Sujal Ghosh

Interleukin-2-Inducible T-Cell Kinase (ITK) Deficiency - Clinical and Molecular Aspects

In patients with underlying immunodeficiency, Epstein-Barr virus (EBV) may lead to severe immune dysregulation manifesting as fatal mononucleosis, lymphoma, lymphoproliferative disease (LPD), lymphomatoid granulomatosis, hemophagocytic lymphohistiocytosis (HLH) and dysgammaglobulinemia. Several newly discovered primary immunodeficiencies (STK4, CD27, MAGT1, CORO1A) have been described in recent years; our group and collaborators were able to reveal the pathogenicity of mutations in the Interleukin-2-inducible T-cell Kinase (ITK) in a cohort of nine patients with most patients presenting with massive EBV B-cell lymphoproliferation. This review summarizes the clinical and immunological findings in these patients. Moreover, we describe the functional consequences of the mutations and draw comparisons with the extensively investigated function of ITK in vitro and in the murine model.

Memory B-cells in healthy and antibody-deficient children

Recently it has become clear that a reduction of IgD-CD27+ memory B-cells in adult CVID patients correlates with clinical aspects of the disease. However, little is known about B-cell dysregulation in pediatric antibody deficiency. Reference values are essential for the interpretation of B-cell subpopulations in children. We present the clinical and immunophenotypical characterization of 16 children and adolescents with CVID and hypogammaglobulinemia. Reference values for IgD+CD27-, IgD+CD27+ and IgD-CD27+ B-cells in healthy children were established for five age groups. In healthy controls we found a continuous increase in IgD-CD27+ B-cell percentage with age from 1.35-5% of B-cells in the second year of life to 4.1-18.7% in adolescents. Interestingly, in 12/14 antibody-deficient patients memory B-cells are significantly below the age-related 10th percentile. We conclude that the reduction of memory B-cells is a useful additional marker for the detection of children with CVID hypogammaglobulinemia and may contribute to the early presentation.

Memory B Cell Function in HIV-Infected Children—Decreased Memory B Cells Despite ART

B cell dysfunction is a well-studied complication of HIV infection in adults. Data on B cell differentiation in normal and HIV-infected children are lacking. We show the distribution of B cell subsets and immunoglobulin levels in HIV-infected children compared with controls. Furthermore, we observe the long-term B cell reconstitution of vaccine-specific immunity after antiretroviral therapy (ART). Phenotype of B cells (naive, non-switched memory, switched memory) was analyzed in 48 infected children and 62 controls. In nine HIV-infected children, functional reconstitution was quantified by tetanus-specific antibodies and by performing a lymphocyte transformation test (LTT) in a longitudinal approach. Switched memory B cells are significantly reduced in HIV-infected children. Vaccine-specific antibodies and response to LTT increase after initiation of ART. Our data indicate a significant dysfunction in the B cell system, despite effective ART. Partial reconstitution of humoral immunity may have therapeutic implications in a subset of HIV-infected children.

Gene therapy for monogenic disorders of the bone marrow

Ex‐vivo gene transfer of autologous haematopoietic stem cells in patients with monogenic diseases of the bone marrow has emerged as a new therapeutic approach, mainly in patients lacking a suitable donor for transplant. The encouraging results of initial clinical trials of gene therapy for primary immunodeficiencies were tempered by the occurrence of genotoxicity in a number of patients. Over the last decade, safer viral vectors have been developed to overcome the risk of insertional mutagenesis and have led to impressive clinical outcomes with considerably improved safety. We review the efforts in specific immunodeficiencies including adenosine deaminase deficiency, X‐linked severe combined immunodeficiency, chronic granulomatous disease and Wiskott Aldrich syndrome. Major recent progress has also been made in haemoglobinopathies, such as beta‐thalassaemia, sickle cell disease and Fanconi anaemia, and also specific lysosomal storage diseases, which, although not strictly bone marrow specific conditions, have been effectively treated by bone marrow‐based treatment. The success of these recent studies and the advent of new technologies, such as gene editing, suggest that gene therapy could become a more generally applied treatment modality for a number of haematopoietic disorders.

Outcome of hematopoietic cell transplantation for DNA double-strand break repair disorders

Background: Rare DNA breakage repair disorders predispose to infection and lymphoreticular malignancies. Hematopoietic cell transplantation (HCT) is curative, but coadministered chemotherapy or radiotherapy is damaging because of systemic radiosensitivity. We collected HCT outcome data for Nijmegen breakage syndrome, DNA ligase IV deficiency, Cernunnos–XRCC4‐like factor (Cernunnos‐XLF) deficiency, and ataxia‐telangiectasia (AT). Methods: Data from 38 centers worldwide, including indication, donor, conditioning regimen, graft‐versus‐host disease, and outcome, were analyzed. Conditioning was classified as myeloablative conditioning (MAC) if it contained radiotherapy or alkylators and reduced‐intensity conditioning (RIC) if no alkylators and/or 150 mg/m2 fludarabine or less and 40 mg/kg cyclophosphamide or less were used. Results: Fifty‐five new, 14 updated, and 18 previously published patients were analyzed. Median age at HCT was 48 months (range, 1.5‐552 months). Twenty‐nine patients underwent transplantation for infection, 21 had malignancy, 13 had bone marrow failure, 13 received pre‐emptive transplantation, 5 had multiple indications, and 6 had no information. Twenty‐two received MAC, 59 received RIC, and 4 were infused; information was unavailable for 2 patients. Seventy‐three of 77 patients with DNA ligase IV deficiency, Cernunnos‐XLF deficiency, or Nijmegen breakage syndrome received conditioning. Survival was 53 (69%) of 77 and was worse for those receiving MAC than for those receiving RIC (P = .006). Most deaths occurred early after transplantation, suggesting poor tolerance of conditioning. Survival in patients with AT was 25%. Forty‐one (49%) of 83 patients experienced acute GvHD, which was less frequent in those receiving RIC compared with those receiving MAC (26/56 [46%] vs 12/21 [57%], P = .45). Median follow‐up was 35 months (range, 2‐168 months). No secondary malignancies were reported during 15 years of follow‐up. Growth and developmental delay remained after HCT; immune‐mediated complications resolved. Conclusion: RIC HCT resolves DNA repair disorder–associated immunodeficiency. Long‐term follow‐up is required for secondary malignancy surveillance. Routine HCT for AT is not recommended.

A prospective study on the natural history of patients with profound combined immunodeficiency: An interim analysis

Background: Absent T‐cell immunity resulting in life‐threatening infections provides a clear rationale for hematopoetic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency (SCID). Combined immunodeficiencies (CIDs) and “atypical” SCID show reduced, not absent T‐cell immunity. If associated with infections or autoimmunity, they represent profound combined immunodeficiency (P‐CID), for which outcome data are insufficient for unambiguous early transplant decisions. Objectives: We sought to compare natural histories of severity‐matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome. Methods: In this prospective and retrospective observational study, we recruited nontransplanted patients with P‐CID aged 1 to 16 years to compare natural histories of severity‐matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome. Results: A total of 51 patients were recruited (median age, 9.6 years). Thirteen of 51 had a genetic diagnosis of “atypical” SCID and 14 of 51 of CID. About half of the patients had less than 10% naive T cells, reduced/absent T‐cell proliferation, and at least 1 significant clinical event/year, demonstrating their profound immunodeficiency. Nineteen patients (37%) underwent transplantation within 1 year of enrolment, and 5 of 51 patients died. Analysis of the HSCT decisions revealed the anticipated heterogeneity, favoring an ongoing prospective matched‐pair analysis of patients with similar disease severity with or without transplantation. Importantly, so far neither the genetic diagnosis nor basic measurements of T‐cell immunity were good predictors of disease evolution. Conclusions: The P‐CID study for the first time characterizes a group of patients with nontypical SCID T‐cell deficiencies from a therapeutic perspective. Because genetic and basic T‐cell parameters provide limited guidance, prospective data from this study will be a helpful resource for guiding the difficult HSCT decisions in patients with P‐CID.

Class Switch Recombination Process in Ataxia Telangiectasia Patients with Elevated Serum Levels of IgM

Ataxia telangiectasia (AT) is a rare primary immunodeficiency disorder with various clinical manifestations. Increased serum levels of IgM and recurrent infections, mainly sinopulmonary infections, can be the presenting feature in a number of AT patients and may be initially misdiagnosed as hyper-IgM (HIgM) syndrome. This study was designed to investigate class switch recombination (CSR) as a critical mechanism in B lymphocytes’ maturation to produce different isotypes of antibody in response to antigen stimulation in AT cases with HIgM presentation. Quantitative IgE production after stimulation by IL-4 and CD40L was considered as an indicator for CSR function. We also compared their results with sex and age matched AT patients without HIgM presentation. We report four AT patients with recurrent infections during infancy and high serum levels of IgM. Laboratory evaluations revealed defective CSR while none of the three AT patients without HIgM presentation had a defect in the CSR process. The characterized defect in AT is a mutation in the ataxia telangiectasia mutated (ATM) gene. This gene may result in CSR defects due to impaired DNA break repair. A special association between AT and HIgM may indicate a new subgroup of AT patients according to their clinical phenotype and CSR condition.

Efficient control of pandemic 2009 H1N1 virus infection with intravenous zanamivir despite the lack of immune function

A teenager who acquired 2009 H1N1 influenza A lower respiratory tract infection during total bone marrow and lymphoid aplasia, in the setting of human leukocyte antigen‐haploidentical hematopoietic stem cell transplantation, was successfully treated with intravenous zanamivir. This case demonstrates efficient control of pandemic influenza infection by intravenous zanamivir in the absence of any functional immune system, thus suggesting profound antiviral activity.

Gene Therapy Approaches to Immunodeficiency

Transfer of gene-corrected autologous hematopoietic stem cells in patients with primary immunodeficiencies has emerged as a new therapeutic approach. Patients with various conditions lacking a suitable donor have been treated with retroviral vectors and a gene-addition strategy. Initial promising results were shadowed by the occurrence of malignancies in some of these patients. Current trials, developed in the last decade, use safer viral vectors to overcome the risk of genotoxicity and have led to improved clinical outcomes. This review reflects the progresses made in specific disorders, including adenosine deaminase deficiency, X-linked severe combined immunodeficiency, chronic granulomatous disease, and Wiskott-Aldrich syndrome.

Editorial: Current Challenges in Immune and Other Acquired Cytopenias of Childhood

The Editorial on the Research Topic Current Challenges in Immune and Other Acquired Cytopenias of Childhood Chronic immune thrombocytopenia (ITP), Evans syndrome (ES), aplastic anemia, etc., are descriptive terms for immune-mediated cytopenias in pediatric hematology that may be summarized as a group of disorders of immune dysregulation based on ill-defined poly- and epigenetic diatheses toward autoimmunity and monogenic primary immunodeficiencies (PIDs); a growing number of which is being identified with next-generation sequencing technologies. The historical view, that cytopenia in the context of these diseases is “acquired,” “idiopathic,” or termed “primary” because cytopenia may be the first and only manifestation at early age, is challenged by the emerging recognition of underlying pathomechanisms and predispositions. Similarly, patients with congenital bone marrow failure syndromes may present without previous syndromic features later during childhood or adolescence and contribute to the wide spectrum of differential diagnosis of cytopenia in childhood. In addition to the diagnostic complexity and prognostic uncertainty, the therapeutic approach to patients with these conditions remains undetermined in many situations. In severe cases, allogeneic hematopoietic stem cell transplantation may be the option of choice; other conditions might require temporary immunosuppression or even no treatment at all. Although the indication for stem cell transplantation will always predominantly depend on the clinical course and status of a patient, the definition of a clear-cut pathomechanism potentially offers a guidance toward targeted therapy approaches. Autoimmunity and cytopenias may occur in the context of many types of PID, whether belonging to the combined immunodeficiencies, to those with predominant antibody deficiency, to syndromic PIDs (e.g., Wiskott–Aldrich or 22q11 deletion syndrome), to the category of PIDs with immune dysregulation [e.g., autoimmune lymphoproliferative syndrome (ALPS), immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome], as well as to humoral and cellular defects of the innate immune system (1, 2). In addition to classical PIDs highly associated with autoimmunity and autoimmune cytopenias such as ALPS or common variable immunodeficiency (CVID), an increasing number of PID syndromes have been described recently to be associated with immune dysregulation and symptoms of autoimmunity such as immune cytopenia. The phenotype in 9 of 13 newly discovered monogenic PIDs published within the last year include cytopenia, immune dysregulation, autoimmunity, and/or autoinflammation as their main manifestation (1, 3, 4), adding to at least 8 of 19 novel PIDs the year before [(5); Al-Herz et al.]. Yet, this fact has not universally been translated into the diagnostic work-up of cytopenia in childhood. The present research topic on immune and other acquired cytopenias of childhood at Frontiers in Pediatric Hematology and Immunology encompasses reviews, original studies, and single-case observations as well as management strategy papers that address the current challenge how to deal with these disorders. Erlacher and Strahm review in depth aspects and differential diagnosis of pancytopenia in childhood, ranging from inherited bone marrow failure syndromes to extrinsically acquired (e.g., infection-associated) defects of hematopoiesis; from autoimmunity to hemophagocytic syndromes and malignancies (e.g., acute lymphoblastic leukemia and myelodysplastic syndromes). Classical inherited bone marrow failure syndromes, e.g., Fanconi anemia, Dyskeratosis congenita, Shwachman Bodian Diamond Syndrome, and Diamond Blackfan anemia, are based on defects of DNA and telomere maintenance or ribosome function. However, immune dysregulation and autoimmunity often have a rather unclear and heterogenous etiology. Polygenetic defects or polymorphisms as well as an array of environmental factors are known to contribute to a predisposition to autoimmunity. In susceptible individuals, autoantibodies may be produced secondarily to infections or other exogenous triggers due to cross-reactivity (molecular mimicry). Pathologic processing of cell debris can lead to presentation of self-antigens to the immune system like anti-glycoprotein IIa/IIIb antibodies in ITP or anti-double strand DNA antibodies in systemic lupus erythematosus (SLE). Among several PID disorders that lead to a predisposition toward autoimmunity, ALPS, most often due to defective Fas-mediated lymphocyte apoptosis and impaired T cell maturation, is a classical PID leading to autoimmune cytopenia, splenomegaly, and lymphoproliferation, with splenic sequestration sometimes contributing to cytopenia. Furthermore, the heterogeneous group of CVID is highly associated with autoimmune cytopenia due to autoantibody formation based on defective B cell selection and maturation. CVID and ALPS may be ruled out on the basis of relatively routine basic immunological tests. The large group of combined immunodeficiencies is generally associated with a lack of naive T cells and an oligoclonal T cell repertoire, which predisposes these patients to autoimmunity in addition to infections. Wiskott–Aldrich and 22q11 deletion syndromes are linked to defective regulatory T (Treg) cells and impaired T cell development, and may be excluded by detection of additional clinical syndromic features, other routine laboratory parameters, impaired in vitro lymphocyte proliferation, and molecular genetic tests; whereas patients with IPEX-(like) syndromes have a primary Treg defect and most often present with enteropathy, multi-organ autoimmunity, and show reduced or absent Treg cell function and diminished STAT5 phosphorylation. Recently, homozygous loss-of-function mutations in the LRBA gene (3, 6, 7) as well as haploinsufficiency of CTLA-4 (8, 9) gain-of-function of PI-3-kinase (10, 11) or of STAT3 (12, 13) showed in part ALPS-like phenotypes with autoimmune cytopenias. In line with these observations, an increasing number of patients with autoimmunity including cytopenias will be referred to genetic analysis to find new causative genes. Rao highlights the experience of the NIH with one of the largest ALPS cohorts in the world, emphasizing the need of effective immune suppression. One of the main lessons from the past decades taught us to avoid splenectomy. Furthermore, Aladjidi et al. report the results from French OBS’CEREVANCE, an observational cohort gathering data on children with ES, chronic ITP and autoimmune hemolytic anemia (AIHA). One hundred fifty-six patients with ES were analyzed; interestingly, in 13 patients SLE was diagnosed, but ALPS was diagnosed only in 3 patients. Thirty percent of all patients were classified as “primary” forms because cytopenia remained the only symptom; in 60% of the patients, the authors observed additional clinical or biochemical features to term this fraction as “unclassified” (Aladjidi et al.). One major red flag for pediatricians: 10% of all patients died at a median age of 14.3 years either due to hemorrhage or infections with the unknown participating role of immunosuppressive treatment. Thus, a “wait-and-see” strategy in ES seems not to be justified for a long period. Phenotypic variations of diseases linked to (pan-)cytopenia are shown in the case report of Karastaneva et al. Two unrelated patients with Fanconi anemia developed rather untypical ITP, but showing a normal marrow. Management of ITP was accomplished with intravenous immunoglobulins (IVIG) and danazol. This rather simple and non-toxic ITP treatment warrants evaluation of autoimmune phenomena in other bone marrow failure syndromes. Although in most patients with ITP first-line treatment usually leads to remission, the application of thrombopoietin agonist is warranted in patients with refractory and chronic forms of ITP. Garzon and Mitchell review the use of Eltrombopag and Romiplostim, which depicts a change in treatment paradigm, as an immunosuppressive regime is avoided, and these drugs are well tolerated. Finally, Koster et al. report the experience of a single hematooncological unit with two cases of a rare infectious disease, namely Leishmaniosis that led to severe cytopenia. The reported incidence of Leishmaniosis among pediatric patients with cytopenia is extremely low, having said that the experience of the reporting hematooncologial unit in central Germany with two cases in 10 years shows how coincidental rare events might happen. Hemophagocytosis (detected after repeated marrow puncture) and polyclonal B cell activation (false positive antibodies for other infectious diseases) are hallmarks of Leishmaniosis. This research topic has collected a range of contributions focusing on diagnostic and therapeutic challenges, reaching from scientific progress in the immunological and hematological context of cytopenias to clinical challenges such as the experience with novel treatment approaches and lessons from patient registries. Despite tremendous progress has been made with next-generation sequencing techniques applied in the diagnostic work-up and the availability of novel potent immunosuppressive drugs and hematopoiesis stimulating agents for the treatment recently, clinical challenges remain for those dealing with the so-called “acquired” cytopenias of childhood.