Altered functional and biochemical response by CD8+ T cells that remain after tolerance

Anwar Murtaza, C. Thomas Nugent2, Pankaj Tailor3, Valerie C. Asensio1, Judith A. Biggs, Iain L. Campbell1 and Linda A. Sherman

1 Departments of Immunology and Neuropharmacology, The Scripps Research Institute, 10550 North Torrey Pines Road, IMM-15, La Jolla, CA 92037, USA
2 Hybritech Inc., a subsidiary of Beckman Coulter Inc., PO Box 269006, San Diego, CA 92196, USA
3 Department of Biochemistry, Room 904, McIntyre Medical Building, 3655 Drummond Street, McGill University, Montreal, Quebec H3G 1Y6, Canada

Correspondence to: L A. Sherman


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
To further define the molecular basis of tolerance to a peripherally expressed antigen we have correlated differences in functional capacity with biochemical events in hemagglutinin (HA)-specific cytotoxic T lymphocyte (CTL) clones derived either from a conventional B10.D2 mouse that is not tolerant to HA (D2 Clone 6) or from an InsHA mouse that is tolerant to HA (InsHA Clone 12). D2 Clone 6, but not InsHA Clone 12, triggers diabetes following in vivo transfer into irradiated InsHA hosts. This diabetogenic clone shows complete and sustained phosphorylation of TCR {zeta}{zeta} chain and ZAP-70 following stimulation with HA-pulsed antigen-presenting cells. In contrast, InsHA Clone 12 showed only partial phosphorylation of TCR {zeta}{zeta} and no phosphorylation of ZAP-70. There was no defect in activation or recruitment of Lck to the TCR complex in both the clones following stimulation with the cognate antigen. This deficiency in the proximal signaling in the InsHA Clone 12 could be overcome by increasing the strength of signal through the CD3–TCR complex, indicating that the signaling machinery of InsHA Clone 12 was functional. These data demonstrate that the HA-responsive CD8+ T cells that can be retrieved from InsHA mice after tolerance induction respond to HA as a partial agonist/antagonist.

Keywords: diabetes, signal transduction, TCR, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Extensive research has shown that many potentially autoreactive T cells are deleted in the thymus (14). However, additional mechanisms appear to be required to minimize T cell responses to peripheral antigens by potentially autoreactive T cells that escape thymic deletion by virtue of their low avidity for self-antigen or because the self-antigen is uniquely expressed in the periphery (5,6).

To study the mechanisms of peripheral tolerance, a number of groups have utilized models in which defined antigens are expressed by murine pancreatic ß cells under the control of the rat insulin promoter (7,8). Our laboratory has used this promoter to produce transgenic InsHA mouse that expresses the influenza virus hemagglutinin (HA) at high levels on the pancreatic ß cells. InsHA mice possess a T cell repertoire tolerant to HA, even after immunization with influenza virus (9). Studies from our group have demonstrated the elimination of CD8+ T cells that have high affinity for the dominant Kd-restricted epitope from HA. Persisting in the repertoire are HA-specific CD8+ T cells that express TCR receptors with low-affinity for this same epitope and require high concentrations of antigen to become activated (10). Upon immunization with influenza virus some of these cells are capable of homing to the pancreas and cause peri-insulitis; however, they do not induce ß cell destruction and diabetes (10). Thus, it appears that tolerance to HA affects the affinity of the TCR of HA-specific T cells. This may be a critical factor in the initiation and potentiation of an autoimmune response. To further investigate the underlying mechanism(s) that govern the functional differences between T cells derived from non-tolerized (B10.D2) or tolerized (InsHA) mice, we selected Kd HA cytotoxic T lymphocyte (CTL) clones which were derived either from the conventional B10.D2 mice (D2 Clone 6 also referred to as Clone 6) or from InsHA mice (InsHA Clone 12 also referred to as Clone 12). Both D2 Clone 6 and InsHA Clone 12 were obtained subsequent to in vivo stimulation with influenza virus. However, previous studies have shown that D2 Clone 6 expresses a higher affinity TCR than InsHA Clone 12 (10).

In this study we have addressed how the inherent difference in the affinity of the TCR between D2 Clone 6 and InsHA Clone 12, as a result of tolerance, affects the pathogenic potential of these HA-specific clones, and correlated this with the proximal signaling events that occur upon encounter with cognate antigen. Our results demonstrate that the diabetogenic capacity of HA-specific CD8+ T cells correlates with the strength of signaling through the TCR.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
The B10.D2 mice were purchased from the breeding colony of The Scripps Research Institute (La Jolla, CA). The InsHA transgenic mice were generated and characterized as previously described (10). All mice were bred and maintained under specific pathogen-free conditions in The Scripps Research Institute's vivarium. All experimental procedures were conducted in strict accordance with the guidelines laid out in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All mice used in these experiments were at least 8 weeks of age. For adoptive transfer experiments mice were irradiated (600 rad) before i.v. injection of the specified number of CTL.

Peptide
The HA peptide (518IYSTVASSL526), which is presented by H-2Kd (11), was synthesized by The Scripps Research Institute core facility using a 430A peptide synthesizer (Applied Biosystems, Foster City, CA). Purity was >85%, as determined by mass spectrophotometry and reverse-phase HPLC analysis on a C18 column (Vydac, Hesperia, CA). Peptide was resuspended in DMSO and stored at –70°C at a concentration of 1 mM.

Generation of Kd HA-specific CTL clones
Generation of Kd HA-specific CTL lines and clones has been described elsewhere (10). Briefly, mice were immunized with 1200 HA units of PR8 in the form of allantoic fluid and boosted with Vac-Kd HA in PBS. Single-cell suspensions were prepared from the spleens of the immunized mice and seeded with 6x106 cells/well in 1 ml of RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (Gemini Bio-Products Calabas, CA), 2 mM glutamine (Life Technologies, Grand Island, NY), 5x10–5 M ß-mercaptoethanol (Sigma, St Louis, MO) and 50 mg/ml gentamycin (Gemini BioProducts), and 6x106 cells/well irradiated (3000 rad) syngeneic antigen-presenting cells (APC) pulsed with a final concentration of 5x10–6 M HA peptide for InsHA CTL or 25x10–9 M for D2 CTL in 1 ml RPMI 1640 complete medium. The cell lines were maintained at 37°C in a humidified incubator with 5% CO2. The InsHA and D2 CTL lines that had been maintained for at least eight passages were cloned by limiting dilution. Cultures were checked weekly, and wells showing cell growth were sequentially expanded into 48- and 24-well plates, and tested for cytolytic activity. Two clones, D2 Clone 6 and InsHA Clone 12 obtained from B10.D2 and InsHA mice, were identified and selected for further study.

Flow cytometry
CD8 and TCR expression was examined on day 10 activated CTL clones to correlate with in vitro signaling experiments, whereas other cell surface markers were assessed on day 4 CTL to correlate with adoptive transfer experiments. CTL (1x106) clones were resuspended in 50 µl of FACS buffer [HBSS containing 1% (w/v) BSA and 0.02% (w/v) sodium azide] and incubated for 20 min on ice with the indicated antibodies, which were either directly conjugated with FITC or biotinylated (PharMingen, La Jolla, CA). When biotinylated antibodies were used, cells were washed 3 times after incubation with the antibody and then incubated with FITC–streptavidin (PharMingen) on ice for 20 min. Cells were fixed in 1 % paraformaldehyde (Sigma, St Louis, MO) and analyzed on a FACScan (Becton Dickinson, Mountain View, CA) using Macintosh CellQuest software.

Cytokine/chemokine production by HA-specific CTL
To assess the cytokine/chemokine production by the CTL used in this study, RNA was extracted from 50x106 CTL on day 4 following stimulation with antigen-pulsed APC. D2 Clone 6 was stimulated with APC pulsed with 8x10–9 M Kd HA peptide, whereas InsHA Clone 1 and InsHA Clone 12 CTL were stimulated with APC pulsed with 8x10–6 M. RNase protection assays were performed as described previously (12). The RNA samples were hybridized with [32P]UTP-labeled multiprobe sets for the detection of various cytokine (probe set ML-11) (13) or chemokine (probe set CK1) (14) RNAs. For both probe sets, a fragment of the RPL32-4A gene (15) served as an internal loading control. For quantitation, autoradiographs were scanned and analyzed by densitometry using NIH Image 1.47. The densitometric value for each transcript was expressed as a ratio to the L32 RNA.

In vivo activity of HA-specific CTL clones
For adoptive transfer experiments, day 4 activated CTL clones were resuspended in HBSS and 10x106 cells/animal were injected into irradiated (600 rad) InsHA mice i.v. The blood glucose level was monitored periodically using a chemistrip and glucometer (Boehringer Mannheim, Indianapolis, IN) and mice that had a blood glucose levels >300 mg/dl were considered diabetic.

Histochemistry
Pancreata were excised, fixed in 10% (v/v) formalin solution (Sigma) and processed for paraffin embedding. Paraffin-embedded tissue was cut using a microtome and sections were placed onto saline-coated Superfrost slides for processing (Fisher Scientific, Pittsburg, PA). Tissue sections were deparaffinized in xylene and gradually rehydrated in graded aqueous/alcohol solutions, beginning with 100% ethanol and ending in distilled water. Serial sections of paraffin-embedded tissue were stained with eosin and all slides were counterstained with Mayer's hematoxylin (both reagents from Sigma).

Immunoprecipitation and phosphorylation assays
To examine the pattern of protein tyrosine phosphorylations in CTL clones, 10x106 CTL (day 10 activated CTL clones were used in all the signaling experiments unless otherwise mentioned in the text) were stimulated with 10x106 B10.D2 fibroblast cells alone or these same cells that were previously pulsed with 5 µg of HA peptide (final peptide concentration was 5 µM in all the signaling experiments unless otherwise mentioned in the text) at 37°C for each time point specified in the text and figures. In experiments where anti-CD3 (2C11) was used for stimulating T cells, 10x106 CTL were stimulated with 5 µg of 2C11 alone or with 5 µg of 2C11 and 10 µg rabbit anti-hamster antibody (PharMingen) at 37°C for the time periods indicated in the text and figure. The cells were washed twice in lysis buffer without Nonidet P-40 and then lysed in lysis buffer containing a final concentration of 1% Nonidet P-40, 2 mM Tris–Cl, pH 7.4, 5 mM EDTA, 150 mM sodium chloride, 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 2 mM sodium vandate, 5 µg/ml each aprotonin and leupeptin, and 1 mM PMSF (all of these were purchased from Sigma). Cell lysates were subjected to immunoprecipitation using optimal amounts of either anti-TCR {zeta} (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-ZAP-70 (Upstate Biotechnology, Lake Placid, NY) antibodies respectively followed by incubation with 20 µl of Sepharose A/G (Santa Cruz Biotechnology) for 30 min at 4°C. The beads were extensively washed with lysis buffer, and the proteins bound to the beads were eluted by adding 2xSDS buffer and boiling the samples at 95°C for 5 min. The proteins were resolved by 10% SDS–PAGE, transferred to nitrocellulose (BioRad, Hercules, CA), immunoblotted with anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology) or protein-specific antibodies followed by incubation with horseradish peroxidase- conjugated secondary antibody (BioRad) and developed by the enhanced chemiluminescence system (Amersham, Arlington, IL).

Functional activity of Lck
CTL clones (10x106) were stimulated with 3x106 B10.D2 fibroblasts alone or these same cells that were pulsed with 5 µg of HA peptide for the time period specified in the text and figure. The cells were then processed as described above. The cell lysates were then subjected to immunoprecipitation with 10 µg of anti-mouse TCR ß antibody H57-597 (PharMingen) bound to Sepharose A/G beads (Santa Cruz Biotechnology) for 4 h at 4°C on a rocker. The immunoprecipitates were washed 3 times with wash buffer containing 1% Nonidet P-40, 2 mM Tris–Cl, pH 7.4, 5 mM EDTA, 150 mM sodium chloride, 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 2 mM sodium vandate, 5 µg/ml each aprotonin and leupeptin, 1 mM PMSF (all of these were purchased from Sigma), and 2 times with kinase buffer without ATP. The immunoprecipitates were resuspended in kinase buffer containing a final concentration of HEPES 10 mM, MnCl2 5 mM, MgCl2 5 mM, DTT 5 mM, Nonidet P- 40 0.1%, 1 µM ATP and 10 µCi [32P]ATP (New England Nuclear, Boston, MA). The reaction was carried out at 30°C for 15 min and terminated by addition of 5xSDS buffer. The proteins were eluted from the beads by boiling at 95°C for 5 min, and fractionated by SDS–PAGE, transferred to nitrocellulose membrane and developed by autoradiography. The blot was reprobed with anti-Lck antibody, 3A5 (Santa Cruz Biotechnology), to confirm the presence of Lck in the TCR complex immunoprecipitates.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Phenotypic and functional characterization of high- and low-affinity CTL clones
The two clones selected for this study, B10.D2-derived D2 Clone 6 and InsHA-derived Clone 12, have been previously described (10). We have previously shown that cell surface molecules that could potentially affect in vivo function such as CD11{alpha}, CD25, CD44, CD49d and CD62L were expressed at similar levels on D2 and InsHA-derived CTL (10). These clones express similar levels of TCR and CD8 on their cell surfaces as assessed by flow cytometry (Fig. 1Go). Each clone was found to express a single TCR, yet they differed significantly with respect to their avidity for HA, as assessed by the amount of antigen required to induce cytolytic activity and production of IFN-{gamma} (10). They also differed in their ability to bind tetrameric Kd HA molecules, which is an indicator of the affinity of the TCR expressed by these clones (10,1619).



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Fig. 1. Flow cytometric analysis of cell surface markers. D2 Clone 6 and InsHA Clone 12 CTL clones were stained for TCR and CD8 expression on their surface as described in Methods. Ten days after antigenic stimulation, CTL were incubated for 20 min with the indicated FITC-conjugated antibody (PharMingen). Negative controls included cells stained with matched isotype antibody directly conjugated with FITC or cells stained with streptavidin–FITC wherever biotinylated antibodies were used. Analysis of samples was conducted using a FACScan and CellQuest software (Becton Dickinson).

 
In order to determine if there were qualitative differences in the cytokine/chemokine production by these clones that could potentially affect their in vivo function, the clones were assessed for expression of a panel of cytokines and chemokines (Fig. 2a and bGo). We also included another low-affinity CTL clone, InsHA Clone 1, in these assays. The high- and low-affinity CTL clones were stimulated in vitro with APC pulsed with the optimum concentration of peptide required for their propagation and effector function, 8x10–9 and 8x10–6 M peptide respectively. No difference was found in these clones with respect to the specific cytokines and chemokines they transcribed at day 4. Thus, our results show that there was no defect per se in the low-affinity clones to transcribe these cytokines and chemokines following in vitro stimulation with Kd HA peptide. This time point was selected as it was to be used to assess in vivo activity of these same clones, as described below. Taken together with our previous findings (10) these results indicate that when normalized for differences in the concentration of the antigen required to trigger activation, no qualitative differences were observed between the high- and low-avidity HA-specific CTL with respect to expression of key activation and effector molecules.




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Fig. 2. (A) Depiction of relative levels of cytokine gene transcription in day 4 activated CTL clone using RNase protection assay. (B) Depiction of relative levels of gene transcription for a panel chemokines in day 4 activated CTL clones using RNase protection assay.

 
Diabetogenic potential of D2 Clone 6 and InsHA Clone 12
D2 Clone 6 and InsHA Clone 12 were compared with respect to their ability to induce diabetes in InsHA mice. D2 Clone 6 and InsHA Clone 12 were stimulated in vitro with APC-pulsed 8x10–9 and 8x10–6 M peptide. Four days following in vitro activation, 10x106 D2 Clone 6 or InsHA Clone 12 were adoptively transferred into irradiated InsHA recipients. Animals were monitored for blood glucose levels. On day 4 after transfer, 75% (thrre of four) of the animals that had received D2 Clone 6 were diabetic (>300 mg/dl blood glucose). In contrast, the mice that had received InsHA Clone 12 did not develop disease (Table 1Go). By day 8, 100% (four of four) of the animals that had received D2 Clone 6 were diabetic, whereas all the animals that received D2 Clone 12 remained healthy (Table 1Go). The InsHA Clone 12 recipients were further monitored up to day 21 for diabetes and remained euglycemic (data not shown). On day 8 following transfer of cells, pancreata were removed and sections prepared for histology. Islets either remained clear or had few infiltrating cells in animals that had received InsHA Clone 12 (Fig. 3AGo), whereas strong infiltration of islets was observed in animals that had received D2 Clone 6 (Fig. 3BGo). It should be noted that InsHA Clone 12 was not able to induce diabetes even when the number of T cells used in adoptive transfer experiments was increased by 10-fold (data not shown).


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Table 1. D2 Clone 6 but not InsHA Clone 12 induces diabetes in syngeneic recipients
 


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Fig. 3. Differences in lymphocyte infiltrates in pancreata of InsHA recipients following adoptive transfer of D2 Clone 6 and InsHA Clone 12. (A) Pancreata of InsHA mice that received InsHA Clone 12 were processed and stained for infiltration of islets 8 days post transfer. (B) Pancreata of InsHA mice that received D2 Clone 6 were processed as described above. Arrows indicate the infiltration of lymphocytes in the islets.

 
D2 Clone 6 and InsHA Clone 12 differ in their ability to phosphorylate TCR {zeta} chain and ZAP-70 following stimulation with the cognate ligand
In an attempt to correlate the diabetogenic potential of each clone with the strength of its response to HA, we investigated the proximal signaling events that occurred in each clone as a result of encounter with the antigen. Ten days after antigenic stimulation, quiescent CTL were incubated for the indicated time points with 10x106 B10.D2 fibroblasts or these same cells previously pulsed with 5 µM of HA peptide (Fig. 4Go). This high concentration of antigen was selected to assure maximal stimulation of the low-affinity InsHA Clone 12. As shown in Fig. 4Go[a, phosphotyrosine (pY) Blot, lanes 2–4], the complete phosphorylation of the TCR {zeta} chain was observed in D2 Clone 6 as both the p21 and the p23 forms were observed following stimulation with the peptide. In contrast, only the p21 form of the TCR {zeta} chain was observed to be phosphorylated when the InsHA Clone 12 was stimulated with the cognate peptide, indicating that only partial phosphorylation of the TCR {zeta} chain had occurred (Fig. 4aGo, pY Blot, lanes 6–8). Moreover, we could not detect complete phosphorylation of the TCR {zeta} chain in InsHA Clone 12 following peptide stimulation for up to 20 min, whereas a complete and sustained phosphorylation of TCR {zeta} chain was observed in D2 Clone 6 (data not shown). Lanes 1 and 5 in the phosphotyrosine blot in Fig. 4(a)Go depict unstimulated controls. Both clones expressed comparable amounts of TCR {zeta} as shown by reprobing the phosphotyrosine immunoblot with the anti-TCR {zeta} antibody (Fig. 4aGo, Zeta Blot).






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Fig. 4. Differences in TCR {zeta} and ZAP-70 phosphorylation in D2 Clone 6, InsHA Clone 12 and D2 Clone 1 following stimulation with the cognate HA peptide. (A) For each sample 10x106 D2 Clone 6 or InsHA Clone 12 were stimulated for the time points indicated (30 s to 5 min) with 10x106 APC alone (lanes 1 and 5) or these same APC pulsed with 5 µg peptide (lanes 2–4 and 6–8). Tyrosine phosphorylation of the {zeta} chain in the anti-TCR {zeta} immunoprecipitated complexes was visualized by anti-phosphotyrosine immunoblotting (pY Blot). All sample contained comparable levels of TCR {zeta} as shown in the Zeta Blot. (B) D2 Clone 6 and InsHA Clone 12 were stimulated as described in (A) for 2–20 min (lanes 2–5 and 7–10). Lanes 1 and 6 depict CTL clones stimulated with APC alone. Tyrosine phosphorylation of ZAP-70 in anti-ZAP-70 immunoprecipitated complexes was visualized by anti-phosphotyrosine immunoblotting (pY Blot). The total amount of ZAP-70 in all the samples is shown in the ZAP-70 Blot. (C) Tyrosine phosphorylation of the {zeta} chain in the anti-TCR {zeta} immunoprecipitated complexes from D2 Clone 6 and InsHA Clone 1 respectively following treatment as described in (A). Complexes were visualized by anti-phosphotyrosine immunoblotting (pY Blot). Lanes 1 and 5 depict CTL clones stimulated with APC alone. (D) Tyrosine phosphorylation of ZAP-70 in anti-ZAP-70 immunoprecipitated complexes from D2 Clone 6 and InsHA Clone 1 following treatment as described in (A). Immunoprecipitated complexes were visualized by anti-phosphotyrosine immunoblotting (pY Blot). Lanes 1 and 5 depict CTL clones stimulated with APC alone.

 
We next examined phosphorylation of ZAP-70 in the two clones following stimulation with peptide. As shown in Fig. 4Go(b, pY blot), phosphorylation of ZAP-70 was observed in D2 Clone 6 following stimulation of the clone with the peptide. The phosphorylation of ZAP-70 could be observed from 2 to 20 min in D2 Clone 6, suggesting a sustained response to the peptide (Fig. 4bGo, pY Blot, lanes 2–5). In contrast, we could not detect phosphorylation of ZAP-70 in the InsHA Clone 12 following stimulation with cognate antigen (Fig. 4bGo, pY Blot, lanes 7–10). Lanes 1 and 6 in the phosphotyrosine blot in Fig. 4(b)Go depict unstimulated controls. Both of the clones expressed the ZAP-70 protein as indicated by reprobing the immunoblot with anti-ZAP-70 antibody (Fig. 4bGo, ZAP-70 Blot). Thus, our results clearly demonstrate profound differences in the proximal signaling events in the two clones.

To rule out the possibility that the deficiency in proximal signaling observed in the InsHA Clone 12 was not limited to one low-affinity clone but rather a general characteristic of all low-affinity clones, we tested another low-affinity clone, InsHA Clone 1 (also referred to as Clone 1) that was derived from a different InsHA mouse. As shown in Fig. 4Go(c and d, pY Blot), following stimulation of Clone 1 with the peptide, incomplete TCR {zeta} and no ZAP-70 phosphorylation could be observed. In contrast, complete proximal signaling was observed in D2 Clone 6 following stimulation with the HA peptide (Fig. 4c and dGo, pY Blot). Therefore, both HA-specific clones derived from InsHA mice demonstrate the same phenotype.

D2 Clone 6 and InsHA Clone 12 have functionally active Lck associated with TCR
Lck, a member of the src-kinase family, is responsible for the proximal signaling cascade in T cells that follows antigenic stimulation (20,21). Furthermore, Lck activity has been shown to have a profound affect on T cell development and function (2324). Thus, we wanted to exclude the possibility that a defect in the Lck activity in InsHA Clone 12 was responsible for the defective upstream signaling events observed in this clone. Both the high- and low-affinity clones respectively were stimulated as before for the time periods indicated in Fig. 5Go with either APC alone or APC pulsed with 5 µg peptide. Following stimulation, the TCR from these two clones was immunoprecipitated and then subjected to a standard in vitro kinase assay (IVK) (Fig. 5Go). In D2 Clone 6, as well as InsHA Clone 12, Lck was functionally active as demonstrated by its ability to undergo autophosphorylation following peptide stimulation (compare APC + T cell Alone and APC + T cell + Peptide lanes, Fig. 5Go, IVK). Both clones had Lck associated with their TCR as assessed by reprobing the immunoblot with anti-Lck antibody, 3A5 (Fig. 5Go, anti-Lck Blot). Small amounts of Lck were constitutively associated with the TCR of both clones (Fig. 5Go, APC + T cell Alone lanes). These results clearly indicate that in InsHA Clone 12 there was no defect in either the recruitment of Lck to the TCR or its ability to undergo in vitro autophosphorylation.



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Fig. 5. Recruitment of active Lck to the TCR complexes of D2 Clone 6 and InsHA Clone 12 following peptide stimulation. D2 Clone 6 and InsHA Clone 12 were compared for Lck autophosphorylation in a standard IVK. This assay determines the functional activity of Lck that was recruited to the TCR complexes of the CTL clones. In this assay 10x106 D2 Clone 6 or InsHA Clone 12 were stimulated with 3x106 APC alone (Lanes 1–3 and 7–9) or pulsed with 5 µg HA peptide (lanes 4–6 and 10–12) for 30 s to 5 min (IVK). The lysates were mixed with immunoprecipitating anti-TCR antibody and Sepharose A/G beads for 4 h at 4°C, washed and used for IVK, and the proteins were resolved on SDS–PAGE, transferred to nitrocellulose membrane and developed by autoradiography. The blot was reprobed with anti-Lck antibody 3A5 to confirm the presence of Lck (Anti LCK Blot) in all the samples tested.

 
Increasing the strength of signal through the TCR can restore ZAP-70 phosphorylation in InsHA Clone 12
The results obtained thus far are consistent with the interpretation that the incomplete signaling in the InsHA Clone 12 that was derived from the tolerized InsHA mouse was due to low affinity of its TCR for the cognate antigen. Accordingly, it would be anticipated that by enhancing the signal through TCR–CD3 complex we could obtain phosphorylation of ZAP-70 in this clone. To determine if this was the case, we increased the strength of signal by ligating the CD3{varepsilon} of the CTL clones with anti-CD3 antibody. D2 Clone 6 and InsHA Clone 12 were stimulated with anti-CD3{varepsilon} (2C11) alone or with anti-CD3 and rabbit anti-hamster antibody for the time points shown in Fig. 6Go. As shown, stimulation of the clones with anti-CD3 cross-linked by the secondary antibody induced a rapid and sustained phosphorylation of ZAP-70 in D2 Clone 6 (Fig. 6Go, pY Blot) as well as InsHA Clone 12 (Fig. 6Go, pY Blot). In fact anti-CD3 produced a stronger signal in InsHA Clone 12 than D2 Clone 6. Both the CTL clones expressed similar levels of ZAP-70 protein (Fig. 6Go, ZAP-70 Blot). This result confirmed that the proximal signaling machinery in InsHA Clone 12 was fully functional.



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Fig. 6. Anti-CD3 stimulation induces phosphorylation of ZAP-70 in D2 Clone 6 and InsHA Clone 12. 10x106 cells of D2 Clone 6 or InsHA Clone 12 were stimulated with anti-CD3 alone for 2 min (lanes 1 and 6) or with anti-CD3 + rabbit anti-hamster antibody for 30 s to 10 min (lanes 2–5 and 7–10). Tyrosine phosphorylation of ZAP-70 in anti-ZAP-70 immunoprecipitated complexes was visualized by anti-phosphotyrosine immunoblotting (pY Blot). All the samples contained similar levels of ZAP-70 as shown in the ZAP-70 Blot.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have attempted to define the molecular basis of tolerance in the InsHA model by correlating differences in the ability of HA-specific CD8+ T cells from non-tolerant (D2 Clone 6) and tolerant (InsHA Clone 12) mice to initiate diabetes in vivo with the biochemical events that occur upon recognition of HA in vitro. The high- and low-affinity clones used in this study closely reflected the in vivo and in vitro characteristics of the populations from which they were derived (10).

Although D2 Clone 6 and InsHA Clone 12 express similar levels of TCR and CD8 on their cell surface and each express a single TCR {alpha}ß heterodimer, we have previously shown that the affinity of the TCR expressed by these clones varies significantly (10). The TCR of D2 Clone 6 has a higher affinity than that of InsHA Clone 12 for the HA epitope. As a consequence, D2 Clone 6 is able to lyse targets pulsed with lower amounts (10–10 M) of HA peptide than is required to achieve lysis by InsHA Clone 12 (10–8 M) (10). Moreover, this in vitro lysis was perforin mediated and independent of Fas–Fas ligand pathway (10). Also, previous work from our laboratory has shown that D2 Clone 6 can produce significantly higher amounts of IFN-{gamma} than InsHA Clone 12 when challenged with a limiting concentration of peptide (10). However, at high peptide concentrations InsHA Clone 12 was able to produce levels of IFN-{gamma} comparable to D2 Clone 6. We have now extended this analysis of the pattern of cytokines/chemokines expression by the high- and low-affinity CTL following in vitro activation with APC pulsed with optimum amounts of peptide. These results suggest no qualitative differences between these clones. Most significantly, we now show that adoptive transfer of D2 Clone 6 into irradiated InsHA recipients results in the rapid induction of diabetes, whereas transfer of InsHA Clone 12 is unable to induce disease in these animals. Disease was correlated with strong islet infiltration in animals that received D2 Clone 6 as demonstrated by histochemistry, whereas islets remained free of infiltrates in animals that had received InsHA Clone 12. Taken together, these results indicate that the affinity of the TCR is an important parameter in determining the pathogenicity of HA-specific CD8+ T cells in the InsHA mice.

To further understand the mechanism(s) that governed functional differences in CTL response to the HA peptide we studied the proximal TCR signaling events in these two clones following antigenic stimulation. Our results showed that TCR engagement by the cognate ligand in D2 Clone 6 led to a complete and sustained phosphorylation of the TCR {zeta} chain and ZAP-70. In contrast, InsHA Clone 12 and InsHA Clone 1 demonstrated only partial phosphorylation of the TCR {zeta} chain and with no phosphorylation of ZAP-70. The altered biochemical events observed in the low-affinity CTL clones correlated with in vivo and in vitro function.

Lck has been strongly implicated in the initiation of proximal T cell signaling events such as ZAP-70 phosphorylation (20,21), and has also been shown to be essential for activation and development of T cells (2324). Our results showed that these differences in phosphorylation were not due to a deficiency in Lck activity in InsHA Clone 12 as Lck was recruited to the TCR following peptide stimulation and was capable of autophosphorylation in both clones. However, it is tempting to speculate that the sub-optimal signals generated by the engagement of the InsHA Clone 12 TCR by HA peptide may lead to the recruitment of negative regulators of proximal signaling such as phosphatase SHP-1 to the TCR which would then dephosphorylate Lck and ZAP-70, and attenuate the proximal signaling events in this clone. A recent study has demonstrated the recruitment of SHP-1 to a TCR that had been engaged by an antagonist ligand (25).

Further evidence that there was no defect per se in the proximal signaling machinery of InsHA Clone 12 was obtained by signaling the cells with anti-CD3. By increasing the strength of signal through the CD3–TCR complex with anti-CD3 we were able to restore the proximal signaling events in InsHA Clone 12. This result also correlates well with earlier results from our laboratory that showed that the D2 and InsHA-derived populations were comparable in their ability to lyse P815 targets coated with varying amounts of anti-CD3 antibody (10). Hence, a stronger signal through the CD3–TCR complex was needed to initiate these signaling events in InsHA Clone 12 due to the lower affinity of the TCR. Thus, the tolerant T cells that express a lower affinity TCR have a higher threshold of activation and consequently require a stronger strength of signal through the TCR as compared to the higher-affinity T cells in order to express similar function. Considering that a stronger TCR signal, as delivered by anti-CD3, can restore the complete proximal signaling events in InsHA Clone 12, it is tempting to speculate that if the low-affinity HA-specific T cell repertoire could be hyperstimulated by a superagonist ligand it may be possible to potentiate an autoimmune response to HA in the InsHA mice as has been demonstrated by a recent study using low-affinity CD4+ T cells (26).

The partial signaling observed upon incubation of Clone 12 with high concentrations of HA is similar to results obtained in studies that have used low-affinity antagonist peptides to signal T cells. Although a number of mechanisms have been proposed to explain the functional and biochemical differences between antagonist and agonist peptides, recent studies show that antagonists have a faster off-rate as compared to the agonist ligands (2730). Consequently, this rapid dissociation of TCR–ligand complex results in only a partial biochemical and functional response (3133). However, we would conclude that HA-specific T cells from mice tolerant to HA respond to this self-antigen as a partial agonist/antagonist. Interestingly, a recent study using CD4 T cells has shown that partially phosphorylated TCR {zeta} can inhibit T cell activation by perhaps inhibiting the activity of positively acting kinases (34). In our study we observed the recruitment of Lck into the TCR complex of the low-affinity CTL following activation with the peptide but did not observe the subsequent generation of proximal signaling events. Thus, our results may be more consistent with studies that show recruitment of a negative regulators such as SHP-1 into the TCR complex due to sub-optimal ligation of the TCR, which could dephosphorylate the Lck and consequently attenuate the generation of optimum proximal signaling events in the low-affinity clones.

Our system differs from that previously described with antagonist peptides in that the differences in the strength of signaling are due to differences in the affinity of the TCR rather than differences in the peptide sequence used for activation. This study is also novel with respect to the fact that we have attempted to correlate the proximal signaling events in T cells from tolerant and non-tolerant mice with their ability to cause disease in vivo.

We previously demonstrated that peripheral tolerance in the InsHA mice affects the affinity of the TCR of HA-specific CD8+ T cells. Consequently, this results in an altered functional response in vivo as assessed by the inability of these T cells to induce diabetes in syngeneic hosts, which correlates with incomplete proximal signaling events such as partial TCR {zeta} and no ZAP-70 phosphorylation in vitro. Of interest, a correlation between the avidity of T cells specific for an islet antigen has recently been demonstrated to be important in the breakdown of tolerance in the diabetes-prone NOD mouse (35).

In conclusion, our studies have correlated the strength of signal transduction through the TCR with the diabetogenic potential of a CD8+ T cell. Tolerance in InsHA mice affects the affinity of T cells which leads to altered functional and biochemical responses by the HA-specific T cells that remain in the repertoire.


    Acknowledgments
 
We thank J. Hernandez and Dr A. Altman at the La Jolla Institute of Allergy & Immunology for their help. This work was supported by National Institutes of Health grants: 2 T32 AG 00080-19, DK57644-01 and DK50824. V. C. A. was a post-doctoral fellow of the National Multiple Sclerosis Society. I. L. C. was supported by MH 50426.


    Abbreviations
 
APC antigen-presenting cell
CTL cytotoxic T lymphocyte
HA hemagglutinin
IVK in vitro kinase assay
ZAP-70 {zeta}-associated protein 70 kDa

    Notes
 
Transmitting editor: S. L. Swain

Received 19 February 2001, accepted 16 May 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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