CD4+ T cells drive corneal nerve damage but are dispensable for corneal epitheliopathy development in dry eye disease

Dry eye disease (DED) is a disorder characterized by a dysfunctional tear film in which the corneal epithelium and its abundant nerves are affected by ocular desiccation, inflammation, and the local immune response. Although adaptive immunity and specifically CD4+ T cells play a role in DED pathogenesis, the exact contribution of these cells to corneal epithelial and neural damage remains undetermined. To address this, we explored the progression of a surgical DED model in wild-type (WT) and T cell-deficient mice. We observed that adaptive immune-deficient mice developed all aspects of DED comparably to WT mice except for the absence of functional and morphological corneal nerve changes, nerve damage-associated transcriptomic signature in the trigeminal ganglia, and sustained tear cytokine levels. Adoptive transfer of CD4+ T cells from DED WT mice to T cell-deficient mice reproduced corneal nerve damage but not epitheliopathy. Conversely, T cell-deficient mice reconstituted solely with naive CD4+ T cells developed corneal nerve impairment and epitheliopathy upon DED induction, thus replicating the WT DED phenotype. Collectively, our data show that while corneal neuropathy is driven by CD4+ T cells in DED, corneal epithelial damage develops independently of the adaptive immune response. These findings have implications for T cell-targeting therapies currently in use for DED.


Introduction
The ocular surface is the mucosal microenvironment that encircles the cornea and keeps it moist and protected for proper sight (1,2).Ocular surface homeostasis depends on a delicate balance between all of its components: the cornea, the conjunctiva, the tear film, the eyelids, the lacrimal and meibomian glands, the ocular surface immune system, and ocular surface innervation.
Dry eye disease (DED) is an increasingly prevalent ocular surface disorder characterized by a dysfunctional tear film, visual disturbance, local inflammation and tissue damage, neurosensory abnormalities, and symptoms ranging from ocular discomfort to overt pain (3).The diverse components of the ocular surface are varyingly affected in DED, leading to heterogeneous clinical presentations.While some patients have overt ocular surface epitheliopathy accompanied by discomfort or pain, others present mainly with symptoms derived from neurosensory alterations and little or no ocular epithelial damage (4,5).Within the multifactorial pathophysiology of DED, ocular surface inflammation plays a pivotal role in perpetuating the disease by favoring tissue damage, which in turn elicits more inflammation and creates a vicious cycle (6)(7)(8).However, if differences in the ocular surface inflammatory and immune responses lead to the DED clinical spectrum is unclear.
A large body of evidence indicates that CD4 + T cells become key drivers of DED-associated ocular inflammation by orchestrating the local adaptive immune response (7,8).Clinical studies show that DED patients have higher numbers of conjunctival CD4 + T cells (9) and at least two FDA-approved drugs (cyclosporine and lifitegrast) for topical anti-inflammatory therapy selectively block T cell activation in the ocular surface (7,10).In animal models, the adoptive transfer of CD4 + T cells from DED mice into T cell-deficient recipients reproduces the conjunctival goblet cell loss and lacrimal gland infiltration observed in donor mice (11,12) while depletion of ocular surface antigen-presenting cells prevents local CD4 + T cell activation and goblet cell loss (13).Moreover, DED increases the number of T-helper (Th)1 and Th17 CD4 + T cells in the eye-draining lymph nodes (14,15) of mice, and consistently, both interferon (IFN)-γ and interleukin (IL)-17 are increased in the tears and conjunctiva of DED patients (16)(17)(18)(19).Dysfunctional regulatory CD4 + T cells also contribute to DED pathogenesis by not restraining effector CD4 + T cells (20)(21)(22).
Effector CD4 + T cells in DED are mostly Th1 and Th17 (14,15), which promote type 1 and type 3 immunity, respectively (23), by releasing proinflammatory cytokines and recruiting other immune cells (23,24).IFN-γ, a hallmark type 1 cytokine, exerts a deleterious effect on the ocular surface by promoting both the apoptosis of conjunctival goblet cells and corneal epithelial cells and the squamous metaplasia of corneal epithelial cells (12,(25)(26)(27)(28)(29).IL-17, a hallmark type 3 cytokine, induces corneal epithelial cells to secrete matrix metalloproteinases, which degrade intercellular junctions and lead to corneal barrier disruption (19,30).IL-17 also favors corneal lymphangiogenesis, which facilitates the migration of corneal antigen-presenting cells and, thus, pathogenic immune responses (31).IFN-γ and IL-17 have additional effects on corneal and conjunctival epithelial, stromal, and immune cells that amplify ocular surface inflammation and tissue damage (29).However, it should be noted that CD4 + T cells are not the only source of the pathogenic IFN-γ and IL-17 in the ocular surface because CD8 + T cells, γδ T cells, NK cells, and other innate lymphoid cells also secrete these cytokines (32,33).In the setting of DED, NK cells contribute early to IFN-γ levels while γδ T cells also produce IL-17 (34)(35)(36)(37).Thus, while the pathogenicity of type 1 and 3 immunity in DED-associated corneal epitheliopathy is firmly established, it may be mediated by CD4 + T cells and innate immune cells (35).
Contrasting the ample evidence on the pathogenic mechanisms that lead to ocular surface epitheliopathy, the pathophysiology of neurosensory abnormalities in DED has not been explored in such detail (38,39).The intraepithelial endings of the corneal nerves are almost in direct contact with the tear film and are the most impacted corneal nerve segments in DED.Since these nerves rely entirely on corneal epithelial cells for support (40), become affected early in the course of the disease (41), and their impairment coincides with corneal epitheliopathy development, it is assumed that corneal nerve damage in DED is secondary to corneal epitheliopathy and caused by the same mechanisms.However, the heterogeneous presentations of DED include a neuropathic form with minimal corneal epitheliopathy and ocular surface inflammation but overt symptoms and detectable corneal nerve alterations (5).In line with this, we observed in a murine tear hyperosmolarity model that corneal nerve changes can ensue in the absence of corneal barrier disruption (42), and more recently, that corneal neuropathy may develop independently of corneal epitheliopathy if a type 1 immune response is present in the ocular surface (43).However, since both models lack ocular desiccation, the defining disease feature that initiates corneal pathology (3,4), their conclusions may not apply to DED because additional mechanisms could be at play.The disease setting determines the pathogenic impact (or lack thereof) of the local immune response on the corneal nerves: herpetic keratitis and ocular graft-versus-host-disease but not ocular allergy exhibit corneal neuropathy despite all three being immune-driven ocular surface disorders (44).In the case of DED, whether local CD4 + T cell-coordinated adaptive immunity in the context of ocular desiccation contributes to corneal neuropathy has not been fully explored.
We hypothesized that the CD4 + T cell-coordinated adaptive immune response in DED may induce corneal neuropathy independently from corneal epitheliopathy.Therefore, we set out to explore the relative contribution of the adaptive immune response, and more specifically of CD4 + T cells, to corneal epithelial and nerve damage in a murine model of DED.

2.
Materials and methods and consistently, female mice develop worse DED than their male counterparts (46), we performed all experiments using equal numbers of female and male animals to consider this difference and we included sex as a variable in the analysis.

Reagents and antibodies
Unless otherwise specified, all chemical and biological reagents were from Sigma-Aldrich (Buenos Aires, Argentina).Table 1 lists all antibodies and the most significant reagents.

Lacrimal gland excision surgery
Mice were anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed on a heated pad.Excision surgery comprised four steps: 1) a 3-mm-long incision was made along the middle third of the line joining the lateral canthus of the ear and the pinna; 2) the superior pole of the extraorbital lacrimal gland was exposed by incising the ensheathing fibrous capsule; 3) the lacrimal gland was pulled out gently and excised taking special care not to damage the blood vessels next to its inferior pole; 4) the skin was closed using 6-0 nylon thread.The glands from both sides were excised sequentially.Sham surgery consisted only of steps 1 and 4. In all cases, a single dose of 10 mg/kg diclofenac sodium was injected s.c. in the scruff for postoperative analgesia, and ciprofloxacin ointment was applied over the wound once the surgery was completed.The eyes were protected from desiccation with sodium hyaluronate 0.4% (Dropstar LC, Laboratorio Poen, Argentina) until the mice recovered from anesthesia.

Tear production measurements
Tear production was measured by inserting a 1 mm-wide phenol-red impregnated filter paper strip in the inferior conjunctival fornix adjacent to the lateral canthus, where it was held in place for 60 seconds while restraining the mouse gently and allowing for normal blinking.The wetted length of the right eye of each mouse was measured (in mm) and used as one data point.

Assessment of corneal epithelial barrier function
Corneal fluorescein uptake was measured as previously described (43,47).In brief, 0.5 μl of dextran-fluorescein isothiocyanate (average molecular weight 3000-5000, 10 mg/ml in PBS) was applied to each eye and then the mouse was returned to its cage.After 3 min, a 10-second-long video of each eye under blue light was captured with the aid of a fluorescence-adapted dissection microscope (NightSea SFA-RB, Electron Microscopy Sciences, Hatfield, PA, USA).For analysis, a masked observer (AV) exported a representative video frame as an image and selected the exposed corneal area suitable for analysis as the region of interest by excluding reflections and other artifacts using ImageJ software (version 2.15.0, https://imagej.net/software/fiji/).Then, the green channel was extracted and the mean fluorescence intensity within the region of interest was calculated after background subtraction (50-pixel rolling ball radius), and the average of both eyes was used for analysis.

Assessment of corneal mechanical sensitivity
Mechanical thresholds were determined using a mouse-adapted version of Cochet-Bonnet esthesiometry (41,48).Nylon 6-0 monofilament was cut into segments of varying lengths (1.0 to 5.5 cm in 0.5 cm steps).With the mouse held firmly in one hand, the cornea was touched six times with each filament, starting with the longest segment.A positive response was defined as blinking and retracting the eye in reaction to at least three of the six tries.The longest segment yielding a positive response was used as the sensitivity threshold, and the average of both eyes was used for analysis.Corneal sensitivity was measured in the morning (8-11 AM) before any other experimental manipulation.

Spleen cell collection and CD4 + T cell purification
Spleens were collected in 5 ml of serum-free RPMI 1640 medium and rendered into cell suspensions by gently pressing through nylon mesh using a syringe plunger; then, red blood cells were lysed with ammonium chloride-potassium buffer, and finally, CD4 + T cells were isolated by negative selection with the aid of magnetic beads (MojoSort™ Mouse CD4+ T Cell Isolation Kit, BioLegend #480033) following the manufacturer's instructions.Cell purity was confirmed through flow cytometry CD4 + immunostaining (>98% CD4 + cells).Each Rag1KO mouse received 1x10 6 cells in 0.5 ml PBS through intraperitoneal injection for adoptive transfer.Image acquisition was performed with an Olympus IX83 inverted motorized microscope (Olympus, Tokyo, Japan) equipped with a UPlanSapo 10x/0.4objective and a Disk Scanning Unit.Due to the irregularity in the distribution of conjunctival goblet cells within the conjunctival surface, composite images spanning the entire conjunctival strips were obtained using the multiple image alignment module of the cellSens Dimensions software (Olympus).

Lymph node cells and flow cytometry analysis
Images were acquired as Z-stacks with a 3-μm step size to ensure that all goblet cells within the conjunctival epithelium were included.To assess the area occupied by goblet cells, maximum intensity projections including all epithelial slices within the stacks were created using ImageJ.
Then, the entire conjunctival surface was demarcated as the area of interest using the polygon selection tool, a background correction (20-pixel rolling ball radius) was applied, the image was thresholded, and finally, the percentage of the selected area occupied by the wheat germ agglutinin-derived signal was measured by the software.

Corneal immunostaining and confocal laser scanning microscopy acquisition
Eyes were processed as described by Yun et al (50).In brief, eyes were fixed in a pre-chilled formaldehyde-containing buffer for 75 min, washed, and stored in methanol at -20 °C until processed for staining.Then, the fixed corneas were cut from the back of the eye under a dissection microscope, washed 3 times for 10 min and permeabilized with 1% Triton X-100 in PBS for 1 h, blocked overnight with 1% BSA, 0.3% Triton X-100 and Tween 0.01% in PBS, and stained overnight with Alexa 488-conjugated Alexa Fluor® 488 anti-tubulin β3, Alexa Fluor® 594 anti-mouse/human Ki-67 and Alexa Fluor® 647 anti-mouse/human CD324 (E-cadherin) antibodies (#801203, # 151213 and #147308, BioLegend).Each batch of anti-tubulin β3 antibody was titrated before use to minimize background staining, usually resulting in 0.5 μl antibody/100 μl buffer/cornea (2.5 μg/ml) as optimal.The stained corneas were washed three times for 60 min in PBS-Tween 0.1%, counterstained with 1 μg/ml DAPI, mounted flat with the aid of relaxing cuts in Aqua-Poly/Mount (PolySciences), and stored at 4 °C until imaged.
Image acquisition was performed with a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan) equipped with Plapon 60X/1.42 and UPlanSapo 20X/0.75objectives.Z stacks (0.5-μm step size) spanning the entire corneal epithelium (approximately 30 μm) were obtained at two opposite located at 600 μm from the center (defined as the center of the nerve whorl or as the center of the disorganized area in those samples with highly disrupted nerve whorls).Corneal nerve analysis was performed at three different levels within the corneal epithelium.For subapical nerve terminals, the first section located entirely beneath the apical epithelial squamous cells (1-1.5 μm deep, usually the third or fourth slice in the stack) was selected.Then, the image was thresholded after background subtraction (10-pixel rolling ball radius), and the percentage area occupied by nerve endings was determined by the corresponding ImageJ function.For mid-epithelial nerve terminals, the middle section between the apical-and basal-most epithelial sections from each stack was chosen.Then, the number of nerve endings was assessed after background subtraction (10-pixel rolling ball radius) by a masked observer (AV) using the Cell Counter ImageJ function.Data are shown as the number of terminals / 60X field (423.94µm2 area).To analyze the complexity of the subbasal epithelial nerves, the Sholl plugin in ImageJ software was used.In brief, a maximum intensity projection of the 10 sections encompassing the corneal subbasal nerve mat was created, then the background was subtracted (50-pixel rolling ball radius), and the image was thresholded.
Finally, 10 concentric circles with a 10-µm radius step size were traced at the center of the final image, and the resulting sum of intersections of tubulin β3 nerves for each concentric circle was calculated using the software and used for analysis (51).
For epithelial cell turnover analysis, a blind observer (AV) selected a single section from the Z-stack encompassing the basal epithelial cells and manually counted the number of Ki-67 + cells.
Side views where obtained using the Orthogonal Views function of ImageJ.

RNA isolation from trigeminal ganglia and RNA-Seq analysis
The trigeminal ganglia were dissected after euthanasia and cardiac perfusion with PBS to remove contaminating blood cells (52), collected in ice-cold TRI Reagent, and stored at -80°C until processing.For RNA isolation, both trigeminal ganglia from one mouse were homogenized in 1 ml of TRI Reagent, and then 0.2 ml of isopropanol was added.After centrifugation, RNA was purified from the aqueous phase using the Direct-zol RNA MiniPrep kit (Cat #R2052, Zymo Research, Irvine, CA, USA) following the manufacturer's instructions.The concentration and purity of RNA were assessed with a NanoDrop 1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).RNA-Seq was performed by NovoGene (Sacramento, CA, USA) using the Illumina NovaSeq platform to generate 150 bp paired-end reads.The sequenced reads were mapped to the mouse reference genome (assembly GRCm38/mm10) using STAR v2.7.11a, and the quantification of reads per gene was estimated by RSEM v1.

Tear cytokine levels
To collect a tear washing sample, each mouse was manually restrained, then 5 µl of PBS+BSA 0.1% was applied on one eye using a 10-µl pipette tip, and finally the same volume was gently collected 20 seconds later from the ocular surface.The right and left eye washings from one mouse were pooled as one sample and stored at -80°C until processing.Cytokine levels were determined using a bead-based multiplex assay (LEGENDplex, #741048, BioLegend) following the manufacturer's instructions and are expressed as mean±SEM (pg/ml).

Statistical analysis
Student's t-test and one-or two-way analysis of variance (ANOVA) with Sidak's post hoc tests were used to compare the means of two or more samples, respectively.Significance was set at p<0.05 and two-tailed tests were used in all experiments.All data are shown as mean±SEM.

Impact of adaptive immunity deficiency on DED phenotype development
CD4 + T cells are pathogenic in DED because they enhance desiccation-induced ocular surface inflammation by coordinating the local adaptive immune response (7,(9)(10)(11)(12)14,15).However, whether a CD4 + T cell-coordinated adaptive immune response is required for either corneal epitheliopathy or neuropathy to develop in this disorder remains undetermined.To address this question, we compared the progression of the disease phenotype in immunocompetent wild-type (WT) and adaptive immunity-deficient Rag1KO (lacking CD4 + and CD8 + T, NKT, and B cells) mice using a surgical DED model (Figure 1A).This model involves the bilateral excision of the extraorbital lacrimal gland, the largest of the three glands that contribute to tear secretion in rodents.The resulting desiccation of the ocular surface then activates a pathogenic immune response, thus mimicking aqueous-deficient DED (53,54).All in vivo measurements were performed at baseline and on days 5 and 10 to assess progression, after which mice were euthanized because ocular signs of DED and the accompanying CD4 + T cell response are fully developed at this time point (55,56).
First, we verified that the tear deficiency 5 days after surgery was comparable in both strains (Figure 1B) to ensure the validity of the Rag1KO DED model.Baseline tear production did not differ between strains nor between female and male mice that underwent either sham-or DED-inducing surgery (sex factor, <2% total variation, p=0.50).Also, the external eye phenotype in WT and Rag1KO did not appreciably change after 10 days of DED induction (Figure 1C).Next, we quantified conjunctival goblet cells because their loss is a clinically validated finding in DED that correlates with disease severity (57,58).Since the distribution of these cells within the conjunctiva is not uniform(59), we measured the total area occupied by goblet cells in low-magnification images obtained from conjunctival whole mounts (Figure 1D and top panels of E).As shown in Figure 1D, the total conjunctival goblet cell area did not differ between control mice (while DED induction led to a comparable loss of goblet cells in both strains.The DED-induced loss was more readily evident but comparable in magnitude when restricting the analysis to the goblet cell-rich area of the conjunctiva from both strains (Figure 1E, bottom panels).Altogether this data shows that both mouse strains develop comparable DED in terms of tear production and conjunctival goblet cell loss, and therefore, that the Rag1KO mouse is a valid model for assessing how the lack of adaptive immunity impacts the corneal aspects of the disease.

Adaptive immune deficiency does not impede DED-associated corneal epitheliopathy development
Corneal epitheliopathy is a hallmark clinical finding in DED, characterized by epithelial barrier dysfunction and accelerated cell turnover(4).Although it is accepted that type 1 and type 3 immunity contribute to corneal epithelial damage (12,19,25,28,30), whether the adaptive (T and B-cell dependent) component of the immune response is required for corneal epitheliopathy to develop in DED is unclear.To answer this question, we first measured corneal dye uptake as a means to probe corneal barrier function (4,60).In DED, increased epithelial cell shedding and matrix metalloproteinase activity disrupt tight junctions, thus allowing the intercellular diffusion of fluorescein-tagged dextran (60).As shown in Figures 2A and 2B, DED WT mice developed a time-dependent increase in corneal dye uptake with the typical punctate staining pattern observed in patients (4,60).Remarkably, DED Rag1KO mice showed corneal dye uptake that was comparable to that of DED WT mice on days 5 and 10 of disease induction (Figure 2A) and with no appreciable differences in the staining pattern (Figure 2B).Sub-analysis by sex of the DED groups (Supplementary Figure 1A, 3-way ANOVA with factors strain, sex, and time) revealed that while time had the largest effect on corneal epitheliopathy development (60.7% of total variation, p<0.0001), female mice had worse epithelial disease than male mice of the same strain (sex factor accounted for 9.1% of total variation, p<0.0001) and strain had no significant overall effect (1.4% of total variation, p=0.08).Female WT mice with DED had more corneal dye uptake on day 5 (p=0.01)than female Rag1KO mice with DED but this difference disappeared by day 10 (p=0.59),suggesting a faster tempo (Supplementary Figure 1A).
Next, we quantified the number of proliferating corneal epithelial cells as an indicator of cell turnover rate, which increases in DED (61).As shown in Figures 2C and Supplementary Figure 1B, both WT and Rag1KO mice with DED had more proliferating (Ki67 + ) corneal epithelial cells with no statistically significant difference between strains.Of note, proliferating cells were restricted to the basal epithelial layer in control mice while they also extended to the suprabasal layer in DED mice of both strains (Supplementary Figure 1C), a pattern consistent with DED-induced corneal epitheliopathy (61).These results suggest that the absence of an adaptive immune response does not condition the development of corneal epitheliopathy in this DED model.

Adaptive immune deficiency prevents DED-associated corneal neuropathy development
We have previously shown that antigen-driven CD4 + T cell activation in an ocular surface that is not exposed to desiccation is sufficient to cause corneal nerve damage (43).However, other factors may also determine whether corneal nerve damage occurs in the context of an ocular surface immune response since CD4 + T cell activation drives corneal nerve damage in herpetic keratitis and ocular graft-versus-host disease but not in allergic conjunctivitis mouse models (44).
DED involves ocular desiccation and innate immune activation followed by an adaptive immune response.Thus, if local adaptive immunity is required for corneal nerve dysfunction to develop in DED is unknown.To address this question, we first measured corneal sensitivity to mechanical and capsaicin stimulation to probe two different types of nerve fibers in WT and Rag1KO with DED.
At baseline, Rag1KO mice showed slightly reduced corneal mechanosensitivity (-4.4±0.9%,Supplementary Figure 2A) and increased sensitivity to capsaicin (Supplementary Figure 2B) compared to WT mice.Sex had no significant effect (p=0.58, Supplementary Figure 2C) on baseline mechanosensitivity thresholds in both strains.Therefore, when evaluating the effects of DED in each strain, the results were normalized to each strain-specific baseline (Figures 2D and   2E).DED WT mice developed a progressive reduction in corneal sensitivity and increased capsaicin sensitivity, as previously described (41,47), but this was not observed in DED Rag1KO mice.In contrast to corneal epitheliopathy where female sex was the strongest determinant of differences, sub-analysis by sex of the DED groups (Supplementary Figure 2D, 3-way ANOVA with factors strain, sex, and time) revealed that strain had the largest effect (22.3% of total variation, p<0.0001) on mechanosensitivity followed by time (14.3% of total variation, p<0.0001) while sex had a small significant effect (2.9% of total variation, p=0.03).Although the drop in corneal mechanosensitivity was greater in the females, WT mice of both sexes displayed reduced sensitivity on days 5 and 10 of DED induction (Supplementary Figure 2D).
Since corneal mechanosensitivity reduction in mouse DED models correlates with structural changes (38,39), we evaluated corneal nerve morphology on day 10 (Figures 2F-I and Supplementary Figure 2E) at three different levels within the intraepithelial innervation (43,47).DED WT mice had reduced corneal nerve density at the subapical, mid-epithelial, and subbasal levels.
By contrast, nerve density in Rag1KO mice with DED did not significantly change at any of the three corresponding levels of intraepithelial corneal innervation (Figure 2F).The magnitude of corneal nerve changes in DED WT mice decreased from the superficial subapical level to the deeper subbasal level, and DED Rag1KO mice showed a similar but not significant trend (Supplementary Figure 2F).Altogether, the data indicates that corneal nerve damage requires an adaptive immune response to develop in the context of DED.
3.4 DED induces a trigeminal transcriptomic signature related to corneal nerve damage only in adaptive immune-sufficient mice Corneal nerve fibers are the terminal projections of the somatosensory neuronal bodies located in the trigeminal ganglion, and many studies have shown that injury to the peripheral nerve endings of extraocular tissue-innervating trigeminal neurons leads to reactive gene expression changes (62)(63)(64).To further study the impact of adaptive immune deficiency on corneal neuropathy development, we analyzed the transcriptional profiles of the trigeminal ganglia of the same mouse groups by bulk RNA-sequencing.We determined the effect of DED on trigeminal gene expression separately in the two strains using the same-strain sham-treated group as a reference, and then we compared samples from WT DED and Rag1KO DED mice.A complete list of differentially expressed genes can be found in Supplementary File 1.
After 10 days of DED induction, there were 172 differentially expressed genes (DEGs) in WT mice, of which 77 were up-regulated and 95 were down-regulated, whereas there were only 7 DEGs (5 up-and 2 down-regulated) in Rag1KO mice (Figures 3A and 3B).Up-regulated genes in WT DED mice included: Atf3, Sprr1a, and Fos, three canonical regeneration-associated genes induced by axonal injury in peripheral nervous system neurons(65); Mbp, which encodes myelin basic protein and is induced during the Schwann cell response to nerve injury (66); and Sparc, a matricellular protein secreted by glial cells that modulates axonal regeneration (67).
Down-regulated genes in WT DED mice included: Trpm8, Trpa1, and Piezo2, three cation channels involved in corneal thermo-and polymodal nociception and mechanosensation (38); Asic1 and Asic3, acid-sensing cation channels expressed in trigeminal neurons that participate in nociception (68); Pirt, a positive regulator of transient receptor potential vanilloid-1 channel activity in nociceptive neurons(69); Kcnk18, Scn10a, Scn4b, and several other voltage-gated potassium and sodium channels that determine sensory neuron excitability and are involved pain syndromes (70); and L1cam and Sema4f, which guide axonal growth (41).Cd74 was the only DEG that was up-regulated in both WT and Rag1KO samples: it encodes the macrophage-inhibitory-factor receptor and its expression in the trigeminal ganglion has been mapped to macrophages and neutrophils (71).The remaining 4 up-regulated DEGs in Rag1KO mice were: Smoc2 (a matricellular protein related to Sparc) and S100a4 (a cytoskeleton-interacting protein), both of which are induced by nerve injury, and Pmp2, which encodes peripheral myelin protein 2 and its regulation is linked to S100a4 (72).
Analysis of DEGs between trigeminal ganglia from WT and Rag1KO mice with DED revealed 34 up-regulated and 39 down-regulated genes in WT mice (Supplementary Figure 3).were also down-regulated relative to sham WT mice, and the axon-guiding semaphorin Sema3f.
Gene set enrichment analysis found that DED affected 189 pathways in WT mice and 24 pathways in Rag1KO mice (Supplementary Figure 4).In WT mice, the 10 most significantly activated pathways pertained to pre-and postsynaptic translation, the humoral immune response, and antigen processing and presentation while the 10 most significantly suppressed pathways related to microtubule-based transport, exocytosis, detection of abiotic stimuli, and those involved in sensory perception of pain.In Rag1KO mice, the 10 most significantly pathways activated by DED related to presynaptic translation and regulation of antigen processing and presentation whereas the 10 most suppressed ones were dendrite development, regulation of transporter activity, synapse assembly, and potassium ion transport, albeit the statistical significance was 10-fold lower than in WT mice.Altogether, these findings indicate that DED induces trigeminal gene expression changes that are more profound and more related to corneal nerve damage in WT than in Rag1KO mice, which is consistent with the lack of corneal neuropathy development in the latter group.
3.5 DED leads to sustained proinflammatory cytokine levels in tears only in adaptive immune-sufficient mice The adaptive immune response, and specifically its CD4 + T cells, sustain ocular surface inflammation in DED by releasing Th1 and Th17-associated pathogenic cytokines (13,15,19,31,56,(74)(75)(76).Having observed that the corneal nerves were spared by DED in adaptive immune-deficient mice, we hypothesized that the specific lack of CD4 + T cells and their associated cytokines coordinating the local immune response in this strain could explain the absence of DED-associated corneal neuropathy.Therefore, we analyzed cytokine levels in tear washings from WT and Rag1KO mice on day 10 of DED induction, a time at which the CD4 + T cell response has fully ensued(11,56)(Figure 4).In WT mice, DED increased the levels of 6 of the 7 tested cytokines (IFN-γ, TNF, IL-6, IL-10, IL-17A, and IL-22).By contrast, Rag1KO mice with DED did not have increased tear levels of any of the 7 cytokines on day 10 when compared to control mice of the same strain.Subanalysis by sex of the WT strain data revealed that female DED mice had higher tear levels of IL-17A (p=0.03) and a similar trend for IFN-γ (p=0.07) while the other tested cytokines did not differ between sexes.Our data highlights the pivotal role that the adaptive immune response plays in amplifying and sustaining ocular surface inflammation in DED through cytokine production.

Adoptive transfer of CD4 + T cells from DED mice recapitulates ocular phenotype and corneal nerve damage but not epitheliopathy in the recipients
Our results so far supported our hypothesis that the adaptive immune response drives corneal nerve damage in DED.Based on our previous observation that isolated ocular CD4 + T cell activation is capable of inducing corneal neuropathy (43), we hypothesized that the lack of CD4 + T cells in Rag1KO mice was responsible for their resistance to develop corneal nerve damage in the context of DED.To test this, we isolated splenic CD4 + T cells from control or DED WT mice 10 days after surgery and transferred them into sex-matched naïve Rag1KO mice, which were then assessed weekly for DED pathology (Figure 5A).First, we validated the model: CD4 + T cells from control or DED WT did not significantly differ in the proportion of Th1 or Th17 cells, although there was a trend towards more IFN and IL-17A production in the latter group (Supplementary Figure 5A).Also, to rule out a potentially confounding effect of contaminating CD8 + T or B cells that may proliferate rapidly in the lymphopenic host, we used flow cytometry in lymph nodes of the recipient mice 4 weeks after transfer to quantify these cells.We confirmed that the recipient Rag1KO mice were effectively reconstituted only with CD4 + T cells (Figure 5B).Then, we verified the functionality of the transferred CD4 + T cells by assessing their migration to the ocular surface.As previously reported (11), DED-induced CD4 + T cells were more efficient than control CD4 + T cells at homing to the conjunctiva in the recipient mice (Figure 5C and Supplementary Figure 5B).Despite the conjunctival infiltration, no appreciable changes in external eye phenotype (Figure 5D) were observed, as is the case for the surgically induced DED model (Figure 1B).As a final validation step, we evaluated the pathogenicity of transferred CD4 + T cells by assessing their effect on tear production and conjunctival goblet cells.Rag1KO mice that received CD4 + T cells from DED WT donors had decreased tear production compared to Rag1KO mice that received control CD4 + T cells (Figure 5E), in line with the increased lacrimal gland infiltration described in a similar model (11).In addition, DED CD4 + T cell-recipient mice exhibit decreased conjunctival goblet cell density compared to control recipient mice (Figures 5F and 5G), thus reproducing all the non-corneal findings observed in WT and Rag1KO with surgically-induced DED (Figure 1).
Once we established the validity of this adoptive transfer model, we assessed the capacity of the pathogenic CD4 + T cells to cause corneal epithelial damage in the recipient mice in the absence of desiccating stress.As shown in Figures 6A-B, neither group of recipient mice (control and DED-induced CD4 + T cells) showed a change from baseline levels of corneal dextran-FITC uptake.Sub-analysis by sex (Supplementary Figure 6A, 3-way ANOVA with factors transferred cells, sex, and time) confirmed that no factor had a significant effect on corneal epitheliopathy development (<5% of total variation, p>0.05).Moreover, both groups of recipient mice had comparable numbers of proliferating corneal epithelial cells (Figures 6C and Supplementary Figure 6B) that were restricted to the basal layer (Supplementary Figure 6C), as observed in homeostatic conditions.Thus, these findings indicate that in the absence of desiccating stress, CD4 + T cells from DED mice are not sufficient to induce corneal epitheliopathy in Rag1KO recipient mice.
In parallel to corneal epitheliopathy assessments, we evaluated corneal sensitivity as an indicator of corneal nerve function.Contrasting with corneal epithelial barrier function results, corneal mechanosensitivity significantly decreased in the DED-induced CD4 + T cell-recipient mice as early as 7 days after transfer (Figure 6D).Of note, in this group the sensitivity thresholds did not worsen significantly from day 7 until the end of the experiment (day 28).Moreover, sub-analysis by sex (Supplementary Figure 6D, 3-way ANOVA with factors group, sex, and time) revealed that sex had no effect (p=0.88) on mechanosensitivity, in contrast with the findings from the surgical DED model (Supplementary Figure 2A).Finally, we analyzed corneal nerve morphology on day 28 post transfer and found reduced intraepithelial nerve density in the DED-induced CD4 + T cell-recipient mice at the three levels analyzed: subapical nerve endings, mid-epithelial vertical fibers, and subbasal nerves (Figures 6E-H).The pattern of change in DED-induced CD4 + T cell-recipient mice was similar to that observed in WT mice with DED: a larger relative reduction in the subapical nerve endings than in the subbasal corneal nerves (Supplementary Figure 6E).Altogether these results show that adoptive transfer of pathogenic DED-induced CD4 + T cells reproduces corneal neuropathy but not epitheliopathy in recipient mice.

Adaptive immune-deficient mice reconstituted with naive CD4 + T cells gain the ability to develop corneal nerve damage upon DED induction
To confirm that CD4 + T cells drive corneal nerve damage in DED, we tested if reconstitution of Rag1KO mice with naive CD4 + T cells was sufficient to enable corneal neuropathy development upon surgical DED induction.To this aim, we isolated CD4 + T cells from naive WT mice and transferred them by i.p. injection into Rag1KO mice that, one week later, underwent either bilateral extraorbital lacrimal gland excision or sham surgery as control (Figure 7A).First, we confirmed that T cell reconstitution in the cervical lymph nodes was effective and limited to CD4 + T cells (Figure 7B), and then, we tested for the development of corneal epitheliopathy and neuropathy after surgical DED induction.We found that corneal barrier disruption progressively increased on days 5 and 10 of DED induction (Figure 7C and 7D) and to an extent comparable to WT and Rag1KO mice with DED (Figure 2A).However, and contrasting with Rag1KO mice lacking T and B cells, CD4 + -reconstituted DED Rag1KO mice developed progressive loss of corneal mechanical sensitivity (Figure 7E).Concordantly, we found reduced corneal nerve density at the subapical, mid-epithelial, and subbasal levels in CD4 + -reconstituted mice with DED compared to CD4 + -reconstituted mice that underwent sham surgery (Figures 7F-I).The pattern of change in CD4 + -reconstituted mice with DED showed a larger relative reduction in the subapical nerve endings than in the subbasal corneal nerves (Figure 7J).These results altogether show that reconstitution of Rag1KO mice with naïve CD4 + T cells from WT mice followed by surgical DED induction enables corneal neuropathy onset and progression, thus confirming that CD4 + T cells are required for corneal nerve damage to develop in the setting of DED disease.

Discussion
Our current understanding of DED involves dysfunctional tear film-instigated corneal epitheliopathy and neurosensory abnormalities as defining clinical features and ocular surface inflammation as a key driver of the disease process (3,4,6).Contrasting with the numerous studies into the pathogenesis of corneal epitheliopathy in DED, the mechanisms underlying corneal nerve damage are poorly understood (38).A CD4 + T cell-driven adaptive immune response perpetuates ocular surface inflammation(8) and thus interfering with CD4 + T activation is the target of several approved therapies for DED in patients.However, whether CD4 + T cells are required for either DED-associated corneal epitheliopathy or neuropathy to occur is undetermined.Herein we show that while DED-associated corneal epitheliopathy develops unimpeded in the absence of CD4 + T cells, corneal nerve damage does not.Thus, our findings indicate that the mechanisms behind these two defining aspects of the disease are different and that therapies that target one may not necessarily tackle the other in the multifactorial pathogenesis of the DED.
Our finding of corneal epitheliopathy developing independently of CD4 + T cell activation might seem to contradict the literature on DED pathogenesis (7,8), but a meticulous analysis of the published reports reveals otherwise.The contribution of a CD4 + T cell-driven adaptive immune response to DED progression was established almost two decades ago by showing that the adoptive transfer of CD4 + T cells from DED mice reproduces lacrimal gland-and conjunctival infiltration in T cell-deficient recipients (11).This study, however, did not report corneal barrier disruption in the recipient mice, a hallmark of corneal epitheliopathy.Subsequent investigations demonstrated the pathogenic relevance of type 1 (Th1) and type 3 (Th17) immunity in many aspects of DED including corneal epitheliopathy (13,15,19,30,31,34,56,(74)(75)(76)(77), but none has indicated that corneal epitheliopathy does not occur in the absence of CD4 + T cell activation.One study indicated that local depletion of dendritic cells and macrophages prevents conjunctival CD4 + T cell infiltration and goblet cell loss but did not describe any effect on corneal epitheliopathy-related signs (13).Our findings are in complete agreement with these reports: adoptive transfer of CD4 + T cells from WT mice with DED reproduces lacrimal gland dysfunction, conjunctival infiltration and conjunctival goblet cell loss in T cell-deficient recipient mice (Figures 5   and 6).Another landmark study showed that the adoptive transfer of effector memory CD4 + T cells from female mice with DED into naïve recipients leads to more severe corneal epitheliopathy than when naïve or effector CD4 + T cells from the same donor mice are transferred, but in all cases corneal epithelial changes developed only after desiccating stress exposure (56).Our findings are also in line with this report, as female WT mice (with CD4 + T cells) in our study developed more severe corneal epitheliopathy than female Rag1KO mice (without CD4 + T cells) on day 5 of DED induction, but the differences disappeared by day 10 among strains and sexes (Supplementary Figure 1).Another report observed no corneal barrier disruption in Rag1KO mice subjected to desiccating stress for 5 days, and this resistance to corneal epitheliopathy development was overcome by prior adoptive transfer of Th17 but not Th1 CD4 + T cells from WT mice with DED (78).
However, this study employed milder and shorter ocular desiccation treatment (5 days), potentially selecting for the least pathogenic conditions.At any rate, none of the studies outlined above reported that the adoptive transfer of CD4 + T cells without additional ocular desiccation leads to corneal epitheliopathy development in the recipient mice.Consistently, the present study indicates that CD4 + T cells are dispensable for DED-associated corneal epitheliopathy onset, but these cells may accelerate or worsen this aspect of the disease in females.
Our data showing that CD4 + T cells are not required for corneal epithelial damage to occur in DED does not imply that type 1 and 3 immunity are not involved in this aspect of the disease.
IFN-γ and IL-17 are pathogenic to the corneal epithelium (19,27,29), and conjunctival NK cells and other innate immune cells represent a significant source of these cytokines in the ocular surface (30,(34)(35)(36)(37). Using a non-surgical DED model, Coursey et al. (35) showed that conjunctival IFN-γ and Th1-associated chemokines in Rag1KO mice increase after 5 days of desiccating stress but return to baseline levels by day 10.These authors also observed conjunctival goblet cell loss in Rag1KO mice after 5 days of DED induction, indicating that early IFN-γ production by innate immune cells in the ocular surface is sufficient to cause this typical DED finding (26,35).The present study complements these prior reports by showing that DED-induced conjunctival goblet cell apoptosis, which is induced mainly by IFN-γ (12,26), occurs unimpeded in the absence of CD4 + T cells (Figures 1D and 1E).Of note, we did not detect increased tear levels of IFN-γ and IL-17 in T cell-deficient mice after 10 days of DED induction (as opposed to WT mice, Figure 5), indicating that CD4 + T cells are required for sustained production of these pathogenic cytokines in the DED-affected ocular surface, as previously shown by others (56).Thus, our findings also suggest that the early innate components of type 1 and type 3 responses are competent to prime the proinflammatory ocular surface state and conjunctival goblet cell dysfunction that contribute to corneal epithelial damage.
In contrast to corneal epitheliopathy, we observed that the onset of corneal nerve damage in the setting of DED is entirely dependent on the presence of CD4 + T cells.Basal corneal epithelial cells ensheath the subbasal nerves within infoldings of their cell membrane, thus serving as surrogate Schwann cells to the intraepithelial corneal fibers by providing physical and metabolic support (40).Considering the intimate relationship between corneal epithelial cells and nerve fibers, it is striking that desiccation-induced pathologic changes in the former may occur while the latter remain unaffected in the absence of CD4 + T cells.However, we have found that the converse is also true: corneal nerves may be damaged by CD4 + T cell activation in the ocular surface while the corneal epithelium remains unaffected in the absence of desiccating stress (43).Thus, the present findings extend the notion of an epithelial-neural divide in corneal pathology to DED disease.Since the pathogenic mechanisms that drive the corneal epithelial aspect of the disorder are clearly different from the ones that cause its neural manifestations, this might translate to variations in the immune and non-immune components of DED contributing to the diverse presentations of the disease.In line with this, one study found little to no difference in the extent of corneal epithelial changes (as assessed by in vivo confocal microscopy) between Sjögren's and non-Sjögren's syndrome DED patients (79).By contrast, two studies reported worse changes in corneal nerve morphology (also by in vivo confocal microscopy) in Sjögren's than in non-Sjögren's syndrome DED patients (79,80).The sensitivity of corneal nerves to CD4 + T cell activation could also explain the corneal neuropathy findings that correlate with the severity of the systemic autoimmune disease in rheumatoid arthritis patients (81) or that are observed after recovery in COVID-19 patients (82).
The trigeminal transcriptomic signatures observed after 10 days of DED induction are consistent with the extent of corneal nerve damage in wild-type and Rag1KO mice (Figure 3 and Supplementary Figures 3 and 4).Gene expression changes in DED WT mice but not in Rag1KO mice correspond to the common transcriptomic signatures of nerve injury and regeneration and neuropathic pain: up-regulation of transcription factors Atf3 and Fos and neuropeptide Y, and altered ion channel expression (65,83).Based on the number of DEGs and the fold-change in expression, the magnitude of the DED-induced transcriptomic signature in the trigeminal ganglion is smaller than those reported for other models of trigeminal nerve injury (62)(63)(64)84).However, most models involve whole nerve ligation or injection of inflammatory agents whereas DED targets the peripheral nerve endings of only 2% of trigeminal neurons (38).A combined analysis of bulkand single-cell RNA-seq studies of dorsal root ganglia in neuropathic pain or nerve injury models showed that most gene expression changes take place in nociceptor neurons, and that the reduced magnitude of expression changes in whole ganglion studies probably relates to the diluting effect of other cell types (83).In light of this, the qualitatively comparable trigeminal transcriptomic signature observed after 10 days of DED in WT mice in our study probably indicates a pain maintenance period as that reported after 10 days of partial infraorbital nerve transection (84).Functional impairment of mechanical sensitivity, capsaicin hypersensitivity, spontaneous ocular pain, and morphological alterationsin corneal nerve fibers are well established at this time point in our model (47).These findings are consistent with the gene expression changes involving detection of noxious stimuli, neuronal excitability and signal transduction, nerve repair, and the immune response found in the trigeminal ganglion of DED WT mice, which are associated with a neuropathic pain state in other models (83,84).Supporting this hypothesis, the up-regulation of the macrophage-specific marker Cd74 in both DED WT and Rag1KO mice probably reflects the increase in neuron-associated macrophages in the trigeminal ganglion upon axonal injury that relates to neuropathic pain (71,85).Thus, our data support the notion that adaptive immune response contributes to the development of neuropathic pain in the setting of DED.
Although our study does not delve into the exact process by which CD4 + T cells promote corneal neuropathy, it is tempting to speculate that the elevated tear cytokine levels in WT but not in Rag1KO mice after 10 days of desiccating stress play a role (Figure 5).CD4 + T cells coordinate immune responses in tissues mostly by secreting cytokines that have diverse effects on innate immune cells and non-immune cells (23,24).In DED, conjunctival CD4 + T cells increase in number and constitute a likely source of the Th1 and Th17 cytokines that reach the cornea through the tear film.We observed more severe disruption of the superficial subapical nerve endings than of subbasal nerve fibers in the three models tested, one of which lacked ocular desiccation and relied entirely on CD4 + T cell activation for ocular pathogenicity.This pattern of nerve fiber impairment is compatible with a soluble factor diffusing from the tear film into the corneal epithelium, which could be an action-at-distance effect of conjunctival CD4 + T cells.In line with this hypothesis, we observed increased conjunctival infiltration after transferring DED-induced CD4 + T cells into Rag1KO mice (Figure 6C).Nonetheless, a smaller number of T cells patrol the corneal epithelium under homeostatic conditions in humans and mice (86) and may even establish corneal tissue residency in pathologic settings (87).Therefore, it remains to be established whether CD4 + T cells must enter the cornea to exert their pathogenic action on corneal nerves or if they do so at a distance from the surrounding conjunctiva in DED disease, and the exact role of inflammatory tears in this process.
Finally, female sex is the most influential risk factor for DED in patients (88,89), and our findings also shed light on this association: female mice develop worse DED in terms of corneal epithelial and neural impairment in part due to greater pathogenic activity of CD4 + T cells in this sex (Supplementary Figures 1 and 2).However, the difference in disease severity between sexes cannot be solely attributed to these cells because female mice adoptively transferred with CD4 + T cells from female DED mice do not develop worse corneal neuropathy than their male counterparts (Supplementary Figure 6A), indicating that sex influences other pathogenic factors that are triggered by desiccation in the ocular surface.In line with this, a reduced regulatory role of ocular surface neutrophils in female mice has been linked to an amplified effector CD4 + T cell response in the ocular surface and worse DED phenotype (90).Alternatively, the more severe corneal mechanosensitivity impairment in female mice with DED (Supplementary Figure 2A) may involve greater susceptibility of corneal nerves to immune-mediated damage than in males.Supporting this idea, corneal neural repair mechanisms are conditioned by sex as female mice exhibit faster corneal neuroregeneration than their male counterparts (91).
In summary, the present study contributes to our understanding of the pathophysiology of DED-associated corneal epithelial and nerve damage by showing that desiccation-induced changes in the distal subapical endings of corneal nerves require the activation of CD4 + T cells while those in corneal epithelial cells occur independently of the adaptive immune response.One limitation of our study (and of DED animal models in general) is that the onset of desiccation is abrupt, as opposed to DED patients who typically suffer a progressive impairment of the tear film, which eventually elicits CD4 + T cell-sustained ocular surface inflammation that aggravates the disease (92).Thus, ocular surface CD4 + T cell activity may contribute indirectly to corneal epithelial damage during the initial phases of the disease in patients, as suggested by the faster tempo of corneal epitheliopathy in female WT mice (Supplementary Figure 1A).By contrast, the resistance of corneal nerves to experimental desiccation in the absence of CD4 + T cells indicates that corneal neuropathy in DED is mostly immune-mediated.In line with this, we have previously shown that corneal nerves are particularly sensitive to the activation of Th1 CD4 + T cells in the ocular surface in the absence of desiccating stress (43), and on the other hand, that signaling through transient receptor potential vanilloid-1 channels in the corneal tissue is required to propagate desiccation-initiated corneal nerve damage in DED (47).Therefore, it is likely that in the context of DED, sustained levels of Th1 cytokines from CD4 + T cells prime corneal nerve changes that later propagate proximally through overactivation of transient receptor potential vanilloid-1 channels.
More work is warranted in this direction since the mechanisms that promote corneal nerve alterations vary among ocular surface disorders (44).Nevertheless, our findings have implications for current DED management because they help explain why therapies aimed at reducing or controlling CD4 + T cell recruitment and activation in the ocular surface may be more impactful on certain manifestations of the disease and not on others.At the same time, the present study also highlights the need for further research to elucidate the precise mechanisms underlying corneal epithelial and nerve damage in DED and to identify potential therapeutic targets for these conditions.

Data availability
The data underlying Figs.

Figure 1 -
Figure 1 -Impact of adaptive immune deficiency on dry eye phenotype development.A) Dry eye disease (DED) was surgically induced in wild-type (WT) or recombination-activating gene 1-knockout (Rag1KO) mice of both sexes through bilateral excision of the extraorbital lacrimal gland.Sham-operated animals were included as controls (Ct).B) Tear production on day 5 as measured by phenol red-paper wetting length.C) External eye appearance of Ct and DED mice of both strains 10 days after surgery.D) Total conjunctival area occupied by goblet cells 10 days after DED induction.E) Representative micrographs of wheat germ agglutinin-stained goblet cells in conjunctival whole-mounts used for quantification.Low-magnification micrographs (top) of whole-mounted conjunctival strips used for measurements shown in D, with their corresponding high-magnification insets of the goblet cell-rich area (bottom) provided only as a reference.The actual goblet cell-occupied area is shown in the top right corner of each image.All experiments were performed twice or more with 6 mice/group/experiment.For all experiments, each data point represents one animal and the mean±standard error of measurement is shown.To compare means, two-way ANOVA was used for C and D (strain and treatment) with Sidak's post hoc test.* indicates p<0.05, *** indicates p<0.001, **** indicates p<0.0001, and ns indicates not significant.

Figure 2 -
Figure 2 -Lack of adaptive immunity does not hinder corneal epitheliopathy but prevents corneal neuropathy development in dry eye.Dry eye disease (DED) was surgically induced in wild-type (WT) or recombination-activating gene 1-knockout (Rag1KO) mice of both sexes through bilateral excision of the extraorbital lacrimal gland.Sham-operated animals were included as controls (Ct).A) Cumulative data and B) representative micrographs of corneal dextran-fluorescein uptake in Ct and DED mice from both strains.Data shown as the mean fluorescence intensity (MFI) calculated with ImageJ software (Methods section).C) Number of proliferating (Ki67+) cells within the epithelial basal layer of corneal whole-mounts obtained 10 days after DED induction.D) Corneal mechanosensitivity and E) capsaicin thresholds in Ct and DED mice from both strains on days 0, 5, and 10 of DED induction.F) Representative micrographs and G-H-I) density of intraepithelial corneal innervation analyzed at three different levels by Alexa Fluor 488 anti-tubulin β3 staining: subapical (G, % area occupied by nerve endings) and mid-epithelial (H, count of nerve endings/field) nerve ending density and complexity of subbasal nerves (I, sum of intersections at all Sholl radii).All experiments were performed twice or more with 6 mice/group/experiment.For all experiments, each data point represents one animal and mean±standard error of measurement is shown.To compare means, two-way ANOVA was used for A, D, and E (group and time) and C, G, H, and I (strain and treatment) with Sidak's post hoc test.* indicates p<0.05, *** indicates p<0.001, **** indicates p<0.0001, and ns indicates not significant.

Figure 3 -
Figure 3 -Gene expression changes induced by dry eye disease in the trigeminal ganglion.Dry eye disease (DED) was surgically induced in wild-type (WT) or recombination-activating gene 1-knockout (Rag1KO) mice for 10 days and then the trigeminal ganglia were harvested for bulk RNA-Seq analysis (female mice, n=3 per group).Differential gene expression was calculated between the sham-operated (Ct) and DED mice of each strain.A) Volcano plots of differentially expressed genes in DED vs Ct mice of each strain.Up-regulated genes in DED mice are shown in red and down-regulated genes are shown in blue.B) Heatmaps (normalized counts, Z score) of

Figure 5 -F)
Figure 5 -Adoptive transfer of dry eye-induced CD4 + T cells reproduces ocular surface phenotype.A) Experimental design: CD4 + T cells were isolated from the spleens and lymph nodes of wild-type (WT) mice of both sexes 10 days after surgical induction of dry eye disease (DED) and then adoptively transferred into sex-matched recombination-activating gene 1-knockout (Rag1KO) mice, which were evaluated over the course of 4 weeks.Sham-operated WT mice were used as a source of control (Ct) CD4 + T cells.B) Flow cytometry of cervical lymph node cells (representative example) obtained from a CD4 + T cell-reconstituted Rag1KO mouse 4 weeks after transfer.C) Conjunctival CD4 + T cells in Rag1KO mice 4 weeks after transfer as assessed by flow cytometry

Figure 6 -
Figure 6 -Adoptive transfer of dry eye-induced CD4 + T cells reproduces corneal neuropathy but not epitheliopathy.The experimental design was described in detail in Figure 5A and involved the adoptive transfer of control (Ct) or dry eye disease (DED)-induced CD4 + T cells from WT mice into recombination-activating gene 1-knockout (Rag1KO) mice of the same sex that were subsequently evaluated over 4 weeks.A) Cumulative and B) representative micrographs of corneal dextran-fluorescein uptake in Ct-and DED-induced CD4 + T cell recipients.Data shown as the mean fluorescence intensity (MFI) calculated with ImageJ software (Methods section).The dotted line represents the mean corneal uptake observed in DED WT mice.C) Quantification of proliferating cells within the epithelial basal layer of corneal whole-mounts obtained from Ct-and DED-induced CD4 + T cell-recipient mice.D) Corneal mechanosensitivity thresholds in Ct-and DED-induced CD4 + T cell recipients.E) Representative micrographs and F-H) density of intraepithelial corneal innervation analyzed at three different levels by Alexa Fluor 488 anti-tubulin β3 staining: subapical (F, % area occupied by nerve endings) and mid-epithelial (G, count of nerve endings/field) nerve ending density and complexity of subbasal nerves (H, sum of intersections at all Sholl radii).All experiments were performed twice or more with 6 mice/group/experiment.For all

Figure 7 -
Figure 7 -Reconstitution of T cell-deficient with CD4 + T cells enables corneal neuropathy development upon dry eye induction.A) Experimental design: CD4 + T cells were isolated from the spleens and lymph nodes of wild-type (WT) mice of both sexes and then adoptively transferred into sex-matched recombination-activating gene 1-knockout (Rag1KO) mice, which one week later underwent either sham (Ct) or dry eye disease (DED)-inducing surgery and were evaluated over (45)mice were 6-8 weeks old at the beginning of the experiments and both male and female mice were included.All protocols were approved by the Institute of Experimental Medicine animal ethics committee (approval #084/2020) and adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.Since DED is more prevalent and more severe in women(45), C57BL/6 (C57BL/6NCrl) mice were originally obtained from Charles River Laboratories (Wilmington, MA, USA) and recombination-activating gene 1 (Rag1)-knockout (Rag1KO (B6.129S7-Rag1tm1Mom/J,JAX stock #002216) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).Mice were bred and maintained at the Institute of Experimental Medicine's conventional animal facility.

Table 1 -
Reagents and antibodies 3.1.Subsequent analyses were performed in R (v4.3.1).Gene filtering was performed by selecting features with >=10 reads in at least 3 samples.For quality controls, the filtered data were subjected to a principal component analysis and hierarchical clustering (Ward.D2 method, Euclidean distance) after trimmed mean of M-values normalization (edgeR v4.0.16).Gene information (ENSEMBL ID, external gene name and gene biotype) was extracted using biomaRt (v2.58.2).Differential gene expression analyses were performed by the DESeq2 methodology (v1.42.1).Genes with log2 fold change > 0.378 and adjusted p-value < 0.1 were considered as upregulated, and genes with log2 fold change < -0.378 and adjusted p-value < 0.1 were categorized as downregulated.The list of genes was ranked by log10 adjusted p-value * sign(log2 fold change) and subjected to a Gene Set Enrichment Analysis with the Gene Ontology database, using enrichGO and simplify (clusterProfiler v4.10.1).Enriched Gene Ontology terms with adjusted p-value of 0.05 were considered significant.All raw data files are available at ArrayExpress (https://www.ebi.ac.uk/biostudies/arrayexpress), accession E-MTAB-13945