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The effect of extracorporeal photopheresis on T cell response in chronic graft-versus-host disease a

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Lingqiao Zhu , Daniel R. Couriel & Cheong-Hee Chang

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Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA b

Adult Blood and Marrow Transplant Program, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA Accepted author version posted online: 10 Jun 2015.Published online: 18 Jul 2015.

Click for updates To cite this article: Lingqiao Zhu, Daniel R. Couriel & Cheong-Hee Chang (2015): The effect of extracorporeal photopheresis on T cell response in chronic graft-versus-host disease, Leukemia & Lymphoma, DOI: 10.3109/10428194.2015.1057893 To link to this article: http://dx.doi.org/10.3109/10428194.2015.1057893

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Leukemia & Lymphoma, 2015; Early Online: 1–9 © 2015 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2015.1057893

ORIGINAL ARTICLE: CLINICAL

The effect of extracorporeal photopheresis on T cell response in chronic graft-versus-host disease Lingqiao Zhu1, Daniel R. Couriel2 & Cheong-Hee Chang1 1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA, and 2Adult Blood

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and Marrow Transplant Program, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA

two reactions. Acute GVHD (aGVHD) occurs in the first 3–6 months post allo-HSCT, and mainly affects the skin, gastrointestinal tract and liver. On the other hand, cGVHD imperfectly mimics other autoimmune disorders and appears around 3–6 months after allo-HSCT or later. It can affect almost any organ system, particularly skin, fascia and mucosae. Further details on the complexity of GVHD are beyond the scope of this manuscript, and are covered extensively elsewhere [2,3]. Corticosteroids are used as a first-line therapy to treat both aGVHD and cGVHD but non-responding patients require an additional immunosuppressive therapy. Among the various immunomodulating modalities, extracorporeal photopheresis (ECP) has shown therapeutic efficacy, particularly in patients with cGVHD who failed corticosteroid treatments [2,3]. Another potential advantage of ECP in the therapy of GVHD is that it improves GVHD while possibly preserving the GVT function [4]. The treatment involves the extracorporeal exposure of autologous leukocytes to 8-methoxypsoralen (MOP) and ultraviolet A radiation with subsequent reinfusion, but the underlying mechanisms of action of extracorporeal photopheresis (ECP) are poorly understood. Existing literature indicates that ECP normalizes the inverted CD4/CD8 ratio and increases the number of natural killer (NK) cells [5], shifts dendritic cell (DC) population from DC1 to DC2 [6], and increases the T regulatory (Treg) population [7,8]. In the current study, we investigated the effect of ECP by comparing immune parameters in cGVHD patients treated with this technology to patients receiving other therapies. We then analyzed patients according to their history of aGVHD and the clinical phenotype of cGVHD (sclerotic versus non-sclerotic).

Abstract Extracorporeal photopheresis (ECP) is a safe and effective immunoregulatory therapy for steroid-refractory chronic graftversus-host disease (cGVHD) but its mechanism of action is poorly understood. In this study, we evaluated the effect of ECP in a sample of cGVHD patients. Our data showed that ECP-treated patients had lower CD4 T and B cells, and substantially higher NK cells than untreated patients. T regulatory (Treg) cells were similar between the two groups of patients. Interestingly, Treg cells were higher in ECP-treated patients and ECP-responders who had no history of aGVHD or sclerosis, than in those who had one of them or both. These findings suggest that at least one of the mechanisms of immunomodulation by ECP targets the Treg cell population and that an increase in Treg cells may be associated with response in patients with cGVHD. Together, the results of ECP are different depending on the patients’ clinical condition. Keywords: Extracorporeal photopheresis treatment, chronic graft-versus host disease

Introduction Allogeneic hematopoietic stem-cell transplant (allo-HSCT) provides a potential cure for patients with malignant and nonmalignant disorders [1]. Unfortunately, graft-versus-host disease (GVHD) remains the major barrier, representing a major cause of morbidity and mortality post allo-HSCT. In general, although better supportive care has resulted in improvement of overall survival after allo-HSCT over time, the treatment of both acute and chronic GVHD (cGVHD) is still far from optimal and largely based on corticosteroids. GVHD is caused by donor T cells that mediate host tissue damage via inflammatory mechanisms. At the same time donor T cells also promote the graft-versus-tumor (GVT) effect, essential for the therapeutic efficacy of HSCT. Thus, a key to a good recovery of the patients’ post-HSCT lies on adequate immune recovery with the “right” balance of the

Materials and methods Patient population Patients were consented and enrolled under an IRB-approved protocol in the University of Michigan Blood and Marrow Transplant Program. The sample of patients analyzed under-

Correspondence: Lingqiao Zhu and Cheong-Hee Chang, Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Tel: ⫹ 1 734 764 7550. Fax: ⫹ 1 734 764 3562. E-mail: [email protected] Received 21 November 2014; revised 20 May 2015; accepted 30 May 2015

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went HSCT, developed cGVHD, and came to the transplant clinic for long-term follow-up. At each visit, blood samples were drawn. Patients that received ECP had a diagnosis of moderate to severe cGVHD and were refractory to standard therapy. Chronic GVHD diagnosis and scoring were defined according to the National Institutes of Health (NIH) Consensus Development Project criteria [9]. Clinical data were obtained retrospectively from the University of Michigan Blood and Marrow Transplantation Program. The following parameters were obtained: age, disease, date of blood and marrow transplant (BMT), date of ECP, rounds of ECP went through, donor source, HLA-match, conditioning intensity, day post-BMT of GVHD, overall GVHD grade, GVHD stage in each target organ, relapse, non-relapse mortality, and overall survival. Blood samples from patients who received ECP within 6 months were analyzed. ECP treatments are delivered at different frequencies, approximately from twice weekly to once monthly. A total of 11 patients who had undergone ECP treatment (ECP⫹) and 21 patients without ECP treatment (ECP-) were selected for the study. Patient characteristics were shown in Table I.

ECP treatment protocol ECP treatment was performed using the UVAR TS system together with UVADEX (Therakos, Inc). Each cycle involves the collection, separation and extracorporeal exposure of autologous leukocytes to 8-methoxypsoralen (MOP) and ultraviolet A radiation, with subsequent reinfusion. The active ingredient in UVADEX® (methoxsalen) Sterile Solution is 8-methoxypsoralen (methoxsalen). Methoxsalen is a naturally occurring photoactive substance found in the seed of the Ammimajus (umbelliferae plant). It belongs to a class of compounds known as psoralens or furocoumarins. The chemical name is 9-methoxy-7H-furo[3,2-g][1]-benzopyran-7-one. The formulation of the drug is a sterile liquid at a concentration of 20 μg/ml in a 10 ml vial. A complete description of the pharmacokinetic activity of methoxsalen is available in the Investigator’s Brochure (Therakos, Inc).

Table I. Patients’ characteristics. ECP⫹ (%) Number Gender Female Male History of acute GVHD Sclerotic GVHD Overall grade of cGVHD 1 2 3 Corticosteroids* Other therapies* Tacrolimus Rituximab Cyclosporine Sirolimus Etanercept Imatinib FAM (Fluticasone, azithromycin, and montelukast)

n ⫽ 11 (34)

ECP⫺ (%) n ⫽ 23 (66)

6 (54) 5 (46) 4 (36) 5 (45)

10 (43) 13 (57) 12 (52) 4 (17)

0 4 (36) 7 (64) 11 (100)

8 (35) 10 (43) 3 (14) 11 (52)

7 (63) 1 (9) 1 (9) 1 (9) 1 (9) 1 (9) 2 (18)

13 (62) 1 (5) 0 1 (5) 0 0 2 (9)

*Assessed at the time the blood sample was drawn

Systemic exposure to UVADEX® (methoxsalen) Sterile Solution following ECP is minimal. The total dose of methoxsalen used to inoculate these cells is less than 1/200th of the oral dose used in conjunction with the UVAR XTS® System. Each patient received 2–4 treatments per week at initiation, followed by taper according to response and clinical practice guidelines. As response to therapy in cGVHD is complex and controversial, in this study, we defined response to ECP as clinical improvement plus discontinuation of corticosteroids.

Cell preparation and flow cytometry Blood samples were centrifuged with Ficoll to separate plasma from cells and preserved as described [10]. Peripheral blood mononuclear cells (PBMC) were stained with the following FITC-, PE-, PerCP-, PE-CY7-, APC-, APC-Cy7-, Pacific blueor biotin-conjugated antibodies: CD3, CD4, CD8, CD19, CD45RA, CD45RO and CD56. Antibodies were purchased from BD PharMingen or eBioscience. Cells were also intracellularly stained with antibodies against T-bet and Foxp3. For T-bet and Foxp3 staining, Foxp3 staining buffer set (eBioscience) was used for cell fixation and permeabilization following the company’s protocol. Events were acquired on a FACSCanto flow cytometer (BD), and the data were analyzed with the FlowJo software (Tree Star Inc.).

Single cell cytokine assay for lymphocytes Freshly isolated PBMC were stimulated with phorbol 13-myristic acid (PMA) and ionomycin (Calbiochem) for 4 h. Brefeldin A (Sigma) was added during the last 3 h of stimulation. Cells were then stained with anti-CD3, CD4 and CD8 antibodies to define T cell subsets, fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.2% saponin (Sigma) followed by intracellular staining of IFN-γ, IL-4 and IL-17.

Statistical analysis Analysis of data for statistical significance was conducted using Prism 6 for Macintosh (GraphPad Software, Incorporated). Unpaired t test was used to calculate the p values. Due to the small number of patients in the ECP-treated group, a p value smaller than 0.1 was considered statistically significant.

Results Different composition of circulating lymphocytes of ECPⴙ and ECPⴚ patients We compared the frequency of immune cell types with freshly isolated PBMC from ECP-treated (ECP⫹) (n ⫽ 11) or untreated (ECP⫺) cGVHD patients (n ⫽ 21). Cells were stained with antibody cocktails for different lymphocyte subsets. From the total lymphocytes in the FSC/SSC plot, we analyzed for CD3 ⫹ T cells, CD56 ⫹ NK cells, and CD19 ⫹ B cells. CD3 ⫹ T cells were further divided into CD4 ⫹ and CD8 ⫹ cells. Staining profiles of two representative patients (Patients 1 and 2) from each group are shown in Figure 1A. ECP⫹ patients had significantly lower percentages of B cells but higher NK cells compared to the ECP⫺ group,

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Figure 1. The lymphocyte composition of patients with or without ECP treatment. (A) Total leukocytes were stained with the cocktails of antibodies to determine the type of cells. The staining profiles of two patients in each group are shown. (B) Foxp3 expressing CD4 T cells were compared between the two groups of patients. (C) Summary of all patients in ECP– (n ⫽ 21) and ECP⫹ (n ⫽ 11) patients. Numbers in the graph are p values and the values lower than 0.1 are shown.

while CD3 ⫹ T cells were comparable between the two groups (Figure 1 A and C). Among CD3 ⫹ cells, the percentage of CD4 ⫹ cells was lower in ECP⫹ patients than ECP⫺ group (Figure 1C, middle panel). Accordingly, the ECP⫹ group showed the lower CD4/CD8 ratio than the ECP- group although this did not reach the statistical significance (Figure 1C, right panel). Some reports indicate that ECP increases the levels of circulating regulatory T cells in ECP-treated patients. However, we found that neither the frequency (Figure 1 B and C) nor the cell number (data not shown) of Foxp3 ⫹ CD4 T cells between the two groups were significantly different. To determine whether ECP treatment changes the composition of memory and naïve T cells, the expression of CD45RA and CD45RO in CD4 T cells was analyzed. The ratio of CD45RA/CD45RO was not different between the ECP⫹ and ECP⫺ groups (Figure 2A). Although we did not observe a significant difference in total Treg populations with or without ECP (Figure 1 B and C), it is possible that ECP treatment may change the distribution of Treg. When the expression of Foxp3 in CD45RA⫹ and CD45RO⫹ CD4 T cells was compared, Foxp3-expressing cells were enriched in CD45RO⫹ cells and this pattern was similar between ECP- and ECP⫹ patients (Figure 2B). Together, these data demonstrate that ECP-treated cGVHD patients have a different lymphocyte

Figure 2. The naïve versus memory profile of CD4 T cells among patients. (A) Representative profiles of CD45RA and CD45RO expression of CD4 T cells in the two groups of patients. The right panel shows the ratio of naive over memory CD4 T cells from ECP– (n ⫽ 6) and ECP⫹ (n ⫽ 7) patients as indicated. (B) Foxp3 expressing cells were enriched in CD45RO⫹ cells in both groups of patients.

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composition compared to the ECP⫺ ones but the constitution of naïve and memory CD4 T cells as well as Treg cells are similar between the two groups.

IL-13, TGF-β, and TNF-α, were not significantly different either (data not shown). Therefore, ECP treatment does not seem to change the effector cell types of circulating T cells.

ECP does not affect the cytokine production by T cells

Lymphocyte profiles are different in patients with a history of aGVHD or sclerosis

Next, we studied whether ECP affected T cell function using two different approaches. First, we measured the cytokine expression of T cells after short stimulation of PBMC with PMA and ionomycin followed by intracellular cytokine staining as described in the Materials and methods section. This short stimulation activates cytokine expression in cells that have been activated and committed to effectors and thus allow us to determine the type of Th cells in patients. If ECP controls the T cell function via cytokine expression, T cells from ECP⫹ patients are expected to express a different pattern of cytokines. CD4 or CD8 T cell subsets were analyzed for the expression of IFN-γ, IL-4 and IL-17 that are the signature cytokines of Th1, Th2 and Th17, respectively. Although the percentage of cytokine-expressing cell populations varied among individual patients, our analysis did not show a significant difference between the two groups in either CD4 (Figure 3A) or CD8 (Figure 3B) T cell subsets. Nevertheless, IFN-γ expressing CD4 and CD8 T cells were dominant with a few IL-4 ⫹ cells (Figure 3). IL-17 ⫹ cells were not easily detectable in either group (Figure 3). As a second and an alternative approach, we measured the levels of cytokines and chemokines in the plasma using ELISA. Consistent with intracellular staining of cytokines, IL-17A was undetectable and IL-4 and IFN-γ levels were comparable between the ECP⫹ and ECP- group (data not shown). Other cytokines, including IL-1β, IL-2, IL-6, IL-10, IL-12,

A large percentage of cGVHD patients have a history of aGVHD, as these patients are more prone to cGVHD [11]. The immune responses in aGVHD and cGVHD are very different, with aGVHD being predominantly inflammatory [12] and cGVHD displaying autoimmune and fibrotic characteristics [13]. We hypothesized that a history of aGVHD would make a difference in the lymphocyte composition of ECP⫹ and ECP⫺ patients. Therefore, we first divided patients into aGVHD⫺ and aGVHD⫹ groups, and each group was further subdivided into ECP⫹ and ECP⫺. In patients without a history of aGVHD (aGVHD⫺), higher percentages of CD56 ⫹ NK cells were detected in ECP⫹ compared with ECP⫺ ones (Figure 4A, top panel). In the group with a history of aGVHD, the NK cell subpopulation seemed to be increased in the ECP⫹ group but the difference was not significant. Instead, percentages of CD8 T cells and CD19 B cells were increased and decreased, respectively, in ECP⫹ patients (Figure 4A, lower panel). Therefore, a history of aGVHD correlates with the lymphocyte composition and may have an impact on response to ECP. We then examined patients using sclerosis, a clinical phenotype of cGVHD, as a variable. ECP-treated patients with sclerosis showed the same increase in NK cells observed in patients without a previous history of aGVHD (compare the top panel in Figure 4A with the bottom panel in Figure 4B). Furthermore, ECP treatment in Sclerosis- patients was associated with lower

Figure 3. Comparison of the cytokine production of T cells between ECP⫹ and ECP– patients. Representative profiles of IL-4, IFN-γ, and IL-17 expressing CD4 (A) and CD8 T cells (B) are shown. Two patients from each group are compared. PBMC were stimulated with PMA and ionomycin for 4 h followed by intracellular staining of indicated cytokines.

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chemokine-mediated recruitment of T cells all of which contribute to the pathogenesis of cGVHD [14–23]. Because our data in Figure 3 clearly showed a dominant Th1 immune response in patients regardless of ECP treatment, we focused on the Th1 response. To further investigate the presence or generation of Th1 type T cells in cGVHD patients, we examined the expression of T-bet. T-bet is the master transcription factor for Th1 cell differentiation [24]. It is also reported that other cell types such as CD8 T cells, NK cells, and B cells express T-bet [24–26], and all of these cell types control the type of immunity. To this end, we compared T-bet expression in CD4 T cells, CD8 T cells, B cells, and NK cells among four groups of patients based on the history of aGVHD and ECP treatment: aGVHD-ECP⫺, aGVHD⫺ ECP⫹, aGVHD⫹ ECP⫺ and aGVHD⫹ ECP⫹. Patients without aGVHD history showed decreased T-bet⫹ cells in CD8 T cells but not other cell types if they received ECP treatments (Figure 5A and B). By contrast, patients with a history of aGVHD (aGVHD⫹) showed a significant increase in T-bet expressing CD4 T and B cells (Figure 5A and B). Almost all NK cells expressed T-bet and they were similar among the four groups (Figure 5A and B). Next, we examined patients that were grouped based on sclerosis and ECP treatment. Unlike a history of aGVHD, the presence of sclerosis was not associated with changes of T-bet⫹ cells in any subgroup (Figure 5C). In order to assess the concurrent influence of a history of aGVHD and sclerosis, we divided ECP-treated patients into four groups: aGVHD⫺ Sclerosis⫺, aGVHD⫺ Sclerosis⫹, aGVHD⫹ Sclerosis⫺, and aGVHD⫹ Sclerosis⫹. As shown in Figure 6A, aGVHD⫺ Sclerosis⫺ group showed the highest frequency of Treg than the rest of the groups albeit a small number of patients. But the same group of patients did not have as many T-bet⫹ cells compared to others. Instead, the aGVHD⫹ Sclerosis⫺ patients tended to have higher T-bet⫹ CD4, CD8 T cells and B cells (Figure 6B). Therefore, our study revealed the combinatorial effect of aGVHD and sclerosis on immune responses of ECP⫹ patients.

Immune profile is different between ECP responders and non-responders

Figure 4. aGVHD history and the presence of sclerosis results in a difference in the lymphocyte composition. (A) All patients were first divided into aGVHD– and aGVHD⫹ groups and then each group of patients was further subdivided into ECP– and ECP⫹ groups. Indicated cell types were analyzed as in Figure 1. aGVHD–ECP– (n ⫽ 9), aGVHD– ECP⫹ (n ⫽ 7), aGVHD⫹ ECP– (n ⫽ 12), aGVHD⫹ ECP⫹ (n ⫽ 4). (B) Same patients were used as in (A) except that patients were subdivided based on the presence of sclerosis. Sclerosis–ECP– (n ⫽ 17), Sclerosis– ECP⫹ (n ⫽ 6), Sclerosis⫹ ECP– (n ⫽ 4), Sclerosis⫹ ECP⫹ (n ⫽ 5). Numbers in the graphs are p values.

CD4 and higher CD8 T cells (Figure 4B, top panel). NK cells were comparable between the two groups.

History of aGVHD and sclerosis influences T-Bet expression Studies have shown that cGVHD is initiated and modulated by multiple factors including different Th cell responses and

Next, we wanted to study the changes in the immune responses of patients who responded to ECP treatment compared to those who did not. For this purpose, 11 ECP⫹ patients were divided into responders and non-responders and their immune parameters were compared. As response to therapy in cGVHD is complex and controversial, we defined response to ECP as clinical improvement plus discontinuation of corticosteroids. Among the ECP⫹ patients, seven were responders based on our criteria. Responders had a significantly lower frequency of CD8 T cells than nonresponders, while B cells were elevated (Figure 7A, left panel). In addition, there was a trend towards higher Foxp3 ⫹ CD4 T cells in ECP responders. Other cell populations showed no significant differences (Figure 7A, left panel). T-bet expression was comparable in all cell types between ECP responders and non-responders (Figure 7A, right panel). Responders were subsequently grouped according to history of aGVHD and sclerosis and their immune

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Figure 5. aGVHD history of patients correlates with the frequency of T-bet expressing T cells. (A) T-bet expression profiles in indicated cell types from four groups of patients were compared. Staining was carried out as in Figure 1 and T-bet expression was assessed as described in the Materials and methods. A representative profile from each group is shown. (B and C) The same groups of patients were subdivided based on aGVHD (B) or sclerosis (C) to assess T-bet⫹ cells. NA, not available. Numbers in the graphs are p values.

responses were compared. Of note, all aGVHD⫹ Sclerosis⫹ patients in our group were non-responders, so the analysis was performed on three groups: aGVHD⫺ Sclerosis⫺, aGVHD⫺ Sclerosis⫹, aGVHD⫹ Sclerosis⫺. As illustrated in Figure 7B, immune responses varied among the three subgroups of responders to ECP. In addition, there was a clear trend towards higher Treg in aGVHD⫺ Sclerosis⫺ patients than the other two groups (first panel in Figure 7C), whereas T-bet⫹ cells were similar among three groups (Figure 7C).

Discussion The pathophysiology of cGVHD and the mechanism of action of ECP are both complex and multifactorial. Studies have shown contradicting Th1 vs. Th2 response after ECP treatment in cGVHD patients. An early report showed a shift of the DC population from DC1 to DC2 together with a concordant shift from a predominantly Th1 (IL-2, IFN-γ) to Th2 (IL-4, IL-10) cytokine profile in cGVHD patients after ECP treatment [6]. Other groups demonstrated an increase of IFN-γ generating CD4 T cells after ECP in cGVHD patients [27] and cutaneous cGVHD patients [28]. These results sug-

gest an increase of Th1 cells. Differences among many studies likely stemmed from several factors including the different experimental settings, patient characteristics and therapies, variations in measured immune parameters, and differences in data analysis. Although we had a small number of patients, we chose our patients with strict parameters to minimize heterogeneity of patients and disease-dependent variables. Some of our analyses did not show statistical significance despite showing a trend, which is likely related to the sample size. Nevertheless, we made important observations. When we compared the patients based on ECP treatment alone, we did not see a significant difference in IFN-γ in CD4 and CD8 T cells in cellular or plasma levels. Furthermore, the pattern of T-bet expression in major lymphocyte populations and Treg cells were comparable. However, differences emerged when patients were analyzed in subgroups based on clinically relevant parameters. When we assessed T-bet expressing cells, which would indicate Th1 immunity, between two patient groups using ECP treatment as a single criterion, we did not find a significant difference (data not shown). Furthermore, history of

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Figure 6. ECP-treated patients with neither aGVHD nor Sclerosis have more Treg cells than others. Eleven patients who received ECP were selected and grouped based on their history of aGVHD together with the presence of sclerosis. Expression of Foxp3 (A) and T-bet in CD4, CD8 and CD19 cells (B) were assessed and compared. aGVHD–Sclerosis– (n ⫽ 2 or 3), aGVHD–Sclerosis– (n ⫽ 3 or 4), aGVHD–Sclerosis– (n ⫽ 3), aGVHD– Sclerosis– (n ⫽ 1).

aGVHD alone irrespective of ECP treatment did not differentiate the two groups of patients regarding T-bet⫹ cells (data not shown). When we considered the two together, there were significant differences in T-bet expression (Figure 5B). ECP⫹

patients with aGVHD had high T-bet⫹ CD4 and CD19 cells, whereas T-bet⫹ CD8 T cells were reduced in ECP⫹ patients without aGVHD. However, changes in T-bet expressing cells did not correlate with the patients’ response to ECP treatment.

Figure 7. Changes in ECP responsive patients. (A) ECP-treated patients (n ⫽ 11) were subdivided into responders (n ⫽ 7) and non-responders (n ⫽ 4) to the treatment and the immune parameters were compared. Summary graphs of indicated cell types (left graph) and the percentages of T-bet⫹ cells (right graph) are shown. (B and C) Responders were further subdivided into aGVHD–Sclerosis– (n ⫽ 2 or 3), aGVHD–Sclerosis– (n ⫽ 2), and aGVHD– Sclerosis– (n ⫽ 2) groups and compared for the frequency of cell types (B), and Foxp3 and T-bet expression (C). Numbers in the graph are p values.

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Perhaps Th1 immunity established during the aGVHD phase maintains even after the patients develop cGVHD. This is consistent with recent findings that Th1 response plays a role in initiating or modulating cGVHD [18,19]. We observed a substantially higher proportion of NK cells in ECP-treated patients than the ECP⫺ ones (Figure 1A and C). This is consistent with an earlier observation that ECP treatment in cGVHD patients leads to an increase of the NK cell number [5]. The same report also showed that the two patients who had no response to ECP already had high NK cell reconstitution before therapy. When we examined our ECP-treated patients more closely, the ratios of NK cells were comparable between responders and non-responders (Figure 7A). Therefore it is possible that the increase of NK cells is the result of ECP treatment without affecting the responsiveness although we cannot rule out the possibility that the modulation of the NK cell population by ECP is one of its working mechanisms. A longitudinal study on these ECP patients would provide an insight to understand the role of NK cells. In contrast to previously published reports that circulating Treg cells were increased in GVHD patients after ECP treatment [7,29–32], we observed little difference in the frequency of Foxp3 ⫹ CD4⫹ T cells between the ECP⫹ and ECP⫺ groups (Figure 1B and C). However, the assessment of immune response according to history of aGVHD and presence of sclerosis, the fibrotic form of cGVHD, showed some differences. Treg cells were elevated in ECP⫹ patients without a history of aGVHD or sclerosis (Figure 6). These findings imply that Treg levels could be affected by different clinical variables in a heterogeneous disease like cGVHD. Many studies reported the changes of Treg frequencies with ECP treatments but functional tests are limited. Because the numbers of Treg cells isolated from patients are low, it is difficult to test suppressive function. It has been demonstrated in the mouse model that the absence of Treg cells is responsible for autoimmunity in cGVHD [21]. Therefore, the increase in the Treg population may contribute to the efficacy of ECP in the treatment of cGVHD [8], so studying the function in addition to frequency of Treg cells would be highly informative. The clinical implications of our finding in Treg cell levels according to different variables in the setting of ECP therapy remains to be defined. As selection and grouping of patients may also influence the trends observed, these results should be interpreted with caution. The inclusion of clinically relevant and well-defined parameters in larger patient samples with a longer and thorough follow-up will provide better insights and a more accurate and better understanding of the potential mechanisms of action of ECP, patients’ responses to therapy, and their immune recovery. Our study shows that the effect of ECP treatment may vary depending on clinical manifestations of cGVHD, which may explain the contradicting results of published studies and also highlights the complexity of translational research in cGVHD.

Acknowledgements We thank Maggie Kennel and Bryan Fiema for patient’s sample collections and the data management, respectively.

This work was supported in part by Michigan Institute for Clinical and Health Research and MCubed both of which were funded by the University of Michigan to Drs Couriel and Chang, and also in part by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR000433 to Dr Chang.

Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal

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The effect of extracorporeal photopheresis on T cell response in chronic graft-versus-host disease.

Extracorporeal photopheresis (ECP) is a safe and effective immunoregulatory therapy for steroid-refractory chronic graft-versus-host disease (cGVHD) b...
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