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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3222-3233
Irradiated Donor Leukocytes Promote Engraftment of Allogeneic Bone
Marrow in Major Histocompatibility Complex Mismatched Recipients
Without Causing Graft-Versus-Host Disease
By
Edmund K. Waller,
Alan M. Ship,
Stephen Mittelstaedt,
Timothy W. Murray,
Richard Carter,
Irina Kakhniashvili,
Sagar Lonial,
Jeannine
T. Holden, and
Michael W. Boyer
From the Bone Marrow and Stem Cell Transplantation Center, Emory
University, Atlanta, GA.
 |
ABSTRACT |
Graft rejection in allogeneic bone marrow transplantation (BMT) can
occur when donor and recipient are mismatched at one or more major
histocompatibility complex (MHC) loci. Donor T cells can prevent graft
rejection, but may cause fatal graft-versus-host disease (GVHD). We
tested whether irradiation of allogeneic donor lymphocytes would
preserve their graft-facilitating activity while inhibiting their
potential for GVHD. Infusions of irradiated allogeneic T cells did not
cause GVHD in MHC-mismatched SJL  (SJL × C57BL6) F1, C57BL6 B10.RIII, and C57BL6 B10.BR mouse donor recipient BMT pairs.
The 60-day survival among MHC-mismatched transplant recipients
increased from 2% (BM alone) to up to 75% among recipients of BM plus
irradiated allogeneic splenocytes. Optimal results were obtained using
50 × 106 to 75 × 106 irradiated donor
splenocytes administered in multiple injections from day  1 to day
+1. Recipients of an equal number of nonirradiated MHC-mismatched
donor splenocytes uniformly died of acute GVHD. The graft facilitating
activity of the irradiated allogeneic splenocytes was mediated by donor
T cells. Irradiation to 7.5 Gy increased nuclear NF B in T cells and
their allospecific cytotoxicity. Irradiated T cells survived up to 3 days in the BM of MHC-mismatched recipients without proliferation.
Recipients of irradiated allogeneic splenocytes and allogeneic BM had
stable donor-derived hematopoiesis without a significant representation
of donor splenocytes in the T-cell compartment. Irradiated allogeneic T
cells thus represent a form of cellular immunotherapy with time-limited
biologic activity in vivo that can facilitate allogeneic BMT without
causing GVHD.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
GRAFT REJECTION AND graft-versus-host
disease (GVHD) represent two potentially lethal immunological side
effects of transferring hematopoietic cells between genetically
nonidentical individuals.1-5 Graft rejection represents an
immune response by host immune cells against donor stem cells. GVHD
represents a complementary immune response by donor T cells against
host cells and tissues. One approach to prevent fatal GVHD in
allogeneic bone marrow transplantation (BMT) has been to remove
immunocompetent T cells from the graft. Currently used methods of
T-cell depletion include elutriation,6,7 absorption to
lectin,8 application of specific monoclonal antibodies with
complement or immunotoxin,9-11 or by specific selection of
CD34+ donor stem cells.12 The potential to
successfully transplant T-cell-depleted (TCD) allografts from
antigenically mismatched donors without graft rejection or an increased
rate of leukemia relapse would greatly extend the availability of
allogeneic transplantation to patients without an antigenically matched
sibling donor.13
One potential method of enhancing engraftment by allogeneic stem cells
in transplantation is to add a population of donor immune cells that
are incapable of causing GVHD, but retain graft-facilitating activity.
Ionizing radiation has been shown to limit the capacity of lymphocytes
to proliferate while preserving their cytotoxicity against tumor
cells14 and their activity as veto cells against host
immune cells.15 A human T-cell line (TALL-104) has been shown to be effective in eliminating clonogenic human leukemic cells
when mixtures of irradiated T cells and leukemia cells were infused
into human bone xenografts in immunodeficient severe combined immunodeficiency (SCID)-hu mice.16 In theory, irradiated
donor lymphocytes could have all of the desirable short-term effects of
donor T-cell infusions without causing GVHD.
We reasoned that the presence of irradiated donor leukocytes could
counteract the ability of host cells to reject donor stem cells. Graft
rejection of TCD allogeneic transplants appears to be
mediated by a radioresistant host cell(s) that exerts a (transient) alloreactive cytotoxic effect against donor stem
cells.2,17-19 We hypothesized that irradiated allogeneic
donor T cells could facilitate BM engraftment by counteracting the host
cytotoxic cells thereby allowing the survival of repopulating donor
hematopoietic stem cells.
To test these hypotheses, we have used a model of graft failure for MHC
mismatched allogeneic BMT in mice. Recipients of BM allografts and
irradiated allogeneic spleen cells were successfully engrafted with and
repopulated by T cells derived from the donor BM cells, with a minimal
contribution from the spleen cell graft and without developing GVHD.
These studies provide the preclinical basis for adoptive immunotherapy
using multiple doses of irradiated donor lymphocytes in transplant
recipients without producing cumulative toxicity.
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MATERIALS AND METHODS |
Animals.
SJL (H2s), (SJL × C57.BL6) F1 (H2s/b),
B10.RIII (H2r), B10.BR (H2k), and CD45.1/CD45.2
congenic strains of C57.BL6 (H2b) mice aged 8 to 10 weeks
were purchased from Charles River/Jackson Laboratories (Bar Harbor,
ME). BA (Thy 1.1) mice on C57.BL6 (H2b) background were
obtained from Dr Miriam Lieberman (Stanford University, Palo Alto,
CA). CB17 scid/scid (Balb/c,
H2d background) mice were purchased from the Emory
University Animal Care Facility. Mice were given acidified sterile
water and maintained in Micro isolator cages (Lab Products Inc,
Maywood, NJ) at the Emory University Animal Care Facility. Experiments
were performed in conformance with the Guide for the Care and Use of
Laboratory Animals published by the National Academy Press, Washington,
DC, 1996, and approval by the Emory University Institutional Animal Care and Use Committee (IACUC).
Donor cell preparations.
BM cells were harvested from mice by removing the femora and tibia and
flushing the cells out of the BM shaft with sterile Hanks' balanced
salt solution (HBSS) containing 3% heat-inactivated fetal bovine serum
(HBSS/FBS) using a 25-gauge needle. Splenocytes were harvested by
perfusing the spleen with sterile HBSS/FBS.
Cytotoxicity assays.
Cytotoxicity assays were performed using the CytoTox 96 Assay
(Promega Corp, Madison, WI) following the manufacturer's protocol. Lysis of a fixed number of leukemia cells using a range of effector cell concentrations was calculated using the formula: % Cytotoxicity = 100 × (Experimental Effector Spontaneous Target
Spontaneous)/(Target Maximum Target Spontaneous). Separate
determinations of the spontaneous release from effector cells were
performed at each effector cell concentration.
T-cell depletion and enrichment of splenocytes.
Harvested spleen cells were suspended in phosphate-buffered saline
(PBS) containing 5 mmol/L EDTA and 0.1% bovine serum albumin (BSA) and
incubated with anti-FcR iii/ii antibody (Pharmingen, San Diego, CA) to
block nonspecific antibody binding. Monoclonal antibodies were obtained
from Pharmingen. The splenocytes were then incubated with saturating
concentrations of either (1) biotinylated anti-CD3 antibody (positive
selection) or (2) a combination of biotinylated anti-MAC-1,
anti-Gr-1, and anti-C19 antibodies (negative depletion). After
antibody staining, cells were washed once in HBSS/FBS, then resuspended
with 125 µL Streptavidin Microbeads (Miltenyi Biotech
Gmbh, Bergisch Gladbach, Germany) per 2.5 × 108
cells. The T-cell and non-T-cell fractions were separated by immunomagnetic chromatography using the Vario MACS magnetic separation column (Miltenyi Biotech, Gmbh). Using either the method of positive selection or the method of negative depletion, the final purity of T
cells was 75% to 85%. TCD splenocytes were the unbound cell fractions
from the anti-CD3 column.
Irradiation and reconstitution.
Recipient mice were exposed to 10 Gy or 11 Gy of radiation from a
137Cs source, delivered in 2 equal fractions 5 hours apart
at a dose rate of 1.24 Gy/min. Lethally irradiated mice were maintained on oral aqueous antibiotics (1.1 mg/mL neomycin sulfate and 1,000 U/mL
polymyxin sulfate) for 3 days before irradiation and for 4 weeks after
BMT. Splenocytes were irradiated using a single fraction from the same
137Cs source. All intravenous administrations were
performed using a 25-gauge needle to inject 0.2 mL of a cell suspension
prepared in HBSS/FBS into the retroorbital sinus into mice anesthetized with metofane or into a tail vein of recipient mice. BM transplant recipients were irradiated 1 to 2 days before BMT and received a single
injection of BM or the appropriate mixture of BM and splenocytes on day
0. Recipients of allogeneic splenocytes received 1 to 3 injection(s)
within the range of day 2 to day +1.
Analysis of hematopoietic engraftment of transplant recipients.
Mice were anesthetized with metofane and 0.2 mL peripheral blood was
collected from the retroorbital venous sinus at 1 to 4 months after
transplant. Red blood cells were depleted by 1G sedimentation using 3%
Dextran T500 in HBSS followed by hypotonic saline lysis.20
Fluorescence-activated cell sorting (FACS) analysis enumerated host and
donor leukocytes and T cells using a variety of monoclonal antibodies
specific for H2b and H2k MHC, as well as
specific leukocyte markers (Thy 1.1, Thy 1.2, CD45.1, and CD45.2;
Pharmingen). Propidium iodide was added at a concentration of 1 µg/mL and dead cells were electronically excluded.
Assessment of GVHD in transplant recipients.
All transplant recipients were evaluated for the presence of clinical
GVHD as manifested by weight loss, alopecia and/or ruffled fur,
diarrhea, and a decreased level of activity associated with a
"hunched over" appearance. Necropsy was performed after
euthanasia of moribund mice and of mice at predetermined time points.
Hematoxylin-eosin-stained tissue sections of liver, gastrointestinal
tract, and skin were examined microscopically for histologic evidence
of GVHD and scored according to published criteria.21
Abnormalities noted that were consistent with GVHD included single cell
apoptosis in the epidermis (acute GVHD) and epithelial thickening and
loss of adnexal structures of the skin (chronic GVHD). Cutaneous GVHD
was graded on a scale of 0-1; gastrointestinal GVHD was graded on a
scale of 0-2; and hepatic GVHD was graded on a scale of 0-3. An overall GVHD histologic score was calculated from the sum of individual scores
for skin, intestine, and liver. Normal tissue, with no evidence of
acute or chronic GVHD was given a score of 0. Severe GVHD, with
apoptosis and/or epithelial thickening and adnexal loss of the skin,
extensive sloughing of intestinal epithelium, and extensive hepatic
parenchymal injury and inflammation, was given a (maximal) score of 6.
Nuclear extraction and electrophoretic mobility shift assay (EMSA).
The CD3+ enriched cell fractions from mouse splenocytes
were selected with the Mini MACS system as described above and washed with PBS, then irradiated at 0, 7.5, or 30 Gy. After 6 hours incubation at 37°C, nuclear extracts were obtained according to the method of
Schulze-Ostoff.22 Briefly, the cell pellets were lysed with HMK buffer A (1 mol/L  EPES,
1 mol/L  gCl2, 1 mol/L
 Cl) containing 0.1% (wt/vol) Triton X at
4°C. Intact cell nuclei were collected by centrifugation, washed
with HMK buffer, then lysed in 20 mmol/L HEPES, 25% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA (HMK-NaCl)
buffer. The protein content of the nuclear extracts supernatants was
determined by Micro BCA Protein Assay. Constant amounts of nuclear
protein were mixed with gel-shift binding buffer and
32P-labeled oligonucleotide strands specific for NFB
(CD28RE, B, AP-1, and Nf-interleukin-2 [IL-2]).
The complex between the oligonucleotide and NFB was revealed by
autoradiography in an EMSA on a polyacrylamide gel in 0.5 × TBE
buffer run at 100 V. Quantitative autoradiography was performed using
the BETASCOPE 603 blot analyzer (Bio-Rad Model GS-700 Imaging
Densitometer, Hercules, CA).23
Flow cytometric analysis of intracellular cytokine expression.
The effect of irradiation on the levels of intracytoplasmic IL-2,
 -interferon (IFN), tumor necrosis factor (TNF), and IL-4 protein in
mouse T cells was measured by intracellular staining using flow
cytometry with fluorescent-conjugated monoclonal antibodies to specific
cytokines and T cells. Irradiated splenocytes were irradiated,
incubated at 37°C for 4 to 20 hours, then treated with Brefeldin A
(Sigma-Aldrich Corp, St Louis, MO) for an additional 4 hours before
fixation, permeabilization, and flow cytometric analysis.24
Statistical analyses.
Survival differences between different groups were calculated with the
Cox test comparing different survival curves in a pair-wise fashion.
Differences between mean values were compared using the Student's
t-test. Differences between the fraction of animals surviving
at day 30 were determined using the Fisher's test.25
| |
RESULTS |
Complete H2 disparity in mice is a major barrier to successful
allogeneic BMT.
Mice transplanted with 0.5 × 106 BM cells from
allogeneic MHC mismatched donors died of graft failure. Moribund
animals had marked cytopenias of the peripheral blood (mean leukocyte
counts of 0.2 ± 0.3 × 109/L, hematocrits of 5.3% ± 2.2%, and platelet counts of 19,000 ± 11,000). Less than 5%
of peripheral blood and BM cells were donor-derived. In contrast, 80%
to 90% of mice receiving an equal number of congenic MHC identical BM
cells survived transplants (Table 1). Large
doses (5 × 106 to 25 × 106 cells)
of allogeneic C57.BL6 BM were needed to successfully engraft B10.BR and
B10.RIII recipients (Table 1). Thus, the allogeneic barrier to
transplantation using MHC mismatched donor-recipient pairs could be
overcome by increasing the number of donor cells.26,27
T cells lose proliferative capacity after exposure to greater than 5 Gy ionizing radiation, but survive transiently in vivo.
Mouse spleen cells were exposed to a single fraction of radiation
between 0 Gy and 20 Gy and then cultured in the presence of anti-CD3
monoclonal antibody and IL-2. These culture conditions model an in vivo
environment in which alloreactive T cells are stimulated in a
MHC-mismatched recipient.28 Radiation doses of 7.5 Gy or
more effectively prevented T-cell proliferation
(Fig 1A). The effect of radiation on the
proliferation of allogeneic T cells in vivo was determined by injecting
groups of 2 to 3 Balb/c scid recipients with 15 × 106 allogeneic C57.BL6 splenocytes treated with 0 to 20 Gy
irradiation. Aliquots of their peripheral blood were aliquots for the
presence of donor H2b T cells on days 1, 2, 5, and 7 posttransplant. Nonirradiated Thy 1.1+, H2b donor T cells
were easily detectable at each time point and increased in number as
they proliferated in recipient SCID mice over 5 to 7 days, producing
clinically evident acute GVHD by day 5 to 7 posttransplant. Mice that
received allogeneic splenocytes irradiated to 2.5 Gy developed clinical
GVHD 7 to 30 days after injection (Fig 1B). Mice that received
allogeneic splenocytes irradiated at doses of 5 Gy and higher had
declining numbers of detectable donor cells on 2, 5, and 7 days after
injection and remained free of GVHD.29,30
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| Fig 1.
The proliferative capacity of mouse T cells was inhibited
by radiation doses of 5 Gy or greater. (A) Murine splenocytes were
irradiated with a single dose between 0 and 20 Gy and then cultured in
the presence of immobilized anti-CD3 monoclonal antibody in RPMI media
containing 50 U/mL IL-2 and 10% vol/vol FBS. The numbers of viable T
cells were counted after 2 to 15 days of culture. (B) Groups of 2 to 5 SCID mice (H2d) received intravenous injections of 15 × 106 splenocytes prepared from a CD45.1+
C57.BL6 (H2b) donor and irradiated at the doses shown. One,
2, 5, and 7 days after injection, 200 µL peripheral blood were
analyzed for the presence of donor-derived T cells by FACS using
antibodies to CD45.1, CD3, and propidium iodide 1 µg/mL to exclude
nonviable cells. The mean (n = 2 to 4) percentage of donor-derived
CD3+, CD45.1+ cells among the nucleated
blood cells at each time point is shown. *, Some of the mice that
received nonirradiated allogeneic splenocytes were killed because of
the development of GVHD at day 5 or day 7 postsplenocyte infusion.
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The half-life of irradiated allogeneic T cells in vivo was estimated by
measuring the survival of donor splenocytes in Balb/c scid mice
(CD45.2, H2d). After sublethal irradiation, scid
mice were injected with 25 × 106 of MHC-mismatched
C57.BL6 splenocytes (Thy 1.2, CD45.1, H2b) irradiated to 0 Gy, 7.5 Gy, or 30 Gy and an equal number of nonirradiated C57.BL6
splenocytes (Thy 1.1, CD45.1, H2b). Two mice from each 3 groups of 6 scid were killed at 1, 4, and 36 hours. The
frequency of CD45.1+ donor cells in the peripheral blood
and BM of recipient mice was determined using flow cytometry. Overall,
CD45.1+ donor cells were detected at frequencies between
0.1% and 1% for up to 36 hours (data not shown). Donor T cells
irradiated to 7.5 Gy (Thy 1.2+) disappeared from the blood
with a half-life of 7 hours and disappeared from the BM with a
half-life of 83 hours. In contrast, nonirradiated Thy 1.1+
donor T cells proliferated in the blood and BM during the same period.
The overall frequency of irradiated (7.5 Gy) donor T cells in the BM 1 hour after injection was 0.004% (1 irradiated donor T cell per 25,000 host BM cells). The effect of a 30-Gy dose of radiation was to increase
the rate of disappearance of the irradiated allogeneic T cells from
both the blood and BM (data not shown).
Irradiated splenocytes retain alloreactive cytotoxicity
in vitro.
B10.BR splenocytes were cultured with allogeneic C1498 leukemia cells
(derived from an H2b C57.BL6 mouse) for 14 days.
Alloreactive T cells were then examined for cytotoxicity against C1498
leukemia cell targets 4 hours after exposure to 0 to 30 Gy irradiation.
Figure 2 presents typical data from 1 of 3 experiments performed and is consistent with earlier reports of the
effect of radiation on enhancing allospecific cytotoxicity after mixed
lymphocyte culture.31 Higher doses of radiation (30 Gy)
blunted the cytotoxic effect of the alloprimed T cells, and doses of 60 Gy eliminated their cytotoxic activity (data not shown).
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| Fig 2.
Specific cytotoxicity of H2k effectors
against H2b targets. CD8+ cells were selected
from B10.BR splenocytes and lymph nodes by immunomagnetic beads. Cells
were cultured in the presence of IL-2, IL-7, IL-12, and C1498 leukemia
cells irradiated to 60 Gy (Boyer49). After 14 days of
culture, the effector cells were irradiated between 0 and 30 Gy with a
137Cs source. The irradiated and nonirradiated effector
cells were incubated with nonirradiated C1498 target cells and specific
cytotoxicity of the cells was measured with the CytoTox 96 Assay
following the manufacturer's protocol. The mean (±SD) of specific
cytotoxicity for quadruplicate samples is shown for E:T ratios of 0.3:1
to 10:1.
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Mouse T cells irradiated to 7.5 Gy have increased levels of NFB,
but their profile of IL-2, IL-4, IFN, and TNF cytokine
expression are not significantly changed.
Irradiation to 7.5 Gy increased intranuclear NFB in unfractionated
splenocytes and caused a mean 4-fold increase in T cells that had been
enriched using negative depletion of Mac-1/Gr-1/CD19+ cells
(Fig 3). Maximal levels of NFB in
irradiated lymphocytes were seen 4 to 6 hours after radiation exposure
(data not shown). Irradiated T cells incubated for 6 hours at either
4°C or 37°C had similar levels of intranuclear NFB (Fig 3).
T cells that were enriched from splenocytes by positive selection using
an anti-CD3+ antibody had levels of NFB that were
significantly increased compared with T cells enriched by negative
depletion and were only slightly increased by radiation (Fig 3). The
activating effect of radiation was dose-dependent. A 30-Gy dose of
irradiation did not increase intranuclear NFB as much as irradiation
to 7.5 Gy, probably due to increased rates of cell death at the higher
radiation dose (data not shown). The temperature-independence of NFB
induction by radiation suggests that new protein synthesis may not be
required for the activation of T cells by radiation. The temporal
increase in intranuclear NFB after radiation implies that an
enzymatic or a nonenzymatic modification of existing nuclear or
cytoplasmic proteins may be involved. Irradiation to 7.5 Gy did not
change the pattern or magnitude of type 1 cytokine (IL-2, TNF, or
IFN) or type 2 cytokine (IL-4) expression among T cells from C57.BL6 mice in response to B10.BR allogeneic stimulators after incubation at
37°C for 6 to 24 hours (data not shown).
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| Fig 3.
Enhanced NFB in nuclear extracts of mouse T cells
after exposure to ionizing radiation. T cells, enriched by either
negative depletion or positive selection, or unfractionated splenocytes
were irradiated at a dose rate of 5 Gy/min to 7.5 Gy or 30 Gy.
Irradiated cells were cultured in RPMI + 10% FCS at 4°C or
37°C for 6 hours. Nuclear extracts were prepared and the relative
level of nuclear NFB determined by incubating 10 µg of nuclear
protein with a 32P-labeled oligonucleotide containing
NFB binding sequence. The far left lane labeled "NC" contained
water with no nuclear extract; lane "PC" contained a Hela cell
nuclear extract (positive control for NFB) incubated with the
32P-labeled oligonucleotide alone; "PC + SC"
contained a combination of the Hela cell nuclear extract, the
32P-labeled oligonucleotide, and an excess of the unlabeled
specific oligonucleotide competitor; and "PC + NSC" contained a
combination of the Hela cell nuclear extract, the
32P-labeled oligonucleotide, and an excess of an unlabeled
nonspecific oligonucleotide. The next 3 lanes from the left contained
nuclear extracts of unfractionated splenocytes irradiated at 0 Gy, 7.5 Gy, or 30 Gy then incubated at 37°C. The mean increase in the upper
NFB band of nonirradiated T cells compared with T cells irradiated
to 7.5 Gy at 37°C was 4-fold (n = 5 EMSA from 2 separate
isolations of T cells). Three lanes contained nuclear extracts of T
cells enriched by negative selection irradiated at 0 Gy incubated at
37°C, 7.5 Gy incubated at 37°C, or 7.5 Gy incubated at 4°C.
Two lanes on the right contained nuclear extracts of T cells enriched
by positive selection irradiated at 0 Gy incubated at 37°C, or 7.5 Gy incubated at 37°C.
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Splenocytes irradiated to 7.5 Gy have diminished GVHD potential in
parental F1 transplant model.
Five of 6 (83%) of (SJL × C57.BL6) F1 mice that received 1 × 106 TCD parental SJL BM cells survived to day +51
posttransplant. These mice appeared healthy and active, with a median
weight of 35 ± 1.4 g. All 7 F1 mice that received TCD SJL BM and
30 × 106 irradiated (7.5 Gy) SJL
splenocytes survived without evident clinical signs of GVHD. These mice
appeared healthy and had a median weight of 35 ± 1.8 g. Seven of 8 (87.5%) F1 mice, which received a combination of SJL TCD BM and 30 × 106 nonirradiated SJL splenocytes, survived to day
+51. However, these mice were wasted with a median weight of 24.4 ± 3.4 g and had patchy alopecia and diarrhea
(Table 2). Necropsy of all surviving animals at day +51 showed lymphoid hypoplasia among recipients of
nonirradiated allogeneic splenocytes. These animals had spleens with
mean weights of 30 ± 20 mg compared with normal spleen weights among recipients of TCD BM alone (160 ± 30 mg, P < .0001)
or TCD BM plus irradiated allogeneic splenocytes (150 ± 30 mg,
P < .0001). Histology examination of samples of skin, liver,
and intestine from transplanted mice showed variable degrees of
lymphocytic infiltration of the liver, epithelial thickening, and
autolysis and sloughing of intestinal epithelium. Evidence for acute
GVHD was minimal among recipients of TCD BM alone
(Fig 4A) or irradiated allogeneic
lymphocytes (Fig 4B). Histologic examination of the liver from
recipients of nonirradiated allogeneic splenocytes showed acute and
chronic inflammation of the portal tract (Fig 4C) and focal hepatic
parenchymal injury. The hepatic GVHD score (on a scale of 0 to 3) and
overall GVHD score (on a scale of 0 to 6) for each animal was
calculated according to the Materials and Methods section. Recipients
of nonirradiated splenocytes had higher GVHD scores than the group that
received irradiated splenocytes and the group that received TCD BM
alone (Table 2).
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| Fig 4.
Histology sections of liver from recipients of MHC
mismatched TCD BM and allogeneic splenocytes. Sections of liver from
(C57.BL6 × SJL) F1 mice that were transplanted on day 0 with 1 × 106 SJL TCD BM cells alone (A) or TCD BM in combination
with 30 × 106 irradiated (7.5 Gy) splenocytes from SJL
donors (B) or with 30 × 106 nonirradiated splenocytes
(C). Mice were euthanized at day +51 and samples of liver tissue were
fixed in 10% formalin, embedded in paraffin, and sections were stained
with hematoxylin and eosin. Original magnification is ×165 in all 3 photomicrographs.
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Irradiated allogeneic splenocytes overcome graft failure in
allogeneic BMT across the H2b H2r
major MHC barrier.
B10.RIII mice (n = 24) transplanted with 0.5 × 106
C57.BL6 allogeneic BM cells alone had 0% survival at 30 days
posttransplant. In contrast, recipients of an equal number of syngeneic
B10.RIII BM cells had 80% 30-day survival (n = 15, Table 1). The
addition of 5 × 106 irradiated (10 Gy) allogeneic
C57.BL6 spleen cells increased survival among B10.RIII recipients of
C57.BL6 BM to 20% (n = 5). Mice that received a total of 10 × 106 C57.BL6 spleen cells irradiated to 10 Gy had 41%
survival at 30 days posttransplant (n = 17, P < .0001 compared with the survival of recipients of allogeneic BM cells alone).
Greater than 85% of all blood leukocytes were donor-derived among
B10.RIII mice that survived transplant with C57.BL6 allogeneic BM and
irradiated splenocytes (data not shown).
The effects of the dose of irradiation and number of allogeneic
splenocytes in facilitating H2b H2k
transplants.
Experimental groups of 10 to 15 lethally irradiated B10.BR mice were
transplanted with 0.5 × 106 C57.BL6 allogeneic BM
cells on day 0 or BM in combination with 1 or more infusions of
irradiated allogeneic spleen cells. Injection of a total of 10 × 106 allogeneic splenocytes irradiated to 5 Gy, 7.5 Gy, or
10 Gy produced similar effects on graft facilitation, with 60-day
survival rates of 22%, 33%, and 30%, respectively. In contrast, all
mice that received allogeneic BM plus 30 × 106
irradiated (30 Gy) allogeneic splenocytes died of graft failure by day
+60. Larger numbers of allogeneic splenocytes irradiated to 7.5 Gy were
the most effective in promoting survival after lethal total body
irradiation and transplantation with allogeneic bone marrow. As shown
in Fig 5, recipients of allogeneic bone plus 75 × 106 irradiated allogeneic splenocytes had a
60-day survival posttransplant of 60% (28 mice from 3 experiments
administering injections of irradiated splenocytes on day 1, 0, and +1). More recipients of irradiated splenocytes survived to 60 days
than did recipients of BM alone (2% survival, n = 60, P < 1 × 10 6). In addition, more recipients
of irradiated splenocytes survived to day 60 than did recipients of BM
plus 75 × 106 nonirradiated allogeneic splenocytes
(0% survival, n = 12, P < 1 × 106). Figure 6
summarizes 13 different experiments in which 141 B10.BR mice received
between 2.5 and 120 × 106 irradiated and
nonirradiated allogeneic C57.BL6 splenocytes. The 60-day survival was
roughly proportional to the cell dose of irradiated splenocytes, while
recipients of more than 25 × 106 nonirradiated
allogeneic splenocytes uniformly died of acute GVHD.
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| Fig 5.
Survival of B10.BR mice transplanted with C57.BL6 BM
cells was enhanced by irradiated C57.BL6 splenocytes. Data are pooled
from 3 experiments in which groups of 8 to 10 B10.BR mice were
irradiated to 10 Gy on day -2 and transplanted with 0.5 × 106 C57.BL6 allogeneic BM cells or 0.5 × 106
syngeneic B10.BR BM cells on day 0. One group of 8 B10.BR mice received
3 injections of 25 × 106 nonirradiated C57.BL6 allogeneic
splenocytes on day 1, day 0, and day +1 ( ), while a second
group of 28 B10.BR mice received 3 injections of 25 × 106
irradiated (7.5 Gy) allogeneic C57.BL6 spleen cells on day 1, day 0, and day +1 ( ). A control group of 60 B10.BR mice received
allogeneic BM cells alone ( ). Another control group of 20 B10.BR
mice received irradiation without a transplant ( ) and 26 B10.BR mice
were transplanted with syngeneic H2k BM cells ( ).
Significant differences in survival using the Cox statistic were found
between the group of mice receiving multiple infusions of irradiated
allogeneic splenocytes and the group that received nonirradiated
splenocytes (P = 3 × 106); the mice that
received irradiated allogeneic splenocytes and mice that received
allogeneic BM alone (P = 1 × 102); and the
mice that received multiple infusions of nonirradiated allogeneic
splenocytes and mice that received allogeneic BM alone (P
= 3 × 107).
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| Fig 6.
Sixty-day survival of B10.BR mice transplanted with
allogeneic C57.BL6 BM and splenocytes. Recipient mice received 10 Gy
irradiation in 2 equal fractions on day 2 or day 1 and
transplanted with 0.5 × 106 BM cells from C57.BL6 donors
on day 0. The 60-day survival of groups of 7 to 21 mice in 14 separate
experiments receiving various numbers of irradiated (7.5 Gy) () or
nonirradiated ( ) allogeneic splenocytes given in 1 to 3 injections
on consecutive days around the day of BM transplant (day 0). Best-fit
lines are shown for the survival of mice receiving different doses of
irradiated allogeneic splenocytes ( ) (r2 = .49)
and unirradiated allogeneic lymphocytes ( ).
|
|
Irradiated allogeneic lymphocytes did not produce GVHD in the first
week after transplantation into allogeneic recipients.
Three groups of B10.BR mice received either (1) 0.5 × 106 C57.BL6 allogeneic BM alone; (2) allogeneic BM plus 75 × 106 irradiated allogeneic lymphocytes; or (3)
allogeneic BM plus 75 × 106 nonirradiated allogeneic
lymphocytes. Mice were euthanized during the first week posttransplant,
and necropsy was performed to determine whether acute GVHD had
developed. Mice that received allogeneic marrow and nonirradiated
allogeneic splenocytes had clinical and histological evidence of acute
GVHD as evidenced by mean 15% loss of body weight by day +5 and a mean
24% weight loss by day +6 posttransplant. The peripheral blood on day
+5 showed leukopenia (total leukocyte count of 0.9 ± 0.4 × 106 /L), but with relatively large numbers of donor T cells
in the blood (14.4% of blood leukocytes). Histological examination of the livers of these recipients showed evidence of acute GVHD, with mean
GVHD scores of 1 ± 1 on day +5 and 1.8 ± 0.5 on day +6. In
contrast, mice which had received transplants containing allogeneic BM
alone or BM plus irradiated allogeneic splenocytes had no significant
weight loss and showed no histological evidence of GVHD (mean histology
scores of 0). Pancytopenia was present (mean leukocyte counts of 0.3 ± 0.3 and 0.4 ± 0.3 × 106/L, respectively),
but peripheral blood T cells derived from the irradiated donor
splenocytes were not observed (<0.3% of leukocytes). Necropsies of
moribund mice at 3 weeks posttransplant that had received allogeneic BM
and irradiated allogeneic splenocytes showed marked pancytopenia and
absence of donor-derived T cells in their peripheral blood.
Irradiated allogeneic splenocytes did not contribute significantly to
donor-derived hematopoiesis in H2b H2k transplants.
We used congenic strains of C57.BL6 mice expressing either Thy 1.1 versus Thy 1.2 or CD45.1 versus CD45.2 as BM and splenocyte donors. The
origin of T cells in recipient mice was determined by the expression of
the donor spleen or donor BM specific marker. Among the B10.BR mice
that received injections of C57.BL6 splenocytes irradiated to 7.5 Gy,
the overall mean (±standard deviation [SD]) fraction of
peripheral blood cells that were donor-derived (H2b) was
49% ± 35% at day +30 to day +45. The mean frequency of
donor-derived cells was relatively constant across multiple experiments
involving different doses of irradiated splenocytes in combination with a fixed dose of allogeneic BM (Fig 7).
Mixed chimerism was present in the T-cell compartment in all mice that
received various doses of irradiated splenocytes and BM. The overall
mean percentages of day +30 peripheral blood T cells in B10.BR
transplant recipients that were derived from allogeneic donor BM was
25% ± 31%, while 11% ± 17% of peripheral blood T cells were
derived from the irradiated allogeneic donor splenocytes. Significant
numbers of blood T cells derived from the infusions of irradiated
allogeneic splenocytes were only seen at the highest doses of
splenocytes infused. Recipients of 75 × 106, or 120 × 106 irradiated allogeneic splenocytes had 9% ± 14% and 35% ± 22%, respectively, of peripheral blood T cells
derived from the irradiated donor splenocytes at day +30.
Interestingly, the fraction of T cells derived from the irradiated
splenocytes declined to nearly nondetectable levels at 3 months
posttransplant, even among recipients of the largest number of
irradiated splenocytes. A typical flow cytometric plot of the blood of
a recipient of the highest dose (120 × 106) of
irradiated splenocytes analyzed at day +30 and day +112 is shown in
Fig 8. In this animal, 88% of blood
leukocytes were donor-derived at day +30 and 96% of blood leukocytes
donor-derived by day +112 (Fig 8, upper panels). The T-cell compartment
of the blood had mixed donor (11% of nucleated blood cells) and
host-type T cells (8% of the blood cells) at day +30. By day +112,
27% of blood cells were donor-derived T cells and only 4% were of
host type (Fig 8, upper panels). Thirty-five percent of donor-derived T cells were derived from the irradiated splenocytes at day +30, while
spleen-derived T cells were undetectable at day +112 (Fig 8, lower
panels).
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| Fig 7.
Recipients of allogeneic BM and irradiated allogeneic
splenocytes had mixed donor and host type hematopoiesis. The mean
percentage of peripheral blood leukocytes expressing H2b
(donor type; ) or H2k (host type; ) from B10.BR
(H2k) mice surviving to day +30 after transplantation
with C57.BL6 (H2b) BM and irradiated (7.5 Gy) splenocytes
is shown. The conditions of the transplant are described in the legend
to Fig 6. The error bars depict the SD of the mean.
|
|
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| Fig 8.
Flow cytometric analysis of peripheral blood
leukocytes from recipients of allogeneic TCD BM and irradiated
allogeneic splenocytes. Peripheral blood was obtained from B10.BR
recipients of 0.5 × 106 CD45.2+
C57.BL6 TCD BM cells and 50 × 106 irradiated
CD45.1+ C57.BL6 splenocytes. The conditions of the
transplant are described in the legend to Fig 6. Upper panels: the
total percentages of donor T cells (upper right quadrant),
host-type T cells (lower right quadrant), and host non-T cells
(lower left quadrant) and donor-type non-T cells (upper left quadrant)
are shown for 2 representative animals bled on day +30 and day +112
posttransplant. Lower panels: the fraction of donor T cells derived
from the irradiated splenocytes (CD45.1+, upper right
quadrant) or from the donor BM (CD45.1, lower right
quadrant) are shown.
|
|
Enhanced survival and donor hematopoiesis among recipients of
irradiated allogeneic splenocytes requires donor-type T cells.
Splenocytes were fractionated into T-cell-enriched (TCE) or TCD
subsets using magnetic bead separation and negative depletion with
antibodies against either T-cell (CD3) or B-cell, monocyte, and
granulocyte markers (CD19/Gr-1/MAC-1). The initial content of T cells
in the spleen was 15% to 20%; the TCE fraction contained a mean of
90% to 98% T cells, while the TCD fractions contained 0.5% to 1.5%
T cells. The TCD fraction was mainly B cells (78.8%). The content of
natural killer (NK) 1.1+ cells were
slightly reduced in both the TCD (2.85%) and TCE (2.02%) fractions
compared with the frequency in unfractionated splenocytes (4.4%). The
reduction of NK 1.1+ cells is consistent with the
coexpression of CD3 and MAC-1 on minor populations of NK
1.1+ cells. Three experiments, involving 114 mice, were
performed in which B10.BR mice were transplanted with mixtures of
C57.BL6 allogeneic BM cells and TCD, TCE, or unfractionated C57.BL6
irradiated splenocytes. The number of T cells injected in the group
that received TCE splenocytes was equivalent to the number of T cells present in 75 × 106 unfractionated spleen cells (15 to 20 × 106 T cells). Mice injected with TCD
splenocytes received the same number of non-T cells as present in 75 × 106 unfractionated spleen cells (60 × 106 non-T cells). The survival of mice that received TCE
irradiated splenocytes was equivalent to mice that received
unfractionated irradiated splenocytes and superior to recipients of
irradiated TCD splenocytes (Fig 9). Forty
percent of the B10.BR recipients of allogeneic BM and unfractionated
irradiated splenocytes, as well as 41% of the B10.BR recipients of TCE
irradiated splenocytes, survived 40 days posttransplant. In contrast,
only 7% or 8%, respectively, of the recipients of allogeneic BM or
allogeneic BM and TCD splenocytes survived transplant (Fig 9, P = .006 comparing survival between recipients of TCD and TCE
splenocytes).
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| Fig 9.
Enhanced survival of B10.BR mice transplanted with
C57.BL6 BM cells and irradiated C57.BL6 splenocytes depended on the
presence of splenic T cells. Data are pooled from 3 experiments in
which groups of 8 to 10 B10.BR mice were irradiated with 10 Gy on day
2 and transplanted with 0.5 × 106 C57.BL6 allogeneic
BM cells and splenocytes or 0.5 × 106 syngeneic B10.BR BM
cells on day 0. Allogeneic splenocytes were prepared daily from C57.BL6
mice, TCD, or TCE, as described in Materials and Methods, and
irradiated to 7.5 Gy immediately before injection on days 1, 0, and
+1. One group of 5 B10.BR mice received syngeneic B10.BR BM
( ); a second group of 25 B10.BR mice
received a total of 75 × 106 C57.BL6 irradiated
splenocytes with C57.BL6 BM (); a third group of 16 B10.BR mice received C57.BL6 BM alone (____); a fourth
group of 25 B10.BR mice received a total of 60 × 106
C57.BL6 TCD irradiated splenocytes with C57.BL6 BM (- -);
a fifth group of 29 B10.BR mice received a total of 15 × 106 C57.BL6 TCE irradiated splenocytes with C57.BL6 BM
(); and a sixth group of 15 mice received a total of 75 × 106 nonirradiated C57.BL6 splenocytes with C57.BL6 BM
(- - -). A significantly superior survival was found for both the
groups of mice receiving multiple infusions of irradiated allogeneic
splenocytes and the group receiving TCE irradiated allogeneic
splenocytes compared with the group that received nonirradiated
splenocytes (P < .01); the group that
received TCD allogeneic splenocytes (P  .01); and the group
that received syngeneic BM (P < .05).
|
|
In a second series of 2 experiments, recipient B10.BR mice were treated
with a larger, more immunosuppressive dose of radiation (11 Gy), as
well as a larger dose of C57.BL6 BM cells (1 × 106).
These conditions permitted recipients of BM alone to survive and
allowed us to evaluate the extent to which irradiated donor lymphocytes
facilitate donor chimerism independently of their radioprotective
effect. All of the recipients that received BM alone survived to day
+60, with autologous hematopoietic recovery observed in 12 of 18 mice.
In contrast, recipients of allogeneic BM plus 75 × 106 irradiated allogeneic splenocytes had 61% survival at
60 days posttransplant, with 82% of surviving mice showing greater
than 90% donor-derived hematopoiesis
(Table 3). Seven of 10 mice that received
allogeneic BM plus TCD splenocytes survived to 60 days, but only 2 of 7 of these mice had donor hematopoiesis (Table 3). The median percentage
(±SD) of donor cells among recipients of irradiated allogeneic
splenocytes was 100% ± 42% compared with a median value of 5% ± 48% among recipients of allogeneic BM and TCD irradiated
splenocytes (P = .04; Table 3). In addition, the median
percentage of peripheral blood T cells that were derived from donor BM
was 100% ± 42% among recipients of irradiated splenocytes (Table
3). This value was significantly higher than the percentage of
BM-derived T cells among recipients TCD irradiated splenocytes (13% ± 32%, P = .02, Table 3) and higher than recipients of BM alone (0% ± 47%, P = .02, Table 3). In these
experiments, the contribution of the donor splenocytes to T-cell
reconstitution was minimal (a median of 5% of blood T cells was
derived from the irradiated donor splenocytes, Table 3).
Lymphocytes from recipients of irradiated allogeneic splenocytes are
tolerant to both donor and host MHC types and do not produce GVHD when
transferred to secondary BMT recipients.
The GVHD potential of lymphocytes from chimeric H2b
H2k BM recipients of the highest dose (120 × 106) of irradiated splenocytes were compared with
the GVHD potential of naive MHC mismatched H2b and
H2k lymphocytes. Spleen cells from BM transplant recipients
were serially transplanted into mice of both donor and host strains. B10.BR mice that had received C57.BL6 allogeneic BM and irradiated (7.5 Gy) C57.BL6 allogeneic splenocytes were killed at day +112. At this
time, the mice were fully chimeric (>95% of blood cells were
donor-derived). Splenocytes from the chimeric mice (>98% of T cells
were derived from donor BM) were serially transplanted into lethally
irradiated C57.BL6 or B10.BR mice in combination with BM that was
syngeneic with the recipient strain. Splenocytes from the transplanted
chimeric mice (that were predominantly H2b) did not cause
clinically detectable GVHD or graft failure in animals of either
H2b or H2k strains. Transplantation using naive
MHC mismatched splenocytes lead to severe GVHD, which was fatal by day
+30 in 3 of 4 recipients (Table 4). Flow
cytometric analysis of blood, spleen, and BM from recipients of either
tolerant or naive MHC mismatched splenocytes showed that MHC-mismatched
T cells from recipients of irradiated splenocytes failed to proliferate
in the secondary recipients. In contrast, transplanted naive MHC
mismatched T cells proliferated and produced acute GVHD (Table 4).
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|
Table 4.
Secondary Transfer of Splenocytes From B10.BR
(H2k) Mice Transplanted With C57.BL6 Allogeneic BM and
Irradiated (7.5 Gy) Splenocytes Into C57.BL6 and B10.BR Recipients
|
|
| |
DISCUSSION |
In this report, we have studied the ability of irradiated allogeneic
leukocytes to facilitate engraftment of allogeneic stem cells without
causing GVHD. In the H2b H2k, and
H2b H2r BM transplant
model systems, transplantation with 0.5 × 106
allogeneic BM cells led to graft failure and death among more than 95%
of recipient mice (Table 1 and Fig 5). The rare surviving mouse had
pancytopenia and a predominance of host type T cells, implicating a
population of relatively radioresistant host T cells in graft
rejection. Doses of 5 Gy or greater inhibited the proliferative capacity of mouse T cells (Fig 1)31 and splenocytes
irradiated to 7.5 Gy did not produce GVHD in MHC mismatched recipients
(Table 2).30 Irradiated donor T cells could be detected in
the BM of recipient SCID mice for more than 3 days. In addition,
irradiated cytotoxic T-lymphocyte (CTL) effector cells
had enhanced allospecific cytolytic activity compared with
nonirradiated CTL effector cells (Fig 3).31,32 Based on
these findings, we tested the effect of adding irradiated splenocytes
to BM transplants across a major MHC barrier. The addition of
allogeneic splenocytes irradiated between 5 Gy and 10 Gy facilitated
engraftment by allogeneic BM cells without producing clinically evident
GVHD, even when very large doses of allogeneic splenocytes were
administered. The engraftment promoting effect of irradiated allogeneic
lymphocytes was dependent on the dose of radiation used and the number
of irradiated splenocytes infused (Fig 6). Multiple infusions of
25 × 106 irradiated splenocytes given
around the day of transplantation were more effective than a
single injection of 25 × 106 irradiated
splenocytes given on day 0 (data not shown). The effect of irradiated
splenocytes administered at the time of transplant was to favor
donor-derived hematopoiesis rather than simply enhancing radioprotection. T cells present in the allogeneic splenocytes were
necessary for this effect, but did not contribute directly to
donor-derived hematopoiesis (Table 3).
The present study is in contrast to the two previous reports using
animal models of BM transplantation that did not show clear evidence of
the ability of irradiated allogeneic lymphocytes to facilitate
engraftment. Gratwohl et al33 studied the role of irradiated allogeneic leukocytes in BMT using antigenically mismatched genetically unrelated donor-recipient pairs of rabbits transplanted with a combination of allogeneic BM and irradiated autologous BM. Deeg
et al34 studied irradiated allogeneic lymphocytes in the
outbred dog BMT model. In the study by Gratwohl, a series of 5 infusions of irradiated (15 Gy) donor lymphocytes from day 1 to day 10 did not prevent graft rejection (0 of 5 animals engrafted). The
addition of cyclosporine to a combination of TCD allogeneic BM,
irradiated autologous BM, and irradiated allogeneic buffy coat cells
increased the rate of engraftment to 80%, but 40% of these recipients
died of GVHD.33 In the report by Deeg, infusions of
irradiated (20 Gy) donor lymphocytes from dog leukocyte antigens (DLA)
mismatched BM donors did not prevent fatal graft failure.34 The addition of viable donor lymphocytes to the allogeneic DLA mismatched BM graft resulted in an increased frequency of stable hematopoietic engraftment, but recipient dogs uniformly died of GVHD.35 In the dog model system, gamma irradiation (20 Gy)
of whole blood products transfused before DLA-mismatched allogeneic BMT
prevented sensitization to alloantigens, but did not induce allospecific tolerance.36
There is limited clinical experience using irradiated donor lymphocytes
to treat human patients. Infusions of allogeneic donor lymphocytes
irradiated to 15 to 20 Gy to allogeneic transplant recipients at high
risk for graft rejection may reduce the risk of graft failure when
given after BMT.8,37,38 In these studies, the contribution
of the irradiated donor lymphocytes to hematopoietic engraftment or
clinical GVHD was unclear. There was no way to distinguish the
irradiated lymphocytes from the variable number of nonirradiated
lymphocytes contained within the BM graft. There are two significant
differences between the present study and earlier reports of using this
strategy in animal transplant models and in patients. First, we used a
lower dose of radiation to treat the allogeneic donor cells (7.5 Gy
v 15 to 20 Gy). Second, we administered the irradiated
allogeneic cells before and concomitant with the BM cells rather than
as multiple infusions after the BM graft. In addition, we used donor
strains that were congenically marked to determine whether donor T
cells originated from the BM or the spleen cell transplant.
Irradiated splenocytes could potentially enhance engraftment by
supplying sufficient radioresistant donor hematopoietic progenitor cells to achieve short-term hematopoietic engraftment and
radioprotection. The irradiated hematopoietic progenitors would
complement the limited number of stem cells in the allogeneic BM graft
and allow animals to survive with autologous recovery. Studies by Till
and McCulloch39 demonstrated that radiation doses of 7.5 Gy
led to a 2.5 log reduction in the colony-forming unit
(CFU) content of BM cells. However,
H2b H2k transplants using
irradiated (7.5 Gy) allogeneic donor splenocytes alone did not result
in hematopoietic engraftment of recipients (0 of 5 mice survived to day
+30 (data not shown). CD45.1+ myeloid and monocytic progeny
of irradiated allogeneic CD45.1 spleen cells were not detected in the
blood of B10.BR recipients of C57.BL6 CD45.2 allogeneic BM and C57.BL6
CD45.1 irradiated spleen cells (data not shown). Therefore, the
engraftment promoting effect of irradiated donor splenocytes was not
due to an increased dose of radioresistant splenic hematopoietic stem cells.
The mechanisms for the graft facilitating activity of irradiated
allogeneic lymphocytes may include the retention of cytotoxic or
"veto" activity by irradiated donor cells that inhibit or
eliminate residual host T cells.31,40 While radiation
enhanced the cytotoxic activity of allostimulated CTL (Fig 2), a
measurable effect of radiation on the allospecific cytolytic activity
of noncultured splenocytes was not observed (data not shown). Thus, the
data presented in Fig 2 demonstrate retention of biologic activity after irradiation, but do not directly demonstrate that graft facilitation of irradiated splenocytes is due to enhanced allospecific cytotoxicity. Second, a large number of allogeneic donor cells either
overwhelm cytotoxic capacity of radioresistant host cells ("cold
target inhibition") or induce anergy among cytotoxic host cells and
thereby permit the survival of allogeneic donor stem cells.41 Third, elaboration of cytokines by irradiated
allogeneic lymphocytes could either inhibit graft rejection by host
lymphocytes or promote the growth of allogeneic donor stem
cells.26,33,42,43 While our study is potentially consistent
with all 3 hypothetical mechanisms, the present data favor a central
role for T cells in this process. The failure of TCD splenocytes to
facilitate engraftment militates against a simple "cold target
inhibition" mechanism.
The low frequency of allospecific T cells among unprimed splenocytes
might argue against the role of antigen-specific T cells in enhancing
engraftment after irradiation. Anti-H2d allospecific,
cytolytic T cells are present at a frequency of 1/570 in C57.BL6
splenocytes.44 If anti-H2k allospecific CTL are
present at a similar frequency, then 75 million donor splenocytes
(circa 20 million T cells) would contain approximately 35,000 allospecific anti-H2k CTL or 1 million allospecific T
cells/kg. Such a small number of allospecific lymphocytes might be
inadequate to promote engraftment if they have lost the capacity for
proliferation in vivo. However, new DNA synthesis is not needed for the
generation of allospecific CTL,45 and an equivalent number
of FACS-purified CD8+ BM "facilitating cells" have
been shown to enhance engraftment of MHC mismatched c-kit+,
Sca-1+, Lin stem cells.46
The critical time for engraftment of allogeneic stem cells across a
major MHC barrier is likely in the first few days after
BMT.47 Thus, even when nonirradiated allogeneic T cells are
administered during the initial engraftment period, there is relatively
little time for significant clonal expansion of allospecific T cells.
Preliminary data suggest that immunotherapy using irradiated donor
lymphocytes have antileukemic activity in animal and human clinical
model systems. Multiple infusions of irradiated allogeneic cells may be
safely given to patients who have experienced relapse of leukemia or
lymphoma after allogeneic BMT, with an antitumor response observed in
25% of patients (Waller et al48).
Multiple infusions of irradiated allogeneic lymphocytes that retain
short-term allospecific cytotoxicity and lack the potential for clonal
expansion in vivo can be considered as a novel form of immunotherapy
with defined pharmacokinetics. This method of immunotherapy might have clinical applications for facilitating TCD allogeneic hematopoietic stem cell transplants, as well as mediating a direct anticancer effect
in transplant recipients.
| |
ACKNOWLEDGMENT |
We acknowledge Sylvia D. Ennis, Senior Research Project Coordinator for
her editorial assistance with this manuscript and Richard Lopez for
helpful discussions.
| |
FOOTNOTES |
Submitted November 19, 1998; accepted June 24, 1999.
Supported in part by National Cancer Institute (NCI)
Grant No. 1RO1CA74364 and the Leukemia Research Foundation (to E.K.W.). R.C. was supported by the Aplastic Anemia Foundation.
A.M.S., S.M., and T.W.M. contributed equally to this work.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Edmund K. Waller, MD, PhD, Emory University
School of Medicine, Division of Hematology and Oncology, Suite
1003, 1639 Pierce Dr, Atlanta, GA 30322; e-mail:
ewaller{at}emory.edu.
| |
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