Comparison of the frequency of peptide-specific cytotoxic T lymphocytes restricted by self- and allo-MHC following in vitro T cell priming
Tian-Hui Yang1,
Matthew Lovatt2,
Matthias Merkenschlager2 and
Hans J. Stauss1
1 Department of Immunology, Imperial College of Science Technology and Medicine, and 2 Lymphocyte Development Group, MRC Clinical Sciences Centre, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK
Correspondence to: H. J Stauss; E-mail: h.stauss{at}ic.ac.uk
Transmitting editor: M. Feldmann
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Abstract
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T cell recognition of antigenic peptides is thought to occur preferentially in the context of self-MHC. Here, we have tested the ability of four different Kbpeptide combinations to stimulate self- and allo-restricted CTL responses in three different mouse stains. Responder T cells were primed in vitro with peptide-loaded stimulator cells, followed by limiting dilution assays to measure the number of peptide-specific cytotoxic T lymphocytes (CTL). For three peptides the number of CTL restricted by self-MHC was higher than for allo-MHC-restricted responses, although the difference was surprisingly small (3- to 5-fold). For the fourth peptide there was no detectable difference in the number of self- and allo-restricted CTL. Peptide titration experiments revealed that high avidity CTL were present in both the self- and allo-restricted setting. These data showed that the bias for preferred peptide recognition in the context of self-MHC imposed by positive thymic selection seems marginal. This raised the possibility that the TCR repertoire is inherently biased towards MHC restriction, independent of MHC-guided thymic selection. This was supported by the analysis of mature T cells generated from the thymus of MHC-deficient mice by lectin stimulation. Kb-restricted CTL were found amongst these T cells at numbers similar to those of allo-restricted CTL. In summary, the data suggest that MHC-restricted peptide recognition is an inherent feature of the TCR repertoire and does not require thymic selection by MHC molecules.
Keywords: cytotoxic T lymphocyte, MHC, TCR, thymus
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Introduction
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We have previously demonstrated that the allo-MHC-restricted T cell repertoire can serve as a source of cytotoxic T lymphocytes (CTL) specific for tumour-associated proteins to which autologous CTL were tolerant (13). It has remained unclear, however, whether allo-restricted CTL represent rare exceptions to the general rule that CTL recognition is self-MHC restricted (4). Thymic education is thought to select CTL capable of recognizing peptides presented by self-MHC class I molecules (5). Nevertheless, the phenomenon of allo-reactivity clearly demonstrates that CTL can recognize peptides presented in the context of allogeneic class I molecules (613). Since allogeneic MHC class I molecules normally present several thousand peptides derived from various cellular proteins, it has been impossible to measure the frequency of CTL specific for any one defined peptide epitope. Current concepts of positive thymic selection predict that peptide recognition in the context of self-MHC is much more efficient than peptide recognition in the context of allogeneic MHC molecules. In the past, experiments in bone marrow chimeric mice and/or thymus transplantation provided support for the notion that thymic MHC molecules determine the MHC-restriction specificity of mature CTL (14). However, in some experiments CTL restricted by MHC alleles not expressed in the thymic epithelium were readily detected (1519). These contradictory results are difficult to reconcile, and may in part reflect that parameters such as the nature of the antigen-presenting cells (APC) and the immunogencity of the peptide epitopes presented by two different MHC alleles were usually not well defined. Thus, strong CTL responses against MHC-A + peptide-x and weak responses against MHC-B + peptide-y might be due to APC competition and immunodominance mechanisms operating at the level of CTL induction rather than reflecting positive thymic selection (20).
In this study we have used strictly defined in vitro stimulation conditions to determine the impact of positive thymic selection on the MHC-restriction preference of mature CTL. Responder T cells from normal donor mice of three different MHC haplotypes were stimulated for 5 days in vitro with the same APC presenting a defined MHCpeptide combination, followed by limiting dilution analysis (LDA) to determine the number of peptide-specific CTL. A preference for recognition in the context of self over allo-MHC was seen for three peptides, whilst one peptide stimulated similar numbers of self- and allo-restricted CTL. Experiments with T cells artificially generated from pre-selection thymocytes of MHC-deficient mice indicated that the TCR repertoire is intrinsically biased towards MHC-restricted peptide recognition and does not require MHC encounter during thymic selection.
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Methods
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Peptides
Four peptides were used in this study: OVA (SIINFEKL), mOVA (SIIAFEKL), SV9 (FAPGNYPAL) and VSV-2A (RAYVYQGL). All peptides were synthesized at the local peptide synthesis service, HPLC purified and tested by MALDI mass spectrometry.
Cell lines
RMA-S cells were derived from RMA cells after mutagenesis with ethylmethane-sulphonate followed by five rounds of selection with anti-H-2 alloantisera and rabbit complement treatment to obtain cells with decreased levels of MHC class I expression (21). RMA-S cells were found to have a point mutation at nucleotide 97 of the TAP2 gene, which generates a premature stop codon (22). The human cell line T2 is a fusion hybrid of a B-lymphoblastoid cell line and a T-lymphoblastoid cell. These cells have no TAP transporter genes, and express reduced levels of HLA-A2 and no detectable endogenous HLA-B5 (23). T2 cells transfected with murine H2-Kb expressed Kb molecules at levels that were similar to the levels of HLA-A2. The T2-Kb cells were a gift from Dr E. Cerundulo (John Radcliffe Hospital, Oxford).
Antibodies
Y3 is a mouse mAb specific for the
1/
2 domain of the murine Kb molecule (24).
Peptide-binding assays
For MHC class I-binding assays, RMA-S cells were cultured at 26°C for 18 h. The medium used for the binding assay consisted of RPMI, 5% FCS which had been heated for 10 min at 100°C in a water bath to inactivate proteases. Serial dilution of peptides were prepared in assay medium in 96-well, round-bottomed, non-tissue culture-treated assay plates (Falcon 3910). The temperature-induced RMA-S cells were washed and resuspended at 1 x 106 cells/ml in assay medium. Cells (1 x 105) were added to each peptide-containing well in the assay plate. Wells containing RMA-S cells but no peptide were used as negative control. A sample of temperature induced RMA-S cells was withheld and kept on ice as a positive control. Binding of peptides to H2-Kb was allowed to proceed for 2 h at 37°C. After 2 h cells were washed and stained by indirect immunofluorescence with FITC-labeled antibodies for surface Kb (Y3 antibody) expression. FACS analysis was performed on a Becton Dickinson (Oxford, UK) FACScan flow cytometer. For each sample the mean fluorescence FL1 was determined using CellQuest software. Half-maximal binding was determined as the concentration of peptide needed to obtain 50% of the maximum FL1 signal for each individual peptide with the mean FL1 signal of RMA-S cells incubated at 37°C without any peptide defining the baseline.
Decay of peptide-stabilized Kb MHC class I molecules
RMA-S cells were cultured at 25°C overnight and for the last 2 h 10 µg/ml of BFA and 200 µmol of the relevant Kb-binding peptides were added. At time point 0 cells were shifted to 37°C after they were washed 3 times and resuspended in media containing BFA but no peptides. Samples of cells were removed at the indicated time points and stained with Kb-specific Y3 antibodies. The level of peptide-stabilized and temperature-induced class I molecules at time 0 was defined as 100%.
Generation of mature T lymphocytes by treatment of thymic organ cultures from MHC class I- and II-deficient mice with mitogenic lectins
Thymic lobes from newborn MHC/ mice were cut into three or four fragments with a scalpel blade, placed on 0.8 µm Nucleopore filters floating on DMEM (10% heat-inactivated FCS, 2 x 105 M 2-mercaptoethanol, glutamine and 50 µg/ml gentamycin) and cultured at 37°C, 5% CO2. Succinyl concanavalin A (Sigma, Poole, UK) was added at a concentration of 10 µg/ml. Thymocytes were recovered after 6 days, counted, and used for phenotypic and functional studies. The thymocytes used as responder cells in this study were 30% CD8+ and 9% CD4+.
Measurement of CTL frequency
Self- and allo-restricted CTL were generated by in vitro stimulation of naive C57BL/6, C6.C-H2bm10 and B10.A(4R) responder splenocytes with peptide-coated RMA-S stimulator cells. In order to measure the frequency of peptide-specific CTL, responder splenocytes were first immunized in vitro with peptide-loaded stimulator cells followed by LDA. The frequency of peptide-specific CTL in naive splenocytes prior to immunization was too low to be reliably detectable in LDA assays. The primary immunization was done by stimulating 2 x 106 splenocytes in 24-well plates with 2 x 105 RMA-S cells coated with OVA, mOVA, SV9 or VSV-2A peptides. The culture media consisted of RPMI with 10% FCS, penicillin, streptomycin, glutamine, 2-mercaptoethanol and no IL-2. After 5 days, decreasing numbers of responder cells (range 105300) taken from the bulk cultures were stimulated under limiting dilution conditions (24 replicas per responder cell number) in 96-well plates with irradiated peptide-loaded T2-Kb stimulator cells (104/well) and syngeneic splenocytes (2 x 105/well) in 200 µl microcultures containing 10 U/ml recombinant IL-2. After 10 days microcultures were re-stimulated using the same number of stimulator cells and feeder cells, and after 5 days each well was tested in a 51Cr-release killing assay against T2-Kb target cells coated with the immunizing peptide or a Kb-binding control peptide. T2-Kb cells coated with control peptides were used to determine the level of peptide non-specific, allo-reactive killing activity, which varied considerably between microculture. Thus, for each microculture the level of allo-reactive killing was subtracted from the level of killing observed against T2-Kb cells coated with the immunizing peptide. Microcultures were scored positive if the subtracted value showed >10% peptide-specific CTL killing of T2-Kb cells coated with the immunizing peptide. The frequency of peptide-specific CTL was calculated using zero-linear regression analysis and all data had statistically acceptable
2 < 15 and P > 0.02.
The same protocol was used to measure the frequency of peptide-specific CTL in responder T cells obtained from thymic organ cultures of MHC-deficient mice.
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Results
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Characterization of Kb-binding peptides
Four Kb-binding peptides were selected for this study. The ovalbumin-derived peptide SIINFEKL (OVA) (25) and the Sendai-virus-derived peptide FAPGNYPAL (SV9) (26) represent known immunodominant epitopes in C57BL/6 mice. The mOVA peptide differs from the OVA peptide by a 1 amino acid substitution (N
A at position 4), abolishing CTL recognition of this peptide without affecting Kb-binding (27). Conse quently, immunodominant CTL recognizing the native OVA peptide cannot participate in immune responses against the mOVA peptide. Similarly, the VSV-2A peptide differs from an immunodominant epitope derived from vesicular stomatitis virus by 1 amino acid, resulting in loss of CTL recognition without affecting Kb binding (28). Thus, in this study the mOVA and VSV-2A peptides served as epitopes of unknown immunogenicity in the self-restricted response, which is similar to the situation in allo-restricted responses where the immunogenicity of the four selected peptides was unknown.
First, the ability of the four peptides to stabilize H2-Kb class I molecules on the TAP-deficient RMA-S cells was analyzed. Following overnight incubation at 25°C these cells express a substantial amount of peptide-deficient Kb class I molecules that are unstable at 37°C. However, in the presence of Kb-binding peptides the conformation of the class I molecules is stabilized, resulting in recognition by conformation-dependent antibodies. Titration experiments showed that 10 nM of OVA and mOVA, and 100 nM of SV9 and VSV-2A peptides were required for half-maximal stabilization of Kb class I molecules (Fig. 1A). Analysis of the half-life of the peptideKb complexes at 37°C showed that it was >5.5 h for all peptides, although complexes formed with OVA and mOVA were more stable than those formed with SV9 and VSV-2A peptides (Fig. 1 B).

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Fig. 1. Binding studies of selected peptides using H2-Kb stabilization assays. (A) Temperature-induced RMA-S cells were cultured with the indicated concentrations of Kb-binding peptides as described in Methods. Peptide-stabilized Kb class I molecules were detected by FACS analysis using the Kb-specific antibody Y3. (B) The half-life of peptide stabilization was measured using temperature-induced RMA-S cells pretreated to express a maximal number of Kbpeptide complexes (see Methods). At time point 0 cells were washed and cultured in the absence of peptide. Samples of cells were removed at the indicated time points and the level of peptide-stabilized Kb molecules was determined by FACS analysis. The levels of peptide-stabilized and temperature-induced class I molecules at time 0 was defined as 100%. The stability of temperature-induced Kb molecules without peptide is shown for comparison.
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Analysis of self- and allo-restricted CTL
For the generation of self- and allo-restricted CTL we used T cells from C57BL/6, C6.C-H2bm10 and B10.A(4R) mice. These strains were selected because they have the same genetic background and express identical MHC class I molecules except for differences at the H2-K locus, thus allowing us to link differences in the frequency of peptide-specific CTL to differences in the H2-K locus class I molecules, excluding effects of modifier genes present in the genetic background. In order to measure the number of peptide-specific CTL, responder splenocytes were first primed in vitro with peptide-loaded stimulator cells followed by LDA. The number of peptide-specific CTL precursors in naive splenocytes prior to in vitro priming was too low for reliable detection in LDA assays. The Kb molecules expressed by C57BL/6 mice differ by 28 amino acids from the Kk molecules of B10.A(4R) mice and by 5 amino acids from Kbm10 molecules of C6.C-H2bm10 mice. Thus, positive selection in C6.C-H2bm10 mice was expected to result in a larger number of CTL capable of recognizing peptides presented by Kb class I molecules compared to B10.A(4R) mice.
To test these predictions, responder splenocytes were primed in bulk using peptide-loaded RMA-S cells and after 5 days graded numbers of responder lymphocytes were seeded in limiting dilution 96-well plates using peptide-loaded T2-Kb as stimulator cells. Microcultures were re-stimulated once, and then the CTL activity of each individual well was analyzed against T2-Kb coated with the immunizing peptide or a control peptide to distinguish between peptide-specific and MHC-reactive CTL. Each dot in Fig. 2 shows the CTL activity of individual microcultures. Microcultures above the shaded area were scored as positive since they displayed >10% specific lysis of T2-Kb coated with the immunizing peptide compared with T2-Kb coated with a control peptide. Preferential killing of control T2-Kb targets was rare (microcultures below the shaded area), indicating that the assay measured CTL that responded to peptide immunization. Furthermore, expansion of microcultures resulted in CTL lines with peptide-specific killing activity (see Fig. 4). Figure 2 demonstrates that Kb-restricted CTL specific for OVA, VSV-2A and mOVA were readily detectable in cultures derived from C57BL/6, C6.C-H2bm10 and B10.A(4R) responders.

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Fig. 2. Peptide-specific CTL are detectable in limiting dilution conditions. Responder splenocytes from C57BL/6, C6.C-H2bm10 and B10.A(4R) mice were primed in bulk cultures with peptide-loaded stimulator cells as described in Methods. After 5 days responder lymphocytes were stimulated with the same peptides using LDA conditions to detect microcultures with peptide-specific CTL activity. In the top panels the stimulating peptide was SV9, in the middle panels it was VSV-2A and in the bottom panels it was mOVA. Each data point in the panels shows the killing activity of individual microcultures against T2-Kb target cells coated with the immunizing peptide or a control peptide. Microcultures above the shaded area lysed targets coated with the immunizing peptide at least 10% more efficiently than targets coated with control peptides. These microcultures were considered as peptide specific.
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Fig. 4. Avidity comparison of self- and allo-restricted CTL. Microcultures that showed SV9 peptide-specific killing in the experiments shown in Fig. 2 were expanded to test the avidity of the CTL. All expanded CTL were tested for their ability to kill RMA-S target cells coated with decreasing concentrations of the SV9 peptide. The broken lines indicate the peptide concentration required for half-maximal killing of individual CTL lines.
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In order to determine the number of peptide-specific CTL, the LDA data were subjected to zero-linear regression analysis (Table 1). The mean frequency of SV9-specific CTL in cultures from C57BL/6 mice was 1/27,746 compared to a mean frequency of 1/158,661 and 1/115,580 in C6.C-H2bm10 and B10.A(4R) mice respectively. Thus, self-restricted CTL against this peptide were more frequent than allo-restricted CTL, but only by a factor of 35. Similar results were obtained with the OVA and VSV-2A peptides. Here, the mean frequency of self-restricted CTL specific for OVA (1/80,803) was
3-fold higher than the frequency of allo-restricted CTL in C6.C-H2bm10 (1/246,604) and B10.A(4R) (1/219,661) responders. Similarly, self-restricted CTL specific for VSV-2A (1/23,345) were more frequent than allo-restricted CTL [1/122,436 for B10.A(4R) and 1/230,000 for C6.C-H2bm10]. In contrast, analysis of the mOVA peptide revealed that the frequency of allo-restricted CTL in C6.C-H2bm10 responders (1/73,292) was actually higher than that of self-restricted CTL (1/232,326). The frequency in B10.A(4R) (1/204,438) responders was similar to the frequency of self-restricted CTL.
Analysis of T lymphocytes generated from pre-selection thymocytes in the absence of MHC molecules
Thymocytes from MHC-deficient mice show a thymic maturation block at the CD4/CD8 double-positive stage (29). We have recently demonstrated that the addition of mitogenic plant lectins such as succinyl concanavalin A and maackia amurensis to MHC-deficient thymi in organ culture results in the maturation of functionally competent, single-positive CD4 and CD8 T cells (30). This allowed us to test whether mature T cells generated in the absence of MHC molecules can mount specific CTL responses against the SV9 peptide presented by Kb. Lectin-selected T cells from MHC-deficient thymic organ cultures were co-cultured with the peptide-loaded stimulator cells for 5 days followed by LDA analysis of responder T cells. Figure 3 shows that SV9-specific CTL were readily detected in the LDA analysis. The frequency of SV9-specific CTL was 1/83,954, which is similar to the frequency found in the allo-restricted CTL response against this peptide (Table 1).

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Fig. 3. Peptide-specific CTL are present in an MHC-unselected repertoire. T cells generated in thymic organ cultures in the presence of lectins and in the absence of MHC molecules were primed in vitro with SV9 peptide-loaded RMA-S stimulator cells for 5 days, and then analysed in LDA conditions as described in Fig. 2. The killing activity of individual microcultures against T2-Kb target cells coated with the immunizing SV9 peptide or a Kb-binding control peptide is shown. Microcultures above the shaded area lysed targets coated with the immunizing peptide at least 10% more efficiently than targets coated with the control peptide. These microcultures were considered as peptide-specific. Linear regression analysis indicated that the frequency of SV9-peptide-specific CTL was 1/83,594 ( 2 = 0.0515; P = 1).
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Avidity of self- and allo-restricted CTL
In order to characterize the avidity of self- and allo-restricted CTL, microcultures with specificity for SV9 peptides (Fig. 2) were expanded to obtain sufficient CTL for peptide titration experiments. In total, 13 SV9-specific, Kb-restricted CTL lines were analyzed: five from B10.A(4R) donors, three from C6.C-H2bm10 donors and five from C57BL/6 donors (Fig. 4). The highest avidity was displayed by an allo-restricted CTL line derived from B10.A(4R) donors showing half-maximal target cell lysis at 30 pM peptide (Fig 4A). This was followed by the avidity of a self-restricted CTL line requiring 50 pM peptide for half-maximal lysis (Fig 4C). Although the number of CTL lines was relatively small, the avidity range of self-restricted CTL (50500 pM) was tighter than that of allo-restricted CTL (307000 pM). Thus, thymic selection events appeared to narrow the avidity range of self-MHC-restricted CTL.
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Discussion
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We have tested the immunogenicity of defined antigenic peptides presented by self- and allo-MHC, and found that following in vitro priming three out of four peptides stimulated more self-restricted CTL and one peptide stimulated more allo-restricted CTL. For two peptides that were known to represent immunodominant self-restricted epitopes, the number of self-restricted CTL was 3- to 5-fold greater than that of allo-restricted CTL. However, one of the remaining two peptides of unknown immunogenicity stimulated a 3-fold larger number of CTL in allogeneic B6.C-Hbm10 responders compared to syngeneic C57BL/6 responders. The other peptide stimulated 5-fold more CTL in C57BL/6 mice compared to allogeneic responders. The LDA assay used in this study was not sufficiently sensitive to detect the CTL precursor frequency in naive T cell populations. Instead, we measured the number of CTL in T cell populations after 5 day in vitro priming, leading to an expansion of peptide-specific CTL. Although identical in vitro stimulation conditions were used, preferential expansion of self- or allo-restricted CTL cannot be excluded. Consequently, the presented data measure the magnitude of peptide-specific CTL responses following CTL precursor priming and expansion, a valid indicator of the strength of CTL responses.
Recent elegant studies with peptide libraries have demonstrated that allo-restricted CTL generated in a human (31) and murine (32) system display a similar level of diversity as self-restricted CTL. These experiments also demonstrated that high-avidity CTL were present in the allo-restricted repertoire, although a direct comparison with self-restricted responses was not possible because self- and allo-restricted CTL recognized different peptides present in the library. The analysis of the human responses revealed that self-restricted CTL specific for the peptide library were only 2-fold more frequent than allo-restricted CTL (31), whilst in murine experiments a much greater difference was observed between self- and allo-restricted CTL (32). The latter result might be related to an in vitro depletion protocol designed to remove unwanted allo-reactive CTL, which may have removed some peptide-specific, allo-restricted CTL. Together, the data obtained with peptide libraries are compatible with those of our study using single, defined peptide epitopes.
One possible explanation for the high frequencies of allo-restricted CTL is that the residues in the
1 and
2 helices of MHC class I molecules that are contacted by the TCR are frequently conserved between class I alleles (3). Kb, the CTL restriction element employed in our current study, differs from Kbm10 by five residues in the
2 domain, and from Kk by 18 residues in the
1 domain and 10 residues in the
2 domain. Structural and functional studies of three different Kb-restricted TCR have identified 18 contact residues in the
helices lining the peptide-binding groove of the Kb molecule. Surprisingly, only three residues were contacted by all three TCR. This suggests that individual TCR use a subset of 18 potential contact points for Kb-restricted peptide recognition. These contact residues are frequently conserved amongst MHC alleles. For example, the Kk and Kbm10 molecules used in this study share with Kb 16 and 17 of the 18 TCR contact points respectively. A similar level of conservation is seen in Kd, Dk and Db molecules which share respectively 14, 15 and 17 of the 18 TCR contacts with Kb. This substantial conservation of TCR contacts provides one possible explanation for the high frequency of Kb-restricted CTL in the B10.A(4R) and C6.C-H2bm10 repertoire. Kb-restricted CTL might occur in the B10.A(4R) and C6.C-H2bm10 repertoire as a consequence of positive selection on structurally related MHC molecules.
As an alternativeand not mutually exclusiveexplanation for Kb-restricted CTL, we considered the possibility that MHC-restricted recognition might be an intrinsic feature of the TCR repertoire. We tested this idea by stimulating Kb-restricted CTL among T cells that had been generated from pre-selection thymocytes of ß2-microglobulin/, Aß/ mice (30). Interest ingly, the number of Kb-restricted CTL after in vitro priming of MHC-unselected T cells was similar to that seen in the Kk- and Kbm10-selected repertoire, demonstrating that positive selection on MHC molecules is probably not required for the generation of peptide-specific, allo-restricted CTL. It is possible that ß2-microglobulin/, Aß/ mice express low level of class I heavy chain (33) that might affect T cell repertoire selection. However, this is unlikely since we have previously shown that functional CTL are detectable in thymic organ cultures from MHC-deficient mice only after lectin induced maturation (30), indicating that residual class I heavy chain expression was insufficient to produce detectable numbers of functional CTL in our system.
Our results with CD8 T cells from MHC-deficient mice are reminiscent of observations that CD4 T cells generated in the absence of MHC-driven selection are MHC reactive with frequencies similar to those with which MHC-selected T cells respond to allogeneic MHC molecules (34,35). The data in this study show that not only MHC reactivity but also MHC restriction is an intrinsic property of the TCR repertoire prior to positive and negative selection. We may underestimate the benefits of positive selection by focussing on CTL numbers. An important function of positive selection may be to select CTL with a useful avidity (5). This may explain our observation that self-restricted CTL lines appeared to recognize SV9 peptides within a relative narrow concentration range of 50500 pM. In contrast, the concentration range covered by allo-restricted CTL was much wider and included low-avidity (probably useless) as well as high-avidity (potentially harmful) CTL.
High-avidity allo-restricted CTL can be raised against tumour-associated peptide epitopes to which autologous CTL are tolerant. Thus, the allo-restricted repertoire provides a source of tumour-reactive CTL which are absent in the autologous repertoire. Such CTL can be used for adoptive immunotherapy of leukemia patients undergoing allogeneic bone marrow transplantation. In addition, TCR from allo-restricted CTL can be inserted into viral vectors capable of infecting autologous CTL (36,37), thereby providing them with tumour-reactive specificities which they do not normally possess.
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Acknowledgements
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This work was supported by the Cancer Research Campaign and the Leukaemia Research Fund (T. H. Y. and H. J. S.) and the Medical Research Council, UK (M. L. and M. M).
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Abbreviations
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APCantigen-presenting cell
CTLcytotoxic T lymphocyte
LDAlimiting dilution analysis
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