Endogenous T Cell Receptor Chains Research Articles

Facilitating matched pairing and expression of TCR chains introduced into human T cells

AbstractAdoptive transfer of T lymphocytes is a promising treatment for a variety of malignancies but often not feasible due to difficulties generating T cells that are reactive with the targeted antigen from patients. To facilitate rapid generation of cells for therapy, T cells can be programmed with genes encoding the α and β chains of an antigen-specific T-cell receptor (TCR). However, such exogenous α and β chains can potentially assemble as pairs not only with each other but also with endogenous TCR α and β chains, thereby generating αβTCR pairs of unknown specificity as well as reducing the number of exogenous matched αβTCR pairs at the cell surface. We demonstrate that introducing cysteines into the constant region of the α and β chains can promote preferential pairing with each other, increase total surface expression of the introduced TCR chains, and reduce mismatching with endogenous TCR chains. This approach should improve both the efficacy and safety of ongoing efforts to use TCR transfer as a strategy to generate tumor-reactive T cells.

Redirecting Human T Lymphocytes toward Tumor-Associated Antigens by T Cell Receptor (TCR) Replacement.

The gene transfer of alpha and beta chains derived from a defined TCR has been successfully applied to endow T cells with specificities directed against tumor-associated antigens. However, it is still unclear if the transfer of TCR genes into T cells that already express an endogenous TCRalpha and beta chain leads to engineered T cells expressing four different TCR complexes on their cell surface. Mixed TCR heterodimers composed of endogenous and exogenous TCR chains may acquire new specificities, which may cause unwanted reactions in patients following adoptive T cell transfer. We examined the possibility of mixed TCR heterodimer formation using defined conditions of single TCR chain transfer into human cytotoxic T cell (CTL) clones specific for CMV and Melan-A, respectively. After stimulation for three days CTLs were retrovirally transduced with the beta chain derived from a gp100-specific TCR. The expression of the exogenous (transduced) and the endogenous beta chain was distinguished by flow cytometry using antibodies against the different Vbeta motives. Indeed, CTLs that had been transduced with the single beta chain expressed this chain on the cell surface indicating the formation of mixed TCRs, because the expression of the exogenous TCRbeta chain requires the pairing with the endogenous TCRalpha chain. Furthermore, we transduced the CTL clones with both the TCRalpha and beta chain derived from a gp100-specific TCR. The transduced T cells were positively stained with an A2/gp100 multimer documenting the correct formation of the exogenous TCR chains. Functionality of transduced CTL clones was tested by antigen-specific IFN-gamma release and cytolytic activity. The TCR-transduced T cells were sorted with the A2/gp100 multimer and expanded for two weeks. Double staining with HLA multimers for the endogenous and the transduced TCRs showed the downregulation of the endogenous TCRs in three different CTL clones. In one CTL clone, the endogenous TCR was even replaced by the exogenous TCR as documented by flowcytometry and antigen-specific T cell function. In conclusion, transfer of single TCR chains in CTL clones can result in the formation of TCR heterodimers. However, our results also show that complete TCRs are predominantly expressed or can even replace other TCRs following transfer. The development of dominant TCRs will facilitate the therapeutical approaches of adoptive transfer regimens based on TCR-transduced T cells, because dominant TCRs can be selected or TCRs can be modified to be more dominant.

Immune Regulatory Mechanisms Influence Early Pathology in Spinal Cord Injury and in Spontaneous Autoimmune Encephalomyelitis

Injuries to the central nervous system (CNS) trigger an inflammatory reaction with potentially devastating consequences. In this report we compared the characteristics of the inflammatory response on spinal cord injury (SCI) caused by a stab wound between the T7 and T9 vertebrae and spontaneous experimental autoimmune encephalomyelitis (EAE). SCI and EAE were compared in two types of myelin basic protein Ac1-11-specific T-cell receptor transgenic mice: T/R+ mice harbor regulatory T cells, and T/R- mice lack regulatory T cells. Our results show that 8 days after SCI, T/R- mice developed a strong T-cell infiltrate in the spinal cord, with remarkable down-modulation of CD4 expression that was accompanied by a local increase in Mac-3+ and F4/80+ reactivity and diffuse local and distal astrogliosis. In contrast, T/R+ mice exhibited a modest increase in CD4+ cells localized to the site of injury, without CD4 down-modulation; focal astrogliosis was restricted to the site of the lesion, although Mac-3+ and F4/80+ cells were also present. Similarly to T/R- mice that underwent SCI, T cells displaying down-modulated CD4 expression were found in the CNS of older T/R- mice afflicted by spontaneous EAE. Overall, our results suggest that common mechanisms regulate T-cell accumulation in CNS lesions of different causes, such as mechanic lesion or autoimmune-mediated damage.

Anti-class II antibodies, but not cytotoxic T-lymphocyte antigen 4-immunoglobulin hybrid molecules, prevent rejection of major histocompatibility complex class II-negative myeloma in T-cell receptor-transgenic mice.

We have previously shown that tumour-specific CD4+ T cells protect against subcutaneous injections of major histocompatibility complex (MHC) class II-negative MOPC315 myeloma cells. Here, we have interfered with the immunologic events that lead to successful rejection of MOPC315 challenges in T-cell receptor (TCR)-transgenic mice. The CD4+ T cells have a transgene-encoded TCR specific for a MOPC315 V-region idiotypic (Id) peptide presented on the MHC class II molecule E(d). A side-by-side comparison indicated that DNA-recombination-deficient TCR-transgenic mice were better protected against MOPC315 tumour development than recombination-sufficient counterparts, suggesting that B cells or endogenous TCR chains might facilitate tumour progression in this model. Intraperitoneal injections of E(d)-specific antibodies over a period of initial 24 days, abrogated protection against tumours in both strains of mice. By contrast, injections of anticostimulatory molecules (cytotoxic T-lymphocyte antigen 4-immunoglobulin hybrid molecules) had no effect. The findings demonstrate that tumour rejection depends on the presence of MHC class II molecules, despite the fact that MOPC315 tumour cells themselves do not express them. The results are consistent with the idea that secreted myeloma protein is processed and presented by class II+ antigen-presenting cells to Id-specific naïve CD4+ T cells that become activated and kill the myeloma cells by a bystander mechanism. While Id presentation on class II molecules is absolutely required for tumour rejection, costimulatory CD80/CD86 molecules might be dispensible in this process.

Genetic engineering of T cell specificity for immunotherapy of cancer

The ultimate goal of immunotherapy of cancer is to make use of the immune system of patients to eliminate malignant cells. Research has mainly focused on the generation of effective antigen specific T-cell responses because of the general belief that T-cell immunity is essential in controlling tumor growth and protection against viral infections. However, the isolation of antigen specific T cells for therapeutic application is a laborious task and it is often impossible to derive autologous tumor specific T cells to be used for adoptive immunotherapy. Therefore, strategies were developed to genetically transfer tumor specific immune receptors into patients T cells. To this end, chimeric receptors were constructed that comprise antibody fragments specific for tumor associated antigens, linked to genes encoding signaling domains of the T-cell receptor (TCR) or Fc receptor. T cells expressing such chimeric antibody receptors recapitulate the immune specific responses mediated by the introduced receptor. Recently, we introduced chimeric TCR genes into primary human T lymphocytes and demonstrated that these T cell transductants acquired the exquisite major histocompatibility complex (MHC) restricted tumor specificity dictated by the introduced TCR. Importantly, the introduction of chimeric TCR bypasses problems associated with the introduction of nonmodified TCR genes, such as pairing of introduced TCR chains with endogenous TCR chains and unstable TCRα expression. A novel strategy which is completely independent of available tumor specific T-cell clones for cloning of the TCR genes was recently used to transfer MHC restricted tumor specificity to T cells. Human “TCR-like” Fab fragments obtained by in vitro selection of Fab phages on soluble peptide/MHC complexes were functionally expressed on human T lymphocytes, resulting in MHC restricted, tumor specific lysis and cytokine production. In addition, affinity maturation of the antibody fragment on Fab phages allows improvement of the tumor cell killing capacity of chimeric Fab receptor engrafted T cells. Developments in retroviral transfer technology now enables the generation of large numbers of antigen specific T cells that can be used for adoptive transfer to cancer patients. In this article we summarize the developments in adoptive T cell immunogenetic therapy and discuss the limitations and perspectives to improve this technology toward clinical application.