Amé-Thomas et al. Intratumoral TFH in follicular lymphoma Characterization of intratumoral follic... more Amé-Thomas et al. Intratumoral TFH in follicular lymphoma Characterization of intratumoral follicular helper T cells in follicular lymphoma: role in the survival of malignant B cells
Distinguishing chronic lymphoproliferative disorders of NK cells (CLPD-NK) from reactive NK-cell ... more 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, ...
Follicular lymphoma (FL), the most frequent indolent non-Hodgkin’s B cell lymphoma, is considered... more Follicular lymphoma (FL), the most frequent indolent non-Hodgkin’s B cell lymphoma, is considered as a prototypical centrocyte-derived lymphoma, dependent on a specific microenvironment mimicking the normal germinal center (GC). In agreement, several FL genetic alterations affect the crosstalk between malignant B cells and surrounding cells, including stromal cells and follicular helper T cells (Tfh). In our study, we sought to deconvolute this complex FL supportive synapse by comparing the transcriptomic profiles of GC B cells, Tfh, and stromal cells, isolated from normal versus FL tissues, in order to identify tumor-specific pathways. In particular, we highlighted a high expression of IL-6 and IL-7 in FL B cells that could favor the activation of FL Tfh overexpressing IFNG, able in turn to stimulate FL B cells without triggering MHC (major histocompatibility) class II expression. Moreover, the glycoprotein clusterin was found up-regulated in FL stromal cells and could promote FL B...
Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1/PRAD... more Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1/PRAD-1 gene overexpression. Some aggressive variants have been recognized with a blastic or large cell morphology, higher proliferative activity, and shorter survival. p53 gene mutations in lymphoid neoplasms have been detected mainly in high grade lymphomas and have been associated with tumor progression in follicular and small lymphocytic lymphomas. To determine the role of p53 alterations in MCL, we examined 35 typical and 8 aggressive variants (5 blastic and 3 large cell) of MCLs by a combination of immunohistochemistry, single-strand conformational polymorphism analysis of genomic DNA and/or cDNA obtained by reverse transcriptase-polymerase chain reaction, denaturing gradient gel electrophoresis, and sequencing. Of the 8 aggressive MCLs, 3 (38%) contained missense point mutations in axon 8 codon 278 (Pro --> Leu), exon 8 codon 273 Arg --> His), and exon 5 codon 151 (Pro --> Ser...
Although the cause(s) of clinical drug resistance in non-Hodgkin's lymphomas (NHL) is unknown... more Although the cause(s) of clinical drug resistance in non-Hodgkin's lymphomas (NHL) is unknown, in vitro studies suggest that abnormalities of the p53 gene, bcl-2 overexpression, and low tumor proliferation rates may increase chemotherapy resistance. We analyzed tumor tissue from 75 patients with relapsed/refractory NHL (Working Formulation A through H) for p53 mutation/overexpression (abnormality), bcl-2 expression, and tumor proliferation and correlated them with multiple clinical characteristics, response to therapy, disease-free survival, and overall survival (OS). All tumor biopsy specimens were obtained within 6 weeks of treatment with EPOCH (infusional etoposide, vincristine, and doxorubicin and bolus prednisone and cyclophosphamide) chemotherapy. Overall, 16 (21%) tumors had a p53 abnormality. Of 13 tumors with overexpression, mutations were confirmed by sequence analysis in 11, and, in 44 tumors without overexpression, 3 showed mutations. A multivariate analysis showed t...
Purpose: Follicular lymphoma arises from a germinal center B-cell proliferation supported by a bi... more Purpose: Follicular lymphoma arises from a germinal center B-cell proliferation supported by a bidirectional crosstalk with tumor microenvironment, in particular with follicular helper T cells (Tfh). We explored the relation that exists between the differentiation arrest of follicular lymphoma cells and loss-of-function of CREBBP acetyltransferase. Experimental Design: The study used human primary cells obtained from either follicular lymphoma tumors characterized for somatic mutations, or inflamed tonsils for normal germinal center B cells. Transcriptome and functional analyses were done to decipher the B- and T-cell crosstalk. Responses were assessed by flow cytometry and molecular biology including ChIP-qPCR approaches. Results: Conversely to normal B cells, follicular lymphoma cells are unable to upregulate the transcription repressor, PRDM1, required for plasma cell differentiation. This defect occurs although the follicular lymphoma microenvironment is enriched in the potent i...
Plasma cell differentiation is a tightly regulated process that requires appropriate T cell helps... more Plasma cell differentiation is a tightly regulated process that requires appropriate T cell helps to reach the induction threshold. To further understand mechanisms by which T cell inputs regulate B cell fate decision, we investigate the minimal IL-2 stimulation for triggering human plasma cell differentiation in vitro. Here we show that the timed repression of BACH2 through IL-2-mediated ERK/ELK1 signalling pathway directs plasma cell lineage commitment. Enforced BACH2 repression in activated B cells unlocks the plasma cell transcriptional program and induces their differentiation into immunoglobulin M-secreting cells. RNA-seq and ChIP-seq results further identify BACH2 target genes involved in this process. An active regulatory region within the BACH2 super-enhancer, under ELK1 control and differentially regulated upon B-cell activation and cellular divisions, helps integrate IL-2 signal. Our study thus provides insights into the temporal regulation of BACH2 and its targets for co...
The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events durin... more The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events during B-cell differentiation including somatic hypermutation and class switch recombination which mark the end of immunoglobulin gene maturation in germinal centers of secondary lymph nodes. These two events rely on the activity of activation-induced cytidine deaminase (AID) which requires DNA double strand breaks be created, a potential danger to the cell. Applying 3D-fluorescence in situ hybridization coupled with immunofluorescence staining to a previously described experimental system recapitulating normal B-cell differentiation ex vivo, we have kinetically analyzed the radial positioning of the two IGH gene loci as well as their proximity with the nucleolus, heterochromatin and γH2AX foci. Our observations are consistent with the proposal that these IGH gene rearrangements take place in a specific perinucleolar "recombination compartment" where AID could be sequestered thus li...
American journal of respiratory and critical care medicine, Aug 1, 2017
Sepsis induces a sustained immune dysfunction responsible for poor outcome and nosocomial infecti... more Sepsis induces a sustained immune dysfunction responsible for poor outcome and nosocomial infections. Myeloid-derived suppressor cells (MDSCs) described in cancer and inflammatory processes may be involved in sepsis-induced immune suppression but their clinical impact remains poorly defined. To clarify phenotype, suppressive activity, origin, and clinical impact of MDSCs in septic patients. Peripheral blood transcriptomic analysis was performed on 29 septic patients and 15 healthy donors. A second cohort of 94 consecutive septic patients, 11 severity-matched intensive care patients and 67 healthy donors was prospectively enrolled for flow cytometry and functional experiments. Genes involved in MDSC suppressive functions, including S100A12, S100A9, MMP8 and ARG1, were upregulated in the peripheral blood of septic patients. CD14(pos)HLA-DR(low/neg) monocytic (M)-MDSCs were expanded in intensive care septic and non-septic patients and CD14(neg)CD15(pos) low-density granulocytes/ granul...
Follicular lymphoma (FL) is the most frequent indolent lymphoma and is characterized by the accum... more Follicular lymphoma (FL) is the most frequent indolent lymphoma and is characterized by the accumulation of germinal center-derived malignant B cells engaged in a bidirectional crosstalk with their supportive microenvironment in invaded lymph nodes (LN) and bone marrow (BM). T follicular helper cells (TFH) and infiltrating stromal cells have been shown to favor FL B-cell growth but the mechanisms of their protumoral effect and how LN/BM microenvironment is converted into a lymphoma-permissive cell niche remain poorly understood. We demonstrated here that FL-infiltrating LN and BM stromal cells overexpressed CXCL12 in situ IL-4(hi) FL-TFH, unlike FL B cells themselves, triggered CXCL12 upregulation in human stromal cell precursors. In agreement, expression of CXCL12 was associated with IL-4 expression and signaling within the FL BM and LN niches. This IL-4/CXCL12 axis was amplified in activated lymphoid stromal cells as shown in our in vitro model of human lymphoid stroma differentia...
In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils rep... more In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils represents an independent prognostic factor. These cell subsets include monocytic and granulocytic myeloid derived suppressor cells (M- and G-MDSCs) defined by their ability to suppress T-cell responses. MDSCs are a heterogeneous population described in inflammatory and infectious diseases as well as in numerous tumors including multiple myeloma, chronic lymphocytic leukemia and DLBCL. However, their mechanisms of action remain unclear. We broadly assessed the presence and mechanisms of suppression of MDSC subsets in DLBCL. First, a myeloid suppressive signature was identified by gene expression profiling in DLBCL peripheral blood. Accordingly, we identified in a cohort of 66 DLBCL patients an increase in circulating G-MDSC (Lin(neg)HLA-DR(neg)CD33(pos)CD11b(pos)) and M-MDSC (CD14(pos)HLA-DR(low)) counts. Interestingly, only M-MDSC number was correlated with the International Prognostic Ind...
Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the othe... more Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We investigated the prevalence, specificity and clinical relevance of mutations of XPO1, which encodes a member of the karyopherin-β nuclear transporters, in a large cohort of PMBL. PMBL cases defined histologically or by gene expression profiling (GEP) were sequenced and the XPO1 mutational status was correlated to genetic and clinical characteristics. The XPO1 mutational status was also assessed in DLBCL, Hodgkin lymphoma (HL) and mediastinal gray-zone lymphoma (MGZL).The biological impact of the mutation on Selective Inhibitor of Nuclear Export (SINE) compounds (KPT-185/330) sensitivity was investigated in vitro. XPO1 mutations were present in 28/117 (24%) PMBL cases and in 5/19 (26%) HL cases but absent/rare in MGZL (0/20) or DLBCL (3/197). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observe...
Both tumor-associated neutrophils (TAN) and cancer-associated fibroblasts (CAFs) display specific... more Both 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 th...
Amé-Thomas et al. Intratumoral TFH in follicular lymphoma Characterization of intratumoral follic... more Amé-Thomas et al. Intratumoral TFH in follicular lymphoma Characterization of intratumoral follicular helper T cells in follicular lymphoma: role in the survival of malignant B cells
Distinguishing chronic lymphoproliferative disorders of NK cells (CLPD-NK) from reactive NK-cell ... more 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, ...
Follicular lymphoma (FL), the most frequent indolent non-Hodgkin’s B cell lymphoma, is considered... more Follicular lymphoma (FL), the most frequent indolent non-Hodgkin’s B cell lymphoma, is considered as a prototypical centrocyte-derived lymphoma, dependent on a specific microenvironment mimicking the normal germinal center (GC). In agreement, several FL genetic alterations affect the crosstalk between malignant B cells and surrounding cells, including stromal cells and follicular helper T cells (Tfh). In our study, we sought to deconvolute this complex FL supportive synapse by comparing the transcriptomic profiles of GC B cells, Tfh, and stromal cells, isolated from normal versus FL tissues, in order to identify tumor-specific pathways. In particular, we highlighted a high expression of IL-6 and IL-7 in FL B cells that could favor the activation of FL Tfh overexpressing IFNG, able in turn to stimulate FL B cells without triggering MHC (major histocompatibility) class II expression. Moreover, the glycoprotein clusterin was found up-regulated in FL stromal cells and could promote FL B...
Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1/PRAD... more Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1/PRAD-1 gene overexpression. Some aggressive variants have been recognized with a blastic or large cell morphology, higher proliferative activity, and shorter survival. p53 gene mutations in lymphoid neoplasms have been detected mainly in high grade lymphomas and have been associated with tumor progression in follicular and small lymphocytic lymphomas. To determine the role of p53 alterations in MCL, we examined 35 typical and 8 aggressive variants (5 blastic and 3 large cell) of MCLs by a combination of immunohistochemistry, single-strand conformational polymorphism analysis of genomic DNA and/or cDNA obtained by reverse transcriptase-polymerase chain reaction, denaturing gradient gel electrophoresis, and sequencing. Of the 8 aggressive MCLs, 3 (38%) contained missense point mutations in axon 8 codon 278 (Pro --> Leu), exon 8 codon 273 Arg --> His), and exon 5 codon 151 (Pro --> Ser...
Although the cause(s) of clinical drug resistance in non-Hodgkin's lymphomas (NHL) is unknown... more Although the cause(s) of clinical drug resistance in non-Hodgkin's lymphomas (NHL) is unknown, in vitro studies suggest that abnormalities of the p53 gene, bcl-2 overexpression, and low tumor proliferation rates may increase chemotherapy resistance. We analyzed tumor tissue from 75 patients with relapsed/refractory NHL (Working Formulation A through H) for p53 mutation/overexpression (abnormality), bcl-2 expression, and tumor proliferation and correlated them with multiple clinical characteristics, response to therapy, disease-free survival, and overall survival (OS). All tumor biopsy specimens were obtained within 6 weeks of treatment with EPOCH (infusional etoposide, vincristine, and doxorubicin and bolus prednisone and cyclophosphamide) chemotherapy. Overall, 16 (21%) tumors had a p53 abnormality. Of 13 tumors with overexpression, mutations were confirmed by sequence analysis in 11, and, in 44 tumors without overexpression, 3 showed mutations. A multivariate analysis showed t...
Purpose: Follicular lymphoma arises from a germinal center B-cell proliferation supported by a bi... more Purpose: Follicular lymphoma arises from a germinal center B-cell proliferation supported by a bidirectional crosstalk with tumor microenvironment, in particular with follicular helper T cells (Tfh). We explored the relation that exists between the differentiation arrest of follicular lymphoma cells and loss-of-function of CREBBP acetyltransferase. Experimental Design: The study used human primary cells obtained from either follicular lymphoma tumors characterized for somatic mutations, or inflamed tonsils for normal germinal center B cells. Transcriptome and functional analyses were done to decipher the B- and T-cell crosstalk. Responses were assessed by flow cytometry and molecular biology including ChIP-qPCR approaches. Results: Conversely to normal B cells, follicular lymphoma cells are unable to upregulate the transcription repressor, PRDM1, required for plasma cell differentiation. This defect occurs although the follicular lymphoma microenvironment is enriched in the potent i...
Plasma cell differentiation is a tightly regulated process that requires appropriate T cell helps... more Plasma cell differentiation is a tightly regulated process that requires appropriate T cell helps to reach the induction threshold. To further understand mechanisms by which T cell inputs regulate B cell fate decision, we investigate the minimal IL-2 stimulation for triggering human plasma cell differentiation in vitro. Here we show that the timed repression of BACH2 through IL-2-mediated ERK/ELK1 signalling pathway directs plasma cell lineage commitment. Enforced BACH2 repression in activated B cells unlocks the plasma cell transcriptional program and induces their differentiation into immunoglobulin M-secreting cells. RNA-seq and ChIP-seq results further identify BACH2 target genes involved in this process. An active regulatory region within the BACH2 super-enhancer, under ELK1 control and differentially regulated upon B-cell activation and cellular divisions, helps integrate IL-2 signal. Our study thus provides insights into the temporal regulation of BACH2 and its targets for co...
The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events durin... more The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events during B-cell differentiation including somatic hypermutation and class switch recombination which mark the end of immunoglobulin gene maturation in germinal centers of secondary lymph nodes. These two events rely on the activity of activation-induced cytidine deaminase (AID) which requires DNA double strand breaks be created, a potential danger to the cell. Applying 3D-fluorescence in situ hybridization coupled with immunofluorescence staining to a previously described experimental system recapitulating normal B-cell differentiation ex vivo, we have kinetically analyzed the radial positioning of the two IGH gene loci as well as their proximity with the nucleolus, heterochromatin and γH2AX foci. Our observations are consistent with the proposal that these IGH gene rearrangements take place in a specific perinucleolar "recombination compartment" where AID could be sequestered thus li...
American journal of respiratory and critical care medicine, Aug 1, 2017
Sepsis induces a sustained immune dysfunction responsible for poor outcome and nosocomial infecti... more Sepsis induces a sustained immune dysfunction responsible for poor outcome and nosocomial infections. Myeloid-derived suppressor cells (MDSCs) described in cancer and inflammatory processes may be involved in sepsis-induced immune suppression but their clinical impact remains poorly defined. To clarify phenotype, suppressive activity, origin, and clinical impact of MDSCs in septic patients. Peripheral blood transcriptomic analysis was performed on 29 septic patients and 15 healthy donors. A second cohort of 94 consecutive septic patients, 11 severity-matched intensive care patients and 67 healthy donors was prospectively enrolled for flow cytometry and functional experiments. Genes involved in MDSC suppressive functions, including S100A12, S100A9, MMP8 and ARG1, were upregulated in the peripheral blood of septic patients. CD14(pos)HLA-DR(low/neg) monocytic (M)-MDSCs were expanded in intensive care septic and non-septic patients and CD14(neg)CD15(pos) low-density granulocytes/ granul...
Follicular lymphoma (FL) is the most frequent indolent lymphoma and is characterized by the accum... more Follicular lymphoma (FL) is the most frequent indolent lymphoma and is characterized by the accumulation of germinal center-derived malignant B cells engaged in a bidirectional crosstalk with their supportive microenvironment in invaded lymph nodes (LN) and bone marrow (BM). T follicular helper cells (TFH) and infiltrating stromal cells have been shown to favor FL B-cell growth but the mechanisms of their protumoral effect and how LN/BM microenvironment is converted into a lymphoma-permissive cell niche remain poorly understood. We demonstrated here that FL-infiltrating LN and BM stromal cells overexpressed CXCL12 in situ IL-4(hi) FL-TFH, unlike FL B cells themselves, triggered CXCL12 upregulation in human stromal cell precursors. In agreement, expression of CXCL12 was associated with IL-4 expression and signaling within the FL BM and LN niches. This IL-4/CXCL12 axis was amplified in activated lymphoid stromal cells as shown in our in vitro model of human lymphoid stroma differentia...
In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils rep... more In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils represents an independent prognostic factor. These cell subsets include monocytic and granulocytic myeloid derived suppressor cells (M- and G-MDSCs) defined by their ability to suppress T-cell responses. MDSCs are a heterogeneous population described in inflammatory and infectious diseases as well as in numerous tumors including multiple myeloma, chronic lymphocytic leukemia and DLBCL. However, their mechanisms of action remain unclear. We broadly assessed the presence and mechanisms of suppression of MDSC subsets in DLBCL. First, a myeloid suppressive signature was identified by gene expression profiling in DLBCL peripheral blood. Accordingly, we identified in a cohort of 66 DLBCL patients an increase in circulating G-MDSC (Lin(neg)HLA-DR(neg)CD33(pos)CD11b(pos)) and M-MDSC (CD14(pos)HLA-DR(low)) counts. Interestingly, only M-MDSC number was correlated with the International Prognostic Ind...
Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the othe... more Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We investigated the prevalence, specificity and clinical relevance of mutations of XPO1, which encodes a member of the karyopherin-β nuclear transporters, in a large cohort of PMBL. PMBL cases defined histologically or by gene expression profiling (GEP) were sequenced and the XPO1 mutational status was correlated to genetic and clinical characteristics. The XPO1 mutational status was also assessed in DLBCL, Hodgkin lymphoma (HL) and mediastinal gray-zone lymphoma (MGZL).The biological impact of the mutation on Selective Inhibitor of Nuclear Export (SINE) compounds (KPT-185/330) sensitivity was investigated in vitro. XPO1 mutations were present in 28/117 (24%) PMBL cases and in 5/19 (26%) HL cases but absent/rare in MGZL (0/20) or DLBCL (3/197). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observe...
Both tumor-associated neutrophils (TAN) and cancer-associated fibroblasts (CAFs) display specific... more Both 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 th...
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