A Novel Mutation in the NBD Domain of NLRC4 Causes Mild Autoinflammation With Recurrent Urticaria

BackgroundNOD-like receptor family CARD-containing 4 protein (NLRC4) is a cytosolic protein that forms an inflammasome in response to flagellin and type 3 secretion system (T3SS) proteins from invading Gram-negative bacteria. NLRC4 mutations have been recently identified in early-onset severe autoinflammatory disorders. In this study, we reported a novel mutation in NLRC4 in two Chinese patients, who manifested with recurrent urticaria and arthralgia.MethodsWe summarized the clinical data of the two patients. Gene mutations were identified by whole-exome sequencing (WES). Swiss-PdbViewer was used to predict the pathogenicity of the identified mutations. Cytokine levels and caspase-1 activation were detected in the patient PBMCs with lipopolysaccharide (LPS) stimulation. All previously published cases with NLRC4 mutations were reviewed.ResultsWe identified a missense heterozygous mutation (c.514G>A, p.Gly172Ser), which was located in the highly conserved residue of nucleotide-binding domain (NBD) of NLRC4. The mutation did not alter the expression of NLRC4 protein, but induced considerably much higher production of IL-1β and IL-6 in patient PBMCs than in healthy controls after LPS stimulation. Four NLRC4 inflammasomopathy phenotypes have been described, with severe inflammatory diseases including macrophage activation syndrome, enterocolitis and NOMID in patients with mutations in the NBD and HD1 domains, whereas a mild clinical phenotype was associated with two mutations in the WHD domain of NLRC4.ConclusionWe identified a novel mutation in the NBD domain, and the patients just presented with a mild inflammatory phenotype. Thus, our findings reinforce the diversity of NLRC4 mutations and expand the clinical spectrum of associated diseases.

Autoinflammatory disease with corneal and mucosal dyskeratosis caused by a novel NLRP1 variant

Objectives Here we investigated a patient with inflammatory corneal intraepithelial dyskeratosis, mucosal inflammation, tooth abnormalities and, eczema to uncover the genetic and immunological basis of the disease. Methods On suspicion of an autoinflammatory condition, Sanger sequencing of nucleotide-binding oligomerization domain-like, leucine-rich repeat pyrin domain containing 1 (NLRP1) was performed and combined with an in vitro inflammasome reconstitution assay to measure caspase-1-mediated IL-1β cleavage, stimulation of patient peripheral blood mononuclear cells (PBMCs) and whole blood to measure IL-1β, IL-18 production and quantification of apoptosis-associated speck-like protein containing CARD (ASC) speck formation as a measure of inflammasome activation by flow cytometry. Results Sanger sequencing revealed a novel mutation (c.175G>C, p.A59P; NM_33004.4) in the inflammasome molecule NLRP1 segregating with disease, although with incomplete penetrance, in three generations. We found that patient PBMCs produced increased IL-1β in response to inflammatory stimuli, as well as increased constitutive levels of IL-18. Moreover, we demonstrate that expression of the identified NLRP1 A59P variant caused spontaneous IL-1β cleavage to mature IL-1β. In addition, patient PBMCs responded to NLRP1 stimulation with increased ASC speck formation as a reflection of elevated inflammasome activity. Conclusion We demonstrate that this novel NLRP1 A59P variant caused increased activation of the NLRP1 inflammasome, resulting in constitutively and inducibly elevated IL-1β and IL-18 synthesis. We suggest the NLRP1 mutation underlies the pathogenesis of this rare autoinflammatory dyskeratotic disease inherited in an autosomal dominant manner with incomplete penetrance in the patient and within the family for several generations.

Cellular immune biomarkers to prognosticate for survival to adoptive T-cell therapy in advanced nasopharyngeal cancer.

6047 Background: We have published a Phase II clinical trial in 35 advanced incurable stage 4c NPC patients employing upfront first line chemo-immunotherapy of four cycles of gemcitabine and carboplatin, followed by six cycles of adoptive transfer autologous EBV-specific cytotoxic T lymphocytes (CTL). The 2- and 3-year overall survival rates were 62.9 and 37.1% respectively, which represent the best reported survival outcome for first-line treatment of advanced NPC when compared to historical clinical trials. Methods: From this Phase II trial, we completed serial multiple deep immune phenotyping analyses including flow cytometry, nanostring, and multiplex ELISA assays. We then investigated how these factors influence and determine successful therapy using 2-year survival rates. Results: Longitudinal modular transcriptome analysis of patient PBMCs revealed significant increases in T cell, NK, and B cell modules post chemotherapy within the 2-year survivor group. Administration of CTLs restored expression of lymphocyte transcripts in the non-survivor group at 2-weeks post infusion. However, as immunotherapy proceeded, 2-year survivors displayed significant increases in multiple lymphocyte module associated transcripts. These immune correlates were associated with increased IFN-γ and decreased myeloid chemokine concentrations in peripheral sera during immunotherapy in 2-year survivors. Longitudinal immunophenotyping of patient PBMCs at serial timepoints showed that 2-year survivors displayed significant decreased amounts of monocytic myeloid-derived suppressor cells (mMDSCs), compared to non-survivors after chemotherapy, which subsequently determined successful CTL immunotherapy survival benefit. Conclusions: This is the first report in human cancer patients that successful adoptive T cell immunotherapy correlates with lower mMDSCs following chemotherapeutic preconditioning. We have also identified potential prognostic immune biomarkers that effectively predicts efficacy of CTL immunotherapy, following upfront combination chemotherapy, and thus survival. Lymphodepletion with chemotherapy appears to be vital for adoptive T cell therapy efficacy.

MicroRNA Mir-155 and Myb Proto-Oncogene Family Members Cooperate in Pathogenesis of Chronic Lymphocytic Leukemia.

Abstract 58 Introduction: Role of small non-coding microRNAs (miRNA) in hematopoiesis has been recently established by studies demonstrating increased levels of miR-155 in chronic lymphocytic leukemia (CLL) (Fulci 2007, Marton 2008). PU.1 is an ETS family transcription factor controling myelo-lymphoid differentiation and is directly negatively regulated by miR-155 (Vigorito 2007). Our aim of this study is to determine mechanisms of miR-155 upregulation in CLL pathogenesis and the role of PU.1 downregulation in the process. Methods: miRNA and mRNA levels were determined by qPCR and Affymetrix mRNA expression profiling in peripheral blood mononuclear cells (PBMC) and in purified B cells. Control (N=13) and CLL patients (N=66) were studied. All patients were subgrouped according to cytogenetics (FISH) and Rai status. Protein-DNA localization assays were done using chromatin immunoprecipitation. Results: miR-155 is significantly upregulated in both CLL patient PBMCs and a subset of sorted B-cells whereas PU.1 and its target genes are repressed in all CLL subgroups. Indeed, expression profiling analysis of CLL samples identified a broad repression of ∼80 miR-155 targets (among them key transcriptional regulators FOS, SATB1, MEF2A, MYBL1, SIRT1, MECP2 and CEBPB) and ∼380 repressed PU.1 target genes, among them regulators of hematopoietic homeostasis (FOS, CSF1R, CSF2R, IL4R, IL21R) and apoptosis (BID, BIRC3). Next, we have studied the mechanism of miR-155 gene (also known as BIC) upregulation in CLL. Wehave newly identified a regulatory CpG island (BIC-CpG) upstream of miR-155 BIC gene that contains DNA binding motifs for E-box transcription factors and is not mutated in CLL patients. Two E-box-binding proteins, MYB and MYBL2, are significantly upregulated in CLL patient PBMCs as well as in a subset of sorted B-cells in all CLL subgroups. Furthermore in primary CLL cells, MYB protein presence is significantly enriched at BIC-CpG alongside a marked enrichment with transcriptionally active chromatin mark histone H3K9Acetyl. To further study the role of MYB in transactivation of the BIC-CpG we have prepared reporter constructs and found that MYB indeed activates BIC-CpG and downstream transcription. Apart from miR-155/BIC, expression profiling analysis identified additional ∼50 upregulated MYB targets, among them cancer-related genes such as CA1, MCM4, BCL2, PDCD4, and CXCR4. Functional assays using siRNA inhibition of PU.1 in normal PBMCs result in upregulation of miR-155 and MYB, indicating that silencing of PU.1 and upregulation of MYB and its target miR-155 may represent an important mechanism of CLL pathogenesis. Conclusion: Our data propose a mechanistic relationship between PU.1 and its negative regulator miR-155 in CLL. Our data also demonstrate that miR-155 is transcriptionally activated by MYB family of E-box binding proteins. Manipulation of these mechanistic relationships may harbor a potential for molecular therapies against CLL. (Grants NR9021-4, 10310-3, 2B06077) Disclosures: No relevant conflicts of interest to declare.

Reconstitution of STAT4 Restores Defective Interferon-gamma Production and Allows Normal IFN-gamma-Dependent Responses after Autologous Stem Cell Transplantation.

Production of IFN-gamma is critical for optimal antitumor immune responses in several preclinical animal models. Patients with lymphoma who have undergone autologous hematopoietic stem cell transplantation exhibit profoundly diminished IFN-gamma production in vivo during IL-12 therapy (Clin Cancer Res.2002; 8: 3383). Furthermore, post-transplant patient peripheral blood mononuclear cells (PBMCs) stimulated directly in vitro with IL-12 secrete little if any IFN-gamma. To determine if restoration of IFN-gamma production would be sufficient to reconstitute IFN-gamma-dependent responses after transplantation, the integrity of IFN-gamma signaling was assessed in post-transplant patient PBMCs. Compared to control subject PBMCs, post-transplant patient PBMCs expressed equivalent levels of CD119 (IFN-gamma receptor alpha) and STAT1. Moreover, tyrosine phosphorylation of STAT1 in response to IFN-gamma did not differ in post-transplant patient as compared to control PBMCs. Thus, IFN-gamma signaling is intact after transplantation. Our prior studies have shown that STAT4 protein levels are decreased by ~97% in post-transplant patient PBMCs, whereas levels of Jak2, Tyk2, and STAT3 are similar to control PBMCs (Blood2005; 106: 963). We wished to demonstrate directly that STAT4 deficiency is the mechanism of defective IFN-gamma production after autologous transplantation. Enriched CD4+ T cells were isolated from post-transplant patient and control PBMCs and cultured under conditions that promote Th1 differentiation. STAT4 deficiency persisted in post-transplant patient CD4+ T cells cultured under Th1 conditions, whereas these cells expressed normal levels of T-bet. Compared to control Th1 cells, post-transplant patient CD4+ T cells cultured under Th1 conditions exhibited markedly reduced IFN-gamma production after restimulation with CD3 mAb. These results are consistent with expected impairment of Th1 differentiation due to profound STAT4 deficiency. STAT4 cDNA was transiently transfected into Th1-cultured CD4+ T cells from post-transplant patients. After stimulation with CD3 mAb plus IL-12, the amounts of IFN-gamma secreted by STAT4-transfected patient cells were equivalent to or higher than those secreted by control subject cells. These data indicate that reconstitution of STAT4 expression is sufficient to restore IFN-gamma production by post-transplant patient PBMCs. As IFN-gamma signaling is normal post-transplant, circumventing STAT4 deficiency should therefore restore IFN-gamma-dependent antitumor immune responses after autologous transplantation. Future studies will determine the molecular mechanisms of STAT4 deficiency after transplantation and develop clinically feasible methods to circumvent this deficiency.

Phase I Study of the Sequential Combination of Interleukin-12 and Interferon Alfa-2b in Advanced Cancer: Evidence for Modulation of Interferon Signaling Pathways by Interleukin-12

Purpose To evaluate the safety of sequentially administered recombinant (r) human (h) interleukin-12 (IL-12) and interferon alfa-2b (IFN-α-2b) in patients with advanced cancer and to determine the effects of endogenously produced IFN-γ on Janus kinase-signal transducer and activator of transcription (Jak-STAT) signal transduction in patient peripheral-blood mononuclear cells (PBMCs). Patients and Methods Forty-nine patients with metastatic cancer received rhIL-12 on day 1 and IFN-α-2b on days 2 to 6 of either a 14-day (n = 43) or a 7-day treatment cycle (n = 6). rhIL-12 was initially administered subcutaneously at a dose of 100 ng/kg, whereas IFN-α-2b was escalated from 1 to 10 million units (MU) in cohorts of three patients (1, 3, 5, 7, or 10 MU). rhIL-12 was subsequently administered intravenously (IV) in escalating doses (100 to 500 ng/kg) to achieve greater IFN-γ production. Peripheral blood was drawn for measurement of plasma IFN-γ and the induction of Jak-STAT signal transduction in PBMCs. Results No IL-12–or IFN-α–related dose-limiting toxicities were observed. There were no responses in 41 assessable patients. Five patients exhibited stable disease lasting 6 months or longer while on therapy. Optimal induction of IFN-γ by IL-12 occurred after an IV dose of 250 ng/kg. Patient PBMCs exhibited increased levels of STAT1 after IL-12 administration. The peak level of IFN-γ achieved with IL-12 therapy correlated with the peak level of intracellular STAT1 in patient PBMCs (r = 0.38, P = .021). Conclusion The combination of rhIL-12 and IFN-α-2b can be administered sequentially with minimal toxicity. IV administration of rhIL-12 modulates IFN-α–induced Jak-STAT signal transduction in patient PBMCs.

Impaired interferon-γ production as a consequence of STAT4 deficiency after autologous hematopoietic stem cell transplantation for lymphoma

Production of interferon γ (IFN-γ) is critical for optimal antitumor immunotherapy in several preclinical animal models. Interleukin-12 (IL-12)–induced IFN-γ production is markedly defective after autologous stem cell transplantation. Quantitative deficiency in CD4 T cells, relative increase in CD25+CD4+ T cells, and bias toward T helper 2 (Th2) differentiation are not the primary mechanisms of defective IFN-γ production. IL-12 receptor β1 (IL-12Rβ1) and IL-12Rβ2 are expressed at equivalent or higher levels on posttransplantation patient peripheral blood mononuclear cells (PBMCs) as compared with control PBMCs. IL-12–induced tyrosine phosphorylation of signal transducer and activator of transcription 4 (STAT4) was undetectable or barely detectable in posttransplantation patient PBMCs, whereas IL-4–induced tyrosine phosphorylation of STAT6 did not differ in posttransplantation patient and control PBMCs. Levels of STAT4 protein were decreased by 97% in posttransplantation patient PBMCs. Levels of STAT4 mRNA were also significantly decreased in posttransplantation patient PBMCs. Incubation with IL-12 and IL-18 in combination partially reversed the defective IFN-γ production by posttransplantation patient PBMCs. IFN-γ production in response to IL-12 plus IL-18 did not require increased expression of STAT4 but was dependent on the activity of p38 mitogen-activated protein kinase (MAPK). These results indicate that defective IFN-γ production is due to an intrinsic deficiency in STAT4 expression by posttransplantation patient lymphocytes and suggest strategies for circumventing this deficiency in cancer immunotherapy.

Impaired Interferon-Gamma Production as a Consequence of Profound STAT4 Deficiency in Lymphocytes of Lymphoma Patients after Autologous Hematopoietic Stem Cell Transplantation.

Production of IFN-gamma has been found to be critical for optimal antitumor immunotherapy in several preclinical animal models. IL-12-induced IFN-gamma production is markedly defective in patients with lymphoma who have undergone autologous hematopoietic stem cell transplantation (AHSCT). We have investigated the mechanism of defective IFN-gamma production after AHSCT. Increasing the number of CD4 T cells in IFN-gamma assays did not substantially improve IFN-gamma production by post-transplant patient peripheral blood mononuclear cells (PBMCs). Thus, the quantitative deficiency in CD4 T cells observed for up to a year after AHSCT was not the primary mechanism of defective IFN-gamma production. Post-transplant patient T cells and NK cells expressed the IL-12 receptor beta1 subunit at significantly higher levels than did control PBMCs. Moreover, IL-12 receptor beta2 was expressed by a higher proportion of NK cells from post-transplant patients as compared to control subjects. Thus, down-regulation of IL-12 receptor subunits did not account for defective IFN-gamma production by post-transplant patient PBMCs. Levels of Jak2 and Tyk2 were comparable in control and post-transplant patient PBMCs. In contrast, IL-12-induced tyrosine phosphorylation of STAT4 was undetectable or barely detectable in post-transplant patient PBMCs, whereas it was readily detectable in control PBMCs. The total levels of STAT4 protein were also markedly decreased (P<0.027) in post-transplant patient PBMCs (3275+/−1692; mean+/−SE by densitometric analysis) as compared to control PBMCs (8644+/−732). This profound STAT4 deficiency persists for at least 6 months following AHSCT. Incubation of post-transplant patient PBMCs with IL-12 and IL-18 in combination partially reversed the defective IFN-gamma production seen after stimulation with IL-12 alone. IFN-gamma production in response to IL-12 plus IL-18 was not associated with increased expression of STAT4 but was dependent on the activity of p38 MAP kinase. These results indicate that defective IFN-gamma production is due to an intrinsic deficiency in STAT4 expression by post-transplant patient lymphocytes and suggest strategies for circumventing this deficiency in post-transplant cancer immunotherapy.