T-cell Receptor Excision Circle Assay Research Articles

Клінічний випадок комбінованого імунодефіциту, діагностованого за допомогою визначення TREC

Early diagnosis and timely treatment of combined immunodeficiency (CID) is extremely important for the child’s future life. At birth, children often have no specific signs of the disease, but from the first months of life CID manifests by severe infections that can impact on the results of further treatment. Detection of T-cell lymphopenia by determining the number of T-cell receptor excision circles (TRECs) in a dry spot after the birth of a child formed the basis for newborn screening for severe CID. KREC (kappa-deleting recombination excision circle) assay is used for detection of B-cell lymphopenia. Clinical case. We report the first case of CID (T-B+NK+) suspected by detection of TREC in Ukraine in a 2.5-month-old child. Immunodeficiency was manifested by severe complicated pneumonia at the age of 2 months, which had an atypical course and was resistant to treatment. A screening study to detect T- and B-lymphopenia using the TREC/KREC assay twice showed a zero TREC value and a KREC number of 3.12×105 per 106 cells, indicating severe CID. Subsequent immunological studies confirmed the deficiency of T-cell immunity. Venous thrombosis and increasing multiorgan failure led to the death of the child. Thus, newborn screening using TREC assay will allow the timely detection of severe combined immunodeficiencies with further adequate therapy and measures to prevent life-threatening infections, including SARS-CoV-2. The research was carried out in accordance with the principles of the Helsinki Declaration. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

Future Perspectives of Newborn Screening for Inborn Errors of Immunity.

Newborn screening (NBS) programs continue to expand due to innovations in both test methods and treatment options. Since the introduction of the T-cell receptor excision circle (TREC) assay 15 years ago, many countries have adopted screening for severe combined immunodeficiency (SCID) in their NBS program. SCID became the first inborn error of immunity (IEI) in population-based screening and at the same time the TREC assay became the first high-throughput DNA-based test in NBS laboratories. In addition to SCID, there are many other IEI that could benefit from early diagnosis and intervention by preventing severe infections, immune dysregulation, and autoimmunity, if a suitable NBS test was available. Advances in technologies such as KREC analysis, epigenetic immune cell counting, protein profiling, and genomic techniques such as next-generation sequencing (NGS) and whole-genome sequencing (WGS) could allow early detection of various IEI shortly after birth. In the next years, the role of these technical advances as well as ethical, social, and legal implications, logistics and cost will have to be carefully examined before different IEI can be considered as suitable candidates for inclusion in NBS programs.

Recurrent Fevers in a Boy Toddler with Dysmorphic Features and Hypodontia.

Recurrent Fevers in a Boy Toddler with Dysmorphic Features and Hypodontia.

Newborn Screening for Severe Combined Immunodeficiency Using the Multiple of the Median Values of T-Cell Receptor Excision Circles.

All newborn screening programs screen for severe combined immunodeficiency by measurement of T-cell receptor excision circles (TRECs). Herein, we report our experience of reporting TREC assay results as multiple of the median (MoM) rather than using conventional copy numbers. This modification simplifies the assay by eliminating the need for standards with known TREC copy numbers. Furthermore, since MoM is a measure of how far an individual test result deviates from the median, it allows normalization of TREC assay data from different laboratories, so that individual test results can be compared regardless of the particular method, assay, or reagents used.

A Novel De Novo PGM3 Pathogenic Mutation Identified in an Infant Presenting with Abnormal TREC Assay and Severe Neutropenia

Phosphoglucomutase 3 (PGM3) deficiency is an autosomal recessive congenital glycosylation disorder associated with a variable clinical phenotype including immunodeficiency, atopy and progressive bone marrow failure. We present an infant with compound heterozygous pathogenic PGM3 mutations, including a novel de novo variant, who initially presented with an abnormal T-cell receptor excision circles (TREC) assay on newborn screen.

PP574 Cost-Effectiveness Of Newborn Screening Of Primary Immunodeficiency Diseases: A Systematic Review And Economic Evaluation

IntroductionPrimary immunodeficiency diseases (PIDs) are a heterogeneous group of over 200 disorders with defects in the function and/or development of the immune system. Although early screening is imperative for improving therapeutic efficiency and preventing disease-associated morbidity, its widespread use has been limited, owing to the low incidence of PIDs. It is particularly important to evaluate the cost-effectiveness of PIDs screening for newborns. The aim of this study was to provide an overview of the existing cost-effectiveness evidence on newborn screening of PIDs and to provide reference for decision-makers in China and other developing countries.MethodsWe conducted a systematic review using three electronic databases (PubMed, CNKI, and CSPD) of cost and cost-effectiveness studies of PIDs screening published during 2000–2019. Two reviewers independently searched databases and screened titles, abstracts and full texts; a third reviewer resolved disputes when necessary. The initial search returned 124 references, of which 10 full articles were included in the review. Five of the studies conducted analyses using model-based techniques.ResultsSevere combined immunodeficiency (SCID) was the predominantly studied condition (80%). Most studies (70%) examined the T-cell receptor excision circle (TREC) assay. A healthcare system's perspective was commonly used (50%) for cost calculations, and most studies (50%) were US-based. The majority (67%) of the studies found the TREC assay an effective screening tool for SCID, but the incremental cost-effectiveness ratio (ICER) varied across screening test specificity and disease incidence.ConclusionsEvidence from the published literature demonstrated that newborn screening for PIDs generally appeared to be cost-effective, and most importantly, it is lifesaving and allows children with PIDs an opportunity to live a healthier life. However, the type of PIDs included in this study were limited and most studies were done in developed countries whose health systems are different from low-/middle-income countries (LMIC). Further research is required to identify the cost-effectiveness of PIDs screening both in developed and developing countries.

Establishing Simultaneous T Cell Receptor Excision Circles (TREC) and K-Deleting Recombination Excision Circles (KREC) Quantification Assays and Laboratory Reference Intervals in Healthy Individuals of Different Age Groups in Hong Kong.

The clinical experience gathered throughout the years has raised awareness of primary immunodeficiency diseases (PIDD). T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) assays for thymic and bone marrow outputs measurement have been widely implemented in newborn screening (NBS) programs for Severe Combined Immunodeficiency. The potential applications of combined TREC and KREC assay in PIDD diagnosis and immune reconstitution monitoring in non-neonatal patients have been suggested. Given that ethnicity, gender, and age can contribute to variations in immunity, defining the reference intervals of TREC and KREC levels in the local population is crucial for setting up cut-offs for PIDD diagnosis. In this retrospective study, 479 healthy Chinese sibling donors (240 males and 239 females; age range: 1 month−74 years) from Hong Kong were tested for TREC and KREC levels using a simultaneous quantitative real-time PCR assay. Age-specific 5th–95th percentile reference intervals of TREC and KREC levels (expressed in copies per μL blood and copies per 106 cells) were established in both pediatric and adult age groups. Significant inverse correlations between age and both TREC and KREC levels were observed in the pediatric age group. A significant higher KREC level was observed in females than males after 9–12 years of age but not for TREC. Low TREC or KREC levels were detected in patients diagnosed with mild or severe PIDD. This assay with the established local reference intervals would allow accurate diagnosis of PIDD, and potentially monitoring immune reconstitution following haematopoietic stem cell transplantation or highly active anti-retroviral therapy in the future.

Recurrent microdeletions at chromosome 2p11.2 are associated with thymic hypoplasia and features resembling DiGeorge syndrome

Thymic hypoplasia/aplasia occurs as a part of DiGeorge syndrome, which has several known genetic causes, and with loss-of-function mutations in forkhead box N1 (FOXN1). We sought to determine the cause of selective T-cell lymphopenia with inverted kappa/lambda ratio in several kindreds. Patients were identified through newborn screening for severe combined immunodeficiency using the T-cell receptor excision circle assay. Those found to have selective T-cell lymphopenia underwent testing with chromosomal microarray analysis. Three-week-old mice heterozygous for a loss-of-function mutation in forkhead box I3 (FOXI3), a candidate gene within the common deleted region found in patients, were compared with wild-type littermates. Assessments included body and organ weights, flow cytometric analysis of thymocytes and splenocytes, and histologic/transcriptomic analyses of thymic tissue. Five kindreds with similar immunophenotypes that included selective T-cell lymphopenia had overlapping microdeletions at chromosome 2p11.2 that spanned FOXI3 and, in most cases, the immunoglobulin kappa light chain locus. Studies in a mouse knockout strain for FOXI3 revealed smaller body weights and relatively lower thymus weights in heterozygous compared with wild-type animals. Histology and flow cytometry on spleens and thymi from 3-week-old pups for T- and B-cell subsets and epithelial cells did not show any significant qualitative or quantitative differences. Transcriptomic analysis of thymic RNA revealed divergence in global transcriptomic signatures, and Ingenuity Pathway Analysis revealed predicted dysfunction in epithelial adherens junctions. Microdeletions at chromosome 2p11.2 are associated with T-cell lymphopenia and probable thymic hypoplasia in human subjects, and haploinsufficiency for FOXI3, a candidate gene within the deleted region, is the likely underlying cause.

Combination of TREC Measurement and Next-Generation Sequencing in Newborn Screening for Severe Combined Immunodeficiency: A Pilot Program in Japan

INTRODUCTIONSevere combined immunodeficiency disease (SCID) is the most severe form of primary immunodeficiency disorders (PIDs). Impaired cellular and humoral immunity renders the affected infants susceptible to various infections and results in death within the first 2 years of life. Affected infants are asymptomatic at birth, untreated disease leads to death, and prompt treatment (i.e., hematopoietic stem cell transplantation, gene therapy, or enzyme replacement therapy) is linked to significant improvement in outcome. Thus, SCID meets the disease criteria for newborn screening (NBS). The T-cell receptor excision circle (TREC) is an excellent marker of recently formed T cells, and quantitative PCR-based measurement of TREC is an excellent tool in population-based NBS for SCID.Recent progress in next-generation sequencing (NGS) has enabled the simultaneous sequencing of numerous nucleic acids, detecting single nucleotide changes as well as copy number variants. We launched a pilot newborn optional screening program for SCID, combining the measurement of TREC and NGS in Japan.PATIENTS AND METHODSWe measured TREC copy number using the Enlite™ Neonatal TREC assay (Perkin Elmer, Turku, Finland), which utilizes the duplex amplification of TREC and beta-actin in the same reaction for each specimen. We used TREC negative cutoffs as follows: TREC copy number of <30 copies/μL and beta-actin copy number of ≥50 copies/μL. In patients with TREC negative results, genomic DNA was subjected to DNA capture designed using SureDesign (Agilent, Santa Clara, USA), covering a total of 349 genes associated with PIDs, inherited bone marrow failure syndromes, and the 22q11.2 region. Target capture, enrichment, and indexing were performed according to the manufacturer's instructions. Generated libraries were sequenced using a HiSeq 2500 platform (Illumina, San Diego, USA). This study was approved by the ethical committees of the Nagoya University Graduate School of Medicine and Fujita Health University.RESULTSFrom April 2017 to March 2018, we screened a total of 22,865 newborns, covering 57% of the total number of births in the Aichi prefecture, Japan. We identified 48 (0.21%) newborns with TREC negative results. These newborns were referred to the Nagoya University Hospital or Fujita Health University Hospital and received thorough immunological examination, including target capture sequencing. Among them, 12 (25%) newborns had background diseases, including Down syndrome (n = 4), gastrointestinal defects (n = 3), congenital diaphragmatic hernia (n = 2), congenital chylothorax (n = 2), and severe congenital heart anomaly (n = 1).Immunological assessment identified 11 (23%) infants with lymphocytopenia (<1500 /μL). These infants avoided live vaccines and received appropriate interventions to prevent infection. Using target sequencing analyses, we identified four patients with PIDs, including 22q11.2 deletion syndrome (n = 2), Wiskott‒Aldrich syndrome (n = 1), and combined immunodeficiency with an unknown causative gene (n = 1).CONCLUSIONWe successfully launched a pilot newborn optional screening program for SCID, combining the measurement of TREC and NGS in Japan. We did not identify typical SCID patients probably because of the relatively small sample size. However, this newborn screening program, incorporating an NGS assay as a second test, achieved early accurate diagnoses of patients with other PIDs with TREC negative results. Consequently, this program may facilitate patient management and optimize treatment outcomes. DisclosuresNo relevant conflicts of interest to declare.

CAUSES OF T-CELL LYMPHOPENIA IDENTIFIED ON NEWBORN SCREENING IN VIRGINIA 2015-2018: A SINGLE CENTER EXPERIENCE

Introduction Virginia instituted newborn screening (NBS) for severe combined immunodeficiency (SCID) in June of 2015. The NBS uses a T-cell receptor excision circle (TREC) assay to evaluate for T-cell lymphopenia. The objective of this study was to identify underlying causes of T-cell lymphopenia in infants who have an abnormal screen but do not have confirmed SCID. Methods Retrospective chart review of patients with abnormal or critical TREC assays was performed. A screen was considered abnormal if the TREC cycle threshold was between 35.51 - 38.3, and critical if it was 0 or greater than 38.3. Results There were 78 patients evaluated with either an abnormal or critical initial or repeat NBS. Of those 78 patients, 11 had a normal initial NBS and subsequent abnormal repeats. These patients did not require further workup. Repeat NBS was normal for 27 patients who had an initial abnormal or critical screen. These patients did not require flow cytometry. Flow cytometry was performed for 40 patients with an abnormal or critical screen and it was normal in 30 patients. Of the 10 abnormal flow cytometry results, one patient had SCID. One patient had Jacobsen syndrome. Two patients had partial DiGeorge Syndrome. Lastly, there were 6 patients with idiopathic CD4+ lymphopenia. Conclusions Newborn screening for SCID is also able to identify non-SCID T-cell lymphopenia. It is important to recognize these patients to ensure necessary management and follow up. In addition, changes in screening guidelines may be needed to decrease the number of false positive results.

Newborn Screening for Severe Combined Immunodeficiency: Analytic and Clinical Performance of the T Cell Receptor Excision Circle Assay in France (DEPISTREC Study).

Severe combined immunodeficiency (SCID) is characterized by a major T cell deficiency. Infants with SCID are asymptomatic at birth but die from infections in the first year of life if not treated. Survival rates are better for early treatment. SCID therefore meets criteria for newborn screening (NBS). T cell receptor excision circle (TREC) quantification is a reliable marker of T cell deficiency and can be performed using Guthrie cards. The DEPISTREC project was designed to study the feasibility, clinical utility, and cost-effectiveness of generalized SCID screening in France. About 200,000 babies from all over the country were screened at birth with a commercial kit. We determined assay performance and proposed a cutoff for classification of results. Our findings suggest that, given clearly established validation rules and decision-making procedures, the TREC assay is a suitably specific and sensitive method for high-throughput SCID screening. Clinical Trials: NCT02244450.

Development of a Population-Based Newborn Screening Method for Severe Combined Immunodeficiency in Manitoba, Canada.

The incidence of Severe Combined Immunodeficiency (SCID) in Manitoba, (1/15,000), is at least three to four times higher than the national average and that reported from other jurisdictions. It is overrepresented in two population groups: Mennonites (ZAP70 founder mutation) and First Nations of Northern Cree ancestry (IKBKB founder mutation). We have previously demonstrated that in these two populations the most widely utilized T-cell receptor excision circle (TREC) assay is an ineffective newborn screening test to detect SCID as these patients have normal numbers of mature T-cells. We have developed a semi-automated, closed tube, high resolution DNA melting procedure to simultaneously genotype both of these mutations from the same newborn blood spot DNA extract used for the TREC assay. Parallel analysis of all newborn screening specimens utilizing both TREC analysis and the high-resolution DNA procedure should provide as complete ascertainment as possible of SCID in the Manitoba population.

Newborn Screening for Severe Combined Immunodeficiency.

This review provides a brief history of newborn screening (NBS) for severe combined immunodeficiency (SCID), discusses the theoretical basis for the T cell receptor excision circle (TREC) assay, highlights the results of recent studies using the TREC, and provides practical advice for the evaluation of infants with an abnormal TREC assay. Currently, all but three states perform NBS for SCID in the USA. NBS using the TREC assay is highly sensitive in identifying infants with SCID and may also identify infants with T cell lymphopenia due to other causes such as congenital syndromes, multiple congenital anamolies, and some combined immunodeficiencies. Regardless of the genetic etiology, all forms of SCID are characterized by a severe deficiency of naïve T cells. TRECs are a biomarker of newly formed, naïve T cells that have recently left the thymus. Consequently, the TREC assay identifies infants with SCID and other causes of severe T cell lymphopenia.

High Incidence of Severe Combined Immunodeficiency Disease in Saudi Arabia Detected Through Combined T Cell Receptor Excision Circle and Next Generation Sequencing of Newborn Dried Blood Spots.

Severe combined immunodeficiency disease (SCID) is the most severe form of primary immunodeficiency disorders (PID). T-cell receptor excision circle (TREC) copy number analysis is an efficient tool for population-based newborn screening (NBS) for SCID and other T cell lymphopenias. We sought to assess the incidence of SCID among Saudi newborn population and examine the feasibility of using targeted next generation sequencing PID gene panel (T-NGS PID) on DNA isolated from dried blood spots (DBSs) in routine NBS programs as a mutation screening tool for samples with low TREC count. Punches from 8,718 DBS collected on Guthrie cards were processed anonymously for the TREC assay. DNA was extracted from samples with confirmed low TREC count, then screened for 22q11.2 deletion syndrome by real-time polymerase chain reaction and for mutations in PID-related genes by T-NGS PID panel. Detected mutations were confirmed by Sanger sequencing. Sixteen out of the 8,718 samples were confirmed to have low TREC copy number. Autosomal recessive mutations in AK2, JAK3, and MTHFD1 were confirmed in three samples. Two additional samples were positive for the 22q11.2 deletion syndrome. In this study, we provide evidence for high incidence of SCID among Saudi population (1/2,906 live births) and demonstrate the feasibility of using T-NGS PID panel on DNA extracted from DBSs as a new reliable, rapid, and cost-effective mutation screening method for newborns with low TREC assay, which can be implemented as part of NBS programs for SCID.

A Practical Approach to Newborn Screening for Severe Combined Immunodeficiency Using the T Cell Receptor Excision Circle Assay.

Severe combined immunodeficiency (SCID) is a life-threatening condition of newborns and infants caused by defects in genes involved in T cell development. Newborn screening (NBS) for SCID using the T cell receptor excision circle (TREC) assay began in Wisconsin in 2008 and has been adopted or is being implemented by all states in 2017. It has been established that NBS using the TREC assay is extremely sensitive to detect SCID in the newborn period. Some controversies remain regarding how screening positives are handled by individual states, including when to perform confirmatory flow cytometry, what is the necessary diagnostic workup of patients, what infection prophylaxis measures should be taken, and when hematopoietic stem cell transplantation should occur. In addition, the TREC can also assay detect infants with T cell lymphopenia who are not severe enough to be considered SCID; management of these infants is also evolving.

Guidelines for Screening, Early Diagnosis and Management of Severe Combined Immunodeficiency (SCID) in India.

Severe combined immunodeficiency (SCID) is one of the most severe and fatal forms of inherited primary immunodeficiency. Early diagnosis of SCID improves the outcome of life before and after hematopoietic stem cell transplant (HSCT). SCID fulfills the internationally-established criteria for a condition to be screened for at birth. T cell receptor excision circle (TREC) assay is commonly used in western countries as part of newborn blood spot screening (NBS) program as the assay has high sensitivity and specificity to identify SCID infants, allowing early intervention and curative bone marrow (BM) transplantation. In India, the blood spot based screening programs are yet to mature into a full-fledged national program. Moreover, TREC assay, a PCR based test, is not widely available and may cost USD 5-7 per test; thus limiting its applicability for screening newborns in Indian scenario. Most of the SCID patients have lymphopenia at birth and routine evaluation for absolute lymphocyte count (ALC) on cord blood samples can help in pre-symptomatic detection and early intervention for neonates with SCID. Although ALC count lacks the sensitivity and specificity of TREC assay; its lower cost and widespread availability makes it an attractive option for identifying newborns with lymphopenia during the post-partum hospital stay. BCG vaccine and other live attenuated vaccines (e.g., oral polio vaccine) should be withheld in lymphopenic infants until SCID is excluded by clinical and/or immunological work-up. A diagnosis of SCID warrants immediate care to prevent and treat infections and wherever feasible, early stem cell transplantation for disease free survival.

Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency.

Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn blood spot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well. An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members. DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range. SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID.

Newborn Screening for Severe Combined Immunodeficiency

Newborn screening (NBS) for severe combined immunodeficiency (SCID) utilizing the T-cell receptor excision circle assay is a sensitive and specific method to detect T-cell lymphopenia in early infancy. Starting in 2008, several programs have implemented SCID NBS with successful detection of SCID-affected infants; currently over two thirds of U.S. infants receive SCID NBS. Population-based, unbiased screening has established an incidence of SCID to be 1 in 58,000 births, and has revealed a distribution of SCID genotypes different from prior reports from specific SCID treatment centers. Detecting SCID-affected infants in the newborn period allows for timely implementation of protective measures and optimal definitive treatment prior to the onset of life-threatening infections. Infants with non-SCID T-cell lymphopenia also detected by NBS may have one of several recognized syndromes in which lymphocyte development may be impaired, as well as other conditions associated with secondary T-cell lymphopenia.

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.

Cellular signalling pathways in immune aging and regeneration.

The thymus is one of the cornerstones of an effective immune system. It produces new T-cells for the naïve T-cell pool, thus refreshing the peripheral repertoire. As we age, the thymus atrophies and there is a decrease in the area of active T-cell production. A decline in the output of the thymus eventually leads to changes in the peripheral T-cell pool which includes increases in the number of cells at or near their replicative limit and contraction of the repertoire. Debate about the age-associated changes in the thymus leading to functional decline centres on whether this is due to problems with the environment provided by the thymus or with defects in the progenitor cell compartment. In mice, the evidence points towards problems in the epithelial component of the thymus and the production of IL-7 (interleukin 7). But there are discussions about how appropriate mouse models are for human aging. We have developed a simple system that utilizes both human keratinocyte and fibroblast cell lines arrayed on a synthetic tantalum-coated matrix to provide a permissive environment for the maturation of human CD34+ haemopoietic progenitor cells into mature CD4+ or CD8+ T-lymphocytes. We have characterized the requirements for differentiation within these cultures and used this system to compare the ability of CD34+ cells derived from different sources to produce mature thymocytes. The TREC (T-cell receptor excision circle) assay was used as a means of identifying newly produced thymocytes.