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. Author manuscript; available in PMC: 2014 Aug 26.
Published in final edited form as: J Invest Dermatol. 2011 Aug 18;132(1):179–187. doi: 10.1038/jid.2011.255

Whole-Body UVB Irradiation during Allogeneic Hematopoietic Cell Transplantation Is Safe and Decreases Acute Graft-versus-Host Disease

Marina Kreutz 1, Sigrid Karrer 2, Petra Hoffmann 1, Eva Gottfried 1, Rolf-Markus Szeimies 2, Joachim Hahn 1, Matthias Edinger 1, Michael Landthaler 2, Reinhard Andreesen 1, Miriam Merad 3, Ernst Holler 1
PMCID: PMC4144809  NIHMSID: NIHMS610490  PMID: 21850024

Abstract

Depletion of host Langerhans cells (LCs) prevents cutaneous graft-versus-host disease (GvHD) in mice. We analyzed whether UVB irradiation is tolerated during the course of human allogeneic hematopoietic cell transplantation and whether depletion of LCs by broadband UVB could improve GvHD outcome. A total of 17 patients received six whole-body UVB irradiations with 75% of the individually determined minimal erythemal dose after conditioning with a reduced intensity protocol. LCs, dermal dendritic cells (DCs), and macrophages were analyzed before and after UVB irradiation by immunohistochemical analysis. Circulating blood cells and serum factors were analyzed in parallel. In striking contrast to previous data, our irradiation protocol was well tolerated in all patients. UVB treatment decreased the number of LCs and also affected dermal DCs. UVB-treated patients also had significantly higher 25-hydroxyvitamin D3 serum levels and higher numbers of circulating CD4+ FoxP3+ regulatory T cells. Strikingly, nine out of nine patients with complete LC depletion (<1 LC per field) developed only grade I GvHD or no GvHD up to day 100. Our results strongly suggest that prophylactic UVB irradiation post transplant is safe and should be further explored as a clinical strategy to prevent acute (skin) GvHD.

INTRODUCTION

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for patients with high-risk leukemia, lymphoma, and myeloma. The success of allo-HCT is largely based on immunological graft-versus-tumor effects. This beneficial effect is counterbalanced by graft-versus-host disease (GvHD), which occurs in 20–75% of transplant recipients. GvHD manifests as inflammation of the skin, liver, and gut, with the skin being the most frequently affected organ. Therefore, a major objective in allo-HCT is the prevention of GvHD.

The importance of recipient dendritic cells (DCs) in initiating GvH responses has been established in animal models (Shlomchik et al., 1999; Zhang et al., 2002). Donor DCs have a supplementary role and are able to augment acute GvHD through cross-presentation of host tissue (Matte et al., 2004). We have shown that epidermal DCs, also called Langerhans cells (LCs), survive the irradiation regimen in mice that receive allo-HCT (Merad et al., 2002), and that persistent recipient LCs are sufficient to generate cutaneous GvHD upon donor lymphocyte infusion in a major mismatch allo-HCT mouse model (Merad et al., 2004). Similar results were observed in a minor mismatch allo-HCT model (Durakovic et al., 2006). However, a new publication states that LCs are not required for GvHD induction when other host antigen-presenting cells are present (Li et al., 2011).

During conditioning, chemotherapy or total-body irradiation leads to eradication of the patient’s residual disease and elimination of host immune cells. However, in humans, LCs have been shown to survive the conditioning therapy in significant numbers compared with blood DCs and monocytes (Auffermann-Gretzinger et al., 2002; Fagnoni et al., 2004; Collin et al., 2006). The persistence of recipient LCs in the skin of patients who received allo-HCT has been reported more than 20 years ago (Perreault et al., 1985).

Our studies in mice also showed that exposure to UV light before donor lymphocyte infusion can deplete remaining host LCs, induce their replacement with donor-derived LCs, and improve cutaneous GvHD outcome (Merad et al., 2004). Importantly, exposure to UVB light is a potent inhibitor of cutaneous hypersensitivity response in mice and humans (Aubin, 2003). The mechanisms that control UVB-induced immunosuppression remain unclear. In mice, the depletion of LCs (Toews et al., 1980), modulation of LC antigen-presenting function via UVB-induced release of immunosuppressive soluble factors (Ullrich, 1994; Dai and Streilein, 1997), and the induction of T regulatory cells by UV-exposed LCs have been implicated in this process (Schwarz et al., 2004).

Similarly, in humans, low-dose UVB irradiation leads to LC depletion from the epidermis (Murphy et al., 1993) and the impairment of LC antigen presentation and allostimulatory function (Cooper et al., 1992). UVB exposure also induces the recruitment of IL-10 secreting immunosuppressive macrophages to the dermis, which may also contribute to UVB-mediated immunosuppression (Kang et al., 1994). In addition, soluble factors induced upon UVB exposure, such as IL-10, cis-urocanic acid, and vitamin D3, have been shown to directly modulate LC antigen-presentation function (Ullrich, 1994; Beissert et al., 2001; Penna et al., 2007). 1,25-dihydroxyvitamin D3, the physiologically active form of vitamin D3, induces the development of T regulatory cells in mice (Gorman et al., 2007), and administration of 1,25-dihydroxyvitamin D3 analog improves autoimmune diabetes in NOD mice (Gregori et al., 2002).

In this study, we examined whether allo-HCT patients tolerate prophylactic exposure to UVB at the time of transplantation, and analyzed a potential effect on (cutaneous) GvHD. Our results on the first 17 patients suggest that UVB therapy can be safely incorporated to the transplantation regimen and has the potential to improve (cutaneous) GvHD outcome in patients treated with allo-HCT.

RESULTS

Using a clinically relevant mouse model of allo-HCT, we have shown that in vivo depletion of LCs by UVC irradiation prevents cutaneous GvHD (Merad et al., 2004). To analyze whether prophylactic UVB irradiation is tolerated and can improve cutaneous GvHD outcome, we irradiated 17 allo-HCT patients with low-dose UVB starting from day 1 after transplantation (schematic view see Figure 1a). Patients received 75% of their individual minimal erythemal dose, six times every second day. In spite of the older age and the high-risk group of patients, UVB prophylaxis did not result in an increased incidence of neutropenic fever or manifest infections as compared with historical controls. Full donor engraftment was achieved in 16/17 patients. One chronic lymphocytic leukemia patient engrafted only partially and then developed a secondary graft failure. All other patients showed an uneventful engraftment, the mean time to recovery of >500 neutrophil counts was 15 days with a range of 10 to 28 days. Mean time to recovery of >20,000 platelets was 14 days (range 10–27 days). One patient showed skin toxicity during UVB prophylaxis, most likely due to concomitant administration of the photosensitizing anti-fungal drug voriconazole. In all other patients, UVB irradiation was well tolerated and led to a complete depletion of CD1a-positive LCs in 9 out of 17 patients (Table 1). None of the patients with complete LC depletion developed a grade II GvHD up to day 100. Accordingly, the number of CD1a+ LCs in the epidermis of UVB patients correlated with the development of acute GvHD grade less than day 100 (Table 1, r = 0.47, P value = 0.05). In contrast, five out of eight patients with incomplete depletion developed GvHD ≥grade II. Only 2/17 patients (12%) developed acute cutaneous GvHD≥grade II in contrast to about 45% in historical controls treated with identical reduced intensity conditioning regimen. After day 100, the incidence of severe chronic or delayed acute GvHD did not significantly differ between both groups, but median time to maximal-severe GvHD in general was significantly delayed in the group with complete depletion (Table 1).

Figure 1. Analysis of skin biopsies: Langerhans cell (LC) depletion.

Figure 1

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Six whole-body irradiations were given every second day starting from day 1 after allogeneic hematopoietic cell transplantation (allo-HCT). Control patients received the same conditioning regime but no UVB irradiation. Biopsies were taken before conditioning (biopsy between day −15 and −7), after conditioning/before transplantation (biopsy day–1/0), and after UVB exposure (biopsy between day +10 and +15) (a) Skin biopsies were analyzed by immunohistochemical staining. (b) CD1a expression of two representative samples (patient #9, #11; bar = 100 μm). (c) The mean absolute numbers of CD1a+ cells in each skin biopsy isolated from all 17 UVB-treated patients and 9 control patients. Data of UVB patients were grouped in patients with complete depletion (<1 LC per field, “complete”) and patients with incomplete depletion (≥1 LC per field, “incomplete”). (d, e) The individual course of CD1a+ cells in skin biopsies. NS, not significant.

Table 1.

Patient characteristics and incidence of GvHD in UVB patients and control patients

GvHD
Donor1 Diagnosis Conditioning2 UVB dose
(J cm−2)		 CD1a+3
(cells per field)		 <Day 100 >Day 100 Time4
UVB patients: complete depletion
#1 sibl NHL FBM 0.34 0 0 3 Chronic 812
#2 sibl AML FBM 0.18 0 0 2 Chronic
#3 urd MM FBM 0.09 0 0 2 Chronic
#4 urd CLL FBM 0.18 0 NE NE
#5 sibl ALL FBM 0.24 0 0 3 Chronic 340
#6 sibl NHL RIC 0.19 0 0 0
#7 sibl NHL FBM 0.18 0.4 0 1 Chronic
#8 urd NHL FBM 0.18 0 1 3 190
#9 urd ALL FBM 0.24 0.2 0 4 234
Median 0.18 0 287
UVB patients: incomplete depletion
#10 sibl AML FBM 0.12 1 1 1 Chronic
#11 sibl AML FBM 0.18 5.5 0 0
#12 urd CLL FBM 0.12 5 0 0
#13 urd AML FBM 0.18 2 2 3 100
#14 urd CLL FBM 0.24 1 3 3 80
#15 sibl NHL FBM 0.22 2 4 3 Chronic 61
#16 urd CML FBM 0.2 6 2 1 Chronic
#17 sibl MM FBM 0.18 85 4 NE 31
Median 0.18 3.5 71
Control patients
#1 sibl MDS FBM 0 0.2 0 0
#2 sibl AML FBM 0 5 3 NE 55
#3 sibl MM FBM 0 2 3 3 Chronic 53
#4 sibl MM FBM 0 6 0 0
#5 urd MDS FBM 0 4 0 2 Chronic
#6 urd AML FBM 0 5 0 3 197
#7 sibl MM RIC 0 0.2 0 0
#8 urd AML RIC 0 9 1 1 Chronic
#9 sibl MM RIC 0 0 4 NE 63
Median 0 4 59
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Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; CLL, chronic lymphocytic leukemia; CML, chronic myelogenous leukemia; GvHD, graft-versus-host disease; MDS, myelodysplastic syndrome; MM, myeloma; NE, not evaluable; NHL, non-Hodgkin lymphoma; RIC, reduced intensity conditioning; sibl, sibling; urd, unrelated donor.

1

Donor cells were obtained from sibl or urd.

2

Patients received either conditioning consisting of fludarabin/bis-chloroethylnitrosourea or carmustine and melphalan (FBM) or were treated with comparable RIC consisting of fludarabin and high-dose treosulfane or high-dose melphalan.

3

Biopsies were taken between day 10 and day 15 and the number of CD1a+ cells was determined by immunohistochemical staining.

4

Time to maximal severe GvHD was significantly (P=0.028) delayed in comparison to patients with incomplete depletion. Severe GvHD is defined as acute (<day 100) or acute delayed (>day 100) GvHD grade 3–4 or severe chronic GvHD; chronic GvHD is graded according to the National Institutes of Health as mild (1), moderate (2), or severe (3).

5

The number of CD1a+ cells per field correlated to GvHD <day 100 in UVB patients (r=0.47, P=0.026).

In control patients without UVB irradiation, we also found a decrease in LC numbers around day 14, but this reduction was not significant when compared with LC numbers before the conditioning regimen (Figure 1c). However, as we did not monitor the individual LC numbers of the control group after conditioning at the day of HCT, it cannot be excluded that these patients showed differences to the UVB group in the number of CD1a+ cells at this time point. Figure 1b shows two representative samples of skin biopsies isolated at different time points during transplantation. UVB irradiation decreased the number of LCs in all but one patient; however, to a variable degree. A complete depletion was found in nine patients (Figure 1b, patient #9). Incomplete depletion was found in eight patients (Figure 1b, patient #11). Figure 1d and e shows the individual course of CD1a+ cells of patients with complete depletion (Figure 1d) and incomplete depletion (Figure 1e). Conditioning resulted in a decrease in CD1a+ cells in seven out of nine patients in the “complete depletion” group. However, even those patients with high numbers of CD1a+ cells after conditioning responded with a complete LC depletion after UVB irradiation. In contrast, in the group with incomplete depletion, even patients with low LC numbers before UVB treatment did not respond with a complete depletion. Therefore, UVB responsiveness and LC depletion is not only determined by LC numbers before UVB treatment or responsiveness to conditioning but also seems to depend on other factors.

CD14+ and CD14 HLA-DR+ dermal antigen-presenting cells were not significantly influenced by the conditioning regimen (Figure 2a and b). However, by day 14 post transplantation, the number of dermal CD14+ cells dropped significantly in control patients who did not receive UVB, whereas it remained unchanged in the UVB group (Figure 2a and b). CD14−/HLA-DR+ DCs decreased significantly during conditioning regimen, but their number was not significantly affected by UVB irradiation (Figure 2c). Finally, the number of CD3+ T cells or regulatory T cells in the skin was not affected by the conditioning regimen or by UVB irradiation (data not shown).

Figure 2. Enrichment of HLA-DR+ CD14+ dermal cells in UVB-irradiated patients.

Figure 2

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Six whole-body irradiations were given every second day with 75% of the minimal erythemal dose starting from day 1 after allogeneic hematopoietic cell transplantation (allo-HCT). Control patients received the same conditioning regime but no UVB irradiation. Skin biopsies were analyzed by immunohistochemical staining using anti-HLA-DR and anti-CD14. The absolute numbers of CD14+/HLA-DR+ cells (a), CD14+/HLA-DR–cells (b), and CD14−/HLA-DR+ cells (c) show an enrichment of CD14+/HLA-DR+ double-positive cells in patients treated with UVB. Statistical analysis was performed with two-tailed Student’s t-test. NS, not significant.

The number of T cells in peripheral blood is reduced to about 1% of the initial cell number after conditioning. When we analyzed the reconstitution of blood CD4+ T cells and regulatory T cells around 5 weeks post transplantation, UVB-exposed patients showed significantly higher levels of regulatory T cells compared with control patients (Figure 3a and data not shown), whereas no other blood leukocyte populations, e.g., CD8+ T cells, CD56+ NK cells, or CD14+ monocytes, were significantly influenced by the UVB treatment (Figure 3c and data not shown). However, the percentage of circulating CD14+ cells was significantly higher in the subgroup of UVB patients with complete depletion (Figure 3d). No significant difference between both subgroups was seen for regulatory T cells (Figure 3b).

Figure 3. Flow cytometry analysis of immune cells in peripheral blood after UVB irradiation.

Figure 3

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Mononuclear cells were isolated from blood samples before conditioning, after transplantation, and at two different time points after UVB irradiation (between days 14–22 and days 38–69). Stainings were performed with CD4+/Foxp3+ (a) and CD14+ (c) at each time point for each patient. Data of UVB patients were further analyzed and grouped in patients with complete depletion of CD1a+ Langerhans cell (LC; <1 LC per field, “complete”) and patients with incomplete depletion (≥1 LC per field, “incomplete”, b, d). Statistical analysis was performed with two-tailed Student’s t-test. NS, not significant.

Because the secretion of soluble factors may also be involved in the protective effect of UVB irradiation, we next investigated serum 25-hydroxyvitamin D3 levels and circulating cytokines. UVB patients had significantly higher 25-hydroxyvitamin D3 serum levels, as compared with control patients (Figure 4a), but similar levels of 1,25-dihydroxyvitamin D3 and the complement factor C3c. With regard to circulating cytokines, our results did not reveal any differences in IL-10 and macrophage colony-stimulating factor (M-CSF) serum levels between control and UVB patients, but macrophage migration inhibitory factor serum levels were significantly reduced in the UVB group (Figure 4b). However, high M-CSF serum levels significantly correlated with high 25-hydroxyvitamin D3 serum levels (Figure 4c), suggesting that 25-hydroxyvitamin D3 might modulate M-CSF release. Furthermore, high 25-hydroxyvitamin D3 serum levels were especially found in patients with low-grade GvHD 0–1 (Figure 4d).

Figure 4. Determination of cytokines, 25-hydroxyvitamin D3 (25(OH)D3), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and C3c in the serum of UVB and control patients.

Figure 4

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Serum samples were collected before conditioning (cond), after transplantation, and around day 14 after UVB exposure. Complement C3c and vitamin D metabolites were analyzed with a routine procedure in the Department of Clinical Chemistry, University of Regensburg (a). Serum macrophage migration inhibitory factor (MIF), M-CSF, and IL-10 levels were measured by commercially available ELISA (b). Statistical analysis was performed with two-tailed Student’s t-test. Graph (c) shows the correlation between 25-hydroxyvitamin D3 and M-CSF serum levels at day 14 after UVB irradiation (r = 0.53, P = 0.05). For further analysis UVB patients were grouped according to their acute graft-versus-host disease (GvHD) severity (GvHD 0–1 vs. 2–4, d). M-CSF, macrophage colony-stimulating factor; NS, not significant.

DISCUSSION

GvHD is a potentially life-threatening complication of allo-HCT, and several experimental and clinical efforts focus on GvHD prevention. In a previous study, Yuksel et al. (2006) tried to decrease the incidence and severity of GvHD by peritransplant UVB irradiation. The results were disappointing and revealed that UVB irradiation is detrimental to the outcome of allo-HCT as seven of eight patients developed severe acute GvHD including severe GvHD of the skin. In striking contrast to these data, our irradiation protocol was well tolerated in all patients. Most likely, the reason for this discrepancy in the results is due to differences in the irradiation protocol, as in this study UVB irradiation was initiated 10 days before transplantation, thus interfering with conditioning. Furthermore, Yuksel et al. (2006) used narrowband UVB, whereas in our study patients were irradiated with broadband UVB. Our data in mice have clearly shown that exposure to UVC leads to massive recruitment of circulating monocytes to the skin between 18 hours and 4 days after irradiation (Merad et al., 2004). It is thus likely that in this published study, patients’ exposure to UVB at this early time point led to a recruitment of host monocyte-derived DCs and to an increased number of host DCs at the time of transplant, which may explain the severe GvHD outcome in these patients.

In our protocol, UVB treatment decreased the number of LCs in 15 out of 17 patients, and the number of CD1a+ epidermal DCs correlated with GvHD severity in the UVB group. It has long been hypothesized that LCs are essential for the immune response in the skin; however, murine model systems have meanwhile shown that LCs are neither absolutely required for contact hypersensitivity nor for T cell-mediated GvHD as long as other antigen-presenting cells are present (Li et al., 2011). Although our data in UVB-treated patients indicate an important role of LCs in the development of GvHD, one patient in the control group developed GvHD grade IV despite complete LC depletion (0 CD1a+ cells per field). Therefore, the number of LCs seems to be only one determinant for a lower GvHD risk in UVB patients. It is most likely that other factors and/or cell types contribute to the protective effect.

Dermal CD14+ cells dropped only significantly in control patients who did not receive UVB, suggesting that UVB restrains or recruits CD14+ cells in the dermis. This is in line with data from Kang et al. (1994) showing that UVB leads to an infiltration of immunosuppressive macrophages to the dermis. These immunosuppressive dermal cells could also contribute to the protective effect of UVB irradiation.

UVB-treated patients also had significantly higher numbers of circulating CD4+ FoxP3+ regulatory T cells compared with control patients, indicating that exposure to UVB facilitates the reconstitution of the regulatory T compartment in patients, whereas it does not influence the reconstitution of other leukocyte populations in the blood. As T regulatory cells are critical for the prevention of GvHD and can also reverse established GvHD in a mouse model of allo-HCT (Edinger et al., 2003; Gatza et al., 2008), part of the positive effect of UVB could be mediated through the induction of T regulatory cells. In mice, exposure to UVB leads to the expansion of T regulatory cells, and UV-induced LC migration into lymph nodes is thought to be required for the induction of a T regulatory cell response (Schwarz et al., 2005). Furthermore, topically applied 1,25-dihydroxyvitamin D3 has also been shown to induce the suppressive activity of T regulatory cells in draining lymph nodes (Gorman et al., 2007), suggesting that UVB-induced 25-hydroxyvitamin D3 in the skin may create a local immunosuppressive environment leading to the generation of tolerogenic DCs and increased numbers of circulating regulatory T cells. Accordingly, UVB irradiation also led to a significant increase in 25-hydroxyvitamin D3 serum levels, and lower 25-hydroxyvitamin D3 levels were associated with a higher risk of severe GvHD in UVB patients. In contrast, all control patients without UVB irradiation had low 25-hydroxyvitamin D3 levels after allo-HCT. This is in line with data from our group showing that 25-hydroxyvitamin D3 serum levels decrease after HCT and remain low in patients who develop severe GvHD, whereas they rise again in patients without GvHD (Kreutz et al., 2004). As vitamin D analogs are known to prevent acute GvHD in rat bone marrow transplantation (Pakkala et al., 2001), and polymorphism of the vitamin D receptor gene correlated with GvHD outcome (Middleton et al., 2002), it is possible that the increase in 25-hydroxyvitamin D3 release in UV-treated patients has a key role in the modulation of GvHD in these patients. We also analyzed the correlation between 25-hydroxyvitamin D3 serum levels and circulating blood cells and cytokines, and found that 25-hydroxyvitamin D3 correlated positively with M-CSF levels. These results are consistent with in vitro data showing that 1,25-dihydroxyvitamin D3 increases M-CSF secretion from human circulating monocytes in vitro (Kaneki et al., 1994).

Altogether, our data show that low-dose UVB irradiation is safe and well tolerated in allo-HCT patients. Our data also show that successful depletion of LCs mediated by prophylactic broadband UVB exposure correlates with a lower risk for acute GvHD. Exposure to UVB also leads to elevated serum levels of immunosuppressive factors such as 25-hydroxyvitamin D3 and increased number of circulating T regulatory cells. The exact contribution of these immunosuppressive mechanisms to GvHD outcome, as well as the exact role of LCs in UVB-mediated immunosuppression, remains to be examined. These results suggest that prophylactic UVB exposure at the time of transplant should be explored as a clinical strategy to prevent acute GvHD. Further studies are also required to optimize the timing and dosage of UVB to achieve complete LC depletion in all patients.

PATIENTS AND METHODS

Patients

All patients were included after giving written informed consent. The study had been approved by the local ethics committee at the University Medical Centre of Regensburg and was conducted according to the Declaration of Helsinki Principles.

A total of 17 patients received UVB prophylaxis: 8 patients were female and 9 were male; the median age at SCT was 54 years (range 34–64 years). Underlying diseases were acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, low-grade lymphoma or chronic lymphocytic leukemia, high-grade lymphoma (non-Hodgkin lymphoma) and myeloma (see Table 1). All patients were in a more advanced stage of disease (only partial remission of leukemia or ≥2nd complete remission). Donors were HLA-identical siblings in nine, 10/10 antigen-matched unrelated donors in six, and 9/19 antigen-matched unrelated donors in two. Patients received reduced intensity conditioning consisting of fludarabin/bis-chloroethylnitrosourea or carmustine and melphalan (FBM) as described previously (Table 1, Marks et al., 2008); four patients were treated with reduced intensity conditioning consisting of fludarabin (30 mg m−2 for 5 days) and high-dose treosulfane (14 g m−2 for 3 days) or high-dose mephalan (Table 1). Immunosuppressive prophylaxis included ciclosporin starting from day −2, and mycophenolate mofetil was started at the day of SCT 6 hours after infusion of peripheral blood stem cells. Antithymocyte globulin was used as pretransplant serotherapy in a dose of 10 mg kg−1 for 3 days in all unrelated donors. All patients received peripheral blood stem cell grafts; the mean number of CD34 cells infused was 4.8 (±1.2) × 106 per kg. Patients receiving the identical conditioning protocol without additional UVB exposure served as controls.

Acute and chronic GvHD were graded and staged according to clinical criteria as previously described (Marks et al., 2008). To summarize the combined effects on acute, delayed acute, and chronic GvHD, severe GvHD was defined as either acute GvHD grade III or IV or severe chronic GvHD, and time to maximal occurrence of severe GvHD was calculated for all patients.

Irradiation protocol

The patients’ individual minimal erythemal dose was determined 1 day before stem cell transplantation by a sequence of seven trial exposures made on normal skin on the buttocks using incremental series of UVB irradiations (UVB doses: 14-20-28-40-56-80 and 112 mJ cm−2). The minimal erythemal dose was taken to be the dose given to the aperture showing just perceptible erythema.

One day after allogeneic stem cell transplantation, broadband UVB (wavelength 280–320 nm) therapy was started using a whole-body irradiation cabinet (UV 7001 K Waldmann Medizintechnik, Villingen-Schwenningen, Germany) containing 13 Waldmann low-pressure lamps (F85/100 W–UV21) for broadband UVB irradiation. The cabinet contains UV-sensor systems that automatically and continuously measure the light intensity (mW cm−2) and the light dose (J cm−2). Treatment was given every second day for a total of six whole-body UVB irradiations with 75% of the minimal erythemal dose.

Skin biopsies and immunohistochemistry/immunofluorescence

Punch biopsies (5 mm) were taken from clinically normal skin from the medial side of the upper arm before conditioning, on the day before transplantation and on day 12 after transplantation after the last UVB irradiation. Biopsies from control patients not receiving UVB irradiation were taken at days 10–14.

Immunohistochemistry of epidermal cells was performed with formalin-fixed paraffin-embedded 2-μm thick sections using CD1a (Clone 010, Zytomed) at a previously titrated optimal concentration and the Zytochem Plus Broad Spectrum HRP-Kit (Zytomed, Berlin, Germany). Sections were counterstained with AEC (Dako, Hamburg, Germany). Negative controls were performed by omitting incubation with the primary antibody. Immunoreactive cells in at least five randomly selected areas were counted by two blinded investigators and expressed as positive cells per high-power field.

Immunofluorescence staining of dermal cells was performed on 6-μm frozen skin sections using rabbit polyclonal anti-CD14 (AbCam, San Francisco, CA), mouse anti-HLA-DR (Dako), donkey anti-mouse biotin, donkey anti-rabbit FITC, and Streptavidine-Cy5 (Jackson Immunoresearch, West Grove, PA). Staining with secondary antibodies alone was used as negative control. Nuclear cells were counted in 10 randomly selected fields.

Flow cytometry of mononuclear cells

Mononuclear cells were isolated from freshly drawn blood samples by density gradient centrifugation over Ficoll/Hypaque (Biochrom AG, Berlin, Germany). Stainings were performed with 2–10 × 105 cells per 100 μl PBS/2% FCS using the following antibodies at previously titrated optimal concentrations: CD3-FITC (SK7), CD3-APC (UCHT1), CD4-FITC (SK3), CD8-APC (SK1), CD14-PE (M0P9), CD19-FITC (4G7), CD25-PE and -APC (2A3), and CD56-PE (B159; all from BD Biosciences, Heidelberg, Germany). Propidium iodide (2.5 μg ml−1) was added to unfixed samples to exclude dead cells. FOXP3 stains were performed with PE-conjugated antibodies (PCH101) and respective isotype control from eBioscience (San Diego, CA) according to the manufacturer’s instruction. Flow cytometric data were acquired on a FACSCalibur (BD Biosciences) and analyzed with FlowJo software (Treestar, Ashland, OR).

Determination of cytokines and vitamin D3 metabolites in serum

Blood samples were taken before allo-HCT and at day 14. Cytokines (macrophage migration inhibitory factor-1, M-CSF, and IL-10) were measured in the serum samples by commercially available ELISA (R&D Systems, Abingdon, UK). Complement C3c was measured by nephelometry and vitamin D metabolites were analyzed by radioimmunoassay in the Department of Clinical Chemistry, University of Regensburg, Germany.

Abbreviations

allo-HCT

allogeneic hematopoietic cell transplantation

DC

dendritic cell

GvHD

graft-versus-host disease

LC

Langerhans cell

Footnotes

CONFLICT OF INTEREST The authors state no conflict of interest.

REFERENCES

  1. Aubin F. Mechanisms involved in ultraviolet light-induced immunosuppression. Eur J Dermatol. 2003;13:515–23. [PubMed] [Google Scholar]
  2. Auffermann-Gretzinger S, Lossos IS, Vayntrub TA, et al. Rapid establishment of dendritic cell chimerism in allogeneic hematopoietic cell transplant recipients. Blood. 2002;99:1442–8. doi: 10.1182/blood.v99.4.1442. [DOI] [PubMed] [Google Scholar]
  3. Beissert S, Ruhlemann D, Mohammad T, et al. IL-12 prevents the inhibitory effects of cis-urocanic acid on tumor antigen presentation by Langerhans cells: implications for photocarcinogenesis. J Immunol. 2001;167:6232–8. doi: 10.4049/jimmunol.167.11.6232. [DOI] [PubMed] [Google Scholar]
  4. Collin MP, Hart DN, Jackson GH, et al. The fate of human Langerhans cells in hematopoietic stem cell transplantation. J Exp Med. 2006;203:27–33. doi: 10.1084/jem.20051787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cooper KD, Oberhelman L, Hamilton TA, et al. UV exposure reduces immunization rates and promotes tolerance to epicutaneous antigens in humans: relationship to dose, CD1a-DR+ epidermal macrophage induction, and Langerhans cell depletion. Proc Natl Acad Sci USA. 1992;89:8497–501. doi: 10.1073/pnas.89.18.8497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dai R, Streilein JW. Ultraviolet B-exposed and soluble factor-pre-incubated epidermal Langerhans cells fail to induce contact hypersensitivity and promote DNP-specific tolerance. J Invest Dermatol. 1997;108:721–6. doi: 10.1111/1523-1747.ep12292099. [DOI] [PubMed] [Google Scholar]
  7. Durakovic N, Bezak KB, Skarica M, et al. Host-derived Langerhans cells persist after MHC-matched allografting independent of donor T cells and critically influence the alloresponses mediated by donor lymphocyte infusions. J Immunol. 2006;177:4414–25. doi: 10.4049/jimmunol.177.7.4414. [DOI] [PubMed] [Google Scholar]
  8. Edinger M, Hoffmann P, Ermann J, et al. CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med. 2003;9:1144–50. doi: 10.1038/nm915. [DOI] [PubMed] [Google Scholar]
  9. Fagnoni FF, Oliviero B, Giorgiani G, et al. Reconstitution dynamics of plasmacytoid and myeloid dendritic cell precursors after allogeneic myeloablative hematopoietic stem cell transplantation. Blood. 2004;104:281–9. doi: 10.1182/blood-2003-07-2443. [DOI] [PubMed] [Google Scholar]
  10. Gatza E, Rogers CE, Clouthier SG, et al. Extracorporeal photopheresis reverses experimental graft-versus-host disease through regulatory T cells. Blood. 2008;112:1515–21. doi: 10.1182/blood-2007-11-125542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorman S, Kuritzky LA, Judge MA, et al. Topically applied 1,25-dihydroxyvitamin D3 enhances the suppressive activity of CD4+CD25+ cells in the draining lymph nodes. J Immunol. 2007;179:6273–83. doi: 10.4049/jimmunol.179.9.6273. [DOI] [PubMed] [Google Scholar]
  12. Gregori S, Giarratana N, Smiroldo S, et al. A 1alpha,25-dihydroxyvitamin D(3) analog enhances regulatory T-cells and arrests autoimmune diabetes in NOD mice. Diabetes. 2002;51:1367–74. doi: 10.2337/diabetes.51.5.1367. [DOI] [PubMed] [Google Scholar]
  13. Kaneki M, Inoue S, Hosoi T, et al. Effects of 1 alpha,25-dihydroxyvitamin D3 on macrophage colony-stimulating factor production and proliferation of human monocytic cells. Blood. 1994;83:2285–93. [PubMed] [Google Scholar]
  14. Kang K, Hammerberg C, Meunier L, et al. CD11b+ macrophages that infiltrate human epidermis after in vivo ultraviolet exposure potently produce IL-10 and represent the major secretory source of epidermal IL-10 protein. J Immunol. 1994;153:5256–64. [PubMed] [Google Scholar]
  15. Kreutz M, Eissner G, Hahn J, et al. Variations in 1alpha,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) serum levels during allogeneic bone marrow transplantation. Bone Marrow Transplant. 2004;33:871–3. doi: 10.1038/sj.bmt.1704448. [DOI] [PubMed] [Google Scholar]
  16. Li H, Kaplan DH, Matte-Martone C, et al. Langerhans cells are not required for graft-versus-host disease. Blood. 2011;117:697–707. doi: 10.1182/blood-2010-07-299073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marks R, Potthoff K, Hahn J, et al. Reduced-toxicity conditioning with fludarabine, BCNU, and melphalan in allogeneic hematopoietic cell transplantation: particular activity against advanced hematologic malignancies. Blood. 2008;112:415–25. doi: 10.1182/blood-2007-08-104745. [DOI] [PubMed] [Google Scholar]
  18. Matte CC, Liu J, Cormier J, et al. Donor APCs are required for maximal GVHD but not for GVL. Nat Med. 2004;10:987–92. doi: 10.1038/nm1089. [DOI] [PubMed] [Google Scholar]
  19. Merad M, Hoffmann P, Ranheim E, et al. Depletion of host Langerhans cells before transplantation of donor alloreactive T cells prevents skin graft-versus-host disease. Nat Med. 2004;10:510–7. doi: 10.1038/nm1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Merad M, Manz MG, Karsunky H, et al. Langerhans cells renew in the skin throughout life under steady-state conditions. Nat Immunol. 2002;3:1135–41. doi: 10.1038/ni852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Middleton PG, Cullup H, Dickinson AM, et al. Vitamin D receptor gene polymorphism associates with graft-versus-host disease and survival in HLA-matched sibling allogeneic bone marrow transplantation. Bone Marrow Transplant. 2002;30:223–8. doi: 10.1038/sj.bmt.1703629. [DOI] [PubMed] [Google Scholar]
  22. Murphy GM, Norris PG, Young AR, et al. Low-dose ultraviolet-B irradiation depletes human epidermal Langerhans cells. Br J Dermatol. 1993;129:674–7. doi: 10.1111/j.1365-2133.1993.tb03330.x. [DOI] [PubMed] [Google Scholar]
  23. Pakkala I, Taskinen E, Pakkala S, et al. MC1288, a vitamin D analog, prevents acute graft-versus-host disease in rat bone marrow transplantation. Bone Marrow Transplant. 2001;27:863–7. doi: 10.1038/sj.bmt.1702873. [DOI] [PubMed] [Google Scholar]
  24. Penna G, Amuchastegui S, Giarratana N, et al. 1,25-Dihydroxyvitamin D3 selectively modulates tolerogenic properties in myeloid but not plasmacytoid dendritic cells. J Immunol. 2007;178:145–53. doi: 10.4049/jimmunol.178.1.145. [DOI] [PubMed] [Google Scholar]
  25. Perreault C, Pelletier M, Belanger R, et al. Persistence of host Langerhans cells following allogeneic bone marrow transplantation: possible relationship with acute graft-versus-host disease. Br J Haematol. 1985;60:253–60. doi: 10.1111/j.1365-2141.1985.tb07411.x. [DOI] [PubMed] [Google Scholar]
  26. Schwarz A, Maeda A, Kernebeck K, et al. Prevention of UV radiation-induced immunosuppression by IL-12 is dependent on DNA repair. J Exp Med. 2005;201:173–9. doi: 10.1084/jem.20041212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schwarz A, Maeda A, Wild MK, et al. Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity. J Immunol. 2004;172:1036–43. doi: 10.4049/jimmunol.172.2.1036. [DOI] [PubMed] [Google Scholar]
  28. Shlomchik WD, Couzens MS, Tang CB, et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science. 1999;285:412–5. doi: 10.1126/science.285.5426.412. [DOI] [PubMed] [Google Scholar]
  29. Toews GB, Bergstresser PR, Streilein JW. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J Immunol. 1980;124:445–53. [PubMed] [Google Scholar]
  30. Ullrich SE. Mechanism involved in the systemic suppression of antigen-presenting cell function by UV irradiation. Keratinocyte-derived IL-10 modulates antigen-presenting cell function of splenic adherent cells. J Immunol. 1994;152:3410–6. [PubMed] [Google Scholar]
  31. Yuksel M, Baron E, Camouse M, et al. Peritransplant use of ultraviolet-B irradiation (UV-B) therapy is detrimental to allogeneic stem cell transplantation outcome. Biol Blood Marrow Transplant. 2006;12:665–71. doi: 10.1016/j.bbmt.2006.02.004. [DOI] [PubMed] [Google Scholar]
  32. Zhang Y, Louboutin JP, Zhu J, et al. Preterminal host dendritic cells in irradiated mice prime CD8+ T cell-mediated acute graft-versus-host disease. J Clin Invest. 2002;109:1335–44. doi: 10.1172/JCI14989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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