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AN ABSTRACT OF THE THESIS OF: Mark Hall for the degree of Honors Baccalaureate of Science in Biochemistry and Biophysics presented on April 19th, 2011. Title: Activation of Co-stimulatory Molecule OX40 Increases Memory T-cell Function and Cytokine Production in Uveitis Abstract approved: _________________________________________ Kevin Ahern Uveitis is a serious ophthalmological disorder characterized by intraocular infiltration of inflammatory cells. In most cases, uveitis is derived from the adaptive immune response. More specifically, CD4+ T lymphocytes play an important role in the pathogenesis of uveitis by recognizing uveitogenic antigen and orchestrating the immune response. While it is known that OX40 costimulatory molecule increases ocular inflammation via stimulation of naïve CD4+ T lymphocytes, OX40 augmentations of effector T cell re-stimulation and infiltration into the eye is not fully characterized. Our overall goal is to elucidate the function and mechanism of OX40 (a critical T cell costimulatory molecule) in orchestrating an abnormal immune response during uveitis. Our lab employed an adoptive transfer mouse model as an in vivo model for controlled immunity transference. We used DO11.10 strains of BALB/c background whose transgenic T cell receptor (TCR) is modified to specifically recognize ovalbumin. The novel ovalbumin (OVA) adoptive transfer model was developed in our lab and allowed for us to study the transference of immunity from our donor T cell transgenic mice to our in vivo recipient antigen-challenged model mice which would allow us to observe the increased severity with OX40 stimulation. T cells were harvested from donor DO11.10 mice and stimulated with OVA peptide along with OX40 activating antibody in vitro, and then transferred to recipient mice without transgenic T cell. OVA peptide was administered via intraocular injections and inflammation was assessed 24 hours post-antigen challenge. CD4+ T cells of the host mice do not naturally respond to OVA, therefore response to OVA needs to be mediated by donor T cells. Furthermore, via RT-PCR methods, OX40 function was strongly correlated with enhanced chemokine and cytokine production including IL-17, IFN-γ, and CCL20. Lastly, neutralization experiments of IL17 and IFN-γ showed decreased overall ocular inflammation. This study not only helped to further advance our understanding of this important co-stimulatory molecule in activated T lymphocyte function, but also helped us better understand the role of OX40 in ocular inflammation. We were also able to understand functionality of important cytokines and chemokines in the pathogenesis of uveitis. This study can ultimately help in developing a clinical therapeutic approach to uveitis. Keywords: OX40, CD4+ T cell, uveitis, adoptive transfer, IL-17, IFN-γ Corresponding e-mail address: [email protected] Copyright by Mark Hall On April 19th, 2011 All Rights Reserved Activation of Co-stimulatory Molecule OX40 Increases Memory T-cell Function and Cytokine Production in Uveitis Mark Hall A Project Submitted to Oregon State University University Honors College In fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Biochemistry and Biophysics Presented April 11th, 2011 Commencement June 2011 Honors Baccalaureate of Science in Biochemistry and Biophysics project of Mark Hall presented on April 11th, 2011. APPROVED: Kevin Ahern, representing Biochemistry and Biophysics Zili Zhang, representing Immunology Jeff Greenwood, representing Biochemistry and Biophysics Chair, Department of Biochemistry and Biophysics Dan Arp, University Honors College I understand that my project will become part of the permanent collection of Oregon State University, University Honors College. My signature below authorizes release of my project to any reader upon request. Mark Hall, Author Acknowledgments I would like to thank Dr. Zili Zhang, Wenwei Zhong, and Dr. Xuimei Wu for teaching me proper research technique and practice in the lab and for guidance on this project. I would also like to thank Dr. Jim Rosenbaum, Dr. Stephen Planck, and Dr. Doran Spencer for their resources and help on this project. I would also like to thank Kevin Ahern for mentoring me while writing this honors thesis. Table of Contents Abstract Background Materials and Methods o Mice o Cell Culture and Stimulation/Isolation Methods o Uveitis Model o Adoptive Transfer Protocol o Flow Cytometry o Intravital Microscopy o Real Time PCR o Intravital Microscopy Quantification and Calculations o Statistics/Analysis Method Results o OVA stimulated mice cause up regulation of OX40 (CD 134) o Stimulation by OX40 increases uveitis o OX40 activation enhances cytokine production o OX40 activation of mature T-cells further increases uveitis o Neutralization of IFN-γ and IL-17ab showed decreased inflammatory response Discussion References Tables and Figures 1 2 4 4 5 5 6 6 7 7 8 9 9 9 10 12 13 14 15 19 21 List of Figures 1. RT-PCR: Fold increase of OX40 expression 2. Flow Cytometry: Characterization of CD4+ population. 3. Flow Cytometry: Characterization of OX40 expression population. 4. Intravital Microscopy: 24 hours post-adoptive transfer. 5. Intravital Microscopy: 72 hours post-adoptive transfer. 6. Intravital Quantification: Leukocyte roller flow rate. 7. Intravital Quantification: Leukocyte sticker concentration. 8. Intravital Microscopy: Comparison of inflammation in naïve and antigen experienced adoptive transfer models. 9. RT-PCR: Fold increase of cytokine and chemokine mRNA expression of OX40 stimulated CD4+ T cells over naive T cells. 10. RT-PCR: Fold increase of cytokine and chemokine mRNA expression of antigen experienced OX40 stimulated T cells over naïve OX40 stimulated T cells. 11. Flow Cytometry: Time profiling of OX40 population in naïve and antigen experienced mouse models. 12. Intravital Quantification: Leukocyte roller flow rate of neutralization experiment. 13. Intravital Quantification: Leukocyte sticker concentration of neutralization experiment. 14. Intravital Microscopy: Neutralization control group. 15. Intravital Microscopy: 24 hours post-adoptive transfer α-IL17 neutralization group. 16. Intravital Microscopy: 24 hour post-adoptive transfer α-IFN-γ neutralization group. University Honors College Copyright Release Form We are planning to release this Honors Thesis in one or more electronic forms. I grant the right to publish my thesis entitled, Activation of Co-stimulatory Molecule OX40 Increases Memory T-cell Function and Cytokine Production in Uveitis in the Honors College OSU Library’s Digital Repository (D-Space), and its employees the nonexclusive license to archive and make accessible, under conditions specified below. The right extends to any format in which this publication may appear, including but not limited to print and electronic formats. Electronic formats include but are not limited to various computer platforms, application data formats, and subsets of this publication. I, as the Author, retain all other rights to my thesis, including the right to republish my thesis all or part in other publications. I certify that all aspects of my thesis which may be derivative have been properly cited, and I have not plagiarized anyone else’s work. I further certify that I have proper permission to use any cited work which is included in my thesis which exceeds the Fair Use Clause of the United States Copyright Law, such as graphs or photographs borrowed from other articles or persons. Signature: ______________________________________________ Printed Name: ___________________________________________ Date: __________________________________________________ Abstract: Uveitis is a serious ophthalmological disorder characterized by intraocular infiltration of inflammatory cells. In most cases, uveitis is derived from the adaptive immune response. More specifically, CD4+ T lymphocytes play an important role in the pathogenesis of uveitis by recognizing uveitogenic antigen and orchestrating the immune response. While it is known that OX40 co-stimulatory molecule increases ocular inflammation via stimulation of naïve CD4+ T lymphocytes, OX40 augmentations of effector T cell re-stimulation and infiltration into the eye is not fully characterized. Our overall goal is to elucidate the function and mechanism of OX40 (a critical T cell co-stimulatory molecule) in orchestrating an abnormal immune response during uveitis. Our lab employed an adoptive transfer mouse model as an in vivo model for controlled immunity transference. We used DO11.10 strains of BALB/c background whose transgenic T cell receptor (TCR) is modified to specifically recognize ovalbumin. The novel ovalbumin (OVA) adoptive transfer model was developed in our lab and allowed for us to study the transference of immunity from our donor T cell transgenic mice to our in vivo recipient antigenchallenged model mice which would allow us to observe the increased severity with OX40 stimulation. T cells were harvested from donor DO11.10 mice and stimulated with OVA peptide along with OX40 activating antibody in vitro, and then transferred to recipient mice without transgenic T cell. OVA peptide was administered via intraocular injections and inflammation was assessed 24 hours post-antigen challenge. CD4+ T cells of the host mice do not naturally respond to OVA, therefore response to OVA needs to be mediated by donor T cells. Furthermore, via RT-PCR methods, OX40 function was strongly correlated with enhanced 2 chemokine and cytokine production including IL-17, IFN-γ, and CCL20. Lastly, neutralization experiments of IL-17 and IFN-γ showed decreased overall ocular inflammation. This study not only helped to further advance our understanding of this important costimulatory molecule in activated T lymphocyte function, but also helped us better understand the role of OX40 in ocular inflammation. We were also able to understand functionality of important cytokines and chemokines in the pathogenesis of uveitis. This study can ultimately help in developing a clinical therapeutic approach to uveitis. Background: Uveitis is a term associated with many different types of severe inflammation of the eye. Over 100 cases per 100,000 occur in the United States and is on the same order as diabetes as a leading cause of blindness (8). This constitutes a significant healthcare burden and is caused by genetic, immunological and environmental factors. Although its etiology is not fully understood, due to its multivariable complexity, what is known is leukocyte infiltration into the ocular tissue is a major cause for uveitis (2). More specifically, lymphocytes are known to be involved in the pathology of this disease. Uveitis can affect all parts of the eye including the anterior chamber, posterior chamber, and more (1,2,6). The immune system consists of an innate and adaptive response. The adaptive response is the focus of this study. Lymphocytes are manufactured in the bone marrow initially as pluripotent hematopoietic stem cells. In the bone marrow and thymus, also known as the primary lymphoid organ, they differentiate into a common lymphoid progenitor. While B cells mature in the bone marrow, T lymphocytes migrate to the thymus to continue their maturation. 3 After maturation in their respective primary lymphoid organs, they travel to the peripheral lymphoid organs via the blood-stream. B and T lymphocytes continually circulate between the blood stream and lymphatic vessels and tend to collect in the lymph nodes waiting for antigen stimulation. B and T cells that have matured and associate in the lymph nodes, but have not encountered antigen are considered naïve cells. However, once antigen presenting cells (APC’s) or more specifically dendritic cells (another white blood cell derived from the same type of hematopoietic stem cells) encounter antigen in the periphery, they transport from the site of infection through the afferent lymphatic vessels into the draining lymph nodes. Once antigen from dendritic cells is presented to T and B cells, these lymphocytes undergo activation into effector cells via proliferation and differentiation and travel to the site of infection through the efferent lymphatic vessels (3). Lymphocytes including CD4+, CD8+, and NK cells are vital to adaptive immunity and are believed to be essential to the pathogenesis of uveitis. CD4+ cells are a subset of T cells known as T helper cells which aid in the activation of B cells and examples include Th-1, Th-2, and a newer subset known as Th-17 (1,4,5). ICAM-1 (on antigen presenting cells) and LFA-1 (on T cell) form an adhesion to the MHC class II molecule (with bound antigen) to present to the T cell receptor (TCR) which facilitates a signal cascade within the T cell. Other co-stimulatory molecules are crucial in augmenting this adhesion, presenting and eventual proliferation phase. One of these co-stimulatory molecules is the focus of this paper: OX40 (CD 134) (1,4). The focus of this paper is on the impact of OX40 on pathogenic T cell activation and function in uveitis. It is a co-stimulating molecule in the augmentation of effector T lymphocytes. It is a member of the TNFR superfamily. OX40 signal triggers the NF-B signaling pathway which activates PKC and further transcription (1). Unlike similar co- 4 stimulatory molecules (e.g. CD28) that are responsible for initial T cell activation, OX40 is an inducible co-stimulatory molecule, and is preferentially up-regulated in activated CD4+, CD8+, and NKT cells (1). OX40 is believed to not only be involved in the initial stimulation of T cells upon antigen presentation by APC’s, but is also potentially capable of “a second wave” of costimulation. Although OX40 has been implicated in many T cell-mediated diseases, such as experimental autoimmune encephalomyelitis and multiple sclerosis, little is known of OX40 during the up-regulation of uveitogenic effector lymphocytes and the development of uveitis. It is believed that OX40 is not only implicated in activation of naïve T cells, but also an important factor in re-activation of memory T cells that have already been antigen-experienced. Based on previous experimentation with the EAU model, other understandings, and the research in this study, we showed that OX40 is an effective co-stimulatory molecule in uveitis. Not only does OX40 increase naïve T cell proliferation, but also augments re-activation of memory effector T cells and consequently increased inflammatory response to the intraocular region of the eye with the in vivo mouse model. Further, neutralization experiments of chemokine and cytokine molecules show possibly effective clinical therapeutic methods and also allow for better characterization of the signal pathway. Materials and Methods Mice Six to eight-week-old BALB/c (no transgenic T cell receptor) and DO11.10 mice (with transgenic T cell receptor) on a BALB/c background (Jackson Laboratory, Bar Harbor, Maine) were used for the experiments. The animal experimental protocols are in accordance with the 5 ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and have been approved by our institutional animal care and use committee. Mice also bred to express phenotype with actin GFP tags. Cell Culture and Stimulation/Isolation Methods After DO11.10 mice were sacrificed, their spleens were harvested and single cell suspensions were prepared by homogenizing the tissue through a 70 m cell strainer (BD Biosciences, Mountain View, CA). Red blood cells (RBC) were lysed with 1X RBC lysis buffer (Sigma, St Louis, MO) at room temperature for 5 min. The cell suspension was washed twice with RPMI, and then cultured in RPMI with 10% fetal bovine serum (FBS) in an atmosphere of 95% air and 5% CO2 at 37°C. These cells were cultured in the presence of OVA323-339 peptide (Ile-Ser-Gln-Ala-ValHis-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg) (AnaSpec, Fremont, CA) with and without OX40 activating antibody (Clone OX84) for 72 hours. Prior to adoptive transfer, the lymphocytes were further purified using Lympholyte®-M (Cedar Lane Laboratories, Burlington, NC) according to the manufacturer’s instruction. After Lympholyte®-M purification, CD4+ cells were further isolated via EasySep Mouse CD4+ selection kit from StemCell Technologies (Vancouver, BC). Uveitis Model To generate uveitis in direct response to antigen stimulation, transgenic DO11.10 mice were injected with antigen in 2 l phosphate-buffered saline (PBS) and PBS alone as a control into the vitreous chamber of each eye. The injections were performed with ultra-thin, pulled 6 borosilicate glass needles (outer diameter about 50 µm) and Hamilton syringes under direct visualization through a surgical microscope. These mice received 100 µg of OVA peptide (Sigma, St. Louis, MO). Adoptive Transfer Protocol For the adoptive transfer protocol, donor OVA-activated DO11.10 CD4+ lymphocytes were injected into wildtype BALB/c mice at a total amount of 1.5x10^7 cells per recipient BALB/c mouse. Donor cells conferred effector T cell immunity to recipient mice via tail vein injection. The OVA-activated DO11.10 CD4+ lymphocytes were previously cultured for 72 hours (as indicated in Cell Culture and Stimulation Methods) with and without OX40 activating antibody. The recipient mice were given intravitreal injections with 0.5 µg E. coli strain 055:B5 lipopolysaccharide (Sigma) and 100 µg OVA in PBS. Flow Cytometry Flow cytometry was utilized to define OX40 positive CD4+ T cell subset population. DO11.10 splenocytes were suspended in PBS containing 2% FBS and 0.1% sodium azide. AntiCD4 FITC (Clone RM4-5) and anti-OX40 PE antibodies to label these cell surface markers for 1 hour at 40C. After PBS wash, the cells were fixed for analysis. Data acquisition was performed on a FACSCalibur flow cytometer, and data were analyzed using CellQuest software. 7 Intravital Microscopy Rhodamine was intraperitoneally administered into recipient mice 24 and 72 hours post adoptive transfer and antigen challenge. One hundred fifty µl of rhodamine (0.2% in PBS) was administered to label intravascular leukocyte at least 5 minutes before, but not more than 10 minutes before video/ data acquisition. The iris and ciliary region of each eye of the recipient mice were observed via intravital epifluorescence videomicroscopy. Mice were anesthetized for microscopy with isofluorane. The microscope was a modified DM-LFS microscope (Leica, Bannockburn, IL) and used either CF 84/NIR Black and white camera (Kappa, Gleichen, Germany) or a color Optronics DEI-750CE camera (Optronics International, Chelmsford, MA). Real time videos were saved as NTSC video format, recorded in 10 second intervals, and 3-4 videos were taken of each eye in each recipient. Inflammation was observed by leukocytes that were either rolling or adhering to vascular walls. Inflammation was quantified in ImageJ (McMaster Biophotonics). Real Time PCR Total RNA from eye homogenates was isolated with RNAeasy Mini kit (Qiagen, Valencia, CA). First-strand cDNA synthesis was accomplished with oligo (dT)-primed Omniscript reverse transcriptase kit (Qiagen, Valencia, CA). Gene-specific cDNA was amplified by PCR using mouse specific primer pairs. IFN- sense: 5’-TCA AGT GGC ATA GAT GTG GAA GAA-3’, and IFN- anti-sense: 5’TGG CTC TGC AGG ATT TTC ATG-3’ IL-17A sense: 5'-GTG GCG GCT ACA GTG AAG GCA-3', and IL-17A antisense: 5'- GAC AAT CGA GGC CAC GCA GGT-3' 8 IL-21 sense: 5’-ACC AGA CCA AGG CCC TGT C-3’, and IL-21 anti-sense: 5’-TGG GCT CTT GTT GAG TTG AGA TT-3’ IL-22 sense: 5’-TCA GAC AGG TTC CAG CC-3’, and IL-22 antisense: 5'-TCC AGT TCC CCA ATC GCC-3' CCL20 sense: 5’-TTT TGG GAT GGA ATT GGA CAC-3’, and CCL20 anti-sense: 5’- TGC AGG TGA AGC CTT CAA CC-3’ -actin sense, 5'-ATG CCA ACA CAG TGC TGT CT-3', and -actin antisense, 5'-AAG CAC TTG CGG TGC ACG AT- 3'). OX40 primers were commercially purchased from SABiosciences (Frederick, MD). The realtime PCR was performed using a Real-time PCR Master mix (SABiosciences, Fredrick, MD), and running for 40 cycles at 95°C for 15 sec and 55°C for 40 sec. The mRNA levels of investigating genes in each sample were normalized to β-actin mRNA and quantified using a formula; 2 [(Ct/β-actin – Ct/gene of testing gene)]. The result was expressed as fold difference in the cells stimulated with OVA and OX40 activating antibody compared to the group treated with OVA alone. Intravital Microscopy Quantification and Calculations All cells that were counted in inflammation were at least 5 µm in diameter (measurable in ImageJ) and passed through the vascular area of interest at some point during a 10 second video. The two straightest and largest vessels in each video were quantified and had a planar focal area of 2000µm² to 4000µm². Cells that rolled along vasculature and adhered to vasculature were counted in either the flow rate (cells/min./mm² vessel) or the sticker concentration (cells/mm²). 9 Stickers were calculated in an excel spreadsheet as =IF((vessel area in µm²)="","",1000000*(# of stickers)/(( vessel area in µm²)*3.14)) and rollers were calculated as =IF((vessel area in µm²)="","",60000000*(# of rollers)/(10 seconds)/( ( vessel area in µm²)*3.14)). Sticker concentration and roller flow rate were averaged over 2 vessels per image capture, then three to four videos per eye, then both eyes per recipient mouse, then finally averaged over all recipient mice in each group. Statistics/Analysis Method Data are expressed as the average ± SD. Statistical probabilities were evaluated by Student’s t test, with a value of p < 0.05 considered significant. Results: Using the protocols described in the methods section, three different adoptive transfer experiments were performed to answer all the major questions concerning this study. Our lab wanted to confirm that OX40 augmented naïve effector T cell function in ocular inflammation. We then set out to learn if OX40 augmented mature T cell function in ocular inflammation, and also if introduction of monoclonal anti-chemokines would neutralize OX40 stimulated T lymphocytes. OVA stimulated mice cause up regulation of OX40 (CD 134) Two groups of DO11.10 transgenic mice were selected for this initial experiment to test the affinity for OVA antigen challenge and if antigen stimulation can up regulate OX40. One 10 group of DO11.10 mice was injected with 100µg OVA323-339 peptide in PBS and the other group of mice was injected with bovine serum albumin (BSA) as a control (n=3 per group). Twenty four hours after intravitreal injection of OVA or BSA, eyes were harvested from both groups and analyzed for OX40 production. Real time PCR was used to determine OX40 expression. The OVA peptide group showed a three-fold increase of OX40 mRNA expression over the BSA control group (Fig. 1). Flow cytometry was also employed to characterize OX40 expression and CD4+ expression with OVA vs. no OVA stimulation. CD4+ FITC and OX40 PE labels illustrate a 20% increase of OX40 expression in the total cell population among the OVA stimulated DO11.10 eye tissue as opposed to the non-OVA stimulated cells (Fig 2). This characterization and understanding of expression of OX40 after OVA peptide stimulation allowed for further use of the adoptive transfer protocol. Stimulation by OX40 increases uveitis To isolate our study for direct OX40 expression and augmentation of effector T lymphocytes, we again utilized the DO11.10 transgenic mice in an in vivo adoptive transfer model protocol developed in Dr. Zhang’s lab. DO11.10 strain has a T cell receptor specific for OVA peptide. Spleens of DO11.10 were harvested and grown in vitro according to the cell culture section in materials and methods. Splenocytes were grown in culture with 10% FBS and either had 10µg/ml. OVA peptide only (control group) or OVA + 4µg/ml. OX40 activating antibody. Cells were cultured for 72 hours, and then CD4+ cells were selectively isolated via Lympholyte®-M (Cedar Lane Laboratories) and then EasySep Mouse CD4+ selection kit (StemCell Technologies). 1.5x10^7 cells suspended in PBS were transferred to recipient 11 BALB/c mice (n=2) via tail vein injections. 100 µg. OVA peptide (in PBS) challenge with 0.5 μg Escherichia coli strain 055:B5 lipopolysaccharide (Sigma) was administered to vitreous chamber of each mouse eye. Twenty four and 72 hours post antigen challenge, 150 µl rhodamine was injected intraperitoneally immediately before video microscopy and inflammation of the uvea was assessed and quantified 24 hours and 72 hours post OVA challenge. This adoptive transfer model allows for isolation of studying OX40 with limited variables. The use of transgenic DO11.10 mice allows for antigen recognition to be limited to OVA peptide. In vitro priming of donor cells with OVA and OX40 eliminates many variables associated with in vivo priming. Also, the isolation and purification of lymphocytes via Lympholyte-M and CD4+ selection kit allows for the adoptive transfer of only the adaptive immunity of that very specific T cell subset of CD4+ OVA (and OX40) stimulated effector T cells. This use of transgenic mice and CD4+ lymphocytes eliminates variables associated with other immune cell function and physiological adaptations. This protocol allows for an OX40 focused pathogenesis. Consistent with earlier studies, intravitreal introduction of OVA increases leukocyte migration to the eye of the recipient BALB/c mice and in turn increases overall inflammation of eye. Similar to previous studies, the greatest augmentation of inflammation occurred 24 hours post antigen challenge. This study showed substantial inflammation at the 24 hour point, as well as lingering inflammation at the 72 and 96 hour post-OVA challenge (96 hour data not shown). Rollers and stickers continue to adhere to the iris vasculature. The BALB/c recipient mice with the OX40 in vitro primed T cells showed severe uveitis and increased overall leukocyte traffic. The difference in inflammation between the OVA control group and the OVA + OX40 group 12 increases over time until the 72 hour mark when no leukocyte “stickers” are seen in the control group. These results indicate two things. Firstly, OX40 has a significant effect on augmenting effector T cell function and secondly, OX40 continues an inflammatory response over a longer period of time then with just OVA peptide antigen stimulation. Although it is well understood that OX40 expression occurs mainly in activated CD4+ T cell subsets, we made sure to characterize the lymphocyte population after OX40 activating antibody expression. Donor DO11.10 lymphocytes were isolated to characterize these different lymphocyte populations. Cell surface expression of CD4, CD8 and OX40 were analyzed via flow cytometry with gated OX40 to compare CD4 and CD8. The CD4 was labeled with CY7 and CD8 was labeled with FITC. The characterization of the T cell subsets was as predicated with the vast majority of the post-OVA antigen stimulated lymphocytes being CD4+ cells (Fig 3). OX40 activation enhances cytokine production After initial analysis of the intravital microscopy data from the previous immunity adoptive transfer protocol, we further characterized the inflammatory process by analyzing cytokine production of T helper cells after in vitro priming and compared the OVA stimulated group with the OVA + OX40 stimulated group. Using real time PCR, our lab probed the change in eye of Th1, Th2, and Th17 cytokine transcription. All Th cells are CD4+ subsets of T lymphocytes. Cytokines are extracellular signaling molecules that are found to be important in immunoregulation. Chemokines are a subset of cytokines that act as chemoattractants for immune cell migration. Interleukins (IL-17A, IL-21, etc) are a family of cytokines that are 13 expressed by CD4+ T helper cells which signal for stimulation of development and migration of T and B cells to the site of inflammation (i.e. ocular tissue of the eye after OVA peptide challenge) (3). We measured the increase of mRNA for IL-17A, IL-21, IL-22, IFN-γ and CCL20. The OVA + OX40 group was measured in “fold increase” over the OVA only group. Results show a 3.5X to 7X increase in cytokine production for all probed cytokines (Fig 9). Adoptive transfer of OX40 activating antibody stimulated donor CD4+ cells not only increased leukocyte migration and inflammation in eye tissue, but also augmented T helper cell cytokine expression, which, in turn, can act in a positive feedback loop to contribute to further leukocyte infiltration into the eye during uveitis. OX40 activation of mature T-cells further increases uveitis While the previous adoptive transfer experiment provided good characterization of the effect of OX40 on an immune response in our DO11.10 transgenic and OVA challenge model and increased our knowledge base on the co-stimulatory molecule OX40, still to be better understood is the effect of OX40 on effector T cell function in uveitis. It has been shown in previous studies that OX40 is implicated in re-activation of antigen-experienced T cells. We proposed that OX40 (+ OVA) stimulation of mature T cells will produce an increased immune response over naïve effector T cells also stimulated with OX40 (+ OVA). To test this, we utilized a similar adoptive transfer protocol. DO11.10 mice (n=7-8) were primed with 100 µg. OVA peptide via intraperitoneal injection for 4 to 5 weeks. After this time period, spleen cells were harvested from OVA challenged mice, as well as spleen cells from non-OVA challenged mice (with naïve lymphocytes). Both groups were treated with OVA + OX40 activating antibody to stimulate both naïve and antigen-experienced T cells. Following 72 hours of in vitro 14 culture, CD4+ lymphocytes were isolated, purified and adoptively transferred by previously explained techniques. Uveitis was assessed via intravital microscopy and the OVA primed effector T cell group with OX40 stimulation showed substantially more inflammation over the OX40 stimulated naïve T cell group (Fig. 8). Then, we compared the profile of OX40 expression between naïve and antigen-experienced CD4+ lymphocytes of DO11.10 mice in response to OVA. As determined by flow cytometry, OX40 expression of DO11.10 lymphocytes was similar between naïve cells and mature antigen experienced cells (data not shown). Not only did the mature (antigen-experienced) T cells elicit a greater inflammatory response, but effector function was more rapid then with naïve T cells (Fig. 11). Characterization of the OX40 profile of re-activated T cells was performed against naïve cell expression. For this, mRNA from ocular tissue was isolated to examine OX40 augmentation of effector molecules (IFN-γ, CCL20, IL-17A) between the naïve T cells with OVA+OX40 ab. and the OVA primed T cells with OVA+OX40 ab. The real time PCR expression of IFN-γ, IL17A, IL-21, IL-22, and CCL20 was analyzed between the two groups. The OVA primed group showed a minimum of 4-5 fold increase in cytokine expression among IL-22, CCL20, and IFN-γ and with a maximum of ~30 fold increase in IL-21 expression. Neutralization of IFN-γ and IL-17ab showed decreased inflammatory response The last argument regarding stimulation of effector molecules was tested via a neutralization experiment. For this we compared the impact of these effector molecules (and in particular IFN-γ and IL-17A) in the pathogenesis of uveitis. Primed DO11.10 T lymphocytes were isolated and grown in vitro in two groups: one group with OVA + OX40 activating antibody and the other group with OVA alone. Upon adoptive transfer to BALB/c mice, each of 15 the two previously described groups were further sub-divided into three separate groups of mice received OVA + 100 µg. of α-IL-17A, OVA + 100 µg. of α-IFN-γ monoclonal antibody (R&D Systems) or just OVA treatment to give a total of six different groups of mice. Intravital microscopy assessed ocular leukocyte traffic 24 hours post antigen challenge and adoptive transfer. The results show significant decrease in an inflammatory response with introduction of anti-IL-17A and anti-IFN-γ to both the OVA + OX40 and OVA only groups (Fig.12, Fig. 13) for both rolling and adhering leukocyte. Intravital imaging shows noticeable decrease in inflammation with rhodamine labeled leukocytes (Fig. 14, 15, 16). This quantification allows for further characterization of the importance of cytokines in the T cell signaling pathways in uveitis. Discussion: In this study we demonstrate that up-regulation of OX40 augments the effector function of naïve and mature antigen-experienced lymphocytes which, in turn, increases the severity of OVA-induced uveitis. T cell activation, differentiation and migration to the eye via OX40 stimulation is integral to the ocular inflammation. The eye possesses a blood-ocular barrier which subdues and sometimes completely eliminates an immune response. It is only during a great physical disruption of this barrier by trauma, infection or severe internal ocular inflammation, such as the OVA antigen stimulation, that this blood-ocular barrier can be overcome and an immune response can be delivered to the site of antigen presentation (8). With severe immunological stimulation, leukocytes can penetrate into the ocular tissue. Lymphocytes in particular are crucial to immunological responses including the development of uveitis. 16 The adoptive transfer protocol used simulated the negative selection process of T cells in the thymus as they would if they were derived from the same mouse. Adoptive transfer of CD4+ cells in tail vein injections simulated the pathological process of lymphocytes penetrating the blood-ocular barrier, and the intraocular injection of OVA peptide into the vitreous chamber of the eye simulated trauma or infection. T cell activation and differentiation occur via signaling of both the T cell receptor (TCR) and a secondary co-stimulatory molecule. TCR is activated by presentation of antigen fragments by major histocompatibility complex (MHC class II) transmembrane proteins delivered on dendritic cells and other antigen presenting cells (1,3). The co-binding of TCR with CD4 increases the intracellular signal strength. Also, for helper T cells (CD4+) and cytotoxic T cells (CD8+), co-binding of CD4 or CD8 with TCR increases intracellular signaling. However, as was determined in preliminary experimentation in the DO11.10 mice, the T cell population with OX40 expression in the uveitis model was almost exclusively CD4+ cells. The secondary co-stimulatory molecule in this study that augments effector function of CD4+ and CD8+ cells after initial activation by antigen presentation to TCR is OX40 (CD134). OX40 ultimately activates the NF-κB signaling pathway to induce more robust proliferation and survival in cells (1). NF-κB signaling ultimate activates PKC which functions to allow NF-κB to act as a transcription factor in eliciting an adaptive immune response. Despite the current knowledge associated with OX40 acting as a co-stimulatory molecule in T cell stimulation and proliferation, less was known before this study regarding lymphocyte function, with OX40 costimulation in ocular inflammation. Experimentally, the adoptive transfer protocol fits our desired pathological means. The DO11.10 mice have transgenic TCR to specifically bind to OVA peptide. What was learned is that OX40 increases activation and proliferation of T helper 17 cell populations in vitro, and this stimulated effector function augmented an immune response upon tail vein adoptive transfer and ocular OVA challenge to recipient BALB/c mice. Not only did the OX40 stimulated immune response show increased inflammation, but also an increase in duration of the immune response. Furthermore, T helper cell cytokine production from ocular tissue was increased among the group with OX40 activation and can be attributed in part to the NF-κB signaling pathway (1). Some of these cytokines include IL-17A and IFN-γ and increased expression of cytokines further elicits lymphocyte migration to the site of inflammation. Not only does OX40 augment initial T cell effector function in the DO11.10 adoptive transfer protocol, but it is also known to protect T cells during the contraction phase and eventual cell death. After inflammation has subsided, the majority of T cells that would perform cellular apoptosis would survive under conditions with OX40 molecule present. Therefore, a larger number of mature (antigen experienced) T cells can be delivered more readily upon subsequent future events of uveitis. This was demonstrated in the studies performed in which the OVA primed T cells from DO11.10 mice showed not only a greater overall inflammatory response, but also a much quicker response (Fig. 11) to re-develop effector function and ocular inflammation. This may explain the clinical observation of how uveitis patients are commonly known to develop recurrent episodes of eye inflammation. Furthermore, cytokine expression among reactivated T cells shows increased expression. Further sub-categories of T helper cells include Th1, and Th17 cells which are known in the literature to emphasize production of IFN-γ and IL-17A cytokines respectively (1). This increased cytokine production is observed in the results of both adoptive transfer studies (naïve OVA only vs. naïve OVA+ OX40 and naïve vs. mature T cells). Interestingly, while it was originally thought that Th17 cells inhibited Th1 cells (and subsequent expression of IFN-γ), this 18 turns out to not be the case (1). Recent studies have demonstrated that some Th17 cells coexpress IFN-γ and do not inhibit Th1 expression (in fact, it has been found that Th1 cells are required for Th17 cell proliferation.). OX40 acts as an anti-apoptotic agent during the contraction phase of lymphocytes, it augments T cell proliferation and migration to sites of inflammation (as seen in the initial adoptive transfer study) and it signals for increased cytokine production among CD4+ T helper cells, and in particular Th1 and Th17 lymphocytes. In summary, we have demonstrated that increased OX40 increased overall effector function in lymphocytes. If CD4+ T cells were stimulated by OX40 and adoptively transferred to non-transgenic BALB/c mice, a greater inflammatory response was observed in OVA peptide antigen-induced uveitis. Whereas inhibition of effector cytokine, such as IFN- and IL-17A with monoclonal anti-IFN-γ and anti-IL-17A reduced ocular inflammation showing the importance of cytokine production in the pathogenesis of uveitis. Thus, further validation and characterization of the role of OX40 in the proliferation, migration, cell survival and reactivation of memory T cells has an important implication for understanding the pathology of uveitis and more generally lymphocyte biology. 19 References: 1. Z. Zhang, W. Zhong, D. Hinrichs, X. Wu, A. Weinberg, M. Hall, D. Spencer, K. Wegmann, and J.T. Rosenbaum. Activation of OX40 Augments Th17 Cytokine Expression and Antigen Specific Uveitis. American Journal of Pathology 177(6):2912-2920, 2010. 2 . Z. Zhang, W. Zhong, M.J. Hall, P. Kurre, D. Spencer, A. Skinner, S. O’Neill, Z. Xia, and J.T. Rosenbaum. CXCR4 but not CXCR7 is mainly implicated in ocular leukocyte trafficking during ovalbumin-induced acute uveitis. Experimental Eye Research 89(4):52231, 2009. 3 . Janeway, Charles, Paul Travers, Mark Walport, Mark Shlomchik. Immunobiology. 5th. New York, NY: Garland Publishing, 2001. Print. 4 . W. Zhong, Z. Zhang (Corresponding and co-first author), D. Hinrichs, M. Hall, Z. Xia, and J.T. Rosenbaum. OX40 induces CCL20 expression in the context of antigen stimulation: An expanding role of co-stimulatory molecules in chemotaxis. Cytokine 50(3):253-259, 2010. 5 . Z. Zhang, J.T. Rosenbaum, W. Zhong, C. Lim, and D.J. Hinrichs. Co-stimulation of Th17 cells. Seminars in Immunopathology 32(1):55-70, 2010. 6 . Zhang, W. Zhong, D. Spencer, H. Chen, H. Lu, and J.T. Rosenbaum. Interleukin-17 causes neutrophil mediated inflammation in ovalbumin-induced uveitis in DO11.10 mice. Cytokine 46:79-91, 2009. 7 . W. Zhong, J. Kolls, H. Chen, F. McAllister, P. Oliver, and Z. Zhang. Chemokines orchestrate the crescendo of leukocyte trafficking in inflammatory bowel Disease. Frontiers in Bioscience 13:1654-1664, 2008. 8. Nussenblatt RB: The natural history of uveitis. Int Ophthalmol 1990, 14:303–308 20 9. Becker MD, Adamus G, Davey MP, Rosenbaum JT: The role of T cells in autoimmune uveitis. Ocul Immunol Inflamm 2000, 8:93–100 21 Figures: 5 Fold increase (OVA vs control) 4 3 2 1 0 OX40/beta-actin mRNA 1 Figure 1: Fold increase of OX40 expression of OVA peptide stimulated (100µg OVA) uveitis in eye tissue of BALB/c mice vs. non-OVA stimulated (BSA) eye tissue. Figure 2: Flow cytometry characterization of T cell CD4+ subset prior to adoptive transfer protocol. The left flow chart is with no OVA stimulation of DO11.10 transgenic T cells and little expression of CD4 and OX40 is present. However, after OVA stimulation, the population of T lymphocytes expressing CD4+ and OX40 (CD 134) increased in total cell population percentage by 20%. This indicates proper and substantial stimulation of T cell development. 22 Data.009 OVA 72h 4 10 98.58% 0.29% 3 CD4 CY7 10 2 10 1 10 0.40% 0.73% 0 10 0 10 1 10 2 10 3 10 4 10 CD8 FITC Figure 3: Analysis and characterization of lymphocyte populations post adoptive transfer and antigen stimulation of effector T cells. CD4 was labeled with CY7 and CD8 was labeled FITC. T cell subset is vastly CD4+ at >98.5% and minimally CD8+ cells. This shows the correct understanding of T cell subset and further characterization of OX40 to further the study. Figure 4: Black and white intravital microscopy images taken 24 hours post-adoptive transfer and i.p. antigen challenge of mouse recipients with rhodamine labeled cells. The left image results from OVA stimulation only, while the right image is effector T cells stimulated with OVA peptide + OX40 activating antibody. 23 Figure 5: Black and white intravital microscopy images taken 72 hours post-adoptive transfer and i.p. antigen challenge of mouse recipients with Rhodamine labeled cells. The left image results from OVA stimulation only, while the right image is effector T cells stimulated with OVA peptide + OX40 activating antibody. Inflammation is decreased after 4 days. Rollers Flow Rate (cells/mm^2 vessel/min.) 2500 Comparative Concentration of Leukocyte Rollers 2000 1500 OVA Control 1000 OVA + OX40 500 0 24 Hours 72 Hours Hours since Antigen Challenge Figure 6: Initial adoptive transfer protocol to determine relative inflammation of OVA vs. OVA + OX40 stimulation of T cells. Inflammation was quantified via intravital microscopy, ImageJ software analysis and quantification protocol (explained in Materials and Methods). At both time points (24 and 72 hours) OVA + OX40 stimulation have increased inflammation and rollers flow rate. Stickers Concentration (Cells/mm^2) 24 Comparative Concentration of Leukocyte Stickers 250 200 OVA Control 150 OVA + OX40 100 50 0 24 Hours 72 Hours Hours since Antigen Challenge Figure 7: Similar to Figure 6. Initial adoptive transfer protocol to determine relative inflammation of OVA vs. OVA + OX40 stimulation of T cells based on leukocyte sticker concentration. Inflammation quantified via intravital microscopy, ImageJ software analysis and quantification protocol (explained in Materials and Methods). At both time points (24 and 72 hours) OVA + OX40 stimulation have much greater inflammation. 25 Fold Increase of OVA + OX40 over OVA Figure 8: Black and white intravital microscopy images taken 24 hours post-adoptive transfer and i.p. antigen challenge of mouse recipients with rhodamine labeled cells. The left image results from OVA + OX40 stimulation of naïve T cells, while the right image is effector T cells stimulated with OVA peptide + OX40 activating antibody. Inflammation is greatly increased with adoptive transfer of mature effector T cell immunity. OVA+OX40Ab vs OVA 9 8 7 6 5 4 3 2 1 0 IL-17A IL-21 IL-22 IFN-r CCL20 mRNA expression Figure 9: Real time PCR comparative study. Ocular tissue mRNA was harvested and the effector molecules IL-17A, IL-21, IL22, IFN-γ, and CCL20 were analyzed with primers described in Materials and Methods; Real Time PCR section. Bars show a fold increase of effector molecules mRNA expressed in the in vitro stimulated OVA+OX40 group over the OVA only in vitro stimulated group. 26 Fold increase ( Memory vs naive T cells) 35 30 Memory vs. Naive T cells treated with OVA + OX40 Ab. 25 20 15 10 5 0 IL-17A IL-21 IL-22 IFN-r CCL20 mRNA expression Figure 10: Similar to Figure 9. Same protocol followed as outlined in Materials and Methods; Real Time PCR section. However, this study is the fold increase of effector molecule mRNA of the re-activated memory T cells over the naïve T cells (both groups treated with OVA + OX40). 27 % total CD4+ population 70 Naïve 60 OVA primed 50 40 30 20 10 0 0 6 12 24 48 Hours Figure 11: Profiling of OX40 expression in naïve and antigen experienced CD4+ T cells from DO11.10 mice. Some DO11.10 mice were challenged with 100 µg OVA intraperitoneally. Four weeks later, splenocytes were isolated from the antigen experienced mice and naïve animals for a comparison. These cells were further stimulated with OVA peptide (5 µg/ml) for up to 48 hours. Cell surface CD4 and OX40 expression were analyzed by flow cytometry. The plot represents the mean of the percentage of OX40+CD4+ cells from two independent studies. 28 Leukocyte Roller Flow Rate of Anti-Chemokine Study Roller Flow Rate (#cells/min/mm^2) 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 OVA + OX40 OVA + OX40 OVA + OX40 OVA control control + α-IFN-γ + α-IL17 OVA + α-IFN-γ OVA + α-IL17 OVA vs. OVA + OX40 Variables Figure 12: Neutalization experiment intravital microscopy quantification. Roller flow rate was calculated according to Materials and Methods; Intravital Microscopy Quantifications and Calculations section. There were two main groups (OVA vs. OVA+OX40 group) with 3 subgroups each. OVA+OX40 control group was compared to the OVA +OX40 +anti-IFN-γ and the OVA +OX40 +anti-IL-17A. Similarly, the OVA control group was directly compared to the OVA+ anti-IFN-γ and the OVA+ antiIL-17A group. Leukocyte Sticker Concentration of Anti-Chemokine Study Sticker Concentration (#cells/mm^2) 1200 1000 800 600 400 200 0 OVA + OX40 OVA + OX40 OVA + OX40 OVA control OVA + α-IFN- OVA + α-IL17 control + α-IFN-γ + α-IL17 γ OVA vs. OVA + OX40 Variables Figure 13: Similar to Figure 11. However, this is quantification of the vasculature adhering cells. 29 Figure 14: Intravital microscopy images of the neutralization experiment. This is the OVA control group and the OVA + OX40 control group. Compared to Figures 14 and 15 for analysis. Figure 15: Intravital microscopy images of the neutralization experiment. This is the OVA+ anti-IL-17A group and the OVA + OX40+ anti-IL-17A group. Compared to Figures 13 and 15 for analysis. 30 Figure 16: Intravital microscopy images of the neutralization experiment. This is the OVA+ anti-IFN-γ group and the OVA + OX40+ anti-IFN-γ group. Compared to Figures 13 and 14 for analysis.