Defective FAS-Mediated Apoptosis and Immune Dysregulation in Gaucher Disease.

BACKGROUND: Gaucher disease (GD) is a rare disorder characterized by defective function of ß-glucocerebrosidase, which leads to progressive accumulation of its substrate in various organs, particularly the mononuclear phagocyte system. Hepatosplenomegaly and cytopenia represent the disease's most common features, but patients with GD also show hyperinflammation, hypergammaglobulinemia, and immune dysregulation involving B, T, and natural killer cells. As clinical phenotype can be underhand, symptoms can overlap with autoimmune lymphoproliferative syndrome (ALPS) or other ALPS-like disorders. OBJECTIVE: To evaluate the ALPS-like immunological pattern and apoptosis function in patients with GD. METHODS: We evaluated lymphocyte subsets and immunophenotypic and serological features of ALPS (double-negative T cells [DNTs], B220+DNTs, CD27+, T-reg/HLA-DR ratio, IL-10, IL-18, vitamin B12) in a population of patients with GD. Moreover, we tested FAS/TRAIL-induced apoptosis and CASP8/CASP10/PARP function in patients showing an immune-dysregulation pattern. RESULTS: A total of 41 patients (33 treated, 8 treatment-naïve) were studied. Nine (21%) and 7 (17%) of 41 patients had high DNT and B220+DNT counts, respectively. Overall, 10 of 41(24%) patients showed immunological features suggestive of ALPS that were more frequent in treatment-naïve subjects (P = .040 vs P = .031) and in those with early onset of the disease (P = .046 vs P = .011), respectively. FAS-induced apoptosis and caspase activation were further evaluated in these 10 patients and were found to be defective in 7 of them. CONCLUSIONS: We show that patients with GD may have ALPS-like features and FAS-mediated apoptosis defects that are more pronounced in treatment-naïve subjects and in patients with early onset of the disease. Therefore, diagnostic workup of patients with an ALPS-like phenotype should include screening for GD.

Outcomes and Treatment Strategies for Autoimmunity and Hyperinflammation in Patients with RAG Deficiency.

BACKGROUND: Although autoimmunity and hyperinflammation secondary to recombination activating gene (RAG) deficiency have been associated with delayed diagnosis and even death, our current understanding is limited primarily to small case series. OBJECTIVE: Understand the frequency, severity, and treatment responsiveness of autoimmunity and hyperinflammation in RAG deficiency. METHODS: In reviewing the literature and our own database, we identified 85 patients with RAG deficiency, reported between 2001 and 2016, and compiled the largest case series to date of 63 patients with prominent autoimmune and/or hyperinflammatory pathology. RESULTS: Diagnosis of RAG deficiency was delayed a median of 5 years from the first clinical signs of immune dysregulation. Most patients (55.6%) presented with more than 1 autoimmune or hyperinflammatory complication, with the most common etiologies being cytopenias (84.1%), granulomas (23.8%), and inflammatory skin disorders (19.0%). Infections, including live viral vaccinations, closely preceded the onset of autoimmunity in 28.6% of cases. Autoimmune cytopenias had early onset (median, 1.9, 2.1, and 2.6 years for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively) and were refractory to intravenous immunoglobulin, steroids, and rituximab in most cases (64.7%, 73.7%, and 71.4% for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively). Evans syndrome specifically was associated with lack of response to first-line therapy. Treatment-refractory autoimmunity/hyperinflammation prompted hematopoietic stem cell transplantation in 20 patients. CONCLUSIONS: Autoimmunity/hyperinflammation can be a presenting sign of RAG deficiency and should prompt further evaluation. Multilineage cytopenias are often refractory to immunosuppressive treatment and may require hematopoietic cell transplantation for definitive management.

SCID genotype and 6-month posttransplant CD4 count predict survival and immune recovery.

The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4+ and CD4+CD45RA+ cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4+ cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

Primary Immune Deficiency Treatment Consortium (PIDTC) report.

The Primary Immune Deficiency Treatment Consortium (PIDTC) is a network of 33 centers in North America that study the treatment of rare and severe primary immunodeficiency diseases. Current protocols address the natural history of patients treated for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, and chronic granulomatous disease through retrospective, prospective, and cross-sectional studies. The PIDTC additionally seeks to encourage training of junior investigators, establish partnerships with European and other International colleagues, work with patient advocacy groups to promote community awareness, and conduct pilot demonstration projects. Future goals include the conduct of prospective treatment studies to determine optimal therapies for primary immunodeficiency diseases. To date, the PIDTC has funded 2 pilot projects: newborn screening for SCID in Navajo Native Americans and B-cell reconstitution in patients with SCID after hematopoietic stem cell transplantation. Ten junior investigators have received grant awards. The PIDTC Annual Scientific Workshop has brought together consortium members, outside speakers, patient advocacy groups, and young investigators and trainees to report progress of the protocols and discuss common interests and goals, including new scientific developments and future directions of clinical research. Here we report the progress of the PIDTC to date, highlights of the first 2 PIDTC workshops, and consideration of future consortium objectives.

Assays for B cell and germinal center development.

The study of B cell development is highly relevant for a better understanding of human disorders in which B cells are involved. B cell development in humans appears to proceed largely along a linear pathway and occurs in three compartments: bone marrow, peripheral blood, and lymphoid tissue. The focus of this unit is on the detailed immunophenotypic evaluation of cell suspensions obtained from these compartments. These protocols are based on routine methodology, commonly used by clinical flow laboratories. Special emphasis will be placed on the approach to the study of B cell development, including current knowledge regarding immunophenotypic identification of B cell subsets. Multiparameter flow cytometry provides powerful analytical tools, as long as attention is paid to careful design and proper execution of flow cytometric acquisition and analysis. The goal of this unit is to provide a guide to the flow cytometric study of B cell development in humans.

Autoimmune lymphoproliferative syndrome (ALPS).

In patients with ALPS, defective homeostasis of lymphocytes is reflected in abnormal accumulation of lymphocytes, leading to lymphadenopathy, (hepato)splenomegaly and hypersplenism, autoimmunity due to a failure to remove autoreactive lymphocytes, and inappropriate survival of lymphocytes associated with an increased occurrence of lymphoma. Several of the laboratory findings are unique for ALPS and reflect defective Fas-mediated apoptosis and abnormal immune regulation. Much has been learned about the molecular mechanisms that underlie defective Fas-mediated apoptosis and the complex relationship between genotype, phenotype and disease penetrance. Family studies strongly suggest the contribution of one or more additional factors to the pathogenesis of ALPS. This may pertain to defective immunoregulation by an altered IL-2/IL-2 receptor system, reflected in the specific loss of CD4+/CD25+ T cells, and/or by the highly increased IL-10 levels, but other factors may equally be involved. Treatment strategies remain mostly targeted at the disease manifestations, but more specific therapies directed at the primary pathogenic defects themselves might become possible in the future. Continued efforts directed at both careful clinical follow-up and basic scientific investigation are needed to increase our understanding of the incidence, natural history, and pathogenesis of ALPS. In return, this may prove of benefit for the understanding of autoimmune disease in general.

Common expression of an unusual CD45 isoform on T cells from patients with large granular lymphocyte leukaemia and autoimmune lymphoproliferative syndrome.

Patients with T-cell large granular lymphocyte (T-LGL) leukaemia and autoimmune lymphoproliferative syndrome (ALPS) share many features, including autoimmunity and an expansion of (cytotoxic) T cells, which in ALPS patients express an unusual (B220) isoform of CD45, corresponding to an altered O-glycosylation profile. Here we showed that T-LGL leukaemia cells also expressed this B220 isoform. We hypothesize that B220+ T cells constitute proliferating T cells that have become competent to undergo apoptosis, but that constitutive (ALPS) or functional (T-LGL) defects prevent this process. Altered O-glycosylation of the extracellular domains of CD45 may have consequences for this tyrosine phosphatase as a regulator of cell proliferation and survival.

Human B cells express a CD45 isoform that is similar to murine B220 and is downregulated with acquisition of the memory B-cell marker CD27.

BACKGROUND: Differences between human and murine B cells exist at all stages of B-cell development, including the stage of memory B-cell formation. B cells in mice are identified with the pan-B-cell-specific CD45 isoform, B220. In initial studies in humans, it appeared that B220 expression did not include all B cells. This study was performed to expand on those preliminary findings. METHODS: Multiparameter flow cytometric detection of B220 expression on B cells was combined with a variety of B-cell markers. RESULTS: In contrast to mice, B220 was not a pan-B-cell marker in humans but was downregulated in the majority of B cells that acquired the human memory B-cell marker, CD27, whereas a minor memory B-cell subset remained B220(+), suggesting differences in differentiation. CONCLUSIONS: The B220 isoform in humans is developmentally regulated in humans, tied to the acquisition of a memory phenotype, and as such can be used as a differentiation-specific CD45 isoform, akin to the use of CD45 isoforms to distinguish between naive and memory T-cell subsets. Patients with immunodeficiency disorders, associated with defective memory B-cell generation and absent or reduced CD27(+) B cells, showed a corresponding lack of B220 downregulation consistent with altered differentiation of B-cell subsets.

A composite picture of TcR alpha/beta(+) CD4(-)CD8(-) T Cells (alpha/beta-DNTCs) in humans with autoimmune lymphoproliferative syndrome.

The discovery of an unusual T-cell subset characterized by the expression of the alpha/beta T-cell receptor without expression of either CD4 or CD8 [alpha/beta-double-negative T cells (alpha/beta-DNTCs)] provided critical insights in the evaluation of a "new" lymphoproliferative disorder known as autoimmune lymphoproliferative syndrome (ALPS). ALPS is a disorder of defective Fas-mediated lymphocyte apoptosis, manifested by accumulation of alpha/beta-DNTCs and other lymphocyte subsets, leading to lymphadenopathy and splenomegaly, autoimmunity, and an increased risk of lymphoma. The expanded population of alpha/beta-DNTCs from ALPS patients has a remarkable uniform phenotype that is for the most part similar to alpha/beta-DNTCs from mice with defective Fas (lpr) or Fas ligand (gld). This is in contrast to the minor alpha/beta-DNTC compartment in healthy individuals that contains multiple, immunophenotypically distinct subpopulations. Current data indicate that alpha/beta-DNTCs from ALPS patients are derived from cytotoxic CD8(+) T cells, chronically activated in vivo but anergic in vitro. Their anergic state may be related to persistent modifications of O-linked carbohydrates on cell surface molecules, such as CD43 and CD45, as well as to the increased presence of interleukin-10. Although largely consistent with a model of (linear) CD8(+) cytotoxic T-cell differentiation, the expression patterns of certain surface molecules, such as CD27 and CD28, are not consistent with this model. This may be the result of the perturbed homeostasis of lymphocytes in ALPS, thereby revealing pathways of differentiation and immunophenotypes, including phenotypes pertaining to cell surface glycosylation that are hidden from view in healthy individuals.