Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: J Immunol. 2016 Apr 22;196(11):4544–4552. doi: 10.4049/jimmunol.1600219

Inflammatory signals regulate IL-15 in response to lymphodepletion1

Scott M Anthony *,‡,2, Sarai C Rivas ‡, Sara L Colpitts †,3, Megan E Howard ‡, Spencer W Stonier *,‡,4, Kimberly S Schluns *,‡
PMCID: PMC4875792  NIHMSID: NIHMS774697  PMID: 27183627

Abstract

Induction of lymphopenia has been exploited therapeutically to improve immune responses to cancer therapies and vaccinations. Whereas IL-15 has well-established roles in stimulating lymphocyte responses after lymphodepletion, the mechanisms regulating these IL-15 responses are unclear. We report that cell surface IL-15 expression is upregulated during lymphopenia induced by total body irradiation (TBI), cyclophosphamide, or Thy1 Ab-mediated T cell depletion, as well as in RAG-/- mice; interestingly, the cellular profile of surface IL-15 expression is distinct in each model. In contrast, soluble(s) IL-15 complexes are upregulated only after TBI or αThy1Ab. Analysis of cell specific IL-15Rα conditional knock out mice revealed that macrophages and DCs are important sources of sIL-15 complexes after TBI but provide minimal contribution in response to Thy1 Ab treatment. Unlike with TBI, induction of sIL-15 complexes by αThy1 Ab is sustained and only partially dependent on type I Interferons. The Stimulator of IFN Genes (STING) pathway was discovered to be a potent inducer of sIL-15 complexes and was required for optimal production of sIL-15 complexes in response to Ab-mediated T cell depletion and TBI suggesting products of cell death drive production of sIL-15 complexes after lymphodepletion. Lastly, we provide evidence that IL-15 induced by inflammatory signals in response to lymphodepletion drives lymphocyte responses, as memory CD8 T cells proliferated in an IL-15-dependent manner. Overall, these studies demonstrate that the form in which IL-15 is expressed, it's kinetics and cellular sources, and the inflammatory signals involved are differentially dictated by the manner in which lymphopenia is induced.

Introduction

IL-15 responses by T cells are elevated after the depletion of lymphocytes by total body irradiation (TBI) or high dose chemotherapy regimens and in lymphocyte deficient RAG-/- mice (1-3). These enhanced responses to IL-15 during lymphopenia have been exploited therapeutically to improve the immune responses to cancer therapies and vaccinations (2,4-6). For instance, lymphodepletion is used as a conditioning regimen for adoptive T cell therapies of melanoma in mouse models and in human patients (2,3). Lymphopenia induces the proliferation of adoptively-transferred T cells as well as promotes a loss of tolerance against self-antigens, leading to enhanced anti-tumor responses (7); all of these responses are dependent on IL-15. Despite the clear importance of IL-15 in enhancing T cell responses during lymphopenia, the mechanisms regulating IL-15 during lymphopenia are not clear, particularly with respect to the realization that IL-15 can mediate responses through multiple mechanisms, including transpresentation and soluble cytokine-receptor complexes. Furthermore, the cellular source for either form of IL-15 during lymphopenia has not been elucidated.

The mechanism of lymphopenia-enhanced IL-15 expression was originally considered a passive process, whereby the availability of a constant low level of IL-15 is enhanced simply due to the loss or lack of endogenous lymphocytes utilizing this IL-15; this theory is often referred to as the cytokine sink (3). However, a later report demonstrated that the anti-tumor effect induced by TBI was instead mediated by the presence of microbial components that leak through the intestines (8). More recent studies found that soluble IL-15/IL-15Rα complexes (sIL-15 complexes) are transiently increased in mice treated with total body irradiation (TBI) or Cyclophosphamide (CTX) and in the sera of patients undergoing lymphodepletion regimens (9). These transiently induced sIL-15 complexes are one potential factor enhancing T cell responses after lymphodepletion as recombinant sIL-15 complexes are robust mediators of T cell and NK cell proliferation (10,11). Interestingly, the transient induction of sIL-15 complexes in mice after TBI precedes the presence of microbial components and LPS (8) suggesting the early induction of sIL-15 complexes is mediated by an additional mechanism. Our recent studies have found that type I IFNs are an important pathway stimulating the production of sIL-15 complexes after TBI (12) providing further support for the idea that active inflammatory signals upregulate sIL-15 complexes. Therefore, the objective of this study was to further elucidate the mechanisms regulating IL-15 during lymphopenia. Using multiple mouse models of lymphopenia, we investigated the factors and cell types required for the generation of lymphopenia-induced IL-15 and sIL-15 complexes.

Materials and Methods

Mice

C57Bl/6 (wild-type;Wt) mice were purchased from NCI/Charles River (Frederick, MD). RAG1-/- mice, IL-15Rαfl/fl (13), CD11cCre (14), LysM-Cre (15), and Tmem173-/- mice (16) were purchased from Jackson Laboratories (Bar Harbor, MN). IL-15Rα-/- knockout (Rko) mice (17) were originally generated and obtained by Dr. Averil Ma through Leo Lefrancois and backcrossed to the C57Bl/6 line. Thy1.1+ Pmel-1 TCR Transgenic (specific for Gp100 peptide 25-33 in the context of H2-Db) mice were provided by Dr. Willem Overwijk (18). IFNAR1-/- mice were provided by Dr. Paul W. Dempsey (Department Of Microbiology and Molecular Genetics, University of California, Los Angeles and Dr. Tadatsugu Taniguchi, Department of Immunology, Tokyo University, Japan) to W. Overwijk and crossed to the C57Bl/6 background (19). IL-15 transcriptional reporter mice were generated by Dr. Leo Lefrancois (Department of Immunology, University of Connecticut, Farmington, CT) (20); experiments utilizing these mice were performed at the University of Connecticut Health Science Center. All other mice described were maintained under specific pathogen-free conditions at the institutional animal facility. The animal facility is fully accredited by the Association of Assessment and Accreditation of Laboratory Animal Care International. All animal procedures were conducted on mice between 6-10 weeks of age, in accordance with the animal care and use protocols (100409934) approved by the Institutional Animal Care and Use Committee at the UT MD Anderson Cancer Center.

To generate BM chimeras, BM was collected from the tibia and femurs of IL-15Rα-/- (CD45.1) and WT (CD45.2) mice and depleted of T cells as previously described (21). IL-15Rα-/- (CD45.2) and WT (CD45.2) recipients were irradiated with 1,000 RADs and injected i.v. with 3 ×106 BM cells. After complete BM reconstitution (8-12 weeks later), serum was collected from peripheral blood and analyzed for levels of sIL-15 complexes.

In vivo injections

For Poly I:C stimulation, mice were administered Poly I:C i.p. (150 μg, Sigma, St. Louis, MO). For stimulation of the STING pathway, mice were administered c-di-GMP i.v. (Invivogen, San Diego, CA) at the indicated doses. For chemotherapy treatments, mice were administered cyclophosphamide dissolved in saline i.p. (200mg/kg). For TBI, mice were exposed to a cesium irradiation source at the indicated doses. Complement-mediated depletion of T cells was performed by injecting i.p. anti-Thy1.2/CD90.2 (300ug, clone 30H12), anti-CD8α (clone 2.43), or anti-CD4 (GK1.5) (all from BioXcell, Lebanon, NH). Depletion was confirmed with flow cytometric analysis of the spleen. For serum isolations, peripheral blood was collected from sacrificed mice via cardiac puncture and on some occasions from the retro-orbital cavity prior to treatment from the same mice. Blood was allowed to clot and centrifuged to separate serum.

Analysis of Cytokine and LPS Expression

ELISA specific for murine soluble IL-15Rα/IL-15 complexes (eBioscience, San Diego, CA) was performed according to manufacturer's recommendations. The limit of detection of this ELISA in our hands was 1pg/ml. Limulus amebocyte lysate (LAL) chromogenic Endotoxin quantitation assay (ThermoScientific, Rockford, IL) was performed according to manufacturer's recommendations. Cell surface IL-15 was detected in splenic myeloid cells isolated directly ex vivo as previously described (22). Briefly, cell surface IL-15 was detected with polyclonal rabbit anti-IL-15-biotin (Peprotech, Rocky Hill, NJ) followed by streptavidin-APC (Jackson ImmunoResearch). Background staining was determined by staining analogous populations with a biotinylated Ig control (Jackson ImmunoResearch). The following mAbs were purchased from BD Biosciences (San Jose, CA), eBiosciences, or BioLegend: CD19, CD3, DX5, CD11b, CD11c, B220, CD19, CD90.1, CD90.2, CD8, NK1.1, and CD44. Lineage+ cells were identified as CD19+, CD3+, or NK1.1+. For analysis of the IL-15 transcriptional reporter mice, the following cell populations were gated based on FSC, SSC, and exclusion of a Live/Dead dye and phenotypically defined as follows: CD11b+DCs (lin-CD45+F4/80-Ly6C-CD11c+MHC Class II+CD11b+ ), monocytes (lin-CD45+CD11b+Ly6G-Ly6C+), CD8+DCs (lin-CD45+F4/80-Ly6C-CD11c+MHC Class II+CD8+), Neutrophils (lin-CD45+CD11b+Ly6G+), and CD103+ DCs (lin-CD45+F4/80-Ly6C-CD11c+MHC Class IIhighCD103+). At greater than 30 days post infection, the expression of CD8α, CD90.1 and high CD44 expression was used to define memory Pmel-1 T cells. Flow cytometric data were acquired with a LSRII (BD Biosciences) or LSR Fortessa (BD Biosciences) and analyzed with Flowjo software version 9.7.6 (Flowjo LLC, Ashland). RNA was isolated from sorted cell populations using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. cDNA was synthesized using the Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). iQ SYBR supermix (Bio-rad, Hercules, CA) was used to conduct quantitative PCR assays in triplicate. Expression of IL-15 was determined by the following formula, applied to each reaction: 1.8 (CT β-actin-CT IL-15) ×106.

Bone marrow derived dendritic cells (BMDCs)

BMDCs were generated using Flt3L as previously described (23). Briefly, BM cells were flushed from femurs of indicated mice, dissociated, and treated with Tris ammonium chloride to lyse red blood cells. BM cells were then cultured in RPMI Complete Medium (CM) at a concentration of 1×106 cells/mL. CM RPMI contains 2.5, mM HEPES, 5.5×10-5 M 2-ME, 100U/ml penicillin, 100 μg/ml streptomycin, 5mM glutamine and 10% fetal calf serum supplemented with 200ng/mL FLT-3L (R&D Systems, Minneapolis, MN) at 37° C with 5% CO2 for 9 days. BMDCs were seeded at 1-2.5×106 cells/mL and stimulated with IFNα (300U/mL, PBL Laboratories, Piscataway,NJ), or the STING agonist c-di-GMP (25μg/mL, Invivogen, San Diego, CA). Supernatants were collected after 24 hours and analyzed for sIL-15 complexes using ELISA.

Analysis of lymphopenia-induced proliferation on memory CD8 T cells

4×105 Naïve (CD44-) Thy1.1+ (CD90.1) Pmel-1 TCR transgenic CD8 T cells were adoptively transferred to Thy1.2+ Wt recipients and one day later, infected with recombinant VSV expressing Gp100 (18,24). Greater than 35 days post infection, total CD8 T cells (containing Thy1.1+memory Pmel-1 T cells) were negatively enriched using the Dynal CD8 enrichment kit (Invitrogen, Carlsbad, CA), labeled with 2mM CFSE as previously described (22,25) and injected intravenously into Thy1.2+ Wt or IL-15Rα-/- mice (between 0.1-0.5 ×106 Pmel-1 CD8 T cells/mouse). Isolated splenic memory Pmel-1 T cells were CD44hi, with ∼40-60% expressing CD62L suggesting this population contained a mixture of effector and central memory T cells. Five days after αThy1.2 injection, lymphocytes in spleen and lymph nodes were analyzed for the presence of Pmel-1 T cells and CFSE dilution.

Statistical Analysis

Statistical differences were determined by a two-tailed Students t test. * indicates p<0.05. Analyses were performed using GraphPad Prism, version 6 (GraphPad Software, San Diego, CA) or Microsoft Excel 2010 (Redmond, WA)

Results

Expression of sIL-15 complexes and surface IL-15 in various models of lymphopenia

IL-15 responses by T cells and NK cells are increased during lymphopenic conditions (3,9). Since IL-15 responses can be mediated by both transpresentation and sIL-15 complexes, we examined the expression of sIL-15 complexes and cell surface IL-15 during lymphodepletion. Similar to previous reports, TBI increased the levels of sIL-15 complexes in peripheral blood serum 24h post treatment as previously described (9,12) (Figure 1A). In contrast to previous reports (9), significant increases in serum sIL-15 complexes were not observed 72 hrs after CTX treatment (Figure 1A) in multiple experiments as well as during earlier time points (Figure 1B). This lack of up-regulation of sIL-15 complexes after CTX treatment in our hands is not clear but was not due to insufficient drug delivery as abundant lymphopenia was observed, similar to the level of depletion observed in previous studies (Supplemental Figure 1) (9). Despite RAG-/- mice being endogenously lymphopenic, the levels of sIL-15 complexes were equivalent to those of untreated WT mice (Figure 1A). To determine if baseline levels of sIL-15 complexes in untreated Wt and RAG-/- mice were above the level of detection of the ELISA, serum from IL-15Rα-/- mice was analyzed and found to contain no detectable levels of sIL-15 complexes (Figure 1A). These results reveal sIL-15 complexes are circulating at low levels during the steady state and are not upregulated in all forms of lymphopenia.

Figure 1. Differential regulation of IL-15 in various models of lymphopenia.

Figure 1

Open in a new tab

A) Levels of sIL-15 complexes in serum isolated from peripheral blood of Wt mice subjected to TBI (800 RADS, 24 hrs) or CTX (200mg/kg, 72 hrs) or in untreated RAG-/-, or IL-15Rα-/- mice as determined by ELISA. n=3-4 mice/group. Data is one representative of three experiments. B) Serum levels of sIL-15 complexes in Wt mice subjected to CTX (200mg/kg) at the indicated times post treatment or after Poly I:C (150μg i.p., 24 hr), n=3 mice/group. C) Levels of LPS in serum of mice at the indicated times post TBI (1000RADS) as measured using limulus amebocyte lysate assay. n=2-4 mice/group, one representative of two experiments is shown. D) Representative flow cytometric histogram depicting staining with control Ig (black histogram) and anti-IL-15 Ab (grey histograms) in splenic DCs (lin-CD11c+CD11b+) and monocytes/macrophages (lin-CD11c-CD11b+) isolated from mice treated as indicated. E) Graph shows average MFI of IL-15 expression in DCs and monocytes/macrophages from indicated mice. n=3 mice/group, one representative of three experiments is shown. Error bars represent SD. F) Sorted splenic DCs (Lin-CD11Chi) from the indicated mice were analyzed by qtPCR for expression of IL-15 transcripts and β-actin. Results are representative of two independent experiments.

Since previous studies provided evidence that anti-tumor therapies involving TBI can lead to damage of the intestinal lining resulting in presence of commensal bacteria in the mesenteric lymph nodes and enhanced levels of circulating LPS (8), we wanted to examine the levels of LPS present at early times post TBI when sIL-15 complexes are generated. Baseline LPS levels in untreated mice were equivalent to those observed in the aforementioned study (8). While we observe sIL-15 complexes as early as 16 hrs post TBI (data not shown), levels of serum LPS levels were not increased at this time, or at 24, 72, or 168 hrs post TBI (Figure 1C). Therefore, these findings suggest the early induction of sIL-15 complexes is not mediated by the presence of commensal bacteria in the circulation.

We also examined how transpresentation of IL-15 is affected by varying types of lymphopenia. In general, cell surface IL-15 expression by myeloid cells was enhanced, relative to untreated WT mice, under all lymphopenic conditions examined; however, differential cell-type expression was noted (Figure 1D,E). DCs (lin-CD11b+CD11c+) from RAG-/- mice and CTX-treated mice, but not mice subjected to TBI displayed a significant increase in cell-surface IL-15 expression (Figure 1D,E). In contrast, IL-15 expression by monocytes/macrophages (lin-CD11b+ CD11c-) was increased after TBI and in RAG-/- mice, but not CTX treatment (Figure 1D,E). To determine if increased levels of cell surface IL-15 in DCs of RAG-/- mice coincides with increased IL-15 transcription, splenic DCs were sorted and IL-15 transcription was measured using qtPCR. Levels of IL-15 transcripts were not significantly different in DCs isolated from WT and RAG-/- mice (Figure 1F) indicating increased surface IL-15 in RAG-/- mice occurs independent of transcription. The differential induction of sIL-15 complexes and increases in cell surface IL-15 suggest that distinct mechanisms regulate IL-15 after TBI versus chemotherapy. Furthermore, these results suggest increased surface IL-15 is common to lymphopenia, while sIL-15 complexes are induced by inflammatory signals.

Cell specific expression of IL-15 after TBI

Since we found sIL-15 complexes are produced during the steady state, we examined the cellular source of sIL-15 complexes during homeostasis in various combinations of IL-15Rα BM chimeras. After complete reconstitution of the hematopoietic compartment (∼8-12 weeks after BM transfer), Wt control BM chimeras (Wt BM→ Wt recipients) expressed similar levels of sIL-15 complexes as unmanipulated WT mice while control IL-15Rα-/- BM chimeras (Rko BM→ Rko recipients) expressed undetectable levels of sIL-15 complexes (Figure 2A). The levels of sIL-15 complexes were significantly decreased in BM chimeras lacking IL-15Rα in the hematopoietic compartment (Rko BM→ Wt recipients) but not in chimeras lacking expression of IL-15Rα by radiation-resistant parenchymal cells (Wt BM→ Rko recipients) indicating most of the sIL-15 complexes produced during the steady state is derived from hematopoietic cells (Figure 2A).

Figure 2. Cell specific expression of IL-15 during the steady state and after TBI.

Figure 2

Open in a new tab

A) Levels of sIL-15 complexes in the serum of IL-15Rα-/- BM chimeras 8-12 weeks after irradiation and BM reconstitution. One of two experiments is shown. B) Levels of serum sIL-15 complexes in control IL-15Rαfl/fl (Black), CD11cCre+/+ × IL-15Rαfl/fl (White) and LysMCre+/+ × IL-15Rαfl/fl (Grey) mice before and 24 hours post TBI (1000 RADS) (n=2-5 mice/group), one experiment of three is shown. C) IL-15-GFP transcriptional reporter mice were left untreated (cont.) or subjected to TBI (800 RADS). GFP intensity as a measure of IL-15 transcriptional activity was examined in various populations in spleen and peripheral LNs using immunofluorescence staining and flow cytometry. Data shown is average fold difference in MFI of GFP expression in TBI-treated vs. untreated littermate control mice for each of the respective cell types analyzed. n=6-7 mice/group, data is compiled from two experiments. Error bars represent SEM. * indicates p<0.05.

To determine the cellular requirements of TBI-induced sIL-15 complexes, we examined cell-specific IL-15Rα deficient mice. Serum was collected from CD11cCre+/+ × IL-15Rαfl/fl, LysMCre+/+ × IL-15Rαfl/fl, and IL-15Rαfl/fl mice prior to and 24 hours after TBI (1000 RADS) and levels of soluble IL-15 complexes were examined by ELISA. During the steady state, modest decreases in sIL-15 complexes were observed in both CD11c-Cre × IL-15Rαfl/fl mice and LysM-Cre × IL-15Rαfl/fl mice (Figure 2B); however, these trends were not statistically significant. In response to TBI, induction of sIL-15 complexes was substantially hampered in both the CD11cCre+/+ × IL-15Rαfl/fl and LysMCre+/+ × IL-15Rαfl/fl × mice than in the control IL-15Rαfl/fl mice (Figure 2B). Overall, these experiments demonstrate that both CD11c+ and LysM+ cells, which consist mostly as dendritic cells and macrophages respectively, contribute minimally to the production of sIL-15 complexes during the steady state but are substantial sources in response to TBI.

To specifically identify the type of myeloid cells expressing IL-15 after TBI as well as identify additional cell types that may be contributing to IL-15 production, we utilized an IL-15 transcriptional reporter mouse (developed in the laboratory of Dr. Leo Lefrancois) that expresses GFP under the control of the IL-15 promoter (20). Cells were isolated 24 hours after TBI from spleens and peripheral LNs of Wt and transgenic mice. TBI upregulated GFP expression in CD11b+ DCs, monocytes, CD8α+ DCs, and even in CD45- cells (Figure 2C). Unfortunately, GFP expression analysis of macrophages after TBI was inconclusive as changes in GFP were inconsistent (data not shown). Although neutrophils have been previously shown to express IL-15 mRNA at baseline and low levels of IL-15 protein upon in vitro stimulation (26), IL-15 reporter expression by neutrophils was not increased in response to TBI (Figure 2C). Furthermore, reporter expression was not increased by CD103+DCs in the PLNs (Figure 2C) showing that specific subsets of DCs are responsive to this type of stimulation. These results indicate that IL-15 transcriptional activity is induced by TBI in specific cells among both the hematopoietic and parenchymal compartments.

Expression of IL-15 after Ab-mediated T cell depletion

Since Ab-mediated lymphocyte depletion is more specific to lymphocytes than chemotherapy or irradiation and should not affect the integrity of the intestinal lining, we next examined how this model of lymphodepletion impacts production of sIL-15 complexes. Hence, mice were depleted of T cells using complement fixing Ab, anti-Thy1 mAb (30H12, 300 μg, i.p.). After Ab treatment, levels of serum sIL-15 complexes significantly increased at 24 hrs, but unlike after TBI, decreased slowly over time (Figure 3A). Treatment with either depleting anti-CD4 Ab (GK1.5) or anti-CD8 Ab (2.43) alone had little effect on levels of sIL-15 complexes (Figure 3B) indicating the amount of lymphocyte death occurring with these treatments is insufficient to induce sIL-15 complexes. Since complement-mediated depletion is an active process that leads to systemic inflammation (27), we examined the role of Type I IFN signaling in the αThy1-mediated induction of sIL-15 complexes. In mice that are unable to respond to Type I IFN (IFNAR-/-), induction of sIL-15 complexes in response to αThy1 treatment was significantly reduced, corresponding to an approximate 50% reduction (Figure 3B). Hence, while both TBI and complement-mediated depletion of T cells lead to a substantial amount of cell death, their reliance on Type I IFN signaling to mediate induction of sIL-15 complexes varied (12). To examine cellular sources of sIL-15 complexes produced in response to αThy1-mediated T cell depletion, we again utilized cell-specific IL-15Rα deficient mice. Levels of soluble IL-15 complexes were measured in serum of IL-15Rαfl/fl, CD11cCre+/+ × IL-15Rαfl/fl and LysMCre+/+ × IL-15Rαfl/fl mice prior to and 24 hours after treatment with αThy1 Ab. A significant induction of sIL-15 complexes was observed in response to αThy1 Ab in all groups (Figure 3C); however, the induction was only slightly reduced in the absence of IL-15 derived from DCs or monocyte/macrophage compared to Wt controls (Figure 3C). These results indicate that DCs, macrophages, and monocytes contribute to the production, but are not major sources of sIL-15 complexes in response to αThy1-mediated T cell depletion.

Figure 3. Specific depletion of T cells leads to production of sIL-15 complexes.

Figure 3

Open in a new tab

A) Levels of sIL-15 complexes in serum of Wt mice at different times after αThy1.2 injection (300μg, i.p.) as determined by ELISA. n=2-3 mice/group, one representative of three experiments. B) Level of sIL-15 complexes in serum of Wt mice 24 hrs after treatment with αCD4 Ab, αCD8 Ab, and αThy1 Ab and in IFNAR-/- mice with and without treatment with αThy1 Ab. C) Serum levels of sIL-15 complexes in control IL-15Rαfl/fl (Black), CD11cCre+/+ × IL-15Rαfl/fl (White) and LysMCre+/+ × IL-15Rαfl/fl (Grey) mice before (untreated) and 24 hours post TBI (1000 RADS) (n=2-5 mice/group), one representative experiment of three is shown. D) Cell surface IL-15 expression after Thy1 Ab treatment. Graph shows average MFI of IL-15 staining by respective cell types in spleens at 0, 1, 2, 4, and 10 days after αThy1 Ab treatment. Populations were defined by the following gating: CD11b+ DCs (lineage-CD11b+CD11c+F480-), CD8+ DCs (lineage-CD11b-CD11c+CD8α+), monocytes (lineage-CD11b+CD11c-F480-) macrophages (lineage-CD11b+CD11c-F480+). Data showing IL-15 expression by CD8+ DCs was derived from pooled samples. Error bars represent SD.* indicates p<0.05.

To determine how the cell surface IL-15 expression changed with Ab-mediated T cell depletion, splenocytes isolated at different times post Thy1 Ab treatment were stained for surface IL-15 along with other cell surface markers to delineate different myeloid cell populations as previously described. Increases in cell surface IL-15 by CD8+ DCs, CD11b+ monocytes, and macrophages was observed after Thy1 Ab treatment but increases were not present in CD11b+DCs at any time point (Figure 3D). In the monocytes and macrophages, increases in surface IL-15 were apparent 1, 2 and 4 days after Ab treatment, while increases by CD8+ DCs were not evident until 2 days post treatment (Figure 3D). These results demonstrate that increases in IL-15 transpresentation can be concurrent with increases in sIL-15 complexes, similar to that observed after TBI. In addition, treatment with αThy1 Ab leads to a specific array of myeloid cells transpresenting IL-15. Altogether, these results directly show that specific depletion of T cells leads to the generation of sIL-15 complexes, which is partially driven by type I IFNs, as well as the upregulation of surface IL-15 by distinct myeloid cells.

Regulation of sIL-15 complexes by the STING pathway

As both complement activation and TBI lead to a significant amount of cell death, we next asked whether immunogenic cell death may be an important signal leading to induction of sIL-15 complexes. The Stimulator of Interferon genes (STING) serves as an important recognition of cytoplasmic DNA leading to the induction of Type I IFNs (28-31). As Type I IFN signaling is an important regulator of sIL-15 complexes during lymphopenia (12), we investigated the role of the STING pathway in the induction of sIL-15 complexes. Injection of STING agonist, c-di-GMP (25μg, i.v.) induced high levels of circulating sIL-15 complexes as early as 12 hours post treatment, remained elevated at 24 hours, and returned to almost baseline levels at 48 hours (Figures 4A). To determine if STING agonist could directly induce sIL-15 complexes in DCs, Flt3L-derived BMDCs were treated with c-di-GMP. Indeed, STING agonist robustly induced production of sIL-15 complexes by DCs, which was more potent than IFN-α alone (Figure 4B). Furthermore, in vivo induction of sIL-15 complexes by STING agonist was significantly impaired in both CD11cCre+/+ × IL-15Rαfl/fl and LysMCre+/+ × IL-15Rαfl/fl compared to control mice (Figure 4C) showing both DCs and macrophages are major sources of sIL-15 complexes in response to this type of stimulation. Type I IFNs are a required intermediate in STING-induced sIL-15 complexes as serum sIL-15 complexes failed to be induced in IFNAR-/- mice in response to treatment with the STING agonist, c-di-GMP (Figure 4D). Collectively, these findings are the first demonstration that stimulation of the STING pathway, via the induction of type I IFNs, leads to the induction of sIL-15 complexes.

Figure 4. Regulation of sIL-15 complexes by the STING pathway.

Figure 4

Open in a new tab

A) STING agonist c-di-GMP (25μg) was administered i.v. and the levels of sIL-15 complexes in serum were analyzed at the indicated times post injection. B) Flt3L BMDCs were generated from Wt or IL-15Rα-/- mice and left unstimulated, stimulated with IFN-α (300U/mL), or with c-di-GMP (25μg/mL) for 24 hours. Cell culture supernatants were isolated and measured for the levels of sIL-15 complexes by ELISA. n=2 wells/group, one representative of two experiments is shown. C) Levels of serum sIL-15 complexes in control IL-15Rαfl/fl (Black), CD11c-Cre+/+ × IL-15Rαfl/fl (White) and LysM-Cre+/+ × IL-15Rαfl/fl (Grey) mice before and 24 hours treatment with c-di-GMP (25μg, i.v.). D) Levels of sIL-15 complexes in serum of Wt or IFNAR-/-mice treated with c-di-GMP (25μg, i.v. 12 hrs). n=3 mice/group, one representative of two experiments is shown. Error bars represent SD. * indicates p<0.05. E, F) Levels of sIL-15 complexes in serum from Wt and Tmem173-/- mice prior to and 24 hours after treatment with (E) TBI (1000 RADS) or (F) one and two days after αThy1Ab (300mg, i.p.). n=3-4 mice/group; data is one representative of 3 experiments. Error bars represent SD. * indicates p<0.05.

As our previous studies have shown that type I IFNs are important for induction of sIL-15 complexes after TBI (12) and substantial cell death is induced by TBI, we investigated the requirement for STING signaling in TBI-induced sIL-15 complexes. We obtained commercially available Tmem173-/- mice that are incapable of stimulating the STING pathway (16) and examined the ability of TBI to induce sIL-15 complexes in the absence of STING signaling. In Tmem173-/- mice, induction of sIL-15 complexes by TBI were reduced by approximately 50% (Figure 4E) indicating optimal induction of sIL-15 complexes by TBI requires STING signaling. Since Type I IFN signaling is completely required for TBI-induced sIL-15 complexes (12), these results also suggest other STING-independent pathways are driving production of type I IFNs after TBI. In response to αThy1-mediated T cell depletion, the early induction of sIL-15 complexes was unaffected in Tmem173-/- mice whereas the production of sIL-15 complexes (2 days after Thy1Ab) was impaired (Figure 4F) suggesting that STING signaling is required for the sustained generation of sIL-15 complexes in response to αThy1 treatment. Overall, these results show that stimulation of the STING pathway significantly contributes to the induction of sIL-15 complexes in response to TBI and Ab-mediated T cell depletion.

Effects of inflammatory IL-15 on memory CD8 T cells

Thus far, we have examined the regulation of lymphopenia-induced IL-15 expression at the levels of transcription, transpresentation and production of sIL-15 complexes. These findings provide evidence that a complex set of inflammatory signals are involved in the upregulation of IL-15 that is specifically hallmarked by the production of sIL-15 complexes. We suspect that the sIL-15 complexes produced after lymphodepletion contribute to enhanced responses by IL-15-responsive lymphocytes, such as memory CD8 T cells and NK cells. Because presently there is no model to distinguish the effects of sIL-15 complexes from those mediated by transpresented IL-15, we are unable to conclusively determine the functions of lymphopenia-induced sIL-15 complexes on responding lymphocytes. Nevertheless, we can address the role of IL-15 induced by inflammatory signals after αThy1Ab-mediated T cell depletion, which is dominated by the expression of sIL-15 complexes. Specifically, we investigated the effects of αThy1-induced inflammatory IL-15 on memory CD8 T cell proliferation.

To generate memory CD8 T cells that are resistant to αThy1.2-mediated depletion, naïve Thy1.1+ TCR transgenic Pmel-1 CD8 T cells (specific for the melanocyte differentiation antigen gp100 peptide 25-33 in the context of H2-Db) were adoptively transferred to Wt recipients (Thy1.2+) followed by infection with recombinant VSV expressing gp100 one day later (18,24). After the Pmel-1 T cells were allowed to differentiate into memory T cells (>30 days post infection), total CD8 T cells (containing Thy1.1+ memory CD8 T cells) from the spleen were enriched via negative selection, CFSE-labeled, and adoptively transferred to Thy1.2+ Wt or IL-15Rα-/- mice. One day post transfer, all mice were injected with αThy1.2 to deplete host T cells and induce inflammatory IL-15 expression. Since the donor Thy1.1+ memory CD8 T cells are resistant to depletion by Thy1.2 Ab treatment, they can respond to the IL-15 upregulated within the environment. As expected, one day post Ab treatment, sIL-15 complexes were elevated in blood from Wt mice but not in IL-15Rα-/- mice (data not shown). After five days, the majority of Thy1.1+ T cells (between 75-95%) proliferated at least 1-2 divisions in the spleen and LNs of Wt mice (Figure 5A,B). This depletion-induced T cell proliferation was fully dependent upon inflammatory IL-15, as few of the memory CD8 T cells (<25%) underwent proliferation in the IL-15Rα-/- mice (Figure 5A,B). The increase in proliferation also translated to an increase in the number of donor memory CD8 T cells (Figure 5C); although the increased cell numbers were consistent, this effect was not statistically significant. These data directly confirm that inflammatory signals induced by Ab-mediated T cell depletion lead to the proliferation and accumulation of memory CD8 T cells in an IL-15 dependent manner. These findings reveal an unappreciated role of inflammatory signals in the regulation of IL-15 after lymphodepletion.

Figure 5. Lymphopenia-induced T cell proliferation is dependent on inflammatory IL-15.

Figure 5

Open in a new tab

Memory CD8 T cells (>30 days post infection) on the Thy1.1 (CD90.1) background were negatively enriched from SPs, CFSE-labeled, and adoptively transferred to Wt or IL-15Rα hosts on Thy1.2 background (CD90.2) (between 0.1-0.5 ×106 Pmel-1 T cells/mouse). One day later, all mice were subjected to depletion of endogenous T cells via injection αThy1.2 (30H12, 300μg i.p.). A) Histogram depicts CFSE dilution of Thy1.1+ CD8 T cells in spleen of Wt or IL-15Rα-/- mice. B) Average percentage of donor Thy1.1+ CD8 T cells that had undergone at least one proliferation in the spleen, peripheral LN, and mesenteric LN among each group of mice. n=3 mice/group. * indicates significant differences between Wt mice (p<0.05). C) The average number of donor Thy1.1+ CD8 T cells recovered for the indicated tissues. Error bars represent SD. * indicates p<0.05. One of three experiments is shown.

Discussion

Induction of lymphopenia is an effective strategy to enhance T cell responses in various types of immunotherapies that works in part by increasing responses to homeostatic cytokines, such as IL-15 and IL-7 (1,3,32). Surprisingly, the mechanism(s) responsible for enhancing IL-15 responses during lymphopenia, until now, were unclear. This is attributed to the unique and recently appreciated mechanisms that are utilized by IL-15 to induce responses, which include transpresentation and sIL-15 complexes. While transpresentation is believed to be responsible for driving IL-15 responses during homeostasis (13,25,33,34), the production of sIL-15 complexes is associated with immune activation or inflammatory signals (12,35). Herein, this study demonstrates that both transpresentation and sIL-15 complexes are distinctly regulated during lymphopenia, with each differentially affected by the manner in which lymphopenia is induced. Moreover, we provide clear evidence that inflammatory signals are an integral component in the regulation of IL-15, particularly in the induction of sIL-15 complexes after TBI and Ab-mediated lymphodepletion. These inflammatory signals are heterogeneous among the different methods of inducing lymphodepletion, with pathways utilizing STING and type I IFNs but not completely dependent on any one pathway. Lastly, we provide evidence that elevated sIL-15 complexes are contributing to enhanced T cell responses after lymphodepletion. Our discovery mirrors the results described by the Harty Lab showing an important role for inflammatory IL-15 in the selective stimulation of memory CD8 T cells (36,37).

Our results comparing showing expression of sIL-15 complexes is limited to TBI- and Thy1-treated mice has lead us to conclude that induction of sIL-15 complexes is related to the amount of inflammatory signals present in each model. Previous studies have reported that the cytotoxicity of CTX is due to the induction of apoptosis (38,39), which is a type of cell death that avoids the release of cellular contents. We suspect that sIL-15 complexes were not generated in CTX-treated mice, despite the abundant amount of cell death because the type of cell death involved (apoptotic versus non apoptotic) did not sufficiently ignite an inflammatory response. In the context of a weak inflammatory response, mouse colonies housed in different locations and thus harboring a different composition of microbiota could possess a higher or lower threshold of immune activation; this could be an explanation for why other groups clearly observed an induction of sIL-15 complexes with CTX (9) and we did not. In contrast to cell death induced by CTX, apoptosis is not the main process of cell death induced by irradiation (40) and complement-mediated cell death is clearly associated with an inflammatory response (27). This knowledge together with our observation that STING signaling contributes to the production of sIL-15 complexes, provides the first evidence that products of cell death induced by TBI or αThy1 Ab directly regulate IL-15 expression. Our inability to correlate increases in sIL-15 complexes with systemic LPS provides additional evidence that the presence of microbial products is not required for the upregulation of sIL-15 complexes. We don't discount the role of LPS in potentially driving production of sIL-15 complexes in other models, such as HIV (41) and some tumor models, which utilize numerous approaches to induce immune stimulation and/or that disrupt immune homeostasis (8). Overall, our findings support the idea that abundant cell death and the immune system's subsequent response to cellular byproducts is an important inflammatory pathway regulating IL-15 after lymphodepletion.

Although sIL-15 complexes were not increased in RAG-/- mice, we and others (42) find cell surface IL-15 expression by DCs is increased suggesting lymphopenia alone is sufficient to upregulate IL-15. Because there is no overt immune stimulation in RAG-/- mice, we postulate the upregulated surface IL-15 is due to a passive mechanism, such as the presence of a cytokine sink. This theory is supported by the lack of upregulated IL-15 transcription we observe in DCs isolated from RAG-/- mice. Cell surface IL-15 is also increased in the other models of lymphopenia, which could be mediated by both active inflammatory signals and passive mechanisms. At least with TBI, our observation that IL-15 transcription is increased in multiple cell types indicates the presence of an activating signal. Signaling via Type I IFNs is at least one potential inflammatory mechanism increasing surface IL-15 expression levels as type I IFNs are produced after TBI and directly increase IL-15 transcription and cell surface expression (12,43-45). Nevertheless, some increased surface IL-15 could also be due to the presence of a cytokine sink as these mice are lymphocyte deficient. The increase in surface IL-15 along with the absence of sIL-15 complexes in RAG-/- mice suggests the enhanced IL-15 responses reported in these mice are driven by enhanced transpresentation of IL-15. As such, increased levels of IL-15 transpresentation in any model of lymphopenia have the potential to modulate lymphocytes responses. Alternatively, increasing the levels of surface IL-15 could be an additional mechanism to increase IL-15 expression prior to cleavage, thus enhancing the production of sIL-15 complexes. Unfortunately, for circumstances where both transpresentation and sIL-15 complexes are increased, there currently are no model systems that can be used to discern the contribution of each mechanism in driving IL-15 responses.

Whereas increases in IL-15 transpresentation are observed to some degree in each model of lymphopenia, the type of cells transpresenting IL-15 under these different conditions varied. This differential cellular expression likely reflects the diverse signals regulating IL-15 among the different model systems, which again could involve both passive and active forces. A previous study determined that the loss of DC specific IL-15 expression predominantly affected CD8 T cells with a central memory phenotype while loss of macrophage specific IL-15 expression resulted in a specific loss of effector memory CD8 T cells (13). If similar effects were observed during adoptive T cell therapy, then one would predict that CTX treatment would better support central memory T cells while TBI-treatment would better support effector memory T cells.

In addition to differences in cellular IL-15 expression, we revealed unique kinetics in the production of sIL-15 complexes among models of lymphopenia that could influence T cell responses in adoptive T cell therapy. Studies investigating differences in the signaling of IL-2 and IL-15 have found evidence that the duration of cytokine signal can differentially affect T cell responses (46,47). Specifically in infection models, IL-15 signaling promotes differentiation of short-lived effector T cells over memory precursor T cells (48,49) while later in the response, it supports the maintenance of memory CD8 T cells. Hence, similar effects on differentiation may be observed during adoptive T cell therapy. Alternatively, with the chronic stimulation associated with a tumor setting, a more continuous exposure to sIL-15 complexes may contribute to the exhaustion of tumor-specific T cells. Future studies should be carried out to better characterize the different kinetics of IL-15 after lymphodepletion regimens in human patients while also taking into consideration how treatment with recombinant formulations of IL-15 or IL-15 agonists may compliment the changing levels of endogenous sIL-15 complexes.

In summary, this study reveals that the regulation of IL-15 after lymphodepletion is far more complex than previously appreciated and how expression of sIL-15 complexes and transpresented IL-15 are each independently regulated by a variety of mechanisms and/or pathways. By showing that both transpresented IL-15 and sIL-15 complexes are upregulated during lymphopenia, we provide evidence that IL-15 responses mediated during lymphopenic conditions could be elicited by transpresented IL-15, sIL-15 complexes, or the combination of both. Much of the regulation of IL-15 during lymphodepletion is driven by inflammatory signals that involves the immune system's response to products of cell death and type I IFNs. During our investigations, we also identified the STING pathway as another inflammatory pathway regulating sIL-15 complexes, which can contribute to enhanced T cell responses in circumstances associated with release of cytosolic DNA. Since STING agonists are currently being pursued as adjuvants in antitumor immune responses, it is likely that IL-15 induced by this treatment is contributing to enhanced CD8 T cell responses. Overall, the unique expression profile of IL-15 in each model could impact IL-15-responsive lymphocytes in a distinct way, which provides a rationale to better investigate the effects of lymphodepletion on T cell differentiation and its corresponding effects on T cell effector functions and persistence. Understanding these differences and their impact could be one approach towards fine-tuning T cell responses during immunotherapy for enhanced anti-tumor responses.

Supplementary Material

1

Acknowledgments

The authors wish to thank Dr. Willem Overwijk for helpful comments and suggestions and for sharing IFNAR1-/- and Pmel-1 Tg mice and Dr. Lynn Puddington for mentoring support.

Footnotes

1

This research was supported by National Institutes of Health NIH predoctoral training grant CA009598 (to S.M.A and S.W.S.), a postdoctoral fellowship PF-11-152-01-LIB from the American Cancer Society (to S.L.C.), and a seed fund from the Center for Inflammation and Cancer at the MD Anderson Cancer Center and MD Anderson Bridge funding (to K.S.S).

References

  • 1.Sandau MM, Winstead CJ, Jameson SC. IL-15 is required for sustained lymphopenia-driven proliferation and accumulation of CD8 T cells. J Immunol. 2007;179:120–125. doi: 10.4049/jimmunol.179.1.120. [DOI] [PubMed] [Google Scholar]
  • 2.Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R, Kammula U, Robbins PF, Huang J, Citrin DE, Leitman SF, Wunderlich J, Restifo NP, Thomasian A, Downey SG, Smith FO, Klapper J, Morton K, Laurencot C, White DE, Rosenberg SA. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol. 2008;26:5233–5239. doi: 10.1200/JCO.2008.16.5449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Gattinoni L, Finkelstein SE, Klebanoff CA, Antony PA, Palmer DC, Spiess PJ, Hwang LN, Yu Z, Wrzesinski C, Heimann DM, Surh CD, Rosenberg SA, Restifo NP. Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J Exp Med. 2005;202:907–912. doi: 10.1084/jem.20050732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Dudley ME, Wunderlich JR, Yang JC, Sherry RM, Topalian SL, Restifo NP, Royal RE, Kammula U, White DE, Mavroukakis SA, Rogers LJ, Gracia GJ, Jones SA, Mangiameli DP, Pelletier MM, Gea-Banacloche J, Robinson MR, Berman DM, Filie AC, Abati A, Rosenberg SA. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol. 2005;23:2346–2357. doi: 10.1200/JCO.2005.00.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Bracci L, Moschella F, Sestili P, La SV, Valentini M, Canini I, Baccarini S, Maccari S, Ramoni C, Belardelli F, Proietti E. Cyclophosphamide enhances the antitumor efficacy of adoptively transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic proliferation, and specific tumor infiltration. Clin Cancer Res. 2007;13:644–653. doi: 10.1158/1078-0432.CCR-06-1209. [DOI] [PubMed] [Google Scholar]
  • 6.Koike N, Pilon-Thomas S, Mule JJ. Nonmyeloablative chemotherapy followed by T-cell adoptive transfer and dendritic cell-based vaccination results in rejection of established melanoma. J Immunother. 2008;31:402–412. doi: 10.1097/CJI.0b013e31816cabbb. [DOI] [PubMed] [Google Scholar]
  • 7.Oelert T, Papatriantafyllou M, Pougialis G, Hammerling GJ, Arnold B, Schuler T. Irradiation and IL-15 promote loss of CD8 T-cell tolerance in response to lymphopenia. Blood. 2010;115:2196–2202. doi: 10.1182/blood-2009-06-227298. [DOI] [PubMed] [Google Scholar]
  • 8.Paulos CM, Wrzesinski C, Kaiser A, Hinrichs CS, Chieppa M, Cassard L, Palmer DC, Boni A, Muranski P, Yu Z, Gattinoni L, Antony PA, Rosenberg SA, Restifo NP. Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling. J Clin Invest. 2007;117:2197–2204. doi: 10.1172/JCI32205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Bergamaschi C, Bear J, Rosati M, Kelly BR, Alicea C, Sowder R, Chertova E, Rosenberg SA, Felber BK, Pavlakis GN. Circulating interleukin-15 (IL-15) exists as heterodimeric complex with soluble IL-15 receptor alpha (IL-15Ralpha) in human serum. Blood. 2012;120:1–8. doi: 10.1182/blood-2011-10-384362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Rubinstein MP, Kovar M, Purton JF, Cho JH, Boyman O, Surh CD, Sprent J. Converting IL-15 to a superagonist by binding to soluble IL-15R{alpha} Proc Natl Acad Sci U S A. 2006;103:9166–9171. doi: 10.1073/pnas.0600240103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Stoklasek TA, Schluns KS, Lefrancois L. Combined IL-15/IL-15Ralpha immunotherapy maximizes IL-15 activity in vivo. J Immunol. 2006;177:6072–6080. doi: 10.4049/jimmunol.177.9.6072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Anthony SM, Howard ME, Hailemichael Y, Overwijk WW, Schluns KS. Soluble interleukin-15 complexes are generated in vivo by type I interferon dependent and independent pathways. PLoS One. 2015;10:e0120274. doi: 10.1371/journal.pone.0120274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Mortier E, Advincula R, Kim L, Chmura S, Barrera J, Reizis B, Malynn BA, Ma A. Macrophage- and dendritic-cell-derived interleukin-15 receptor alpha supports homeostasis of distinct CD8(+) T cell subsets. Immunity. 2009;31:811–822. doi: 10.1016/j.immuni.2009.09.017. [DOI] [PubMed] [Google Scholar]
  • 14.Gillgrass AE, Chew MV, Krneta T, Ashkar AA. Overexpression of IL-15 promotes tumor destruction via NK1.1+ cells in a spontaneous breast cancer model. BMC Cancer. 2015;15:293. doi: 10.1186/s12885-015-1264-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 1999;8:265–277. doi: 10.1023/a:1008942828960. [DOI] [PubMed] [Google Scholar]
  • 16.Sauer JD, Sotelo-Troha K, von MJ, Monroe KM, Rae CS, Brubaker SW, Hyodo M, Hayakawa Y, Woodward JJ, Portnoy DA, Vance RE. The N-ethyl-N-nitrosourea-induced Goldenticket mouse mutant reveals an essential function of Sting in the in vivo interferon response to Listeria monocytogenes and cyclic dinucleotides. Infect Immun. 2011;79:688–694. doi: 10.1128/IAI.00999-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Lodolce JP, Boone DL, Chai S, Swain RE, Dassopoulos T, Trettin S, Ma A. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity. 1998;9:669–676. doi: 10.1016/s1074-7613(00)80664-0. [DOI] [PubMed] [Google Scholar]
  • 18.Overwijk WW, Tsung A, Irvine KR, Parkhurst MR, Goletz TJ, Tsung K, Carroll MW, Liu C, Moss B, Rosenberg SA, Restifo NP. gp100/pmel 17 is a murine tumor rejection antigen: induction of “self”-reactive, tumoricidal T cells using high-affinity, altered peptide ligand. J Exp Med. 1998;188:277–286. doi: 10.1084/jem.188.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Muller U, Steinhoff U, Reis LF, Hemmi S, Pavlovic J, Zinkernagel RM, Aguet M. Functional role of type I and type II interferons in antiviral defense. Science. 1994;264:1918–1921. doi: 10.1126/science.8009221. [DOI] [PubMed] [Google Scholar]
  • 20.Colpitts SL, Stoklasek TA, Plumlee CR, Obar JJ, Guo C, Lefrancois L. Cutting Edge: The role of IFN-alpha receptor and MyD88 signaling in induction of IL-15 expression in vivo. J Immunol. 2012;188:2483–2487. doi: 10.4049/jimmunol.1103609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Castillo EF, Stonier SW, Frasca L, Schluns KS. Dendritic cells support the in vivo development and maintenance of NK cells via IL-15 trans-presentation. J Immunol. 2009;183:4948–4956. doi: 10.4049/jimmunol.0900719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Stonier SW, Ma LJ, Castillo EF, Schluns KS. Dendritic cells drive memory CD8 T cell homeostasis via IL-15 trans-presentation. Blood. 2008:4546–4554. doi: 10.1182/blood-2008-05-156307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Frasca L, Stonier SW, Overwijk WW, Schluns KS. Differential mechanisms of memory CD8 T cell maintenance by individual myeloid cell types. J Leukoc Biol. 2010 doi: 10.1189/jlb.1209816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Hailemichael Y, Dai Z, Jaffarzad N, Ye Y, Medina MA, Huang XF, Dorta-Estremera SM, Greeley NR, Nitti G, Peng W, Liu C, Lou Y, Wang Z, Ma W, Rabinovich B, Sowell RT, Schluns KS, Davis RE, Hwu P, Overwijk WW. Persistent antigen at vaccination sites induces tumor-specific CD8(+) T cell sequestration, dysfunction and deletion. Nat Med. 2013;19:465–472. doi: 10.1038/nm.3105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Schluns KS, Klonowski KD, Lefrancois L. Transregulation of memory CD8 T-cell proliferation by IL-15R alpha(+) bone marrow-derived cells. Blood. 2004;103:988–994. doi: 10.1182/blood-2003-08-2814. [DOI] [PubMed] [Google Scholar]
  • 26.Colpitts SL, Stonier SW, Stoklasek TA, Root SH, Aguila HL, Schluns KS, Lefrancois L. Transcriptional regulation of IL-15 expression during hematopoiesis. J Immunol. 2013;191:3017–3024. doi: 10.4049/jimmunol.1301389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Markiewski MM, Lambris JD. The role of complement in inflammatory diseases from behind the scenes into the spotlight. Am J Pathol. 2007;171:715–727. doi: 10.2353/ajpath.2007.070166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Ishikawa H, Ma Z, Barber GN. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature. 2009;461:788–792. doi: 10.1038/nature08476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ishikawa H, Barber GN. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature. 2008;455:674–678. doi: 10.1038/nature07317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Deng L, Liang H, Xu M, Yang X, Burnette B, Arina A, Li XD, Mauceri H, Beckett M, Darga T, Huang X, Gajewski TF, Chen ZJ, Fu YX, Weichselbaum RR. STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors. Immunity. 2014;41:843–852. doi: 10.1016/j.immuni.2014.10.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Woo SR, Fuertes MB, Corrales L, Spranger S, Furdyna MJ, Leung MY, Duggan R, Wang Y, Barber GN, Fitzgerald KA, Alegre ML, Gajewski TF. STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity. 2014;41:830–842. doi: 10.1016/j.immuni.2014.10.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Schluns KS, Kieper WC, Jameson SC, Lefrancois L. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nature Immunology. 2000;1:426–432. doi: 10.1038/80868. [DOI] [PubMed] [Google Scholar]
  • 33.Burkett PR, Koka R, Chien M, Chai S, Boone DL, Ma A. Coordinate Expression and Trans Presentation of Interleukin (IL)-15R{alpha} and IL-15 Supports Natural Killer Cell and Memory CD8+ T Cell Homeostasis. J Exp Med. 2004;200:825–834. doi: 10.1084/jem.20041389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Huntington ND, Legrand N, Alves NL, Jaron B, Weijer K, Plet A, Corcuff E, Mortier E, Jacques Y, Spits H, Di Santo JP. IL-15 trans-presentation promotes human NK cell development and differentiation in vivo. J Exp Med. 2009;206:25–34. doi: 10.1084/jem.20082013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Mortier E, Woo T, Advincula R, Gozalo S, Ma A. IL-15Ralpha chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation. J Exp Med. 2008;205:1213–1225. doi: 10.1084/jem.20071913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Nolz JC, Harty JT. IL-15 regulates memory CD8+ T cell O-glycan synthesis and affects trafficking. J Clin Invest. 2014;124:1013–1026. doi: 10.1172/JCI72039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Richer MJ, Pewe LL, Hancox LS, Hartwig SM, Varga SM, Harty JT. Inflammatory IL-15 is required for optimal memory T cell responses. J Clin Invest. 2015;125:3477–3490. doi: 10.1172/JCI81261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Davidoff AN, Mendelow BV. Cell-cycle disruptions and apoptosis induced by the cyclophosphamide derivative mafosfamide. Exp Hematol. 1993;21:922–927. [PubMed] [Google Scholar]
  • 39.Hemendinger RA, Bloom SE. Selective mitomycin C and cyclophosphamide induction of apoptosis in differentiating B lymphocytes compared to T lymphocytes in vivo. Immunopharmacology. 1996;35:71–82. doi: 10.1016/0162-3109(96)00124-5. [DOI] [PubMed] [Google Scholar]
  • 40.Eriksson D, Stigbrand T. Radiation-induced cell death mechanisms. Tumour Biol. 2010;31:363–372. doi: 10.1007/s13277-010-0042-8. [DOI] [PubMed] [Google Scholar]
  • 41.Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, Kazzaz Z, Bornstein E, Lambotte O, Altmann D, Blazar BR, Rodriguez B, Teixeira-Johnson L, Landay A, Martin JN, Hecht FM, Picker LJ, Lederman MM, Deeks SG, Douek DC. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med. 2006;12:1365–1371. doi: 10.1038/nm1511. [DOI] [PubMed] [Google Scholar]
  • 42.Do JS, Min B. IL-15 produced and trans-presented by DCs underlies homeostatic competition between CD8 and {gamma}{delta} T cells in vivo. Blood. 2009;113:6361–6371. doi: 10.1182/blood-2008-12-192997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Mattei F, Schiavoni G, Belardelli F, Tough DF. IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation. J Immunol. 2001;167:1179–1187. doi: 10.4049/jimmunol.167.3.1179. [DOI] [PubMed] [Google Scholar]
  • 44.Zhang X, Sun S, Hwang I, Tough DF, Sprent J. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity. 1998;8:591–599. doi: 10.1016/s1074-7613(00)80564-6. [DOI] [PubMed] [Google Scholar]
  • 45.Sikora AG, Jaffarzad N, Hailemichael Y, Gelbard A, Stonier SW, Schluns KS, Frasca L, Lou Y, Liu C, Andersson HA, Hwu P, Overwijk WW. IFN-alpha enhances peptide vaccine-induced CD8+ T cell numbers, effector function, and antitumor activity. J Immunol. 2009;182:7398–7407. doi: 10.4049/jimmunol.0802982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Castro I, Yu A, Dee MJ, Malek TR. The basis of distinctive IL-2- and IL-15-dependent signaling: weak CD122-dependent signaling favors CD8+ T central-memory cell survival but not T effector-memory cell development. J Immunol. 2011;187:5170–5182. doi: 10.4049/jimmunol.1003961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Obar JJ, Lefrancois L. Early signals during CD8 T cell priming regulate the generation of central memory cells. J Immunol. 2010;185:263–272. doi: 10.4049/jimmunol.1000492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Joshi NS, Cui W, Chandele A, Lee HK, Urso DR, Hagman J, Gapin L, Kaech SM. Inflammation directs memory precursor and short-lived effector CD8(+) T cell fates via the graded expression of T-bet transcription factor. Immunity. 2007;27:281–295. doi: 10.1016/j.immuni.2007.07.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Rubinstein MP, Lind NA, Purton JF, Filippou P, Best JA, McGhee PA, Surh CD, Goldrath AW. IL-7 and IL-15 differentially regulate CD8+ T-cell subsets during contraction of the immune response. Blood. 2008;112:3704–3712. doi: 10.1182/blood-2008-06-160945. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS774697-supplement-1.pdf (73KB, pdf)

RESOURCES