Peripheral Deletion of Antigen-Specific T Cells Leads to Long-Term Tolerance Mediated by CD8+ Cytotoxic Cells
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免疫学杂志 2005年第7期
Abstract
Peripheral deletion is one mechanism by which potentially self-reactive clones are removed whether they escape thymic deletion. We have examined the consequences of deleting Ag-specific T cells by i.v. injection of soluble Ag. Deletion of DO11.10 T cells by peptide was mediated predominately via a Fas/FasL mechanism. Animals that underwent deletion were tolerant to subsequent immunization with Ag, even when tolerant mice were given fresh Ag-specific DO11.10 T cells before immunization. Tolerance was mediated by CD8+ T cells that killed the DO11.10-transgenic T cells in vivo. These data demonstrate that the programmed cell death of large numbers of T cells leads to peripheral tolerance mediated by CD8+ CTLs.
Introduction
The immune response is dependent on programmed cell death at every point during development and activation. The consequences of cell death in the thymus are clonal deletion, while apoptosis in the periphery can lead to productive immunity (clonal selection) or tolerance (peripheral tolerance). Peripheral deletion is one mechanism by which the immune system eliminates self-reactive T cells that escaped thymic deletion. For example, in response to soluble tissue Ags released during infection or tissue damage, the immune system could eliminate potential self-reactive clones to avoid autoimmunity. Injection of superantigen into normal mice (1) or soluble peptide Ag into TCR-transgenic mice (2, 3) has provided evidence for deletion as a mechanism to control reactive clones. In a number of models, peripheral deletion can occur through specific death receptors (1, 3, 4, 5) or by cell autonomous mechanisms involving Bcl-2 family members such as the up-regulation of proapoptotic molecules (6).
Apoptosis of lymphocytes can result in immune tolerance (7, 8). Mechanisms proposed to account for the tolerogenic nature of apoptosis include deletion of reactive clones, anergy (clonal inactivation) (2), immune deviation (Th2 T cells over Th1) (9), and active regulation (T regulatory or T cells) (10). Apoptosis of lymphocytes can prevent autoimmunity (11, 12, 13) and sustain allografts (14). Apoptotic cells can also have significant consequences during important physiological processes such as bacterial (15) and parasitic infections (16).
Fas/FasL-induced apoptosis is an important mechanism of cell death that can lead to tolerance. This has been demonstrated for immune-privileged sites (17), the skin (18), and thyroid (19). We have recently shown that tolerance induced by the injection of Ag-coupled spleen cells required Fas/FasL-mediated apoptosis and resulted in the induction of regulatory T cells produced when the apoptotic cells were presented to immune system via the cross-priming pathway (10). Because relatively large numbers of apoptotic cells were required to induce cross-tolerance, we wondered whether other systems where there was substantial Fas/FasL-mediated T cell apoptosis would also produce an active tolerogenic state. In the present studies, we have examined the consequences of the T cell apoptosis that occurs when soluble peptide Ags encounter TCR-transgenic T cells. Our studies show that deletion of T cells leads to infectious tolerance mediated by CD8+ killer cells that specifically delete Ag-reactive T cell clones. These data suggest that apoptosis of large numbers of T cells can lead to regulation of systemic immunity, perhaps to prevent subsequent immune responses.
Materials and Methods
Mice
BALB/c were purchased from the National Cancer Institute. The gld and lpr mutations were bred onto the BALB/c background by crossing B6-gld and B6-lpr (originally obtained from The Jackson Laboratory) to BALB/c for a minimum of 10 generations. Mice were screened by PCR as described (20). Because the mutations were derived from B6 mice, these strains are designated C.B6-lpr and C.B6-gld. DO11.10-lpr mice were bred in our facility by crossing C.B6-lpr mice by the DO11.10-transgenic mouse. Groups usually consist of five mice and experiments were repeated at least twice. Statistical significance between groups was determined by Student’s t test using a 95% (p < 0.05). All animal studies were approved by the Washington University Animal Studies Committee.
Immune response to protein Ags
To generate an immune response to OVA or -galactosidase (-gal),3 mice were immunized s.c. with 0.2 ml of 100 μg of OVA or -gal (Sigma-Aldrich) emulsified 1:1 in CFA. Seven days later, mice were challenged in the right footpad with 100 μg of heat-aggregated OVA or -gal in 0.033 ml of PBS and in the left footpad with 0.033 ml of PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge).
In vivo cytotoxicity
Purified DO11.10 T cells or normal CD4+ T cells were suspended at 1 x 107 cells/ml in warmed PBS/0.1% BSA. CFSE (1.25 μl/ml of a 5-mM stock) was added to the DO11.10 for the CFSEhigh and a 10-fold dilution was used for the CFSElow population (reference population). Cells were incubated 10 min at 37°C (water bath). The reaction was stopped by the addition of ice-cold PBS containing 10% FBS. Cells were washed three times with PBS/FBS, counted, and resuspended to the appropriate volume. Ten million target cells (CFSEhigh) and 10 million reference cells (CFSElow) were injected into naive or tolerant BALB/c recipients that had been immunized with CFA/OVA 7 days earlier. Spleens were harvested 18 h later and analyzed by flow cytometry. A total of 3–5000 events in the reference population were collected and the number of target cells recovered was enumerated. Unimmunized BALB/c mice were used as controls. The percent reduction in the number of recovered CFSEhigh cells in the unimmunized vs the tolerant mice was considered the percent killing. Individual mice were analyzed and the percent killing in each group of mice was determined.
Purification of T cells
Spleens were removed from both DO11.10 and BALB/c mice. These cells were then passed over nylon wool columns to enrich for T cells. Resultant cells were then stained with KJ-126 and CD4 to determine the number of KJ-126+, CD4+ cells in the DO.11.10 preparation or the number of CD4+ T cells in the BALB/c spleen population. The percentage of KJ-126+ cells ranged between 50 and 80% depending on the specific experiment. The percentage of CD4+ T cells isolated from BALB/c spleen ranged from 80 to 90%. However, all mice were injected with 1 x 107 KJ-126+ cells and 1 x 107 CD4+ T cells.
For the adoptive transfer experiment, spleens and lymph nodes were isolated from tolerant mice and were then treated with anti-CD4 (clone RL174.2), CD8 (clone 53.6.72), or both for 45 min on ice. Cells were then washed one time in cold PBS and a 1/16 dilution of rabbit complement (Pel-Frez) was added for 30 min at 37°C. Cells were washed three times in PBS and resuspended in PBS. Mice received 2 x 107 cells or their equivalence in 0.2 ml i.v.
Cytokine production
T cells from tolerant, immunized, and naive mice were removed and purified as described above. T cells (5 x 106/ml) were placed in culture with an equal number of irradiated BALB/c spleen cells and pulsed with OVA. At daily intervals, supernatants were analyzed for the presence of cytokines by BD Cytometric Bead Array kits (BD Pharmingen).
Peptide-induced deletion of DO11.10 T cells
Five million DO11.10 or DO11.10-lpr cells purified from transgenic mice were injected into naive BALB/c mice. Twenty-four hours later, mice received an i.p. injection of 300 μg of OVA323–339 peptide. At various times following peptide injection, several mice were killed and the percentage of KJ-126+ cells was determined in the spleen and lymph nodes by flow cytometry. V6+ T cells were monitored as a reference population.
Results
Recent studies have shown that peripheral deletion of superantigen and peptide-specific TCR-transgenic T cells was mediated, at least in part, by FasL/Fas interactions (1, 3). We confirmed this using TCR-transgenic T cells from DO11.10 mice by seeding BALB/c mice with purified CD4+ DO11.10+ T cells from DO11.10 or DO11.10-lpr mice. Mice were then given peptide and the number of KJ-126+ T cells was monitored in the spleen and lymph nodes (Fig. 1). BALB/c mice given DO11.10 T cells plus peptide deleted the KJ-126+ cells from the spleen and lymph nodes over the 28 days of the experiment. When DO11.10+ T cells were given to C.B6-gld mice, however, significant deletion was not observed. Similarly, there was limited deletion when DO11.10-lpr cells were transferred into wild-type BALB/c mice. Thus, deletion of DO11.10+ T cells by peptide administration is largely Fas/FasL-dependent, as has been observed for T cells with other TCR specificities (3, 4).
FIGURE 1. Fas/FasL-mediated deletion of DO11.10 T cells. DO11.10 or DO11.10-lpr cells purified from transgenic mice were injected into naive BALB/c mice or C.B6-gld mice. Twenty-four hours later, mice received an i.p. injection of 300 μg of OVA323–339 peptide. At various times following peptide injection, several mice were killed and the percentage of KJ-126+ cells were determined in the spleen and lymph nodes.
The immune response to OVA was examined in mice that had deleted the KJ-126+ T cells. BALB/c mice were seeded with DO11.10 or DO11.10-lpr T cells and injected with peptide. One month later, the mice were immunized with OVA and the immune response was tested. As shown in Fig. 2, BALB/c mice in which transferred DO11.10 T cells were deleted by peptide injection were not capable of generating a delayed-type hypersensitivity (DTH) response to OVA (Group (Grp) 4). This observation contrasts with results obtained using C.B6-gld as recipients for DO11.10 T cells (Grp 7) or when DO11.10-lpr cells were seeded to BALB/c mice (Grp 6). In these cases where deletion did not occur, a DTH response was generated to OVA. Tolerance was not the result of peptide administration (Grp 2), showing that soluble peptide alone cannot induce tolerance in BALB/c mice without the presence of DO11.10. We conclude that Fas/FasL-mediated deletion is important for tolerance to develop.
FIGURE 2. Tolerance in mice deleting DO11.10. DO11.10 (DO) or DO11.10-lpr (DO-lpr) cells purified from transgenic mice were injected into naive BALB/c mice or C.B6-gld mice. Twenty-four hours later, some groups received an i.p. injection of 300 μg of OVA323–339 peptide. One month later, mice were immunized s.c. with OVA/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). *, Significantly different from the immune control (Grp 1).
During normal immune responses that result in productive immunity, apoptosis of T cells nevertheless occurs (21, 22); but such apoptosis need not result in tolerance. This suggested that the tolerance induced in the present system might be dependent on the number of apoptotic cells that were present. We examined this by determining the number of DO11.10 T cells that must be present during peptide-induced deletion for tolerance to be established. BALB/c mice were given decreasing doses of DO11.10 T cells before peptide injection. Two weeks later, mice were immunized and DTH was examined. These data (Fig. 3) show that a minimum of 1 x 106 DO11.10 T cells must be seeded (and deleted) to generate tolerance to OVA. Deletion of 5 x 105 T cells was not tolerogenic. This suggests that there must be a threshold of apoptosis that leads to tolerance.
FIGURE 3. Minimum number of DO11.10 that must be deleted for tolerance. Varying numbers of DO11.10 T cells purified from transgenic mice were injected into naive BALB/c mice. Twenty-four hours later, some groups of mice received an i.p. injection of 300 μg of OVA323–339 peptide. One month later, mice were immunized s.c. with OVA/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). *, Significantly different from the immune control.
Although our data suggest that deletion of DO11.10 cells is required for tolerance, it is still formally possible that the lack of response in recipient mice is simply the removal or anergy of endogenous OVA-reactive clones. Therefore, we examined the ability of tolerant mice (that had deleted DO11.10 T cells) to support the response of naive DO11.10 T cells. Groups of BALB/c or C.B6-gld mice were seeded with 1 x 106 DO11.10 or DO11.10-lpr T cells (minimum needed to show tolerance, see Fig. 3) and these mice were given peptide. One month later, the mice were given a second infusion of naive DO11.10 T cells ranging from 1 to 10 x 106 (numbers on the x-axis). These recipients were immunized with OVA/CFA and the immune response was examined 1 wk later (Fig. 4A). When BALB/c mice had undergone a previous "episode" of peptide-induced deletion of DO11.10 T cells (?), up to 5 x 106 fresh naive DO11.10 cells could be reinjected and tolerance was still observed. When no peptide was administered (i.e., there was no deletion) (), or whether the first deletion was prevented by using C.B6-gld recipients () or DO11.10-lpr donor T cells (), immunity was established when DO11.10 T cells were reinjected before immunization with OVA/CFA. Fig. 4B shows that tolerance does not target the response to another Ag. Mice rendered tolerant by deletion of DO11.10 cells produced a normal immune response to -gal, even when the response was measured 24 and 48 h postchallenge. Thus, the peptide-induced deletion of the DO11.10 T cell population leads to the establishment of an Ag-specific tolerant state. Tolerance can prevent subsequent responses by fresh T cells of the same specificity.
FIGURE 4. Tolerant mice cannot support an anti-OVA immune response by naive DO11.10 T cells. A, DO11.10 or DO11.10-lpr cells purified from transgenic mice were injected into naive BALB/c mice or C.B6-gld mice. Twenty-four hours later, some groups received an i.p. injection of 300 μg of OVA323–339 peptide. One month later, mice received the indicated numbers (x-axis, second infusion) of freshly isolated, naive DO11.10 T cells. On the same day as the second infusion, these mice were immunized s.c. with OVA/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). The dotted line represents the immune response of BALB/c mice that received only a CFA/OVA immunization. *, Significantly different from the immune control. B, Mice rendered tolerant by peptide-induced deletion of DO11.10 T cells (1 mo earlier) were immunized s.c. on with OVA/CFA or -gal/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA or -gal and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). Bkg represents the response of naive mice challenges with Ag. *, Significantly different from the immune control.
Recently it was shown that peptide-induced deletion of DO11.10 resulted in a residual population of cells that could regulate immunity (23). Another study found that following deletion there were residual DO11.10 T cells that were unable to respond (anergic) to the Ag (2). However, it seemed to us that apoptosis in this system, which proceeded by Fas/FasL interaction, might lead to a more active form of tolerance as we have observed in other systems (10, 17). To rule out any effects of residual TCR-transgenic T cells we tested whether DO11.10 T cells induced to die by another method could substitute for the apoptotic cells generated during peptide-induced deletion. Decreasing doses of irradiated, naive DO11.10 T cells were injected into BALB/c mice. Two weeks later, mice were given 1 x 106 fresh DO11.10 T cells and immunized with OVA. Fig. 5 shows that injection of 1 x 106 apoptotic DO11.10 T cells can substitute for T cells deleted via peptide administration. This suggests that apoptosis of T cells is critical to the induction of tolerance. Residual regulatory DO11.10 T cells could not be involved as none are available when irradiated DO11.10 T cells were used to induce tolerance. We then determined whether apoptotic cells could be used to tolerize C.B6-gld mice. As shown in Fig. 5B, apoptotic (irradiated) DO11.10 T cells induced potent tolerance in both BALB/c and C.B6-gld mice. This suggests that FasL may not be an effector molecule in this system. These results are similar to what we found in other systems where apoptosis led to tolerance (10, 17).
FIGURE 5. Irradiated DO11.10 T cells substitute for peptide-induced deletion. A, BALB/c mice received the indicated numbers of irradiated (3000R) or nonirradiated DO11.10 T cells i.v. Two weeks later, mice were immunized s.c. with OVA/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Mice that received DO11.10 plus peptide we use as the tolerance control. B, BALB/c or C.B6-gld mice received 2 x 106 irradiated DO11.10 T cells i.v. Three days later, they were immunized s.c. with OVA/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values (A and B) are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). *, Significantly different from the immune control.
That naive DO11.10 cells could not produce an immune response in tolerant mice suggests that these fresh T cells might be rendered anergic or were actively deleted upon immunization. When we examined cytokine production in tolerant mice (Fig. 6), we found that tolerant mice had substantially reduced IFN- and IL-4 production, consistent with either of these mechanisms. This is consistent with either a deletion or anergy mechanism. These results also suggest that tolerance is not the result of an immune deviation (9), as both IFN- and IL-4 are inhibited.
FIGURE 6. Cytokine production in tolerant mice. DO11.10 T cells purified from transgenic mice were injected into naive BALB/c mice. Twenty-four hours later, recipient mice received an i.p. injection of 300 μg of OVA323–339 peptide. One month later, mice were immunized s.c. with OVA/CFA. Seven days later, mice spleens and lymph nodes (5 x 106 cells/ml) were harvested and placed in culture with 10 μg/ml OVA323–339 peptide. Supernatants were harvested 48 h later and the amount of IFN- (A) and IL-4 (B) determined by flow cytometry.
We then explored the possibility of deletion in tolerant mice using an in vivo cytotoxicity assay. This assay monitored eradication of an adoptively transferred target population (DO11.10 T cells) in tolerant and nontolerant mice. BALB/c recipient mice were rendered tolerant by injection of 1 x 106 DO11.10 T cells and peptide. As controls, mice were untreated or given DO11.10-lpr cells (plus peptide). Two weeks later, these mice were immunized with OVA/CFA. One week following immunization, mice were infused with fresh DO11.10 T cells labeled with a high concentration of CFSE (target cells, CFSEhigh) and with syngeneic BALB/c CD4+ T cells that were labeled with less CFSE (reference population, CFSElow) at a ratio of 1:1. Eighteen hours later, the remaining DO11.10 cells were analyzed by comparing their numbers to the reference population. Fig. 7A shows that when CFSEhigh and CFSElow cells were infused into immune mice, DO11.10 T cells were recovered in similar numbers to the reference population (0% killing). Similarly, when CFSE-labeled cells were given to mice that had not deleted the DO11.10 T cells (DO11.10-lpr), no killing was observed (0% killing). However, when labeled cells were given to tolerant mice (that had previously undergone peptide-induced deletion), the CFSEhigh DO11.10 T cells were rapidly eliminated (69% killing). Thus, peptide-induced deletion activates a mechanism that eliminates (or kills) the infused DO11.10 T cell population.
FIGURE 7. In vivo cytotoxicity of DO11.10 T cells. DO11.10 or DO11.10-lpr cells T cells purified from transgenic mice were injected into naive BALB/c mice. Twenty-four hours later, recipient mice received an i.p. injection of 300 μg of OVA323–339 peptide. Two weeks later, recipient mice received 1 x 107 DO11.10 T cells and 1 x 107 CD4+ BALB/c T cells labeled with CFSE. Eighteen hours later, spleens and lymph nodes were harvested and the relative numbers of DO11.10 (CFSEhigh) and CD4+ T cells (CFSElow) were determined by flow cytometry. Numbers were compared with unimmunized mice (n = 5). Percent killing shown on the graph represents the average of mice in that group; immune control (0%) (n = 5), DO11.10-lpr (0%) (n = 7), DO11.10 (69 ± 4%) (n = 12).
The cell type responsible for killing was explored in Fig. 8. T cells from spleens and lymph nodes of tolerant BALB/c mice were separated into CD4+ and CD8+ fractions. These cells were then injected into naive BALB/c mice that were immunized with OVA/CFA. One week later, they were tested for DTH. These data show that the CD8+ (not CD4+ or CD4– CD8–) T cells prevent the response to OVA. Thus, tolerance in this system is mediated by a CD8+ cytotoxic T cell.
FIGURE 8. Adoptive transfer of tolerance. BALB/c mice were rendered tolerant by injection of DO11.10 T cells and 300 μg of OVA323–339 peptide. Two weeks later, mice were immunized s.c. with OVA/CFA. One week later, spleen and lymph nodes were removed, separated into CD4+ and CD8+ T cells and these cells were transferred to naive BALB/c mice. Recipient mice were immunized s.c. with OVA/CFA, and seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in mircometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). Bkg represents the response of naive mice challenges with Ag. *, Significantly different from the immune control.
Discussion
Apoptosis and tolerance are biological responses clearly linked to the control of the peripheral immune response (24). Apoptosis of tissue cells preserves organ homeostasis by transducing tolerogenic signals though the dendritic cells and the cross-priming pathway to prevent autoimmunity (25, 26). Apoptosis in the immune system promotes peripheral deletion and the removal of dangerous autoreactive T cells (27, 28) by using cross-priming to actively silence responsive clones (29). Studies with peptide-induced and superantigen-induced deletion of T cells have helped us understand the mechanisms of peripheral deletion and its importance to the induction of tolerance. Our results presented here extend current paradigms of peripheral tolerance demonstrating that following deletion of Ag-reactive T cells there may be an additional level of control put into play when relatively large numbers of peripheral T cells delete by apoptosis. We demonstrate that deletion of TCR-transgenic T cells via the Fas/FasL pathway leads to the establishment of a state of active tolerance. This tolerance extends to naive T cells of the same specificity and is mediated by activation of CD8+ T cells that kill their T cell targets. Our data suggest that this active immunoregulation is directed toward the Ag-reactive cells for the purpose of controlling any self responses that might be generated when large numbers of T cells die and release potentially dangerous autoantigens and cytokines. This contrasts apoptosis that occurs during normal turnover of T cells where tolerance is not established following deletion, allowing the progression to a productive immune response.
The role of apoptosis, specifically the apoptotic cell, in the induction of tolerance has recently been the subject of much study. Interestingly, tolerance induced by apoptotic cells can have positive and negative effects on immunity. For example, massive apoptosis during parasitic (16) and bacterial (15) infections can enhance pathology during these diseases. In experimental trypanosome infection, apoptotic lymphocytes fuel parasite growth. These studies revealed that an interaction between apoptotic T cells (generated during the infection) and macrophages promoted an anti-inflammatory response. Interestingly, necrotic cells did not have this function. Similar results were observed in an experimental model of bacterial sepsis. In this study, sepsis-induced lymphocyte apoptosis decreased the chances of survival for the infected host. If apoptosis was prevented with caspase inhibitors, or if T cell apoptosis was prevented by a Bcl-2 transgene, survival was significantly improved. Thus, during these infections it would seem that all the danger signals elicited by the infection are overcome by the presence of apoptotic lymphocytes.
In contrast to these findings, apoptosis that occurs during the experimental induction of graft tolerance can have positive consequences for the organ and tissue homeostasis. It was recently demonstrated that long-lasting graft tolerance could be induced with an immunosuppressive regimen consisting of combined CD40 and B7 blockade coupled with rapamycin treatment (14). Interestingly, this tolerance required T cell apoptosis as overexpression of Bcl-XL in the T cells blocked the effect. Tolerance was also not established whether rapamycin (which facilitates the apoptotic signal process in T cells) was replaced with cyclosporin A (which prevents apoptosis in T cells). The long-term nature of this tolerance could not be explained by simple deletion, suggesting that tolerance is infectious and mediated by regulatory cells. Perhaps the tolerance observed in that system is directed toward reactive T cells, as we have observed in this study, rather than to a specific Ag.
Immune privilege sites also use Fas/FasL-induced apoptosis to promote tolerance to control inflammatory responses. In the eye, constitutive FasL expression induces apoptosis of invading lymphoid cells (30) leading to systemic tolerance (17) mediated by regulatory T cells. Both deletion and tolerance protect vision from the damaging consequences of inflammation.
The specificity of the CD8+ T cell that mediates tolerance in the present system appears to be the T cell rather than the specific Ag. We know this because the tolerance (cytotoxicity) is directed toward the TCR-transgenic T cells and it can be induced with apoptotic T cells in the absence of Ag. These results are similar to those observed in systems studying T cell vaccination as prevention for autoimmune disease (31, 32, 33). In these models, regulation is directed toward the TCR rather than the specific Ag. Other reports show that T cell-derived peptides are the target of the regulatory cells (33, 34, 35), suggesting an anti-Id response directed toward the TCR of DO11.10 might be responsible. Studies demonstrating the induction of suppressor T cells following staphylococcal enterotoxin B-induced deletion of V8+ T cells also support this idea (36), however, further study will be required. In addition, Fas/FasL-mediated deletion of the class I-restricted CD8+ T cell (4) in response to OVA expressed in the pancreas has been observed. Whether this leads to an active tolerance directed toward the T cell is not known.
We do not yet know the mechanism by which the CD8+ T cells kill their targets in vivo. Possibilities included FasL or the perforin/granzyme (37) system. CD8+ cytotoxic lymphocytes, NK cells, and lymphokine-activated killer cells depend primarily on the perforin/granzyme system to kill their targets, while CD4+ T cells use Fas and other mechanisms to induce cell death. We believe that the FasL pathway is not involved in the present system for three reasons: 1) When trinitrophenyl-coupled (TNP)-spl are injected into the anterior chamber of the eye, tolerance develops because the spleen cells undergo FasL-mediated apoptosis. FasL-defective gld mice do not develop tolerance, unless the TNP-spl are induced to undergo apoptosis before they are injected (17). 2) Tolerance following i.v injection of TNP-spl also depends on Fas/FasL-mediated apoptosis. Again, gld mice cannot be tolerized, unless the TNP-spl are irradiated before injection (10). 3) Irradiated DO11.10 cells can induce tolerance in C.B6-gld mice (see Fig. 5B). However, because we have not formally ruled out any mechanisms for CD8+ killer cell activity, this will require further study.
Data presented in this paper suggest that apoptosis of a clonal population of T cells mediated by Fas/FasL can lead to a state of tolerance. Tolerance induction requires the death of a relatively large number of cells, suggesting it does not occur during the activation of the adaptive immune response. The tolerance, mediated by cytotoxic CD8+ killer cells, is specifically directed toward the T cell clone. We propose that this mechanism may be an adaptation to control potential antiself responses that could occur when large numbers of T cells die by apoptosis and their remnants enter the Ag-processing pathways. Our system examines the results of eliminating a large number of CD4+-transgenic T cells. We do not know whether similar results are obtained with CD8+ CTLs, as it has been shown that apoptosis of virus-specific cells can lead to memory (38). Consequently, the relationship between our finding and other naturally occurring tolerance pathways will require further study.
Disclosures
The authors have no financial conflict of interest.
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 National Institutes of Health Grants EY12826 (to T.A.F.), EY06765 (to T.A.F.), and EY12707 (to P.M.S.), the Department of Ophthalmology and Visual Sciences Core Grant (EY08972), and an Arthritis Foundation Biomedical Science Grant (to P.M.S.). Support was also received from The Foundation for Fighting Blindness and from Research to Prevent Blindness.
2 Address correspondence and reprint requests to Dr. Thomas A. Ferguson, Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110. E-mail address: Ferguson@vision.wustl.edu
3 Abbreviations used in this paper: -gal, -galactosidase; DTH, delayed-type hypersensitivity; Grp, group; TNP, trinitrophenyl.
Received for publication December 20, 2004. Accepted for publication January 21, 2005.
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Peripheral deletion is one mechanism by which potentially self-reactive clones are removed whether they escape thymic deletion. We have examined the consequences of deleting Ag-specific T cells by i.v. injection of soluble Ag. Deletion of DO11.10 T cells by peptide was mediated predominately via a Fas/FasL mechanism. Animals that underwent deletion were tolerant to subsequent immunization with Ag, even when tolerant mice were given fresh Ag-specific DO11.10 T cells before immunization. Tolerance was mediated by CD8+ T cells that killed the DO11.10-transgenic T cells in vivo. These data demonstrate that the programmed cell death of large numbers of T cells leads to peripheral tolerance mediated by CD8+ CTLs.
Introduction
The immune response is dependent on programmed cell death at every point during development and activation. The consequences of cell death in the thymus are clonal deletion, while apoptosis in the periphery can lead to productive immunity (clonal selection) or tolerance (peripheral tolerance). Peripheral deletion is one mechanism by which the immune system eliminates self-reactive T cells that escaped thymic deletion. For example, in response to soluble tissue Ags released during infection or tissue damage, the immune system could eliminate potential self-reactive clones to avoid autoimmunity. Injection of superantigen into normal mice (1) or soluble peptide Ag into TCR-transgenic mice (2, 3) has provided evidence for deletion as a mechanism to control reactive clones. In a number of models, peripheral deletion can occur through specific death receptors (1, 3, 4, 5) or by cell autonomous mechanisms involving Bcl-2 family members such as the up-regulation of proapoptotic molecules (6).
Apoptosis of lymphocytes can result in immune tolerance (7, 8). Mechanisms proposed to account for the tolerogenic nature of apoptosis include deletion of reactive clones, anergy (clonal inactivation) (2), immune deviation (Th2 T cells over Th1) (9), and active regulation (T regulatory or T cells) (10). Apoptosis of lymphocytes can prevent autoimmunity (11, 12, 13) and sustain allografts (14). Apoptotic cells can also have significant consequences during important physiological processes such as bacterial (15) and parasitic infections (16).
Fas/FasL-induced apoptosis is an important mechanism of cell death that can lead to tolerance. This has been demonstrated for immune-privileged sites (17), the skin (18), and thyroid (19). We have recently shown that tolerance induced by the injection of Ag-coupled spleen cells required Fas/FasL-mediated apoptosis and resulted in the induction of regulatory T cells produced when the apoptotic cells were presented to immune system via the cross-priming pathway (10). Because relatively large numbers of apoptotic cells were required to induce cross-tolerance, we wondered whether other systems where there was substantial Fas/FasL-mediated T cell apoptosis would also produce an active tolerogenic state. In the present studies, we have examined the consequences of the T cell apoptosis that occurs when soluble peptide Ags encounter TCR-transgenic T cells. Our studies show that deletion of T cells leads to infectious tolerance mediated by CD8+ killer cells that specifically delete Ag-reactive T cell clones. These data suggest that apoptosis of large numbers of T cells can lead to regulation of systemic immunity, perhaps to prevent subsequent immune responses.
Materials and Methods
Mice
BALB/c were purchased from the National Cancer Institute. The gld and lpr mutations were bred onto the BALB/c background by crossing B6-gld and B6-lpr (originally obtained from The Jackson Laboratory) to BALB/c for a minimum of 10 generations. Mice were screened by PCR as described (20). Because the mutations were derived from B6 mice, these strains are designated C.B6-lpr and C.B6-gld. DO11.10-lpr mice were bred in our facility by crossing C.B6-lpr mice by the DO11.10-transgenic mouse. Groups usually consist of five mice and experiments were repeated at least twice. Statistical significance between groups was determined by Student’s t test using a 95% (p < 0.05). All animal studies were approved by the Washington University Animal Studies Committee.
Immune response to protein Ags
To generate an immune response to OVA or -galactosidase (-gal),3 mice were immunized s.c. with 0.2 ml of 100 μg of OVA or -gal (Sigma-Aldrich) emulsified 1:1 in CFA. Seven days later, mice were challenged in the right footpad with 100 μg of heat-aggregated OVA or -gal in 0.033 ml of PBS and in the left footpad with 0.033 ml of PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge).
In vivo cytotoxicity
Purified DO11.10 T cells or normal CD4+ T cells were suspended at 1 x 107 cells/ml in warmed PBS/0.1% BSA. CFSE (1.25 μl/ml of a 5-mM stock) was added to the DO11.10 for the CFSEhigh and a 10-fold dilution was used for the CFSElow population (reference population). Cells were incubated 10 min at 37°C (water bath). The reaction was stopped by the addition of ice-cold PBS containing 10% FBS. Cells were washed three times with PBS/FBS, counted, and resuspended to the appropriate volume. Ten million target cells (CFSEhigh) and 10 million reference cells (CFSElow) were injected into naive or tolerant BALB/c recipients that had been immunized with CFA/OVA 7 days earlier. Spleens were harvested 18 h later and analyzed by flow cytometry. A total of 3–5000 events in the reference population were collected and the number of target cells recovered was enumerated. Unimmunized BALB/c mice were used as controls. The percent reduction in the number of recovered CFSEhigh cells in the unimmunized vs the tolerant mice was considered the percent killing. Individual mice were analyzed and the percent killing in each group of mice was determined.
Purification of T cells
Spleens were removed from both DO11.10 and BALB/c mice. These cells were then passed over nylon wool columns to enrich for T cells. Resultant cells were then stained with KJ-126 and CD4 to determine the number of KJ-126+, CD4+ cells in the DO.11.10 preparation or the number of CD4+ T cells in the BALB/c spleen population. The percentage of KJ-126+ cells ranged between 50 and 80% depending on the specific experiment. The percentage of CD4+ T cells isolated from BALB/c spleen ranged from 80 to 90%. However, all mice were injected with 1 x 107 KJ-126+ cells and 1 x 107 CD4+ T cells.
For the adoptive transfer experiment, spleens and lymph nodes were isolated from tolerant mice and were then treated with anti-CD4 (clone RL174.2), CD8 (clone 53.6.72), or both for 45 min on ice. Cells were then washed one time in cold PBS and a 1/16 dilution of rabbit complement (Pel-Frez) was added for 30 min at 37°C. Cells were washed three times in PBS and resuspended in PBS. Mice received 2 x 107 cells or their equivalence in 0.2 ml i.v.
Cytokine production
T cells from tolerant, immunized, and naive mice were removed and purified as described above. T cells (5 x 106/ml) were placed in culture with an equal number of irradiated BALB/c spleen cells and pulsed with OVA. At daily intervals, supernatants were analyzed for the presence of cytokines by BD Cytometric Bead Array kits (BD Pharmingen).
Peptide-induced deletion of DO11.10 T cells
Five million DO11.10 or DO11.10-lpr cells purified from transgenic mice were injected into naive BALB/c mice. Twenty-four hours later, mice received an i.p. injection of 300 μg of OVA323–339 peptide. At various times following peptide injection, several mice were killed and the percentage of KJ-126+ cells was determined in the spleen and lymph nodes by flow cytometry. V6+ T cells were monitored as a reference population.
Results
Recent studies have shown that peripheral deletion of superantigen and peptide-specific TCR-transgenic T cells was mediated, at least in part, by FasL/Fas interactions (1, 3). We confirmed this using TCR-transgenic T cells from DO11.10 mice by seeding BALB/c mice with purified CD4+ DO11.10+ T cells from DO11.10 or DO11.10-lpr mice. Mice were then given peptide and the number of KJ-126+ T cells was monitored in the spleen and lymph nodes (Fig. 1). BALB/c mice given DO11.10 T cells plus peptide deleted the KJ-126+ cells from the spleen and lymph nodes over the 28 days of the experiment. When DO11.10+ T cells were given to C.B6-gld mice, however, significant deletion was not observed. Similarly, there was limited deletion when DO11.10-lpr cells were transferred into wild-type BALB/c mice. Thus, deletion of DO11.10+ T cells by peptide administration is largely Fas/FasL-dependent, as has been observed for T cells with other TCR specificities (3, 4).
FIGURE 1. Fas/FasL-mediated deletion of DO11.10 T cells. DO11.10 or DO11.10-lpr cells purified from transgenic mice were injected into naive BALB/c mice or C.B6-gld mice. Twenty-four hours later, mice received an i.p. injection of 300 μg of OVA323–339 peptide. At various times following peptide injection, several mice were killed and the percentage of KJ-126+ cells were determined in the spleen and lymph nodes.
The immune response to OVA was examined in mice that had deleted the KJ-126+ T cells. BALB/c mice were seeded with DO11.10 or DO11.10-lpr T cells and injected with peptide. One month later, the mice were immunized with OVA and the immune response was tested. As shown in Fig. 2, BALB/c mice in which transferred DO11.10 T cells were deleted by peptide injection were not capable of generating a delayed-type hypersensitivity (DTH) response to OVA (Group (Grp) 4). This observation contrasts with results obtained using C.B6-gld as recipients for DO11.10 T cells (Grp 7) or when DO11.10-lpr cells were seeded to BALB/c mice (Grp 6). In these cases where deletion did not occur, a DTH response was generated to OVA. Tolerance was not the result of peptide administration (Grp 2), showing that soluble peptide alone cannot induce tolerance in BALB/c mice without the presence of DO11.10. We conclude that Fas/FasL-mediated deletion is important for tolerance to develop.
FIGURE 2. Tolerance in mice deleting DO11.10. DO11.10 (DO) or DO11.10-lpr (DO-lpr) cells purified from transgenic mice were injected into naive BALB/c mice or C.B6-gld mice. Twenty-four hours later, some groups received an i.p. injection of 300 μg of OVA323–339 peptide. One month later, mice were immunized s.c. with OVA/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). *, Significantly different from the immune control (Grp 1).
During normal immune responses that result in productive immunity, apoptosis of T cells nevertheless occurs (21, 22); but such apoptosis need not result in tolerance. This suggested that the tolerance induced in the present system might be dependent on the number of apoptotic cells that were present. We examined this by determining the number of DO11.10 T cells that must be present during peptide-induced deletion for tolerance to be established. BALB/c mice were given decreasing doses of DO11.10 T cells before peptide injection. Two weeks later, mice were immunized and DTH was examined. These data (Fig. 3) show that a minimum of 1 x 106 DO11.10 T cells must be seeded (and deleted) to generate tolerance to OVA. Deletion of 5 x 105 T cells was not tolerogenic. This suggests that there must be a threshold of apoptosis that leads to tolerance.
FIGURE 3. Minimum number of DO11.10 that must be deleted for tolerance. Varying numbers of DO11.10 T cells purified from transgenic mice were injected into naive BALB/c mice. Twenty-four hours later, some groups of mice received an i.p. injection of 300 μg of OVA323–339 peptide. One month later, mice were immunized s.c. with OVA/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). *, Significantly different from the immune control.
Although our data suggest that deletion of DO11.10 cells is required for tolerance, it is still formally possible that the lack of response in recipient mice is simply the removal or anergy of endogenous OVA-reactive clones. Therefore, we examined the ability of tolerant mice (that had deleted DO11.10 T cells) to support the response of naive DO11.10 T cells. Groups of BALB/c or C.B6-gld mice were seeded with 1 x 106 DO11.10 or DO11.10-lpr T cells (minimum needed to show tolerance, see Fig. 3) and these mice were given peptide. One month later, the mice were given a second infusion of naive DO11.10 T cells ranging from 1 to 10 x 106 (numbers on the x-axis). These recipients were immunized with OVA/CFA and the immune response was examined 1 wk later (Fig. 4A). When BALB/c mice had undergone a previous "episode" of peptide-induced deletion of DO11.10 T cells (?), up to 5 x 106 fresh naive DO11.10 cells could be reinjected and tolerance was still observed. When no peptide was administered (i.e., there was no deletion) (), or whether the first deletion was prevented by using C.B6-gld recipients () or DO11.10-lpr donor T cells (), immunity was established when DO11.10 T cells were reinjected before immunization with OVA/CFA. Fig. 4B shows that tolerance does not target the response to another Ag. Mice rendered tolerant by deletion of DO11.10 cells produced a normal immune response to -gal, even when the response was measured 24 and 48 h postchallenge. Thus, the peptide-induced deletion of the DO11.10 T cell population leads to the establishment of an Ag-specific tolerant state. Tolerance can prevent subsequent responses by fresh T cells of the same specificity.
FIGURE 4. Tolerant mice cannot support an anti-OVA immune response by naive DO11.10 T cells. A, DO11.10 or DO11.10-lpr cells purified from transgenic mice were injected into naive BALB/c mice or C.B6-gld mice. Twenty-four hours later, some groups received an i.p. injection of 300 μg of OVA323–339 peptide. One month later, mice received the indicated numbers (x-axis, second infusion) of freshly isolated, naive DO11.10 T cells. On the same day as the second infusion, these mice were immunized s.c. with OVA/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). The dotted line represents the immune response of BALB/c mice that received only a CFA/OVA immunization. *, Significantly different from the immune control. B, Mice rendered tolerant by peptide-induced deletion of DO11.10 T cells (1 mo earlier) were immunized s.c. on with OVA/CFA or -gal/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA or -gal and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). Bkg represents the response of naive mice challenges with Ag. *, Significantly different from the immune control.
Recently it was shown that peptide-induced deletion of DO11.10 resulted in a residual population of cells that could regulate immunity (23). Another study found that following deletion there were residual DO11.10 T cells that were unable to respond (anergic) to the Ag (2). However, it seemed to us that apoptosis in this system, which proceeded by Fas/FasL interaction, might lead to a more active form of tolerance as we have observed in other systems (10, 17). To rule out any effects of residual TCR-transgenic T cells we tested whether DO11.10 T cells induced to die by another method could substitute for the apoptotic cells generated during peptide-induced deletion. Decreasing doses of irradiated, naive DO11.10 T cells were injected into BALB/c mice. Two weeks later, mice were given 1 x 106 fresh DO11.10 T cells and immunized with OVA. Fig. 5 shows that injection of 1 x 106 apoptotic DO11.10 T cells can substitute for T cells deleted via peptide administration. This suggests that apoptosis of T cells is critical to the induction of tolerance. Residual regulatory DO11.10 T cells could not be involved as none are available when irradiated DO11.10 T cells were used to induce tolerance. We then determined whether apoptotic cells could be used to tolerize C.B6-gld mice. As shown in Fig. 5B, apoptotic (irradiated) DO11.10 T cells induced potent tolerance in both BALB/c and C.B6-gld mice. This suggests that FasL may not be an effector molecule in this system. These results are similar to what we found in other systems where apoptosis led to tolerance (10, 17).
FIGURE 5. Irradiated DO11.10 T cells substitute for peptide-induced deletion. A, BALB/c mice received the indicated numbers of irradiated (3000R) or nonirradiated DO11.10 T cells i.v. Two weeks later, mice were immunized s.c. with OVA/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Mice that received DO11.10 plus peptide we use as the tolerance control. B, BALB/c or C.B6-gld mice received 2 x 106 irradiated DO11.10 T cells i.v. Three days later, they were immunized s.c. with OVA/CFA. Seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values (A and B) are expressed in micrometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). *, Significantly different from the immune control.
That naive DO11.10 cells could not produce an immune response in tolerant mice suggests that these fresh T cells might be rendered anergic or were actively deleted upon immunization. When we examined cytokine production in tolerant mice (Fig. 6), we found that tolerant mice had substantially reduced IFN- and IL-4 production, consistent with either of these mechanisms. This is consistent with either a deletion or anergy mechanism. These results also suggest that tolerance is not the result of an immune deviation (9), as both IFN- and IL-4 are inhibited.
FIGURE 6. Cytokine production in tolerant mice. DO11.10 T cells purified from transgenic mice were injected into naive BALB/c mice. Twenty-four hours later, recipient mice received an i.p. injection of 300 μg of OVA323–339 peptide. One month later, mice were immunized s.c. with OVA/CFA. Seven days later, mice spleens and lymph nodes (5 x 106 cells/ml) were harvested and placed in culture with 10 μg/ml OVA323–339 peptide. Supernatants were harvested 48 h later and the amount of IFN- (A) and IL-4 (B) determined by flow cytometry.
We then explored the possibility of deletion in tolerant mice using an in vivo cytotoxicity assay. This assay monitored eradication of an adoptively transferred target population (DO11.10 T cells) in tolerant and nontolerant mice. BALB/c recipient mice were rendered tolerant by injection of 1 x 106 DO11.10 T cells and peptide. As controls, mice were untreated or given DO11.10-lpr cells (plus peptide). Two weeks later, these mice were immunized with OVA/CFA. One week following immunization, mice were infused with fresh DO11.10 T cells labeled with a high concentration of CFSE (target cells, CFSEhigh) and with syngeneic BALB/c CD4+ T cells that were labeled with less CFSE (reference population, CFSElow) at a ratio of 1:1. Eighteen hours later, the remaining DO11.10 cells were analyzed by comparing their numbers to the reference population. Fig. 7A shows that when CFSEhigh and CFSElow cells were infused into immune mice, DO11.10 T cells were recovered in similar numbers to the reference population (0% killing). Similarly, when CFSE-labeled cells were given to mice that had not deleted the DO11.10 T cells (DO11.10-lpr), no killing was observed (0% killing). However, when labeled cells were given to tolerant mice (that had previously undergone peptide-induced deletion), the CFSEhigh DO11.10 T cells were rapidly eliminated (69% killing). Thus, peptide-induced deletion activates a mechanism that eliminates (or kills) the infused DO11.10 T cell population.
FIGURE 7. In vivo cytotoxicity of DO11.10 T cells. DO11.10 or DO11.10-lpr cells T cells purified from transgenic mice were injected into naive BALB/c mice. Twenty-four hours later, recipient mice received an i.p. injection of 300 μg of OVA323–339 peptide. Two weeks later, recipient mice received 1 x 107 DO11.10 T cells and 1 x 107 CD4+ BALB/c T cells labeled with CFSE. Eighteen hours later, spleens and lymph nodes were harvested and the relative numbers of DO11.10 (CFSEhigh) and CD4+ T cells (CFSElow) were determined by flow cytometry. Numbers were compared with unimmunized mice (n = 5). Percent killing shown on the graph represents the average of mice in that group; immune control (0%) (n = 5), DO11.10-lpr (0%) (n = 7), DO11.10 (69 ± 4%) (n = 12).
The cell type responsible for killing was explored in Fig. 8. T cells from spleens and lymph nodes of tolerant BALB/c mice were separated into CD4+ and CD8+ fractions. These cells were then injected into naive BALB/c mice that were immunized with OVA/CFA. One week later, they were tested for DTH. These data show that the CD8+ (not CD4+ or CD4– CD8–) T cells prevent the response to OVA. Thus, tolerance in this system is mediated by a CD8+ cytotoxic T cell.
FIGURE 8. Adoptive transfer of tolerance. BALB/c mice were rendered tolerant by injection of DO11.10 T cells and 300 μg of OVA323–339 peptide. Two weeks later, mice were immunized s.c. with OVA/CFA. One week later, spleen and lymph nodes were removed, separated into CD4+ and CD8+ T cells and these cells were transferred to naive BALB/c mice. Recipient mice were immunized s.c. with OVA/CFA, and seven days later, mice were challenged in the right footpad with 100 μg of OVA and in the left footpad with PBS. Measurements were taken 24 h later using an engineer’s micrometer. Values are expressed in mircometers (± SE) and represent the difference between the right footpad (Ag challenge) and the left footpad (PBS challenge). Bkg represents the response of naive mice challenges with Ag. *, Significantly different from the immune control.
Discussion
Apoptosis and tolerance are biological responses clearly linked to the control of the peripheral immune response (24). Apoptosis of tissue cells preserves organ homeostasis by transducing tolerogenic signals though the dendritic cells and the cross-priming pathway to prevent autoimmunity (25, 26). Apoptosis in the immune system promotes peripheral deletion and the removal of dangerous autoreactive T cells (27, 28) by using cross-priming to actively silence responsive clones (29). Studies with peptide-induced and superantigen-induced deletion of T cells have helped us understand the mechanisms of peripheral deletion and its importance to the induction of tolerance. Our results presented here extend current paradigms of peripheral tolerance demonstrating that following deletion of Ag-reactive T cells there may be an additional level of control put into play when relatively large numbers of peripheral T cells delete by apoptosis. We demonstrate that deletion of TCR-transgenic T cells via the Fas/FasL pathway leads to the establishment of a state of active tolerance. This tolerance extends to naive T cells of the same specificity and is mediated by activation of CD8+ T cells that kill their T cell targets. Our data suggest that this active immunoregulation is directed toward the Ag-reactive cells for the purpose of controlling any self responses that might be generated when large numbers of T cells die and release potentially dangerous autoantigens and cytokines. This contrasts apoptosis that occurs during normal turnover of T cells where tolerance is not established following deletion, allowing the progression to a productive immune response.
The role of apoptosis, specifically the apoptotic cell, in the induction of tolerance has recently been the subject of much study. Interestingly, tolerance induced by apoptotic cells can have positive and negative effects on immunity. For example, massive apoptosis during parasitic (16) and bacterial (15) infections can enhance pathology during these diseases. In experimental trypanosome infection, apoptotic lymphocytes fuel parasite growth. These studies revealed that an interaction between apoptotic T cells (generated during the infection) and macrophages promoted an anti-inflammatory response. Interestingly, necrotic cells did not have this function. Similar results were observed in an experimental model of bacterial sepsis. In this study, sepsis-induced lymphocyte apoptosis decreased the chances of survival for the infected host. If apoptosis was prevented with caspase inhibitors, or if T cell apoptosis was prevented by a Bcl-2 transgene, survival was significantly improved. Thus, during these infections it would seem that all the danger signals elicited by the infection are overcome by the presence of apoptotic lymphocytes.
In contrast to these findings, apoptosis that occurs during the experimental induction of graft tolerance can have positive consequences for the organ and tissue homeostasis. It was recently demonstrated that long-lasting graft tolerance could be induced with an immunosuppressive regimen consisting of combined CD40 and B7 blockade coupled with rapamycin treatment (14). Interestingly, this tolerance required T cell apoptosis as overexpression of Bcl-XL in the T cells blocked the effect. Tolerance was also not established whether rapamycin (which facilitates the apoptotic signal process in T cells) was replaced with cyclosporin A (which prevents apoptosis in T cells). The long-term nature of this tolerance could not be explained by simple deletion, suggesting that tolerance is infectious and mediated by regulatory cells. Perhaps the tolerance observed in that system is directed toward reactive T cells, as we have observed in this study, rather than to a specific Ag.
Immune privilege sites also use Fas/FasL-induced apoptosis to promote tolerance to control inflammatory responses. In the eye, constitutive FasL expression induces apoptosis of invading lymphoid cells (30) leading to systemic tolerance (17) mediated by regulatory T cells. Both deletion and tolerance protect vision from the damaging consequences of inflammation.
The specificity of the CD8+ T cell that mediates tolerance in the present system appears to be the T cell rather than the specific Ag. We know this because the tolerance (cytotoxicity) is directed toward the TCR-transgenic T cells and it can be induced with apoptotic T cells in the absence of Ag. These results are similar to those observed in systems studying T cell vaccination as prevention for autoimmune disease (31, 32, 33). In these models, regulation is directed toward the TCR rather than the specific Ag. Other reports show that T cell-derived peptides are the target of the regulatory cells (33, 34, 35), suggesting an anti-Id response directed toward the TCR of DO11.10 might be responsible. Studies demonstrating the induction of suppressor T cells following staphylococcal enterotoxin B-induced deletion of V8+ T cells also support this idea (36), however, further study will be required. In addition, Fas/FasL-mediated deletion of the class I-restricted CD8+ T cell (4) in response to OVA expressed in the pancreas has been observed. Whether this leads to an active tolerance directed toward the T cell is not known.
We do not yet know the mechanism by which the CD8+ T cells kill their targets in vivo. Possibilities included FasL or the perforin/granzyme (37) system. CD8+ cytotoxic lymphocytes, NK cells, and lymphokine-activated killer cells depend primarily on the perforin/granzyme system to kill their targets, while CD4+ T cells use Fas and other mechanisms to induce cell death. We believe that the FasL pathway is not involved in the present system for three reasons: 1) When trinitrophenyl-coupled (TNP)-spl are injected into the anterior chamber of the eye, tolerance develops because the spleen cells undergo FasL-mediated apoptosis. FasL-defective gld mice do not develop tolerance, unless the TNP-spl are induced to undergo apoptosis before they are injected (17). 2) Tolerance following i.v injection of TNP-spl also depends on Fas/FasL-mediated apoptosis. Again, gld mice cannot be tolerized, unless the TNP-spl are irradiated before injection (10). 3) Irradiated DO11.10 cells can induce tolerance in C.B6-gld mice (see Fig. 5B). However, because we have not formally ruled out any mechanisms for CD8+ killer cell activity, this will require further study.
Data presented in this paper suggest that apoptosis of a clonal population of T cells mediated by Fas/FasL can lead to a state of tolerance. Tolerance induction requires the death of a relatively large number of cells, suggesting it does not occur during the activation of the adaptive immune response. The tolerance, mediated by cytotoxic CD8+ killer cells, is specifically directed toward the T cell clone. We propose that this mechanism may be an adaptation to control potential antiself responses that could occur when large numbers of T cells die by apoptosis and their remnants enter the Ag-processing pathways. Our system examines the results of eliminating a large number of CD4+-transgenic T cells. We do not know whether similar results are obtained with CD8+ CTLs, as it has been shown that apoptosis of virus-specific cells can lead to memory (38). Consequently, the relationship between our finding and other naturally occurring tolerance pathways will require further study.
Disclosures
The authors have no financial conflict of interest.
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 National Institutes of Health Grants EY12826 (to T.A.F.), EY06765 (to T.A.F.), and EY12707 (to P.M.S.), the Department of Ophthalmology and Visual Sciences Core Grant (EY08972), and an Arthritis Foundation Biomedical Science Grant (to P.M.S.). Support was also received from The Foundation for Fighting Blindness and from Research to Prevent Blindness.
2 Address correspondence and reprint requests to Dr. Thomas A. Ferguson, Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110. E-mail address: Ferguson@vision.wustl.edu
3 Abbreviations used in this paper: -gal, -galactosidase; DTH, delayed-type hypersensitivity; Grp, group; TNP, trinitrophenyl.
Received for publication December 20, 2004. Accepted for publication January 21, 2005.
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