Signal conversion as tumor micro environment (TME) specifically activated cytokine.

Targeted cytokine delivery has been gaining popularity in cancer immunotherapy since systemic recombinant cytokine treatment has not been successful due to low response rate and systemic toxicities in the clinical studies. In order to address these issues, we propose a new concept that cytokine signal is specifically activated at tumor-micro-environment (TME) by delivering two protein subunits of heterodimeric cytokine fused with a tumor targeting antibody respectively to TME and by bridging the two subunits into active heterodimeric form.Interleukin-12 (IL-12) is one of the major cytokines which can induce immune activation. IL-12 consists of two protein subunits which are p35 and p40. IL-12 signaling is initiated when it forms as the heterodimeric protein and binds to IL-12 receptor complex. We made fusion proteins of both IL-12p35 and IL-12p40 targeting specific tumor associated antigens (TAAs) and demonstrated the formation of bioactive IL12p70 with TME targeting antibody toward both p35 and p40 to form as the active molecule. We describe our concept validation in an in vitro based functional assay.

Effects of APRIL (TNFSF13) polymorphisms and splicing isoforms on the secretion of soluble APRIL.

Functional APRIL (TNFSF13) is a secreted trimer generated by furin protease cleavage. We previously reported the association of APRIL haplotypes formed by two nonsynonymous polymorphisms, Gly67Arg and Asn96Ser, with systemic lupus erythematosus. Here, we tested the hypothesis that polymorphisms and/or alternative splicing may influence the generation of soluble APRIL (sAPRIL). HEK 293T cells were transfected with plasmids containing one of the six combinations of splicing isoforms (α or ß) and haplotypes (susceptible, neutral, or protective). APRIL concentrations were quantitated in the cell lysates and supernatants using an enzyme-linked immunosorbent assay (ELISA). The association between splicing efficiency and polymorphisms was analyzed using quantitative reverse transcription polymerase chain reaction (RT-PCR). The efficiency of cleavage by furin protease was analyzed using western blotting. Although both splicing isoforms were cleaved by furin protease, sAPRIL was not detected in the supernatant of the cells transfected with the ß isoform, regardless of the haplotype. This suggested that, similarly to B-cell activating factor (BAFF), one of the major APRIL splicing isoforms may not be secreted as a functional molecule. Furthermore, the secretion of sAPRIL was decreased in the transfectants expressing the protective haplotype. An association between the polymorphisms and splicing efficiency or furin cleavage efficiency was not detected. In conclusion, these observations suggested that both alternative splicing and polymorphisms may affect the generation of functional sAPRIL.

Association of a functional polymorphism in the 3'-untranslated region of SPI1 with systemic lupus erythematosus.

OBJECTIVE: SPI1, also referred to as PU.1, is an Ets family transcription factor that interacts with IRF2, IRF4, and IRF8. In view of the significance of the type I interferon pathway in systemic lupus erythematosus (SLE), this study was undertaken to investigate a possible association between SPI1 polymorphisms and SLE. METHODS: A case-control association study was performed using 6 tag single-nucleotide polymorphisms (SNPs), as well as a SNP located upstream of SPI1 previously found to be associated with acute myelogenous leukemia, in 400 Japanese patients with SLE and 450 healthy controls. Resequencing of all exons and known regulatory regions was performed to identify functional polymorphisms. Association of genotype and SPI1 expression was examined using the GENEVAR database and reporter assays. RESULTS: A significant association was detected in 2 SNPs in intron 2 (rs10769258 and rs4752829) (P = 0.005 and P = 0.008, respectively, under the dominant model). The association was stronger in patients with nephropathy. Resequencing identified a potentially functional polymorphism in the 3'-untranslated region (3'-UTR), rs1057233, which was in strong linkage disequilibrium with the SNPs in intron 2. The number of risk alleles at rs1057233 was strongly correlated with SPI1 messenger RNA (mRNA) level in the database analysis (P = 0.0002), and was confirmed by a reporter assay. Interestingly, rs1057233 alters a target sequence for microRNA hsa-miR-569 (miR-569). Transfection experiments demonstrated that miR-569 inhibits expression of a reporter construct with the 3'-UTR sequence containing the nonrisk allele but not the risk allele. CONCLUSION: Our findings indicate that a SNP in the 3'-UTR of SPI1 is associated with elevated SPI1 mRNA level and with susceptibility to SLE.

Association of a functional polymorphism in the IRF5 region with systemic sclerosis in a Japanese population.

OBJECTIVE: Interferon regulatory factor 5, an established susceptibility factor for systemic lupus erythematosus (SLE), plays a role in type I interferon and proinflammatory cytokine induction. A recent study showed association of a functional single-nucleotide polymorphism (SNP) in intron 1 of IRF5, rs2004640, with systemic sclerosis (SSc) in a European French population. We undertook the present study to determine whether IRF5 polymorphisms are also associated with a predisposition to SSc in Japanese. METHODS: A case-control association study was performed for rs2004640 as well as for rs10954213 and rs2280714, all of which were previously reported to be associated with SLE, in 281 SSc patients and 477 healthy controls. Patients with SSc complicated by SLE or Sjögren's syndrome were excluded. Association of the rs2280714 genotype with messenger RNA (mRNA) levels of IRF5 and adjacently located transportin 3 (TNPO3) was examined using the GENEVAR database. RESULTS: All 3 SNPs were significantly associated with SSc, with the rs2280714 A allele having the strongest association (allele frequency P=0.0012, odds ratio 1.42 [95% confidence interval 1.15-1.75]). Association was preferentially observed in subsets of patients with diffuse cutaneous SSc (dcSSc) and anti-topoisomerase I antibody positivity. Conditional analysis revealed that rs2280714 could account for most of the association of these SNPs, while an additional contribution of rs2004640 was also suggested for dcSSc. The genotype of rs2280714 was strongly associated with IRF5 mRNA expression, while only marginal association was detected with TNPO3 mRNA expression. CONCLUSION: Association of IRF5 with SSc was replicated in a Japanese population. Whether the causal SNP is different among populations requires further investigation.

Role of STAT4 polymorphisms in systemic lupus erythematosus in a Japanese population: a case-control association study of the STAT1-STAT4 region.

INTRODUCTION: Recent studies identified STAT4 (signal transducers and activators of transcription-4) as a susceptibility gene for systemic lupus erythematosus (SLE). STAT1 is encoded adjacently to STAT4 on 2q32.2-q32.3, upregulated in peripheral blood mononuclear cells from SLE patients, and functionally relevant to SLE. This study was conducted to test whether STAT4 is associated with SLE in a Japanese population also, to identify the risk haplotype, and to examine the potential genetic contribution of STAT1. To accomplish these aims, we carried out a comprehensive association analysis of 52 tag single nucleotide polymorphisms (SNPs) encompassing the STAT1-STAT4 region. METHODS: In the first screening, 52 tag SNPs were selected based on HapMap Phase II JPT (Japanese in Tokyo, Japan) data, and case-control association analysis was carried out on 105 Japanese female patients with SLE and 102 female controls. For associated SNPs, additional cases and controls were genotyped and association was analyzed using 308 SLE patients and 306 controls. Estimation of haplotype frequencies and an association study using the permutation test were performed with Haploview version 4.0 software. Population attributable risk percentage was estimated to compare the epidemiological significance of the risk genotype among populations. RESULTS: In the first screening, rs7574865, rs11889341, and rs10168266 in STAT4 were most significantly associated (P < 0.01). Significant association was not observed for STAT1. Subsequent association studies of the three SNPs using 308 SLE patients and 306 controls confirmed a strong association of the rs7574865T allele (SLE patients: 46.3%, controls: 33.5%, P = 4.9 x 10(-6), odds ratio 1.71) as well as TTT haplotype (rs10168266/rs11889341/rs7574865) (P = 1.5 x 10(-6)). The association was stronger in subgroups of SLE with nephritis and anti-double-stranded DNA antibodies. Population attributable risk percentage was estimated to be higher in the Japanese population (40.2%) than in Americans of European descent (19.5%). CONCLUSIONS: The same STAT4 risk allele is associated with SLE in Caucasian and Japanese populations. Evidence for a role of STAT1 in genetic susceptibility to SLE was not detected. The contribution of STAT4 for the genetic background of SLE may be greater in the Japanese population than in Americans of European descent.

Association of IL-10 receptor 2 (IL10RB) SNP with systemic sclerosis.

Interleukin-10 (IL-10) signaling has been suggested to play a role in systemic sclerosis (SSc). IL10RB codes for IL-10 receptor 2 (IL-10R2), a component shared in receptor complexes for IL-10, IL-22, IL-26 and interferon (IFN)-lambda. In this study, we examined association of IL10RB polymorphism with susceptibility to SSc. Genotype A/A at rs2834167 (47K/K) was significantly increased in diffuse cutaneous SSc (dcSSc) (41.3% in dcSSc, 20.9% in controls, P=0.0018, odds ratio=2.67). A SNP in the 5' flanking region of IL10RB, rs999788, also showed association with dcSSc; however, this association was shown to be secondarily caused by linkage disequilibrium with rs2834167. Significant association was not observed in limited cutaneous SSc (lcSSc). Presence of anti-topoisomerase I antibody was also associated with rs2834167A/A genotype (P=0.0019). Serum IL-10 level was significantly associated with the number of rs2834167A allele (P=0.007). These findings suggested that signaling through IL-10R2 may play a causative role in dcSSc.

Association of IRF5 polymorphisms with systemic lupus erythematosus in a Japanese population: support for a crucial role of intron 1 polymorphisms.

OBJECTIVE: To determine whether the IRF5 gene, which encodes interferon regulatory factor 5, is associated with systemic lupus erythematosus (SLE) in a Japanese population. METHODS: A case-control study was performed in 277 SLE patients and 201 healthy controls. Associations between the IRF5 genotype and levels of messenger RNA (mRNA) for interferon (IFN) pathway genes were examined using an mRNA expression database of HapMap samples. RESULTS: Carriers of the rs2004640T single-nucleotide polymorphism (SNP) were slightly increased among SLE patients (58.8%) as compared with controls (50.2%). When data from our Japanese population were combined with previously published data from a Korean population, the T allele frequency was found to be significantly increased in SLE patients (P = 8.3 x 10(-5)). While no association was observed for the rs10954213 SNP or the exon 6 insertion/deletion, significant associations with 3 intron 1 SNPs (-4001, rs6953165, and rs41298401) were found. The allele frequency of rs41298401G was significantly decreased in SLE patients (13.0% versus 18.7% in controls; P = 0.017), and the allele frequency of rs6953165G, which was in absolute linkage disequilibrium with -4001A, was increased in SLE patients (8.8% versus 5.2% in controls; P = 0.034). The Caucasian risk haplotype was not present; instead, a protective haplotype carrying rs2004640G, rs41298401G, the deletion in exon 6, and rs10954213A was identified. SNP rs10954213, but not intron 1 SNPs, was associated with IRF5 at the mRNA level; nevertheless, intron 1 SNPs were also associated with levels of mRNA for several IFN pathway genes, suggesting a functional role. CONCLUSION: IRF5 was found to be associated with SLE in Asian populations. Intron 1 SNPs, rather than exon 6 and 3'-untranslated region polymorphisms, appeared to play a crucial role.

Molecular genetic analyses of human NKG2C (KLRC2) gene deletion.

Human NKG2A, NKG2C and NKG2E genes are located on 12p13 in the NK gene complex. We recently identified deletion of NKG2C in a Japanese population. This study was performed to identify the breakpoint, and to examine the association of NKG2C deletion with susceptibility to rheumatoid arthritis and systemic lupus erythematosus. The location of the breakpoint was determined to be 1.5-1.8 kb telomeric from the 3' end of NKG2A by comparing sequences of the intergenic segments upstream and downstream of the NKG2C gene in the common haplotype with the intergenic sequence between NKG2A and NKG2E in the deletion haplotype. Based on this information, a genotyping system was developed. The frequency of NKG2C deletion haplotype was 20.2% in Japanese and 20.0% in Dutch populations. The frequency of homozygous deletion was 4.1% in Japanese and 3.8% in Dutch. Evidence for an association with rheumatic diseases was not detected. These results indicated that NKG2C deletion is commonly present in Japanese and Dutch, suggesting that NKG2C is not essential for survival and reproduction, and is not associated with rheumatic diseases.