Gene-Modified CD8+ T Cells Undergo Functional Polarization to Effector and Central Memory Cells in Response to Antigen Exposure.
Authors: Neeson Paul , Shin Amanda , Tai Tsin , Chen Karen , Davis Joanne , Tainton Kellie , Darcy Phil , Kershaw Michael H. , Peinert Stefan , Guru Preethi , Harrison Simon , Prince H. Miles , Ritchie D. 
Published: April 2020

Abstract
Adoptive transfer (AT) of autologous T cells genetically-redirected against tumor antigens has considerable potential as cancer immunotherapy [Kershaw, Nat Rev Immunol. 2005]. However, the in vivo persistence of AT T cells is critical for tumor control and requires the development (in vitro or in vivo) of a memory T cell subset. We investigated the generation of memory T cell subsets in a novel chimeric T cell receptor-expressing T cell product prior to, and after exposure to cognate antigen. Gene-modified T cells (LeY-T) express a chimeric receptor comprising a single chain variable fragment (scFv) specific for Lewis Y (LeY) antigen coupled to the intracellular signaling domains of CD3 zeta and CD28, capable of inducing T cell effector granule release and target killing [Westwood PNAS 2005]. To produce LeY-T cells, PBMC from healthy donors (n=20) or multiple myeloma patients (n=2) were cultured with anti-OKT3 (30ng/ml) and IL-2 (600IU/ml) for three days, followed by two rounds of transduction with retroviral supernatant. Subsequently, T cells were expanded in high dose IL-2 (600IU/ml) from day 5 onwards. T cells were harvested for this study on culture days 10–12, CD8+ and CD4+ T cells expressed the chimeric protein (50–60)%. LeY CD8+ T cell subsets were assessed as naïve (N), central memory (CM), effector memory (EM) or effector (E) based on three features:-
phenotype (CD45RA, CCR7, CD28, CD27 and perforin); homeostatic cytokine (IL-15/IL-7) proliferation; response to Lewis antigen contact including cell proliferation and cytokine secretion.
We repeatedly observed that CD8+ LeY-T cells analyzed directly from the initial expansion culture demonstrate an effector memory (EM) phenotype (CD45RA−/CCR7−/CD28+/perforinhi and variable CD27 expression) (Figure 1A). Furthermore in vitro expanded LeY CD8 T cells express IL- 15R beta (CD122) and the common gamma chain (CD132), they proliferate in response to IL-15 (86% cell division, division index 1.82), but less with IL-7 (30% cell division, division index 0.56). Baseline expanded CD8+ LeY-T cells respond to the presence of LeY antigen by proliferating and secreting IFN-gamma (4–8% of CD8 T cells) but not IL-2. Importantly, no IFN-gamma secretion was seen in control T cells transduced with empty vector (Figure 1B, OVCAR cells). Furthermore, no IFN-gamma was secreted by the control or the CD8+ LeY-T cells in response to the Lewis antigen negative cell line (Figure 1C, HCT116 cells). To explore the memory component further, we examined the functional status of the CD8+ LeY-T cells seven and 30 days following a 48-hour exposure to LeY antigen (OVCAR cells), and compared this to CD8+ LeY-T cell functional status at baseline. Thus, direct from transduction, expansion culture LeY CD8+ T cells were largely EM phenotype (95%) a small population of cells (1–5)% had a CM phenotype (CD45RA−/CCR7+/CD28+/perforinlo). In contrast, seven days after Lewis antigen contact the EM cells had decreased to (76–88)% and CM increased to (10–21)%; this distribution was retained up to day 30 post-antigen exposure. In addition, seven days after Lewis antigen exposure, CD8+ LeY-T cells retain the capacity to proliferate in response to Lewis antigen and to secrete IFN-gamma, at no stage do these cells secrete IL-2. In conclusion, the CD8+ LeY-T cells produced by in vitro transduction and expansion culture have an EM functional status direct from in vitro culture indicating that they are an appropriate starting population for in vivo adoptive transfer. After exposure to LeY expressed on tumor cell lines in vitro, CD8+ LeY T cells show further polarization to either EM or CM cells. These results suggest that the LeY-chimeric T cells have the potential to form long-term memory populations in vivo after adoptive transfer.
Figure 1. LeY T cells have an effector memory phenotype and respond to Lewis antigen expressing cell lines by secreting IFN-gamma. Following the transduction culture, the CD8+ LeY-T cells (A) expressed high levels of perforin and an EM phenotype. In (B), LeY T or empty vector control T cells were co-cultured with tumour cells overnight and intracellular cytokine secretion assay performed. The LeY CD8+ T cells responded to Lewis antigen expressing OVCAR cells by secreting IFN-gamma, whereas no response was observed with the negative cell line HCT-116. Figure 1. LeY T cells have an effector memory phenotype and respond to Lewis antigen expressing cell lines by secreting IFN-gamma. Following the transduction culture, the CD8+ LeY-T cells (A) expressed high levels of perforin and an EM phenotype. In (B), LeY T or empty vector control T cells were co-cultured with tumour cells overnight and intracellular cytokine secretion assay performed. The LeY CD8+ T cells responded to Lewis antigen expressing OVCAR cells by secreting IFN-gamma, whereas no response was observed with the negative cell line HCT-116.