HY peptides modulate transplantation responses to skin allografts

Edward James1, Diane Scott1, Jian-Guo Chai1, Maggie Millrain1, Phillip Chandler1,2 and Elizabeth Simpson1

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


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Injection of female C57BL/6 mice with immature female bone marrow-derived dendritic cells (BMDC) pulsed with a single immunodominant HYDb Uty peptide, WMHHNMDLI, induces prolonged survival of syngeneic male skin grafts. In contrast, injection of immature female BMDC pulsed with a single MHC class I-restricted HYAb Dby peptide, NAGFNSNRANSSRSS, causes immunization similar to that following injection of male cells. Tolerance induced by HYDb Uty peptide pretreatment is not characterized by clonal deletion: long-term tolerant mice maintain circulating HYDb Uty tetramer+ T cells which expand following exposure to male cells in vivo or in vitro. Tolerance to male skin grafts can be adoptively transferred into neonatal females with splenocytes from tolerant donors. Tolerance is specific—third-party skin grafts are rejected. We propose that tolerance in this model is initiated by cognate interaction of HYDb Uty-specific CD8+ T cells with their ligand, presented either on the injected immature BMDC or on recipient DC. This interaction leads to incomplete activation of the CD8+ T cells resulting in diminished responsiveness of CD4+ and CD8+ T cells specific for HY peptide epitopes subsequently presented on the male graft.

Keywords: dendritic cell, epitope, immunoregulation, tetramer analysis, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Minor histocompatibility (H) antigens remain a barrier to the transplantation of organs and tissues between individuals matched for the strong antigens encoded by the major histocompatibility complex (MHC). Minor H antigens are composed of peptides derived from polymorphic intracellular proteins. They function to stabilize MHC molecules during biosynthesis for expression at the cell surface. These antigens are encoded by both autosomal and Y chromosome genes (1). Bone marrow grafts between HLA-matched siblings are often complicated by graft-versus-host disease (GVHD) directed at minor H disparities, including the male-specific antigen, HY (2). Such GVH responses can be alleviated by treatment with immunosuppressive drugs, but this exposes patients to the risk of infection. The ability to induce specific tolerance to minor H antigens would be clinically advantageous and there is a need to provide pre-clinical data with manipulable experimental models.

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 results—some 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.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
C57BL/6J (B6) mice (6–8 weeks old) were purchased from Harlan Olac (Bicester, UK). C57BL/6EiJ (B6/EiJ) mice (a closely related histocompatible subline of C57BL/6J) were bred and maintained in the Biological Services Unit at the Hammersmith Hospital, where they were available to set up timed matings. All mice were housed under specific pathogen-free conditions.

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 10–5 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 6–8 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 6–7 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 cell–Dynabead 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-CD4–allophycocyanin and CD8–PerCp (BD PharMingen, Oxford, UK) antibodies together with one of the following: anti-CD69–FITC, CD44–FITC, CD25–FITC, CD45RB–FITC, CD3{epsilon}–FITC or CD62L–FITC (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-{gamma} (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-{gamma} (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 streptavidin–alkaline 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 2–3 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).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell surface phenotype of female BMDC after in vitro culture with GM-CSF
Comparative cytometric analysis of female BMDC, taken after 8 days in vitro culture in the presence of 75 ng/ml GM-CSF and splenic DC is shown in Fig. 1. Approximately 80% of the BMDC were judged to be DC, as indicated by CD11c expression (Fig. 1A) and all the CD11c+ cells also express CD11b (Fig. 1G). All BMDC expressed low levels of the co-stimulatory markers CD80, CD86, CD40 and MHC class II (Fig. 1B–F). This is in contrast to the more mature splenic DC that expressed much higher levels of CD80, CD86 and MHC class II co-stimulatory molecules. These results indicate that the BMDC are of immature phenotype.



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Fig. 1. Comparison of cell surface markers expressed by BMDC (day 8) and splenic DC. Expression by BMDC (8 days) (dark grey) and splenic DC (light grey) of (A) CD11c, (B) CD80, (C) CD86, (D) CD40 (BMDC only), (E) MHC class I (BMDC only), (F) MHC class II and (G) CD11b. Filled histograms represent negative controls.

 
Class I- and II-restricted HY peptides have immunomodulatory effects on skin graft rejection
Female BMDC pulsed with the HYDb Uty peptide, WMHHNMDLI, or the HYAb Dby peptide, NAGFNSNRANSSRSS, were injected into the footpads of groups of B6 females. Figure 2 shows the aggregate male skin graft survival times of female recipients in 15 experiments performed over a period of 3 years. These include 10 control females receiving no pretreatment, 68 control females given footpad injection of female BMDC alone, 92 pretreated with female BMDC pulsed with the HYDb Uty peptide, 22 pretreated with HYAb Dby peptide pulsed BMDC and 14 given male BMDC. All the mice received test male grafts 21 days after pretreatment. Preliminary experiments indicated that this was the optimal time for establishing immunomodulation. Female mice receiving grafts 14 days after pretreatment did not show prolonged graft survival, whilst those receiving grafts 28 days after pretreatment showed a slightly lower proportion of mice with graft prolongation (data not shown). Essentially similar results have been reported by Waldmann et al. who showed that it took some time (5 weeks) to establish tolerance in their CD4 antibody model of transplantation tolerance (23). The median survival time (MST) of the grafts of the two control groups, untreated and female BMDC pretreated, was 44 and 45 days respectively. This is slightly longer than has been previously reported (24), and is likely to be the result of natural variation seen between different housing conditions, microbiological status and diet. There is a subset of females within the large group pretreated with female BMDC pulsed with the HYDb Uty peptide that reject, with a similar tempo as the controls and a group that exhibit prolonged survival of the male grafts, most retaining their graft to at least 100 days, some to beyond 200 days (MST > 100 days). In marked contrast, mice pretreated with the HYAb Dby peptide showed significantly shorter MST (14.5 days) compared with the control groups (P < 0.001). The sensitization provided by the HYAb Dby peptide is equivalent to that following pretreatment with male BMDC (MST 12.5 days).



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Fig. 2. The effect of pretreatment of B6 females with male BMDC or peptide-pulsed female BMDC on the survival of test male grafts. Graft survival of syngeneic male grafts placed onto B6 female mice that had been pretreated by footpad injection with either male BMDC (triangles, 14 recipients), unpulsed female BMDC (circles, 68 recipients), HYDb Uty peptide-pulsed female BMDC (squares, 92 recipients) or HYAb Dby peptide-pulsed female BMDC (diamonds, 22 recipients) all taken after 8 days in vitro culture. A further group of mice were left untreated (upside down triangles, 10). All mice received a B6 male skin graft 21 days after pretreatment.

 
Phenotype of HY-specific T cells during responses to skin grafts modulated by peptides
A time course experiment was carried out to measure the appearance of T cells specific for class I- and II-restricted HY epitopes in groups of mice pretreated with female BMDC alone or pulsed with the HYDb Uty peptide (tolerance inducing) or the HYAb Dby peptide (stimulatory). Mice from each group were then test-grafted with male skin on day 21. HY peptide-specific cells were measured at intervals following peptide pretreatment and after skin grafting.

Analysis of HY peptide-specific cells by tetramer (HYDb Uty) and by IFN-{gamma} 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-{gamma} 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-{gamma} 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-{gamma} 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-{gamma} 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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Fig. 3. Ex vivo analysis of splenic T cells from BMDC-pretreated mice. BMDC were injected into B6 female mice 21 days prior to placement of a B6 male skin graft. (A) Mice pretreated with unpulsed (squares), HYDb Uty-pulsed (triangles) or HYAb Dby-pulsed (circles) female BMDC were sacrificed at 10, 15, 20, 29, 42, 49, 55 and 63 days after skin grafting, and the percentage of HYDb Uty-specific CD8+ T cells in spleens was assessed. (B) Representative FACS plots of the activation state of HYDb Uty tetramer+ CD8+ T cells from two mice pretreated with HYDb Uty-pulsed BMDC sacrificed at days 20 and 63 respectively. (C) The number of IFN-{gamma}-secreting cells per 2 x 105 from spleens of BMDC-pretreated mice was evaluated ex vivo following 24-h culture with either irradiated male or female spleen cells pulsed with the HYAb Dby peptide.

 
In contrast, the group of mice pretreated with the HYAb Dby peptide-pulsed female BMDC showed high levels of HYAb Dby-specific CD4+ T cell responses assessed by IFN-{gamma} ELISPOT analysis 10 days after grafting (Fig. 3C). At this time point, there were no HYDb Uty tetramer+ CD8+ T cells in these mice (Fig. 3A). By day 15, mice in this group had rejected their grafts and as well as raised levels of HYAb Dby-specific T cells, there were now detectable levels of HYDb Uty tetramer+ CD8+ T cells (Fig. 3A and C). The tetramer+ cells showed up-regulation of CD44 and down-regulation of CD62L (not shown), again consistent with activation/memory phenotype. Whether the appearance of the HY-specific class I-restricted T cells in these mice contributed to the graft rejection, together with the class II-restricted HYAb Dby-specific cells or whether they were a consequence of presence of the graft and the release of antigenic material is unclear. Day 15 was the last time point for which mice from this group were analysed.

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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Fig. 4. Abrogation of the induction of non-responsiveness. The results show skin graft survival from B6 female mice that received various pretreatments. Two groups were pretreated only with unpulsed (circles) or HYDb Uty peptide-pulsed BMDC (squares). Three groups received HYDb Uty-pulsed BMDC and 7 days after the initial injection were given an additional injection of HYAb Dby peptide-pulsed BMDC (diamonds) male BMDC (upside down triangles) or BMDC pulsed with a low dose (10 µM) of HYAb Dby peptide (triangles). All mice received a male skin graft 21 days after the second (or only) injection.

 
Phenotype and function of T cells from long-term skin graft tolerant mice
Spleen cells from 10 long-term tolerant mice pretreated with female BMDC pulsed with the HYDb Uty peptide that had retained male grafts for between 98 and 325 days were examined for the presence of HY-reactive T cells. The percentages of HYDb Uty tetramer+ CD8+ T cells varied between 0.06 and 0.6% (Table 1). These levels are similar to those found in mice several months after immunization with male tissues, which retain the ability to mount rapid ‘memory’ responses on exposure to male cells in vivo and in vitro [(14) and our unpublished data]. The cell surface phenotype of HYDb Uty tetramer+ cells from these long-term tolerant mice was variable with respect to activation/memory markers. The levels of CD25 expression were, with only two exceptions, uniformly low, and the HYDb Uty tetramer+ cells showed up-regulation of CD44 and down-regulation of CD62L and CD3{epsilon}, indicating antigen exposure. This cell surface phenotype is similar to that of the HYDb Uty tetramer+ cells in mice that had just rejected their grafts. In vitro re-stimulation of splenocytes from long-term tolerant mice with male cells resulted in expansion of the percentage of HYDb Uty tetramer+ T cells (6- to 40-fold). This indicates that the HYDb Uty peptide-specific T cells present in these tolerant mice were able to respond to the male antigen in vitro, a finding which is in contrast to naive mice which, although also exhibiting very low levels of tetramer+ cells, do not show expansion on in vitro re-stimulation [(14) and our unpublished data]. In addition, the tetramer+ cells from these naive mice gave a very consistent pattern of cell surface phenotype, exhibiting low levels of CD69, CD25 and CD44, and high levels of CD3{epsilon} and CD62L, typical of naive cells.


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Table 1. Percentage of HYDb Uty-specific CD8+ T cells in spleen cell populations taken from tolerant mice that had retained their male skin grafts until the time of sacrifice
 
In vivo re-stimulation of non-responsive mice, that had tolerated their male test grafts for between 300 and 350 days, by i.p. injection of 5 x 106 male spleen cells, also resulted in a significant expansion of HYDb Uty tetramer+ CD8+ T cells. Analysis of HYDb Uty tetramer+ cells in the PBL of these mice 15 days after injection showed that six of the seven tolerant mice had increased the percentage of HYDb Uty tetramer+ cells to between 2 and almost 20% (Fig. 5). The mouse with very high numbers of tetramer+ cells did eventually reject its previously tolerated graft, but not until 42 days after treatment. The remainder retained their grafts >100 days, despite the significant in vivo clonal expansions of HYDb Uty specific T cells.



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Fig. 5. Effects of injection of male cells into tolerant mice. B6 females that had retained their B6 male skin grafts for >200 days were injected i.p. with 5 x 106 male spleen cells. The mice were bled at 2-week intervals following injection and analysed for the presence of HYDb Uty-specific CD8+ T cells. The percentages of tetramer+ cells for mouse 1 (squares), 2 (triangles), 3 (upside down triangles), 4 (diamonds), 5 (circles), 6 (asterix) and 7 (crosses) are shown.

 
Regulatory cells can transfer tolerance
Unseparated spleen cells containing small numbers of HYDb Uty tetramer+ CD8+ T cells from long-term skin graft tolerant mice were injected into groups of four to nine female neonatal mice. When the recipients reached 6 weeks of age they were test-grafted with syngeneic male skin. Figure 6(A) shows the results of the first adoptive transfer experiment. One donor transferred lasting graft acceptance to five of the seven recipients (MST > 100 days), whereas transfer of spleen cells from the other two donors led to only two out of seven and one out of four recipients respectively with prolonged graft acceptance (MSTs of 52 and 36 days respectively). This result suggests that, whilst regulatory cells are present in the spleens of long-term tolerant mice, the balance between them and potential effector cells varies between mice, perhaps being disturbed by the adoptive transfer procedure itself.



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Fig. 6. Adoptive transfer of tolerance. (A) Graft survival of female B6/EiJ neonates (<3 days old) that had been injected i.p. with 5 x 106 unseparated spleen cells from three individual tolerant donors (indicated by squares, triangles and upside down triangles) or with 5 x 106 spleen cells from a naive female (circles). (B) Combined results of graft survival from three adoptive transfer experiments using three tolerant donor mice. Female B6/EiJ neonates were injected with 5 x 106 unseparated (squares, 15 recipients), CD8+ T cell-depleted (upside down triangles, 20 recipients) or CD4+ T cell-depleted (diamonds, 12 recipients) donor spleen cells from tolerant donors. A further group received naive B6 female spleen cells (circles, 16 recipients). All recipients were given a B6/EiJ male skin graft at 6 weeks of age.

 
In subsequent experiments, the spleen cells from long-term skin graft tolerant mice were depleted of CD8+ or CD4+ subpopulations before injection into neonatal female recipients. For each donor, a control population of unseparated splenocytes was injected into at least five recipients, to ascertain whether they contained the regulatory population(s). Figure 6(B) shows the aggregate survival time, from three experiments, of male skin grafts following adoptive transfer of unseparated (15 recipients), CD8+ T cell-depleted (20 recipients) or CD4+ T cell-depleted (12 recipients) spleen cells from long-term tolerant donors. The results from a group of mice receiving spleen cells from a naive female mouse (16 recipients) are also shown. In another cohort (four recipients) given splenocytes from a donor that had been pretreated with female BMDC and had rejected its graft 50 days previously, recipients rejected their grafts between days 35 and 50 (not shown).

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.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tolerance is induced by the HYDb Uty peptide
The induction of a state of non-responsiveness or tolerance in high responder H2b females to syngeneic male skin grafts following in vivo administration of the HYDb Uty HY peptide pulsed onto immature female BMDC is the central finding of this report (Fig. 2). Such a model showing induction of antigen-specific tolerance to a single MHC class I-restricted minor H epitope, that is then able to induce non-responsiveness to second MHC class I and a potent helper MHC class II-restricted epitope, is similar to the linked suppression originally described by Waldmann et al. (19). It is also one that, if applicable to other H antigens, might provide an effective alternative to the ‘blanket’ immunosuppression currently used in the treatment of acute and chronic graft rejection. Other groups have also reported that for both HY (26) and other tumour or minor H antigens (16,18,27) presentation of a single MHC class I-restricted epitope failed to induce an antigen-specific T cell response. Rather it induced non-responsiveness even when the class I determinant was subsequently presented in the presence of a helper epitope.

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 I–peptide 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 3–5 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.


    Acknowledgements
 
The authors thank Justin Locke and Geeta Patel for making the synthetic peptides, David Gray for the GM-CSF-secreting hybridoma, X63-Ag8, Mrs Vivien Tikerpae for help with the preparation, and Robert Lechler for critically reading the manuscript. We thank Jonathan Silk and Hamlata Dewchand for their help throughout.


    Abbreviations
 
APC—antigen-presenting cell

BMDC—bone marrow-derived dendritic cells

DC—dendritic cell

GM-CSF—granulocyte macrophage colony stimulating factor

GVHD—graft versus host disease

H—histocompatibility

HYDb Uty, HYDb Smcy and HYAb Dby

—H2Db- and H2Ab-restricted HY peptides derived fromthe Uty, Smcy and Dby genes respectively

MST—mean survival time


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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