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Th2-oriented profile of male offspring T cells present in women with systemic sclerosis and reactive with maternal major histocompatibility complex antigens.

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ARTHRITIS & RHEUMATISM
Vol. 46, No. 2, February 2002, pp 445–450
DOI 10.1002/art.10049
© 2002, American College of Rheumatology
Published by Wiley-Liss, Inc.
Th2-Oriented Profile of Male Offspring T Cells Present in
Women With Systemic Sclerosis and Reactive With
Maternal Major Histocompatibility Complex Antigens
Cristina Scaletti,1 Alessandra Vultaggio,1 Stefania Bonifacio,2 Lorenzo Emmi,1
Francesca Torricelli,2 Enrico Maggi,1 Sergio Romagnani,1 and Marie-Pierre Piccinni1
Objective. To characterize the cytokine production profile of male-offspring T cells reactive against
maternal major histocompatibility complex (MHC) antigens present in the peripheral blood and/or skin from
women with systemic sclerosis (SSc).
Methods. T cell clones were generated from peripheral blood and/or skin biopsy specimens from 3
women with SSc of recent onset and from peripheral
blood from 3 healthy women, all of whom had 1 male
child. All clones were screened for their proliferative
response in vitro to maternal MHC antigens by measuring 3H-thymidine uptake and for their expression of Y
chromosome by using fluorescence in situ hybridization.
The concentrations of interferon-␥ and interleukin-4
(IL-4) released by T cell clones in response to maternal
MHC antigens were evaluated in culture supernatants,
using appropriate enzyme-linked immunosorbent assays.
Results. Thirty-nine of 202 T cell clones generated
from women with SSc and 11 of 312 from healthy women
proliferated in vitro in response to maternal MHC
antigens. Seven MHC-reactive T cell clones obtained
from women with SSc and 1 obtained from healthy
women exhibited the Y chromosome, thus indicating
that the clones were derived from T cells of male
offspring. All clones generated from male-offspring T
cells of SSc women (but not from those of healthy
women) produced significantly higher levels of IL-4 in
response to stimulation with maternal MHC antigens
than did all other clones generated from the same
women. The other clones proliferated in response to
maternal or allogeneic MHC antigens but did not
exhibit the Y chromosome.
Conclusion. Male-offspring T cells that are
present in the blood and skin of women with SSc and
react with maternal MHC antigens exhibit a Th2oriented profile, supporting the possibility that a
chronic graft-versus-host reaction attributable to longterm microchimerism plays a pathogenic role in SSc.
Systemic sclerosis (SSc) is a connective tissue
disease characterized by inflammatory, microvascular,
and fibrotic changes affecting the skin (scleroderma)
and a variety of internal organs, including the gastrointestinal tract, lungs, heart, and kidney, and by the
production of autoantibodies, most notably anticentromere, antitopoisomerase, and anti–RNA polymerase
antibodies (1,2). In recent years, the pathogenic mechanisms responsible for the alterations seen in patients
with SSc have been partially clarified. Increased numbers of CD4⫹ T cells have been found in skin lesions, as
well as in other organs, of patients with early-stage SSc
(1,2). Cytokines capable of altering endothelial cell
function and/or inducing fibrosis have been detected in
SSc sera and tissues (1), suggesting that cytokines secreted by T cells or other cells of the immune system
may play an important pathogenic role in this disease
(1,2).
Recent advances in investigations of the immune
response led to identification of subpopulations of
CD4⫹ T helper cells, termed type 1 (Th1) and type 2
(Th2), based on their profiles of cytokine production,
which are associated with different patterns of immuno-
Supported by grants from the Italian Association for Cancer
Research and the European Economic Community.
1
Cristina Scaletti, MD, Alessandra Vultaggio, MD, Lorenzo
Emmi, MD, Enrico Maggi, MD, Sergio Romagnani, MD, Marie-Pierre
Piccinni, PhD: University of Florence, Florence, Italy; 2Stefania Bonifacio, PhD, Francesca Torricelli, PhD: Azienda Ospedaliera di Careggi, Florence, Italy.
Address correspondence and reprint requests to Sergio Romagnani, MD, Dipartimento di Medicina Interna, Viale Morgagni, 85,
Florence 50134, Italy. E-mail: s.romagnani@dfc.unifi.it.
Submitted for publication September 26, 2000; accepted in
revised form September 3, 2001.
445
446
logic reactions (3,4). Th1 cells produce interferon-␥
(IFN␥) and tumor necrosis factor ␤ (TNF␤) and are
responsible for phagocyte-dependent host responses
(characterized by delayed-type hypersensitivity reactions
or a granulomatous pattern), which are involved in
protection against intracellular parasites. Th2 cells,
which produce interleukin-4 (IL-4), IL-5, and IL-13, are
responsible for phagocyte-independent, antibodymediated responses, and are implicated in protection
against gastrointestinal nematodes and in allergic conditions (3,4).
We and other investigators have reported the
existence of a predominant activation of IL-4–producing
Th2-like T cells in patients with SSc, which may account
for the major alterations that occur in this disease (5–8).
The origin of the Th2-dominated immune response in
SSc is still unclear. The clinical features of SSc are
similar to those of chronic graft-versus-host disease
(cGVHD) (9), a chimeric disorder that occurs in recipients of allogeneic stem cell transplants and in which
Th2-type responses also predominate (10,11).
Identification of fetal DNA and cells in skin
lesions and blood from women with SSc has been
previously reported (12), suggesting that a microchimerism established by fetal T cells and the activation of such
cells may induce a graft-versus-host reaction manifesting
as SSc (12,13). However, subsequent studies showed no
difference in the frequency of microchimerism with fetal
cells between healthy women and women with SSc;
therefore, the causal link between microchimerism and
disease pathogenesis remains uncertain (14).
In the current study, we attempted to characterize the cytokine profile of male-offspring T cells reactive
against maternal major histocompatibility complex
(MHC) antigens present in the blood and skin of women
with SSc. To do this, T cell clones were generated from
peripheral blood (PB) and skin biopsy specimens from 3
women with SSc of recent onset and from blood from 3
healthy women; all 6 women had a male child. The
maternal T cell clones were then screened for their
ability to proliferate in vitro in response to autologous
non–T cells, and proliferating clones were examined for
expression of the Y chromosome.
Seven maternal T cell clones derived from
women with SSc and 1 derived from healthy women
proliferated in response to autologous non–T cells and
exhibited the Y chromosome, which demonstrated that
they were derived from male-offspring T cells. Of note,
all clones generated from male-offspring T cells of SSc
women produced significantly higher levels of IL-4 in
response to stimulation with maternal non–T cells than
SCALETTI ET AL
did all other clones generated from the same women.
The other clones responded to the same stimulation but
did not exhibit the Y chromosome. These data suggest
that male-offspring T cells that are present in blood
and/or skin of women with SSc and are reactive against
maternal MHC antigens exhibit a Th2-oriented profile,
thus supporting their possible role in the cGVHD that
may occur in women with SSc.
PATIENTS AND METHODS
Patients. Three women (ages 38, 46, and 50 years) with
recent-onset SSc participated throughout the study. Three
healthy women of comparable ages (36, 42, and 52 years)
served as controls. All 6 women were selected because each
had 1 male child, and none had received either blood transfusions or allografts. The procedures followed in this study were
in accordance with the ethical standards of the Regional
Committee on Human Experimentation.
Generation of T cell clones. T cell clones were generated from PB mononuclear cells (PBMC) and skin biopsy
specimens from SSc patients, as described in detail elsewhere
(5). Briefly, PBMC obtained from 10-ml samples were cultured
for 3 days, and small (2-mm diameter) skin fragments were
cultured for 7–10 days in RPMI 1640 supplemented with 2 mM
glutamine, 20 ␮M ␤-mercaptoethanol, and 10% fetal calf
serum in the presence of recombinant IL-2 (rIL-2) (20 units/
ml). T cell blasts from both PB and skin lymphocyte cultures
were then cloned under limiting dilution conditions (0.3 cell/
well) in the presence of phytohemagglutinin (1% volume/
volume), as described previously (5). Growing microcultures
were expanded at weekly intervals, using rIL-2 (50 units/ml)
and 105 irradiated (6,000 rads) allogeneic PBMC pooled from
3 different healthy women as feeder cells. Cell-surface marker
analysis of T cell clones was performed on a Cytoron absolute
cytofluorimeter (Ortho, Raritan, NJ), using fluorescein
isothiocyanate– and phycoerythrin-conjugated anti-CD4 and
anti-CD8 monoclonal antibodies (mAb) (Becton Dickinson,
Mountain View, CA).
Mixed lymphocyte cultures. The proliferative response
of T cell clones to MHC antigens was assessed by mixed
lymphocyte culture reaction. T cell blasts (5 ⫻ 104) were
cultured for 5 days in 0.2 ml of medium in the absence or
presence of 105 irradiated (6,000 rads) non–T cells, which were
obtained by removing CD3⫹ cells, as described (5). Sixteen
hours before harvesting, cultures were pulsed with 0.5 ␮Ci of
3
H-thymidine (Amersham, Little Chalfont, UK), and radionuclide uptake was measured by scintillation counting, as previously reported. T cell clones were considered reactive to MHC
antigens when the mitogenic index (ratio between counts per
minute obtained in the presence and cpm in the absence of
non–T cells) was ⬎10.
Fluorescence in situ hybridization (FISH). The FISH
technique was used to analyze T cell clones for the presence of
Y chromosome–positive cells. To this end, T cell blasts for
each clone (106/ml) were fixed in ethanol/acetic acid (3:1),
according to standard procedures (15). They were denatured
in a 70% formamide/2⫻ saline–sodium citrate (SSC) solution
at 80°C for 2 minutes and then rehydrated with an increasing
PATHOGENIC ROLE OF LONG-TERM FETAL MICROCHIMERISM IN SSc
series of alcohol at ⫺20°C. Samples were then hybridized
simultaneously with 2 probes: the one specific for the centromeric region of X chromosome was labeled with spectrum
green, and the one specific for the centromeric region of Y
chromosome was labeled with spectrum orange. Probe denaturation was performed at 73°C for 5 minutes. Hybridization
was performed at 37°C in a moist chamber overnight. After
hybridization, samples were washed twice in 0.1⫻ SSC/0.3%
Nonidet P40 (NP40) at 73°C and then once in 2⫻ SSC/0.1%
NP40 at room temperature. Samples were counterstained with
4⬘,6-diamidino-2-phenylindole (DAPI) (0.5 gm/ml) and
mounted with antifade solution. Sample analysis was performed using a fluorescence microscope equipped with a
triple-band filter for DAPI, spectrum green, and spectrum
orange. For each sample, 80–100 nuclei were analyzed, as well
as metaphases, if present.
Detection of cytokine production by T cell clones. The
ability of T cell clones to produce cytokines was evaluated after
T cell blasts (106/ml) were stimulated for 36 hours with phorbol
myristate acetate (PMA) (20 ng/ml; Sigma, St. Louis, MO) and
anti-CD3 mAb (100 ng/ml; Ortho), or after T cell blasts (5 ⫻
105) were incubated for 5 days with autologous or allogeneic
non–T cells (5 ⫻ 105) in a total 0.2-ml volume. Cell-free
supernatants were assessed for IFN␥ and IL-4 content by
enzyme-linked immunosorbent assay (ELISA). The quantitative determination of IFN␥ and IL-4 was performed with
ELISAs made in-house, as described elsewhere (5). Cytokine
levels that were 5 SD above the mean cytokine levels of control
supernatants (derived from irradiated feeder cells alone) were
regarded as positive.
MHC typing and statistical analysis. The sequencebased typing method (16) was used to perform MHC typing.
The Mann-Whitney U nonparametric test and the chi-square
test were used to analyze samples.
RESULTS
T cell clones were generated from both PB and
skin biopsy specimens from 3 women with SSc of recent
onset and from PB of 3 healthy women, all of whom had
a male child. All clones were screened for their proliferative response to irradiated non–T cells from the same
women. A total of 29 (18%) of 161 T cell clones
generated from the PB and 10 (24%) of 41 from the skin
of the 3 SSc women, but only 11 (3.5%) of 312 from the
PB of healthy women, showed a proliferative response to
autologous non–T cells. T cell clones could not be
generated from irradiated feeder cells cultured alone,
thus excluding the possibility that clones could be derived from T lymphocytes present in feeder cells. Table
1 shows the total numbers of T cell clones derived from
PB and skin of the 6 women, as well as the number of
clones that were reactive with autologous non–T cells
from each woman.
Next, the FISH technique was used to screen the
reactive T cell clones for the presence of Y chromosome.
447
Table 1. Reactivity of T cell clones generated from peripheral blood
(PB) or skin of women with systemic sclerosis (SSc) or from PB of
healthy women to major histocompatibility complex (MHC) antigens
from the same women*
T cell clones,
no. reactive/no. tested (%)
SSc women
1
2
3
Total
Healthy women
1
2
3
Total
PB
Skin
11/55 (20)
8/45 (18)
10/61 (16)
29/161 (18)
2/10 (20)
4/13 (30)
4/18 (22)
10/41 (24)
1/118 (0.8)
6/102 (5.8)
4/93 (4.3)
11/312 (3.5)
ND
ND
ND
* T cell blasts (5 ⫻ 104) for each clone were stimulated for 5 days with
irradiated non–T cells (1 ⫻ 105) obtained from the same subject from
which the T cell clones were generated, and their proliferative
response was assessed by measuring the incorporation of labeled
thymidine. T cell clones were considered reactive to MHC antigens
when the mitogenic index was ⬎10, as described in Patients and
Methods. ND ⫽ not done.
All T cell blasts from 7 (18%) of 39 reactive clones from
the SSc women (4 from the first, 2 from the second, and
1 from the third) and 1 (9%) of 11 from the healthy
women showed Y chromosome (Figure 1). Because
none of the women had previously received any transfusion or allograft, this finding indicated that these
clones were derived from male-offspring T cells, and,
therefore, that they were alloreactive clones specific for
maternal MHC antigens rather than autoreactive clones.
This hypothesis was confirmed by comparing the MHC
sequence-based typing of 2 T cell clones from woman 1
(A*2601/0101;B*4501/0801;C*0602/0701;DRB1*0701/
1302;DRB4*0101101; DRB3*0301;DQA1*0201/0102;
DQB1*0202/0604;DPB1*⫺/02012) with her MHC
typing (A*0101/2605;B*0801/0801;C*0701/0702;
DRB1*1302/03011;DRB3*0301/0202;DQA1*0102/
05011;DQB1*0604/0201;DPB1*02012/0401), and that of
her son (A*2601/0101;B*4501/0801;C*0602/0701;
DRB1*0701/1302;DRB4*0101101;
DRB3*0301;
DQA1*0201/0102;DQB1*0202/0604;DPB1*⫺/02012)
and husband (A*03011/2601;B*1801/4501;C*0701/0602;
DRB1*11011/0701;DRB3*0202; DRB4*0101101;
DQA1*0505/0201;DQB1*0301/0202;DPB1*02012/⫺).
In women with SSc, 4 of 7 clones were obtained
from PB and 3 from skin, suggesting the presence of
male-offspring–reactive T cells in both the circulation
and target tissues. Moreover, the fact that these clones
448
SCALETTI ET AL
Figure 1. Presence of Y chromosome in T cell clones generated from the skin of women with systemic sclerosis. T cell blasts
from each clone were cohybridized simultaneously with 1 probe specific for the centromeric region of X chromosome (labeled
with spectrum green) and the other specific for the centromeric region of Y chromosome (labeled with spectrum orange),
distinguishing male cells (yellow ⫹ red signal) from female (2 yellow signals) cells. A, T cell blasts from 1 clone showing Y
chromosome. B, A mitotic T cell blast from the same clone. C, T cell blasts from 1 clone showing no Y chromosome. D, Mitotic
T cell blasts from the same clone.
were truly reactive against maternal MHC antigens was
fully supported by the observation that their in vitro
proliferative response to maternal non–T cells could be
completely blocked by the addition of anti–MHC class II
mAb D.1/12 (data not shown).
The ability of clones showing Y chromosome to
produce IFN␥ and IL-4 in response to either polyclonal
stimulation with PMA plus anti-CD3 mAb or specific
stimulation with maternal MHC antigens (non–T cells)
was also examined. As control, all reactive T cell clones
showing no Y chromosome were tested for their ability
to produce IFN␥ and IL-4 under the same experimental
conditions, as well as in response to non–T cells from 10
different donors who had incompatible MHC antigens.
Among the different groups of clones, there were no
significant differences in the production of IFN␥ or IL-4
following polyclonal stimulation (data not shown). Following MHC stimulation, however, the clones derived
from male-offspring T cells from SSc women produced
reduced amounts of IFN␥ (1.8 ⫾ 0.8 ng/ml) compared
with T cell clones that showed no Y chromosome from
the same women (5.2 ⫾ 1.6 ng/ml), even though the
difference was not statistically significant. However, the
male-offspring clones produced significantly higher levels of IL-4 than did the clones showing no Y chromosome (3.3 ⫾ 1.1 versus 0.26 ⫾ 0.1 ng/ml) (Z, corrected
for ties, ⫺3.8, P ⬍ 0.0001) when they were stimulated
with either maternal MHC antigens (Figure 2) or allogeneic non–T cells (data not shown). In contrast, the
only MHC-reactive clone generated from male-offspring
PATHOGENIC ROLE OF LONG-TERM FETAL MICROCHIMERISM IN SSc
449
T cells of healthy women produced low concentrations
of both IFN␥ and IL-4 in response to stimulation with
maternal MHC antigens (Figure 2).
DISCUSSION
SSc is an autoimmune disease with a strong
predilection in women, a peak incidence in the years
after childbearing, and clinical and immunologic similarities to cGVHD (1,2). Both SSc and cGVHD are
characterized by skin, lung, and esophageal involvement
and intense fibrosis (1,2). Immunologic similarities include lymphocytic infiltration of affected tissues, the
presence of Scl-70 and PM-Scl serum autoantibodies,
and up-regulation of a series of cytokines (1,2,5–8).
Moreover, several reports have indicated that IL-4 production by CD4⫹ T cells infiltrating SSc and cGVHD
lesions is prominent in both animal models and humans,
suggesting a Th2-polarized phenotype of the specific
immune response in both conditions (5–8,10,11).
Recent reports have suggested that SSc may be
the result of a graft-versus-host reaction caused by
persistent feto-maternal microchimerism, which has
been detected in large numbers of childbearing women
and their immunocompetent offspring, respectively
(12,13). If this hypothesis is correct, the offspring T cells
engrafted in the PB or skin of women with SSc should
have a Th2-polarized phenotype, such as that responsible for cGVHD in bone marrow allograft recipients.
To address this possibility, we generated T cell
clones from both PB and skin of 3 women with recentonset SSc, as well as from the PB of 3 healthy women, all
of whom had 1 male child. T cell clones that proliferated
in response to maternal MHC antigens were then selected and screened for the presence of Y chromosome.
T cell clones showing the Y chromosome were
obviously derived from male-offspring–engrafted T cells,
as was also demonstrated by their MHC typing. In
contrast, the clones that were reactive with maternal
non–T cells but showed XX chromosomes were the
progeny of maternal autoreactive T cells. Of note, the
number of clones that proliferated in response to autologous non–T cells was significantly higher in women
with SSc than in healthy women, independent of
whether they expressed the XY or the XX chromosome.
This finding suggests the presence of an increased
proportion of both autoreactive maternal and offspringengrafted T cells in women with SSc. The increased
proportions of clones derived from male offspring found
Figure 2. High interleukin-4 (IL-4) production by male-offspring T
cells present in the peripheral blood (PB) or skin of women with
systemic sclerosis (SSc) in response to maternal major histocompatibility complex (MHC) antigens. T cell clones were generated
from PB and skin of women with SSc and from PB of healthy
women, all of whom had male children, and were selected for
their ability to proliferate in response to maternal MHC antigens.
The ability of MHC-reactive T cell clones showing Y chromosome (E)
and of those showing no Y chromosome (F) to produce IL-4
and interferon-␥ (IFN␥) following stimulation with irradiated
non–T cells from the PB of the same women was compared. IL-4
production and IFN␥ production by individual T cell clones are
shown.
450
in this study may be consistent with other studies that
showed higher levels of fetal DNA in the blood or skin
of women with SSc compared with healthy women
(12,13). However, the mechanisms responsible for the
increased proportions of autoreactive T cell clones in
women with SSc remain unclear.
When the ability of MHC-reactive clones to
produce IFN␥ and IL-4 in response to polyclonal stimulation was assessed, no difference in the cytokine
production profiles among the different groups of clones
was found. However, clones derived from male-offspring
T cells of women with SSc showed significantly higher
IL-4 production in response to MHC stimulation than
did maternal autoreactive clones, whereas the only clone
derived from male-offspring T cells of healthy women
produced low concentrations of both IFN␥ and IL-4. In
male-offspring T cell clones derived from women with
SSc, the high production of IL-4 in response to stimulation with maternal MHC antigens was not attributable
simply to the different type of stimulation (autoreactive
versus alloreactive). When T cell clones lacking the Y
chromosome were stimulated with allogeneic cells, the
amount of IL-4 produced was comparable with that
produced by autoreactive maternal T cell clones but was
significantly lower than that of male-offspring T cell
clones showing alloreactivity toward maternal MHC
antigens.
Certainly, interpretation of these data is problematic because of the small number of subjects and the
fact that the number of clones being compared (i.e., 7
from SSc women and 1 from healthy women) was
different. However, the results suggest that maleoffspring T cells present in PB and/or skin of women
with SSc exhibit a functional profile prevalently oriented
toward Th2 rather than Th1 cytokine production in
response to stimulation with maternal MHC antigens.
This finding supports the concept that fetal cells found
in the PB and/or skin of women with SSc are functionally
similar to those responsible for cGVHD in experimental
animal models (9,10).
The selected CD4⫹ T cell population derived
from fetal cell engraftment may be at least partially
included in the population of T cells expressing IL-4 that
are found in the skin of patients with SSc (5), and it is
probably responsible for the enhanced levels of IL-4 in
the biologic fluids of these patients (6–8). Thus, although the results of this study need further investiga-
SCALETTI ET AL
tion, they provide support for the hypothesis that a fetal
antimaternal cGVHD may be an immunopathogenic
mechanism in the development of SSc in some women.
ACKNOWLEDGMENT
The authors thank Roberto Accolla, University of
Varese, Varese, Italy, for the kind gift of the D.1/12 anti–MHC
class framework mAb.
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