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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 713-720
Extreme Leukoreduction of Major Histocompatibility Complex Class II
Positive B Cells Enhances Allogeneic Platelet Immunity
By
John W. Semple,
Edwin R. Speck,
Donna Cosgrave,
Alan H. Lazarus,
Victor S. Blanchette, and
John Freedman
From the Divisions of Hematology, St. Michael's Hospital and The
Hospital for Sick Children, The University of Toronto and The Toronto
Platelet Immunobiology Group, Toronto, Ontario, Canada.
 |
ABSTRACT |
In a murine model of platelet alloimmunization, we examined the
definitive role that mononuclear cells (MC) have in modulating platelet
immunity by using platelets from severe combined immunodeficient (SCID)
mice. CB.17 (H-2d) SCID or BALB/c (H-2d) mouse
platelets were transfused weekly into fully allogeneic CBA
(H-2k) mice and antidonor antibodies measured by flow
cytometry. MC levels in BALB/c platelets were 1.1 ± 0.6/µL and SCID
mouse platelets could be prepared to have significantly lower
(<0.05/µL) MC numbers. Transfusions with 108 BALB/c
platelets (containing  100 MC/transfusion) stimulated IgG antidonor
antibodies in 100% of the recipients by the fifth transfusion, whereas
108 SCID mouse platelets (containing 5 MC/transfusion)
stimulated higher-titered IgG alloantibodies by the second
transfusion. When titrations of BALB/c peripheral blood MC were added
to the SCID mouse platelets, levels approaching 1 MC/µL reduced SCID
platelet immunity to levels similar to BALB/c platelets.
Characterization of the alloantibodies showed that the low levels of MC
significantly influenced the isotype of the antidonor IgG; the presence
of 1 MC/µL was associated with induction of noncomplement fixing IgG1 antidonor antibodies, whereas platelet transfusions, devoid of MC
(<0.05/µL), were responsible for complement-fixing IgG2a
production. When magnetically sorted defined subpopulations of MC were
added to the SCID platelets, major histocompatability complex (MHC) class II positive populations, particularly B cells, were found to be
primarily responsible for the reduced SCID mouse platelet immunity. The
presence of low numbers of MC within the platelets was also associated
with an age-dependent reduction in platelet immunogenicity; this
relationship however, was not observed with SCID mouse platelets devoid
of MC. The results suggest that a residual number of MHC class II
positive B cells within allogeneic platelets are required for maximally
reducing alloimmunization.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PRODUCTION OF ANTIDONOR major
histocompatibility complex (MHC) class I antibodies is a frequent
complication of platelet transfusions. This response can be initiated
by direct allorecognition where the T-cell receptors of recipient
CD4+ T helper (Th) cells directly recognize intact donor
MHC class II molecules on the surface of donor antigen-presenting cells (APC).1-10 Hence, leukoreduction strategies such as
leukofiltration have been used to remove the contaminating APC from
platelet concentrates.11-21 A number of clinical
studies11-21 including the recent large multicenter Trial
to Reduce Alloimmunization against Platelets (TRAP),20 have
confirmed that leukofiltration of platelets significantly reduces
alloimmunization, although a percentage of transfusion recipients still
become alloimmunized. In the patients receiving leukofiltered blood
products, the mechanism of alloimmunization is unknown, but because the
TRAP study showed few failed leukofiltration episodes, it suggests that
MHC class I positive, class II negative platelets may, in themselves,
stimulate MHC alloimmunization.
It remains unclear what is the actual extent of leukocyte removal
required to achieve maximum benefit to the recipient. In this case,
benefit can be defined by the clinical outcome of reduced alloimmunization and platelet refractoriness. In an early experimental murine model, Claas et al22 showed that allogeneic
platelets could induce IgG alloantibody formation only if at least
103 contaminating leukocytes were present. Based on an
approximate murine blood volume of 2 mL, this dosage translated to a
human transfusion of 2.5 × 106 leukocytes. Clinical
studies subsequently similarly suggested that the minimal threshold of
leukocyte contamination in human platelet concentrates to prevent
alloimmunization should be less than 1 to 5 × 106
leukocytes.21 There is however, little experimental
evidence to support whether this level of leukoreduction is optimal for completely preventing alloimmunization or whether lower levels may be
required. Several reports have indicated that in healthy mice, rats and
humans transfused with leukoreduced allogeneic platelets, leukocyte
levels as low as 1/µL can activate recipient T cells23
and stimulate IgG antidonor alloantibody responses.24-27 Thus, an experimental model in which platelets for transfusion can be
consistently prepared with none, or few, leukocytes would be
advantageous to understanding the relationship between platelets and
leukocytes in modulating recipient immunity.
One potential way to achieve this is to use platelets derived from
genetically immunodeficient animals. CB.17 severe combined immunodeficient (SCID) mice were derived from BALB/c mice and have a
point mutation on chromosome 16, which inhibits their ability to repair
double-stranded DNA breaks.28 Because the proper gene rearrangements for T-cell and B-cell receptors is critically dependent on this DNA repair process, these cells are deleted early in ontogeny. Although myeloid cell numbers are relatively normal, these mice contain
no detectible T or B cells in their peripheral blood and can be further
depleted of natural killer (NK) cells and some monocytes by in vivo
treatment with anti-asialo Gm1 (AsGm 1) antibody. Thus, platelets
prepared from the platelet-rich-plasma of antibody-treated SCID mouse
blood can be consistently rendered extremely leukoreduced (<0.05
mononuclear cells [MC]/µL). We present evidence that SCID mouse
platelets, despite greater MC reduction, are significantly more
immunogenic than BALB/c platelets in allogeneic CBA recipients and that
levels of approximately 1 MC/µL are optimal for maximal suppression
of platelet immunity.
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MATERIALS AND METHODS |
Animals and cell lines.
CBA (H-2k) female mice, 8 weeks of age were used as the
transfusion recipients and female BALB/c (H-2d) and CB.17
(H-2d) SCID mice were used as donors and purchased from
commercial breeders. EL-4 (H-2b) C57BL/6 thymoma, P815
(H-2d) DBA mastocytoma, and RT 1.1 (H-2k) CBA
lymphoma cell lines were used for serological typing of the recipient
sera. All cell lines and cell culture assays were maintained in
RPMI-1640 with 5% fetal calf serum (FCS), 100 µg/mL penicillin/streptomycin/fungizone, 100 mmol/L L-glutamine and 5 × 10-5 mol/L 2-mercaptoethanol.
Antibodies.
Antiasialo Gm1 (AsGm 1) antibody was obtained from Waco Laboratories
(Waco, TX). Fluorescein isothiocyanate (FITC)-labeled antimurine-CD45,
-CD3, -CD4 and -F4/80 antibodies were obtained from Cedarlane
Laboratories (Hornsby, Ontario). Phycoerythrin (PE)-labeled antimurine-CD45, -CD8, -B220, -Ly 6G (CD89) and -CD16 antibodies were obtained from PharMingen (Cedarlane Laboratories). Monoclonal FITC- and PE-labeled isotype control reagents were obtained
from Cedarlane Laboratories. Biotinylated antimurine-CD3, -B220 and
-I-Ad MHC class II antibodies were obtained from PharMingen
and biotinylated antimurine-NK (catalog #CL8994B) antibody was obtained
from Cedarlane Laboratories.
SCID mouse peripheral blood characterization and treatment.
To ensure complete penetrance of the SCID mutation, peripheral blood
was screened for residual B and T cells by flow cytometry. Briefly,
mice were bled via a tail-nick procedure into microvettes (Sarstedt,
Montreal, Quebec) containing 1.0% (vol/vol) EDTA and 15 U/mL heparin
(final concentrations) in saline. Blood counts were performed on a Toa
hematology analyzer (Kobe, Japan) calibrated to rodent
settings. Red blood cells (RBC) within the whole blood were removed by incubation with RBC lysing solution (Becton Dickinson, Mississauga, Ontario), and the leukcocytes were labeled with the indicated combinations of FITC- and PE-labeled monoclonal antibodies for 45 minutes at room temperature in the dark. The labeled blood was
then analyzed by flow cytometry. In indicated experiments, to
additionally deplete peripheral blood NK cells and some monocytes, SCID
mice were injected intraperitoneally with 100 µg of anti-AsGm1 antibody 48 to 72 hours before bleeding.
Table 1 summarizes the peripheral blood
leukocyte composition of the murine platelet donors.
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Table 1.
Flow Cytometric Analysis of Peripheral
Blood and Percolled Leukocyte Subpopulations (Percentage ± SD of
Total Leukocytes)
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Platelet preparation.
Mice were bled by the procedure described above. The whole blood was
pooled, centrifuged at 120xg, and platelet rich plasma (PRP)
aspirated off; care was taken not to disturb the buffy coat. The
platelets were washed once in EDTA/heparin-saline,
adjusted to 109 cells/mL (stock solution), and leukocytes
were enumerated. The stock platelet solutions were stored for 18 hours
at room temperature before transfusion. In some experiments, platelets
were transfused fresh; within 4 hours of collection. By flow cytometry,
there was <0.01% of RBC and CD89+ granulocytes were not
detected in the stock solutions of platelets. Murine RBC were not
immunogenic at these levels (JWS, unpublished).
Enumeration of contaminating leukocytes.
Contaminating MC in the platelet concentrates were enumerated by flow
cytometry using hypotonic lysis, propidium iodide (PI), and counting
beads as an internal standard. Briefly, 100 µL of platelets
(109/mL) were incubated with 95 µL of buffer (1 mg/mL
sodium citrate, 0.03% (vol/vol) Triton X-100, 50 µg/mL PI, 10 mg/mL
RNase), and 5 µL of counting beads (Flow Count Fluorospheres, Coulter
Electronics, Hialeah, FL) at 25°C in the dark. Within 30 minutes,
the suspension was acquired on a FACSort flow cytometer with an
electronic gate set around the counting beads and acquisition was
stopped when 5,000 beads were acquired. Standard curves were generated
using 10-fold dilutions of BALB/c Percolled MC (5 × 103/µL to 0.005/µL). For analysis, gates were set
around the MC nuclei based on forward scatter and FL2 (PI)
fluorescence. The number of contaminating MC/µL was determined by the
formula:
This
method could consistently detect as low as 0.05 MC/µL. The average
nucleated MC contamination in the stock solutions of BALB/c platelets
(109 platelets/mL) was 1.1 ± 0.6 MC/µL (n = 67),
<0.3 MC/µL (n = 20) for CB.17 SCID mice and <0.05 MC/µL (n = 20) in the anti-AsGm1-treated SCID mice.
Transfusion protocol.
In each transfusion protocol, groups of 10 mice were bled 24 hours
before the first transfusion and then injected with 100 µL of
platelets weekly via the tail vein. Sera were collected at weekly
intervals and tested for the presence of antidonor MHC alloantibodies.
MC preparation.
For SCID mouse platelet dosing, MC were prepared from the peripheral
blood of BALB/c mice by centrifugation on a 1.077 g/mL Percoll cushion
at 2,800g for 30 minutes. The collected MC were washed twice
before use. Flow cytometric analysis of the MC are shown in Table 1.
Depletion of selected MC by magnetic activated cell sorter.
To analyze the effect of MC subpopulations on platelet immunity, BALB/c
MC were first depleted of the indicated MC populations by a magnetic
activated cell sorter (MACS, Miltenyi Biotech, Auburn, CA) using
biotinylated antibodies and streptavidin-magnetic beads (Becton-Dickinson) as previously described.29 Briefly,
106 MC were incubated with 5 µg of antibody for 45 minutes at 4°C, washed once, and then incubated with a 10 µL of
streptavidin beads for 30 minutes at 4°C. The labeled cells were
passed over a cooled MACS column (Miltenyi Biotech), and the unbound
cells collected and analyzed by flow cytometry. For all of the
depletions, this method removed >90% of the positively selected
cells. Depletion with anti-MHC class II also depleted >90% of B220 B
cells. The unbound MC cells were washed twice, adjusted to
105/mL, and 10 µL were added to 990 µL of
109 SCID mouse platelets (to make a final concentration of
approximately 1 MC/µL).
Flow cytometric analyses.
For detection and characterization of antidonor antibodies,
105 donor MC were incubated with serial dilutions of
recipient sera for 45 minutes at 20°C, washed once,
and labeled with FITC-conjugated goat antimouse IgG (Fc-specific,
Cedarlane Laboratories) for 30 minutes at 20°C in the dark. Cells
were analyzed on a FACSort flow cytometer (Becton Dickinson, San Jose,
CA) operating with an argon ion laser at 15 mW; 10,000 events were
acquired using an electronic cellular (lymphocyte) gate based on
forward and side scatter and were analyzed using LYSYS II software
(Becton Dickinson). Matched prebleed serum was used as the negative
control in all experiments. Antidonor specificity of the antibodies was confirmed by positive reactivity with donor cells, but absence of
reactivity with recipient or third party typing cells. Isotype characterization of the antidonor antibodies was performed using FITC-conjugated goat antimouse IgG1, 2a, 2b and 3 antibodies (Cedarlane Laboratories).
To measure MHC class I expression on SCID and BALB/c mouse platelets,
flow cytometric analysis was performed using PE-labeled anti-Dd MHC class I antibody (Cedarlane Laboratories).
Briefly, 106 platelets were incubated with 5 µL of the
antibody for 45 minutes at room temperature and cells were analyzed on
markers set with fluorescently-labeled isotype control antibodies as
previously described.25
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RESULTS |
The immunogenicity of BALB/c and SCID mouse platelets.
Serial 10-fold dilutions of either BALB/c or SCID mouse platelets were
transfused into allogeneic CBA mice and the number of transfusions,
which induced antidonor antibodies in 100% of the recipients, was
compared. CBA mice did not develop antibodies after transfusions with
either syngeneic platelets or syngeneic MC at any time during the
8-week transfusion protocol. In control allogeneic experiments, weekly
transfusions of either 106 or 105 BALB/c MC
induced high titered IgG antidonor antibodies in all mice by the second
transfusion (Fig 1A), whereas a dose of
103 MC/transfusion did not induce antibodies during the
protocol (Fig 1A). For platelet transfusions, significant changes in
antidonor immunity were observed depending on the platelet donor. When
titrations of BALB/c platelets were transfused into CBA mice,
108 platelets/transfusion (containing 100
MC/transfusion) induced IgG antidonor antibodies in all mice by the
fifth transfusion (Fig 1B). In contrast, 108 platelets
(containing approximately 5 MC/transfusion) from SCID mice induced IgG
antibodies in all recipient mice by the second transfusion (Fig 1C).
The lowest SCID mouse platelet dose capable of inducing IgG antibodies
in all recipient mice was eight transfusions of 106
platelets (containing <0.05 MC/transfusion, Fig 1C). Anti-AsGm 1 treatment of the SCID mouse donors did not affect their platelet immunity (Fig 1C).
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| Fig 1.
SCID mouse platelets are more immunogenic compared with
the platelets of BALB/c mice. CBA recipient mice were transfused weekly
with titrations of either (A) control transfusions with BALB/c MC, (B)
BALB/c mouse platelets, or (C) platelets from anti-AsGm1-treated SCID
mice and their sera tested for antidonor antibodies. The data is
expressed as the percentage of mice responding (n = 20) by detection
of IgG antidonor antibody during the transfusion protocol. Each line
represents a group of mice receiving: (A) ( ) 106, ( )
105, ( ) 104, and ( ) 103
control MC per transfusion; (B) () 108, ()
107, and () 106 BALB/c platelets per
transfusion; (C) () 108, () 107 and
() 106 antibody-treated SCID platelets per transfusion,
(X) 108 platelets from antibody nontreated SCID mice. For B
and C, the numbers in brackets correspond to the number of MC
transfused with the platelets.
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Characterization of the serum IgG antibodies showed that they reacted
strongly with donor MHC-matched typing cells, but not with recipient or
third party MHC-matched cell lines. Th1 and Th2 associated-isotype
analysis of IgG2a and IgG1, respectively, showed that the control MC
transfusions (105/transfusion) induced predominantly IgG1
antidonor antibodies with detectible, but lower, levels of IgG2a
(Th2>Th1, Fig 2A), whereas BALB/c
platelets induced a strong IgG2a response with weaker IgG1 antibodies
(Th1, Fig 2B). The platelets from SCID mice, on the other hand,
stimulated the production of predominantly IgG2a antibodies with only
small amounts of IgG1 detected (Th1>Th2, Fig 2C). IgG2b, but not IgG3
antidonor antibodies, were detected, but at a lower level compared with
IgG2a. The increased SCID mouse platelet immunity was not due to
increased MHC antigen dosing, as all of the platelet preparations had
similar levels of high MHC class I expression. Additionally, none of
the platelet preparations had any APC, which could stimulate, at least,
proliferation of recipient spleen cells in mixed lymphocyte cultures.
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| Fig 2.
MC affect platelet-induced IgG antidonor isotype
production. CBA recipient mice (n = 10) were transfused weekly with
either (A) control transfusions with 105 BALB/c MC, (B)
108 BALB/c mouse platelets, or (C) 108
platelets from SCID mice and their sera tested for the presence of IgG1
and IgG2a antidonor antibodies. The data are representative flow
cytometric histogram analyses of IgG1 and IgG2a antidonor isotypes in a
recipient's serum (1/25) at week 8 transfusion; all mice gave similar
results. In all panels, prebleed IgG antidonor (),
IgG1 (- - -), and IgG2a () reactivity.
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The role of contaminating MC in modulating platelet immunity.
To determine the effect of MC numbers on the enhanced immunogenicity of
SCID mouse platelets and the IgG antidonor isotype modulation,
titrations of BALB/c MC were added to platelets prepared from
anti-AsGm1-treated SCID mice and transfused into CBA recipients. Compared with SCID platelets alone, the addition of MC at 1/µL prolonged the time to formation of antidonor antibodies in all mice to
7 weeks (Fig 3). As the levels of added MC
approached 1/µL, platelet-induced antidonor IgG titers were reduced
to levels similar to those induced by BALB/c platelets
(Fig 4). Additionally, the presence of MC
(at 1/µL) was associated with the production of IgG1 antidonor
antibodies at levels similar to those in BALB/c platelet recipient mice
(not shown). Thus, MC at levels of 1/µL suppressed recipient immunity
against allogeneic platelets and was primarily responsible for inducing
noncomplement fixing IgG1 antidonor antibodies.
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| Fig 3.
MC can reduce recipient IgG antidonor platelet immunity
in CBA mice. The data is presented as the percentage of mice (n = 10), which become IgG antidonor antibody positive during the
transfusion protocol. () Transfusions of 108 BALB/c
mouse platelets; () transfusions with 108 platelets from
anti-AsGm1-treated SCID mice; () transfusion with 108
platelets from anti-AsGm1-treated SCID mice with added BALB/c MC (at
1/µL).
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| Fig 4.
MC at 1/µL maximally inhibit platelet-induced IgG
antidonor antibody titers. Titrations of BALB/c MC were added to stock
platelets from anti-AsGm1-treated SCID mice and transfused into CBA
recipients weekly. Control transfusions with 108 BALB/c
platelets or 105 BALB/c MC are shown. The data is expressed
as the mean ± standard deviation (SD) of week 8 titers from
10 recipient CBA mice.
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To identify the MC subpopulation(s) responsible for the reduced
immunity, BALB/c MC were first depleted of various MC by MACS before
being added to the SCID mouse platelets. SCID platelet immunity was
reduced only when B220+ B cells
(Fig 5, columns 2 and 3,) were present. The
presence or absence of CD3+ T cells or asialo Gm
1+ NK cells had no affect on the SCID mouse platelet
immunity (Fig 5). Depletion of MHC class II+ MC (which also
depleted B cells) also did not affect the SCID platelet immunity (Fig
5). Thus, MHC class II positive B cells were primarily responsible for
reducing allogeneic platelet immunity.
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| Fig 5.
Residual MHC class II positive B cells are responsible
for the reduced platelet immunity. BALB/c MC were depleted (-) of
either T cells (second column), NK cells (third column), B cells
(fourth column), or MHC class II+ cells (fifth column) by
MACS, added to platelets (at 1/µL) from anti-AsGm1-treated SCID
mice, and transfused weekly into CBA recipients. The data is expressed
as the mean ± SD of the week 8 titers from 10 recipient mice. The
first column represents control transfusions with anti-AsGm1-treated
SCID mouse platelets alone.
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To determine if storage of the platelets had an effect on their
immunogenicity, they were either transfused fresh (within 4 hours of
collection) or after 18 hours of storage. Fresh BALB/c platelets were
more immunogenic compared with 18-hour stored BALB/c platelets
(Fig 6A). This was not seen when either
fresh or stored platelets from SCID mice were transfused (Fig 6B).
Thus, the presence of MC at levels of approximately 1/µL is
associated with a storage-induced reduction in platelet immunity.
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| Fig 6.
Residual MC are associated with a storage-induced
reduction in platelet immunity. A total of 108 platelets
from (A) BALB/c mice or (B) anti-AsGm1-treated SCID mice were either
transfused fresh (within 4 hours of collection, ) of after 18 hours
of storage () into CBA recipients (n = 10). Data is expressed as
the percentage of mice which become IgG antidonor antibody positive
during the weekly transfusion protocol.
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DISCUSSION |
Alloimmunization induced by platelet transfusions is defined by the
presence of anti-MHC class I antibodies and is thought to be due to the
direct recognition of donor MC within the platelet concentrates.
Leukoreduction of platelet concentrates has been shown to be effective
in reducing the incidence of alloimmunization.11-21 Nonetheless, some patients still become alloimmunized, and there is
evidence that leukodepletion may be ineffective for reducing alloimmunization in those patients previously sensitized against HLA
(eg, due to pregnancy),16 although this is
controversial.20 It has been suggested that the minimum
immunizing dose of contaminating MC within a 300-mL pooled platelet
concentrate is approximately 3 to 17 MC/µL (1 to 5 × 106 cells/concentrate) and leukofilters have been designed
to remove MC to levels below this concentration. Although there is
little experimental data that has confirmed these MC numbers, a
previous murine study demonstrated that allogeneic platelets could only induce antidonor alloantibodies if 103 contaminating MC
were present.22 Based on comparative blood volumes,
103 MC in mice translates to 2.5 × 106 MC
in humans. However, several reports have demonstrated that in healthy
recipients, leukoreduced allogeneic platelets stimulate the formation
of IgG antidonor alloantibodies23-27 due to an indirect allorecognition mechanism.25,26 The current study was
designed to characterize the relationship between allogeneic platelets and contaminating MC in modulating recipient immunity. The results indicate that as MC levels are reduced to levels of 1 MC/µL, the recipient's immune response against platelets is suppressed, whereas further MC reduction to levels approaching <0.05/µL enhances
allogeneic platelet immunity.
When serial 10-fold dilutions of the allogeneic platelets were
transfused into recipient mice, SCID mouse platelets were found to be
significantly more immunogenic than BALB/c mouse platelets (Fig 1).
Removing NK cells from the SCID mouse platelet donors with anti-AsGm 1 antibody did not affect their higher immunity (Fig 1C), suggesting that
NK cells, at least, are not responsible. Furthermore, the increased
recipient immunity was not due to differences in the amount of MHC
class I expression on the murine mouse platelets, but appeared to be
due to the absence of MHC class II positive B cells. The recipient
immune mechanism(s) responsible for the altered antiplatelet immunity
induced by the added MC is unknown, but several possibilities exist.
One explanation may be due to the difference in host immunity against a
purely MHC class I positive product versus one that contains small
numbers of MHC class II positive APC, which can directly interact with
recipient CD4+ T helper cells. In support of this, there is
accumulating evidence in experimental transplantation, which suggests
that the type and quantity of passenger leukocytes in MHC class I
positive allografts is important in determining the outcome of the
recipient's antidonor immune response, which can significantly affect
the graft's survival. For example, MHC class I positive liver grafts,
which are comparatively rich in leukocytes, are generally considered to
be the least immunogenic of organ allografts.30-35 In many
species, fully allogeneic liver allografts are permanently accepted
without any requirement for recipient immunosuppression,32
whereas prior deletion of the passenger leukocytes by irradiation
results in enhanced antidonor immunity and rapid graft
rejection.33,34 Recently, Steptoe et al35 have
demonstrated that dendritic cell numbers were responsible for
modulating liver rejection; this suggests that modulating the number of
MHC class II APC within allogenic tissue significantly influences the
recipient antidonor immune response. Our results with MHC class I
positive allogeneic platelets suggests an analogous pattern in that MHC
class II positive B cell numbers within the platelets are critical for
maximally inhibiting platelet-induced immunity (Figs 4 and
5).
Other mechanisms have been postulated to be responsible for the effect
of passenger leukocytes on recipient immune responsiveness. These
include the long-term engraftment of donor-derived hematopoietic cells
(microchimerism) or, alternatively, the transfer of potentially toleragenic costimulatory molecule-deficient APC resulting in operational tolerance via clonal anergy.36,37 Possibly
related to operational tolerance is the data in Fig 6. In the platelet preparations which contained MC, fresh (within 4 hours of collection) transfused platelets were significantly more immunogenic than aged
platelets (18 hours). This was not seen for the SCID mouse platelets
devoid of MC. This suggests that not only are a certain number of MC
cells required for modulating platelet immunity but, in addition,
storage may enhance their inhibitory effect; it may be that storage
induces age-related changes in APC, which causes them to induce a
recipient immune tolerant-like state. We are currently studying this
possibility.
The data in Fig 2 show that the contaminating MC differentially
regulated the recipient IgG isotype response; SCID mouse platelets, devoid of MC, induced high-titered complement-fixing IgG2a antidonor antibodies, whereas the presence of low numbers of MC (1/µL) within the platelets were associated with the production noncomplement-fixing IgG1 antibodies. We have previously shown that these types of platelet-induced IgG2a/IgG1 antidonor isotypes can induce a transient pancytopenia when infused into donor mice indicating their ability to
cause thrombocytopenia.38 Furthermore, because murine IgG2a and IgG1 antidonor isotype responses are strongly associated with the
presence of Th1 and Th2 cytokines respectively,39 our data also support previous results showing an increase in recipient serum
levels of interferon (IFN)- associated with leukoreduced platelet
transfusions28 and suggest that residual MC populations within platelets may be important in modulating Th1 and Th2 alloimmune responses. More striking, however, was that as the MC levels approached 1/µL, maximal suppression of both IgG1 and IgG2a antidonor immunity occurred. Overall, these results suggest that the platelet:MC ratio
transfused may be critical to the quantitative (titer) and qualitative
(isotype) outcome of the recipient antidonor immune response. For
example, compared with platelets at 20 × 106
platelets:1 MC (eg, anti-AsGm1-treated SCID mouse platelets), those at
106 platelets:1MC (eg, BALB/c mouse platelets) are optimal
for maximally suppressing platelet immunity. Whether these findings in
our murine model can be applied to human transfusions remains unknown,
but suggests, at least, that in immunocompetent recipients, excessive leukoreduction may be undesirable.
Although no animal model of transfusion will completely mimic the human
situation, these data suggest that in mammalian species with a
functioning immune system, allogeneic platelets, independently of
leukocytes, are potent immunogens with respect to MHC class I antigens.
The data further suggest that residual numbers of MHC class II positive
B cells within allogeneic platelets significantly modulate both the
quality (isotype) and quantity (titer) of the recipient's antidonor
immunity. These findings may have relevance to the development of
technologies capable, for example, of selective leukoreduction, which
may have benefit for those recipients who become alloimmunized despite
receiving standard leukodepleted products. More importantly, however,
we have established a murine model of MC-independent platelet immunity
which will allow for the examination of antigen-specific
immunotherapies for platelet alloimmunization.
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ACKNOWLEDGMENT |
We thank Dr Fraser Wright (Connaught Laboratories, Willowdale, Ontario)
for his invaluable discussions.
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FOOTNOTES |
Submitted January 22, 1998;
accepted September 16, 1998.
Supported by Grant No. TO. 22929 from the Canadian Red Cross Blood
Services.
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 John W. Semple, PhD, Division of
Hematology, St. Michael's Hospital, 30 Bond St, Toronto, Ontario,
Canada, M5B 1W8.
| |
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Abstract
Introduction