| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Usage Without Superantigenic Activity1
*
Department of Immunology, Guy’s, King’s and St. Thomas’ School of Medicine, London, United Kingdom;
Rheumatology Unit, Institute of Child Health, UCL, London, United Kingdom; and
Division of Medicine, University of Bristol, Bristol, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain selection
among peripheral blood and intraislet T lymphocytes. To examine these
issues, we analyzed TCR V chain-specific up-regulation of the early
T cell activation marker, CD69, on CD4 T cells after incubation with
Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the V chains 2, 7,
and 8 were the most frequently activated by CVB4. Up-regulation of CD69
by different TCR families was significantly more frequent in new onset
type 1 DM patients (p = 0.04), 100% of whom
(n = 8) showed activation of CD4 T cells bearing
V8, compared with 50% of control subjects (n =
8; p = 0.04). T cell proliferation after incubation
with CVB4 Ags required live, nonfixed APCs, suggesting that the
selective expansion of CD4 T cells with particular V chains resulted
from conventional antigen processing and presentation rather than
superantigen activity. Heteroduplex analysis of TCR V chain usage
after CVB4 stimulation indicated a relatively polyclonal, rather than
oligo- or monoclonal response to viral Ags. These results provide
evidence that new-onset patients with type 1 DM and healthy controls
are primed against CVB4, and that CD4 T cell responses to the virus
have a selective TCR V chain usage which is driven by viral Ags
rather than a superantigen. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells. Studies
showing geographical variability in disease prevalence, as well as
increasing incidence of type 1 DM despite a stable gene pool, suggest
that environmental factors play a major role in the development of this
autoimmune disease (1, 2). In particular, numerous studies
have suggested an association between enterovirus infections and the
development of type 1 DM. This association is based on virus isolation
from the pancreas of newly diagnosed patients (3), several
epidemiological and prospective studies showing that some cases of type
1 DM are strongly associated with enterovirus infections
(4, 5, 6), and case-control studies that show an increased
prevalence and levels of IgM Abs to Coxsackievirus B (CVB) in newly
diagnosed patients (7). Initially, Gamble and Cumming
(8) reported an association with CVB, but more recently
there has been evidence for the involvement of a wider range of
enteroviruses, including Coxsackievirus A and echoviruses
(9, 10, 11, 12).
Several mechanisms have been proposed to explain this link between
virus infections and type 1 DM, among them the so-called "molecular
mimicry" theory (13, 14), as well as bystander
activation of autoreactive T cells by virus-specific T cells during an
ongoing infection (15). More recently, it has been
suggested that viral protein(s) with superantigenic activity
(16) may be active in the development of type 1 DM. In
studies on two children with recent onset type 1 DM, a marked
overrepresentation of mRNA encoding the TCR V7 chain among islet
infiltrating lymphocytes was observed (16). In a more
recent study of PBLs (17), TCR V7 mRNA was found to
predominate in type 1 DM patients at diagnosis compared with healthy
individuals and patients with long-term diabetes. To date, there has
been no adequate explanation for this TCR V skewing
(18, 19, 20, 21, 22, 23, 24). However, a potential clue to the etiology of the
TCR V skewing in type 1 DM patients may be found in a study on T
cells infiltrating the hearts of children with proven CVB3-associated
acute myocarditis (25). The study showed skewing of TCR
V mRNA toward expression of V transcripts (7, 3, and 13.1)
similar to those seen in islets from diabetic children
(16), thus providing a link between Coxsackieviruses and
TCR V skewing.
In some of the above studies, spectratype analysis indicated that
expansion of selected TCR V chain transcripts was poly rather than
oligo- or monoclonal. This argued against an Ag-driven T cell expansion
and in favor of the possibility that during CVB infection, protein(s)
drives TCR V skewing through a superantigen effect. In the present
study, we investigated this possibility directly by examining TCR
V-specific up-regulation of the early activation marker, CD69, on
CD4 and CD8 T cells, following exposure to CVB4 viral proteins. Our
results show that under these conditions CVB4 proteins activate mainly
CD4 T cells compared with CD8 T cells. We show that the activation does
indeed target selected T cells according to V chain expression,
including V chains previously shown to be preferentially represented
in islets of Langerhans at diagnosis of type 1 DM. However, we provide
evidence that this TCR V activation requires viral Ag processing and
is therefore unlikely to be the result of superantigen activity.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Sixteen subjects were studied. There were eight (six males, two females) healthy nondiabetic donors without family history of the disease and eight (five males, three females) newly diagnosed type 1 DM patients (all within 2 wk of diagnosis). Mean age (±SD) for the healthy donors was 34.4 ± 6.8 years and for the diabetic patients 30.4 ± 5.7. Blood was drawn with the consent of all subjects and appropriate permission from the Institute’s Ethical Review Board. The matched healthy controls and type 1 DM patients were tested during the same time period to control for seasonal variations in exposure to CVB4. HLA genotyping was performed by PCR-single-strand conformation polymorphism.
CD69 up-regulation studies
PBMCs were obtained by Ficoll-Hypaque centrifugation of
heparinized blood, washed twice in HBSS, and resuspended at 1 x
106 cells/ml in RPMI 1640 medium (Life
Technologies, Paisley, U.K.) supplemented with 10%
AB+ human serum (PAA Laboratories, Linz,
Austria), 50 IU/ml penicillin, and 50 µg/ml streptomycin (Life
Technologies). Suspended PBMCs were distributed in a 24-well tissue
culture plate (1 ml/well) supplemented with medium alone, toxic shock
syndrome toxin-1 (TSST-1; 100 ng/ml; Toxin Technology, Sarasota,
FL), or either control uninfected or CVB4-infected Vero cell
lysate (JVB strain; 5 µl/ml; 
0.2 µg of protein lysate/ml;
Institute Virion, Zurich, Switzerland) and incubated for 5 h at
37°C. Cell lysates were prepared as previously described
(26). In control experiments, influenza-infected and
control cell lysates (Institute Virion) were used as a source of
Ags.
After incubation, cells were collected and washed (300 x
g for 10 min at 4°C) twice with PBS containing 5% FCS
(PAA Laboratories). After washing, cells were resuspended in 1 ml of
PBS-FCS, aliquoted, and stained for 30 min at 4°C with CD4-APC (clone
SK3), CD8-PerCP (clone SK1; both from BD Biosciences, Erembodegem,
Belgium), CD69-PE (clone CH/4; Serotec, Oxford, U.K.), and 1 of 16
FITC-labeled V Abs: anti-human V1 (clone BL37.2), 2 (clone
MPB2D5), 3.1 (clone 8F10), 5.1 (clone LC4), 6.7 (clone OT145), 7 (clone
ZOE), 8 (56C5.2), 11 (clone C21), 12 (clone VER2.32.1), 13.1 (clone
IMMU222), 14 (clone CASS1.1.3), 16 (clone TAMAYA1.2), 17 (clone C1), 20
(clone ELL1.4), 21.3 (clone IG125), and 22 (clone IMMU546; all from
Serotec). Cells were also stained with the corresponding fluorochrome
and isotype control Abs (either from BD Biosciences or Serotec), and
nonspecific staining subtracted was from the appropriate populations.
After staining, cells were washed twice with PBS-FCS and resuspended in
PBS for analysis using a FACSCalibur flow cytometer (BD Biosciences).
Ten thousand CD4-positive events and 5000 CD8-positive events were
collected. Data were analyzed using the CellQuest program (BD
Biosciences). The panel of 16 V-specific mAbs we used represents the
major TCR V families and stains 
60–70% of TCR-positive
cells.
Detection of CD69 up-regulation on specific V+ T
cells
To demonstrate that T cells bearing a specific V chain showed
CD69 up-regulation after exposure to CBV Ags, we used two separate
analyses. This approach was first validated in preliminary studies
using TSST-1 as the stimulus, as discussed in Results. Only
those V families that showed significant CD69 up-regulation as
judged by both analyses were considered positive. The two separate
analyses are illustrated in Fig. 1
and are based on a direct comparison
between PBMCs incubated with CVB-infected and uninfected lysates.
|
+
cells was considered positive if the proportion of a specific V
subset expressing CD69 was higher after stimulation with the
CVB-infected lysate compared with the control uninfected lysate. The
proportion of a specific V subset expressing CD69 is given by the
formula quadrant 2/quadrant 2 + 4 (Fig. 1
).
In the second analysis, we examined the contribution of each V
family to the total number of cells activated (i.e., total
CD69+) by CVB. The total population of cells
activated to express CD69 by a given stimulus is represented by
quadrant 1 + quadrant 2. For each patient and control, a mean value for
quadrant 1 + quadrant 2 was obtained from the 16 separate V
analyses. This value was used as the denominator, and values in
quadrant 2 for each V analysis were used as the numerator. This is
given by the formula quadrant 2/(quadrant 1 +
2)MEAN. Activation of a specific V family was
considered to contribute to total CD69 up-regulation when quadrant
2/(quadrant 1 + 2)MEAN equated to greater
than zero.
Only those V families in which there was evidence of CD69
up-regulation by CVB4 proteins (first analysis) and evidence that this
up-regulation contributed to total CD69 up-regulation by CVB4 (second
analysis) were considered positive. The rationale for using a
combination of two separate analyses is that it takes into account
those V families that are poorly represented numerically, in which
small changes in the numbers of activated cells appear as large
proportional changes, although overall these cells represent a very
small percentage of cells activated by the virus. Reproducibility of
this analytical approach was assessed by preliminary experiments
conducted on samples 3–4 wk apart obtained from healthy control
subjects. A representative example is subject C7, who showed positive
activation of TCR V families 1, 2, 5, 6, 11, 16, and 17 on first
analysis, with the remaining families negative. An identical pattern of
positivity and negativity was seen in the second sample.
Proliferation assays
PBMCs (105 cells/well) were cultured in
triplicate in U-bottom 96-well plates with medium alone, 100 ng/ml
tetanus toxoid (TT; Pasteur Merieux MSD, Berkshire, U.K.), 10 ng/ml
staphylococcal enterotoxin B (SEB) (Toxin Technology) and 5
µl/ml (0.2 µg of protein lysate/ml final concentration) of
either uninfected or CVB4-infected Vero cell lysate. These
concentrations were shown to give optimal proliferation in preliminary
and previous studies (26). After 6 days at 37°C, 0.5
µCi/well [3H]thymidine was added and the
cultures were harvested after a further 18 h of incubation onto
glass fiber filters. Proliferation was measured as
[3H]thymidine incorporation determined by
direct beta plate counting in a Matrix 9600 counter (Packard
Instrument, Pangbourne, U.K.). Results are expressed as a stimulation
index (SI: mean counts with Ag/mean counts with relevant control
preparation (i.e., uninfected Vero cell lysate). For SEB and TT
responses, the SI was calculated using the formula: mean counts with
Ag/mean counts with medium alone. An SI above 3 is considered positive
by convention. Mean counts in the presence of uninfected lysate were
684 cpm (range, 206-2417) and in the presence of medium alone 489 cpm
(range, 61–1532).
To study the possibility that a superantigen is encoded in the CVB4 virus genome, a responder population (CD3+HLA-DR- cells) and an APC population (CD19+) were isolated from total PBMCs by positive selection using microbeads as suggested by the manufacturer (Miltenyi Biotec, Surrey, U.K.). Briefly, cells were resuspended at 107/80 µl in PBS containing 0.5% BSA and 2 mM EDTA. Twenty microliters of the corresponding magnetic beads (CD3 or CD19) was added to the cells and incubated at 6–12°C for 15 min. Cells were isolated using a type MS+/RS+ column, prewashed with PBS/BSA/EDTA, and placed in a magnetic MACS separator. The cell suspension was applied to the column and washed three times with 500 µl buffer. The column was then removed from the magnet and retained cells were eluted in 1 ml of PBS/BSA/EDTA. The CD3+ population was depleted of HLA-DR+ cells using HLA-DR Dynabeads (Dynal, Wirral, U.K.) according to the manufacturer’s instructions, and the CD3/HLA-DR- population was washed with HBSS and resuspended at 106 cells/ml in RPMI 1640 medium (RPMI 1640/10% AB+ serum). The CD19+ cells were resuspended at 4 x 106 cells/ml in RPMI 1640 medium and divided into two tubes. One tube was left unmanipulated (APCs nonfixed) and to the second the same volume of 2% paraformaldehyde in PBS was added to the cells, incubated for 10 min at room temperature, washed twice, and resuspended in RPMI 1640 medium at 4 x 106 cells/ml. The purities of the isolated populations were assessed by flow cytometry and for the responder population (CD3+HLA-DR- cells) was 95% or greater, with only 0.35–2% of CD19+ cells (B cells), and for the APC population (CD19+) purity was 87–99%.
To set up the proliferation assays, responder cells (105 cells/well) and APCs (4 x 104 cells/well), either nonfixed or fixed with paraformaldehyde, were cultured in triplicate in U-bottom 96-well plates with the corresponding Ag (SEB, TT, CVB4-infected lysate, or no Ag) in a total volume of 200 µl. Total unmanipulated PBMCs were also incubated with the same Ags. After 5 days in culture, the cells were labeled with [3H]thymidine and harvested as described above. Results are expressed as a SI (mean counts with Ag/mean counts with medium alone).
Heteroduplex analysis of TCR usage
A detailed description of this analysis can be seen in the study
by Maini et al. (28). In the context of the current study,
this technique is able to provide a direct visualization of clonal
expansion of T cells according to particular V usage. Briefly, PBMCs
(4–6 x 106) were harvested after 6 days in
culture with either uninfected or CVB4-infected cell lysates. Total RNA
was extracted using the SV Total RNA Isolation System kit (Promega,
Southampton, U.K.) and cDNA was synthesized using the RETROscript
(Ambion, Oxon, U.K.).
Twenty-six different PCRs, for both cells incubated with uninfected and
CVB4-infected lysates, were performed using a consensus internal C
primer (5'-CACCCACGAGCTCAGCTCCACGTGGTC-3') and 26 TCRBV primers
as described before (29) in a final volume of 50 µl.
Hot-start PCR was conducted with initial denaturation at 95°C for 5
min, followed by 30 cycles of 95°C for 30 s, 58°C for 30
s, and 72°C for 1 min, with a final extension period at 72°C for 10
min. PCR products were visualized on a 1% agarose gel. DNA carriers
consisting of cDNAs encoding BV1–24 cloned from T cell clones or lines
as described previously and provided by G. Casorati (Unitá
d’Immunochimica, DBIT, Istituto Scientifico, Milan, Italy)
(29) were amplified using the same TCRBV primers and
conditions as those for the sample cDNAs, but using a different C
primer, external C primer
(5'-TGCTGACCCCACTGTCGACCTCCTTCCCATT-3') which is 30-bp 3' to the
internal C. A 20-µl aliquot of each sample PCR product was mixed
with 400 ng of the appropriate BV-matched carrier product, denatured at
95°C for 5 min, and allowed to reanneal at 50°C for 1 h. The
mixtures were loaded on a 12% nondenaturing polyacrylamide gel and
resolved at 10 mA for 16 h at 4°C. For initial visualization,
heteroduplex gels were stained with ethidium bromide for 30 min and
then blotted onto nylon Hybond N+ membranes in
20x SSC. The DNA was denatured and fixed to the membrane by a 20-min
incubation in 0.4 M NaOH. The external C primer was biotin-labeled
using the BrightStar Psoralen-Biotin kit (Ambion). Membranes were
prehybridized for at least 2 h at 42°C with prehybridization
buffer (6x SSC containing 5x Denhardt’s, 0.1% SDS, and 100 µg/ml
sonicated salmon sperm DNA; Sigma, Poole, U.K.) and hybridized for
16 h at 42°C with hybridization buffer (6x SSC, 0.1% SDS)
containing 10 ng/ml labeled probe. Membranes were washed in 5x
SSC/0.1% SDS for 20 min at room temperature and then twice in 1x
SSC/0.1% SDS for 15 min each at 42°C. To detect the biotinylated
probe, we used a chemiluminescent detection kit (BrightStar BioDetect;
Ambion) following the manufacturer’s instructions. Filters were
exposed to x-ray films at room temperature for 2–16 h.
Statistical analysis
Comparisons of the frequency of activation of different V
chains was made using Fisher’s exact test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
skewing by expression of the early activation
marker CD69
Up-regulation of the early activation marker CD69 has been used
previously to identify superantigen activity in EBV (27).
Using a similar approach, we incubated PBMCs with TSST-1, a
superantigen known to favor TCR V 2 expansion, for 5 h and,
after harvesting, cells were stained for CD4, CD69, and different V
chains. According to the two criteria described (see Materials
and Methods and Fig. 1), T cells
carrying V2 chains were activated by TSST-1 as well as some V
3+ cells (data not shown). These data confirm
that this experimental approach is able to identify superantigenic
activity.
Activation of T cells with CVB4 Ags elicits CD69 expression on
cells bearing selected TCR V chains
A typical example of the results obtained in our analyses on
PBMCs from patients with type 1 DM and control subjects, using CVB4
lysate as the antigenic stimulus, is shown in Fig. 1. As stated above,
two criteria were used to determine whether CVB4 activates specific
V populations: 1) an increase in the proportion of cells of a
specific V population expressing CD69 in the presence of CVB4 Ags
and 2) an increase in specific
V+CD69+ cells as a
proportion of the total number of cells induced to express CD69 by
CVB4 Ags.
In the case depicted in Fig. 1 for CD4 T cells from a type 1 DM patient
(D1, Table I), several V families show
an increase in their percentage of CD69 expression after incubation
with CVB4 Ags compared with uninfected lysates. Increases vary from
29.8% for V6 to 2.4% for V20, (Fig. 1A). Some V
families (1, 7, 12) show no increase compared with the uninfected
lysates.
|
families analyzed). In the case shown, the major component of this
increase is from the V8 and V6 families (Fig. 1B),
although other V cells also contribute. Several other V families
also contribute to the increase in CD69 expression after incubation
with CVB4, the exceptions being TCR V 1, 7, 12, 14, 16, 17, and 21
(Fig. 1B). This analysis indicates that in this patient, CD4
T cells having TCR V chains 2, 3, 5, 6, 8, 11, 13, 20, and 22 were
activated by the CVB4-infected lysate and also contribute to the total
virus-activated CD69 population. In this individual, therefore, these
V chains were considered positive.
We went on to analyze eight patients with new-onset type 1 DM and eight
healthy nondiabetic control subjects in the same way, applying the
two-step criteria to identify V families in which CD69 was
up-regulated by CVB4. In the CD4 T cell compartment, considering all
the subjects tested together (16 in total), we observed that certain
V families showed CD69 up-regulation more frequently. CD69
up-regulation on TCR V8 cells was seen in 75% (12 of 16 subjects)
and V2, 7, 11, and 12 in 56.3% (9 of 16), 62.5% (10 of 16), 69.2%
(9 of 13), and 60% (9 of 15) subjects, respectively (Table I). T cells
expressing remaining V chains were activated in 50% of cases
or less.
However, when we compared the two groups of subjects, we observed
differences in the V activation patterns of type 1 DM patients
compared with healthy control subjects. Among type 1 DM patients, 100%
(8 of 8) subjects showed clear up-regulation of CD69 CD4 T cells
bearing TCR V8 chains compared with 50% (4 of 8) of healthy
subjects (p = 0.04; Table I). CD4 T cells with
other TCR V chains were more frequently activated in patients than
control subjects, most notably in the cell populations expressing V2
(75% in type 1 DM patients vs 37.5% in control subjects), V3 (62.5
vs 25%), V7 (75 vs 50%), and V21 (62.5 vs 37.5%), although
these differences in frequency failed to reach statistical significance
(Table I). In contrast, V13-expressing cells were more frequently
activated in control subjects (62.5 vs 25% in type 1 DM patients),
although this difference did not reach statistical significance.
Overall, coculture of CVB4 Ags with PBMCs from type 1 DM patients
induced V chain-specific CD69 up-regulation more frequently (70
positive of 125 tests, 56%) than PBMCs from control subjects (56 of
125 tests, 45%; p = 0.04). Although the number of
cases studied is small, there did not appear to be any relationship
between HLA-DR genotype and V-specific CD69 up-regulation
(Table I).
In the CD8 compartment, CD69 up-regulation was lower in magnitude and
frequency than for CD4 T lymphocytes (Table II). Considering all the subjects, only
CD8 T cells bearing TCR V1 chains were activated in >50% of all
test subjects. There were no significant differences between type 1 DM
patients and control subjects in the frequency of CD8 V
chain-specific activation.
|
TCR-related activation we
observed, we also analyzed CD69 up-regulation after coculture with
influenza viral cell lysates, compared with the appropriate control
cell lysates, when sufficient cells were available. The pattern of
V-specific CD69 up-regulation on CD4 T cells by influenza viral cell
lysates was consistently different than that seen with CVB4 lysates, as
shown for subject C7 in Fig. 2.
|
-bearing T cells requires
processing of CVB4 Ags
In the light of evidence that CVB4 Ags induce CD69 expression on
selected populations of T cells according to V chain expression, our
next step was to examine whether this could be due to a superantigen
effect. Superantigens bind directly to MHC class II molecules, outside
the conventional peptide binding groove, and stimulate T cells
regardless of Ag specificity via engagement with the TCR V chain.
Their interaction with MHC class II molecules and subsequent T cell
activation does not require conventional Ag processing and presentation
by APCs. We designed an assay to distinguish between T cell activation
driven by superantigen and that driven by conventionally processed Ag.
In this assay, APCs and responder T cells (HLA-DR depleted) were
purified and the APCs either fixed with paraformaldehyde or left
unmanipulated. Responder T cells were stimulated to proliferate in the
presence of the superantigen SEB irrespective of whether fixed or
nonfixed APCs were used (SIs, 54.6 and 47.1, respectively; Fig. 3A). In contrast, as expected,
the conventional recall Ag TT, which requires processing by APCs for T
cell recognition, only induced T cell proliferation in the presence of
live, unfixed APCs (Fig. 3B). Under identical conditions,
the ability of CVB4 Ag lysates to induce T cell proliferation was
examined. In the case of CVB4, the results were similar to those for
the recall Ag TT, in that proliferation only occurred in the presence
of live, unfixed APCs (Fig. 3C). None of the cases tested
showed significant proliferation to CVB4 Ags in the presence of fixed
APCs (SI < 2.0). These data were acquired in three separate
experiments on three healthy individuals (cases C6, C7, and C8 in Table I) and argue against the possibility that the CVB4 lysates contain Ags
capable of inducing T cell proliferation or V skewing through a
superantigen effect.
|
families that show CD69
up-regulation
Expression of CD69 is a marker of early T cell activation,
typically seen within 6 h of stimulation, and as such it is
frequently used for analysis of Ag-specific T cell activation. However,
its expression gives limited information on the functional fate of the
activated cell. To examine whether patterns of TCR V-specific T cell
proliferation taking place over several days mirror those seen using
CD69 analysis, we used heteroduplex analysis of TCR V expression.
This approach is able to identify clonal and oligoclonal T cell
expansion within a specific V family after antigenic stimulation
(28, 29, 30).
After 6 days in culture with either uninfected or CVB4-infected
lysates, cDNA prepared from PBMCs was analyzed using this technique.
One limitation of an RT-PCR based TCR detection technique is that for
CD4+ cells, in vitro culture is often required to
expand clones to a size that is readily detectable. As we and others
have previously described (26, 31), T cell proliferative
responses to CVB4 are typically CD4 T cell dominated and only
detectable at low levels in healthy individuals and type 1 DM patients.
Proliferation assays performed on PBMCs from subjects in the present
study showed a similar pattern of low reactivity. For this reason,
sufficient cDNA for heteroduplex analysis of TCR V usage was
available on only a limited number of subjects.
Healthy donor 7 shows CD69 up-regulation of CD4 T cells bearing V
chains 1, 2, 5, and 6 (Table I), but not V7 or 8 cells. As shown in
Fig. 4A, numerous
CVB4-specific CD4 T cell clones are generated in this subject after 6
days of in vitro stimulation with CVB4. After CVB4 Ag stimulation, the
highest frequency of novel clones is seen among T cells bearing TCR
V1, 5.1, 5.2, and 6, mirroring those V families up-regulating
CD69 in this individual. Similarly, V families not induced to
express CD69 (V chains 7 and 8) are associated with fewer novel
CVB4-specific clones.
|
B shows similar results for two patients with type 1
DM. Subject D7 shows CD69 up-regulation among CD4 T cells with TCR
V5 and 8 chains (Table I). Heteroduplex analysis showed that,
although there are fewer novel clonal bands after virus stimulation
compared with the healthy donor, there is a correlation between CD69
up-regulation and the appearance of CVB4-specific clonal bands
associated with the V8 and 5 families (Fig. 4B). Fainter
bands could also be seen sporadically in TCR V chains that do not
show CD69 up-regulation, probably due to a lower level of activation of
some V-bearing T cells by CVB4 proteins or bystander activation
(Fig. 4B). A similar picture emerges from analysis of the
second type 1 DM patient (D4). In this subject, CD69 up-regulation is
detected in CD4 T cells with TCR V chains 1 and 8 (Table I). Again,
strong CVB4-specific T cell clonal bands are detected in association
with the same TCR V chains that up-regulate CD69 (Fig. 4B).
Analysis of the number of Ag-specific novel TCR clonal bands across the
repertoire suggested that overall the response to CVB4 Ags involved
multiple T cell clones. The pattern is typical of an oligoclonal,
Ag-driven response and is quite distinct from heteroduplex analysis of
TCR V expression by T cells stimulated with a superantigen such as
SEB or a mitogen such as PHA. Under these conditions of polyclonal
stimulation, a smear is seen, representing many hundreds or thousands
of distinct clones (32).
The correlation we observed between the analysis of V-specific CD69
up-regulation and TCR V expansion by heteroduplex analysis suggests
that clones bearing selected TCR V chains and showing early evidence
of activation by CVB4 Ags proceed to expansion in vitro.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain usage by T cells specific for CVB4
and whether the CVB4 genome encodes a protein with superantigen
activity. Our results indicate that the host response to CVB4 typically
involves recruitment of CD4 T cells bearing numerous different V
chains. In particular, CVB4-activated T cells used V chains 2, 7,
and 8 in the majority of subjects. Analysis of CD69 up-regulation and
use of the heteroduplex technique to analyze TCR V usage associated
with clonal CD4 T cell proliferation indicate that CVB4 induces both
early activation and proliferation of clones bearing the same V
chain. The most important finding in this study, however, is that using
a proliferation assay designed to assess the requirement for Ag
processing in T cell activation, we demonstrate that CVB4 Ags only
induce T cell proliferation after conventional Ag processing and
presentation by APCs, rendering it unlikely that the CVB4 genome
encodes a protein with superantigenic activity.
Our work was prompted by consideration of three separate studies
published in recent years. First, in the study of Conrad et al.
(16), an expansion of T cells bearing V7 transcripts
was noted in the islets of Langerhans of two children who died at
diagnosis of type 1 DM. Subsequently, the same group reported modest
but significant expansions of similar V7 cells in the peripheral
blood of patients with type 1 DM and those at risk of developing the
disease (17). A possible link between these cells and
Coxsackievirus was made in a report describing overrepresentation of
selected V transcripts, including V7, in association with CVB3 in
the diseased hearts of children with proven Coxsackie-mediated
myocarditis (25). Our study was designed to test the
hypothesis that CVB4 viral Ags induce V-specific T cell activation
and to examine whether this is achieved through a superantigenic
effect.
After the initial report of V skewing in the pancreas of type 1 DM
patients at diagnosis (16), the effect of a superantigen
was invoked as a possible explanation (33). Having
demonstrated selective TCR V activation in the current study, we
examined whether such effects could be due to a CVB4-encoded
superantigen. However, after treatment of CD19+ B
lymphocytes with paraformaldehyde, which abrogates Ag uptake and
processing, these APCs could not sustain CVB4-induced T cell
proliferation. These experiments argue against the existence of a
superantigen effect operating on those V chains we were able to
study. However, there remains the possibility that during natural
infection with CVB4, proteolytic processing of the viral polyprotein in
infected cells generates polypeptide fragments with superantigenic
effects. A similar dependence on proteolytic processing to display
superantigenic activity has been demonstrated for the prototypic viral
superantigen, the mouse mammary tumor virus superantigen
(34). In this study, it was demonstrated that proteolytic
processing of the viral superantigen 7 by furin was essential for the
protein to display superantigenic activity. However, given the general
ubiquity of intracellular expression of furin and other proteases, we
consider it unlikely that lack of protease activity has masked a CVB4
superantigen. Our interpretation that presentation of CVB4 Ags by
conventional means to T cells may induce preferential use of particular
TCR V chains is supported by previous reports, such as the
preferential usage of T cells bearing TCR V17 in recognition of the
influenza A virus matrix peptide 57–68 (35).
Our results show that patients with type 1 DM exhibit a high frequency
of V-associated CD69 up-regulation after incubation with CVB4 Ags.
In addition, we noted that some V chains were more frequently
up-regulated in new onset type 1 DM patients than control subjects.
However, the number of cases we have studied to date is small, and this
precludes us from demonstrating with confidence whether patients with
type 1 DM and healthy controls differ in the frequency and selectivity
of V-associated CD69 up-regulation. We found that among CD4 T cells,
those having the TCR V2, 7, and 8 chains showed the highest
frequency of CD69 up-regulation by CVB4. In the case of V8, all
newly diagnosed type 1 DM patients show activation of CD4 T cells
bearing this TCR V, and this frequency is significantly higher than
in healthy control subjects. Although these analyses were performed on
small numbers of subjects and require confirmation in larger cohorts,
two preliminary conclusions can be drawn. First, our data suggest that
the CD4 T cell response to CVB4 is dominated by the V8 family of T
cells. Second, the higher frequency of CVB4-induced V8 CD4 T cell
activation seen in patients with type 1 DM could reflect a higher
frequency of primed T cells in the peripheral blood at the time of
diagnosis, consistent with a recent exposure to the virus. There is a
large body of work examining serological responses to enteroviruses in
association with type 1 DM, and numerous studies are consistent with
there being a higher prevalence of enterovirus infections near to
diagnosis. Epidemiological studies and case reports also support a role
for enteroviruses in the disease pathogenesis (4, 5, 6, 7, 8, 9, 10).
However, very few studies have examined T cell responses to the
virus.
Preferential activation of CD4 T cells bearing selected TCR V chains
is of potential importance in the light of postmortem studies in which
TCR V usage has been analyzed in the pancreas of patients with
new-onset type 1 DM. In particular, Hanninen et al. (36)
showed that T cells having TCR V8 chains were the most highly
represented V family (8–14 cells per islet) in the islet infiltrate
of a pancreas obtained at diagnosis. In a similar study by Somoza et
al. (37), only three distinct V chains were clearly
detectable by RT-PCR in the intrapancreatic lymphocyte preparation, of
which one was V8. These studies supporting a role for
V8-expressing T cells in intraislet pathogenic events in type 1 DM
are balanced by postmortem studies in which V7 T cells were
overrepresented (16), and analysis of pancreatic biopsy
specimens in Japanese type 1 DM patients in which no dominant V
usage could be seen (38). These differences could be
due to disease heterogeneity or differences in methodology. The
tentative hypothesis that the CVB4-induced V8 expansion we observe
reflects intrapancreatic events such as infection with pancreas-tropic
enteroviruses will therefore require further experimental
confirmation.
Other studies have examined V skewing of T cells in the peripheral
blood, although the approaches used are not strictly comparable to the
current work. TCR V7 chain expansion was found in a pilot study of
type 1 DM patients and first-degree relatives at risk of the disease,
and a temporal relationship between this observation and enterovirus
infection was suggested by the data (17). Our results show
that CD4 T cells with V7 chains are among the three V T cell
families most frequently activated by CVB4 in vitro.
In conclusion, our data show that CVB4 induces a skewed activation of
CD4 T cells, but only following processing and presentation of viral
Ags by APCs. This suggests that CVB4 is not capable of interacting with
T cells via superantigen effects. Additional studies will be required
to extend our preliminary finding that V selection by CVB4 may be
more frequent in patients with type 1 DM. The fact that some of the
V chains utilized by the virus have been detected in infiltrated
islets of Langerhans supports the proposal that enteroviruses such as
CVB4 may have a role in type 1 DM pathogenesis.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Mark Peakman, Department of Immunology, Guy’s, King’s and St. Thomas’ School of Medicine, Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, U.K. E-mail address: mark.peakman{at}kcl.ac.uk
3 Abbreviations used in this paper: DM, diabetes mellitus; CVB/CVB4, Coxsackievirus B/Coxsackievirus B4; TSST-1, toxic shock syndrome toxin-1; TT, tetanus toxoid; SEB, staphylococcal enterotoxin B; SI, stimulation index.
Received for publication April 9, 2001. Accepted for publication July 10, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene expression and enterovirus infection in type I diabetes: a pilot study. Diabetologia 43:1484.[Medline]
and chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc. Natl. Acad. Sci. USA 88:8987. usage, and cytokine profile. J. Immunol. 153:1360.[Abstract]
-chain clonotypes and interferon-
are expressed in the pancreas of patients with recent-onset insulin-dependent diabetes mellitus. Diabetes Res. Clin. Pract. 34:37.[Medline]This article has been cited by other articles:
|
|
 |
|
  R. Varela-Calvino, A. Skowera, S. Arif, and M. Peakman Identification of a Naturally Processed Cytotoxic CD8 T-Cell Epitope of Coxsackievirus B4, Presented by HLA-A2.1 and Located in the PEVKEK Region of the P2C Nonstructural Protein J. Virol., December 15, 2004; 78(24): 13399 - 13408. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Varela-Calvino, R. Ellis, G. Sgarbi, C. M. Dayan, and M. Peakman Characterization of the T-Cell Response to Coxsackievirus B4: Evidence That Effector Memory Cells Predominate in Patients With Type 1 Diabetes Diabetes, June 1, 2002; 51(6): 1745 - 1753. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Control, uninfected lysates; 
,
CVB4-infected lysates.
