Evaluation of Cytokines after Platelet-Lysate-Expanded Mesenchymal Stromal Cell Therapy for Steroid Refractory Acute Graft-Versus-Host Disease
Authors: Takahashi Yoshiyuki , Kojima Daiei , Kawashima Nozomu , Murakami Norihiro , Suzuki Kyogo , Sekiya Yuko , Narita Atsushi , Kamei Michi , Nishio Nobuhiro , Okuno Yusuke , Muramatsu Hideki , Hama Asahito , Kojima Seiji 
Published: October 2019

Abstract
Introduction: Bone marrow-derived mesenchymal stromal cells (MSCs) exhibit in vitro immunomodulatory properties and have been effectively used to treat patients with acute GVHD following hematopoietic stem cell transplantation (HSCT). Expansion of MSCs involves the use of fetal bovine serum (FBS), although regulatory guidelines recommend that alternatives be used. Therefore, expansion of MSCs where FBS has been replaced by platelet lysates (PL) has been reported. Previous phase I studies of PL expanded third party, bone marrow derived MSCs have been reported showing no acute toxicity and 66 to 71% overall response rate. Although a uniform mechanism has not yet discovered, the available data have revealed immunomodulatory effects of MSCs therapy on T lymphocytes, B lymphocytes, NK cells, dendritic cells and macrophages. Therefore, we planned comprehensive evaluation of cytokines in patients who received PL expanded MSC therapy for steroid refractory acute GVHD.
Materials and Methods: We conducted the phase 1 clinical trial of PL expanded MSC therapy for steroid resistant acute GVHD in children and young adults. Steroid resistance was defined as lack of clinical improvement after 7days treatment of methylprednisolone (2mg/kg/day) or GVHD progression within 3 days from steroid onset. MSCs were harvested frombone marrow donors and were expanded in 5% PL-containing medium. MSCs product had to satisfy all the release criteria including absence of bacteria, fungi, mycoplasma, endotoxin level < 5 EU/kg, > 70% viability, > 70% positivity for CD44, CD73 and CD105, and < 10% contamination by CD14, CD34, and CD45 hematopoietic cells. MSCs were injected intravenously immediately after thawing. Each MSC infusions aimed at reaching 1-2 x 10(6) cells/kg recipient body weight. Further MSC administrations could be provided with one week interval. Serum samples were collected before steroid treatment, before MSCs therapy, 1, 7, 14, 21, 28 days after MSCs therapy. A panel of 64 different cytokines was measured using Milliplex map Human Cytokine/Chemokine Magnetic Bead Panel (Millipore, Billerica, MA) to quantify the serum cytokine levels. The assay was performed according to the manufacturer's instructions.
Results: Four patients (aged 2, 5, 15, 25 years) received intravenous (i.v.) MSCs for grade III-IV acute GVHD (liver: two cases, gut: two cases) on two occasions following HLA haploidentical HSCT or unrelated HLA matched SCT. The median dose of infusions was 1.2 x 10(6)/kg (range: 0.7-2.7 x 10(6)/kg). No acute side effects after infusions were observed. Clinical response was recognized within 7days and complete response was obtained in all four patients within 3 weeks from the administration of MSCs. Relapse of GVHD was not observed. Reactivation of cytomegalovirus (n = 3), Epstein-Barr virus (n = 1), adenovirus (n = 1), BK virus (n = 1), and fungal infection (n = 1) were observed in the four patients following the administration of MSCs. At a median follow-up of 21 months (range: 10-38 months), three of the four patients survived although one patient died of relapse of leukemia. The serum concentrations of the interleukins (IL)-6, interferon (IFN)g and Flt-3L were significantly elevated in all four patients before steroid therapy. Although Flt-3L level was decreased after steroid therapy in all the patients, IL-6 and IFNg were kept in high in two patients with liver GVHD grade 4. Serum level of IL-6 and IFNg were significantly decreased on day7 and day14 after MSCs therapy in both patients concomitant with 52.6-54.1% and 81.7-86.5% reduction of total bilirubin level on day7 and day14 respectively.
Conclusion: This study supports the safety and efficacy of PL-expanded MSCs in the treatment of steroid-resistant GVHD in children and young adults. Reduction of inflammatory cytokines such as IL-6 and IFNg on day7 after MSCs infusion and observation of clinical response within 7days suggested the immunomodulatory effects of MSCs therapy on activated T lymphocytes as therapeutic mechanism of MSCs for acute GVHD rather than differentiation potential and regenerative effects of MSCs on damaged tissues.

Disclosures
No relevant conflicts of interest to declare.