Hypomorphic RAG deficiency: impact of disease burden on survival and thymic recovery argues for early diagnosis and HSCT.

Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and 30% of the patients, respectively. Grafts were T-cell depleted in 15 cases (25%). Overall survival at 1 and 4 years was 77.5% and 67.5% (median follow-up of 39 months). Infection was the main cause of death. In univariable analysis, active infection, organ damage pre-HSCT, T-cell depletion of the graft, and transplant from a mismatched family donor were predictive of worse outcome, whereas organ damage and T-cell depletion remained significant in multivariable analysis (hazard ratio [HR] = 6.01, HR = 8.46, respectively). All patients diagnosed by newborn screening or family history survived. Cumulative incidences of acute and chronic graft-versus-host disease were 35% and 22%, respectively. Cumulative incidences of new-onset autoimmunity was 15%. Immune reconstitution, particularly recovery of naïve CD4+ T cells, was faster and more robust in patients transplanted before 3.5 years of age, and without organ damage. These findings support the indication for early transplantation.

Acute chest syndrome in children with sickle cell disease: Data from a national AIEOP cohort identify priority areas of intervention in a hub-and-spoke system.

Acute chest syndrome (ACS) is a frequent cause of hospitalization in sickle cell disease (SCD). Despite advances in acute care, many settings still lack knowledge about ACS best practices. After the AIEOP Guidelines were published in 2012, suggesting standardized management in Italy, a retrospective study was performed to assess the diagnostic and therapeutic pathways of ACS in children. From 2013 to 2018, 208 ACS episodes were presented by 122/583 kids in 11 centres. 73 were male, mean age 10.9 years, 85% African, 92% HbSS or Sß°. In our hub-and-spoke system, a good adherence to Guidelines was documented, but discrepancies between reference centres and general hospitals were noted. Improvement is needed for timely transfer to reference centres, use of incentive spirometry, oxygen therapy and pain management.

Diagnosis and management of acquired aplastic anemia in childhood. Guidelines from the Marrow Failure Study Group of the Pediatric Haemato-Oncology Italian Association (AIEOP).

Acquired aplastic anemia (AA) is a rare heterogeneous disorder characterized by pancytopenia and hypoplastic bone marrow. The incidence is 2-3 per million population per year in the Western world, but 3 times higher in East Asia. Survival in severe aplastic anemia (SAA) has improved significantly due to advances in hematopoietic stem cell transplantation (HSCT), immunosuppressive therapy, biologic agents, and supportive care. In SAA, HSCT from a matched sibling donor (MSD) is the first-line treatment. If a MSD is not available, options include immunosuppressive therapy (IST), matched unrelated donor, or haploidentical HSCT. The purpose of this guideline is to provide health care professionals with clear guidance on the diagnosis and management of pediatric patients with AA. A preliminary evidence-based document prepared by a group of pediatric hematologists of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Hemato-Oncology (AIEOP) was discussed, modified and approved during a series of consensus conferences that started online during COVID 19 and continued in the following years, according to procedures previously validated by the AIEOP Board of Directors.

Hypomorphic Rag1 mutations alter the preimmune repertoire at early stages of lymphoid development.

Hypomorphic RAG1 mutations allowing residual T- and B-cell development have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (CID-G/AI) and abnormalities of the peripheral T- and B-cell repertoire. To examine how hypomorphic Rag1 mutations affect the earliest stages of lymphocyte development, we used CRISPR/Cas9 to generate mouse models with mutations equivalent to those found in patients with CID-G/AI. Immunological characterization showed partial development of T and B lymphocytes, with persistence of naïve cells and preserved serum immunoglobulin but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with CID-G/AI. By using high-throughput sequencing, we identified marked skewing of Igh V and Trb V gene usage in early progenitors, with a bias for productive Igh and Trb rearrangements after selection occurred and increased apoptosis of B-cell progenitors. Rearrangement at the Igk locus was impaired, and polyreactive immunoglobulin M antibodies were detected. This study provides novel insights into how hypomorphic Rag1 mutations alter the primary repertoire of T and B cells, setting the stage for immune dysregulation frequently seen in patients.

A mutation in caspase-9 decreases the expression of BAFFR and ICOS in patients with immunodeficiency and lymphoproliferation.

Lymphocyte apoptosis is mainly induced by either death receptor-dependent activation of caspase-8 or mitochondria-dependent activation of caspase-9. Mutations in caspase-8 lead to autoimmunity/lymphoproliferation and immunodeficiency. This work describes a heterozygous H237P mutation in caspase-9 that can lead to similar disorders. H237P mutation was detected in two patients: Pt1 with autoimmunity/lymphoproliferation, severe hypogammaglobulinemia and Pt2 with mild hypogammaglobulinemia and Burkitt lymphoma. Their lymphocytes displayed defective caspase-9 activity and decreased apoptotic and activation responses. Transfection experiments showed that mutant caspase-9 display defective enzyme and proapoptotic activities and a dominant-negative effect on wild-type caspase-9. Ex vivo analysis of the patients' lymphocytes and in vitro transfection experiments showed that the expression of mutant caspase-9 correlated with a downregulation of BAFFR (B-cell-activating factor belonging to the TNF family (BAFF) receptor) in B cells and ICOS (inducible T-cell costimulator) in T cells. Both patients carried a second inherited heterozygous mutation missing in the relatives carrying H237P: Pt1 in the transmembrane activator and CAML interactor (TACI) gene (S144X) and Pt2 in the perforin (PRF1) gene (N252S). Both mutations have been previously associated with immunodeficiencies in homozygosis or compound heterozygosis. Taken together, these data suggest that caspase-9 mutations may predispose to immunodeficiency by cooperating with other genetic factors, possibly by downregulating the expression of BAFFR and ICOS.

Contact investigation based on serial interferon-gamma release assays (IGRA) in children from the hematology-oncology ward after exposure to a patient with pulmonary tuberculosis.

BACKGROUND: Interferon-gamma release assays (IGRAs) have high specificity and sensitivity for the diagnosis of tuberculosis (TB) infection. However, their role as a screening tool in children with immunodeficiency disorders is still unclear. In the present study, we performed a contact investigation using serial IGRAs on children with immunodeficiency conditions exposed to a contagious TB patient. METHODS: Children who were exposed to a contagious TB case underwent serial QuantiFERON(®) TB Gold In-Tube (QFT-GIT) and T-SPOT(®).TB (T-SPOT) testing. RESULTS: Eighteen children were tested. At the first testing, only two children (11 %) were positive to T-SPOT. Indeterminate results were more frequent with QFT-GIT (35 %) than with T-SPOT (12 %). In the multivariable analysis, a statistically significant association of lymphocyte count <500 cells/mm(3) (p < 0.00005) and low age (p = 0.03) with indeterminate results for the QFT-GIT test but not for T-SPOT (p = 0.10 and p = 0.88, respectively) was found. At the end of October 2012, 15 of the 18 children were alive and none developed active TB disease. CONCLUSION: T-SPOT provided more determinate results and was less influenced by low age and lymphocytopenia than QFT-GIT in this population of immunodeficient children. These findings suggest that T-SPOT is a more accurate test for the identification of TB infection in young children with lymphocytopenia and should be preferred to QFT-GIT under such specific conditions.

X-linked thrombocytopenia and Wiskott-Aldrich syndrome are allelic diseases with mutations in the WASP gene.

X-linked thrombocytopenia (XLT) is a rare recessive hereditary disorder characterized by isolated thrombocytopenia with small-sized platelets. The XLT locus has been located to chromosome Xp11 by linkage analysis, which is also where the recently cloned Wiskott-Aldrich syndrome (WAS) gene, maps. The relationship between XLT and WAS has long been debated; they might be due to different mutations of the same gene or to mutations in different genes. We now show that mutations in the WAS gene, different from those found in WAS patients, are present in three unrelated male patients with isolated thrombocytopenia and small-sized platelets. Our results demonstrate that XLT and WAS are allelic forms of the same disease, but the causes of the differences need to be further investigated.

Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis.

Osteopetrosis includes a group of inherited diseases in which inadequate bone resorption is caused by osteoclast dysfunction. Although molecular defects have been described for many animal models of osteopetrosis, the gene responsible for most cases of the severe human form of the disease (infantile malignant osteopetrosis) is unknown. Infantile malignant autosomal recessive osteopetrosis (MIM 259700) is a severe bone disease with a fatal outcome, generally within the first decade of life. Osteoclasts are present in normal or elevated numbers in individuals affected by autosomal recessive osteopetrosis, suggesting that the defect is not in osteoclast differentiation, but in a gene involved in the functional capacity of mature osteoclasts. Some of the mouse mutants have a decreased number of osteoclasts, which suggests that the defect directly interferes with osteoclast differentiation. In other mutants, it is the function of the osteoclast that seems to be affected, as they show normal or elevated numbers of non-functioning osteoclasts. Here we show that TCIRG1, encoding the osteoclast-specific 116-kD subunit of the vacuolar proton pump, is mutated in five of nine patients with a diagnosis of infantile malignant osteopetrosis. Our data indicate that mutations in TCIRG1 are a frequent cause of autosomal recessive osteopetrosis in humans.

Comparison of American and European practices in the management of patients with primary immunodeficiencies.

Primary immunodeficiency diseases (PIDs) comprise a heterogeneous group of rare disorders. This study was devised in order to compare management of these diseases in the northern hemisphere, given the variability of practice among clinicians in North America. The members of two international societies for clinical immunologists were asked about their management protocols in relation to their PID practice. An anonymous internet questionnaire, used previously for a survey of the American Academy of Allergy, Asthma and Immunology (AAAAI), was offered to all full members of the European Society for Immunodeficiency (ESID). The replies were analysed in three groups, according to the proportion of PID patients in the practice of each respondent; this resulted in two groups from North America and one from Europe. The 123 responses from ESID members (23·7%) were, in the majority, very similar to those of AAAAI respondents, with > 10% of their practice devoted to primary immunodeficiency. There were major differences between the responses of these two groups and those of the general AAAAI respondents whose clinical practice was composed of < 10% of PID patients. These differences included the routine use of intravenous immunoglobulin therapy (IVIg) for particular types of PIDs, initial levels of IVIg doses, dosing intervals, routine use of prophylactic antibiotics, perceptions of the usefulness of subcutaneous immunoglobulin therapy (SCIg) and of the risk to patients' health of policies adopted by health-care funders. Differences in practice were identified and are discussed in terms of methods of health-care provision, which suggest future studies for ensuring continuation of appropriate levels of immunoglobulin replacement therapies.

Production and persistence of specific antibodies in COVID-19 patients with hematologic malignancies: role of rituximab.

The ability of patients with hematologic malignancies (HM) to develop an effective humoral immune response after COVID-19 is unknown. A prospective study was performed to monitor the immune response to SARS-CoV-2 of patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), chronic lymphoproliferative disorders (CLD), multiple myeloma (MM), or myelodysplastic/myeloproliferative syndromes (MDS/MPN). Antibody (Ab) levels to the SARS-CoV-2 nucleocapsid (N) and spike (S) protein were measured at +1, +3, +6 months after nasal swabs became PCR-negative. Forty-five patients (9 FL, 8 DLBCL, 8 CLD, 10 MM, 10 MDS/MPS) and 18 controls were studied. Mean anti-N and anti-S-Ab levels were similar between HM patients and controls, and shared the same behavior, with anti-N Ab levels declining at +6 months and anti-S-Ab remaining stable. Seroconversion rates were lower in HM patients than in controls. In lymphoma patients mean Ab levels and seroconversion rates were lower than in other HM patients, primarily because all nine patients who had received rituximab within 6 months before COVID-19 failed to produce anti-N and anti-S-Ab. Only one patient requiring hematological treatment after COVID-19 lost seropositivity after 6 months. No reinfections were observed. These results may inform vaccination policies and clinical management of HM patients.

PTX3 genetic variations affect the risk of Pseudomonas aeruginosa airway colonization in cystic fibrosis patients.

Cystic fibrosis (CF) is a common life-threatening autosomal recessive disorder in the Caucasian population, and the gene responsible is the CF transmembrane conductance regulator (CFTR). Patients with CF have repeated bacterial infection of the airways caused by Pseudomonas aeruginosa (PA), which is one of the predominant pathogen, and endobronchial chronic infection represents a major cause of morbidity and mortality. Pentraxin 3 (PTX3) is a gene that encodes the antimicrobial protein, PTX3, which is believed to have an important role in innate immunity of lung. To address the role of PTX3 in the risk of PA lung colonization, we investigated five single nucleotide polymorphisms of PTX3 gene in 172 Caucasian CF patients who were homozygous for the F508del mutation. We observed that PTX3 haplotype frequencies were significantly different between patients with PA colonization, as compared with noncolonized patients. Moreover, a protective effect was found in association with a specific haplotype (odds ratio 0.524). Our data suggest that variations within PTX3 affect lung colonization of Pseudomonas in patients with CF.

In vitro correction of JAK3-deficient severe combined immunodeficiency by retroviral-mediated gene transduction.

Mutations affecting the expression of the Janus family kinase JAK3 were recently shown to be responsible for autosomal recessive severe combined immunodeficiency (SCID). JAK3-deficient patients present with a clinical phenotype virtually indistinguishable from boys affected by X-linked SCID, a disease caused by genetic defects of the common gamma chain (gamma c) that is a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. The specific interaction of JAK3 and gamma c represents the biochemical basis for the similarities between these two immunodeficiencies. Both forms of SCID are characterized by recurrent, severe infections leading to death in infancy unless successfully treated by allogeneic bone marrow transplantation. Because of the potentially lethal complications associated with allogeneic bone marrow transplantation and the frequent lack of suitable marrow donors, the development of alternative forms of therapy is highly desirable. To this end, we investigated a retroviral-mediated gene correction approach for JAK3-deficiency. A vector carrying a copy of JAK3 cDNA was constructed and used to transduce B cell lines derived from patients with JAK3-deficient SCID. We demonstrate restoration of JAK3 expression and phosphorylation upon IL-2 and IL-4 stimulation. Furthermore, patients' cells transduced with JAK3 acquired the ability to proliferate normally in response to IL-2. These data indicate that the biological defects of JAK3-deficient cells can be efficiently corrected in vitro by retroviral-mediated gene transfer, thus providing the basis for future investigation of gene therapy as treatment for JAK3-deficient SCID.

NTB-A [correction of GNTB-A], a novel SH2D1A-associated surface molecule contributing to the inability of natural killer cells to kill Epstein-Barr virus-infected B cells in X-linked lymphoproliferative disease.

In humans, natural killer (NK) cell function is regulated by a series of receptors and coreceptors with either triggering or inhibitory activity. Here we describe a novel 60-kD glycoprotein, termed NTB-A, that is expressed by all human NK, T, and B lymphocytes. Monoclonal antibody (mAb)-mediated cross-linking of NTB-A results in the induction of NK-mediated cytotoxicity. Similar to 2B4 (CD244) functioning as a coreceptor in the NK cell activation, NTB-A also triggers cytolytic activity only in NK cells expressing high surface densities of natural cytotoxicity receptors. This suggests that also NTB-A may function as a coreceptor in the process of NK cell activation. Molecular cloning of the cDNA coding for NTB-A molecule revealed a novel member of the immunoglobulin superfamily belonging to the CD2 subfamily. NTB-A is characterized, in its extracellular portion, by a distal V-type and a proximal C2-type domain and by a cytoplasmic portion containing three tyrosine-based motifs. NTB-A undergoes tyrosine phosphorylation and associates with the Src homology 2 domain-containing protein (SH2D1A) as well as with SH2 domain-containing phosphatases (SHPs). Importantly, analysis of NK cells derived from patients with X-linked lymphoproliferative disease (XLP) showed that the lack of SH2D1A protein profoundly affects the function not only of 2B4 but also of NTB-A. Thus, in XLP-NK cells, NTB-A mediates inhibitory rather than activating signals. These inhibitory signals are induced by the interaction of NTB-A with still undefined ligands expressed on Epstein-Barr virus (EBV)-infected target cells. Moreover, mAb-mediated masking of NTB-A can partially revert this inhibitory effect while a maximal recovery of target cell lysis can be obtained when both 2B4 and NTB-A are simultaneously masked. Thus, the altered function of NTB-A appears to play an important role in the inability of XLP-NK cells to kill EBV-infected target cells.

X-linked lymphoproliferative disease. 2B4 molecules displaying inhibitory rather than activating function are responsible for the inability of natural killer cells to kill Epstein-Barr virus-infected cells.

2B4 is a surface molecule involved in activation of the natural killer (NK) cell-mediated cytotoxicity. It binds a protein termed Src homology 2 domain-containing protein (SH2D1A) or signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), which in turn has been proposed to function as a regulator of the 2B4-associated signal transduction pathway. In this study, we analyzed patients with X-linked lymphoproliferative disease (XLP), a severe inherited immunodeficiency characterized by critical mutations in the SH2D1A gene and by the inability to control Epstein-Barr virus (EBV) infections. We show that, in these patients, 2B4 not only fails to transduce triggering signals, but also mediates a sharp inhibition of the NK-mediated cytolysis. Other receptors involved in NK cell triggering, including CD16, NKp46, NKp44, and NKp30, displayed a normal functional capability. However, their activating function was inhibited upon engagement of 2B4 molecules. CD48, the natural ligand of 2B4, is highly expressed on the surface of EBV(+) B cell lines. Remarkably, NK cells from XLP patients could not kill EBV(+) B cell lines. This failure was found to be the consequence of inhibitory signals generated by the interaction between 2B4 and CD48, as the antibody-mediated disruption of the 2B4-CD48 interaction restored lysis of EBV(+) target cells lacking human histocompatibility leukocyte antigen (HLA) class I molecules. In the case of autologous or allogeneic (HLA class I(+)) EBV(+) lymphoblastoid cell lines, restoration of lysis was achieved only by the simultaneous disruption of 2B4-CD48 and NK receptor-HLA class I interactions. Molecular analysis revealed that 2B4 molecules isolated from either XLP or normal NK cells were identical. As expected, in XLP-NK cells, 2B4 did not associate with SH2D1A, whereas similar to 2B4 molecules isolated from normal NK cells, it did associate with Src homology 2 domain-containing phosphatase 1.

Immunodeficiencies.

Primary immunodeficiencies (PIDs) are uncommon, chronic and severe disorders of the immune system in which patients cannot mount a sufficiently protective immune response, leading to an increased susceptibility to infections. The treatment of choice for PID patients with predominant antibody deficiency is intravenous immunoglobulin (Ig) replacement therapy. Despite major advances over the last 20 years in the molecular characterization of PIDs, many patients remain undiagnosed or are diagnosed too late, with severe consequences. Various strategies to ensure timely diagnosis of PIDs are in place, and novel approaches are being developed. In recent years, several patient registries have been established. Such registries shed light on the pathology and natural history of these varied disorders. Analyses of the registry data may also reveal which patients are likely to respond well to higher Ig infusion rates and may help to determine the optimal dosing of Ig products. Faster infusion rates may lead to improved convenience for patients and thus increase patient compliance, and may reduce nursing time and the need for hospital resources. Data from two recent studies suggest that Gamunex and Privigen are well tolerated at high infusion rates. Nevertheless, careful selection of patients for high infusion rates, based on co-morbid conditions and tolerance of the current infusion rate, is advisable. Based on the available data, intravenous Ig offers broad protection against encapsulated organisms. As vaccine trends change, careful monitoring of specific antibody levels in the general population, such as those against pneumococcal and meningococcal bacteria, should be implemented.

Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- immunodeficient patients.

Adenosine deaminase (ADA) deficiency results in severe combined immunodeficiency, the first genetic disorder treated by gene therapy. Two different retroviral vectors were used to transfer ex vivo the human ADA minigene into bone marrow cells and peripheral blood lymphocytes from two patients undergoing exogenous enzyme replacement therapy. After 2 years of treatment, long-term survival of T and B lymphocytes, marrow cells, and granulocytes expressing the transferred ADA gene was demonstrated and resulted in normalization of the immune repertoire and restoration of cellular and humoral immunity. After discontinuation of treatment, T lymphocytes, derived from transduced peripheral blood lymphocytes, were progressively replaced by marrow-derived T cells in both patients. These results indicate successful gene transfer into long-lasting progenitor cells, producing a functional multilineage progeny.

Stem cell transplantation for primary immunodeficiencies.

BMT is curative in almost 75% of children affected by severe primary immunodeficiencies (PIDs). Recently, the chance of cure has increased thanks to the availability of matched unrelated donors (MUDs). Nevertheless, besides the conventional indications to BMT (profound or absent T-cell function, profound or absent natural killer function, known syndromes with T-cell deficiencies), indications to BMT for PIDs affecting the quality of life or having an expectation of life that does not exceed the third-fourth decade remain unclear. Infact, if it is evident that the survival rate in an infant grafted for a PID with a MUD is expected to be more than 80%, alternative treatments such as gene therapy are now available.

Healthy twins delivered after oocyte cryopreservation and bilateral ovariectomy for ovarian cancer.

Anti-neoplastic treatments have significantly increased the survival of cancer patients, but female patients risk premature menopause. Oocyte cryopreservation has been proposed as a fertility-saving option. This report describes the first live birth achieved with autologous cryopreserved oocytes in an ovariectomized borderline cancer patient. A patient with a borderline ovarian tumour asked for oocyte cryopreservation after a right adnexectomy. Ovulation induction resulted in the retrieval and cryopreservation of seven mature oocytes. Thirty-nine months after a left ovariectomy, the patient asked for oocyte thawing and embryo transfer. Endometrial growth was induced using hormone replacement treatment. Three of the seven cryopreserved oocytes were thawed; they survived and, after insemination, normal fertilization took place. Three embryos were transferred into the patient's uterus. A twin pregnancy was achieved with the birth of two healthy females. Oocyte cryopreservation may be a reliable option for preserving fertility in young cancer patients who risk premature menopause due to surgery, chemotherapy or radiotherapy.

Jak3 deficiency blocks innate lymphoid cell development.

Loss-of-function mutations in the tyrosine kinase JAK3 cause autosomal recessive severe combined immunodeficiency (SCID). Defects in this form of SCID are restricted to the immune system, which led to the development of immunosuppressive JAK inhibitors. We find that the B6.Cg-Nr1d1tm1Ven/LazJ mouse line purchased from Jackson Laboratories harbors a spontaneous mutation in Jak3, generating a SCID phenotype and an inability to generate antigen-independent professional cytokine-producing innate lymphoid cells (ILCs). Mechanistically, Jak3 deficiency blocks ILC differentiation in the bone marrow at the ILC precursor and the pre-NK cell progenitor. We further demonstrate that the pan-JAK inhibitor tofacitinib and the specific JAK3 inhibitor PF-06651600 impair the ability of human intraepithelial ILC1 (iILC1) to produce IFN-γ, without affecting ILC3 production of IL-22. Both inhibitors impaired the proliferation of iILC1 and ILC3 and differentiation of human ILC in vitro. Tofacitinib is currently approved for the treatment of moderate-to-severely active rheumatoid arthritis. Both tofacitinib and PF-06651600 are currently in clinical trials for several other immune-mediated conditions. Our data suggest that therapeutic inhibition of JAK may also impact ILCs and, to some extent, underlie clinical efficacy.