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1.
Cytotherapy ; 21(6): 612-618, 2019 06.
Article in English | MEDLINE | ID: mdl-31056424

ABSTRACT

INTRODUCTION: Cell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34+ quantification; (ii) percentage of CD34+ viability and (iii) evaluation of HSC functional ability to form colony forming unit-granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34+, using a Fluorescent Labeled Inhibitor of CAspases (FLICA). METHODS: Caspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34+, CD3+, CD14+ and CD56+ cells. Viable 7AAD-/FLICA+ cells were then correlated with various parameters. RESULTS: We showed a significant caspase pathway activation, with 23% CD34+/7AAD-/FLICA+ cells after thawing, compared with the 2% described in fresh CD34+ cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3+, CD56+ and CD14+ cells. We also report a significant correlation between the rate of CD34+/7AAD-/FLICA+ cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004). DISCUSSION: Our results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3+ and CD56+ subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).


Subject(s)
Antigens, CD34/metabolism , Cryopreservation/methods , Granulocytes/physiology , Peripheral Blood Stem Cell Transplantation/methods , Peripheral Blood Stem Cells/cytology , Adolescent , Adult , Aged , Apoptosis/physiology , Autografts , Caspases/metabolism , Female , Flow Cytometry , Granulocytes/cytology , Humans , Lymphoma/pathology , Lymphoma/therapy , Male , Middle Aged , Multiple Myeloma/pathology , Multiple Myeloma/therapy , Peripheral Blood Stem Cells/physiology , Transplantation, Autologous , Young Adult
2.
BMC Cancer ; 14: 727, 2014 Sep 29.
Article in English | MEDLINE | ID: mdl-25265970

ABSTRACT

BACKGROUND: Mutations in the activation segment of the v-raf murine sarcoma viral oncogene homolog B (BRAF) gene are present in approximately 50% of melanomas. The selective BRAF inhibitor vemurafenib has demonstrated significant clinical benefits in patients with melanomas harboring the most common mutations (V600E, V600K and V600R). However, the clinical activity of BRAF inhibitors in patients with rare mutations of codon 600 and the surrounding codons has not been documented. CASE PRESENTATION: We used the BRAF inhibitor vemurafenib to treat a patient presenting a rare p.V600_K601delinsD-mutated melanoma. An objective response was evidenced by two months of progression-free survival. By cloning and sequencing BRAF exon 15, we confirmed that a dual mutation was present on a single allele and thus resulted in a BRAFV(600DK601del) mutant protein. We also performed an in silico crystal structure analysis of the mutated protein, in order to characterize the nature of the putative interaction between vemurafenib and the mutant protein. CONCLUSION: This clinical experience suggests that (i) patients with BRAFV(600DK601del)-mutation-positive melanoma can be treated successfully with the oral BRAF inhibitor vemurafenib and (ii) molecular screening in this context should encompass rare and complex mutations.


Subject(s)
Antineoplastic Agents/administration & dosage , Indoles/administration & dosage , Melanoma/drug therapy , Melanoma/genetics , Proto-Oncogene Proteins B-raf/genetics , Sulfonamides/administration & dosage , Aged , Antineoplastic Agents/therapeutic use , Female , Humans , Indoles/therapeutic use , Models, Molecular , Neoplasm Metastasis/drug therapy , Point Mutation , Protein Structure, Tertiary , Proto-Oncogene Proteins B-raf/chemistry , Sequence Deletion , Sulfonamides/therapeutic use , Treatment Outcome , Vemurafenib
3.
Front Immunol ; 5: 292, 2014.
Article in English | MEDLINE | ID: mdl-24982661

ABSTRACT

B-cell chronic lymphocytic leukemia (CLL) is the most frequent adult leukemia in the Western world. It is a heterogeneous disease characterized by clonal proliferation and the accumulation of CD5(+) mature B lymphocytes. However, the normal counterpart from which the latter cells arise has not yet been identified. CD27 expression and gene expression profiling data suggest that CLL cells are related to memory B-cells. In vitro, memory B-cells differentiate into plasma cells when stimulated with CpG oligodeoxynucleotide (CpG). The objective of the present study was therefore to investigate the ability of CpG, in the context of CD40 ligation, to induce the differentiation of CLL B-cells into antibody-secreting cells (ASCs). CD20(+)CD38(-) CLL B-cells were stimulated with a combination of CpG, CD40 ligand and cytokines (CpG/CD40L/c) in a two-step, 7-day culture system. We found that the CpG/CD40L/c culture system prompted CLL B-cells to differentiate into CD19(+)CD20(+)CD27(+)CD38(-)ASCs. These cells secreted large amounts of IgM and had the same shape as plasma cells. However, only IgMs secreted by ASCs that had differentiated from unmutated CLL B-cells were poly/autoreactive. Class-switch recombination (CSR) to IgG and IgA was detected in cells expressing the activation-induced cytidine deaminase gene (AICDA). Although these ASCs expressed high levels of the transcription factors PRDM1 (BLIMP1), IRF4, and XBP1s, they did not downregulate expression of PAX5. Our results suggest that CLL B-cells can differentiate into ASCs, undergo CSR and produce poly/autoreactive antibodies. Furthermore, our findings may be relevant for (i) identifying the normal counterpart of CLL B-cells and (ii) developing novel treatment strategies in CLL.

4.
PLoS One ; 9(6): e99601, 2014.
Article in English | MEDLINE | ID: mdl-24927415

ABSTRACT

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. We recently characterized for the first time the expression of Signaling Lymphocyte Activating Molecule 3 (SLAMF3) in human hepatocytes and here, we report that SLAMF3 interacts with the HCV viral protein E2 and is implicated in HCV entry process. We found a strong correlation between SLAMF3 expression level and hepatocyte susceptibility to HCV infection. The use of specific siRNAs to down-modulate SLAMF3 expression and SLAMF3-blocking antibodies both decreased the hepatocytes susceptibility to HCV infection. Moreover, SLAMF3 over-expression significantly increased susceptibility to HCV infection. Interestingly, experiments with peptides derived from each SLAMF3 domain showed that the first N-terminal extracellular domain is essential for interaction with HCV particles. Finally, we showed that recombinant HCV envelop protein E2 can bind SLAMF3 and that anti-SLAMF3 antibodies inhibited specifically this interaction. Overall, our results revealed that SLAMF3 plays a role during HCV entry, likely by enhancing entry of viral particle within hepatocytes.


Subject(s)
Antigens, CD/metabolism , Hepacivirus/physiology , Hepatitis C/virology , Viral Envelope Proteins/metabolism , Antigens, CD/chemistry , Antigens, CD/genetics , Antigens, CD/immunology , Cell Line, Tumor , Gene Knockdown Techniques , Hepatitis C/genetics , Hepatitis C/metabolism , Hepatocytes/metabolism , Hepatocytes/virology , Humans , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signaling Lymphocytic Activation Molecule Family , Tetraspanin 28/pharmacology , Viral Envelope Proteins/immunology
5.
Immunol Cell Biol ; 92(7): 591-604, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24797583

ABSTRACT

In this study, we investigated the capacity of chronic lymphocytic leukemia (CLL) B cells to undergo terminal differentiation into Ig-secreting plasma cells in T cell-independent and T cell-dependent responses. We used a two-step model involving stimulation with phorbol myristate acetate (PMA) and CD40L, together with cytokines (PMA/c and CD40L/c), for 7 days. We describe immunophenotypic modifications, changes in the levels of mRNA and protein for transcription factors and morphological and functional events occurring during the differentiation of CLL B cells into antibody-secreting cells (ASCs). The induction of differentiation differed significantly between the CD40L/c and PMA/c culture systems. The PMA/c culture system allowed CLL B cells to differentiate into IgM-secreting cells with an immunophenotype and molecular profile resembling those of preplasmablasts. By contrast, CD40L/c-stimulated cells had a phenotype and morphology similar to those of activated B cells and resembling those of the CLL B cells residing in the lymph node and bone marrow. These data suggest that the CLL B cells are not frozen permanently at a stage of differentiation and are able to differentiate into ASCs as appropriate stimulation are provided. The data presented here raise questions about the molecular processes and stimulation required for CLL B-cell differentiation and about the inability of CD40 ligand to induce differentiation of the CLL B cells.


Subject(s)
Antibody Formation/immunology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , CD40 Ligand/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Tetradecanoylphorbol Acetate/immunology , Aged , Aged, 80 and over , Antigens, Surface/metabolism , B-Lymphocytes/pathology , Cell Differentiation , Cytokines/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Immunoglobulin M/biosynthesis , Immunophenotyping , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged , Phenotype , Transcription, Genetic
6.
Immunology ; 141(2): 268-75, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24134819

ABSTRACT

In B cells, B-cell receptor (BCR) immunoglobulin revision is a common route for modifying unwanted antibody specificities via a mechanism called VH replacement. This in vivo process, mostly affecting heavy-chain rearrangement, involves the replacement of all or part of a previously rearranged IGHV gene with another germline IGHV gene located upstream. Two different mechanisms of IGHV replacement have been reported: type 1, involving the recombination activating genes complex and requiring a framework region 3 internal recombination signal; and type 2, involving an unidentified mechanism different from that of type 1. In the case of light-chain loci, BCR immunoglobulin editing ensures that a second V-J rearrangement occurs. This helps to maintain tolerance, by generating a novel BCR with a new antigenic specificity. We report that human B cells can, surprisingly, undergo type 2 replacement associated with κ light-chain rearrangements. The de novo IGKV-IGKJ products result from the partial replacement of a previously rearranged IGKV gene by a new germline IGKV gene, in-frame and without deletion or addition of nucleotides. There are wrcy/rgyw motifs at the 'IGKV donor-IGKV recipient chimera junction' as described for type 2 IGHV replacement, but activation-induced cytidine deaminase (AID) expression was not detected. This unusual mechanism of homologous recombination seems to be a variant of gene conversion-like recombination, which does not require AID. The recombination phenomenon described here provides new insight into immunoglobulin locus recombination and BCR immunoglobulin repertoire diversity.


Subject(s)
Cytidine Deaminase/physiology , Homologous Recombination , Immunoglobulin Variable Region/genetics , Receptors, Antigen, B-Cell/genetics , Amino Acid Sequence , Base Sequence , Cell Line , Gene Rearrangement , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin kappa-Chains/genetics , Interleukin-1/pharmacology , Lipopolysaccharides/pharmacology , Molecular Sequence Data
7.
J Biol Chem ; 286(7): 5034-42, 2011 Feb 18.
Article in English | MEDLINE | ID: mdl-21106534

ABSTRACT

STAT5a and -5b (signal transducers and activators of transcription 5a and 5b) proteins play an essential role in hematopoietic cell proliferation and survival and are frequently constitutively active in hematologic neoplasms and solid tumors. Because STAT5a and STAT5b differ mainly in the carboxyl-terminal transactivation domain, we sought to identify new proteins that bind specifically to this domain by using a bacterial two-hybrid screening. We isolated hTid1, a human DnaJ protein that acts as a tumor suppressor in various solid tumors. hTid1 interacts specifically with STAT5b but not with STAT5a in hematopoietic cell lines. This interaction involves the cysteine-rich region of the hTid1 DnaJ domain. We also demonstrated that hTid1 negatively regulates the expression and transcriptional activity of STAT5b and suppresses the growth of hematopoietic cells transformed by an oncogenic form of STAT5b. Our findings define hTid1 as a novel partner and negative regulator of STAT5b.


Subject(s)
HSP40 Heat-Shock Proteins/metabolism , STAT5 Transcription Factor/metabolism , Animals , COS Cells , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Chlorocebus aethiops , HSP40 Heat-Shock Proteins/genetics , Hematopoietic Stem Cells/metabolism , Humans , Jurkat Cells , Mice , Protein Structure, Tertiary , STAT5 Transcription Factor/genetics , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
8.
Biochem J ; 390(Pt 1): 359-66, 2005 Aug 15.
Article in English | MEDLINE | ID: mdl-15833084

ABSTRACT

The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel-JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA-Binding Proteins/metabolism , Milk Proteins/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , ras Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line , Cell Proliferation , GRB2 Adaptor Protein , Mutation , Protein Binding , Proto-Oncogene Proteins c-akt , STAT5 Transcription Factor , Signal Transduction
9.
Oncogene ; 23(17): 3040-7, 2004 Apr 15.
Article in English | MEDLINE | ID: mdl-15048088

ABSTRACT

The transcription factors STAT5A and STAT5B (STAT: signal transducer and activator of transcription) play a major role in the signaling events elicited by a number of growth factor and cytokine receptors. In this work, we aimed to investigate the role of STAT5 in human precursor B cell survival by introducing dominant-negative (DN) forms of STAT5A or STAT5B in the 697 pre-B cell line. All clones expressing DN forms of either transcription factor exhibited a higher spontaneous apoptotic rate that was massively enhanced upon interleukin-7 (IL-7) stimulation. This was associated with caspase 8 cleavage, mitochondrial transmembrane potential disruption and caspase 3 activation. However, the DN forms of STAT5 did not alter the expression of Bcl-2, Bax, Bcl-x, Bim, A1 and Mcl1 proteins in IL-7-stimulated cells. The pancaspase inhibitor Z-Val-Ala-Asp-fluoromylmethyl ketone partially suppressed IL-7-mediated mitochondrial transmembrane potential disruption and cell death, suggesting that IL-7 induced the death of DN STAT5 expressing 697 cells through caspase-dependent and -independent mechanisms that both require mitochondrial activation.


Subject(s)
Apoptosis/immunology , B-Lymphocytes/immunology , Caspases/metabolism , DNA-Binding Proteins/genetics , Interleukin-7/pharmacology , Milk Proteins , Trans-Activators/genetics , Apoptosis/drug effects , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , B-Lymphocytes/physiology , Caspase 3 , Cell Death/drug effects , Cell Line , Humans , Mitochondria/drug effects , Mitochondria/immunology , STAT5 Transcription Factor , Tumor Suppressor Proteins
10.
Eur J Immunol ; 33(5): 1372-81, 2003 May.
Article in English | MEDLINE | ID: mdl-12731064

ABSTRACT

We have previously shown that Fas-induced apoptosis is markedly enhanced by IL-7 in human pre-B but not pro-B cell lines. In addition, pre-B cell receptor (pre-BCR) ligation significantly potentiates the IL-7 effects on Fas-triggered pre-B cell death. We show herein that transforming growth factor (TGF)-beta 1 sharply reduces Fas-induced death rate of pre-B but not pro-B cells. TGF-beta 1 causes inhibition of Fas-mediated disruption of mitochondrial transmembrane potential and cleavage of caspase 8, Bid and caspase 3. Bcl2 expression is markedly increased in TGF-beta 1-treated pre-B cells, whereas cellular FLICE-like inhibitory protein long (c-FLIPL), Bcl-XL, Bax, and Bad expression remains unchanged. TGF-beta 1 causes a selective growth arrest of pre-B cells in G0/G1 phase of the cell cycle and induces a partial down-modulation of both Fas and pre-BCR expression. All TGF-beta 1-mediated effects, but Bcl2 up-regulation, can be reproduced by the LY294002 phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor but not by inhibitors of the MAPK/ERK (MEK) and Janus kinase (Jak)/STAT pathways, which promote cell death. Akt phosphorylation is strongly inhibited by TGF-beta1 in pre-B but not pro-B cells and is not modified by Fas engagement. Altogether, our findings suggest that TGF-beta1 prevents Fas-induced apoptosis of pre-B lines by inhibiting PI3K pathway and by enhancing expression of Bcl2. They also suggest that the PI3K/Akt pathway is involved in the control of Fas and pre-BCR expression, a checkpoint in B cell development.


Subject(s)
Apoptosis/drug effects , B-Lymphocytes/physiology , Hematopoietic Stem Cells/physiology , Protein Serine-Threonine Kinases , Transforming Growth Factor beta/pharmacology , fas Receptor/physiology , B-Lymphocytes/drug effects , Cell Division/drug effects , Cell Line , Hematopoietic Stem Cells/drug effects , Humans , Mitochondria/physiology , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-bcl-2/analysis , Transforming Growth Factor beta1
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