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The Journal of Immunology, 2005, 174: 6499-6508.
Copyright © 2005 by The American Association of Immunologists

Absence of Recipient CCR5 Promotes Early and Increased Allospecific Antibody Responses to Cardiac Allografts 1

Hiroyuki Amano*,§, Alice Bickerstaff, Charles G. Orosz, Andrew C. Novick*, Hiroshi Toma§ and Robert L. Fairchild2,*,{dagger},{ddagger}

* Glickman Urological Institute and {dagger} Department of Immunology, Cleveland Clinic Foundation, Cleveland, OH 44195; {ddagger} Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106; § Department of Urology, Tokyo Women’s Medical School, Tokyo, Japan; and Department of Surgery, Transplantation Division, Ohio State University College of Medicine, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Acute rejection is mediated by T cell infiltration of allografts, but mechanisms mediating the delayed rejection of allografts in chemokine receptor-deficient recipients remain unclear. The rejection of vascularized, MHC-mismatched cardiac allografts by CCR5–/– recipients was investigated. Heart grafts from A/J (H-2a) donors were rejected by wild-type C57BL/6 (H-2b) recipients on day 8–10 posttransplant vs day 8–11 by CCR5–/– recipients. When compared with grafts from wild-type recipients, however, significant decreases in CD4+ and CD8+ T cells and macrophages were observed in rejecting allografts from CCR5-deficient recipients. These decreases were accompanied by significantly lower numbers of alloreactive T cells developing to IFN-{gamma}-, but not IL-4-producing cells in the CCR5–/– recipients, suggesting suboptimal priming of T cells in the knockout recipients. CCR5 was more prominently expressed on activated CD4+ than CD8+ T cells in the spleens of allograft wild-type recipients and on CD4+ T cells infiltrating the cardiac allografts. Rejecting cardiac allografts from wild-type recipients had low level deposition of C3d that was restricted to the graft vessels. Rejecting allografts from CCR5–/– recipients had intense C3d deposition in the vessels as well as on capillaries throughout the graft parenchyma similar to that observed during rejection in donor-sensitized recipients. Titers of donor-reactive Abs in the serum of CCR5–/– recipients were almost 20-fold higher than those induced in wild-type recipients, and the high titers appeared as early as day 6 posttransplant. These results suggest dysregulation of alloreactive Ab responses and Ab-mediated cardiac allograft rejection in the absence of recipient CCR5.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A key event during the allograft rejection process is the recruitment of alloantigen-activated T cells into the graft, where they are activated to express the effector functions that mediate graft tissue destruction. This recruitment is directed by both adhesion molecules and chemokines and their respective receptors. T cell attractant chemokines including CXCL9/monokine induced by IFN-{gamma} (Mig), 3 CXCL10/IFN-{gamma}-inducible protein-10, and CCL5/RANTES are expressed during rejection, and several clinical studies have indicated a correlation between the expression of these chemokines in graft biopsies with ongoing rejection episodes (1, 2, 3, 4). The importance of chemokines in rejection is further indicated by studies in animal models demonstrating prolonged allograft survival when donors or recipients with targeted deletions in genes encoding specific chemokines or receptors are used or when recipients are treated with Abs to these molecules (5, 6, 7, 8, 9, 10). However, in complete MHC-mismatched donor to recipient strain combinations, targeting a single chemokine or receptor in the absence of adjunctive therapy has only resulted in delayed rejection of allografts.

The rejection of allografts when a specific chemokine or chemokine receptor is antagonized has been interpreted to indicate the redundancy in the chemokine system and the ability of other chemokine/receptor interactions to replace the target. Previous studies from this laboratory indicated the prolonged survival of cardiac allografts when recipients were treated with Abs to the T cell chemoattractant Mig (9). At the time of rejection, there were many CCR5-expressing cells in the cardiac allografts, suggesting that mononuclear cell expression of CCR5 may substitute for Mig-CXCR3 interactions in directing T cells into the grafts. A recent study reported the prolonged survival of cardiac allografts in CCR5–/– recipients (11). In clinical renal transplantation, recipients expressing a homozygous mutation encoding a nonfunctional CCR5 have better graft outcomes than recipients expressing functional receptors (12). These studies suggest that expression of CCR5 may play an important role in directing T cells into allografts to mediate acute rejection. In addition to directing T cell recruitment, in vitro studies have indicated that CCR5 ligands, including MIP-1{alpha}, MIP-1{beta}, and RANTES, amplify T cell activation during culture with anti-CD3 mAb or APCs (13, 14, 15). In light of these in vitro studies, we have investigated the activation of alloantigen-specific T cells as well as mechanisms of rejection in CCR5-deficient recipients of MHC-mismatched cardiac allografts. The results indicate decreased T cell infiltration into rejecting allografts in CCR5–/– recipients that is due to reduced effector T cell priming to alloantigens, and suggest that alternative mechanisms mediate cardiac allograft rejection in CCR5–/– recipients.


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

A/J (H-2a), BALB/c (H-2d), P/J (H-2p), and C57BL/6 (H-2b) mice were obtained through C. Reeder at the National Cancer Institute (Frederick, MD). CCR5–/– mice were obtained from The Jackson Laboratory. Adult males of 8–12 wk of age were used throughout this study.

Antibodies

The following Abs were used for immunohistological and flow cytometry analyses: FITC-conjugated goat anti-mouse IgG Ab (Pierce); FITC-conjugated rat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM Abs, PerCP-conjugated GK1.5 (rat anti-mouse CD4 mAb) and 53-6.7 (rat anti-mouse CD8 mAb), FITC-conjugated PgP-1, Ly-24 (anti-mouse CD44 mAb), biotinylated and PE-conjugated rat anti-mouse CCR5 mAb, and FITC-conjugated goat anti-mouse IgM and IgG Ab (BD Pharmingen); rat anti-mouse macrophage mAb, F4/80 (Serotec); goat anti-mouse CXCR3 (C20) and goat anti-mouse CCR5 antiserum (Santa Cruz Biotechnology); PE-conjugated donkey anti-goat IgG Ab (Jackson ImmunoResearch Laboratories); and FITC-conjugated rabbit anti-C3d Ab (DakoCytomation).

Preparation of bone marrow-derived dendritic cells (DC)

Bone marrow cells were flushed from the femurs and tibia of A/J mice and cultured for 7 days in medium containing 10 ng/ml GM-CSF and 10 ng/ml IL-4 to generate mature DC. After the culture, the DC were isolated by positive selection using CD11c+ MACS cell separation magnetic beads (Miltenyi Biotec), and 1.6 x 106 DC were injected i.v. into wild-type C57BL/6 and B6.CCR5–/– mice.

Heterotopic cardiac transplant

Cardiac transplants were performed using the method of Corry et al. (16). Briefly, donor and recipient mice were anesthetized with phenobarbital. Donor hearts were harvested and placed in chilled lactated Ringer’s solution while the recipient mice were prepared. The donor heart was anastamosed to the recipient abdominal aorta and vena cava using microsurgical techniques. Upon completion of the anastamoses and organ perfusion, the transplanted hearts resumed spontaneous contraction. The strength and quality of cardiac impulses were graded by palpation each day, as previously described (9). Rejection of cardiac grafts was considered complete by the cessation of impulse and was confirmed visually for each graft by laparotomy. In C57BL/6 recipients, complete rejection of A/J cardiac grafts occurs between 8 and 10 days after transplantation. Cardiac isografts in C57BL/6 recipients functioned for >100 days. The significance in allograft survival between recipient groups was analyzed by log rank test, and p < 0.01 was considered a significant difference between groups.

Immunohistology

Heart grafts were retrieved from recipients at the time of rejection, embedded in OCT compound (Sakura Finetek), and frozen at –80°C. Sections were cut at 8 µm and mounted onto slides. For immunohistochemistry, sections were fixed in acetone for 10 min and air dried. Slides were immersed in PBS for 10 min and then in 0.03% H2O2 for 10 min to eliminate endogenous peroxidase activity. The slides were then stained for 1 h with 5 µg/ml anti-CD4 mAb (GK1.5), anti-CD8 mAb (53-6.7), or anti-macrophage F4/80 mAb in 0.05 M Tris-HCl with 1% BSA. Control slides were incubated with rat IgG as the primary Ab. After three washes in PBS for 5 min each, slides were incubated for 20 min with biotinylated goat anti-rat IgG diluted 1/300 in PBS. After three washes in PBS, slides were incubated with streptavidin-HRP (DakoCytomation) for 20 min and washed another three times. To prepare the substrate-chromagen solution, a 10 mg tablet of 3,3'-diaminobenzidine (Sigma-Aldrich) was dissolved in 15 ml of PBS plus 12 µl of 30% H2O2. The solution was applied to the slides, which were incubated for 3–7 min and then rinsed in dH2O to stop the reaction. The slides were counterstained with hematoxylin for 3 min and rinsed with tap water. The slides were dehydrated and viewed under light microscopy, and the images were captured using ImagePro Plus (Media Cybernetics). Numbers of cells staining positive were counted in eight random fields from three different tissue sections from three different grafts, and the significance between mean numbers of positive cells per field in different treatment groups was tested using Mann-Whitney U test. To investigate colocalization of CCR5 with graft-infiltrating CD4 and CD8 T cells and macrophages, adjacent sections were cut and placed on separate slides as mirror images (17). One slide was stained with goat anti-mouse CCR5 antiserum and the other with Ab to detect infiltrating CD4 or CD8 T cells or macrophages, as above.

For immunofluorescent staining of C3d, 8-µm frozen sections were fixed in acetone for 10 min and air dried. Slides were immersed in PBS for 10 min, and FITC-conjugated rabbit anti-C3d Ab diluted 1/100 in 1% BSA/PBS was applied for 30 min in a humid chamber. After three washes with PBS, the slides were mounted with VECTASHIELD with 4',6'-diamidino-2-phenylindole (Vector Laboratories) and viewed under a fluorescent microscope.

ELISPOT assay

Priming of alloantigen-specific T cells from heart allograft recipients was investigated by enumerating IFN-{gamma}-producing and IL-4 T cells using ELISPOT assays, as previously described (18, 19). Briefly, ELISA spot plates (Unifilter 350; Polyfiltronics) were coated with 2 µg/ml IFN-{gamma}- or IL-4-specific mAb and incubated overnight at 4°C. The plates were blocked with 1% BSA/PBS and then washed four times with PBS. Spleen cell suspensions from graft recipients were prepared on day 7 posttransplant and used as responder cells. Spleen cells from A/J, C57BL/6, and DBA mice were prepared and treated with mitomycin C for use as stimulator cells in the assay, as described above. Responder and stimulator cells (1:2) were cultured in serum-free HL-1 medium (BioWhittaker) supplemented with 1 mM L-glutamine. After 24 h of cell culture at 37°C in 5% CO2, cells were removed from the plate by extensive washing with PBS. Biotinylated anti-IFN-{gamma} mAb (2 µg/ml) or anti-IL-4 (4 µg/ml) was added, and the plate was incubated for 6 h at room temperature. The plate was washed three times with PBS/0.05% Tween 20, and streptavidin-conjugated alkaline phosphatase was added to each well. After 2 h at room temperature, the plates were washed with PBS, and NBT-5-bromo-4-cloro-3-indolyl substrate (Kirkegaard & Perry Laboratories) was added for the detection of IFN-{gamma}- or IL-4-producing cells. The resulting spots were counted with an ImmunoSpot Series I analyzer (Cellular Technology) that was designed to detect ELISA spots with predetermined criteria for spot size, shape, and colorimetric density.

Flow cytometry

The expression of CXCR3 and CCR5 on CD4+ and CD8+ T cells in the spleens of naive mice and heart allograft recipients and on graft-infiltrating T cells was tested by flow cytometry. Spleen cell suspensions were prepared, and 106 cell aliquots were washed three times with staining buffer (Dulbecco’s PBS with 2% FCS/0.2% NaN3) and stained with PerCP rat anti-mouse CD4 or anti-mouse CD8{alpha} mAb, FITC anti-mouse CD44 mAb, and goat anti-mouse CXCR3 or biotinylated anti-mouse CCR5 Ab for 25 min on ice. The cells were washed twice, and the cells were stained with PE-streptavidin to detect CCR5+ cells or PE anti-goat IgG to detect CXCR3+ cells. After washing five times, the cells were analyzed by flow cytometry using a FACScan (BD Biosciences). The CD4+ or CD8+ T cells were gated and analyzed for expression of CD44 and CXCR3 or CCR5. To isolate allograft-infiltrating cells, the allograft was retrieved on day 7 posttransplant, cut into small pieces, and incubated at 37°C in 20 ml of a mixture containing 1 mM sodium pyruvate, 10 mM HEPES, 1000 U/ml collagenase, 100 µg/ml DNase, and 1000 U/ml hyaluronidase in HBSS. After 1 h, the tissue was pressed through a cell strainer, the cells were collected and washed three times with HBSS, and then 106 cell aliquots were stained with anti-CD4, anti-CD8, and anti-CCR5 Abs.

Flow cytometry to detect and measure donor-specific Abs in cardiac allograft recipient serum was performed using a modification of the method described by Wasowska et al. (20). Thymocyte suspensions from naive A/J, P/J, and C57/BL6 mice were prepared in HBSS, and 50-µl aliquots containing 1.5 x 105 thymocytes were incubated with 50 µl of 4-fold dilutions of sera from naive wild-type C57BL/6 and wild-type and CCR5-deficient heart allograft recipients for 1 h on ice. The cells were washed three times and suspended in 50 µl of staining buffer (Dulbecco’s PBS with 2% FCS/0.2% NaN3) containing a mixture of FITC-conjugated goat Abs specific for mice IgG and IgM (BD Pharmingen) for 30 min on ice. The cells were washed twice, fixed, and analyzed by flow cytometry. The mean channel fluorescence of each dilution of each serum sample was determined, and the dilution that returned the mean channel fluorescence to the level observed when A/J thymocytes were stained with a 1/4 dilution of normal wild-type serum was divided by two and reported as the titer. Serum from groups of five cardiac allograft recipients was tested, and differences in mean titer between wild-type and CCR5–/– recipients were determined using Mann-Whitney U test.

To test for the presence of donor-reactive IgM and IgG isotypes in naive and cardiac allograft recipient serum, 106 A/J thymocytes were incubated with a 1/20 dilution of serum. The cells were washed three times and stained with FITC-conjugated Abs to mouse IgM and IgG isotypes for 30 min on ice. The cells were washed three times and analyzed by flow cytometry. Results shown are the percentage of A/J thymocytes that bound detectable naive and allograft recipient Ab.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Delayed rejection, but decreased T cell infiltration into heart allografts in CCR5–/– recipients

To begin to investigate the potential role of CCR5 in cardiac allograft rejection, the rejection of complete MHC-mismatched A/J heart grafts by wild-type C57BL/6 and CCR5–/– recipients was compared. Wild-type recipients rejected the allografts at day 8–10 posttransplant (Fig. 1). A modest prolongation to day 8–11 was observed in CCR5-deficient recipients, but this prolongation was not statistically significant. Tissue sections prepared from rejecting allografts retrieved from wild-type recipients indicated the intense mononuclear cell infiltration typical of acute rejection (Fig. 2). In contrast, rejecting allografts retrieved from CCR5–/– recipients had a clear decrease in mononuclear cell infiltration. When frozen sections were stained to assess infiltration by CD4+ and CD8+ T cells and macrophages, there was a striking decrease in infiltration by all three leukocyte populations into the rejecting allografts retrieved from CCR5-deficient recipients (Fig. 3). When random sections of slides were counted, a 4-fold decrease in infiltrating CD4+ and CD8+ T cells (p < 0.01) was observed in theCCR5-deficient recipients when compared with infiltration by these T cells in rejecting grafts from wild-type recipients at the time of rejection (Fig. 4). The number of infiltrating macrophages was ~2-fold less in the CCR5–/– recipient (p < 0.05).



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  		FIGURE 1. Rejection of MHC-mismatched A/J cardiac allografts by wild-type C57BL/6 vs CCR5–/– recipients. Groups of C57BL/6 wild-type (n = 8) and CCR5–/– (n = 7) mice received cardiac allografts from A/J donors, and the grafts were monitored daily by palpation for rejection. Allograft rejection was confirmed visually in all recipients by laparatomy. Isografts were maintained >100 days.

 


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  		FIGURE 2. Histological analysis of rejecting allografts from wild-type C57BL/6 vs CCR5–/– recipients. At the time of rejection, A/J cardiac allografts from wild-type C57BL/6 (a) and CCR5–/– (b) recipients were retrieved, and formalin-fixed sections were prepared and stained with H&E and viewed under light microscopy. Magnification, x200.

 


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  		FIGURE 3. Immunohistological analysis of rejecting allografts from wild-type C57BL/6 and CCR5–/– recipients. At the time of rejection, A/J cardiac allografts were retrieved from wild-type C57BL/6 (d–f) and CCR5–/– (a–c) recipients, and frozen sections were prepared and stained with Abs to CD4 (a and d), CD8 (b and e), and F4/80 to detect macrophages (c and f). Magnification, x200.

 


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  		FIGURE 4. Mononuclear cell infiltration into cardiac allografts from wild-type vs CCR5–/– recipients. Rejecting A/J allografts from wild-type and CCR5–/– recipients were retrieved, and frozen sections were prepared and stained with specific Abs to detect CD4+ (a) and CD8+ T cells (b) and macrophages (c). The slides were viewed at x200, and the number of positively staining cells was counted in eight random fields of three different frozen sections from five different grafts. The number of graft-infiltrating CD4+ and CD8+ T cells (p < 0.01, Mann-Whitney U test) and macrophages (p < 0.05) was significantly less for each cell population in grafts from CCR5–/– recipients ({blacksquare}) when compared with cell infiltration into wild-type recipients ({cjs2112}).

 
Expression of CCR5 by heart allograft-infiltrating cells

A potential mechanism underlying the decreased infiltration of mononuclear cells into heart allografts in CCR5–/– recipients was the need for CCR5 expression to direct this infiltration. To investigate this, the colocalization of CCR5 on infiltrating CD4+ and CD8+ T cells and macrophages in rejecting heart allografts retrieved from wild-type recipients was analyzed. Adjacent, mirror-image sections were prepared from the heart allografts at the time of rejection, and one was stained to detect CCR5 expression and the other stained with Abs to detect the phenotype of the infiltrating cell populations. Infiltrating CD4+ T cells clearly expressed CCR5 (Fig. 5a). In contrast, graft-infiltrating CD8+ T cells and macrophages did not colocalize with CCR5 staining, whereas vascular smooth muscle cells known to constitutively express CCR5 (21) stained positively (Fig. 5, b and c).



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  		FIGURE 5. Colocalization of CCR5 with allograft-infiltrating CD4+ T cells. On day 8 posttransplant, rejecting A/J allografts were retrieved from C57BL/6 recipients. Mirror-image frozen sections were prepared and stained to colocalize CCR5 expression (b, d, and f) with infiltrating CD4 (a) or CD8 (c) T cells and macrophages (e) by immunohistochemistry.

 
The expression of CCR5 on allograft-infiltrating CD4+ and CD8+ T cell populations was further investigated using another approach. The allografts were retrieved on day 7 posttransplant, the allograft tissue was digested, and aliquots of the cells were stained with Abs and analyzed by flow cytometry. Although allograft infiltration by CD4+ T cells was much less intense than infiltration by CD8+ T cells (Figs. 4 and 5), 50% of the infiltrating CD4+ T cells expressed CCR5, whereas only 31% of the infiltrating CD8+ T cells expressed CCR5 (Fig. 6).



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  		FIGURE 6. Expression of CCR5 on allograft-infiltrating CD4+ and CD8+ T cells. On day 7 posttransplant, A/J allografts were retrieved and digested, and the isolated cells were stained with Abs to detect CCR5 expression on gated graft-infiltrating CD4 (a) and CD8 (b) T cells. The number in each panel indicates the percentage of graft-infiltrating CD4 or CD8 T cells staining positively for CCR5.

 
Temporal expression of CXCR3 and CCR5 on T cells from recipients of heart allografts

The low expression of CCR5 on allograft-infiltrating CD8+ T cells and macrophages suggested that suboptimal trafficking into the allograft was unlikely to account for the reduced cellular infiltration observed in rejecting heart allografts in CCR5–/– recipients. The expression of chemokine receptors on recipient T cells during priming for rejection of heart allografts has remained largely undefined. To investigate this aspect of the T cell response, the expression of the chemokine receptors CXCR3 and CCR5 was compared on CD4+ and CD8+ T cells in the spleen of allograft recipients. In addition, the expression of CD44 as an indicator of cellular activation was used to test chemokine receptor expression on activated T cell populations in the allograft recipients. Low numbers of CXCR3-expressing CD4+ T cells were observed in naive mice, and this expression was restricted to the CD44high population (Fig. 7a). In heart allograft recipients, these numbers increased slightly on day 4 posttransplant and then increased much more on days 6 and 8, with the expression again restricted to the CD44high cell population. Although more CXCR3+ CD8+ than CD4+ T cells were observed in the spleens of naive mice, similar results were observed with CD8+ T cell expression of CXCR3 during progression to allograft rejection (Fig. 7b). CD4+ T cell expression of CCR5 followed a similar pattern as CXCR3 expression, with slight increases at day 4 posttransplant and further increases afterward (Fig. 7c). In contrast, expression of CCR5 was not as prominent on CD8+ T cells in allograft recipients and was expressed at lower levels than observed on CD4+ T cells (Fig. 7d). At day 4 posttransplant, CCR5 expression was observed only on CD44high cells for each T cell population, but was observed on populations of CD44low and CD44high CD4+ and CD8+ T cells at days 6 and 8 posttransplant.



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  		FIGURE 7. Expression of CXCR3 and CCR5 on splenic T cells from C57BL/6 recipients of A/J cardiac allografts. C57BL/6 mice received cardiac grafts from A/J donors, and at the indicated times posttransplant aliquots of spleen cell suspensions from allograft recipients and naive mice were stained with Abs to CD4 (a and c) or CD8 (b and d), CXCR3 or CCR5, and CD44. The cells were analyzed by three-color flow cytometry, and the CD4+ and CD8+ cell populations were gated to analyze expression of CXCR3 or CCR5 vs CD44. Data shown are from a single experiment and are representative of two individual experiments.

 
Decreased alloreactive T cell priming in CCR5–/– allograft recipients

The role of CCR5 in alloreactive T cell priming was investigated by comparing the development of alloreactive T cells from wild-type and CCR5–/– heart allograft recipients with IFN-{gamma}- and IL-4-producing cells using ELISPOT assays. The number of IFN-{gamma}-producing cells in the CCR5–/– recipient spleen was less than half that observed in recipients of wild-type grafts at day 7 posttransplant (Fig. 8). The number of alloantigen-specific T cells producing IL-4 was similar in each set of recipients, suggesting that immune deviation did not account for the decreased priming of alloreactive T cells to IFN-{gamma}-producing cells observed in CCR5–/– recipients.



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  		FIGURE 8. Frequency of donor-specific IFN-{gamma}- and IL-4-producing cells in the spleen of wild-type and CCR5–/– recipients of A/J cardiac allografts. On day 7 posttransplant, spleen cells from each recipient and from naive wild-type mice were analyzed by ELISPOT to enumerate the number of alloantigen-specific T cells producing IFN-{gamma} (a) and IL-4 (b). Spleens from each set of recipients had similar numbers of T cells as analyzed by flow cytometry and total cell counts. The results are representative of two individual experiments.

 
The possibilities that the decreased priming of alloreactive T cells in CCR5-deficient recipients of heart allografts was due to a direct requirement for CCR5 for T cell priming vs an inhibitory effect of passenger leukocytes in the allograft on the priming of the CCR5-deficient T cells were addressed. First, purified A/J DC were administered to wild-type and CCR5–/– recipients, and ELISPOT assays were performed 4 days later (Fig. 9a). The number of alloreactive T cells producing IFN-{gamma} in CCR5-deficient recipients of the DC was 50% that induced in wild-type recipients. Second, heart allograft donor A/J mice were subjected to 1100 rad whole body irradiation to deplete the donors of radiation-sensitive passenger leukocytes, and 2 days later the hearts were transplanted to wild-type and CCR5–/– recipients. On day 7 posttransplant, graft recipient spleen cells were tested for levels of alloreactive T cell priming using IFN-{gamma} ELISPOT assays (Fig. 9b). Again, the number of alloreactive T cells producing IFN-{gamma} in the CCR5-deficient recipients was almost 50% that observed in wild-type recipients of the heart allografts from the irradiated donors. Collectively, the results in Figs. 8 and 9 indicate the reduced priming of T cells to alloantigen in the absence of CCR5.



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  		FIGURE 9. Frequency of donor-specific IFN-{gamma}-producing cells in response to irradiated allografts and to donor DC. a, Wild-type C57BL/6 and CCR5–/– mice received 1.6 x 106 bone marrow-derived DC i.v., and 4 days later spleen cells from each recipient and from naive wild-type mice were analyzed by ELISPOT to enumerate the number of alloantigen-specific T cells producing IFN-{gamma}. b, A/J mice were treated with 1100 rad whole body irradiation, and 2 days later the hearts were transplanted to wild-type C57BL/6 and CCR5–/– mice. On day 7 posttransplant, spleen cells from each recipient and from naive wild-type mice were analyzed by ELISPOT to enumerate the number of alloantigen-specific T cells producing IFN-{gamma}.

 
Increased alloantigen-specific Ab production in CCR5–/– allograft recipients

Because CCR5-deficient recipients had decreased alloreactive T cell priming and infiltration into the allograft at rejection, the potential role of alloantigen-specific Ab in the rejection of cardiac allografts in these recipients was investigated. First, sections of rejecting allografts were prepared and stained to detect deposition of C3d as an indication of Ab reactivity in the allograft. As a positive control, wild-type C57BL/6 mice that had previously rejected an A/J skin allograft and had also been primed with two injections of A/J spleen cells to induce high levels of donor-reactive Abs were transplanted with A/J cardiac allografts that were rejected on day 5 and were stained for C3d deposition. Whereas isografts had no detectable C3d staining, allografts from donor-sensitized recipients had intense C3d staining around all large vessels as well as on capillaries throughout the heart allograft parenchyma (Fig. 10, a vs b). Low, but detectable C3d deposition was observed in allografts retrieved from wild-type recipients, and this staining was restricted to the large vessels (Fig. 10c). In contrast, C3d deposition in rejecting allografts retrieved from CCR5–/– recipients was much more intense around the large vessels and included C3d-positive capillaries throughout the graft parenchyma (Fig. 10d) similar to staining observed in the allografts retrieved from donor-sensitized wild-type recipients.



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  		FIGURE 10. Deposition of C3d in grafts from CCR5–/– recipients of A/J cardiac allografts. Prepared frozen section from a, day 10 isografts; b, rejecting A/J cardiac allografts from donor-sensitized wild-type C57BL/6 recipients on day 5 posttransplant; c, rejecting cardiac allografts on day 8 posttransplant from wild-type C57BL/6 recipients; and d, rejecting allografts on day 9 posttransplant from CCR5–/– recipients were stained with FITC anti-C3d and viewed by immunofluorescent microscopy.

 
The levels of donor-reactive Ab in the serum of groups of five wild-type vs CCR5–/– recipients were compared at days 6 and 8 posttransplant. In wild-type heart allograft recipients, donor-reactive Ab titers were at low levels at day 6 (281.6 ± 210.3) posttransplant and were slightly increased by day 8 (435.2 ± 171.7), the day of allograft rejection (Fig. 11, a and b). In contrast, high titers of donor-reactive Abs were observed in CCR5–/– recipients as early as day 6 posttransplant (7372.8 ± 1831.7), and at day 8 posttransplant (8192 ± 0.0) were 18.8 times the levels observed in wild-type recipients. No reactivity against third-party P/J allogeneic targets was observed in serum from either allograft recipient group (data not shown). Although increases in donor-reactive IgG Ab of all isotypes were observed in the serum of CCR5-deficient allograft recipients, no differences in donor-specific IgG isotype production were observed at day 8 posttransplant in serum from CCR5-deficient allograft recipients when compared with wild-type recipients (Fig. 11c).



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  		FIGURE 11. Production of donor-reactive Ab in wild-type C57BL/6 and CCR5–/– recipients of A/J cardiac allografts. On days 6 (a) and 8 (b) posttransplant, serum from groups of wild-type (n = 5) and CCR5-deficient (n = 4) allograft recipients and from naive wild-type mice was diluted, and each dilution was assayed by flow cytometry for activity against A/J thymocytes to determine the titer of each serum. Activity against C57BL/6 and P/J thymocytes was negative. Individual titers are shown with the mean represented by the bar. For each test day posttransplant, p < 0.01 when comparing titers of donor-reactive Abs between wild-type and CCR5–/– recipients. c, Serum was obtained from wild-type ({blacksquare}) and CCR5-deficient ({blacktriangleup}) cardiac allograft recipients at day 8 posttransplant and from naive wild-type mice (•). The serum was diluted 1/20 and incubated with A/J thymocytes. After washing the cells, the isotype of the A/J-reactive Abs was tested using FITC-conjugated Abs specific for mouse IgM and IgG isotypes and flow cytometry. The data are reported as percentage of A/J thymocytes positively stained for each subclass or isotype-developing Ab.

 
Finally, potential differences in alloantibody responses to immunization with A/J spleen cells in wild-type vs CCR5–/– mice were tested. Groups of wild-type and CCR5–/– mice were immunized with two injections of A/J spleen cells, and 14 days after the second injection the titers of A/J-reactive Abs in the serum were tested (Fig. 12). Similar to the induction of donor-reactive Abs in heart allograft recipients, CCR5-deficient mice produced much higher titers of donor-reactive Abs than wild-type mice immunized with the A/J spleen cells (1536.0 ± 868.8 vs 213.3 ± 73.9).



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  		FIGURE 12. Induction of higher alloreactive Ab responses in CCR5-deficient than wild-type mice to immunization with A/J splenocytes. Groups of four wild-type C57BL/6 and CCR5–/– mice were immunized with 4 x 107 A/J spleen cells i.p. on days 0 and +7, and 14 days later serum from immunized and naive mice was diluted and tested for reactivity to A/J and C57BL/6 thymocytes to determine titers of A/J-reactive Ab. Values of p < 0.05 when titers of wild-type and CCR5–/– mice are compared.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Chemokines are important mediators directing leukocyte trafficking to tissue sites of inflammation and cellular positioning during the development of lymphoid tissue architecture (22, 23). In addition to these functions, chemokines such as IL-8 activate integrin function and promote leukocyte adhesion to the vascular endothelium (24, 25). In vitro studies have indicated that RANTES augments the activation of T cells by anti-CD3 Ab to produce IL-2 and proliferate (13, 14). The role of chemokines in the activation of T cells during the initiation of in vivo responses remains poorly understood.

The key characteristic of acute allograft rejection is alloantigen-primed T cell and macrophage infiltration into the allograft. This infiltration is largely mediated by the coordinated functions of adhesion molecules and chemokines. A great deal of interest has centered on the ability of strategies neutralizing the function of these sets of molecules to inhibit cellular infiltration into allografts and promote long-term graft survival. Studies from several laboratories have indicated that antagonism of specific chemokines or their receptors delays or inhibits rejection of skin, cardiac, islet, and small bowel allografts in rodent models (6, 26, 27, 28). Much of the early work was focused on CXCR3 and its ligands IFN-{gamma}-inducible protein-10 and Mig, potent chemoattractants for Ag-primed T cells. Studies from Hancock et al. (7) indicated prolonged survival of MHC-mismatched cardiac allografts from 7 to 8 days in wild-type recipients to beyond 60 days posttransplant in CXCR3–/– recipients. Previous studies from this laboratory reported the absence of class II MHC disparate skin allograft rejection in recipients treated with Abs to Mig (26). Treatment with Mig-specific Abs also significantly prolongs the survival of MHC-mismatched cardiac allografts to day 18–25 posttransplant (9). At the time of cardiac allograft rejection in Mig-Ab-treated recipients, high expression of CCR5 was observed in the grafts, suggesting a potential compensation for Mig antagonism by CCR5-binding chemokines in directing T cell graft infiltration.

The role of CCR5 and its primary ligands, MIP-1{alpha}, MIP-1{beta}, and RANTES, in allograft rejection has generated a considerable amount of recent interest. In human renal transplant patients, expression of a nonfunctional CCR5, CCR5{Delta}32, is associated with decreases in acute rejection and better graft outcomes (12). Improved lung allograft survival in rat recipients is observed by treatment with anti-RANTES Abs (5). In a mouse model, allogeneic islets in wild-type recipients are rejected by day 10 posttransplant, whereas 80% of islet allografts are rejected by day 30 in CCR5–/– recipients and the remainder survive beyond day 100 (27).

A recent study indicated prolongation of BALB/c cardiac allograft survival from day 7–8 posttransplant in C57BL/6 wild-type recipients to day 20 in CCR5–/– recipients (11). Comparison of allograft histology at day 7 posttransplant indicated significant reductions in T cell and macrophage infiltration into BALB/c allografts in CCR5-deficient recipients when compared with rejecting allografts in wild-type recipients. The data in the current study indicate a more modest prolongation of A/J cardiac allograft survival in CCR5–/– recipients. This difference in survival is not apparently due to the use of A/J mice as the allograft donor, as we have also observed rejection of BALB/c cardiac allografts at day 10/11 posttransplant in CCR5–/– recipients. Although survival of A/J cardiac allografts is only extended 2–3 days in CCR5-deficient recipients, there are striking decreases in T cell and macrophage infiltration at the time of rejection when compared with rejecting allografts in wild-type recipients that raised questions regarding mechanisms underlying this rejection in CCR5–/– recipients.

Rejecting cardiac allografts in CCR5–/– recipients stained intensely with Abs to C3d, an indication of potential Ab deposition in the grafts. The histology of the allografts appeared identical with rejecting allografts from donor-sensitized wild-type recipients with heavy deposition of C3d around the large vessels and capillaries of the allograft. Consistent with this staining, CCR5–/– allograft recipients had high serum levels of donor-specific Ab, with titers >20-fold that observed in wild-type recipients. The presence of this Ab response appeared within 6 days after transplantation at a time when CCR5 expression was detectable on activated CD4+ T cells in wild-type recipients of the cardiac allografts. These results are strongly suggestive of an Ab-mediated rejection mechanism in the absence of recipient CCR5.

Several recent studies are supportive of a potential role of CCR5 in the regulation of Ab responses. The addition of RANTES and MIP-1{alpha} to human B cell cultures has been shown to up-regulate IgG4 and IgE production (29), although the requirement for CCR1 or CCR5 for this effect remains unclear. Immunization of CCR5–/– mice with T-dependent Ags also enhances Ig responses (30), but the consequence of this increase on an ongoing immune response such as during an infection or to an allograft has not been previously addressed. In conjunction with these studies, the current results suggest dysregulation of Ab responses in the absence of CCR5. The mechanism underlying this dysregulation remains unclear. In contrast to activated CD4+ T cells, we could not detect expression of CCR5 on B cells from C57BL/6 recipients of cardiac allografts, suggesting that CCR5-mediated regulation of Ig responses is mediated through T cells. The absence of CCR5 on B cells has also been observed in BALB/c mice, whereas B cells from NOD mice experiencing active diabetes expressed CCR5 (31). The alloreactive CD4 T cell response in CCR5-deficient recipients did not deviate to an IL-4-producing phenotype, suggesting that another CCR5-expressing T cell population may regulate this Ab response to the cardiac allograft. An intriguing possibility arises from studies indicating that a population of {gamma}{delta} T cells restricts the magnitude of Ab responses in lpr mice and CCR5 is expressed by many populations of these T cells (32, 33). In preliminary studies, we have observed the presence of CCR5+ {gamma}{delta} T cells in the spleens of wild-type recipients of cardiac allografts. Experiments to test the potential role of these T cells in regulating alloantibody responses to cardiac allografts as well as the absence of such cells in CCR5-deficient recipients are in progress.

Two potential explanations were considered to account for the decreased T cell and macrophage infiltration into cardiac allografts in CCR5–/– recipients. First, there could be defective recruitment of alloantigen-primed T cells into the allograft that would support a role for CCR5 in directing this graft infiltration. Based on the demonstrated role of CCR5-binding chemokines on T cell activation in vitro (13, 14), an alternative mechanism could be an alteration in T cell priming to graft alloantigens. Previous studies using mixed lymphocyte reactivity as an assessment of alloreactive T cell priming in cardiac allograft recipients indicated no difference between wild-type and CCR5–/– recipients (11). However, in allogeneic islet recipients, a significant decrease in the number of alloreactive T cells producing IFN-{gamma} was observed in CCR5-deficient recipients (27). This decrease was apparently not due to defective alloreactive T cell priming, as the number of alloreactive T cells producing IL-4 was increased in CCR5–/– recipients, suggesting a switch to a type 2 cytokine-producing phenotype in the absence of CCR5. In the current study, CCR5–/– recipients of heart allografts also had decreases in the numbers of alloantigen-reactive T cells producing IFN-{gamma}, but this was not compensated by an increase in the number of IL-4-producing cells. It is also worth noting studies by several laboratories indicating infiltration and rejection of cardiac allografts by alloreactive T cells producing type 2 cytokines (34, 35, 36). These results suggest that alloreactive T cell deviation to type 2 cytokine production is unlikely to account for the decreased cellular infiltration into the cardiac allografts observed in CCR5-deficient recipients.

The results of the current study indicate a striking enhancement of donor-specific Ab responses in CCR5-deficient recipients of cardiac allografts and Ab deposition in the graft. In light of the low levels of mononuclear cell infiltration observed in these allografts at the time of rejection, the high levels of alloreactive Ab induced in the absence of CCR5 are likely to be a factor in mediating rejection of the graft. There is a considerable interest in the potential use of chemokine receptor antagonists to inhibit allograft rejection in clinical transplantation. Differences in alloreactive responses and allograft rejection are to be expected in wild-type recipients treated with chemokine receptor antagonists vs chemokine receptor-deficient recipients. Nevertheless, the results of the current study raise caution regarding the use of CCR5 antagonism as a therapeutic strategy in which administration of such an antagonist may promote Ab responses and allograft rejection.


   Acknowledgments
 
We thank Tarek El-Sawy, Peter Heeger, Anna Valujskikh, Wink Baldwin, and Angus Thomson for helpful suggestions concerning these studies; Danielle Kish for help with generating bone marrow-derived DC; and the staff of the Cleveland Clinic Foundation Biological Resources Unit for excellent care of the animals used in this study.


   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 National Institutes of Health Grants AI40459 and AI51620. Back

2 Address correspondence and reprint requests to Dr. Robert L. Fairchild, NB3-79, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-0001. E-mail address: fairchr{at}ccf.org Back

3 Abbreviations used in this paper: Mig, monokine induced by IFN-{gamma}; DC, dendritic cell. Back

Received for publication March 30, 2004. Accepted for publication March 9, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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