Natural killer (NK) cells inhibit early stages of tumor formation, recurrence, and metastasis. Here, we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to interleukin 15 (IL-15) released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphologic feature of being densely loaded with periodic acid-Schiff–positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL-15 receptor α on cancer cells was needed to efficiently induce granulated NK cells, and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL-15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors. Cancer Res; 72(8); 1964–74. ©2012 AACR.

Natural killer (NK) cells were originally discovered as peripheral blood lymphocytes that killed certain cancer cells in vitro (1–4). In vivo, NK cells inhibited primary tumor formation, tumor recurrence, and metastasis, but usually only at incipient stages [for review see (5)]. In patients, intratumoral NK cells may reduce metastatic seeding thereby leading to longer survival (6). Transformed cells often have decreased MHC class I surface expression or have induced expression of ligands for activating receptors on NK cells (7, 8), and thus are targets of NK cells and their receptors. However, the lack of studies showing NK cells eliminating established solid tumors has cast doubts on the clinical usefulness of NK cells in the therapy of advanced solid tumors.

NK cells can be activated with high doses of interleukin (IL) 2 to proliferate and kill a broad panel of cancer targets and this is being exploited clinically in selected groups of cancer patients (9–11); however, adoptive transfer of NK cells activated in vitro with IL-2 failed to cause tumor regression in melanoma patients (12). Interleukin 15 (IL-15), discovered in 1994 (13), also activates human NK cells to kill cancer cells by a perforin-dependent mechanism (14, 15). In mice, IL-15 is required for the development and survival of NK cells, and these IL-15 effects depend on expression of the IL-15 receptor α (IL-15Rα) on cells other than the NK cell (16). Similar to IL-2-induced NK cells, IL-15-induced cells also can prevent cancer development and metastasis (17–19). In addition to activating NK cells, IL-15 also activates T cells and thereby induces certain T cell–mediated anticancer responses [for review see (20)]. Nevertheless, whether IL-15 was expressed as a transgene by the cancer cells (21–24) or given as treatment (25–28), anticancer effects remained limited to the prevention of cancer development and metastasis or reduced tumor growth. Therefore, it certainly has remained unclear whether NK cells, when properly activated by IL-15, could have an effect on solid tumors once grown to clinically relevant sizes. Here, we show that, even in the complete absence of T-cell immunity, NK cells can be induced to eradicate large solid tumors.

Mice

Perforin-deficient (Jackson Laboratory) and IL-15Rα-deficient mice (provided by A. Ma, University of California, San Francisco, CA) were crossed to Rag1−/− mice (Jackson Laboratories) and Rag2−/−γc−/− (Taconic), C3H/HeN (Charles River Laboratories), and IL-15-transgenic mice [DQ8-Dd-IL-15, (29)] were maintained under specific pathogen-free conditions at the University of Chicago facilities. The Institutional Animal Care and Use Committee (IACUC) at the University of Chicago approved all animal experiments.

Cells

All cells were cultured in Dulbecco's Modified Eagle's Medium, 5% fetal calf serum. P. Ohashi (University of Toronto, Canada) with permission of H. Hengartner (University Hospital, Switzerland) provided the MC57G fibrosarcoma cell line. 8215 is an MCA-induced cancer line generated in an IL-15Rα-deficient mouse (125 μg MCA). 9604 is a UV-induced cancer line of a Kb–/−Db–/− mouse (ref. 30; 3 UV exposures per week for half a year; mice provided by A. Chervonsky, The University of Chicago, IL). Both are primary lines that have never been passaged in a mouse. The spontaneous line AG104A of C3H/HeN origin was described (31). All cell lines were passaged only a few times (less than 1 month) after thawing of working batch freezings, which were generated shortly after obtaining cell lines, to reduce total number of passages to a minimum. Cell lines were authenticated by morphology and growth rate and were Mycoplasma free.

To transduce the 4 cell lines, we used MFG-IL-15HS-IRES-ECFP, MFG-IL-15Rα, or the empty amphotropic virus. Cultures were flow sorted to enrich for positive cells (see Supplementary Materials and Methods for plasmids and details).

ELISA and flow cytometry

M-IL-15 or M-control cells were plated at 4 × 104 cells/mL in a T25 culture flask. Forty-eight–hour supernatants were assayed with the mouse IL-15 Ready-SET-Go! Kit (eBioscience).

Single-cell suspensions from mouse tissue or trypsinized MC57 cells were first blocked with anti-CD16/anti-CD32 antibody then stained with conjugated antibodies (see Supplementary Materials and Methods for antibodies). Flow cytometry data were collected on a FACSCalibur or LSRII, and sorting was carried out on a FACS Aria (all BD Biosciences) and analyzed with FlowJo software (Treestar Inc.).

Tumor challenges, measurements, and reisolations

Mice were injected s.c. with 2.5 × 106 to 5 × 106 cells in 100 μL of phosphate-buffered saline, unless otherwise indicated. Tumor volume was measured every 2 to 5 days with calipers along 3 orthogonal axes and calculated by abc/2. Statistical significance of tumor eradication was determined by the Fisher exact probability test.

Anti-IL-15 (clone M96, provided by Amgen, Seattle, WA) was injected once at 45 μg before cancer cell inoculation and 95 μg weekly thereafter. To deplete NK cells, mice received 100 μg anti-NK1.1 (clone PK136) every 3 to 4 days; NK cell depletion was confirmed by flow cytometry of peripheral blood samples with anti-NK1.1 and anti-CD122. For bone marrow transfers, 3 × 107 cells were injected into each recipient mouse. Details are found in Supplementary Materials and Methods.

Microscopy

Images were taken on the Leitz Laborlux D microscope with a Leitz 63×/1.4 objective and Zeiss 20×/0.25 and 6.3×/0.16 objectives using the QImaging Retiga 2000R camera and QCapture software. Macroscopic images were taken with the Nikon Coolpix 6000 camera.

Histology and immunohistochemistry

Tissue was frozen in ornithine carbamyl transferase with isopentane in dry ice. Sections (4 μm) were cut, dried, mounted, and fixed in acetone. Before staining, endogenous peroxidase was blocked (Dako dual endogenous enzyme block). Anti-NK1.1-FITC (FITC, fluorescein isothiocyanate) and anti-granzyme B-FITC were used at 1 μg/mL and 2.5 μg/mL, respectively. Anti-FITC (Pierce) was used as recommended by the manufacturers. Horseradish peroxidase was developed with DAB (Dako) for 10 minutes. Slides were counterstained with hematoxylin, dehydrated in alcohol, and mounted in mounting medium (Sakura Finetek). Details are found in Supplementary Materials and Methods.

IL-15-secreting cancer cells form large tumors in mice lacking NK and T cells

MC57 fibrosarcoma cells were transduced with the murine IL-15 gene preceded by the IL-2 leader sequence; levels of ECFP fluorescence were used for selecting cells secreting large amounts of IL-15 (M-IL-15; Fig. 1A, Supplementary Fig. S1). As control, MC57 cells were transduced with the empty ECFP vector (M-control). M-IL-15 cells did not form tumors in Rag1−/− mice (Fig. 1B, left). However, M-IL-15 and M-control cells grew at similar rates and formed large tumors in Rag2−/−γc−/− mice (Fig. 1B, middle), which are unable to respond to IL-15. Tumors also grew in Rag1−/− mice while these were given a neutralizing anti-IL-15 antibody (Fig. 1B, right). Figure 1C shows that peripheral NK cells were absent in both types of mice in which M-IL-15 grew.

Large solid tumors regress after anti-IL-15 application is stopped

Surprisingly, large M-IL-15 tumors (>1 cm in diameter or >500 mm3 in volume, Fig. 1B) were rejected completely after withdrawal of the anti-IL-15 antibody (Fig. 1D, left; Supplementary Table SI). By contrast, most M-control tumors continued to grow at the same rate. Regression of large IL-15-secreting tumors did not stop the growth of contralateral M-control tumors suggesting that the effect of IL-15 is largely restricted to the local microenvironment of the IL-15-secreting tumor (Fig. 1D, middle).

Densely granulated cells found in the regressing tumor

M-control tumors growing in anti-IL-15–treated Rag1−/− mice were densely packed with viable cancer cells, many in mitosis; only the centers of these M-control tumors were necrotic (Fig. 2A). Cross-sections of regressing M-IL-15 tumors 12 days after final injection of anti-IL-15 showed mostly necrotic tumor tissue (Fig. 2B). The few partially viable areas were packed with densely granulated cells and it was difficult to identify any remaining viable cancer cells. The granules stained bright magenta with periodic acid-Schiff (PAS) and were resistant to pretreatment with diastase. Some of these granulated cells in the regressing tumors were undergoing mitosis suggesting that the granulated cells represented infiltrating host cells rather than thanatosomes derived of dying cancer cells (Fig. 2B right, arrow; Fig. 5, left middle; ref. 32).

Transfer of Rag1−/− bone marrow cells causes regression of M-IL-15 tumors in Rag2−/−γc−/− mice

Rag2−/−γc−/− mice reconstituted with Rag1−/− bone marrow were resistant to outgrowth of M-IL-15 cancer cells (Fig. 3A). Next, we treated Rag2−/−γc−/− mice bearing M-control or M-IL-15 tumors with bone marrow from Rag1−/− mice 2 weeks after cancer cell inoculation (tumor volumes between 200 and 500 mm3). Although the bone marrow transfer had no apparent influence on the growth of the M-control tumors, the bone marrow–derived cells eradicated M-IL-15 tumors in 50% of the mice (Fig. 3B), suggesting that γc+ bone marrow–derived cells can reject established tumors. As observed in M-IL-15 tumors after anti-IL-15 treatment was stopped, numerous cells filled with PAS+ diastase-resistant granules were found in the tumors (Fig. 3C). These cells were absent in the tumors before the bone marrow transfer. Adoptive transfers of spleen cells from Rag1−/− mice also had an effect on M-IL-15 but not M-control tumors, which was however not as strong as seen for bone marrow transfers (data not shown).

Densely granulated NK1.1+ cells are essential for tumor eradication

Immunohistochemical analyses showed that the majority of tumor-infiltrating cells in regressing tumors expressed NK1.1 and granzyme B (Fig. 4A). To confirm that the densely granulated cells found in the regressing tumors were tumor-infiltrating NK cells, we sorted NK1.1+ cells also expressing Dolichos biflorus agglutinin (DBA), a marker of densely granular uterine NK (uNK) cells, from M-IL-15 tumors and prepared cytospins. Although some of the granules seemed to have collapsed or been extruded during the isolation process, the reisolated and sorted NK1.1+DBA+ cells were packed with PAS+ granules (Fig. 4B). To determine whether NK cells were indeed required for the rejection of the M-IL-15 tumors, we depleted NK cells from anti–IL-15-treated Rag1−/− mice after withdrawal of the antibody. In accordance with our hypothesis, we found that in the absence of NK cells, tumors expressing IL-15 were not rejected (Fig. 4C, Supplementary Table SI). Flow cytometry revealed that the tumor-infiltrating NK cells expressed normal levels of NKp46, 2B4, and CD122 but reduced levels of DX5 and NKG2D, compared with splenic NK cells (Fig. 4D); differences in marker expression were not due to differences in sample preparation (data not shown). The infiltrating NK cells also had decreased CD27, an immature NK cell marker, and increased 4-1BB, CD11b, and TRAIL expression, characteristic of activated NK cells (33, 34). Despite the similarities to activated NK cells, we found no PAS+ NK cells in spleens from poly I:C activated wild-type mice (data not shown), nor in short-term cultures of NK cells activated with IL-15 (Supplementary Fig S2A). Also, mice with heavily infiltrated M-IL-15 tumors had only very few granulated NK cells in their spleens (Supplementary Fig S2B). Microscopic analysis from serial sacrifices showed that the NK cells took time to mature into granular PAS+ cells and could not be detected in tumors as early as 3 days post cancer cell injection but were present by day 12 (Supplementary Fig. S3).

An analysis of cytokines that may have synergized with IL-15 to promote the differentiation of densely granular NK cells was conducted. CD11b+ stromal cells in growing M-IL-15 tumors expressed IL-12 and IL-10, but no detectable IL18 and TGF-β (Supplementary Fig. S4). Cancer cells obtained from growing tumors or cultured in vitro did not express any of these cytokines at detectable levels (Supplementary Fig. S4 and data not shown, respectively).

Eradication depends on perforin released by infiltrating NK cells and destruction of cancer cells at the tumor margin

Given the prominent granules found in M-IL-15 tumors, we hypothesized that perforin was involved in tumor rejection. We evaluated the rejection of M-IL-15 tumors grown in Rag1−/−Prf1−/− mice with anti-IL-15 antibody after antibody treatment was stopped. Indeed, in the absence of perforin, we found M-IL-15 tumors grew with similar kinetics to viable M-control tumors (compare left with right of Fig. 1D; Supplementary Table SI). Surprisingly, the tumors growing in Rag1−/−Prf1−/− mice were mostly necrotic (Fig. 5, left; Supplementary Fig S3 for non–anti-IL-15–treated Rag1−/−Prf1−/− mice). Control tumors only displayed a small area of necrosis typical for fast growing tumors (Fig. 2A). The rim of M-IL-15 tumors in Rag1−/−Prf1−/− mice was heavily infiltrated with densely granulated proliferating NK cells intermingled with proliferating cancer cells.

We also analyzed the appearance of M-IL-15 tumors growing in Rag1−/− mice shortly after cessation of anti-IL-15 treatment (Fig. 5, right), when the tumor had not yet begun to shrink and no NK cells were detectable in the peripheral blood. The margins of the tumors lacked PAS+ cells but contained dividing cancer cells, while further inside the tumor, there was heavy infiltration of PAS+ cells and cancer cell destruction. The center remained vascularized, however, which contrasts to the center of the tumor in the Rag1−/−Prf1−/− mouse in which no tumor vessels were detected. This indicated that the anti-IL-15 antibody exerted its neutralizing activity mostly at the tumor margin, and that this effect was sufficient to allow tumor growth.

Stromal IL-15Rα expression is not required for temporary regression but is required for tumor eradication

Unlike most cytokines, IL-15 is presented in trans on IL-15Rα-expressing cells. MC57 cells express high levels of IL-15Rα (Fig. 6A) and thus can present IL-15 to NK cells. To determine whether IL-15Rα expression by cancer cells and not the stroma was sufficient for tumor rejection, we grew M-IL-15 cells in Rag1−/−IL-15Rα−/− mice. Although Rag1−/−IL-15Rα−/− mice are initially deficient in NK cells, M-IL-15 tumors decreased in size after cessation of anti-IL-15 treatment (Fig. 6B). Similarly, when Rag1−/−IL-15Rα−/− mice were injected with M-IL-15 cancer cells without being pretreated with anti-IL-15 antibody, tumors initially grew and then regressed almost completely. However, most tumors eventually relapsed, except for one (Fig. 6B, Supplementary Table S1). Analysis of Rag1−/−IL-15Rα−/− mice–bearing M-IL-15 tumors showed that NK cells can be found in the peripheral blood early but not later, when tumors began to relapse (Fig. 6C). Taken together, IL-15Rα expression seems to be required on stromal cells to achieve complete tumor eradication.

A mixture containing 5% of non–IL-15-secreting cancer cells never formed tumors (Supplementary Fig. S5A). With higher numbers of nonsecreting cancer cells (10%–20% of cancer cells), there was outgrowth of tumors, probably due to no sufficient IL-15 to be transpresented on IL-15Rα of nonsecreting cancer and stromal cells, resulting in the incapability of NK cells to kill. As shown by the selection of nonsecreting cells (Supplementary Fig S5B), all IL-15-secreting cells were specifically deleted.

Several cancer cell lines induce densely granulated NK cells

We transduced 3 other cancer cell lines to express IL-15 and/or IL-15Rα at high levels (Supplementary Fig. S6). Both cancer lines expressing IL-15 and Rα, C3H/HeN-derived AG104A-IL-15-Rα, and C57BL/6-derived 9604-IL-15-Rα formed viable tumors when injected in syngeneic mice pretreated with anti-IL-15 antibody. Histology of tumors from nontreated animals showed destruction of cancer cells and infiltration by densely granulated NK cells (Fig. 7A and B).

The IL-15Rα-deficient cancer line 8215 was transduced to express IL-15 only (Supplementary Fig. S6). This line also formed viable tumors in Rag1−/−Prf1−/− mice (Fig. 7C). In contrast to M-IL-15 tumors growing in Rag1−/−Prf1−/− mice, these tumors did not show extensive central necrosis and infiltration of only very few granular NK cells. Taken together, densely granular NK cells are induced in different cancer lines expressing both high levels of IL-15Rα and of secreted IL-15. IL-15Rα is needed on cancer cells to efficiently induce densely granulated NK cells.

We show that IL-15-induced NK cells can eliminate large established solid tumors in a host completely devoid of T and B cells. This unprecedented effect was apparently caused by massive influx of a unique type of very densely granulated NK cells that resemble a NK subtype described in the mammalian uterus during pregnancy (uNK; ref. 35) that also depends on IL-15 (36). Unlike granulocytes, which are end stage cells, these heavily granulated NK cells were still clearly proliferating at the tumor site. Other mature NK cells can also undergo blastogenesis such as virally induced NK cells in the spleen (37) and uNK cells in the uterus (38). As described for mature uNK cells, the granules of the IL-15-induced NK cells in the tumor tissue were PAS-positive and diastase resistant and their cytoplasmic membrane stained with the lectin DBA, which binds to glycoconjugates containing N-acetyl-d-galactosamine. However, it may be inappropriate to draw further analogies between the densely granulated NK cells described here and uNK because the tumor-infiltrating IL-15-induced NK cells are a cytolytic population generated by manipulating the tumor microenvironment. Also, although perforin is required for the eradication of tumors by these granulated NK cells, uNK do not need perforin for successful pregnancies (39).

Transgenic mice overexpressing secreted IL-15 do not show densely granulated NK cells in their spleen, liver, or gut (data not shown), nor do short-term cultures of wild-type or IL-15-transgenic splenic NK cells stimulated with high-dose IL-15. The difference to IL-15-secreting tumors might be the level of IL-15 secretion and/or IL-15Rα expression, but also the presence of other cytokines. IL-12 was shown to increase the production of NK effector cytokines and IFN-γ while it suppressed target killing (40, 41). IL-10, in contrast, was recently shown to enhance cytolytic activity of NK cells (42). High local levels of IL-15 were, nevertheless, critical for the generation of densely granulated NK cells; cancer lines not expressing IL-15 induced only very few granulated cells (Supplementary Fig S2C). Furthermore, expression of IL-15Rα by cancer cells was needed for effective induction of PAS+ cells. Finally, infiltration of granulated NK cells required time. Taken together, a specific tumor milieu with high IL-15 and IL-15Rα expression and several days of maturation were needed for the differentiation of densely granulated NK cells with antitumor function.

We have several lines of experiments indicating that these IL-15-induced densely granulated NK cells were indeed the effectors eradicating the large tumors: (i) tumors formed in mice deficient in NK cells such as Rag2−/−γc−/− mice and Rag1−/− mice given anti-IL-15 antibody to deplete NK cells but not in Rag1−/− mice that contain NK cells; (ii) transfer of bone marrow or spleen cells from Rag1−/− mice into tumor-bearing Rag2−/−γc−/− or cessation of NK depletion with anti-IL-15 led to tumor rejection accompanied by tumor infiltration of these heavily granulated cells; (iii) cells isolated from the tumors had characteristic peripheral NK markers when stained by immunohistochemistry or analyzed by flow cytometry; (iv) infiltrating cells sorted for NK1.1 and DBA showed PAS+ granules in cytospins; (v) only IL-15-secreting but not contralateral nonsecreting control tumors were eradicated; (vi) the anti-NK1.1 antibody administration abolished rejection; (vii) densely granulated NK cells were completely absent from the tumor margin during systemic application of anti-IL-15 antibody.

Several methodologic differences to other studies expressing IL-15 in cancer cells could explain why such strong antitumor effects have not been described earlier. For example, earlier studies used human IL-15, which was not fused to any signaling peptide needed for effective secretion; comparison of ELISA data suggests up to 7,000-fold higher secretion of IL-15 (22). Furthermore, it is not clear whether the cancers expressed IL-15Rα. More recent studies employing highly secreted IL-15 do show NK-dependent inhibition of tumor outgrowth, similar to what we find; whether rejection of established tumors was studied in these models has not been described (17, 18, 24). We were able to study rejection of established tumors by growing them under protection of anti-IL-15 antibody, as transpresented IL-15 is needed for (i) augmenting susceptibility to killing (18) and (ii) survival of circulating NK cells. Interestingly, NK cells survived in the center of tumors growing in anti–IL-15-treated mice, consistent with the notion that antibodies cannot effectively diffuse into solid tumors (43). The rim of these tumors remained NK cell free and viable, even shortly after cessation of the antibody treatment. Tumors began to regress approximately 10 days after the last dose of antibody when circulating and infiltrating NK cells were detected. It remains unclear whether these NK cells originated from the surviving NK cells within the tumor or whether they infiltrated from the periphery and matured in the tumor.

Tumors in mice lacking perforin had vast central necrosis while the rim remained viable. It is tempting to speculate that the destruction of the greater mass of the tumor was caused by IFN-γ and TNF still being produced by the perforin-deficient IL-15-induced NK cells, as IFN-γ was shown to be an important effector molecule in adoptively transferred NK cells (44). Furthermore, infiltrated NK cells had upregulated 4-1BB, which has been implicated with elevated IFN-γ secretion (45). Although likely, it needs to be confirmed that NK cells caused the central necrosis in Prf−/− mice and the effector molecules involved need to be defined. Nevertheless, massive central necrosis and dense infiltration by perforin-deficient NK cells at the tumor margin did not change the growth rate of the cancer. The similarly growing IL-15-secreting tumors in perforin-competent mice receiving anti-IL-15 antibody looked histologically different, but also showed central necrosis and a viable tumor rim. These results indicate that the viability of the margins of tumors, not of the bulk tumor beneath this layer, determine whether eradication occurs.

Interestingly, tumors relapsed after nearly complete destruction when the hosts lacked IL-15Rα. As discussed for cancer cells above, the tumor stroma may need to express IL-15Rα to become a target for NK cells and killing of stroma may be needed for complete eradication (46). Because IL-15Rα expression on non-NK cells is required for maturation of NK cells from precursors (16), IL-15Rα expression on cancer cells growing in the IL-15Rα-deficient hosts must have sufficed for developing and maintaining fully mature NK cells from precursors. The existence of such precursors has previously been reported [for review see (47)]. However, after the NK cells have killed the cancer cells, this support was obviously eliminated. Accordingly, relapse of IL-15-secreting tumors in IL-15Rα−deficient hosts correlated with the absence of peripheral NK cells.

Now the greatest challenge for using IL-15 in cancer therapeutics is targeting the malignant tissue. Based on the combined findings, we think IL-15 and IL-15Rα must be localized to the tumor. Localizing IL-15 to the tumor is daunting, but ongoing research suggests that linking IL-15 and its stabilizing IL-15Rα (25–28) to a tumor-specific antibody could be an effective approach. Although this strategy eliminates the appeal of NK cells in not requiring identification of antigens, it aims to take advantage of the unprecedented destructive ability of IL-15-activated NK cells for large established tumors. Kroemer and colleagues showed that soluble IL-15/IL-15Rα conjugates could stimulate NK cells in vivo to reject skin allografts in the absence of adaptive immune cells (48). Once a proper delivery approach is developed, these effector NK cells need to be induced only in the microenvironment of the tumor margin sparing the rest of the body from the side effects and suffering that accompany many available therapies today.

No potential conflicts of interest were disclosed.

The authors thank Michael Caligiuri for reagents and Jennifer Stone and David Kranz for technical support and discussions; Mark Smyth and Laurence Zitvogel for advice on phenotypic analysis; Sigrid Dubois, Nicole Fortenbery, Patricia González-Greciano, Jose Moyano, Vinay Kumar, and David Raulet for helpful suggestions; and the staff of the University of Chicago Flow Cytometry Core Facility and Elsamma Chettiath, Cezary Ciszewski, Dorothy Kane, and Christy Schmehl for technical assistance.

This work was supported by NIH grants P01-CA97296, R01-CA22677, and R01-CA37516 to H. Schreiber, R01-DK67180 to B. Jabri, the Digestive Disease Research Core Center (P30-DK42086) and the Cancer Center at the University of Chicago, the University of Chicago Committee on Immunology training grant (T32 AI 0709) to R.B. Liu, and a Research Fellowship of the German Research Foundation (DFG, EN 703/3-1) to B. Engels.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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