Gwen Knapp Center for Lupus and Immunology Research,
1 Department of Pathology,
2 Ben May Institute for Cancer Research and
3 Committee on Immunology, University of Chicago, 924 E. 57th Street, R414, IL 60637, USA
Correspondence to: P. G. Ashton-Rickardt
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: MHC, T cell differentiation, thymocyte development
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
T cells acquire the capacity for MHC-restricted antigen recognition through two types of selection in the thymus (9). Positive selection (10,11) ensures that T cells leaving the thymus express TCR that are capable of MHC-restricted antigen recognition. Engagement of TCR on immature thymocytes with self-peptide/MHC molecules, expressed by thymic epithelial cells, rescues cells from programmed cell death (12,13). Thymocytes expressing TCR specific for class II MHC differentiate into CD4+ Th cells and those with TCR specific for class I MHC differentiate into cytotoxic T lymphocytes (CTL) (14). Negative selection eliminates those T cells that are potentially autoreactive through clonal deletion (15) and is thought to involve engagement of TCRs on immature thymocytes with self-peptide/MHC molecules expressed on the surface of bone marrow-derived thymic stromal cells (16,17). A central paradox is how similar interactions that take place between TCR and self-peptide/MHC complexes trigger different cell fates during the two types of thymic selection, and so give rise to T cells that recognize MHC-restricted antigen but are largely tolerant to self.
The nature of the MHC ligand that triggers the selection of CD8+ T cells has been studied in experiments using cultured fetal thymi from class I MHC-deficient mice (1822). In transporter of antigen-1 (TAP1)-deficient mice the surface expression of class I MHC is impaired, which results in a block in the positive selection of CD8+ cells (23). The surface expression of MHC class I molecules can be partially restored in cultured fetal thymi from TAP1 mice by the addition of peptide which stabilizes `empty' class I MHC molecules on thymic stromal cells (18). It was observed that the positive selection of CD8+ thymocytes was specific for the peptide presented by class I MHC on thymic stromal cells, which led to the view that the positive selection of CD8+ cells depends on the specific recognition of thymic self-peptides (18,24). Further studies indicated that the ability of non-thymic, antigen-variant peptides to induce the positive selection of CD8+ T cells, when added to cultured fetal thymi from TAP1 mice, was influenced by the density of peptide on stromal cells and the innate ability of peptides to stimulate the TCR (19). This led to a model of T cell selection in which the avidity of the TCR:peptide/MHC interaction is a critical parameter in determining thymocyte cell fate (19,25,26). Avidity is defined as the overall strength of interaction and is the product of the affinities of interaction between TCR (including CD4 or CD8 co-receptors) and peptide/MHC and their respective cell densities. High avidity interactions give negative selection and low-avidity interactions give positive selection. This model predicts that the affinity of self-peptide/class I MHC for thymocyte TCR determines whether a cell undergoes positive or negative selection. Furthermore, the model implies that self-peptide specificity of CD8+ cells selection would be a result of differences in affinity between TCR and self-peptide/class I MHC molecules. However, to date there is no direct evidence that the affinity between TCR and self-peptide/MHC molecules, which are actually expressed in the thymus, determines the developmental fate of thymocytes.
To examine the role of thymic self-peptides in positive and negative selection of CD8+ cells, we measured the binding of thymic self-peptide/H-2Db molecules to a TCR expressed by CD8+ cells. The B6.2.16 TCR (27) expressed on CD8+ cells recognizes a male-specific, self-peptide negative (HY, human Y) that is expressed in the thymus (28,29). We demonstrate the high-affinity binding of multimeric HY/H-2Db tetramers to B6.2.16 on CD8+ cells (referred to as B6.2.16 CD8 cells hereafter). The B6.2.16 TCR bound non-antigenic, thymic self-peptide/H-2Db tetramers over a range of affinity that was significantly lower than that of the TCR for the antigenic HY/H-2Db tetramer. We examined the consequences of TCR binding to self-peptide/H-2Db on the positive and negative selection of B6.2.16 CD8 cells in fetal thymi from TAP1-deficient mice. We found that the relative affinity of self-peptide/H-2Db for the B6.2.16 TCR determined the ability of the self-peptides to induce the positive or negative selection of B6.2.16 CD8 thymocytes. The self-peptides in our experiments are expressed at high levels in the thymus; therefore, our findings are of general physiological relevance. High-affinity binding of B6.2.16 TCR by the HY/H-2Db resulted in negative selection and low-affinity binding by non-agonist, self-peptide/H-2Db molecules resulted in positive selection. Relatively small differences in self-peptide/MHC affinity for thymocyte TCR determined whether self-peptides could induce positive selection and so would account for the self-peptide specificity of B6.2.16 CD8 cell positive selection.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein expression
Inclusion bodies of human ß2-microglobulin (hß2m) or the mouse H-2Dbheavy chain with a biotinylation tag (biotH-2Dbheavy chain) were recovered from Escherichia coli using a modified version of a previously described procedure (31). In brief, BL21 (DE3) pLysS cells were grown at 37°C in LuriaBertani medium containing 50 µg/ml carbenicillin (hß2m) or 50 µg/ml kanamycin (biotH-2Dbheavy chain). Isopropyl ß-D-thiogalactopyranoside (IPTG; Sigma, St Louis, MO) was added to a final concentration of 0.5 mM when the culture reached an OD600 of ~0.6. After 4 h, the bacteria were harvested and lysed by repeated sonication in lysis buffer [20 mM TrisHCl (pH 8.0), 23% sucrose, 1 mM EDTA] containing 0.5% Triton X-100 and 40 µg/ml DNase I (Boehringer Mannheim, Mannheim, Germany). After two washes with lysis buffer, the purified inclusion bodies were dissolved in 20 mM TrisHCl (pH 8.0), 4 M urea.
Refolding and purification tetrameric peptide/H-2Db complexes
Peptides were dissolved in 20 mM Tris (pH 8.0), 4 M urea and then mixed with biotH-2Dbheavy chain and hß2m at a molar ratio of 3:3:1. The mixtures were dialyzed against 20 mM TrisHCl (pH 8.0), 150 mM NaCl at 4°C in Spectra Por CE dialysis tubing (Spectra, mol. wt cut of 500 kDa; Spectrum, Houston, TX) for 48 h. The soluble fraction was incubated for 1 h at 37°C and centrifuged at 15,000 g to remove precipitates. Correctly folded peptide/H-2Db complexes were purified using an anti-H-2Db mAb affinity column (B22-249R1) that was coupled at a density of 10 mg/ml to Protein A resin (Pharmacia, Uppsala, Sweden) with dimethylsuberimidate (Sigma) using standard procedures (32). Upon elution with 100 mM triethylamine (pH 11.5) and neutralization with 1 M sodium phosphate buffer (pH 6.8), the trimeric complexes were dialyzed into PBS and stored at 4°C or directly used for experiments. All purified complexes were subjected to analytical size exclusion FPLC on a Superdex G-200 column (Pharmacia) and showed a homogeneous peak (>95% purity) with the retention time of an ~45 kDa protein.
For biotinylation, the peptide/hß2m/biotH-2Dbheavy chain complexes were dialyzed overnight into 20 mM TrisHCl (pH 8.0), 50 mM NaCl. Typically, 12 mg of protein at a concentration of 12 mg/ml was incubated overnight with 3 µg of the enzyme BirA (Avidity, Denver, CO) in biotinylation buffer [20 mM TrisHCl (pH 8.0), 50 mM NaCl, 50 mM Bicine (pH 8.3), 10 mM ATP, 10 mM Mg acetate, 40 µM biotin, 1 mM pefabloc (Boehringer Mannheim), 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin (Sigma)]. The uncoupled biotin was removed by dialysis against PBS and the efficiency of the biotinylation checked by Western blotting with streptavidin (SA) horseradish peroxidase (HRP; PharMingen, San Diego, CA). To generate tetrameric peptide/MHC complexes, the biotinylated MHC complexes were mixed in a molar ratio of 8:1 with R-phycoerythrin (PE)-labeled SA (PharMingen). The formation of the tetrameric complex was confirmed by analytical size exclusion FPLC on a Superdex G-200 column. Under these conditions, tetramer formation (> 200 kDa) was complete because we did not observe any uncoupled SAPE (60 kDa), or monomers (105 kDa), dimers (150 kDa) or trimers (195 kDa) of H-2DbSAPE. Therefore, for several different MHC tetramers prepared, each contained the same amount of SAPE.
Flow cytometric analysis
The following mAb were used for staining: anti-CD8 [allophycocyanin (APC)], anti-CD4 (PE), anti-CD44 (PE), anti-I-Ab (PE) and rat anti-mouse IgG1 isotype control (PharMingen) and anti-H-2Db B22.249R1 (1 domain specific) (5). The anti-B6.2.16 TCR clonotypic mAb (IgG1) T3.70 (33) was affinity purified on Protein G (Pharmacia) and FITC labeled using standard protocols (34). Staining of thymocytes was performed as described previously (26). Live cells were identified on the basis of forward and sideward light scattering properties. The log10 fluorescence resulting from mAb staining was determined on gated live cells using predetermined standard calibration of the flow cytometer. Data were analyzed using Cell Quest v1.2 software (Becton Dickinson, Mountain View, CA).
Intracellular IFN- expression by B6.2.16 CD8 cells was detected by staining with T3.70FITC and anti-CD8-APC. Upon fixation in 1% paraformaldehyde, the cells were permeabilized and stained in 0.3% saponin in PBS containing anti-IFN-
PE mAb (IgG2a) or the PE-labeled IgG2a isotype mAb control. After three washes in 0.03% saponin (PBS), IFN-
expression of B6.2.16 CD8 cells was analyzed by FACS.
MHC tetramer staining
Tetrameric PE-labeled peptide/MHC complexes were used for staining at the concentrations indicated. The staining was performed for 2 h at 25°C in FACS buffer (2% FCS, 0.1% sodium azide in PBS) and the cells were washed once with FACS buffer 10 min before analysis. CD8+ spleen cells from the following transgenic mice were analyzed by MHCtetramer staining and FACS: B6.2.16 recombination activation gene 1 (RAG1) (anti-HY/H-2Db) (17), D7 TCRa [anti-Listeria monocytogenes LemA 1-6 (LemA/H-2M3wt)] (35) and P14 RAG1 (anti-LCMVH-2Db) (30). To measure equilibrium binding of peptide/H-2Db molecules to the B6.2.16 TCR, splenocytes from B6.2.16 RAG1 mice were stained with MHC tetramers or the SAPE control at the same concentration. The samples were directly analyzed by FACS, without washing, and the mean staining intensity of MHC tetramerPE staining on CD8+ splenocytes determined. The mean specific fluorescence intensity (log10) (mean MHC tetramerPE fluorescence mean fluorescence of SAPE control) was plotted against the concentration of MHC-tetramers.
Agonist assays
Male-specific B6.2.16 CTL were generated as described previously (29) and used at an effector to target ratio of 10:1 with 51Cr-labeled RMA cells (H-2b) (36). After 4 h, the degree of specific 51Cr release was determined in the presence of HY, BP, RR or HH peptides (1014 to 105 M) as (experimental release spontaneous release)/(maximum release spontaneous release) x 100, in triplicate. The maximum specific release obtained with the HY peptide was 60% of the maximum release. The specific release with the BP, RR or HH peptides up to concentrations of 105 M was <2%. In all experiments, the spontaneous release was 7% of the maximum release.
To measure antigenic activation, lymph node cells from B6.2.16 RAG1+ transgenic mice were seeded in 96-well round-bottom plates (2 x 105) and incubated in cRPMI-10 (RPMI 1640 containing 10% FCS, non-essential amino acids, 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, 100 µg/ml penicillinstreptomycin, 5.5 x 105 M ß-mercaptoethanol) for 6 days in the absence or presence of peptide (3 x 105 M). B6.2.16 CD8 cells were analyzed after each day of culture and analyzed by FACS for the expression of CD44 or intracellular INF-.
Half-life of peptide/H-2Db complexes
Purified H-2Db complexes at a concentration of 0.3 mg/ml in PBS/1% BSA were incubated at 37°C. Aliquots were taken at t=0 and at various time points during the incubation (up to 360 h). We determined the amount of peptide/H-2Db that reacted with the B22-249R1 anti-H-2Db mAb, which is conformation dependent, and so distinguishes between correctly and incorrectly folded H-2Db molecules (5). The different samples were subjected to a 2 h immunoprecipitation with an excess of B22.249R1-coupled Protein A resin followed by six washes with PBS containing 1% NP-40. The samples were taken up in SDSPAGE sample buffer with ß-mercaptoethanol, boiled for 5 min and separated on a 15% SDSPAGE gel. Upon staining with Coomassie blue, the gel was directly analyzed by densitometry, and the intensity of the H-2Dbheavy chain and the antibody light chain band was determined using Image Quant software (Molecular Dynamics, Sunnyvale, CA). To control for equal loading, the densitometric values of the H-2Dbheavy chain were corrected by the following factor: highest value of the antibody light chain band density of a sample on the gel/antibody light chain band of the analyzed sample. Assuming that the dissociation of H-2Db follows first- order kinetics, the percentage of correctly folded peptide/ H-2Db molecules was calculated: (the intensity of the H-2Dbheavy chain bands at a given time background intensity of gel)/(the intensity of the sample at t=0 background intensity of gel) x 100.
Fetal thymus organ culture (FTOC)
Mice expressing the B6.2.16 TCR (C57BL/6/129Sv) were crossed with TAP1-deficient mice (23) TAP1/, C57BL/6/129Sv) to generate B6.2.16+/+ TAP1/ mice. B6.2.16+/+ TAP1/ males were mated with TAP1/ or TAP1+/+ (C57BL/6) females to produce B6.2.16+/ TAP1/ and B6.2.16+/ TAP1+/ fetal thymic, and FTOC performed as described before (19). In brief, dissected fetal thymi (day 16 post-coitum) were separated into individual lobes and cultured for 10 days on sponges (Gelfoam; Upjohn, Kalamazoo, MI) and polycarbonate filters (Millipore 0.8 µm; Fisher Scientific, Pittsburgh, PA) in cRPMI-10 medium alone or in cRPMI-10 containing the indicated amount of peptide. The media with or without peptide were changed every 2 days.
Peptide/MHC class I complex stabilization analyses
Stabilization of peptide/MHC complexes on TAP1 thymic stromal cells was analyzed as described previously (30). In brief, day 16 fetal thymi were cultured for 3 days with BP, RR or HH (300 µM) or HY peptide (150 µM). On day 3, the cultured lobes were digested with trypsin (Gibco/BRL, Gaithersburg, MD). The remaining cell suspensions were stained first with anti-H-2Db (B22.249R1) followed by goat anti-mouse immunoglobulin G FITC (Southern Biotechnology Asso- ciates, Birmingham, AL) and then stained with anti-I-AbPE (PharMingen) and analyzed by FACS. The relative level of fluorescence intensity of anti-H-2Db staining on I-Ab-positive cells of TAP1 FTOC incubated with peptide was determined as the percentage of the level observed on stromal cells of the TAP1 FTOC minus the level observed on stromal cells of TAP1 FTOC incubated alone.
Anti-TCR/CD3 phosphotyrosine blots
A single-cell suspension of thymocytes from B6.2.16 RAG1 female mice was prepared and thymocytes (108) incubated for 10 min at 37°C in cRPMI-10 alone or in media containing MHC-tetramer (50 µg/ml), or the 145-2C11 mAb (anti-TCR CD3-chain; 20 µg) (37) in a total volume of 0.5 ml. The cells were lysed in lysis buffer [20 mM TrisHCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100] to which 50 mM sodium fluoride, 1 mM sodium vanadate, 30 mM sodium pyrophosphate, 0.5 mM phenylmethylsulfonyl fluoride was added. The cleared supernatant was immunoprecipitated with 1 µg of Protein A-bound mAb 145-2C11. After six washes with lysis buffer, the samples were taken up in SDSPAGE sample buffer with ß-mercaptoethanol, boiled for 5 min and separated on a 15% SDSPAGE gel. The gel was immunoblotted and probed with anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology, Lake Placid, NY) then by Protein AHRP (Amersham, Piscataway, NJ). Phosphorylated proteins were detected using chemoluminescence detection (ECL; Amersham). To control for equal loading the blot was stripped and reprobed using rabbit anti-TCR
chain serum (generous gift from P. Allen, Washington University, St Louis, MO) followed by anti-rabbit FcHRP (ICN, Costa Mesa, CA).
Quantitation of anti-HY CTL precursors
Thymocytes from B6.2.16 TAP1+ or B6.2.16 TAP1 FTOC were seeded in limiting dilution assays as described before (26). Limiting dilutions of cells were plated at doses that ranged from 1000 to 30,000 (24 replicate wells per cell dose) and co-cultured with irradiated female C57BL/6 spleen cells (1 x 106) in cRPMI-containing rIL-2 (10 u/ml; Gibco/BRL). At the beginning of the culture and every second day, HY peptide was added at a concentration of 107 M. After 7 days, the micro-cultures were assayed for cytolytic activity to 51Cr-labeled RMA target cells pulsed with HY peptide (107 M). The cytolytically positive cultures were defined as those with 51Cr release values exceeding the mean spontaneous release by >3 SD. The CTL precursor frequencies were calculated using the Poisson distribution (26).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
We next examined the contribution of TCR and CD8 to staining of cells by self-peptide/H-2Db tetramers (Fig. 2). To do this we compared the level of CD8 and TCR expression on CD8+ cells were different transgenic mice that expressed class I MHC restricted TCR. We found that CD8+ cells from mice expressing the D7 TCR, which is specific for the LemA peptide (fMIGWII), presented by the non-classical class I molecule H-2M3wt (35), expressed the same level of CD8 and CD3 as B6.2.16 CD8 cells (Fig. 2A
). We then determined the extent of peptide/H-2Db tetramer staining on the surface of D7 CD8 cells. To avoid possible cross-reaction of non-transgenic TCR on D7 CD8 cells with self-peptide/H-2Db tetramers, spleen cells were purified from D7 transgenic mice that lack the expression of endogenous TCR
chain, thus ensuring that the CD8 cells express only the D7 TCR (42). We observed no specific staining over background of D7 CD8 cells with HY/H-2Db or self-peptide/H-2Db tetramers (Fig. 2B
). Since the level of TCR was the same on CD8 cells expressing either the D7 (H-2M3wt restricted) or B6.2.16 (H-2Db restricted) TCR, we conclude that binding of self-peptide/H-2Db tetramers to B6.2.16 CD8 cells is specific for the B6.2.16 TCR. Furthermore, the expression of CD8 on D7 CD8 cells was not sufficient to mediate self-peptide/H-2Db staining, implying that the low level staining of B6.2.16 CD8 cells by these tetramers cannot be entirely due to weak interactions between CD8 and the
3 domain of H-2Db. Our findings demonstrate that the staining of B6.2.16 CD8 cells by self-peptide/H-2Db tetramers is a consequence of the binding of B6.2.16 TCR
ß chains to self-peptide and/or the
1/
2 domains of H-2Db.
|
|
|
The affinity of thymic self-peptides for the B6.2.16 TCR determines the positive selection of B6.2.16 CD8 cells
We wanted to determine whether the affinity between the B6.2.16 TCR and self-peptide/H-2Db molecules, expressed on thymic epithelial cells, determines the positive selection of B6.2.16 CD8 cells. To this end we analyzed the positive selection of B6.2.16 CD8 cells in TAP1+ and TAP1 cultured fetal thymi. After 10 days of culture we observed that the number of B6.2.16 CD8 cells recovered from female B6.2.16 TAP1 FTOC was 10 times less than female B6.2.16 TAP1+ FTOC, indicating a block in positive selection due to low class I MHC molecule expression (18,23) (Fig. 4A and B). Limiting dilution analysis revealed a correspondingly low frequency of anti-HY CTL precursors from female B6.2.16 TAP1 FTOC (Table 3
).
|
|
|
The inability of HH to induce B6.2.16 CD8 cell positive selection was not due to low density on TAP1 thymic stromal cells because the level of HH/H-2Db was about the same as BP/H-2Db and higher than RR/H-2Db (Table 4). Therefore, since the affinity of HH/H-2Db for the B6.2.16 TCR was lower than RR or BP, we conclude that the affinity of thymic self-peptide/H-2Db molecule for the B6.2.16 TCR is a critical parameter in determining whether self-peptides trigger the positive selection of B6.2.16 CD8 cells.
High-affinity binding of HY/H-2Db induces the negative selection of B6.2.16 CD8 cells
We next examined whether the affinity of thymic self-peptide/H-2Db binding to B6.2.16 TCR was a critical parameter in determining whether B6.2.16 CD8 cells underwent positive or negative selection. In male H-2b mice, thymocytes that express the B6.2.16 TCR undergo negative selection which results in a decrease in the number of CD4+CD8+ thymocytes in B6.2.16 transgenic mice (17). In male B6.2.16 TAP1+ FTOC, we observed a substantial decrease (14-fold) in the number of CD4+CD8+ thymocytes compared to female B6.2.16 TAP1+ FTOC, which is indicative of negative selection (Fig. 5A and B). The addition of HY peptide (150 µM) to B6.2.16 TAP1 FTOC resulted in a substantial decrease in the number of CD4+CD8+ (400-fold decrease) and B6.2.16 CD8 (3-fold) thymocytes (data not shown), whereas neither of the non-agonist self-peptides, which have a low affinity for B6.2.16 TCR, decreased the numbers of these thymocytes (Fig. 5B
). Thus the expression of HY/H-2Db but not BP, RR or HH self-peptide/H-2Db on thymic stromal cells induced the negative selection of B6.2.16 CD8 cells in B6.2.16 TAP1 FTOC. As shown in Table 4
, the level of HY/H-2Db on TAP1 thymic stromal cells that triggered negative selection of B6.2.16 CD8 cells was significantly lower than that of the other three self-peptides. Therefore the lack of negative selection after the addition of BP, RR or HH was not due to low expression on thymic stromal cells. We conclude that the negative selection of B6.2.16 CD8 cells is triggered by the high affinity binding of the B6.2.16 TCR to HY/H-2Db molecules on TAP1 stromal thymic cells.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiments with multivalent self-peptide/H-2Db tetramers revealed that the B6.2.16 TCR binds thymic self-peptide/H-2Db (Fig. 1). Antibody blocking experiments with the clonotypic T3.70 mAb (specific for the B6.2.16 TCR
chain) showed that the antigen-binding domain of the TCR interacts with the H-2Db. The potential reactivity self-peptide/H-2Db tetramers for CD8 was not sufficient to account for the staining of B6.2.16 CD8 cells (Fig. 2
). Thus, the binding of self-peptide/H-2Db tetramers was specific for the B6.2.16 TCR. The B6.2.16 TCR bound non-agonist, self-peptide/H-2Db tetramers at different affinities, i.e. the B6.2.16 TCR binding to H-2Db tetramers was self-peptide specific. This implies that the B6.2.16 TCR either interacts to some extent with the self-peptide entrenched in the peptide binding pocket of H-2Db, or that self-peptide indirectly influences TCR binding to the
1/
2 domains of H-2Db (45).
We found that the B6.2.16 TCR could bind three different non-agonist, self-peptide/H-2Db tetramers despite the fact that the potential TCR contact residues (P1P4; P6P8) of each self-peptide are dissimilar (Figs 1 and 3). Therefore, we conclude that the binding of the B6.2.16 TCR to self-peptide/H-2Db is quite degenerative (cross-reactive). The plasticity of TCR:self-peptide/H-2Db binding may be explained by the X-ray crystal structures of TCR:MHC class I complexes. These studies revealed a high proportion of contacts between TCR and MHC
1/
2 domain residues, which are independent of antigen or self-peptide entrenched in the binding pocket (3,4,46). Therefore, one could postulate that contacts between TCR and
1/
2 domains provide the necessary low-affinity binding that drives positive selection. The contribution of TCR CDR3 binding to self-peptide would be quite small and so a variety of different self-peptides can be recognized by the same TCR (46).
We found that a minimal affinity of self-peptide binding to the B6.2.16 TCR is required to trigger B6.2.16 CD8 cell positive selection. The BP and RR self-peptides had a higher affinity than HH for the B6.2.16 TCR. Only BP or RR, but not HH, could induced the positive selection of B6.2.16 CD8 cells, despite the fact that the selecting peptide on HH was present at a level equal to or higher than TAP1 thymic stromal cells (Table 1 and Fig. 4D
). Although RR had a higher affinity than BP for the B6.2.16 TCR, we found that the degree of B6.2.16 CD8 cell positive selection triggered by each self-peptide was about the same (Fig. 4D
). This is probably due to the fact that after the addition of self-peptides to B6.2.16 TAP1 FTOC the expression of RR on TAP1 thymic stromal cells was lower than that of BP (Table 4
) and is consistent with previous observations that peptide/MHC density, on thymic stromal cells, as well as affinity for thymocyte TCR determines the extent of positive selection (25).
The binding of the P14 TCR to H-2Db tetramers was more specific for the RR self-peptide than the binding of the B6.2.16 TCR (Table 2). This explains our earlier finding that, unlike B6.2.16 CD8 cell positive selection, the positive selection of P14 CD8 cells was only induced by RR in TAP1 FTOC (30). Taken together, these studies imply that the degree of specificity of TCR binding to thymic self-peptide/class I MHC determines the self-peptide specificity of CD8 cell positive selection.
The self-peptides used in our studies are among the most abundant peptides eluted from H-2Db molecules expressed on thymic epithelium and together account for 1223% of total H-2Db-presented self-peptide (30). Therefore, the differences in TCR:self-peptide/H-2Db affinity that control CD8+ cell positive selection may be of general physiological relevance. Previous work using antigen-variant peptides, which are not normally expressed in the thymus, also indicated that the affinity between peptide/MHC molecules and TCR determines the outcome of thymocyte selection (21,47). It was reported that ovalbumin (OVA) antigen peptide and agonist variants bound the H-2Kb-restricted anti-OVA TCR (OT-1) with high affinity and induced negative selection of OT-1 CD8 cells, and lower affinity OVA variants induced positive selection.
The level of H-2Db stabilization by self-peptides on thymic stromal cells in B6.2.16 TAP1 FTOC was relatively high; indeed BP and HH led to the rescue of ~50% of the normal TAP1+ level of H-2Db (Table 4). Despite this, the number of B6.2.16 CD8+ cells generated was never more than twice (17% of B6.2.16 TAP1 FTOC level) the level of unsupplemented B6.2.16 TAP1 lobes (8% of B6.2.16 TAP1+ FTOC level) (Fig. 4D
). It is likely that positive selection in normal thymi requires TCR binding to certain self-peptide/H-2Db molecules that have a higher affinity for the TCR than those studied in this paper. However, studies with antigen-variant peptides suggest that the density of `high-affinity' self-peptides, on normal thymic stromal cells, will probably be lower than that of the self-peptides in our study, in order to ensure that the overall avidity of interaction is in the range for positive rather than negative selection (26,48).
The finding that self-peptide/MHC affinity for TCR determines the outcome of thymic selection supports the view that the avidity of TCR:self-peptide/MHC interaction is a critical parameter determining whether thymocytes undergo positive or negative selection (25). Our findings are also consistent with earlier models of T cell selection that predicted that high-affinity TCR:MHC interactions drives negative selection and low-affinity interactions positive selection (49). Since the Smcy gene, which encodes the HY self-peptide, is expressed in the thymus then the negative selection of B6.2.16 CD8 cells by this self-peptide is likely to be of physiological relevance in ensuring central tolerance in males to self.
The binding of non-agonist self-peptides to the B6.2.16 TCR were of a low affinity and did not induce the hyper-phosphorylation of TCR chain, in contrast to the high-affinity binding of HY/H-2Db (Fig. 6
). A lack of overt TCR
hyper-phosphorylation has also been observed in thymocytes undergoing positive selection, on a wild-type repertoire of self-peptides, in non-transgenic adult thymi (44). One caveat of these findings, and of the results in Fig. 6
, is that since the number of thymocytes undergoing positive selection is low, it may be difficult to detect TCR
hyper-phosphorylation. Our findings support the view that the signals transduced by the TCR that lead to positive or negative selection differ at the level of TCR
chain phosphorylation. Therefore, high-affinity self-peptide binding to TCR leads to the transduction of signals that are qualitatively different from those that result from low-affinity self-peptide binding. These different signals in turn lead to activation induced cell death (negative selection) or rescue from apoptosis (positive selection).
![]() |
Acknowledgments |
---|
![]() |
Abbreviations |
---|
APC allophycocyanin |
ß2m ß2-microglobulin |
CTL cytotoxic lymphocytes |
FTOC fetal thymus organ culture |
HRP horseradish peroxidase |
IPTG isopropyl ß-D-thiogalactopyranoside |
OVA ovalbumin |
PE phycoerythrin |
RAG1 recombination activation gene |
SA streptavidin |
TAP1 transporter associated with antigen processing |
![]() |
Notes |
---|
Received 30 March 2000, accepted 6 June 2000.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|