Scand. J. Immunol. 45, 688–697, 1997
Circulating T Cells of Patients with Active Psoriasis Respond to
Streptococcal M-Peptides Sharing Sequences with Human
Epidermal Keratins
H. SIGMUNDSDOTTIR*, B. SIGURGEIRSSON†, M. TROYE-BLOMBERG‡, M. F. GOOD§,
H. VALDIMARSSON* & I. JONSDOTTIR*
Departments of *Immunology and †Dermatology, The National University Hospital, Reykjavik, Iceland; ‡Department of Immunology,
University of Stockholm, Sweden; and §Molecular Immunology Unit, Queensland Institute of Medical Research, Brisbane, Australia
(Received 4 December 1996; Accepted in revised form 5 February 1997)
Sigmundsdottir H, Sigurgeirsson B, Troye-Blomberg M, Good MF, Valdimarsson H, Jonsdottir I. Circulating
T Cells of Patients with Active Psoriasis Respond to Streptococcal M-Peptides Sharing Sequences with
Human Epidermal Keratins. Scand J Immunol 1997;45:688–697
Psoriasis is a T-cell mediated inflammatory skin disease which has been associated with group A, bhaemolytic streptococcal infections. Four 20 a.a. long M6-peptides sharing 5–6 a.a. sequences with human
epidermal keratins were identified. To investigate the role of potentially cross-reactive T cells in the
pathogenesis of psoriasis, interferon-g (IFN-g) and interleukin-4 (IL-4) responses of circulating T cells to
these peptides were analysed by ELISPOT and RT-PCR in 14 psoriatic patients, 12 healthy individuals and six
patients with atopic dermatitis (AD). Untreated psoriatic patients’ responses were significantly higher to these
peptides than healthy and AD controls, while responses to a control M6-peptide, not sharing sequences with
keratin, were negligible in all groups. No difference was found in response to streptokinase/streptodornase
(SK/SD). M6-protein and peptides exclusively elicited IFN-g production, with little IL-4 production, even in
AD patients. Interferon-g responses to all the M6-peptides were abolished after successful treatment of
psoriatic patients, but responses to SK/SD were unaffected. The results indicate that active psoriasis is
associated with Th1-like cells responding to streptococcal M6-peptides sharing sequences with human
epidermal keratin. This is consistent with the hypothesis that psoriasis may be induced and exacerbated in
susceptible individuals by M-protein specific Th1-like cells that cross-react with human epidermal keratin.
Ingileif Jonsdottir, Department of Immunology, National University Hospital, Landspitalinn, 101 Reykjavik,
Iceland
INTRODUCTION
Psoriasis is a T-cell mediated inflammatory skin disease which
affects about 2% of Caucasians. The characteristic skin lesion is
persistent, erythematous and scaly, reflecting infiltration of
inflammatory cells, increased proliferation and turnover of
keratinocytes. It has been reported that eruption of psoriatic
lesions coincides with epidermal influx and activation of CD4+ T
cells [1]. The infiltrates in the dermis and the deeper layers of
epidermis consist mostly of macrophages and T lymphocytes and
a large proportion of the T cells are activated [2, 3]. The evidence
that T cells play a key role in the pathogenesis of psoriasis [4] is
now compelling. Cyclosporin A [5] and anti-CD4 antibodies [6]
are very effective in clearing psoriatic lesions and there is no
688
evidence that antibodies or immune complexes contribute significantly to the pathogenic process. More recently, administration of low dose interleukin-2 (IL-2) toxin has been reported to
induce clinical improvement in psoriasis [7] and typical psoriatic
changes have been induced by injecting activated autologous
mononuclear cells into uninvolved psoriatic skin transplanted on
severe combined immune deficiency (SCID) mice [8].
Throat infections with group A, b-haemolytic streptococci
have been associated with onset of acute psoriasis and exacerbation of chronic psoriasis [9–11]. M-protein is a major virulence factor of b-haemolytic streptococci and forms fibrillar ahelical coiled-coil dimers protruding from the surface of the
bacteria [12]. Multiple serotypes of M-protein vary in the aminoterminal half and protective antibodies are serotype specific [13]
q 1997 Blackwell Science Ltd
Psoriatic T Cells Respond to M-Peptides Sharing Sequences with Keratin
and T cell epitopes are distributed throughout both aminoterminal and conserved carboxy-terminal regions of the Mprotein [14]. Cross-reaction between M-proteins and human
epidermal keratin has been postulated to be involved in the
pathogenesis of psoriasis [15, 16]. No particular M-protein
serotypes have been associated with psoriasis suggesting that
the conserved region of M-protein may be involved.
Keratin belongs to the family of coiled-coil proteins structurally related to streptococcal M-protein [12] and an extensive
amino acid sequence homology with 50 kDa (K14) type I human
epidermal keratin has been reported [15]. Increased expression of
this keratin has also been found in psoriatic patients [17–19], as
well as of K6, K16 and K17 [20]. Cross-reactions between
streptococcal M-protein and human keratin have been demonstrated with monoclonal antibodies [21]. Patients with acute and
chronic psoriasis showed increased proliferative responses to
streptococcal antigens [22] which were sensitive to trypsin,
indicating that M-protein might be involved [23], and increased
proliferative responses were also demonstrated with purified M5protein [24]. It has been possible to isolate T cell lines reacting
with streptococcal antigens from psoriatic lesions [25]. Similarly, cellular immune responses to group A streptococcal antigens play a major role in the pathogenesis of rheumatic fever
[26]. Increased proliferative responses to streptococcal antigens
have been demonstrated [27], as has infiltration of rheumatic
heart lesions by T cells [28]. The specificity of infiltrating T cells
in the heart is not known, but T cells responding to epitopes
shared between streptococcal M-protein and cardiac myosin
have been cloned. However, such T cells could be obtained
from control donors with no history of heart disease as well as
patients with rheumatic heart disease [29].
Human CD4+ T lymphocytes (Th cells) can be functionally
distinguished according to their cytokine secretion pattern. It has
been shown that Th1 cells, characterized by interferon-gamma
(IFN-g) production, are responsible for cell mediated immunity
and inflammatory responses, while Th2 cells, characterized by
IL-4, are involved in the switching of immunoglobulin M (IgM)
to IgE and are associated with allergic diseases [30]. In psoriatic
lesions, the Th1 type of cytokine pattern has been demonstrated
[31, 32], and accumulation of various cytokine-releasing T cell
subsets in psoriatic epidermis may regulate the inflammatory
process and keratinocyte hyperplasia [16]. T cell lines derived
from psoriatic lesions were found to produce predominantly Th1like cytokines [25], but a similar frequency of Th1- and Th2-like
clones, with the majority of clones producing low amounts of
both IFN-g and IL-4, has also been reported [33]. Several studies
suggest that IFN-g plays an important role in the pathogenesis of
psoriasis. Proliferative response of psoriatic keratinocytes to
IFN-g was decreased compared to normal keratinocytes in
vitro [34, 35] and expression of IFN-g receptors on keratinocytes
is decreased in psoriatic lesions [36]. Recently, a unique cytokine
pattern was described in psoriatic lesions [37] and both stimulatory and inhibitory activity of supernatants from lesional T cell
clones keratinocyte growth was dependent on IFN-g [38].
The principal objective of this project was to test the
689
hypothesis that psoriatic lesions are caused and maintained by
T lymphocytes that recognize amino acid sequences that are
common to streptococcal M-proteins and human epidermal
keratins [16]. To investigate the role of potentially cross-reactive
T cells, M6-peptides sharing amino acid sequences with human
epidermal keratins were identified and synthesized for analysis of
T cell responses in psoriatic patients, healthy controls and
patients with atopic dermatitis (AD). The nature of the responses
was determined by analysis of cytokine production.
MATERIALS AND METHODS
Study subjects. Fourteen untreated patients with plaque psoriasis were
recruited and with two exceptions healthy individuals paired for age and
sex were tested simultaneously. The severity of the disease was determined by the Psoriasis Area and Severity Index (PASI) [39] and ranged
from 4.0–49.7 (mean = 14.6) before treatment. For comparison six
untreated patients with atopic dermatitis were tested. Nine of the
psoriatic patients were tested again after treatment consisting of ultraviolet B (UVB) combined with bathing in a geothermal lagoon three
times per week for 6–8 weeks. This has shown to be an effective
treatment in psoriasis [40]. Their average PASI score was 17.3 (range
6.4–49.7) before treatment and 0.7 (range 0–2.9) after treatment. The
study was approved by the Ethical Committee of the National University
Hospital.
Antigens. We isolated M-protein from Streptococcus pyogenes serotype 6 (Public Health Laboratory Services, London, England) as
described by Pruksakorn et al. [41]. The bacteria were cultured on
blood agar, incubated in Todd-Hewitt broth (without salt) at 378C for
18 h, and washed with phosphate buffered saline (PBS) before boiling in
0.2 M HCl, pH 2. The pH was adjusted to pH 7, using NaOH, and the
bacteria removed by centrifugation. Sterility of the supernatant containing the M6-protein was checked by seeding on blood agar and purity of
the protein was tested using SDS and Western blotting. The M6-protein
was further purified using fast preparative liquid chromatography
(FPLC) on column MonoQ (Pharmacia AS, Copenhagen, DenmarkPharmacia) to a single component of < 60,000 Da. Human epidermal
keratin isolated from the calluses of the feet (Sigma, St Louis, MD, USA)
was dialyzed against PBS and kept frozen. Streptokinase/streptodornase
(SK/SD) was purchased from Behringwerke AG, Marburg, Germany.
M6-peptides:M6-protein sequences shared with epidermal keratin were
identified by multiple alignment construction and analysis workbench
(MACAW) program (NCBI, Bethesda, MD, USA). Four 20 a.a. long
M6-peptides containing shared sequences (peptides 145, 146, 149 and
150) and one control peptide not sharing sequences with keratins
(peptide 159) were synthesized by the ‘tea bag’ method [42] and
purity checked by high performance liquid chromatography (HPLC).
The sequences of the M6-peptides selected (Table 1) are identical to the
respective M5-peptides [41].
Isolation of T lymphocytes. Peripheral blood mononuclear cells
(PBMC) were isolated from 50 ml of heparinized blood by Ficoll–
Hypaque (Sigma USA) centrifugation [52]. Human monocytes were
isolated from PBMC by plastic adherence at 378C for 1 h in tissue culture
medium (TCM): RPMI-1640 (Gibco, BRL, Life Technologies, Paisley,
UK) with 2 mM glutamin (Gibco), 100 U/ml penicillin/100 mg streptomycin (Gibco) supplemented with 50% human AB serum. The monocytes were later used as antigen-presenting cells (APC). The PBMC were
incubated with carbonyl-iron at 378C for 30 min and neutrophiles
removed by magnetic attraction. The lymphocytes were washed and
q1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 45, 688–697
690 H. Sigmundsdottir et al.
resuspended in tissue culture media (TCM supplemented with 0.2%
human serum albumin (HSA) (Pharmacia AS). The T cells were isolated
by E-rosetting [53] of lymphocytes with sheep red blood cells (SRBC)
treated with neuraminidase (Sigma). Then they were separated from the
non-rosetting cells by Ficoll–Hypaque (Sigma) centrifugation and the
SRBC lysed with sterile, distilled H2O for 35 s. The T cells were washed
and adjusted to 1 × 106 cells/ml TCM with 10% heat-inactivated human
AB serum adding 5 × 104 autologous monocytes/ml (5%).
T cell stimulation. The T cells and 5% autologous monocytes were
stimulated with an optimal concentration of M6-protein (3 mg/ml),
human epidermal keratin (1 mg/ml) or SK/SD (20 U/ml) in 10 ml
tubes at 378C and humidified 5% CO2. Stimulation with M6-peptides
(2.5 mg/ml) was performed in the presence of a sub-threshold concentration (0.05 mg/ml) of phytohaemagglutinin (PHA: Glaxo Wellcome,
Middlesex, UK), which enhances the response of precommitted T
cells. At this concentration PHA did not stimulate T cells above background with TCM only. The synergistic effect of sub-threshold PHA has
been demonstrated in allergy to birch antigen [54].
Determination of IFN-g and IL-4 production. ELISPOT assay [55]
was used to determine the frequency of cytokine producing cells,
essentially as described elsewhere [54, 56]. The assays were optimized
using tetanus toxoid (10 mg/ml) and PHA (5 mg/ml) for T cell stimulation. Nitrocellulose-bottomed 96-well Millititer HA plates (Millipore
Co., Bedford, MA, USA) were coated with 15 mg/ml of monoclonal
antibodies to IFN-g (1-D1K) and IL-4 (82–4) (Mabtech AB, Stockholm,
Sweden) at 48C overnight and unbound antibodies removed. A total of
150,000 T cells prestimulated in tubes for 5 h were transferred in 150 ml
to each antibody-coated well and the ELISPOT plates were incubated at
378C and humidified in 5% CO2 for 3 days. The cells were removed and
biotin-conjugated monoclonal antibodies (Mabtech AB) to IFN-g or
IL-4 (1 mg/ml) were added. The plates were incubated for 3 h, followed
by incubation with strepavidin–alkaline phosphatase (Mabtech AB)
(1 mg/ml) for 1 h. Wells were washed with PBS between incubation
steps. The reaction was developed by BCIP/NBT (5-bromo-4-chloro-3indolyl phosphate/nitroblue tetrazolium) substrate solution (Bio-Rad
Laboratories, Hercules, CA, USA) and incubated until bluish spots
appeared (< 1 h) and the reaction stopped by washings with tap
water. The plates were dried and the number of blue spots in each
well counted using a dissection microscope. One spot represents cytokine production from one cell. Results are expressed as number of spots
per 150,000 T cells above background (< 10 spots/150,000 T cells)
cultures with TCM only or TCM + 0.05 mg/ml PHA for M6-peptide
stimulation. No difference was found between 12 paired cultures with
TCM + 0.05 mg/ml PHA and TCM only (P = 0.7). Positive Th1-like
response was arbitrarily defined as > 10 IFN-g spots/150,000 T cells,
above background.
RNA extraction. A total of 106 T cells were removed from cultures
after stimulation for 4, 7 and 20 h and washed three times with
diethylpyrocarbonate (DEPC)-treated PBS (0.1% DEPC). All fluid was
removed and the pellet evaporated and kept at –708C until RNA
extraction [57]. Briefly, cells (1–3×106) were lysed in solution D (4 M
guanidinum thiocyanate, 25 mM sodium citrate pH 7, 0.5% sarcosyl,
0.1 M 2-mercaptoetanol) and 2 M sodium acetate NaAc (pH 4), with
phenol and chloroform:isoamylalcohol [49:1] added sequentially, vortexed for 2 min, centrifugated at 10,000 g for 20 min at 48C, and the
water phase transferred to new tubes. One volume of isopropanol was
added and RNA precipitated at –208C for at least 1 h before
centrifugation at 10,000 g for 20 min at 48C. The pellet was again
lysed in solution D and the precipitation repeated once before the RNA
was resuspended in 75% EtOH and centrifugated at 10,000 g for 15 min
at 48C. It was then evaporated in a vacuum desiccator and redissolved in
distilled water. To prevent RNAse activity, RNAse inhibitor (Scandinavian Diagnostic Services, Falkenberg, Sweden) was added to the RNA
and the amount of total RNA was determined photochemically at
260 nm. Extracted RNA was used for polymerase chain reaction
(PCR) amplification.
Determination of IFN-g and IL-4 gene expression. The RT-PCR
amplifications of cytokine mRNA were performed with the Gene Amp
PCR kit (Perkin Elmer Cetus, Norwalk, CT, USA) as described by the
manufacturer. Total RNA (300 ng) was reverse transcribed in 20 ml
reactions containing 5 U rTth DNA polymerase, 0.5 mM Tris-HCl (pH
8.3), 4.5 mM KCl, 1.0 mM MnCl2, 800 mM dioxynucleotide triphosphate
(dNTP) and 75 pM of the respective 30 primers. Reverse transcription was
performed at 55–608C for 10–15 min (depending on the primers). Then
PCR was carried out in a buffer supplemented with 25 mM MgCl2 and
75 pM of the respective 50 primers. The following primers were used:
IFN-g (355 bp), 50 – AGT-TAT-ATC-TTG-GCT-TTT-CA and 30 –
ACC-GAA-TAA-TTA-GTC-ACC-TT; IL-4 (317 bp), 50 – CTT-CCCCCT-CTG-TTC-TTC-CT and 30 – TTC-CTG-TCG-AGC-CGT-TTCAG. Samples were amplified (38 cycles) in a DNA Thermo Cycler 2400
(Perkin Elmer) and the PCR products were electrophoresed on 1.5%
agarose gels, visualized by ethidium bromide and photodocumented.
Statistical analysis. Mann–Whitney Rank Sum Test was used to
compare the T cell responses between psoriatic patients and healthy
controls or AD patients. Kruskal–Wallis was used to compare psoriatic
patients with both control groups. Responses of psoriatic patients before
and after treatment were compared by paired t-test. Frequency of
psoriatic patients and healthy controls responding to one or more of
the M6-peptides was compared with Fisher’s exact test. Pearson’s
Correlation was used to analyze the relationship between responses of
individual patients to different stimuli and to PASI score.
RESULTS
T cell responses to streptococcal M6-protein, human epidermal
keratin and SK/SD
In all subjects tested the response to streptococcal M6-protein,
human epidermal keratin and SK/SD was predominantly of the
Th1 type characterized by IFN-g production (Fig. 1). Untreated
psoriatic patients showed a higher frequency of IFN-g producing
cells responding to M6-protein than the control groups, but the
difference was not significant (P = 0.054). The PASI scores [39]
of untreated psoriatic patients did not correlate with their
responses to the M6-protein. There was no difference between
psoriatic patients and control groups in IFN-g response to keratin
or the control antigen SK/SD, which elicited much stronger
responses than the other antigens. Interleukin-4 production was
absent or low (1–6 IL-4+/150,000 T cells in 18 of 123 cultures, or
14.6%) in response to all the antigens and no difference was
found between the groups.
T cell responses to streptococcal M6-peptides
To investigate whether M6-protein reactive T cells of psoriatic
patients were potentially cross-reactive with human epidermal
keratins, peripheral blood T cells were stimulated with
q1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 45, 688–697
Psoriatic T Cells Respond to M-Peptides Sharing Sequences with Keratin
Fig. 1. T cell responses to streptococcal M6-protein, keratin and
SK/SD. The number of IFN-g producing cells per 150,000 T cells is
shown for healthy controls (O), patients with psoriasis (A) or atopic
dermatitis (D) evaluated by ELISPOT. T cells with 5% monocytes
were stimulated for 3 days with M6-protein (3 mg/ml) and keratin
(1 mg/ml) as shown on the Y-axis, left; and SK/SD (20 U/ml) as
shown on the Y-axis, right. Responses to each antigen were compared
between the groups by Mann–Whitney rank sum test: * P < 0.05,
** P < 0.01, *** P < 0.001.
M6-peptides sharing sequences with keratins. The amino acid
sequences of streptococcal M6-protein and published sequences
of human epidermal keratins were compared using the MACAW
program. Three shared sequences of 5–6 amino acids were
identified and four 20 amino acid M6-peptides containing these
sequences and one control M6-peptide without shared sequences
were synthesized. These M6-peptide sequences [29] are shown in
Table 1 along with the keratin types sharing sequences with each
of the peptides.
The T cell responses to all M6-peptides were predominantly of
the Th1 type with IFN-g production in patients with psoriasis,
atopic dermatitis and healthy controls, while IL-4 production was
absent or minimal (1–6 IL-4+/150,000 T cells in 21 of 165
cultures, or 12.7%) in all groups. Although the IFN-g response to
the different M6-peptides varied, the psoriatic patients showed
higher responses to the peptides sharing sequences with keratin
than the healthy controls and AD patients (Fig. 2). This difference between psoriasis patients and the control groups was
significant for peptides 145 (P < 0.01), 146 (P < 0.0001) and
150 (P < 0.001). The M6-peptide 146, which shares six a.a. with
several keratins (Table 1) was the peptide that most frequently
induced response in psoriatic patients. Also, five out of 14
psoriatic patients showed the highest response to this peptide,
while none of the controls showed any response (Fig. 2). Two of
the M6-peptides, 149 and 150, are overlapping, sharing the same
sequence in different locations, and the responses of psoriatic
patients to these peptides were usually concordant. It is also
worth noting that the psoriatic patients who expressed the
strongest response to peptide 149 showed the highest response
Table 1. Sequences of streptococcal M6-peptides shared with keratins
M6-peptide sequencesa
Keratins
References for
keratin sequences
LRRDLDASREAKKQVEKALE
K10
K13
K14
K17
K19
43
44
45
46
47
146
AKKQVEKALEEANSKLAALE
K9
K13
K14
K15
K16
K17
K19
48
44
45
49
50
46
47
149
KLTEKEKAELQAKLEAEAKA
K18
51
150
QAKLEAEAKALKEQLAKQAE
K18
51
159
MATAGVAAVVKRKEEN
—
M6-peptide
145
a
691
Ref. 29.
q1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 45, 688–697
692 H. Sigmundsdottir et al.
Fig. 2. T cell responses to streptococcal M6-peptides. The number of IFN-g producing cells per 150 000 T cells is shown for healthy controls
(O), patients with psoriasis (A) or atopic dermatitis (D) evaluated by ELISPOT after stimulation with M6-peptides 145, 146, 149 and 150 sharing
sequences with keratins and the control peptide 159. T cells and 5% monocytes were cultured for 3 days with the M6-peptides at 2.5 mg/ml in the
presence of sub-threshold PHA (0.05 mg/ml). Responses to each antigen were compared between the groups by Mann–Whitney rank sum test:
* P < 0.05, ** P < 0.01, *** P < 0.001.
to keratin. Only two of the healthy controls responded weakly to
the M6-peptide 149 and one to M6-peptide 150, but the AD
patients did not respond to any of the peptides with either IFN-g
production (Fig. 2 and Table 2) or IL-4 production. The IFN-g
response to the control M6-peptide 159 was low in all groups
(Fig. 2).
The majority of the psoriatic patients responded to at least two
peptides (Table 2), but neither the number of peptides eliciting
Table 2. Frequency of individuals responsing to M6-peptides
Positive responsea to one or more peptides
Healthy controls
Untreated patients with
psoriasis
Patients with psoriasis
after treatment
Untreated patients with
atopic dermatitis
a
$1
peptide
$2
peptides
$3
peptides
4
peptides
2/12
17%
12/14**
86%
3/9
33%
0/6
0%
1/12
8%
8/14*
57%
2/9
22%
0/6
0%
0/12
0%
6/14*
43%
1/9
11%
0/6
0%
0/12
0%
3/14
21%
0/9
0%
0/6
0%
response nor the frequency of responding T cells correlated with
the PASI score (data not shown). However, the number of M6peptides eliciting a positive response in each psoriatic patient
correlated with the frequency of IFN-g producing cells to M6protein (r = 0.59, P = 0.026) and keratin (r = 0.75, P = 0.002).
We also tested proliferative responses in four psoriatic patients
and paired healthy controls. The proliferative responses were
low, but in agreement with IFN-g production the psoriatic
patients responded better to the M6-peptides (stimulation index
or SI range: 0.2–3.7) than the controls (SI range: 0.5–2.1). The
difference was most pronounced for peptides 145 (mean SI: 1.8
and 1.3, respectively) and 146 (mean SI: 1.6 and 1.1). No
difference was observed in response to M6-protein (mean SI:
0.8 and 0.7, respectively).
Taken together, the results indicate that in active psoriasis the
frequency of circulating Th1-like cells recognizing streptococcal
M-peptides sharing sequences with human keratins may be
increased.
Expression of IFN-g and IL-4 genes in T cells stimulated with
streptococcal M6-peptides and keratin
Positive response is arbitrarily defined as >10 IFN-g producing cells
per 150,000 T cells. Comparison of untreated patients with psoriasis and
healthy controls by Fisher’s exact test: P < 0:05 (*) and P < 0:01 (**).
In eight psoriatic patients IFN-g and IL-4 gene expression was
also measured by RT-PCR in cultures stimulated with keratin or
M6-peptides. No IL-4 mRNA expression was detected in
response to any of the antigens (data not shown) which is in
agreement with results obtained by ELISPOT, as IL-4 producing
cells were absent or very few. Generally high expression of
IFN-g mRNA was detected in samples from psoriatic patients
q1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 45, 688–697
Psoriatic T Cells Respond to M-Peptides Sharing Sequences with Keratin
693
Table 3. Comparison of IFN-g production before treatment measured by ELISPOT and RT-PCR
Keratin
PS1
PS2
PS5
PS6
PS7
PS8
PS10
PS12
Peptide 145
ELISPOT
PCR
ELISPOT
28.5
54.5
0
14
0
0
0
2.5
++
++
++
ND
++
+
–
ND
15
18.5
0
11.5
6.5
1
12.5
0
Peptide 146
Peptide 149
Peptide 150
Peptide 159
Stimulation
without antigen
PCR ELISPOT
PCR
ELISPOT
PCR
ELISPOT
PCR
ELISPOT
PCR
ELISPOT
PCR
+++
+
++
++
+
–
+
–
+++
+
+++
+++
+
++
–
+
38.5
34
0
19
0
1.5
4
0
++
+
++
+++
–
+
+
–
14.5
21
0
39.5
21
17
5
12
++
++
–
–
+
+
+
+
ND
ND
ND
ND
ND
ND
6.5
0.5
ND
ND
ND
ND
ND
ND
–
–
2.5
2.5
51
9
4
4
2.5
9
ND
+
+
–
ND
ND
–
ND
31
21
77.5
56.5
1
33
3
7.5
IFN-g production of psoriatic patients (PS). T cells with 5% monocytes were stimulated for 3 days with 1 mg/ml of keratin, or 2.5 mg/ml of M6peptides 145, 146, 149 and 150 sharing sequences with keratins or the control peptide 159 in the presence of sub-threshold PHA (0.05 mg/ml).
ELISPOT results are expressed as the number of IFN-g producing cells per 150,000 T cells, after subtraction of background stimulation without
antigen. RNA extraction and RT-PCR were performed after culturing for 4, 7 and 20 h. Bands were graded visually. Results for background stimulation
is shown in the last two columns. ND ¼ not done.
with a high frequency of IFN-g producing cells and weak
expression was observed where the frequency of IFN-g producing cells was low, both in eight psoriatic patients (Table 3) and
five healthy controls (data not shown). The findings were discordant in only one patient instance (PS5) where IFN-g mRNA
expression was strong but the frequency of IFN-g producing cells
low. This individual showed spontaneous IFN-g production
(background) in cultures without antigen detected both in RTPCR and ELISPOT. Thus, consistent results were obtained with
RT-PCR and ELISPOT, demonstrating the predominance of
Th1-like cell responding to streptococcal M-peptides in active
psoriasis.
Effect of treatment on T cell responses of psoriatic patients to
streptococcal M6-peptides, M6-protein, keratin and SK/SD
Nine psoriatic patients were treated by UVB combined with
bathing in a geothermal lagoon for 6–8 weeks until clinical
remission was achieved. Their mean PASI score was 17.3 (range
6.4–49.7) before treatment, compared to 0.7 (range 0–2.9) after
treatment. The T cell responses of these patients were tested
before and at the end of the treatment.
After treatment the psoriasis patients showed markedly
reduced IFN-g production in response to all the M6-peptides
sharing sequences with keratin and this was significant for
peptides 146 and 150 (P < 0.01). Thus, when clinical remission
was achieved, most of the patients had become unresponsive to
the M6-peptides (Fig. 3), and their responses did not differ from
the controls in terms of frequency of IFN-g producing cells
responding to the M6-peptides (Figs 2 and 3) or in the number of
M6-peptides eliciting a positive response (Table 2). It is worth
noting that one patient with severe psoriasis (PASI = 26.2)
responded only to peptide 146 before treatment (77 spots/
150,000 T cells), but in spite of rapid clinical improvement
this patient still showed a fairly strong response to peptide 146
(27 spots/150,000 T cells) after the treatment (PASI = 0) and had
acquired responses to peptides 145 and 149 (32.5 and 13.5 spots/
150,000 T cells, respectively: see Fig. 3). Interestingly, this
patient relapsed within 1 week while none of the others relapsed
during an observation time of 4 weeks. After treatment only two
patients showed a marked decrease in response to M6-protein
and one to keratin and no difference was observed in response to
SK/SD before and after treatment in seven of the nine psoriatic
patients tested (Fig. 4). Six of the healthy controls were tested
again together with the psoriatic patients after treatment and their
responses to the M6-protein, keratin and M6-peptides were
consistently low or absent (data not shown).
DISCUSSION
In the majority of patients with active psoriasis, Th1 type
responses to one or more of the M6-peptides 145, 146, 149 and
150, which share 5–6 a.a. with human epidermal keratins, were
demonstrated and these T cells disappeared from the blood
during clinical remission, while responses of healthy controls
and AD patients were low or absent. These peptides are from the
conserved carboxy-terminal region of the M-protein. Strikingly,
peptide 146, with the longest shared sequence, was most
frequently and strongly recognized by the T cells from the
psoriatic patients while none of the controls responded to this
peptide. In agreement with these findings, peptides 145 and 146
were found to be immunodominant in individuals with a
history of rheumatic fever or frequent exposure to group A
streptococcal infections and cross-reaction of T cell lines specific
for these peptides with homologous myosin peptides has been
demonstrated [29].
q1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 45, 688–697
694 H. Sigmundsdottir et al.
Fig. 3. T cell responses of psoriatic patients to streptococcal M6peptides before and after treatment. The number of IFN-g producing
cells per 150,000 T cells is shown for psoriatic patients before and
after treatment evaluated by ELISPOT after stimulation with M6peptides 145, 146, 149 and 150. T cells and 5% monocytes were
cultured for 3 days with the M6-peptides at 2.5 mg/ml in the presence
of sub-threshold PHA (0.05 mg/ml). Statistical analysis was performed
with the paired t-test.
Streptococcal infections are common and it is therefore not
unexpected that adults have circulating T cells that can respond
to epitopes on streptococcal M6-proteins. Although the psoriatic
patients tended to respond more frequently and strongly to the
M6-protein and keratin than the healthy controls, the differences
were not significant. However in the control groups, responses to
the M6-peptides which share sequences with keratins were
absent or minimal.
In psoriatic patients there was a relationship between the
intensity of the T cell responses to M6-protein, keratin and
M6-peptides eliciting a positive response. However, patients
responding strongly to one or more of the M6-peptides did not
always respond well to the M6-protein. One possible explanation
may be that processing of the M6-protein may not always result
in effective presentation or a sufficient number of T cell epitopes
[58]. It is not known whether such peptides are presented in vivo
and the importance of human leucocyte antigen (HLA) genotypes for presentation of the M6-peptides remains to be studied.
However, the clear difference between psoriatic patients and
controls in the response to the M6-peptides and the marked
decrease associated with clinical remission indicates that T cell
Fig. 4. T cell responses of psoriatic patients to M6-protein, keratin
and SK/SD before and after treatment. The number of IFN-g
producing cells per 150,000 T cells was evaluated by ELISPOT. T
cells with 5% monocytes were stimulated for 3 days with M6-protein
(3 mg/ml) and keratin (1 mg/ml), shown by the Y-axis, left; and SK/SD
(20 U/ml), shown by the Y-axis, right. Statistical analysis was
performed with the paired t-test.
responses to these M6-protein sequences are relevant for the
pathogenesis of psoriasis. Some patients responded well to M6peptides without responding to keratin. The content of different
keratins in the keratin preparation used is not known and keratins
containing the shared sequences may not be processed and
presented in sufficient density to stimulate cross-reactive T
cells. Peptides 145 and 146 share sequences with K14, K16
and K17 (Table 1) which are present in low amounts in normal
skin. In psoriasis, increased expression of K14 (50 kDa) [17–19],
K17 and the hyperproliferative keratins K6 and K16 [20] has
been demonstrated.
Atopic dermatitis is an inflammatory skin disease associated
with infiltration of T cells without keratinocyte hyperproliferation and has, in contrast to psoriasis, been considered to be a Th2
type of disorder. The difference in Th1/Th2 profiles does not
explain the lack of response in AD, as none of the AD patients
responded with IL-4 production to any of the M6-peptides.
Similar frequencies of Th1- and Th2-like cells have been
found in atopic skin although Th0-like cells were predominating
[59]. It has also been suggested that Th2-like cytokines are
crucial for the initiation of AD while the expression of Th1like cytokines may be critical for the progressive chronic course
of the disease since IFN-g was down-regulated after successful
therapy [60].
It has previously been shown that UVB combined with bathing
in a geothermal lagoon is an effective treatment for psoriasis [40]
and all the patients participating in this study had clinical
remission after 6–8 weeks. This improvement coincided with
q1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 45, 688–697
Psoriatic T Cells Respond to M-Peptides Sharing Sequences with Keratin
marked decreases in T cell responses to the M6-protein and in
particular to M6-peptides sharing sequences with keratin. Interestingly, the only patient who after treatment still showed strong
responses to the M6-peptide 146 and had acquired responses to
the M6-peptides 145 and 149, relapsed within 1 week while the
improvement was sustained for at least 4 weeks in all the other
patients. This suggests that the presence of circulating T cells
specific for M6-peptides sharing sequences with keratin may
precede or coincide with the appearance of psoriatic lesions. It is
interesting that the T cell response to SK/SD was not affected by
the treatment indicating that it did not induce non-specific
suppression. This antigen was used as a positive control because
it is derived from streptococci, but in contrast to M-peptide
reactive T cells, it was considered unlikely that SK/SD specific
cells would be preferentially recruited to skin lesions in patients
with chronic psoriasis. Previous studies have shown that activated T cells disappear from the epidermis and dermis during
spontaneous or treatment induced resolution of skin lesions [61,
62]. It has recently been confirmed that T cells disappear from
the epidermis of psoriasis patients during UVB treatment and
furthermore reported that T cells are tenfold more sensitive to
apoptotic effects of UVB than keratinocytes [63]. The M6peptide specific T cells may therefore have disappeared from
the blood as a result of anergy or apoptosis in the skin. It is
unlikely however, that this reflects a permanent unresponsiveness, as most psoriatic patients eventually relapse after successful
treatment. It has been suggested that clearance of autoimmune
inflammation in the nervous system may be accomplished by
apoptosis of antigen specific T cells within the lesions [64, 65].
Previous studies on responses of psoriatic patients to Mproteins have mostly been based on proliferation to crude
streptococcal antigen preparations [22, 23]. A higher proliferative response to purified M5-protein in psoriatic patients compared to normal subjects has been reported but the level of
response was low [24], which is in agreement with our findings.
It has been demonstrated that discordant responses may be
observed by proliferation and IFN-g production, indicating that
the two assays may measure primed T cells belonging to different
subsets [66]. In the present study the intensity and the nature of T
cell responses was evaluated by cytokine production. The frequency of T cells responding to individual 20 a.a. long M6peptides was expected to be low resulting in low proliferation;
thus the sensitivity and easy quantitation of ELISPOT was an
advantage. The sensitivity of the technique was increased by
testing the responses to the M6-peptides in the presence of subthreshold concentrations of PHA, which did not increase the
background but enhances the response of precommitted T cells.
Strong expression of IFN-g mRNA detected by RT-PCR closely
paralleled a high frequency of IFN-g producing cells enumerated
by ELISPOT while no IL-4 mRNA expression was detected,
which is consistent with the lack of IL-4 producing cells. Thus,
results obtained by these two complementary methods suggested
that peripheral T cell responses to streptococcal M6-protein and
its peptides were restricted to Th1-like cells. Whether they exhibit the unique cytokine-pattern recently described in psoriatic
695
lesions [36] can be evaluated by RT-PCR analysis of additional
cytokines in our samples.
We have postulated that in streptococcal infection superantigens may stimulate T cells to home to the skin and M-protein
specific T cells thus recruited may be maintained in the epidermis
due to the presence of cross-reactive epitopes [16]. Superantigens have recently been shown to induce a selective expression of the skin homing receptor cutaneous-lymphocyte
associated antigen, CLA [67]. Furthermore, superantigen activated mononuclear cells have been reported to induce psoriatic
change in skin grafts on SCID mice [8]. In this study a strong
association of active psoriasis with increased frequency of
circulating Th1-like cells specific for M6-peptides sharing
sequences with keratin has been demonstrated. This is consistent
with the hypothesis that M-protein specific Th1-like cells crossreacting with human epidermal keratins play an important role in
the pathogenesis of psoriasis. Although we cannot exclude the
possibility that T cell responses to the M6-peptides sharing
sequences with keratins are fortuitous, it is well documented
that streptococcal infections often precede initiation of guttate
psoriasis and worsening of plaque type psoriasis [9–11]. Also the
patients enrolled in the study were not selected because of a
history of recent or recurrent streptococcal infections, only
because they were untreated and had an active disease. Whether
T cells responding to streptococcal M-proteins and/or keratins
are directly pathogenic is difficult to prove as appropriate animal
models are lacking and the presence of such potentially crossreactive T cells in the skin of psoriatic patients is still to be
demonstrated.
ACKNOWLEDGMENTS
The authors acknowledge the help of Bergljot Magnadottir and
Dr Sigurdur Magnusson with purification and analysis of the M6protein.
This work was supported by The Research Funds of University
of Iceland, the National University Hospital and The Icelandic
Research Council. Hekla Sigmundsdottir was the recipient of a
grant from The Icelandic Research Fund for Graduate Students.
REFERENCES
1 Baker BS, Swain AF, Valdimarsson H, Fry L. T-cell subpopulations
in the blood and skin of patients with psoriasis. Br J Dermatol
1984;110:37–44.
2 Baker BS, Swain AF, Fry L, Valdimarsson H. Epidermal Tlymphocytes and HLA-DR expression in psoriasis. Br J Dermatol
1984;110:555–64.
3 Bjerke JR, Krough HK, Matre R. Characterisation of mononuclear
cell infiltrate in psoriatic lesions. J Invest Dermatol 1978;71:340.
4 Valdimarsson H, Baker BS, Jónsdóttir I, Fry L. Psoriasis: a disease of
abnormal proliferation induced by T lymphocytes. Immunol Today
1986;7:256–9.
5 Griffiths CEM, Powles AV, Leonard JN, Fry L, Baker BS, Valdimarsson H. Clearance of psoriasis with low dose cyclosporin. Br
Med J 1986;293:731–2.
q1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 45, 688–697
696 H. Sigmundsdottir et al.
6 Morel P, Revillard J-P, Nicolas J-F, Wijdenes J, Rizova H, Thivolet
J. Anti-CD4 monoclonal antibody therapy in severe psoriasis. J
Autoimmunity 1992;5:465–77.
7 Wrone-Smith T, Nickoloff BJ. Dermal injection of immunocytes
induces psoriasis. J Clin Invest 1996;98:1878–87.
8 Gottlieb SL, Gilleaudeal P, Johnson R et al. Response of psoriasis to
a lymphocyte-selective toxin (DAB389IL-2) suggests a primary
immune, but not keratinocyte, pathogenesis basis. Nature Medicine
1996;1:442–7.
9 Whyte HJ, Baughman RD. Acute guttate psoriasis in streptococcal
infections. Arch Dermatol 1964;89:350–6.
10 Norrlind R. Significance of infection in origin of psoriasis. Acta
Rheumatol Scand 1955;1:135–44.
11 Telfer NR, Chalmers RJG, Whale K, Colman G. The role of
streptococcal infection in the initiation of guttate psoriasis. Arch
Dermatol 1992;128:39–42.
12 Manjula BN, Trus BL, Fishetti VA. Presence of the distinct regions
in the coiled-coil structure of the streptococcal PepM5 protein:
Relationship to mammalian coiled-coil proteins and implications to
its biological properties. Proc Natl Acad Sci USA 1985;82: 1064–8.
13 Kehoe MA. Group A streptococcal antigens and vaccine potential.
Vaccine 1991;9:797–806.
14 Robinson JH, Atherton MC, Goddacre JA, Pinkney M, Weightman
H, Kehoe MA. Mapping T-cell epitopes in group A streptococcal
type 5 M-protein. Infect Immun 1991;59:4324–31.
15 McFadden J, Valdimarsson H, Fry L. Cross-reactivity between
streptococcal M surface antigen and human skin. British Journal of
Dermatology 1991;125:443–7.
16 Valdimarsson H, Baker BS, Jónsdóttir I, Powles AP, Fry L. Psoriasis
—a T-cell mediated autoimmune disease induced by streptococcal
superantigens? Immunol Today 1995;16:145–9.
17 Aoki S, Yaoita H, Kitajima Y. An elevated level of autoantibodies
against 48- to 50- kd keratins in the serum of patients with psoriasis. J
Invest Dermatol 1989;92:179–83.
18 Wongwaisayawan H, Yoshiike T, Aikawa Y, Briggaman RA, Ogawa
H. Antikeratin 14 monoclonal antibody staining in psoriasis and
seborrhoeic keratosis: immunofluorescence and two colour FACS
studies. Arch Dermatol Res 1991;283:405–10.
19 McGuire J, Osber M, Lightfoot L. Two keratins MW 50.000 and
56.000 are synthesized by psoriatic epidermis. Br J Dermatol
1984;111:27–37.
20 Leigh IM, Navsaria H, Purkis PE, Bowden PE, Riddle PN. Keratins
(K16 and K17) as markers of keratinocyte hyperproliferation in
psoriasis in vivo and in vitro. Br J Dermatol 1995;133:501–11.
21 Swerlick RA, Cunningham MW, Hall NK. Monoclonal antibodies
cross-reactive with group A streptococci and normal and psoriatic
skin. J Invest Dermatol 1986;87:367–71.
22 Baker BS, Powles AV, Malkani AK, Lewis H, Valdimarsson H, Fry
L. Altered cell-mediated immunity to group A haemolytic streptococcal antigens in chronic plaque psoriasis. Br J Immunol
1991;125:38–42.
23 Baker BS, Bokth SB, Garioch JJ et al. Decreased T cell reactivity to
trypsinized group A, type 22 streptococci in psoriasis. Acta Derm
Venerol 1994;74:276–8.
24 McFadden JP, Quinn DG, Camp RDR. In vitro lymphocyte response
to streptococcal M protein in psoriasis. Biochem Soc Trans
1991;19:11S.
25 Baker BS, Bokth S, Powles A et al. Group A streptococcal antigenspecific T-lymphocytes in guttate psoriatic lesions. Br J Dermatol
1993;128:493–9.
26 Carapetis JR, Currie BJ, Good MF. Towards understanding the
pathogenesis of rheumatic fever. Scand J Rheumatol 1996;25:127–
31.
27 Read SW, Zabrisky JB, Fischetti V, Utermohlen V, Falk R. Cellular
reactivity studies to streptococcal antigens. Migration inhibition
studies in patients with streptococcal infections and rheumatic
fever. J Clin Invest 1974;54:439.
28 Kemeny E, Grieve T, Marcus R, Sareli P, Zabriesky JB. Identification of mononuclear cells and T-cell subsets in rheumatic valvulitis.
Immunol Immunopathol 1989;52:225.
29 Pruksakorn S, Currie B, Brandt E et al. Identification of T-cell
autoepitopes that cross-react with the carboxyterminal segment of
the M protein of group A streptococci. Int Immunol 1994;6:1235–44.
30 Romagnani S. Lymphokine production by human T cells in disease
stages. Annu Rev Immunol 1994;12:227–57.
31 Schlaak JF, Buslau M, Jochum W et al. T cells involved in psoriasis
vulgaris belong to the Th1 subset. J Invest Dermatol 1994;102:
145–9.
32 Uyemura K, Yamamura M, Fivenson DF, Modlin RL, Nickoloff BJ.
The cytokine network in lesional and lesional-free psoriatic skin is
characterized by a T-helper type 1 cell-mediated response. J Invest
Dermatol 1993;101:701–5.
33 Barna M, Snidjewint FGM, van der Heijden FL, Bos JD, Kapsenberg
ML. Characterization of lesional psoriatic skin T lymphocyte clones.
Acta Derm Venereol 1991;186:9–11.
34 Baker BS, Powles AV, Valdimarsson H, Fry L. An altered response
by psoriatic keratinocytes to gamma interferon. Scand J Immunol
1988;28:735–40.
35 Nickoloff BJ, Mitra RS, Elder RT, Ficher GJ, Voorhees JJ.
Decreased growth inhibition by recombinant transforming growth
factor-a in keratinocytes cultured from psoriatic lesions. Br J
Dermatol 1989;121:161–74.
36 Scheynius A, Fransson J, Johansson C et al. Expression of interferon-gamma receptors in normal and psoriatic skin. J Invest
Dermatol 1992;98:255–8.
37 Vollmer S, Mensen A, Trommler P, Schendel D, Prinz JC. Tlymphocytes derived from skin-lesions of patients with psoriatic
vulgaris express a novel cytokine pattern that is distinct from that of
T helper type 1 and T helper type 2 cells. Eur J Immunol
1994;24:2377–83.
38 Prinz JC, Gross B, Vollmer S et al. T cell clones from psoriasis skin
lesions can promote keratinocyte proliferation in vitro via secreted
products. Eur J Immunol 1994;24:593–8.
39 Frederikson T, Petterson U. Severe psoriasis-oral therapy with a new
retinoid. Dermalogica 1978;157:238–44.
40 Olafsson JH, Sigurgeirsson B, Palsdottir R. The effect bathing in a
thermal lagoon in Iceland has on psoriasis. J Eur Acad Dermatol
Venerol 1994;3:460–4.
41 Pruksakorn S, Galbraith A, Houghton RA, Good MF. Conserved T
and B cell epitopes on the M-protein of group A streptococci. J
Immunol 1992;148:2729–35.
42 Houghten RA. General method for rapid solid-phase synthesis of
large numbers of peptides: specificity of antigen–antibody interaction at the level of individual amino acids. Proc Natl Acad Sci
USA 1988;82:5131–5.
43 Rieger M, Franke WW. Identification of an orthologous mammalian
cytokeratin gene. High degree in intron sequence conservation during
evolution of human cytokeratin 10. J Mol Biol 1988;204:841–56.
44 Kuruc N, Leube RE, Moll I, Bader BL, Franke WW. Synthesis of
cytokeratin 13, a component characteristic of internal stratified
q1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 45, 688–697
Psoriatic T Cells Respond to M-Peptides Sharing Sequences with Keratin
45
46
47
48
49
50
51
52
53
54
55
56
epithelia, is not induced in human epidermal tumors. Differentiation
1989;42:111–23.
Marchuk D, McCronon S, Fuchs E. Remarkable conservation of
structure among intermediate filament genes. Cell 1984;39:491–8.
Troyanovsky SM, Leube RE, Franke WW. Characterization of the
human gene encoding cytokeratin 17 and its expression pattern. Eur J
Cell Biol 1992;59:127–37.
Rasmussen HH, van Damme J, Puype M, Gesser B, Celis JE,
Vandekerchove J. Microsequence of 145 protein recorded in the
two-dimensional gel protein database of normal human epidermal
keratinocytes. Electrophoresis 1992;13:960–9.
Langbein L, Heid HW, Moll I, Franke WW. Molecular characterization of the body-site specific human epidermal cytokeratin 9: cDNA
cloning amino acid sequences, and tissue specificity of gene expression. Differentiation 1993;55:57–72.
Leube RE, Bader BL, Nosch FX, Zimbelmann R, Achtstaetter T,
Franke WW. Molecular expression of the stratification-related cytokeratins 4 and 15. J Cell Biol 1991;106:1249–61.
Rosenberg M, RayChaudhury A, Shows TB, Le Beau MM, Fuchs E.
A group of type 1 keratin genes on human chromosome 17;
characterization and expression. Mol Cell Biol 1988;8:722–36.
Kulesh DA, Oshima RG. Complete structure of the gene for human
keratin 18. Mol Cell Biol 1988;8:1540–50.
Perlmann H, Perlmann P, Pape GR, Hallden G. Purification and
assay of antibody dependent lymphocytic effector cells (K-cells) in
human blood. Scand J Immunol 1976;5:57–63.
Jonsdottir I, Dillner-Centerlind ML, Perlmann H, Perlmann P. Antibody dependent cellular cytotoxicity and mitogen responsiveness of
human peripheral blood lymphocytes differing in avidity for sheep
erythrocytes. Scand J Immunol 1979;10:525–33.
Gabrielsson S, Paulis S, Rak S et al. Specific induction of interleukin-4-producing cells in response to in vitro allergen stimulation
in atopic individuals. Clin Exp Allergy 1997 (in press).
Czerkinsky C, Anderson G, Ekre HP, Nilson LA, Klareskog L,
Ouchterlony Ö. Reverse ELISPOT assay for clonal analyses of
cytokine production. J Immunol Methods 1988;110:29–36.
Kabilan L, Anferson G, Lolli F, Ekre H, Troye-Blomberg M.
57
58
59
60
61
62
63
64
65
66
67
q1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 45, 688–697
697
Detection of intracellular expression and secretion of IFNg at the
single cell level after activation of human T-cells with tetanus toxoid
in vitro. Eur J Immunol 1990;20:1085–9.
Chomczynski P, Sacchi N. Single-step method of RNA isolation by
acid guanidinium thiocyanate-phenol-chloroform extraction. Anal
Biochem 1987;162:156–9.
Lanzavecchia A. How can cryptic epitopes trigger autoimmunity? J
Exp Med 1995;181:1945–8.
Del Prete G, Maggi E, Romagnani S. Biology of disease. Human Th1
and Th2 cells: functional properties, mechanism of regulation, and
role in disease. Lab Invest 1994;70:299–306.
Grewe M, Gyufko K, Schöpf E, Krutmann J. Lesional expression of
interferon-g in atopic eczema. Lancet 1995;343:25–6.
Baker BS, Griffiths CEM, Lambert S et al. The effects of cyclosporin
A on lymphocytes and dendritic sub-populations in psoriasis. Br J
Dermatol 1987;116:503–10.
Baker BS, Swain AF, Griffiths CEM, Leonard JN, Fry L, Valdimarsson H. The effects of topical treatment with steroids and
dithranol on epidermal T-lymphocytes and dendritic cells in
psoriasis. Scand J Immunol 1985;22:471–7.
Krueger JG, Wolfe JT, Nabeya RT et al. Successful ultraviolet B
treatment of psoriasis in accompanied by reversal of keratinocyte
pathology and by selective depletion of intradermal T-cells. J Exp
Med 1995;182:2057–68.
Schmied M, Breitskopf H, Gold R et al. Apoptosis of T-lymphocytes
in experimental autoimmune encephalomyelitis. Am J Pathol
1996;143:446–52.
Bauer J, Wekerle H, Lassmann H. Apoptosis in brain-specific
autoimmune disease. Curr Op Immunol 1995;7:839–43.
Kabilan L, Troye-Blomberg M, Perlmann H et al. T-cell epitopes in
Pf155/RESA, a major candidate for a Plasmodium falciparum
malaria vaccine. Proc Natl Acad Sci 1988;85:5659–63.
Leung DYM, Gately M, Trumble A, Ferguson-Darnell B, Schlievert
PM, Picker LJ. Bacterial superantigens induce T cell expression of
the skin-selective homing receptor, the cutaneous lymphocyteassociated antigen, via stimulation of interleukin 12 production. J
Exp Med 1995;181:747–53.