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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.

Plasmacytoid dendritic cells (pDC) were identified about 20 years ago, based on their unique ability to rapidly produce copious amounts of all subsets of type I and type III interferon (IFN-I/III) upon virus sensing, while being refractory to infection. Yet, the identity and physiological functions of pDC are still a matter of debate, in a large part due to their lack of specific expression of any single cell surface marker or gene that would allow to track them in tissues and to target them in vivo with high specificity and penetrance. Indeed, recent studies showed that previous methods that were used to identify or deplete pDC also targeted other cell types, including pDC-like cells and transitional DC (tDC) that were proposed to be responsible for all the antigen presentation ability previously attributed to steady state pDC. Hence, improving our understanding of the nature and in vivo choreography of pDC physiological functions requires the development of novel tools to unambiguously identify and track these cells, including in comparison to pDC-like cells and tDC. Here, we report successful generation of a pDC-reporter mouse model, by using an intersectional genetic strategy based on the unique co-expression of Siglech and Pacsin1 in pDC. This pDC-Tomato mouse strain allows specific ex vivo and in situ detection of pDC. Breeding them with Zbtb46GFP mice allowed side-by-side purification and transcriptional profiling by single cell RNA sequencing of bona fide pDC, pDC-like cells and tDC, in comparison to type 1 and 2 conventional DC (cDC1 and cDC2), both at steady state and during a viral infection, revealing diverging activation patterns of pDC-like cells and tDC. Finally, by breeding pDC-Tomato mice with Ifnb1EYFP mice, we determined the choreography of pDC recruitment to the micro-anatomical sites of viral replication in the spleen, with initially similar but later divergent behaviors of the pDC that engaged or not into IFN-I production. Our novel pDC-Tomato mouse model, and newly identified gene modules specific to combinations of DC types and activations states, will constitute valuable resources for a deeper understanding of the functional division of labor between DC types and its molecular regulation at homeostasis and during viral infections.

International audience; 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.

Follicular lymphoma (FL) derives from malignant transformation of germinal center (GC) B cells. FL malignant B cells are heterogeneous and diverge from their GC B cell-of-origin, but the diversity, function, and location of malignant B cell states remain to be addressed. Based on integrative single-cell RNA-seq, we identified and studied recurrent FL malignant B cell states and dynamics. Most FL B cells spanned a continuum of states from proliferating GC-like to quiescent memory (Mem)-like cell states. That GC-to-Mem axis was the main source of intra-tumor transcriptional heterogeneity. While FL B cell states were independent from subclonal B cell receptor genetics divergence, T follicular helper (TFH) cell-derived signals controlled the transition from Mem-like to GC-like states. GC-like, TFH-activated and Mem-like FL B cells tended to occupy distinct niches within and around tumor follicles. Our study characterizes novel malignant cell states recurrent in B cell lymphomas, and highlights the functional plasticity of malignant B cells.

SummaryNatural killer (NK) cells are innate cytotoxic lymphoid cells (ILCs) involved in the killing of infected and tumor cells. Among human and mouse NK cells from the spleen and blood, we previously identified by single-cell RNA sequencing (scRNAseq) two similar major subsets, NK1 and NK2. Using the same technology, we report here the identification, by single-cell RNA sequencing (scRNAseq), of three NK cell subpopulations in human bone marrow. Pseudotime analysis identified a subset of resident CD56bright NK cells, NK0 cells, as the precursor of both circulating CD56dim NK1-like NK cells and CD56bright NK2-like NK cells in human bone marrow and spleen under physiological conditions. Transcriptomic profiles of bone marrow NK cells from patients with acute myeloid leukemia (AML) exhibited stress-induced repression of NK cell effector functions, highlighting the profound impact of this disease on NK cell heterogeneity. Bone marrow NK cells from AML patients exhibited reduced levels of CD160, but the CD160high group had a significantly higher survival rate.

SummaryLung-resident memory B cells (MBCs) provide localized protection against reinfection in the 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. scRNA-seq analysis and confocal imaging revealed that two main transcriptionally distinct subsets of MBCs colonize the lung peribronchial niche after infection. These subsets arise from different progenitors and are both class-switched, somatically mutated and intrinsically biased in their differentiation fate towards plasma cells. Combined analysis of antigen-specificity and B cell receptor repertoire unveiled a highly permissive selection process that segregates these subsets into “bona fide” virus-specific MBCs and “bystander” MBCs with no apparent specificity for eliciting viruses. 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.

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.

International audience; Conventional type 1 dendritic cells (cDC1s) are critical for antitumor immunity. They acquire antigens from dying tumor cells and cross-present them to CD8 + T cells, promoting the expansion of tumor-specific cytotoxic T cells. However, the signaling pathways that govern the antitumor functions of cDC1s in immunogenic tumors are poorly understood. Using single-cell transcriptomics to examine the molecular pathways regulating intratumoral cDC1 maturation, we found nuclear factor κB (NF-κB) and interferon (IFN) pathways to be highly enriched in a subset of functionally mature cDC1s. We identified an NF-κB–dependent and IFN-γ–regulated gene network in cDC1s, including cytokines and chemokines specialized in the recruitment and activation of cytotoxic T cells. By mapping the trajectory of intratumoral cDC1 maturation, we demonstrated the dynamic reprogramming of tumor-infiltrating cDC1s by NF-κB and IFN signaling pathways. This maturation process was perturbed by specific inactivation of either NF-κB or IFN regulatory factor 1 (IRF1) in cDC1s, resulting in impaired expression of IFN-γ–responsive genes and consequently a failure to efficiently recruit and activate antitumoral CD8 + T cells. Last, we demonstrate the relevance of these findings to patients with melanoma, showing that activation of the NF-κB/IRF1 axis in association with cDC1s is linked with improved clinical outcome. The NF-κB/IRF1 axis in cDC1s may therefore represent an important focal point for the development of new diagnostic and therapeutic approaches to improve cancer immunotherapy.

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Targeting immune checkpoints, such as Programmed cell Death 1 (PD1), has improved survival in cancer patients by restoring antitumor immune responses. Most patients, however, relapse or are refractory to immune checkpoint blocking therapies. Neuropilin-1 (NRP1) is a transmembrane glycoprotein required for nervous system and angiogenesis embryonic development, also expressed in immune cells. We hypothesized that NRP1 could be an immune checkpoint co-receptor modulating CD8

International audience; The antibody response and B cell memory development are major effector arms of the immune system. They both rely on the selection of B cells in specific anatomical structures called germinal centers (GC). GCs are not only sites for clonal B cell expansion but also the places where the antibody repertoire diversifies following somatic hypermutations and class switch recombination. CD4+ T cells that are present in GCs guide the stringent cell selection of high-affinity B cell variants. They can be divided in two functional cell subsets: T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Furthermore, follicular B and T cell subsets are in close contact with specialized stromal cells allowing cell migration, adhesion, and participating in B cell selection mechanisms. Importantly, on top of their essential role as orchestrators of protective immune responses against infections, GCs have also been involved in diverse pathological conditions including autoimmunity, allergy, and cancer. The great complexity of these structures means that GC aficionados encompass researchers working on a very large palette of topics including in depth analysis of the immune cell types cited above, characterization of cell interactions between GC immune and/or mesenchymal cell subsets, as well as deciphering disease physiopathology. The great versatility of these research foci means that the GC field is extremely dynamic and multidisciplinary. However, because there are many scientific topics addressed when studying GC and despite a consequent number of teams interested in this field, events centered only on GCs allowing researcher interactions are quite rare.

Follicular lymphoma (FL) is an indolent yet challenging disease. Despite a generally favorable response to immunochemotherapy regimens, a fraction of patients does not respond or relapses early with unfavorable prognosis. For the vast majority of those who initially respond, relapses will repeatedly occur with increasing refractoriness to available treatments. Addressing the clinical challenges in FL warrants deep understanding of the nature of treatment-resistant FL cells seeding relapses, and of the biological basis of early disease progression. Great progress has been made in the last decade in the description and interrogation of the (epi)genomic landscape of FL cells, of their major dependency to the tumor microenvironment (TME), and of the stepwise lymphomagenesis process, from healthy to subclinical disease and to overt FL. A new picture is emerging, in which an ever-evolving tumor-TME duo sparks a complex and multilayered clonal and functional heterogeneity, blurring the discovery of prognostic biomarkers, patient stratification and reliable designs of risk-adapted treatments. Novel technological approaches allowing to decipher both tumor and TME heterogeneity at the single-cell level are beginning to unravel unsuspected cell dynamics and plasticity of FL cells. The upcoming drawing of a comprehensive functional picture of FL within its ecosystem holds great promise to address the unmet medical needs of this complex lymphoma.

Conventional type 1 dendritic cells (cDC1s) are critical for anti-tumor immunity. They acquire antigens from dying tumor cells and cross-present them to CD8+ T cells, promoting the expansion of tumor-specific cytotoxic T cells. However, the signaling pathways that govern the anti-tumor functions of cDC1s are poorly understood. We mapped the molecular pathways regulating intra-tumoral cDC1 maturation using single cell RNA sequencing. We identified NF-κB and IFN pathways as being highly enriched in a subset of functionally mature cDC1s. The specific targeting of NF-κB or IFN pathways in cDC1s prevented the recruitment and activation of CD8+ T cells and the control of tumor growth. We identified an NF-κB-dependent IFNγ-regulated gene network in cDC1s, including cytokines and chemokines specialized in the recruitment and activation of cytotoxic T cells. We used single cell transcriptomes to map the trajectory of intra-tumoral cDC1 maturation which revealed the dynamic reprogramming of tumor-infiltrating cDC1s by NF-κB and IFN signaling pathways. This maturation process was perturbed by specific inactivation of either NF-κB or IRF1 in cDC1s, resulting in impaired expression of IFN-γ-responsive genes and consequently a failure to efficiently recruit and activate anti-tumoral CD8+ T cells. Finally, we demonstrate the relevance of these findings to cancer patients, showing that activation of the NF-κB/IRF1 axis in association with cDC1s is linked with improved clinical outcome. The NF-κB/IRF1 axis in cDC1s may therefore represent an important focal point for the development new diagnostic and therapeutic approaches to improve cancer immunotherapy.One Sentence SummaryNF-κB and IRF1 coordinate intra-tumoral cDC1 maturation and control of immunogenic tumor growth.

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., Author summary Mice reconstituted with human hematopoietic stem cells (hu-mice) are a powerful tool for the study of human immune function in vivo and can be useful as a pre-clinical model to rank vaccination strategies. However, in hu-mice, human B cell-development and function are impaired and cells fail to efficiently transition from IgM B cells to IgG B cells. One finding of our study is that CD40-targeted vaccination combined with CpG-B adjuvant overcomes the usual defect of human B-cell switch and maturation in hu-mouse models. We further reported that the HIV-1 envelope-specific IgG+ hu-B cells elicited in hu-mice by the anti-CD40.Env vaccine used more VH3 and VH4 family genes and displayed higher rates of somatic hypermutations than the non-specific IgG+ hu-B-cell counterpart. VH3 antibodies are essential for antiviral immunity. We also showed that monitoring ICOS+ circulating Tfh cells seven days after the last booster immunization is a surrogate marker for vaccine-induced HIV-1-specific B-cell responses. Overall, we report important results, both in the setting of this hu-mouse model and for a prophylactic HIV vaccine.

International audience; 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.

International audience; 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.

Background & AimsIn 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 or SepSecs) autoantibodies is associated with significantly reduced overall survival, but the associated autoreactive CD4 T cells have not been characterized yet. Here we isolated and deeply characterized SLA-specific CD4 T cells in AIH patients.MethodsWe used brief ex vivo restimulation with overlapping SLA-derived peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterize their transcriptome and TCR repertoire in n=5 AIH patients. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing, to identify their phenotypic niche. We further characterized disease-associated peripheral blood T cells by high content flow cytometry in an additional cohort of n=46 AIH patients and n=18 non-alcoholic steatohepatitis (NASH) controls.ResultsAutoreactive 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 (IL21, IFNG, TIGIT, CTLA4, NR3C1, CD109, KLRB1 and CLEC2D). Autoreactive TCR clonotypes were restricted to the memory PD-1+CXCR5− CD4 T cells. This subset was significantly increased in the blood of AIH patients and supported B cell differentiation through IL-21. Finally, we identified a specific phenotype (PD-1+CD38+CD27+CD127−CXCR5−) of CD4 T cells linked to disease activity and IgG response during AIH.ConclusionsThis work provides for the first time a deep characterization of rare circulating autoreactive CD4 T cells and the identification of their peripheral reservoir in AIH. We also propose a generic phenotype of pathogenic CD4 T cells related to AIH disease activity.

Single-cell RNA sequencing (scRNA-seq) allows the identification, characterization, and quantification of cell types in a tissue. When focused on B and T cells of the adaptive immune system, scRNA-seq carries the potential to track the clonal lineage of each analyzed cell through the unique rearranged sequence of its antigen receptor (BCR or TCR, respectively), and link it to the functional state inferred from transcriptome analysis. Here we introduce FB5P-seq, a FACS-based 5’-end scRNA-seq method for cost-effective integrative analysis of transcriptome and paired BCR or TCR repertoire in phenotypically defined B and T cell subsets. We describe in details the experimental workflow and provide a robust bioinformatics pipeline for computing gene count matrices and reconstructing repertoire sequences from FB5P-seq data. We further present two applications of FB5P-seq for the analysis of human tonsil B cell subsets and peripheral blood antigen-specific CD4 T cells. We believe our novel integrative scRNA-seq method will be a valuable option to study rare adaptive immune cell subsets in immunology research.

Introduction Angioimmunoblastic T-cell lymphoma (AITL) is the most frequent of nodal peripheral T-cell lymphomas. AITL results from the transformation of T follicular helper (T FH) cells and is characterized by chemo-resistance and poor survival (5-year OS around 30%). Recent data from prospective clinical trials suggest that disease outcome may be impacted by factors other than genomic features, such as the tumor microenvironment (TME) and overall intra-tumoral heterogeneity. Our understanding of AITL intra-tumoral genetic, transcriptional and functional heterogeneity is limited because most molecular data generated so far have come from bulk analyses. Single-cell RNA sequencing (scRNA-seq) enables fine characterization of cell types and functional cell states. When focused on T or B cells, 5'-end scRNA-seq also yields the TCR or BCR sequences that allow tracking clonally related cells. Here we studied the intra-tumor heterogeneity of AITL tumors using integrative scRNA-seq. Methods We analyzed lymph node live cell suspensions from AITL patients (n=10) using droplet-based 10x Genomics 5'-end scRNA-seq. Malignant T cells from 4 AITL samples were also analyzed by FACS index sorting and plate-based 5'-end scRNA-seq to link cell surface phenotype and gene expression profile. We identified malignant T cell clones by intersecting the gene expression and TCR sequencing data, and performed separate focused analyses of TME subsets and malignant T cells. We compared subsets of malignant T cells from all patients using marker gene-based metaclustering to identify AITL T cell states conserved across patients. We explored the genetic heterogeneity of malignant T cells by mapping RHOA G17V mutations and inferring copy number variation (CNV) subclones from scRNA-seq data. In select cases, we performed in situ analysis by immunohistochemistry (IHC) or spatial transcriptomics to characterize the spatial distribution of malignant T cell subsets identified by scRNA-seq. Results Based on gene expression, malignant T cells grouped in patient-specific clusters, while non-malignant T, B and myeloid TME cells from all patients clustered by cell type or cell state. Among TME cells, we identified 7 subsets of B cells (including activated B cells, plasma cells, and one patient-specific monoclonal B cell proliferation), 6 subsets of myeloid cells (including macrophages, conventional and plasmacytoid dendritic cells), and 8 subsets of non-malignant T cells (including activated cytotoxic T lymphocytes (CTL) with clonal expansions). Patient-specific malignant T cells were heterogeneous and divided into several gene-expression based clusters. Metaclustering of malignant T cell subsets identified T central memory (T CM)-like and T FH-like states in 10/10 samples. We also identified in 3/10 samples clusters of CTL-like malignant T cells expressing characteristic marker genes (including NKG7, GNLY, GZMK, PRF1). We observed an intra-sample continuum of gene expression states from quiescent T CM-like to proliferating T FH-like states. T FH-like cells were larger in size and expressed higher levels of surface PD1 and ICOS than T CM-like and CTL-like subsets. We detected the RHOA G17V mutation in malignant T cells of 4/4 mutated cases, with no evidence of subclonal heterogeneity for that mutation. We detected clonal and subclonal CNV in most AITL malignant T cells. CTL-like states were enriched in specific CNV subclones, but the T CM-like to T FH-like continuum was observed in all CNV subclones, suggesting that functional plasticity and subclonal genetic evolution may occur independently. In situ staining of markers for T FH-like (PD1, ICOS, CD200) and CTL-like (GZMK, GZMA) cells showed that T FH-like and CTL-like cells occupied distinct tissue niches within the tumor. In spatial transcriptomics analysis, T FH-like cells mapped to follicular dendritic cell (FDC)-rich areas, while T CM-like cells were associated with T-zone reticular cells. Conclusions Our analyses recapitulate known characteristics of AITL TME, and uncover previously unrecognized heterogeneity among malignant T cells across multiple patients. The distinct gene expression programs, phenotypes, genetics, and locations of T FH-like, T CM-like and CTL-like states suggest that AITL malignant T cells undergo significant functional plasticity and genetic divergence, which could influence response to therapy and overall clinical course. Figure 1 Figure 1. Disclosures Lemonnier: Institut Roche: Research Funding; Gilead: Other: travel grant. Gaulard: Gilead: Consultancy; Innate Pharma: Research Funding; Sanofi: Research Funding; Alderaan: Research Funding; Takeda: Consultancy, Honoraria. Milpied: Institut Roche: Research Funding; Innate Pharma: Research Funding; Bristol Myers Squibb: Research Funding.

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.

International audience; 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.

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. 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. 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.

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.

Introduction Follicular Lymphoma (FL), the second most frequent lymphoma in adults, often presents as a disseminated disease at diagnosis. Despite a generally slow progression and a median overall survival of more than 15 years with current chemo-immunotherapies, FL patients often suffer from multiple relapses. Yet, the biological mechanisms promoting FL dissemination, progression and relapse are still poorly understood. FL, like most B-cell lymphomas, originates from germinal centers (GC) where B-cells physiologically undergo clonal expansion, antibody affinity maturation, and differentiation into antibody-producing plasma cells (PC) or recirculating memory (Mem) B-cells. Recently, we provided evidence that FL B-cells are not blocked in a GC B-cell state but might adopt new dynamic modes of functional diversity (Milpied et al., Nature Immunology 2018), yet the main sources of intratumoral heterogeneity within FL remained to be identified. Methods Frozen live cell suspensions were obtained from the CeVi collection of the Institute Carnot/Calym (ANR, France). We initially applied a plate-based 5'-end single-cell RNAseq (scRNAseq) method for deep integrative single-cell analyses of transcriptome, B-cell receptor (BCR) sequence, and surface phenotype on FACS-sorted FL B-cells (4 patients, lymph node biopsies) and their non-malignant counterparts (6 adult healthy donors, spleen and tonsil samples). We confirmed our findings on additional FL samples with high-throughput droplet-based 3'-end scRNAseq (9 patients, lymph node biopsies), and 5'-end scRNAseq paired with BCRseq (5 patients, lymph node biopsies). Custom and existing bioinformatics analysis pipelines were combined for quality control and cell filtering, dimensionality reduction (PCA, t-SNE, UMAP), clustering, pseudo-time analysis, BCR sequence analysis and integrative data analysis. We further validated our transcriptomic data with FACS-based surface and intracellular protein analysis (8 patients, lymph node biopsies). Results Consistent with our previous findings, FL B-cells were transcriptionally diverse, with most cells exhibiting a patient-specific gene expression profile distinct from PC, GC and Mem cells. Challenging the mainstream view of a differentiation blockade in FL, we identified rare FL B-cells carrying a PC-like profile (including low expression of MS4A1/CD20, high expression of XBP1, MZB1, PRDM1). PC-like FL B-cells expressed high levels of the tumor clonal BCR heavy and light chain mRNA, and BCR sequence phylogenetic analysis revealed that those cells did not branch out from a specific tumor subclone. Most importantly, we found that the molecular profiles of the vast majority of FL B-cells spanned a continuum of transitional states between proliferating GC-like and quiescent Mem-like gene expression states. Principal component analysis and pseudo-time reconstruction revealed that pseudo-immune differentiation axis was consistently the main source of intra-sample transcriptional heterogeneity. On top of cell cycle related genes, GC-like FL B-cells notably expressed AICDA, BCL6, RGS13, NANS, CD81, and CD38 genes. By contrast, Mem-like FL B-cells expressed CD44, GPR183, CD69, CXCR4, CCR7, SELL, KLF2, suggesting that those cells may not be confined to the FL follicles. Flow cytometry analysis of dissociated FL tumors confirmed that only the CD38hiCD81hi subset of FL B-cells (GC-like cells), expressed Ki67 and high levels of Bcl6, whereas only CD38negCD81neg FL B-cells (Mem-like cells) consistently contained CD44+ and GPR183+ cells. Conclusions Our study suggests that FL B-cells hijack the physiological GC differentiation process to dynamically alternate between GC-like and Mem-like states that might be responsible for FL progression and dissemination, respectively. We anticipate that such FL-specific clonal dynamics may be orchestrated by extrinsic signals delivered by tumor-infiltrating T cells. Disclosures Milpied: Innate Pharma: Research Funding; Institut Roche: Research Funding.

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.

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.

Follicular lymphoma (FL) is a prototypical example of germinal center (GC) derived B-cell lymphoma. Using a mouse model recapitulating the sporadic occurrence of the FL hallmark BCL2/IGH translocation in healthy individuals, our previous work demonstrated that FL genesis is a dynamic process that requires multiple re-entries of BCL2+ memory B-cells into the GC to ultimately accumulate in lymphoid organs. In line with this, using single-cell gene expression analysis of human FL vs normal GC B-cells, we recently discovered that FL cells are not 'frozen' at a particular GC maturation stage but instead exhibit a major desynchronization of the GC-specific expression program (Milpied et al. Nat Immunol in press). Since KMT2D loss-of-function mutations and BCL2 translocations are the 2 main alterations in FL, we hypothesized these 2 genetic events might explain the GC program desynchronization we observe in humans. To explore the in vivo consequences of Kmt2d inactivation with Bcl2 overexpression in regulating GC/memory dynamics, we transduced bone marrow progenitors carrying B-cell-specific conditional Cd19-Cre Kmt2dflox/flox alleles with a retrovirus encoding human BCL2 or reporter alone, followed by iv transplantation into lethally irradiated WT recipients. Only double-mutant Kmt2d-/-Bcl2+ mice manifested with GC-derived lymphomas in chronically challenged animals, recapitulating histological and phenotypic features associated with human FL progression from early preneoplastic lesions to overt FL-like tumors. We used integrative single-cell analysis of surface phenotype (10-color panel), gene expression (88-gene panel by microfluidic RT-qPCR) and IGH clonality to deconvolute cellular heterogeneity of flow-sorted GC and memory B-cells after acute (day 10) or chronic T-cell dependent immunization in double-mutant vs. single Bcl2+, Kmt2d-/- or WT mice, retaining >4000 cells for downstream analysis. Populations of WT GC B-cells were molecularly heterogeneous and spanned a cyclic continuum of transitional B-cell states polarized between the light and dark zone where synchronized expression of gene modules characterized mouse GC functional identity. In acute responses, single and double-mutant mice formed GC similar to control chimera mice, and single GC B-cells from all genotypes clustered together with WT GCs, suggesting unperturbed GC transcriptional dynamics upon first antigen encounter. However, preneoplastic/tumoral Kmt2d-/-Bcl2+ mice after chronic challenge showed massive GC hyperplasia and single B-cells expressed a distinct transcriptional signature that clustered separately from WT GC or memory cells. Specifically, murine lymphoma B-cells sorted with a GC-like phenotype accumulated in transitional cell states where the synchrony of most GC-specific co-expression patterns was progressively lost whereas expression of cytokines (Lta) or surface markers linked to GC to memory transition (Gpr183, Cxcr3) became markedly expressed, indicating that Kmt2d-deficient lymphomas were not blocked at a particular GC stage. Given the importance of T-cell help for the fate 'decisions' of GC-to-memory B-cells, we further explored whether tumor cell-intrinsic factors may affect immune cell phenotypes thereby supporting the GC gene expression desynchronization. Using flow cytometry, we found that Kmt2d inactivation instructed a progressive remodeling of the tumor microenvironment with an increased recruitment of CD4+ T-follicular helper (TFH) (n=21, p9000 cells), we revealed a TFH cluster with an activation signature distinct from normal TFH and a concomitant expansion of exhausted CD8+ T-cells strongly co-expressing inhibitory receptors (Lag3, Tim3, Pdcd1), thereby indicating that Kmt2d inactivation in B-cells may favor lymphoma formation by shaping the FL tumor supportive niche and contributing to immune evasion. In summary, our integrative single-cell analyses in murine lymphomas revealed that Kmt2d mutations in FL not only instruct B-cell intrinsic effects involving the desynchronization of the normal GC expression program, but also trigger a concomitant re-education of a tumor-supportive immune microenvironment, establishing for the first time a key link between the most frequent epigenetic alteration and the FL microenvironment. Disclosures Salles: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding; Janssen: Honoraria, Other: Advisory Board; Novartis: Consultancy, Honoraria; Takeda: Honoraria; Servier: Honoraria, Other: Advisory Board; Epizyme: Honoraria; Merck: Honoraria; BMS: Honoraria, Other: Advisory Board; Morphosys: Honoraria; Acerta: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Other: Advisory Board; Pfizer: Honoraria; Servier: Honoraria; Abbvie: Honoraria.

Treg are immune cells that play a critical role in the regulation of the immune response. Although the transcription factor Foxp3 is widely accepted as the standard marker of Treg, specific surface markers are needed to better characterize these cells and decipher their mechanisms of action. Neuropilin-1 (Nrp-1), a membrane protein primarily involved in the nervous system, was identified as a specific marker of murine Treg, but its expression has not been rigorously investigated in human Treg. Here we show that in contrast to murine Treg and regardless of their origins (blood, thymus, spleen, lymph node or tonsil), human Foxp3(+) Treg do not specifically express Nrp-1. However, a population of Foxp3(-) Nrp-1(+) T cells can be detected in human secondary lymphoid organs, and Nrp-1 expression is induced on peripheral blood T lymphocytes upon in vitro activation. We conclude that Nrp-1 cannot be used as a specific marker of human Treg, but might represent a novel activation marker of human T cells both in vitro and in vivo.

Some molecules described in the nervous system are also expressed in cells involved in the control of the immune response, suggesting they have a role as common mechanisms between neuroendocrine and immune systems. In this review, we focus on the expression and role of neuropilins (NPs) and their soluble ligands class 3 semaphorins in thymus physiology, particularly migration of developing thymocytes. We also discuss the concept of multivectorial thymocyte migration, including semaphorins, as a new individual cell migration vector.

Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions.

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.

Treg are immune cells that play a critical role in the regulation of the immune response. Although the transcription factor Foxp3 is widely accepted as the standard marker of Treg, specific surface markers are needed to better characterize these cells and decipher their mechanisms of action. Neuropilin-1 (Nrp-1), a membrane protein primarily involved in the nervous system, was identified as a specific marker of murine Treg, but its expression has not been rigorously investigated in human Treg. Here we show that in contrast to murine Treg and regardless of their origins (blood, thymus, spleen, lymph node or tonsil), human Foxp3(+) Treg do not specifically express Nrp-1. However, a population of Foxp3(-) Nrp-1(+) T cells can be detected in human secondary lymphoid organs, and Nrp-1 expression is induced on peripheral blood T lymphocytes upon in vitro activation. We conclude that Nrp-1 cannot be used as a specific marker of human Treg, but might represent a novel activation marker of human T cells both in vitro and in vivo.

Purpose: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are two incurable B-cell lymphoid neoplasms characterized by distinct clinical presentation and evolution. Bendamustine hydrochloride is a multifunctional, alkylating agent with a purine-like ring system that exhibits activity in multiple cancer models, including CLL and MCL, but whose mechanism is only partially described. Our aim was to analyze the apoptotic pathways activated by bendamustine in CLL and MCL together with the relevance of p53 mutation in determining the response to this drug. Experimental Design: Thirteen CLL/MCL cell lines and primary tumor cells from 8 MCL and 25 CLL patients were cultured for up to 24 h with bendamustine followed by cytotoxic assays, flow cytometry, immunofluorescence, and Western blot analysis of p53 response pathway and apoptosis-related factors. Results: Bendamustine displayed cytotoxic activity on most CLL and MCL primary cells and cell lines irrespective of ZAP-70 expression and p53 status. Bendamustine was found to act synergistically with nucleoside analogues in both CLL and MCL, this combination being effective in p53 mutated cases resistant to standard chemotherapy. Bendamustine cytotoxicity was mediated by the generation of reactive oxygen species and triggering of the intrinsic apoptotic pathway involving up-regulation of PUMA and NOXA, conformational activation of BAX and BAK, and cytosolic release of caspase-related and caspase-unrelated mitochondrial apoptogenic proteins. Conclusions: Our findings support the use of bendamustine as a therapeutic agent, alone or in combination, for CLL and MCL with p53 alterations and describe the molecular basis of its activity in these entities.

T follicular helper (Tfh) cells play an essential role in the development of antigen-specific B cell immunity. Tfh cells regulate the differentiation and survival of activated B cells outside and inside germinal centers (GC) of secondary lymphoid organs. They act through cognate contacts with antigen-presenting B cells, but there is no current marker to specifically identify those Tfh cells which productively interact with B cells. Here we show that neuropilin 1 (Nrp1), a cell surface receptor, is selectively expressed by a subset of Tfh cells in human secondary lymphoid organs. Nrp1 expression on Tfh cells correlates with B cell differentiation in vivo and in vitro, is transient, and can be induced upon co-culture with autologous memory B cells in a cell contact-dependent manner. Comparative analysis of ex vivo Nrp1(+) and Nrp1(-) Tfh cells reveals gene expression modulation during activation. Finally, Nrp1 is expressed by malignant Tfh-like cells in a severe case of angioimmunoblastic T-cell lymphoma (AITL) associated with elevated terminal B cell differentiation. Thus, Nrp1 is a specific marker of Tfh cells cognate activation in humans, which may prove useful as a prognostic factor and a therapeutic target in neoplastic diseases associated with Tfh cells activity.

Abstract 346 Introduction: Invariant Natural Killer T (iNKT) cells express a highly restricted T cell receptor (TCR) repertoire composed of a single invariant Va14Ja18 chain in mice and a Va24Ja18 chain in humans. In contrast to their conventional counterpart, iNKT cells recognize CD1d-bound glycolipids rather than peptides. iNKT cells have elicited a lively interest in the last years because of their implication in several immune responses including experimental graft-versus-host-disease (GVHD). In this study, we addressed the question whether human iNKT cells could be associated with the outcome of allogeneic haematopoietic stem cell transplantation (HSCT). Materials and methods: Forty-seven patients allografted for diverse hematological malignancies in our institution entered prospectively the study. We sequentially analyzed the reconstitution of peripheral blood CD1d tetramer + iNKT, CD56+ NK, and CD4+, CD8+, CD4+CD25high T cells for 90 days after allogeneic HSCT by flow cytometry. Results were correlated to the clinical evolution of allogeneic HSCT. Results: We observed two groups of patients according to their iNKT/106 T cell ratios on days 30, 60 and 90 after HSCT. Patients with at least one point above the normal ratio of 1000 iNKT/106 T cells were considered in the iNKT high group (n=17), while those with a poor reconstitution, defined by all points below that threshold, were analyzed in the iNKT low group (n=30). iNKT reconstitution was the only significant difference in terms of immune reconstitution between the two groups since reconstitution of T CD4+, CD8+, or CD4+CD25high and NK cells was similar at all time points in both groups. Chimerism, analyzed by PCR amplification of short tandem repeat markers, showed that in all cases iNKT displayed a 100% donor origin. Pre-transplant characteristics of patients were similar between the two groups except for the conditioning regimen and the source of stem cells. Patients in the iNKT high group had more often received a reduced-intensity conditioning (80.5% versus 46.7%, p=0.004) and peripheral blood stem cells (88.5% versus 53.6%, p=0.015). Occurrence and severity of acute GVHD was significantly reduced in the iNKT high group (23.5 % grade I-II and 0% grade III-IV) compared to the iNKT low group (66.7 % grade I-II and 26.6% grade III-IV), (p Conclusion: Our data suggest that donor-derived iNKT cell reconstitution after allogeneic HSCT may represent a predictive factor of acute GVHD and a prognostic factor of transplant outcome. Disclosures: No relevant conflicts of interest to declare.

Abstract 1166 Poster Board I-188 Introduction: The complement system consists of several serum proteins and cell membrane receptors that can be activated by three possible pathways leading to the production of pro-inflammatory factors such as the anaphylatoxins C3a and C5a and the formation of the membrane attack complex. The complement system has been implicated in the pathophysiology of several immune diseases and we previously reported that it was activated after allogeneic haematopoietic stem cell transplantation (HSCT) both in humans and mice. It's activation after allogeneic HSCT following a myeloablative conditioning could predict the development of gastrointestinal (GI) GVHD in humans. We now present updated data with long term follow up on an enlarged series of patients who did or did not activate complement after allogeneic HSCT. We also discuss the preliminary results of the inhibition of complement activation on experimental models of GVHD. Materials and methods: Complement activation was determined by measurment of complement proteins before and once a week up to 3 months after allogeneic HSCT in 34 patients allografted for diverse haematological malignancies in our institution following conventional myeloablative conditioning. Results were correlated to the clinical evolution of allogeneic HSCT. Preclinically, inhibition of mouse complement activation by C1 inhibitor esterase or an anti-C5 monoclonal antibody was tested in a parent (C57BL/6, H-2b) to F1 [(C57BL/6xDBA2), H-bd] GVHD mouse model. Lethally irradiated (9.75 to 11 Gy) mice reconstituted with either syngeneic or allogeneic bone marrow cells and splenocytes (107 and 13 to 20 ×106 cells/recipient, respectively) were treated with complement inhibitors between day-7 to Day 21 or day 1 to Day 28 post-HSCT. Clinical Results: Fifteen (44%) patients showed an activation of the classical pathway, as defined by a decrease of C3 and C4 proteins below normal values and of at least 50% of their pre-HSCT levels. Activation occurred during the 4 first weeks following HSCT in 11 patients, between 6 and 8 weeks in 2 cases and later, after withdrawal of immunosuppression, in 2 patients. Pre-transplant characteristics (age at transplant, sex, underlying haematological disease, conditioning regimen, use of ATG, type of donor and CMV risk) of patients who showed an activation of complement were comparable to those who did not. We confirmed that activation of complement was significantly associated with acute GI GVHD (80 % in the activated group versus 5.3% in the non activated, p=0.0028) and occurred from 1 to 3 weeks before the appearance of clinical GVHD. Patients in the activated group had a significantly increased incidence of capillary leak syndrome concomitant to the conditioning toxicity or GVHD (66.7% in the activated group vs 10.5% in the non activated, p=0.019). There was no significant difference between the groups in terms of skin or hepatic acute GVHD, TMA, VOD and chronic GVHD. In a landmark analysis by day 100 post-HSCT, overall survival was significantly impaired in the group of patients with complement activation (p=0.008) because of increased toxicity related mortality (p=0.02) and relapse (p=0.01) risks in comparison to the patients without complement activation. Preclinical Results: Complement inhibitors capable of blocking the first pathway (C1 inhibitor esterase) or terminal complement activation (anti-C5 mAb) were used in a mouse model of GVHD. These inhibitors were administrated before and/or during the phase of activation of complement which starts on day 4 post-HSCT. However, GVHD clinical signs and mortality did not appear to be improved following complement inhibitor administration in this mouse model. Conclusion: Activation of complement after allogeneic HSCT following a myeloablative conditioning appears as a predictive marker of acute GI GVHD and a pejorative prognostic factor of transplant outcome. However, inhibition of complement activation in a mouse experimental model of allogeneic HSCT did not allow the control of GVHD. It is not clear, therefore, that this mouse model is useful as a model of human GVHD. Disclosures: No relevant conflicts of interest to declare.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are two different types of mature B-cell non-Hodgkin’s lymphoma (NHL). CLL has an indolent natural history and patients are very responsive to frontline chemotherapy. Unfortunately, multiple relapses are inevitable, and ultimately, no regimen or treatment strategy offers a distinct survival benefit over another. In contrast, patients with MCL generally experience a more aggressive course, with rapid disease progression and also without specific therapeutic options. Bendamustine hydrochloride (Treanda™) is a multifunctional, alkylating agent that exhibits single-agent activity in multiple hematologic and solid tumors. Recently, the combination of bendamustine with rituximab has demonstrated to be a highly active regimen in the treatment of low-grade lymphomas and MCL. However, very little is known about its mode of action. The ability of bendamustine to induce apoptosis in vitro in MCL and CLL cells and the mechanisms implicated in bendamustine-evoked cell death signaling were investigated. Bendamustine exerted cytostatic and cytotoxic effects in 11 MCL cell lines and primary tumor cells from 7 MCL patients and 10 CLL patients independent of their p53 status, and other gene alterations. In vitro treatment of cells with bendamustine induced activation of both p53-dependent and -independent signaling pathways that converged in all cases to the activation of the pro-apoptotic protein Noxa, conformational changes of Bax and Bak, and mitochondrial depolarization. These events led to cytosolic release of the mitochondrial apoptogenic factors cytochrome c, Smac/DIABLO and AIF, and activation of both caspase -dependent and -independent cell death. Genotoxic stress and caspase-independent cell death are often associated with the generation of reactive oxygen species (ROS). We observed that ROS production was a key step in the induction of apoptosis by bendamustine, since pre-incubation of tumor cells with ROS scavengers reverted all the typical hallmarks of apoptosis. Furthermore, bendamustine exerted a cytotoxic effect in p53 deleted CLL cases that were resistant to fludarabine treatment. These findings support the use of bendamustine as a therapeutic agent in MCL and CLL cells and also establish the basis for the use of bendamustine in lymphoid malignancies that show resistance to classic genotoxic agents that depend on cellular p53 status.

Summary 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., Graphical Abstract, Highlights • scRNA-seq on spleen and blood NK cells reveals organ-specific signatures • scRNA-seq reveals the heterogeneity of NK cells in the blood and spleen • scRNA-seq on NK cells defines NK1 as human CD56dim and mouse CD27−CD11b+ NK cells • scRNA-seq on NK cells defines NK2 as human CD56bright and mouse CD27+CD11b− NK cells, Several NK cell subsets have been reported in humans and mice, but their heterogeneity remains poorly characterized. Using high-throughput single-cell RNA-seq, Crinier et al. provide conserved tissue-specific gene signatures of NK cells from spleen and blood and identified two major NK cell subsets transcriptionally similar across organs and species.