Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands
1 Department of Clinical Immunology, Royal Free and University College Medical School (Royal Free Campus), London NW3 2PF, UK
Correspondence to: W. Van Eden
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: anergy, autoreactivity, B7-2, IL-4, IL-10, tolerance
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For hsp60 as a self-antigen, evidence in favor of positive selection was obtained in a transgenic mouse model where mouse hsp60 was overexpressed using the MHC class II promoter, leading to abundant thymic expression in conjunction with an increased peripheral T cell reactivity (5). Also, other groups described the presence of cross-reactive T cells recognizing self-hsp60-derived peptides and their mycobacterial homologues in the periphery, indicating that tolerance for this self-antigen was not complete (69). In addition, the inherent degeneracy of TCR recognition (7,10) would easily jeopardize self/non-self discrimination, especially in the case of conserved proteins like heat shock proteins, given the fact that bacterial heat shock proteins are amongst the most immunogenic proteins present in microbial organisms (11,12). Also, mechanisms described for inducing and maintaining active peripheral tolerance to self-antigens present in the periphery are held to be related to the quality of TCR signaling, and such mechanisms can lead to the induction of self-reactive T cells which are rather regulatory than pathogenic. These latter mechanisms include the induction of anergy (13,14), generation of IL-10 (Tr1) (15,16) and/or transforming growth factor-ß producing cells (Th3) (17) or a switch from Th1- to Th2-type responses resulting in immune deviation (18).
In the case of self-hsp60-reactive T cells, expansion of T cells recognizing a conserved epitope of Mycobacterium tuberculosis hsp60 (M256270) induced resistance to active disease in the models of adjuvant arthritis (AA) and avridine-induced arthritis (1921). These T cells were cross-reactive in recognizing the corresponding rat homologue peptide R256270 and this cross-reactivity was restricted to T cells specific for the conserved mycobacterial core-peptide M256265 (21) (Paul et al., submitted). As in vivo these T cells may see the self-antigen (over-)expressed at the surface of stressed cells at sites of inflammation or at sites of immune activation (2224), it is possible that this leads to activation of such cells. If so, the resulting inhibition of disease development could be a consequence of the quality of TCR signaling induced by the self-antigen, producing a regulatory functional phenotype in these self-responsive T cells. In this study we therefore tested the functional phenotype of M256270-specific T cells upon recognition of the rat homologue of the core-epitope M256265, peptide R256265. We found this peptide to be recognized as a partial agonist, that skews the cytokine profile in the direction of a Th2 phenotype and that induces the preferential expression of B7-2 and down-modulates the expression of LFA-1 on M256270-specific T cells. As we previously described that M256270-specific T cells inhibit the development of AA (21), we suggest that the B7-2 expression on T cells cross-reactive with self-hsp might be involved in the regulation of AA.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antigens and adjuvants
Dimethyl dioctadecyl ammonium bromide (DDA; Eastman Kodak, Rochester, NY) (25) was used as adjuvant. DDA was prepared as a 20 mg/ml suspension in PBS, and sonicated and heated to produce a gel which was mixed 1:1 with 2 mg/ml peptide solution before immunization. M. tuberculosis hsp60 peptide M256270 (ALSTLVVNKIRGTFK), M256265 (ALSTLVVNKI) and rat hsp60 peptide R256265 (ALSTLVLNRL) were produced by solid-phase peptide synthesis (26) and contained an amide group at the C-terminus. Peptides were checked by reversed-phase HPLC and by fast atom bombardment mass spectrometry.
MHC binding assay
The MHCpeptide binding assay studies were performed using a direct binding assay as described (27). Briefly, Rat RT1.Bl molecules were affinity purified from cell lysates of the concanavalin A-activated MHC class II+ Z1a T cell clone using the mAb OX6 coupled to Sepharose-4B beads. For competition studies purified MHC class II molecules (2 µM) were incubated with 200 nM of biotinylated peptide myelin basic protein (MBP) 7285 and a dose range (0256 µM) of competitor peptides for 40 h at room temperature and pH 5 in the presence of a protease inhibitor mix. The MHCpeptide mixtures were analyzed via non-reducing SDSPAGE, followed by Western blotting (Hybond-ECL; Amersham, Little Chalfont, UK). Biotinylated peptides were visualized on preflashed films (Hyperfilm; Amersham), through enhanced chemiluminescence (Western Blot ECL kit; Amersham). Spots on the films were quantified by image processing.
Generation of peptide-specific T cell lines
Rats were lightly anesthetized using ether and immunized with 50 µg of synthetic peptide in 50 µl PBS/DDA in each hind footpad (i.e. 100 µg/rat). Ten days later, draining popliteal lymph nodes were removed, disaggregated, washed twice and used as a source of primed lymph node cells (PLNC). T cell lines were generated by culturing PLNC at 5x106/ml in culture medium (IMDM; Gibco/BRL, Gaithersburg, MD) plus 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco/BRL) and 5x105 M 2-mercaptoethanol) supplemented with 2% naive inactivated rat serum (NRS) in the presence of 10 µg/ml peptide. After 3 days, viable cells were harvested using a Ficoll-Isopaque gradient and expanded for 4 days in culture medium supplemented with 10% EL-4 supernatant (as a source of IL-2) and 10% FCS. Seven days after initial stimulation, 4x105 T cells/ml were re-stimulated with 1x106/ml irradiated (3000 rad) syngeneic splenocytes as a source of APC and 10 µg/ml peptide in culture medium supplemented with 2% NRS. Lines were maintained in this 7 day re-stimulation cycle. The M256270-specific T cell lines were generated by immunization and re-stimulation with peptide M256270.
T cell proliferation assay
T cell lines were cultured in triplicate in 200 µl flat-bottomed microtiter wells (Costar, Cambridge, MA) at 2x104 cells/well with or without antigen and 2x105 irradiated syngeneic splenocytes/well as APC in culture medium supplemented with 2% NRS. Responses to a dose range of individual peptides were tested; concanavalin A (2.5 µg/ml) and human rIL-2 (10 U/ml; PharMingen, San Diego, CA) were used as positive controls. Cultures were incubated for 96 h at 37°C, 5% CO2 and pulsed with [3H]thymidine, 0.4 µCi/well (sp. act. 1 Ci/mmol; Amersham) for the final 1618 h. [3H]Thymidine incorporation was measured using a liquid scintillation ß counter (Wallac, Turku, Finland). Results are expressed as mean c.p.m. ± SD of triplicate wells.
Induction of anergy in the presence of APC
M256270-specific T cells (4x105 /ml) were incubated for 2024 h with 2x106/ml APC in culture medium supplemented with 2% NRS and 10 or 100 µg/ml of peptide in a six-wells plate (Costar). As a control, only culture medium supplemented with 2% NRS was added. After 2024 h, viable T cells were collected by Ficoll-Isopaque gradient centrifugation and cultured additionally for 3 days in culture medium supplemented with 2% NRS at 4x105/ml. Viable T cells were then recovered by Ficoll-Isopaque gradient centrifugation and tested in a lymphocyte proliferation assay as described above.
Flow-cytometry
Expression of cell-surface molecules on M256270-specific T cells was analyzed after 2024 h of incubation in the presence of irradiated splenocytes as APC, as described above for induction of anergy with a few modifications. APC were first labeled with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester [BCECF-AM; Boehringer Mannheim, Almere, The Netherlands; measured in the FL1-channel of a FACScan] by incubating 1x107 splenocytes/ml for 20 min at 37°C, 5% CO2 atmosphere, with 0.1 µM BCECF-AM. After washing 3 times, APC were pulsed with different concentrations (1, 10 or 100 µg/ml) of peptides M256265 or R256265 by incubating 1.3x107 APC/ml for 1.5 h with peptide at 37°C, 5% CO2. After washing twice, 4x105 M256270-specific T cells/ml in culture medium supplemented with 2% NRS were added 7 days after the last re-stimulation to 2x106/ml labeled APC in a six-well plate (Costar).
After 2024 h of incubation following the protocol described above, viable cells were collected by Ficoll-Isopaque gradient centrifugation, and washed twice with culture medium, counted and reconstituted to 4x105 cells/ml. Cells were washed once with FACS buffer (PBS containing 1% BSA and 0.1% NaN3 and 4% NRS) and incubated for 30 min on ice at 1:2 in FACS buffer diluted mouse-anti-rat specific cell-surface marker. Cells were washed twice and incubated for 30 min at 1:300 in FACS buffer-diluted phycoerythrin (PE)-conjugated goat anti-mouse total IgG antibody (PharMingen). Cells were washed twice and resuspended in FACS buffer without NRS. Residual dead cells were excluded based on the FSC/SSC by raising the threshold on FSC and the PE fluorescence of the T cell population was measured in the FL2-channel of a FACScan (Becton Dickinson, Mountain View, CA) after gating on the FL1-negative population (when incubated with BCECF-AM-labeled APC). Data were analyzed using CellQuest software (Becton Dickinson). The following mAb were used: IgG1 isotype mAb: R73 (anti-TCR ß), W3/25 (anti-CD4), OX39 (anti-CD25 or anti-IL2R ), 3H5 (anti-CD80 or B7-1), 24F (anti-CD86 or B7-2), IA29 (anti-CD54 or ICAM-1); IgG2a isotype mAb: WT.1 (anti-CD11a or LFA-1); IgG2b isotype mAb: OX40 (anti-CD134 or OX40 antigen). Isotype control mAb were UD15 [IgG1, anti-choramphenicol (28)] and IE7.4H11 [IgG2a, anti-ovine IgE (29)]; for IgG2b no isotype control mAb was available, therefore only secondary antibody served as control. R73 was a kind gift from Professor T. Hunig; 3H5 and 24F hybridomas were kind gifts from Dr H. Yagita.
Cytokine analysis
To assess the cytokines produced by the M256270-specific T cells, they were stimulated with a dose range (0.1100 µg/ml) of peptides M256265 or R256265, or medium alone in the presence of APC under the conditions described above for lymphocyte proliferation assays. For detection of cytokine mRNA levels, RT-PCR analysis was performed. Total RNA was isolated from cell pellets by extraction with RNAzol (Gibco/BRL). The isolated RNA and 0.5 µg oligo(dT)1218 was heated at 55°C for 10 min and cooled to room temperature. First strand synthesis was performed by incubating 1 µg RNA in a reaction mixture (total volume 20 µl) containing 5 mM Tris-HCl, pH 8.3 at 42°C, 50 mM KCl, 10 mM MgCl2, 2 mM dithiothreitol, 1 mM of dATP, dCTP, dGTP and dTTP each, and 25 U reverse transcriptase. The mixture was incubated at 42°C for 1.5 h. The mixture was cooled on ice and diluted 6 times in H2O. cDNA was incubated in a mixture (total volume of 20 µl) containing 0.1 mM of dATP, dCTP, dGTP and dTTP each, 50 mM KCl, 10 mM TrisHCl, pH 9.0 at 25°C, 1.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100, 50 ng of the desired 5' and 3' primers each, and 0.625 U Taq DNA polymerase I (Perkin-Elmer, Nieuwerkerk aan den Ussel, The Netherlands). The mixture was subjected to 2 min of 94°C, followed by 35 cycles of 10 s at 94°C, 30 s at 60°C and 30 s at 72°C, followed by 10 min at 72°C, in a PCR apparatus (9600; Perkin-Elmer). For detection of IL-4 mRNA, a nested PCR was performed with a first amplification for 35 cycles with full-length primers followed by a second amplification for 25 cycles with nested primers. PCR products were visualized on a 2% ethidium bromide-stained agarose gel. The following rat-specific primer pairs were used: G3PDH (452 bp fragment): 5'-ACC ACA GTC CAT GCC ATC AC, 3'-TCC ACC ACC CTG TTG CTG TA; IL-4 (first PCR round 460 bp fragment, including XbaIBamHI restriction sites as indicated in small type): 5'-CCg gat ccA TGG GTC TCA GCC CCC ACC T, 3'-GCt cta gaT TAG GAC ATG GAA GTG CAG GAC T and for nested PCR (238 bp fragment): 5'-ATG CAC CGA GAT GTT TGT ACC, 3'-TTT CAG TGT TCT GAG CGT GGA; IL-10 (127 bp fragment): 5'-TGC CAA GCC TTG TCA GAA ATG ATC AAG, 3'-GTA TCC AGA GGG TCT TCA GCT TCT CTC; IFN- (419 bp fragment): 5'-CCC TCT CTG GCT GTT ACT GC, 3'-CTC CTT TTC CGC TTC CTT AG.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Similar to peptide M256265, peptide R256265 induced a 2- to 3-fold down-regulation of TCR expression (Fig. 3) as well as down-regulation of CD3 (data not shown). Moreover, peptides M256265 and R256265 induced a significant up-regulation of the IL-2 receptor and up-regulation of the adhesion molecule ICAM-1. Also, the expression of OX40 antigen was up-regulated compared to T cells which had been incubated with non-pulsed APC (Fig. 3
). The amount of up-regulation of the IL-2 receptor, OX40 and ICAM-1 upon stimulation with peptide R256265 was less than that seen upon stimulation with peptide M256265, which induced high proliferative responses. However, the level of TCR down-regulation was similar upon stimulation with peptides M256265 and R256265 (Fig. 3
). Up-regulation of the IL-2 receptor/OX40 and down-regulation of the TCR was also found with lower doses of peptide R256265 (110 µg/ml; data not shown).
|
The rat homologue peptide R256265 does not induce unresponsiveness
As the sequence of the rat homologue peptide R256 265 differed slightly from the mycobacterial peptide M256265 (see Methods), we investigated whether peptide R256265 could act as an anergy-inducing APL for M256270-specific T cells. Therefore M256270-specific T cells were incubated with peptide M256270 or with peptide R256265 in the presence of APC. T cells incubated with APC only served as a control. After 2024 h of incubation, viable T cells were collected and maintained in culture for 3 days in the absence of IL-2 in order to allow re-expression of TCRCD3. Subsequently proliferative responses to peptide M256270 in the presence of freshly added APC were examined. Figure 4 shows that a supraoptimal dose (100 µg/ml) of peptide M256270 induced unresponsiveness. The unresponsiveness induced was not the result of a decreased viability, as concanavalin A and IL-2 responses were found to be similar to those of T cells which did not become unresponsive (Fig. 4
). Furthermore this was not due to a down-regulation of the TCRCD3, since after 3 days of culturing expression levels were similar to T cells incubated with APC only (data not shown). In contrast to this, peptide R256265 did not induce unresponsiveness in M256270-specific T cells at peptide concentrations of 100 or 10 µg/ml (Fig. 4
). Also, we found no differences in the IL-2 production, upon rechallenge with peptide M256265 and freshly added APC, between T cells incubated for 2024 h with 10 or 100 µg/ml R256265 and APC or APC only, as measured by using the IL-2-dependent CTLL16 cell line (data not shown).
|
Upon stimulation with peptide M256265, the levels of IFN- and IL-10 mRNA increased with increasing peptide concentrations used for stimulation. IL-4 mRNA was induced to similar levels at all peptide concentrations, with the exception of the weaker signal seen at 25 µg/ml (Fig. 5
, columns 25). Clearly IL-4 mRNA levels showed no correlation with the peptide concentrations used for stimulation of the T cells. In contrast, stimulation with peptide R256265 induced a different pattern, i.e. a peptide concentration-dependent decrease in the induction of IFN-
and (less clearly) IL-10 mRNA (Fig. 5
, columns 69) compared to the levels produced upon stimulation with APC only (Fig. 5
, column 1). Interestingly, stimulation with low concentrations of peptide R256265 induced IL-4 mRNA and this effect was lost at higher peptide concentrations.
|
Peptide R256265 down-regulates LFA-1, and preferentially induces B7-2 and not B7-1
Subsequently we investigated whether the activation of M256270-specific T cells by peptide R256265, which did not elicit proliferative responses, led to a differential expression of cell-surface markers.
When we analyzed the expression of the adhesion molecule LFA-1 (CD11a) on M256270-specific T cells we found that peptide M256265 up-regulated LFA-1 expression (Fig. 6a, upper panel), whereas peptide R256265 induced a down-regulation of LFA-1 (Fig. 6a
, lower panel). This down-regulation was also found for lower doses (110 µg/ml) of peptide R256265 (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
T cells with specificity for self are now known to be a normal constituent of the healthy immune system (3,4). Having escaped central tolerance induction, they are either subjected to mechanisms of peripheral tolerance (reviewed in 30,31) or they can be mediators of peripheral tolerance (3236). As the self-hsp60-reactive T cells were capable of adoptively transferring their protective effect, they can be such mediators of peripheral tolerance themselves. Since the amino acid sequences of the rat and mycobacterial hsp60 peptide differed slightly, the rat peptide R256265 could be seen as an APL (1). APL can induce T cell responses that differ qualitatively from wild-type peptide-induced responses (2,37). Amongst these are peripheral tolerance-promoting functional phenotypes such as anergy or T cells producing anti-inflammatory cytokines (3842). Therefore self-derived peptides, when recognized as APL, may be able to mediate peripheral tolerance by means of these mechanisms.
First of all, we determined if the differences in amino acid sequence between the mycobacterial peptide and its rat homologue affected MHC binding. The self-peptide R256265 was found to have a 5-fold lower MHC-binding affinity as compared to its mycobacterial homologue M256265. This was compatible with earlier observations that most peptides inducing autoreactive T cells were relatively weak MHC binders (43). Due to a resulting low-avidity interaction with the TCR these T cells may have escaped negative selection (44,45), explaining their presence in the repertoire. However, despite a 5-fold lower MHC-binding affinity, we found that proliferative responses could not be induced with peptide R256265 even with a 10- to 100-fold increase in antigen concentration compared to the mycobacterial homologue (Fig. 1). This indicated the presence of differences in TCR contact residues, in addition to differences in MHC contact residues, between peptide R256265 and peptide M256265, and the ability of the rat homologue to be recognized as an APL. That peptide R256265 was indeed recognized as an APL was shown by the absence of proliferative responses accompanied by TCRCD3 down-regulation and profound up-regulation of activation markers, as also described by others (38,46). Therefore we determined if peptide R256265 could induce a functional phenotype capable of promoting peripheral tolerance. First we tested its ability to induce anergy in the presence of APC (38). Preincubation with a high dosage of the wild-type peptide M256270 induced unresponsiveness, possibly reflecting a post-activation refractory period. However, under the test conditions as used for inducing anergy by APL (38), peptide R256265 did not induce anergy. Thus our in vitro data were not supportive of an anergy-inducing mechanism in our self-hsp60-specific T cells, upon encountering self-hsp60 in vivo, to account for the protective effect of these T cells in arthritis. However, we cannot exclude that this mechanism might occur in vivo by, for instance, the recognition of self-hsp60 on non-professional APC (13,47) or by a more continuous engagement of the TCR (48).
Secondly, we determined whether peptide R256265 could induce anti-inflammatory cytokines in M256270-specific T cells. We found that, compared to peptide M256265, the self-peptide did not up-regulate IL-2, IFN- or IL-10 but still induced low levels of IL-4. Thus, although peptide R256265 did not induce the production of cytokines other than induced by the mycobacterial homologue, stimulation with this peptide led to a relative skewing towards a Th2 phenotype. As peptide R256265 had a lower MHC-binding affinity, described to preferentially induce a Th2-phenotype (4951), this could have accounted for our observed induction of IL-4. However, this seems unlikely, as peptide M256265 which had a higher MHC binding affinity, also induced IL-4. Higher concentrations of peptide R256265 inhibited the production of IL-4, IL-10 and IFN-
in contrast to the induction of these cytokines by the mycobacterial peptide M256265. Thus, it argues more for a qualitatively different TCR signal induced by peptide R256265 rather than the effects being related to a reduced amount of MHCpeptide complexes.
Further analysis of cell-surface markers revealed that peptide R256265 preferentially induced the expression of B7-2 on M256270-specific T cells. This was not seen for peptide M256265 (Fig. 6c). At all the concentrations tested peptide R256265 did not induce the expression of B7-1 on M256270-specific T cells, in contrast to that seen upon stimulation with peptide M256265 (Fig. 6b
). So far no data on the differential expression of B7-1 and B7-2 on T cells after activation with altered peptides or self-epitopes have been described. However, several reports suggested a role for B7-2, expressed on T cells, in the down-modulation of T cell responses, including anti-T cell T cell responses (52). Greenfield et al. reported that B7-2-transfected T cell tumors did not provide co-stimulation to other T cells in vitro and, in fact, inhibited the induction of anti-tumor immunity in vivo. In addition, they showed that the B7-2 expressed on the T cell tumors preferentially bound to CTLA-4 and this was also observed for the B7-2 expressed on a subpopulation of normal murine T cells (53), indicating that the B7-2 expressed on T cells may have different qualities than B7-2 expressed on APC. Indeed, it was shown that the B7-2 expressed on human T cells had a different glycosylation form with no detectable binding to CD28, but still binding to CTLA-4 (54). In addition, the interaction of B7-2 expressed on murine T cells with CTLA-4 was shown to be responsible for down-modulation of T cell responses in vitro (52). Recently it was reported that the B7-2 expressed on T cell tumors was responsible for suppression of antitumor immunity, with IL-4 and/or IL-10 producing CD4+ T cells playing a critical role in the suppression (55).
Thus, the up-regulation of B7-2 on M256270-specific T cells, induced upon recognition of self-hsp on APC, may well provide such T cells with a mechanism to control pathogenic T cells in vivo. As activated pathogenic rat T cells do express MHC class II molecules (56) and enhanced levels of self-hsp60 (22), they can present the self-hsp60-peptide to M256270-specific T cells expressing B7-2. Also, in a more direct way, this TT presentation of self-hsp may induce the expression of B7-2 on the M256270-specific T cells and in support of this we found that TT presentation of peptide R256265 also increased B7-2 expression (data not shown). In both cases, B7-2 expression would lead to a negative signal upon interaction with CTLA-4 expressed on the activated pathogenic T cells contributing to regulation. In addition to studies of B7-2 expression on professional APC, that showed B7-2 signaling to be required for the development of IL-4 and IL-10 producing T cells in vitro and in vivo (57), we describe here that peptide R256265 preferentially induced B7-2 expression on T cells at (low) peptide concentrations when induction of IL-4 and IL-10 was also observed. It has been reported that blockade of LFA-1/ICAM-1 signaling promoted Th2 development (58), which also seems in agreement with our finding of decreased LFA-1 expression upon stimulation with peptide R256265.
Thus, in the case of T cells recognizing self-hsp60 as an APL, they can discriminate between self or non-self by detecting qualitatively distinct signals through the TCR. Importantly, such APL-like recognition of self might lead to a potential regulatory phenotype which could well be triggered in vivo upon recognition of self-hsp60 on cells leading to down-modulation of pathogenic T cells. In particular, up-regulation of activation markers, down-regulation of the TCR/LFA-1, and induction of the anti-inflammatory cytokines IL-4 and IL-10 together with the induction of B7-2 were observed at those (low) peptide concentrations of R256265 which may be physiologically relevant. Therefore, this ability of a self-hsp60 peptide to be recognized as an APL not only can promote positive selection of these self-reactive T cells in the thymus, but may also ensure that upon recognition in the periphery these T cells are tolerized into a regulatory mode, thereby controlling peripheral tolerance to other self-antigens.
![]() |
Acknowledgments |
---|
![]() |
Abbreviations |
---|
AA adjuvant arthritis |
APC antigen-presenting cell |
APL altered peptide ligand |
BCECF-AM 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester |
DDA dimethyl dioctadecyl ammonium bromide |
MBP myelin basic protein |
NRS naive inactivated rat serum |
PE phycoerythrin |
PLNC primed lymph node cells |
![]() |
Notes |
---|
Received 12 January 2000, accepted 21 March 2000.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|