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*
Microbiology and Tumor Biology Center and
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
Department of Oncology, IGT Laboratory, Karolinska Hospital, Stockholm, Sweden; and
§
Department of Chemistry, University of Virginia, Charlottesville, VA 22901
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(EF1). EF1 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
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
peptides. Thus, low avidity T cells specific for these self peptides
escape tolerance by deletion, despite expression of both EF1
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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
(EF1). 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 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, EF11 and
EF12, which differ at position 5 of the peptide and in their
expression pattern (35, 36). The EF11 is expressed in
most tissues, while EF12 has been found to replace, at least
partially, EF11 in skeletal muscle and brain (35, 36, 37).
We expected a complete tolerance by deletion to the abundantly
expressed EF11, but how would the immune system handle EF12, an
almost identical self protein with a more restricted tissue expression?
We found that CD8+ T cells with the capacity to
recognize the EF1-derived self peptides are present in the mature
repertoire. Low avidity CTLs could be raised against both isoforms of
the EF1 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-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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 11 (EF11) 412–420 ESFSDYPPL
(EF11p); EF12 412–420 ESFSQYPPL (EF12p); 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
EF11p and the EF12p 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 
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 mAb 53-6.7 (PharMingen,
San Diego, CA) and the PE-conjugated anti-TCRß mAb H57-597
(PharMingen), respectively. Staining for LFA-1 expression was done
using the anti-LFA-1 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-EF11p 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
EF11 and EF12 were modified from the corresponding human
primers (47); mEF11-F
5'-TCTTAATCAGTGGTGGAAG-3' and mEF11-R 5'-TTTGGTCAAGTTGTTTCC-3'
amplified a fragment of 187 bp. mEF12-F 5'-CTACGTCAGCGACTGGAT-3'
and mEF12-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 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
). Database searches identified the
peptide sequence as aa 412–420 of EF1, 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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protein is the second most abundant intracellular protein,
and occurs as two isoforms, EF11 and EF12, which differ at
position 5 of the 412–420 peptide. The EF11 isoform is expressed in
most tissues, while EF12 has been found to replace EF11 in
skeletal muscle and brain (35, 36).
Molecular modeling of the EF11 412–420 peptide (EF11p) and the
EF12 412–420 peptide (EF12p) 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. 2). 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 EF11p and
EF12p at position 5 is thus likely to affect recognition by the
TCR.
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1/2 412–420 self peptides
Having identified the EF11p as a naturally processed self
peptide presented by the H-2Kb restriction
element, we next investigated whether CTL responses could be generated
against the EF11p and EF12p 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 EF11p and
EF12p using peptide-pulsed RMA-S, RMA, and DC as target cells (Fig. 3, 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 EF11 412–420 peptide was originally isolated (Fig. 3, 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. 3C).
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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
isoform variant, while CTLs generated against the control SVp required
only 10-5 µM for
efficient lysis (Fig. 3
D). Thus, the tolerance to cells
expressing EF1 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 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 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 EF11p and SVp (Fig. 4). CD8 expression was consistently
higher in CTLs specific for EF11 (Fig. 4). 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 EF11p 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. 5, A and
C), while binding of tetrameric
EF11p-H-2Kb to EF11p-specific CTLs was
weaker and lost upon dilution of the tetramer (Fig. 5, 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-derived self peptide is due to low TCR affinity.
The relative increase in CD8 expression in EF11p-specific CTLs may
suggest that levels of coreceptor can be modulated to compensate for
low TCR affinity.
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isoforms in DCs and peripheral tissues
With the evidence for expression of H-2Kb
complexed with EF1-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 EF11 was
strongly expressed in the thymus, while EF12 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. 6). The bone marrow-derived DCs were
sorted by FACS for CD11c expression to obtain a homogenous DC
population. The EF11 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. 6). Expression of the EF12
isoform was restricted to brain and muscle, with only very weak
expression in lymphoid organs. Most interestingly, however, EF12
mRNA was readily detected in purified DCs (Fig. 6). Thus, both isoforms
of the EF1 protein are expressed in the APC believed to be
responsible for negative selection (5, 6, 7, 8).
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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 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 EF11p, 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 EF11p-specific CTLs,
while they were still less sensitive than CTLs specific for the
non-self OVAp (Fig. 7) and SVp (Fig. 3).
Interestingly, the doubly substituted F1E-R5Dp generated CTLs with
peptide sensitivity similar to those generated against the SVp and OVAp
(Fig. 7). 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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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 EF11p and EF12p were most efficient in stabilizing
H-2Kb, while R5Dp and SVp were somewhat less
efficient especially at lower concentrations (Fig. 8). Because the EF1 peptides exhibited
the strongest binding to the restricting MHC class I molecule, we
conclude that the high peptide dose requirement of CTLs specific for
EF11p and EF12p 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 peptides is a
property intrinsic to the CD8+ T cells specific
for self peptide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have investigated the patterns of CD8+ T
cell reactivity and tolerance to the abundantly expressed self protein
EF1. 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 exists
as two differentially expressed isoforms, EF11 and EF12.
Intracellular degradation of EF11 produces a nine-mer peptide
fragment, EF11p, which we have shown is presented by the MHC class I
molecule H-2Kb. EF11p 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 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 peptide in complex with
H-2Kb. Furthermore, the low avidity of
EF11p-specific CTLs correlates with low TCR affinity for the
H-2Kb-EF11p complex. Most importantly, these
CTLs do not lyse bone marrow-derived DCs, although both isoforms of
EF1 are expressed in such APC. These data indicate that
CD8+ T cells with high avidity to the
EF1-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-specific T cells does not occur, despite the fact
that both EF1 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
EF12 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-derived peptides by
deletion of all cells with the capacity to recognize EF11p or
EF12p? 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 EF1p (Fig. 3D), it can be estimated that these CTLs
need at least 300 H-2Kb-EF1p complexes to
reach the triggering threshold. CTLs against the modified EF1
peptides R5Dp and R1Ep display intermediate avidity (Fig. 7). We would
like to interpret these data within an avidity pit model of T cell
tolerance, which is summarized in Fig. 9.
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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Implications for T cell reactivity to self peptides in immunotherapy against cancer and in autoimmunity
The present data show that EF1 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
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 play a role
in adult atopic dermatitis (62).
EF12 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 EF12 isoform of EF1, and the
muscular and neurological defects of the wasted mouse could then be due
to lack of the EF12 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 EF12 expression
in organs such as spleen (36). We now show that EF12
mRNA is expressed in bone marrow-derived DCs (Fig. 6). 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 EF12 in DC development. It should be
noted that apart from its function in protein synthesis, EF1 has
also been found to associate with actin filaments, suggesting a role in
cytoskeleton organization and cell mobility (66).
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Acknowledgments |
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Footnotes |
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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.
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.
Received for publication December 27, 1999. Accepted for publication March 28, 2000.
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