Patients suffering from acute graft-versus-host disease after bone-marrow transplantation have functional CD4+CD25hiFoxp3+ regulatory T cells.

Acute Graft-Versus-Host Disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell (HSC) transplantation. Naturally-occurring CD4(+)CD25(hi)(Foxp3(+)) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine graft models. In this report, we investigated their role in the prevention of aGVHD in patients transplanted with bone-marrow-derived HSC. When CD4(+)CD25(hi)Foxp3(+) T cells were isolated from bone-marrow grafts, they showed no suppressive activity. The analysis of their function in patients suffering from aGVHD after transplantation revealed a gain of suppressive activity indicating their inability to control the aGVHD induction. Thus, our findings clearly demonstrate that CD4(+)CD25(+) and CD4(+)CD25(hi)Foxp3(+) T cells, when administered in steady-state physiological conditions, do not influence the outcome of aGVHD after bone-marrow transplantation.

Soluble human leucocyte antigen-G molecules in peripheral blood haematopoietic stem cell transplantation: a specific role to prevent acute graft-versus-host disease and a link with regulatory T cells.

Haematopoietic stem cell transplantation is often complicated by the life-threatening graft-versus-host disease (GVHD) which consists of an allogeneic reaction of the graft cells against the host organs. The aim of this study was to investigate the putative involvement of soluble human leucocyte antigen (sHLA) class I molecules, and particularly sHLA-G molecules, in the occurrence and/or prevention of acute GVHD (aGVHD) in allogeneic peripheral blood stem cell (PSC) transplantation. Whole sHLA class I molecules seem to be involved in aGVHD pathogenesis because detection of a high concentration of these molecules in the first month post allograft is correlated with aGVHD occurrence. Conversely, a high level of sHLA-G molecules before and after allograft could indicate good prognosis in PSC allograft transplantation. sHLA-G molecules seem to be involved in aGVHD prevention, not only because they are enriched in plasma of patients without aGVHD, but also because: (i) a positive correlation has been found between sHLA-G level and CD4+ CD25+ CD152+ natural regulatory T cell (T(reg)) frequency in the blood of transplanted patients; and (ii) the presence of CD4+ CD25+ CD152+ natural T(reg) is correlated with increased sHLA-G expression in in vitro mixed leucocyte reaction cultures. Altogether, these results support the immunomodulatory function of sHLA-G molecules that might create a regulatory network together with the natural T(reg) to foster the induction of a tolerogenic environment and improve PSC transplantation favourable outcome.

High multidrug resistance protein activity in acute myeloid leukaemias is associated with poor response to chemotherapy and reduced patient survival.

Multidrug resistance protein (MRP) activity was investigated in 44 newly diagnosed acute myeloid leukaemia (AML) patients using a functional assay based on efflux of carboxy-2',7'-dichlorofluorescein, an anionic dye handled by both MRP1 and MRP2. Elevated MRP transport was detected in 29% of cases, but was not significantly correlated with sex, age, white blood cell count at diagnosis or karyotype. In contrast, it was associated with secondary AML (P = 0.002), CD34 positivity (P = 0.041) and P-glycoprotein activity (P = 0.01). There was a lower rate of complete remission in MRP-positive patients versus MRP-negative patients (23% versus 81%; P = 0.001); overall survival was also better for MRP-negative patients (P = 0.004). These data indicate a probable role for MRP activity in the clinical outcome of AML.

Differential expression of the efflux pumps P-glycoprotein and multidrug resistance-associated protein in human monocyte-derived dendritic cells.

P-glycoprotein (P-gp), an ATP-binding cassette (ABC) drug efflux pump, has been recently shown to play an important role in the physiology of Langherans cells, a subtype of dendritic cells (DC) found in the skin. The present study was designed to investigate expression and activity of P-gp and of multidrug resistance-associated protein (MRP), another ABC efflux pump sharing numerous substrates with P-gp, in human monocyte-derived DC. Immunolabeling experiments and dye efflux assays indicated that such cells displayed elevated levels of MRP activity and expression when compared to those present in parental monocytes. Generation of DC from monocytes in the presence of the MRP inhibitor indomethacin did not, however, alter the capacity of DC to stimulate allogeneic T cells proliferation in mixed lymphocyte reaction. In addition, indomethacin did not inhibit the up-regulation of the CD1a, a marker occurring during the differentiation of monocytes into DC. In contrast to that of MRP, functional expression of P-gp was not detected in monocyte-derived DC. Such antigen presenting cells that constitute a promising tool for antitumor vaccinal therapy therefore display differential expression of the efflux pumps P-gp and MRP.

Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from peripheral blood.

Multidrug resistance proteins (MRPs) such as MRP1, MRP2 and MRP3 are membrane efflux pumps involved in multidrug resistance and handling organic anions. In the present study, MRP activity was investigated in normal mature leucocytes and CD34-positive hematopoietic cells from peripheral blood using the flow cytometric carboxy-2',7'-dichlorofluorescein (CF) efflux assay. Basal and similar cellular exports of CF, an anionic fluorescent dye substrate for MRP1 and MRP2 transporters, were evidenced in lymphocytes whatever their subsets (CD3, CD4, CD8, CD20 and CD56 cells), in CD14 monocytes and in CD15 granulocytes whereas higher CF efflux was found in CD34 cells. Such outwardly-directed transports of CF were inhibited by known blockers of MRP function such as probenecid whereas the P-glycoprotein modulator verapamil did not alter the retention of the dye in the blood leukocytes. Peripheral mature blood leukocytes were moreover found to express MRP1 mRNAs and MRP1 protein as assessed by Northern-blot and Western-blot analyses, whereas MRP2 and MRP3 transcripts were not present or only at very low levels. Mature leukocytes therefore display basal constitutive MRP-related transport activity regardless of cell lineage and likely related to MRP1 expression whereas higher MRP-related efflux can be detected in peripheral CD34 hematopoietic cells.

HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC.

Professional APC are characterized by their ability to present peptide via HLA class II in the presence of costimulatory molecules (CD40, CD80, and CD86). The efficiency of Ag presentation can be classed as follows: mature dendritic cells (DC) are most efficient, immature DC and macrophages are intermediate, and monocytes are considered poor APC. There is a large body of evidence demonstrating that HLA-DR transmits signals in the APC. In this study, we have addressed the question of the outcome of HLA-DR signals on APC of the monocyte/DC lineages throughout their differentiation from immature to mature APC. DC were generated from both monocytes and CD34+ cells of the same individual, macrophages were differentiated from monocytes. Immunophenotypical analysis clearly distinguished these populations. HLA-DR-mediated signals led to marked apoptosis in mature DC of either CD34 or monocytic origin. Significantly less apoptosis was observed in immature DC of either origin. Nonetheless, even immature DC were more susceptible to HLA-DR-mediated apoptosis than macrophages, whereas monocytes were resistant to HLA-DR-mediated apoptosis. The mechanism of HLA-DR-mediated apoptosis was independent of caspase activation. Taken together, these data lead to the notion that signals generated via HLA-DR lead to the demise of mature professional APC, thereby providing a means of limiting the immune response.

Molecular cloning of a lipolysis-stimulated remnant receptor expressed in the liver.

The lipolysis-stimulated receptor (LSR) is a lipoprotein receptor primarily expressed in the liver and activated by free fatty acids. Antibodies inhibiting LSR functions showed that the receptor is a heterotrimer or tetramer consisting of 68-kDa (alpha) and 56-kDa (beta) subunits associated through disulfide bridges. Screening of expression libraries with these antibodies led to identification of mRNAs derived by alternate splicing from a single gene and coding for proteins with molecular masses matching that of LSR alpha and beta. Antibodies directed against a synthetic peptide of LSR alpha and beta putative ligand binding domains inhibited LSR activity. Western blotting identified two liver proteins with the same apparent molecular mass as that of LSR alpha and beta. Transient transfections of LSR alpha alone in Chinese hamster ovary cells increased oleate-induced binding and uptake of lipoproteins, while cotransfection of both LSR alpha and beta increased oleate-induced proteolytic degradation of the particles. The ligand specificity of LSR expressed in cotransfected Chinese hamster ovary cells closely matched that previously described using fibroblasts from subjects lacking the low density lipoprotein receptor. LSR affinity is highest for the triglyceride-rich lipoproteins, chylomicrons, and very low density lipoprotein. We speculate that LSR is a rate-limiting step for the clearance of dietary triglycerides and plays a role in determining their partitioning between the liver and peripheral tissues.