BO LR OI GO ID NCAOLM PA OR NT EI CN LT ES Pathogen reduction by ultraviolet C light effectively inactivates human white blood cells in platelet products Petra Pohler,1 Meike Müller,2 Carla Winkler,2 Dirk Schaudien,2 Katherina Sewald,2 Thomas H. Müller,1 and Axel Seltsam1

BACKGROUND: Residual white blood cells (WBCs) in cellular blood components induce a variety of adverse immune events, including nonhemolytic febrile transfusion reactions, alloimmunization to HLA antigens, and transfusion-associated graft-versus-host disease (TAGVHD). Pathogen reduction (PR) methods such as the ultraviolet C (UVC) light-based THERAFLEX UV-Platelets system were developed to reduce the risk of transfusion-transmitted infection. As UVC light targets nucleic acids, it interferes with the replication of both pathogens and WBCs. This preclinical study aimed to evaluate the ability of UVC light to inactivate contaminating WBCs in platelet concentrates (PCs). STUDY DESIGN AND METHODS: The in vitro and in vivo function of WBCs from UVC-treated PCs was compared to that of WBCs from gamma-irradiated and untreated PCs by measuring cell viability, proliferation, cytokine secretion, antigen presentation in vitro, and xenogeneic GVHD responses in a humanized mouse model. RESULTS: UVC light was at least as effective as gamma irradiation in preventing GVHD in the mouse model. It was more effective in suppressing T-cell proliferation (>5-log reduction in the limiting dilution assay), cytokine secretion, and antigen presentation than gamma irradiation. CONCLUSIONS: The THERAFLEX UV-Platelets (MacoPharma) PR system can substitute gamma irradiation for TA-GVHD prophylaxis in platelet (PLT) transfusion. Moreover, UVC treatment achieves suppression of antigen presentation and inhibition of cytokine accumulation during storage of PCs, which has potential benefits for transfusion recipients.


athogen reduction (PR) technologies are increasingly used for labile blood products. They increase blood safety by limiting the exposure of patients to potentially infectious blood. PR technologies that use ultraviolet (UV) light with or without photosensitizers or photoreagents target nucleic acids and thus inactivate pathogens without destroying the membranes of cellular therapeutic products.1,2 Since they cause substantial damage to nucleic acids, all UV-based PR technologies also have the potential to inactivate contaminating white blood cells (WBCs). Contaminating viable T cells in cellular blood products are considered the primary cause of transfusion-associated graft-versus-host disease (TA-GVHD) in susceptible patients. TA-GVHD occurs after transfusion of whole blood and cellular blood components when viable donor T-lymphocytes proliferate and engraft, eliciting an immune response in the recipient unable to mount an immune response to allogeneic donor cells due to human leukocyte antigen (HLA) compatibility or immunosuppression. As UV-based PR technologies impair WBC function by DNA modification, they are potential alternatives to gamma irradiation, the current

ABBREVIATIONS: 7-AAD = 7-aminoactinomycin; APC(s) = antigen-presenting cell(s); HS = human serum; LDA(s) = limiting dilution assay(s); LPS = lipopolysaccharide; MLC = mixed lymphocyte culture; PC = platelet concentrate; PHA = phytohemagglutinin; PR = pathogen reduction; TA-GVHD = transfusion-associated graft-versus-host disease. From the 1German Red Cross Blood Service NSTOB, Institute Springe, Springe, Germany; and 2Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany. Address reprint requests to: Axel Seltsam, MD, MHBA, German Red Cross Blood Service NSTOB, Institute Springe, Eldagsener Strasse 38, 31832 Springe, Germany; e-mail: [email protected] Received for publication June 20, 2014; revision received July 18, 2014, and accepted July 21, 2014. doi: 10.1111/trf.12836 © 2014 AABB TRANSFUSION **;**:**-**. 2015;55:337–347.

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standard of care for TA-GVHD prevention in patients at risk. By targeting nucleic acids, such PR treatments may also influence the occurrence and development of other donor antirecipient reactions associated with the transfer of allogeneic donor WBCs, such as alloimmunization, nonhemolytic febrile transfusion reactions, and transfusion-related immunomodulation. Gamma irradiation and leukoreduction of blood products before transfusion were shown to reduce but not eliminate these undesirable WBC-mediated immunologic effects.3-6 THERAFLEX UV-Platelets (MacoPharma, Mouvaux, France), a novel PR system that works without photoactive substances, is currently undergoing clinical efficacy and safety testing.7,8 This system uses shortwave UVC light (254 nm), which directly interacts with nucleic acids, resulting in the formation of pyrimidine dimers that block the elongation of nucleic acid transcripts.9 Because UVC light selectively targets the nucleic acids of pathogens and WBCs, this technology is suited for the treatment of cellular blood products. UVC light significantly reduces the infectivity of platelet concentrates (PCs) contaminated by disease-causing viruses, bacteria, and protozoa.7,8,10 The objective of this study was to assess the effects of PR treatment with the THERAFLEX UV-Platelets system on the in vitro and in vivo function of PC-contaminating WBCs compared to those of gamma irradiation, the standard of care for treating blood products for transfusion to immunocompromised patients and patient populations susceptible to TA-GVHD.

MATERIALS AND METHODS Platelet preparation and treatment Leukoreduced plasma-reduced PCs were prepared from pooled buffy coats from five healthy donors, suspended in SSP+ (MacoPharma), and stored at 22 ± 2°C under gentle agitation. The approximate values were as follows: volume 350 mL, platelet (PLT) concentration 1 × 109/mL, plasma content 35%, and residual WBC content not more than 1 × 106 per unit. PCs were spiked with peripheral blood mononuclear cells (PBMNCs) prepared from buffy coats by density centrifugation. Three ABO-compatible PCs were pooled and spiked with PBMNCs derived from a single donor at a concentration of approximately 1 × 106 PBMNCs/mL PC. The PC pool was split again into three single full-dose PLT units (volume 350 mL, PLT concentration 1 × 109/mL, plasma content 35%), which were stored until further treatment with either UVC light, 30 Gy gamma irradiation, or no treatment (controls). In the first group, PCs were UVC treated on a UV device (MacoTronic, MacoPharma) using the THERAFLEX UV-Platelets disposable set (REF XUV 4005 XU, MacoPharma)8 at a dose of 0.2 J/cm2 (animal study) or 338 TRANSFUSION Volume Volume February 2015 2 TRANSFUSION **,55, ** **

with two partial doses of 0.1 J/cm2 in close succession to yield a total dose of 0.2 J/cm2 (in vitro studies).

In vitro characterization of PBMNC response PBMNCs were reisolated from PCs by centrifugation and used for in vitro assays on the same day. Viability of the cells was assessed by trypan blue exclusion.

Immunophenotype A panel of antibodies was used to define various subpopulations of PBMNCs by flow cytometry (Cytomics FC500, Beckman Coulter, Krefeld, Germany). The origin of the lymphocytes was determined by expression of either murine or human CD45 (panleukocyte marker). Expression levels of CD4 (T-helper cells), CD8 (cytotoxic T cells and natural killer [NK] cells), CD3 (pan T cells), CD11a (panleukocytes), CD19 (B cells), CD14 (monocytes), CD16/CD56 (NK cells), and CD25 (T-cell activation) were measured using fluorescence-labeled monoclonal antibodies (MoAbs; Beckman Coulter).

Proliferation The ability of PBMNCs to proliferate in response to a mitogen (phytohemagglutinin [PHA]) or to anti-CD3 plus anti-CD28 (anti-T-cell receptor antibodies) bound to Dynabeads (Life Technologies, Darmstadt, Germany) was tested in in vitro cultures. PBMNCs (8 × 105/mL) were stimulated in the presence of anti-CD3 and anti-CD28 Dynabeads (4 × 107/mL), PHA (10 μg/mL), or medium only. The proliferative response was quantified by measuring [3H]thymidine uptake by liquid scintillation counting with a radioactivity counter (Perkin Elmer TopCount, Canberra Packard, Frankfurt, Germany).

Mixed lymphocyte culture The ability of UVC light–treated PBMNCs to act as antigen-presenting cells (APCs; stimulator cells) was investigated using the mixed lymphocyte culture (MLC) assay, in which lymphocytes of incompatible individuals are cocultured and stimulate each other to significant proliferation mediated by major histocompatibility complex incompatibility. The capacity of the stimulator cells to proliferate upon stimulation is inactivated by gamma irradiation, while the responder cells are left untreated and maintain their ability to proliferate. Thus, the proliferation of responder cells in the MLC assay is indicative of the ability of these cells to proliferate but also demonstrates the ability of the stimulator cells to act as APCs. Stimulator PBMNCs (5 × 105/well) were cocultured with 1 × 105 responder PBMNCs. Cocultures of untreated responder cells with gamma-irradiated stimulator cells served as positive controls. Proliferative alloimmune responses were quantified by measuring [3H]thymidine uptake by liquid scintillation counting.


Limiting dilution assay

Membrane integrity and apoptosis of WBCs in PCs

Limiting dilution assays (LDAs) were conducted to quantify the effectiveness of the treatments on the ability of T cells to respond by defining the frequency of responder T cells in spiked PCs after treatment with UVC light or gamma irradiation. Based on the results of the MLC assay (see above), the PC-derived PBMNCs (responder cells) used were either untreated (positive controls) or treated (UVC light or gamma irradiation) and exposed to a pool of gamma-irradiated (inactivated) lymphocytes of HLAincompatible individuals (stimulator cells). The formation of cell clones in the wells signaled the proliferative response of alloreactive T cells. The LDA is a dose– response assay that allows detection of an all-or-none (positive or negative) immune response in each individual culture. Therefore, the number of negative cultures detected in the experiment allows for determination of the cell frequency in the original population. Serial dilutions of responder cells were added to wells containing allogeneic stimulator cells and stimulatory factors like interleukin (IL)-2 and PHA. Proliferation was measured based on [3H]thymidine incorporation. Responder T-cell frequencies were calculated by minimum chi-square analysis based on a Poisson distribution using computer software (L-Calc, Vancouver, British Columbia, Canada) as outlined in Table 1.11,12

The effect of UVC light on the viability of contaminating WBCs in PCs was investigated by determination of cell membrane integrity (7-aminoactinomycin [7-AAD] staining) and expression of the apoptosis marker CD95. WBCs isolated from PCs on different days during storage were identified by CD11a expression and costained for 7-AAD and CD95 to identify WBCs with impaired cell membrane integrity (CD11a+/7-AAD+), apoptotic WBCs (CD11a+/ CD95+), and WBCs with intact cell membranes (CD11a+/ 7-AAD–) by flow cytometry (Beckman Coulter).

Cytokine secretion Levels of cytokines produced by PBMNCs isolated from spiked PCs in response to lipopolysaccharide (LPS) or released upon exposure to anti-CD3 and anti-CD28 in culture supernatants were measured using MSD multiplex technology (Meso Scale Discovery, Rockville, MD). The effect of UVC light on IL-8 secretion by contaminating WBCs into the supernatant of stored PCs was also investigated using an enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions (R&D Systems, Wiesbaden, Germany).

Xenogeneic GVHD model The objective was to investigate whether UVC irradiation of human PBMNCs can prevent the development of xenogeneic GVHD in nonobese diabetic/severe combined immunodeficient recipient mice—NOD.CgPrkdcscidIl2rgtm1WjlISzJ mice, commonly known as NOD/ SCIDgamma (NSG) mice (Charles River Laboratories, Sulzfeld, Germany)—after intraperitoneal injection of PBMNCs.13 This xenogeneic mouse model allowed us to use human WBCs from PCs prepared and pathogen reduced under blood bank conditions. Approximately 6to 8-week-old, age-matched, nulliparous, and nonpregnant female mice were treated by whole-body irradiation with 200 cGy 1 day before injection to ensure complete NK-cell inactivation to achieve faster engraftment of human PBMNCs and faster development of xenogeneic GVHD.13 Three groups of 12 mice each received equal numbers of untreated, gamma-irradiated, or UVC-treated PBMNCs (2.5 × 107 cells) from one of four donors. Control mice (n = 12) were injected with phosphate-buffered saline (PBS) containing 0.5% human serum (HS). After cell transfer or sham treatment, the mice were monitored and weighed for a period of 61 days. They were euthanized at the end of the observation period or when they became moribund, as defined by signs of severe illness such as

TABLE 1. Quantification of T-cell proliferation after UVC treatment T-cell frequency (f)* Donor 1 2 3 4 5 Mean SD

fcontrol 1/163 1/198 1/71 1/139 1/75 1/129 ±1/55

f30Gy 5.1 0.3

* T-cell frequencies (f) of proliferating irradiated (f0.1 UVC; f0.2 UVC; f30 Gy) and nonirradiated (fcontrol) responder cells from five different donors, calculated from the results of the LDA. Responder cells were isolated from spiked PCs and cocultured with gamma-irradiated stimulator cells. Log reduction in responder T-cell frequency in relation to T-cell frequency of nonirradiated cells (controls) is indicated. The mean frequency of proliferating (alloreactive) lymphocytes in untreated PBMNCs was calculated from one in 129 plated responder cells.

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hunched position, scrubby fur, rough skin, and/or loss of more than 10% body weight. At the time of euthanization, peripheral blood, spleen cells, and marrow cells were collected and stained with antibodies for flow cytometric analysis. A histopathologic examination of the skin, marrow (sternum), spleen, lung, liver, and gastrointestinal tract was conducted in all animals. Trimming and slide preparation was done according to current guidelines.14-16 Immune histologic staining was performed using anti-human CD3 antibody to show human lymphocytes in peripheral organs.

Statistical analysis Results are expressed as mean ± standard deviation (SD) unless otherwise indicated. Statistical analysis was performed by a nonparametric Dunnett’s test (Prism 4 Version 4.03, GraphPad Software, Inc., La Jolla, CA). Differences between treatment groups and controls were considered significant at the p level of less than 0.05.

RESULTS Viability testing and immunophenotyping of WBCs from spiked PCs PBMNCs isolated from treated and untreated PCs up to 2 hours after spiking showed less than 3% dead cells in every sample, independent of the type of treatment. This indicates a high viability of the WBCs used for in vitro experiments or transferred into NSG mice. The fact that surface marker expression profiles were similar in all treatment groups suggests that UVC light and gamma irradiation did not significantly alter the composition of the lymphocyte populations in the WBCs (Fig. S1, available as supporting information in the online version of this paper).

WBC inactivation Proliferation of WBCs in response to T-cell stimuli WBCs from five human donors were spiked in and reisolated from PCs after UVC light or gamma irradiation. Strong proliferation was observed in untreated WBCs after stimulation (Fig. 1). No proliferation was observed in UVC light–treated WBCs, independent of whether the half dose (0.1 J/cm2) or full dose (0.2 J/cm2) was used. These results indicate that the half dose is sufficient to completely abolish the proliferative capacity of contaminating WBCs. In contrast, WBCs isolated from gamma-irradiated PCs still showed (in two of five experiments) some low proliferative activity (Experiment 1, 63 ± 24 cpm without stimulation, 3391 ± 184 cpm with PHA, and 935 ± 172 cpm with anti-CD3 and anti-CD28; Experiment 4, 50 ± 33 cpm 340 TRANSFUSION Volume Volume February 2015 4 TRANSFUSION **,55, ** **

Fig. 1. Proliferative response. WBCs isolated from untreated, UVC-irradiated (0.1 or 0.2 J/cm2), or gamma-irradiated (30 Gy) spiked PCs were tested for their ability to proliferate in response to anti-CD3 and anti-CD28 or PHA by [3H]thymidine incorporation. Proliferation was corrected by subtracting background levels of cells in medium from the response of cells in the presence of a stimulus. Mean ± SD of triplicate measurements are indicated (n = 5). ( ) Medium; ( ) CD3 and CD28; ( ) PHA.

without stimulation, 1110 ± 47 cpm with PHA, and 1261 ± 28 cpm with anti-CD3 and anti-CD28).

Ability of WBCs to act as APCs In contrast to WBCs from gamma-irradiated PCs, those from spiked PCs treated with 0.1 and 0.2 J/cm2 UVC light were unable to induce proliferation of HLA-mismatched responder WBCs. This suggests that lymphocytes lose their capability to act as APCs in an allogeneic setting when exposed to a low UVC dose of 0.1 J/cm2 (Fig. 2).

Proliferation of WBCs in response to allogeneic stimulator cells LDAs were performed to quantify the proliferation of viable T cells (Table 1). Cells from nonirradiated spiked PCs were found to generate clones at a frequency of one in 129 WBCs. No T-cell clones were detected in WBC samples from spiked PCs irradiated with a UVC dose of 0.2 J/cm2. Log reduction in these samples reached the limit of detection of the assay, as indicated by “or greater.” The mean growth was less than one in 1.5 × 107 plated cells, representing a greater than 5.1-log reduction. A few clones appeared in the LDA in three of the five donors when using WBCs from spiked PCs irradiated with a UVC dose of 0.1 J/cm2; no T-cell growth was detected in the other two donors (Donors 1 and 3). Thus, the mean T-cell growth for WBCs irradiated with 0.1 J/cm2 UVC light was calculated


Fig. 2. Allostimulation. WBCs isolated from UVC-irradiated (0.1 or 0.2 J/cm2) or gamma-irradiated (30 Gy) spiked PCs were tested for their ability to induce proliferation in untreated allogeneic responder cells (allostimulation). Proliferation, as measured by [3H]thymidine incorporation, was corrected by subtracting background levels measured for cells in medium from the response of cells in the presence of the stimulus. Data are presented as mean ± SD (n = 7). (□) Stimulator cells alone; (■) MLC.

as one in 5.9 × 106 plated cells, corresponding to a mean reduction of 4.5 log, which was comparable to the frequency of alloreactive lymphocytes from gammairradiated spiked PCs (mean reduction, 4.3 log). A previously published study showed somewhat higher inactivation levels of gamma-irradiated WBCs (>5.5 log) in the LDA.12 This difference in WBC inactivation levels measured after gamma irradiation is most probably due to the different readout methods used for determination of WBC inactivation capacity in this study ([3H]thymidine uptake by liquid scintillation counting) and the previous study (microscopic readout).

Fig. 3. Cytokine secretion. WBCs isolated from untreated (◆), UVC-irradiated ( ; 0.2 J/cm2), or gamma-irradiated ( ; 30 Gy) spiked PCs were stimulated with (A) 0.1 μg/mL LPS for 24 hours or (B) with 25 μL of anti-CD3 and anti-CD28 beads for 72 hours (n = 5). Cytokine levels were measured in the supernatants of stimulated cells. Replicates with WBCs cultured in medium only served as negative controls. Significant differences compared to untreated controls: *p < 0.05, ***p < 0.001. GM-CSF = granulocyte-macrophage–colony-stimulating factor.

Cytokine levels after stimulation UVC light significantly inhibited cytokine release of PC-contaminating WBCs upon exposure to T-cell– stimulating agents. LPS induced the level of a number of cytokines in the supernatant of cultured WBCs isolated from untreated and gamma-irradiated spiked PCs (Fig. 3A). WBCs isolated from spiked PCs after UVC light exposure showed a strongly reduced response to LPS. This strong inhibitory effect was already achieved at a UVC light dose of 0.1 J/cm2 and was even more pronounced at a dose of 0.2 J/cm2 (data only shown for 0.2 J/cm2). Stimulation with anti-CD3 and anti-CD28 beads also significantly induced cytokine secretion in untreated WBCs. However, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-10 levels were significantly reduced after

gamma irradiation and to an even higher extent after UVC exposure (Fig. 3B).

IL-8 levels in stored PCs In this experiment, IL-8 was used as a representative WBCderived cytokine because it is known to be released in PCs in measurable quantities during storage. UVC light dosedependently reduced IL-8 levels in spiked PCs during storage compared to levels in untreated and gammairradiated spiked PCs (Fig. 4).

Viability of WBCs in PCs during storage Membrane integrity, apoptosis, and proliferation capacity were investigated to assess the viability of the WBCs in PCs Volume 55, February Volume 2015 **, ** **TRANSFUSION TRANSFUSION341 5


Fig. 4. IL-8 secretion in PCs during storage. IL-8 levels were measured in the supernatants of untreated ( ), UVCirradiated (0.1 [ ] or 0.2 [ ] J/cm2), or gamma-irradiated ( ; 30 Gy) spiked PCs during 7 days of storage. Data are presented as mean ± SD (n = 5). Significant differences between treatment groups and untreated controls: *p < 0.05.

during storage. Independent of treatment group, the membrane integrity of WBCs was almost unaffected until Day 3 of storage and then strongly decreased until Day 7 (Fig. S2, available as supporting information in the online version of this paper). The impairment of membrane integrity was stronger for WBCs from gamma irradiation– and UVC light–treated PCs than for those from untreated PCs. On Days 4 to 7 of storage, the percentage of WBCs with intact membranes was lower after UVC irradiation than after gamma irradiation. On Day 7, no lymphocytes with intact membranes were detected in UVC-irradiated PCs, whereas approximately 20% of WBCs with intact membranes were found in untreated and gammairradiated PCs, respectively. There was no difference in loss of membrane integrity between WBCs from PCs irradiated at a UVC dose of 0.1 J/cm2 versus 0.2 J/cm2. This indicates that the lower dose (half of the standard UVC dose) also had the maximum effect on cell membrane integrity. CD95 expression on WBCs, a marker of apoptosis, remained constant and did not differ significantly between the treatment groups (data not shown). The proliferative response of untreated WBCs to mitogenic stimulation continuously decreased during storage. UVC treatment completely abolished the proliferation capacity of WBCs, which did not recover during storage (Fig. 5). Gamma-irradiated WBCs also showed significantly lower proliferation levels than untreated WBCs.

GVHD prevention Health status All mice that received untreated PBMNCs developed physical signs of GVHD (Table 2). Their health status dete342 TRANSFUSION Volume Volume February 2015 6 TRANSFUSION **,55, ** **

Fig. 5. Proliferative response to mitogenic stimulation during storage. WBCs isolated on Days 1, 3, 4, 5, and 7 from untreated ( ), UVC-irradiated (0.1 [ ] or 0.2 [ ] J/cm2), or gammairradiated ( ; 30 Gy) spiked PCs were tested for their ability to proliferate in response to PHA by [3H]thymidine incorporation. Proliferation was corrected by subtracting background levels of cells in medium from the response of cells in the presence of a stimulus. Data are presented as mean ± SD (n = 5). Significant differences between treatment groups and untreated controls: ***p < 0.001.

riorated continuously until Day 14, and their weight started to decline shortly after PBMNC injection (Fig. 6A). The survival rate of these mice was only 50% on day 17 and rapidly declined to zero by Day 25 (Fig. 6B). In contrast, none of the recipient mice injected with PBMNCs isolated from UVC-treated or gamma-irradiated PCs developed physical signs of GVHD.

Immunophenotypic analysis The majority of PBMNCs in mice with an ongoing GVHD immune response were of human origin. The proportion of human WBCs in the marrow was lower than that in the spleen and the blood (Fig. 7). Immunophenotypic analysis revealed that these compartments were composed mainly of human T and B lymphocytes (Fig. S3, available as supporting information in the online version of this paper). The blood and spleen contained higher proportions of T cells than the marrow. Most of the T cells in the blood, spleen, and marrow were CD8+ cytotoxic T cells. High proportions of activated lymphocytes (CD25+) were detected in the blood, spleen, and marrow. The ratio of CD4+ to CD8+ cells was 0.6 to 0.8, indicating a bias toward cytotoxic T cells. NK cells (CD16+/CD56+) were also detected in the blood, spleen, and marrow, whereas relevant quantities of B cells (CD19+) were only detected in the spleen. Human PBMNCs were not found in mice injected with UVC-irradiated PBMNCs. This indicates that UVC irradiation efficiently inactivates human PBMNCs,


TABLE 2. GVHD development in a xenogeneic mouse model: summary of histopathologic and clinical findings Treatment group (n = 12 per group) Untreated UVC irradiation Gamma irradiation

Number of deaths 12/12 0/12 0/12

Survival days

Pathogen reduction by ultraviolet C light effectively inactivates human white blood cells in platelet products.

Residual white blood cells (WBCs) in cellular blood components induce a variety of adverse immune events, including nonhemolytic febrile transfusion r...
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