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Originally published online as doi:10.1189/jlb.0205059 on April 7, 2005

Published online before print April 7, 2005
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(Journal of Leukocyte Biology. 2005;78:43-50.)
© 2005 by Society for Leukocyte Biology

Fas-mediated death and sensory adaptation limit the pathogenic potential of autoreactive T cells after strong antigenic stimulation

Kelli R. Ryan, David McCue and Stephen M. Anderton1

University of Edinburgh, Institute of Immunology and Infection Research, School of Biological Sciences, United Kingdom

1 Correspondence: University of Edinburgh, Institute of Immunology and Infection Research, School of Biological Sciences, Kings Buildings, West Mains Road, Edinburgh EH9 3JT, UK. E-mail: steve.anderton{at}ed.ac.uk


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ABSTRACT
 
The ability of autoreactive T cells to induce autoimmune pathology is dependent on their ability to respond to the level of autoantigen presented in the target organ. Emerging evidence suggests that at the population level, T cell sensitivity for self can be reduced by deletion of those cells bearing high-affinity T cell receptors (TCRs) or by sensory adaptation of individual cells. Here, we have investigated the mechanisms that prevent the induction of experimental autoimmune encephalomyelitis (EAE) when myelin basic protein (MBP)-reactive T cells are exposed to a strong, antigenic stimulus. Stimulation of MBP-reactive TCR transgenic T cells with a superagonist peptide led to extensive activation-induced cell death (AICD) through Fas signaling. Using T cells lacking Fas, we found that disruption of this deletional mechanism only partially increased EAE in response to superagonist, failing to restore susceptibility to the level found in response to the wild-type MBP peptide. A significant fraction of the MBP-reactive T cells was able to avoid AICD in response to superagonist, but these cells had a reduced sensitivity for an antigen that correlated with elevated levels of CD5. Therefore, when TCR affinity is fixed, autoreactive T cell sensitivity can be shifted to below a threshold for harm by a combination of AICD and sensory adaptation.

Key Words: autoimmunity • multiple sclerosis • T cells • tolerance • CD5


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INTRODUCTION
 
Cross-reactive T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHC) means that a large proportion of the peripheral T cell repertoire will have a level of reactivity for self-pMHC. An absolute, deletion-based mechanism of maintaining self-tolerance would therefore produce a porous T cell repertoire, compromising adaptive immunity [1 ]. Recent data clearly indicate that a large, self-reactive T cell repertoire can be maintained in the presence of a cognate self-antigen [2 , 3 ]. The key is that these T cells display relatively low-sensitivity for self-pMHC and as a result, do not cause pathology. These data led us to propose a "threshold for harm" model in which the T cell repertoire is conditioned to be insensitive to physiological levels of self-pMHC [4 ]. This threshold can be maintained by two broad mechanisms: a selective deletion of T cells bearing TCRs with high-affinity for self during the expansion-phase of a peripheral immune response (i.e., negative selection in the periphery) and adaptation of the individual cell in response to antigen, through loss of cell surface receptors (TCR or coreceptor) or through up-regulation of inhibitory receptors and/or signaling molecules, resulting in a requirement for a stronger antigenic stimulus to induce T cell activation and effector function [4 5 6 7 ].

Adaptation at the single-cell level is a central feature of the tunable activation threshold model [5 , 6 ], and such effects (chiefly at the level of the TCR and coreceptor expression) were evident in early studies of T cell tolerance [8 9 10 ]. We recently provided evidence for negative selection in the periphery from our analysis of the myelin basic protein (MBP)-reactive T cell repertoire in response to altered peptide ligands (APL) with increased MHC class II binding affinities and therefore, superagonist properties [11 ]. These APL failed to induce experimental autoimmune encephalomyelitis (EAE), as the T cells that were selectively expanded expressed low-affinity TCRs and, therefore, required levels of the wild-type antigen for activation that apparently could not be achieved in the central nervous system (CNS). The T cells expressing high-affinity MBP-reactive TCRs, which would normally induce EAE, were deleted from the repertoire by activation-induced cell death (AICD) in response to immunization with these strongly antigenic, superagonist APL.

Clearly, the biochemical "tuning" processes and selective deletion may be active within a heterogeneous population of antigen-reactive T cells in response to antigenic challenge in vivo. Understanding the processes involved in the decision of a T cell to "tune" or to die will be valuable in any attempt to modulate an immune response. Here, we have investigated the impact of tuning and deletion on the response to the Ac1-9 peptide of MBP using a TCR transgenic system in which TCR affinity is fixed. First, using an in vitro model, we show that superagonist-induced AICD requires Fas-Fas lignad (FasL) interactions between T cells. In vivo, however, disruption of Fas signaling in encephalitogenic T cells leads to only a partial restoration of disease susceptibility in response to a high-dose, superagonist peptide. We find that the TCR transgenic T cells that survive APL-induced death (in vitro and in vivo) are desensitized to antigenic stimulation (i.e., "tuned"), and this correlates with elevated levels of the inhibitory receptor CD5. These results indicate that within a homogeneous, self-reactive T cell population, Fas-mediated cell death and increased CD5 signaling may act in concert to maintain antigen sensitivity below the threshold for harm.


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MATERIALS AND METHODS
 
Mice
Tg4 mice expressing a transgenic TCR that reacts with the Ac1-9:Au pMHC complex [12 ] were kindly provided by Professor D. Wraith (University of Bristol, UK). Fas– and FasL– mice were produced by backcrossing the Tg4 and the syngeneic B10.PL strains with lpr [13 ] and gld [14 ] mutant mice (kindly provided by Professor David Gray, University of Edinburgh, UK). For some in vivo experiments, T cells from Tg4 mice bearing the Ly5.1 marker were used. All mice were maintained under specific pathogen-free conditions at the Institute of Immunology and Infection Research, University of Edinburgh (UK).

Media and reagents
Cells were cultured in tissue-culture medium consisting of RPMI-1640 medium [supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5x10–5 M 2-mercaptoethanol (all from Gibco/Invitrogen, Paisley, UK) and 5% fetal calf serum (FCS; Sigma, Poole UK)]. Serum-free wash media contained all of the above except for FCS. Fluorescence-activated cell sorter (FACS) staining was performed in 1x phosphate-buffered saline (PBS; Gibco/Invitrogen) plus 0.1% bovine serum albumin (Sigma).

Antigens, immunizations, and induction of EAE
The 4Lys (Ac-ASQKRPSQR) and 4Tyr (Ac-ASQYRPSQR) peptides were synthesized using standard F-moc chemistry at the Advanced Biotechnology Centre, Imperial College (London, UK). Mice were injected subcutaneously (s.c.) with 100 nmol 4Lys or 4Tyr (unless otherwise stated) in 100 µl total volume of complete Freund’s adjuvant (CFA; Sigma), emulsified in PBS at a 1:1 ratio. For the induction of EAE, mice also received 200 ng pertussis toxin (ECACC, Wiltshire, UK) intraperitoneally on days 0 and 2 in 0.5 ml PBS. EAE was monitored daily from day 7 through to days 28–30, and mice were scored from 0 to 6 as follows: 0, no disease; 1, flaccid tail; 2, impaired gait or impaired righting reflex; 3, partial hind-limb paralysis; 4, total hind-limb paralysis; 5, front and hind-limb paralysis; 6, moribund. Disease burdens were compared among groups using two-tailed Mann-Whitney U testing.

In vitro Tg4 T cell death
CD4+ T cells were purified from Tg4, Tg4Fas–, or Tg4FasL– splenocytes by positive magnetic separation through a magnetic cell sorter LS+ column, according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+ cells were then labeled with 1 µM carboxyfluorescein succinyl ester (CFSE; Molecular Probes, Eugene, OR) in serum-free wash medium at 107 per ml and incubated at 37°C for 15 min. The reaction was then quenched by washing with RPMI 5 containing 5% FCS. Labeled CD4+ cells (5x105) were then cultured with 2 x 106 irradiated (30 Gy) splenocytes from the CD4-negative fraction plus 4Lys or 4Tyr. At various time-points after activation, cultures were stained with anti-CD4-allphycocyanin (APC), Annexin V-phycoerythrin (PE), and 7-amino-actinomycin D (BD PharMingen, Oxford, UK) and analyzed by flow cytometry on a FACSCalibur (Becton Dickinson, San Jose, CA). In some experiments, blocking monoclonal antibodies to FasL or tumor necrosis factor receptor 1 [TNFR1; both hamster immunoglobulin G1, BD PharMingen) were added on the day cultures were set up and 2 days later, at 5 µg/ml.

Desensitization of Tg4 cells in vivo
Tg4 x Ly5.1+ splenocytes (5x106) were injected intravenously into B10.PL x C57BL/6 mice 1 day prior to immunization with 100 nmol 4Lys or 4Tyr as above. After 7 days, spleen cells were stained with anti-CD4-APC, anti-Ly5.1-fluorescein isothiocyanate, and anti-CD5-PE or labeled with CFSE as above and restimulated in vitro with 4Lys or 4Tyr at varying concentrations for 3 days. Cultures were then stained with anti-CD4-APC, anti-CD5-PE, and anti-Ly5.1-biotin plus avidin-peridinin chlorophyll protein (BD PharMingen). All samples were analyzed by gating on the CD4+ Ly5.1+ population.

Generation and analysis of Tg4 T cell lines (TCL)
TCL were generated from Tg4 or Tg4-recombinase activating gene 2–/– (RAG2–/–) splenocytes by primary in vitro stimulation with the indicated concentrations of 4Lys or 4Tyr. Cells were stimulated three times with peptide using a 7-day stimulation/expansion cycle in which splenocytes were initially stimulated in 24-well plates (5x106 cells/well) for 48 h in tissue-culture medium. T cell blasts were then isolated using Nycoprep 1.077A (Axis-Shield PoC AS, Oslo, Norway) and expanded in tissue-culture medium supplemented with 2.5% concanavalin A-activated rat spleen supernatant as a source of T cell growth factors. T cell restimulations were carried out in 24-well plates (106 T cells/well) with peptide in the presence of irradiated (30 Gy), syngeneic spleen antigen-presenting cells (3x106/well). T cell blasts were isolated and expanded as above.

TCL (2x104 cells/well) were cultured in duplicate in a 96-well, flat-bottomed microtiter plates (BD Biosciences, San Jose, CA) with irradiated (30 Gy) B10.PL splenocytes (2.5x105 cells/well) and the indicated concentrations of peptide in tissue-culture medium. 3H-Thymidine (dThd; Amersham International, Amersham, UK) was added (0.5 µCi/well) during the final 18 h of a 72-h incubation prior to harvesting and measurement of 3H-dThd incorporation using a liquid scintillation ß-counter (Wallac, Turku, Finland).


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RESULTS
 
Superagonist-induced AICD is quantitative
Immunization with the Ac1-9 peptide of MBP provokes EAE in H-2u mice. The lysine residue at position 4 of Ac1-9 interacts suboptimally with the hydrophobic P4 pocket in the peptide-binding cleft of the Au MHC class II molecule, producing a pMHC complex of weak affinity [15 16 17 18 19 ]. To compensate for this, the T cells that recognize the Ac1-9-Au complex generally express TCRs of medium- to high-affinity [20 ]. The Tg4 transgenic mouse provides a source of naive T cells expressing a TCR with high-affinity for the Ac1-9-Au complex [11 , 12 ]. Substitution of a tyrosine for the native lysine at position 4 increases the MHC binding affinity by approximately 1 million-fold [11 , 15 ], creating a superagonist ligand (4Tyr) for Tg4 T cells in vitro. We have previously reported that paradoxically, the 4Tyr peptide does not induce EAE in wild-type H-2u mice and leads to extensive deletion of moderate- to high-affinity T cells (including Tg4 cells) in vivo through AICD [11 ]. The remaining T cells that are productively expanded in response to 4Tyr immunization use different, low-affinity TCRs and have a sensitivity for the wild-type 4Lys peptide, which is apparently too low to provoke disease in response to presentation of MBP in the CNS.

To investigate the mechanisms of superagonist-induced AICD, we established an in vitro assay to study the responses of naïve Tg4 T cells to stimulation with 4Lys or 4Tyr. Purified CD4+ Tg4 T cells were labeled with CFSE and then cultured with antigen-presenting cells plus varying doses of the 4Lys and 4Tyr peptides. A kinetic analysis using Annexin V binding as a measure of early apoptosis revealed a clear amplification of Tg4 cell death in response to the superagonist 4Tyr compared with the wild-type 4Lys peptide. At high-peptide concentrations, levels of apoptosis were similar with either peptide, but at lower concentrations, apoptosis was far more evident on culture with the 4Tyr peptide (Fig. 1A ). We conclude that superagonist-induced T cell death is quantitative, as lowering the concentration of peptide allows efficient T cell activation in the absence of significant levels of AICD.



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  		Figure 1. Tg4 T cells undergo Fas-mediated AICD in response to superagonist. Purified CD4+ Tg4 T cells were stimulated in vitro with CD4-depleted APC and varying concentrations of 4Lys or 4Tyr. (A) Apoptosis was measured at various time-points after stimulation (day 4 shown here). (B) Expression levels of Fas and FasL were measured over the course of 72 h after activation with 0.1 µM of either peptide. The Fas-FasL interaction was inhibited by adding anti-FasL antibodies into the culture at days 0 and 2 (C) or through the use of Fas-deficient, FasL-deficient Tg4 cells or APC (D and E). All cultures were stained with anti-CD4-APC and Annexin V-PE. Results shown are the percentage of CD4+ cells that stained positive for Annexin V. Data are from two of several experiments that gave consistent results. Ag, Antigen.

Immunization with low doses of superagonist induces EAE
A quantitative model of superagonist-induced AICD would predict that immunization with less superagonist would allow high-affinity encephalitogenic T cells to survive the encounter with antigen and cause EAE. Previous studies had compared 4Lys with 4Tyr using an immunizing dose of 100–200 nMol per mouse. Given that in vitro activation studies with these two peptides show that femtomolar concentrations of 4Tyr induce equivalent responses to those seen when 4Lys is used in the nanomolar range, we performed a wide titration, lowering 4Tyr by tenfold increments. We were somewhat surprised, therefore, that lowering the immunizing dose of 4Tyr to 10–20 nMol was sufficient to induce EAE (Table 1 ). Doses of 4Tyr between 0.1 and 20 nMol gave similar disease kinetics (day of onset, maximum score, and cumulative disease burden) to those induced with 100–200 nMol 4Lys. Mice did not develop EAE when doses of 4Tyr below 0.1 nMol were used. We also saw a mild, late-onset form of disease in some mice when 100 but not 200 nMol 4Tyr was used. We conclude that the normal course of EAE can be induced after exposure to an approximate 2-log dose range of the superagonist peptide, which must be high enough to expand the autoreactive T cells but low enough to avoid AICD. These EAE data do not mirror the differences described for in vitro dose responses, as measured by proliferation (approximately 6 logs) [11 ], but are in agreement with the increase in sensitivity to AICD when 4Tyr is used. Thus, using 4Tyr at 2- to 3-log lower concentrations than 4Lys induces equivalent levels of apoptosis in vitro (Fig. 1A) and EAE in vivo (Table 1) . We conclude that a quantitative model can explain the ability of the superagonist ligand to induce or prevent EAE in this model.


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  		Table 1. EAE in Response to Low Doses of 4Tyr

Fas-dependent AICD of Tg4 T cells
The most prominent and well-characterized mechanism of AICD involves the Fas pathway [21 ]. Naïve Tg4 T cells up-regulated their expression of Fas and FasL over time upon in vitro culture with antigen (4Lys or 4Tyr; Fig. 1B ). Expression of Fas peaked earlier, and higher numbers of cells expressed FasL in response to 4Tyr. Disrupting the Fas-FasL interaction by including an anti-FasL blocking antibody in our cultures decreased Tg4 cell death by up to 75% (Fig. 1C) . Consistent with this, Tg4 cells that lacked Fas were resistant to antigen-driven apoptosis (Fig. 1D) . Culture of wild-type or FasL– Tg4 cells with wild-type or FasL– APC revealed that Tg4 apoptosis was blocked only when the T cells lacked functional FasL (Fig. 1E) . We conclude that upon strong antigenic stimulation, Tg4 cells kill one another through the up-regulation of FasL.

The TNFR is associated with many of the same downstream signaling molecules as Fas [21 22 23 ]. However, anti-TNFR antibody did not inhibit T cell death (data not shown). Type I interferons (IFNs) have been reported to protect T cells from death [24 ], but again, addition of IFN-{alpha} did not reduce the levels of Tg4 cell death (data not shown).

Disruption of Fas-mediated death in Tg4 cells only partially restores their encephalitogenic potential in response to superagonist
We next tested whether removing Fas signaling was sufficient to allow Ac1-9-reactive T cells to induce EAE in response to strong, superagonist stimulation. This question is complicated by the fact that the Fas-FasL interaction is crucial to the pathological process of EAE within the CNS. Hence Fas- and FasL-deficient mice are resistant to EAE, and myelin-reactive T cells, which lack FasL, are not encephalitogenic [25 , 26 ]. We therefore used a system in which the encephalitogenic T cells lacked Fas but not FasL, so that Fas signaling to the CNS oligodendrocytes was intact. Tg4 or Tg4Fas– T cells were transferred in B10.PL x C57BL/6 mice. These recipient mice have two important advantages over B10.PL mice. First, there is no risk of rejection of the transferred T cells (the Fas deficiency was introduced by crossing with lpr mutant mice on the C57BL/6 background). It is more important that we found that B10.PL x C57BL/6 mice are almost totally resistant to EAE induced with Ac1-9 but develop severe disease after transfer of Tg4 cells, and the transferred cells comprise greater than 70% of CNS-infiltrating mononuclear cells at the peak of disease (data not shown). Therefore, any effects on EAE severity could be attributed to the transferred Tg4 population. Tg4 or Tg4Fas– cells were transferred 1 day before immunization with 4Lys or 4Tyr. Mice that received Tg4 cells developed severe EAE upon immunization with 4Lys, whereas 4Tyr induced a much milder disease (Fig. 2A and 2B ). As shown in Figure 2A , Tg4 or Tg4Fas– cells induced a similar disease after immunization with 4Lys. In contrast, after immunization with the 4Tyr peptide, mice that had received Tg4Fas– cells developed more severe disease than those that received Fas-sufficient Tg4 cells (Fig. 2B) . Although this effect reached significance, it did not fully restore susceptibility to EAE to the level seen when using the 4Lys peptide. Consistent with previous reports [27 ], mice that received Tg4FasL– cells did not develop EAE, even after immunization with the wild-type 4Lys peptide (data not shown). We conclude that disruption of Fas signaling cannot fully restore pathogenic potential in cells stimulated with superagonist in vivo.



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  		Figure 2. Adoptive transfer of Fas-deficient Tg4 T cells increases disease severity induced by the superagonist 4Tyr. B10.PL mice received 5 x 106 splenocytes from Tg4 or Tg4Fas– mice on day –1 and were primed s.c. the next day with 100 nmol 4Lys (A) or 4Tyr (B). Disease burden was significantly higher (P, 0.0007) after 4Tyr immunization when using transfer of Tg4Fas– cells (four of five mice reaching EAE grade above 2) compared with Tg4 cells (one of five mice reaching EAE grade above 2). Data are from one of two experiments that gave consistent results.

Functional desensitization of Tg4 cells in response to superagonist
If Fas-mediated AICD was not alone in controlling the pathogenic potential of Tg4 cells in vivo, what other mechanisms might be at play? An indication that other factors may be involved came from our attempts to generate long-term TCL using Tg4 cells. We have done this successfully by stimulating the Tg4 cells repeatedly with 1 µM 4Lys. In contrast, 4Tyr at this dose led to extensive T cell death and failed to generate stable, long-term TCL. However, a significant number of cells did persist through several rounds of restimulation, allowing us to characterize their response patterns and surface phenotype in comparison with TCL raised against 1 µM 4Lys or lower doses of 4Tyr (outlined in Fig. 3A ).



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  		Figure 3. TCL raised against high concentrations of 4Tyr are less responsive to peptide. (A) Tg4 splenocytes were stimulated three times in vitro with 1 µM 4Lys or a fixed concentration of 4Tyr. (B) Ten days after the third stimulation, individual TCL (2x104) were cultured with irradiated B10.PL splenocytes and increasing concentrations of 4Lys or 4Tyr, and proliferation was measured. Data are from one of several experiments giving consistent results. cpm, Counts per minute. (C) TCL raised against 1 µM 4Lys, 1 µM 4Tyr, or 0.001 µM 4Tyr were evaluated for TCR-ß chain and CD5 expression levels on the same day as the proliferation assays were set up (i.e., 10 days since previous exposure to antigen). Data are from one of three experiments giving consistent results.

We found that the cells that persisted after stimulation with 1 µM 4Tyr were desensitized, requiring tenfold higher concentrations of 4Lys for activation compared with cells raised against 4Lys (Fig. 3B) . When these two TCL were assayed using 4Tyr, this effect was even more pronounced with a 3- to 4-log shift in sensitivity (Fig. 3B) . Tg4 cells initially stimulated with low doses of 4Tyr showed restored responsiveness to 4Lys and 4Tyr to the extent that TCL generated against 0.001 µM 4Tyr showed response patterns similar to those generated against 1 µM 4Lys (again, a 3-log shift). These shifts in sensitivity were found in proliferative responses and in cytokine production (not shown). All of these Tg4-derived TCL produced interleukin (IL)-2, IL-4, and IFN-{gamma} in response to 4Lys or 4Tyr. In contrast to previous reports [28 ], we found no evidence that primary in vitro exposure to high doses of 4Tyr selectively promotes differentiation into cells producing IL-4 rather than IFN-{gamma}.

A phenotypic analysis of the different TCL revealed that the shifts in sensitivity did not correlate with changes in the surface expression of TCR (Fig. 3C) or CD4 (not shown). We saw no difference in the speed or level of TCR down-regulation within the first 24 h after stimulation with 4Lys compared with 4Tyr (not shown). However, TCL generated against 1 µM 4Tyr showed a four- to fivefold elevation of CD5 levels compared with those generated against 1 µM 4Lys (Fig. 3C) . Furthermore, this CD5 up-regulation was gradually reduced in line with the dose of 4Tyr used until 0.001 µM again produced similar levels to those seen with the high dose of 4Lys.

It is important that we found the above effects when using T cells derived from Tg4RAG2+/+ mice or from Tg4RAG2–/– mice, excluding the possibility that the desensitized cells, which were expanded in response to high-dose 4Tyr, were expressing endogenous TCR-{alpha} chains. We conclude that the cells that escape AICD in vitro do so because of a functional desensitization that correlates with enhanced CD5 expression.

Functional desensitization of Tg4 T cells after in vivo exposure to superagonist
We tested whether in vivo exposure to 4Tyr could also have a desensitizing effect using a T cell transfer model. To identify Tg4 cells in mixed populations, we generated Tg4 mice expressing the Ly5.1 marker and transferred these cells into B10.PL x C57BL/6 (Ly5.2+) recipients. After immunization with 4Lys or 4Tyr, splenocytes were isolated, labeled with CFSE, and cultured in vitro with a dose range of the 4Lys peptide. This allowed analysis of Tg4 T cell proliferation (by gating on the CD4+ Ly5.1+ population, Fig. 4A ). After 3 days of culture, CFSE profiles revealed that in the presence of a high dose (10 µM) of 4Lys, 4Lys- and 4Tyr-primed Tg4 cells had divided to an equivalent extent. However, when 0.1 or 1 µM 4Lys was added to cultures, the Tg4 cells from mice immunized with 4Tyr had a clear deficit in their ability to proliferate. Furthermore, phenotypic analysis of the Tg4 cells on the day of isolation revealed the same pattern that was seen in vitro, with no differences in TCR or CD4 levels evident (not shown) but a marked increase in CD5 expression in the 4Tyr-immunized group (Fig. 4B) . Thus, we conclude that immunization with the 4Tyr superagonist can allow the expansion of cells bearing the moderate-affinity Tg4 TCR, but these cells have reduced functional sensitivity that correlates with increased CD5 expression.



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  		Figure 4. In vivo desensitization of Tg4 T cells following immunization with 4Tyr. Naïve B10.PL x C57BL/6 mice received 5 x 106 Tg4 Ly5.1+ splenocytes 1 day prior to immunization with 100 nmol 4Lys or 4Tyr. (A) Spleen cells isolated 7 days later were labeled with CFSE and restimulated for 3 days with 4Lys at the indicated doses. (B) Spleen cells were stained ex vivo with anti-CD5, anti-CD4, and anti-Ly5.1 antibodies. All results are shown after gating on the CD4+ Ly5.1+ population. Solid histograms show cells from mice immunized with 4Lys or 4Tyr; dotted histograms show cells from control mice immunized with CFA alone. These data are from one of two experiments giving consistent results.


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DISCUSSION
 
Our data show that a large fraction of encephalitogenic T cells dies in response to superagonist. However, a significant number of cells persist but show a greatly impaired ability to provoke CNS autoimmunity. These cells show a sensory adaptation that correlates with elevated expression of CD5 and that presumably serves to shift their sensitivity for self to below the threshold for harm. After in vivo exposure to 4Tyr, Tg4 T cell responses to the wild-type 4Lys peptide were diminished greatly at 1 µM and are absent at 0.1 µM (Fig. 4) . Through analysis of heterogeneous Ac1-9-reactive T cell populations from wild-type mice, we previously showed that selection of low-affinity TCRs in response to superagonist meant the responding T cell population had low sensitivity for wild-type Ac1-9 [11 ]. Hence, in this selective model, little or no EAE developed when the expanded T cells required concentrations of 4Lys above 1 µM for their activation in vitro. The present data therefore essentially show that this same in vivo shift in sensitivity can be observed in T cells with a fixed, moderate-affinity TCR. It is interesting that this shift was never quite so profound in our in vitro systems, and sensitivity to 4Lys was lowered by only approximately tenfold after exposure to 1 µM 4Tyr (Fig. 4) . This indicates that the adaptation process may be more powerful when 4Tyr is given in vivo or may simply reflect a greater ability of the in vitro TCL assay to detect T cell activation. A further surprising finding using the in vitro system was that the desensitization after culture with 4Tyr was more evident when 4Tyr was used for recall responses, showing at least a 1000-fold loss in sensitivity compared with the tenfold shift to 4Lys described above. Although APL-specific tuning has also been reported in models of thymic selection using TCR transgenic systems [29 , 30 ], it is not clear why we also see this here. These previous studies used partial agonist/antagonist APL that had alterations at a major TCR contact residue, rather than at a MHC contact, as we have used here.

A previous study reported that in vitro exposure to high doses of a superagonist APL (with a TCR contact substitution) led to functional desensitization of TCR transgenic cells recognizing a peptide based on the 139–151 epitope of the proteolipid protein (PLP) [31 ]. This loss of sensitivity involved alterations in T cell signaling that differed from "classical" anergy [32 ] (reduced p21ras and mitogen-activated protein kinase activity but also reduced Ca++ signaling and not reversible by exogenous IL-2) [7 , 31 ]. These cells showed selective loss of IL-2 production, with IFN-{gamma} production remaining intact, unlike our findings that all cytokine production was diminished after exposure to 4Tyr. CD5 was not investigated in the previous report. There are similarities but also clear differences, therefore, in the effects of these two superagonists. This most likely reflects some qualitative effect on TCR triggering in the PLP system (with a TCR-contact substitution), whereas the dominant effect of the 4Tyr superagonist appears to be purely quantitative as a result of enhanced MHC binding. Unfortunately, the previous study [31 ] was unable to test the relevance of T cell desensitization to disease, as the transgenic T cells used already had low sensitivity for the wild-type PLP epitope and were therefore intrinsically nonencephalitogenic.

Two recent reports have assessed the roles of CD5 in the context of EAE. One study reports that presentation of the myelin oligodendrocyte glycoprotein peptide [MOG(35-55)] by steady-state dendritic cells leads to antigen-specific unresponsiveness that is associated with and dependent on up-regulation of CD5 on the T cell [33 ]. However, this study did not investigate the role of CD5 in terms of altering antigenic strength or of T cell activation thresholds. The second study reports that CD5–/– mice are relatively resistant to the induction of EAE with MOG(35-55) [34 ]. At face value, this is a surprising result, as lack of CD5 should lower the threshold for T cell activation and therefore lead to increased priming of encephalitogenic T cells. However, the absence of CD5 was shown to enhance initial T cell activation but then, to also promote substantially greater AICD at later time-points. This higher T cell death could account for the reduction in EAE seen and would fit well with our own observations. If CD5 signaling limits AICD, perhaps through activation of casein kinase 2 [35 ], its absence could inhibit disease in response to wild-type antigen. In contrast, up-regulation of CD5, which we see, might well prevent AICD, but the concomitant reduction in positive T cell signaling would shift the sensitivity of the T cells for Ac1-9 to a level below the threshold for harm.

A model of thymic selection has been proposed in which an inverse correlation in the level of CD5 and TCR affinity serves to position developing thymocytes within the window for positive selection. Hence, high CD5 reduces positive selection when TCR affinity for a selecting pMHC complex is low, whereas low CD5 will increase negative selection if TCR affinity is high [36 ]. A similar model has been proposed for the maintenance of T cell sensitivity in peripheral homeostasis. If a T cell receives a strong pMHC stimulus, its expression of CD5 will increase, reducing subsequent sensitivity to T cell triggering [37 ]. These models fit well with our present data about the moderate-affinity Tg4 TCR in response to 4Lys or 4Tyr. Tg4 T cell sensitivity for 4Lys is moderate; therefore, there is little death and no need to increase CD5 in response to moderate doses of 4Lys. In contrast, sensitivity for 4Tyr is high, leading to increased AICD and increased levels of CD5 on those cells that persist (i.e., increased CD5 correlates with avoidance of negative selection). This was evident in vitro and in vivo, in the latter case, correlating with the inability of 4Tyr to expand Tg4 cells effectively with pathogenic potential (as a result of their functional desensitization). At this point, it is difficult to determine definitively whether the increase in CD5 after exposure to the superagonist results from a selective expansion of cells that already express high levels of CD5 (those cells with lower levels being sensitive to AICD) or a generalized shift in expression in response to strong stimulation. We found elevated CD5 levels in vitro within 24 h of primary exposure to 4Tyr (data not shown), and this probably points to the latter alternative. Nevertheless, it is clear from the increased levels of AICD in response to 4Tyr that a significant fraction of the Tg4 cells is unable to use CD5 signaling to avoid death.

Recent evidence points to a role for adaptation in T cell sensitivity in maintaining peripheral tolerance [32 , 38 ]. This results in higher doses of antigen being required to provoke T cell activation subsequently ex vivo (when TCR transgenic T cells are used) or in vivo using immunization protocols. The net result is functional unresponsiveness to concentrations of antigen normally encountered in vivo [4 , 6 ]. The clear advantage of such a mechanism is that the T cell remains as a potentially active part of the immune repertoire to respond to foreign antigens for which it may have higher sensitivity. Perhaps the best example of the value of this comes from studies showing that T cells recognizing a transgenic antigen expressed in the pancreas do not elicit diabetes but remain capable of clearing tumor cells expressing the same antigen [39 ]. An important principal to these adaptive models is that when the tuning stimulus is removed, T cell sensitivity can increase.

Our results indicate that the strength of antigenic stimulus can influence T cell sensitivity during an in vivo immune response with full inflammatory signals. In response to a superagonist stimulus, a T cell has two options: to adapt or die. We find that when the TCR affinity is fixed, some cells can avoid AICD in response to a strong, antigenic stimulus, as they have a sensitivity below the threshold for harm, and this correlates with increased levels of CD5. Understanding these processes and the point at which death must override adaptation will tell us a great deal about the continuum of checkpoints that control T cell autoaggression.


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ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Wellcome Trust. S. M. A. is a Medical Research Council Senior Research Fellow.

Received February 1, 2005; revised March 3, 2005; accepted March 4, 2005.


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