Biomedical subjects
Daniel Olive
Publications and source records attributed to Daniel Olive.
Deficient expression of NCR in NK cells from acute myeloid leukemia: Evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction.
Natural killer (NK) cells play an important role in tumor-cell clearance, particularly against leukemia, as shown by killer cell inhibitory receptor (KIR)-mismatched allogeneic stem cell transplantation. Analysis of in vitro IL-2-expanded NK cells from patients with myelocytic/monocytic acute myeloid leukemia (AML-NK cells) has revealed poor cytolytic functions because of deficient expression of pivotal activation molecules-the natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46. To exclude the possibility that this observation was caused by the in vitro amplification of a small NCR(dull) population, we analyzed the AML-NK phenotype directly, without any in vitro expansion. We first confirmed that the NCR(dull) phenotype was not an in vitro artifact. Moreover, analysis of a large population of AML patients allowed us to demonstrate that phenotype was not restricted to a French-American-British (FAB) subtype and was not associated with a particular cytogenetic abnormality. Our longitudinal study of AML patients showed that the NCR(dull) phenotype was acquired during leukemia development because we observed its complete (for NKp46) or partial (for NKp30) reversibility in patients achieving complete remission (CR). Reversibility of the NCR(dull) phenotype after CR suggested that leukemia cells might be involved in NCR down-regulation. In agreement with this hypothesis, direct contact between leukemic blasts and NK cells (but not leukemia-cell supernatants) induced loss or decrease in NKp30 and NKp46 expression while impeding NKp44 induction by IL-2. We excluded the major implication of TGF-beta in NCR down-regulation. Although the clinical antitumor value of NK cells is clearly demonstrated in allogeneic stem cell transplantation, the role of NK cells in autologous transplantation is not proved. Interestingly, we observed a correlation between the NCR(dull) phenotype and poor survival in AML patients, suggesting that NK-deficient activation caused by NCR down-regulation could play a role in patient outcome. The prognostic value of NCR expression is discussed, and pathophysiologic implication of the NCR phenotype will be further investigated in a larger study.
Active transcription of the human FASL/CD95L/TNFSF6 promoter region in T lymphocytes involves chromatin remodeling: role of DNA methylation and protein acetylation suggest distinct mechanisms of transcriptional repression.
Fas ligand (FasL/CD95L/TNFSF6), a member of the tumor necrosis factor family, initiates apoptosis in lymphoid and nonlymphoid tissues by binding to its receptor Fas (CD95/TNFRSF6). Although the transcriptional control of TNFSF6 gene expression is subjected to intense study, the role of its chromatin organization and accessibility to the transcriptional machinery is not known. Here, we determined the chromatin organization of TNFSF6 gene 5' regulatory regions. Using the indirect end-labeling technique, a unique region named HSS1 and encompassing nucleotides -189 to +185 according to the transcriptional start site, was identified throughout a 20-kilobase nucleosomal DNA domain surrounding the promoter. The HSS1 region displayed hypersensitivity to in vivo DNase I digestion in TNFSF6-expressing cells only, including upon T cell activation. Hypersensitivity to micrococcal nuclease digestion and to specific restriction enzyme digestion suggested the precise positioning of two nucleosomes across the transcription start site and minimal promoter region, likely interfering with TNFSF6 active transcription in T lymphocytes. Indeed, HSS1 hypersensitivity to nuclease digestion strictly correlated with TNFSF6 transcription, including in primary and leukemia T cells. HSS1 chromatin remodeling preceded detectable TNFSF6 mRNA accumulation and was blocked by cycloheximide that also prevented TNFSF6 transcription. However, DNA methylation levels of the TNFSF6 HSS1 region did not correlate with transcriptional activation. Induction of global protein acetylation by treatment with histone deacetylase inhibitors was not accompanied by HSS1 chromatin remodeling and/or TNFSF6 transcription. We conclude that chromatin remodeling is a primary event in the activation of TNFSF6 expression in primary and leukemia T cells and that mechanisms independent of protein deacetylation and of DNA methylation of the TNFSF6 promoter region are involved in the repression of TNFSF6 gene expression.
The co-expression of 2B4 (CD244) and CD160 delineates a subpopulation of human CD8+ T cells with a potent CD160-mediated cytolytic effector function.
Within human CD8+ T lymphocytes, the CD27-CD45RAhigh or CD56+ phenotypes contribute to precisely define the cells with CTL effector function. Novel markers were demonstrated to correlate with CTL properties, such as the 2B4 (CD244) receptor, a member of the CD2 subset of the immunoglobulin superfamily or the glycosylphosphatidylinositol-anchored CD160 receptor. We performed a study of these markers to further define the population of effectors with CTL functions. Here we show that cytotoxic subpopulations defined by surface markers CD160, CD56 and CD57 are mostly contained in the 2B4+CD8+ T cell population. Expression of CD160 identifies two populations in the 2B4+ population. The 2B4+CD160+ subset expresses a bona fide CTL phenotype. The co-expression of 2B4 and CD160 defines T cells containing high amounts of perforin and granzyme B. During CTL ontogeny, an up-regulation of 2B4 and CD160 is observed from a naive to a terminally differentiated phenotype. Finally, we demonstrated that CD160 triggering failed to induce cytotoxicity per se, but costimulated CD3-redirected killing. We conclude that the co-expression of 2B4 and CD160 defines a CD8+ T lymphocyte subpopulation with high CTL activity.
[Lymphocyte coreceptors].
The activation of the immune system is tightly regulated by positive and negative receptors that allow the fine tuning of the immune cells. This regulation relies on receptors that were initially described in T lymphocytes, but have now been identified on cells from both innate and acquired immunity. The co-stimulatory receptors can allow cell activation or amplify it, regulate cell suvival and determine their effector functions. The co-inhibitory receptors can either prevent, decrease of inhibit the activation and differentiation process. The co-stimulatory and co-inhibitory molecules belong mainly to the so-called Ig superfamily and historically were called << CD28 and B7 family >>. The members of the tumor necrosis factor receptor (TNFR) family devoid of intra-cytoplasmic death domain but binding TNF receptor associated factors (TRAF) are also important but are up to now mainly co-stimulatory. The prototypical co-stimulatory molecules belonging to CD28 family are CD28 and ICOS, whereas the co-inhibitory molecules identified so far are CTLA-4, PD-1 and BTLA. Their receptors belong in most instances to the B7 family. For instance, B7.1/CD80 and B7.2/CD86 interact both with CD28 and CTLA-4 ; PDL1 and PDL2 bind to PD-1. The exception being so far BTLA which interacts with the TNFR family member HVEM (Herpes virus entry mediator). Three other B7 family members B7-H3, B7-H4 and BT3.1 are orphan receptors until now. The basis of co-inhibition rely on distinct mechanisms, one that has been postulated being the ability of the intracytoplasmic domain of PD-1 and BTLA to bind to the protein tyrosine phosphatases SHP-1 and SHP-2. The pathways used by the co-stimulatory receptors are also not completely understood and rely for CD28 both on signal similar to the one elicited by TcR and consequently increasing the overall signal and other more specific, elicited by the activation of PI3-OH kinase, vav1 and rearrangement of cytoskeleton. Recently, reverse signaling has been described for B7 family members which further increases the spectrum of functions elicited by these families. Co-stimulation and co-inhibition are among the most promising molecules and pathways to be targeted by mAbs, recombinant proteins and drugs in auto-immune diseases, transplantation and cancer.
Role for the CD28 molecule in the control of Coxiella burnetii infection.
Q fever is an infectious disease caused by Coxiella burnetii, an obligate intracellular bacterium that replicates in macrophages. As cell-mediated immune response to microbial pathogens requires signals mediated by T-cell receptors and costimulatory molecules such as CD28, we wondered if CD28 is involved in protection against C. burnetii infection. CD28-deficient (CD28-/-) mice were inoculated with C. burnetii by intraperitoneal and intravenous routes. With both wild-type and CD28-/- mice, C. burnetii organisms were detected exclusively in spleen and liver. The antibody response against C. burnetii was impaired in CD28-/- animals, but, surprisingly, the lack of CD28 decreased C. burnetii burden in the infected tissues, whatever the manner of inoculation of bacteria. The CD28 deficiency had no effect on either granuloma formation, which reflects cell-mediated immunity against C. burnetii, or the production of gamma interferon and tumor necrosis factor, two cytokines known to be involved in granuloma formation. On the other hand, the production of interleukin-10 (IL-10) by peritoneal macrophages was highly impaired in CD28-/- mice. The results suggest that CD28 initiates a signal that favors C. burnetii replication through the modulation of the IL-10 pathway.
T-cell: section report.
The antibodies submitted to the T-cell section were evaluated by different laboratories. We focused our studies on antibodies that reacted to defined molecules to the CD28 family and their ligands B7/butyrophilin family.
Inflammatory cytokines and acute graft-versus-host disease after reduced-intensity conditioning allogeneic stem cell transplantation.
This study investigated the role of inflammatory cytokines in acute graft-versus-host disease (aGVHD) incidence and severity in 113 patients who underwent reduced-intensity conditioning (RIC) allogeneic stem cell transplantation (allo-SCT). Among all tested cytokines in the first 3 months after allo-SCT, only interleukin-12 p70 (IL-12p70) levels in the first month were significantly associated with grades II to IV aGVHD development (P < .001). IL-12p70 levels were directly correlated with aGVHD severity grade (P < .001). Before aGVHD onset, blood monocytes, the main precursor pool of IL12p70-secreting dendritic cells, recovered more rapidly in patients with grades II to IV aGVHD (P = .005). Similarly, at the effector level, there was a more robust reconstitution of naive CD3+CD4+CD45RA+CD27+ T cells in patients developing grades II to IV aGVHD (P = .006). In multivariate analysis, IL-12p70 level measured in the first month was the strongest predictive factor for aGVHD development (P < .001). These findings, reconstituting a T(H)1 loop, support a model in which aGVHD reflects a type 1 alloreaction after RIC allo-SCT.
CD molecules 2005: human cell differentiation molecules.
The immune system works through leukocytes interacting with each other, with other cells, with tissue matrices, with infectious agents, and with other antigens. These interactions are mediated by cell-surface glycoproteins and glycolipids. Antibodies against these leukocyte molecules have provided powerful tools for analysis of their structure, function, and distribution. Antibodies have been used widely in hematology, immunology, and pathology, and in research, diagnosis, and therapy. The associated CD nomenclature is commonly used when referring to leukocyte surface molecules and antibodies against them. It provides an essential classification for diagnostic and therapeutic purposes. The most recent (8th) Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), held in Adelaide, Australia, in December 2004, allocated 95 new CD designations and made radical changes to its aims and future operational strategy in order to maintain its relevance to modern human biology and clinical practice.
Defective killing of dendritic cells by autologous natural killer cells from acute myeloid leukemia patients.
At the frontier between innate and adaptive immunity, dendritic cells (DCs) secrete numerous cytokines and express costimulatory molecules that initiate or enhance natural killer (NK) and T-lymphocyte responses. NK cells also regulate DC physiology by killing immature DCs (iDCs), thus limiting inflammation and inappropriate T-lymphocyte tolerization. In a previous study, we have reported that NK cells from acute myeloid leukemia patients (AML-NK cells) have deficient natural cytotoxicity receptor (NCR) expression. Herein, we analyzed the consequences of such a defect regarding the regulatory role of AML-NK cells in DC physiology. We show that NK cells display poor cytolytic capacities against DCs derived from healthy donor monocytes or derived from autologous leukemic blasts. These data point to a novel defect in the regulation of adaptive immune responses initiated by DCs in AML patients. This may lead to specific T-lymphocyte tolerization by spontaneous or ex vivo expanded iDCs expressing leukemia-derived antigens.
Imatinib: the narrow line between immune tolerance and activation.
Despite the rapid success of imatinib as a targeted cancer therapy, there seems to be some controversy about its influence on immune function. Research relating to the immunological effects of imatinib has aimed to gain insight into this paradoxical picture, whereby imatinib appears to be on the edge between immune tolerance and immune defence. In addition to the flexibility of several immune effectors, subtle but important experimental variations might explain the seemingly conflicting data, pointing to the possible use of imatinib to modulate immune responses against tumours. The clinical use of imatinib, and its putative immunomodulatory properties, represent challenging issues still to be resolved.
Current update on reduced intensity regimens for allogeneic stem cell transplantation.
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Tec kinase migrates to the T cell-APC interface independently of its pleckstrin homology domain.
Tec is the prototypical member of the Tec tyrosine kinases family, which plays an important role in T cell signaling. We show in this study that Tec translocates to the immunological synapse when a T cell contacts a dendritic cell. Surprisingly, the presence of the pleckstrin homology (PH) domain of Tec is not required for this accumulation, and despite a strong activation of 3'-phosphorylated phosphoinositide lipids synthesis during the synapse formation, the Tec PH domain is not redistributed to the T cell plasma membrane. In contrast, we demonstrate that an active Src homology 3 domain is absolutely required, underlining the essential role played by this part of the molecule in the recruitment and/or stabilization of Tec at the immunological synapse. Our results nevertheless suggest that the PH domain controls the kinase activity of the molecule in vivo. We finally demonstrate that the two domains are necessary to trigger transcriptional events following Ag presentation. These data support a model in which the plasma membrane recruitment of the PH-containing protein Tec is not dependent on the production of 3'-phosphorylated phosphoinositide lipids by the PI3K, but rather on an intact Src homology 3 domain.
Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment.
The integral membrane adaptor protein linker for activation of T cells (LAT) couples the T-cell receptor (TCR) with downstream signalling and is essential for T-cell development and activation. Here, we investigate the dynamic distribution of LAT-GFP fusion proteins by time-lapse video imaging of live T lymphocytes interacting with antigen-presenting cells. We show that LAT forms two distinct cellular pools, one at the plasma membrane and one that co-distributes with transferrin-labelled intracellular compartments also containing the TCR/CD3-associated zeta chain. The distribution of LAT between these two pools is dependent on LAT intracytoplasmic residues. Whereas plasma membrane-associated LAT is recruited to immune synapses after a few seconds of cell conjugate formation, the intracellular pool is first polarized and then recruited after a few minutes. We further show that LAT intracytoplasmic amino acid residues, particularly the Tyr136, 175, 195 and 235 residues, are required for its own recruitment to the immune synapse and that a herein-identified juxtamembrane LAT region (amino acids 32-104) is involved in the localization of LAT in intracellular pools and in T-cell signalling. Altogether, our results demonstrate that LAT controls its own recruitment at the immune synapse, where it is required as a scaffold protein for the signalling machinery. The results also suggest that the intracellular pool of LAT might be required for T-cell activation.
Functional interaction of RasGAP-binding proteins Dok-1 and Dok-2 with the Tec protein tyrosine kinase.
The Dok adaptor family of proteins binding to RasGAP, consisting of Dok-1 and Dok-2, are critical regulators in cell proliferation. These molecules are partners and/or substrates of different protein tyrosine kinases considered as oncoproteins. Here, we show that Dok-1 and Dok-2 are the major tyrosine-phosphorylated proteins associated to Tec, a protein tyrosine kinase expressed in T cells. Furthermore, we evaluate the effect of Dok-1 or Dok-2 on Tec-mediated signalling pathways in T cells. Here, we provide evidence that Dok-1 and Dok-2 proteins are involved in a negative feedback regulation of Tec via a downregulation of its tyrosine phosphorylation and downstream signalling pathways including the Ras pathway. Either Dok-1 or Dok-2 therefore represents a mean of potent retrograde control for protein tyrosine kinase signalling, and then possibly of tumor development.
Intensive sequential chemotherapy with hematopoietic growth factor support for non-Hodgkin lymphoma in patients infected with the human immunodeficiency virus.
BACKGROUND: Optimal treatment of human immunodeficiency virus (HIV)-associated non-Hodgkin lymphoma (NHL) has yet to be defined, because chemotherapy could exacerbate immunodeficiency, with subsequent adverse effects for patients. METHODS: The authors investigated the feasibility of an intensive chemotherapy regimen for HIV-associated NHL. Thirty-eight patients were treated with a first course of cyclophosphamide (Cy), vincristine, and prednisone; followed by 3 courses of high-dose Cy (2000 mg/m2), doxorubicin (Doxo; 50 mg/m2), vincristine, and prednisone (modified high-dose CHOP); 1 course of high-dose methotrexate (MTX; 8000 mg/m2); and 1 course of high-dose cytarabine (8000 mg/m2). Radiotherapy was added to the treatment regimen for patients with bulky disease or residual tumor. Chemotherapy was administered in conjunction with granulocyte-colony-stimulating factor and antiretroviral therapy. RESULTS: Patients received 91.5%, 93%, 66%, and 63% of the scheduled doses of Cy, Doxo, MTX, and cytarabine, respectively. The complete response rate was 60.5%, with a total response rate of 79%. The 40-month overall survival rate was 43%, the disease-free survival rate was 65%, and the recurrence-free survival rate was 39%. Both an International Prognostic Index score of 0 or 1 and Burkitt-type histology had positive effects on survival, whereas CD4-positive lymphocyte counts, viral burden, and previous highly active antiretroviral therapy did not. CD4-positive T lymphocyte levels decreased from 0.197 +/- 0.156 x10(9)/L before treatment to 0.152 +/- 0.1 x10(9)/L at 6 months after the end of treatment. A decrease in viral load, from 380,000 +/- 785,000 copies/mL before treatment to 25,000 +/- 43,000 copies/mL at 6 months after the end of treatment, also was observed. CONCLUSIONS: The results of the current study indicate that intensive chemotherapy is effective and tolerable for patients with HIV-associated NHL.
Imatinib and plasmacytoid dendritic cell function in patients with chronic myeloid leukemia.
Plasmacytoid dendritic cells (PDCs) are crucial effectors in innate immunity. In this study, we show that imatinib, a potent inhibitor of BCR/ABL tyrosine kinase activity, in the presence of Flt3-Ligand, could induce CD34+ progenitors from chronic myeloid leukemia (CML) to give rise in vitro to typical BDCA-2+ type I interferon-producing PDCs. The effect of imatinib on PDC generation was related to up-regulation of Flt3 on leukemic CD34+ progenitors. Moreover, patients with chronic myeloid leukemia (CML) who were in complete cytogenetic or molecular response after imatinib treatment restored their blood PDCs both quantitatively and functionally comparable to healthy donors, in contrast to patients not responding to imatinib, further confirming that disease response to imatinib is accompanied by restoration of PDC function in vivo. These findings provide evidence that response to imatinib is capable to restore some DC-related immune functions in CML that might be beneficial for long-term disease control.
The SH3 domain of Tec kinase is essential for its targeting to activated CD28 costimulatory molecule.
The Tec family of protein tyrosine kinases plays an important role in T cell signaling. Tec, the prototypical member of this kinase family, can interact with CD28, which is a costimulatory molecule. However, the regulation of Tec upon CD28 stimulation remains poorly understood. Here we show that CD28-B7-mediated interactions are likely involved in the relocalization of Tec at the contact zone between T cells and APC. Upon CD28 ligation with specific antibodies or natural ligands, Tec translocates to the plasma membrane where it colocalizes with the CD28 molecule. The Src-homology 3(SH3) domain of Tec and the two proline-rich motifs of CD28 are involved in this process. Furthermore, we show that CD28 signaling requires the SH3 domain of Tec as well as proline residues present in the intracytoplasmic tail of CD28. These results should provide new insights into the complex regulation of Tec kinases in T cells.