A novel sensitive approach for frequency analysis of measles virus-specific memory T-lymphocytes in healthy adults with a childhood history of natural measles

Ralph Nanan1, Andrea Rauch1, Eckhart Kämpgen2, Stefan Niewiesk3 and Hans Wolfgang Kreth1

Children’s 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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Measles virus (MV), a single-stranded negative-sense RNA virus, is an important pathogen causing almost 1 million deaths annually. Acute MV infection induces immunity against disease throughout life. The immunological factors which are responsible for protection against measles are still poorly understood. However, T-cell-mediated immune responses seem to play a central role. The emergence of new single-cell methods for quantification of antigen-specific T-cells directly ex vivo has prompted us to measure frequencies of MV-specific memory T-cells. As an indicator for T-cell activation IFN-{gamma} production was measured. PBMC were analysed by intracellular staining and ELISPOT assay after stimulation with MV-infected autologous B-lymphoblastoid cell lines or dendritic cells. T-cell responses were exclusively seen with PBMC from MV-seropositive healthy adults with a history of natural measles in childhood. The median frequency of MV-specific T-cells was 0·35% for CD3+CD4+ and 0·24% for the CD3+CD8+ T-cell subset. These frequencies are comparable with T-cell numbers reported by other investigators for persistent virus infections such as Epstein–Barr virus, cytomegalovirus or human immunodeficiency virus. Hence, this study illustrates that MV-specific CD4+ and CD8+ T-cells are readily detectable long after the acute infection, and thus are probably contributing to long-term immunity. Furthermore, this new approach allows efficient analysis of T-cell responses from small samples of blood and could therefore be a useful tool to further elucidate the role of cell-mediated immunity in measles as well as in other viral infections.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Measles is an acute, highly contagious viral disease causing almost 1 million deaths annually. The main complications of measles are pneumonia, encephalitis and secondary bacterial infections due to transient immunosuppression (reviewed in Katz, 1995 ). Despite the strong immunosuppressive effects in vivo, acute measles virus (MV) infection induces life-long immunity. This was first observed during an epidemic in the Faroe Islands when only inhabitants who had had measles 65 years earlier escaped infection (Panum, 1940 ). In contrast, immunization with attenuated measles vaccine does not seem to induce life-long protection as documented by outbreaks in populations that had been vaccinated 15 to 20 years earlier (Gustafson et al., 1987 ; Chen et al., 1990 ).

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 MHC–peptide 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-{gamma} 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 Epstein–Barr 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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Donor population.
The donors in this study were eight healthy MV-seropositive adults, 25 to 42 years old, with no history of chronic disease or immunodeficiency. They were free from infection and received no drug therapy. In addition, cord blood was taken from three healthy full-term infants with no signs of congenital viral or bacterial infection. Blood was taken with the consent of the donors or their parents after the nature and possible consequences of the study had been fully explained. The study was approved by the ethical committee of the University of Würzburg.

Serology was assessed by standard enzyme immunoassay (EIA) for MV and EBV (Institute für Virologie und Immunobiologie, Würzburg, Germany).

{blacksquare} 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 Ficoll–Hypaque 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 Ficoll–Hypaque (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 granulocyte–macrophage 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 ).

{blacksquare} 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.

{blacksquare} Intracellular staining for IFN-{gamma}.
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-{gamma} 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.

{blacksquare} IFN-{gamma}-specific ELISPOT assay.
ELISPOT assays for IFN-{gamma} 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-{gamma} 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-{gamma} 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-{gamma}-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-{gamma} 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-{gamma}-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).

{blacksquare} 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 manufacturer’s 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.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
In vitro activation of specific T-cells with MV-infected APC
For stimulation of MV-specific T-cells, an autologous cell system is required. MV has a tropism for cells of the lymphocytic and monocytic lineage (Esolen et al., 1993 ; Joseph et al., 1975 ). Whereas DC as professional APC are most effective for induction of primary and secondary T-cell responses, B-LCL are readily available and have also been efficiently used as APC to induce secondary CD4+ and CD8+ T-cells. Therefore, DC and B-LCL were potential candidates as APC. However, after infection DC rapidly developed syncytia and decreased in numbers to less than 5%, due to the lytic effects of MV. Therefore, to avoid formation of syncytia, fusion inhibitory peptide had to be added to cultures (Richardson & Choppin, 1983 ). Still, more than 100 ml of venous blood per experiment was needed when using DC as APC. In contrast, B-LCL were generated from 3 to 5 ml of venous blood. Once established they could be used indefinitely. Furthermore, under the conditions used more than 80% of B-LCL were infected after 24 h whereas only 12% of DC were positive after 72 h (Fig. 1).



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Fig. 1. Specific activation of T-cells with MV-infected APC. PBMC from donor C, a 32-year-old healthy MV- and EBV-seropositive adult with a childhood history of natural measles, were cultured for 16 h with autologous DC or B-LCL. APC were either uninfected or infected with MV (m.o.i.=1), as indicated. Intracellular and surface markers were stained following fixation and permeabilization of cells. Lymphocytes were gated in side-scatter versus forward-scatter light and for CD3+ cells. Dot plots show frequencies of IFN-{gamma}-positive cells as a percentage of CD3+CD8+ or CD3+CD8- T-lymphocytes. Additionally, surface expression of MV haemagglutinin protein in uninfected (filled histogram) and MV-infected (open histogram) APC are compared in histogram overlays. B-LCL were infected with MV (m.o.i.=1) and incubated for 24 h. DC were infected with MV (m.o.i.=1) and incubated for 72 h in the presence of 10 µg/ml fusion inhibitory protein.

 
Next, we tested the ability of MV-infected B-LCL and DC to activate MV-specific T-cells. PBMC in the absence of APC produced no IFN-{gamma} (data not shown). As shown in Fig. 1, with MV-infected DC specific CD3+CD4+ and CD3+CD8+ T-cells could be induced to produce IFN-{gamma}. In parallel experiments with MV-infected B-LCL as APC there was also an MV-specific T-cell response in both T-cell subsets. However, uninfected B-LCL also induced IFN-{gamma} production in PBMC. Most likely, B-LCL express latent EBV antigens which activate EBV-specific T-cells. Therefore, when calculating MV-specific T-cell frequencies EBV-specific T-cells have to be subtracted. Consistently, frequencies of MV-specific T-cells obtained with MV-infected B-LCL as APC were comparable to frequencies assessed with DC for both CD3+CD4+ and CD3+CD8+ T-lymphocyte subsets. Representative results from three experiments are given in Fig. 1.

Frequencies of IFN-{gamma}-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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Fig. 2. Detection of MV-specific T-cells in longitudinal blood samples. Blood samples were collected at 4- to 6-week-intervals from two healthy MV- and EBV-seropositive adults [donors A (•) and F ({blacksquare})], with a childhood history of natural measles. MV-specific T-cells were activated with B-LCL as APC. IFN-{gamma}-positive cells were determined by IFN-{gamma}-specific ELISPOT assays with 1x105 unseparated PBMC. Means of triplicates (±1 SD) were calculated from experiments of longitudinal blood samples for each donor.

 
MHC restriction and virus-specificity of IFN-{gamma}-positive cells
We further investigated MHC restriction of MV-specific T-cells using 174xCEM.T2 cell lines as APC. These cells are deficient for endogenous MHC class I peptide loading and express no MHC class II molecules. As shown in Fig. 3, there was no MV-specific T-cell response when these cells were used as APC.



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Fig. 3. MHC restriction and specificity of CD3+CD4+ and CD3+CD8+ T-lymphocyte subsets were tested (donor A) by intracellular IFN-{gamma} staining. Autologous B-LCL were used as APC. These cells were either uninfected, infected with MV, mock-infected with Vero cell supernatant or incubated with inactivated MV (heat-treated for 60 min at 56 °C). Cells of the 174xCEM.T2 cell line were also used uninfected or MV-infected as APC.

 
In addition, we tested whether replication of virus was needed for induction of CD8+ T-cells. For this purpose, autologous B-LCL were incubated with heat-inactivated MV and used as APC. As shown, there was a clear response of MHC class II-restricted CD4+ T-cells which recognize non-replicative antigens, but not of MHC class I-restricted CD8+ T-cells for which de novo synthesized antigens are required (Fig. 3).

Comparison of T-cell frequencies by intracellular IFN-{gamma} staining and ELISPOT assay
Both CD3+CD4+ T-cells as well as CD3+CD8+ T-cells were analysed for IFN-{gamma} expression by intracellular staining. To verify intracellular IFN-{gamma} production by an alternative approach, IFN-{gamma} 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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Fig. 4. Comparison of MV-specific T-cell frequencies determined by ELISPOT assay and intracellular staining. (a) To analyse T-cell subset frequencies by IFN-{gamma}-specific ELISPOT assays CD4+ and CD8+ T-lymphocyte subsets were enriched from PBMC of donor D by indirect immunomagnetic sorting. CD4+-enriched PBMC contained 93% CD3+CD4+ and 4% CD3+CD8+ lymphocytes, CD8+-enriched PBMC contained 95% CD3+CD8+ and 1·5% CD3+CD4+ lymphocytes as determined by flow cytometry. Triplicates of 1x105 enriched T-cell subsets were incubated with 5x104 uninfected or MV-infected B-LCL in an IFN-{gamma}-specific ELISPOT assay for 24 h. MV-specific T-cell frequencies were calculated for each subset and are given in % as mean values of triplicate determinations. (b) In parallel experiments PBMC were analysed by intracellular staining for IFN-{gamma}.

 
Specificity of IFN-{gamma} secretion by MV-specific T-cells
To exclude a non-specific stimulation of MV-specific T-cells by B-LCLs the following controls were performed (Table 1). Cord blood lymphocytes from healthy full-term infants without contact with either EBV or MV did not display a virus-specific response. T-cells from two EBV-seronegative, MV-seropositive donors secreted IFN-{gamma} after stimulation with MV-infected stimulator cells but not after stimulation with B-LCLs alone. We also observed a strict association between seropositivity and T-cell response to both EBV and MV. Indeed, the correlation between MV-specific CD4+ T-cells and MV-specific antibody titres was significant (linear regression analysis, r=0·8) (Table 1).


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Table 1. Virus-specific T-cells in seropositive and seronegative donors

 
Frequency of MV-specific T-cells in healthy adults with a childhood history of measles
MV-specific T-cells were evaluated in a group of eight MV-seropositive individuals with a known history of natural measles in childhood (Table 1). Six of these donors were also EBV-seropositive. MV-seropositive adult donors had a median frequency for MV-specific CD3+CD4+ T-cells of 0·35%, ranging from 0·08 to 0·85%. For the CD3+CD8+ T-cell subset the median frequency was 0·24%, ranging from 0·05 to 0·48%.

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%.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
In this study we quantified CD3+CD4+ and CD3+CD8+ MV-specific T-lymphocytes in PBMC. T-cell responses strictly correlated with seropositivity and were clearly antigen-specific. They occurred in the appropriate T-cell subset and depended on MHC-restricted antigen presentation.

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-{gamma} 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-{gamma}-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-{gamma} 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.


   Acknowledgments
 
We are grateful to Dirk Busch (Institute für Hygiene, Mikrobiologie und Immunobiologie, Technische Universität München) for helpful discussions.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 25 October 1999; accepted 6 January 2000.