1. Role of trans-cellular IL-15 presentation in the activation of NK cell–mediated killing, which leads to enhanced tumor immunosurveillance
by Hisataka Kobayashi, Sigrid Dubois, Noriko Sato, Helen Sabzevari, Yoshio Sakai, Thomas A. Waldmann, and Yutaka Tagaya
Blood
Volume 105(2):
January 15, 2005
©2005 by American Society of Hematology

2. (A) Survival of IL-15 Tg mice following injection with MC38 cells.
(A) Survival of IL-15 Tg mice following injection with MC38 cells. (A) Survival curve describing MC-38–injected IL-15 Tg mice. Two million MC38 cells,23 a murine colon carcinoma cell line, were injected via the tail vain into C57BL/6 mice or IL-15 Tg mice11 (n = 7), and their survival was monitored over 8 months. (B) MC38 cells formed lung metastases in the IL-15 Tg mouse following the NK-cell depletion. Anti-asialo GM1 antibody was injected into IL-15 Tg mice every 3 days (from day -9 to day 45) to deplete NK cells, which were occasionally monitored by analyzing the peripheral blood cells from mice by flow cytometry. Micro-MR (magnetic resonance) images were collected using a 1.5-Tesla superconductive magnet unit (Signa LX, WI). The arrows indicate the location of metastatic MC38 tumor masses in the lungs of an NK-depleted IL-15 Tg mouse.
Hisataka Kobayashi et al. Blood 2005;105:
©2005 by American Society of Hematology

3. Flow cytometry analyses of CD8 T cells from mice challenged by parental MC38 and its IL-15Rα–transfected clone.
Flow cytometry analyses of CD8 T cells from mice challenged by parental MC38 and its IL-15Rα–transfected clone. Lymphocytes were separated (day 1, day 4, day 7, and day 14 after injection) from spleens of wild-type mice challenged by MC38 cells or their IL-15Ra transfected clone (mIL-15Rα+ MC38) and were analyzed for their expression of surface markers (CD8 versus CD69 or CD8 versus Asialo GM1) by flow cytometry.
Hisataka Kobayashi et al. Blood 2005;105:
©2005 by American Society of Hematology

4. Evidence that IL-15 does not directly modulate the proliferation of MC38 cells.
Evidence that IL-15 does not directly modulate the proliferation of MC38 cells. (A) No expression of IL-15Rα or IL-2/IL-15Rβ in MC38 cells. Expression of these molecules in MC38 cells was assessed by RT-PCR. Lanes: DNA marker (1-kb ladder; Invitrogen, Carlsbad, CA), MC38, CTLL2 (from left to right). Marker band size (from top to bottom) was 517/506 (doublet), 396, 344, 298, 220/201 (doublet). Due to the presence of multiple splice variants of the IL-15Rα transcript expressed by CTLL2 cells, at least 3 amplicons were detectable. In contrast, no band was amplified from MC38 mRNA. Amplification of the β-actin fragment from cDNA of MC38 or CTLL2 transcripts was confirmed as a loading control. Primer sets were IL-15Rα: SN, 5′-ATGGCCTCGCCGCAGCTCCGG-3′, AS, 5′-TAGAGATGGCCACTTTCGTCATTTTGG-3′; IL-2/IL-15Rβ: SN, 5′-CTT GGA GAT GCT GAT CCC TAG TAC-3′, AS, 5′-GGG AGC TCA GCT GGG AGA AGA ACT-3′. (B) Proliferative responses of MC38 cells and their IL-15Rα–transfected clones to IL-15. No significant proliferative responses were observed in the presence or absence of IL-15 with parental MC38 cells and their IL-15Rα–transfected clones.
Hisataka Kobayashi et al. Blood 2005;105:
©2005 by American Society of Hematology

5. Lack of IL-15Rα expression on NK cells.
Lack of IL-15Rα expression on NK cells. (A) Flow cytometry of IL-15Rα expression on ex vivo NK cells. NK cells were expanded from bone marrow precursors from C57BL/6 mice in the presence of 70 nM IL-15 for 10 days. The cells were stripped of any bound cytokine by treating briefly in acetic acid–phosphate-buffered saline (PBS; pH 3)22 and then stained using anti–IL-15Rα polyclonal antisera (Santa Cruz), followed by a staining with phycoerythrin (PE) anti–goat IgG (Sigma, St Louis, MO). As a positive control, dendritic cells were expanded from bone marrow precursors in the presence of 1 nM GM-CSF (PeproTech) and were stimulated by lipopolysaccharide (LPS; 1μgmL-1) and interferon-γ (10 nM) for 24 hours to augment their expression of IL-15Rα. (B) Lack of proliferative responses of NK cells to low doses of IL-15. NK cells expanded by IL-15 as described in panel A (> 90% were NK1.1+, data not shown) were incubated with the indicated concentrations of IL-15 for 20 hours and pulsed with 1 μCi (0.037 MBq) [3H]-thymidine for 4 hours to measure their DNA synthesis. More than 10 nM IL-15 was needed to induce meaningful proliferative responses in these NK cells, although less than 100 pM IL-2 induced an almost maximum proliferative response from these cells. (C) Induction of NK proliferation by the IL-15trans-presentation. PT-18 cells24,25 were transfected with a human IL-15Rα expression construct, and clones expressing this molecule were selected using G418. The IL-15Rα+ PT-18 cells were first irradiated by 50 Gy of x-ray on the previous day of the experiment and then incubated with indicated concentrations of soluble IL-15 for 60 minutes at 37°C. After an extensive wash with PBS, of these IL-15–bearing PT-18 cells were added to equal numbers of NK cells and incubated for 20 hours, followed by a pulse with 1μCi (0.037 MBq)3[H] thymidine for 4 hours. A similar experiment was undertaken by incubating IL-15Rα+ MC38 cells with effector NK cells, but those MC38 cells were lysed almost instantaneously, so that we could not observe NK cell proliferation (data not shown). (D) Strong induction of NK-cell mediated killing of IL-15Rα+ MC38 cells. IL-15Rα+ or IL-15Rα- (parental) MC38 cells were incubated with 0.5 nM IL-15 for 2 hours concurrent with the 51Cr labeling prior to the cytotoxicity assay. The cells were then extensively washed in PBS to remove any free IL-15. Twenty-five thousand labeled (with IL-15 and 51Cr) cells were mixed with various numbers of effector NK cells for 6 hours, and the cell-bound and released radioactivity were measured.
Hisataka Kobayashi et al. Blood 2005;105:
©2005 by American Society of Hematology

6. Survival curves of wt or NK-depleted C57BL/6 mice injected with parental and IL-15Rα-transfected MC38 cells.
Survival curves of wt or NK-depleted C57BL/6 mice injected with parental and IL-15Rα-transfected MC38 cells. (A) Expression levels of IL-15Rα on MC38 transfectants. Flow cytometry data showing the expression levels of mouse IL-15Rα on MC38 transfectants. (Left) Parental MC38/isotype control staining of all MC38 clones; (middle) MC38-mouse IL-15Rα (moderate); (right) MC38-mouse IL-15Rα (high). (B) Survival of wt mice challenged by IL-15Rα–transfected MC38 cells. Two million parental or IL-15Rα (moderate or high expression)–transfected MC38cells were injected via the tail vain into C57BL/6 mice (n = 11 for parental MC38 injected mice, and n = 6 for mice injected with IL-15Rα+[moderate] or IL-15Rα+MC38 cells, n = 4 for NK-depleted wt mice injected with IL-15Rα+MC38), and their survival was monitored for 60 days. The survival of mice injected with IL-15Rα+ (high) MC38 cells was significantly longer (P < .001) than that of mice injected with the parental MC38 cells. The left panel shows the levels of IL-15Rα expression on these MC38 transfectants as assessed by flow cytometry. When the IL-15Rα+ MC 38 cells were injected into mice that had been depleted of either NK cells or CD8 T cells, only NK-depleted mice allowed the development of IL-15Rα+ (high) fatal MC38 cell tumors.
Hisataka Kobayashi et al. Blood 2005;105:
©2005 by American Society of Hematology

7. No pulmonary metastasis in C57BL/6 mice injected with IL-15Rα–transfected MC38 cells.
No pulmonary metastasis in C57BL/6 mice injected with IL-15Rα–transfected MC38 cells. Micro-MR images of lungs of C57/BL6 mice injected with parental or IL-15Rα–transfected (high or moderate expression) MC38 cells are shown. Parental MC38 cells formed fatal pulmonary metastases and almost filled the lungs of all of the injected mice (n = 11) (left). In contrast, no visible metastatic masses were observed in lungs of all mice (n = 6) injected with IL-15Rα+MC38 (images were taken at 28 days after injection; T indicates tumor).
Hisataka Kobayashi et al. Blood 2005;105:
©2005 by American Society of Hematology

8. A schematic representation of the MC38-mediatedtrans-activation of NK cells.
A schematic representation of the MC38-mediatedtrans-activation of NK cells. In IL-15 Tg mice, the constitutive presence of IL-15 activates NK (and CTL) cells to enhance cytokine-induced lysis of MC38 cells, resulting in the efficient rejection of these tumor cells (top). If the MC38 cells express IL-15Rα on their surface, the endogenously produced IL-15 in mice can be captured and be presented intrans to NK/CTL cells in the neighborhood (left). This scenario explains the reduced growth or lack of growth of MC38/IL-15Rα transfectants in normal mice. Hypothetically, it is plausible that administration of dendritic cells that have been engineered or activated to express IL-15Rα may activate NK/CTLs, and thus leads to activation of immune-surveillance mechanism against tumor cell development in vivo.
Hisataka Kobayashi et al. Blood 2005;105:
©2005 by American Society of Hematology