Hemifacial myohyperplasia is due to somatic muscular PIK3CA gain-of-function mutations and responds to pharmacological inhibition.

Hemifacial myohyperplasia (HFMH) is a rare cause of facial asymmetry exclusively involving facial muscles. The underlying cause and the mechanism of disease progression are unknown. Here, we identified a somatic gain-of-function mutation of PIK3CA in five pediatric patients with HFMH. To understand the physiopathology of muscle hypertrophy in this context, we created a mouse model carrying specifically a PIK3CA mutation in skeletal muscles. PIK3CA gain-of-function mutation led to striated muscle cell hypertrophy, mitochondria dysfunction, and hypoglycemia with low circulating insulin levels. Alpelisib treatment, an approved PIK3CA inhibitor, was able to prevent and reduce muscle hypertrophy in the mouse model with correction of endocrine anomalies. Based on these findings, we treated the five HFMH patients. All patients demonstrated clinical, esthetical, and radiological improvement with proof of target engagement. In conclusion, we show that HFMH is due to somatic alteration of PIK3CA and is accessible to pharmacological intervention.

PIK3CA gain-of-function mutation in adipose tissue induces metabolic reprogramming with Warburg-like effect and severe endocrine disruption.

PIK3CA-related overgrowth syndrome (PROS) is a genetic disorder caused by somatic mosaic gain-of-function mutations of PIK3CA. Clinical presentation of patients is diverse and associated with endocrine disruption. Adipose tissue is frequently involved, but its role in disease development and progression has not been elucidated. Here, we created a mouse model of PIK3CA-related adipose tissue overgrowth that recapitulates patient phenotype. We demonstrate that PIK3CA mutation leads to GLUT4 membrane accumulation with a negative feedback loop on insulin secretion, a burst of liver IGFBP1 synthesis with IGF-1 sequestration, and low circulating levels. Mouse phenotype was mainly driven through AKT2. We also observed that PIK3CA mutation induces metabolic reprogramming with Warburg-like effect and protein and lipid synthesis, hallmarks of cancer cells, in vitro, in vivo, and in patients. We lastly show that alpelisib is efficient at preventing and improving PIK3CA-adipose tissue overgrowth and reversing metabolomic anomalies in both animal models and patients.

Alpelisib administration reduced lymphatic malformations in a mouse model and in patients.

Lymphatic cystic malformations are rare genetic disorders mainly due to somatic gain-of-function mutations in the PIK3CA gene. These anomalies are frequently associated with pain, inflammatory flares, esthetic deformities, and, in severe forms, life-threatening conditions. There is no approved medical therapy for patients with lymphatic malformations. In this proof-of-concept study, we developed a genetic mouse model of PIK3CA-related lymphatic malformations that recapitulates human disease. Using this model, we demonstrated the efficacy of alpelisib, an approved pharmacological inhibitor of PIK3CA in oncology, in preventing lymphatic malformation occurrence, improving lymphatic anomalies, and extending survival. On the basis of these results, we treated six patients with alpelisib, including three children, displaying severe PIK3CA-related lymphatic malformations. Patients were already unsuccessfully treated with rapamycin, percutaneous sclerotherapies, and debulking surgical procedures. We assessed the volume of lymphatic malformations using magnetic resonance imaging (MRI) for each patient. Alpelisib administration was associated with improvements in the six patients. Previously intractable vascular malformations shrank, and pain and inflammatory flares were attenuated. MRI showed a decrease of 48% in the median volume of lymphatic malformations over 6 months on alpelisib. During the study, two patients developed adverse events potentially related to alpelisib, including grade 1 mucositis and diarrhea. In conclusion, this study supports PIK3CA inhibition as a promising therapeutic strategy in patients with PIK3CA-related lymphatic anomalies.

Immunodynamics of explanted human tumors for immuno-oncology.

Decision making in immuno-oncology is pivotal to adapt therapy to the tumor microenvironment (TME) of the patient among the numerous options of monoclonal antibodies or small molecules. Predicting the best combinatorial regimen remains an unmet medical need. Here, we report a multiplex functional and dynamic immuno-assay based on the capacity of the TME to respond to ex vivo stimulation with twelve immunomodulators including immune checkpoint inhibitors (ICI) in 43 human primary tumors. This "in sitro" (in situ/in vitro) assay has the potential to predict unresponsiveness to anti-PD-1 mAbs, and to detect the most appropriate and personalized combinatorial regimen. Prospective clinical trials are awaited to validate this in sitro assay.

Hip Fracture Leads to Transitory Immune Imprint in Older Patients.

Background: Hip fracture (HF) is common in the geriatric population and is associated with a poor vital and functional prognosis which could be impacted by immunological changes. The objective here is to decipher immune changes occurring in the 1st days following HF and determine how phenotype, function, and regulation of innate and adaptive compartments adapt during acute stress event. Methods: We included HF patients, aged over 75 years. For each patient, blood samples were taken at five different timepoints: four in the perioperative period (day 0 to hospital discharge) and one at long term (6-12 months). Phenotypical and functional analysis were performed longitudinally on fresh blood or cryopreserved PBMCs. Clinical data were prospectively collected. Results: One-hundred HF patients and 60 age-matched controls were included. Innate compartment exhibits pro-inflammatory phenotypes (hyperleukocytosis, increase of CD14+ CD16+ proportion and CCR2 expression), maintaining its ability to produce pro-inflammatory cytokines. Adaptive compartment extends toward a transitory immunosuppressive profile (leucopenia) associated with an active T-cell proliferation. Furthermore, increases of LAG-3 and PD-1 and a decrease of 2-B4 expression are observed on T-cells, reinforcing their transitory suppressive status. Of note, these immune changes are transitory and sequential but may participate to a regulation loop necessary for homeostatic immune control at long term. Conclusion: HF is associated with several transitory immunological changes including pro-inflammatory phenotype in innate compartment and immunosuppressive profile in adaptive compartment. A comprehensive assessment of immune mechanisms implicated in the patient's prognosis after HF could pave the way to develop new immune therapeutics strategies.

Phenotypic and Functional Differences between Human Herpesvirus 6- and Human Cytomegalovirus-Specific T Cells.

Human herpesvirus 6 (HHV-6) infects >90% of the population and establishes a latent infection with asymptomatic episodes of reactivation. However, HHV-6 reactivation is associated with morbidity and sometimes mortality in immunocompromised patients. To date, control of the virus in healthy virus carriers and the failure to control it in patients with disease remain poorly understood. In particular, knowledge of HHV-6-specific T-cell responses is limited. Here, we characterized HHV-6A- and HHV-6B-specific CD4+ and CD8+ T-cell responses from peripheral blood mononuclear cells (PBMCs) of healthy donors. We studied the phenotype of effector HHV-6-specific T cells ex vivo, as well as of induced specific suppressive regulatory CD4+ T cells in vitro poststimulation, in comparison to human cytomegalovirus (HCMV) responses. Compared to that for HCMV, we show that ex vivo T-cell reactivity in peripheral blood is detectable but at very low frequency, both for HHV-6A and -6B viruses. Interestingly, the phenotype of the specific T cells also differs between the viruses. HHV-6A- and HHV-6B-specific CD4+ T lymphocytes are less differentiated than HCMV-specific T cells. Furthermore, we show a higher frequency of HHV-6-specific suppressive regulatory T cells (eTregs) than HCMV-specific eTregs in coinfected individuals. Despite the strong similarity of HHV-6 and HCMV from a virologic point of view, we observed immunological differences, particularly in relation to the frequency and phenotype of effector/memory and regulatory virus-specific T cells. This suggests that different immune factors are solicited in the control of HHV-6 infection than in that of HCMV infection.IMPORTANCE T cells are central to an effective defense against persistent viral infections that can be related to human cytomegalovirus (HCMV) or human herpesvirus 6 (HHV-6). However, knowledge of HHV-6-specific T-cell responses is limited. In order to deepen our knowledge of T-cell responses to HHV-6, we characterized HHV-6A- and HHV-6B-specific CD4+ and CD8+ T-cell responses directly ex vivo from healthy coinfected blood donors. Despite the strong similarity of HHV-6 and HCMV from a virologic point of view, we observed immunological differences, particularly in relation to the frequency and phenotype of effector/memory and regulatory virus-specific T cells. This suggests that different immune factors are solicited in the control of HHV-6 infection than in that of HCMV infection. Our findings may encourage immunomonitoring of patients with viral replication episodes to follow the emergence of effector versus regulatory T cells.

New Insights into Lymphocyte Differentiation and Aging from Telomere Length and Telomerase Activity Measurements.

αß CD8+, γδ, and NK lymphocytes are fundamental effector cells against viruses and tumors. These cells can be divided into multiple subsets according to their phenotype. Based on progressive telomere attrition from naive to late effector memory cells, human CD8+ T cell subsets have been positioned along a pathway of differentiation, which is also considered as a process of lymphocyte aging or senescence. A similar categorization has not been clearly established for γδ and NK cell populations. Moreover, the distinction between the aging of these populations due to cellular differentiation or due to the chronological age of the donor has not been formally considered. In this study, we performed systematic measurements of telomere length and telomerase activity in human αß CD8+, γδ, and NK lymphocytes based on subset division and across age to address these points and better understand the dichotomy between differentiation and temporal aging. This approach enables us to position phenotypically distinct γδ or NK subsets along a putative pathway of differentiation, such as for CD8+ T cells. Moreover, our data show that both cellular differentiation and donor aging have profound but independent effects on telomere length and telomerase activity of lymphocyte subpopulations, implying distinct mechanisms and consequences on the immune system.

B7-H6-mediated downregulation of NKp30 in natural killer cells contributes to HIV-2 immune escape.

OBJECTIVE: HIV-1 and HIV-2 differ notably in their epidemiology, with worldwide HIV-1 spread and HIV-2 mainly confined to West Africa. Natural killer (NK) cells are critical antiviral effectors of the immune system; however, limited information is available about these innate effector cells during HIV-2 infection. METHOD: In this study, 24 untreated HIV-2-infected patients were analyzed and compared with 21 long-term nonprogressor and 10 controller HIV-1 patients, and healthy donors. Extensive phenotype and functional NK-cell characteristics, as well as ligands of activating NK receptors involved in NK lysis were determined by flow cytometry. RESULTS: We report in HIV-2 patients a very significant reduced expression of the activating NKp30 receptor (P < 0.0001) on NK cells, much higher than observed in HIV-1 patients. The impaired expression of NKp30 is correlated negatively with HLA-DR (r = -0.5970; P = 0.0002), and positively with both NKG2A (r = 0.5324; P < 0.0001) and Siglec-7 (r = 0.5621; P = 0.0004). HIV-2 patients with NKp30 NK cells displayed overproduction of IFN-γ (P < 0.0001) associated with impaired cytolytic function when tested against target cells expressing surface B7-H6. This cellular ligand of NKp30 is strongly detectable as a surface molecule on CD4 T cells infected by HIV-2. CONCLUSION: Altogether, our data suggested that the defective expression of NKp30 may be induced by the chronic engagement of this receptor by B7-H6 expressed on HIV-2-infected target cells. This represents a novel mechanism by which the chronic ligand exposure by the viral environment may subvert NK-cell-mediated function to establish persistent HIV-2 infection.

An Optimized Lentiviral Vector Efficiently Corrects the Human Sickle Cell Disease Phenotype.

Autologous transplantation of hematopoietic stem cells transduced with a lentiviral vector (LV) expressing an anti-sickling HBB variant is a potential treatment for sickle cell disease (SCD). With a clinical trial as our ultimate goal, we generated LV constructs containing an anti-sickling HBB transgene (HBBAS3), a minimal HBB promoter, and different combinations of DNase I hypersensitive sites (HSs) from the locus control region (LCR). Hematopoietic stem progenitor cells (HSPCs) from SCD patients were transduced with LVs containing either HS2 and HS3 (ß-AS3) or HS2, HS3, and HS4 (ß-AS3 HS4). The inclusion of the HS4 element drastically reduced vector titer and infectivity in HSPCs, with negligible improvement of transgene expression. Conversely, the LV containing only HS2 and HS3 was able to efficiently transduce SCD bone marrow and Plerixafor-mobilized HSPCs, with anti-sickling HBB representing up to ∼60% of the total HBB-like chains. The expression of the anti-sickling HBB and the reduced incorporation of the ßS-chain in hemoglobin tetramers allowed up to 50% reduction in the frequency of RBC sickling under hypoxic conditions. Together, these results demonstrate the ability of a high-titer LV to express elevated levels of a potent anti-sickling HBB transgene ameliorating the SCD cell phenotype.

Elderly human hematopoietic progenitor cells express cellular senescence markers and are more susceptible to pyroptosis.

The maintenance of effective immunity over time is dependent on the capacity of hematopoietic stem cells (HSCs) to sustain the pool of immunocompetent mature cells. Decline of immune competence with old age may stem from HSC defects, including reduced self-renewal potential and impaired lymphopoiesis, as suggested in murine models. To obtain further insights into aging-related alteration of hematopoiesis, we performed a comprehensive study of blood hematopoietic progenitor cells (HPCs) from older humans. In the elderly, HPCs present active oxidative phosphorylation and are pressed to enter cell cycling. However, p53-p21 and p15 cell senescence pathways, associated with telomerase activity deficiency, strong telomere attrition, and oxidative stress, are engaged, thus limiting cell cycling. Moreover, survival of old HPCs is impacted by pyroptosis, an inflammatory form of programmed cell death. Lastly, telomerase activity deficiency and telomere length attrition of old HPCs may be passed on to progeny cells such as naive T lymphocytes, further highlighting the poor hematopoietic potential of the elderly. This pre-senescent profile is characteristic of the multiple intrinsic and extrinsic factors affecting HPCs in elderly individuals and represents a major obstacle in terms of immune reconstitution and efficacy with advanced age.

Elevated Neopterin Levels Predict Early Death in Older Hip-fracture Patients.

Our society faces a major challenge concerning management of the health and socio-economic burden caused by acute physical stress in the older population (+75years). In particular, hip-fracture surgery (HFS) represents a major health care preoccupation, affecting 1.6 million patients worldwide, resulting in a significant drop in life quality and autonomy. The trauma is associated with 20-30% one-year mortality in the elderly. In the present study, we aim to identify factors, which influence and/or predict the outcome of elderly hip- fracture patients (HFP) post-surgery. Our objective was to identify biomarkers with a prognostic capacity of one-year mortality. We employed an observational cohort of HFP (n=60) followed-up longitudinally during the first year post fracture. Clinical and biological data (n=136), collected at arrival to hospital, were then compared to healthy controls (n=42) and analyzed using a regularized logistic regression model with lasso penalty followed by 10-fold cross-validation of variables. We show that plasmatic neopterin levels, a molecule released by IFN-γ-activated macrophages, is predictive of mortality in HFP (ROC-AUC=0.859). Moreover, neopterin measured at arrival to the hospital correlated negatively with the time of survival after HFS. Neopterin therefore represents a biomarker, which enables better follow-up of patients at risk of early death.

Reduced Oxidative Burst by Primed Neutrophils in the Elderly Individuals Is Associated With Increased Levels of the CD16bright/CD62Ldim Immunosuppressive Subset.

The aim of our study was to analyze polymorphonuclear neutrophil (PMN) functions in elderly individuals compared with those in healthy young participants, directly in whole blood to avoid issues with data interpretation related to cell isolation procedures. Despite the presence of increased circulating levels of proinflammatory cytokines, resting PMNs from the elderly individuals were not activated as shown by normal CD62L and CD11b expression at the PMN surface and normal constitutive reactive oxygen species (ROS) production. However, suboptimal stimulation induced modulations of CD62L and CD11b expression, which positively correlated with the interleukin-6 circulating level, suggesting a possible in vivo preactivation of old PMNs by this cytokine. In addition, PMN phagocytosis of opsonized Escherichia Coli was decreased in elderly individuals. Furthermore, upon preincubation of elderly whole-blood samples with tumor necrosis factor-α or Toll Receptor agonists, we observed a reduced PMN oxidative burst in response to formyl peptides. Elderly participants also exhibited an increased percentage of the immunosuppressive CD16bright/CD62Ldim PMN subpopulation, which was characterized by a lower phagocytic index and a reduced ROS production compared with the CD16bright/CD62Lbright subset. Thus, the reduced phagocytosis and ROS production associated with an expansion of immunosuppressive CD16bright/CD62Ldim PMN subpopulation might be involved in the increased susceptibility to bacterial and fungal infections with old age.

Preservation of Lymphopoietic Potential and Virus Suppressive Capacity by CD8+ T Cells in HIV-2-Infected Controllers.

Compared with HIV-1, HIV-2 infection is characterized by a larger proportion of slow or nonprogressors. A better understanding of HIV-2 pathogenesis should open new therapeutic avenues to establish control of HIV-1 replication in infected patients. In this study, we studied the production of CD8(+) T cells and their capacity for viral control in HIV-2 controllers from the French ANRS CO5 HIV-2 cohort. HIV-2 controllers display a robust capacity to support long-term renewal of the CD8(+) T cell compartment by preserving immune resources, including hematopoietic progenitors and thymic activity, which could contribute to the long-term maintenance of the CD8(+) T cell response and the avoidance of premature immune aging. Our data support the presence of HIV-2 Gag-specific CD8(+) T cells that display an early memory differentiation phenotype and robust effector potential in HIV-2 controllers. Accordingly, to our knowledge, we show for the first time that HIV-2 controllers possess CD8(+) T cells that show an unusually strong capacity to suppress HIV-2 infection in autologous CD4(+) T cells ex vivo, an ability that likely depends on the preservation of host immune resources. This effective and durable antiviral response probably participates in a virtuous circle, during which controlled viral replication permits the preservation of potent immune functions, thus preventing HIV-2 disease progression.

Coordinated expansion of both memory T cells and NK cells in response to CMV infection in humans.

NK cells are key players in the fight against persistent viruses. Human cytomegalovirus (HCMV) infection is associated with the presence of a population of CD16(+) CD56(dim) NKG2C(+) NK cells in both acutely and latently infected individuals. Here, we studied the nature of these terminally differentiated NK cells in different human populations infected with HCMV: healthy donors stratified by age, thymectomized individuals, pregnant women suffering from primary CMV infection, and lung transplant patients. Both CD16(+) CD56(dim) NK- and CD8 T-cell phenotypes as well as functional capacities were determined and stratified according to age and/or CMV event. Similarly to T-cell responsiveness, we observe an accumulation over time of NKG2C(+) NK cells, which preferentially expressed CD57. This accumulation is particularly prominent in elderly and amplified further by CMV infection. Latent HCMV infection (without replication) is sufficient for NKG2C(+) CD57(+) NK cells to persist in healthy individuals but is not necessarily required in old age. Collectively, the present work supports the emerging concept that CMV shapes both innate and adaptive immunity in humans.

Vitamin D supplementation is associated with reduced immune activation levels in HIV-1-infected patients on suppressive antiretroviral therapy.

BACKGROUND: A majority of HIV-1-infected patients present a severe deficit in vitamin D, which predicts short-term mortality. Vitamin D is a naturally synthesized hormone, with important immunomodulatory functions. In the general population, its deficit has been associated with increased markers of inflammation. Vitamin D deficit may therefore play a role in the establishment of elevated systemic immune activation, which persists despite suppressive antiretroviral therapy (ART) in HIV-infected patients, and is predictive of disease progression; and vitamin D supplementation may be beneficial in this context. METHODS: We performed both a cross-sectional study (vitamin D deficit versus normal level) and a longitudinal study (upon vitamin D supplementation for 6 to 12 months) of HIV-1-infected patients receiving suppressive ART. The primary outcome measure was the percentage of activated memory CD8(+) T cells in blood, which is a robust marker associated with disease progression. Secondary outcomes included general T-lymphocyte and B-lymphocyte phenotype. RESULTS: Although vitamin D deficiency had no influence on T-cell and B-cell subset distribution, we found an association between vitamin D and immune activation levels in HIV-1-infected patients. Vitamin D supplementation in vitamin D-deficient patients resulted in reduced immune activation levels. CONCLUSION: The present data support the rationale of vitamin D supplementation in the routine clinical management of HIV-1-infected patients, in order to decrease immune activation levels and possibly improve long-term survival.