Molecular characterization of the microenvironment in CLL-like monoclonal B cell lymphocytosis and early-stage chronic lymphocytic leukemia

The analysis of the microenvironment in CLL-like monoclonal B cell lymphocytosis (MBL) and early-stage chronic lymphocytic leukemia (CLL) is relevant for understanding the natural history of CLL. To this end, a total of 58 MBL, 54 early-stage CLL and 31 healthy subjects were extensively characterized by various immunological and molecular methods. Purified CD4+ and CD8+ mononuclear cells from peripheral blood were subjected to gene expression studies and T cell receptor (TR) repertoire analysis, whereas cytokine immunoassays were performed in serum samples. Gene expression studies in CD4+ cells revealed increased cytotoxic and inflammatory pathways, which were higher in MBL than in early-stage CLL. Gene dysregulation was not remarkable in CD8+ cells. Increased serum levels of cytokines such as IL8, IFNγ and TNFα were also observed in MBL, while early-stage CLL generally displayed lower cytokine levels, especially amongst cases bearing somatically hypermutated IGHV genes. TR analysis demonstrated oligoclonality in both entities with persisting T cell clones over time and increasing clonality within CD4+ T cells concurrently with the expansion of neoplastic B cells. Besides, identical T cell clonotypes were identified in different MBL/CLL cases. All these findings implicate inflammatory processes and antigenic elements in the immune background of CLL, whose effects are significantly altered during progression from MBL to CLL.El estudio del microambiente en la linfocitosis B monoclonal (LBM) de tipo LLC y en estadios iniciales de la leucemia linfática crónica (LLC) tiene gran relevancia para entender la historia natural de la enfermedad. Con este objetivo se caracterizaron 58 casos de LBM, 54 de LLC en fases iniciales y 31 sujetos sanos. Se analizó la expresión génica y el repertorio del receptor de células T (TR) en fracciones de células mononucleares CD4+ y CD8+ purificadas a partir de sangre periférica. Además, se realizaron inmunoensayos para medir niveles de citoquinas en suero. Los estudios de expresión génica revelaron patrones citotóxicos e inflamatorios aumentados en células CD4+, superiores en LBM con respecto a LLC en fases iniciales. En las células CD8+ no se observó ninguna disfunción remarcable en la expresión génica. Se detectaron niveles aumentados de citoquinas como IL8, IFNγ y TNFα en sueros de sujetos con LBM, mientras que en LLC en fases iniciales los niveles de citoquinas fueron generalmente inferiores, principalmente debido a los casos con hipermutaciones del gen IGHV. El análisis del TR mostró la existencia de oligoclonalidad en ambas entidades y de clones T persistentes en el tiempo, así como niveles de clonalidad en la fracción T CD4+ que aumentan conjuntamente con la expansión de las células B malignas. Asimismo, se identificaron clonotipos comunes en diferentes casos con LBM/LLC. Todos estos hallazgos implican un papel clave de los procesos inflamatorios y de los elementos antigénicos desde las etapas más tempranas de la enfermedad, cuyos efectos varían notablemente durante la progresión desde LBM a LLC

Molecular characterization of the microenvironment in CLL-like monoclonal B cell lymphocytosis and early-stage chronic lymphocytic leukemia

The analysis of the microenvironment in CLL-like monoclonal B cell lymphocytosis (MBL) and early-stage chronic lymphocytic leukemia (CLL) is relevant for understanding the natural history of CLL. To this end, a total of 58 MBL, 54 early-stage CLL and 31 healthy subjects were extensively characterized by various immunological and molecular methods. Purified CD4+ and CD8+ mononuclear cells from peripheral blood were subjected to gene expression studies and T cell receptor (TR) repertoire analysis, whereas cytokine immunoassays were performed in serum samples. Gene expression studies in CD4+ cells revealed increased cytotoxic and inflammatory pathways, which were higher in MBL than in early-stage CLL. Gene dysregulation was not remarkable in CD8+ cells. Increased serum levels of cytokines such as IL8, IFNγ and TNFα were also observed in MBL, while early-stage CLL generally displayed lower cytokine levels, especially amongst cases bearing somatically hypermutated IGHV genes. TR analysis demonstrated oligoclonality in both entities with persisting T cell clones over time and increasing clonality within CD4+ T cells concurrently with the expansion of neoplastic B cells. Besides, identical T cell clonotypes were identified in different MBL/CLL cases. All these findings implicate inflammatory processes and antigenic elements in the immune background of CLL, whose effects are significantly altered during progression from MBL to CLL.El estudio del microambiente en la linfocitosis B monoclonal (LBM) de tipo LLC y en estadios iniciales de la leucemia linfática crónica (LLC) tiene gran relevancia para entender la historia natural de la enfermedad. Con este objetivo se caracterizaron 58 casos de LBM, 54 de LLC en fases iniciales y 31 sujetos sanos. Se analizó la expresión génica y el repertorio del receptor de células T (TR) en fracciones de células mononucleares CD4+ y CD8+ purificadas a partir de sangre periférica. Además, se realizaron inmunoensayos para medir niveles de citoquinas en suero. Los estudios de expresión génica revelaron patrones citotóxicos e inflamatorios aumentados en células CD4+, superiores en LBM con respecto a LLC en fases iniciales. En las células CD8+ no se observó ninguna disfunción remarcable en la expresión génica. Se detectaron niveles aumentados de citoquinas como IL8, IFNγ y TNFα en sueros de sujetos con LBM, mientras que en LLC en fases iniciales los niveles de citoquinas fueron generalmente inferiores, principalmente debido a los casos con hipermutaciones del gen IGHV. El análisis del TR mostró la existencia de oligoclonalidad en ambas entidades y de clones T persistentes en el tiempo, así como niveles de clonalidad en la fracción T CD4+ que aumentan conjuntamente con la expansión de las células B malignas. Asimismo, se identificaron clonotipos comunes en diferentes casos con LBM/LLC. Todos estos hallazgos implican un papel clave de los procesos inflamatorios y de los elementos antigénicos desde las etapas más tempranas de la enfermedad, cuyos efectos varían notablemente durante la progresión desde LBM a LLC

Validation and functional characterization of GWAS-identified variants for chronic lymphocytic leukemia: a CRuCIAL study

This work was partially supported by the European Union’s Horizon 2020 research and innovation program (grant No 856620); grants from the Instituto de Salud Carlos III (Madrid, Spain; PI17/02256 and PI20/01845); Consejería de Economía, Conocimiento, Empresas y Universidad (Granada, Spain; A-CTS-448-UGR18); Consejería de Transformación Económica, Industria, Conocimiento y Universidades (Sevilla, Spain; PY20/01282); Generalitat de Catalunya (17SGR437); Gilead Sciences Fellowship (GLD17/00282); the “Xarxa de Bancs de tumors“ sponsored by Pla Director d’Oncologia de Catalunya (XBTC); the Associazione Italiana per la Ricerca sul Cancro and Fondazione Cariplo (TRIDEO 16923 and AIRC IG21436); the Spanish Association Against Cancer (AECC) Scientific Foundation grant GCTRA18022MORE; and the Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), action Genrisk. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Chronic lymphocytic leukemia-like monoclonal B-cell lymphocytosis exhibits an increased inflammatory signature that is reduced in early-stage chronic lymphocytic leukemia

Several studies in chronic lymphocytic leukemia (CLL) patients have reported impaired immune cell functions, which contribute to tumor evasion and disease progression. However, studies on CLL-like monoclonal B-cell lymphocytosis (MBL) are scarce. In the study described here, we characterized the immune environment in 62 individuals with clinical MBL, 56 patients with early-stage CLL, and 31 healthy controls. Gene expression arrays and quantitative reverse transcription polymerase chain reaction were performed on RNA from CD4+ peripheral blood cells; serum cytokines were measured with immunoassays; and HLA-DR expression on circulating monocytes, as well as the percentages of Th1, cytotoxic, exhausted, and effector CD4+ T cells, were evaluated by flow cytometry. In addition, cell cultures of clonal B cells and CD14-enriched or -depleted cell fractions were performed. Strikingly, MBL and early-stage CLL differed in pro-inflammatory signatures. An increased inflammatory drive orchestrated mainly by monocytes was identified in MBL, which exhibited enhanced phagocytosis, pattern recognition receptors, interleukin-8 (IL8), HMGB1, and acute response signaling pathways and increased pro-inflammatory cytokines (in particular IL8, interferon γ [IFNγ], and tumor necrosis factor α). This inflammatory signature was diminished in early-stage CLL (reduced IL8 and IFNγ levels, IL8 signaling pathway, and monocytic HLA-DR expression compared with MBL), especially in those patients with mutations in IGHV genes. Additionally, CD4+ T cells of MBL and early-stage CLL exhibited a similar upregulation of Th1 and cytotoxic genes and expanded CXCR3+ and perforin+ CD4+ T cells, as well as PD1+ CD4+ T cells, compared with controls. Cell culture assays disclosed tumor-supporting effects of monocytes similarly observed in MBL and early-stage CLL. These novel findings reveal differences in the inflammatory environment between MBL and CLL, highlighting an active role for antigen stimulation in the very early stages of the disease, potentially related to malignant B-cell transformation

Restricted T cell receptor repertoire in CLL-like monoclonal B cell lymphocytosis and early stage CLL

Analysis of the T cell receptor (TR) repertoire of chronic lymphocytic leukemia-like monoclonal B cell lymphocytosis (CLL-like MBL) and early stage CLL is relevant for understanding the dynamic interaction of expanded B cell clones with bystander T cells. Here we profiled the T cell receptor β chain (TRB) repertoire of the CD4+ and CD8+ T cell fractions from 16 CLL-like MBL and 13 untreated, Binet stage A/Rai stage 0 CLL patients using subcloning analysis followed by Sanger sequencing. The T cell subpopulations of both MBL and early stage CLL harbored restricted TRB gene repertoire, with CD4+ T cell clonal expansions whose frequency followed the numerical increase of clonal B cells. Longitudinal analysis in MBL cases revealed clonal persistence, alluding to persistent antigen stimulation. In addition, the identification of shared clonotypes among different MBL/early stage CLL cases pointed towards selection of the T cell clones by common antigenic elements. T cell clonotypes previously described in viral infections and immune disorders were also detected. Altogether, our findings evidence that antigen-mediated TR restriction occurs early in clonal evolution leading to CLL and may further increase together with B cell clonal expansion, possibly suggesting that the T cell selecting antigens are tumor-related.This work has been supported by the following grants: PI11/01621, PI15/00437, Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness; 2014/SGR585, Generalitat de Catalunya; Fundació La Caixa; H2020 No. 692298 project “MEDGENET, Medical Genomics and Epigenomics Network” by the EU; H2020 “AEGLE, An analytics framework for integrated and personalized healthcare services in Europe”, by the EU

Sézary syndrome patient-derived models allow drug selection for personalized therapy

Current therapeutic approaches for Sézary syndrome (SS) do not achieve a significant improvement in long-term survival of patients, and they are mainly focused on reducing blood tumor burden to improve quality of life. Eradication of SS is hindered by its genetic and molecular heterogeneity. Determining effective and personalized treatments for SS is urgently needed. The present work compiles the current methods for SS patient-derived xenograft (PDX) generation and management to provide new perspectives on treatment for patients with SS. Mononuclear cells were recovered by Ficoll gradient separation from fresh peripheral blood of patients with SS (N = 11). A selected panel of 26 compounds that are inhibitors of the main signaling pathways driving SS pathogenesis, including NF-kB, MAPK, histone deacetylase, mammalian target of rapamycin, or JAK/STAT, was used for in vitro drug sensitivity testing. SS cell viability was evaluated by using the CellTiter-Glo_3D Cell Viability Assay and flow cytometry analysis. We validated one positive hit using SS patient-derived Sézary cells xenotransplanted (PDX) into NOD-SCID-γ mice. In vitro data indicated that primary malignant SS cells all display different sensitivities against specific pathway inhibitors. In vivo validation using SS PDX mostly reproduced the responses to the histone deacetylase inhibitor panobinostat that were observed in vitro. Our investigations revealed the possibility of using high-throughput in vitro testing followed by PDX in vivo validation for selective targeting of SS tumor cells in a patient-specific manner

Reduced expansion of CD94/NKG2C + NK cells in chronic lymphocytic leukemia and CLL-like monoclonal B-cell lymphocytosis is not related to increased human cytomegalovirus seronegativity or NKG2C deletions

Data de publicació electrònica: 22-02-2021Introduction: Dysregulated NK cell-mediated immune responses contribute to tumor evasion in chronic lymphocytic leukemia (CLL), although the NK cell compartment in CLL-like monoclonal B-cell lymphocytosis (MBL) is poorly understood. In healthy individuals, human cytomegalovirus (HCMV) induces the expansion of NK cells expressing high levels of CD94/NKG2C NK cell receptor (NKR) specific for HLA-E. Methods: We analyzed the expression of NKG2A, NKG2C, ILT2, KIR, CD161, and CD57 in 24 MBL and 37 CLL. NKG2C was genotyped in these patients and in 81 additional MBL/CLL, while NKG2C gene expression was assessed in 26 cases. In 8 CLL patients with increased lymphocytosis (≥20 × 109 /L), tumor HLA-E and HLA-G expression was evaluated. Results: NKR distribution did not significantly differ between MBL and CLL patients, although they exhibited reduced NKG2C+ NK cells compared with a non-CLL group (4.6% vs 12.2%, P = .012). HCMV+ patients showed increased percentages of NKG2C+ NK cells compared with HCMV- (7.3% vs 2.9%, P = .176). Frequencies of NKG2C deletions in MBL/CLL were similar to those of the general population. Low/undetectable NKG2C expression was found among NKG2C+/- (45%) and NKG2C+/+ (12%) patients. CLL cases with increased lymphocytosis displayed especially reduced NKG2C expression (1.8% vs 8.1%, P = .029) and tumor cells with high HLA-E (>98%) and variable HLA-G expression (12.4%, range: 0.5-56.4). CLL patients with low NKG2C expression (<7%) showed shorter time to first treatment (P = .037). Conclusion: Reduced percentages of CD94/NKG2C+ NK cells were observed in CLL and MBL patients independently of HCMV serostatus and NKG2C zygosity, particularly in CLL patients with increased lymphocytosis, which could potentially be related to the exposure to tumor cells

Restricted T cell receptor repertoire in CLL-like monoclonal B cell lymphocytosis and early stage CLL

Analysis of the T cell receptor (TR) repertoire of chronic lymphocytic leukemia-like monoclonal B cell lymphocytosis (CLL-like MBL) and early stage CLL is relevant for understanding the dynamic interaction of expanded B cell clones with bystander T cells. Here we profiled the T cell receptor β chain (TRB) repertoire of the CD4+ and CD8+ T cell fractions from 16 CLL-like MBL and 13 untreated, Binet stage A/Rai stage 0 CLL patients using subcloning analysis followed by Sanger sequencing. The T cell subpopulations of both MBL and early stage CLL harbored restricted TRB gene repertoire, with CD4+ T cell clonal expansions whose frequency followed the numerical increase of clonal B cells. Longitudinal analysis in MBL cases revealed clonal persistence, alluding to persistent antigen stimulation. In addition, the identification of shared clonotypes among different MBL/early stage CLL cases pointed towards selection of the T cell clones by common antigenic elements. T cell clonotypes previously described in viral infections and immune disorders were also detected. Altogether, our findings evidence that antigen-mediated TR restriction occurs early in clonal evolution leading to CLL and may further increase together with B cell clonal expansion, possibly suggesting that the T cell selecting antigens are tumor-related.This work has been supported by the following grants: PI11/01621, PI15/00437, Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness; 2014/SGR585, Generalitat de Catalunya; Fundació La Caixa; H2020 No. 692298 project “MEDGENET, Medical Genomics and Epigenomics Network” by the EU; H2020 “AEGLE, An analytics framework for integrated and personalized healthcare services in Europe”, by the EU