Targeting Dipeptidyl Peptidase IV (CD26) Suppresses Autoimmune Encephalomyelitis and Up-Regulates TGF-1 Secretion In Vivo 1 Andreas Steinbrecher, 2 * Dirk Reinhold, ‡ Laura Quigley,* Ameer Gado,* Nancy Tresser, § Leonid Izikson,* Ilona Born, ¶ Ju ¨ rgen Faust, ¶ Klaus Neubert, ¶ Roland Martin, ² Siegfried Ansorge, ‡ and Stefan Brocke 3 * CD26 or dipeptidyl peptidase IV (DP IV) is expressed on various cell types, including T cells. Although T cells can receive activating signals via CD26, the physiological role of CD26/DP IV is largely unknown. We used the reversible DP IV inhibitor Lys[Z(NO 2 )]-pyrrolidide (I40) to dissect the role of DP IV in experimental autoimmune encephalomyelitis (EAE) and to explore the therapeutic potential of DP IV inhibition for autoimmunity. I40 administration in vivo decreased and delayed clinical and neuropathological signs of adoptive transfer EAE. I40 blocked DP IV activity in vivo and increased the secretion of the immu- nosuppressive cytokine TGF-1 in spinal cord tissue and plasma during acute EAE. In vitro, while suppressing autoreactive T cell proliferation and TNF- production, I40 consistently up-regulated TGF-1 secretion. A neutralizing anti-TGF-1 Ab blocked the inhibitory effect of I40 on T cell proliferation to myelin Ag. DP IV inhibition in vivo was not generally immunosuppressive, neither eliminating encephalitogenic T cells nor inhibiting T cell priming. These data suggest that DP IV inhibition represents a novel and specific therapeutic approach protecting from autoimmune disease by a mechanism that includes an active TGF-1-mediated antiinflammatory effect at the site of pathology. The Journal of Immunology, 2001, 166: 2041–2048. O ur current model for the initiation of T cell-mediated inflammatory disease of the CNS includes peripheral Ag-specific T cell activation and Th1 differentiation (1– 3). A peripheral T cell activation step appears to be required for autoreactive T cells to enter the CNS via the blood-brain barrier (4). The process of lesion formation is further governed by a com- plex pattern of cyto- and chemokine expression on restimulation of autoreactive T cells in situ (5, 6). It is widely accepted that Th1 cells, critical for cell-mediated immunity by their production of IL-2, IFN-, TNF-, and lymphotoxin are involved in the immu- nopathology of organ-specific autoimmune disease (7–9). A role as regulators has been suggested for Th2 cells (10 –12) and cells pro- ducing TGF- (13–16). In addition, the requirement for costimu- latory signals in T cell activation and their potential for modulating T cell-mediated autoimmunity have been clearly established (17, 18). Recent evidence suggests that the cell surface dipeptidyl pep- tidase IV (DP IV, 4 EC 3.4.14.5, CD26) may have a role in T cell activation and homeostasis (19, 20). DP IV is a highly conserved type II integral membrane protein, constitutively expressed on a wide variety of epithelial, endothe- lial, and lymphoid cell types (21). It corresponds to the leukocyte differentiation Ag CD26. On CD4 + T cells, CD26/DP IV is tightly regulated, depending on the state of activation (22), and it is found on T cells activated in vivo and memory T cells (23). DP IV acts as a serine peptidase catalyzing the cleavage of N-terminal dipep- tides from peptides and proteins carrying proline, hydroxyproline, or alanine in the penultimate position (24). A possible role of CD26/DP IV in T cell-mediated immunity is suggested by: 1) its potent costimulatory activity for T cells activated via the TCR (20); 2) its capacity to interact with extracellular matrix molecules (25, 26); and 3) the suggestion that cleavage by DP IV might regulate the function of numerous immunologically relevant pep- tides and proteins, including cytokines and chemokines that carry an X-Pro-motif at the N terminus (24). Clinical observations also link CD26/DP IV to autoimmunity. Elevated numbers of CD26 + CD4 + T cells were described in peripheral blood and ce- rebrospinal and synovial fluids from patients with multiple scle- rosis (27–29), and clinically active rheumatoid arthritis (30, 31), respectively. Recently, the reversible, competitive DP IV inhibi- tors, Lys[Z(NO 2 )]-pyrrolidide (I40) and Lys[Z(NO 2 )]-thiazolidide (I49) have been extensively analyzed in vitro. They specifically and dose-dependently suppress proliferation and secretion of var- ious cytokines by human and murine T cells (32–34). Interestingly, it is well documented that these inhibitors also induce a 3- to 4-fold *Neurological Diseases and ² Cellular Immunology Sections, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; ‡ Institute of Experimental Internal Medicine, Depart- ment of Internal Medicine, Otto-von-Guericke University, Magdeburg, Germany; § Office of the Clinical Director, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; and ¶ Institute of Bio- chemistry, Department of Biochemistry and Biotechnology, Martin-Luther-Univer- sity Halle-Wittenberg, Halle (Saale), Germany Received for publication December 7, 1999. Accepted for publication November 6, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 A.S. was a postdoctoral fellow (Ste 813/1-1) of the Deutsche Forschungsgemein- schaft. D.R., I.B., J.F., K.N., and S.A. were supported by the Deutsche Forschungs- gemeinschaft Grant SFB 387. 2 Address correspondence and reprint requests to Dr. Andreas Steinbrecher, Depart- ment of Neurology, University of Regensburg, Universitaetsstrasse 84, 93053 Re- gensburg, Germany. E-mail address: andreas.steinbrecher@klinik.uni-regensburg.de 3 Current address: Department of Pathology, Hadassah Medical School, The Hebrew University, P.O. Box 12272, Jerusalem 91120, Israel. 4 Abbreviations used in this paper: DP IV, dipeptidyl peptidase IV; EAE, experimen- tal autoimmune encephalomyelitis; MBP, myelin basic protein; PLP, proteolipid pro- tein; LNC, lymph node cells; Z(NO 2 ), 4-nitrobenzyloxycarbonyl; I40, Lys[Z(NO 2 )]- pyrrolidide; I49, Lys[Z(NO 2 )]-thiazolidide; KLH, keyhole limpet hemocyanin. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00