CD28 Fails to Costimulate Tumor-Specific Activation of Epstein Barr-Virus-Specific Memory Effector T Cells.

Genetic modification of cytotoxic T cells with tumor antigen-specific chimeric receptors (chRec) specifically redirects their effector functions towards tumor cells. Integration of the signaling domains of the costimulatory molecule CD28 into chRec enhances antigen-specific proliferation of peripheral blood-derived polyclonal human T cell populations. While CD28 plays an essential role in the priming of naïve CD4+ T cells, its contribution to effector memory cytotoxic T cell (CTL) responses is controversial. We investigated the function of chRec containing the signaling domains of CD28 in vitro expanded T cells with specificity for Epstein-Barr-virus (EBV). Chimeric T cell receptors containing an extracellular single-chain antibody domain directed against the tumor ganglioside antigen G_D2 fused to the intracellular signaling domains of both the CD28 and the T cell receptor (TCR) ζ chain (14.G2a-CD28ζ), or TCRζ alone (14.G2a-ζ) were expressed in EBV-specific cytotoxic T cell (CTL) lines from three seropositive donors and in peripheral blood T cells preactivated by CD3-/CD28-specific antibody crosslinking by retroviral gene transfer. Following transduction with the chRec genes, 14.G2a-ζ and 14.G2a-CD28ζ EBV-CTL had comparable levels of chRec surface expression (21–28% versus 26–40%). EBV-CTL had an immunophenotype characteristic of memory effector T cells, coexpressing CD3 and CD45RO in the absence of CD45RA and CD27 surface expression. The transduced CTL maintained their capacity to specifically lyse autologous EBV targets in 4 hr ⁵¹Cr release assays and to proliferate after stimulation with autologous EBV targets. Intracellular cytokine staining demonstrated efficient and comparable antigen-specific IFN-γ secretion by both 14.G2a-ζ and 14.G2a-CD28ζ-transduced CTL in response to EBV and tumor targets. Furthermore, G_D2+ neuroblastoma targets were lysed in a comparable and antigen-specific manner. However, while antigen engagement by 14.G2a-CD28ζ efficiently induced expansion of non-specifically activated polyclonal peripheral blood lymphocytes in an antigen-dependent manner, CD28 signaling failed to overcome the failure of transduced EBV-CTL to specifically proliferate in response to G_D2+ tumor cells. Thus, the costimulatory requirement for the highly efficient proliferative activation response of EBV-specific CTL to viral antigen can not be mimicked by combined CD28 and ζ signaling. Exploring the mechanisms and costimulatory requirements of effector memory T cell reactivation may help to exploit the full potential of this highly cytolytic T cell population for adoptive immunotherapy of cancer.