By
From the * Transplantation Biology Research Center, Surgical Service, Massachusetts General
Hospital/Harvard Medical School, Boston, Massachusetts 02129; and BioTransplant, Inc.,
Charlestown, Massachusetts 02129
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Abstract |
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Xenotransplantation could overcome the severe shortage of allogeneic organs, a major factor
limiting organ transplantation. Unfortunately, transplantation of organs from pigs, the most
suitable potential donor species, results in hyperacute rejection in primate recipients, due to the
presence of anti-Gal1-3Gal (Gal) natural antibodies (NAbs) in their sera. We evaluated the
ability to tolerize anti-Gal NAb-producing B cells in
1,3-galactosyltransferase knockout (GalT KO) mice using bone marrow transplantation (BMT) from GalT+/+ wild-type (WT)
mice. Lasting mixed chimerism was achieved in KO mice by cotransplantation of GalT KO
and WT marrow after lethal irradiation. The levels of anti-Gal NAb in sera of mixed chimeras were reduced markedly 2 wk after BMT, and became undetectable at later time points. Immunization with Gal+/+ xenogeneic cells failed to stimulate anti-Gal antibody production in
mixed chimeras, whereas the production of non-Gal-specific antixenoantigen antibodies was
stimulated. An absence of anti-Gal-producing B cells was demonstrated by enzyme-linked immunospot assays in mixed KO+WT
KO chimeras. Thus, mixed chimerism efficiently induces anti-Gal-specific B cell tolerance in addition to T cell tolerance, providing a single
approach to overcoming both the humoral and the cellular immune barriers to discordant xenotransplantation.
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Introduction |
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Although modern immunosuppressive therapies have improved the success of clinical organ transplantation, a severe shortage of allogeneic organs currently limits the number of transplants performed (1, 2). Given the urgent need for donor organs and the problems associated with the use of nonhuman primates, interest has become focused on the potential use of nonprimates as organ donors for humans. The species generally believed to be most suitable for this purpose is the pig (2). However, xenotransplantation from evolutionarily distant species such as the pig poses formidable obstacles. One particularly imposing challenge arises from the presence of preexisting, or "natural," xenoreactive antibodies (NAbs)1 at high titers in the sera of all humans (5). These NAbs are major effectors of hyperacute rejection. Furthermore, even if NAbs are absorbed or in some way removed before xenogeneic organ transplantation, their return is associated with the phenomenon of delayed xenograft rejection/acute vascular rejection (2, 6).
Most of the NAb activity against porcine cells in human
sera is directed against the Gal1-3Gal
1-4GlcNAc-R
epitope, which is widely expressed on glycoproteins and
glycolipids of most mammalian species, including swine (9-
11). Although a number of strategies have been used to
promote successful xenotransplantation by targeting different steps in the progression of NAb-mediated rejection, none has proved entirely successful. The elimination of
anti-Gal NAb from recipients by immunoabsorption by
donor-species organ hemoperfusion (12) or immunoaffinity columns of synthetic oligosaccharides (13, 14), or by
treatment with anti-Ig antibodies (15) has been found effective in preventing or delaying hyperacute rejection (13-
15). However, the efficiency of such treatments is short-lived, as NAb levels return rapidly to their original levels
and participate in the delayed rejection of xenografts. In
view of the ability of NAb to initiate complement-independent changes in endothelium, to participate in antibody-dependent cell-mediated cytotoxicity against porcine
cells, and to play a role in delayed xenograft rejection/acute
vascular rejection, inactivation of the recipient complement
system (2, 8, 10) or approaches to reducing Gal epitope
density (16) alone would be unlikely to permit long-term xenoengraftment.
Previous studies have demonstrated that induction of a
state of mixed hematopoietic chimerism can lead to permanent tolerance of T cells to allogeneic and concordant xenogeneic antigens, with excellent immunocompetence (19).
In addition, reductions in mouse IgM NAbs capable of
binding to rat bone marrow cells (BMC) were observed in
rat mouse mixed chimeras (24), suggesting that NAb-forming B cells might also be tolerized by this approach. Recently, mice homozygous for a null
1,3-galactosyltransferase allele (GalT KO) have been generated by targeted disruption of the murine
1,3GalT gene (25). As in
humans, sera of these animals contain anti-Gal NAb, thus
providing a model in which to evaluate methods of inducing anti-Gal NAb tolerance. We have now evaluated the
possibility of achieving mixed chimerism in GalT KO mice
by transfer of GalT+/+ wild-type (WT) mouse BMC with
GalT KO mouse marrow to lethally irradiated GalT KO
mouse recipients, in order to determine the potential of mixed
chimerism to tolerize anti-Gal NAb-producing B cells.
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Materials and Methods |
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Animals.
GalT KO (GalBone Marrow Transplantation.
BMC were T cell-depleted (TCD) using anti-CD4 and anti-CD8 mAbs and rabbit complement as described (26, 27). Recipients were treated with 9.75 Gy whole body irradiation from a 137Cs source (0.97 Gy/min), followed within 4-6 h by i.v. infusion of TCD BMC.Flow Cytometric Analysis of Chimerism.
Single-cell suspensions were incubated with anti-WT mouse H-2Kb 5F1-FITC and PE-labeled anti-CD19 (for B cell chimerism), or anti-CD4-PE plus anti-CD8-PE (for T cell chimerism) (for two-color flow cytometric [FCM] analysis), or with 5F1-FITC, anti-CD19-PE, and anti-CD4-Bio plus anti-CD8-Bio (for three-color FCM analysis) mAbs (PharMingen, San Diego, CA) as described (27). Nonspecific FcELISA for Detecting Mouse NAb Reactive with Gal.
96-well microtiter ELISA plates (Corning Glass Works, Corning, NY) were coated overnight at 4°C with 100 µl of 5 µg/ml ofFCM Analysis of Anti-Gal, Anti-rabbit RBC, and Anti-pig PBMC Antibodies.
Indirect immunofluorescence staining of WT mouse cells after incubation with GalEnzyme-linked Immunospot Assay for Detecting Anti-Gal Antibody-producing Cells.
Enzyme-linked immunospot (ELISPOT) assays were performed as described (Xu, Y. and A.D. Thall, manuscript in preparation). In brief, cell suspensions were serially diluted (four fivefold dilutions, beginning with 8 × 105 cells/ well) and placed in triplicate wells in MultiScreen-HA plates (Millipore Corp., Bedford, MA) precoated withStatistical Analysis.
Student's t test for comparison of means was used for statistical analysis. A P value <0.05 was considered to be significant. ![]() |
Results |
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To determine whether anti-Gal NAb-producing B cells in GalT KO mice would be rendered tolerant in
the presence of WT hematopoietic cells, experiments were
conducted in which GalT KO mice were lethally irradiated
(9.75 Gy) and reconstituted with 26 × 106 TCD WT BMC
alone, or with 6.5 × 106 TCD GalT KO plus 26 × 106
TCD WT BMC. GalT KO mice injected with 6.5 × 106
TCD GalT KO BMC, WT mice injected with 26 × 106 TCD
WT BMC, and uninjected GalT KO and WT mice served
as control groups. Mixed chimerism was detected in GalT
KO recipients of a mixture of WT and KO BMC at all
time points studied. The proportion of WT donor cells increased in these mice between 2 and 8 wk after bone marrow transplantation (BMT), and was subsequently maintained at a steady state. As is shown for a representative
chimera in Fig. 1 A and summarized in Fig. 1 B, mixed
chimerism was observed among both B and T cells in the
PBLs of these animals. Mixed chimerism, including B and
T as well as myeloid lineages, was also detected in the bone
marrow, spleen, and peritoneal cavity at the time of killing
19 wk after BMT (Fig. 1 C, and data not shown). As expected, at each time point, T and B cells in PBLs of syngeneic BMT recipients of KO marrow alone (KO KO) or
WT marrow alone (WT
WT) were fully of KO and WT
origin, respectively. B cells in GalT KO recipients of WT
BMC alone (WT
KO) were almost fully WT in origin
(Fig. 1).
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To determine whether or not anti-Gal NAb persisted in the serum after lethal irradiation, GalT KO mice were lethally irradiated, and their serum levels of anti-Gal NAb were measured at various times. As is shown in Fig. 2, no decline in the serum levels of anti-Gal NAb was observed in irradiated GalT KO mice, even in animals not receiving BMT, by 14 d after irradiation. Since all lethally irradiated mice not receiving BMT appeared sick and began to succumb by 10 d after irradiation, no data were obtained beyond 2 wk after irradiation. The absence of a significant difference in the levels of anti-Gal NAb between lethally irradiated GalT KO mice that did or did not receive reconstituting KO BMC (Fig. 2) suggests that newly developed B cells derived from BMT inocula were not a major source of NAb at early time points up to 14 d after irradiation. The relatively constant levels of anti-Gal NAb in sera of these mice suggest either that the anti-Gal NAbs are long-lived Igs, or that the irradiation dose of 9.75 Gy does not eradicate all host anti-Gal NAb-producing B cells.
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Levels of anti-Gal NAb were determined by Gal-specific ELISA assay and FCM analysis. Similar results were
obtained with both assays. As is shown in Fig. 3, sera of
GalT KO mice receiving KO BMC contained levels of
anti-Gal NAb that were similar to those of untreated KO
mice at all time points studied. In contrast, levels of anti-Gal NAb in sera of WT+KO KO recipients were reduced significantly by 2 wk, and declined further to become undetectable by 4 wk after BMT (Fig. 3 B). A similar
reduction in serum levels of anti-Gal NAb was also observed in lethally irradiated WT
KO recipients. As expected, sera of WT
WT recipients and of normal WT
control mice did not contain anti-Gal NAb (Fig. 3). Similar results were observed in two repeat experiments in which
anti-Gal NAb became undetectable in sera of 12 of 12 lethally irradiated GalT KO recipients of mixed WT and KO
BMC, all of which showed mixed chimerism, with proportions of PBL B cells that were WT ranging from 1 to
50% (data not shown).
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The reduced NAb levels detected in mixed chimeras
could be due to downregulation of anti-Gal NAb production by GalT KO B cells in the presence of WT hematopoietic cells, or might reflect adsorption of NAb onto WT
hematopoietic cells. FCM analyses did not provide any evidence for absorption of NAb on WT hematopoietic cells
in chimeras (data not shown). Furthermore, we looked directly for the presence of anti-Gal NAb-producing B cells
in these animals using an ELISPOT assay. To increase the
sensitivity of the assay and to determine whether or not B
cells of mixed chimeras were tolerant to the Gal epitope on
xenogeneic cells, animals were immunized by intraperitoneal injection of 109 rabbit RBCs, which express large
amounts of Gal, 19 wk after BMT. Spleen cells, BMC, and
peritoneal cavity cells were analyzed 8 d later for the capacity to produce anti-Gal antibodies as measured by ELISPOT
assay. B cells producing anti-Gal antibodies (both IgM and
IgG) were undetectable in all three tissues of mixed chimeras, whereas large numbers of these cells were detected in
normal GalT KO mice and GalT KO recipients of KO
BMC. Results in mixed chimeras resembled those from
WT KO recipients and, most importantly, those from
normal WT mice (Fig. 4) in which anti-Gal-forming B cells were not detected. These results show definitively that mixed WT+KO
KO chimeras are fully tolerant of the
Gal epitope at the B cell level, and rule out the possibility
that the reduced anti-Gal NAb levels in sera of mixed or
fully WT
KO chimeras were caused by adsorption of the
NAb on WT cells.
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To determine whether or not mixed
chimeras were capable of producing antibodies against antigens other than Gal, sera from rabbit RBC-immunized
mixed chimeras were tested for the development of anti-rabbit antibodies. As is shown in Fig. 5 A, sera of KO KO
recipients but not of WT mice or chimeras contained both
anti-Gal and anti-rabbit RBC antibodies after immunization.
However, rabbit RBC-immunized WT mice, WT
KO
chimeras, and WT+KO
KO chimeras showed an increase
in the level of anti-rabbit RBC serum antibodies, but not
with anti-Gal specificity (Fig. 5 A).
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Since expression of Gal on porcine cells is a major barrier
to xenotransplantation from pigs to humans, we evaluated
the ability of mixed chimeras to remain tolerant to Gal after
immunization with pig cells. Normal GalT KO mice,
mixed KO+WT KO chimeras, and control KO
KO
and WT
KO BMT recipients were immunized three times with 106 pig PBMCs at 15, 16, and 22 wk after
BMT. Serum levels of anti-pig and anti-Gal antibodies
were determined 3 wk after the last injection by FCM and
ELISA, respectively. Again, anti-Gal IgM were detected only in sera of normal GalT KO mice and KO
KO recipients but not in sera of mixed or fully WT chimeras. In
contrast, increased serum levels of anti-pig IgM were observed in all pig PBMC-sensitized mice (Fig. 5, B and C).
An absence of functional anti-Gal-forming B cells in these
mixed chimeras after immunization with pig PBMCs was
further demonstrated by ELISPOT assays (data not shown).
These results confirm that B cells recovering in mixed
WT+KO
KO chimeras are functional and specifically
tolerant to the Gal epitope.
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Discussion |
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While the broad species distribution of the -galactosyl
carbohydrate residue (Gal) has previously limited the analysis of anti-Gal NAb to primate species, the recent development of GalT KO mice, which do not express Gal, now
permits evaluation of anti-Gal NAb in a small animal model
system. We have used GalT KO mice to explore the possibility that mixed chimerism could induce specific tolerance
of anti-Gal-producing B cells. We have demonstrated recently that xenoreactive anti-pig NAb can be rapidly restored in C.B-17 scid mice by transfer of immunocompetent adult mouse BMC, indicating that marrow-derived B
cells are efficient producers of IgM NAb (28a). Therefore,
in this study, we reconstituted lethally irradiated GalT KO
mice with GalT KO mouse BMC to restore the potential
to produce anti-Gal NAb, and to evaluate the potential of
cotransplanted WT marrow to tolerize anti-Gal-producing
B cells.
The results of our studies demonstrate that successful induction of mixed bone marrow chimerism leads to tolerance of anti-Gal NAb-producing B cells. This conclusion was supported by the results of ELISPOT assays, which demonstrated definitively the absence of functional anti-Gal-producing B cells in mixed chimeras. Furthermore, B cells in these animals are capable of producing Ig with specificities other than anti-Gal, indicating that specific tolerance of anti-Gal-producing B cells was achieved by the induction of mixed chimerism.
Our ELISPOT data are consistent with the possibility
that tolerance of anti-Gal-producing B cells was induced
by either clonal deletion or anergy, or by both mechanisms. Experiments using transgenic mice have shown that
immature self-reactive B cells can be eliminated by apoptosis (clonal deletion) and/or alteration of receptor antigenic specificity (receptor editing) via signals induced through
surface Ig cross-linking (29). Since the Gal epitope is
recognized as a self antigen in mixed GalT WT+KO KO
chimeras, the above mechanisms of tolerance induction of
self-reactive B cells would explain the observed tolerance
among NAb-producing B cells resulting from induction of
mixed chimerism. These studies of the important Gal specificity provide the first demonstration that BMT can induce B cell tolerance among a polyclonal population of nontransgenic NAb-producing B cells with a known specificity.
Although the above mechanisms of B cell tolerance appear to depend on a signal induction cascade applicable to
immature but not mature B cells, cell surface-associated
antigens are also capable of inducing tolerance among peripheral mature B cells (29, 35). Experiments using
transgenic mice have shown that cross-linking of cell surface IgM is able to induce mature B cell tolerance via apoptotic cell death (deletion) (30, 37). In the present study,
because BMT recipients were lethally irradiated before
BMT and only a limited number of mature Gal/
B cells
was included in the BMT inoculum, the majority of GalT KO B cells in tolerized mixed chimeras developed in the
presence of WT hematopoietic cells. However, the persistence of anti-Gal IgM NAb in nonreconstituted, irradiated
GalT KO mice (Fig. 2) suggests that anti-Gal NAb-producing B cells might be radioresistant. Since IgM has an average half-life of only 2 d in the serum of adult mice (38),
the constant level of anti-Gal NAb in these mice over a 2-wk
period likely reflects the ongoing production of these NAb
by radioresistant B cells. The reduction in anti-Gal NAb
levels observed as early as 2 wk after BMT in recipients of WT BMC is consistent with the possibility that preexisting
anti-Gal NAb-forming B cells were also tolerized in these
mice. To address the possibility that mixed chimerism can
lead to tolerance of preexisting mature B cells, mixed Gal
chimerism is now being induced in mice conditioned with
a nonmyeloablative conditioning regimen.
These studies demonstrate that mixed chimerism has the potential to induce specific tolerance of anti-Gal NAb- producing B cells, in addition to the T cell tolerance to xenoantigens demonstrated previously (21, 39). To our knowledge, mixed chimerism is the first approach shown to achieve efficient and permanent inhibition of polyclonal antidonor NAb production. These findings suggest that this approach may ultimately contribute to the successful use of discordant xenogeneic organs in clinical transplantation. The potential of this strategy to induce both B and T cell tolerance, and thus to permit solid organ xenograft acceptance in a pig-to-primate species combination, is currently under investigation.
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Footnotes |
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Address correspondence to Megan Sykes, Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital, MGH East, Bldg. 149-5102, 13th St., Boston, MA 02129. Phone: 617-726-4070; Fax: 617-724-9892; E-mail: sykes{at}helix.mgh.harvard.edu
Received for publication 15 January 1998.
1 Abbreviations used in this paper: BMC, bone marrow cells; BMT, bone marrow transplantation; ELISPOT, enzyme-linked immunospot; FCM, flow cytometric; Gal, GalThe authors thank Drs. David D.K.C. Cooper, Julia Greenstein, and Henry Winn for helpful review of the manuscript, and Dr. David H. Sachs for his advice and encouragement. We also thank Ms. Guiling Zhao for outstanding animal care, and Ms. Diane Plemenos for expert assistance with the manuscript.
This work was supported by a sponsored research agreement between Massachusetts General Hospital and BioTransplant, Inc., and by National Heart, Lung, and Blood Institute grant R01 HL-49915. Dr. M. Sykes is a consultant to BioTransplant, Inc.
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