Regulatory T cells (Treg) are important in maintaining tolerance to self tissues. As both CD28 and CTLA-4 molecules are implicated in the function of Treg, we investigated the ability of their two natural ligands, CD80 and CD86, to influence the Treg-suppressive capacity. During T cell responses to alloantigens expressed on dendritic cells, we observed that Abs against CD86 potently enhanced suppression by CD4+CD25+ Treg. In contrast, blocking CD80 enhanced proliferative responses by impairing Treg suppression. Intriguingly, the relative expression levels of CD80 and CD86 on dendritic cells are modulated during progression from an immature to a mature state, and this correlates with the ability of Treg to suppress responses. Our data show that CD80 and CD86 have opposing functions through CD28 and CTLA-4 on Treg, an observation that has significant implications for manipulation of immune responses and tolerance in vivo.

The controlled manipulation of immune responses is an important therapeutic goal. The ability to prevent T cell activation and establish long term tolerance is desirable in both transplantation and autoimmune disease settings. In contrast, heightened T cell activation is seen as a potential tumor therapy. The CD28-CTLA-4 pathway holds considerable promise for immunotherapy because it is clear that CD28 is a key activator, whereas CTLA-4 is a crucial attenuator, of T cell responses. CD28 is known to provide important stimulatory signals for T cells resulting in enhanced proliferation, cytokine production, and antiapoptotic signals. However, CD28 functions are opposed by the related receptor CTLA-4 (CD152) (see Refs. 1, 2, 3, 4 for review). The importance of CTLA-4 in limiting T cell activation is demonstrated by the fatal hyperactivation of T cells seen in CTLA-4-deficient mice (5), resulting in the destruction of a variety of tissues. This suggests that under conditions of normal homeostasis, engagement of CTLA-4 is important in preventing T cell activation to self-Ags. It is therefore somewhat paradoxical that CTLA-4 shares its only known ligands, CD80 and CD86, with CD28.

A major gap in our understanding of the CD28/CTLA-4 system has been the role played by CD80 and CD86. Both ligands are found on APCs and are known to provide efficient costimulation via CD28; however, they have distinct patterns of expression. In general, CD86 is thought to be more widely expressed and at higher levels than CD80 (6, 7, 8, 9, 10). In addition, the more immunodeficient phenotype of the CD86 knockout mouse has generated a strong perception that CD86 is the more important costimulator of T cell activation (11, 12). However, this view does not incorporate the role of CTLA-4 as a negative regulator, and an alternative possibility is that the absence (or decreased levels) of CD86 might affect the function of CTLA-4 via its alternative ligand CD80 (1). Thus, the relative levels of CD80 and CD86 expressed on APCs might affect the balance between CD28- and CTLA-4-dependent outcomes.

Recently, it has become clear that, uniquely, both CD28 and CTLA-4 are expressed constitutively on regulatory T cells (Treg)3 and can have substantial influence on their function (13, 14, 15, 16). As Treg are thought to suppress a variety of autoimmune diseases (17) and be involved in the tolerance to allografts (18, 19), understanding how these cells are regulated is of considerable significance. Intriguingly, there have been suggestions that the state of DC maturation can influence the development and function of some types of regulatory T cells (20) and promote T cell tolerance (21, 22).

With these issues in mind, we investigated the function of CD80 and CD86 in modulating the function of Treg via CD28 and CTLA-4. In response to allogeneic dendritic cells (DCs) we observed that blockade of CD86 potently inhibited CD4+ T cell responses, whereas anti-CD80 enhanced the responses. Surprisingly, the inhibitory effect of anti-CD86 required the presence of CD4+CD25+ T cells, and the removal of these cells prevented suppression. This suggested that stimulation of Treg via CD86 inhibits their suppressive function. Consistent with this view, alloresponses to cultured mature DCs (mDCs) that expressed high levels of CD86 were resistant to Treg suppression, whereas responses to immature DCs (iDCs) that expressed lower levels of CD86 were inhibited. Taken together, our data show that CD80 and CD86 have opposite roles in the functioning of Treg via CD28 and CTLA-4.

Human monocytes were purified from PBMC by negative selection using human monocyte enrichment mixture and magnetic colloid according to the instructions of the manufacturer (StemCell, Meylan, France). Briefly, PBMC were isolated from fresh buffy coats (provided by the National Blood Transfusion Service (Birmingham, U.K.)) using a Ficoll-Paque gradient. Cells were washed twice with PBS, then resuspended at 1 × 108 cells/ml in isolation buffer, and incubated with the monocyte enrichment Ab mixture at 4°C for 30 min. The cells were washed and subsequently incubated with magnetic colloid at 4°C for 30 min. Unlabeled monocytes passed through the MACS column and were collected. To generate iDCs, monocytes were cultured in RPMI 1640 medium containing 10% FCS and antibiotics with GM-CSF (PeproTech, Rocky Hill, NJ; 800 U/ml) and IL-4 (PeproTech; 500 U/ml) at concentration of 2 × 106 cells/ml. Half the medium was replaced every other day with GM-CSF- and IL-4-containing medium. Mature DCs were generated by stimulating iDCs with LPS (026:B6; Sigma-Aldrich, St. Louis, MO; 1 μg/ml) on day 5 for an additional 24 h.

CD4+CD25+ and CD4+CD25 T cells were separated using specific anti-CD25 microbeads (Miltenyi Biotec, Auburn, CA) and positive or negative selection, respectively. Initially, CD4+ T cells were purified by negative selection by incubating PBMC with human CD4+ T cell enrichment mixture and magnetic colloid according to the manufacturer’s instructions (Stemsep). CD4+ T cells were then resuspended in MACS buffer, incubated with CD25+ microbeads on ice for 30 min, washed, and loaded on the column. CD4+CD25 T cells, which did not bind to the column, were collected from the flow-through and washed before use. CD4+CD25+ T cells were subsequently retrieved from the column and washed before use.

For analysis of DC phenotype, DCs were collected in cold PBS and preincubated in 100 μl of rabbit serum at 37°C for 30 min to block FcRs. mAbs directly conjugated to FITC or PE were subsequently used against CD80, CD86, HLA-DR CD14, CD40, and CD83 (BD PharMingen, San Diego, CA). In time-course experiments, cells were collected at different time points during the culture of monocytes in GM-CSF and IL-4 and stained with FITC-labeled CD80 and CD86. Stained cells were analyzed on a FACScan flow cytometer using CellQuest software (BD Biosciences, Mountain View, CA). In cell sorting experiments CD4+CD25+ T cells were stimulated with PMA (5 ng/ml) for 2 h to induce CTLA-4 recycling. Cells were stained for CTLA-4 expression at 37°C using CTLA-4-PE (BN13; BD PharMingen) and sorted into positive and negative populations using a MoFlow cell sorter. Sorted cells were added to alloresponses as detailed below.

Primary DC-stimulated MLR was conducted in 96-well, U-bottom tissue culture plates in 200 μl of RPMI 1640 containing 10% FCS and antibiotics. DCs were mixed with 1 × 105 allogeneic total CD4+ T cells or CD4+CD25 T cells at a ratio between 1:10 and 1:100 DC:T cells. Cultures were also conducted in the presence or the absence of neutralizing mAbs: anti-human CD80 and CD86 (R&D Systems, Minneapolis, MN), anti-CD28 Fab′ (9.3; a gift from C. June (University of Pennsylvania, Philadelphia, PA)) or anti-CTLA-4 F(ab′)2 (Alexis) and used at 10 μg/ml. Assays were incubated for 5 days, and during the last 16 h [3H]thymidine was added at 1 μCi/well. [3H]thymidine incorporation was measured by scintillation counting, and proliferative responses were expressed as the mean [3H]thymidine incorporation (counts per minute) of triplicate wells ± SD. Counts due to DCs alone were routinely <1000 cpm. Results shown are representative examples of a minimum of five experiments performed.

CD4+CD25+ T cells (1 × 105) were preincubated with DCs (1 × 104) for 18 h in the presence of anti-CD80, anti-CD86, anti-CD28, anti-CTLA-4, or control mouse IgG (10 μg/ml) as shown. Cells were then washed and transferred to CD4+CD25 T cells (1 × 105), stimulated with allogeneic DCs (at a ratio of either 1:10 to 1:100), and incubated for 5 days. CD25+ and CD25 cells were always from the same donor. As controls, CD4+CD25 T cells were preincubated with DCs and then transferred to into CD4+CD25 alloresponses for 5 days. Proliferative responses were assessed by 3H incorporation) as detailed above. Results shown are representative examples of a minimum of six experiments performed.

To determine proliferation, Treg cells were washed twice with PBS and incubated with 2.5 μM CFSE for 10 min at room temperature, agitating gently every 2–3 min. The reaction was quenched by the addition of an equal volume of RPMI 1640 containing 10% FCS and incubation for 1 min. The tube was filled with PBS, and the cells were washed by centrifugation three times. The final cell pellet was made up to 2 × 106 cell/ml.

Chinese hamster ovary (CHO) cells transfected with CD80 and CD86 were generated and used as previously described (15). Before use, cells were fixed with 0.025% glutaraldehyde in PBS for 2–3 min, washed extensively with medium containing 10% FCS, and recounted.

To investigate the functional effects of CD80 and CD86 expression on DCs, we used allostimulation assays in which CD4+ T cells were stimulated with cultured DCs in the presence of blocking Abs. As shown in Fig. 1,a, although CD86 blockade was highly effective at inhibiting stimulation by mDCs, CD80 blockade was ineffective and even enhanced responses in a number of experiments. These effects were observed at two different DC:T cell ratios. Given the expression levels of CD80 and CD86 (Fig. 1,a, inset), these data seemed inconsistent with a simple model in which anti-CD86 blocked CD28 costimulation, because in the absence of CD86 interactions, CD80 should be able to compensate and provide costimulation through CD28. In control experiments both anti-CD80 and anti-CD86 Abs completely and specifically abolished costimulation by CD80 or CD86 transfectants, demonstrating their blocking ability over a range of Ab concentrations (Fig. 1,b). Furthermore, despite using doses of anti-CD80 that were 20-fold in excess of that required to block costimulation by transfectants, we did not observe inhibition using DCs (Fig. 1,c). We therefore investigated whether the differing effects of CD86 and CD80 blockade on DCs were due to effects other than blockade of CD28 costimulation. CD4+CD25+ cells were therefore depleted from the total CD4+ population. Strikingly, the inhibitory effect of CD86 blockade (Fig. 1 d) was abrogated in the absence of Treg. Thus, rather than simply inhibiting CD28 costimulation of CD25 T cells, CD86 blockade appeared to enhance the inhibitory function of Treg.

FIGURE 1.

Anti-CD86 inhibits alloresponses to cultured DCs by affecting CD4+ CD25+ T cells. Total CD4+ (a) or CD4+ CD25 (d) T cell proliferation to allogeneic mDCs was measured at 6 days by [3H]thymidine incorporation. Cultures were conducted at two different DC:T cell ratios and treated with control Ig (□) or Abs to CD80 (▪) or CD86 (▨) at 10 μg/ml. Bars represent the mean of triplicate wells ± SD. [3H]thymidine incorporation by DCs alone was routinely <1000 cpm. Analysis of CD80 and CD86 expression on mDC by FACS is shown in the inset to a. Filled histograms represent specific Ab staining compared with isotype controls (open histograms). b and c, Titrations of anti-CD80 and -CD86 Abs were performed to confirm costimulation blockade. Purified CD4 T cells were stimulated with anti-CD3 (1 μg/ml) and transfectants expressing either CD80 or CD86 in the presence of blocking Abs (b). c, Alloresponses stimulated by mDCs were incubated in the presence of the blocking Abs shown. Responses in b and c are shown as a percentage of the maximal [3H]thymidine incorporation without Ab blockade. The experiments shown are representative of three to five independent experiments.

FIGURE 1.

Anti-CD86 inhibits alloresponses to cultured DCs by affecting CD4+ CD25+ T cells. Total CD4+ (a) or CD4+ CD25 (d) T cell proliferation to allogeneic mDCs was measured at 6 days by [3H]thymidine incorporation. Cultures were conducted at two different DC:T cell ratios and treated with control Ig (□) or Abs to CD80 (▪) or CD86 (▨) at 10 μg/ml. Bars represent the mean of triplicate wells ± SD. [3H]thymidine incorporation by DCs alone was routinely <1000 cpm. Analysis of CD80 and CD86 expression on mDC by FACS is shown in the inset to a. Filled histograms represent specific Ab staining compared with isotype controls (open histograms). b and c, Titrations of anti-CD80 and -CD86 Abs were performed to confirm costimulation blockade. Purified CD4 T cells were stimulated with anti-CD3 (1 μg/ml) and transfectants expressing either CD80 or CD86 in the presence of blocking Abs (b). c, Alloresponses stimulated by mDCs were incubated in the presence of the blocking Abs shown. Responses in b and c are shown as a percentage of the maximal [3H]thymidine incorporation without Ab blockade. The experiments shown are representative of three to five independent experiments.

Close modal

To directly assess the effect of CD80 and CD86 blockade on Treg function, we performed experiments on purified CD25+ T cells. CD4+CD25+ T cells were therefore preincubated in the presence (Fig. 2,a) or the absence (Fig. 2,b) of DCs and blocking CD80 or CD86 Abs. These pretreated Treg were then washed to remove the Ab and tested for their capacity to suppress alloresponses. The results of this experiment (Fig. 2 a) revealed that the addition of stimulated Treg that had not been treated with blocking Ab (control Ig) modestly inhibited the responses of CD25 cells. However, pretreatment of Treg with anti-CD86 Ab enhanced suppression, whereas pretreatment with anti-CD80 Ab diminished the inhibitory effects of Treg. Both these effects required the presence of DCs, ruling out the possibility that the CD80 and CD86 Abs acted directly on the Treg. Pretreatment of DCs with Abs in the absence of Treg did not modulate suppression (data not shown), indicating that the effects were not due to Ab carried over into the second culture or to alterations in DC phenotype due to Ab-induced signaling. We therefore concluded that the interaction of Treg with CD86 on DCs inhibited their function (as CD86 blockade enhanced suppression) and that CD80 interaction with Treg promoted suppression (as CD80 blockade reversed suppression). As the Abs were only present during the initial 18-h Treg contact with DCs, this suggested that the ability of Treg to suppress was acquired during this period.

FIGURE 2.

CD80 and CD86 Abs directly affect CD25+ T cell:DC interactions. CD25+ T cells were incubated in the presence (a) or the absence (b) of stimulating mDCs in the presence of the Abs shown for 18 h. Cells were then washed and added to a CD25 alloresponse at a ratio of 1:1 CD25+:CD25 T cells. The CD25 alloresponse with control CD25 cells added is shown (CD25). Proliferation was measured by [3H]thymidine incorporation on day 6. Bars represent the mean of triplicate wells ± SD. The experiment is representative of more than five experiments performed.

FIGURE 2.

CD80 and CD86 Abs directly affect CD25+ T cell:DC interactions. CD25+ T cells were incubated in the presence (a) or the absence (b) of stimulating mDCs in the presence of the Abs shown for 18 h. Cells were then washed and added to a CD25 alloresponse at a ratio of 1:1 CD25+:CD25 T cells. The CD25 alloresponse with control CD25 cells added is shown (CD25). Proliferation was measured by [3H]thymidine incorporation on day 6. Bars represent the mean of triplicate wells ± SD. The experiment is representative of more than five experiments performed.

Close modal

Given that CD80 and CD86 appeared to be differentially controlling Treg function, we investigated how this might be mediated by their receptors, CD28 and CTLA-4. Treg were therefore primed with DCs in the presence of anti-CTLA-4 and anti-CD28 Abs (either whole or F(ab′)2), washed, and used to suppress alloresponses as before. This demonstrated that blocking CTLA-4 using F(ab′)2, during Treg contact with DCs, reversed their suppressive activity, whereas whole anti-CTLA-4 Ab had no blocking activity (Fig. 3,a). In contrast, blocking anti-CD28 Fab′ Ab potentiated the suppressive activity of Treg in a manner similar to that of anti-CD86 (Fig. 3,b). As the expression of CTLA-4 on purified CD25+ cells is not homogeneous, we hypothesized that CTLA-4 expression should correlate with suppressive activity. We therefore performed cell-sorting experiments in which CD4+ CD25+ T cells were stimulated to induce surface CTLA-4 expression and then sorted into CTLA-4-positive and -negative populations (Fig. 3 c). The suppressive functions of these cells were then assessed in alloresponses as before. This experiment clearly demonstrated that CTLA-4+ Treg were substantially more suppressive than CTLA-4 Treg, consistent with a role for CTLA-4 in Treg function. Together, our data demonstrated that blocking CD86 or CD28 enhanced Treg function and, conversely, that blocking CD80 or CTLA-4 limited the suppressive activity of Treg. We therefore concluded that CD80 and CD86 have distinct functional interactions with CTLA-4 and CD28 on Treg

FIGURE 3.

CTLA-4 and CD28 modulate suppression by CD25+ cells. CD25+ T cells were pretreated for 18 h with DCs and anti-CTLA-4 Abs (a) or anti-CD28 Abs (b) and compared with anti-CD80 or anti-CD86 blockade. All Abs were used at at10 μg/ml. Pretreated cells were washed and tested for suppression of an alloresponses between CD25 T cells and DCs (using a ratio of 1:1 CD25+:CD25 T cells). c, For CTLA-4 sorting CD4+CD25+ T cells were stimulated with PMA (5 ng/ml) for 2 h, labeled with anti-CTLA-4-PE, and sorted into positive and negative populations as shown. The upper panel shows CTLA-4 expression before sorting (bold line, stimulated cells; dashes, unstimulated; thin line, isotype control), and the lower panel shows the positive and negative populations after sorting. Sorted cells were added to a CD25 alloresponse at the above ratio. DCs were present at 1:10 or 1:100 T cells. Proliferation was measured by [3H]thymidine incorporation. Bars represent the mean of triplicate wells ± SD. Data are representative of three experiments performed.

FIGURE 3.

CTLA-4 and CD28 modulate suppression by CD25+ cells. CD25+ T cells were pretreated for 18 h with DCs and anti-CTLA-4 Abs (a) or anti-CD28 Abs (b) and compared with anti-CD80 or anti-CD86 blockade. All Abs were used at at10 μg/ml. Pretreated cells were washed and tested for suppression of an alloresponses between CD25 T cells and DCs (using a ratio of 1:1 CD25+:CD25 T cells). c, For CTLA-4 sorting CD4+CD25+ T cells were stimulated with PMA (5 ng/ml) for 2 h, labeled with anti-CTLA-4-PE, and sorted into positive and negative populations as shown. The upper panel shows CTLA-4 expression before sorting (bold line, stimulated cells; dashes, unstimulated; thin line, isotype control), and the lower panel shows the positive and negative populations after sorting. Sorted cells were added to a CD25 alloresponse at the above ratio. DCs were present at 1:10 or 1:100 T cells. Proliferation was measured by [3H]thymidine incorporation. Bars represent the mean of triplicate wells ± SD. Data are representative of three experiments performed.

Close modal

The above data suggested a model in which the relative expression levels of CD80 and CD86 on DCs could potentially modulate the potency of Treg via differential interactions with CD28 and CTLA-4. We therefore analyzed the expression patterns of CD80 and CD86 during the culture and maturation of monocyte-derived DCs to determine whether this might influence Treg behavior. This revealed clear changes in the expression patterns of these molecules (Fig. 4). Firstly, we observed that the initial PBMC population was CD86 positive and CD80 negative. However, in culture with GM-CSF and IL-4, we noted that by 24 h CD80 began to be expressed, whereas CD86 expression began to diminish. By 96 h the cells resembled iDCs and expressed substantial levels of CD80, but lower levels of CD86. To establish that these cells could progress further into mature DCs, the cells were stimulated with LPS. This clearly showed substantial up-regulation of CD86 by 24 h (Fig. 4,a) and that the mature cells up-regulated CD83 as well as CD40 and MHC class II (Fig. 4 b).

FIGURE 4.

Expression patterns of CD80 and CD86 on iDCs and mDCs. a, DCs were derived from PBMC, and CD80 and CD86 expression was measured at various times in culture in GM-CSF and IL-4. DCs were matured by the addition of LPS for 24 h (mDC). Cells were stained using specific Abs shown (filled histograms), compared with a fluorochrome-matched isotype control Ab (open histograms), and analyzed by FACS analysis. b, iDC and mDC were assessed for CD83, CD40, and MHC class II molecule expression. Data are representative of >10 independent experiments performed.

FIGURE 4.

Expression patterns of CD80 and CD86 on iDCs and mDCs. a, DCs were derived from PBMC, and CD80 and CD86 expression was measured at various times in culture in GM-CSF and IL-4. DCs were matured by the addition of LPS for 24 h (mDC). Cells were stained using specific Abs shown (filled histograms), compared with a fluorochrome-matched isotype control Ab (open histograms), and analyzed by FACS analysis. b, iDC and mDC were assessed for CD83, CD40, and MHC class II molecule expression. Data are representative of >10 independent experiments performed.

Close modal

Based on this expression pattern, we reasoned that the suppressive capacity of Treg should be greater when stimulated by iDCs than by mDCs, because the levels of CD86 are lower. To test this hypothesis, alloresponses were established using CD25 T cells and either iDC or mDC. Treg were then added to these cultures at different ratios of Treg:T cells. The results of this experiment revealed that adding Treg to iDC (Fig. 5,a) significantly suppressed their proliferation at a ratio of 1:1 Treg:T cells. In contrast, using mDCs (Fig. 5,b), little suppression was observed. To confirm that CD86 was involved in abrogating suppression by mDC, blocking Abs were added. This revealed (Fig. 5 c) that blockade of CD86 on mDC could indeed restore Treg suppression. Overall, this indicated that Treg suppression is significantly influenced by DC maturation and reflects the balance between CD80 and CD86 expression.

FIGURE 5.

Alloresponses stimulated by iDCs are more effectively suppressed by CD25+ T cells than those of mDCs. Six-day alloresponses were established using CD25 T cells iDCs (a) or mDCs (b). CD25+ T cells were titrated into these cultures at the ratios of CD25+ T cells:CD25 T cells shown to assess potency. Experiments were conducted at a DC:T cell ratio of either 1:10 or 1:100. Responses were measured by [3H]thymidine incorporation. Bars represent the mean of triplicate wells ± SD. c, mDC were used to stimulate responses as described in b at a ratio of 1:1 CD25:CD25+ in the presence of Abs to CD80 (▪), CD86 (▨), or control Ig (□). Data are representative of between three and five experiments.

FIGURE 5.

Alloresponses stimulated by iDCs are more effectively suppressed by CD25+ T cells than those of mDCs. Six-day alloresponses were established using CD25 T cells iDCs (a) or mDCs (b). CD25+ T cells were titrated into these cultures at the ratios of CD25+ T cells:CD25 T cells shown to assess potency. Experiments were conducted at a DC:T cell ratio of either 1:10 or 1:100. Responses were measured by [3H]thymidine incorporation. Bars represent the mean of triplicate wells ± SD. c, mDC were used to stimulate responses as described in b at a ratio of 1:1 CD25:CD25+ in the presence of Abs to CD80 (▪), CD86 (▨), or control Ig (□). Data are representative of between three and five experiments.

Close modal

Recent reports suggest that CD25+ Treg can proliferate in response to DCs (23, 24). We therefore investigated whether CD80 or CD86 blockade might also influence this response. Treg were labeled with CFSE, and their division was measured in response to DCs. This revealed (Fig. 6) that mDC induced more T cells to divide than iDC. However, substantial numbers of T cells remained undivided, consistent with the nature of allostimulation. However, when mDC were used in the presence of blocking CD80 and CD86 Abs, we observed that blocking CD86 inhibited division, whereas blocking CD80 enhanced Treg division. These findings suggested that CD80 and CD86 engagement had opposite effects on Treg division as well as suppressive function.

FIGURE 6.

CD80 and CD86 differentially modulate CD25+ T cell division. CFSE-labeled Treg cells were incubated with mDC or iDC in an allostimulation assay for 6 days (a). The effects of anti-CD80 and anti-CD86 Abs (10 μg/ml) on division stimulated by mDC were studied (b). Cell division was monitored by flow cytometry after 6 days.

FIGURE 6.

CD80 and CD86 differentially modulate CD25+ T cell division. CFSE-labeled Treg cells were incubated with mDC or iDC in an allostimulation assay for 6 days (a). The effects of anti-CD80 and anti-CD86 Abs (10 μg/ml) on division stimulated by mDC were studied (b). Cell division was monitored by flow cytometry after 6 days.

Close modal

It is clear that T cell tolerance to self-Ags is not purely a consequence of T cell deletion in the thymus, but that other mechanisms exist to allow discrimination between pathogen-associated TCR signaling events and those generated by our own tissues. Considerable evidence suggests that this relates to the recognition of pathogen-associated molecules, or so-called danger signals, which are recognized by cells of the innate immune system such as DCs. DC maturation occurs as a consequence of recognition of these pathogen-associated molecules via a variety of Toll-like receptors (25, 26), but exactly how this information is subsequently communicated to T cells is not entirely clear. It is generally thought that increased expression of MHC class II and costimulatory molecules such as CD86 is important in this process. However, CD86 and its relative, CD80, bind to both stimulatory (CD28) and inhibitory (CTLA-4) receptors on T cells, raising the significant question of how a stimulatory CD28 signal, as opposed to an inhibitory CTLA-4 signal, is ensured. The present study demonstrates that CD86 expression regulates this decision not as a result of enhanced costimulation, but by diminishing the capacity of Treg to suppress responses.

Our data support a model in which the relative expression levels of CD80 and CD86 on DCs could dictate the balance between stimulatory and inhibitory outcomes by modifying the potency of Treg. This scenario is consistent with the fact that CD86 is highly responsive to danger signals such as LPS, CFA, and many other inflammatory stimuli (12) that enhance T cell responses. Recently, others have also suggested that DC maturation can influence the inhibitory effects of Treg (27). However, in contrast to our findings, these studies showed that cytokine signals, possibly mediated by IL-6, could limit the ability of the responder T cells to be regulated by Treg. Combined with our data this suggests that the ability of Treg to suppress T cell responses may be dictated by both signals received by the Treg themselves and the status of the responder T cells.

Our experiments clearly indicate distinct functions for CD80 and CD86. The differential functions of these molecules have been the subject of considerable study, with most data suggesting that the two ligands share substantially overlapping functions (4, 12, 28). However, it seems likely that such studies measured predominantly CD28-dependent activation of CD25 T cells, in which both CD80 and CD86 appear to perform similarly. There is, however, increasing evidence to support the view that CD80 may be a more effective ligand for CTLA-4 than CD86. Our own studies, directly comparing the ability of CD80 and CD86 transfectants to stimulate T cell responses, indicate that CTLA-4 inhibition is only observed with CD80 as the ligand (15). In addition, in transplantation models it is clear that CD80 is the effective ligand for CTLA-4 in preventing rejection (29, 30). Furthermore, other evidence, such as the ability of Abs to CD80 to exacerbate disease in NOD mice (31) and the tolerogenic potential of CD80-expressing, but not CD86-expressing, tumors (32), is consistent with the view that CD80 may be the more effective ligand for CTLA-4.

Our data also revealed that both CD28 and CD86 blockade had similar effects on Treg function, indicating that CD28 is primarily involved in sensing the signals from CD86. Consistent with this observation Sakaguchi et al. (33) showed that CD28 stimulation could abolish the inhibitory capacity of CD25+ regulatory T cells. It therefore seems likely that CD86 represents a CD28-biased ligand. In contrast, although CD80 is certainly an effective CD28 ligand, its stimulatory effects are substantially opposed by its interactions with CTLA-4, which is expressed on Treg after contact with DCs. This interpretation also receives strong support from recent biophysical data (34) showing that CD80-CTLA-4 interactions are likely to be highly favored compared with CD86. Further data consistent with a role for CD28 on Treg have been obtained from NOD×CD28−/− mice (16), which have exacerbated diabetes due to a lack of Treg. Thus, CD28 signals are probably important for the expansion and/or survival of Treg, a role that we would suggest requires CD86 engagement. This concept gains further support from our observation that mDCs, which express higher levels of CD86 than iDCs, are more effective at driving Treg proliferation. Here again, our experiments revealed that CD80 and CD86 have opposing roles in influencing Treg proliferation.

In contrast to the roles of CD86 and CD28, we observed that both CD80- and CTLA-4-blocking Abs enhanced T cell responses to DCs, indicating that CD80 acts as a ligand for CTLA-4 on Treg and is involved in their suppressive function. The role of CTLA-4 in Treg function is somewhat controversial, because it appears to play a role in some models (13, 14, 18, 19), but not in others (35, 36). In our experiments both CD80 and CTLA-4 Abs inhibited suppression by Treg. However, these effects were less pronounced than the enhancement of suppression observed with CD28 and CD86 Abs. We believe that this is due to the difficulty of disrupting CD80-CTLA-4 interactions. Firstly the affinity of this interaction is 100-fold greater than that of CD86-CD28 and possibly as great as 10,000-fold if the bivalent nature of CTLA-4 binding is taken into account (34). Secondly, delivery of CTLA-4 directly from an intracellular compartment to the immune synapse (37) makes it very inaccessible to blockade by Ab. Accordingly, in our experience only F(ab′)2, but not whole CTLA-4 Abs, are effective at blockade, because these have sufficient overall avidity combined with the small physical size required for access to the immune synapse. Our demonstration that CTLA-4+ Treg are more suppressive than CTLA-4 Treg provides further strong support for a role for CTLA-4 in the function of Treg. Finally, consistent with a role for CD80 and CTLA-4 in Treg function, it is interesting to note that CD80-deficient NOD mice have exacerbated diabetes, where it is known that Treg play a role in protection (2).

The CD28-CTLA-4 pathway has been extensively targeted in immunotherapy and is therefore of considerable clinical relevance (38, 39, 40). The most well-developed reagent, CTLA-4-Ig, a soluble antagonist of both CD80 and CD86, has now been used in human clinical trials (41, 42). However, a potential limitation of this reagent is that it has the capacity to interfere with the natural inhibitory functions of CTLA-4 as well as inhibit the activating function of CD28. Thus, CTLA-4-Ig may be less effective than reagents that leave the natural inhibitory function of CTLA-4 intact. In other strategies, CD80 and CD86 have been targeted to tumors in an attempt to stimulate T cell responses. However, as there is evidence that CTLA-4 is involved in suppressing responses to tumors (43, 44, 45, 46), a knowledge of how CD80 and CD86 ligands interact with CD28 and CTLA-4 is essential to avoid immune suppression rather than stimulation. Thus, there is a need to clearly define the roles of CD80 and CD86 and their contributions to both stimulating and inhibiting T cell responses. Our data now present clear evidence that for human T cells, CD86-CD28 interactions represent a potent signal that interferes with the inhibitory function of Treg, whereas CD80 ligation promotes regulatory function by interacting with CTLA-4. These observations provide further evidence of differential functions of CD80 and CD86 and suggest approaches for the development of more rational immunotherapies.

We thank Drs. Peter Lane and Lucy Walker for helpful comments.

1

This work was supported by the Wellcome Trust (to Y.Z.) and the Arthritis Research Campaign (to D.M.S., C.N.M., and M.L.). D.M.S. is an Arthritis Research Campaign Senior Fellow. K.M. is an Medical Research Council Ph.D. student.

3

Abbreviations used in this paper: Treg, regulatory T cell; CHO, Chinese hamster ovary; DC, dendritic cell; mDC, mature DC.

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