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peptide tetramer-based studies of T cell function
Journal of Immunological Methods 268 (2002) 21 – 28 www.elsevier.com/locate/jim
Review
MHC/peptide tetramer-based studies of T cell function Xiao-Ning Xu*, Gavin R. Screaton MRC Human Immunology Unit, The Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK Received 18 October 2001; accepted 14 January 2002
Abstract Direct visualization and quantification of antigen-specific T cells using major histocompatibility complex (MHC)/peptide tetramer technology offers a powerful means to study specific T cell populations of interest. In combination with functional assays, this technology has already provided many new insights into several long-standing immunological concepts in basic science as well as clinical settings. Published by Elsevier Science B.V. Keywords: Tetramer; CTL; Apoptosis; Tumour
1. Introduction Since Altman et al. (1996) published the first paper in 1996 describing the use of major histocompatibility complex (MHC) class I/peptide tetrameric technology (the tetramer) for direct visualisation and quantification of antigen-specific cytotoxic T cells, there have been many tetramer-based studies in both basic and clinical immunology. The success of this technique lies with the increased avidity of soluble MHC/peptide complex (the monomer) for the peptide-specific T cell receptor (TCR) using biotinylated MHC/peptide
Abbreviations: CTL, cytotoxic T lymphocytes; AICD, activation-induced cell death; TCR, T cell receptor; mAb, monoclonal antibodies. * Corresponding author. Tel.: +44-1865-222623; fax: +441865-222502. E-mail address: [email protected] (X.-N. Xu). 0022-1759/02/$ - see front matter. Published by Elsevier Science B.V. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 1 9 6 - 5
monomer bound to avidin or streptavidin, which has four biotin-binding sites. The binding of the tetramer to the TCR can be easily detected by flow cytometry if a fluorochrome-labelled avidin or streptavidin is used. The tetramer is primarily a tool to determine the frequency of antigen-specific (tetramer-positive) T cells. Recently, the use of this technology has been extended to evaluate functional aspects of T cells. For example, tetramer staining can be combined with intracellular detection of cytokines (IFN-g), chemokines (MIP-1a), and cytotoxins (perforin/granzymes) following antigen/mitogen stimulation in vitro to assess the functional status of tetramer-positive T cells (Appay et al., 2000). In addition, the tetramer-positive T cells can be sorted and expanded in in vitro cell culture for any further functional analysis (Dunbar et al., 1998). In this review, we will discuss several innovative and clinically relevant applications of the tetramer for T cell function, including the use of the
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tetramer to study T cell activation, apoptosis, and its therapeutic potentials.
2. T cell activation and apoptosis Successful contact between a T cell and antigenexpressing cell sets in train a series of rearrangements in the cell membrane, which leads to the formation of the immunological synapse (Grakoui et al., 1999). This has now been visualised in CD4 + cells and contains a number of molecules in addition to the T cell receptor complex such as adhesion molecules and CD45. The signal initiated from the TCR leads to a wide range of downstream events. These include cytokine release, proliferation, and, in the case of cytotoxic T lymphocytes (CTL), release of lytic granules (Valitutti et al., 1995). It is known that the cross-linking of TCR complex by a specific antibody alone (i.e., anti-CD3) can cause T cell activation. However, it has remained controversial whether the binding of a single TCR by its natural ligand is sufficient enough to trigger T cell activation. Also, the number of TCR molecules that are required to produce the necessary signalling unit for T cell activation is unclear. To address these questions, Boniface et al. (1998) took advantage of tetramer technology and constructed defined oligomers of soluble MHC class II/peptide complex molecules, namely monomer, dimer, trimer, and tetramer. Using these oligomers to stimulate antigenspecific T cells in vitro, they found that the potency of these oligomers from strongest to nonstimulatory was tetramer>trimer>dimer>monomer. This is consistent with data from MHC class I/peptide-restricted CTL where monomers or dimers of MHC class I/peptide complex induced minimal TCR zeta chain phosphorylation (Xu et al., 2001), which characterizes one of the early events of T cell activation (Fig. 1). These studies suggest that stabilization and colocalization of, at least, two or three TCRs on the T cell surface per se are required for signalling T cell activation. Signalling via the TCR can also induce T cells to undergo apoptosis, in part mediated by Fas/Fas ligand (FasL) interaction (activation-induced cell death, AICD). AICD is an important immunological control mechanism for peripheral tolerance (Lenardo et al., 1999). Most studies on AICD in human T cells were
Fig. 1. Activation of T cells using oligomers. (A) CD4 + T cell response (acid release) to oligomers of MHC class II/peptide as indicated (modified from Boniface et al., 1998). (B) CTL response (TCR zeta chain phosphorylation) to oligomers of MHC class I/ peptide complex as indicated.
performed by cross-linking TCR with either anti-CD3 or anti-TCR a/h chain-specific monoclonal antibodies (mAb) or using peptide-pulsed target cells. It is unknown whether the number of TCRs engaged by its ligand for cellular activation is the same as is required for the triggering of apoptosis. Even more complicated is the case of CD8 + CTL, as compared with CD4 + T helper cells, because activation of CTL can elicit two different outcomes: perforin-dependent killing of the target cells (forward killing), or death of the CTL mediated by upregulation of FasL (AICD, backward killing). FasL expression on CTL is a twoedged sword, which induces apoptosis of Fas + target cells as well as CTL itself. We have used tetramers to evaluate the signalling thresholds for CTL target killing and CTL apoptosis (Xu et al., 2001). The advantage of using tetramers was that it enabled us to produce a target cell-free system to assess the contribution of any adhesion/accessory molecules to the death of CTL. In addition, we constructed tetramers using MHC class I heavy chain complexed with a
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peptide/b2M fusion protein (peptide covalently linked to h2M). Tetramers fused with peptide can overcome the problem of CTL killing each other, induced by free peptide derived from unstably folded tetramers. Multimers of MHC class I/peptide complex were made by mixing avidin-coated magnetic beads with the biotin-monomeric MHC/peptide complex to form multiple tetramers on the surface of beads. The benefit of using tetramer beads for functional studies is to allow us to remove agents toxic to cells (i.e., bacterial endotoxin, proteinase inhibitors, and sodium azide), which can either be inherited from the process of making the complex or added to preserve their stability. When cocultured with CTL, tetramer beads were able to trigger the death of CTL in a peptide-specific manner and showed a dose –response curve similar to that obtained using live cells stably transfected with peptide/b2M fusion construct (Fig. 2A). We varied the loading of the avidin-coated beads by varying the ratio of two monomers containing either agonist peptide or an irrelevant peptide. Exposure of CTL to beads more densely coated with agonist MHC complexes led to an increase in CTL apoptosis (Fig. 2B). These data suggest that the death of CTL can be
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triggered by its natural ligand in the absence of additional molecules. The magnitude of this response depends on the density of the ligand, which is consistent with the finding that increasing the stoichiometry of the MHC monomer, avidin, increased T cell activation signalling (Boniface et al., 1998). To search for molecules in addition to TCR that could regulate the balance between forward and backward killing of CTL, we examined the role of the coreceptor CD8 since binding of MHC class I/peptide complex to TCR also involves binding to CD8 molecules. Most human CTL kill their targets in a CD8dependent fashion (i.e., blocking CD8 blocks/reduces CTL killing). First, we examined the effect of CD8 on the binding of the tetramer to the CTL clone using either anti-CD8-blocking mAb or a mutant tetramer where two amino acid substitutions (aa 227/228, from DT to KA ) in the a3 domain abolish the interaction with CD8 as determined by protein – protein interaction using Biacore analysis (Purbhoo et al., 2001). Blocking CD8 interaction using anti-CD8 or the a3 mutant tetramer showed that the MHC tetramers were still able to stain the antigen-specific CTL clone, although to a reduced intensity (Fig. 3A). However, in spite of the weaker binding, beads loaded with the
Fig. 2. Induction of apoptosis by tetramer-coated beads. (A) Apoptosis of CTL was induced by either tetramer beads or target cells expressing the MHC/peptide complex. (B) Density-dependent induction of CTL death. Mixed tetramers were formed by loading avidin-coated beads with different molar ratios of two monomers (agonist and irrelevant).
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Fig. 3. (A) FACS analysis of a Flu-specific CTL clone. Interaction of CD8 with the tetramer was blocked with anti-CD8 mAb or by a mutation in the a3 domain of A2. (B) Apoptosis of CTL induced by treatment with wild-type or a3 domain mutant tetramers.
mutant tetramers were still fully competent to induce the death of CTL, and this showed a similar dose response to beads loaded with the wild-type tetramers (Xu et al., 2001) (Fig. 3B). This was a surprising result as it has been previously shown that CD8 coreceptor is required to generate a productive signal from the TCR and most CTL clones will not kill if CD8/MHC contacted is blocked using a monoclonal antibody. This spurred us to examine tyrosine phosphorylation induced by TCR engagement with either the wild type
of a3 domain mutant tetramers. As expected, the wildtype tetramers induced considerable tyrosine phosphorylation of the TCR zeta chain (Fig. 1B), yet the mutant tetramers that were fully competent to induce CTL apoptosis induced a signal hardly above background (not shown). Thus, our studies using tetramer technology have demonstrated that the signalling thresholds required for activation and death are distinct and can be dissociated by the binding of CD8 (Fig. 4). This may provide a useful approach for the deletion of
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Fig. 4. The role of CD8 in regulation of death of a CTL and its target. Full activation induced by TCR and CD8 engagement leads to perforin/granzyme release and the expression of FasL on CTL, whereas a3 domain mutants of MHC class I abolish CD8 engagement and lead to a reduced TCR signal that upregulates FasL expression but does not lead to perforin/granzymes release.
antigen-specific CTL without the concomitant T cell activation, which normally causes end-organ damage.
3. Immunotherapy potentials 3.1. Autoimmunity and graft rejection Selective deletion of auto- or alloreactive T cells is a major goal for the treatment of autoimmune disease, or to prevent the rejection of transplanted organs. Although effective in many cases, conventional therapy relies upon broad-spectrum immunosuppression with the consequent risk of opportunistic infection or tumourigenesis. Therefore, it would clearly be desir-
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able to manipulate the immune system to eliminate only the disease-specific T cells. One obvious approach as a targeted immunotherapy would be to employ the specific interaction between MHC/peptide complex and TCR to induce tolerance (anergy or apoptosis) of reactive T lymphocytes. Although soluble MHC has been used to suppress alloreactive T cells in some transplantation models, its efficacy is limited largely due to low affinity of soluble MHC binding to the cognate TCR (Wang et al., 1996). Obviously, multimeric MHC/peptide complexes with increased affinity to TCR would be the ideal candidates. Indeed, recently, two groups have reported that using multimeric soluble MHC/peptide complex in either dimeric or tetrameric form can induce tolerance of antigen-specific T cells in vivo. Using dimeric MHC/peptide complex, O’Herrin et al. (2001) showed that the soluble MHC dimers induced modulation of surface expression of TCR and inhibited T cell cytolytic activity at nanomolar concentration in vitro. This is associated with phosphorylation of TCR zeta chain and Zap-70, the signalling complex required for T cell activation. When they injected the soluble dimers into alloreactive TCR transgenic mice, they found that there was a significant inhibition of the alloreactive T cell activity, which resulted in consequent outgrowth of an allogeneic tumour (see Schneck’s section). Similarly, Maile et al. (2001) were able to activate naı¨ve male-specific CD8 + T cells by injection of HY class I tetramer in vivo in a female HYTCR transgenic mice model. However, multiple injections of the tetramers led to the elimination of male-specific T cells by either induction of anergy or AICD and subsequently enhanced the survival of male skin grafts in female mice (Fig. 5). Thus, soluble multimeric MHC/peptide complexes may provide a useful tool for antigen-specific deletion of ‘unwanted’ T cells. However, the deletion of T cells by any full agonist peptide/MHC complex should be used with caution, in particular for therapeutic interventions in vivo, because self-reactive T cells could potentially be activated by the agonist and gain full effector function before the death of these cells is induced. The release of cytokines, chemokines, and cytotoxins as a consequence of activation can lead to severe damage to the lymphoid organs and to general immunosuppression (Aichele et al., 1997). Other strategies must be
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Fig. 5. Increasing survival of skin grafts by multiple injection of tetrameric MHC/peptide complex (modified from Maile et al., 2001).
developed to overcome these problems. Although selective signalling ligands such as altered (partial agonist) peptides have been used to regulate T cell deletion without immune damage (Combadiere et al.,
1998), this approach has a major limitation since, in many cases, the responsible peptide epitopes have not yet been identified. Therefore, soluble multimeric MHC/peptide complexes created with mutant heavy chain lacking CD8 interaction would not lead to this detrimental T cell activation and may allow antigenspecific deletion of the disease-mediating CTL (Fig. 4). The other advantage of this approach is that it can allow the deletion of CTL without the need to identify the antigenic peptide. So, for instance, in our experiments, we were able to induce apoptosis of allospecific CTL by expressing an a3 domain mutant in cells where it is loaded with a variety of endogenous peptides (Xu et al., 2001). It would also be possible to produce soluble forms of these molecules loaded with unknown host peptides by expression in mammalian cells and conceivably cells derived from the patient for whom therapy was planned. We are currently testing the utility of these approaches in the treatment of CD8-mediated autoimmunity models and organ transplantation.
Fig. 6. Redirected killing of tumour cells using the tetramer. For example, Flu-specific CTL can kill tumour cells targeted using antitumourspecific antibody conjugated with MHC class I/Flu peptide tetramers by either direct (A) or indirect linking (B) with avidin.
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3.2. Redirected killing of tumour cells Although the CTL response plays an important role in antitumour activity, tumour growth implies that CTL either fail to be elicited or are inadequate. Many tumour cells have defects in antigen presentation due to mutations in their h2M gene, leading to loss of MHC I expression at the cell surface and escape from CTL surveillance (Romero et al., 1998). On the other hand, many tumour cells express tumour-associated antigens that can be recognised by antibodies (Riethmuller and Johnson, 1992). The use of monoclonal antibodies as antitumour therapies, so-called magic bullets, has been evaluated extensively in both animal models and clinical practices (Levy, 2000). Other antibody-based antitumour strategies include bispecific antibodies, one arm of the antibody binding to tumour antigen and the other to TCR, which bring the T cells close to tumour cells and redirect nontumourspecific T cells to kill tumour cells (Guo et al., 1997). Recently, Ogg et al. (2000) and Robert et al. (2000) have described another new antitumour strategy in which they target tumour cells using a tumour-specific antibody conjugated with MHC class I/peptide tetramer. The antibody – tetramer complexes were made by mixing biotinylated antitumour-specific mAb with avidin- and biotin-conjugated MHC/peptide monomers. This was done at relative concentrations to form an avidin –biotin complex, with one site binding to the antibody and other three sites binding to biotin – MHC/peptide monomers (Ogg et al., 2000). The complex could also be made by directly chemically coupling avidin with mAb and then mixing this with biotin –MHC/peptide monomers to form antibody– MHC/peptide tetramer complexes (Robert et al., 2000). Both complexes were able to bind to the surface of tumour cells and sensitise them to lysis by tetramerspecific CTL (Fig. 6). Thus, antibody-guided tetramers can localize TCR ligands on the surface of tumour cells and also trigger the activation of antigenspecific T cell effector functions. There are several advantages of this technique as compared with others. Firstly, it can increase tumour immunogenicity and overcome the problem of absent or low-level expression of MHC molecules found in many tumours. Secondly, induction of tumour cell apoptosis may enhance cross-priming (uptake by dendritic cells or
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macrophages) and subsequently initiate and amplify T cell responses against tumour-specific peptide antigens. Finally and more importantly, if multispecific tetramers are applied, antitumour activities could be accelerated even more by redirecting existing effective CTL such as those specific to EBV, CMV, and influenza, which are known to present at high frequencies in the circulation and generally with an activated and/or memory phenotype. However, the utility of this technique in vivo for the treatment of tumours needs to be confirmed and modified, aimed at increasing stability and reducing the size of the complex.
References Aichele, P., Brduscha-Riem, K., Oehen, S., Odermatt, B., Zinkernagel, R.M., Hengartner, H., Pircher, H., 1997. Peptide antigen treatment of naive and virus-immune mice: antigen-specific tolerance versus immunopathology. Immunity 6, 519. Altman, J.D., Moss, P.A.H., Goulder, P.J.R., Barouch, D.H., McHeyzer-Williams, M.G., Bell, J.I., McMichael, A.J., Davis, M.M., 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94. Appay, V., Nixon, D.F., Donahoe, S.M., Gillespie, G.M., Dong, T., King, A., Ogg, G.S., Spiegel, H.M., Conlon, C., Spina, C.A., Havlir, D.V., Richman, D.D., Waters, A., Easterbrook, P., McMichael, A.J., Rowland-Jones, S.L., 2000. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. J. Exp. Med. 192, 63. Boniface, J.J., Rabinowitz, J.D., Wulfing, C., Hampl, J., Reich, Z., Altman, J.D., Kantor, R.M., Beeson, C., McConnell, H.M., Davis, M.M., 1998. Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/ MHC ligands. Immunity 9, 459. Combadiere, B., Sousa, C.R., Trageser, C., Zheng, L.X., Kim, C.R., Lenardo, M.J., 1998. Differential TCR signaling regulates apoptosis and immunopathology during antigen responses in vivo. Immunity 9, 305. Dunbar, P.R., Ogg, G.S., Chen, J., Rust, N., van der Bruggen, P., Cerundolo, V., 1998. Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood. Curr. Biol. 8, 413. Grakoui, A., Bromley, S.K., Sumen, C., Davis, M.M., Shaw, A.S., Allen, P.M., Dustin, M.L., 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221. Guo, Y.J., Che, X.Y., Shen, F., Xie, T.P., Ma, J., Wang, X.N., Wu, S.G., Anthony, D.D., Wu, M.C., 1997. Effective tumor vaccines generated by in vitro modification of tumor cells with cytokines and bispecific monoclonal antibodies. Nat. Med. 3, 451. Lenardo, M., Chan, K.M., Hornung, F., McFarland, H., Siegel, R., Wang, J., Zheng, L., 1999. Mature T lymphocyte apoptosis-
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immune regulation in a dynamic and unpredictable antigenic environment. Annu. Rev. Immunol. 17, 221. Levy, R., 2000. A perspective on monoclonal antibody therapy: where we have been and where we are going. Semin. Hematol. 37, 43. Maile, R., Wang, B., Schooler, W., Meyer, A., Collins, E.J., Frelinger, J.A., 2001. Antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers. J. Immunol. 167, 3708. Ogg, G.S., Dunbar, P.R., Cerundolo, V., McMichael, A.J., Lemoine, N.R., Savage, P., 2000. Sensitization of tumour cells to lysis by virus-specific CTL using antibody-targeted MHC class I/peptide complexes. Br. J. Cancer 82, 1058. O’Herrin, S.M., Slansky, J.E., Tang, Q., Markiewicz, M.A., Gajewski, T.F., Pardoll, D.M., Schneck, J.P., Bluestone, J.A., 2001. Antigen-specific blockade of t cells in vivo using dimeric MHC peptide. J. Immunol. 167, 2555. Purbhoo, M.A., Boulter, J.M., Price, D.A., Vuidepot, A.L., Hourigan, P.R., Dunbar, P.R., Olson, K., Dawson, S.J., Phillips, R.E., Jakobsen, B.K., Bell, J.I., Sewell, A.K., 2001. The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain. J. Biol. Chem. 276, 32786. Riethmuller, G., Johnson, J.P., 1992. Monoclonal antibodies in the detection and therapy of micrometastatic epithelial cancers. Curr. Opin. Immunol. 4, 647.
Robert, B., Guillaume, P., Luescher, I., Romero, P., Mach, J.P., 2000. Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes. Eur. J. Immunol. 30, 3165. Romero, P., Dunbar, P.R., Valmori, D., Pittet, M., Ogg, G.S., Rimoldi, D., Chen, J.L., Lienard, D., Cerottini, J.C., Cerundolo, V., 1998. Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes. J. Exp. Med. 188, 1641. Valitutti, S., Muller, S., Cella, M., Padovan, E., Lanzavecchia, A., 1995. Serial triggering of many T cell receptors by a few peptide – MHC complexes. Nature 375, 148. Wang, M., Stepkowski, S.M., Wang, M.E., Tian, L., Qu, X., Tu, Y., He, G., Kahan, B.D., 1996. Induction of specific allograft immunity by soluble class I MHC heavy chain protein produced in a baculovirus expression system. Transplantation 61, 448. Xu, X.N., Purbhoo, M.A., Chen, N., Mongkolsapaya, J., Cox, J.H., Meier, U.C., Tafuro, S., Dunbar, P.R., Sewell, A.K., Hourigan, C.S., Appay, V., Cerundolo, V., Burrows, S.R., McMichael, A.J., Screaton, G.R., 2001. A novel approach to antigen-specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex. Immunity 14, 591.
Review
MHC/peptide tetramer-based studies of T cell function Xiao-Ning Xu*, Gavin R. Screaton MRC Human Immunology Unit, The Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK Received 18 October 2001; accepted 14 January 2002
Abstract Direct visualization and quantification of antigen-specific T cells using major histocompatibility complex (MHC)/peptide tetramer technology offers a powerful means to study specific T cell populations of interest. In combination with functional assays, this technology has already provided many new insights into several long-standing immunological concepts in basic science as well as clinical settings. Published by Elsevier Science B.V. Keywords: Tetramer; CTL; Apoptosis; Tumour
1. Introduction Since Altman et al. (1996) published the first paper in 1996 describing the use of major histocompatibility complex (MHC) class I/peptide tetrameric technology (the tetramer) for direct visualisation and quantification of antigen-specific cytotoxic T cells, there have been many tetramer-based studies in both basic and clinical immunology. The success of this technique lies with the increased avidity of soluble MHC/peptide complex (the monomer) for the peptide-specific T cell receptor (TCR) using biotinylated MHC/peptide
Abbreviations: CTL, cytotoxic T lymphocytes; AICD, activation-induced cell death; TCR, T cell receptor; mAb, monoclonal antibodies. * Corresponding author. Tel.: +44-1865-222623; fax: +441865-222502. E-mail address: [email protected] (X.-N. Xu). 0022-1759/02/$ - see front matter. Published by Elsevier Science B.V. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 1 9 6 - 5
monomer bound to avidin or streptavidin, which has four biotin-binding sites. The binding of the tetramer to the TCR can be easily detected by flow cytometry if a fluorochrome-labelled avidin or streptavidin is used. The tetramer is primarily a tool to determine the frequency of antigen-specific (tetramer-positive) T cells. Recently, the use of this technology has been extended to evaluate functional aspects of T cells. For example, tetramer staining can be combined with intracellular detection of cytokines (IFN-g), chemokines (MIP-1a), and cytotoxins (perforin/granzymes) following antigen/mitogen stimulation in vitro to assess the functional status of tetramer-positive T cells (Appay et al., 2000). In addition, the tetramer-positive T cells can be sorted and expanded in in vitro cell culture for any further functional analysis (Dunbar et al., 1998). In this review, we will discuss several innovative and clinically relevant applications of the tetramer for T cell function, including the use of the
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tetramer to study T cell activation, apoptosis, and its therapeutic potentials.
2. T cell activation and apoptosis Successful contact between a T cell and antigenexpressing cell sets in train a series of rearrangements in the cell membrane, which leads to the formation of the immunological synapse (Grakoui et al., 1999). This has now been visualised in CD4 + cells and contains a number of molecules in addition to the T cell receptor complex such as adhesion molecules and CD45. The signal initiated from the TCR leads to a wide range of downstream events. These include cytokine release, proliferation, and, in the case of cytotoxic T lymphocytes (CTL), release of lytic granules (Valitutti et al., 1995). It is known that the cross-linking of TCR complex by a specific antibody alone (i.e., anti-CD3) can cause T cell activation. However, it has remained controversial whether the binding of a single TCR by its natural ligand is sufficient enough to trigger T cell activation. Also, the number of TCR molecules that are required to produce the necessary signalling unit for T cell activation is unclear. To address these questions, Boniface et al. (1998) took advantage of tetramer technology and constructed defined oligomers of soluble MHC class II/peptide complex molecules, namely monomer, dimer, trimer, and tetramer. Using these oligomers to stimulate antigenspecific T cells in vitro, they found that the potency of these oligomers from strongest to nonstimulatory was tetramer>trimer>dimer>monomer. This is consistent with data from MHC class I/peptide-restricted CTL where monomers or dimers of MHC class I/peptide complex induced minimal TCR zeta chain phosphorylation (Xu et al., 2001), which characterizes one of the early events of T cell activation (Fig. 1). These studies suggest that stabilization and colocalization of, at least, two or three TCRs on the T cell surface per se are required for signalling T cell activation. Signalling via the TCR can also induce T cells to undergo apoptosis, in part mediated by Fas/Fas ligand (FasL) interaction (activation-induced cell death, AICD). AICD is an important immunological control mechanism for peripheral tolerance (Lenardo et al., 1999). Most studies on AICD in human T cells were
Fig. 1. Activation of T cells using oligomers. (A) CD4 + T cell response (acid release) to oligomers of MHC class II/peptide as indicated (modified from Boniface et al., 1998). (B) CTL response (TCR zeta chain phosphorylation) to oligomers of MHC class I/ peptide complex as indicated.
performed by cross-linking TCR with either anti-CD3 or anti-TCR a/h chain-specific monoclonal antibodies (mAb) or using peptide-pulsed target cells. It is unknown whether the number of TCRs engaged by its ligand for cellular activation is the same as is required for the triggering of apoptosis. Even more complicated is the case of CD8 + CTL, as compared with CD4 + T helper cells, because activation of CTL can elicit two different outcomes: perforin-dependent killing of the target cells (forward killing), or death of the CTL mediated by upregulation of FasL (AICD, backward killing). FasL expression on CTL is a twoedged sword, which induces apoptosis of Fas + target cells as well as CTL itself. We have used tetramers to evaluate the signalling thresholds for CTL target killing and CTL apoptosis (Xu et al., 2001). The advantage of using tetramers was that it enabled us to produce a target cell-free system to assess the contribution of any adhesion/accessory molecules to the death of CTL. In addition, we constructed tetramers using MHC class I heavy chain complexed with a
X.-N. Xu, G.R. Screaton / Journal of Immunological Methods 268 (2002) 21–28
peptide/b2M fusion protein (peptide covalently linked to h2M). Tetramers fused with peptide can overcome the problem of CTL killing each other, induced by free peptide derived from unstably folded tetramers. Multimers of MHC class I/peptide complex were made by mixing avidin-coated magnetic beads with the biotin-monomeric MHC/peptide complex to form multiple tetramers on the surface of beads. The benefit of using tetramer beads for functional studies is to allow us to remove agents toxic to cells (i.e., bacterial endotoxin, proteinase inhibitors, and sodium azide), which can either be inherited from the process of making the complex or added to preserve their stability. When cocultured with CTL, tetramer beads were able to trigger the death of CTL in a peptide-specific manner and showed a dose –response curve similar to that obtained using live cells stably transfected with peptide/b2M fusion construct (Fig. 2A). We varied the loading of the avidin-coated beads by varying the ratio of two monomers containing either agonist peptide or an irrelevant peptide. Exposure of CTL to beads more densely coated with agonist MHC complexes led to an increase in CTL apoptosis (Fig. 2B). These data suggest that the death of CTL can be
23
triggered by its natural ligand in the absence of additional molecules. The magnitude of this response depends on the density of the ligand, which is consistent with the finding that increasing the stoichiometry of the MHC monomer, avidin, increased T cell activation signalling (Boniface et al., 1998). To search for molecules in addition to TCR that could regulate the balance between forward and backward killing of CTL, we examined the role of the coreceptor CD8 since binding of MHC class I/peptide complex to TCR also involves binding to CD8 molecules. Most human CTL kill their targets in a CD8dependent fashion (i.e., blocking CD8 blocks/reduces CTL killing). First, we examined the effect of CD8 on the binding of the tetramer to the CTL clone using either anti-CD8-blocking mAb or a mutant tetramer where two amino acid substitutions (aa 227/228, from DT to KA ) in the a3 domain abolish the interaction with CD8 as determined by protein – protein interaction using Biacore analysis (Purbhoo et al., 2001). Blocking CD8 interaction using anti-CD8 or the a3 mutant tetramer showed that the MHC tetramers were still able to stain the antigen-specific CTL clone, although to a reduced intensity (Fig. 3A). However, in spite of the weaker binding, beads loaded with the
Fig. 2. Induction of apoptosis by tetramer-coated beads. (A) Apoptosis of CTL was induced by either tetramer beads or target cells expressing the MHC/peptide complex. (B) Density-dependent induction of CTL death. Mixed tetramers were formed by loading avidin-coated beads with different molar ratios of two monomers (agonist and irrelevant).
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Fig. 3. (A) FACS analysis of a Flu-specific CTL clone. Interaction of CD8 with the tetramer was blocked with anti-CD8 mAb or by a mutation in the a3 domain of A2. (B) Apoptosis of CTL induced by treatment with wild-type or a3 domain mutant tetramers.
mutant tetramers were still fully competent to induce the death of CTL, and this showed a similar dose response to beads loaded with the wild-type tetramers (Xu et al., 2001) (Fig. 3B). This was a surprising result as it has been previously shown that CD8 coreceptor is required to generate a productive signal from the TCR and most CTL clones will not kill if CD8/MHC contacted is blocked using a monoclonal antibody. This spurred us to examine tyrosine phosphorylation induced by TCR engagement with either the wild type
of a3 domain mutant tetramers. As expected, the wildtype tetramers induced considerable tyrosine phosphorylation of the TCR zeta chain (Fig. 1B), yet the mutant tetramers that were fully competent to induce CTL apoptosis induced a signal hardly above background (not shown). Thus, our studies using tetramer technology have demonstrated that the signalling thresholds required for activation and death are distinct and can be dissociated by the binding of CD8 (Fig. 4). This may provide a useful approach for the deletion of
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Fig. 4. The role of CD8 in regulation of death of a CTL and its target. Full activation induced by TCR and CD8 engagement leads to perforin/granzyme release and the expression of FasL on CTL, whereas a3 domain mutants of MHC class I abolish CD8 engagement and lead to a reduced TCR signal that upregulates FasL expression but does not lead to perforin/granzymes release.
antigen-specific CTL without the concomitant T cell activation, which normally causes end-organ damage.
3. Immunotherapy potentials 3.1. Autoimmunity and graft rejection Selective deletion of auto- or alloreactive T cells is a major goal for the treatment of autoimmune disease, or to prevent the rejection of transplanted organs. Although effective in many cases, conventional therapy relies upon broad-spectrum immunosuppression with the consequent risk of opportunistic infection or tumourigenesis. Therefore, it would clearly be desir-
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able to manipulate the immune system to eliminate only the disease-specific T cells. One obvious approach as a targeted immunotherapy would be to employ the specific interaction between MHC/peptide complex and TCR to induce tolerance (anergy or apoptosis) of reactive T lymphocytes. Although soluble MHC has been used to suppress alloreactive T cells in some transplantation models, its efficacy is limited largely due to low affinity of soluble MHC binding to the cognate TCR (Wang et al., 1996). Obviously, multimeric MHC/peptide complexes with increased affinity to TCR would be the ideal candidates. Indeed, recently, two groups have reported that using multimeric soluble MHC/peptide complex in either dimeric or tetrameric form can induce tolerance of antigen-specific T cells in vivo. Using dimeric MHC/peptide complex, O’Herrin et al. (2001) showed that the soluble MHC dimers induced modulation of surface expression of TCR and inhibited T cell cytolytic activity at nanomolar concentration in vitro. This is associated with phosphorylation of TCR zeta chain and Zap-70, the signalling complex required for T cell activation. When they injected the soluble dimers into alloreactive TCR transgenic mice, they found that there was a significant inhibition of the alloreactive T cell activity, which resulted in consequent outgrowth of an allogeneic tumour (see Schneck’s section). Similarly, Maile et al. (2001) were able to activate naı¨ve male-specific CD8 + T cells by injection of HY class I tetramer in vivo in a female HYTCR transgenic mice model. However, multiple injections of the tetramers led to the elimination of male-specific T cells by either induction of anergy or AICD and subsequently enhanced the survival of male skin grafts in female mice (Fig. 5). Thus, soluble multimeric MHC/peptide complexes may provide a useful tool for antigen-specific deletion of ‘unwanted’ T cells. However, the deletion of T cells by any full agonist peptide/MHC complex should be used with caution, in particular for therapeutic interventions in vivo, because self-reactive T cells could potentially be activated by the agonist and gain full effector function before the death of these cells is induced. The release of cytokines, chemokines, and cytotoxins as a consequence of activation can lead to severe damage to the lymphoid organs and to general immunosuppression (Aichele et al., 1997). Other strategies must be
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Fig. 5. Increasing survival of skin grafts by multiple injection of tetrameric MHC/peptide complex (modified from Maile et al., 2001).
developed to overcome these problems. Although selective signalling ligands such as altered (partial agonist) peptides have been used to regulate T cell deletion without immune damage (Combadiere et al.,
1998), this approach has a major limitation since, in many cases, the responsible peptide epitopes have not yet been identified. Therefore, soluble multimeric MHC/peptide complexes created with mutant heavy chain lacking CD8 interaction would not lead to this detrimental T cell activation and may allow antigenspecific deletion of the disease-mediating CTL (Fig. 4). The other advantage of this approach is that it can allow the deletion of CTL without the need to identify the antigenic peptide. So, for instance, in our experiments, we were able to induce apoptosis of allospecific CTL by expressing an a3 domain mutant in cells where it is loaded with a variety of endogenous peptides (Xu et al., 2001). It would also be possible to produce soluble forms of these molecules loaded with unknown host peptides by expression in mammalian cells and conceivably cells derived from the patient for whom therapy was planned. We are currently testing the utility of these approaches in the treatment of CD8-mediated autoimmunity models and organ transplantation.
Fig. 6. Redirected killing of tumour cells using the tetramer. For example, Flu-specific CTL can kill tumour cells targeted using antitumourspecific antibody conjugated with MHC class I/Flu peptide tetramers by either direct (A) or indirect linking (B) with avidin.
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3.2. Redirected killing of tumour cells Although the CTL response plays an important role in antitumour activity, tumour growth implies that CTL either fail to be elicited or are inadequate. Many tumour cells have defects in antigen presentation due to mutations in their h2M gene, leading to loss of MHC I expression at the cell surface and escape from CTL surveillance (Romero et al., 1998). On the other hand, many tumour cells express tumour-associated antigens that can be recognised by antibodies (Riethmuller and Johnson, 1992). The use of monoclonal antibodies as antitumour therapies, so-called magic bullets, has been evaluated extensively in both animal models and clinical practices (Levy, 2000). Other antibody-based antitumour strategies include bispecific antibodies, one arm of the antibody binding to tumour antigen and the other to TCR, which bring the T cells close to tumour cells and redirect nontumourspecific T cells to kill tumour cells (Guo et al., 1997). Recently, Ogg et al. (2000) and Robert et al. (2000) have described another new antitumour strategy in which they target tumour cells using a tumour-specific antibody conjugated with MHC class I/peptide tetramer. The antibody – tetramer complexes were made by mixing biotinylated antitumour-specific mAb with avidin- and biotin-conjugated MHC/peptide monomers. This was done at relative concentrations to form an avidin –biotin complex, with one site binding to the antibody and other three sites binding to biotin – MHC/peptide monomers (Ogg et al., 2000). The complex could also be made by directly chemically coupling avidin with mAb and then mixing this with biotin –MHC/peptide monomers to form antibody– MHC/peptide tetramer complexes (Robert et al., 2000). Both complexes were able to bind to the surface of tumour cells and sensitise them to lysis by tetramerspecific CTL (Fig. 6). Thus, antibody-guided tetramers can localize TCR ligands on the surface of tumour cells and also trigger the activation of antigenspecific T cell effector functions. There are several advantages of this technique as compared with others. Firstly, it can increase tumour immunogenicity and overcome the problem of absent or low-level expression of MHC molecules found in many tumours. Secondly, induction of tumour cell apoptosis may enhance cross-priming (uptake by dendritic cells or
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macrophages) and subsequently initiate and amplify T cell responses against tumour-specific peptide antigens. Finally and more importantly, if multispecific tetramers are applied, antitumour activities could be accelerated even more by redirecting existing effective CTL such as those specific to EBV, CMV, and influenza, which are known to present at high frequencies in the circulation and generally with an activated and/or memory phenotype. However, the utility of this technique in vivo for the treatment of tumours needs to be confirmed and modified, aimed at increasing stability and reducing the size of the complex.
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