Research ArticleImmunotherapy

IL-2Rα mediates temporal regulation of IL-2 signaling and enhances immunotherapy

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Science Translational Medicine  28 Oct 2015:
Vol. 7, Issue 311, pp. 311ra170
DOI: 10.1126/scitranslmed.aac8155
  • Fig. 1. IL-2/mAb but not IL-15/sIL-15Rα complexes induce potent effector T cell responses in tumor-bearing mice.

    (A) Treatment scheme for B6 mice injected subcutaneously with B16 melanoma tumor cells 7 days before the adoptive transfer of 3 × 106 pmel-1 Tc1 cells. Mice were then treated with human IL-2 (hIL-2)/mAb (clone 5355) or hIL-15/sIL-15Rα complexes. (B) Tumor volume from (A) (n = 9 per group); each line represents one mouse. On the basis of a log-rank test and time to sacrifice (at 400 mm2) for analysis, mice treated with IL-2/mAb complexes had significantly improved outcomes versus each other condition (*P < 0.001 for each comparison). The average tumor areas when treatment was initiated ranged from 15 to 20 mm2 between the four groups. (C) Frequency of donor Tc1 cells in the blood of mice (n = 4 per group) treated as in (A) but in the absence of tumor. Each point represents the average, and bars indicate SE. (D) Frequency of donor OT-I Tc1 cells in the blood of mice (n = 5 per group) treated with mouse IL-2 (mIL-2)/mAbCD122 (clone S4B6) or mIL-2/mAbCD25 (clone 1A12). Each point represents the average, and bars indicate SE. (E) Frequency of donor polyclonal T cells in the blood of mice (n = 5 per group) treated with hIL-2/mAb (clone 5355) complexes or vehicle alone. Each point represents the average, and bars indicate SE. **P < 0.001, significant difference between indicated and other conditions (C to E). Random effects linear regression was used for modeling data and calculating P values comparing conditions. All results are representative of at least two independent experiments.

  • Fig. 2. IL-2Rα mediates sustained signaling in effector CD8+ T cells after withdrawal of IL-2.

    (A) Diagram of the standard cytokine assay in which effector cells are assayed after incubation with titrated cytokine. (B and C) Levels of phosphorylated STAT5 (pSTAT5) in Tc1 and Tc0 cells that were cultured with increasing amounts of mIL-2 or mIL-15 for 1 hour. (D) As in (B), except Tc1 cells were incubated as indicated for up to 2 hours with cytokine (200 ng/ml) and assayed for pSTAT5. (E) Diagram of the cytokine pulse assay in which effector cells are incubated with saturating amounts of cytokine (200 ng/ml). Cells are then washed thoroughly, recultured at 37°C without additional cytokine, and assayed for pSTAT5. (F) Levels of pSTAT5 in Tc1 cells that were pulsed with mIL-2 with or without anti–IL-2Rα mAb (PC61 clone) for 1 hour, washed, and recultured at 37°C for the times indicated. (G and H) Levels of pSTAT5 in polyclonal effector T cells from wild-type (WT) (IL-2Rα+/+) or IL-2Rα+/− mice that were pulsed for 1 hour with mIL-2 or mIL-15 and assayed as described in (E). Except for (G) and (H), all effector cells were generated from pmel-1 mice. All results are representative of at least three independent experiments.

  • Fig. 3. IL-2Rα facilitates sustained IL-2 signaling through creation of an extracellular reservoir and recycling.

    (A) The presence of IL-2 on the surface of polyclonal T cells depends on IL-2Rα. Polyclonal effector CD8+ T cells were pulsed for 2 hours with or without mIL-2. Before (and during) the pulse, T cells were incubated with anti–IL-2Rα mAb (PC61). Cells were then washed and stained for surface IL-2. (B) Time course of surface IL-2 on polyclonal T cells after reculture at 37°C. (C) Levels of pSTAT5 in Tc1 cells that were pulsed with IL-2, washed, and recultured at 37°C with or without anti–IL-2 mAb (clone S4B6 or 1A12). (D) Recycling of IL-2 on effector T cells. Pmel-1 Tc1 cells were incubated with hIL-2 or mIL-2 at 37°C for 2 hours. As indicated, cells were then acid-washed and recultured at 37°C for 90 min in the presence of anti–hIL-2 mAb conjugated to Alexa 647. Cells were then washed, fixed, and assayed by flow cytometry. (E) Recycling of IL-2 on pulsed cells while mixed with nonpulsed cells. Pmel-1 Tc1 cells were pulsed with hIL-2 at either 4°C or 37°C for 2 hours and then acid-washed. Cells were then mixed with nonpulsed carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled Tc1 cells. The mixed cells were recultured at 37°C for 45 min in the presence of anti–hIL-2 mAb conjugated to Alexa 647. Cells were then washed, fixed, and assayed by flow cytometry. (F) Internalized IL-2 leads to sustained pSTAT5 signaling. Pmel-1 Tc1 cells were pulsed with hIL-2 at either 4°C or 37°C for 2 hours and then acid-washed. Cells were then recultured at 37°C and assayed for pSTAT5. (G) Subcellular localization of hIL-2 and IL-2Rα. Pmel-1 Tc1 cells were pulsed with hIL-2 (or medium alone) for 1 hour at 37°C and stained for hIL-2 and IL-2Rα. Cells were then imaged by confocal microscopy. (H) Subcellular localization of hIL-2 and Rab5. As in (G), except cells were stained for Rab5. Results are representative of three independent experiments.

  • Fig. 4. IL-2Rα on donor T cells is critical for persistence in lymphoreplete but not lymphodepleted hosts.

    (A) WT and IL-2Rα+/− effector CD8+ T cells show similar persistence with or without IL-2 therapy. Effector T cells from WT and IL-2Rα+/− mice were activated, mixed, and injected into recipient mice (n = 3 to 5 per group). Mice received injections of hIL-2/mAb (clone 5355) complexes, hIL-15/sIL-15Rα complexes, or vehicle alone. The proportion of IL-2Rα+/− T cells among all donor T cells in the spleen was determined before and after transfer. Each triangle represents one mouse, and bars indicate the mean. (B) Total number of donor T cells per spleen for the experiment shown in (A). Bars indicate the mean. **P < 0.001, significant difference between indicated conditions. (C) Total number of donor T cells in the spleen for the experiment in (A). Mice (n = 5 per group) were given total body irradiation (600 rads) 1 day before adoptive transfer of 1 × 107 Tc1 (pmel-1) cells and then treated with hIL-2/mAb (clone 5355) or hIL-15/sIL-15Rα complexes. The frequency of donor T cells in the blood of mice was determined at the indicated time points. Each point represents the average, and bars indicate SE. **P < 0.001, significant difference between control and indicated conditions. All results are representative of two independent experiments.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/311/311ra170/DC1

    Fig. S1. IL-2/mAb complexes selectively enhance the persistence of donor T cells.

    Fig. S2. In the absence of donor T cells, hIL-2/mAb and IL-15/sIL-15Rα complexes mediate comparable antitumor immunity.

    Fig. S3. Treatment with IL-2/mAb, IL-2/mAbCD25, and IL-15/sIL-15Rα complexes induces differential expansion of CD8+ memory-phenotype T cells, NK cells, and T regulatory cells.

    Fig. S4. Tc1 but not Tc0 effector CD8+ T cells show preferential responsiveness to IL-2/mAb complexes.

    Fig. S5. Blockade of IL-2Rα has minimal impact on Tc1 cells in response to titrated IL-2 or IL-15.

    Fig. S6. Tc1 effector CD8+ T cells exhibit comparable functional sensitivity to IL-2 and IL-15 in vitro.

    Fig. S7. Tc1 effector CD8+ T cells pulsed with IL-2 mediate sustained cytokine signaling.

    Fig. S8. IL-2–mediated sustained cytokine signaling is IL-2Rα–dependent in 11 independent experiments.

    Fig. S9. Tc1 effector CD8+ T cells pulsed with IL-2 exhibit IL-2Rα–dependent proliferation after cytokine withdrawal.

    Fig. S10. Human IL-2 mediates sustained cytokine signaling on mouse Tc1 effector CD8+ T cells.

    Fig. S11. Human effector CD8+ T cells pulsed with IL-2 mediate sustained IL-2Rα–dependent signaling.

    Fig. S12. Human IL-2/mAb (clone 5355), but not mouse IL-2/mAbCD122 (clone S4B6), complexes are permissive to IL-2Rα–dependent sustained signaling in vitro.

    Fig. S13. Antibodies for mouse and human IL-2 are species-specific.

    Fig. S14. Detection of hIL-2 by confocal microscopy is species-specific and dependent on pulsing cells with cytokine at 37°C.

    Fig. S15. Detection of mIL-2 by confocal microscopy.

    Fig. S16. Colocalization of hIL-2 with EEA1 and LAMP-1 by confocal microscopy.

    Fig. S17. In lymphodepleted mice, IL-15/sIL-15Rα and hIL-2/mAb mediate comparable engraftment of Tc1 effector CD8+ T cells.

    Table S1. Raw data for main and supplementary figures.

  • Supplementary Material for:

    IL-2Rα mediates temporal regulation of IL-2 signaling and enhances immunotherapy

    Ee W. Su, Caitlin J. Moore, Samantha Suriano, Christopher Bryce Johnson, Neizel Songalia, Alicia Patterson, Daniel J. Neitzke, Kristina Andrijauskaite, Elizabeth Garrett-Mayer, Shikhar Mehrotra, Chrystal M. Paulos, Andrew L. Doedens, Ananda W. Goldrath, Zihai Li, David J. Cole, Mark P. Rubinstein*

    *Corresponding author. E-mail: markrubinstein{at}musc.edu

    Published 28 October 2015, Sci. Transl. Med. 7, 311ra170 (2015)
    DOI: 10.1126/scitranslmed.aac8155

    This PDF file includes:

    • Fig. S1. IL-2/mAb complexes selectively enhance the persistence of donor T cells.
    • Fig. S2. In the absence of donor T cells, hIL-2/mAb and IL-15/sIL-15Rα complexes mediate comparable antitumor immunity.
    • Fig. S3. Treatment with IL-2/mAb, IL-2/mAbCD25, and IL-15/sIL-15Rα complexes induces differential expansion of CD8+ memory-phenotype T cells, NK cells, and T regulatory cells.
    • Fig. S4. Tc1 but not Tc0 effector CD8+ T cells show preferential responsiveness to IL-2/mAb complexes.
    • Fig. S5. Blockade of IL-2Rα has minimal impact on Tc1 cells in response to titrated IL-2 or IL-15.
    • Fig. S6. Tc1 effector CD8+ T cells exhibit comparable functional sensitivity to IL-2 and IL-15 in vitro.
    • Fig. S7. Tc1 effector CD8+ T cells pulsed with IL-2 mediate sustained cytokine signaling.
    • Fig. S8. IL-2–mediated sustained cytokine signaling is IL-2Rα–dependent in 11 independent experiments.
    • Fig. S9. Tc1 effector CD8+ T cells pulsed with IL-2 exhibit IL-2Rα–dependent proliferation after cytokine withdrawal.
    • Fig. S10. Human IL-2 mediates sustained cytokine signaling on mouse Tc1 effector CD8+ T cells.
    • Fig. S11. Human effector CD8+ T cells pulsed with IL-2 mediate sustained IL-2Rα–dependent signaling.
    • Fig. S12. Human IL-2/mAb (clone 5355), but not mouse IL-2/mAbCD122 (clone S4B6), complexes are permissive to IL-2Rα–dependent sustainedsignaling in vitro.
    • Fig. S13. Antibodies for mouse and human IL-2 are species-specific.
    • Fig. S14. Detection of hIL-2 by confocal microscopy is species-specific and dependent on pulsing cells with cytokine at 37°C.
    • Fig. S15. Detection of mIL-2 by confocal microscopy.
    • Fig. S16. Colocalization of hIL-2 with EEA1 and LAMP-1 by confocal microscopy.
    • Fig. S17. In lymphodepleted mice, IL-15/sIL-15Rα and hIL-2/mAb mediate comparable engraftment of Tc1 effector CD8+ T cells.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data for main and supplementary figures.

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