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Natural Killer Cells Degenerate Intact Sensory Afferents following Nerve Injury

Summary Sensory axons degenerate following separation from their cell body, but partial injury to peripheral nerves may leave the integrity of damaged axons preserved. We show that an endogenous ligand for the natural killer (NK) cell receptor NKG2D, Retinoic Acid Early 1 (RAE1), is re-expressed in adult dorsal root ganglion neurons following peripheral nerve injury, triggering selective degeneration of injured axons. Infiltration of cytotoxic NK cells into the sciatic nerve by extravasation occurs within 3 days following crush injury. Using a combination of genetic cell ablation and cytokine-antibody complex stimulation, we show that NK cell function correlates with loss of sensation due to degeneration of injured afferents and reduced incidence of post-injury hypersensitivity. This neuro-immune mechanism of selective NK cell-mediated degeneration of damaged but intact sensory axons complements Wallerian degeneration and suggests the therapeutic potential of modulating NK cell function to resolve painful neuropathy through the clearance of partially damaged nerves.


INTRODUCTION
Injury to a peripheral nerve results in the destruction of severed axons distal to the injury site via Wallerian degeneration (Conforti et al., 2014) thereby facilitating subsequent debris removal by immune cells (Dekkers et al., 2013). Simultaneously, a genetic program of axonal outgrowth is then initiated to allow the reinnervation of targets by healthy nerves (Chandran et al., 2016), which is in turn supported by the permissive environment created by infiltrating immune cells (Gaudet et al., 2011). While this adaptive response to axonal injury facilitates the phagocytosis of debris and ultimately regeneration of axons, it also contributes to the maladaptive symptom of neuropathic pain (Costigan et al., 2009). As a consequence, neuro-immune signaling mechanisms of hypersensitivity following injury to the adult peripheral nervous system (PNS) are the subject of intense research (Chavan et al., 2017).
Axon degeneration is generally regarded as neuron autonomous; however, discrepancies in the timing of axon degeneration after injury (Neukomm and Freeman, 2014) and recent evidence pointing to the role of as-yet-unknown extrinsic factors in axon fragmentation (Gamage et al., 2017) open up the possibility of a role for immune cells with cytotoxic action in the degeneration process itself.
Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) typically involved in the destruction of tumor or virusinfected target cells. NK cell activity is coordinated by a balance of stimulatory and inhibitory signals mediated via a distinct receptor repertoire (Vivier et al., 2011). One of the best-characterized activating receptors is NK group 2D (NKG2D), which is expressed on NK cells and some T cells. Endogenous ligands for NKG2D are normally absent from most tissues but are expressed in disease states such as tumorigenesis and infection (Raulet et al., 2013). NK cells are found among the immune cells that infiltrate the damaged nerve following injury in adult animals (Cui et al., 2000), and the presence of intra-neural NK cells correlates with disease severity in a form of peripheral neuropathy (Lackowski et al., 1998). NK cells have previously been shown to be involved in guanethidine toxicity to sympathetic neurons (Hickey et al., 1992). In addition, adult murine NK cells are directly cytotoxic to immature embryonic dorsal root ganglion (DRG) neurons in vitro (Backströ m et al., 2000). However, NK cells at this stage of primary sensory neuron development appear to be immature (Tang et al., 2012), and thus the consequences of this NK cell interaction for the adult are unclear.
We investigated the response and functional consequences of NK cell interactions in the context of peripheral nerve injury in the adult mouse. Our results identify cytotoxic NK cells as a novel cellular trigger for the specific degeneration of damaged primary afferent axons that are still connected to their cell bodies in the injured PNS. Enhancing this NK cell-dependent degeneration mechanism increased clearance of damaged axons resulting in lower levels of mechanical hypersensitivity post-injury. This discovery provides new insight into immune mechanisms of damaged axon clearance and suggests the therapeutic potential of interventions that modulate NK cell function.

Activated NK Cells Induce Cytotoxicity in Embryonic
Sensory Neurons by an RAE1-Mediated Mechanism NK cells are classically activated by the cytokine interleukin (IL)-2, which primes them for cytotoxic attack by increasing intracellular content of granzyme B ( Figure S1A). We examined the effects of IL-2-stimulated NK cells on DRG neurons acutely isolated from embryonic (E15) and adult mice (cultured less than 24 h in vitro), using freshly isolated, unstimulated splenic NK cells as a control ( Figure S1B). Confirming original reports (Backströ m et al., 2000), embryonic DRG neurons were highly susceptible to NK cell-mediated cytotoxicity (Figures 1A and 1B). Separating NK cells and embryonic DRG with a transwell membrane prevented neurite fragmentation ( Figure S1C) despite the presence of nanogram amounts of granzyme B in the culture media (Figure S1D). In contrast, we found no evidence of cell lysis of acutely isolated adult DRG neurons by lactate dehydrogenase (LDH)-release cytotoxicity assay, even at much higher effectorto-target (E:T) ratios ( Figure 1B), although some neurites appeared truncated ( Figure 1A).
Time-lapse confocal imaging of DiI-labeled DRG neurons co-cultured with IL-2-stimulated NK cells expressing yellow fluorescent protein (YFP) revealed that stimulated NK cells were highly motile, enabling multiple direct cell-cell contacts between NK cells and sensory neurons. In embryonic DRG, this led to neurite destruction and cell death (Video S1); however, acutely cultured adult DRG neurite membranes remained largely intact (Video S2). NK cell contacts were often synchronous with intracellular Ca 2+ events in embryonic DRG neurites (Figures 1C and 1D;Video S3), leading to neurite fragmentation. While Ca 2+ events were also observed in adult DRG after NK cell contact (Figures 1C and 1D) and occasionally led to neurite fragmentation (Video S4), they occurred at a much lower frequency than in embryonic neurons ( Figure 1D).
Retinoic Acid Early inducible protein 1 (RAE1), encoded by the Raet1 gene family (a,b,g,d,ε), acts as a membrane-bound ligand for the activating receptor NKG2D (Cerwenka et al., 2000), which has previously been implicated in NK cell-mediated lysis of embryonic DRG (Backströ m et al., 2003). First, we confirmed that NK cytotoxicity against embryonic DRG could be attenuated by an NKG2D receptor blocking antibody ( Figure S1E). Using universal primers, we observed Raet1 transcripts in acutely dissociated embryonic and adult DRG neurons ( Figure 1E), but they were 17 times more abundant in embryonic DRG relative to adult DRG when assayed by qPCR ( Figure 1F). Western blot using a pan-RAE1 antibody revealed a band at approximately 40-50 kDa in embryonic but not adult DRG tissue ( Figure 1G). To assess the functional contribution of Raet1 in embryonic DRG neurons, we selectively knocked down all Raet1 isoforms using small interfering RNA (siRNA) ( Figure 1H), which, compared to negative control, led to a 20% reduction in NKmediated lysis ( Figure 1I).

Nerve Injury Drives RAE1 Expression in Adult Sensory
Neurons, Allowing Cytotoxic Attack by Activated NK Cells Adult mouse DRG neurons were cultured in a microfluidic chamber for 5 days in vitro. Following exposure to stimulated NK cells, we saw a 25% loss of neurite coverage relative to freshly isolated control NK cells (Figures 2A and 2B). Raet1 mRNA was timedependently upregulated in adult DRG cultures ( Figure 2C), with corresponding de novo expression of RAE1 protein after 2 days in vitro ( Figure 2D). Subsequently, adult DRG neurons cultured for 2 days displayed over a 10-fold increase in neurite fragmentation relative to controls in the presence of stimulated NK cells (Figures 2E and 2F). Transfection of dissociated adult DRG neurons with Raet1 siRNA prior to culture delayed Raet1 upregulation ( Figure 2G) and reduced the ability of stimulated NK cells to fragment DRG neurites relative to negative control siRNA (Figures 2H and 2I). We also confirmed that fragmentation of adult DRG neurites (2 days in vitro) can be reduced by blocking the NKG2D receptor on NK cells prior to co-culture (Figures S2A and S2B).
We next asked whether Raet1 upregulation in dissociated DRG in vitro occurs in injured cells in vivo by cutting the spinal nerve of the fifth lumbar DRG (L5x) in adult mice ( Figure 3A). An injurydependent upregulation of Raet1 was revealed by qPCR using RNA isolated from L5 DRG at 4 and 7 days after L5x injury (Figure 3B). The difference in Raet1 transcript levels between ipsilateral and contralateral L5 DRG 7 days after L5x injury was maintained over the first 24 h in culture ( Figure 3C). Injured (L5x) DRG exhibited 3.5-fold more fragmentation of neurites when exposed to IL-2-stimulated NK cells (Figures 3F and 3G) than DRG from uninjured (sham) animals ( Figures 3D and 3E), an effect that was attenuated by prior blocking of NKG2D receptors on NK cells (Figures S2C and S2D). No difference was seen in the total DRG cell body area between different groups ( Figure S2E) suggesting that the fragmentation was neurite specific.
Cytotoxic NK Cells Respond to Nerve Injury In Vivo and Target RAE1-Expressing Axons To characterize the response and function of NK cells in vivo, we crossed Ncr1 icre mice (Narni-Mancinelli et al., 2011) with reporter mice that express either YFP (Rosa26 eyfp ) or the diphtheria toxin (DTx) receptor (Rosa26 dtr ; DTR) following Cre-mediated recombination. Ncr1 encodes the NKp46 receptor, which is expressed on all NK cells, as well as tissue-resident ILCs, including group 1 ILCs (ILC1s) and a subset of group 3 ILCs (NCR1 + ILC3s). This genetic approach allowed us to identify (Figures S3A and S3B) or systemically deplete NKp46 + cells in vivo ( Figure S3C). We did not observe YFP-positive NK cells in sciatic nerve in naive mice (data not shown) or sham surgery mice ( Figures 3H and 3I). In contrast, L5x injury led to the marked recruitment of YFP + NK cells to the injured sciatic nerve (Figures 3H and 3I) and was paralleled by an increase in granzyme B content at 7 days post-injury ( Figure 3J), which was significantly reduced by prior systemic depletion of NK cells ( Figure 3K). L5x injury led to a reduction in the mechanical withdrawal threshold in the ipsilateral hind paw of NKp46-DTR mice treated with PBS ( Figure S3D) that was not affected by prior depletion of NK cells using DTx ( Figure S3D). The sensitivity of the uninjured contralateral paw remained unaffected by either treatment ( Figure S3E).
Consistent with the effect of nerve transection, crush injury ( Figure 4A) also drove Raet1 expression in ipsilateral DRG as observed by qPCR ( Figure 4B). In situ hybridization revealed Raet1 transcripts within individual DRG neurons (Figures 4C and 4D) and across a broad range of cell sizes (Figures S4A and S4B). RAE1 is known to be controlled by post-transcriptional modification (Raulet et al., 2013); therefore, we performed western blot on lysates of sciatic nerve and DRG tissues. Crush injury induced a 45-kDa band in ipsilateral injured sciatic nerve ( Figure 4E), indicating an increase in the glycosylated mature form of RAE1 (Arapovic et al., 2009) in the nerve, but not in Figure 1. Acutely Cultured Embryonic but Not Adult DRG Neurons Reveal Susceptibility to NK-Mediated Cytotoxicity by RAE1 (A) Immunolabeling of co-culture (4 h) between embryonic (top) or adult (bottom) DRG neurons (b-tubulin, magenta) and either freshly isolated (control) or IL-2-stimulated natural killer (NK) cells (NKp46, green). The inset shows a high-magnification image of NK cell in contact with embryonic DRG neurite. (B)  . Student's unpaired t test; t = 9.060, p = 0.0008. n = 3 mice, or replicates per group. (I) LDH-release cytotoxicity assay of negative control or Raet1-selective siRNA knockdown embryonic DRG. Three replicate co-cultures for each siRNA group. Matched two-way ANOVA F(1,10) = 133.85, p < 0.0001). See also Figure S1. the DRG ( Figure 4F). Chronic tight ligation of the sciatic nerve, to block axonal transport, revealed RAE1 immunolabeling within crushed axons proximal to the ligation site (Figures 4G, 4H, and S4B) and greater amounts of RAE1 protein at the ligation site relative to within the axons proximal to the ligation ( Figure S4C).
Crush injury also raised levels of granzyme B in the sciatic nerve ( Figure S5D). Using two-photon imaging of the sciatic nerve in vivo 3 days after crush injury, we observed NKp46-YFP cells ''rolling'' along the wall of intra-neural blood vessels (Video S5). Within the sciatic nerve itself, NK cells were highly motile and displayed multipolar morphology (Video S6), reminiscent of stimulated NK cells in vitro. No YFP + cells were observed by two-photon imaging in the nerves of sham injured mice (data not shown).

Endogenous NK Response Attenuates Post-injury Sensitivity
Depletion of NK cells by DTx treatment in NKp46-DTR mice (Figure S5E) reduced NKp46 + cell infiltration (Figures S5F and S5G) and granzyme B content in the crushed nerves ( Figure S5H). Control (PBS-treated) NKp46-DTR mice were almost completely insensitive to pinprick stimulation of the hind paw until a relatively rapid heel-to-toe pattern of recovery beginning in the second week after injury ( Figures S5I and S5J, PBS) consistent with axon regeneration through the foot (Latremoliere et al., 2018).
(legend continued on next page) injection (Figures 5B, 5C, and 5D, IL-2 complex). This was paralleled by a 10-fold increase in the numbers of NKp46-YFP + cells in the injured nerve at the crush site and distal to the injury, but not proximal to the crush site in IL-2-complex-treated mice ( Figures 5E and 5F). Treated mice also displayed enlargement of the spleen ( Figure 5G). Contralateral nerves did not display any infiltration of NKp46-YFP + cells ( Figures 5E and 5F) and contralateral paws remained fully sensitive to pinprick throughout the experiment (data not shown).
Flow analysis of peripheral blood after recovery (day 16) confirmed depletion of NKp46 + DX5 + NK cells by an anti-NK1.1 antibody in wild-type mice ( Figure 5H) and by DTx in NKp46-DTR but not wild-type mice ( Figure S5K). No difference was observed in CD3 + CD8 + T cell populations with either method of depletion ( Figures 5H and S5L). Anti-NK1.1 antibody was also sufficient to deplete the majority of NKp46-YFP + cells from the peripheral blood of NKp46-YFP mice ( Figure 5I). In isotype-control-treated mice, IL-2 complex treatment again resulted in acute loss of the pinprick response (Figures 5J-5L, Isotype), whereas NK cell-depleted mice maintained a constant level of ''retained'' sensitivity throughout the paw (Figures 5J-5L, Anti-NK1.1). Similar results were obtained in DTx-treated wild-type and NKp46-DTR mice ( Figures  S5M and S5N).
IL-2 complex-stimulated wild-type mice treated with the anti-NKG2D blocking antibody, which did not deplete NK cells (Figures S6A), reduced specific NKG2D labeling on peripheral blood NK cells by >60% ( Figures S6B and S6C), and attenuated the sensory loss after IL-2 complex treatment compared to isotype-control-treated mice ( Figures S6D and S6E).
The density of b-tubulin III-labeled axon fibers in the sciatic nerve 6 days after partial crush was reduced within the crush site and distal region by IL-2 complex treatment (Figures 6A and 6B, insets b and c) but not the proximal nerve region ( Figures  6A and 6B, inset a). Labeling for microtubule-associated protein stathmin 2 (STMN2), expressed by injured DRG neurons (Cho et al., 2013), was observed throughout the crushed nerve and was also reduced by IL-2 complex treatment at the crush site and distal regions relative to IgG control ( Figures 6C and 6D, regions b and c). STMN2 also co-localized with RAE1 immunolabeling at the injury site (Figures S4B).
To assess axon integrity directly, we employed transmission electron microscopy of the sciatic nerve injury site 6 days after partial sciatic nerve crush ( Figure 6E). Myelinated axons, likely to conduct the sensory response to pinprick (Ziegler et al., 1999), were categorized according to their myelin and axoplasm integrity ( Figure 6F). Injured nerves from IL-2-complex-treated mice contained significantly more degenerated myelinated axons than IgG-only controls ( Figures 6E and 6G, degenerated) and less abnormal axon-Schwann cell units ( Figure 6G, abnormal), suggesting that such axon fibers-a feature of clinically relevant peripheral neuropathies (Sander et al., 2000)may be targeted for degeneration by activated NK cells. Depletion of NK cells with an anti-NK1.1 antibody prior to IL-2 complex stimulation prevented these changes ( Figure 6G).

Clearance of Partially Damaged Axons Alleviates Pain Hypersensitivity after Injury
We next compared the effects of partial and full crush models in the absence of immune modulation ( Figures 7A-7C). After recovery of pinprick sensation, paw withdrawal thresholds were significantly lower (i.e., more mechanically sensitive) in mice with previous partial crush injury relative to those that received a full crush, a difference that lasted at least 30 days after the original injury ( Figure 7D). IL-2 complex treatment after partial crush resulted in higher hind-paw mechanical thresholds compared to control (IgG) animals at days 15-16 after injury ( Figure 7E), an effect that was prevented by either prior depletion of NK cells ( Figure 7F) or blockade of the NKG2D-RAE1 interaction (Figure S6F). Combined data revealed a positive correlation between post-crush pinprick sensitivity and the resulting mechanical sensitivity of the injured limb ( Figure 7G).
Finally, we examined the effect of expression of the RAE1 ligand in sensory neurons in the absence of injury. We used a Trpv1-driven Cre mouse line to conditionally overexpress Raet1 in a population of thermal nociceptive DRG neurons (Figure S7A). These mice displayed an impaired ability to sense noxious heat but demonstrated no change in mechanical thresholds compared to littermates and wild-type controls (Figures S7B and S7C).

Recruitment and Cytotoxicity of Injury-Responsive NK Cells
Using two photon imaging in vivo, we observed YFP-expressing NK cells rolling and attaching to the blood vessel wall within the injured nerve, suggesting that NK cells enter the nerve via extravasation from the blood, a process known to be chemokine dependent (Sheikh et al., 2004). Consistent with that possibility, the chemokines CCL2, CCL3, and CX3CL1 are all highly expressed after peripheral nerve injury (Ren and Dubner, 2010) with the corresponding chemokine receptors, CCR2, CCR5, and CX3CR1 expressed by NK cells (Loux et al., 2010). Within the nerve tissue itself, YFP-expressing NK cells displayed a ''stop-go'' behavior, previously observed by live imaging in other tissues (Garrod et al., 2007), allowing NK cells to effectively survey the damaged nerve for injured axons.
In injured sciatic nerve, we detected elevated levels of granzyme B, a serine protease that is essential for NK cell cytotoxic functions (Voskoboinik et al., 2015). Systemic depletion of NK cells reduced levels of granzyme B detected 7 days after nerve injury, suggesting that NK cells are functional in the injured peripheral nerve. In vitro, NK cell contact was followed within minutes by intra-axonal Ca 2+ flux and subsequent neurite fragmentation. This time course is well within the time frame of immune synapse formation between NK and target cells (Lopez et al., 2013) and suggests that targeted delivery of cytotoxic enzymes to the injured axon is sufficient to trigger neurite degeneration (Wang et al., 2012). Further studies will be required to determine whether these NK cell-derived degenerative signals cooperate with the known components of ''intrinsic'' axon degeneration distal to the injury site, including SARM1 and calpain activation (Gerdts et al., 2016).

NK Cell-Mediated Neurodegeneration
Wallerian degeneration in the sciatic nerve can be induced by multiple types of crush injury (Bridge et al., 1994). In our study, a conventional forceps crush injury was sufficient to cause the total loss of pinprick sensation from 1 day after nerve injury in all mice, with recovery of evoked sensation beginning more than 1 week later (Latremoliere et al., 2018). However, in NK cell-depleted mice a significant restoration of pinprick response was observed just days following complete crush. This latent sensitivity is in keeping with early electron microscopic studies of Wallerian degeneration, which report that some fibers may survive more than 2 or 3 days and well beyond an early period of axonal degeneration (Dyck and Hopkins, 1972). Our results therefore suggest that certain sciatic nerve crush injuries may not be sufficient to axotomize all fibers and that degeneration of the surviving axons requires an additional extrinsic trigger.
The rapid return of pinprick sensation we observed in the first week after partial crush in control animals is consistent with the functional recovery present in the ''moderate'' injury model of optic nerve crush (Duvdevani et al., 1990). The increase in b-tubulin III-labeling we observed distal to the partial crush site is further consistent with either sprouting or axon swelling typical after a contusion injury, whereby axons that do not fragment can survive intact long term (Williams et al., 2014). Our electron microscopy data suggest that many axons do indeed survive in the sciatic nerve after partial crush.
IL-2/anti-IL-2 monoclonal (S4B6) antibody complex treatment has been successfully used to expand NK cells in vivo for the treatment of cancer (Votavova et al., 2014). This strategy revealed an acute period of NK cell-dependent sensory loss, which correlated with a lower density of STMN2-expressing axons as well as a reduction in abnormally myelinated axons and a corresponding increase in the number of degenerated myelinated axons in sciatic nerve cross-sections. These results suggest that IL-2-activated NK cells further remove damaged axons that would otherwise have been spared. Critically, the simultaneous recovery of pinprick sensation in both groups of mice (by day 15) suggests that successfully regenerating axon fibers are not among those targeted by NK cells.

Consequences for Post-injury Sensation
After recovery of pinprick responses, partially nerve crushed mice showed hypersensitivity to mechanical stimulation, a surrogate marker of chronic neuropathic pain. The mechanical hypersensitivity that appears 2 weeks after injury may be due to damaged but functionally intact sensory axons, as well as other damaged axons that have not re-innervated their targets but remain within the nerve (Xie et al., 2017). We therefore hypothesize that the active targeting of NK cells to remove partially injured fibers early after injury helps to leave the nerve uncontaminated by previously damaged axons and thus enable successful regeneration without the aberrant sensation and pain often associated with nerve injury (Costigan et al., 2009).
Overexpression of Raet1 in TRPV1-lineage sensory neurons (Mishra et al., 2011) in otherwise naive mice was sufficient for a loss of sensation specific to noxious heat. This points to the potential for neuronal stressors initiating a detrimental action of NK cells on previously uninjured peripheral nerves (Hickey et al., 1992) by upregulation of NK cell ligands (Backströ m et al., 2007;Greene et al., 2016). NK cells may therefore be a therapeutic target in multiple die-back peripheral neuropathies that are often co-morbid with metabolic stress in patients with neuropathic pain (Bennett et al., 2014).

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

STAR+METHODS CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Seog Bae Oh (odolbae@snu.ac.kr).  ) were a kind gift from Dr Lewis Lanier. All mice are originally on a C57BL/6 background except Trpv1 cre , which are on a mixed B6 and 129 background, and Rosa26 eyfp mice which were backcrossed onto the C57BL/6 background for at least 10 generations. All mice were maintained as homozygous stocks in a specific pathogen free (SPF) facility and transferred to a conventional room at least one week before experiments. Double heterozygote Nrc1 icre/wt ;rosa26 eyfp/wt mice (abbreviated to NKp46-YFP) and Nrc1 icre/wt ;rosa26 dtr/wt mice (abbreviated to NKp46-DTR) were bred from single crosses. Trpv1 cre/wt ;rosa26 raet1e/wt mice (abbreviated to TrpV1-Rae1) and Trpv1 cre/wt ;rosa26 wt/wt mice (littermates) were the product of crosses between Trpv1 cre/cre and rosa26 raet1e/wt mating pairs. Mice were maintained on a 12 h:12 h light/dark cycle (lights on at 8:00 a.m.), housed 4-6 mice per cage on wood chip bedding with nestlets and provided with standard laboratory feed and water ad libitum. DRG neurons and NK cells were prepared from male C57BL/6 mice (6-8 weeks); embryonic DRG neurons were prepared from embryos on day 15 in utero (E15) removed from euthanized female C57BL/6 mice. Nerve injury experiments were performed on male mice age 7-9 weeks old of indicated genotype. Animals were killed in accordance with Schedule 1 of the UK Animals (Scientific Procedures) Act 1986 by inhalation of a rising lethal concentration of isoflurane followed by decapitation for tissue culture, or a rising lethal concentration of carbon dioxide at the end of experiments.

METHOD DETAILS
NK cell depletion NKp46-DTR mice were treated with diphtheria toxin (DTx; 100 ng) or sterile PBS vehicle (100 ml) intravenously by retro-orbital injection. Under isoflurane anesthesia (3% induction, 1%-2% maintenance in 100% O 2 ) an ophthalmic solution (0.5% proparacaine, Alcon, Belgium) was applied to the eye as a local anesthetic. An insulin syringe (0.3ml, BD Biosciences) was inserted bevel-down behind the eye into the retro-orbital sinus and 100 ml slowly injected. Injections were alternated between eyes. Injections were administered blind to the content of the syringe starting one day prior to surgery and continuing every four to five days for the duration of the study. All mice were blood sampled at the end of experiments for depletion efficiency check by flow cytometry. For antibody depletion of NK cells, wild-type C57BL/6 mice were injected with 100 mg of LEAF purified anti-mouse NK1.1 (clone PK136) (Biolegend, cat no. 108712, RRID:AB_313399) or LEAF purified rat IgG2a,k isotype control (clone MOPC-173) (Biolegend, cat no. 400224, RRID:AB_326472) intravenously by retro-orbital injection one day before nerve injury.

L5 spinal nerve transection (L5x)
Male mice age 7-9 weeks old were placed under isoflurane inhalation, the dorsal lumbar region was shaved, treated with an iodine solution (Potadine) and a unilateral incision made parallel to the L6 vertebrate. Under a x20 dissection microscope illuminated by a cold light source the musculature was parted by blunt forceps dissection to reveal the L6 transverse process, which was then cut and removed. The L5 spinal nerve, which runs immediately below the L6 process, was carefully freed of connective tissue and cut with fine spring scissors; 1 mm of the nerve was removed to prevent nerve regeneration. The wound was irrigated with sterile saline and closed in two layers with 6-0 silk sutures (Ailee, Korea) and 9mm skin clips (MikRon Precision, CA, USA) Mice were placed in a warm, darkened cage to recover from surgery.

Sciatic nerve crush
Adult male mice (7-9 weeks old) received a single unilateral crush injury to the sciatic nerve (Bridge et al., 1994). Briefly, under isoflurane anesthesia the right thigh was shaved and iodine treated and an incision was made mid-thing length. The sciatic nerve exposed as it emerges from the sciatic foramen by parting the muscle with blunt forceps dissection. The nerve was carefully freed of connective tissue and fully crushed for 15 s using fine, mirror-finished forceps (No 5, Dumont, Fine Science Tools, Germany). The wound was closed in two layers with two sutures of the overlying muscle facia and a single skin clip to close. Complete crush of the sciatic nerve was deemed successful by a sensory score of zero using the pinprick assay on the day following surgery; any mice with a pinprick response less than 24 h after surgery were excluded from analysis.
For partial crush the sciatic nerve an ultra-fine hemostat (Cat no. 13020-12, Fine Science Tools, Germany) was fitted with a custom spacer created from two layers of aluminum foil (15 mm thick) to create a gap 30 mm thick when fully closed. The sciatic nerve was carefully freed of connective tissue and placed between spacers on the hemostat (2-3 mm from the tip) by gently lifting the nerve using a fire-polished glassed rod. The hemostat was then closed on the first locking position and held for 15 s before careful release of the nerve. Full sciatic nerve crush was performed in the same way except no spacer was used. The wound was closed in two layers with two sutures of the overlying muscle facia and a single skin clip. Mice were recovered in a warm, darkened cage. All tools were autoclaved prior to surgery and strict aseptic technique was maintained throughout.

IL-2/anti-IL-2 antibody complex treatment
Recombinant mouse interleukin-2 (Cat no. 212-12, lot no. 0608108; Peprotech, Rocky Hill, NJ, USA) was prepared as a stock at 0.1 mg/ml in PBS (without carrier protein) and stored at 4 C for up to one week, according to the manufacturer's instructions. On the day of treatment, IL-2 (1.5 mg per mouse) was pre-mixed with anti-mouse IL-2 monoclonal antibody (50 mg per mouse) (S4B6-1 clone) (BioXCell; RRID:AB_1107705) and incubated at room temperature for 15 min. The bound cytokine/antibody complex was further diluted in sterile PBS to a total volume in 500 ml and injected intraperitoneally. Injections were given once daily (evening time) for four days. For control experiments, mice were injected with an equal amount of rat IgG2a isotype peptide (clone 2A3) (BioXCell; RRID:AB_1107769). The efficacy of the IL-2 antibody complex treatment was confirmed by enlargement of the spleen compared to PBS injected mice one day after the final injection (Boyman et al., 2006).

Behavioral testing
All sensory testing was performed between the hours of 09.00 and 18.00 in an isolated room maintained at 22 ± 2 C and 50 ± 10% humidity. For mechanical threshold (von Frey filament) testing, mice were brought from the animal colony and placed in transparent plastic boxes on a metal mesh floor with 5x5 mm holes (Ugo Basile, Italy). The mice were then habituated for at least 30 min prior to testing. To assess mechanical sensitivity, the withdrawal threshold of the affected hind paw was measured using a series of von Frey filaments (0.20,0.40,0.70,1.6,3.9,5.9,9.8 and 13.7 mN,Stoelting,Wood Dale,IL,USA;equivalent in grams to 0.02,0.04,0.07,0.16,0.40,0.60 , 1.0 and 1.4). The 50% withdrawal threshold was determined using the 'up-down' method and calculated using Up-Down Reader software (Gonzalez-Cano et al., 2018). A brisk hind paw lift or flinch in response to von Frey filament stimulation was regarded as a withdrawal response. The 0.4 g filament was the first stimulus to be used, and, when a withdrawal response was obtained, the next weaker filament was used. This process was repeated until no response was obtained, at which time the next stronger filament was administered. All behavioral testing was performed by an investigator who was blind to the treatment of the mice.
Pinprick sensory recovery testing was performed as previously described (Latremoliere et al., 2018) with slight modification. Mice were habituated on an elevated mesh in separate compartments. The lateral side of the affected hind paw was separated into five regions from the toe to the heel and stimulated with a stainless steel Austerlitz insect pin (Size 000, FST, Germany). A sensory response was confirmed by rapid lifting or flinching of the paw. The number of responses to two consecutive pin applications to the skin was recorded per region providing a score out of 10. Responses due to direct movement of the paw or hind limb (indicative of extraterritorial proprioception) were excluded. Testing was carried out daily (09.00 -14.00) until full sensory recovery (score of 10) was observed for all mice.
For hot plate testing, mice were placed on a metallic plate heated at 49 C within an acrylic container (Bioseb, France), and the latency to flinching, licking one of the hind paws or jumping was measured. A cut-off time of 60 s was used to minimize the possibility of cutaneous tissue damage.
For injured DRG experiments, ipsilateral L5 DRG were rapidly dissected on ice-cold HBSS from adult mice 7 days after L5x or sham surgery, pooled (n = 3 DRG per group) and dissociated to a single cell suspension as above. DRG were then seeded onto poly-D-lysine and laminin-coated glass bottom dishes (10 3 cells per dish) and cultured overnight in neurobasal medium containing NGF (50 ng/ml).
For microfluidic co-cultures, DRG neurons isolated from adult mice (as above) were suspended in neurobasal medium and seeded (10 4 cells) into the somal reservoir of a microfluidic device (Xona Microfluidics, CA, USA) previously coated with poly-D-lysine (10 mg/ml) and laminin (10 mg/ml) (Sigma). NGF (100 ng/ml) was added to the media in the neurite reservoir. Neurons were cultured for 5 days during which neurites grew along 3 mm x 500 mm channels connected to the neurite reservoir.
NK cell isolation and stimulation in vitro NK cells were prepared from adult male C57BL/5 mouse spleens (6-8 weeks old). Spleens were homogenized by sequentially passing through 70 mm and 40 mm cell strainers (Falcon, BD Biosciences). Red blood cells were lysed by incubation for 2 min in ACK lysis buffer (in mM: 150 NH 4 Cl, 10 KHCO 3 , 0.1 Na 2 EDTA, pH 7.3). Single-cell suspensions were then passed through nylon wool columns (Polysciences, Warrington, PA) for the depletion of adherent populations, consisting of B cells and macrophages. Eluted cells were re-suspended in 0.01 M phosphate buffered saline (PBS) plus 2 mM EDTA and 2% FBS. NK cells were enriched using a magnetic associated cell sorting (MACS) method in combination with a negative selection protocol (Mouse NK cell isolation Kit II, cat no. 130-096-892, Miltenyi Biotech GmbH, Germany) according to the manufacturer's instructions. Briefly, cell suspensions were sequentially incubated at 4 C with a cocktail of biotin-conjugated monoclonal antibodies against non-NK cells followed by anti-biotin microbeads. The cell suspension was then passed through an LS column placed in a magnetic field (MidiMACS Separator, Miltenyi Biotech GmbH). Bead-conjugated non-NK cells remained in the columns while non-labeled NK cells passed through in the eluent. Enriched NK cell were either used directly (control) or stimulated with recombinant murine interleukin (IL)-2 (Cat no. 212-12, lot no. 0608108; Peprotech, Rocky Hill, NJ, USA) at 1000 U/ml for two days before use in experiments. NK cells were cultured at 2x10 6 cells per ml in RPMI 1640 medium (GIBCO, Life Technologies) supplemented with fetal bovine serum (FBS) (10%) and penicillin/ streptomycin (100 U/ml) in 96-well U-bottomed (Falcon, BD Biosciences) plates for 48 h. The purity of NK cells (NKp46+DX5+) in the eluent was checked by flow cytometry to be consistently above 90%. The cells were then harvested and used as effector cells in co-culture and cytotoxicity experiments.

DRG -NK co-cultures
Control or IL-2 stimulated NK cells were harvested, washed in RPMI and re-suspended in neurobasal media. DRG were washed once in neurobasal media (NGF-free) and NK were added to the neurite compartment (5x10 5 cells) for microfluidic cultures or seeded (2.5x10 5 cells) directly over the DRG for glass coverslip cultures, and co-cultured for 4 h at 37 C and 5% CO 2 . Co-cultures were carefully washed once in warm HBSS and fixed with 2%-4% PFA in 0.01M PBS (pH 7.4) for 30 min at RT, followed by washing in PBS (3 3 10 mins) at storage at 4 C prior to immunolabeling for b-tubulin III and NKp46 (see Immunofluorescence).
For transwell experiments, glass coverslip cultures of DRG were transferred to a 24-well plate in 500 ml Neurobasal media. NK cells (2.5x10 5 per well) were seeded onto 6.5 mm diameter polycarbonate transwell membrane inserts with 0.4 mm pore size (Corning) and incubated for 4 h at 37 C and 5% CO 2 before removal of the membrane and fixation as above.

LDH-release cytotoxicity
Effector NK cells were assayed for cytotoxicity against DRG neuron targets by measuring the release of lactate dehydrogenase (LDH) into the culture medium using an LDH Cytotoxicity Assay Kit (Thermo Scientific Pierce, IL, US). Control or IL-2 stimulated NK cells were harvested, washed in RPMI and added to DRG cultures in 96 well plates (Nunclon, Thermo Scientific) in neurobasal medium at various ratios and cultured for 4 h before sampling of media supernatant which was assayed for LDH activity according to manufacturer's instructions. Absorbance values were acquired on a microplate spectrophotometer (BioTek Instruments, VT, US). Each ratio was determined in triplicate. Specific cytotoxicity was calculated as follows: % cytotoxicity = [(experimental releasespontaneous release)/(maximum release -spontaneous release)] x 100. The optimum number of target cells (DRG neurons) for maximum LDH release detection was determined prior to experiments (8x10 3 embryonic DRG neurons; 10 3 adult DRG neurons).

Live confocal imaging
Adult or embryonic mouse DRG (10 3 and 8x10 3 neurons, respectively) cultured for one day on PDL and laminin-coated glass-bottom Petri dishes were fluorescently labeled with Vybrant DiI (Molecular Probes, cat no. V-22886) or loaded with the Ca 2+ indicator rhodamine 3-AM (Molecular Probes, cat no. R10145) according to the manufacturer's instructions. NK cells previously isolated from NKp46-YFP mice and stimulated IL-2 (1000U/ml) for 48 h were suspended in neurobasal media and seeded onto the coverslip (2.5x10 5 cells per dish). Dishes containing DRG-NK co-cultures were immediately transferred to a confocal microscope (LSM 700, Zeiss) and maintained in a humidified atmosphere at 37 C and 5% CO 2 (Live Cell Instruments, Seoul Korea). A time series of single z section images (512 3 512) were acquired using a multitrack setting (488 nm and 555 nm fluorescence emission and differential interference contrast (DIC) bright-field) at 30-60 s intervals and multiple positions under the control of Definite Focus. Images were acquired up to 3h and exported as a sequential time-lapse in AVI format.
In vivo two photon sciatic nerve imaging Male NKp46-YFP mice (8-9 weeks old) received unilateral sciatic nerve crush or sham surgery. On day 3 mice were anaesthetized with pentobarbital (80 mg/kg, i.p.) supplemented with 20 mg/kg immediately prior to recording. Some mice additionally received Dextran-Texas red (neutral 40,000 m.w.) (Molecular Probes) given via retro-orbital injection (100 ml, 10 mg/ml) to visualize the vasculature and confirm maintenance of blood flow during recording. The sciatic nerve was re-exposed and bathed in sterile saline. Mice were placed on a warm pad maintained at 35 C for the duration of the imaging. The sciatic nerve was carefully lifted with two curved glass rods held via a micromanipulator at both distal sides of the exposed nerve to mechanically isolate the nerve from the body and minimize the breathing-induced motion artifacts. A W plan-Apochromat 20X water immersion lens was lowered to the nerve surface 1-2 mm distal from the crush site to identify the blood vessels and YFP-positive cells. After the Ti-sapphire laser (Chameleon, COHERENT) was tuned to the wavelength of 900 nm for excitation of both YFP and Texas Red, 3D time-lapse imaging was performed using a laser scanning confocal microscope (LSM 7MP, Zeiss) as follow: 1024 3 512 pixels per a single image, 0.79 ms of pixel dwell, 2 mm x 25 sections per a z stack (total 50 mm depth) at 30 s interval. The two-photon laser power was compensated according to the depth across 5 -8% of total power. The image was rendered into 2D video using imaging software (Zeiss Efficient Navigation 2012).

DRG small interference RNA gene knockdown
Acute isolated adult or E15 embryonic DRG were transfected with siRNA by electroporation (Neon, Invitrogen) according to the manufacturer's instructions. Briefly, single cell suspensions of DRG were suspended in an electroporation medium containing siRNA oligonucleotide and drawn into a tip containing a gold-plated electrode (5x10 4 cells per 10ml). Tips of cells were placed into a tube containing electrolytic buffer, electroporated (1500V, 20ms) and immediately ejected into penicillin/streptomycin-free neurobasal media containing 50 ng/ml NGF and cultured for 48h at 37 C, 5% CO 2 until assessment of assessment of knockdown or functional experiments. Prior to experiments transfection efficiency was optimized by electroporation of DRG with a cDNA plasmid encoding green fluorescent protein (GFP) (0.5mg) and examination of GFP fluorescence after two days in culture. Two siRNA oligonucleotides per target were tested for knockdown efficiency by real time PCR; siRNA oligos that reduced mRNA expression by more than 70% compared to a negative control siRNA oligonucleotide were used for functional experiments. Gapdh siRNA oligonucleotides (10nM) (Silencer Select, Ambion, Life Technologies, cat no. 4390849) were used as a positive control. siRNA oligonucleotides used for cytotoxicity assays were: Raet1a-e (10nM) (sense) 5 0 -CAAUGGUUACCCACAUUUAtt-3 0 ; (antisense) 5 0 -UAAAUGUGGGUAACCAUUGgt-3 0 (Silencer Select, Ambion, Life Technologies, cat no. 4390771, ID s234333); Negative control #1 (10nM) (Silencer Select, Ambion, Life Technologies, cat no. 4390843).

Peripheral blood lymphocyte isolation
Systemic depletion of NK cell was confirmed at the end of experiments in a sample of peripheral blood obtained by retro-orbital bleed. Briefly, under isoflurane anesthesia a 15ml glass Pasteur pipette was inserted into the retro-orbital sinus and gently twisted to disrupt the orbital venous plexus. 50-100 ml of blood was removed by capillary action and ejected into heparin-coated tubes (Idexx Laboratories, USA). Blood samples were diluted with an equal volume of serum-free RPMI, floated on top of a lymphocyte separation medium (Lympholyte Mammal, Cedarlane Labs, Canada) and centrifuged at 1000 g for 20 min with a slow acceleration gradient. Peripheral blood mononuclear cells were collected from the monolayer, washed in RPMI and suspended in FACS buffer (5% FBS, 0.002% NaN 3 in 0.01M PBS, pH 7.4) for flow cytometry analysis.

Preparation of sciatic nerve suspensions and flow cytometry
For flow cytometry analysis of NK cells present in sciatic nerve, NKp46-YFP mice were deeply anaesthetized with pentobarbital (100mg/kg, i.p.) followed by trans-cardiac perfusion with PBS (0.01 M, pH 7.4) at various time points after peripheral nerve injury to remove peripheral blood from the circulation. Bilateral sciatic nerves were rapidly removed to ice-cold Ca 2+ -and Mg 2+ -free HBSS (Welgene) (including 20mM HEPES) and cut into 1-2 mm pieces. Tissues were transferred to 15 mL tubes and centrifuged at 500 g for 5 min. HBSS was replaced with collagenase A (1 mg/ml) and dispase II (2.4 U/ml) (Roche, Switzerland) and incubated 90 min at 37 C with frequent gentle agitation. Additional digestion was carried out for 5 min in trypsin (0.25%) and stopped with a trypsin inhibitor (2.5 mg/ml) (Sigma, T9003) in PBS followed by washing in RPMI containing 10% serum (GIBCO, Life Technologies).
Nerves were dissociated by trituration with a fire-polished glassed pipette in RPMI containing DNase I (125 U/ml), passed through a 30 mm separation filter to remove debris (Miltenyi) and resuspended in FACS buffer in 5 mL round bottom tubes. Prior to sciatic nerve flow cytometry, CD45+ lymphocyte and NKp46-YFP cell gating was set on peripheral blood lymphocytes from wild-type and NKp46-YFP mice. Sciatic nerve samples were run on the slow setting until the total event count was less than 50 per second, or gated events were less than one per 5 s. NK cells were identified by lymphocyte FSC-SSC scatter profile and YFP fluorescence; in some experiments total lymphocytes were additionally labeled with an APC-conjugated anti-mouse CD45 antibody (1:400; eBioscience, cat no. 17-0451. RRID:AB_469393).

Tissue preparation
At specific time points after nerve injury or sham surgery, mice were deeply anaesthetised with pentobarbital (100 mg/kg, i.p.) followed by exsanguination by trans-cardiac perfusion with PBS (0.01 M, pH 7.4) containing heparin (500 U/L). Whole DRG (lumbar L3-L5) and full length sciatic nerve (spinal nerves to peripheral trifurcation) tissues were dissected and collected in sample tubes which were immediately frozen on liquid nitrogen and stored at À70 C for later molecular analysis. For immunohistochemistry PBS perfusion was followed by a paraformaldehyde fixative (4% PFA, 0.2% picric acid in 0.1M PBS, pH 7.4). Sciatic nerves were post-fixed up to 24h and cryoprotected in sucrose solution (30% sucrose in 0.01M PBS, pH 7.4) at 4 C. Fixed sciatic nerve tissues were embedded in frozen section compound (Optimal Cutting Temperature, Leica), sectioned on a cryostat (14mm) and thawmounted on glass microscope slides (Superfrost Plus, Fischer Scientific).

Immunofluorescence
After washing in PBS (3 3 10 mins) cells and tissues were blocked and permeabilized in 10% normal donkey serum (NDS, Jackson ImmunoResearch) and 0.1%-0.3% Triton X-100 in PBS for 1h at RT. Primary antibodies were applied in 1% NDS, 0.01%-0.03% Triton X-100 in PBS and incubated overnight at 4 C in a humidified chamber. Primary antibodies used were: Rabbit anti-b-tubulin III (1:400-500; Sigma, cat no. Thermo Scientific, cat no. S11222). For RAE1 double labeling with b-tubulin III and STMN2, primary and secondary labeling was performed sequentially (RAE1 first) for each antibody with a repeat blocking stage between. Fluorescence images (1024 3 1024) were acquired sequentially on a laser-scanning confocal microscope (LSM 700 Zeiss) with Zen 2012 software (v8.1 SP1, Zeiss). Coverslips were imaged in a single z section. Tissue sections were mounted with DAPI-containing hard-setting aqueous mounting medium (Vectorsheild, Vector Laboratories) and imaged as a stack of 4-5 3 2mm z sections and exported as a maximum intensity projection TIFF.
In situ hybridization Male C57BL/6 mice (aged 7-8 weeks) received a unilateral sciatic nerve partial crush injury as described. On day 6 post-injury mice were perfused with 0.01M PBS followed by 4% PFA in 0.1M PBS buffer. Bilateral lumbar DRG were dissected and post-fixed overnight at 4 C followed by cryopreservation in 30% sucrose in PBS for 5 days at 4 C. DRG tissues were embedded in OCT compound, 10 mm sections were cut on a cryostat and thaw-mounted to Superfrost slides and stored at À80 C. Sections from the ipsilateral and contralateral DRG of each mouse were mounted on the same slide. In situ hybridization (ISH) was performed with RNA Scope 2.5 HD assay (Red) (322350) according to the manufacturer's instructions (Advanced Cell Diagnostics, Bio-Techne). Slides were allowed to reach room temperature (RT) then washed once in PBS to remove OCT compound followed by hydrogen peroxide treatment 10 min at RT. Sections the underwent antigen retrieval by submerging in boiling retrieval buffer for 2 min followed by wash in distilled water. After ethanol (100%) treatment sections were treated with Protease Plus reagent (ACD) for 30 min at 40 C in a humidified chamber. Slides were then incubated with probe Mm-Raet1 (448121; targets all Raet1 isoforms) or positive and negative control probes for 2 h at 40 C. Following thorough washes sections were incubated with six rounds of amplification reagents followed by signal development with fast red. Sections were then processed for NeuN immunohistochemistry. Briefly, sections were blocked with 10% normal goat serum, 0.3% triton-x in 0.01M PBS (1 h, RT) followed by incubation with a chicken anti-NeuN antibody (1:400) overnight at 4 C. The next day sections underwent sequential labeling with biotinylated goat anti-chicken antibody (1:200) and pacific blue-conjugated streptavidin (1:200).

Transmission electron microscopy
On day 6 after partial crush mice were perfused with 0.1 M sodium cacodylate trihydrate and 0.1 M sucrose buffer, pH 7.3, followed by 4% paraformaldehyde and 2.5% glutaraldehyde (EM grade) in 0.1 M sodium cacodylate buffer, pH 7.3. Bilateral sciatic nerves were dissected and post-fixed in same fixative and washed three times in 0.1 M sodium cacodylate (SC) buffer only. Samples were carefully trimmed down in a proximal direction toward the crush site. Samples were washed 1x 20 min with 50mM glycine in 0.1M SC buffer with rotation followed by 1x 10-20 min with 0.1M SC buffer. Staining was performed with 1.5% potassium ferrocyanide + 1.5% osmium in 0.1M SC buffer for 4hrs at 4 C with vigorous rotation followed by wash 3x 10 min and 1x30 min with MQ water. Finally, sampled were stained with 0.5% uranyl acetate overnight at 4 C and washed 3x 60 min with water. The dehydration and resin infiltration was performed in the Leica AMW microwave. Briefly, this consisted of 1-2min steps in each 30%, 50%, 70%, 90%, 95% and 100% ethanol at 37 C with constant 25W power, followed by 100% acetone with 3x changes over 5 min. Resin infiltration was performed with 3:1, 1:1, 1:3 acetone:epoxy resin (Ducrupan) in 3 min steps at 37 C and 10W. Samples were then taken through 5x changes 100% resin over 80 mins at 45 C, 12 W, with the final resin step performed at 50 C. Nerves were then embedded in fresh Durcupan resin in flat dish molds at 60 C for 48 hr. Semithin (500nm) and ultrathin sections (90nm) were taken approximately 400 mm in from the proximal end of the crush site of each nerve. Semithin sections were transferred to glass slides and stained with Toludine blue. Ultrathins were transferred to formvar coated 50 mesh copper grids and post-stained for 5 mins with lead citrate. Grids were imaged at 120 kV in a FEI Tecnai 12 TEM using a Gatan OneView camera.
Myelinated axons were classified as follows: Normal -Uniform compact myelin sheath with continuous axoplasm; Abnormal -Axons surrounded by myelin abnormalities including tomaculae, myelin out-foldings and recurrent myelin loops (Goebbels et al., 2012); Degenerated -Absent or vacuous axoplasm surrounded by myelin debris. The number of each axon category per field of view (1471 mm 2 ) was counted by an observer blind to the treatment group.

Western blot
Tissues and cells were homogenized in RIPA buffer (Millipore, Cat# 20-188) containing protease inhibitor cocktail (Sigma, P8340) and phosphatase inhibitor cocktail (Gendepot, Cat# P3200). Cultured cells were washed with warmed HBSS and collected in protein lysis buffer by scraping; frozen tissues were disrupted either in a Minilys bead homogenizer (Precellys, Bertin, France) or glass grinder. Homogenized samples were then sonicated (3 3 10 s, 25% amplitude) on ice and then spun at high speed (10,000 g) for 10 min at 4 C after 40 min incubation on ice and pellet discarded. An equal volume of 5X SDS sample buffer was added to the sample lysates which were boiled at 9 C heat block for 5 min. Protein content was determined by colorimetric assay (Lowry, BioRad). Equal amounts of protein (25-40 mg) and protein size markers were separated by SDS-polyacrylamide gel electrophoresis (5% stacking gel, 10% resolving gel) followed by transfer to a PVDF membrane. Membranes were blocked in a 5% skimmed milk solution containing Tris buffered saline and 0.1% tween-20 (TBS-T) at room temperature for 1h and subsequently incubated with goat anti-mouse Pan-RAE1 antibody (1:500; R&D systems, cat no. AF1136. RRID:AB_2238016) overnight at 4 C in blocking solution. Blots were washed with TBS-T (3 3 10 min) and then incubated with anti-goat HRP-conjugated secondary antibody (1:10,000; Santa Cruz, cat no. sc-2020. RRID:AB_631728) for 1 h at room temperature. After washing with TBS-T blots were developed by application of western ECL substrate (BioRad, cat no. 1705061) according to the manufacturer's instructions and images of sequential exposure times were acquired digitally (ChemiDoc, BioRad). Blots were then stripped with stripping buffer at 50 C for 30 min followed by TBS-T washing and subsequently incubated with mouse anti-beta-actin (1:10,000; Sigma, cat no. A5441. RRID:AB_476744) and rabbit anti-N-cadherin (1:10,000; Millipore, cat no. 04-1126. RRID:AB_1977064) in blocking solution. Goat anti-mouse polyclonal (1:10.000; Komabiotech, cat no. K-0211589. RRID:AB_2636911) or goat anti-rabbit (1:10,000; Santa Cruz, cat no. sc-2004. RRID:AB_631746) HRP-conjugated secondary antibodies were applied, respectively, and then exposed after ECL treatment. Embryonic mouse head tissue was used as positive control for RAE1.
For RAE1 expression in tightly ligated sciatic nerve 7 days after injury, mice were perfused with PBS and ipsilateral sciatic nerve was cut into two 5 mm sections: one flanking the crush site and one proximal to the spinal nerves. Tissues were snap frozen in liquid nitrogen and stored at À80 C for later analysis.
Enzyme-linked immunosorbant assay (ELISA) Granzyme B content of sciatic nerves was determined by ELISA according to the manufacturer's instructions (DuoSet, R&D Systems, cat no. DY1865). Briefly, 96 well plates were coated with capture antibody overnight at RT, washed and blocked 1h. Frozen tissues were homogenized on a shaker with 1.4 mm zirconium beads (Precellys, Bertin) in RIPA lysis buffer (Millipore) and lysates were centrifuged 13,000 rpm for 5 min. Supernatants diluted 1:1 with reagent diluent, added in duplicate to coated wells along with a granzyme B standard series and incubated at RT for 2 h. Plates were washed thoroughly before addition of Streptavidin-HRP, followed by substrate buffer. Stop solution was applied and absorbance (450 nm minus 540 nm correction) read on a microplate spectrophotometer (BioTek Instruments, VT, US). Granzyme B levels in samples were analyzed using the four parameter logistic curve fitting algorithm by reference to standard curve values and performed using online software (https://elisaanalysis.com/).

Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from L5 DRG from L5x or sham mice (two mice pooled per sample) and L3-L5 DRG from sciatic crush or sham mice (one mouse per sample). Tissues were disrupted in RTL Plus lysis buffer (QIAGEN) including 1% b-mercaptoethanol with a mini glass mortar and pestle on ice and further homogenized using a Minilys bead homogenizer (Precellys, Bertin, France); DRG cultures were washed once in warm HBSS prior to lysis by pipetting; frozen cell pellets were vortexed in lysis buffer. RNA was purified from lysed samples, including genomic DNA elimination, by on-column extraction (RNeasy Plus, QIAGEN) according to the manufacturer's instructions. RNA was eluted in RNase-free water and analyzed for purity (260/280 nm ratios of approximately 2.0 were maximum intensity projection TIFs for image analysis. Images were processed and analyzed in ImageJ. A 500 mm2 sample area of each DRG section was selected and cropped. Blue channel (NeuN) images were used to guide manual selection of individual neuron outlines, which were saved to the region of interest (ROI) manager. ROI selections were then applied to the red channel (in situ), which was converted to an 8-bit black/white image with threshold set at 1-255. The number of particles per NeuN+ ROI (larger than 1 mm 2 to eliminate background spots) were counted using the Particles Analysis function in ImageJ and exported to a spreadsheet (Microsoft Excel). A total of n = 2138 NeuN+ ROI from ipsilateral DRG and n = 1565 NeuN+ ROI from contralateral DRG were included; n = 3 mice, n = 3 sections per DRG per mouse. Data are presented as a frequency distribution of the number of in situ spots per neuron (NeuN+ ROI), and versus neuronal cell size (mm 2 ).

Statistical Analysis
Comparisons between two groups of data were made with Student's t test (paired or unpaired) or Mann-Whitney test for non-normal distributions (confirmed with Kolmogorov-Smirnov test). Comparisons between three or more groups of data were made with One-way ANOVA. All tests are two-sided unless indicated. Two-way ANOVA tests were used to compare the effects of treatments on molecular, cellular and behavioral assays over time. Data are presented as mean ± standard error of the mean (SEM) unless otherwise stated. ns, not significant. Analyses were performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, https://www.graphpad.com/. No statistical methods were used to predetermine sample sizes, which were based on previous literature and availability of animals. For behavior tests experimenters were blinded either to the treatment or genotype of the mice during both surgery and sensory testing. Treatments were assigned to littermates at random by an independent observer. p < 0.05 was considered significant.