We have previously reported that anti-Gal-α1,3Gal (Gal) IgG3 mAbs mediate a classical complement-dependent hyperacute rejection (HAR), while anti-Gal IgG1 mAbs mediate HAR that is dependent on complement, the Fc-γ receptors FcγRII/III (CD32/CD16), and NK cells. IgG2a and IgG2b subclasses can activate complement and have FcγR binding properties in vitro. Whether these IgG subclasses can mediate HAR in vivo and the mechanisms by which they would do so are not known. In this study, we isolated spontaneous IgG switch mutants from an anti-Gal IgG1 hybridoma. In vitro complement-mediated hemolytic assays with mouse complement indicate that both anti-Gal IgG2a and IgG2b mAbs were more potent compared with the parent anti-Gal IgG1. In vivo administration of anti-Gal IgG2a and IgG2b mAbs into Gal−/− mice induced HAR of rat cardiac xenografts. HAR induced by anti-Gal IgG2a and IgG2b was dependent on complement activation and the presence of NK cells. Using FcγRIII-deficient (Gal−/−CD16−/−) recipients, we observed that HAR mediated by different anti-Gal IgG subclasses was variably dependent on FcγRIII, with IgG1 > IgG2b ≫ IgG2a = IgG3. Using FcγRI-deficient (Gal−/−CD64−/−) recipients, we observed that HAR mediated by anti-Gal IgG1, IgG2a, and IgG2b, but not by anti-Gal IgG3, was dependent on FcγRI. Collectively, these studies demonstrate the necessity and sufficiency of complement in IgG3-mediated HAR and the necessity of both complement and FcγR, especially FcγRI, in IgG1-, IgG2a-, and IgG2b-mediated HAR.

Affinity maturation and isotype switching are hallmarks of a mature Ab response; however, the effects of these events on the activities of graft-specific Abs have not been characterized in the setting of hyperacute rejection (HAR)3 of xenografts. Following the transplantation of pig organs into humans, anti-Gal-α1,3Gal (Gal) IgM titers increase by 4- to 20-fold while the anti-Gal IgG titers increase by 20- to 100-fold (1, 2, 3). Anti-Gal IgG2, IgG3, and IgG4 have been reported in the preimmune serum, whereas all IgG subclasses, including IgG1, are present in the postimmune serum (3, 4) The ability of complement-fixing IgM to induce HAR has been well characterized. It is less clear what effects anti-Gal IgG have in vivo, especially since the ability to fix complement (huIgG3 > huIgG1 > huIgG2 >≫ huIgG4; muIgG3 ≥ muIgG2a = muIgG2b ≫ muIgG1; where hu stands for human and mu stands for murine) and to bind FcγR varies significantly among the IgG subclasses (5, 6, 7, 8, 9, 10, 11). For instance IgG1 binds with low affinity to FcγRII and FcγRIII and with very low affinity to FcγRI; IgG2a binds with high affinity to FcγRI and with low affinity to FcγRIII and FcγRIV; IgG2b binds with low affinity to all FcγRs but with the highest affinity for FcγRIV (5). IgG3 has extremely low affinity for all FcγRs (5).

The α1,3-galactosyltransferase (Gal−/−) knockout mouse, which lacks the expression of the Gal epitope, has been used as a recipient for studying anti-Gal responses to Gal+/+ cardiac xenografts (12, 13, 14, 15, 16). Preformed anti-Gal Abs are primarily IgM, whereas the elicited anti-Gal IgG Abs following xenotransplantation of Lewis rat hearts were restricted to the IgG3 and IgG1 subclasses (14). Molecular characterization of the B cell hybridomas producing anti-Gal IgM and IgG Abs derived from naive mice or early in the immune response indicated the use of a variety of VH genes in a germline configuration for the anti-Gal IgM, while anti-Gal IgG derived 21 days after transplant were encoded by a single VHJ606 14A gene family with evidence of somatic mutation. Of the nine anti-Gal IgG hybridomas isolated, eight produced IgG3 and one IgG1, whereas no IgG2a or IgG2b clones were isolated (14). The restriction in anti-Gal IgG subclasses elicited in Gal−/− mice contrasts with the baboon or human responses in which all IgG subclasses were elicited by porcine xenografts (4, 17, 18, 19, 20). The most likely explanation for this difference is that the concordant Lewis heart grafts were unable to induce the class switch to T-dependent IgG2a and IgG2b, as Gal−/− mice were able to produce all IgG subclasses following multiple injections with pig kidney membranes or rabbit erythrocytes (21, 22).

In this study we describe a modified method of clonal sib selection to isolate spontaneous IgG switch mutants from an anti-Gal IgG1 hybridoma (23, 24). The advantage of using these IgG class-switched mAbs is that potential differences in affinity and specificities are minimized, and the effects of the different IgG subclasses can be investigated. Using a xenotransplantation model of baby Lewis rat hearts into immunocompetent Gal−/− recipients, these mAbs have allowed us to define the in vitro and in vivo activities of anti-Gal Abs of different IgG subclasses.

Anti-Gal IgG, anti-NK1.1, and anti-FcγRII/III (2.4G2) hybridomas were cultured in protein-free hybridoma medium (Invitrogen Life Technologies). Ammonium sulfate was used to precipitate the mAbs from the supernatants. Following extensive dialysis, the purity of the precipitated Ab (>95%) was confirmed by SDS-PAGE and the Ab concentration was determined by UV spectrometry (280 nm).

The Gal-ELISA using Gal-α1,3Gal-BSA (Gal-BSA) disaccharide was performed as previously reported (21, 22). Briefly, Gal-BSA was diluted to 5 μg/ml in PBS while anti-Gal IgG mAbs were diluted in 1% BSA/PBS, and the binding of anti-Gal IgG subclasses was quantified using biotinylated anti-IgG1, IgG2a, IgG2b, or IgG3 mAbs (BD Biosciences). It should be noted that the anti-mouse IgG2a mAb (R19-15) reacts with both IgG2aa and IgG2ab isotypic genes, corresponding to the original γ2a and γ2c genes in wild mouse populations (25). In accordance with common nomenclature, we have referred to both IgG2aa and IgG2ab as IgG2a. The specificity of anti-Gal IgG binding was confirmed by inhibition with 10 mM free Gal-α1,3Gal-trisaccharide (26).

Anti-Gal mAbs were assayed for the ability to bind to and activate complement in vitro using flow cytometry and a hemolysis assay. Briefly, different dilutions of anti-Gal mAbs were incubated with pig RBC (5 × 105) on ice for 30 min. In some experiments, the cells were washed and then incubated with mouse serum (1/5 dilution in 1% BSA/PBS) followed by FITC-conjugated anti-mouse C3b (Cappel Organon Teknika). Anti-Gal mAbs were also assayed for the ability to induce complement-mediated hemolysis in the presence of mouse serum (1/5 dilution) in a hemoglobin release assay as previously reported (27).

Gal−/− mice on a C57BL/6 background were a generous gift from Dr. R. Beulow (Sangstat, Fremont, CA) and were bred and maintained at the University of Chicago (Chicago, IL), an American Association for the Accreditation of Laboratory Animal Care-accredited facility. FcγRI−/− and FcγRIII−/− mice (28, 29) were backcrossed 10 generations to C57BL/6 mice and then crossed with Gal−/− mice to generate double-knockout mice (known as Gal−/−CD64−/− and Gal−/−CD16−/−). C6−/− mice were generously provided by Dr. G. Stahl (Guy’s Hospital, London, UK) (30), backcrossed to C57BL/6 mice for four generations, and then to Gal−/− mice to generate double-knockout mice (known as Gal−/−C6−/−).

Ten- to 16-day-old Lewis rats (Harlan) were used as heart donors. Lewis rat hearts were heterotopically transplanted into the abdomen of recipient mice by anastomosing the donor aorta to the recipient abdominal aorta and the donor pulmonary artery to recipient inferior vena cava, as previously described (31). Anti-Gal mAbs were injected i.v. on the day of transplant. Cobra venom factor (CVF; Quidel) was administered at 3.5 U/mouse on day −1 and 1.0 U/mouse on days 0, 1, and 2 posttransplantation. Anti-asialo GM1 (Waco Chemicals) was administered at 50 μl/mouse on days −2, 0, and 2 posttransplantation. Anti-NK1.1 (PK136; 1 mg/mouse) was administered i.v. on days −2, −1, and 0 relative to heart transplantation. Anti-FcγRII/III (clone 2.4G2) was administered at 125 μg/mouse on day −1 and 2 h before the anti-Gal mAbs. All heart grafts were monitored half-hourly for the first 4 h and then daily. Rejection is defined as the complete cessation of heart pulsation.

Transplanted hearts were surgically removed before the complete cessation of heartbeat and snap frozen in Tissue-Tec OCT (Sakura Finetek). Immunohistochemistry was based on a modified avidin-biotin peroxidase method as previously published (27). Briefly, for the identification of complement deposition, polyclonal goat-anti-human C3 and goat-anti-human C5 were used (Quidel). These Abs are listed to be specific for native C3 and C5, and we infer that they recognize C3b, iC3b, and/or C3d as well as cleaved C5b. For the identification of platelet-fibrin microthrombosis, sections were incubated with rabbit-anti-human von Willebrand’s factor (vWF) and anti-human fibrinogen (DakoCytomation) polyclonal Abs. All sections were then incubated with biotinylated rabbit anti-goat IgG or biotinylated goat anti-rabbit IgG (Vector Laboratories) followed by HRP-streptavidin (Zymed Laboratories). To detect anti-Gal IgG deposition, a panel of biotinylated rat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (BD Biosciences) was applied followed by HRP-streptavidin.

We isolated spontaneous class switch mutants from an anti-Gal IgG1 hybridoma (GT4-31.1) by initial enrichment with anti-IgG2a- or anti-IgG2b-coated Miltenyi bead selection. After four cycles of enrichment, the IgG2a- or IgG2b-enriched hybridoma cells were subcloned by limiting dilution and the highest producer was isolated and used for all subsequent studies. The percentage of IgG2a- and IgG2b-secreting hybridoma cells in the sib-selected clones was determined by flow cytometry and observed to be >99% (Fig. 1,a). Additionally, the purity of the IgG subclass from the sib-selected clones was confirmed with a solid phase Gal ELISA and mAbs specific for IgG subclasses (Fig. 1 b).

FIGURE 1.

Identification and specificity of sib-selected anti-Gal IgG2a and IgG2b hybridomas. a, Sib selection was performed to isolate IgG2a and IgG2b hybridomas from the parental anti-Gal IgG1 hybridoma. Flow cytometric analyses with biotinylated anti-IgG1, anti-IgG2a, and anti-IgG2b were performed on the sib-selected hybridoma. b, Gal-ELISA was performed with the purified anti-Gal IgG1, IgG2a, IgG2b, and IgG3 using IgG subclass-specific mAbs to demonstrate the purity of the sib-selected mAbs. Data are presented as mean OD ± SE of means.

FIGURE 1.

Identification and specificity of sib-selected anti-Gal IgG2a and IgG2b hybridomas. a, Sib selection was performed to isolate IgG2a and IgG2b hybridomas from the parental anti-Gal IgG1 hybridoma. Flow cytometric analyses with biotinylated anti-IgG1, anti-IgG2a, and anti-IgG2b were performed on the sib-selected hybridoma. b, Gal-ELISA was performed with the purified anti-Gal IgG1, IgG2a, IgG2b, and IgG3 using IgG subclass-specific mAbs to demonstrate the purity of the sib-selected mAbs. Data are presented as mean OD ± SE of means.

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We first tested the ability of class-switched anti-Gal IgG mAbs to bind to Gal-BSA by ELISA (Fig. 2,a). As expected, comparable binding profiles were observed for anti-Gal IgG1, IgG2a, and IgG2b to Gal-BSA (Fig. 2,a). This anti-Gal IgG binding to Gal-BSA was completely abrogated by 10 mM free Gal-α1,3Gal trisaccharide, confirming the specificity of the anti-Gal IgG binding (Fig. 2 a).

FIGURE 2.

Functional analysis of sib-selected anti-Gal IgG1, IgG2a, and IgG2b. a, Binding of anti-Gal IgG mAbs was quantified by ELISA using Gal-BSA as substrate. Experiments were performed in triplicate in at least three independent assays, and data are presented as mean OD ± SE of means. b and c, Indicated concentrations of anti-Gal mAbs were incubated with pig RBC, and mAb binding (b) and mouse C3 deposition (c) were quantified by flow cytometry using FITC-conjugated anti-mouse IgG and FITC-conjugated anti-C3 Abs. Data are presented as mean channel fluorescence (MCF) ± SE of means. inh, Inhibitor (10 mM Gal trisaccharide). d, The ability of the mAbs to induce complement-mediated lysis of pig RBC, using mouse serum (1:5 dilution) as a source of complement, is presented. Data are presented as percentage of lysis ± SE of means. All experiments were performed in triplicates and independently repeated three or four times.

FIGURE 2.

Functional analysis of sib-selected anti-Gal IgG1, IgG2a, and IgG2b. a, Binding of anti-Gal IgG mAbs was quantified by ELISA using Gal-BSA as substrate. Experiments were performed in triplicate in at least three independent assays, and data are presented as mean OD ± SE of means. b and c, Indicated concentrations of anti-Gal mAbs were incubated with pig RBC, and mAb binding (b) and mouse C3 deposition (c) were quantified by flow cytometry using FITC-conjugated anti-mouse IgG and FITC-conjugated anti-C3 Abs. Data are presented as mean channel fluorescence (MCF) ± SE of means. inh, Inhibitor (10 mM Gal trisaccharide). d, The ability of the mAbs to induce complement-mediated lysis of pig RBC, using mouse serum (1:5 dilution) as a source of complement, is presented. Data are presented as percentage of lysis ± SE of means. All experiments were performed in triplicates and independently repeated three or four times.

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We next quantified the ability of each of the anti-Gal IgG subclasses to bind and induce mouse C3 deposition on pig erythrocytes, as well as its ability to induce cell lysis in vitro (Fig. 2, b–d). Pig erythrocytes were incubated with the indicated concentrations of anti-Gal IgG Abs, then with FITC-conjugated anti-mouse IgG or anti-C3b, and subjected to flow cytometric analysis. Anti-Gal IgG1 and IgG2a bound comparably to pig erythrocytes while the anti-Gal IgG2b had consistently lower levels of binding (Fig. 2 b). We speculate that the differences in the IgG binding to pig erythrocytes reflect variations in the affinity or the level of biotin conjugation of the secondary mAbs for the IgG subclasses or differences in the fine specificity of the Gal IgG subclasses as a result of different Fc (10, 32).

All four IgG subclasses induced mouse C3 deposition, with IgG2a being the most efficient at stimulating C3 deposition compared with IgG2b or IgG1 (Fig. 2,c). We next compared the ability of the different anti-Gal IgG subclasses to induce the lysis of pig erythrocytes in a standard hemolysis assay by using mouse serum as a source of complement. We observed that anti-Gal IgG2a and IgG2b demonstrated comparable abilities to mediate the hemolysis of pig erythrocytes (p > 0.05). Both anti-Gal IgG2a and IgG2b were significantly less efficient compared with anti-Gal IgG3 (p < 0.01; Fig. 2,d) but were significantly more potent than the parental anti-Gal IgG1 at inducing the lysis of pig erythrocytes (p < 0.001; Fig. 2 d).

To compare the in vivo activities of different anti-Gal IgGs, we used a xenotransplantation model involving heterotopic baby Lewis rat hearts transplanted into the abdomens of immunocompetent Gal−/− mice. Anti-Gal IgG mAbs were injected i.v. on the day of heart transplant. In this model, rat hearts were rejected in 5–7 days as a result of elicited xenoantibody and T cell responses without the administration of anti-Gal mAbs. We observed that all of the IgG subclasses had comparable abilities at inducing HAR (Fig. 3).

FIGURE 3.

Role of complement in HAR mediated by anti-Gal IgGb. Lewis rat hearts were transplanted into Gal−/− or Gal−/−C6−/− mice and then anti-Gal IgG (IgG1, 300 μg/mouse; IgG2a, 200 μg/ml; IgG2b, 250 μg/mouse; and IgG3, 250 μg/mouse) was administered i.v. CVF was administered on days 0–2 posttransplantation as described in Materials and Methods. Lewis rat hearts were monitored for 24 h after anti-Gal administration. In the absence of anti-Gal administration, Lewis rat hearts survived for 5–7 days. All data are presented as percentage of graft survival. WT, Wild type.

FIGURE 3.

Role of complement in HAR mediated by anti-Gal IgGb. Lewis rat hearts were transplanted into Gal−/− or Gal−/−C6−/− mice and then anti-Gal IgG (IgG1, 300 μg/mouse; IgG2a, 200 μg/ml; IgG2b, 250 μg/mouse; and IgG3, 250 μg/mouse) was administered i.v. CVF was administered on days 0–2 posttransplantation as described in Materials and Methods. Lewis rat hearts were monitored for 24 h after anti-Gal administration. In the absence of anti-Gal administration, Lewis rat hearts survived for 5–7 days. All data are presented as percentage of graft survival. WT, Wild type.

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We had previously reported that anti-Gal IgG3 (GT4-31) and IgG1 (GT6-27) mediated the HAR of xenografts by different mechanisms (33). In those studies Rag−/− mice were used as recipients, raising the possibility that a role of T and B cells may have been overlooked. In this study we confirm that IgG3 mAb-mediated HAR in immunocompetent Gal−/− recipients was dependent on complement activation and was inhibited by CVF (Fig. 3). CVF pretreatment was also able to inhibit HAR mediated by anti-Gal IgG2a, IgG2b, and IgG1 (Fig. 3).

To test the importance of the terminal activation of complement in mediating HAR by all four subclasses of anti-Gal IgGs, baby Lewis rat hearts were transplanted into Gal−/−C6−/− mice before anti-Gal IgG challenge. HAR mediated by IgG1, IgG2a, IgG2b, and IgG3 was abrogated in Gal−/−C6−/− recipients (Fig. 3), demonstrating that HAR induced by all four subclasses of anti-Gal IgGs is dependent on the terminal activation of complement in vivo.

Our previous study (33) revealed that the blocking 2.4G2 specific for FcγRII/III, effectively inhibited anti-Gal IgG1-mediated but not IgG3-mediated HAR in Gal−/−Rag−/− recipients. We confirm in immunocompetent Gal−/− recipients that the 2.4G2 mAb did not inhibit IgG3-mediated HAR (Fig. 4,a) but inhibited HAR mediated by IgG1, IgG2a, and IgG2b, with 100, 75, and 100% xenograft survival, respectively (Fig. 4 a). These results suggest that FcγRII, FcγRIII, or both participate in anti-Gal IgG1-, IgG2a-, and IgG2b-mediated, but not in IgG3-mediated, HAR.

FIGURE 4.

The role of FcγRII/III in HAR mediated by anti-Gal IgG. a, Lewis rat hearts were transplanted into Gal−/− mice followed by the i.v. administration of anti-Gal IgG (IgG1, 300 μg/mouse; IgG2a, 200 μg/ml; IgG2b, 250 μg/mouse; and IgG3, 250 μg/mouse). Anti-FcγRII/III (2.4G2) was administered at a dose of 125 μg/mouse 1 day and 2 hours before the anti-Gal IgG2a injection. All data are presented as percentage of graft survival. WT, Wild type. b, Histology and immunohistochemical analysis of HAR mediated by anti-Gal IgG2a. Heart grafts were removed 30 min after anti-Gal IgG2a injection, before the complete cessation of heartbeat. The histology (H&E) and immunohistochemistry were performed as described in Materials and Methods. Treatment groups are indicated on the left and the histology (HE) (A and F) and immunohistochemical stains for IgG2a (B and G), C3 (C and H), C5 (D and I), and vWF (E and J) are indicated at the top. The figures are representative of three recipients per group. Similar results were observed with anti-Gal IgG2b-mediated HAR (data not shown).

FIGURE 4.

The role of FcγRII/III in HAR mediated by anti-Gal IgG. a, Lewis rat hearts were transplanted into Gal−/− mice followed by the i.v. administration of anti-Gal IgG (IgG1, 300 μg/mouse; IgG2a, 200 μg/ml; IgG2b, 250 μg/mouse; and IgG3, 250 μg/mouse). Anti-FcγRII/III (2.4G2) was administered at a dose of 125 μg/mouse 1 day and 2 hours before the anti-Gal IgG2a injection. All data are presented as percentage of graft survival. WT, Wild type. b, Histology and immunohistochemical analysis of HAR mediated by anti-Gal IgG2a. Heart grafts were removed 30 min after anti-Gal IgG2a injection, before the complete cessation of heartbeat. The histology (H&E) and immunohistochemistry were performed as described in Materials and Methods. Treatment groups are indicated on the left and the histology (HE) (A and F) and immunohistochemical stains for IgG2a (B and G), C3 (C and H), C5 (D and I), and vWF (E and J) are indicated at the top. The figures are representative of three recipients per group. Similar results were observed with anti-Gal IgG2b-mediated HAR (data not shown).

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Histology and immunohistochemistry performed on the xenografts revealed the deposition of C3, C5, and vWF expression in the grafts undergoing IgG2a-mediated HAR. The anti-C4 and anti-C5 polyclonal Abs were raised against native C3 and C5, and we infer that they are recognizing C3b, inactivated C3b, and/or C3d, as well as cleaved C5b, deposited on the endothelium of the rejected xenografts. Complement and vWF deposition, as well as fibrin deposition, was significantly reduced by the administration of anti-FcγRII/III mAbs (Fig. 4 b, data not shown). Similar results were observed for anti-Gal IgG2b (data not shown). These results suggest that FcγRII/III-mediated interactions synergize with complement activation to induce anti-Gal IgG2a- and IgG2b-mediated HAR.

To define the phenotype of the FcγR-bearing cells contributing to IgG2a- and IgG2b-mediated HAR, we tested the effect of anti-NK1.1 mAb and anti-asialo GM1 polyclonal Abs (34). As illustrated in Fig. 5, the anti-NK1.1 mAb fully protected against anti-Gal IgG2a- and IgG2b-mediated HAR, while anti-asialo GM1 partially protected against anti-Gal IgG2a- and IgG2b-mediated HAR. The efficacy of NK cell depletion by the two reagents is likely to be the explanation for the slight differences in outcomes. Consistent with our previous report (33), both anti-NK1.1 and anti-asialo GM1 completely protected xenografts against anti-Gal IgG1-induced HAR. Neither anti-NK1.1 nor anti-asialo GM1 treatment protected xenografts from anti-Gal IgG3-mediated HAR. Collectively, these data suggest that HAR mediated by anti-Gal IgG2a and IgG2b resembles anti-Gal IgG1 in that both complement activation and FcγR/NK cells are necessary for mediating HAR.

FIGURE 5.

Role of NK cells in HAR mediated by anti-Gal IgG. Lewis rat hearts were transplanted into Gal−/− mice followed by the i.v. administration of anti-Gal IgG (IgG1, 300 μg/mouse; IgG2a, 200 μg/ml; IgG2b, 250 μg/mouse; and IgG3, 250 μg/mouse). Some recipients were pretreated with anti-NK1.1 mAb (1 mg/mouse) or anti-asialo GM1 (Anti-aGM1) Abs (50 μg/mouse) as described in Materials and Methods. All data are presented as percentage of graft survival. WT, Wild type.

FIGURE 5.

Role of NK cells in HAR mediated by anti-Gal IgG. Lewis rat hearts were transplanted into Gal−/− mice followed by the i.v. administration of anti-Gal IgG (IgG1, 300 μg/mouse; IgG2a, 200 μg/ml; IgG2b, 250 μg/mouse; and IgG3, 250 μg/mouse). Some recipients were pretreated with anti-NK1.1 mAb (1 mg/mouse) or anti-asialo GM1 (Anti-aGM1) Abs (50 μg/mouse) as described in Materials and Methods. All data are presented as percentage of graft survival. WT, Wild type.

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To confirm the role of FcγRIII in IgG1-, IgG2a-, and IgG2b-mediated HAR, Lewis baby rat hearts were transplanted into Gal−/−CD16−/− mice before anti-Gal IgG challenge (Fig. 6). Consistent with the data with the use of anti-FcγRII/III mAbs, Gal−/−CD16−/− recipients did not hyperacutely reject xenografts following the administration of anti-Gal IgG1. Unexpectedly, Gal−/−CD16−/− recipients were able to mediate HAR by anti-Gal IgG2a as well as to partially mediate HAR by anti-Gal IgG2b. Gal−/−CD16−/− recipients were also able to mediate HAR by anti-Gal IgG3. These data indicate that HAR induced by anti-Gal IgG (IgG1 > IgG2b > IgG2a = IgG3) was variably dependent on CD16-mediated events.

FIGURE 6.

Role of FcγRIII (CD16) in HAR mediated by anti-Gal IgG. Lewis rat hearts were transplanted into Gal−/− or Gal−/−CD16−/− mice followed by the i.v. administration of anti-Gal IgG (IgG1, 300 μg/mouse; IgG2a, 200 μg/ml; IgG2b, 250 μg/mouse; and IgG3, 250 μg/mouse). To further investigate the role of FcγRIII-expressing NK cells in HAR, some recipients received either whole or NK cell-depleted Gal−/− spleen cells (4.0 × 107/recipient) 1 day before anti-Gal IgG1 challenge. All data are presented as percent graft survival. WT, Wild type.

FIGURE 6.

Role of FcγRIII (CD16) in HAR mediated by anti-Gal IgG. Lewis rat hearts were transplanted into Gal−/− or Gal−/−CD16−/− mice followed by the i.v. administration of anti-Gal IgG (IgG1, 300 μg/mouse; IgG2a, 200 μg/ml; IgG2b, 250 μg/mouse; and IgG3, 250 μg/mouse). To further investigate the role of FcγRIII-expressing NK cells in HAR, some recipients received either whole or NK cell-depleted Gal−/− spleen cells (4.0 × 107/recipient) 1 day before anti-Gal IgG1 challenge. All data are presented as percent graft survival. WT, Wild type.

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NK cells express FcγRIII, and NK cell activation occurs upon the engagement of multivalent immune complexes with FcγRIII (35). To test the importance of FcγRIII-bearing NK cells in xenograft rejection, we transferred 4.0 × 107 Gal−/− spleen cells into Gal−/−CD16−/− recipients 1 day before anti-Gal IgG1 challenge. Reconstitution of FcγRIII-expressing spleen cells from Gal−/− mice restored HAR by anti-Gal IgG1 (Fig. 6). For negative controls, spleen cells were derived from Gal−/− mice that had received 100 μl/mouse anti-asialo GM1 i.v. for two consecutive days, and the depletion of NK cells was confirmed by flow cytometry (data not shown). No graft rejection was observed when 4.0 × 107 NK cell-depleted Gal−/− spleen cells was infused (Fig. 6). These experiments suggest that FcγRIII-bearing NK cells play an important role in mediating FcγRIII-dependent IgG1-mediated HAR.

Because of the lack of or incomplete dependence of IgG2a and IgG2b on FcγRIII, we tested the role of FcγRI in IgG-mediated HAR. FcγRI is a high affinity receptor that is activated following binding to monomeric IgG2a ≫ IgG3,1,2b (5, 7). As illustrated in Fig. 7, ≥70% of xenografts transplanted into Gal−/−CD64−/− mice did not succumb to HAR following anti-Gal IgG1, IgG2a, and IgG2b challenge. However, Gal−/−CD64−/− mice were able to hyperacutely reject rat cardiac xenografts following anti-Gal IgG3 challenge. Administration of 4.0 × 107 Gal−/− spleen cells into Gal−/−CD64−/− mice restored, either partially or completely, the ability of these mice to induce HAR following anti-Gal IgG1, IgG2a, and IgG2b challenge (Fig. 7). These results indicate that FcγRI-bearing cells play an important role in facilitating anti-Gal IgG1-, IgG2a-, and IgG2b-mediated HAR.

FIGURE 7.

The role of FcγRI (CD64) in HAR mediated by anti-Gal IgG. Lewis rat hearts (Gal+/+) were transplanted into either Gal−/− or Gal−/−CD64−/− mice followed by the i.v. administration of anti-Gal IgG (IgG1, 300 μg/mouse; IgG2a, 200 μg/ml; IgG2b, 250 μg/mouse; and IgG3, 250 μg/mouse). In some recipients of anti-Gal IgG1, IgG2a, and IgG2b, 4.0 × 107 Gal−/− spleen cells were transferred One day before anti-Gal IgG challenge. All data are presented as percentage of graft survival. WT, wild type.

FIGURE 7.

The role of FcγRI (CD64) in HAR mediated by anti-Gal IgG. Lewis rat hearts (Gal+/+) were transplanted into either Gal−/− or Gal−/−CD64−/− mice followed by the i.v. administration of anti-Gal IgG (IgG1, 300 μg/mouse; IgG2a, 200 μg/ml; IgG2b, 250 μg/mouse; and IgG3, 250 μg/mouse). In some recipients of anti-Gal IgG1, IgG2a, and IgG2b, 4.0 × 107 Gal−/− spleen cells were transferred One day before anti-Gal IgG challenge. All data are presented as percentage of graft survival. WT, wild type.

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Naturally occurring anti-Gal Abs in humans and Old World monkeys bind to Gal epitopes expressed on pig cells, where they initiate a rapid complement-driven HAR of pig organs when transplanted into primates. When HAR is prevented, the grafts are rejected within a few days to weeks by a vascular rejection process termed acute vascular rejection (AVR) or delayed xenograft rejection. There is increasing evidence that elicited anti-Gal IgG may cause acute vascular rejection by an as yet unidentified mechanism (4, 36, 37).

We have previously used the Gal−/− mouse for investigating anti-Gal responses to Gal+/+ cardiac xenografts; however, this model yielded anti-Gal IgG Abs that were primarily of the IgG3 subclass (14). We have now generated a series of spontaneous anti-Gal IgG-subclass switch mutants from a single anti-Gal IgG1 (GT4-31.1)-producing hybridoma. This has provided us with the opportunity to investigate the in vitro and in vivo activities of anti-Gal IgG subclasses independently of effects arising from alterations in affinity as a result of different variable region usages. Specifically, we have investigated the effect of IgG subclasses on their ability to induce HAR of xenografts. Izui and colleagues (38, 39) have used a similar approach to demonstrate that IgG switch variants had different abilities to induce autoimmune hemolytic anemia in vivo.

We previously described the ability of IgG1 and IgG3 mAbs to induce HAR in vivo in Gal−/−Rag−/− recipients (33). The current experimental model involves the use of immunocompetent Gal−/− mice as recipients of Gal+/+ rat cardiac xenografts, thus permitting the possible contribution of T and B cells to HAR. The ability of anti-Gal IgG to induce HAR was characterized following a single bolus infusion of purified mAb. We acknowledge that this model does not resemble the physiological conditions where a polyclonal natural anti-Gal IgG pre-exists before xenotransplantation or where the production of an elicited anti-Gal IgG follows more delayed kinetics. Indeed, we recently observed that repeated dosing with lower doses of anti-Gal IgG1 might not result in rejection but in graft accommodation (40). Notwithstanding these caveats, this experimental system does provide us with an elegant model to investigate the in vivo properties of anti-Gal IgG subclasses without the contribution of different affinities.

In these studies, we extend previous observations (33) with a second anti-Gal IgG1 mAb clone and with immunocompetent C57BL/6 recipients and confirm that IgG1-mediated HAR was dependent on both complement and FcγR-mediated interactions, whereas IgG3-mediated rejection was only dependent on complement activation. We also demonstrate that IgG2a and IgG2b mAbs induce HAR by the same dual mechanisms of complement activation and FcγR-mediated interactions. HAR mediated by both IgG2a and IgG2b was prevented following a blockade with the 2.4G2 mAb. This mAb was historically defined as specific for FcγRII/III; however, it has been recently been reported that 2.4G2 also blocks FcγRIV mAbs (41). In addition, Kurlander et al. (42) suggested that 2.4G2 can block FcγRI via its Fc portion. Thus, the evaluation of the role of each class of FcγR required the use of mice genetically deficient in single classes of FcγR.

An important cell type previously identified to be important in mediating IgG1-HAR in mice is the NK cell, because deletion with either anti-asialo GM1 or NK1.1 mAbs abrogated the ability of anti-Gal IgG1 mAbs to induce HAR. We also observed that NK cell depletion with an anti-asialo GM1 Ab or an anti-NK1.1 mAb prevented HAR induced by anti-Gal IgG2a and IgG2b mAbs. Because NK cells only express the activating FcγRIII but not inhibitory FcγRII, we examined whether HAR following anti-Gal IgG1, IgG2a, and IgG2b challenge was mediated by FcγRIII-expressing NK cells by using Gal−/−CD16−/− mice. As expected, FcγRIII-deficiency protected grafts from HAR mediated by anti-Gal IgG1, while the administration of Gal−/− spleen cells but not NK-depleted Gal−/− spleen cells restored HAR. These observations confirm the role of NK cells and CD16 in anti-Gal IgG1-mediated HAR. In contrast to the essential role of FcγRIII in HAR mediated by anti-Gal IgG1, FcγRIII-deficiency minimally altered the ability of anti-Gal IgG2b and IgG2a to induce HAR. These observations suggest that the effect of the 2.4G2 mAb may be due to its binding to FcγRIV or possibly FcγRI. They also prompted us to speculate that anti-NK1.1 and anti-asialo GM1 may have other unanticipated effects such as the depletion of complement or other cell types in addition to NK cells in vivo. Indeed, monocytes, neutrophils, eosinophils, and mast cells have been reported to express asialo GM1 (43). Alternatively, compensatory mechanisms may have evolved in FcγRIII-deficient mice to facilitate HAR mediated by IgG2a and IgG2b.

IgG2a is reported to bind with the highest affinity to the activating receptor FcγRI (5, 7), and both IgG2a and IgG2b bind to the activating receptor FcγRIV (6). Indeed, recent studies by Nimmerjahn et al. (6) suggest a predominant role for FcγRIV in the in vivo pathogenesis of IgG2a and IgG2b. The role of the high affinity FcγRI in mediating the pathogenic effects of IgG in vivo is somewhat controversial. Ravetch and colleagues (7) indicated that the deletion of FcγRI minimally impaired IgG activity in many model systems and reasoned that because FcγRI binds to monomeric IgG2a with the same affinity as it binds immune complexes, FcγRI is likely to be saturated with monomeric IgG2a and is likely to be unavailable for immune complex binding in vivo. Conversely, others have reported that FcγRI is critical for mediating IgG-mediated effector functions in vivo in the setting of arthritis, hypersensitivity responses, Ab-mediated tumor regression, and protection from bacterial infection, as well as in a variety of immunological and inflammatory responses (29, 44, 45, 46).

We have examined the role of the FcγRI, and observed that it plays an important role in anti-Gal IgG2a- and IgG2b-mediated HAR and an unexpectedly important role in anti-Gal IgG1-mediated HAR. The ability of the high-affinity FcγRI to bind to monomeric mouse IgG2a is well described (47) and, thus, the dependence of IgG2a-mediated HAR is not unexpected. However, FcγRI binds to immune complexes of IgG2a with low affinity and to immune complexes of IgG1 with even lower affinity (5). We therefore speculate that under conditions of HAR for rat cardiac xenografts, anti-Gal IgG1 andIgG2b form immune complexes with Gal epitopes expressed on the vascular endothelium of the cardiac xenograft in a manner that is capable of binding to FcγRI. As expected, the absence of FcγRI had no effect on IgG3-mediated, HAR. Collectively, these experiments with FcγR-deficient mice and the blocking of anti-FcγRII/III mAbs support a role for FcγR in HAR mediated by three of the four IgG subclasses.

It is well accepted that IgG2a,2b,3 subclasses are potent activators of complement, whereas mouse IgG1 is a weak activator complement in vitro or in vivo (39, 48, 49). However, studies with arthritogenic IgG mAbs in the K/BxN model indicate that combinations of IgG1 mAbs specific for glucose-6-phosphate isomerase can induce arthritis that is dependent on FcγR and the terminal activation of complement via the alternative pathway (50, 51). These data are consistent with the notion that the anti-GPI IgG1 mAbs can activate complement in vitro. We observed that anti-Gal IgG1 could lyse pig erythrocytes in vitro when mouse complement was used, although significantly less efficiently than the anti-Gal IgG2a, 2b, and 3 subclasses. Despite the modest lytic activity in vitro, we observed that HAR mediated by anti-Gal IgG1 was dependent on terminal activation, similar to that observed for the anti-Gal IgG2a, 2b, and 3 subclasses. Thus, HAR mediated by all IgG subclasses was dependent on the terminal activation of complement. Taken together, our data suggest that complement activation is necessary and sufficient for IgG3-mediated HAR and necessary, but not sufficient, for IgG1, IgG2a, and IgG2b-mediated HAR.

The synergistic interactions between FcγR-mediated interactions and complement components have been previous reported in models of autoimmunity and hypersensitivity (51, 52, 53). Indeed, it has been reported that the complement components C3a and C5a have potent biological activities, including the promotion of leukocyte chemotaxis, enhancement of vascular permeability, and granule secretion by mast cells, basophils, and phagocytes, and also stimulating the production and release of several chemokines and cytokines (52). Furthermore, it has recently been reported that C5a ligation to C5aR on macrophages causes the transcriptional up-regulation of activating FcγRIII and the down-regulation of inhibitory FcγRII (54, 55), whereas the ligation of activating FcγRs by Ag-antibody complexes induces C5a production (56). We speculate that it is less likely that such transcriptional events are at play in our model because of the rapid kinetics of HAR; nonetheless, these published studies collectively support the existence of a complex regulatory network of multiple interactions between FcγRs and C5a:C5aR that can promote inflammatory responses and the development of autoimmune disease.

Immunohistochemical analyses of the xenografts that underwent IgG2a and IgG2b- mediated HAR revealed significantly reduced complement C3 and C5 deposition in the absence of FcγRII/III-mediated interactions. These observations suggested that a positive feedback loop exists between the events induced by complement activation and FcγR. We hypothesize that immune complexes activate the early components of complement, resulting in the generation of C3a and C5a and the up-regulated expression of adhesion molecules that promote leukocyte recruitment (57). This recruitment promotes the engagement of FcγR on recruited mononuclear cells and the secretion of inflammatory mediators as well as Ab-dependent cell-mediated cytotoxicity (ADCC). Thus FcγR/cell-mediated events and the terminal activation of complement can synergistically cause acute endothelial cell injury, activation of the coagulation pathway, and ultimately HAR (illustrated in Fig. 8).

FIGURE 8.

Dual role for complement activation and FcγRs in mediating HAR by anti-Gal IgG1, IgG2a, and IgG2b mAbs. IgG immune complexes can activate the early components of complement, resulting in the generation of the biologically active C3a and C5a and the up-regulated expression of adhesion molecules that promote leukocyte recruitment. The engagement of FcγR on the recruited leukocytes promotes the secretion of inflammatory mediators as well as Ab-dependent cell-mediated cytotoxicity. These events feed back to further activate and injure endothelial cells (EC), which ultimately promote the loss of the anti-thrombotic state, platelet aggregation, thrombosis, and ultimately HAR.

FIGURE 8.

Dual role for complement activation and FcγRs in mediating HAR by anti-Gal IgG1, IgG2a, and IgG2b mAbs. IgG immune complexes can activate the early components of complement, resulting in the generation of the biologically active C3a and C5a and the up-regulated expression of adhesion molecules that promote leukocyte recruitment. The engagement of FcγR on the recruited leukocytes promotes the secretion of inflammatory mediators as well as Ab-dependent cell-mediated cytotoxicity. These events feed back to further activate and injure endothelial cells (EC), which ultimately promote the loss of the anti-thrombotic state, platelet aggregation, thrombosis, and ultimately HAR.

Close modal

In summary, our studies confirm and extend our previous observations that there are two forms of HAR: one that is dependent only on complement activation and is mediated by anti-Gal IgG3 mAbs, and a second that is dependent on complement activation and FcγR and is elicited by three subclasses of anti-Gal IgG Abs, namely IgG1, IgG2a, and IgG2b. We observed an essential role for FcγRIII in IgG1-mediated HAR and for FcγRI in IgG1-, IgG2a-, and IgG2b-mediated HAR. Thus, the process of HAR by IgG is proving to be more complex that previously defined for HAR induced by IgM, and the phenotype of the FcγR-expressing cells and the mechanism by which FcγRs facilitate HAR require further definition.

We thank Lucy Deriy, Agnes Pierwola, Jing Xu, Jamie Kim, and Matthew Smetts for their technical assistance and Dr. Ian Boussy for helpful discussion in the preparation of the manuscript.

The authors have no financial conflict of interest.

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 a grant from the National Institutes of Health (R01 AI52464) to A.S.C.

3

Abbreviations used in this paper: HAR, hyperacute rejection; CVF, cobra venom factor; Gal, Gal-α1,3Gal; vWF, von Willebrand’s factor.

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