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Supplementary Information for

Major histocompatibility complex class I molecules protect motor neurons from

astrocyte-induced toxicity in amyotrophic lateral sclerosis

SungWon Song, Carlos J. Miranda, Lyndsey Braun, Kathrin Meyer, Ashley E. Frakes,

Laura Ferraiuolo, Shibi Likhite, Adam K. Bevan, Kevin D. Foust, Michael J. McConnell,

Christopher M. Walker, Brian K. Kaspar

To whom correspondence should be addressed:

Brian.Kaspar@NationwideChildrens.org

This file includes:

Supplementary Figures 1 to 12

Supplementary Tables 1 to 4

Supplementary Videos 1 to 4

Nature Medicine: doi:10.1038/nm.4052

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Supplementary Figure 1 MHCI expression levels in spinal MNs before and after disease onset in SOD1G93A mice

Supplementary Figure 2 Astrocyte cell lines used are highly enriched for astrocytic markers and show little to no presence of other glia types or cells from the immune system.

Supplementary Figure 3 IPS cell derived MNs share gene expression profile with MNs derived from ES cells

Supplementary Figure 4 Reduction in MHCI expression found in MNs but not in GABAergic neurons when SOD1 mutations are expressed in MNs or astrocytes

Supplementary Figure 5 Down-regulation of MHCI in MNs is observed in the presence of SOD1G93A astrocyte conditioned medium and a endoplasmic reticulum stressor

Supplementary Figure 6 Lentiviral transduction of MNs with H2kb allows sustained MHCI expression in MNs despite co-culture with SOD1G93A astrocytes

Supplementary Figure 7 H2kb knockdown in MNs and GABAergic neurons increases their susceptibility to SOD1G93A astrocytes toxicity

Supplementary Figure 8 CNS delivery of AAV9 at birth results in efficient spinal MN transduction

Supplementary Figure 9 H2kb expression in SOD1G93A astrocytes does not protect MNs from SOD1G93A astrocyte toxicity

Supplementary Figure 10 MHCI inhibitory receptors are expressed in SOD1G93A astrocytes and cytotoxic T lymphocytes

Supplementary Figure 11 Robust HLA-F expression is observed in human ES-derived MN cells upon transduction with LV-HLA-F:GFP

Supplementary Figure 12 Knockdown of KIR3DL2 in ALS astrocytes leads to an increase in the rate and toxicity level of ALS astrocytes

Supplementary Table 1 Summary of the demographic information associated with human spinal cord tissues used for immunostaining

Supplementary Table 2 Summary of the demographic information associated with NPC derived astrocytes lines used in this study

Supplementary Table 3 List of primers used for RT-PCR analysis

Supplementary Table 4 Antibodies and details associated with their use in this study

Supplementary Video 1 Age 117 days. Activity and behaviour of AAV9-H2k treated SOD1G93A mouse.

Supplementary Video 2 Age 131 days. Activity and behaviour of AAV9-H2k treated SOD1G93A mouse.

Supplementary Video 3 Age 141 days. Activity and behaviour of AAV9-H2k treated SOD1G93A mouse.

Supplementary Video 4 Age 159 days. Activity and behaviour of AAV9-H2k treated SOD1G93A mouse.

Nature Medicine: doi:10.1038/nm.4052

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Supplementary Figure 1. MHCI expression levels in spinal MNs before and after disease onset in SOD1G93A mice. (a) In situ hybridization image for MHCI (H2-db) along with β2m obtained from the lumbar spinal cord of a 56-day old wild type mouse. Images were obtained by searching the interactive database of gene expression from the Allen Spinal Cord Atlas (Seattle, WA), available on http://mousespinal.brain-map.org. (b) H2-db transcripts analysis performed by us in the spinal MNs of a 60-day old wild-type mouse and age matched H2kb and H2db -deficient mouse. In dark-filed micrographs, in situ hybridized riboprobes appear white. (c-d) Representative immunofluorescence image (from three analyzed) of MNs MHCI (H2-kbdb) expression levels found in cervical (c) and thoracic (d) spinal cord sections from wild-type and SOD1G93A mice at the age of 30 and 125 days. A total of 199, 277, 208, 212 of MNs in (c) and a total of 99, 110, 142, 110 of MNs in (d) were evaluated corresponding to graph columns 1-4, respectively. WT, wild-type. SOD1, SOD1G93A. (e) Representative immunofluorescence image (from three analyzed) of MNs MHCI (H2-kbdb) expression levels found in sciatic nerve axons fibers of wild-type and SOD1G93A mice at the age of 30 and 125 days. MN, motor neurons. Scale bars, 500 µm (a), 400 μm (b), 100 µm (a insets), 20 µm (c,e).

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Supplementary Figure 2. Astrocyte cell lines used are highly enriched for astrocytic markers and show little to no presence of other glia types or cells from the immune system. (a) Immunofluorescence marker analysis performed in wild-type and SOD1G93A neuronal progenitor cells (NPC) derived astrocytes lines to detect expression of prototypic astrocytic markers. (b,c) Quantitative RT-PCR analysis of wild-type and SOD1G93A NPC derived astrocyte lines for microglia (b) and oligodendrocyte markers (c). (d-g) Quantitative RT-PCR analysis of mouse (d,e) and human (e,f) NPCs derived astrocytes to evaluate presence of cytotoxic T cell (CTL) (d,f) and natural killer (NK) (e,g) cell contaminants. SC, Spleenocytes. WT, wild-type. SOD1, SOD1G93A. Scale bar, 1mm.

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Supplementary Figure 3. IPS cell derived MNs share gene expression profile with MNs derived from ES cells. (a) Schematic representation of the process used to generate MNs from Hb9:GFP iPS and ES cells. MN cultures were purified by FACS prior to experimental use. (b-c) Representative images (from experiments run in triplicate) of SOD1WT and SOD1G93A iPS cell derived MNs compared to MNs derived from ES cells (b) and RT-PCR analysis of prototypic MNs mark performed in iPS

derived MN cultures (c). Data show the mean s.e.m fold induction of triplicates.

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Supplementary Figure 4. Reduction in MHCI expression found in MNs but not in GABAergic neurons when SOD1 mutations are expressed in MNs or astrocytes. (a) Immunofluorescence images (left panel) of MHCI expression in MNs expressing SOD1 mutations for 72 hours, and quantification their MHCI levels (right panel) as observed at 24, 72 and 120 hours. Data is shown as mean ± s.e.m of MHCI fluorescence intensity found in MNs was displayed relative to WT MNs at the time point 24 hours. One-Way ANOVA, *P<0.05; n.s., non-significant P≥0.05. (b) Representative image (n = 3) of MNs expression SOD1 mutations when co-cultured with astrocytes for 24 and 120 hours (left panel), and corresponding quantification (right panel). Data is shown as mean ± s.e.m of MHCI fluorescence intensity found in MNs was displayed relative to WT MNs cultured in the absence of astrocytes. One-Way ANOVA, *P<0.05; n.s., non-significant P≥0.05. (c) Representative image (n = 3) (left panel) and respective quantification of MHCI levels (right panel) found in GABAergic neurons cultured in the presence of SOD1G93A mutation compared to wild type control astrocytes. Data shown as mean ± s.e.m of MHCI fluorescence intensity in GAD67+ cells. MHCI levels shown in graph are displayed as relative to WT, 24hours. Each dot in the graphs represents MHCI level found per GAD67+ cell. One-Way ANOVA, n.s., non-

significant P≥0.05. WT, wild-type astrocytes or MNs. In MNs SOD1, iPSC derived MNs

expressing SOD1G93A mutation. Scale bars 10 µm (a,b), 5 μm (c).

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Supplementary Figure 5. Down-regulation of MHCI in MNs is observed in the presence of SOD1G93A astrocyte conditioned medium and an endoplasmic reticulum stressor. (a) Relative MHCI (H2-kbdb) levels found in MNs cultured in the presence of conditioned medium derived from wild-type or SOD1G93A astrocytes. (b) Representative images (n = 3 per group) of MHCI expression found in MNs cultured in the presence of molecules known to be secreted by SOD1G93A astrocytes and can cause MN death. (c) Quantification of MN MHCI expression levels found when cultured in the presence of stressor molecules as in (c). Data shown was collected 24 hours post astrocyte conditioned medium incubation in (a) and 9 h post drugs administration in (b). Data shown is a representative of three independent experiments with triplicates and shown as mean ± s.e.m of MHCI fluorescence intensity found in MNs. MHCI levels shown in graphs (a) and (c) are displayed as relative to each control. Each dot in the graphs represents MHCI level found per MN (One-Way ANOVA, *P<0.05; ****P<0.0001; ns, non-significant P≥0.5). WT, wild-type. SOD1, SOD1G93A. CM, conditioned medium. Scale bars 10 µm.

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Supplementary Figure 6. Lentiviral transduction of MNs with H2kb allows sustained MHCI expression in MNs despite co-culture with SOD1G93A astrocytes. (a) RT-PCR analysis for H2Kb and actin expression in 293 cells transduced with lentivirus particles encoding either RFP or H2Kb. (b) Levels of RFP expression at 72 hours in Hb9:GFP MNs transduced with lenti-RFP. (c) MHCI levels observed in MNs transduced with either Lv-RFP or Lv-H2K and cultured in the presence of SOD1G93A astrocytes for 72 hours.(d) Quantification of MHCI levels as shown in (c). Data represents one of three experiments performed independently with triplicates and shows mean ± s.e.m of MHCI fluorescence intensity in MNs. MHCI levels shown in d are displayed as relative to WT. Each dot in the graph represents MHCI level found per

MN cell. (One-Way ANOVA, ***P<0.001; ns, non-significant P≥0.5). WT, wild-type.

SOD1, SOD1G93A. Ast, astrocytes. Scale bars, 200 µm (b), 20 µm (c).

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Supplementary Figure 7. H2kb knockdown in MNs and GABAergic neurons increases their susceptibility to SOD1G93A astrocytes toxicity. (a) Quantification of cell survival, of MNs lacking H2-Kb expression, when cultured in the presence of the ER stressor molecule thapsigargin or the reactive oxygen species generating molecule menadione. (b) Quantification of cell survival observed in MN cultures previously treated with a lentivirus expressing H2-Kb or a scrambled (scr) shRNA and cultured for 120 hours in the presence of wild-type or SOD1G93A astrocytes. Statistical analysis was performed for the comparison between red and purple (Two-Way ANOVA, *P<0.05; **P<0.01). (c) RNA (upper panel) and immunofluorescence analysis (lower panel)) of H2-kb of GABAergic neurons transduced with Lv-H2k. (d) Quantification of MHCI levels observed in MNs after Lv-H2k transduction. Data presented as mean ± s.e.m of MHCI fluorescence intensity in GAD67+ cells performed in three independent experiments. MHCI levels were displayed as relative to scr. Each dot in the graph represents MHCI level found per GAD67+ cell. (unpaired t- test, ***P<0.001). (e) Quantification of number of GAD67 positive cells present per well when GABAergic neurons were treated with H2-kb shRNA and cultured in the presence of wild-type or SOD1G93A astrocytes for 120

hours. Representative data of three independent experiments shown by the mean s.e.m of counts found in 3 wells per group (One-Way ANOVA, ***P<0.001; ns, non-significant P≥0.5). WT, wild-type. SOD1, SOD1G93A. Ast, astrocytes. scr, scrambled. Scale bars 5 μm.

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Supplementary Figure 8. CNS delivery of AAV9 at birth results in efficient spinal MN transduction. (a) Immunofluorescent images illustrating spinal cord MNs transduction levels achieved after CNS direct injection at postnatal day 1 with AAV-GFP. ChAT immunostaining was used to visualize MNs. RNA analysis for H2kb and actin in spinal cord of mice transduced treated with AAV9-H2k or AAV9-H2d. Scale bar

50 m.

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Supplementary Figure 9. H2kb expression in SOD1G93A astrocytes does not protect MNs from SOD1G93A astrocyte toxicity. (a) Representative (n = 3) immunofluorescent image of wild-type SOD1G93A and astrocytes transduced with Lv-RFP and cultured in the presence of Hb9:GFP expressing MNs. Quantification of cell survival on MNs cultured in the presence of wild-type SOD1G93A and astrocytes transduced with either Lv-RFP or Lv-H2k. Data represents one of three independent

experiments and is shown as the mean s.e.m of counts found in 3 wells per experimental group. (One-Way ANOVA, ****P<0.0001). WT, wild type. SOD1, SOD1G93A. Scale bar 200 µm.

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Supplementary Figure 10. MHCI inhibitory receptors are expressed in SOD1G93A astrocytes and cytotoxic T lymphocytes. (a-c) LY49C and LY49I expression at disease end-stage in wild-type and SOD1G93A mice as determined by immunohistochemistry (a,b) and quantified from the lumbar spinal cord (c). Evaluation of LY49C and LY49I expression in CD8A positive CTLs that infiltrated in SOD1 spinal cords. Arrowhead indicates LY49C/I positive CTL. WT, wild-type. SOD1, SOD1G93A.

Scale bars 10 m (b) 20 m (d).

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Supplementary Figure 11. Robust HLA-F expression is observed in human ES-derived MN cells upon transduction with LV-HLA-F:GFP. Immunofluorescence images of human ES-derived MN progenitor cells transduced with a Lv-HLA-F:GFP. Transduced cells identified by green fluorescent protein expression (GFP; green),

display high levels of HLA-F expression (red, lower panel). Scale bar 20 m.

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Supplementary Figure 12. Knockdown of KIR3DL2 in ALS astrocytes leads to an increase in the rate and toxicity level of ALS astrocytes. (a) RT-PCR analysis probing for KIR3DL2 and ACTB performed in human ALS astrocytes transduced with a Lv- KIR3DL2 shRNA. b) Levels of MN death observed at both 7 and 14 days post co-culture with human ALS astrocytes lacking KIR3DL2 expression compared to scramble shRNA control. FALS, familial ALS. SALS, sporadic ALS. scr, scrambled.

Nature Medicine: doi:10.1038/nm.4052

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Supplementary Table 1. Summary of the demographic information associated with human spinal cord tissues used for immunostaining.

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Supplementary Table 2. Summary of the demographic information associated with NPC derived astrocytes lines used in this study.

ID Diagnosis Age(yr.) Gender Time from Diagnosis to Death

Non-ALS_1 Non-ALS 61 Male N/A

Non-ALS_2 Non-ALS N/A N/A N/A

Non-ALS_3 Non-ALS 87 Male N/A

fALS_1 Familial ALS 57 Male 8 Months

sALS_1 Sporadic ALS 70 Male 20 Months

sALS_2 Sporadic ALS 55 Male 14 Months

sALS_3 Sporadic ALS 64 Male 14 Months

sALS_4 Sporadic ALS 70 Female 60 Months

sALS_5 Sporadic ALS 67 Male 9 Months

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Supplementary Table 3. List of primers used for RT-PCR analysis.

Species Gene Forward Primer (5'-3') Reverse Primer (5'-3') Product Size (bp) References

Ly49c TCCCACGATGAGTGAGCCA TACCTTTAACTCTAGTTGGAAAA 831 McQueen et al

Ly49i GATGAATGAGCCGGAGGTC TTTCACTGTTCCATCTGTCCT 389 Lee et al

Ly49l GGTGAGGCTTGAGGAGACAG CTTCCCACAAATACAGATGAATGATTTATCACATTTATC 710 Makrigiannis et al

Ly49v GGATTGACAATCACCCATCTAAG GAGAACATTCCAAAAATCTTCAG 318 Stevenaert et al

Ly49w GAGACAGGGAAGCCTCAAAAAG TGGTGCTGCACTTATCGTGG 193

H2-k1 CGAAGCAGAACGATTGCGAG TGGGAGGCCCTGATGATACA 349

H2-k1 CGCCCTGGCTCCGACTCAGAC GAGGGTCATGAACCATCACTT 1061

H2-d1 TGGAACCTTCCAGAAGTGGG TCTTCACACTGAACCCAAGCTC 433

H2-l GGAGTATTGGGAGCGGATCAC GCGTTCCCGTTCTTCAGGTA 360

Nkg2d ACTCAGAGATGAGCAAATGCC CAGGTTGACTGGTAGTTAGTGC 105

Nkp46 AATGGAAACTCGGTGAACATCTG GGGGTTGCTCGACTTTGAC 216

Cd3e TCAGCCTCCTAGCTGTTGG GTCAACTCTACACTGGTTCCTG 88

Cd8a AAGAAAATGGACGCCGAACTT AAGCCATATAGACAACGAAGGTG 139

Iba1 GGATTTGCAGGGAGGAAAAG TGGGATCATCGAGGAATTG 92 Chen at al

Cd11b ATTCGGTGATCCCTTGGATT GTTTGTTGAAGGCATTTCCC 98

Mbp ATGGCATCACAGAAGAGACCCTCA TAAAGAAGCGCCCGATGGAGTCAA 133 Chew et al

Plp1 AGTCAGCCGCAAAACAGACT GCCCCTACCAGACATCTAGC 99

Actb GTGGGCCGCCCTAGGCACCA CTCTTTGATGTCACGCACGATTTC 540

Olig2 SA Biosciences, PPM04752A-200

Ngn2 SA Biosciences, PPM28944A-200

Hb9 SA Biosciences, PPM30673A-200

ChAt SA Biosciences, PPM57674A-200

IIl1 SA Biosciences, PPM04504A-200

Lhx3 SA Biosciences, PPM25822E-200

Nkx6.1 SA Biosciences, PPM32738E-200

NeuroD1 SA Biosciences, PPM05527B-200

HLA-F CGCAGTATTGGGAGTGGACC AGATCCTCCAAAGGCACACGTT 398

CD3E GGGGCAAGATGGTAATGAAG CCAGGATACTGAGGGCATGT 102

CD8A GGGTCCTTCTCCTGTCACTG GTCTCCCGATTTGACCACAG 98

CD56 CAGCCAGCAGATTACAATGC TGGCTGGGAACAATATCCAC 99

CD16 CGGTGCAGCTAGAAGTCCAT TTCCAGCTGTGACACCTCAG 100

ACTB CGCGAGAAGATGACCCAGATC TTGCTGATCCACATCTGCTGG 731

Human

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Supplementary Table 4. Antibodies and details associated with their use in this study.

ICC, Immunocytochemistry; IHC, Immunohistochemistry; PFA, Paraformaldehyde. LY49C/I and CD8A staining in tissues was performed on frozen sections

Company Catalog Number Host Dilution Used Application Tissue Fixation

Mouse Specimen

Ly49c/i BD Pharmagin 553273 Mouse 1:100 ICC/IHC No

MHC Class I (ER-HR52) AbD Serotec mca2398 Rat 1:100 ICC/IHC PFA

Gfap Abcam ab4674 Chicken 1:500 ICC/IHC PFA

Glast Abcam ab416 Rabbit 1:200 IHC PFA

Eaat2 Gift from Dr. Jeffrey Rothstein Rabbit 1:200 ICC PFA

Iba1 Wako NC9288364 Rabbit 1:400 ICC PFA

Mbp Abcam ab106583 Chicken 1:200 ICC PFA

ChAT Millipore ab144p Goat 1:200 ICC/IHC PFA

Gfp Abcam ab13970 Chicken 1:500 ICC/IHC PFA

Rfp Abcam ab34771 Rabbit 1:1600 ICC PFA

S100β Swant 37A Rabbit 1:500 ICC PFA

Cd8a Abcam ab22378 1:200 ICC/IHC No

Gad67 Millipore MAB5406 Mouse 1:200 ICC PFA

Smi32 Covance SMI-32R Mouse 1:500 IHC PFA

Tuj1 Covance PRB-435P Rabbit 1:400 ICC PFA

Human Specimen

KIR3DL2 Abcam ab95303 Rabbit 1:200 ICC/IHC Formalin

MHC Class I (EMR8-5) Abcam ab70328 Mouse 1:50 IHC Formalin

GFAP Abcam ab4674 Chicken 1:500 ICC/IHC Formalin

ChAT Millipore AB143 Rabbit 1:200 ICC PFA

HB9 Abcam ab26128 Rabbit 1:250 ICC PFA

SMI32 Covance SMI-32R Mouse 1:500 ICC PFA

GFP Abcam ab13970 Chicken 1:500 ICC PFA

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Supplementary Movie 1.

Age 117 days. Activity and behavior of a AAV9-H2K treated SOD1G93A mouse (dots on

tail) compared with a same age littermate treated with AAV9-empty (no dots on tail).

Supplementary Movie 2.

Age 131 days. Activity and behavior of a AAV9-H2K treated SOD1G93A mouse (dots on

tail) compared with a same age littermate treated with AAV9-empty (no dots on tail).

Supplementary Movie 3.

Age 141 days. Activity and behavior of a AAV9-H2K treated SOD1G93A mouse (dots on

tail) compared with a same age littermate treated with AAV9-empty (no dots on tail).

Supplementary Movie 4.

Age 159 days. Activity and behavior of a AAV9-H2K treated SOD1G93A mouse (dots on

tail). Littermate treated with AAV9-empty has died prior to this time point.

Nature Medicine: doi:10.1038/nm.4052