Mucosal-associated Invariant Cells are Deficient in Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease, characterized by fibrosis of the skin and other organs, vascular impairment and deficient immune responses. Mucosal-associated invariant T cells (MAIT) have been involved in various inflammatory and autoimmune diseases. The aims of this study were to determine the frequencies of MAIT cells in the blood of patients with systemic sclerosis (SSc) and to compare their distribution in different types of SSc. Blood samples from patients with SSc and healthy controls were examined by flow cytometer to analyse the frequencies of MAIT and γδ T cells. We demonstrate that in SSc the frequencies and absolute numbers of MAIT and γδ T cells are significantly reduced in comparison with healthy controls. MAIT and γδ T cells did not correlate with C-reactive protein, BNP, pulmonary involvement or median skin fibrosis scale, steroid amount or disease duration. In addition, MAIT and γδ T cells decrease did not stratify with gender, interstitial lung disease or active digital ulcers. Functional studies are necessary to determine the signification of MAIT cells decrease in systemic sclerosis.

ATG-induced accelerated immune senescence: clinical implications in renal transplant recipients.

Persistent ATG-induced CD4(+) T cell lymphopenia is associated with serious clinical complications. We tested the hypothesis that ATG induces accelerated immune senescence in renal transplant recipients (RTR). Immune senescence biomarkers were analyzed at transplant and one-year later in 97 incident RTR -62 patients receiving ATG and 35 receiving anti-CD25 mAb (α-CD25). This consisted in: (i) thymic output; (ii) bone marrow renewal of CD34(+) hematopoietic progenitor cells (CD34(+) HPC) and lymphoid (l-HPC) and myeloid (m-HPC) progenitor ratio; (iii) T cell phenotype; and (iv) measurement of T cell relative telomere length (RTL) and telomerase activity (RTA). Clinical correlates were analyzed with a 3 year follow-up. Thymic output significantly decreased one-year posttransplant in ATG-treated patients. ATG was associated with a significant decrease in l-HPC/m-HPC ratio. Late stage differentiated CD57(+) /CD28(-) T cells increased in ATG-treated patients. One-year posttransplant T cell RTL and RTA were consequently lower in ATG-treated patients. ATG is associated with accelerated immune senescence. Increased frequency of late differentiated CD4(+) T cell frequency at transplantation tended to be predictive of a higher risk of subsequent opportunistic infections and of acute rejection only in ATG-treated patients but this needs confirmation. Considering pretransplant immune profile may help to select those patients who may benefit from ATG to prevent severe infections and acute rejection.

A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma.

Human melanoma MZ2-MEL expresses several distinct antigens that are recognized by autologous cytolytic T lymphocytes (CTL). Some of these antigens are encoded by genes MAGE-1, MAGE-3, and BAGE, which are expressed in a large fraction of tumors of various histological types but are silent in normal adult tissues with the exception of testis. We report here the identification of the gene coding for MZ2-F, another antigen recognized by autologous CTL on MZ2-MEL cells. This gene, which was named GAGE-1, is not related to any presently known gene. It belongs to a family of genes that are expressed in a variety of tumors but not in normal tissues, except for the testis. Antigenic peptide YRPRPRRY, which is encoded by GAGE-1, is recognized by anti-MZ2-F CTL on class I molecule HLA-Cw6. The two genes of the GAGE family that code for this peptide, namely GAGE-1 and GAGE-2, are expressed in a significant proportion of melanomas (24%), sarcomas (25%), non-small cell lung cancers (19%), head and neck tumors (19%), and bladder tumors (12%). About 50% of melanoma patients carry on their tumor at least one of the presently defined antigens encoded by the MAGE, BAGE, and GAGE genes.

A new antigen recognized by cytolytic T lymphocytes on a human kidney tumor results from reverse strand transcription.

By stimulating blood lymphocytes from a renal cell carcinoma patient in vitro with the autologous tumor cells, we obtained cytolytic T lymphocyte (CTL) clones that killed several autologous and allogeneic histocompatibility leukocyte antigen (HLA)-B7 renal carcinoma cell lines. We identified the target antigen of these CTLs by screening COS cells transfected with the HLA-B7 cDNA and with a cDNA library prepared with RNA from the tumor cells. The antigenic peptide recognized by the CTLs has the sequence LPRWPPPQL and is encoded by a new gene, which we named RU2. This gene is transcribed in both directions. The antigenic peptide is not encoded by the sense transcript, RU2S, which is expressed ubiquitously. It is encoded by an antisense transcript, RU2AS, which starts from a cryptic promoter located on the reverse strand of the first intron and ends up on the reverse strand of the RU2S promoter, which contains a polyadenylation signal. This mechanism of antigen expression is unprecedented and further illustrates the notion that many peptides recognized by T cells cannot be predicted from the primary structure of the major product of the encoding gene. Antisense transcript RU2AS is expressed in a high proportion of tumors of various histological types. It is absent in most normal tissues, but is expressed in testis and kidney, and, at lower levels, in urinary bladder and liver. Short-term cultures of normal epithelial cells from the renal proximal tubule expressed significant levels of RU2AS message and were recognized by the CTLs. Therefore, this antigen is not tumor specific, but corresponds to a self-antigen with restricted tissue distribution.

Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes.

Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, that codes for one of these antigens named MZ2-E. We show here that antigen MZ2-D, which is present on the same tumor, is encoded by another member of the MAGE gene family named MAGE-3. Like MAGE-1, MAGE-3 is composed of three exons and the large open reading frame is entirely located in the third exon. Its sequence shows 73% identity with MAGE-1. Like MZ2-E, antigen MZ2-D is presented by HLA-A1. The antigenic peptide of MZ2-D is a nonapeptide that is encoded by the sequence of MAGE-3 that is homologous to the MAGE-1 sequence coding for the MZ2-E peptide. Competition experiments using single Ala-substituted peptides indicated that amino acid residues Asp in position 3 and Tyr in position 9 were essential for binding of the MAGE-1 peptide to HLA-A1. Gene MAGE-3 is expressed in many tumors of several types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma and breast carcinoma, but not in normal tissues except for testes. It is expressed in a larger proportion of melanoma samples than MAGE-1. MAGE-3 encoded antigens may therefore have a wide applicability for specific immunotherapy of melanoma patients.

An alternative open reading frame of the human macrophage colony-stimulating factor gene is independently translated and codes for an antigenic peptide of 14 amino acids recognized by tumor-infiltrating CD8 T lymphocytes.

We show that cytotoxic T lymphocytes (CTLs) infiltrating a kidney tumor recognize a peptide encoded by an alternative open reading frame (ORF) of the macrophage colony-stimulating factor (M-CSF) gene. Remarkably, this alternative ORF, which is translated in many tumors concurrently with the major ORF, is also translated in some tissues that do not produce M-CSF, such as liver and kidney. Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected. The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues. Peptide elution indicated that tumor cells naturally present this 14 mer, which is the longest peptide known to be recognized by CTLs. Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.

[Towards the use of intravenous apoptotic leukocyte infusion as a cell-based therapy approach?]. / Vers l'utilisation thérapeutique de l'administration intraveineuse de leucocytes apoptotiques du donneur en thérapie cellulaire ?

In the past few years, significant advances have been performed in the field of cell-based therapies. This concerns mainly regenerative medicine with the use of stem cells, as well as the modulation of immune responses. In order to modulate allogeneic immune responses after transplantation, we have developed a cell therapy approach based on the immunomodulatory properties of intravenous donor apoptotic cell infusion. In allogeneic hematopoietic cell transplantation settings, we reported that intravenous apoptotic leukocyte infusion, simultaneously to allogeneic bone marrow grafts, favors hematopoietic engraftment, prevents alloimmunization and delays graft-versus-host disease. Here, we review the different factors and cells implicated in the immunomodulatory properties of apoptotic cells. Then, we discuss the potential significance of such observations in transfusion practice.

[Unexplained recurrent early miscarriages: Role of immunomodulation?] / Fausses couches précoces récurrentes inexpliquées : quelle est la place de l'immunomodulation ?

About 1-3% of women experience early recurrent miscarriages, defined by ≥3 fetal loss before 14 weeks of gestation. About half of these recurrent early miscarriages could be related to a genetic cause. Pre-implantation genetic diagnosis is used in several European countries, but it is still prohibited in France except for couples at risk for transmission of severe genetic diseases. The immune dysregulation, and in particular allo-immune excessive response, could be responsible for fetal loss in remaining cases, although currently we lack biomarker to confirm the immune-mediated fetal loss. Several immunosuppressive and immunomodulatory treatments have been the subject of small studies in patients with early recurrent miscarriages. The available data do not allow to define the treatment recommendations in this topic, and further studies are necessary.

Impact of plasmacytoid dendritic cells on outcome after reduced-intensity conditioning allogeneic stem cell transplantation.

The reconstitution of the plasmacytoid dendritic cells (PDCs) compartment might influence outcome after allogeneic stem cell transplantation (allo-SCT). Thus, we investigated the impact of blood PDCs measured at the third month after reduced-intensity conditioning (RIC) in 54 patients who received an HLA-identical sibling allo-SCT. The absence of grade II-IV acute graft-versus-host-disease (GVHD) was associated with an improved PDC count at 3 months after RIC-allo-SCT (P=0.003; OR=6.4; 95% CI, 1.9-22). The CD34+ stem cell dose and other lymphoid subsets infused with the allograft did not affect PDC recovery. Although PDC count could not predict death from progression or relapse, patients with a "high" PDC recovery profile had an improved overall survival (OS; P=0.03), in contrast to patients with a "low" PDC recovery profile who had an increased incidence of nonrelapse mortality (GVHD, infections) (P=0.03). The overall incidence of late infections (viral, fungal and bacterial) was significantly higher in the "low" PDC recovery group as compared to the "high" PDC recovery group (59 vs 19%; P=0.002). In a multivariate analysis, only a "high" PDC count was significantly predictive of a decreased risk of death (P=0.04; RR=0.34; 95% CI, 0.12-0.96). Monitoring of PDCs at 3 months after RIC-allo-SCT may be a useful indicator predictor of long-term outcome.

Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities.

The destruction of cells capable of initiating and maintaining leukemia challenges the treatment of human acute myeloid leukemia. Recently, CD34+/CD38- leukemia progenitors have been defined as new leukemia-initiating cells less mature than colony-forming cells. Here we show that CD34+/CD38- leukemia precursors have reduced in vitro sensitivity to daunorubicin, a major drug used in leukemia treatment, in comparison with the CD34+/CD38+ counterpart, and increased expression of multidrug resistance genes (mrp/lrp). These precursors show lower expression of Fas/Fas-L and Fas-induced apoptosis than CD34+/CD38+ blasts. Moreover, the CD34+/CD38- leukemic subpopulation induces a weaker mixed leukocyte reaction of responding T-lymphocytes than the CD34+/CD38+ leukemic counterpart, either in a MHC-unmatched or MHC-matched settings. This weaker immunogenicity could be linked to lower expression on CD34+/CD38- leukemia precursors of major immune response molecules (MHC-DR, LFA-3, B7-1, or B7-2) than CD34+/CD38+ leukemic cells. Nonetheless, the susceptibility of the immature CD38- precursors to cytotoxicity was not different from the sensitivity of the CD38+ counterpart. Finally, CD34+/CD38- leukemia precursors, in contrast with CD38+ precursors, failed, under appropriate conditions, to differentiate into dendritic cells, a central step for antigen recognition. This is to our knowledge the first demonstration that the very immature phenotype of CD34+/CD38- leukemic progenitors confers both chemotherapy resistance and decreased capacities to induce an immune response. Because the susceptibility of the immature leukemia cells as cytotoxic targets is maintained, our data underline the importance of improving the initial steps of leukemia recognition, more particularly by defining optimal conditions of dendritic cell transformation of the very immature hematopoietic precursors.

Translational opportunities for targeting the Th17 axis in acute graft-vs.-host disease.

Allogeneic stem cell transplantation (allo-SCT) is a curative therapy for different life-threatening malignant and non-malignant hematologic disorders. Acute graft-vs.-host disease (aGVHD) and particularly gastrointestinal aGVHD remains a major source of morbidity and mortality following allo-SCT, which limits the use of this treatment in a broader spectrum of patients. Better understanding of aGVHD pathophysiology is indispensable to identify new therapeutic targets for aGVHD prevention and therapy. Growing amount of data suggest a role for T helper (Th)17 cells in aGVHD pathophysiology. In this review, we will discuss the current knowledge in this area in animal models and in humans. We will then describe new potential treatments for aGVHD along the Th17 axis.

Donor interleukin-22 and host type I interferon signaling pathway participate in intestinal graft-versus-host disease via STAT1 activation and CXCL10.

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation, limiting the success of this therapy. We previously reported that interleukin-22 (IL-22) participates to aGVHD development, but the underlying mechanisms of its contribution remain poorly understood. In this study, we analyzed the mechanism of the pathological function of IL-22 in intestinal aGVHD. Ex-vivo colon culture experiments indicated that IL-22 was able to induce Th1-like inflammation via signal transducer and activator of transcription factor-1 (STAT1) and CXCL10 induction in the presence of type I interferon (IFN). To evaluate a potential synergy between IL-22 and type I IFN in aGVHD, we transplanted recipient mice, either wild-type (WT) or type I IFN receptor deficient (IFNAR(-/-)), with bone marrow cells and WT or IL-22 deficient (IL-22(-/-)) T cells. We observed a decreased GVHD severity in IFNAR(-/-) recipient of IL-22(-/-) T cells, which was associated with a lower level of STAT1 activation and reduced CXCL10 expression in the large intestine. Finally, immunohistochemistry staining of STAT1 performed on gastrointestinal biopsies of 20 transplanted patients showed exacerbated STAT1 activation in gastrointestinal tissues of patients with aGVHD as compared with those without aGVHD. Thus, interfering with both IL-22 and type I IFN signaling may provide a novel approach to limit aGVHD.

Dendritic cells: interfaces with immunobiology and medicine. A report from the Keystone Symposia Meeting held in Keystone, 3-8 March 2003.

Dendritic cells (DCs) are the most potent antigen-presenting cells in vitro and in vivo. They play a key role in the initiation of the immune response and are considered promising targets for immunotherapy. The recent DC Keystone Symposia, organized by Ralph M Steinman, Anne O'Garra and Jacques Banchereau, was held on 3-8 March 2003 in Keystone (CO, USA). This multidisciplinary meeting developed various areas related to the DC biology including: (i) DC and the control of immunity vs tolerance; (ii) DC maturation and manipulation for immunotherapy in vivo; and (iii) antigen capture by DC in situ and interactions with microbial pathogens. The aim of this report is to present some of the highlights developed during the meeting and debated among the DC community.

Leukemic dendritic cells: potential for therapy and insights towards immune escape by leukemic blasts.

Dendritic cells (DCs) are a system of potent antigen-presenting cells (APCs) specialized to initiate primary immune responses. DCs are considered important elements in the induction of specific antitumor cytotoxic effectors. At present, because of potential therapeutic implications, the critical role of DCs in cancer patients is under intensive investigation. Interactions between DCs and acute myeloid leukemia cells represent an attractive model for the study of DC physiology. Moreover, DCs can be a valuable therapeutic tool for the adjuvant treatment of leukemic patients. However, DC subsets in vivo may also be affected by leukemogenesis and may contribute to the escape of leukemia from immune control. The aim of this review is to shed further light on this paradoxical picture where the line between immune tolerance and immune defense is narrow.

Abundant expression of fibronectin is a major feature of leukemic dendritic cells differentiated from patients with acute myeloid leukemia.

Dendritic cells (DCs) are the most potent antigen-presenting cells responsible for the initiation of primary immune responses, playing a key role in eliciting effective antitumor immune responses. We reported previously that leukemic blasts from selected patients with acute myeloid leukemia (AML) were able to differentiate in vitro into cells with DC features. In order to identify genes differentially expressed in leukemia-derived DCs (AML-DCs), a polymerase chain reaction (PCR)-based subtraction approach was applied using cDNA from AML-DCs and monocyte-derived DCs from healthy donors as competitors. In the 548 sequences analyzed, 80% corresponded to fibronectin (FN) gene fragments. Overexpression of FN in AML-DCs was demonstrated both by semiquantitative PCR analysis and by immunostaining. In addition, we could show that FN was secreted by AML-DCs. Indeed, FN overexpression was already detectable in AML blasts of M4 and M5 subtype, and was significantly induced during DC differentiation after culture. Although the molecular events leading to overexpression of FN and the in vivo relevance of this phenomenon remain to be resolved, leukemic DCs appear to have specific patterns of differentiation, warranting stringent biological and cellular monitoring for the development and testing of leukemic DC-based immunotherapeutic strategies.

Features of large granular lymphocytes (LGL) expansion following allogeneic stem cell transplantation: a long-term analysis.

Large granular lymphocyte (LGL) proliferation typically follows a chronic course during which major features are cytopenia and immune abnormalities. Elevated numbers of LGL were reported in a few cases following allogeneic stem cell transplantation (allo-SCT). In this report, we present a retrospective analysis of LGL cases that occurred following allo-SCT in a cohort of 201 consecutive patients transplanted over a period of 7 years. Six cases were identified and LGL expansion occurred more frequently following a reduced fludarabine and anti-T lymphocyte globulin-based preparative regimen (4 cases/49), than after a conventional myeloablative regimen (2 cases/152). Expansion of LGL was seen between 3 and 15 months following allo-SCT. Hematopoiesis, with mild to severe cytopenia, was a favored target for LGL. Autoimmune manifestations including polyarthritis and hypergammaglobulinemia were also observed. LGL proliferation was observed in the context of chronic antigenic stimulation associated with recurrent viral infections especially CMV. Moreover, five out of these six high risk patients achieved a long-term complete remission concomitant or following LGL expansion. These data suggest that LGL might be a subset of effector lymphocytes which may participate to the graft-versus-tumor effect.

Identification of precursors of leukemic dendritic cells differentiated from patients with acute myeloid leukemia.

Dendritic cells (DC) can facilitate immune responses that might help in the induction of effective antitumor T cell responses. We reported previously that leukemic blasts from selected patients with acute myeloid leukemia (AML) were able to differentiate in vitro into cells with mature DC features. However, despite the use of a wide variety of cytokine combinations, leukemic DC could not be obtained from all AML patients. In this study, we investigated in a wide range of AML patients (n = 30), the nature and functional characteristics of the blast compartment that can be induced to acquire DC features in vitro. Our results demonstrate that leukemic DC generated in the presence of GM-CSF, IL-4 and matured with CD40L, are composed of two major subsets: DC derived from CD14(+) leukemic cells and leukemic DC derived from in vivo expanded circulating blood myeloid DC (MDC). Leukemic DC of both subsets exhibited DC morphology, had a phenotype of mature DC, and could induce a potent proliferative response of naive CD4(+) T cells. Moreover, both subsets produced large amounts of IL-12p70 and leukemic CD14(+)-derived DC could induce a potent Th1 response. These results can be considered as a prerequisite before the design of vaccine immunotherapy protocols for the adjuvant treatment of AML patients.

Isolation of homologous and heterologous complexes between catalytic and regulatory components of adenylate cyclase by forskolin-Sepharose.

Homologous and heterologous complexes between catalytic and GTP-binding components can be isolated by means of immobilized succinyldeacetylforskolin (forskolin-Sepharose). A heterologous complex is formed by reconstitution of forskolin-Sepharose bound catalytic function from rabbit myocardial membranes with the homogenous [3H]methyl-GTP-binding protein from duck erythrocyte membranes. Analysis of the reconstituted complex by sodium dodecyl sulfate polyacrylamide gelelectrophoresis reveals that only the Mr 42 000 component of the GTP-binding protein's Mr 42 000/Mr 35 000 heterodimer contributes to the formation of active adenylate cyclase.

Cytomegalovirus-specific immune recovery following allogeneic HLA-identical sibling transplantation with reduced-intensity preparative regimen.

Cytomegalovirus (CMV) represents a major cause of morbidity after allogeneic stem cell transplantation (allo-SCT). Using interferon-gamma-enzyme-linked immunospot (ELISPOT) assay and HLA-peptide tetramers, we analysed 54 patients who received a reduced-intensity conditioning regimen, including fludarabine, busulphan and antithymocyte globulin (ATG), with the aim of defining essential elements of protective immunity to CMV. The cumulative incidence of CMV positive antigenaemia was 37% occurring at a median of 43 days (range, 7-104) after allo-SCT. In univariate analysis, conditioning regimen (ATG dose) and graft characteristics (graft source and CD3+ T-cell dose) significantly influenced CMV-specific immune recovery. A significant correlation (P=0.000002) was found between CMV-specific T cells detected by IFN-gamma ELISPOT assay and pp65-specific CD8+ T-cell frequency quantified by tetramers. CMV-specific CD8+ T cells presented a phenotype of effector cells (perforin and 2B4 positive). In multivariate analysis, bone marrow (BM) as a graft source was the only variable associated with an increased risk of CMV positive antigenaemia (P=0.0001) in line with the ELISPOT assay showing a higher frequency of functional CMV-specific effectors within peripheral blood stem cell grafts as compared to BM. Thus, early monitoring of CMV-specific immune recovery using sensitive new tools might prove useful for patient management after allo-SCT.

Large granular lymphocytes (LGL) following non-myeloablative allogeneic bone marrow transplantation: a case report.

We report here the first case of large granular lymphocytes (LGL) expansion following non-myeloablative allo-BMT for chronic myeloid leukemia. We characterized the morphologic, phenotypic and functional features of the LGL subset amplified in vivo 14 months after allo-BMT. Our results indicate that LGL can mediate in vitro a cytolytic activity on tumor cells. In vivo, the timing of the LGL expansion was associated with a sustained complete molecular remission. These observations suggest that LGL are a subset with the properties of effector lymphocytes which may contribute to the graft-versus-tumor effect.