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The Journal of Immunology, 2000, 165: 25-33.
Copyright © 2000 by The American Association of Immunologists

T Cell Tolerance Based on Avidity Thresholds Rather Than Complete Deletion Allows Maintenance of Maximal Repertoire Diversity1

Johan K. Sandberg2,*, Lars Franksson*,{ddagger}, Jonas Sundbäck*, Jakob Michaelsson*, Max Petersson*,{ddagger}, Adnane Achour*, Robert P. A. Wallin*, Nicholas E. Sherman§, Tomas Bergman{dagger}, Hans Jörnvall{dagger}, Donald F. Hunt§, Rolf Kiessling*,{ddagger} and Klas Kärre*

* Microbiology and Tumor Biology Center and {dagger} Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; {ddagger} Department of Oncology, IGT Laboratory, Karolinska Hospital, Stockholm, Sweden; and § Department of Chemistry, University of Virginia, Charlottesville, VA 22901


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Given the flexible nature of TCR specificity, deletion or permanent disabling of all T cells with the capacity to recognize self peptides would severely limit the diversity of the repertoire and the capacity to recognize foreign Ags. To address this, we have investigated the patterns of CD8+ CTL reactivity to a naturally H-2Kb-presented self peptide derived from the elongation factor 1{alpha} (EF1{alpha}). EF1{alpha} occurs as two differentially expressed isoforms differing at one position of the relevant peptide. Low avidity CTLs could be raised against both variants of the EF1{alpha} peptide. These CTLs required 100-fold more peptide-H-2Kb complexes on the target cell compared with CTLs against a viral peptide, and did not recognize the naturally expressed levels of EF1{alpha} peptides. Thus, low avidity T cells specific for these self peptides escape tolerance by deletion, despite expression of both EF1{alpha} isoforms in dendritic cells known to mediate negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity. However, self peptide-specific CTLs expressed elevated levels of CD8. Furthermore, CTLs generated against altered self peptide variants displayed intermediate avidity, indicating cross-reactivity in induction of tolerance. We interpret these data, together with results previously published by others, in an avidity pit model based on avidity thresholds for maintenance of both maximal diversity and optimal self tolerance in the CD8+ T cell repertoire.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class I molecules present a wide array of peptides derived from endogenous proteins to which the CD8+ T cell repertoire must not react, and several mechanisms are operating to avoid such autoreactive T cell responses. Negative selection in the thymus eliminates thymocytes that recognize self peptide-MHC complexes (1, 2, 3, 4), presented by bone marrow-derived dendritic cells (DCs)3 (5, 6, 7, 8). The endogenous proteins most likely to impose self tolerance onto the T cell repertoire by this process are ubiquitously expressed housekeeping gene products and proteins specifically expressed in bone marrow-derived cells. Negative selection cannot completely solve the problem of T cell tolerance, because not all self proteins of the host are expressed in the thymus. A number of mechanisms operate to ensure tolerance to tissue-specific proteins. These include immunological ignorance (9, 10), induction of unresponsiveness (anergy) (11, 12), and down-regulation of cell surface receptors (13, 14). The common denominator for these mechanisms is that T cell activation depends on the specific milieu of secondary lymphoid organs (15). Without the cytokines and costimulatory molecules expressed by APC in lymph nodes, T cell recognition of specific MHC-peptide complexes results in no or aberrant activation.

However, recognition of endogenous peptide-MHC complexes also plays an important positive role in regulation of T cell development and function in the periphery. T cell recognition of self MHC complexes during positive thymic selection is crucial to promote thymocyte survival, and there appears to be a role for specific MHC-bound self peptides in this process (16, 17, 18). Interestingly, recent evidence indicates that mature T cells in the periphery require interaction with MHC molecules for long-term survival (19, 20, 21, 22), and that recognition of self MHC modulates T cell peptide specificity (23).

The number of MHC-presented self peptides that induce T cell deletion or inactivation can be expected to be large (24, 25). It is also known that a single T cell has the capacity to productively interact with a number of different peptide ligands sharing varying degrees of homology (26, 27, 28, 29, 30, 31). Furthermore, the repertoire of TCRs used in the response against a given antigenic peptide is often diverse (32, 33). Given this considerable flexibility and diversity in the interaction between TCRs and peptide-MHC ligands, it could be expected that deletion and inactivation of T cells, which can recognize self peptide-MHC complexes, would severely limit the diversity of the mature T cell repertoire. How does the CD8+ T cell repertoire retain the capacity to recognize and eliminate a diverse flora of pathogens while maintaining self tolerance?

To address this issue, we have isolated a naturally processed and H-2Kb-presented peptide derived from the ubiquitously expressed ribosomal cofactor elongation factor 1{alpha} (EF1{alpha}). The peptide was identified by acid elution of material from immunoprecipitated H-2Kb molecules, followed by HPLC fractionation, capillary zone electrophoresis (CZE) fractionation, and sequencing by mass spectrometry. The EF1{alpha} protein is interesting in relation to development of T cell tolerance for at least two reasons. First, it is one of the most abundant intracellular proteins (34). Second, it occurs as two isoforms, EF1{alpha}1 and EF1{alpha}2, which differ at position 5 of the peptide and in their expression pattern (35, 36). The EF1{alpha}1 is expressed in most tissues, while EF1{alpha}2 has been found to replace, at least partially, EF1{alpha}1 in skeletal muscle and brain (35, 36, 37). We expected a complete tolerance by deletion to the abundantly expressed EF1{alpha}1, but how would the immune system handle EF1{alpha}2, an almost identical self protein with a more restricted tissue expression? We found that CD8+ T cells with the capacity to recognize the EF1{alpha}-derived self peptides are present in the mature repertoire. Low avidity CTLs could be raised against both isoforms of the EF1{alpha} peptide, despite the fact that both isoforms were expressed in the DCs believed to be responsible for negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity, rather than TCR/LFA-1 expression levels or peptide affinity for H-2Kb. Surprisingly, CD8 expression was consistently higher in EF1{alpha}-specific T cells as compared with CTL specific for a viral epitope. Other experimental peptide variants with nonconservative amino acid substitutions induced T cells with intermediate to high avidity. Together with previously published results, the data are interpreted within an avidity pit model of T cell tolerance against self peptides expressed in bone marrow-derived APC. In this model, only T cells with the capacity to recognize self peptides with high avidity are deleted, saving T cells that do not respond against the normal levels of expression of self peptide in vivo. In this way, functional T cell tolerance is accomplished while maintaining maximal diversity in the T cell repertoire. The avidity pit model has implications both for immunotherapy against tumors in which immunity against self Ag is desired, and for autoimmune conditions in which such immunity is unwanted.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and mice

RMA is a subline of the Rauscher virus-induced B6 lymphoma RBL-5, and RMA-S is a TAP 2-deficient variant of RMA (38). Cell lines were maintained at 37°C and 5% CO2 in RPMI 1640 tissue culture medium supplemented with 5% FCS, 50 µg/ml streptomycin, 100 µg/ml penicillin, and 2 mM L-glutamine. Con A-activated blasts were generated by culture of splenocytes in 5 µg/ml of Con A for 2 days in tissue culture medium, as described above, with 10% FCS. DCs were generated as described (39). In brief, bone marrow cells were cultured for 10 days in DMEM supplemented with 20% FCS and rGM-CSF. C57BL/6 (B6) mice of the H-2b haplotype were bred at the Microbiology and Tumor Biology Center, Karolinska Institutet. Animal care was in accordance with institutional guidelines.

Synthetic peptides

The following peptides were purchased from Research Genetics (Huntsville, AL): Elongation factor 1{alpha}1 (EF1{alpha}1) 412–420 ESFSDYPPL (EF1{alpha}1p); EF1{alpha}2 412–420 ESFSQYPPL (EF1{alpha}2p); ESFSRYPPL (R5Dp); FSFSRYPPL (F1E-R5Dp); RSFSDYPPL (R1Ep); Sendai virus nucleoprotein 324–332 FAPGNYPAL (SVp) (40); and OVA 257–264 SIINFEKL (OVAp) (41). Synthetic peptides were analyzed by reversed phase HPLC (RP-HPLC) and mass spectrometry.

Peptide immunizations

Peptide-specific CD8+ CTL was elicited in B6 mice by peptide immunization, as described (42). Briefly, 100 µg peptide was dissolved and mixed with CFA, and injected s.c. in the base of the tail. Twelve days after immunization, 25 x 106 immune spleen cells were cocultured with 25 x 106 2000 rad irradiated B6 splenocytes in the presence of 0.05 µM peptide in 12 ml complete medium (RPMI 1640 supplemented with 10% FCS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 5 x 10-5 M 2-ME, 2 mM L-glutamine, 50 µg/ml streptomycin, and 100 µg/ml penicillin) at 37°C and 5% CO2. After 5–7 days, these cells were used as effector cells in a 51Cr release assay. Long-term CTL lines were maintained in complete medium further supplemented with HEPES buffer and 10 IU/ml IL-2. CTL lines were restimulated in 15-day intervals with 2000 rad irradiated splenocytes in the presence of 0.05 µM of peptide.

Molecular modeling

Models of H-2Kb complexed with the EF1{alpha}1p and the EF1{alpha}2p were based on the published coordinates of the H-2Kb-SVp crystallographic structure (43), and were produced by mutating the relevant amino acid residues. Models were refined by energy minimizations using SYBYL, version 6.5 (Tripos Associates, St. Louis, MO). Relevant amino acid residues were mutated in the H-2Kb-SVp complex. Keeping the backbone of the MHC class I molecule fixed, geometry optimization was performed to a convergence point of 0.05 rms deviations in the total energy term using the Powell algorithm. The dielectric model consisted of a distance-dependent dielectric cutoff of 9Å and an {epsilon} value of 4 to simulate solvent effects with charges taken from the internal dictionary. Prolines were substituted without difficulty in the trans configuration in all cases. Atomic coordinates for the models are available upon request.

Isolation and sequence analysis of a H-2Kb-presented peptide

H-2Kb molecules were immunoaffinity purified from 109 RMA cells. The bound peptides were extracted, and separated on RP-HPLC, as described (44). A total of 5 nl of one of the RP-HPLC fractions was separated by CZE on a Beckman P/ACE 2100 instrument. Briefly, the sample was loaded by pressure on a 75-µm/57-cm capillary, separated at 20,000 V for 5 min, and fractionated by pressure in 80-s fractions, using a 50 mM phosphate, pH 2.5, running buffer. Mass spectra from a CZE fraction rich in material according to the CZE electropherogram were recorded on a Finnigan-MAT TSQ-70 (San Jose, CA) triple quadrupole mass spectrometer equipped with an electrospray ion source and a C-18 microcapillary-HPLC column, as previously described (24). Collision-activated dissociation mass spectra for one abundant peptide species in this CZE fraction were recorded with and without pretreatment of the sample with methanolic-HCl (to convert peptides to their corresponding methyl esters) (24). Subtraction of m/z values for any two fragments that differ by a single amino acid generates a value that specifies the mass and thus the identity of the extra residue in the larger fragment and allows the deduction of the primary structure of the peptide.

mAbs, FACS analysis, and sorting

For measurement of CD8 and TCR expression, CTLs were stained for 30 min with the FITC-conjugated anti-CD8{alpha} mAb 53-6.7 (PharMingen, San Diego, CA) and the PE-conjugated anti-TCR{alpha}ß mAb H57-597 (PharMingen), respectively. Staining for LFA-1 expression was done using the anti-LFA-1{alpha} mAb IC21, followed by secondary FITC-conjugated goat anti-rat Ig (Southern Biotechnology Associates, Birmingham, AL). H-2Kb tetrameric complexes were used for staining in dilutions of a 0.127 mg/ml preparation. After washing, analysis was performed utilizing a FACScan (Becton Dickinson, Sunnyvale, CA) using CellQuest software. For purification of DC, bone marrow cultures were stained with the FITC-conjugated anti-CD11c mAb N418. After washing, sorting was performed using a FACS Vantage (Becton Dickinson). For the H-2Kb peptide stabilization assay, RMA-S cells were incubated overnight at 26°C in complete medium with titrated concentrations of peptide, followed by a 45-min chase at 37°C to remove nonspecific background. Cells were then stained with FITC-conjugated anti-H-2Kb Ab AF6-88.5 (PharMingen) and analyzed for H-2Kb expression by flow cytometry using a FACScan.

H-2Kb tetramers

Production of soluble H-2Kb heavy chain fused with a BirA substrate peptide (H-2Kb-Bsp) and murine ß2m (mß2m), and the in vitro refolding of the H-2Kb-Bsp/mß2m/peptide complex have been described (45, 46). Briefly, the H-2Kb-Bsp, mß2m, and peptide were refolded by dilution in the presence of leupeptin (2 µg/ml), pepstatin A (2 µg/ml), and PMSF (0.2 mM) for 48 h. The refolded complexes were purified by size exclusion chromatography on a Superose 12 column 10/30 (Amersham Pharmacia Biotech, Uppsala, Sweden), enzymatically biotinylated by incubation with BirA enzyme and biotin (Avidity, Denver, CO), according to the instructions of the manufacturer. Free biotin was removed by gel filtration using NAP-5 desalting columns (Amersham Pharmacia Biotech). The MHC complexes were then quickly frozen and stored at -70°C. A new set of tetramers was made for every staining by mixing biotinylated H-2Kb complexes with streptavidin-PE (Molecular Probes, Eugene, OR) at a 4:1 molar ratio. To ascertain comparable quality of tetramers, the percentage of biotinylated MHC molecules in each preparation was assessed by a gel-shift assay and determined to be equal in both H-2Kb-EF1{alpha}1p and H-2Kb-SVp tetramers. The protein concentration of the two H-2Kb/mß2m/peptide preparations was determined in parallel. Both tetramers were prepared simultaneously and used immediately in FACS staining.

PCR analysis of mRNA expression

Total RNA was extracted from either 50 mg tissues or 5 x 106 cultured cells using the QuickPrep total RNA extraction kit (Amersham Pharmacia Biotech), according to the protocol. First-strand cDNA was synthesized using the first-strand cDNA synthesis kit (Amersham Pharmacia Biotech), according to the protocol. Briefly, 20 µl (3 µg) of the total RNA preparation and 0.2 µg NotI-d(T)18 primer were used for the synthesis. Primers to amplify the 3' untranslated regions of mouse EF1{alpha}1 and EF1{alpha}2 were modified from the corresponding human primers (47); mEF1{alpha}1-F 5'-TCTTAATCAGTGGTGGAAG-3' and mEF1{alpha}1-R 5'-TTTGGTCAAGTTGTTTCC-3' amplified a fragment of 187 bp. mEF1{alpha}2-F 5'-CTACGTCAGCGACTGGAT-3' and mEF1{alpha}2-R 5'-GGGTCGCTCAGTTTATTGGG-3' amplified a 124-bp fragment. The PCR was done in 50 µl with 25 pmol of the forward and reverse primers, 0.2 mM dNTPs, 2 U Taq polymerase (Boehringer Mannheim GmbH, Germany), supplied PCR buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3), and 1.5 mM MgCl2), and 5 µl of the first-strand cDNA, all final concentrations. Conditions for the reaction were: 95°C, 1 min; 95°C, 30 s; 48°C, 30 s; 72°C, 45 s (for 35 cycles); 72°C, 7 min. PCR products were analyzed using ethidium bromide (EtBr)-stained 1.5% agarose gels. The quality of the cDNA was confirmed by parallel PCR amplification of a 332-bp GAPDH gene fragment, using the same conditions as for PCR amplification of EF1{alpha} but 28 cycles, and the primers 5'-CCCTCCGGGAAACTGTGGCGT-3' (GAPDH-F) and 5'-ATGCCAGCCCCAGCGTCAAAG-3' (GAPDH-R). All PCRs were repeated at least twice.

CTL assay

CTL activity was measured in a standard 51Cr release assay. Briefly, peptide-coated target cells were prepared by incubating cells with indicated concentrations of peptide for 1 h at 37°C. Sendai virus-infected cells were prepared by infecting target cells in 0.2 ml (5000 hemagglutination U/ml) for 1.5 h at 37°C. After washing, infected target cells were further incubated for 2 h. Peptide-coated or infected cells were labeled with 10 µl 10 mCi/ml 51Cr for 1 h at 37°C. Titrated numbers of effector cells were incubated with 3 x 103 51Cr-labeled target cells for 4 h at 37°C, 5% CO2. After incubation, released radioactivity was measured and specific lysis was calculated according to the formula: percentage of specific release = [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation, sequence analysis, and structural analysis of a ubiquitously expressed self peptide presented by H-2Kb

To study how the peripheral CD8+ T cell repertoire is shaped by the endogenous flora of self peptides, we set out to identify an MHC class I-presented peptide derived from a ubiquitously expressed self protein. H-2Kb molecules were immunoaffinity purified from RMA cells. Associated peptides were extracted under acidic conditions, fractionated by RP-HPLC, and separated by CZE. One of the final CZE fractions was analyzed by electrospray mass spectrometry. Collision-activated dissociation spectra were recorded for one abundant peptide mass, and the sequence ESFSDYPPLxx could be deduced (Fig. 1Go). Database searches identified the peptide sequence as aa 412–420 of EF1{alpha}, a highly conserved protein involved in the transfer of aminoacyl-tRNA to ribosome-bound mRNA (34, 37). Synthetic ESFSDYPPL coeluted in the correct RP-HPLC and CZE fractions (data not shown).



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  		FIGURE 1. Collision-activated mass spectrum of peptide (M+H)+ ions at m/z 1054. Predicted masses for fragment ions of type b are shown above, and masses for ions of type y below the deduced amino acid sequence. Subtraction of m/z values for any two fragments that differ by a single amino acid generates a value that specifies the mass and thus the identity of the extra residue in the larger fragment. Because isoleucine and leucine are of equal mass, they cannot be differentiated on the triple quadropole instrument, and are denoted with Lxx.

 
The EF1{alpha} protein is the second most abundant intracellular protein, and occurs as two isoforms, EF1{alpha}1 and EF1{alpha}2, which differ at position 5 of the 412–420 peptide. The EF1{alpha}1 isoform is expressed in most tissues, while EF1{alpha}2 has been found to replace EF1{alpha}1 in skeletal muscle and brain (35, 36).

Molecular modeling of the EF1{alpha}1 412–420 peptide (EF1{alpha}1p) and the EF1{alpha}2 412–420 peptide (EF1{alpha}2p) in complex with H-2Kb, using the crystal structure of SVp bound to H-2Kb as template, indicated the approximate positions of amino acid side chains in the peptides (Fig. 2Go). Positions 1 and 5 appear to be exposed to the TCR, while positions 6 and 9 are buried in the H-2Kb molecule acting as anchors, in accordance with the known binding motif of H-2Kb-binding peptides. The one amino acid difference between the EF1{alpha}1p and EF1{alpha}2p at position 5 is thus likely to affect recognition by the TCR.



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  		FIGURE 2. The two isoforms of the H-2Kb-presented self peptide derived from EF1{alpha}. Molecular modeling of EF1{alpha}1p and EF1{alpha}2p self peptide variants in complex with the H-2Kb restriction element. Models were refined by energy minimizations using SYBYL, as described in Materials and Methods.

 
Incomplete deletion of CD8+ T cells that recognize the EF1{alpha}1/2 412–420 self peptides

Having identified the EF1{alpha}1p as a naturally processed self peptide presented by the H-2Kb restriction element, we next investigated whether CTL responses could be generated against the EF1{alpha}1p and EF1{alpha}2p peptides. B6 mice were primed s.c. with the corresponding synthetic peptide in adjuvant, and splenic CTL activity was assessed after restimulation in vitro. Peptide-specific CTL responses were readily detectable against both EF1{alpha}1p and EF1{alpha}2p using peptide-pulsed RMA-S, RMA, and DC as target cells (Fig. 3Go, A and B). However, these CTLs failed to kill RMA-S cells and DC in the absence of exogenously added peptide. They also failed to kill RMA lymphoma cells from which the EF1{alpha}1 412–420 peptide was originally isolated (Fig. 3Go, A and B). CTLs generated against the H-2Kb-restricted non-self nuclear protein 324–332 epitope from Sendai virus were able to recognize and kill Sendai virus-infected target cells, demonstrating that CTLs generated against synthetic peptide using our protocol were not inherently unable to recognize naturally processed and presented peptides (Fig. 3GoC).



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  		FIGURE 3. Patterns of CD8+ T cell reactivity and tolerance to EF1{alpha}-derived self peptides. CTLs generated by peptide immunization (as described in Materials and Methods) with EF1{alpha}1p (A) and EF1{alpha}2p (B) in B6 mice were tested against RMA-S, RMA, and B6 DC loaded with the specific peptide or no peptide in a 51Cr release CTL assay. C, CTLs generated by peptide immunization with SVp were tested against RMA-S, RMA, B6 Con A blasts, Sendai virus-infected RMA cells, and RMA-S loaded with either SVp or control peptide. D, The peptide requirement of CTLs specific for EF1{alpha}1p, EF1{alpha}2p, and SVp was compared by loading RMA-S target cells with titrated concentrations of peptide.

 
We next tested the sensitivity, in terms of absolute peptide requirement, of EF1{alpha}p-specific CTL for their cognate MHC-peptide ligands. Approximately 10-3 µM peptide was required to sensitize RMA-S targets for CTLs specific for either EF1{alpha} isoform variant, while CTLs generated against the control SVp required only 10-5 µM for efficient lysis (Fig. 3GoD). Thus, the tolerance to cells expressing EF1{alpha} peptides complexed with H-2Kb correlates with a 100-fold difference in sensitivity for peptide Ag. Taken together, these data indicate that CD8+ T cells with high avidity to the EF1{alpha} self peptides have been deleted during selection, while low avidity T cells that do not recognize the levels of self peptide expressed on normal cells are allowed to persist in the peripheral repertoire.

CD8+ T cells specific for EF1{alpha} peptide display low TCR affinity and increased CD8 expression

We next investigated possible differences between T cells specific for self and non-self peptides, in terms of cell surface expression levels and affinity of Ag receptors. FACS analysis confirmed that all CTLs were TCR+ and LFA-1+, and expression levels of these receptors did not differ between T cells generated against EF1{alpha}1p and SVp (Fig. 4Go). CD8 expression was consistently higher in CTLs specific for EF1{alpha}1 (Fig. 4Go). Thus, the low avidity in self peptide-specific CTLs could not be attributed to low expression of TCR, CD8, or LFA-1. We next assessed TCR binding to peptide-MHC complexes in these T cells using soluble H-2Kb tetramers. T cells specific for EF1{alpha}1p or SVp were stained with serial dilutions of tetrameric H-2Kb complexed with the corresponding peptide, and binding was visualized by FACS. Binding of SVp-H-2Kb tetramer to SVp-specific CTLs was efficient and clearly detectable using even a 1/1000 dilution of tetramer (Fig. 5Go, A and C), while binding of tetrameric EF1{alpha}1p-H-2Kb to EF1{alpha}1p-specific CTLs was weaker and lost upon dilution of the tetramer (Fig. 5Go, B and C). Because the strength of tetramer binding is known to correlate with TCR affinity for the MHC-peptide complex (48), these data indicate that the low avidity in CTL recognition of EF1{alpha}-derived self peptide is due to low TCR affinity. The relative increase in CD8 expression in EF1{alpha}1p-specific CTLs may suggest that levels of coreceptor can be modulated to compensate for low TCR affinity.



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  		FIGURE 4. Expression of cell surface receptors in EF1{alpha}1p-specific T cells. FACS analysis of CD8{alpha}, TCR{alpha}/ß, and LFA-1 expression in CTL elicited against the EF1{alpha}1p (solid line) and SVp (dotted lines).

 


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  		FIGURE 5. T cells specific for the EF1{alpha}1 self peptide have low affinity TCR. FACS analysis of specific H-2Kb tetramer binding to CTL lines specific for SVp (A) and EF1{alpha}1p (B). C, Represents the data in A and B as a diagram. Histograms in A and B show H-2Kb tetramer staining in CD8+ cells, using 1/10 (thick line), 1/100 (solid line), 1/300 (dotted line), and 1/1000 (dashed line) dilutions of 0.127 mg/ml tetramer preparations. Unstained cells were included as a negative control (weak dotted line). Quality of the tetrameric H-2Kb complexes was assessed and ensured, as described in Materials and Methods.

 
Expression of EF1{alpha} isoforms in DCs and peripheral tissues

With the evidence for expression of H-2Kb complexed with EF1{alpha}-derived peptides and data on the presence of low avidity T cells that can react against these peptides at hand, we decided to reexamine the expression patterns in lymphoid tissues of the two isoforms. Our initial experiments had shown that EF1{alpha}1 was strongly expressed in the thymus, while EF1{alpha}2 was not (not shown). In an extended RT-PCR analysis, we now also included mRNA from purified DCs, in addition to mRNA from thymus, spleen, whole bone marrow, brain, skeletal muscle, and the RMA cell line (Fig. 6Go). The bone marrow-derived DCs were sorted by FACS for CD11c expression to obtain a homogenous DC population. The EF1{alpha}1 mRNA was found to be expressed in all tissues and cells tested, which was expected because of the central role this protein plays in protein synthesis (Fig. 6Go). Expression of the EF1{alpha}2 isoform was restricted to brain and muscle, with only very weak expression in lymphoid organs. Most interestingly, however, EF1{alpha}2 mRNA was readily detected in purified DCs (Fig. 6Go). Thus, both isoforms of the EF1{alpha} protein are expressed in the APC believed to be responsible for negative selection (5, 6, 7, 8).



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  		FIGURE 6. Expression of EF1{alpha} isoforms in DCs and lymphoid tissues. RT-PCR of the 3' untranslated region of EF1{alpha}1 (top) and EF1{alpha}2 (middle). DC, CD11c+-sorted cells from bone marrow culture. Non-DC, sorted CD11c- cells from the bone marrow culture. mRNA and cDNA integrity was confirmed using primers specific for GAPDH (bottom).

 
CTLs generated against altered self peptides are of intermediate avidity

We next hypothesized that T cells with ability to recognize ligands with similarity to endogenous self peptides would be partly affected by tolerance induction. If this were true, EF1{alpha} self peptide variants modified by amino acid substitution to become altered self would generate CTLs with intermediate avidity. To test this, the glutamate at position 1 and the aspartate at position 5, indicated by modeling to be TCR contacts in EF1{alpha}1p, were substituted to generate altered self peptides, and these peptides were used to immunize B6 mice. The CTLs specific for the R5Dp and R1Ep altered self variants displayed greater peptide sensitivity than the EF1{alpha}1p-specific CTLs, while they were still less sensitive than CTLs specific for the non-self OVAp (Fig. 7Go) and SVp (Fig. 3Go). Interestingly, the doubly substituted F1E-R5Dp generated CTLs with peptide sensitivity similar to those generated against the SVp and OVAp (Fig. 7Go). Thus, CD8+ T cells that recognize self peptide ligands altered at one position were indeed of intermediate avidity, indicating cross-reactivity in tolerance induction.



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  		FIGURE 7. T cells specific for altered self peptides are of intermediate avidity. CTLs generated by peptide immunization (as described in Materials and Methods) in B6 mice were tested in a 51Cr release CTL assay against RMA-S loaded with titrated amounts of peptide. Each CTL population was tested against the specific peptide used for immunization.

 
Peptides from EF1{alpha} isoforms bind H-2Kb with high affinity

The observed differences in peptide dose requirements between CTLs specific for self, altered self, and non-self peptides could possibly be influenced by differences in peptide binding to the H-2Kb. To assess peptide binding, we used peptide-induced stabilization of empty cell surface H-2Kb molecules on RMA-S cells. Peptides were added to cultures of RMA-S maintained at 26°C for 12 h, followed by a 45-min chase at 37°C, after which the binding capacity of the peptides was assessed by FACS. All peptides formed stable complexes with H-2Kb, indicating strong binding. However, the EF1{alpha}1p and EF1{alpha}2p were most efficient in stabilizing H-2Kb, while R5Dp and SVp were somewhat less efficient especially at lower concentrations (Fig. 8Go). Because the EF1{alpha} peptides exhibited the strongest binding to the restricting MHC class I molecule, we conclude that the high peptide dose requirement of CTLs specific for EF1{alpha}1p and EF1{alpha}2p was not due to inferior peptide binding to H-2Kb. Thus, the low avidity in the interaction between targets and CTLs in recognition of EF1{alpha} peptides is a property intrinsic to the CD8+ T cells specific for self peptide.



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  		FIGURE 8. Relative peptide affinity determined by H-2Kb cell surface stabilization. Flow-cytometric analysis of peptide-induced stabilization of cell surface on RMA-S. Data are expressed as mean fluorescence intensity (MFI) after subtraction of MFI in the absence of peptide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Self Ags expressed in DCs induce only partial T cell deletion

We have investigated the patterns of CD8+ T cell reactivity and tolerance to the abundantly expressed self protein EF1{alpha}. This protein is the second most abundant intracellular protein, and fulfills an essential function in guiding the aminoacyl-tRNA to the ribosome during protein synthesis (34, 37). EF1{alpha} exists as two differentially expressed isoforms, EF1{alpha}1 and EF1{alpha}2. Intracellular degradation of EF1{alpha}1 produces a nine-mer peptide fragment, EF1{alpha}1p, which we have shown is presented by the MHC class I molecule H-2Kb. EF1{alpha}1p is therefore an example of a peptide to which the immune system cannot be allowed to react. Despite this, we observe that CD8+ T cells in the normal repertoire can generate a CTL response against both isoforms of the EF1{alpha} peptide. These CTLs are of low avidity, require a comparably high dose of peptide to sensitize target cells, and do not kill normal cells that express the EF1{alpha} peptide in complex with H-2Kb. Furthermore, the low avidity of EF1{alpha}1p-specific CTLs correlates with low TCR affinity for the H-2Kb-EF1{alpha}1p complex. Most importantly, these CTLs do not lyse bone marrow-derived DCs, although both isoforms of EF1{alpha} are expressed in such APC. These data indicate that CD8+ T cells with high avidity to the EF1{alpha}-derived self peptides have been deleted during tolerance induction, while low avidity T cells that do not react to the levels expressed on normal cells, and Ag-presenting DCs, are allowed to persist in the peripheral repertoire. It is noteworthy that complete deletion of EF1{alpha}-specific T cells does not occur, despite the fact that both EF1{alpha} isoforms are expressed by CD11c+ DCs. Our suspicion that tolerance patterns might differ between the two isoforms due to their differential expression in whole lymphoid organs was not confirmed. This is, however, not surprising, in light of our observation that both isoforms are expressed in DCs. The expression of EF1{alpha}2 in DCs may be related to the need for elimination of high avidity CTLs reactive with this isoform of the peptide, but it is equally possible that this isoform of the protein has an important function in DCs (discussed further below).

The avidity pit model of T cell tolerance

Why has not the CD8+ T cell compartment been rendered completely unresponsive to the EF1{alpha}-derived peptides by deletion of all cells with the capacity to recognize EF1{alpha}1p or EF1{alpha}2p? One probable explanation relates to the requirement for a diverse T cell repertoire to combat the diverse flora of pathogens. To maintain a repertoire, which is diverse enough to recognize all possible Ags while still avoiding autoreactivity, is a major challenge to the immune system. Complete removal of all cells with the capacity to recognize self peptides presented by MHC molecules would lead to a multitude of holes in the T cell repertoire. Considering the large number of self peptides to which the system has to be tolerant, this would severely restrict the diversity of the repertoire, even if only peptides expressed by Ag-presenting and tolerance-inducing DCs would induce such holes. However, our data indicate that only T cells with the capacity to recognize self peptides with high avidity are deleted, saving the low avidity T cells.

T cells specific for the non-self SVp epitope can be expected to require as few as 3–10 H-2Kb-SVp complexes to lyse a target cell (49). Given the 100-fold increase in peptide dose requirement for efficient recognition by the CTLs specific for EF1{alpha}p (Fig. 3GoD), it can be estimated that these CTLs need at least 300 H-2Kb-EF1{alpha}p complexes to reach the triggering threshold. CTLs against the modified EF1{alpha} peptides R5Dp and R1Ep display intermediate avidity (Fig. 7Go). We would like to interpret these data within an avidity pit model of T cell tolerance, which is summarized in Fig. 9Go. This figure illustrates the difference in T cell repertoire avidity for self peptides, altered self peptides, and non-self peptides. The avidity pit is distinct from a hole in the repertoire because only T cells with the capacity to recognize self peptides with high avidity are deleted, saving T cells that are not triggered by normal levels of expression of self peptide. In this way, the T cell repertoire maintains both high diversity and functional tolerance to self. Indeed, data in the literature support that similar patterns exist for other T cell populations with capacity to recognize self ligands. In an early study by Schild et al., it was first observed that CTL could be generated against a number of peptides from self proteins (50). In a more recent study, low avidity MHC class II-restricted T cells could be generated against the endogenous myelin basic protein in comparison with T cells from myelin basic protein-deficient shiverer mice (51). In the case of p53-derived self epitopes, the pattern was found to be mixed, with complete tolerance to one epitope, while low avidity T cells were found against another peptide (52). In the hen egg-white lysozyme transgenic model, hen egg-white lysozyme-specific CD4+ T cells required 2 log more Ag to proliferate when compared with nontransgenic littermates (53). Also, self-reactive CD4+ T cells can escape deletion as a result of low affinity binding of self peptide to MHC class II (54).



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  		FIGURE 9. The avidity pit in the T cell repertoire as the basis for self tolerance to EF1{alpha} peptides presented by H-2Kb. Approximate threshold of activation in terms of estimated numbers of MHC-peptide complexes required to trigger CTL lysis of the target cell. T cells expressing TCRs specific for the self peptide sequence represent the bottom of the avidity pit, whereas T cells that recognize altered self peptides depict the wall. The SVp epitope is representative for the non-self antigenic peptide.

 
How can T cells recognizing self peptides with low avidity make a useful contribution to immune defense against pathogens? The answer to this question probably relates to the degenerate specificity of the TCR. Although T cells display specificity for an antigenic peptide (55), it is also clear that a given TCR can productively recognize a large continuum of related ligands (26, 27, 30, 31, 56, 57), and even peptides without apparent homology to the nominal antigenic peptide (29). Thus, T cells that recognize self peptides with low avidity may be able to respond to other non-self peptides with high avidity. In this way, it is possible to retain a T cell repertoire of maximal diversity while being functionally tolerant to the physiological expression levels of self. This possibility is supported by the observation that T cells tolerized to a low avidity superantigen were still able to recognize their specific MHC-restricted Ag and fulfill functions in immune defense (58).

Implications for T cell reactivity to self peptides in immunotherapy against cancer and in autoimmunity

The present data show that EF1{alpha} induce only partial deletion of potentailly autoreactive T cells, despite the fact that this protein is amply expressed in all cells, including the professional Ag-presenting DCs. This finding is encouraging to the prospects of developing immunotherapy against cancer, in which the potential tumor Ags are often normal self proteins, albeit aberrantly expressed (59, 60); tolerance induction saves low avidity/affinity T cells even if the self Ag is expressed in DCs. We also observe that altered self peptide variants generate T cells with intermediate avidity, indicating that a more high avidity response can be expected in case the tumor epitope is a mutated variant of self. Recent evidence suggests that such altered self peptide ligands can also stimulate low avidity T cells specific for the cognate self peptide (61). Experiments to evaluate the potential antitumor potency of EF1{alpha} 412–420 and the altered variants are in progress.

With regard to autoimmunity, we observe that the CTLs elicited against the self peptides and variant peptides do not react against the expression levels of self peptide-H-2Kb complexes on Con A-activated blasts, DCs, and RMA cells in vitro. Furthermore, mice appear healthy for several months after immunization. These data suggest that the avidity pit in the mature T cell repertoire induced by self peptides expressed in DCs assures functional tolerance to avoid CD8+ T cell-mediated autoimmunity. However, recent results indicate that autoantibodies against EF1{alpha} play a role in adult atopic dermatitis (62).

EF1{alpha}2 expression in DCs in relation to the wasted mouse phenotype

In 1982, Shultz et al. (63) described the wasted mouse with a spontaneous autosomal recessive defect that gives rise to a set of abnormalities. These include muscle wasting (hence the name of the mutation), weight loss, progressive paralysis, and neural degeneration. The Wst gene was recently identified as Eef1a2, which encodes the EF1{alpha}2 isoform of EF1{alpha}, and the muscular and neurological defects of the wasted mouse could then be due to lack of the EF1{alpha}2 protein, normally expressed in these tissues (37, 64). However, the wasted mouse also displays abnormal immunological features, such as decreased numbers of circulating lymphocytes and decreased ratios of the spleen, thymus, and lymph nodes to the body weight. These immunological abnormalities have been hard to explain, because investigators have failed to detect EF1{alpha}2 expression in organs such as spleen (36). We now show that EF1{alpha}2 mRNA is expressed in bone marrow-derived DCs (Fig. 6Go). Although these cells constitute a small subpopulation of all cells in lymphoid tissues, they play a key role in T cell development and activation (65). This finding may not only provide a possible explanation for the immunological abnormalities of the wasted mouse, but may suggest a role for EF1{alpha}2 in DC development. It should be noted that apart from its function in protein synthesis, EF1{alpha} has also been found to associate with actin filaments, suggesting a role in cytoskeleton organization and cell mobility (66).


   Acknowledgments
 
We thank Drs. Rickard Glas, Hans-Gustaf Ljunggren, Benedict Chambers, and Hidde Ploegh for discussions and helpful comments on the manuscript. We further thank Manuel Pataroyo for supplying the IC21 mAb.


   Footnotes
 
1 This work was supported by funds from the Swedish Cancer Foundation, the Swedish Medical Research Council, the Swedish Society for Medical Research, the Cancer Society of Stockholm, and the King Gustaf V Jubilee Fund. D.F.H. is supported by National Institutes of Health Grant AI33993. Back

2 Address correspondence and reprint requests to Dr. Johan Sandberg at the current address: Aaron Diamond AIDS Research Center, The Rockefeller University, 455 1st Avenue, 7th Floor, New York, NY 10016. Back

3 Abbreviations used in this paper: DC, dendritic cell; Bsp, BirA substrate peptide; CZE, capillary zone electrophoresis; EF1, elongation factor 1; mß2m, murine ß2-microglobulin; OVAp, OVA 257–264; RP-HPLC, reversed phase HPLC; SVp, Sendai virus nucleoprotein 324–332. Back

Received for publication December 27, 1999. Accepted for publication March 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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