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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1166-1172
TRANSPLANTATION
A novel application of cyclosporine A in
nonmyeloablative pretransplant host conditioning for allogeneic BMT
Boris Nikolic,
Guiling Zhao,
Kirsten Swenson, and
Megan Sykes
From the Bone Marrow Transplantation Section, Transplantation
Biology Research Center, Surgical Service, Massachusetts General
Hospital/Harvard Medical School, Boston, MA.
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Abstract |
The treatment of mice with anti-CD4 and anti-CD8 monoclonal
antibodies (mAbs) on day  5, plus 3 Gy whole body irradiation (WBI)
and 7 Gy thymic irradiation (TI) on day 0, allows fully major-histocompatibility-complex-mismatched allogeneic bone marrow engraftment and the induction of immunologic tolerance. TI is required
in this model to overcome alloreactivity and possibly to make
"space" in the recipient thymus so that lasting central tolerance
can be achieved. In addition to suppressing mature T cells in the
periphery, Cyclosporine A (CYA) and glucocorticoids have a
powerful influence on the thymus. In this study, we evaluated whether
the administration of CYA to recipient mice for 12 days prior to bone
marrow transplant (BMT), of glucocorticosteroids on the day of BMT, or
a combination of both, could create space and overcome alloresistance
in the thymus by specifically depleting immature and mature thymocytes
prior to BMT. High levels of multilineage donor hematopoietic
repopulation and specific transplantation tolerance were achieved in
mice treated from days 15 to 3 with CYA (20 mg/kg/d
subcutaneously), anti-CD4/CD8 mAbs on day 5, followed by 3 Gy WBI
and 15 × 106 allogeneic bone marrow cells
on day 0. V analysis suggested a central deletional tolerance
mechanism. The same treatment without CYA pretreatment allowed only
transient chimerism, without tolerance. Corticosteroid treatment
abolished the engraftment-promoting and tolerance-inducing effects of
CYA. These results demonstrate a novel pretransplantation-only
application of CYA, which facilitates allogeneic marrow engraftment
with minimal conditioning, by creating thymic space and/or overcoming
intrathymic alloresistance.
(Blood. 2000;96:1166-1172)
© 2000 by The American Society of Hematology.
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Introduction |
The induction of immunological tolerance has become a
major goal of transplantation research because modern immunosuppressive therapy has not improved chronic rejection rates and is associated with
significant side effects. A state of donor-specific tolerance would
obviate the risks of both acute and chronic rejection while eliminating
the need for chronic immunosuppressive therapy, thereby permitting
normal immunocompetence. Successful allogeneic bone marrow engraftment
results in a state of specific transplantation tolerance. However, most
applications of this approach to tolerance induction have involved the
use of supralethal whole body irradiation (WBI) or high doses of total
lymphoid irradiation followed by injection of allogeneic bone marrow
cells (BMCs).1-3 Unfortunately, the severe
toxic side effects of such regimens have precluded their use in humans
as a means of inducing tolerance.
Several years ago, Cobbold et al4 developed a
nonmyeloablative regimen for induction of permanent chimerism and
transplantation tolerance that involved host treatment with depleting
anti-CD4 and CD8 monoclonal antibodies (mAbs) and 6 Gy of WBI. Our
group subsequently showed that similar results could be achieved with an even lower WBI dose, if thymic irradiation (TI) was added to the
regimen.5 This nonmyeloablative regimen includes
administration of depleting doses of anti-CD4 and anti-CD8 mAbs on day
5, 3 Gy WBI and 7 Gy TI on day 0, followed by injection of
1.5 × 107 fully major-histocompatibility-complex
(MHC)-mismatched allogeneic BMCs on day 0. Mice treated in this way
develop permanent mixed chimerism and donor-specific skin allograft
tolerance.5 We have shown that the mechanism of tolerance
in these animals primarily involves central deletion of donor-reactive
T-cell clones resulting from the presence of donor-type
antigen-presenting cells in the thymus, and that the maintenance of
tolerance in this model depends upon the continuous intrathymic
presence of donor hematopoietic cells.6-8 While animals
receiving this conditioning regimen without BMT show no mortality and
maintain excellent health, the mixed chimerism/central deletion
approach to tolerance induction would have greater appeal if
irradiation, with its associated risks, could be eliminated from the
conditioning treatment.
We have recently reported that an additional treatment with depleting
anti-CD4 and anti-CD8 mAbs can overcome the requirement for TI in order
to achieve consistent allogeneic marrow engraftment, mixed chimerism,
and specific transplantation tolerance.9 When 2 mAb
injections were given before transplantation and 3 Gy WBI was given on
day 0, most animals demonstrated high early levels of donor T-cell
repopulation, lasting mixed chimerism, and specific skin graft
acceptance.9 In contrast, when a single mAb injection was
administered either on day 1 or on day 5, administration of TI was essential for the reliable induction of lasting chimerism and
tolerance.5,6,9 A single administration of mAbs 5 days before BMT leads to almost complete depletion of T cells in the periphery of mice. However, alloreactive cells persist in the thymus
and are thought to be responsible for the loss of chimerism and
tolerance if no further treatment is given10 (also, B.N. and M.S., unpublished data, 1996). Consistent with this
hypothesis, animals receiving a single mAb injection without TI have
shown high early levels of chimerism in the periphery,5
with an absence of chimerism in the thymus and a failure to delete
donor-reactive host thymocytes.10 These animals may undergo
intrathymic rejection of donor progenitors. Despite the presence of an
initially successful hematopoietic graft, these animals later lose
peripheral chimerism, presumably owing to the emergence of nontolerant
host T cells from the thymus. The administration of a second mAb
injection or TI seems to effectively overcome the persisting
intrathymic alloresistance, thus allowing long-term chimerism to be achieved.
Additional studies in the Ly-5 congenic system suggest
that thymic "space" for hematopoietic engraftment may be
regulated independently from hematopoietic space11 and that
specific measures are required to permit donor marrow to repopulate the
recipient thymus early, even when very large numbers of donor marrow
cells are administered. Therefore, it is possible that TI or a second mAb injection might be necessary not only to eliminate preexisting donor-reactive thymocytes, but also to create "thymic space," which assures the intrathymic engraftment of large numbers of donor-derived cells and an early high level of donor-derived T-cell recovery.
Repeated injections of Cyclosporine A (CYA) have been
shown to induce marked but reversible thymic
dysfunction.12,13 CYA treatment blocks differentiation of
immature CD4+CD8+ thymocytes into
single-positive (SP) cells and induces thymic medullary involution with
destruction of the medullary dendritic cells.12-15 After
discontinuation of CYA, the immigration of dendritic cell precursors to
the thymus is facilitated.16 Similarly, steroid hormones,
such as glucocorticoids, have been shown to selectively deplete
double-positive (DP) thymocytes and to affect several T
lymphocyte functions.17 We hypothesized that combined
steroid and CYA treatment might help to create thymic space by
depleting the majority DP population, enhance the early migration of
donor dendritic cells to the thymus, and also help to overcome
alloresistance by specifically depleting mature SP thymocytes prior to
BMT. Therefore, we hypothesized that host CYA pretreatment, injection
of hydrocortisone on the day of BMT, or a combination of both
treatments would enhance donor thymic repopulation and allow induction
of permanent tolerance without TI or a second mAb injection.
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Materials and methods |
Animals
Female C57BL/6 (B6: H-2b; Kb,
I-Ab, I-E-, Db), B10.A (B10.A:
H-2a; Kk, I-Ak, I-Ek,
Dd), and SJL (H-2s) mice were purchased from
Harley-Sprague Dawley via Frederick Cancer Research Center, Frederick,
MD. All mice were maintained in sterilized microisolator cages, in
which they received autoclaved feed and autoclaved acidified drinking
water. Recipients in each experiment were age-matched and were 10 to 12 weeks old.
Conditioning and BMT
Recipient B6 mice were treated with mAbs intraperitoneally on the
indicated days (see below). Each
injection18,19 consisted of 1.1 mg of purified
rat antimouse CD4 immunoglobulin (Ig) G2b mAb GK1.5 and 0.7 mg of purified rat antimouse CD8 IgG2b mAb 2.43. On day 0, we administered 3 Gy WBI to mAb-treated animals, as described.5 At 4 to 6 hours after completion
of conditioning on day 0, we intravenously administered
1.5 × 107 untreated BMCs from B10.A mice.
Pretreatment with Cyclosporine A and corticosteroids
Recipient B6 mice were pretreated with daily subcutaneous injections
containing 20 mg/kg CYA (Sandimmune purchased from Sandoz, East
Hanover, NJ) dissolved in sterile olive oil from day 15 to day
3 prior to BMT. Two groups of mice received 1 intraperitoneal injection of 20 mg/kg hydrocortisone acetate (Hydrocortone purchased from Merck Sharp & Dohme, West Point, PA) 4 hours prior to irradiation.
Flow cytometric analysis of chimerism
The level of allogeneic donor T-cell and non-T-cell reconstitution
was evaluated by 2-color flow cytometric analysis on a FACScan or
FACSort (Becton Dickinson, Mountain View, CA) as previously described.20 Briefly, forward-angle and 90°
light-scatter properties were used to distinguish lymphocytes,
granulocytes, and monocytes in peripheral white blood cells (WBCs).
Two-color fluorescence cytometry (FCM) was used to distinguish donor
and host cells expressing particular lineage markers, and the
percentage of donor cells was calculated by subtracting control
staining from quadrants containing donor and host cells expressing a
particular lineage marker and by dividing the net percentage of donor
cells by the total net percentage of donor plus host cells of that
lineage. Nonspecific Fc R binding was
blocked21 with 10 µL of
undiluted culture supernatant containing rat antimouse FcR mAb
2.4G2. Biotinylated anti-H-2Dd mAb 34-2-12 and control mAb
HOPC1 were incubated22 with
phycoerythrin-streptavidin (PEA). Fluorescein isothiocyanate
(FITC)-conjugated mAbs were anti-CD4, anti-CD8, and
anti-B220 (all purchased from Pharmingen, San Diego, CA) and MAC1
(Caltag, San Francisco, CA). Negative control mAb HOPC1-FITC, with no
reactivity to mouse cells, was prepared in our laboratory. Dead cells
were excluded by gating out low forward-angle light scatter/high
propidium iodide-retaining cells.
T-cell receptor analysis
Peripheral blood lymphocytes (PBLs) and spleen cells were stained
with FITC-conjugated anti-T-cell receptor (TCR) V11, V8.1/8.2, and V5.1/5.2 mAbs (Pharmingen). For 2-color analysis, PBLs were labeled with FITC-conjugated HOPC-1 or anti-TCR V11, V8.1/8.2, or
V5.1/5.2 mAbs, and phycoerythrin-conjugated antimouse
CD4 and CD8 mAbs (Pharmingen). At least 5000 gated CD4+
cells were collected for V analysis.
To determine the percentage of mature host thymocytes that were
V11+, V8.1/8.2+, or
V5.1/5.2+, we collected 5000 gated H-2 Kb
class Ihigh cells (distinguished by bright staining above
negative control biotinylated mAb HOPC-1/PEA) for analysis of staining
with FITC-conjugated anti-V mAb. Thymocytes were also
stained with negative control FITC-conjugated HOPC-1
versus biotinylated anti-Kb mAb23 5F1. The
percentage of gated H-2 Kb class Ihigh cells
staining with control mAb HOPC-1 was subtracted from the percentage of
gated H-2 class Ihigh cells staining with anti-V11,
V8.1/8.2, or V5.1/5.2 mAbs or with FITC-conjugated anti-panTCR
mAb, respectively.
Immunohistochemical staining
Indirect immunoperoxidase staining of thymic sections (4 µm) was
performed as we have previously described.10 For detection of mouse I-Ab+ cells, mouse IgG2a
anti-I-Ab mAb 25-9-17 was used24
with a biotinylated rat antimouse IgG2a mAb as secondary
reagent. For detection of mouse I-E+ cells, mouse
IgG2b anti-I-E mAb ISCR-3 was
used25 with a biotinylated rat antimouse
IgG2b mAb as secondary reagent. In each case, staining was
compared with that using isotype-matched negative control mAbs HOPC-1
(mouse IgG2a isotype control) and 74-11-10 (mouse IgG2b
isotype control) with the same secondary reagent. Staining was
developed with the use of the Vectastain ABC kit (Vector Corp, Burlingame, CA), as described.10
Skin grafting
Initial skin grafting was performed 9 weeks following BMT as
previously described.5 Square full-thickness tail skin
grafts (1 cm2) were prepared from donors and grafted onto
the right and left lateral thoracic wall of recipient mice. The first
inspection was carried out on the seventh day, followed by daily
inspection for the first month and then 2 to 3 times per week
thereafter. Grafts were defined as rejected at the time of complete
sloughing or when they formed a dry scab.
Statistical analysis
Statistical significance was determined by means of the Student
t test. A P value less than .05 was considered to be
statistically significant.
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Results |
Pretreatment with CYA from day 15 to day 3 reduces the number
of SP thymocytes
A 2- to 7-week course of treatment with CYA has been shown to
decrease the absolute number of thymocytes and to most markedly decrease the number of SP mature thymocytes.13,14 To
confirm this result with our treatment regimen, B6 mice were pretreated with daily subcutaneous injections containing 20 mg/kg CYA dissolved in
sterile olive oil from day 15 to day 3 with respect to
the time of BMT planned for future studies. In order to avoid the impairment of negative selection associated with BMT plus CYA treatment,14,26-28 we stopped the administration of CYA on
day 3 prior to BMT. It was anticipated that this period of time
would allow the clearance of CYA from the circulation prior to BMT. Mice treated only with olive oil were compared at the time of sacrifice
on day 0 to mice that received CYA from days 15 to 3. A
greater than twofold reduction in total thymocyte number, a 14-fold
reduction in the absolute number of SP CD4+ thymocytes
(sevenfold reduction in percentage of CD4 SP, Figure 1), and a fourfold reduction in the
absolute number of SP CD8+ cells thymocytes (twofold
reduction in percentage of CD8 SP, Figure 1) were detected in the
thymuses of CYA-treated mice compared with animals treated only
with olive oil (Figure 1). Consistent with previous data, the majority
of cells that disappeared with CYA treatment were mature SP T
cells expressing high levels of TCRs (Figure 1, lower left and right)
and high levels of class I MHC (not shown).12,13
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| Fig 1.
Effects of CYA on developing thymocytes.
Mice were treated with daily subcutaneous injections containing sterile
olive oil alone (upper and lower left, CYA), or
20mg/kg CYA dissolved in sterile olive oil from day 15 to day
3 (upper and lower right, CYA+). Mice were killed
at day 0. Age- and sex-matched experimental and control mice were
treated and analyzed simultaneously. Unfractionated thymocytes were
harvested and stained as indicated.
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CYA but not corticosteroid pretreatment permits engraftment of
donor pluripotent hematopoietic stem cells (PPHSCs)
Groups of recipient B6 mice were prepared as indicated in Table
1. Allogeneic repopulation was evaluated 2 weeks and later after BMT by staining peripheral WBCs with a mAb
(34-2-12; anti-H-2Dd) that recognizes donor class I MHC.
Early and lasting chimerism was observed in 4 of 5 recipients treated
with anti-CD4 plus anti-CD8 mAbs on days 1 and 6, and 3 Gy WBI prior to injection of B10.A BMCs (Table 1, Group A), as
described previously.9 In contrast, exclusion of the second
mAb injection was associated with the initial development of chimerism
in 4 of 5 animals, but none of the animals developed long-term
chimerism (Table 1, Group B). These results confirmed previous results
indicating that a second mAb injection was essential for the consistent
induction of lasting chimerism in mice that did not receive TI prior to
BMT.10
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Table 1.
CYA pretreatment permits engraftment of B10.A bone
marrow cells in B6 mice conditioned without thymic
irradiation or a second mAb injection
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We evaluated the possibility that the requirement for a second mAb
injection or TI9 might be overcome by pretreating recipient mice with CYA, corticosteroids, or a combination of both. As is shown
in Table 1, pretreatment of mice with CYA from day 15 to day
3, a single injection of anti-CD4 plus anti-CD8 mAbs on day
5, and 3 Gy WBI prior to BMT resulted in the induction of mixed
chimerism in all animals, and this state persisted longer than 22 weeks
(Table 1, Group C). The addition of CYA pretreatment led to a higher
level of donor chimerism (from 4 to 22 weeks) compared with recipients
of the protocol that includes a second mAb injection on day 1.
The administration of a single dose of corticosteroids on day 0, in
addition to mAbs on day 5 and 3 Gy WBI prior to BMT, resulted in
early chimerism in 3 of 5 animals at 2 weeks post-BMT, with lasting
chimerism in only 1 of 5 animals (Table 1, Group D). The addition of
corticosteroids to CYA pretreatment, anti-CD4 plus anti-CD8 mAbs, and 3 Gy WBI prior to BMT resulted in lasting chimerism in only
1 of 4 animals (Table 1, group E), suggesting that corticosteroid
treatment had a deleterious effect on donor marrow engraftment.
We followed the levels of donor chimerism in the T-cell (Figure
2A-D), granulocyte (Figure 2E-H), and
monocyte (not shown) lineages. Animals treated with only 1 injection of
mAbs on day 5 (Group B from Table 1) developed only initial,
transient T-cell chimerism with fewer than 20% donor-derived cells
(Figure 2A). Of 5 animals that received a second mAb
injection, 4 developed stable long-lasting chimerism in the T-cell,
granulocyte, and monocyte lineages (not shown). Animals receiving
pretreatment with CYA in addition to 1 mAb injection (Group C in Table
1) showed high levels of early donor T-cell repopulation, which
increased over time (Figure 2B). The addition of 1 dose of
corticosterids on day 0 to 1 dose of mAbs on day 5 (Figure 2C)
or to 1 dose of mAbs on day 5 and CYA on days 15 to
3 (Figure 2D) resulted in a complete lack of T-cell chimerism in
all, except for 1 mouse per group. The lower panel of Figure 2 shows
the level of donor chimerism in the granulocyte lineage, which in most
of the animals followed the same pattern as T-cell chimerism.
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| Fig 2.
T-cell (A-D) and granulocyte (E-H) chimerism in
peripheral blood.
Donor representation among various hematopoietic lineages was
determined at multiple time points after BMT by FACS. Each line
represents 1 animal. Results from 1 of 2 similar experiments are shown.
All mice received TCD mAbs on day 5, 3 Gy WBI (day 0), and
15 × 106 BMCs (day 0). Animals in Group B from
Table 1 (n = 4; panels A and E) received no additional treatment.
Group C (n = 5; panels B and F) received pretreatment with CYA from
day 15 to day 3; Group D (n = 5; panels C and G)
received 1 dose of corticosteroids on day 0; and Group E (n = 4;
panels D and H) received pretreatment with CYA from day 15 to
day 3 and 1 dose of corticosteroids on day 0.
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At 41 weeks after BMT, the animals were killed and examined for
chimerism. Long-term chimeras conditioned with CYA pretreatment, mAbs
on day 5, and 3 Gy of WBI, had high levels of multilineage donor
cell chimerism in both the spleen (52.9% to 63% donor-derived class
I+ cells) and the bone marrow (40% to 70% donor-derived
class I+ cells), whereas animals pretreated with only 1 mAb
injection with or without corticosteroids had no detectable chimerism
by FACS (not shown).
Skin graft survival
To evaluate tolerance in chimeric mice, donor B10.A and third-party
SJL (H-2s) skin was grafted 9 weeks post-BMT.
All grafted animals rejected third-party SJL skin grafts within 30 days
(Figure 3B). In 4 of 4 recipients prepared
with the regimen that included mAbs on day 5 only and 3 Gy WBI,
B10.A skin grafts were rejected within 20 days (Figure 3A). In
contrast, in 3 of 4 recipients prepared with the regimen that
included mAbs on both day 5 and day 1, plus 3 Gy WBI,
B10.A skin grafts were accepted for longer than 100 days. In 5 of 5 recipients of CYA pretreatment and mAbs only on day 5, and 3 Gy
WBI, B10.A skin grafts were accepted for longer than 100 days,
indicating that donor-specific skin allograft tolerance had been
induced (Figure 3B). The administration of corticosteroids in addition
to CYA pretreatment prevented the induction of skin graft acceptance in
the majority of animals, with an outcome similar to that achieved in
mice receiving corticosteroids without CYA (Figure 3A).
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| Fig 3.
Donor and third-party skin graft survival.
(A) Donor B10.A skin graft survival for groups that received CYA
pretreatment and a single mAb injection (n = 5,  ); a single mAb
injection and corticosteroids (n = 4,  ); CYA pretreatment, a
single mAb injection, and corticosteroids (n = 4,  ); 2 mAb
injections (mAbs 2 ×, n = 4,  ); and a single mAb injection
only (n = 4,  ). (B) Third-party skin graft survival for the same
groups. Donor B10.A and third-party SJL (H-2s) skin was
grafted 9 weeks post-BMT. Results from the first of 2 similar
experiments are shown.
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Clonal deletion of donor-reactive host thymocytes in mice receiving
CYA pretreatment, mAbs on day 5, and 3 Gy WBI
The marrow donor used in our studies, B10.A, expresses I-E, the
class II MHC molecule that most efficiently presents Mtv8- and
Mtv9-associated superantigens that are endogenous to the C57BL background. As a result, deletion of developing thymocytes whose TCRs
contain V11 and V5, which binds to these superantigens, is
observed in B10.A mice.29,30 The B6 strain, on the other hand, cannot express the I-E molecules to which these superantigens bind and therefore does not delete V11+ and
V5+ T cells from its repertoire.31 To
determine whether or not intrathymic clonal deletion could also be the
basis for T-cell tolerance in B6 mice receiving B10.A BMCs after
pretreatment with CYA on days 15 to 3, anti-CD4 and
anti-CD8 mAbs on day 5, and 3 Gy WBI on day 0, we measured the
percentages of V11+ and V5+ host
(ie, B6) mature thymocytes at 10 months following BMT. To select for mature host-type T cells, we measured these V for gated
H-2Kb high thymocytes, since this population
consists mainly of mature, SP recipient T cells. When B10.A control
mice were studied, gated H-2Dd high
thymocytes were analyzed instead. As a nondeleted TCR
family control, V8.1/2-bearing thymocytes were also studied.
Consistent with the maturity of thymocyte populations selected in this
manner, only high-intensity staining with anti-V mAbs was observed,
indicating that TCRlow immature DP thymocytes
had been gated out.
Mature thymocytes from B10.A mice were appropriately deleted, with
V11+ T cells composing only 0.33%, and
V5+ T cells composing only 0.38%, of thymocytes. In
contrast, V11+ and V5+ cells composed 3%
and 5.3%, respectively, of mature thymocytes in normal B6 mice (Figure
4A). Thymuses from B6 mice receiving CYA
pretreatment, anti-CD4 plus anti-CD8 mAbs on day 5, and 3 Gy WBI
prior to B10.A BMT showed marked deletion of V11+
(average of 0.34%) and V5+ (average of
0.8%) cells among mature B6 thymocytes (Figure 4A). In
contrast to these results, intrathymic clonal deletion of
V11+ and V5+ B6 thymocytes was not
observed in B6 mice that had received B10.A BMT after being conditioned
with 1 injection of anti-CD4 plus anti-CD8 mAbs, and 3 Gy WBI; lasting
mixed chimerism was not successfully induced in these mice. Similarly,
intrathymic clonal deletion of V11+ and
V5+ T cells was not observed in B6 mice that had
received corticosteroids alone or in combination with CYA (not shown).
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| Fig 4.
Deletion at 41 weeks post-BMT of (A) donor-reactive
mature host thymocytes and (B) CD4+ splenocytes.
Results are shown as mean ± standard deviation.
Values are for normal age-matched B6 mouse ( ), normal B10.A mouse
( ), day 5 mAb-injected group (, n = 4), and day 5
mAb-injected group receiving CYA pretreatment ( , n = 2). Values
are compared with those of age-matched B6 mice. *P < .05.
**P < .005, ***P < .0005.
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Figure 4B shows that this intrathymic deletion was reflected among the
CD4 SP T cells in the spleen. An absence of V11+ and
V5+ CD4 T cells was observed in the spleens of untreated
B10.A mice and in long-term chimeras prepared with CYA pretreatment,
anti-CD4 plus anti-CD8, and 3 Gy WBI (Figure 4B). However, there was no deletion of these V families in control B6 mice, which received only
conditioning without BMT, or in mice treated with mAbs on day 5
and with 3 Gy WBI and B10.A BMCs, which demonstrated only early, not
lasting chimerism. Similarly, deletion of splenic V11+
and V5+ CD4+ T cells was not observed in
transplanted B6 mice that had received corticosteroids alone or in
combination with CYA (not shown).
At the time of sacrifice (41 weeks post-BMT), immunohistochemical
staining was performed to examine the presence of donor-derived class
II+ cells in the thymuses of chimeras. In all chimeras that
received CYA pretreatment from day 15 to day 3, anti-CD4
plus anti-CD8 on day 5, and 3 Gy WBI prior to BMT, a large
number of donor-derived class II+ cells with a dendritic
morphology was detected in the corticomedullary and medullary regions
(not shown). Mice pretreated with only 1 injection of mAbs on day
5 and 3 Gy WBI, and a control mouse receiving the conditioning
without BMT, were negative for donor class II+ cells (not
shown). The presence of donor class II+ cells correlated
with the deletion of donor-reactive cells in the thymuses of chimeras,
further supporting the conclusion that intrathymic deletion was a major
mechanism of tolerance.
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Discussion |
We demonstrate here that pretreatment of recipients with CYA can
overcome the requirement for thymic irradiation or for an additional
dose of depleting anti-CD4 and anti-CD8 mAbs in order to achieve
consistent allogeneic marrow engraftment, mixed chimerism, and specific
transplantation tolerance in mice receiving 3 Gy WBI and a peripheral
T-cell-depleting mAb treatment. This, to our knowledge, is the first
such application of CYA, in which the drug is not used to suppress the
immune response after donor grafting, but is instead used to condition
the thymus to make it permissive for donor engraftment. These results
advance our effort to develop a more specific, targeted approach to
recipient conditioning that uses the minimal amount of myelosuppression and immunosuppression required to permit partial reconstitution by an
allogeneic marrow donor.
CYA is an important immunosuppressive drug that is widely used in
transplantation medicine. In addition to suppressing mature T cells in
the periphery, CYA has a powerful influence on the thymus. Short
treatment with CYA decreases the absolute number of thymocytes
(approximately 50%) and reduces the SP thymocyte number by
90%.12 CYA exerts many of its effects via specific inhibition of TCR-mediated T-cell activation. The precise point at
which CYA blocks thymocyte maturation is unclear. However, the relative
lack of CYA-mediated effects on the development of CD4 8 (double
negative) and CD4+8+ (DP)
cells,12,14 as well as the lack of a CYA-mediated effect on
TCR gene rearrangement,12 suggests that the inhibition
occurs at a later stage in thymocyte development, perhaps at the
transition of DP to SP cells.12
With TI, intrathymic engraftment of large numbers of donor-derived
progenitors and, hence, high initial levels of donor-derived T-cell
reconstitution are achieved. TI can be omitted if a second injection of
anti-CD4 and anti-CD8 mAbs is administered 1 day prior to
BMT.9 In support of the hypothesis that administration of a
second mAb injection depletes or inactivates residual host thymocytes
that are capable of causing intrathymic rejection of donor
hematopoietic cells even when peripheral engraftment is achieved, we
have detected residual cytotoxic lymphocyte and mixed lymphocyte
reaction alloreactivity in thymuses of animals receiving a single mAb injection and 3 Gy of WBI. In contrast, no alloreactivity was detected in thymuses of animals receiving mAbs on days 6 and
1 and 3 Gy WBI (B.N. and M.S., unpublished data).
Therefore, CYA pretreatment-induced depletion or inactivation of
mature alloreactive SP thymocytes may enhance early donor thymic
repopulation and induction of permanent tolerance by depleting the SP
thymocytes that are presumably responsible for this alloreactivity in
recipients of a single mAb injection.
In addition to suppressing positive selection and SP thymocyte
maturation, CYA treatment blocks the deletion of potentially autoreactive T-cell clones among the small number of  T cells that do mature in its presence.12,14 Clonal deletion is
prevented by interference with TCR-mediated signal transduction and by
reduction of I-A molecule expression on bone marrow-derived
elements.12,32 A short course of CYA treatment induces
thymic involution with destruction of the medullary dendritic cells
(DCs) and medullary epithelium. In contrast, the cortex of CYA-treated
rodents is relatively intact, and the thymus consists almost entirely
of cortical tissue.32 The autoreactivity of T cells that
escape clonal deletion is not manifested until the withdrawal of CYA, which relieves the block in lymphokine production and initiates the autoimmune process.33 Indeed, irradiated hosts
transplanted with syngeneic bone marrow and then treated with and
withdrawn from CYA develop autoimmunity. This phenomenon, which is
observed in rats,27 mice,26,28 and
humans,34,35 is characterized by a graft-versus-host
disease (GVHD)-like syndrome that is transferable to naive recipients
by T cells.36 To prevent failure of negative selection of
donor and host-reactive thymocytes in our model, we stopped the
administration of CYA on day 3 prior to BMT. Also, the
administration of mAbs, which remain detectable in the circulation for
the first 2 weeks post-BMT,10 and of 3 Gy of WBI on day 0, makes the occurrence of clinical GVHD or antidonor reactivity as a
consequence of our treatment very unlikely. We have followed animals
for a year after BMT, and no signs of GVHD or loss of donor-specific
tolerance have been observed.
The development of central tolerance depends on the appropriate
presentation of antigens to developing thymocytes. The DCs represent
the principal blood-borne antigen-presenting cells in the thymus. A
short course of CYA depletes the thymus of the medullary DCs. In one
study, Brown Norway (BN) rats received (LEW × BN) F1 splenocytes followed by a short course
of CYA.16 After CYA was stopped, the F1 cells were rapidly
recruited to the thymus, and by 10 days after CYA, they were localized
to the corticomedulary junction, the natural location of thymic
dendritic cells.16 We hypothesized that the facilitated
recruitment of new donor-derived DCs into the host thymus might
therefore enhance deletion of alloreactive host-derived T cells and
tolerance induction to alloantigens. Since the host and not the donor
hematopoietic progenitors are subjected to 3 Gy WBI, donor progenitors
might have an advantage over those of the host for early repopulation
of the thymus. This is suggested by the increased levels of donor
T-cell reconstitution observed at relatively early time points in
CYA-pretreated mice.
Although corticosteroids are known to enhance immunosuppression and to
deplete DP thymocytes, therefore potentially creating thymic space,
they did not mediate any beneficial effect on donor marrow engraftment
or tolerance induction. Surprisingly, the addition of corticosteroids
to CYA pretreatment impeded the engraftment of donor cells and the
induction of tolerance. In a recent study, dexamethasone, which induces
cortical involution, did not lead to thymic recruitment of
(LEWxBN) F1 DCs when administered to BN
rats.16 CYA, which induces medullary involution, had the opposite effect.16 Accordingly, by depleting the medulla of DCs, CYA may create space for the homing of new DCs to the medulla during thymic regeneration. In contrast, the cortical involution induced by steroids fails to create space in the medulla for new DCs.
Our data suggest that the detrimental effect of corticosteroids on
tolerance induction may be due to impairment of engraftment of donor
hematopoietic cells. As shown in Figure 2, the animals that received
corticosteroids showed a lack of chimerism as early as 5 weeks
post-BMT, suggesting an initial failure to engraft. We do not know of
any reason to expect such an effect or of any precedent for it, but the
result emphasizes the necessity for a careful analysis of the effects
of administering of corticosteroids around the time of BMT.
Overall, our studies indicate that specific measures are required to
permit donor progenitors to repopulate the recipient thymus at high
levels. CYA pretreatment could be a valuable, relatively nontoxic, and
highly specific treatment for creating a permissive thymic environment
for donor-derived cells. Instead of receiving CYA for a prolonged
period, a recipient would receive only a short course of CYA
followed by BMT from a living donor as a lifelong source of
donor-derived DCs. Donor hematopoietic stem cell engraftment would
result in a robust state of central deletional tolerance to organ
grafts from the same donor. This approach might also be potentially
applicable to xenotransplantation, in which the host could be
pretreated with CYA for several weeks in anticipation of marrow and
organ xenotransplantation.
| |
Acknowledgments |
The authors thank Diane Plemenos for assistance in preparing
the manuscript; Drs Dave Anderson, Hugh Auchincloss, and Joey Kurtz for
helpful review of the manuscript; and Dr David H. Sachs for helpful advice.
| |
Footnotes |
Submitted January 27, 2000; accepted March 30, 2000.
Supported by National Institutes of Health Grant ROI HL49915 and
by a sponsored research agreement between Massachusetts General Hospital and BioTransplant, Inc. Supported in part (B.N.) by The Daland
Fellowship for Research in Clinical Medicine (American Philosophical
Society) and an American Society of Transplant
Physicians-Novartis Fellowship in Transplantation.
Reprints: Megan Sykes, Bone Marrow Transplantation Section,
Transplantation Biology Research Center, Massachusetts General
Hospital, MGH East, Building 149-5102, 13th St, Boston, MA 02129;
e-mail: megan.sykes{at}tbrc.mgh.harvard.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction