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Cytokine Profiles of Pre-Engraftment Syndrome after Single-Unit Cord Blood Transplantation for Adult Patients
Accepted Manuscript Title: Cytokine Profiles of Pre-Engraftment Syndrome after Single-Unit Cord Blood Transplantation for Adult Patients Author: Takaaki Konuma, Chisato Kohara, Eri Watanabe, Motoko Mizukami, Etsuko Nagai, Maki Oiwa-Monna, Susumu Tanoue, Masamichi Isobe, Seiko Kato, Arinobu Tojo, Satoshi Takahashi PII: DOI: Reference:
S1083-8791(17)30611-0 http://dx.doi.org/doi: 10.1016/j.bbmt.2017.07.020 YBBMT 54743
To appear in:
Biology of Blood and Marrow Transplantation
Received date: Accepted date:
27-4-2017 16-7-2017
Please cite this article as: Takaaki Konuma, Chisato Kohara, Eri Watanabe, Motoko Mizukami, Etsuko Nagai, Maki Oiwa-Monna, Susumu Tanoue, Masamichi Isobe, Seiko Kato, Arinobu Tojo, Satoshi Takahashi, Cytokine Profiles of Pre-Engraftment Syndrome after Single-Unit Cord Blood Transplantation for Adult Patients, Biology of Blood and Marrow Transplantation (2017), http://dx.doi.org/doi: 10.1016/j.bbmt.2017.07.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cytokine profiles of pre-engraftment syndrome after single-unit cord blood transplantation for adult patients
Running title: Cytokine profiles of PES after CBT
Takaaki Konuma1, Chisato Kohara1, Eri Watanabe2, Motoko Mizukami3, Etsuko Nagai3, Maki Oiwa-Monna1, Susumu Tanoue1, Masamichi Isobe1, Seiko Kato1, Arinobu Tojo1, and Satoshi Takahashi1.
1
Department of Hematology/Oncology, The Institute of Medical Science, The University of
Tokyo, Tokyo, Japan 2
Department of IMSUT Clinical Flow Cytometry Laboratory, The Institute of Medical
Science, The University of Tokyo, Tokyo, Japan 3
Department of Laboratory Medicine, The Institute of Medical Science, The University of
Tokyo, Tokyo, Japan
Corresponding
author:
Takaaki
Konuma,
M.D.,
Ph.D.,
Department
of
Hematology/Oncology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Tel: +81-3-3443-8111, Fax: +81-3-5449-5429, Email: [email protected]
1 Page 1 of 19
Highlights We examined the relationship between serum cytokine profiles and pre-engraftment syndrome (PES) in 44 adult patients who received cord blood transplantation (CBT). Serum levels of 21 cytokines were measured by multiplex bead assays using a flow cytometer. The elevations of interleukin (IL)-5 and IL-6 at 2 weeks were associated with PES. The conversion from the naïve to the memory phenotype of T cells at 4 and 8 weeks was observed in PES. Abstract Clinical manifestation of high grade fever and skin rash prior to neutrophil engraftment, which is called pre-engraftment syndrome (PES) or pre-engraftment immune reaction, has been frequently observed after cord blood transplantation (CBT). The pathophysiology of PES is poorly understood, but cytokine storm during the early phase of CBT is thought to be one of the main cause of PES. However, the cytokine profiles of PES after CBT are unclear. Therefore, we examined the relationship between serum cytokine profiles and PES in 44 adult patients who received CBT in our institute between February 2013 and June 2016. Serum levels of 21 cytokines, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-17F, IL-18, IL-21, IL-22, IL-23, IL-33, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α were measured by multiplex bead assays using a flow cytometer. The median time until the absolute neutrophil count was >0.5×109/L was 21 days (range, 15–41 days). The cumulative incidence of PES was 79.6% (95% confidence interval, 63.3% to 88.5%) at 60 days after CBT. Serum levels of IL-5 (P=0.009) and IL-6 (P=0.01) at 2 weeks were significantly higher in patients who developed PES compared with those who did not develop PES. The conversion from the naïve to the effector or central memory phenotype of T cells was observed in PES. These data indicate that elevations of IL-5 and IL-6 around the time of clinical manifestation may be possible biomarkers for PES after CBT.
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Keyword; pre-engraftment syndrome, pre-engraftment immune reaction, cord blood transplantation, cytokine, interleukin-5, interleukin-6
Introduction
Cord blood transplantation (CBT) is widely used for patients without human leukocyte antigen (HLA)-matched related or unrelated donors for adult patients. Although the lower incidence and severity of acute graft-versus-host disease (GVHD) is one of the most important advantages of CBT, a unique clinical manifestation with high grade fever and skin rash prior to neutrophil engraftment, which is called pre-engraftment syndrome (PES) or pre-engraftment immune reaction, has been described after CBT [1–11]. The pathophysiology of PES is poorly understood, but cytokine storm during the early phase of CBT is thought to be one of the main causes of PES. However, the cytokine profiles of PES after CBT are unclear. Moreover, because the diagnosis of PES is usually based on only clinical symptoms, identifying specific serum cytokines would clearly be a valuable diagnostic tool for PES. Therefore, we examined the relationship between the levels of 21 serum cytokines and PES in 44 adult CBT recipients.
Patients and methods
Patients and transplant procedures
Forty-nine patients who had been treated with single-unit CBT in our institute between February 2013 and June 2016 were included in this study. The characteristics of patients 3 Page 3 of 19
and CBT are shown in Table 1. One patient who received a second CBT was analyzed twice as a separate patient. All patients received a total body irradiation (TBI) or busulfan-based myeloablative conditioning regimen, which was defined according to the Center for International Blood and Marrow Transplant Research (CIBMTR) criteria [12]. The most common conditioning regimen was TBI 12 Gy, cyclophosphamide, and cytosine arabinoside with or without granulocyte-colony stimulating factor (G-CSF) for myeloid malignancies or lymphoid malignancies, respectively [13,14]. All patients received cyclosporine (CSP)-based GVHD prophylaxis. CSP was given intravenously every day starting day -1 at a dose of 3mg/kg/day, and was tapered beginning between 5 and 8 weeks after CBT. CSP dose was not adjusted for specific trough level. Methotrexate (MTX) was given intravenously on day1 at a dose of 15mg/m2 and on day 3 and 6 at a dose of 10mg/m2. Mycophenolate mofetil (MMF) was given orally at a dose of 30mg/kg/day from day 0 to 27. Conditioning regimen and GVHD prophylaxis were determined by the treating physician. All patients received G-CSF by intravenous infusion starting on day 1 until durable granulocyte recovery was achieved. Cord blood units were obtained from the Japan Cord Blood Bank Network. The institutional Review Board of the Institute of Medical Science, The University of Tokyo approved this study.
Serum cytokine analysis
Serum samples were collected at 2, 4, and 8 weeks after CBT and stored at -80°C until analysis. Serum levels of 21 cytokines, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-17F, IL-18, IL-21, IL-22, IL-23, IL-33, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-α, IFN-γ and tumor necrosis factor (TNF)-α, were measured using multiplex bead assays with a LEGENDplexTM Human Th Cytokine Panel (Cat. No. 740001, BioLegend) and Human Inflammation Panel (Cat. No. 4 Page 4 of 19
740118, BioLegend) according to the manufacturer’s instructions. Because the median intra-assay coefficient of variation was 3.5 % (range, 0.3-18.9 %) for 65 samples assayed in duplicate, the remaining samples were run in a single assay. For data analysis, levels of cytokines below the lower limit of detection were marked as the values of the halfway point between the lower limit of detection and zero.
T cell subsets analysis in peripheral blood
Peripheral blood samples were collected at 4 and 8 weeks after CBT. Peripheral blood mononuclear cells were obtained by LymphoprepTM (Axis-Shield PoC, Oslo, Norway) density gradient centrifugation and stored in cryopreservation medium Bambanker TM (Nippon Genetic Co. Ltd., Tokyo, Japan) until analysis. Cells were stained with Alexa Fluor 700-conjugated
anti
510-conjugated
anti
human human
CD3 CD4
(clone
UCHT-1,
(clone
BioLegend),
RPA-T4,
Brilliant
BioLegend),
Violet
fluorescein
isothiocyanate-conjugated anti human CD8 (clone RPA-T8, Tonbo Biosciences), allophycocyanin (APC)-Cy7-conjugated anti human CD45RA (clone HI100, BioLegend), Brilliant Violet 421-conjugated anti human CCR7 (clone G043H7, BioLegend) antibodies. The analysis was performed on a FACS Aria II (BD Bioscience).
Definitions
PES was defined as an unexplained fever >38.3°C not associated with documented infection and unresponsive to antimicrobial administrations and an unexplained erythematous skin rash resembling acute GVHD that occur prior to neutrophil engraftment, as previously described [1]. Neutrophil engraftment was defined as being achieved on the first of three consecutive days when the absolute neutrophil count was >0.5×10 9/L. Acute 5 Page 5 of 19
GVHD was graded by the treating physician according to the standard criteria [15]. Skin biopsies were not performed in any of the patients for diagnosis of PES and skin GVHD, although colon biopsies were performed to confirm diagnosis of gut GVHD in 3 patients.
Statistical analysis
Continuous variables were compared with the Mann–Whitney U test. Categorical variables were compared with the chi-square test. Estimation of the probabilities of PES and GVHD were based on a cumulative incidence method to accommodate competing risks, and the groups were compared using Gray’s test. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [16], a graphical user interface for the R 3.0.2 software program (R Foundation for Statistical Computing, Vienna, Austria) or GraphPad Prism 6 for Mac OS X (GraphPad Software, San Diego, California). All P-values were two-sided, and P-values of <0.05 were considered to be statistically significant.
Results
Engraftment, PES, and acute GVHD
All patients achieved neutrophil recovery after CBT. The median time until the absolute neutrophil count was >0.5×109/L was 21 days (range, 15–41 days). The cumulative incidences of PES, grade II to IV acute GVHD, and grade III to IV acute GVHD were 79.6% (95% confidence interval [CI], 63.3% to 88.5%), 75.0% (95% CI, 58.9% to 85.5%), and 14.0% (95% CI, 5.6% to 26.1%) at 60 days after CBT, respectively (Figure 1A). The median times to the development of PES, grade II to IV acute GVHD, and grade III to IV 6 Page 6 of 19
acute GVHD were 12 days (range, 4–18 days), 19.5 days (range, 10–50 days), and 43 days (range, 29–57 days), respectively. Because acute GVHD of the skin could not be distinguished from preceding PES in 18 patients, the median time to the development of grade II to IV acute GVHD was faster than the median time of neutrophil engraftment. No patient received corticosteroids for treatment for PES, although seven patients received more than 1 mg/kg of prednisolone (PSL) on a median of 36 days (range, 29–58 days) for treatment of severe acute GVHD. There were no significant differences in the baseline characteristics between patients who did and did not developed PES (Supplementary Table 1).
Serum cytokine profiles during the first 8 weeks after CBT
Five serum cytokines, IL-1β, IL-12p70, IL-23, IL-33, and IFN-α, were below the lower limit of detection in more than 80% of samples. Therefore, we did not evaluate these five cytokines for further analysis. The kinetics of each cytokine during the 8 weeks after CBT are shown in Table 2. Seven cytokine levels were significantly different during the course of CBT. Significant peak levels of IL-5, IL-6, and MCP-1 were observed at 2 weeks after CBT, whereas significant peak levels of IL-9, IL-18, and TNF-α were observed at 4 weeks after CBT (Supplementary Figure 1). All these cytokines levels returned to the probable baseline at 8 weeks after CBT. Serum levels of IL-5 (P=0.009) and IL-6 (P=0.01) at 2 weeks were significantly higher in patients who developed PES compared with those who did not develop PES (Figure 1B,1C). Serum levels of IL-5 (P=0.005), IL-6 (P=0.01), IL-8 (P=0.02), IL-9 (P=0.04), and IFN-γ (P=0.006) at 2 weeks were significantly higher in patients who developed grade II to IV acute GVHD compared with those who did not develop it (Supplementary Figure 2A–E). Serum levels of IL-4 (P=0.007), IL-6 (P=0.04), IL-10 (P=0.005), IL-21 (P=0.007), 7 Page 7 of 19
and TFN-α (P=0.02) at 2 weeks were significantly higher in patients who developed grade III to IV acute GVHD compared with those who did not develop it (Supplementary Figure 2F–J). All cytokine levels at 4 and 8 weeks after CBT were not significantly associated with PES, grade II to IV acute GVHD, or grade III to IV acute GVHD. The serum level of MCP-1 at 8 weeks was significantly lower in patients who received PSL compared with those who did not receive PSL (P<0.001). Among patients who developed PES (n=35), grade II to IV and grade III to IV acute GVHD occurred in 30 and 6 patients, respectively. We also evaluated the effects of cytokine profiles on development of grade II to IV or grade III to IV acute GVHD among patients who developed PES. Serum levels of IL-5 (P=0.006), IL-6 (P=0.04), and IFN-γ (P=0.02) at 2 weeks were significantly higher in patients who developed grade II to IV acute GVHD compared with those who did not develop it (Supplementary Figure 3A–C). Serum levels of IL-4 (P=0.01), IL-10 (P=0.01), IL-17F (P=0.02), IL-21 (P=0.003), and TFN-α (P=0.03) at 2 weeks were significantly higher in patients who developed grade III to IV acute GVHD compared with those who did not develop it (Supplementary Figure 3D–H). Because infections including bacterial blood stream infection (BSI) and cytomegalovirus (CMV) reactivation are commonly observed in patients following CBT, we assessed whether bacterial BSI and CMV reactivation affected the cytokine profiles after CBT. Bacterial BSI occurred in 8 patients (18%) during study period. Bacterial BSI was not associated with the cytokine levels, which were measured within 3 days after the onset of BSIs, at 2 weeks after CBT (Supplementary Table 2). CMV reactivation, which was evaluated by an antigenemia assay using the monoclonal antibodies C10/C11, occurred in 35 patients (80%) during study period. CMV reactivation was not associated with the cytokine levels, which were measured within 3 days after CMV reactivation, at 4 and 8 weeks after CBT (Supplementary Table 2). 8 Page 8 of 19
T cell subsets during the first 8 weeks after CBT
In order to clarify the balance of naïve, central, and effector memory cells in patients who developed PES, we evaluated the expression of CD45RA and CCR7 on CD3+CD4+ and CD3+CD8+ T cells in 24 patients at 4 and 8 weeks after CBT (Figure 2A). At 4 weeks after CBT, central memory (CD45RA-, CCR7+) CD4+ T cells were significantly higher and CD45RA+ effector memory (CD45RA+, CCR7-) CD4+ T cells were significantly lower in patients who developed PES compared with those who did not develop PES (Figure 2B,2C). At 8 weeks after CBT, naïve (CD45RA+, CCR7+) CD4+ and CD8+ T cells were significantly lower and effector memory (CD45RA-, CCR7-) CD4+ and CD8+ T cells were significantly higher in patients who developed PES compared with those who did not develop PES (Figure 2D,E). As for grade II to IV acute GVHD, conversion from the naïve to the effector memory phenotype was observed only in CD4+ T cells at 8 weeks after CBT (Figure 3A-D). PSL treatment did not affect the balance of naïve, central, and effector memory cells on CD4+ and CD8+ T cells at 8 weeks after CBT. We assessed whether the peripheral blood absolute lymphocyte and eosinophil counts (ALC and AEC, respectively) were associated with PES. However, ALC and AEC at 4 and 8 weeks after CBT were not significantly associated with PES, grade II to IV acute GVHD, or grade III to IV acute GVHD (Supplementary Figure 4).
Discussion
Several studies have demonstrated that PES is frequently observed after single and double unit CBT in pediatric and adult patients [1–11]. Although the incidence of PES after CBT has been reported to range from 20% to 78%, it depended on the conditioning 9 Page 9 of 19
regimen, GVHD prophylaxis, and the definition used. In our study, no patient received corticosteroids in GVHD prophylaxis or antithymocyte globulin in the conditioning regimen, and CSP with MTX was commonly administered for GVHD prophylaxis. The incidence of PES in our study was 79.6%, using the definition of PES by Patel et al. [1]. On the other hand, using the definition of PES by Kishi et al. [2], the incidence of PES decreased to 63.6% in our cohort, which was lower than that in the previous report by Kishi et al. [2]. The pathophysiology of PES is poorly understood. However, cytokine storm during the early phase of CBT is thought to be one of the main causes of PES. Morita–Hoshi et al. examined the expression profile of serum proteins using a surface-enhanced
laser
desorption/ionization
time-of-flight
mass
spectroscopy
(SELDI-TOF MS) system in CBT recipients and found that serum amyloid A (SAA) was a candidate biomarker for PES after CBT [3]. In the present study, we investigated 21 serum cytokine levels during the first 8 weeks after CBT to clarify the relationship between serum cytokine levels and PES. Our data showed that dynamic changes in cytokine profiles after CBT were possibly caused by the conditioning regimen and alloimmune reactions. Importantly, elevations of IL-5 and IL-6 at 2 weeks after CBT were significantly associated with PES after CBT. IL-5 is one of the Th2 type cytokines and is a major activator of eosinophils. IL-6 is one of the proinflammatory cytokines and plays an important role in various events during inflammation. In addition, IL-6 is induced by SAA, although IL-6 alone does not induce SAA in vitro [17]. Several studies have demonstrated that levels of IL-5 [18–20] and IL-6 [20–23] are increased in patients with engraftment syndrome and acute GVHD. In addition, the addition of MTX to calcineurin inhibitor has been reported to reduce the incidence of PES after CBT [4, 24]. Terakura et al. also reported that the relative risk of grade II to IV and grade III to IV acute GVHD was significantly lower in CBT recipients receiving calcineurin inhibitor and MTX compared with those receiving calcineurin inhibitor and MMF for GVHD prophylaxis [25]. Interestingly, our data showed 10 Page 10 of 19
that significant suppression of IL-5 and IL-6 was observed by addition of MTX for GVHD prophylaxis. Therefore, GVHD prophylaxis might be associated with the development of PES after CBT. These data suggested the possibility that a similar mechanism might be involved in the development of PES and acute GVHD, although PES is a syndrome that is distinguished from acute GVHD based on onset timing and clinical manifestations. The specific subsets, naïve, central, and effector memory phenotypes, of CD4+ and CD8+ T cells have been reported to play an important role in the development of acute and chronic GVHD [26–28], but their role in PES after CBT is unclear. Matsuno et al. reported that rapid conversion from naïve to memory T cells on both CD4+ and CD8+ T cells was observed in patients who developed PES at 2 weeks after CBT [5]. However, we could not evaluate whether T cells converted to different subsets at 2 weeks after CBT, because of low cell numbers. Meanwhile, our data showed a significant elevation of central memory CD4+ T cells at 4 weeks and elevation of effecter memory CD4+ and CD8+ T cells at 8 weeks in patients with PES after CBT. Importantly, conversion from the naïve to the memory phenotype of T cells was observed in PES. However, although regulation of these T cell subsets might be partially dependent on several cytokines and prolonged alloantigen stimulation, further analysis will be important for understanding the aberrant immunological status in PES. Our study had several limitations. First, this study was based on data from a single-institute in Japan, and the number of patients was small. However, all patients were adults with hematological malignancies and were treated with single-unit CBT following myeloablative conditioning regimens and a cyclosporine-based GVHD prophylaxis. Second, the incidence of grade II to IV acute GVHD might have been overestimated, because acute GVHD of the skin could not be distinguished from preceding PES in 18 patients in our cohort. Therefore, immunological status including serum cytokines and T cell subsets was quite similar between PES and grade II to IV acute GVHD. Because it is 11 Page 11 of 19
difficult to completely separate the diagnosis of PES and acute GVHD, it remains controversial whether a similar immunological profile is involved in the development of PES and acute GVHD. In conclusion, our data demonstrated that elevations of IL-5 and IL-6 around the time of clinical manifestation of high grade fever and skin rash were associated with PES after CBT. Therefore, IL-5 and IL-6 may be possible biomarkers for PES after CBT.
Acknowledgements
The authors thank all of the physicians and staff at the hospital and the cord blood bank in Japan for their help in this study. This work was supported in part by grants-in-aid from Japanese Society for the Promotion of Science (JSPS), and Takeda Science Foundation.
Conflict of Interest
The authors declare no competing financial interests.
References
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Bone Marrow Transplant. 2010;45:1594-1601. 4. Narimatsu H, Terakura S, Matsuo K, et al. Short-term methotrexate could reduce early immune reactions and improve outcomes in umbilical cord blood transplantation for adults. Bone Marrow Transplant. 2007;39:31-39. 5. Matsuno N, Yamamoto H, Watanabe N, et al. Rapid T-cell chimerism switch and memory T-cell expansion are associated with pre-engraftment immune reaction early after cord blood transplantation. Br J Haematol. 2013;160:255-258. 6. Frangoul H, Wang L, Harrell FE Jr, Ho R, Domm J. Preengraftment syndrome after unrelated cord blood transplant is a strong predictor of acute and chronic graft-versus-host disease. Biol Blood Marrow Transplant. 2009;15:1485-1488. 7. Uchida N, Wake A, Nakano N, et al. Mycophenolate and tacrolimus for graft-versus-host disease prophylaxis for elderly after cord blood transplantation: a matched pair comparison with tacrolimus alone. Transplantation. 2011;92:366-371. 8. Wang X, Liu H, Li L, et al. Pre-engraftment syndrome after unrelated donor umbilical cord blood transplantation in patients with hematologic malignancies. Eur J Haematol. 2012;88:39-45. 9. Kanda J, Kaynar L, Kanda Y, et al. Pre-engraftment syndrome after myeloablative dual umbilical cord blood transplantation: risk factors and response to treatment. Bone Marrow Transplant. 2013;48:926-931. 10. Park M, Lee SH, Lee YH, et al. Pre-engraftment syndrome after unrelated cord blood transplantation: a predictor of engraftment and acute graft-versus-host disease. Biol Blood Marrow Transplant. 2013;19:640-646. 11. Brownback KR, Simpson SQ, McGuirk JP, et al. Pulmonary manifestations of the pre-engraftment syndrome after umbilical cord blood transplantation. Ann Hematol. 2014 May;93(5):847-54. 12. Giralt S, Ballen K, Rizzo D, et al. Reduced-intensity conditioning regimen workshop: 13 Page 13 of 19
defining the dose spectrum. Report of a workshop convened by the center for international blood and marrow transplant research. Biol Blood Marrow Transplant. 2009;15:367-369. 13. Konuma T, Kato S, Ooi J, et al. Single-unit cord blood transplantation after granulocyte colony-stimulating
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cyclophosphamide. Leuk Lymphoma. 2015;56:1148-1150. 15. Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant. 1995;15:825-828. 16. Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transplant. 2013;48:452-458. 17. De Buck M, Gouwy M, Wang JM, et al. The cytokine-serum amyloid A-chemokine network. Cytokine Growth Factor Rev. 2016;30:55-69. 18. Imoto S, Oomoto Y, Murata K, et al. Kinetics of serum cytokines after allogeneic bone marrow transplantation: interleukin-5 as a potential marker of acute graft-versus-host disease. Int J Hematol. 2000;72:92-97. 19. Jordan WJ, Brookes PA, Szydlo RM, Goldman JM, Lechler RI, Ritter MA. IL-13 production by donor T cells is prognostic of acute graft-versus-host disease following unrelated donor stem cell transplantation. Blood. 2004;103:717-724. 20. Fujii N, Hiraki A, Aoe K, et al. Serum cytokine concentrations and acute graft-versus-host disease after allogeneic peripheral blood stem cell transplantation: concurrent measurement of ten cytokines and their respective ratios using cytometric bead array. Int J Mol Med. 2006;17:881-885. 14 Page 14 of 19
21. Schots R, Kaufman L, Van Riet I, et al. Proinflammatory cytokines and their role in the development of major transplant-related complications in the early phase after allogeneic bone marrow transplantation. Leukemia. 2003;17:1150-1156. 22. Malone FR, Leisenring WM, Storer BE, et al. Prolonged anorexia and elevated plasma cytokine levels following myeloablative allogeneic hematopoietic cell transplant. Bone Marrow Transplant. 2007;40:765-772. 23. Khandelwal P, Mellor-Heineke S, Rehman N, et al. Cytokine Profile of Engraftment Syndrome in Pediatric Hematopoietic Stem Cell Transplant Recipients. Biol Blood Marrow Transplant. 2016;22:690-697. 24. Iguchi A, Terashita Y, Sugiyama M, et al. Graft-versus-host disease (GVHD) prophylaxis by using methotrexate decreases pre-engraftment syndrome and severe acute GVHD, and accelerates engraftment after cord blood transplantation. Pediatr Transplant. 2016;20:114-119. 25. Terakura S, Wake A, Inamoto Y, et al. Exploratory research for optimal GvHD prophylaxis after single unit CBT in adults: short-term methotrexate reduced the incidence of severe GvHD more than mycophenolate mofetil. Bone Marrow Transplant. 2017;52:423-430. 26. Mohty M, Blaise D, Faucher C, et al. Inflammatory cytokines and acute graft-versus-host disease after reduced-intensity conditioning allogeneic stem cell transplantation. Blood. 2005;106:4407-4411. 27. Yamashita K, Choi U, Woltz PC, et al. Severe chronic graft-versus-host disease is characterized by a preponderance of CD4(+) effector memory cells relative to central memory cells. Blood. 2004;103:3986-3988. 28. D'Asaro M, Dieli F, Caccamo N, Musso M, Porretto F, Salerno A. Increase of CCR7CD45RA+ CD8 T cells (T(EMRA)) in chronic graft-versus-host disease. Leukemia. 2006;20:545-547. 15 Page 15 of 19
Figure 1. Cumulative incidences of PES and acute GVHD after CBT (A). Serum levels of IL-5 and IL-6 after CBT according to the development of PES (B,C) and GVHD prophylaxis (D,E). Figure 2. Characteristics of CD4+ and CD8+ T cell subsets after CBT according to the development of PES. Figure 3. Characteristics of CD4+ and CD8+ T cell subsets after CBT according to the development of grade II to IV acute GVHD.
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Table 1. Characteristics of the patients, cord blood units, and transplantations Entire cohort Number of CBTs 44 Age at CBT, median (range), years 47 (21-68) Body weight, median (range), kg 56.1 (38.6-92.4) CMV seropositive recipients 38 (86%) Disease AML 20 (45%) ALL 8 (18%) MDS 7 (16%) CML 3 (7%) CMML 3 (7%) NHL 1 (2%) ATL 2 (5%) Disease risk index Low , intermediate 28 (64%) High, very high 16 (36%) Numbers of allogeneic HCT 1 39 (89%) ≥2 5 (11%) Conditioning regimen TBI12Gy+CY+Ara-C/G-CSF 23 (52%) TBI12Gy+CY+Ara-C 9 (20%) TBI10Gy+CY 1 (2%) BU+CY+FLU 4 (9%) TBI4Gy+BU+FLU+ Ara-C/G-CSF 6 (14%) TBI4Gy+MEL140+FLU 1 (2%) GVHD prophylaxsis CSP+MTX 33 (75%) CSP+MMF 8 (18%) CSP 3 (7%) 7 TNC, median (range), ×10 /kg 2.47 (1.65-4.64) 5 CD34+cells, median (range), ×10 /kg 1.04 (0.46-2.40) 3 CFU-GM, median (range), ×10 /kg 31.14 (14.54-77.31) HLA disparities 0 4 (9%) 1 8 (18%) 2 30 (68%) 3 2 (5%) ABO incompatibility Match 10 (23%) Minor mismatch 11 (25%) Major mismatch 17 (39%) Bidirectional mismatch 6 (14%) 17 Page 17 of 19
Sex incompatibility Female donor to male recipient 15 (34%) Others 29 (66%) CBT: cord blood transplantation, CMV: cytomegalovirus, AML: acute myeloid leukemia, ALL: acute lymphoblastic leukemia, MDS: myelodysplastic syndrome, CML: chronic myelogenous leukemia, CMML: chronic myelomonocytic leukemia, NHL: non-Hodgkin's lymphoma, ATL: Adult T-cell leukemia, HCT: hematopoietic cell transplantation, TBI: total body irradiation, CY: cyclophosphamide, Ara-C: cytosine arabinoside, G-CSF: granulocyte colony-stimulating factor, BU: busulfan, FLI: fludarabine, MEL: melphalan, GVHD: graft-versus-host disease, CSP: cyclosporine A, MTX: methotrexate, MMF: mycophenolate mofetil, TNC: total nucleated cell, CFU-GM: colony-forming unit for granulocyte/macrophage, HLA: human leukocyte antigen.
18 Page 18 of 19
Table 2. Serum cytokine profiles at 2, 4, and 8 weeks after CBT Week 2 Week 4
Week 8
P-value (the entire group) IL-2 41.9±6.7 47.8±7.2 37.1±4.4 0.72 IL-4 7.8±0.6 10.5±1.2 9.9±1.0 0.13 IL-5 244.9±92.9 100.0±30.4 20.7±3.5 <0.0001 IL-6 322.5±200.0 170.1±51.5 61.2±10.9 0.0039 IL-8 262.0±61.6 1020.0±290.7 465.9±160.4 0.35 IL-9 7.3±0.8 9.9±0.9 9.5±1.0 0.03 IL-10 155.9±113.8 28.6±4.7 21.4±3.6 0.07 IL-13 47.6±5.8 54.9±6.8 48.1±4.2 0.45 IL-17A 108.9±7.9 118.6±8.8 122.3±9.1 0.48 IL-17F 48.5±16.7 149.4±94.0 75.9±33.2 0.51 IL-18 514.0±64.3 902.0±147.2 389.9±71.9 <0.0001 IL-21 61.9±5.6 74.4±6.3 69.1±4.7 0.15 IL-22 128.4±5.2 143.7±5.7 150.6±5.7 0.007 MCP-1 6794.0±923.2 619.3±70.4 818.1±201.3 <0.0001 IFN-γ 75.1±12.7 100.0±15.3 70.4±11.0 0.16 TNF-α 42.4±11.3 455.8±124.7 282.8±66.9 <0.0001 IL: interleukin, MCP: monocyte chemoattractant protein, IFN: interferon, TNF: tumor necrosis factor. Data (pg/ml) are shown as the mean ± standard error of the mean.
P-value (week 2 vs week 4) 0.59 0.12 0.05 0.47 0.91 0.02 0.40 0.34 0.28 0.25 0.01 0.07 0.11 <0.0001 0.09 <0.0001
P-value (week 2 vs week 8) 0.96 0.05 <0.0001 0.0013 0.15 0.03 0.04 0.24 0.30 0.53 0.04 0.12 0.001 <0.0001 0.99 <0.0001
P-value (week 4 vs week 8) 0.41 0.73 <0.0001 0.01 0.30 0.76 0.08 0.79 0.90 0.60 <0.0001 0.89 0.16 0.49 0.10 0.40
19 Page 19 of 19
S1083-8791(17)30611-0 http://dx.doi.org/doi: 10.1016/j.bbmt.2017.07.020 YBBMT 54743
To appear in:
Biology of Blood and Marrow Transplantation
Received date: Accepted date:
27-4-2017 16-7-2017
Please cite this article as: Takaaki Konuma, Chisato Kohara, Eri Watanabe, Motoko Mizukami, Etsuko Nagai, Maki Oiwa-Monna, Susumu Tanoue, Masamichi Isobe, Seiko Kato, Arinobu Tojo, Satoshi Takahashi, Cytokine Profiles of Pre-Engraftment Syndrome after Single-Unit Cord Blood Transplantation for Adult Patients, Biology of Blood and Marrow Transplantation (2017), http://dx.doi.org/doi: 10.1016/j.bbmt.2017.07.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cytokine profiles of pre-engraftment syndrome after single-unit cord blood transplantation for adult patients
Running title: Cytokine profiles of PES after CBT
Takaaki Konuma1, Chisato Kohara1, Eri Watanabe2, Motoko Mizukami3, Etsuko Nagai3, Maki Oiwa-Monna1, Susumu Tanoue1, Masamichi Isobe1, Seiko Kato1, Arinobu Tojo1, and Satoshi Takahashi1.
1
Department of Hematology/Oncology, The Institute of Medical Science, The University of
Tokyo, Tokyo, Japan 2
Department of IMSUT Clinical Flow Cytometry Laboratory, The Institute of Medical
Science, The University of Tokyo, Tokyo, Japan 3
Department of Laboratory Medicine, The Institute of Medical Science, The University of
Tokyo, Tokyo, Japan
Corresponding
author:
Takaaki
Konuma,
M.D.,
Ph.D.,
Department
of
Hematology/Oncology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Tel: +81-3-3443-8111, Fax: +81-3-5449-5429, Email: [email protected]
1 Page 1 of 19
Highlights We examined the relationship between serum cytokine profiles and pre-engraftment syndrome (PES) in 44 adult patients who received cord blood transplantation (CBT). Serum levels of 21 cytokines were measured by multiplex bead assays using a flow cytometer. The elevations of interleukin (IL)-5 and IL-6 at 2 weeks were associated with PES. The conversion from the naïve to the memory phenotype of T cells at 4 and 8 weeks was observed in PES. Abstract Clinical manifestation of high grade fever and skin rash prior to neutrophil engraftment, which is called pre-engraftment syndrome (PES) or pre-engraftment immune reaction, has been frequently observed after cord blood transplantation (CBT). The pathophysiology of PES is poorly understood, but cytokine storm during the early phase of CBT is thought to be one of the main cause of PES. However, the cytokine profiles of PES after CBT are unclear. Therefore, we examined the relationship between serum cytokine profiles and PES in 44 adult patients who received CBT in our institute between February 2013 and June 2016. Serum levels of 21 cytokines, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-17F, IL-18, IL-21, IL-22, IL-23, IL-33, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α were measured by multiplex bead assays using a flow cytometer. The median time until the absolute neutrophil count was >0.5×109/L was 21 days (range, 15–41 days). The cumulative incidence of PES was 79.6% (95% confidence interval, 63.3% to 88.5%) at 60 days after CBT. Serum levels of IL-5 (P=0.009) and IL-6 (P=0.01) at 2 weeks were significantly higher in patients who developed PES compared with those who did not develop PES. The conversion from the naïve to the effector or central memory phenotype of T cells was observed in PES. These data indicate that elevations of IL-5 and IL-6 around the time of clinical manifestation may be possible biomarkers for PES after CBT.
2 Page 2 of 19
Keyword; pre-engraftment syndrome, pre-engraftment immune reaction, cord blood transplantation, cytokine, interleukin-5, interleukin-6
Introduction
Cord blood transplantation (CBT) is widely used for patients without human leukocyte antigen (HLA)-matched related or unrelated donors for adult patients. Although the lower incidence and severity of acute graft-versus-host disease (GVHD) is one of the most important advantages of CBT, a unique clinical manifestation with high grade fever and skin rash prior to neutrophil engraftment, which is called pre-engraftment syndrome (PES) or pre-engraftment immune reaction, has been described after CBT [1–11]. The pathophysiology of PES is poorly understood, but cytokine storm during the early phase of CBT is thought to be one of the main causes of PES. However, the cytokine profiles of PES after CBT are unclear. Moreover, because the diagnosis of PES is usually based on only clinical symptoms, identifying specific serum cytokines would clearly be a valuable diagnostic tool for PES. Therefore, we examined the relationship between the levels of 21 serum cytokines and PES in 44 adult CBT recipients.
Patients and methods
Patients and transplant procedures
Forty-nine patients who had been treated with single-unit CBT in our institute between February 2013 and June 2016 were included in this study. The characteristics of patients 3 Page 3 of 19
and CBT are shown in Table 1. One patient who received a second CBT was analyzed twice as a separate patient. All patients received a total body irradiation (TBI) or busulfan-based myeloablative conditioning regimen, which was defined according to the Center for International Blood and Marrow Transplant Research (CIBMTR) criteria [12]. The most common conditioning regimen was TBI 12 Gy, cyclophosphamide, and cytosine arabinoside with or without granulocyte-colony stimulating factor (G-CSF) for myeloid malignancies or lymphoid malignancies, respectively [13,14]. All patients received cyclosporine (CSP)-based GVHD prophylaxis. CSP was given intravenously every day starting day -1 at a dose of 3mg/kg/day, and was tapered beginning between 5 and 8 weeks after CBT. CSP dose was not adjusted for specific trough level. Methotrexate (MTX) was given intravenously on day1 at a dose of 15mg/m2 and on day 3 and 6 at a dose of 10mg/m2. Mycophenolate mofetil (MMF) was given orally at a dose of 30mg/kg/day from day 0 to 27. Conditioning regimen and GVHD prophylaxis were determined by the treating physician. All patients received G-CSF by intravenous infusion starting on day 1 until durable granulocyte recovery was achieved. Cord blood units were obtained from the Japan Cord Blood Bank Network. The institutional Review Board of the Institute of Medical Science, The University of Tokyo approved this study.
Serum cytokine analysis
Serum samples were collected at 2, 4, and 8 weeks after CBT and stored at -80°C until analysis. Serum levels of 21 cytokines, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-17F, IL-18, IL-21, IL-22, IL-23, IL-33, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-α, IFN-γ and tumor necrosis factor (TNF)-α, were measured using multiplex bead assays with a LEGENDplexTM Human Th Cytokine Panel (Cat. No. 740001, BioLegend) and Human Inflammation Panel (Cat. No. 4 Page 4 of 19
740118, BioLegend) according to the manufacturer’s instructions. Because the median intra-assay coefficient of variation was 3.5 % (range, 0.3-18.9 %) for 65 samples assayed in duplicate, the remaining samples were run in a single assay. For data analysis, levels of cytokines below the lower limit of detection were marked as the values of the halfway point between the lower limit of detection and zero.
T cell subsets analysis in peripheral blood
Peripheral blood samples were collected at 4 and 8 weeks after CBT. Peripheral blood mononuclear cells were obtained by LymphoprepTM (Axis-Shield PoC, Oslo, Norway) density gradient centrifugation and stored in cryopreservation medium Bambanker TM (Nippon Genetic Co. Ltd., Tokyo, Japan) until analysis. Cells were stained with Alexa Fluor 700-conjugated
anti
510-conjugated
anti
human human
CD3 CD4
(clone
UCHT-1,
(clone
BioLegend),
RPA-T4,
Brilliant
BioLegend),
Violet
fluorescein
isothiocyanate-conjugated anti human CD8 (clone RPA-T8, Tonbo Biosciences), allophycocyanin (APC)-Cy7-conjugated anti human CD45RA (clone HI100, BioLegend), Brilliant Violet 421-conjugated anti human CCR7 (clone G043H7, BioLegend) antibodies. The analysis was performed on a FACS Aria II (BD Bioscience).
Definitions
PES was defined as an unexplained fever >38.3°C not associated with documented infection and unresponsive to antimicrobial administrations and an unexplained erythematous skin rash resembling acute GVHD that occur prior to neutrophil engraftment, as previously described [1]. Neutrophil engraftment was defined as being achieved on the first of three consecutive days when the absolute neutrophil count was >0.5×10 9/L. Acute 5 Page 5 of 19
GVHD was graded by the treating physician according to the standard criteria [15]. Skin biopsies were not performed in any of the patients for diagnosis of PES and skin GVHD, although colon biopsies were performed to confirm diagnosis of gut GVHD in 3 patients.
Statistical analysis
Continuous variables were compared with the Mann–Whitney U test. Categorical variables were compared with the chi-square test. Estimation of the probabilities of PES and GVHD were based on a cumulative incidence method to accommodate competing risks, and the groups were compared using Gray’s test. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [16], a graphical user interface for the R 3.0.2 software program (R Foundation for Statistical Computing, Vienna, Austria) or GraphPad Prism 6 for Mac OS X (GraphPad Software, San Diego, California). All P-values were two-sided, and P-values of <0.05 were considered to be statistically significant.
Results
Engraftment, PES, and acute GVHD
All patients achieved neutrophil recovery after CBT. The median time until the absolute neutrophil count was >0.5×109/L was 21 days (range, 15–41 days). The cumulative incidences of PES, grade II to IV acute GVHD, and grade III to IV acute GVHD were 79.6% (95% confidence interval [CI], 63.3% to 88.5%), 75.0% (95% CI, 58.9% to 85.5%), and 14.0% (95% CI, 5.6% to 26.1%) at 60 days after CBT, respectively (Figure 1A). The median times to the development of PES, grade II to IV acute GVHD, and grade III to IV 6 Page 6 of 19
acute GVHD were 12 days (range, 4–18 days), 19.5 days (range, 10–50 days), and 43 days (range, 29–57 days), respectively. Because acute GVHD of the skin could not be distinguished from preceding PES in 18 patients, the median time to the development of grade II to IV acute GVHD was faster than the median time of neutrophil engraftment. No patient received corticosteroids for treatment for PES, although seven patients received more than 1 mg/kg of prednisolone (PSL) on a median of 36 days (range, 29–58 days) for treatment of severe acute GVHD. There were no significant differences in the baseline characteristics between patients who did and did not developed PES (Supplementary Table 1).
Serum cytokine profiles during the first 8 weeks after CBT
Five serum cytokines, IL-1β, IL-12p70, IL-23, IL-33, and IFN-α, were below the lower limit of detection in more than 80% of samples. Therefore, we did not evaluate these five cytokines for further analysis. The kinetics of each cytokine during the 8 weeks after CBT are shown in Table 2. Seven cytokine levels were significantly different during the course of CBT. Significant peak levels of IL-5, IL-6, and MCP-1 were observed at 2 weeks after CBT, whereas significant peak levels of IL-9, IL-18, and TNF-α were observed at 4 weeks after CBT (Supplementary Figure 1). All these cytokines levels returned to the probable baseline at 8 weeks after CBT. Serum levels of IL-5 (P=0.009) and IL-6 (P=0.01) at 2 weeks were significantly higher in patients who developed PES compared with those who did not develop PES (Figure 1B,1C). Serum levels of IL-5 (P=0.005), IL-6 (P=0.01), IL-8 (P=0.02), IL-9 (P=0.04), and IFN-γ (P=0.006) at 2 weeks were significantly higher in patients who developed grade II to IV acute GVHD compared with those who did not develop it (Supplementary Figure 2A–E). Serum levels of IL-4 (P=0.007), IL-6 (P=0.04), IL-10 (P=0.005), IL-21 (P=0.007), 7 Page 7 of 19
and TFN-α (P=0.02) at 2 weeks were significantly higher in patients who developed grade III to IV acute GVHD compared with those who did not develop it (Supplementary Figure 2F–J). All cytokine levels at 4 and 8 weeks after CBT were not significantly associated with PES, grade II to IV acute GVHD, or grade III to IV acute GVHD. The serum level of MCP-1 at 8 weeks was significantly lower in patients who received PSL compared with those who did not receive PSL (P<0.001). Among patients who developed PES (n=35), grade II to IV and grade III to IV acute GVHD occurred in 30 and 6 patients, respectively. We also evaluated the effects of cytokine profiles on development of grade II to IV or grade III to IV acute GVHD among patients who developed PES. Serum levels of IL-5 (P=0.006), IL-6 (P=0.04), and IFN-γ (P=0.02) at 2 weeks were significantly higher in patients who developed grade II to IV acute GVHD compared with those who did not develop it (Supplementary Figure 3A–C). Serum levels of IL-4 (P=0.01), IL-10 (P=0.01), IL-17F (P=0.02), IL-21 (P=0.003), and TFN-α (P=0.03) at 2 weeks were significantly higher in patients who developed grade III to IV acute GVHD compared with those who did not develop it (Supplementary Figure 3D–H). Because infections including bacterial blood stream infection (BSI) and cytomegalovirus (CMV) reactivation are commonly observed in patients following CBT, we assessed whether bacterial BSI and CMV reactivation affected the cytokine profiles after CBT. Bacterial BSI occurred in 8 patients (18%) during study period. Bacterial BSI was not associated with the cytokine levels, which were measured within 3 days after the onset of BSIs, at 2 weeks after CBT (Supplementary Table 2). CMV reactivation, which was evaluated by an antigenemia assay using the monoclonal antibodies C10/C11, occurred in 35 patients (80%) during study period. CMV reactivation was not associated with the cytokine levels, which were measured within 3 days after CMV reactivation, at 4 and 8 weeks after CBT (Supplementary Table 2). 8 Page 8 of 19
T cell subsets during the first 8 weeks after CBT
In order to clarify the balance of naïve, central, and effector memory cells in patients who developed PES, we evaluated the expression of CD45RA and CCR7 on CD3+CD4+ and CD3+CD8+ T cells in 24 patients at 4 and 8 weeks after CBT (Figure 2A). At 4 weeks after CBT, central memory (CD45RA-, CCR7+) CD4+ T cells were significantly higher and CD45RA+ effector memory (CD45RA+, CCR7-) CD4+ T cells were significantly lower in patients who developed PES compared with those who did not develop PES (Figure 2B,2C). At 8 weeks after CBT, naïve (CD45RA+, CCR7+) CD4+ and CD8+ T cells were significantly lower and effector memory (CD45RA-, CCR7-) CD4+ and CD8+ T cells were significantly higher in patients who developed PES compared with those who did not develop PES (Figure 2D,E). As for grade II to IV acute GVHD, conversion from the naïve to the effector memory phenotype was observed only in CD4+ T cells at 8 weeks after CBT (Figure 3A-D). PSL treatment did not affect the balance of naïve, central, and effector memory cells on CD4+ and CD8+ T cells at 8 weeks after CBT. We assessed whether the peripheral blood absolute lymphocyte and eosinophil counts (ALC and AEC, respectively) were associated with PES. However, ALC and AEC at 4 and 8 weeks after CBT were not significantly associated with PES, grade II to IV acute GVHD, or grade III to IV acute GVHD (Supplementary Figure 4).
Discussion
Several studies have demonstrated that PES is frequently observed after single and double unit CBT in pediatric and adult patients [1–11]. Although the incidence of PES after CBT has been reported to range from 20% to 78%, it depended on the conditioning 9 Page 9 of 19
regimen, GVHD prophylaxis, and the definition used. In our study, no patient received corticosteroids in GVHD prophylaxis or antithymocyte globulin in the conditioning regimen, and CSP with MTX was commonly administered for GVHD prophylaxis. The incidence of PES in our study was 79.6%, using the definition of PES by Patel et al. [1]. On the other hand, using the definition of PES by Kishi et al. [2], the incidence of PES decreased to 63.6% in our cohort, which was lower than that in the previous report by Kishi et al. [2]. The pathophysiology of PES is poorly understood. However, cytokine storm during the early phase of CBT is thought to be one of the main causes of PES. Morita–Hoshi et al. examined the expression profile of serum proteins using a surface-enhanced
laser
desorption/ionization
time-of-flight
mass
spectroscopy
(SELDI-TOF MS) system in CBT recipients and found that serum amyloid A (SAA) was a candidate biomarker for PES after CBT [3]. In the present study, we investigated 21 serum cytokine levels during the first 8 weeks after CBT to clarify the relationship between serum cytokine levels and PES. Our data showed that dynamic changes in cytokine profiles after CBT were possibly caused by the conditioning regimen and alloimmune reactions. Importantly, elevations of IL-5 and IL-6 at 2 weeks after CBT were significantly associated with PES after CBT. IL-5 is one of the Th2 type cytokines and is a major activator of eosinophils. IL-6 is one of the proinflammatory cytokines and plays an important role in various events during inflammation. In addition, IL-6 is induced by SAA, although IL-6 alone does not induce SAA in vitro [17]. Several studies have demonstrated that levels of IL-5 [18–20] and IL-6 [20–23] are increased in patients with engraftment syndrome and acute GVHD. In addition, the addition of MTX to calcineurin inhibitor has been reported to reduce the incidence of PES after CBT [4, 24]. Terakura et al. also reported that the relative risk of grade II to IV and grade III to IV acute GVHD was significantly lower in CBT recipients receiving calcineurin inhibitor and MTX compared with those receiving calcineurin inhibitor and MMF for GVHD prophylaxis [25]. Interestingly, our data showed 10 Page 10 of 19
that significant suppression of IL-5 and IL-6 was observed by addition of MTX for GVHD prophylaxis. Therefore, GVHD prophylaxis might be associated with the development of PES after CBT. These data suggested the possibility that a similar mechanism might be involved in the development of PES and acute GVHD, although PES is a syndrome that is distinguished from acute GVHD based on onset timing and clinical manifestations. The specific subsets, naïve, central, and effector memory phenotypes, of CD4+ and CD8+ T cells have been reported to play an important role in the development of acute and chronic GVHD [26–28], but their role in PES after CBT is unclear. Matsuno et al. reported that rapid conversion from naïve to memory T cells on both CD4+ and CD8+ T cells was observed in patients who developed PES at 2 weeks after CBT [5]. However, we could not evaluate whether T cells converted to different subsets at 2 weeks after CBT, because of low cell numbers. Meanwhile, our data showed a significant elevation of central memory CD4+ T cells at 4 weeks and elevation of effecter memory CD4+ and CD8+ T cells at 8 weeks in patients with PES after CBT. Importantly, conversion from the naïve to the memory phenotype of T cells was observed in PES. However, although regulation of these T cell subsets might be partially dependent on several cytokines and prolonged alloantigen stimulation, further analysis will be important for understanding the aberrant immunological status in PES. Our study had several limitations. First, this study was based on data from a single-institute in Japan, and the number of patients was small. However, all patients were adults with hematological malignancies and were treated with single-unit CBT following myeloablative conditioning regimens and a cyclosporine-based GVHD prophylaxis. Second, the incidence of grade II to IV acute GVHD might have been overestimated, because acute GVHD of the skin could not be distinguished from preceding PES in 18 patients in our cohort. Therefore, immunological status including serum cytokines and T cell subsets was quite similar between PES and grade II to IV acute GVHD. Because it is 11 Page 11 of 19
difficult to completely separate the diagnosis of PES and acute GVHD, it remains controversial whether a similar immunological profile is involved in the development of PES and acute GVHD. In conclusion, our data demonstrated that elevations of IL-5 and IL-6 around the time of clinical manifestation of high grade fever and skin rash were associated with PES after CBT. Therefore, IL-5 and IL-6 may be possible biomarkers for PES after CBT.
Acknowledgements
The authors thank all of the physicians and staff at the hospital and the cord blood bank in Japan for their help in this study. This work was supported in part by grants-in-aid from Japanese Society for the Promotion of Science (JSPS), and Takeda Science Foundation.
Conflict of Interest
The authors declare no competing financial interests.
References
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cyclophosphamide. Leuk Lymphoma. 2015;56:1148-1150. 15. Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant. 1995;15:825-828. 16. Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transplant. 2013;48:452-458. 17. De Buck M, Gouwy M, Wang JM, et al. The cytokine-serum amyloid A-chemokine network. Cytokine Growth Factor Rev. 2016;30:55-69. 18. Imoto S, Oomoto Y, Murata K, et al. Kinetics of serum cytokines after allogeneic bone marrow transplantation: interleukin-5 as a potential marker of acute graft-versus-host disease. Int J Hematol. 2000;72:92-97. 19. Jordan WJ, Brookes PA, Szydlo RM, Goldman JM, Lechler RI, Ritter MA. IL-13 production by donor T cells is prognostic of acute graft-versus-host disease following unrelated donor stem cell transplantation. Blood. 2004;103:717-724. 20. Fujii N, Hiraki A, Aoe K, et al. Serum cytokine concentrations and acute graft-versus-host disease after allogeneic peripheral blood stem cell transplantation: concurrent measurement of ten cytokines and their respective ratios using cytometric bead array. Int J Mol Med. 2006;17:881-885. 14 Page 14 of 19
21. Schots R, Kaufman L, Van Riet I, et al. Proinflammatory cytokines and their role in the development of major transplant-related complications in the early phase after allogeneic bone marrow transplantation. Leukemia. 2003;17:1150-1156. 22. Malone FR, Leisenring WM, Storer BE, et al. Prolonged anorexia and elevated plasma cytokine levels following myeloablative allogeneic hematopoietic cell transplant. Bone Marrow Transplant. 2007;40:765-772. 23. Khandelwal P, Mellor-Heineke S, Rehman N, et al. Cytokine Profile of Engraftment Syndrome in Pediatric Hematopoietic Stem Cell Transplant Recipients. Biol Blood Marrow Transplant. 2016;22:690-697. 24. Iguchi A, Terashita Y, Sugiyama M, et al. Graft-versus-host disease (GVHD) prophylaxis by using methotrexate decreases pre-engraftment syndrome and severe acute GVHD, and accelerates engraftment after cord blood transplantation. Pediatr Transplant. 2016;20:114-119. 25. Terakura S, Wake A, Inamoto Y, et al. Exploratory research for optimal GvHD prophylaxis after single unit CBT in adults: short-term methotrexate reduced the incidence of severe GvHD more than mycophenolate mofetil. Bone Marrow Transplant. 2017;52:423-430. 26. Mohty M, Blaise D, Faucher C, et al. Inflammatory cytokines and acute graft-versus-host disease after reduced-intensity conditioning allogeneic stem cell transplantation. Blood. 2005;106:4407-4411. 27. Yamashita K, Choi U, Woltz PC, et al. Severe chronic graft-versus-host disease is characterized by a preponderance of CD4(+) effector memory cells relative to central memory cells. Blood. 2004;103:3986-3988. 28. D'Asaro M, Dieli F, Caccamo N, Musso M, Porretto F, Salerno A. Increase of CCR7CD45RA+ CD8 T cells (T(EMRA)) in chronic graft-versus-host disease. Leukemia. 2006;20:545-547. 15 Page 15 of 19
Figure 1. Cumulative incidences of PES and acute GVHD after CBT (A). Serum levels of IL-5 and IL-6 after CBT according to the development of PES (B,C) and GVHD prophylaxis (D,E). Figure 2. Characteristics of CD4+ and CD8+ T cell subsets after CBT according to the development of PES. Figure 3. Characteristics of CD4+ and CD8+ T cell subsets after CBT according to the development of grade II to IV acute GVHD.
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Table 1. Characteristics of the patients, cord blood units, and transplantations Entire cohort Number of CBTs 44 Age at CBT, median (range), years 47 (21-68) Body weight, median (range), kg 56.1 (38.6-92.4) CMV seropositive recipients 38 (86%) Disease AML 20 (45%) ALL 8 (18%) MDS 7 (16%) CML 3 (7%) CMML 3 (7%) NHL 1 (2%) ATL 2 (5%) Disease risk index Low , intermediate 28 (64%) High, very high 16 (36%) Numbers of allogeneic HCT 1 39 (89%) ≥2 5 (11%) Conditioning regimen TBI12Gy+CY+Ara-C/G-CSF 23 (52%) TBI12Gy+CY+Ara-C 9 (20%) TBI10Gy+CY 1 (2%) BU+CY+FLU 4 (9%) TBI4Gy+BU+FLU+ Ara-C/G-CSF 6 (14%) TBI4Gy+MEL140+FLU 1 (2%) GVHD prophylaxsis CSP+MTX 33 (75%) CSP+MMF 8 (18%) CSP 3 (7%) 7 TNC, median (range), ×10 /kg 2.47 (1.65-4.64) 5 CD34+cells, median (range), ×10 /kg 1.04 (0.46-2.40) 3 CFU-GM, median (range), ×10 /kg 31.14 (14.54-77.31) HLA disparities 0 4 (9%) 1 8 (18%) 2 30 (68%) 3 2 (5%) ABO incompatibility Match 10 (23%) Minor mismatch 11 (25%) Major mismatch 17 (39%) Bidirectional mismatch 6 (14%) 17 Page 17 of 19
Sex incompatibility Female donor to male recipient 15 (34%) Others 29 (66%) CBT: cord blood transplantation, CMV: cytomegalovirus, AML: acute myeloid leukemia, ALL: acute lymphoblastic leukemia, MDS: myelodysplastic syndrome, CML: chronic myelogenous leukemia, CMML: chronic myelomonocytic leukemia, NHL: non-Hodgkin's lymphoma, ATL: Adult T-cell leukemia, HCT: hematopoietic cell transplantation, TBI: total body irradiation, CY: cyclophosphamide, Ara-C: cytosine arabinoside, G-CSF: granulocyte colony-stimulating factor, BU: busulfan, FLI: fludarabine, MEL: melphalan, GVHD: graft-versus-host disease, CSP: cyclosporine A, MTX: methotrexate, MMF: mycophenolate mofetil, TNC: total nucleated cell, CFU-GM: colony-forming unit for granulocyte/macrophage, HLA: human leukocyte antigen.
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Table 2. Serum cytokine profiles at 2, 4, and 8 weeks after CBT Week 2 Week 4
Week 8
P-value (the entire group) IL-2 41.9±6.7 47.8±7.2 37.1±4.4 0.72 IL-4 7.8±0.6 10.5±1.2 9.9±1.0 0.13 IL-5 244.9±92.9 100.0±30.4 20.7±3.5 <0.0001 IL-6 322.5±200.0 170.1±51.5 61.2±10.9 0.0039 IL-8 262.0±61.6 1020.0±290.7 465.9±160.4 0.35 IL-9 7.3±0.8 9.9±0.9 9.5±1.0 0.03 IL-10 155.9±113.8 28.6±4.7 21.4±3.6 0.07 IL-13 47.6±5.8 54.9±6.8 48.1±4.2 0.45 IL-17A 108.9±7.9 118.6±8.8 122.3±9.1 0.48 IL-17F 48.5±16.7 149.4±94.0 75.9±33.2 0.51 IL-18 514.0±64.3 902.0±147.2 389.9±71.9 <0.0001 IL-21 61.9±5.6 74.4±6.3 69.1±4.7 0.15 IL-22 128.4±5.2 143.7±5.7 150.6±5.7 0.007 MCP-1 6794.0±923.2 619.3±70.4 818.1±201.3 <0.0001 IFN-γ 75.1±12.7 100.0±15.3 70.4±11.0 0.16 TNF-α 42.4±11.3 455.8±124.7 282.8±66.9 <0.0001 IL: interleukin, MCP: monocyte chemoattractant protein, IFN: interferon, TNF: tumor necrosis factor. Data (pg/ml) are shown as the mean ± standard error of the mean.
P-value (week 2 vs week 4) 0.59 0.12 0.05 0.47 0.91 0.02 0.40 0.34 0.28 0.25 0.01 0.07 0.11 <0.0001 0.09 <0.0001
P-value (week 2 vs week 8) 0.96 0.05 <0.0001 0.0013 0.15 0.03 0.04 0.24 0.30 0.53 0.04 0.12 0.001 <0.0001 0.99 <0.0001
P-value (week 4 vs week 8) 0.41 0.73 <0.0001 0.01 0.30 0.76 0.08 0.79 0.90 0.60 <0.0001 0.89 0.16 0.49 0.10 0.40
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