Interleukin-7 mediates selective expansion of tumor-redirected cytotoxic T lymphocytes (CTLs) without enhancement of regulatory T-cell inhibition - PubMed (original) (raw)
Interleukin-7 mediates selective expansion of tumor-redirected cytotoxic T lymphocytes (CTLs) without enhancement of regulatory T-cell inhibition
Serena K Perna et al. Clin Cancer Res. 2014.
Erratum in
- Clin Cancer Res. 2014 Aug 15;20(16):4413
Abstract
Purpose: The antitumor activity of chimeric antigen receptor (CAR)-redirected CTLs should be enhanced if it were possible to increase their proliferation and function after adoptive transfer without concomitantly increasing the proliferation and function of regulatory T cells (Treg). Here, we explored whether the lack of IL-7Rα in Treg can be exploited by the targeted manipulation of the interleukin-7 (IL-7) cytokine-cytokine receptor axis in CAR-engrafted Epstein-Barr Virus-specific CTLs (EBV-CTLs) to selectively augment their growth and antitumor activity even in the presence of Treg.
Experimental design: We generated a bicistronic retroviral vector encoding a GD2-specific CAR and the IL-7Rα subunit, expressed the genes in EBV-CTLs, and assessed their capacity to control tumor growth in the presence of Treg in vitro and in vivo when exposed to either interleukin-2 (IL-2) or IL-7 in a neuroblastoma xenograft.
Results: We found that IL-7, in sharp contrast with IL-2, supports the proliferation and antitumor activity of IL-7Rα.CAR-GD2(+) EBV-CTLs both in vitro and in vivo even in the presence of fully functional Treg.
Conclusions: IL-7 selectively favors the survival, proliferation, and effector function of IL-7Rα-transgenic/CAR-redirected EBV-CTLs in the presence of Treg both in vitro and in vivo. Thus, IL-7 can have a significant impact in sustaining expansion and persistence of adoptively CAR-redirected CTLs.
Conflict of interest statement
Competing Financial Interest
The Center for Cell and Gene Therapy has a research collaboration with Celgene and bluebird bio. All authors reviewed the manuscript and approved the final version of the manuscript.
Figures
Figure 1. EBV-CTLs are effectively transduced with the bicistronic vector encoding both the IL-7Rα and the CAR-GD2
Panel A illustrates the schema of the bicistronic γ-retroviral vector encoding the IL-7Rα and GD2-specific CAR linked through a 2A (TAV) sequence. Panel B shows the expression of CAR-GD2 (top histogram) and IL-7Rα (bottom histogram) evaluated by FACS analysis day 7 after transduction. The dotted line indicates control EBV-CTLs while the bold line indicates the transduced EBV-CTLs. The graph represents mean ± SD of 5 donors. Panel C illustrates IL-7Rα expression in four IL-7Rα.CAR-GD2+ EBV-CTLs generated (upper panels) and STAT5 phosphorylation (lower panels) in the absence of cytokines (thin black line), in response to IL-2 (dotted line) or IL-7 (black bold line). Panel D shows the progressive enrichment in cells expressing the two transgenes IL-7Rα and CAR-GD2 when IL-7Rα.CAR-GD2+ EBV-CTLs were expanded in the presence of IL-7. S3, S4, S5 and S6 indicate the transgene expression detected week 3 (S3), week 4 (S4), week 5 (S5) and week 6 (S6), respectively after transduction. Graph represents mean ± SEM of 4 different EBV-CTL lines.
Figure 2. IL-7 supports the proliferation and effector function of IL-7Rα.CAR-GD2+ EBV-CTLs
Panel A shows a representative CFSE-based proliferation assay of control and IL-7Rα.CAR-GD2+ EBV-CTLs. Control and IL-7Rα.CAR-GD2+ EBV-CTLs were activated in the presence of autologous irradiated LCLs and either IL-2 or IL-7. CFSE dilution was evaluated on day 7 using FACS analysis. The graph represents mean ± SD of 5 independent experiments. Panel B illustrates a representative co-culture experiment in which control and IL-7Rα.CAR-GD2+ EBV-CTLs were co-cultured with CHLA-255 GFP-tagged tumor cells (at ratio 1:2) in the presence of IL-2 or IL-7. Residual tumor cells were enumerated by flow cytometry on day 7 of culture. The graph shows mean ± SD of 5 independent experiments. *p<0.001.
Figure 3. Ex vivo expanded Tregs do not respond to IL-7
Panel A shows a CFSE-based assay to illustrate the inhibitory activity of ex vivo expanded Tregs. PBMCs labeled with CFSE were activated in the absence (left histograms) or in the presence of Tregs (right histograms) at a ratio of 1:1. CFSE dilution was measured on day 7 of culture by flow cytometry. The graph represents mean ± SEM of 6 independent experiments. Panel B illustrates the expression of IL-7Rα in ex vivo expanded Tregs in a representative experiment. The plot on the left shows the isotype control, while the plot on the right the IL-7Rα profile. * p< 0.001. Panel C shows the phosphorylation of STAT5 in Tregs not stimulated (dotted lines) or stimulated with IL-2 (left panel) or IL-7 (right panel). Panel D illustrates the proliferative response of Tregs exposed to IL-2 or IL-7. Tregs were labeled with CFSE and stimulated in the presence of IL-2 (left panel) or IL-7 (right panel). CFSE dilution was evaluated on day 7 by flow cytometry. The solid and dotted lines represent the CFSE dilution of Tregs stimulated with or without cytokines, respectively.
Figure 4. IL-7, unlike IL-2, supports in vitro the proliferation and function of IL-7Rα.CAR-GD2+ EBV-CTLs in the presence of Tregs
Panel A. IL-7Rα.CAR-GD2+ EBV-CTLs were co-cultured with CHLA-255 GFP-tagged cells (ratio 1:2) in the presence of IL-2 or IL-7, with or without Tregs. The percentage of residual tumor cells was measured by flow cytometry on day 7 of culture. The plots on the left show a representative experiment, while the graph on the right summarizes mean ± SD of 5 independent experiments. Panel B. IL-7Rα.CAR-GD2+ EBV-CTLs were labeled with CFSE and activated with autologous LCLs in the presence of IL-2 (upper plots) or IL-7 (lower plots) with or without Tregs. CFSE dilution was measured at day 7 of culture by flow cytometry. The plots on the left show a representative experiment, while the graph represents mean ± SD of 5 independent experiments. * p<0.01; ** p=0.005
Figure 5. IL-7, but not IL-2, supports in vivo anti-tumor activity of IL-7Rα.CAR-GD2+ EBV-CTLs in the presence of Tregs
NSG mice engrafted i.p. with CHLA-255 cells tagged with FFLuc were infused with IL-7Rα.CAR-GD2+ EBV-CTLs and received IL-2 ± Tregs or IL-7 ± Tregs. Tumor growth was monitored using an in vivo imaging system (Xenogen – IVIS imaging system). A group of mice received control EBV-CTLs or tumor cells only (Control). Panel A shows the images of different groups of mice. Panel B illustrates the mean ± SD of photons for 8 mice/group in two independent experiments. * p<0.05.
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