T cell receptor-transgenic primary T cells as a tool for discovery of leukaemia-associated antigens

Abstract

Identification of a broad array of leukaemia-associated antigens is a crucial step towards immunotherapy of haematological malignancies. However, it is frequently hampered by the decrease of proliferative potential and functional activity of T cell clones used for screening procedures. Transfer of the genes encoding the T cell receptor (TCR) alpha and beta chains of leukaemia-specific clones into primary T cells may help to circumvent this obstacle. In this study, transfer of two minor histocompatibility antigen (minor H antigen)-specific TCRs was performed and the feasibility of the use of TCR-transgenic T cells for identification of minor H antigens through cDNA library screening was investigated. We found that TCR-transgenic cells acquired the specificity of the original clones and matched their sensitivity. Moreover, the higher scale of cytokine-production by TCR-transgenic T cells permits the detection of either small amounts of antigen-positive cells or cells expressing low amounts of an antigen. When applied in equal numbers, TCR-transgenic T cells and the original T cell clones produced similar results in the screening of a cDNA library. However, the use of increased numbers of TCR-transgenic T cells allowed detection of minute amounts of antigen, barely discernible by the T cell clone. In conclusion, TCR-transfer generates a large amount of functional antigen-specific cells suitable for screening of cDNA expression libraries for identification of cognate antigens.

Fig. 1

Generation and purification of T cell receptor (TCR)-transgenic T cells. FACS-analysis of donor T cells transduced with TCR^RPS4Y (a, b) or TCR^UTY (c, d) before (a, c) and after (b, d) purification of NGFR + cells with paramagnetic beads. The y-axis shows Vβ gene expression, the x-axis shows NGFR-expression (Vα).

Fig. 2

T cell receptor (TCR)-transgenic T cells are superior to T cell clones in their sensitivity and the amplitude of interferon (IFN)-γ response. (a) Reactivity of the YKIII.8 T cell clone and TCR^YKIII.8-transgenic T cells towards decreasing amounts of EBV-LCL of patient origin (EBV_p) was tested in an interferon (IFN)-γ enzyme-linked immunosorbent assay (ELISA). EBV_p were progressively diluted with EBV_d to achieve the presence of 3 × 10⁴ target cells in each well. (b) The same experiment was performed with the YKII.39 T cell clone and TCR^YKII.39-transgenic T cells. (c) An IFN-γ ELISA was used to measure IFN-γ production by YKIII.8 and TCR^YKIII.8-transgenic T cells in response to stimulation with EBV_d loaded with decreasing (10⁻⁵–10⁻⁹ M) concentrations of the TIRYPDPVI peptide. As controls, both cell populations were tested against EBV_p (first bars for each cell type).

Fig. 3

T cell receptor (TCR)-transgenic T cells produce higher amounts of various cytokines compared to the original clones. The multiplex cytokine assay system was used to measure cytokine production by 3 × 10³ cells of either YKIII.8 or TCR^YKIII.8-transgenic T cells in response to overnight stimulation with 3 × 10⁴ EBV-LCL of patient origin (EBV_p). Average results for duplicate measurements and their standard deviations are shown only for those cytokines, which were present in detectable concentrations in at least one of the samples.

Fig. 4

T cell receptor (TCR)-transgenic T cells are specific and sensitive tools for cDNA library screening. 3 × 10³ YKIII.8 (a) or 3 × 10³ TCR^YKIII.8-transgenic T cells (b) were co-cultured overnight with HLA-B*5201⁺ 293-EBNA cells transfected with cDNA pools 685–706 from the Epstein–Barr virus of patient origin (EBV_p) cDNA library. Interferon (IFN)-γ production was measured with an IFN-γ enzyme-linked immunosorbent assay (ELISA). (c) HLA-B*5201⁺ 293-EBNA cells were transfected with either undiluted cDNA pool 46 from the EBV_p cDNA library or its 1: 2 and 1: 4 dilutions. Reactivity of 3 × 10³ YKIII.8 or 3 × 10⁴ TCR^YKIII.8-transgenic T cells towards transfected and non-transfected cells was measured in an IFN-γ ELISA.