Biallelic variants in ribonuclease inhibitor (RNH1), an inflammasome modulator, are associated with a distinctive subtype of acute, necrotizing encephalopathy.

PURPOSE: Mendelian etiologies for acute encephalopathies in previously healthy children are poorly understood, with the exception of RAN binding protein 2 (RANBP2)-associated acute necrotizing encephalopathy subtype 1 (ANE1). We provide clinical, genetic, and neuroradiological evidence that biallelic variants in ribonuclease inhibitor (RNH1) confer susceptibility to a distinctive ANE subtype. METHODS: This study aimed to evaluate clinical data, neuroradiological studies, genomic sequencing, and protein immunoblotting results in 8 children from 4 families who experienced acute febrile encephalopathy. RESULTS: All 8 healthy children became acutely encephalopathic during a viral/febrile illness and received a variety of immune modulation treatments. Long-term outcomes varied from death to severe neurologic deficits to normal outcomes. The neuroradiological findings overlapped with ANE but had distinguishing features. All affected children had biallelic predicted damaging variants in RNH1: a subset that was studied had undetectable RNH1 protein. Incomplete penetrance of the RNH1 variants was evident in 1 family. CONCLUSION: Biallelic variants in RNH1 confer susceptibility to a subtype of ANE (ANE2) in previously healthy children. Intensive immunological treatments may alter outcomes. Genomic sequencing in children with unexplained acute febrile encephalopathy can detect underlying genetic etiologies, such as RNH1, and improve outcomes in the probands and at-risk siblings.

Clinical Practice Guidelines for the Immunological Management of Chromosome 22q11.2 Deletion Syndrome and Other Defects in Thymic Development.

Current practices vary widely regarding the immunological work-up and management of patients affected with defects in thymic development (DTD), which include chromosome 22q11.2 microdeletion syndrome (22q11.2del) and other causes of DiGeorge syndrome (DGS) and coloboma, heart defect, atresia choanae, retardation of growth and development, genital hypoplasia, ear anomalies/deafness (CHARGE) syndrome. Practice variations affect the initial and subsequent assessment of immune function, the terminology used to describe the condition and immune status, the accepted criteria for recommending live vaccines, and how often follow-up is needed based on the degree of immune compromise. The lack of consensus and widely varying practices highlight the need to establish updated immunological clinical practice guidelines. These guideline recommendations provide a comprehensive review for immunologists and other clinicians who manage immune aspects of this group of disorders.

Introducing thymus for promoting transplantation tolerance.

Establishing tolerance remains a central, if elusive, goal of transplantation. In solid-organ transplantation, one strategy for inducing tolerance has been cotransplantation of various forms of thymic tissue along with another organ. As one of the biological foundations of central tolerance, thymic tissue carries with it the ability to induce tolerance to any other organ or tissue from the same donor (or another donor tissue-matched to the thymic tissue) if successfully transplanted. In this review, we outline the history of this approach as well as work to date on its application in organ transplantation, concluding with future directions. We also review our experience with allogeneic processed thymus tissue for the treatment of congenital athymia, encompassing complete DiGeorge syndrome and other rare genetic disorders, and consider whether allogeneic processed thymic tissue implantation may offer a novel method for future experimentation with tolerance induction in organ transplantation.

Experience with cultured thymus tissue in 105 children.

BACKGROUND: Currently, there are no approved therapies to treat congenital athymia, a condition of immune deficiency resulting in high early mortality due to infection and immune dysregulation. Multiple syndromic conditions, such as complete DiGeorge syndrome, 22q11.2 deletion syndrome, CHARGE (coloboma, heart defects, choanal atresia, growth or mental retardation, genital hypoplasia, and ear anomalies and/or deafness) syndrome, diabetic embryopathy, other genetic variants, and FOXN1 deficiency, are associated with congenital athymia. OBJECTIVE: Our aims were to study 105 patients treated with cultured thymus tissue (CTT), and in this report, to focus on the outcomes of 95 patients with treatment-naive congenital athymia. METHODS: A total of 10 prospective, single-arm open-label studies with patient enrollment from 1993 to 2020 form the basis of this data set. Patients were tested after administration of CTT for T-cell development; all adverse events and infections were recorded. RESULTS: A total of 105 patients were enrolled and received CTT (the full analysis set). Of those patients, 10 had diagnoses other than congenital athymia and/or received prior treatments. Of those 105 patients, 95 patients with treatment-naive congenital athymia were included in the efficacy analysis set (EAS). The Kaplan-Meier estimated survival rates at year 1 and year 2 after administration of CTT in the EAS were 77% (95% CI = 0.670-0.844) and 76% (95% CI = 0.657-0.834), respectively. In all, 21 patients died in the first year before developing naive T cells and 1 died in the second year after receipt of CTT; 3 subsequent deaths were not related to immunodeficiency. A few patients developed alopecia, autoimmune hepatitis, psoriasis, and psoriatic arthritis after year 1. The rates of infections, autologous graft-versus-host-disease manifestations, and autoimmune cytopenias all decreased approximately 1 year after administration of CTT. CONCLUSION: Treatment with CTT led to development of naive T cells with a 1-year survival rate of 77% and a median follow-up time of 7.6 years. Immune reconstitution sufficient to prevent infections and support survival typically develops 6 to12 months after administration of CTT.

Care of Children with DiGeorge Before and After Cultured Thymus Tissue Implantation.

BACKGROUND: Children with complete DiGeorge anomaly (cDGA) have congenital athymia plus a myriad of other challenging clinical conditions. The term cDGA encompasses children with congenital athymia secondary to 22q11.2DS, CHARGE syndrome (coloboma, heart defects, choanal atresia, growth or mental retardation, genital abnormalities, and ear abnormalities and/or deafness), and other genetic abnormalities. Some children have no known genetic defects. Since 1993, more than 100 children with congenital athymia have been treated with cultured thymus tissue implantation (CTTI). Naïve T cells develop approximately 6 to 12 months after CTTI. Most of the children had significant comorbidities such as heart disease, hypoparathyroidism, and infections requiring complex clinical care post cultured thymus tissue implantation (CTTI). OBJECTIVE: The purpose of this guidance is to assist multidisciplinary teams in caring for children with cDGA both before and after CTTI. METHODS: Thirty-one specialists, in addition to the authors, were asked to share their experience in caring for children with cDGA at Duke University Health System, before and after CTTI. These specialists included physicians, nurses, dentists, therapists, and dieticians. RESULTS: The goal of a multidisciplinary approach is to have children in the best possible condition for receiving CTTI and provide optimal care post CTTI through development of naïve T cells and beyond. The CTT (cultured thymus tissue) must be protected from high doses of steroids which can damage CTT. Organs must be protected from adverse effects of immunosuppression. CONCLUSION: Creating a multidisciplinary team and a detailed plan of care for children with cDGA is important for optimal outcomes.

Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants.

Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop T cells similar to those of recipient that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell-depleted Lewis rats. The rats were then given cultured thymus tissue from F1 (Lewis × Dark Agouti ) under the kidney capsule and vascularized Dark Agouti (DA) heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naive and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with LWxDA CTTI rejected the third-party BN hearts (mean survival time 10 days); controls did not. CTTI recipients produced antibody against third-party BN donor but not against the DA thymus donor, demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus, with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.

Histopathologic assessment of cultured human thymus.

The maintenance and propagation of complex mixtures of cells in vitro in the form of native organs or engineered organoids has contributed to understanding mechanisms of cell and organ development and function which can be translated into therapeutic benefits. For example, allogeneic cultured postnatal human thymus tissue has been shown to support production of naïve recipient T cells when transplanted into patients with complete DiGeorge anomaly and other genetic defects that result in congenital lack of a thymus. Patients receiving such transplants typically exhibit reversal of their immunodeficiency and normalization of their peripheral blood T cell receptor V-beta repertoire, with long-term survival. This study was designed to assess the histopathologic changes that occur in postnatal human thymus slices when cultured according to protocols used for transplanted tissues. Results showed that as thymic organ cultures progressed from days 0 through 21, slices developed increasing amounts of necrosis, increasing condensation of thymic epithelium, and decreasing numbers of residual T cells. The architecture of the thymic epithelial network remained generally well-preserved throughout the 21 days of culture, with focal expression of cytokeratin 14, a putative biomarker of thymic epithelial cells with long-term organ-repopulating potential. All organ slices derived from the same donor thymus closely resembled one another, with minor differences in size, shape, and relative content of cortex versus medulla. Similarly, slices derived from different donors showed similar histopathologic characteristics when examined at the same culture time point. Taken together, these results demonstrate that diagnostic criteria based on structural features of the tissue identifiable via hematoxylin and eosin staining and cytokeratin immunohistochemistry can be used to evaluate the quality of slices transplanted into patients with congenital athymia.

FOXN1 compound heterozygous mutations cause selective thymic hypoplasia in humans.

We report on 2 patients with compound heterozygous mutations in forkhead box N1 (FOXN1), a transcription factor essential for thymic epithelial cell (TEC) differentiation. TECs are critical for T cell development. Both patients had a presentation consistent with T-/loB+NK+ SCID, with normal hair and nails, distinct from the classic nude/SCID phenotype in individuals with autosomal-recessive FOXN1 mutations. To understand the basis of this phenotype and the effects of the mutations on FOXN1, we generated mice using CRISPR-Cas9 technology to genocopy mutations in 1 of the patients. The mice with the Foxn1 compound heterozygous mutations had thymic hypoplasia, causing a T-B+NK+ SCID phenotype, whereas the hair and nails of these mice were normal. Characterization of the functional changes due to the Foxn1 mutations revealed a 5-amino acid segment at the end of the DNA-binding domain essential for the development of TECs but not keratinocytes. The transcriptional activity of this Foxn1 mutant was partly retained, indicating a region that specifies TEC functions. Analysis of an additional 9 FOXN1 mutations identified in multiple unrelated patients revealed distinct functional consequences contingent on the impact of the mutation on the DNA-binding and transactivation domains of FOXN1.

Newborn Screening for Severe Combined Immunodeficiency and T-cell Lymphopenia in California, 2010-2017.

OBJECTIVES: Newborn screening for severe combined immunodeficiency (SCID) was instituted in California in 2010. In the ensuing 6.5 years, 3 252 156 infants in the state had DNA from dried blood spots assayed for T-cell receptor excision circles (TRECs). Abnormal TREC results were followed-up with liquid blood testing for T-cell abnormalities. We report the performance of the SCID screening program and the outcomes of infants who were identified. METHODS: Data that were reviewed and analyzed included demographics, nursery summaries, TREC and lymphocyte flow-cytometry values, and available follow-up, including clinical and genetic diagnoses, treatments, and outcomes. RESULTS: Infants with clinically significant T-cell lymphopenia (TCL) were successfully identified at a rate of 1 in 15 300 births. Of these, 50 cases of SCID, or 1 in 65 000 births (95% confidence interval 1 in 51 000-1 in 90 000) were found. Prompt treatment led to 94% survival. Infants with non-SCID TCL were also identified, diagnosed and managed, including 4 with complete DiGeorge syndrome who received thymus transplants. Although no cases of typical SCID are known to have been missed, 2 infants with delayed-onset leaky SCID had normal neonatal TREC screens but came to clinical attention at 7 and 23 months of age. CONCLUSIONS: Population-based TREC testing, although unable to detect immune defects in which T cells are present at birth, is effective for identifying SCID and clinically important TCL with high sensitivity and specificity. The experience in California supports the rapid, widespread adoption of SCID newborn screening.

Functional variants in TBX2 are associated with a syndromic cardiovascular and skeletal developmental disorder.

The 17 genes of the T-box family are transcriptional regulators that are involved in all stages of embryonic development, including craniofacial, brain, heart, skeleton and immune system. Malformation syndromes have been linked to many of the T-box genes. For example, haploinsufficiency of TBX1 is responsible for many structural malformations in DiGeorge syndrome caused by a chromosome 22q11.2 deletion. We report four individuals with an overlapping spectrum of craniofacial dysmorphisms, cardiac anomalies, skeletal malformations, immune deficiency, endocrine abnormalities and developmental impairments, reminiscent of DiGeorge syndrome, who are heterozygotes for TBX2 variants. The p.R20Q variant is shared by three affected family members in an autosomal dominant manner; the fourth unrelated individual has a de novo p.R305H mutation. Bioinformatics analyses indicate that these variants are rare and predict them to be damaging. In vitro transcriptional assays in cultured cells show that both variants result in reduced transcriptional repressor activity of TBX2. We also show that the variants result in reduced protein levels of TBX2. Heterologous over-expression studies in Drosophila demonstrate that both p.R20Q and p.R305H function as partial loss-of-function alleles. Hence, these and other data suggest that TBX2 is a novel candidate gene for a new multisystem malformation disorder.

Thymus transplantation for complete DiGeorge syndrome: European experience.

BACKGROUND: Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syndrome (cDGS). METHODS: Twelve patients with cDGS underwent transplantation with allogeneic cultured thymus. OBJECTIVE: We sought to confirm and extend the results previously obtained in a single center. RESULTS: Two patients died of pre-existing viral infections without having thymopoiesis, and 1 late death occurred from autoimmune thrombocytopenia. One infant had septic shock shortly after transplantation, resulting in graft loss and the need for a second transplant. Evidence of thymopoiesis developed from 5 to 6 months after transplantation in 10 patients. Median circulating naive CD4 counts were 44 × 106/L (range, 11-440 × 106/L) and 200 × 106/L (range, 5-310 × 106/L) at 12 and 24 months after transplantation and T-cell receptor excision circles were 2,238/106 T cells (range, 320-8,807/106 T cells) and 4,184/106 T cells (range, 1,582-24,596/106 T cells). Counts did not usually reach normal levels for age, but patients were able to clear pre-existing infections and those acquired later. At a median of 49 months (range, 22-80 months), 8 have ceased prophylactic antimicrobials, and 5 have ceased immunoglobulin replacement. Histologic confirmation of thymopoiesis was seen in 7 of 11 patients undergoing biopsy of transplanted tissue, including 5 showing full maturation through to the terminal stage of Hassall body formation. Autoimmune regulator expression was also demonstrated. Autoimmune complications were seen in 7 of 12 patients. In 2 patients early transient autoimmune hemolysis settled after treatment and did not recur. The other 5 experienced ongoing autoimmune problems, including thyroiditis (3), hemolysis (1), thrombocytopenia (4), and neutropenia (1). CONCLUSIONS: This study confirms the previous reports that thymus transplantation can reconstitute T cells in patients with cDGS but with frequent autoimmune complications in survivors.

Disseminated Mycobacterium kansasii disease in complete DiGeorge syndrome.

PURPOSE: Complete DiGeorge syndrome (cDGS) describes a subset of patients with DiGeorge syndrome that have thymic aplasia, and thus are at risk for severe opportunistic infections. Patients with cDGS and mycobacterial infection have not previously been described. We present this case to illustrate that patients with cDGS are at risk for nontuberculous mycobacterial infections and to discuss further antimicrobial prophylaxis prior to thymic transplantation. METHODS: A 13-month old male was identified as T cell deficient by the T cell receptor excision circle (TREC) assay on newborn screening, and was subsequently confirmed to have cDGS. He presented with fever and cough, and was treated for chronic aspiration pneumonia as well as Pneumocystis jirovecii infection without significant improvement. It was only after biopsy of mediastinal lymph nodes seen on CT that the diagnosis of disseminated Mycobacterium kansasii was made. We reviewed the literature regarding atypical mycobacterial infections and prophylaxis used in other immunocompromised patients, as well as the current data regarding cDGS detection through TREC newborn screening. RESULTS: Multiple cases of cDGS have been diagnosed via TREC newborn screening, however this is the first patient with cDGS and disseminated mycobacterial infection to be reported in literature. Thymic transplantation is the definitive treatment of choice for cDGS. Prophylaxis with either clarithromycin or azithromycin has been shown to reduce mycobacterial infections in children with advanced human immunodeficiency virus infection. CONCLUSIONS: Children with cDGS should receive thymic transplantion as soon as possible, but prior to this are at risk for nontuberculous mycobacterial infections. Severe, opportunistic infections may require invasive testing for diagnosis in patients with cDGS. Antimicrobial prophylaxis should be considered to prevent disseminated mycobacterial infection in these patients.

Clinical course and outcome predictors of critically ill infants with complete DiGeorge anomaly following thymus transplantation.

OBJECTIVES: To identify risk factors for PICU admission and mortality of infants with complete DiGeorge anomaly treated with thymus transplantation. We hypothesized that age at transplantation and the presence of congenital heart disease would be risk factors for emergent PICU admission, and these factors plus development of septicemia would increase morbidity and mortality. DESIGN: Retrospective review. SETTING: Academic medical-surgical PICU. PATIENTS: All infants with complete DiGeorge anomaly treated with thymus transplantation between January 1, 1993, and July 1, 2010. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Consent was obtained from 71 infants with complete DiGeorge anomaly for thymus transplantation, and 59 infants were transplanted. Median age at transplantation was 5.0 months (range, 1.1-22.1 mo). After transplantation, 12 of 59 infants (20%) required 25 emergent PICU admissions. Seven of 12 infants (58%) survived to PICU discharge with six surviving 6 months posttransplantation. Forty-two of 59 infants (71%) transplanted had congenital heart disease, and 9 of 12 (75%) who were admitted to the PICU had congenital heart disease. In 15 of 25 admissions (60%), intubation and mechanical ventilation were necessary. There was no difference between median ventilation-free days between infants with and without congenital heart disease (33 d vs 23 d, p = 0.544). There was also no correlation between ventilation-free days and age of transplantation (R, 0.17; p = 0.423). Age at transplantation and the presence of congenital heart disease were not associated with risk for PICU admission (odds ratio, 0.95; 95% CI, 0.78-1.15 and odds ratio, 1.27; 95% CI, 0.30-5.49, respectively) or PICU mortality (odds ratio, 0.98; 95% CI, 0.73-1.31 and odds ratio, 0.40; 95% CI, 0.15-1.07, respectively). CONCLUSIONS: Most transplanted infants did not require emergent PICU admission. Age at transplantation and the presence of congenital heart disease were not associated with PICU admission or mortality.

Complete thymectomy in adult rats with non-invasive endotracheal intubation.

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of naïve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3- T cells in complete DiGeorge anomaly.

The development of T cells with a regulatory phenotype after thymus transplantation has not been examined previously in complete DiGeorge anomaly (cDGA). Seven athymic infants with cDGA and non-maternal pretransplantation T cell clones were assessed. Pretransplantation forkhead box protein 3 (Foxp3)(+) T cells were detected in five of the subjects. Two subjects were studied in greater depth. T cell receptor variable ß chain (TCR-Vß) expression was assessed by flow cytometry. In both subjects, pretransplantation FoxP3(+) and total CD4(+) T cells showed restricted TCR-Vß expression. The development of naive T cells and diverse CD4(+) TCR-Vß repertoires following thymic transplantation indicated successful thymopoiesis from the thymic tissue grafts. Infants with atypical cDGA develop rashes and autoimmune phenomena before transplantation, requiring treatment with immunosuppression, which was discontinued successfully subsequent to the observed thymopoiesis. Post-transplantation, diverse TCR-Vß family expression was also observed in FoxP3(+) CD4(+) T cells. Interestingly, the percentages of each of the TCR-Vß families expressed on FoxP3(+) and total CD4(+) T cells differed significantly between these T lymphocyte subpopulations before transplantation. By 16 months post-transplantation, however, the percentages of expression of each TCR-Vß family became significantly similar between FoxP3(+) and total CD4(+) T cells. Sequencing of TCRBV DNA confirmed the presence of clonally amplified pretransplantation FoxP3(+) and FoxP3(-) T cells. After thymus transplantation, increased polyclonality was observed for both FoxP3(+) and FoxP3(-) cells, and pretransplantation FoxP3(+) and FoxP3(-) clonotypes essentially disappeared. Thus, post-transplantation thymic function was associated with the development of a diverse repertoire of FoxP3(+) T cells in cDGA, corresponding with immunological and clinical recovery.

Diagnosis of 22q11.2 deletion syndrome and artemis deficiency in two children with T-B-NK+ immunodeficiency.

Two infants are described who presented with 22q11.2 deletion and a T(-)B(-)NK(+) immune phenotype. For both infants, the initial diagnosis was athymia secondary to complete DiGeorge anomaly. The first infant underwent thymus transplantation but 6 months after transplantation had circulating thymus donor T cells; the patient did not develop recipient naïve T cells. Genetic analyses revealed that both patients had Artemis deficiency, a rare form of severe combined immunodeficiency (SCID). Both infants have subsequently undergone bone marrow transplantation. These cases illustrate the importance and paradox of differentiating SCID from complete DiGeorge anomaly because hematopoietic stem cell transplantation (HSCT) is the preferred treatment for SCID but is ineffective for complete DiGeorge anomaly. However, if the thymus is completely absent, donor stem cells from a HSCT would not be able to be educated.