unaltered graft survival and intragraft lymphocytes infiltration in the cardiac allograft of cxcr3...

American Journal of Transplantation 2008; 8: 1593–1603Wiley Periodicals Inc.

C© 2008 The AuthorsJournal compilation C© 2008 The American Society of

Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2008.02250.x

Unaltered Graft Survival and Intragraft LymphocytesInfiltration in the Cardiac Allograft of Cxcr3−/−

Mouse Recipients

J. Kwuna, S. M. Hazinedaroglua, E. Schaddea,

H. A. Kayaoglua, J. Fechnera, H. Z. Hua,

D. Roenneburga, J. Torrealbab, L. Shiaoc,

X. Hongc, R. Pengc, J. W. Szewczykd,

K. A. Sullivane, J. DeMartinoe

and S. J. Knechtlea,∗

aDivision of Transplantation,Department of Surgery,University of Wisconsin-MadisonbDepartment of Pathology, Clinical Science Center,University of Wisconsin, Madison, WIcDepartments of Laboratory Animal ResourcesdMedicinal ChemistryeImmunology, Merck Research Laboratories, Merck,Rahway, NJ∗Corresponding author: Stuart J. Knechtle,[email protected]. M. Hazinedaroglu is supported in part by The Scientificand Technical Research Council of Turkey (TUBITAK) andAnkara University, TurkeyJ. Kwun and S. M. Hazinedaroglu contributed equally tothis study.Supported in part by Merck.The authors declare that they have no potential conflictsof interest.

Previous studies showed that absence of chemokinereceptor Cxcr3 or its blockade prolong mouse cardiacallograft survival. We evaluated the effect of the CXCR3receptor antagonist MRL-957 on cardiac allograft sur-vival, and also examined the impact of anti-CXCR3mAb in human CXCR3 knock-in mice. We found only amoderate increase in graft survival (10.5 and 16.6 days,p < 0.05) using either the antagonist or the antibody,respectively, compared to control (8.7 days). We re-evaluated cardiac allograft survival with two differ-ent lines of Cxcr3−/− mice. Interestingly, in our hands,neither of the independently derived Cxcr3−/− linesshowed remarkable prolongation, with mean graft sur-vival of 9.5 and 10.8 days, respectively. There was nodifference in the number of infiltrating mononuclearcells, expansion of splenic T cells or IFN-c productionof alloreactive T cells. Mechanistically, an increasedother chemokine receptor fraction in the graft infil-trating CD8 T cells in Cxcr3−/− recipients comparedto wild-type recipients suggested compensatory T-celltrafficking in the absence of Cxcr3. We conclude Cxcr3may contribute to, but does not govern, leukocyte traf-ficking in this transplant model.

Key words: Acute rejection, cardiac allograft, cell traf-ficking, mouse, T-cell graft infiltration

Received 7 November 2007, revised 26 February 2008and accepted for publication 13 March 2008

Introduction

Chemokines are small heparin-binding proteins that playan important role in trafficking of leukocytes (1–3). Duringallograft rejection host mononuclear cells (effector, mem-ory T cells and monocytes) enter the graft by interactionof adhesion molecules on endothelial cells and their re-ceptors as well as chemokines and their receptors (4). An-timigration therapy could help to prevent both acute andchronic rejection. The molecular mechanism by which al-loreactive leukocytes enter and persist in the graft is notfully understood. However, there is descriptive evidencefor the expression of chemokines and their receptors inrodent models and human biopsies of allograft rejection(5–8). Studies examining graft survival of heterotopic hearttransplants in knock-out mice have shown that lack of Ccr5or Cxcr3 have an impact on graft survival (4). Studies usingblocking antibodies and small molecular antagonists target-ing Ccr5 or/and Cxcr3 in the same model demonstrated asurvival benefit as well (9,10). Interestingly, a retrospectiveepidemiologic study has shown a correlation between thelack of human CCR5 due to an inborn polymorphism andbeneficial outcomes in renal transplantation (11).

However, more recent studies have not fully confirmedthese results. Graft survival was not significantly prolongedin Ccr5−/− mice (12,13). Similarly, unaltered lymphocyte in-filtration into the area of inflammation in an animal model ofautoimmunity was reported in mice genetically deficient inCxcr3 (14) where Cxcr3−/− mice exhibited increased sever-ity of EAE compared to wild-type mice and no differencewas observed in CNS infiltrating leukocytes. In models ofinfectious disease, an identical degree of leukocyte infiltra-tion was observed in Cxcr3−/− mice during influenza infec-tion (15) and even more T-cell infiltration was observed inM. tuberculosis-infected lung (16).

Confoundingly however, only beneficial effects have beenreported as a consequence of genetically targeting Cxcr3

1593

Kwun et al.

in mice in experimental transplantation models (17–21). Totry to reconcile these different findings related to the role ofCxcr3 in various disease models, we employed test drugsand antibodies specific for the human CXCR3 molecule,and we examined their effectiveness in preventing graftrejection in human CXCR3 (hCXCR3) knock-in (KI) mice.

We found significant but surprisingly only moderate prolon-gation of graft survival using anti-hCXCR3 mAb (1C6) and apotent and selective small molecular antagonist (MRL-957,Merck, NJ) in hCXCR3 KI mice. These unexpected resultswarranted a reexamination of the effect of Cxcr3 deficiencyin the major mismatch heart transplant model, which wethus investigated in two independently derived Cxcr3−/−

lines (both backcrossed 10 generations onto C57BL/6 back-ground; H-2b) available to us. Different from previous re-ports, we found no remarkable prolonged graft survivalin Cxcr3−/− recipients compared to wild-type recipients.Neither the histologic appearance of the grafts, the typeof infiltrates nor expansion of T cells and IFN−c produc-tion via MLR showed difference in Cxcr3−/− recipients.However, an increased proportion of Ccr5+ CD8 T cellsin the absence of Cxcr3 might suggest trafficking into theinflamed site occurs via compensation by other chemokinereceptors.

Materials and Methods

Animals

All control mice, 6–8-week-old male, BALB/c (H-2d) and C57BL/6 (H-2b) wereobtained from Taconic (Hudson, NY). The human CXCR3 KI mice were gen-erated at Merck research laboratories (Rahway, NJ) with ES cell targetingas previously described (22). A homozygous colony was established by in-terbreeding. The Cxcr3−/− mouse lines used were line 1891, generatedby B Lu, at Massachusetts Children’s Hospital (Boston, MA) and line 3347generated at Deltagen (San Carlos, CA). All strains of mice (Cxcr3−/− andhCXCR3 KI) were backcrossed 10 generations onto a C57BL/6 backgroundand are maintained at Taconic. For the purpose of experimentation, micewere housed in individually ventilated cages under specific pathogen-free(SPF) conditions. Experiments were performed in accordance with the Na-tional Institutes of Health and U.S. Department of Agriculture guidelines,after approval of University of Wisconsin Institutional Animal Care and UseCommittee.

Heart transplantation

Recipient aorta and vena cava were prepared before the donor operation.Donor hearts were transplanted immediately into the abdominal cavity ofthe recipients after a short period of cold ischemia in Euro-Collins solu-tion. Donor aorta and pulmonary artery were anastomosed with running10/0 nonabsorbable monofilament sutures in an end-to-side fashion to theinfrarenal aorta and vena cava as the inflow and outflow vessels for circu-lation. Anti-human CXCR3 (1C6) purchased from PharMingen (San Diego,CA) was administered every other day subcutaneously starting on the dayof transplant (Days 0, 2, 4, 6, 8, 10, 12, 14). It was given i.p. at 50 lg/mL(>200 lL/injection). The antibodies were dissolved in PBS and filter steril-ized. It also tested negative for endotoxin. Small molecular antagonist (MRL-957, Merck), a thiazole derivative described in WO2007064553, for CXCR3was developed by Merck research laboratories and formulated in rodentchow and provided ad libitum. To support recovery after surgery, animals

were dosed with MRL-957 by oral gavage. Low-dose cyclosporine (CsA,10 mg/kg; Ben Venue Labs, Inc., OH) was delivered subcutaneously withmicro-osmotic pump (Model 1002; 0.25 lL/h, 14 days) purchased from Alzet(Cupertino, CA). Survival of the grafts were assessed daily by palpation. Re-cipients were sacrificed either after the cessation of heart beating or on adesignated date.

Immunohistochemistry

Explanted mouse heart grafts from C57BL/6 wild-type, hCXCR3 KI andCxcr3−/− recipients and were divided and submitted for paraffin and frozensectioning and staining. Paraffin blocks were sectioned and stained withroutine H&E. Frozen samples embedded in optical coherence tomog-raphy (OCT) were sectioned at 5 lm, and fixed with acetone or 4%paraformaldehyde for subsequent immunohistochemical staining. Sectionswere blocked for nonspecific staining using 10% BSA in TBS for 1 h fol-lowed by 5% nonfat milk in PBS, 30 min. Rat anti-mouse primary anti-bodies against T-cell markers CD4 and CD8 (BD Pharmingen) were in-cubated overnight at 4◦C. Endogenous peroxidase quenching was per-formed post primary antibody incubation with 3% hydrogen peroxide inTBS, 10 min. A donkey anti-rat IgG-HRP conjugated secondary antibodyfrom Jackson Laboratories was used for detection and incubated for 45min at room temperature. The signal was visualized using DAB chromogen(DakoCytomation) followed by counterstaining with hematoxylin. Slideswere viewed using light microscopy. International Society for Heart andLung Transplantation classification was used for scoring the presence andthe degree of rejection (23). Acute rejection was characterized by infiltra-tion, myocyte damage, inflammation, necrosis, edema, hemorrhage andvasculitis.

Flow cytometry

Spleen, lymph node and blood were harvested on the day of rejection oron the designated day and placed into a single cell suspension in RPMI1640 (Life Technologies, Grand Island, NY) by passing through a cell strainer(Becton Dickinson Labware, Franklin Lakes, NJ). Explanted heart graftswere minced and digested for 30 min in liberase CI-purified enzyme blend(0.45 mg/mL, Roche Diagnostics, Indianapolis, IN) in RPMI 1640 media at37◦C. Mononuclear cells were isolated via ACK Lysing Buffer (Cambrex,Walkersville, MD) according to the manufacturer’s instructions. Cells wereresuspended in FACS buffer (PBS containing 2% FBS and 0.09% NaN3) ata concentration of 1 × 106/100 lL. Cells were stained with anti-CD4 FITC(H129.19; RDI, NJ), anti-CD8 FITC (53–6.7, RDI, NJ), anti-CD4 PE (GK1.5),anti-CD8 PE (53–6.7), anti-hCXCR3 PE (1C6), anti-Ccr5 PE (C34–3448), anti-Ccr5 Biotin (HM-Ccr5, Biolegend), anti-CD3 PerCP (145–2C11), anti-CD4APC (RM4–5), anti-mCxcr3 APC (220803, R&D systems) and isotype con-trols all from BD pharmingen (San Diego, CA) unless it is designated. Cy-tometric analysis was performed using a FACSCaliber cytometer (BD Bio-science, San Jose, CA) and analyzed using Cell quest (BD Bioscience) andFlowJo (Tree Star, San Carlos, CA) software.

IFN-c kinetics assay

The IFN-c expression kinetics assay was performed using a modification ofthe methods described by us (24). Recipient splenocytes (10 × 106) wereresuspended in culture medium (RPMI 1640 supplemented with 20mMHEPES, 10mM sodium pyruvate, 2mM l-glutamin, 1× MEM-vitamin solu-tion and 15% fetal bovine serum). 4 × 105cells/100 lL of splenocytes wereplaced and cocultured with irradiated (200 rad) donor cells (BALB/c; 4 × 105

cells/100lL) in a 96 well plate (15 wells per recipient) for 5 days at 37◦Cin a 5% CO2 incubator. Culture supernatant was collected daily for 5 days.The concentration of IFN-c in the culture supernatant was measured withmouse IFN-c ELISA kit (R&D systems, Minneapolis, MN).

1594 American Journal of Transplantation 2008; 8: 1593–1603

Compensatory Trafficking of T Cells Without Cxcr3

LightCycler real-time RT-PCR

RNA was isolated from cardiac allograft tissue by using Trizol reagent (Invit-rogen, Carlsbad, CA). Primers (TIB MoBiol, NJ) used in PCR were previouslydescribed (25): Mig (Cxcl9; 5′-gaa cgg aga tca aac ctg cct-3′ and 5′-tgt agtctt cct tga acg acga-3′), IP-10 (Cxcl10; 5′-cga tga cgg gcc agt gag aatg-3′ and5′-tca aca cgt ggg cag gat agg ct-3′), I-TAC (Cxcl11; 5′-aat tta ccc gag taacgg ctg-3′ and 5′-att atg agg cga gct tgc ttg-3′), human CXCR3 (5′-ccg tccagt ggg tct ttg g-3′ and 5′-agg gct cct gcg tag aag ttg-3′) and GAPDH (5′-caatgt gtc cgt cgt gga-3′ and 5′-gat gcc tgc ttc acc acc-3′). RNA quantificationwas carried out by real-time RT-PCR with a LightCycler RNA amplificationkit SYBR Green I adapted for one-step RT-PCR in glass capillaries using aLightCycler instrument (Roche Diagnostics, Mannheim, Germany). The rel-ative expression ratio was calculated by a ‘Delta-delta method′ {Ratio =2−[dCp(sample−control)] = 2−ddCp}. The amount of mRNA for each gene wasnormalized to the amount of mRNA for GAPDH in each sample. The ratiobetween each gene of interest and GAPDH is used for comparison.

Statistics

Survival was analyzed with a log-rank test. Difference in measured vari-ables between transgenic mice and the wild-type were assessed using theStudent’s t-test. Data are expressed as the mean ± SD where applicable.P-value less than 0.05 was considered statistically significant.

Results

Generation of human CXCR3 knock-in mice

Human CXCR3 KI mice were generated using a target-ing construct, which employed large flanking sequences ofthe murine CXCR3 gene (3.8 Kb at the 5′ end and 3.2 Kbat the 3′ end) in order to ensure high-targeting frequency.

Figure 1: Generation of human

CXCR3 knock-in mice. (A) schematicrepresentation of the hCXCR3 target-ing strategy. The top line shows theloxP-flanked PGKneo cassette target-ing vector, with upstream and down-stream CXCR3 genomic DNA. Thesecond line depicts the wild-typeCxcr3 locus, with restriction enzymesites, locations of Cxcr3 coding region(gradient box). The configuration ofthe properly targeted allele created byhomologous recombination in ES cellsis shown on the thrid line. (B) South-ern blot analysis of the hCXCR3 KI orwild-type mice. The left panel showsEcoRI-digested genomic DNA fromKI or wild-type mice that hybridizedwith probe A, confirming correct 5′ tar-geting. The right panel shows AccI-digested genomic DNA from KI orwild-type mice that hybridized withprobe B, confirming proper 3′ target-ing. (C) Human CXCR3 expressionwas assessed by RT-PCR in wild-type(WT), knock-in (KI) and heterozyous(HET) littermates.

The human CXCR3 gene (2.8 Kb) was cloned into this vec-tor so that exon 1 of the human gene was fused near thehomologous position in the murine CXCR3 gene and thusexpression of the human gene in mice was under directcontrol of the mouse promoter. The construct also con-tained PGK neo (flanked by two loxP sites) and HSVtk ex-pression cassettes to permit positive/negative selection ofcorrectly targeted ES cells. ES cell (AB2.1) targeting wasconducted as previously described (Figure 1A). Targeted EScell clones were screened by Southern blot hybridizationusing both 5′ and 3′ probes prepared from gene sequencesflanking those used in vector construction. Five indepen-dent founders were generated employing this construct.Male chimeric mice were bred to C57BL/6 females andgermline transmission of the targeted allele was confirmedby Southern blot hybridization (Figure 1B, C). A homozy-gous colony, which was evaluated by PCR (Figure 1D) wasestablished by interbreeding and currently held at Taconic.Human CXCR3 expression was upregulated by SEB treat-ment of splenocytes from hCXCR3 KI mice in vitro (SupplFigure 1A). We found that murine CXCR3 ligands were fullyfunctional and bound with high affinity to human CXCR3.In in vitro studies, 1C6 antibody has been shown to stainRBL/hCXCR3 cells. 1C6 blocks 125I-IP-10 (CXCL10) bindingwith SEB activated splenocytes (Day 6) down to the level of45% and 70% inhibition at 10−8 – 10−7 (M) concentrationsrespectively when compared to isotype control antibody(Suppl Figure 1B). To test effectiveness of targeting CXCR3in a transplant model, we tested the anti-human CXCR3

American Journal of Transplantation 2008; 8: 1593–1603 1595

Kwun et al.

Figure 2: Targeting hCXCR3 prolongs graft survival moderately in hCXCR3 Knock-in recipients. BALB/c (H-2d) donor hearts weretransplanted into hCXCR3 KI C57BL/6 (H-2b) recipients. (A) Survival rates of cardiac allograft. Targeting hCXCR3 in KI recipients withanti-hCXCR3 mAb (1C6) treatment (�, MST = 15.8 ± 2.5 days; n = 11) and MRL-957 (�, MST = 10.5 ± 0.8 days; n = 6) showedonly modest prolongation compared to untreated hCXCR3 KI (�, MST = 8.7 ± 2.1 days; n = 8) and wild-type C57BL/6 recipients(�, MST = 8.6 ± 0.5 days; n = 8). Syngeneic combination (�, MST > 100 days; n = 7) showed no complication. (B) Receptor Occupancyby anti-hCXCR3 (1C6) was evaluated by flow cytometry using flurochrome conjugated anti-hCXCR3 mAb. Blood samples were obtainedfrom anti-hCXCR3 mAb (1C6)-treated or untreated heart transplant recipient at day 7 after transplantation. Isolated leukocytes wereimmunostained with FITC-anti-CD3 and PE-anti-hCXCR3 mAbs, then analyzed by flow cytometry. Data shown are from one experiment,and are representative of six independent experiments (a). H&E staining was used to identify degree of acute rejection in anti-hCXCR3mAb (1C6)-treated (b) and untreated (c) hCXCR3 KI recipients. Original maginification ×600. (C) Intragraft chemokine Mig (Cxcl9), IP-10(Cxcl10) and I-TAC (Cxcl11) expression in hCXCR3 KI recipients. Cardiac allografts of hCXCR3 KI recipients were analyzed on 7 days aftertransplantation and compared with date-matched isografts. Relative Mig, IP-10 and I-TAC mRNA expression to control GAPDH mRNA isindicated. ∗p < 0.05. Data shown are representative of five independent experiments (D) RT-PCR analysis of human CXCR3 and GAPDHmRNA in the graft. Data are representative of five independent experiments.

antibody (1C6) and the antihuman CXCR3 small molec-ular antagonist (MRL-957) in a genetically altered mousestrain that expresses human CXCR3 and not mouse Cxcr3(KI mice). We found that mouse anti-human CXCR3 (1C6)monoclonal antibody treatment diminished delayed typehypersensitivity responses (DTH) significantly in compari-son to isotype antibody; furthermore, 1C6 partially blockedCD3+ T-cell recruitment to the peritoneum (48%) followingfoot-pad injection (Unpublished data, Merck). MRL-957 and1C6 effectively blocked the receptor at the targeted con-centrations and blocked chemotaxis of splenocytes fromhCXCR3 KI mice (Suppl Figure 2).

Targeting human chemokine receptor CXCR3 does

not prolong graft survival remarkably in hCXCR3

knock-in cardiac allograft recipients

To investigate the effect of blocking human CXCR3 onallograft rejection, we used hCXCR3 KI mice in a hearttransplantation model. In this model, we transplanted vas-cularized BALB/c (H-2d) hearts to (1) C57BL/6 (H-2b) wild-

type mice, (2) hCXCR3 KI B6 mice without treatment, (3)hCXCR3 KI B6 mice with neutralizing anti-human CXCR3(1C6, BD pharmingen) treatment, as this antibody dimin-ished DTH responses significantly in CXCR3 KI mice (un-published data) and (4) hCXCR3 KI B6 mice with MRL-957treatment. Graft survival is shown in Figure 2A. Isograftcontrols (C57BL/6 to C57BL/6) were sacrificed at day 100with strongly beating grafts. Untreated hCXCR3 KI recip-ients showed similar graft survival to wild-type C57BL/6allogeneic controls (8.33 ± 2.07 vs. 8.62 ± 0.74 days;p > 0.05). We measured intragraft chemokines for Cxcr3in hCXCR3 KI recipients. As shown in Figure 2C, mRNAexpression levels of chemokines Mig (Cxcl9) and IP-10(Cxcl10) but not I-TAC (Cxcl11) were significantly increasedcompared to isograft controls. We also found that intragrafthuman CXCR3 was only in hCXCR3 KI recipients. In otherwords, recruitment derived hCXCR3 positive cells werepresent in the graft (Figure 2D). Analysis of splenocyteswith flow cytometry revealed that 1C6 effectively boundto hCXCR3+ cells in hCXCR3 KI mice at 50 lg/mg doses(Figure 2B). The level of hCXCR3 receptor availability in



Figure 3: Combination treatment of hCXCR3 blockade with

low dose CsA (10mg/kg) prolongs graft survival marginally.

Human CXCR3 KI mice were grafted with BALB/c hearts with notreatment (�, n = 8), CsA alone (�, n = 11), 1C6 alone (•, n =6), 1C6+CsA (◦, n = 7), MRL-957 alone (�, n = 6) and MRL-957+CsA (�, n = 7). (A) Low dose CsA alone prolonged graftsurvival significantly compared to untreated KI recipients (10.7 ±2.5 vs. 8.7 ± 2.1 days, p < 0.01). (B) anti-hCXCR3 mAb (1C6) +CsA treatment (18.7 ± 1.7 days, p < 0.05) and (C) MRL-957+ CsA(15.3 ± 4.4 days, p < 0.01) showed increased graft survival com-pared to anti-hCXCR3 mAb (1C6) or MRL-957 alone groups (15.8 ±2.5 or 10.5 ± 0.8 days, respectively). (D) Mice receiving combina-tion treatment of anti-hCXCR3 mAb (18.7 ± 1.7 vs. 10.7±2.5 days,p = 0.0001) or MRL-957 (15.3 ± 4.4 vs. 10.7 ± 2.5 days, p = 0.016)with low-dose CsA had prolonged graft survival compared to CsAalone indicates synergistic effect.

anti-CXCR3 (1C6)-treated groups was around 1%, whereashCXCR3 KI mice without anti-CXCR3 mAb (1C6) treatmentshowed a higher level of hCXCR3 availability. In accordancewith this, treatment with 1C6 prolonged graft survival aswell as MRL-957 (16.57 days vs. 11 days, p < 0.05). How-ever, histologic examination of cardiac allografts showedsimilar grades of acute rejection and lymphocytic infiltra-tion in both untreated and treated hCXCR3 KI recipients(Figure 2B). These data indicate that targeting hCXCR3 withantibody or a small molecular inhibitor only moderately pro-longed graft survival. However, acute graft rejection wasnot prevented, and leukocyte trafficking into the graft per-sisted in hCXCR3 KI mice. Previous reports indicated thattransplants in Cxcr3−/− recipients in this strain combinationhad markedly prolonged graft survival (9).

Since the prolongation of cardiac graft survival was notas long as expected, we evaluated synergistic effectsof CXCR3 blockade with low dose cyclosporine (CsA,

10 mg/kg). We found that codosing with MRL-957 didnot alter CsA levels and MRL-957 levels were not alteredby CsA (data not shown). We transplanted BALB/c heartsto hCXCR3 KI recipients treated with (1) CsA alone, (2)CsA + 1C6 and (3) CsA + MRL-957. Here, addition of lowdose CsA (10 mg/kg) to anti-hCXCR3 mAb increased MSTfrom 15.8 ± 2.5 to 18.7 ± 1.7 (p < 0.05) and 10.5 ± 0.8 to15.3 ± 4.4 for MRL-957 (p < 0.01) (Figure 3B, C). Increasedmean survival time of both combination treatment groupscompared to CsA alone suggested additive or synergisticeffects between two treatments (Figure 3D). Even thoughgraft survival was statistically significantly prolonged, thebenefit was marginal.

Cardiac allograft survival was only marginally

prolonged in Cxcr3−/− recipients

To further evaluate the role of Cxcr3 in graft rejection andleukocyte accumulation, we independently examined graftsurvival in Cxcr3−/− mice. BALB/c (H-2d) hearts were trans-planted into C57BL/6 wild-type or two different lines ofCxcr3−/− C57BL/6 recipients (Line# 1891 and 3347). Sur-prisingly, only a modest prolongation was observed both inCxcr3−/− line 1891 (MST = 9.54 ± 1.36, n = 11) and line3347 (MST = 10.75 ± 0.95, n = 6) compared to wild-type(MST = 8.62 ± 0.74, n = 8). Histologic examination of car-diac allografts confirmed extensive lymphocytic infiltrationwith myocyte damage in both Cxcr3−/− lines at the time ofrejection as well as post transplant day 7 compared to wild-type recipients (Figure 4). We treated Cxcr3−/− (#1891) re-cipients with low dose CsA (10mg/kg) to evaluate synergis-tic effects of targeting Cxcr3 and calcineurin inhibition. Lowdose CsA treatment prolonged graft survival in Cxcr3−/− re-cipients (14.3 ± 0.95, n = 7, p < 0.0001). However, thiscyclosporine dose was not potent enough to prevent acuterejection. Taken together, these results indicated that ab-sence of Cxcr3 does not prevent acute rejection in MHCmismatched cardiac transplantation.

Cxcr3−/− exhibit no effect on T-cell recruitment

to the allograft and T-cell localization

in immune compartments

To address the mechanisms responsible for graft rejectionin the absence of Cxcr3, we studied leukocyte traffickingin Cxcr3−/− recipients. First, we found no evidence thatabsence of Cxcr3 impacted its ligands expression in thegraft at 7 days (Figure 5A). We compared the percentageof CD4 and CD8 T cells between wild-type and Cxcr3−/−

recipients in four anatomical compartments (lymph node,spleen, blood and graft). Flow cytometric analysis showedthat CD4 and CD8 T cells exist in similar percentages inlymph node, spleen, peripheral blood and graft in Cxcr3−/−

recipients compared to wild type (Figure 5A, B). Im-munohistochemical analysis showed similar infiltration ofCD4 and CD8 T-cell populations in the rejecting allografts(Figure 5C). To quantify the actual cell numbers in spleen,lymph nodes and graft, we stained the cells with CD3, CD4and CD8 mAb for flow cytometric analysis. Neither the ratio


Kwun et al.

Figure 4: The absence of Cxcr3 does not affect lymphocyte infiltration in MHC mismatched heart transplantation. (A) BALB/callografts into wild-type C57BL/6 were rejected within 10 days (� , MST = 8.6 ± 0.5 days; n = 8). Heart transplantation of BALB/cinto Cxcr3-deficient C57BL/6 (� , line 1891; MST = 9.54; n = 11) and other Cxcr3−/− C57BL/6 (•, line 3347; MST = 13.42; n = 6)showed modest prolongation compared to wild-type recipient (p = 0.02 and p = 0.002, respectively). Syngeneic (C57BL/6 to C57BL/6)combination showed no sign of rejection (� , MST > 100 days; n = 7). (B) Representative graft status in mice with cardiac transplant byH&E staining. Grafts were harvested at day 7 after transplantation and fixed with 10% buffered formalin. Sections were prepared andstained by H&E staining as described in Materials and Methods. An isograft (a) in a mouse was accepted without any complications.Grafts from a wild-type mouse (b), line #1891 Cxcr3−/− mouse (c) and line #3347 Cxcr3−/− mouse (d) shown undergoing acute rejectionon day 7 posttransplant. There is a severe perivascular lymphocytic infiltration (arrows) with myocyte damage. Original magnification×600.

nor the actual numbers of CD4 and CD8 T cells were sig-nificantly different in the graft of Cxcr3−/− recipients com-pared to wild-type recipients (Figure 5D). It is believed thatthe chemokine receptor Cxcr3 plays an important role inregulating the immune response not only by influencingT-cell migration but also T-cell activation (26,27). However,CD4 and CD8 T-cell numbers in the spleen (representingsplenic T-cell expansion after heart transplantation) werenot significantly different in Cxcr3−/− recipients comparedto wild-type recipients (p > 0.1) (Figure 5D). These resultsindicated that the Cxcr3 deficiency did not significantly altereither T-cell trafficking or T-cell activation after fully MHCmismatched heart transplantation.

IFN-c production is reduced in primary MLR

responses but normal in secondary MLR responses

in the absence of Cxcr3

Spleens from the cardiac allograft recipients were har-vested 7 days after transplantation and processed to sin-gle cell suspension. The splenocytes from Cxcr3−/− recipi-ents showed similar numbers of IFN-c producing cells afterpolyclonal activation (PMA/Ionomycin) or donor-specific ac-tivation (MLR) with cytometric analysis (data not shown).To determine subtle differences in alloreactive T cells, weassessed IFN-c production by a IFN-c kinetics assay de-scribed by us (24). Recipient splenocytes were coculturedwith irradiated donor splenocytes in 96 well plates andIFN-c concentration was evaluated from the culture super-natant daily for 5 days. Spleen cells derived from pretrans-plant wild-type mice showed typical naı̈ve T-cell responsesagainst BALB/c simulator cells (primary response). Inter-estingly, splenocytes from pretransplant Cxcr3−/− mice

showed altered IFN-c expression patterns. Furthermore,significantly reduced amounts of IFN-c were detected onday 3 (p < 0.05) compared to wild-type (Figure 6A). How-ever, IFN-c expression kinetics by splenocytes from post-transplant recipients showed similar amounts and patternsof IFN-c production in the two groups (Figure 6B). Thesedata suggest that Cxcr3 is important for producing IFN-cfor naı̈ve T cells during MLR but plays a less significant rolein activated T cells.

Compensatory trafficking via Ccr5 in the absence

of Cxcr3

To investigate the effect of allogeneic stimulation onchemokine receptor expression dynamics, the chemokinereceptor Cxcr3 and Ccr5 expression on splenic T cellsbefore and after heart transplantation was examined byflow cytometry. Splenic T cells from naı̈ve C57BL/6 miceshowed that 13.8 ± 10.8% of CD4 and 31.2 ± 8.9% ofCD8 T cells expressed Cxcr3 (Suppl Figure 3). Expressionof Cxcr3 was dramatically increased on splenic CD8 T cells(45.7 ± 4.0) at 7 days after heart transplantation. However,Cxcr3 expression on CD4 T cells was not significantly af-fected (17.5 ± 1.7) by transplantation. On the other hand,Ccr5 is rarely expressed on pretransplant naı̈ve C57BL/6mouse T cells and only a slight increase of Ccr5 expres-sion was identified on both CD4 (3.51 ± 1.5%) and CD8T cell (12.5 ± 4.7%) at POD 7 (Suppl Figure 3). To inves-tigate the role of Ccr5 expressing T cells in this cardiacallograft model, we evaluated the Ccr5 positive cell frac-tion in graft infiltrating CD4 and CD8 T cells. Both wild-type and Cxcr3−/− recipients were sacrificed 7 days aftertransplantation and Cxcr3 or Ccr5 expressing T cells were



Figure 5: The absence of Cxcr3 does not alter lymphocyte distribution after heart transplantation. (A) Intragraft chemokine Mig(Cxcl9), IP-10 (Cxcl10) and I-TAC (Cxcl11) expression in Cxcr3−/− recipients. Cardiac allografts of Cxcr3−/− recipients were analyzed on7 days after transplantation and compared with date-matched wild-type control. Relative Mig, IP-10 and I-TAC mRNA expression to controlGAPDH mRNA is indicated. Data shown are representative of five independent experiments. (B) Cell surface phenotype of lymphocytesin different anatomical compartments of wild-type and Cxcr3−/− C57BL/6 recipients at POD 7. Percentages of CD4 and CD8 T cells werenot different between wild-type and KO mice. Each dot represents one animal. (C) Immunohistological analysis of wild-type and Cxcr3−/−mice. On day 7 after transplantation, grafts were retrieved from wild-type (upper) and Cxcr3−/− recipients (lower), and frozen sectionswere prepared and stained with CD4 or CD8 mAbs. Original magnification ×400. (D) Absolute cell numbers of CD4 and CD8 T cells in thegraft evaluated. Data are plotted on the basis of CD4 T cell and CD8 T-cell subpopulation gated by CD3+CD4+ and CD3+CD8+ staining.Total yield was calculated on the basis of total lymphocytes Results are representative of six mice. Graft infiltrating CD4 and CD8 T cellsshowed no difference in Cxcr3−/− recipients compare to wild-type recipients. Values represent mean ± SD of five animals.

measured in the graft. We found that the Ccr5 expressinggraft infiltrating T cells were increased in Cxcr3-/- recipi-ents compared to wild type (Figure 7A). Interestingly, theproportion of the Ccr5+ fraction in intragraft CD8 T cell wassignificantly increased (p < 0.01) in Cxcr3−/− recipients(Figure 7B). Increased absolute numbers of Ccr5+CD8+

T cells in the graft confirmed that more Ccr5-driven CD8T cells traffick in the absence of Cxcr3. These observa-tions suggested that the less dominant chemokine recep-tor might compensate with respect to trafficking in theabsence of Cxcr3.

Discussion

The question addressed by the present study was whetherthe abolishment of Cxcr3-driven chemotaxis affects allo-graft survival and the magnitude of lymphocyte traffick-ing into cardiac allografts. The main findings of the studyare that a lack of Cxcr3-driven trafficking does not affectlymphocyte infiltration into the graft. Furthermore, graftsurvival in a heterotopic vascularized heart transplantationmodel across full MHC disparities is not profoundly altered.

It seems plausible that Cxcr3 and its ligands would be im-plicated in the pathogenesis of allograft rejection by direct-ing pathogenic T and NK cells to sites of inflammation.Cxcr3 and Ccr5 have been reported to be critical chemokinereceptors to prolong allograft survival in the experimentalsetting in mouse heterotopic heart transplantation (9,28).However, in a cardiac allograft model, we only found mod-erate prolongation of survival in the 1C6 and CXCR3 an-tagonist (MRL-957)-treated recipients (Figure 2). Combina-tion with low dose CsA (10 mg/kg) produced synergisticeffects and increased graft survival, but was not potentenough to prevent rejection (Figure 3). We presumed thatthe altered physiology of the KI mouse was responsiblefor the limited effect of Cxcr3 blockade. We therefore re-examined two independently derived Cxcr3−/− mice avail-able to us. We performed heterotopic vascularized hearttransplants in two independently derived lines of Cxcr3−/−

mice. However, we could not demonstrate remarkable pro-longation of graft survival in either line (Figure 4). Onlymarginal prolongation was achieved by low dose CsA inCxcr3−/− recipients. These results contradicted previousreports in which heart grafts in Cxcr3−/− recipients (equiv-alent to line 1891 in this study) survived to a mean of


Kwun et al.

Figure 6: IFN-c production is reduced compared to the pri-

mary response but effector/memory response intact in the

absence of Cxcr3. (A) IFN-c expression kinetics of pretransplantor (B) posttransplant recipients was measured. Pretransplant naı̈veand transplant POD7 C57BL/6 wild-type or Cxcr3−/− recipient’ssplenocytes were obtained. Cells were co-cultured with irradiatedBALB/c donor splenocytes. Culture supernatant was collected forfive consecutive days and IFN-c was measured with ELISA. Val-ues are the Mean ± SD. Statistical evaluation was performed usingthe Student’s t-test: ∗, p < 0.05; wild-type vs. Cxcr3−/− mice.

58 days with quantitative histological analysis demonstrat-ing significantly reduced cell numbers for CD4 as well asCD8 T cells (9). However, our histology confirmed similardegrees of graft destruction in both Cxcr3−/− and wild-type recipients. Immunohistology confirmed similar lev-els of CD4 and CD8 T cells in the grafts of wild-type andCxcr3−/− recipients (Figure 5). Flow cytometry data fromfour immune compartments confirmed similar levels of T-cell distribution, expansion and graft infiltration (Figures 5and 6).

Several possible explanations could address the discrep-ancy between our findings and those reported previously.First, we used mice generated from a different degree ofbackcrossing and, therefore which may have a somewhatdifferent genetic background. The Cxcr3−/− mice used byHancock et al. (9) were described as having undergone

at least six generations of backcrosses to B6 mice whilewe used 10 generations of backcrossed mice. Therefore,Cxcr3−/− mouse strains (both #1891 and #3347) we usedwere more stringent. Different levels of cleanliness be-tween animal facilities may also contribute to differencesby affecting the basal level of chemokine receptor expres-sion. Variable degrees of disruption of cardiac lymphaticsdue to different surgical techniques could also affect theoutcome of lymphocyte graft infiltration. However, we feelthese simple explanations are unlikely, as our experiencewith CXCR3 blockade (anti-hCXCR3 mAb) in a nonhumanprimate kidney transplantation model showed similar re-sults to our murine data (manuscript in preparation).

Anti-Cxcr3 therapy was initially and simplistically thoughtof as a means to block mononuclear cell trafficking intothe area of inflammation. This idea was based on the as-sumptions that (1) chemotaxis was the sole function medi-ated by Cxcr3 and (2) that no functional redundancy existedamong chemokines/chemokine receptors for Cxcr3-drivenchemotaxis on activated T cells. Several reports showedboth assumptions might not be true. A positive feedbackloop between Cxcr3 and IFN-c production (29) suggestsinvolvement of Cxcr3 in T-cell effector function. Bromleyet al. showed that signaling through CXCR3 can overridethe T-cell receptor (TCR) signal (27). We also found evi-dence of cross talk between CXCR3 and TCR signaling (30)and increased immunodominant minor H-antigen-specificCD8 T cells after heart transplantation in the BALB/b toC57BL/6 combination (31). These collective data suggestmultifunctional roles of CXCR3. However, we found thatneither splenic T-cell expansion nor IFN-c producing cellswere altered in Cxcr3−/− recipients 7 days after transplan-tation (Figure 5A). Since the possible positive feed backloop between Cxcr3 and IFN-c production was reported(29,32,33), we evaluated by flow cytometry the numbersof IFN-c expressing T cells following polyclonal activationor donor allospecific activation. IFN-c producing T-cell num-bers were not different between wild-type and Cxcr3−/−

recipients′ cells (data not shown). Interestingly, IFN-c ex-pression kinetics showed less production of IFN-c during5 day MLR in pretransplant Cxcr3−/− mice than in wild-type mice. Reduced IFN-c production from pretransplantmouse splenocytes (primary response) indicates a reducedcapacity to produce IFN-c in the absence of Cxcr3. How-ever, restoration of IFN-c production against donor fromposttransplant splenocytes suggests that once they areactivated, T cells (effector/memory response) are indepen-dent of a Cxcr3 positive amplification loop to produce IFN-c(Figure 6).

Since we have not found any signs of retention in im-mune compartments, we suspected compensatory traf-ficking of T cells in the absence of Cxcr3. In support ofthis hypothesis, transplantation with chemokine receptor-deficient mice has previously documented delayed acuterejection, but that eventually T cells trafficked into thegraft. Islet transplantation in Ccr2−/− mice and small bowel



Figure 7: Increased proportion or number of Ccr5 expressing CD8+ graft infiltrating cells in Cxcr3−/− recipient. (A) Graft infiltrationof chemokine receptor Cxcr3 or Ccr5 expressing T cells in wild-type and Cxcr3 -/- recipients. Grafts were recovered from wild-type andCxcr3−/− (line#1891) recipients 7 days after transplantation. Graft infiltrating cells were isolated by using liberase CI (0.45mg/mL) and werestained with anti-CD4, anti-CD8 and either anti-Cxcr3 or anti-Ccr5. Abolishment of Cxcr3 expressing CD8 T cells in Cxcr3−/− recipientswas noted. Data shown are from one experiment, and are representative of six independent experiments with six mice per group ineach case. Solid area showed isotype control. (B) Increased proportion of Ccr5+ cells in CD8 T cells in the graft infiltration. Bar graphrepresents the average frequency of Ccr5 expressing CD4 or CD8 T cells in the graft. (C) Increased intragraft infiltration of Ccr5+ CD8 Tcells in the absence of Cxcr3. Actual number of Ccr5 expressing CD4 or CD8 T cells in the graft was evaluated based on flow cytometricdata plot gated by Ccr5+CD4+ and Ccr5+CD8+ staining. Total yield was calculated on the basis of total intragraft lymphocytes counts.Values represent mean ± SD of six animals. ∗∗p < 0.01, ∗p < 0.05 by Student’s t-test; compared to wild type.

transplantation in Cxcr3−/− are such examples (34,35). Per-haps the immune response to fully mismatched allografts,characterized by a high number of alloreactive T cells, withthe addition of ischemic injury and a heterogeneity of T-cellpopulations within a complex chemokine/cytokine milieueffectively overwhelms the effect of abolishing Cxcr3.

Moderate levels of Cxcr3 surface expression (20 to 40%)on CD4 and CD8 T cells have been reported (36–38). In ourstudy, Cxcr3 was expressed by ∼30% of CD8 T cells andby ∼15% of CD4 T cells in pretransplant wild-type mousespleens (Suppl Figure 3). A significant expansion of theCxcr3+ fraction of CD8 T cells occurred in spleens of trans-planted mice, whereas the Cxcr3+ fraction of CD4 T cellswas not significantly increased. Ccr5 is another candidate

receptor for mediating intragraft infiltration of alloreactiveT cells. However, Ccr5 expression was not increased asmuch as Cxcr3 after transplantation on CD8 T cells in wild-type mice.

Interestingly, we observed an increased intragraft Ccr5+

CD8+ T cell fraction in Cxcr3−/− recipients compared towild-type recipients (Figure 7B). Given comparable abso-lute numbers of infiltrating cells this suggests compen-satory trafficking via Ccr5 in the absence of Cxcr3. An alter-native trafficking pathway has been proposed by Fairchildand colleagues during anti-Cxcr3 treatment of cardiacallografts based on high mRNA expression of Ccr5 and itsligands (MIP-1a, RANTES) from anti-Mig-treated allografts(39).


Kwun et al.

In summary, our study demonstrates that the previouslypostulated role of Cxcr3 in a fully MHC mismatchedheart graft model needs critical reevaluation. It is knownthat a multiplicity of chemokines can be found in therejecting allograft (40) The hierarchy and redundant roleof chemokine receptors on effector cells in the richchemokine milieu like a rejecting allograft needs to be care-fully reevaluated. It may be difficult to block leukocyte traf-ficking during explosive expansion and accumulation intothe allograft as seen during acute rejection by blocking asingle chemokine/chemokine receptor or by resorting to asingle gene deficiency. Our conclusion is that acute rejec-tion is a complex process involving mixed subsets of effec-tor cells that can use different trafficking signals to accu-mulate in the same target tissue. Given the complexity andcompensatory nature of chemokine networks, conflictingresults may be expected from single or even double knockout models (41). Attenuated alloresponses with a smallerand less heterogeneous population of alloreactive T cellsby matching degree of MHC might provide more permis-sive models to demonstrate effects of chemokine receptorblockade.

Acknowledgments

The authors would like to thank Dr. Bruno Luckow (Klinikum der UniversitatMunchen, Medizinische Poliklinik, Germany) for critical review of the article.

References

1. Luster AD. Chemokines — Chemotactic cytokines that mediateinflammation. N Engl J Med 1998; 338: 436–445.

2. Gerard C, Rollins BJ. Chemokines and disease. Nat Immunol 2001;2: 108–115.

3. Luster AD, Alon R, von Andrian UH. Immune cell migration in in-flammation: Present and future therapeutic targets. Nat Immunol2005; 6: 1182–1190.

4. Hancock WW. Chemokine receptor-dependent alloresponses. Im-munol Rev 2003; 196: 37–50.

5. Grone HJ, Weber C, Weber KS et al. Met-RANTES reduces vas-cular and tubular damage during acute renal transplant rejection:Blocking monocyte arrest and recruitment. FASEB J 1999; 13:1371–1383.

6. Panzer U, Reinking RR, Steinmetz OM et al. CXCR3 and CCR5positive T-cell recruitment in acute human renal allograft rejection.Transplantation 2004; 78: 1341–1350.

7. Akalin E, Hendrix RC, Polavarapu RG et al. Gene expression anal-ysis in human renal allograft biopsy samples using high-densityoligoarray technology. Transplantation 2001; 72: 948–953.

8. Hoffmann U, Segerer S, Rummele P et al. Expression of thechemokine receptor CXCR3 in human renal allografts—a prospec-tive study. Nephrol Dial Transplant 2006; 21: 1373–1381.

9. Hancock WW, Lu B, Gao W et al. Requirement of the chemokinereceptor CXCR3 for acute allograft rejection. J Exp Med 2000; 192:1515–1520.

10. Akashi S, Sho M, Kashizuka H et al. A novel small-molecule com-pound targeting CCR5 and CXCR3 prevents acute and chronic al-lograft rejection. Transplantation 2005; 80: 378–384.

11. Fischereder M, Luckow B, Hocher B et al. CC chemokine receptor5 and renal-transplant survival. Lancet 2001; 357: 1758–1761.

12. Luckow B, Joergensen J, Chilla S et al. Reduced intragraft mRNAexpression of matrix metalloproteinases Mmp3, Mmp12, Mmp13and Adam8, and diminished transplant arteriosclerosis in Ccr5-deficient mice. Eur J Immunol 2004; 34: 2568–2578.

13. Amano H, Bickerstaff A, Orosz CG, Novick AC, Toma H, FairchildRL. Absence of recipient CCR5 promotes early and increasedallospecific antibody responses to cardiac allografts. J Immunol2005; 174: 6499–6508.

14. Liu L, Huang D, Matsui M et al. Severe disease, unaltered leuko-cyte migration, and reduced IFN-gamma production in CXCR3-/- mice with experimental autoimmune encephalomyelitis. J Im-munol 2006; 176: 4399–4409.

15. Wareing MD, Lyon AB, Lu B, Gerard C, Sarawar SR. Chemokineexpression during the development and resolution of a pulmonaryleukocyte response to influenza A virus infection in mice. J LeukocBiol 2004; 76: 886–895.

16. Chakravarty SD, Xu J, Lu B, Gerard C, Flynn J, Chan J. Thechemokine receptor CXCR3 attenuates the control of chronic my-cobacterium tuberculosis infection in BALB/c mice. J Immunol2007; 178: 1723–1735.

17. Duffner U, Lu B, Hildebrandt GC et al. Role of CXCR3-induceddonor T-cell migration in acute GVHD. Exp Hematol 2003; 31: 897–902.

18. Baker MS, Chen X, Rotramel AR et al. Genetic deletion ofchemokine receptor CXCR3 or antibody blockade of its ligand IP-10 modulates posttransplantation graft-site lymphocytic infiltratesand prolongs functional graft survival in pancreatic islet allograftrecipients. Surgery 2003; 134: 126–133.

19. Agostini C, Calabrese F, Rea F et al. Cxcr3 and its ligand CXCL10are expressed by inflammatory cells infiltrating lung allografts andmediate chemotaxis of T cells at sites of rejection. Am J Pathol2001; 158: 1703–1711.

20. Jiankuo M, Xingbing W, Baojun H et al. Peptide nucleic acid an-tisense prolongs skin allograft survival by means of blockade ofCXCR3 expression directing T cells into graft. J Immunol 2003;170: 1556–1565.

21. Hildebrandt GC, Corrion LA, Olkiewicz KM et al. Blockade ofCXCR3 receptor: Ligand interactions reduces leukocyte recruit-ment to the lung and the severity of experimental idiopathic pneu-monia syndrome. J Immunol 2004; 173: 2050–2059.

22. Shiao LL, Cascieri MA, Trumbauer M, Chen H, Sullivan KA. Gen-eration of mice expressing the human glucagon receptor with adirect replacement vector. Transgenic Res 1999; 8: 295–302.

23. Winters GL, Marboe CC, Billingham ME. The International Societyfor Heart and Lung Transplantation grading system for heart trans-plant biopsy specimens: Clarification and commentary. J HeartLung Transplant 1998; 17: 754–760.

24. Kwun J, Knechtle SJ, Hu H. Determination of the functional statusof alloreactive T cells by interferon-gamma kinetics. Transplanta-tion 2006; 81: 590–598.

25. Christensen JE, de Lemos C, Moos T, Christensen JP, ThomsenAR. CXCL10 is the key ligand for CXCR3 on CD8+ effector T cellsinvolved in immune surveillance of the lymphocytic choriomenin-gitis virus-infected central nervous system. J Immunol 2006; 176:4235–4243.

26. Taub DD, Turcovski-Corrales SM, Key ML, Longo DL, Murphy WJ.Chemokines and T lymphocyte activation: I. Beta chemokines cos-timulate human T lymphocyte activation in vitro. J Immunol 1996;156: 2095–2103.

27. Bromley SK, Peterson DA, Gunn MD, Dustin ML. Cutting edge:Hierarchy of chemokine receptor and TCR signals regulating T cellmigration and proliferation. J Immunol 2000; 165: 15–19.

28. Gao W, Faia KL, Csizmadia V et al. Beneficial effects of targetingCCR5 in allograft recipients. Transplantation 2001; 72: 1199–1205.



29. Campbell JD, Gangur V, Simons FE, HayGlass KT. Allergic humansare hyporesponsive to a CXCR3 ligand-mediated Th1 immunity-promoting loop. FASEB J 2004; 18: 329–331.

30. Dar WA, Knechtle SJ. CXCR3-mediated T-cell chemotaxis involvesZAP-70 and is regulated by signalling through the T-cell receptor.Immunology 2007; 120: 467–485.

31. Kwun J, Hu H, Schadde E et al. Altered distribution of H60 minorH antigen-specific CD8 T cells and attenuated chronic vasculopa-thy in minor histocompatibility antigen mismatched heart trans-plantation in Cxcr3−/− mouse recipients. J Immunol 2007; 179:8016–8025.

32. Gangur V, Simons FE, HayGlass KT. Human IP-10 selectivelypromotes dominance of polyclonally activated and environmen-tal antigen-driven IFN-gamma over IL-4 responses. FASEB J 1998;12: 705–713.

33. Gangur V, Simons FE, HayGlass KT. IP-10 mediated reinforcementof human type 1 cytokine synthesis to environmental allergensamong non-atopic subjects. Int Arch Allergy Immunol 1999; 118(2-4): 387–390.

34. Abdi R, Means TK, Ito T et al. Differential role of CCR2in islet and heart allograft rejection: Tissue specificity ofchemokine/chemokine receptor function in vivo. J Immunol 2004;172: 767–775.

35. Zhang Z, Kaptanoglu L, Tang Y et al. IP-10-induced recruitment ofCXCR3 host T cells is required for small bowel allograft rejection.Gastroenterology 2004; 126: 809–818.

36. Moser B, Loetscher M, Piali L, Loetscher P. Lymphocyte re-sponses to chemokines. Int Rev Immunol 1998; 16(3-4): 323–344.

37. Loetscher M, Gerber B, Loetscher P et al. Chemokine receptorspecific for IP10 and mig: Structure, function, and expression inactivated T-lymphocytes. J Exp Med 1996; 184: 963–969.

38. Qin S, Rottman JB, Myers P et al. The chemokine receptors CXCR3and CCR5 mark subsets of T cells associated with certain inflam-matory reactions. J Clin Invest 1998; 101: 746–754.

39. Miura M, Morita K, Kobayashi H et al. Monokine induced by IFN-gamma is a dominant factor directing T cells into murine cardiacallografts during acute rejection. J Immunol 2001; 167: 3494–3504.

40. Chen D, Ding Y, Schroppel B et al. Differential chemokine andchemokine receptor gene induction by ischemia, alloantigen, andgene transfer in cardiac grafts. Am J Transplant 2003; 3: 1216–1229.

41. de Lemos C, Christensen JE, Nansen A et al. Opposing effectsof CXCR3 and CCR5 deficiency on CD8+ T cell-mediated in-flammation in the central nervous system of virus-infected mice.J Immunol 2005; 175: 1767–1775.

Supplementary Material

The following supplementary material is available for thisarticle:

Material and methods

Splenocytes Cells were harvested and cultured in RPMI1640 media contained 10% FBS, 2 mM L-glutamine,1 mM sodium pyruvate, 10 mM HEPES, 55 lM 2-mercaptoethanol, 100 lM nonessential amino acid and

100 units/mL of penicillin-streptomycin. For activation cellswere adjusted to 2 × 106/mL with RPMI 1640 media andeither 1 lg/mL of Staphylococcus aureus enterotoxin typeB (SEB, Sigma), or 4 lg/mL Concanavalin A (MP Biomed-icals) was added. After 3–4 days incubation at 37◦C, cellswere harvested for assay.

Binding assay Ligand binding assays were performed aspreviously described (Weng et al., 1998) using 50 pM of125I-hIP-10 (NEN, 2200 Ci/mmol) and 5 × 105 cells. All datawere analyzed with GraphPad Prism software. In vitro bind-ing studies demonstrated binding and signaling of murineCXCL9, CXCL10 and CXCL11 through the human KI recep-tor.

Figure S1: (A) CXCR3 expression is up-regulated by

SEB treatment of splenocytes in vitro. (B) MurineCXCR3 ligands are fully functional and bind with high affin-ity to human CXCR3. Human IP-10 and human MIG havesimilar affinity for hCXCR3 (RBL cells) in both binding andfunctional assays.

Figure S2: Neutralizing Ab (1C6) and Compound A,

specific for the human receptor, blocks chemotaxis of

splenocytes from hCXCR3 KI mice. (A) SEB activatedsplenocytes from human CXCR3 KI were incubated withanti-human CXCR3 Ab (blue) or isotype control (red). (B)

MRL-957 (MRK A) blocks chemotaxis.

Figure S3: Dynamics of Cxcr3 and Ccr5 expression on

T lymphocytes after full MHC mismatched heart trans-

plantation. FACS analysis on splenocytes from the pre-transplant or posttransplant recipients showed increasedof Cxcr3 and Ccr5 expression on T cells after transplanta-tion. Pretransplant naı̈ve or posttransplant day 7 recipientspleen was harvested from both wild-type and Cxcr3−/−

mice. Isolated cells were stained with anti-CD4, anti-CD8and anti-Cxcr3 or anti-Ccr5. Data shown are from one ex-periment and are representative of six independent exper-iments with six mice per group in each case. Solid areashows isotype control.

This material is available as part of the online articlefrom: http://www.blackwellpublishing.com/doi/abs/10.1111/j.1600-6143.2008.02250.x

(This link will take you to the article abstract)

Please note: Blackwell Publishing is not responsible forthe content or functionality of any supplementary materialssupplied by the authors. Any queries (other than missingmaterial) should be directed to the corresponding authorfor the article.


unaltered graft survival and intragraft lymphocytes infiltration in the cardiac allograft of cxcr3...

Documents