Itai M. Pessach

Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE). We tested the hypothesis that the pathogenesis of HSE involves non-haematopoietic CNS-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of interferon-β (IFN-β) and/or IFN-λ1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-λ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-α or IFN-β ( IFN-α/β) but not IFN-λ1. Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3-pathway deficiencies.

A novel primary human immunodeficiency due to deficiency in the WASP-interacting protein WIP.

A homozygous mutation that gave rise to a stop codon in the WIPF1 gene resulted in WASP protein destabilization and in symptoms resembling those of Wiskott-Aldrich syndrome

Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes

Cytosolic phospholipase A2 (cPLA2)–generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.

Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro–B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP–keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4+ T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell–activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations.

Recent Advances in Primary Immunodeficiencies: Identification of Novel Genetic Defects and Unanticipated Phenotypes

Primary immunodeficiencies (PIDs) have traditionally been defined according to their immunologic phenotype. Far from being concluded, the search for human genes that, when mutated, cause PID is actively being pursued. During the last year, four novel genetic defects that cause severe combined immunodeficiency and severe congenital neutropenia have been identified. At the same time, the immunologic definition of primary immunodeficiencies has been expanded by the recognition that genetic defects affecting innate immunity may result in selective predisposition to certain infections, such as mycobacterial disease, herpes simplex encephalitis, and invasive pneumococcal infections. Studies of genetically determined susceptibility to infections have recently shown that immunologic defects may also account for novel infectious phenotypes, such as malaria or leprosy. Finally, a growing body of evidence indicates that primary immunodeficiencies may present with a noninfectious clinical phenotype that may be restricted to single organs, as in the case of atypical hemolytic uremic syndrome or pulmonary alveolar proteinosis. Overall, these achievements highlight the importance of human models, which often differ from the corresponding animal models.

Cytosolic phospholipase A2 is responsible for prostaglandin E2 and leukotriene B4 formation in phagocyte-like PLB-985 cells: studies of differentiated cPLA2-deficient PLB-985 cells

Our previously established model of cytosolic phospholipase A2 (cPLA2)‐deficient, differentiated PLB‐985 cells (PLB‐D cells) was used to determine the physiological role of cPLA2 in eicosanoid production. Parent PLB‐985 (PLB) cells and PLB‐D cells were differentiated toward the monocyte or granulocyte lineages using 5 × 10−8 M 1,25 dihydroxyvitamin D3 or 1.25% dimethyl sulfoxide, respectively. Parent monocyte‐ or granulocyte‐like PLB cells released prostaglandin E2 (PGE2) when stimulated by ionomycin, A23187, opsonized zymosan, phorbol 12‐myristate 13‐acetate, or formyl‐Met‐Leu‐Phe (fMLP), and monocyte‐ or granulocyte‐like PLB‐D cells did not release PGE2 with any of the agonists. The kinetics of cPLA2 translocation to nuclear fractions in monocyte‐like PLB cells stimulated with fMLP or ionomycin was in correlation with the kinetics of PGE2 production. Granulocyte‐like PLB cells, but not granulocyte‐like PLB‐D cells, secreted leukotriene B4 (LTB4) after stimulation with ionomycin or A23187. Preincubation of monocyte‐like parent PLB cells with 100 ng/ml lipopolysaccharide (LPS) for 16 h enhanced stimulated PGE2 production, which is in correlation with the increased levels of cPLA2 detected in these cells. LPS preincubation was less potent in increasing PGE2 and LTB4 secretion and did not affect cPLA2 expression in granulocyte‐like PLB cells, which may be a result of their lower levels of surface LPS receptor expression. LPS had no effect on monocyte‐ or granulocyte‐like PLB‐D cells. The lack of eicosanoid formation in stimulated, differentiated cPLA2‐deficient PLB cells indicates that cPLA2 contributes to stimulated eicosanoid formation in monocyte‐ and granulocyte‐like PLB cells.

Modeling altered T-cell development with induced pluripotent stem cells from patients with RAG1-dependent immune deficiencies.

Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-β (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.

Out of breath: GM-CSFRα mutations disrupt surfactant homeostasis

Pulmonary alveolar proteinosis (PAP) is a rare disorder in which surfactant homeostasis in the lung is impaired, causing respiratory distress and, in severe cases, respiratory failure. Most cases of PAP are associated with the formation of autoantibodies against the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF), which is required for normal surfactant homeostasis and lung function. New studies now identify three patients in whom PAP was caused by mutations in the gene encoding the ligand-binding α chain of the GM-CSF receptor.

Thymic and bone marrow output in individuals with 22q11.2 deletion syndrome

Background:The 22q11.2 deletion syndrome (22q11.2DS) is a congenital multisystem anomaly characterized by typical facial features, palatal anomalies, congenital heart defects, hypocalcemia, immunodeficiency, and cognitive and neuropsychiatric symptoms. The aim of our study was to investigate T- and B-lymphocyte characteristics associated with 22q11.2DS.Methods:Seventy-five individuals with 22q11.2DS were tested for T and B lymphocytes by examination of T-cell receptor rearrangement excision circles (TRECs) and B-cell κ-deleting recombination excision circles (KRECs), respectively.Results:The 22q11.2DS individuals displayed low levels of TRECs, while exhibiting normal levels of KRECs. There was a significant positive correlation between TREC and KREC in the 22q11.2DS group, but not in controls. Both TREC and KREC levels showed a significant decrease with age and only TREC was low in 22q11.2DS individuals with recurrent infections. No difference in TREC levels was found between 22q11.2DS individuals who underwent heart surgery (with or without thymectomy) and those who did not.Conclusion:T-cell immunodeficiency in 22q11.2DS includes low TREC levels, which may contribute to recurrent infections in individuals with this syndrome. A correlation between T- and B-cell abnormalities in 22q11.2DS was identified. The B-cell abnormalities could account for part of the immunological deficiency seen in 22q11.2DS.

The Relationship of X-Linked Primary Immune Deficiencies and Autoimmunity

It is well-known that autoimmunity is significantly more prevalent in females. Growing evidence indicates that genes located on the X chromosome may play a role in autoimmunity and immune dysregulation, as also indicated by the frequent association of autoimmune phenomena in patients with X-linked primary immune deficiencies (PIDs). Hence, this group of genetic disorders is of particular interest to study PID-causing genes in the setting of more complex autoimmune disorders. This review focuses on the mechanisms leading to the autoimmune phenomena that are associated with the different X-linked PIDs, and on the intriguing interplay between immune dysregulation and immune deficiency in this unique setting.