The Journal of Immunology
CD103 Is a Marker for Alloantigen-Induced Regulatory CD8ⴙ
T Cells
Elena Uss,1*§ Ajda T. Rowshani,§ Berend Hooibrink,† Neubury M. Lardy,‡
René A. W. van Lier,* and Ineke J. M. ten Berge§
The ␣E␤7 integrin CD103 may direct lymphocytes to its ligand E-cadherin. CD103 is expressed on T cells in lung and gut and on
allograft-infiltrating T cells. Moreover, recent studies have documented expression of CD103 on CD4ⴙ regulatory T cells. Approximately 4% of circulating CD8ⴙ T cells bear the CD103 molecule. In this study, we show that the absence or presence of
CD103 was a stable trait when purified CD103ⴚ and CD103ⴙCD8ⴙ T cell subsets were stimulated with a combination of CD3 and
CD28 mAbs. In contrast, allostimulation induced CD103 expression on ⬃25% of purified CD103ⴚCD8ⴙ T cells. Expression of
CD103 on alloreactive cells was found to be augmented by IL-4, IL-10, or TGF-␤ and decreased by addition of IL-12 to MLCs.
The alloantigen-induced CD103ⴙCD8ⴙ T cell population appeared to be polyclonal and retained CD103 expression after restimulation. Markedly, in vitro-expanded CD103ⴙCD8ⴙ T cells had low proliferative and cytotoxic capacity, yet produced considerable
amounts of IL-10. Strikingly, they potently suppressed T cell proliferation in MLC via a cell-cell contact-dependent mechanism.
Thus, human alloantigen-induced CD103ⴙCD8ⴙ T cells possess functional features of regulatory T cells. The Journal of Immunology, 2006, 177: 2775–2783.
I
ncreasing evidence exists for a protective role for regulatory
T cells (Tregs)2 in autoimmune diseases, allergic diseases,
and allograft rejection, where they control the potential damaging activity of effector Th1 cells, Th2 cells, or CTLs (1–3).
Tregs can be classified into natural (constitutive) and induced regulatory cells. To the latter category belong CD4⫹ T regulatory 1
(Tr1) cells and CD4⫹ Th3 cells, as well as regulatory CD8⫹ T
cells (4).
Natural CD4⫹CD25⫹ Tregs have first been described by Sakaguchi et al. (5) as thymic-derived cells that have multiple immunoregulatory properties, including active suppression of self-Agreactive T cells, promotion of tolerance to allogeneic bone marrow
grafts, and suppression of antitumor immune reactivity.
CD4⫹CD25⫹ T cells have a low proliferative capacity after allogeneic or polyclonal stimulation and express CTLA4, which delivers a negative signal for T cell activation. Their mechanism of
suppression includes cell-cell contact, but secreted cytokines such
as TGF-␤ and IL-10 may also play a role (6). Expression of the
transcription factor Foxp3, a critical regulator of CD4⫹CD25⫹
Treg cell development and function, seems the best marker to identify natural CD4⫹ Tregs (7). Two subsets of inducible CD4⫹
Tregs have been recognized: Ag-specific Tr1 cells that secrete
large amounts of IL-10 and Th3 cells, mainly producing TGF-␤
(8 –10).
*Department of Experimental Immunology and †Department of Cell Biology and
Histology, Academic Medical Center, Amsterdam, The Netherlands; ‡HLA-Diagnostics Laboratory, Sanquin, Amsterdam, The Netherlands; and §Division of Nephrology, Department of Internal Medicine, Academic Medical Center, Amsterdam, The
Netherlands
Received for publication December 5, 2005. Accepted for publication May 17, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Elena Uss, Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, L1110, 1105 AZ Amsterdam, The Netherlands. E-mail address: [email protected]
2
Abbreviations used in this paper: Treg, T regulatory cell; Tr1, T regulatory 1; pen/
strep, penicillin/streptomycin; Tc, T cytotoxic.
Copyright © 2006 by The American Association of Immunologists, Inc.
Although in the past so-called CD8⫹ T suppressor cells were
supposed to play a regulatory role in autoimmune diseases, transplantation, and in protection against cancer (11, 12), only over the
last 10 years has this concept re-emerged (13–15). Until then, efforts to understand the cellular and molecular mechanisms underlying CD8 T cell-mediated immunosuppression were hampered by
difficulties in isolating these cells and by a lack of defining markers. The cell surface marker profile described for CD8⫹ Tregs,
such as CD28⫺, CD45RClow, and CTLA-4⫹ indicates more that
these cells are in an “activated” or “memory” state than that they
are associated with a regulatory function. CD8⫹ Tregs are reported
to mediate Ag-specific suppression by production of the cytokines
IL-10 and/or TGF-␤ and/or by a direct inhibitory action on dendritic cells (16).
The ␣E␤7 integrin CD103 has initially been described to be
expressed on both murine and human CD8⫹ T lymphocytes localized in intestine, bronchoalveolar fluid, and allograft tissues
(17–19). An important function of this molecule appears to be
directing lymphocytes to their ligand E-cadherin, expressed on epithelial cells (20, 21). Although the CD103 molecule can be expressed on alloactivated, graft-infiltrating lymphocytes, this seems
not a prerequisite for the cytotoxic function of these cells as in
vitro cytotoxicity against alloantigens exerted by sorted
CD103⫺CD8⫹ T cells equaled that of sorted CD103⫹CD8⫹ T
cells (22). Moreover, although CD8⫹ T cells from CD103 knockout mice cannot reach the renal epithelial cells, they show normal
cytotoxic capacity (23). Recently, CD103 was shown to be a target
of FoxP3 (24) and was found to be expressed on CD4⫹ Tregs (25,
26). Whether CD103⫹CD8⫹ T cells can also exert regulatory
functions is currently unknown.
In this study, we show that human CD103⫹CD8⫹ T cells can be
induced by stimulation with alloantigens in vitro. These cells possess potent suppressive activity in MLC, which is cell-cell contact
dependent. CD103⫹CD8⫹ T cells secrete IL-10 rather than IFN-␥
and maintain their phenotype after restimulation with alloantigen.
Thus, CD103 represents a novel marker for a subset of alloantigeninduced regulatory CD8⫹ T cells.
0022-1767/06/$02.00
CD103⫹ ALLOANTIGEN-INDUCED CD8⫹ Tregs
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Materials and Methods
Cell-sorting experiments
Cells
T cell subpopulations were purified by FACS sorting. Briefly, unstimulated
PBMC or CFSE-labeled PBMC stimulated for 5 days with irradiated allogeneic stimulator PBMC were labeled with CD103-PE, CD4-PerCP
Cy5.5, and CD8-allophycocyanin. To identify alloresponsive T cells, a gate
was set on the CFSElow population. Next, CD103⫹CD8⫹, CD103⫺CD8⫹,
CD103⫹CD4⫹, and CD103⫺CD4⫹ subsets were separated with the Aria
FACS (BD Biosciences). All sorted subsets were ⬎95% pure.
PBMC were isolated from whole heparinized blood obtained from 14
healthy donors by Ficoll-Paque density centrifugation (Pharmacia Biotech). The current study was approved by the local medical ethics committee of the Academic Medical Center.
Monoclonal Abs
Biotinylated CD103 mAb and V␤3-FITC were purchased from Immunotech and CD103-PE from Caltag Laboratories. CD4⫺ and CD8-PerCP
Cy5.5, and CD27-, CD28-, CCR7-, IFN-␥-, IL-4-, IL-10-, streptavidin-PE,
mIgG1-PE, and CD8-allophycocyanin were all purchased from BD Immunocytometry Systems. CD45RA-RD1 and streptavidin-allophycocyanin
were acquired from BD Pharmingen.
CFSE labeling
PBMC were washed once with PBS containing antibiotics (100 U/ml sodium penicillin (Brocades Pharma) and 100 ␮g/ml streptomycin sulfate
(Invitrogen Life Technologies)) at room temperature before staining. One
microliter of CFSE (Molecular Probes; 5 ␮M stock concentration) was
added in 1 ml of PBS-penicillin/streptomycin (pen/strep)/107 cells. Cells
were subsequently incubated for 12 min at 37°C. Next, cells were washed
three times in PBS-pen/strep at 4°C and resuspended in 1 ml of culture
medium.
Culture and stimulation of the cells
All culture experiments were performed in culture medium consisting of
IMDM (Invitrogen Life Technologies) containing 10% heat-inactivated autologous human serum, pen/strep, and 2-ME (0.0035% v/v; Merck). MLCs
were performed with selected responder-stimulator combinations that gave
high proliferative responses in classical MLCs, detected by [3H]thymidine
incorporation. Responder PBMC were labeled with CFSE and cultured
with irradiated allogeneic stimulator PBMCs or with irradiated autologous
PBMCs. When indicated, responder PBMC were stimulated in the presence of CD3 mAb (soluble CLB-T3/4E) and CD28 mAb (CLB-CD28/1).
The precursor frequency (percentage of cells in the initial subset that has
undergone one or more divisions after culture) was calculated as follows:
[⌺n ⱖ 1(Pn/2n)]/[⌺n ⱖ 0(Pn/2n)], where n is the division number that cells
have gone through and Pn is the number of the cells in division n (27).
To analyze the effect of cytokines on CD103 expression, MLCs were
performed in the absence or presence of IL-7 (10 ng/ml; Strathmann),
IL-15 (10 ng/ml), IL-4 (10 ng/ml), IL-12 (10 ng/ml), TNF-␣ (10 ng/ml),
TGF-␤ (1 ng/ml) (all from R&D Systems), IL-21 (50 ng/ml; Zymogenetics), IL-10 (10 ng/ml) or IL-17 (10 ng/ml) (both from Sanquin). After 5
days of culture, cells were analyzed by FACSCalibur flow cytometer (BD
Biosciences). Responder PBMC stimulated with irradiated autologous cells
in the presence of the above-mentioned cytokines were used as controls.
Anti-TGF-␤-neutralizing mAb was purchased from R&D Systems and
used in a final concentration of 2 ␮g/ml.
TCR-V␤ spectratyping
cDNA was synthesized from RNA from equal numbers of sorted
CD103⫹CD8⫹ and CD103⫺CD8⫹ cells. PCR was performed in single
TCR-V␤ PCRs with a TCR-C␤ primer labeled with a fluorescent dye (Invitrogen Life Technologies) (CAG GCA CAC CAG TGT GGC-FAM) as
described previously (28, 29).
Flow cytometric analyses
Immunofluorescent staining and flow cytometry were performed on day 0
and after 5 days of culture. A total of 3 ⫻ 105 PBMC were incubated with
fluorescent-labeled conjugated mAbs (concentrations according to manufacturer’s instructions) for 30 min at 4°C protected from light. In some
cases, this was followed by incubation with a second-step reagent (streptavidin-PE, -allophycocyanin) for 30 min at 4°C. For the cytokine staining,
cells were first stimulated in a 24-well plate at 1 ⫻ 106 cells/well in 0.5 ml
of culture medium with PMA (1 ng/ml), ionomycin (1 ␮g/ml), and monensin (1 ␮M) (all from Sigma-Aldrich) for 4 h at 37°C. Cells were
washed, and then the same staining procedure as described above was
performed using anti-IL-10-PE, anti-IL-4-PE, or anti-IFN-␥-PE Abs.
Evaluation of cytokine production by ELISA
Determination of cytokine concentrations in culture supernatants was performed by ELISA according to manufacturer’s protocols. Reagents for
IL-10, IL-2, IFN-␥ measurements were obtained from Sanquin. Reagents
for TGF-␤ measurements were purchased from R&D Systems.
In vitro suppressor assay
Responder PBMC were labeled with CFSE and cocultured with irradiated
allogeneic stimulator PBMC. Purified alloreactive CD103⫹CD8⫹,
CD103⫺CD8⫹, CD103⫹CD4⫹, or CD103⫺CD4⫹ cells from the same donor were labeled with 1,3-dichloro-9,9-dimethylacridin-2-one succinimidyl
ester (DDAO-SE; Molecular Probes) and added to the MLC at different
regulator:responder ratios. The assay was performed in round-bottom 96well microtiter plates. After 5 days of culture, precursor frequency was
determined and cells were stained for CD103, CD8, and CD4 to determine
the phenotype of proliferating lymphocytes. Blocking Abs were used at the
following concentrations: 1 ␮g/ml anti-IL-10 (BD Pharmingen), 2 ␮g/ml
anti-TGF␤ (R&D Systems), which had been tested for optimal activity in
previous experiments.
Transwell assay
To evaluate the role of cell-cell contact in suppressive activity, 24-well
plates equipped with a Transwell insert (Costar) consisting of a 200-␮l
upper well separated from an 800-␮l bottom well by a 0.4-␮m microporous
polycarbonate membrane that had not been pretreated in tissue culture
medium were used. The distance between the Transwell membrane and the
well bottom was 1 mm. All the cells were resuspended in culture medium.
Responder PBMC labeled with CFSE and cocultured with irradiated allogeneic stimulator PBMC were plated in the bottom well of the Transwell
system at a concentration of 5 ⫻ 105 cells/ml. The top well insert was
inoculated with culture medium alone or CD103⫹CD8⫹ or CD103⫺CD8⫹
T cell population as indicated. After 5 days of culture, cells were stained
for CD103 and CD8 and analyzed by FACS.
51
Cr-release assay
MLC were performed as described previously (30). Briefly, MLC consisting of responder PBMC and irradiated stimulator cells were used to generate effector cells. After 5 days of proliferation, CD103⫹CD8⫹ and
CD103⫺CD8⫹ alloreactive T cells were sorted and used as effectors. An
8-h 51Cr-release assay was used to detect lysis of stimulator or autologous
target cells at varying E:T ratios. Lymphocytes, cultured for 5 days, were
used as target cells. The specific percentage of lysis was calculated according to the formula: (ER ⫺ SR)/(MR ⫺ SR) ⫻100, where ER is the
experimental chromium release, SR represents the spontaneous chromium
release of target cells in medium alone, and MR equals the maximum
chromium release of target cells in 5% saponin solution. In these experiments, the percentage of specific lysis was derived from the E:T ratio of
5:1. All determinations were done in duplicate.
Statistical analysis
The Mann-Whitney U test was used for comparison of two independent
groups of observations. The two-tailed Kruskal-Wallis test was used for
comparisons of more than two means. Values of p below 0.05 were considered statistically significant.
Results
CD103⫹CD8⫹ T cells derive from CD103⫺CD8⫹ T cells upon
allostimulation
As reported previously, CD103 defines a subset of CD8⫹ T cells
(31). We found that on average ⬃4% of freshly isolated CD8⫹
lymphocytes expressed the CD103 molecule (Fig. 1A). These cells
appeared to have a primed phenotype as they lacked CD45RA,
CD62L, and chemokine receptor CCR7 (data not shown). To investigate the stability of CD103 expression on CD8⫹ T cells, we
purified CD103⫹CD8⫹ and CD103⫺CD8⫹ subsets from PBMC
and stimulated them with a combination of CD3 and CD28 mAb
for 3 days. Both populations maintained their phenotype (Fig. 1B).
In contrast, in alloantigen-stimulated cultures, an up-regulation of
the CD103 molecule was found on CD103⫺CD8⫹ T cells since
The Journal of Immunology
FIGURE 1. CD103⫹CD8⫹ T cells derive from CD103⫺CD8⫹ T cells
upon allostimulation. A, CD103 defines a subset of circulating resting
CD8⫹ T cells. The dot plots represent T cells gated on CD8⫹ T cells. One
representative experiment of nine is shown. B, CD103 is up-regulated by
allostimulation. Purified CD103⫹CD8⫹ and CD103⫺CD8⫹ T cells were
labeled with CFSE and stimulated with a combination of CD3 and CD28
mAb (anti-CD3/anti-CD28) for 3 days, or with alloantigen for 5 days as
indicated. Dot plots (gated on CD8⫹ T cells) represent the phenotype of the
subsets in nondivided (left panel) and divided (middle panel) populations
based on CFSE dilution on days 3 and 5, respectively. The x-axis shows log
fluorescence of CD8-allophycocyanin, the y-axis shows log fluorescence of
CD103-PE. The right panel shows the divisions of these cells. One representative experiment of three is shown. C, Up-regulation of CD103 is
dependent on soluble factors. Purified CD103⫺CD8⫹ T cells were labeled
with CFSE and stimulated with a combination of CD3 and CD28 mAb for
3 days. To determine the influence of alloantigen-induced soluble factors,
responder CD103⫺CD8⫹ T cells were plated in the top insert of a Transwell system at a concentration of 2 ⫻ 105 cells/200 ␮l. The bottom well
was inoculated with culture medium alone (I) or with PBMC cocultured
with irradiated allogeneic stimulator PBMC (II). Purified CD103⫺CD8⫹ T
cells stimulated with alloantigen for 5 days were used as a control (III). In
parallel, purified CD103⫺CD8⫹ T cells stimulated with CD3/CD28 mAb
were cultured in the presence of the supernatant from a 5 day alloculture
(IV). Dot plots represent the phenotype of CD8⫹ T cells (one of two independent experiments). The x-axis shows log fluorescence of CD8-allophycocyanin; the y-axis shows log fluorescence of CD103-PE.
31.44 ⫾ 8.49% (mean ⫾ SD of nine donors tested) of these cells
acquired CD103 expression. Additionally, Fig. 1B shows that both
CD103⫺ and CD103⫹ CD8⫹ T can respond to alloantigens, albeit
CD103⫺ cells have a more potent proliferative response. To address the influence of soluble factors on expression of CD103 by
CD103⫺CD8⫹ T cells, we conducted a Transwell assay.
2777
CD103⫺CD8⫹ T cells stimulated with a combination of CD3 and
CD28 mAb, and separated with a semipermeable membrane from
allostimulated PBMC, up-regulated CD103 (6.8 ⫾ 2.3% of CD8⫹
T cells became CD103⫹) (Fig. 1CII), although to a lesser extent
than when they were stimulated with alloantigen in control cultures without semipermeable membrane (14.7 ⫾ 3.9% of
CD103⫹CD8⫹ T cells) (Fig. 1CIII). Similar results were obtained
when CD103⫺CD8⫹ T cells were stimulated with CD3/CD28
mAb and to which supernatant from a 5-day allostimulated culture
was added (Fig. 1CIV).
To obtain more insight into the details of alloantigen-induced
up-regulation of CD103, MLCs starting with unseparated PBMC
were performed (Fig. 2, A and B). Because the previous experiment showed that CD103⫹ T cells represented only a minute subset of the circulating CD8⫹ T cell pool and CD103⫺CD8⫹ T cells
had a superior mitogenic response upon allostimulation, these experiments largely reflected de novo expression of CD103 on formerly CD103⫺ lymphocytes. Following stimulation with irradiated allogeneic cells, CD8⫹ T cells already up-regulated CD103 at
day 3 when 26.33 ⫾ 14.97% (mean ⫾ SD) expressed the CD103
molecule (Fig. 2C). Expression increased gradually with a peak on
day 5 (53.63 ⫾ 13.81%, Fig. 2C). As we described previously,
alloreactive CD8⫹ T cells bear the phenotype of effector T cells
(32). Next to this, CD4⫹ cells up-regulated CD103 less significantly ( p ⬍ 0.05), because only 10.77 ⫾ 5.23% of alloreactive
CD4⫹ T cells became CD103 positive at day 5 (Fig. 2B). Thereafter, the expression of CD103 in both T cell subsets slightly decreased, yielding 37.77 ⫾ 16.09% CD103⫹CD8⫹ (Fig. 2C) and
8.53 ⫾ 3.71% CD103⫹CD4⫹ T cells on day 7 of culture (data not
shown). Neither CD4⫹ nor CD8⫹ T cells expressed CD103 after
autologous stimulation (data not shown). Based on the diminution
of CFSE fluorescence, expression of CD103 on CD8⫹ alloreactive
T cells increased with the number of cell divisions (Fig. 2D).
CD103 expression on alloreactive CD8⫹ T cells is increased in
the presence of TGF-␤, IL-4, and IL-10 but is down-regulated
by IL-12 in MLCs
To assess the influence of cytokines on CD103 expression, standard MLCs were performed in the presence or absence of different
cytokines. In preliminary experiments, the optimal concentration
of these cytokines had been determined (data not shown). Confirming previous studies (31), addition of TGF-␤ to MLC increased the percentage of CD8⫹ T cells expressing CD103 to
⬎90% (Fig. 3A). Only two of the other cytokines tested also had
a significant effect on CD103 expression on alloreactive cells. IL-4
increased the percentage of CD103⫹-expressing CD8⫹ T cells
from 45.41 ⫾ 10.61% to 75.69 ⫾ 21.91% (four donors, p ⬍ 0.05,
Fig. 3B). Expression of CD103 on alloreactive CD8⫹ T cells stimulated in the presence of IL-12 was significantly lower than in the
control cultures (17.6 ⫾ 12.1% vs 45.41 ⫾ 10.61%). Addition of
IL-10 did not have a significant effect on the frequency of T cells
expressing CD103 (Fig. 3B); however, the amount of CD103 expressed per cell was clearly increased as evidenced by the higher
intensity of CD103 staining (Fig. 3C). To assess the role of TGF-␤
in the elevated expression of CD103 induced by IL-4 and IL-10,
MLCs were performed in the presence of both the aforementioned
cytokines and a neutralizing anti-TGF-␤ mAb (final concentration
2 ␮g/ml). As a control, MLCs were performed in the presence of
rTGF-␤ in a final concentration of 1 ng/ml together with the antiTGF-␤ mAb in a final concentration of 2 ␮g/ml. Addition of the
blocking anti-TGF-␤ mAb did not influence the amount of CD103
expressed on alloreactive T cells induced by IL-4 or IL-10. Likewise, TGF-␤-production was not influenced by the addition of
2778
FIGURE 2. After allogeneic stimulation in vitro, CD103 is mainly expressed on divided CD8⫹ T lymphocytes. PBMC were labeled with CFSE
and cultured with irradiated allogeneic or autologous PBMC. Flow cytometric analysis was performed on day 5 of culture. The subset distribution
was assessed by staining with CD4-PerCP Cy5.5, CD8-PerCP Cy5.5,
CD27-PE, and CD103-bio-allophycocyanin. A, Dot plots represent alloreactive T cells gated on CD4⫹ T cells at days 0 and 5 of culture. Analysis
was done in undivided and divided subsets. The x-axis shows log fluorescence of CD103-bio-allophycocyanin; the y-axis shows log fluorescence of
CD27-PE. B, The mean percentage of CD103⫹ cells ⫾ SD from 14 independent experiments is shown. f, The mean percentage of CD103⫹ cells
within the CD4⫹ alloreactive T cells; u, the mean percentage of CD103⫹
cells within the CD8⫹ subset. Differences in the percentage of CD103⫹
cells within alloreactive CD4⫹, respectively, CD8⫹ cells between days 0
and 5 of culture were significant (p ⬍ 0.05), as was the difference between
CD103⫹ cells in the divided CD4⫹ and CD8⫹ T cell subsets at day 5,
indicated by an asterisk (ⴱ). C, Time course of alloantigen-induced CD103
expression. Bars represent the mean percentage of CD103⫹CD8⫹ alloreactive T cells ⫾ SD (y-axis) of three independent experiments as determined
on days 3–7 after initiation of the MLC (x-axis). D, Expression of CD103
on CD8⫹ alloreactive T cells increases with the number of cell divisions.
The x-axis shows log fluorescence of CFSE; the y-axis shows log fluorescence of CD103-PE.
rIL-12 at a final concentration of 10 ng/ml. (data not shown). Addition of other cytokines such as IL-7, IL-15, IL-21, TNF-␣, or
IL-17 did not significantly affect CD103 expression (Fig. 3B).
Functional properties of CD103⫹CD8⫹ and
CD103⫺CD8⫹subsets
Having established that CD103⫹CD8⫹ T cells specifically expand
upon alloantigenic stimulation, subsequent experiments were performed to determine the V␤ usage, stability, and functional properties of this subset.
CD103⫹ ALLOANTIGEN-INDUCED CD8⫹ Tregs
FIGURE 3. CD103 expression on alloactivated CD8⫹ T cells is increased when cocultured in the presence of TGF-␤, IL-4, or IL-10 and
decreased by IL-12. A, Expression of CD103 on alloreactive CD8⫹ T cells
on day 5 of MLC, performed in the absence or presence of several cytokines. x-axis, Log fluorescence CD103-PE; filled histograms, isotype control; bold histograms, CD8⫹ alloreactive T cells. One representative experiment of four experiments is shown. B, The bars represent the mean
percentage ⫾ SD of CD103⫹CD8⫹ T cells at day 5 of MLC in the absence
or presence of different cytokines (four experiments). Statistical significance is indicated by an asterisk (ⴱ, p ⬍ 0.05). C, The bars represent mean
fluorescence intensity (MFI) of CD103 on CD103⫹CD8⫹ T cells on day 5
of MLC in the absence or presence of different cytokines. The mean percentage ⫾ SD from four independent experiments is shown. Statistical
significance is indicated by an asterisk (ⴱ, p ⬍ 0.05).
The TCR V␤ repertoire of sorted CD103⫹CD8⫹ T cells was
determined and compared with that of CD103⫺CD8⫹ T cells to
investigate the clonal composition of the CD103⫹CD8⫹ T population. As shown in Fig. 4A, although somewhat less diverse than
the CD103⫺CD8⫹ fraction, CD103⫹CD8⫹ T cells used a broad
spectrum of V␤ families inferring that this population contained
multiple clones that have expanded in response to alloantigen
exposure.
As compared with their expression in the CD103⫺CD8⫹ T cell
population, expression of V␤3, V␤10, and V␤20 was significantly
reduced in the CD103⫹CD8⫹ T cell population (Fig. 4A). The
lower expression of V␤3 by CD103⫹CD8⫹ T cells was confirmed
The Journal of Immunology
2779
FIGURE 4. CD103⫹CD8⫹ T cells are a polyclonal
population with low proliferative capacity. A, TCR
V␤ repertoire of sorted CD103⫹CD8⫹ and
CD103⫺CD8⫹ T cells. After allostimulation for 5
days, CD8⫹ cells were sorted into CD103⫹CD8⫹ and
CD103⫺CD8⫹ subsets. The TCR V␤ repertoire of
sorted CD103⫹CD8⫹ T cells was determined and
compared with that of CD103⫺CD8⫹ T cells. ⴱ, V␤
expressed differentially between CD103⫹CD8⫹
and CD103⫺CD8⫹ populations. B, The difference in
V␤3 expression between CD103⫹CD8⫹ and
CD103⫺CD8⫹ T cells was confirmed by FACS
analysis of alloreactive T cells. Dot plots represent
alloreactive CD8⫹ T cells. The x-axis shows log fluorescence of V␤3-FITC; the y-axis shows log fluorescence of CD103-PE. One representative experiment of two is shown. C and D, Analysis of stability
of CD103 expression. CD103⫹CD8⫹ and CD103⫺CD8⫹
subsets, sorted after 5 days of allostimulation, were
labeled with CFSE and restimulated with alloantigen.
After 5 days of restimulation, FACS analysis was performed. Dot plots of the left panel represent sorted
CD103⫹CD8⫹ T cells (C) and sorted CD103⫺CD8⫹
T cells (D) restimulated with alloantigen, showing
maintenance of phenotype. The right panels show
hardly any proliferative capacity of the CD103⫹CD8⫹ T
cell subset compared with the CD103⫺CD8⫹ T cell subset. One representative experiment of three is shown.
by FACS analysis (Fig. 4B). To examine the stability of CD103⫹
expression on alloantigen-expanded CD8⫹ T cells, sorted
CD103⫹CD8⫹ T cells were labeled with CFSE and restimulated
with alloantigen or a combination of CD3 and CD28 mAb. Irrespective of the stimulus, acquired CD103 expression appeared to
be a stable trait as sorted CD103⫹CD8⫹ remained predominantly
CD103⫹; purified CD103⫺CD8⫹ T cells also retained their phenotype (Fig. 4, C and D). On day 7 after restimulation, CD103
expression on the sorted CD103⫹ T cells subsequently went down,
but no changes were observed regarding CD103 expression on the
sorted CD103⫺ subset (data not shown). Remarkably, based on the
diminution of CFSE fluorescence, CD103⫹CD8⫹ T cells hardly
proliferated, whereas CD103⫺CD8⫹ cells divided up to four times
after allogeneic restimulation (Fig. 4, C and D).
Next, the ability of CD103⫹CD8⫹ T cells to produce inflammatory and tolerogenic cytokines was investigated. To this end,
CD8⫹ T cells from MLC after 5 days of stimulation were sorted
into CD103⫹ and CD103⫺ subsets and restimulated with alloantigen for 5 days. Production of the cytokines by the purified subsets
as measured by ELISA showed that IL-10 and TGF-␤ were produced by both subsets, whereas IFN-␥ was significantly higher in
the supernatants of CD103⫺CD8⫹ T cells (Fig. 5A). In accordance
with this observation, we found that production of IFN-␥, determined by FACS, predominantly correlated with the absence of
CD103 expression on T cells (53.87 ⫾ 8.03% of CD103⫺CD8⫹
cells produced IFN-␥ vs 14.89 ⫾ 4.91% of CD103⫹CD8⫹ T cells,
Fig. 5B).
Finally, we investigated whether CD103⫹CD8⫹ and
CD103⫺CD8⫹ populations differed in cytotoxic potential. After
separation of the alloreactive cells into CD103⫹CD8⫹ and
CD103⫺CD8⫹ populations, their lytic capacity against allogeneic
target cells was tested (see Materials and Methods). As shown in
Fig. 5C, CFSElow-negCD103⫺CD8⫹ T cells are superior in cytotoxic activity compared with CD103⫹CD8⫹ T cells.
The CD103⫹CD8⫹ subset possesses regulatory activity
Given that the CD103⫹CD8⫹ subset was able to produce IL-10,
but no IFN-␥, combined with the observation that CD103⫹CD8⫹
T cells had low proliferative capacity, we examined the possible
regulatory capacity of this subset. Alloexpanded purified subsets
were tested for their suppressive activity in MLCs. Responder
PBMC were labeled with CFSE and stimulated with irradiated
allogeneic PBMC for 5 days. Addition of CD103⫹CD8⫹ T cells,
autologous to the responder PBMC, from a regulator:responder
ratio of 1:10, considerably decreased the precursor frequency from
3.88 ⫾ 1.81% in control allostimulated cultures to 2.16 ⫾ 1.89%
in the experimental cultures ( p ⬍ 0.005, Fig. 6A). Addition of
CD103⫺CD8⫹ cells to the culture did not influence the precursor
frequencies of allostimulated cells (Fig. 6A). Notably,
CD103⫹CD4⫹ T cells also showed regulatory capacity as has been
reported previously (26). Added at a 1:2 ratio to the MLC,
CD103⫹CD4⫹ T cells significantly inhibited the proliferation of
alloreactive cells (precursor frequency 1.03 ⫾ 0.48%) compared
with control cultures (precursor frequency 3.46 ⫾ 0.45%) (Fig.
2780
CD103⫹ ALLOANTIGEN-INDUCED CD8⫹ Tregs
FIGURE 5. Functional properties of alloreactive
CD103⫹CD8⫹ and CD103⫺CD8⫹subsets. A, Cytokine
profiles of the alloreactive CD103⫹CD8⫹ and
CD103⫺CD8⫹ T cell subsets. Sorted alloreactive
CD103⫹CD8⫹ or CD103⫺CD8⫹ T cell subsets were
restimulated with alloantigen for 5 days. The production
of the tolerogenic cytokines IL-10 and TGF-␤ and the
inflammatory cytokine IFN-␥ by purified subsets was
detected by ELISA. The bars represent mean ⫾ SD of
the concentration of IL-10, IFN-␥, or TGF-␤ in the culture supernatants. The mean percentage ⫾ SD from
three independent experiments is shown. Statistical significance is indicated by an asterisk (ⴱ, p ⬍ 0.05). B,
Intracellular cytokine expression. PBMC were labeled
with CFSE and stimulated with alloantigen. After 5 days
of culture, expression of the tolerogenic cytokine IL-10
and the inflammatory cytokine IFN-␥ was analyzed by
FACS. Dot plots represent alloreactive T cells gated on
CD8⫹ T cells. The x-axis shows log fluorescence of
CD103-bio-allophycocyanin; the y-axis shows log fluorescence of IL-10- and IFN-␥-PE. One representative
experiment of four is shown. C, Cytotoxic capacity of
CD103⫺CD8⫹ and CD103⫹CD8⫹ alloreactive T cells.
Cytotoxicity was measured by the percentage of 51Cr
release, as depicted on the y-axis. Alloreactive CTL
were generated in MLC after 5 days of allogeneic stimulation and CD103⫹CD8⫹ (⽧) and CD103⫺CD8⫹ alloreactive T cells (f) were sorted and used as effectors.
The cytotoxic activity of the total alloreactive lymphocyte pool is shown as Œ. Different numbers of alloreactive effector cells are depicted on the x-axis. One representative experiment of three is shown.
6B). Thus, the CD103⫹CD8⫹ subset showed a similar inhibitory
effect in in vitro suppressor assay as CD103⫹CD4⫹ cells. To examine whether suppressive capacity is an intrinsic characteristic of
alloactivated CD103⫹CD8⫹ T cells or whether it is a feature of all
CD8⫹ T cells bearing the CD103 molecule, we purified
CD103⫹CD8⫹ T cells from freshly isolated PBMC and tested
their inhibitory ability. Freshly isolated CD103⫹CD8⫹ T cells significantly inhibited proliferation of polyclonally stimulated PBMC
(combination of CD3 and CD28 Abs) at a regulator:responder ratio
1:2 whereas CD103⫺CD8⫹ T cells did not (data not shown).
We next analyzed whether the inhibitory effect of the
CD103⫹CD8⫹ subset is mediated by the immunosuppressive cytokines IL-10 and/or TGF-␤. Addition of anti-IL-10 or anti-TGF␤-neutralizing Abs to the in vitro suppressive assay did not affect
the inhibition of proliferation of alloactivated cells by the
CD103⫹CD8⫹ subset (Fig. 6C). In previous experiments, antiIL-10 and anti-TGF-␤ mAbs were shown to inhibit the function of
IL-10 and TGF-␤, respectively (data not shown). To address the
involvement of membrane-bound compounds, we studied the effect of cell-cell interaction on suppressive activity of
CD103⫹CD8⫹ T cells. We found that the inhibitory function of
this population depends on cell-cell contact as no suppression of T
cell proliferation was observed when the regulatory cells were separated from MLC by a Transwell insert (Fig. 6D).
In vitro-activated CD103⫹CD8⫹ T cells are Ag nonspecific in
their ability to suppress T cell proliferation
To investigate whether suppression by alloactivated
CD103⫹CD8⫹ T cells is Ag specific, we designed the following
experiments. For the generation of CD103⫹CD8⫹ regulatory cells,
we used PBMC from completely HLA-mismatched donors. Two
MLC combinations were used (responder X stimulated with Y and
responder X stimulated with Z), from which two types of regulatory CD103⫹CD8⫹ T cells were isolated by cell sorting (XY
CD103⫹CD8⫹ and XZ CD103⫹CD8⫹). Next, a suppressor assay
was performed in which responder PBMC (X) were labeled with
CFSE and cultured with irradiated allogeneic stimulator PBMC
(Y) for 5 days. When XY CD103⫹CD8⫹ T cells were added in
several regulator:responder ratios, the alloresponse was inhibited
by 60 – 80%. This alloresponse could also be inhibited in the presence of XZ CD103⫹CD8⫹ T cells, although to a lower extent (Fig.
6E), indicating that suppression of the immune response by
CD103⫹CD8⫹ cells was not dependent upon the presence of a
specific allopeptide in the culture. Addition of XZ CD103⫺CD8⫹
T lymphocytes as well as addition of XY CD103⫺CD8⫹ T cells
hardly affected the precursor frequency of allostimulated cells
(Fig. 6E).
Discussion
In the present study, we show that the integrin CD103 is up-regulated on human CD8⫹ T cells after stimulation by alloantigen in
vitro, which is dependent on soluble factors. In contrast, the upregulation of CD103 was far less after nonspecific polyclonal stimulation, suggesting a role for monocyte-derived factors in the induction of CD103 expression. CD103 appeared to be mainly
expressed on dividing CD8⫹ T cells, which, as we previously
showed, bear the phenotype of effector T cells (32). Up-regulation
of CD103 on in vitro-alloactivated T cells was augmented by
TGF-␤, IL-4, or IL-10 and diminished by IL-12. Secretion of the
tolerogenic cytokine IL-10 was slightly higher in the sorted
CD103⫹CD8⫹ T cell subset than in the CD103⫺CD8⫹ T cell subset, but CD103⫺CD8⫹ T cells secreted considerably higher
amounts of IFN-␥. CD103⫹CD8⫹ T cells were polyclonal, had a
low proliferative and cytotoxic capacity compared with
The Journal of Immunology
2781
FIGURE 6. CD103⫹CD8⫹ T cells have a strong regulatory capacity in vitro, which is not mediated by soluble IL-10 or TGF-␤ but appeared cell-cell
contact dependent and Ag-nonspecific. Purified CD103⫹CD8⫹, CD103⫺CD8⫹, CD103⫹CD4⫹, and CD103⫺CD4⫹ T cells were tested for their suppressive
activity in in vitro MLC. A, Responder PBMC were labeled with CFSE and cocultured with irradiated allogeneic stimulator PBMC. Purified CD103⫹CD8⫹
(u) or CD103⫺CD8⫹ (o) were added to the MLC at different regulator:responder ratios, namely 1:20, 1:10, 1:5, 1:2, and 1:1 (x-axis). As a positive control,
PBMC were stimulated with alloantigen only (f). On the y-axis, the mean percentage of precursor frequencies ⫾ SD from five separate experiments is
represented. Statistical significance is indicated by an asterisk (ⴱ, p ⬍ 0.05). B, Purified CD103⫹CD4⫹ (u) or CD103⫺CD4⫹ (o) T cells were added to
MLC at a regulator:responder ratio of 1:2. As a positive control, PBMC were stimulated with alloantigen only (f). The y-axis shows the mean percentage
of precursor frequencies ⫾ SD from two separate experiments. C, CFSE-labeled PBMC were cocultured in MLC together with purified CD103⫹CD8⫹ T
cells (u) at a regulator:responder ratio of 1:2 in the presence or absence of neutralizing Abs against IL-10 or TGF-␤. As a positive control, PBMC were
stimulated with alloantigen (f). The y-axis shows the mean percentage of precursor frequencies ⫾ SD from three independent experiments. D, Responder
PBMC labeled with CFSE and cocultured with irradiated allogeneic stimulator PBMC were plated in the bottom well of a Transwell system at a
concentration of 5 ⫻ 105 cells/ml. The top well insert was inoculated with culture medium alone or with purified CD103⫹CD8⫹ or CD103⫺CD8⫹ T cell
population as indicated. The y-axis shows precursor frequencies ⫾ SD from three independent experiments. Statistical significance is indicated by an
asterisk (ⴱ, p ⬍ 0.05). E, To evaluate Ag specificity of the CD103⫹CD8⫹ regulatory T cells, purified XY CD103⫹CD8⫹(u), XZ CD103⫹CD8⫹(䡺), XY
CD103⫺CD8⫹ (o), and XZ CD103⫺CD8⫹ (1) T lymphocytes were added to the MLC at different regulator/responder ratios, as depicted on the x-axis
(see explanation in Results). As a positive control, PBMC were stimulated with alloantigen only (f). The y-axis shows the mean percentage of precursor
frequencies ⫾ SD from three independent experiments. Statistical significance is indicated by an asterisk (ⴱ, p ⬍ 0.05).
CD103⫺CD8⫹ T cells, but displayed a strong suppressive activity
in the MLC. This suppressive effect of CD103⫹CD8⫹ T cells appeared not to be mediated by IL-10 or TGF-␤, but was found to be
cell-cell contact dependent. Addition of CD103⫹CD8⫹ T cells
stimulated by third-party cells inhibited the proliferative capacity
of alloactivated cells, though to a lesser extent than did alloantigen-specific CD103⫹CD8⫹ T cells.
CD103 is the receptor for E-cadherin, which is expressed on
epithelial cells, including renal tubular epithelial cells. As such, in
renal allografts CD103 may direct graft-infiltrating lymphocytes to
tubular epithelial cells, which are the main target for apoptotic cell
death during allograft rejection (33, 34). Independent from its role
in migration of immune cells, CD103 is also a marker of T cell
activation (35). In support of this, effector-memory tonsil-resident
2782
CD103⫹CD8⫹ T cells were found to be more reactive to their
cognate Ag than the CD103⫺CD8⫹ T cells, which permits rapid
recall responses at even low Ag concentrations, suggesting a role
of CD103 not only in homing and retention of T cells at epithelial
sites but also in promoting T cell function (36). Whether the
CD103 molecule also directly contributes to the regulatory function of natural Tregs (26) and CD8⫹ Tregs (this manuscript) is
unknown. Recently, CD103 was demonstrated to be a biomarker
for murine CD8⫹ suppressor T cells. These cells required IFN-␥ to
suppress CD4⫹ T cell division (37). A putative regulatory function
for CD103-expressing CD8⫹-alloreactive T cells appears to accord with the absence of CD103⫹CD8⫹ T cells in biopsies from
rat renal allografts undergoing unmodified acute rejection (38) and
from patients with acute rejection (39). In contrast, CD103⫹CD8⫹
T cells are mainly present in late allograft rejection, occurring
beyond the first 6 mo after transplantation, as well as in biopsies
with signs of chronic rejection (40). Thus, during ongoing, smouldering alloactivation in a predominant Th2 microenvironment, as
is the case during chronic allograft rejection (41), negative regulatory signals may prevent the alloresponse from inducing an acute
inflammatory reaction. In this respect, studies to a possible involvement of CD103⫹CD8⫹ T cells in subclinical allograft rejection, in which no deterioration of allograft function occurs, despite
the presence of a clear inflammatory cellular infiltrate, will be of
particular interest (34).
Whereas about half of the in vitro alloactivated CD8⫹ T cells
expressed the CD103 molecule, the tolerogenic cytokines TGF-␤,
IL-4, and IL-10 markedly increased the percentage and/or the intensity of CD103 expression. Other in vitro studies have also indicated an important role for TGF-␤ in the up-regulation of CD103
expression on CD8⫹ effector T cells (31). Hadley and colleagues
(42) demonstrated that up-regulation of CD103 expression by alloreactive CD8⫹ cells occurred subsequent to entry into the graft,
which was dependent on the local level of TGF-␤. The enhancing
effect of IL-4 on CD103 expression of allogeneic-stimulated
CD8⫹ T cells is in line with the described up-regulation of CD103
on both CD4⫹ and CD8⫹ cells after in vitro stimulation by CD3
mAbs in the presence of this cytokine (43). IL-4 may exert its
effect on CD103 expression directly or indirectly by induction of
TGF-␤ production as has been described for murine T cells (44).
However, we did not find a blocking effect of anti-TGF-␤ mAb on
the expression of CD103 induced by IL-4. Our finding of downregulation of CD103 expression on CD8⫹ T cells after allogeneic
stimulation in vitro in the presence of IL-12 was also observed
after stimulation of T cells with CD3 mAbs in the presence of this
cytokine (43). Altogether, these findings indicate that a Th2 more
than a Th1 microenvironment favors CD103 expression, which
supports its role in late and chronic rejection more than in acute
renal allograft rejection. CD103 might well be differentially expressed on T cytotoxic (Tc)1 and Tc2 CD8⫹ T cells, where
CD103-expressing CD8⫹ CTLs, possibly of the Tc2 type, are
more involved in a regulatory function than in a cytotoxic one.
Indeed, in agreement with data from the literature (22), we showed
that the cytotoxic capacity of CD103⫹CD8⫹ T cells against alloantigens is less than that of CD103⫺CD8⫹ T cells, indicating no
role for CD103 in allocytotoxic effector function.
In this study, we show that expression of V␤3, V␤10, and V␤20
was significantly reduced in the CD103⫹CD8⫹ alloreactive T cell
population as compared with that in the CD103⫺CD8⫹ T cell population. Thus, not all specificities are present within the alloreactive CD103⫹CD8⫹ T cell population. Several studies have provided evidence for a restricted V␤ gene usage in response to DR
synthetic peptides presented in the context of self MHC molecules,
i.e., allostimulation via the indirect pathway (45, 46). Preferential
CD103⫹ ALLOANTIGEN-INDUCED CD8⫹ Tregs
activation of T cell subsets by specific DR alleles may play an
important role in alloresponses as occur in MLRs and in organ
transplantation.
Tregs may exert their suppressive function by several mechanisms. Regardless of their mechanism of action, Tregs may operate
both at the level of T effector cells and at the level of APCs by
decreasing the expression of MHC class II and costimulatory molecules (6). Regarding CD8⫹ Tregs, IL-10 has been described to be
involved in their capacity to suppress T cell proliferation (47). The
CD103-bearing CD8⫹ alloreactive lymphocytes that we here describe produced IL-10 and TGF-␤, but could not be inhibited in
their immunosuppressive activity by anti-IL-10 or anti-TGF-␤
mAbs, suggesting an IL-10- and TGF-␤-independent mechanism
of suppression. In contrast, the suppressive effect of
CD103⫹CD8⫹ T cells appeared to be cell-cell contact dependent.
Recent reports have re-examined a role of cell death as a mechanism of suppression by Treg (48, 49). Grossman et al. (50) indicates that human CD4⫹CD25⫹ Treg mediate their suppressive effects via death induced by a granzyme A-perforin-dependent
mechanism. Granzyme B was shown to be involved in contactmediated suppression by murine CD4⫹CD25⫹ Treg (51). We
demonstrate that CD103⫹CD8⫹ alloreactive T cells were less cytotoxic than CD103⫺CD8⫹ T cells and therefore cytotoxicity is
not likely to be involved in the mechanism of suppression by
CD103⫹CD8⫹ T cells. Further studies are needed to elucidate
their mechanism of action. The absence of an appropriate cosignal,
the presence of immature or plasmacytoid dendritic cells, or the
presence of specific cell surface or soluble molecules may all be
operational (6). In contrast, high cytotoxicity of the CD103⫺CD8⫹
T cells might be an explanation for a slightly decreased proliferative capacity of allostimulated PBMC in the presence of sorted
allogeneic CD103⫺CD8⫹ T cells.
It is still unsolved whether Tregs need a second encounter with
the same peptide/Ag to become functional. In this study, we demonstrate that once activated, CD103⫹CD8⫹ alloreactive Tregs are
able to suppress T cell reactivity. It may be conceived that during
a local response, CD103 initially serves to retain alloantigen-induced CD8⫹ Treg cells at the graft site and later functions on
Tregs that exert their role as soon as alloantigen is recognized.
Acknowledgments
We thank Si-La Yong for her excellent technical support. We are grateful
to Drs. Eddy Wierenga and Ester M. M. van Leeuwen for critical reading
of the manuscript.
Disclosures
The authors have no financial conflict of interest.
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