The antigen specificity of T lymphocytes is dictated solely by the T cell receptor (TCR) alpha and beta chains. Consequently, genetic transfer of TCR chains may be an appealing strategy with which to impose a desirable virus- or tumor-antigen specificity onto cytotoxic or helper T cell populations. We describe here the genetic introduction of a virus-specific TCR into peripheral T cells in a mouse model system. These experiments showed that T cells redirected by TCR gene transfer expanded upon viral infection of mice and efficiently homed to effector sites. In this setting, TCR gene transfer was not associated with any significant autoimmune pathology. In addition, small numbers of TCR-transduced T cells promoted the rejection of antigen-expressing tumors in vivo. These data suggest that the redirection of T cells by TCR gene transfer is a viable strategy for the rapid induction of virus- or tumor-specific immunity.

Adoptive transfer of T-cell receptor (TCR) genes has been proposed as an attractive approach for immunotherapy in cases where the endogenous T-cell repertoire is insufficient. While there are promising data demonstrating the capacity of TCRmodified T cells to react to foreign antigen encounter, the feasibility of targeting tumor-associated self-antigens has not been addressed. Here we demonstrate that T-cell receptor gene transfer allows the induction of defined self-antigenspecific T-cell responses, even when the endogenous T-cell repertoire is nonreactive. Furthermore, we show that adoptive transfer of T-cell receptor genes can be used to induce strong antigen-specific T-cell responsiveness in partially MHCmismatched hosts without detectable graft versus host disease. These results demonstrate the feasibility of using a collection of "off the shelf" T-cell receptor genes to target defined tumor-associated self-antigens and thereby form a clear incentive to test this immunotherapeutic approach in a clinical setting. IntroductionMajor histocompatibility complex (MHC) molecules present peptides on the cell surface irrespective of whether they are derived from foreign proteins or from self-proteins. Different tissue types within the body each express a unique set of proteins, and peptide epitopes derived from such tissue-specific proteins can in principle be used as tumor-rejection antigens. 1 However, because most of these tumor-associated antigens (TAAs) are nonmutated selfantigens, the T-cell repertoire specific for such antigens is generally small in size and low in avidity. Indeed, both preclinical studies and clinical trials have provided evidence that a lack of T cells with the required reactivity is a major factor limiting T-cell-based immunotherapy. For instance, murine studies have demonstrated that tumor-specific T-cell responses against foreign tumor-associated antigens can readily be induced by vaccination. However, when the same tumor-associated antigen is considered "self" by the available T-cell repertoire, reactivity to these antigens is highly reduced. 2,3 In line with this, replacement of the endogenous T-cell compartment through a combination of allogeneic stem cell transplantation (allo-SCT) and donor lymphocyte infusion (DLI) forms an effective treatment strategy for patients with hematologic malignancies such as chronic myeloid leukemia (CML). 4 Importantly, the antileukemic effect of allo-SCT/DLI is dependent on the recognition of minor histocompatibility antigens (MiHAgs) of the recipient as "nonself" by the infused donor lymphocytes, and the development of T-cell responses against such antigens is predictive of remission. 5 The effects of DLI following allo-SCT provide an excellent example of how an endogenous antigen can become foreign by introduction of a novel T-cell compartment and how recognition of endogenous antigens by this exogenous T-cell compartment is associated with remission. The major drawback of this treatment protocol is that the introduced T-cell reactivity against self...

The diversity of the T cell receptor (TCR) repertoire is limited, because of the processes of positive and negative T cell selection. To obtain T cells with specificities beyond the immune system's capacity, we have developed a strategy for retroviral TCR display. In this approach, a library of T cell variants is generated in vitro and introduced into a TCR-negative murine T cell line by retroviral transfer. We document the value of TCR display by the creation of a library of an influenza A-specific TCR and the subsequent in vitro selection of TCRs that either recognize the parental influenza epitope or that have acquired a specificity for a different influenza A strain. The resulting in vitro selected TCRs induce efficient T cell activation after ligand recognition and are of equal or higher potency than the in vivo generated parent receptor. TCR display should prove a useful strategy for the generation of high-affinity tumor-specific TCRs for gene transfer purposes.T cells, the prime mediators of adaptive cellular immunity, specify their action through the T cell receptor (TCR)-mediated recognition of a peptide epitope bound to a MHC molecule. 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, self-tolerance leads to the removal of the highaffinity T cell repertoire specific for self antigens (1, 2), and this removal will include T cells with desirable specificities, such as many self antigens expressed on tumor tissues. Because of the potential therapeutic value of TCRs with such tumor͞lineage specificities, we set out to develop an in vitro strategy for TCR selection that can be used to bypass in vivo tolerance.For the in vitro isolation and generation of mAbs, antibodyphage display has proven to be a useful technology to replace hybridoma technology and animal immunization (3). Analogous to this technology, TCRs have been expressed as singlechain fragments (scTCRs) on the surface of both phage (4) and yeast (5). 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 affinity for an allogeneic peptide-MHC target (6). Such high-affinity 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 TCRs. Specifically, the ability of T cells to discriminate between closely related ligands seems to be related directly to the property of TCR͞CD3 complexes to cluster after encountering their cognate ligands (7,8), and it may prove difficult to copy this process in these systems. We present here a T cell-based in vitro TCR selection strategy that can be used to isolate ␣␤ heterodimeric TCRs with increased affinities or altered specificities. This strategy for TCR display closely mimics the in vivo situation by retrov...

CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine whether Th cell responses can be generated by redirecting CD4+ T cells to MHC class I ligands, we have introduced MHC class I-restricted TCRs into postthymic murine CD4+ T cells and examined CD4+ T cell activation and helper function in vitro and in vivo. These experiments indicate that Ag-specific CD4+ T cell help can be induced by the engagement of MHC class I-restricted TCRs in peripheral CD4+ T cells but that it is highly dependent on the coreceptor function of the CD8β-chain. The ability to generate Th cell immunity by infusion of MHC class I-restricted Th cells may prove useful for the induction of tumor-specific T cell immunity in cases where MHC class II-associated epitopes are lacking.