The Generation of CD25+CD4+ Regulatory T Cells That Prevent Allograft Rejection Does Not Compromise Immunity to a Viral Pathogen
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免疫学杂志 2005年第6期
Abstract
In all but a small minority of cases, continued survival of solid organ grafts after transplantation depends on lifelong, nonselective immunosuppression that, although effective, results in increased rates of infection, cancer, and vascular disease. Therapeutic strategies that engage or mimic self-tolerance may allow prolonged allograft survival without the disadvantages of nonspecific immunotherapy. Pretreatment of recipient mice with donor alloantigen combined with transient modulation of the peripheral T cell pool with anti-CD4 Ab leads to the indefinite survival of MHC-incompatible cardiac allografts without further therapy. Tolerance is dependent on CD25+CD4+ regulatory T cells that arise from naive CD25– precursors and regulate rejection via both IL-10 and CTLA-4. Although these cells are clearly effective at controlling rejection, the proven ability of recently activated CD25+ cells to mediate bystander regulation raises the possibility that tolerized individuals might also have a reduced capacity to respond to environmental pathogens. We have examined anti-influenza responses in tolerized primary heart recipients, secondary recipients following adoptive transfer of regulatory populations, and tolerized mice in which bystander regulation has been deliberately induced. Neither virus-specific CTL activity in vitro nor the clearance of virus in vivo was significantly diminished in any of these treatment groups compared with infected unmanipulated controls. The data suggest that the induction of dominant allograft tolerance dependent on regulatory T cells does not necessarily result in attenuated responses to pathogens providing further support for the development of tolerance induction protocols in clinical transplantation.
Introduction
The immune system contains peripheral regulatory mechanisms that play an essential role in the prevention of autoimmunity. Although as yet it has not been possible to define a single cell surface marker that is unique to regulatory cells, particular attention has been focused on CD4 cells expressing high levels of CD25, the -chain of the IL-2 receptor. CD25+CD4+ T cells have been shown to play an important regulatory role in several animal models of autoimmune disease including colitis (1), diabetes (2, 3), gastritis (4, 5), and thyroiditis (4). The fact that CD25+CD4+ T cells with regulatory capacity, at least in vitro, can also be found in human peripheral blood (6, 7) suggests that a breakdown of regulation could be an important factor in human autoimmune disease.
Such studies in autoimmune models have prompted a re-evaluation of the role of regulatory/suppressor cells in transplantation tolerance and accumulating evidence indicates that significant parallels exist between naturally occurring peripheral T cell regulation and the regulation of T cell responses toward a transplanted allograft following tolerance induction. For example, we and others (8, 9, 10) have demonstrated that tolerant animals with long-surviving allografts contain populations of CD25+CD4+ T cells that prevent allograft rejection in adoptive transfer transplant models. More recently, we have shown that these cells can be generated in vivo from naive precursors in a thymus-independent process (11) by exposure to alloantigen either with (12) or without (13) concomitant anti-CD4 Ab therapy and that these cells regulate anti-graft responses in vivo using mechanisms shared with naturally occurring regulatory cells (12).
The specific regulation of destructive T cell responses remains an attractive potential solution to the problem of rejection in clinical transplantation. Current clinical regimens rely on lifelong, nonspecific immunosuppression, and although these are effective in the short term, the dual problems of undesirable side effects and delayed graft loss due to chronic rejection continue to stimulate a search for alternative therapies. The generation of regulatory cells before transplant in the clinical setting could give the graft protection from the outset, provide a route to operational tolerance and allow long-term graft survival without chronic immunosuppression. However, little is known of the impact that these generated regulatory cells might have on protective T cell responses.
In the alloantigen/anti-CD4 Ab tolerance induction protocol, we have described (14) the CD25+CD4+ regulatory T cells generated by the pretreatment protocol regulate in a donor alloantigen-specific manner (12), but an important in vitro study has shown that once activated through their TCR, CD25+CD4+ T cells can also regulate without Ag specificity. Using T cells isolated from two distinct TCR transgenic mice specific for different peptides and restricted by either I-Ed or I-Ek, Thornton and Shevach (15) demonstrated that CD25+CD4+ T cells isolated from either cell donor could inhibit the proliferation of CD25– responders isolated from the corresponding TCR transgenic mouse. However, more importantly, once activated, the same CD25+CD4+ population could also inhibit the proliferation of CD25– responder cells isolated from the second TCR transgenic mouse in a manner that was both Ag and MHC independent, a phenomenon referred to as "bystander suppression". In the transplant setting where tolerance is dependent on regulatory T cells, the presence of the graft has been shown to be essential for maintenance of the tolerant state (16) probably by promoting a continual reactivation of the regulatory T cell population. We considered it possible that reactivation of regulatory cells by the graft could lead to nonspecific bystander suppression in vivo and that although it may be possible to generate donor-reactive CD25+CD4+ T cells that regulate responses toward the allograft, an effect on responses to environmental pathogens cannot be ruled out. Therefore, the aim of this study was to use a well-defined model of transplantation tolerance where continued graft survival is known to depend on CD25+CD4+ T cells and ask whether the presence of these cells has any impact on immunity against a well-characterized pathogen, influenza virus. The hypothesis tested was that tolerant mice might show attenuated antiviral responses.
Materials and Methods
Induction of tolerance and heart transplantation
CBA (H-2k) mice were pretreated with 200 μg of the IgG2a anti-CD4 Ab YTS 177.9 (17) on days –28 and –27 and with a donor-specific transfusion (DST) 5 of C57BL/10 (H-2b) blood on day –27 and day 0 (anti-CD4/DST + reboost protocol). Five days after the second DST (day +5), the tolerized mice were either transplanted with donor-specific hearts or used as cell donors for adoptive transfer experiments. Specific details are given in the relevant figure legends. Cardiac transplantation was conducted under general anesthesia according to the method outlined by Corry et al. (18). Briefly, hearts were isolated from exsanguinated heparinized donors, and the ascending aorta and pulmonary artery were anastomosed end-to-side to the recipient abdominal aorta and inferior vena cava, respectively. Recipient animals were maintained in specific pathogen-free conditions to minimize exposure to environmental pathogens. Heart function was followed by daily palpation and the quality of each graft was determined at 100 days posttransplant as described below.
Enumeration of arterial intimal proliferation as a measure of graft outcome
At 100 days posttransplant, hearts were removed from recipients under terminal anesthesia and snap-frozen in OCT embedding compound (Bayer) in liquid nitrogen. Cryostat sections (8 μm) were taken from six planes each 200 μm apart in a method based on that of Armstrong et al. (19). Sections were then fixed in PBS/10% formaldehyde and stained using Miller’s elastin stain, which identifies elastic fibers combined with Van Gieson counter staining. The degree of intimal proliferation in arteries >80 μm in diameter was then determined as described (19) using pixel-counting software (Zeiss) to measure the area of the vascular lumen relative to the area circumscribed by the internal elastic lamina. Data are presented as percentage of vascular occlusion.
Infection with influenza virus and determination of antiviral responses
Mice were infected intranasally with five hemagglutinin (HA) units of influenza virus A/PR/8/34 (PR8) as shown in the figures. Spleens were harvested for flu-specific CTL assays 11 days after infection, and lungs were harvested for the determination of viral load.
CTL assays
Single spleen cell suspensions from infected mice were plated at 4 x 106 per well (24-well plate) in T cell medium (RPMI 1640 supplemented with 10–5 M 2-ME, 20 mM sodium pyruvate, 2 mM glutamine, 45 μg/ml penicillin, 45 μg/ml streptomycin, 90 μg/ml kanamycin, and 10% FCS). The cells were restimulated in vitro by the addition of irradiated peptide-pulsed stimulators (1 x 106 per well) to the culture wells. Stimulators were prepared by incubating naive CBA spleen cells for 1 h at 37°C with the immunodominant H-2Kk-restricted peptide of PR8 nucleoprotein SDYEGRLI (20) at a concentration of 10–4 M. The cells were then irradiated, washed twice, and resuspended in T cell medium. After 2 days of incubation at 37°C, rIL-2 was added to the cultures at a final concentration of 10 U/ml. After a further 3 days, equal numbers of wells from individual mice were resuspended in 700 μl, and 3-fold dilutions of the effector cells were performed. Chromium release assays were performed as described previously (21) using 51Cr-labeled H-2k-positive L cell targets (L929) either unpulsed or pulsed with the SDYEGRLI peptide at a final concentration of 10–5 M. Specific lysis was determined as described previously (21). Total available 51Cr was defined by incubation of targets with 5% detergent (Triton X-100), and spontaneous chromium release was determined by incubation of targets with medium alone. Typically, spontaneous release was <15% of the available total. It is important to note that spleen cells exposed to the nucleoprotein peptide SDYEGRLI in vitro without prior viral infection in vivo make negligible CTL responses demonstrating that the assay system used in these experiments provides a true reflection of the initial priming event in vivo.
Analysis of lung viral load
Relative viral load was determined by a modified ELISA. Lungs of infected and control uninfected mice were harvested into 2 ml of RPMI 1640 and stored at –80°C. After thawing, the lungs were homogenized on ice and centrifuged. Doubling dilutions of lung homogenate were made in duplicate in RPMI 1640/10% FCS in 24-well plates. Each well then received 1.4 x 105 Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection) in a total volume of 400 μl. After 48 h of incubation at 37°C, supernatants were removed, wells were washed with 400 μl of PBS, and the adherent MDCK cells were fixed with 400 μl of 10% formaldehyde for 30 min at 20°C. The wells were washed with PBS, and the cells were permeabilized with 400 μl of 0.5% Triton X-100 for 5 min. After two PBS washes, nonspecific binding sites were blocked for 90 min at 20°C with PBS/10% FCS. After washing with PBS, 150 μl of anti-PR8 HA primary Ab (a kind gift of Dr. K. Gould, Imperial College, London, U.K.) was added for 90 min. After five washes with PBS, 150 μl of HRP-conjugated rabbit anti-mouse secondary Ab (A-4416; Sigma-Aldrich) was added and incubated for 90 min at 20°C. The wells were washed five times with PBS then incubated at 20°C with 300 μl of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (A-1888; Sigma-Aldrich) substrate in citric acid buffer containing 0.03% H2O2. Aliquots from each well were taken after 10 min and read at 450 nm on an ELISA plate reader. A positive control calibration curve was constructed using 10-fold dilutions of stock PR8 virus starting at 50 HA U/ml.
Results
Experimental models
Pretreatment of CBA mice with a single DST plus the anti-CD4 Ab YTS 177.9 28 days before transplant leads to the indefinite survival of primary cardiac allografts (14), which is dependent on a population of CD25+CD4+ T cells that can also prevent skin allograft rejection in a sensitive adoptive transfer model (12). To test the impact of the presence of these alloreactive regulatory cells on responses against a model pathogen influenza virus, two alternative transplant models were employed. In the primary heart model, CBA mice pretreated with the tolerogenic 177/DST protocol were given a second DST 5 days before transplantation with the specific aim of reactivating the regulatory population to increase the likelihood of detecting any effect on antiviral responses (see Fig. 2a). Preliminary results in an analogous anti-CD4/DST model showed that Ag rechallenge between 3 and 5 days before transplant improves the quality of graft function significantly compared with the anti-CD4/single DST protocol consistent with regulatory cell reactivation (data not shown). In the second model, CBA mice pretreated and rechallenged with a second DST as above were used as adoptive transfer cell donors (see Fig. 5a). Adoptive transfer of spleen cells from tolerized mice to naive syngeneic secondary recipients allows the indefinite survival of cardiac allografts without immunosuppression. Since these secondary recipients have a completely intact immune system modified neither by Ab therapy nor alloantigen challenge, we believe that this model represents an extremely stringent test of tolerance. Our hypothesis was that if regulation of allograft responses also resulted in attenuated antiviral responses, this second model might provide the most sensitive detection system. In both models, transplant recipients were infected with influenza 7 days posttransplant then assayed for CTL activity and viral load 11 days later at the peak of antiviral responses.
FIGURE 2. CBA mice (H-2k) were pretreated and transplanted with B.10 cardiac allografts as in Fig. 1. The mice were infected intranasally 7 days posttransplant with 5 HA units of influenza A/PR/8/34. Naive untransplanted mice were infected in the same way. Spleens and lungs were harvested for flu-specific CTL and viral load assays 11 days later, respectively (a). Lysis of flu-peptide-sensitized target cells is shown for each animal in the four treatment groups (b) and for each group ± the SEM (c). Horizontal lines within each bar indicate the mean level of nonspecific killing ± SEM as determined by lysis of target cells in the absence of peptide.
FIGURE 5. Adoptive transfer recipients (177/DST + reboost) as in Fig. 4 were infected intranasally 7 days posttransplant with 5 HA units of influenza A/PR/8/34. Untreated CBA mice transplanted with B.10 hearts and naive untransplanted CBA mice were infected in the same way and housed under identical conditions. All mice were harvested for CTL and viral load assays 11 days after infection (a). Lysis of flu-peptide-sensitized target cells (mean ± SEM) is shown for each group (b). Horizontal dotted lines in each bar indicate the mean level of nonspecific killing as determined by lysis of target cells in the absence of peptide. Relative viral load in the lungs of the adoptive transfer mice was little different from that of the control animals reflecting efficient clearance of the virus despite the regulation of allograft responses (c).
The anti-CD4/DST plus reboost protocol in primary allograft recipients leads to indefinite graft survival with only modest levels of vasculopathy
To examine the effectiveness of the anti-CD4/DST plus DST reboost protocol, pretreated CBA mice were transplanted with B.10 hearts. Control mice pretreated with anti-CD4 Ab (YTS 177) only plus a DST challenge on day 0 or with DST only plus a DST challenge on day 0 rejected their grafts with median survival times (MST) of 48 and 14 days, respectively (Fig. 1a). In contrast, mice given the combined anti-CD4/DST pretreatment plus a DST reboost on day 0 all accepted their B.10 cardiac allografts beyond 100 days with little decline in cardiac function as assessed by palpation. The integrity of these hearts was confirmed by histological evaluation 120 days posttransplant which revealed little myocardial damage and low levels of vasculopathy (mean vascular occlusion of 28%, Fig. 1b). This compares with occlusion levels of up to 75% in transplanted mice tolerized by the anti-CD4/DST protocol then given anti-GITR or anti-CD25 Abs to perturb the function of putative regulatory cells (data not shown).
FIGURE 1. CBA mice (H-2k) were given 200 μg of anti-CD4 Ab (YTS 177.9; days –28 and –27, i.v. + a C57BL/10 (B.10, H-2b) DST, day –27; DST alone (day –27) or YTS 177.9 alone (days –28 and –27). On day 0, all mice received a B.10 DST "reboost" and were transplanted with fully vascularized B.10 cardiac allografts 5 days later. No further treatment was given. Graft survival was assessed by palpation (a). Cardiac vasculopathy in the 177/DST + reboost group was determined histologically 100 days posttransplant and compared with that in syngeneic heart allografts 100 days posttransplant (b).
Anti-influenza CTL responses and viral clearance are unaffected by allograft tolerance in primary heart recipients
CBA mice were pretreated with the anti-CD4/DST plus DST reboost protocol, transplanted with B.10 cardiac allografts then infected with influenza virus 7 days posttransplant (Fig. 2a). This time point was chosen for influenza infection because day 7 represents a balance between recipient sensitization and possible graft adaptation after transplantation. Previous work in the mouse cardiac allograft model has shown that heart-derived donor dendritic leukocytes can be detected in the recipient spleen 1 day posttransplant and that this migration is maintained for at least 4 days (22). In untreated mice, this results in graft rejection by day 8–10. The anti-CD4/DST protocol, known to generate CD25+CD4+ regulatory cells (12), converts this into indefinite graft survival (Fig. 1a) indicating that at least by 7 days posttransplant, dominant regulation in the presence of ongoing sensitization must be occurring. Indeed, in an analogous model, regulatory T cells can be generated in vivo by exposure to nominal Ag plus anti-CD4 Ab and are also capable of preventing skin allograft rejection by bystander regulation (46). These CD25+CD4+ regulatory T cells are present in the spleen as early as 1 day after Ag rechallenge and persist for at least a further 14 days (data not shown). Infection at later time points posttransplant was rejected as a strategy for looking at the effect of regulation on virus responses because of the possibility that graft adaptation rather than regulation might be responsible for continued graft survival.
Naive CBA infected with influenza made good CTL responses with 35% specific lysis at the highest effector to target cell ratio (Fig. 2b). Importantly, CTL responses in tolerized transplanted mice (177/DST + heart) were essentially identical to those from naive mice indicating that the presence of dominant regulation has no detrimental effect on protective CTL immunity (Fig. 2, b and c). Interestingly, CTL responses in mice given either of the control pretreatment regimens (177 only or DST only) were considerably augmented compared with those from naive mice possibly due to the provision of nonspecific cytokine help driven by the early stages of graft rejection (Fig. 1a).
To examine the effects of regulation on pathogen clearance from the lung, a modified ELISA assay was used to detect influenza HA expression by cell monolayers exposed to lung homogenates. This approach was validated by exposure of MDCK cells to serial dilutions of stock virus (Fig. 3a). As a positive control for clearance of the virus in vivo, naive untransplanted mice were infected with 5 HA units of influenza PR8 then lungs were harvested 1 h and 2, 4, and 7 days later for determination of relative viral load (n = 2 mice per time point). Viral titer reached a peak at day 4 and returned almost to background levels 7 days postinfection demonstrating that there was effective clearance of the virus (Fig. 3b). Fig. 3c shows viral load data from the tolerized transplanted and control mice shown in Fig. 2. When assayed 11 days after infection, the lungs of naive mice contained significant levels of virus but more importantly, mice tolererized by the anti-CD4/DST protocol cleared the virus as shown by the low HA signals, which were practically identical to those from uninfected mice representing essentially the background of the assay. Taken together these data demonstrate that despite the presence of CD25+CD4+ cells capable of regulating rejection responses (12) protective anti-flu responses appear to be unaffected.
FIGURE 3. Viral load in the lungs of mice in Fig. 2 was determined by ELISA for viral HA produced by MDCK cell monolayers exposed to lung homogenate. All data represent the mean of triplicate wells. The assay was validated using 10-fold dilutions of stock virus starting from 50 HA units/ml (a), and its suitability for detection of viral clearance in vivo confirmed using naive mice infected with 5 HA units of virus, n = 2 per time point (b). Relative viral load in the lungs of mice shown in Fig. 2 is shown (c). Data shown are for individual naive infected mice; the remainder represents mean ± SEM of replicate animals. Viral clearance in transplanted tolerized primary heart recipients was unaffected by the presence of dominant T cell regulation.
Tolerance can be transferred from anti-CD4/DST-pretreated mice to naive recipients resulting in long-term allograft survival with little obstructive vasculopathy
In the primary heart model described above, long-term graft survival is dependent on the combined anti-CD4/DST pretreatment. However, pretreatment with either of the control regimens (177 only + reboost or DST only + reboost) leads to some prolongation of allograft survival (Fig. 1a) indicating that each of these components has an independent attenuating effect on rejection responses. Therefore, it could be argued that the additional effect of the regulatory cells generated by the anti-CD4/DST protocol in these primary recipients is only rather small and that, if this is the case, the model is not well suited for assessing the impact of dominant regulation on antiviral responses. To address this possibility an alternative model was used in which tolerance is transferred from pretreated mice to naive CBA secondary recipients. In this situation, regulatory cells in the transferred population are required to prevent rejection mediated by an otherwise completely intact immune repertoire. As shown in Fig. 4, adoptive transfer of spleen cells from mice pretreated with either of the control regimens resulted in acute cardiac allograft rejection, whereas transfer of cells from mice pretreated with the tolerogenic anti-CD4/DST + reboost protocol resulted in long-term graft survival (Fig. 4a). The effectiveness of regulation in this system is emphasized not only by the survival of these hearts but also by the paucity of vascular lesions such that the overall vascular occlusion was <15% (Fig. 4b). One potential explanation for the reduced level of vasculopathy in these hearts compared with that seen in primary recipients (Fig. 1b) is that in the secondary recipients, adoptive transfer of regulatory cells resulted in the recruitment of naive cells into the regulatory pool by "infectious tolerance" (23).
FIGURE 4. CBA cell donors (H-2k) were pretreated as in Fig. 1. On day 0, all mice received a B.10 DST reboost. Five days later, spleen cells from these donors (5 x 107) were adoptively transferred to naive unmodified CBA recipients. These cell recipients were transplanted with fully vascularized B.10 cardiac allografts 24 h later. No further treatment was given. Graft survival was assessed by palpation (a). The level of vasculopathy in the adoptive transfer recipients was determined histologically 100 days posttransplant, and vascular occlusion compared with that in syngeneic heart allografts 100 days posttransplant (b).
Protective immunity to influenza virus is unaffected by the presence of dominant regulation
CBA cell donors were pretreated with the anti-CD4/DST induction protocol then rechallenged with a second DST at day 0. Spleen cells were adoptively transferred to naive CBA recipients 5 days later, which were transplanted with B.10 cardiac allografts then infected with influenza PR8 (Fig. 5a). The mice were harvested 11 days later for CTL and viral load assays. Influenza-specific CTL activity in these adoptive transfer recipients was comparable with that from naive untransplanted mice or from naive recipients rejecting B.10 cardiac allografts (Fig. 5b). This equivalence in antiviral responses was also reflected in the viral load assay where the adoptive transfer recipients had levels of virus that were almost identical to those in control mice (Fig. 5c). Taken together these data indicate that the presence of regulation efficient enough to give the graft almost complete protection from damage (Fig. 4) does not influence immunity to influenza virus.
Recent Ag rechallenge sufficient to drive bystander regulation does not impair antiviral responses
In addition to the anti-CD4/DST protocol described above, we have also shown that CD25+CD4+ regulatory cells can be generated by pretreatment with unrelated "random" blood transfusion given either with (single transfusion) or without (five transfusions) anti-CD4 Ab (13). Our working hypothesis is that random transfusion generates regulatory cells that can protect skin allografts either via a mechanism of cross-reactivity or via bystander suppression. We currently favor the latter explanation because we have recently shown that pretreatment of cell donors with an unrelated non-cross-reactive Ag combined with anti-CD4 Ab generates CD25+CD4+ cells that can prevent skin allograft rejection in an adoptive transfer model.6 Significantly, these cells only regulate in vivo if they are restimulated with Ag before transfer, a result that is in complete accordance with in vitro observations using peptide-specific T cells (15). If bystander suppression is a feature of recently activated regulatory cells only, it could be argued that we were unable to detect an effect on viral immunity because by day 7 posttransplant (the time of infection, Figs. 2 and 5) regulation had become focused due to continual reactivation of the regulatory cells by the graft itself such that regulation was relatively specific either in terms of regulatory cell function or recruitment. To examine the impact of recent regulatory T cell activation on antiviral responses, CBA mice were pretreated with the tolerogenic anti-CD4/DST protocol then rechallenged with a second DST at day 0. These mice were then infected with influenza 1 day after rechallenge and harvested 11 days later (Fig. 6a). Day +1 was chosen for the infection interval because in an analogous model we have clear evidence that Ag rechallenge 1 day before adoptive transfer activates bystander regulation powerful enough to prevent skin graft rejection, which is Ag nonspecific.6 However, despite this reactivation, there was no evidence of attenuated anti-flu responses either in terms of CTL activity (Fig. 6b) or viral clearance from the lung (Fig. 6c).
FIGURE 6. CBA mice (H-2k) were pretreated with the tolerizing 177/DST (B.10) protocol, reboosted with an additional B.10 blood transfusion on day 0, infected with 5 HA units of influenza PR8 1 day later, and 11 days after infection, spleens and lungs were harvested for flu-specific CTL and viral load assays, respectively (a). Spleen cells were restimulated in vitro with syngeneic splenocytes pulsed with the immunodominant H-2Kk-restricted peptide of PR8 nucleoprotein, SDYEGRLI, expanded with IL-2 and incubated with 51Cr-labeled peptide-pulsed H-2k-positive L cell targets. Naive CBA mice infected and harvested in exactly the same way served as controls (b). The lungs from these mice were assessed for relative viral load using a modified ELISA for viral HA (c).
Discussion
Despite enormous improvements during the past three decades in the control of acute allograft rejection, it is becoming clear that current immunosuppression regimens are much less successful in preventing delayed graft loss or dysfunction (24). Although immunosuppression protocols are being developed and refined continually, it is likely that long-term graft survival independent of chronic immunosuppression will depend ultimately on the generation of some form of dominant regulation. Proof of principle support has come from a large number of diverse protocols in rodent transplant models, and although it may prove to be more difficult to induce regulation in primates and by implication in humans than in rodents (25, 26), there are good grounds for optimism that it should be possible to engage natural mechanisms of regulation in the clinical situation. Indeed, the blood transfusion effect, which was so influential on the outcome of cadaveric kidney transplantation before the introduction of cyclosporine (27), might be explained by the generation of CD25+CD4+ regulatory T cells (13). Although elective blood transfusion as a pretransplant strategy is now used in very few centers, the administration of donor bone marrow as a route to mixed chimerism is currently attracting a great deal of attention (28) and has proven effective in a small number of patients (29). By far the greatest experience with bone marrow protocols has been obtained in the mouse where bone marrow infusion is usually combined with cytoreductive conditioning and inhibition of peripheral T cell function (28, 30, 31, 32, 33). This approach leads to mixed chimerism and in many cases central deletion of donor-reactive T cells. However, there is evidence that even in these deletional models, regulatory T cells make an essential contribution to continued allograft survival (A. B. Adams and C. P. Larsen, unpublished observations). 6 Thus, in models that are considered prototypic for regimens that may be introduced more widely in clinical transplantation, regulation appears to make at least a contribution to the maintenance of tolerance. Therefore, an understanding of the impact of T cell-mediated regulation on pathogen clearance is highly relevant.
The clearance of influenza virus from infected mice involves both CD4+ and CD8+ T cell responses (34), and T cells of both subsets are susceptible to regulation by CD25+CD4+ regulatory T cells (12, 35) making influenza an appropriate pathogen for the study of the effect of alloantigen-driven regulatory T cells on viral immunity. Our experiments conducted in models in which there is a demonstrable generation of CD25+CD4+ regulatory T cells (11, 12) revealed no adverse effect of transplantation tolerance on influenza-specific CTL or on clearance of the virus in vivo. This was something of a surprise given that regulation of rejection in these models appears to be dependent on IL-10 (12), a cytokine first identified because of its immunosuppressive properties on T cell activation and synthesis of Th1 cytokines (36). The most simple explanation for this lack of effect is that following tolerance induction and transplantation, the majority of regulatory cells migrate to and are sequestered within the graft (37) thus limiting any systemic effect. Thus, even though influenza-specific CTL were obtained from the spleens of transplanted animals (Figs. 2 and 5), a compartment we have shown to contain regulatory cells after anti-CD4/DST pretreatment (11, 12), it could be argued that the presence of the graft may have selectively depleted the spleen of regulatory cells thereby allowing antiviral responses to develop relatively normally. However, this explanation cannot apply to the experiments shown in Fig. 6 where tolerized mice remained untransplanted and were infected 1 day after a second alloantigen challenge. Influenza-specific CTL with apparently normal activity were obtained from the spleens of these mice (Fig. 6b) despite the fact that alloantigen-driven regulatory cells remain in the spleen for at least 5 days after rechallenge (Fig. 4). It could also be argued that any perturbation of CTL responses caused by ongoing regulation was masked in our experiments because exogenous IL-2 was added during the in vitro expansion of CTL precursors. This is a potentially valid criticism because regulation in vitro can often be overcome by the addition of exogenous IL-2 (38, 39, 40, 41). However, viral clearance from the lungs of infected mice, which is an entirely in vivo phenomenon involving no experimental manipulation, was also unaffected by the presence of regulatory cells (Figs. 3, 5, and 6), supporting the CTL data.
Several studies have addressed the effect that viral infection has on transplantation tolerance, and the overall view is that both prior and concurrent exposure to viral pathogens can prevent tolerance induction. Although previous viral exposure leads to memory T cells (predominantly CD8+) that appear to be resistant to tolerance induction (25, 26), CD8+ T cells driven by peri-transplant viral exposure have also been implicated in rejection of the graft itself (42, 43). As far as the converse situation is concerned, previous studies have examined the clearance of viral pathogens in mixed allogeneic chimeras (44), and although mice containing both host and donor restricted T cells may have more difficulty clearing chronic infections (45), mixed chimeras seem to clear acute viral infections at similar rates to their normal counterparts. However, much less attention has been paid to the effect that transplantation tolerance based on active T cell regulation might have on protective immunity. This is an important area because it is hoped that tolerance induction will circumvent many of the problems associated with current nonspecific immunosuppression. To our knowledge, this is the first study to examine the effects of alloantigen-driven CD25+CD4+ regulatory cells on responses to a viral pathogen. We have found no diminution of antiviral responses in three different situations, and although the data reflect responses to a single pathogen in one particular transplant setting, we believe our data give grounds for cautious optimism that bystander suppression may be less of a problem in transplantation than might be imagined. We are currently extending our studies to other model pathogens and transplant models to determine whether this optimism is well placed.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We are grateful to Prof. Herman Waldman (Sir William Dunn School of Pathology, University of Oxford) for providing the hybridoma YTS 177.9 and to Dr. Paul Klenerman (Nuffield Department of Medicine, University of Oxford) for comments on the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by The Wellcome Trust. A.G. is a Medical Research Council Senior Research Fellow. K.W. holds a Royal Society Wolfson Research Merit Award.
2 Address correspondence and reprint requests to Dr. Andrew R. Bushell, Nuffield Department of Surgery, John Radcliffe Hospital, Oxford OX3 9DU, U.K. E-mail address: andrew.bushell{at}nds.ox.ac.uk
3 Current address: The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K.
4 Current address: Department of Medical Biochemistry and Immunology, Cardiff University, Cardiff CF14 4XN, U.K.
5 Abbreviations used in this paper: DST, donor-specific transfusion; MDCK, Madin-Darby canine kidney; HA, hemagglutinin.
6 S. Bigenzahn, P. Blaha, Z. Koporc, E. Selzer, C. Heusser, K. Wagner, F. Muehlbacher, and T. Wekerle. 2003. The role of regulation for induction and maintenance of tolerance through BMT plus costimulation blockade. Presented at Basic Sciences Symposium of the Transplantation Society, September 14th-17th, 2003, Keystone, Colorado.
Received for publication August 26, 2004. Accepted for publication November 18, 2004.
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In all but a small minority of cases, continued survival of solid organ grafts after transplantation depends on lifelong, nonselective immunosuppression that, although effective, results in increased rates of infection, cancer, and vascular disease. Therapeutic strategies that engage or mimic self-tolerance may allow prolonged allograft survival without the disadvantages of nonspecific immunotherapy. Pretreatment of recipient mice with donor alloantigen combined with transient modulation of the peripheral T cell pool with anti-CD4 Ab leads to the indefinite survival of MHC-incompatible cardiac allografts without further therapy. Tolerance is dependent on CD25+CD4+ regulatory T cells that arise from naive CD25– precursors and regulate rejection via both IL-10 and CTLA-4. Although these cells are clearly effective at controlling rejection, the proven ability of recently activated CD25+ cells to mediate bystander regulation raises the possibility that tolerized individuals might also have a reduced capacity to respond to environmental pathogens. We have examined anti-influenza responses in tolerized primary heart recipients, secondary recipients following adoptive transfer of regulatory populations, and tolerized mice in which bystander regulation has been deliberately induced. Neither virus-specific CTL activity in vitro nor the clearance of virus in vivo was significantly diminished in any of these treatment groups compared with infected unmanipulated controls. The data suggest that the induction of dominant allograft tolerance dependent on regulatory T cells does not necessarily result in attenuated responses to pathogens providing further support for the development of tolerance induction protocols in clinical transplantation.
Introduction
The immune system contains peripheral regulatory mechanisms that play an essential role in the prevention of autoimmunity. Although as yet it has not been possible to define a single cell surface marker that is unique to regulatory cells, particular attention has been focused on CD4 cells expressing high levels of CD25, the -chain of the IL-2 receptor. CD25+CD4+ T cells have been shown to play an important regulatory role in several animal models of autoimmune disease including colitis (1), diabetes (2, 3), gastritis (4, 5), and thyroiditis (4). The fact that CD25+CD4+ T cells with regulatory capacity, at least in vitro, can also be found in human peripheral blood (6, 7) suggests that a breakdown of regulation could be an important factor in human autoimmune disease.
Such studies in autoimmune models have prompted a re-evaluation of the role of regulatory/suppressor cells in transplantation tolerance and accumulating evidence indicates that significant parallels exist between naturally occurring peripheral T cell regulation and the regulation of T cell responses toward a transplanted allograft following tolerance induction. For example, we and others (8, 9, 10) have demonstrated that tolerant animals with long-surviving allografts contain populations of CD25+CD4+ T cells that prevent allograft rejection in adoptive transfer transplant models. More recently, we have shown that these cells can be generated in vivo from naive precursors in a thymus-independent process (11) by exposure to alloantigen either with (12) or without (13) concomitant anti-CD4 Ab therapy and that these cells regulate anti-graft responses in vivo using mechanisms shared with naturally occurring regulatory cells (12).
The specific regulation of destructive T cell responses remains an attractive potential solution to the problem of rejection in clinical transplantation. Current clinical regimens rely on lifelong, nonspecific immunosuppression, and although these are effective in the short term, the dual problems of undesirable side effects and delayed graft loss due to chronic rejection continue to stimulate a search for alternative therapies. The generation of regulatory cells before transplant in the clinical setting could give the graft protection from the outset, provide a route to operational tolerance and allow long-term graft survival without chronic immunosuppression. However, little is known of the impact that these generated regulatory cells might have on protective T cell responses.
In the alloantigen/anti-CD4 Ab tolerance induction protocol, we have described (14) the CD25+CD4+ regulatory T cells generated by the pretreatment protocol regulate in a donor alloantigen-specific manner (12), but an important in vitro study has shown that once activated through their TCR, CD25+CD4+ T cells can also regulate without Ag specificity. Using T cells isolated from two distinct TCR transgenic mice specific for different peptides and restricted by either I-Ed or I-Ek, Thornton and Shevach (15) demonstrated that CD25+CD4+ T cells isolated from either cell donor could inhibit the proliferation of CD25– responders isolated from the corresponding TCR transgenic mouse. However, more importantly, once activated, the same CD25+CD4+ population could also inhibit the proliferation of CD25– responder cells isolated from the second TCR transgenic mouse in a manner that was both Ag and MHC independent, a phenomenon referred to as "bystander suppression". In the transplant setting where tolerance is dependent on regulatory T cells, the presence of the graft has been shown to be essential for maintenance of the tolerant state (16) probably by promoting a continual reactivation of the regulatory T cell population. We considered it possible that reactivation of regulatory cells by the graft could lead to nonspecific bystander suppression in vivo and that although it may be possible to generate donor-reactive CD25+CD4+ T cells that regulate responses toward the allograft, an effect on responses to environmental pathogens cannot be ruled out. Therefore, the aim of this study was to use a well-defined model of transplantation tolerance where continued graft survival is known to depend on CD25+CD4+ T cells and ask whether the presence of these cells has any impact on immunity against a well-characterized pathogen, influenza virus. The hypothesis tested was that tolerant mice might show attenuated antiviral responses.
Materials and Methods
Induction of tolerance and heart transplantation
CBA (H-2k) mice were pretreated with 200 μg of the IgG2a anti-CD4 Ab YTS 177.9 (17) on days –28 and –27 and with a donor-specific transfusion (DST) 5 of C57BL/10 (H-2b) blood on day –27 and day 0 (anti-CD4/DST + reboost protocol). Five days after the second DST (day +5), the tolerized mice were either transplanted with donor-specific hearts or used as cell donors for adoptive transfer experiments. Specific details are given in the relevant figure legends. Cardiac transplantation was conducted under general anesthesia according to the method outlined by Corry et al. (18). Briefly, hearts were isolated from exsanguinated heparinized donors, and the ascending aorta and pulmonary artery were anastomosed end-to-side to the recipient abdominal aorta and inferior vena cava, respectively. Recipient animals were maintained in specific pathogen-free conditions to minimize exposure to environmental pathogens. Heart function was followed by daily palpation and the quality of each graft was determined at 100 days posttransplant as described below.
Enumeration of arterial intimal proliferation as a measure of graft outcome
At 100 days posttransplant, hearts were removed from recipients under terminal anesthesia and snap-frozen in OCT embedding compound (Bayer) in liquid nitrogen. Cryostat sections (8 μm) were taken from six planes each 200 μm apart in a method based on that of Armstrong et al. (19). Sections were then fixed in PBS/10% formaldehyde and stained using Miller’s elastin stain, which identifies elastic fibers combined with Van Gieson counter staining. The degree of intimal proliferation in arteries >80 μm in diameter was then determined as described (19) using pixel-counting software (Zeiss) to measure the area of the vascular lumen relative to the area circumscribed by the internal elastic lamina. Data are presented as percentage of vascular occlusion.
Infection with influenza virus and determination of antiviral responses
Mice were infected intranasally with five hemagglutinin (HA) units of influenza virus A/PR/8/34 (PR8) as shown in the figures. Spleens were harvested for flu-specific CTL assays 11 days after infection, and lungs were harvested for the determination of viral load.
CTL assays
Single spleen cell suspensions from infected mice were plated at 4 x 106 per well (24-well plate) in T cell medium (RPMI 1640 supplemented with 10–5 M 2-ME, 20 mM sodium pyruvate, 2 mM glutamine, 45 μg/ml penicillin, 45 μg/ml streptomycin, 90 μg/ml kanamycin, and 10% FCS). The cells were restimulated in vitro by the addition of irradiated peptide-pulsed stimulators (1 x 106 per well) to the culture wells. Stimulators were prepared by incubating naive CBA spleen cells for 1 h at 37°C with the immunodominant H-2Kk-restricted peptide of PR8 nucleoprotein SDYEGRLI (20) at a concentration of 10–4 M. The cells were then irradiated, washed twice, and resuspended in T cell medium. After 2 days of incubation at 37°C, rIL-2 was added to the cultures at a final concentration of 10 U/ml. After a further 3 days, equal numbers of wells from individual mice were resuspended in 700 μl, and 3-fold dilutions of the effector cells were performed. Chromium release assays were performed as described previously (21) using 51Cr-labeled H-2k-positive L cell targets (L929) either unpulsed or pulsed with the SDYEGRLI peptide at a final concentration of 10–5 M. Specific lysis was determined as described previously (21). Total available 51Cr was defined by incubation of targets with 5% detergent (Triton X-100), and spontaneous chromium release was determined by incubation of targets with medium alone. Typically, spontaneous release was <15% of the available total. It is important to note that spleen cells exposed to the nucleoprotein peptide SDYEGRLI in vitro without prior viral infection in vivo make negligible CTL responses demonstrating that the assay system used in these experiments provides a true reflection of the initial priming event in vivo.
Analysis of lung viral load
Relative viral load was determined by a modified ELISA. Lungs of infected and control uninfected mice were harvested into 2 ml of RPMI 1640 and stored at –80°C. After thawing, the lungs were homogenized on ice and centrifuged. Doubling dilutions of lung homogenate were made in duplicate in RPMI 1640/10% FCS in 24-well plates. Each well then received 1.4 x 105 Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection) in a total volume of 400 μl. After 48 h of incubation at 37°C, supernatants were removed, wells were washed with 400 μl of PBS, and the adherent MDCK cells were fixed with 400 μl of 10% formaldehyde for 30 min at 20°C. The wells were washed with PBS, and the cells were permeabilized with 400 μl of 0.5% Triton X-100 for 5 min. After two PBS washes, nonspecific binding sites were blocked for 90 min at 20°C with PBS/10% FCS. After washing with PBS, 150 μl of anti-PR8 HA primary Ab (a kind gift of Dr. K. Gould, Imperial College, London, U.K.) was added for 90 min. After five washes with PBS, 150 μl of HRP-conjugated rabbit anti-mouse secondary Ab (A-4416; Sigma-Aldrich) was added and incubated for 90 min at 20°C. The wells were washed five times with PBS then incubated at 20°C with 300 μl of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (A-1888; Sigma-Aldrich) substrate in citric acid buffer containing 0.03% H2O2. Aliquots from each well were taken after 10 min and read at 450 nm on an ELISA plate reader. A positive control calibration curve was constructed using 10-fold dilutions of stock PR8 virus starting at 50 HA U/ml.
Results
Experimental models
Pretreatment of CBA mice with a single DST plus the anti-CD4 Ab YTS 177.9 28 days before transplant leads to the indefinite survival of primary cardiac allografts (14), which is dependent on a population of CD25+CD4+ T cells that can also prevent skin allograft rejection in a sensitive adoptive transfer model (12). To test the impact of the presence of these alloreactive regulatory cells on responses against a model pathogen influenza virus, two alternative transplant models were employed. In the primary heart model, CBA mice pretreated with the tolerogenic 177/DST protocol were given a second DST 5 days before transplantation with the specific aim of reactivating the regulatory population to increase the likelihood of detecting any effect on antiviral responses (see Fig. 2a). Preliminary results in an analogous anti-CD4/DST model showed that Ag rechallenge between 3 and 5 days before transplant improves the quality of graft function significantly compared with the anti-CD4/single DST protocol consistent with regulatory cell reactivation (data not shown). In the second model, CBA mice pretreated and rechallenged with a second DST as above were used as adoptive transfer cell donors (see Fig. 5a). Adoptive transfer of spleen cells from tolerized mice to naive syngeneic secondary recipients allows the indefinite survival of cardiac allografts without immunosuppression. Since these secondary recipients have a completely intact immune system modified neither by Ab therapy nor alloantigen challenge, we believe that this model represents an extremely stringent test of tolerance. Our hypothesis was that if regulation of allograft responses also resulted in attenuated antiviral responses, this second model might provide the most sensitive detection system. In both models, transplant recipients were infected with influenza 7 days posttransplant then assayed for CTL activity and viral load 11 days later at the peak of antiviral responses.
FIGURE 2. CBA mice (H-2k) were pretreated and transplanted with B.10 cardiac allografts as in Fig. 1. The mice were infected intranasally 7 days posttransplant with 5 HA units of influenza A/PR/8/34. Naive untransplanted mice were infected in the same way. Spleens and lungs were harvested for flu-specific CTL and viral load assays 11 days later, respectively (a). Lysis of flu-peptide-sensitized target cells is shown for each animal in the four treatment groups (b) and for each group ± the SEM (c). Horizontal lines within each bar indicate the mean level of nonspecific killing ± SEM as determined by lysis of target cells in the absence of peptide.
FIGURE 5. Adoptive transfer recipients (177/DST + reboost) as in Fig. 4 were infected intranasally 7 days posttransplant with 5 HA units of influenza A/PR/8/34. Untreated CBA mice transplanted with B.10 hearts and naive untransplanted CBA mice were infected in the same way and housed under identical conditions. All mice were harvested for CTL and viral load assays 11 days after infection (a). Lysis of flu-peptide-sensitized target cells (mean ± SEM) is shown for each group (b). Horizontal dotted lines in each bar indicate the mean level of nonspecific killing as determined by lysis of target cells in the absence of peptide. Relative viral load in the lungs of the adoptive transfer mice was little different from that of the control animals reflecting efficient clearance of the virus despite the regulation of allograft responses (c).
The anti-CD4/DST plus reboost protocol in primary allograft recipients leads to indefinite graft survival with only modest levels of vasculopathy
To examine the effectiveness of the anti-CD4/DST plus DST reboost protocol, pretreated CBA mice were transplanted with B.10 hearts. Control mice pretreated with anti-CD4 Ab (YTS 177) only plus a DST challenge on day 0 or with DST only plus a DST challenge on day 0 rejected their grafts with median survival times (MST) of 48 and 14 days, respectively (Fig. 1a). In contrast, mice given the combined anti-CD4/DST pretreatment plus a DST reboost on day 0 all accepted their B.10 cardiac allografts beyond 100 days with little decline in cardiac function as assessed by palpation. The integrity of these hearts was confirmed by histological evaluation 120 days posttransplant which revealed little myocardial damage and low levels of vasculopathy (mean vascular occlusion of 28%, Fig. 1b). This compares with occlusion levels of up to 75% in transplanted mice tolerized by the anti-CD4/DST protocol then given anti-GITR or anti-CD25 Abs to perturb the function of putative regulatory cells (data not shown).
FIGURE 1. CBA mice (H-2k) were given 200 μg of anti-CD4 Ab (YTS 177.9; days –28 and –27, i.v. + a C57BL/10 (B.10, H-2b) DST, day –27; DST alone (day –27) or YTS 177.9 alone (days –28 and –27). On day 0, all mice received a B.10 DST "reboost" and were transplanted with fully vascularized B.10 cardiac allografts 5 days later. No further treatment was given. Graft survival was assessed by palpation (a). Cardiac vasculopathy in the 177/DST + reboost group was determined histologically 100 days posttransplant and compared with that in syngeneic heart allografts 100 days posttransplant (b).
Anti-influenza CTL responses and viral clearance are unaffected by allograft tolerance in primary heart recipients
CBA mice were pretreated with the anti-CD4/DST plus DST reboost protocol, transplanted with B.10 cardiac allografts then infected with influenza virus 7 days posttransplant (Fig. 2a). This time point was chosen for influenza infection because day 7 represents a balance between recipient sensitization and possible graft adaptation after transplantation. Previous work in the mouse cardiac allograft model has shown that heart-derived donor dendritic leukocytes can be detected in the recipient spleen 1 day posttransplant and that this migration is maintained for at least 4 days (22). In untreated mice, this results in graft rejection by day 8–10. The anti-CD4/DST protocol, known to generate CD25+CD4+ regulatory cells (12), converts this into indefinite graft survival (Fig. 1a) indicating that at least by 7 days posttransplant, dominant regulation in the presence of ongoing sensitization must be occurring. Indeed, in an analogous model, regulatory T cells can be generated in vivo by exposure to nominal Ag plus anti-CD4 Ab and are also capable of preventing skin allograft rejection by bystander regulation (46). These CD25+CD4+ regulatory T cells are present in the spleen as early as 1 day after Ag rechallenge and persist for at least a further 14 days (data not shown). Infection at later time points posttransplant was rejected as a strategy for looking at the effect of regulation on virus responses because of the possibility that graft adaptation rather than regulation might be responsible for continued graft survival.
Naive CBA infected with influenza made good CTL responses with 35% specific lysis at the highest effector to target cell ratio (Fig. 2b). Importantly, CTL responses in tolerized transplanted mice (177/DST + heart) were essentially identical to those from naive mice indicating that the presence of dominant regulation has no detrimental effect on protective CTL immunity (Fig. 2, b and c). Interestingly, CTL responses in mice given either of the control pretreatment regimens (177 only or DST only) were considerably augmented compared with those from naive mice possibly due to the provision of nonspecific cytokine help driven by the early stages of graft rejection (Fig. 1a).
To examine the effects of regulation on pathogen clearance from the lung, a modified ELISA assay was used to detect influenza HA expression by cell monolayers exposed to lung homogenates. This approach was validated by exposure of MDCK cells to serial dilutions of stock virus (Fig. 3a). As a positive control for clearance of the virus in vivo, naive untransplanted mice were infected with 5 HA units of influenza PR8 then lungs were harvested 1 h and 2, 4, and 7 days later for determination of relative viral load (n = 2 mice per time point). Viral titer reached a peak at day 4 and returned almost to background levels 7 days postinfection demonstrating that there was effective clearance of the virus (Fig. 3b). Fig. 3c shows viral load data from the tolerized transplanted and control mice shown in Fig. 2. When assayed 11 days after infection, the lungs of naive mice contained significant levels of virus but more importantly, mice tolererized by the anti-CD4/DST protocol cleared the virus as shown by the low HA signals, which were practically identical to those from uninfected mice representing essentially the background of the assay. Taken together these data demonstrate that despite the presence of CD25+CD4+ cells capable of regulating rejection responses (12) protective anti-flu responses appear to be unaffected.
FIGURE 3. Viral load in the lungs of mice in Fig. 2 was determined by ELISA for viral HA produced by MDCK cell monolayers exposed to lung homogenate. All data represent the mean of triplicate wells. The assay was validated using 10-fold dilutions of stock virus starting from 50 HA units/ml (a), and its suitability for detection of viral clearance in vivo confirmed using naive mice infected with 5 HA units of virus, n = 2 per time point (b). Relative viral load in the lungs of mice shown in Fig. 2 is shown (c). Data shown are for individual naive infected mice; the remainder represents mean ± SEM of replicate animals. Viral clearance in transplanted tolerized primary heart recipients was unaffected by the presence of dominant T cell regulation.
Tolerance can be transferred from anti-CD4/DST-pretreated mice to naive recipients resulting in long-term allograft survival with little obstructive vasculopathy
In the primary heart model described above, long-term graft survival is dependent on the combined anti-CD4/DST pretreatment. However, pretreatment with either of the control regimens (177 only + reboost or DST only + reboost) leads to some prolongation of allograft survival (Fig. 1a) indicating that each of these components has an independent attenuating effect on rejection responses. Therefore, it could be argued that the additional effect of the regulatory cells generated by the anti-CD4/DST protocol in these primary recipients is only rather small and that, if this is the case, the model is not well suited for assessing the impact of dominant regulation on antiviral responses. To address this possibility an alternative model was used in which tolerance is transferred from pretreated mice to naive CBA secondary recipients. In this situation, regulatory cells in the transferred population are required to prevent rejection mediated by an otherwise completely intact immune repertoire. As shown in Fig. 4, adoptive transfer of spleen cells from mice pretreated with either of the control regimens resulted in acute cardiac allograft rejection, whereas transfer of cells from mice pretreated with the tolerogenic anti-CD4/DST + reboost protocol resulted in long-term graft survival (Fig. 4a). The effectiveness of regulation in this system is emphasized not only by the survival of these hearts but also by the paucity of vascular lesions such that the overall vascular occlusion was <15% (Fig. 4b). One potential explanation for the reduced level of vasculopathy in these hearts compared with that seen in primary recipients (Fig. 1b) is that in the secondary recipients, adoptive transfer of regulatory cells resulted in the recruitment of naive cells into the regulatory pool by "infectious tolerance" (23).
FIGURE 4. CBA cell donors (H-2k) were pretreated as in Fig. 1. On day 0, all mice received a B.10 DST reboost. Five days later, spleen cells from these donors (5 x 107) were adoptively transferred to naive unmodified CBA recipients. These cell recipients were transplanted with fully vascularized B.10 cardiac allografts 24 h later. No further treatment was given. Graft survival was assessed by palpation (a). The level of vasculopathy in the adoptive transfer recipients was determined histologically 100 days posttransplant, and vascular occlusion compared with that in syngeneic heart allografts 100 days posttransplant (b).
Protective immunity to influenza virus is unaffected by the presence of dominant regulation
CBA cell donors were pretreated with the anti-CD4/DST induction protocol then rechallenged with a second DST at day 0. Spleen cells were adoptively transferred to naive CBA recipients 5 days later, which were transplanted with B.10 cardiac allografts then infected with influenza PR8 (Fig. 5a). The mice were harvested 11 days later for CTL and viral load assays. Influenza-specific CTL activity in these adoptive transfer recipients was comparable with that from naive untransplanted mice or from naive recipients rejecting B.10 cardiac allografts (Fig. 5b). This equivalence in antiviral responses was also reflected in the viral load assay where the adoptive transfer recipients had levels of virus that were almost identical to those in control mice (Fig. 5c). Taken together these data indicate that the presence of regulation efficient enough to give the graft almost complete protection from damage (Fig. 4) does not influence immunity to influenza virus.
Recent Ag rechallenge sufficient to drive bystander regulation does not impair antiviral responses
In addition to the anti-CD4/DST protocol described above, we have also shown that CD25+CD4+ regulatory cells can be generated by pretreatment with unrelated "random" blood transfusion given either with (single transfusion) or without (five transfusions) anti-CD4 Ab (13). Our working hypothesis is that random transfusion generates regulatory cells that can protect skin allografts either via a mechanism of cross-reactivity or via bystander suppression. We currently favor the latter explanation because we have recently shown that pretreatment of cell donors with an unrelated non-cross-reactive Ag combined with anti-CD4 Ab generates CD25+CD4+ cells that can prevent skin allograft rejection in an adoptive transfer model.6 Significantly, these cells only regulate in vivo if they are restimulated with Ag before transfer, a result that is in complete accordance with in vitro observations using peptide-specific T cells (15). If bystander suppression is a feature of recently activated regulatory cells only, it could be argued that we were unable to detect an effect on viral immunity because by day 7 posttransplant (the time of infection, Figs. 2 and 5) regulation had become focused due to continual reactivation of the regulatory cells by the graft itself such that regulation was relatively specific either in terms of regulatory cell function or recruitment. To examine the impact of recent regulatory T cell activation on antiviral responses, CBA mice were pretreated with the tolerogenic anti-CD4/DST protocol then rechallenged with a second DST at day 0. These mice were then infected with influenza 1 day after rechallenge and harvested 11 days later (Fig. 6a). Day +1 was chosen for the infection interval because in an analogous model we have clear evidence that Ag rechallenge 1 day before adoptive transfer activates bystander regulation powerful enough to prevent skin graft rejection, which is Ag nonspecific.6 However, despite this reactivation, there was no evidence of attenuated anti-flu responses either in terms of CTL activity (Fig. 6b) or viral clearance from the lung (Fig. 6c).
FIGURE 6. CBA mice (H-2k) were pretreated with the tolerizing 177/DST (B.10) protocol, reboosted with an additional B.10 blood transfusion on day 0, infected with 5 HA units of influenza PR8 1 day later, and 11 days after infection, spleens and lungs were harvested for flu-specific CTL and viral load assays, respectively (a). Spleen cells were restimulated in vitro with syngeneic splenocytes pulsed with the immunodominant H-2Kk-restricted peptide of PR8 nucleoprotein, SDYEGRLI, expanded with IL-2 and incubated with 51Cr-labeled peptide-pulsed H-2k-positive L cell targets. Naive CBA mice infected and harvested in exactly the same way served as controls (b). The lungs from these mice were assessed for relative viral load using a modified ELISA for viral HA (c).
Discussion
Despite enormous improvements during the past three decades in the control of acute allograft rejection, it is becoming clear that current immunosuppression regimens are much less successful in preventing delayed graft loss or dysfunction (24). Although immunosuppression protocols are being developed and refined continually, it is likely that long-term graft survival independent of chronic immunosuppression will depend ultimately on the generation of some form of dominant regulation. Proof of principle support has come from a large number of diverse protocols in rodent transplant models, and although it may prove to be more difficult to induce regulation in primates and by implication in humans than in rodents (25, 26), there are good grounds for optimism that it should be possible to engage natural mechanisms of regulation in the clinical situation. Indeed, the blood transfusion effect, which was so influential on the outcome of cadaveric kidney transplantation before the introduction of cyclosporine (27), might be explained by the generation of CD25+CD4+ regulatory T cells (13). Although elective blood transfusion as a pretransplant strategy is now used in very few centers, the administration of donor bone marrow as a route to mixed chimerism is currently attracting a great deal of attention (28) and has proven effective in a small number of patients (29). By far the greatest experience with bone marrow protocols has been obtained in the mouse where bone marrow infusion is usually combined with cytoreductive conditioning and inhibition of peripheral T cell function (28, 30, 31, 32, 33). This approach leads to mixed chimerism and in many cases central deletion of donor-reactive T cells. However, there is evidence that even in these deletional models, regulatory T cells make an essential contribution to continued allograft survival (A. B. Adams and C. P. Larsen, unpublished observations). 6 Thus, in models that are considered prototypic for regimens that may be introduced more widely in clinical transplantation, regulation appears to make at least a contribution to the maintenance of tolerance. Therefore, an understanding of the impact of T cell-mediated regulation on pathogen clearance is highly relevant.
The clearance of influenza virus from infected mice involves both CD4+ and CD8+ T cell responses (34), and T cells of both subsets are susceptible to regulation by CD25+CD4+ regulatory T cells (12, 35) making influenza an appropriate pathogen for the study of the effect of alloantigen-driven regulatory T cells on viral immunity. Our experiments conducted in models in which there is a demonstrable generation of CD25+CD4+ regulatory T cells (11, 12) revealed no adverse effect of transplantation tolerance on influenza-specific CTL or on clearance of the virus in vivo. This was something of a surprise given that regulation of rejection in these models appears to be dependent on IL-10 (12), a cytokine first identified because of its immunosuppressive properties on T cell activation and synthesis of Th1 cytokines (36). The most simple explanation for this lack of effect is that following tolerance induction and transplantation, the majority of regulatory cells migrate to and are sequestered within the graft (37) thus limiting any systemic effect. Thus, even though influenza-specific CTL were obtained from the spleens of transplanted animals (Figs. 2 and 5), a compartment we have shown to contain regulatory cells after anti-CD4/DST pretreatment (11, 12), it could be argued that the presence of the graft may have selectively depleted the spleen of regulatory cells thereby allowing antiviral responses to develop relatively normally. However, this explanation cannot apply to the experiments shown in Fig. 6 where tolerized mice remained untransplanted and were infected 1 day after a second alloantigen challenge. Influenza-specific CTL with apparently normal activity were obtained from the spleens of these mice (Fig. 6b) despite the fact that alloantigen-driven regulatory cells remain in the spleen for at least 5 days after rechallenge (Fig. 4). It could also be argued that any perturbation of CTL responses caused by ongoing regulation was masked in our experiments because exogenous IL-2 was added during the in vitro expansion of CTL precursors. This is a potentially valid criticism because regulation in vitro can often be overcome by the addition of exogenous IL-2 (38, 39, 40, 41). However, viral clearance from the lungs of infected mice, which is an entirely in vivo phenomenon involving no experimental manipulation, was also unaffected by the presence of regulatory cells (Figs. 3, 5, and 6), supporting the CTL data.
Several studies have addressed the effect that viral infection has on transplantation tolerance, and the overall view is that both prior and concurrent exposure to viral pathogens can prevent tolerance induction. Although previous viral exposure leads to memory T cells (predominantly CD8+) that appear to be resistant to tolerance induction (25, 26), CD8+ T cells driven by peri-transplant viral exposure have also been implicated in rejection of the graft itself (42, 43). As far as the converse situation is concerned, previous studies have examined the clearance of viral pathogens in mixed allogeneic chimeras (44), and although mice containing both host and donor restricted T cells may have more difficulty clearing chronic infections (45), mixed chimeras seem to clear acute viral infections at similar rates to their normal counterparts. However, much less attention has been paid to the effect that transplantation tolerance based on active T cell regulation might have on protective immunity. This is an important area because it is hoped that tolerance induction will circumvent many of the problems associated with current nonspecific immunosuppression. To our knowledge, this is the first study to examine the effects of alloantigen-driven CD25+CD4+ regulatory cells on responses to a viral pathogen. We have found no diminution of antiviral responses in three different situations, and although the data reflect responses to a single pathogen in one particular transplant setting, we believe our data give grounds for cautious optimism that bystander suppression may be less of a problem in transplantation than might be imagined. We are currently extending our studies to other model pathogens and transplant models to determine whether this optimism is well placed.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We are grateful to Prof. Herman Waldman (Sir William Dunn School of Pathology, University of Oxford) for providing the hybridoma YTS 177.9 and to Dr. Paul Klenerman (Nuffield Department of Medicine, University of Oxford) for comments on the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by The Wellcome Trust. A.G. is a Medical Research Council Senior Research Fellow. K.W. holds a Royal Society Wolfson Research Merit Award.
2 Address correspondence and reprint requests to Dr. Andrew R. Bushell, Nuffield Department of Surgery, John Radcliffe Hospital, Oxford OX3 9DU, U.K. E-mail address: andrew.bushell{at}nds.ox.ac.uk
3 Current address: The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K.
4 Current address: Department of Medical Biochemistry and Immunology, Cardiff University, Cardiff CF14 4XN, U.K.
5 Abbreviations used in this paper: DST, donor-specific transfusion; MDCK, Madin-Darby canine kidney; HA, hemagglutinin.
6 S. Bigenzahn, P. Blaha, Z. Koporc, E. Selzer, C. Heusser, K. Wagner, F. Muehlbacher, and T. Wekerle. 2003. The role of regulation for induction and maintenance of tolerance through BMT plus costimulation blockade. Presented at Basic Sciences Symposium of the Transplantation Society, September 14th-17th, 2003, Keystone, Colorado.
Received for publication August 26, 2004. Accepted for publication November 18, 2004.
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