T-cell Receptor Excision Circles Research Articles

Determining the relationship of p16INK4a and additional molecular markers of aging with clinical frailty in hematologic malignancy.

Older adults with hematologic malignancies (HM) have unique challenges due to age and fitness. The primary aim of this pilot study was to benchmark the ability of multiple biomarkers of aging (p16, epigenetic clocks, T cell gene expression profiles, and T cell receptor excision circles (TREC) to identify frailty as measured by a clinical impairment index (I2) in patients with HM. 70 patients newly diagnosed with HM had peripheral blood T lymphocytes (PBTL) analyzed for p16INK4a expression using the OSU_Senescence Nanostring CodeSet. PBTL epigenetic age was measured using 7 epigenetic clocks, and TREC were quantified by qRT-PCR. A composite clinical impairment index (I2) was generated by combining values from 11 geriatric metrics (Independent Activities of Daily Living (iADL), physical health score, Short Physical Performance Battery (SPPB), Body Mass Index (BMI), Eastern Cooperative Oncology Group (ECOG) performance status, self-reported KPS, Blessed Orientation Memory Concentration (BOMC), polypharmacy, Mental Health Inventory (MHI)-17, Medical Outcomes Study (MOS) subscales). Clinical frailty was defined as a score of 7 or greater on the I2. Age-adjusted p16INK4a was similar in newly diagnosed patients and healthy controls (p > 0.1). PBTL p16INK4a levels correlated positively with the Hannum [r = 0.35, 95% CI (0.09-0.75); p adj. = 0.04] and PhenoAge [r = 0.37, 95% CI (0.11-0.59); p adj. = 0.04] epigenetic clocks. The discrimination ability of the I2 model was calculated using the area under the receiver operating characteristic curve (AUC). After adjusting for chronologic age and disease group, baseline p16INK4a [AUC = 0.76, 95% CI (0.56-0.98); p = 0.01], Hannum [AUC = 0.70, 95% CI (0.54-0.85); p = 0.01], PhenoAge [AUC = 0.71, 95% CI (0.55-0.86); p = 0.01], and DunedinPACE [AUC = 0.73, 95% CI (0.57-0.88); p = < 0.01] measures showed the greatest potential to identify clinical frailty using the I2. Our pilot data suggest that multiple blood-based aging biomarkers have potential to identify frailty in older adults with HM. We developed the I2 index to quantify impairments across geriatric domains and discovered that PBTL p16, Hannum, PhenoAge, and DunedinPACE are promising indicators of frailty in HM.

Expanded Newborn Screening for Inborn Errors of Immunity: The Experience of Tuscany

Inborn errors of immunity (IEIs) include 485 inherited disorders characterized by an increased susceptibility to life threatening infectious diseases, autoimmunity and malignant diseases with a high mortality rate in the first years of life. Severe Combined Immunodeficiency is the most severe of the IEIs and its detection should be a primary goal in a newborn screening (NBS) program. The term "actionable" has recently been used for all IEIs with outcomes that can be demonstrably improved through early specialized intervention. to evaluate the results of the expanded NBS strategy for IEIs in Tuscany Region (Italy), based on TREC (T-cell Receptor Excision Circles), KREC (Kappa Recombining Excision Circles) and Tandem Mass-based assays. This is a retrospective study collecting data from all infants born in Tuscany from October 10, 2018, to October 10, 2022. Tandem mass assay to identify Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency, together with TREC and KREC molecular analysis were conducted on dried blood spot (DBS) from the newborns' Guthrie Cards. A new DBS and evaluation by an immunologist were carried out when the results of the first test were outside the diagnostic cut-offs. 94,319 newborns were evaluated. Referral rates for TREC (0.031%) and KREC (0.074%) in this study are in line with the data available in literature. The results from the expanded NBS strategy revealed an incidence rate of 1/9,431 affected newborns. This work represents the first description of a sustainable and real-life based expanded NBS program for IEIs with a high diagnostic incidence facilitating prompt management of identified patients.

Newborn Screening for Severe T and B Cell Lymphopenia Using TREC/KREC Detection: A Large-Scale Pilot Study of 202,908 Newborns.

Newborn screening (NBS) for severe inborn errors of immunity (IEI), affecting T lymphocytes, and implementing measurements of T cell receptor excision circles (TREC) has been shown to be effective in early diagnosis and improved prognosis of patients with these genetic disorders. Few studies conducted on smaller groups of newborns report results of NBS that also include measurement of kappa-deleting recombination excision circles (KREC) for IEI affecting B lymphocytes. A pilot NBS study utilizing TREC/KREC detection was conducted on 202,908 infants born in 8 regions of Russia over a 14-month period. One hundred thirty-four newborns (0.66‰) were NBS positive after the first test and subsequent retest, 41% of whom were born preterm. After lymphocyte subsets were assessed via flow cytometry, samples of 18 infants (0.09‰) were sent for whole exome sequencing. Confirmed genetic defects were consistent with autosomal recessive agammaglobulinemia in 1/18, severe combined immunodeficiency - in 7/18, 22q11.2DS syndrome - in 4/18, combined immunodeficiency - in 1/18 and trisomy 21 syndrome - in 1/18. Two patients in whom no genetic defect was found met criteria of (severe) combined immunodeficiency with syndromic features. Three patients appeared to have transient lymphopenia. Our findings demonstrate the value of implementing combined TREC/KREC NBS screening and inform the development of policies and guidelines for its integration into routine newborn screening programs.

EFFECT OF ORAL VITAMIN D3 ON THYMIC INDEX IN MALNOURISHED CHILDREN

Objectives: To compare the thymic Index, vitamin D3 level, and T cell receptor excision circle (TREC) of healthy and malnourished infants. To evaluate the effect of oral vitamin D3 supplementation on the Thymic Index and TREC in malnourished children. Material and Methods: This quasi-experimental study (non-equivalent control group design) included 44 infants within the age range of 2 to 6 months. The cases were 22 malnourished infants, and 22 were healthy infants (controls) based on WHO Z scores classification (weight for length). Weight, age, thymic Index (TI), Vitamin D levels, and TRECs were recorded for cases and controls(pre-test). The cases were given Vitamin D supplementation for 8 weeks. The weight for length (z-score), thymic Index (TI), vitamin D levels, and TRECs were checked in these malnourished children on follow-up (post-test). Thymic Index (TI) was checked by ultrasonography (length multiplied by breadth). ELISA was performed to check Vitamin D levels and RT-PCR was done to compare TRECs. Results: The malnourished infants had a mean thymic index of 8.015 ±3.2192 as compared to controls of 11.667 ±3.2730. Post Vit D supplementation TI increased (8.459 ±3.2191) in cases. Weight, length, and Z scores were found to be directly related to TI values. Pre and Post supplementation TI were strongly correlated to each other (r= 0.985, p= &lt;0.001). The thymic index increased significantly (p= 0.001) after intervention. There was a statistically non-significant rise in TRECS (809 vs 4242) (p= 0.075) after vitamin D supplementation. The Pearson Correlation showed no significant correlation of TRECS to any of the variables including the Thymic index. Conclusion: TI (thymic size) and vitamin D levels increase with vitamin D supplementation in malnourished children. However, Vitamin D supplementation has no significant effect on TRECS. Keywords: Malnourished, Vitamin D3, T cell receptor excision circles, Thymic Index, TRECs.

Increased mortality in infants with abnormal T-cell receptor excision circles.

T-Cell Receptor Excision Circles based newborn screening (TREC-NBS) allows for early detection of T-cell lymphopenia in infants with primary immunodeficiency disorders (PIDD). The utility of abnormal TREC-NBS in infants without PIDD is not well studied. We sought to evaluate the association of abnormal TREC-NBS with mortality. 365,207 TREC-NBS from October 2011 to December 2014 were reviewed. 467 newborns had abnormal screens and did not meet the criteria for a PIDD diagnosis. Cases were matched to controls (1:3) based on gestational age, birth weight, neonatal intensive care unit status (NICU), and race. Data were obtained through NBS, birth and death certificates records from the Michigan Department of Health and Human Services (MDHHS) databases. Infants with abnormal TREC-NBS had higher mortality even when PIDD was ruled-out. Transient abnormal TREC-NBS was not associated with higher mortality, but unresolved or late abnormal TREC-NBS was associated with higher mortality. Infants with late abnormal TREC-NBS had severe prematurity, lower birth weight, lower Apgar scores, and higher percentage of congenital anomalies. Infants with abnormal TREC-NBS may be at a higher risk of morbidity and mortality and should be carefully followed, especially if discharged home before a repeat screen can be completed. This study explores the risk factors and mortality for newborns with secondary T-cell lymphopenia captured on T-Cell Receptor Excision Circles based newborn screening (TREC-NBS). Abnormal TREC-NBS allows for prompt life-saving interventions for primary immunological conditions such as Severe Combined Immunodeficiency (SCID), but can also be associated with non-immunologic conditions. Unresolved and late abnormal TREC-NBS is associated with higher mortality even without primary immunodeficiency, likely detected in infants with more severe prematurity, lower birth weight, and congenital anomalies. TREC-NBS positive infants with secondary T-cell lymphopenia require special attention and close monitoring.

Multiplex Real-Time PCR-Based Newborn Screening for Severe Primary Immunodeficiency and Spinal Muscular Atrophy in Osaka, Japan: Our Results after 3 Years.

In newborn screening (NBS), it is important to consider the availability of multiplex assays or other tests that can be integrated into existing systems when attempting to implement NBS for new target diseases. Recent developments in innovative testing technology have made it possible to simultaneously screen for severe primary immunodeficiency (PID) and spinal muscular atrophy (SMA) using quantitative real-time polymerase chain reaction (qPCR) assays. We describe our experience of optional NBS for severe PID and SMA in Osaka, Japan. A multiplex TaqMan qPCR assay was used for the optional NBS program. The assay was able to quantify the levels of T-cell receptor excision circles and kappa-deleting recombination excision circles, which is useful for severe combined immunodeficiency and B-cell deficiency screening, and can simultaneously detect the homozygous deletion of SMN1 exon 7, which is useful for NBS for SMA. In total, 105,419 newborns were eligible for the optional NBS program between 1 August 2020 and 31 August 2023. A case each of X-linked agammaglobulinemia and SMA were diagnosed through the optional NBS and treated at early stages (before symptoms appeared). Our results show how multiplex PCR-based NBS can benefit large-scale NBS implementation projects for new target diseases.

Differentiation of regulatory myeloid and T-cells from adult human hematopoietic stem cells after allogeneic stimulation.

Donor hematopoietic stem cell (DHSC) infusions are increasingly being studied in transplant patients for tolerance induction. To analyze the fate of infused DHSCs in patients, we developed an in vitro culture system utilizing CD34+DHSCs stimulated with irradiated allogeneic cells in cytokine supplemented medium long-term. Flow cytometric analyses revealed loss of the CD34 marker and an increase in CD33+ myeloid and CD3+ T-cell proportion by 10.4% and 72.7%, respectively, after 21 days in culture. T-cells primarily expressed TcR-αβ and were of both CD4+ and CD8+ subsets. Approximately 80% of CD3+ T cells lacked expression of the co-stimulatory receptor CD28. The CD4+ compartment was predominated by CD4+CD25+CD127-FOXP3+ Tregs (>50% CD4+CD127- compartment) with <1% of all leukocytes exhibiting a CD4+CD127+ phenotype. Molecular analyses for T-cell receptor excision circles showed recent and increased numbers of TcR rearrangements in generated T cells over time suggesting de novo differentiation from DHSCs. CD33+ myeloid cells mostly expressed HLA-DR, but lacked expression of co-stimulatory receptors CD80 and CD83. When studied as modulators in primary mixed lymphocyte reactions where the cells used to stimulate the DHSC were used as responders, the DHSC-lines and their purified CD8+, CD4+, CD33+ and linage negative subsets inhibited the responses in a dose-dependent and non-specific fashion. The CD8+ cell-mediated inhibition was due to direct lysis of responder cells. Extrapolation of these results into the clinical situation would suggest that DHSC infusions into transplant recipients may generate multiple subsets of donor "chimeric" cells and promote recipient Treg development that could regulate the anti-donor immune response in the periphery. These studies have also indicated that T cell maturation can occur in vitro in response to allogeneic stimulation without the pre-requisite of a thymic-like environment or NOTCH signaling stimulatory cell line.

Predictors for low T-cell receptor excision circles (TREC) in infants without severe combined immunodeficiency (SCID) or thymic defects

Predictors for low T-cell receptor excision circles (TREC) in infants without severe combined immunodeficiency (SCID) or thymic defects

Newborn Screening for Severe Combined Immunodeficiency: Lessons Learned from Screening and Follow-Up of the Preterm Newborn Population.

Newborn screening (NBS) for Severe Combined Immunodeficiency (SCID) by measurement of T-cell receptor excision circles (TRECs) successfully identifies newborns with SCID and severe T-cell lymphopenia, as intended. At the same time, NBS programs face the challenge of false positive results, with a disproportionately high number in the premature newborn population. This study evaluates TREC values and SCID screening outcomes in premature newborns and elucidates evidence-based SCID screening practices that reduce unnecessary follow-up activities in this population. De-identified individual SCID newborn screening data and aggregate SCID screening data were obtained from seven states across the US for babies born between 2018 and 2020. Relevant statistics were performed on data pooled from these states to quantify screening performance metrics and clinical impact on various birth and gestational age categories of newborns. The data were normalized using multiples-of-the-median (MoM) values to allow for the aggregation of data across states. The aggregation of NBS data across a range of NBS programs highlighted the trajectory of TREC values over time, both between and within newborns, and provides evidence for improved SCID screening recommendations in the premature and low birth weight population.

Assessment of trec and krec levels in COVID-19 patients with varying disease severity

According to the WHO data, the number of infected people exceeded 765.2 million people during the COVID-19 pandemic. The severity of patient’s condition is determined by immune system hyperactivation. Activation of T- and B-lymphocyte subsets plays a prominent role in the control of infectious process. A content of small circular DNA molecules — T-cell receptor excision circles (TREC — T-cell receptor excision circles) and B-cell (“kappa”) excision rings (KREC — Kappa-deleting recombination excision circles) — in the peripheral blood can be used as a marker of the functionally active T and B cells maturation. The purpose of this work is to quantify peripheral blood TREC and KREC level in patients with the new coronavirus infection COVID-19 of varying severity.Materials and methods.The material consisted of 1028 blood samples from patients with a confirmed diagnosis of COVID-19, as well as 717 blood samples from apparently healthy volunteers. The content of TREC and KREC DNA fragments in the total DNA fraction was assessed by quantitative Real-time PCR using the “TREC/KREC-AMP PS” test system (St. Petersburg Pasteur Institute, Russia). Blood cell phenotyping was carried out using flow cytometry.Results.TREC/KREC levels were significantly reduced in COVID-19 patients (p 0.0001 at 95% CI). A significant direct correlation was established between the levels of peripheral blood TREC molecules and level of CD45+CD3+CD19–T cells (r = 0.59, p 0.0001), as well as between KREC level and count of CD45+CD3–CD19+B cells (r = 0.66, p 0.0001). The level of TREC molecules in patients with severe vs. moderate infection was significantly reduced in patients aged 30–39 years old (p = 0.0404) and 40–49 years old (p = 0.0356). The negative correlation between severity of COVID-19 clinical manifestations and TREC level in the blood of patients in 30–49 year age group indicates about an opportunity of using this analyte as a diagnostic and prognostic laboratory marker of patient’s condition. A simple PCR analysis algorithm makes it relevant to use the described method for assessing a state of immunity in coronavirus patients in the context of systemic negative impact of the SARS-CoV-2 virus on human organism.

In-Depth Immune Profiling in Children with BCP-ALL within the AIEOP-BFM ALL 2017 Study

The international multicenter frontline therapy trial AIEOP-BFM ALL 2017 prospectively randomizes CD19-targeting T-cell engager blinatumomab (BLN) against standard of care (SOC) to improve outcomes in pediatric patients (pts) with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) stratified for high (HR) and medium (MR) risks of relapse. In HR arm BLN is given earlier, directly after intensified HR therapy block, while in the MR arm, it is given towards the end of chemotherapy, before maintenance. The immune composition prior to BLN might thus differ between both treatment groups (HR vs MR) which may impact BLN-mediated anti-leukemic activity. Within the Germany-wide immune monitoring program accompanying the AIEOP-BFM ALL 2017 trial we addressed this question and studied a total of 1056 longitudinal peripheral blood samples collected between 11/2018 and 05/2022 from 381 BCP-ALL pts. Samples were prospectively analyzed with multicolor flow cytometry to study immune cell composition (Panel 1) at the time of BLN application (d0) and under ongoing immunotherapy within first 24h (d1), at day 14 (d14), at the end of the cycle I (d28) and end of cycle 2 (day28_II), if applicable (Figure 1A). In-depth T-cell profiling was performed in the BLN arm, utilizing 4 additional 12-color panels concentrated on markers associated with T-cell functionality (measured with standardized cytometer settings). Data were analyzed using classical 2D-manual gating strategies (&amp;gt; 2000 variables) and completed with R-based machine learning algorithms; namely partial Least-Squares Discriminant Analysis (PLS-DA), a multivariate dimensionality-reduction tool for identifying HR- and MR-defining immune signatures and PhenoGraph algorithm for unsupervised T-cell cluster detection. T-cell receptor excision circles (TRECs) and TR-repertoire analysis were also performed among paired samples of 117 BLN-treated pts. Baseline immune composition differed notably between HR and MR pts directly prior to BLN therapy, as shown by PLS-DA analysis on manually gated immune subsets. HR pts showed higher neutrophil counts and lower lymphocyte and monocyte counts compared to MR pts. Interestingly, MR pts had higher levels of B- and T-cells, which are crucial for BLN-mediated T-cell activation. HR pts displayed an elevated CD4+/CD8+ ratio, shifted naïve/memory T-cell phenotypes and increased proportions of immunosuppressive T-regulatory cells. These results suggest that more intensive therapy protocols directly prior to BLN influence the immune composition towards compromised states in the HR arm, potentially affecting BLN-mediated T-cell leukemia control. BLN effectively depleted CD19+ cells in 221 out of 224 pts. We observed significantly stronger T-cell activation/expansion in MR pts compared to HR pts under BLN therapy. This expansion that was primarily attributed to naïve CD8+ T-cell populations, resulting from clonal T-cell proliferation independent of thymic contribution, as supported by TREC and TR-repertoire analysis. The PhenoGraph clustering also identified this expanding naïve CD8+ T-cell populations, surprisingly marked by loss of adhesion molecule DNAM1 (panel 1), normally involved in NK and T-cell mediated cytotoxicity and by gaining the expression of TIM-3 (panel 3), an inhibitory molecule usually co-expressed with PD1 on exhausted T-cells with memory phenotypes. In this setting however, TIM3 was not co-expressed with PD1 (Figure 1B) and its expression was correlated with the B-cell levels at baseline and was strongest in MR pts under BLN therapy. Our data suggest an until now undescribed association between DNAM1 and TIM3 with BLN-mediated T-cell activation, hinting that they may serve as a measure of T-cell activation strength or an early T-cell exhaustion marker. In conclusion our study provides clinically relevant insights in how chemotherapy prior BLN might affect the patients´ immune system, possibly influencing the BLN anti-leukemic activity, and helps to determine the optimal place of immunotherapy with BLN in multimodal treatment schedules. Here, we report until know undescribed effect of BLN on T-cell compartment. Upcoming clinical data, which will be correlated with obtained immune profiles will answer the open question regarding the clinical relevance of these findings, and shed light into the mechanisms determining response to BLN, for its maximum benefit in childhood ALL.

Immune Signature in Telomere-Biology Disorders

Excessive telomere shortening caused by pathogenic germline variants in telomere-biology genes may result in bone marrow failure, hematopoietic malignancy and extramedullary complications, such as idiopathic pulmonary fibrosis (IPF), liver cirrhosis, and solid tumors. Patients with short telomeres also may develop immunodeficiency with low counts of T-cell receptor excision circles (TRECs), kappa-deleting excision circles (KRECs), and CD4 + T cells. The aim of the present study was to comprehensively determine the immune system changes in human patients with telomeropathies by deep-phenotyping peripheral blood mononuclear cells (PBMCs) and quantifying cytokine serum levels. We applied a 39-marker panel of metal-labeled antibodies and employed mass cytometry (CyTOF) to assess the subsets of lymphocytes, monocytes, and dendritic cells (DC) in 20 patients and 10 healthy individuals. High-dimensional analysis was conducted using automated clustering ( Flow-SOM) to identify subpopulations and cell states, whereas t-SNE was employed as a visualization tool. A total of 32 serum cytokines, chemokines and growth factors were quantified by a bead-based immunoassay (Luminex™). First, we confirmed previous findings of low CD4/CD8 ratio (0.73±0.56 vs. 1.3±0.69 in controls, p=0.05), and reduced absolute naïve (CD45RA +CCR7 +) CD4 + and CD8 + cell counts ( p&amp;lt;0.01), with effector memory cell accumulation in patients with telomere diseases, and the current study further showed a reduction in naïve B (CD19 +IgD +CD27 -) and in recent thymic emigrants (RTE), characterized by CD31 +CD45RA + ( p&amp;lt;0.01), indicating a consistent aged B and T cell profile. T H1, T H17 and T H17.1 CD4 + lymphocytes were reduced ( p&amp;lt;0.05), whereas the T H2/T H1 ratio was increased (1.6±0.83 vs. 0.82±0.39 in controls; p=0.013), demonstrating a dysregulated T helper cell diversity. T regs, on the other hand, did not appear to be affected. Plasmacytoid DCs (CD3 -CD141 +CD123 +CD1c -), which are known inducers of T helper differentiation, were reduced in patients ( p=0.014). A double negative T cell (DNT) population, characterized as CD3 +CD4 -CD8 -Vd2 -Tbet +Granzyme B +, was significantly increased ( p=0.0002). Activated effector memory and TEMRA CD4 + cells, also expressing exhaustion markers PD-1 and TIGIT (CD4 +CD45RA +/-CCR7 -CD38 +HLADR +Ki67 +PD-1 +TIGIT low) and proliferating switched memory B cells (CD3 -CD19 +IgD -CD27 +Ki67 +) were higher ( p=0.046 and p=0.041, respectively) suggesting the occurrence of an environment prone to immune activation, also supported by the prevalence of CD14 +CD16 +CXCR3 +CCR4 + classical monocytes ( p=0.0042). Additionally, mucosal-associated invariant T (MAIT) cells (CD3 +CD161 highVɑ7.2 +) were markedly reduced in patients ( p&amp;lt;0.0001), possibly due to their recruitment to other tissues as a response to inflammation. Conversely, immature NK cells (CD3 -CD19 -CD56 brightCD16 -) were more frequent in patients as compared to controls ( p=0.026), and individuals with the shortest telomere lengths had an even higher frequency of these cells. In addition to the previously described CD4 + immunodeficiency, the predominance of immature NK cells may potentially impact the immunosurveillance of malignant transformations. When an unsupervised clustering algorithm using cell subpopulations as variables was applied, patients and controls were correctly identified in 100% of cases. Telomere length positively correlated with some cytokine levels in serum. This was observed for angiopoietin-1, IL-1 superfamily, IL-3, IL-4, IL-7, IL-23, IL-27, M-CSF, MCP-1, MIP-3a and PDGF-BB. Taken together, our results reveal a distinct immunophenotype in telomeropathies, which comprises deficient or skewed maturation of certain subsets, an aged phenotype of B and T cells, aberrant lymphocyte activation and very low levels of circulating MAIT cells, which might be indicators of ongoing inflammatory processes, tied with dysregulated cytokine levels. This immune perturbance may contribute to the clinical onset of telomeropathies and increase the risk of infections, cirrhosis, IPF and cancer development.

Improved T- and B-Cell Neogenesis in Children with Acute Leukemia Given an Alpha-Beta T-Cell Depleted Haplo-HSCT Combined with the Infusion of Donor T-Cells Genetically Modified with Inducible Caspase 9 Suicide Gene (Rivo-cel)

Background αβ T- and B-cell depleted HLA-haploidentical HSCT (αβhaplo-HSCT) is a suitable transplant option promptly available and virtually applicable to any children in need of an allograft. However, the suboptimal recovery of adaptive immunity still represents a limitation of the approach. In order to better deconvolute the reconstitution of adaptive immunity, in this study we measured T-cell receptor and kappa chain-deleting recombination excision circles (TREC and KREC), which represent reliable surrogates of T and B-cell neogenesis, respectively. We also analyzed a cohort of patients who received after the allograft infusion of a titrated number of donor T-cells genetically modified with inducible caspase 9 (iC9) suicide gene (Rivo-cel) as part of a prospective clinical trial (NCT02065869) with the goal of accelerating the recovery of thymus-independent adaptive immunity. Patients and study design We retrospectively analyzed 134 patients (≤ 25 years) with acute lymphoblastic (ALL) or myeloid leukemia (AML) who received a αβ haplo-HSCT between 2014 and 2021; 65 of them received post-transplant infusion of Rivo-cel, at the dose of 1x10 6 cells/kg, after a median time of 26 days (range: 11-87) post-transplant. No patient received post-transplantation pharmacological GVHD prophylaxis (Locatelli et al. Blood 2017). Details on patient and donor characteristics, as well as on graft composition are reported in Table 1. T-cell neogenesis was investigated by quantifying signal joint- (sj-) and beta (b-) TREC while B-cell neogenesis was analyzed by measuring coding-joint (cj-) and sj- KREC. We performed real-time quantitative PCR, as previously described (Arruda et al. 2018), on genomic DNA extracted from PBMC collected at 6 different time points (before and 1, 3, 6, 12 and 18 months after the allograft). At the same time-points peripheral blood of patients was also evaluated by flow-cytometry to phenotypically characterize the pool of leukocytes. Results With a median follow-up of 6 years (range 1-9) the overall (OS) and disease-free survival (DFS) probabilities of the entire cohort were 88,1% and 82%, respectively. The cumulative incidence (CI) of relapse was 15%. CI of grade II-IV acute Graft-versus-Host Didease (GvHD) was 19%, while that of chronic GvHD (cGvHD) was 7%. In the Rivo-cel cohort, sjTREC and bTREC were consistently detectable as early as one month post-HSCT, while started to recover by 3 months after αβhaplo-HSCT alone, and were significantly higher at +3 ( p=0,0006 and p=0,02, respectively) and +6 ( p=0,0003 and p= 0,001) months post-HSCT as compared with αβhaplo-HSCT. sjTREC and bTREC reached pre-transplant values at 6 and 12 months in Rivo-cel and αβhaplo-HSCT cohort, respectively. B-cell neogenesis, assessed by sj and cjKRECs, showed a superimposable recovery kinetics. A higher number of sjKRECs was observed in Rivo-cel cohort at + 1, +3 and +6 months after transplantation as compared with αβhaplo-HSCT ( p=0,007 , p=0,02 and p=0,04for sjKRECs ; p=0,005 , p=0,092 p=0,035forcjKRECs, respectively) . Furthermore, in the Rivo-cel cohort, patients with detectable sj and bTREC values at +3 months after transplant showed a significantly higher overall survival ( p=0,01 p=0,02, respectively). Also in this cohort, aGvHD was confirmed to play a detrimental effect on T-cell neogenesis, at 3 and 6 months after transplant. The same negative effect was documented for chronic GvHD at +6, +12 and +18 months, also affecting B-cell neogenesis at +6 months after transplant. Type of leukemia did not affect T and B-cell neogenesis. Conclusions In patients given αβhaplo-HSCT, T-cell and B-cell neogenesis were shown to be significantly accelerated by the post-transplant infusion of a titrated number of donor T-cells genetically modified with the iC9 suicide gene. Further studies are needed to elucidate the biologic mechanisms responsible for this favorable effect on recovery of adaptive immunity. Our data confirm the detrimental effect of both acute and chronic GvHD in recovery of thymic function and B-cell neogenesis. In this perspective, the possibility to successfully control/abrogate GvHD through the apoptosis of Rivo-cel triggered by the infusion of the dimerizing agent (AP1903) offers also the advantage of, at least partly, preventing the detrimental effect played by this complication on immune recovery.

Shorter birth length and decreased T-cell production and function predict severe infections in children with non–severe combined immunodeficiency cartilage–hair hypoplasia

Cartilage-hair hypoplasia (CHH) is a syndromic inborn error of immunity caused by variants in the RMRP gene. Disease manifestations vary, and their ability to predict outcome is uncertain. The optimal management of infants with CHH who do not fulfill classical severe combined immunodeficiency (SCID) criteria is unknown. We described longitudinal changes in lymphocyte counts during childhood and explored correlations of early childhood clinical and laboratory features with clinical outcomes on long-term follow-up of CHH patients. Immunologic laboratory parameters, birth length, the presence of Hirschsprung disease, and severe anemia correlated to the primary end points of respiratory and severe infections. We implemented traditional statistical methods and machine learning techniques. Thirty-two children with CHH were followed up for 2.7 to 22.1 years (median, 8.2 years, in total 331.3 patient-years). None of the patients had classical SCID. Median lymphocyte subclass counts, apart from CD16+/56+ cells, were subnormal throughout childhood, but did not show age-related decline seen in healthy children. Low immunoglobulin levels were uncommon and often transient. Respiratory and/or severe infections developed in 14 children, 8 of whom had low naive T-cell counts, absent T-cell receptor excision circles, and/or partial "leaky" SCID-level lymphopenia. Shorter birth length correlated with lower lymphocyte counts and the occurrence of infections. Of the laboratory parameters, decreased naive T-cell counts and abnormal lymphocyte proliferation responses contributed most to the development of severe infections. In addition, all participants with absent T-cell receptor excision circles developed severe infections. Opportunistic infections occurred only in children with leaky SCID-level lymphopenia. Shorter birth length and a combination of laboratory abnormalities can predict the development of severe infections in children with CHH.

Severe combined immunodeficiency with a homozygous c.464T&gt;C (p.Leu155Pro) mutation in the RAG2 gene in a country without neonatal screening: Report of two clinical cases

Introduction: Severe combined immunodeficiency (SCID) is a disorder belonging to the broad spectrum of inborn errors of immunity (IEI), characterized by a predisposition to develop recurrent, severe, difficult-to-treat opportunistic infections. This disorder is associated with abnormalities in genes of the adaptive immune system and should be suspected in patients with infections during the first months of life, mainly due to fungal pathogens and intracellular agents. Case Presentation: Two cases of SCID diagnosed at the General Hospital with Specialties "Juan María de Salvatierra” in Baja California Sur (BCS), México in 2022 are described. The first patient presented persistent candidiasis stomatitis with no response to oral treatment. The second patient presented mycotic dermatitis and oropharyngeal candidiasis compatible with Candida albicans and multiple episodes of multi-treated airway infection with partial remission. The diagnosis was completed with a genetic panel that showed a mutation in RAG2 homozygous variant c.464T&gt;C (p.Leu155Pro) in both patients. Conclusions: SCID should be considered in patients with recurrent and difficult-to-treat infectious diseases in the first months of life. For its timely detection, implementing neonatal screening quantifying of T-cell receptor excision circles (TRECs) is recommended. This method is not available in Mexico, so it has been relevant to have physicians trained in the timely clinical identification of the disease. Thanks to the prompt referral of both patients to our hospital, we could treat the comorbidities associated with SCID, provide a specific molecular diagnosis, and transfer the patients to a hospital specialized in bone marrow transplantation for definitive treatment.

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.

Newborn screening for severe combined immunodeficiency and inborn errors of immunity.

Severe combined immune deficiency (SCID) is the most devastating genetic disease of the immune system with an unfavorable outcome unless diagnosed early in life. Newborn screening (NBS) programs play a crucial role in facilitating early diagnoses and timely interventions for affected infants. SCID marked the pioneering inborn error of immunity (IEI) to undergo NBS, a milestone achieved 15 years ago through the enumeration of T-cell receptor excision circles (TRECs) extracted from Guthrie cards. This breakthrough has revolutionized our approach to SCID, enabling not only presymptomatic identification and prompt treatments (including hematopoietic stem cell transplantation), but also enhancing our comprehension of the global epidemiology of SCID. NBS is continuing to evolve with the advent of novel diagnostic technologies and treatments. Following the successful implementation of SCID-NBS programs, a call for the early identification of additional IEIs is the next step, encompassing a broader spectrum of IEIs, facilitating early diagnoses, and preventing morbidity and mortality.

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.

Complete fatty degeneration of thymus associates with male sex, obesity and loss of circulating naïve CD8+ T cells in a Swedish middle-aged population

BackgroundFatty degeneration of thymus (or thymus involution) has long been considered a normal ageing process. However, there is emerging evidence that thymic involution is linked to T cell aging, chronic inflammation and increased morbidity. Other factors, aside from chronological age, have been proposed to affect the involution rate. In the present study, we investigated the imaging characteristics of thymus on computed tomography (CT) in a Swedish middle-aged population. The major aims were to establish the prevalence of fatty degeneration of thymus and to determine its associations with demographic, lifestyle and clinical factors, as well as inflammation, T cell differentiation and thymic output.ResultsIn total, 1 048 randomly invited individuals (aged 50–64 years, 49% females) were included and thoroughly characterized. CT evaluation of thymus included measurements of attenuation, size and a 4-point scoring system, with scale 0–3 based on the ratio of fat and soft tissue. A majority, 615 (59%) showed complete fatty degeneration, 259 (25%) predominantly fatty attenuation, 105 (10%) half fatty and half soft-tissue attenuation, while 69 (6.6%) presented with a solid thymic gland with predominantly soft-tissue attenuation. Age, male sex, high BMI, abdominal obesity and low dietary intake of fiber were independently associated with complete fatty degeneration of thymus. Also, fatty degeneration of thymus as well as low CT attenuation values were independently related to lower proportion of naïve CD8+ T cells, which in turn was related to lower thymic output, assessed by T-cell receptor excision circle (TREC) levels.ConclusionAmong Swedish middle-aged subjects, nearly two-thirds showed complete fatty degeneration of thymus on CT. This was linked to depletion of naïve CD8+ T cells indicating that CT scans of thymus might be used to estimate immunological aging. Furthermore, our findings support the intriguing concept that obesity as well as low fiber intake contribute to immunological aging, thereby raising the possibility of preventive strategies.

Postnatal corticosteroid treatment as a risk factor for false positivity in severe combined immunodeficiency newborn screening.

From 2011, 37 children were referred to a hospital due to low levels of T cell receptor excision circles (TRECs) from newborn screening. Among them, three children were immunologically characterized and followed up to show that postnatal corticosteroid usage may be among the causes of false positivity in TRECs screening.