Feasibility of commercial interferon-γ-based methods for the diagnosis of latent Mycobacterium tuberculosis infection in Finland, a country of low incidence and high bacille Calmette–Guérin vaccination coverage

The performances of the QuantiFERON-TB Gold in Tubes (QFGT), T SPOT-TB (ELISPOT) and the Mantoux test were compared for the diagnosis of latent tuberculosis infection in Finland, a country of low tuberculosis incidence. In Cohort A (16 students), freshly isolated peripheral blood mononuclear cells (PBMCs), and in Cohort B (21 school children), cryopreserved PBMCs, were used for the ELISPOT assay. Cryopreservation of cells in fetal calf serum, but not in serum-free medium, produced false-positive results. Discrepancies between the results of the assays were observed. It was concluded that the accuracy of these ex-vivo methods needs additional evaluation.     

BK Virus‐Specific Immunity Kinetics: A Predictor of Recovery From Polyomavirus BK‐Associated Nephropathy

Impaired BKV‐specific immunity is associated with development of BKV‐associated nephropathy. Suitable immunological parameters to identify patients at risk, however, are still debated. We monitored 18 kidney‐transplant recipients through the course of self‐limited BKV‐reactivation (n = 11) and BKV‐associated nephropathy (n = 7). BKV‐specific cellular immunity directed to nonstructural small and Large T‐antigen, and structural VP1–3 was analyzed in an interferon‐γ Elispot assay. BKV‐specific IgM and IgG were measured using an enzyme‐linked immunosorbent assay simultaneously. BKV‐specific cellular immunity directed to five BKV‐proteins increased significantly from diagnosis to resolution of BKV‐reactivation (p < 0.001). Patients with self‐limited BKV‐reactivation developed BKV‐specific T cells without therapeutic interventions, and cleared BKV‐reactivation within a median period of 1 month. Patients with BKV‐associated nephropathy, however, showed BKV‐specific T cells after a median period of 5 months after therapeutic interventions only, and cleared BKV‐reactivation after a median period of 8 months. Anti‐structural T cells were detected earlier than anti‐nonstructural T cells, which coincided with BKV‐clearance. Patients with BKV‐associated nephropathy showed the highest frequencies of BKV‐specific T cells at recovery, the highest increase in BKV‐specific IgG and persistence of increased IgM levels (p < 0.05). Our results suggest prognostic values of BKV‐specific immune monitoring to identify those patients at risk of BKV‐associated nephropathy and to aid in the management of therapeutic interventions.     

Serial re-challenge with influenza vaccine as a tool to study individual immune responses

With new treatments of inflammatory diseases targeting key inflammatory pathways follows an increased risk for infections. The aim of the present study was to identify an immunological readout where consecutive immunizations induce reproducible immune responses. Such a method could be used as a tool to assess drug-induced immunomodulation in individual patients by comparing responses to the immunizations before and after introduction of a specific treatment. Importantly, the vaccine is merely used as a model antigen and protective immunity is not the primary aim of the method. Eleven volunteers were immunized with influenza vaccine three times, four weeks apart. In order to find the optimal readout for the method, immune responses to the immunizations were measured as circulating antigen-specific B-cells, serum antibody titers and avidity, T-cell proliferation and cytokine secretion. The first exposure to the influenza vaccine induced a stronger B- and T-cell responses than the consecutive immunizations. The second and third immunizations induced comparable but lower B-cell responses as measured by ELISPOT. In summary, we have measured immune responsiveness by using repeated immunizations with influenza virus vaccine as the model antigen. The induction of comparable B-cell responses after the second and third serial immunizations provides a possibility to investigate effects on immune responsiveness by immunomodulatory drugs. The method also allows humoral memory and immune competence per se to be studied on a cellular level in different patient groups.     

Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity

Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the T_H1, T_H2, and T_H17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood mononuclear cell (PBMC). Using the enzyme-linked Immunospot^® assay (ELISPOT) that excels in low frequency measurements, we have established these in a sizable cohort of healthy HCMV controllers. Cytokine recall responses were seen in all seropositive donors. Specifically, interferon (IFN)-γ and/or interleukin (IL)-17 were seen in isolation or with IL-4 in all test subjects. IL-4 recall did not occur in isolation. While the ratios of T_H1, T_H2, and T_H17 cells exhibited substantial variations between different individuals these ratios and the frequencies were relatively stable when tested in samples drawn up to five years apart. IFN-γ and IL-2 co-expressing polyfunctional cells were seen in most subjects. Around half of the HCMV-specific CD4 cells were in a reversible state of exhaustion. The data provided here established the T_H1, T_H2, and T_H17 characteristic of the CD4 cells that convey immune protection for successful immune surveillance against which reactivity can be compared when the immune surveillance of HCMV fails.     

寻常型天疱疮患者抗原特异性Th细胞的研究

【摘要】 目的 探讨寻常型天疱疮（pemphigus vulgaris,PV ）患者抗原特异性Th1和Th2细胞在不同疾病阶段的变化,进一步了解自身反应性T细胞在疾病中的作用。 方法 收集24例PV患者资料,合成桥粒芯糖蛋白抗原肽段DG3（96 ～ 112）。体外用该肽段分别刺激患者的外周血单一核细胞（PBMC）,用酶联免疫斑点（ELISPOT）方法检测干扰素（IFN）-γ+ Th1细胞和白介素（IL）-4+ Th2细胞的数量,以及记忆性B细胞的数量。用t检验或单因素方差分析（one-way ANOVA）对各组数值进行比较,用Pearson相关系数对Th细胞、记忆性B细胞及抗桥粒芯糖蛋白3（Dsg3）抗体滴度进行相关性分析。 结果 24例PV患者男女比例1.67 ∶ 1,平均年龄（56.59 ± 14.66）岁。5 × 105个PBMC中,PV患者特异性IFN-γ+ Th1细胞绝对数为420.18 ± 350.29,高于健康对照145.12 ± 86.56,差异有统计学意义（P < 0.05）。PV患者特异性IL-4+ Th2的细胞数（366.76 ± 192.44）与健康对照（335.88 ± 164.96）之间差异无统计学意义。PV患者外周血中特异性IL-4+ Th2占总Th2细胞的百分率为37.03% ± 23.44%,特异性IFN-γ+ Th1细胞为23.62% ± 16.77%;7例患者进行了治疗前后的自身对照比较。特异性IL-4+ Th2细胞的数量在治疗后（241.68 ± 160.60）较治疗前（452.82 ± 199.29）明显下降,差异有统计学意义（t = 2.48,P < 0.05）。特异性Th细胞、记忆性B细胞及抗Dsg3抗体滴度之间无统计学相关性（均P > 0.05）。结论 抗原肽段DG3（96 ～ 112）含有被PV患者特异性Th细胞识别的致病性抗原表位;特异性IFN-γ+ Th1细胞和IL-4+ Th2细胞均在疾病中发挥一定的作用;特异性IL-4+ Th2细胞在疾病活动中可能起到了更重要的致病作用。     

Granzyme B ELISPOT assay for ex vivo measurements of T cell immunity

A major goal in immunodiagnostics has been the development of assay systems that can measure CD8⁺ T cell immunity in humans, directly ex vivo, at high resolution, and with high throughput. We established granzyme B (grB) enzyme-linked immunospot assay (ELISPOT) in conjunction with image analysis to this end. Using grB transfected and untransfected Chinese hamster ovary (CHO) cells and T cell lines, we show that the antibody pair utilized was grB-specific and that only activated T cells secrete grB. GrB release began within 4 h after antigen stimulation and stopped within 40 h. Side-by-side comparison showed grB ELISPOT assays to have a higher resolution than classic chromium-release assays in terms of signal-to-noise ratio. The linearity of the relation of the number of CD8⁺ effector T cells plated to grB spots detected suggests that grB ELISPOT assays measure the frequencies of grB-secreting cells directly. Reactivity to HIV peptides was seen in grB ELISPOT assays of freshly isolated PBMC from HIV patients, consistent with the detection of peptide-specific memory cells. The higher resolution and lower labor and material investment should make grB ELISPOT assays an attractive alternative to chromium-release assays in monitoring the clonal sizes of specific CD8 memory cells in vivo.     

Mucosal and systemic immune responses to plasmid protein pgp3 in patients with genital and ocular Chlamydia trachomatis infection

The circulating and cervical B cell responses to Chlamydia trachomatis plasmid protein pgp3 were characterized in children and adults with ocular or genital chlamydial infection using the enzyme-linked immunospot assay (ELISPOT) and ELISA. No pgp3-specific ASCs were detected in healthy controls, but predominantly IgA ASCs were detected in UK adults with uncomplicated cervicitis or urethritis (P = 0.03, 0.019). In patients with extragenital complications or pelvic inflammatory disease a mixed response with more IgG and IgM ASCs was evident, suggesting a breach of mucosal immune compartmentalization with more extensive infection. In women with chlamydial cervicitis, ASCs secreting predominantly IgA, but also IgG, to pgp3 were present in cervix at presentation, with a frequency 30-50 times higher than blood. Cervical ASC numbers, especially IgG, fell markedly six weeks after antibiotic treatment. We detected principally IgA pgp3-specific antibody secreting cells (ASCs) in children resident in a Gambian endemic area, with a trend towards suppression of IgA responses during intense trachomatous inflammation (P = 0.06), as previously reported for other chlamydial antigens, and in keeping with the findings in genital disease. These data provide a rationale for further studies of immune responses to pgp3 in humans and animal models of chlamydia-induced disease, and its potential use in diagnostic assays and protective immunization strategies.     

Solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of IgG rheumatoid factor-secreting cells

Although IgG rheumatoid factor may play an essential role in the pathogenesis of rheumatoid arthritis, there is no precise method for its specific detection at the cellular level. A modification of the recently developed enzyme-linked immunospot assay has been devised for enumeration of cells secreting IgG rheumatoid factor (IgG RF) and simultaneous quantitation of the IgG RF secreted. Specific, sensitive and simple, this new assay should provide a valuable tool for study of isotype-specific RF secretion by single cells.     

CTL responses to regulatory proteins Tat and Rev in HIV-1 B'/C virus-infected individuals

Objective To characterize HIV-1 specific CTL responses to regulatory proteins Tat and Rev in HIV-B＇/C virus-infected ART-naive individuals. Methods HIV-1-specific CTL responses were analyzed by IFN-7 ELISPOT assay using overlapping peptides spanning the consensus sequences of HIV-1 clade C Tat and Rev proteins. Statistical analysis and graphical presentation were performed using SIGMAPLOT 10.0 and SIGMASTAT 3.5. For samples with a positive response, the magnitude of CTL responses was compared between HIV-1 C proteins by Wilcoxon rank sum test, and the significance threshold was P〈0.05. Results Tat and Rev were frequently recognized, with 23% and 52% of the tested individuals having detectable responses to these proteins, respectively. Several immunodominant regions were detected in Rev. No significant correlation was observed between the magnitude and breadth of CTL responses to regulatory proteins and the control of virus replication in this study. Conclusion Tat and Rev can serve as targets for HIV-l-specific CTL, and several immunodominant regions are detectable in Rev. Further characterization of epitopes and their role in virus control may shed light on pathogenesis of HIV- 1 natural infection and also be useful for the design and testing of candidate vaccines.     

HCV-specific lymphocyte responses in individuals with positive anti-HCV but negative HCV-RNA

BackgroundHepatitis C virus (HCV) status cannot be reliably predicted in anti-HCV positive/HCV-RNA negative individuals who may either have recovered spontaneously or have a false-positive test due to antibody cross-reaction. Investigating T lymphocyte responses in individuals with different HCV status may help understand the cellular immune mechanisms underlying spontaneous recovery, treatment response, and chronicity.ObjectiveWe aimed to determine whether anti-HCV positive, HCV-RNA negative individuals are truly spontaneous recoverers from acute HCV infection.Study designWe used enzyme-linked immunosorbent spot (ELISPOT) assay to compare HCV-specific lymphocyte response among anti-HCV positive/HCV-RNA negative individuals, patients with sustained virological response to interferon-γ/ribavirin treatment, and patients with chronic HCV infection.ResultsWe found that 83% of anti-HCV positive/HCV-RNA negative individuals without a past medical history of acute icteric hepatitis had an HCV-specific T lymphocyte response in peripheral blood. Lymphocyte responses in these individuals were similar in magnitude to treatment responders unlike patients with chronic HCV whose virus-directed immunity was significantly suppressed.ConclusionsDetection of HCV-specific T lymphocyte responses using ELISPOT is a feasible method to ascertain past asymptomatic acute HCV infection.