1 Division of Infectious Diseases and Clinical Immunology and 2 Division of Endocrinology, Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, 89081 Ulm, Germany
The first two authors contributed equally to this work
Correspondence to: B. J. Manfras; E-mail: burkhard.manfras{at}medizin.uni-ulm.de
Transmitting editor: S. H. E. Kaufmann
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Abstract |
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Keywords: alveolar echinococcosis, cytokine, cytokine flow cytometry, ELISPOT assay, T cell memory
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Introduction |
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Several studies have reported elevated levels of Th2-type cytokines in patients with AE (1013). Activation of Th1 cells has been associated with the control of the infection, although experimental data are limited to murine models. For example, in vivo treatment with IL-12 has been reported to control larval growth and prevent secondary infection (14).
Cells of the innate immune system and lymphocytes both primarily rely on cytokines for mediating their effector and regulatory functions. Naive T cells do not produce cytokines such as IL-4 and IFN-, but learn to express them via an instructed differentiation process (15,16). The generation of Th2 cytokine-producing memory T cells (Th2 cells) requires production of IL-4, either by the cells of the innate immune system or by T cells during the primary antigen encounter (17).
Judgment on the induction of Th1/Th2 immunity has relied primarily on measurements of antigen-induced cytokines in supernatants or lysates of mixed cell populations with the cellular source of the cytokine not directly detectable. However, only cytokines produced by T cells (cognate cytokine) reflect long-term commitment by the immune system and hence the sought-after information.
Immunity mediated by T cells can be comprehensively defined by the clonal size (frequency) of the antigen-reactive memory cell pool and the type of cytokines that these T cells produce. In this study, we provide information on both parameters.
Assessment of T cell responsiveness has been greatly facilitated by the development of two improved detection methods of antigen-specific memory T cells allowing for a more accurate characterization of phenotypic and biological properties of memory CD4+ T cells. Technical improvements in ELISPOT analysis have been introduced that include membranes improving the signal-to-noise ratio (18). Cytokine flow cytometry, a technique that also allows precise determination of the frequency of T cells reacting to a given antigen, additionally provides precise discrimination of different cell populations secreting the same cytokine (19). Previously used techniques for detection of the cytokine response upon antigenic stimulation (i.e. cytokine ELISA) involve extended stimulation periods (up to 5 days), long enough for priming naive T cells. Furthermore, interpretations of results were hampered for those cytokines that are not exclusively produced by memory T cells.
To gain a better understanding of the shaping of the adaptive immune response, and to clarify the role of Th1 and Th2 cytokines in a chronic helminthic infection, we performed a cross-sectional study comparing T cell memory and its cytokine commitment of the E. multilocularis antigen-specific immune response in patients at different disease stages of AE.
We specifically studied IFN-, IL-2, tumor necrosis factor (TNF)-
, IL-4, IL-5 and IL-10 secretion by peripheral blood lymphocytes isolated from patients with progressive disease and inactive disease, patients after surgical elimination of the parasite (cured patients), and from healthy control subjects.
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Methods |
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For assessment of active/progressive disease, imaging was repeated once every year for the last 2 years before entry into the study. In addition, in 22 patients the [14F]deoxyglucose positron emission tomography (PET) scan was used to assess glucose utilization of liver lesions as described previously (21). For immunodiagnosis, a commercially available Em2+ ELISA (Bordier Affinity Products, Crissier, Switzerland) was used (22). AE patients with detectable parasitic masses were stratified into two groups referred to as active (progressive) AE (N = 16; mean age 53 years; range 3183) and inactive (non-progressive) AE (N = 10; mean age 62 years; range 3782) according to the level of evidence for parasite activity as assessed by changes of the parasitic mass, extent of calcification and, when available activity in the [14F]deoxy glucose PET scan. In addition, 12 patients (mean age 53 years; range 2382) were recruited for the study who had been previously diagnosed with AE and had remained free of detectable parasitic masses for at least 2 years after complete surgical removal of a liver lesion, and therefore are referred to as cured AE patients. Clinical data are summarized in Table 1. There was no significant difference between the three patient groups with respect to age, gender and duration of the disease after the initial diagnosis, and localization and volume of the parasitic mass. All patients with active AE except one were treated with a benzimidazole (albendazole or mebendazole) (23). The control group consisted of 20 healthy individuals (mean age 52.9 years; range 2282 years) without any history of chronic diseases. Informed consent was obtained from each patient and control subject. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki as reflected in approval by the local ethical committee. Cytomegalovirus (CMV) serological status was determined by ELISA at the Ulm Clinical Virology Laboratory.
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ELISPOT assays
Immobilon-P MultiScreen plates (Millipore, Eschborn, Germany) were coated with cytokine capture antibodies overnight at 4°C with capture antibodies dissolved in PBS (50 µl/well) that were specific for either IFN- (mAb M700A-E, 2 µg/ml; Endogen, Biozol, Eching, Germany) or IL-5 (mAb TRFK5, 5 µg/ml; BD PharMingen, Heidelberg, Germany). Cells derived from peripheral blood mononuclear cells (PBMC) were subjected to a pre-activation culture in 96-well flat-bottom tissue culture plates (Costar Life Sciences, New York, NY) at 5 x 105 cells/well in 200 µl of complete RPMI 1640 medium (Life Technologies, Karlsruhe, Germany) supplemented with 2 mmol/l L-glutamine and 10% heat-inactivated ABO serum (PAA, Cölbe, Germany). Antigen-specific stimulation was performed with vesicular fluid antigen at 20 µg/ml, and CMV antigen and PPD at 10 µg/ml. All reactions were carried out in duplicates. Cells were incubated for 812 h at 37°C in a 5% CO2 incubator. Antibody-coated ELISPOT plates were washed 3 times with sterile PBS. The plates were then blocked with BSA (Serva, Heidelberg, Germany) (10 g/l in PBS) for 2 h and washed 3 times with PBS. Cells from the pre-activation cultures were carefully resuspended and 1 x 105 PBMC were transferred to each of the capture antibody-coated plates. The remaining cells were further incubated for proliferation assays. After 24 h for IFN-
assays or after 48 h for IL-5 assays, the plates were washed and biotinylated detection antibodies were added for 12 h at 4°C. The secondary antibodies specific for IFN-
were mAb M701 from Endogen (2 µg/ml) and for IL-5, mAb 18522D (1.5 µg/ml) from PharMingen; they were in 10 g/l BSA/PBS with 0.5% Tween (Sigma-Aldrich, Deisenhofen, Germany). Subsequently, streptavidinhorseradish peroxidase (BD Biosciences, Heidelberg, Germany; 1/2000 dilution) in PBS/BSA/Tween was added for 2 h at room temperature. The spots were developed using AEC (Sigma-Aldrich) solution: 10 mg/ml AEC dissolved in 1 ml N,N-dimethyl formamide (Fuka, Neu-Ulm, Germany), of which 1 ml was freshly diluted into 30 ml of 0.1 M sodium acetate buffer (pH 5). This solution was then filtered and mixed with 15 µl H2O2 to provide the final AEC development solution, of which 200 µl was plated per well. The plates were developed for 510 min; the reaction was stopped by rinsing with tap water when clear spots became visible. The membranes were air dried overnight before the plates were subjected to analysis.
Spot numbers were determined manually under a microscope and by using an ImmunoSpot image analyzer (Cellular Technology, Cleveland, OH) specifically designed for the ELISPOT assay. Digitized images were analyzed using criteria based on spot size and color intensity to identify cytokine production by cells. The number of specific T cells was calculated by subtracting the negative (medium) control values and expressed as the number of s.f.u./105 PBMC. IFN- and IL-5 ELISPOT assays measured the accurate frequencies of recall antigen-specific CD4+ memory cells with a detection limit at
1/100,000 cells. An antigen-specific response was counted when the number of s.f.u. exceeded the 3-fold negative control value.
Whole-blood antigenic stimulation
Whole-blood antigenic stimulation was carried out as previously described in detail by Suni et al. (25) with some modifications. Briefly, ammonium heparinized venous blood was aliquoted into 5-ml polypropylene tubes (Becton Dickinson, Heidelberg, Germany) at 500 µl/tube. The co-stimulatory mAb CD28 (clone CD28.2; BD PharMingen) was added at 2 µg/ml and CD49d (clone HP2/1; Beckmann Coulter, Unterschleissheim, Germany) at 1 µg/ml. Antigens were subsequently added at optimal stimulatory concentrations. The culture tubes were incubated upright in a humidified 37°C, 5% CO2 incubator for a total of 6 h. The secretion inhibitor Brefeldin A was added after 1 h of incubation at 10 µg/ml. At 6 h, reactions were stopped by adding 50 µl 20 mM EDTA and 10 min incubation at room temperature. Blood samples were then lysed and fixed in 3.5 ml FACS lysing solution (BD Biosciences) for 10 min at room temperature. Cells were washed in PBS + 1% BSA and frozen in a freezing medium containing 10% DMSO and 90% FCS.
Cytokine flow cytometry
After staining with fluorescent-conjugated mAb in a total staining volume of 100 µl, cells were washed in PBS with 0.1% BSA and analyzed on a FACSCalibur flow cytometer using CellQuest software (both from BD Biosciences). At least 20,000 cells were analyzed on the flow cytometer after single gating on lymphoid cells. CD4+ T cell subsets were consecutively gated with a lymphocyte gate in a SSC/FSC plot and a CD3+ (PerCP) gate. Activated cytokine-producing T cells were gated for staining with anti-CD69 antibody (allophycocyanin conjugated) and antibodies to one of the six cytokines (IFN-, IL-2, IL-4, IL-5, IL-10, TNF-
; FITC, phycoerythrin or allophycocyanin conjugated). Antibodies were all from BD Biosciences. Subset analysis was performed using CellQuest software (BD Biosciences). Unstimulated samples (medium controls) were analyzed to determine background cytokine responses. Numbers of cytokine-positive cells were given after background subtraction. Data files were analyzed with CellQuest software.
Statistical analysis
The MannWhitney U-test test and the standard 2-test were used to compare groups with regard to continuous and discrete variables respectively. Differences with P < 0.05 were considered significant. In ELISPOT assays, numbers of spots in duplicate wells were compared with controls.
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Results |
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We studied circulating memory T cells in human AE in patients with and without apparent metacestode activity, as well as in AE patients after complete surgical removal of the parasite (cured patients). Assessment of metacestode activity was based on repetitive imaging of parasitic lesions and the course of the serological markers over the follow-up periods of many years. The frequency of circulating E. multilocularis-specific CD4+ memory T cells was determined ex vivo based on cytokine production upon antigen stimulation. In order to gain a comprehensive view of the function of these specific memory cells, we tested for the production of two Th1 cytokines (IFN- and IL-2) and three Th2 cytokines (i.e. IL-4, IL-5 and IL-10). In addition, we tested for the production of the pro-inflammatory cytokine TNF-
. Most importantly, the results of the two independent single-cell assays revealed a low frequency of specific CD4+ memory T cells. However, an E. multilocularis-specific response was clearly detectable in the majority of patients (68.5%). While 81% (13 of 16) of patients with progressive AE exhibited a response at a level >1000 per 106 CD4+ memory T cells. This level of response was detectable in 70% (seven of 10) of patients with inactive disease. In comparison, only 50% (six of 12) of the cured AE patients revealed a response at this level. Memory CD4+ T cells producing IFN-
or IL-2 upon stimulation with E. multilocularis-VF antigen, and therefore regarded as Th1 cells, were detectable in the range of 0.52.0% CD4+ T cells. IL-4-, IL-5- or IL-10-producing CD4+ memory T cells specific for E. multilocularis were detected at comparable frequencies in the 0.52.0% range (Fig. 1A and B). This distinct pattern of cytokine commitment of E. multilocularis-specific memory CD4+ T cells was invariably seen in all three patient groups, and therefore independent of the presence and activity of metacestode tissue.
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Recall antigen T cell reactivity and response upon polyclonal stimulation
Twenty-eight of the 38 AE patients (74%) showed a significant (>0.1%) PPD recall response. In the control group, 65% of the individuals showed a PPD response at this level. The frequency of the Th1 cytokine-producing memory cells was in the range of 0.14% of CD4+ T cells without significant differences between patient groups and healthy controls. Furthermore, the ratio between PPD-specific Th1 and Th2 memory CD4+ T cells was not shifted in AE patients. Polyclonal stimulation of cells was performed, yielding no significant differences in the proportion of activated cells as measured by CD69 expression and cytokine response (data not shown). These results rule out general hyporesponsiveness of peripheral memory T cells in AE patients.
Comparison of E. multilocularis-reactivity with CMV reactivity
Among chronic infections of the human, the importance of memory CD4+ T cells is best appreciated for CMV. This virus infects a large proportion of adults, usually following clinically benign primary infection in either early childhood or adolescence, and remains as a latent or low-level infection without end organ disease in immunocompetent individuals (26). Based on seropositivity, analysis of the cytokine profile of CMV-reactive memory CD4+ T cells was possible in eight (50%) AE patients with active disease, seven (70%) patients with inactive AE and three (25%) cured AE patients. The cytokine response was Th1 dominated and yielded precursor frequencies in the range 0.47%.
Taken together, the frequencies of E. multilocularis-VF-responding Th1 CD4+ memory T cells were significantly lower than of CMV-responding cells (P = 0.006). In contrast, Th2 response was at the same level (no significant differences, P = 0.36) (Fig. 1A and B).
Comparison between cytokine ELISPOT assay and cytokine cytometry
To test whether the mode of in vitro stimulation of T cells might influence the determined frequencies of antigen-specific cells, we used two techniques in parallel. While for the ELISPOT assay it is necessary to isolate PBMC, whole-blood stimulation has the advantage that cells remain almost untouched. Cytokine production of stimulated T cells is thought to depend on many factors such as antigen processing and duration of stimulation. The cytokine ELISPOT assays were performed according to an improved protocol proven to reliably detect cytokine-producing cells at the single-cell level (Fig. 2A). Nevertheless, since E. multilocularis-VF is supposed to represent a complex mixture of parasite antigens and is expected to stimulate cells of the innate immune system, its application in this study is limited to IFN- and IL-5. Their production can be assigned to T cells, while IL-4, IL-10 and TNF-
can also be secreted by cells of the innate immune system (e.g. granulocytes, monocytes). In order to extend the analysis of cytokine production by memory T cells to these cytokines, we used whole-blood stimulation (Fig. 2B). Results from both experimental approaches correlated well for single event frequencies >0.2% (r = 0.75), while a poor correlation was observed for lower precursor frequencies.
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Discussion |
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To provide an independent measure of CD4+ cell-produced cytokines, we determined the secretion of cytokines at the single-cell level and by direct visualization of CD4+ T cells. We examined the cytokine signatures of freshly isolated individual cells using ELISPOT assays. In this approach, T cells are challenged with antigen for 24 or 48 h respectively, a time period sufficient to induce cytokine expression in differentiated T cells, but insufficient to permit proliferation and cytokine differentiation in vitro (16,29). Nevertheless, its application with complex antigens is limited to IFN-, IL-2 and IL-5 because IL-4, IL-10 and TNF-
can also be secreted by cells of the innate immune system (e.g. granulocytes, monocytes). Therefore, we also applied cytokine flow cytometry. This method allows simultaneous cytokine detection and phenotyping of cells at the single-cell level. It has been successfully applied to detection and enumeration of CMV antigen-specific cells in peripheral blood using antigen preparations from lysates of CMV-infected cells as well as peptides (25,30). A profound difference between these two methods lies in the culture conditions and duration of stimulation. Nevertheless, in our hands, results from both experimental approaches compared well for precursor frequencies >0.5%. Importantly, antigen-stimulated CD4+ T cells displayed a comparable expression profile for type 1 and type 2 cytokines. In a direct comparison to the magnitude of CD4+ memory T cell response to CMV and the recall antigen PPD, the overall frequencies of E. multilocularis-specific memory cells allow for the hypothesis that the establishment of an appropriate Th1 immune response against the pathogen during the chronic phase of infection is averted. As in other helminthic infections, the underlying mechanisms behind down-regulation of the cellular immune response during infection with E. multilocularis remain unknown. An effective response of the adaptive immune system can be impeded at two major checkpoints. First, antigen processing and presentation can be hampered by parasite-specific masking of epitopes by carbohydrates (e.g. mucin-type glycosylation as seen for the Em2 antigen) (31). Second, appropriate priming of naive T cells and establishment of T cell memory might be hampered by regulatory cells (e.g. regulatory T cells) and cytokines (e.g. IL-10). One could suppose that antigen-specific T cells were constantly removed from the circulation by homing to the liver lesion and subsequently undergoing apoptosis or necrosis, thus giving rise to misleading interpretation of their frequencies. However, this potential error could be ruled out by examining cured patients in whom the frequencies of antigen-specific T cells did not increase significantly after surgical elimination of the liver lesion. Our current research effort is directed toward identifying hostparasite interactions preventing a stronger Th1 response.
Evidence from experimental models suggests a role for regulatory immune cells (i.e. dendritic cells, regulatory T cells) for reducing the severity of immunopathology and providing protection against organ destruction (32). IL-10 plays a major role in the regulation of the intensity of both Th1 and Th2 responses during helminth infections (12,33). Previous studies from our group and others have indicated a Th2-dominated E. multilocularis antigen-specific cytokine response during infection with E. multilocularis (10,11). However, these studies were centered on ELISA analyses of culture supernatants derived from PBMC stimulated with parasite antigens and could not define the number of antigen-specific CD4+ memory T cells in disease because several different lymphocyte populations can contribute to cytokine production upon stimulation. Previous attempts to establish recall antigen-specific T cell memory in freshly isolated cellular material from patients relied on readout systems that do not measure the frequency of the responding cells (cytokine ELISA, RT-PCR, proliferation); other studies utilized T cell clones and in vitro expanded cell lines, which also do not provide data on the frequencies or state of differentiation and function of antigen-specific cells in vivo.
Our data suggest that IL-5-producing memory T cells might disappear faster than any other from circulation after complete removal of the metacestode. A role of IL-5 in the innate and adaptive immune response to parasites was recently demonstrated (34), but other than that no data on the kinetics of the memory function is currently available. Previous studies reported that IL-5 synthesis was specifically increased in PBMC from AE patients upon stimulation with parasitic antigens (10). This IL-5 production was attributed exclusively to activated CD4+ T lymphocytes by our group and others (11,12,35). IL-5 is considered to be a Th2-type cytokine involved in B cell growth and in antibody synthesis.
The role of IL-10 in chronic AE remains obscure, although it could be clearly demonstrated that a higher basal production of IL-10 by PBMC was associated with progressive AE, while significantly lower IL-10 levels were measured in patients with abortive AE. A regulatory role of IL-10 in the immunopathology of AE (12) was suggested.
In addition to this emphasis on quantitative and functional characterization of the T cell memory induced in AE, we made every endeavor to assess the activity of the metacestode. Other than long-term follow-up of patients with multiple imaging techniques including PET scans, there is no reliable marker for metacestode activity. We have evaluated our approach for assessment of metacestode activity previously (21).
The comparison of active (progressive) AE with inactive (non-progressive) AE and with the situation in cured patients (after complete surgical removal of the metacestode tissue) indicates that there is a balanced expansion of Th1 and Th2 cells throughout the course of E. multilocularis infection. In contrast to studies on hepatitis C virus, numbers of antigen-specific memory cells did not expand after elimination of the pathogen (36,37). Thus, our study provides strong evidence supporting a view that human immune responses to E. multilocularis are markedly different from those to viral and bacterial pathogens.
The limitation of this study to the compartment of circulating T cells is given by the facts that AE in humans is a rare disorder and that liver biopsies are generally contraindicated. Therefore, in most cases only the circulating T cell compartment can be investigated. On the positive side, however, we were able to include 38 patients with this rare disorder into the study.
Helminths, in comparison to viruses and bacteria, have a much more complex genome with marked differences in the number of expressed genes. Unfortunately, no information on immunodominant T cell epitopes is available for E. multilocularis. Therefore, currently it is not possible to characterize the immune response by methods such as tetramer staining allowing for a specific characterization of the CD8+ memory T cells. The combined frequencies of Th1 and Th2 cytokine-producing precursor cells might be overestimated by the fact that in our experimental setting we did not account for T cells secreting more than one cytokine. However, the majority of cells express a single cytokine only, as indicated by control experiments (data not shown).
In conclusion, we have presented the first detailed examination of antigen-specific cellular memory in a human chronic helminth infection. Most importantly, a balanced establishment of Th1 and Th2 memory cells throughout the course of E. multilocularis infection was observed lacking a dominating Th1 memory function. One particularity of the study was that patients with a chronic parasitic infection were unexposed to other parasites causing chronic infection, which have the potential to shift the Th1/Th2 cytokine balance. The recent use of an improved ELISPOT technology and whole-blood stimulation assay enabled new advances in the study of antigen-specific responses at the single-cell level. Whether the antigen-specific cellular hyporesponsiveness with impeded Th1 CD4+ memory T cell generation is a cause or a result of the progressive metacestode activity remains to be determined. The implication of the present study is that immunotherapeutic approaches enhancing generation of Th1 memory cells might be beneficial in host defense against chronic infection with E. multilocularis.
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Acknowledgements |
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Abbreviations |
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CMVcytomegalovirus
PBMCperipheral blood mononuclear cell
PETpositron emission tomography
PPDpurified protein derivative
TNFtumor necrosis factor
VFvesicular fluid
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References |
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