Background CD27 is a lymphocyte costimulatory molecule that regulates T-cell, natural killer (NK) cell, B-cell, and plasma cell function, survival, and differentiation. On the basis of its function ...and expression pattern, we considered CD27 a candidate gene in patients with hypogammaglobulinemia. Objective We sought to describe the clinical and immunologic phenotypes of patients with genetic CD27 deficiency. Methods A molecular and extended immunologic analysis was performed on 2 patients lacking CD27 expression. Results We identified 2 brothers with a homozygous mutation in CD27 leading to absence of CD27 expression. Both patients had persistent symptomatic EBV viremia. The index patient was hypogammaglobulinemic, and immunoglobulin replacement therapy was initiated. His brother had aplastic anemia in the course of his EBV infection and died from fulminant gram-positive bacterial sepsis. Immunologically, lack of CD27 expression was associated with impaired T cell–dependent B-cell responses and T-cell dysfunction. Conclusion Our findings identify a role for CD27 in human subjects and suggest that this deficiency can explain particular cases of persistent symptomatic EBV viremia with hypogammaglobulinemia and impaired T cell–dependent antibody generation.
Background Human hypomorphic nuclear factor-κB essential modulator (NEMO) mutations cause diverse clinical and immunologic phenotypes, but understanding of their scope and mechanistic links to immune ...function and genotype is incomplete. Objective We created and analyzed a database of hypomorphic NEMO mutations to determine the spectrum of phenotypes and their associated genotypes and sought to establish a standardized NEMO reconstitution system to obtain mechanistic insights. Methods Phenotypes of 72 individuals with NEMO mutations were compiled. NEMO L153R and C417R were investigated further in a reconstitution system. TNF-α or Toll-like receptor (TLR)–5 signals were evaluated for nuclear factor-κB activation, programmed cell death, and A20 gene expression. Results Thirty-two different mutations were identified; 53% affect the zinc finger domain. Seventy-seven percent were associated with ectodermal dysplasia, 86% with serious pyogenic infection, 39% with mycobacterial infection, 19% with serious viral infection, and 23% with inflammatory diseases. Thirty-six percent of individuals died at a mean age of 6.4 years. CD40, IL-1, TNF-α, TLR, and T-cell receptor signals were impaired in 15 of 16 (94%), 6 of 7 (86%), 9 of 11 (82%), 9 of 14 (64%), and 7 of 18 (39%), respectively. Hypomorphism-reconstituted NEMO-deficient cells demonstrated partial restoration of NEMO functions. Although both L153R and C417R impaired TLR and TNF-α–induced NF-κB activation, L153R also increased TNF-α–induced programmed cell death with decreased A20 expression. Conclusion Distinct NEMO hypomorphs define specific disease and genetic characteristics. A reconstitution system can identify attributes of hypomorphisms independent of an individual's genetic background. Apoptosis susceptibility in L153R reconstituted cells defines a specific phenotype of this mutation that likely contributes to the excessive inflammation with which it is clinically associated.
Background Dedicator of cytokinesis 8 (DOCK8) mutations are responsible for a rare primary combined immunodeficiency syndrome associated with severe cutaneous viral infections, increased IgE levels, ...autoimmunity, and malignancy. Natural killer (NK) cells are essential for tumor surveillance and defense against virally infected cells. NK cell function relies on Wiskott-Aldrich syndrome protein for filamentous actin (F-actin) accumulation at the lytic NK cell immunologic synapse. DOCK8 activates cell division cycle 42, which, together with Wiskott-Aldrich syndrome protein, coordinates F-actin reorganization. Although abnormalities in T- and B-cell function have been described in DOCK8-deficient patients, the role of NK cells in this disease is unclear. Objectives We sought to understand the role of DOCK8 in NK cell function to determine whether NK cell abnormalities explain the pathogenesis of the clinical syndrome of DOCK8 deficiency. Methods A cohort of DOCK8-deficient patients was assembled, and patients' NK cells, as well as NK cell lines with stably reduced DOCK8 expression, were studied. NK cell cytotoxicity, F-actin content, and lytic immunologic synapse formation were measured. Results DOCK8-deficient patients' NK cells and DOCK8 knockdown cell lines all had decreased NK cell cytotoxicity, which could not be restored after IL-2 stimulation. Importantly, DOCK8 deficiency impaired F-actin accumulation at the lytic immunologic synapse without affecting overall NK cell F-actin content. Conclusions DOCK8 deficiency results in severely impaired NK cell function because of an inability to form a mature lytic immunologic synapse through targeted synaptic F-actin accumulation. This defect might underlie and explain important attributes of the DOCK8 deficiency clinical syndrome, including the unusual susceptibility to viral infection and malignancy.
In melanocytes Rab27a interacts with melanophilin to mediate the melanosome transport required for pigmentation.1 In natural killer (NK) cells and cytotoxic T lymphocytes, degranulation and thereby ...cell-mediated cytotoxicity require interaction of the active form of Rab27a with Munc13-4.2,3 Mutations in the human RAB27A gene cause the autosomal recessive Griscelli syndrome type 2 (GS2) with diluted pigmentation and immunodeficiency, which can progress to hemophagocytic lymphohistiocytosis (HLH).4 Recently, 6 patients with GS2 and normal pigmentation have been reported, identifying Rab27a mutations that selectively disrupt Munc13-4 binding when re-expressed in HEK293 cells.5 Here we present a new case of GS2 with no dilution of pigmentation and extend prior biochemical results through physiologic cell expression studies. Using NK and melanoma cell lines, we have investigated the binding activity of the mutations to the endogenously expressed interaction partners in comparison with wild-type Rab27a. ...we confirm the previously described biology in a truly physiologic context using cells that have full ability to mediate the functions of cytotoxicity or pigmentation.
Background DiGeorge syndrome affects more than 3.5 million persons worldwide. Partial DiGeorge syndrome (pDGS), which is characterized by a number of gene deletions in chromosome 22, including the ...chicken tumor virus number 10 regulator of kinase (Crk)–like (CrkL) gene, is one of the most common genetic disorders in human subjects. To date, the role of natural killer (NK) cells in patients with pDGS remains unclear. Objective We sought to define the effect of pDGS-related Crk haploinsufficiency on NK cell activation and cytotoxic immunological synapse (IS) structure and function. Methods Inducible CrkL-silenced NK cells were used to recapitulate the pDGS, CrkL-haploinsufficient phenotype. Findings were validated by using NK cells from patients with actual pDGS. Ultimately, deficits in the function of NK cells from patients with pDGS were restored by lentiviral transduction of CrkL. Results Silencing of CrkL expression inhibits NK cell function. Specifically, pDGS haploinsufficiency of CrkL inhibits accumulation of activating receptors, polarization of cytolytic machinery and key signaling molecules, and activation of β2 -integrin at the IS. Reintroduction of CrkL protein restores NK cell cytotoxicity. Conclusion CrkL haploinsufficiency causes functional NK deficits in patients with pDGS by disrupting both β2 -integrin activation and activating receptor accumulation at the IS. Our results suggest that NK cell IS quality can directly affect immune status, providing a potential target for diagnosis and therapeutic manipulation in patients with pDGS and in other patients with functional NK cell deficiencies.
Earlier identification and intervention with HSCT is associated with fewer short-term complications and improved long-term survival.1 Long-term follow-up studies of post-transplant SCID patients ...document sustained T cell engraftment that is nonetheless complicated by respiratory disease, growth and neurodevelopmental impairment, and susceptibility to chronic infections.2 Severe, chronic human papillomavirus (HPV) infections have been noted in subsets of SCID patients post-transplant.3,4 This has been confined to only those having γc or JAK3 mutations. Appendix  Patient 1 Patient 2 Patient 3 Patient 4 SCID phenotype T-B+NK- T-B+NK- T-B+NK- T-B+NK- Specific diagnosis γc γc γc γc Age at transplant (d) 22 120 14 219 Type of transplant MMRD mother MMRD mother MRD sister MRD sister Pretransplant conditioning None None None None T-cell depletion None None None None Engraftment Mixed (T+B-) Mixed (T+B-) Mixed (T+B-) Mixed (T+B-) T cell engraftment (%) 100 99 98 100 Time of onset of warts after transplant (y) 7.75 9.4 7 5.75 Location of warts Face, trunk Trunk, arms Face, hands, feet, arms, legs Face, hands, feet, arms, legs Other long-term comorbidities PLE, asthma, on immunoglobulin replacement None None None Most recent ALC cells/mm3 1755 2014 1650 1482 Most recent CD3 cells/mm3 1615 (92%) 1672 (83%) 1518 (92%) 1289 (87%) Most recent CD4 cells/mm3 562 (32%) 467 (23%) 726 (45%) 888 (60%) Most recent CD19 cells/mm3 123 (7%) 282 (14%) 99 (6%) 133 (9%) Most recent IgG mg/dL 599 873 1139 1690 Table I Demographic, clinical, and immunologic characteristics of 4 patients with severe HPV disease ALC, Absolute lymphocyte count; MRD, matched related transplant; MMRD, mismatched related transplant; PLE, protein losing enteropathy.
Background Autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity ...in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. Objective To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of β-glucan through the Dectin-1 pathway, which is required for defense against Candida species. Methods Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. Results PBMCs from patients with APECED syndrome had reduced TNF-α responses after Dectin-1 ligation but in part used a Raf-1–mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-α release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain–containing protein 9 after receptor ligation and localized with Dectin-1 at the cell membrane. Conclusion AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome.
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