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The Journal of Immunology, 2002, 168: 5352-5358.
Copyright © 2002 by The American Association of Immunologists

Intact Active Bone Transplantation Synergizes with Anti-CD40 Ligand Therapy to Induce B Cell Tolerance1

Dengping Yin, LianLi Ma, Anncy Varghese, JiKun Shen and Anita S.-F. Chong2

Department of General Surgery, Rush Presbyterian Hospital, St. Luke’s Medical Center, Chicago, IL 60612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Blockade of T cell costimulatory pathways can result in the prolongation of allograft survival through the suppression of Th1 responses; however, late allograft rejection is usually accompanied by an emerging allograft-specific humoral response. We have recently determined that intact active bone (IAB) fragments transplanted under the kidney capsule can synergize with transient anti-CD40 ligand (CD40L) treatment to induce robust donor-specific allograft tolerance and suppress the alloantibody response. In this study, we take advantage of the ability of galactosyltransferase-deficient knockout (GT-Ko) mice to respond to the carbohydrate epitope, galactose-{alpha}1,3-galactose (Gal), to investigate whether IAB plus transient anti-CD40L therapy directly tolerize B cell responses. GT-Ko mice tolerized to Gal-expressing C3H hearts and IAB plus transient anti-CD40L therapy were challenged with pig kidney membranes that express high levels of Gal. The anti-Gal IgM and IgG responses were significantly suppressed in IAB-tolerant mice compared with controls, while the non-Gal anti-pig Ab responses were comparable. The anti-pig T cell cytokine response (IFN-{gamma} and IL-4) was comparable in IAB-tolerant and control mice. The tolerant state for the anti-Gal IgM response could be reversed with repeated immunization, whereas the tolerant state for the IgG response was robust and resisted repeated immunization. These observations provide an important proof-of-concept that adjunct therapies can synergize with anti-CD40L Abs to tolerize B cell responses independent of their effects on T cells. This model, which does not require mixed chimerism, provides a unique opportunity for investigating the mechanism of peripheral tolerance in a clinically relevant population of carbohydrate-specific B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Blockade of the CD40-CD40 ligand (CD40L)3 or CD28-B7 costimulatory pathways can result in the prolongation of allograft survival in rodents and nonhuman primates, but is unable to induce durable allograft tolerance in the rigorous nonhuman primate models (1, 2, 3, 4, 5, 6, 7). A consistent observation in the primate model is the inability of costimulation blockade to inhibit alloantibody production (3, 4, 5, 6, 7). In the case of allogeneic renal transplantation, alloantibodies were detected during therapy with anti-CD40L or anti-CD80 and anti-CD86 (3, 4, 5). In islet transplantation, alloantibody production was suppressed during anti-CD40L therapy, but rapidly increased upon cessation of therapy (6, 7). Because the appearance of alloantibodies generally precedes rejection, it has been postulated that alloantibodies play an initiating role in the rejection of the long-surviving allografts. Thus, adjunct therapies that are capable of synergizing with costimulation blockade to inhibit humoral responses and of inducing stable/indefinite allograft survival would be clinically significant.

We have recently determined that intact active bone (IAB) fragments transplanted under the kidney capsule can synergize with transient anti-CD40L treatment (250 µg/mouse daily from days 0 to 3, then every other day from days 5 to 13) to induce robust donor-specific allograft tolerance (8). Tolerant mice accepted a second donor-specific heart (transplanted on days 60–90) and also donor-specific skin (transplanted on days 90–120), but rejected third-party skin (transplanted on days 90–120). Tolerance was observed in the absence of macrochimerism, but in the presence of donor microchimerism in the recipient peripheral blood and bone marrow. The histology of the transplanted allografts, as late as 270 days posttransplant, revealed normal histology with minimal cellular infiltration and an absence of coronary artery disease. Most significantly, there was minimal deposited alloantibodies in the allograft, and the circulating alloantibody levels were also significantly suppressed. However, it was unclear from those studies whether suppression of alloantibody production was due to inhibition of T cells necessary for providing B cell help or whether it was due to direct inhibition of alloantibody-producing B cells.

In this study, we take advantage of the ability of galactosyltransferase-deficient knockout (GT-Ko) mice to respond to the carbohydrate epitope, galactose-{alpha}1,3-galactose (Gal), to investigate whether IAB plus transient anti-CD40L therapy can tolerize anti-Gal B cell responses. GT-Ko mice were tolerized to Gal-expressing C3H hearts and IAB plus transient anti-CD40L therapy, then challenged with pig kidney membranes that express high levels of Gal (9). We reasoned that our tolerant GT-Ko mice were tolerized only to C3H Ags and their anti-pig T cell responses should be normal, thus any defect in anti-Gal Ab production following immunization with pig Ags should reflect tolerance in the anti-Gal B cell population. Here, we report that this tolerizing strategy of IAB plus transient anti-CD40L can directly induce B cell tolerance.


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

GT-Ko, produced by homologous recombination with a defective GGTA1 gene on a background of C57BL/6, DBA/2, and 129SvSn strains (H-2bxd), were obtained from Dr. J. Lowe (Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI) (10, 11) and maintained at the Rush Presbyterian St. Luke’s Medical Center. Six- to 10-wk-old C3H mice (H-2k) were used as heart donors. Heterotopic mouse hearts were transplanted into the abdomen of the recipient by anastomosing the donor aorta and recipient aorta, and the donor pulmonary artery and recipient inferior vena cava. Baby Lewis rat (10–18 days old) heart grafts were transplanted into the cervical area of the recipient by anastomosing the donor aorta and recipient carotid artery, and the donor pulmonary artery and recipient external jugular vein (end-to-side). The heart grafts were monitored daily until rejection unless otherwise indicated and rejection was defined as complete cessation of pulsation.

IAB and bone marrow cell transplantation

The knee joints containing the heads of tibiae and femora from the hind legs of C3H mice were harvested and cleaned of connective tissue. Each knee joint was cut with scissors into six to eight small fragments, and the fragments of one to two knee joints were transplanted under the kidney capsule of each recipient mouse on the day of heart transplantation. Transplanted knee joints contained ~2–2.5 x 107 bone marrow cells.

Immunization with pig kidney membranes

Pig kidneys were homogenized with a tissue homogenizer for 30 s on ice, then membranes were washed three times by centrifugation for 30 min at 38,000 x g. Membranes were resuspended in PBS, and 100 mg/mouse was used to immunize i.p. at 2-wk intervals.

Antibodies

Anti-CD40L mAbs (MR1) were purified from protein-free culture supernatants by 45% ammonium sulfate precipitation and dialyzed in PBS (Ligocyte, Bozeman, MT). Anti-CD40L was administered at a dose of 250 µg/mouse, i.v. daily from days 0 to 3, then i.p., every other day from days 5 to 13 after transplant.

Analysis of donor-reactive and anti-Gal Abs alloantibody titers

Donor-reactive Abs were determined by flow cytometry as previously reported (12, 13). Briefly, 1/100 dilutions of mouse serum were incubated with C3H lymph node cells for 1 h at 4°C, then cells were washed and incubated with PE-conjugated anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) or fluorescein-conjugated anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL). The mean channel fluorescence of the stained samples was determined by flow cytometry (FACScan; BD Biosciences, Mountain View, CA).

Anti-Gal Ab titers in sera were determined by ELISA using BSA-Gal (V-Labs, Covington, LA) as specific substrate (10 µg/ml) and BSA (Sigma-Aldrich, St. Louis, MO) as nonspecific control (12, 13). BSA-Gal- or BSA-coated plates (Costar, Corning, NY) were preblocked with 1% BSA/PBS, then serum was added. After 1 h, plates were washed, blocked with 1% BSA/PBS, and then incubated with HRP-conjugated anti-mouse IgM or anti-mouse IgG (Jackson ImmunoResearch Laboratories). The OD were determined on an ELISA plate reader (Bio-Rad, Richmond, CA), and the results are presented as mean relative OD. OD = OD (BSA-Gal) - OD (BSA). For identifying the IgG subclasses, biotinylated isotype-specific mAbs and streptavidin-FITC (BD PharMingen, San Diego, CA) were used in the ELISA. Standards consisted of serial dilutions of immobilized, purified mAbs of the respective IgG subclass.

Anti-pig non-Gal Ab titers were determined by ELISA using pig kidney membranes as substrate. To absorb anti-Gal Abs; serum samples were incubated with fixed rabbit RBC (33% v/v solution) at 4°C for 18 h. Serum samples were harvested, serially diluted, and then added to ELISA plates with adhered kidney membranes (1 mg/ml) and preblocked with 1% BSA/PBS. After 1 h, plates were washed then incubated with HRP-conjugated anti-mouse IgM or anti-mouse IgG (Jackson ImmunoResearch Laboratories). The OD were determined on an ELISA plate reader (Bio-Rad), and the results are presented as mean relative OD.

IFN-{gamma} and IL-4 ELISA spot assay

The IL-4 and IFN-{gamma} ELISA spot assays were performed as previously reported (14). Briefly, ELISA spot plates were coated overnight with 11B11 (8 µg/ml) and R46A1 (8 µg/ml) for IL-4 and IFN-{gamma}, respectively (BD PharMingen). The plates were blocked with PBS/1% BSA, then splenocytes (5 x 105/well) were added with stimulators comprising 2 x 105/well 50 cGy irradiated pig kidney cell line, PK15, in a total volume of 200 µl/well HL-1 medium (BioWhittaker, Walkersville, MD). After 24 h, the plates were washed with PBS/0.025% Tween 20 and probed with biotinylated anti-IL-4 and anti-IFN-{gamma} (BVD-24G2 or XMG1.2, respectively). After 25 h, the plates were washed and incubated with alkaline phosphatase-conjugated anti-biotin (Vector Laboratories, Burlingame, CA) for 2 h. The plates were developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich), and the resulting spots were counted by a computer-assisted Immunospot image analyzer (Cellular Technology, Cleveland, OH).

Anti-Gal ELISA spot assay

The anti-Gal ELISA spot assay to determine the frequency of anti-Gal IgM- and IgG Ab-producing B cells was quantified following a modified procedure previously described by Odhan et al. (15). Briefly, 94-well Immulon 2 plates (Dynatech Laboratories, Chantilly, VA) were coated with 25 µg/ml BSA or BSA-Gal. Serial dilutions of splenocytes from 5 x 107/ml protein-free hybridoma medium (Life Technologies, Grand Island, NY) were incubated for 24 h in ELISA plates preblocked with PBS/1% BSA. Plates were washed with PBS/0.05% Tween 20, then incubated with alkaline phosphatase-conjugated anti-IgM or anti-IgG (Southern Biotechnology Associates). Plates were washed and 100 µl/well 0.6% low melting point agarose in Tris buffer (0.1 M Tris base (pH 9.5), 5 mM MgCl2, and 0.1 M NaCl) was added. Then 100 µl/well 5-bromo-4-chloro–3-indolyl phosphate (1 mg/ml in Tris buffer) was added, and the resulting spots were counted under a magnification glass. Each experiment was performed in duplicate, and the frequency of specific anti-Gal B cells were calculated using the formula: (mean numbers of spots in BSA-Gal wells) - (mean numbers of spots in BSA wells)/(number of cells per well) x 106.

Histology and immunohistochemistry

Heart grafts were surgically removed and snap frozen in Tissue-Tek OCT (Sakura Finetek USA, Torrence, CA) using liquid nitrogen. All hearts were sectioned (5 µm) and stained with H&E. Other sections for immunohistochemical staining were subjected to the standard avidin-biotin peroxidase method as previously described (16). Primary Abs of anti-mouse IgM (R4-22) and anti-mouse IgG (R3-34) were purchased from BD PharMingen and biotinylated goat anti-mouse IgG from Jackson ImmunoResearch Laboratories. For identification of complement deposition, sections were serially incubated with goat anti-C3 or anti-C5 polyclonal Abs (Quidel, San Diego, CA), biotinylated rabbit anti-goat IgG (Vector Laboratories), and HRP-conjugated-streptavidin (Zymed Laboratories, South San Francisco, CA). Immunostaining was developed with chromogen, 3,3'-diaminobenzidine solution, and counterstained with Mayer’s hematoxylin.

Statistical analysis

Statistical significance was determined by ANOVA using StatView (Abacus Concepts, Berkeley, CA) using ANOVA and post hoc Student-Newman-Keuls tests. A p value of <0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of alloantibody and anti-Gal Abs with IAB plus anti-CD40L

A crucial observation made in nonhuman primates is the inability of costimulation blockade to tolerize the alloantibody response (3, 4, 5, 6, 7). In most cases, increased alloantibody titers were observed while on anti-CD40L therapy or upon cessation of therapy. We used a completely major and minor histocompatibility-mismatched allogeneic cardiac transplantation model, GT-Ko mice (H-2dxb) as recipients of Gal-expressing C3H hearts (H-2k), in which allograft hearts are rejected in 7–9 days. In anti-CD40L-treated recipients, allograft rejection was observed in 52–67 days, while anti-CD40L and IAB cotransplantation resulted in graft survival of >128 days. We observed that alloantibody and anti-Gal Ab titers were increased at the time of rejection (day 60 posttransplantation) in untreated and anti-CD40L-treated recipients (Fig. 1Go). In contrast, mice transplanted with donor IAB and transient anti-CD40L exhibited significantly depressed alloantibody and anti-Gal Ab responses (Student-Newman-Keuls test, p < 0.05). Both IgM and IgG responses were significantly reduced in the recipients of IAB and anti-CD40L, although the IgG responses were more profoundly suppressed (Fig. 1Go).



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  		FIGURE 1. Tolerant GT-Ko mice receiving IAB and transient anti-CD40L mAb have significantly reduced alloantibody (a and b) and anti-Gal Ab (c and d) levels compared with untreated or anti-CD40L (MR1)-treated recipients. Alloantibodies were quantified by flow cytometry and data as described in Materials and Methods, and are presented as mean channel fluorescence (MCF). Anti-Gal Ab titers were determined by ELISA and data are presented as mean OD after subtraction for nonspecific binding to BSA-coated wells. Data are the means of >=5 recipients per group and error bars are SEM. {square}, Untreated; {diamondsuit}, anti-CD40L only; •, IAB- plus anti-CD40L-treated groups.

 
Previous studies involving immunohistochemical analyses indicated minimal IgM and IgG deposition in the grafts (8). In addition, the frequency of donor cells derived from IAB in recipients, other than the heart allograft, was low and could only be detected by PCR but not by immunohistochemistry or flow cytometry (data not shown; Ref. 8). These observations collectively argue against adsorption of alloantibodies by graft and IAB-derived cells as the reason for low alloantibody titers and support a conclusion that IAB can synergize with anti-CD40L to suppress Ab production.

Suppression of anti-Gal IgM responses by IAB plus anti-CD40L

To address the question of whether IAB and anti-CD40L directly tolerized B cell responses, instead of indirectly by tolerizing T cell responses, we took advantage of the ability of GT-Ko mice to respond to the Gal epitope. We had previously determined that immunization of GT-Ko mice with pig kidney membranes elicits a vigorous T-independent anti-Gal IgM response 2 wk after the first immunization and a T-dependent IgG response 2 wk after the second immunization (17). We reasoned that challenging tolerant GT-Ko mice with pig kidney membranes would allow us to address the question of whether anti-Gal B cells were directly affected since a naive repertoire of pig-specific T cells should be normally activated to provide T cell help to anti-Gal B cells. Thus, we immunized either naive or IAB-tolerized GT-Ko mice with 100 mg/mouse pig kidney membranes at 2-wk intervals for a total of three immunizations. Serum was harvested before each immunization and ELISA was used to detect the levels of anti-Gal Abs.

As presented in Fig. 2Go, the anti-Gal IgM response was maximally elevated within 2 wk for the first immunization with pig kidney membranes (Fig. 2Goa). At this time point (week 2), anti-Gal IgM titers were significantly lower in the tolerant, compared with the control, mice (Student-Newman-Keuls, p < 0.05). Similar, statistically significant, suppression of the IgM response in the tolerant GT-Ko mice relative to the control mice was also observed 2 wk following the second immunization. By the third immunization, there was no statistically significant difference between the tolerant and control GT-Ko mice.



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  		FIGURE 2. Tolerant GT-Ko mice receiving IAB and transient anti-CD40L mAb ({blacksquare}) have significantly reduced anti-Gal Ab levels compared with naive GT-Ko mice ({square}) following immunization with pig kidney membranes. Immunization with pig kidney membranes was initiated 60–80 days after transplantation and anti-CD40L treatment, and all animals were immunized at 2-wk intervals for a total of three immunizations. Serum was collected at the indicated times after first immunization. Anti-Gal IgM (a and c) and IgG (b and d) titers were determined by ELISA, and data are presented as mean OD after subtraction for nonspecific binding to BSA-coated wells. Data are the means of 8–12 recipients per group and error bars are SEM (a and b), while c and d present the individual response of tolerant and control mice.

 
Careful examination of individual anti-Gal IgG responses revealed two important trends: first, the tolerant mice were either completely hyporesponsive (11 of 14) or they had normal responses (3 of 14), and, second, repeated challenge with pig kidney membranes appeared to break the hyporesponsive state and increased the portion of mice that could produce anti-Gal IgM (Fig. 2Goc). These observations suggested that IAB plus anti-CD40L therapy directly tolerized anti-Gal IgM-producing B cells; however, repeated immunizations resulted in the reversal of tolerance. To further test this possibility, the frequency of anti-Gal IgM-producing B cells in tolerant mice receiving one or three immunizations was measured by an ELISPOT assay (Fig. 3Go). The percentage of B cells from the spleens of tolerized, control, and naive GT-Ko mice were comparable at 67.7, 56.2, and 60.1%, respectively. The frequency of anti-Gal IgM-producing B cells in tolerant mice after one immunization was lower than that of control mice (1.81 vs 16.2 per 106 spleen cells; Fig. 3Goa). After three immunizations, the frequency of anti-Gal IgM-producing B cells in tolerant mice was comparable to control mice (39.2 vs 41.4 per 106 spleen cells; Fig. 3Gob).



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  		FIGURE 3. The frequency of anti-Gal IgG-producing cells from tolerant GT-Ko mice receiving IAB and transient anti-CD40L mAb are significantly reduced compared with that of naive GT-Ko mice immunized three times with pig kidney membranes. Immunization with pig kidney membranes was initiated 60–80 days after transplantation and anti-CD40L treatment, and the mice were sacrificed on the 7–10 days after the first (a) or third (b and c) immunization. The frequency of anti-Gal IgM (a and b)- and IgG (c)-producing cells were determined by ELISPOT. Each experiment was performed in duplicate, and data represent the means of 4–5 (a) and 7–10 recipients (b and c) per group ± SEM.

 
Long-term suppression of anti-Gal IgG responses by IAB plus anti-CD40L

The most dramatic difference between tolerant and control GT-Ko mice was in their anti-Gal IgG response following immunization with pig kidney membranes (Fig. 2Gob). As previously reported, the titers of anti-Gal IgG in control GT-Ko mice, 2 wk after the second immunization, were maximally increased and remained at the same level 1 wk after the third immunization. In contrast, the titers of anti-Gal IgG was significantly suppressed in all but one tolerant GT-Ko mice 2 wk after the second immunization and 1 wk after the third immunization (Fig. 2Gob). Careful examination of the individual anti-Gal IgG responses revealed that after the second immunization the tolerant mice were either completely hyporesponsive (n = 9) or they had a normal response (n = 1). After a third challenge with pig kidney membranes, only two of seven hyporesponsive mice developed marginally elevated anti-Gal IgG titers (Fig. 2God).

To confirm that tolerant mice were not capable of producing anti-Gal IgG Abs, we quantified the frequency of anti-Gal IgG-producing B cells in tolerant vs control GT-Ko mice following three immunizations with pig kidney membranes. The frequency of anti-Gal IgG-producing cells in the spleens of control GT-Ko mice immunized three times with pig kidney membranes was 6.6 per 107 spleen cells (Fig. 3Goc). Despite the vigorous challenge with pig xenoantigens, the frequency of anti-Gal IgG in IAB-tolerized GT-Ko mice was significantly suppressed (0.6 per 107 spleen cells) compared with control mice (Student-Newman-Keuls; p < 0.05). These data suggest that hyporesponsiveness in the humoral response elicited by IAB and transient anti-CD40L treatment was due, in part, to tolerance of the B cell population.

To confirm that mice receiving anti-CD40L and IAB had reduced production of anti-Gal Abs, we performed a complete titration of anti-Gal Abs in the sera from either control or tolerized mice after three immunizations with pig kidney membranes. The anti-Gal IgG titers in the tolerized GT-Ko mice were significantly suppressed while the anti-Gal IgM were only marginally reduced (Fig. 4Go, a and b). In addition, we confirmed the specificity of the suppressed response by measuring the non-Gal anti-pig Ab titers. As expected, the non-Gal anti-pig response was comparable in tolerant and control GT-Ko mice when assayed after three immunizations with pig kidney membranes (Fig. 4Go, c and d). These observations suggest that the effect on anti-Gal B was specific and directly related to the tolerizing regiment with Gal-expressing C3H heart and IAB graft plus transient anti-CD40L treatment.



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  		FIGURE 4. Tolerant GT-Ko mice receiving IAB and transient anti-CD40L mAb ({square}) have significantly reduced anti-Gal Ab levels, but equivalent non-Gal anti-pig Ab responses, compared with control immunized GT-Ko mice ({diamondsuit}). Immunization with pig kidney membranes was initiated 60–80 days after transplantation and anti-CD40L treatment, and all animals were immunized at 2-wk intervals for a total of three immunizations. Serum was tested at 5 wk after the first immunization with pig kidney membranes. Anti-Gal (a and b) and non-Gal anti-pig (c and d) Ab titers were determined by ELISA, and data are presented as mean OD after subtraction for nonspecific binding to BSA-coated wells. Data are the means of five recipients per group and error bars are SEM. a and c are IgM, while b and d are IgG.

 
Anti-pig T cell cytokine responses in tolerant GT-Ko mice

We confirmed that the isotype of anti-Gal Abs in control GT-Ko mice following three immunizations with pig kidney membranes to be predominantly IgG3 >> IgG2a = IgG2b = IgG1 (Fig. 5Goa). These anti-Gal IgG responses have previously been shown to be T cell-dependent and were completely inhibited by anti-CD40L mAb therapy (17). IgG2a, IgG2b, and IgG3 Ab responses have previously been reported to be regulated by IFN-{gamma}, while IgG1 responses are dependent on IL-4 (Ref. 18 and our unpublished data). We therefore tested whether suppression of T-dependent anti-Gal IgG responses was due to suppression of the anti-pig cytokine responses. We compared the frequency of IFN-{gamma}- and IL-4-producing cells from tolerant vs control GT-Ko mice when stimulated with irradiated PK15 cells in vitro. As presented in Fig. 5Gob, the frequency of IFN-{gamma}-producing cells in control GT-Ko mice stimulated with irradiated PK15 cells was 247.5 per 5 x 105 splenocytes, a significantly higher frequency than those from naive mice (Student-Newman-Keuls, p < 0.05). The frequency of IFN-{gamma}-producing splenocytes from tolerant GT-Ko mice was 309 per 5 x 105 splenocytes, comparable to the frequency observed with control mice (Student-Newman-Keuls, p > 0.05). Likewise, the frequency of IL-4-producing cells in control GT-Ko mice stimulated with irradiated PK15 cells was 692 per 5 x 105 splenocytes, a significantly higher frequency than splenocytes from naive mice (Student-Newman-Keuls, p < 0.05). The frequency of IL-4-producing splenocytes from tolerant GT-Ko mice was 606 per 5 x 105 splenocytes, comparable to the frequency observed with control mice (Student-Newman-Keuls, p > 0.05). Thus, it appears that priming of anti-pig Th1 and Th2 responses was normal in GT-Ko mice tolerant to Gal-expressing C3H hearts.



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  		FIGURE 5. a, Control GT-Ko mice respond to immunization with pig kidney membranes with an anti-Gal-IgG response that includes all IgG subclasses with IgG3 being dominant. The symbols indicate individual responses of GT-Ko mice after three immunizations with pig kidney membranes. The frequency of IFN-{gamma} (b)- and IL-4 (c)-producing cells in the spleens of tolerant and control GT-Ko mice after three immunizations with pig kidney membranes. The frequency of cytokine-producing cells was determined by ELISPOT assays; each experiment was performed in triplicate, and data represent the means of four mice per group ± SEM. {square}, Naive; , control-immunized; {blacksquare}, tolerant-immunized groups.

 
Tolerant mice are unable to hyperacutely reject Lewis heart grafts

To test whether differences in levels of anti-Gal Abs were functionally significant, we transplanted Lewis rat hearts into tolerant or control mice that had been immunized three times with pig kidney membranes. Naive GT-Ko mice generally reject Lewis rat hearts in 4–5 days, whereas in control immunized mice, the Lewis rat hearts were hyperacutely rejected in <60 min (Table IGo). In contrast, tolerant mice immunized three times with pig kidney membranes were unable to hyperacutely reject Lewis rat hearts, and these hearts survived for >24 h and were sacrificed for immunohistological examination. Transplanted Lewis hearts from the tolerant GT-Ko recipients had reduced IgM deposition relative to control GT-Ko recipients (Fig. 6Go, a and d). There was minimal deposition of IgG in the Lewis hearts from tolerant and control mice (data not shown). More importantly, there was minimal C3 and C5 deposition in the Lewis rat hearts from tolerant mice 1 day (Fig. 6Go, e and f) after transplantation, but extensive C3 and C5 deposition in the Lewis rat hearts from control mice (Fig. 6Go, b and c) at the time of hyperacute rejection (8–15 min posttransplantation). Thus, cotransplantation of IAB and transient anti-CD40L treatment can significantly alter the anti-Gal Ab response and inhibit the ability of GT-Ko mice to elicit hyperacute rejection.


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  		Table I. Xenografts transplanted into tolerant GT-Ko mice do not succumb to hyperacute rejection

 


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  		FIGURE 6. Lewis rat hearts transplanted into control GT-Ko mice immunized three times with pig kidney membranes exhibit classic features of hyperacute rejection including extensive deposition of IgM (a), C3 (b), and C5 (c). In contrast Lewis rat hearts transplanted into tolerant GT-Ko mice that had been immunized three times with pig kidney membranes did not succumb to hyperacute rejection and exhibited reduced IgM (d) and no C3 (e) or C5 (f) deposition at 24 h after transplantation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is increasing evidence that alloantibodies contribute to the process of hyperacute, acute, and chronic rejection of allografts (19, 20, 21, 22, 23, 24). Yet most experimental models of allotransplantation have concentrated on the role of cellular immune responses in acute allograft rejection, while most investigations into the induction of allograft tolerance have focused on the regulation of the cellular and cytokine responses. Because alloantibodies can have significant pathological effects on the allografts, the absence of such data in rodent models makes it difficult to assess whether tolerance with indefinite allograft survival can occur in the presence of alloantibodies.

In this article, we report that treatment with anti-CD40L alone resulted in only a transient suppression of alloantibody production and that the alloantibody titers rapidly increased upon cessation of therapy. This is similar to observations with allogeneic islet transplantation in nonhuman primates suppressed with anti-CD40L (6, 7). A simple regimen of heart allograft and IAB cotransplantation synergize with transient anti-CD40L treatment to induce B cell tolerance. Interestingly, transplantation of C3H IAB and transient anti-CD40L alone, in the absence of cardiac allograft, did not induce B cell tolerance (our unpublished data). Following a single challenge with pig kidney membranes immunization, where only anti-Gal IgM responses were elicited, we observed that the majority (11 of 14) of our tolerized mice were tolerant. When challenged with additional injections of pig membranes, tolerance of the anti-Gal IgM response was lost and an increasing portion of mice acquired normal responsiveness to Gal.

These observations that anti-CD40L can synergize with IAB without requiring stable mixed chimerism is novel and are complementary to those reported by Sykes and her colleagues (15, 25, 26, 27) that lasting multilineage mixed chimerism can result in tolerance of the anti-Gal Ab response. In those studies, GT-Ko mice were subject to either lethal irradiation or sublethal irradiation plus T cell depletion to induce lasting multilineage mixed chimerism through the engraftment of Gal+/+ bone marrow cells. The mechanism of tolerance elicited by the mixed chimeric state was proposed to be central deletion or receptor editing, as newly emerging anti-Gal B cells would be deleted or modified in the presence of Gal-expressing cells in the bone marrow. Under the nonlethal condition regimen, preexisting mature anti-Gal B cells also appeared to rapidly lose their ability to produce Abs. The mechanism for this rapid loss in ability to produce anti-Gal Abs by the preexisting B cells was not defined, although tolerization through Ag receptor cross-linking leading to anergy and/or apoptosis or rapid cell turnover were suggested possibilities (15). It will be important to test whether the mechanism involved in the tolerance of preexisting B cells in their model is related to the mechanism of B cell hyporesponsiveness in our model of IAB and anti-CD40L.

A unique aspect of our studies is the dramatic effect of our tolerizing regiment with C3H heart and IAB plus anti-CD40L on the anti-Gal IgG response. Even after three immunizations with pig kidney membrane, when the anti-Gal IgG response was maximal in control mice, the anti-Gal IgG response was significantly suppressed in the tolerant mice. This conclusion was reached by measuring the concentration of anti-Gal IgG in the serum, determining the frequency of anti-Gal IgG-producing cells, and checking for Ab deposition on the C3H heart and Lewis xenografts. We illustrated the specificity of the B cell hyporesponsiveness induced by our regimen by confirming that the non-Gal anti-pig Ab responses, including the IgG response, were comparable in tolerant and control mice. We have previously shown that the anti-Gal IgG response was completely T cell dependent (17), while others have shown that Th1 (IFN-{gamma}) and Th2 (IL-4) cytokine responses differently regulate the production of IgG isotypes (18). The observation that the numbers of IFN-{gamma}- and IL-4-producing cells in the spleen from tolerant mice were comparable to control mice suggest that altered, porcine-specific, Th1 or Th2 responses is not the reason for the anti-Gal IgG hyporesponsiveness observed in our model of tolerance.

Several mechanisms of B cell tolerance have been reported, including deletion at the immature B cell stage, B cell anergy, regulation of T cell help, receptor editing, follicular competition, Fas-mediated elimination by T cells, and censoring in the germinal centers (28, 29, 30, 31). Any number of these mechanisms could mediate the anti-Gal tolerance induced by IAB and anti-CD40L. The differential ability of our tolerizing regimen with C3H heart and IAB plus anti-CD40L to control anti-Gal IgM vs IgG responses are not understood at present. It is clear that two different populations of B cells contribute to the anti-Gal Ab response. The first is a T-independent B-1B cell subset that is responsible for T-independent anti-Gal IgM production (32), and the second is a conventional T-dependent B-2 population that produces anti-Gal IgG (17, 33, 34, 35). It has recently been reported that the signaling thresholds for B-1 and B-2 cells differ, and differentiation of B-0 and B-2 cells to B-1 cells that have increased activation thresholds can result in functional B cell tolerance (36, 37, 38). Activation thresholds can be affected by the affinity of the B cell receptor itself or by alterations in the levels of coreceptors such as CD19, CD22, or CD5 (38, 39, 40). Identification of anti-Gal B cells from tolerant and control mice, characterizing the expression of B cell coreceptors and comparing their signaling abilities, should allow us to test whether differentiation of B-0 and B-2 cells to B-1 cells that have increased activation thresholds is the basis of anti-Gal B cell tolerance in our model.

A number of groups, including ours, have shown that anti-Gal IgM tend to be polyreactive and their affinity for Gal are considerably lower that of anti-Gal IgG (35, 41). In addition, we observed that the VH gene usage for anti-Gal IgM Abs is unrestricted, while the VH gene usage for anti-Gal IgG is highly restricted to a singleVH606 family (41). It is, therefore, possible that B cells capable of producing high-affinity anti-Gal IgG become tolerized by the regimen of IAB and anti-CD40L. In addition, the preexisting repertoire of anti-Gal IgM B cells capable of giving rise to high-affinity anti-Gal IgG-producing B cells may also have become tolerized. Multiple immunizations with pig kidney membranes may elicit a new repertoire of IgM-producing B cells capable of cross-reacting with Gal, but incapable of generating IgG with sufficient affinity for Gal. Indirect support for the latter possibility come from observations with tolerant (after three immunizations) and control GT-Ko mice challenged with Lewis rat hearts. Despite comparable titers of circulating anti-Gal IgM and frequencies of anti-Gal IgM-producing B cells (Figs. 2Go and 4Go), tolerant mice were unable to induce hyperacute rejection of Lewis rat hearts. Immunohistochemistry revealed lower levels of IgM and C3 and C5 deposition in the Lewis rat hearts transplanted into tolerant mice compared with those transplanted into control immunized GT-Ko mice. These observations suggest that anti-Gal IgM Abs secreted by the tolerant mice may be of lower affinity and have reduced ability to fix complement. Studies comparing the VH usage and affinity of anti-Gal IgM from tolerant mice after repeated immunizations of pig kidney membranes with control immunized mice should provide a vigorous test of this possibility.

In summary, we have described the ability of Gal-expressing allogeneic heart and IAB grafts to synergize with transient anti-CD40L treatment to induce B cell tolerance for both anti-Gal IgM and IgG. The tolerant state for the anti-Gal IgM response was reversed with repeated immunization, whereas the tolerant state for the IgG response was robust and resisted repeated immunization. These observations provide an important proof-of-concept that adjunct therapies can synergize with anti-CD40L Abs to tolerize B cell responses. This model provides a unique opportunity for studying the mechanism of tolerance, in a non-mixed chimerism setting, of a clinically relevant population of carbohydrate-specific B cells.


   Acknowledgments
 
We thank Drs. Uri Galili and Ian Boussy for helpful critique of this study. We also thank Dr. Keith Bishop and Sherry Chan (University of Michigan, Ann Arbor, MI) as well as Dr. Peter Heeger (Cleveland Clinic, Cleveland, OH) for assistance in the cytokine ELISPOT assays and the long-term loan of the Immunospot image analyzer.


   Footnotes
 
1 This study was supported in part by Grant R01 43631 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Anita S.-F. Chong, Department of General Surgery, Rush Presbyterian St. Luke’s Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. E-mail address: achong{at}rush.edu Back

3 Abbreviations used in this paper: CD40L, CD40 ligand; IAB, intact active bone; GT-Ko, galactosyltransferase-deficient knockout; Gal, galactose-{alpha}1,3-galactose. Back

Received for publication January 9, 2002. Accepted for publication March 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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