NK Cell Terminal Differentiation: Correlated Stepwise Decrease of NKG2A and Acquisition of KIRs

BACKGROUND: Terminal differentiation of NK cells is crucial in maintaining broad responsiveness to pathogens and discriminating normal cells from cells in distress. Although it is well established that KIRs, in conjunction with NKG2A, play a major role in the NK cell education that determines whether cells will end up competent or hyporesponsive, the events underlying the differentiation are still debated. METHODOLOGY/PRINCIPAL FINDINGS: A combination of complementary approaches to assess the kinetics of the appearance of each subset during development allowed us to obtain new insights into these terminal stages of differentiation, characterising their gene expression profiles at a pan-genomic level, their distinct surface receptor patterns and their prototypic effector functions. The present study supports the hypothesis that CD56dim cells derive from the CD56bright subset and suggests that NK cell responsiveness is determined by persistent inhibitory signals received during their education. We report here the inverse correlation of NKG2A expression with KIR expression and explore whether this correlation bestows functional competence on NK cells. We show that CD56dimNKG2A-KIR+ cells display the most differentiated phenotype associated to their unique ability to respond against HLA-E+ target cells. Importantly, after IL-12+IL-18 stimulation, reacquisition of NKG2A strongly correlates with IFN-gamma production in CD56dimNKG2A- NK cells. CONCLUSIONS/SIGNIFICANCE: Together, these findings call for the reclassification of mature human NK cells into distinct subsets and support a new model, in which the NK cell differentiation and functional fate are based on a stepwise decrease of NKG2A and acquisition of KIRs

Induction of Plasmodium falciparum-Specific CD4+ T Cells and Memory B Cells in Gabonese Children Vaccinated with RTS,S/AS01E and RTS,S/AS02D

The recombinant circumsporozoite protein (CS) based vaccine, RTS,S, confers protection against Plasmodium falciparum infection in controlled challenge trials and in field studies. The RTS,S recombinant antigen has been formulated with two adjuvant systems, AS01 and AS02, which have both been shown to induce strong specific antibody responses and CD4 T cell responses in adults. As infants and young children are particularly susceptible to malaria infection and constitute the main target population for a malaria vaccine, we have evaluated the induction of adaptive immune responses in young children living in malaria endemic regions following vaccination with RTS,S/AS01(E) and RTS,S/AS02(D). Our data show that a CS-specific memory B cell response is induced one month after the second and third vaccine dose and that CS-specific antibodies and memory B cells persist up to 12 months after the last vaccine injection. Both formulations also induced low but significant amounts of CS-specific IL-2(+) CD4(+) T cells one month after the second and third vaccine dose, upon short-term in vitro stimulation of whole blood cells with peptides covering the entire CS derived sequence in RTS,S. These results provide evidence that both RTS,S/AS01(E) and RTS,S/AS02(D) induced adaptive immune responses including antibodies, circulating memory B cells and CD4(+) T cells directed against P. falciparum CS protein.ClinicalTrials.gov NCT00307021

Multiple 17-OHP Cutoff Co-Variates Fail to Improve 21-Hydroxylase Deficiency Screening Accuracy

To improve the positive predictive value (PPV) of newborn screening for 21-hydroxylase deficiency (21OHD), co-variates have been used to modify 17-hydroxyprogesterone (17OHP) cutoffs. The objective of this study is to evaluate whether 17OHP screening cutoffs adjusted for both collection time (CT) and birth weight (BW) improved the sensitivity and PPV of 21OHD screening. Unaffected newborn screening samples were stratified based on BW and CT to establish 17OHP concentration cutoffs at the 95th and 99th percentile. These cutoffs were applied to a cohort of confirmed cases of 21OHD to determine the sensitivity and PPV of the modified screening parameters. 17OHP cutoffs at the 99th percentile, adjusted for BW and CT, had a sensitivity of 96.3% and a specificity of 98.9%, but a relatively low PPV (0.130) for the identification of 21OHD and did not detect all cases. Use of the 95th percentile further increased sensitivity to 98.1% but resulted in a notably lower PPV (0.027). Alternative approaches that do not rely exclusively on 17OHP are needed to improve newborn screening accuracy for 21OHD

Screening for Methylmalonic and Propionic Acidemia: Clinical Outcomes and Follow-Up Recommendations

Wisconsin’s newborn screening program implemented second-tier testing on specimens with elevated propionylcarnitine (C3) to aid in the identification of newborns with propionic and methylmalonic acidemias. The differential diagnosis for elevated C3 also includes acquired vitamin B12 deficiency, which is currently categorized as a false positive screen. The goal of this study was to summarize screening data and evaluate their effectiveness at establishing diagnoses and categorizing false positive cases. All Wisconsin newborns born between 2013 and 2019 with a positive first-tier screen for C3 were included in this study. For each case the first- and second-tier newborn screening data and confirmatory test results were compiled. The clinical determination for each case was reviewed and categorized into groups: inborn error of metabolism, maternal B12 deficiency, infant B12 deficiency, and false positive. A review of the screening data showed a significant overlap in the concentration of biomarkers for newborns with genetic versus acquired disease. Additionally, a review of confirmatory test results showed incomplete ascertainment of maternal vitamin B12 status. The Wisconsin newborn screening program recommended a confirmatory testing algorithm to aid in the diagnosis of inborn errors of metabolism and acquired vitamin B12 deficiency

Improving Reproducibility to Enhance Scientific Rigor through Consideration of Mouse Diet

Animal husbandry conditions, including rodent diet, constitute an example highlighting the importance of reporting experimental variables to enhance scientific rigor. In the present study, we examine the effects of three common rodent diets including two chows (Purina 5015 and Teklad 2019) and one purified ingredient diet (AIN-76A) on growth anthropometrics (body weight), behavior (nest building, actigraphy, passive avoidance) and blood biomarkers (ketones, glucose, amino acid profiles) in male and female C57BL/6J mice. We find increased body weight in response to the chows compared to purified ingredient diet albeit selectively in male mice. We did not find significantly altered behavior in female or male wild type C57BL/6J mice. However, amino acid profiles changed as an effect of sex and diet. These data contribute to a growing body of knowledge indicating that rodent diet impacts experimental outcomes and needs to be considered in study design and reporting

Wisconsin’s Screening Algorithm for the Identification of Newborns with Congenital Adrenal Hyperplasia

Newborn screening for congenital adrenal hyperplasia (CAH) has one of the highest false positive rates of any of the diseases on the Wisconsin panel. This is largely due to the first-tier immune assay cross-reactivity and physiological changes in the concentration of 17-hydroxyprogesterone during the first few days of life. To improve screening for CAH, Wisconsin developed a second-tier assay to quantify four different steroids (17-hydroxyprogesterone, 21-deoxycortisol, androstenedione, and cortisol) by liquid chromatography–tandem mass spectrometry (LC–MSMS) in dried blood spots. From validation studies which included the testing of confirmed CAH patients, Wisconsin established its own reporting algorithm that incorporates steroid concentrations as well as two different ratios—the birth weight and the collection time—to identify babies at risk for CAH. Using the newly developed method and algorithm, the false positive rate for the CAH screening was reduced by 95%. Patients with both classical forms of CAH, salt-wasting and simple virilizing, were identified. This study replicates and expands upon previous work to develop a second-tier LC–MSMS steroid profiling screening assay for CAH. The validation and prospective study results provide evidence for an extensive reporting algorithm that incorporates multiple steroids, birth weight, and collection times

Analytical Validation of Familial Hypercholesterolemia Biomarkers in Dried Blood Spots

Heterozygous familial hypercholesterolemia (HeFH) is a common, treatable genetic disorder characterized by premature atherosclerosis and cardiovascular disease, yet the majority of affected individuals remain undiagnosed. Newborn screening could play a role in identification of at-risk individuals and provide an opportunity for early intervention, prior to the onset of symptoms. The objective of this study was to develop and validate assays for quantification of candidate HeFH biomarkers in dried blood spots (DBS). Commercially available enzyme assay kits for quantification of serum total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) were modified for high-throughput analysis of DBS. Apolipoprotein B (ApoB) concentrations in DBS were measured using an immunoassay with modifications from published studies. All three assays were validated according to the College of American Pathologists guidelines for clinical laboratories. The performance of TC, LDL-C, and ApoB assays was assessed by precision, recovery, limit of quantification (LOQ) and linearity. Precision studies yielded coefficients of variation (CV) of less than 15%, with recovery greater than 75% for all three assays. The determined LOQ and linearity were comparable to serum-based assays. In a direct comparison between serum and DBS concentrations, positive correlations were demonstrated for TC, LDL-C, and ApoB. Additionally, the initial evaluation of the three biomarker concentrations within the unaffected population was similar to values obtained in previous published studies. This study reports on methods for quantification of TC, LDL-C, and ApoB in DBS. Assay validation results were within acceptable limits for newborn screening. This is an important first step toward the identification of newborns with HeFH

Clinical relevance of the discrepancy in phenylalanine concentrations analyzed using tandem mass spectrometry compared with ion-exchange chromatography in phenylketonuria

Introduction: Metabolic control of phenylketonuria (PKU) and compliance with the low-phenylalanine (phe) diet are frequently assessed by measuring blood phe concentrations in dried blood spots (DBS) collected by patients instead of plasma phe concentrations. Objective: Our objective was to investigate the difference in blood phe concentrations in DBS collected by subjects and analyzed using either a validated newborn screening tandem mass spectrometry (MS/MS) protocol or ion-exchange chromatography (IEC) compared to plasma phe concentrations obtained simultaneously and analyzed using IEC. Design: Three to four fasting blood samples were obtained from 29 subjects with PKU, ages 15–49 years. Capillary blood was spotted on filter paper by each subject and the DBS analyzed using both MS/MS and IEC. Plasma was isolated from venous blood and analyzed using IEC. Results: Blood phe concentrations in DBS analyzed using MS/MS are 28% ± 1% (n = 110, p 600 μmol/L. Due to the large variability across subjects of 13.2%, a calibration factor to adjust blood phe concentrations is not recommended. Analysis of DBS using IEC reduced the discrepancy to 15 ± 2% lower phe concentrations compared to plasma analyzed using IEC (n = 38, p = 0.0001). This suggests that a major contributor to the discrepancy in phe concentrations is the analytical method. Conclusion: Use of DBS analyzed using MS/MS to monitor blood phe concentrations in individuals with PKU yields significantly lower phe levels compared to plasma phe levels analyzed using IEC. Optimization of current testing methodologies for measuring phe in DBS, along with patient education regarding the appropriate technique for spotting blood on filter paper is needed to improve the accuracy of using DBS to measure phe concentrations in PKU management

Harmonizing Newborn Screening Laboratory Proficiency Test Results Using the CDC NSQAP Reference Materials

Newborn screening (NBS) laboratories cannot accurately compare mass spectrometry-derived results and cutoff values due to differences in testing methodologies. The objective of this study was to assess harmonization of laboratory proficiency test (PT) results using quality control (QC) data. Newborn Screening Quality Assurance Program (NSQAP) QC and PT data reported from 302 laboratories in 2019 were used to compare results among laboratories. QC materials were provided as dried blood spot cards which included a base pool and the base pool enriched with specific concentrations of metabolites in a linear range. QC data reported by laboratories were regressed on QC data reported by the Centers for Disease Control and Prevention (CDC), and laboratory’s regression parameters were used to harmonize their PT result. In general, harmonization tended to reduce overall variation in PT data across laboratories. The metabolites glutarylcarnitine (C5DC), tyrosine, and phenylalanine were displayed to highlight inter- and intra-method variability in NBS results. Several limitations were identified using retrospective data for harmonization, and future studies will address these limitations to further assess feasibility of using NSQAP QC data to harmonize PT data. Harmonizing NBS data using common QC materials appears promising to aid result comparison between laboratories