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Neural stem cells sustain natural killer cells that dictate recovery from brain inflammation

Nature Neuroscience volume 19, pages 243–252 (2016)Cite this article

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Abstract

Recovery from organ-specific autoimmune diseases largely relies on the mobilization of endogenous repair mechanisms and local factors that control them. Natural killer (NK) cells are swiftly mobilized to organs targeted by autoimmunity and typically undergo numerical contraction when inflammation wanes. We report the unexpected finding that NK cells are retained in the brain subventricular zone (SVZ) during the chronic phase of multiple sclerosis in humans and its animal model in mice. These NK cells were found preferentially in close proximity to SVZ neural stem cells (NSCs) that produce interleukin-15 and sustain functionally competent NK cells. Moreover, NK cells limited the reparative capacity of NSCs following brain inflammation. These findings reveal that reciprocal interactions between NSCs and NK cells regulate neurorepair.

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Figure 1: Retention of NK cells in the SVZ of MS patients.
Figure 2: Preferential accumulation of NK cells in the SVZ during progression of brain inflammation.
Figure 3: NSCs have a distinct cytokine profile during progression of brain inflammation.
Figure 4: NSC-derived IL-15 is necessary to maintain NK cell proliferation, survival and IFN-γ production.
Figure 5: Coordinated upregulation of IL-15 and IL-15Rα in NSCs during progression of brain inflammation.
Figure 6: IL-15Rα upregulation in NSCs during the late stages of brain inflammation.
Figure 7: NK cells eliminate NSCs during the late stages of brain inflammation.
Figure 8: Altered expression of MHC class I molecules in NSPCs and effects of NK cell removal on EAE.

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Acknowledgements

We thank Y. Yan for technical help. This study was supported in part by National Basic Research Program of China grant 2013CB966900; National Science Foundation of China grants 81230028, 81301044 and 81471535; the National Key Clinical Specialty Construction Program of China; US National Institutes of Health grants R01NS082745, R01CA175391 and R01DK081536; and American Heart Association grant 16SDG27250236; and a National Multiple Sclerosis Society research grant.

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Authors and Affiliations

  1. Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China

    Qiang Liu, Wei-Na Jin & Fu-Dong Shi

  2. Division of Neurology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, Arizona, USA

    Qiang Liu, Wei-Na Jin & Fu-Dong Shi

  3. Barrow Brain Tumor Research Center, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, Arizona, USA

    Nader Sanai

  4. Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California, USA

    Antonio La Cava

  5. Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Luc Van Kaer

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Contributions

Q.L., N.S. and F.-D.S. designed the studies; Q.L. and W.-N.J. performed the studies; A.L.C. and L.V.K. advised on design, execution of experiments and interpretation of results; and Q.L., A.L.C., L.V.K. and F.-D.S. wrote and edited the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Fluorescence minus one (FMO) controls for the identification of CNS-infiltrating NK cells.

Single cell suspensions were prepared from CNS tissues of Cd1d−/− or Cd1d−/−NK1.1-tdTomato+ mice at day 30 of EAE and stained with fluorochrome-conjugated antibodies. a-b. Representative flow cytometry dot plots show the identification of gating boundaries of CD3 (FITC), td-Tomato and IFN-γ (APC) with corresponding FMO controls.

Supplementary Figure 2 Reduced expression of proinflammatory factors during the recovery phase of EAE.

CNS homogenates were prepared from EAE mice of the indicated groups at 14, 30, 40, 60 dpi. Concentrations were measured by a Multi-Analyte ELISArray kit. Average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 3.5 ± 0.7 at 14 dpi, 2.5 ± 0.7 at 30 dpi, 2.3 ± 0.8 at 40 dpi and 2.1 ± 0.8 at 60 dpi. n = 9 per group. IFN-γ: P < 0.001 (14 dpi versus 30 dpi), P < 0.001 (14 dpi versus 40 dpi), P < 0.001 (14 dpi versus 60 dpi). F (3, 32) = 8.30, one-way ANOVA. IL-17A: P < 0.001 (14 dpi versus 30 dpi), P < 0.001 (14 dpi versus 40 dpi), P < 0.001 (14 dpi versus 60 dpi). F (3, 32) = 6.11, one-way ANOVA. TNF-α: P < 0.001 (14 dpi versus 30 dpi), P < 0.001 (14 dpi versus 40 dpi), P < 0.001 (14 dpi versus 60 dpi). F (3, 32) = 6.83, one-way ANOVA. IL-1β: P < 0.001 (14 dpi versus 30 dpi), P < 0.001 (14 dpi versus 40 dpi), P < 0.001 (14 dpi versus 60 dpi). F (3, 32) = 7.16, one-way ANOVA. IL-6: P < 0.001 (14 dpi versus 30 dpi), P < 0.001 (14 dpi versus 40 dpi), P < 0.001 (14 dpi versus 60 dpi). F (3, 32) = 5.54, one-way ANOVA. MCP-1: P < 0.001 (14 dpi versus 30 dpi), P < 0.001 (14 dpi versus 40 dpi), P < 0.001 (14 dpi versus 60 dpi). F (3, 32) = 3.39, one-way ANOVA. Results are from three individual experiments. Error bars represent s.e.m.; ** P < 0.01.

Supplementary Figure 3 FMO controls for the identification of SVZ type B, type C and type A cells.

Single cell suspensions were prepared from dissociated SVZ tissues of WT adult mice and stained with fluorochrome-conjugated antibodies. a. Representative flow cytometry dot plots show the identification of gating boundaries of GFAP (PE), CD24 (FITC) and EGFR (AF647) with corresponding FMO controls. b. Representative flow cytometry dot plots show SVZ type B (GFAP+EGFR+CD24−), type C (GFAP−EGFR+CD24−) and type A (GFAP−EGFR−CD24low) cell types from WT mice before sorting. c. Representative flow cytometry dot plots show purity of SVZ type B, type C and type A cell types (>98%) after sorting.

Supplementary Figure 4 Representative factors produced by CNS cells.

a. ELISA for IL-15, VEGF, IL-4, and MCP-1 from lysates of sorted astrocytes, microglia, neurons and NSCs show that IL-15 is a major factor produced by NSCs as compared to other CNS cells during the recovery phase of EAE (30 dpi). b-c. Immunostaining shows that most IL-15-expressing cells in SVZ are GFAP+, whereas only a small portion (<10%) of IL-15+ cells are microglia/macrophages (Iba1+). Average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 2.3 ± 0.6 at 30 dpi. n = 12 per group. Data represent three individual experiments. Error bars represent s.e.m.

Supplementary Figure 5 Removal of IL-15 from NSCs prevents NK cell retention during the late stage of brain inflammation.

a. Schematic map of the nestin-specific shIL-15 lentiviral vector. b. Intraventricular injection of 1 x 107 lentivirus particles containing shIL-15 or control sequence into recipient mice. c. Intraventricular injection of a lentivirus containing a shIL-15 sequence results in loss of IL-15 in nestin-expressing cells in the SVZ of Cd1d−/− mice. n = 12 per group. P < 0.001, t (22) = 5.58, unpaired t-test. d. Intraventricular injection of a lentivirus containing shIL-15 sequence at 20 dpi prevents NK cell retention in SVZ of Cd1d−/− mice at EAE 30 dpi, but not in striatum. Absolute numbers of cells per dissected SVZ or striatum tissue are shown. Average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 2.5 ± 0.6 in lenti-control group and 1.7 ± 0.5 in lenti-shIL-15 group at 30 dpi. n = 12 per group. P < 0.001 (Lenti-control SVZ versus Lenti-control striatum), P < 0.001 (Lenti-control SVZ vs. Lenti-sh-IL-15 SVZ). F (1, 44) = 5.5, two-way ANOVA. e. To determine BrdU incorporation in NK cells in vivo, EAE mice were given i.p. injections of 120 mg kg−1 BrdU in PBS daily, and were sacrificed 10 days after the last injection at 30 dpi. Representative flow cytometry plots show that intraventricular injection of a lentivirus containing a shIL-15 sequence reduces BrdU+ NK cell counts in SVZ of Cd1d−/− mice at 30 dpi. Bar graphs summarize the decreased counts of BrdU+ NK cells after intraventricular injection of a lentivirus containing a shIL-15 sequence at 30 dpi. Absolute numbers of cells per dissected SVZ tissue are shown. n = 12 per group. P = 0.006, t (22) = 2.23, unpaired t-test. f. Intraventricular injection of a lentivirus containing a shIL-15 sequence at 20 dpi reduces the counts of IFN-γ-expressing NK cells in SVZ of Cd1d−/− mice at EAE 30 dpi. Bar graphs summarize the decreased counts of IFN-γ-expressing NK cells after intraventricular injection of a lentivirus containing a shIL-15 sequence at 30 dpi. Absolute numbers of cells per dissected SVZ tissue are shown. n = 12 per group. P = 0.007, t (22) = 2.11, unpaired t-test. In e-f, average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 2.6 ± 0.5 in lenti-control group and 1.8 ± 0.5 in Lenti-shIL-15 group at 30 dpi. Data represent three independent experiments. Error bars represent s.e.m.; ** P < 0.01.

Supplementary Figure 6 FMO controls and gating strategy for BrdU+, IFN-γ+ or annexin V+ NK cells.

NK cells were cultured with flow cytometry-sorted NSCs from EAE SVZ (30 dpi) in transwell plates and counted by flow cytometry in the same well (with cell contact) or spatially separated (no cell contact) by a membrane (0.4 μm pores). Representative flow cytometry dot plots show FMO controls and gating strategy for BrdU+, IFN-γ+ or annexin V+ NK cells.

Supplementary Figure 7 Full-length blots for Figure 6.

Supplementary Figure 8 Depletion of NK cells increases the number of proliferating NSCs, but does not affect the numbers of proliferating SVZ type C and A cells during EAE recovery.

a. “Long-term” BrdU injection was used to determine the number of SVZ-retaining type B cells. Anti-NK1.1 mAb was used to deplete NK cells in Cd1d−/− mice devoid of natural killer T (NKT) cells. b. Increased numbers of SVZ GFAP+BrdU+ cells in EAE Cd1d−/− mice treated with anti-NK1.1 mAb as compared to mice injected with IgG control at 30 dpi. c. Counts of GFAP+BrdU+ cells were increased in EAE Cd1d−/− mice treated with NK1.1 mAb during the late stages of EAE (30, 40, 60 dpi) but not in the peak phase (14 dpi). n = 6 mice per group at each time point. P = 0.003 (30dpi), P = 0.002 (40dpi), P = 0.005 (60dpi). F (3, 40) = 2.82, two-way ANOVA. d. Decreased SVZ GFAP+caspase-3+ cells in EAE Cd1d−/− mice treated with NK1.1 mAb as compared to those injected with IgG control at 30 dpi. e. Counts of GFAP+caspase-3+ cells were significantly decreased in EAE Cd1d−/− mice treated with NK1.1 mAb as compared to IgG-treated control during the late stages of EAE (30, 40, 60 dpi) but not in the peak phase (14 dpi). n = 6 mice per group at each time point. P = 0.005 (30dpi), P = 0.004 (40dpi), P = 0.008 (60dpi). F (3, 40) = 2.23, two-way ANOVA. In c and e, average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 3.5 ± 0.7 (IgG) and 3.3 ± 0.7 (NK 1.1 mAb) at 14 dpi, 2.6 ± 0.6 (IgG) and 1.6 ± 0.5 (NK 1.1 mAb) at 30 dpi, 2.3 ± 0.6 (IgG) and 1.5 ± 0.5 (NK 1.1 mAb) at 40 dpi and 2.2 ± 0.6 (IgG) and 1.3 ± 0.5 (NK 1.1 mAb) at 60 dpi. f. Anti-NK1.1 mAbs were injected after EAE induction, and “short-term” BrdU labeling was used to birthdate rapidly dividing SVZ progenitors in Cd1d−/− mice. g. Representative images show MASH1+BrdU+ or DCX+BrdU+ cells in EAE Cd1d−/− mice at 30 dpi. h. Unchanged counts of MASH1+BrdU+ or DCX+BrdU+ cells in EAE Cd1d−/− mice as compared to IgG-injected controls after pretreatment with anti-NK1.1 mAbs. In h, average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 3.2 ± 0.6 (IgG) and 3.3 ± 0.5 (NK 1.1 mAb) at 14 dpi, 2.5 ± 0.7 (IgG) and 1.7 ± 0.5 (NK 1.1 mAb) at 30 dpi, 2.5 ± 0.7 (IgG) and 1.6 ± 0.6 (NK 1.1 mAb) at 40 dpi and 2.3 ± 0.5 (IgG) and 1.5 ± 0.6 (NK 1.1 mAb) at 60 dpi. n = 6 mice per group at each time point. Data represent three independent experiments. Scale bars: 40 μm, 20 μm (inset). Error bars represent s.e.m.; ** P < 0.01.

Supplementary Figure 9 Removal of NK cells is associated with proliferation of SVZ NSPCs during the late stages of EAE.

a. Protocol of anti-NK1.1mAb injection, EAE induction and BrdU injection. b. Depletion of NK cells using anti-NK1.1 mAb in EAE Cd1d−/− mice is associated with increased numbers of BrdU+ cells in SVZ at 30 dpi. Control: 0 dpi. c. Quantitation of BrdU+ cells in EAE Cd1d−/− mice pretreated with IgG (control) or anti-NK1.1 mAb. n = 6 mice per group at each time point. P = 0.008 (30dpi), P = 0.006 (40dpi), P = 0.007 (60dpi). F (3, 40) = 7.73, two-way ANOVA. d. Depletion of NK cells is associated with increased numbers of DCX+ cells in SVZ of EAE Cd1d−/− mice at 30 dpi. Control: 0 dpi. e. Quantitation of DCX+ cells in EAE Cd1d−/− mice pretreated with IgG or anti-NK1.1 mAb. n = 6 mice per group at each time point. P = 0.007 (30dpi), P = 0.012 (40dpi), P = 0.013 (60dpi). F (3, 40) = 5.35, two-way ANOVA. In b-e, average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 3.5 ± 0.7 (IgG) and 3.3 ± 0.7 (NK 1.1 mAb) at 14 dpi, 2.7 ± 0.5 (IgG) and 1.8 ± 0.7 (NK 1.1 mAb) at 30 dpi, 2.6 ± 0.8 (IgG) and 1.5 ± 0.8 (NK 1.1 mAb) at 40 dpi and 2.5 ± 0.6 (IgG) and 1.3 ± 0.5 (NK 1.1 mAb) at 60 dpi. f. EAE induction and BrdU injection in Rag2−/−or Rag2−/−γc−/− Cd1d−/− mice that received 2D2 Cd1d−/− T cells (devoid of NKT cells). g. Deletion of NK cells prevents reduction of SVZ-proliferating (BrdU+) cells independently of T, B, and NKT cells at 30 dpi. Control: 0 dpi. h. Quantitation of BrdU+ cells in SVZ from Rag2−/− and Rag2−/−γc−/− mice during EAE. n = 6 mice per group at each time point. P = 0.008 (30dpi), P = 0.006 (40dpi), P = 0.008 (60dpi). F (3, 40) = 8.36, two-way ANOVA. i. Deficiency of NK cells is associated with increased numbers of DCX+ cells in Rag2−/− and Rag2−/−γc−/− mice at 30 dpi. Control: 0 dpi. j. Quantitation of DCX+ cells in SVZ from Rag2−/− and Rag2−/−γc−/− mice. n = 6 mice per group at each time point. P = 0.043 (30dpi), P = 0.036 (40dpi), P = 0.009 (60dpi). F (3, 40) = 5.65, two-way ANOVA. In g-j, average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 3.2 ± 0.6 (Rag2−/−) and 3.4 ± 0.7 (Rag2−/−γc−/−) at 14 dpi, 2.5 ± 0.8 (Rag2−/−) and 1.7 ± 0.7 (Rag2−/−γc−/−) at 30 dpi, 2.3 ± 0.7 (Rag2−/−) and 1.5 ± 0.8 (Rag2−/−γc−/−) at 40 dpi and 2.2 ± 0.6 (Rag2−/−) and 1.3 ± 0.5 (Rag2−/−γc−/−) at 60 dpi. Experiments were repeated three times. Scale bars: 40 μm. Error bars represent s.e.m.; * P < 0.05; ** P < 0.01.

Supplementary Figure 10 NK cell–induced cytotoxicity against SVZ type B cells.

a. Cytotoxicity against NSCs by NK cells was measured by 51Cr-release assay. Target cells (cultured NSCs) were labeled with 51Cr. 104 NSCs/well from naïve control, EAE 14 dpi and EAE 30 dpi SVZ, were co-cultured with activated NK cells at different effector:target ratios (50:1, 10:1; 5:1; 1:1, and 1:10) for 24 h. Activated NK cells were prepared by IL-2 (10 μg ml−1), IL-15 (10 μg ml−1), and LPS (5 μg ml−1) stimulation for 96 h prior to co-culture. Cytotoxicity was measured and data shown are expressed as percentage of naïve controls from four independent experiments with each value repeated in triplicate. Average EAE disease grade for mice used for NSC isolation at the time of tissue harvesting was 3.6 ± 0.7 at 14 dpi and 2.6 ± 0.6 at 30 dpi. n = 12 per group. P < 0.001 (50:1), P < 0.001 (10:1), P < 0.001 (5:1), P < 0.001 (1:1). F (8, 165) = 6.26, two-way ANOVA. b. A representative image of Fura-2AM-loaded NSCs and selected region of interest (ROI). Scale bar, 20 µm. c. Increased Ca2+ influx signals in NSCs isolated from EAE SVZ at 30 dpi and treated with activated NK cells. Ca2+ influx signals were measured by the ratio of 340/380nm in the selected ROIs of NSCs. d. Averaged Ca2+ influx signals in ROIs selected from NSCs from the indicated groups, which were treated or untreated with activated NK cells for 12 h. Average EAE disease grade for mice used for NSC isolation at the time of tissue harvesting was 3.5 ± 0.6 at 14 dpi and 2.3 ± 0.8 at 30 dpi. n = 8 per group. P = 0.005 (30 dpi NSCs + NK cells versus 0 dpi NSCs), P = 0.008 (30 dpi NSCs + NK cells versus 14 dpi NSCs + NK cells). F (2, 21) = 2.78, one-way ANOVA. In a-d, data represent four independent experiments. e. NSCs were sorted from EAE SVZ at 30 dpi by flow cytometry and cultured in vitro for 48 h. Immunostaining shows increased counts of GFAP+caspase-3+ cells relative to total GFAP+ cells after treatment with NK cells for 24 h. n = 12 per group. P = 0.006 (30 dpi NSCs + NK cells 1:1 versus 30 dpi NSCs), P = 0.003 (30 dpi NSCs + NK cells 1:10 versus 30 dpi NSCs). F (2, 33) = 5.88, one-way ANOVA. f. Cell proliferation was determined by in vitro BrdU incorporation and co-labeling with GFAP. Counts of BrdU+GFAP+ cells relative to total GFAP+ cells in NSC cultures were unaffected by treatment with NK cells. Results in e-f are from three independent experiments. n = 12 per group. Average EAE disease grade for mice used for NSC isolation at the time of tissue harvesting was 2.5 ± 0.6 at 30 dpi. Error bars represent s.e.m.; ** P < 0.01.

Supplementary Figure 11 Construction of and treatment with adenoviral vectors encoding Qa1.

a. Full-length Qa1 cDNA was amplified by RT-PCR from RNA isolated from splenocytes of C57BL/6 mice. After verification of the sequence, cDNA was ligated into the DUAL-CCM+ shuttle vector with a C-terminal Flag epitope tag under the control of a CMV promoter. The recombinant adenovirus was custom generated by Vector Biolabs. b. A representative image of SVZ type B cells (GFAP+EGFR+DAPI+) freshly isolated and sorted by flow cytometry from Cd1d−/− mice at 30 dpi. Scale: 10 μm. c−d. SVZ type B cells freshly isolated and sorted by flow cytometry from Cd1d−/− mice at 30 dpi were cultured prior to infection with 1X107 pfu adenovirus without Qa1 sequence (control, c) or 1X107 pfu adenovirus containing Qa1 sequence (Ad-Qa1, d). Cells were fixed, permeabilized, blocked with anti-Flag Ab and stained with an Alexa Fluor secondary Ab. Scale: 20 μm. e. Intraventricular injection of adenovirus containing Qa1 sequence at 20 dpi prevents loss of Qa1 expression in SVZ of Cd1d−/− mice at 30 dpi, Scale: 50 μm. f. Intraventricular injection of adenovirus containing Qa1 sequence at 20 dpi prevents loss of Qa1 expression in SVZ, but not in striatum (STM) or cortex (CTX) of Cd1d−/− mice at 30 dpi, Scale: 40 μm. n = 12 mice per group. P = 0.001 (Ad-control SVZ versus Ad-Qa1 SVZ), t (22) = 3.83, unpaired t-test. In e-f, average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 2.6 ± 0.5 in Ad-control group and 1.5 ± 0.5 in Ad-Qa1 group at 30 dpi. Data represent three independent experiments. Error bars represent s.e.m.; ** P < 0.01.

Supplementary Figure 12 Qa1 overexpression in the SVZ promotes the survival of NSCs during the late stages of brain inflammation.

a. “Long-term” BrdU injection was used to determine the number of SVZ-retaining type B cells. Representative images and bar graph show increased BrdU+ cells in EAE SVZ (30 dpi) of Cd1d−/− mice receiving intraventricular injection of adenovirus containing Qa1 sequence at 20 dpi. EAE mice receiving adenovirus containing null control sequence were used as control (Ad-control). Control: 0 dpi. Average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 2.4 ± 0.5 in Ad-control group and 1.5 ± 0.4 in Ad-Qa1 group at 30 dpi. Scale: 50 μm. n = 12 mice per group. P = 0.02 (EAE + Ad-control versus EAE + Ad-Qa1), t (22) = 2.42, unpaired t-test. b. Cytotoxicity against NSCs by NK cells was measured by 51Cr-release assay. Target cells (NSCs) were obtained from naïve control SVZ, EAE 30 dpi SVZ from Cd1d−/− mice receiving intraventricular injection of adenovirus containing Qa1 or null control sequence at 20 dpi. Target cells were labeled with 51Cr. 104 NSCs/well were then co-cultured with activated NK cells at different effector:target ratios (50:1, 10:1; 5:1; 1:1, and 1:10) for 24 h. Activated NK cells were prepared by IL-2 (10 μg ml−1), IL-15 (10 μg ml−1), and LPS (5 μg ml−1) stimulation for 96 h prior to co-culture. Cytotoxicity was measured and data shown are expressed as percentage of naïve controls from four independent experiments with each value repeated in triplicate. Average EAE disease grade for mice used for NSC isolation at the time of tissue harvesting was 2.6 ± 0.7 in Ad-control group and 1.4 ± 0.6 in Ad-Qa1 group at 30 dpi. n = 12 per group. P < 0.001 (50:1), P < 0.001 (10:1), P < 0.001 (5:1), P < 0.001 (1:1). F (8, 165) = 3.73, two-way ANOVA. c. Qa1 overexpression attenuates the increase of Ca2+ influx signals in NSCs treated with activated NK cells. Ca2+ influx signals were measured by the ratio of 340/380nm in the selected ROIs of NSCs. NSCs were obtained from naïve control SVZ, EAE 30 dpi SVZ of Cd1d−/− mice receiving intraventricular injection of adenovirus containing Qa1 or null control sequence at 20 dpi. Averaged Ca2+ influx signals in ROIs selected from NSCs from the indicated groups, which were treated with activated NK cells for 12 h. Average EAE disease grade for mice used for NSC isolation at the time of tissue harvesting was 2.5 ± 0.6 in Ad-control group and 1.5 ± 0.5 in Ad-Qa1 group at 30 dpi. Control: 0 dpi. n = 8 per group. P = 0.003 (30 dpi NSCs + Ad-control versus Control), P = 0.006 (30 dpi NSCs +Ad-control versus 30 dpi NSCs + Ad-Qa1). F (2, 21) = 9.37, one-way ANOVA. d. NSCs were sorted from naïve control SVZ, EAE 30 dpi SVZ of Cd1d−/− mice receiving intraventricular injection of adenovirus containing Qa1 or null control sequence at 20 dpi. Sorted NSCs were then cultured in vitro for 48 h. Immunostaining shows that Qa1 overexpression reduced the increase of GFAP+caspase-3+ cell counts relative to total GFAP+ cells after treatment with NK cells for 24 h. Control: 0 dpi. Average EAE disease grade for mice used for NSC isolation at the time of tissue harvesting was 2.3 ± 0.5 in Ad-control group and 1.4 ± 0.4 in Ad-Qa1 group at 30 dpi. n = 12 per group. P = 0.003 (30 dpi NSCs + Ad-control versus Control), P = 0.02 (30 dpi NSCs + Ad-control versus 30 dpi NSCs + Ad-Qa1). F (2, 33) = 3.12, one-way ANOVA. Data represent four independent experiments. Error bars represent s.e.m.; * P < 0.05, ** P < 0.01.

Supplementary Figure 13 Removal of NK cells or overexpression of Qa1 in the SVZ promotes oligodendrogenesis around the SVZ.

EAE was induced in Cd1d−/−, Rag2−/− and Rag2−/−γ c−/− mice. To induce EAE, Rag2−/− and Rag2−/−γc−/− mice were transferred with 5X106 2D2 T cells on day 0. To deplete NK cells, Cd1d−/− mice received anti-NK1.1 mAb injection (i.p.) at two days prior to MOG immunization and every 5 days thereafter until termination of experiments. EAE mice received intraventricular injection of adenovirus containing Qa1 or null control sequence at 20 dpi and SVZ tissues from EAE mice were harvested at 30 dpi. a. To determine the generation of neural/glial antigen 2 (NG2)/ BrdU+ oligodendrocyte progenitors, EAE mice were given i.p. injections of 120 mg kg−1 BrdU in PBS every 2 h for 10 h, and were sacrificed 1 week (7 d washout time) after the last injection at 30 dpi. Quantitation of BrdU+NG2+ cells in the SVZ of EAE mice (30 dpi) is expressed as the total number of positive cells per area measured (cells per mm2). n = 12 mice per group. P = 0.004 (IgG versus NK1.1 mAb), t (22) = 2.36. P = 0.004 (Ad-control versus Ad Qa1), t (22) = 2.28. P = 0.006 (Rag2−/− versus Rag2−/−γc−/−), t (22) = 2.03. P = 0.007 (Rag2−/− + Ad-control versus Rag2−/−+ Ad-Qa1), t (22) = 1.96, unpaired t-test. b. To determine newly generated mature cyclic nucleotide phosphohydrolase (CNPase)/ BrdU+ oligodendrocytes, mice were injected i.p. with 150 mg kg−1 BrdU twice per day for 2 days and were killed 2 weeks (14 d washout time) after the last injection. Quantitation of BrdU+CNPase+ cells in the SVZ of EAE mice (30 dpi) is expressed as the total number of positive cells per area measured (cells per mm2). In a-b, average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 2.6 ± 0.6 in IgG group, 1.3 ± 0.5 in NK1.1-mAb group, 2.5 ± 0.5 in Ad-control group, 1.4 ± 0.6 in Ad-Qa1 group, 2.3 ± 0.5 in Rag2−/− group, 1.2 ± 0.6 in Rag2−/−γc−/− group, 2.4 ± 0.7 in Rag2−/− + Ad-control group, 1.3 ± 0.6 in Rag2−/− + Ad-Qa1 group at 30 dpi, Scale bars: 40 μm. n = 12 mice per group. P = 0.003 (IgG versus NK1.1 mAb), t (22) = 2.83. P = 0.003 (Ad-control versus Ad Qa1), t (22) = 3.01. P = 0.007 (Rag2−/− versus Rag2−/−γc−/−), t (22) = 2.05. P = 0.006 (Rag2−/− + Ad-control versus Rag2−/−+ Ad-Qa1), t (22) = 2.12, unpaired t-test. Data represent three independent experiments. Error bars represent s.e.m.; ** P < 0.01.

Supplementary Figure 14 Removal of NK cells promotes oligodendrogenesis in the spinal cord.

EAE was induced in Cd1d−/−, Rag2−/− and Rag2−/−γc−/− mice. To deplete NK cells, Cd1d−/− mice received anti-NK1.1 mAb injection (i.p.) at two days prior to MOG immunization and every 5 days thereafter until termination of experiments. Spinal cord tissues were obtained at 30 dpi from EAE mice. Counts of NG2+ or CNPase+ cells were measured by flow cytometry analysis. a. Representative flow cytometry plots show the identification of gating boundaries of NG2 (FITC) with corresponding FMO control. b. Representative flow cytometry plots show the identification of gating boundaries of CNPase (FITC) with corresponding FMO control. c. Quantitation of NG2+ cells in the spinal cord of EAE mice (30 dpi) is expressed as the total number of positive cells per mouse spinal cord. n = 12 mice per group. P = 0.006 (IgG versus NK1.1 mAb), t (22) = 2.06. P = 0.008 (Rag2−/− versus Rag2−/−γc−/−), t (22) = 1.92, unpaired t-test. d. Quantitation of CNPase+ cells in the spinal cord of EAE mice (30 dpi) is expressed as the total number of positive cells per mouse spinal cord. n = 12 mice per group. P = 0.007 (IgG versus NK1.1 mAb), t (22) = 1.98. P = 0.006 (Rag2−/− versus Rag2−/−γc−/−), t (22) = 2.23, unpaired t-test. In c-d, average EAE disease grade for mice used for tissue analysis at the time of tissue harvesting was 2.7 ± 0.6 in IgG group, 1.5 ± 0.6 in NK1.1-mAb group, 2.4 ± 0.6 in Rag2−/− group and 1.3 ± 0.4 in Rag2−/−γc−/− group at 30 dpi. Data represent three independent experiments. Error bars represent s.e.m.; ** P < 0.01.

Supplementary Figure 15 Oligodendrocyte progenitors are resistant to NK cell killing.

a. Representative flow cytometry plots and immunostained image of oligodendrocyte progenitor cells show that oligodendrocyte progenitor cells express Qa1. Scale bar, 20 µm. b. Quantitation of Qa1+ oligodendrocyte progenitor cells shows unaltered expression of Qa1 in WT controls (0 dpi) or EAE mice at 14 or 30 dpi. Average EAE disease grade for mice used for cell isolation at the time of tissue harvesting was 3.3 ± 0.8 at 14 dpi and 2.6 ± 0.7 at 30 dpi. n = 6 mice per data plot. Results were obtained from three independent experiments. c. Cytotoxicity against oligodendrocyte progenitor cells by NK cells was measured by 51Cr-release assay. Target cells (oligodendrocyte progenitor cells) were obtained from naïve control, EAE 14 or 30 dpi CNS tissues from WT mice by sorting NG2+ cells from CNS tissues. Target cells were labeled with 51Cr. 1 x 104 oligodendrocyte progenitor cells s/well were then co-cultured with activated NK cells at different effector:target ratios (50:1, 10:1; 5:1; 1:1, and 1:10) for 24 h. Activated NK cells were prepared by IL-2 (10 μg ml−1), IL-15 (10 μg ml−1), and LPS (5 μg ml−1) stimulation for 96 h prior to coculture. Cytotoxicity was measured and data shown are expressed as percentage of naïve controls from four independent experiments with each value repeated in triplicate. Average EAE disease grade for mice used for cell isolation at the time of tissue harvesting was 3.5 ± 0.7 at 14 dpi and 2.5 ± 0.8 at 30 dpi. n = 12 per group. d. NSCs were sorted from SVZ of WT mice and oligodendrocyte progenitor cells (NG2+) were sorted from UBC-GFP mice respectively by flow cytometry at 30 dpi. Thereafter, oligodendrocyte progenitor cells and NSCs were co-cultured with or without IL-15 mAb in vitro for 48 h. Oligodendrocyte progenitor cells cultured alone without IL-15 mAb treatment were used as controls. Immunostaining shows unaltered counts of GFP+NG2+ cells relative to those in controls after treatment with IL-15 mAb or co-cultured with NSCs. n = 12 per group. e. NSCs were sorted from SVZ of WT mice and oligodendrocytes (CNPase+) were sorted from UBC-GFP mice respectively by flow cytometry at 30 dpi. Thereafter, oligodendrocytes and NSCs were co-cultured with or without IL-15 mAb in vitro for 48 h. Oligodendrocytes cultured alone without IL-15 mAb treatment were used as controls. Immunostaining shows unaltered counts of GFP+CNPase+ cells relative to those in controls after treatment with IL-15 mAb or co-cultured with NSCs. n = 12 per group. Average EAE disease grade for mice used for cell isolation at the time of tissue harvesting was 3.5 ± 0.8 (WT) and 3.2 ± 0.7 (UBC-GFP) at 14 dpi, 2.5 ± 0.7 (WT) and 2.3 ± 0.8 (UBC-GFP) at 30 dpi. Results were obtained from four independent experiments. Error bars represent s.e.m.

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Liu, Q., Sanai, N., Jin, WN. et al. Neural stem cells sustain natural killer cells that dictate recovery from brain inflammation. Nat Neurosci 19, 243–252 (2016). https://doi.org/10.1038/nn.4211

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