Impact of photodynamic treatment with meso-substituted porphyrin on the immunomodulatory capacity of white blood cell-containing red blood cell products.

Abstract

After transfusion, the presence of contaminating white blood cells (WBC) in blood components may result in either deleterious or positive immunological responses. We have previously reported that photodynamic treatment (PDT) with meso-substituted mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin (Tri-P(4)) and red light can inactivate pathogens in red blood cell (RBC) products. The present study explored the effect of PDT on contaminating WBC in RBC products with varying hematocrit (Hct). After PDT, we evaluated adaptive and innate immunomodulation through allogeneic and mitogenic stimulation. PDT resulted in decreased T-cell proliferation which was more pronounced with lower Hct. Dark effect of porphyrin Tri-P(4) was remarkable on antigen-presenting cells affecting expression of co-stimulatory molecules CD80/CD86. Finally, cytokine profile after PDT revealed a mixed Th1/Th2 type response while surface antigen expression supported the development of alternatively activated macrophages (AAM phi or Type 2 macrophages) instead of dendritic cells. In conclusion, PDT with Tri-P(4) altered proliferation, allo-stimulation, cell surface antigen expression and cytokine profiles of the cells. These results suggest that PDT may be potentially useful in preventing transfusion-associated graft-versus-host disease and alloimmunization. It seems worthwhile to further explore PDT-induced immunomodulation to optimize conditions which may result in allo-tolerance by AAM phi.

Full text

Page 1

Impact of Photodynamic Treatment with Meso-substituted Porphyrin
on the Immunomodulatory Capacity of White Blood Cell-containing
Red Blood Cell Products
Laurence Trannoy*1, Dave Roelen2, Karin Koekkoek2and Anneke Brand1,2
1Department of Research and Education, Sanquin Blood Bank South-west, Leiden, The Netherlands
2Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden,
The Netherlands
Received 20 February 2009, accepted 23 July 2009, DOI: 10.1111⁄j.1751-1097.2009.00624.x
ABSTRACT
After transfusion, the presence of contaminating white blood cells
(WBC) in blood components may result in either deleterious or
positive immunological responses. We have previously reported
thatphotodynamictreatment(PDT)withmeso-substitutedmono-
phenyl-tri-(N-methyl-4-pyridyl)-porphyrin (Tri-P(4)) and red
light can inactivate pathogens in red blood cell (RBC) products.
The present study explored the effect of PDT on contaminating
WBCinRBCproductswithvaryinghematocrit(Hct).AfterPDT,
we evaluated adaptive and innate immunomodulation through
allogeneic and mitogenic stimulation. PDT resulted in decreased
T-cell proliferation which was more pronounced with lower Hct.
Dark effect of porphyrin Tri-P(4) was remarkable on antigen-
presenting cells affecting expression of co-stimulatory molecules
CD80/CD86. Finally, cytokine profile after PDT revealed a
mixed Th1/Th2 type response while surface antigen expression
supportedthedevelopmentofalternativelyactivatedmacrophages
(AAM/ or Type 2 macrophages) instead of dendritic cells. In
conclusion, PDT with Tri-P(4) altered proliferation, allo-stimu-
lation, cell surface antigen expression and cytokine profiles of the
cells. These results suggest that PDT may be potentially useful in
preventing transfusion-associated graft-versus-host disease and
alloimmunization. It seems worthwhile to further explore PDT-
induced immunomodulation to optimize conditions which may
result in allo-tolerance by AAM/.
INTRODUCTION
Risks associated with blood transfusion have dramatically
decreased since the implementation of careful donor selection
and the development of sensitive diagnostic tests; however, a
certain risk for complications by contaminating pathogens still
remains (1,2).
Not only are viruses, bacteria and protozoa considered as
pathogenic, allogeneic white blood cells (WBC) present in
blood components can also be recognized as pathogenic
contaminants, inducing immune responses in the recipient,
sometimes with deleterious effects. Such WBC-induced com-
plications of blood transfusion include transfusion-associated
graft-versus-host disease (GvHD), transfusion-related immu-
nosuppression, which can result in enhanced susceptibility to
postoperative infection and multiple-organ-dysfunction syn-
drome, nonhemolytic febrile transfusion reactions (NHFTR)
and immunization to human leukocyte antigens (HLA) (3).
Although the mechanism is yet to be unraveled, the presence of
allogeneic WBC in transfusion products can be beneficial in
reducing the risk of allograft rejection and of spontaneous
recurrent abortion (3).
Anumberofapproacheshavebeenimplementedtominimize
the pathogenic role of contaminating WBC. Leukodepletion of
bloodcomponentsbyfiltrationhasresultedinareductioninthe
frequency of NHFTR and HLA immunization (2). Gamma
irradiationiseffectiveinpreventingWBCproliferationbutdoes
notimpairthestimulatorycapabilities ofthesecells(4).Someof
these techniques, aimed to inactivate pathogen in blood prod-
ucts, such as Mirasol-Pathogen-Reduction technology (5) and
Inactine technology using Pen 110 (6) have been shown to also
reduce the proliferative capacity of the WBC as well as their
ability to present antigens. Furthermore, it has been shown that
photodynamic treatment (PDT) can have immunomodulatory
effects in various experimental systems. In vivo studies showed
that PDT with verteporfin was effective in alleviating immune
pathology in murine models of arthritis, contact hypersensi-
tivity, experimental allergic encephalomyelitis and retention of
allogeneic skin grafts (7,8). PDT of peripheral blood mononu-
clear cells (PBMC) with a rhodamine derivative TH9402 may
induce tolerogenic dendritic cells (DC) (9), whereas treatment
with extracorporeal therapy using 8-methoxypsoralen was
foundto reverse GvHD (10). Investigations into themechanism
involved in immunomodulatory PDT have shown that PDT
selectively eliminates certain cell populations, disrupts T-cell
response by affecting antigen-presenting cells (APC) by lower-
ing expression of key cell surface molecules, including MHC I
and II, CD80, CD86 and CD54 and modulates the cytokine
network (11–13).
PDT utilizes mainly porphyrin and phthalocyanine-based
photosensitizers which accumulate somewhat selectively in
rapidly dividing cells such as activated cells of the immune
system (14–16). Exposure to sublethal doses of PDT may result
in the modification of cell surface receptor expression levels
andcytokinerelease and
behavior (8).
consequentlyinfluencecell
*Corresponding author email: l.trannoy@zonnet.nl (Laurence Trannoy)
?2009TheAuthors.JournalCompilation.TheAmericanSocietyofPhotobiology 0031-8655/10
Photochemistry and Photobiology, 2010, 86: 223–230
223

Page 2

Mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin
(Tri-P(4)) is a positively charged amphiphilic porphyrin that
can be activated with red light to generate singlet oxygen
responsible for oxidative damage to vicinal molecules. We
have previously demonstrated that this photosensitizer was
superior to other similar porphyrin photosensitizers in its
action to efficiently inactivate a wide range of viruses and
bacteria with limited damage to red blood cells (RBC) (17,18).
PDT with Tri-P(4) was also shown to have limited effects on
cord blood-derived stem cells and progenitor cells (19). These
observations raised the question as to whether this treatment
may be able to modulate WBC present in RBC product to
reduce immunological posttransfusion reactions or to enhance
beneficial tolerizing effects of blood transfusion.
We therefore explored the potential immunological effects
of PDT with Tri-P(4) on contaminating WBC in RBC
products with varying hematocrit (Hct). For this, we evaluated
the effect of the treatment on in vitro adaptive and innate
immunological parameters, such as proliferation in response to
mitogens and to allogeneic stimulator cells, the ability to
stimulate proliferation of healthy untreated responder cells,
survival, expression of constitutive and inducible cell surface
antigens and production of soluble mediators (cytokines).
chloride
MATERIALS AND METHODS
Materials. Photosensitizer
pyridyl)-porphyrin chloride) was kindly provided by Buchem b.v.
(Apeldoorn, The Netherlands). Stock solutions of 1 mM
prepared in PBS and stored in the dark at 2–6?C. PBS (pH 7.4)
and Ficoll gradient were provided by the Leiden University Medical
Centre (LUMC) Pharmacy (Leiden, The Netherlands). SAG-M is a
red cell storage solution containing 150 mmol L)1
mmol L)1adenine, 50 mmol L)1glucose and 29 mmol L)1mannitol
(FreseniusHemocare,Emmer-Compascuum,
Iscove’s modified Dulbecco’s medium and RPMI-glutamine was
purchased from Invitrogen (Breda, The Netherlands). Penicillin and
streptomycin (pen⁄strep) were purchased from Bio-Whittaker (Ver-
viers, Belgium). Pools of 40 individual AB sera, screened for the
absence of HLA antibodies and disturbance in mixed lymphocyte
reaction (MLR), were obtained from Sanquin Blood Bank South-
west (Rotterdam,TheNetherlands).
lipopolysaccharide (LPS) and b-mercapto-ethanol were purchased
from Sigma-Aldrich (Zwijndrecht, The Netherlands). Phycoerythrin
(PE)-conjugated anti-human CD14, CD16, CD33, CD54, CD80,
CD83, CD86, HLA-DR monoclonal antibodies (MoAb), PE-
conjugatedisotype-matchedmouse
isothiocyanate (FITC)-conjugated anti-human CD45 MoAb, FITC-
conjugated isotype-matched mouse IgG1 MoAb, propidium iodide
(PI), IOTest3 lysis solution and the Flow-count fluoropheres were
purchased from Beckman Coulter (Mijdrecht, The Netherlands).
Blood products and processing. Whole blood-derived buffy coats
wereprovidedbyhealthyHLA-typeddonorsfromSanquinBloodBank
supply (Leiden, the Netherlands). The buffy coats were washed twice at
500 gfor10 minwithSAG-Mtoremoveplasma.Afterthelastwash,the
supernatant and the WBC layer were transferred into a new tube and
centrifuged at 850 g for 10 min. The WBC pellet was resuspended in
10 mLSAG-Mforcounting.Subsequently,theWBCandSAG-Mwere
added to the washed RBC to obtain a final product with a WBC
concentration 20.106cell mL)1and an Hct of 20 ± 4% or 55 ± 5%.
Hct and cell concentrations were measured by Act 10 blood analyzer
(Beckman Coulter).
The final product obtained is referred to as WBC-enriched RBC
(WBC-RBC).AllsampleswereDNAtypedatlowresolutionfortheloci
HLA-A, -B, -C, -DRB1 and -DQB1 by polymerase chain reac-
tion⁄sequence-specific oligonucleotide using a reverse dot-blot method
(20). HLA typing was performed at the national reference laboratory
for histocompatibility testing (LUML).
Tri-P(4) (mono-phenyl-tri-(N-methyl-4-
M were
NaCl, 1.25
TheNetherlands).
Phytohemagluttin (PHA),
IgG2aMoAb,fluorescein
Light source. For all illuminations, the light source was a 300 W
halogen lamp (Philips, Eindhoven, The Netherlands) combined with a
cut-off filter (Kodak, wratten nr. 23A, Rochester, NY) only transmit-
ting light >600 nm. The fluence rate at the level of the petri dish was
adjusted to 100 W per m2, as measured with an IL1400A radiometer
with a SEL033 detector (International Light, Newburyport, MA). To
avoid heating of the samples, the light was passed through a 1 cm thick
water layer. The temperature did never exceed 25?C.
Photodynamic treatment. Tri-P(4) was added to the blood compo-
nents to a final concentration of 25 lM
were thoroughly mixed. Subsequently 3 mL samples were transferred
into 6 cm diameter petri dishes (tissue culture grade; Greiner, Alphen
a⁄d Rhijn, The Netherlands), resulting in a cell layerthickness of 1 mm.
The dishes were agitated at room temperature on a horizontal circular
shaker (75 rpm) for 5 min in the dark and subsequently illuminated
under continuous agitation for 30 min corresponding to a light of
180 kJ m)2. The light control corresponded to WBC-RBC illuminated
without addition of photosensitizer. The dark control corresponded to
WBC-RBC spiked with photosensitizer but kept in the dark during the
whole procedure (including preincubation and illumination time).
Mitogenic stimulation. To study proliferation, mononuclear cells
(MNC) were isolated from WBC-RBC immediately after PDT by
centrifugation on Ficoll density gradient and subsequently, the cells
were resuspended in culture medium containing RPMI-glutamine
supplemented with 10% human serum and 100 lg mL)1Pen⁄strep.
The cells (106cell mL)1) were stimulated with 1 lg mL)1PHA in
U-bottom 96-well plates. Cell proliferation was quantified by incubat-
ing the cells during the last 18 h of 4-day cultures with 1 lCi
[3H]thymidine. Before being pulsed, supernatants were collected and
stored at )40?C. Proliferation was measured as [3H]thymidine incor-
poration by liquid scintillation spectroscopy using a beta plate (Wallac,
Turku, Finland). Results are expressed as percentage of light control.
Mixed lymphocyte reaction. To study allo-reactivity, MNC isolated
from WBC-RBC after PDT by Ficoll isolation, as described above,
were co-cultured with MNC isolated from a completely HLA-
mismatched buffy coat (allogeneic cells). Equal numbers of responder
and stimulator cells (100 lL of 106cells mL)1) were cultured in
U-bottom 96-well plates (Costar, Cambridge, MA). Stimulator cells
were obtained by irradiation at 3000 Rad. Tri-P(4)-treated cells,
controls and allogeneic cells were used both as responders and as
stimulators. As a control for spontaneous proliferation, the responder
andstimulatorcellswereculturedinmediumonly.Cellproliferationwas
quantified by incubating the cells during the last 18 h of 5-day cultures
with 1 lCi [3H]thymidine (Amersham International, Amersham, UK).
Assays were performed in triplicate. Before being pulsed, supernatants
were collected and stored at )40?C. Proliferation was measured as
described above. Results are expressed as percentage of light control.
LPS stimulation. After PDT, WBC-RBC was washed four times at
500 g for 10 min to remove the photosensitizer and subsequently
diluted 10-fold with IMDM supplemented with 100 lg mL)1Pen⁄-
strep and 50 lM
suspension (150 lL⁄well) was plated in triplicate in plate-bottom plate
(Costar) and 50 lL of LPS (final concentration 250 pg mL)1) or
culture medium was added. After 20 h of incubation at 37?C, 5% CO2,
samples were pooled and then centrifuged at 540 g for 5 min. The
supernatant was harvested and frozen at )40?C until use. The
supernatant from the non-LPS-treated cell suspensions was harvested
and frozen to be used as reference values before stimulation.
Cytokine analysis. Harvested supernatants from MLR and LPS
stimulation assays were tested for a total of 17 cytokines at once
(interleukin-1beta [IL-1b], IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,
IL-12p70,IL-13,IL-17,granulocyte-colonystimulatingfactor[G-CSF],
granulocyte-monocyte-CSF [GM-CSF], interferon-gamma [IFN-c],
monocyte chemoattractant protein-1 [MCP-1], macrophage inflamma-
tory protein-1b [MIP-1b] and tumornecrosis factor-a[TNF-a]) with the
Bio-Plex human Cytokine panel following the manufacturer’s descrip-
tion.SampleswereanalyzedusingaBio-Plexarrayreaderequippedwith
Bio-Plex software (Bio-Rad Laboratories,Veenendaal,TheNetherlands).
Immunophenotyping. After PDT application of WBC-RBC, samples
were washed four times with PBS at 150 g for 10 min to remove the
photosensitizer. Cells were incubated with anti-human MoAb for
20 min at room temperature. After red cell lysis with lysis solution
IOTest3, samples were analyzed on a four-laser cytometer EpicsXL-
MCL flow cytometer using Expo32 software (Beckman Coulter).
M. The WBC-RBC suspensions
M beta-mercapto-ethanol. For stimulation with LPS, cell
224Laurence Trannoy et al.

Page 3

Cell viability. Using PI labeling and flow cytometry, cell viability
was assayed in cell cultures from MLR at days 1, 4 and 6, day 0 being
the day of input. Unstained cells and cells labeled with PE-conjugated
isotype-matched mouse IgG2a and FITC-conjugated IgG1 of irrele-
vant specificity were used as negative controls.
Statistics. For statistical analysis, repeated-measures analysis of
variance (ANOVA) or Friedman test were used when appropriate,
dependingonthetypeofdistribution.Pvaluesarefromcomparisonwith
lightcontrolsanddeterminedbyTukey’sorNewman–Keuls’posttestsas
indicated in legends. P values <0.05 were considered as statistically
significant. All data are expressed as mean ± standard deviation (SD).
RESULTS
Effect of PDT on proliferation
After PDT application, MNC were tested for their capacity to
proliferate in response to PHA (Fig. 1A), and to allogeneic
stimulation (Fig. 1B). PDT induced a significant reduction in
proliferation when samples were treated in low Hct level. After
stimulation with PHA, no significant difference was observed
between light and dark controls. However, after PDT at low
Hct level and allogeneic stimulation, we observed a significant
increase in proliferation in dark controls compared with light
controls. The spontaneous proliferation of the phototreated
cells and dark control cells did not differ from the light
controls (data not shown).
Effect of PDT on the ability of MNC to present antigen
After PDT application, MNC were tested for their capacity to
present antigen and thereby stimulate a proliferative response
in untreated HLA-mismatched allogeneic cells. As can be seen
from Fig. 1C, the proliferative response induced by photody-
namically treated stimulator cells was significantly reduced by
approximately 50% of the light controls when the cells were
treated in lowHct level.In highHct level, nodifference between
the samples was seen. In the dark samples, no difference in the
proliferative response was seen compared with light controls.
The spontaneous proliferation of the irradiated stimulator cells
did not differ between the samples (data not shown).
Effect of PDT on cell viability
Immediately after PDT of WBC-RBC, MNC were isolated by
Ficoll centrifugation. Cell viability was determined during the
MLR cultures at days 1, 4 and 6 of culture to check that the
proliferation inhibition induced by PDT may not be due to
apoptosis induction. PDT did not affect the overall cell
viability. The percentage of PI-positive cells was 10–15% at
day1andincreased to25–30%atday6ofthe MLRculture and
was similar to untreated controls. There was no difference in
nonviable cells in cultures where photodynamically treated cells
were used as responders or in cultures where the treated cells
wereusedasstimulatorcells.Inaddition,nodifferencewasseen
between light controls and dark controls (data not shown).
Effect of PDT on the release of cytokines by allo-activated
phototreated responder cells in MLR
The cytokine pattern produced in MLR by photodynami-
cally treated MNC used as responders was determined. As
the effect of PDT on the proliferation was dependent on the
Hct level of the treated product, a similar relationship was
observed with the cytokines released during the cultures.
When MNC were treated in low Hct level (<30), a
significant negative correlation was observed between proli-
feration and the release of IL-4, IL-10, IL-13, IL-12, TNF-a,
MIP-1b and INF-c (Fig. 2); the lower the proliferation, the
more cytokines were released. In contrast, the release of IL-2
was positively correlated with proliferation; the lower the
proliferation, the lower IL-2 was released. In high Hct level
(>50), no significant change in cytokine release in PDT
Figure 1. Proliferation capacity of mononuclear cells (MNC) after
phytohemagluttin (PHA) stimulation (A), and allogeneic stimulation
(B) after photodynamic treatment (PDT) of WBC-RBC in low (?20%)
and high (?50%) hematocrit (Hct). (C) Capacity of phototreated
MNC to stimulate proliferation in MLR. Proliferation is expressed as
percentage of light control. Mean with standard deviation are shown.
Light controls (black bars), Dark samples (gray bars), PDT samples
(white bars). Statistical differences between the three groups of samples
within each Hct level were performed using ANOVA and Tukey post
tests. Comparison of each group between the two Hct levels was
performed using Student’s t-test. *P < 0.05; **P < 0.01.
Photochemistry and Photobiology, 2010, 86225

Page 4

samples was seen compared with light controls. Moreover,
for IL-1b, IL-5, IL-6, IL-7, IL-8, IL-17, G-CSF, GM-CSF
and MCP-1, no significant difference with light controls was
observed at both Hct levels (data not shown). Finally, no
difference in cytokine pattern between dark and light
controls was seen (data not shown).
Effect of PDT on the release of cytokines by Tri-P(4)-treated
stimulator cells in MLR
The cytokine pattern produced in MLR by allogeneic cells
after antigen presentation by Tri-P(4)-treated MNC was
determined. In concordance with the proliferation results, the
level of cytokines released did not correlate with the Hct level
of the product during PDT. However, as can be seen from
Fig. 3, significant negative correlations between proliferation
and the release of IL-1b, IL-6, INF-c, TNF-a, MIP-1b and
GM-CSF were observed. Striking was that the cytokine levels
mostly exceeded the levels of light controls (>100%) when
proliferation was the most inhibited, except for IL-2 showing a
significant positive correlation with proliferation. In addition,
no difference in cytokine pattern between dark and light
controls was seen (data not shown).
Effect of PDT on LPS-stimulated cells
After PDT application, WBC-RBC were stimulated with LPS
for 20 h, after which cytokine production and antigen expres-
sion were determined. As can be seen in Fig. 4, in dark and
PDT samples, a significant reduction of nearly 50% in release
of IL-8 and MIP-1b compared with light controls was
observed (graph in Fig. 4) whereas for the other cytokines
tested no difference with the light controls was observed
(frame in Fig. 4).
The proportion of cells expressing surface antigen was
determined (Fig. 5). PDT induced an increase in numbers of
CD14, HLA-DR and CD83-expressing cells compared to
light and dark controls. The increase in the amount of
HLA-DR and CD83-positive cells was only observed after
LPS stimulation, while the increase in the amount of
CD14 positive cells was LPS independent. Moreover, 4 h
after PDT application and before LPS stimulation, the
amount of CD80 and CD86-expressing cells decreased
significantly compared to light controls as well in the dark
samples as in PDT samples. No PDT effect and LPS
effect was observed in the amounts of CD33, CD54 and
CD16-expressing cells (Fig. 5).
Changes in cellular expression of cell surface markers
were determined by measuring mean fluorescence intensities
(MFI) of each marker (Table 1). The MFI of CD14 and
HLA-DR expressions increased upon PDT as compared to
light and dark controls and both were LPS independent. In
contrast, MFI of CD83 expression increased upon PDT only
without LPS, while after LPS stimulation, MFI remained
similar between the three groups of samples. No significant
change in MFI of CD33, CD16, CD54, CD80 and CD86
expressions was observed after PDT compared to light and
dark controls.
DISCUSSION
PDT with Tri-P(4) and red light has been shown to inactivate
a number of pathogens including viruses and bacteria in
RBC (18) and cord blood products (19). As contaminating
Figure 2. Correlation between proliferation and cytokine release in mixed lymphocyte reaction wherein mononuclear cells were used as responders
after photodynamic treatment (PDT) of WBC-RBC in Hct <30 (circle) or in Hct >50 (square). Both level of released cytokine and proliferation
rate are expressed as percentage of light control. Only PDT samples are represented. Results shown are mean of triplicates of 6 independent
experiments, each one represented by one dot. Correlations are indicated by Pearson r and P-value. Fit lines were established by linear regression.
226Laurence Trannoy et al.

Page 5

WBC in RBC are implicated as a causative factor for an
immune response after transfusion (3,21), we were interested
to determine the effect of Tri-P(4)-mediated PDT on these
cells.
PDT with Tri-P(4) had no effect on the spontaneous
proliferation and cell viability of the treated WBC. However,
photodynamically treated WBC showed impaired proliferation
afterstimulation withPHA anduponco-culture withallogeneic
stimulator cells. The degree of impairment of proliferative
capacity increased when WBC were exposed to PDT in lower
Hct. Although cytokine production was reduced at a certain
levelofproliferation inhibition,strongerproliferation
inhibition resulted in less decrease in cytokine production
(Fig. 2). PDT also impaired the capacity of APC to stimulate
the T-cell proliferation in MLR; however, this effect was less
influenced by Hct.
Moreover, the reduction in the stimulation capabilities of
APC was associated with increased production of cytokines
suggestive of active inhibition. We also found by using LPS
that the APC response was affected by the presence of Tri-P(4)
only, in the absence of light.
The dark effect of the photosensitizer Tri-P(4) on APC was
compatible with our observations that Tri-P(4) binds noncov-
alently but differentially to leukocytes with a preference for
IL-8MIP-1beta
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
Cytokine release (pg/ml)
*
*
*
*
*
*
Figure 4. Effect of photodynamic treatment on the release of cytokines in lipopolysaccharide-stimulated cells. Cytokine releases are expressed in
pg mL)1. Mean ± SD of six independent experiments are shown. Asterisks indicate statistical differences with the light controls as determined by
ANOVA and Tukey posttest. *P < 0.05; **P < 0.01. Graph legend: Light controls (black bars); Dark controls (gray bars); PDT (white bars).
Figure 3. Correlation between proliferation and cytokine release in mixed lymphocyte reaction wherein mononuclear cells were used as stimulators
after photodynamic treatment (PDT) of WBC-RBC. Both level of released cytokine and proliferation rate are expressed as percentage of light
control. Only PDT samples are represented. Results shown are mean of triplicates of five or six independent experiments, each one represented by
one dot. Correlations are indicated by Pearson r and P-value. Fit lines were established by linear regression.
Photochemistry and Photobiology, 2010, 86227

Page 6

monocytes⁄macrophages and granulocytes (L.L. Trannoy,
unpublished). We observed that release of both chemoattrac-
tants IL-8 and MIP-1b were reduced as well in dark and in
PDT samples. It has been reported that the release of these
cytokines has in common a signaling pathway (22,23). Further
studies are required to identify the membrane receptor which
may interact with Tri-P(4) disrupting simultaneously the
release of IL-8 and MIP-1b. Moreover, the expression of
co-stimulatory molecules CD80 and CD86 on APC was also
reduced both in dark and PDT samples (Fig. 5). These results
suggest that Tri-P(4) affects the antigen presentation function-
ality of APC by interacting directly with monocytes⁄macro-
phages even in the absence of light.
The Hct of the blood product influences the photody-
namic effect on T cells. Increasing the Hct caused higher
pigmentation of the blood product and may hinder proper
light penetration to the WBC. Thereby PDT may affect
differently the WBC. High-dose and low-dose PDT has been
shown to generate different results as high doses resulted in
necrotic or apoptotic cells, while low-dose PDT or sublethal
dose PDT may result in the modification of cell surface
receptor expression and consequently influence cell activities
(8,24).
Another possible explanation is the capacity of RBC to
scavenge reactive oxygen species (ROS) (25). Tri-P(4) is a
porphyrin that remains extracellular by binding exclusively
to the cell membrane of living and resting cells (L.L.
Trannoy, unpublished). Illumination of porphyrin Tri-P(4)
at appropriate wavelength generates ROS, in particularly
singlet oxygen (1O2) which reacts with substances in close
vicinity. ROS is also a natural product, produced by
neutrophils in host defense to kill invading pathogens and
involved inintercellular and
homeostasis,cell proliferation
inflammatory and immune responses (26). Generation of
ROS by light activation of Tri-P(4) can cause lipid and
amino acid peroxidation, affecting cell membrane-associated
targets and may produce a number of effects on the immune
cells. RBC may play a protective role by scavenging PDT-
generated extracellular ROS and varying the Hct of RBC
may affect the degree of photodamage induced by ROS.
intracellular
and
signaling of
differentiation and
Figure 5. Effect of photodynamic treatment (PDT) on the cell surface
expression on lipopolysaccharide (LPS)-stimulated WBC-RBC. Cell
surface antigen expression is expressed as percentage of positive cells
from the CD45+population. Day 0 corresponds to unstimulated
WBC-RBC 4 h after PDT. Medium and LPS corresponded to WBC-
RBC cultured after PDT without and with LPS respectively for 20 h.
Light controls (black bars), Dark controls (gray bars), PDT (white
bars). Mean ± SD of eight independent experiments are shown.
Asterisks indicate statistical differences with the light controls in their
respective culture media as determined by ANOVA and Tukey
posttest. *P < 0.05; **P < 0.01; n.d. = not detectable.
Table 1. Intensities of cell surface marker expression (MFI) on WBC after PDT.
MediumLPS
Light control DarkPDT Light control DarkPDT
HLA-DR
CD83
CD14
CD33
CD16
CD54
CD80
CD86
107 ± 89
30 ± 12
729 ± 234
39 ± 12
32 ± 12
87 ± 95
62 ± 98
69 ± 29
114 ± 63
33 ± 7.6
886 ± 271
39 ± 17
31 ± 7.4
106 ± 130
42 ± 29
97 ± 73
210 ± 139*
41 ± 6.7*
1016 ± 404†
35 ± 16
31 ± 6.6
76 ± 57
45 ± 19
110 ± 55
136 ± 129
26 ± 12
773 ± 187
52 ± 24
32 ± 8.4
215 ± 272
34 ± 3.7
59 ± 48
99 ± 73
26 ± 11
709 ± 249
49 ± 29
31 ± 12
147 ± 178
27 ± 6.7
52 ± 27
208 ± 157*
28 ± 8.7
1174 ± 411†
48 ± 20
40 ± 23
166 ± 160
41 ± 7.4
66 ± 24
Data indicated are mean fluorescence intensity (MFI) ± SD of eight independent experiments. WBC = white blood cell; LPS = lipopolysac-
charide; HLA = human leukocyte antigen. Statistical differences were determined with ANOVA and Newman–Keuls’ posttests or with Friedman
test depending on the type of distribution. Tests were performed in respective medium. Significant difference with light controls (P < 0.05):
*ANOVA; †Friedman test.
228 Laurence Trannoy et al.

Page 7

Further research would determine whether illumination with
a lower light dose in the absence of RBC may show the
same effects on WBC after PDT with Tri-P(4).
In our study, T-cell response and IL-2 release correlated
with the Hct level of the photodynamically treated WBC-RBC
products, as seen after PDT when Tri-P(4)-treated cells were
used as responders in MLR and after PHA stimulation. This
indicated that T cells are target for ROS-mediated PDT and
that the presence of RBC during PDT application may protect
the T cells against the damaging effect of singlet oxygen.
Possible targets of PDT may be the TCR and CD28 signaling
pathways which were found to be sensitive to oxidative stress
induced, e.g. by BSO (DL-buthionine-(S,R)-sulfoximine)
depleting intracellular antioxidant-reduced glutathione (27)
and after PUVA treatment (28). Both treatments result in
T-cell hyporesponsiveness.
Our results showed that PDT with Tri-P(4) acts both on
APC and on T cells, on the one hand through a dark effect of
Tri-P(4) down-regulating the costimulatory molecules CD80
and CD86 and on the other hand through a photodynamic
effect inhibiting the responder and stimulatory potentials of
WBC in MLR. Therefore we suggest that the immunomod-
ulatory effect of PDT may result in an impaired interaction
between T cells and APC. Blockage of CD80⁄CD86-CD28
co-stimulation was shown to result in T-cell hyporesponsive-
ness and in generation of alternatively activated macrophages
(AAM/), also referred to as Type 2 macrophages (29–31). The
induction of this type of macrophages is supported on the one
hand by the PDT-induced increase in cell number and cellular
expression of HLA-DR and CD14 on APC (Table 1), and on
the other hand by the refractoriness of CD80⁄CD86 up-
regulation to LPS stimulation (Table 1). These cellular events
are partly characteristic of AAM/ as demonstrated by Xu
et al. (32) and Verreck et al. (30). As these cells are unable to
prime T cells, they may contribute to the PDT-induced T-cell
hyporesponsiveness (31,33). An additional indication pointing
toward the involvement of APC after PDT is the fact that
unstimulated WBC-RBC cultures of phototreated cells showed
an up-regulation of CD83 which was refractory to LPS
stimulation (Table 1). Indeed, monocytes are precursors of DC
and macrophages, with each cell type having the capability to
convert into the other until late in differentiation⁄maturation
process (34). Hence, PDT seems to activate monocytes to
differentiate into DC as suggested by the high expression of
CD83 and HLA-DR (Table 1); however, LPS stimulation
skews this polarization favoring Type 2 macrophage develop-
ment (lower CD83, higher CD14 expression) (30).
Finally, at a level of PDT-induced proliferation reduction
up to 40%, it appeared that the allo-stimulated cells released
increasing cytokine levels proportionally to the degree of
inhibition of proliferation, cytokine levels exceeding even
these in light control cultures (Fig. 3). These results strongly
suggest an immunomodulatory event. Moreover, the cytokine
profile revealing a mixed Th1⁄Th2 type response suggests
that several types of APC may be synergically or simulta-
neously involved.
In conclusion, in addition to inactivating pathogens, PDT
with Tri-P(4) may be able to steer the immune response to
T-cell hyporesponsiveness by functionally affecting both T
cells and APC. These dual effects may present an advantage on
gamma irradiation which was shown to inhibit proliferation of
the MNC without affecting the antigen-presenting capabilities
of the irradiated cells (35). Consequently, we may consider the
possibility of using PDT with Tri-P(4) to deviate an immune
response toward tolerance or to prevent undesired transfusion-
related immunological reactions.
Acknowledgements—This work was supported by ZonNW (grant no.
28-24140), The Netherlands. The authors thank Aine Honohan for
technical support and helpful English editing. The authors also thank
Johan Lagerberg for the critical review of this article.
REFERENCES
1. Barbara, J. (2004) Why ‘‘Safer than ever’’ may not be quite safe
enough. Transfus. Med. Hemother. 31, 2–10.
2. Andreu, G., P. Morel, F. Forestier, J. Debeir, D. Rebibo, G.
Janvier and P. Herve (2002) Hemovigilance network in France:
Organization and analysis of immediate transfusion incident re-
ports from 1994 to 1998. Transfusion 42, 1356–1364.
3. Blajchman, M. A., S. Dzik, E. C. Vamvakas, J. Sweeney and E. L.
Snyder (2001) Clinical and molecular basis of transfusion-induced
immunomodulation: Summary of the proceedings of a state-of-
the-art conference. Transfus. Med. Rev. 15, 108–135.
4. Fast, L. D. (2005) Control of immune responses induced by the
transfer of allogeneic white blood cells during transfusion.
Transfusion 45, 44s–50s.
5. Fast, L. D., G. DiLeone, J. Li and R. P. Goodrich (2006) Func-
tional inactivation of white blood cells by Mirasol treatment.
Transfusion 46, 642–648.
6. Fast, L. D., G. DiLeone, C. M. Edson and A. Purmal (2002)
PEN110 treatment functionally inactivates the PBMNCs present
in RBC units: Comparison to the effects of exposure to gamma
irradiation. Transfusion 42, 1318–1325.
7. Obochi, M. O., L. G. Ratkay and J. G. Levy (1997) Prolonged
skin allograft survival after photodynamic therapy associated with
modification of donor skin antigenicity. Transplantation 63, 810–
817.
8. Ratkay, L. G., J. D. Waterfield and D. W. Hunt (2000) Photo-
dynamic therapy in immune (non-oncological) disorders: Focus
on benzoporphyrin derivatives. BioDrugs 14, 127–135.
9. Broady, R., J. Yu and M. K. Levings (2008) Pro-tolerogenic
effects of photodynamic therapy with TH9402 on dendritic cells.
J. Clin. Apher. 23, 82–91.
10. Heshmati, F. (2003) Mechanisms of action of extracorporeal
photochemotherapy. Transfus. Apher. Sci. 29, 61–70.
11. Hunt, D. W. and A. H. Chan (2000) Influence of photodynamic
therapy on immunological aspects of disease—An update. Expert
Opin. Investig. Drugs 9, 807–817.
12. Gollnick, S. O., X. Liu, B. Owczarczak, D. A. Musser and B. W.
Henderson (1997) Altered expression of interleukin 6 and inter-
leukin 10 as a result of photodynamic therapy in vivo. Cancer Res.
57, 3904–3909.
13. King, D. E., H. Jiang, G. O. Simkin, M. O. Obochi, J. G. Levy
and D. W. Hunt (1999) Photodynamic alteration of the surface
receptor expression pattern of murine splenic dendritic cells.
Scand. J. Immunol. 49, 184–192.
14. Jiang, H., D. J. Granville, B. M. McManus, J. G. Levy and D. W.
Hunt (1999) Selective depletion of a thymocyte subset in vitro with
an immunomodulatory photosensitizer. Clin. Immunol. 91, 178–
187.
15. Hunt, D. W., H. Jiang, D. J. Granville, A. H. Chan, S. Leong and
J. G. Levy (1999) Consequences of the photodynamic treatment of
resting and activated peripheral T lymphocytes. Immunopharma-
cology 41, 31–44.
16. Jiang, H., D. J. Granville, J. R. North, A. M. Richter and D. W.
Hunt (2002) Selective action of the photosensitizer QLT0074 on
activated human T lymphocytes. Photochem. Photobiol. 76, 224–
231.
17. Trannoy, L. L., J. W. M. Lagerberg, T. M. A. R. Dubbelman, J. J.
Schuitmaker and A. Brand (2004) Positively charged porphyrins:
Photochemistry and Photobiology, 2010, 86229

Page 8

A new series of photosensitizers for sterilization of RBCs.
Transfusion 44, 1186–1196.
18. Trannoy, L. L., F. G. Terpstra, D. de Korte, J. W. M. Lagerberg,
A. J. Verhoeven, A. Brand and F. A. C. van Engelenburg (2006)
Differential sensitivities of pathogens in red cell concentrates to
Tri-P(4)-photoinactivation. Vox Sang. 91, 111–118.
19. Trannoy, L. L., Y. van Hensbergen, J. W. Lagerberg and A.
Brand (2008) Photodynamic treatment with mono-phenyl-tri-
(N-methyl-4-pyridyl)-porphyrin for pathogen inactivation in cord
blood stem cell products. Transfusion 48, 2629–2637.
20. Verduyn, W., I. I. Doxiadis, J. Anholts, J. J. Drabbels, A. Naipal,
J. D’Amaro, G. G. Persijn, M. J. Giphart and G. M. Schreuder
(1993)BiotinylatedDRB sequence-specific
Comparison to serologic HLA-DR typing of organ donors in
eurotransplant. Hum. Immunol. 37, 59–67.
21. Vamvakas, E. C. (1998) Meta-analysis of randomized controlled
trials of the efficacy of white cell reduction in preventing HLA-
alloimmunization and refractoriness to random-donor platelet
transfusions. Transfus. Med. Rev. 12, 258–270.
22. Lakshmanan, U. and A. G. Porter (2007) Caspase-4 interacts with
TNF receptor-associated factor 6 and mediates lipopolysaccha-
ride-induced NF-{kappa}B-dependent production of IL-8 and CC
chemokineligand4(macrophage-inflammatory
J. Immunol. 179, 8480–8490.
23. Lucchi, N. W. and J. M. Moore (2007) LPS induces secretion of
chemokines by human syncytiotrophoblast cells in a MAPK-
dependent manner. J. Reprod. Immunol. 73, 20–27.
24. Moor, A. C. (2000) Signaling pathways in cell death and survival
after photodynamic therapy. J. Photochem. Photobiol. B, Biol. 57,
1–13.
25. Fonseca, A. M., G. Porto, K. Uchida and F. A. Arosa (2001) Red
blood cells inhibit activation-induced cell death and oxidative
stress in human peripheral blood T lymphocytes. Blood 97, 3152–
3160.
26. Fialkow, L., Y. Wang and G. P. Downey (2007) Reactive oxygen
and nitrogen species as signaling molecules regulating neutrophil
function. Free Radic. Biol. Med. 42, 153–164.
oligonucleotides.
protein-1).
27. Gringhuis, S. I., E. A. Papendrecht-van der Voort, A. Leow, E. W.
Nivine Levarht, F. C. Breedveld and C. L. Verweij (2002) Effect of
redox balance alterations on cellular localization of LAT and
downstream T-cell receptor signaling pathways. Mol. Cell. Biol.
22, 400–411.
28. Legitimo, A., R. Consolini, S. Di, W. Bencivelli and F. Mosca
(2002) Psoralen and UVA light: An in vitro investigation of
multiple immunological mechanisms underlying the immunosup-
pression induction in allograft rejection. Blood Cells Mol. Dis. 29,
24–34.
29. Fujihara, M., T. A. Takahashi, M. Azuma, C. Ogiso, T. L.
Maekawa, H. Yagita, K. Okumura and S. Sekiguchi (1996)
Decreased inducible expression of CD80 and CD86 in human
monocytes after ultraviolet-B irradiation: Its involvement in
inactivation of allogenecity. Blood 87, 2386–2393.
30. Verreck, F. A., T. de Boer, D. M. Langenberg, L. van der Zanden
and T. H. Ottenhoff (2006) Phenotypic and functional profiling of
human proinflammatory type-1 and anti-inflammatory type-2
macrophages in response to microbial antigens and IFN-gamma-
and CD40L-mediated costimulation. J. Leukoc. Biol. 79, 285–293.
31. Tzachanis, D., A. Berezovskaya, L. M. Nadler and V. A. Bous-
siotis (2002) Blockade of B7⁄CD28 in mixed lymphocyte reaction
cultures results in the generation of alternatively activated mac-
rophages, which suppress T-cell responses. Blood 99, 1465–1473.
32. Xu, W., N. Schlagwein, A. Roos, T. K. van den Berg, M. R. Daha
and C. van Kooten (2007) Human peritoneal macrophages show
functional characteristics of M-CSF-driven anti-inflammatory
type 2 macrophages. Eur. J. Immunol. 37, 1594–1599.
33. Haugen, T. S., B. Nakstad, O. H. Skjonsberg and T. Lyberg (1998)
CD14 expression and binding of lipopolysaccharide to alveolar
macrophages and monocytes. Inflammation 22, 521–532.
34. Palucka, K. A., N. Taquet, F. Sanchez-Chapuis and J. C.
Gluckman (1998) Dendritic cells as the terminal stage of monocyte
differentiation. J. Immunol. 160, 4587–4595.
35. Fast, L. D., G. DiLeone, C. M. Edson and A. Purmal (2002)
Inhibition of murine GVHD by PEN110 treatment. Transfusion
42, 1326–1332.
230 Laurence Trannoy et al.