Marie Tosolini

FOXP3-enriched infiltrates associated with better outcome in renal allografts with inflamed fibrosis

BACKGROUND FOXP3-expressing regulatory T cells (Tregs) play a crucial role in maintaining allogeneic transplant tolerance in experimental models. In clinical transplantation, there are few data about their role in chronic inflammation. We hypothesized that Tregs might accumulate within the graft since enrichment of Tregs has been frequently described in chronically inflamed tissues. METHODS Sixty-seven biopsies, indicated by a rise in creatinine level, were studied. Thirty-four biopsies showing acute T-cell-mediated rejection and 33 displaying inflamed fibrosis were selected. Tregs frequency was calculated for each infiltrate by counting FOXP3+ and CD3+ cells on two contiguous serial sections. RESULTS A total of 121 infiltrates were scored with a mean of 309 CD3+ cells per infiltrate (range: 50-700). Tregs were enriched within allografts exhibiting inflamed fibrosis versus acute cellular rejection (10.6 +/- 6.8% versus 5.5 +/- 2.6%, respectively, P = 0.005). In those with inflammation within scarred areas, the subset of patients with a low FOXP3/CD3 ratio (below the median value) displayed a lower frequency of B-cell-enriched nodular cell clusters (20% versus 86%, P = 0.001) and had a significantly lower graft survival (log-rank, P = 0.02). In multivariate analysis, the low FOXP3/CD3 ratio remained an independent indicator of outcome (P = 0.03). Consistently, the FOXP3+/IL-17+ cell ratio was higher in nodular than in diffuse infiltrates. CONCLUSIONS Our results suggest that Tregs may dampen the graft injury in chronic (versus acute) inflammation and stress the importance of devising strategies to enhance Tregs efficiency.

Human Monocyte Recognition of Adenosine-Based Cyclic Dinucleotides Unveils the A2a Gαs Protein-Coupled Receptor Tonic Inhibition of Mitochondrially Induced Cell Death

ABSTRACT Cyclic dinucleotides are important messengers for bacteria and protozoa and are well-characterized immunity alarmins for infected mammalian cells through intracellular binding to STING receptors. We sought to investigate their unknown extracellular effects by adding cyclic dinucleotides to the culture medium of freshly isolated human blood cells in vitro. Here we report that adenosine-containing cyclic dinucleotides induce the selective apoptosis of monocytes through a novel apoptotic pathway. We demonstrate that these compounds are inverse agonist ligands of A2a, a Gαs-coupled adenosine receptor selectively expressed by monocytes. Inhibition of monocyte A2a by these ligands induces apoptosis through a mechanism independent of that of the STING receptors. The blockade of basal (adenosine-free) signaling from A2a inhibits protein kinase A (PKA) activity, thereby recruiting cytosolic p53, which opens the mitochondrial permeability transition pore and impairs mitochondrial respiration, resulting in apoptosis. A2a antagonists and inverse agonist ligands induce apoptosis of human monocytes, while A2a agonists are antiapoptotic. In vivo, we used a mock developing human hematopoietic system through NSG mice transplanted with human CD34+ cells. Treatment with cyclic di-AMP selectively depleted A2a-expressing monocytes and their precursors via apoptosis. Thus, monocyte recognition of cyclic dinucleotides unravels a novel proapoptotic pathway: the A2a Gαs protein-coupled receptor (GPCR)-driven tonic inhibitory signaling of mitochondrion-induced cell death.

Mechanisms of PD-1/PD-L1 expression and prognostic relevance in non-Hodgkin lymphoma: a summary of immunohistochemical studies

Immune checkpoint blockade therapeutics, notably antibodies targeting the programmed death 1 (PD-1) receptor and its PD-L1 and PD-L2 ligands, are currently revolutionizing the treatment of cancer. For a sizeable fraction of patients with melanoma, lung, kidney and several other solid cancers, monoclonal antibodies that neutralize the interactions of the PD-1/PD-L1 complex allow the reconstitution of long-lasting antitumor immunity. In hematological malignancies this novel therapeutic strategy is far less documented, although promising clinical responses have been seen in refractory and relapsed Hodgkin lymphoma patients. This review describes our current knowledge of PD-1 and PD-L1 expression, as reported by immunohistochemical staining in both non-Hodgkin lymphoma cells and their surrounding immune cells. Here, we discuss the multiple intrinsic and extrinsic mechanisms by which both T and B cell lymphomas up-regulate the PD-1/PD-L1 axis, and review current knowledge about the prognostic significance of its immunohistochemical detection. This body of literature establishes the cell surface expression of PD-1/PD-L1 as a critical determinant for the identification of non-Hodgkin lymphoma patients eligible for immune checkpoint blockade therapies.

Cyclic dinucleotides modulate human T-cell response through monocyte cell death

Cyclic dinucleotides, a class of microbial messengers, have been recently identified in bacteria, but their activity in humans remains largely unknown. Here, we have studied the function of cyclic dinucleotides in humans. We found that c‐di‐AMP and cGAMP, two adenosine‐based cyclic dinucleotides, activated T lymphocytes in an unusual manner through monocyte cell death. c‐di‐AMP and cGAMP induced the selective apoptosis of human monocytes, and T lymphocytes were activated by the direct contact with these dying monocytes. The ensuing T‐cell response comprised cell‐cycle exit, phenotypic maturation into effector memory cells and proliferation arrest, but not cell death. This quiescence was transient since T cells remained fully responsive to further restimulation. Together, our results depict a novel activation pattern for human T lymphocytes: a transient quiescence induced by c‐di‐AMP‐ or cGAMP‐primed apoptotic monocytes.

nwCompare and AutoCompare Softwares for Proteomics and Transcriptomics Data Mining – Application to the Exploration of Gene Expression Profiles of Aggressive Lymphomas

The global protein and gene expression profiling technologies have revolutionized the study of normal and malignant cells. Transcriptomes permitted to delineate subtypes of B-cell lymphomas which were otherwise histologically and clinically undistinguishable. Although the data mining of proteomes or transcriptomes from these malignant cells can unveil new aspects of their biology, tools to simultaneously compare several samples are scarce. Here we depict nwCompare and Autocompare, two new freewares we developed with this aim, and examplify their use for the comparative data mining of transcriptomes from normal human B cells and B cell lymphomas such as follicular lymphomas (FL) and diffuse large Bcell lymphomas (DLBCL).

Long non-coding RNA expression profile in cytogenetically normal acute myeloid leukemia identifies a distinct signature and a new biomarker in NPM1-mutated patients

Long non-coding RNAs are defined as transcripts larger than 200 nucleotides but without protein-coding potential. There is growing evidence of the important role of long non-coding RNAs in cancer initiation, development and progression. In this study, we sought to evaluate the long non-coding RNA expression profile of patients with cytogenetically normal acute myeloid leukemia (AML). RNA-sequencing of 40 cytogenetically normal AML patients allowed us to quantify 11,036 long non-coding RNAs. Among these, more than 8000 were previously undescribed long non-coding RNAs. Using unsupervised analysis, we observed a specific long non-coding RNA expression profile dependent on the mutational status of the NPM1 gene. Statistical analysis allowed us to identify a minimal set of 12 long non-coding RNAs capable of discriminating NPM1-mutated from NPM1-wild-type patients. These results were validated by qRT-PCR on an independent cohort composed of 134 cytogenetically normal AML patients. Furthermore, we have identified one putative biomarker, the long non-coding RNA XLOC_109948 whose expression pattern predicts clinical outcome. Interestingly, low XLOC_109948 expression indicates a good prognosis especially for NPM1-mutated patients. Transient transfection of GapmeR against XLOC_109948 in NPM1-mutated OCI-AML3 cell line treated with Ara-C or ATRA enhances apoptosis suggesting XLOC_109948 plays a role in drug sensitivity. This study improves our knowledge of the long non-coding RNA transcriptome in cytogenetically normal AML patients. We observed a distinct long non-coding RNA expression profile in patients with the NPM1 mutation. The newly identified XLOC_109948 long non-coding RNA emerged as a strong prognostic factor able to better stratify NPM1-mutated patients.

Nurse-like cells promote CLL survival through LFA-3/CD2 interactions

In the tumoral micro-environment (TME) of chronic lymphocytic leukemia (CLL), nurse-like cells (NLC) are tumor-associated macrophages which play a critical role in the survival and chemoresistance of tumoral cells. This pro-survival activity is known to involve soluble factors, but few data are available on the relative role of cells cross-talk. Here, we used a transcriptome-based approach to systematically investigate the expression of various receptor/ligand pairs at the surface of NLC/CLL cells. Their relative contribution to CLL survival was assessed both by fluorescent microscopy to identify cellular interactions and by the use of functional tests to measure the impact of uncoupling these pairs with blocking monoclonal antibodies. We found for the first time that lymphocyte function-associated antigen 3 (LFA-3), expressed in CLL at significantly higher levels than in healthy donor B-cells, and CD2 expressed on NLC, were both key for the specific pro-survival signals delivered by NLC. Moreover, we found that NLC/CLL interactions induced the shedding of soluble LFA-3. Importantly, in an exploratory cohort of 60 CLL patients receiving frontline immunochemotherapy, increased levels of soluble LFA-3 were found to correlate with shorter overall survival. Altogether, these data suggest that LFA-3/CD2 interactions promote the survival of CLL cells in the tumor microenvironment.In the tumoral micro-environment (TME) of chronic lymphocytic leukemia (CLL), nurse-like cells (NLC) are tumor-associated macrophages which play a critical role in the survival and chemoresistance of tumoral cells. This pro-survival activity is known to involve soluble factors, but few data are available on the relative role of cells cross-talk. Here, we used a transcriptome-based approach to systematically investigate the expression of various receptor/ligand pairs at the surface of NLC/CLL cells. Their relative contribution to CLL survival was assessed both by fluorescent microscopy to identify cellular interactions and by the use of functional tests to measure the impact of uncoupling these pairs with blocking monoclonal antibodies. We found for the first time that lymphocyte function-associated antigen 3 (LFA-3), expressed in CLL at significantly higher levels than in healthy donor B-cells, and CD2 expressed on NLC, were both key for the specific pro-survival signals delivered by NLC. Moreover, we found that NLC/CLL interactions induced the shedding of soluble LFA-3. Importantly, in an exploratory cohort of 60 CLL patients receiving frontline immunochemotherapy, increased levels of soluble LFA-3 were found to correlate with shorter overall survival. Altogether, these data suggest that LFA-3/CD2 interactions promote the survival of CLL cells in the tumor microenvironment.

RND1 regulates migration of human glioblastoma stem-like cells according to their anatomical localization and defines a prognostic signature in glioblastoma

Despite post-operative radio-chemotherapy, glioblastoma systematically locally recurs. Tumors contacting the periventricular zone (PVZ) show earlier and more distant relapses than tumors not contacting the PVZ. Since glioblastoma stem-like cells (GSCs) have been proposed to play a major role in glioblastoma recurrence, we decided to test whether GSC migration properties could be different according to their anatomical location (PVZ+/PVZ–). For that purpose, we established paired cultures of GSCs from the cortical area (CT) and the PVZ of glioblastoma patient tumors. We demonstrated that PVZ GSCs possess higher migration and invasion capacities than CT GSCs. We highlighted specific transcriptomic profiles in PVZ versus CT populations and identified a down-regulation of the RhoGTPase, RND1 in PVZ GSCs compared to CT GSCs. Overexpression of RND1, dramatically inhibited PVZ GSC migration and conversely, downregulation of RND1 increased CT GSC migration. Additionally, transcriptomic analyses also revealed a down-regulation of RND1 in glioblastoma compared to normal brain. Using the glioblastoma TCGA database, low levels of RND1 were also shown to correlate with a decreased overall survival of patients. Finally, based on signaling pathways activated in patients with low levels of RND1, we identified an RND1low signature of six genes (MET, LAMC1, ITGA5, COL5A1, COL3A1, COL1A2) that is an independent prognostic factor in glioblastoma. These findings contribute to explain the shorter time to progression of patients with PVZ involvement and, point out genes that establish the RND1low signature as key targets genes to impede tumor relapse after treatment.

EBV infection determines the immune hallmarks of plasmablastic lymphoma

ABSTRACT Despite recent therapeutic progress, plasmablastic lymphoma (PBL), a distinct entity of high grade B cell lymphoma, is still an aggressive lymphoma with adverse prognosis. PBL commonly occurs in patients with HIV infection and PBL cells frequently express Epstein Barr virus (EBV) genome with type I latency. Occasionally however, PBL may develop in patients with an immunodepressed status without EBV and HIV infection. The aim of this study was to determine which PBL patients may benefit from the emerging strategies of immune checkpoint blockade. Here, we produced and analyzed the transcriptomic profiles of such tumors to address this question. Unsupervised hierarchical clustering analysis of PBL samples revealed they segregate according to their tumor EBV-status. Moreover, EBV+ PBL displays abundant leucocyte infiltrates and T-cell activation signatures, together with high expression levels of mRNA and protein markers of immune escape. This suggests that EBV infection induce an anti-viral cytotoxic immunity which progressively exhausts T lymphocytes and promotes the tolerogenic microenvironment of PBL. Hence, most EBV+ PBL patients presenting an early stage of cancer immune-editing process appear as the most eligible patients for immune checkpoint blockade therapies.

EBV infection promotes tumor infiltrating leucocyte and immune escape in plasmablastic lymphoma according to gene expression profiling

chemo‐resistant patients compared to chemosensitive patients. Tryptase expression showed a positive correlation with MVD, supporting a role for mast cells in DLBCL tumor angiogenesis, while the CD68 correlation with MVD was not significant, indicating a different role for TAMs rather than angiogenesis in DLBCL. A statistically significant difference was observed in the expression of CD3 in patients with bulky disease. Specifically, a higher expression of CD3 was observed in nonbulky disease patients (mean expression 52.91%, n = 20) compared to bulky disease patients (mean expression 34.9%, n = 9), P value < .05. The reduction in T cells in bulky disease patients contributes to loosen the immune control over the tumor, resulting in an increased cell proliferation, leading to large tumor cell masses, which are predictive of poor prognostic and clinical outcomes. CD3 showed a positive correlation with tryptase and MVD, while multiple regression analysis efficaciously predicted MVD depending on CD3 and tryptase as predictors, supporting a complex interplay between these cells in sustaining tumor angiogenesis in DLBCL patients. The improved understanding of tumor biology and of the role of the tumor microenvironment has led to advances in the diagnosis, classification, prognostics, as well as novel treatments of patients with hematologic malignancies. In particular, translational research, leading to drugs that target the interaction between the tumor microenvironment and malignant cells, has provided many promising new approaches to cancer therapy. Ongoing dynamic and correlation studies of tumor biology and the contribution of the tumor microenvironment should be promoted in the context of novel drug development in order to identify optimal therapies for various lymphomas and improve the curability of these diseases.