Integrin Dependent 2β L α But Not 1β4 α Renal Allografts Is T ...

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of April 4, 2018. This information is current as Integrin Dependent 2 β L α But Not 1 β 4 α Renal Allografts Is T Lymphocytes to + Antigen-Reactive CD4 Homing of In Vitro-Generated Donor Kupiec-Weglinski Ritter, Hans-Dieter Volk, Ana J. Coito and Jerzy W. Markus H. Hammer, Yuan Zhai, Masamichi Katori, Thomas http://www.jimmunol.org/content/166/1/596 doi: 10.4049/jimmunol.166.1.596 2001; 166:596-601; ; J Immunol References http://www.jimmunol.org/content/166/1/596.full#ref-list-1 , 6 of which you can access for free at: cites 29 articles This article average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved. Copyright © 2001 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on April 4, 2018 http://www.jimmunol.org/ Downloaded from by guest on April 4, 2018 http://www.jimmunol.org/ Downloaded from

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Integrin Dependent2βLα But Not 1β4αRenal Allografts Is

T Lymphocytes to+Antigen-Reactive CD4Homing of In Vitro-Generated Donor

Kupiec-WeglinskiRitter, Hans-Dieter Volk, Ana J. Coito and Jerzy W. Markus H. Hammer, Yuan Zhai, Masamichi Katori, Thomas

http://www.jimmunol.org/content/166/1/596doi: 10.4049/jimmunol.166.1.596

2001; 166:596-601; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/166/1/596.full#ref-list-1

, 6 of which you can access for free at: cites 29 articlesThis article

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Every submission reviewed by practicing scientistsNo Triage! •    

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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Homing of In Vitro-Generated Donor Antigen-Reactive CD41

T Lymphocytes to Renal Allografts Is a4b1 But Not aLb2

Integrin Dependent1

Markus H. Hammer,* † Yuan Zhai,* Masamichi Katori,* Thomas Ritter, † Hans-Dieter Volk,†

Ana J. Coito,2* and Jerzy W. Kupiec-Weglinski2,3*

The extravasation and sequestration of Ag-reactive T lymphocytes into vascularized organ allografts depend on a cascade ofcomplex interactions among circulating lymphocytes, endothelial cells, and extracellular matrix proteins. Ag-activated donor-specific CD4 T cells are major initiators and effectors in the allograft rejection response. Interfering with the intragraft homingof activated CD4 T cells may represent a novel therapeutic approach in transplant recipients. We have developed a FACS-basedshort-term homing assay that allows tracing in vitro-generated Ag-reactive CD4 T cells after adoptive transfer in test rat recip-ients. Allospecific cell lines were preincubated with anti-a4b1 or anti-aLb2 mAb, because of enhanced expression of both integrinreceptors after alloactivation. The pretreated LewisBN lymphocytes were carboxyfluorescein diacetate succinimidyl ester labeledand adoptively transferred into Lewis rat recipients of Brown Norway kidney allografts. The injection of equal numbers ofPKH-26-labeled untreated cells allowed quantitative comparison of both populations in the same animal. Ex vivo treatment withanti-a4b1 mAb diminished intragraft infiltration of adoptively transferred T cells by 85% in a donor-specific fashion. In contrast,treatment with anti- aLb2 mAb did not affect intragraft cell sequestration. Hence, blockinga4b1 integrin interactions representsa novel strategy in preventing local intragraft recruitment of Ag-reactive CD4 T cells in transplant recipients. The Journal ofImmunology,2001, 166: 596–601.

D onor-reactive CD4 T lymphocytes are major initiatorsand effectors in acute and chronic allograft rejection (1,2). The immune rejection cascade starts immediately af-

ter organ transplantation, and involves activation of intragraft APCthat migrate to the host-draining lymph nodes (LN)4 and/or spleen.The APC prime donor-reactive T cells for cytokine-dependentclonal expansion and differentiation into effector T cells. The hom-ing pattern of effector cells is distinct from that of naive T cells (3).Different adhesion molecule expression profiles allow recirculatingcells to migrate into tissues, particularly into the graft, and inter-acting with adhesion molecule counterparts or chemokines ex-pressed on inflamed endothelial cells and extracellular matrix(ECM). The role of the Ag in the effector cell homing into thetarget tissue remains relatively ill-defined. In a rat model of ex-perimental autoimmune encephalomyelitis, it was shown that bothautoantigen (myelin basic protein)-specific and unrelated activatedT cells transmigrate into the brain, but only myelin basic protein-

specific T cells persisted there for more than 24 h (4), suggestingthat Ag is involved in trapping of T cells in the tissue. The role ofalloantigen specificity in homing to the graft is less well studied.

In the past, conventional immunosuppressive drugs were de-signed either to deplete circulating T cells (ATG, irradiation,OKT3) or to inhibit the process of Ag priming, cytokine produc-tion/action, and clonal expansion (calcineurin inhibitors, antipro-liferative drugs, corticosteroids, rapamycine) of harmful alloreac-tive cells. However, interfering with the homing of alloreactive Tcells and other effector cells into the graft is a new approach forpreventing and combating graft-deteriorating immune processes.The inhibitory impact of conventional immunosuppressive drugscan be easily screened in vitro by measuring immune features suchas diminished NF-kB activation, free Ca21 levels, cytokine pro-duction, or proliferation after T cell activation (5). In contrary, theeffects of agents that modulate the homing are more difficult toaddress.

In the syngeneic situation, the highly inflamed transplant,caused by ischemia/reperfusion injury, attracts mainly polymor-phonuclear cells and monocytes/macrophages; Ag-activated lym-phocytes are only secondary in the pathogenesis of the very earlyultimate graft loss. Indeed, short-term treatment with agents thatbind to homing receptors (e.g., P-selectin glycoprotein ligand-1Ig)prevents ischemia/reperfusion injury in syngeneic grafts, stressingthe importance of early Ag-nonspecific immune response (6). Ini-tial Ag-nonspecific graft injury and alloantigen priming of T cellsin the secondary immune organs promote accumulation of allo-reactive effector cells in the graft, which in turn initiates acuterejection. CD4 T cell-derived cytokines (e.g., IFN-g) and surfacereceptors (e.g., CD40 ligand) also attract and activate monocytes/macrophages, thereby initiating localized delayed-type hypersen-sitivity reaction (7, 8). Although continuous immunosuppressionusually prevents early acute rejection, increased Ag-specific Th

*Dumont-University of California, Los Angeles Transplant Center, Department ofSurgery, University of California, Los Angeles School of Medicine, Los Angeles, CA90095; and†Institute of Medical Immunology, Charite, Humboldt-University, Berlin,Germany

Received for publication August 2, 2000. Accepted for publication October 4, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institute of Health Grants RO1 AI42223 andRO1 AI23847 (to J.W.K.-W.), American Heart Association Grant 0030006N (toA.J.C.), and the Dumont Research Foundation. M.H.H. was supported partially by theBiomedical Exchange Program.2 A.J.C. and J.W.K.-W. are co-senior authors.3 Address correspondence and reprint requests to Dr. J. W. Kupiec-Weglinski, TheDumont-UCLA Transplant Center, Room 77-120 CHS, 10833 Le Conte Avenue, LosAngeles, CA 90095. E-mail address: [email protected] Abbreviations used in this paper: LN, lymph node; BN, Brown Norway; ECM,extracellular matrix; TCM, T cell medium; WF, Wistar Furth.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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frequencies in peripheral blood reflect donor-directed reactivityduring later rejection episodes in transplant recipients (9, 10).

The rolling, firm adhesion, and extravasation of activated lym-phocytes is a highly controlled multistep process (11). In the caseof activated type 1 T cells, P- and E-selectin ligands are believedto be responsible for the first loose rolling attachment (12). Thisslower movement allows the interaction of cellular chemokine andintegrin receptors as well as CD44 with their endothelial counter-parts. This firm adhesion promotes the diapedesis of lymphocytesinto the tissue, where ECM proteins provide framework for se-questration and intercellular migration (13). Integrin-, selectin-,chemokine-, and CD44-dependent steps in T cell homing representpotential targets in blocking the sequestration of activated Ag-re-active T lymphocytes into the allografts. Thea4b1 (VLA-4) andaLb2 (LFA-1) integrins are of particular importance. Indeed, bothhave specific endothelial counterreceptors (VCAM-1, ICAM-1/2/3, respectively), besides their affinity toward common bindingsites of ECM proteins (e.g., RGD motif) and specific binding sites(e.g., CS-1 splicing motif on fibronectin) (14). Moreover,a4b1 isa versatile integrin, which can support both rolling and firm adhe-sion of leukocytes to VCAM-1 (15); its binding ligand affinity issignificantly up-regulated in activated (CD25high1) CD4 lympho-cytes (16). Interestingly, mAbs againsta4b1 integrin have beendemonstrated to inhibit the onset of experimental autoimmune en-cephalomyelitis initiated by Th1 lymphocytes specific for the my-elin basic protein (17). mAb targeting adhesion molecules repre-sent an interesting tool for studying cellular interactions in T cellhoming in vivo. However, adhesion molecules are not only in-volved in homing of T cells, but also in their activation by deliv-ering costimulatory signals (18) as well as in regulating Th1-typecytokine network (19). This makes the interpretation of their invivo action difficult. The use of in vitro preactivated T cells for invivo homing studies circumvents this problem. Moreover, the abil-ity of resting cells to sequester at the graft site is limited as com-pared with that of alloactivated cells (20).

In the present study, we investigated the role ofa4b1 andaLb2

integrin expression in the trafficking of donor-specific CD4 T cellsin rat renal allograft recipients. In addition, the impact of the TCRspecificity on CD4 lymphocyte recruitment to renal allografts wasaddressed.

Materials and MethodsAnimals and grafting techniques

Inbred male adult rats (200–250 g) were used (Harlan Sprague Dawley,Indianapolis, IN). Lewis (LEW, RT1l) or Brown Norway (BN, RT1n) kid-neys were transplanted into the abdominal great vessels of LEW recipientsby using standard microvascular techniques. Wistar Furth (WF, RT1u) ratsserved as third-party donors to generate control cell lines.

Generation of alloantigen-specific T lymphocytes

The generation of Ag-reactive T lymphocytes has been described else-where (21). Briefly, LN cells from LEW rats (33 105 cells/well) wereincubated withg-irradiated (5000 rad) LN cells from BN donors (33 105

cells/well). The cells were grown in T cell medium (TCM) containingDMEM with 2 mM L-glutamine, 2 mML-asparagine, antibiotics, and 2%heat-inactivated autologous LEW serum. After 4 days in culture, T cellblasts were harvested using rat Ficoll gradient centrifugation and propa-gated for 6–8 days in IL-2-conditioned medium. This medium was basedon TCM supplemented with supernatant produced by Con A-stimulated ratsplenocytes. Contaminating Con A was removed by addition of methylmannopyranoside (Sigma, St. Louis, MO). For the following restimulation,resting T lymphocytes were incubated withg-irradiated (5000 rad) synge-neic LEW thymocytes (23 105/well) in the presence ofg-irradiated (5000rad) BN thymocytes (43 105 cells/well). T lymphocyte blasts were trans-ferred into IL-2-conditioned medium and propagated. Alloantigen speci-ficity of T lymphocytes was tested with a standard proliferation assay mea-suring [3H]TdR incorporation.

FACS analysis

Phenotype analysis of cell lines was performed by using mouse mAbsagainst rat CD4, CD8, CD25, CD45RC, and L-selectin (Biosource Inter-national, Camarillo, CA; PharMingen, San Diego, CA). mAbs againsta4b1

and aLb2 integrin receptors were directly labeled with Alexa Fluor 488Protein Labeling kit according to manufacturer’s protocol (MolecularProbes, Eugene, OR). For indirect Ab staining ofa4b1 andaLb2 preincu-bated lymphocytes, cells were incubated at 4°C for 20 min in 0.1 ml PBSwith 1% goat serum and a PE-conjugated goat anti-mouse Ig (Zymed, SanFrancisco, CA). After washing twice in PBS, cells were analyzed by aFACScan (Becton Dickinson, Franklin Lakes, NJ), and data were evaluatedwith FCS-Express software (De Novo Software,Thornhill, Ontario,Canada).

For intracellular cytokine staining, the cells were harvested 3 days afteralloantigen-dependent restimulation (IFN-g) or after stimulation with PMA(10 ng/ml) and ionomycin (400 ng/ml) at 2.53 106/ml for 4 h, respectively(IL-4). GolgiStop (4ml/6 ml; PharMingen) was added for the last 4 h. Forintracellular cytokine staining, the cells were restimulated with PMA (10ng/ml) and ionomycin (400 ng/ml) at 2.53 106/ml for 4 h in the presenceof GolgiStop (4ml/6 ml; PharMingen). The stimulated cells were collectedand fixed/permeabilized using the CytoStain kit (PharMingen). Normal ratIgG (10mg) was added to 1 million cells to block nonspecific binding sites,followed by mouse anti-rat IFN-g (DB-1) or IL-4 (OX-81) mAb conju-gated with R-PE (PharMingen). The stained cells were washed and thenanalyzed by a three-color FACS. CD41 lymphocytes were gated for cy-tokine expression. PMA1 ionomycin-driven stimulation does not alterintracellular cytokine expression profile seen after allostimulation.

TCR Vb RT-PCR

RNA was extracted from lymphocytes using a Qiagen RNase Mini kit(Qiagen, Valencia, CA). A total of 0.2–1mg of RNA was reverse tran-scribed into cDNA (40ml) by 20 U of Moloney murine leukemia virusreverse transcriptase (Roche Molecular Biochemicals, Indianapolis, IN). Atotal of 0.5ml of cDNA was amplified in 25ml using a fixed downstreamprimer (Cb1) derived from TCRb-chain C region and one of the 22 dif-ferent Vb-specific upstream primers. The PCR mixture was set up as fol-lows: cDNA, 0.4 mM of each primers, 0.2 mM of each dNTP, 2 mMMgCl2, and 0.725 U of AmpliTaq Gold (Perkin-Elmer, Foster City, CA) in13 GeneAmp buffer II. Amplifications were conducted in a GeneAmpPCR System 9700 (Perkin-Elmer). The reactions started with a 10-mindenaturation at 94°C, followed by 35 cycles of 30 s at 94°C, 20 s at 60°C,and 30 s at 72°C, and ended with an elongation step of 5 min at 72°C. Sixmicroliters of PCR products was analyzed in a 2% NuSieve-agarose gel(FMC, Rockland, ME).

Fluorescent labeling and adoptive transfer studies

Three to five times restimulated cell lines were washed in PBS and labeledwith CFSE (Molecular Probes) or PKH-26 (Sigma). For CFSE staining,cells were washed in serum-free DMEM and incubated in 2 nM CFSE/PBSfor 30 min. The staining reaction was stopped by adding 10% horse serumin TCM. PKH-26 labeling (25ml 2 3 1024 M dye) was performed ac-cording to manufacturer’s protocol, and cell viability was determined bytrypan blue (Sigma). A total of 100mg/ml anti-a4b1 mAb (clone GG5/3;mouse IgG1) or anti-aLb2 mAb (clone WT-1; mouse IgG2A; PharMingen)was added to CFSE-labeled cell lines and incubated for 30 min at 37°C in5% CO2. Cells were washed with TCM/5% serum and injected into LEWrats 1 day after kidney engraftment.

In some experiments, LEWBN T lymphocytes (1003 106) were infusedi.v. into lightly g-irradiated (450 rad) LEW rats that were then challenged24 h later with cardiac allografts from BN donors. The immune potentialof transferred cells can be accurately determined in such “test-tube” ratrecipients (22).

Tracing labeled cells by fluorescent microscopy and FACSanalysis

For histological analysis of PKH-26-labeled T cell distribution, organswere frozen in OCT. The 5-mm sections were mounted on gelatinizedslides and dried for 60 min at room temperature. For fluorescence analysis,we used a laser microscope (Leica, Deerfield, IL). For flow-cytometricdetection of fluorescent T cells, kidneys were removed, homogenized, andfiltered through a 100-mm strainer. After washing twice in PBS, the pelletwas resuspended with 10 ml DMEM (Life Technologies) containing 10mgliver collagenase type V (Sigma). Incubation was done at 37°C for 30 min.Leukocytes were isolated by Ficoll density separation (Life Technologies).For Ab staining, cells were incubated at 4°C for 20 min in 0.1 ml PBS with1% bovine serum and mouse Cy-Chrome conjugated anti-rat CD4 mAb.

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Arterial blood samples were lysed and fixed in 4% paraformaldehyde inPBS. Secondary staining was done using a PE-conjugated goat anti-mouseIg (Zymed).

Results and DiscussionGeneration and characterization of Ag-specific T cell lines

To determine the effects of alloantigen and integrin receptors on Tcell homing, we generated LEW T cell lines with BN or WF al-lospecificity. Allospecificity was confirmed in a proliferation assay([3H]TdR incorporation) against syngeneic, allogeneic, or third-party g-irradiated splenocytes. LEWBN T lymphocytes showedvigorous proliferation only after stimulation with BN, but notLEW or WF cells, whereas LewWF T lymphocytes responded pri-marily against WF splenocytes (data not shown). The cell linesresembled a memory cell phenotype, with.95% being TCR1,CD41, CD82, a4b1 integrin1, aLb2 integrin1, L-selectinlow, andCD45RClow (Fig. 1). Moreover, in vitro-generated cells showedtype 1 T effector phenotype, as evidenced by readily measurableamounts of IFN-g after restimulation without any further poly-clonal sitmulation. On the other hand, intracellular IL-4 accumu-lation was not detectable even after PMA plus ionomycin stimu-lation (Fig. 1). No predominant Vb usage despite clonalproliferation during restimulations could be detected. By usingRT-PCR analysis, all 22 known rat TCR Vb chains were readilydetectable in LEWBN T lymphocytes (Fig. 1). Thus, despite itsalloantigen specificity, the cell line consists of polyclonal T cells.

To address the relevance of allospecific T cell lines in the graftrejection response, we adoptively transferred LEWBN T lympho-cytes into lightly g-irradiated LEW rat recipients of BN heartgrafts. The rejection time in this model varies between 14 and 21days (22). However, i.v. infusion of LEWBN T cells triggered anaccelerated rejection of cardiac allografts within 6 days, therebyconfirming the operational in vivo activity of in vitro generatedcells.

Distribution of PKH-26-labeled LEWBN T lymphocytes afteradoptive transfer

Homing of donor Ag-specific T lymphocytes to organ allografts isa complex and highly regulated process (20, 23). It depends on the

complementary interactions between endothelial cells, lymphocytesurface molecules, and local ECM proteins. Although most of li-gand-receptor interactions appear to be redundant, some single fac-tors carry critical nonredundant functions in the recruitment ofcertain lymphocyte subtypes toward inflamed tissues. The donorAg is one of these mandatory factors in the process. It has beenrecently shown that the recognition of alloantigens presented byendothelial MHC class II complexes enhanced the transmigrationof Ag-specific lymphocytes through endothelial cell layers in vitro(24). Interestingly, the inflammatory response due to ischemia/reperfusion injury after transplantation has led to up-regulation ofMHC class II molecules on the endothelial surface (25). In thisstudy, the distribution of alloreactive T lymphocytes was evaluatedat different time points. One day after transplantation, 153 106

PKH-26-labeled cells were injected into groups of allogeneic (BNto LEW) and syngeneic (LEW to LEW) kidney-grafted rats. Weused cells from the same lines and batches for each group of syn-geneic and allogeneic transplanted rats. This allowed the quanti-fication of differences due to graft-related effects. Several organswere harvested 12, 24, and 48 h after cell transfer, and the distri-bution of fluorescence-stained lymphocytes was monitored bycounting 5-mm tissue sections (Fig. 2). Time-dependent decreaseof fluorescent light emission of PKH-26 did not allow us to dis-tinguish between labeled cells and autofluorescence background atlater than 48 h. The highest number of labeled cells was foundthroughout in recipients’ lung and liver. LEWBN T lymphocytes dorecirculate through secondary lymphatic organs, as shown by theappearance of labeled cells in peripheral LNs and spleens. No Tcell homing to contralateral nontransplanted kidneys was found.An increased recruitment of labeled T cells was detectable only inthe allograft, whereas the number of cells infiltrating syngeneickidney grafts and other organs remained stable. Fig. 3 depicts dis-tinct homing patterns of adoptively transferred lymphocytes insyngeneic vs allogeneic rat renal graft recipients. The presence ofalloantigens (BN to LEW graft) did not affect the homing ofLEWBN T lymphocytes into recipients’ lung, liver, or spleen, asnearly equal numbers of cells could be detected in both syngeneicand allogeneic recipient groups. However, renal allografts at-tracted significantly higher numbers of LEWBN T cells as com-pared with syngeneic control grafts. Differences were seen fromthe earliest timepoints studied and increased with time probablydue to the intragraft proliferation, apoptosis of donor-reactive Tcells outside the transplant, and/or trapping of recirculating cells,perhaps by direct allorecognition, inside the graft.

The role of alloantigens on intragraft homing of Ag-activated Tlymphocytes

The increased recruitment of LEWBN T lymphocytes to BN ratherthan LEW grafts may not necessarily be due to Ag-specific cellhoming. Ongoing acute rejection could possibly amplify the in-flammatory reaction in the allogeneic (BN) kidney. The attractionof more cells by highly inflamed endothelium could result in dif-ferent infiltration pattern in allo/syngeneic grafts. To test our hy-pothesis that alloantigen is a critical factor in recruiting T lym-phocytes to the graft, we generated a WF-specific cell line as athird-party control. A total of 253 106 of CFSE-labeled LEWWF

T cells and 253 106 PKH-26-labeled LEWBN T cells was simul-taneously injected into LEW rats 24 h post-BN kidney engraft-ment. This protocol enabled us to evaluate the effects of alloanti-gen alone by keeping the inflammatory state of the transplantedgraft identical for both cell lines. The organs containing sufficientnumbers of infiltrating cells for FACS analysis (lung, liver, renal

FIGURE 1. Characterization of LEWBN T lymphocytes.A, FACS sur-face and intracellular cytokine staining of three to five times restimulatedLEWBN T lymphocytes.B, TCR Vb expression pattern after PCR ampli-fication of three to five times restimulated LEWBN T lymphocytes (n5 3).

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graft) were harvested 24 h after the transfer. Isolated graft-infil-trating cells were stained with anti-rat CD4 mAb, and FACS anal-ysis was performed. Dot blots representing the ratio of green-la-beled WF- to red-labeled BN-specific cells in lung and in thetransplanted kidney are shown in Fig. 4. The ratio of injected cellsmeasured before injection was found to be identical with the ratioin lung or liver. In contrast, a 40% increase in numbers of BN-specific lymphocytes as compared with WF-specific cells was de-tected in the BN graft. We also injected a mixture of equal num-

bers of PKH-26- and CFSE-labeled LEWBN T cells into LEWrecipients of BN renal grafts. These controls did not show anysignificant changes in the red to green ratio in all tested organs,implying that alloantigen itself may represent a factor in recruitingthe lymphocytes to the graft. The impact of alloantigen can beexplained by three not mutually elusive principles. First, alloanti-gen-specific lymphocytes home into the graft in an Ag-specificmanner (24). High MHC class II expression following transplan-tation might initiate the cross-talk between graft endothelium and

FIGURE 2. PKH-26-labeled LEWBN T lymphocytes in vivo. The digital images of PKH-26-labeled (red) cells in various tissues 48 h after adoptive Tcell transfer in rat renal allograft recipients.A, Allogeneic transplanted kidney tubular region; tubulus emitted yellow autofluorescence signal (originalmagnification,3100).B, allogeneic transplanted kidney glomerula region (original magnification,3100).C, lung (original magnification,340). D, liver(original magnification,340). Representative of four distinct experiments.

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adoptively transferred CD41 T lymphocytes (25). The donor-de-rived endothelial cells present the alloantigen via the direct path-way. The protocol, used to generate allospecific cell lines, pro-motes this direct interaction because TCR activation during invitro priming and expansion was performed by donor (BN)-de-rived MHC class II molecules. Second, clonal expansion inside thegraft or in secondary lymphatic organs may change the total num-ber of alloreactive T lymphocytes. Third, random homing of acti-vated T cells to organ grafts was followed by enhanced apoptosisin the absence of alloantigens (26).

Pretreatment with anti-a4b1 but notaLb2 mAb inhibitsintragraft homing of Ag-specific T cells

Ag-activated and naive T lymphocytes exhibit distinct in vivo re-circulation and homing patterns (3, 20, 23). Although these dif-ferences may not be absolute, activated cells have a higher poten-tial to transmigrate and accumulate at local inflamed tissues,whereas naive T lymphocytes recirculate between peripheral lym-phatic compartments. This may be due, at least in part, upon theexpression of distinct homing receptors on their surface. Thea4b1,a member of theb1 integrin family, andaLb2, a b2 integrin, areboth highly expressed on activated, but not quiescent T lympho-cytes (13). It has been shown that T cell activation dramaticallyenhances the binding affinity betweena4b1 and its endothelialcounterreceptor VCAM-1 (16). These features support the role ofa4b1 andaLb2 as potential targets for therapeutic intervention intransplant recipients. Indeed, we have recently shown that target-

ing a4b1 in vivo interactions significantly prolongs the survival ofcardiac allografts in rats (27). Although other studies showed onlymarginal effects of anti-a4b1 or anti-aLb2 mAb monotherapy, co-treatment with both Abs often resulted in an indefinite survival ofmurine heart and islet grafts (28, 29).

We have then attempted to evaluate the role ofa4b1 andaLb2

upon the homing of activated alloreactive CD4 T lymphocytes inallograft recipients. CFSE-labeled LEWBN T lymphocytes wereincubated with anti-a4b1 or anti-aLb2 mAb to block interactionswith their counterreceptors. Preincubation had no influence ona4b1 or aLb2 surface receptor density, as shown by comparingdirect staining of control lymphocytes and secondary staining ofpreincubated cells. After removing unbound Ab by extensivewashing, 253 106 mAb-treated CFSE1 cells were injected inconcert with an equal number of nontreated PKH-26-labeledLEWBN T lymphocytes into rat recipients of renal allografts. Wedid not mix both populations before injection to avoid binding ofcontaminating Ab to nontreated PKH-26 control cells. Inhibition/augmentation of cell sequestration was calculated by defining theCFSE/PKH-26 ratio in lungs as the injection ratio. The organswere harvested 24 h after the transfer, and graft-infiltrating cellswere isolated/stained, as described above. Although we were ableto detect labeled cells in spleen, peripheral LNs, and blood, thepercentage in comparison with hosts native CD4 lymphocytes wastoo low (,0.05%) to quantify distribution differences after 24 h.Fig. 5 shows the distribution of pretreated and untreated labeledcells in rat kidney allografts. Ex vivo treatment with anti-a4b1

FIGURE 4. Influence of alloantigen on T lympho-cyte homing. Lung-infiltrating cells (A) and kidneygraft-infiltrating cells (B) were isolated 24 h afteradoptive transfer of 253 106 PKH-26-labeledLEWBN T lymphocytes and 253 106 CFSE-labeledLEWWF T lymphocytes into allogeneic kidney-trans-planted rats. The isolated cells were stained for CD4expression. The dot blots illustrate CFSE and PKH-26events of CD41 cells by FACS analysis. Data are ex-pressed as ratio between PKH-26- and CFSE-positiveevents.n 5 3.

FIGURE 3. Distribution kinetics of LEWBN T lymphocytes inrat renal transplant recipients. A total of 153 106 of LEWBN Tlymphocytes was injected into syngeneic and allogeneic trans-planted rats. Tissues were harvested 12, 24, and 48 h after adoptivetransfer, and fluorescent cells were counted on tissue sections. Dataare expressed as ratio between cells in allogeneic and syngeneictransplanted rats (n5 3).

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mAb inhibited infiltration of renal allografts with CFSE-labeledcells by 85% (p , 0.05). In contrast, no significant inhibition ofanti-aLb2 mAb-treated cells was detectable. The intralung andliver ratios independent of any kind of treatment were nearly iden-tical in all treatment groups. In addition, adoptive transfer studieswith two dyes and without any Abs showed no different homingbehavior into the graft or into other organs (e.g., lung), implyingthat the two dyes do not appear to differentially affect cell migra-tion patterns. Moreover, the difference in graft infiltration aftera4b1 andaLb2 mAb treatment was not due to different mAb den-sities on cell surfaces, or isotype-dependent clearance of mAb-coated cells. Indeed, equal numbers of treated and untreated cellssequestered in hosts’ spleens and livers. Both mAb preparationshad no effect on the surface phenotype of transferred T cells, theiractivation (CD25 expression), proliferation ([3H]TdR incorpora-tion), and apoptosis (annexinV staining) after alloantigen-specificactivation in vitro; no receptor down-regulation was noted follow-ing secondary staining againsta4b1 or aLb2 (data not shown).

The limited number of circulating adoptively transferred cells atlater time points (,0.02% after 24 h) favors the hypothesis that thefirst hours after leaving of lymphoid organs are most critical forselective trafficking of activated alloreactive lymphocytes into theallograft. During this early time frame, anti-a4b1 mAb was detect-able on the cell surface (data not shown), suggesting a direct in-terfering in the rolling and transmigration process inside the donorgraft as causal mechanism of the blocked intragraft mononuclearcell infiltration. On the other hand, the beneficial effects of anti-aLb1 mAb therapy in some transplantation models (28, 29) maynot be due to diminished intragraft recruitment of activated allo-reactive CD4 cells. Perhaps direct signaling effects through LFA-1/ICAM-1 molecule interaction during T cell activation and prim-ing play a role. It has been shown that ICAM-1 stimulation of Tlymphocytes enhances thet1/2 of inducible IL-2, thereby bypassingcyclosporine-based inhibition of T cell activation (30). We favorthe notion that anti-aLb2 mAb treatment interferes with priming/activation of alloreactive CD41 T lymphocytes, whereas anti-a4b1

mAb blocks homing of primed cells into the graft. Thea4b1 in-tegrin-blocking therapy with mAbs or small molecules may thusrepresent a useful therapeutic option in transplant recipients.

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FIGURE 5. Influence of anti-a4b1 and anti-aLb2 mAb on intragraft Tlymphocyte homing. LEWBN T lymphocytes were preincubated with anti-a4b1 or anti-aLb2 mAb and adoptively transferred into LEW recipients ofBN renal allografts. Kidney allograft and lung-infiltrating cells were har-vested, and the ratio between PKH-26- and CFSE-labeled cells was cal-culated. Data are expressed as percentage of inhibited infiltration calculatedbetween lung and renal graft ratio. WF cell line: see Fig. 4 (n5 3); anti-aLb2 mAb pretreatment (n5 3); anti-a4b1 mAb pretreatment (n5 4).

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