Gonadotropins: A new concept in keratoconus

AuthorBlock: Dimitrios Karamichos1, Brayden Barrientez1, Sarah E. Nicholas1, Symon Ma1, Lyly Van1, Sashia Bak-Nielsen2, Jesper Hjortdal2 1Ophthalmology, OUHSC, Dean McGee Institute, Oklahoma City, Oklahoma, United States; 2Aarhus University, , Denmark;

DisclosureBlock: Dimitrios Karamichos, University of Oklahoma Health Sciences Center Code P (Patent), Brayden Barrientez, None; Sarah E. Nicholas, None; Symon Ma, None; Lyly Van, None; Sashia Bak-Nielsen, None; Jesper Hjortdal, None;

Purpose Keratoconus (KC) is the most common ectatic corneal disease with significant visual acuity burden. Despite recent advancements, KC pathobiology remains unclear. In recent years, our focus has been on unravelling the role of sex hormones in KC. The current study presents evidence, for the first time, that sex hormone imbalances in KC initiated from the anterior pituitary and the secretion of gonadotropins.Methods The purpose of this study was to investigate the role of gonadotropins in KC. We recruited 86 KC patients (63 male, 23 female), and 45 healthy controls (22 male, 23 female). Plasma samples were collected and analyzed using enzyme-linked immunosorbent assay. Corneal stromal cells from KC and healthy controls and human epithelial corneal cells were also investigated for gonadotropin-related markers.Results Our findings showed a significantly lower LH/FSH ratio in KCs when compared to healthy controls, in both males and females. The lowest LH/FSH ratio was seen in KC-Females at the 15-29y/o and ≥46y/o age groups and was directly correlated with increased KC severity. Stromal cells from both healthy and KC expressed LH, LHR, and FSHR, but not FSH. Corneal epithelial cells only expressed FSHR.Conclusions Our study is the first to demonstrate the role of LH/FSH in KCs and expands the list of organs known to express gonadotropins to include the human . Our findings suggest an intriguing mechanism for the onset and/or progression of KC.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Corneal nerve ablation abolishes ocular immune privilege through a SP-mediated down-regulation of CD103 and interferon gamma receptor expression on T regulatory cells.

AuthorBlock: Jerry Y. Niederkorn1, Sudha neelam1 1Ophthalmology, Univ Texas Southwestern Med Ctr, Dallas, Texas, United States;

DisclosureBlock: Jerry Y. Niederkorn, None; Sudha neelam, None;

Purpose Severing corneal nerves during orthotopic corneal transplantation elicits the elaboration of the neuropeptide substance P (SP), which induces the generation of CD11c+ contrasuppressor (CS) cells. CS cells disable T regulatory cells (Tregs) that are induced when antigens enter the anterior chamber, either by direct injection or by orthotopic corneal transplantation. This study examined the crucial cell surface molecules on T regulatory cells that are adversely affected by CS cells that are generated by severing corneal nerves.Methods CS cells were induced by severing corneal nerves with a 2.0 mm trephine in the of BALB/c mice. Anterior chamber-associated immune deviation (ACAID) CD8+ Tregs were generated by injecting ovalbumin into the anterior chamber (AC). We analyzed the effects of CS cells and SP on the expression and function of two cell surface molecules (CD103 and the receptor of interferon-gamma; IFN-γR), that are crucial for the induction and function of ACAID T regs. CS cells were co-cultured with ACAID Tregs and the expression of CD103 and IFN-γR was analyzed by flow cytometry. Transwell cultures were used to determine if the effect of CS cells on CD103 and IFN-γR expression was contact- dependent.Results Severing corneal nerves resulted in a 66% reduction in the expression of CD103 and a 70% reduction in the interferon-gamma receptor (IFN-γR) on CD8+ ACAID Tregs isolated from the spleens of trephined mice. These effects could be mimicked in vitro by co-culturing CD8+ ACAID T regs with either CS cells or with SP. Down regulation of CD103 and IFN-γR on ACAID Tregs by CS cells was found to be contact-dependent.Conclusions Severing corneal nerves resulted in a 66% reduction in the expression of CD103 and a 70% reduction in the interferon-gamma receptor (IFN-γR) on CD8+ ACAID Tregs isolated from the spleens of trephined mice. These effects could be mimicked in vitro by co-culturing CD8+ ACAID T regs with either CS cells or with SP. Down regulation of CD103 and IFN-γR on ACAID Tregs by CS cells was found to be contact-dependent.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. The effect of the diabetic state in donors on resident corneal immune cells and graft survival

AuthorBlock: Hayate Nakagawa1, Tomas Blanco1, Sonia Anchouche1, Rohan Bir Singh1, Hamid Alemi1, Jia Yin1, Reza Dana1 1Schepens Eye Research Institute of Mass. Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States;

DisclosureBlock: Hayate Nakagawa, None; Tomas Blanco, None; Sonia Anchouche, None; Rohan Bir Singh, None; Hamid Alemi, None; Jia Yin, None; Reza Dana, None;

Purpose The diabetic state promotes infiltration and maturation of antigen presenting cells (APC) in the cornea. The effect of these corneal APCs on outcomes of corneal transplantation is yet to be elucidated. In this study, we investigate the effect of altered immune milieu in from diabetic donors transplanted to normal recipients, using a validated murine model of corneal allotransplantation.Methods Type I diabetes mellitus was induced in C57BL/6 mice by injecting 50mg/kg streptozocin (STZ) for 5 days. The altered glycemic state was confirmed by a self-monitoring glucometer. On day 25, mice with blood glucose levels of 300mg/dL were considered diabetic. The corneas from diabetic mice were harvested and grafted onto normal BALB/c recipient mice, or used for flow cytometry analysis. Recipient mice were followed up for 8 weeks using slit-lamp biomicroscopy. Two weeks after the transplantation, both cornea and draining lymph node of recipient mice were analyzed using flow cytometry.Results A significantly higher frequency of CD11b+ cells (82±2.5%, p=0.0009) and higher expression of MHC- II (fold increase p=0.048) were observed in corneas from diabetic donors compared to normal mice. The allograft rejection was faster in recipients of diabetic donor corneas compared to recipients with normal donors. CD11b+ cells frequency (2.58±0.2%;p=0.014) as well as their maturation markers such as MHC-II (p=0.006), CD80 (p=0.007) and CD86 (p=0.03), were significantly increased in recipients corneas with diabetic donors compared to recipients with normal donors. The frequency of CD11b+APCs (0.9±0.06%; p=0.047) and IFNγ+ T-cells (2±0.18%; p=0.005) were significantly higher in dLNs of mice receiving grafts from diabetic donors compared to recipients with normal donors. Conversely, the frequency of FoxP3+ Tregs (13.3±0.2%; p=0.012), and their expression of IL-10 (p=0.07) and TGFβ1 (p=0.0085) were significantly reduced in mice receiving grafts from diabetic donors compared to recipients with normal donors.Conclusions Our data suggest that type I diabetes mellitus induces maturation of resident corneal immune cells and; consequently, diabetic donor corneas may precipitate graft immune rejection in recipient mice.Layman Abstract (optional): Provide a 50-200 word description of your work that non- scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. PRE-ACTIVATED MESENCHYMAL STROMAL CELLS INDUCE REGULATORY IMMUNE POPULATIONS IN VIVO AND PROLONG CORNEAL ALLOGRAFT SURVIVAL

AuthorBlock: Thomas Ritter1, Oliver Treacy2,1, Xizhe Chen1, Nick Murphy1, Paul Lohan1, Md Nahidul Islam1, Ellen Donohoe1, Matthew Griffin1, Luke Watson3, Steven McLoughlin1, Grace O'Malley2, Kevin Lynch2,1, Aideen Ryan2 1Medicine, Nt'l Univ of Ireland, Galway, Galway, Ireland; 2Discipline of Pharmacology and Therapeutics, National University of Ireland Galway, Galway, Ireland; 3Orbsen Therapeutics, Galway, Ireland;

DisclosureBlock: Thomas Ritter, None; Oliver Treacy, None; Xizhe Chen, None; Nick Murphy, None; Paul Lohan, None; Md Nahidul Islam, None; Ellen Donohoe, None; Matthew Griffin, None; Luke Watson, None; Steven McLoughlin, None; Grace O'Malley, None; Kevin Lynch, None; Aideen Ryan, None;

Purpose To investigate a novel licensing strategy to significantly improve the immunosuppressive capacity of MSCs in vitro and their therapeutic efficacy in vivo in a fully allogeneic murine corneal transplant model.Methods Murine mesenchymal stromal cells (Balb/c) were pre-conditioned with TGF-β1 (TGF-β MSC) and tested for immunomodulatory properties in vitro in T cell co-culture assays. Moroever we tested if TGF-β MSC had an enhanced ability to prolong rejection free survival in a fully allogeneic mouse cornea transplant model (C57BL/6 to Balb/c). Immune cell populations were analysed in the draining lymph nodes, spleens and lungs of TGF-β MSC treated mice. Finally, contact-dependent and contact- independent signalling mechanisms of TGF-β MSC were investigated to identify the mechanism of action of enhanced immunomodulation.Results Pre-conditioning of murine MSC with TGF-β1 (TGF-β MSC) significantly improved their ability to modulate innate and adaptive immune cells in vitro and in vivo. We found that TGF-β MSCs significantly suppressed the proliferation of stimulated CD3+CD4+ and CD3+CD8+ T lymphocytes while significantly increasing the numbers of CD3+CD4+FoxP3+ regulatory T lymphocytes (Tregs) following co-culture assays. We tested if TGF-β MSC had an enhanced ability to prolong rejection free survival in a fully allogeneic mouse cornea transplant model (C57BL/6 to Balb/c). TGF-β MSC treated mice presented with a rejection free survival rate of 70% (n=13) compared to 25% (n=14) in the non- activated MSC treatment group. Prolongation of graft survival was associated with; (i) increased Treg populations in the draining lymph nodes (dLNs) and lungs of TGF-β MSC treated mice, and (ii) decreases in antigen presenting cell populations in the dLNs, lungs and spleens of TGF-β MSC treated mice. Finally, we confirmed that (i) TGF-β MSC mediated their therapeutic effects via canonical Smad2/3 signalling, that (ii) the potent immunosuppressive effects mediated by TGF-β MSC were contact-dependent and (iii) prostaglandin E2 (PGE2) (via prostaglandin EP4 receptor) plays a vital role in TGF-β MSC mediated immunosuppression of T lymphocytes.Conclusions We have shown for the first time that pre-treatment of MSC with TGF-β significantly enhances their immunosuppressive capacity in vitro and therapeutic efficacy in a murine corneal transplant model.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Tear hyperosmolarity acts as a disruptor of ocular surface immune tolerance in the context of dry

AuthorBlock: Jeremias Gaston Galletti1, Maximiliano S. Miglio1, Mauricio Guzmán1, Irene Keitelman1, Florencia Sabbione1, Mirta N. Giordano1, Analía Trevani1 1Innate Immunity Lab, Institute of Experimental Medicine/CONICET, National Academy of Medicine, Buenos Aires, BUENOS AIRES, Argentina;

DisclosureBlock: Jeremias Gaston Galletti, None; Maximiliano S. Miglio, None; Mauricio Guzmán, None; Irene Keitelman, None; Florencia Sabbione, None; Mirta N. Giordano, None; Analía Trevani, None;

Purpose Tear hyperosmolarity is commonly detected in dry eye disease (DED), but its exact role in disease progression is incompletely understood. On the other hand, breakdown of ocular surface tolerance (homeostasis) has been reported in DED models but the trigger remains unknown. The purpose of this work was to evaluate whether hyperosmolar stress could affect ocular surface homeostasis in mice.Methods 8-to-12 week-old Balb/c mice were instilled isoosmolar (0.3 Osm) or hyperosmolar (3 Osm) saline on both 3 times daily for 5 days to induce hyperosmolar stress (HS). Ovalbumin (OVA) was also instilled as a surrogate antigen or OVA-pulsed dendritic cells (DCs) were subconjunctivally injected at different time points. Induced T cell responses were measured after subcutaneous immunization with OVA by the delayed-type hypersensitivity (DTH) assay. In addition, T cells in draining lymph nodes (day 5) were evaluated by flow cytometry.Results Compared to non-instilled immunized mice, OVA-instilled mice developed reduced DTH responses (i.e. were tolerized), as did their OVA+saline instilled cage mates (p<0.05). By contrast OVA+HS mice exhibited full DTH responses (i.e. were not tolerized). In the latter, T cells in draining lymph nodes showed increased expression of activation (CD69, CD25) and memory (CD44) markers. Local injection of immature dendritic cells (DCs) in HS mice, but not in saline-instilled mice, induced increased T cell proliferation in the draining lymph nodes. Conjunctivae from HS mice showed higher levels of epithelial NF-κB activation and increased numbers of DCs, and conversely, topical NF-κB inhibition prevented ocular surface tolerance disruption in HS mice. Consistently, supernatants from epithelial cells exposed to HS for 4 h, but not to control medium, had higher interleukin-1β and -6 levels and favored DC maturation and T cell proliferation in vitro.Conclusions HS is sufficient to abolish conjunctival immune tolerance to a harmless antigen in Balb/c mice, suggesting that HS by itself can skew the immune response at the ocular surface. The HS-induced proinflammatory mucosal environment favors local maturation of DCs and T cell responses in the draining lymph nodes. These results altogether suggest that hyperosmolar stress plays a role in dry eye initiation as an immune disruptor.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. The inflammation that keeps dry eye going might begin by the detrimental effect that increased tear evaporation has on the immune system of the ocular surface, the part of the eye that is exposed to the environment. We here report some very early changes in the way that the ocular immune system reacts that could explain the origin of dry eye and at the same time, we suggest potentially new treatment strategies for this all too common disease. Anti-apoptotic and anti-inflammatory effect of PD-L1 in Dry Eye Mouse Model

AuthorBlock: Jin Hyoung Park1,2, Sae-Byeok Hwang1, Soon-Suk Kang2, Hungwon Tchah3,2, Jae Yong Kim3,2 1Ophthalmology, Miso Eye Clinic, Sujeong-gu, Seongnam-si, Korea (the Republic of); 2Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Asan Medical Center,, Seoul, Korea (the Republic of); 3Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea (the Republic of);

DisclosureBlock: Jin Hyoung Park, None; Sae-Byeok Hwang, None; Soon-Suk Kang, None; Hungwon Tchah, None; Jae Yong Kim, None;

Purpose To examine the effects of programmed death-ligand1 (PD-L1) on apoptosis and inflammation through T-cell suppression in scopolamine- induced dry eye model.Methods We induced desiccation stress in female C57BL/6 mouse using scopolamine. The C57BL/6 mouse was divided into 3 groups: “Control”, “Desiccation”, and “Desiccation + PD-L1”; topical recombinant mouse PD-L1 was applied three times daily for 5 days. To investigate the effects of PD-L1 on T-cell in desiccation stress induced dry eye model, we used immunohistochemical analysis for CD3e and PD- L1, immunofluorescent staining for CD4 andIL-17 with 3 groups of mouse cornea. To evaluate the effect of PD-L1 on desiccation stress induced apoptosis, we performed Tunnel assay and measured expression of Bax by western blot usingprotein extracts of mouse cornea. Moreover, to determine the effects of PD-L1 on inflammation, we quantified the expression of nuclear factor-κB (NF-κB) activation and phosphorylated NF-κB inhibitor α (IκB-α) using western blot analysis with protein extracts of mouse cornea.Results CD3e, CD4, and IL-17 levels further increased by scopolamine induced desiccation stress, and induction of the T-cell markers was significantly decreased by PD-L1. In the desiccation stress induced mouse cornea, apoptotic and inflammatory proteins, Bax, phosphorylated NF-κB and phosphorylated IκB-α were also enhanced.. while these expreesion levels were markedly reduced by PD- L1.Conclusions PD-L1 had potent anti-inflammatory and anti-apoptotic effects in desiccation stress through T-cell suppression. Therefore, PD-L1 may represent a novel therapeutic approach to managing dry eye disease.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Trigeminal ganglion transcriptome unravels the RICTOR/mTORC2 pathway fostering corneal nerve regeneration after a novel RvD6 isomer treatment.

AuthorBlock: Thang Luong PHAM1, Azucena H. Kakazu1, Jiucheng He1, Haydee E P Bazan1 1Neuroscience Center, Louisiana State University Health New Orleans, New Orleans, Louisiana, United States;

DisclosureBlock: Thang Luong PHAM, None; Azucena H. Kakazu, None; Jiucheng He, None; Haydee E P Bazan, None;

Purpose Recently, our laboratory discovered a novel stereospecific Resolvin D6-isomer (RvD6si) released in tears that is activated by pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) upon corneal injury. RvD6si promotes corneal functional recovery by restoring the high-density innervation. Here, we explored the stimulation by RvD6si of the transcriptome of the trigeminal ganglion (TG) to elucidate the mechanism that fosters corneal nerve regeneration.Methods Eight-week-old male CD1 mice were used. Corneal epithelium from the right eye was removed from a 2mm diameter surface under anesthesia. Then, corneas were treated topically with vehicle, RvD6, and RvD6si three times per day. Two weeks after treatment, the ipsilateral TG was dissected and analyzed for gene expression using RNA-sequencing. Differential gene expression analysis was done using DESeq2 and significantly changed genes were subjected to Ingenuity Pathway Analysis (IPA).Results Among treatment conditions, the RvD6 and RvD6si shared 58 upregulated genes and 36 downregulated genes compared to vehicle-treated corneas. The upstream predicted results from IPA software unraveled a strong induction by RvD6si of a transcriptional factor named Rictor, a key component of the rapamycin-insensitive complex 2 of mTOR (mTORC2). There were 39 genes modulated by RICTOR significantly changed by the treatment. Among those genes, 37 (95%) genes matched the IPA knowledge collected from published data. Additionally, the RNA-seq also demonstrated that RvD6 treatment (both RvD6 and RvD6si) reduced the gene expression of two major neuropeptides, tachykinin precursor 1 and calcitonin-related polypeptide beta, that were regarded as major pain-induced mediators in migraines and other primary headaches and increased the expression of transient receptor potential melastatin 8 (TRPM8), a cold receptor important to maintaining tear secretion and control neuropathic pain after surgery.Conclusions This study unraveled the role of Rictor signaling in the cell bodies of TG to foster the nerve innervation in the cornea after injury and treatment with RvD6si. Furthermore, the expression of genes related to neuropathic pain are decreased while TRPM8 is increased. The new RvD6si uncovers new therapeutic avenues for corneal pathologies that affect tissue innervation.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. The miR-183/96/182 Cluster Regulates the Functions of Myeloid-derived Corneal Resident Innate Immune Cells

AuthorBlock: Shunbin Xu1, Ardian Coku1, Linda D. Hazlett1, Sharon Ann McClellan1, Ahalya Pitchaikannu1 1Ophthalmology, Visual and Anatomical Sciences, School of Medicine Wayne State University, Detroit, Michigan, United States;

DisclosureBlock: Shunbin Xu, None; Ardian Coku, None; Linda D. Hazlett, None; Sharon Ann McClellan, None; Ahalya Pitchaikannu, None;

Purpose Myeloid-derived corneal resident innate immune cells (MDCRIICs) play significant roles in the homeostasis of the cornea, including ocular immune privilege, transplant graft survival, wound healing, corneal nerve regeneration and inflammatory/immune responses following tissue damage and/or microbial infection and orchestrate adaptive immune responses. However, the roles of microRNAs (miRNAs) in MDCRIICs are utterly unknown. The purpose of this project is to determine the role of the miR-183/96/182 cluster (miR-183/96/182) in the regulation of MDCRIICs.Methods Young adult (8-18 weeks old) miR-183/96/182 knockout (ko) and wild-type control (wt) mice were used. To trace MDCRIICs, we also produced miR-183/96/182 ko and wt mice on the background Csf1r- EGFP (Jackson Laboratory), which express enhanced green fluorescent protein (EGFP) in MDCRIICs. To detail the dynamics of these cells, central corneas of anesthetized mice were scarred and 5 ml of 1X106 CFU/ml of Pseudomonas aeruginosa (PA. Strain 19660; ATCC) was topically applied. Naïve and PA-infected corneas were harvested 3 and 6 hours post-infection (hpi) for flat-mount confocal microscopic studies. In addition, corneas of naïve mice were dissociated for fluorescence activated cell sorting (FACS). Total RNA was isolated from FACS-purified resident macrophages (ResMΦ) for quantitative (q)RT-PCR analysis.Results 1) Inactivation of miR-183/96/182 resulted in an increased number (~54%) of steady-state corneal resident innate immune cells, including MDCRIICs and ResMΦ; 2) MDCRIICs are highly dynamic in PA- infected and non-infected contralateral eyes. At 3 hpi, the number of MDCRIICs was significantly decreased (~82%) in PA-infected eyes of both ko and wt mice; however, at 6 hpi, ko vs. wt mice showed significantly enhanced “come-back” in the infected eyes (~217% vs. 124%); 3) Corneal ResMΦ simultaneously express IL-10 and IL-17F; miR-183/96/182 regulates IL-10 and IL-17F production by targeting the genes encoding key transcriptional regulators.Conclusions Corneal ResMΦ are innate IL-10- and IL-17F-producing cells. miR-183/96/182 plays important roles in corneal innate immunity by regulating the number, dynamics and functions of MDCRIICs.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. The cornea contains resident immune cells, which play important roles in the homeostasis of the cornea, including ocular immune privilege, transplant graft survival, wound healing, corneal nerve regeneration and inflammatory/immune responses following tissue damage and/or microbial infection and orchestrate adaptive immune responses. However, the roles of microRNAs (miRNAs)- a new class of regulators of gene expression, in corneal resident immune cells are utterly unknown. The purpose of this project is to determine the role of the miR-183/96/182 cluster (miR-183/96/182) in the regulation of MDCRIICs. We discovered the miR-183/96/182 regulates the number, dynamics and functions of corneal resident immune cells, suggesting that this miRNA cluster may modulate many physiological and pathological processes in health and disease of the cornea and the eye. Functional enrichment analysis of the tear proteome supports the role of immunological processes in MGD

AuthorBlock: Pali P. Singh1, Rose Mathew1, Martha A. Cady1, Nikolai Skiba1, Chen Yu1, Victor L. Perez Quinones1, Daniel R. Saban1 1Duke University, Durham, North Carolina, United States;

DisclosureBlock: Pali P. Singh, None; Rose Mathew, None; Martha A. Cady, None; Nikolai Skiba, None; Chen Yu, None; Victor L. Perez Quinones, Novartis Code F (Financial Support), Alcon Code C (Consultant), Kala Code C (Consultant), Daniel R. Saban, Novartis Code F (Financial Support)

Purpose The immune response has recently been shown to play a major etiological role in Meibomian gland dysfunction (MGD). In the AED mouse model, Th17 mediated neutrophils have been shown to cause MGD. Likewise, in humans, tear neutrophils have been correlated with an increase in MGD severity. Here, we use functional enrichment analysis of the human tear proteome in a subject with MGD as compared to a healthy control to investigate the molecular underpinnings of MGD.Methods Tear samples were obtained by Schirmer strip from human subjects, one with MGD and the other a healthy control. Proteins were eluted into PBS, alkylated, purified via paramagnetic beads, and trypsin digested for label-free data-dependent mass spectrometry. Returned proteins of both conditions were entered into STRING for pathway enrichment analysis.Results From the MGD tear sample, we identified 846 unique proteins with high confidence and 871 from the healthy control. Overall, STRING enrichment analysis of the identified proteins in both conditions emphasized immune and metabolic pathways. The top 25 Reactomes, sorted by increasing False Discovery Rate (FDR), showed greater enrichment of immune pathways in MGD based on percent of the pathway identified. These enriched pathways include innate immune system (21% MGD v 14% control), cytokine signaling (15% MGD v 8% control), and neutrophil degranulation (28% MGD v 19% control). The regulation of complement cascade Reactome was decreased in MGD (26%) as compared to healthy control (40%).Conclusions Identification of proteins in human tears via an unbiased label-free proteomics approach emphasizes immune Reactomes in MGD, which is consistent with a contribution of the immune response to the etiology as described previously. Enrichment of the neutrophil degranulation Reactome in the MGD condition also supports the role of neutrophils in MGD. How these results compare to the tear proteome in MGD of greater severity is yet to be determined. Future directions include increasing the sample size and quantitative proteomics to determine up- or down-regulation of proteins between conditions.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. CD103+ Conventional Dendritic Cells Are Critical for Corneal Allograft Survival

AuthorBlock: Tomas Blanco1, Hayate Nakagawa1, Rohan Bir Singh1, Yukako Taketani1, Hong yan Ge1, Nai-Wen Fan1, Yihe Chen1, Jia Yin1, Sunil Chauhan1, Reza Dana1 1Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States;

DisclosureBlock: Tomas Blanco, None; Hayate Nakagawa, None; Rohan Bir Singh, None; Yukako Taketani, None; Hong yan Ge, None; Nai-Wen Fan, None; Yihe Chen, None; Jia Yin, None; Sunil Chauhan, None; Reza Dana, None;

Purpose Conventional type 1 CD103+ dendritic cells (cDC1) induce immune tolerance by promoting conversion of naïve CD4+T cells to Foxp3+ regulatory T cells (Tregs). Herein, we examined the role of CD103+ DCs in corneal allograft survival.Methods Allogeneic corneal transplantation was performed using C57BL/6J mice as donors and BALB/c as recipients. CD103+ DCs were depleted in recipients with subconjunctival injection of anti-CD103- saporin conjugated (SAP). On day 3, naïve Tregs, antigen specific Tregs or CD4+CD25- T cells were subconjunctivally injected (1x105, syngeneic ) to graft recipients and PBS injections were used as controls. Bone marrow derived induced-CD103+DCs (iCD103+, 1x104) where subconjunctivally injected to evaluate their tolerogenic efficacy, and iCD11b DCs injected mice were used as negative controls. Animals were followed up for 8 weeks. Corneas and dLNs were assessed by flow cytometry. Additionally, CD103+ DCs were FACS sorted and evaluated by PCRResults CD103+ DCs were successfully depleted in cornea(<1%,p<0.001), conjunctiva(<1%, p<0.001) and dLN(<1%,p<0.001) of SAP treated mice compared to controls by day 7. Allografts were rejected in SAP treated mice (100%, p<0.001) compared to controls (55%). Injection of Tregs moderately extended graft survival compared to SAP, but <20% (naïve,p<0.001) or <35% (antigen- specific,p<0.001) grafts survived after 8 weeks compared to controls. All the grafts were rejected in CD4+CD25- T cell injected mice (p<0.001). Adoptive transfer of iCD103 DCs significantly increased graft survival to 90% (p<0.001), whereas 100% of grafts were rejected in mice inkected witht iCD11b- (100%, p<0.001). Mice treated with iCD103 DCs showed significantly increased Treg frequency (14.3%, p<0.01) and FoxP3 expression [1.5-fold (p<0.01)] compared to either iCD11b (10.3%) or PBS (11%) treated controls. IFNγ expression was significantly decreased in dLNs (0.40%, p<0.01) of iCD103 recipient mice compared to iCD11b (2.48%) or PBS (0.90%) controls. Additionally, mRNA expression of Irf8, Itgb8, Itgae, Clec9a, Cd274 and Xcr1 genes in cornea, and dLN was significantly upregulated in transplanted mice (p<0.001) compared to normal.Conclusions These results signify the critical role of CD103+ DCs in promoting tolerance and allograft survival. These findings suggest a possible therapeutic potential of adoptively transferred CD103+ cells in preventing allograft rejection.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details.