Transgenic Expression of IL15 Selectively Expands Antigen Specific Cytotoxic T Cells (CTLs) Enhancing Their Anti-Tumor Effect In Vivo.
Authors: Vera J. , Quintarelli C. , Savoldo B. , Giordano G. , Foster A. , Pule M. , Heslop Helen , Rooney C. , Brenner Malcolm , Dotti G. 
Published: October 2019

Abstract
The anti-tumor effect of adoptively transferred tumor-specific CTLs is impaired by the limited capacity of these cells to expand within the tumor microenvironment. Administration of IL2 has been used to overcome this limitation, but the systemic toxicity and the potential expansion of unwanted cells including regulatory T cells limit the clinical value of this strategy. To discover whether transgenic expression of lymphokines by the CTLs themselves might overcome these limitations, we compared the effects of transgenic expression of IL2 and IL15 in our model of tumor antigen-Epstein Barr Virus-specific CTLs (EBV-CTLs). Bicistronic retroviral vectors were generated encoding hIL2 or hIL15 and a truncated form of the human CD34 molecule (DCD34) as a marker to track transgenic cells. The two genes were linked using a 2A peptide cleavable sequence. Based on the expression of CD34, the transduction rate was 29±6%, 26±12% and 34±19% respectively for CTLs/IL2v, CTLs/IL15v and CTLs/DCD34 alone. Twenty eight days after stimulation with the antigen (LCLs) at E:T ratio 1:1, we observed significant expansion of CTLs/IL2v (2283 fold expansion, range 410–5131) and CTLs/IL15v (1194, range 41–3420) compared to CTLs/DCD34 without cytokines (<2 fold expansion). The expansion rate was antigen-dose dependent, and was significantly reduced when a 4:1 E:T ratio was used. Cytokine release after antigenic stimulation was 5.8±3 pg/mL and 79±25 pg/mL for IL15 and IL2, respectively. Transgenic CTLs maintained the same phenotype as control T cells (>90% CD3+/CD8+) and remained polyclonal as assessed by staining for the TCRVb repertoire. Antigenic specificity was maintained since CTLs/IL2v and CTLs/IL15v killed autologous LCLs (40±10% at E:T ratio 20:1) but not allogeneic LCLs or K562 cells (<15%). Tetramer staining for Class I HLA-restricted EBV-peptides also confirmed that antigen specificity was retained. Since transgenic cells expressed CD34 on the cell surface we also followed the expression of this marker overtime. We found that the percentage of CD34+ cells increased with time for CTLs/IL15v (from 24±4% to 65±13% by day 21 of culture), whilst remaining unchanged for CTLs/IL2v (from 38±6% to 19±12% by day 21 of culture) suggesting that CTLs/IL15v produced and consumed the cytokine primarily in an autocrine rather than a paracrine mechanism, while sufficient transgenic IL2 was produced for paracrine growth stimulation of non-transduced cells. Additional experiments using CTLs labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and intracellular staining for BCL2 indicated that the selection of CTLs/IL15v was likely determined both by their higher proliferative rate and their increased resistance to apoptosis. In vivo experiments using bioluminescent imaging of a SCID mouse model engrafted with LCLs and injected with transgenic CTLs marked with the firefly-luciferase, showed that cytokine-transduced CTLs had improved expansion and persistence within the tumor compared to control CTLs. Moreover, their anti-tumor activity at 40–45 days after CTL was increased − 5/5 mice receiving control CTLs had tumor size >0.8–1 cm, while 5/7 mice receiving CTLs/IL15v or CTLs/IL2v had undetectable tumor. In conclusion, our data indicate that transgenic expression of either IL2 or IL15 improves the expansion and anti-tumor effect of EBV-CTLs in vivo. However, IL15 preferentially expands transduced CTLs, and may avoid an increase of unwanted cells in the tumor microenvironment.