Treatment of chronic GvHD with mesenchymal stromal cells induces durable responses: A phase II study

Abstract

Steroid-refractory chronic graft-vs-host disease (cGvHD) contributes to morbidity after allogeneic hematopoietic stem cell transplantation. Here, we report on 11 patients with severe, refractory cGvHD treated with repeated infusions of allogeneic bone marrow-derived mesenchymal stromal cells (MSC) over a 6- to 12-month period. Six patients responded to MSC treatment following National Institutes of Health response criteria, accompanied by improvement in GvHD-related symptoms and quality of life. This response was durable, with systemic immunosuppressive therapy withdrawn from two responders, and a further two free from steroids and tapering calcineurin inhibitors. All responders displayed a distinct immune phenotype characterized by higher levels of naïve T cells and B cells before treatment compared with the nonresponders, and a significantly higher fraction of CD31+ naïve CD4+ T cells. MSC treatment was associated with significant increases in naïve T cells, B cells, and Tregs 7 days after each infusion. Skin biopsies showed resolution of epidermal pathology. CXCL9 and CXCL10 showed differential responses in responder and nonresponder patients. Our data support the use of MSC infusions as treatment for steroid-refractory cGvHD with durable responses. We propose CXCL9 and CXCL10 as early biomarkers for responsiveness to MSC treatment. Our results highlight the importance of the MSC recipient immune phenotype in promoting treatment response. This trial was registered at www.ClinicalTrials.gov as #NCT01522716.

Keywords: cellular therapy; clinical trials; hematopoietic stem cell transplantation; mesenchymal stem cells; thymus.

Conflict of interest statement

The authors declared no potential conflicts of interest.

© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Figures

FIGURE 1

Heatmap of individual responses: NIH organ score, National Institutes of Health (NIH) global score, range of motion (ROM), and body surface area (BSA) percentage involved with sclerosis. Time points are at study enrolment and at the end of MSC treatment. Black boxes denote organs with response and red boxes denote organs with progression. CNI, calcineurin inhibitors; ECP, extracorporeal photopheresis; F, fascia; J, joints; MMF, mycophenolate mofetil; M, muscles; P, prednisolone; Tac, tacrolimus

FIGURE 2

Responders have higher absolute and relative levels of naïve CD4+ T cells and naïve B cells compared with nonresponders. A and B, Representative plots of the flow cytometry analysis. CD4+ T cells were divided into naïve, CM, EM, and T‐EMRA based on CD27 and CD45RA. 32 Naïve B cells were defined as CD19+CD27‐IgD+CD21hi. CD21 was used to exclude CD27‐CD21low B cells, a population previously suggested to contain aberrant B cells in cGvHD. 33 C and D, Relative and absolute numbers of naïve CD4+ T cells were higher in R compared with NR throughout the study. There were no differences in CM CD4+ T‐cell numbers. E and F, Memory B cells were not different between R and NR. Absolute numbers of naïve B cells were higher in R compared with NR throughout the study. P‐values in the absolute number graphs represent the P‐values for the Responder factor in the linear mixed effects analysis. P‐values for relative numbers with Wilcoxon rank‐sum test. *P < .05. Error bars show mean ± SEM. CM, central memory T‐cell; EM, effector memory T‐cell; T‐EMRA, terminally differentiated effector memory T cells

FIGURE 3

Mesenchymal stromal cell (MSC) treatment induces improvements in inflammatory cytokine profile in responders and in epidermal skin pathology in both responders and nonresponders. A, Multiplex analysis of plasma samples taken before each MSC infusion is presented. Levels of several pro‐inflammatory chemokines, as well as TNFα and IL6 increased in NR and decreased in R after two and six MSC infusions (1 and 5 months after treatment start, respectively), compared to before the first infusion. P‐values for within group changes using repeated measures ANOVA. P‐values for comparing NR and R at different time points with t test. *P < .05, **P < .005, ***P < .0005. B, Blinded pathological review of skin biopsies taken before and after treatment revealed improvements with mainly epidermal changes in both R and NR after treatment. For details, see Table S5. Representative images of one R (patient 5) and one NR (patient 10) showing mononuclear infiltration (asterisks), extensive vacuolization of KC (black arrows) as well as apoptotic and Civatte bodies in the epithelium (white arrows) before treatment. C, The epidermal pathology regressed in both R and NR after treatment with little or no remaining vacuolization and few apoptotic cells, but in the NR dermal sclerosis progressed (X). CXCL, chemokine (C‐X‐C motif) ligand; IL, interleukin; KC, keratinocytes; LP, lamina propria; MCP, monocyte chemotactic protein; NR, nonresponder; R, responder; TNF, tumor necrosis factor

FIGURE 4

Short‐term increases in naïve miRNA, T cells, and Tregs after infusion. A, Four circulating miRNAs were significantly up regulated in R and NR plasma at 1 hour after mesenchymal stromal cell (MSC) infusion 1 and returned to baseline levels 24 hours after infusion. t Test with Benjamini‐Hochberg correction. B, Representative plots of the flow cytometry analysis. Tregs are defined as CD4+ FoxP3+ T cells. Naïve CD4+ T cells are defined as in Figure 2 (CD4+ CD45RA+ CD27+). C and D, Absolute number of naïve CD4+ T cells and Tregs increase in R 1 and 7 days after each MSC infusion compared with before infusion. These short‐term increases are not seen in NR. X‐axes display infusion number. The P‐values are derived from the mixed effects model and represent the significance of the factor Days since last infusion. *P < .05, **P < .005. Error bars show mean ± SEM. R, responder; NR, nonresponder; Treg, regulatory T cell

FIGURE 5

Thymic function and proliferation of naïve CD4+ T cells. A, Thymic function was assessed by measuring the proportion of CD31+ cells among naïve CD4+ T cells. We found a higher percentage of CD31+ naïve CD4+ T cells in R compared to NR before infusion, indicating better thymic function. B, Proliferation of naïve CD4+ T cells was assessed using levels of Ki67. The levels of Ki67 were not different between the responders and the nonresponders. Data from infusion 3. P‐values calculated using the Wilcoxon rank‐sum test. *P < .05. Error bars show mean ± SEM