Inclusion of the Bovine Neutrophil Beta-Defensin 3 with Glycoprotein D of Bovine Herpesvirus 1 in a DNA Vaccine Modulates Immune Responses of Mice and Cattle

Bovine herpesvirus 1 (BoHV-1) causes recurrent respiratory and genital infections in cattle and predisposes them to lethal secondary infections. While modified live and killed BoHV-1 vaccines exist, these are not without problems. Development of an effective DNA vaccine for BoHV-1 has the potential to address these issues. As a strategy to enhance DNA vaccine immunity, a plasmid encoding the bovine neutrophil beta-defensin 3 (BNBD3) as a fusion with truncated glycoprotein D (tgD) and a mix of two plasmids encoding BNBD3 and tgD were tested in mice and cattle. In mice, coadministration of BNBD3 on the separate plasmid enhanced the tgD-induced gamma interferon (IFN-γ) response but not the antibody response. BNBD3 fused to tgD did not affect the antibody levels or the number of IFN-γ-secreting cells but increased the induction of tgD-specific cytotoxic T lymphocytes (CTLs). In cattle, the addition of BNBD3 as a fusion construct also modified the immune response. While the IgG and virus-neutralizing antibody levels were not affected, the number of IFN-γ-secreting cells was increased after BoHV-1 challenge, specifically the CD8⁺ IFN-γ⁺ T cells, including CD8⁺ IFN-γ⁺ CD25⁺ CTLs. While reduced virus shedding, rectal temperature, and weight loss were observed, the level of protection was comparable to that observed in pMASIA-tgD-vaccinated animals. These data show that coadministration of BNBD3 with a protective antigen as a fusion in a DNA vaccine strengthened the Th1 bias and increased cell-mediated immune responses but did not enhance protection from BoHV-1 infection.     

Distinct Contributions of CD4+ and CD8+ T Cells to Pathogenesis of Trypanosoma brucei Infection in the Context of Gamma Interferon and Interleukin-10

Although gamma interferon (IFN-γ) and interleukin-10 (IL-10) have been shown to be critically involved in the pathogenesis of African trypanosomiasis, the contributions to this disease of CD4⁺ and CD8⁺ T cells, the major potential producers of the two cytokines, are incompletely understood. Here we show that, in contrast to previous findings, IFN-γ was produced by CD4⁺, but not CD8⁺, T cells in mice infected with Trypanosoma brucei. Without any impairment in the secretion of IFN-γ, infected CD8^−/− mice survived significantly longer than infected wild-type mice, suggesting that CD8⁺ T cells mediated mortality in an IFN-γ-independent manner. The increased survival of infected CD8^−/− mice was significantly reduced in the absence of IL-10 signaling. Interestingly, IL-10 was also secreted mainly by CD4⁺ T cells. Strikingly, depletion of CD4⁺ T cells abrogated the prolonged survival of infected CD8^−/− mice, demonstrating that CD4⁺ T cells mediated protection. Infected wild-type mice and CD8^−/− mice depleted of CD4⁺ T cells had equal survival times, suggesting that the protection mediated by CD4⁺ T cells was counteracted by the detrimental effects of CD8⁺ T cells in infected wild-type mice. Interestingly, CD4⁺ T cells also mediated the mortality of infected mice in the absence of IL-10 signaling, probably via excessive secretion of IFN-γ. Finally, CD4⁺, but not CD8⁺, T cells were critically involved in the synthesis of IgG antibodies during T. brucei infections. Collectively, these results highlight distinct roles of CD4⁺ and CD8⁺ T cells in the context of IFN-γ and IL-10 during T. brucei infections.     

Reduced interferon-α production by dendritic cells in type 1 diabetes does not impair immunity to influenza virus

The increased risk and persistence of infections in diabetic condition is probably associated with defects in the cellular immune responses. We have previously shown a decrease in the production of interferon (IFN)-α by dendritic cells (DCs) in diabetic subjects. The basal level of IFN-α in splenic plasmacytoid DCs (pDCs) is also lower in non-obese diabetic (NOD) mice compared to prediabetic mice. The objective of this study was to analyse the ability of diabetic mice to mobilize innate and CD8⁺ T cell-mediated immune response to influenza A virus (IAV) with the live influenza A/Puerto Rico/8/1934 H1N1 (PR8) strain or with its immunodominant CD8⁺ T cell epitopes. We found that following immunization with IAV, the level of IFN-α in diabetic mice was increased to the level in prediabetic mice. Immunization of NOD mice with the immunodominant IAV PR8 peptide induced clonal expansion of IFN-γ-producing CD8⁺ T cells similar to the response observed in prediabetic mice. Thus, diabetic and prediabetic NOD mice have a similar capacity for IFN-α and IFN-γ production by pDCs and CD8⁺ T cells, respectively. Therefore, the DC-related immune defect in diabetic NOD mice does not impair their capacity to develop an effective immune response to IAV. Our results suggest that reduced IFN-α production by diabetic human and mouse DCs is not an impediment to an effective immunity to IAV in type 1 diabetic subjects vaccinated with live attenuated influenza vaccine.     

Depletion of γδ+ T cells increases CD4+ FoxP3 (T regulatory) cell response in coxsackievirus B3-induced myocarditis

Coxsackievirus B3 (CVB3) causes severe myocarditis in BALB/c mice which depends upon CD4⁺ T helper type 1 [Th1; i.e. interferon-γ⁺ (IFN-γ⁺)] and γδ⁺ cells. Depleting γδ⁺ cells using anti-γδ antibody suppresses myocarditis and CD4⁺ IFN-γ⁺ cell numbers in the spleen and heart of infected mice while increasing CD4⁺ FoxP3⁺ cells. Mice deficient in γδ⁺ cells have increased numbers of naïve (CD44^lo CD62L^hi) and fewer effector (CD44^hi CD62^lo) memory CD4⁺ cells than infected γδ⁺-cell-sufficient mice. Virus neutralizing antibody titres are not significantly different between γδ⁺ T-cell-sufficient and -deficient animals. To confirm that the memory cell response differs in acutely infected mice lacking γδ⁺ cells, CD4⁺ cells were purified and adoptively transferred into naïve recipients, which were rested for 4 weeks then infected with CVB3. Recipients given either 0·5 × 10⁶ or 1·0 × 10⁶ CD4⁺ from infected donors developed over twice the severity myocarditis and 10-fold less cardiac virus titre compared with recipients given equivalent numbers of CD4⁺ cells from infected and γδ⁺-cell-depleted donor animals. Additionally, to show that more functionally active T regulatory cells are present in γδ⁺ T-cell-depleted mice, CD4⁺ CD25⁺ and CD4⁺ CD25⁻ cells were isolated and adoptively transferred into infected recipients. Mice receiving CD4⁺ CD25⁺ cells from γδ⁺ T-cell-depleted donors developed significantly less myocarditis and CD4⁺ Th1 cell responses compared with mice receiving equal numbers of CD4⁺ CD25⁺ cells from infected γδ⁺ T-cell-sufficient animals. This study shows that γδ⁺ cells promote CD4⁺ IFN-γ⁺ acute and memory responses by limiting FoxP3⁺ T regulatory cell activation.     

Assessment of the Phenotype and Functionality of Porcine CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus (CSFV) and Virulent CSFV Challenge

Vaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has been associated with virus-specific T cell gamma interferon (IFN-γ) responses. In this study, we employed flow cytometry to characterize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3⁺ CD4⁻ CD8^hi T cell population was the first and major source of CSFV-specific IFN-γ. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-α). To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3⁺ CD4⁺ CD8α⁺) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44^hi CD62L⁻ and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-γ⁺ CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface, and subpopulations that coexpressed TNF-α and interleukin 2 (IL-2) were identified. While it is hoped that these data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines.     

Reduced Frequency of Memory T Cells and Increased Th17 Responses in Patients with Active Tuberculosis

Phenotypic and functional alterations in Mycobacterium tuberculosis T cell subsets have been reported in patients with active tuberculosis. A better understanding of these alterations will increase the knowledge about immunopathogenesis and also may contribute to the development of new diagnostics and prophylactic strategies. Here, the ex vivo phenotype of CD4⁺ and CD8⁺ T cells and the frequency and phenotype of gamma interferon (IFN-γ)- and interleukin 17 (IL-17)-producing cells elicited in short-term and long-term cultures following CFP-10 and purified protein derivative (PPD) stimulation were determined in noninfected persons (non-TBi), latently infected persons (LTBi), and patients with active tuberculosis (ATB). Phenotypic characterization of T cells was done based on the expression of CD45RO and CD27. Results show that ATB had a reduced frequency of circulating CD4⁺ CD45RO⁺ CD27⁺ T cells and an increased frequency of CD4⁺ CD45RO⁻ CD27⁺ T cells. ATB also had a higher frequency of circulating IL-17-producing CD4⁺ T cells than did LTBi after PPD stimulation, whereas LTBi had more IFN-γ-producing CD4⁺ T cells than did non-TBi. The phenotype of IFN-γ-producing cells at 24 h differs from the phenotype of IL-17-producing cells with no differences between LTBi and ATB. At 144 h, IFN-γ- and IL-17-producing cells were mainly CD45RO⁺ CD27⁺ T cells and they were more frequent in ATB. These results suggest that M. tuberculosis infection induces alterations in T cells which interfere with an adequate specific immune response.     

Mycobacterium bovis BCG Vaccination Induces Divergent Proinflammatory or Regulatory T Cell Responses in Adults

Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ⁺) interleukin 2-positive (IL-2⁺) tumor necrosis factor α-positive (TNF-α⁺) polyfunctional CD4⁺ T cells concurrent with CD4⁺ IL-17A⁺ and CD8⁺ IFN-γ⁺ T cells or, in contrast, virtually absent cytokine responses with induction of CD8⁺ regulatory T cells. Significant induction of polyfunctional CD4⁺ IFN-γ⁺ IL-2⁺ TNF-α⁺ T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8⁺ T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8⁺ regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4⁺ T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.     

Protective and Pathological Functions of CD8+ T Cells in Leishmania braziliensis Infection

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a strong Th1 response that leads to skin lesion development. In areas where L. braziliensis transmission is endemic, up to 15% of healthy subjects have tested positive for delayed-type hypersensitivity to soluble leishmania antigen (SLA) and are considered to have subclinical (SC) infection. SC subjects produce less gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) than do CL patients, but they are able to control the infection. The aim of this study was to characterized the role of CD8⁺ T cells in SC infection and in CL. Peripheral blood mononuclear cells (PBMC) were stimulated with SLA to determine the frequencies of CD4⁺ IFN-γ⁺ and CD8⁺ IFN-γ⁺ T cells. Monocytes from PBMC were infected with L. braziliensis and cocultured with CD8⁺ T cells, and the frequencies of infected monocytes and levels of cytotoxicity markers, target cell apoptosis, and granzyme B were determined. The frequency of CD8⁺ IFN-γ⁺ cells after SLA stimulation was higher for SC individuals than for CL patients. The frequency of infected monocytes in SC cells was lower than that in CL cells. CL CD8⁺ T cells induced more apoptosis of infected monocytes than did SC CD8⁺ T cells. Granzyme B production in CD8⁺ T cells was higher in CL than in SC cells. While the use of a granzyme B inhibitor decreased the number of apoptotic cells in the CL group, the use of z-VAD-FMK had no effect on the frequency of these cells. These results suggest that CL CD8⁺ T cells are more cytotoxic and may be involved in pathology.     

Neither Classical nor Alternative Macrophage Activation Is Required for Pneumocystis Clearance during Immune Reconstitution Inflammatory Syndrome

Pneumocystis is a respiratory fungal pathogen that causes pneumonia (Pneumocystis pneumonia [PcP]) in immunocompromised patients. Alveolar macrophages are critical effectors for CD4⁺ T cell-dependent clearance of Pneumocystis, and previous studies found that alternative macrophage activation accelerates fungal clearance during PcP-related immune reconstitution inflammatory syndrome (IRIS). However, the requirement for either classically or alternatively activated macrophages for Pneumocystis clearance has not been determined. Therefore, RAG2^−/− mice lacking either the interferon gamma (IFN-γ) receptor (IFN-γR) or interleukin 4 receptor alpha (IL-4Rα) were infected with Pneumocystis. These mice were then immune reconstituted with wild-type lymphocytes to preserve the normal T helper response while preventing downstream effects of Th1 or Th2 effector cytokines on macrophage polarization. As expected, RAG2^−/− mice developed severe disease but effectively cleared Pneumocystis and resolved IRIS. Neither RAG/IFN-γR^−/− nor RAG/IL-4Rα^−/− mice displayed impaired Pneumocystis clearance. However, RAG/IFN-γR^−/− mice developed a dysregulated immune response, with exacerbated IRIS and greater pulmonary function deficits than those in RAG2 and RAG/IL-4Rα^−/− mice. RAG/IFN-γR^−/− mice had elevated numbers of lung CD4⁺ T cells, neutrophils, eosinophils, and NK cells but severely depressed numbers of lung CD8⁺ T suppressor cells. Impaired lung CD8⁺ T cell responses in RAG/IFN-γR^−/− mice were associated with elevated lung IFN-γ levels, and neutralization of IFN-γ restored the CD8 response. These data demonstrate that restricting the ability of macrophages to polarize in response to Th1 or Th2 cytokines does not impair Pneumocystis clearance. However, a cell type-specific IFN-γ/IFN-γR-dependent mechanism regulates CD8⁺ T suppressor cell recruitment, limits immunopathogenesis, preserves lung function, and enhances the resolution of PcP-related IRIS.     

CD8+ T Cell Exhaustion,Suppressed Gamma Interferon Production,and Delayed Memory Response Induced by Chronic Brucella melitensis Infection

Brucella melitensis is a well-adapted zoonotic pathogen considered a scourge of mankind since recorded history. In some cases, initial infection leads to chronic and reactivating brucellosis, incurring significant morbidity and economic loss. The mechanism by which B. melitensis subverts adaptive immunological memory is poorly understood. Previous work has shown that Brucella-specific CD8⁺ T cells express gamma interferon (IFN-γ) and can transition to long-lived memory cells but are not polyfunctional. In this study, chronic infection of mice with B. melitensis led to CD8⁺ T cell exhaustion, manifested by programmed cell death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) expression and a lack of IFN-γ production. The B. melitensis-specific CD8⁺ T cells that produced IFN-γ expressed less IFN-γ per cell than did CD8⁺ cells from uninfected mice. Both memory precursor (CD8⁺ LFA1^HI CD127^HI KLRG1^LO) and long-lived memory (CD8⁺ CD27^HI CD127^HI KLRG1^LO) cells were identified during chronic infection. Interestingly, after adoptive transfer, mice receiving cells from chronically infected animals were able to contain infection more rapidly than recipients of cells from acutely infected or uninfected donors, although the proportions of exhausted CD8⁺ T cells increased after adoptive transfer in both challenged and unchallenged recipients. CD8⁺ T cells of challenged recipients initially retained the stunted IFN-γ production found prior to transfer, and cells from acutely infected mice were never seen to transition to either memory subset at all time points tested, up to 30 days post-primary infection, suggesting a delay in the generation of memory. Here we have identified defects in Brucella-responsive CD8⁺ T cells that allow chronic persistence of infection.     

Performance of the QuantiFERON-Cytomegalovirus (CMV) Assay for Detection and Estimation of the Magnitude and Functionality of the CMV-Specific Gamma Interferon-Producing CD8+ T-Cell Response in Allogeneic Stem Cell Transplant Recipients

The performance of the QuantiFERON-cytomegalovirus (CMV) assay was compared to that of a flow cytometry intracellular cytokine staining (ICS) method for the detection of CMV-specific gamma interferon (IFN-γ)-producing CD8⁺ T-cell responses in allogeneic stem cell transplant (allo-SCT) recipients and for estimations of their magnitude and functionality. A total of 90 whole-blood specimens from 23 allo-SCT recipients was analyzed by both methods. Overall, the percentage of specimens that yielded concordant results by both methods was 68.8% (κ = 0.691; 95% confidence interval [CI], 0.548 to 0.835), and the sensitivity of the QuantiFERON-CMV assay for the detection of positive IFN-γ T-cell responses (>0.2 IU/ml), taking the ICS method as the reference, was 76.3%. The magnitude of IFN-γ-producing CD8⁺ T-cell responses to CMV-specific peptides measured with the QuantiFERON-CMV assay correlated significantly (σ = 0.695; P = <0.001) with that of the total IFN-γ-producing CD8⁺ T cells and dual-functional (IFN-γ/tumor necrosis factor alpha [TNF-α] [σ = 0.652; P = <0.001] and IFN-γ/CD107a [σ = 0.690; P = <0.001]) and trifunctional (IFN-γ/TNF-α/CD107a [σ = 0.679; P = >0.001]) CMV-specific CD8⁺ T-cell responses, as quantitated by ICS. In summary, the data indicated that the QuantiFERON-CMV assay is less sensitive than the ICS method for the detection of CMV-specific IFN-γ-producing CD8⁺ T-cell responses in the allo-SCT setting. Nevertheless, it allowed the estimation of the total and polyfunctional CMV-specific IFN-γ-producing CD8⁺ T-cell responses in specimens that tested positive by both methods.     

Heparin-Binding Hemagglutinin Induces IFN-γ+ IL-2+ IL-17+ Multifunctional CD4+ T Cells during Latent but Not Active Tuberculosis Disease

The mycobacterial heparin-binding hemagglutinin (HBHA) protein induces a potent gamma interferon (IFN-γ) response in latent tuberculosis (TB) infection and is a candidate vaccine and diagnostic antigen. We have assessed HBHA-specific intracellular IFN-γ, interleukin-2 (IL-2), and IL-17 production by CD4⁺ T cells in TB cases and household contacts (HHCs) as well as the level of secreted IFN-γ in whole-blood culture supernatant. HHCs were further classified as tuberculin skin test (TST) positive or negative, and the group was also divided as HIV positive or negative. Our study revealed that HBHA induces multifunctional IFN-γ-, IL-2-, and IL-17-coexpressing CD4⁺ T cells in HHCs but not in active TB cases; however, IFN-γ levels in culture supernatant did not differ between participant groups. Further studies are needed to completely understand how HBHA induces immune responses in different disease groups.     

Measurement of Phenotype and Absolute Number of Circulating Heparin-Binding Hemagglutinin,ESAT-6 and CFP-10, and Purified Protein Derivative Antigen-Specific CD4 T Cells Can Discriminate Active from Latent Tuberculosis Infection

The tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation with Mycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 [IL-2], interleukin-17A [IL-17A], interleukin-22 [IL-22], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNF-α]) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154⁺ TNF-α⁺ cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic [ROC] curve of 0.9277; P < 0.0001). Also, the proportions and absolute numbers of HBHA-specific CD4 T cells were significantly higher in those with latent TB infection, particularly CD154⁺ TNF-α⁺ IFN-γ⁺ IL-2⁺ and CD154⁺ TNF-α⁺ CXCR3⁺. Finally, we found that the ratio of ESAT-6- and CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers of M. tuberculosis-specific CD4 cells which differentiate between active and latent TB.     

Taurocholic acid inhibits the response to interferon-α therapy in patients with HBeAg-positive chronic hepatitis B by impairing CD8+ T and NK cell function

Pegylated interferon-alpha (PegIFNα) therapy has limited effectiveness in hepatitis B e-antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. However, the mechanism underlying this failure is poorly understood. We aimed to investigate the influence of bile acids (BAs), especially taurocholic acid (TCA), on the response to PegIFNα therapy in CHB patients. Here, we used mass spectrometry to determine serum BA profiles in 110 patients with chronic HBV infection and 20 healthy controls (HCs). We found that serum BAs, especially TCA, were significantly elevated in HBeAg-positive CHB patients compared with those in HCs and patients in other phases of chronic HBV infection. Moreover, serum BAs, particularly TCA, inhibited the response to PegIFNα therapy in HBeAg-positive CHB patients. Mechanistically, the expression levels of IFN-γ, TNF-α, granzyme B, and perforin were measured using flow cytometry to assess the effector functions of immune cells in patients with low or high BA levels. We found that BAs reduced the number and proportion and impaired the effector functions of CD3⁺CD8⁺ T cells and natural killer (NK) cells in HBeAg-positive CHB patients. TCA in particular reduced the frequency and impaired the effector functions of CD3⁺CD8⁺ T and NK cells in vitro and in vivo and inhibited the immunoregulatory activity of IFN-α in vitro. Thus, our results show that BAs, especially TCA, inhibit the response to PegIFNα therapy by impairing the effector functions of CD3⁺CD8⁺ T and NK cells in HBeAg-positive CHB patients. Our findings suggest that targeting TCA could be a promising approach for restoring IFN-α responsiveness during CHB treatment.     

Cytolytic mechanisms and T-cell receptor Vβ usage by ex vivo generated Epstein–Barr virus-specific cytotoxic T lymphocytes

Ex-vivo-generated Epstein–Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable β-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0·0001) and ethylene glycol-bis tetraacetic acid (P < 0·0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-γ and TNF-α. Granzyme B, perforin and Fas ligand were detected in CD8⁺ and CD4⁺ cells in all CTL; however, a greater proportion of CD8⁺ than CD4⁺ T cells expressed granzyme B (P < 0·0001) and more granzyme B was detected in CD8⁺ T cells than in CD4⁺ T cells (P = 0·001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.     

Ex-vivo whole blood secretion of interferon (IFN)-γ and IFN-γ-inducible protein-10 measured by enzyme-linked immunosorbent assay are as sensitive as IFN-γ enzyme-linked immunospot for the detection of gluten-reactive T cells in human leucocyte antigen (HLA)-DQ2·5+-associated coeliac disease

T cell cytokine release assays are used to diagnose infectious diseases, but not autoimmune or allergic disease. Coeliac disease (CD) is a common T cell-mediated disease diagnosed by the presence of gluten-dependent intestinal inflammation and serology. Many patients cannot be diagnosed with CD because they reduce dietary gluten before medical workup. Oral gluten challenge in CD patients treated with gluten-free diet (GFD) mobilizes gluten-reactive T cells measurable by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) or major histocompatibility complex (MHC) class II tetramers. Immunodominant peptides are quite consistent in the 90% of patients who possess HLA-DQ2·5. We aimed to develop whole blood assays to detect gluten-specific T cells. Blood was collected before and after gluten challenge from GFD donors confirmed to have CD (n = 27, all HLA-DQ2·5⁺), GFD donors confirmed not to have CD (n = 6 HLA-DQ2·5⁺, 11 HLA-DQ2·5⁻) and donors with CD not following GFD (n = 4, all HLA-DQ2·5⁺). Plasma IFN-γ and IFN-γ inducible protein-10 (IP-10) were measured by enzyme-linked immunosorbent assay (ELISA) after whole blood incubation with peptides or gliadin, and correlated with IFN-γ ELISPOT. No T cell assay could distinguish between CD patients and controls prior to gluten challenge, but after gluten challenge the whole blood IFN-γ ELISA and the ELISPOT were both 85% sensitive and 100% specific for HLA-DQ2·5⁺ CD patients; the whole blood IP-10 ELISA was 94% sensitive and 100% specific. We conclude that whole blood cytokine release assays are sensitive and specific for detection of gluten-reactive T cells in CD; further clinical studies addressing the utility of these tests in patients with an uncertain diagnosis of CD is warranted.     

Expression of Regulatory T Cells in Jejunum,Colon, and Cervical and Mesenteric Lymph Nodes of Dogs Naturally Infected with Leishmania infantum

Using flow cytometry, we evaluated the frequencies of CD4⁺ and CD8⁺ T cells and Foxp3⁺ regulatory T cells (Tregs) in mononuclear cells in the jejunum, colon, and cervical and mesenteric lymph nodes of dogs naturally infected with Leishmania infantum and in uninfected controls. All infected dogs showed chronic lymphadenitis and enteritis. Despite persistent parasite loads, no erosion or ulcers were evident in the epithelial mucosa. The colon harbored more parasites than the jejunum. Frequencies of total CD4⁺, total Foxp3, and CD4⁺ Foxp3⁺ cells were higher in the jejunum than in the colon. Despite negative enzyme-linked immunosorbent assay (ELISA) serum results for cytokines, levels of interleukin-10 (IL-10), gamma interferon (IFN-γ), transforming growth factor beta (TGF-β), and tumor necrosis factor alpha (TNF-α) were higher in the jejunum than in the colon for infected dogs. However, IL-4 levels were higher in the colon than in the jejunum for infected dogs. There was no observed correlation between clinical signs and histopathological changes or immunological and parasitological findings in the gastrointestinal tract (GIT) of canines with visceral leishmaniasis. However, distinct segments of the GIT presented different immunological and parasitological responses. The jejunum showed a lower parasite load, with increased frequencies and expression of CD4, Foxp3, and CD8 receptors and IL-10, TGF-β, IFN-γ, and TNF-α cytokines. The colon showed a higher parasite load, with increasing expression of IL-4. Leishmania infantum infection increased expression of CD4, Foxp3, IL-10, TGF-β, IFN-γ, and TNF-α and reduced CD8 and IL-4 expression in both the jejunum and the colon.     

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) induced in human CD4⁺ and CD8⁺ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4⁺ cells can make IL-2 than the other two cytokines, and that there are more TNF-α-positive CD4⁺ cells than cells with IFN-γ. For normal CD8⁺ cells the highest production of cytokine is of IFN-γ (up to 50% of the cells) especially at longer times (10–20h) of stimulation. For CD8⁺ cells, IL-2-positive cells exceed those with TNF-α. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4⁺ or CD8⁺ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-α but significantly raised levels of production of IFN-γ in both CD4⁺ and CD8⁺ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.     

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8⁻ T cells, IL-2 and interferon-gamma (IFN-γ) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8⁺ T cells IL-2 was optimally induced after 10 h and IFN-γ after 6 h. The levels of IL-2 and IFN-γ in CD8⁺ and CD8⁻ T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2⁺ (range 9.7–41.3) and CD3/IFN-γ⁺ cells (10.1–44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3⁺/CD8⁺ peripheral blood T cells expressing IFN-γ and an increased percentage of CD3⁺/CD8⁻ T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.