Number Of T-cell Receptor Excision Circles Research Articles

Importance of TREC and KREC as molecular markers for immunological evaluation of down syndrome children

Recurrent and severe infections occurred in children with Down Syndrome (DS) due to immunological parameter defects have been reported. The aim of the study is to evaluate the importance of using T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) as molecular markers for immunological investigation of children with DS. The study included 40 non-disjunction trisomy 21 confirmed DS children, and 25 healthy controls. Peripheral blood (PB) was analyzed for lymphocyte subpopulations by flow cytometry, serum immunoglobulin levels, and TREC and KREC copy numbers using quantitative real-time PCR. DS patients showed significantly lower absolute counts of PB T lymphocytes, T helper lymphocytes, T cytotoxic lymphocytes, B lymphocytes, and Natural killer cells, and lower serum IgA, IgG, and IgM levels compared to healthy controls. Copy number of TREC and KREC showed no significant differences between DS patients and healthy controls. There is a significant positive correlation between TREC copy number with a percentage and absolute count of helper T lymphocytes in patients. Also, the KREC copy number was significantly negatively correlated with the age of patients. These findings suggest that copy numbers of TREC and KREC could be useful as molecular markers for immunological evaluation of patients with DS.

Screening newborns for low T cell receptor excision circles (TRECs) Fails to Detect Immunodeficiency, Centromeric Instability, and Facial Anomalies Syndrome.

Assessment of T-cell receptor excision circles (TRECs) in dried blood spots of newborns allows the detection of severe combined immunodeficiency (SCID) (T cells <300/μL at birth) with a presumed sensitivity of 100%. TREC screening also identifies patients with selected combined immunodeficiency (CID) (T cells >300/μL, yet <1500/μL at birth). Nevertheless, relevant CIDs that would benefit from early recognition and curative treatment pass undetected. We hypothesized that TREC screening at birth cannot identify CIDs that develop with age. We analyzed the number of TRECs in dried blood spots in archived Guthrie cards of 22 children who had been born in the Berlin-Brandenburg area between January 2006 and November 2018 and who had undergone hematopoietic stem-cell transplantation (HSCT) for inborn errors of immunity. All patients with SCID would have been identified by TREC screening, but only 4 of 6 with CID. One of these patients had immunodeficiency, centromeric instability, and facial anomalies syndrome type 2 (ICF2). Two of 3 patients with ICF whom we have been following up at our institution had TREC numbers above the cutoff value suggestive of SCID at birth. Yetall patients with ICF had a severe clinical course that would have justified earlier HSCT. In ICF, naïve T cells may be present at birth, yet they decline with age. Therefore, TREC screening cannot identify these patients. Early recognition is nevertheless crucial, as patients with ICF benefit from HSCT early in life.

Monitoring T-Cell Kinetics in the Early Recovery Period of Lung Transplantation Cases by Copy Number Levels of T-Cell Receptor Excision Circle.

Lung transplantation is a life-saving procedure for patients with end-stage lung diseases. T-Cell receptor excision circle (TREC) is circular DNA produced during T-cell receptor gene rearrangement in the thymus and indicates naive T-cell migration from the thymus. Therefore, its levels represent thymic T-cell output. Post-transplant lymphocyte kinetics correlate with graft tolerance. The aim of this study was to investigate T-lymphocyte kinetics in the early recovery period after lung transplantation. For this purpose, copy numbers of TREC were determined in patients with a lung transplant. In addition, TREC copy numbers were evaluated according to age, diagnosis and the forced expiratory volume in 1 second (FEV1) of lung transplant patients. Peripheral blood samples were taken from patients aged 23 to 59 years who underwent lung transplantation at the Thoracic Surgery Clinic, Kartal-Koşuyolu High Specialization Educational and Research Hospital. This study included peripheral blood samples from 11 lung transplant patients (comprising four with chronic obstructive pulmonary disease, three with idiopathic pulmonary fibrosis, one with cystic fibrosis, one with silicosis and two with bronchiectasis; three females in total). Samples were taken at three different timepoints: Before transplant, and 24 hours and 7 days post transplant. TREC copy numbers were analyzed with real time reverse transcriptase-polymerase chain reaction. Post-transplant TREC numbers and density values were higher compared to pre-transplant values, although these differences were statistically insignificant. TREC copy numbers were found to be significantly higher in patients younger than 45 years compared to patients older than 45 years. At 24 hours after the transplant, the average TREC copy number/peripheral blood mononuclear cells of the cases with an FEV1 value of or below 50% was found to be statistically significantly higher than that of cases with an FEV1 value above 50% (p=0.046). There was no statistically significant difference in TREC copy numbers between male and female patients or by diagnostic group. TREC copy numbers can be evaluated as a prognostic marker for lung transplantation. There is a need for multicenter studies with more patients.

Immune function in newborns with in-utero exposure to anti-TNFα therapy.

Background and aimAnti-TNFα is measurable in infants exposed in utero up to 12 months of age. Data about the exposure effect on the infant’s adaptive immunity are limited. We aimed to prospectively evaluate the distribution and function of T and B cells, in infants of females with inflammatory bowel disease, in utero exposed to anti-TNFα or azathioprine.MethodsA prospective multi-center study conducted 2014–2017. Anti-TNFα levels were measured in cord blood, and at 3 and 12 months. T-cell repertoire and function were analyzed at 3 and 12 months by flow-cytometry, expression of diverse T cell receptors (TCR) and T-cell receptor excision circles (TREC) quantification assay. Serum immunoglobulins and antibodies for inactivated vaccines were measured at 12 months. Baseline clinical data were retrieved, and 2-monthly telephonic interviews were performed regarding child infections and growth.Results24 pregnant females, age 30.6 (IQR 26.5–34.5) years were recruited, 20 with anti-TNFα (infliximab 8, adalimumab 12), and 4 with azathioprine treatment. Cord blood anti-TNFα was higher than maternal blood levels [4.3 (IQR 2.3–9.2) vs. 2.5 (IQR 1.3–9.7) mcg/ml], declining at 3 and 12 months. All infants had normal number of B-cells (n = 17), adequate levels of immunoglobulins (n = 14), and protecting antibody levels to Tetanus, Diphtheria, Hemophilus influenza-B and hepatitis B (n = 17). All had normal CD4+, CD8+ T-cells, and TREC numbers. TCR repertoire was polyclonal in 18/20 and slightly skewed in 2/20 infants. No serious infections requiring hospitalization were recorded.ConclusionWe found that T-cell and B-cell immunity is fully mature and immune function is normal in infants exposed in utero to anti-TNFα, as in those exposed to azathioprine. Untreated controls and large-scale studies are needed to confirm these results.

Determining T and B Cell development by TREC/KREC analysis in primary immunodeficiency patients and healthy controls.

T cell receptor excision circles (TRECs) and kappa-deleting excision circles (KRECs) are DNA fragments potentially indicative of T and B cell development, respectively. Recent thymic emigrants (RTEs) are a subset of peripheral cells that may also represent thymic function. Here, we investigated TREC/KREC copy numbers by quantitative real-time PCR in the peripheral blood of patients with primary immunodeficiencies (PIDs, n=145) and that of healthy controls (HCs, n=86) and assessed the correlation between RTEs and TREC copy numbers. We found that TREC copy numbers were significantly lower in children and adults with PIDs (P<.0001 and P<.002, respectively) as compared with their respective age-matched HCs. A moderate correlation was observed between TREC copies and RTE numbers among children with PID (r=.5114, P<.01), whereas no significant correlation was detected between RTE values and TREC content in the HCs (r=.0205, P=.9208). Additionally, we determined TREC and KREC copy numbers in DNA isolated from the Guthrie cards of 200 newborns and showed that this method is applicable to DNA isolated from both peripheral blood samples and dried blood spots, with the two sample types showing comparable TREC and KREC values. We further showed that RTE values are not always reliable markers of T cell output. Although additional confirmatory studies with larger cohorts are needed, our results provide thresholds for TREC/KREC copy numbers for different age groups.

Increased Naive, Stem Cell Memory and Terminally Differentiated CD8+ T Cells in Chronic Graft Versus Host Disease

BACKGROUND AND AIMSDespite improved clinical outcomes of patients undergoing Hematopoietic Stem Cell Transplantation (HSCT) in recent years, chronic GVHD (cGVHD) and its associated complications have a significant impact on non-relapse mortality and quality of life. Loss of immune tolerance in the post-transplant period plays an important role in the emergence of cGVHD, and while imbalances in CD4+ T cell subsets, such as regulatory T cells, are known to contribute to its pathobiology, the role of different subsets of CD8+ T cells is poorly characterized.PATIENTS AND METHODSWe prospectively monitored a group of 56 patients who underwent unrelated donor HSCT after reduced intensity conditioning containing ATG. GVHD prophylaxis consisted of cyclosporine plus mycophenolate mofetil for all patients. 15 patients were excluded due to early disease relapse and/or death secondary to infection or acute GVHD in the first 9 months post-HSCT. cGVHD diagnosis and staging was performed according to the 2014 NIH criteria. Patients were divided into 2 groups according to cGVHD status: 17 patients (41%) with cGVHD (cGVHD+) and 24 patients (59%) without cGVHD (cGVHD-).Peripheral blood was analyzed by flow cytometry to characterize T cell subsets at fixed time points over a 2 year period. CD8+ T subsets were identified as follows: True Naïve cells as CD45RA+CD62L+CD95- (TN), Stem Cell Memory cells as CD45RA+CD62L+CD95+ (SCM), Central Memory cells as CD45RA-CD62L+ (CM), Effector Memory cells as CD45RA-CD62L- (EM) and terminally differentiated effector cells as CD45RA+CD62L- (EMRA).Frozen PBMC from patients at months 9 and 12 post-HSCT were used to purify CD8+ T cells by cell sorting to quantify Signal-Joint T Cell Receptor Excision Circles (TREC) by Real-Time PCR and to evaluate TCR diversity by TCR Vbeta complementarity-determining region 3 spectratyping.RESULTSPatients with cGVHD had significantly increased percentages and absolute counts of TN CD8 in the first 6 months post-HSCT compared with cGVHD- patients (p≤0.04 at months 1 and 3 for percentages; p≤0.02 for months 1-6 in absolute counts).Furthermore, cGVHD+ patients had increased SCM CD8 cells throughout the post-HSCT period (significances of p<0.05 at months 6 and 12 for percentages and 18 and 24 for counts). The increased prevalence of these two subsets was not associated with increased proliferation as assessed by Ki67, which was reduced in cGVHD+ in the total CD45RA+CD62L+ gate (TN plus SCM cells) (p≤0.04 at months 6, 12 and 18). No significant differences were observed in susceptibility to apoptosis as measured by BCL2 or CD95 expression levels.CM and EM subset counts were reduced in cGVHD+ as compared to cGVHD- (p=0.04 and 0.02 at month 12). EM CD8 were the dominant population in cGVHD- patients throughout the follow up. Significant increases in the percentage of EM CD8 cells in cGVHD- were observed at months 2, 9, 12 and 18 (p≤0.04).cGVHD+ patients had increased percentages of EMRA (p<0.04 at months 9, 12 and 18) and absolute counts (p<0.04 in months 2, 9 and 24). This was not correlated to altered Ki67, Bcl-2 or CD95 levels.The increase in TN in cGVHD+ patients was not associated with increased TREC numbers within CD8+ T cells. TREC content was significantly reduced in both patient groups at month 9 as compared healthy controls (HC) (p<0.05). This could be due to the low prevalence of these cells and/or to rapid differentiation into other memory subsets such as the SCM and EMRA. Despite the differences in subset prevalence in the two patient groups, we did not observe any significant differences in TCR Vbeta diversity in CD8+T cells. Reduced TCR diversity was only observed when patients were compared to HC (p=0.0001).DISCUSSIONOur observations of increased TN, SCM and EMRA CD8 subsets very early after HSCT in patients who subsequently develop cGVHD suggest the possibility that these may be relevant biomarkers for cGVHD development. SCM T cells have previously been shown to cause lethal GVHD in mouse models and to represent a reservoir of antigen-specific T cells capable of differentiating in vivo into other memory subsets with effector function. We speculate that thymic tissue damage resulting from conditioning regimens and/or acute GVHD, together with other yet unidentified factors, may lead to the emergence of self-reactive naïve CD8+ T cells that differentiate to SCM and later to EMRA cells potentially mediating cGVHD. DisclosuresNo relevant conflicts of interest to declare.

T-cell Receptor Excision Circles in Newborns with Heart Defects

In the fetus, the cardiac neural crest gives rise to both the thymus and the conotruncus of the heart. In newborn screening for severe T-cell lymphopenia neonates with congenital heart defects may be detected. In this study, we investigated the occurrence of T-cell lymphopenia in neonates with or without 22q11.2 deletion syndrome (del) suffering from heart defects. This retrospective cohort study included 125 patients with heart defects. T-cell receptor excision circles (TRECs), a measure for T-cell lymphopenia, were quantified by RT-PCR using stored newborn screening blood spots. Three patient groups were compared: non-conotruncal defects (n = 57), conotruncal defects (n = 42), and 22q11.2 del with conotruncal defects (n = 26). Significantly lower TREC values were detected in patients with 22q11.2 del and conotruncal heart defects compared to those with non-syndromic conotruncal (p < 0.001) and non-conotruncal (p < 0.001) defects. In contrast, no significant difference was found between patients with non-syndromic conotruncal and non-conotruncal heart defects (p = 0.152). Low TREC levels were obtained in neonates treated with heart surgery/intervention within 2 weeks after birth and in those with a fatal outcome (p = 0.02) independent of patient group. A correlation was found between low TREC numbers and oxygen saturation, SpO2 below 95% (p = 0.017). The SpO2 was significantly lower in the non-syndromic conotruncal group compared to non-conotruncal (p < 0.001) and 22q11.2 del group (p = 0.015). No correlation was found between low neonatal TRECs and infections needing hospitalization later in life (p = 0.135). Patients with 22q11.2 del and conotruncal defects have significantly lower TREC levels compared to patients with heart defects without this syndrome.

Determining Laboratory Reference Values of TREC and KREC in Different Age Groups of Iranian Healthy Individuals

Assessment of the number of T-cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) copies has been recently described as biomarkers of newly formed T and B cells respectively. In this study, we aimed to explore the effects of demographic variables including age, gender, weight, height and ethnicity on these two episomal DNA molecules. Second, for the first time in our country, we determined the reference values of TREC and KREC copy numbers in different age groups of Iranian healthy individuals as a threshold for identifying T cell and B cell lymphopenia.The TREC and KREC copy numbers were evaluated in 251 dried blood spot (DBS) samples from healthy volunteers (age range: 0-60 years). Six primary immunodeficiency (PID) patients were included as disease controls. TREC and KREC copies were markedly reduced with increasing age. Although the levels of TREC and KREC were higher in females than males, this difference did not reach statistical significance. These findings suggest that demographic variables including age should be considered for interpretation results of the TREC/KREC assay.

COMPLEMENT DEFICIENCY IN TWO PATIENTS WITH ABSENCE OF T-CELL RECEPTOR EXCISION CIRCLES ON NEWBORN SCREENING

Introduction Most states use T-Cell Receptor Excision Circle (TREC) assays in routine newborn screening to identify infants with T cell defects. Some of these low TREC infants have normal T cell counts and function, without evidence of clinically significant disease. However, some of these low TREC infants without evidence of T cell dysfunction may develop serious infections, necessitating further immune evaluation. Case Description We present two infants whose initial newborn screens were flagged for low TREC numbers but had normal T cell number and function on further evaluation. These infants had significant infectious history – Patient 1 was an ex-36 week gestational age female who had necrotizing enterocolitis and intraabdominal abscess requiring resection, while Patient 2 was a term female who had streptococcal bacteremia leading to shock soon after birth, E. Coli tracheitis and recurrent ear infections in the first year of life. Further evaluation demonstrated complement deficiecy, in both – Patient 1 had low Mannose Bending lectin, while Patient 2 had low AH50 and properidin. Discussion These infants highlight the importance of using clinical history to guide the medical testing for all patients. Although both infants had low TRECs on their newborn screens, this could have been related to stress from their initial illness. The low TREC referral facilitated timely evaluation by clinical immunologists, even though their ultimate immunodefiency was in another arm of the immune system. An unintended consequence of routine TREC newborn screening may be earlier identification of primary immunodeficiency patients with non-T cell defects. Most states use T-Cell Receptor Excision Circle (TREC) assays in routine newborn screening to identify infants with T cell defects. Some of these low TREC infants have normal T cell counts and function, without evidence of clinically significant disease. However, some of these low TREC infants without evidence of T cell dysfunction may develop serious infections, necessitating further immune evaluation. We present two infants whose initial newborn screens were flagged for low TREC numbers but had normal T cell number and function on further evaluation. These infants had significant infectious history – Patient 1 was an ex-36 week gestational age female who had necrotizing enterocolitis and intraabdominal abscess requiring resection, while Patient 2 was a term female who had streptococcal bacteremia leading to shock soon after birth, E. Coli tracheitis and recurrent ear infections in the first year of life. Further evaluation demonstrated complement deficiecy, in both – Patient 1 had low Mannose Bending lectin, while Patient 2 had low AH50 and properidin. These infants highlight the importance of using clinical history to guide the medical testing for all patients. Although both infants had low TRECs on their newborn screens, this could have been related to stress from their initial illness. The low TREC referral facilitated timely evaluation by clinical immunologists, even though their ultimate immunodefiency was in another arm of the immune system. An unintended consequence of routine TREC newborn screening may be earlier identification of primary immunodeficiency patients with non-T cell defects.

Detection of newly produced T and B lymphocytes by digital PCR in blood stored dry on nylon flocked swabs

BackgroundA normal number of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) is considered a biomarker for adequate new T- and B-cell production. In newborns, detection of TRECs and KRECs by real time PCR from dried blood spotted on filter paper is used for the screening of severe immunodeficiency. In adults, elderly and during diseases, where the number of TRECs is lower than in newborns and children, a large amount of DNA and a sensitive method of amplification are necessary to identify newly produced lymphocytes.MethodsDNA was prepared from blood of 203 healthy adults (range: 18–91 years old) absorbed for 10 s on flocked swabs and let to dry, or from peripheral blood mononuclear cells. DNA was subjected to digital PCR and to well established conventional real time PCR-based method using TREC- and KREC-specific primers and probes. The number of TRECs and KRECs was expressed per mL of blood. Statistical analysis was performed by nested ANOVA, Pearson coefficient of determination, and by linear regression tests.ResultsThe novel method for the storage of dried blood on nylon flocked swabs and the use of digital PCR allow quantification of TRECs and KRECs with high degree of sensitivity, specificity, accuracy, and precision. TRECs and KRECs were amplified by digital PCR in all tested blood samples, including those obtained from elderly individuals (>70 years old) and that were negative by real time PCR. Furthermore, values of TRECs and KRECs obtained by digital PCR were in the range of those acquired by real time PCR.ConclusionsOur findings demonstrate that DNA isolation from dried blood on flocked swabs followed by digital PCR-based analysis represents a useful tool for studying new lymphocyte production in adults and elderly individuals. This suggests the potential use of the methodology when monitoring of clinical variables is limited by the number of molecules that can be amplified and detected, such as in patients with immunodeficiency or under immunosuppressive therapies.

Thymus activity measured by T-cell receptor excision circles in patients with different severities of respiratory syncytial virus infection

BackgroundRespiratory syncytial virus (RSV) infection is an important cause of hospitalization in previously healthy infants. Immunological mechanisms predisposing infants to severe disease are poorly understood. Early biomarkers for disease severity may assist clinical decisions. We investigated T-cell receptor excision circles (TREC), episomal DNA made during thymic T-cell receptor rearrangement, and a marker for thymus activity, both during disease and in neonatal screening cards as a risk factor for RSV disease severity.MethodsOne hundred thirteen patients hospitalized with RSV infection <12 months of age, grouped by disease severity, were available for this investigation, in which we conducted both a prospective and a case-control study. The prospective study included 47 RSV positive infants (mild n = 13, moderate n = 10, severe n = 24). TREC counts were determined by PCR of DNA extracted from EDTA-blood collected on hospitalization, and corrected for lymphocytes using ANCOVA. The case-control study included 85 newborns who later in infancy became RSV positive (mild n = 32, moderate n = 24, severe n = 29) and 47 newborns who never developed RSV disease as healthy controls included from health centres in the same catchment area. TRECs were measured using DNA extracted from dry blood spots from stored neonatal screening cards, followed by PCR. Student’s T-test compared patients with controls, ANOVA compared disease severity groups.ResultsDuring RSV infection patients in the severe disease group had significantly lower (p = 0.017) TREC/200 μL blood compared to the other two disease groups, after correction for lymphocyte count. Newborn TREC levels, were significantly higher in RSV patients compared to controls (p < 0.0001). No significant differences in TREC copies at birth were found between disease severities.ConclusionDuring acute RSV infection a lower number of TREC is found in the severe disease group. TREC has potential as an immunological marker for severe RSV infection. Higher neonatal TREC counts indicate that infants later presenting with severe RSV do not have reduced thymic activity at birth and probably no congenital T-cell defect.

Newborn Screening for Severe Primary Immunodeficiency Diseases in Sweden\u2014a 2-Year Pilot TREC and KREC Screening Study

Newborn screening for severe primary immunodeficiencies (PID), characterized by T and/or B cell lymphopenia, was carried out in a pilot program in the Stockholm County, Sweden, over a 2-year period, encompassing 58,834 children. T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) were measured simultaneously using a quantitative PCR-based method on DNA extracted from dried blood spots (DBS), with beta-actin serving as a quality control for DNA quantity. Diagnostic cutoff levels enabling identification of newborns with milder and reversible T and/or B cell lymphopenia were also evaluated. Sixty-four children were recalled for follow-up due to low TREC and/or KREC levels, and three patients with immunodeficiency (Artemis-SCID, ATM, and an as yet unclassified T cell lymphopenia/hypogammaglobulinemia) were identified. Of the positive samples, 24 were associated with prematurity. Thirteen children born to mothers treated with immunosuppressive agents during pregnancy (azathioprine (n = 9), mercaptopurine (n = 1), azathioprine and tacrolimus (n = 3)) showed low KREC levels at birth, which spontaneously normalized. Twenty-nine newborns had no apparent cause identified for their abnormal results, but normalized with time. Children with trisomy 21 (n = 43) showed a lower median number of both TREC (104 vs. 174 copies/μL blood) and KREC (45 vs. 100 copies/3.2 mm blood spot), but only one, born prematurely, fell below the cutoff level. Two children diagnosed with DiGeorge syndrome were found to have low TREC levels, but these were still above the cutoff level. This is the first large-scale screening study with a simultaneous detection of both TREC and KREC, allowing identification of newborns with both T and B cell defects.

Rapid molecular diagnostics of severe primary immunodeficiency determined by using targeted next-generation sequencing

Primary immunodeficiency diseases (PIDDs) are inherited disorders of the immune system. The most severe form, severe combined immunodeficiency (SCID), presents with profound deficiencies of Tcells, Bcells, or both at birth. If not treated promptly, affected patients usually do not live beyond infancy because of infections. Genetic heterogeneity of SCID frequently delays the diagnosis; a specific diagnosis is crucial for life-saving treatment and optimal management. We developed a next-generation sequencing (NGS)-based multigene-targeted panel for SCID and other severe PIDDs requiring rapid therapeutic actions in a clinical laboratory setting. The target gene capture/NGS assay provides an average read depth of approximately 1000×. The deep coverage facilitates simultaneous detection of single nucleotide variants and exonic copy number variants in one comprehensive assessment. Exons with insufficient coverage (<20× read depth) or high sequence homology (pseudogenes) are complemented by amplicon-based sequencing with specific primers to ensure 100% coverage of all targeted regions. Analysis of 20 patient samples with low T-cell receptor excision circle numbers on newborn screening or a positive family history or clinical suspicion of SCID or other severe PIDD identified deleterious mutations in 14 of them. Identified pathogenic variants included both single nucleotide variants and exonic copy number variants, such as hemizygous nonsense, frameshift, and missense changes in IL2RG; compound heterozygous changes in ATM, RAG1, and CIITA; homozygous changes in DCLRE1C and IL7R; and a heterozygous nonsense mutation in CHD7. High-throughput deep sequencing analysis with complete clinical validation greatly increases the diagnostic yield of severe primary immunodeficiency. Establishing a molecular diagnosis enables early immune reconstitution through prompt therapeutic intervention and guides management for improved long-term quality of life.

Simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) by real-time PCR.

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/10(6) cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Defect in B Cell Production Driven By GATA2 Mutation Results in Their Absolute Reduction and Mature Phenotype in Pediatric Patients

IntroductionGermline mutations in GATA2 were recently identified as causative for several overlapping syndromes: MonoMAC (monocytopenia, mycobacterial infections), DCML (dendritic cells, monocytes, B and NK cells deficiency), Emberger syndrome (lymphedema, sensorineural deafness, multiple warts) and familiar myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Of note, GATA2 mutations were also found in children and young adults with “primary” MDS. Aplastic anemia (AA) constitutes an important differential diagnosis to pediatric MDS, particularly in patients with normal cytogenetics.Because of heterogeneous phenotype of GATA2 mutated patients, defining a set of typical findings would help in their earlier identification and understanding the natural course of the disease. Therefore we aimed to analyze monocytes and lymphocyte subpopulations with the emphasis on B cell lineage by flow cytometry (FC) and polymerase chain reaction (PCR) in all pediatric patients with GATA2 mutation diagnosed in the Czech Republic.Patients and methodsEleven pediatric patients were found to harbor GATA2 mutations in the Czech Republic so far. Three mutations were intronic. There was a clear male predominance (9/11). In 7 patients the disease manifested with MDS in childhood, 2 female patients were followed for immunodeficiency and developed MDS in adulthood. One another patient was diagnosed with interstitial lung disease and chronic EBV infection. His brother, carrying the same mutation, has mild neutropenia. Bone marrow (BM) and peripheral blood (PB) samples were analyzed by FC. The level of intronRSS-Kde recombination excision circles (KREC) and T-cell receptor excision circles (TREC) for assessment of proliferation history of B and T cells was examined by PCR. The control group comprised 26 GATA2 wild-type MDS (“other MDS”) patients and 36 AA patients.ResultsDisturbance of B cell compartment was the most frequently observed anomaly in the patients with GATA2 mutation. We observed a decrease of absolute and relative B cell numbers in PB and BM (n=9/11). In BM there was a decrease of immature CD10pos B cells (n=10) with proportional increase of plasma cells. Peripheral blood B cell immunophenotype was shifted towards memory B cells (n=5/7). Presence of normal B cell precursors CD19pos10pos34pos in BM was observed only in 1 patient in part of follow-up samples. Atypical malignant B lymphoblasts were present in another patient, whose MDS quickly progressed to AML with a clear switch to B lymphoid phenotype. Despite significantly reduced number of B cells the levels of IgG were normal in majority of patients. Only 2 patients had IgG hypogammaglobuliemia, in one patient with chronic active EBV infection IgG hypergammaglobulinemia was present. Slightly decreased IgA level was present in 6 patients. Although B cell numbers in other MDS control patients were significantly lower compared to AA, still the decrease was less prominent in comparison with GATA2.The decrease of immature and naive B cells in patients with GATA2 mutation was reflected in very low level of KREC in PB and BM. Stored newborn dry blood spots from 4 patients were evaluated for TREC and KREC numbers. Strikingly, only one patient had negative KREC levels (the youngest patient from our cohort with MDS diagnosed at age 4). The remaining 3 patients had normal TREC and KREC levels at birth. Thus, the deterioration of de novo production of B cells occurred supposedly postnatally in most patients. Low KREC levels were also present in some patients with other MDS (n=5).Relative monocytopenia was found in 2 patients, low NK cells were present in 6 patients. T cells were mostly of naive non-activated phenotype.ConclusionsChanges in B cell compartment are the most characteristic feature in patients with GATA2 mutation. Decreased number of B cells together with a shift towards mature phenotype and decreased level of KREC reflect history of substantial B cell proliferation in an environment of impaired production. This process appears to happen postnatally and resemble normal ageing process, which is accelerated due to progenitor cell impairment. Immunophenotyping is a useful tool in identifying patients for GATA2 sequencing.Supported by GAUK 802214, IGA NT/14534-3, NT/13462-4, UNCE 204012, GAČR P301/10/1877 DisclosuresNo relevant conflicts of interest to declare.

Primary immunodeficiencies screening: neonatal screening for T/B cell disorders - a triplex PCR method for quantitation of TRECs and KRECs in newborns.

Primary immunodeficiency disorders (PID) comprise a group of more than 200 different diseases. Major efforts are currently being undertaken to develop methods for detection of the clinically most severe forms in the neonatal period. Polymerase chain reaction (PCR)-based detection of signal joint T cell receptor excision circles (TRECs), extracted from Guthrie cards, has already proved to be a valuable tool for identifying children with T cell lymphopenia, including patients with severe combined immunodeficiencies (SCID) 1, and is now carried out routinely in selected states in the United States. Recently, a similar method for analysis of B cell κ-deleting excision circles (KRECs) was developed 2, allowing identification of patients with B cell lymphopenia for the detection of patients with X-linked agammaglobulinaemia (XLA). To evaluate the possibilities for neonatal screening for different forms of PID, we combined the TREC and KREC assays into a triplex PCR method and evaluated its potential in a large number of anonymized control samples and retrospective samples from patients with a variety of PIDs 3. Retrospective samples from patients with SCID and XLA were identified easily, and the former could also be assigned to subgroups (T−B+, T−B−). In addition, patients with ataxia–telangiectasia, dedicator of cytokinesis 8 (DOCK8) mutations and Nijmegen breakage syndrome showed low frequencies of TRECs and/or KRECs. However, patients with selective immunoglobulin (Ig)A deficiency, hyper-IgM or common variable immunodeficiency could not be identified 3, 4. ‘Additional positive’ results were observed in a proportion of prematurely born children and in those with trisomy 21 (Down syndrome) 4 and DiGeorge syndrome 5. The reduced number of TRECs in approximately one-third of the latter patients appeared to correlate with disease severity. Second-tier tests were also developed to identify the two latter disorders. Selected patients with Wiskott–Aldrich syndrome also showed reduced TREC/KREC numbers 6. Initial screening of 20 000 anonymized Guthrie cards from Swedish and German children helped to define suitable cut-off values that would allow identification of patients with suspected PID, and an open prospective pilot trial in Sweden was initiated in November 2013. This trial will run for 3 years, followed by an evaluation of the results. It currently covers approximately one-third of all newborn children in Sweden (the Stockholm region) and will subsequently, if successful, be implemented nationwide. In conclusion, neonatal screening for severe forms of PID is feasible and fulfils the Wilson–Jungner criteria 7, 8. This screening will help to identify newborn children with a variety of PID, allowing early diagnosis and therapy. The presented work has been performed mainly by Stephan Borte and the samples have been obtained from the Swedish Screening Centre for Metabolic Diseases (CMMS; Ulrika von Döbeln). Part of the costs for the ongoing trial is being covered by an unrestricted research grant from Baxter Healthcare.

Newborn screening for severe T and B cell lymphopenia identifies a fraction of patients with Wiskott–Aldrich syndrome

The lack or marked reduction of recently formed T and B cells provides a basis for neonatal screening for severe combined immunodeficiencies (SCID) and X-linked agammaglobulinemia (XLA). Newborns with other conditions are also identified if a severe T or B cell lymphopenia is present at birth. We retrospectively analyzed Guthrie card samples from 11 children with Wiskott–Aldrich syndrome (WAS), a rare disease that requires early diagnosis and treatment, to determine whether combined T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) screening could identify these patients. 4 of 11 patients showed markedly reduced TREC or KREC copy numbers in their DBS as compared to storage-time matched controls and prospectively screened Swedish and German newborns. No correlation was observed between the WAS gene mutations, the clinical severity/course and the result of the screening assay. A diagnosis of WAS should thus be considered in newborns with positive TREC or KREC screening results.

Combined newborn screening for familial hemophagocytic lymphohistiocytosis and severe T- and B-cell immunodeficiencies

Combined newborn screening for familial hemophagocytic lymphohistiocytosis and severe T- and B-cell immunodeficiencies

Inverse Associations between Obesity Indicators and Thymic T-Cell Production Levels in Aging Atomic-Bomb Survivors

Reduction of the naive T-cell population represents a deteriorating state in the immune system that occurs with advancing age. In animal model studies, obesity compromises the T-cell immune system as a result of enhanced adipogenesis in primary lymphoid organs and systemic inflammation. In this study, to test the hypothesis that obesity may contribute to the aging of human T-cell immunity, a thousand atomic-bomb survivors were examined for obesity status and ability to produce naive T cells, i.e., T-cell receptor excision circle (TREC) numbers in CD4 and CD8 T cells. The number of TRECs showed a strong positive correlation with naive T cell numbers, and lower TREC numbers were associated with higher age. We found that the TREC number was inversely associated with levels of obesity indicators (BMI, hemoglobin A1c) and serum CRP levels. Development of type-2 diabetes and fatty liver was also associated with lower TREC numbers. This population study suggests that obesity with enhanced inflammation is involved in aging of the human T-cell immune system. Given the fact that obesity increases the risk of numerous age-related diseases, attenuated immune competence is a possible mechanistic link between obesity and disease development among the elderly.

Newborn screening for severe combined immunodeficiency and T-cell lymphopenia in California: Results of the first 2 years

Assay of T-cell receptor excision circles (TRECs) in dried blood spots obtained at birth permits population-based newborn screening (NBS) for severe combined immunodeficiency (SCID). We sought to report the first 2 years of TREC NBS in California. Since August 2010, California has conducted SCID NBS. A high-throughput TREC quantitative PCR assay with DNA isolated from routine dried blood spots was developed. Samples with initial low TREC numbers had repeat DNA isolation with quantitative PCR for TRECs and a genomic control, and immunophenotyping was performed within the screening program for infants with incomplete or abnormal results. Outcomes were tracked. Of 993,724 infants screened, 50 (1/19,900 [0.005%]) had significant T-cell lymphopenia. Fifteen (1/66,250) required hematopoietic cell or thymus transplantation or gene therapy; these infants had typical SCID (n = 11), leaky SCID or Omenn syndrome (n = 3), or complete DiGeorge syndrome (n = 1). Survival to date in this group is 93%. Other T-cell lymphopenic infants had variant SCID or combined immunodeficiency (n = 6), genetic syndromes associated with T-cell impairment (n = 12), secondary T-cell lymphopenia (n = 9), or preterm birth (n = 8). All T-cell lymphopenic infants avoided live vaccines and received appropriate interventions to prevent infections. TREC test specificity was excellent: only 0.08% of infants required a second test, and 0.016% required lymphocyte phenotyping by using flow cytometry. TREC NBS in California has achieved early diagnosis of SCID and other conditions with T-cell lymphopenia, facilitating management and optimizing outcomes. Furthermore, NBS has revealed the incidence, causes, and follow-up of T-cell lymphopenia in a large diverse population.