ALPS, FAS, and beyond: from inborn errors of immunity to acquired immunodeficiencies.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune regulatory disorder characterized by benign or malignant lymphoproliferation and autoimmunity. Classically, ALPS is due to mutations in FAS and other related genes; however, recent research revealed that other genes could be responsible for similar clinical features. Therefore, ALPS classification and diagnostic criteria have changed over time, and several ALPS-like disorders have been recently identified. Moreover, mutations in FAS often show an incomplete penetrance, and certain genotypes have been associated to a dominant or recessive inheritance pattern. FAS mutations may also be acquired or could become pathogenic when associated to variants in other genes, delineating a possible digenic type of inheritance. Intriguingly, variants in FAS and increased TCR αß double-negative T cells (DNTs, a hallmark of ALPS) have been identified in multifactorial autoimmune diseases, while FAS itself could play a potential role in carcinogenesis. These findings suggest that alterations of FAS-mediated apoptosis could trespass the universe of inborn errors of immunity and that somatic mutations leading to ALPS could only be the tip of the iceberg of acquired immunodeficiencies.

Case Report: FOXP3 Mutation in a Patient Presenting With ALPS.

ALPS and IPEX are two well-characterized inborn errors of immunity with immune dysregulation, considered as two master models of monogenic auto-immune diseases. Thus, with autoimmunity as their primary clinical manifestation, these two entities may show clinical overlap. Traditionally, immunological biomarkers are used to establish an accurate differential diagnosis. Herein, we describe a patient who presented with clinical features and biomarkers fulfilling the diagnostic criteria of ALPS. Severe apoptotic defect was also shown in the patient's cell lines and PHA-activated peripheral blood lymphocytes. Sanger sequencing of the FAS gene did not reveal any causal mutation. NGS screening revealed a novel deleterious variant located in the N terminal repressor domain of FOXP3 but no mutations in the FAS pathway-related genes. TEMRA cells (terminally differentiated effector memory cells re-expressing CD45RA) and PD1 expression were increased arguing in favor of T-cell exhaustion, which could be induced by unrestrained activation of T effector cells because of Treg deficiency. Moreover, defective FOXP3 observed in the patient could intrinsically induce increased proliferation and resistance to apoptosis in T effector cells. This observation expands the spectrum of FOXP3 deficiency and underscores the role of NGS in detecting mutations that induce overlapping phenotypes among inborn errors of immunity with immune dysregulation. In addition, these findings suggest a potential link between FOXP3 and FAS pathways.

Primary Immune Regulatory Disorders With an Autoimmune Lymphoproliferative Syndrome-Like Phenotype: Immunologic Evaluation, Early Diagnosis and Management.

Primary immune regulatory disorders (PIRD) are associated with autoimmunity, autoinflammation and/or dysregulation of lymphocyte homeostasis. Autoimmune lymphoproliferative syndrome (ALPS) is a PIRD due to an apoptotic defect in Fas-FasL pathway and characterized by benign and chronic lymphoproliferation, autoimmunity and increased risk of lymphoma. Clinical manifestations and typical laboratory biomarkers of ALPS have also been found in patients with a gene defect out of the Fas-FasL pathway (ALPS-like disorders). Following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA), we identified more than 600 patients suffering from 24 distinct genetic defects described in the literature with an autoimmune lymphoproliferative phenotype (ALPS-like syndromes) corresponding to phenocopies of primary immunodeficiency (PID) (NRAS, KRAS), susceptibility to EBV (MAGT1, PRKCD, XIAP, SH2D1A, RASGRP1, TNFRSF9), antibody deficiency (PIK3CD gain of function (GOF), PIK3R1 loss of function (LOF), CARD11 GOF), regulatory T-cells defects (CTLA4, LRBA, STAT3 GOF, IL2RA, IL2RB, DEF6), combined immunodeficiencies (ITK, STK4), defects in intrinsic and innate immunity and predisposition to infection (STAT1 GOF, IL12RB1) and autoimmunity/autoinflammation (ADA2, TNFAIP3,TPP2, TET2). CTLA4 and LRBA patients correspond around to 50% of total ALPS-like cases. However, only 100% of CTLA4, PRKCD, TET2 and NRAS/KRAS reported patients had an ALPS-like presentation, while the autoimmunity and lymphoproliferation combination resulted rare in other genetic defects. Recurrent infections, skin lesions, enteropathy and malignancy are the most common clinical manifestations. Some approaches available for the immunological study and identification of ALPS-like patients through flow cytometry and ALPS biomarkers are provided in this work. Protein expression assays for NKG2D, XIAP, SAP, CTLA4 and LRBA deficiencies and functional studies of AKT, STAT1 and STAT3 phosphorylation, are showed as useful tests. Patients suspected to suffer from one of these disorders require rapid and correct diagnosis allowing initiation of tailored specific therapeutic strategies and monitoring thereby improving the prognosis and their quality of life.

Type I interferon activation in RAS-associated autoimmune leukoproliferative disease (RALD).

RAS-associated autoimmune leukoproliferative disease (RALD) is a rare immune dysregulation syndrome caused by somatic gain-of-function mutations of either NRAS or KRAS gene in hematopoietic cells. We describe a 27-year-old patient presenting at 5 months of age with recurrent infections and generalized lymphadenopathy who developed a complex multi-organ autoimmune syndrome with hypogammaglobulinemia, partially controlled with oral steroids, hydroxichloroquine, mofetil mycophenolate and IVIG prophylaxis. Activation of type I interferon pathway was observed in peripheral blood. Since 18 years of age, the patient developed regenerative nodular hyperplasia of the liver evolving into hepatopulmonary syndrome. Whole exome sequencing analysis of the peripheral blood DNA showed the NRAS p.Gly13Asp mutation validated as somatic. Our report highlights the possibility of detecting somatic NRAS gene mutations in patients with inflammatory immune dysregulation and type I interferon activation.

Next Generation Sequencing for Detecting Somatic FAS Mutations in Patients With Autoimmune Lymphoproliferative Syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune regulatory disorder clinically defined by chronic and benign lymphoproliferation, autoimmunity and an increased risk of lymphoma due to a genetic defect in the FAS-FASL apoptotic pathway. Genetic defects associated with ALPS are germinal and somatic mutations in FAS gene, in addition to germinal mutations in FASLG, FADD, CASP8 and CASP10 genes. The accumulation of CD3+TCRαß+CD4-CD8- double negative T-cells (DNT) is a hallmark of the disease and 20-25% of ALPS patients show heterozygous somatic mutations restricted to DNT in the FAS gene (ALPS-sFAS patients). Nowadays, somatic mutations in the FAS gene are detected through Sanger sequencing in isolated DNT. In this study, we report an ALPS-sFAS patient fulfilling clinical and laboratory ALPS criteria, who was diagnosed through NGS with a targeted gene panel using DNA from whole blood. Data analysis was carried out with Torrent Suite Software and variant detection was performed by both germinal and somatic variant caller plugin. The somatic variant caller correctly detected other six ALPS-sFAS patients previously diagnosed in the authors' laboratories. In summary, this approach allows the detection of both germline and somatic mutations related to ALPS by NGS, avoiding the isolation of DNT as the first step. The reads of the somatic variants could be detected even in patients with DNT in the cut off limit. Thus, custom-designed NGS panel testing may be a faster and more reliable method for the diagnosis of new ALPS patients, including those with somatic FAS mutations (ALPS-sFAS).

Autoimmune Cytopenias and Dysregulated Immunophenotype Act as Warning Signs of Inborn Errors of Immunity: Results From a Prospective Study.

Inborn errors of immunity (IEI) are genetic disorders characterized by a wide spectrum of clinical manifestations, ranging from increased susceptibility to infections to significant immune dysregulation. Among these, primary immune regulatory disorders (PIRDs) are mainly presenting with autoimmune manifestations, and autoimmune cytopenias (AICs) can be the first clinical sign. Significantly, AICs in patients with IEI often fail to respond to first-line therapy. In pediatric patients, autoimmune cytopenias can be red flags for IEI. However, for these cases precise indicators or parameters useful to suspect and screen for a hidden congenital immune defect are lacking. Therefore, we focused on chronic/refractory AIC patients to perform an extensive clinical evaluation and multiparametric flow cytometry analysis to select patients in whom PIRD was strongly suspected as candidates for genetic analysis. Key IEI-associated alterations causative of STAT3 GOF disease, IKAROS haploinsufficiency, activated PI3Kδ syndrome (APDS), Kabuki syndrome and autoimmune lymphoproliferative syndrome (ALPS) were identified. In this scenario, a dysregulated immunophenotype acted as a potential screening tool for an early IEI diagnosis, pivotal for appropriate clinical management and for the identification of new therapeutic targets.

Autoimmune Lymphoproliferative Syndrome in Children with Nonmalignant Organomegaly, Chronic Immune Cytopenia, and Newly Diagnosed Lymphoma

This study investigated the frequency of and predictive factors for autoimmune lymphoproliferative syndrome (ALPS) in children with lymphoma, chronic immune cytopenia, and nonmalignant organomegaly. Thirty-four children with suspected ALPS (n=13, lymphoma; n=12, immune cytopenia; n=9, nonmalignant organomegaly) were included. Double-negative T-cells, lymphocyte apoptosis, and genetic findings were analyzed. Patients were stratified into two groups as proven/probable ALPS and clinically suspected patients according to the ALPS diagnostic criteria. Of the 34 patients, 18 (53%) were diagnosed with proven/probable ALPS. One patient had a mutation (c.652-2A>C) in the FAS gene. The remaining 16 (47%) patients were defined as clinically suspected patients. Predictive factors for ALPS were anemia and thrombocytopenia in patients with lymphoma, splenomegaly and lymphadenopathy in patients with immune cytopenia, and young age in patients with nonmalignant organomegaly. ALPS may not be rare in certain risk groups. Our study indicates that screening for ALPS may be useful in children having lymphoma with cytopenia at diagnosis, in those having nonmalignant organomegaly with immune cytopenia, and in those having chronic immune thrombocytopenic purpura or autoimmune hemolytic anemia with organomegaly developing during follow-up.

Defective glycosylation and multisystem abnormalities characterize the primary immunodeficiency XMEN disease.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαß+ T cells (αßDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

Superficial and Deep Cutaneous Involvement by RAS-Associated Autoimmunne Leukoproliferative Disease (RALD Cutis): A Histologic Mimicker of Histiocytoid Sweet Syndrome.

RAS-associated autoimmune leukoproliferative disease (RALD) is a recently described noninfectious and nonmalignant clinical syndrome characterized by autoimmune disorders, massive splenomegaly, modest lymphadenopathy, and monocytosis. On the molecular level, RALD is defined by somatic mutations of either NRAS or KRAS gene in a subset of hematopoietic cells. To date, there is a dearth of well-documented histopathologic description of cutaneous involvement by RALD in the literature. In the current case report, a 43-year-old female patient with a history of RALD presented with clinical pictures of sepsis and an erythematous rash in the left lower extremity. Histologic examination revealed a dense perivascular and interstitial infiltrate of immature myeloid cells admixed with scattered neutrophils involving the dermis and subcutaneous adipose tissue, imparting a panniculitis-like histologic pictures. There was a strong angiocentric propensity of the immature hematopoietic cells as well as extensive extravasation of red blood cells, even in the subcutaneous adipose tissue. Immunohistochemically, the immature hematopoietic cells were positive for CD43, CD4, and CD68, but negative for CD34, CD117, and myeloperoxidase. Overall, the histologic and cytologic findings were highly reminiscent of histiocytoid Sweet syndrome. Review of the English literature revealed cutaneous involvements by RALD only in patients with KRAS mutation compared with none of its NRAS counterparts. However, larger clinicopathologic studies on cutaneous involvement by RALD are warranted. The term "RALD cutis" with its histologic and molecular features is suggested to serve as a potential groundwork for future studies of this rare phenomenon.

Paradoxical CD4 Lymphopenia in Autoimmune Lymphoproliferative Syndrome (ALPS).

Autoimmune lymphoproliferative syndrome (ALPS) is caused by germline or somatic loss of function FAS mutations resulting in impaired apoptosis and consequent expansion of T-lymphocytes causing organomegaly and autoimmune anemia, neutropenia and thrombocytopenia. Herein, we report on a case of disseminated varicella zoster infection after post-partum vaccination in a patient found to have CD4 lymphopenia and eventually diagnosed with ALPS caused by a novel germline missense mutation in FAS death-domain. A subsequent retrospective analysis of 169 patients of the NIH ALPS-FAS cohort, revealed that CD4-T-cells lymphopenia (< 300 cells/µl) may occur in 5% of ALPS-FAS patients irrespectively of the underlying genetic defect, organomegaly or immunosuppressive treatment. Although immunophenotyping did not show depletion of specific CD4-T-cells subpopulations, CD4-lymphopenic ALPS-FAS subjects had an expansion of a subset of circulating T-follicular-helper (cTfh) cells, associated with autoantibody production (CCR7lowPD-1high). Furthermore, autoantibodies binding on CD4-T-cells were detected in 50% of the CD4-lymphopenic ALPS-FAS patients and caused cytotoxicity in a natural killer (NK)-mediated antibody-dependent-cellular cytotoxicity assay. Such autoantibodies can therefore be associated with CD4-T-cell death, impaired activation induced proliferation or impaired trafficking. The expansion of autoreactive T-cells in ALPS-FAS is known to be associated with autoimmune clinical manifestations, however our study reveals that ALPS-FAS can also be associated with a paradoxical depletion of CD4-T-cells due to the presence of autoantibodies on the surface of CD4-T-cells which can in turn result in increased susceptibility to opportunistic infections. These novel findings have implications for the diagnosis, clinical monitoring, and management of patients with ALPS-FAS.

Lack of IL-17 Receptor A signaling aggravates lymphoproliferation in C57BL/6 lpr mice.

Defects in Fas function correlate with susceptibility to systemic autoimmune diseases like autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosus (SLE). C57BL/6 lpr (B6/lpr) mice are used as an animal model of ALPS and develop a mild SLE phenotype. Involvement of interleukin-17A (IL-17A) has been suggested in both phenotypes. Since IL-17 receptor A is part of the signaling pathway of many IL-17 family members we investigated the role of IL-17 receptor signaling in disease development in mice with a B6/lpr background. B6/lpr mice were crossed with IL-17 receptor A deficient (IL-17RA KO) mice and followed over time for disease development. IL-17RA KO/lpr mice presented with significantly enhanced lymphoproliferation compared with B6/lpr mice, which was characterized by dramatic lymphadenomegaly/splenomegaly and increased lymphocyte numbers, expansion of double-negative (DN) T-cells and enhanced plasma cell formation. However, the SLE phenotype was not enhanced, as anti-nuclear antibody (ANA) titers and induction of glomerulonephritis were not different. In contrast, levels of High Mobility Group Box 1 (HMGB1) and anti-HMGB1 autoantibodies were significantly increased in IL-17RA KO/lpr mice compared to B6/lpr mice. These data show that lack of IL-17RA signaling aggravates the lymphoproliferative phenotype in B6/lpr mice but does not affect the SLE phenotype.

Novel insights into FAS defects underlying autoimmune lymphoproliferative syndrome revealed by studies in consanguineous patients.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immunodeficiency disease due to impaired Fas-Fas ligand apoptotic pathway. It is characterized by chronic nonmalignant, noninfectious lymphadenopathy and/or splenomegaly associated with autoimmune manifestations primarily directed against blood cells. Herein, we review the heterogeneous ALPS molecular bases and discuss recent findings revealed by the study of consanguineous patients. Indeed, this peculiar genetic background favored the identification of a novel form of AR ALPS-FAS associated with normal or residual protein expression, expanding the spectrum of ALPS types. In addition, rare mutational mechanisms underlying the splicing defects of FAS exon 6 have been identified in AR ALPS-FAS with lack of protein expression. These findings will help decipher critical regions required for the tight regulation of FAS exon 6 splicing. We also discuss the genotype-phenotype correlation and disease severity in AR ALPS-FAS. Altogether, the study of ALPS molecular bases in endogamous populations helps to better classify the disease subgroups and to unravel the Fas pathway functioning.

ALPS-Like Phenotype Caused by ADA2 Deficiency Rescued by Allogeneic Hematopoietic Stem Cell Transplantation.

Adenosine deaminase 2 (ADA2) deficiency is an auto-inflammatory disease due to mutations in cat eye syndrome chromosome region candidate 1 (CECR1) gene, currently named ADA2. The disease has a wide clinical spectrum encompassing early-onset vasculopathy (targeting skin, gut and central nervous system), recurrent fever, immunodeficiency and bone marrow dysfunction. Different therapeutic options have been proposed in literature, but only steroids and anti-cytokine monoclonal antibodies (such as tumor necrosis factor inhibitor) proved to be effective. If a suitable donor is available, hematopoietic stem cell transplantation (HSCT) could be curative. Here we describe a case of ADA2 deficiency in a 4-year-old Caucasian girl. The patient was initially classified as autoimmune neutropenia and then she evolved toward an autoimmune lymphoproliferative syndrome (ALPS)-like phenotype. The diagnosis of ALPS became uncertain due to atypical clinical features and normal FAS-induced apoptosis test. She was treated with G-CSF first and subsequently with immunosuppressive drugs without improvement. Only HSCT from a 9/10 HLA-matched unrelated donor, following myeloablative conditioning, completely solved the clinical signs related to ADA2 deficiency. Early diagnosis in cases presenting with hematological manifestations, rather than classical vasculopathy, allows the patients to promptly undergo HSCT and avoid more severe evolution. Finally, in similar cases highly suspicious for genetic disease, it is desirable to obtain molecular diagnosis before performing HSCT, since it can influence the transplant procedure. However, if HSCT has to be performed without delay for clinical indication, related donors should be excluded to avoid the risk of relapse or partial benefit due to a hereditary genetic defect.

Autoimmune Lymphoproliferative Syndrome Masquerading as Posttransplant Lymphoproliferative Disorder.

We present a case of a 2-year-old female presenting with diffuse lymphadenopathy 2 years following orthotopic heart transplant. Initially, she was diagnosed with posttransplant lymphoproliferative disease based on clinical presentation and pathology and she was treated accordingly. Because of persistent lymphadenopathy following the completion of chemotherapy and new onset of autoimmune cytopenias, repeat flow of the lymph node showed an elevated double negative T-cell population prompting evaluation for autoimmune lymphoproliferative syndrome (ALPS). A complete workup was confirmative of a germline Fas mutation consistent with ALPS-FAS. This case emphasizes the importance of considering ALPS-FAS in a patient with lymphadenopathy of unknown cause.

STAT5B: A Differential Regulator of the Life and Death of CD4+ Effector Memory T Cells.

Understanding the control of Ag restimulation-induced T cell death (RICD), especially in cancer immunotherapy, where highly proliferating T cells will encounter potentially large amounts of tumor Ags, is important now more than ever. It has been known that growth cytokines make T cells susceptible to RICD, but the precise molecular mediators that govern this in T cell subsets is unknown until now. STAT proteins are a family of transcription factors that regulate gene expression programs underlying key immunological processes. In particular, STAT5 is known to favor the generation and survival of memory T cells. In this study, we report an unexpected role for STAT5 signaling in the death of effector memory T (TEM) cells in mice and humans. TEM cell death was prevented with neutralizing anti-IL-2 Ab or STAT5/JAK3 inhibitors, indicating that STAT5 signaling drives RICD in TEM cells. Moreover, we identified a unique patient with a heterozygous missense mutation in the coiled-coil domain of STAT5B that presented with autoimmune lymphoproliferative syndrome-like features. Similar to Stat5b-/- mice, this patient exhibited increased CD4+ TEM cells in the peripheral blood. The mutant STAT5B protein dominantly interfered with STAT5-driven transcriptional activity, leading to global downregulation of STAT5-regulated genes in patient T cells upon IL-2 stimulation. Notably, CD4+ TEM cells from the patient were strikingly resistant to cell death by in vitro TCR restimulation, a finding that was recapitulated in Stat5b-/- mice. Hence, STAT5B is a crucial regulator of RICD in memory T cells in mice and humans.