1 Transplantation Biology Group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College of Science, Technology & Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK 2 Present address: Department of Molecular Immunology, Institute of Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-03175, USA
Correspondence to: E. Simpson; E-mail: elizabeth.simpson{at}csc.mrc.ac.uk
Transmitting editor: T. Hünig
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Abstract |
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Keywords: dendritic cell, epitope, immunoregulation, tetramer analysis, tolerance
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Introduction |
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The experimental model that we have chosen to examine is that of the graft rejection response mediated by the male-specific transplantation antigen, HY, in mice. The reasons for this are that the response to HY can be studied in isolation from autosomal antigens, since in every inbred strain, males differ from females only by virtue of the Y chromosome genes. Furthermore, in the mouse, most of the genes that have been shown to be involved in the response to HY, and the MHC class I- and II-restricted peptides that they encode have been identified (37). Similarly, in humans, many of the genes and MHC class I-restricted peptides have been identified (813). In addition, the identification of MHC class I-restricted HY peptides has permitted the use of tetramers to enumerate ex vivo the proportion of CD8+ T cells making HY responses, both in mice (14) and humans (15).
In vivo administration of peptides from tumour-associated antigens has been used in attempts to induce anti-tumour responses; these have met with variable resultssome provoking immunity (16), others exacerbation of tumour growth (17). Increase in tumour growth might be attributable to induction of tolerance, since exposure to an individual MHC class I-restricted tumour peptide in the absence of a helper determinant has been shown to induce tolerance to skin grafts bearing that antigen (18). These findings suggested to us the approach of pretreatment of females of the H2b haplotype which are high responders to HY, with selected HY peptides before placing a test male skin graft.
Our approach was further informed by the observations of Waldmann et al. that induction of tolerance to a set of minor alloantigens under the umbrella of anti-CD4 and anti-CD8 treatment could also induce tolerance to a second alloantigen when it was presented as a subsequent F1 graft to the tolerant animal, thus leading to the concept of linked suppression (19).
This paper describes conditions under which a single class I-restricted minor H peptide can be used to induce tolerance to test grafts expressing both the inducing and additional MHC class I- as well as class II-restricted epitopes. This tolerance does not appear to be associated with clonal deletion or anergy. In vivo transfer of tolerance implicates regulatory mechanism(s) involving interaction between CD4+ and CD8+ cells. In contrast the single class II peptide presented in vivo in an identical fashion causes immunization.
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Methods |
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Tissue culture media
RPMI 1640 Medium (Gibco/BRL, Paisley, UK) was supplemented with 10% FCS (Bioclear, Calne, UK), HEPES (10mM), penicillin (100 IU/ml) and 100 µg/ml streptomycin (Gibco/BRL), 5 x 105 M 2-mercaptoethanol and 2 mM/ml L-glutamine (Gibco/BRL).
Peptides and tetramers
Peptides were synthesized by the Central Research Resources unit at the Hammersmith Hospital. This was performed using FMOC-protected amino acids and [2-(1-H-benzotriazol-2-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] activation chemistry. After purification by HPLC, the fidelity of synthesis was confirmed by mass spectrometry. Synthetic peptides were made up as 1 mM stocks in PBS and filter-sterilized.
MHC class I tetramers were produced using a modification of the method of Altman et al. (20) as described previously (14).
Generation and phenotypic analysis of bone marrow-derived dendritic cells (BMDC) and splenic DC
For the preparation of BMDC, bone marrow cells were isolated from the femur and tibia of donor mice. The bones were excised and cleaned of muscle and tendon and cut at the epiphysis at each end. The bone marrow was flushed out with 5 ml BSS/10%FCS using a syringe with a 25 gauge needle. The bone marrow cells were resuspended at 2 5 x 105 cells/ml in complete RPMI medium containing 5% granulocyte macrophage colony stimulating factor (GM-CSF) supernatant (75 ng/ml GM-CSF) from the GM-CSF-secreting hybridoma X63-Ag8, a kind gift from David Gray, and dispensed at 6 ml/well in six-well plates. The cells were incubated at 37°C and 5% CO2 for 68 days.
Single-cell suspensions of spleens from donor mice were made in PBS, centrifuged at 1500 r.p.m. for 7 min, washed once in PBS and resuspended in RPMI medium (15 ml for every two spleen equivalents). After incubation at 37°C for 5 min, the cells were transferred to a 150-mm Petri dish and incubated at 37°C for 90 min. Non-adherent cells were washed off with warm RPMI medium and 15 ml of fresh RPMI medium was added to the adherent population. The cells were incubated overnight at 37°C. The non-adherent DC were harvested and cell surface phenotype assessed.
Pretreatment of mice before test skin grafting
BMDC were harvested at the appropriate time. Aliquots of 67 x 106 cells were pelleted (1700 r.p.m. for 5 min) and resuspended in 1 ml RPMI medium containing 15 µl peptide (HYDb Uty peptide, WMHHNMDLI or HYAb Dby peptide, NAGFNSNRANSSRSS: 1 mM in PBS). The cells were incubated at 37°C for 90 min and washed 3 times in RPMI medium (1200 r.p.m. for 5 min). Non-pulsed and peptide-pulsed DC were resuspended to 2 x 108 BMDC/ml in PBS, and 25 µl injected into the footpad of recipient mice (giving 5 x 106 cells/recipient).
Dynabead depletion
Spleen cells from experimental mice were washed in RPMI resuspended in 3 ml RPMI For depletion of B cells, 0.3 ml of sheep anti-mouse IgG Dynabeads (Dynal, Bromborough, UK) were washed twice in BSS and resuspended in 2 ml cold RPMI. For depletion of CD4+ or CD8+ T cells, 0.15 ml of either anti-CD4 or anti-CD8 Dynabeads (Dynal) were washed as described above. The spleen cells were mixed with the Dynabeads and incubated at 4°C for 30 min with rotation. The cellDynabead suspensions were placed on a magnet for 5 min. Unbound cells were then removed, transferred to a fresh tube and placed on the magnet for a further 5 min. The unbound cells were again transferred into a fresh tube and centrifuged at 1200 r.p.m. for 5 min. The cells were either resuspended at 5 x 107cells/ml for injection or 1 x 107cells/ml for flow cytometric analysis.
FACS staining and analysis of T cells
Spleen cells
Spleen cells (5 x 106) were depleted of B cells by incubation with sheep anti-mouse IgG Dynabeads (Dynal) and washed in 10ml PBS containing 5% FCS (PBS wash) and centrifuged at 1200 r.p.m. for 5 min. The resuspended cell pellet was incubated with the HYDb Uty tetramer (14) for 20 min at room temperature in the dark and washed 3 times. The cells were resuspended in 100 µl PBS wash, incubated with anti-CD4allophycocyanin and CD8PerCp (BD PharMingen, Oxford, UK) antibodies together with one of the following: anti-CD69FITC, CD44FITC, CD25FITC, CD45RBFITC, CD3FITC or CD62LFITC (BD PharMingen) for 30 min at 4°C in the dark. The samples were washed 3 times in PBS wash and analysed by flow cytometry using a FACSCalibur (Becton Dickinson).
Peripheral blood lymphocyte staining
Mice were tail-bled into Eppendorf tubes containing 200 µl blood buffer (10 mM EDTA and 100 U heparin/ml in PBS) and mixed well. Immediately after mixing, 1 ml red blood cell lysis buffer (Puregene) was added. The cell suspension was mixed by inverting 6 times, incubated at room temperature for 15 min and centrifuged at 3500 r.p.m. for 4 min. The supernatant was removed, and the cell pellet was resuspended in 50 µl PBS wash and transferred into tubes for staining. Tetramer and antibody staining was performed as described above.
ELISPOT assays
These were carried out as described previously (14). Briefly, plates (MultiScreen-IP; Millipore, Bedford, MA) were coated with anti-mouse IFN- (BD PharMingen). Peptide-pulsed stimulator cells [107 female splenocytes incubated with 10 µM peptide (HYDb Uty, HYDb Smcy or HYAb Dby) 37°C for 2 h and washed 3 times] or unpulsed male and female cells were plated at 2 x 105 cells/well. Responder cells were also plated at 2 x 105 cells/well in a final volume of 200 µl. The cells were incubated overnight at 37°C, water lysed and washed with PBS/0.1% Tween 20. Then 100 µl biotinylated anti-mouse IFN-
(20 ng/ml in PBS/1% BSA) was added and incubated at 4°C overnight. The plates were washed, incubated for 1 h at room temperature with 1 µg/ml streptavidinalkaline phosphatase (Sigma, Poole, UK) and developed with 5-bromo-4chloro-3-indolyl phosphate/nitroblue tetrazolium substrate (Sigma).
Cell transfer in vivo
B6/EiJ females time-mated with syngeneic males were used to produce neonates. Female neonates were injected i.p. within 3 days of birth with either 5 x 106 undepleted donor spleen cells from tolerant or control mice or the remainder from 5 x 106 spleen cells following depletion of the selected population. At 6 weeks of age the recipients were grafted with syngeneic male tail skin.
Skin grafting
Skin grafting was conducted as described previously (21) and after removal of the plaster casts, the grafts were observed every 23 days. Grafts were scored as being rejected when <10% viable tissue was visible. Statistical analysis of graft survival was by the log-rank method (22).
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Results |
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Analysis of HY peptide-specific cells by tetramer (HYDb Uty) and by IFN- ELISPOT (HYDb Uty, HYDb Smcy and HYAb Dby) analysis, in spleens from mice sacrificed on days 7, 14, 21 and 28 after peptide pretreatment, but not grafted, showed no HY peptide-specific T cells (results not shown). This suggests that the immunoregulation being induced at this time did not involve detectable numbers of HY peptide-specific T cells, at least in the splenic population.
Tetramer (Fig. 3A) and IFN- ELISPOT (not shown) analysis of the control group pretreated with female BMDC alone and the immunomodulated group pretreated with HYDb Uty peptide-pulsed female BMDC showed no evidence for the presence of HY-specific cells at day 10 after grafting. However, by day 20 both tetramer+ CD8+ T cells and T cells reactive to the three HY peptides identified by ELISPOT analysis could be detected. The percentage of HYDb Uty tetramer+ cells was similar,
1%, at days 20 and 29 in both control and HYDb Uty peptide-pretreated groups, as was their cell surface phenotype. This showed up-regulation of CD44 and down-regulation of CD62L, a pattern consistent with an activation/memory phenotype (Fig. 3B). IFN-
ELISPOT analysis of HY peptide-specific cells at these time points also showed increased responses, but again there was little difference between the responses of the control and HYDb Uty peptide-pulsed groups. Figure 3(C) shows the IFN-
ELISPOT results from cells stimulated in vitro with the HYAb Dby peptide. Overall, similar results were obtained using the HYDb Uty and HYDb Smcy peptides (not shown), i.e. there was little difference between mice pretreated with unpulsed female BMDC and those pulsed with the HYDb Uty peptide. By day 42, the magnitude of the HY response had diminished as measured by tetramer analysis (Fig. 3A) and the responses remained low at days 49 (not shown) and 55 (Fig. 3A). Even at day 63, when some of the mice were showing signs of skin graft rejection, the numbers of HY tetramer and peptide-specific T cells in the spleen remained low (not shown). In both groups at the later time points the tetramer+ cells showed a broad spectrum of levels of cell surface expression of CD44 and some cells had down-regulated CD62L (Fig. 3B). IFN-
ELISPOT analysis of HY peptide-specific cells at the later time points gave similar results to the tetramer analysis, and no difference between the control and HYDb Uty peptide pretreated groups was observed.
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These results indicate that the immunomodulation of male graft rejection induced by pretreatment with the HYDb Uty peptide did not appear to be associated with deletion of T cells specific for this epitope. Nor did the tetramer+ cells in these mice have cell surface markers that distinguished them from tetramer+ cells from the control group of mice pretreated with female DC alone. In addition, staining with anti-CD25 and CD69 showed uniformly low levels of expression on the majority of CD4+, CD8+ and HYDb Uty tetramer+ cells (data not shown), indicating that at the time points examined the cells were considerably beyond their early activation stage. The finding that there was no significant difference in the results of tetramer and ELISPOT analysis between the HYDb Uty peptide-treated and control BMDC-only-treated groups may reflect the fact that the graft rejection responses in these groups follow similar kinetics up to day 63. It might also be argued that because only 50% of the mice went on to reject their grafts and the time points chosen were before this was demonstrable, half of the experimental group might be expected to show similar responses to the control group. In the experiments reported here, none of the experimental group (from a total of 18 mice tested) was different from the control (18 mice also tested). In contrast, the accelerated rejection of test male grafts in HYAb Dby peptide-pretreated mice was associated with early appearance of T cells to this strong helper epitope and followed by the expansion of class I-restricted HYDb Uty-specific CD8+ cells at the time of graft rejection.
Blocking induction of tolerance
Interference with the induction of non-responsiveness, following pretreatment with HYDb Uty peptide-pulsed female BMDC on day 0, was tested by footpad injection of BMDC pulsed with the stimulatory HYAb Dby peptide. Three groups of mice received HYAb Dby peptide-pulsed BMDC on day 7, male BMDC or female BMDC pulsed with a much lower dose of HYAb Dby peptide. This lower dose of 10 µg had been previously shown to stimulate an HYAb Dby peptide-specific clone (B9) (7,25) in vitro to a similar level as male cells. All mice received a test male skin graft 21 days after the final BMDC injection. Mice in each of the HYAb Dby-treated groups rejected their grafts rapidly, with a second set rejection time (Fig. 4); thus not only was tolerance abrogated, but sensitization occurred instead. In contrast, in the control group receiving just HYDb Uty peptide-pulsed BMDC >50% of mice retained test skin grafts over 100 days.
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The graft survival time of the mice receiving unseparated splenocytes from tolerant donors (MST > 100 days) is significantly longer than those receiving splenocytes from naive donors (MST 59.5 days) (P < 0.001). Mice receiving CD4+-depleted spleen cells rejected their grafts with a tempo that was not significantly different from those receiving naive cells (MST 48 days). Those receiving CD8+-depleted spleen cells showed a slight prolongation of graft survival (MST 63 days), but this was not significantly different from the population that received spleen cells from naive female donors. The P value between mice receiving the CD8+-depleted and unseparated populations from tolerant donors was P < 0.006. These results therefore indicate that both CD8+ and CD4+ cells appear to be important in the adoptive transfer of tolerance. Placement of a second male graft together with a third-party graft >100 days after the first resulted in the retention of the second male graft on recipients that had maintained their original graft, whilst the third-party grafts were all rejected rapidly.
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Discussion |
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Contrasting effect of the HYAb Dby peptide.
In contrast to the results obtained with the HYDb Uty peptide, in vivo treatment of female H2b mice with female BMDC pulsed with the HYAb Dby peptide that is recognized solely by the CD4+ T cell population, causes a rapid second set rejection of male grafts, similar to that induced by immunization with male cells (Fig. 2). Thus it appears that the same population of immature BMDC is able to induce either tolerance or immunity, depending on the peptide with which the cells have been pre-pulsed. Evidence from the time course analysis indicates that following pretreatment with the HYAb Dby peptide there is an initial expansion of the HYAb Dby-specific CD4+ T cells (Fig. 3C) followed by the appearance of HYDb Uty tetramer+ cells only at the time of graft rejection (Fig. 3A). Thus the HYAb Dby peptide-pulsed BMDC population is able to activate CD4+ cells which, on encountering the same epitope subsequently presented directly or indirectly by the male graft, are able to provide help for the HYDb Uty-specific CD8+ T cell populations which expand as the grafts are rejected.
Mechanisms of tolerance induction
Induction of non-responsiveness in the model investigated here depends on the use of immature female BMDC to present the HYDb Uty peptide. This was demonstrated by the finding that lipopolysaccharide-activated BMDC pulsed with the HYDb Uty peptide failed to induce non-responsiveness (James, PhD Thesis, University of London, 2001). Immature BMDC generated with very low concentrations of GM-CSF (0.1 ng/ml) have been shown both to induce allo-unresponsiveness in vitro and to induce prolonged cardiac allograft survival when used to pretreat the recipients of completely MHC-mismatched heart grafts (28). The concentrations of GM-CSF used here were much higher (75 ng/ml). Nevertheless the BMDC used in the experiments here do express an immature phenotype as shown by the low level expression of MHC class II and the co-stimulatory molecules, CD40, CD80 and CD86, compared with mature splenic DC (Fig. 1).
Presentation of the HYDb Uty peptide by the injected immature BMDC could be direct or via uptake and transfer of the MHC class Ipeptide complex to host DC in the draining popliteal lymph node. Such a transfer of antigen onto host DC by a process known as cross-presentation has been shown to be potentially tolerogenic both for MHC class I (29)- and II (30)-restricted peptides. It has further been demonstrated that the uptake of apoptotic cells by DC can induce tolerance and this has been proposed as a mechanism whereby tolerance to self-antigens is maintained (31,32), and we have evidence (unpublished observations) that the injected BMDC do indeed contain a significant proportion of apoptotic cells.
The observation that the same population of immature female BMDC pulsed with the HYAb Dby peptide causes immunization shows clearly that these cells can present antigen in an immunogenic fashion in vivo. The lack of response to the HYDb Uty peptide therefore presumably results from incomplete activation of the CD8+ T cells in the absence of help, in the form of HY peptide-specific CD4+ T cells, resulting in the failure to establish a CD8+ effector population. The finding that the induction of tolerance can be prevented by treatment of the mice with female BMDC pulsed with the HYAb Dby peptide 7 days after the initial pretreatment with the potentially tolerizing HYDb Uty-pulsed female BMDC (Fig. 4) shows that the absence of help to a class II-restricted epitope, subsequently presented on the test graft, during the induction of tolerance, is crucially important.
It might be argued that tolerance induction is a result of localized expansion followed by peripheral deletion of the high-affinity HYDb Uty peptide-specific CD8+ T cells, as has been shown in several transgenic models (33,34). The postulate would then be that the remaining low-avidity HYDb Uty-specific CD8+ T cells detected by tetramer and ELISPOT analysis, together with the HYDb Smcy-specific CD8+ T cells would not be able to mount the necessary cytotoxic T lymphocyte response required for eliciting graft rejection. Evidence against this is 2-fold. Firstly, after peptide pretreatment and prior to grafting we could find no evidence for expansions of HY peptide-specific CD8+ T cells, at least in the splenic population. Secondly, treatment of H2b females with depleting anti-CD4 and/or anti-CD8 antibodies showed that, whilst giving both antibodies together at the time of grafting abrogates rejection for a prolonged period, either anti-CD4 or CD8 antibody given separately merely slows down rejection by a few days (Simpson et al., unpublished). These results imply that clonal expansion of naive cells in either the CD4+ or CD8+ population can provide effectors for male skin graft rejection.
We would favour the postulate that HYDb Uty-specific CD8+ T cells, after receiving an incomplete activation signal from the peptide-pulsed immature BMDC, following placement of the male graft, interact either directly with antigen-presenting cells (APC) from the graft, or indirectly with reprocessed male antigen on the recipient APC and prevent them from expressing their full stimulatory capacity. When such APC, expressing the range of HY peptides derived from male cells, then interact with the HY peptide-specific CD4+ and CD8+ T cells they in turn are not able to activate these cells and instead induce a regulatory population of cells. Evidence in favour of this model is from recent in vitro findings in which human anergic, albeit CD4+, T cells were able to cause suppression of antigen presentation by immature and mature DC (35).
Mechanism of maintenance of tolerance
Since potentially antigen-reactive cells are clearly demonstrable in the tolerant mice, the state of non-responsiveness must be being maintained by some manner of non-deletional mechanism. One possibility is that the HY-specific cells that may have been rendered anergic as a result of incomplete activation due to TCR triggering by the HYDb Uty peptide-pulsed female BMDC remain so in the presence of the male graft. The second possibility is that the potentially HY-reactive cells are being held in check by a regulatory population of cells. The small numbers of HYDb Uty tetramer+ T cells found in spleens of mice pretreated with HYDb Uty-pulsed female BMDC and bearing longstanding male skin grafts can be expanded by in vitro culture with male APC (Table 1). This is similar to the results obtained from mice that have rejected their grafts (14) and suggests that the HY peptide-specific cells in these tolerant mice are not anergic. Perhaps even more convincing are the in vivo findings of clonal expansions (Fig. 5). Injection of male spleen cells into long-term tolerant mice caused a significant expansion of HYDb Uty tetramer+ cells in six of the seven mice tested. This is the first report of such expansions of antigen-specific CD8+ T cells in tolerant mice in response to antigenic challenge. In only one instance, where the percentage of tetramer+ cells increased to almost 20%, did this immunization protocol result in graft rejection by these tolerant animals and, even then, the graft rejection was slow. These results suggest that maintenance of tolerance is not a result of an anergic CD8+ T cell population.
Direct evidence for the presence of regulatory cells comes from the finding that adoptive transfer of splenocytes from tolerant mice into neonatal recipients is able to cause transfer of non-responsiveness. The nature of the regulatory cells is not known. Depletion of either CD4+ or CD8+ populations resulted in considerable loss of the ability to transfer tolerance. Although CD4+ depletion appears more effective, CD8+ depletion also caused almost as significant a loss in ability to transfer tolerance.
Neonatal recipients were used in these adoptive transfer experiments for two reasons. One was the number of cells that need to be transferred. Only 5 x 106 cells were required for each neonatal recipient compared with 35 x 107 that would be necessary for transfer into adults. Since individual tolerant donors variably transfer tolerance, transfer of small numbers of cells allows the inclusion of a control group of recipients receiving unseparated cells for each donor. The second reason was that the use of neonates enabled in vivo expansion of the potential regulatory cell population, whereas transfer into adult recipients would not. However, it might be argued that the tolerance observed in the neonatal recipients was a result of chimerism due to the transfer of male cells in the spleen cell population from the long-term tolerant female donors that had maintained their male skin graft. This is extremely unlikely since the extent of chimerism in the donors was undetectable. The original work of Weissman et al. showed that at least 1 x 106 male splenocytes are required to cause tolerance in neonatal female recipients (36). This would mean 20% of the transferred tolerant splenocytes would have to be of male origin. Furthermore, from a report by Matzinger et al. it is clear that the only recipients of skin grafts to become chimeric with donor cells are those that are immunoincompetent at the time of grafting (37). According to this, one would not expect the tolerant donors to be chimeric with male cells.
The results reported here at some level mirror those obtained by Waldmann and Cobbold (38) who, using certain strain combinations, were able to induce tolerance to multiple minor H antigens under an umbrella of anti-CD4 and/or CD8 antibody treatment. Once established, this tolerance could be adoptively transferred and was dependent on CD4+ T cells. The substantial genetic differences between the MHC-matched strains they used would include large numbers of both class I- and II-restricted minor H epitopes. Nevertheless, CD4+ T cells alone permitted adoptive transfer. In these experiments it is therefore the same subpopulation that is implicated in both the induction and maintenance stage of the non-responsiveness, despite the fact that presumably both MHC class I- and II-restricted T cells would be involved in the rejection response per se. The ability of the transferred cells to cause linked suppression (19) and infectious tolerance (39) has led this group to propose that regulation possibly occurs as a result of incapacitation of the APC by the primary or secondary tolerant cells (40). Although in the experiments reported here, the regulation must initially involve CD8+ T cells, since during tolerance induction the only antigen present is the class I-restricted HYDb Uty peptide, once established the regulation is maintained by both CD8+ and CD4+ cells. The maintenance of tolerance may indeed be via incapacitated/tolerogenic APC.
In summary, this paper presents evidence that peptide epitopes of minor H antigens can be introduced in vivo in such a way as to induce tolerance or immunity. The choice of the HY model system has allowed analysis of the relevant peptide-specific T cell populations during the induction and effector phase of immunomodulated allograft responses. The results provide evidence for a regulatory network which includes both CD4+ and CD8+ cells, co-existing with allo-antigen reactive T cells capable of becoming effectors. This experimental system may model normal physiological regulation of immune responses to pathogens and autoantigens.
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Acknowledgements |
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Abbreviations |
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BMDCbone marrow-derived dendritic cells
DCdendritic cell
GM-CSFgranulocyte macrophage colony stimulating factor
GVHDgraft versus host disease
Hhistocompatibility
HYDb Uty, HYDb Smcy and HYAb Dby
H2Db- and H2Ab-restricted HY peptides derived fromthe Uty, Smcy and Dby genes respectively
MSTmean survival time
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References |
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