Total soluble HLA class I and soluble HLA-G in multiple myeloma and monoclonal gammopathy of undetermined significance.

Serum beta2-microglobulin, the light chain of the HLA class I molecular complex, remains one of the best survival prognostic factors in multiple myeloma, but other HLA class I molecules might be of interest in monoclonal gammopathies. In this study, we evaluate total soluble HLA class I (HLA-Is) and soluble HLA-G (HLA-Gs) in 103 patients with newly diagnosed multiple myeloma, 30 patients with monoclonal gammopathy of undetermined significance (MGUS), and 30 healthy subjects, studying their prognostic value in multiple myeloma. In multiple myeloma patients, HLA-Is and HLA-Gs median values were 0.8 microg/mL and 28 ng/mL, respectively. Median HLA-Is concentration was higher in stage II and III multiple myeloma patients than in stage I multiple myeloma, MGUS, and control patients. Median HLA-Gs was significantly lower in healthy controls than in MGUS and multiple myeloma patients. A high level of HLA-Is (> or =2.1 microg/mL) was predictive of short survival (P = 0.017). For each given level of beta2-microglobulin, the relative risk of death was higher for patients with HLA-Is > or = 2.1 microg/mL than in patients with a lower level (P = 0.047). HLA-Gs, a marker of monoclonal gammopathy, was of no prognostic value, but the addition of HLA-Is to beta2-microglobulin produced an efficient prognostic score (P < 0.0001). HLA-Is is a new marker of multiple myeloma tumor load and provides additional survival prognostic information to beta2-microglobulin.

MHC class II signaling function is regulated during maturation of plasmacytoid dendritic cells.

Dendritic cells (DC) play a central role in the immune response, linking innate and adaptative responses to pathogens. Myeloid DC (MDC) produce interleukin-12 in response to bacterial stimuli, whereas plasmacytoid DC (PDC) produce high levels of type I interferon upon viral infection. Human leukocyte antigen (HLA)-DR engagement has been shown to induce apoptosis in various antigen-presenting cells (APC). We now report the consequences of HLA-DR molecule engagement in human PDC, which had thus far not been studied as a result of the difficulty in isolating such cells. HLA-DR engagement on PDC, obtained using a two-step, immunomagnetic separation, led to recruitment of HLA-DR molecules at the site of engagement in mature but not immature PDC. In contrast, relocalization of protein kinase C (PKC) isoenzymes, indicating PKC activation, was observed at the site of HLA-DR engagement and was accompanied by relocalization of a lipid raft marker, the ganglioside M1 staining, in immature and mature PDC. Similar to MDC, HLA-DR-mediated apoptosis was regulated throughout PDC maturation. Freshly isolated PDC were resistant, whereas CD40 ligand-matured PDC were sensitive to HLA-DR-mediated apoptosis. Neither caspase activation nor PKC activation was required for HLA-DR-mediated apoptosis. However, the intrinsic pathway of apoptosis was implicated as mature PDC underwent mitochondrial depolarization in response to HLA-DR engagement. These data provide further arguments for considering HLA-DR-mediated apoptosis as a conserved mechanism of regulating survival of diverse APC and support the ongoing development of humanized ligands for HLA class II molecules as therapeutic tools for use in lymphoproliferative disease.

Capacity of myeloid and plasmacytoid dendritic cells especially at mature stage to express and secrete HLA-G molecules.

Human leukocyte antigen (HLA-G), a class Ib major histocompatibility complex molecule, is potentially relevant in the immune response through its various immune cell functions. Its expression noticed in some malignancies has also been shown on macrophages and dendritic cells (DC) in tumoral and inflammatory diseases. As DC constitute a key component in the immune response, this work aimed at assessing the expression of HLA-G at transcriptional and proteic levels during differentiation and maturation of the different DC subsets. We show that HLA-G transcription was induced during CD34+-derived DC differentiation and is associated with a cell-surface expression in half of cases and with a substantial secretion of soluble HLA-G in all cases. Results were very similar for monocyte-derived DC, but there was still a weak HLA-G cell-surface expression and a lower level of secretion. On the contrary, HLA-G transcription was weak in plasmacytoid DC without any HLA-G cell-surface expression and with a basal level of secretion. The mechanisms involved in HLA-G expression appear transcriptional and post-transcriptional. However, the amount of HLA-G transcripts and the expression of the protein are not related. HLA-G expression or secretion by DC may have negative consequences on the function of effective immune cells and also on DC themselves via the interaction with inhibitory receptors expressed by these cells. The capacity of DC to express or secrete HLA-G should be studied in the context of cellular therapy using DC in addition to its suppressive action in immune response.

Soluble HLA-G inhibits human dendritic cell-triggered allogeneic T-cell proliferation without altering dendritic differentiation and maturation processes.

The role of the nonclassical human leukocyte antigen (HLA) class Ib molecule HLA-G in immune tolerance was first reported at maternofetal interface. This immunomodulating role could be exerted more generally in tumoral or post-transplantation situations in inhibiting natural killer (NK) and T-lymphocyte mediated lysis. Among the different transcripts resulting from alternative splicing, the mainly secreted isoform, HLA-G5, corresponds to complete molecule and has been demonstrated to be elevated in melanomas and in serum from heart-transplanted patients. As dendritic cells expressed ILT4, an inhibitory receptor capable of interacting with HLA-G, we have studied the effect of soluble HLA-G (HLA-G5) on differentiation, maturation, apoptosis and function of monocyte or CD34+-derived dendritic cells (DC). Soluble HLA-G did not alter differentiation, maturation or apoptosis of DC whatever their origin. On the other hand, an inhibitory effect of HLA-G5 on T lymphocytes proliferation was found in 53% of mixed leukocyte reactions (MLR) and was variable in intensity. These data demonstrate an indirect way of HLA-G5 action on DC occurring via T lymphocytes that reinforces the immune inhibitory role of soluble HLA-G capable to be secreted during tumoral malignancies or following heart transplantation.

Soluble HLA-G molecules are increased in lymphoproliferative disorders.

The immunomodulatory properties of soluble human leukocyte antigen G (sHLA-G) explain its potential interest in malignancies. HLA-G frequently transcribed in lymphoproliferative disorders is rarely expressed at cell surface. In this article, we will demonstrate that the plasmatic level of soluble HLA-G was significantly increased in 70% of B chronic lymphocytic leukemia, 53% of non-Hodgkin B lymphoma (B-NHL), and 45% of T-NHL. To explain this variable secretion, the HLA-G secreting cell was searched and was identified as tumoral T4 lymphocytes only in one patient with Sezary syndrome. To approach the mechanisms involved in sHLA-G secretion, the potential role of cytokines has been studied in vitro on T lymphomas. A significant increase of sHLA-G level is observed after activation by cytokines associated with a small increase in the quantity of transcripts using real-time polymerase chain reaction, suggesting an involvement of both transcriptional and post-transcriptional mechanisms. Western Blot analysis reveals no evident variation of the protein expression whatever the conditions, suggesting a continuous secretion and a low intracellular storage. The frequency of the sHLA-G secretion associated to its inhibiting role on T cells and natural killer cells during tumoral lymphoid malignancies suggests a potential role of these molecules as escape mechanism from antitumoral response.

Early circulating lymphocyte apoptosis in human septic shock is associated with poor outcome.

The role of lymphocyte apoptosis in septic shock remains a controversial issue. Using Annexin V and flow cytometry analysis on freshly isolated cells, we evaluated circulating lymphocyte apoptosis in 23 septic shock, 25 sepsis without shock, 7 nonseptic critically ill, and 25 control patients. In patients with sepsis, we compared day 1 lymphocyte apoptosis (i.e., within 3 days of the onset of infection) with that observed 5-7 days after (day 6) according to shock state, mortality, and seventy factors. At day 1, patients in septic shock exhibited higher lymphocyte apoptosis than that present in controls (16.5% +/- 3.5% vs. 3% +/- 0.5%, respectively, P = 0.0001). At day 6, patients with sepsis without shock restored undamaged CD4+ T and CD8+ T lymphocyte counts, whereas patients in septic shock increased only CD4+ T cells. Similarly, survivors restored undamaged lymphocyte count at day 6 (+70%, P < 0.001), whereas nonsurvivors did not. Day 6 undamaged lymphocyte count negatively correlated with day 1 SAPS II, day 6 LOD score, mechanical ventilation, and ICU stay duration. We observed no apoptotic effect of septic shock plasma or septic shock circulating mononuclear cells on target lymphoid cell lines. We found no alteration in any death receptors Fas, TRAIL-R1, TRAIL-R2, or in their ligands on circulating blood cells. Catecholamines and interleukin 10 levels significantly increased in patients with septic shock, but did not correlate with apoptosis levels. We conclude that lymphocyte apoptosis is rapidly increased in blood of patients in septic shock and that lymphocyte apoptosis leads to a profound and persistent lymphopenia associated with poor outcome. These results suggest that lymphocyte apoptosis is one of the main components of human septic shock immune dysfunction and could be related more to microcirculatory disturbance than to circulating factors.

Soluble HLA-G molecules increase during acute leukemia, especially in subtypes affecting monocytic and lymphoid lineages.

Human leukocyte antigen G (HLA-G) molecules exhibit immunomodulatory properties corresponding to nonclassic class I genes of the major histocompatibility complex. They are either membrane-bound or solubly expressed during certain tumoral malignancies. Soluble human leukocyte antigen G (sHLA-G) molecules seem more frequently expressed than membrane-bound isoforms during hematologic malignancies, such as lymphoproliferative disorders. Assay of these molecules by enzyme-linked immunosorbent assay in patients suffering from another hematologic disorder (acute leukemia) highlights increased sHLA-G secretion. This increased secretion seems more marked in acute leukemia subtypes affecting monocytic and lymphoid lineages such as FABM4 and FABM5, as well as both B and T acute lymphoblastic leukemia (ALL). Moreover, this study uses in vitro cytokine stimulations and reveals the respective potential roles of granulocyte-macrophage colony-stimulating factor and interferon-gamma in increasing this secretion in FABM4 and ALL. Correlations between sHLA-G plasma level and clinical biologic features suggest a link between elevated sHLA-G level and 1) the absence of anterior myelodysplasia and 2) high-level leukocytosis. All these findings suggest that sHLA-G molecules could be a factor in tumoral escape from immune survey during acute leukemia.

Polycyclic aromatic hydrocarbons affect functional differentiation and maturation of human monocyte-derived dendritic cells.

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are environmental carcinogens exhibiting potent immunosuppressive properties. To determine the cellular bases of this immunotoxicity, we have studied the effects of PAHs on differentiation, maturation, and function of monocyte-derived dendritic cells (DC). Exposure to BP during monocyte differentiation into DC upon the action of GM-CSF and IL-4 markedly inhibited the up-regulation of markers found in DC such as CD1a, CD80, and CD40, without altering cell viability. Besides BP, PAHs such as dimethylbenz(a)anthracene and benzanthracene also strongly altered CD1a levels. Moreover, DC generated in the presence of BP displayed decreased endocytic activity. Features of LPS-mediated maturation of DC, such as CD83 up-regulation and IL-12 secretion, were also impaired in response to BP treatment. BP-exposed DC poorly stimulated T cell proliferation in mixed leukocyte reactions compared with their untreated counterparts. In contrast to BP, the halogenated arylhydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin, which shares some features with PAHs, including interaction with the arylhydrocarbon receptor, failed to phenotypically alter differentiation of monocytes into DC, suggesting that binding to the arylhydrocarbon receptor cannot mimic PAH effects on DC. Overall, these data demonstrate that exposure to PAHs inhibits in vitro functional differentiation and maturation of blood monocyte-derived DC. Such an effect may contribute to the immunotoxicity of these environmental contaminants due to the major role that DC play as potent APC in the development of the immune response.

Cytochrome P450-dependent toxicity of environmental polycyclic aromatic hydrocarbons towards human macrophages.

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are potent immunosuppressive environmental contaminants acting on lymphocytes and monocytes. To establish whether differentiated macrophages, which play a crucial role in innate and acquired immunity, can also constitute major cellular targets, we have characterized PAH effects towards primary human macrophages. BP-treatment was found to dramatically alter their functional capacities and to trigger a caspase- and mitochondrion-related apoptosis, associated with down-regulation of the survival factors c-FLIP(L) and Bcl-X(L) and up-regulation of the pro-apoptotic factor p53. Such deleterious effects were associated with BP metabolite production, whose inhibition by the cytochrome P-450 1A1 inhibitor alpha-naphthoflavone fully abolished BP toxicity. In contrast to BP, the related halogenated arylhydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin, known to be poorly metabolized if any, only minimally affected macrophages. Overall, these data provide evidence for a cytochrome P-450-dependent toxicity of PAHs towards human differentiated macrophages, which may contribute to their immunosuppressive effects.

Major histocompatibility complex abnormalities in non-Hodgkin lymphomas.

An optimal antitumoral immune response requires the participation of both CD8 and CD4 T lymphocytes, which are activated by peptide antigen presentation via human leucocyte antigen (HLA) class I and class II molecules respectively. Loss of HLA molecules has been observed in different malignancies, and provides a mechanism for escape from immune surveillance. Furthermore, HLA-G, a class Ib molecule, is considered to be an immune tolerance-inducing molecule. HLA-G expression on tumour cells could provide a further mechanism for immune escape. To determine the frequency and the pattern of HLA defects in non-Hodgkin lymphomas (NHL), HLA expression was prospectively studied in 614 NHL cases, using flow cytometry. Furthermore, HLA-G expression was tested in 50 cases, including 20 cases selected on the basis of their defective HLA class I expression. In 64 cases (10.4%), lymphomatous cells exhibited lower HLA class I mean fluorescence intensity compared with reactive cells. Their characteristics were (1) the diversity of histological entities; (2) the significant frequency of relapse or transformation; (3) the increased incidence of high-grade NHL compared with low-grade; and (4) the severity of the class I defect in 50% of the cases, mainly in high-grade NHL. A defect in HLA-DR expression was always associated with a severe class I defect (12 cases; 2%). The HLA-G protein was detected in three class I defective cases. These HLA alterations frequently appeared as a secondary event at relapse or at transformation, suggesting a direct role in lymphomagenesis.

Polycyclic aromatic hydrocarbons inhibit differentiation of human monocytes into macrophages.

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are ubiquitous environmental carcinogenic contaminants exerting deleterious effects toward cells acting in the immune defense such as monocytic cells. To investigate the cellular basis involved, we have examined the consequences of PAH exposure on macrophagic differentiation of human blood monocytes. Treatment by BP markedly inhibited the formation of adherent macrophagic cells deriving from monocytes upon the action of either GM-CSF or M-CSF. Moreover, it reduced expression of macrophagic phenotypic markers such as CD71 and CD64 in GM-CSF-treated monocytic cells, without altering cell viability or inducing an apoptotic process. Exposure to BP also strongly altered functional properties characterizing macrophagic cells such as endocytosis, phagocytosis, LPS-triggered production of TNF-alpha and stimulation of allogeneic lymphocyte proliferation. Moreover, formation of adherent macrophagic cells was decreased in response to PAHs distinct from BP such as dimethylbenz(a)anthracene and 3-methylcholanthrene, which interact, like BP, with the arylhydrocarbon receptor (AhR) known to mediate many PAH effects. In contrast, benzo(e)pyrene, a PAH not activating AhR, had no effect. In addition, AhR was demonstrated to be present and functional in cultured monocytic cells, and the use of its antagonist alpha-naphtoflavone counteracted inhibitory effects of BP toward macrophagic differentiation. Overall, these data demonstrate that exposure to PAHs inhibits functional in vitro differentiation of blood monocytes into macrophages, likely through an AhR-dependent mechanism. Such an effect may contribute to the immunotoxicity of these environmental carcinogens owing to the crucial role played by macrophages in the immune defense.

Monocyte human leukocyte antigen-DR transcriptional downregulation by cortisol during septic shock.

Monocyte deactivation has been identified as a major factor of immunosuppression in sepsis and is associated with a loss of surface human leukocyte antigen-DR (HLA-DR) expression on circulating monocytes. Using flow cytometry, quantitative reverse transcription-polymerase chain reaction, we investigated this phenomenon in septic patients. We confirmed the early loss of monocyte HLA-DR expression in all infected patients and demonstrated that this persistent lowered expression at Day 6 correlated with severity scores, secondary infection, and death. This phenomenon occurred at a transcriptional level via a decrease in the class II transactivator A (CIITA) transcription. Furthermore, these abnormalities correlated with the high cortisol levels observed in sepsis and not with those of other putative factors such as catecholamines or interleukin-10. Finally, in vitro studies evidenced that glucocorticoids decrease HLA-DR expression at a transcriptional level via a decrease in CIITA mRNA levels, mainly by down modulating its isoforms I and III. We conclude that in human sepsis, the loss of HLA-DR expression on circulating monocytes is associated with a poor outcome. We suggest that the high endogenous cortisol level observed in septic shock may be a possible new factor involved in the loss of HLA-DR expression on monocytes via its effect on HLA-DR and CIITA transcription.

Potassium antimonyl tartrate induces caspase- and reactive oxygen species-dependent apoptosis in lymphoid tumoral cells.

The metalloid salt potassium antimonyl tartrate (PAT), previously used as an antiparasitic agent, has recently been shown to exert cytotoxicity towards acute promyelocytic leukaemia cells like arsenical compounds. In this study, we have investigated its effects towards human lymphoid malignant cells and compared them with those of arsenic trioxide (As2O3). Like As2O3, PAT was found to inhibit cell growth of various lymphoid cell lines, deriving from either acute lymphoid leukaemias (Jurkat, Molt-4 and Nalm-6) or lymphomas (Daudi, Raji and Rec1). PAT toxicity was linked, at least in part, to induction of apoptosis in both Daudi and Jurkat cells, which was dependent on caspase activity. This apoptotic process was also associated, similarly to that triggered by As2O3, with loss of mitochondrial potential and enhanced cellular production of reactive oxygen-related species. It was enhanced by co-treatment with the pro-oxidant buthionine sulphoximine and abolished in response to the antioxidant N-acetylcysteine, thus underlining that PAT toxicity, similarly to that of As2O3, is probably modulated by the redox status of the cells. PAT, used at concentrations in the micromolar range that are thought to be clinically achievable, was also demonstrated to markedly decrease the viability of primary cultured tumoral B cells that originated from 18 patients suffering from chronic lymphoid leukaemia whereas normal lymphocytes were less sensitive. These data therefore suggest that PAT may deserve to be evaluated in the treatment of some lymphoid malignancies.

MHC class II-mediated apoptosis of mature dendritic cells proceeds by activation of the protein kinase C-delta isoenzyme.

The mature dendritic cell (DC) is considered to be the most potent antigen-presenting cell. Regulation of the DC, particularly its survival, is therefore critical. Mature DC are markedly more sensitive to HLA-DR-mediated apoptosis than immature DC. To further characterize this key survival difference, we compared the intracellular signals initiated via HLA-DR in mature versus immature DC. Apoptosis was unchanged by inhibition of tyrosine kinases or phosphatases. HLA-DR-mediated re-localization of protein kinase C (PKC)-delta to the nucleus was detected in mature DC by confocal microscopy and by immunoblotting. Activation of PKC-delta in mature DC was revealed by the detection of the PKC-delta catalytic fragment in the nuclear fraction isolated from mature DC which had been stimulated via HLA-DR. The broad-spectrum PKC inhibitor, Calphostin C, as well as the PKC-delta-selective inhibitor, Rottlerin, inhibited HLA-DR-mediated apoptosis of mature cells. Taken together, these data reveal a role for the PKC-delta isoenzyme in regulating HLA class II-mediated apoptosis of mature DC. Thus, the lifespan of the mature DC could be controlled by signals generated in the course of antigen presentation, and thereby prevent DC persistence and prolonged stimulation of T and B lymphocytes.

HLA-G and lymphoproliferative disorders.

The immunomodulatory properties of the HLA-G molecule explain its relevance in malignancies. Our investigations in lymphoproliferative disorders show (i) a frequent and variable distribution of alternatively spliced HLA-G mRNA isoforms, (ii) a rare cell surface expression in diffuse large cell lymphomas with HLA class I loss in half of cases, and (iii) an increased serum level of sHLA-G in half of cases. The potential role of the microenvironment and/or tumoral process in HLA-G expression is discussed in the light of these data. HLA-G rather through its soluble isoform might provide a new way of immune evasion for lymphoid proliferations.

Loss of heterozygosity, a frequent but a non-exclusive mechanism responsible for HLA dysregulation in non-Hodgkin's lymphomas.

The frequent alteration of human leucocyte antigen (HLA) class I molecule expression observed in non-Hodgkin's lymphomas (NHL), similarly to solid tumours, has been reported to favour tumoral escape from the immune system. In order to identify the underlying mechanisms, we analysed 15 HLA defective NHL including partial (n = 10) and total class I (n = 5) loss, as well as HLA class II defects (n = 5). The HLA defect concerned HLA-A and -B antigens in 14 of 15 cases. In the cases with partial defect, the use of specific allelic monoclonal antibodies detected a defect of both alleles of A or B loci in six of seven tested cases. Allelic reverse transcription polymerase chain reaction (RT-PCR) demonstrated defects in six of nine cases, including four alterations of both A and B mRNA alleles. Real-time quantitative RT-PCR (RQ-PCR) did not detect the HLA-DR transcript in the two negative HLA-DR lymphomas, contrasting with the presence of CMH II transactivator (CIITA) transcript. Loss of heterozygosity (LOH) was detected in nine of 14 cases through variable pattern of nine microsatellites markers of the HLA locus. Taken together, these findings demonstrate the complexity and the variability of the mechanisms underlying HLA protein deficiencies with a high frequency of LOH. The diversity of these mechanisms indicates the importance of positive selection of HLA altered clones in the development of these NHL cases.