TL1A Expression in Human IBD and Animal Models Bala Manickam Pfizer ACVP-STP fellow, Anatomic pathology resident University of Georgia, Athens GA

TL1A & DR3 - Biology TL1A (aka TNFSF15) is a recently identified member of TNF superfamily. Expressed by monocytes, macrophages, dendritic cells, synovial fibroblasts, chondrocytes and endothelial cells Increased expression when stimulated by cytokines, immune complexes and micro-organisms. Its Cell surface receptor DR3 is (aka TNFSF25, WSL-1, TRAMP ands LARD) is mainly expressed by T cells TL1A-DR3 interaction amplifies the DR3 expression and mediates the inflammatory effects. DR3 is a type I cytokine receptor with an intracytoplasmic death domain DcR3 is the decoy receptor that does not have an intracytplasmic domain and hence inhibits the downstream signaling Nat Rev Rheumatol. 2010 Feb;6(2):67-8 Biochem Pharmacol. 2011 Apr 1;81(7):838-47

Inflammatory Bowel Disease Human IBD IBD represents an important chronic disease affecting the GI tract of man and domesticated animal species. The 2 IBD entities in humans are Crohn’s disease (CD) and Ulcerative colitis (UC). Immune mediated - responds to immunomodulatory drugs The pathogenesis of IBD involves: Failure of immune regulation. Genetic susceptibility. Environmental triggers (microbial flora). Disruption of the mucosal barrier. Mouse models of IBD DSS (Dextran sulphate) colitis: Oral administration of this sulphated polysaccharide to mice induces a self limiting colitis TNBS (Trinitrobenzene sufonic acid) colitis: Colonic inflammation is induced by intrarectal administration of TNBS dissolved in 50% ethanol Canine IBD Small intestine: Lymphocytic-plasmacytic enteritis (LPE), eosinophilic enteritis and eosinophilic gastro-enteritis (EGE) Large intestine; Lymphocytic-plasmacytic colitis, eosinophilic colitis, histiocytic ulcerative colitis (HUC) (mainly PAS-positive macrophages), and regional granulomatous colitis (mainly PAS-negative macrophages)

TL1A & DR3 expression in human IBD Healthy Person CD UC TL1A TL1A: CD- lymphocytes and macrophages; UC- Plasma cells DR3: Increased numbers of immunoreactive cells were detected in the lamina propria lymphocytes from involved areas of IBD patients DR3 J Immunol 2003;171;4868-4874

TL1A & DR3 : Evidence of Efficacy Gut bacteria Control anti TL1A TNBS colitis DSS colitis Fig 1: Attenuation of TNBS colitis by anti-TL1A mAb. Fig 2: DSS model colitis; both prophylactic and therapeutic Fig 3: TL1A transgenic mice spontaneously develop colitis; DR3 KO attenuates colitis Proposed mechanism Control DR3 KO GASTROENTEROLOGY 2008;135:552–567 Mucosal Immunology (2011) 4, 172-185;

Rationale for current study Up-regulation of TL1A and DR3 in human CD and UC and in rodent IBD models TL1A and DR3 deficiency attenuates murine IBD IBD occurs spontaneously in dogs. Because of this, and because it shares a similar immunopathology, it may provide valuable mechanistic insight into the disease in humans. Studying IBD in spontaneous and induced models may provide insights into how we model the disease in laboratory mice

Hypothesis By characterizing and comparing expression profile of TL1A and other critical immunological markers in murine IBD to people, we may further validate (and perhaps even uncover) therapeutic targets for human IBD.

Objectives Compare and characterize the staining of TL1A & DR3 in intestinal tissues obtained from DSS and TNBS mouse colitis models, human IBD, canine IBD and normal colons from cynomolgus monkeys. Compare the staining characteristics of mast cells in intestinal tissues obtained from DSS and TNBS mouse colitis models, human IBD and canine IBD. Compare the expression of T&B cells in different models of IBD.

Experimental design Groups Number of subjects Number of slides/subject Total number of slides DSS (Mouse) 5 10 50 TNBS (Mouse) Normal colon (Mouse) UC (Human) 6 36 CD (Human) Normal colon (Human) Normal colon (Primate) 25 IBD (Dog) Normal colon (Dog)

Reagents Anti-human TL1A mAb (made by Pfizer, optimized for IHC) Homology to mouse, rat: 92% Homology to dog (predicted): 86% Anti-human DR3 mAb (made by Novus Biol) The immunogenic peptide shares 44% homology with canine DR3 (XP_546752 ) 88% homology with primate DR3 (XM_003274297 CD3, CD45RB220, CD20, Toluidine blue Pfizer Internal Use

Results: TL1A expression Normal CD UC Human Naïve DSS TNBS Scattered cells within the lamina propria of the normal/naïve are positive for TL1A TL1A expression is increased in IBD conditions in both human cases as well as in murine models. A moderate to variably strong staining of the cytoplasm and the membrane of the Non T, non B, Non neutrophilic cell type was consistently present within the inflamed areas in mice and humans Mice

Results: TL1A expression Cynomolgus Human Mice Vasculature (+) Germinal center (-) TL1A expression in the vasculature (endothelial cells) were consistent in cynomolgus, humans and mice tissues and served as an internal control GALT did not stain positive for TL1A in both mice and human IBD tissues DR3: The lymphocytes infiltrating the lamina propria and the submucosa in crohn’s disease were strongly positive for DR3. The staining was localized to the nucleus, cytoplasm and the cell membrane CD Lamina propria DR3

Mast cells in IBD – Dog, Mice Naive IBD H&E T-blue Dog: Only rare mast cells are present within the control and the IBD dogs Mice: Only rare mast cells are present in Naïve, DSS and TNBS colitis; There is no difference in the expression of Mast cells between the groups Naive DSS TNBS

B-lymphocytes-Human IBD Normal CD UC Germinal center Lamina propria GALT hyperplasia is present in patients with CD and UC Increased numbers of B-lymphocytes are present within the lamina propria of the CD and UC patients when compared to normal individual

B-lymphocytes-Mice IBD Naive DSS TNBS Germinal center Lamina propria GALT hyperplasia is present in DSS and TNBS colitis Increased numbers of B-lymphocytes are present within the lamina propria of the CD and UC patients when compared to normal individual Basal level expression of B cells within the normal colon was similar in mice and humans B & T cells within the GALT and the lamina propria were markedly increased in IBD patients when compared to healthy controls that was consistent with mice models

T-lymphocytes-Human IBD Normal CD UC Germinal center Lamina propria GALT hyperplasia is present in patients with CD and UC Increased numbers of T-lymphocytes are present within the lamina propria of the CD and UC patients when compared to normal individual

T-lymphocytes-Mice IBD Naive DSS TNBS Germinal center Lamina propria Increased numbers of B-lymphocytes are present within the lamina propria of the CD and UC patients when compared to normal individual Basal level expression of T cells within the normal human colon was increased when compared to the mice models

B & T lymphocytes- Cynomolgus B-cells T-cells Germinal center There are few scattered B-lymphocytes within the lamina propria of naïve Cynomolgus colon Germinal center stains positive for B-lymphocytes There are few scattered T-lymphocytes within the lamina propria of naïve Cynomolgus colon Lamina propria

Conclusion TL1A: B&T-lymphocytes: Mast cells: The expression of TL1A was similar and consistent across the species TL1A was up regulated in IBD when compared to the Naive/healthy patients This data not only validates the existing murine IBD models but also indicates that TL1A could be a druggable target in IBD We also uncovered that Cynomolgus shares similar staining characteristics with human and murine IBD tissues and thus can probably be a good model to study human IBD B&T-lymphocytes: Increased numbers of both B & T cells in the lamina propria in cases of IBD, suggests a potential role for immune activation in IBD Mast cells: In our study, We could not detect any difference in the expression of mast cells in both naïve and or IBD patients in both murine and Dog cases.

Acknowledgement Dr. Timothy LaBranche (Industry Mentor) Dr. Elizabeth Howerth (Academic Mentor) Dr. Zaher Radi (Pfizer) Dr. Shawn O’ Neil (Pfizer) Jameel Syed (Pfizer) Zachery Stewart (Pfizer) University of Georgia ACVP-STP Coalition Studies were funded and supported by DRSD, Pfizer

Thank you! Pfizer Internal Use