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‘Anergic’ T cells Modulate the T-cell Activating Capacity of Antigen-presenting Cells
doi:10.1006/jaut.2000.0372, available online at http://www.idealibrary.com on
Journal of Autoimmunity (2000) 14, 335–341
‘Anergic’ T cells Modulate the T-cell Activating Capacity of Antigen-presenting Cells Leonie S. Taams*, Elmieke P. J. Boot, Willem van Eden and Marca H. M. Wauben Institute of Infectious Diseases and Immunology, Department of Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht, The Netherlands
Received 20 January 2000 Accepted 28 February 2000 Key words: anergy, tolerance, immunoregulation, APC, Lewis rat
Nowadays there is compelling evidence for immunoregulation by T cells. Recently, we showed that so-called ‘anergic’ T cells are not functionally inert but can act as regulatory cells by actively suppressing other T cell responses. We now show that ‘anergic’ T cells mediate this suppressive effect via modulation of the T-cell activating capacity of the antigen-presenting cell (APC). Upon removal of the ‘anergic’ T cells, the suppressive APC phenotype persisted, indicating that ‘anergic’ T cells conditioned the APC to become a mediator of T cell suppression. The inhibitory signal delivered by ‘anergic’ T cells depended on the presence of the cognate ligand for the ‘anergic’ T cell, and appeared to be dominant since previously activated APC were rendered inhibitory as well. These findings imply that APC upon cross-talk with T cells can adopt distinct functional phenotypes ranging from T-cell stimulatory to T-cell suppressive. The contribution of ‘anergic’ T cells to the functional tuning of APC offers an explanation for the maintenance of ‘anergic’ T cells in the repertoire, and for their role in immunoregulation. © 2000 Academic Press
Introduction
immunoregulatory effects, such as Th2 cells [15], IL-10 or TGF- producing cells [16–19], CD45RClow cells [20] and CD4+CD25+T cells [21, 22]. Recently, it was shown that so-called ‘anergic’ T cells can function as regulatory T cells in vitro and in vivo by suppressing the responses of other T cells [23–27]. Until now it was unclear whether ‘anergic’ T cells mediate their immunoregulatory effects via direct inhibition of the responder T cells or via modulation of the stimulatory capacity of the APC. Here we show that CD4+T cells rendered ‘anergic’ through T-T presentation actively downregulate the T cell stimulatory capacity of the APC in an antigen (Ag)-specific manner. Upon removal of the ‘anergic’ T cells, the suppressive phenotype of APC persisted, indicating that ‘anergic’ T cells turned the APC into a tolerogenic mode. We propose that ‘anergic’ T cells are of critical importance in the immune repertoire for the functional tuning of APC, and that the APC after cross-talk with the surrounding cells and factors act as central regulator of the immune response.
Downregulation of immune responses is essential in order to prevent chronic inflammation and to maintain peripheral tolerance. Several mechanisms can contribute to the resolution of the immune response such as T cell apoptosis [1], induction of T cell suppressive cytokines (e.g. IL-10) [2], and T cell anergy [3]. Besides T cells, antigen-presenting cells (APC) may also play a key role in limiting the immune response. Indeed, evidence is accumulating that tolerogenic APC exist [4–6]. Such APC might be induced by factors such as IL-4, IL-10, IL-13 or TGF- in the microenvironment, leading to tolerogenic APC exhibiting a localized suppressive effect, e.g. in the lung or the eye [7–9]. Furthermore, it has been shown that pathogenic micro-organisms can modulate APC function in order to evade their eradication [10–12]. Interestingly, tolerogenic APC might also be induced due to cross-talk between professional antigenpresenting cells (APC), responder T cells and regulatory T cells present in the same cellular cluster [13,14]. Several T cell subsets have been proposed to mediate
Materials and Methods Correspondence to: Dr M.H.M. Wauben, Institute of Infectious Diseases and Immunology, Department of Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508 TD Utrecht, the Netherlands. Fax: +31 30 253 3555. E-mail: [email protected] * Present working address: Dept. Clinical Immunology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK.
Rats, T cells and Ag Seven to ten-week-old specific pathogen free male inbred Lewis rats (RT1.L) were obtained from the University of Limburg (Maastricht, the Netherlands). The isolation, maintenance and properties of the CD4+T cell clones A2b, specific for Mycobacterium 335
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tuberculosis (Mt) 65 kD heat shock protein (HSP65) peptide 176–190, and Z1a, specific for myelin basic protein (MBP) peptide MBP72–85 and the stronger MHC class II (RT1.BL) binding peptide analog MBP72–85S79A (S79A), have been described previously [28–31]. In vitro culture of APC and ‘anergic’ T cells Anergy was induced through T-T presentation as described previously [25]. In brief, A2b or Z1a T cells (3×106 cells/ml) were incubated with their respective stimulatory peptides 176–190 (10 g/ml) or S79A (50 g/ml) in the absence of professional APC. After overnight culture, viable T cells were collected by Ficoll-Isopaque gradient centrifugation, and maintained in culture medium (Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with L-Glutamine (2 mM), -mercaptoethanol (50 M), penicillin (50 U/ml), streptomycin (50 U/ml) and 2% heat-inactivated normal rat serum, or 10% heatinactivated fetal calf serum) in the absence of exogenous rIL-2. ‘Anergic’ T cells were added to APC cultures 3–7 days after anergy induction. As APC source, splenocytes from naive Lewis rats were used after Ficoll-Isopaque gradient centrifugation. Splenocytes (1.2×107 cells/ml) were pulsed with peptide 176–190 (10 g/ml) or S79A (50 g/ml) for 1–2 h at 37°C. Peptide-pulsed splenocytes (2×106/ml) were cultured with non-anergic or ‘anergic’ T cells (4×105/ml) at a 5:1 ratio in 5-ml cultures in 6-well plates (Costar) for 16–20 h in culture medium. In some experiments, splenocytes were cultured with both non-anergic and ‘anergic’ T cells at a 5:1:1.5 ratio, added either simultaneously, or ‘anergic’ T cells were added 6 h after non-anergic T cells. T cell depletion and testing of the T-cell activating capacity of APC M-450 goat-anti-mouse IgG Dynabeads (Dynal) in PBS (2×108/ml) were coated overnight at 4°C with 0.1 mg/ml purified R73 (anti-TCR mAb) [32] and 0.5 v/v of OX34 (anti-CD2 mAb) [33] hybridoma cell culture supernatant in PBS+1% rat serum. For T-cell depletion, APC/T-cell cultures were incubated with mAb-coated beads (five beads per target) for 30–45′ at 4°C while rolling, followed by magnetic depletion. Depletion of T cell clones A2b and Z1a was confirmed by FACS analysis (data not shown). Upon depletion, the majority of the APC population (>95%) consisted of OX33+cells (B cells). No differences in recovery, composition, or viability of isolated APC were observed between splenocytes precultured with nonanergic or ‘anergic’ T cells. Isolated APC (1×105 per well) were added in triplicate cultures to responder T cells (2×104 per well) in flat-bottomed 96-well plates in culture medium without or with additional Ag, and proliferative responses were assessed by [3H]thymidine incorporation during the last 18 h of a 96 h assay. For analysis of adherent APC, after overnight APC/T-
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cell culture, non-adherent cells were removed by resuspension, and adherent APC were washed twice with IMDM (37°C). A2b responder cells were added at different concentrations to the adherent cells in culture medium. After 3 days, A2b cells were collected, transferred to 96 wells plates and [3H]thymidine was added for 16 h. For polyclonal ex vivo responses, inguinal LN cells (ILNC) were isolated 14 days after immunization of Lewis rats with 100 l Mt in IFA (10 mg/ml) in the base of the tail. Popliteal LN cells (PLNC) were isolated 10 days after immunization of Lewis rats with 50 l of a 1:1 emulsion of MBP72–85 (1 mg/ml) in CFA (Mt, 4 mg/ml) in each hind footpad. Ex vivo polyclonal responses were measured by culturing LNC (2×105 per well) with Mt (10 g/ml) or MBP72–85 (10 g/ml) in the absence or presence of non-anergic or ‘anergic’ T cells (2×104 or 6×104 per well).
Statistics Data are expressed as means of triplicate values ±standard error of the mean (SEM), and analysed using a Student’s t-test for comparison between two groups. A probability value of P<0.05 was considered statistically significant.
Results ‘Anergic’ T cells down-regulate the T-cell activating capacity of APC in an Ag-specific manner, and induce linked suppression To investigate the effect of ‘anergic’ T cells on APC function, anergy was induced in the rat CD4+T cell clones A2b and Z1a through T-T presentation, as described previously [25,34]. After a rest period of 3–7 days after anergy induction, (non-) ‘anergic’ T cells were added to peptide-pulsed APC (splenocytes) derived from naive Lewis rats. After overnight culture, APC were isolated and added to fresh responder T cells, in the absence or presence of additional Ag. After 4 days, T cell proliferation was measured by [3H]thymidine incorporation. As shown in Figure 1A, the T-cell activating capacity of APC precultured with ‘anergic’ A2b cells was significantly reduced, as compared to APC precultured with non-anergic A2b. The APC modulation was Ag-specific, as ‘anergic’ Z1a cells, specific for peptide S79A, did not modulate peptide 176–190-pulsed APC (Figure 1B). Importantly, the observed difference between APC precultured with non-anergic and ‘anergic’ A2b cells was not due to up-regulation of the T-cell activating capacity of APC by non-anergic A2b cells, since the T-cell activating capacity of 176–190-pulsed APC precultured with (non) ‘anergic’ Z1a cells or non-anergic A2b cells was comparable. Furthermore, the addition of peptide 176–190 during the culture did not restore the stimulatory capacity of APC precultured with ‘anergic’ A2b (Figure 1A), indicating that the decreased stimulatory
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Second culture with:
176–190 176–190
Non-anergic A2b Anergic A2b
A2b + med A2b + med
176–190
Non-anergic A2b
A2b + 176–190
176–190
Anergic A2b
A2b + 176–190
A
*
Pre-cultured with:
*
APC pulsed with:
0
50
100
150 B
176–190 176–190
Non-anergic Z1a Anergic Z1a
A2b + med A2b + med
176–190
Non-anergic Z1a
A2b + 176–190
176–190
Anergic Z1a
A2b + 176–190 0
50
100
150 C
Non-anergic A2b Anergic A2b
Z1a + med Z1a + med
176–190
Non-anergic A2b
Z1a + S79A
176–190
Anergic A2b
Z1a + S79A
*
176–190 176–190
0
20
40
60 D
Z1a + med Z1a + med
S79A
Non-anergic Z1a
Z1a + S79A
S79A
Anergic Z1a
Z1a + S79A
*
Non-anergic Z1a Anergic Z1a
*
S79A S79A
0
5
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15 E
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S79A
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S79A
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A2b + 176–190
*
S79A S79A
0
25
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Proliferation (cpm × 10–3)
Figure 1. ‘Anergic’ T cells downregulate the T-cell activating capacity of APC in an Ag-specific manner, and induce linked suppression. Splenocytes were prepulsed with peptide 176–190 (A–C) or peptide S79A (D, E) and cultured overnight with non-anergic ( ) or ‘anergic’ ( ) A2b (A, C) or Z1a cells (B, D, E). After overnight culture, T cells were depleted, and the T-cell activating capacity of APC was assessed in a lymphocyte proliferation assay using A2b (A, B, E) or Z1a (C, D) cells as responder T cells in the absence (med) or presence of exogenously added peptide. A, B, D, Ag-specific down-regulation of the T-cell activating capacity of APC. One representative experiment out of four independent experiments is shown. C–E, Induction of linked suppression via downregulation of the T-cell activating capacity of APC. The data are representative for two independent experiments with each T cell clone. *P<0.05 as compared to APC precultured with non-anergic T cells.
capacity was not due to internalization or shedding of preformed MHC/176–190 complexes. Previously, it has been described that regulatory T cells can induce linked suppression, i.e. regulatory T cells can inhibit T cells with another Ag specificity provided that both Ag are presented by the same cell [25, 35, 36]. In Figure 1 we show that this phenomenon is mediated via downregulation of the T-cell activating capacity of
APC. Besides a decreased capacity of APC to stimulate T cells specific for the same ligand present during preculture with ‘anergic’ T cells (Figure 1A, D), also a decreased capacity to stimulate T cells specific for a different peptide (Figure 1C, E) was observed. The expression of MHC, co-stimulatory and adhesion molecules on isolated APC was investigated by FACS analysis in five independent experiments. We
30 *# *# 20
*
*
10
0
t=0
t=6
Figure 2. ‘Anergic’ T cells downregulate the T-cell activating capacity of previously in vitro activated APC. Peptide 176– 190-pulsed splenocytes were cultured with non-anergic ( ) or ‘anergic’ ( ) A2b cells, or with a mix of non-anergic: ‘anergic’ A2b cells (1:1.5, h). In mixed cultures, ‘anergic’ T cells were added simultaneously with non-anergic T cells (t=0) or 6 h later (t=6). After overnight culture, T cells were depleted, and the T-cell activating capacity of APC was investigated using A2b cells as responder cells. One out of two independent experiments is shown. *P<0.05 as compared to APC precultured with non-anergic T cells. # indicates that there was no significant difference between the 1:1.5 conditions at t=0 and t=6.
Table 1. ‘Anergic’ T cells downregulate the T cell-activating capacity of adherent APC Proliferative A2b response (cpm±SD)‡ Preculture with#: 2×105 A2b/ml 4×105 A2b/ml 8×105 A2b/ml
non-anergic A2b
‘anergic’ A2b
2,676±384 67,929±3,375 32,802±1,688
235±88* 480±105* 425±136*
# Splenocytes were pulsed with peptide 176–190, and cultured with non-anergic or ‘anergic’ A2b cells in six-well plates. After overnight culture, non-adherent cells were removed. Adherent cells were washed twice with Iscove’s medium (37°C), and A2b responder cells were added at the indicated concentrations. ‡ After 3 days of culture, A2b T-cells were collected, and the proliferative response was measured by [3H]thymidine incorporation for 16 h. * P<0.05 compared to APC precultured with non-anergic A2b cells.
did not observe any downregulation of RTl.BL (the MHC class II molecule to which the peptides used in this study bind), RTl.DL, or the class I molecule RTI.AL after incubation of APC with ‘anergic’ T cells (data not shown). Furthermore no significant alterations were observed in the expression of the costimulatory molecules CD80 (B7–1) and CD86 (B7–2), or the adhesion molecules CD11a (LFA-1) or CD54 (ICAM-1) (data not shown). ‘Anergic’ T cells down-regulate the T-cell activating capacity of previously in vitro activated APC Recent studies have shown that helper T cells can activate APC directly, e.g. through CD40L-CD40 inter-
ILNC proliferation (cpm × 10–3)
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PLNC proliferation (cpm × 10–3)
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70
A
35 * *#
0
50
Non-anergic
Anergic A2b
B
25 * *# 0
Non-anergic
Anergic Z1a
Figure 3. ‘Anergic’ T cells downregulate the T-cell activating capacity of previously in vivo activated APC. A, Lewis rats were immunized in the base of the tail with Mt/IFA. Ex vivo ILNC responses to Mt (10 g/ml) were measured in the absence ( ) or presence (1:0.1, h; 1:0.3 ) of (non-) ‘anergic’ A2b cells. B, Lewis rats were immunized with MBP72–85 in CFA in each hind footpad. Ex vivo PLNC responses to MBP72–85 (10 g/ml) were measured in the absence ( ) or presence (1:0.1, h; 1:0.3 ) of (non-) ‘anergic’ Z1a cells. Results are representative for one out of nine different Mt/IFA-immunized animals, and one out of three different MBP72–85/CFA-immunized animals. *P<0.05 as compared to APC cultured with non-‘anergic’ T cells. # P<0.05 as compared to the culture with a 1:0.1 ratio of LNC to ‘anergic’ T cells.
actions [14, 37, 38]. We investigated whether ‘anergic’ T cells could modulate previously activated APC as well as non-activated APC. To activate APC, peptide 176–190-pulsed APC were precultured with nonanergic, helper, A2b cells. ‘Anergic’ A2b cells were added to these cultures either simultaneously (t=0) or after 6 h (t=6), and after overnight culture the T-cell activating capacity of APC was investigated. Previously, we have shown that 6 h of incubation is sufficient to fully activate both T cells and APC [25]. Furthermore, CD40L is expressed optimally at 6 h after T cell activation [39, 40], and this period is sufficient to activate APC via CD40L-CD40 interactions [40]. Figure 2 shows that when ‘anergic’ T cell were added 6 h after non-anergic A2b T cells, they were still able to modulate the T-cell activating capacity of APC (t=6, open bars), indicating that ‘anergic’ T cells overruled the activation signals previously delivered by helper T cells, and that even in the presence of fully activated helper T cells, ‘anergic’ T-cell mediated modulation of the activated APC could occur.
‘Anergic’ T cells Modulate the T-cell Activating Capacity
The experiments described so far were performed with mostly B cells as APC. In addition, we found that adherent APC (macrophages) precultured with ‘anergic’ T cells also displayed a strongly reduced T-cell activating capacity (Table 1).
‘Anergic’ T cells downregulate the T-cell activating capacity of previously in vivo activated APC Finally, we investigated whether ‘anergic’ T cells were able to down-regulate in vivo activated APC. Lewis rats were immunized with Mycobacterium tuberculosis (Mt) in IFA, containing the HSP65 176–190 epitope recognized by clone A2b. After 14 days, the draining inguinal lymph nodes were removed, and the T-cell activating capacity of in vivo activated APC was investigated either in the absence or presence of ‘anergic’ A2b cells. Figure 3A shows that the capacity of in vivo activated APC to stimulate polyclonal responses to Mt was strongly reduced in the presence of ‘anergic’ A2b cells. Similar results were found when Lewis rats were immunized with MBP72–85 in CFA. The T-cell activating capacity of in vivo activated APC to stimulate polyclonal responses to MBP72–85 was completely abrogated by addition of MBP72–85 specific ‘anergic’ Zla cells (Figure 3B).
Discussion Recently, we and others showed that ‘anergic’ T cells can actively suppress the responses of other T cells, and that this suppressive effect was not mediated through passive competition for ligand or IL-2, or via production of inhibitory soluble factors such as IL-4, IL-10 or TGF, but was dependent on cell-cell contact between ‘anergic’ T cells, APC and non-anergic T cells [22, 23, 25–27, 41, 42]. Here we show that ‘anergic’ T cells exert this suppressive effect via modulation of the T-cell activating capacity of APC in an Ag-dependent manner leading to both Ag-specific and linked suppression. Since, the suppressive APC phenotype persisted upon removal of ‘anergic’ T cells, it is suggested that ‘anergic’ T cells condition APC to become suppressive. However, although the suppressive effect mediated via such modulated APC is clear (approximately 50% inhibition of the proliferative response), it is noteworthy that the inhibition of T cell proliferation in the presence of ‘anergic’ T cells is always more pronounced (>50% inhibition) (Figure 3, [25]). This could indicate that several mechanisms work in concert. Currently, we are investigating the effect on the production of different cytokines, and the expression of cell surface markers in the absence or presence of ‘anergic’ T cells. The APC populations preincubated with ‘anergic’ or non-anergic T cells did not differ in the expression of MHC, B7–1/2, LFA-1 or ICAM-1 as evaluated by FACS analysis. However, we can not fully exclude that a minor APC population present in the recovered APC samples containing
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>95% B cells had an altered expression pattern. Furthermore, it is possible that a minor very potent APC population, e.g. splenic DC, forming stable conjugates with ‘anergic’ T cells, was removed from the nonadherent APC fraction during the depletion of ‘anergic’ T cells. However, the fact that the differences in the T cell activating capacity of APC preincubated with ‘anergic’ or non-anergic T cells were also observed with overnight adherent spleen cells, which in general will no longer contain splenic DC, argues against the latter argument. To circumvent these problems, we are currently analysing the effect of ‘anergic’ T cells on highly purified APC sub populations. Importantly, as ‘anergic’ T cells were able to modulate APC previously activated in vitro or in vivo, we argue that the suppressive APC phenotype was not due to the lack of APC activation by ‘anergic’ T cells (e.g. via CD40L, OX40), but in contrast was actively induced via cell–cell contact between ‘anergic’ T cells and APC. Although at present we do not know the nature of the ligand, we suggest that the ligand involved in these molecular interactions should be present on different APC, as we found that downregulation of the T-cell activating capacity of APC occurred irrespective of the nature of the APC such as, splenic B cells, splenic macrophages, and LN derived APC. T-cell anergy has been defined previously as a state in which the T lymphocyte is alive but fails to display certain functional responses, e.g. proliferation and IL-2 production, upon exposure to its specific Ag under otherwise stimulatory conditions [43]. As such the T cells used in this study fulfilled the anergy criteria, however the fact that they actively modulate the APC function indicates that such cells are not non-functional but have a regulatory function. Therefore, we temporarily have designated these cells as ‘anergic’. It has been shown that ‘anergic’ T cells can persist in vivo provided the presence of their Ag [44–48]. One important question in the case of self-reactive T cells is why such potentially harmful cells are maintained in the repertoire? Our present findings that ‘anergic’ T cells can contribute to the resolution of an (auto)immune response and can induce tolerance via their APC-modulatory effect might offer an explanation for the maintenance and function of certain self-reactive T cells. The specificity of the modulatory effect is secured by the fact that ‘anergic’ T cells only modulate APC which present their cognate ligand. Furthermore, the ratio between ‘anergic’ and non-anergic T cells in the same T-cell/APC cluster will determine whether the APC will become T-cell stimulatory or suppressive [25, 49]. In addition we have shown recently that the suppressive capacity of ‘anergic’ T cells depends on their level of T cell anergy which will lead to a further fine-tuning of the APC modulation [49]. The immune response will then be determined by the ratio between stimulatory and suppressive APC. In conclusion, ‘anergic’ T cells should be regarded not only as tolerized cells, but also as active tolerizing cells due to their capacity to switch APC into a suppressive mode. The suppressive APC
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subsequently might function as a temporal bridge for T cell suppression, in analogy with the recent findings of a conditioned APC being a temporal bridge for T cell activation [14]. Thus, whereas helper CD4+T cells serve to initiate an immune response, ‘anergic’ CD4+T cells play a role in controlling an ongoing immune response and maintaining peripheral tolerance via APC modulation. Further understanding of the mechanism of the Ag-specific induction of suppressive APC by ‘anergic’ T cells may offer novel means to restore the immunological balance in chronic inflammatory conditions, such as occur during autoimmune diseases, allergies and transplant rejections.
Acknowledgements The authors wish to thank Prof Dr H. Ploegh, Dr S. Albani and Dr R. Offringa for critical review of the manuscript, and Dr G. Verheijden (Organon) for discussion. Professor I.R. Cohen is acknowledged for donating T cell clones A2b and Zla, and P. van Kooten for mAb purification. The work of Dr M.H.M. Wauben has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. This work was supported by N.V. Organon, the Netherlands, and by an EC Network grant (APTNET, No. BIO4– CT97–2151).
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Journal of Autoimmunity (2000) 14, 335–341
‘Anergic’ T cells Modulate the T-cell Activating Capacity of Antigen-presenting Cells Leonie S. Taams*, Elmieke P. J. Boot, Willem van Eden and Marca H. M. Wauben Institute of Infectious Diseases and Immunology, Department of Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht, The Netherlands
Received 20 January 2000 Accepted 28 February 2000 Key words: anergy, tolerance, immunoregulation, APC, Lewis rat
Nowadays there is compelling evidence for immunoregulation by T cells. Recently, we showed that so-called ‘anergic’ T cells are not functionally inert but can act as regulatory cells by actively suppressing other T cell responses. We now show that ‘anergic’ T cells mediate this suppressive effect via modulation of the T-cell activating capacity of the antigen-presenting cell (APC). Upon removal of the ‘anergic’ T cells, the suppressive APC phenotype persisted, indicating that ‘anergic’ T cells conditioned the APC to become a mediator of T cell suppression. The inhibitory signal delivered by ‘anergic’ T cells depended on the presence of the cognate ligand for the ‘anergic’ T cell, and appeared to be dominant since previously activated APC were rendered inhibitory as well. These findings imply that APC upon cross-talk with T cells can adopt distinct functional phenotypes ranging from T-cell stimulatory to T-cell suppressive. The contribution of ‘anergic’ T cells to the functional tuning of APC offers an explanation for the maintenance of ‘anergic’ T cells in the repertoire, and for their role in immunoregulation. © 2000 Academic Press
Introduction
immunoregulatory effects, such as Th2 cells [15], IL-10 or TGF- producing cells [16–19], CD45RClow cells [20] and CD4+CD25+T cells [21, 22]. Recently, it was shown that so-called ‘anergic’ T cells can function as regulatory T cells in vitro and in vivo by suppressing the responses of other T cells [23–27]. Until now it was unclear whether ‘anergic’ T cells mediate their immunoregulatory effects via direct inhibition of the responder T cells or via modulation of the stimulatory capacity of the APC. Here we show that CD4+T cells rendered ‘anergic’ through T-T presentation actively downregulate the T cell stimulatory capacity of the APC in an antigen (Ag)-specific manner. Upon removal of the ‘anergic’ T cells, the suppressive phenotype of APC persisted, indicating that ‘anergic’ T cells turned the APC into a tolerogenic mode. We propose that ‘anergic’ T cells are of critical importance in the immune repertoire for the functional tuning of APC, and that the APC after cross-talk with the surrounding cells and factors act as central regulator of the immune response.
Downregulation of immune responses is essential in order to prevent chronic inflammation and to maintain peripheral tolerance. Several mechanisms can contribute to the resolution of the immune response such as T cell apoptosis [1], induction of T cell suppressive cytokines (e.g. IL-10) [2], and T cell anergy [3]. Besides T cells, antigen-presenting cells (APC) may also play a key role in limiting the immune response. Indeed, evidence is accumulating that tolerogenic APC exist [4–6]. Such APC might be induced by factors such as IL-4, IL-10, IL-13 or TGF- in the microenvironment, leading to tolerogenic APC exhibiting a localized suppressive effect, e.g. in the lung or the eye [7–9]. Furthermore, it has been shown that pathogenic micro-organisms can modulate APC function in order to evade their eradication [10–12]. Interestingly, tolerogenic APC might also be induced due to cross-talk between professional antigenpresenting cells (APC), responder T cells and regulatory T cells present in the same cellular cluster [13,14]. Several T cell subsets have been proposed to mediate
Materials and Methods Correspondence to: Dr M.H.M. Wauben, Institute of Infectious Diseases and Immunology, Department of Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508 TD Utrecht, the Netherlands. Fax: +31 30 253 3555. E-mail: [email protected] * Present working address: Dept. Clinical Immunology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK.
Rats, T cells and Ag Seven to ten-week-old specific pathogen free male inbred Lewis rats (RT1.L) were obtained from the University of Limburg (Maastricht, the Netherlands). The isolation, maintenance and properties of the CD4+T cell clones A2b, specific for Mycobacterium 335
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tuberculosis (Mt) 65 kD heat shock protein (HSP65) peptide 176–190, and Z1a, specific for myelin basic protein (MBP) peptide MBP72–85 and the stronger MHC class II (RT1.BL) binding peptide analog MBP72–85S79A (S79A), have been described previously [28–31]. In vitro culture of APC and ‘anergic’ T cells Anergy was induced through T-T presentation as described previously [25]. In brief, A2b or Z1a T cells (3×106 cells/ml) were incubated with their respective stimulatory peptides 176–190 (10 g/ml) or S79A (50 g/ml) in the absence of professional APC. After overnight culture, viable T cells were collected by Ficoll-Isopaque gradient centrifugation, and maintained in culture medium (Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with L-Glutamine (2 mM), -mercaptoethanol (50 M), penicillin (50 U/ml), streptomycin (50 U/ml) and 2% heat-inactivated normal rat serum, or 10% heatinactivated fetal calf serum) in the absence of exogenous rIL-2. ‘Anergic’ T cells were added to APC cultures 3–7 days after anergy induction. As APC source, splenocytes from naive Lewis rats were used after Ficoll-Isopaque gradient centrifugation. Splenocytes (1.2×107 cells/ml) were pulsed with peptide 176–190 (10 g/ml) or S79A (50 g/ml) for 1–2 h at 37°C. Peptide-pulsed splenocytes (2×106/ml) were cultured with non-anergic or ‘anergic’ T cells (4×105/ml) at a 5:1 ratio in 5-ml cultures in 6-well plates (Costar) for 16–20 h in culture medium. In some experiments, splenocytes were cultured with both non-anergic and ‘anergic’ T cells at a 5:1:1.5 ratio, added either simultaneously, or ‘anergic’ T cells were added 6 h after non-anergic T cells. T cell depletion and testing of the T-cell activating capacity of APC M-450 goat-anti-mouse IgG Dynabeads (Dynal) in PBS (2×108/ml) were coated overnight at 4°C with 0.1 mg/ml purified R73 (anti-TCR mAb) [32] and 0.5 v/v of OX34 (anti-CD2 mAb) [33] hybridoma cell culture supernatant in PBS+1% rat serum. For T-cell depletion, APC/T-cell cultures were incubated with mAb-coated beads (five beads per target) for 30–45′ at 4°C while rolling, followed by magnetic depletion. Depletion of T cell clones A2b and Z1a was confirmed by FACS analysis (data not shown). Upon depletion, the majority of the APC population (>95%) consisted of OX33+cells (B cells). No differences in recovery, composition, or viability of isolated APC were observed between splenocytes precultured with nonanergic or ‘anergic’ T cells. Isolated APC (1×105 per well) were added in triplicate cultures to responder T cells (2×104 per well) in flat-bottomed 96-well plates in culture medium without or with additional Ag, and proliferative responses were assessed by [3H]thymidine incorporation during the last 18 h of a 96 h assay. For analysis of adherent APC, after overnight APC/T-
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cell culture, non-adherent cells were removed by resuspension, and adherent APC were washed twice with IMDM (37°C). A2b responder cells were added at different concentrations to the adherent cells in culture medium. After 3 days, A2b cells were collected, transferred to 96 wells plates and [3H]thymidine was added for 16 h. For polyclonal ex vivo responses, inguinal LN cells (ILNC) were isolated 14 days after immunization of Lewis rats with 100 l Mt in IFA (10 mg/ml) in the base of the tail. Popliteal LN cells (PLNC) were isolated 10 days after immunization of Lewis rats with 50 l of a 1:1 emulsion of MBP72–85 (1 mg/ml) in CFA (Mt, 4 mg/ml) in each hind footpad. Ex vivo polyclonal responses were measured by culturing LNC (2×105 per well) with Mt (10 g/ml) or MBP72–85 (10 g/ml) in the absence or presence of non-anergic or ‘anergic’ T cells (2×104 or 6×104 per well).
Statistics Data are expressed as means of triplicate values ±standard error of the mean (SEM), and analysed using a Student’s t-test for comparison between two groups. A probability value of P<0.05 was considered statistically significant.
Results ‘Anergic’ T cells down-regulate the T-cell activating capacity of APC in an Ag-specific manner, and induce linked suppression To investigate the effect of ‘anergic’ T cells on APC function, anergy was induced in the rat CD4+T cell clones A2b and Z1a through T-T presentation, as described previously [25,34]. After a rest period of 3–7 days after anergy induction, (non-) ‘anergic’ T cells were added to peptide-pulsed APC (splenocytes) derived from naive Lewis rats. After overnight culture, APC were isolated and added to fresh responder T cells, in the absence or presence of additional Ag. After 4 days, T cell proliferation was measured by [3H]thymidine incorporation. As shown in Figure 1A, the T-cell activating capacity of APC precultured with ‘anergic’ A2b cells was significantly reduced, as compared to APC precultured with non-anergic A2b. The APC modulation was Ag-specific, as ‘anergic’ Z1a cells, specific for peptide S79A, did not modulate peptide 176–190-pulsed APC (Figure 1B). Importantly, the observed difference between APC precultured with non-anergic and ‘anergic’ A2b cells was not due to up-regulation of the T-cell activating capacity of APC by non-anergic A2b cells, since the T-cell activating capacity of 176–190-pulsed APC precultured with (non) ‘anergic’ Z1a cells or non-anergic A2b cells was comparable. Furthermore, the addition of peptide 176–190 during the culture did not restore the stimulatory capacity of APC precultured with ‘anergic’ A2b (Figure 1A), indicating that the decreased stimulatory
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Second culture with:
176–190 176–190
Non-anergic A2b Anergic A2b
A2b + med A2b + med
176–190
Non-anergic A2b
A2b + 176–190
176–190
Anergic A2b
A2b + 176–190
A
*
Pre-cultured with:
*
APC pulsed with:
0
50
100
150 B
176–190 176–190
Non-anergic Z1a Anergic Z1a
A2b + med A2b + med
176–190
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A2b + 176–190
176–190
Anergic Z1a
A2b + 176–190 0
50
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150 C
Non-anergic A2b Anergic A2b
Z1a + med Z1a + med
176–190
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Z1a + S79A
176–190
Anergic A2b
Z1a + S79A
*
176–190 176–190
0
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60 D
Z1a + med Z1a + med
S79A
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S79A
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*
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Proliferation (cpm × 10–3)
Figure 1. ‘Anergic’ T cells downregulate the T-cell activating capacity of APC in an Ag-specific manner, and induce linked suppression. Splenocytes were prepulsed with peptide 176–190 (A–C) or peptide S79A (D, E) and cultured overnight with non-anergic ( ) or ‘anergic’ ( ) A2b (A, C) or Z1a cells (B, D, E). After overnight culture, T cells were depleted, and the T-cell activating capacity of APC was assessed in a lymphocyte proliferation assay using A2b (A, B, E) or Z1a (C, D) cells as responder T cells in the absence (med) or presence of exogenously added peptide. A, B, D, Ag-specific down-regulation of the T-cell activating capacity of APC. One representative experiment out of four independent experiments is shown. C–E, Induction of linked suppression via downregulation of the T-cell activating capacity of APC. The data are representative for two independent experiments with each T cell clone. *P<0.05 as compared to APC precultured with non-anergic T cells.
capacity was not due to internalization or shedding of preformed MHC/176–190 complexes. Previously, it has been described that regulatory T cells can induce linked suppression, i.e. regulatory T cells can inhibit T cells with another Ag specificity provided that both Ag are presented by the same cell [25, 35, 36]. In Figure 1 we show that this phenomenon is mediated via downregulation of the T-cell activating capacity of
APC. Besides a decreased capacity of APC to stimulate T cells specific for the same ligand present during preculture with ‘anergic’ T cells (Figure 1A, D), also a decreased capacity to stimulate T cells specific for a different peptide (Figure 1C, E) was observed. The expression of MHC, co-stimulatory and adhesion molecules on isolated APC was investigated by FACS analysis in five independent experiments. We
30 *# *# 20
*
*
10
0
t=0
t=6
Figure 2. ‘Anergic’ T cells downregulate the T-cell activating capacity of previously in vitro activated APC. Peptide 176– 190-pulsed splenocytes were cultured with non-anergic ( ) or ‘anergic’ ( ) A2b cells, or with a mix of non-anergic: ‘anergic’ A2b cells (1:1.5, h). In mixed cultures, ‘anergic’ T cells were added simultaneously with non-anergic T cells (t=0) or 6 h later (t=6). After overnight culture, T cells were depleted, and the T-cell activating capacity of APC was investigated using A2b cells as responder cells. One out of two independent experiments is shown. *P<0.05 as compared to APC precultured with non-anergic T cells. # indicates that there was no significant difference between the 1:1.5 conditions at t=0 and t=6.
Table 1. ‘Anergic’ T cells downregulate the T cell-activating capacity of adherent APC Proliferative A2b response (cpm±SD)‡ Preculture with#: 2×105 A2b/ml 4×105 A2b/ml 8×105 A2b/ml
non-anergic A2b
‘anergic’ A2b
2,676±384 67,929±3,375 32,802±1,688
235±88* 480±105* 425±136*
# Splenocytes were pulsed with peptide 176–190, and cultured with non-anergic or ‘anergic’ A2b cells in six-well plates. After overnight culture, non-adherent cells were removed. Adherent cells were washed twice with Iscove’s medium (37°C), and A2b responder cells were added at the indicated concentrations. ‡ After 3 days of culture, A2b T-cells were collected, and the proliferative response was measured by [3H]thymidine incorporation for 16 h. * P<0.05 compared to APC precultured with non-anergic A2b cells.
did not observe any downregulation of RTl.BL (the MHC class II molecule to which the peptides used in this study bind), RTl.DL, or the class I molecule RTI.AL after incubation of APC with ‘anergic’ T cells (data not shown). Furthermore no significant alterations were observed in the expression of the costimulatory molecules CD80 (B7–1) and CD86 (B7–2), or the adhesion molecules CD11a (LFA-1) or CD54 (ICAM-1) (data not shown). ‘Anergic’ T cells down-regulate the T-cell activating capacity of previously in vitro activated APC Recent studies have shown that helper T cells can activate APC directly, e.g. through CD40L-CD40 inter-
ILNC proliferation (cpm × 10–3)
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70
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Non-anergic
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Non-anergic
Anergic Z1a
Figure 3. ‘Anergic’ T cells downregulate the T-cell activating capacity of previously in vivo activated APC. A, Lewis rats were immunized in the base of the tail with Mt/IFA. Ex vivo ILNC responses to Mt (10 g/ml) were measured in the absence ( ) or presence (1:0.1, h; 1:0.3 ) of (non-) ‘anergic’ A2b cells. B, Lewis rats were immunized with MBP72–85 in CFA in each hind footpad. Ex vivo PLNC responses to MBP72–85 (10 g/ml) were measured in the absence ( ) or presence (1:0.1, h; 1:0.3 ) of (non-) ‘anergic’ Z1a cells. Results are representative for one out of nine different Mt/IFA-immunized animals, and one out of three different MBP72–85/CFA-immunized animals. *P<0.05 as compared to APC cultured with non-‘anergic’ T cells. # P<0.05 as compared to the culture with a 1:0.1 ratio of LNC to ‘anergic’ T cells.
actions [14, 37, 38]. We investigated whether ‘anergic’ T cells could modulate previously activated APC as well as non-activated APC. To activate APC, peptide 176–190-pulsed APC were precultured with nonanergic, helper, A2b cells. ‘Anergic’ A2b cells were added to these cultures either simultaneously (t=0) or after 6 h (t=6), and after overnight culture the T-cell activating capacity of APC was investigated. Previously, we have shown that 6 h of incubation is sufficient to fully activate both T cells and APC [25]. Furthermore, CD40L is expressed optimally at 6 h after T cell activation [39, 40], and this period is sufficient to activate APC via CD40L-CD40 interactions [40]. Figure 2 shows that when ‘anergic’ T cell were added 6 h after non-anergic A2b T cells, they were still able to modulate the T-cell activating capacity of APC (t=6, open bars), indicating that ‘anergic’ T cells overruled the activation signals previously delivered by helper T cells, and that even in the presence of fully activated helper T cells, ‘anergic’ T-cell mediated modulation of the activated APC could occur.
‘Anergic’ T cells Modulate the T-cell Activating Capacity
The experiments described so far were performed with mostly B cells as APC. In addition, we found that adherent APC (macrophages) precultured with ‘anergic’ T cells also displayed a strongly reduced T-cell activating capacity (Table 1).
‘Anergic’ T cells downregulate the T-cell activating capacity of previously in vivo activated APC Finally, we investigated whether ‘anergic’ T cells were able to down-regulate in vivo activated APC. Lewis rats were immunized with Mycobacterium tuberculosis (Mt) in IFA, containing the HSP65 176–190 epitope recognized by clone A2b. After 14 days, the draining inguinal lymph nodes were removed, and the T-cell activating capacity of in vivo activated APC was investigated either in the absence or presence of ‘anergic’ A2b cells. Figure 3A shows that the capacity of in vivo activated APC to stimulate polyclonal responses to Mt was strongly reduced in the presence of ‘anergic’ A2b cells. Similar results were found when Lewis rats were immunized with MBP72–85 in CFA. The T-cell activating capacity of in vivo activated APC to stimulate polyclonal responses to MBP72–85 was completely abrogated by addition of MBP72–85 specific ‘anergic’ Zla cells (Figure 3B).
Discussion Recently, we and others showed that ‘anergic’ T cells can actively suppress the responses of other T cells, and that this suppressive effect was not mediated through passive competition for ligand or IL-2, or via production of inhibitory soluble factors such as IL-4, IL-10 or TGF, but was dependent on cell-cell contact between ‘anergic’ T cells, APC and non-anergic T cells [22, 23, 25–27, 41, 42]. Here we show that ‘anergic’ T cells exert this suppressive effect via modulation of the T-cell activating capacity of APC in an Ag-dependent manner leading to both Ag-specific and linked suppression. Since, the suppressive APC phenotype persisted upon removal of ‘anergic’ T cells, it is suggested that ‘anergic’ T cells condition APC to become suppressive. However, although the suppressive effect mediated via such modulated APC is clear (approximately 50% inhibition of the proliferative response), it is noteworthy that the inhibition of T cell proliferation in the presence of ‘anergic’ T cells is always more pronounced (>50% inhibition) (Figure 3, [25]). This could indicate that several mechanisms work in concert. Currently, we are investigating the effect on the production of different cytokines, and the expression of cell surface markers in the absence or presence of ‘anergic’ T cells. The APC populations preincubated with ‘anergic’ or non-anergic T cells did not differ in the expression of MHC, B7–1/2, LFA-1 or ICAM-1 as evaluated by FACS analysis. However, we can not fully exclude that a minor APC population present in the recovered APC samples containing
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>95% B cells had an altered expression pattern. Furthermore, it is possible that a minor very potent APC population, e.g. splenic DC, forming stable conjugates with ‘anergic’ T cells, was removed from the nonadherent APC fraction during the depletion of ‘anergic’ T cells. However, the fact that the differences in the T cell activating capacity of APC preincubated with ‘anergic’ or non-anergic T cells were also observed with overnight adherent spleen cells, which in general will no longer contain splenic DC, argues against the latter argument. To circumvent these problems, we are currently analysing the effect of ‘anergic’ T cells on highly purified APC sub populations. Importantly, as ‘anergic’ T cells were able to modulate APC previously activated in vitro or in vivo, we argue that the suppressive APC phenotype was not due to the lack of APC activation by ‘anergic’ T cells (e.g. via CD40L, OX40), but in contrast was actively induced via cell–cell contact between ‘anergic’ T cells and APC. Although at present we do not know the nature of the ligand, we suggest that the ligand involved in these molecular interactions should be present on different APC, as we found that downregulation of the T-cell activating capacity of APC occurred irrespective of the nature of the APC such as, splenic B cells, splenic macrophages, and LN derived APC. T-cell anergy has been defined previously as a state in which the T lymphocyte is alive but fails to display certain functional responses, e.g. proliferation and IL-2 production, upon exposure to its specific Ag under otherwise stimulatory conditions [43]. As such the T cells used in this study fulfilled the anergy criteria, however the fact that they actively modulate the APC function indicates that such cells are not non-functional but have a regulatory function. Therefore, we temporarily have designated these cells as ‘anergic’. It has been shown that ‘anergic’ T cells can persist in vivo provided the presence of their Ag [44–48]. One important question in the case of self-reactive T cells is why such potentially harmful cells are maintained in the repertoire? Our present findings that ‘anergic’ T cells can contribute to the resolution of an (auto)immune response and can induce tolerance via their APC-modulatory effect might offer an explanation for the maintenance and function of certain self-reactive T cells. The specificity of the modulatory effect is secured by the fact that ‘anergic’ T cells only modulate APC which present their cognate ligand. Furthermore, the ratio between ‘anergic’ and non-anergic T cells in the same T-cell/APC cluster will determine whether the APC will become T-cell stimulatory or suppressive [25, 49]. In addition we have shown recently that the suppressive capacity of ‘anergic’ T cells depends on their level of T cell anergy which will lead to a further fine-tuning of the APC modulation [49]. The immune response will then be determined by the ratio between stimulatory and suppressive APC. In conclusion, ‘anergic’ T cells should be regarded not only as tolerized cells, but also as active tolerizing cells due to their capacity to switch APC into a suppressive mode. The suppressive APC
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subsequently might function as a temporal bridge for T cell suppression, in analogy with the recent findings of a conditioned APC being a temporal bridge for T cell activation [14]. Thus, whereas helper CD4+T cells serve to initiate an immune response, ‘anergic’ CD4+T cells play a role in controlling an ongoing immune response and maintaining peripheral tolerance via APC modulation. Further understanding of the mechanism of the Ag-specific induction of suppressive APC by ‘anergic’ T cells may offer novel means to restore the immunological balance in chronic inflammatory conditions, such as occur during autoimmune diseases, allergies and transplant rejections.
Acknowledgements The authors wish to thank Prof Dr H. Ploegh, Dr S. Albani and Dr R. Offringa for critical review of the manuscript, and Dr G. Verheijden (Organon) for discussion. Professor I.R. Cohen is acknowledged for donating T cell clones A2b and Zla, and P. van Kooten for mAb purification. The work of Dr M.H.M. Wauben has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. This work was supported by N.V. Organon, the Netherlands, and by an EC Network grant (APTNET, No. BIO4– CT97–2151).
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