Stromal Cell Contribution to Human Follicular Lymphoma Pathogenesis

Follicular lymphoma (FL) is the prototypical model of indolent B cell lymphoma displaying a strong dependence on a specialized cell microenvironment mimicking normal germinal center. Within malignant cell niches in invaded lymph nodes and bone marrow, external stimuli provided by infiltrating stromal cells make a pivotal contribution to disease development, progression, and drug resistance. The crosstalk between FL B cells and stromal cells is bidirectional, causing activation of both partners. In agreement, FL stromal cells exhibit specific phenotypic, transcriptomic, and functional properties. This review highlights the critical pathways involved in the direct tumor-promoting activity of stromal cells but also their role in the organization of FL cell niche through the recruitment of accessory immune cells and their polarization to a B cell supportive phenotype. Finally, deciphering the interplay between stromal cells and FL cells provides potential new therapeutic targets with the aim to mobilize malignant cells outside their protective microenvironment and increase their sensitivity to conventional treatment

Comparison of two enzymatic immunoassays, high resolution mass spectrometry method and radioimmunoassay for the quantification of human plasma histamine

International audienceHistamine (HA) is one of the main immediate mediators involved in allergic reactions. HA plasma concentration is well correlated with the severity of vascular and respiratory signs of anaphylaxis. Consequently, plasma quantification of HA is useful to comfort the diagnosis of anaphylaxis. Currently, radioimmunoassay (RIA) is the gold standard method to quantify HA due to its high sensitivity, but it is time consuming, implicates specific formations and cautions for technicians, and produces hazardous radioactive wastes. The aim of this study was to compare two enzymatic immunoassays (EIA) and one in-house liquid chromatography high-resolution mass spectrometry method (LC-HRMS) with the gold standard method for HA quantification in plasma samples of patients suspected of anaphylaxis reactions. Ninety-two plasma samples were tested with the 4 methods (RIA, 2 EIA and LC-HRMS) for HA quantification. Fifty-eight samples displayed HA concentrations above the positive cut-off of 10 nM evaluated by RIA, including 18 highly positive samples (>100 nM). Our results showed that Immunotech® EIA and LC-HRMS concentrations were highly correlated with RIA values, in particular for samples with a HA concentration around the positive cut-off. In our hands, plasma concentrations obtained with the Demeditec Diagnostics® EIA correlated less with results obtained by RIA, and an underestimation of plasma HA levels led to a lack of sensitivity. In conclusion, this study demonstrates that Immunotech® EIA and LC-HRMS method could be used instead of RIA to assess plasma HA in human diagnostic use

The yin and the yang of follicular lymphoma cell niches: Role of microenvironment heterogeneity and plasticity.

International audienceFollicular lymphoma (FL) results from the malignant transformation of germinal center B cells and is characterized by recurrent genetic alterations providing a direct growth advantage or facilitating interaction with tumor microenvironment. In agreement, accumulating evidences suggest a dynamic bidirectional crosstalk between FL B cells and surrounding non-malignant cells within specialized tumor niches in both invaded lymph nodes and bone marrow. Infiltrating stromal cells, macrophages, and T/NK cell subsets either contribute to anti-tumor immune response, or conversely form a tumor supportive network promoting FL B cell survival, growth, and drug resistance. This review depicts the phenotypic heterogeneity and functional plasticity of the most important FL cell partners and describes their complex interplay. We also unravel how malignant B cells recruit and subvert accessory immune and stromal cells to trigger their polarization toward a supportive phenotype. Based on these observations, innovative therapeutic approaches have been recently proposed, in order to benefit from local anti-tumor immunity and/or to selectively target the protective cell niche

Impact of Chronic Viral Infection on T-Cell Dependent Humoral Immune Response

During the last decades, considerable efforts have been done to decipher mechanisms supported by microorganisms or viruses involved in the development, differentiation, and function of immune cells. Pathogens and their associated secretome as well as the continuous inflammation observed in chronic infection are shaping both innate and adaptive immunity. Secondary lymphoid organs are functional structures ensuring the mounting of adaptive immune response against microorganisms and viruses. Inside these organs, germinal centers (GCs) are the specialized sites where mature B-cell differentiation occurs leading to the release of high-affinity immunoglobulin (Ig)-secreting cells. Different steps are critical to complete B-cell differentiation process, including proliferation, somatic hypermutations in Ig variable genes, affinity-based selection, and class switch recombination. All these steps require intense interactions with cognate CD4+ helper T cells belonging to follicular helper lineage. Interestingly, pathogens can disturb this subtle machinery affecting the classical adaptive immune response. In this review, we describe how viruses could act directly on GC B cells, either through B-cell infection or by their contribution to B-cell cancer development and maintenance. In addition, we depict the indirect impact of viruses on B-cell response through infection of GC T cells and stromal cells, leading to immune response modulation

Flow cytometric detection and isolation of human tonsil or lymph node T follicular helper cells.

International audienceT-follicular helper (Tfh) cells have emerged as an independent CD4(+) helper T-cell lineage required for antigen-selected germinal center B-cell survival, class switch recombination, and differentiation into long-lived plasma cells. The quantification and function of Tfh subsets are currently extensively explored in humans with infectious diseases, cancer, or autoimmune disorders. Reliable methods to identify and isolate human Tfh cells in patients and healthy donors are necessary to perform these studies. Here, we propose a classical and robust flow cytometric method to detect and isolate Tfh cells from human secondary lymphoid organs based on the expression of CXCR5, PD-1, and CD25 in the CD4(+) T-cell population. An alternative protocol using anti-ICOS and anti-Bcl-6 antibodies and requiring fixation and permeabilization steps without a decrease of detection of membrane markers is also described

Impact of Chronic Viral Infection on T-Cell Dependent Humoral Immune Response

International audienceDuring the last decades, considerable efforts have been done to decipher mechanisms supported by microorganisms or viruses involved in the development, differentiation, and function of immune cells. Pathogens and their associated secretome as well as the continuous inflammation observed in chronic infection are shaping both innate and adaptive immunity. Secondary lymphoid organs are functional structures ensuring the mounting of adaptive immune response against microorganisms and viruses. Inside these organs, germinal centers (GCs) are the specialized sites where mature B-cell differentiation occurs leading to the release of high-affinity immunoglobulin (Ig)-secreting cells. Different steps are critical to complete B-cell differentiation process, including proliferation, somatic hypermutations in Ig variable genes, affinity-based selection, and class switch recombination. All these steps require intense interactions with cognate CD4 helper T cells belonging to follicular helper lineage. Interestingly, pathogens can disturb this subtle machinery affecting the classical adaptive immune response. In this review, we describe how viruses could act directly on GC B cells, either through B-cell infection or by their contribution to B-cell cancer development and maintenance. In addition, we depict the indirect impact of viruses on B-cell response through infection of GC T cells and stromal cells, leading to immune response modulation

Histamine quantification in human plasma using High Resolution Accurate Mass LC-MS technology

International audienceBackground Histamine (HA) is a small amine playing an important role in anaphylactic reactions. In order to identify and quantify HA in plasma matrix, different methods have been developed but present several disadvantages. Here, we developed an alternative method using liquid chromatography coupled with an ultra-high resolution and accurate mass instrument, Q ExactiveTM (Thermofisher) (LCHRMS). Methods The method includes a protein precipitation of plasma samples spiked with HA-d4 as internal standard (IS). LC separation was performed on a C18 Accucore column (100*2.1 mm, 2.6 μm) using a mobile phase containing nonafluoropentanoic acid (3 nM) and acetonitrile with 0.1% (v/v) formic acid on gradient mode. Separation of analytes was obtained within 10 min. Analysis was performed from full scan mode and targeted MS2 mode using a 5 ppm mass window. Ion transition monitored for targeted MS2 mode were 112.0869 > 95.0607 m/z for HA and 116.1120 > 99.0855 m/z for HAd4. Calibration curves were obtained by adding standard calibration dilution at 1 to 180 nM in TrisBSA. Results Elution of HA and IS occurred at 4.1 min. The method was validated over a range of concentrations from 1 nM to 100 nM. The intra- and inter-run precisions were < 15% for quality controls. Human plasma samples from 30 patients were analysed by LCHRMS, and the results were highly correlated with those obtained using the gold standard Radioimmunoassay (RIA) method. Conclusion Overall, we demonstrate here that LCHRMS is a sensitive method for histamine quantification in biological human plasmas, suitable for routinely use in medical laboratories. In addition, LCHRMS is less time-consuming than RIA, avoid the use of radioactivity, and could then be considered as an alternative quantitative method

Human Lymphoid Stromal Cells Contribute to Polarization of Follicular T Cells Into IL-4 Secreting Cells

International audienceFibroblastic reticular cells (FRCs) are the specialized lymphoid stromal cells initially identified as triggering T-cell recruitment and dynamic motion in secondary lymphoid organs. Interestingly, FRCs also display antigen presentation capacities and support lymphocyte survival. CXCR5(+)CD4(+)follicular T cells are important players of B-cell maturation and antibody response. Our study reported thatin vitro-differentiated FRC-like cells enhanced the growth of the whole CXCR5(+)CD4(+)T-cell compartment, while enhancing IL-4 secretion specifically by the PD1(dim)CXCR5(+)CD4(+)cell subset, in a Notch- and ICAM1/LFA1-dependent manner. In addition, we revealed that in follicular lymphoma (FL) tissues, previously identified as enriched for PD1(hi)CXCR5(hi)CD4(+)mature follicular helper T cells, PD1(dim)CXCR5(+)CD4(+)T cells displayed an enrichment for Notch and integrin gene signatures, and a Notch and ICAM-1-dependent overexpression of IL-4 compared to their non-malignant counterparts. These findings suggest that the crosstalk between FRCs and CXCR5(+)PD1(dim)CD4(+)T cells may contribute to the FL IL-4 rich environment, thus providing new insights in FL lymphomagenesis

In vitro functional defects of bone marrow-derived CD34+ progenitors from newly diagnosed mature B-cell malignancies with bone marrow tumor involvement.

International audienceOBJECTIVE: We hypothesized that the presence of tumor cells in bone marrow (BM) could alter hematopoietic progenitor cell functions. Therefore, we evaluated phenotypic and in vitro functional properties of BM-derived CD34+ progenitors issued from untreated and newly diagnosed patients presenting a mature B-lymphoproliferative disorder (LPD) involving the BM (Inv+). PATIENTS AND METHODS: In vitro proliferation and differentiation capacities of primitive and committed progenitors were evaluated by cobblestone area-forming cell (CAFC) and colony-forming cell (CFC) assays, and ex vivo cell expansion. Migratory capacities of CD34+ cells were explored by chemotaxis assays using a CXCL12alpha gradient. RESULTS: Our results showed that CD34+ cells from Inv+ patients overexpressed CD117 and had a significant decrease of week-3 and -6 CAFC, and CFC frequencies, compared to cells obtained from healthy volunteers and LPD patients without BM involvement (Inv-). In addition, progenitors from Inv+ patients maintained a significantly decreased CFC capacity after ex vivo cell expansion, compared to healthy volunteers. However, the former cells held their migratory capacity in response to CXCL12alpha. CONCLUSION: Functional defects of primitive and committed CD34+ progenitors detected among LPD patients with BM tumor involvement suggest either that tumor cells may induced bystander effects on progenitors or that "unusual" CD34+ cells may exist in the BM that could belong to the proliferating tumor tissue

Neutrophils trigger a NF-κB dependent polarization of tumor-supportive stromal cells in germinal center B-cell lymphomas

International audienceBoth tumor-associated neutrophils (TAN) and cancer-associated fibroblasts (CAFs) display specific phenotypic and functional features and contribute to tumor cell niche. However, their bidirectional crosstalk has been poorly studied, in particular in the context of hematological malignancies. Follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL) are two germinal center-derived lymphomas where various cell components of infiltrating microenvironment, including TAN and CAFs, have been demonstrated to favor directly and indirectly malignant B-cell survival, growth, and drug resistance. We show here that, besides a direct and contact-dependent supportive effect of neutrophils on DLBCL B-cell survival, mediated through the BAFF/APRIL pathway, neutrophils and stromal cells cooperate to sustain FL B-cell growth. This cooperation relies on an overexpression of IL-8 by lymphoma-infiltrating stromal cells that could thereafter efficiently promote neutrophil survival and prime them to neutrophil extracellular trap. Conversely, neutrophils are able to activate stromal cells in a NF-κB-dependent manner, inducing their commitment towards an inflammatory lymphoid stroma phenotype associated with an increased capacity to trigger malignant B-cell survival, and to recruit additional monocytes and neutrophils through the release of CCL2 and IL-8, respectively. Altogether, a better understanding of the lymphoma-supporting effects of neutrophils could be helpful to design new anti-tumor therapeutic strategie