Abstract

Systemic administration of IL-2 may improve the expansion and persistence of adoptively transferred anti-tumor CTLs. However, significant toxicity and concomitant expansion of regulatory T cells (Tregs) limit the clinical value of this strategy. IL-7 plays a crucial role in maintaining T-cell homeostasis, and administration appears well tolerated and not to expand Tregs. Nonetheless, the value of IL-7 for promoting proliferation of tumor-specific CTLs after adoptive transfer remains questionable, due to the lack of expression of the IL-7 receptor (IL-7Rα) on effector CTLs. Thus we have found IL-7Rα to be essentially undetectable in our model of EBV-specific CTL lines (EBV-CTLs) due to downregulation of IL-7Rα transcripts. To determine whether transgenic expression of IL-7Rα can restore the capacity of established EBV-CTLs to respond to IL-7, the full-length of IL-7Rα was cloned in the SFG retroviral vector (SFG/IL-7Rα). We transduced EBV-CTLs generated from 5 healthy donors and evaluated their growth kinetics and antigen specificity. As control, EBV-CTLs were transduced with the SFG retroviral vector encoding for a truncated form of the CD34 molecule (SFG.ΔCD34). After transduction with SFG/IL-7Rα, IL-7Rα was detectable in 58% to 76% of EBV-CTLs. The transgenic IL-7Rα was functional since addition of IL-7 (2ng/mL) to transgenic cells induced phosphorylation of STAT5 within 10 min. To evaluate whether rhIL-7 sustained the expansion of EBV-CTLs/IL-7Rα+, transduced CTLs were stimulated weekly with autologous EBV-LCLs in the presence of IL-2 (50U/mL) or IL-7 (2ng/mL). Control EBV-CTLs and EBV-CTLs/IL-7Rα+ expanded equally well with IL-2. However, only EBV-CTL/IL-7Rα+ significantly proliferated in the presence of IL-7 [from 1×106 cells to 103×106 cells (range, 38-286×106)] over a period of five weeks. CTL expansion remained antigen dependent, and antigen withdrawal led to cessation of CTL growth. EBV-CTLs/IL-7Rα+ showed the expected growth advantage in the presence of IL-7, increasing from 55%±15% to 79%±5% of the total within 5 weeks. In contrast, the proportion of IL-7Rα+ cells marginally declined when the cells were expanded with IL-2 (from 64%±14% to 40%±11%). Importantly, EBV-CTLs/IL7Rα+ expanded with IL-7 retained their ability to respond to other common-γ-chain cytokines. EBV-CTLs/IL7Rα+ grown with IL-7 for more than 2 weeks, expanded further when exposed to both antigen and IL-2 or IL-15. Control CTLs grown with IL-2 and EBV-CTLs/IL-7Rα+ grown with IL-7 both remained polyclonal, as assessed by VβTCR repertoire analysis, and were mostly CD3+/CD8+ (> 90±8%) with an effector-memory profile. EBV-CTLs/IL-7Rα+ also retained antigen specificity measured by tetramer staining, by IFNγ release in response to EBV-peptides and by MHC-restricted killing of autologous LCLs (52%±18% at a ratio 20:1 vs 9±17% of allogeneic LCL); this killing was comparable to the activity of control CTLs cultured in IL-2 (51%±27%). Preliminary studies indicate that EBV-CTLs/IL-7Rα+ also expand in vivo in response to IL-7 and maintain their anti-tumor activity in a SCID mouse xenograft model.

Author notes

Disclosure: Research Funding: NIH, Leukemia & Lymphoma Society and Doris Duke Charitable foundation.