Childrens Hospital, University of Würzburg1 and Department of Dermatology2, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany
Institute für Virologie und Immunobiologie, Versbacherstr. 7, D-97080 Würzburg, Germany3
Author for correspondence: Ralph Nanan. Fax +49 931 201 3720. e-mail nanan{at}mail.uni-wuerzburg.de
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Abstract |
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Introduction |
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The immunological factors which determine the outcome of the disease and which lead to life-long immunity against measles are poorly understood. However, T-cell-mediated immunity seems to play a central role in the clearance of established MV infection and in protection against reinfection. This assumption is strongly supported by indirect evidence: although MV infection induces both humoral and cell-mediated responses, agammaglobulinaemic children recover from infection and develop immunity against measles, whereas those with T-cell anomalies suffer the most severe complications (Burnet, 1968 ).
However, cell-mediated immunity against MV has been difficult to investigate. The reason for this is the strong immunosuppressive effects exhibited by infectious MV in vitro. Peripheral blood lymphocytes from patients with acute measles as well as in vitro infected PBMC exhibit a state of unresponsiveness to mitogenic stimulation. Cell cycle analysis has shown that both T- and B-lymphocytes are arrested in late G1 when stimulated with mitogen in the presence of infectious MV (McChesney & Oldstone, 1989 ; Engelking et al., 1999
).
Despite experimental difficulties due to immunosuppression, there is also direct evidence for the involvement of CD4+ and CD8+ T-lymphocytes in immunity to measles (Jacobson et al., 1984 ; Van Binnendijk et al., 1990
; Nanan et al., 1995
; Mongkolsapaya et al., 1999
). However, the techniques employed have been limited in their ability to provide precise quantitative information on MV-specific memory T-cells. Due to low precursor frequencies, enumeration of virus-specific memory T-cells in peripheral blood has been difficult. A prerequisite for detection of precursors was prior expansion of T-cell lines or clones before performing proliferation or cytotoxicity assays.
Recently, novel methods for quantifying antigen-specific T-lymphocytes at a single-cell level have been described. The use of tetrameric MHCpeptide complexes which bind specifically to appropriate T-cell receptors allows direct quantitative analysis of T-cell responses (Altman et al., 1996 ; Busch et al., 1998
; Murali-Krishna et al., 1998
; Crawford et al., 1998
; Doherty, 1998
). Alternatively, T-cells can be stimulated with epitope peptides and their IFN-
production measured by ELISPOT assay or by intracellular flow cytometry (Scheibenbogen et al., 1997
; Waldrop et al., 1997
; Kern et al., 1998
; Pitcher et al., 1999
). T-cell numbers detected with these tests were comparable to those determined by tetramer staining (Murali-Krishna et al., 1998
; Flynn et al., 1998
). Experiments using one or more of these approaches to test humans infected with human immunodeficiency virus (HIV) or EpsteinBarr virus (EBV) and mice infected with lymphocytic choriomeningitis virus have shown very clearly that earlier methods depending on expansion of T-cells underestimated the prevalence of virus-specific T-cells by a factor of at least 10 (Altman et al., 1996
; Butz & Bevan, 1998
; Murali-Krishna et al., 1998
; Tan et al., 1999
; Pitcher et al., 1999
).
These new techniques allow analysis of either CD4+ T-cells with selected viral antigens or of CD8+ T-cells relying heavily on the knowledge of viral epitopes. In this study we describe a modified single-cell approach to simultaneously quantify immune responses of both virus-specific CD4+ and CD8+ T-cells which is independent of preselecting viral antigens or epitopes. This technique was used to quantify the frequency of memory T-cells after MV infection and compare it to EBV-specific memory T-cells.
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Methods |
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Serology was assessed by standard enzyme immunoassay (EIA) for MV and EBV (Institute für Virologie und Immunobiologie, Würzburg, Germany).
Cell preparation.
Samples (5 to 20 ml) of heparinized venous blood (20 U/ml heparin) were drawn and processed within 6 h of collection. Mononuclear cells were isolated on FicollHypaque density gradients (Pharmacia). Aliquots of isolated lymphocytes were stored in liquid nitrogen in the presence of 10% DMSO and 20% foetal calf serum.
Autologous B-lymphoblastoid cell lines (B-LCL) were established for each donor, 6 to 8 weeks before testing, from 5x106 PBMC by using the B95-8 strain of EBV and the L-leucine methyl ester procedure (Thiele & Lipsky, 1985 ).
For preparation of peripheral blood dendritic cells (DC) PBMC were isolated by FicollHypaque (Pharmacia) density gradient centrifugation from 100 ml of heparinized venous blood obtained from healthy adult donors. DC were isolated by adherence for 1 h in culture dishes (Becton Dickinson). DC were cultured in RPMI 1640 medium containing 10% autologous serum with 800 units/ml granulocytemacrophage colony-stimulating factor and 1000 units/ml IL-4 for 7 days. Cells were harvested and infected in the same culture medium.
174xCEM.T2 cell lines were used with written consent from P. Cresswell (Yale University, New Haven, CT, USA). T2 cells are unable to present endogenously synthesized peptides due to a deficiency of TAP1 and TAP2 transporter genes and are MHC class II antigen negative (Salter & Cresswell, 1986 ).
MV infection.
The Edmonston strain of MV was grown in Vero cells (continuous African green monkey kidney cells). Virus stocks contained 106 p.f.u./ml. To prepare MV-infected antigen-presenting cells (APC), cells were infected with MV at an m.o.i. of 1. B-LCL were incubated for 24 h and DC for 72 h before they were used as APC. In order to get a recovery of more than 10% from MV-infected DC, MV fusion inhibitory peptide (Sigma) (Richardson & Choppin, 1983 ) had to be added. Infection rate was monitored by cell surface expression of MV haemagglutinin protein using MAb clone L177.
Intracellular staining for IFN-
.
The protocol adopted was essentially that of P. Openshaw (Openshaw et al., 1995 ). Briefly, PBMC were cultured in 96-well (round bottom) plates at a concentration of 2x105 cells per well without or with 1x105 B-LCL or 2x104 DC as APC. Brefeldin A (Sigma) at 2 µg/ml was added for the last 4 h of culture to stop cytokine secretion. After 16 h of culture cells were harvested, washed once by centrifugation in PBS with 0·02% NaN3 and 0·2% BSA (FACS buffer), and then stained with R-phycoerythrin (RPE)-conjugated anti-CD8 or anti-CD4 (Dako) and biotin-conjugated anti-CD3 (Immunotech). After removing unbound antibody cell-bound biotinylated anti-CD3 was stained with streptavidin Cy-Chrome (Pharmingen). After further washing cells were fixed in 2% formaldehyde for 20 min and then washed again in FACS buffer. Permeabilization was then performed by incubating cells in PBS with 0·5 % saponin and 10% BSA in PBS for 10 min. For intracellular staining a murine monoclonal fluorescein isothiocyanate (FITC)-conjugated anti-IFN-
MAb clone, 15.45 (Hölzel Diagnostik, Cologne, Germany), was used. For blocking experiments unconjugated antibody of the same clone was used. After intracellular staining cells were washed once in saponin buffer and then in FACS buffer. Samples were analysed on a FACScan flow cytometer (Becton Dickinson, Immunocytometry system). Lymphocytes were gated in side-scatter versus forward-scatter light; CD3+ T-lymphocytes were gated in fluorescence 3. For frequency analysis 50000 to 150000 events per sample were measured.
IFN-
-specific ELISPOT assay.
ELISPOT assays for IFN- were performed with slight modifications after established protocols (Fujihashi et al., 1993
; Sarawar & Doherty, 1994
). Briefly, 96-well nitrocellulose-based microtitre plates (Millititre HA; Millipore) were coated overnight at 4 °C with the anti-IFN-
MAb clone 1-D1-K at a concentration of 15 µg/ml in PBS (Hölzel Diagnostik). After washing with PBS, 1x105 lymphocytes were added to the wells without or with 5x104 APC and incubated for 24 h at 37 °C. After the wells had been washed biotinylated anti-IFN-
MAb clone 7-b6-1 (Hölzel Diagnostik) was added, and incubated overnight at 4 °C. Plates were washed in PBS and anti-biotin MAb conjugated with streptavidin (ALP-PQ; Hölzel Diagnostik) was added, followed by 2 h incubation at room temperature. Spots representing individual IFN-
-secreting cells were visualized by developing with an alkaline phosphatase conjugate substrate kit (Bio-Rad). Well-plates were photographed with a Wild Fotomakroskop M400 camera. Spots were counted after slide projection. All assays were performed in triplicate.
Optimal culture conditions were extensively studied when establishing the IFN- ELISPOT assay. Reproducible spots were obtained with an absolute number of 1 to 2x105 cells per well and with an effector to target ratio of 2:1. Furthermore, kinetic studies revealed that the number of IFN-
-secreting cells did not increase when PBMC were incubated in our assay for periods progressively longer than 12 h (up to 30 h). However, the quality of spots was best after 24 h. At earlier periods spots were small and sometimes difficult to discriminate from background phenomena. Later, spots became fuzzy and confluent (data not shown).
Enrichment of CD4+ and CD8+ cell subsets.
Separation was achieved with the MiniMACS separation system (Miltenyi Biotec). A suspension of up to 5x107 PBMC was incubated for 15 min at 4 °C with anti-CD4- or anti-CD8-conjugated magnetic beads (Miltenyi Biotec) and then separated by using the MiniMACS separation column according to the manufacturers instructions. Cell-surface marker analysis with Cy-Chrome-conjugated anti-CD3 MAb, PE-conjugated anti-CD8 MAb and FITC-conjugated anti-CD4 MAb was performed on enriched cells.
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Results |
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Frequencies of IFN--positive cells were highly reproducible from multiple independent assays performed by splitting individual blood samples as well as from longitudinal samples, as shown in Fig. 2
. Thus, for further experiments B-LCL were used as APC.
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Comparison of T-cell frequencies by intracellular IFN- staining and ELISPOT assay
Both CD3+CD4+ T-cells as well as CD3+CD8+ T-cells were analysed for IFN- expression by intracellular staining. To verify intracellular IFN-
production by an alternative approach, IFN-
ELISPOT assays were performed. CD4+- and CD8+-enriched T-cells were analysed separately. Frequencies of MV-specific T-cell subsets were comparable in both assays in multiple independent experiments (Fig. 4
).
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In parallel, EBV-specific T-cells were evaluated also. The median frequency of EBV-specific CD4+ T-cells was 0·08%, with a range of 0·01 to 0·2%, and for CD8+ T-cells 0·14%, with a range of 0·08 to 0·32%.
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Discussion |
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Our modified single-cell quantitative approach has several important advantages over earlier methods to analyse T-cell frequencies which required previous expansion of T-cells. First, this assay identifies and quantifies antigen-responsive T-cells within 16 h of culture and is independent of T-cell proliferation. Thus, cell cycle arrest induced by infectious MV in vitro becomes irrelevant for this assay.
Second, synthetic peptides have previously been used in a tetramer technique as well as with IFN- ELISPOT and intracellular staining assays. However, these techniques mostly rely on the knowledge of epitopes in the context of selected MHC molecules and involve laborious construction of the appropriate reagents. In contrast, in our approach using MV-infected autologous APC, immune responses against viral antigens in the context of all MHC alleles are measured. This has the practical consequence that MHC typing can be omitted, allowing clinical investigation of T-cell responses of virtually any donor population from small samples of blood.
Third, in our experiments whole virus was used as antigenic challenge. Thus, viral proteins go through the antigen-processing machinery and might be presented in a more physiological context on APC than when adding exogenous peptide in artificially high concentrations.
Furthermore, using MV-infected APC allows simultaneous analysis of MHC class I- and II-restricted T-cells. Thus, subset assignment of antigen-specific responses allows the co-evaluation of CD4+ and CD8+ T-cells.
For humans, frequency data are available from individuals with persistent virus infections such as EBV, CMV and HIV. For detection of virus-specific CD4+ T-cells, most investigators used intracellular cytokine staining. Flow cytometric assays exhibited a median frequency of 0·71% (range 0·15 to 2·3%) IFN--producing CD4+ T-cells in normal CMV-positive subjects (Waldrop et al., 1997
; Kern et al., 1998
). In nonprogressive HIV patients CD4+ memory T-cells were found with a median frequency of 0·40% (range 0·1 to 1·7%) (Pitcher et al., 1999
).
Frequency analysis of virus-specific CD8+ memory T-cells was mainly performed by tetramer staining and by intracellular IFN- staining. Here, for CMV-derived dominant peptide epitopes memory CD8+ T-cell frequencies were between 0·3 and 3 % (Kern et al., 1998
) and for EBV-specific CD8+ T-cells between 0·03 and 3·8% (Callan et al., 1998
; Tan et al., 1999
; Dalod et al., 1999a
, b
).
In contrast to persistent viral infections, information on human memory T-cell frequencies for non-persistent infections are mainly based on studies using limiting dilution assays, which markedly underscored T-cell frequencies. In this study, for the first time, virus-specific memory T-cell subsets were simultaneously analysed for a non-persistent virus infection with a single-cell approach. Surprisingly, the frequencies of MV-specific CD4+ and CD8+ memory T-cells found are comparable to those previously reported for persistent virus infections such as EBV, CMV or HIV. Thus, even decades after acute measles both the CD4+ and CD8+ T-cell pools contain high levels of MV-specific memory T-cells. This indicates that cell-mediated immunity to MV is long-lived and might be independent from virus persistence.
Whereas in persistent virus infections both CD4+ and CD8+ T-cells have been shown to be critical for maintenance of memory, the interdependence of these T-cell subsets for lasting immunity to non-persistent infections has not been studied (Kalams & Walker, 1998 ; Zajac et al., 1998
). It is therefore of special interest that after natural MV infection comparable levels of MV-specific memory CD4+ and CD8+ T-cells were found. Thus, we suggest that immunity to measles also requires a balanced ratio of virus-specific T-helper to cytotoxic memory T-cells. However, further investigations will reveal whether comparable frequencies of MV-specific T-cell subsets are also detected after immunization with live attenuated MV or when analysing T-cell subsets in patients with measles complications such as acute encephalitis or subacute sclerosing panencephalitis. Hence, this approach will be useful in determining the immunological factors that decide the outcome of the disease and which lead to lifelong immunity in measles.
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Acknowledgments |
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Received 25 October 1999;
accepted 6 January 2000.