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Circumventing T-cell Tolerance to Tumour Antigens
Biologicals (2001) 29, 277–283 doi:10.1006/biol.2001.0300, available online at http://www.idealibrary.com on
Circumventing T-cell Tolerance to Tumour Antigens H. W. H. G. Kessels, K. E. de Visser, A. M. Kruisbeek and T. N. M. Schumacher* Department of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands
Abstract. During past decades, many attempts have been made to induce or enhance tumour-specific T-cell immunity in cancer patients by vaccination. However, it has become apparent that in a large number of cases the naturally occurring tumour-specific T-cell repertoire is of low affinity and therefore inefficient in mediating tumour rejection. Because of the potential therapeutic value of high affinity TCRs with tumour/ lineage specificities, we set out to develop a number of new technologies that can be used to create improved tumour-specific T-cell immunity. These strategies entail: (i) the efficient expansion of low affinity T cells specific for self antigens through the use of variant peptides with improved TCR-binding characteristics; (ii) a retroviral library-based technology to improve the affinity of (self-specific) T-cell receptors in vitro, and (iii) proof of principle for the feasibility of TCR gene transfer as a means to generate T-cell populations with a desired antigen-specificity in vivo. Collectively this toolbox should allow us to create improved T-cell receptors for human tumour antigens, which can subsequently be used to impose © 2001 The International Association for Biologicals tumour-reactivity on to peripheral T cells. Key words: T-cell receptor, tumour antigens, tolerance, gene therapy.
Introduction In the past five to 10 years a large number of human tumour antigens has been identified by expression, cloning and biochemical purification of peptides.1 The T-cell epitopes identified with these strategies belong to two di#erent categories. The first category is formed by neo-antigens and includes antigens formed by somatic mutation of normal gene products. Because these antigens are non-self, the relevant T-cell populations in these patients have generally not been tolerized. Consequently a high a#inity T-cell repertoire for these antigens is available, and this T-cell repertoire may be targeted by immunotherapeutic interventions. However, the frequency of neo-antigens in di#erent tumour types is rather limited, possibly because only tumour cells that lack strong rejection antigens grow out to form established tumours.2 In addition, most of the tumour-derived neo-antigens are patient-specific, and it is di#icult to design broadly applicable strategies that target such antigens. Contrary to expectations, the molecular definition of human tumour antigens revealed that the majority of human tumour antigens are derived *To whom correspondence should be addressed. E-mail: [email protected] 1045–1056/01/090277+07 $35.00/0
from non-mutated proteins that are not entirely tumour-specific. This category is formed by peptides derived from normal cellular proteins that are often expressed in a restricted set of cell lineages. These T-cell epitopes include the melanocytedi#erentiation antigens, for which tolerance induction is only partial (see below), and proteins for which the normal expression pattern is restricted to immunologically privileged sites, such as testis.1 An attractive feature of this class of antigens (referred to as tumour-lineage antigens) is that these antigens are shared between large patient groups. Since their identification, the role of melanocyte di#erentiation antigens (MDAs) as rejection antigens has been further suggested by the finding that (skin homing) T cells with MDA specificity are found in patients with auto-immune vitiligo and melanoma. More direct evidence for a role of MDA-specific T cells comes from the observation that vaccination with MDA-derived peptides can result in immune selection in vivo, and objective cancer responses in patients that have been vaccinated with a gp100derived epitope have been observed. Despite these early successes, many studies have suggested that self-tolerance may result in suboptimal (low avidity) recognition of self antigens by cytotoxic T cells.3,4 For instance, many groups have 2001 The International Association for Biologicals
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isolated MDA-specific T-cell lines from melanoma patients that e#iciently lyse peptide-coated target cells, but that are only moderately e#ective against tumour cells. In such cases T-cell activation can be improved by the exogenous addition of peptide to the melanoma cells, both in CTL assays and in NFAT-based reporter assays for T-cell activation. Importantly, it has been directly demonstrated that the avidity of this interaction also has a major impact on the in vivo e#icacy of tumour-specific CTL.5 Animal studies have directly established the factors that a#ect the avidity of the interaction between TCRs and peptide-MHC complexes and thereby a#ect T-cell tolerance to self antigens. These factors include the expression level/cellular processing of the self antigen, the stability of the peptide-MHC complex, and the a#inity of individual T cells for the self antigen involved. Collectively, these animal models lead to the conclusion that self-tolerance appears absolute for epitopes that are well expressed/processed and that bind MHC with high a#inity. For self epitopes that are presented in lower numbers, tolerance induction appears to a#ect high a#inity T cells, but not low a#inity T cells. Because of the limitations on the naturally occurring tumour-specific T-cell responses, the development of in vitro strategies to create more robust tumour-specific T-cell receptors is of critical significance. In this paper we describe three parallel lines of research in mouse model systems that collectively should allow the creation of enhanced tumour-specific T-cell immunity through TCR gene therapy.
line 293T, by Fugene or pfx-2 lipid transfection (Invitrogen). Forty-eight hours after transfection retroviral supernatants were collected and used for transductions, through a recombinant human fibronectin fragments-based transduction procedure. In brief, non-tissue culture treated Falcon Petri dishes (3 cm diameter) (Becton Dickinson) were coated with 2 ml of 30 mg/ml recombinant human fibronectin fragment CH-296 (RetroNectin ; Takara, Otsu, Japan) at room temperature for two h. The CH-296 solution was removed and replaced with 2 ml 2% BSA (Sigma) in PBS for 30 min at room temperature. The target cells were plated on RetroNectinTM coated dishes (0·5106 cells/Petri dish) in one ml of retroviral supernatant. Cells were cultured at 37 C for 24 h, washed and transferred to 25 cm2 culture flasks (Falcon plastics, Becton Dickinson). For transduction of mouse spleen cells the same procedure was used with the following modifications. Plasmid DNA was transfected into Phoenix-E cells and the target cells obtained following culture in Con A and IL-7 were transduced by two rounds of spin infection. Mice
C57BL/10 mice and RAG / mice were obtained from the experimental animal department of the Netherlands Cancer Institute. Influenza A NP transgenic mice on a C57BL/10 background (B10NP mice) were a kind gift of Dr D. Kioussis, NIMR, London, U.K. Mice were handled in accordance with institutional guidelines.
Results Materials and methods Peptide synthesis and preparation of H-2Db tetramers
Peptides were produced using standard FMoc chemistry on a Syro 60-channel peptide synthesizer. Soluble allophycocyanin (APC)-labelled H-2Db tetramers were produced as described6,7 and stored frozen in Tris-bu#ered saline/16% glycerol/0·5% bovine serum albumin (BSA). Cell lines and retroviral transduction
The 34·1L cell line is a derivative of the day 14 foetal thymus-derived prethymocyte cell line 34·1L, and was obtained by transduction with the murine CD3 chain.8 For transduction of 34·1L cells, plasmid DNA was transfected into Phoenix-A cells, a derivative of the human embryonic kidney cell
Effects of self tolerance on the self-specific T-cell repertoire
In a series of elegant experiments by Davis and coworkers, it was first shown that fluorescently labelled oligomeric MHC class I molecules (MHC class I tetramers) can be used to specifically stain antigen-specific T cells known to recognize a given combination of peptide and MHC class I.6 We and many other groups have expanded on this technique with the assembly of tetramers of mouse MHC molecules complexed with various antigenic peptides, and the resulting reagents now form an indispensable tool for the detection of polyclonal populations of antigen-specific T cells during viral or bacterial infections. To assess the e#ects of self tolerance on the antigen-specific T-cell repertoire we have compared the antigen-specific T-cell
Circumventing T-cell Tolerance
response against the same antigen when present either as a foreign antigen or as a self antigen.9 In these experiments wild type mice or mice transgenic for a fragment of influenza A nucleoprotein (B10NP mice) were infected with influenza A/NT/60/68 and the CD8 + T-cell response against the nucleoprotein epitope NP366 (sequence ASNENMDAM) was monitored by MHC tetramer technology. These experiments reveal that whereas a prominent antigen-specific T-cell response can be detected in wild type mice infected with influenza A, the magnitude of this response is significantly reduced in NP-transgenic mice. More importantly, the NP-specific T-cell population in B10NP mice binds the relevant MHC tetramers less avidly (Fig. 1) and only exerts T-cell e#ector functions at high antigen concentrations, compared to NP-specific T cells in non-transgenic mice. These data are consistent with a model in which high avidity self-specific T cells have been deleted and only low avidity self-specific T cells remain.10 These low avidity T cells have a demonstrable cytotoxic capacity, albeit only at high antigen concentrations. In contrast, as a consequence of this low avidity the in vitro expansion of these self-specific T cells upon encounter of the NP366 ligand is severely impaired both at low and high antigen concentrations.11 Peptide ligands bound to MHC class I molecules can be considered to carry two functional groups. With one set of side-chain and main chain atoms the peptide is bound to the relevant MHC class I molecule and a second set of side-chain and main chain atoms is contacted by the T-cell receptor. Peptide ligands that are insu#iciently strong immunogens can therefore in theory be optimized by altering either functional moiety. Specifically, peptides may be modified with the aim of improving their MHC class I-binding characteristics, or alternatively may be modified to improve their interaction with antigen-specific T-cell receptors. It has previously been documented that for self antigens that bind poorly to the relevant MHC class I molecule, altered peptide ligands can be produced that bind with a markedly increased a#inity, and as a consequence form superior immunogens. In contrast, with a single exception,12 relatively little is known about the e#ects of manipulating TCR-interacting residues on the induction of self-specific T-cell responses. To address this issue we have generated a large set of variants of the NP-epitope in which TCR-interacting residues have been altered. Subsequently, these altered peptide ligands have been tested for their capacity to promote the expansion
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Figure 1. Low avidity NP-specific T cells in NP-transgenic mice. B10 and B10NP mice were infected with influenza A/NT/60/68 and 14 days following infection, lungs were isolated and cells were stained with anti-CD8 antibody, NP366-MHC tetramers and anti-TCR antibody.9 The middle panels show reduced levels of MHC tetramer binding in influenza A-infected NP-transgenic mice as compared to influenza A-infected non-transgenic animals. The bottom panels show that T-cell receptor expression on NP366-MHC tetramerhi and NP366-MHC tetramerlo cells in B10 and B10NP mice respectively is identical.
of the self-specific NP366 T-cell repertoire in NP-transgenic mice. These experiments reveal that a single altered peptide ligand can form a higher a#inity ligand for most if not all self-specific NP366 T cells and as a consequence can be used to induce dramatic expansion of self-specific T cells in a situation in which the self ligand is incapable of mediating this e#ect.11 This type of altered peptide ligands, which strongly induce the expansion of self-specific T cells, may form a useful tool to obtain
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large amounts of self-specific T cells for adoptive transfer of T cells or T-cell receptors (see below), in case of self antigens that are over-expressed on tumour tissue. Alternatively, the resulting low a#inity self-specific repertoire may form a useful starting point to obtain high a#inity self-specific T-cell receptors through the use of in vitro methods (see below).
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In vitro modification of T-cell receptors by retroviral TCR display
As outlined above and in several other studies, self-tolerance leads to the removal of the high a#inity T-cell repertoire specific for self antigens, including self antigens expressed on tumour tissues. Because of the potential therapeutic value of TCRs with such tumour/lineage specificities, we set out to develop an in vitro strategy for T-cell receptor selection that can be used to bypass in vivo tolerance.8 For the in vitro isolation and generation of monoclonal antibodies, antibody phage display has proven to be a useful technology to replace hybridoma technology and animal immunization. Analogous to this technology, TCRs have been expressed as single chain fragments (scTCR) on the surface of both phage and yeast. Recently, yeast TCR display was shown to be a successful strategy for the in vitro selection of variant scTCR fusion proteins with a dramatic increase in a#inity for an allogeneic peptide/MHC target.13 Such high a#inity scTCRs may be of significant use as probes for the detection of specific peptide/MHC complexes. However, it is unclear whether yeast or phage-based TCR display systems will prove equally useful to change the fine-specificity of T-cell receptors. Specifically, the ability of T cells to discriminate between closely related ligands appears to be directly related to the property of TCR/CD3 complexes to cluster upon encounter of their cognate ligands14,15 and it may prove di#icult to copy this process in these systems. We have developed a T-cell-based in vitro TCR selection strategy that can be used to isolate heterodimeric TCRs with increased a#inities or altered specificities. This strategy for TCR display closely mimics the in vivo situation, by insertion of libraries of T-cell receptor genes into a TCRnegative T-cell host (Fig. 2). Such T-cell linedisplayed TCR libraries not only allow the selection of desirable TCRs by biochemical means, but also o#er the possibility of directly testing the functional behaviour of selected TCRs.
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Figure 2. Schematic representation of the generation and screening of retroviral TCR display libraries.
As a host for a T-cell line-displayed TCR library, an immature T-cell line named 34·1L that does not express endogenous T-cell receptor and chains but does express all CD3 components required for TCR assembly was created. As a first target for retroviral TCR display we used a high a#inity murine TCR of which the antigen specificity is well established. This F5 T-cell receptor (V4; V11) specifically recognizes the immunodominant H-2Dbrestricted CTL epitope NP366–374 (ASNENMDAM) of the influenza A/NT/60/68 nucleoprotein.16 Following introduction of the F5 TCR into the 34·1L cell line by retroviral transduction, the transduced cell line expresses high levels of the introduced F5 TCR as measured by anti-TCR and MHC tetramer flow cytometry. To test the feasibility of in vitro selection of TCRs with defined specificities, we aimed to isolate novel T-cell receptors with either the same specificity as the parental TCR, or receptors that have acquired a specificity for a variant influenza epitope. To modify the peptide specificity of TCRs without generating variant TCRs that are broadly cross-reactive, mutation of only those areas of the TCR that interact primarily with the antigenic peptide is preferred. Structural analysis of four di#erent human and mouse TCRs complexed with their cognate peptide/MHC class I all point to the CDR3 loops of the TCR and chain as the major determinants of peptide specificity. Because in the current set of experiments we were primarily interested in obtaining TCRs that can discriminate between epitopes that di#er in the C-terminal half of the peptide (see below), a TCR library
Circumventing T-cell Tolerance
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was manufactured such that its structural diversity is directed towards the TCR CDR3 loop exclusively. To examine whether variant TCRs could be obtained that retain the ligand specificity of the parental F5 TCR, the T-cell library was screened for binding of tetrameric H-2Db complexes containing the A/NT/60/68 nucleoprotein CTL epitope (ASNENMDAM) (Fig. 2B). Following two rounds of in vitro selection, two di#erent clones persisted: the parental F5 clone and a variant clone named NT-1. The CDR3 DNA sequence of the NT-1 TCR contains five mutations that result in three conservative amino acid substitutions.8 This variant TCR binds A/NT/60/68 NP366–374 tetramers with similar e#iciency as the F5 TCR (data not shown). The TCR CDR3 library was subsequently screened for the presence of T-cell receptors that bind H-2Db tetramers containing a variant influenza A nucleoprotein epitope. This variant NP366–374 epitope (ASNENMETM), derived from the influenza A/PR8/34 strain, di#ers from the A/NT/60/68 CTL epitope by two conservative amino acid substitutions in the C-terminal half of the peptide and is not recognized by the F5 T-cell receptor. The TCR CDR3 library was subjected to four rounds of selection with H-2Db tetramers that contain the variant epitope, to select for the TCR clone(s) that exhibit highest a#inity for this epitope. From this TCR library selection a single TCR clone emerged (named PR-1) that binds avidly to the A/PR8/34 NP366–374 tetramers. Sequence analysis of the PR-1 TCR reveals seven nucleotide mutations in its CDR3 DNA sequence compared to the parental F5 TCR. These mutations result in four conservative amino acid changes and one non-conservative Arg to Trp substitution.8 To examine whether in vitro selected variant TCRs can evoke T-cell activation upon peptide recognition, ligand-induced IL-2 gene transcription was measured. To this purpose we used a selfinactivating (SIN) retroviral vector containing multiple NFAT binding sites upstream of a minimal IL-2 promoter and the reporter gene YFP. 34.1L cells expressing the F5, NT-1 or PR-1 TCR were virally transduced with the NFAT-YFP reporter construct and the transduced cells were exposed to target cells in the presence of di#erent concentrations of either the A/NT/60/68 or A/PR8/34 T-cell epitope. Both variant clones NT-1 and PR-1 e#iciently induce T-cell activation upon specific antigen recognition with an absolute specificity for the epitope used during the in vitro selections (Fig. 3).
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Figure 3. Signaling function of in vitro selected TCRs. TCR expressing 34.1L cells transduced with the NFATYFP construct were exposed to EL4 target cells in the presence of di#erent concentrations of either the A/NT/ 60/68 (open squares) or A/PR8/34 (filled circles) T-cell epitope.8 Sensitivity and specificity of the di#erent TCRs was determined by flow cytometric analysis of the percentage of YFP expressing 34.1L cells. T-cell activation upon stimulation with PMA (10 ng/ml) and ionomycin (1.67 mg/ml) results in 60–65% YFP expressing cells (not shown). The data shown are means of triplicates S.D.
These experiments show that in vitro selection of variant T-cell receptors by retroviral TCR display can yield receptors with high potency, as revealed by both biochemical means and functional assays. T-cell receptors that are isolated from such libraries should be useful for the creation of redirected T-cell populations, through gene transfer of peripheral T lymphocytes.
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TCR gene transfer
a pronounced improvement in tumour rejection, without resulting in overt auto-immunity. Collectively, these experiments provided the first proof of principle of TCR gene transfer as a realistic immunotherapeutic option
Isolate peripheral T cells Introduce TCR-encoding genes in vitro Infuse redirected T cells
Figure 4. Redirecting T cells through TCR gene transfer.
Immunotherapy through TCR gene transfer
Because the specificity of T cells is entirely contained within the alpha and beta chains of the TCR, it should in theory be possible to impose a desired antigenic specificity onto cytotoxic T cells by introduction of TCR-encoding genes with this desirable specificity. Furthermore, this type of TCR gene therapy would form a useful strategy to endow T cells with in vitro modified T-cell receptors, geared towards an optimal recognition of antigens expressed on tumour tissues. This novel therapeutic strategy would entail the introduction of TCR genes into peripheral T cells of patients ex vivo and subsequent re-introduction of the T cells that have now obtained a desirable antigen specificity (Fig. 4). To assess the feasibility of TCR gene transfer for the creation of T-cell populations with a desirable antigen specificity we have developed an animal model system. In this model system, mouse peripheral T cells were infected (transduced) with retroviruses expressing the influenza A NP-specific F5 T-cell receptor. While murine T cells are generally refractory to infection with Moloney-based retroviruses, upon culture of primary mouse T cells with Con A and IL-7 transduction e#iciencies of 5–10% can be achieved. We have subsequently used such TCRtransduced T-cell populations to examine their in vivo behaviour. These experiments have established that TCR-transduced T cells react to antigen in vivo and expand dramatically in number (H.W.H.G.K. et al., unpublished data). Importantly, the introduction of small numbers of TCR-modified T-cell populations into immunodeficient RAG / mice leads to
Discussion The immune system contains a large collection of T cells that covers a broad range of peptide/MHC specificities and thereby can identify subtle changes in MHC epitope presentation. However, selftolerance leads to the removal of the high a#inity T-cell repertoire specific for self antigens, and this will include T cells with desirable specificities, such as for many self antigens expressed on tumour tissues. Because of the potential therapeutic value of TCRs with such tumour/lineage specificities, we set out to develop strategies for T-cell receptor selection and transfer that can be used to bypass in vivo tolerance. In a first set of experiments we have demonstrated how self tolerance leads to the selective deletion of high avidity T cells from the T-cell repertoire. Furthermore, we document the use of altered peptide ligands with improved TCRbinding characteristics as a strategy to promote the expansion of low avidity self-specific T cells. For tumour antigens that are highly over-expressed on tumour tissues such as HER2/neu, adoptive transfer of self-specific T-cell populations obtained through the use of such altered peptide ligands may form a viable therapeutic strategy. However, because of the reduced antigen sensitivity of low avidity selfspecific T cells, infusion of these cells is unlikely to be successful in cases in which tumour/lineage antigens are not, or only modestly over-expressed in tumour tissue. In these cases, optimization of T-cell receptors by retroviral TCR display may form a useful strategy to obtain more potent tumourspecific T-cell receptors. Finally, we have set up a model system that provides the first proof of principle for the feasibility of TCR gene therapy. This strategy of TCR gene transfer should be useful to introduce in vitro-modified T-cell receptor genes specific for tumour antigens. However, it is apparent that in certain human conditions, introduction of unmodified T-cell receptors through TCR gene transfer may be equally appealing. For instance, the infusion of EBV-specific T cells in patients with post-transplant lymphoma has previously been shown to result in marked tumour regression. The infusion of TCR-modified, EBV-directed T cells in this setting may provide an interesting alternative
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strategy that circumvents the requirement for the conventional large scale in vitro generation of EBV-specific T-cell populations. Conclusions
It is becoming increasingly clear that the ine#ectiveness of tumour-specific T-cell responses is in large part due to the e#ects of self tolerance. To address this issue we have set out to develop new techniques that can be used to improve tumourspecific T-cell immunity. Through the use of these techniques we seek to create T-cell receptors that recognize antigens expressed on tumour tissue with high a#inity. Introduction of such in vitro-modified T-cell receptors into peripheral T-cell populations by gene transfer forms an attractive strategy to enhance tumour-specific T-cell immunity in settings where the endogenous T-cell response is insu#icient. Note added in proof
Data described as ‘‘(H.W.H.G.K. et al., unpublished data)’’ are now in press with Nature Immunology. Acknowledgements This research was supported by grants from the Dutch Cancer Society (NKB 97-1442 and NKB 99-2036) and the Dutch Foundation for Scientific Research (NWO Pioneer 00-03). We thank M. Toebes and M. van den Boom for excellent technical support throughout this research. References 1. Gilboa E. The makings of a tumour rejection antigen. Immunity 1999; 11: 263–270. 2. Shankaran V, Ikeda H, Bruce AT, White JM, Swanson PE, Old LJ et al. IFN-gamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature 2001; 410: 1107–1111. 3. Kawakami Y, Rosenberg SA. Immunobiology of human melanoma antigens MART-1 and gp100 and their use for immuno-gene therapy. Int Rev Immunol 1997; 14: 173–192.
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4. Yee C, Savage PA, Lee PP, Davis MM, Greenberg PD. Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers. J Immunol 1999; 162: 2227–2234. 5. Zeh HJ, 3rd, Perry-Lalley D, Dudley ME, Rosenberg SA, Yang JC. High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumour e#icacy. J Immunol 1999; 162: 989–994. 6. Altman JD, Moss PAH, Goulder PJR, Barouch DH, McHeyzer-Williams MG, Bell JI et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996; 274: 94–96. 7. Haanen JB, Toebes M, Cordaro TA, Wolkers MC, Kruisbeek AM, Schumacher TN. Systemic T cell expansion during localized viral infection. Eur J Immunol 1999; 29: 1168–1174. 8. Kessels HW, van Den Boom MD, Spits H, Hooijberg E, Schumacher TN. Changing T cell specificity by retroviral T cell receptor display. Proc Natl Acad Sci USA 2000; 97: 14578–14583. 9. de Visser KE, Cordaro TA, Kioussis D, Haanen JB, Schumacher TN, Kruisbeek AM. Tracing and characterization of the low-avidity self-specific T cell repertoire. Eur J Immunol 2000; 30: 1458–1468. 10. Theobald M, Biggs J, Hernandez J, Lustgarten J, Labadie C, Sherman LA. Tolerance to p53 by A2.1restricted cytotoxic T lymphocytes. J Exp Med 1997; 185: 833–841. 11. de Visser KE, Cordaro TA, Kessels HW, Tirion FH, Schumacher TN, Kruisbeek AM. Low-avidity selfspecific t cells display a pronounced expansion defect that can be overcome by altered peptide ligands. J Immunol 2001; 167: 3818–3828. 12. Slansky JE, Rattis FM, Boyd LF, Fahmy T, Ja#ee EM, Schneck JP et al. Enhanced antigen-specific antitumour immunity with altered peptide ligands that stabilize the MHC-peptide-TCR complex. Immunity 2000; 13: 529–538. 13. Holler PD, Holman PO, Shusta EV, O’Herrin S, Wittrup KD, Kranz DM. In vitro evolution of a T cell receptor with high a#inity for peptide/MHC. Proc Natl Acad Sci USA 2000; 97: 5387–5392. 14. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 1998; 395: 82–86. 15. Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM et al. The immunological synapse: a molecular machine controlling T cell activation. Science 1999; 285: 221–227. 16. Townsend AR, Gotch FM, Davey J. Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell 1985; 42: 457–467.
Circumventing T-cell Tolerance to Tumour Antigens H. W. H. G. Kessels, K. E. de Visser, A. M. Kruisbeek and T. N. M. Schumacher* Department of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands
Abstract. During past decades, many attempts have been made to induce or enhance tumour-specific T-cell immunity in cancer patients by vaccination. However, it has become apparent that in a large number of cases the naturally occurring tumour-specific T-cell repertoire is of low affinity and therefore inefficient in mediating tumour rejection. Because of the potential therapeutic value of high affinity TCRs with tumour/ lineage specificities, we set out to develop a number of new technologies that can be used to create improved tumour-specific T-cell immunity. These strategies entail: (i) the efficient expansion of low affinity T cells specific for self antigens through the use of variant peptides with improved TCR-binding characteristics; (ii) a retroviral library-based technology to improve the affinity of (self-specific) T-cell receptors in vitro, and (iii) proof of principle for the feasibility of TCR gene transfer as a means to generate T-cell populations with a desired antigen-specificity in vivo. Collectively this toolbox should allow us to create improved T-cell receptors for human tumour antigens, which can subsequently be used to impose © 2001 The International Association for Biologicals tumour-reactivity on to peripheral T cells. Key words: T-cell receptor, tumour antigens, tolerance, gene therapy.
Introduction In the past five to 10 years a large number of human tumour antigens has been identified by expression, cloning and biochemical purification of peptides.1 The T-cell epitopes identified with these strategies belong to two di#erent categories. The first category is formed by neo-antigens and includes antigens formed by somatic mutation of normal gene products. Because these antigens are non-self, the relevant T-cell populations in these patients have generally not been tolerized. Consequently a high a#inity T-cell repertoire for these antigens is available, and this T-cell repertoire may be targeted by immunotherapeutic interventions. However, the frequency of neo-antigens in di#erent tumour types is rather limited, possibly because only tumour cells that lack strong rejection antigens grow out to form established tumours.2 In addition, most of the tumour-derived neo-antigens are patient-specific, and it is di#icult to design broadly applicable strategies that target such antigens. Contrary to expectations, the molecular definition of human tumour antigens revealed that the majority of human tumour antigens are derived *To whom correspondence should be addressed. E-mail: [email protected] 1045–1056/01/090277+07 $35.00/0
from non-mutated proteins that are not entirely tumour-specific. This category is formed by peptides derived from normal cellular proteins that are often expressed in a restricted set of cell lineages. These T-cell epitopes include the melanocytedi#erentiation antigens, for which tolerance induction is only partial (see below), and proteins for which the normal expression pattern is restricted to immunologically privileged sites, such as testis.1 An attractive feature of this class of antigens (referred to as tumour-lineage antigens) is that these antigens are shared between large patient groups. Since their identification, the role of melanocyte di#erentiation antigens (MDAs) as rejection antigens has been further suggested by the finding that (skin homing) T cells with MDA specificity are found in patients with auto-immune vitiligo and melanoma. More direct evidence for a role of MDA-specific T cells comes from the observation that vaccination with MDA-derived peptides can result in immune selection in vivo, and objective cancer responses in patients that have been vaccinated with a gp100derived epitope have been observed. Despite these early successes, many studies have suggested that self-tolerance may result in suboptimal (low avidity) recognition of self antigens by cytotoxic T cells.3,4 For instance, many groups have 2001 The International Association for Biologicals
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isolated MDA-specific T-cell lines from melanoma patients that e#iciently lyse peptide-coated target cells, but that are only moderately e#ective against tumour cells. In such cases T-cell activation can be improved by the exogenous addition of peptide to the melanoma cells, both in CTL assays and in NFAT-based reporter assays for T-cell activation. Importantly, it has been directly demonstrated that the avidity of this interaction also has a major impact on the in vivo e#icacy of tumour-specific CTL.5 Animal studies have directly established the factors that a#ect the avidity of the interaction between TCRs and peptide-MHC complexes and thereby a#ect T-cell tolerance to self antigens. These factors include the expression level/cellular processing of the self antigen, the stability of the peptide-MHC complex, and the a#inity of individual T cells for the self antigen involved. Collectively, these animal models lead to the conclusion that self-tolerance appears absolute for epitopes that are well expressed/processed and that bind MHC with high a#inity. For self epitopes that are presented in lower numbers, tolerance induction appears to a#ect high a#inity T cells, but not low a#inity T cells. Because of the limitations on the naturally occurring tumour-specific T-cell responses, the development of in vitro strategies to create more robust tumour-specific T-cell receptors is of critical significance. In this paper we describe three parallel lines of research in mouse model systems that collectively should allow the creation of enhanced tumour-specific T-cell immunity through TCR gene therapy.
line 293T, by Fugene or pfx-2 lipid transfection (Invitrogen). Forty-eight hours after transfection retroviral supernatants were collected and used for transductions, through a recombinant human fibronectin fragments-based transduction procedure. In brief, non-tissue culture treated Falcon Petri dishes (3 cm diameter) (Becton Dickinson) were coated with 2 ml of 30 mg/ml recombinant human fibronectin fragment CH-296 (RetroNectin ; Takara, Otsu, Japan) at room temperature for two h. The CH-296 solution was removed and replaced with 2 ml 2% BSA (Sigma) in PBS for 30 min at room temperature. The target cells were plated on RetroNectinTM coated dishes (0·5106 cells/Petri dish) in one ml of retroviral supernatant. Cells were cultured at 37 C for 24 h, washed and transferred to 25 cm2 culture flasks (Falcon plastics, Becton Dickinson). For transduction of mouse spleen cells the same procedure was used with the following modifications. Plasmid DNA was transfected into Phoenix-E cells and the target cells obtained following culture in Con A and IL-7 were transduced by two rounds of spin infection. Mice
C57BL/10 mice and RAG / mice were obtained from the experimental animal department of the Netherlands Cancer Institute. Influenza A NP transgenic mice on a C57BL/10 background (B10NP mice) were a kind gift of Dr D. Kioussis, NIMR, London, U.K. Mice were handled in accordance with institutional guidelines.
Results Materials and methods Peptide synthesis and preparation of H-2Db tetramers
Peptides were produced using standard FMoc chemistry on a Syro 60-channel peptide synthesizer. Soluble allophycocyanin (APC)-labelled H-2Db tetramers were produced as described6,7 and stored frozen in Tris-bu#ered saline/16% glycerol/0·5% bovine serum albumin (BSA). Cell lines and retroviral transduction
The 34·1L cell line is a derivative of the day 14 foetal thymus-derived prethymocyte cell line 34·1L, and was obtained by transduction with the murine CD3 chain.8 For transduction of 34·1L cells, plasmid DNA was transfected into Phoenix-A cells, a derivative of the human embryonic kidney cell
Effects of self tolerance on the self-specific T-cell repertoire
In a series of elegant experiments by Davis and coworkers, it was first shown that fluorescently labelled oligomeric MHC class I molecules (MHC class I tetramers) can be used to specifically stain antigen-specific T cells known to recognize a given combination of peptide and MHC class I.6 We and many other groups have expanded on this technique with the assembly of tetramers of mouse MHC molecules complexed with various antigenic peptides, and the resulting reagents now form an indispensable tool for the detection of polyclonal populations of antigen-specific T cells during viral or bacterial infections. To assess the e#ects of self tolerance on the antigen-specific T-cell repertoire we have compared the antigen-specific T-cell
Circumventing T-cell Tolerance
response against the same antigen when present either as a foreign antigen or as a self antigen.9 In these experiments wild type mice or mice transgenic for a fragment of influenza A nucleoprotein (B10NP mice) were infected with influenza A/NT/60/68 and the CD8 + T-cell response against the nucleoprotein epitope NP366 (sequence ASNENMDAM) was monitored by MHC tetramer technology. These experiments reveal that whereas a prominent antigen-specific T-cell response can be detected in wild type mice infected with influenza A, the magnitude of this response is significantly reduced in NP-transgenic mice. More importantly, the NP-specific T-cell population in B10NP mice binds the relevant MHC tetramers less avidly (Fig. 1) and only exerts T-cell e#ector functions at high antigen concentrations, compared to NP-specific T cells in non-transgenic mice. These data are consistent with a model in which high avidity self-specific T cells have been deleted and only low avidity self-specific T cells remain.10 These low avidity T cells have a demonstrable cytotoxic capacity, albeit only at high antigen concentrations. In contrast, as a consequence of this low avidity the in vitro expansion of these self-specific T cells upon encounter of the NP366 ligand is severely impaired both at low and high antigen concentrations.11 Peptide ligands bound to MHC class I molecules can be considered to carry two functional groups. With one set of side-chain and main chain atoms the peptide is bound to the relevant MHC class I molecule and a second set of side-chain and main chain atoms is contacted by the T-cell receptor. Peptide ligands that are insu#iciently strong immunogens can therefore in theory be optimized by altering either functional moiety. Specifically, peptides may be modified with the aim of improving their MHC class I-binding characteristics, or alternatively may be modified to improve their interaction with antigen-specific T-cell receptors. It has previously been documented that for self antigens that bind poorly to the relevant MHC class I molecule, altered peptide ligands can be produced that bind with a markedly increased a#inity, and as a consequence form superior immunogens. In contrast, with a single exception,12 relatively little is known about the e#ects of manipulating TCR-interacting residues on the induction of self-specific T-cell responses. To address this issue we have generated a large set of variants of the NP-epitope in which TCR-interacting residues have been altered. Subsequently, these altered peptide ligands have been tested for their capacity to promote the expansion
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Figure 1. Low avidity NP-specific T cells in NP-transgenic mice. B10 and B10NP mice were infected with influenza A/NT/60/68 and 14 days following infection, lungs were isolated and cells were stained with anti-CD8 antibody, NP366-MHC tetramers and anti-TCR antibody.9 The middle panels show reduced levels of MHC tetramer binding in influenza A-infected NP-transgenic mice as compared to influenza A-infected non-transgenic animals. The bottom panels show that T-cell receptor expression on NP366-MHC tetramerhi and NP366-MHC tetramerlo cells in B10 and B10NP mice respectively is identical.
of the self-specific NP366 T-cell repertoire in NP-transgenic mice. These experiments reveal that a single altered peptide ligand can form a higher a#inity ligand for most if not all self-specific NP366 T cells and as a consequence can be used to induce dramatic expansion of self-specific T cells in a situation in which the self ligand is incapable of mediating this e#ect.11 This type of altered peptide ligands, which strongly induce the expansion of self-specific T cells, may form a useful tool to obtain
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large amounts of self-specific T cells for adoptive transfer of T cells or T-cell receptors (see below), in case of self antigens that are over-expressed on tumour tissue. Alternatively, the resulting low a#inity self-specific repertoire may form a useful starting point to obtain high a#inity self-specific T-cell receptors through the use of in vitro methods (see below).
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In vitro modification of T-cell receptors by retroviral TCR display
As outlined above and in several other studies, self-tolerance leads to the removal of the high a#inity T-cell repertoire specific for self antigens, including self antigens expressed on tumour tissues. Because of the potential therapeutic value of TCRs with such tumour/lineage specificities, we set out to develop an in vitro strategy for T-cell receptor selection that can be used to bypass in vivo tolerance.8 For the in vitro isolation and generation of monoclonal antibodies, antibody phage display has proven to be a useful technology to replace hybridoma technology and animal immunization. Analogous to this technology, TCRs have been expressed as single chain fragments (scTCR) on the surface of both phage and yeast. Recently, yeast TCR display was shown to be a successful strategy for the in vitro selection of variant scTCR fusion proteins with a dramatic increase in a#inity for an allogeneic peptide/MHC target.13 Such high a#inity scTCRs may be of significant use as probes for the detection of specific peptide/MHC complexes. However, it is unclear whether yeast or phage-based TCR display systems will prove equally useful to change the fine-specificity of T-cell receptors. Specifically, the ability of T cells to discriminate between closely related ligands appears to be directly related to the property of TCR/CD3 complexes to cluster upon encounter of their cognate ligands14,15 and it may prove di#icult to copy this process in these systems. We have developed a T-cell-based in vitro TCR selection strategy that can be used to isolate heterodimeric TCRs with increased a#inities or altered specificities. This strategy for TCR display closely mimics the in vivo situation, by insertion of libraries of T-cell receptor genes into a TCRnegative T-cell host (Fig. 2). Such T-cell linedisplayed TCR libraries not only allow the selection of desirable TCRs by biochemical means, but also o#er the possibility of directly testing the functional behaviour of selected TCRs.
(1) Tranduce α -chain (-library)
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Figure 2. Schematic representation of the generation and screening of retroviral TCR display libraries.
As a host for a T-cell line-displayed TCR library, an immature T-cell line named 34·1L that does not express endogenous T-cell receptor and chains but does express all CD3 components required for TCR assembly was created. As a first target for retroviral TCR display we used a high a#inity murine TCR of which the antigen specificity is well established. This F5 T-cell receptor (V4; V11) specifically recognizes the immunodominant H-2Dbrestricted CTL epitope NP366–374 (ASNENMDAM) of the influenza A/NT/60/68 nucleoprotein.16 Following introduction of the F5 TCR into the 34·1L cell line by retroviral transduction, the transduced cell line expresses high levels of the introduced F5 TCR as measured by anti-TCR and MHC tetramer flow cytometry. To test the feasibility of in vitro selection of TCRs with defined specificities, we aimed to isolate novel T-cell receptors with either the same specificity as the parental TCR, or receptors that have acquired a specificity for a variant influenza epitope. To modify the peptide specificity of TCRs without generating variant TCRs that are broadly cross-reactive, mutation of only those areas of the TCR that interact primarily with the antigenic peptide is preferred. Structural analysis of four di#erent human and mouse TCRs complexed with their cognate peptide/MHC class I all point to the CDR3 loops of the TCR and chain as the major determinants of peptide specificity. Because in the current set of experiments we were primarily interested in obtaining TCRs that can discriminate between epitopes that di#er in the C-terminal half of the peptide (see below), a TCR library
Circumventing T-cell Tolerance
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was manufactured such that its structural diversity is directed towards the TCR CDR3 loop exclusively. To examine whether variant TCRs could be obtained that retain the ligand specificity of the parental F5 TCR, the T-cell library was screened for binding of tetrameric H-2Db complexes containing the A/NT/60/68 nucleoprotein CTL epitope (ASNENMDAM) (Fig. 2B). Following two rounds of in vitro selection, two di#erent clones persisted: the parental F5 clone and a variant clone named NT-1. The CDR3 DNA sequence of the NT-1 TCR contains five mutations that result in three conservative amino acid substitutions.8 This variant TCR binds A/NT/60/68 NP366–374 tetramers with similar e#iciency as the F5 TCR (data not shown). The TCR CDR3 library was subsequently screened for the presence of T-cell receptors that bind H-2Db tetramers containing a variant influenza A nucleoprotein epitope. This variant NP366–374 epitope (ASNENMETM), derived from the influenza A/PR8/34 strain, di#ers from the A/NT/60/68 CTL epitope by two conservative amino acid substitutions in the C-terminal half of the peptide and is not recognized by the F5 T-cell receptor. The TCR CDR3 library was subjected to four rounds of selection with H-2Db tetramers that contain the variant epitope, to select for the TCR clone(s) that exhibit highest a#inity for this epitope. From this TCR library selection a single TCR clone emerged (named PR-1) that binds avidly to the A/PR8/34 NP366–374 tetramers. Sequence analysis of the PR-1 TCR reveals seven nucleotide mutations in its CDR3 DNA sequence compared to the parental F5 TCR. These mutations result in four conservative amino acid changes and one non-conservative Arg to Trp substitution.8 To examine whether in vitro selected variant TCRs can evoke T-cell activation upon peptide recognition, ligand-induced IL-2 gene transcription was measured. To this purpose we used a selfinactivating (SIN) retroviral vector containing multiple NFAT binding sites upstream of a minimal IL-2 promoter and the reporter gene YFP. 34.1L cells expressing the F5, NT-1 or PR-1 TCR were virally transduced with the NFAT-YFP reporter construct and the transduced cells were exposed to target cells in the presence of di#erent concentrations of either the A/NT/60/68 or A/PR8/34 T-cell epitope. Both variant clones NT-1 and PR-1 e#iciently induce T-cell activation upon specific antigen recognition with an absolute specificity for the epitope used during the in vitro selections (Fig. 3).
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Figure 3. Signaling function of in vitro selected TCRs. TCR expressing 34.1L cells transduced with the NFATYFP construct were exposed to EL4 target cells in the presence of di#erent concentrations of either the A/NT/ 60/68 (open squares) or A/PR8/34 (filled circles) T-cell epitope.8 Sensitivity and specificity of the di#erent TCRs was determined by flow cytometric analysis of the percentage of YFP expressing 34.1L cells. T-cell activation upon stimulation with PMA (10 ng/ml) and ionomycin (1.67 mg/ml) results in 60–65% YFP expressing cells (not shown). The data shown are means of triplicates S.D.
These experiments show that in vitro selection of variant T-cell receptors by retroviral TCR display can yield receptors with high potency, as revealed by both biochemical means and functional assays. T-cell receptors that are isolated from such libraries should be useful for the creation of redirected T-cell populations, through gene transfer of peripheral T lymphocytes.
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TCR gene transfer
a pronounced improvement in tumour rejection, without resulting in overt auto-immunity. Collectively, these experiments provided the first proof of principle of TCR gene transfer as a realistic immunotherapeutic option
Isolate peripheral T cells Introduce TCR-encoding genes in vitro Infuse redirected T cells
Figure 4. Redirecting T cells through TCR gene transfer.
Immunotherapy through TCR gene transfer
Because the specificity of T cells is entirely contained within the alpha and beta chains of the TCR, it should in theory be possible to impose a desired antigenic specificity onto cytotoxic T cells by introduction of TCR-encoding genes with this desirable specificity. Furthermore, this type of TCR gene therapy would form a useful strategy to endow T cells with in vitro modified T-cell receptors, geared towards an optimal recognition of antigens expressed on tumour tissues. This novel therapeutic strategy would entail the introduction of TCR genes into peripheral T cells of patients ex vivo and subsequent re-introduction of the T cells that have now obtained a desirable antigen specificity (Fig. 4). To assess the feasibility of TCR gene transfer for the creation of T-cell populations with a desirable antigen specificity we have developed an animal model system. In this model system, mouse peripheral T cells were infected (transduced) with retroviruses expressing the influenza A NP-specific F5 T-cell receptor. While murine T cells are generally refractory to infection with Moloney-based retroviruses, upon culture of primary mouse T cells with Con A and IL-7 transduction e#iciencies of 5–10% can be achieved. We have subsequently used such TCRtransduced T-cell populations to examine their in vivo behaviour. These experiments have established that TCR-transduced T cells react to antigen in vivo and expand dramatically in number (H.W.H.G.K. et al., unpublished data). Importantly, the introduction of small numbers of TCR-modified T-cell populations into immunodeficient RAG / mice leads to
Discussion The immune system contains a large collection of T cells that covers a broad range of peptide/MHC specificities and thereby can identify subtle changes in MHC epitope presentation. However, selftolerance leads to the removal of the high a#inity T-cell repertoire specific for self antigens, and this will include T cells with desirable specificities, such as for many self antigens expressed on tumour tissues. Because of the potential therapeutic value of TCRs with such tumour/lineage specificities, we set out to develop strategies for T-cell receptor selection and transfer that can be used to bypass in vivo tolerance. In a first set of experiments we have demonstrated how self tolerance leads to the selective deletion of high avidity T cells from the T-cell repertoire. Furthermore, we document the use of altered peptide ligands with improved TCRbinding characteristics as a strategy to promote the expansion of low avidity self-specific T cells. For tumour antigens that are highly over-expressed on tumour tissues such as HER2/neu, adoptive transfer of self-specific T-cell populations obtained through the use of such altered peptide ligands may form a viable therapeutic strategy. However, because of the reduced antigen sensitivity of low avidity selfspecific T cells, infusion of these cells is unlikely to be successful in cases in which tumour/lineage antigens are not, or only modestly over-expressed in tumour tissue. In these cases, optimization of T-cell receptors by retroviral TCR display may form a useful strategy to obtain more potent tumourspecific T-cell receptors. Finally, we have set up a model system that provides the first proof of principle for the feasibility of TCR gene therapy. This strategy of TCR gene transfer should be useful to introduce in vitro-modified T-cell receptor genes specific for tumour antigens. However, it is apparent that in certain human conditions, introduction of unmodified T-cell receptors through TCR gene transfer may be equally appealing. For instance, the infusion of EBV-specific T cells in patients with post-transplant lymphoma has previously been shown to result in marked tumour regression. The infusion of TCR-modified, EBV-directed T cells in this setting may provide an interesting alternative
Circumventing T-cell Tolerance
strategy that circumvents the requirement for the conventional large scale in vitro generation of EBV-specific T-cell populations. Conclusions
It is becoming increasingly clear that the ine#ectiveness of tumour-specific T-cell responses is in large part due to the e#ects of self tolerance. To address this issue we have set out to develop new techniques that can be used to improve tumourspecific T-cell immunity. Through the use of these techniques we seek to create T-cell receptors that recognize antigens expressed on tumour tissue with high a#inity. Introduction of such in vitro-modified T-cell receptors into peripheral T-cell populations by gene transfer forms an attractive strategy to enhance tumour-specific T-cell immunity in settings where the endogenous T-cell response is insu#icient. Note added in proof
Data described as ‘‘(H.W.H.G.K. et al., unpublished data)’’ are now in press with Nature Immunology. Acknowledgements This research was supported by grants from the Dutch Cancer Society (NKB 97-1442 and NKB 99-2036) and the Dutch Foundation for Scientific Research (NWO Pioneer 00-03). We thank M. Toebes and M. van den Boom for excellent technical support throughout this research. References 1. Gilboa E. The makings of a tumour rejection antigen. Immunity 1999; 11: 263–270. 2. Shankaran V, Ikeda H, Bruce AT, White JM, Swanson PE, Old LJ et al. IFN-gamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature 2001; 410: 1107–1111. 3. Kawakami Y, Rosenberg SA. Immunobiology of human melanoma antigens MART-1 and gp100 and their use for immuno-gene therapy. Int Rev Immunol 1997; 14: 173–192.
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4. Yee C, Savage PA, Lee PP, Davis MM, Greenberg PD. Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers. J Immunol 1999; 162: 2227–2234. 5. Zeh HJ, 3rd, Perry-Lalley D, Dudley ME, Rosenberg SA, Yang JC. High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumour e#icacy. J Immunol 1999; 162: 989–994. 6. Altman JD, Moss PAH, Goulder PJR, Barouch DH, McHeyzer-Williams MG, Bell JI et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996; 274: 94–96. 7. Haanen JB, Toebes M, Cordaro TA, Wolkers MC, Kruisbeek AM, Schumacher TN. Systemic T cell expansion during localized viral infection. Eur J Immunol 1999; 29: 1168–1174. 8. Kessels HW, van Den Boom MD, Spits H, Hooijberg E, Schumacher TN. Changing T cell specificity by retroviral T cell receptor display. Proc Natl Acad Sci USA 2000; 97: 14578–14583. 9. de Visser KE, Cordaro TA, Kioussis D, Haanen JB, Schumacher TN, Kruisbeek AM. Tracing and characterization of the low-avidity self-specific T cell repertoire. Eur J Immunol 2000; 30: 1458–1468. 10. Theobald M, Biggs J, Hernandez J, Lustgarten J, Labadie C, Sherman LA. Tolerance to p53 by A2.1restricted cytotoxic T lymphocytes. J Exp Med 1997; 185: 833–841. 11. de Visser KE, Cordaro TA, Kessels HW, Tirion FH, Schumacher TN, Kruisbeek AM. Low-avidity selfspecific t cells display a pronounced expansion defect that can be overcome by altered peptide ligands. J Immunol 2001; 167: 3818–3828. 12. Slansky JE, Rattis FM, Boyd LF, Fahmy T, Ja#ee EM, Schneck JP et al. Enhanced antigen-specific antitumour immunity with altered peptide ligands that stabilize the MHC-peptide-TCR complex. Immunity 2000; 13: 529–538. 13. Holler PD, Holman PO, Shusta EV, O’Herrin S, Wittrup KD, Kranz DM. In vitro evolution of a T cell receptor with high a#inity for peptide/MHC. Proc Natl Acad Sci USA 2000; 97: 5387–5392. 14. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 1998; 395: 82–86. 15. Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM et al. The immunological synapse: a molecular machine controlling T cell activation. Science 1999; 285: 221–227. 16. Townsend AR, Gotch FM, Davey J. Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell 1985; 42: 457–467.