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Clinical and Experimental Immunology
O R I G I N A L A RT I C L E
doi:10.1111/cei.12331
Frequency and clonality of peripheral γδ T cells in psoriasis patients receiving anti-tumour necrosis factor-α therapy
J. Kelsen,*† A. Dige,* M. Christensen,‡ F. D’Amore§ and L. Iversen¶ *Gastro-Immuno Research Laboratory (GIRL), Department of Hepatology and Gastroenterology, ‡ Institute of Pathology, §Department of Hematology and ¶Department of Dermatology, Aarhus University Hospital, and †Department of Medicine, Randers Regional Hospital, Randers, Denmark
Accepted for publication 5 March 2014 Correspondence: J. Kelsen, Department of Medicine, Randers Regional Hospital, Skovlyvej 1, DK-8930 Randers NO, Denmark. E-mail:[email protected]
Summary Hepatosplenic γδ T cell lymphoma (HSTCL) has been observed in patients with Crohn’s disease (CD) who received anti-tumour necrosis factor (TNF)-α agents and thiopurines, but only one case was reported in a psoriasis patient worldwide. This difference could be due to differences in either the nature of the inflammatory diseases or in the use of immunomodulators. We investigated the impact of anti-TNF-α agents on the level and repertoire of γδ T cells in peripheral blood from psoriasis patients. Forty-five men and 10 women who were treated with anti-TNF-α agents for psoriasis were monitored for a median 11 months for the level and clonality of γδ T cells via flow cytometry and polymerase chain reaction (PCR) analysis of T cell receptor gamma (TCR-γ) gene rearrangements. Seventeen men had a repeated analysis within 48 h of the infliximab infusion to reveal a possible expansion of γδ T cells, as observed previously in CD patients. Ten psoriasis patients who were never exposed to biologicals and 20 healthy individuals served as controls. In the majority of psoriasis patients, the level and clonal pattern of γδ T cells was remarkably stable during infliximab treatment. A single male patient repeatedly experienced a significant increase in the level of γδ T cells after infliximab infusions. A monoclonal γδ T cell repertoire in a polyclonal background tended to be more frequent in anti-TNF-α-treated patients than naive patients, suggesting that anti-TNF-α therapy may promote the clonal selection of γδ T cells in psoriasis patients. Keywords: HSTCL, infliximab, lymphoma, psoriasis, γδ T cells
Introduction Gamma-delta (γδ) T cell lymphoma is a distinct form of peripheral T cell lymphoma that is divided into hepatosplenic T cell lymphoma (HSTCL) and non-HSTCL [1–3]. Approximately 200 cases of HSTCL have been reported worldwide, usually with fatal outcomes [2,4]. HSTCL was observed in a disproportionately high number of male Crohn’s disease (CD) patients who were treated with anti-tumour necrosis factor (TNF)-α therapy and thiopurines. In 2006, the Food and Drug Administration (FDA) mandated a warning due to the suspected association of this treatment with the development of HSTCL [5,6]. It remains unclear whether lymphomagenesis is driven partially by the disease or primarily by the treatment [7,8]. Due to the rare incidence of HSTCL, conventional epidemiological causality assessment tools are not applicable [9,10]. 142
According to the recent REFURBISH study, only one case of HSTCL in a psoriasis patient was reported worldwide; this patient was also receiving anti-TNF-α therapy (i.e. etanercept) in combination with methotrexate and cyclosporin [11,12]. It remains unknown why psoriasis patients appear to have a lower risk of developing HSTCL. The widespread use of thiopurines (i.e. azathioprine and 5-mercaptopurine) in inflammatory bowel disease (IBD) patients, in whom methotrexate is used less frequently than in psoriasis patients, may play a role. Alternatively, psoriasis patients may be less prone to HSTCL due to differences in the natures of these diseases [13]. The level of γδ T cells in healthy individuals is relatively stable, and γδ T cells normally comprise 3–5% of the peripheral T cell population. Lymphomagenesis is believed to be a multi-step process in which transforming events lead to the gradual selection of malignant clones [1]. In this context, we previously observed a significant expansion of γδ T cells in selected CD
© 2014 British Society for Immunology, Clinical and Experimental Immunology, 177: 142–148
Gamma-delta T cells in psoriasis patients Table 1. Characteristics of psoriasis patients and healthy controls.
Number Age, median (range) Female Male Anti-TNF-treatment Infliximab Adalimumab Etanercept Current use of immunomodulators Methotrexate Hydroxyurea Observation period, median months (range) Number of infliximab infusions received, median (range)
Anti-TNF
Naive
Healthy controls
55 48 (24, 76) 10 45
10 50 (34, 66) 4 6
20 34 (23, 57) 8 12
3 (0, 8)
7 (0, 16)
31 18 6
14 1 11 (0, 29) 26 (5, 55)
Flow cytometry For flow cytometric staining, 100 μl of venous blood was incubated for 20 min at room temperature with optimised amounts of the following fluorescence-conjugated antibodies: anti-αβ-T cell receptor [TCR–fluorescein isothiocyanate (FITC) (clone WT-31], anti-Vδ2-phycoerythrin (PE) (clone B6), anti-CD3-peridinin chlorophyll (PerCP) (clone SK7), anti-CD8-PE-cyanin (Cy)7 (clone RPA-T8), anti-γδ-TCRallophycocyanin (APC) (clone B1) and anti-CD4-APC-Cy7 (clone RPA-T4). All antibodies were from BD Biosciences (San Diego, CA, USA). Six-colour flow cytometry was performed within 4 h of staining on a fluorescence activated cell sorter (FACS)Canto flow cytometer (BD Biosciences). For each sample, 30 000 events were recorded in the forward-/side-scatter live lymphocyte gate. All γδ T cell frequencies were calculated as a proportion of the total CD3+ T cells. The data were analysed using FACSDiva version 5·1 software (BD Biosciences).
TNF = tumour necrosis factor.
TCR-γ gene rearrangement analysis patients within 2 days of infliximab infusion. This phenomenon was observed preferentially in men with extraordinarily high baseline levels of γδ T cells, which may characterise individuals at increased risk of developing HSTCL [14]. In this study, we investigated the impact of anti-TNF-α therapy on the levels and the clonality of γδ T cells in a population of psoriasis patients to address the causative role of the underlying inflammatory disease in the development of HSTCL.
Materials and methods Patients Due to the risk profile of HSTCL, which primarily affects men, we focused upon male patients receiving anti-TNF-α agents (Table 1). Forty-five men and 10 women with psoriasis were recruited in the out-patient clinic of the Department of Dermatology at Aarhus University Hospital. Blood samples were drawn at scheduled visits in a 3-year period with a median observation period of 11 months. Seventeen men had taken paired blood samples within 48 h of infliximab infusion, and of these, two patients were monitored again with paired samples 12 months later for confirmation of the primary findings. According to the clinical algorithm, the patients had diverse medical histories and had been treated with a variety of immunosuppressants, including cyclosporin, efalizumab, hydroxyurea, methotrexate and one or more of the three anti-TNF-α agents. In addition, samples from 10 psoriasis patients who had never been treated with immunomodulators or anti-TNF-α agents and 20 healthy individuals were analysed on two occasions.
Genomic DNA was extracted from peripheral blood using a MagNaPure Compact (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Two regions of the TCR-γ gene were amplified by multiplex polymerase chain reaction (PCR) with primers covering the variable segments Vγ1-8 in combination with the joining segments Jγ1·1/2·1 and Jγ1·3/2·3. The joining primers were fluorescence-labelled. Jγ1·1/2·1 was TETlabelled (green) and Jγ1·3/2·3 was FAM-labelled (blue). Beta-globin was included as control for the PCR reaction. The primers used in this study were previously recommended in the BIOMED-2 protocol [15]. All primers were purchased from MWG-Biotech (Ebersberg, Germany). A non-template PCR and a polyclonal sample served as negative and positive controls, respectively. PCR was performed using a GeneAmp®9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Fragment analysis to assess clonality was performed via high-resolution capillary electrophoresis using an ABI 310 Sequence Analyser (Applied Biosystems). All PCR products were analysed with a TAMRA-500 DNA size marker. The lengths of the Vγ1-8/Jγ1·3/2·3 and Vγ1-8/Jγ1·1/2·1 PCR products were 195–230 base pairs (bp) and 230–255 bp, respectively. Data were analysed using the GeneMapper program, version 4·1 (Applied Biosystems).
Clonality of γδ T cells We estimated the relative height of the peaks to exclude pseudo spikes. To be considered genuine monoclonal peaks, the height of the peaks should have a minimum ratio of at least twice the height of the normal distribution of the
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Percentage γδ T cells of peripheral T cells
35 30 25 20 15 10 5 0
Anti-TNF
Naive
Healthy
Fig. 1. Frequencies of γδ T cells. The level of γδ T cells in 55 psoriasis patients who were treated with anti-tumour necrosis factor (TNF)-α therapy, 10 anti-TNF-α-naive psoriasis patients and 20 healthy individuals. No statistically significant differences were observed between the groups. Each individual contributed a single measurement, which was obtained when entering the study.
polyclonal peaks [16]. The different patterns of γδ T cell clonality were defined as polyclonal, oligoclonal or monoclonal in a polyclonal background [17].
Statistical analysis Unpaired comparisons of continuous variables were performed using Student’s t-test. Differences for which P < 0·05 were considered statistically significant. All statistical analyses were performed using spss version 11·0 software.
Ethics All participants provided written informed consent, and the study was approved by the Central Denmark Region Committee on Biomedical Research Ethics (j. no. 22559).
Results The level of γδ T cells is stable in psoriasis patients and healthy individuals We monitored the level of γδ T cells in peripheral blood by flow cytometry (Fig. 1). The 55 psoriasis patients who were treated with antiTNF-α agents had a mean γδ T cell level of 5·2% of all CD3+ T cells, with a range of 0·5–29·4%. The mean level was calculated based on a single baseline determination per patient, when the patients entered the study. Levels tended 144
to be higher in male patients (mean 5·7%) than in female patients (mean 3·1%) (P = 0·18). In the 10 psoriasis patients who were naive to anti-TNF-α agents, the mean level of γδ T cells was 5·6%. This value was particularly influenced by a single patient with 20·2% γδ T cells. The 20 healthy controls had a mean level of 3·4% γδ T cells, with a range of 1–12%. These results were not significantly different from those of the anti-TNF-α-treated patients (P = 0·13). Individual γδ T cell levels were subject to little variation over time, and our observations indicate the existence of a relatively narrow person-specific interval. The anti-TNF-α treated patients were followed for a median 11 months. Among these, the infliximab-treated patients had received a median 26 infusions when recruited to the study. We found no signs that γδ T cell levels gradually increased with an increasing number of infliximab infusions. We further investigated the distribution of Vδ subtypes and differentiated them into Vδ1 and Vδ2 subtypes. Despite large variations between individuals, the distribution of subtypes and the total number of γδ T cells within individual patients were stable. Anti-TNF-α-treated patients had the same relative distribution of Vδ subtypes as healthy controls, i.e. a relative predominance of Vδ2-positive T cells; mean 66 versus 34% Vδ1-positive T cells. The distribution in the small group of naive patients was equal. All patients whose total γδ T cell levels were above the mean exhibited a relative predominance of Vδ2 subtypes.
Monoclonal TCR-γ populations in a polyclonal background are observed frequently in anti-TNF-α-treated psoriasis patients We assessed clonality in two regions of the TCR-γ gene: Vγ1-8/Jγ1·3/2·3 and Vγ1-8/Jγ1·1/2·1. As described previously, the γδ T cell clonality patterns were divided into three categories [17]: first, polyclonal γδ T cell populations that had an even distribution of TCR-γ subtypes, reflecting the presumed normal γδ T cell distribution in a healthy population; secondly, oligoclonal TCR-γ-populations that had an uneven distribution of peaks (i.e. fewer than five peaks); and thirdly, monoclonal TCRγ-populations (i.e. a dominant peak) in a polyclonal background. Clonality analysis using the Vγ1-8/Jγ1·3/2·3 region revealed that an oligoclonal γδ T cell repertoire or a monoclonal γδ T cell repertoire in a polyclonal background was only observed in psoriasis patients who were receiving antiTNF-α agents. Psoriasis patients who were naive to biologicals and healthy controls uniformly displayed a polyclonal pattern (Fig. 2). In the Vγ1-8/Jγ1·1/2 region, the results were more heterogeneous. Oligoclonal TCR-γ populations were observed with equal frequencies in the three groups. A monoclonal pattern in a polyclonal background was observed frequently in anti-TNF-α-treated patients, but this pattern was not
© 2014 British Society for Immunology, Clinical and Experimental Immunology, 177: 142–148
Gamma-delta T cells in psoriasis patients
Region Vg1-8/Jg 1·3/2·3
Region Vg1-8/Jg 1·1/2·
Monoclonal in a polyclonal background Polyclonal γδ-T cell repertoire Oligoclonal γδ-T cell repertoire Fig. 2. Distribution of clonality patterns. The γδ T cell clonality patterns were divided into three categories: polyclonal γδ T cell populations, oligoclonal γδ T cell populations and a monoclonal γδ T cell population in a polyclonal background. The relative distribution of these clonality patterns was not significantly different among the three groups of individuals. However, there was a tendency towards a higher prevalence of a monoclonal γδ T cell population in a polyclonal background in anti-TNF-α treated psoriasis patients.
observed in naive patients. A monoclonal pattern with a single dominant peak was also observed occasionally in healthy individuals (Fig. 2). No association between clonality patterns and a specific anti-TNF-α agent (i.e. etanercept, infliximab or adalimumab) could be identified. We observed no compelling signs that the γδ T cell repertoire of an individual patient gradually narrowed during the observation period; however, the patient with the longest history of infliximab treatment (55 infusions) presented with a monoclonal peak in a polyclonal background (Fig. 3).
Seventeen male patients were monitored for the level of γδ T cells immediately before infliximab infusion and again within 48 h of infusion (Fig. 4). This relatively narrow window of observation was chosen to increase the likelihood of observing a causal relationship between infliximab infusion and changes in γδ T cell levels. Fifteen patients displayed the same or slightly lower γδ T cell levels after infliximab treatment. However, two patients experienced a notable increase in γδ T cell levels. In one case, an initial increase from 29·4 to 37·5% γδ T cells was observed and a repeated increase from 16·2 to 21·5% γδ T cells was observed 18 months later (Fig. 5). In the other case, an increase from 8·7 to 10·7% γδ T cells was observed (Fig. 6). However, the latter increase was observed only once in this patient and thus did not represent an invariable response to infliximab infusions. It is worth noting that among the 17 male psoriasis patients, only the two patients with the highest baseline levels of γδ T cells experienced an increase. Both these patients received combination therapy with methotrexate and infliximab. In contrast, only four of the 15 patients in the group without γδ T cell expansion received this combination therapy. In addition, both patients who experienced a significant increase in γδ T cells displayed a stable γδ T cell clonality pattern, without overt signs of newly arising γδ T cell clones or a narrowing of the original clonality pattern towards dominant γδ T cell clones (Figs 5 and 6).
Discussion HSTCL has been observed primarily in patients receiving immunosuppressants post-transplantation and in CD
0 102 103 104 105 –79
0·8%
0 102 103 104 105 –43
0 103 104 105
0 103 104 105
0·8%
December 2011
–1,109
–2,587
0 103 104 105
October 2011 Before infliximab After infliximab
γδ–TCR
Fig. 3. Flow cytometry and analysis of T cell receptor (TCR)-γ gene rearrangement. The upper panel shows flow cytometric analysis in a 48-year-old male psoriasis patient who was treated with methotrexate and infliximab in October 2011, December 2011 and December 2012 (infusion number 55). In October 2011, paired samples were obtained within 48 h and levels of γδ T cells were stable, with a range from 0·4 to 0·8% of peripheral T cells. Lower panel: high-resolution capillary electrophoreses of TCR-γ gene rearrangements showing a stable single dominant γδ T cell clone. The peak height indicates the relative fluorescence intensity. The x-axis indicates the size of the fragments in base pairs. Rearrangements in the Vγ1-8/Jγ1·3/2·3 (blue) and Vγ1-8/Jγ1·1/2·1 (green) regions are shown. The red peaks represent the DNA size marker.
Expansion of γδ T cells in response to infliximab therapy is observed in selected psoriasis patients
0·8%
0 102 103 104 105 –78
December 2012 0 103 104 105
Healthy controls
–1,557
Naive to anti-TNF-α agents
–3,766
Psoriasis patients
0·4%
0 102 103 104 105 –90
αβ–TCR
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5
0
After Ifx
Fig. 4. Response to infliximab infusion. Changes in the levels of γδ T cells in 17 male patients in response to infliximab infusion are shown. The samples were obtained immediately before infliximab infusion and 48 h later. The two patients with the highest baseline level exhibited a notable increase in the level of γδ T cells, and the levels of the remaining 15 patients were stable or decreased.
patients treated with thiopurines and anti-TNF-α agents [18]. In this study, we aimed to investigate the striking difference in HSTCL susceptibility between CD patients and psoriasis patients. We hypothesised that psoriasis patients respond to anti-TNF-α therapy without expansion of γδ T
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February 2013 Before infliximab After infliximab
M 0 –304
103 104 105
37·5%
M 0 –304
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0 103 104 105
29·4%
–2,460
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0 103 104 105 –2,460
γδ–TCR
September 2011 Before infliximab After infliximab
16·2%
M 0 –304
103 104 105
21·5%
M 0 –304
103 104 105
M 0 103 104 105 –2,460
0 103 104 105
0 103 104 105
M 0 103 104 105 –1,969
–318
M 0 103 104 105 –2,460
–226
0 103 104 105
–226
0 103 104 105
αβ–TCR
Vδ2–TCR
Fig. 5. Flow cytometry and analysis of T cell receptor (TCR)-γ gene rearrangement. The upper panel shows flow cytometric analysis of a 45-year-old male psoriasis patient who was treated with methotrexate and infliximab, monitored in September 2011(infusion number 42) and February 2013. On both occasions, the patient displayed a noticeable increase in the level of γδ T cells within 48 h of the infusion. Middle panel: flow cytometric characterisation of Vδ-subtypes revealed that the expansion had occurred in the Vδ-2-positive population. Lower panel: an assessment of γδ T cell clonality at the same time-points revealed an unchanged oligoclonal distribution of γδ T cell populations. High-resolution capillary electrophoresis of TCR-γ gene rearrangements is shown. The peak height indicates the relative fluorescence intensity. The x-axis indicates the size of the fragments in base pairs. Rearrangements in the Vγ1-8/Jγ1·3/2·3 (blue) and Vγ1-8/Jγ1·1/2·1 (green) regions are shown. The red peaks represent the DNA size marker.
–2,460
Before Ifx
0 103 104 105
15
–2,460
20
cells or narrowing of the clonality pattern of γδ T cells in peripheral blood. In the majority of psoriasis patients, well-defined stable individual levels of γδ T cells were observed during a median observation period of nearly 1 year. Oligoclonal γδ T cell populations were observed frequently, and selected psoriasis patients exhibited a stable and narrow TCR-γ repertoire, with monoclonal γδ T cells in a polyclonal background. This type of distribution was also observed in a patient who had received 55 infusions of infliximab. The significance of these findings is difficult to assess. However, monoclonal γδ T cells in a polyclonal background appear to be relatively common in this population of psoriasis patients, and cannot be viewed as evidence of premalignant lesions. We preferentially monitored male patients, who account for more than 80% of HSTCL cases [19]. In particular, one male psoriasis patient paralleled our previous observations in a cohort of CD patients. Specifically, in a background of high baseline levels of γδ T cells (15–30%), this patient experienced a significant expansion of γδ T cells within 48 h of infliximab infusion. This patient exhibited this phenomenon on two occasions with an 18-month interval, when monitored in the context of infliximab infusions. Importantly, in this specific patient, the γδ T cell repertoire remained oligoclonal throughout the observation period and the baseline level of γδ T cells tended to decrease between infusions. In our previous study, the short time-interval between these events led us to assume that causality existed between
–218
Percentage γδ T cells of peripheral T cells
25
–2,599
0 103 104 105
Vδ1–TCR
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7·5%
–3,565
0 103 104 105
0102 103 104 105 –180
–4,080
0102 103 104 105 –178
October 2010 Before infliximab After infliximab
10·6%
–1,909
0 103 104 105
8·6%
–1,217
γδ–TCR
September 2010 Before infliximab After infliximab
0102 103 104 105 –157
αβ–TCR
Fig. 6. Flow cytometry and analysis of T cell receptor (TCR)-γ gene rearrangement. The upper panel shows flow cytometric analysis of a 47-year-old male psoriasis patient who was treated with methotrexate and infliximab, monitored in September 2010 (infusion number 10) and October 2011. On only the first occasion, the patient displayed an increase in the level of γδ T cells within 48 h of the infusion. Lower panel: an assessment of γδ T cell clonality showed an unchanged polyclonal distribution of γδ T cell populations. High-resolution capillary electrophoresis of TCR-γ gene rearrangements is shown. The peak height indicates the relative fluorescence intensity. The x-axis indicates the size of the fragments in base pairs. Rearrangements in the Vγ1-8/Jγ1·3/2·3 (blue) and Vγ1-8/Jγ1·1/2·1 (green) regions are shown. The red peaks represent the DNA size marker.
infliximab treatment and γδ T cell proliferation. Thus, we proposed that infliximab could be a transforming event in lymphomagenesis. We estimated that approximately 5% of CD patients repeatedly expand the level of γδ T cells when exposed to infliximab. Based on these findings, we expected to potentially identify one patient in the psoriasis population in this study. As described previously in healthy individuals, the level of γδ T cells in peripheral blood was slightly higher in men. As expected, we observed that the majority of γδ T cells were of the Vδ2 subtype [20]. It has been argued that the relative predominance of Vδ2 T cells in peripheral blood and skin may protect psoriasis patients from the development of HSTCL, which is reported mainly in the Vδ1 subtype [13]. Due to the casuistic nature of HSTCL, we aimed to address the impact of anti-TNF-α therapy on individual patients. This approach is hampered by the fact that psoriasis patients often have a medical history that includes treatment with several different biologicals. Ideally, a study should be conducted with selective inclusion of psoriasis patients who are naive to biologicals and immunomodulators. Inherently, patients with severe inflammation will be treated with immunomodulators and biologicals, not allowing conclusions about the relative impacts of disease and medication. Therefore, we included a group of psoriasis patients who were not suitable for anti-TNF-α therapy due to comorbidity or personal preference, but were eligible for climate therapy due to severe disease. In this selected group of psoriasis patients, we also observed an oligoclonal TCR-γ repertoire, but no examples of a monoclonal pattern in a polyclonal background.
Lymphomagenesis is believed to result from the gradual selection of ultimately malignant γδ T cell clones. In the group of anti-TNF-α-treated patients, a monoclonal γδ T cell repertoire in a monoclonal background was more common. In individual patients, we did not observe a narrowing of the γδ T cell repertoire or a general increase in the level of γδ T cells during the course of anti-TNF-α therapy. With the exception of the presumed infliximab-induced expansion of γδ T cells in two male psoriasis patients, we found no evidence that anti-TNF-α therapy exerts a proliferative effect on γδ T cells.
Conclusions A well-defined individual γδ T cell level and a stable γδ T cell repertoire was observed in the vast majority of psoriasis patients. It remains unknown why selected young male patients with CD and psoriasis experience infliximabinduced expansion of γδ T cells without sharing the same risk of developing HSTCL.
Acknowledgements This study was supported by research funding from the Danish Council for Independent Research and the Danish Colitis–Crohn Foundation.
Disclosure L. I. has served as a consultant and/or paid speaker for and/or participated in clinical trials sponsored by compa-
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nies that manufacture biologicals used for the treatment of psoriasis, including AbbVie, Janssen-Cilag, MSD and Pfizer. 10
Author contributions J. K. designed the project, performed experiments and drafted the manuscript. A. D. performed flow cytometry, analysed the data and revised the manuscript. M. C. performed the clonality analysis. F. D. took part in study design and edited and revised the manuscript. L. I. recruited the patients and drafted the manuscript.
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© 2014 British Society for Immunology, Clinical and Experimental Immunology, 177: 142–148
Clinical and Experimental Immunology
O R I G I N A L A RT I C L E
doi:10.1111/cei.12331
Frequency and clonality of peripheral γδ T cells in psoriasis patients receiving anti-tumour necrosis factor-α therapy
J. Kelsen,*† A. Dige,* M. Christensen,‡ F. D’Amore§ and L. Iversen¶ *Gastro-Immuno Research Laboratory (GIRL), Department of Hepatology and Gastroenterology, ‡ Institute of Pathology, §Department of Hematology and ¶Department of Dermatology, Aarhus University Hospital, and †Department of Medicine, Randers Regional Hospital, Randers, Denmark
Accepted for publication 5 March 2014 Correspondence: J. Kelsen, Department of Medicine, Randers Regional Hospital, Skovlyvej 1, DK-8930 Randers NO, Denmark. E-mail:[email protected]
Summary Hepatosplenic γδ T cell lymphoma (HSTCL) has been observed in patients with Crohn’s disease (CD) who received anti-tumour necrosis factor (TNF)-α agents and thiopurines, but only one case was reported in a psoriasis patient worldwide. This difference could be due to differences in either the nature of the inflammatory diseases or in the use of immunomodulators. We investigated the impact of anti-TNF-α agents on the level and repertoire of γδ T cells in peripheral blood from psoriasis patients. Forty-five men and 10 women who were treated with anti-TNF-α agents for psoriasis were monitored for a median 11 months for the level and clonality of γδ T cells via flow cytometry and polymerase chain reaction (PCR) analysis of T cell receptor gamma (TCR-γ) gene rearrangements. Seventeen men had a repeated analysis within 48 h of the infliximab infusion to reveal a possible expansion of γδ T cells, as observed previously in CD patients. Ten psoriasis patients who were never exposed to biologicals and 20 healthy individuals served as controls. In the majority of psoriasis patients, the level and clonal pattern of γδ T cells was remarkably stable during infliximab treatment. A single male patient repeatedly experienced a significant increase in the level of γδ T cells after infliximab infusions. A monoclonal γδ T cell repertoire in a polyclonal background tended to be more frequent in anti-TNF-α-treated patients than naive patients, suggesting that anti-TNF-α therapy may promote the clonal selection of γδ T cells in psoriasis patients. Keywords: HSTCL, infliximab, lymphoma, psoriasis, γδ T cells
Introduction Gamma-delta (γδ) T cell lymphoma is a distinct form of peripheral T cell lymphoma that is divided into hepatosplenic T cell lymphoma (HSTCL) and non-HSTCL [1–3]. Approximately 200 cases of HSTCL have been reported worldwide, usually with fatal outcomes [2,4]. HSTCL was observed in a disproportionately high number of male Crohn’s disease (CD) patients who were treated with anti-tumour necrosis factor (TNF)-α therapy and thiopurines. In 2006, the Food and Drug Administration (FDA) mandated a warning due to the suspected association of this treatment with the development of HSTCL [5,6]. It remains unclear whether lymphomagenesis is driven partially by the disease or primarily by the treatment [7,8]. Due to the rare incidence of HSTCL, conventional epidemiological causality assessment tools are not applicable [9,10]. 142
According to the recent REFURBISH study, only one case of HSTCL in a psoriasis patient was reported worldwide; this patient was also receiving anti-TNF-α therapy (i.e. etanercept) in combination with methotrexate and cyclosporin [11,12]. It remains unknown why psoriasis patients appear to have a lower risk of developing HSTCL. The widespread use of thiopurines (i.e. azathioprine and 5-mercaptopurine) in inflammatory bowel disease (IBD) patients, in whom methotrexate is used less frequently than in psoriasis patients, may play a role. Alternatively, psoriasis patients may be less prone to HSTCL due to differences in the natures of these diseases [13]. The level of γδ T cells in healthy individuals is relatively stable, and γδ T cells normally comprise 3–5% of the peripheral T cell population. Lymphomagenesis is believed to be a multi-step process in which transforming events lead to the gradual selection of malignant clones [1]. In this context, we previously observed a significant expansion of γδ T cells in selected CD
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Gamma-delta T cells in psoriasis patients Table 1. Characteristics of psoriasis patients and healthy controls.
Number Age, median (range) Female Male Anti-TNF-treatment Infliximab Adalimumab Etanercept Current use of immunomodulators Methotrexate Hydroxyurea Observation period, median months (range) Number of infliximab infusions received, median (range)
Anti-TNF
Naive
Healthy controls
55 48 (24, 76) 10 45
10 50 (34, 66) 4 6
20 34 (23, 57) 8 12
3 (0, 8)
7 (0, 16)
31 18 6
14 1 11 (0, 29) 26 (5, 55)
Flow cytometry For flow cytometric staining, 100 μl of venous blood was incubated for 20 min at room temperature with optimised amounts of the following fluorescence-conjugated antibodies: anti-αβ-T cell receptor [TCR–fluorescein isothiocyanate (FITC) (clone WT-31], anti-Vδ2-phycoerythrin (PE) (clone B6), anti-CD3-peridinin chlorophyll (PerCP) (clone SK7), anti-CD8-PE-cyanin (Cy)7 (clone RPA-T8), anti-γδ-TCRallophycocyanin (APC) (clone B1) and anti-CD4-APC-Cy7 (clone RPA-T4). All antibodies were from BD Biosciences (San Diego, CA, USA). Six-colour flow cytometry was performed within 4 h of staining on a fluorescence activated cell sorter (FACS)Canto flow cytometer (BD Biosciences). For each sample, 30 000 events were recorded in the forward-/side-scatter live lymphocyte gate. All γδ T cell frequencies were calculated as a proportion of the total CD3+ T cells. The data were analysed using FACSDiva version 5·1 software (BD Biosciences).
TNF = tumour necrosis factor.
TCR-γ gene rearrangement analysis patients within 2 days of infliximab infusion. This phenomenon was observed preferentially in men with extraordinarily high baseline levels of γδ T cells, which may characterise individuals at increased risk of developing HSTCL [14]. In this study, we investigated the impact of anti-TNF-α therapy on the levels and the clonality of γδ T cells in a population of psoriasis patients to address the causative role of the underlying inflammatory disease in the development of HSTCL.
Materials and methods Patients Due to the risk profile of HSTCL, which primarily affects men, we focused upon male patients receiving anti-TNF-α agents (Table 1). Forty-five men and 10 women with psoriasis were recruited in the out-patient clinic of the Department of Dermatology at Aarhus University Hospital. Blood samples were drawn at scheduled visits in a 3-year period with a median observation period of 11 months. Seventeen men had taken paired blood samples within 48 h of infliximab infusion, and of these, two patients were monitored again with paired samples 12 months later for confirmation of the primary findings. According to the clinical algorithm, the patients had diverse medical histories and had been treated with a variety of immunosuppressants, including cyclosporin, efalizumab, hydroxyurea, methotrexate and one or more of the three anti-TNF-α agents. In addition, samples from 10 psoriasis patients who had never been treated with immunomodulators or anti-TNF-α agents and 20 healthy individuals were analysed on two occasions.
Genomic DNA was extracted from peripheral blood using a MagNaPure Compact (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Two regions of the TCR-γ gene were amplified by multiplex polymerase chain reaction (PCR) with primers covering the variable segments Vγ1-8 in combination with the joining segments Jγ1·1/2·1 and Jγ1·3/2·3. The joining primers were fluorescence-labelled. Jγ1·1/2·1 was TETlabelled (green) and Jγ1·3/2·3 was FAM-labelled (blue). Beta-globin was included as control for the PCR reaction. The primers used in this study were previously recommended in the BIOMED-2 protocol [15]. All primers were purchased from MWG-Biotech (Ebersberg, Germany). A non-template PCR and a polyclonal sample served as negative and positive controls, respectively. PCR was performed using a GeneAmp®9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Fragment analysis to assess clonality was performed via high-resolution capillary electrophoresis using an ABI 310 Sequence Analyser (Applied Biosystems). All PCR products were analysed with a TAMRA-500 DNA size marker. The lengths of the Vγ1-8/Jγ1·3/2·3 and Vγ1-8/Jγ1·1/2·1 PCR products were 195–230 base pairs (bp) and 230–255 bp, respectively. Data were analysed using the GeneMapper program, version 4·1 (Applied Biosystems).
Clonality of γδ T cells We estimated the relative height of the peaks to exclude pseudo spikes. To be considered genuine monoclonal peaks, the height of the peaks should have a minimum ratio of at least twice the height of the normal distribution of the
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Percentage γδ T cells of peripheral T cells
35 30 25 20 15 10 5 0
Anti-TNF
Naive
Healthy
Fig. 1. Frequencies of γδ T cells. The level of γδ T cells in 55 psoriasis patients who were treated with anti-tumour necrosis factor (TNF)-α therapy, 10 anti-TNF-α-naive psoriasis patients and 20 healthy individuals. No statistically significant differences were observed between the groups. Each individual contributed a single measurement, which was obtained when entering the study.
polyclonal peaks [16]. The different patterns of γδ T cell clonality were defined as polyclonal, oligoclonal or monoclonal in a polyclonal background [17].
Statistical analysis Unpaired comparisons of continuous variables were performed using Student’s t-test. Differences for which P < 0·05 were considered statistically significant. All statistical analyses were performed using spss version 11·0 software.
Ethics All participants provided written informed consent, and the study was approved by the Central Denmark Region Committee on Biomedical Research Ethics (j. no. 22559).
Results The level of γδ T cells is stable in psoriasis patients and healthy individuals We monitored the level of γδ T cells in peripheral blood by flow cytometry (Fig. 1). The 55 psoriasis patients who were treated with antiTNF-α agents had a mean γδ T cell level of 5·2% of all CD3+ T cells, with a range of 0·5–29·4%. The mean level was calculated based on a single baseline determination per patient, when the patients entered the study. Levels tended 144
to be higher in male patients (mean 5·7%) than in female patients (mean 3·1%) (P = 0·18). In the 10 psoriasis patients who were naive to anti-TNF-α agents, the mean level of γδ T cells was 5·6%. This value was particularly influenced by a single patient with 20·2% γδ T cells. The 20 healthy controls had a mean level of 3·4% γδ T cells, with a range of 1–12%. These results were not significantly different from those of the anti-TNF-α-treated patients (P = 0·13). Individual γδ T cell levels were subject to little variation over time, and our observations indicate the existence of a relatively narrow person-specific interval. The anti-TNF-α treated patients were followed for a median 11 months. Among these, the infliximab-treated patients had received a median 26 infusions when recruited to the study. We found no signs that γδ T cell levels gradually increased with an increasing number of infliximab infusions. We further investigated the distribution of Vδ subtypes and differentiated them into Vδ1 and Vδ2 subtypes. Despite large variations between individuals, the distribution of subtypes and the total number of γδ T cells within individual patients were stable. Anti-TNF-α-treated patients had the same relative distribution of Vδ subtypes as healthy controls, i.e. a relative predominance of Vδ2-positive T cells; mean 66 versus 34% Vδ1-positive T cells. The distribution in the small group of naive patients was equal. All patients whose total γδ T cell levels were above the mean exhibited a relative predominance of Vδ2 subtypes.
Monoclonal TCR-γ populations in a polyclonal background are observed frequently in anti-TNF-α-treated psoriasis patients We assessed clonality in two regions of the TCR-γ gene: Vγ1-8/Jγ1·3/2·3 and Vγ1-8/Jγ1·1/2·1. As described previously, the γδ T cell clonality patterns were divided into three categories [17]: first, polyclonal γδ T cell populations that had an even distribution of TCR-γ subtypes, reflecting the presumed normal γδ T cell distribution in a healthy population; secondly, oligoclonal TCR-γ-populations that had an uneven distribution of peaks (i.e. fewer than five peaks); and thirdly, monoclonal TCRγ-populations (i.e. a dominant peak) in a polyclonal background. Clonality analysis using the Vγ1-8/Jγ1·3/2·3 region revealed that an oligoclonal γδ T cell repertoire or a monoclonal γδ T cell repertoire in a polyclonal background was only observed in psoriasis patients who were receiving antiTNF-α agents. Psoriasis patients who were naive to biologicals and healthy controls uniformly displayed a polyclonal pattern (Fig. 2). In the Vγ1-8/Jγ1·1/2 region, the results were more heterogeneous. Oligoclonal TCR-γ populations were observed with equal frequencies in the three groups. A monoclonal pattern in a polyclonal background was observed frequently in anti-TNF-α-treated patients, but this pattern was not
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Gamma-delta T cells in psoriasis patients
Region Vg1-8/Jg 1·3/2·3
Region Vg1-8/Jg 1·1/2·
Monoclonal in a polyclonal background Polyclonal γδ-T cell repertoire Oligoclonal γδ-T cell repertoire Fig. 2. Distribution of clonality patterns. The γδ T cell clonality patterns were divided into three categories: polyclonal γδ T cell populations, oligoclonal γδ T cell populations and a monoclonal γδ T cell population in a polyclonal background. The relative distribution of these clonality patterns was not significantly different among the three groups of individuals. However, there was a tendency towards a higher prevalence of a monoclonal γδ T cell population in a polyclonal background in anti-TNF-α treated psoriasis patients.
observed in naive patients. A monoclonal pattern with a single dominant peak was also observed occasionally in healthy individuals (Fig. 2). No association between clonality patterns and a specific anti-TNF-α agent (i.e. etanercept, infliximab or adalimumab) could be identified. We observed no compelling signs that the γδ T cell repertoire of an individual patient gradually narrowed during the observation period; however, the patient with the longest history of infliximab treatment (55 infusions) presented with a monoclonal peak in a polyclonal background (Fig. 3).
Seventeen male patients were monitored for the level of γδ T cells immediately before infliximab infusion and again within 48 h of infusion (Fig. 4). This relatively narrow window of observation was chosen to increase the likelihood of observing a causal relationship between infliximab infusion and changes in γδ T cell levels. Fifteen patients displayed the same or slightly lower γδ T cell levels after infliximab treatment. However, two patients experienced a notable increase in γδ T cell levels. In one case, an initial increase from 29·4 to 37·5% γδ T cells was observed and a repeated increase from 16·2 to 21·5% γδ T cells was observed 18 months later (Fig. 5). In the other case, an increase from 8·7 to 10·7% γδ T cells was observed (Fig. 6). However, the latter increase was observed only once in this patient and thus did not represent an invariable response to infliximab infusions. It is worth noting that among the 17 male psoriasis patients, only the two patients with the highest baseline levels of γδ T cells experienced an increase. Both these patients received combination therapy with methotrexate and infliximab. In contrast, only four of the 15 patients in the group without γδ T cell expansion received this combination therapy. In addition, both patients who experienced a significant increase in γδ T cells displayed a stable γδ T cell clonality pattern, without overt signs of newly arising γδ T cell clones or a narrowing of the original clonality pattern towards dominant γδ T cell clones (Figs 5 and 6).
Discussion HSTCL has been observed primarily in patients receiving immunosuppressants post-transplantation and in CD
0 102 103 104 105 –79
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October 2011 Before infliximab After infliximab
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Fig. 3. Flow cytometry and analysis of T cell receptor (TCR)-γ gene rearrangement. The upper panel shows flow cytometric analysis in a 48-year-old male psoriasis patient who was treated with methotrexate and infliximab in October 2011, December 2011 and December 2012 (infusion number 55). In October 2011, paired samples were obtained within 48 h and levels of γδ T cells were stable, with a range from 0·4 to 0·8% of peripheral T cells. Lower panel: high-resolution capillary electrophoreses of TCR-γ gene rearrangements showing a stable single dominant γδ T cell clone. The peak height indicates the relative fluorescence intensity. The x-axis indicates the size of the fragments in base pairs. Rearrangements in the Vγ1-8/Jγ1·3/2·3 (blue) and Vγ1-8/Jγ1·1/2·1 (green) regions are shown. The red peaks represent the DNA size marker.
Expansion of γδ T cells in response to infliximab therapy is observed in selected psoriasis patients
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After Ifx
Fig. 4. Response to infliximab infusion. Changes in the levels of γδ T cells in 17 male patients in response to infliximab infusion are shown. The samples were obtained immediately before infliximab infusion and 48 h later. The two patients with the highest baseline level exhibited a notable increase in the level of γδ T cells, and the levels of the remaining 15 patients were stable or decreased.
patients treated with thiopurines and anti-TNF-α agents [18]. In this study, we aimed to investigate the striking difference in HSTCL susceptibility between CD patients and psoriasis patients. We hypothesised that psoriasis patients respond to anti-TNF-α therapy without expansion of γδ T
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February 2013 Before infliximab After infliximab
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γδ–TCR
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21·5%
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0 103 104 105
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–318
M 0 103 104 105 –2,460
–226
0 103 104 105
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0 103 104 105
αβ–TCR
Vδ2–TCR
Fig. 5. Flow cytometry and analysis of T cell receptor (TCR)-γ gene rearrangement. The upper panel shows flow cytometric analysis of a 45-year-old male psoriasis patient who was treated with methotrexate and infliximab, monitored in September 2011(infusion number 42) and February 2013. On both occasions, the patient displayed a noticeable increase in the level of γδ T cells within 48 h of the infusion. Middle panel: flow cytometric characterisation of Vδ-subtypes revealed that the expansion had occurred in the Vδ-2-positive population. Lower panel: an assessment of γδ T cell clonality at the same time-points revealed an unchanged oligoclonal distribution of γδ T cell populations. High-resolution capillary electrophoresis of TCR-γ gene rearrangements is shown. The peak height indicates the relative fluorescence intensity. The x-axis indicates the size of the fragments in base pairs. Rearrangements in the Vγ1-8/Jγ1·3/2·3 (blue) and Vγ1-8/Jγ1·1/2·1 (green) regions are shown. The red peaks represent the DNA size marker.
–2,460
Before Ifx
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–2,460
20
cells or narrowing of the clonality pattern of γδ T cells in peripheral blood. In the majority of psoriasis patients, well-defined stable individual levels of γδ T cells were observed during a median observation period of nearly 1 year. Oligoclonal γδ T cell populations were observed frequently, and selected psoriasis patients exhibited a stable and narrow TCR-γ repertoire, with monoclonal γδ T cells in a polyclonal background. This type of distribution was also observed in a patient who had received 55 infusions of infliximab. The significance of these findings is difficult to assess. However, monoclonal γδ T cells in a polyclonal background appear to be relatively common in this population of psoriasis patients, and cannot be viewed as evidence of premalignant lesions. We preferentially monitored male patients, who account for more than 80% of HSTCL cases [19]. In particular, one male psoriasis patient paralleled our previous observations in a cohort of CD patients. Specifically, in a background of high baseline levels of γδ T cells (15–30%), this patient experienced a significant expansion of γδ T cells within 48 h of infliximab infusion. This patient exhibited this phenomenon on two occasions with an 18-month interval, when monitored in the context of infliximab infusions. Importantly, in this specific patient, the γδ T cell repertoire remained oligoclonal throughout the observation period and the baseline level of γδ T cells tended to decrease between infusions. In our previous study, the short time-interval between these events led us to assume that causality existed between
–218
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–1,909
0 103 104 105
8·6%
–1,217
γδ–TCR
September 2010 Before infliximab After infliximab
0102 103 104 105 –157
αβ–TCR
Fig. 6. Flow cytometry and analysis of T cell receptor (TCR)-γ gene rearrangement. The upper panel shows flow cytometric analysis of a 47-year-old male psoriasis patient who was treated with methotrexate and infliximab, monitored in September 2010 (infusion number 10) and October 2011. On only the first occasion, the patient displayed an increase in the level of γδ T cells within 48 h of the infusion. Lower panel: an assessment of γδ T cell clonality showed an unchanged polyclonal distribution of γδ T cell populations. High-resolution capillary electrophoresis of TCR-γ gene rearrangements is shown. The peak height indicates the relative fluorescence intensity. The x-axis indicates the size of the fragments in base pairs. Rearrangements in the Vγ1-8/Jγ1·3/2·3 (blue) and Vγ1-8/Jγ1·1/2·1 (green) regions are shown. The red peaks represent the DNA size marker.
infliximab treatment and γδ T cell proliferation. Thus, we proposed that infliximab could be a transforming event in lymphomagenesis. We estimated that approximately 5% of CD patients repeatedly expand the level of γδ T cells when exposed to infliximab. Based on these findings, we expected to potentially identify one patient in the psoriasis population in this study. As described previously in healthy individuals, the level of γδ T cells in peripheral blood was slightly higher in men. As expected, we observed that the majority of γδ T cells were of the Vδ2 subtype [20]. It has been argued that the relative predominance of Vδ2 T cells in peripheral blood and skin may protect psoriasis patients from the development of HSTCL, which is reported mainly in the Vδ1 subtype [13]. Due to the casuistic nature of HSTCL, we aimed to address the impact of anti-TNF-α therapy on individual patients. This approach is hampered by the fact that psoriasis patients often have a medical history that includes treatment with several different biologicals. Ideally, a study should be conducted with selective inclusion of psoriasis patients who are naive to biologicals and immunomodulators. Inherently, patients with severe inflammation will be treated with immunomodulators and biologicals, not allowing conclusions about the relative impacts of disease and medication. Therefore, we included a group of psoriasis patients who were not suitable for anti-TNF-α therapy due to comorbidity or personal preference, but were eligible for climate therapy due to severe disease. In this selected group of psoriasis patients, we also observed an oligoclonal TCR-γ repertoire, but no examples of a monoclonal pattern in a polyclonal background.
Lymphomagenesis is believed to result from the gradual selection of ultimately malignant γδ T cell clones. In the group of anti-TNF-α-treated patients, a monoclonal γδ T cell repertoire in a monoclonal background was more common. In individual patients, we did not observe a narrowing of the γδ T cell repertoire or a general increase in the level of γδ T cells during the course of anti-TNF-α therapy. With the exception of the presumed infliximab-induced expansion of γδ T cells in two male psoriasis patients, we found no evidence that anti-TNF-α therapy exerts a proliferative effect on γδ T cells.
Conclusions A well-defined individual γδ T cell level and a stable γδ T cell repertoire was observed in the vast majority of psoriasis patients. It remains unknown why selected young male patients with CD and psoriasis experience infliximabinduced expansion of γδ T cells without sharing the same risk of developing HSTCL.
Acknowledgements This study was supported by research funding from the Danish Council for Independent Research and the Danish Colitis–Crohn Foundation.
Disclosure L. I. has served as a consultant and/or paid speaker for and/or participated in clinical trials sponsored by compa-
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nies that manufacture biologicals used for the treatment of psoriasis, including AbbVie, Janssen-Cilag, MSD and Pfizer. 10
Author contributions J. K. designed the project, performed experiments and drafted the manuscript. A. D. performed flow cytometry, analysed the data and revised the manuscript. M. C. performed the clonality analysis. F. D. took part in study design and edited and revised the manuscript. L. I. recruited the patients and drafted the manuscript.
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