Interferon-γ Enzyme-linked Immunospot Assay Research Articles

Cellular immune response following pre-exposure and postexposure rabies vaccination by intradermal and intramuscular routes.

PurposeImmunization against rabies in humans induces protective neutralizing antibodies; however, the induction of type 1 or type 2 cytokine mediated cellular immune responses following rabies vaccination is not understood. Hence, the present study investigated cellular cytokine responses in vaccinated individuals.Materials and MethodsThe study groups included healthy rabies antigen naive controls (n=10), individuals who received intradermal primary (n=10) or booster pre-exposure vaccination (n=20) and subjects who received postexposure rabies vaccination either by intradermal (n=18) or intramuscular (n=20) routes. The antigen specific cellular responses were analyzed by stimulating peripheral blood mononuclear cells with a rabies vaccine antigen in the interferon-γ (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunospot (ELISpot) assay. These responses were compared to the rabies virus neutralizing antibody (RVNA) titers that were measured by rapid fluorescent focus inhibition test.ResultsWe observed that cellular and humoral immune responses to primary intradermal rabies vaccination could be greatly enhanced by a booster vaccine; and both type 1 and type 2 cytokine responses were significantly elevated. The magnitude of type 1 and type 2 cytokine responses did not differ significantly among the intramuscular and intradermal routes of postexposure vaccination. The number of cells producing IFN-γ and IL-4 correlated significantly with the levels of RVNA.ConclusionBoth type 1 and type 2 cellular cytokine responses are strongly induced after rabies vaccination and directly correlate with levels of RVNA titers. The neutralizing antibody as well as the type 1 and type 2 cytokine responses may be important for vaccine induced protective responses against rabies.

Altered cellular and humoral immunity to varicella-zoster virus in patients with autoimmune diseases.

Patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and granulomatosis with polyangiitis (Wegener's) (GPA) have a 3-20-fold increased risk of herpes zoster compared to the general population. The aim of this study was to evaluate if susceptibility is due to decreased levels of cellular and/or humoral immunity to the varicella-zoster virus (VZV). A cross-sectional study of VZV-specific immunity was performed in 38 SLE patients, 33 GPA patients, and 51 healthy controls. Levels of IgG and IgM antibodies to VZV were measured using an in-house glycoprotein enzyme-linked immunosorbent assay (ELISA). Cellular responses to VZV were determined by interferon-γ (IFNγ) enzyme-linked immunospot (ELISpot) assay and carboxyfluorescein succinimidyl ester (CFSE) dye dilution proliferation assay. Levels of IgG antibodies to VZV were increased in SLE patients as compared to healthy controls, but levels of IgM antibodies to VZV were not. Antibody levels in GPA patients did not differ significantly from levels in healthy controls. In response to stimulation with VZV, decreased numbers of IFNγ spot-forming cells were found among SLE patients (although not GPA patients) as compared to healthy controls. Proliferation of CD4+ T cells in response to stimulation with VZV was decreased in SLE patients but not GPA patients. SLE patients have increased levels of IgG antibodies against VZV, while cellular immunity is decreased. In GPA patients, antibody levels as well as cellular responses to VZV were comparable to those in healthy controls. These data suggest that increased prevalence of herpes zoster in SLE patients is due to a poor cellular response. Vaccination strategies should aim to boost cellular immunity against VZV.

Feasibility and Safety of Systemic rAAV9-hNAGLU Delivery for Treating Mucopolysaccharidosis IIIB: Toxicology, Biodistribution, and Immunological Assessments in Primates

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease caused by autosomal recessive defect in α-N-acetylglucosaminidase (NAGLU). In anticipation of a clinical gene therapy treatment for MPS IIIB in humans, we tested the rAAV9-CMV-hNAGLU vector administration to cynomolgus monkeys (n=8) at 1E13 vg/kg or 2E13 vg/kg via intravenous injection. No adverse events or detectable toxicity occurred over a 6-month period. Gene delivery resulted in persistent global central nervous system and broad somatic transduction, with NAGLU activity detected at 2.9-12-fold above endogenous levels in somatic tissues and 1.3-3-fold above endogenous levels in the brain. Secreted rNAGLU was detected in serum. Low levels of preexisting anti-AAV9 antibodies (Abs) did not diminish vector transduction. Importantly, high-level preexisting anti-AAV9 Abs lead to reduced transduction in liver and other somatic tissues, but had no detectable impact on transgene expression in the brain. Enzyme-linked immunoabsorbent assay showed Ab responses to both AAV9 and rNAGLU in treated animals. Serum anti-hNAGLU Abs, but not anti-AAV9 Abs, correlated with the loss of circulating rNAGLU enzyme. However, serum Abs did not affect tissue rNAGLU activity levels. Weekly or monthly peripheral blood interferon-γ enzyme-linked immunospot assays detected a CD4(+) T-cell (Th-1) response to rNAGLU only at 4 weeks postinjection in one treated subject, without observable correlation to tissue transduction levels. The treatment did not result in detectable CTL responses to either AAV9 or rNAGLU. Our data demonstrate an effective and safe profile for systemic rAAV9-hNAGLU vector delivery in nonhuman primates, supporting its clinical potential in humans.

Interferon-γ Enzyme-Linked Immunospot Assay in Patients with Tuberculosis and Healthy Adults

BackgroundInterferon-γ assays based on tuberculosis (TB)-specific antigens have been utilized for diagnosing and ruling out latent TB and active TB, but their utility is still limited for TB incidence countries. The aim of this study is to understand the clinical utility of enzyme-linked immunospot (ELISpot) assays among patients with clinically suspected TB and healthy adults in clinical practices and community-based settings.MethodsThe ELISpot assays (T SPOT.TB, Oxford Immunotec, UK) were prospectively performed in 202 patients. After excluding those with indeterminate results, 196 were included for analysis: 41 were TB patients, 93 were non-TB patients, and 62 were healthy adults.ResultsThe sensitivity and negative predictive values of the T SPOT.TB assays for the diagnosis of TB were 87.8% and 89.1%, respectively, among patients with suspected TB. The agreement between the tuberculin skin test (10-mm cutoff) and the T SPOT.TB assay was 66.1% (kappa=0.335) in all participants and 80.0% (kappa=0.412) in TB patients. Among those without TB (n=155), a past history of TB and fibrotic TB scar on chest X-rays were significant factors that yielded positive T SPOT.TB results. There was a significant difference in the magnitude of T SPOT.TB spot counts between TB patients and non-TB patients or healthy adults.ConclusionThe T SPOT.TB assay appeared to be a useful test for the diagnostic exclusion of TB. A positive result, however, should be cautiously interpreted for potential positives among those without active TB in intermediate TB incidence areas.

Self-Reactive CFTR T Cells in Humans: Implications for Gene Therapy

Cystic fibrosis (CF) is one of the most common autosomal recessive lethal disorders affecting white populations of northern European ancestry. To date there is no cure for CF. Life-long treatments for CF are being developed and include gene therapy and the use of small-molecule drugs designed to target specific cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations. Irrespective of the type of molecular therapy for CF, which may include gene replacement, exon skipping, nonsense suppression, or molecular correctors, because all of these modulate gene expression there is an inherent risk of activation of T cells against the wild-type version of CFTR. Here we report the validation of the human interferon-γ enzyme-linked immunospot assay and its application for the analysis of CFTR-specific T cell responses in patients with CF and in non-CF subjects. We found non-CF subjects with low levels of self-reactive CFTR-specific T cells in the United States and several patients with CF with low to high levels of self-reactive CFTR-specific T cells in both the United States and the United Kingdom.

Detection of Epstein–Barr virus‐specific memory CD4+T cells using a peptide‐based cultured enzyme‐linked immunospot assay

Approaches to evaluate T-cell responses to Epstein-Barr virus (EBV) include enzyme-linked immunospot (ELISPOT), which quantifies cells capable of immediate interferon-γ secretion upon antigen stimulation. However, evaluation of expandable EBV-specific memory T cells in an ELISPOT format has not been described previously. We quantified EBV-specific T-cell precursors with high proliferative capacity by using a peptide-based cultured interferon-γ ELISPOT assay. Standard and cultured ELISPOT responses to overlapping peptide pools (15-mers overlapping by 11 amino acids) covering the lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) EBV proteins were evaluated in 20 healthy subjects with remote EBV infection and, for comparison, in four solid organ transplant recipients. Cultured ELISPOT responses to both lytic and latent EBV antigens were significantly higher than standard ELISPOT responses. The distribution of EBV-specific T-cell responses detected in healthy virus carriers showed more consistent cultured ELISPOT responses compared with standard ELISPOT responses. T-cell responses quantified by cultured ELISPOT were mainly mediated by CD4+ T cells and a marked pattern of immunodominance to latent-phase antigens (EBNA1 > EBNA3 family antigens > LMP2 > LMP1) was shown. Both the magnitude and distribution of EBV-specific T-cell responses were altered in solid organ transplant recipients; in particular, cultured ELISPOT responses were almost undetectable in a lung-transplanted patient with EBV-associated diseases. Analysis of T-cell responses to EBV by ELISPOT assays might provide new insights into the pathogenesis of EBV-related diseases and serve as new tools in the monitoring of EBV infection in immunocompromised patients.

Granulocyte-Macrophage Colony-Stimulating Factor-Armed Oncolytic Measles Virus Is an Effective Therapeutic Cancer Vaccine

Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased antitumor activity, and are currently under investigation in clinical phase 1 trials. Approaches with other viral vectors have shown that insertion of immunomodulatory transgenes enhances the therapeutic potency. In this study, we engineered MV for expression of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). For the first time, therapeutic efficacy and adaptive immune response in the context of MV oncolysis could be evaluated in the previously established immunocompetent murine colon adenocarcinoma model MC38cea. MC38cea cells express the human carcinoembryonic antigen (CEA), allowing for infection with retargeted MV. Intratumoral application of MV-GMCSF significantly delayed tumor progression and prolonged median overall survival compared with control virus-treated mice. Importantly, more than one-third of mice treated with MV-GMCSF showed complete tumor remission and rejected successive tumor reengraftment, demonstrating robust long-term protection. An enhanced cell-mediated tumor-specific immune response could be detected by lactate dehydrogenase assay and interferon-γ enzyme-linked immunospot assay. Furthermore, MV-GMCSF treatment correlated with increased abundance of tumor-infiltrating CD3(+) lymphocytes analyzed by quantitative microscopy of tumor sections. These findings underline the potential of oncolytic, GM-CSF-expressing MV as an effective therapeutic cancer vaccine actively recruiting adaptive immune responses for enhanced therapeutic impact and tumor elimination. Thus, the treatment benefit of this combined immunovirotherapy approach has direct implications for future clinical trials.

Role of IL28B Gene Polymorphism and Cell-Mediated Immunity in Spontaneous Resolution of Acute Hepatitis C

A single-nucleotide polymorphism (SNP; rs12979860) near the IL28B gene has been associated with spontaneous and treatment-induced hepatitis C virus clearance. We investigated predictors of spontaneous disease resolution in a cohort of patients with acute hepatitis C (AHC), analyzing epidemiological, clinical and virological parameters together with IL28B.rs12979860 genotypes and cell-mediated immunity (CMI). Fifty-six symptomatic AHC patients were enrolled and followed prospectively. CMI was measured in 31 patients at multiple time points by interferon-γ enzyme-linked immunospot assay and was correlated to the IL28B.rs12979860 SNP. Eighteen patients had a self-limiting AHC that was associated with female sex (P = .028), older age (P = .018), alanine aminotransferase level >1000 U/L (P = .027), total bilirubin level >7 mg/dL (P = .036), and IL28B.rs12979860 genotype CC (P = .030). In multivariate analysis, only CC genotype was independently associated with self-limiting AHC (odds ratio, 5.3; 95% confidence interval, 1.1-26.5). Patients with the CC genotype with self-limiting AHC had a stronger (P = .02) and broader (P = .013) CMI than patients with the CT genotype with chronically evolving AHC. In patients with chronically evolving disease, CC genotype was associated with a broader CMI compared to CT genotype (P = .028). A negative CMI was more frequently associated with CT genotype among persistently infected patients (P = .043) and with persistent infection among CT patients (P = .033). . Self-limiting AHC was independently associated with CC genotype. The correlation between IL28B.rs12979860 genotypes and CMI is suggestive of a possible important role of CMI in favoring hepatitis C virus clearance in CC patients.

Enhancement of tumor-associated antigen-specific T cell responses by radiofrequency ablation of hepatocellular carcinoma

Radiofrequency ablation (RFA) is one of the treatments for hepatocellular carcinoma (HCC) and is known to enhance host immune response. However, the epitopes to which enhanced immune responses occur, the impact on patient prognosis, and the functions and phenotype of T cells induced are still unclear. To address these issues, we analyzed immune responses before and after RFA in 69 HCC patients using 11 tumor-associated antigen (TAA)-derived peptides that we identified to be appropriate to analyze HCC-specific immune responses. The immune responses were analyzed using enzyme-linked immunospot (ELISPOT) assay and tetramer assays using peripheral blood mononuclear cells. An increase in the number of TAA-specific T cells detected by interferon-γ ELISPOT assays occurred in 62.3% of patients after RFA. The antigens and their epitope to which enhanced T cell responses occur were diverse, and some of them were newly induced. The number of TAA-specific T cells after RFA was associated with the prevention of HCC recurrence, and it was clarified to be predictive of HCC recurrence after RFA by univariate and multivariate analyses. The number of TAA-specific T cells after RFA was inversely correlated with the frequency of CD14+ HLA-DR(-/low) myeloid-derived suppressor cells (MDSCs). The modification of T cell phenotype was observed after RFA. The number of TAA-specific T cells at 24 weeks after RFA was decreased. Although RFA can enhance various TAA-specific T cell responses and the T cells induced contribute to the HCC recurrence-free survival of patients, besides immunosuppression by MDSCs, the memory phenotype and lifetime of TAA-specific T cells are not sufficient to prevent HCC recurrence completely. Additional treatments by vaccine or immunomodulatory drugs might be useful to improve the immunological effect of RFA.

HLA-A2 and B35 Restricted Hantaan Virus Nucleoprotein CD8+ T-Cell Epitope-Specific Immune Response Correlates with Milder Disease in Hemorrhagic Fever with Renal Syndrome

BackgroundHantaan virus (HTNV) infection in humans is a serious public health concern in Asia. A potent T cell activation peptide vaccine from HTNV structure protein represents a promising immunotherapy for disease control. However, the T cell epitopes of the HTNV restricted by the HLA alleles and the role of epitope-specific T cell response after HTNV infection remain largely unexplored.Methodology/Principal FindingsFive well-conserved novel CD8+ T-cell epitopes of the HTNV nucleoprotein restricted by the most popular HLA alleles in Chinese Han population were defined with interferon-γ enzyme-linked immunospot assay in 37 patients infected with HTNV during hospitalization. Two epitopes aa129–aa137 and aa131–aa139 restricted by HLA-A2 and B35, respectively, were selected to evaluate the epitope-specific CD8+ T-cell response. HLA-peptide pentamer complex staining showed that the frequency of single epitope-specific CD8+ T cell could be detected in patients (95% confidence interval for aa129–aa137: 0.080%–0.208%; for aa131–aa139: 0.030%–0.094%). The frequency of epitope-specific pentamer+ CD8+ T-cell response was much higher in mild/moderate patients than in severe/critical ones at the acute stage of the disease. Moreover, the frequency of epitope-specific CD8+ T cells at acute stage was inversely associated with the peak level of serum creatinine and was positively associated with the nadir platelet counts during the hospitalization. The intracellular cytokine staining and the proliferation assay showed that the effective epitope-specific CD8+ T cells were characterized with the production of interferon-γ, expression of CD69 and the strong capacity of proliferation.Conclusion/SignificanceThe novel HLA class I restricted HTNV nucleoprotein epitopes-specific CD8+ T-cell responses would be closely related with the progression and the severity of the disease, which could provide the first step toward effective peptide vaccine development against HTNV infection in humans.

Which patients respond best to hepatitis B vaccination after a hepatitis B virus-related liver transplantation?

BackgroundA combination of hepatitis B immunoglobulin and nucleos(t)ide analogues is the current standard of care for controlling hepatitis B recurrence after orthotopic liver transplantation (OLT). However, frequent immunoglobulin treatment is expensive and inconvenient. This study investigated the efficacy of hepatitis B virus (HBV) vaccination in preventing the recurrence of hepatitis B after living donor OLT.MethodsTwenty-seven patients who had undergone living donor OLT participated in the study; five had acute HBV infected liver failure (ALF-OLT) and 22 had HBV related liver cirrhosis (LC-OLT). Hepatitis B surface antigen (HBsAg)-containing vaccine was administered to them for at least 1 year after transplantation and continued once monthly for up to 36 months post-OLT. Patients who had anti-HBs antibody titers above 100 mIU/mL for a minimum of 6 months without immunoglobulin administration were defined as good responders; the others were defined as poor responders. Interferon-γ enzyme-linked immunospot assays against HBs and HBc antigens were used to assay cellular immune responses.ResultsAll five of the ALF-OLT patients had good responses after a median of four (range 2.5–5) vaccinations. Nine of the 22 LC-OLT patients had good responses after a median of 19 (range 11.5–30) vaccinations. Among the LC-OLT group, those with livers donated by relatively higher-aged, marital and high-titer anti-HBs antibody donors were good responders. LC-OLT patients classed as good responders showed interferon-γ responses comparable to those of the ALF-OLT patients.ConclusionsThe ALF-OLT and LC-OLT patients who received livers from relatively higher-aged, marital, high-titer anti-HBs antibody donors were the best candidates for HBV vaccine administration. Boosting donors before transplantation may facilitate later vaccine response of the recipients.

The Potential of Using Enzyme-linked Immunospot to Diagnose Cephalosporin-induced Maculopapular Exanthems

There is no reliable test to diagnose cephalosporin-induced maculopapular exanthems (MPE). This study aimed to evaluate the role of enzyme-linked immunospot assay in the diagnosis of cephalosporin-induced MPE compared with skin testing. A total of 25 patients with a history of cephalosporin-induced MPE were skin tested and the frequencies of cephalosporin-specific interferon-γ-, interleukin-5-, and interleukin-10-releasing cells/10(6) peripheral blood mononuclear cells were measured after stimulating with the culprit drug, compared with 20 non-allergic controls. Values greater than means+2 standard deviations of the values in non-allergic controls were considered diagnostic. The study showed that the combination of interferon-γ and interleukin-5 enzyme-linked immunospot assays was more sensitive than skin testing to diagnose cephalosporin allergy (40% vs. 8%, p = 0.008) and sensitivity increased to 57.1% when the test was performed within 2 years of the drug reaction. Enzyme-linked immunospot assay is a promising tool for confirming the diagnosis of cephalosporin-induced MPE.

The antitumor effect of human cord blood-derived dendritic cells modified by the livin α gene in lung cancer cell lines

The growth of malignant tumors is associated with mechanisms of immune escape and inhibition of apoptosis. Livin is a novel member of the inhibitors of apoptosis (IAP) protein family that inhibits cell apoptosis. Livin is specifically expressed by the majority of tumor cells, but it is not expressed in normal adult tissues. In this study, we used umbilical cord blood (UCB)-derived dendritic cells (DCs) infected with a recombinant adenovirus encoding the livin gene as a vaccine to activate effector cells such as cytotoxic T lymphocytes (CTLs) to recognize and kill livin-expressing cancer cells in vitro as an improved strategy for overcoming the ability of these cancer cells to escape apoptosis and antitumor immune responses. We employed interferon-γ (IFN-γ) enzyme-linked immunospot assays to confirm that our immunization strategy induced an antigen-specific reaction to livin and flow cytometric analysis of staining with Annexin V and PI to measure the cytotoxic activity of the effector cells against the livin-expressing lung cancer cell lines A549 and H460. Our results show that the recombinant adenovirus was able to promote the maturation of the UCB-derived DCs. This DC vaccine could activate antigen-specific T cells to produce IFN-γ upon recognition of livin peptide in the context of the appropriate HLA molecule. The antigen-specific T cells mediate significant cytotoxicity against the cancer cells, but are unlikely to cause an autoimmune reaction against the human bronchial epithelia cells (HBE), which do not express livin.

HLA-B7–Restricted Islet Epitopes Are Differentially Recognized in Type 1 Diabetic Children and Adults and Form Weak Peptide-HLA Complexes

The cartography of β-cell epitopes targeted by CD8+ T cells in type 1 diabetic (T1D) patients remains largely confined to the common HLA-A2 restriction. We aimed to identify β-cell epitopes restricted by the HLA-B7 (B*07:02) molecule, which is associated with mild T1D protection. Using DNA immunization on HLA-B7–transgenic mice and prediction algorithms, we identified GAD and preproinsulin candidate epitopes. Interferon-γ (IFN-γ) enzyme-linked immunospot assays on peripheral blood mononuclear cells showed that most candidates were recognized by new-onset T1D patients, but not by type 2 diabetic and healthy subjects. Some epitopes were highly immunodominant and specific to either T1D children (GAD530–538; 44% T cell–positive patients) or adults (GAD311–320; 38%). All epitopes displayed weak binding affinity and stability for HLA-B7 compared with HLA-A2–restricted ones, a general feature of HLA-B7. Single-cell PCR analysis on β-cell–specific (HLA-B7 tetramer–positive) T cells revealed uniform IFN-γ and transforming growth factor-β (TGF-β) mRNA expression, different from HLA-A2–restricted T cells. We conclude that HLA-B7–restricted islet epitopes display weak HLA-binding profiles, are different in T1D children and adults, and are recognized by IFN-γ+TGF-β+CD8+ T cells. These features may explain the T1D-protective effect of HLA-B7. The novel epitopes identified should find valuable applications for immune staging of HLA-B7+ individuals.

Maximizing Tumor Immunity With Fractionated Radiation

Technologic advances have led to increased clinical use of higher-sized fractions of radiation dose and higher total doses. How these modify the pathways involved in tumor cell death, normal tissue response, and signaling to the immune system has been inadequately explored. Here we ask how radiation dose and fraction size affect antitumor immunity, the suppression thereof, and how this might relate to tumor control. Mice bearing B16-OVA murine melanoma were treated with up to 15 Gy radiation given in various-size fractions, and tumor growth followed. The tumor-specific immune response in the spleen was assessed by interferon-γ enzyme-linked immunospot (ELISPOT) assay with ovalbumin (OVA) as the surrogate tumor antigen and the contribution of regulatory T cells (Tregs) determined by the proportion of CD4(+)CD25(hi)Foxp3(+) T cells. After single doses, tumor control increased with the size of radiation dose, as did the number of tumor-reactive T cells. This was offset at the highest dose by an increase in Treg representation. Fractionated treatment with medium-size radiation doses of 7.5 Gy/fraction gave the best tumor control and tumor immunity while maintaining low Treg numbers. Radiation can be an immune adjuvant, but the response varies with the size of dose per fraction. The ultimate challenge is to optimally integrate cancer immunotherapy into radiation therapy.

Immunology of Diabetes Society T‐Cell Workshop: HLA class I tetramer‐directed epitope validation initiative T‐Cell Workshop Report—HLA Class I Tetramer Validation Initiative

Identification of T-cell reactivity to β-cell antigen epitopes is an important goal for studying pathogenesis and for designing and monitoring of immunotherapeutic interventions in type 1 diabetes (T1D). We performed a multicentre validation of known human leukocyte antigen (HLA) class I CD8+ T-cell epitopes. To this end, peripheral blood T-cell responses were measured in 35 recently (<2 years) diagnosed HLA-A*02:01+ T1D patients using blind-coded HLA-A2 tetramers (TMrs) and pentamers (PMrs), encompassing two epitopes of preproinsulin (PPI; PPIA12-20 and PPIB10-18) and two epitopes of glutamic acid decarboxylase (GAD; GAD114-122 and GAD536-545). We also compared the readout of TMrs and PMrs with a CD8+ T-cell interferon-γ enzyme-linked immunospot assay. Despite the minute frequencies of autoreactive cells detected by TMrs/PMrs, most (73-77%) T1D patients had responses to one or more of the epitopes used. All four epitopes were recognized by T1D patients, with a prevalence ranging from 5 to 25%. TMrs and PMrs detected more positive responses to the β-cell epitopes than CD8+ T-cell interferon-γ enzyme-linked immunospot. However, concordance between positive responses to TMrs and PMrs was limited. Using a multicentre blind-coded setup and three different T-cell assays, we have validated PPI and GAD epitopes as commonly recognized CD8+ T-cell targets in recently diagnosed T1D patients. Both TMrs and PMrs showed higher detection sensitivity than the CD8+ T-cell interferon-γ enzyme-linked immunospot assay. However, there are some important methodological issues that need to be addressed in using these sensitive techniques for detecting low frequency responses.

Radiofrequency ablation for hepatocellular carcinoma induces glypican-3 peptide-specific cytotoxic T lymphocytes

Glypican-3 (GPC3), a carcinoembryonic antigen, is an ideal target for anticancer immunotherapy against hepatocellular carcinoma (HCC). In this study, we attempted to compare the induction of the GPC3-specific T-cell-mediated immune response after locoregional therapies in HCC patients and tumor-bearing mice. Twenty-seven HCC patients treated with locoregional therapies, including radiofrequency ablation (RFA), surgical resection and transcatheter arterial chemo-embolization (TACE), were prospectively enrolled in this study. Additionally, we performed RFA experiments using a mouse model. GPC3-specific T-cell response was investigated pre-treatment and post-treatment by an interferon-γ enzyme-linked immunospot assay using peripheral blood mononuclear cells from HCC patients and lymph node cells from tumor-bearing mice. Circulating GPC3-specific cytotoxic T lymphocytes (CTLs) were increased in 5 of 9 patients after RFA and in 4 of 9 patients after TACE, but in only 1 of 9 patients after surgical resection. All 7 patients with GPC3-expressing HCCs exhibited an increase in GPC3-specific CTLs after RFA or TACE, whereas none of the 7 patients did after surgical resection. The number of increased GPC3-specific CTLs after RFA was significantly larger than that after surgical resection (P=0.023). Similarly, the frequency of GPC3-specific CTLs after RFA was significantly greater than that after surgical resection in the mouse model (P=0.049). We validated for the first time the stronger effect on the immune system brought by RFA compared with surgical resection for HCC patients and tumor-bearing mice. Combined treatment of RFA and immunotherapy is a reasonable strategy against HCC.

Diagnostic performance of whole-blood interferon-γ assay and enzyme-linked immunospot assay for active tuberculosis,

The aim of this study was to compare the diagnostic performance of 2 interferon-γ release assays, an enzyme-linked immunospot assay (T-SPOT.TB; Oxford Immunotec Ltd., Oxford, UK) and the QuantiFERON-TB Gold in-Tube assay (QFT-GIT; Cellestis Ltd., Carnegie, Australia), in patients with suspected active tuberculosis (TB). From October 2009 to October 2011, a total of 200 patients with suspected TB were enrolled. Clinical and microbiological characteristics of the patients were collected and blood samples were obtained for T-SPOT.TB and QFT-GIT assays. Among the 200 subjects, 98 (49%) had culture-confirmed TB, 18 (9%) had probable TB, and the remaining 84 (42%) subjects did not have TB. The sensitivity, specificity, positive predictive value, and negative predictive value for active TB diagnosis by the T SPOT. TB were 83%, 71%, 81%, and 75%, respectively. For QFT-GIT, the sensitivity, specificity, positive predictive value, and negative predictive value for active TB diagnosis were 66%, 76%, 80%, and 62%, respectively. The QFT-GIT assay resulted in more indeterminate and false-negative results than the T-SPOT.TB assay, especially in immunocompromised patients. In conclusion, T-SPOT.TB had a higher sensitivity and resulted in fewer indeterminate results than the QFT-GIT assay for diagnosing active TB.

CD4+ T-cell response against human papillomavirus type 16 E6 protein is associated with a favorable clinical trend

The association between the CD8+ T-cell responses to human papillomavirus type 16 (HPV-16) E6 protein and a favorable clinical trend has been demonstrated previously. The roles of human papillomavirus (HPV)-specific CD4+ T-cell responses and of regulatory T-cells (Tregs) were examined. Subjects with a recent history of abnormal Papanicolaou smear were eligible, and colposcopy-guided biopsy was performed at enrollment. Interferon-γ enzyme-linked immunospot assay and fluorescent-activated cell sorter analysis to measure the frequencies of Tregs were performed. Subjects with histological diagnoses of cervical intraepithelial neoplasia 1, 2, or 3 were considered to have short-term persistence of cervical abnormality and were called "persistors" (n=51) while those of normal histology were designated to be "regressors" (n=33). A significantly higher percentage CD4+ T-cell response was detected in the regressors (15/33 or 45.5%) compared with the persistors (10/51 or 19.6%) (P=.015) for the E6 peptides but not for the E7 peptides. The CD4+ responses to certain E6 regions [E6(16-40), E6(91-115), E6(106-130), and E6(136-158)] were also significantly higher in the regressors. Although there was no difference in the frequencies of Tregs between the two groups, low frequencies of Tregs were significantly associated with positive CD4+ T-cell responses within certain E6 regions [E6(16-40), E6(31-55), E6(76-100), E6(91-115), and E6(106-130)]. The CD4+ and CD8+ T-cell responses to the HPV-16 E6 protein are associated with a favorable clinical trend. The HPV-16 E6 protein should be incorporated in the design of an HPV therapeutic vaccine.

Diagnostic value of an enzyme-linked immunospot assay for interferon-γ in cutaneous tuberculosis

The aim of this study was to evaluate the diagnostic performance of an enzyme-linked immunospot (ELISPOT) assay (T-SPOT.TB; Oxford Immunotec, Oxford, UK) for interferon-γ in patients with suspected cutaneous tuberculosis (TB). From March 2007 to June 2010, a total of 45 patients with suspected cutaneous TB were enrolled. Data on clinical characteristics of the patients and conventional laboratory results were collected, and blood samples were obtained for ELISPOT assay. Ten subjects (22.2%) had culture-confirmed TB, 2 (4.4%) subjects had probable TB, and the remaining 33 (73.3%) subjects did not have TB. Twenty-one patients with mycobacterial infection had available biopsy or surgical specimens for histopathologic examination and 16 (76.2%) specimens had pathologic features (granulomatous inflammation, caseating necrosis, or acid-fast bacilli) consistent with mycobacterial infection. Among these 16 patients, all 6 patients with TB and 3 patients with Mycobacterium marinum infection had positive ELISPOT results, but none of the patients with Mycobacterium abscessus, Mycobacterium chelonae, or Mycobacterium avium infection had positive ELISPOT results. Overall, 9 of 10 patients with culture-confirmed TB and both patients with probable TB had positive ELISPOT assay. For patients with nontuberculous mycobacteria infections, ELISPOT assay was positive only in patients with M. marinum and M. kansasii infection. The sensitivity, specificity, positive predictive value, and negative predictive value for cutaneous TB diagnosis by the ELISPOT assay were 91.6% (95% CI, 64.6–98.5%), 75.8% (95% CI, 59.0–87.2%), 57.9% (95% CI, 34.0–78.9%), and 96.2% (95% CI, 59.0–87.2%), respectively. In conclusion, ELISPOT assay can provide useful support in the diagnosis of cutaneous TB.