Valérie Guilloux

Modulation of HLA-G Antigens Expression by Human Cytomegalovirus: Specific Induction in Activated Macrophages Harboring Human Cytomegalovirus Infection

After infection, human CMV (HCMV) establishes a latent and persistent infection in immature myeloid progenitors and peripheral blood monocytes. Completion of the HCMV life cycle is possible upon maturation of monocytes to tissue macrophages and under permissive circumstances, e.g., immunosuppression. We investigated the hypothesis that HLA-G molecules could be induced during HCMV reactivation in activated macrophages to favor virus dissemination. In this study, we provide evidence that HLA-G Ags are produced during viral reactivation in macrophages generated after allogeneic stimulation of HCMV latently infected monocytes. While HLA-G surface expression is up-regulated, classical MHC-I molecules are partially down-regulated by HCMV. In vivo, bronchoalveolar macrophages collected from patients suffering from acute HCMV pneumonitis also express HLA-G molecules. The direct correlation between HLA-G Ag induction and HCMV infection was confirmed in U-373 MG astrocytoma cells. Soluble HLA-G expression is stimulated upon HCMV infection, and this modulation depends on the cooperative action of the two immediate-early-1 pp72 and immediate-early-2 pp86 products. Because HLA-G transcription is active in macrophages and U-373 MG astrocytoma cells, it is likely that the modulation of HLA-G protein expression during HCMV replication occurs at a post-transcriptional level. Our data suggest that induction of HLA-G molecules could be an additional mechanism that helps HCMV to subvert host defenses.

Lung macrophages and dendritic cells express HLA-G molecules in pulmonary diseases

HLA-G is selectively expressed in extravillous trophoblast of human placenta, which does not express classical HLA-A and -B molecules. Several studies report the role of HLA-G as a molecule involved in immune tolerance. By interacting with NK and T cells inhibitory receptors, HLA-G may downregulate their cytotoxicity functions. To appreciate the biologic and clinical relevance of HLA-G expression in lung diseases, HLA class I and HLA-G expression were analyzed in a panel of 36 ex vivo neoplastic tissues and 8 non-neoplastic lung tissues. Immunohistochemical analysis was performed using a pan-HLA class I antibody (W6/32) and three different specific anti-HLA-G antibodies (87G, MEMG/9 and 4H84). These findings demonstrated that HLA-G products were not expressed in pulmonary structural cells. However, HLA-G molecules were detected in activated macrophages and dendritic cells infiltrating lung carcinomas (33%) and nontumoral pulmonary diseases (25%). HLA-G expression was not correlated with classical HLA alterations. No statistical correlation was found between HLA-G expression and clinical or biologic parameters except high tumor size. The expression of HLA-G in myelo-monocytic cells infiltrating lung pathologic tissues could alter antigenic presentation and contribute to decrease immune response efficiency, subsequently favoring the progression of tumoral or inflammatory processes.

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.

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.

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.

Down-modulation of granulocyte macrophage-colony stimulating factor receptor on monocytes during human septic shock.

Objective:Loss of surface human leukocyte antigen-DR (HLA-DR) on monocytes is a major factor of immunosuppression in sepsis. Granulocyte macrophage-colony stimulating factor (GM-CSF) up-regulates HLA-DR expression on monocytes via the GM-CSF receptor (GM-CSFr) through a transcriptional mechanism involving the class II transactivator factor (CIITA). We investigated monocyte GM-CSFr expression and its relationship with HLA-DR in septic patients. Design:Prospective clinical experimental study. Setting:University hospital intensive care unit and research facility. Patients:Septic patients with and without septic shock, control patients. Interventions:Flow cytometry and real-time quantitative reverse polymerase chain reaction were used to characterize GM-CSFr expression and transcription in septic patients and in ex vivo stimulated healthy monocytes. Measurements and Main Results:We showed an early GM-CSFr down-modulation in patients with septic shock compared with those without septic shock and controls. A persistent low GM-CSFr expression was observed in patients who acquired secondary infections or in those who died, and this persistent defect correlated with severity scores. We demonstrated that GM-CSFr down-modulation occurs at a posttranscriptional level since we observed no alteration in GM-CSFr transcription in monocytes isolated from septic patients. Furthermore, we demonstrated that GM-CSFr expression levels on monocytes correlated not only with HLA-DR expression and transcription levels but also with RNA levels of its main transcriptional factor CIITA. Because we previously showed in septic patients a relationship between high cortisol plasma level and low monocyte HLA-DR expression, we investigated the effects of glucocorticoids on monocyte GM-CSFr expression and observed a similar posttranscriptional down-modulation of GM-CSFr by steroids. However, the in vivo putative role of steroids in HLA-DR down-regulation via GM-CSFr down-modulation needs further investigation. Conclusion:Monocyte GM-CSFr down-modulation occurred in septic shock, was associated with severity, and might be either another manifestation of monocyte deactivation linked to sepsis or an additional mechanism participating in immunosuppression.

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.

HLA-G Expression in Guillain-Barré Syndrome Is Associated with Primary Infection with Cytomegalovirus

GUILLAIN-BARRE SYNDROME (GBS) is an acute peripheral neuropathy characterized by a symmetric, usually ascending paralysis due to an acute inflammatory demyelination. GBS is considered the prototype of postinfectious illness with immunopathologic features that include an inadequate immune response against persistent foreign epitopes which cross-react with self-epitopes of peripheral nerves. Regarding this hypothesis, progressive understanding of the immunopathological phenomena leading to the persistence of infective agents in host may provide a rationale for targeting immunotherapy in GBS. In the field of immune tolerance, recent studies have focused on the role of the Human Leukocyte Antigen–G (HLA-G) molecules, which belong to the non-classical HLA class Ib molecules. HLA-G proteins differ from classical HLA-Ia molecules by their low polymorphism, their restricted tissue distribution in tumors (1), and pulmonary (5) or muscle inflammation (6). Furthermore, they exert immunotolerant functions in feto-maternal tolerance, heart-graft acceptance, and tumor immunoevasion (1). Lastly, in a previous report, we evidenced HLA-G expression on monocyte-macrophages during human cytomegalovirus (HCMV) reactivation. We proposed this phenomenon as a new mechanism that could help HCMV eluding host defences and persisting in human body (4). Consequently, and because CMV infection is the most common viral disease causing GBS (2), we examined HLA-G expression on peripheral monocytes in a patient with a GBS induced by HCMV primary infection. A 41-year-old man presented with a bilateral ascending paresthesias and a progressive weakness in lower limbs. On admission, he was afebrile, presented with areflexia of the lower limbs, and had progressively developed a flaccid ascending tetraparesis. GBS was confirmed by elevated CSF protein concentration (0.6g/L) without cellular response and an absence of spinal cord compression on CT scan. Electrophysiological studies showed slow motor conduction, increased distal latencies, an abolition of F waves, and a normal sensory parameters. Acute HCMV infection was proved by high anti-CMV IgM and IgG antibodies, and positive blood PCR (3). Primary infection was demonstrated by an urea denaturation test showing predominance of low-avidity IgG produced early in infection. Tests for other viruses (HSV, HIV, HAV, HBV, and HCV) remained negative. Despite treatment by immunoglobulins and ganciclovir, the patient developed respiratory failure and required mechanical ventilation for 2 months. Recovery was slow, and he was discharged 3 months after admission. We evidenced HLA-G expression on some of the patient’s blood monocytes by allogeneic stimulation. Briefly, monocytes were isolated at the onset of the disease by Ficoll density gradient centrifugation and