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Medical Research Council Center for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom
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Abstract |
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Introduction |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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 x 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.
Purification of human CD4+CD25+ and CD4+CD25- T cells
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.
Flow cytometry
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.
Allostimulation
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 x 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 cell assays
CD4+CD25+ T cells (1 x 105) were preincubated with DCs (1 x 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 x 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.
CFSE labeling
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 x 106 cell/ml.
Transfectants
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.
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Results |
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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. 1a, 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. 1a, 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. 1b). 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. 1c). 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. 1d) 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.
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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. 3a). In contrast, blocking anti-CD28 Fab' Ab potentiated the suppressive activity of Treg in a manner similar to that of anti-CD86 (Fig. 3b). 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. 3c). 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
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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. 4a) and that the mature cells up-regulated CD83 as well as CD40 and MHC class II (Fig. 4b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 NODxCD28-/- 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David M. Sansom, Medical Research Council Center for Immune Regulation, University of Birmingham Medical School, Vincent Drive, Birmingham, U.K. B15 2TT. E-mail address: d.m.sansom{at}bham.ac.uk
3 Abbreviations used in this paper: Treg, regulatory T cell; CHO, Chinese hamster ovary; DC, dendritic cell; mDC, mature DC.
Received for publication August 25, 2003. Accepted for publication December 18, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) 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.
