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Activated leukocyte cell adhesion molecule (ALCAM/CD166) affects immune responses in ovalbumin-induced food allergy Yun Seon Kim Department of Medical Science The Graduate School, Yonsei University

Activated leukocyte cell adhesion molecule (ALCAM/CD166) affects immune responses in ovalbumin-induced food allergy Directed by Professor Myung Hyun Sohn The Master s Thesis submitted to the Department of Medical Science, the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Master of Medical Science Yun Seon Kim December 2016

This certifies that the Master s Thesis of Yun Seon Kim is approved. Thesis Supervisor : Myung Hyun Sohn Thesis Committee Member#1 : Kyung Won Kim Thesis Committee Member#2 : Hyoung-Pyo Kim The Graduate School Yonsei Unuversity December 2016

감사의글 설렘과두려움을안고시작했던약 3 년간의석사과정을마무리하게되었습니다. 학위논문이나오기까지도움주신많은분들께감사의인사를드리고자합니다. 먼저지도교수님이신손명현교수님께감사드립니다. 실수가있어도넓은마음으로받아주시고가르쳐주신덕분에무사히졸업할수있었습니다. 항상관심가져주시고, 능동적으로공부할수있도록많은깨우침을주신김규언교수님, 바쁘신가운데에도제학위논문을심사해주시고따뜻한조언을해주신김경원교수님과김형표교수님께도감사드립니다. 연구나인생에대한아낌없는조언으로저를이끌어주신이경은선생님과조금더좋은결과를낼수있도록조언을해주신홍정연선생님, 제연구주제에대해같이고민해주시고연구실생활에잘적응할수있도록도와주신김미나선생님께감사드립니다. 처음실험을하는저에게많은실험을알려주신허원일선생님과연구의동반자로서, 때로는선의의경쟁자로서, 또친구로서항상옆을지켜준미선언니에게도감사한마음을전합니다. 떨어져있어서자주만나지는못해도전화하면반갑게받아준경래, 항상웃게해주고언니처럼다독여주는수진이, 늘가까이있다고느껴지는오랜친구다희에게도고마운마음전합니다. 고민상담도해주고항상신나게놀아주는희경이, 학부때만나평생철없이놀것같았지만어느새대학원졸업도하고, 열심히사회생활하고 있는 보라, 영진오빠, 지수오빠, 호균오빠, 현민오빠, 혜수, 늘

듬직하게곁을지켜주며안좋은일이생기면위로해주고, 좋은일이생기면같이기뻐해주고응원해준용범이오빠한테도고마운마음을전합니다. 연구실생활에대해공감해주며도움을준수언언니, 깊은대화로새로운깨달음을주는여경언니, 소윤이, 송희에게고마운마음전합니다. 제가잘되도록하늘에서지켜보셨을할아버지, 할머니, 큰고모부, 둘째고모, 그리고잘지내는지늘신경써주시는큰고모, 큰아빠, 큰엄마, 둘째고모부, 막내고모, 막내고모부, 둘째큰아빠, 둘째큰엄마와사촌언니, 오빠, 형부, 새언니들께감사드리고, 타지생활에적응하고마음편히학교다닐수있도록뒷바라지해주신외할아버지, 외할머니와학위생활에관심가지고응원해주신 큰이모, 큰이모부, 작은이모, 작은이모부, 외삼촌, 외숙모와 동생들에게도감사의말씀전합니다. 사촌 마지막으로제학위생활이잘끝나도록전전긍긍하시며격려와위로해주시고후에더멋진사람이되길바라며늘기도해주시는부모님과, 학위생활에바빠언니로서신경써주지못했지만이해해주고오히려저를챙겨준윤지에게도감사합니다. 가족들의뒷받침이없었더라면불가능했을것입니다. 사랑합니다. 2016 년 12 월 김윤선

TABLE OF CONTESTS ABSTRACT 1 Ⅰ. INTRODUCTION 4 Ⅱ. MATERIALS AND METHODS 7 1. Subjects 7 2. Animals 8 3. Antibodies and reagents 8 4. Experimental food allergy 8 5. Measurement of parameters of food allergy 9 6. Isolation and culture of immune cells 9 7. Cell counting kit 8 10 8. Determination of the concentrations of cytokines in splenocyte cultured media 11 9. Flow cytometric analysis 11 10. Measurement of cytokines by real-time PCR 12 11. Histological evaluation 12 12. Electron microscopy 13 13. Quantification of the serum concentrations of total IgE and OVAspecific IgE (ELISA) 14 14. Statistical analysis 14 Ⅲ. RESULTS 15 1. Increased ALCAM levels in serum of OVA-challenged food allergy model 15

2. Attenuated immune responses in ALCAM -/- mice model 19 3. Diminished T cell responses in ALCAM -/- mice 24 4. Increased levels of ALCAM in serum of children with food allergy 31 Ⅳ. DISCUSSION 34 Ⅴ. CONCLUSION 38 REFERENCES 39 ABSTRACT (IN KOREAN) 45

LIST OF FIGURES Figure1. Increased levels of ALCAM in serum of food allergyinduced wild type mice with ovalbumin 16 Figure2. Attenuated immune responses in food allergy-induced ALCAM deficient mice with ovalbumin 20 Figure3. Decreased T cell proliferation in food allergy-induced ALCAM deficient mice with ovalbumin 26 Figure4. Diminished T cell population and T cell activation in food allergy-induced ALCAM deficient mice with ovalbumin 28 Figure5. Enhanced levels of ALCAM in serum of children with food allergy 32

LIST OF TABLES Table1. Characteristics of subjects 33

ABSTRACT Activated leukocyte cell adhesion molecule (ALCAM/CD166) affects immune responses in ovalbumin-induced food allergy Yun Seon Kim Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Myung Hyun Sohn) Food allergy is an abnormal immune response resulting from failing to establish oral tolerance and considered one of the worldwide major health problems. Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of immunoglobulin superfamily transmembrane glycoproteins and express on surface of antigen presenting cells (APCs), especially dendritic cells (DCs). CD6 is known for co-stimulatory molecule and binds with ALCAM. There are many studies that long term interaction of CD6 and ALCAM affects immune response. However, there are currently no studies identified functions of ALCAM in food allergy. So, 1

this study aimed to identify the functions of ALCAM in OVA-induced food allergy. Female BALB/c wild type (WT) mice and ALCAM mutant (ALCAM -/- ) mice were intraperitoneally sensitized and orally fed with ovalbumin (OVA). After last challenge, mice were assessed parameters of experimental food allergy, including clinical and diarrhea scores, and core temperature. Mice were sacrificed and analyzed systemic immune responses such as expression of T H2 cytokines, levels of serum total IgE and OVA-specific IgE, injury scores of H&E stained intestine, and T cell responses. Food allergy-induced WT mice showed decreased expression of ALCAM mrna in small intestine and increased levels of ALCAM in serum. Clinical and diarrhea scores, serum total IgE and OVA-specific IgE levels, mrna expressions of T H2 cytokines (IL-4, IL-5, IL-13), and injury scores of H&E stained small intestine were enhanced in OVA-challenged WT mice against control mice. In contrast, these parameters and immune responses of food allergy were attenuated in OVA-challenged ALCAM -/- mice. T cell proliferation of total cells from spleen and mesenteric lymph node (mln), and T H2 cytokine levels of splenocytes cultured media were diminished in OVA-challenged ALCAM -/- mice. Furthermore, proportion of CD3 + CD4 + T cell population and activated T cell of spleen, mln, small intestine were dropped in OVA-challenged ALCAM -/- mice. Lastly, levels of serum ALCAM increased in children with food allergy against healthy control subjects. 2

In conclusion, this study suggests that ALCAM affects immune response in experimental food allergy. That is, ALCAM regulates allergic disease and affects immune responses by alternation of T cell activation and T H2 response in OVA-induced food allergy. Key words : activated leukocyte cell adhesion molecule, ALCAM, CD166, food allergy, food allergy murine model 3

Activated leukocyte cell adhesion molecule (ALCAM/CD166) affects immune responses in ovalbumin-induced food allergy Yun Seon Kim Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Myung Hyun Sohn) Ⅰ. INTRODUCTION The prevalence of food allergy has significantly increased and considered as a lethal disease. 1-3 Although food allergy is a serious public health problem, mechanism that systemic allergic immune response to food allergens in the gastrointestinal tract remains limited. 4,5 Gastrointestinal tract, the main organ of food allergy, is the largest immunologic barrier in the body; it constantly encounters microbiota antigens and dietary proteins. Therefore, the balance between tolerance and allergic sensitization is crucial for induction of food allergy. 6-10 When maintenance of the balance is failed, orally ingested food allergens are permeated 4

and processed as small fragment and then presented to T cells by antigen presenting cells (APCs). 11 These interactions lead to T cell proliferation and T H2 cytokine secretion such as interleukin (IL)-4, IL-5, and IL-13. These T H2 cytokines can promote the production of food allergen-specific immunoglobulin (Ig) E from B cells. IgE antibodies bind to FcεRⅠ on the surface of mast cells and induce degranulation of mast cells. Finally, inflammatory mediators from mast cells, such as histamine, prostaglandin, and leukotrienes and immune cells present symptoms of food allergy. 12,13 It is important that APCs present allergens to T cells in the immune response. A synapse is a specialized structure that forms when the plasma membranes of two cells come into close apposition to transmit signals. Formation of synapse between T cells and APCs called immunological synapses and maintenance of this interaction is crucial for T cell activation. And there are many co-stimulatory molecules on immune cells that engage in prolonged maintenance of immunological synapse. 14 Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the immunoglobulin superfamily (IgSF). 15 There are many studies that ALCAM is presented on APCs, especially dendritic cells (DCs) and has five immunoglobulin (Ig) domains, include two membrane-distal variable(v)-type and three membrane-proximal constant(c 2)-type Ig folds. 15,16 And ALCAM is involved in maintenance of tissue architecture, immune response and tumor progression. 17 5

Previous studies demonstrated that expression of ALCAM is correlated with aggressive disease in a variety of cancers, including melanoma, prostate, breast, ovarian, esophageal, bladder, and intestinal cancers and used as prognostic marker in human. 18-24 ALCAM engages in homophilic interactions with ALCAM or heterophilic interactions with CD6 on T cells in immunological synapse. 25 The heterophilic interactions are stronger than homophilic interactions. CD6, ligand for ALCAM, belongs to the scavenger receptor cysteine-rich protein superfamily (SRCRSF) and plays a role in T cell activation as an accessory molecule in immunological synapse. 26-28 Interaction of ALCAM and CD6 is the first characterized the interaction of IgSF and SRCRSF and they play a role during T cell activation as co-stimulatory molecules. 15,25,29 ALCAM mediates immune responses as co-stimulatory molecule by the long-term interactions with CD6, 29 and plays a role in pathogenesis of disease, such as cancer. However, there are few studies that effects of ALCAM in allergic disease, which is related to immune response. Therefore, this study aimed to identify the effects of ALCAM in allergic disease, especially food allergy. 6

Ⅱ. MATERIALS AND METHODS 1. Subjects A total of 143 children among who visited the Severance Children s hospital for work-up or treatment of food allergy or routine health check-up between April, 2012 and September, 2014 were enrolled in this study. Food allergy was defined according to the guidelines of the National Institute of Allergy and Infectious Diseases Expert panel report on 2010. 30 Thorough medical history including previous adverse food reactions and physical examination were performed at the first visit. Patients who diagnosed as other allergic diseases such as atopic dermatitis, chronic urticaria, allergic rhinitis or asthma were excluded considering potential effect on allergic sensitization. Blood sampling were done on the same day after obtaining consent. After blood sample was taken, serum samples were stored at -20 and we measured the serum levels of total IgE and specific IgE levels by the Pharmacia CAP assay (Uppsala, Sweden). A specific IgE test was performed for individual suspicious food allergens and if not specified, for five most common food allergens in Korea, including cow milk, egg white, wheat, peanut, soybean, and peanut. Serum ALCAM levels were assessed with a commercially available enzyme-linked immunosorbent assay kit (R&D systems, Minneapolis, USA), according to the manufacturer s instructions. This study was approved by the Institutional Review Board of Severance Hospital (protocol no. 4-2004-0036). Written informed consent was obtained from the participants and their parents. 7

2. Animals Four- to six-wk-old female BALB/c mice were purchased from OrientBio Inc. (Kyeonggi, Korea). ALCAM mutant (ALCAM -/- ) mice with C57BL/6 background were purchased from JAX Laboratories (Bar Harbor, ME, USA) and backcrossed to BALB/c background for more than seven generations. All animal experiments were performed in compliance with Korea Research Institute of Bioscience and Biotechnology and approved by the institutional review boards of Yonsei University College of Medicine Council of Science and Technology. 3. Antibodies and reagents For Flow cytometric analysis, cells were stained with allophycocyanin (APC) - conjugated anti-cd3, fluorescein isothiocyanate (FITC) - conjugated anti-cd4, phycoerythrin (PE) - conjugated anti-cd44, and APC conjugated anti-cd62 ligand (CD62L) antibodies. These antibodies were obtained from ebioscience (San Diego, CA, USA). Ovalbumin (grade Ⅴ) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cholera toxin was purchased from List Biologicals (Campbell, CA, USA). 4. Experimental food allergy Wild type mice and ALCAM mutant mice were sensitized twice within a 2-wk interval by intraperitoneal (i.p.) injection with 50 μg ovalbumin (OVA) and 10 μg cholera toxin (CT) as adjuvants. Then all mice were orally challenged with OVA 8

(50 mg in 200 μl saline) six times within a one-day interval. Control mice were sensitized and challenged with PBS alone. They were sacrificed one day after the final challenge and subjected to analysis of allergic responses in the small intestine. (Figure1. A) 5. Measurement of parameters of food allergy At last challenge, mice were measured core temperature before and 15, 30, and 60 min after challenge in the rectum with a digital thermometer. (Testo, Lenzkirch, Germany) Mice were also observed for 2 hrs to record diarrhea and clinical sores after the final challenge as previously described. 12,31 Briefly, Diarrhea was scored as follows: 0, normal stools; 1, a few wet and unformed stools; 2, a number of wet and unformed stools with moderate perianal staining of the coat; 3, severe and watery stool with severe perianal staining of the coat. The clinical response was also scored as follows: 0, no symptom; 1, scratching and rubbing around the nose, ear and head; 2, puffiness around the eyes and mouth, reduced activity, and/or increased respiratory rate; 3, labored respiration; 4, no activity after prodding, or convulsion; 5, death. 6. Isolation and culture of immune cells Spleen cells, mesenteric lymph node (mln) cells, and lamina propria mononuclear cells were isolated from mice. Spleen and mln were homogenized using a syringe plunger in a cell strainer (BD Falcon; 40 μm ). The cells were centrifuged and 9

washed with Roswell Park Memorial Institute (RPMI) medium supplemented with 5% fetal bovine serum (FBS) and then red blood cells (RBCs) were lysed with ACK lysis buffer (0.15 M NH 4Cl, 0.1 mm KHCO 3, 0.1 mm Na 2-EDTA in distilled water; ph 7.2). Cells were washed with RPMI containing 5% FBS. The cells were cultured 96-well flat bottom plate in the absence or presence of 100 μl ovalbumin(1 mg / ml ). Small intestine (jejunum) lamina propria mononuclear cells were isolated as previously described. 32 Briefly, jejunum was removed from mice and placed into cold RPMI containing 10 % FBS. Jejunum was gently flushed with PBS, cut into 3-4 inch segments, removed residual fat by rolling, and inverted inside out by curved forceps. Place the tissue segments in extraction media (500 rpm, 15 min, 37 ), and digestion media (500 rpm, 30 min, 37 ) after mince the tissue. After digestion, filter the digested tissue and centrifuge that solution with RPMI containing 10 % FBS twice. After 72 hrs of culture, cells with medium were collected and centrifuged. The supernatant and centrifuged cells were frozen separately. 7. Proliferation assay Isolated cells from spleen and mln were cultured 96-well flat bottom plate in the absence or presence of 50 μl ovalbumin (10 mg / ml ) for 5 days. At the fifth day, 10 μl of cell counting kit-8 (CCK-8) solution was added per well with cultured cells and incubated two-to-four hrs in incubator. And then, 96-well flat bottom culture plates were read at 450 nm with ELISA reader. 10

8. Determination of the concentrations of cytokines in splenocyte cultured media The concentrations of IL-4, IL-5 and IL-13 in cell-culture supernatants were determined by ELISA (R&D Systems, Minneapolis, MN) as manufacturer s instructions. Briefly, ELISA plates were coated with purified anti-cytokine antibodies and blocked with 1 % BSA/PBS for 1 hr. Samples and dilution standards were loaded and incubated for 2 hrs at room temperature. The bounded cytokine was detected with anti-mouse-cytokine antibodies for 2 hrs, followed by streptavidin-hrp for 20 min. The plates were developed by addition of the substrate a tetramethylbenzidine (TMB) (KPL, Gaithersburg, MD) and stopped with 2 N sulfuric acid. Plates were read at 250 nm. 9. Flow cytometric analysis Single-cell suspensions (1 10 6 to 2 10 6 cells) were resuspended in 100 μl of FACS buffer (0.5 % FBS in PBS) and dead cells excluded by Viability dye efluor780 (ebioscience, San Diego, CA, USA). Cells were simultaneously stained with optimal concentrations of monoclonal antibodies specific for CD3, CD4, CD44, and CD62 ligand (CD62L). Therefore, population of T cell was selected by CD3, and CD4 and activation of T cell was selected by CD4, CD44, and CD62L. Cells were stained for 30 min at 4, washed twice with cold staining buffer (PBS, 2 % FCS, and 0.02 % sodium azide). Samples were run on a FACSVerse flow cytometer (BD Biosciences), and data were analyzed using FlowJo (Tree Star, Ashlnd, OR). 11

10. Measurement of cytokines by real-time PCR Total RNA was prepared with small intestine (jejunum) by TRIzol reagent (Invitirogen). First strand cdna was synthesized using superscript with random hexamer primers (Invitrogen). Real-time PCR was performed with Exicycler 96 (Bioneer, Korea), and mrna levels were quantified using the AccuPower GreenStar qpcr Master Mix (Bioneer), according to the manufacturer s instructions. Amplification was conducted for 45 cycles. Quantification values were normalized to the mean value of housekeeping gene; β-actin. Based on the cycle threshold (C t) values obtained, a relative and normalized mrna expression level was determined for each gene using ΔC t method. The C t value for each gene was corrected by the mean C t value of the two housekeeping genes. The results were calculated as a relative expression using the following formula: 2 -ΔCt K, where K is a 10 6 factor, and expressed as arbitrary units. Fold increase in the expression levels of the experimental groups was normalized to those of the negative control group. 11. Histologic evaluation Small intestine (jejunum) sections were stained with hematoxyline and eosin (H&E). Briefly, jejunum was fixed with 10 % buffered formalin, embedded in paraffin and cut into 5 μm sections for H&E staining to assess gut inflammation respectively. Microphotographs were taken by Nikon microscope (Elipes 90i) with camera. Histological damaged score was measured as follow; 33 0, no damage; 1, low amount of damage is seen with distinct structural components (epithelial layer 12

and lamina propria); 2, structural components can still be differentiated but the epithelial layer is noticeably separating from the lamina propria; 3, disorganization of the villi is beginning and differentiating between structural components is difficult; 4, organization of the villi is chaotic; 5, structure of villi is chaotic and many villi are completely destroyed down to the basal layers of tissue. 12. Electron microscopy Small intestine (jejunum) specimens were pre-fixed in Karnovsky s fixative solutions (2 % glutaraldehyde paraformaldehyde in 0.1 M phosphate buffer for 30 min, they were post fixed with 1 % osmium teroxide (OsO 4) dissolved in 0.1 M phosphate buffer for 2 hrs and dehydrated in ascending gradual series (50 ~ 100 %) of ethanol and infiltrated with propylene oxide. Specimens were embedded by Poly/Bed 812 kit (Polysciences Inc., Warrington, PA, USA). Pure resin embedding and polymerization was carried out at 60 electron microscope oven (TD-700, DOSAKA, Tokyo, Japan) for 24 hrs. Three hundred fifty nm thick section were initially cut and stained with toluidine blue for light microscope. Seventy nm thin sections were double stained with 7 % uranyl acetate and lead citrate for contrast staining. These sections were cut by LEICA Ultracut UCT Ultra-microtome (Leica Microsystems, Vienna, Austria). All of the thin sections were observed by JEM-1011 transmission electron microscopy (JEOL, Tokyo, Japan) at the acceleration voltage of 80 kv. The images were obtained with a MegaView Ⅲ camera (Olympus, Tokyo, Japan). 13

13. Quantification of the serum concentrations of total IgE and specific IgE (ELISA) Blood was collected by cardiac puncture. Serum total immunoglobulin E (IgE) and ovalbumin-specific IgE level was measured using mouse IgE ELISA assay kit (BD Bioscience, San Diego, CA) according to the manufacturer s instructions. Microplates were coated with anti-ige antibody in coating buffer. Serum samples and diluted standards were added and incubated for 2 hrs at room temperature. After washing, detection antibody and SAv-HRP reagent were applied onto plate for 2 hrs at room temperature. The reaction was developed with a TMB (KPL) and stopped with 2 N sulfuric acid. Plates were read at 450 nm. For measurement of ovalbumin-specific IgE, microplates were coated with anti-ige antibody and ovalbumin in coating buffer. 14. Statistical analysis Data were expressed as mean ±SD. Significant differences between two groups were estimated using unpaired Student s t-test. Statistical significance was set at P 0.05. 14

Ⅲ. RESULTS 1. Increased ALCAM levels in serum of OVA-challenged food allergy model To address the effects of ALCAM in food allergy, we investigated ALCAM level in OVA-challenged wild type (WT) mouse model. WT mice was intraperitoneally sensitized, and orally challenged with OVA, (Figure 1. A) and evaluated with parameters of food allergy. Clinical and diarrhea scores were increased, and core temperature was decreased in OVA-challenged WT mice. (Figure 1. B-D) Levels of serum OVA-specific IgE were also enhanced in OVA-challenged WT mice against to control mice. (Figure 1. E) Unlike increased levels of ALCAM in serum, mrna expression of ALCAM decreased in small intestine (jejunum) of food allergy-induced mice. (Figure 1. F) 15

A BALB/c Sacrifice 0 14 28 30 32 34 36 38 39 (Day) 50 μg of OVA with 10 μg of CT Challenge with 50 mg of OVA B C Symptom Score (points) 5 4 3 2 1 0 PBS ** OVA Diarrhea Score (points) 4 3 2 1 0 PBS ** OVA D Core Temperature ( ) 38 36 34 32 PBS OVA ** ** ** 0 15 30 45 60 Time (min) 16

E OVA-specific IgE (O.D.) 6 ** 4 2 0 PBS OVA F Small intestine Serum ALCAM fold-change 3 2 1 * ALCAM Level (ng/ml) 6 4 2 ** 0 PBS OVA 0 PBS OVA 17

Figure 1. Increased levels of ALCAM in serum of food allergy- induced wild type mice with ovalbumin. (A) Experimental protocol for (OVA)-induced food allergy model. Mice were sensitized with OVA/CT and challenged with OVA. Clinical score (B), diarrhea score (C), and core temperature (D) were measured after the last challenge. (E) Level of OVA-specific IgE in mice serum. (F) ALCAM levels were measured in small intestine, and serum. Mean data were estimated from triplicated experiments. * P < 0.05, ** P < 0.01. 18

2. Attenuated immune responses in ALCAM -/- mice model To identify contributions of ALCAM to OVA-induced food allergy, we induced food allergy to WT and ALCAM mutant (ALCAM -/- ) mice with OVA. Levels of the parameters of food allergy, T H2 cytokines, total IgE, OVA-specific IgE, and histological injury of small intestine were increased in OVA-challenged WT mice against to control mice. (Figure 2.) In OVA-challenged ALCAM -/- mice, clinical and diarrhea scores were decreased against OVA-challenged WT mice. (Figure 2. A, B) Level of core temperature was less dropped in OVA-challenged ALCAM -/- mice than OVA-challenged WT mice. (Figure 2. C) In addition, mrna expressions of T H2 cytokines such as IL-4, IL-5, and IL-13 were diminished in small intestine of OVA-challenged ALCAM -/- mice. (Figure 2. D) Levels of serum total IgE, and OVA-specific IgE were also attenuated in OVA-challenged ALCAM -/- mice. (Figure 2. E) In OVA-challenged ALCAM -/- mice, the characteristics of injured small intestine, such as damaged villi and infiltrated cells were milder than OVA-challenged WT mice. (Figure 2. F, G) Chen T. et al. identified that enlarged intestinal permeability and damaged intestinal tight junction in sensitized juvenile rat with OVA. 34 So, we observed intestinal tight junction through electron microscopy (EM). The intestinal tight junction and desmosomes of OVA-challenged WT mice smeared and spread against to control mice. However, these phenomena were weakened in OVA-challenged ALCAM -/- mice. (Figure 2. H) Put together, ALCAM -/- mice showed diminished immune response. 19

A B Symptom Score (points) 5 4 3 2 1 0 WT ALCAM -/- PBS ** ## OVA Diarrhea Score (points) 3 ** ## 2 1 0 PBS OVA C Core Temperature ( ) 40 38 36 34 32 WT/PBS ALCAM -/- /PBS ## ## ** ** WT/OVA ALCAM -/- /OVA ## ** 0 15 30 45 60 Time (min) 20

D 40 WT ALCAM -/- IL-4 fold-change 30 20 10 ** ## 0 PBS OVA IL-5 fold-change 6 ** ## 4 2 IL-13 fold-change 20 15 10 5 ** ## 0 PBS OVA 0 PBS OVA E Total IgE (pg/ml) 3000 2500 2000 1500 1000 500 0 WT ALCAM -/- PBS ** ## OVA OVA-specific IgE (O.D.) 3 2 1 0 PBS ** ## OVA 21

F G WT/PBS WT/OVA Injury Score (points) 5 4 3 2 1 ** ## 0 PBS OVA ALCAM -/- /PBS ALCAM -/- /OVA H WT/PBS WT/OVA ALCAM -/- /PBS ALCAM -/- /OVA 22

Figure 2. Attenuated immune responses in food allergy-induced ALCAM deficient mice with ovalbumin. After last challenge, clinical score (A), diarrhea score (B), and core temperature (C) were measured in OVA-challenged WT and ALCAM -/- mice. (D) Expression of T H2 cytokines mrna (IL-4, IL-5, and IL-13) in small intestine of mice. (E) Levels of total IgE and O.D. values of OVA-specific IgE were measured in mice serum by ELISA. Histological observations of small intestine (G) and its injury score (F). (H) Morphological observation of intestinal tight junction using electron microscopy (EM). Mean data were estimated from triplicated experiments. ** means P < 0.01 (WT/PBS vs. WT/OVA). ## means P < 0.01 (WT/OVA vs. ALCAM -/- /OVA). Magnification; (F, x200); (I, x50k). 23

3. Diminished T cell responses in ALCAM -/- mice It is well known that ALCAM on APCs and CD6 on T cells interact in immunological synapse and play roles as co-stimulatory molecule. 35 Therefore, we figured out the role of ALCAM in T cell responses through identifying T cell responses. Splenocytes and mesenteric mln cells were isolated from WT mice and ALCAM -/- mice, and cultured with OVA. And then T cell proliferation was measured with CCK-8. The rate of total T cell was diminished in both cell types from OVA-challenged ALCAM -/- mice compared with those from OVA-challenged WT mice. (Figure 3. A) Levels of T H2 cytokines (IL-4, IL-5, IL-13) were decreased in splenocytes cultured media of OVA-challenged ALCAM -/- mice against to OVA-challenged WT mice. (Figure 3. B) The change of T cell proliferation in whole cell from spleen and mln was identified. (Figure 3.) To address more specific changes of T cell responses in immune tissues, splenocytes, mesenteric lymph nodes (mln) cells, and lamina propria mononuclear cells were isolated and cultured from spleen, mln, and small intestine. For identify T cell population, CD3, and CD4 were used as markers. Number of CD3 + CD4 + T cells enhanced in immune systems, especially in small intestine, of OVA-challenged WT mice against that of control mice. However, the number of CD3 + CD4 + T cells decreased in OVA-challenged ALCAM -/- mice than OVA-challenged WT mice. We obtained similar data in T cell activation with markers, CD4, CD44, and CD62 ligand (CD62L). Number of activated cells 24

(CD4 + CD44 high CD62L low ) decreased in OVA-challenged ALCAM -/- mice than OVA-challenged WT mice. (Figure 4. A, B) Graphs represent populations of cells. (Figure 4. C, D) These results suggest that ALCAM has an effect on T cell population and activation in the immune systems by interaction with CD6 on T cells. 25

A Proliferation (O.D.) 1.0 0.8 0.6 0.4 0.2 Splenocyte WT ALCAM -/- ** ## Proliferation (O.D.) 0.8 0.6 0.4 0.2 mln ** ## 0.0 CON OVA 0.0 CON OVA B WT ALCAM -/- IL-4 Level (pg/ml) 30 ** ## 20 10 0 CON OVA 20 IL-5 Level (pg/ml) 60 40 20 ** ## IL-13 Level (pg/ml) 15 10 5 ** ## 0 CON OVA 0 CON OVA 26

Figure 3. Decreased T cell proliferation in food allergy-induced ALCAM deficient mice with ovalbumin. Spleen and mln were removed and cells were isolated from these tissues. Total cells of spleen and mln were cultured in the absence (Control; CON) or presence of OVA for 5days. T cell proliferation was assessed with CCK-8. The supernatant of splenocyte was used for measurement of released T H2 cytokine level. (A) Values of total cell proliferation in spleen and mln. (B) T H2 cytokine levels in supernatant of splenocytes cultured media by ELISA. ** means P < 0.01 (WT/CON vs. WT/OVA). ## means P < 0.01 (WT/OVA vs. ALCAM -/- /OVA). 27

A Control WT/OVA ALCAM -/- / OVA 36.9 52.9 49.8 mln 13.4 23.4 15.7 Spleen CD3 12.3 36.6 23.5 Small intestine CD4 B CD3 + CD4 + (x10 4 ) 120 100 80 60 40 20 0 Control WT ALCAM -/- ** ## mln ** ## ** ## Spleen Small intestine 28

C Control WT/OVA ALCAM -/- / OVA mln Spleen CD44 Small intestine CD62L D CD4 + CD44 high CD62L low (x10 4 ) 20 15 10 5 0 Control WT ALCAM -/- ** ## mln ** ## Spleen ** # Small intestine 29

Figure 4. Diminished T cell population and T cell activation in food allergy-induced ALCAM deficient mice with ovalbumin. Single cells of spleen, mesenteric lymph node (mln), and small intestine (jejunum) were gated with CD3, and CD4 or CD4, CD44, and CD62 ligand (CD62L) by flow cytometry. (A) CD3 + CD4 + T cell populations of splenocytes, mln cells and small intestine lamina propria mononuclear cells of OVA-challenged WT and ALCAM -/- mice. (B) Data of flow cytometry (A) showed as a graph. (C) Activated T cells were measuredand gated with CD4 + CD44 high CD62L low in lymphatic tissues of OVA-challenged WT and ALCAM -/- mice. (D) Data of flow cytometry (C) showed as a graph. Mean data were estimated from triplicated experiments. ** means P < 0.01 (WT/Control vs. WT/OVA). # means P < 0.05, ## means P < 0.01 (WT/OVA vs. ALCAM -/- /OVA). 30

4. Increased levels of ALCAM in serum of children with food allergy To determine the level of ALCAM in children with food allergy, we evaluated 143 children aged 0.5 10.8 years, with 53 and 90 in the food allergy and control groups, respectively. The clinical characteristics of the subjects are summarized in Table 1. There was no significant difference in gender, while mean age was higher in the control group than that in the food allergy group (7.4 ± 1.44 vs. 2.24 ± 1.9, P < 0.0001). Serum total IgE levels were significantly increased in children with food allergy compared to healthy controls (260.8 ± 294.6 vs. 40.4 ± 30.5, P < 0.0001). ALCAM levels in serum were significantly higher in those with food allergy than in healthy controls (35.4 ± 4.7 vs. 28.1 ± 4.2, P < 0.0001). (Figure 5.) A multiple regression analysis performed to evaluate whether ALCAM level relates to food allergy after controlling for age demonstrated similar results that ALCAM level was significantly increased in the food allergy group. (β = 5.75, P < 0.0001) 31

ALCAM levels of human serum (ng/ml) 50 40 30 20 10 0 ** Control Food allergy (n=90) (n=53) Figure 5. Enhanced levels of ALCAM in serum of children with food allergy. The ALCAM level of human serum was measured in children with food allergy (n=53) and healthy control subjects (n=90) by ELISA. ** P < 0.01. 32

Table 1. Subject characteristics Characteristics Control (n=90) Food allergy (n=53) P value Age, yr 7.4 ± 1.44 2.24 ± 1.9 P < 0.0001 Sex, M (%) 42 (47) 32 (60) 0.158 Serum total IgE, ku/l 40.4 ± 30.5 260.8 ± 294.6 P < 0.0001 Values are expressed as number (percentage), mean ± SD. IgE, immunoglobulin E. 33

Ⅳ. DISCUSSION Allergenic food proteins may induce not production of IgE but activation of special subsets of T cells to establish food allergy. 36 It means that the most common form of food allergy is mediated by IgE antibodies, but food allergy is also induced by T cells. 37 In small intestine, the main tissue of food allergy, lymphocytes are found at various locations in the gut mucosa. T cells especially resident in the epithelium. 38 When APCs, such as DCs, are present food allergens to T cells, the immunological synapse is formed. And there are many co-stimulatory molecules on immune cells engage in prolonged maintenance of immunological synapse. 14 ALCAM, one of co-stimulatory molecules, is expressed by DCs and interacts with CD6 on T cells. Many studies demonstrated that ALCAM mediates immune responses by interaction with CD6 29 and affects pathogenesis of cancers. One of the studies, they characterized ALCAM as intestinal cancer stem cell marker in the human and mouse gastrointestinal tract. 39 This study identified that enhanced ALCAM levels in serum of OVA-challenged WT mice and children with food allergy against to control. These findings indicate that ALCAM has an effect on OVA-induced food allergy and correlate to studies that addressed the role of ALCAM in murine model or human disease. Hansen AG. et al. identified that ALCAM is overexpressed in murine model of metastatic prostate cancer and patient with prostate cancer. 40 Moreover, levels of ALCAM increased in patients with a variety of cancers, including melanoma, prostate, breast, 34

ovarian, esophageal, bladder, and intestinal cancers. 18-24,41 Here, this study show that the attenuated symptoms of food allergy in OVA-challenged ALCAM -/- mice compared with WT mice, such as decreased levels of T H2 cytokines, injury of small intestine, and scores of parameters. It means ALCAM promotes immune response in food allergy and these data correlate with other studies that demonstrated the role of ALCAM in disease. 42-44 von Bauer R. et al. demonstrated that ALCAM similar to structure of receptor for advanced glycation end products (RAGE) and plays a role in delayed type hypersensitivity (DTH) as a pattern recognition receptor. 45 They induced DTH to mice and identified that decreased clinical score of allergic disease in DTH-induced ALCAM -/- mice against to DTH-induced WT mice. Li XM et al. characterized T cell response to peanut allergen in a murine model of peanut allergy. 46 Authors identified that levels of allergen-specific IgE and systemic anaphylaxis score are significantly enlarged in peanut-induced mice compared to control mice. Moreover, T cell proliferation of splenocytes which were stimulated by peanut increased in peanut-induced mice. In another study, authors induce food allergy to BALB/c mice by sensitization with OVA. 47 OVA-challenged mice exhibited symptoms of food allergy such as incidence of diarrhea, decreased body temperature, increased levels of OVA-specific IgE, and expanded the number of mast cells. In addition, levels of T H2 cytokines, and T cell proliferation of mln and splenocytes were enhanced in OVA-challenged mice. Another previous studies identified that CD6, ligand for ALCAM, is a co-stimulatory molecule in T cell 35

activation. 28 Both ALCAM and CD6 are actively recruited and contribute to stabilization of the immunological synapse. Moreover, it is already known that ALCAM-CD6-mediated adhesion contributes to both early and later stages of DC-induced T cell activation and proliferation. 29,35 Aukje W. Zimmerman demonstrated that CD6 and ALCAM form a key adhesive receptor-ligand pair that is not only involved in early DC-T-cell binding but also in sustaining DC-induced T-cell proliferation long after the initial contact has been established. 29 These studies mean that study of T cell biology is the most widely used to investigate the role of ALCAM. For these reasons, we measured total T cell proliferation, CD4+T cell population, and activated T cells of systemic immune tissues to identify the reasons of attenuated symptoms in OVA-challenged ALCAM -/- mice in this study. As a result, total T cell proliferation of cultured mln and splenocytes was diminished in OVA-challenged ALCAM -/- mice. More specifically, CD3 + CD4 + T cell population and T cell activation were decreased in mln, spleen, and small intestine of OVA-challenged ALCAM -/- mice than OVA-challenged WT mice. These results suggest that defected ALCAM draw attenuated T cell activation by diminished interactions with CD6 on T cells. In this study, ALCAM levels of serum enlarged in OVA-challenged WT mice and children with food allergy, and OVA-challenged ALCAM -/- mice showed attenuated symptoms of food allergy. Diminished T cell responses in OVA-challenged ALCAM -/- mice indicate that ALCAM affects immune responses in food allergy 36

through change T cell activation and T H2 response. Although more mechanism studies are required to specifically address the role of ALCAM in food allergy, this study suggests that ALCAM affects immune response in OVA-induced food allergy. 37

Ⅴ. CONCLUSION In this study, expression of ALCAM in small intestine and levels of ALCAM in serum were changed in OVA-challenged WT mice compared to control mice. ALCAM levels in serum of children with food allergy were also enhanced compared to healthy control subjects. In OVA-challenged ALCAM -/- mice, immune responses were attenuated against to OVA-challenged WT mice. Total T cell proliferation of spleen and mln, CD3 + CD4 + T cell population, and CD4 + CD44 high CD62L low activated T cell of spleen, mln, small intestine were also diminished in OVA-challenged ALCAM -/- mice compared to OVA-challenged WT mice. In conclusion, this study suggests that ALCAM affects immune response in experimental food allergy. That is, ALCAM regulates allergic disease and affects immune responses by alternation of T cell activation and T H2 response in OVA-induced food allergy. 38

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ABSTRACT (in Korean) 난백알부민에의해유도된식품알레르기의면역반응에서 활성백혈구부착분자 (ALCAM/CD166) 의역할 < 지도교수손명현 > 연세대학교대학원의과학과 김윤선 식품알레르기는경구면역관용에실패함으로써나타나는비정상적인면역반응으로, 전세계적으로주요하게다뤄지는건강문제중하나의질병으로손꼽힌다. 활성백혈구부착분자 (ALCAM/CD166) 는면역글로불린상과 (IgSF) 에속하는막관통당단백질로, T 세포의표면에존재하는 CD6와상호작용하여면역반응에서역할을하는것으로알려져있다. 또한 ALCAM은여러가지기관에서발병되는암의발생에서역할을한다고알려져있다. 그러나, 현재이러한 ALCAM이알레르기질병의면역반응에미치는영향이나기능을확인한논문은없는것으로알려져있다. 따라서본연구는알레르기질병중하나인식품알레르기모델에서 ALCAM의기능을확인하기위하여진행되었다. 45

BALB/c 야생형마우스와 ALCAM 결핍마우스에난백알부민을복강주사하여감작시키고경구투여했다. 그후마우스에서증상에대한정도, 체온, 소장의형태, 혈청면역글로불린 E, T H 2 면역반응등을포함한식품알레르기모델의척도를확인했다. 식품알레르기를유발한야생형마우스의소장에서는 A LC A M 의 발현량이감소하였고혈청에존재하는 ALCAM의양은증가한다는것을확인했다. 또한대조군보다난백알부민으로식품알레르기를유발한야생형마우스에서증상에대한정도, 혈청에존재하는총면역글로불린 E와난백알부민에특이적인면역글로불린 E의양, T H 2 사이토카인 ( 인터류킨-4, 인터류킨-5, 인터류킨-13) 의발현량, 헤마톡실린 & 에오신염색을한소장의손상정도등이증가한것을확인하였다. 그에반해서, 이러한식품알레르기에대한척도가 ALCAM 결핍마우스에서억제되어나타나는것을확인했다. T 세포의보조자극신호분자로알려진 CD6가그의리간드인 ALCAM에어떠한영향을미치는지알아보기위해마우스의비장과림프절에서 T 세포의급증을확인하였을때, 식품알레르기가일어난야생형마우스보다 ALCAM 결핍마우스에서더감소하는현상을확인하였다. ALCAM이관여하는 T 세포의변화를좀더특이적으로확인하기위하여마우스의비장과림프절, 소장에서세포를분리하고염색하여 C D 3 + C D 4 + T 세포의분포와 CD4 + CD44 high CD62L low 에해당하는활성된 T 세포의수를유세포분석으로측정하였다. 그결과 T 세포의분포와활성정도역시 ALCAM 결핍 46

마우스의비장, 림프절, 소장에서감소하는것을확인했다. 마지막으로, 마우스에서확인한결과와같이건강한대조군보다식품알레르기증상을보이는환아의혈청에서도 ALCAM의양이증가하는것을확인하였다. 결론적으로, 본연구에서진행한실험결과를통해 ALCAM이 T 세포의표면에존재하는 CD6와상호작용하여 T 세포의급증, 분포, 활성등을유도하고, T H 2 면역반응에영향을미침으로서식품알레르기모델에연관되어있다는것을확인하였다. 핵심되는말 : 식품알레르기, 식품알레르기마우스모델, 활성백혈구부착분자, ALCAM, CD166 47