CD28 co-stimulation via tumour-specific chimaeric receptors induces an incomplete activation response in Epstein-Barr virus-specific effector memory T cells

Abstract

Expression of tumour antigen-specific chimaeric receptors in T lymphocytes can redirect their effector functions towards tumour cells. Integration of the signalling domains of the co-stimulatory molecule CD28 into chRec enhances antigen-specific proliferation of polyclonal human T cell populations. While CD28 plays an essential role in the priming of naive CD4(+) T cells, its contribution to effector memory T cell responses is controversial. We compared the function of the chRec with and without the CD28 co-stimulatory domain, expressing it in peripheral blood T cells or Epstein-Barr virus (EBV)-specific T cell lines. The chimaeric T cell receptors contain an extracellular single-chain antibody domain, to give specificity against the tumour ganglioside antigen G(D2). The transduced cytotoxic T lymphocytes (CTL) maintained their specificity for autologous EBV targets and their capacity to proliferate after stimulation with EBV-infected B cells. Intracellular cytokine staining demonstrated efficient and comparable antigen-specific interferon (IFN)-gamma secretion by CTL following engagement of both the native and the chimaeric receptor, independent of chimaeric CD28 signalling. Furthermore, tumour targets were lysed in an antigen-specific manner by both chRec. However, while antigen engagement by CD28 zeta chRec efficiently induced expansion of polyclonal peripheral blood lymphocytes in an antigen-dependent manner, CD28 signalling did not induce proliferation of EBV-CTL in response to antigen-expressing tumour cells. Thus, the co-stimulatory requirement for the efficient activation response of antigen-specific memory cells cannot be mimicked simply by combining CD28 and zeta signalling. The full potential of this highly cytolytic T cell population for adoptive immunotherapy of cancer requires further exploration of their co-stimulatory requirements.

Fig. 1

Immunophenotypes of non-transduced, 14.G2aζ- and 14.G2a–CD28ζ-transduced Epstein–Barr virus–cytotoxic T lymphocytes (EBV–CTL) following five to six stimulations with autologous lymphoblastoid cell lines (LCL) and expansion in the presence of rhIL2 (50 U/ml). Shown are the mean percentages of fluorescence-positive cells after staining of three cell lines with fluorescence-marked antibodies (a), and a representative experiment (b).

Fig. 2

Intracellular interferon (IFN)-γ and tumour necrosis factor (TNF)-α secretion by 14.G2aζ (a,b,c) and 14.G2a–CD28ζ (d,e,f) transduced Epstein–Barr virus–cytotoxic T lymphocytes (EBV–CTL) in response to autologous lymphoblastoid cell lines (LCL) (b,e), the G_D2⁺ target cell line LAN-5 (c,f), or the G_D2^– target cell line A204 (a,d). To exclude non- transduced CTL within the cultures from analysis, the gate was set on F(ab′)2⁺ cells. Shown is one representative experiment of four (IFN-γ), and two, (TNF-α) respectively. The lower percentage of chRec-positive T cells in these experiments when compared to the transduction efficiencies is explained by antigen-specific down-regulation of chRec.

Fig. 3

(a) 14.G2a-ζ and 14.G2a–CD28ζ-transduced Epstein–Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) lines specifically lyse G_D2⁺ tumour targets. Non-transduced (A), 14.G2a-ζ (B), and 14.G2a–CD28ζ (C)-tranduced CTL were tested against ⁵¹Cr-labelled autologous G_D2⁺ neuroblastoma tumour cells (LAN-1, LAN-5), G_D2^– rhabdomyosarcoma cells (A-204) or K-562 cells. Shown is one representative experiment of three. (b) Flow cytometric analysis of CD107a expression of 14.G2aζ (A, B, C) and 14.G2a–CD28ζ (D, E, F) transduced EBV–CTL in response to autologous lymphoblastoid cell lines (LCL) (B, E), the G_D2⁺ target cell line LAN-5 (C, F), or the G_D2^– target cell line A204 (A, D). To exclude non-transduced CTL within the cultures from analysis, the gate was set on F(ab′)2⁺ cells. Shown is one representative experiment of three.

Fig. 4

(a) Tumour-specific CD28ζ signalling in non-specifically prestimulated peripheral blood T cells mediates T cell expansion. Non-transduced (A), 14.G2a-ζ (B) or 14.G2a–CD28ζ-transduced peripheral blood-derived T cell cultures received weekly stimulations with irradiated G_D2⁺ (LAN-5) or G_D2^– (A-204) tumour cell targets (40 Gy) at a 1 : 1 stimulator : responder ratio. Cells were fed twice-weekly with medium containing rhuIL-2 (40 IU/ml), and their growth kinetics were assessed. Shown is one representative experiment of four. (b) Proliferative responses of 14.G2aζ and 14.G2a–CD28ζ transduced peripheral blood mononuclear cells to 5 days’ stimulation with irradiated G_D2⁺ (LAN-5) or G_D2^– (A-204) tumour cell targets (40 Gy) at a 1 : 1 stimulator : responder ratio assessed by flow cytometric enumeration. Shown is one representative experiment of two.

Fig. 5

(a) Neither 14.G2a-ζ nor 14.G2a–CD28ζ transduced cytotoxic T lymphocytes (CTL) expand in response to tumour targets. Non-transduced (A), 14.G2a-ζ (B) or 14.G2a–CD28ζ (C)-transduced Epstein–Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) cultures received weekly stimulations with irradiated autologous lymphoblastoid cell lines (LCL) (40 Gy), or with irradiated G_D2⁺ tumour cell targets (40 Gy) at a 1 : 1 stimulator : responder ratio. Cells were fed twice-weekly with medium containing rhuIL-2 (40 IU/ml), and their growth kinetics were assessed. Shown is one representative experiment of two. (b) Proliferative responses of 14.G2aζ and 14.G2a–CD28ζ transduced EBV–CTL to 5 days’ stimulation with irradiated autologous LCL (auto-LCL) or with G_D2⁺ (LAN-1, LAN-5) or G_D2^– (A-204) tumour cells at a 1 : 1 stimulator : responder ratio assessed by flow cytometric enumeration. Shown is one representative experiment of two. (c) Target-specific proliferative responses assessed by measurement of [³H]-thymidine uptake. Transduced and non-transduced EBV-specific CTL were stimulated with irradiated autologous LCL, or with G_D2⁺ (LAN-1, LAN-5) or G_D2^– (A-204, K-562) tumour cells at a 1 : 4 stimulator : responder ratio. Shown is one representative experiment of three.