Α-chain Gene Research Articles

Rhesus Macaques: VNTR Polymorphism of the FCGRT Gene.

Variable number tandem repeat (VNTR) polymorphisms of the human neonatal IgG Fc receptor α-chain gene (FCGRT) are known to influence the expression levels of FCGRT and IgG in serum. Monkeys are considered to be a relevant biological model for studying the effects of immunobiological drugs. The study determined the functional VNTR polymorphisms of the FCGRT gene in 109 male rhesus macaques from the nursery of the Kurchatov Complex of Medical Primatology. PCR amplification of samples was carried out followed by electrophoretic separation of DNA fragments in a 2% agarose gel. Individual DNA amplification products were sequenced (according to Sanger system) in forward and reverse directions to confirm the specificity. The genotyping showed that the VNTR polymorphism of the FCGRT gene in the studied population of rhesus macaques is presented by 9 variants. The frequency of the VNTR5 allele associated with lower IgG levels was 14.2%, and the most common one was the VNTR7 allele (25.2%). We also identified alleles that have not been previously reported: VNTR3, VNTR4, VNTR6, VNTR8, and VNTR9. The study allows to consider rhesus macaques as a potential model for studying the immunological response depending on the genetic VNTR variant of FCGRT.

Static electromagnetic field and recombinant human fibroblasts encoding miR-451 and miR-16 increased cell trans-differentiation to CD71+ and CD235a+ erythroid like progenitor.

Ex vivo blood production is an urgent need of most countries, and creating production protocols can save the lives of many patients. Despite the recent advances in blood production in ex vivo conditions, its high-scale production is not yet possible, and requires further studies. Therefore, by transfecting fibroblast cells with miR-16, and miR-451 genes, as well as applying low frequency electromagnetic fields (ELF-EMF) treatment, we tried to increase the differentiation of these cells into CD71+ and CD235a+ erythroid like progenitors. After preparation, and cultivation of human dermal transgenic fibroblast cells, they were transfected by Plenti3-hsa-miR451, Plenti3-hsa-miR16 and Plenti3-backbone inserted into E. coli Stbl4 genome. Then, transgenic fibroblast cells were treated with 10mT ELF-EMF every day for 20 minutes for 7 days. Using a flow cytometer, the expressions of CD71, and CD235a were studied in these cells, and the expressions of genes involved in hematopoiesis were studied using the RT-PCR technique. The results indicated an increase in the differentiation of fibroblast cells treated with 10mT ELF-EMF to erythroid like progenitors. Furthermore, the percentage of CD71+ and CD235a+ cells was the highest in irradiated cells encoding miR-16 and miR-451, which indicates their differentiation into erythroid like progenitors. Also, in the transgenic cells treated with ELF-EMF, an increase in the expressions of α-chain, β-chain, γ-chain and GATA1 genes was observed, which indicates the potential of these cells for hematopoiesis. However, there was no significant difference in the expression of CD34 and CD38 genes in these cell lines. Both ELF-EMF and upregulations of miR-16 and miR-451 lead to improved differentiation of fibroblast cells into erythroid like progenitors.

Association of fibrinogen and plasmin inhibitor, but not coagulation factor XIII gene polymorphisms with coronary artery disease.

BackgroundIn the final phase of clot formation, fibrinogen constitutes frame, whereas factor XIII (FXIII) active form is responsible for the covalent cross-linking of fibrin fibres and plasmin inhibitor (PI), thus contributing to clot stability. It could be expected that any change of coagulation factors' structure affects the clot formation and modulates the atherothrombotic risk. The aim was to determine the frequency of four single nucleotide polymorphisms: (i) A > G in codon 312 of the fibrinogen α-chain gene (rs6050, Thr312AlaFGA), (ii) C > T at position 10034 of the 3 - untranslated region in the fibrinogen γ-chain gene (rs2066865, 10034C > T FGG), (iii) C > T in codon 564 of the FXIII-A subunit gene (rs5982, Pro564LeuFXIII-A), and (iv) C > T in codon 6 of the plasmin inhibitor gene (rs2070863, Arg6TrpPI) in Croatian patients and their association with coronary artery disease (CAD).MethodsWe performed the unrelated case-control association study on the consecutive sample of patients 18 years old, who had undergone coronary angiography for investigation of chest pain and suspected CAD. The cases were patients with confirmed CAD (N=201), and the controls were the subjects with no CAD (N=119). Samples were genotyped using PCR-RFLP analysis.ResultsObserved frequencies of the rare alleles of Thr312Ala FGA, 10034C > T FGG, Leu564Pro FXIII-A and Arg6Trp PI polymorphisms were 21%, 17%, 14%, 20%, respectively. Patients with 10034C > T FGG CC genotype had 3.5 times (95% CI 1.02-12.03) higher adjusted odds for CAD than patients with 10034C > T FGG TT genotype. Patients with Arg6Trp PI CC genotype had 3.86 times (95% CI 1.23-12.12) higher odds for CAD than patients with Arg6Trp PI TT genotype. It seems that those genotype-related higher odds are also male-gender related. No difference was observed regarding any other investigated polymorphism.ConclusionsOur finding suggests that 10034C > T FGG and Arg6Trp PI are associated with CAD.

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis

AbstractPlasma fibrinogen molecules comprise 2 copies of Aα, Bβ, and γ chains folded into a hexameric protein. A minor fibrinogen isoform with an extended Aα chain (AαE) is more abundant in newborn human blood than in adults. Larval zebrafish produce predominantly AαE-containing fibrinogen, but its functional significance is unclear. In 3-day-old zebrafish, when hemostasis is reliant on fibrinogen and erythrocyte-rich clotting but is largely thrombocyte-independent, we measured the time to occlusion (TTO) in a laser-induced venous thrombosis assay in 3 zebrafish strains (AB, TU, and AB × TL hybrids). AB larvae showed delayed TTO compared with the TU and AB × TL strains. Mating AB with TU or TL produced larvae with a TU-like TTO. In contrast to TU, AB larvae failed to produce fibrinogen AαE, due to a mutation in the AαE-specific coding region of fibrinogen α-chain gene (fga). We investigated whether the lack of AαE explained the delayed AB TTO. Transgenic expression of AαE, but not Aα, shortened the AB TTO to that of TU. AαE rescued venous occlusion in fibrinogen mutants or larvae with morpholino-targeted fibrinogen α-chain messenger RNA, but Aα was less effective. In 5-day-old larvae, circulating thrombocytes contribute to hemostasis, as visualized in Tg(itga2b:EGFP) transgenics. Laser-induced venous thrombocyte adhesion and aggregation is reduced in fibrinogen mutants, but transgenic expression of Aα or AαE restored similar thrombocyte accumulation at the injury site. Our data demonstrate a genetic modifier of venous thrombosis and a role for fibrinogen AαE in early developmental blood coagulation, and suggest a link between differentially expressed fibrinogen isoforms and the cell types available for clotting.

Interaction between functional polymorphisms in FCER1A and TLR2 and the severity of atopic dermatitis

Dendritic cell toll-like receptors (TLRs) and the high-affinity immunoglobulin E (IgE) receptor (FcεRI) may biologically interact with regard to atopic dermatitis (AD) development and, especially, severity. Our aim here was to test if such interaction can be detected on the genetic level. The combined effect of the TLR2 gene (TLR2) rs4696480 and the FcεRI α-chain gene (FCER1A) rs2252226 and rs2251746 polymorphisms on the AD severity as measured by SCORAD was assessed. The FCER1A rs2252226 and TLR2 rs4696480 polymorphisms interacted with regard to SCORAD. Higher SCORAD was observed in patients being the TLR2 rs4696480 major homozygotes and carrying at the same time the FCER1A rs2252226 minor allele, compared to those characterized by (any other of) the remaining combined rs2252226 and rs4696480 genotypes. The observation of the epistatic effect of TLR2 and FCER1A genetic variants on SCORAD is in line with the involvement of the interaction TLRs-FcεRI in the pathophysiology of AD.

Organ Transplantation in Hereditary Fibrinogen A α-Chain Amyloidosis: A Case Series of French Patients

Fibrinogen A α-chain amyloidosis (AFib amyloidosis) is a form of amyloidosis resulting from mutations in the fibrinogen A α-chain gene (FGA), causing progressive kidney disease leading to kidney failure. Treatment may include kidney transplantation (KT) or liver-kidney transplantation (LKT), but it is not clear what factors should guide this decision. The aim of this study was to characterize the natural history and long-term outcomes of this disease, with and without organ transplantation, among patients with AFib amyloidosis and various FGA variants. Case series. 32 patients with AFib amyloidosis diagnosed by genetic testing in France between 1983 and 2014, with a median follow-up of 93 (range, 4-192) months, were included. Median age at diagnosis was 51.5 (range, 12-77) years. Clinical presentation consisted of proteinuria (93%), hypertension (83%), and kidney failure (68%). Manifestations of kidney disease appeared on average at age 57 (range, 36-77) years in patients with the E526V variant, at age 45 (range, 12-59) years in those with the R554L variant (P<0.001), and at age 24.5 (range, 12-31) years in those with frameshift variants (P<0.001). KT was performed in 15 patients and LKT was performed in 4. In KT patients with the E526V variant, recurrence of AFib amyloidosis in the kidney graft was less common than with a non-E526V (R554L or frameshift) variant (22% vs 83%; P=0.03) and led to graft loss less frequently (33% vs 100%). Amyloid recurrence was not observed in patients after LKT. Analyses were based on clinically available historical data. Small number of patients with non-E526V and frameshift variants. Our study suggests phenotypic variability in the natural history of AFib amyloidosis, depending on the FGA mutation type. KT appears to be a viable option for patients with the most common E526V variant, whereas LKT may be a preferred option for patients with frameshift variants.

1663-P: Gene Editing of the Human T-Cell Receptor Locus Enables Optimized In Vitro Testing of Autoreactive T-Cell Function in Type 1 Diabetes

Gene editing of the human T cell receptor locus enables optimized in vitro testing of autoreactive T cell function in type 1 diabetes. The generation of antigen specific T cells and subsequent implementation as “living drugs” has garnered great interest in the immunotherapy space. One of the main caveats to this approach is the potential for heterologous chain pairing between the α- and β-chains of the endogenous and de-novo T cell receptors (TCRs). The consequences of chain mispairing can range from reduced antigen specific functionality to off-target autoimmune reactivity. The latter is an important consideration when designing therapies for autoimmune and inflammatory diseases, specifically with regards to the introduction of regulatory T cells (Treg) adoptive cell therapy. To date, infusions of polyclonal Tregs have demonstrated safety but have failed to reverse established type 1 diabetes (T1D). This demonstrates the need for the generation of safe and effective antigen-specific Tregs capable of recognizing β-cells in T1D. We have previously shown the ability to generate GAD-specific Tregs which maintain lineage stability and suppressive phenotype. To extend this work, we deleted the endogenous TCR α-chain gene (TRAC) via CRISPR/cas9 in CD8+T cells. We achieved &amp;gt;90% editing efficiency through ribonucleoprotein (RNP) complex mediated single guide RNA delivery. Moreover, we found TRAC-KO CD8+ T cells displayed increased peptide MHC-dextramer staining, CD69 and CD25 upregulation after stimulation with cognate antigen, and increased antigen-specific killing of β-cell lines when compared to mock guide treated T cells. We believe this workflow can be applied to generate antigen specific Tregs, which we expect to provide increased suppressive capacity. Hence, we present a TCR-gene editing protocol in primary human T cells that results in efficient deletion of the endogenous TCR α-chain and robust enhancement of antigen-specific T cell function. Disclosure L.D. Peters: None. A.L. Posgai: None. T.M. Brusko: Board Member; Self; OneVax, LLC. Consultant; Self; Caladrius Biosciences, Inc., Sanofi-Aventis. Speaker’s Bureau; Self; CSL Behring. Funding National Institutes of Health (P01AI42288, R01DK106191, T325T32DK108736-03HIRN, UC4 P0063595)

Abstract 3585: T cell receptor-stabilized regenerated CD8ab cytotoxic T lymphocytes for cancer immunotherapy

Abstract The use of antigen-specific T cell derived induced iPSCs as a cell source for regeneration of cytotoxic T lymphocytes (CTLs) have advantages in rejuvenation profile, and reproducible number of CTLs. However for the safe and efficient regenerated T cell immunotherapy, strict antigen specificity is essential. Here we report CD8αβ T cells differentiated from T cell-derived iPSCs underwent additional rearrangement of the T cell receptor (TCR) α-chain gene at the CD4/CD8 double positive stage during in vitro differentiation and lost antigen specificity. CRISPR knockout of a recombinase gene in the T-iPSCs successfully prevented this additional TCR rearrangement. Moreover, when CD8αβ T cells were differentiated from non-T cell-derived but antigen-specific TCR-transduced iPSCs, they showed monoclonal expression of the transduced TCR. TCR stabilized regenerated CD8αβ T cells effectively inhibit tumor growth in xenograft cancer models. These approaches could contribute to safe and effective regenerative T cell immunotherapies. Citation Format: Atsutaka Minagawa, Akitsu Hotta, Yohei Kawai, Yutaka Yasui, Yasushi Uemura, Masaki Yasukawa, Tetsuya Nakatsura, Shin Kaneko. T cell receptor-stabilized regenerated CD8ab cytotoxic T lymphocytes for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3585.

Genomic structure, polymorphism, and expression analysis of the spotted scat Scatophagus argus major histocompatibility complex class I alpha gene

The complete complementary DNA (cDNA) sequence of the major histocompatibility complex (MHC) class I α gene in Scatophagus argus was obtained by homology-based cloning and rapid amplification of cDNA ends polymerase chain reaction (PCR. The intron sequences, polymorphism, and tissue expression of this gene were then analyzed. The gene cDNA sequence is 2020 base pairs (bp) in length, including a 86-bp 5′-untranslated region (UTR), 866-bp 3′-UTR and 1068-bp open reading frame. It contains seven exons and six introns, of which there are two types of the fourth intron with respective lengths of 655 and 691 bp; it also has microsatellite (T)n fragments, and the sixth intron has a separate 3′-UTR. From 42 individuals, 20 alleles of the class I α-chain gene were identified, called here “Scar-UA*0101–Scar-UA*2001,” which demonstrate high polymorphism. Reverse transcription-PCR analysis shows its expression in ten tissues, with high expression levels in the liver, spleen, and kidney, and low levels of expression in the stomach, intestines, and muscle. A tree depicting the molecular relationships built using the neighbor-joining method shows that Scatophagus argus has a close genetic relationship to Perciformes fish such as Sparus aurata, Dicentrarchus labrax, and a more distant relationship to salmonid and cyprinid fishes, amphibians, birds, and mammals.

Hypochromic microcytic anemia: a clincopathological cross-sectional study

Background Iron-deficiency anemia, the most common cause of microcytic anemia, is a worldwide nutritional problem; its prevalence in the developing countries is three times higher than in the developed countries. It is a common disease in Egypt with a very high prevalence rate. It affects all age groups and all socioeconomic levels of the society. Identification of infants who are at risk for iron deficiency anemia (IDA) is vital as it impairs psychomotor development and growth and reduces physical activity and resistance to infection. Aim The aim of this work was to assess the prevalence of IDA among infants aged 6 months and 2 years presenting with hypochromic microcytic anemia and to determine the risk factors of iron deficiency among this age group. Participant and methods A total of 40 infants (6 months and 2 years) presenting with hypochromic microcytic anemia attending Alexandria University Children’s Hospital were included in this study. All were subjected to complete blood count, iron profile [serum ferritin (SF), serum iron, and total iron-binding capacity], and finding occult blood in stool. Those having normal iron profile were further subjected to capillary hemoglobin (Hb) electrophoresis and PCR for α-chain gene mutations to exclude β-thalassemias and α-thalassemias. Results Overall, 77.5% (31/40) of the studied infants had IDA, which represents the main cause of microcytic anemia in this study, whereas 17.5% (7/40) had β-thalassemia trait, diagnosed by increased HbA2 more than 3.5% by capillary Hb electrophoresis, and 5% (2/40) had α-thalassemia trait of the homozygous α3.7 (−α3.7/−α3.7) deletional type, detected by PCR for α-gene mutations. Moreover, this study showed that infants with IDA had significantly higher frequencies of preterm deliveries (48.38%) compared with β-thalassemia trait (14.28%), and α-thalassemia trait (0%), P=0.023, KWχ2=4.849. A significant positive correlation was found between Hb, serum iron, and SF levels of infants with IDA and both maternal Hb during pregnancy (r=0.937, P 6 months) (n=26), whose SF levels ranged between 2.0 and 5.9 μg/l, with a mean±SD of 4.04±1.10, P=0.001. Conclusion High prevalence of IDA was detected among infants presenting with microcytic hypochromic anemia (77.5%). The main risk factors for developing IDA include inadequate iron intake, preterm delivery, delayed onset of weaning, occult blood loss in stools owing to either early introduction of cow’s milk or parasitic infestations, and finally, infants born to young or anemic mothers.

The Forgotten: Identification and Functional Characterization of MHC Class II Molecules H2-Eb2 and RT1-Db2.

In this article, we report the complete coding sequence and to our knowledge, the first functional analysis of two homologous nonclassical MHC class II genes: RT1-Db2 of rat and H2-Eb2 of mouse. They differ in important aspects compared with the classical class II β1 molecules: their mRNA expression by APCs is much lower, they show minimal polymorphism in the Ag-binding domain, and they lack N-glycosylation and the highly conserved histidine 81. Also, their cytoplasmic region is completely different and longer. To study and compare them with their classical counterparts, we transduced them in different cell lines. These studies show that they can pair with the classical α-chains (RT1-Da and H2-Ea) and are expressed at the cell surface where they can present superantigens. Interestingly, compared with the classical molecules, they have an extraordinary capacity to present the superantigen Yersinia pseudotuberculosis mitogen. Taken together, our findings suggest that the b2 genes, together with the respective α-chain genes, encode for H2-E2 or RT1-D2 molecules, which could function as Ag-presenting molecules for a particular class of Ags, as modulators of Ag presentation like nonclassical nonpolymorphic class II molecules DM and DO do, or even as players outside the immune system.

The influence of interleukin-7 receptor α-chain haplotypes on outcome after allogeneic hematopoietic cell transplantation.

We investigated the influence of IL-7 receptor α-chain (IL-7Rα) gene haplotypes in donors on the outcome of haematopoietic cell transplantation (HCT). Unlike the association between single donor SNPs and HCT outcome found previously, only trends towards association were found here, due to 'dilution' of SNPs into haplotypes.

Polymorphisms in Interleukin-2 and Interleukin-7 Receptor α-Chain Genes and Human Herpes Virus-6 as Risk Factors for Multiple Sclerosis

Polymorphisms in Interleukin-2 and Interleukin-7 Receptor α-Chain Genes and Human Herpes Virus-6 as Risk Factors for Multiple Sclerosis

Molecular cloning and characterization of the α-chain gene of goose immunoglobulin heavy chain

A novel gene encoding the α-chain of goose immunoglobulin heavy-chain (Igα) was cloned by reverse transcription-PCR. The cDNA had 1,760 bp and encompassed a partial V-D-J region of the heavy chain, a constant region (Cα) and 3'-untranslated region of α-chain. The Cα gene contains four constant region domains (CH1-CH4). Phylogenetic analysis indicated that goose IgCα has a close genetic relationship with duck, ostrich and chicken IgCα. Three-dimensional modeling and glycosylation analysis revealed the goose Igα is consistent with the characterization of immunoglobulin. Western blotting suggested the goose IgCα has the same antigenicity to natural IgA. In general, the identification of goose immunoglobulin not only provides insights into the evolution of the Ig heavy-chain gene family, but may also benefit future studies of the avian immune system.

NFκB induces overexpression of bovine FcRn

Among the many functions of the neonatal Fc receptor (FcRn) for IgG, it binds to IgG-opsonized antigen complexes and propagates their traffic into lysosomes where antigen processing occurs. We previously reported that transgenic (Tg) mice and rabbits that carry multiple copies and overexpress FcRn have augmented humoral immune responses. Nuclear factor-kappa B (NFκB) is a critical molecule in the signaling cascade in the immune response. NFκB induces human FcRn expression and our previous in silico analysis suggested NFκB binding sites in the promoter region of the bovine (b) FcRn α-chain gene (FCGRT). Here, we report the identification of three NFκB transcription binding sites in the promoter region of this gene using luciferase reporter gene technology, electromobility shift assay and supershift analysis. Stimulation of primary bovine endothelial cells with the Toll-like receptor-4 ligand lipopolysaccharide (LPS), which mediates its effect via NFκB, resulted in rapid upregulation of the bFcRn expression and a control gene, bovine E-selectin. This rapid bFcRn gene induction was also observed in the spleen of bFcRn Tg mice treated with intraperitoneally injected LPS, analyzed by northern blot analysis. Finally, NFκB-mediated bFcRn upregulation was confirmed at the protein level in macrophages isolated from the bFcRn Tg mice using flow cytometry with a newly developed FcRn specific monoclonal antibody that does not cross-react with the mouse FcRn. We conclude that NFκB regulates bFcRn expression and thus optimizes its functions, e.g., in the professional antigen presenting cells, and contributes to the much augmented humoral immune response in the bFcRn Tg mice.

The DNA Damage- and Transcription-Associated Protein Paxip1 Controls Thymocyte Development and Emigration

Histone 3 lysine 4 trimethylation (H3K4me3) is associated with promoters of active genes and found at hot spots for DNA recombination. Here we have shown that PAXIP1 (also known as PTIP), a protein associated with MLL3 and MLL4 methyltransferase and the DNA damage response, regulates RAG-mediated cleavage and repair during V(D)J recombination in CD4(+) CD8(+) DP thymocytes. Loss of PAXIP1 in developing thymocytes diminished Jα H3K4me3 and germline transcription, suppressed double strand break formation at 3' Jα segments, but resulted in accumulation of unresolved Tcell receptor α-chain gene (Tcra) breaks. Moreover, PAXIP1 was essential for release of mature single positive (SP) αβ Tcells from the thymus through transcriptional activation of sphingosine-1-phosphate receptor S1pr1 as well as for natural killer Tcell development. Thus, in addition to maintaining genome integrity during Tcra rearrangements, PAXIP1 controls distinct transcriptional programs during DP differentiation necessary for Tcra locus accessibility, licensing mature thymocytes for trafficking and natural killer Tcell development.

Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

Epigenetic Modulation to Enable Antigen-specific T-cell Therapy of Colorectal Cancer

Development of specific immunotherapy for colorectal cancer (CRC) will require identification of antigens selectively or exclusively expressed on CRC cells and strategies to induce and enhance immune responses against these antigenic targets. Cancer-testis (C-T) antigens are proving to be excellent targets for immunotherapy of solid tumors such as melanoma, but their clinical utility for treatment of CRC has to date been limited by their infrequent expression in CRC cells. Here we report that the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) induces expression of NY-ESO-1 and other C-T genes in CRC cells both in vitro and in vivo in a dose-dependent manner but has negligible effects on the expression of C-T genes in normal nontransformed cells such as fibroblasts. The induction by DAC of NY-ESO-1 expression in CRC cells persists over 100 days after DAC exposure and is associated with increased levels of NY-ESO-1 protein. CRC cells exposed to DAC at concentrations that can be readily achieved in vivo are rendered susceptible to major histocompatibility complex-restricted recognition by CD8 NY-ESO-1-specific T cells. We also demonstrate that retroviral transduction of polyclonal peripheral blood T cells from a metastatic CRC patient with the T-cell receptor α-chain and β-chain genes encoding a human leukocyte antigen-A2-restricted, NY-ESO-1157-165-specific T-cell receptor can be used to generate both CD8 and CD4 NY-ESO-1157-165-specific T cells that selectively recognize DAC-treated CRC but not nontransformed cells. Collectively, these results suggest that the combination of epigenetic modulation and adoptive transfer of genetically engineered T lymphocytes may enable specific immunotherapy for CRC.

Abrogation of Treg function deteriorates rheumatoid arthritis

An early prognostic indicator which warns of progressive joint destruction of rheumatoid arthritis (RA) was explored using a novel suspension-array technique in moderate (Steinbrocker stage I and II) and severe (Steinbrocker stage IV) RA patients. DNA microarray analysis of peripheral blood lymphocytes showed significant increase of interleukin (IL)-2 receptor α-chain (CD25) gene expression, a regulatory T cell (Treg) surface marker in severe RA patients. In contrast, suspension array, a comprehensive bead-based enzyme-linked immunosorbent assay (ELISA), revealed decreased production of IL-10 and increased production of interferon (IFN)-γ in sera in the incipient stage of the aggressive disease process. Both in moderate and in severe RA patients, the IFN-γ/IL-10 ratio indicated deterioration of the disease with universal validity. Fluorescence-activated cell sorting (FACS) and reverse-transcription polymerase chain reaction (RT-PCR) analysis showed extant CD4+CD25+ regulatory T cells in severe RA patients, however Foxp3, a regulatory T cell-specific transcription factor, gene expression was absent, while glucocorticoid-induced tumor necrosis factor (TNF) receptor family-related protein (GITR), which transmits a signal that abrogates regulatory T cell functions, was elevated. In the current study, we showed the validity of suspension-array analysis for enabling more complete understanding of RA, and showed that IFN-γ/IL-10 ratio can be a prognostic tool for early lesion and more aggressive RA.

Abrogation of Treg function deteriorates rheumatoid arthritis

An early prognostic indicator which warns of progressive joint destruction of rheumatoid arthritis (RA) was explored using a novel suspension-array technique in moderate (Steinbrocker stage I and II) and severe (Steinbrocker stage IV) RA patients. DNA microarray analysis of peripheral blood lymphocytes showed significant increase of interleukin (IL)-2 receptor α-chain (CD25) gene expression, a regulatory T cell (Treg) surface marker in severe RA patients. In contrast, suspension array, a comprehensive bead-based enzyme-linked immunosorbent assay (ELISA), revealed decreased production of IL-10 and increased production of interferon (IFN)-γ in sera in the incipient stage of the aggressive disease process. Both in moderate and in severe RA patients, the IFN-γ/IL-10 ratio indicated deterioration of the disease with universal validity. Fluorescence-activated cell sorting (FACS) and reverse-transcription polymerase chain reaction (RT-PCR) analysis showed extant CD4+CD25+ regulatory T cells in severe RA patients, however Foxp3, a regulatory T cell-specific transcription factor, gene expression was absent, while glucocorticoid-induced tumor necrosis factor (TNF) receptor family-related protein (GITR), which transmits a signal that abrogates regulatory T cell functions, was elevated. In the current study, we showed the validity of suspension-array analysis for enabling more complete understanding of RA, and showed that IFN-γ/IL-10 ratio can be a prognostic tool for early lesion and more aggressive RA.