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*
Division of Pediatric Oncology, Dana-Farber Cancer Institute, and Division of Thoracic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115;
Department of Surgery, Univeristy of Pittsburgh Health System, Pittsburgh, PA 15261; and
Institute for Cellular Therapeutics, University of Louisville, Louisville, KY 40202
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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A) bone marrow chimerism has been
demonstrated to confer donor-specific tolerance in nonsensitized
recipients, but has not been evaluated in the setting of
allosensitization. The current study documents that despite significant
anti-donor sensitization, mixed allogeneic engraftment is possible
and provides a marked advantage over fully allogeneic (BA) models.
Moreover, the acceptance of donor skin grafts and loss of circulating
anti-donor Abs suggest that allosensitization can be abrogated with
the induction of stable mixed allogeneic
chimerism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mixed allogeneic chimerism, induced by the reconstitution of lethally irradiated animals with a mixture of host- and donor-type BM, has been shown to confer permanent donor-specific transplantation tolerance for subsequent skin, heart, and islet grafts in nonsensitized recipients (7, 8, 9, 10). Due to the high incidence of allosensitization in the transplant population, the clinical application of mixed allogeneic chimerism would be significantly limited if chimerism and tolerance were precluded by the presence of preformed donor-reactive Abs or other anti-donor memory responses. In the current study, we have investigated the impact of allosensitization on BM engraftment and the induction of donor-specific tolerance. Although fully allogeneic reconstitution resulted in a high incidence of engraftment failure and death, stable alloengraftment and the induction of donor-specific transplantation tolerance were achieved in sensitized recipients utilizing a model of mixed allogeneic BM chimerism. Survival, alloengraftment, and the induction of tolerance were significantly improved when large numbers (80 x 106) of allogeneic BM cells were administered several months after initial sensitization. Of even greater importance was the abrogation of allosensitization, demonstrated by the permanent loss of circulating anti-donor Abs and the induction of donor-specific tolerance following mixed allogeneic BMT.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male, 6- to 8-wk-old C57BL/10SnJ (B10), B10.BR/SgSnJ (B10.BR), B10.D2, and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed in a specific pathogen-free facility in the University of Pittsburgh Cancer Institute (Biomedical Science Tower, Pittsburgh, PA).
Skin grafting
Skin grafting was performed by a modification of the method of Billingham and Medawar (11). Full thickness skin grafts were harvested from the tails of B10 (H-2b), B10.BR (H-2k), BALB/c, or B10.D2 (H-2d). Full thickness graft beds were surgically prepared in the lateral thoracic wall of anesthetized mice, taking care to preserve the panniculus carnosus. Grafts were covered by a double layer of Vaseline gauze and a plaster cast to prevent shearing. A single donor graft was placed at the time of allosensitization. When assessing donor-specific tolerance, skin grafts from syngeneic, allogeneic donor, and third-party animals were placed on each recipient, leaving a 3-mm skin bridge separating each graft. Casts were removed on the seventh day, and grafts were scored daily for percent rejection. Rejection was considered complete when no residual viable graft could be seen. Graft survivals were calculated by the life-table method, and the median survival time (MST) was derived from the time point at which 50% of grafts were surviving (12).
Ab-dependent complement-mediated 51Cr microcytotoxicity assay
Serum was collected from recipient mice (B10 or B10.BR) before the placement of the sensitizing skin allograft (B10.BR/B10.D2 or B10, respectively) and weekly thereafter, with the exception of a 4-wk period immediately following BMT. Collected sera was stored at -20°C until needed for analysis, at which time it was decomplemented at 50°C for 30 min, serially diluted from 1/2 to 1/1024, and placed in 96-well round-bottom plates. A total of 2.5 x 104 51Cr-labeled donor target splenocytes was added to each well, incubated at 37°C for 30 min, and washed at 1000 rpm. Following a second 30-min incubation at 37°C in the presence of rabbit complement (1/8), supernatants were collected and counted on a gamma counter (Titertek system, Skatron Instruments, Lier, Norway; DP5000, Beckman, Fullerton, CA). Zero and 100% cytotoxicity was determined by spontaneous (media alone) and maximum (Triton X) release of 51Cr, respectively. The percentage of cytotoxicity was determined by the formula: % cytotoxicity = 100% x [(experimental - spontaneous)51Cr/(maximum - spontaneous)51Cr].
Spontaneous 51Cr release for controls with media complement, or Ab alone, was less than 20% of total release. An H-2-specific antiserum (IgM) was used as a positive control, with donor-specific lysis resulting in 65–100% maximum cytotoxicity of splenocytes in this assay.
Statistical analysis of cytotoxic activity between groups of animals was performed utilizing unpaired two-tailed t test analysis.
Chimera preparation
Fully and mixed allogeneic chimeras were prepared by a
modification of the methods previously described (7, 8).
Briefly, inbred B10 or B10.BR male recipients were lethally irradiated
with 950 cGy from a 137Cs source (Nordion,
Ontario, Canada). Using sterile technique, BM was flushed from the
femurs and tibias of donor (B10.BR and B10.D2 or B10, respectively)
mice with Medium 199 (Life Technologies, Grand Island, NY) containing
50 µl/ml of gentamicin, mechanically resuspended, filtered through
sterile nylon mesh, and washed. BM was T cell depleted, when indicated,
by treatment with a 1/60 dilution of rabbit anti-mouse brain (RAMB)
antisera (108 cells/ml) at 4°C for 30 min,
followed by a 1/8 dilution of rabbit complement (Life Technologies) at
37°C for 30 min. Cells were then washed twice and resuspended for
injection. Recipient animals were reconstituted 4–6 h after lethal
irradiation via injection of cells into the lateral tail vein. Fully
allogeneic reconstitution (B10.D2B10; B10.BRB10; B10B10.BR)
was performed using doses of untreated allogeneic marrow ranging from
15 x 106 to 80 x
106 cells/animal. Recipients of mixed allogeneic
marrow received 5 x 106 T cell-depleted
syngeneic BM cells and doses of untreated or T cell-depleted allogeneic
marrow of 5 x 106 to 80 x 106
cells, as indicated (B10 + B10.D2B10; B10 + B10.BRB10; B10.BR +
B10B10.BR). Radiation controls were performed simultaneously to
confirm adequacy of the lethal radiation dose.
Characterization of chimeras by flow cytometry
Recipients were characterized for engraftment using flow cytometry (FACSort; Becton Dickinson, Mountain View, CA) to determine the percentage of PBL bearing H-2b (B10), H-2k (B10.BR), and H-2d (B10.D2) surface Ags, as previously described (13). Briefly, peripheral blood was collected into heparinized plastic serum vials containing 200 µl of Medium 199 and separated over 3 ml of lymphocyte separation medium (LSM; Organon Teknik, Durham, NC) at 37°C at 400 x g for 25 min. The buffy coat layer was aspirated from the saline-LSM interface and washed, and lymphocytes were stained with the appropriate mAb for 45 min at 4°C. Anti-H-2b FITC (PharMingen, San Diego, CA), anti-H-2k FITC (PharMingen), and anti-H-2d FITC (PharMingen) mAb were utilized for anti-class I staining of donor and recipient cells.
Mixed lymphocyte reactions
MLR were performed as previously described (14, 15). Briefly, murine splenocytes were ACK lysed (ammonium chloride potassium carbonate lysing buffer), washed, and reconstituted in DMEM (Life Technologies) supplemented with 0.75% normal mouse serum, 0.09 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.05 mM 2-ME, and 1 mM NG-mono-methyl L-arginine (15). A total of 4 x 105 responders/well was cocultured 1:1 with irradiated stimulators (20 Gy) in a total of 200 µl of media and incubated at 37°C in 5% CO2 for a total of 4 days. Cultures were pulsed on the third day with 1 µCi [3H]thymidine (New England Nuclear, Boston, MA) and harvested on the fourth day with an automated harvester (PHD Cell Harvester Technology, Cambridge, MA).
Cell-mediated lympholysis (CML)
CML assays were performed using a modification of techniques described elsewhere (14, 16, 17). RPMI 1640 medium (Life Technologies) was supplemented as above, except that 10% FCS (Life Technologies) was used in place of normal mouse serum. A total of 4 x 106 responders was cocultured with 4 x 105 irradiated splenocyte stimulators (20 Gy) in 2.5 ml of medium at 37°C for 5 days. Mouse target blasts were stimulated with Con A (Miles Yeda Research Products, Rehovot, Israel) for 2–3 days. After 5 days, responders were harvested, counted, and resuspended at appropriate E:T ratios with 1 x 104 51Cr-labeled, Con A blasts. After 4.5 h, supernatants were harvested with the Titertek supernatant harvesting system, and specific lysis was calculated as follows: specific lysis = (experimental release - spontaneous release)/(maximal HCl release - machine background) x 100. Spontaneous release was <25% of maximum release, unless otherwise indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Donor-specific allosensitization was induced by placement of
B10.BR or B10.D2 skin grafts on B10 recipients or B10 grafts on B10.BR
recipients. Donor skin grafts were completely rejected by naive
recipients with a MST of 12.4 days. Recipient serum was obtained before
placement of the skin allograft and weekly during the rejection period,
until allosensitization was documented for each animal. Serum was
tested for the presence of donor-specific cytotoxic Abs against donor
splenocytes, using a 51Cr microcytotoxicity
assay. Anti-donor cytotoxic Abs were not present before (Fig. 1
A, week 0) or at the onset of
graft rejection (week 2). The rapid rise in anti-donor cytotoxic
activity following allograft rejection (week 3) could be completely
abrogated in vivo by the i.p. administration of cyclophosphamide.
Cytotoxic Abs were donor specific, as evidenced by the absence of
cytotoxicity toward third-party splenocytes, but not tissue specific,
with marked cytotoxic activity against donor alloantigens on cells of
either BM or splenic origin (Fig. 1B).
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The impact of allosensitization on conventional fully allogeneic
BMT was demonstrated with the transplantation of increasing doses of
allogeneic marrow in sensitized and nonsensitized recipients
(B10.D2B10, B10.BRB10, and B10B10.BR). Twelve weeks following
sensitization, recipients were lethally irradiated and reconstituted
with 15–80 x 106 allogeneic BM cells.
Because the inoculum consists of only donor BM cells, long-term
survival of fully allogeneic chimeras is indicative of donor
engraftment. Survival of sensitized recipients increased in proportion
to donor cell number, but remained markedly inferior to nonsensitized
controls (Table I). Transplantation of
30 x 106 donor cells did not rescue
recipients from aplasia, 40 x 106 cells
resulted in a single 30-day survivor (9%), and 80 x
106 donor cells resulted in engraftment in only
25% of sensitized recipients.
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B10; B10 + B10.BRB10; B10.BR +
B10B10.BR). Survival of mixed allogeneic chimeras was 100%,
irrespective of the number of donor cells in the BM inoculum. Donor
engraftment was assessed by the presence of donor lymphocytes in the
peripheral blood (PBL) 1 mo following reconstitution. As evident in
fully allogeneic reconstitution, alloengraftment in mixed chimeras was
decreased in sensitized recipients and directly correlated with the
size of the donor BM inoculum (Fig. 2).
Donor engraftment was only achieved in those sensitized recipients
reconstituted with BM inocula containing 60 x
106 or 80 x 106
allogeneic BM cells. Transplantation of mixed inocula (syngeneic plus
allogeneic BM) significantly increased the incidence of donor
engraftment with 73% (n = 8/11) of sensitized
recipients reconstituted with 80 x 106
allogeneic BM cells exhibiting donor chimerism, as compared with 25%
following fully allogeneic reconstitution. Once engraftment
occurred, however, the level of donor chimerism was high, with mean
chimerism 4–6 wk following mixed reconstitution with 60 x
106 or 80 x 106
allogeneic BM cells being 83.3 ± 16.7% and 98.8 ± 0.6%,
respectively.
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Alloengraftment is dependent on initial recipient Ab response
The importance of the recipient immune status at the time of BMT
on engraftment failure was demonstrated by assessing alloengraftment
when recipient allosensitization was prevented or enhanced.
Cyclophosphamide is a potent inhibitor of B cell-Ab responses, and
inhibits conventional cellular immune responses at higher doses
(22). Administration of this agent before donor Ag
exposure thereby prevents the recipient from mounting humoral or
cellular anti-donor immune responses. Despite the prior placement
of a donor skin graft, alloengraftment readily occurred when
allosensitization was prevented in vivo by the administration of
cyclophosphamide (Table II). Conversely,
alloengraftment was completely prevented when recipients, sensitized 12
wk earlier, were given a single i.v. bolus of 60 x
106 donor splenocytes as a second donor Ag
challenge, 2 days before mixed allogeneic BMT.
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). This was
most notably evident in fully allogeneic chimeras, as all recipients
succumbed to aplasia. The incidence of donor chimerism following mixed
reconstitution was decreased to 33%, but remained superior to fully
allogeneic BMT. The decreased incidence of donor engraftment (33 vs
73%) evident in animals transplanted 5–7 wk following
allosensitization corresponds to a higher Ab titer at the time of BMT
(88 ± 8% maximum cytotoxicity, n = 5) as
compared with animals presensitized 12 wk before BMT (42 ± 6%
maximum cytotoxicity, n = 18, p =
0.0001).
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Mixed allogeneic BMT was performed in 13 recipients, 5–7 wk
following sensitization. Of these, 54% exhibited evidence of donor
engraftment at 1 mo post-BMT. The correlation between failure of
engraftment and sensitization suggested that recipients who failed to
engraft must have exhibited high anti-donor Ab titers at the time
of BMT. To test this hypothesis, the anti-donor cytotoxic activity
just before mixed allogeneic BMT was compared between recipients with
and without donor engraftment (Fig. 3).
Surprisingly, anti-donor cytotoxic activity was not significantly
different between these two populations at 64.3 ± 12.4% and
69.9 ± 11.2%, respectively (unpaired t test;
p = 0.625).
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).
In the first group (n = 4), recipients exhibited high
levels (>90%) of permanent donor chimerism that persisted throughout
the 4-mo follow-up. These chimeras were characterized by a slow rise in
anti-donor cytotoxic activity, reaching peak levels at the time of
BMT (45.8 ± 10.7%). The second group exhibited low levels of
donor chimerism (<5%), which disappeared by the fourth month, and
thus donor marrow failed to engraft in these recipients. Recipients
with transient or failed alloengraftment (n = 9) were
characterized by a rapid generation of high titer cytotoxic activity
that began to slowly decline at the time of BMT (peak cytotoxicity
= 88 ± 7.3%). The peak anti-donor cytotoxicity present in
recipients with long-term donor engraftment never reached the high
degree of cytotoxicity exhibited early in the course of sensitization
by recipients that failed to engraft (unpaired t test;
p = 0.031). Thus, despite similar anti-donor
cytotoxic activity at the time of BMT (5–7 wk), the incidence of donor
chimerism was quite different. These data indicate that anti-donor
cytotoxic activity at the time of BMT is not the sole determinant of
engraftment failure, but that alloengraftment might also be influenced
by recipient immunologic memory mediated by cellular and/or humoral
mechanisms.
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Cellular immune responses were assessed in vitro and in vivo after
mixed allogeneic reconstitution to determine whether recipients
remained immunocompetent and if donor-specific tolerance had been
achieved. In vitro proliferative MLR and cytotoxic CML assays revealed
that lymphocytes from previously sensitized mixed allogeneic chimeras
were functionally tolerant to both donor (B10) and host-strain (B10.BR)
alloantigens (Fig. 5, A and
B). Responses against third-party (BALB/c) alloantigens
remained intact.
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). MHC-disparate
third-party grafts, placed concurrently to confirm immunocompetence in
vivo, were rejected (MST = 10 days). In contrast, sensitized
recipients that failed to develop stable allogeneic chimerism promptly
rejected both donor and third-party allografts (Fig. 6).
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If immunologic memory against donor Ags was truly altered following mixed reconstitution, one would expect circulating anti-donor Abs to be absent. As expected, high levels of donor-specific cytotoxicity persisted in recipients without long-term donor chimerism, 14 wk following BMT (73.5 ± 11.7%; n = 9). In contrast, recipients with stable chimerism had lost anti-donor cytotoxic activity (9.9 ± 7.4%; unpaired t test, p = 0.006). The absence of anti-donor Abs was maintained throughout the 4-mo follow-up period, providing evidence that immunologic memory responses can be altered as a result of successful mixed allogeneic reconstitution.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mouse) has been achieved in the presence
of natural anti-rat BM Abs (7, 10, 24, 25), the impact
of high titer alloantibodies on mixed alloengraftment and tolerance had
not been previously investigated. Due to the high incidence of
allosensitization in the transplant population, the clinical
application of mixed chimerism would be significantly limited if
alloengraftment and/or tolerance were precluded by the presence of
preformed Abs or immunologic memory directed against the donor.
Therefore, we were most interested in determining whether mixed
chimerism could overcome the allosensitization barrier by either
reducing or eliminating the generation of donor-specific Abs and by
abrogating the cellular alloresponse that precludes BM engraftment and
the induction of tolerance. The current report examines both donor and
recipient factors preventing alloengraftment in sensitized recipients.
It demonstrates that, unlike fully allogeneic models, mixed allogeneic
reconstitution can occur in the presence of circulating anti-donor
Abs, resulting in the establishment of allogeneic chimerism,
donor-specific transplantation tolerance, and the abrogation of the
anti-donor Ab response. Following rejection of skin allografts, anti-donor Abs prevented fully allogeneic reconstitution at all doses of donor BM less than 80 x 106. Even then, engraftment failure resulted in the death of 75% of all recipient animals. Several clinical and experimental studies of fully allogeneic BMT, including the initial reports by Barnes, Loutit, and Garver, have documented similarly high rates of engraftment failure in the presence of documented allosensitization (4, 26, 27, 28, 29, 30). Based on these findings, three explanations for engraftment failure in sensitized recipients have been proposed: 1) injected marrow is immediately targeted by circulating anti-donor Abs; 2) marrow is destroyed by a cellular immune response from sensitized radioresistant cells; or 3) both responses are involved. Demonstration that passive transfer of donor-specific antisera to nonsensitized recipients resulted in alloengraftment failure appeared to establish that anti-donor Abs alone were responsible (26, 28). Although subliminal levels of Ab were postulated to allow some donor marrow to survive, the contribution of a recipient cellular immune response to alloengraftment failure could not be assessed in these earlier models because passively transferred serum is short-lived and fails to invoke a memory response, and the mortality following BMT in actively immunized recipients was 100% (26, 31).
Death following fully allogeneic reconstitution is either the result of immediate rejection of the donor marrow, as suggested by Loutit et al. (26), or due to delayed or transient alloengraftment, whereby the recipient dies of aplasia despite the initial presence of viable donor marrow. In contrast, mixed allogeneic reconstitution provides hematologic support in the form of syngeneic BM that is neither delayed nor decreased by donor-specific Ab, thus assuring recipient survival and providing sufficient time for viable donor BM to engraft. This approach decreased engraftment failure 3-fold compared with fully allogeneic BMT. Evidence of donor chimerism at doses of 60 x 106 allogeneic cells suggests that some donor BM escapes the initial anti-donor cytotoxic response and remains viable in sensitized recipients. Although supported in a mixed allogeneic environment, surviving stem cells are insufficient to rescue fully allogeneic recipients. In addition to limited numbers of viable allogeneic stem cells, TCD with RAMB antiserum has been demonstrated to concurrently remove facilitating marrow subpopulations (e.g., T cell subsets or facilitating cells), resulting in increased engraftment failure even in nonsensitized recipients (32). Facilitation of allogeneic stem cell engraftment appears to be equally important in sensitized recipients, as reconstitution with RAMB-treated donor BM decreased the incidence of donor chimerism by nearly half. The results of these studies demonstrate that by supporting recipient survival and permitting the delayed engraftment of allogeneic BM not immediately removed by circulating anti-donor Abs, mixed reconstitution provides the means to actively study alloengraftment in the sensitized recipient.
Although mixed allogeneic reconstitution with large doses of donor marrow resulted in alloengraftment in the majority of sensitized recipients, 25% either failed to initially exhibit donor chimerism or lost chimerism over the next few months. These failures highlight the importance of both: 1) the titer of circulating anti-donor cytotoxic Ab at BMT, and 2) the induction of an anti-donor memory response.
The incidence of donor chimerism was dramatically affected by differences in anti-donor cytotoxicity at the time of allogeneic BMT. Alloengraftment readily occurred when the initial anti-donor response was prevented with the administration of cyclophosphamide, but was significantly decreased in situations of high anti-donor cytotoxicity, such as with a shortened transplant interval or second donor Ag challenge. However, anti-donor cytotoxic activity at the time of BMT was not the sole determinant of engraftment failure, demonstrating little difference between those recipients that went on to engraft with donor marrow and those that did not. The original claims that engraftment failure resulted from the presence of alloantibody alone were based on models in which recipient memory responses could not be assessed in situ (26, 31). Memory, or secondary responses, requires a long-term Ag-specific memory stem cell population to give rise to shorter-lived progeny each time Ag is encountered (33). Several studies have shown that the magnitude of a memory response is a function of the number of immune cells recruited during the primary response (34, 35, 36). The importance of this fact is illustrated in the current study by the higher and earlier peak in anti-donor cytotoxic activity that is evident in sensitized recipients that subsequently fail to engraft. Thus, despite similar Ab titers at the time of BMT, recipients with the greatest premium of Ab activity at the initial response, and thus greater Ab production during a secondary memory response, exhibit increased rates of engraftment failure.
Immunologic memory decays in a biphasic fashion (34, 35, 36).
The initial decline over the first 40 days is rapid, reflecting the
loss of Ag-specific progeny, or Ab-producing cells. The later phase is
akin to the senescence of unstimulated memory cells and has a
t1/2 of 
100–200 days
(34, 35, 36, 37). The marked difference in successful
alloengraftment between recipients transplanted at 5 or 
12 wk
following sensitization parallels these two phases of immunologic
memory. Due to the presence of large numbers of activated memory and
Ab-forming cells, peak Ab titers are significantly greater if Ag
restimulation occurs during the early phase (34). This
explanation may account for the increase in engraftment failure noted
in recipients transplanted early after sensitization, despite the fact
that circulating Ab titers were similar in recipients transplanted
later. When restimulation occurs during the secondary phase, as when
BMT is delayed, the secondary Ab response is decreased because
Ab-forming cells must first be derived from the remaining memory cells
before producing Ab. The delay in BMT did allow the peripheral Ab
response to decay, but it did not disappear completely and immunologic
memory was not lost before BMT. This was evident in the poor survival
following delayed fully allogeneic reconstitution and the absence of
alloengraftment following a second donor Ag challenge given immediately
before BMT. Therefore, although high donor cell numbers and decaying Ab
titers may permit the initial engraftment of viable donor marrow to
occur in the sensitized host, under the guise of syngeneic hematologic
support, the induction of multilineage chimerism and donor-specific
tolerance must re-educate the recipient immunologic memory to promote
the maintenance of long-term alloengraftment.
Stable donor chimerism following mixed allogeneic reconstitution was uniquely characterized by the loss of anti-donor Abs and the induction of donor-specific transplantation tolerance assessed in vivo and in vitro. The resurgence of anti-donor cytotoxic activity in lethally irradiated recipients that failed to engraft with donor marrow lends support to the hypothesis that memory must be maintained within a radioresistant recipient population. The follicular dendritic cell has been postulated to be the in vivo source of antigenic stimulation necessary for the maintenance of memory, and interestingly, also the induction of tolerance (38, 39, 40, 41). Positive and negative selection pathways, documented to be functional following mixed reconstitution, may prove to be critical to the success of mixed allogeneic BMT in sensitized recipients (8, 42, 43, 44).
In the current study, mixed allogeneic reconstitution achieved alloengraftment even early after allosensitization in a significant number of recipients. This feat was not possible, despite lethal conditioning, with fully allogeneic BMT. Although it is possible that mixed chimerism may merely provide syngeneic support for subsequent allogeneic engraftment in the sensitized recipient rather than a true reversal of the sensitized state, it is important to recognize that some of the sensitized recipients, despite the presence of syngeneic BMT and transient alloengraftment, failed to exhibit long-term engraftment of the allogeneic BM component, donor-specific tolerance, or loss of circulating anti-donor Abs. This observation suggests that the mere presence of syngeneic support is not in and of itself sufficient to permit allogeneic engraftment in a sensitized recipient. It also supports the hypothesis that the sensitized state must be abrogated to permit a stable state of alloengraftment. Although the conditioning regimen itself, and not the state of mixed chimerism per se, may be hypothesized to blunt or eliminate the memory response, it must be noted that only those animals that achieve mixed allogeneic chimerism concurrently attain a state of donor-specific tolerance. Recipients that undergo an identical conditioning and transplant regimen, but fail to demonstrate mixed allogeneic chimerism, maintain an alloresponse and reject donor skin grafts. Unlike earlier conclusions, based on models of fully allogeneic reconstitution, these results suggest that the ability to achieve donor BM engraftment in a sensitized host is determined by both the circulating Ab titer at BMT and the status of the recipient immunologic memory response. We anticipate that success may be further enhanced in those recipients with extremely vigorous anti-donor responses by combining the therapies of immunoabsorption, immunosuppression, and mixed reconstitution. It is our hope that the clinical application of mixed allogeneic chimerism will eventually result in a drug-free state of donor-specific transplantation tolerance and recipient immunocompetence, that will extend the application of BM and solid organ transplantation to include sensitized recipients currently denied this potentially life-saving therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yolonda L. Colson, Division of Pediatric Oncology, Dana-Farber Cancer Institute, Mayer 615, 44 Binney Street, Boston, MA 02115.
3 Abbreviations used in this paper: BM, bone marrow; BMT, bone marrow transplantation; CML, cell-mediated lympholysis; MST, median survival time; RAMB, rat anti-mouse brain; TCD, T cell depletion.
Received for publication December 14, 1999. Accepted for publication April 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mouse) to induce donor-specific tolerance to sequential or simultaneous islet xenografts. Transplantation 57:592.[Medline]
B10). J. Immunol. 136:28.[Abstract]
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globulin (antibody) in several species. J. Exp. Med. 96:313.[Abstract]
P1) irradiation chimeras. Cell. Immunol. 121:185.[Medline]
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