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*
Department of Pediatrics, Division of Bone Marrow Transplantation, and
Department of Therapeutic Radiology, University of Minnesota Hospital and Clinic, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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+ than CD4+ T cell-mediated murine GVHD.
To determine how RAPA inhibited GVHD, thoracic duct lymphocytes (TDL)
were isolated from recipients of allogeneic donor grafts. Compared with
controls, RAPA-treated recipients had a marked decrease in donor TDL T
cell number between days 5 and 24 posttransplant. CD8+ T
cell expansion was preferentially inhibited. RAPA inhibited Th1 or Th1
cytotoxic (Tc1) cytokines, but not Th2 or Tc2, cell generation. In situ
mRNA hybridization also showed that TDL T cells from RAPA-treated mice
had a lower frequency of granzyme B+ cells, indicating that
RAPA inhibited the generation of CTL capable of mediating cytolysis
through the release of granzyme B. In another system, RAPA was found to
inhibit the GVL response of delayed donor lymphocyte infusions. Since
CD8+ T cells are the primary effectors in this system,
these data suggest that RAPA directly interfered with GVL effector cell
expansion or function. We conclude that RAPA is effective in inhibiting
Th1 or Tc1 cytokine production and CD8+ and
TCR+ T cell-mediated GVHD, but abrogates GVL. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In several of the GVHD model systems, RAPA-treated recipients had mean
weight values identical with recipients of pan-T cell-depleted (TCD)
donor grafts (12, 13). It is possible that RAPA administration prevents
GVHD lethality by reducing the expansion of alloreactive donor T cells,
such that a critical threshold of donor T cells is not reached.
Alternatively, RAPA may modify GVHD reactivity by inducing
counter-regulatory cells such as Th2 or Tc2 (IL-4- and IL-10-producing)
cells or at least skewing alloreactive T cell responses away from T
cell responses generally associated with acute GVHD lethality, i.e.,
Th1 or Tc1 (IL-2- and IFN--producing) cells (16, 17, 18, 19, 20, 21). In addition,
RAPA may inhibit the in vivo capacity of cytolytic donor
CD8+ T cells or CD4+ T cells to mediate the
tissue-destructive effects of GVHD (22, 23, 24, 25, 26). The effect of RAPA on
CD4+ vs CD8+ T cell-mediated GVHD is
unknown.
The present study was undertaken to determine 1) the T cell types
(CD4+, CD8+, TCR +) that are
most susceptible to RAPA administration, 2) the effect of RAPA on Th1
or Tc1 and Th2 or Tc2 induction in heavily irradiated allogeneic BMT
recipients, and 3) whether the graft-vs-leukemia (GVL) effect mediated
by the delayed post-BMT donor splenocyte infusion was adversely
affected by RAPA administration. Together, these studies provide the
first detailed examination of how RAPA administration alters donor
anti-host T cell responses in irradiated BMT recipients of
allogeneic inocula.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.BR/SgSnJ (H2k), C.H-2bm1
(termed bm1), and C.H2bm12 (termed bm12) mice were
purchased from The Jackson Laboratory (Bar Harbor, ME). B6
(H2b: CD45.2) mice and B6-CD45.1 congenic mice were
purchased from the National Institutes of Health (Bethesda, MD). Adult
H-2d/d TCR -expressing transgenic mice (G8) were
originally generated by the cross of a homozygote G8 transgenic male to
BALB/c females and then backcrossed for five generations before
establishing a breeding colony (27). Mice were housed at the University
of Minnesota Hospital in a specific pathogen-free facility in
microisolator cages. Donors and recipients were 8 to 10 wk of age at
the time of BMT.
Bone marrow transplantation
For examining the effects of RAPA on inhibiting GVHD induced by
either CD4+ T cells or CD8+ T cells, MHC
class II (bm12) or class I (bm1) disparate recipients were sublethally
irradiated on day 0 with 6.0 Gy of TBI from a 137cesium
source at a dose rate of 85 cGy/min (28, 29). Four to six hours after
TBI, recipients were injected with highly purified lymph node (LN)
B6-CD45.2 or B6-CD45.1 CD4+ T cells (105 cells)
or CD8+ T cells (0.3, 1, or 3 x 106
cells). In other experiments, the donor and recipient strain
combination was reversed, and the same procedure was followed. To
examine the effects of RAPA on modifying GVHD induced by TCR
+ T cells, B6 recipients were sublethally irradiated
with 7.0 Gy of TBI on day 0 then injected with purified G8 TCR
+ T cells (2 x 106) (30) as
described below. For TCR + GVHD experiments only,
mice were given anti-CD8 and anti-NK1.1 (hybridoma PK-136, rat
IgG2a, provided by Dr. Gloria Koo, Merck-Sharpe-Dohme, Rahway, NJ) mAbs
(400 µg weekly from days -1 through day 28 post-BMT) to prevent host
cells from rejecting the BALB/c T cell inocula. Hematocrit values were
obtained at periodic intervals as an indicator of the possible bone
marrow-destructive effects of infused T cells (28, 29, 30, 31). Although T
cell-mediated hemopoietic destruction contributes to the lethality
effect of donor T cells in these three different systems, recipients
have histologic evidence of GVHD, indicating that the donor T cells
have tissue-destructive capabilities as well (28, 29, 30).
To purify LN cells, single cell suspensions of axillary, mesenteric,
and inguinal LN cells were obtained (as a source of GVHD-causing
effector cells) by passing minced LN through a wire mesh and collecting
them into PBS supplemented with 2% FCS. Cell preparations were
depleted of NK cells and enriched for CD4+,
CD8+, or TCR + T cells by depletions with
anti-CD8 (hybridoma 2.43, rat IgG2b, provided by Dr. David Sachs,
Cambridge, MA), anti-CD4 (hybridoma GK1.5, rat IgG2b, provided by
Dr. Frank Fitch, Chicago, IL), or both mAb, respectively. mAb-coated T
cells were passaged through a goat anti-mouse and goat anti-rat
Ig-coated column (Biotex, Edmonton, Canada). The final composition of T
cells in the donor graft was determined by flow cytometry and was
always found to be 
94% T cells of the desired phenotype.
GVL studies
Our GVL induction procedure has been described in detail previously (32). In brief, mice undergoing BMT for GVL assessment were conditioned with 800 cGy irradiation from an x-ray source on day -1 and infused with either 8 x 106 T cell-depleted (anti-Thy1.2 (clone 30-H-12, provided by Dr. David Sachs, Cambridge, MA) and complement-treated) allogeneic B10.BR BM on day 0. Donor B10.BR splenocytes were administered i.v. at a dose of 25 x 106 on day 21 post-BMT. On day 28 post-BMT, mice were challenged with an MHC class I+, class II- acute myeloid leukemia line, C1498 (32). C1498 (American Type Culture Collection, Rockville, MD), originally derived as a spontaneous tumor from a B6 mouse, was grown in RPMI (Life Technologies, Grand Island, NY) with 10% heat-inactivated FBS (HyClone, Ogden, UT), 2 mmol/l L-glutamine, MEM amino acids, 1 mmol/l sodium pyruvate, 50 mmol/l 2-ME, and penicillin/streptomycin (33, 34). Frozen stocks were used for experiments. C1498 cells (2 x 105) were given via the i.v. route. All available animals were examined for evidence of GVHD and tumor, and were extensively examined by necropsy. Representative mice were examined for histologic evidence of tumor or GVHD as previously described (32).
Thoracic duct cannulation studies
To induce lethal GVHD mediated by CD4+ and CD8+ T cells, B10.BR recipients were irradiated with 8.0 Gy of TBI delivered by x-ray at a dose rate of 0.41 cGy/min as previously described (12). Recipients were given 10 x 106 splenocytes along with 8 x 106 B6 bone marrow cells treated with anti-Thy1.2 (antibody 30-H-12, rat IgG2b, provided by Dr. David Sachs, Boston, MA) plus complement (Nieffenegger, Woodland, CA) as described previously (12). The bone marrow plus splenocyte inoculum was administered via the caudal vein in a 0.5-ml volume. For TDL isolation, cannulae were inserted in the thoracic duct of recipients at the indicated times post-BMT, and TDL were collected over a period of 18 h (28, 29).
RAPA and cyclosporin A (CsA) administration
RAPA was administered as a suspension in carboxymethylcellulose (CMC; Sigma, St. Louis, MO) (12). A stock solution of 2.5 mg/ml rapamycin in CMC was prepared. For GVHD experiments, RAPA was resuspended in CMC at a final concentration of 0.2% CMC for injections and administered at doses of 1.5 mg/kg/dose i.p. beginning on the day of or the day before BMT and continuing daily for 14 days, and then three times per week until 21 or 28 days post-BMT, as indicated. This RAPA dose and schedule have been shown to be optimal in preventing GVHD in each donor-recipient strain combination tested to date. CMC was administered to controls. CsA, provided by Dr. Arcesse (Sandoz Pharmaceuticals, Hanover, NJ), was prepared from lyophilized material (SandImmune, lot 3905 692; 50 mg/ml), resuspended in 0.2% CMC and administered at doses of 10 mg/kg (13) according to the schedule described above for RAPA. For GVL experiments, RAPA was administered daily for 14 days (days 20–34), then three times per week for an additional 2 wk (through day 50) as described for GVHD experiments.
Flow cytometry
The T cell, B cell, and granulocyte/macrophage constituency of
splenocytes or TDL effluent was measured using mAb directed toward CD4
or CD8, CD45R/B220, and Mac1, respectively. All fluorochrome-labeled
mAb, unless indicated, were obtained from PharMingen (San Diego, CA).
To determine donor or host origin of B10.BR recipients of B6 donor
grafts, single cell suspensions were costained with
H2k FITC-labeled mAb or
H2b-phycoerythrin mAb. For bm1 recipients of B6-CD45.2
CD8+ T cells, splenocyte suspensions were monitored with
CD45.2 (clone 104-2, rat IgG2a) and CD45.1 (clone A20-1.7, rat
IgG2a), both provided by Dr. U. Hammerling (New York, NY). For splenic
flow cytometry studies, cells were first incubated with 2.4G2 to block
Fc receptors and then incubated with an optimal concentration of
fluorochrome-labeled mAb for 45 min at 4°C. Cells were washed three
times and resuspended for analysis. TDL were also assessed for the
expression of activation Ags (CD25 (IL-2R -chain), CD40 ligand
(gp39) CD69, CD80 (B7-1), CD86 (B7-2), CD132 (OX40), and CD152
(CTLA-4)) or Ags associated with an effector/memory cell phenotype
(CD62 ligand (L-selectinlow)) by two- or three-color flow
cytometry using FITC-, phycoerythrin-, or biotin (along with
SA-PerCP)-conjugated mAb purchased from PharMingen or Becton Dickinson
(Mountain View, CA). Irrelevant mAb control values were subtracted from
values obtained with relevant mAbs. All results were obtained using a
FACScan (Becton Dickinson). Forward and side scatter settings were
gated to exclude red cells and debris, and 0.7 to 1 x
104 cells were analyzed for each determination.
In vitro MLR cultures
For quantifying alloantigen responses, TDL T cells from post-BMT
or non-BMT B6 controls were mixed with irradiated (30 Gy) Thy1.2
plus complement-treated splenocyte stimulators from B10.BR or B6 mice
(29). Cells were suspended in DMEM (BioWhittaker, Walkersville, MD),
10% FCS (HyClone, Logan, UT), 2-ME (5 x 10-5
M; Sigma), 10 mM HEPES buffer, 1 mM sodium pyruvate (Life
Technologies), and amino acid supplements (1.5 mM
L-glutamine, L-arginine, and
L-asparagine; Sigma), antibiotics (penicillin, 100 U/ml;
streptomycin, 100 µg/ml; Sigma). Cell responders (105)
and irradiated stimulators (5 x 105) were plated into
96-well round-bottom (Costar, Cambridge, MA) plates and placed at
37°C and 10% CO2 for 1 to 4 days. 
counts per minute
were calculated by subtracting the syngeneic proliferative response
from the allogeneic proliferative response. Supernatants were harvested
at the indicated times and analyzed for IL-2, IL-4, IL-10, and IFN-
levels using ELISA kits (R&D Systems, Minneapolis, MN) according to the
manufacturer’s recommendations.
Histology and in situ antisense:sense mRNA hybridization of tissues
Mice were sacrificed and autopsied, and tissues were taken for
histopathologic analysis. Samples were placed in either 10% neutral
buffered formalin and then embedded in paraffin or were embedded in OCT
compound and snap-frozen in liquid nitrogen. Organs were scored
positive for GVHD if there was single cell necrosis (skin, colon),
crypt dropout (colon), periportal infiltrate with acute necrosis
(liver), or endothelialitis with a lymphocytic infiltrate (lung) (35).
In previous studies, these features were present only in mice with
active GVHD and not in irradiated recipients of either syngeneic or
Thy1.2 plus complement-treated fully allogeneic BM (35). For in situ
hybridization, TDL cytospins were hybridized with riboprobes specific
for IFN- bases (1–270), IL-10 (392–937), granzyme A (80–910), and
granzyme B (239–775) mRNA. The mRNA:RNA probe hybrids were detected
using an alkaline phosphatase -digoxigenin F(ab')2
Ab and 5-bromo-4-chloro-3-indoyl-phosphate/nitro blue tetrazolium color
substrate. The in situ hybridization technique has been described
previously (36, 37).
Statistical analyses
Group comparisons of continuous data were made using Student’s
t test. Survival data were analyzed by lifetable methods
using the Mantel-Peto-Cox summary of 
2 (38). Actuarial
survival and relapse rates are shown. p < 0.05 was
considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether RAPA could inhibit CD4+ T
cell-mediated GVHD, sublethally irradiated bm12 recipients
(n = 8/group) were given the minimal uniformly
lethal dose of purified CD4+ T cells (105).
RAPA-treated recipients had a significantly higher actuarial survival
rate than controls (Fig. 1
A). However, only 12%
of RAPA-treated recipients survived the first month posttransfer,
indicating that the benefits of RAPA were modest. While day 14 mean
hematocrit values in RAPA-treated mice were significantly higher than
those in controls (34 vs 18%, respectively), RAPA-treated recipients
developed GVHD-induced aplasia, manifested by a progressive anemia (day
21 mean hematocrit values <25% in RAPA-treated mice). Therefore,
despite the fact that we have previously shown that RAPA administration
increases hematocrit values above those in non-BMT control mice, RAPA
administration could not prevent CD4+ T cell-mediated
aplasia. Mean weight curves, reflective of survival rates and
hematocrit, demonstrated a 1-wk delay in the onset of a severe
GVHD-induced weight loss (Fig. 1B).
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bm12 system
and to optimize the likelihood for finding a beneficial effect of RAPA,
we changed our GVHD model to the somewhat less aggressive bm12B6
GVHD model system. Sublethally irradiated B6 recipients were infused
with bm12 CD4+ T cells (105), and a cohort of
recipients was given RAPA. Two replicate experiments were performed
with similar results, and the data were pooled for analysis. Although
RAPA-treated recipients had a significantly (p
= 0.0089) higher actuarial survival rate than the controls (25 vs 0%,
respectively; n = 16/group), only a minority of
RAPA-treated recipients survived long term (data not shown). Compared
with controls, RAPA-treated mice had day 14 mean hematocrit values (26
vs 19%) and mean weight curves (not shown) that were only modestly
superior. Experiments in both model systems are consistent with the
fact that RAPA administration is unable to prevent the majority of
sublethally irradiated recipients of CD4+ T cells from
succumbing to GVHD-induced lethality.
In contrast to CsA, RAPA inhibits cytokine responses, leaving cytokine
production unimpaired. CsA coadministered with RAPA had a dramatic
effect on inhibiting CD4+ T cell-mediated GVHD-induced
mortality (Fig. 2). While none of the
recipients given RAPA or CsA survived beyond 1 mo posttransfer, 75% of
RAPA- plus CsA-treated recipients survived the 3-mo observation period.
Day 14 mean hematocrit values were highest in the RAPA- plus
CsA-treated group than in the recipients given RAPA, CsA, or neither
agent (34 vs 28 vs 16 vs 19%, respectively). By day 28, mean
hematocrit values in the RAPA- plus CsA-treated group had normalized to
45%, and only one of the mice in the other three groups (mean
hematocrit value = 8%) survived to this time point for analysis.
Consistent with the survival and hematocrit data, RAPA- or CsA-treated
recipients experienced severe GVH-induced weight loss before death (not
shown). In contrast, the mean body weight values in RAPA- plus
CsA-treated mice exceeded pre-BMT body weights by 2 mo posttransfer.
Splenic flow cytometry of RAPA- plus CsA-treated mice showed marked T
and B cell lymphopenia (Table I). Mice
also had histologic evidence of GVHD predominantly involving the lung
and liver and, to a far lesser extent, the colon and skin.
Collectively, these data indicate that inhibition of cytokine responses
by RAPA alone is insufficient to inhibit CD4+ T
cell-mediated GVHD. CsA coadministration is necessary to down-regulate
cytokine production. When administered together, RAPA and CsA are
highly effective in inhibiting CD4+ T cell-mediated GVHD
lethality.
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+ T cells
infused into sublethally irradiated MHC class I disparate recipients
To determine whether CD8+ T cell-mediated GVHD
lethality was susceptible to RAPA inhibition, sublethally irradiated
bm1 recipients were given a uniformly lethal CD8+ T cell
dose (106) from B6 donors. Two replicate experiments with
pooled actuarial survival data (n = 16
mice/group) were performed with similar results, showing that RAPA is
highly effective in preventing CD8+ T cell-mediated GVHD
lethality (Fig. 3A).
RAPA-treated recipients had a significantly (p
= 0.0002) higher actuarial survival rate than controls (94 vs 0%,
respectively). Day 14 mean hematocrit values in RAPA-treated mice were
significantly higher than control values (38 vs 10%, respectively).
Although RAPA-treated recipients regained their pre-BMT body weight by
day 46 posttransfer (Fig. 3B), mice were not entirely
GVHD free by clinical assessment, as confirmed by histologic assessment
at this time (not shown). Splenic flow cytometry of RAPA-treated
recipients (n = 4) analyzed 2 mo posttransfer
showed a mean of 8.5 x 106 donor CD8+ T
cells, representing at least an eightfold expansion over input cell
number and a dramatic CD4+ T cell lymphopenia (Table I
).
Thus, RAPA did not preclude donor CD8+ T cell expansion or
GVHD target tissue damage.
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B6 model. Sublethally irradiated B6 recipients
(n = 8/group) were given 0.3, 1, or 3 x
106 purified donor bm1 CD8+ T cells. Cohorts of
mice were treated with RAPA. In the bm1B6 system, the administration
of RAPA was associated with a significant increase in actuarial
survival rates. There was a statistical trend
(p = 0.068) toward a higher actuarial survival
rate in RAPA-treated recipients of 3 x 106
CD8+ T cells compared with control recipients of
106 (75 vs 38%, respectively), indicating at least a
threefold reduction in the capacity of CD8+ T cells to
mediate lethal GVHD. Day 14 hematocrits were significantly
(p < 0.001) higher in RAPA-treated recipients
compared with their respective controls. Consistent with both the
survival and mean body weight data, RAPA-treated recipients of 3
x 106 CD8+ T cells had significantly
(p = 0.001) higher day 14 hematocrit values
than control recipients of 106 CD8+ T cells (37
vs 28%, respectively).
In other studies, the effect of RAPA on inhibiting CD8+ T
cell-mediated GVHD lethality was quantified using the more aggressive
B6bm1 system. RAPA-treated recipients had a significantly higher
actuarial survival rate than their respective CD8+ T cell
dose controls (n = 8/group). RAPA-treated
recipients of 3 x 106 CD8+ T cells had a
significantly (p = 0.018) higher actuarial
survival rate than control recipients of a 10-fold lower
CD8+ T cell dose (3 x 105
CD8+ T cells; 80 vs 0%, respectively). RAPA-treated
recipients of 1 x 106 CD8+ T cells had
lower mean weight curves and a significantly (p
= 0.030) lower 6.5-mo actuarial survival rate than control recipients
of 1 x 105 CD8+ T cells (40 vs 80%,
respectively). Day 14 mean hematocrit values were 8 and 4% in control
recipients and 36 and 26% in RAPA-treated recipients of 1 or 3 x
106 CD8+ T cells, respectively. Control
recipients of 0.1 x 106 CD8+ T cells had
a day 14 mean hematocrit value equivalent to that in RAPA-treated
recipients of 106 CD8+ T cells (35 vs 36%,
respectively). Flow cytometry results of day 159 posttransfer
splenocytes were similar in the latter two groups
(n = 3–4 mice/group; Table I). Recipients in
both groups were noted to have modest T and B cell lymphopenia and
histologic evidence of GVHD. Similar flow cytometric and histologic
findings were observed in both groups, suggesting an equivalent
severity of GVHD. Taken together, these data suggest that RAPA
administration led to an approximately 10-fold reduction in the GVHD
lethality capacity of CD8+ T cells, while the lethality
capacity of donor CD4+ T cells was reduced <3-fold.
To determine whether RAPA could inhibit the GVHD-induced lethality of a
different type of CTL population, TCR + T cells, we
used our previously described sublethal TBI model system in which the
infusion of highly purified allogeneic (H2d) G8 TCR
+ T cells into B6 recipients results in lethal GVHD
(30). Compared with control recipients of TCR + T
cells, RAPA-treated recipients of G8 transgenic TCR +
T cells (2 x 106) had a significantly
(p = 0.00096) higher actuarial survival rate,
similar (p = 0.16) to that in recipients not
receiving donor T cells (0 vs 88 vs 100%, respectively; Fig. 4A). Although tissues
were not available for assessment of GVHD pathology, RAPA-treated mice
had clinical and body weight evidence of transient GVHD (Fig. 4B). Compared with control recipients of TCR
+ T cells, RAPA-treated recipients had significantly
higher day 14 mean hematocrit values comparable to those in irradiation
controls (15 vs 25 vs 27%, respectively). Flow cytometry of
splenocytes from long term recipients did not reveal the presence of
donor TCR + T cells (Table I). In earlier studies in
which high dose TBI was used to generate TCR + T
cell-mediated GVHD in this strain combination, splenic flow cytometry
performed on day 9 post-BMT revealed a high proportion of splenic
donor-derived TCR + T cells (12%) and more total TCR
+ T cells that localized to the spleen at that time
than initially infused (30). The current data suggest that RAPA
interfered with donor TCR + expansion post-BMT.
Compared with the irradiation controls, RAPA-treated recipients also
had no evidence of T or B cell lymphopenia. RAPA-treated mice, in fact,
had an unexplained relative increase in the number of B220+
cells. Thus, RAPA treatment markedly reduced GVHD-induced lethality,
but did not prevent a transient GVH reaction.
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In previous studies involving heavily irradiated recipients, we
have observed that RAPA administration significantly increased
actuarial survival rates in each of four donor-recipient strain
combinations differing at MHC class I and II loci, such that 50 to
100% of RAPA-treated mice vs 0 to 12% of controls survived 2 mo
post-BMT (12, 13). Our data, described above, show that
CD8+ T cell-mediated GVHD lethality is more
susceptible to RAPA inhibition than is GVHD induced by CD4+
T cells. Because CD4+ and CD8+ T cells both
expand during the GVHD response against MHC class I and II disparate
recipients, we next asked whether RAPA administration prevented GVHD
lethality in full MHC disparate recipients by inhibiting only
CD8+ T cell expansion or by inhibiting both
CD4+ and CD8+ T cell expansion.
To accomplish this goal, TDL were isolated by thoracic duct cannulation, which was performed at sequential time periods post-BMT. There is a high degree of recirculation of T cells through the lymphatics in normal mice (28, 29, 39, 40, 41). In BMT mice, alloreactive T cells gain access to the lymphatics and can be isolated in high numbers via overnight cannulation. TDL cells isolated early post-BMT are comprised almost exclusively of donor T cells. Since there is a continual migration of alloreactive donor T cells into the lymphatics, TDL T cell enumeration provides the most accurate estimate of T cell expansion possible post-BMT. Moreover, TDL T cell function can be directly assessed without further T cell purification, as would be necessary if splenocytes were analyzed.
To quantify donor T cell expansion, heavily irradiated RAPA or
vehicle-treated control B10.BR recipients were given B6 BM plus
supplemental splenocytes containing an average of 2.4 x
106 CD4+ T cells and 1.4 x
106 CD8+ T cells. At various time periods
post-BMT, mice were cannulated for quantification of TDL T cell
production. Two replicate experiments were performed, and data were
pooled for analysis (Fig. 5). RAPA
administration inhibited the generation of CD4+ and
CD8+ TDL T cells, which was particularly evident on days 5
and 7 post-BMT, the time of peak donor T cell expansion. For example,
on day 5 post-BMT, control recipients produced about 14.8 x
106 TDL T cells/day (0.62 x 106 TDL T
cells/ml/h), while RAPA-treated mice produced only 0.9 x
106 TDL T cells per day, which was a 94% reduction in TDL
T cell number per day (Fig. 5A). Compared with
controls, CD4+ TDL T cell production was reduced by 91%
(Fig. 5B) and CD8+ TDL T cell production
was reduced by 95% (Fig. 5C) in RAPA-treated
recipients on day 5 post-BMT. However, by day 7 post-BMT,
CD4+ TDL T cell expansion was reduced by only 26%, while
CD8+ TDL T cell content was reduced by 82% in RAPA-treated
mice compared with controls. The greater degree of inhibition of donor
CD8+ vs CD4+ T cell expansion in RAPA-treated
mice compared with controls was evident through the first 15 days
post-BMT. Thus, RAPA administration had a longer lasting effect on
inhibiting donor CD8+ vs CD4+ T cell
expansion.
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). Both CD4+ and
CD8+ TDL from vehicle-treated controls and RAPA-treated
recipients had high levels of each activation Ag examined (CD25 (IL-2),
CD80 (B7-1), CD86 (B7-2), CD134 (OX40), CD152 (CTLA-4)). In addition,
CD40 ligand was up-regulated on CD4+ TDL T cells, and CD69
was up-regulated on both CD4+ and CD8+ TDL T
cells (data not shown). The only evident difference between the control
and RAPA-treated recipients was the lack of CD62 ligand (L-selectin)
down-regulation in the latter group. Each of these findings was
observed on days 7 and 9 post-BMT and was entirely replicated in a
second experiment (data not shown).
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) were primed to host alloantigens
in vivo, as evidenced by the high proliferative response of TDL T cells
to host splenocytes, which peaked on day 1 or 2 of culture (Fig. 6). No
apparent differences in the proliferative responses between
RAPA-treated and vehicle-treated TDL were observed despite the fact
that similar proportions of CD4+ T cells were plated per
well in the two groups (56 and 49%, respectively). To determine
whether TDL T cells from these two groups had different Th or Tc
profiles, cytospins of TDL T cells used for the MLR studies were
analyzed by in situ mRNA hybridization. The frequency of TDL T cells
from RAPA-treated recipients that expressed IFN- (Fig. 7) mRNA was lower than that in TDL T
cells obtained from vehicle-treated controls. In contrast, the
frequency of TDL T cells from RAPA-treated recipients that expressed
IL-10 mRNA (Fig. 7) was higher than that in vehicle-treated controls.
Consistent with the in situ hybridization findings, TDL T cells
isolated from RAPA-treated, but not vehicle-treated control, recipients
were skewed away from a Th1 or Tc1 phenotype, as determined by MLR
supernatant ELISA assays for IL-2 and IFN- (Th1) and IL-4 and IL-10
(data not shown).
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; no increases in the frequency
of IL-10 mRNA-positive TDL T cells in the RAPA-treated group over the
relative high frequency in the control group were noted. Collectively,
the in situ mRNA hybridization and ELISA studies indicate that RAPA
treatment skews TDL T cells away from a Th1 or Tc1 phenotype. In vivo administration of RAPA inhibits the GVL effect of delayed lymphocyte infusion (DLI) against AML cells after allogeneic BMT
Because RAPA was highly effective at inhibiting GVHD lethality in
a number of different donor-recipient strain combinations differing at
MHC class I and II loci, we asked whether GVL also would be diminished
by RAPA. For this purpose, we used a B10.BRB6 GVL system in which
donor splenocytes given on day 21 post-BMT permit the recipient to
resist a supralethal dose of AML cells given 1 wk later. DLI has become
a mainstay of therapy for leukemia patients relapsing post-BMT
(43, 44, 45, 46).
Previously, we had shown that RAPA significantly reduces GVHD lethality
in this strain combination, such that 52% of RAPA-treated recipients
given 25 x 106 donor splenocytes on day 0
post-BMT survived 2 mo post-BMT vs none of the controls (13). In the
present studies, B6 recipients were heavily irradiated (day -1),
infused with B10.BR TCD BM (day 0), and then given B10.BR donor
splenocytes (25 x 106 cells) on day 21 post-BMT with
or without concurrent RAPA (1.5 mg/kg/dose daily from days 20–34
post-BMT then three times weekly through day 50 post-BMT) (Fig. 8).
Recipients given supralethal doses of C1498 (2 x 105)
cells iv on day 28 post-BMT all died of leukemia by day 51 post-BMT
(Fig. 8, A and
C). Compared with mice given C1498 alone, mice given
DLI and C1498 cells had a significantly (p =
0.00069) higher actuarial survival rate, with a prolongation of the
median survival time from 49 to 75 days. RAPA administration removed
most, but not all, of the GVL effect of DLI in this model system. As
control recipients of DLI and C1498, mice also given RAPA had a
significantly (p = 0.0012) lower survival rate
and a higher incidence of leukemia (Fig. 8, A andC). A slight GVL effect was still evident, however, since
recipients given DLI, C1498, and RAPA had a higher
(p = 0.0044) actuarial survival rate than
controls given C1498 without either DLI or RAPA. However, the
difference in median survival time between these two groups was only
2 days.
|
B) evidence of
GVHD. Interestingly, RAPA administration prevented the GVH-induced
weight loss of DLI. Collectively, these data indicate that RAPA
administration reduces both the anti-AML GVL- and GVH-related side
effects of DLI after allogeneic BMT. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ T
cell more so than CD4+ T cell-mediated lethality in
sublethally irradiated allogeneic recipients. The coadministration of
CsA, required for the down-regulation of cytokine production, is
necessary to inhibit CD4+ T cell-mediated GVHD. 2) RAPA
administration reduces the expansion of donor CD8+ and, to
a lesser extent, CD4+ T cells in vivo. 3) RAPA
administration skews donor TDL T cells away from a Th1 or Tc1
phenotype, which also is associated with a reduction in the frequency
of cells expressing granzyme B mRNA. Although Th2 cell generation is
favored, TDL T cells exposed to RAPA are stimulated by host
alloantigen-expressing cells, as evidenced by the up-regulation of T
cell activation Ags and in vitro response to host-type stimulator
cells. 4) RAPA administration markedly diminishes the GVL capacity
of DLI.
The greater biologic efficacy of RAPA in preventing GVHD lethality
mediated by CD8+ and TCR + T cells than
by CD4+ T cells is most consistent with the greater
dependency of the former than the latter cell types on paracrine growth
factor release. Although CD8+ and TCR + T
cells can produce low levels of cytokines, these cells typically
mediate cytolysis and are dependent upon CD4+ T cells for
help with activation and expansion. CD4+ T cells respond to
triggering stimuli via the release of cytokines that drive their own
activation and expansion as well as that of other cell types, including
CD8+ and TCR + T cells. The inhibition of
T cell cytokine responsiveness by RAPA can be overcome at high
concentrations of IL-2 (47). Autocrine growth factor production by
CD4+ T cells should result in higher local concentrations
of cytokines to CD4+ T cells than those produced by
paracrine stimulation of CD8+ and TCR + T
cells. The fact that CsA coadministration markedly increased the
efficacy of RAPA suggests that in situations in which cytokines are
limiting, as would occur with CsA inhibition of cytokine production,
the effect of RAPA on inhibiting GVHD is most pronounced. Moreover, in
a BMT setting in which donor CD4+ and CD8+ T
cells can expand, the inhibition of donor CD4+ T cell
expansion in RAPA-treated recipients was transient, being most evident
only on day 5 post-BMT. In contrast, in the same RAPA-treated
recipients, donor CD8+ T cell expansion was consistently
suppressed for 15 days post-BMT. These data support the hypothesis that
CD8+ T cells are more readily inhibited than
CD4+ T cells by RAPA due either to the greater dependence
upon or the lower levels of available paracrine growth factors for
CD8+ T cell responses.
It is possible that the cells capable of migrating into the thoracic duct lymphatics represent the small population of T cells that have escaped RAPA-mediated inhibition. Consistent with that hypothesis, detailed flow cytometric analysis of CD4+ and CD8+ T cells isolated between days 5 and 24 post-BMT did not show that RAPA administration interfered with the activation of either T cell subset as denoted by the up-regulation of CD25 (IL-2R), CD80 (B7-1), CD86 (B7-2), and CD134 (OX40). Consistent with the flow cytometry data, RAPA administration did not prevent the in vivo priming of donor T cells to host alloantigens, as assessed in an in vitro MLR culture. On the surface, these data differ somewhat from those of other studies demonstrating that T cells exposed to RAPA can be rendered tolerant. Recent studies by Boussiotis also show that RAPA induces tolerance in alloantigen-specific CD4+ T cell clones (48). Other studies by Chen et al. have shown that CD8+ T cells exposed to MHC class I disparate heart allografts under the cover of RAPA have compromises in intracellular signaling pathways dependent upon calcium flux after mitogen stimulation and on tyrosine phosphorylation upon TCR cross-linking (49).
Differences in the effect of RAPA on inhibiting the expansion of
CD4+ vs CD8+ T cells also could not be
accounted for by differences in the expression of CD152 (CTLA-4), a
molecule that can dampen T cell responses. CTLA-4 expression was
up-regulated on a similar proportion of CD4+ and
CD8+ T cells in RAPA-treated recipients (Table II).
Although TDL T cells isolated from vehicle-treated controls
down-regulated the expression of CD62 ligand (L-selectin) on both T
cell subsets throughout the day 5 to 24 period in each experiment,
RAPA-treated recipients maintained a high level of L-selectin
expression. High L-selectin expression could be associated with a
reduced capacity of these cells to migrate through the appropriate
endothelial cell barriers to gain access to relevant GVHD target
organs. The reason for the lack of L-selectin down-regulation in
RAPA-treated recipients is unknown.
RAPA administration precluded the up-regulation of Th1 or Tc1 cytokine
and granzyme B mRNA in TDL T cells. These findings may explain how RAPA
administration reduces the GVHD response mediated by two distinct types
of T cell populations typically characterized by CTL activity,
CD8+ T cells and TCR + T cells. The
frequency of TDL T cells isolated from RAPA-treated recipients that are
capable of producing IFN- was reduced compared with that of
vehicle-treated controls. Conversely, the frequency of TDL T cells
capable of producing IL-10 was increased in RAPA-treated recipients
(see Fig. 7). Supernatants obtained from MLR cultures established using
TDL T cells from RAPA- or vehicle-treated control recipients and
irradiated, T cell-depleted, host-type stimulators showed a
preferential skewing toward Th2 or Tc2 (IL-10-producing) cells in the
former and Th1 or Tc1 (IFN--producing) cells in the latter (not
shown). Acute GVHD has been classified as a Th1 or Tc1 disease process
in most studies (16, 17, 18, 19, 20, 21). The administration of donor T cells skewed ex
vivo toward a Th2 or a Tc2 phenotype can reduce the GVHD capacity of
nonmanipulated or Th1-containing donor T cell inocula (16, 17, 18, 19, 20).
Granulocyte CSF mobilization of allogeneic peripheral blood cells is
associated with the generation of donor T cells with a Th2 phenotype
and a reduced in vivo GVHD capacity compared with those of nontreated
cells (21).
The TDL T cell in situ hybridization data are consistent with in vivo studies in RAPA-treated recipients of solid organ grafts, demonstrating a skewing toward a Th2/Tc2 and away from a Th1/Tc1 phenotype (50, 51, 52). Th2 or Tc2 skewing may have resulted in the down-regulation of harmful T cell responses. The higher frequency of TDL T cells expressing IL-10 mRNA and the higher IL-10 protein content of MLR supernatants in RAPA compared with controls may be responsible in part for the GVHD protective effect of RAPA. Although the infusion of exogenous IL-10 in two different BMT model systems can accelerate GVHD lethality under certain conditions (53, 54), it is unknown what the operative mechanism(s) is for these effects. More compelling and direct evidence of the T cell immunosuppressive properties has been derived from studies of human (55) and murine T cells exposed to IL-10 in vitro (56). In an MLR reaction, the addition of exogenous IL-10 to a human bulk culture induced anergy that was long lasting (55). In other studies, the exposure of T cells to exogenous IL-10 has been shown to suppress the proliferation of Ag-specific CD4+ T cells in vitro and to prevent colitis induced by pathogenic CD4+ T cells in vivo (56).
Another important finding of our studies is that TDL T cells isolated from RAPA-treated allogeneic recipients post-BMT have a reduced frequency of granzyme B mRNA expression. Granzyme B, a serine protease released from CTL and NK cells, can cause apoptosis in target cells (42). The inhibition of CTL induction by RAPA has recently been shown to be associated with a reduction in granzyme B mRNA expression and enzymatic activity in mouse CTL induced in vitro with anti-CD3 mAb (57). Granzyme B expression in rat cardiac allograft recipients also is reduced by RAPA administration (58). The lack of granzyme B induction in TDL T cells isolated from RAPA-treated recipients may account at least in part for the preferential inhibitory effect of RAPA on CD8+ vs CD4+ T cell-mediated GVHD. In support of this hypothesis, Ley and coworkers have found that MHC class I-dependent GVHD is inhibited when granzyme B-deficient donor CD8+ T cells are infused, whereas MHC class II-dependent GVHD was not altered by infusing granzyme B-deficient CD4+ effectors (22, 26). Fas ligand-mediated lysis of Fas (APO-1: CD95)-expressing GVHD target cells is the primary effector cell mechanism for CD4+-mediated GVHD lethality in MHC class II disparate recipients (22, 26). Therefore, the relative inefficacy of RAPA in inhibiting CD4+ T cell-mediated GVHD compared with CD8+ T cell-mediated GVHD may be due to the differential inhibitory effects of RAPA on granzyme B vs Fas ligand induction.
The RAPA-induced suppression of the GVL effects of DLI also may be related to a shift away from a Th1/Tc1 phenotype, as manifested by the cytokine and granzyme B mRNA expression data. In the DLI model system used, the GVL effect against MHC class I+, class II- C1498 cells is mediated by CD8+ T cells (32). Therefore, GVHD preventive strategies such as RAPA administration that preclude CD8+ T cell expansion or function should have adverse effects on the GVL response of DLI. Fowler and Gress have shown that Tc1-mediated GVL is more potent than Tc2-mediated GVL against myeloid leukemia in mice (59, 60). At the same time, Tc2 recipients had essentially no histologic evidence of GVHD, whereas Tc1 recipients had mild to moderate GVHD.
In summary, we have shown that RAPA administration is highly effective
in preventing GVHD mediated by CD8+ and TCR
+ T cells, while CD4+ T cell-mediated
GVHD is more resistant to RAPA inhibition. RAPA administration
inhibited the GVL effect of DLI. These findings are consistent with the
potent ability of RAPA to inhibit CD8+ T cell expansion and
granzyme B induction as well as to skew TDL T cells toward a Th2/Tc2
phenotype. Our data have important implications for designing human
clinical trials of RAPA in the context of allogeneic BMT.
|
Acknowledgments |
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|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Bruce R. Blazar, University of Minnesota Hospital, Box 109, Mayo Building, 420 S.E. Delaware St., Minneapolis, MN 55455.
3 Abbreviations used in this paper: RAPA, rapamycin; BMT, bone marrow transplantation; GVHD, graft-vs-host disease; TCD, T cell depleted; Tc, T cytotoxic GVL, graft-vs-leukemia; TBI, total body irradiation; LN, lymph node; TDL, thoracic duct lymphocytes; CsA, cyclosporin A; CMC, carboxymethylcellulose; low, low level; DLI, delayed lymphocyte infusion; AML, acute myeloid leukemia.
Received for publication December 2, 1997. Accepted for publication February 5, 1998.
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