Effector T cells have a lower ligand affinity threshold for activation than naive T cells

Kazuhiko Kimachi1, Katsuji Sugie2 and Howard M. Grey2

1 The Chemo-Sero Therapeutic Research Institute, Kikuchi Laboratory, Kawabe Kyokushi Kikuchi, Kumamoto 869-1298, Japan 2 Division of Immunochemistry, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA

Correspondence to: K. Kimachi; E-mail: kimachi{at}kaketsuken.or.jp
Transmitting editor: D. R. Green


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
It has been previously established that effector and memory T cells are more sensitive to antigen stimulation than naive T cells. In this study, we compared the effect of ligand affinity on the activation of naive and effector T cells derived from pigeon cytochrome c (PCC)-specific TCR transgenic mice by stimulating these cells with a variety of ligands with widely differing antigenicity. The data obtained indicated the following. (i) The differences in antigen dose requirements for activation of naive and effector cells widened as the affinity of the antigen decreased. Most dramatically, peptides that were TCR antagonists for naive T cells were recognized as agonists by effector T cells. (ii) While both naive and effector T cells were activated by the bacterial superantigen staphylococcal enterotoxin A, specific for the transgenic TCR Vß3 chain, effector, but not naive, T cells were stimulated to proliferate by toxic shock syndrome toxin-1, a superantigen not previously described to be stimulatory for Vß3 T cells. (iii) Effector T cells, but not naive cells, proliferated in response to endogenous self-peptides presented by antigen-presenting cells in a syngeneic mixed lymphocyte reaction. Taken together these data indicate that effector T cells have a lower affinity threshold for activation than naive T cells. Further studies demonstrated that the heightened reactivity of effector T cells to low-affinity ligands declined progressively with repeated stimulations by antigen such that after repeated stimulation effector T cells were no longer stimulated by low-affinity ligands but recognized them as TCR antagonists similar to naive T cells.

Keywords: cellular differentiation, TCR antagonist


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
Naive T cells, once activated by antigen, proliferate and differentiate into effector and/or memory T cells that differ from naive cells with respect to faster kinetics of the response (1,2), a decreased dependency on co-stimulation (3,4) and the ability to carry out effector functions (58). It has also been shown that effector and memory cells are more sensitive to antigen than naive cells in that they can be activated by lower concentrations of antigen than that required for the activation of naive cells (912). In a previous study, we determined the number of antigen–MHC complexes required for the activation of naive and recently primed effector T cells. It was found that ~40 antigen–MHC complexes per antigen-presenting cell (APC) were sufficient to stimulate a proliferative response from effector cells while naive T cells required ~400 complexes per APC. This increased sensitivity of effector T cells to antigen was correlated with an enhanced ability to express IL-2 and IL-2 receptors at low antigen concentrations (12). Left unexplored by this and other studies that explored the function of naive and effector T cells was the question of whether naive and effector T cells also differ with respect to the affinity of interaction with ligand that is required to achieve activation. Previous studies that suggest that qualitative differences in ligand recognition of naive and previously primed T cells may exist come from studies of virus-infected animals in which it was shown that CD8 memory T cells generated by one viral infection acquired the ability to react to heterologous viruses (13,14), mutated strains of virus (15,16) and alloantigens (17,18). Such studies are suggestive that previously primed cells may have a lower threshold of activation such that they can respond to altered peptide ligands (APL) that are not recognized by naive T cells. However, a major problem in interpretation of these types of studies is that they were conducted with polyclonal T cell populations and therefore it is possible that the results reflected changes in T cell repertoire, rather than a lowering of the affinity threshold for activation of effector and memory T cells at clonal level.

In this study we have further analyzed how naive and effector T cells differ with respect to the ligand affinity required for activation. For this purpose naive and effector T cells were derived from pigeon cytochrome c (PCC)-specific TCR transgenic mice that expressed a single homogeneous TCR. As antigen, we used a panel of APL that varied in affinity as indicated by the differences in the concentration required to activate T cell clones. Included in the panel were non-stimulatory, TCR antagonists. We have also compared the ability of naive and effector T cells to be stimulated by different superantigens and to endogenous self-peptides as a further measure of their ability to respond to low-affinity ligands. Our results indicate that effector T cells are capable of responding to lower-affinity ligands than are naive cells, but this capacity to recognize low-affinity ligands is relatively transient and is lost upon repeated stimulation with antigen.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
Mice
AD10 TCR transgenic mice on an H-2a (B10.A) background were produced as described elsewhere (19) and bred in our animal facility. All mice were used at 2–6 months of age.

Antigens and antibodies
Peptide PCC 88–104, moth cytochrome c (MCC) 88–103 and analog peptides were synthesized, purified and analyzed as previously described (20). PCC protein was purchased from Sigma (St Louis, MO). Staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST)-1 were purchased from Toxin Technology (Sarasota, FL). mAb to mouse cell-surface antigens that were used in flow cytometry included FITC-conjugated anti-CD62L (MEL-14), phycoerythrin-conjugated anti-Vß3 (KJ25) and biotinylated anti-V{alpha}11 (RR8-1). Anti-CD4 (RM4-5), CD8a (63-6.7), I-Ek (14-4-4s), I-Ak (11-5.2) and Kk (36-7-5) were used as inhibitory antibodies in the mixed lymphocyte reaction (MLR) assay. Biotinylated anti-I-Ak (11.5.2), I-Ek (14-4-4s), CD8a (63-6.7), CD11b (M1/70), CD62L (MEL-14) were used for purification of naive T cells.

Cell fractionation and generation of effector T cells
To purify naive CD4+ T cells from AD10 transgenic mice, lymph nodes and spleens were isolated and single-cell suspensions were prepared in Click’s medium (Irvine Scientific, Santa Ana, CA), supplemented with 2% FCS. Cells were passed over nylon wool columns, incubated with anti-CD8, anti-Mac-1, anti-I-Ek, anti-I-Ak and anti-mouse Ig biotinylated antibodies, followed by incubation with streptavidin-conjugated magnetic beads (MACS; Miltenyi Biotec, Auburn, CA). The purified CD4+ T cells obtained from this procedure were then enriched for naive cells by positive selection using biotinylated anti-CD62L antibody and streptavidin-conjugated magnetic beads. Naive CD4+ T cells purified in this manner were >95% CD4+ and <1% CD44high. For the generation of effector T cells, 1 x 106 naive T cells were incubated with 1 x 107 irradiated (2000 rad) B10.A splenocytes in the presence of 30 µg/ml PCC protein for 4 days. They were then split 1:8 in IL-2 (20 U/ml)-containing Click’s medium with 10% FCS and cultured for an additional 8–10 days to allow the cells to return to a resting state. Effector T cells were further stimulated every 14 days with PCC in the same manner to generate re-stimulated effector T cells.

T cell stimulation assay
CH27 B lymphoma cells (5 x 106/ml) were pulsed with PCC or APL at 37°C for 3 h followed by washing. Mitomycin C (100 µg/ml; Sigma) was added during the last hour of incubation. In some experiments, lipopolysaccharide (LPS) blast cells that were prepared by stimulating T cell-depleted B10.A spleen cells with 25 µg/ml of LPS and dextran sulfate (Sigma, St Louis, MO) for 3 days were used as APC. Naive and effector T cells (2.5 x 104) were incubated in triplicate with peptide-pulsed APC (2.5 x 104) in round-bottom 96-well plates in 0.2 ml. Proliferation was measured by incorporation of [3H]thymidine (1 µCi/well) between 48 and 66 h. For TCR antagonist assays, APC were pre-pulsed with 0.5 µg/ml PCC and treated with mitomycin C. T cells and APC were mixed, and soluble K99R peptide was added at various concentrations. In the autologous MLR assay, naive and effector T cells (2 x 105) were cultured with irradiated (2000 rad) T-depleted B10.A spleen cells (2 x 106) in RPMI 1640:DMEM:F12 (2:1:1) medium supplemented with 10 µg/ml transferrin, 5 µg/ml insulin, 10 nM selenious acid, 25 µM monoethanolamine and 1% normal mouse serum. Proliferation was measured by incorporation of [3H] thymidine (1 µCi/well) between 24 and 42 h. Antibodies used for inhibition were added at 2 µg/ml at the start of the culture.

Conjugate formation assay
For conjugate formation, CH27 were membrane stained with lipophilic green fluorochrome DiOC18 and T cells with red fluorochrome DilC18. CH27 were pulsed with peptides, distributed in round-bottom plates (2 x 105 cells/well) and mixed with T cells at a 1:1 ratio. After 30 min of incubation at 37°C, cells were vigorously pipetted to disrupt non-specific conjugates and immediately analyzed on a flow cytometer (FACScan; Becton Dickinson, San Jose, CA), and the percentage of total APC that formed conjugates with T cells was calculated.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
Differences in responsiveness between naive and effector T cells increase as ligand affinity decreases
The purpose of this study was to compare the activation threshold of naive and antigen-stimulated effector T cells. Several studies have been previously performed that focused on antigen dose (ligand density on APC) requirements for stimulating naive and previously primed T cells, but few have investigated the capacity of these cell types to respond to ligands of different affinity for the TCR. To this end we compared effector and naive T cells for their capacity to respond to APL of cytochrome c that had similar binding affinity for I-Ek but that varied in their affinity for the AD10 TCR as defined by the amount of peptide required to stimulate a proliferative response (Table 1). Antigenicity was expressed as the relative stimulatory capacity of each peptide compared with the cognate PCC peptide when assayed on the AD10 T cell clone that was the origin of the TCR transgenic mice used in this study. Included in the panel were two APL, T102G and K99R, that were TCR antagonists as determined by their inability to stimulate a proliferative response and their capacity to inhibit the proliferation induced by an agonist peptide (21).


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Table 1. Antigenicity of peptides
 
Naive and primary effector T cells (obtained 10–14 days after in vitro stimulation with antigen-pulsed APC) were compared for their capacity to proliferate to the different APL (Fig. 1). Both T cell populations proliferated vigorously in response to the strong antigenic peptides, PCC and MCC, although ~10-fold more antigen was required to stimulate naive T cells to the same level as effector cells. The difference in antigen dose requirements for naive and effector T cells widened when peptides with weaker antigenicity were studied (Fig. 1, middle panel). Naive T cells required at least 100-fold more of these lower-affinity antigens to achieve the same proliferative response as effector T cells. Finally, and most strikingly, was the data obtained with the two TCR antagonists, K99R and T102G (Fig. 1, lower panel). Effector T cells recognized both peptides as agonists by mounting a significant proliferative response, while, as expected, naive T cells did not respond to the TCR antagonists even at the highest concentration tested (500 µg/ml).



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Fig. 1. Proliferation of naive and effector T cells in response to cytochrome c peptides and APL. Naive (open symbols) and effector T cells (closed symbols) were stimulated with CH27 APC pulsed with a variety of cytochrome c APL of differing affinity. DNA synthesis was measured during the last 18 h of a 3-day culture. With decreasing affinity of peptides for the TCR, the enhanced ability of effector T cells to respond relative to naive T cells markedly increased. The results are representative of five independent experiments performed.

 
This set of peptides was also analyzed for their capacity to stimulate expression of the early marker of activation, CD69, with similar results. Naive and effector cells responded similarly to MCC and PCC with naive cells requiring ~3-fold more antigen to induce CD69 expression on 50% of the T cells. This difference widened to 10- to 100-fold for the group of antigens with weak stimulatory capacity and with the TCR antagonists, significant induction of CD69 expression was only observed with effector cells (data not shown).

We also compared the sensitivity of naive and effector T cells to stimulation by ligands of different affinity with respect to the physical interactions between T cells and APC that lead to the formation of stable conjugates between these two cell types. Maximum numbers of conjugates were formed with 1–10 µg of PCC peptide, while 100 µg of the TCR antagonist was needed in order to induce significant numbers of conjugates. As shown in Fig. 2, while both naive and effector cells formed similar numbers of conjugates to the cognate peptide, PCC, only effector cells were able to form stable conjugates with the TCR antagonist, T102G.



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Fig. 2. Conjugate formation of naive and effector T cells induced by PCC and a low-affinity TCR antagonist peptide. Antigen-dependent conjugate formation was measured by flow cytometry. CH27 cells were stained with green dye, then pulsed with PCC 88–104 (at 10 µg/ml) or the APL T102G (at 100 µg/ml) for 2 h and incubated with naive or effector T cells pre-stained with red dye (T:APC = 1:1). After 30 min incubation, the conjugates of T–APC were detected as double-positive cell conjugates. The results are from one representative experiment of three that were performed.

 
V{alpha} expression in naive and effector T cells
Since the AD10 transgenic mice used in this study were not bred on a RAG-deficient background, there was the possibility that TCRs on some cells that expressed endogenous V{alpha} chains had a higher affinity for the TCR antagonists K99R and T102G than those that expressed only the V{alpha}11 TCR transgene. These endogenous V{alpha}-expressing T cells, if selectively expanded in the course of generating effector cells, could have contributed to the proliferative response obtained following stimulation with the TCR antagonists. To examine this possibility, we analyzed the expression of the transgenic TCR chains, V{alpha}11 and Vß3, on naive and effector T cells (Table 2). CD62L+ naive T cells contained ~10–15% of V{alpha}11low cells that presumably co-expressed unidentified endogenous V{alpha} chains. In contrast, TCRs on effector cells were more homogeneous, expressing very few V{alpha}11low cells. These data indicate that cells expressing only the TCR V{alpha}11 transgene were selectively expanded during the course of antigen stimulation, making it highly unlikely that the stimulation of effector T cells by TCR antagonist peptides was due to expression of novel endogenous V{alpha} chains which when associated with the Vß3 transgene had a higher affinity for the APL than the original V{alpha}11/Vß3 TCR transgene.


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Table 2. V{alpha}11low cells in naive and effector T cell population
 
Response of naive and effector cells to bacterial superantigens
As an independent test of the capacity of effector T cells to respond to ligands of low affinity we tested naive and effector cells for their ability to respond to two bacterial superantigens, SEA and TSST-1. TSST-1 has been reported to specifically stimulate murine T cells that express Vß15 or Vß16, whereas SEA preferentially stimulates Vß3-expressing T cells (22). The response of naive and effector T cells to these two superantigens is shown in Fig. 3. SEA stimulated both naive and effector cells very well, with naive cells requiring ~10-fold more SEA than effector cells to achieve the same level of stimulation; a similar profile to that observed with the high-affinity PCC or MCC peptides. In contrast, TSST-1 stimulated the Vß3-expressing effector cells very well, albeit requiring microgram quantities to do so, but there was no significant proliferation of naive cells observed at any of the concentrations of TSST-1 tested. These data are consistent with the data obtained with peptide antigens of varying affinities for the AD10 TCR and support the conclusion that effector T cells have a lower affinity threshold than naive cells for stimulation by antigen.



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Fig. 3. Effector, but not naive, T cells are capable of proliferating in response to TSST-1 superantigen. Naive (open circles) and effector T cells (closed circles) were stimulated by SEA (specific for transgenic Vß3; bottom) and TSST-1 (specific for Vß15 and Vß16; top). DNA synthesis was measured during the last 18 h of a 3-day culture. The results are from one representative experiment of two performed.

 
Effector, but not naive, T cells proliferate to irradiated spleen cells in a syngeneic MLR
Since effector T cells proliferated, expressed CD69 and formed stable conjugates in response to TCR antagonists, but naive T cells did not, we also wished to determine if effector T cells were sufficiently sensitive to low-affinity ligands to be able to respond to the endogenous self-antigens presented by syngeneic APC in a MLR assay. Naive and effector T cells were co-cultured with irradiated B10.A spleen cells in the presence of 1% normal B10.A serum to avoid the introduction of foreign antigens by the use of FCS. As shown in Fig. 4, while naive T cells co-cultured with syngenic spleen cells showed no evidence of APC-mediated proliferation, in contrast, effector T cells gave a significant, albeit modest, proliferative response when co-cultured with syngeneic spleen cells. That this response was mediated through the specific interaction between the TCR and class II MHC on the APC was shown by the finding that anti-I-Ek and anti-CD4 antibodies inhibited the response, whereas anti-I-Ak and control antibodies (anti-Kk and anti-CD8) had no effect.



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Fig. 4. Effector, but not naive, T cells proliferate to irradiated spleen cells in a syngeneic MLR. Naive and effector T cells were co-cultured with irradiated B10.A T-depleted spleen cells in the presence of 1% normal B10.A serum and various mAb. Proliferation was measured during the last 18 h of a 2-day incubation. Each antibody was added at a final concentration of 2 µg/ml. The results are from one representative experiment of three performed.

 
Effect of repeated stimulation on the capacity of effector T cells to respond to low-affinity ligands
The APL K99R and T102G were initially defined as TCR antagonists by their ability to inhibit agonist-induced proliferation of the AD10 T cell clone (23,24). Since this clone was maintained by repeated stimulation with antigen and could be considered as an effector T cell, we questioned why K99R and T102G were antagonistic to the AD10 T clone, but acted as weak agonists to primary effector T cells. To explore this apparent discrepancy we decided to determine the effects of repeated rounds of stimulation of T cells on their capacity to respond to low-affinity ligands. For this purpose, naive T cells were stimulated with PCC and irradiated spleen cells every 14 days, thereby generating a panel of T cells that had been stimulated 1, 2, 3 or 4 times by antigen. Figure 5(a and b) shows the results of antigenicity and antagonism assays obtained with these different effector cells. With respect to their responsiveness to the cognate antigen PCC, the heightened responsiveness of primary effector cells declined progressively with subsequent stimulations such that after the third stimulation, the dose–response curve was similar to that of naive cells and after the fourth stimulation, the effector T cells were less responsive than naive T cells. The response to the low-affinity ligand, K99R, was even more dramatically altered by repeated stimulation. Whereas primary effector cells were effectively stimulated, secondary effector cells responded very weakly, and tertiary and quaternary effector cells not at all. This lack of responsiveness of repeatedly stimulated cells coincided with the reversion of K99R to a TCR antagonist by the third round of re-stimulation (Fig. 5b). Thus with respect to both antigen-dose responsiveness and TCR antagonism, repeatedly stimulated effector T cells resembled naive cells in their response profiles.



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Fig. 5. Effect of repeated stimulation on the capacity of effector T cells to respond to low-affinity ligands. (a) Proliferation assay: naive, primary and repeatedly stimulated effector T cells were stimulated by MCC 88–103 or K99R. Proliferation was measured during the final 18 h of a 3-day culture. (b) TCR antagonism assay: T cells were stimulated by APC pre-pulsed with suboptimal amounts of MCC 88–103. K99R peptide was added at various concentrations. K99R inhibited the proliferation of naive and repeatedly stimulated effector T cells, but increased the proliferation of primary effector cells. The results are from one representative experiment of three (a) or two (b) that were performed.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
In this report naive and effector T cells were compared with respect to their ability to be activated by low-affinity ligands for the TCR. The conclusion, supported by findings obtained with three different types of ligands, was that primary effector T cells can be stimulated to proliferate by low-affinity TCR ligands that are incapable of stimulating a proliferative response in naive T cells. First, by stimulating TCR transgenic T cells with a panel of APL of varying affinity for the TCR, it was found that while both naive and effector T cells required increasing amounts of antigen to be activated as the ligand affinity decreased, this effect was much more marked in naive than in primary effector T cells. This was most dramatically illustrated by the capacity of effector T cells to recognize as agonists, peptides that were TCR antagonists for naive T cells. The capacity of effector T cells to be activated by low-affinity ligands that naive T cells were unresponsive to was also demonstrated by the finding that primary effector T cells could be stimulated to proliferate by a superantigen, TSST-1, that was non-stimulatory for naive T cells bearing the same TCR. Finally, primary effector T cells were stimulated by endogenous self-peptides presented by APC in a syngeneic MLR, whereas naive T cells failed to proliferate when incubated with the same APC. Thus, not only do primary effector T cells respond to lower concentrations of agonist ligands than naive T cells (12), they also have a lower affinity threshold for activation, enabling them to respond to ligands of an affinity too low to stimulate a proliferative response in naive T cells. This conclusion is consistent with a recent report that studied the ability of naive and activated TCR transgenic CD8 T cells to respond to a panel of analog peptides derived from a combinatorial peptide library (25). It was found that half of the 20 peptide analogs tested that were non-stimulatory for naive T cells could stimulate IFN-{gamma} production and cytotoxic activity in effector T cells.

In the course of our studies it was also shown that the heightened reactivity of effector T cells to low-affinity ligands was not a stable characteristic of previously primed cells and that the response to low-affinity ligands declined progressively with repeated antigenic stimulations such that after three rounds of stimulation, T cells no longer responded to a TCR antagonist peptide as an agonist, but rather responded in a manner similar to that of naive cells and recognized it as an antagonist. Although we have no ready explanation for these findings, they have provocative implications for the means by which escape mutants are generated during the course of chronic viral infections such as HIV (26) and HBV (27). That is, if the immune system cannot completely clear a virus within the first few weeks following infection, then mutations within immunodominant regions of the virus that lead to lower affinity interactions with a major portion of the virus-specific T cell repertoire may result in the failure of these T cells to mount effector cell responses due to the higher affinity threshold that accompanies repeated stimulation of T cells.

Several possible mechanisms may be invoked to explain the lowered affinity threshold for activation exhibited by primary effector T cells. For instance, it has been reported that the TCR of effector T cells show an increased ability to bind dimer antigen–MHC complexes compared with the same TCR expressed on naive T cells (28). Analysis of the binding reaction led to the conclusion that the avidity of TCR on effector T cells was 20- to 50-fold greater than that of naive T cells. This increased avidity was sensitive to cholesterol depletion, suggesting that TCR on effector cells may be constitutively clustered in lipid rafts making it easier for the two ligand copies in the dimer antigen–MHC molecule to be simultaneously engaged by the TCRs of effector cells than the TCRs of naive cells. Another possible mechanism to explain the lower affinity threshold of effector T cells would be the expression on effector cells of co-stimulatory receptors that were not expressed, expressed at lower density or more slowly expressed on naive T cells. Potential candidates for such molecules include OX-40, 4-1BB and LFA-1 among others (29,30). Of particular interest are recent studies with the integrin-associated protein CD47. An agonist antibody to this molecule not only co-stimulated T cells that had been stimulated with suboptimal concentrations of antigen or anti-CD3, but was also capable of converting TCR antagonist APL into agonists (31). In a similar manner, the expression of CD4 has been shown to preferentially augment the activating capacity of low-affinity ligands (32). If CD4 function or its associated kinase, Lck, was augmented in effector T cells, this could explain our observations. The report that Lck is localized to the plasma membrane to a greater extent in effector and memory cells than in naive T cells is consistent with this possibility (33). A third possible mechanism that could explain the lower ligand affinity threshold of effector cells involves the observations that effector T cells are not as dependent on certain co-stimulatory signaling pathways as naive T cells. If low-affinity TCR ligands are not able to activate these co-stimulatory pathways, this could explain why such ligands may not be able to stimulate naive cells, but are able to stimulate effector cells. The co-stimulation provided by CD28 may fit into this category, since under certain circumstances effector cells have been shown to be relatively independent of CD28 mediated co-stimulation (34,35), and there is also evidence to support the concept that TCR antagonists and weak agonists cannot activate a CD28 co-stimulatory pathway since they fail to induce the localization to the immunologic synapse of CD28 and one of its important down stream effector molecules, protein kinase C{theta} (36,37).

The demonstration in this report that previously stimulated T cells can be activated by low-affinity ligands that are not recognized as antigens by naive T cells may be relevant to the mechanisms of induction of autoimmunity. For instance, we have previously shown that T cell tolerance to an antigen could be broken by immunization with a higher-affinity ligand. As a consequence, some of the effector T cells specific for the high-affinity ligand could be shown to be activated by the tolerized antigen, whereas naive tolerant T cells were unresponsive (38,39). Also, a study of diabetogenic CD8 T cells demonstrated that effector T cells were capable of responding to ligands that their naive counterparts could not (25,40). Both of these studies are consistent with the suggestion that, either by molecular mimicry or bystander activation, T cells with a low-affinity recognition of self-antigen, upon activation, can be re-stimulated by the low-affinity self-ligand and cause autoimmune tissue damage to the organ that expresses the self-antigen.


    Acknowledgments
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
This work was supported in part by a grant from the NIH (AI18634). We are grateful to Ms Nancy Martorana for help in the preparation of the manuscript.


    Abbreviations
 
APC—antigen-presenting cells

APL—altered peptide ligands

LPS—lipopolysaccharide

MCC—moth cytochrome c

MLR—mixed lymphocyte reaction

PCC—pigeon cytochrome c

SEA—staphylococcal enterotoxin A

TSST—toxic shock syndrome toxin


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 

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