A multiscale model of interleukin-6-mediated immune regulation in Crohn's disease and its application in drug discovery and development.

In this study, we have developed a multiscale systems model of interleukin (IL)-6-mediated immune regulation in Crohn's disease, by integrating intracellular signaling with organ-level dynamics of pharmacological markers underlying the disease. This model was linked to a general pharmacokinetic model for therapeutic monoclonal antibodies and used to comparatively study various biotherapeutic strategies targeting IL-6-mediated signaling in Crohn's disease. Our work illustrates techniques to develop mechanistic models of disease biology to study drug-system interaction. Despite a sparse training data set, predictions of the model were qualitatively validated by clinical biomarker data from a pilot trial with tocilizumab. Model-based analysis suggests that strategies targeting IL-6, IL-6Rα, or the IL-6/sIL-6Rα complex are less effective at suppressing pharmacological markers of Crohn's than dual targeting the IL-6/sIL-6Rα complex in addition to IL-6 or IL-6Rα. The potential value of multiscale system pharmacology modeling in drug discovery and development is also discussed.CPT: Pharmacometrics & Systems Pharmacology (2014) 3, e89; doi:10.1038/psp.2013.64; advance online publication 8 January 2014.

Utility of animal models for identification of potential therapeutics for rheumatoid arthritis.

Animal models of rheumatoid arthritis (RA) are widely used for testing potential new therapies for RA. However, the question of which animal model is most predictive of therapeutic efficacy in human RA commonly arises in data evaluation. A retrospective review of the animal models used to evaluate approved, pending RA therapies, and compounds that were discontinued during phase II or III clinical trials found that the three most commonly used models were adjuvant-induced arthritis (AIA) in rats and collagen-induced arthritis (CIA) in rats and mice. Limited data were found for more recently developed genetically modified animal models. Examination of the efficacy of various compounds in these animal models revealed that a compound's therapeutic efficacy, rather than prophylactic efficacy, in AIA and CIA models was more predictive of clinical efficacy in human RA than data from either model alone.

Correlation of the epitopes defined by anti-CD26 mAbs and CD26 function.

To clarify the different anti-CD26 mAbs corresponding different functions of CD26, the correlation of the epitopes defined by anti-CD26 mAbs and the functions of CD26 have been studied. Using truncated, human-rat CD26 swap mutants and cross-blocking studies, 13 anti-CD26 mAbs were divided into 5 separate groups. These 5 epitopes were localized between the 1-247th, 248-358th, 359-449th (closer to the 359th amino acid), 450-577th and 359 653th amino acid regions. MAbs against two of these five epitopes, the 248-358th and 359-449th amino acid regions, were associated with inducing modulation of CD26 and T-cell costimulation through the CD3 pathway. Furthermore, mAbs against one of these epitopes, the 359-449th amino acid region, appeared to encompass the ADA binding domain. Analysing the avidity of each mAb to the CD26 molecule using DPPIV enzymatic activity as an indicator, we found that the function of CD26 had little correlation with the avidity of anti-CD26 mAbs, suggesting that distinct epitopes defined by anti-CD26 mAbs appeared to be associated with different functions of CD26. These results will be very useful in the further definition of the functional domains of CD26.

Cross-linking of CD26 by antibody induces tyrosine phosphorylation and activation of mitogen-activated protein kinase.

CD26, a T-cell activation antigen that has dipeptidyl peptidase IV activity in its extracellular domain and has also been shown to play an important role in T-cell activation. The earliest biochemical events seen in stimulated T lymphocytes activated through the engagement of the T-cell receptor (TCR) is the tyrosine phosphorylation of a panel of cellular proteins. In this study we demonstrate that antibody-induced cross-linking of CD26-in CD26-transfected Jurkat cells induced tyrosine phosphorylation of several intracellular proteins with a similar pattern to that seen after TCR/CD3 stimulation. Herbimycin A, an inhibitor of the src family protein tyrosine kinases dramatically inhibited this CD26-mediated effect on tyrosine phosphorylation. Major tyrosine phosphorylated proteins were identified by immunoblotting, and included p56lck, p59fyn, zeta associated protein-tyrosine kinase of 70,000 MW (ZAP-70), mitogen-activated protein (MAP) kinase, c-Cb1, and phospholipase C gamma. CD26-induced tyrosine phosphorylation of MAP kinase correlated with increased MAP kinase activity. In addition, CD26 was costimulatory to CD3 signal transduction since co-cross-linking of CD26 and CD3 antigens induced prolonged and increased tyrosine phosphorylation in comparison with CD3 activation alone. We therefore conclude that CD26 is a true costimulatory entity that can up-regulate the signal transducing properties of the TCR.

Determination of adenosine deaminase binding domain on CD26 and its immunoregulatory effect on T cell activation.

CD26, a 110-kDa cell surface glycoprotein, exhibits dipeptidyl peptidase IV enzyme activity and plays an important role in T cell costimulation. In the present study, we examined both the exact adenosine deaminase (ADA) binding domain on CD26 and the functional consequences of mutated CD26 transfectants that were deficient for cell surface ADA. Using CD26 deletion, human-rat swap, and point mutations, we found that the residues of L340, V341, A342, and R343 on the CD26 molecule were essential amino acids for ADA binding. When these amino acids were mutated and transfected into Jurkat cells, the resultant CD26 transfectants expressed only CD26, not ADA, on the cell surface. The amount of IL-2 produced by wild-type and mutated CD26 transfectants was almost the same following stimulation with anti-CD3 plus PMA. However, the mutated CD26 transfectants were much more sensitive to the inhibitory effect of adenosine on IL-2 production than were the wild CD26 transfectants. These data suggest that ADA on the cell surface does not directly involve T cell activation. Conversely, CD26 alone does not result in modulating the inhibitory effect of adenosine. Only the ADA bound to CD26 on the cell surface was functional and could counteract the inhibitory effect of elevated extracellular adenosine.

Requirement of Fas(CD95), CD45, and CD11a/CD18 in monocyte-dependent apoptosis of human T cells.

Our previous studies demonstrated that upon activation, monocytes (Mo) were able to sensitize peripheral blood T (PBT) cells to apoptosis induced by treatment with PMA. However, it is unknown what gene products provide the death signal to the sensitized PBT cells and how activated Mo enable PBT cells to become susceptible to apoptosis. Here, we show that PBT cells, but not Mo, express functional Fas ligand upon treatment with PMA. Moreover, this Mo-dependent T cell apoptosis could be blocked by a Fas-Ig fusion protein, as well as by a nonlytic mAb against Fas molecule. These results strongly suggest involvement of Fas-Fas ligand interaction in the death of PBT cells. Unlike Fas-induced apoptosis, however, Mo-dependent T cell death was completely inhibited by overexpression of the Bcl-2 protein, and PMA alone was sufficient to trigger apoptosis in T cells when Mo were included in culture. Furthermore, anti-CD11a, anti-CD18, or anti-CD45/CD45RA mAbs; could prevent PBT cells from death triggered by PMA plus Mo, suggesting that these Ags participate in the apoptotic process. The participation of CD45RA in the death of PBT cells was further demonstrated by the observation that the J45.01 cell line, a CD45-deficient variant of Jurkat cells, did not undergo apoptosis by this Mo-dependent mechanism. When transfected with cDNA encoding CD45RA, J45.01 cells acquired apoptotic response to PMA stimulation in the presence of Mo to a similar, but lesser, degree than normal Jurkat cells.

Characterization of adenosine deaminase binding to human CD26 on T cells and its biologic role in immune response.

CD26, a T cell activation Ag, also known as dipeptidyl peptidase IV, is directly associated with adenosine deaminase (ADA) on the surface of T cells and T cell lines. In the present study, we examined both the binding of ADA and CD26 and the functional consequences of this interaction. We found that ADA was associated with CD26 on T cell lines lacking either ADA or dipeptidyl peptidase IV enzymatic activity, indicating that the association between dipeptidyl peptidase IV and ADA did not require enzymatic activity. Moreover, using immunoelectron microscopy, we demonstrated that CD26 and ADA co-localized on the cell surface, but not inside cells, suggesting that CD26 did not transport ADA to the surface. In keeping with this observation, we showed that human CD26-transfected murine pre-B cell lines lacking human ADA acquired ADA from an extracellular source. More importantly, adenosine in the absence of cell surface ADA inhibited T cell proliferation and IL-2 production induced by various stimuli. On the other hand, cells expressing ADA and CD26 on the surface were much more resistant to the inhibitory effect of adenosine. These data suggest that ADA on the cell surface is involved in an important immunoregulatory mechanism by which released ADA binds to cell surface CD26, and this complex is capable of reducing the local concentration of adenosine.

Enzymatic activity of CD26 (dipeptidylpeptidase IV) is not required for its signalling function in T cells.

CD26 is a proteolytic enzyme (dipeptidylpeptidase IV) expressed on the T cell surface that defines an alternative activation signal for human T lymphocytes. Crosslinking of CD26 via monoclonal antibodies triggers proliferation and cytotoxicity in preactivated T cells. In this study, we used highly specific competitive and irreversible inhibitors of dipeptidylpeptidase IV to study the role of the enzymatic activity in activation of CD26-transfected T cells as well as of CD26-expressing normal human T cell clones. These inhibitors at concentrations that blocked up to 95% of the enzymatic activity, did not specifically inhibit T cell activation neither via TCR/CD3 nor via CD26 itself. This demonstrates that the enzymatic activity of CD26 is not required for its T cell activating properties.

Function of dipeptidyl peptidase IV (CD26, Tp103) in transfected human T cells.

CD26 (Tp103) is a proteolytic enzyme (dipeptidyl peptidase IV) expressed on the T cell surface that defines an alternative activation signal for human T lymphocytes. It is absent from or present in only low amounts on resting T cells but it is expressed strongly after activation. Crosslinking of CD26/Tp103 via the monoclonal antibody CB.1 triggers functional activities in preactivated T cells. To study the molecular requirements for T cell activation via CD26 we transfected a cDNA encoding CD26 into several CD26-negative cells. In Jurkat T cell leukemia cells that normally do not express the CD26 antigen, the transfected CD26 molecule is functional because the monoclonal antibody CB.1 induces an increase of cytosolic Ca2+ concentration and IL-2 production. For this stimulatory effect a crosslinking of the monoclonal antibody CB.1 is necessary. After modulation of the TCR/CD3 complex the transfected Jurkat cells were insensitive to triggering via CD26. Moreover, a CD26-transfected TCR-negative variant of Jurkat cells did not respond to CD26 triggering despite high levels of expression of the molecule on their surface. These data demonstrate that the function of CD26/Tp103 is dependent on the expression of the T cell receptor complex. In search of a physiological function of CD26 we found a costimulatory effect of mAb CB.1 in combination with the nonstimulatory anti-CD3 antibody BMA030 and an additive effect in the response to the superantigen staphylococcal enterotoxin E. Transfected Jurkat cells, however, did not show a reproducibly enhanced responsiveness to the superantigen compared to that of untransfected cells.

The T cell triggering molecule Tp103 is associated with dipeptidyl aminopeptidase IV activity.

Tp103 is a 103-kDa T cell activation molecule that defines an alternative activation signal for human T lymphocytes. It is absent from or present in only low amounts on resting T cells but is expressed strongly after activation. Cross-linking of Tp103 via a mAb CB.1 leads to triggering of functional activities in preactivated CD3+ T lymphocytes. By using mAb CB.1 in immunohistology we found that Tp103 is expressed in epithelial cells of various tissues, such as kidney, prostate, epidermis and on endothelia of liver, spleen, lungs, and most vessels, and in bile duct canaliculi in the liver. We found a carcinoma cell line expressing Tp103 and could precipitate a 110-kDa molecule from the surface of these cells. We considered several known molecules of similar distribution and molecular mass for identity with Tp103 and show here that Tp103 is probably identical to the proteolytic enzyme dipeptidyl aminopeptidase IV. When we purified Tp103 by affinity chromatography, typical dipeptidyl aminopeptidase IV activity copurified with Tp103. On activated T cells the enzymatic activity associated with Tp103 is expressed on the outside of the cell. We show that mAb CB.1 recognizes the same molecule as the anti-CD26 mAb anti-Ta1. The anti-Ta1 mAb was found to have T cell-activating activity too, but to differ in its requirements for triggering of T lymphocytes.