Correction: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

[This corrects the article DOI: 10.1371/journal.ppat.1008560.].

Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

Human cytomegalovirus (HCMV) causes serious complications to immune compromised hosts. Dendritic cells (iDCgB) expressing granulocyte-macrophage colony-stimulating factor, interferon-alpha and HCMV-gB were developed to promote de novo antiviral adaptive responses. Mice reconstituted with a human immune system (HIS) were immunized with iDCgB and challenged with HCMV, resulting into 93% protection. Immunization stimulated the expansion of functional effector memory CD8+ and CD4+ T cells recognizing gB. Machine learning analyses confirmed bone marrow T/CD4+, liver B/IgA+ and spleen B/IgG+ cells as predictive biomarkers of immunization (≈87% accuracy). CD8+ and CD4+ T cell responses against gB were validated. Splenic gB-binding IgM-/IgG+ B cells were sorted and analyzed at a single cell level. iDCgB immunizations elicited human-like IgG responses with a broad usage of various IgG heavy chain V gene segments harboring variable levels of somatic hypermutation. From this search, two gB-binding human monoclonal IgGs were generated that neutralized HCMV infection in vitro. Passive immunization with these antibodies provided proof-of-concept evidence of protection against HCMV infection. This HIS/HCMV in vivo model system supported the validation of novel active and passive immune therapies for future clinical translation.

Mutations of the gene FNIP1 associated with a syndromic autosomal recessive immunodeficiency with cardiomyopathy and pre-excitation syndrome.

AMPK (adenosine monophosphate-activated protein kinase) is phosphorylated (AMPK-P) in response to low energy through allosteric activation by Adenosine mono- or diphosphate (AMP/ADP). Folliculin (FLCN) and the FLCN-interacting proteins 1 and 2 (FNIP1, 2) modulate AMPK. FNIP1 deficiency patients have a AMPK-P gain of function phenotype with hypertrophic cardiomyopathy, Wolff-Parkinson-White pre-excitation syndrome, myopathy of skeletal muscles and combined immunodeficiency.

CAMT-MPL: congenital amegakaryocytic thrombocytopenia caused by MPL mutations - heterogeneity of a monogenic disorder - a comprehensive analysis of 56 patients.

Congenital amegakaryocytic thrombocytopenia caused by deleterious homozygous or compound heterozygous mutations in MPL (CAMT-MPL) is a rare inherited bone marrow failure syndrome presenting as an isolated thrombocytopenia at birth progressing to pancytopenia due to exhaustion of hematopoietic progenitors. The analysis of samples and clinical data from a large cohort of 56 patients with CAMT-MPL resulted in a detailed description of the clinical picture and reliable genotype-phenotype correlations for this rare disease. We extended the spectrum of CAMT causing MPL mutations regarding number (17 novel mutations) and impact. The clinical courses showed a great variability with respect to the severity of thrombocytopenia, the development of pancytopenia and the consequences from bleedings. The most severe clinical problems were (1) intracranial bleedings pre- and perinatally and the resulting long-term consequences, and (2) the development of aplastic anemia in the later course of the disease. An important and new finding was that thrombocytopenia was not detected at birth in a quarter of the patients. The rate of non-hematological abnormalities in CAMT-MPL was higher than described so far. Most of the anomalies were related to the head region (brain anomalies, ocular and orbital anomalies) and consequences of intracranial bleedings. The present study demonstrates a higher variability of clinical courses than described so far and has important implications on diagnosis and therapy. The diagnosis CAMT-MPL has to be considered even for those patients who are inconspicuous in the first months of life or show somatic anomalies typical for other inherited bone marrow failure syndromes.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells.

T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4(+)CD8(+) double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1(-/-) mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.

FcγRIII (CD16)-mediated ADCC by NK cells is regulated by monocytes and FcγRII (CD32).

Monocytes are known to engage in reciprocal crosstalk with NK cells but their influence on NK-cell-associated antibody-dependent cellular cytotoxicity (ADCC) is not well understood. We demonstrate that in humans FcγRIII (CD16)-dependent ADCC by NK cells is considerably enhanced by monocytes, and that this effect is regulated by FcγRII (CD32) crosslinking in healthy individuals. It is known that during HIV-1 infection, NK cells are known to express low levels of CD16 and exhibit reduced ADCC. We show that immune regulation of CD16-mediated NK-cell cytotoxicity by monocytes through CD32 engagement is substantially disturbed in chronic progressive HIV-1 infection. Expression of activating isoform of CD32 represented a compensatory mechanism for reduced expression of CD16 on NK cells during HIV-1 infection. As a result, the regulation of NK-cell-associated ADCC by monocytes is skewed and eventually constitutes a novel factor that contributes to HIV-1-associated immune deficiency, dysregulation and pathogenesis. Our data therefore provide evidence, for the first time, that in humans monocytes act as a rheostat for FcγRIII-mediated NK-cell functions maintaining a well-balanced immune response.

High frequencies of polyfunctional CD8+ NK cells in chronic HIV-1 infection are associated with slower disease progression.

UNLABELLED: Natural killer (NK) cells are effector and regulatory innate immune cells and play a critical role in the first line of defense against various viral infections. Although previous reports have indicated the vital contributions of NK cells to HIV-1 immune control, nongenetic NK cell parameters directly associated with slower disease progression have not been defined yet. In a longitudinal, retrospective study of 117 untreated HIV-infected subjects, we show that higher frequencies as well as the absolute numbers of CD8(+) CD3(-) lymphocytes are linked to delayed HIV-1 disease progression. We show that the majority of these cells are well-described blood NK cells. In a subsequent cross-sectional study, we demonstrate a significant loss of CD8(+) NK cells in untreated HIV-infected individuals, which correlated with HIV loads and inversely correlated with CD4(+) T cell counts. CD8(+) NK cells had modestly higher frequencies of CD57-expressing cells than CD8(-) cells, but CD8(+) and CD8(-) NK cells showed no differences in the expression of a number of activating and inhibiting NK cell receptors. However, CD8(+) NK cells exhibited a more functional profile, as detected by cytokine production and degranulation. IMPORTANCE: We demonstrate that the frequency of highly functional CD8(+) NK cells is inversely associated with HIV-related disease markers and linked with delayed disease progression. These results thus indicate that CD8(+) NK cells represent a novel NK cell-derived, innate immune correlate with an improved clinical outcome in HIV infection.

Deciphering the impact of parameters influencing transgene expression kinetics after repeated cell transduction with integration-deficient retroviral vectors.

Lentiviral and gammaretroviral vectors are state-of-the-art tools for transgene expression within target cells. The integration of these vectors can be deliberately suppressed to derive a transient gene expression system based on extrachromosomal circular episomes with intact coding regions. These episomes can be used to deliver DNA templates and to express RNA or protein. Importantly, transient gene transfer avoids the genotoxic side effects of integrating vectors. Restricting their applicability, episomes are rapidly lost upon dilution in dividing target cells. Addressing this limitation, we could establish comparably stable percentages of transgene-positive cells over prolonged time periods in proliferating cells by repeated transductions. Flow cytometry was applied for kinetic analyses to decipher the impact of individual parameters on the kinetics of fluoroprotein expression after episomal retransduction and to visualize sequential and simultaneous transfer of heterologous fluoroproteins. Expression windows could be exactly timed by the number of transduction steps. The kinetics of signal loss was affected by the cell proliferation rate. The transfer of genes encoding fluoroproteins with different half-lives revealed a major impact of protein stability on temporal signal distribution and accumulation, determining optimal retransduction intervals. In addition, sequential transductions proved broad applicability in different cell types and using different envelope pseudotypes without receptor overload. Stable percentages of cells coexpressing multiple transgenes could be generated upon repeated coadministration of different episomal vectors. Alternatively, defined patterns of transgene expression could be recapitulated by sequential transductions. Altogether, we established a methodology to control and adjust a temporally defined window of transgene expression using retroviral episomal vectors. Combined with the highly efficient cell entry of these vectors while avoiding integration, the developed technology is of great significance for a broad panel of applications, including transcription-factor-based induced cell fate conversion and controlled transfer of genetically encoded RNA- or protein-based drugs.

MYH9-related disease: a novel prognostic model to predict the clinical evolution of the disease based on genotype-phenotype correlations.

MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in the gene for nonmuscle myosin heavy chain IIA (NMMHC-IIA). MYH9-RD is characterized by a considerable variability in clinical evolution: patients present at birth with only thrombocytopenia, but some of them subsequently develop sensorineural deafness, cataract, and/or nephropathy often leading to end-stage renal disease (ESRD). We searched for genotype-phenotype correlations in the largest series of consecutive MYH9-RD patients collected so far (255 cases from 121 families). Association of genotypes with noncongenital features was assessed by a generalized linear regression model. The analysis defined disease evolution associated to seven different MYH9 genotypes that are responsible for 85% of MYH9-RD cases. Mutations hitting residue R702 demonstrated a complete penetrance for early-onset ESRD and deafness. The p.D1424H substitution associated with high risk of developing all the noncongenital manifestations of disease. Mutations hitting a distinct hydrophobic seam in the NMMHC-IIA head domain or substitutions at R1165 associated with high risk of deafness but low risk of nephropathy or cataract. Patients with p.E1841K, p.D1424N, and C-terminal deletions had low risk of noncongenital defects. These findings are essential to patients' clinical management and genetic counseling and are discussed in view of molecular pathogenesis of MYH9-RD.

The spectrum of ELANE mutations and their implications in severe congenital and cyclic neutropenia.

Neutrophil elastase gene (ELANE) mutations are responsible for the majority of cases of severe congenital neutropenia (CN) and cyclic neutropenia (CyN). We screened CN (n = 395) or CyN (n = 92) patients for ELANE mutations and investigated the impact of mutations on mRNA expression, protein expression, and activity. We found 116 different mutations in 162 (41%) CN patients and 26 in 51 (55%) CyN patients, 69 of them were novel. CyN-associated mutations were predicted to be more benign than CN-associated mutations, but the mutation severity largely overlapped. The frequency of acquired CSF3R mutations, malignant transformation, and the need for hematopoietic stem cell transplantation was significantly higher in CN patients with ELANE mutation than in ELANE mutation negative patients. Cellular elastase activity was reduced in neutrophils from CN/CyN patients, irrespective of the mutation status. In CN, enzymatic activity was significantly lower in patients with ELANE mutations compared with those with wild-type ELANE. Despite differences in the spectrum of mutations in CN or CyN, type or localization of mutation only partially determine the clinical phenotype. Specific ELANE mutations have limited predictive value for leukemogenesis; the risk for leukemia was correlated with disease severity rather than with occurrence of an ELANE mutation.

Activated human hepatic stellate cells induce myeloid derived suppressor cells from peripheral blood monocytes in a CD44-dependent fashion.

BACKGROUND & AIMS: Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells associated with the suppression of immunity. However, little is known about how or where MDSCs are induced and from which cells they originate. The liver is known for its immune regulatory functions. Here, we investigated the capacity of human hepatic stellate cells (HSCs) to transform peripheral blood monocytes into MDSCs. METHODS: We cultured freshly isolated human monocytes from healthy donors on primary human HSCs or an HSC cell-line and characterized the phenotype and function of resulting CD14(+)HLA-DR(-/low) monocytes by flow cytometry, quantitative PCR, and functional assays. We analyzed the molecular mechanisms underlying the induction and function of the CD14(+)HLA-DR(-/low) cells by using blocking antibodies or knock-down technology. RESULTS: Mature peripheral blood monocytes co-cultured with HSCs downregulated HLA-DR and developed a phenotypic and functional profile similar to MDSCs. Only activated but not freshly isolated HSCs were capable of inducing CD14(+)HLA-DR(-/low) cells. Such CD14(+)HLA-DR(-/low) monocyte-derived MDSCs suppressed T-cell proliferation in an arginase-1 dependent fashion. HSC-induced development of CD14(+)HLA-DR(-/low) monocyte-derived MDSCs was not mediated by soluble factors, but required physical interaction and was abrogated by blocking CD44. CONCLUSIONS: Our study shows that activated human HSCs convert mature peripheral blood monocytes into MDSCs. As HSCs are activated during chronic inflammation, the subsequent local induction of MDSCs may prevent ensuing excessive liver injury. HSC-induced MDSCs functionally and phenotypically resemble those isolated from liver cancer patients. Thus, our data suggest that local generation of MDSCs by liver-resident HSCs may contribute to immune suppression during inflammation and cancer in the liver.

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia.

Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl(-/-)) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl(-/-) bone marrow cells into Mpl(-/-) mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl(-/-) cells had increased long-term repopulating potential, with a marked increase in lineage(-)Sca1(+)cKit(+) cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage(-)Sca1(+)cKit(+) cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Congenital amegakaryocytic thrombocytopenia (CAMT) presenting as severe pancytopenia in the first month of life.

Congenital amegakaryocytic thrombocytopenia (CAMT) is characterised by neonatal thrombocytopenia, with reduced or absent bone marrow megakaryocytes, leading eventually to pancytopenia. The mean age for progression to bone marrow failure is four years, with the earliest reported being six months. We describe a CAMT patient with compound heterozygous mutations of the causative MPL gene (one being a previously unreported splice site mutation in intron 11) who developed pancytopenia within the first month of life. This report emphasises the importance of considering CAMT in the differential diagnosis of congenital aplastic anaemia or idiopathic aplastic anaemia in babies.

THROMKIDplus Patient Registry and Biomaterial Banking for Children with Inherited Platelet Disorders.

Inherited platelet disorders (IPDs) represent a heterogeneous group of disorders that include both quantitative (thrombocytopenia or thrombocytosis) and qualitative (thrombocytopathy) defects. To gain better knowledge about the prevalence, pathogenesis, and clinical consequences of specific diseases, to improve diagnosis and treatment of patients with IPD, and to support translational research on a genetic, molecular, and physiological basis, the THROMKIDplus study group currently comprising 24 sites in Germany, Austria, and Switzerland decided to establish a patient registry with associated biomaterial banking for children. This registry is designed as a retrospective-prospective, multicenter observational study and supposed to launch in the second half of 2023. Blood smears, plasma, platelet pellets, and DNA of patients will be stored in certified biomaterial banks for future translational research projects. The main inclusion criteria are (1) diagnosis of or highly suspected IPD after assessment of a THROMKIDplus competence center and (2) patients aged 0 to 17 years. Initial and follow-up data on patient history, laboratory parameters, standardized documentation of bleeding tendency, and congenital defects are collected according to good clinical practice and current data protection acts by using the MARVIN platform, a broadly used data management system supported by the German Society for Pediatric Oncology Hematology (GPOH). The THROMKIDplus study group intends to enroll ∼200 patients retrospectively and an annual amount of ∼50 patients prospectively.

Multiple extrathymic precursors contribute to T-cell development with different kinetics.

T-cell development in the thymus depends on continuous supply of T-cell progenitors from bone marrow (BM). Several extrathymic candidate progenitors have been described that range from multipotent cells to lymphoid cell committed progenitors and even largely T-lineage committed precursors. However, the nature of precursors seeding the thymus under physiologic conditions has remained largely elusive and it is not known whether there is only one physiologic T-cell precursor population or many. Here, we used a competitive in vivo assay based on depletion rather than enrichment of classes of BM-derived precursor populations, thereby only minimally altering physiologic precursor ratios to assess the contribution of various extrathymic precursors to T-lineage differentiation. We found that under these conditions multiple precursors, belonging to both multipotent progenitor (MPP) and common lymphoid progenitor (CLP) subsets have robust T-lineage potential. However, differentiation kinetics of different precursors varied considerably, which might ensure continuous thymic output despite gated importation of extrathymic precursors. In conclusion, our data suggest that the thymus functions to impose T-cell fate on any precursor capable of filling the limited number of progenitor niches.

Robust Salmonella metabolism limits possibilities for new antimicrobials.

New antibiotics are urgently needed to control infectious diseases. Metabolic enzymes could represent attractive targets for such antibiotics, but in vivo target validation is largely lacking. Here we have obtained in vivo information about over 700 Salmonella enterica enzymes from network analysis of mutant phenotypes, genome comparisons and Salmonella proteomes from infected mice. Over 400 of these enzymes are non-essential for Salmonella virulence, reflecting extensive metabolic redundancies and access to surprisingly diverse host nutrients. The essential enzymes identified were almost exclusively associated with a small subgroup of pathways, enabling us to perform a nearly exhaustive screen. Sixty-four enzymes identified as essential in Salmonella are conserved in other important human pathogens, but almost all belong to metabolic pathways that are inhibited by current antibiotics or that have previously been considered for antimicrobial development. Our comprehensive in vivo analysis thus suggests a shortage of new metabolic targets for broad-spectrum antibiotics, and draws attention to some previously known but unexploited targets.

Down-regulation of the fetal stem cell factor SOX17 by H33342: a mechanism responsible for differential gene expression in breast cancer side population cells.

Human solid tumors contain rare cancer side population (SP) cells, which expel the fluorescent dye Hoechst 33342 (H33342) and display cancer stem cell characteristics. Transcriptional profiling of cancer SP cells isolated by H33342 fluorescence analysis is a newly emerging approach to discover cancer stem cell markers and aberrant differentiation pathways. Using Affymetrix expression microarrays and quantitative reverse transcription-PCR, we investigated differential gene expression between SP and non-SP (NSP) cells isolated from human mammary carcinoma cell lines. A total of 136 genes were up-regulated in breast cancer SP relative to NSP cells, one of which was the fetal stem cell factor and Wnt/beta-catenin signaling pathway target SOX17. Strikingly, we discovered that SOX17 was down-regulated by H33342 in a dose-dependent manner. In SP cells, which expel H33342, down-regulation of SOX17 was less pronounced than in NSP cells, which retain H33342. As a result of this, SOX17 displayed a 10-20-fold overexpression in cancer SP relative to NSP cells. Similar results were obtained for further stemness-related genes, namely EPC1 and SPRY1. These findings establish a previously unidentified gene-regulatory impact of H33342 as a novel mechanism responsible for differential gene expression in cancer SP cells. This has significant implications for the future interpretation of cancer SP cells.

Acute-phase protein α1-antitrypsin inhibits neutrophil calpain I and induces random migration.

A rapid recruitment of neutrophils to sites of injury or infection is a hallmark of the inflammatory response and is required for effective host defense against pathogenic stimuli. However, neutrophil-mediated inflammation can also lead to chronic tissue destruction; therefore, a better understanding of the mechanisms underlying neutrophil influx and activation is of critical importance. We have previously shown that the acute phase protein α1-antitrypsin (AAT) inhibits neutrophil chemotaxis. In this study, we examine mechanisms related to the effect of AAT on neutrophil responses. We report a previously unknown function of AAT to inactivate calpain I (µ-calpain) and to induce a rapid cell polarization and random migration. These effects of AAT coincided with a transient rise in intracellular calcium, increase in intracellular lipids, activation of the Rho GTPases, Rac1 and Cdc42, and extra-cellular signal-regulated kinase (ERK1/2). Furthermore, AAT caused a significant inhibition of nonstimulated as well as formyl-met-leu-phe (fMLP)-stimulated neutrophil adhesion to fibronectin, strongly inhibited lipopolysaccharide-induced IL-8 release and slightly delayed neutrophil apoptosis. The results presented here broaden our understanding of the regulation of calpain-related neutrophil functional activities, and provide the impetus for new studies to define the role of AAT and other acute phase proteins in health and disease.

HIV infection is associated with a preferential decline in less-differentiated CD56dim CD16+ NK cells.

HIV-1 infection is characterized by loss of CD56(dim) CD16(+) NK cells and increased terminal differentiation on various lymphocyte subsets. We identified a decrease of CD57(-) and CD57(dim) cells but not of CD57(bright) cells on CD56(dim) CD16(+) NK cells in chronic HIV infection. Increasing CD57 expression was strongly associated with increasing frequencies of killer immunoglobulin-like receptors (KIRs) and granzyme B-expressing cells but decreasing percentages of cells expressing CD27(+), HLA-DR(+), Ki-67(+), and CD107a. Our data indicate that HIV leads to a decline of less-differentiated cells and suggest that CD57 is a useful marker for terminal differentiation on NK cells.