Newborn screening for severe combined immunodeficiency; the Wisconsin experience (2008-2011).

Severe combined immunodeficiency is a life-threatening primary immune deficiency characterized by low numbers of naïve T cells. Early diagnosis and treatment of this disease decreases mortality. In 2008, Wisconsin began newborn screening of infants for severe combined immunodeficiency and other forms of T-cell lymphopenia by the T-cell receptor excision circle assay. In total, 207,696 infants were screened. Seventy-two infants had an abnormal assay. T-cell numbers were normal in 38 infants, abnormal in 33 infants, and not performed in one infant, giving a positive predictive value for T-cell lymphopenia of any cause of 45.83% and a specificity of 99.98%. Five infants with severe combined immunodeficiency/severe T-cell lymphopenia requiring hematopoietic stem cell transplantation or other therapy were detected. In summary, the T-cell receptor excision circle assay is a sensitive and specific test to identify infants with severe combined immunodeficiency and severe T-cell lymphopenia that leads to life-saving therapies such as hematopoietic stem cell transplantation prior to the acquisition of severe infections.

Granzyme B regulates antiviral CD8+ T cell responses.

CTLs and NK cells use the perforin/granzyme cytotoxic pathway to kill virally infected cells and tumors. Human regulatory T cells also express functional granzymes and perforin and can induce autologous target cell death in vitro. Perforin-deficient mice die of excessive immune responses after viral challenges, implicating a potential role for this pathway in immune regulation. To further investigate the role of granzyme B in immune regulation in response to viral infections, we characterized the immune response in wild-type, granzyme B-deficient, and perforin-deficient mice infected with Sendai virus. Interestingly, granzyme B-deficient mice, and to a lesser extent perforin-deficient mice, exhibited a significant increase in the number of Ag-specific CD8(+) T cells in the lungs and draining lymph nodes of virally infected animals. This increase was not the result of failure in viral clearance because viral titers in granzyme B-deficient mice were similar to wild-type mice and significantly less than perforin-deficient mice. Regulatory T cells from WT mice expressed high levels of granzyme B in response to infection, and depletion of regulatory T cells from these mice resulted in an increase in the number of Ag-specific CD8(+) T cells, similar to that observed in granzyme B-deficient mice. Furthermore, granzyme B-deficient regulatory T cells displayed defective suppression of CD8(+) T cell proliferation in vitro. Taken together, these results suggest a role for granzyme B in the regulatory T cell compartment in immune regulation to viral infections.

Efficacy, safety, and pharmacokinetics of a 10% liquid immune globulin preparation (GAMMAGARD LIQUID, 10%) administered subcutaneously in subjects with primary immunodeficiency disease.

A multi-center, prospective, open-label study was conducted in primary immunodeficiency disease patients to determine the tolerability and pharmacokinetics of a 10% liquid IgG preparation administered subcutaneously. Forty-nine subjects (3-77 years old) were enrolled. Pharmacokinetic equivalence of subcutaneous treatment was achieved at a median dose of 137% of the intravenous dose, with a mean trough IgG level of 1,202 mg/dL at the end of the assessment period. The overall infection rate during subcutaneous treatment was 4.1 per subject-year. Three acute serious bacterial infections were reported, resulting in a rate of 0.067 per subject-year. A low overall rate of temporally associated adverse events (8%), and a very low rate of infusion site adverse events (2.8%), was seen at volumes up to 30 mL/site and rates ≤ 30 mL/h/site. Thus, subcutaneous replacement therapy with a 10% IgG preparation proved effective, safe and well-tolerated in our study population of subjects with primary immunodeficiency disease.

Impact of trough IgG on pneumonia incidence in primary immunodeficiency: A meta-analysis of clinical studies.

Primary immunodeficiency disease (PIDD) associated with hypogammaglobulinemia is typically treated with immunoglobulin replacement therapy. When administered as intravenous immunoglobulin (IVIG), an IgG trough occurs prior to the next replacement dose. While frequently measured, IgG trough levels required to minimize infection risk are not established. To address this question, all available studies evaluating trough IgG and pneumonia incidence in PIDD patients with hypogammaglobulinemia receiving IVIG were quantitatively combined by meta-analysis. Seventeen studies with 676 total patients and 2,127 patient-years of follow-up were included. Pneumonia incidence declined by 27% with each 100mg/dL increment in trough IgG (incidence rate ratio, 0.726; 95% confidence interval, 0.658-0.801). Pneumonia incidence with maintenance of 500 mg/dL IgG trough levels (0.113 cases per patient-year) was 5-fold that with 1000 mg/dL (0.023 cases per patient-year). This meta-analysis provides evidence that pneumonia risk can be progressively reduced by higher trough IgG levels up to at least 1000 mg/dL.

Implementing routine testing for severe combined immunodeficiency within Wisconsin's newborn screening program.

Severe combined immunodeficiency (SCID) is the result of genetic defects that impair normal T-cell development. SCID babies typically appear normal at birth, but acquire multiple life-threatening infections within a few months. Early diagnosis and treatment with a bone-marrow transplant markedly improves long-term outcomes. On January 1, 2008, the newborn screening (NBS) program in Wisconsin became the first in the world to routinely test all newborns for SCID. A realtime quantitative polymerase chain reaction assay measures T-cell receptor excision circles (TRECs), which are formed during the maturation of normal T-cells. A lack or very low number of TRECs is consistent with T-cell lymphopenia. The development and validation of the TREC assay and the results of the first year of screening have been published. This article describes the process used to add SCID to the NBS panel, the establishment of follow-up capacity, and the integration of SCID screening into routine NBS workflows. The development of this expanded NBS program is described so that other states might benefit from the processes used in Wisconsin.

Multiplexed quantitative real-time PCR to detect 22q11.2 deletion in patients with congenital heart disease.

22q11.2 Deletion syndrome (22q11.2 DS) [DiGeorge syndrome type 1 (DGS1)] occurs in ∼1:3,000 live births; 75% of children with DGS1 have severe congenital heart disease requiring early intervention. The gold standard for detection of DGS1 is fluorescence in situ hybridization (FISH) with a probe at the TUPLE1 gene. However, FISH is costly and is typically ordered in conjunction with a karyotype analysis that takes several days. Therefore, FISH is underutilized and the diagnosis of 22q11.2 DS is frequently delayed, often resulting in profound clinical consequences. Our goal was to determine whether multiplexed, quantitative real-time PCR (MQPCR) could be used to detect the haploinsufficiency characteristic of 22q11.2 DS. A retrospective blinded study was performed on 382 subjects who had undergone congenital heart surgery. MQPCR was performed with a probe localized to the TBX1 gene on human chromosome 22, a gene typically deleted in 22q11.2 DS. Cycle threshold (C(t)) was used to calculate the relative gene copy number (rGCN). Confirmation analysis was performed with the Affymetrix 6.0 Genome-Wide SNP Array. With MQPCR, 361 subjects were identified as nondeleted with an rGCN near 1.0 and 21 subjects were identified as deleted with an rGCN near 0.5, indicative of a hemizygous deletion. The sensitivity (21/21) and specificity (361/361) of MQPCR to detect 22q11.2 deletions was 100% at an rGCN value drawn at 0.7. One of 21 subjects with a prior clinical (not genetically confirmed) DGS1 diagnosis was found not to carry the deletion, while another subject, not previously identified as DGS1, was detected as deleted and subsequently confirmed via microarray. The MQPCR assay is a rapid, inexpensive, sensitive, and specific assay that can be used to screen for 22q11.2 deletion syndrome. The assay is readily adaptable to high throughput.

Immunosuppressive effects in infants treated with corticosteroids for infantile hemangiomas.

BACKGROUND: Infantile hemangiomas are the most common benign tumors of infancy. Up to 38% of hemangiomas require treatment with systemic medications because of complications. Corticosteroids have been the mainstay for treating such hemangiomas. However, prospective studies evaluating their immunosuppressive effects in infants with hemangiomas are lacking. OBSERVATIONS: Sixteen patients who presented to the Birthmark and Vascular Anomalies Center at the Children's Hospital of Wisconsin from November 1, 2006, through February 28, 2008, were enrolled in the study. A significant reduction in the numbers of all B- and T-lymphocyte subpopulations was observed after corticosteroid administration. CD19(+) B lymphocytes and CD4(+) T cells were significantly reduced by 8 weeks of corticosteroid therapy, whereas CD8(+) T cells were reduced at week 16 compared with baseline. Immune function was also affected because 13 and 5 patients had protective diphtheria titers and tetanus titers, respectively, 3 months after discontinuation of corticosteroid therapy compared with baseline. CONCLUSIONS: These results demonstrate that corticosteroids measurably affect both lymphocyte cell numbers and function in this patient population. Prophylaxis with the combination of trimethoprim and sulfamethoxazole should be considered in infants treated with corticosteroids for infantile hemangiomas. We also recommend that tetanus and diphtheria antibodies be checked in patients who receive oral corticosteroids during the immunization period and that additional immunization be administered if the titers are not protective after corticosteroid therapy.

Statewide newborn screening for severe T-cell lymphopenia.

CONTEXT: A newborn blood screening (NBS) test that could identify infants with a profound deficiency of T cells may result in a reduction in mortality. OBJECTIVE: To determine if quantitating T-cell receptor excision circles (TRECs) using real-time quantitative polymerase chain reaction on DNA extracted from dried blood spots on NBS cards can detect infants with T-cell lymphopenia in a statewide program. DESIGN, SETTING, AND PARTICIPANTS: Between January 1 and December 31, 2008, the Wisconsin State Laboratory of Hygiene screened all infants born in Wisconsin for T-cell lymphopenia by quantitating the number of TRECs contained in a 3.2-mm punch (approximately 3 microL of whole blood) of the NBS card. Flow cytometry to enumerate the number of T cells was performed on full-term infants and preterm infants when they reached the equivalent of at least 37 weeks' gestation with TREC values of less than 25/microL. Infants with T-cell lymphopenia were evaluated by a clinical immunologist. MAIN OUTCOME MEASURES: The number of infants with TREC values of less than 25/microL with T-cell lymphopenia confirmed by flow cytometry. RESULTS: Exactly 71,000 infants were screened by the TREC assay. Seventeen infants aged at least 37 weeks' gestation had at least 1 abnormal TREC assay (TREC values < 25/microL), 11 of whom had samples analyzed by flow cytometry to enumerate T cells. Eight infants demonstrated T-cell lymphopenia. The causes of the T-cell lymphopenia included DiGeorge syndrome (n = 2), idiopathic T-cell lymphopenia (n = 2), extravascular extravasation of lymphocytes (n = 3), and a Rac2 mutation (n = 1). The infant with the Rac2 mutation underwent successful cord blood transplantation. CONCLUSION: In a statewide screening program, use of the TREC assay performed on NBS cards was able to identify infants with T-cell lymphopenia.

Development of a routine newborn screening protocol for severe combined immunodeficiency.

BACKGROUND: Severe combined immunodeficiency (SCID) is characterized by the absence of functional T cells and B cells. Without early diagnosis and treatment, infants with SCID die from severe infections within the first year of life. OBJECTIVE: To determined the feasibility of detecting SCID in newborns by quantitating T-cell receptor excision circles (TRECs) from dried blood spots (DBSs) on newborn screening (NBS) cards. METHODS: DNA was extracted from DBSs on deidentified NBS cards, and real-time quantitative PCR (RT-qPCR) was used to determine the number of TRECs. Positive controls consisted of DBS from a 1-week-old T(-)B(-)NK(+) patient with SCID and whole blood specimens selectively depleted of naive T cells. RESULTS: The mean and median numbers of TRECs from 5766 deidentified DBSs were 827 and 708, respectively, per 3.2-mm punch ( approximately 3 muL whole blood). Ten samples failed to amplify TRECs on initial analysis; all but 1 demonstrated normal TRECs and beta-actin amplification on retesting. No TRECs were detected in either the SCID or naive T-cell-depleted samples, despite the presence of normal levels of beta-actin. CONCLUSIONS: The use of RT-qPCR to quantitate TRECs from DNA extracted from newborn DBSs is a highly sensitive and specific screening test for SCID. This assay is currently being used in Wisconsin for routine screening infants for SCID.

Cellular and genetic basis of primary immune deficiencies.

Knowledge of the genetic mutations of primary immune deficiency syndromes has grown significantly over the last 30 years. In this article the authors present an overview of the clinical aspects, laboratory evaluation, and genetic defects of primary immunodeficiencies, with an emphasis on the pathophysiology of the known molecular defects. This article is designed to give the primary pediatrician a general knowledge of this rapidly expanding field.

Hemophagocytic lymphohistiocytosis: diagnosis, pathophysiology, treatment, and future perspectives.

Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease in which the immune system becomes overactive due to its inability to effectively respond to infections and/or shut down the immune response to such infections. The discovery of genetic defects in the secretory pathway of natural killer (NK) cells and cytotoxic T cells in some patients with this disease has raised important questions of the role of cytotoxic cells in the control of infections and in immune regulation. This review will give a brief overview of the clinical presentation and accepted treatment of HLH. Furthermore, it will give an in-depth review into the known genetic defects and current knowledge of the pathophysiology of this disorder, and will highlight recent evidence suggesting that cytotoxic defects in CD4+ T regulatory cells may contribute to the pathogenesis of HLH.

Allergic dysregulation and hyperimmunoglobulinemia E in Foxp3 mutant mice.

BACKGROUND: Regulatory T cells have been proposed to play an important role in regulating allergic inflammation. The transcription factor Foxp3 is a master switch gene that controls the development and function of natural and adaptive CD4(+)CD25(+) regulatory T (T(R)) cells. In human subjects loss-of-function Foxp3 mutations trigger lymphoproliferation, autoimmunity, and intense allergic inflammation in a disease termed immune dysregulation polyendocrinopathy enteropathy-X-linked syndrome. OBJECTIVE: We sought to examine the evolution and attributes of allergic inflammation in mice with a targeted loss-of-function mutation in the murine Foxp3 gene that recapitulates a known disease-causing human Foxp3 mutation. METHODS: Foxp3 mutant mice were generated by means of knock-in mutagenesis and were analyzed for histologic, immunologic, and hematologic abnormalities. The role of signal transducer and activator of transcription 6 (Stat6) in disease pathogenesis was analyzed by using Stat6 and Foxp3 double-mutant mice. RESULTS: Foxp3 mutant mice developed an intense multiorgan inflammatory response associated with allergic airway inflammation, a striking hyperimmunoglobulinemia E, eosinophilia, and dysregulated T(H)1 and T(H)2 cytokine production in the absence of overt T(H)2 skewing. Concurrent Stat6 deficiency reversed the hyperimmunoglobulinemia E and eosinophilia and delayed mortality, which is consistent with a pathogenic role for allergic inflammation in Foxp3 deficiency. CONCLUSION: Allergic dysregulation is a common and fundamental consequence of loss of CD4(+)CD25(+) T(R) cells caused by Foxp3 deficiency in different species. Abnormalities affecting T(R) cells might contribute to a variety of allergic diseases.

Development of hemophagocytic lymphohistiocytosis in triplets infected with HHV-8.

Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder of immune dysregulation, characterized by end-organ damage from lymphocytic infiltration and macrophage activation. All known mutations associated with the HLH occur in genes critical in the perforin-granzyme pathway. Herein, we report HLH occurring in 2 female triplet infants who also had associated human herpesvirus type 8 (HHV-8) infections. The subjects had identical novel compound-heterozygous mutations in the Perforin alleles, resulting in undetectable perforin expression and NK-cell cytotoxicity. Both infants also had evidence of infection with HHV-8. These reports are, to our knowledge, the first cases of HLH in triplets and the first reported cases of HHV-8 infection associated with HLH in non-renal transplant and non-HIV-infected subjects.

Granzyme B and the downstream granzymes C and/or F are important for cytotoxic lymphocyte functions.

Although the functions of granzyme A (GzmA) and GzmB are well-defined, a number of orphan granzymes of unknown function are also expressed in cytotoxic lymphocytes. Previously, we showed that a targeted loss-of-function mutation for GzmB was associated with reduced expression of several downstream orphan granzyme genes in the lymphokine-activated killer cell compartment. To determine whether this was caused by the retained phosphoglycerate kinase I gene promoter (PGK-neo) cassette in the GzmB gene, we retargeted the GzmB gene with a LoxP-flanked PGK-neo cassette, then removed the cassette in embryonic stem cells by transiently expressing Cre recombinase. Mice homozygous for the GzmB null mutation containing the PGK-neo cassette (GzmB-/-/+PGK-neo) displayed reduced expression of the closely linked GzmC and F genes in their MLR-derived CTLs and lymphokine-activated killer cells; removal of the PGK-neo cassette (GzmB-/-/DeltaPGK-neo) restored the expression of both genes. Cytotoxic lymphocytes derived from mice with the retained PGK-neo cassette (GzmB-/-/+PGK-neo) had a more severe cytotoxic defect than those deficient for GzmB only (GzmB-/-/DeltaPGK-neo). Similarly, GzmB-/-/+PGK-neo mice displayed a defect in the allogeneic clearance of P815 tumor cells, whereas GzmB-/-/DeltaPGK-neo mice did not. These results suggest that the retained PGK-neo cassette in the GzmB gene causes a knockdown of GzmC and F expression, and also suggest that these granzymes are relevant for the function of cytotoxic lymphocytes in vitro and in vivo.

Human T regulatory cells can use the perforin pathway to cause autologous target cell death.

Cytotoxic T lymphocytes and natural killer cells use the perforin/granzyme pathway to kill virally infected cells and tumor cells. Mutations in genes important for this pathway are associated with several human diseases. CD4(+) T regulatory (Treg) cells have emerged as important in the control of immunopathological processes. We have previously shown that human adaptive Treg cells preferentially express granzyme B and can kill allogeneic target cells in a perforin-dependent manner. Here, we demonstrate that activated human CD4(+)CD25(+) natural Treg cells express granzyme A but very little granzyme B. Furthermore, both Treg subtypes display perforin-dependent cytotoxicity against autologous target cells, including activated CD4(+) and CD8(+) T cells, CD14(+) monocytes, and both immature and mature dendritic cells. This cytotoxicity is dependent on CD18 adhesive interactions but is independent of Fas/FasL. Our findings suggest that the perforin/granzyme pathway is one of the mechanisms that Treg cells can use to control immune responses.

Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the perforin/granzyme pathway as a major mechanism to kill pathogen-containing cells and tumor cells.(1,2) Dysregulation of this pathway results in several human diseases, such as hemophagocytic lymphohistiocytosis. Here we characterize the single-cell expression pattern of granzymes A and B in human lymphocytes using a flow cytometry-based assay. We demonstrate that most circulating CD56(+)8(-) NK cells, and approximately half of circulating CD8(+) T lymphocytes, coexpressed both granzymes A and B. In contrast, few circulating CD4(+) T lymphocytes expressed granzymes A or B. Activation of CD8(+) T lymphocytes with concanavalin A (ConA)/interleukin-2 (IL-2), and activation of CD4(+) T lymphocytes with antibodies to CD3/CD28 or CD3/CD46 (to generate T regulatory [Tr1] cells), induced substantial expression of granzyme B, but not granzyme A. Naive CD4(+)CD45RA(+) cells stimulated with antibodies to CD3/CD46 strongly expressed granzyme B, while CD3/CD28 stimulation was ineffective. Finally, we show that granzyme B-expressing CD4(+) Tr1 cells are capable of killing target cells in a perforin-dependent, but major histocompatibility complex (MHC)/T-cell receptor (TCR)-independent, manner. Our results demonstrate discordant expression of granzymes A and B in human lymphocyte subsets and T regulatory cells, which suggests that different granzymes may play unique roles in immune system responses and regulation.