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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 3140-3147
Prevention of Transfusion-Associated Graft-Versus-Host Disease by
Photochemical Treatment
By
Joshua A. Grass,
Tamim Wafa,
Aaron Reames,
David Wages,
Laurence Corash,
James L.M. Ferrara, and
Lily Lin
From the Cerus Corporation Concord, CA; the University of
Michigan Cancer Center Ann Arbor, MI; and the Baxter Healthcare
Corporation, Fenwal Division, Viral Inactivation, Round Lake, IL.
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ABSTRACT |
Photochemical treatment (PCT) with the psoralen S-59 and long
wavelength ultraviolet light (UVA) inactivates high titers of contaminating viruses, bacteria, and leukocytes in human platelet concentrates. The present study evaluated the efficacy of PCT to
prevent transfusion-associated graft-versus-host disease (TA-GVHD) in
vivo using a well-characterized parent to F1 murine
transfusion model. Recipient mice in four treatment groups were
transfused with 108 splenic leukocytes. (1) Control group
mice received syngeneic splenic leukocyte transfusions; (2) GVHD group
mice received untreated allogeneic splenic leukocytes; (3) gamma
radiation group mice received gamma irradiated (2,500 cGy) allogeneic
splenic leukocytes; and (4) PCT group mice received allogeneic splenic
leukocytes treated with 150 µmol/L S-59 and 2.1 J/cm2
UVA. Multiple biological and clinical parameters were used to monitor
the development of TA-GVHD in recipient mice over a 10-week posttransfusion observation period: peripheral blood cell levels, spleen size, engraftment by donor T cells, thymic cellularity, clinical
signs of TA-GVHD (weight loss, activity, posture, fur texture, skin
integrity), and histologic lesions of liver, spleen, bone marrow, and
skin. Mice in the control group remained healthy and free of detectable
disease. Mice in the GVHD group developed clinical and histological
lesions of TA-GVHD, including pancytopenia, marked splenomegaly,
wasting, engraftment with donor derived T cells, and thymic hypoplasia.
In contrast, mice transfused with splenic leukocytes treated with
(2,500 cGy) gamma radiation or 150 µmol/L S-59 and 2.1 J/cm2 UVA remained healthy and did not develop detectable
TA-GVHD. Using an in vitro T-cell proliferation assay, greater than
105.1 murine T cells were inactivated by PCT. Therefore, in
addition to inactivating high levels of pathogenic viruses and bacteria in PC, these data indicate that PCT is an effective alternative to
gamma irradiation for prevention of TA-GVHD.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
A PHOTOCHEMICAL TREATMENT process (PCT)
using the psoralen S-59 and long wavelength ultraviolet light (UVA, 320 to 400 nm) has been developed to inactivate viral and bacterial
pathogens in platelet concentrates (PC) while retaining in vitro and in vivo platelet function.1 Because S-59 is nucleic acid
specific, T cells contaminating PC preparations are highly susceptible
targets for inactivation. Human T cells have been shown to be extremely sensitive to S-59 PCT when evaluated by a sensitive in vitro limiting dilution assay (LDA) and other molecular analyses.2 The
concentration of S-59 used to inactivate viruses and bacteria in PC is
approximately 3,000-fold higher than that required to inactivate
greater than 5 log10 of T cells in a clonogenic T-cell
proliferation assay.2
Contaminating leukocytes in PC provide no hemostatic benefit and can
cause a number of adverse immune reactions in PC recipients. Donor T
cells are known to initiate transfusion-associated graft-versus-host disease (TA-GVHD).3 TA-GVHD, a life-threatening T-cell
mediated immune reaction, is 80% to 90% fatal and there is no
effective therapy.4 Many PC transfusion recipients are at
risk for TA-GVHD. Although the risk is greater for immunocompromised
patients, TA-GVHD has been reported in immunocompetent patients as
well.5
Although we previously have shown the inactivation of T cells using
sensitive in vitro assays, in vivo inactivation of T cells with PCT has
not yet been established. In this study we evaluated the efficacy of
PCT to prevent TA-GVHD in a well-characterized parent to F1
murine transfusion model.6 Affected mice exhibit clinical
signs and findings analogous to human TA-GVHD. In this model, parental
A mice were used as donors and hybrid offspring B6AF1
(C57BL/6 × A) mice as recipients. Strain A donor mice are homozygous at the H-2 locus and the recipient B6AF1 mice
are heterozygous. Donor A cells recognize the B6 antigens on recipient
cells as foreign and initiate acute TA-GVHD, while the recipient
B6AF1 host cells recognize the donor cells as self and fail
to reject them. Transfusion of 1.0 × 108 leukocytes
results in acute TA-GVHD that closely resembles human TA-GVHD. PCT was
compared with gamma radiation, the current method of TA-GVHD
prophylaxis, for its ability to prevent TA-GVHD in this model. The
dynamic range of murine T-cell inactivation also was determined using
an in vitro T-cell proliferation assay.
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MATERIALS AND METHODS |
Mice.
Strain A and B6AF1 mice were purchased from Jackson
Laboratory (Bar Harbor, ME). All mice were housed in rodent HEPA-vented condominiums with filter top cages and autoclaved bedding (Northeastern Products Corp, Columbia City, OR). Mice were maintained on a diet of
gamma radiation sterilized mouse chow (PMI Nutritional International, St Louis, MO) and autoclaved water. Bedding and water were changed daily. Donor (A) and recipient (B6AF1) mice were 8 to 12 weeks old at the time of transfusion.
Preparation of donor splenic leukocytes (splenocytes).
Donor mice, A or B6AF1, were anesthetized with metophane
(Mallinkrodt, Chicago, IL) and killed by cervical dislocation. For each
transfusion, 2 to 3 spleens from donor mice were used. The spleens were
removed under aseptic conditions and teased through 70-µm nylon mesh
filters in 5 mL of isotonic phosphate buffered saline (PBS)
supplemented with 1% bovine serum albumin (PBS/1% BSA). Five mL of
splenocyte suspension was overlayed onto 5 mL of lympholyte M (Accurate
Chemical, Westbury, NY) and centrifuged at 1,000g for 20 minutes at 22°C. The mononuclear cells were isolated and washed twice
with PBS 1% BSA (250g, 10 minutes).
Gamma irradiation.
One third of the donor A splenocytes were resuspended in 30 mL of
PBS/1% BSA and transferred into small PL 2410 plastic containers (Baxter Healthcare Corp, Fenwal Division, Deerfield, IL). The splenocytes were transported on ice (4°C) to the Alameda Contra Costa
Blood Bank and irradiated with 2,500 cGy (Nordion Gamma Cell-1000
Irradiator; Nordion Inc, Kanata, Ontario, Canada). The irradiated cells
were pelleted by centrifugation at 250g for 10 minutes at
22°C and resuspended in PBS/1% BSA to a final concentration of 1.0 × 108 cells in 200 µL to 400 µL.
Photochemical treatment.
One third of the donor A splenocytes were resuspended in 30 mL of
PBS/1% BSA and transferred into small PL 2410 plastic containers. The
volume to surface ratio resulted in a fluid layer of approximately 1 cm. The psoralen S-59 (Cerus Corp, Concord, CA) was added to the
splenocyte suspension to a final concentration of 150 µmol/L. The
structure and synthesis of S-59 has been described.7 The splenocytes were illuminated with 2.1 J/cm2 of UVA on a UVA
illumination device (Baxter Healthcare Corp). The fluence of the UVA
device was 15 to 20 mW/cm2. A dose of 2.1 J/cm2
was delivered in approximately 2 to 3 minutes. After PCT, the treated
cells were pelleted by centrifugation at 250g for 10 minutes at
22°C and resuspended in PBS/1% BSA to a final concentration of 1.0 × 108 cells in a final volume of 200 µL to 400 µL.
Transfusion.
The splenocytes were counted on a hematology analyzer (Biochem
Immunosystems, Allentown, PA). Approximately 1.0 × 108
cells were transfused through the lateral tail veins of recipient mice
anesthetized with metophane. Donor splenocytes were transfused into
recipients according to the four experimental groups (Table 1).
Peripheral blood cell counts.
One day before transfusion and weekly after transfusion, blood samples
were obtained from recipient mice by retro-orbital venipunture.
Capillary tubes (Biochem Immunosystems) were used to collect 40 µL of
blood that was immediately diluted into 10 mL Haema Line Diff Silos
(Biochem Immunosystems). White blood cell (WBC), red blood cell (RBC),
and platelet (PLT) levels were enumerated with an automated hematology
analyzer (Biochem Immunosystems).
Clinical scores.
One day before transfusion and weekly after transfusion, recipient mice
were weighed and scored for clinical signs of TA-GVHD as previously
described.8 Body weight, posture, activity, fur texture,
and skin integrity were scored from 0 to 2 for a total possible score
of 10.
Assessment of splenomegaly.
Spleens of recipient mice were removed 2 to 3 weeks after transfusion.
Spleens were weighed in preweighed tubes containing PBS. The spleen
index is defined as (spleen weight/body weight) × 1,000.
Assessment of donor T-cell engraftment.
Recipient spleens were removed 2 weeks after transfusion, processed
into single-cell suspensions by teasing through a 70-µm nylon mesh
screen in PBS, and prepared for two-color flow cytometric analysis.
Splenocytes (1.0 × 106 cells) were labeled
with 1.0 µg of anti-CD3-PE (pan T cell) and anti-H-2Kb-FITC (recipient major
histocompatibility complex [MHC] class I) antibodies (Pharmingen, San
Diego, CA) for 30 minutes on ice. The cells were then washed by
centrifugation (250g for 10 minutes at 22°C) and resuspended
in 1.0 mL of PBS. The cells were analyzed on a flow cytometer with
lysis II software (Becton Dickinson, San Jose, CA) using forward and
side scatter to gate on the leukocytes. A dot plot of 1.0 × 104 cells was generated with FL1 (horizontal axis)
measuring fluorescence intensity of recipient MHC class I
H-2Kb+ cells and FL2 (vertical axis) measuring fluorescence
intensity of CD3+ T cells (Fig 1). Cells in
the upper left quadrant, positive for CD3 and negative for
H-2Kb, were classified as donor T cells and were quantified
as a percentage of the total number of cells analyzed.
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| Fig 1.
Engraftment of donor T cells in spleens of recipient
mice. Analysis of recipient spleens for the presence of donor-derived T
cells was performed 2 weeks after transfusion. Splenocytes were
analyzed using two-color flow cytometry with antibodies for
CD3+ cells on the vertical axis and recipient specific
MHC class I H-2b on the horizontal axis. Donor T cells
appear in the upper left-hand quadrant. Spleens of two mice in each
group were analyzed and the figure shown is representative of both mice
in their respective group. As expected, mice in the control group had
no allogeneic donor T cells present in their spleens 2 weeks after
transfusion. Mice in the GVHD group had 37% donor-derived T cells.
Mice in the PCT and Gamma groups had no evidence of donor-derived T
cells in their spleens, similar to the control group.
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Assessment of TA-GVHD induced immune suppression.
GVHD-induced immune suppression, measured by thymic cellularity, was
quantified 2 to 3 weeks after transfusion. At the time of death, thymus
glands were removed from recipient mice and macerated between the
frosted surfaces of two glass microscope slides. The thymocytes were
resuspended in 5 mL of PBS with 1% BSA and thoroughly mixed. The cells
were filtered through 70-µm nylon filters to obtain
single cell suspensions. The number of thymocytes was quantified using
an automated hematology analyzer (Biochem Immunosystems).
Histology.
Sections of liver, spleen, skin, and bone marrow were removed at
various times after transfusion. Each section was preserved in formalin
(Shandon, Pittsburgh, PA) embedded in paraffin, mounted on slides, and
stained with hematoxylin and eosin. The slides were scored in blinded
fashion by a single observer using a scoring system (Table
2). Histology data were obtained from three
separate experiments (Table 3) over 6 months
using a total of 30 mice. The number of mice in each treatment group
ranged from 6 to 11.
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Table 3.
The Number of Recipient Mice Used per Study Group in
Each Experiment and the Time at Which Each Measurement Was Made
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Statistical analysis.
Data for the spleen index, WBC counts, RBC counts, PLT counts, donor
T-cell engraftment, and thymic counts were analyzed by Student's
t-test for significant differences between the control group
and the test groups. A P value of <.05 was considered significant.
In vitro inactivation of murine T cells.
Using the same murine strain combinations as for the preceding in vivo
experiments, T-cell inactivation by PCT was determined by an in vitro
T-cell proliferation assay. Assays were performed in U-bottom 96-well
microtiter plates. Mixed lymphocyte cultures were prepared using
splenocytes from B6AF1 mice as stimulators and splenocytes
from A mice as responders. Donor splenocytes (strain A) were prepared
and subjected to PCT as described above.
Two experiments were conducted. Stimulator cells were irradiated with
2,000 cGy to prevent proliferation and 2 × 105 cells were
plated per well. In one experiment (experiment 4), untreated donor
responder cells were plated at levels of 20, 40, and 80 cells per well
(24 wells per cell level) containing stimulator cells. Approximately 1 × 105 PCT donor responder cells were plated into 80 wells
containing stimulator cells. In another experiment (experiment 5),
untreated donor responder cells were plated at levels of 200, 400, and
800 cells per well (32 wells per cell level). PCT donor responder cells
were plated at levels of 5 × 104 and 1 × 105 per well (32 wells per cell level).
The mixed splenocytes were cultured in a volume of 200 µL of RPMI
1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/L
L-glutamine, 1 mmol/L sodium pyruvate, nonessential amino acids, 5 × 10 5 mol/L 2-mercaptoethanol, penicillin and
streptomycin, lipopolysaccharide (Sigma Chemical Co, St Louis, MO), 2.5 µg/mL Con A, and 40 units/mL hIL2. Cells were cultured
in humidified 5% CO2 at 37°C for 7 days and pulsed with
1 µCi/well of 3H-thymidine (5 Ci/mg; Amersham) for the
last 12 to 18 hours of culture. On day 8, cells were harvested onto
glass fiber filters with an automated cell harvester (Cambridge
Technology, Cambridge, MA). Incorporated radioactivity was measured by
scintillation counting. Wells were scored either positive or negative.
Only wells that were three standard deviations above the background were scored positive. The T-cell frequencies and the log10
T-cell reduction were calculated by minimum chi-square analysis based on a Poisson distribution as previously described.2
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RESULTS |
Overview.
Three separate experiments were performed using a total of 40 mice as
transfusion recipients. Separate experiments were conducted to assess
the spectrum of parameters associated with TA-GVHD. Both biological and
clinical parameters were measured to monitor the development and
severity of TA-GVHD in transfusion recipients. The number of recipient
mice used per study group and the time post-transfusion when each
biological measurement was made were summarized (Table 3).
Experiment 1 was designed to measure biological parameters and clinical
scores for up to 3 weeks after transfusion. Peripheral blood cell
levels (WBC, RBC, and PLT) were monitored weekly. Clinical scores
including body weight, skin integrity, fur texture, activity, and
posture were scored weekly. On week 3, mice were killed. Thymic cellularity and splenomegaly were evaluated. Sections of liver, skin,
spleen, and bone marrow were analyzed and scored for histopathologic evidence of TA-GVHD.
In experiment 2, the recipient mice were observed for up to 10 weeks
after transfusion. All but two mice survived the 10-week evaluation
period. Two mice in the GVHD group died 1 to 2 weeks after transfusion
because of severe TA-GVHD. Weekly peripheral blood cell levels and
clinical scores were obtained. Mice were killed on week 10 for
preparation of tissue sections and scored for histopathologic evidence
of TA-GVHD.
In experiment 3, two mice from each study group were killed on week 2 for analysis of splenomegaly, thymic cellularity, and engraftment of
donor T cells. The remaining mice in each study group were monitored
for a total of 9 weeks for weekly measurement of peripheral blood cell
levels and clinical scores. Mice were killed on week 9 for tissue
histopathologic evaluation of TA-GVHD.
The data from each experiment were pooled for analysis. Statistical
comparisons were made among the four study groups (control, GVHD,
gamma, and PCT).
Splenomegaly.
Splenomegaly has been shown to be a reliable measure of the severity of
GVHD (Table 4).9
Mice in the control group received syngeneic splenic leukocyte
transfusions and had normal spleen sizes with an average spleen index
of 3.67 ± 0.2 (SE). Mice in the GVHD group had grossly enlarged
spleens with an average spleen index of 18.75 ± 1.3. Mice that
received PCT or gamma-irradiated splenocyte transfusions, however, had
spleen indices comparable with the control, 3.66 ± 0.3 and 3.64 ± 0.4, respectively. The spleen indices for the PCT, gamma, and control
groups were not significantly different from each other
(P > .05). The GVHD group was statistically different
(P < .05) from the PCT, gamma, and control groups.
Donor T-cell engraftment.
Proliferation of donor T cells is a key initiating event in the onset
of TA-GVHD.4 The number of donor T cells in recipient spleens was measured by flow cytometric analysis. This assay used the
MHC class I difference between donor and recipient mice to identify
cell phenotype. Cells in the upper left quadrant of the dot plot (Fig
1) represent donor-derived T cells (CD3+,
Kb ). Mice in the control group that received syngeneic
splenocyte transfusions contained no evidence of donor T cells. Mice in
the GVHD group that received untreated allogeneic splenocyte
transfusions had an average ± standard error of 34.34% ± 2.2%
(Table 4) donor T cells in their spleens 2 to 3 weeks after
transfusion, indicative of GVHD.9 Additionally, they also
contained donor-derived non-T cells typical of GVHD (lower left
quadrant). The control group and mice that received PCT or gamma
radiation-treated splenocytes did not have a significant percentage of
donor T cells in their spleens. These results indicate that PCT or
gamma irradiation protected mice from engraftment by viable donor T cells.
Thymic cellularity Immune suppression.
Immune suppression is a well-documented sign of TA-GVHD.9
Thymic cellularity has been used to measure the severity of GVHD in
murine systems. For each experimental group, the average number of
thymocytes ± the standard error was measured 2 to 3 weeks after transfusion. In this study, mice in the GVHD group had consistently low
numbers of thymocytes (1.2 ± 0.2 × 107 cells) in
their thymus glands 2 to 3 weeks after transfusion, indicative of an
immunosuppressed state (Table 4). Mice in the control, PCT, or gamma
groups had normal thymic cellularity, 5.5 ± 0.9 × 107 cells, 3.8 ± 0.6 × 107 cells, and 3.8 ± 0.6 × 107 cells, respectively. Thymic cellularity of
mice in the GVHD group was statistically different from thymic
cellularity in the PCT, gamma, and control groups when analyzed by
Student's t-test (P < .05). Although thymic
cellularity of the PCT and gamma groups was less than that of the
control group, this difference was not statistically significant
(P > .05).
Peripheral blood cell levels.
Peripheral blood samples of each mouse were obtained the day before
donor leukocyte transfusion and on a weekly basis after transfusion.
WBC counts, RBC counts, and PLT counts were monitored up to 10 weeks
after transfusion or until the mice were killed for other analyses.
Data for each group up to 3 weeks after transfusion are shown (Fig
2). Peripheral blood cell levels in the
syngeneic transfusion control mice remained stable for the course of
the study. Mice that received PCT- or gamma-treated splenocyte
transfusions had peripheral blood cell counts that were not
significantly different from the controls (P > .05). In
contrast, the peripheral blood cell counts of all three hematopoietic
lineages were significantly less (P < .05) in the GVHD
group. Three weeks after transfusion, PLT counts in the GVHD group
recovered. This rebound coincided with an increase in megakaryocytes
observed in spleen sections of these mice. In mice, when the bone
marrow becomes aplastic, the spleen can become a site of
hematopoiesis.10 This observation suggests the platelet
counts in the GVHD group rebounded because of splenic hematopoiesis,
but these mice had persistent acute GVHD documented in other tissues.
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| Fig 2.
Peripheral blood cell counts. Peripheral blood was
sampled from each mouse 1 day before and weekly after transfusion until
death. Average counts for WBC (A) RBC (B) and PLT (C) were plotted for
each week. Error bars represent the standard error for each group. Mice
in the GVHD group (squares) developed pancytopenia 2 to 3 weeks after
transfusion. Levels of WBC, RBC, and PLT for mice in the control
(diamonds), PCT (triangles), and gamma (circles) groups were not
statistically different at any time after transfusion
(P > .05) when analyzed by Student's t-test.
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Body weight.
The average body weight of mice in each group over a 10-week period
after transfusion, was determined (Fig 3).
Mice in the GVHD group gained an average of
only 1 g of body weight in the 10-week period consistent with active
TA-GVHD. Mice in the control, PCT, and gamma groups gained an average
of 7 g of body weight as expected for healthy mice of this age. The
body weights of mice in the control, gamma, and PCT groups were not
significantly different from each other (P > .05)
throughout the observation period. In contrast, after week 4, mice in
the GVHD group weighed significantly less (P < .05) than
all other groups.
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| Fig 3.
Body weight. Body weight was monitored on a weekly basis
for each transfusion group. The average of body weight in the control
group (diamonds) increased by 7 g as expected for healthy mice of this
age. Mice in the PCT (triangles) and gamma (circles) groups also gained
weight, similar to the control group. Mice in the GVHD (squares) group
that received untreated transfusions failed to gain weight, an
observation consistent with the development of TA-GVHD. Error bars
represent the standard error for each group.
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Clinical scores.
Recipient mice were monitored for clinical signs of TA-GVHD 1 week
before transfusion and weekly after transfusion for up to 10 weeks.
Body weight, posture, activity, fur texture, and skin integrity were
scored on a scale from 0 to 2 for severity with a total possible
cumulative score of 10 (Table 2). The average and standard error for
each group at each week was calculated (Fig 4).
Similar to the syngeneic controls, mice
that received gamma- or PCT-treated splenocyte transfusions remained
healthy and free of clinical signs of TA-GVHD. The average cumulative clinical scores for these groups remained below 1.0 for the duration of
the study. Mice that received untreated allogeneic splenocyte transfusion in the GVHD group, however, displayed progressively more
severe signs of TA-GVHD as indicated by the increase in clinical scores
over time. Mice in the GVHD group had significantly higher scores
(P < .05) than all other groups at several time points; progressive mortality in the GVHD group, however, reduced the number of
evaluable mice over time and contributed to increased variance. The
trend for mice in the GVHD was indicative of increasingly worse
clinical scores while mice in all other groups exhibited no clinical
deterioration. The average cumulative clinical score for the GVHD group
was 5.0 (out of a maximal score of 10) 10 weeks after transfusion.
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| Fig 4.
Clinical scores. Clinical assessments of body weight,
activity, posture, fur texture, and skin integrity were scored weekly.
The average and standard deviations of weekly clinical scores over a
10-week period after transfusion for each group were calculated. The
error bars represent the standard error for each weekly average. Mice
in the GVHD (squares) group had progressively higher clinical scores
indicative of TA-GVHD, while mice in the control (diamonds), PCT
(triangles), and gamma (circles) groups remained healthy and free of
clinical signs of TA-GVHD.
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Histology.
The liver, spleen, bone marrow, and cheek (skin) tissue sections of
selected animals from each group of transfusion recipients were
evaluated for histologic evidence of GVHD (Table 5).
Mice that received PCT- or gamma-irradiated
splenocyte transfusions did not develop histologic lesions
characteristic of TA-GVHD. They had average cumulative scores of 1.2 ± 0.5 and 1.6 ± 0.7, respectively, out of a total possible
cumulative histologic score of 13. Histology scores of mice in the PCT
and gamma groups were not significantly different when analyzed by
Student's t-test (P > .05) from mice in the
control group (1.0 ± 0.3) that received syngeneic transfusions. In
contrast, mice in the GVHD group that received untreated allogeneic
spleen-cell transfusions developed severe histologic abnormalities
indicative of TA-GVHD. The average cumulative score for the GVHD group
was 9.0 ± 0.6. Scores of the GVHD group were statistically different
from the control, PCT and gamma groups (P < .05).
In vitro murine T-cell inactivation.
The extent of T-cell inactivation by PCT was measured in two
experiments (Table 6). Untreated donor T
cells in both experiments showed viability at a frequency of 1/51 and
greater than 1/61 T cells. After PCT, donor T cells showed viability at
a frequency of less than 1/7,950,093 and less than 1/4,759,209 T cells,
respectively. The levels of T-cell inactivation by PCT were estimated
at greater than 105.2 for Experiment 4 and greater than
104.9 for Experiment 5, with a mean inactivation of greater
than 105.1 T cells.
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DISCUSSION |
Gamma irradiation of PC is the current method used for TA-GVHD
prophylaxis for platelet transfusion. Many years of clinical use have
shown that gamma irradiation can prevent TA-GVHD. An in vitro LDA that
measures T-cell viability indicated that 2,500 cGy was required to
inactivate at least 105 T cells.11 The LDA has
shown that the dose response curve for gamma irradiation T-cell
inactivation is steep. A fivefold decrease in the dose of gamma (from
2,500 cGy to 500 cGy) affected the T-cell inactivation efficacy. Only
101.1-1.3 T cells were inactivated by treatment with 500 cGy of gamma.11 Although infrequent, failure of gamma
irradiation to prevent TA-GVHD has been reported with doses of 1,500 to
2,000 cGy.12 In contrast, the S-59 concentration used in
PCT can be reduced 3,000-fold while maintaining high levels of T-cell
inactivation. The combination of 150 µmol/L S-59 and 3 J/cm2 UVA used to inactivate viruses and bacteria in PC was
shown to inactivate greater than 105.4
T-cells.2 A 3,000-fold decrease in the dose of S-59 (from 150 µmol/L to 0.05 µmol/L) did not affect the T-cell inactivation efficacy by PCT. The combination of 0.05 µmol/L S-59 and 1 J/cm2 UVA inactivated (to the limit of detection) greater
than 104.1 T cells.2
PCT has the potential to greatly improve the safety of platelet
transfusion by inactivating viruses and bacteria contaminating PC and
potentially blood-borne pathogens that remain undetected. Clinical
studies in humans have shown that PCT of PC results in adequate
retention of in vivo platelet recovery and lifespan.13 The
present study suggests that PCT can be used independently of gamma
irradiation for the prevention of TA-GVHD. Because PCT has a large dose
range for the inactivation of T cells, it offers the potential for a
new robust means to prevent TA-GVHD. Thus, viral and bacterial
inactivation by PCT, and TA-GVHD prophylaxis could be achieved with a
single procedure.
Cytokines secreted by leukocytes that accumulate during PC storage have
been shown to cause febrile nonhemolytic transfusion reactions.14 Using conditions described in this report, PCT inhibited cytokine accumulation during PC storage.2,15 This is not unexpected, given the mechanism of S-59 plus UVA photochemistry with modification of leukocyte DNA at a frequency of one adduct per 83 base pairs, effectively inhibiting nucleic acid synthesis and
transcription.2 Gamma irradiation before PC storage, at the
doses used for TA-GVHD prohylaxis, only partially inhibited cytokine
accumulation during PC storage.15 Therefore, inhibition of
cytokine synthesis during storage of PC is another potential clinical
benefit that PCT can provide to PC transfusion recipients.
Donor leukocytes in PC potentiate HLA alloimmunization that can lead to
platelet transfusion refractoriness in recipients of multiple PC
transfusions.16-18 In vitro studies have shown that leukocytes treated with 8-methoxypsoralen (8-MOP) and UVA are unable to
stimulate proliferation of untreated allogeneic leukocytes in mixed
lymphocyte reaction assays.19 Other studies have shown that
contaminating T cells in PC treated with 4'-aminomethyl
4,5'8-trimethylpsoralen and UVA cannot upregulate the early activation
antigen CD69 when stimulated with phorbyl myristate
ester.20 These results suggest that PCT treated leukocytes
are unlikely to become activated or serve as effective
antigen-presenting cells in vivo, a pathway thought to be important for
donor recognition followed by host alloantibody production.
Furthermore, in vivo studies have shown that treatment with 8-MOP and
UVA of allogeneic mouse PC containing leukocytes reduced
alloimmunization in recipient mice.21
The current study augments previous investigations using in vitro
biological and molecular assays to show inactivation of leukocytes by
S-59 and UVA.2 Mice that received lymphocyte transfusions
treated with S-59 and UVA remained healthy and free of detectable
TA-GVHD, comparable with mice that received gamma-treated lymphocyte
transfusions. The dynamic range of T-cell inactivation using the in
vivo experimental murine model was limited by the number of T cells
that could be transfused intravenously. This limitation arose because
of the increased viscosity of the highly concentrated leukocyte
suspensions required for induction of TA-GVHD. To show a greater range
of T-cell inactivation, we utilized an in vitro T-cell proliferation
assay, similar to our prior study with human T cells, to expand the
dynamic range of PCT inactivation. With this in vitro assay, we were
able to show a greater than 105.1-fold reduction in viable
murine T cells. This level of inactivation was similar to our previous
data with human T cells. Thus, the in vitro inactivation data, in
conjunction with the in vivo data, further indicate the potential of
PCT for prophylaxis of TA-GVHD in addition to inactivation of
contaminating viruses and bacteria in PC.
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ACKNOWLEDGMENT |
We thank Prof John E. Hearst for his constant encouragement and
valuable suggestions throughout this study, Dr Chu Lin for his
supervision of animal care, Margaret Rheinschmidt for her assistance in
the fluorescence-activated cell sorter analysis of antibody-labeled
mouse leukocytes, Dr Peyton Metzel for providing the UVA illumination
device and disposables, Dr Don Buchholz for reviewing the manuscript,
and John Hull for reviewing the experimental data. We also thank the
Blood Bank of Alameda Contra Costa County for making the Nordion Gamma
Cell-1000 Irradiator available for gamma irradiation of cellular samples.
| |
FOOTNOTES |
Submitted March 31, 1998; accepted January 4, 1999.
Supported in part by Grants No. HL 3340 and CA 34952 from the National
Heart, Lung and Blood Institute. Presented in part at the American
Society of Hematology, December 1996.
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 Lily Lin, PhD, Cerus Corporation, 2525 Stanwell Dr, Concord, CA 94520.
| |
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