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Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Building on the concept of mixed cellular chimerism, we have shown that tolerance can be induced using gene therapy by expressing retrovirally transduced allogeneic MHC genes in host bone marrow-derived cells (3, 4). Reconstitution of lethally irradiated mice with host bone marrow infected with retroviruses carrying the allogeneic MHC class I gene H-2Kb resulted in lifelong expression of Kb on the surface of multiple bone marrow-derived hematopoietic lineages, resulting in a state of molecular chimerism. T cells from molecular chimeras reconstituted with H-2Kb-transduced bone marrow were specifically tolerant to Kb. In addition, mice reconstituted with H-2Kb-transduced bone marrow exhibited long-term acceptance of Kb disparate skin allografts with no additional immunosuppression. Thus, molecular chimerism can induce stable tolerance. In addition, because the induction of molecular chimerism relies on the transfer of genes rather than immunocompetent cells, problems of graft-vs-host disease and engraftment failure associated with allogeneic bone marrow transplants should be avoidable, making this approach clinically attractive.
It has been suggested for a number of years that bone marrow-derived APCs that populate the thymus are perhaps most important for inducing tolerance to MHC Ags (5, 6). However, in mixed cellular chimeras it has been suggested that relatively high levels of donor type T cell chimerism correlates with maintenance of long-term donor-specific tolerance (7). Indeed, thymocyte precursors have been shown to be able to induce tolerance to MHC class I Ags (8), and MHC class I Ags expressed only in CD2+ cells induce CD8 T cell tolerance (9). Thus, T cells also have the potential to be tolerogenic. To define the requirements to induce tolerance after the induction of molecular chimerism, we set out to evaluate the ability of distinct hematopoietic cell lineages expressing a retrovirally transduced allogeneic MHC class I Ag to induce tolerance. Expression of H-2Kb in bone marrow-derived cells from recombinase-activating gene (RAG)3-1 mutant mice deficient in mature B and T cells (10) was not sufficient to induce tolerance to Kb when transplanted into conditioned recipients together with wild-type bone marrow. These data suggest that induction of tolerance in molecular chimeras requires expression of the retrovirally transduced allogeneic MHC Ag on the surface of mature lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.AKM/SnJ (H-2Kk, Ik, Dq) and B10.MBR (H-2Kb, Ik, Dq) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). RAG-1 mutant mice (10) on the 129/Sv background were crossed to B10.AKM/SnJ mice, and the resulting offspring were backcrossed to the B10.AKM/SnJ strain for six generations. At each generation, mice carrying the mutant RAG-1 allele were selected and used for backcrossing. At the sixth generation, mice were intercrossed to generate homozygous mutants deficient in B and T cells. Resulting RAG-1-deficient mice were intercrossed to generate a colony of RAG-1 mice on the B10.AKM H-2 haplotype. Mice were housed using microisolator conditions in autoclaved cages and maintained on irradiated feed and autoclaved acidified drinking water. All sentinel mice housed in the same colony were viral Ab free. Six- to 12-wk-old mice were used in all experiments.
Retroviruses
Construction and production of vesicular stomatitis virus
(VSV)-Kb has been described previously (3). Briefly, the
cDNA encoding H-2Kb
(Kb) was cloned into the MMP retroviral
vector kindly provided by Dr. R. C. Mulligan (Children’s
Hospital, Boston, MA) to generate pMMP-Kb. The
MMP retroviral vector is a derivative of MFG (11).
VSV-G envelope protein pseudotyped viruses were produced by packaging
the pMMP-Kb retroviral vector in 293T cells by
transient transfection (as described in Refs. 12 and
13) to generate VSV-Kb virus. Functional titers of VSV-Kb
retroviral supernatants were determined by analyzing expression of
Kb on NIH-3T3 cells by cell surface staining and
flow cytometry after infection. All virus preparations were made in
affiliation with the Harvard Institute for Human Genetics Gene Therapy
Initiative. The viral titer obtained for the preparation of VSV-Kb used
in this report was 
1–2 x 106 infectious
particles per milliliter.
Bone marrow harvest, transduction, and transplantation
Bone marrow cells were harvested and transduced as described previously (3, 14). Briefly, bone marrow cells from mice treated 7 days prior with 5-fluorouracil (150 mg/kg) were cultured in Retronectin (TaKaRa Biomedicals, Shiga, Japan)-coated tissue culture plates in transduction medium (DMEM containing 15% lot-tested FCS and cytokines to achieve a final concentration of 100 ng/ml human IL-6 (R&D Systems, Minneapolis, MN), 100 ng/ml recombinant mouse stem cell factor (BioSource International, Camarillo, CA), 50 ng/ml recombinant mouse thrombopoietin (R&D Systems), and 50 ng/ml recombinant mouse Flt-3 ligand (R&D Systems)). Bone marrow cells were cultured at a density of 6 x 106 cells/ml and infected with VSV-Kb virus using a multiplicity of infection of at least 1. Viral supernatants and transduction medium was replaced at 24 and 72 h after the start of the transduction. Twenty-four hours after the last round of infection, bone marrow cells were harvested, washed twice in HBSS, and counted. Mock transductions were conducted in the same manner, except viral supernatants were replaced with transduction medium. Bone marrow cells were used to reconstitute conditioned recipients as described in the text.
Flow cytometry
All cell surface staining and flow cytometry were performed as described previously (3, 15). mAbs specific for CD4 (RM4-5), CD8 (53-6.7), CD3 (2C11), H-2Kb (AF6-88.5), Ly-6G (Gr-1, RB6-8C5), CD19 (1D3), CD11b (Mac-1, M1/70), NK cells (DX5), and CD11c (HL3) were obtained from BD PharMingen (San Diego, CA). Polyclonal rabbit anti-asialo-GM1 Ab was purchased from Wako BioProducts (Richmond, VA). Expression of Kb on hematopoietic progenitors was performed as described (3). In all cases, mock-infected samples were used to set flow cytometry analysis gates.
PCR assay
DNA was purified from blood using a QIAamp DNA blood mini kit
according to the manufacturer’s instructions (Qiagen, Valencia, CA).
RNA was prepared from blood cells using a RNeasy mini kit (Qiagen).
Complementary DNA was prepared from RNA with oligo(dT) primers with the
Superscript first-strand synthesis kit (Invitrogen, Carlsbad, CA).
Primer sequences used are as follows: Kb forward
primer, 5'-GCTGATCACCAAACACAAGTG-3'; Kb
reverse primer, 5'-ATGGCGTTACTTAAGCTAGC-3'; 
-actin forward
primer, 5'-AACCCCAAGGCCAACCGCGAGAAGATGACC-3'; -actin reverse
primer, 5'-GGTGATGACCTGGCCGTCAGGCAGCTCGTA-3'; Y-chromosome forward
primer, 5'-CTCCTGATGGACAAACTTTACG-3'; Y-chromosome reverse primer,
5'-TGAGTGCTGATGGGTGACGG-3'.
CTL assays
CTL assays were performed as described (3).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the ability of bone marrow-derived APCs to induce
tolerance, we used RAG-1 mutant mice that were backcrossed to B10.AKM
strain for six generations. RAG-1 mutant mice
(R0) lack mature lymphocytes (10);
therefore, by reconstituting conditioned hosts with a mixture of
H-2Kb-transduced RAG-1 and mock-transduced
wild-type B10.AKM bone marrow it is possible to directly assess the
ability of bone marrow-derived APCs to induce tolerance to
Kb. R0 bone marrow was
harvested from mice treated 7 days prior with 150 mg/kg 5-fluorouracil
and transduced with VSV-G protein enveloped retroviruses carrying the
gene encoding H-2Kb, hereafter referred to
as VSV-Kb, as described (3). Immediately following
transduction with VSV-Kb, 21% of R0 bone
marrow cells expressed Kb on their cell surface
at levels readily detectable by cell surface staining and flow
cytometry (Fig. 1
).
5-Fluorouracil-treated R0 bone marrow was
transduced at the same efficiency observed for wild-type
B10.AKM bone marrow (24%) (Fig. 1). Analysis of
Sca-1+, lineage marker negative
(Sca-1+Lin-)
hematopoietic progenitors (16) from
R0 mice and wild-type B10.AKM controls after
transduction revealed that hematopoietic progenitors from both strains
were transduced at similar efficiencies with VSV-Kb, resulting in
expression of Kb on the surface of 20–30% of
early hematopoietic progenitors (Fig. 2).
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To examine the ability of APCs to induce tolerance,
R0 mice were treated with 6-Gy whole-body
irradiation, as well as a depleting dose of anti-CD4 (GK1.5; Ref.
17), CD8 (2.43; Ref. 18) and
anti-asialo-GM1 Abs, and reconstituted the following
day with 6 x 106 mock-transduced wild-type
B10.AKM, 6 x 106 VSV-Kb-transduced
R0 bone marrow, or a mixture of 6 x
106 mock-transduced B10.AKM and 6 x
106 VSV-Kb-transduced R0
bone marrow cells. Hosts were treated with anti-CD4 and CD8 to
deplete any residual mature T cells from the B10.AKM donor bone marrow
inoculum in vivo. Anti-asialo-GM1 was used to deplete NK
cells from the host. Bone marrow-derived cells expressing
Kb on their surface were detectable in the blood
of R0 mice reconstituted with VSV-Kb-transduced
R0 bone marrow by cell surface staining and flow
cytometry at all time points analyzed over a 22-wk follow-up period
(data not shown). As expected, bone marrow-derived cells expressing
Kb were not detected in the blood of
R0 mice reconstituted with mock-transduced bone
marrow (data not shown). Bone marrow-derived cells expressing
Kb were present in the blood of
R0 mice reconstituted with a mixture of
mock-transduced B10.AKM and VSV-Kb-transduced R0
bone marrow cells early after reconstitution; however, the frequency of
cells expressing Kb in these mice fell to levels
undetectable by cell surface staining and flow cytometry by 4 wk after
bone marrow transplantation (Fig. 3).
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RT-PCR was used to further analyze engraftment of retrovirally
transduced cells in reconstituted mice. R0 mice
reconstituted with VSV-Kb-transduced R0
bone marrow as well as R0 mice reconstituted with
a mixture of mock-transduced wild-type B10.AKM and VSV-Kb-transduced
R0 bone marrow contained in their blood cells
expressing retrovirally encoded Kb for at least
20 wk after transplantation, detectable by RT-PCR using
Kb specific primers (Fig. 4). Expression of retrovirally encoded
Kb was not detected in control
R0 mice reconstituted with mock-transduced
wild-type B10.AKM bone marrow (Fig. 4). Insofar as expression of
Kb on bone marrow-derived cells in mice
reconstituted with a mixture of mock-transduced B10.AKM and
VSV-Kb-transduced R0 bone marrow cells was
undetectable by cell surface staining and flow cytometry, these data
suggest that a relatively low frequency of cells expressing the
retrovirally transduced gene were present in these mice.
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). However, as observed for
R0 mice reconstituted with a mixture of
mock-transduced B10.AKM and VSV-Kb-transduced R0
bone marrow, expression of Kb on bone
marrow-derived cells fell to below our limits of detection by 4 wk
after transplantation of B10.AKM mice with a mixture of mock-transduced
B10.AKM female and VSV-Kb-transduced R0 male bone
marrow (Fig. 5).
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). Similar results were
observed at 12 wk after bone marrow transplantation (data not shown).
These data suggest that R0 bone marrow cells were
able to engraft even though the frequency of cells expressing
Kb rapidly declined by 4 wk after
transplantation. Therefore, the inability to detect
Kb-expressing cells is not due to simply a
failure of R0 bone marrow to engraft.
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To examine whether expression of Kb on bone marrow-derived APCs was sufficient to induce tolerance, we assessed the ability of splenocytes from R0 mice reconstituted with mock-transduced wild-type B10.AKM; VSV-Kb-transduced R0 bone marrow; or a mixture of mock-transduced B10.AKM and VSV-Kb-transduced R0 bone marrow cells to kill Kb-expressing target cells 20 wk after bone marrow transplantation. Similarly, we analyzed the ability of splenocytes from B10.AKM mice reconstituted with mock-transduced B10.AKM, VSV-Kb-transduced B10.AKM, VSV-Kb-transduced R0, or a mixture of mock-transduced B10.AKM and VSV-Kb-transduced R0 bone marrow to lyse Kb-bearing targets 8 wk after bone marrow transplantation. Mice in each group were immunized after bone marrow transplantation with irradiated B10.MBR splenocytes and sacrificed 9 days later, and splenocytes were harvested. Splenocytes were then restimulated in vitro in the presence or absence of 10 U/ml IL-2 for 5 days with irradiated TBA-Kb cells, an Abelson virus-transformed B10.AKM pre-B cell line expressing Kb (3), and CTL assays were performed.
Splenocytes from either R0 or B10.AKM mice
reconstituted with mock-transduced wild-type B10.AKM bone marrow were
able to lyse Kb-expressing targets (Fig. 7). Addition of IL-2 to these cultures
led to increased killing of targets expressing Kb
(Fig. 7). In contrast, splenocytes from either R0
or B10.AKM mice reconstituted with a mixture of mock-transduced
wild-type B10.AKM and VSV-Kb-transduced R0 bone
marrow lysed targets expressing Kb only when IL-2
was provided during in vitro restimulation (Fig. 7). As expected,
splenocytes from either control R0 or B10.AKM
mice reconstituted with VSV-Kb-transduced R0 bone
marrow were unable to lyse Kb-expressing targets
(Fig. 7). In contrast, B10.AKM mice reconstituted with
VSV-Kb-transduced B10.AKM bone marrow cells were unable to lyse targets
expressing Kb even when IL-2 was added during in
vitro restimulation (Fig. 7), consistent with our previous observation
that expression of Kb on wild-type bone
marrow-derived cells results in tolerance (3). Together,
these data suggest that mice reconstituted with a mixture of
mock-transduced wild-type B10.AKM and VSV-Kb-transduced
R0 bone marrow are hyporesponsive rather than
tolerant to Kb.
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To examine why R0 bone marrow failed to
induce tolerance, we analyzed expression of Kb in
the thymus of wild-type B10.AKM recipients reconstituted with
VSV-Kb-transduced R0 bone marrow,
VSV-Kb-transduced B10.AKM bone marrow, or a mixture of
VSV-Kb-transduced R0 and mock-transduced B10.AKM
bone marrow. B10.AKM mice reconstituted with VSV-Kb-transduced B10.AKM
bone marrow contained a significant number of cells expressing
Kb in their thymuses (Fig. 8). While macrophages and NK cells
expressing Kb were detected in the thymus of
these mice, the majority of cells expressing Kb
were CD3+ thymocytes. The frequency of
Kb-expressing cells in the thymus of B10.AKM mice
reconstituted with VSV-Kb-transduced B10.AKM bone marrow was similar to
the overall frequency of cells expressing Kb in
the blood (Fig. 5
). In contrast, B10.AKM mice reconstituted with
VSV-Kb-transduced R0 bone marrow contained
relatively fewer Kb-expressing cells in their
thymus (Fig. 8). Expression of Kb was detected on
NK cells and macrophages in the thymuses of these mice. The frequency
of Kb-expressing cells in the thymus of B10.AKM
mice reconstituted with VSV-Kb-transduced R0 bone
marrow was significantly lower than the frequency observed in the blood
(Fig. 5). We were unable to detect cells expressing
Kb in the thymus of B10.AKM mice reconstituted
with a mixture of VSV-Kb-transduced R0 and
mock-transduced B10.AKM bone marrow (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data suggest that expression of the retrovirally transduced MHC class I gene in all bone marrow-derived lineages except mature B and T cells is not sufficient to induce robust tolerance. Therefore, either expression of Kb on APCs alone is insufficient to induce tolerance or an insufficient number of APCs expressing Kb may have led to hyporesponsiveness rather than tolerance. Mice reconstituted with a mixture of VSV-Kb-transduced R0 and wild-type B10.AKM bone marrow contained only low levels of hematopoietic cells derived from transduced progenitors by 4 wk after transplantation. It is unlikely that inefficient transduction of early hematopoietic progenitors in R0 bone marrow led to a decline in Kb expression in mice reconstituted with a mixture of VSV-Kb-transduced R0 and mock-transduced B10.AKM bone marrow because hematopoietic progenitors from R0 mice were transduced at a frequency similar to that observed for wild-type bone marrow. In addition, R0 and wild-type mice reconstituted with VSV-Kb-transduced R0 bone marrow exhibited expression of Kb on bone marrow-derived cells long term, suggesting that early progenitors were transduced. We suggest that cells expressing Kb in mice reconstituted with a mixture of VSV-Kb-transduced R0 and wild-type B10.AKM bone marrow may have undergone rejection as they developed. The persistent low level of Kb expression detected by RT-PCR may reflect engraftment of H-2Kb-transduced hematopoietic stem cells, which have been suggested to be immunoprivileged (20). Therefore, it is possible that the transduced stem cells persist while progeny expressing Kb are actively rejected. Consistent with this idea, cells expressing Kb were detectable in mice receiving a mixture of VSV-Kb-transduced R0 and wild-type B10.AKM bone marrow only at relatively early time points after bone marrow transplantation, before T cell recovery took place (data not shown). Hyporesponsiveness may result from activation-induced cell death of Kb-reactive T cells. It is also possible that low levels of Kb expressed on resting APCs induced anergy rather than deletional tolerance, which could be overcome by providing exogenous IL-2.
It has previously been observed that relatively inefficient transduction and expression of H-2Kb in bone marrow-derived cells results in hyporesponsiveness to Kb, which can be overcome by providing T cell help (21, 22). Interestingly, Fraser et al. (21) were able to observe expression of Kb on the surface of macrophages and B220+ cells; however, Kb expression was not detected on CD4 or CD8 T cells in the majority of mice reconstituted with H-2Kb-transduced bone marrow. We suggest that the inability to achieve tolerance in that study may have been related to inefficient expression of Kb on T cells, or restricted expression in hematopoietic cell lineages such as myeloid cells, which we show here are unable to induce tolerance.
It has been suggested that bone marrow-derived APCs are critical
mediators of negative selection in the thymus (5, 6). Yet
is has been shown, in mixed cellular chimeras, that a relatively high
level of donor type T cell chimerism correlates with maintenance of
long-term donor-specific tolerance (7), and it has been
shown that thymocyte precursors can induce tolerance to MHC class I Ags
(8). In mice reconstituted with VSV-Kb-transduced
R0 bone marrow, relatively few cells expressing
Kb were present in the thymus of reconstituted
mice. In contrast, the frequency of cells expressing
Kb was significantly higher in the thymus of mice
reconstituted with VSV-Kb-transduced wild-type bone marrow. We suggest
that the requirement for T cells to induce tolerance may be related to
the ability of T lineage cells to deliver Ag to the thymus. Because T
lineage cells comprise 85% of the hematopoietic cells in the
thymus, expression of Kb on T cell precursors or
mature single positive T cells may be required to achieve a threshold
level of Kb expression in the thymus to induce
negative selection of Kb-reactive T cells.
It has recently been described that reconstitution of non-myeloablated recipients with CD4 and CD8 double knockout bone marrow can result in state of mixed host and donor hematopoietic chimerism without inducing tolerance (23). In contrast, we did not observe a dissociation of chimerism and tolerance. Kb was not expressed at significant levels in the periphery of nontolerant mice. Although the results of Umemura et al. (23) are consistent with the notion that mature T cells are required to induce tolerance, in that study it is possible that transplant rejection was related to minor Ag disparities between the knockouts used as bone marrow donors and the skin graft donors. Nevertheless, insofar as tolerance was not achieved in mixed chimeras generated using bone marrow from CD4 and CD8 double knockout mice, we suggest that the failure to induce tolerance may have been related to low levels of alloantigen expressed in the thymus resulting from a lack of bone marrow-derived T cells.
We speculate that mature lymphocytes expressing Kb are required to bring the retrovirally transduced alloantigen to the thymus, establishing a critical threshold of expression that must be achieved to establish negative selection of alloreactive T cells. T lineage cells may be well suited for this purpose because they comprise the vast majority of cells in the thymus, and therefore may have the greatest potential for delivering Ag. We are currently examining this possibility. Defining the minimum expression level required to induce and maintain tolerance after the induction of molecular chimerism could be used to predict whether stable tolerance will be established. Insofar as our results demonstrate that efficient trafficking of cells expressing Kb to the thymus is required to induce tolerance, we suggest that strategies designed to enhance trafficking of Ag to the thymus may facilitate tolerance induction. In addition, we suggest that, in addition to genetic engineering of bone marrow, it may be possible to induce tolerance to transplantation Ags by treating transiently immunosuppressed hosts with autologous peripheral lymphoid cells expressing allogeneic MHC genes introduced using viral delivery vehicles.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John Iacomini, Transplantation Biology Research Center, Massachusetts General Hospital East, Building 149, 13th Street, Boston, MA 02129. E-mail address: iacomini{at}helix.mgh.harvard.edu
3 Abbreviations used in this paper: RAG, recombinase-activating gene; VSV, vesicular stomatitis virus.
Received for publication May 14, 2002. Accepted for publication August 7, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), VSV-Kb-transduced B10.AKM bone marrow (
), a mixture of
VSV-Kb-transduced R0 and mock-transduced B10.AKM bone
marrow (x), or mock-transduced B10.AKM bone marrow
(
). Shown are
the mean values obtained for between five and six mice per time point.
The data are representative of three independent experiments.
),
or a mixture of VSV-Kb-transduced R0 and mock-transduced
B10.AKM bone marrow (