90 days after HSCT and reached greater levels in recipient younger than 45 years (P = .003) and after a reduced-intensity conditioning regimen (P = .03). Low posttransplantation iNKT/T ratios (ie, < 10(-3)) were an independent factor associated with the occurrence of acute GVHD (aGVHD; P = .001). Inversely, reaching iNKT/T ratios > 10(-3) before day 90 was associated with reduced nonrelapse mortality (P = .009) without increased risk of relapse and appeared as an independent predictive factor of an improved overall survival (P = .028). Furthermore, an iNKT/T ratio on day 15 > 0.58 × 10(-3) was associated with a 94% risk reduction of aGVHD. These findings provide a proof of concept that early postallogeneic HSCT iNKT cell recovery can predict the occurrence of aGVHD and an improved overall survival.

IL-17-producing invariant NKT cells in lymphoid organs are recent thymic emigrants identified by neuropilin-1 expression.

Despite increasing knowledge on the mechanisms of invariant natural killer T (iNKT)-cell development in the thymus, the function of recent thymic emigrant (RTE) iNKT cells remains largely unexplored, principally because of a lack of bona fide markers to distinguish RTE from long-lived iNKT cells. Whether the recently described IL-17-producing iNKT cell subset is part of RTE has notably not been addressed. In the present study, we show that neuropilin-1 (Nrp-1), a transmembrane receptor mainly found on T-regulatory (Treg) cells in the murine immune system, is specifically expressed on RTE iNKT cells in naive mice. We used the Nrp-1 marker to discriminate RTE from mature iNKT cells and compare their functions. We show that RTE iNKT cells proliferate more than mature iNKT cells after in vitro activation; that, unlike mature iNKT cells, most RTE iNKT cells fail to rapidly produce IFN-γ and IL-4 after in vivo activation; and, most importantly, that IL-17-producing iNKT cells in lymphoid organs of naive mice are contained within the RTE iNKT cell pool. Our results establish an accurate marker of RTE iNKT cells and reveal that continuous thymic output is required for pro-inflammatory IL-17 secretion, a key function of adult iNKT cells.

Germinal center-dependent and -independent immune responses of tumor-infiltrating B cells in human cancers.

B cells play essential roles in immunity, mainly through the production of high affinity plasma cells (PCs) and memory B (Bmem) cells. The affinity maturation and differentiation of B cells rely on the integration of B-cell receptor (BCR) intrinsic and extrinsic signals provided by antigen binding and the microenvironment, respectively. In recent years, tumor infiltrating B (TIL-B) cells and PCs (TIL-PCs) have been revealed as important players in antitumor responses in human cancers, but their interplay and dynamics remain largely unknown. In lymphoid organs, B-cell responses involve both germinal center (GC)-dependent and GC-independent pathways for Bmem cell and PC production. Affinity maturation of BCR repertoires occurs in GC reactions with specific spatiotemporal dynamics of signal integration by B cells. In general, the reactivation of high-affinity Bmem cells by antigens triggers GC-independent production of large numbers of PC without BCR rediversification. Understanding B-cell dynamics in immune responses requires the integration of multiple tools and readouts such as single-cell phenotyping and RNA-seq, in situ analyses, BCR repertoire analysis, BCR specificity and affinity assays, and functional tests. Here, we review how those tools have recently been applied to study TIL-B cells and TIL-PC in different types of solid tumors. We assessed the published evidence for different models of TIL-B-cell dynamics involving GC-dependent or GC-independent local responses and the resulting production of antigen-specific PCs. Altogether, we highlight the need for more integrative B-cell immunology studies to rationally investigate TIL-B cells as a leverage for antitumor therapies.

ShIVA: a user-friendly and interactive interface giving biologists control over their single-cell RNA-seq data.

Single-cell technologies have revolutionised biological research and applications. As they continue to evolve with multi-omics and spatial resolution, analysing single-cell datasets is becoming increasingly complex. For biologists lacking expert data analysis resources, the problem is even more crucial, even for the simplest single-cell transcriptomics datasets. We propose ShIVA, an interface for the analysis of single-cell RNA-seq and CITE-seq data specifically dedicated to biologists. Intuitive, iterative and documented by video tutorials, ShIVA allows biologists to follow a robust and reproducible analysis process, mostly based on the Seurat v4 R package, to fully explore and quantify their dataset, to produce useful figures and tables and to export their work to allow more complex analyses performed by experts.

A natural protective function of invariant NKT cells in a mouse model of innate-cell-driven lung inflammation.

Activation of invariant natural killer T (iNKT) cells by treatment with their α-galactosyl ceramide ligand provides therapeutic benefits in several immune inflammatory settings. Given the artificial nature of this stimulation, the natural regulatory functions of iNKT remain uncertain. Addressing this issue in a mouse model of innate-cell-driven lung inflammation induced by the cytokine/alarmin IL-33 that targets iNKT cells, we found that eosinophil and neutrophil recruitment was markedly increased in treated iNKT cell-deficient (Jα18 KO) mice, as was the local production of eotaxin and keratinocyte chemoattractant chemokines. By contrast, lung inflammation decreased after adoptive transfer of iNKT cells, which restored the WT inflammatory response in Jα18 KO mice. Finally, we established that this natural anti-inflammatory function of iNKT cells depends on their IFN-γ production and on endogenous IL-12. Our study provides the first evidence of a protective role of iNKT cells during lung inflammation that does not require pharmacological TCR engagement.

Neuroblasts contribute to oligodendrocytes generation upon demyelination in the adult mouse brain.

After demyelinating insult, the neuronal progenitors of the adult mouse sub-ventricular zone (SVZ) called neuroblasts convert into oligodendrocytes that participate to the remyelination process. We use this rare example of spontaneous fate conversion to identify the molecular mechanisms governing these processes. Using in vivo cell lineage and single cell RNA-sequencing, we demonstrate that SVZ neuroblasts fate conversion proceeds through formation of a non-proliferating transient cellular state co-expressing markers of both neuronal and oligodendrocyte identities. Transition between the two identities starts immediately after demyelination and occurs gradually, by a stepwise upregulation/downregulation of key TFs and chromatin modifiers. Each step of this fate conversion involves fine adjustments of the transcription and translation machineries as well as tight regulation of metabolism and migratory behaviors. Together, these data constitute the first in-depth analysis of a spontaneous cell fate conversion in the adult mammalian CNS.