Immunology 1998 95 591-594
`
`FTY720, a novel immunosuppressant, induces sequestration of circulating mature
`lymphocytes by acceleration of lymphocyte homing in rats, III. Increase in
`frequency of CD62L-positive T cells in Peyer's patches by FTY720-induced
`lymphocyte homing
`
`Y. YANAGAWA, Y. MASUBUCHI & K. CHIBA Research Laboratories, Yoshitomi Pharmaceutical Industries Ltd, Iruma,
`Saitama, Japan
`
`SUMMARY
`FTY720, a novel immunosuppressant, sequesters circulating mature lymphocytes, especially
`T cells, within lymph nodes and Peyer's patches by accelerating lymphocyte homing, and thereby
`causes lymphocyte depletion in the blood. The FTY720-induced acceleration of lymphocyte
`homing appears to be mediated by lymphocyte homing receptors including CD62L, CD49d/137,
`and CDl la/CD18. In this study, expressions of CD62L, CD49d and CDl la on T cells in the
`peripheral blood, lymph nodes and Peyer's patches were analysed by flow cytometry in rats given
`FTY720 (1 mg/kg) orally. FTY720 markedly decreased the number of peripheral blood T cells,
`while not affecting CD62L, CD49d and CDl la expressions at 1-3 hr after administration. In
`contrast, both the frequency of CD62L-positive T cells and intensity of CD62L expression on
`T cells were increased in Peyer's patches but not lymph nodes at 3 hr after administration of
`FTY720. CD49d and CDl la expressions on T cells were unaffected by FTY720 in both Peyer's
`patches and lymph nodes at the same point in time. On the other hand, analysis of lymphocyte
`homing with calcein-labelled lymphocytes and anti-CD62L monoclonal antibody (mAb) confirmed
`that FTY720 predominantly increased CD62L-dependent lymphocyte homing to Peyer's patches.
`These findings indicate that FTY720 increases the frequency of CD62L-positive T cells by
`accelerating CD62L-predominant homing in Peyer's patches.
`
`INTRODUCTION
`FTY720 (2-amino-2-[2-(4-octylphenyl )ethyl ]propane-1,3-diol
`hydrochloride), a novel immunosuppressant, was created by
`chemical modification of ISP-I, a derivative of Isaria sinclai-
`rii. 1'2 FTY720 shows more potent immunosuppressive activity
`than cyclosporin A (CsA) and FK506 in skin and cardiac
`allograft models while, unlike CsA and FK506, unaffecting
`lymphocyte proliferation and interleukin-2 (IL-2) production
`vitro.3-
`in rat allogeneic mixed lymphocyte culture in
`In
`addition, FTY720 combined with CsA displays a marked
`synergistic effect in prolonging skin, cardiac and renal allograft
`survival in rats and dogs, suggesting that FTY720 possesses a
`unique mechanism of action.3 9 A striking feature of FTY720
`
`Received 21 May 1998; revised 12 August 1998; accepted 12
`August 1998.
`Abbreviations: PBL, peripheral blood lymphocytes; LN, lymph
`nodes; PLN, peripheral lymph nodes; MLN, mesenteric lymph nodes;
`PP, Peyer's patches; MFI, mean fluorescence intensity; mAb, mono-
`clonal antibody.
`Correspondence:
`Chiba,
`Dr Kenji
`Laboratories,
`Research
`Yoshitomi Pharmaceutical Industries Ltd, 3-7-25, Koyata, Iruma,
`Saitama, 358-0026 Japan.
`© 1998 Blackwell Science Ltd
`
`591
`
`is induction of a marked decrease in the number of circulating
`lymphocytes, especially T cells, at the doses prolonging allo-
`graft survival.5'8 The marked decrease in circulating T cells
`appears to cause a reduction of T-cell recruitment into allo-
`grafts.9 Recently, we reported a mechanism underlying the
`disappearance of circulating lymphocytes in FTY720-treated
`rats.5 As described in our previous report, while markedly
`decreasing the circulating lymphocytes in the bloodstream,
`FTY720 significantly increased the lymphocyte number in
`lymph nodes (LN) and Peyer's patches (PP) at 3-24 hr after
`administration to rats.5 In addition, analysis of lymphocyte
`trafficking using calcein-labelled lymphocytes revealed that
`FTY720 accelerated lymphocyte homing to LN and PP, and
`this acceleration was almost completely inhibited by simul-
`taneous treatment with anti-CD62L, CD49d, and CDl l a
`monoclonal antibodies (mAbs).5 Therefore, we concluded that
`FTY720 sequesters circulating lymphocytes within LN and PP
`by accelerating lymphocyte homing through lymphocyte-
`CD49d/17
`homing
`including
`receptors,
`CD62L,
`and
`CDl la/CD1 8. In this study, to elucidate the relation between
`the acceleration of lymphocyte homing and expressions of
`lymphocyte-homing receptors, we analysed CD62L, CD49d
`and CD lla expressions on T cells in peripheral blood, LN
`and PP by flow cytometry in FTY720-treated rats.
`
`SUN - IPR2017-01929, Ex. 1020, p. 1 of 4
`
`

`

`592
`
`Y. Yanagawa et al.
`
`CD3 mAb (1 F4), biotinylated-anti-rat CD49d mAb (TA-2),
`and streptavidin-Cy-Chrome". CD62L, CD49d and CDlla
`expressions on rat T cells were determined by two-colour flow
`cytometry with EPICS) XL-MCL (Coulter Co. Miami, FL).
`The percentage of positive cells was determined by setting the
`lower limit above the non-specific fluorescence with isotype-
`matched control IgG. The mean fluorescence intensity (MFI)
`was recorded in the linear amplification mode of EPICS® XL.
`
`Analysis oflymphocyte-homing with calcein-labelled
`lymphocytes
`The in vivo lymphocyte-homing was analysed as previously
`described.5 Lymphocytes (1 x 108 cells) from the PLN and
`MLN of F344 rats were labelled by incubation for 30 min on
`ice
`in
`10 ml of RPMI-1640 medium containing 02 ptM
`calcein-AM, as described in previous reports.5 4"5 The calcein-
`labelled lymphocytes were treated with 60,g/ml of hamster
`antirat CD62L mAb (HRL3) or isotype-matched control IgG.
`After treatment with the mAbs, the calcein-labelled lympho-
`cytes (5 x 107 cells/05 ml) were intravenously transfused to
`the rats at 2 5 hr after FTY720-administration. The rats were
`killed at 30 min after the transfusion, and all PP were collected.
`The number of calcein-labelled lymphocytes in PP was deter-
`mined by flow cytometry.
`
`RESULTS
`Effect of FTY720 on T-cell number and CD62L, CD49d and
`CDI la expressions of T cells in peripheral blood
`The number of T cells and expressions of CD62L, CD49d,
`and CDl a on T cells in peripheral blood was analysed by
`flow cytometry at 1-3 hr after oral administration of FTY720
`(1 mg/kg) to rats. The number of T cells in peripheral blood
`
`L .A I
`
`rff.
`
`0
`
`0
`
`0*
`It
`
`IgG
`control
`
`Vehicle
`control
`
`FTY720
`1 mg/kg
`
`MATERIALS AND METHODS
`
`Animals
`Inbred strain male F344 rats (RTIl"l) were purchased from
`Japan Charles River Inc. (Atsugi, Kanagawa, Japan). All rats
`were used at 5-7 weeks of age.
`
`Reagents and monoclonal antibodies
`FTY720 was synthesized by Taito Co., Ltd.' For oral adminis-
`tration, FTY720 was dissolved in distilled water. Control
`animals received the vehicle only. Calcein-AM was obtained
`from Molecular Probes (Eugene, OR). Fluoroscein isothiocy-
`anate (FITC)-conjugated anti-rat CD3 mAb (1 F4)'0
`was
`obtained from Caltag Laboratories (South San Francisco,
`CA). Hamster anti-rat CD62L mAb (HRL-3)" and biotinyl-
`ated mouse anti-rat CD49d (TA-2)12 were purchased from
`Seikagaku-kougyou Ltd. (Tokyo, Japan). Phycoerythrin (PE)-
`conjugated HRL-3, PE-conjugated mouse antirat CDl la mAb
`(WT.1 ),'13 isotype-matched control immunoglobulin G (IgG),
`streptavidin-Cy-ChromeTm
`and
`obtained
`from
`were
`PharMingen (La Jolla, CA).
`
`Flow cytometry
`Peripheral blood was collected from tail vain of F344 rats.
`Peripheral lymph nodes (PLN; axillary lymph nodes), mesen-
`teric lymph nodes (MLN) and PP were removed from rats,
`and single cell suspensions were prepared by mincing and
`passing through stainless mesh. The cells were stained with
`FITC-anti-rat CD3 mAb (1F4), and PE-anti-rat CD62L mAb
`(HRL3) or PE-anti-rat CDl a mAb (WT. 1). For analysis of
`CD49d expression, the cells were stained with FITC-anti-rat
`
`O Control
`* FTY720 1 mg/kg
`
`2
`1
`3
`Hours after administration
`
`(a)
`
`-
`
`( 0
`
`(b)
`
`1
`
`2
`
`3
`
`3
`2
`1
`Hours after administration
`
`1
`
`2
`
`3
`
`Fluorescence Intensity (log)
`
`Figure 1. The number of T cells
`(a) and expressions of CD62L,
`CD49d, and CDl la on T cells (b) in the peripheral blood of control
`and FTY720-treated rats at 1, 2 and 3 hr after administration. Blood
`was collected from the tail vein of the rats at 1, 2 and 3 hr after oral
`administration of FTY720 (1 mg/kg). The number of CD3 + T cells
`and expressions of CD62L, CD49d and CDl la on CD3 + T cells was
`determined by flow cytometry. Each column represents the mean + SE
`of three animals. (**P<0-01, t-test as compared with control.)
`
`Figure 2. Effect of FTY720 on CD62L expression on T cells in PP.
`PP were separated at 3 hr after a single oral administration of FTY720
`(1 mg/kg) to rats. Lymphocytes from PP were stained with FITC-
`anti-rat CD3 (1F4) and PE-anti-rat CD62L mAb (HRL3). As a
`negative control, lymphocytes were treated with isotype-matched
`control IgG. CD62L expression on T cells was determined by two-
`colour flow cytometry. Results are representative of data obtained in
`four pairs of rats.
`
`1998 Blackwell Science Ltd, Immunology, 95, 591-594
`
`SUN - IPR2017-01929, Ex. 1020, p. 2 of 4
`
`

`

`Effects on lymphocyte circulation of FTY720
`
`593
`
`was significantly decreased at 2-3 hr (Fig. la). In contrast,
`FTY720 did not affect CD62L, CD49d and CD lI a expressions
`on peripheral blood T cells at 1-3 hr (Fig. Ib).
`
`Effect of FTY720 on CD62L, CD49d and CDIla expressions of
`T cells in PP and LN
`Expressions of CD62L, CD49d and CD1 la on T cells in PP
`and LN was analysed by flow cytometry at 3 hr after adminis-
`tration of FTY720 (1 mg/kg). In the control rats, the frequency
`of CD62L-positive T cells in PP was lower than in PLN and
`MLN. FTY720 significantly
`the frequency of
`increased
`CD62L-positive T cells in PP but not PLN and MLN (Figs 2
`and 3). Although the data are not shown, all T cells in the
`PLN, MLN and PP were CD49d-and CD1 la-positive in both
`the control and FTY720-treated rats. The intensity of CD62L
`expression on CD62L-positive T cells was also significantly
`increased in PP by FTY720-treatment, while slightly decreasing
`in PLN and MLN (Fig. 4). On the other hand, FTY720 did
`not affect the intensity of CD49d and CD lla expression on
`T cells in PLN, MLN, and PP (Fig. 4).
`
`Effect of anti-CD62L mAb on lymphocyte homing into PP in
`control and FTY720-treated rats
`Lymphocyte homing was assessed by flow cytometry with
`calcein-labelled lymphocytes in rats. Calcein-labelled lympho-
`cytes treated with anti-CD62L mAb were intravenously trans-
`fused into rats 2-5 hr after oral administration of FTY720
`(1 mg/kg). The rats were sacrificed 30 min after the trans-
`fusion, and the number of calcein-labelled lymphocytes in the
`PP was determined by flow cytometry. As shown Fig. 5,
`FTY720 markedly increased lymphocyte homing to PP. When
`treated with anti-CD62L mAb, lymphocyte homing into PP
`was only slightly increased by FTY720. On the other hand,
`anti-CD62L mAb partially decreased lymphocyte homing to
`PP in control rats (by 48%), and markedly decreased it in
`FTY720-trated rats (by 72%). Thus, FTY720 appeared to
`predominantly induce CD62L-dependent lymphocyte-homing
`to PP.
`
`DISCUSSION
`Circulating lymphocytes in the blood home to LN and PP
`through interaction of lymphocyte homing receptors including
`
`0 80
`
`060
`
`13
`CL40
`-jC~4
`~20
`
`0
`
`*
`
`Control IgG
`
`Anti-CD62L mAb
`
`L
`
`TControl
`* FTY720 1 mg/kg
`
`PLN
`
`MLN
`
`PP
`
`10000 20000 30000 40000 60000
`0
`No. of calcein-labelled lymphocytes in PP
`
`O Control U FTY720 I mg/kg
`Figure 3. The percentage of CD62L+ T cells in lymphocytes from
`PLN, MLN and PP of control and FTY720-treated rats at 3 hr after
`oral administration of FTY720. PLN, MLN and PP were separated
`at 3 hr after single oral administration of FTY720 (1 mg/kg) to rats.
`CD62L expressions on CD3+ T cells were determined by two-colour
`flow cytometry. Each column represents the mean+SE of four ani-
`mals. (**P<0 01, t-test as compared with control.)
`
`Figure 5. Effect of anti-CD62L mAb on lymphocyte homing into PP
`in control and FTY720-treated rats. Calcein-labelled lymphocytes
`were treated with 60 gig/ml of hamster anti-rat CD62L mAb or
`isotype-matched control IgG. The calcein-labelled lymphocytes were
`intravenously transfused into the rats at 2-5 hr after administration of
`FTY720 (1 mg/kg). Peyer's patches were collected at 30 min after the
`transfusion, and the number of calcein-labelled lymphocytes in the PP
`was determined by flow cytometry. Each column represents the
`mean + SE of four animals.
`
`Mi
`
`PLN MLN PP
`
`PLN MLN PP
`
`PLN MLN PP
`
`C Control
`* FTY720 I mglkg
`Figure 4. Expressions of CD62L, CD49d and CDl la on T cells in PLN, MLN and PP of control and FTY720-treated rats at 3 hr
`after administration of FTY720. Peripheral lymph nodes, MLN and PP were separated at 3 hr after a single oral administration
`of FTY720 (1 mg/kg) to rats. CD62L, CD49d and CDI la expressions on CD3+ T cells were determined by two-colour flow
`cytometry. Each column represents the mean+SE of four animals. (**P<001, t-test as compared with control.)
`
`© 1998 Blackwell Science Ltd, Immunology, 95, 591-594
`
`SUN - IPR2017-01929, Ex. 1020, p. 3 of 4
`
`

`

`594
`
`Y Yanagawa et al.
`
`CD62L, CD49d/137 and CD1 a/CD 18 to their ligands on
`high endothelial venule (HEV), and then subsequently return
`to the blood again. 16,17 The involvement of respective lympho-
`cyte-homing receptors in lymphocyte trafficking is thought to
`be closely related to the expression pattern of their ligands
`on HEV. glycosylation-dependent cell adhesion molecule-i
`(GlyCAM-1), the CD62L ligand, is expressed on PLN-HEV
`at high level, and on PP-HEV at low level,18 while anti-CD62L
`mAb almost completely inhibits lymphocyte entry into PLN,
`but only partially into PP.'9 Lymphocyte homing into LN is
`more dependent on interaction through CD62L than homing
`molecule-i
`adhesion
`addressin
`cell
`Mucosal
`into
`PP.
`(MAdCAM-1), the CD49d/17 ligand is expressed on PP-HEV
`while lymphocyte entry into PP but not
`but not PLN,20'2
`PLN is markedly inhibited by treatment with anti-CD49d
`anti-07-integrin mAb.'9 Therefore, lymphocyte
`mAb or
`homing into PP is predominantly mediated by CD49d/P7
`rather than CD62L. Consequently, the frequency of CD62L-
`positive lymphocytes in PP is lower than that in LN." In this
`study, FTY720 increased the frequency of CD62L-positive
`T cells in PP (Figs 2 and 3). In addition, FTY720 predomi-
`nantly enhanced CD62L-mediated lymphocyte homing to PP
`when compared with CD62L-independent homing (Fig. 5),
`indicating that FTY720 increased the involvement of CD62L
`in lymphocyte homing to PP. These observations indicate that
`FTY720 increases CD62L-positive T cells in PP by accelerating
`CD62L-predominant lymphocyte homing. On the other hand,
`FTY720 did not affect CD62L expression on T cells, while
`markedly decreasing T-cell numbers in blood (Fig. 1). It is
`possible that the entry of CD62L-positive and CD62L high-
`expressing T cells into PP is enhanced by increase in expression
`CD62L-ligands
`not CD62L in
`of
`on PP-HEV but
`FTY720-treated rats. In contrast to PP, there was no notable
`change in lymphocyte homing receptor expressions on T cells
`in the PLN and MLN of FTY720-treated rats (Figs 3 and 4).
`FTY720 may not affect the involvement of respective lympho-
`cyte-homing receptors in lymphocyte trafficking to PLN and
`MLN, while accelerating lymphocyte homing to these organs.
`Peyer's patches are thought to play a central role in
`immune defence and oral immune tolerance in gut.22'23 In this
`study, we demonstrated that FTY720 modulates the frequency
`of CD62L-possitive T cells in PP, raising the possibility that
`FTY720 affects the immune responses in the gut. The effect
`of FTY720 on T-cell functions in PP are currently being
`analysed. Finally, we believe that FTY720, which has a unique
`mechanism of action, is useful as an immunosuppressive drug
`for organ transplantation and as a tool for investigating
`immune responses.
`
`REFERENCES
`1. ADACHi K., KOHARA T., NAKAO N. et al. (1995) Design, synthesis,
`activity
`relationships
`2-substituted-
`of
`and
`structure
`novel immuno-
`Discovery
`of
`2-amino- 1,3-propanediols:
`a
`suppressant, FTY720. Biomed Chem Lett 5, 853.
`2. FUJITA T., HIROSE R., YONETA M. et al. (1996) Potent immuno-
`suppressants, 2-alkyl-2-aminopropane-1,3-diols. J Med Chem 39,
`4451.
`3. CHIBA K., HOSHINO Y., SUZUKI C. et al. (1996) FTY720, a
`novel immunosuppressant possessing unique mechanisms.
`I.
`Prolongation of skin allograft survival and synergistic effect in
`combination with cyclosporine in rat. Transplant Proc 28, 1056.
`4. HOSHINO Y., SUZUKI C., OHTSUKI M., MASUBUCHI Y., AMANO Y.
`
`& CHIBA K. (1996) FTY720, a novel immunosuppressant
`possessing unique mechanisms. Long-term graft survival induction
`in rat heterotopic cardiac allograft and synergistic effect in combi-
`nation with cyclosporine A. Transplant Proc 28, 1060.
`5. CHIBA K., YANAGAWA Y., MASUBUCHI Y. et al. (1998) FTY720,
`a novel immunosuppressant, induces sequestration of circulating
`mature-lymphocytes by acceleration of lymphocyte homing in
`rats. I. FTY720 selectively decreases the number of circulating
`mature-lymphocytes by acceleration of lymphocyte homing.
`J Immunol 160, 5037.
`6. KAWAGUCHI T., HOSHINO Y., RAHMAN F. et al. (1996) FTY720,
`a novel immunosuppressant possessing unique mechanisms.
`Synergistic prolongation of canine renal allograft survival in
`combination with cyclosporine A. Transplant Proc 28, 1062.
`7. SUZUKI S., ENOSAWA S., KAKEFUDA T., AMEMIYA H., HOSHINO Y.
`& CHIBA K. (1996) Long-term graft acceptance in allografted rats
`and dogs by treatment with a novel immunosuppressant, FTY720.
`Transplant Proc 28, 1375.
`8. SUZUKI S., ENOSAWA S., KAKEFUDA T. et al. (1996) A novel
`immunosuppressant, FTY720, having an unique mechanism of
`action induces long-term graft acceptance in rat and dog allo-
`transplantation. Transplantation 61, 200.
`9. YANAGAWA Y., SUGAHARA K., KATAOKA H., KAWAGUCHI T.,
`MASUBUCHI Y. & CHIBA K. (1998) FTY720, a novel immuno-
`of
`circulating
`sequestration
`induces
`mature-
`suppressant,
`lymphocytes by acceleration of lymphocyte homing in rats. II.
`FTY720 prolongs skin allograft survival by decreasing T cell
`infiltration into grafts but not cytokine production in
`vivo.
`J Immunol 160, 5493.
`10. TANAKA T., MASUKo T., YAGITA H., TAMURA T. & HASHIMOTO
`Y. (1989) Characterization of a CD3-like rat cell surface antigen
`recognized by a monoclonal antibody. J Immunol 142, 2791.
`(1993)
`K.
`T.,
`al.
`F.,
`KITAMURA
`KUIDA
`et
`11. TAMATANI
`Characterization of rat LECAM-1 (L-selectin) by the use of
`monoclonal antibodies and evidence for the presence of soluble
`LECAM-l in rat sera. Eur J Immunol 23, 2181.
`12. ISSEKUTz T.B. (1991) Inhibition of in vivo lymphocyte migration
`to inflammation and homing to lymphoid tissues by the TA-2
`monoclonal antibody. J Immunol 147, 4178.
`13. TAMATANI T., KOTANI M. & MIYASAKA M. (1991) Characterization
`of the rat leukocyte integrin, CD1 l/CD1 8, by the use of LFA-1
`subunit-specific monoclonal antibodies. Eur J Immunol 21, 627.
`14. STEEBER D.A., GREEN N.E., SATO S. & TEDDER T.F. (1996)
`Lymphocyte-migration in L-selectin-deficient mice. Altered subset
`migration and aging of the immune system. J Immunol 157, 1096.
`15. MARTIN D.R. & MILLER R.G. (1989) In vivo administration of
`histoincompatible lymphocytes leads to rapid functional deletion
`of cytotoxic T lymphocyte precursors. J Exp Med 170, 679.
`16. FORD W.L. & GoWANS J.L. (1969) The traffic of lymphocytes.
`Semin Hematol 6, 67.
`17. BUTCHER E.C. & PICKER L.J. (1996) Lymphocyte homing and
`homeostasis. Science 272, 60.
`18. LASKY L.A., SINGER M.S., DOWBENKO D. et al. (1992) An
`endothelial ligand for L-selectin is a novel mucin-like molecule.
`Cell 69, 927.
`(1993)
`M.J., McEvoy L.M. & BUTCHER E.C.
`19. BRISKIN
`MAdCAM-1 has homology to immunoglobulin & mucin-like
`adhesion receptors and to IgA-1. Nature 363, 3.
`20. BERLIN C., BERG E.L., BRISKIN M.J. et al. (1993) a407 integrin
`mediates lymphocyte binding to the mucosal vascular addressin
`MAdCAM-1. Cell 74, 185.
`21. HAMANN A., ANDREW D.P., WESTRICH D.J., HOLZMANN B. &
`BUTCHER E.C. (1994) Role of a4-integrins in lymphocyte homing
`to mucosal tissue in vivo. J Immunol 152, 3282.
`22. BRANDTZAEG P. (1989) Overview of the mucosal immune system.
`Curr Topics Microbiol Immunol 146, 13.
`23. NGAN J. & KIND L.S. (1978) Suppressor T cells for IgE and IgG
`in Peyer's patches of mice made tolerant by the oral administration
`of ovalbumin. J Immunol 120, 861.
`©¢ 1998 Blackwell Science Ltd, Immunology, 95, 591-594
`
`SUN - IPR2017-01929, Ex. 1020, p. 4 of 4
`
`