Diagnostic evaluation of paediatric autoimmune lymphoproliferative immunodeficiencies (ALPID): a prospective cohort study.

BACKGROUND: Lymphoproliferation and autoimmune cytopenias characterise autoimmune lymphoproliferative syndrome. Other conditions sharing these manifestations have been termed autoimmune lymphoproliferative syndrome-like diseases, although they are frequently more severe. The aim of this study was to define the genetic, clinical, and immunological features of these disorders to improve their diagnostic classification. METHODS: In this prospective cohort study, patients were referred to the Center for Chronic Immunodeficiency in Freiburg, Germany, between Jan 1, 2008 and March 5, 2022. We enrolled patients younger than 18 years with lymphoproliferation and autoimmune cytopenia, lymphoproliferation and at least one additional sign of an inborn error of immunity (SoIEI), bilineage autoimmune cytopenia, or autoimmune cytopenia and at least one additional SoIEI. Autoimmune lymphoproliferative syndrome biomarkers were determined in all patients. Sanger sequencing followed by in-depth genetic studies were recommended for patients with biomarkers indicative of autoimmune lymphoproliferative syndrome, while IEI panels, exome sequencing, or genome sequencing were recommended for patients without such biomarkers. Genetic analyses were done as decided by the treating physician. The study was registered on the German Clinical Trials Register, DRKS00011383, and is ongoing. FINDINGS: We recruited 431 children referred for autoimmune lymphoproliferative syndrome evaluation, of whom 236 (55%) were included on the basis of lymphoproliferation and autoimmune cytopenia, 148 (34%) on the basis of lymphoproliferation and another SoIEI, 33 (8%) on the basis of autoimmune bicytopenia, and 14 (3%) on the basis of autoimmune cytopenia and another SoIEI. Median age at diagnostic evaluation was 9·8 years (IQR 5·5-13·8), and the cohort comprised 279 (65%) boys and 152 (35%) girls. After biomarker and genetic assessments, autoimmune lymphoproliferative syndrome was diagnosed in 71 (16%) patients. Among the remaining 360 patients, 54 (15%) had mostly autosomal-dominant autoimmune lymphoproliferative immunodeficiencies (AD-ALPID), most commonly affecting JAK-STAT (26 patients), CTLA4-LRBA (14), PI3K (six), RAS (five), or NFκB (three) signalling. 19 (5%) patients had other IEIs, 17 (5%) had non-IEI diagnoses, 79 (22%) were unresolved despite extended genetics (ALPID-U), and 191 (53%) had insufficient genetic workup for diagnosis. 16 (10%) of 161 patients with a final diagnosis had somatic mutations. Alternative classification of patients fulfilling common variable immunodeficiency or Evans syndrome criteria did not increase the proportion of genetic diagnoses. INTERPRETATION: The ALPID phenotype defined in this study is enriched for patients with genetic diseases treatable with targeted therapies. The term ALPID might be useful to focus diagnostic and therapeutic efforts by triggering extended genetic analysis and consideration of targeted therapies, including in some children currently classified as having common variable immunodeficiency or Evans syndrome. FUNDING: Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy. TRANSLATION: For the German translation of the abstract see Supplementary Materials section.

Non-apoptotic FAS signaling controls mTOR activation and extrafollicular maturation in human B cells.

Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain this phenotype. We tested the hypothesis that defective non-apoptotic FAS signaling may contribute to impaired B cell differentiation in ALPS. We analyzed secondary lymphoid organs of patients with ALPS-FAS and found low numbers of memory B cells, fewer GC B cells, and an expanded extrafollicular (EF) B cell response. Enhanced mTOR activity has been shown to favor EF versus GC fate decision, and we found enhanced PI3K/mTOR and BCR signaling in ALPS-FAS splenic B cells. Modeling initial T-dependent B cell activation with CD40L in vitro, we showed that FAS competent cells with transient FAS ligation showed specifically decreased mTOR axis activation without apoptosis. Mechanistically, transient FAS engagement with involvement of caspase-8 induced nuclear exclusion of PTEN, leading to mTOR inhibition. In addition, FASL-dependent PTEN nuclear exclusion and mTOR modulation were defective in patients with ALPS-FAS. In the early phase of activation, FAS stimulation promoted expression of genes related to GC initiation at the expense of processes related to the EF response. Hence, our data suggest that non-apoptotic FAS signaling acts as molecular switch between EF versus GC fate decisions via regulation of the mTOR axis and transcription. The defect of this modulatory circuit may explain the observed hypergammaglobulinemia and low memory B cell numbers in ALPS.

Single-cell transcriptomics reveal different maturation stages and sublineage commitment of human thymic invariant natural killer T cells.

Invariant natural killer T cells are a rare, heterogeneous T-cell subset with cytotoxic and immunomodulatory properties. During thymic development, murine invariant natural killer T cells go through different maturation stages differentiating into distinct sublineages, namely, invariant natural killer T1, 2, and 17 cells. Recent reports indicate that invariant natural killer T2 cells display immature properties and give rise to other subsets, whereas invariant natural killer T1 cells seem to be terminally differentiated. Whether human invariant natural killer T cells follow a similar differentiation model is still unknown. To define the maturation stages and assess the sublineage commitment of human invariant natural killer T cells during thymic development, in this study, we performed single-cell RNA sequencing analysis on human Vα24+Vß11+ invariant natural killer T cells isolated from thymocytes. We show that these invariant natural killer T cells displayed heterogeneity, and our unsupervised analysis identified 5 clusters representing different maturation stages, from an immature profile with high expression of genes important for invariant natural killer T cell development and proliferation to a mature, fully differentiated profile with high levels of cytotoxic effector molecules. Evaluation of expression of sublineage-defining gene sets revealed mainly cells with an invariant natural killer T2 signature in the most immature cluster, whereas the more differentiated ones displayed an invariant natural killer T1 signature. Combined analysis with a publicly available single-cell RNA sequencing data set of human invariant natural killer T cells from peripheral blood suggested that the 2 main subsets exist both in thymus and in the periphery, while a third more immature one was restricted to the thymus. Our data point to the existence of different maturation stages of human thymic invariant natural killer T cells and provide evidence for sublineage commitment of invariant natural killer T cells in the human thymus.

Autoimmune lymphoproliferative immunodeficiencies (ALPIDs): A proposed approach to redefining ALPS and other lymphoproliferative immune disorders.

Chronic nonmalignant lymphoproliferation and autoimmune cytopenia are relevant manifestations of immunohematologic diseases of childhood. Their diagnostic classification is challenging but important for therapy. Autoimmune lymphoproliferative syndrome (ALPS) is a genetically defined inborn error of immunity combining these manifestations, but it can explain only a small proportion of cases. Diagnostic categories such as ALPS-like disease, common variable immunodeficiency, or Evans syndrome have therefore been used. Advances in genetics and increasing availablity of targeted therapies call for more therapy-oriented disease classification. Moreover, recent discoveries in the (re)analysis of genetic conditions affecting FAS signaling ask for a more precise definition of ALPS. In this review, we propose the term autoimmune lymphoproliferative immunodeficiencies for a disease phenotype that is enriched for patients with genetic diseases for which targeted therapies are available. For patients without a current molecular diagnosis, this term defines a subgroup of immune dysregulatory disorders for further studies. Within the concept of autoimmune lymphoproliferative immunodeficiencies, we propose a revision of the ALPS classification, restricting use of this term to conditions with clear evidence of perturbation of FAS signaling and resulting specific biologic and clinical consequences. This proposed approach to redefining ALPS and other lymphoproliferative conditions provides a framework for disease classification and diagnosis that is relevant for the many specialists confronted with these diseases.

Combined germline and somatic human FADD mutations cause autoimmune lymphoproliferative syndrome.

BACKGROUND: The autoimmune lymphoproliferative syndrome (ALPS) is a noninfectious and nonmalignant lymphoproliferative disease frequently associated with autoimmune cytopenia resulting from defective FAS signaling. We previously described germline monoallelic FAS (TNFRSF6) haploinsufficient mutations associated with somatic events, such as loss of heterozygosity on the second allele of FAS, as a cause of ALPS-FAS. These somatic events were identified by sequencing FAS in DNA from double-negative (DN) T cells, the pathognomonic T-cell subset in ALPS, in which the somatic events accumulated. OBJECTIVE: We sought to identify whether a somatic event affecting the FAS-associated death domain (FADD) gene could be related to the disease onset in 4 unrelated patients with ALPS carrying a germline monoallelic mutation of the FADD protein inherited from a healthy parent. METHODS: We sequenced FADD and performed array-based comparative genomic hybridization using DNA from sorted CD4+ or DN T cells. RESULTS: We found homozygous FADD mutations in the DN T cells from all 4 patients, which resulted from uniparental disomy. FADD deficiency caused by germline heterozygous FADD mutations associated with a somatic loss of heterozygosity was a phenocopy of ALPS-FAS without the more complex symptoms reported in patients with germline biallelic FADD mutations. CONCLUSIONS: The association of germline and somatic events affecting the FADD gene is a new genetic cause of ALPS.

Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing.

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

AKT activity orchestrates marginal zone B cell development in mice and humans.

The signals controlling marginal zone (MZ) and follicular (FO) B cell development remain incompletely understood. Here, we show that AKT orchestrates MZ B cell formation in mice and humans. Genetic models that increase AKT signaling in B cells or abolish its impact on FoxO transcription factors highlight the AKT-FoxO axis as an on-off switch for MZ B cell formation in mice. In humans, splenic immunoglobulin (Ig) D+CD27+ B cells, proposed as an MZ B cell equivalent, display higher AKT signaling than naive IgD+CD27- and memory IgD-CD27+ B cells and develop in an AKT-dependent manner from their precursors in vitro, underlining the conservation of this developmental pathway. Consistently, CD148 is identified as a receptor indicative of the level of AKT signaling in B cells, expressed at a higher level in MZ B cells than FO B cells in mice as well as humans.

Autoimmunity and immunodeficiency associated with monoallelic LIG4 mutations via haploinsufficiency.

BACKGROUND: Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. OBJECTIVES: This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. METHODS: Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed. RESULTS: A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4+ T cells and low TCR-Vα7.2+ T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. CONCLUSIONS: This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.

Revisiting autoimmune lymphoproliferative syndrome caused by Fas ligand mutations.

BACKGROUND: Fas ligand (FasL) is expressed by activated T cells and induces death in target cells upon binding to Fas. Loss-of-function FAS or FASLG mutations cause autoimmune-lymphoproliferative syndrome (ALPS) characterized by expanded double-negative T cells (DNT) and elevated serum biomarkers. While most ALPS patients carry heterozygous FAS mutations, FASLG mutations are rare and usually biallelic. Only 2 heterozygous variants were reported, associated with an atypical clinical phenotype. OBJECTIVE: We revisited the significance of heterozygous FASLG mutations as a cause of ALPS. METHODS: Clinical features and biomarkers were analyzed in 24 individuals with homozygous or heterozygous FASLG variants predicted to be deleterious. Cytotoxicity assays were performed with patient T cells and biochemical assays with recombinant FasL. RESULTS: Homozygous FASLG variants abrogated cytotoxicity and resulted in early-onset severe ALPS with elevated DNT, raised vitamin B12, and usually no soluble FasL. In contrast, heterozygous variants affected FasL function by reducing expression, impairing trimerization, or preventing Fas binding. However, they were not associated with elevated DNT and vitamin B12, and they did not affect FasL-mediated cytotoxicity. The dominant-negative effects of previously published variants could not be confirmed. Even Y166C, causing loss of Fas binding with a dominant-negative effect in biochemical assays, did not impair cellular cytotoxicity or cause vitamin B12 and DNT elevation. CONCLUSION: Heterozygous loss-of-function mutations are better tolerated for FASLG than for FAS, which may explain the low frequency of ALPS-FASLG.

Monogenic early-onset lymphoproliferation and autoimmunity: Natural history of STAT3 gain-of-function syndrome.

BACKGROUND: In 2014, germline signal transducer and activator of transcription (STAT) 3 gain-of-function (GOF) mutations were first described to cause a novel multisystem disease of early-onset lymphoproliferation and autoimmunity. OBJECTIVE: This pivotal cohort study defines the scope, natural history, treatment, and overall survival of a large global cohort of patients with pathogenic STAT3 GOF variants. METHODS: We identified 191 patients from 33 countries with 72 unique mutations. Inclusion criteria included symptoms of immune dysregulation and a biochemically confirmed germline heterozygous GOF variant in STAT3. RESULTS: Overall survival was 88%, median age at onset of symptoms was 2.3 years, and median age at diagnosis was 12 years. Immune dysregulatory features were present in all patients: lymphoproliferation was the most common manifestation (73%); increased frequencies of double-negative (CD4-CD8-) T cells were found in 83% of patients tested. Autoimmune cytopenias were the second most common clinical manifestation (67%), followed by growth delay, enteropathy, skin disease, pulmonary disease, endocrinopathy, arthritis, autoimmune hepatitis, neurologic disease, vasculopathy, renal disease, and malignancy. Infections were reported in 72% of the cohort. A cellular and humoral immunodeficiency was observed in 37% and 51% of patients, respectively. Clinical symptoms dramatically improved in patients treated with JAK inhibitors, while a variety of other immunomodulatory treatment modalities were less efficacious. Thus far, 23 patients have undergone bone marrow transplantation, with a 62% survival rate. CONCLUSION: STAT3 GOF patients present with a wide array of immune-mediated disease including lymphoproliferation, autoimmune cytopenias, and multisystem autoimmunity. Patient care tends to be siloed, without a clear treatment strategy. Thus, early identification and prompt treatment implementation are lifesaving for STAT3 GOF syndrome.

The expansion of human T-bethighCD21low B cells is T cell dependent.

Accumulation of human CD21low B cells in peripheral blood is a hallmark of chronic activation of the adaptive immune system in certain infections and autoimmune disorders. The molecular pathways underpinning the development, function, and fate of these CD21low B cells remain incompletely characterized. Here, combined transcriptomic and chromatin accessibility analyses supported a prominent role for the transcription factor T-bet in the transcriptional regulation of these T-bethighCD21low B cells. Investigating essential signals for generating these cells in vitro established that B cell receptor (BCR)/interferon-γ receptor (IFNγR) costimulation induced the highest levels of T-bet expression and enabled their differentiation during cell cultures with Toll-like receptor (TLR) ligand or CD40L/interleukin-21 (IL-21) stimulation. Low proportions of CD21low B cells in peripheral blood from patients with defined inborn errors of immunity (IEI), because of mutations affecting canonical NF-κB, CD40, and IL-21 receptor or IL-12/IFNγ/IFNγ receptor/signal transducer and activator of transcription 1 (STAT1) signaling, substantiated the essential roles of BCR- and certain T cell­derived signals in the in vivo expansion of T-bethighCD21low B cells. Disturbed TLR signaling due to MyD88 or IRAK4 deficiency was not associated with reduced CD21low B cell proportions. The expansion of human T-bethighCD21low B cells correlated with an expansion of circulating T follicular helper 1 (cTfh1) and T peripheral helper (Tph) cells, identifying potential sources of CD40L, IL-21, and IFNγ signals. Thus, we identified important pathways to target autoreactive T-bethighCD21low B cells in human autoimmune conditions, where these cells are linked to pathogenesis and disease progression.

A distinct CD38+CD45RA+ population of CD4+, CD8+, and double-negative T cells is controlled by FAS.

The identification and characterization of rare immune cell populations in humans can be facilitated by their growth advantage in the context of specific genetic diseases. Here, we use autoimmune lymphoproliferative syndrome to identify a population of FAS-controlled TCRαß+ T cells. They include CD4+, CD8+, and double-negative T cells and can be defined by a CD38+CD45RA+T-BET- expression pattern. These unconventional T cells are present in healthy individuals, are generated before birth, are enriched in lymphoid tissue, and do not expand during acute viral infection. They are characterized by a unique molecular signature that is unambiguously different from other known T cell differentiation subsets and independent of CD4 or CD8 expression. Functionally, FAS-controlled T cells represent highly proliferative, noncytotoxic T cells with an IL-10 cytokine bias. Mechanistically, regulation of this physiological population is mediated by FAS and CTLA4 signaling, and its survival is enhanced by mTOR and STAT3 signals. Genetic alterations in these pathways result in expansion of FAS-controlled T cells, which can cause significant lymphoproliferative disease.

Germline TET2 loss of function causes childhood immunodeficiency and lymphoma.

Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.

Distinct molecular response patterns of activating STAT3 mutations associate with penetrance of lymphoproliferation and autoimmunity.

Germline STAT3 gain-of-function (GOF) mutations have been linked to poly-autoimmunity and lymphoproliferation with variable expressivity and incomplete penetrance. Here we studied the impact of 17 different STAT3 GOF mutations on the canonical STAT3 signaling pathway and correlated the molecular results with clinical manifestations. The mutations clustered in three groups. Group 1 mutants showed altered STAT3 phosphorylation kinetics and strong basal transcriptional activity. They were associated with the highest penetrance of lymphoproliferation and autoimmunity. Group 2 mutants showed a strongly inducible transcriptional reporter activity and were clinically less penetrant. Group 3 mutants were mostly located in the DNA binding domain and showed the strongest DNA binding affinity despite a poor transcriptional reporter response. Thus, the GOF effect of STAT3 mutations is determined by a heterogeneous response pattern at the molecular level. The correlation of response pattern and clinical penetrance indicates a significant contribution of mutation-determined effects on disease manifestations.

CD57 identifies T cells with functional senescence before terminal differentiation and relative telomere shortening in patients with activated PI3 kinase delta syndrome.

Premature T-cell immunosenescence with CD57+ CD8+ T-cell accumulation has been linked to immunodeficiency and autoimmunity in primary immunodeficiencies including activated PI3 kinase delta syndrome (APDS). To address whether CD57 marks the typical senescent T-cell population seen in adult individuals or identifies a distinct population in APDS, we compared CD57+ CD8+ T cells from mostly pediatric APDS patients to those of healthy adults with similarly prominent senescent T cells. CD57+ CD8+ T cells from APDS patients were less differentiated with more CD27+ CD28+ effector memory T cells showing increased PD1 and Eomesodermin expression. In addition, transition of naïve to CD57+ CD8+ T cells was not associated with the characteristic telomere shortening. Nevertheless, they showed the increased interferon-gamma secretion, enhanced degranulation and reduced in vitro proliferation typical of senescent CD57+ CD8+ T cells. Thus, hyperactive PI3 kinase signaling favors premature accumulation of a CD57+ CD8+ T-cell population, which shows most functional features of typical senescent T cells, but is different in terms of differentiation and relative telomere shortening. Initial observations indicate that this specific differentiation state may offer the opportunity to revert premature T-cell immunosenescence and its potential contribution to inflammation and immunodeficiency in APDS.

Corrigendum: Clinical and Molecular Heterogeneity of RTEL1 Deficiency.

[This corrects the article on p. 449 in vol. 8, PMID: 28507545.].

Mitochondrial Priming by CD28.

T cell receptor (TCR) signaling without CD28 can elicit primary effector T cells, but memory T cells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon T cell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). microRNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for T cells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory T cells. Our data show that initial CD28 signals during T cell activation prime mitochondria with latent metabolic capacity that is essential for future T cell responses.

Clinical and Molecular Heterogeneity of RTEL1 Deficiency.

Typical features of dyskeratosis congenita (DC) resulting from excessive telomere shortening include bone marrow failure (BMF), mucosal fragility, and pulmonary or liver fibrosis. In more severe cases, immune deficiency and recurring infections can add to disease severity. RTEL1 deficiency has recently been described as a major genetic etiology, but the molecular basis and clinical consequences of RTEL1-associated DC are incompletely characterized. We report our observations in a cohort of six patients: five with novel biallelic RTEL1 mutations p.Trp456Cys, p.Ile425Thr, p.Cys1244ProfsX17, p.Pro884_Gln885ins53X13, and one with novel heterozygous mutation p.Val796AlafsX4. The most unifying features were hypocellular BMF in 6/6 and B-/NK-cell lymphopenia in 5/6 patients. In addition, three patients with homozygous mutations p.Trp456Cys or p.Ile425Thr also suffered from immunodeficiency, cerebellar hypoplasia, and enteropathy, consistent with Hoyeraal-Hreidarsson syndrome. Chromosomal breakage resembling a homologous recombination defect was detected in patient-derived fibroblasts but not in hematopoietic compartment. Notably, in both cellular compartments, differential expression of 1243aa and 1219/1300aa RTEL1 isoforms was observed. In fibroblasts, response to ionizing irradiation and non-homologous end joining were not impaired. Telomeric circles did not accumulate in patient-derived primary cells and lymphoblastoid cell lines, implying alternative pathomechanisms for telomeric loss. Overall, RTEL1-deficient cells exhibited a phenotype of replicative exhaustion, spontaneous apoptosis and senescence. Specifically, CD34+ cells failed to expand in vitro, B-cell development was compromised, and T-cells did not proliferate in long-term culture. Finally, we report on the natural history and outcome of our patients. While two patients died from infections, hematopoietic stem cell transplantation (HSCT) resulted in sustained engraftment in two patients. Whether chemotherapy negatively impacts on the course and onset of other DC-related symptoms remains open at present. Early-onset lung disease occurred in one of our patients after HSCT. In conclusion, RTEL deficiency can show a heterogeneous clinical picture ranging from mild hypocellular BMF with B/NK cell lymphopenia to early-onset, very severe, and rapidly progressing cellular deficiency.

IgG4-related disease in autoimmune lymphoproliferative syndrome.

A patient with autoimmune lymphoproliferative disorder (ALPS) developed IgG4-related disease. In retrospect, he had high levels of serum IgG4 for several years prior to presenting with IgG4-related pancreatitis. These high IgG4 levels were masked by hypergammaglobulinemia, a common feature of ALPS. We next screened 18 ALPS patients; four of them displayed increased levels of IgG4. Hence, IgG4-related disease should be considered in ALPS patients, especially in those manifesting lymphocytic organ infiltration or excessive hypergammaglobulinaemia. Screening of IgG4-related disease patients for ALPS-associated mutations would provide further information on whether this disease could be a late-onset atypical presentation of ALPS.