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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2135-2141
Stealth Cells: Prevention of Major Histocompatibility
Complex Class II-Mediated T-Cell Activation by Cell
Surface Modification
By
Kari L. Murad,
Edmund J. Gosselin,
John W. Eaton, and
Mark D. Scott
From the Division of Experimental Pathology and the Department of
Microbiology and Immunology, Albany Medical College, Albany, NY;
and the Department of Pediatrics, Baylor College of Medicine, Houston,
TX.
 |
ABSTRACT |
Transfusion or transplantation of T lymphocytes into an allogeneic
recipient can evoke potent immune responses including, in
immunocompromised patients, graft-versus-host disease (GVHD). As our
previous studies demonstrated attenuated immunorecognition of red blood
cells covalently modified with methoxy(polyethylene glycol) (mPEG), we
hypothesized that T-cell activation by foreign antigens might similarly
be prevented by mPEG modification. Mixed lymphocyte reactions (MLR)
using peripheral blood mononuclear cells (PBMC) from HLA class II
disparate donors demonstrate that mPEG modification of PBMC effectively
inhibits T-cell proliferation (measured by 3H-thymidine
incorporation) in a dose-dependent manner. Even slight derivatization
(0.4 mmol/L mPEG per 4 × 106 cells) resulted in a  75%
decrease, while higher concentrations caused 96% decrease in
proliferation. Loss of PBMC proliferation was not due to either
mPEG-induced cytotoxicity, as viability was normal, or cellular anergy,
as phytohemagglutinin (PHA)-stimulated mPEG-PBMC demonstrated normal
proliferative responses. Addition of exogenous interleukin (IL)-2 also
had no proliferative effect, suggesting that the mPEG-modified T cells
were not antigen primed. Flow cytometric analysis demonstrates that
mPEG-modification dramatically decreases antibody recognition of
multiple molecules involved in essential cell:cell interactions,
including both T-cell molecules (CD2, CD3, CD4, CD8, CD28, CD11a,
CD62L) and antigen-presenting cell (APC) molecules (CD80, CD58, CD62L)
likely preventing the initial adhesion and costimulatory events
necessary for immune recognition and response.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE T LYMPHOCYTE plays a central role in
the immune response and under normal circumstances, T-cell activation
is closely regulated.1,2 However, with the advent and
expansion of transfusion and transplantation medicine, the unwanted
activation of donor T cells in blood products has become an issue of
grave clinical concern, especially with regards to graft-versus-host
disease (GVHD).3-5 Consequently, extensive attention has
been given to the removal or inactivation of contaminating (passenger)
T cells, as well as other leukocytes (B lymphocytes, mononuclear cells, and granulocytes) from blood products.
Currently blood banks inactivate/remove passenger leukocytes via gamma
irradiation (inactivation) and/or filtration (removal). While both are
clinically proven leuko-reduction methods, neither is without
problems.6-8 Irradiation has been shown to result in a
decreased storage life of red blood cells (RBC) and may not completely
eliminate the risk of allosensitization as leukocyte membrane fragments
remain after irradiation. Filtration methods meanwhile are not 100%
effective and significantly increase the costs of blood processing due
to the high costs of the filter. Hence even following these methods, a
risk of allosensitization to disparate antigens and/or GVHD remains for
the transfused patient.
Previously, we demonstrated that the modification of RBC membranes with
nonimmunogenic materials (eg, methoxy[polyethylene glycol]; mPEG)
could yield antigenically silent (stealth) cells.9-12 These
stealth cells exhibit decreased/absent antisera-mediated agglutination,
antibody binding, and decreased immunogenicity. Importantly, this
modification procedure has no negative effects on normal cell
structure, function, or viability. Based on these data, we hypothesized
that it would be possible to alter both immune recognition of foreign
tissues (eg, major histocompatability complex [MHC]
class I and II) and prevent the necessary cell:cell interaction of the
T cell and antigen-presenting cell (APC) by the covalent modification
of cell surfaces with mPEG. To investigate this hypothesis, we have
used mixed lymphocyte reaction (MLR) assays using HLA class II
disparate donors.13,14
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MATERIALS AND METHODS |
Cell derivatization.
Peripheral blood mononuclear cells (PBMC) were prepared from healthy
donors by layering whole blood over a Histopaque gradient (Sigma Chemical Co, St Louis, MO) and centrifuging (900g) for 30 minutes. The PBMC were washed 3 times with HEPES/RPMI
media containing 1% human albumin. Cell derivatization was performed using cyanuric chloride activated mPEG,  5 kD (Sigma
Chemical Co, St Louis, MO) with concentrations ranging from 0.2 to 4 mmol/l mPEG per 4 × 106 cells. Washed PBMC were
incubated with activated mPEG for 60 minutes at room temperature in
isotonic alkaline phosphate buffer (PBS; 50 mmol/L
K2HPO4 and 105 mmol/L NaCl; pH
8.0).12 The derivatized cells were washed once in RPMI 1650 media containing 1x HEPES and 1% albumin. Depending on the study being
performed, the modified cells were either washed once in AimV media if
prepared for MLR or 2 times in PBS containing 1.5 mmol/L sodium azide
for antibody binding studies.
MLR.
MLR were conducted using PBMC obtained from HLA class II disparate
donors (Table 1).13,14 Donor
MHC typing (Class I A/B and class II DR) was determined by the Albany
Medical Center Transplantation Laboratory. Stimulator cells were
rendered unresponsive to proliferation by gamma-irradiation (2 Gy).15 Time course experiments were performed and
demonstrated that statistically significant responses were obtained
after 4 days of total incubation time using populations of human PBMC.
Unless stated otherwise, stimulator and responder PBMC (2.5 × 105 cells per well) were suspended in AimV media and
cocultured, in triplicate, in 96-well plates at 37°C, 5%
CO2, for 3 days. At day 3, all wells were pulsed with
3H-thymidine and incubated 24 hours at 37°C, 5%
CO2. Cellular DNA was collected onto filters using a cell
culture harvester (Skatron; Suffolk, UK) and 3H-thymidine
incorporation was determined by scintillation counting. For some
studies exogenous human recombinant interleukin-2 (IL-2; Intergen;
Purchase, NY) was added to each well of the MLR on day 1 at a
concentration of 0.83 U/mL. The viability of the modified cells both
before and after MLR culture was determined by a propidium iodide
exclusion assay on the flow cytometer.
Mitogen and antigen presentation studies.
Control and mPEG-derivatized cells were incubated, in triplicate, with
phytohemagglutinin (PHA) (Sigma; 1 µg per 107 cells) or
tetanus toxoid antigen16 (Sigma; 33.3 µg/mL) in 96-well plates at 37°C, 5% CO2 for 3 days. As in the MLR
studies, all wells were pulsed the 3H-thymidine at day 3 and incubated an additional 24 hours (37°C, 5% CO2)
before filter harvesting and determination of 3H-thymidine incorporation.
PBMC surface marker analysis.
To quantitate the ability of mPEG-derivatization to camouflage T-cell
and APC adhesion and surface molecules, control and derivatized PBMC
were examined by flow cytometry (FACScan, Becton Dickinson & Co,
Mountain View, CA).17,18 For these studies, control and
derivatized PBMC were resuspended into sample tubes at a concentration
of 1.0 × 106 PBMC to which 20 µL of
fluorescently-labeled mouse antihuman CD antibody (Becton Dickinson & Co) was added. The PBMC were incubated on ice for 30 minutes, washed 2 times in PBS containing azide and immediately analyzed for
the fluorescence intensity of T-cell (CD2, CD3, CD4, CD8, CD28, CD11a,
and CD62L) and APC (CD14, CD80, CD58, CD62L, and HLA class II)
molecules. A minimum of 10,000 PBMC per sample were quantitated for the
amount of bound fluorescein isothiocyanate (FITC), phycoerythrin
(PE), and/or PerCp fluorescently-conjugated antibodies.
All samples were analyzed using lymphocyte parameters previously
established for FACScan analysis.
Cytokine assays.
To determine if mPEG-modification altered cytokine profiles of the
PBMC, stimulator and responder PBMC were suspended in AimV media and
cocultured in 96-well plates at 37°C, 5% CO2, for 4 days. On day 4, the supernatant was removed from each well and like
wells were pooled together. IL-2, IL-4, IL-10, tumor necrosis factor
(TNF) and interferon (IFN) sandwich enzyme-linked immunosorbent assay (ELISA) assays (Immunotech; Marseilles, France) were performed according to the manufacturer's specifications.
| |
RESULTS |
In a 1-way MLR, responder TH cells recognize disparate
allogeneic class II MHC molecules on the stimulator cells (irradiated to prevent cell division) and proliferate. The CD4+
TH responder cells are the initial proliferative cells and
later stimulate proliferation of CD8+ cells.19
As shown in Fig 1, the covalent
modification of either the responder or stimulator (insert) PBMC with
mPEG results in virtually complete inhibition of recognition of the
antigenically foreign lymphocytes as denoted by the absence of
3H-thymidine incorporation in the responder cells. The
observed decrease in T-cell proliferation is dependent on the degree of mPEG-derivatization of the responder cells. However, even slight derivatization of the responder population (0.2 mmol/L mPEG/4 × 106 cells) results in a 75% decrease in
3H-thymidine incorporation. Higher degrees of modification
(0.6 to 2.4 mmol/L mPEG/4 × 106 cells) give rise to a
96% decrease in proliferation mimicking the background proliferation
rates seen in cells exposed to no antigenic stimulus. Furthermore, the
loss of proliferation arising from the pegylation of stimulator PBMC
cannot be overcome by the addition of increasing numbers of control
responder cells relative to the same stimulator cell population (Fig 1
insert). Hence, covalent modification of either the stimulator or
responder PBMC population effectively abrogates class II-mediated
proliferation.
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| Fig 1.
Cellular proliferation in MLR is effectively attenuated
by mPEG-modification of either the responder (main graph) or stimulator
(insert) PBMC population. As shown, pegylation of responder cells leads
to a mPEG dose-dependent loss of cellular proliferation in response to
a disparate MHC class II stimulator PBMC population. Insert: Increasing
responder cell numbers does not overcome the antiproliferative effects
of mPEG-modification. Shown is increasing numbers of unmodified
responder cells exposed to 2.5 × 105 pegylated (2.4 mmol/L per 4 × 106 PBMC) stimulator PBMC. Cell
proliferation was determined by 3H-thymidine incorporation
into the DNA of the responder cells after 4 days exposure to stimulator
cells (irradiated to prevent cell replication). Longer incubations (7 days) were also performed on human PBMC MLR yielding similar results,
but higher control counts per minute (CPM). The results
shown are the mean ± standard deviation (SD) of triplicate samples
from a representative experiment from over 20 individual studies. The
main graph uses donors 2 and 3 (responder), while the insert uses
donors 2 and 4 (responder). All values for the mPEG-derivatized cells
different from control PBMC at P < .0001.
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The loss of T-cell stimulation and activation is also evident in
photomicrographs of the MLR. As shown in
Fig 2, massive proliferation, cell
spreading, and extensive foci of responder cells are seen in response
to unmodified stimulator cells (Fig 2A). In contrast, the same
population of responder cells do not recognize mPEG-modified stimulator
cells (Fig 2B, 1.2 mmol/L mPEG/4 × 106 cells) as
evidenced by both their rounded morphology and failure to proliferate
(absence of 3H-thymidine incorporation; Fig 1).
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| Fig 2.
Photomicrograph of lymphocyte proliferation in response
to control (A) and mPEG-derivatized (B: 1.2 mmol/L mPEG/4 × 106 PBMC) stimulator PBMC. In (B), the PBMC remain small
and rounded, a morphological feature characteristic of resting,
nonproliferative lymphocytes, whereas in (A), the PBMC are larger and
exhibit stellate protrusions typical of antigen-stimulated cells. Shown
is the response of donor 4 to donor 1 (stimulator).
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Further supporting an abrogation in cellular response in the
mPEG-treated MLR is the significantly decreased production of proinflammatory cytokines. For example, endogenous IL-2 production from
MLR cocultures containing mPEG-derivatized PBMC (2.4 mmol/L mPEG/4 × 106 cells) exhibits an average decrease of 41.6% ± 12.3% (mean ± standard error of mean [SEM]; n = 4) as
compared with the production of endogenous IL-2 generated by unmodified
PBMC. Similar decreases (mean ± SEM) in the TNF
(69.8% ± 13.2%), IL-4 (89.6% ± 10.4%), and IFN (94.6 ± 3.7) cytokine levels are observed upon mPEG-modification of PBMC.
Furthermore, even suppressor cytokines, such as IL-10, exhibit a
significant decrease (81.7% ± 1.0%) in the pegylated PBMC
indicating maintenance of a resting, naive, population.
Moreover, the loss of proliferation is not solely due to decreased
cytokine production. As shown in Fig 3, the
addition of exogenous IL-2 to the MLR at concentrations normally used
to enhance a T-cell response16 cannot reverse the
attenuated immune response. This lack of IL-2-dependent proliferation
suggests that the mPEG-modified responder T cells are not antigen
primed (ie, are naive T cells) and therefore are unable to respond to
the exogenous IL-2. Importantly, the loss of IL-2 enhancement is also
not due to camouflaging of the IL-2 receptor. Using antibodies directed
against the IL-2 receptor (CD25), it is observed that even at the
highest rates of mPEG-derivatization, a < 35% decrease in receptor
detection is noted immediately after modification (data not shown).
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| Fig 3.
Proliferation of the mPEG-modified PBMC is not affected
by the addition of exogenous IL-2, suggesting that these cells are not
receiving the initial, antigen recognition signals necessary to evoke a
proliferation response. Cell proliferation is measured by
3H-thymidine incorporation into the DNA of responder cells
(2.5 × 105 PBMC) in response to irradiated stimulator
cells (2.5 × 105 PBMC) and 0.83 U/mL IL-2 present for the
duration of the 4-day culture. The results shown are the mean ± SD of
triplicate samples from a representative experiment of 4 independent
studies using pegylated responder cells. All values for the
mPEG-derivatized cells differ from control PBMC at P < .0001.
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The loss of proliferative response is also not due to mPEG-mediated
cytotoxicity. As shown in Table 2,
virtually no difference in the viability of control and mPEG-modified
PBMC is observed by propidium iodide analysis. Immediately after the
preparation of the MLR, 92% of the control and derivatized responder
cells are viable. Similarly, both the control and pegylated PBMC show virtually identical viability ( 60%) after 4 days in MLR culture. Further supporting the lack of mPEG-cytotoxicity is the finding that
derivatization does not alter the proliferative potential of the PBMC.
Stimulation of cellular proliferation of the mPEG-modified PBMC with
the mitogen PHA (1 µg per 107 cells)20
results in a normal proliferative response after 4 days of culture
(Fig 4). Similar results are obtained with
PHA stimulation before the pegylation procedure (data not shown). Hence, these data clearly indicate that loss of cellular viability or
proliferation potential do not underlie the loss of MHC-mediated activation and proliferation.
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| Fig 4.
No differences are observed in T-cell proliferation when
control and mPEG-modified PBMC are exposed to the mitogen PHA. Cell
proliferation is measured by 3H-thymidine incorporation
into the DNA of PBMC (2.5 × 105 PBMC) in response to PHA
stimulation for the duration of the 4-day culture (see Materials and
Methods). The results shown are the mean ± SD of triplicate samples
from a representative experiment of 4 independent studies. Insert:
Photomicrographs of mPEG-derivatized lymphocytes (1.2 mmol/L mPEG per 4 × 106 PBMC) without PHA treatment (A) and with PHA
treatment (B) after 4 days of culture.
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To determine if the inhibition of MHC class II-dependent T-cell
activation occurred as a consequence of altered cell-cell (T-cell APC)
interaction, we investigated the antigenic recognition of adhesion and
costimulatory molecules after cell surface derivatization with mPEG. It
was our hypothesis that weakening of any of these interactions by
mPEG-derivatization would impair both the initial cellular recognition
and binding of the foreign MHC class II molecules to the T-cell
receptor (TCR) complex, as well as the later costimulatory interactions, thus preventing T-cell activation and
proliferation.21-23
Indeed, flow cytometric analysis demonstrates that mPEG-modification
results in a significant reduction in the detectable signal for a
number of surface epitopes involved in cell activation and
proliferation. As shown in Fig 5, mPEG
modification effectively blocks antibody-mediated detection of CD28 (a
costimulatory molecule present on T cells, which binds to CD80), CD3
(associated with the TCR and necessary for signal transduction), CD4
(coreceptor for MHC class II molecules and present on T cells), as well
as CD80 (costimulatory molecule present on B cells and APC). Indeed, while 76.7% of the lymphocytes are initially CD3 positive, treatment with 0.6 and 2.4 mmol/L mPEG per 4 × 106 cells
results in a dramatic decrease in antibody binding, such that only
25.9% and 14.9%, respectively, of the cells are seen as
CD3+. Even more striking is the loss of detection of
CD28+ cells. In the control population, 72.9% are
CD28+, whereas in the 0.6 and 2.4 mmol/L mPEG-modified
cells, only 33.3% and 0.20%, respectively, appeared as
CD28+. Similarly, CD4 is effectively camouflaged by mPEG
modification. While 50.0% of control PBMC are CD4+, only
4.9% of the mPEG-modified (2.4 mmol/L mPEG per 4 × 106) cell were positive. Of further interest was the change
in CD80 (B7) present on B cells and APC. In control cells, 12.1% of
the PBMC are CD80+, whereas after derivatization with 0.6 and 2.4 mmol/L mPEG per 4 × 106 cells, only 0.9% and
0.82% are positive, virtually identical to the isotype control
(0.52%). Furthermore, as noted in Table 3,
comparable findings of decreased antibody binding to surface determinants involved in antigen recognition and response are observed
in both T cell (CD2, CD8, CD11a, and L-selectin) and APC (CD14, CD58,
and L-selectin) populations. Interestingly, the very large HLA class II
molecule on the APC is not significantly blocked by
mPEG-derivatization.
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| Fig 5.
Multiple cell determinants are effectively camouflaged by
cell surface derivatization with mPEG. Shown are the scatter plot
analyses for CD28 (A) costimulatory molecule present on T cells, binds
to CD80; CD3 (B) associated with TCR on T cells and is necessary for
signal transduction; CD4 (C) coreceptor for MHC class II molecules
present on T cells); and CD80 (D) costimulatory molecule present on B
cells and APC. Lane 1 represents the control unmodified PBMC; lane 2 represents the same PBMC population modified with 2.4 mmol/L mPEG (cell
concentration of 4 × 106); while lane 3 represents the
appropriate isotype control for the sample. In all cases, mPEG
modification resulted in a significant decrease (P < .001) in
antibody detection of the indicated cell marker. This decrease in
antibody detection occurs in a dose-dependent manner upon
increasing modification with mPEG (data not shown).
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Table 3.
Percent Binding of Monoclonal Antibodies to the
Surface of Control and mPEG-Modified PBMC (dosage of mPEG per
4 × 106 PBMC)
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While the PBMC proliferative response to MHC class II disparate cells
was shown to be effectively blunted, we were also interested in what
effect mPEG-modification has on exogenous antigen presentation. Consequently, we examined the proliferative response of control and
pegylated PBMC to tetanus toxoid. Interestingly, the proliferative response of human PBMC to tetanus toxoid is invariably a memory response. This is of importance because memory responses can be of
significant consequence in transfusion/transplantation medicine. As
shown in Fig 6, levels of mPEG modification
(eg 2.4 mmol/L per 4 × 106 PBMC) that completely
abrogated recognition of foreign PBMC also has a significant, but much
lesser, effect on the proliferative response to tetanus toxoid
(P < .001). These data indicate that mPEG-derivatization
cannot only significantly attenuate antigen presentation/activation,
but can also inhibit memory cell responses.
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| Fig 6.
T-cell proliferation in response to tetanus toxoid
challenge is decreased after mPEG-derivatization. However, the loss of
proliferation is significantly less than that observed after exposure
to disparate MHC-Class II antigens (see Fig 1; eg, 60% v
>95% at 1.2 mmol/L mPEG per 4 × 106 PBMC). Shown is
the mean ± SEM tetanus toxoid-dependent proliferation of pegylated
PBMC relative to control cells of 4 independent experiments.
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Thus, mPEG modification of either the responder or stimulator PBMC
effectively attenuates/prevents MHC class II-mediated activation and
proliferation of foreign lymphocytes. The loss of proliferation is
likely mediated via altered cell:cell communication between the APC and
T cell arising from the camouflage of crucial cellular determinants
involved in cell:cell adhesion and in the costimulatory signals
required for T-cell activation and proliferation. In addition, these
data suggest that the covalent modification of PBMC can also decrease
antigen-specific and recall antigen-dependent proliferation.
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DISCUSSION |
The MLR is a very sensitive measure of histocompatibility between the
donor and recipient, as it measures the antigenic variance within the
HLA class II complex (the primary antigens responsible for tissue
compatibility in transplants). Indeed, before the use of
immunosuppressive therapy (eg, cyclosporin), this assay, although time
consuming, was perhaps the best indicator of the probability of both
graft survival in an organ recipient and the onset and severity of
GVHD. Consequently, the MLR assay is particularly well suited to
examine whether mPEG modification could effectively attenuate the risk
of GVHD due to lymphocyte contamination of blood products.
Our studies demonstrate that mPEG-derivatization of PBMC effectively
abrogates allospecific, MHC class II-mediated T-cell activation. The
initial T cell-APC communication is dependent on the interaction of
adhesion molecules such as the T cell's CD2 (lymphocyte
function-associated antigen [LFA]-2) ligand binding to
CD58 (LFA-3) on the APC.24 Subsequent to this event,
cell:cell interaction is further stabilized by the binding of
CD11a/CD18 (LFA-1) on the T cell to CD54 (intracellular adhesion
molecule [ICAM]-1) on the APC.25 Initially,
all of these interactions are of relatively low-affinity, but coupling
of the TCR complex (CD3-TCR) and CD4 molecule with the MHC class II
ligands on the APC leads to conformational changes in CD11a resulting
in high-affinity binding to CD54.26-28 Once the TCR-MHC
class II interaction is engaged, the T-cell proliferative response is
fully dependent on secondary costimulatory events such as the
interaction of CD28 (T cell) with CD80 (B7; APC). The CD28-CD80
interaction provides a sufficient secondary signal to induce T-cell
proliferation and cytokine production.
As a consequence of the global camouflage imparted by cell surface
pegylation, the signals necessary to initiate T-cell activation, cytokine production, and subsequent T-cell proliferation are not generated. Indeed, these pegylated cells morphologically resemble resting PBMC even after 4 days of culture in a MLR. The lack of antigen
priming is further suggested by the failure of exogenous IL-2 to
override the mPEG-dependent inhibition. Because IL-2 is largely
responsible for the stimulation and proliferation of activated T cells,
the failure of exogenous IL-2 to induce MLR proliferation in the
mPEG-modified PBMC strongly suggests that the primary immune recognition/interactions (eg, adhesion, TCR-MHC) between the allogeneic APC and T cells have not occurred.
Interestingly, surface camouflage of the PBMC population does not
entirely prevent T activation and proliferation in response to other
antigens (tetanus toxoid) or mitogens (PHA). This clearly demonstrates
that the pegylated PBMC are viable and have the identical proliferation
potential (in the case of PHA) as the unmodified cells. The difference
in the response between a singular antigen, such as tetanus toxoid, and
a complex antigen (eg, HLA disparate cell) perhaps lies in the
phenotype of T-cell response stimulated for each antigen condition. In
a 1-way MLR, T-cell recognition to disparate HLA-class II cells occurs
through direct recognition of foreign APC (foreign MHC and peptide) as
well as some presentation of these cells by responder APC (self MHC and
foreign peptide).29 The responder T-cell population
recognizing the alloantigens are polyclonal naive T cells. In contrast,
the T lymphocytes responding to tetanus toxoid antigen are primarily
clonal memory cells. The differences between initiating a naive versus
memory T-cell response may provide an explanation for the less
effective attenuation of the tetanus toxoid response. For example, the
binding of soluble anticlass II immunoglobulins (eg, lymphocyte
activation gene-3 [LAG-3]-Ig) to PBMC results in
blocked allospecific T-cell proliferation, but normal mitogen and
recall antigen proliferation.30
However, the antiactivation/proliferation mechanism of
mPEG-derivatization of cells is in contrast to other methods of
preventing naive T-cell activation (eg, blocking antibodies
[anti-CD2 and anti-CD3]31 or soluble ligands [LAG-3-Ig
and cytotoxic T-lymphocyte antigen-4 (CTLA-4)
Ig]32). As shown in Fig 5 and Table 3, the immunosuppressive action of PBMC pegylation arises from a global camouflage of the cell and the simultaneous inhibition of the numerous
sites and pathways involved in T-cell activation and proliferation.
This has multiple advantages over the use of a signal blocking antibody
or ligand in that redundant activation pathways may be simultaneously
blocked/inhibited. In addition, the pegylation of blood products
is likely to be much more cost effective than the use of large
quantities of blocking antibodies.
Based on these and our previous findings,9-12 the use of
mPEG-modified blood or blood components could effectively decrease allosensitization rates in transfused patients and diminish the risk of
GVHD in immunosuppressed patients. However, the immunomodulatory effects of mPEG-modification may not be limited to RBC or lymphocytes. Because immunoprotective levels of mPEG-modification do not affect cellular structure, function, or viability of a wide range of cell
types (eg, RBC, lymphocyte, epithelial cells, endothelial cells, and
monocytes),9-12 it may be possible to translate these findings to other instances of tissue transplantation. A particularly attractive application would be the infusion of an activated mPEG solution into the vasculature of organ graft to derivatize the endothelium with covalently bound mPEG. The modified endothelium might
well become antigenically inert and prevent the initial binding of
preformed antibodies (hence, complement activation), as well as
decrease cell-cell interactions (eg, leukocyte rolling, adhesion, and
migration) responsible for hyperacute rejection.10,12 This
would be of potential therapeutic benefit in decreasing or preventing
both hyperacute and acute tissue rejection with a consequent increase
in the pool of available donor organs (both allogeneic and
xenogeneic).12,33-35
In summary, these data demonstrate that mPEG-modification of
lymphocytes can effectively block MHC class II-mediated, allospecific T-cell activation, as well as significantly attenuate antigen-specific and memory cell-dependent activation and proliferation. The loss of
responsiveness is likely due to altered cell-cell interaction via
reduced recognition and binding between the adhesion molecules and
receptor/ligand requirements involved in the early stages of T cell-APC
interaction and activation. These findings suggest that
mPEG-modification of passenger leukocytes still present in blood
products even after leukoreduction may prove to be an effective means
of preventing activation and proliferation of the donor T cells in
response to MHC disparate host tissue. Consequently, the use of
pegylated blood products may prove beneficial in the treatment of
immunosuppressed patients, as well as the chronically transfused.9-12
| |
FOOTNOTES |
Submitted February 10, 1999; accepted May 12, 1999.
Supported by National Institutes of Health Grants No. HL53066 (to
M.D.S.), HL58584 (to M.D.S.), and AI35327 (to E.J.G.) and by Cooley's
Anemia Foundation (K.L.M.).
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 Mark D. Scott, PhD, Division of
Experimental Pathology, Department of Pathology and Laboratory Medicine
MC-81, Albany Medical College, 47 New Scotland Ave, Albany, NY 12208;
e-mail: mscottpath{at}aol.com.
| |
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