Just Accepted by Leukemia & Lymphoma
Modulation of lymphocyte subpopulations by extracorporeal photo-pheresis in patients with acute GvHD or graft rejection Katrin Lorenz, Katharina Rommel, Jiju Mani, Nan Jin, Inken Hilgendorf, Anthony D. Ho, Mathias Freund, Michael Schmitt, Anita Schmitt doi: 10.3109/10428194.2014.931956 Leuk Lymphoma Downloaded from informahealthcare.com by Baylor University on 06/15/14 For personal use only.
ABSTRACT Extracorporeal photopheresis (ECP) constitutes a promising treatment for patients with steroid-refractory acute graft-versus-host disease (aGvHD) after allogeneic stem cell transplantation and for patients with graft rejection after solid organ transplantation (SOT). There is an increasing body of evidence that modulation of lymphocyte subsets might play a crucial role in the mechanism of action in ECP. We therefore analyzed immunological effects concomitantly to clinical findings in patients under ECP therapy with multicolor flow cytometry. In a patient with steroid-refractory aGvHD and a patient with progressive bronchiolitis obliterans syndrome (BOS) after double-lung transplantation, clinical responses to ECP therapy were paralleled by an increase of CD4+CD25hiFoxP3+ regulatory T cells and a decrease of T(EMRA) (CD3+CD8+CD45RA+CD62L+ effector memory T cells) as well as of NKT cells. In summary, immunomonitoring of T cell subsets can elucidate the mechanism of action in ECP.
© 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
Modulation of lymphocyte subpopulations by extracorporeal photo-pheresis in patients with acute GvHD or graft rejection
Katrin Lorenz2, Katharina Rommel1, Jiju Mani2, Nan Jin2, Inken Hilgendorf1,
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Anthony D. Ho2, Mathias Freund1, Michael Schmitt2, Anita Schmitt2
Dept. Internal Medicine III, University Clinic Rostock, Rostock, Germany 2Internal Medi-
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Corresponding author: Anita Schmitt, Cellular Immunotherapy, Dept. Internal Medicine V, University Clinic Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg. Germany. Tel. +49-6221-56-35829. FAX. +49-6221-56-5740. E-Mail: [email protected] ABSTRACT
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Extracorporeal photopheresis (ECP) constitutes a promising treatment for patients with steroid-refractory acute graft-versus-host disease (aGvHD) after allogeneic stem cell transplantation and for patients with graft rejection after solid organ transplantation (SOT). There is an increasing body of evidence that modulation of lymphocyte subsets might play a crucial role in the mechanism of action in ECP. We therefore analyzed immunological effects concomitantly to clinical findings in patients under ECP therapy with multicolor flow cytometry. In a patient with steroid-refractory aGvHD and a patient with progressive bronchiolitis obliterans syndrome (BOS) after double-lung transplantation, clinical responses to ECP therapy were paralleled by an increase of CD4+CD25hiFoxP3+ regulatory T cells and a decrease of T(EMRA) (CD3+CD8+CD45RA+CD62L+ effector memory T cells) as well as of NKT cells. In summary, immunomonitoring of T cell subsets can elucidate the mechanism of action in ECP.
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cine V, University Clinic Heidelberg, Heidelberg, Germany
Key words: Extracorporeal photopheresis (ECP), GvHD, solid organ rejection (SOT), regulatory T cells, effector T cells, NK cells
INTRODUCTION Acute graft-versus-host disease (aGvHD) after allogeneic stem cell transplantation (ASCT) and graft rejection after solid organ transplantation (SOT) presenting as bronchiolitis obliterans syndrome (BOS) constitute lifethreatening complications.[1-3] Recently, extracorporeal photopheresis (ECP)
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has been used for second-line treatment of GvHD and BOS. It is one of the most considered options for the treatment of steroid-refractory aGvHD.[4]
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However immunological data supporting the efficacy are limited.[5-7]
two patients.
MATERIALS AND METHODS
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Patient #1:
A 65 y/o male patient with acute lymphoblastic leukemia (B-ALL) developed a steroid-refractory aGvHD grade IV of the gut 42 days after ASCT. Despite therapy with steroids (2mg/kg/d, 7d) and calcineurin inhibitor he suffered
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from extensive diarrhea histologically compatible with GvHD and negative for microbiological results (Fig.1). Patient #2:
A 33 y/o male patient was double-lung transplanted for cystic fibrosis and presented with BOS 42 months later. Despite triple-drug therapy comprising
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Here we present clinical results and elaborated immunological analysis of
steroids, a calcineurin inhibitor as well as a cell-cycle inhibitor the patient’s lung function deteriorated (Fig. 2). Extracorporeal photopheresis ECP was performed using the UVAR XTS device (Therakos, Exton, PA). In both patients an intensive ECP regimen with two ECP treatments weekly up to 12 weeks was started, followed by tapering out every second week, every third week and monthly. Peripheral blood samples were taken before and in
the course of ECP therapy. Informed consent was obtained from both patients. Flow cytometry Multicolor flow cytometry was performed on a LSRII device using the following
antibodies
anti-CD3*PE,
anti-CD4*FITC,
anti-CD8*Per-CP,
anti-
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CD16*APC-Cy7, anti-CD19*PE, anti-CD20*Per-CP, anti-CD21*APC, antiCD27*FITC, anti-CD45RA*APC, anti-CCR7*PE-Cy7, anti-FOXP3*PE, anti(BD,
Heidelberg,
Germany),
anti-CD25*Alexa647,
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NKG2D*PE
anti-
end, Fell, Germany) and NEAR*APC/Cy7 (Invitrogen, Darmstadt, Germany) (Fig.1, 2).
RESULTS
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Patient #1:
Immunologically we observed a CD19+CD20+CD21+ B cell frequency below normal with 3.4% and a very low CD4+ T cell level with 3.8%, with a
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CD4/CD8 ratio of 0.3, while the CD8+ T cell population ranged about 12.1% with strongly increased values for CD3+CD8+CCR7-CD45RA+ T(EMRA) cells of 60.1% and elevated values for CD16+CD56+CD3+NKG2D+ NKT cells of 11.0%.
Within the first 28 days of intensive ECP treatment the average stool volume
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CD28*PacificBlue, anti-CD56*Alexa488, anti-CD62L*PacificBlue (BioLeg-
declined dramatically and steroids were tapered to 25% of the initial dosis. Immunologically
we
CD4+CD25hiFoxP3+
determined
Tregs
from
a
prominent 9.4%
to
increase 22.1%
of and
CD3+CD8+CCR7+CD45RA-/+ T(CM)/ T(naïve) cells from 0.9% to 11.4% and 15.0% to 43.1% respectively. In contrast T(EMRA) cells decreased from 60.1% to 30.5% and NKT cells from 11.0% to 1.6%. After 218 days of ECP treatment the patient showed a stable clinical situation with normal stool volume and consistence without steroids. The analysis
of lymphocytes showed an increase of NK cells from initially 15.5% up to 39.8% and of CD4+ T cells up to 16.4%. The initial decrease of CD45RA+CD8+ T(EMRA) cells and NKT cells persisted (Fig. 1). The patient remained in complete remission of ALL and GvHD, is alive and well 2.5 years after ASCT.
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Patient #2: At begin of ECP therapy the patient presented with BOS grade III with a vital
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capacity (VC) of 5,100 ml (initial BOS grade 1: VC of 5,940 ml), a forced ex-
FEV1 of 4,930 ml) and exertional dyspnea (Fig. 2). Immunologically the patient presented with a B cell frequency below normal with 2.6%, with low levels for NK/NKT cells about 8.1% and 0.7% and low levels for CD4+ Tregs
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with 1.2%. CD4+ T cells were normal with 31.4% and CD8+ T cells were elevated with 44.8% and a CD4/CD8 ratio of 0.7 going along with elevated levels for T(EMRA) cells of 58.8% and elevated levels for CD62L expres-
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sion.
Within the first 28 days of intensive ECP treatment the FEV1 showed no further decline. Immunologically NK /NKT cells and CD4+ Treg cells remained low, whereas the naïve CD8+ T cells increased from 27.8% to 45.2%. T(EMRA)/ T(EM) cells decreased slightly with a clear reduction of CD62L
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piratory volume in one second (FEV1) of 2,120 ml (initial BOS grade 1:
expression. After 106 days of ECP treatment the lung function measured by spirometry showed a stabilisation with a VC of 5,070 ml and FEV1 of 2,160 ml. These clinical findings paralleled with an increase of CD4+ Treg cells up to 6.7% and of naïve T cells up to 55.6%. The NKT cells still remained unchanged low, while T(EMRA) /T(EM) cells further decreased down to 36.6% and to 3.2% (Fig. 2). Furthermore the lung function even improved to a VC of 5,360 ml and to a FEV1 of 2,410 ml 15 weeks later while the immunosuppressive therapy remained unchanged.
DISCUSSION Interaction of 8-methoxypsoralen (8-MOP) photo-sensibilized wight blood cells (WBC) and ultraviolet A (UVA) light is one putative mechanism of action for ECP therapy. ECP might be based on the treatment of about 10% of total peripheral blood
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leukocytes with the photoactive drug 8-MOP, which leads upon UVA light exposure to crosslinking of pyrimidine bases followed by apoptosis of the [16-18]
ECP was reported to have an impact on both aGvHD and
The pathophysiology of GvHD and rejection compromise the following three phases
[8-10]
: phase 1: activation of antigen-presenting cells (APCs), phase
2: activation, differentiation and migration of T cell eventually developing
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their full effector function in phase 3.
In the first of these three phases dendritic cells (DCs) constitute the most professional APCs which can exert a duo-fold role. On one hand DCs might
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elicit alloreactive or even clonal antigen-specific T cell responses. On the other hand so called tolerogenic DCs might rather exert a desirable immunomodulatory or even immunosuppressive effect on T cells. This might play an important role in tissue damage through activated CD45RA+ T effector memory cells termed T(EMRA) cells in GvHD. [25]
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rejection of SOT.
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treated cells
Within 24 to 48 hours after ECP treatment leukocytes exposed to 8-MOP and UVA light undergo apoptosis. In an in vitro experiment ingestion of apoptotic cells by DCs lead to development of tolerogenic DCs [20] In addition
to this effect on DCs ECP interfered with the production and secretion of proinflammatory cytokines by DCs.
[24]
Moreover ECP was reported to affect
proliferating cells while preserving or even expanding regulatory T cells. [19-22]
In a murine model ECP enhanced the production of IL-10, which could
down-regulate the T helper 1 cell response and also might lead to an increased frequency of Treg cells. [17],[23] Both tolerogenic DCs and increased numbers of Tregs might suppress the proliferation and proper function of CD8+ effector T cells which are considered to constitute, in concert with NKT cells, the cellular drivers in the proc-
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ess of aGvHD and rejection of solid organ allografts.[11-14] Regarding our immunological data from the patients before ECP therapy we
[15]
Furthermore a shift in the CD8+ T cell compartment could be de-
tected in favor of effector T cells when compared to low levels for naïve/central memory T cells (Fig. 1, 2). Moreover, a difference in NKT cell frequencies was observed in both patients: while NKT cells were increased up
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to 11.0% in the patient with aGvHD, the NKT cell level was low with 0.7% in the patient with chronic allograft rejection, as described for chronic GvHD. [15] Furthermore, the B cell population in both patients ranged below normal val-
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ues, maybe due to immunosuppressive therapy.
Both patients responded clinically to ECP therapy. During ECP therapy dynamic changes in lymphocyte subpopulations in the effector phase were observed: In the patient with aGvHD an obvious decrease of effector T cells was observed in parallel with a clear increase of Treg cells under intensive
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cells.
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have seen an imbalance of CD8+ over CD4+ T cells in favor of CD8+ T
ECP therapy in the first month. Furthermore the frequency of different T cell populations as Treg and T (EMRA) cells came down to base level. In the other patient with chronic allograft rejection a rather slow but steady decrease of effector T cells was detected in parallel with an increase of Treg cells and naïve T cell over the whole time of ECP therapy starting at an exceptional low level. Eventually this dynamic lead to normal ratios of T cell subpopulations.
Contrary to the slow changes in allograft rejection an obvious decrease of CD8+ T cells expressing highly the homing receptor L-selectin (CD62L) was observed within the first month of ECP therapy. This may be of interest for the clinical response in BOS as blood vessels that are similar to high endothelial venules may appear in chronically inflamed tissue increasing lympho-
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cyte entry into the tissue however down regulation of lymphocytes expressing L-selectin might counteract the homing.
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Our findings in immune monitoring are in concordance with previous publica-
therapy correlates along with a normalization or even increase of Treg cells in patients with GvHD
[26-28]
and with BOS
[29]
. Moreover the normalization of
previously imbalanced central versus effector memory cell populations was
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associated with the clinical response in patients with chronic GvHD [30-31]. In summary while we are lacking a valid and predictive marker for GvHD and graft rejection in SOT, immunomonitoring of lymphocytes subsets, as per-
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formed in this pilot study, might be of help for the identification of patients responding to ECP.
DECLARATION OF INTERESTS
The authors declare that they have no conflicts of interest with regard to
this work.
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tions: Several different studies demonstrated that clinical response of ECP
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CD4_ and CD8_Central Memory Cells Associated with ChronicGraft-
FIGURE LEGENDS Fig. 1: (A) ECP treatment of a patient with life-threatening steroidrefractory aGvHD grade IV of the gut. The first blood sample was drawn from the patient before initiation of ECP treatment when the patient suffered from severe diarrhea. ECP treatments are marked by vertical bars. ECP
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therapy was eventually stopped, when gut function had been stabilized and the patient produced normal stools. (B-F) Immunomonitoring during ECP
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treatment. (B) Dynamics of all lymphocytes (squares): absolute cell num-
(squares): Percentage of all cells in the lymphocyte gate. (D) Dynamics of the NK and NK-T cell subsets: Lymphocytes were separated into CD3- and CD3+ subsets. NK cells (squares) were detected in the CD3-, and NK-T cells
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(diamonds) in the CD3+ compartment based on a triple positive labeling of CD16, CD56 and NKG2D. Percentage of all cells in the CD3+ or CD3- lymphocyte gate. (E) Dynamics of the CD4+CD25hiFoxP3+Treg (diamonds):
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Percentage of all CD4+ T cells (F) Dynamics of CD8+ T cell subsets: CD8+ cells were separated into four subsets (naive, CM, EM, EMRA) based on CD45RA and CCR7 expression. Naive T cells CD3+CD8+CCR7+CD45RA+ (closed circles), central memory T cells (CM) CD3+CD8+CCR7+CD45RA(triangles), effector memory T cells (EM) CD3+CD8+CCR7-CD45RA- (dia-
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bers (cells/µl) (C) Dynamics of CD19+CD20+CD21+ B cell frequency
monds) and CD3+CD8+CCR7-CD45RA+ effector memory T cells (EMRA)
(squares) are depicted as percentage of all CD8+ T cells.
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Fig. 2: (A) ECP treatment of a patient with life-threatening graft rejection presenting as BOS grade III after double-lung transplantation. Forced expiratory volume in one second (FEV1) measured by spirometry before and during ECP treatment. The first blood sample was drawn from the patient before initiation of ECP treatment, the last time point when lung
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function was already stabilized. The frequency of ECP treatments was decreased. (B-G) Immunomonitoring during ECP treatment. (B) Dynamics
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of all lymphocytes (squares) absolute cell numbers (cells/µl) (C, D) Dynam-
and (C) percentage of all CD4+ T cells.
(E, F, G) Dynamics of CD8+ T cell subsets: (G) CD8+ cells were separated into four subsets (naive, CM, EM, EMRA) based on CD45RA and CCR7 ex-
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pression. (E, F) Naive T cells CD3+CD8+CCR7+CD45RA+ (closed circles), central memory T cells (CM) CD3+CD8+CCR7+CD45RA- (triangles), effector memory
T
cells
(EM)
CD3+CD8+CCR7-CD45RA-
(diamonds)
and
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CD3+CD8+CCR7-CD45RA+ effector memory T cells (EMRA) (squares) are depicted as (F) percentage of all CD8+ T cells and (E) percentage of CD62L+ cells in the four different subsets of CD8+ T cells.
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ics of the Treg (diamonds): (D) Definition of the CD4+CD25hiFoxP3+ Treg
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Modulation of lymphocyte subpopulations by extracorporeal photo-pheresis in patients with acute GvHD or graft rejection Katrin Lorenz, Katharina Rommel, Jiju Mani, Nan Jin, Inken Hilgendorf, Anthony D. Ho, Mathias Freund, Michael Schmitt, Anita Schmitt doi: 10.3109/10428194.2014.931956 Leuk Lymphoma Downloaded from informahealthcare.com by Baylor University on 06/15/14 For personal use only.
ABSTRACT Extracorporeal photopheresis (ECP) constitutes a promising treatment for patients with steroid-refractory acute graft-versus-host disease (aGvHD) after allogeneic stem cell transplantation and for patients with graft rejection after solid organ transplantation (SOT). There is an increasing body of evidence that modulation of lymphocyte subsets might play a crucial role in the mechanism of action in ECP. We therefore analyzed immunological effects concomitantly to clinical findings in patients under ECP therapy with multicolor flow cytometry. In a patient with steroid-refractory aGvHD and a patient with progressive bronchiolitis obliterans syndrome (BOS) after double-lung transplantation, clinical responses to ECP therapy were paralleled by an increase of CD4+CD25hiFoxP3+ regulatory T cells and a decrease of T(EMRA) (CD3+CD8+CD45RA+CD62L+ effector memory T cells) as well as of NKT cells. In summary, immunomonitoring of T cell subsets can elucidate the mechanism of action in ECP.
© 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
Modulation of lymphocyte subpopulations by extracorporeal photo-pheresis in patients with acute GvHD or graft rejection
Katrin Lorenz2, Katharina Rommel1, Jiju Mani2, Nan Jin2, Inken Hilgendorf1,
1
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Anthony D. Ho2, Mathias Freund1, Michael Schmitt2, Anita Schmitt2
Dept. Internal Medicine III, University Clinic Rostock, Rostock, Germany 2Internal Medi-
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Corresponding author: Anita Schmitt, Cellular Immunotherapy, Dept. Internal Medicine V, University Clinic Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg. Germany. Tel. +49-6221-56-35829. FAX. +49-6221-56-5740. E-Mail: [email protected] ABSTRACT
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Extracorporeal photopheresis (ECP) constitutes a promising treatment for patients with steroid-refractory acute graft-versus-host disease (aGvHD) after allogeneic stem cell transplantation and for patients with graft rejection after solid organ transplantation (SOT). There is an increasing body of evidence that modulation of lymphocyte subsets might play a crucial role in the mechanism of action in ECP. We therefore analyzed immunological effects concomitantly to clinical findings in patients under ECP therapy with multicolor flow cytometry. In a patient with steroid-refractory aGvHD and a patient with progressive bronchiolitis obliterans syndrome (BOS) after double-lung transplantation, clinical responses to ECP therapy were paralleled by an increase of CD4+CD25hiFoxP3+ regulatory T cells and a decrease of T(EMRA) (CD3+CD8+CD45RA+CD62L+ effector memory T cells) as well as of NKT cells. In summary, immunomonitoring of T cell subsets can elucidate the mechanism of action in ECP.
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cine V, University Clinic Heidelberg, Heidelberg, Germany
Key words: Extracorporeal photopheresis (ECP), GvHD, solid organ rejection (SOT), regulatory T cells, effector T cells, NK cells
INTRODUCTION Acute graft-versus-host disease (aGvHD) after allogeneic stem cell transplantation (ASCT) and graft rejection after solid organ transplantation (SOT) presenting as bronchiolitis obliterans syndrome (BOS) constitute lifethreatening complications.[1-3] Recently, extracorporeal photopheresis (ECP)
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has been used for second-line treatment of GvHD and BOS. It is one of the most considered options for the treatment of steroid-refractory aGvHD.[4]
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However immunological data supporting the efficacy are limited.[5-7]
two patients.
MATERIALS AND METHODS
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Patient #1:
A 65 y/o male patient with acute lymphoblastic leukemia (B-ALL) developed a steroid-refractory aGvHD grade IV of the gut 42 days after ASCT. Despite therapy with steroids (2mg/kg/d, 7d) and calcineurin inhibitor he suffered
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from extensive diarrhea histologically compatible with GvHD and negative for microbiological results (Fig.1). Patient #2:
A 33 y/o male patient was double-lung transplanted for cystic fibrosis and presented with BOS 42 months later. Despite triple-drug therapy comprising
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Here we present clinical results and elaborated immunological analysis of
steroids, a calcineurin inhibitor as well as a cell-cycle inhibitor the patient’s lung function deteriorated (Fig. 2). Extracorporeal photopheresis ECP was performed using the UVAR XTS device (Therakos, Exton, PA). In both patients an intensive ECP regimen with two ECP treatments weekly up to 12 weeks was started, followed by tapering out every second week, every third week and monthly. Peripheral blood samples were taken before and in
the course of ECP therapy. Informed consent was obtained from both patients. Flow cytometry Multicolor flow cytometry was performed on a LSRII device using the following
antibodies
anti-CD3*PE,
anti-CD4*FITC,
anti-CD8*Per-CP,
anti-
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CD16*APC-Cy7, anti-CD19*PE, anti-CD20*Per-CP, anti-CD21*APC, antiCD27*FITC, anti-CD45RA*APC, anti-CCR7*PE-Cy7, anti-FOXP3*PE, anti(BD,
Heidelberg,
Germany),
anti-CD25*Alexa647,
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NKG2D*PE
anti-
end, Fell, Germany) and NEAR*APC/Cy7 (Invitrogen, Darmstadt, Germany) (Fig.1, 2).
RESULTS
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Patient #1:
Immunologically we observed a CD19+CD20+CD21+ B cell frequency below normal with 3.4% and a very low CD4+ T cell level with 3.8%, with a
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CD4/CD8 ratio of 0.3, while the CD8+ T cell population ranged about 12.1% with strongly increased values for CD3+CD8+CCR7-CD45RA+ T(EMRA) cells of 60.1% and elevated values for CD16+CD56+CD3+NKG2D+ NKT cells of 11.0%.
Within the first 28 days of intensive ECP treatment the average stool volume
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CD28*PacificBlue, anti-CD56*Alexa488, anti-CD62L*PacificBlue (BioLeg-
declined dramatically and steroids were tapered to 25% of the initial dosis. Immunologically
we
CD4+CD25hiFoxP3+
determined
Tregs
from
a
prominent 9.4%
to
increase 22.1%
of and
CD3+CD8+CCR7+CD45RA-/+ T(CM)/ T(naïve) cells from 0.9% to 11.4% and 15.0% to 43.1% respectively. In contrast T(EMRA) cells decreased from 60.1% to 30.5% and NKT cells from 11.0% to 1.6%. After 218 days of ECP treatment the patient showed a stable clinical situation with normal stool volume and consistence without steroids. The analysis
of lymphocytes showed an increase of NK cells from initially 15.5% up to 39.8% and of CD4+ T cells up to 16.4%. The initial decrease of CD45RA+CD8+ T(EMRA) cells and NKT cells persisted (Fig. 1). The patient remained in complete remission of ALL and GvHD, is alive and well 2.5 years after ASCT.
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Patient #2: At begin of ECP therapy the patient presented with BOS grade III with a vital
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capacity (VC) of 5,100 ml (initial BOS grade 1: VC of 5,940 ml), a forced ex-
FEV1 of 4,930 ml) and exertional dyspnea (Fig. 2). Immunologically the patient presented with a B cell frequency below normal with 2.6%, with low levels for NK/NKT cells about 8.1% and 0.7% and low levels for CD4+ Tregs
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with 1.2%. CD4+ T cells were normal with 31.4% and CD8+ T cells were elevated with 44.8% and a CD4/CD8 ratio of 0.7 going along with elevated levels for T(EMRA) cells of 58.8% and elevated levels for CD62L expres-
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sion.
Within the first 28 days of intensive ECP treatment the FEV1 showed no further decline. Immunologically NK /NKT cells and CD4+ Treg cells remained low, whereas the naïve CD8+ T cells increased from 27.8% to 45.2%. T(EMRA)/ T(EM) cells decreased slightly with a clear reduction of CD62L
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piratory volume in one second (FEV1) of 2,120 ml (initial BOS grade 1:
expression. After 106 days of ECP treatment the lung function measured by spirometry showed a stabilisation with a VC of 5,070 ml and FEV1 of 2,160 ml. These clinical findings paralleled with an increase of CD4+ Treg cells up to 6.7% and of naïve T cells up to 55.6%. The NKT cells still remained unchanged low, while T(EMRA) /T(EM) cells further decreased down to 36.6% and to 3.2% (Fig. 2). Furthermore the lung function even improved to a VC of 5,360 ml and to a FEV1 of 2,410 ml 15 weeks later while the immunosuppressive therapy remained unchanged.
DISCUSSION Interaction of 8-methoxypsoralen (8-MOP) photo-sensibilized wight blood cells (WBC) and ultraviolet A (UVA) light is one putative mechanism of action for ECP therapy. ECP might be based on the treatment of about 10% of total peripheral blood
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leukocytes with the photoactive drug 8-MOP, which leads upon UVA light exposure to crosslinking of pyrimidine bases followed by apoptosis of the [16-18]
ECP was reported to have an impact on both aGvHD and
The pathophysiology of GvHD and rejection compromise the following three phases
[8-10]
: phase 1: activation of antigen-presenting cells (APCs), phase
2: activation, differentiation and migration of T cell eventually developing
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their full effector function in phase 3.
In the first of these three phases dendritic cells (DCs) constitute the most professional APCs which can exert a duo-fold role. On one hand DCs might
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elicit alloreactive or even clonal antigen-specific T cell responses. On the other hand so called tolerogenic DCs might rather exert a desirable immunomodulatory or even immunosuppressive effect on T cells. This might play an important role in tissue damage through activated CD45RA+ T effector memory cells termed T(EMRA) cells in GvHD. [25]
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rejection of SOT.
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treated cells
Within 24 to 48 hours after ECP treatment leukocytes exposed to 8-MOP and UVA light undergo apoptosis. In an in vitro experiment ingestion of apoptotic cells by DCs lead to development of tolerogenic DCs [20] In addition
to this effect on DCs ECP interfered with the production and secretion of proinflammatory cytokines by DCs.
[24]
Moreover ECP was reported to affect
proliferating cells while preserving or even expanding regulatory T cells. [19-22]
In a murine model ECP enhanced the production of IL-10, which could
down-regulate the T helper 1 cell response and also might lead to an increased frequency of Treg cells. [17],[23] Both tolerogenic DCs and increased numbers of Tregs might suppress the proliferation and proper function of CD8+ effector T cells which are considered to constitute, in concert with NKT cells, the cellular drivers in the proc-
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ess of aGvHD and rejection of solid organ allografts.[11-14] Regarding our immunological data from the patients before ECP therapy we
[15]
Furthermore a shift in the CD8+ T cell compartment could be de-
tected in favor of effector T cells when compared to low levels for naïve/central memory T cells (Fig. 1, 2). Moreover, a difference in NKT cell frequencies was observed in both patients: while NKT cells were increased up
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to 11.0% in the patient with aGvHD, the NKT cell level was low with 0.7% in the patient with chronic allograft rejection, as described for chronic GvHD. [15] Furthermore, the B cell population in both patients ranged below normal val-
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ues, maybe due to immunosuppressive therapy.
Both patients responded clinically to ECP therapy. During ECP therapy dynamic changes in lymphocyte subpopulations in the effector phase were observed: In the patient with aGvHD an obvious decrease of effector T cells was observed in parallel with a clear increase of Treg cells under intensive
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cells.
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have seen an imbalance of CD8+ over CD4+ T cells in favor of CD8+ T
ECP therapy in the first month. Furthermore the frequency of different T cell populations as Treg and T (EMRA) cells came down to base level. In the other patient with chronic allograft rejection a rather slow but steady decrease of effector T cells was detected in parallel with an increase of Treg cells and naïve T cell over the whole time of ECP therapy starting at an exceptional low level. Eventually this dynamic lead to normal ratios of T cell subpopulations.
Contrary to the slow changes in allograft rejection an obvious decrease of CD8+ T cells expressing highly the homing receptor L-selectin (CD62L) was observed within the first month of ECP therapy. This may be of interest for the clinical response in BOS as blood vessels that are similar to high endothelial venules may appear in chronically inflamed tissue increasing lympho-
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cyte entry into the tissue however down regulation of lymphocytes expressing L-selectin might counteract the homing.
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Our findings in immune monitoring are in concordance with previous publica-
therapy correlates along with a normalization or even increase of Treg cells in patients with GvHD
[26-28]
and with BOS
[29]
. Moreover the normalization of
previously imbalanced central versus effector memory cell populations was
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associated with the clinical response in patients with chronic GvHD [30-31]. In summary while we are lacking a valid and predictive marker for GvHD and graft rejection in SOT, immunomonitoring of lymphocytes subsets, as per-
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formed in this pilot study, might be of help for the identification of patients responding to ECP.
DECLARATION OF INTERESTS
The authors declare that they have no conflicts of interest with regard to
this work.
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tions: Several different studies demonstrated that clinical response of ECP
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CD4_ and CD8_Central Memory Cells Associated with ChronicGraft-
FIGURE LEGENDS Fig. 1: (A) ECP treatment of a patient with life-threatening steroidrefractory aGvHD grade IV of the gut. The first blood sample was drawn from the patient before initiation of ECP treatment when the patient suffered from severe diarrhea. ECP treatments are marked by vertical bars. ECP
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therapy was eventually stopped, when gut function had been stabilized and the patient produced normal stools. (B-F) Immunomonitoring during ECP
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treatment. (B) Dynamics of all lymphocytes (squares): absolute cell num-
(squares): Percentage of all cells in the lymphocyte gate. (D) Dynamics of the NK and NK-T cell subsets: Lymphocytes were separated into CD3- and CD3+ subsets. NK cells (squares) were detected in the CD3-, and NK-T cells
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(diamonds) in the CD3+ compartment based on a triple positive labeling of CD16, CD56 and NKG2D. Percentage of all cells in the CD3+ or CD3- lymphocyte gate. (E) Dynamics of the CD4+CD25hiFoxP3+Treg (diamonds):
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Percentage of all CD4+ T cells (F) Dynamics of CD8+ T cell subsets: CD8+ cells were separated into four subsets (naive, CM, EM, EMRA) based on CD45RA and CCR7 expression. Naive T cells CD3+CD8+CCR7+CD45RA+ (closed circles), central memory T cells (CM) CD3+CD8+CCR7+CD45RA(triangles), effector memory T cells (EM) CD3+CD8+CCR7-CD45RA- (dia-
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bers (cells/µl) (C) Dynamics of CD19+CD20+CD21+ B cell frequency
monds) and CD3+CD8+CCR7-CD45RA+ effector memory T cells (EMRA)
(squares) are depicted as percentage of all CD8+ T cells.
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Fig. 2: (A) ECP treatment of a patient with life-threatening graft rejection presenting as BOS grade III after double-lung transplantation. Forced expiratory volume in one second (FEV1) measured by spirometry before and during ECP treatment. The first blood sample was drawn from the patient before initiation of ECP treatment, the last time point when lung
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function was already stabilized. The frequency of ECP treatments was decreased. (B-G) Immunomonitoring during ECP treatment. (B) Dynamics
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of all lymphocytes (squares) absolute cell numbers (cells/µl) (C, D) Dynam-
and (C) percentage of all CD4+ T cells.
(E, F, G) Dynamics of CD8+ T cell subsets: (G) CD8+ cells were separated into four subsets (naive, CM, EM, EMRA) based on CD45RA and CCR7 ex-
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pression. (E, F) Naive T cells CD3+CD8+CCR7+CD45RA+ (closed circles), central memory T cells (CM) CD3+CD8+CCR7+CD45RA- (triangles), effector memory
T
cells
(EM)
CD3+CD8+CCR7-CD45RA-
(diamonds)
and
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CD3+CD8+CCR7-CD45RA+ effector memory T cells (EMRA) (squares) are depicted as (F) percentage of all CD8+ T cells and (E) percentage of CD62L+ cells in the four different subsets of CD8+ T cells.
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ics of the Treg (diamonds): (D) Definition of the CD4+CD25hiFoxP3+ Treg
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