Transpresentation of interleukin 15 by stromal cell subsets regulates immune cell homeostasis

Stromal cells are important bone marrow (BM) niche components that regulate immune cell homeostasis through the production of cytokines such as interleukin 15 (IL-15). Although stromal-derived IL-15 is known to support lymphocyte survival, it remains unclear which stromal cell subsets are capable of IL-15 transpresentation, and how they influence specific lymphocyte populations. By using conditional IL-15 receptor alpha (IL-15Rα) deletion models, we demonstrate that IL-15Rα expression by BM stromal cells is essential for the maintenance of multiple IL-15-dependent lymphocyte populations. Deletion of IL-15Rα in Lepr⁺ or IL-7⁺ stromal cells selectively reduced central memory CD8⁺ T cells in the BM, whereas deletion of IL-15Rα in Osx⁺ stromal cells resulted in a marked loss of natural killer T (NKT) cells and tissue-resident memory CD8⁺ T cells. Surprisingly, endothelial-specific IL-15Rα deletion did not affect lymphocyte maintenance in the BM, but specifically impaired natural killer (NK) maturation and survival in the periphery, uncovering a role of endothelial IL-15 in mature NK cell maintenance. Together, our findings establish that transpresentation of IL-15 by distinct BM stromal cell subsets creates functionally specialized BM niches to support specific lymphocyte populations.

1 Introduction

The bone marrow (BM) is a semi-solid tissue within the medullar cavity of the bone. It serves as a reservoir for hematopoietic stem and progenitor cells as well as a diverse range of differentiated immune cell subsets, including long-lived memory T cells, natural killer (NK) cells, invariant NKT cells, and innate lymphoid cells (ILCs) (1, 2). These hematopoietic cells rely on survival signals and homeostatic cues from specialized microenvironments within the BM, which can be provided by stromal cell populations (3). Among the key cytokines that specifically sustain certain lymphocyte populations in the BM is interleukin 15 (IL-15), a pleiotropic cytokine of the common gamma chain (γc) family crucial for the survival and function of memory CD8⁺ T cells, NK cells, and NKT cells (4–6). Unlike other γc cytokines, IL-15 is not commonly secreted under physiological conditions but is instead presented in trans by cells expressing IL-15 complexed with its high-affinity receptor alpha chain (IL-15Rα). This unique mode of action, referred to as trans-presentation, requires physical interactions between IL-15-producing cells and responding lymphocytes that express the IL-2/15Rβ and common γc-chain (CD122 and CD132, respectively) (7, 8), resulting in tightly controlled, geographically restricted signaling. This delivery mechanism raises important questions regarding the identity and specialization of IL-15 transpresentation niches within lymphoid and non-lymphoid organs.

The regulation of IL-15 expression itself is complex and operates at multiple levels. At the transcriptional level, IL-15 expression is controlled by various stimuli including type I interferons, TLR ligands, or exposure to IFN-γ, which can differentially induce Il-15 in monocytes, dendritic cells, and stromal cells (9). However, high Il15 mRNA expression does not always correlate with protein production, owing to extensive post-transcriptional and translational control. The 5’ untranslated region (UTR) of IL-15 mRNA contains multiple upstream AUGs that inhibit efficient translation, and the protein is subject to stringent intracellular trafficking and degradation mechanisms (10–13). Similarly, IL-15Rα is regulated not only transcriptionally but also via trans-endosomal recycling, alternative splicing and shedding of its extracellular domain, which can yield soluble IL-15Rα with distinct biological functions (7, 14, 15).

Previous studies have highlighted the importance of IL-15 transpresentation in thymic development (16, 17), NK cell homeostasis (18, 19), and memory T cell survival (5, 20, 21), implicating hematopoietic and non-hematopoietic cells as potential sources of IL-15/IL-15Rα complexes (22). In particular, our recent work demonstrated that mesenchymal stromal cells (MSCs) in the BM produce IL-15 and are required for the maintenance of IL-15-dependent lymphocyte subsets under steady-state conditions (23). However, it is still unclear whether stromal cells merely produce soluble IL-15 or actively trans-present the cytokine via IL-15Rα to adjacent lymphocytes.

In this study, we define the ability of specific BM stromal cell subsets to trans-present IL-15 and to support various lymphocyte compartments in vivo. Our results reveal that IL-15Rα expression in Lepr⁺ and IL-7⁺ MSCs is critical for the maintenance of central memory CD8⁺ T cells, whereas Osx⁺ stromal cells are required for the survival of BM-resident NKT cells and tissue-resident memory CD8⁺ T cells. Endothelial cells, while largely dispensable for lymphocyte survival in the BM, were found to influence peripheral NK cell maintenance. By dissecting the stromal architecture of IL-15 delivery, our work provides new insight into the cellular division of labor that sustains lymphocyte diversity in the BM.

2 Materials and methods

2.1 Experimental animals

Mice were housed in individually ventilated cages (IVCs) under controlled environmental conditions (temperature, humidity, light cycles) with ad libitum access to food and water at the Core Facility Laboratory Animal Breeding and Husbandry (Medical University of Vienna). Animal experiments were performed in accordance with institutional and national guidelines and approved by the Austrian Federal Ministry of Education, Science and Research (66.009/0407-V/3b/2018 and 66.009/0408-V/3b/2018).

IL-15-IRES-EGFP knock-in mice were described previously (23). IL-15^flox mice were generated by Nan-Shih Liao (Academia Sinica, Taiwan; MTA #13T-1050130-16M) (24) and purchased from the Jackson Laboratory (JAX Stock No. 034188). IL-7 Cre mice (25) were generously provided by Ellen R. Ritchie (MD Anderson Cancer Center). Cdh5-CreERT2 mice (26) were kindly provided by Ralf H. Adams (Max Planck Institute for Molecular Biomedicine, Germany, MTA #151520 with CancerTools.org). CXCL12-DsRed mice (JAX No. 022458), IL-15Rα^flox mice (JAX No. 022365), IL-15Rα^KO mice (JAX No. 003723), Prx1-Cre mice (JAX No. 005584), Lepr-Cre mice (JAX No. 008320), Osx1-GFP-Cre mice (JAX No. 006361), Rosa26^{tm14(tdTomato)} (JAX No. 007914) and Rosa26^tm1(EYFP) mice (JAX No. 006148) were all obtained from the Jackson Laboratory.

To induce the expression of Cre in Cdh5-Cre/ERT2 mice, 1 mg of tamoxifen (Sigma-Aldrich, Cat# T5648) was intraperitoneally injected into mice older than 8-weeks for five consecutive days, and tissues were analyzed 3–6 weeks after the last day of injection.

All Cre-loxP-based conditional knockout experiments were conducted using littermate controls matched for age and sex. Age, number and sex distribution of the mice are indicated in detail in the Supplementary Table 2. Cre-driver lines (preferentially males) were backcrossed to floxed mice lacking any Cre-positive ancestry to minimize the risk of unintended recombination. All offspring was systematically screened for germline recombination using deletion-specific genotyping primers (listed in Supplementary Table 1).

The Cre driver lines used (Prx1-Cre, Lepr-Cre, Osx-Cre and Cdh5-Cre/ERT2) have been extensively characterized in prior studies (26–30). We previously validated knockout specificity (23) and additionally verified stromal targeting by flow cytometry and immunofluorescence of reporter mice (Supplementary Figures S6A-E).

2.2 Single cell RNA sequencing and data analysis

For single cell RNA sequencing of CAR cells from mouse long bones, stromal cells were isolated from CXCL12-DsRed^+/- mice. BM was flushed from femur and tibia and bones were crushed and digested in Collagenase IV (1mg/mL, Sigma-Aldrich) for 40 minutes to yield a single-cell suspension. Erythrocytes were lysed using ACK buffer (150mM NH₄Cl, 10mM KHCO₃, 0.1mM Na₂EDTA at pH 7.4). Live cells were stained with Aqua Zombie viability dye and sorted on a FACSMelody cell sorter (BD Biosciences) as CD45^-CD31^-Ter119^-DsRed⁺ stromal cells.

Single-cell libraries were then generated using the 10x Genomics Chromium Single Cell 3′ v3 kit, and sequencing was performed across two independent runs. Raw data were processed using CellRanger (version 7.1.0, 10x Genomics).

Data analysis was performed using the Seurat R package (version 5.0.1). Low-quality cells were excluded based on the number of detected features (cells with <200 or >6000 genes were removed) and high mitochondrial gene expression (>10%). Hematopoietic cells were excluded by filtering out cells expressing Ptprc and Gypa. Datasets were integrated using the Harmony R package, and dimensionality reduction and clustering were performed using UMAP. IL-15 and IL-15Rα-expressing cells were defined as cells with a non-zero expression value for IL-15 or IL-15Rα transcripts, respectively. Clusters with less than 30 cells were excluded from the quantification analysis. Stacked Violin plots were generated using the scCustomize package (31).

Re-analyzed datasets from published bone marrow (32–35) were described previously (23) and derived from Gene Expression Omnibus (GSE156635, GSE122467, GSE108892, GSE128423 and GSE273212).

2.3 Tissue preparation

Bone marrow was harvested by flushing femurs and tibiae with FACS buffer (PBS + 2% FBS + 2mM EDTA), followed by red blood cell lysis using ACK buffer (150mM NH4Cl, 10mM KHCO3, 0.1mM Na2EDTA at pH 7.4) and antibody staining for flow cytometric analysis. The spleen was processed by gentle mechanical disruption through a 70 μm cell strainer, followed by ACK lysis and staining. For isolation of immune cells from the liver, left liver lobes where mechanically disrupted through a 70µm cell strainer and then digested in 1mg/mL Collagenase IV and 1mg/mL DNase I (Sigma-Aldrich) in RPMI-1640 medium for 30 minutes at 37 °C. The resulting cell suspension was then topped up with 5mL FACS buffer and centrifuged at 50g for 3 minutes to de-enrich for hepatocytes. The supernatant was pelleted, resuspended in FACS buffer and layered onto Lymphoprep™ medium (Stemcell Technologies) in a Leukosep™ tube (Greiner) to separate mononuclear cells according to the manufacturer’s instructions. Peripheral blood was obtained by terminal retro-orbital bleeding of isoflurane-anaesthesized mice, followed by two rounds of ACK lysis. For endothelial cell analysis, spleens were digested with Collagenase IV (1mg/mL, Sigma-Aldrich) in RPMI-1640 medium for 30 minutes at 37 °C. Cells were then washed, ACK-lysed, and prepared for flow cytometry. To enrich for pericytes, collagenase-digested bone was stained with anti-CD146-PE for 20 minutes and then subjected to magnetic enrichment using the EasySep Mouse PE Positive Selection Kit (StemCell Technologies).

2.4 Flow cytometry

Single-cell suspensions were stained with commercial fluorescently conjugated antibodies for 40–60 minutes in 100ul FACS buffer per sample. For in vivo labelling of vascular leukocytes, 3µg anti-CD45-PE antibody (Biolegend, 30-F11 Cat#103106) were injected into isoflurane-anaesthesized mice 3–4 minutes before euthanasia and organ isolation (36, 37). The following antibodies were obtained from Biolegend: a4b7 (DATK32, APC, Cat# 120608), CD106 (429, AF647, Cat# 105712), CD117 (2B8, BV785, Cat# 105841); CD117 (ACK2, BV605, Cat# 135122), CD11b (M1/70, PerCP/BV650, Cat# 101230, 101259), CD127 (A7R34, PE, APC, Cat# 135009), CD135 (A2F10, BV421, Cat# 135314), CD144 (BV13, BV421, Cat# 138013), CD146 (ME-9F1, PE, Cat# 134704), CD19 (1D3/CD19, APC-Cy7, Cat# 152412), CD25 (3C7, PE-Cy7, Cat# 101916), CD27 (LG.3A10, FITC, Cat# 124207), CD31 (MEC13.3, PE-Cy7, Cat# 102523), CD3ϵ (145-2C11, BV785, Cat# 100355); CD3ϵ (500A2, APC-Fire750, Cat# 152308), CD3ϵ (17A2, APC, Cat#100236), CD4 (RM4-5, AF700, Cat# 100536), CD44 (IM7, FITC/BV785/BV711, Cat# 103006, 103059, 103057), CD45 (30-F11, AF700/APC-Fire750, Cat# 103128, 103154), CD49a (HM1a, PE-Cy7, Cat# 142608), CD49b (DX5, APC, Cat# 108910), CD54 (YN1/1.7.4, AF647, Cat# 116120), CD62L (MEL-14, PE-Cy7, Cat# 104417), CD69 (H1.2F3, PE/BV605, Cat# 104507), CD71 (R17217, APC-Fire750, Cat# 113828), CD8a (53-6.7, PerCP, Cat# 100732), CXCR3 (CXCR3-173, BV421, Cat# 126522), Eomes (W17001A, PE, Cat# 157705), Gr1 (Rb6-8C5, APC-Cy7, Cat# 108423), KLRG1 (2F1/KLRG1, PE-Cy7, Cat# 138416), NK1.1 (PK136, BV421/APC-Cy7, Cat# 108732, 560618), Sca1 (D7, FITC/BV785, Cat# 108105, 108139), T-bet (4B10, AF488, Cat# 644830), TCRβ (H57-597, APC-Fire750, Cat# 109246), and Ter119 (Ter119, APC-Cy7/FITC/BV421, Cat# 116223, 116206, 116233). CD8a (53-6.7, BB700, Cat# 566410) was obtained from BD Biosciences and LepR (polyclonal biotinylated, Cat# BAF497) from R&D Systems. The Zombie Aqua Viability staining kit (BioLegend, Cat# 423101) was used as a viability dye.

Samples were acquired on a BD LSR Fortessa X-20 or sorted on a FACSMelody cell sorter (BD Biosciences) located at the Center for Cancer Research. Compensation and data analysis was performed using FlowJo (version 10, BD Biosciences). For all samples, quality gates based on size and granularity (FSC-A vs SSC-A gate), doublet exclusion (FSC-A vs FSC-H) and dead cell exclusion (by gating on Zombie Aqua negative cells) were included. The gating strategies for the respective cell types can be found in the Supplementary Figures.

2.5 ELISA

Levels of IL-15/IL-15Rα complexes were quantified from cell lysates prepared from collagenase-digested bones using a non-denaturing lysis buffer (150 mM NaCl, 50 mM Tris-HCl with 1% Halt Protease Inhibitor Cocktail added before use). Total protein concentration of the lysates was measured using a bicinchoninic assay (BCA) kit (Thermo Fisher Scientific, Cat# 23227). Quantification was performed using the Mouse IL-15/IL-15R Complex Uncoated ELISA kit from Invitrogen (catalog no. 88-7215) according to the manufacturer’s protocol. A five-point standard curve was used for quantification, and data were fitted using five-parameter logistic regression (5PL). Concentrations were adjusted to sample total protein concentration. To account for inter-experimental variation, values were then normalized to the mean of littermate controls within each experiment.

2.6 qRT-PCR

RNA was extracted from FACS-sorted cells lysed in RLT Plus lysis buffer supplemented with 1% β-mercaptoethanol using the RNeasy Micro Plus kit from Qiagen. cDNA was synthesized with the RevertAid First Strand cDNA kit from Thermo Fisher Scientific (cat.no. K1621), using oligo-_dT priming. Quantitative reverse transcription PCR (qRT-PCR) was performed on a CFX96 TouchReal-Time PCR Detection System from BioRad using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, cat.no. K0222). Relative expression values were normalized to mouse beta-actin using the comparative threshold cycle method (2−ΔCt). Primers were synthesized by Sigma Aldrich. Primer sequences are listed in Supplementary Table 1.

2.7 Statistical analysis

Data are represented as mean ± standard error of the mean (SEM) of biological replicates. Statistical analyses were performed using GraphPad Prism version 8. For comparisons between two groups, unpaired two-tailed Student’s t-tests were used. A p-value <0.05 was considered statistically significant. Where indicated, p-values between 0.05 and 0.1 are reported above individual graphs. For comparisons involving more than two groups, one-way ANOVA followed by Tukey’s post hoc test was applied. Sample processing order was random and group allocation was determined by genotype.

3 Results

3.1 BM stromal cell subsets differentially express IL-15 and IL-15rα

We have recently shown that deletion of IL-15 from stromal cells using different conditional knockout mouse models results in a significant reduction of NK cell precursor and memory CD8⁺ T cell populations in the BM (23). It remains unclear whether stromal cells can trans-present IL-15 via IL-15rα, especially since deletion in Lepr⁺ MSCs, which are prominent IL-15 producers, had a small effect on IL-15-dependent immune cell populations in the BM. In fact, when comparing the abundance of IL-15- and IL-15rα-expressing stromal cells in an integrated scRNA-seq dataset from published repositories (32–35) (Figure 1A, Supplementary Figure S1A) and IL-15-enriched stroma from IL-15^GFP mice (23) (Figure 1B, Supplementary Figure S1B), it becomes apparent that the percentage of cells expressing IL-15, IL-15rα or both, varies greatly across different stromal cell types in the BM. While chondrocytes and pericytes had a very high IL-15rα/IL-15 ratio, mesenchymal stromal cell (MSC) populations, most prominently the CAR-like Lepr⁺ “MSC_stem”, hardly expressed any IL-15rα detectable by scRNA-seq (Figures 1A, B). Notably, the ratio between IL-15rα and IL-15 expression was higher in endosteal niche-associated stromal subsets like osteo-MSCs compared to CAR-like MSCs (Figures 1C, D). Furthermore, scRNA-seq from sorted Cxcl12⁺ MSCs and endothelial cells using a Cxcl12-DsRed reporter mouse-model indicated that CAR-like MSCs preferentially expressed IL-15 rather than IL-15rα or both IL-15rα and IL-15 (Supplementary Figures S1C, D).

Figure 1

Figure 1. IL-15 and IL-15Rα are differentially expressed by BM stromal cells during the steady state. (A) Public scRNA-seq dataset of mouse bone marrow (32–35) showing abundance of cells expressing IL-15, IL-15Rα or both. (B) IL-15/IL-15Rα expression of stromal cell clusters in a dataset enriched for IL-15 GFP expressing stroma (23). Clusters containing less than 30 cells were omitted in the quantification plots. (C) Fold ratio of IL-15Rα versus IL-15 producers in stromal cell clusters from mouse bone marrow, related to (A, D) Fold ratio of IL-15Rα versus IL-15 producers in stromal cell clusters from IL-15 enriched bone marrow, related to B.

Since scRNA-seq data are biased toward underestimating weakly expressed genes (38), the inherently low expression of the IL-15 and IL-15rα transcripts might not be representative of IL-15 complex protein expression in vivo. We therefore sorted IL-15^GFP+ and IL-15^GFP- BM macrophages and Lineage^-CD45^-CD31^- stromal cells from IL-15^GFP reporter mice and assessed the expression of IL-15 and IL-15rα via qPCR. IL-15 expression strongly correlated with IL-15rα expression in BM macrophages, but also IL-15^hi stromal cells expressed high amounts of IL-15rα mRNA (Supplementary Figure S1E).

In summary, there seems to be at least some variation in stromal IL-15rα expression, which raises the question whether Lepr⁺ stromal cells are able to trans-present IL-15 to immune cells, or whether they rather produce soluble IL-15 for IL-15rα-independent signaling.

3.2 Lepr⁺ MSCs trans-present IL-15 to central memory CD8⁺ T cells in the BM

We hypothesized that if stromal cells trans-present IL-15 to immune cells, deletion of IL-15rα should mimic the phenotypes of IL-15 deletion. Thus, we crossed Lepr-Cre mice to IL-15rα^flox mice for conditional deletion of IL-15rα from BM MSCs. We have previously established a role of MSC-derived IL-15 in specifically supporting central memory CD8⁺ T cells in the BM, and in supporting an age-dependent NK cell accumulation using Lepr-Cre mice (23). Indeed, central memory but not CD62L^- effector memory nor CD69⁺ tissue resident memory CD8⁺ T cells were significantly reduced in the BM of IL-15rα^flox Lepr-Cre mice compared to WT littermates (Figure 2A, B, Supplementary Figures S2A, B). The abundance of NK cells and NKT cells in the BM was unaffected (Supplementary Figures S2C, D).

Figure 2

Figure 2. IL-15Rα deletion in IL-7- and Lepr-expressing stromal cells leads to reduced CD8⁺ central memory T cells. Representative flow cytometry plots (A) and relative quantification (B) of central (T_CM), effector (T_EM) and tissue resident (T_RM) memory CD8⁺ T cells in the bone marrow of IL-15Rα^flox/flox Lepr-Cre mice and IL-15Rα^flox/flox controls. (C) Schematic layout of the generation of an IL-7-Rosa26^YFP fate reporter mouse. (D) Flow cytometry plots showing YFP⁺CD45^- cells in wild type controls and IL-7-Cre Rosa26^YFP mice. (E) Representative flow cytometry histogram and quantification of LepR expression in YFP⁺ cells. Representative flow cytometry plots (F) and relative quantification (G) of central (T_CM), effector (T_EM) and tissue resident (T_RM) memory CD8⁺ T cells in the bone marrow of IL-15Rα^flox IL-7-Cre mice and IL-15Rα^flox/flox littermates. Dot plots show the mean ± SEM with each dot representing a biological replicate. Asterisks above the dot plots indicate P values from unpaired two-tailed Student’s t tests. *P < 0.05, n.s. P > 0.05.

Since central memory T cells depend on both IL-7 and IL-15 for their long term survival (39, 40), we next asked whether IL-7 and IL-15 are provided by the same stromal cells or derived from different stromal niches. In order to track IL-7 expression in the BM, we crossed a Rosa26-YFP strain to IL-7 Cre mice (Figure 2C). Within the BM of these IL-7 fate reporter mice, YFP was exclusively expressed in CD45^- stromal cells (Figure 2D) and the YFP⁺ population strongly overlapped with Lepr⁺ MSCs (Figure 2E). On the other hand, IL7⁺ stromal cells represented a distinct subset of Lepr⁺ MSCs (Supplementary Figure S2E). Accordingly, scRNA-seq data show a small fraction of IL-7/IL-15 double producers within the stem-like MSC population (Supplementary Figure S2F). When crossing IL-15rα^flox mice to IL-7-Cre mice, we observed a reduction in central memory but not CD62L^- effector memory or CD69⁺ tissue resident CD8⁺ T cells (Figures 2F, G), mirroring the Lepr-Cre phenotype and further supporting the observation that CD8 T_CM, but not T_RM, rely on MSC-mediated IL-15 transpresentation. IL-15rα^flox IL-7-Cre mice further showed a slight reduction of mature Eomes⁺ NK1.1⁺ NK cells, but not NKT cells, in the BM (Supplementary Figure S2G). A similar phenotype has previously been observed in aged IL-15^flox Lepr-Cre mice (23).

In summary, a polyfunctional MSC population that is capable of producing both IL-7 and IL-15 throughout development appears to specifically support central memory T cells in the BM.

3.3 IL-15 trans-presentation by endosteal stromal cells supports tissue resident memory CD8⁺ T cells and NKT cells

Since stromal cells targeted by Osx-Cre have been shown to produce IL-15 important for memory CD8⁺ T cell subsets and NKT cells, we next asked whether Osx⁺ stromal cells were able to transpresent IL-15 via IL-15Rα. We therefore crossed IL-15rα^flox with Osx-Cre: GFP mice and assessed immune cell abundances in the BM.

IL-15rα deletion had a pronounced impact on the abundance of CD8⁺ memory T cells, in particular CD69⁺ tissue resident memory (CD8 T_RM) cells, although not reaching the levels of IL-15rα^-/- mice (Figures 3A, B). This phenotype was BM-specific, as neither NK cells nor CD8 T_CM cells were reduced in the spleen or blood of IL-15rα^flox Osx-Cre mice (Figure 3C, Supplementary Figure S3A–C). Similarly, the relative abundance of hematopoietic stem cells (LSK) and common lymphoid progenitors (CLP) was not affected (Supplementary Figures S3D, E), arguing against a general effect on hematopoiesis. CD3⁺NK1.1⁺ NKT cells, but not CD3^-NK1.1⁺Eomes⁺CD127^- NK cells, were also significantly reduced in the BM of IL-15rα^flox Osx-Cre mice compared to WT littermates (Figures 3D, E), mirroring the phenotype observed in IL-15^flox Osx-Cre mice (23). Since the Osx-Cre transgene is associated with reduced overall bone size and cellularity (Supplementary Figure S3F) (41), only relative quantifications have been considered for this strain.

Figure 3

Figure 3. IL-15Rα deletion in Osx-Cre targeted stromal cells leads to loss of memory T cells and NKT cells in the bone marrow. Representative flow cytometry plots (A) and relative quantification (B) of central (T_CM), tissue resident (T_RM), and effector memory CD8⁺ T cells (T_EM) in the bone marrow of IL-15Rα^flox Osx-Cre mice compared to IL-15Rα^flox/flox and total knockout (IL-15Rα^KO/KO) controls. (C) Relative quantification of CD44⁺CD62L⁺ central memory CD8⁺ T cells (CD8 T_CM) in the spleen of IL-15Rα^flox Osx-Cre mice compared to IL-15Rα^flox/flox littermates. Representative flow cytometry plots (D) and relative quantification (E) of NK cells and CD3⁺NK1.1⁺ NKT cells in the bone marrow of IL-15Rα^flox Osx-Cre mice compared to IL-15Rα^flox/flox and total knockout (IL-15Rα^KO/KO) controls. Dot plots show the mean ± SEM of biological replicates. Asterisks above the dot plots indicate P values from Tukey’s multiple comparisons test after One-Way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. P > 0.05.

In summary, Osteo-MSCs displayed a higher IL-15rα/IL-15 ratio and Osx-Cre-mediated IL-15rα deletion resulted in a BM-specific reduction of memory CD8⁺ T cells and NKT cells.

3.4 IL-15 trans-presentation by Prx1⁺ stromal cells supports NK cell maturation

Given that Prx1-Cre targets all mesenchymal cells, we anticipated a greater or at least comparable effect to that observed with the previously used Lepr-Cre and Osx-Cre lines. Similar to IL-15 deletion from Prx1-targeted cells (23), NK cells and NKT cells were not affected in the BM of IL-15rα^flox Prx1-Cre mice (Figure 4A). In contrast to previous observations in IL-15^flox Prx1-Cre mice, we surprisingly observed no reduction of memory CD8 T cells in the BM of IL-15rα^flox Prx1-Cre mice (Figure 4B, Supplementary Figure S4A) (23). In the spleen, there was a trend (not reaching statistical significance) toward less NK cells but not memory CD8⁺ T cells (Figure 4C, Supplementary Figures S4B, C), which fits the phenotype observed with IL-15 deletion (23). Accordingly, no changes in IL-15-dependent lymphocytes were observed in the blood of IL-15rα^flox Prx1-Cre mice (Supplementary Figure S4D). BM NK cells, although not differing in overall levels, also expressed significantly less KLRG1, and less of the NK cells were of the fully mature CD11b⁺CD27^- phenotype (Figures 4D, E, Supplementary Figures S4E, F). Additionally, BM lysates from IL-15rα^flox Prx1-Cre, but not IL-15rα^flox Osx-Cre mice, contained significantly less overall IL-15 complexed to IL-15rα (Figure 4F).

Figure 4

Figure 4. IL-15Rα deletion in Prx1-cre targeted mesenchymal stromal cells affects NK cell maturation. (A) Relative quantification of NK cells and NKT cells in the bone marrow of IL-15Rα^flox Prx1-Cre mice compared to IL-15Rα^flox/flox littermates. (B) Relative quantification of central (CD8 T_CM) and tissue resident memory CD8⁺ T cells (CD8 T_RM) in the bone marrow of IL-15Rα^flox Prx1-Cre mice compared to IL-15Rα^flox/flox littermates. (C) Quantification of central memory CD8⁺ T cells and NK cells in the spleens of IL-15Rα^flox Prx1-Cre mice compared to IL-15Rα^flox/flox littermates. Representative flow cytometry plots (D) and quantification (E) of NK maturation markers. Shown are the relative abundance of the terminally mature CD11b⁺CD27^- NK subpopulation among total bone marrow NK cells (pre-gated on CD45⁺Lin^-SSC-A^lowCD3^-NK1.1⁺) as well as the percentage of Klrg1⁺ NK cells. (F) ELISA comparing IL15/IL15RA complex (quantified as pg/mg total protein and normalized to littermate controls) in the bone marrow of IL-15Rα^flox/flox controls, IL-15Rα^flox/flox Prx1-Cre mice, IL-15Rα^flox/flox Osx-Cre mice and IL-15Rα^KO/KO mice. (G) Representative flow cytometry histogram (left) and quantification (right) of CD146 expression among IL-15^GFP+ stromal cells. (H) Flow cytometry plot from digested bone of IL-15^GFP mice pre-enriched for CD146 (Mcam) and pre-gated on CD31^-Lin^-CD45^- stromal cells showing lack of IL-15-GFP expression in CD146⁺ cells. Dot plots show the mean ± SEM of biological replicates. Asterisks above the dot plots indicate P values from unpaired two-tailed Student’s t tests. *P < 0.05, ****P < 0.0001, n.s. P > 0.05.

Because this partial phenocopy was unexpected, we next explored whether certain stromal subsets targeted by Prx1-Cre but not by Osx-Cre or Lepr-Cre might express IL-15Rα in the absence of IL-15, thereby potentially exerting a cytokine scavenging rather than transpresenting role. IL-15rα expression has previously been shown to be able to exert anti-inflammatory, scavenging effects counteracting the effects of IL-15 (42, 43). Based on our transcriptional data (Figure 1B), chondrocytes and pericytes showed the highest IL-15rα/IL-15 ratios. While chondrocytes have been shown to produce IL-15 (23) and are also targeted by Osx-Cre (44), pericytes appeared as a candidate stromal cell population expressing IL-15Rα without corresponding IL-15 production. Indeed, pericytes were under-represented among IL-15^GFP+ sorted stromal cells (23) (Figure 1B) and IL-15^GFP+ Lin^- CD45^-CD31^- stromal cells were mostly negative for the pericyte marker Mcam (CD146) (Figure 4G). Similarly, in CD146- enriched stromal fractions from digested bone of IL-15^GFP mice, IL-15^GFP expression was absent from the Mcam-positive population, suggesting that BM pericytes are IL-15rα⁺ and IL-15^-, and could thereby locally modulate IL-15 bioavailability (Figure 4H).

3.5 IL-15 transpresentation regulates endothelial-NK cell interaction and NK cell survival

Endothelial cells produce IL-15 in both mice and humans (23, 45, 46). However, deleting mouse IL-15 from endothelial cells has no effect on immune cell abundances in the BM (23), and neither did IL-15rα deletion in the BM when using tamoxifen-inducible Cdh5-iCre mice (Supplementary Figures S5A, B). Accordingly, BM lysates from IL-15rα Cdh5-iCre mice had unchanged total amounts of IL-15/IL-15rα complex (Supplementary Figure S5C). However, endothelial cell-specific deletion of IL-15rα led to significantly reduced NK cells, but not central memory CD8⁺ T cells, in the blood (Figures 5A, B, Supplementary Figures S5D, E) and spleen (Figure 5C, Supplementary Figure S5D). Notably, blood NK cell numbers were further reduced in Cdh5-iCre mice on an IL-15rα^flox/KO background compared to their IL-15rα^flox/KO littermate controls (Figure 5D), suggesting a gene dosage-dependent effect of IL-15rα loss. When comparing circulating NK cells from IL-15^flox conditional knockout mice, Cdh5-iCre littermates showed a lower expression of the cell adhesion marker CD44 (Supplementary Figure S5E), suggesting a potential role for endothelial IL-15 in NK cell tissue retention and/or trafficking. On the other hand, endothelial cells from the spleen of IL-15^flox Cdh5-iCre mice showed a trend towards lower ICAM-1 expression (Supplementary Figures S5F, G). To further explore the potential impact of endothelial IL-15 on NK cell trafficking or maturation, we examined expression of the CXC chemokine receptor 3 (CXCR3), which was increased among total NK cells from IL-15^flox Cdh5-iCre mice across several compartments, including the blood, spleen and bone marrow (Supplementary Figure S5H). However, since Cxcr3 is more prevalent on immature NK cells, this change might represent a compositional shift rather than altered chemokine responsiveness. Consistent with this, the loss of NK cells in the blood specifically affected mature CD11b⁺CD27^- NK cells (Figure 5E). To elucidate whether the decrease in NK cells reflected altered tissue egress or peripheral maintenance, we next assessed possible changes of vascular versus parenchymal NK cells in different tissues using intravenous injection of CD45-PE to label intravascular NK cells. As observed in in IL-15rα^flox Cdh5-iCre mice (Supplementary Figure S5B), the abundance of total NK cells in the bone marrow of IL-15^flox Cdh5-iCre mice was unchanged (Figure 5F). However, vascular PE-labelled NK cells, and among them specifically mature CD11b⁺CD27^- NK cells, were reduced while their parenchymal counterparts remained unaffected (Figures 5G, H, Supplementary Figures S5I, J). In the spleen, parenchymal NK cells were more pronouncedly decreased in the absence of endothelial IL-15 compared to vascular NK cells (Figure 5H, Supplementary Figure S5K). Similarly, total and parenchymal NK cells in the liver of IL-15^flox Cdh5-iCre mice were significantly reduced compared to littermate controls (Figure 5I, Supplementary Figure S5L). A not statistically significant reduction was observed for vascular NK cells in spleen and liver. These results indicate that endothelial IL-15 deletion specifically supports mature NK cell maintenance in the circulation, in the (peri-)vascular niche of the bone marrow and in highly vascularized organs such as the spleen and liver.

Figure 5

Figure 5. Endothelial IL-15Rα regulates peripheral NK cells and adhesion molecule expression. (A) Representative flow cytometry plot and relative quantification of NK cells from the blood of tamoxifen-induced IL-15Rα^flox/flox Cdh5-Cre/ERT2 (Cdh5-iCre) mice compared to IL-15Rα^flox/flox littermates. All mice were injected with 1mg tamoxifen i.p. for 5 consecutive days, and analyzed 3 to 6 weeks later. (B) Relative abundance of central memory CD8⁺ T cells (CD8 T_CM) in the blood of tamoxifen-induced IL-15Rα^flox/flox Cdh5-Cre/ERT2 (Cdh5-iCre) mice compared to IL-15Rα^flox/flox littermates (IL-15Rα^fl). (C) Relative quantification of (NK1.1⁺CD3^-CD49b⁺CD127^-) NK cells and central memory CD8⁺ T cells in the spleen of IL-15Rα^flox/flox Cdh5-Cre/ERT2 (Cdh5-iCre) mice compared to IL-15Rα^flox/flox littermates. (D) Relative quantification of NK cells and central memory CD8⁺ T cells in the blood of IL-15Rα^flox/KO Cdh5-Cre/ERT2 (Cdh5-iCre) mice on a mixed B6/129S background compared to IL-15Rα^flox/KO littermates. (E) Relative abundance of the indicated NK cell maturation stages in the blood of tamoxifen-induced IL-15^flox/flox Cdh5-iCre mice compared to IL-15^flox/flox controls. (F) Representative flow cytometry plot (left) and quantification (right) of bone marrow NK1.1⁺CD3^- NK cells in IL-15^flox/flox and IL-15^flox/flox Cdh5-iCre mice, subdivided into parenchymal (CD45-PE^-) and vascular NK cells (CD45-PE⁺). (G) Relative abundance of parenchymal (CD45-PE^-) and vascular (CD45-PE⁺) mature CD11b⁺CD27^- NK cells in the bone marrow of IL-15^flox/flox Cdh5-iCre mice compared to IL-15^flox/flox littermates. (H) Relative quantification of splenic NK1.1⁺CD3^- NK cells in IL-15^flox/flox versus IL-15^flox/flox Cdh5-iCre mice, subdivided into parenchymal (CD45-PE^-) and vascular NK cells (CD45-PE⁺). (I) Relative quantification of NK1.1⁺ NK cells int the livers of IL-15^flox/flox and IL-15^flox/flox Cdh5-iCre mice, subdivided into parenchymal (CD45-PE^-) and vascular NK cells (CD45-PE⁺). One animal was excluded from the quantification due to loss during the CD45-PE labeling procedure. Dot plots show the mean ± SEM of biological replicates and asterisks above the plots indicate P values from unpaired two-tailed Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. P > 0.05.

In summary, endothelial IL-15/IL-15rα deletion had a pronounced impact on peripheral mature NK cells, while central memory CD8⁺ T cell abundance was unaffected.

4 Discussion

IL-15 is a critical cytokine for the survival and function of several lymphocyte subsets, including memory CD8⁺ T cells, NK cells and iNKT cells (6, 47–49). While it is well established that IL-15 is primarily presented in trans by cells expressing IL-15Rα (7, 50), the precise cellular sources and mechanisms of this transpresentation in the BM remain incompletely understood. In this study, we investigated the contribution of BM stromal cells to IL-15/IL-15Rα-mediated support of lymphocyte populations, with a focus on dissecting the cellular and spatial heterogeneity of this system. We reveal that distinct stromal cell populations exhibit highly variable expression of IL-15 and IL-15Rα, and that different lymphocyte subsets rely on specific stromal niches for IL-15Rα-dependent maintenance and survival.

We and others have previously shown that BM stromal cells, particularly MSCs, are capable of producing IL-15 (51) and that stromal IL-15 contributes to the maintenance of memory CD8⁺ T cells and developing NK cells (23). However, it remains unclear whether these cells are capable of transpresenting IL-15 via IL-15Rα, or whether alternative mechanisms of IL-15 delivery are used. Analysis of scRNA-seq data demonstrated a striking decoupling of IL-15 and IL-15Rα mRNA levels among BM stromal subsets (Figure 1). This would suggest an unevenly distributed capacity for IL-15 transpresentation across the stromal compartment, theoretically requiring intercellular cooperation between IL-15⁺ and IL-15Rα⁺ cells. While transpresentation is likely the primary mode of IL-15 signaling, previous studies also support non-classical or uncoupled mechanisms of IL-15activity, e.g. in the context of tumor growth where IL-15Rα can mediate autonomous survival signaling (52). Likewise, there is some evidence of in cis signaling functions of the IL-15Rα (53), e.g. by supporting stromal function and bone mineralization (54, 55). Similarly, IL-15Rα has been implicated in IL-15-independent in cis signaling of IL-17-producing γδ T cells, suggesting functional plasticity of the receptor (56). Additionally, both IL-15 and IL-15Rα are heavily post-transcriptionally regulated, complicating the interpretation of RNA expression data (15, 57). Lepr⁺ MSCs, a known source of IL-15, showed negligible IL-15Rα expression by scRNA-seq, while other subsets such as osteo-lineage MSCs and pericytes had a higher IL-15Rα/IL-15 ratio. However, scRNA-seq very likely underestimates low-abundance transcripts like IL-15Rα (38), since IL-15-GFP expression still correlated with IL-15Rα mRNA abundance in stromal cells. We further demonstrate that Lepr⁺ MSCs, despite low IL-15Rα mRNA expression, contribute to the maintenance of central memory (T_CM) CD8⁺ T cells via IL-15 transpresentation (Figure 2). Conditional deletion of either IL-15 or IL-15Rα in Lepr⁺ MSCs led to a comparable slight reduction in BM-resident T_CM cells under steady state conditions (23), consistent with the requirement of both components for transpresentation. Fate mapping with IL-7-Cre Rosa26-YFP mice further revealed that a subset of Lepr⁺ MSCs co-expressed IL-7, identifying them as a potential double source of IL-7 and IL-15 signals essential for T_CM survival. Prior studies have emphasized the complementary roles of IL-7 and IL-15 in maintaining CD8⁺ T cell memory, and different memory T cell subsets show varying degrees of IL-7 dependency (40, 58). Notably, the effect of Lepr/IL-7-mediated IL-15Rα deletion was specific to T_CM cells and spared tissue-resident memory (T_RM) cells and NKT cells, supporting the notion of a specific IL-7/IL-15 codependency niche (48). Interestingly, the reduction of T_CM cells upon IL-15Rα deletion in IL-7⁺ MSCs also led to a slight, albeit not statistically significant, increase in effector memory (T_EM) cells, potentially due to niche competition and the reallocation of resources. However, the CD8 T_CM phenotype was subtle under steady-state conditions, especially with regard to absolute quantification. Since the reduction of bone marrow CD8 T_CM in an Lepr-Cre induced IL-15 deletion model has been shown to be age-dependent (23), suggesting that the subtle phenotype observed here in younger adult mice (8–16 weeks) may become more pronounced in older animals. Nonetheless, further studies under conditions of immune activation, like infection or vaccination, are required to determine whether these subtle steady-state alterations have functional consequences or remain without biological relevance.

While Lepr⁺ MSCs predominantly support T_CM cells, we found that endosteal stromal cells targeted by Osx-Cre additionally maintain CD8 T_RM and NKT cells through IL-15 transpresentation (Figure 3). Deletion of IL-15Rα in Osx⁺ cells resulted in a marked reduction of these lymphocyte populations, indicating that endosteal niches are functionally distinct from other BM stromal niches. Surprisingly, deletion of IL-15Rα in mesenchymal stromal cells using Prx1-Cre had only modest effects on CD8⁺ T cells, despite targeting a diverse range of stromal populations (Figure 4). One possible explanation for this attenuated phenotype is the presence of Prx1-targeted stromal subsets, such as IL-15rα⁺ IL-15^- pericytes, that may act as local cytokine “scavengers”. Similar to CD25-mediated IL-2 scavenging by regulatory T cells, IL-15Rα-expressing stromal cells may buffer IL-15 availability, thus modulating the cytokine landscape. This model is supported by literature describing anti-inflammatory functions of (soluble) IL-15Rα and its role in fine-tuning cytokine responses (42, 43). While this hypothesis remains to be functionally tested, the observed expression pattern of IL-15 and IL-15rα in bone marrow pericytes is consistent with a potential modulatory role of these cells in local IL-15 availability. Such an effect could contribute to the attenuated phenotype observed upon IL-15rα deletion in Prx1-Cre mice.

Our data further implicate endothelial cells in the support of NK cells through IL-15 transpresentation (Figure 5). Deletion of IL-15Rα in endothelial cells impaired NK cell maintenance in the periphery, yet had a minor effect on CD8⁺ T cells, which is surprising since endothelial IL-15 deletion has been shown to affect CD8⁺ T cells as well (23). This aligns with previous work suggesting that soluble IL-15 may be sufficient to expand memory CD8⁺ T cells, which are less reliant on cell-bound IL-15Rα transpresentation than NK cells, highlighting differential sensitivity among lymphocyte subsets (59, 60). IL-15 has also been shown to enhance endothelial-NK cell adhesion via LFA-1/ICAM-1 (61) and endothelial IL-15 can influence lymphocyte trans-endothelial migration (45). Along these lines, we also observed subtle changes in adhesion molecule expression on both NK cells and splenic endothelial cells, suggesting a potential role of endothelial IL-15Rα in NK-endothelial cellular interactions.

There is increasing evidence that IL-15 functions in both a homeostatic and an inflammatory mode, which are shaped by differences in dose, cellular source, and context. We showed that, under steady-state conditions, stromal and endothelial cells provide low-level, spatially restricted IL-15 transpresentation that moderately supports NK and CD8⁺ T-cells, whereas during acute activation dendritic cells and monocytes have been shown to rapidly upregulate IL-15 to drive effector responses (5, 7, 62). In line with this model, we have previously shown that NK cells from stromal IL-15-deficient mice retain normal per-cell cytokine production (23), suggesting that stromal IL-15 mainly calibrates homeostatic development rather than acute effector function. Although we would not expect stromal IL-15 deletion to impair early responses during acute infection, future studies will be required to determine whether stromal IL-15 influences immune function in chronic inflammatory settings, including tumor growth, tissue rechallenge, or sustained antigen exposure, where prolonged niche interactions and long-term memory formation may become limiting factors.

Together, our findings support a model in which IL-15 transpresentation is compartmentalized within specialized stromal niches of the BM and the periphery. Not all stromal cells support all lymphocytes equally. Rather, specific subsets are tailored to deliver IL-15 or IL-15Rα/IL-15 to distinct immune cell populations. The observed effects remained moderate, indicating that other cellular sources can compensate for the loss of stromal IL-15/IL-15Rα. This niche specialization ensures the maintenance of immune cell diversity and allows for context-specific modulation of cytokine signaling. Thus, our work highlights the importance of accounting for stromal cell heterogeneity in studies of immune regulation, with implications for regenerative medicine, autoimmune disease research and cancer.

Data availability statement

The original scRNA-seq data generated in the present study have been deposited on the Open Science Framework platform (accessible via https://osf.io/b3tex/overview?view_only=634be02df77449ffbad7b1b41de484a9). Re-analyzed bone marrow datasets from previously published studies were derived from Gene Expression Omnibus (accession numbers GSE156635, GSE122467, GSE108892, GSE128423 and GSE273212).

Ethics statement

The animal study was approved by Austrian Federal Ministry of Education, Science and Research (No. 66.009/0407-V/3b/2018 and 66.009/0408-V/3b/2018). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

CS: Conceptualization, Funding acquisition, Investigation, Formal analysis, Methodology, Visualization, Writing – original draft. RB: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – review & editing. EP: Investigation, Writing – review & editing. AS-B: Investigation, Writing – review & editing. SF: Investigation, Writing – review & editing. NB: Investigation, Writing – review & editing. ER: Writing – review & editing, Resources. MF: Writing – review & editing, Funding acquisition, Investigation. RF: Funding acquisition, Writing – review & editing, Supervision. DH-B: Funding acquisition, Supervision, Writing – review & editing, Conceptualization, Investigation, Project administration.

Funding

The author(s) declared that financial support was received for this work and/or its publication. C.S. was supported by an APART-MINT postdoctoral fellowship of the Austrian Academy of Sciences. R.B. was supported by a DOC fellowship of the Austrian Academy of Sciences. This research was funded in whole or in part by the Austrian Science Fund (FWF) (10.55776/F61) to M. Farlik, and (10.55776/P33340) and (10.55776/P36995) to D.H.-B. The work was supported by the Vienna Science and Technology Fund (WWTF) (10.47379/LS20042) to D.H.-B. and M. Farlik.

Acknowledgments

We would like to thank Nan-Shih Liao (Academia Sinica, Taipei, Taiwan) for sharing Il15^flox/flox mice and Ralf H. Adams (Max Planck Institute for Molecular Biomedicine, Münster, Germany) for sharing Cdh5-Cre^ERT2 mice. We would like to thank Judith Stein, Linda Evangelisti and Cynthia Hughes (Yale University, New Haven, USA) for their help in generating Il15-GFP mice, and Valerie Plajer, Melanie Lietzenmayer and Johannes Reisecker for technical assistance.

Conflict of interest

The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2025.1673309/full#supplementary-material

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