Linking the KIR phenotype with STAT3 and TET2 mutations to identify chronic lymphoproliferative disorders of NK cells.

Distinguishing chronic lymphoproliferative disorders of NK cells (CLPD-NK) from reactive NK-cell expansion is challenging. We assessed the value of killer immunoglobulin-like receptor(KIR) phenotyping and targeted high-throughput sequencing in a cohort of 114 consecutive patients with NK cell proliferation, retrospectively assigned to a CLPD-NK group (n = 46) and a reactive NK group (n = 68). We then developed an NK-cell clonality score combining flow cytometry and molecular profiling with a positive predictive value of 93%. STAT3 and TET2 mutations were respectively identified in 27% and 34% of the patients with CLPD-NK, constituting a new diagnostic hallmark for this disease. TET2-mutated CLPD-NK preferentially exhibited a CD16low phenotype, more frequently displayed a lower platelet count, and was associated with other hematologic malignancies such as myelodysplasia. To explore the mutational clonal hierarchy of CLPD-NK, we performed whole-exome sequencing of sorted, myeloid, T, and NK cells and found that TET2 mutations were shared by myeloid and NK cells in 3 of 4 cases. Thus, we hypothesized that TET2 alterations occur in early hematopoietic progenitors which could explain a potential link between CLPD-NK and myeloid malignancies. Finally, we analyzed the transcriptome by RNA sequencing of 7 CLPD-NK and evidenced 2 groups of patients. The first group displayed STAT3 mutations or SOCS3 methylation and overexpressed STAT3 target genes. The second group, including 2 TET2-mutated cases, significantly underexpressed genes known to be downregulated in angioimmunoblastic T-cell lymphoma. Our results provide new insights into the pathogenesis of NK-cell proliferative disorders and, potentially, new therapeutic opportunities.

SARS-CoV-2-Induced ARDS Associates with MDSC Expansion, Lymphocyte Dysfunction, and Arginine Shortage.

PURPOSE: The SARS-CoV-2 infection can lead to a severe acute respiratory distress syndrome (ARDS) with prolonged mechanical ventilation and high mortality rate. Interestingly, COVID-19-associated ARDS share biological and clinical features with sepsis-associated immunosuppression since lymphopenia and acquired infections associated with late mortality are frequently encountered. Mechanisms responsible for COVID-19-associated lymphopenia need to be explored since they could be responsible for delayed virus clearance and increased mortality rate among intensive care unit (ICU) patients. METHODS: A series of 26 clinically annotated COVID-19 patients were analyzed by thorough phenotypic and functional investigations at days 0, 4, and 7 after ICU admission. RESULTS: We revealed that, in the absence of any difference in demographic parameters nor medical history between the two groups, ARDS patients presented with an increased number of myeloid-derived suppressor cells (MDSC) and a decreased number of CD8pos effector memory cell compared to patients hospitalized for COVID-19 moderate pneumonia. Interestingly, COVID-19-related MDSC expansion was directly correlated to lymphopenia and enhanced arginase activity. Lastly, T cell proliferative capacity in vitro was significantly reduced among COVID-19 patients and could be restored through arginine supplementation. CONCLUSIONS: The present study reports a critical role for MDSC in COVID-19-associated ARDS. Our findings open the possibility of arginine supplementation as an adjuvant therapy for these ICU patients, aiming to reduce immunosuppression and help virus clearance, thereby decreasing the duration of mechanical ventilation, nosocomial infection acquisition, and mortality.

BAFF and CD4+ T cells are major survival factors for long-lived splenic plasma cells in a B-cell-depletion context.

Previous data have suggested that B-cell-depletion therapy may induce the settlement of autoreactive long-lived plasma cells (LLPCs) in the spleen of patients with autoimmune cytopenia. To investigate this process, we used the AID-CreERT2-EYFP mouse model to follow plasma cells (PCs) engaged in an immune response. Multiplex polymerase chain reaction at the single-cell level revealed that only a small fraction of splenic PCs had a long-lived signature, whereas PCs present after anti-CD20 antibody treatment appeared more mature, similar to bone marrow PCs. This observation suggested that, in addition to a process of selection, a maturation induced on B-cell depletion drove PCs toward a long-lived program. We showed that B-cell activating factor (BAFF) and CD4+ T cells play a major role in the PC survival niche, because combining anti-CD20 with anti-BAFF or anti-CD4 antibody greatly reduce the number of splenic PCs. Similar results were obtained in the lupus-prone NZB/W model. These different contributions of soluble and cellular components of the PC niche in the spleen demonstrate that the LLPC expression profile is not cell intrinsic but largely depends on signals provided by the splenic microenvironment, implying that interfering with these components at the time of B-cell depletion might improve the response rate in autoimmune cytopenia.

Emergence of long-lived autoreactive plasma cells in the spleen of primary warm auto-immune hemolytic anemia patients treated with rituximab.

Primary warm autoimmune hemolytic anemia (wAIHA) is a rare autoimmune disease in which red blood cells are eliminated by IgG autoantibodies. We analyzed the antibody-secreting cells in the spleen and the peripheral blood of wAIHA patients in various contexts of treatment. Plasmablasts were observed in peripheral blood of newly diagnosed wAIHA patients and, accordingly, active germinal center reactions were present in the spleen of patients receiving short-term corticosteroid therapy. Long-term corticosteroid regimens markedly reduced this response while splenic plasma cells were able to persist, a fraction of them secreting anti-red blood cell IgG in vitro. In wAIHA patients treated by rituximab and who underwent splenectomy because of treatment failure, plasma cells were still present in the spleen, some of them being autoreactive. By using a set of diagnostic genes that allowed us to assess the plasma cell maturation stage, we observed that these cells displayed a long-lived program, differing from the one of plasma cells from healthy donors or from wAIHA patients with various immunosuppressant treatments, and more similar to the one of normal long-lived bone-marrow plasma cells. Interestingly, an increased level of B-cell activating factor (BAFF) was observed in the supernatant of spleen cell cultures from such rituximab-treated wAIHA patients. These results suggest, in line with our previous report on primary immune thrombocytopenia, that the B-cell depletion induced by rituximab promoted a suitable environment for the maturation and survival of auto-immune long-lived plasma cells in the spleen.

IL-2 requirement for human plasma cell generation: coupling differentiation and proliferation by enhancing MAPK-ERK signaling.

Mature B cell differentiation involves a well-established transcription factor cascade. However, the temporal dynamics of cell signaling pathways regulating transcription factor network and coordinating cell proliferation and differentiation remain poorly defined. To gain insight into the molecular processes and extrinsic cues required for B cell differentiation, we set up a controlled primary culture system to differentiate human naive B cells into plasma cells (PCs). We identified T cell-produced IL-2 to be critically involved in ERK1/2-triggered PC differentiation. IL-2 drove activated B cell differentiation toward PC independently of its proliferation and survival functions. Indeed, IL-2 potentiated ERK activation and subsequent BACH2 and IRF8 downregulation, sustaining BLIMP1 expression, the master regulator for PC differentiation. Inhibition of the MAPK-ERK pathway, unlike STAT5 signaling, impaired IL-2-induced PC differentiation and rescued the expression profile of BACH2 and IRF8. These results identify IL-2 as a crucial early input in mature B cell fate commitment.

Characterization of a transitional preplasmablast population in the process of human B cell to plasma cell differentiation.

The early steps of differentiation of human B cells into plasma cells are poorly known. We report a transitional population of CD20(low/-)CD38(-) preplasmablasts along differentiation of human memory B cells into plasma cells in vitro. Preplasmablasts lack documented B cell or plasma cell (CD20, CD38, and CD138) markers, express CD30 and IL-6R, and secrete Igs at a weaker level than do plasmablasts or plasma cells. These preplasmablasts further differentiate into CD20(-)CD38(high)CD138(-) plasmablasts and then CD20(-)CD38(high)CD138(+) plasma cells. Preplasmablasts were fully characterized in terms of whole genome transcriptome profiling and phenotype. Preplasmablasts coexpress B and plasma cell transcription factors, but at a reduced level compared with B cells, plasmablasts, or plasma cells. They express the unspliced form of XBP1 mRNA mainly, whereas plasmablasts and plasma cells express essentially the spliced form. An in vivo counterpart (CD19(+)CD20(low/-)CD38(-)IL-6R(+) cells) of in vitro-generated preplasmablasts could be detected in human lymph nodes (0.06% of CD19(+) cells) and tonsils (0.05% of CD19(+) cells). An open access "B to Plasma Cell Atlas," which makes it possible to interrogate gene expression in the process of B cell to plasma cell differentiation, is provided. Taken together, our findings show the existence of a transitional preplasmablast population using an in vitro model of plasma cell generation and of its in vivo counterpart in various lymphoid tissues.

Functional specialization of short-lived and long-lived macrophage subsets in human tonsils.

Macrophages play a central role in tissue homeostasis and host defense. However, the properties of human macrophages in non-diseased tissues remain poorly understood. Here, we characterized human tonsil macrophages and identified three subsets with distinct phenotype, transcriptome, life cycle, and function. CD36hi macrophages were related to monocytes, while CD36lo macrophages showed features of embryonic origin and CD36int macrophages had a mixed profile. scRNA-seq on non-human primate tonsils showed that monocyte recruitment did not pre-exist an immune challenge. Functionally, CD36hi macrophages were specialized for stimulating T follicular helper cells, by producing Activin A. Combining reconstruction of ligand-receptor interactions and functional assays, we identified stromal cell-derived TNF-α as an inducer of Activin A secretion. However, only CD36hi macrophages were primed for Activin A expression, via the activity of IRF1. Our results provide insight into the heterogeneity of human lymphoid organ macrophages and show that tonsil CD36hi macrophage specialization is the result of both intrinsic features and interaction with stromal cells.

Modified nanofat grafting: Stromal vascular fraction simple and efficient mechanical isolation technique and perspectives in clinical recellularization applications.

Background: Nanofat grafting (NG) is a simple and cost-effective method of lipoaspirates with inter-syringe passages, to produce stromal vascular fraction (SVF) and isolate adipose-derived stem cells (ASCs). This represents a tremendous interest in the future clinical needs of tissue engineering. In this study, we optimized the NG technique to increase the yield of ASC extractions. Methods: We analyzed three groups of SVF obtained by 20, 30, and 40 inter-syringe passages. The control group was an SVF obtained by enzymatic digestion with Celase. We studied their cell composition by flow cytometry, observed their architecture by confocal microscopy, and observed immunomodulatory properties of the ASCs from each of the SVFs by measuring inflammatory markers of macrophages obtained by an ASC monocyte co-culture. Results: We have established the first cell mapping of the stromal vascular fraction of adipose tissue. The results showed that SVF obtained by 20 inter-syringe passages contains more statistically significant total cells, more cells expressing the ASC phenotype, more endothelial cells, and produces more CFU-F than the SVF obtained by 30 and 40 passages and by enzymatic digestion. Confocal microscopy showed the presence of residual adipocytes in SVF obtained by inter-syringe passages but not by enzymatic digestion. The functional study indicates an orientation toward a more anti-inflammatory profile and homogenization of their immunomodulatory properties. Conclusion: This study places mechanically dissociated SVF in the center of approaches to easily extract ASCs and a wide variety and number of other progenitor cells, immediately available in a clinical setting to provide both the amount and quality of cells required for decellularized tissues.

CXCR4 expression functionally discriminates centroblasts versus centrocytes within human germinal center B cells.

The human germinal center is a highly dynamic structure where B cells conduct their terminal differentiation and traffic following chemokine gradients. The rapidly dividing centroblasts and the nondividing centrocytes represent the two major B cell subsets present in germinal center and also the most common normal counterparts for a majority of lymphomas. CD77 expression was previously associated to proliferating centroblasts undergoing somatic hypermutation, but data from transcriptional studies demonstrate that CD77 is not a reliable marker to discriminate human centroblasts from centrocytes. Herein we were able for the first time to separate these two subpopulations based on the expression of the chemokine receptor CXCR4 allowing their characterization. Phenotypic and functional features were especially explored, giving an accurate definition of CXCR4(+) centroblasts compared with CXCR4(-) centrocytes. We show that CXCR4(+) and CXCR4(-) germinal center B cells present a clear dichotomy in terms of proliferation, transcription factor expression, Ig production, and somatic hypermutation regulation. Microarray analysis identified an extensive gene list segregating these B cells, including highly relevant genes according to previous knowledge. By gene set enrichment analysis we demonstrated that the centroblastic gene expression signature was significantly enriched in Burkitt's lymphomas. Collectively, our findings show that CXCR4 expression can properly separate human centroblasts from centrocytes and offer now the possibility to have purified normal counterparts of mature B cell-derived malignancies.

High-Dimensional Phenotyping of Human Myeloid-Derived Suppressor Cells/Tumor-Associated Macrophages in Tissue by Mass Cytometry.

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are heterogeneous cells that share myeloid markers and are not easily distinguishable in human tumors due to their lack of specific markers. These cells are a major player in the tumor microenvironment and are involved in the prognosis and physiopathology of various tumors. Here is presented a scheme to decipher these cells by mass cytometry.

Mass Cytometry Identifies Expansion of T-bet+ B Cells and CD206+ Monocytes in Early Multiple Sclerosis.

Multiple sclerosis (MS) is an immune-driven demyelinating disease of the central nervous system. Immune cell features are particularly promising as predictive biomarkers due to their central role in the pathogenesis but also as drug targets, even if nowadays, they have no impact in clinical practice. Recently, high-resolution approaches, such as mass cytometry (CyTOF), helped to better understand the diversity and functions of the immune system. In this study, we performed an exploratory analysis of blood immune response profiles in healthy controls and MS patients sampled at their first neurological relapse, using two large CyTOF panels including 62 markers exploring myeloid and lymphoid cells. An increased abundance of both a T-bet-expressing B cell subset and a CD206+ classical monocyte subset was detected in the blood of early MS patients. Moreover, T-bet-expressing B cells tended to be enriched in aggressive MS patients. This study provides new insights into understanding the pathophysiology of MS and the identification of immunological biomarkers. Further studies will be required to validate these results and to determine the exact role of the identified clusters in neuroinflammation.

Functional characterization of PD1+TIM3+ tumor-infiltrating T cells in DLBCL and effects of PD1 or TIM3 blockade.

In diffuse large B-cell lymphoma (DLBCL), tumor-infiltrating T lymphocytes (TILs) are involved in therapeutic responses. However, tumor-specific TILs can be dysfunctional, with impaired effector functions. Various mechanisms are involved in this exhaustion, and the increased expression of programmed cell death receptor 1 (PD1) and TIM3 on dysfunctional cells suggests their involvement. However, conflicting data have been published regarding their expression or coexpression in DLBCL. We evaluated the presence and phenotype of CD4+ and CD8+ TILs in freshly collected tumor tissues in DLBCL and compared the results with those in follicular lymphoma, classical Hodgkin lymphoma, and nonmalignant reactive lymphadenopathy. We found that TILs expressing both PD1 and TIM3 were expanded in DLBCL, particularly in the activated B cell-like subgroup. Isolated PD1+TIM3+ TILs exhibited a transcriptomic signature related to T-cell exhaustion associated with a reduction in cytokine production, both compromising the antitumor immune response. However, these cells expressed high levels of cytotoxic molecules. In line with this, stimulated PD1+TIM3+ TILs from DLBCL patients exhibited reduced proliferation and impaired secretion of interferon-γ, but these functions were restored by the blockade of PD1 or TIM3. In summary, the PD1+TIM3+ TIL population is expanded and exhausted in DLBCL but can be reinvigorated with appropriate therapies.

Comparative immune profiling of acute respiratory distress syndrome patients with or without SARS-CoV-2 infection.

Acute respiratory distress syndrome (ARDS) is the main complication of coronavirus disease 2019 (COVID-19), requiring admission to the intensive care unit (ICU). Despite extensive immune profiling of COVID-19 patients, to what extent COVID-19-associated ARDS differs from other causes of ARDS remains unknown. To address this question, here, we build 3 cohorts of patients categorized in COVID-19-ARDS+, COVID-19+ARDS+, and COVID-19+ARDS-, and compare, by high-dimensional mass cytometry, their immune landscape. A cell signature associating S100A9/calprotectin-producing CD169+ monocytes, plasmablasts, and Th1 cells is found in COVID-19+ARDS+, unlike COVID-19-ARDS+ patients. Moreover, this signature is essentially shared with COVID-19+ARDS- patients, suggesting that severe COVID-19 patients, whether or not they experience ARDS, display similar immune profiles. We show an increase in CD14+HLA-DRlow and CD14lowCD16+ monocytes correlating to the occurrence of adverse events during the ICU stay. We demonstrate that COVID-19-associated ARDS displays a specific immune profile and may benefit from personalized therapy in addition to standard ARDS management.

Nonclassical Monocytes Are Prone to Migrate Into Tumor in Diffuse Large B-Cell Lymphoma.

Absolute count of circulating monocytes has been proposed as an independent prognostic factor in diffuse large B-cell lymphoma (DLBCL). However, monocyte nomenclature includes various subsets with pro-, anti-inflammatory, or suppressive functions, and their clinical relevance in DLBCL has been poorly explored. Herein, we broadly assessed circulating monocyte heterogeneity in 91 DLBCL patients. Classical- (cMO, CD14pos CD16neg) and intermediate- (iMO, CD14pos CD16pos) monocytes accumulated in DLBCL peripheral blood and exhibited an inflammatory phenotype. On the opposite, nonclassical monocytes (ncMOSlanpos, CD14low CD16pos Slanneg and ncMOSlanneg, CD14low CD16pos, Slanneg) were decreased in peripheral blood. Tumor-conditioned monocytes presented similarities with ncMO phenotype from DLBCL and were prone to migrate in response to CCL5 and CXCL12, and presented similarities with DLBCL-infiltrated myeloid cells, as defined by mass cytometry. Finally, we demonstrated the adverse value of an accumulation of nonclassical monocytes in 2 independent cohorts of DLBCL.

Circulating Myeloid Regulatory Cells: Promising Biomarkers in B-Cell Lymphomas.

The monocyte/macrophage lineage has been shown to be involved in the promotion of a protumoral tumor microenvironment and resistance to treatment in B cell lymphomas. However, it is still poorly described at the single cell level, and tissue samples are not easily accessible. Thus, a detailed analysis of the circulating myeloid cell compartment in the different B lymphomas is needed to better understand the mechanisms of resistance to treatment and identify at risk patients. In this Perspective, we review current knowledge on the phenotypic and functional description of the circulating monocytic lineage in B cell lymphomas and provide first insights into the heterogeneity of these cell populations in health and lymphoma, using mass cytometry. Indeed, the monocytic compartment is a continuum more than distinct subpopulations, as demonstrated by our high-resolution approach, explaining the sometimes confusing and contradictory conclusions on the prognostic impact of the different populations, including monocytes and monocytic myeloid derived suppressor cells (M-MDSC). By identifying S100A9high monocytic cells as a potential biomarker in diffuse large B cell lymphoma (DLBCL) in this proof-of-concept preliminary study including a limited number of samples, we underline the potential of circulating myeloid regulatory cells as diagnostic and prognostic biomarkers in B-cell lymphomas.

A splenic IgM memory subset with antibacterial specificities is sustained from persistent mucosal responses.

To what extent immune responses against the gut flora are compartmentalized within mucosal tissues in homeostatic conditions remains a much-debated issue. We describe here, based on an inducible AID fate-mapping mouse model, that systemic memory B cell subsets, including mainly IgM+ B cells in spleen, together with IgA+ plasma cells in spleen and bone marrow, are generated in mice in the absence of deliberate immunization. While the IgA component appears dependent on the gut flora, IgM memory B cells are still generated in germ-free mice, albeit to a reduced extent. Clonal relationships and renewal kinetics after anti-CD20 treatment reveal that this long-lasting splenic population is mainly sustained by output of B cell clones persisting in mucosal germinal centers. IgM-secreting hybridomas established from splenic IgM memory B cells showed reactivity against various bacterial isolates and endogenous retroviruses. Ongoing activation of B cells in gut-associated lymphoid tissues thus generates a diversified systemic compartment showing long-lasting clonal persistence and protective capacity against systemic bacterial infections.

MLPA screening reveals novel subtelomeric rearrangements in holoprosencephaly.

Holoprosencephaly (HPE) is the most common developmental brain anomaly in human, associated with a wide spectrum of presentations. The etiology is heterogeneous, due to environmental and genetic factors. Out of 12 cytogenetic candidate loci previously reported, eight were subtelomeric, including the loci in which two of the four major HPE genes were identified (SHH and TGIF). Recently, we reported that these two genes could be mutated or microdeleted. Therefore, we hypothesized that subtelomeres screening in HPE patients could refine the known subtelomeric candidate loci and identify novel ones. In this study, 181 samples, 72 fetuses and 109 live-born infants, with HPE and a normal karyotype, and 10 patients deleted for SHH or TGIF (3.5 Mb from telomeres) were screened for subtelomeric rearrangements using the multiplex ligation probe-dependent amplification (MLPA) method with two kits. Quantitative PCR was performed when discrepancies were observed between these two kits. We found that known SHH and TGIF microdeletions on 7q and 18p, encompassed their subtelomeric region (3.5 Mb) and were often associated with cryptic gains. Out of the 181 samples, we detected rearrangements in known candidate HPE loci (1q, 20p, and 21q) as well as in other novel subtelomeric locations (1p, 5q, 8p, 17q, 18q, 22q, and Xq) and in the subcentromeric 15q. We also found associations between cryptic subtelomeric gain and loss that may be inherited from a parental balanced translocation, which is helpful for genetic counseling. These findings reinforce the multihit origin for HPE and contribute to the explanation of the wide phenotypic spectrum described in this developmental disorder.

The AID-Cre-ERT2 Model: A Tool for Monitoring B Cell Immune Responses and Generating Selective Hybridomas.

Expression of activation-induced cytidine deaminase (AID) is the hallmark of B cells engaged in an immune response in germinal centers. We designed an inducible fate-mapping reporter mouse in which AID-expressing B cells could be timely and irreversibly marked, by knockin at the Aicda locus of a tamoxifen-inducible Cre recombinase. This mouse model allows notably for the long-term follow-up of memory B cells and plasma cells engaged in an immune response. We describe here a protocol to generate hybridomas from small memory subsets that can be easily traced and identified in this mouse line through Cre-activated fluorescent reporters.

Mass cytometry deep phenotyping of human mononuclear phagocytes and myeloid-derived suppressor cells from human blood and bone marrow.

The monocyte phagocyte system (MPS) includes numerous monocyte, macrophage, and dendritic cell (DC) populations that are heterogeneous, both phenotypically and functionally. In this study, we sought to characterize those diverse MPS phenotypes with mass cytometry (CyTOF). To identify a deep phenotype of monocytes, macrophages, and DCs, a panel was designed to measure 38 identity, activation, and polarization markers, including CD14, CD16, HLA-DR, CD163, CD206, CD33, CD36, CD32, CD64, CD13, CD11b, CD11c, CD86, and CD274. MPS diversity was characterized for 1) circulating monocytes from healthy donors, 2) monocyte-derived macrophages further polarized in vitro (i.e., M-CSF, GM-CSF, IL-4, IL-10, IFN-γ, or LPS long-term stimulations), 3) monocyte-derived DCs, and 4) myeloid-derived suppressor cells (MDSCs), generated in vitro from bone marrow and/or peripheral blood. Known monocyte subsets were detected in peripheral blood to validate the panel and analysis pipeline. Then, using various culture conditions and stimuli before CyTOF analysis, we constructed a multidimensional framework for the MPS compartment, which was registered against historical M1 or M2 macrophages, monocyte subsets, and DCs. Notably, MDSCs generated in vitro from bone marrow expressed more S100A9 than when generated from peripheral blood. Finally, to test the approach in vivo, peripheral blood from patients with melanoma (n = 5) was characterized and observed to be enriched for MDSCs with a phenotype of CD14+HLA-DRlowS100A9high (3% of PBMCs in healthy donors, 15.5% in patients with melanoma, P < 0.02). In summary, mass cytometry comprehensively characterized phenotypes of human monocyte, MDSC, macrophage, and DC subpopulations in both in vitro models and patients.