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Summary LPIN1 mutations are responsible for inherited recurrent rhabdomyolysis, a life-threatening condition with no efficient therapeutic intervention. Here, we conduct a bedside-to-bench-and-back investigation to study the pathophysiology of lipin1 deficiency. We find that lipin1-deficient myoblasts exhibit a reduction in phosphatidylinositol-3-phosphate close to autophagosomes and late endosomes that prevents the recruitment of the GTPase Armus, locks Rab7 in the active state, inhibits vesicle clearance by fusion with lysosomes, and alters their positioning and function. Oxidized mitochondrial DNA accumulates in late endosomes, where it activates Toll-like receptor 9 (TLR9) and triggers inflammatory signaling and caspase-dependent myolysis. Hydroxychloroquine blocks TLR9 activation by mitochondrial DNA in vitro and may attenuate flares of rhabdomyolysis in 6 patients treated. We suggest a critical role for defective clearance of oxidized mitochondrial DNA that activates TLR9-restricted inflammation in lipin1-related rhabdomyolysis. Interventions blocking TLR9 activation or inflammation can improve patient care in vivo., Graphical abstract, Highlights Lipin1 deficiency causes mitophagosome and late endosome dysfunction in human myoblasts Damaged mitochondria trigger TLR9-mediated inflammation and myoblast death Mitochondrial DNA elimination or blockade of TLR9 activation prevent myoblast death Hydroxychloroquine sulfate treatment may prevent rhabdomyolysis episodes in patients, Hamel et al. investigate the mechanism underlying life-threatening rhabdomyolysis in human lipin1 deficiency. They find that endosomal accumulation of mitochondrial DNA in myoblasts induces inflammation and cell death mediated by TLR9 activation in vitro. Treatment of 6 patients with hydroxychloroquine sulfate suggests benefits in preventing rhabdomyolysis episodes.

Introduction Lipin-1 deficiency is a major cause of rhabdomyolysis that are precipitated by febrile illness. The prognosis is poor, with one-third of patients dying from cardiac arrest during a crisis episode. Apart from acute rhabdomyolysis, most patients are healthy, showing normal clinical and cardiac ultrasound parameters. Patients and methods We report cardiac and exercise examinations of 8 children carrying two LPIN1 mutations. The examinations were performed outside of a myolysis episode, but one patient presented with fever during one examination. Results All but one patient displayed normal resting cardiac function, as determined by echocardiography. One patient exhibited slight left ventricular dysfunction at rest and a lack of increased stroke volume during cycle ramp exercise. During exercise, peripheral muscle adaptation was impaired in 2 patients compared to healthy controls: they presented an abnormal increase in cardiac output relative to oxygen uptake: dQ/dVO2 = 8.2 and 9.5 (> 2DS of controls population). One patient underwent 2 exercise tests; during one test, the patient was febrile, leading to acute rhabdomyolysis in the following hours. He exhibited changes in recovery muscle reoxygenation parameters and an increased dQ/dVO2 during exercise compared with that under normothermia (7.9 vs 6), which did not lead to acute rhabdomyolysis. The four patients assessed by cardiac 1H-magnetic resonance spectroscopy exhibited signs of intracardiac steatosis. Conclusion We observed abnormal haemodynamic profiles during exercise in 3/8 patients with lipin-1 deficiency, suggesting impaired muscle oxidative phosphorylation during exercise. Fever appeared to be an aggravating factor. One patient exhibited moderate cardiac dysfunction, which was possibly related to intracardiac stored lipid toxicity.

Homozygous or compound heterozygous IL36RN gene mutations underlie the pathogenesis of psoriasis-related pustular eruptions including generalized pustular psoriasis, palmoplantar pustular psoriasis, acrodermatitis continua of Hallopeau, and acute generalized exanthematous pustular eruption. We identified two unreported IL36RN homozygous mutations (c.41C>A/p.Ser14X and c.420_426del/p.Gly141MetfsX29) in patients with familial generalized pustular psoriasis. We analyzed the impact of a spectrum of IL36RN mutations on IL-36 receptor antagonist protein by using site-directed mutagenesis and expression in HEK293T cells. This enabled us to differentiate null mutations with complete absence of IL-36 receptor antagonist (the two previously unreported mutations, c.80T>C/p.Leu27Pro, c.28C>T/p.Arg10X, c.280G>T/p.Glu94X, c.368C>G/p.Thr123Arg, c.368C>T/p.Thr123Met, and c.227C>T/p.Pro76Leu) from mutations with decreased (c.95A>G/p.His32Arg, c.142C>T/p.Arg48Trp, and c.308C>T/p.Ser113Leu) or unchanged (c.304C>T/p.Arg102Trp and c.104A>G/p.Lys35Arg) protein expression. Functional assays measuring the impact of mutations on the capacity to repress IL-36–dependent activation of the NF-κB pathway showed complete functional impairment for null mutations, whereas partial or no impairment was observed for other mutations considered as hypomorphic. Finally, null mutations were associated with severe clinical phenotypes (generalized pustular psoriasis, acute generalized exanthematous pustular eruption), whereas hypomorphic mutations were identified in both localized (palmoplantar pustular psoriasis, acrodermatitis continua of Hallopeau) and generalized variants. These results provide a preliminary basis for genotype-phenotype correlation in patients with deficiency of the IL-36Ra (DITRA), and suggest the involvement of other factors in the modulation of clinical expression.

Human type 1 diabetes results from a destructive auto-reactive immune response in which CD8(+) T lymphocytes play a critical role. Given the intense ongoing efforts to develop immune intervention to prevent and/or cure the disease, biomarkers suitable for prediction of disease risk and progress, as well as for monitoring of immunotherapy are required. We undertook separate multi-parameter analyses of single naïve and activated/memory CD8(+) T lymphocytes from pediatric and adult patients, with the objective of identifying cellular profiles associated with onset of type 1 diabetes. We observe global perturbations in gene and protein expression and in the abundance of T cell populations characterizing pediatric but not adult patients, relative to age-matched healthy individuals. Pediatric diabetes is associated with a unique population of CD8(+) T lymphocytes co-expressing effector (perforin, granzyme B) and regulatory (transforming growth factor β, interleukin-10 receptor) molecules. This population persists after metabolic normalization and is especially abundant in children with high titers of auto-antibodies to glutamic acid decarboxylase and with elevated HbA1c values. These findings highlight striking differences between pediatric and adult type 1 diabetes, indicate prolonged large-scale perturbations in the CD8(+) T cell compartment in the former, and suggest that CD8(+)CD45RA(-) T cells co-expressing effector and regulatory factors are of interest as biomarkers in pediatric type 1 diabetes.

Natural regulatory T cells (Tregs) may have a great therapeutic potential to induce tolerance in allogeneic cells and organ transplantations. In mice, we showed that alloantigen-specific Tregs (spe-Tregs) were more efficient than polyclonal Tregs (poly-Tregs) in controlling graft-versus-host disease (GVHD). Here we describe a clinical-grade compliant method for generating human spe-Tregs. Tregs were enriched from leukapheresis products with anti-CD25 immunomagnetic beads, primed twice by allogeneic mature monocyte-derived dendritic cells (mDCs), and cultured during 3 weeks in medium containing interleukin 2 (IL-2), IL-15, and rapamycin. After 3 weeks of culture, final cell products were expanded 8.3-fold from the initial CD25+ purifications. Immunophenotypic analyses of final cells indicate that they were composed of 88 ± 2.6% of CD4+ T cells, all expressing Treg-specific markers (FOXP3, Helios, GARP, LAP, and CD152). Spe-Tregs were highly suppressive in vitro and also in vivo using a xeno-GVHD model established in immunodeficient mice. The specificity of their suppressive activity was demonstrated on their ability to significantly suppress the proliferation of autologous effector T cells stimulated by the same mDCs compared to third-party mDCs. Our data provide evidence that functional alloantigen Tregs can be generated under clinical-grade compliant conditions. Taking into account that 130 × 106 CD25+ cells can be obtained at large scale from standard leukapheresis, our cell process may give rise to a theoretical final number of 1 × 109 spe-Tregs. Thus, using our strategy, we can propose to prepare spe-Tregs for clinical trials designed to control HLA-mismatched GVHD or organ transplantation rejection.

International audience; Mutations in the desmoplakin (DSP) and desmoglein-1 (DSG1) genes have been implicated in patients with the inherited inflammatory skin disease known as severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome (MIM#603165, see Tables E1 and E2 in this article's Online Repository at www.jacionline.org).1, 2 The DSP and DSG1 genes encode desmosome components that are critical for the structure of intercellular junctions and maintenance of epithelial barrier integrity. DSP and DSG1 are also key regulators of signaling pathways involved in differentiation, epidermal homeostasis, and carcinogenesis. DSG1 promotes keratinocyte differentiation by inhibiting epidermal growth factor receptor/extracellular signal-regulated kinase signaling through ERBB2-interacting protein (ERBIN), a scaffolding and signaling protein.3 Through characterization of a new syndrome featuring severe allergic dermatitis and DSG1 deficiency, we highlighted the pivotal role of the functional DSG1/ERBIN interaction as an inhibitor of skin inflammation through the nuclear factor κB (NF-κB) signaling pathway.

Rhabdomyolysis results from the rapid breakdown of skeletal muscle fibers, which leads to leakage of potentially toxic cellular content into the systemic circulation. Acquired causes by direct injury to the sarcolemma are most frequent. The inherited causes are: i) metabolic with failure of energy production, including mitochondrial fatty acid ß-oxidation defects, LPIN1 mutations, inborn errors of glycogenolysis and glycolysis, more rarely mitochondrial respiratory chain deficiency, purine defects and peroxysomal α-methyl-acyl-CoA-racemase defect (AMACR), ii) structural causes with muscle dystrophies and myopathies, iii) calcium pump disorder with RYR1 gene mutations, iv) inflammatory causes with myositis. Irrespective of the cause of rhabdomyolysis, the pathology follows a common pathway, either by the direct injury to sarcolemma by increased intracellular calcium concentration (acquired causes) or by the failure of energy production (inherited causes), which leads to fiber necrosis. Rhabdomyolysis are frequently precipitated by febrile illness or exercise. These conditions are associated with two events, elevated temperature and high circulating levels of pro-inflammatory mediators such as cytokines and chemokines. To illustrate these points in the context of energy metabolism, protein thermolability and the potential benefits of arginine therapy, we focus on a rare cause of rhabdomyolysis, aldolase A deficiency. In addition, our studies on lipin-1 (LPIN1) deficiency raise the possibility that several diseases involved in rhabdomyolysis implicate pro-inflammatory cytokines and may even represent primarily pro-inflammatory diseases. Thus, not only thermolability of mutant proteins critical for muscle function, but also pro-inflammatory cytokines per se, may lead to metabolic decompensation and rhabdomyolysis.

International audience; BackgroundIncontinentia pigmenti (IP; MIM308300) is a severe, male-lethal, X-linked, dominant genodermatosis resulting from loss-of-function mutations in the IKBKG gene encoding nuclear factor κB (NF-κB) essential modulator (NEMO; the regulatory subunit of the IκB kinase [IKK] complex). In 80% of cases of IP, the deletion of exons 4 to 10 leads to the absence of NEMO and total inhibition of NF-κB signaling. Here we describe a new IKBKG mutation responsible for IP resulting in an inactive truncated form of NEMO.; ObjectivesWe sought to identify the mechanism or mechanisms by which the truncated NEMO protein inhibits the NF-κB signaling pathway.; MethodsWe sequenced the IKBKG gene in patients with IP and performed complementation and transactivation assays in NEMO-deficient cells. We also used immunoprecipitation assays, immunoblotting, and an in situ proximity ligation assay to characterize the truncated NEMO protein interactions with IKK-α, IKK-β, TNF receptor-associated factor 6, TNF receptor-associated factor 2, receptor-interacting protein 1, Hemo-oxidized iron regulatory protein 2 ligase 1 (HOIL-1), HOIL-1-interacting protein, and SHANK-associated RH domain-interacting protein. Lastly, we assessed NEMO linear ubiquitination using immunoblotting and investigated the formation of NEMO-containing structures (using immunostaining and confocal microscopy) after cell stimulation with IL-1β.; ResultsWe identified a novel splice mutation in IKBKG (c.518+2T>G, resulting in an in-frame deletion: p.DelQ134_R256). The mutant NEMO lacked part of the CC1 coiled-coil and HLX2 helical domain. The p.DelQ134_R256 mutation caused inhibition of NF-κB signaling, although the truncated NEMO protein interacted with proteins involved in activation of NF-κB signaling. The IL-1β-induced formation of NEMO-containing structures was impaired in fibroblasts from patients with IP carrying the truncated NEMO form (as also observed in HOIL-1(-/-) cells). The truncated NEMO interaction with SHANK-associated RH domain-interacting protein was impaired in a male fetus with IP, leading to defective linear ubiquitination.; ConclusionWe identified a hitherto unreported disease mechanism (defective linear ubiquitination) in patients with IP.

Item does not contain fulltext Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metabolism (alpha-oxoglutarate dehydrogenase [alpha-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metabolism (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymatic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been associated with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metabolism. LIPT1 deficiency spares GCS deficiency and has been associated with a biochemical signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals' fibroblasts showed reduced oxygen consumption rates, PDHc, alpha-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was observed after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clinical condition nor activities of PDHc, alpha-KGDHc, or leucine metabolism in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2.

International audience; Lipin-1 is a Mg2+-dependent phosphatidic acid phosphatase (PAP) that in mice is necessary for normal glycerolipid biosynthesis, controlling adipocytes metabolism and adipogenic differentiation. Mice carrying inactivating mutations in the Lpin1 gene display the characteristic features of human familial lipodystrophy. Very little is known on the roles of lipin-1 in human adipocyte physiology. Apparently fat distribution and weight is normal in humans carrying LPIN1 inactivating mutations, but a detailed analysis of adipose tissue appearance and functions in these patients has not been available so far. In this study, we performed a systematic histopathological, biochemical and gene expression analysis of adipose tissue biopsies from human patients harbouring LPIN1 biallelic inactivating mutations and affected by recurrent episodes of severe rhabdomyolysis. We also explored the adipogenic differentiation potential of human mesenchymal cell populations derived from lipin-1 defective patients. White adipose tissue from human LPIN1 mutant patients displayed a dramatic decrease in lipin-1 protein levels and PAP activity, with a concomitant moderate reduction of the adipocyte size. Nevertheless the adipose tissue develops without obvious histological signs of lipodystrophy and with a normal qualitative composition of the storage lipids. The increased expression of key adipogenic determinants such as SREBP1, PPARG and PGC1A shows that specific compensatory phenomena can be activated in vivo in human adipocytes under deficiency of a functional lipin-1.

Cross-priming of CD8+ T cells is of critical importance in triggering CD8+ T cell responses against pathogens and tumors. It is commonly thought that conventional type 1 dendritic cells (cDC1) cross-prime with unequalled efficacy, conferred to them by a proteasome and TAP-dependent pathway. Here we present data challenging the unique cross-priming efficacy of both the cDC1 subset and the proteasome/TAP-dependent pathway. Splenic CD169 macrophages are strategically placed in the marginal zone for capture of blood-borne pathogens. However, they are considered unable to directly cross-present and to depend on antigen transfer to cDC1 for cross-priming. We have developed a protocol for purification of CD169 macrophages. Extensive characterization shows that these cells display morphology, phenotype and gene expression distinguishing them clearly from cDC1, cDC2, plasmacytoid DC and red pulp macrophages. In vitro, CD169 cells cross-present soluble, receptor-targeted and phagocytosed antigen with equal or better efficiency than cDC1. Remarkably, this is achieved through a vacuolar, BFA and epoxomicin-independent pathway. Using intravital biphoton microscopy, 3D light sheet imaging and antigen targeting to CD169, we show that CD169 macrophages establish long-lasting antigen-dependent contacts with cognate CD8+ T cells that result in T cell activation in situ and generation of triple-positive effector as well as memory cells. Therefore, splenic CD169 macrophages represent a second set of myeloid cells equipped for highly efficient cross-priming. The biological role of cross-priming by these cells as well as their cooperation with cDC1 cells are under investigation.

Type 1 diabetes results from the destruction of β-cells by an autoimmune T-cell response assisted by antigen-presenting B cells producing autoantibodies. CD8+ T-cell responses against islet cell antigens, thought to play a central role in diabetes pathogenesis, can be monitored using enzyme-linked immunosorbent spot (ELISpot) assays. However, such assays have been applied to monitoring of adult patients only, leaving aside the large and increasing pediatric patient population. The objective of this study was twofold: 1) to develop a CD8+ T-cell interferon-γ ELISpot assay for pediatric patients and 2) to determine whether zinc transporter 8 (ZnT8), a recently described target of autoantibodies in a majority of patients, is also recognized by autoreactive CD8+ T cells. Using DNA immunization of humanized mice, we identified nine HLA-A2–restricted ZnT8 epitopes. Among 36 HLA-A2+ children with diabetes, 29 responded to ZnT8 epitopes, whereas only 3 of 16 HLA-A2+ control patients and 0 of 17 HLA-A2− control patients responded. Some single ZnT8 epitopes performed as well as the group of epitopes in discriminating between patients and control individuals. Thus, ZnT8 is a major CD8+ T-cell autoantigen, and ELISpot assays display similar performance in adult and pediatric type 1 diabetes.

Regulatory T cells (Treg) play a pivotal role in the control of graft-versus-host disease (GVHD) and might also influence the graft-versus-tumor effect after allogeneic stem cell transplantation. We assessed this role after donor lymphocyte infusions (DLIs) by quantifying Treg in DLI products, using the CD25, Foxp3 but also the recently identified CD127 Treg markers. Compared with others, patients in durable complete remission of their malignancy after DLI had received a lower number of FoxP3CD25, FoxP3CD127, or CD4CD127 Treg cells (P=0.04). The CD4CD127 Treg content of DLI remained significantly correlated with the hematologic response in multivariate analysis (P=0.05). Treg may thus inhibit graft-versus-tumor effect after DLI, a setting where the antitumoral effect observed is only driven by T-cell-mediated cytotoxicity, independently of any other associated treatment. In comparison with the intracytoplasmic Foxp3 marker, the membranous CD4CD127 phenotype of Treg could be particularly relevant to manipulate this cell-population, to increase the antitumoral response in strategies of allogeneic or autologous immunotherapy.

Background Human CD4+CD25+FOXP3+ natural regulatory T-cells (nTreg) have a great therapeutic potential for the induction of tolerance in allo-transplanted patients or for the control of severe auto-immune diseases. However, clinical-grade production of nTreg remains difficult to achieve because of the absence of a truly specific surface marker and of their low frequency that implies a need for their ex vivo expansion. Furthermore, safety issues should be taken into consideration due to the risk of either uncontrolled nTreg-induced immunosuppression or uncontrolled proliferation of autoreactive contaminating T-cells particularly in an auto-immune context. Methods We compared different clinical-grade conditions for immuno-magnetic selection and ex vivo expansion of nTreg. For safety, expanded cells were genetically modified with retroviral vectors co-expressing human CD90 and HSV1 thymidine kinase. The CD90 surface marker and thymidine kinase allow for selection and elimination of transduced cells by ganciclovir, respectively. Results We showed that (i) nTreg could be enriched in a one step using CD25 microbeads, were functionally suppressive and mainly FOXP3+; (ii) using anti-CD28- and anti-CD3-coated beads, interleukin-2 and rapamycin, nTreg were expanded 150–200-fold after 3 weeks. Under these clinical-grade conditions, they remained suppressive, and no major alteration of the TCR repertoire was observed; (iii) after efficient retroviral transduction and CD90 selection, nTreg maintained their suppressive activity; (iv) transduced nTreg could be eliminated by ganciclovir upon activation. Conclusions The efficient procedure reported here for the preparation of nTreg, whose safety has been ensured, is now applicable for further clinical trials. Copyright © 2008 John Wiley & Sons, Ltd.

Острый рабдомиолиз драматичное внезапное разрушение мышечных волокон скелетных мышц. К генетическим этиологическим факторам относят: метаболические расстройства, сопровождаемые дефицитом окисления жирных кислот, дефицитом липина-1, аномалии гликогенолиза и гликолиза, реже дефицит митохондриальной дыхательной цепи, дефицит пурина и пероксизмальный дефицит α-метил-ацил-КоА-рацемазы (α-methyl-acyl-CoA-racemase, AMACR); структурные патологии в рамках дистрофинопатий и миопатий; аномалии кальциевого обмена с мутациями в гене RYR1; воспалительные реакции, ассоциированные с миозитом. Независимо от причины, дефицит аденозинтрифосфата в миоците приводит к повышению содержания внутриклеточного кальция и некрозу мышечных волокон. Провоцирующим фактором рабдомиолиза могут быть экзогенные факторы, среди которых травматизация мышц является самой частой причиной рабдомиолиза метаболического генеза. В случае лихорадки следует учитывать 2 фактора: повышение температуры тела и существование провоспалительных цитокинов. В статье описан случай рабдомиолиза у 3 детей от близкородственного брака, спровоцированный гипертермией и вызванный дефицитом альдолазы А, не сопровождаемой гемолитической анемией. В рассматриваемом случае миоглобинурия была всегда вызвана фебрильной температурой. В свою очередь, фермент альдолаза-А обладает тканеспецифичной термолабильностью: при тестируемых температурах он обнаружен в миобластах, но не в эритроцитах, что объясняет специфическую симптоматику у описываемых пациентов. Существуют предположения, что в клеточной липотоксичности участвуют так называемые жировые капли. В ходе исследований in vitro дефицит альдолазы А был возмещен добавлением аргинина. Другие типы рабдомиолиза метаболического генеза, вероятно, являются провоспалительными заболеваниями.

International audience; Aldolase A deficiency has been reported as a rare cause of hemolytic anemia occasionally associated with myopathy. We identified a deleterious homozygous mutation in the ALDOA gene in 3 siblings with episodic rhabdomyolysis without hemolytic anemia. Myoglobinuria was always triggered by febrile illnesses. We show that the underlying mechanism involves an exacerbation of aldolase A deficiency at high temperatures that affected myoblasts but not erythrocytes. The aldolase A deficiency was rescued by arginine supplementation in vitro but not by glycerol, betaine or benzylhydantoin, three other known chaperones, suggesting that arginine-mediated rescue operated by a mechanism other than protein chaperoning. Lipid droplets accumulated in patient myoblasts relative to control and this was increased by cytokines, and reduced by dexamethasone. Our results expand the clinical spectrum of aldolase A deficiency to isolated temperature-dependent rhabdomyolysis, and suggest that thermolability may be tissue specific. We also propose a treatment for this severe disease.

Cytomegalovirus (CMV) is responsible for significant morbidity and mortality in immunocompromised patients undergoing allogeneic hematopoietic stem cell transplantation. The limitations of antiviral drugs and a better understanding of the cellular immune response to CMV has lead to the development of alternative therapies that restore host cellular immunity to CMV. Infusion of donor T lymphocytes results in variable protection against CMV but a high incidence of graft-versus-host disease in the allogeneic setting. To prevent this complication and further improve anti-CMV immune response, several groups have developed new approaches, such as the introduction of a suicide gene to control alloreactivity against the host or the selective activation of CMV-specific T cells by antigen-presenting cells expressing CMV antigens introduced by gene transfer. Depending on the target cells and the strategy chosen, adenovirus, retrovirus or poxviruses derived vectors are used for gene transfer. The protocols as well as the preclinical and clinical results obtained in the field of anti-CMV immunotherapy using gene transfer are reported and discussed.

Human cytomegalovirus (HCMV) and adenovirus cause significant morbidity and mortality in immunocompromised hosts undergoing allogeneic stem cell transplantation. We have previously established a procedure for the generation of polyclonal CTL with specificity against adenovirus and HCMV using a recombinant adenovirus encoding the HCMV pp65 protein (RAdpp65). However, specific CTL expanded after in vitro culture steps were subjected to several in vitro restimulations and, depending on the protocol adopted, this could lead to a selection bias, compromising the clinical benefit. To determine which part of the memory repertoire is selected after in vitro restimulation, we have followed the specificity and clonal composition of pp65-peptide-specific CD8(+) T cells in HLA-A*201 individuals before and after repeated in vitro restimulation of cells with RAdpp65, combining HLA tetrameric complexes and immunoscope analysis. Tetramer staining showed that, after in vitro restimulation, up to 60% of CD8(+) T cells were virus-specific. Immunoscope analysis showed that the predominant TCRBV diversity of pp65-specific clones was conserved, demonstrating that the memory repertoire was preserved all along the procedure. Altogether, these results suggest that the use of RAdpp65 to induce CMV- and adenovirus-specific CTL maybe appropriate for immunotherapy.

We have attempted to improve retrovirus-mediated gene transfer efficacy into hematopoietic progenitor cells (HPCs) without causing them to lose their lymphoid potential. Highly purified CD34(+) cells on CH-296 fibronectin fragments have been transduced with three different cytokine combinations. Murine CD2 was used as a marker gene. Transgene expression was assayed by FACS analysis shortly after transduction of CD34(+) cells and after long-term culture (LTC) extended by differentiation of various lymphoid lineages: NK cells, B cells, and dendritic cells. Compared with the historical cytokine mix, i.e., SCF (stem cell factor) + IL-3 (interleukin 3) + IL-6, the combination SCF + FL (Flt-3 ligand) + M-GDF (megakaryocyte growth and differentiation factor) + IL-3 significantly improved the total number of viable cells and CD34(+) cells after transduction and the long term-cultured progenitors after 6 weeks. In addition, the combination of SCF + FL + M-GDF + IL-3 maintained more efficiently the lymphoid potential of the progeny of transduced long term-cultured CD34(+) cells, as attested by the significantly higher number of CD56(+), CD19(+), and CD1a(+) cells recovered when FL and M-GDF were added to SCF + IL-3. Thus, even though additional improvements may still be needed in transduction of HPCs, these conditions were adopted for a clinical trial of gene therapy for X-linked severe combined immunodeficiency.

International audience; BACKGROUND: Synthesis and apoenzyme attachment of lipoic acid have emerged as a new complex metabolic pathway. Mutations in several genes involved in the lipoic acid de novo pathway have recently been described, but no mutation was found so far in genes involved in the specific process of attachment of lipoic acid to apoenzymes pyruvate dehydrogenase (PDHc), alpha-ketoglutarate dehydrogenase (alpha-KGDHc) and branched chain alpha-keto acid dehydrogenase (BCKDHc) complexes. METHODS: Exome capture was performed in a boy who developed Leigh disease following a gastroenteritis and had combined PDH and alpha-KGDH deficiency with a unique amino acid profile that partly ressembled E3 subunit (dihydrolipoamide dehydrogenase / DLD) deficiency. Functional studies on patient fibroblasts were performed. Lipoic acid administration was tested on the LIPT1 ortholog lip3 deletion strain yeast and on patient fibroblasts. RESULTS: Exome sequencing identified two heterozygous mutations (c.875C > G and c.535A > G) in the LIPT1 gene that encodes a mitochondrial lipoyltransferase which is thought to catalyze the attachment of lipoic acid on PDHc, alpha-KGDHc, and BCKDHc. Anti-lipoic acid antibodies revealed absent expression of PDH E2, BCKDH E2 and alpha-KGDH E2 subunits. Accordingly, the production of 14CO2 by patient fibroblasts after incubation with 14Cglucose, 14Cbutyrate or 14C 3OHbutyrate was very low compared to controls. cDNA transfection experiments on patient fibroblasts rescued PDH and alpha-KGDH activities and normalized the levels of pyruvate and 3OHbutyrate in cell supernatants. The yeast lip3 deletion strain showed improved growth on ethanol medium after lipoic acid supplementation and incubation of the patient fibroblasts with lipoic acid decreased lactate level in cell supernatants. CONCLUSION: We report here a putative case of impaired free lipoic acid attachment due to LIPT1 mutations as a cause of PDH and alpha-KGDH deficiencies. Our study calls for renewed efforts to understand the mechanisms of pathology of lipoic acid-related defects and their heterogeneous biochemical expression, in order to devise efficient diagnostic procedures and possible therapies.

This Response refers to JGM 1286, which was published in The Journal of Gene Medicine 11:2. Please follow the link below to view the Letter to the Editor. Letter to the Editor

Using a reporter plasmid containing the luciferase gene under the control of the insulin-like growth factor 1 (IGF-1) promoter region [including its 5ʹ untranslated region (UTR)], we demonstrate that a 17-mer oligophosphorothioate containing C-5 propyne pyrimidines is able to inhibit luciferase gene expression in the nanomolar concentration range when the anti-sense oligonucleotide is targeted either to a coding sequence in the luciferase gene or to the 5ʹ UTR of the gene for IGF-1. Inhibition was obtained independently of whether the plasmid and the anti-sense oligonucleotide were co-transfected or transfected separately into hepatocarcinoma cells. However, the efficiency of inhibition by the anti-sense oligonucleotides was 10-fold greater in the first case. The unmodified oligophosphorothioate targeted to the 5ʹ UTR of IGF-1 did not inhibit luciferase gene expression at a 100-fold higher concentration unless its length was increased from 17 to 21 nt, in which case an inhibition of gene expression was obtained and an IC50 of 200 nM was observed.

Abstract 47 Adoptive transfer of natural regulatory T-cells (Tregs) may have a great therapeutic potential for the induction of tolerance in transplantation patients. This concept was demonstrated in murine models of graft versus host disease (GVHD) where alloantigen-specific Tregs (sTregs) were always more efficient to control GVHD than polyclonal Tregs (pTregs). Recently, we reported a procedure for expanding human pTregs in the presence of anti-CD3/anti-CD28 coated beads, cells being cultured in RPMI medium containing 10% human serum, interleukin (IL)-2 and rapamycin (namely TCM for Tregs Culture Medium) during 3 weeks. Whether or not alloantigen sTregs could be generated for the control of GVHD was investigated. In a first set of experiments, we compared the activation of Tregs when stimulated by allogeneic monocyte-derived dendritic cells (DCs) under different conditions. To follow cell divisions, Tregs (CD4+CD25high T-cells) were purified by FACS, stained with CFSE and co-cultured in TCM ± IL-15 in the presence of either immature (i) or mature (m) allogeneic DCs. Data showed that: i) mDCs yielded higher Treg divisions than iDCs; ii) combination of IL-2 + IL-15 triggered better cell division than IL-2 or IL-15 alone; iii) Tregs divided from day-3 to day-10 when stimulated by mDCs and cultured in TCM + IL-15. Next, the alloantigen specificity of divided Tregs was evaluated: Tregs stimulated by allogeneic mDCs (mDC1) were FACS-sorted at day-4 on the basis of CFSE-staining. CFSElow, CFSEintermediate (int) and CFSEhigh cell fractions were separated and expanded in TCM medium IL-15 in the presence anti-CD3/anti-CD28 coated beads. At day-21, they were assayed for their ability to inhibit within 4 days the proliferation of autologous effector T-cells (Teff) stimulated by either mDC1 or a third party of allogeneic mDCs (mDC2), used as control of specificity. In these mixed leukocyte cultures, different Tregs/Teff cell ratio were tested. Results showed that the CFSElow fraction (highly dividing Tregs) was greatly enriched in sTregs, by contrast to the CFSEhigh and CFSEint fractions. From these data, further experiments that are more suitable for clinical application were performed. Tregs selected by FACS or by immunomagnetic selection (MACs), based on CD25 expression, were cultured in TCM + IL-15 and stimulated twice (day-0 and day-10) by allogeneic mDCs (mDC1). At day-21, the alloantigen-specific suppression of sTregs was studied and compared to the suppression activity of pTregs. Results showed that sTregs better suppressed the proliferation of mDC1-stimulated Teff than pTregs. In addition, sTregs better maintained their suppressive activity than pTregs at low Tregs/Teff cell ratio. Under these culture conditions, sTregs can be expanded ∼12 fold within 3-weeks. Our data showed that human sTregs can be generated under clinical grade conditions. Further experiments using a xenogeneic GVHD model are now envisaged to compare the respective capacity for sTregs and pTregs to control GVHD. Disclosures: No relevant conflicts of interest to declare.

In order to gain further insight on the role of Kaposi's sarcoma-associated herpesvirus (KSHV) in classic and endemic Kaposi's sarcoma (KS) pathogenesis, we aimed to determine (i) whether KSHV is detectable in peripheral blood mononuclear cells (PBMCs), (ii) which PBMCs subpopulation harbor the virus, (iii) which clinical, histologic, and immunologic parameters are associated with KSHV viremia in a population of classic and endemic KS. KSHV viremia and various immunologic parameters were screened on 81 patients. KSHV viremia was positive in 58% of the patients. KSHV was detected in B cells, T cells, and monocytes. CD34+ cells depleted in circulating endothelial cells (CECs) were never infected and 50% of the patients tested had CECs infected by KSHV. We observed a significant increase of IL-2 and IFN-gamma production by CD4 T cells and an increase of IFN-gamma production by CD8 T cells compared to control patients. KS progression (P = 0.001) and KS staging (P = 0.03) were significantly and independently associated with positive KSHV viremia. Our results show that there is no specific immunosuppression in classic or endemic KS. We showed that KSHV can be detected within CECs and that KSHV viremia could be an indicator of circulating mature or precursor spindle cells.

Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (Ad) cause significant morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. We have established a procedure to generate polyclonal cytotoxic T lymphocyte (CTL) populations with specificity against Ad and CMV or against Ad and EBV. Healthy donor-derived dendritic cells (DCs) were transduced with recombinant adenovirus encoding either CMV pp65 or EBV EBNA3C and used to stimulate autologous T cells. Stimulated T lymphocytes displayed specific simultaneous cytotoxicity against CMV and adenovirus and to a lesser extent against adenovirus and EBV. Recombinant vaccinia virus encoding individual adenovirus proteins showed that the T cell response to the adenovirus was directed mainly against the capsid protein hexon. The frequency of IFN-gamma-secreting T cells was 0.02% for adenovirus alone, and 0.05 and 0.14% for adenoviruses encoding EBNA3C and pp65, respectively. pp65-specific CTLs killed autologous fibroblasts infected with the laboratory strain CMV AD169. The culture conditions were specific as alloreactive T cells were not expanded. Therefore, this approach could be considered in order to generate efficient virus cytolytic T cells to be used as adoptive immunotherapy in transplanted patients.