Tuning T cell activation threshold and effector function with cross-reactive peptide ligands

Lindsay B. Nicholson, Ana C. Anderson and Vijay K. Kuchroo

Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA

Correspondence to: V. K. Kuchroo


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have generated a panel of cross-reactive T cells by immunizing SJL mice (I-As) with Q144 peptide, an analog of an autoantigenic peptide (W144) of myelin proteolipid protein (PLP) 139–151 (HSLGKWLGHPDKF) in which W was replaced by Q at position 144. Following immunization with Q144, T cells were expanded in vitro with W144, which is a cross-reactive, suboptimal ligand, for Q144-specific T cells. The T cell clones responded to both ligands and grew normally on the peptide W144, but were hyperstimulated when activated by Q144 in vitro. This hyperstimulation results in a heteroclitic proliferative response with secretion of additional cytokines not induced by W144. Thus expansion of T cells by a suboptimal cross-reactive ligand effectively lowers the activation threshold so that the immunizing antigen becomes a hyperstimulating ligand for the clones. Surprisingly, when the T cell clones are grown on the hyperstimulating ligand Q144, some adapt by increasing their activation threshold. This desensitization results in a loss of response to a number of cross-reactive ligands and the appearance of a more specific T cell response. Long-term culture with the hyperstimulating ligand is sometimes associated with down-regulation of CD4 expression. These results provide an explanation for the common finding of T cell heteroclicity, and suggest that although the specificity and hierarchy of the response of T cells to peptides is determined by the TCR, activation threshold and effector functions are modified by exposure to cross-reactive ligands. This observation has implications for the development and regulation of autoimmune disease.

Keywords: superagonists, T cell tuning, TCR cross-reactivity


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The specificity of the T cell-dependent adaptive immune response is conferred by the {alpha} and ß chains of the TCR, which interact with peptides in the context of class I or II MHC molecules (1). Despite its functional specificity, it is apparent that the TCR can cross-react with different peptides presented in the context of the appropriate restriction element (2,3). In comparison to the cognate ligand, the relative response to these different cross-reactive ligands can vary greatly. Some ligands activate the responding T cell as well as the cognate ligand, others can hyperstimulate (46), hypostimulate, antagonize or anergize T cells (711). These different outcomes are probably dictated by the strength and length of binding of the peptide–MHC complex to the TCR and/or by the level of TCR occupancy, although the actual molecular mechanisms responsible for these various outcomes have not been clearly defined (1215). Cross-reactive interactions may be particularly significant in the context of autoimmune disease, when T cells which respond to a foreign determinant may also cross-react with self-antigen. The importance of such cross-reactive interactions is highlighted by studies performed using analogs of autoantigens which may both prevent (16,17) or promote autoimmune disease (18,19) in model systems.

In an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis, which is induced with a peptide derived from myelin proteolipid protein (PLP) 139–151 (referred to here as W144) (20), CD4+, Th1-type T cells are sufficient to induce disease. On the other hand, immunization with an analog of this peptide, Q144 (a tryptophan to glutamine substitution at position 144), induces non-pathogenic cross-reactive T cells which respond to W144 by producing Th2/Th0 cytokines (16). Therefore PLP 139–151-reactive T cells, derived following exposure to different cross-reactive immunogens (W144 versus Q144), have fundamentally different effects on the outcome of an autoimmune disease process. One of the important properties of these protective cells is believed to be their Th2/Th0 phenotype and we therefore wanted to study the cytokine responses of such cross-reactive cells in more detail. Differentiated T cell clones are known to have quite stable phenotypes when they are maintained in culture by re-stimulation with the immunizing ligand (21). However, the characteristics of T cell clones maintained on cross-reactive ligands are less well explored. We wondered whether expanding T cells with different cross-reactive ligands might affect activation threshold and effector functions of T cells leading to adaptation of the immune response.

Adaptation could occur by at least two non-exclusive mechanisms. Heterogeneity in the responding population might allow the differential selection and expansion of a minor component of that population. Alternatively, cells could show changes in their activation thresholds as a result of tuning signaling or surface molecules. The theoretical implications of such a model have been discussed in the context of the tunable activation thresholds model of cell activation both for peripheral lymphocytes (22) and also thymocytes (23), and evidence for a role for tuning of thymocytes has been described recently (24), but in the periphery such models have not been substantiated by experimental data. To investigate some of these possibilities we generated cross-reactive T cells by immunizing SJL (H-2s) mice with Q144 peptide, but expanding and maintaining the cells with the cross-reactive peptide W144. We show here that the responding T cell clones can be activated by both W144 and Q144, but that Q144 hyperstimulates these T cells, inducing heteroclitic proliferative responses and the secretion of new cytokines not seen with W144. Furthermore such cells can tune their growth and cytokine production, when expanded by the hyperstimulatory ligand, by modulating activation thresholds.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Female SJL mice (4–6 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen-free conditions. They were maintained in accordance with the guidelines of the Committee on Animals of Harvard Medical School.

Antigens
Peptide antigens with C-terminal amides were synthesized by Dr Richard Laursen (Boston University) and by Quality Controlled Biochemicals (Hopkinton, MA). Most peptides were >90% pure, as determined by HPLC, and were not purified further. The peptides used in these experiments were W144 (PLP 139–151) (HSLGKWLGHPDKF), Q144 (HSLGKQLGHPDKF), A144 (HSLGKALGHPDKF), S144 (HSLGKSLGHPDKF), L144 (HSLGKLLGHPDKF) and Neuraminidase 101–120 (EALVRQGLAKVAYVYKPNNT). The relative binding of these peptides to I-As compared with W144 is 0.6 (A144), 0.8 (L144), 0.6 (Q144), 0.8 (S144) and 1.3 (Neuraminidase 101–120).

Generation of T cells clones
Mice were injected s.c. at five sites with 100 µg of Q144 peptide antigen emulsified in complete Freund's adjuvant (Difco, Detroit, MI) containing a total of 250 µg Mycobacterium tuberculosis H37 RA. On day 10 lymph nodes were removed and lymph node cells (LNC) prepared from them. LNC (5x106/well) were cultured in 24-well round-bottom plates (Falcon; Becton Dickinson, Lincoln Park, NJ) for 5 days, in the presence of cross-reactive antigen (W144; 20 µg/ml). T cell blasts were purified over a Ficoll-Hypaque gradient, and fed with culture medium containing 0.6% T cell growth factor (T-Stim; Collaborative Biomedical Research, Bedford, MA) and 0.06% recombinant IL-2. Cells were fed every 2–3 days and re-stimulated every 10–18 days with antigen (20 µg/ml) plus irradiated syngeneic spleen cells (5x106 cells/ml) as a source of antigen-presenting cells (APC). T cells were cloned at limiting dilution in round-bottom 96-well tissue culture plates after two and three rounds of stimulation. Clones were expanded to provide cells for in vitro assays.

To expand clones on different ligands, equal numbers of established QW clones were activated with identical numbers of spleen cells and equal concentrations of either Q144 or W144 peptide. Activation of these two lines was always carried out with identical numbers of APC, identical amounts of antigen and at the same time. Proliferation assays were carried out using the same cell numbers and ELISAs were performed with all samples from the same clone, whether grown on Q144 or W144, measured on the same plate.

In vitro proliferation assays and cytokine assays
Rested T cell clones (1–5x104 cells/well) were activated with irradiated (3000 rad) syngeneic splenic APC (5x105/well) and peptide antigens. Proliferation was assessed by pulsing the cells with [3H-methyl]thymidine 1 µC/well 48 h after activation. The cells were harvested 18 h later and the incorporated radioactivity measured in triplicate wells in a Beckman scintillation counter (model LS 5000; Beckman Instruments, Fullerton, CA). Supernatants were collected 40 h after activation and diluted 1:2, then cytokine concentrations were measured by specific capture ELISA according to the manufacturer's instructions (PharMingen, San Diego CA) as previously described (16).

FACS analysis of surface markers
Cells from clones activated with either W144 or Q144 were stained with directly conjugated antibodies specific for the cell surface molecules CD4 (clone RM4-5), CD28 (clone 37.51) or TCR (clone H57-597) (PharMingen). Staining was carried out at saturating concentrations of antibody and with an appropriate matched isotype control for each antibody. Samples were analyzed on a FACSort, calibrated with appropriately stained control T cell clones, using CellQuest software (Becton Dickinson, San Jose, CA).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Proliferative responses of cross-reactive T cell clones
To generate cross-reactive T cells and investigate the impact of cross-reactive ligands on T cell effector functions (proliferation and cytokine production), we immunized SJL mice with Q144 peptide. The lymph node T cells were activated and expanded in culture by the cross-reactive ligand W144 which generally acts as a suboptimal ligand for the Q144-specific T cell repertoire. The T cells were maintained by activation with W144 and cloned at limiting dilution after two to three rounds of re-stimulation in vitro. Therefore, except for the initial in vivo immunization with Q144, the clones were always maintained by re-stimulation with W144. They have been successfully maintained in culture for >1 year using this regimen, and are described as QW clones. When the opposite strategy, immunization with W144 and expansion with Q144 was tried, we were unable to clone T cells for functional analysis.

The clones were all CD4+ and drawn from a diverse population of precursor T cells as judged by their Vß usage (data not shown). All the clones responded to Q144 and W144, which suggests that W144 is expanding T cells induced by Q144, rather than by primary in vitro immunization or by expansion of a high-frequency W144-responsive, memory T cell population. We examined the proliferative response of the clones over a range of antigen (W144 and Q144) concentrations. For all but one T cell clone (14 out of 15), Q144 induced a stronger (heteroclitic) proliferative response when compared to W144 and of these cells we could detect additional Th1 cytokines from eight out of 14 cells. The difference in the proliferative responses between W144 and Q144 varied from clone to clone but the lower responses to W144 did not impair the ability of the cells to grow successfully in culture with this ligand. The proliferative response of four representative cross-reactive clones to W144 or Q144 ligands is shown in Fig. 1Go.



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Fig. 1. Q144 is a heteroclitic ligand for cross-reactive Q144/W144 T cell clones maintained with W144. The T cell clones derived from mice immunized with Q144 but re-stimulated in vitro with W144 (circles) show heteroclitic responses to Q144 (squares). The relative difference in half-maximal proliferation ranges from 2 to 4 log. The results are representative of four experiments.

 
Phenotype of cross-reactive T cell clones
We analyzed the phenotype of the clones based on the pattern of cytokines they secreted following activation with W144 and with Q144. All 15 clones produced Th2 cytokines (IL-4 and/or IL-10); of these clones, five out of 15 clones also produced low levels of Th1 cytokines (e.g. IFN-{gamma}) upon activation with W144 peptide. Since W144 is a suboptimal ligand for these Q144-induced T cell clones, this suggests that in this system suboptimal ligands favor Th2 cytokine production. When we activated the same clones with Q144 we noted that in many cases, in addition to Th2 cytokines, we could also detect Th1 cytokines (IFN-{gamma}, tumor necrosis factor-{alpha} or IL-2); in many cases we did not detect these cytokines when the clones were activated with W144 even at high antigen concentrations. We did not find clones in which the induction of Th1 cytokines by Q144 (or other hyperstimulating ligands) was associated with reduction in their Th2 cytokine production, although some clones showed a lower proliferation in response to the hyperstimulating ligand, probably due to high dose suppression. Data on the cytokine secretion of eight clones is shown in Table 1Go.


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Table 1. Cross-reactive clones responding to Q144 and W144 secrete more Th1 cytokine when activated with Q144
 
This pattern of response is analogous to responses to superagonist peptide ligands that we and others have previously defined (4,6), in that, for clones maintained by re-stimulation with the peptide W144, activation with Q144 elicits a heteroclitic proliferative response and the secretion of cytokines is not detected on activation with the cognate ligand. However, in comparison with the superagonists described previously, where the superagonist ligands were identified by screening analogs of the activating peptide, this series of cross-reactive clones begins to define the basis of heteroclicity and hierarchical relationships among various cross-reactive T cell ligands. Therefore expansion of Q144-specific T cells with the suboptimal ligand W144 in vitro results in the generation of T cells for which W144 acts as an optimal ligand and Q144 acts as a `superagonist ligand'. These results show that expansion by a cross-reactive ligand affects the subsequent T cell response to the immunizing antigen. Immunization with Q144 and expansion with Q144 induces T cells that show a maximal proliferative response at 10–20 µM antigen and produce Th2/Th0 cytokines upon activation in vitro (16). On the other hand, expansion of Q144-specific T cells by a suboptimal cross-reactive ligand, W144, results in a cross-reactive population of T cells with a lower threshold (2–4 log) of T cell activation in response to Q144.

Expansion with hyperstimulatory ligand alters the activation threshold and the effector phenotype
We next investigated the effect of expanding QW T cell clones in vitro with the hyperstimulatory ligand Q144 compared with the same cells activated with W144. We considered several possibilities. First, that clones might be hyperstimulated by activation with Q144 and not expand because of activation induced cell death. Second, that there may be no detectable difference between clones grown on either ligand in terms of activation thresholds and cytokine production, since the phenotype of the T cell has been fixed. Third, that the clones might grow on either ligand but their phenotype would change (adaptation). To determine the effect of expanding Q144/W144 cross-reactive clones with Q144 or W144 directly, we took equal numbers of cloned T cells and expanded them under identical tissue culture conditions by re-stimulation with either W144 or Q144.

Four T cell clones were split and maintained on Q144 or W144 in parallel. After at least three rounds of re-stimulation we determined their phenotype. Two of the clones (QW.1D4 and QW.6D10) had very similar dose–response curves when either ligand was used to maintain them in culture (Fig. 2aGo). In contrast, two other clones had shifted their dose response to Q144 by ~2 log (QW.6F2) or 3 log (QW.7G1) (Fig. 2bGo). As a result their sensitivity had decreased, since they required higher concentrations of Q144 to achieve half maximal proliferation. To see if this shift in dose response had affected the response to other cross reactive ligands, we compared the activation of QW.6F2 and QW.7G1 by other ligands (Fig. 2cGo). The dose–response curves with these ligands were also shifted to the right and QW.7G1, which normally responds to A144 and L144, had essentially lost the ability to respond to these two ligands. Therefore the effect of growing QW.7G1 on Q144 is to both decrease the sensitivity of the T cell clone and at the same time increase its specificity (Fig. 2cGo).



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Fig. 2. Some Q144/W144 T cell clones adapt when they are maintained by re-stimulation with Q144. Equal numbers of QW T cell clones which had been cultured with W144 (closed symbols) or Q144 (open symbols) were activated with either Q144 (squares) or W144 (circles). Chronic re-stimulation had no effect on the proliferative response of some of the clones QW.1D4 and QW.6D10 (a); however, with other clones, QW.6F2 and QW.7G1, there was a shift in the dose–response curves to W144 and Q144 (b). The changes in dose response affected all other cross-reactive peptides (A144, diamonds; L144, triangles; S144, inverted triangles) and led, in some cases, to the complete loss of response to some ligands (c). The results are representative of three experiments.

 
We also analyzed the impact of growing the T cell clones in the presence of Q144 or W144 cross-reactive ligands on cytokine production. We found that the pattern of cytokines secreted by clones grown with the different cross-reactive ligands was altered depending upon whether the clone was expanded with W144 or Q144. IL-4 and IL-10 were relatively insensitive, in that whether they were expanded by Q144 or W144, IL-4 maintained the same response in three out of four clones and IL-10 showed differences in response in two or three clones. In contrast, Th1 cytokines were much more sensitive and we saw significant changes in IFN-{gamma} or IL-2 production when clones were maintained with the hyperstimulating ligand. The phenotype of QW.7G1 was changed completely from producing IL-4, IL-10, IFN-{gamma} and IL-2 to a Th2 clone producing IL-4 and IL-10 when the clone was expanded in the presence of Q144 (Fig. 3Go). This is consistent with a situation in which clones grown on either ligand can reach the threshold for Th2 cytokine stimulation, but only clones grown on W144, which are more sensitive, can reach the threshold necessary for Th1 cytokine secretion. To assess the growth of cells following activation with Q144 and W144, we took cells activated with either W144 or Q144 and compared cell counts for 9 days following activation. There were no significant differences between the two ligands (data not shown).



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Fig. 3. Expansion of QW cross-reactive T cells by hyperstimulating ligands results in adaptation and the loss of Th1 cytokines. The adaptation of T cell clones had a greater effect on the secretion of Th1 cytokines than on the secretion of Th2 cytokines when clones were activated with either Q144 (squares) or W144 (circles). Cytokines were measured by ELISA in culture supernatants obtained 40 h after activation. The results are representative of three experiments.

 
Tuning of activation threshold by two independent mechanisms in response to expansion by hyperstimulating ligand
The mechanism by which the T cell clones adapt and change their proliferative response and cytokine phenotype upon re-stimulation with different cross-reactive ligands is unknown. Since we could demonstrate adaptation after two rounds of activation it seemed likely that it was a short term process and rather unlikely that we were observing the outgrowth of a minor population of cells. To address the possibility that surface molecules might be modulated, we stained for the expression of various surface molecules on T cell clones that were expanded with W144 or Q144. We did not find any change in the level of expression of TCR, CD4 or CD28 (Fig. 4Go) in three out of the four clones that we tested. However, for QW.7G1, we identified a significant decrease in the expression of CD4 (Fig. 4Go). To follow this process, we stained cells over sequential activations and found that the number of CD4 cells approximately doubled at each activation between the sixth and the eighth, and by the 11th, cells were essentially negative for CD4 expression (Fig. 5Go). Therefore T cell clones can adapt to hyperstimulation by the outgrowth of a CD4 population or by tuning CD4 expression. If the loss of CD4 expression from the surface of the clone QW.7G1 was also responsible for the changes in the cytokine production from this clone, then changes in cytokine production should correlate with the loss of CD4 from the surface of this T cell. To address this issue we graphed the changes in cytokine production and loss of CD4 with the number of activations by Q144 through time. As shown in Fig. 5Go, the changes in the Th1 cytokines were complete after two activations, whereas loss of CD4 in response to hyperstimulation is a much slower process and complete loss of CD4 took at least 10 rounds of activation. These data suggest that the changes in cytokine production are acutely tuned and may be independent of loss of CD4 in these cells. Therefore short-term changes in effector functions may be as a result of other mechanisms such as down-regulation of other surface receptors or tuning of intracellular signaling pathways. At least two independent processes appear to take place by which the T cells adapt to hyperstimulatory ligands, acute tuning of effector function and selection of a minor population of T cells (over time) which cannot be hyperstimulated by the superagonist ligand.



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Fig. 4. Adaptation can occur by down-regulation of CD4. Repeated stimulation of QW.7G1 with Q144, but not W144, leads to the outgrowth of a CD4 population of cells. Cells were stained with saturating concentrations of directly conjugated antibody (filled histogram, cells cultured with W144; thick line, cells cultured with Q144) and isotype control (dashed line). The results are from cells stained after seven activations and are representative of at least three experiments.

 


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Fig. 5. Adaptation occurs by two independent mechanisms. The kinetics of change in cytokine profile and levels of CD4 expression of QW.7G1 suggest that these are independent of each other. QW.7G1 cells grown on Q144 or W144 were activated with Q144. Differences in the cytokine secretion of cells grown on Q144, compared to cells grown on W144 (normalized to 100%), and the fraction of CD4 T cells grown on Q144 are shown in terms of the number of activations.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this paper we have described the generation of cross-reactive `QW' T cell clones, from mice immunized with Q144 but expanded in vitro with W144. The clones were of a predominantly Th2 phenotype when re-stimulated with W144. Activation of many of the QW clones with Q144 resulted in the production of proinflammatory Th1 cytokines that we did not detect when the cells were activated with W144. Compared to W144, the proliferative response to Q144 was heteroclitic, and this response is analogous to superagonist ligands which we and others have described previously. The results presented here for several clones now begin to explain the basis for heteroclicity and superagonism. The heteroclitic responses and hierarchical relationship can clearly be related to the immunization and expansion strategy, in that activation in vitro by a suboptimal cross-reactive ligand expands T cells which are hyper-responsive to the immunizing antigen, e.g. activation of QW clones with W144 shifts the dose–response curve to Q144 to the left. This results in hyper-proliferation and also changes in the cytokine production of the responding T cells when they are activated by Q144, compared with W144.

The differences in the cross-reactive phenotype of T cell populations derived from mice immunized with Q144 and expanded either with Q144 or W144 could occur by the selection of a more cross-reactive population of cells from amongst all the cells which respond to Q144 or by the adaptation of cells with Q144-specific TCR so that they are of high avidity for Q144 and are sensitive enough to be activated by W144. While we cannot assess the contribution of selection to the final make-up of the QW clone population, we have been able to show that these cells can be rendered less sensitive by repeated activation with the hyperstimulatory ligand. If low-avidity interactions do maintain T cells in a more reactive state, then the most important implication of this finding is that many different autoreactive T cells may arise by a similar mechanism, i.e. infection by a pathogen followed by `rescue' of autoreactive cells by virtue of their ability to expand when they encounter self-antigen. The mechanism by which activation with low-avidity ligands `rescues' cross-reactive T cell clones is uncertain. The QW clones could be selected from a larger pool of Q144-responsive cells on the basis of their cross-reactivity with W144. The explanation for the heteroclitic responses to Q144 then depends on the proposal that cross-reactive ligands are usually sub-optimal compared with immunizing ligands (12,13). In a population of cells which respond to Q144, W144 will most often be a suboptimal ligand and cells will be more sensitive to Q144 than W144. An alternative explanation is that re-stimulation by W144 causes adaptation/selection of T cells, which shifts the activation threshold, allowing the T cells to be viable when expanded by W144 but hyperstimulated when activated by Q144. These possibilities are not mutually exclusive. Because the T cell response to Q144 and W144 is diverse at the level of the TCR, it is difficult to distinguish between these two possibilities by comparing QW- and Q144-specific cells on the basis of their TCR.

We have attempted to address the question of the mechanism of T cell adaptation by activating cells under identical conditions, except for the a change in one amino acid of the ligand used to re-stimulate them in vitro. Whereas a single activation of cross-reactive QW clones with Q144 results in hyperactivation and production of Th1 cytokines, expansion of the same clones with Q144 causes the T cell clones to adapt. This adaptation affected the function of the clone, leading to a decrease in the sensitivity of the response to Q144 and a loss of Th1 cytokine secretion, occurred within two rounds of stimulation, and was stable for at least a further three rounds of activation. In one clone we also found a down-regulation of CD4 over time, in a manner which is consistent with the outgrowth of a CD4 population under the selection pressure imposed by activation with the hyperstimulating ligand, Q144 (Fig. 5Go). The two different mechanisms that our data readily support are (i) an acute tuning of T cell function and (ii) selection over time of a population of T cells that is less reactive with the hyperstimulatory ligand.

The data presented here shows that the effector functions of a T cell may be tuned by previous exposure of the cell to cross-reactive ligands. This is consistent with experiments using a TCR transgenic mouse that show that the expression of an endogenous antagonist leads to a blunting of the T cells' response (25,26) . This is clearly most important for cells that enter the memory population and our findings are consistent with a model of affinity selection for T cell memory. This type of model, which has been proposed elsewhere (27,28) predicts that memory cells have TCR which cross-react with autoantigens and it is this sub-optimal stimulation which selects them for the memory pool. Based on the predominantly heteroclitic response which we observe to Q144, this mechanism may also serve to select TCR which have a high avidity for the immunogen but low avidity for the autoantigen. Because the interaction with the autoantigen W144 is suboptimal, it favors the production of Th2 cytokines which are often (but not always) non-pathogenic in organ-specific autoimmune disease. Therefore memory cells, which are necessarily cross-reactive with self-antigen, are less pathogenic when stimulated by the autoantigen than by the immunizing peptide.

The observation that long-term adaptation is sometimes associated with a down-regulation of CD4, in a manner which is consistent with the outgrowth of a CD4 population under the selection pressure imposed by activation with hyperstimulating ligand, Q144, is intriguing. Activation thresholds can clearly be modulated by CD4 levels (29,30) and although CD4/CD8 populations are infrequent in normal mice, they are a striking feature of mrl/lpr mice (31). These mice have a defect in Fas-mediated apoptosis which leads to a failure of activation-induced cell death (32). We therefore hypothesize that the accumulation of CD4 cells in these mice represents the down-regulation of CD4 on autoreactive T cells, which would normally be eliminated by Fas-dependent apoptosis.

In summary we have shown how exposure to a cross-reactive ligand leads to a change in activation threshold and effector function of a clonal population of T cells. From our data it appears that the activation thresholds and effector functions of T cells are tunable. We find that T cell recognition has two essential and distinct components: TCRs function to define the specificity and hierarchy of ligand recognition, but T cell function is determined by the activation threshold of the cell, which is in turn defined by the T cell's history. The data presented here shows that the response of two T cells, with identical receptors but which have encountered different antigens, may be qualitatively different. This is consistent with a stochastic model of autoimmunity in which random environmental events determine the outcome of the autoimmune disease process by modulating the activation thresholds of those autoreactive T cells which are selected on a permissive genetic background.


    Acknowledgments
 
We would like to thank Dr Abul Abbas for helpful discussions and Vadim Turchin for technical assistance. This work was supported by grants from the National Institutes of Health (R01NS30843, R01NS35685 and P01AI39671-01A1) to V. K. K. and K08 AI01557-01 to L. B. N., and from the National Multiple Sclerosis Society, New York (RG2571 and RG2320) to V. K. K. A. C. A. is a predoctoral fellow of the Howard Hughes Medical Institute.


    Abbreviations
 
APC antigen-presenting cell
LNC lymph node cells
PLP proteolipid protein
Q144 myelin PLP 139–151 (HSLGKQLGHPDKF)
W144 myelin PLP 139–151 (HSLGKWLGHPDKF)

    Notes
 
Transmitting editor: L. Steinman

Received 29 July 1999, accepted 19 October 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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