Lymphocyte Subgroups and KREC Numbers in Common Variable Immunodeficiency: A Single Center Study.

Common variable immunodeficiency (CVID) results in defective B cell differentiation and impaired antibody production and is the most common symptomatic primary immunodeficiency. Our aim was to evaluate the correlation among B cell subgroups, κ-deleting recombination excision circle (KREC) copy numbers, and clinical and immunological data of the patients with CVID, and evaluate the patients according to classifications currently available to define the role of KREC copy numbers in the diagnosis of CVID. KREC analysis was performed using a quantitative real-time polymerase chain reaction assay, and B cell subgroups were measured by flow cytometry. The median age of the patients (n = 30) was 25 (6-69) years. Parental consanguinity ratio was 33%. The median age at diagnosis was 15 (4-59), and follow-up period was 6 (1-37) years. CD19+ and CD4+ cell counts at the time of diagnosis were low in 66.7% and 46.7% of the patients, respectively. CD19+ cell counts were positively correlated with KREC copy numbers in patients and healthy controls. CD19+ cell counts and KREC copy numbers were significantly reduced in CVID patients compared to healthy controls as expected. KRECs are quantitative markers for B cell defects. We found low CD4+ cell numbers, recent thymic emigrants, and lymphopenia in some of the patients at diagnosis, which reminds the heterogeneity of CVID's etiology. In this study, a positive correlation was shown between CD19+ cell counts and KREC copy numbers. Low KREC copy numbers indicated B cell deficiency; however, high KREC copy numbers were not sufficient to rule out CVID.

Immune profiling of a patient with alemtuzumab-associated progressive multifocal leukoencephalopathy.

A 31-year-old woman affected by multiple sclerosis (MS) experienced generalized tonic-clonic seizures 2 months after the second alemtuzumab cycle. Positive JC virus (JCV)-DNA in cerebrospinal fluid (CSF) and lesion iconography at magnetic resonance imaging (MRI) were suggestive of progressive multifocal leukoencephalopathy (PML). After 1 month, during full-blown immune reconstitution inflammatory syndrome, JCV-DNA became negative and symptoms gradually improved. New T- and B-cell output and T- and B-cell diversity were low and lymphocytes poorly responded to stimulation. This is the first case of an alemtuzumab-treated patient with clinical symptoms and radiological features compatible with PML. The lack of large T- and B-cell diversity, necessary for JCV recognition, is likely to have concurred to PML insurgence.

Immune recovery after in vivo T-cell depletion myeloablative conditioning hematopoietic stem cell transplantation in severe beta-thalassemia children.

BACKGROUND: The clinical outcome of hematopoietic stem cell transplantation (HSCT) in those with severe beta-thalassemia (ß-TM) is closely related to post-transplantation immune reconstitution (IR). However, the data on the IR in these settings are scarce. METHODS: A prospective analysis of the clinical outcome and IR in 47 children with severe ß-TM who underwent in vivo T-cell depletion myeloablative conditioning and matched sibling donor HSCT was performed. Immune reconstitution, including immune cell subset counts, as well as the generation of new T and B cells assays after HSCT, was measured. RESULTS: In the first year after HSCT, bacterial infections and cytomegalovirus (CMV) reactivation were observed in 70.2% and 36.2% of the patients, respectively. In the same period, poor CD4+ T-cell recovery was observed. The B cells recovered within 6 months. Natural killer (NK) cells recovered as early as 1 month, but their function was defective. Cord blood and bone marrow (CB + BM) group had slower T-cell recovery, and higher B cells and NK cells in comparison with peripheral blood and bone marrow (PB + BM) group. CONCLUSIONS: The high incidence of infection within 1 year after in vivo T-cell depletion myeloablative conditioning HSCT in severe ß-TM was consistent with poor IR.

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

BACKGROUND: A 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. METHODS: DNA 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. RESULTS: The 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. CONCLUSIONS: Our 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.

Prospective neonatal screening for severe T- and B-lymphocyte deficiencies in Seville.

BACKGROUND: Early diagnosis of primary immunodeficiency such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) improves outcome of affected children. T-cell-receptor-excision circles (TRECs) and kappa-deleting-recombination-excision circles (KRECs) determination from dried blood spots (DBS) identify neonates with severe T- and/or B-lymphopenia. No prospective data exist of the impact of gestational age (GA) and birth weight (BW) on TRECs and KRECs values. METHODS: TRECs and KRECs determination using triplex RT-PCR (TRECS-KRECS-ß-actin-Assay) from prospectively collected DBS between 02/2014 and 02/2015 in three hospitals in Seville, Spain. Cut-off levels were TRECs < 6/punch, KRECs < 4/punch and -ß-actin>700/punch. Internal (SCID, XLA, ataxia telangiectasia) and external controls (NBS quality assurance program, CDC) were included. RESULTS: A total of 5160 DBS were tested. Re-punch was needed in 77 samples (1.5%) due to insufficient ß-actin (<700 copies/punch). Pre-term neonates (GA<37 weeks) and neonates with a BW<2500 g showed significantly lower TRECs and KRECs levels (p < 0.001). Due to repeat positive results five neonates were re-called (<0.1%): Fatal chromosomopathy (n = 1; TRECs 1/KRECs 4); extreme pre-maturity (n = 2; TRECs 0/KRECs 0 and TRECs 1/KRECs 20 copies/punch); neonates born to mothers receiving azathioprine during pregnancy (n = 2; TRECs 92/KRECs 1 and TRECs 154/KRECs 3 copies/punch). All internal and external controls were correctly identified. CONCLUSIONS: TRECS-KRECS-ß-actin-Assay correctly identifies T- and B-cell lymphopenias. Pre-maturity and low BW is associated with lower TREC and KREC levels. Extreme pre-maturity and maternal immune suppressive therapy may be a cause for false positive results of TRECs and KRECs values, respectively. To reduce the rate of insufficient samples, DBS extraction and storage need to be improved.

Newly produced T and B lymphocytes and T-cell receptor repertoire diversity are reduced in peripheral blood of fingolimod-treated multiple sclerosis patients.

BACKGROUND: Fingolimod inhibits lymphocyte egress from lymphoid tissues, thus altering the composition of the peripheral lymphocyte pool of multiple sclerosis patients. OBJECTIVE: The objective of this paper is to evaluate whether fingolimod determines a decrease of newly produced T- and B-lymphocytes in the blood and a reduction in the T-cell receptor repertoire diversity that may affect immune surveillance. METHODS: Blood samples were obtained from multiple sclerosis patients before fingolimod therapy initiation and then after six and 12 months. Newly produced T and B lymphocytes were measured by quantifying T-cell receptor excision circles and K-deleting recombination excision circles by real-time PCR, while recent thymic emigrants, naive CD8(+) lymphocytes, immature and naive B cells were determined by immune phenotyping. T-cell receptor repertoire was analyzed by complementarity determining region 3 spectratyping. RESULTS: Newly produced T and B lymphocytes were significantly reduced in peripheral blood of fingolimod-treated patients. The decrease was particularly evident in the T-cell compartment. T-cell repertoire restrictions, already present before therapy, significantly increased after 12 months of treatment. CONCLUSIONS: These results do not have direct clinical implications but they may be useful for further understanding the mode of action of this immunotherapy for multiple sclerosis patients.

Clinical relevance of thymic and bone marrow outputs in multiple sclerosis patients treated with alemtuzumab.

Thymic and bone marrow outputs were evaluated in 13 sequential samples of 68 multiple sclerosis patients who initiated alemtuzumab and were clinically followed for 48 months. Three months after alemtuzumab infusions, the levels of new T lymphocytes were significantly reduced, but progressively increased reaching the highest values at 36 months, indicating the remarkable capacity of thymic function recovery. Newly produced B cells exceeded baseline levels as early as 3 months after alemtuzumab initiation. Heterogeneous patterns of new T- and B-cell recovery were identified, but without associations with age, sex, previous therapies, development of secondary autoimmunity or infections, and disease re-emergence. Trial registration version 2.0-27/01/2016.

Early ART in Acute HIV-1 Infection: Impact on the B-Cell Compartment.

HIV-1 infection induces B cell defects, not fully recovered upon antiretroviral therapy (ART). Acute infection and the early start of ART provide unique settings to address the impact of HIV on the B cell compartment. We took advantage of a cohort of 21 seroconverters, grouped according to the presence of severe manifestations likely mediated by antibodies or immune complexes, such as Guillain-Barré syndrome and autoimmune thrombocytopenic purpura, with a follow-up of 8 weeks upon effective ART. We combined B and T cell phenotyping with serum immunoglobulin level measurement and quantification of sj-KRECs and ΔB to estimate bone marrow output and peripheral proliferative history of B cells, respectively. We observed marked B cell disturbances, notably a significant expansion of cells expressing low levels of CD21, in parallel with markers of both impaired bone marrow output and increased peripheral B cell proliferation. This B cell dysregulation is likely to contribute to the severe immune-mediated conditions, as attested by the higher serum IgG and the reduced levels of sj-KRECs with increased ΔB in these individuals as compared to those patients with mild disease. Nevertheless, upon starting ART, the dynamic of B cell recovery was not distinct in the two groups, featuring both persistent alterations by week 8. Overall, we showed for the first time that acute HIV-1 infection is associated with decreased bone marrow B cell output assessed by sj-KRECs. Our study emphasizes the need to intervene in both bone marrow and peripheral responses to facilitate B cell recovery during acute HIV-1 infection.

Newborn Screening for Primary Immunodeficiencies: The Gaps, Challenges, and Outlook for Developing Countries.

Primary immunodeficiency diseases (PIDs) are genetically inherited diseases characterized by an increased susceptibility to infections, autoimmunity, lymphoproliferation, and malignancies. PIDs are under-diagnosed and the registered cases and reported prevalence are far below the estimated numbers especially in countries with large population and high consanguinity rates. Delays in diagnosis yield major morbidities and mortalities with resultant increased economic burden. Newborn screening using TRECs and KRECs, currently being implemented in some countries, is aimed through early diagnosis, to overcome the delays in the diagnosis and hence the poor outcome of some of the severe PIDs. However, the limited resources in developing countries challenges the implementation of newborn PID screening programs. There are considerable gaps in our knowledge that must be bridged. Setting the norms of TRECs and KRECs for each country is needed. Furthermore, some PIDs that might present in the neonatal period could not be detected by the current screening programs, and their diagnosis requires clinical expertise. Not to mention, local guidelines for the management of patients diagnosed by NBS should be set forth. Also, in the absence of NBS, clinicians should be aware of the early manifestations of PID. All these mandate conducting studies genuine to each country, developing programs for raising public awareness and clinical training of physicians to attain the required immunological skills.

Naive B Cell Output in HIV-Infected and HIV-Uninfected Children.

In this study, we aimed to quantify KREC (kappa-deleting recombination excision circle) levels and naive B cell output in healthy HIV-uninfected children, compared with HIV-infected South African children, before and after starting ART (antiretroviral therapy). Samples were acquired from a Child Wellness Clinic (n = 288 HIV-uninfected South African children, 2 weeks-12 years) and the Children with HIV Early Antiretroviral Therapy (CHER) trial (n = 153 HIV-infected South African children, 7 weeks-8 years). Naive B cell output was estimated using a mathematical model combining KREC levels to reflect B cell emigration into the circulation, flow cytometry measures of naive unswitched B cells to quantify total body naive B cells, and their rates of proliferation using the intracellular marker Ki67. Naive B cell output increases from birth to 1 year, followed by a decline and plateau into late childhood. HIV-infected children on or off ART had higher naive B cell outputs than their uninfected counterparts (p = .01 and p = .04). This is the first study to present reference ranges for measurements of KRECs and naive B cell output in healthy and HIV-infected children. Comparison between HIV-uninfected healthy children and HIV-infected children suggests that HIV may increase naive B cell output. Further work is required to fully understand the mechanisms involved and clinical value of measuring naive B cell output in children.

Immune reconstitution after HSCT in SCID-a cohort of conditioned and unconditioned patients.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the effective mean of immune restoration in severe combined immunodefiency (SCID). Usually, HSCT without cytoreductive conditioning is attempted. Nevertheless, conditioning procedures are still preferred in a subset of patients. Herein, we describe the immunological outcome in a cohort of conditioned and unconditioned patients, from diagnosis, through transplantation, to follow-up. This retrospective study was conducted on 17 patients with SCID (10 conditioned, 7 unconditioned) who later underwent HSCT. Immune reconstitution was assessed in the post-transplant year by quantification of T cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs), among additional laboratory and clinical evaluations. Unconditioned patients were diagnosed and transplanted earlier. TREC and KREC quantification showed a gradual increase in both groups, with higher levels in the conditioned group. Engraftment percentages differed drastically between groups, favoring the conditioned group. Unconditioned patients were significantly more dependent on intravenous immunoglobulins (IVIGs). One patient from each group succumbed to disease complications. Conditioning demonstrated superior laboratorial outcomes. Patients with unique characteristics (i.e., consanguinity, Bacillus Calmette-Guérin vaccination, impaired access to IVIG) may require personalized considerations. The effort to implement secondary prevention of SCID with newborn screening should continue.

A call to include severe combined immunodeficiency in newborn screening program.

Quantification of the T cell receptor excision circles (TRECs) has recently emerged as a useful non-invasive clinical and research tool to investigate thymic activity. It allows the identification of T cell production by the thymus. Quantification of TREC copies has recently been implemented as the preferred test to screen neonates with severe combined immunodeficiency (SCID) or significant lymphopenia. Neonatal genetic screening for SCID is highly important in countries with high rates of consanguinous marriages, such as Israel, and can be used for early diagnosis, enabling prompt therapeutic intervention that will save lives and improve the outcome of these patients. TREC measurement is also applicable in clinical settings where T cell immunity is involved, including any T cell immunodeficiencies, HIV infection, the aging process, autoimmune diseases, and immune reconstitution after bone marrow transplantation. TAKE-HOME MESSAGES: Severe combined immunodeficiency, a life-threatening condition, can be detected by neonatal screening.The earlier the detection and the quicker the implementation of appropriate treatment, the greater the likelihood for improved outcome, even cure, for the affected children.TRECs and KRECs quantification are useful screening tests for severe T and B cell immunodeficiency and can be used also to evaluate every medical condition involving T and B cell immunity.

Applying T-cell receptor excision circles and immunoglobulin κ-deleting recombination excision circles to patients with primary immunodeficiency diseases.

BACKGROUND: Biomarkers of T-cell receptor excision circles (TRECs) and immunoglobulin κ-deleting recombination excision circles (KRECs) reflect naïve T and B cell emigrants. This study assessed the biomarkers in patients with primary immunodeficiency diseases (PIDs) to determine the lymphocyte output disturbance and the correlation to lymphocytes. METHODS: A standard plasmid was constructed to calculate TRECs and KRECs in 250 ng genomic DNA from whole blood of PIDs patients. These were correlated to naïve and memory lymphocytes for further classification and adequate treatment. RESULTS: In 69 studied patients, the low TRECs mainly included those with severe combined T and B immunodeficiency (SCID, 7/8), combined immunodeficiency (CID, 4/4), and common variable immunodeficiency (CVID, 6/7). The diminished KRECs was in SCID (4/8), CID (4/4), CVID (7/7), Bruton's tyrosine kinase mutation (Btk, 3/4), anti-B cell deletion (by anti-CD20 antibody in 1), and Behçet syndrome under steroid treatment (1). The TRECs and KRECs positively correlated to absolute naïve T (CD4 + CD45RA+) and naïve B (CD19 + CD27-), and to memory B (CD19 + CD27+) numbers, respectively. CONCLUSION: This study validates that low TRECs and KRECs values reflect low naïve T and B lymphocytes in 'combined immunodeficiencies' and in some CVID patients with the potential to develop the CID phenotype.