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The Journal of Immunology, 2008, 180, 4714-4721
Copyright © 2008 by The American Association of Immunologists, Inc.
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Combined CXCR3/CCR5 Blockade Attenuates Acute and Chronic Rejection1

Gabriel T. Schnickel2,*,§, Sam Bastani2,*,§, George R. Hsieh*,§, Ali Shefizadeh*, Rubina Bhatia*, Michael C. Fishbein{dagger}, John Belperio{ddagger} and Abbas Ardehali3,*,§

* Department of Surgery, {dagger} Department of Pathology and Laboratory Medicine, and {ddagger} Division of Pulmonary and Critical Care Medicine, Division of Cardiothoracic Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095; and § West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA 90073


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chemokine-chemokine receptor interactions orchestrate mononuclear cells recruitment to the allograft, leading to acute and chronic rejection. Despite biologic redundancy, several experimental studies have demonstrated the importance of CXCR3 and CCR5 in acute rejection of allografts. In these studies, deficiency or blockade of CXCR3 or CCR5 led to prolongation of allograft survival, yet allografts were ultimately lost to acute rejection. Given the above findings and the specificity of mononuclear cells bearing CXCR3 and CCR5, we hypothesized that combined blockade of CXCR3 and CCR5 will lead to indefinite (>100 days) graft survival in a full MHC-mismatched murine cardiac allograft model. The donor hearts in the control group were rejected in 6 ± 1 days after transplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival >15-fold vs the control group; all allografts survived for >100 days. More importantly, the donor hearts did not display any intimal lesions characteristic of chronic rejection. Further analysis of the donor hearts in the CXCR3/CCR5 blockade group demonstrated graft infiltration with CD4+CD25+ T cells expressing the Foxp3 gene. Depletion of CD25+ cells in the combined CXCR3 and CCR5 blockade group resulted in acute rejection of the allografts in 22 ± 2 days. Combined CXCR3 and CCR5 blockade also reduced alloantigen-specific T lymphocyte proliferation. Combined CXCR3 and CCR5 blockade is effective in preventing acute and chronic rejection in a robust murine model. This effect is mediated, in part, by CD25+ regulatory T cell recruitment and control of T lymphocyte proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recruitment of mononuclear cells to the graft is an early and key event in the development of acute and chronic rejection in solid organ transplantation (1). Chemokines play an important role in directing the migration of mononuclear cells. Chemokines are a large family of secretory proteins that act on responsive leukocytes via their corresponding G protein-coupled receptor (2). Targeting of specific chemokine-chemokine receptor interaction to block the recruitment of effector cells is an attractive strategy to control acute and chronic rejection due to specificity of the approach and lack of systemic immunosuppressive effects.

In the past decade, several groups have undertaken studies to dissect out the role of chemokine/chemokine receptors in alloimmune responses (3, 4, 5, 6, 7, 8, 9, 10, 11). In experimental models of acute rejection, virtually every chemokine and chemokine receptor is expressed (1). Despite the redundancy within the chemokine-chemokine receptor family, several chemokine receptors have emerged as pivotal in directing activated T lymphocytes in acute rejection. Chief among these chemokine receptors are CXCR3 and CCR5. CXCR3 is expressed on Th1 T cells, some B cells, and NK cells. CXCR3 is a receptor for three ligands: monokine induced by IFN-{gamma} (MIG)4/CXCL9, IFN-{gamma}-inducible protein 10 (IP-10)/CXCL10, and IFN-{gamma}-inducible T cell {alpha}-chemoattractant /CXCL11. CCR5 is expressed on Th1 T cells, macrophages, dendritic cells, and NK cells. The CCR5 ligands are: RANTES/CCL5, MIP-1{alpha}/CCL3, and MIP-1β/CCL4.

Several studies have shown that CXCR3, CCR5, and their corresponding ligands are expressed in both murine and human cardiac allograft rejection (3, 4, 6, 8, 11, 12, 13). In experimental models, the deficiency or blockade of a single chemokine (CXCR3 or CCR5 ligands) did not protect the allograft from acute rejection (except for IP-10/CXCL10 in the graft) (14). However, blockade or absence of CXCR3 or CCR5 prolonged allograft survival in a fully MHC-mismatched model, yet the allografts were eventually lost to acute cellular rejection (4, 6). Given the specificity of T lymphocytes bearing CXCR3 and CCR5, the available experimental data, and the corroborating clinical data, we hypothesized that combined CXCR3 and CCR5 blockade will lead to indefinite (>100 days) graft survival in a full MHC-mismatched mouse heart transplant model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Adult female CCR5–/– (B6;129P-Cmkbr5tm1Kuz) mice (CCR5–/– mice), wild-type B6;129PF2/J mice (wild-type mice), and BALB/c mice, 6–12 wk old, were purchased from The Jackson Laboratory. Wild-type B6;129PF2/J served as control mice for CCR5–/– (B6;129P-Cmkbr5tm1Kuz) mice. All animals received humane care in compliance with Institutional Animal Care-approved criteria and protocols.

Mouse heart transplantation

BALB/c strain donor hearts were transplanted into wild-type recipient mice or CCR5–/– recipient mice. Intra-abdominal heterotopic heart transplantation was performed using a modification of the method outlined by Corry et al. (15). The function of the allografts was assessed daily by abdominal palpation. The animals were sacrificed if the allografts stopped beating or at 24 or 100 days.

Reagent

Anti-CXCR3 Ab was prepared as previously described (16). Briefly, polyclonal goat anti-murine CXCR3 was produced by the immunization of goats with a 16-mer peptide (PYDYGENESDFSDSPP) constituting the NH2 terminus of murine CXCR3. The goat was immunized in multiple intradermal sites with CFA followed by at least three boosts. Direct ELISA was used to evaluate titers. The specificity of the serum was analyzed when titers reached >1/1,000,000. The specificity of anti-CXCR3 Ab was confirmed by Western blot analyses against cells expressing CXCR3 and a panel of murine and human cytokines (IL-1 R{alpha}, IL-1, IL-2, IL-6, IL-4, TNF-{alpha}, IFN-{gamma}, and CXC and CC chemokine families). The potency of anti-CXCR3 Ab was determined by a chemotaxis assay in vitro. Murine splenocytes were stimulated with IL-2 for 10 days with documented CXCR3 expression by FACS analysis. A total of 50 ng/ml IP-10/CXCL10 and MIG/CXCL9 were preincubated with either anti-CXCR3 Ab or control Ab for 30 min at 37°C. Chemotaxis assay was performed as previously described with 5.0-µm polycarbonate filters coated with fibronectin (ICN Biomedicals) (17). The anti-CXCR3 Ab demonstrated neutralizing capacity by inhibiting chemotaxis of IL-2-stimulated, CXCR3-expressing splenocytes to IP-10/CXCL10 and MIG/CXCL9 in this assay (Table I).


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  		Table I. Anti-CXCR3 Ab demonstrated neutralizing capacity by inhibiting chemotaxis of IL-2-stimulated, CXCR3-expressing splenocytes against IP-10/CXCL10 and MIG/CXCL9

 
The potency of this anti-CXCR3 Ab in mice has also been demonstrated by a previous study. In a murine tracheal allograft model, this anti-CXCR3 Ab reduced infiltration of CXCR3- positive leukocytes and attenuated the severity of obliterative bronchiolitis (16). The dose of the anti-CXCR3 Ab in the current study was based on this previously published report.

The anti-CXCR3 Ab administered according to this study’s protocol does not result in CXCR3-positive cell depletion. The percentage of CXCR3-positive splenocytes in the anti-CXCR3 Ab vs control Ab-treated groups were similar (7.9 ± 1.2 vs 8.6 ± 1.9, p = NS). To determine whether this anti-CXCR3 Ab specifically deletes activated T cells in vitro, we also performed the following MLRs (responder cells: 1) wild-type splenocytes plus medium, 2) wild-type splenocytes plus control Ab, and 3) wild-type splenocytes plus anti-CXCR3 Ab; stimulator cells: BALB/c splenocytes). Cells were harvested on days 2 and 4, and the number of viable cells was counted both directly and after addition of trypan blue. The number of nonviable cells was similar, suggesting that this anti-CXCR3 Ab does not lyse activated T cells (data not shown).

The anti-CD25 mAb (rat IgG1, clone PC-61) was purchased from Bioexpress Cell culture services. This Ab, which is specific to IL-2R {alpha}-chain, causes depletion of CD25+ cells in vivo (18, 19).

Experimental group

In the experimental group, BALB/c strain donor hearts were transplanted into CCR5–/– recipient mice. These animals were then treated with 0.5 ml of anti-CXCR3 Ab i.p. every other day, beginning on postoperative day 0. Five animals were sacrificed on day 24 posttransplantation. The remaining five were harvested on day 100 posttransplantation. There were three control groups: BALB/c donor hearts were transplanted into 1) wild-type mice treated with control Ab (n = 5), 2) CCR5–/– mice treated with control Ab (n = 5), and 3) wild-type mice treated with anti-CXCR3 Ab (n = 5). The protocol for administration of the anti-CXCR3 Ab or control Ab was identical to the regimen for the experimental group.

An additional group of animals (identical to the experimental group: BALB/c hearts were transplanted into CCR5–/– recipients and treated with anti-CXCR3 Ab) were used to study the role of CD25+ cells (n = 5). These mice were treated with anti-CD25 Ab (0.5 mg i.p. on day 0 plus 0.25 mg i.p. on days 2, 4, 6, 8, and 10) (20). No immunosuppression was given to any of the recipient mice.

Histology/immunohistochemistry

The basal segments of explanted hearts were either stained with H&E or immunostained. The primary Abs used for immunohistochemistry were as follows: rat anti-mouse CD4 mAb (clone L3T4), rat anti-mouse CD8a mAb (Ly-2; BD Pharmingen), rat anti-mouse CD25 mAb (clone 7D4; BD Pharmingen), and rat anti-mouse MOMA-2 mAb for monocytes/macrophages (Serotec). Immunohistochemistry was performed using the ABC immunoperoxidase technique. A blinded observer graded the perivascular and intimal regions.

Morphometric analyses

The explanted hearts underwent serial sectioning (5-µm thick) from the mid-ventricular level to the base. Verhoeff elastic staining was performed for morphometric analyses of arterial intimal lesions. All coronary arteries (30–350 µm in diameter) were analyzed on a PC using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image/). Three cross- sections of each mouse heart were evaluated. The number of analyzed vessels per heart was 9 ± 2. Luminal and intimal areas were traced and the areas quantitated with Optimas software (Optimas 6.0; Media Cybernetics). Intimal thickening was calculated according to the formula intimal/intimal + luminal and expressed as a percentage.

Graft-infiltrating cell isolation and FACS analysis

Hearts were digested in collagenase D solution. Isolated cells were counted after lysis of erythrocytes. Surface labeling of cells was performed by FITC- and PE-labeled CD4 and CD8 Abs, respectively, and PE-labeled CD25 Ab (BD Pharmingen). FACS analysis of labeled cells was conducted on an EPICS XL-MCL flow cytometer (Coulter).

RT-PCR

Total RNA was isolated from the donor hearts using the TRIzol (Invitrogen Life Technologies) method as previously described (21). Two micrograms of DNase I-treated RNA was then used to synthesize the first strand of cDNA by the cDNA Synthesis Kit (Bio-Rad). TaqMan-based PCR assays were used to measure DNA using an Applied Biosystems Prism 770 Sequence Detection System. A master mix was used consisting of 12.5 µl of iTaq SYBR Green Supermix with Rox (Bio-Rad), 5 µl of 10 µM forward primer, 5 µl of 10 µM reverse primer, and sterile water. The cDNA product was amplified using PCR primers specific for the mouse Foxp3 (forward and reverse primer sequences for Foxp3 are 5'-CCCAGGAAAGACAGCAACCTT-3' and 5'-TTCTCACAACCAGGCCTCTTG-3'). PCR conditions were 95°C for 3 min, 50 cycles of 95°C for 15 s, and 60°C for 60 s. All quantitative PCR assays contained no-template control samples (negative controls) and five samples consisting of mouse genomic DNA added to reactions in duplicate to produce standards. The threshold cycle values from the genomic DNA standards were used to create a standard curve to assess the amount of DNA in samples. All samples were run in duplicate. Data are reported as quantity of transcript (as reported by cycle threshold) per 2 µg of RNA.

Mixed leukocyte reaction

A total of 8 x 105 responder splenocytes (wild-type mice or CCR5–/– mice) were incubated with a similar number of irradiated stimulator cells (BALB/c) for 72 h, followed by pulsing with 0.5 µCi of [3H]thymidine (Amersham Biosciences) for 14 h. The cells were harvested with a semiautomated cell harvester and counted on a beta scintillation counter. Exogenous anti-CXCR3 Ab or control Ab was added to each well at varying concentrations at the start of the MLR. All MLRs were performed in triplicate and repeated three times (using three animals).

ELISPOT

ELISPOT assays for murine IFN-{gamma} were performed according to the manufacturer’s guidelines (BD Biosciences). Briefly, 200,000 cells from a 48-h MLR were plated on 96-well plates that had been previously coated with a goat anti-murine IFN-{gamma} Ab overnight. The cells were incubated for 24 h. The wells were then washed and reacted with a biotinylated goat anti-murine IFN-{gamma} Ab. The spots were visualized with 3-amino-9-ethylcarbazole chromogen (Sigma-Aldrich). Visualization and analysis were performed using Immunospot Series 1 Analyzer (Cellular Technology). All assays were performed in triplicate and were repeated three times.

Statistical analyses

All results were expressed as mean ± SEM. Data were analyzed with a paired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Combined CXCR3 and CCR5 blockade prolongs allograft survival

Wild-type mice receiving a full MHC-mismatched allograft and treated with control Ab (control group) rejected their cardiac allografts at 6 ± 1 days. The survival of the donor hearts in CCR5–/– mice receiving control Ab and in the wild-type mice receiving anti-CXCR3 Ab were prolonged to 29 ± 9 and 34 ± 3 days, respectively (Fig. 1). Histologic examination of the rejected allografts revealed marked infiltration with mononuclear cells, myocyte necrosis, and swelling characteristic of acute cellular rejection (Fig. 2A). In contrast, the donor hearts in CCR5–/– mice treated with anti-CXCR3 Ab (experimental group) survived to 100 days post-transplant. Examination of allografts in the experimental group at both 24 and 100 days revealed intact myocardial architecture, with moderate mononuclear cell infiltration (Fig. 2B).


Figure 1
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  		FIGURE 1. Donor hearts transplanted into control recipients were rejected in 6 ± 1 days. Hearts transplanted into CCR5–/– mice treated with control Ab were rejected in 29 ± 9 days compared with 34 ± 3 days for hearts transplanted into wild-type mice treated with anti-CXCR3 Ab. In contrast, the donor hearts in the experimental group (CCR5–/– mice treated with anti-CXCR3 Ab) survived to 100 days (n = 5 for all groups).

 

Figure 2
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  		FIGURE 2. A, Histologic analyses of control allografts revealed intense infiltration of myocardium with mononuclear cells and myocyte necrosis (H&E stain; original magnification, x40). B, Examination of the donor hearts in experimental groups demonstrated intact myocardial architecture with moderate mononuclear cell infiltration (H&E stain; original magnification, x40). C, The donor hearts in the experimental group, surviving to 100 days, did not display intimal lesions characteristic of chronic rejection (Verhoeff stain; original magnification, x40). Micrographs are representative of five grafts per group.

 
Combined CXCR3 and CCR5 blockade prevents the development of chronic rejection

Vascular intimal lesions, also known as cardiac allograft vasculopathy (CAV), characterize chronic rejection in cardiac allografts. Although the control grafts did not survive long enough to develop intimal lesions, the donor hearts in the experimental group (at 100 days post-transplantation) did not display CAV on histologic examination (Fig. 2C).

Combined CXCR3 and CCR5 blockade and graft-infiltrating mononuclear cells

Analysis and characterization of graft-infiltrating cells was performed by flow cytometry. As expected, acutely rejecting grafts (control) were infiltrated with a large number of CD4 and CD8 lymphocytes (Fig. 3, A and B). There was a reduction in the number of CD4 and CD8 lymphocytes in the experimental group (CCR5–/– mice treated with anti-CXCR3 Ab) at 24 days. However, when the grafts were examined at 100 days post-transplant, the number of graft- infiltrating CD4 and CD8 lymphocytes had increased compared with 24-day allografts (Fig. 3, A and B).


Figure 3
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  		FIGURE 3. Flow cytometric analyses of graft-infiltrating CD4+ (A) and CD8+ cells (B). Explanted donor hearts were digested; infiltrating cells were isolated and stained for flow cytometric examination. The acutely rejecting donor hearts in the control group were infiltrated with a large number of CD4 and CD8 lymphocytes. The donor hearts in the experimental group (CCR5–/–+anti-CXCR3 Ab) at 24 days displayed a moderate number of graft-infiltrating CD4 and CD8 lymphocytes. When the donor hearts in the experimental groups (CCR5–/–+anti-CXCR3 Ab) were examined at 100 days, there was an increase in the number of graft-infiltrating CD4 and CD8 lymphocytes. The numbers were obtained from five animals per group and are represented as mean ± SEM.

 
Combined CXCR3 and CCR5 blockade and regulatory T cells

We next sought to further characterize the phenotype of cells infiltrating the donor hearts in the experimental group at 100 days. CD4+CD25+ T cells in the allografts at 100 days amounted to 30.7 ± 3.7% of CD3+ graft-infiltrating cells (Fig. 4A). For comparison purposes, in acutely rejecting allografts, CD4+CD25+ T lymphocytes were 3.4 ± 0.3% of all graft-infiltrating CD3+ cells. Immunohistochemical studies confirmed the flow findings. There were focal clusters of CD4+CD25+ cells (grade, 1.8 ± 0.1) in the perivascular regions of the donor hearts from the experimental group at 100 days. In contrast, there were sparse CD4+CD25+ cells (grade, 1.0 ± 0.0) scattered throughout the rejected myocardium in the control group.


Figure 4
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  		FIGURE 4. A, Characterization of graft-infiltrating CD4 lymphocytes by flow cytometry revealed that 30.7 ± 3.4% of the graft-infiltrating CD3+ cells were CD4+CD25+ lymphocytes in 100-day surviving allografts in the experimental group. For comparison purposes, 3.4 ± 0.3% of the graft-infiltrating CD3+ cells were CD4+CD25+ lymphocytes in acutely rejecting control allografts (wild-type + control Ab). Numbers were derived from five animals per group and are represented as mean ± SEM. B, Foxp3 gene transcript level per graft-infiltrating CD4 lymphocytes in 100-day surviving allografts in the experimental group was higher when compared with acutely rejecting allografts. Foxp3 gene transcript levels were measured by RT-PCR. The values are from five animals per group and are expressed as mean ± SEM.

 
To further characterize the role of CD25+ cells, we treated an additional group of experimental animals (BALB/c donor hearts transplanted into CCR5–/– recipients treated with anti-CXCR3 Ab) with anti-CD25 Ab. These cardiac allografts were lost in 22 ± 2 days, in comparison to >100 days of survival in the experimental group (p < 0.05; Fig. 1). We also performed MLRs to assess the impact of CD25+ cell neutralization on allogeneic T cell proliferation in vitro. The proliferative response of CCR5–/– splenocytes treated with the anti-CXCR3 Ab (combined CXCR3 and CCR5 blockade group) was 87,105 ± 5,871 cpm; addition of anti-CD25 Ab augmented the proliferative response to 123,101 ± 8,374 cpm (p < 0.05). These observations suggest that CD25+ cell depletion in the setting of combined CXCR3 and CCR5 blockade reduces allograft survival in vivo and attenuates allogeneic T cell proliferation in vitro.

We next analyzed the donor hearts for Foxp3 gene transcript levels. Since Foxp3 is predominantly, if not exclusively, expressed in CD4 lymphocytes (22), we normalized the Foxp3 expression by graft-infiltrating CD4+ lymphocytes. Foxp3 gene transcript level/CD4+ lymphocytes in 100-day-old allografts were 26 ± 8.1 x 10–5 arbitrary units/CD4+ lymphocyte (Fig. 4B). For comparison purposes, Foxp3 gene transcript level/CD4+ lymphocyte in the control group (wild-type plus control Ab) was 1.4 ± 0.6 x 10–5 arbitrary units/CD4+ lymphocytes (p < 0.05).

Combined CXCR3 and CCR5 blockade decreases alloantigen-specific T lymphocyte proliferation and effector cytokine production

To further define the mechanism(s) of allograft survival in the combined CXCR3/CCR5 blockade group, we asked whether combined blockade affected allogeneic T lymphocyte proliferation in vitro. MLR was performed with wild-type or CCR5–/– splenocytes used as responder cells with and without the addition of anti-CXCR3 Ab or control Ab. We found that both the wild-type splenocytes treated with the anti-CXCR3 Ab and the CCR5–/– splenocytes treated with control Ab had a decreased proliferative response when compared with the control group (wild-type mice plus control Ab, p < 0.05; Fig. 5). The splenocytes from CCR5–/– mice treated with the anti-CXCR3 Ab (combined CXCR3 and CCR5 blockade group) had the lowest proliferative response among the studied groups (p < 0.05 when compared with the control group; Fig. 5).


Figure 5
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  		FIGURE 5. In vitro proliferative responses. Specified splenocytes (8 x 105) were incubated with a similar number of irradiated BALB/c stimulator cells for 72 h followed by pulsing with [3H]thymidine. Exogenous anti-CXCR3 Ab or control Ab was added to specified wells at the start of MLR. All MLRs were performed in triplicates and repeated three times. The values are presented as mean ± SEM. CCR5–/– splenocytes treated with control Ab and wild-type splenocytes treated with anti-CXCR3 Ab had decreased allogeneic proliferative responses when compared with wild-type splenocytes treated with the control Ab (p < 0.05). Combined CXCR3 and CCR5 blockade (CCR5–/– + anti-CXCR3 Ab) was associated with the greatest inhibition of allogeneic proliferative response (p < 0.05 when compared with single blockade splenocytes).

 
To assess effector cytokine (IFN-{gamma}) production, we studied the wild-type splenocytes treated with the control Ab and CCR5–/– splenocytes treated with anti-CXCR3 Ab (combined CXCR3 and CCR5 blockade group) by ELISPOT assay. The number of IFN-{gamma}-positive spots per well was lower when the combined CXCR3 and CCR5 blockade group was compared with the wild-type splenocytes (Fig. 6).


Figure 6
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  		FIGURE 6. To determine whether the combined CCR5/CXCR3 blockade also affects T lymphocyte effector functions in vitro, production of IFN-{gamma} in an allogeneic MLR was examined by ELISPOT assay. CCR5–/– splenocytes treated with anti-CXCR3 Ab had a lower number of IFN-{gamma}-positive spots per well when compared with wild-type splenocytes treated with the control Ab (47 ± 2 vs 129 ± 8 spots/well, respectively).

 
To assess the impact of combined CXCR3 and CCR5 blockade on donor-reactive responses in vivo, we sacrificed the following groups of animals on day 7 post-transplant and harvested the splenocytes. The groups were: wild-type plus control Ab (control group), CCR5–/– + control Ab, wild-type plus anti-CXR3 Ab, and CCR5–/– plus anti-CXCR3 Ab (experimental group). It should be noted that some of the wild-type plus control Ab splenocytes had been harvested on day 5 post-transplant, since the donor hearts were rejected by then. The harvested splenocytes were then analyzed for donor- specific proliferative responses via MLR and effector cytokine production via IFN-{gamma} ELISPOT. The splenocytes from the wild-type plus control Ab demonstrated the highest proliferative response and IFN-{gamma}-positive spots (statistically significant when compared with the other groups), while the splenocytes from the CCR5–/– mice treated with the anti-CXCR3 Ab showed the lowest proliferative response and number of IFN-{gamma}-positive spots on ELISPOT (Fig. 7, A and B).


Figure 7
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  		FIGURE 7. This figure represents the proliferative response of splenocytes from transplanted animals. To assess the impact of the combined CXCR3 and CCR5 blockade on donor-reactive responses in vivo, we sacrificed the following groups of animals on day 7 posttransplant and harvested the splenocytes. The groups were: wild-type+control Ab (control group), CCR5–/– + control Ab, wild-type + anti-CXR3 Ab, and CCR5–/–+anti-CXCR3 Ab (experimental group). It should be noted that some of the wild-type + control Ab splenocytes had been harvested on day 5 posttransplant, since the donor hearts were rejected by then. The harvested splenocytes were then analyzed for donor-specific proliferative response via MLR and effector cytokine production via IFN-{gamma} ELISPOT. The splenocytes from the CCR5–/– mice treated with the anti-CXCR3 Ab showed the lowest proliferative response (A) and number of IFN-{gamma}-positive spots on ELISPOT (B). Splenocytes were set up in a MLR in an identical fashion as described in Fig. 5. ELISPOT was performed as described in Materials and Methods. Assays were performed in triplicates from each animal; there were three animals in each group and the data are presented as mean ± SEM.

 
In addition, we set up MLRs with splenocytes from the experimental group harvested at day 100 and compared their proliferative response with splenocytes harvested from the wild-type plus control Ab (on day 7 post-transplant) against donor Ags or third-party stimulator cells. The proliferative response of splenocytes from combined CXCR3 and CCR5 blockade group (at 100 days post-transplant) was markedly lower when compared with control group splenocytes (Fig. 8). The proliferative responses of splenocytes from the combined CXCR3 and CCR5 blockade group (at 100 days) and splenocytes from control group against third-party stimulator cells were similar (Fig. 8).


Figure 8
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  		FIGURE 8. This figure represents the proliferative response of splenocytes from transplanted animals. The splenocytes from the experimental group (CCR5–/– + anti-CXCR3 Ab at 100 days) had a decreased proliferative response against BALB/c stimulator cells when compared with the splenocytes derived from the wild-type mice treated with the control Ab, which had rejected their grafts (p < 0.05). The proliferative response of the splenocytes from both groups to the third-party allostimulators was similar (p = NS). Splenocytes were set up in a MLR in an identical fashion as described in Fig. 5. MLR was performed in triplicates from each animal; there were five animals in each group; the data are presented as mean ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study demonstrates that combined CXCR3 and CCR5 blockade prevents acute and chronic rejection in a fully MHC-mismatched murine model up to 100 days. The long surviving allografts were infiltrated with CD4+CD25+ T cells that expressed Foxp3 gene. CD25+ depletion resulted in acute rejection of the allografts in 22 ± 2 days. Combined CXCR3 and CCR5 blockade was also associated with attenuated allogenic T lymphocyte proliferation.

The fate of a transplanted organ is determined in large part by the number of induced effector T cells. The effector T cell pool size is, in turn, dependent on several factors such as precursor frequency, factors involved in Ag presentation and costimulation, proinflammatory signals produced by innate immune system, and factors that regulate T cell expansion such as induction of regulatory T cells. In a fully MHC-mismatched model, a large pool of alloreactive T cells are stimulated that lead to rapid rejection of the transplanted organ (20). Combined blockade of two chemokine receptors in this study led to marked attenuation of alloimmune response in a full MHC-mismatched model; donor hearts survived >100 days without manifestations of CAV. Previous studies have shown that isolated blockade of CCR5 or CXCR3 are associated with a 3- and 8-fold increase in allograft survival, respectively (4, 6). In contrast, combined blockade of these two chemokine receptors in this study was synergistic and associated with a >15-fold increase in allograft survival.

One possible explanation for this beneficial effect of combined CXCR3 and CCR5 blockade is the induction of regulatory T cells. Regulatory T cells are known to be a critical factor in expansion and activation of effector T cells, which will in turn determine the fate of the transplanted organ. In experimental studies, CD4+CD25+ T cells are shown to inhibit allograft rejection (23, 24, 25, 26, 27). In a MHC class II- mismatched cardiac allograft model, acute rejection of the donor heart grafts was inhibited by CD4+CD25+ regulatory cells that restricted the clonal expansion of alloreactive T cells (27). The findings of the current study suggest that surviving allografts with CXCR3 and CCR5 blockade are infiltrated with a significant number of CD4+ cells expressing regulatory phenotype; depletion of CD25+ cells resulted in early rejection of the allografts. The role of chemokine receptors or its blockade in T regulatory cell development is indeed an intriguing finding and deserves further investigation.

Recently, Heller et al. (28) have shown that blockade of CXCR3-CXCL10 interaction in a model of native vessel atherosclerosis was associated with increased regulatory T cell numbers and activity. There was an increase in the Foxp3 gene transcript as well as other T regulatory-associated cytokines in the atherosclerotic lesions, without any effect on CD4+CD25+ cells or Foxp3 expression in the spleen, the lymph nodes or circulating blood. This observation along with our findings suggest that interruption of the CXCR3 ligand axis may affect the balance of regulatory and effector T cells at sites of inflammation.

Another explanation for the beneficial effect of this strategy is that the combined CXCR3 and CCR5 blockade was able to inhibit clonal T cell expansion and effector cytokine production. Our in vitro and in vivo findings indeed confirm that combined CXCR3 and CCR5 blockade can attenuate T cell proliferative responses and reduce the size of the effector T cell pool. Previous studies have also shown that interaction of CXCR3 with its ligands can enhance both CD4 and CD8 lymphocyte activation and expansion (29). Blockade of CXCR3 or any of its three ligands was associated with a reduction in allogenic T lymphocyte proliferation and effector cytokine production (29).

Two previous reports have examined the role of combined CXCR3 and CCR5 blockade in experimental studies. de Lemos et al. (30) studied CD8 lymphocyte trafficking in the CNS of virally infected mice. In this model, CD8 lymphocyte infiltration into the CNS mediates cell damage. Using CXCR3/CCR5 double-deficient mice, the authors demonstrated that CD8 lymphocyte infiltration was only mildly delayed. Unexpectedly, virus-infected CXCR3/CCR5- deficient mice displayed accelerated CD8 lymphocytes expansion and more severe disease compared with wild-type mice. The differences between the de Lemos et al. study (30) and our study may be due to differences in the animal model of disease and/or double-deficient mice vs Ab- mediated CXCR3 blockade. In another study, Tokuyama et al. studied (31) the impact of CCR2, CCR5, and CXCR3 blockade by a non-peptide chemokine receptor antagonist in a mouse model of colitis. The authors demonstrated that CCR2/CCR5/CXCR3 blockade inhibits mononuclear cell infiltration into the lamina propria of colon and attenuates the disease severity.

Based on the findings of this study, we propose that combined CXCR3 and CCR5 blockade in this model attenuates allogeneic T cell proliferation in the secondary lymphoid tissue. Moreover, lack of CXCR3 and CCR5 on the activated effector cells further diminishes trafficking of these cells into the graft. Meanwhile, the graft is infiltrated with CD4+ lymphocytes expressing Foxp3 and regulatory phenotype. Alternatively, trafficking and/or retention of regulatory T cells may be enhanced. Increased number of regulatory CD4+ cells in association with decreased number of effector T cells in the graft with attenuated ability to produce effector cytokines tips the balance against graft rejection and destruction. The net effect in this robust model is absence of histologic findings of acute and chronic rejection.

Despite combined CXCR3 and CCR5 blockade, the donor hearts harvested at 24 and 100 days post-transplantation were infiltrated with a moderate number of effector T lymphocytes. The CXCR3/CCR5-independent mediators of recruitment of these cells are currently under study. Furthermore, it is unclear how the regulatory CD4+ lymphocytes were infiltrated into the graft. The recruitment of regulatory CD4+ lymphocytes is likely independent of CXCR3 and CCR5. Two studies have shown that regulatory CD4 lymphocyte trafficking is mediated via chemokine receptors CCR4 and CCR8 (32, 33).

Can combined CXCR3/CCR5 blockade lead to donor-specific tolerance? Although this strategy was associated with prolonged allograft survival without manifestations of chronic rejection, we did not test for donor-specific tolerance (such as testing for rejection of third-party donor or for infectious tolerance), because this was not among the goals of the current study. However, given the findings of this study, the tolerogenic properties of combined chemokine blockade deserves further investigation. It is also important to note that this study had used CCR5–/– mice; we did not block CCR5 receptor in wild-type mice. Congenital absence of this receptor influences the development of immune responses and may have impacted the results of the current study.

In conclusion, combined CXCR3 and CCR5 blockade was effective in preventing acute and chronic rejection in a robust murine model. Despite redundancy within the chemokine receptor/chemokine system, this study lends support to anti-chemokine receptor strategies in controlling alloimmune responses. Further elucidation of mechanisms of combined chemokine receptor blockade may pave the way for preclinical studies using this strategy.


   Acknowledgments
 
We are grateful to Benjamin Bonavida for assistance with the flow cytometric studies.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the American Heart Association and Veterans Administration Research services. A.A. is the recipient of an Established Investigator Award from the American Heart Association. Back

2 G.T.S. and S.B. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Abbas Ardehali, Division of Cardiothoracic Surgery, 62-182 CHS, University of California Medical Center, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: aardehali{at}mednet.ucla.edu Back

4 Abbreviations used in this paper: MIG, monokine induced by IFN-{gamma}; IP-10, IFN-{gamma}-inducible protein 10; CAV, cardiac allograft vasculopathy. Back

Received for publication January 12, 2007. Accepted for publication January 28, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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