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Autoimmune Lacrimal KeratoconjunctivitisAutoreactive T Cell-Mediated Experimental Ocular Surface APCs Are Necessary for

Pflugfelder and Michael E. SternPaiva, Larry A. Wheeler, Jerry Y. Niederkorn, Stephen C. Chris S. Schaumburg, Karyn F. Siemasko, Cintia S. De

http://www.jimmunol.org/content/187/7/3653doi: 10.4049/jimmunol.1101442August 2011;

2011; 187:3653-3662; Prepublished online 31J Immunol 

Referenceshttp://www.jimmunol.org/content/187/7/3653.full#ref-list-1

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The Journal of Immunology

Ocular Surface APCs Are Necessary for AutoreactiveT Cell-Mediated Experimental Autoimmune LacrimalKeratoconjunctivitis

Chris S. Schaumburg,* Karyn F. Siemasko,* Cintia S. De Paiva,† Larry A. Wheeler,*

Jerry Y. Niederkorn,‡ Stephen C. Pflugfelder,† and Michael E. Stern*

As specialized sentinels between the innate and adaptive immune response, APCs are essential for activation of Ag-

specific lymphocytes, pathogen clearance, and generation of immunological memory. The process is tightly regulated; however,

excessive or atypical stimuli may ignite activation of APCs in a way that allows self-Ag presentation to autoreactive T cells in

the context of the necessary costimulatory signals, ultimately resulting in autoimmunity. Studies in both animal models and patients

suggest that dry eye is a chronic CD4+ T cell-mediated ocular surface autoimmune-based inflammatory disease. Using a desiccating

stress-induced mouse model of dry eye, we establish the fundamental role of APCs for both the generation and maintenance of

ocular-specific autoreactive CD4+ T cells. Subconjunctival administration of liposome-encapsulated clodronate efficiently dimin-

ished resident ocular surface APCs, inhibited the generation of autoreactive CD4+ T cells, and blocked their ability to cause

disease. APC-dependent CD4+ T cell activation required intact draining cervical lymph nodes, as cervical lymphadenectomy also

inhibited CD4+ T cell-mediated dry eye disease. In addition, local depletion of peripheral conjunctival APCs blocked the ability of

dry eye-specific CD4+ T cells to accumulate within the ocular surface tissues, suggesting that fully primed and targeted dry eye-

specific CD4+ T cells require secondary activation by resident ocular surface APCs for maintenance and effector function. These

data demonstrate that APCs are necessary for the initiation and development of experimental dry eye and support the standing

hypothesis that dry eye is a self-Ag–driven autoimmune disease. The Journal of Immunology, 2011, 187: 3653–3662.

Dry eye, also known as dysfunctional tear syndrome, is acommon ocular surface disease with high prevalence andsignificant morbidity worldwide (1, 2). Patients experi-

ence a variety of symptoms including ocular discomfort, fatigue,and chronic pain, accompanied by blurred and fluctuating vision. Inthe most severe cases, corneal opacification or ulceration may resultin reduced vision and blindness. Emerging evidence suggests thatdry eye is an ocular surface autoimmune-based inflammatory dis-ease. Environmental and/or microbial stress, combined with ge-netically predisposed factors, is thought to perpetuate chronicautoreactive T cell-mediated inflammation and dysfunction of thelacrimal function unit (LFU; cornea, conjunctiva, lacrimal glands,andmeibomian glands) (3). Inflammatory cell infiltration within theLFU tissues correlates with elevated proinflammatory cytokinelevels, increased epithelial cell apoptosis, and diminished mucin-secreting goblet cell numbers, coupled with decreased tear pro-duction in animal models (4) and patients with dry eye (5).CD4+ T cells make a prominent contribution to chronic in-

flammation during the immunopathogenesis of dry eye, under-

scoring its basis as an autoimmune-based inflammatory disease. Thisnotion is supported by several lines of evidence: 1) activated CD4+

T cells are localized within the ocular surface tissues of dry eye

patients (5, 6); 2) CD4+ T cells are sufficient to induce dry eye inmice(4); and 3) compounds that inhibit T cells (e.g., cyclosporine A) at-tenuate dry eye disease in both animals and humans (5, 7). Dry eyedisease, induced in a mouse model of autoimmune lacrimal kerato-conjunctivitis (ALKC), is a valuable tool for defining the underlyingmechanisms of CD4+ T cell-mediated disease (4). Mice exposed toenvironmental stress (i.e., desiccating stress [DS]) and administered

scopolamine develop clinical and histopathological similarities to thehuman disease, including a robust CD4+ T cell infiltrate into the LFU(4). Moreover, CD4+ T cells isolated from the cervical lymph nodes(CLNs) and/or spleen of experimental dry eye mice readily traffic tothe ocular surface, where they mediate full-blown dry eye diseasewhen adoptively transferred to athymic T cell-deficient nude re-cipient mice (4). CD4+ T cell homing and pathogenesiswas restricted

to the ocular surface tissues of the nude recipientmice, indicating thatautoreactive CD4+ T cells are targeted to the ocular surface duringactivation within the CLN and spleen of donor mice exposed to DS.Engagement with APCs is necessary for activation and differen-

tiation of CD4+ T cells within the secondary lymphoid organs (e.g.,interaction with dendritic cells [DCs]) and reactivation and main-

tenance of activated T cells at sites of inflammation (e.g., interactionwith DCs and/or macrophages). Activation of naive T cells bycompetent APCs is well defined (8), but much less is known re-garding the requirements of APC-dependent reactivation of fullyprimed, activated T cells. DCs are among the most potent pro-fessional APCs and in the presence of the proper costimulatorysignals may effectively present self-Ag to autoreactive T cells dur-

ing the development and progression of autoimmune disease (9, 10).In the eye, DCs and macrophages are present within the ocularsurface tissues (11). For example, DCs are present in the healthy

*Biological Sciences, Inflammation Research Program, Allergan, Inc., Irvine, CA92612; †Ocular Surface Center, Cullen Eye Institute, Baylor College of Medicine,Houston, TX 77030; and ‡Department of Ophthalmology, University of Texas South-western Medical Center, Dallas, TX 75390

Received for publication May 19, 2011. Accepted for publication August 1, 2011.

Address correspondence and reprint requests to Dr. Michael E. Stern, BiologicalSciences, Inflammation Research Program, Allergan, Inc., 2525 Dupont Drive,RD3-2D, Irvine, CA 92612. E-mail address: stern_michael@allergan.com

Abbreviations used in this article: ALKC, autoimmune lacrimal keratoconjunctivitis;AX SX, axillary lymphadenectomy; C12MDP-LIP, clodronate liposome; CLN, cer-vical lymph node; CLN SX, bilateral surgical cervical lymphadenectomy; DC, den-dritic cell; DS, desiccating stress; LFU, lacrimal function unit; MHC II, MHC classII; OD, ocular dexter; OS, ocular sinister; PAS, periodic acid-Schiff; Sham SX, shamsurgery.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

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corneal stroma (12, 13), increasing in number and with expression ofcostimulatory molecules following inflammatory insults (14). MHCclass II (MHC II)molecules are required for presentation of antigenicepitopes to CD4+ T cells and are also upregulated on APCs localizedwithin the ocular surface tissues in animalmodels (15) and in patientswith dry eye (16). Homing of CD11c+CD11b+MHC II+ APCs fromthe ocular surface to the CLN is dependent on CCR7–CCL21 sig-naling (13); more recently, homing of mature MHC II+ APCs to thedraining CLNs was shown to be associated with enhanced lym-phoangiogenesis on the ocular surface during DS-induced experi-mental dry eye (17). Nonetheless, the absolute role of APCs in theinitiation and progression of dry eye has not been evaluated.In this report, we demonstrate for the first time, to our knowl-

edge, that APCs are necessary for both the generation and main-tenance of ocular-specific autoreactive CD4+ T cells during theimmunopathogenesis of experimental dry eye. Local depletion ofAPCs in the conjunctiva of mice exposed to DS inhibited thegeneration of autoreactive CD4+ T cells and blocked the ability toadoptively transfer disease to T cell-deficient nude recipient mice.Similarly, surgical removal of the CLN by cervical lymphade-nectomy inhibited CD4+ T cell activation and T cell-mediated dryeye disease. In addition, fully primed and targeted dry eye-specificCD4+ T cells did not readily accumulate and cause disease inAPC-depleted mice, implying that secondary activation by resi-dent ocular surface APCs is required for T cell maintenance andeffector function within the ocular surface tissues. These datademonstrate that ocular surface APCs are necessary for the initi-ation and development of dry eye in a mouse model of autore-active T cell-mediated experimental ALKC and support theparadigm that dry eye is a self-Ag–driven autoimmune disease.

Materials and MethodsDry eye mouse model of ALKC

Female C57BL/6 mice (6–8 wk old) were purchased from Taconic Farms(Oxnard, CA). Induction of dry eye using the mouse model of ALKC wasperformed as previously described (4, 18–20). In brief, micewere exposed toDS in perforated cages with constant airflow from fans positioned on bothsides and room humidity maintained at 30–35%. Injection of scopolaminehydrobromide (0.5 mg/0.2 ml; Sigma-Aldrich, St. Louis, MO) was admin-istered three to four times a day (three times a day: 08:00, 12:00, and 17:00 h;four times a day: 08:00, 12:00, 14:00, and 17:00 h) on alternating hind flanksto augment disease. DS was induced for 5 consecutive d, whereas controlmice were maintained in a nonstressed environment maintained at 50–75%relative humidity without exposure to forced air. All animal experimentswere approved by the institutional animal care and use committees atAllergan (Irvine, CA) and Baylor College of Medicine (Houston, TX). Allstudies adhered to the Association for Research in Vision and Ophthalmol-ogy statement for the Use of Animals in Ophthalmic and Vision Research.

Adoptive transfer

CD4+ T cells were purified from CLNs and spleen isolated from control orDS mice (5 d) and were adoptively transferred (5 3 106 cells suspended in100 ml sterile PBS) via i.p. injection into nude recipient mice as previouslydescribed (4, 21). Mice were sacrificed 3 d posttransfer, and tears andocular surface tissues were collected for analysis.

Preparation of dichloromethylene diphosphonate (clodronate)liposomes

Liposome-encapsulated clodronate has been widely used to depletephagocytic APCs; ingestion of clodronate liposomes leads to intracellularrelease and accumulation of clodronate, which induces apoptosis (22).Clodronate liposomes (C12MDP-LIP) were prepared as previously de-scribed (23). Briefly, 8 mg cholesterol (Sigma-Aldrich) and 86 mg phos-phatidylcholine (Sigma-Aldrich) were dissolved in 10 ml chloroform in around-bottom flask. After low-vacuum rotary evaporation at 40˚C, a thinfilm formed on the inner surface of the flask. Either 10 ml PBS, to makePBS containing liposomes, or 2.5 g C12MDP (clodronate; Sigma-Aldrich)dissolved in 10 ml PBS, to make C12MDP-LIP, was added to the flask.The film was then dispersed by gentle rotation for 18 h and sonicated for 3

min at room temperature to disperse the liposomes. Liposomes werewashed twice by centrifugation in PBS at 10,000 3 g for 20 min andresuspended in 10 ml PBS. C12MDP-LIP and PBS containing liposomeswere stored at 4˚C and used within 14 d of preparation. All liposomesranged in size from 100 nm to 3 mm in diameter. Each 100 ml C12MDP-LIP suspension contained 1.0 mg clodronate.

Depletion of APCs on the ocular surface usingliposome-encapsulated clodronate

Donor mice received subconjunctival injections of 3 ml (two injections/eye)on three separate occasions starting 4 d before exposure to DS and thenagain on days 21 and 3 of DS. Where indicated, nude recipient mice weretreated with clodronate or PBS-loaded liposomes on days 24 and21 priorto receiving DS-specific CD4+ T cells from dry eye mice according to thefollowing permutations: ocular dexter (OD), clodronate; ocular sinister(OS), PBS; OD, clodronate; OS, no treatment; OD, PBS; and OS, notreatment. Injections were placed within the subconjuctival space; theinjections alternated between temporal-nasal and inferior-superior place-ment corresponding to the 3/9 and 6/12 positions on a clock.

Cervical and axillary lymphadenectomy

Cervical or axillary lymph nodes were surgically excised from C57BL/6mice (n = 5/group) under general anesthesia with Avertin (2,2,2-tribromoethanol; Sigma-Aldrich) 7 d prior to induction of DS. Sham sur-gery was performed in a separate group of mice and consisted of surgicalincision and wound closure with staples. All mice were allowed to fullyrecover before being returned to their cage.

Cytokine and chemokine levels in tears during experimental ALKC

Relative levels of select cytokines and chemokines present in the tears wereevaluated over the course of experimental ALKC using Luminex analysis(Luminex, Austin, TX) as previously described (21). In brief, tears werecollected at various time points (e.g., 0 or 5 d post-DS); 1.5ml Beadlyte assaybuffer (Millipore, Bellerica, MA) was placed on each eye, and 1 ml/eye wascollected and combined with 8 ml Beadlyte buffer. Buffer and tear fluid werecollected by capillary action using a 1-ml volume glass capillary tube(Drummond Scientific, Broomall, PA) positioned in the tear meniscus of thelateral canthus. Samples were frozen at280˚C until the time of assay. Proteinlevels were assessed using the appropriate Millipore beads (Millipore) andanalyzed on a Luminex 100 or Luminex FLEXMAP 3D (Luminex).

T cell isolation and flow cytometry

Control and DSmicewere sacrificed at various time points (e.g., days 0, 1, 3,6, 7, and 10) following sustained exposure to DS. CLNs and spleen wereharvested, RBCs were lysed, and single-cell suspensions were generatedaccording to standard protocol. Cells were phenotyped using FITC-conjugated rat anti-mouse CD4, PE-conjugated rat anti-mouse CD69,and APC-conjugated rat anti-mouse MHC II, CD83, CD86, and CCR7. Inall cases, isotype-matched conjugated Abs were used as controls. Cells wereblocked with CD16/32 Ab for 10 min and then incubated with primary Absfor 20–40 min at 4˚C, washed, and analyzed using an FACStar flowcytometer (BD Biosciences, Mountain View, CA) and FlowJo software(Tree Star, Ashland, OR). Frequency data are presented as the percentageof positive cells within the gated population.

Histology

Whole eyes including lids were surgically excised, fixed in 10% formalin,and embedded in paraffin. Eight-micrometer sections were stained withH&E or periodic acid-Schiff (PAS) reagent to evaluate gross inflammatorycell infiltration within the LFU or conjunctival goblet cell density, re-spectively. Sections were viewed by light microscopy Eclipse E400(Nikon, Melville, NY) and photographed using a DS-Fi1 digital camera(Nikon). H&E sections were evaluated for inflammatory cell infiltrationwithin the ocular surface tissues on a scale from 0–3: 0, no inflammatorycell infiltration; 1, mild inflammatory cell infiltration; 2, moderate in-flammatory cell infiltration; and 3, intense inflammatory cell infiltration.Goblet cells were counted in the entire superior and inferior conjunctivastarting at the limbus over the entire length to the tarsal conjunctiva; dataare expressed as the average number of goblet cells per mouse.

Immunohistochemistry

Whole eyes including lids were surgically excised, embedded, and flashfrozen in optimal cutting temperature (OCT compound; VWR, Suwanee,GA). Eight-micrometer sagittal sections were cut with a cryostat (HM 500;Micron, Waldorf, Germany) and placed on glass slides that were stored at

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280˚C. Sections were stained for CD4+, CD11b+, CD11c+, and Iba1+ cellsusing the following mAbs: 1:40 rat anti-mouse CD4 (L3T4; clone H129.9),1:80 rat anti-mouse CD11b (clone M1/70), 1:80 hamster anti-mouse CD11c(BD Biosciences), and 1:500 rabbit anti-mouse Iba1 (Wako, Richmond VA).Polyclonal secondary Abs were biotinylated and included anti-rat IgG(1:50), polyclonal anti-rat IgG (1:50), polyclonal anti-hamster IgG (1:80),and anti-rabbit IgG (1:100) (BD Biosciences). Positive cells were visualizedusing the ABC Vectastain Kit in conjunction with NovaRED Substratekit (Vector Laboratories, Burlingame, CA). For control, sections were alsostained with the primary isotype Abs in conjunction with the biotinylatedsecondary Abs or appropriate secondary Abs alone. Three sections fromeach animal were examined and photographed with a microscope equippedwith a digital camera (Eclipse E400 with a DS-Fi1; Nikon). ConjunctivalCD4+ T cells, CD11b+ monocytes/macrophages, CD11c+ DCs, and Iba1+

macrophages were counted starting at the limbus to the tarsal conjunctivaand to a depth of 75 mm below the epithelial basement membrane; data wereexpressed as the average number of cells per conjunctiva.

Evans blue staining

NonstressedC57BL/6mice (n=3) receivedone 20ml bilateral subconjunctivalinjection of 1% of Evans blue (Sigma-Aldrich) prepared in physiologic saline.Evans blue dye has been usedwith success to trace lymphatic drainage in mice(24). After 30 min, mice were euthanized and photographed using a NikonDS-U2 color camera attached to a Nikon Stereoscope (SMZ1500; Nikon).

Statistics

Statistically significant differences (p # 0.05) were calculated by Student ttest or one-way ANOVAwith Bonferroni’s posttest using GraphPad Prismsoftware (GraphPad, La Jolla, CA).

ResultsAccumulation of mature DCs correlates with CD4+ T cellactivation within the regional CLNs

Previous studies demonstrated that exposure to DS induces pro-duction of proinflammatory cytokines and chemokines (e.g., IL-1b,TNF-a, CCL2, CCL3, CCL5, and CXCL10) (4, 20, 25). Acute-response cytokine production was observed early following expo-sure to DS; for example, TNF-a was significantly (*p # 0.05) el-evated in the tears of dry eye mice by 24 h (44.1 6 9.8 pg/ml)following sustained exposure to DS compared with naive mice(16.2 6 1.5 pg/ml). As DCs are potent APCs that function at theinterface of the innate and adaptive immune response, the kineticsof CD11c+ DC activation was evaluated within the regional drain-ing CLN. Acute cytokine production was associated with an in-creased percentage of CD11c+ DCs (1.95 6 0.3% versus 0.91 60.20%; p# 0.05) within the draining CLNs (Fig. 1A).Moreover, theCD11c+ DCs displayed elevated expression of activation/maturation markers, including MHC II+ (0.94 6 0.21% versus0.55 6 0.11%), CD83+ (1.52 6 0.36% versus 0.38 6 0.10%; p #0.05), CD86+ (1.326 0.20% versus 0.626 0.13%; p # 0.05), andCCR7+ (0.906 0.15% versus 0.486 0.14%) compared with naivemice (day 0) (Fig. 1A). Accumulation of mature DCs precededCD4+ T cell activation, indicated by an increased frequency ofCD4+ T cells bearing the early activation marker CD69+. A sig-nificant increase (p # 0.001) in the frequency of CD4+CD69+

T cells was observed as early as day 3 (15.1 6 1.0%) and peakedby 6 d DS (16.8 6 0.6%) as compared with control (day 0) mice(10.5 6 0.5%) (Fig. 1B). These data are consistent with the tem-poral framework indicative of an Ag-specific immune responseand support the hypothesis that the DS-induced DC activation/maturation mediates expansion of autoreactive lymphocytes withinthe regional lymph nodes during the immunopathogenesis of ex-perimental dry eye.

Liposome-encapsulated clodronate effectively depletes APCswithin the ocular surface tissues

To determine the functional role of APCs during the initiationand development of experimental dry eye, APCs present within the

ocular surface tissues were depleted using liposome-encapsulatedclodronate (22), which has been used successfully to deplete APCsin models of viral infection (26), corneal transplant (27, 28),and autoimmunity (29). Liposomes loaded with clodronate or PBSwere injected (3 ml 3 two injections/eye) into the subconjunctivalspace (alternating between opposing temporal-nasal and inferior-superior injections, corresponding to the 3/9 and 6/12 positionson a clock, respectively) 4 d before exposure to DS and then againon days 21 and 3 of DS. Mice treated with clodronate displayed asignificant (p # 0.001) reduction in the number of CD11b+

monocytes within the conjunctiva (12.5 6 2.1) compared withmice that received PBS liposomes as a control (45.6 6 3.9),without apparent toxicity to the surrounding epithelial cells(Fig. 2A, 2B). Among ocular surface monocytes, the numbersof CD11c+ DCs (1.5 6 0.9) were significantly (p # 0.01)decreased within the conjunctiva of clodronate-treated mice rel-ative to mice that received PBS liposomes (4.2 6 0.9) (Fig. 2C,2D); the total number of Iba+ macrophages followed a similartrend, with significantly (p # 0.01) less cells in clodronate-injected mice (0.5 6 0.2) compared with control (3.7 6 1.0)(data not shown). Therefore, subconjunctival instillation ofliposome-encapsulated clodronate is effective in reducing theoverall number of ocular surface APCs, which includes a broadpopulation of monocytes/macrophages (CD11b+/Iba1+) and DCs(CD11c+).

APC depletion mutes ocular surface inflammation in micefollowing exposure to DS

To determine if depletion of conjunctival APCs impacted theperipheral inflammatory response, proinflammatory cytokine/chemokine production, CD4+ T cell infiltration, and goblet cellnumbers were evaluated. Mice treated with liposome-encapsulatedclodronate showed a slight trend toward decreased levels of APC-derived cytokines (e.g., TNF-a, IL-6, and IL-12) (Fig. 3A) and

FIGURE 1. Temporal activation of DCs and CD4+ T cells during the

development of ALKC. Draining CLNs were isolated from dry eye mice

exposed to DS for various lengths of time and flow cytometry was used to

evaluate the kinetics of DC and CD4+ T cell activation within the draining

CLNs. Exposure toDS resulted in a higher frequency of CD11c+DCs bearing

activation/maturation markers within the CLN compared with naive mice

(day 0) (A), which correlated with activation of CD4+ T cells during initiation

and development of experimental dry eye (B). Data are represented as

average frequency6 SEM. Statistically significant values (*p# 0.05, **p#

0.01, ***p # 0.001) are indicated relative to naive (day 0) control mice.

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prominent T cell-derived cytokines (e.g., IL-2, IFN-g, IL-17, andIL-10) compared with PBS controls, although the change was notsignificant, indicating that the ocular surface tissues were able tomount an acute proinflammatory response despite reduced APCcounts. However, mice exposed to DS and treated with clodronatedisplayed a significant (p # 0.01) decrease in the number of in-filtrating CD4+ T cells (7.96 2.0) within the conjunctiva comparedwith dry eye mice treated with PBS (20.1 6 2.8) as a control (Fig.3B, 3C). Reduced CD4+ T cell infiltration correlated with preser-vation of ocular surface tissues, assessed by a significant increase(p # 0.001) in the numbers of goblet cells in clodronate-treatedmice (77.9 6 6.6) relative to PBS control (47.3 6 5.1), which wasnot significantly different compared with goblet cell numbersfound in naive mice (91.8 6 7.0) (Fig. 3E). These results demon-strate that the absence of a fully intact population of resident ocu-lar surface APCs significantly impacts T cell-mediated immuno-

pathogenesis in dry eye mice, without disrupting the DS-inducedacute proinflammatory cytokine response.

APC depletion attenuates the generation of DS-specificautoreactive T cells and the development of dry eye followingadoptive transfer to T cell-deficient mice

We hypothesized that the absence of a complete repertoire of APCswithin the ocular surface tissues would inhibit generation of ocularsurface-specific autoreactive CD4+ T cells within the regionaldraining CLNs. To this end, the pathogenic capacity of CD4+

T cells isolated from APC-depleted donor mice and exposed to DSfor 5 d was evaluated following adoptive transfer to T cell-deficient nude recipient mice. The current study shows thatCD4+ T cells are activated by 5 d of DS (Fig. 1B), and our pre-vious work demonstrated that CD4+ T cells from untreated DSmice were sufficient to mediate experimental dry eye in the nude

FIGURE 2. Local delivery of clodronate liposomes

depletes ocular surface APCs. A and C, Immunohis-

tochemistry (IHC) using anti-CD11b+ or anti-CD11c+

Abs indicated that subconjunctival injections of lipo-

some encapsulated clodronate reduced the overall

number of APCs within the conjunctiva, without ap-

parent toxicity to the surrounding epithelial cells.

Original magnification 3200. B and D, Quantification

showed a significant decrease in CD11b+ and CD11c+

cells (*p # 0.05, ***p # 0.001) compared with mice

that received PBS-loaded liposomes as a control.

Arrows indicate examples of CD11b+ and CD11c+

cells. Data are shown as average conjunctival cell

counts 6 SEM and representative of three independent

experiments, with an n = 5 to 6 mice/group.

FIGURE 3. Depletion of APCs decreases CD4+ T cell

accumulation within the ocular surface tissues of mice

exposed to DS. Proinflammatory cytokine/chemokine

production, CD4+ T cell infiltration, and goblet cell num-

bers were evaluated on the ocular surface between mice

exposed to DS and treated with either clodronate- or PBS-

loaded liposomes. A, Mice treated with clodronate dis-

played a slight, but nonsignificant, decrease in the levels of

select epithelial cell, APC-derived (e.g., TNF-a, IL-6, and

IL-12), and predominant T cell-derived cytokines (e.g., IL-

2, IFN-g, IL-17, and IL-10)within the tears comparedwith

PBS controls at 5 d of DS. By contrast, clodronate-treated

DS mice displayed a decrease in the number of infiltrating

conjunctival CD4+ T cells by IHC (B) that was statistically

significant compared with PBS controls (C). Decreased

accumulation of CD4+ T cells correlated with increased

numbers of PAS+ cells within the conjunctiva (D), in-

dicating goblet cells were preserved in APC-depletedmice

(E). A, Average concentration of protein (pg/ml) within

a 2 ml tear volume 6 SEM. B, CD4+ T cell staining in

the conjunctiva. Original magnification3200. C, Average

conjunctival (Conj.) CD4+ T cell counts6 SEM.D, PAS+

goblet cell staining in the conjunctiva. Original magnifi-

cation3200. E, Average conjunctival goblet cell counts6SEM. The data are representative of three independent

experiments, with an n = 5 to 6 mice/group. Statistically

significant values (**p # 0.01, ***p # 0.001, ****p #

0.0001) are indicated.

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recipients by 3 d posttransfer (4). By contrast, nude recipient micereceiving CD4+ T cells from clodronate-treated donor mice ex-posed to DS displayed a marked reduction ocular surface in-flammation compared with recipients of CD4+ T cells from DSmice treated with PBS as a control (Figs. 4, 5). For example, asignificant decrease (p # 0.05) in overall inflammatory cell in-filtration was observed within the ocular surface tissues (Fig. 4A),reported as a lower severity score in recipients of CD4+ T cellsfrom clodronate-treated donor mice (0.3 6 0.1) compared withPBS controls (1.2 6 0.4) (Fig. 4B). Moreover, CD4+ T cellsisolated from clodronate-treated donor mice displayed a reducedcapacity to accumulate within the ocular surface tissues of nuderecipients (9.8 6 2.3) relative to PBS controls (58.3 6 12.6) (Fig.4C, 4D). The absence of a CD4+ T cell accumulation correlatedwith preservation of ocular surface tissues (Fig. 4E); goblet cellcounts were significantly (p # 0.01) higher in the conjunctiva ofnude mice receiving CD4+ T cells from clodronate-treated donormice (91.5 6 0.4) compared with controls (42.0 6 9.5) (Fig. 4F).Muted inflammatory cell infiltration was also associated witha striking reduction in the proinflammatory cytokine/chemokineresponse, indicated by decreased IL-1b, TNF-a, IL-2, IL-6,CCL5, CXCL10, IFN-g, and IL-17 levels within the tears ofnude recipients of CD4+ T cells isolated from clodronate-treateddonor mice (Fig. 5). Moreover, there was also a decrease in IL-10

(1126 18 pg/ml) in nude mice receiving CD4+ T cells from APC-depleted mice exposed to DS compared with those receiving cellsfrom PBS-liposome–treated controls (282 6 86 pg/ml), suggest-ing that regulatory T cells derived from donor mice were not in-volved in dampening the pathogenic CD4+ T cell response. Takentogether, these results suggest that the absence of APCs in miceexposed to DS inhibits generation of ocular-specific autoreactiveCD4+ T cells, which otherwise readily traffic to the ocular surfacetissues of nude recipient mice and mediate robust pathologicalchanges resembling dry eye disease.

Local depletion of APCs prevents accumulation of autoreactiveT cells within the ocular surface tissues

To determine if peripheral maintenance of fully primed and tar-geted DS-specific CD4+ T cells requires secondary activation byresident ocular surface APCs, nude recipient mice were treatedwith clodronate on days 24 and 21 prior to receiving pathogenicCD4+ T cells from dry eye mice. Subconjunctival injection ofclodronate-loaded liposomes in the OD eye of nude recipient micesignificantly reduced (p # 0.001) the average number of DS-specific CD4+ T cells (4.4 6 1.4) accumulating within the con-junctiva compared with the contralateral OS eye of the same micereceiving PBS liposomes as an internal control (38.1 6 7.6) (Fig.6A). A similar significant (p # 0.001) decrease in CD4+ T cell

FIGURE 4. APC depletion in mice exposed to DS attenuates the generation of autoreactive CD4+ T cells and blocks the ability to adoptively transfer T

cell-mediated disease to nude recipient mice. CD4+ T cells were isolated from the CLNs and spleen of clodronate- or PBS-treated mice exposed to DS and

adoptively transferred to nude recipient mice to determine if the absence of a full repertoire of APCs within the ocular surface tissues inhibits generation of

ocular surface-specific autoreactive CD4+ T cells. H&E staining (A) showed a significant decrease in overall inflammatory cell infiltration within the ocular

surface tissues of nude recipients of CD4+ T cells from clodronate-treated donor mice compared with PBS controls (3 d postadoptive transfer) (B).

Furthermore, IHC (C) confirmed that CD4+ T cells isolated from clodronate-treated donor mice did not readily accumulate within the conjunctiva, as there

was only trace CD4+ staining, which accounted for a significant decrease in CD4+ T cells (D) and protection from the loss of PAS-positive goblet cells (E,

F). A, H&E staining. Original magnification3200. B, Overall inflammatory score6 SEM (scale 0–3). C, CD4+ T cell staining in the conjunctiva. Original

magnification3200. D, Average conjunctival (Conj.) CD4+ T cell counts6 SEM. E, PAS+ goblet cell staining in the conjunctiva. Original magnification3200.

F, Average conjunctival goblet cell counts 6 SEM. The data are representative of three independent experiments, with n = 5 to 6 mice/group. Statistically

significant values (*p # 0.05, **p # 0.01) are indicated relative to nude recipients of CD4+ T cells from PBS liposome-treated mice.

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accumulation was also observed when clodronate-treated OD eyes(7.1 6 0.9) were compared with untreated OS eyes (36.9 6 7.1)(Fig. 6B). By contrast, there was no difference between thenumber of infiltrating CD4+ T cells between control PBS-treatedOD eyes (25.1 6 3.2) and untreated OS eyes (30.0 6 5.3), con-firming that there is not a bias between right and left eyes withrespect to T cell-mediated inflammation (Fig. 6C). Attenuatedaccumulation of DS-specific CD4+ T cells within the ocular sur-face tissues of the OD eyes from clodronate-liposome–treatednude recipient mice correlated with preservation of conjunctivalgoblet cells indicated by increased numbers compared with PBS-liposome–treated (49.7 6 6.8 versus 33.8 6 7.6) or untreated(44.8 6 7.0 versus 18.8 6 3.5; p # 0.01) internal OS control eyes(Fig. 7). Average goblet cell counts for healthy untouched nudemouse eyes were 80.1 6 9.7, indicating that APC depletion innude recipient mice preserves goblet cells, but not completely tonormal levels. Collectively, these results suggest that DS-specificCD4+ T cells infiltrating the ocular surface tissues need to re-encounter their cognate APCs for peripheral maintenance andeffector function.

Lymphadenectomized mice do not develop T cell-mediatedexperimental dry eye

To further evaluate the role of APC-dependent autoreactive CD4+

T cell activation during the immunopathogenesis of dry eye, a bi-lateral surgical cervical lymphadenectomy (CLN SX) was per-formed 7 d prior to induction of DS. A separate group of controlmice received either sham surgery (Sham SX) or axillary lympha-denectomy (AX SX). In cervical lymphadenectomized mice ex-posed to DS, there were significantly (p # 0.05) less CD4+ T cells

(68.6 6 6.6) in the conjunctiva compared with Sham SX controls(90.16 6.7) (Fig. 8A, 8B). The change correlated with preservationof goblet cell density in cervical lymphadenectomizedmice (79.361.5), which was significantly (p , 0.001) higher relative to ShamSX mice (73.9 6 2.1) and comparable to untreated naive mousecontrol levels (80.6 6 2.9) (Fig. 8C). By contrast, removal of theaxillary lymph nodes did not result in a significant decrease inCD4+ T cells or increase in goblet cell density (Fig. 8B, 8C).We have previously shown that both CLN-derived or splenic

CD4+ T cells from dry eye mice were sufficient to cause dry eyedisease in T cell-deficient nude recipient mice (4). To establishif removal of the CLN impacted CD4+ T cell activation in thespleen, CD4+ T cells from all experimental groups were adop-tively transferred to T cell-deficient mice, and CD4+ T cell infil-tration and PAS+ goblet cell density was assessed at 3 d post-transfer. As noted in the donor mice, nude recipients of CD4+

T cells from DS mice with CLN excision also displayed signifi-cantly (p # 0.001) less infiltrating CD4+ T cells and retention oflive goblet cells (Fig. 9A–C). However, excision of axillary nodesin donor mice exposed to DS also influenced splenic CD4+ T cellactivation; there was significantly (p # 0.05) less infiltration ofdonor-derived CD4+ T cells following adoptive transfer in nuderecipients, which also correlated with a significant (p # 0.001)increase in goblet cell counts (Fig. 9A, 9B), suggesting that theaxillary nodes may also support a degree of Ag and/or APClymphatic drainage from the ocular surface.To resolve and support the finding that removal of axillary lymph

nodes in donor mice exposed to DS resulted in reduced CD4+ T cellinfiltration in nude recipient mice, subconjunctival administrationof 1% Evans blue was used to trace lymphatic drainage from the

FIGURE 5. Depletion ofAPCs inmice exposed

to DS inhibits the CD4+ T cell-mediated proin-

flammatory cytokine response following adoptive

transfer to nude recipient mice. CD4+ T cells iso-

lated from APC-depleted donor mice exposed to

DS did not induce robust production of select acute

response proinflammatory cytokines IL-1b (A),

TNF-a (B), IL-6 (C); chemokines CCL5 (D) and

CXCL10 (E); or predominant T cell-derived

cytokines IL-2 (F), IFN-g (G), and IL-17 (H)

compared with mice receiving CD4+ T cells from

DS mice treated with PBS liposomes at 3 d post-

adoptive transfer. Data are shown as average

concentration of protein (pg/ml) within a 2 ml tear

volume 6 SEM and representative of three in-

dependent experiments, with an n = 5 to 6 mice/

group. Statistically significant values (*p # 0.05)

are indicated relative to tear values of nude mice

receiving CD4+ T cells from donor mice exposed

to DS and treated with PBS liposomes as a control.

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eye (Fig. 10). As anticipated, there was intense drainage of the dyeinto the CLN (Fig. 10C, 10H, 1). However, the axillary nodesshowed faint staining (Fig. 10H, 2), indicating that some level oflymphatic drainage exists from the ocular surface to the axillarynodes. In addition, the face and fore and hind paws also accu-mulated dye compared with naive, untreated mice (Fig. 10D–G).

DiscussionActivation of the immune response is tightly regulated to protect theocular surface from pathogenic challenge while preserving tissueand maintaining tolerance to self-Ags and commensal flora. Aber-rant activation of the innate and adaptive response may breachtolerance and result in autoimmunity to self-Ags localized to theocular surface tissues, leading to chronic inflammation and tissuedamage (3). Prior work showed exposure to desiccating and/orosmotic stress rapidly activates MAPK pathways and stimulatesresident ocular surface cells to secrete proinflammatory cytokines

(4, 20, 30, 31), which precedes infiltration of CD4+ T cells andocular surface pathology (4). The significance of CD4+ T cells indisease was demonstrated when adoptive transfer of DS-specificCD4+ T cells isolated from the CLN and/or spleen of experimen-tal dry eye mice was shown to be sufficient to mediate disease inT cell-deficient nude recipient mice. Collectively, our previousfindings prompted us to hypothesize that APCs provide the funda-mental link between the DS-induced innate response and a self-Ag–driven autoreactive T cell response to ocular surface tissues duringthe immunopathogenesis of dry eye.The current study supports and expands on our earlier work to

demonstrate that APCs are necessary for the initiation and de-velopment of dry eye in the mouse model of ALKC. CD11c+ DCshave long been known to play a predominant role activation ofnaive T cells within the regional lymphoid organs, and CD11c+

CD11b+MHC II+ cells have previously been shown to traffic fromthe ocular surface to the draining CLNs during inflammation (13).In the context of experimental dry eye, exposure to DS resultedin accumulation of mature CD11c+ DCs bearing costimulatorymolecules within the draining CLNs by 24 h that correlated withsubsequent activation of CD4+ T cells, with the kinetics indicativeof an Ag-driven T cell response. Furthermore, this report estab-lishes that subconjunctival injection of liposome-encapsulatedclodronate is a sufficient method to diminish the population ofresident APCs, including both monocytes/macrophages (CD11b+/Iba1+) and DCs (CD11c+) within the ocular surface tissues. De-pletion of ocular surface APCs reduced the number of infiltratingCD4+ T cells and preserved goblet cells within the conjunctiva. Acompensatory increase in anti-inflammatory cytokines (e.g., IL-10) was not observed in clodronate-treated mice, suggesting theabsence of APCs, and not regulatory T cells, is directly re-sponsible for attenuating pathogenic T cells. In addition, there areseveral studies demonstrating that clodronate liposomes do notdirectly affect T cell function and proliferation in vitro and in vivo(32–34), further supporting the requirement for Ag presentationduring the development of experimental dry eye. The observationthat cervical lymphadenectomized mice exposed to DS also dis-played significantly less infiltrating CD4+ T cells and highergoblet cell density corroborates the hypothesis that activation ofautoreactive CD4+ T cells occurs in the draining CLNs via cell-to-cell contact with ocular surface-derived APCs bearing self-Ag.The current findings are also consistent with similar studies us-ing other inflammatory models with a high degree of peripheral

FIGURE 6. APCs are necessary for maintenance of autoreactive T cells

within the ocular surface tissues following adoptive transfer of dry eye-

specific autoreactive CD4+ T cells. To determine if peripheral maintenance

of fully primed and targeted DS-specific CD4+ T cells requires secondary

activation by resident ocular surface APCs, DS-specific CD4+ T cells were

adoptively transferred to nude recipients treated with clodronate- or PBS-

loaded liposomes according to the following permutations: OD, clodro-

nate; OS, PBS; OD, clodronate; OS, no treatment; OD, PBS; and OS, no

treatment. Subconjunctival injection of clodronate-loaded liposomes in the

OD eye of nude recipient mice resulted in a dramatic reduction in the

average number of DS-specific CD4+ T cells compared with the contra-

lateral internal control OS eye treated with PBS liposomes (A), which was

similar when clodronate-treated OD eyes were compared with untreated

OS eyes at 3 d postadoptive transfer (B). By contrast, there was no dif-

ference between the number of infiltrating CD4+ T cells between control

PBS-treated OD eyes and untreated OS eyes (C). Data are shown as av-

erage conjunctival CD4+ T cell counts 6 SEM and representative of two

independent experiments, with an n = 5 to 6 mice/group. Statistically

significant values (***p # 0.001) are indicated relative to internal OS

control eyes that were either untreated or treated with PBS liposomes.

FIGURE 7. The absence of DS-specific CD4+ T cells within the ocular

surface tissues of APC-depleted nude recipient mice correlates with

preservation of conjunctival goblet cells. Adoptive transfer of pathogenic

DS-specific T cells to nude recipient mice treated with liposome-encap-

sulated clodronate did not result in a significant loss in conjunctival goblet

cells compared with contralateral internal control eyes, which were either

treated with PBS-loaded liposomes or left untreated. Data are shown as

average conjunctival goblet cell counts 6 SEM quantified at 3 d post-

adoptive transfer. The data are representative of two independent experi-

ments with an n = 5 to 6 mice/group. Statistically significant values (**p#

0.01) are indicated relative to OS internal control eyes that were either

untreated or treated with PBS liposomes.

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lymphatic drainage to the CLNs. For example, surgical excision ofthe CLNs delayed the onset and reduced clinical disease in theneuroinflammatory model of experimental autoimmune encepha-lomyelitis (35, 36), and cervical lymphadenectomy dramaticallyreversed corneal allograft rejection to a survival rate of 92%compared with 0% survival noted in the eyes of mice with intactCLNs (37).The absence of APCs within the ocular surface tissues of

clodronate-treated mice did not dramatically affect the acuteproinflammatory cytokine response to DS. These data have severalimplications in the face of reduced CD4+ T cell infiltration andgoblet cell preservation in APC-depleted mice exposed to DS.Firstly, it supports the notion that the dry eye is an Ag-drivenautoimmune-mediated disease and not merely the result of non-specific bystander T cell activation in response to a cytokinestorm, described in some cases of bacterial and viral challenge(38–40). In addition, these results imply that alterations in che-mokine ligand levels do not account for the marked reduction inT cell accumulation into the conjunctiva observed in clodronate-treated mice, as there was no difference in CCL5 or CXCL10 tearlevels. As reported previously, it is likely that epithelial cells arealso prominent cytokine/chemokine source within the ocular sur-face tissues in response to DS and/or osmotic stress (4, 20, 30, 31).So, even in the face of attenuated CD4+ T cell activation, thestress-induced proinflammatory cytokine response may proceedas long as the mice are exposed to the desiccating environment.

Along these lines, the mild and nonsignificant decrease in prom-inent Th1- and Th17-derived cytokines (e.g., IL-2, IFN-g, IL-17)in mice exposed to DS suggests other resident and/or infiltratingcell types are a reservoir for IFN-g (e.g., NK cells) (41) and IL-17(e.g., gd T cells) (42) and may contribute to steady-state levelsof tear cytokines during the DS-induced inflammatory response.We have previously shown that Th1 CD4+ T cell infiltration andgoblet cell preservation are inversely proportional during the ex-perimental dry eye (43), and although a significant decrease inIFN-g was not observed in clodronate-treated mice, it is possiblethat IFN-g levels are below the threshold required to compromisegoblet cell integrity after 5 d of DS, or perhaps IFN-g derivedfrom infiltrating CD4+ T cells has a greater impact on goblet cellsthan, for instance, NK cell-derived IFN-g (41). The 5-d time pointwas chosen because this is the period when maximal CD4+ T cellactivation was observed within the draining CLNs; therefore, it isalso possible that Th1- and Th17-derived cytokines may continueto increase on the ocular surface of dry eye mice, further aug-menting the difference between PBS and clodronate-treated miceat later time points.Adoptive transfer studies were used to confirm an absolute role

of APCs in priming and targeting autoreactive CD4+ T cells to theocular surface during experimental dry eye. APC-depleted donormice exposed to DS were unable to transfer CD4+ T cell-mediateddisease to athymic nude recipient mice, whereas CD4+ T cellsfrom PBS-treated control mice maintained pathogenicity and

FIGURE 8. Cervical lymphadenectomized mice do

not develop full-blown T cell-mediated experimental

dry eye. To evaluate the role of the draining CLNs in

APC-dependent autoreactive CD4+ T cell activation,

CLN SX was performed before exposing mice to DS. A

separate group of DS mice received either Sham SX or

AX SX. CLN excision (A) resulted in significantly less

CD4+ T cells in the conjunctiva compared with Sham

SX (B); AX SX did not result in a significant decrease

in CD4+ T cells. C, Goblet cell density was also pre-

served in CLN SX mice, but was not maintained in AX

SX controls. CD4+ T cell staining in the conjunctiva

(A); average conjunctival CD4+ T cell counts 6 SEM

(B); average conjunctival goblet cell counts 6 SEM

(C), quantified at 3 d postadoptive transfer. Original

magnification 3400. The data are representative of

two independent experiments with an n = 5 mice/

group. Statistically significant values (*p # 0.05,

***p # 0.001) are noted.

FIGURE 9. Intact CLNs are required for pathoge-

nicity of DS-specific splenic CD4+ T cells. To further

determine if CLN removal impacted splenic CD4+ T cell

activation, CD4+ T cells from naive mice or those ex-

posed to DS with Sham SX, AX SX, or CLN SX were

adoptively transferred to T cell-deficient mice. Nude

recipients of CD4+ T cells from DS mice with CLN SX

(A) displayed significantly decreased CD4+ T cell in-

filtration (B) and increased the number of goblet cells

(C). A–C, A similar, but intermediate, response was also

observed in nude recipient mice that received CD4+

T cells from DS donor mice with AX SX. CD4+ T cell

staining in the conjunctiva (A); average conjunctival

CD4+ T cell counts6SEM (B); and average conjunctival

goblet cell counts 6 SEM (C), quantified at 3 d post-

adoptive transfer. Original magnification3400. The data

are representative of two independent experiments with

n = 5 mice/group. Statistically significant values (*p #

0.05, **p # 0.01, ***p # 0.001) are indicated.

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caused full-blown dry eye disease. It is interesting to note thatexcision of axillary nodes did not influence CD4+ T cell counts orgoblet cell density in donor mice, but rather exerted moderatedisease when the CD4+ T cells were transferred to nude recipientmice. During the development of experimental dry eye, it ispossible that ocular surface Ags gain access to other lymph nodes,for instance: 1) by soluble transport of Ags into the conduit systemof other lymph nodes, where resident DCs may gain access forpresentation to Ag-specific T cells (44); or 2) DCs activated withinthe ocular surface tissues may transport the Ag to other lymphnodes, or to other DCs, to fuel the autoimmune response (45). Asnoted, DS induces lymphoangiogenesis on the ocular surface,which likely facilitates APC and/or Ag drainage during experi-mental dry eye (17). Indeed, subconjunctival administration ofEvans blue showed intense lymphatic drainage from the ocularsurface to the CLNs, but also mild drainage to axillary nodes,supporting a role for other lymph nodes in the priming of ocularsurface-specific CD4+ T cells. However, the exact mechanisms bywhich axillary lymph nodes influence activation of DS-specificCD4+ T cells in the spleen is not known.This study also suggests that secondary activation or triggering

of DS-specific CD4+ T cells by APCs residing locally within theocular surface is required for peripheral maintenance and effectorfunction. APC-depleted nude recipient mice showed a markedreduction in number of pathogenic DS-specific CD4+ T cells ac-cumulating within the conjunctiva compared with internal controleyes of the same mice, which also correlated with decreased pa-thology. These results suggest that DS-specific CD4+ T cells in-filtrating the ocular surface tissues need to re-encounter theircognate APCs, including DCs and/or macrophages, for peripheralmaintenance and effector function. In support, Goyal et al. (46)used a topical CCR2 antagonist to demonstrate that infiltratingCD11b+ cells were required for T cell-mediated disease in a sim-ilar mouse model of DS-induced dry eye. The role of secondaryT cell activation at peripheral inflammatory sites is further sup-ported in other animal models. Resident macrophages isolatedfrom the CNS were capable of stimulating MBP-reactive T cellsex vivo (47). During the immunopathogenesis of experimentalautoimmune encephalomyelitis, infiltrating encephalitogenic Thcells required restimulation by their cognate APCs to identify theirtarget (48, 49), and whereas activated OVA-specific or purifiedprotein derivative of tuberculin-specific T cell lines also migrated

into CNS, only activated MBP-specific T cells were maintainedwithin in the spinal cord and caused neurologic impairment, in-cluding decreased motor function and partial to complete hindlimb paralysis (50). By and large, compared with the extensivebody of evidence defining the interactions between naive Th cellsand APCs (i.e., DCs within the secondary lymphoid organs), thereis still a limited understanding of the peripheral APC requirementsof fully primed, activated Th cells.These data establish an essential role of APCs in initiating

and maintaining chronic activation of CD4+ T cells during ex-perimental dry eye and support the paradigm that it is a self-Ag–driven autoimmune-based inflammatory disease. Regarding theautoantigen itself, little is known. Type 3 muscarinic acetylcholinereceptor was proposed based on the presence of autoreactive se-rum from dry eye patients (51), and excessive acetylcholine re-ceptor stimulation was demonstrated to expose cryptic type 3muscarinic acetylcholine receptor epitopes to be sampled by APCs(52). Putative autoantigens were also identified from the kalli-krein family, namely Klk13 and Klk1b22 (53, 54). Nonetheless,identifying the specific initiating autoantigen is fundamentallydifficult and still remains elusive among the vast spectrum oforgan-specific autoimmune diseases. Studies focused on definingthe influence of inflammatory stimuli on activation of differentAPC subsets and how these subsets direct activation and differ-entiation of pathogenic lymphocytes will be valuable in devel-oping a greater understanding of the complex mechanisms orches-trating the immunopathogenesis of dry eye disease.

DisclosuresC.S.S., K.F.S., L.A.W., and M.E.S. are employees of Allergan, Inc.

J.Y.N. and S.C.P. are consultants of Allergan, Inc.

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