The αEβ7 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 CD4+ regulatory T cells. Approximately 4% of circulating CD8+ T cells bear the CD103 molecule. In this study, we show that the absence or presence of CD103 was a stable trait when purified CD103− and CD103+CD8+ T cell subsets were stimulated with a combination of CD3 and CD28 mAbs. In contrast, allostimulation induced CD103 expression on ∼25% of purified CD103−CD8+ T cells. Expression of CD103 on alloreactive cells was found to be augmented by IL-4, IL-10, or TGF-β and decreased by addition of IL-12 to MLCs. The alloantigen-induced CD103+CD8+ T cell population appeared to be polyclonal and retained CD103 expression after restimulation. Markedly, in vitro-expanded CD103+CD8+ 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 CD103+CD8+ T cells possess functional features of regulatory T cells.
Increasing 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, 2, 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-Ag-reactive 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-β 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-β (8, 9, 10).
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, 14, 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-β and/or by a direct inhibitory action on dendritic cells (16).
The αEβ7 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, 18, 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-γ and maintain their phenotype after restimulation with alloantigen. Thus, CD103 represents a novel marker for a subset of alloantigen-induced regulatory CD8+ T cells.
Materials and Methods
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.
Biotinylated CD103 mAb and Vβ3-FITC were purchased from Immunotech and CD103-PE from Caltag Laboratories. CD4− and CD8-PerCP Cy5.5, and CD27-, CD28-, CCR7-, IFN-γ-, 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.
7 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
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: [Σn ≥ 1(Pn/2n)]/[Σn ≥ 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).
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 μg/ml), and monensin (1 μM) (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-γ-PE Abs.
Evaluation of cytokine production by ELISA
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.
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+
To evaluate the role of cell-cell contact in suppressive activity, 24-well plates equipped with a Transwell insert (Costar) consisting of a 200-μl upper well separated from an 800-μl bottom well by a 0.4-μm 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.
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.
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.
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. 1⇓A). 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. 1⇓B). In contrast, in alloantigen-stimulated cultures, an up-regulation of the CD103 molecule was found on CD103−CD8+ T cells since 31.44 ± 8.49% (mean ± SD of nine donors tested) of these cells acquired CD103 expression. Additionally, Fig. 1⇓B 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. 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. 1⇓CII), 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. 1⇓CIII). 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. 1⇓CIV).
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. 2⇓C). Expression increased gradually with a peak on day 5 (53.63 ± 13.81%, Fig. 2⇓C). 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. 2⇓B). Thereafter, the expression of CD103 in both T cell subsets slightly decreased, yielding 37.77 ± 16.09% CD103+CD8+ (Fig. 2⇓C) 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. 2⇓D).
CD103 expression on alloreactive CD8+ T cells is increased in the presence of TGF-β, 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-β to MLC increased the percentage of CD8+ T cells expressing CD103 to >90% (Fig. 3⇓A). 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. 3⇓B). 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. 3⇓B); however, the amount of CD103 expressed per cell was clearly increased as evidenced by the higher intensity of CD103 staining (Fig. 3⇓C). To assess the role of TGF-β 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-β mAb (final concentration 2 μg/ml). As a control, MLCs were performed in the presence of rTGF-β in a final concentration of 1 ng/ml together with the anti-TGF-β mAb in a final concentration of 2 μg/ml. Addition of the blocking anti-TGF-β mAb did not influence the amount of CD103 expressed on alloreactive T cells induced by IL-4 or IL-10. Likewise, TGF-β-production was not influenced by the addition of 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-α, or IL-17 did not significantly affect CD103 expression (Fig. 3⇓B).
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 Vβ usage, stability, and functional properties of this subset.
The TCR Vβ 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. 4⇓A, although somewhat less diverse than the CD103−CD8+ fraction, CD103+CD8+ T cells used a broad spectrum of Vβ 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 Vβ3, Vβ10, and Vβ20 was significantly reduced in the CD103+CD8+ T cell population (Fig. 4⇑A). The lower expression of Vβ3 by CD103+CD8+ T cells was confirmed by FACS analysis (Fig. 4⇑B). 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-β were produced by both subsets, whereas IFN-γ was significantly higher in the supernatants of CD103−CD8+ T cells (Fig. 5⇓A). In accordance with this observation, we found that production of IFN-γ, determined by FACS, predominantly correlated with the absence of CD103 expression on T cells (53.87 ± 8.03% of CD103−CD8+ cells produced IFN-γ vs 14.89 ± 4.91% of CD103+CD8+ T cells, Fig. 5⇓B).
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. 5⇑C, 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-γ, 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. 6⇓A). Addition of CD103−CD8+ cells to the culture did not influence the precursor frequencies of allostimulated cells (Fig. 6⇓A). 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. 6⇓B). 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-β. Addition of anti-IL-10 or anti-TGF-β-neutralizing Abs to the in vitro suppressive assay did not affect the inhibition of proliferation of alloactivated cells by the CD103+CD8+ subset (Fig. 6⇑C). In previous experiments, anti-IL-10 and anti-TGF-β mAbs were shown to inhibit the function of IL-10 and TGF-β, 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. 6⇑D).
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. 6⇑E), 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. 6⇑E).
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 up-regulation 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-β, 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-γ. CD103+CD8+ T cells were polyclonal, had a low proliferative and cytotoxic capacity compared with 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-β, 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 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-γ 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-β, 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-β 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-β. 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-β production as has been described for murine T cells (44). However, we did not find a blocking effect of anti-TGF-β mAb on the expression of CD103 induced by IL-4. Our finding of down-regulation 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 Vβ3, Vβ10, and Vβ20 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 Vβ 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 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-β, but could not be inhibited in their immunosuppressive activity by anti-IL-10 or anti-TGF-β mAbs, suggesting an IL-10- and TGF-β-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 contact-mediated 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.
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.
The authors have no financial conflict of interest.
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, L1-110, 1105 AZ Amsterdam, The Netherlands. E-mail address:
↵2 Abbreviations used in this paper: Treg, T regulatory cell; Tr1, T regulatory 1; pen/strep, penicillin/streptomycin; Tc, T cytotoxic.
- Received December 5, 2005.
- Accepted May 17, 2006.
- Copyright © 2006 by The American Association of Immunologists