Semaphorin 3A Expression on Donor and Recipient Regulatory Cells: A Novel Pre-Transplant Biomarker Predicting the Development of Acute Graft-Versus-Host Disease
Authors: Vadas Zahava , Ganon-Elazar Eti , Nov Yuval , Henig Israel , Attias Dina , Haddad Nuhad , Fineman Riva , Rowe Jacob M. , Zuckerman Tsila 
Published: October 2019

Abstract
Introduction: Acute graft-versus-host disease (aGVHD) is a major limitation of allogeneic stem cell transplantation (alloSCT) due to associated morbidity and mortality. A search for biomarkers that predict its occurrence is continuously evolving. Regulatory T cells (T regs) have been shown to suppress the early expansion of alloreactive donor T cells and limit the capacity to induce GVHD without minimizing the graft-versus-leukemia (GVL) effect. Recently, the role of regulatory B cells (B reg) in GHVD was demonstrated, with exacerbation of GVHD in both donor and host mice lacking functional regulatory B cells. Semaphorin 3A (Sema3A) is an immunoregulatory molecule secreted by activated B, T and antigen presenting cells. It enhances suppressor ability of B and T regulatory cells by increased secretion of interleukin-10 (IL-10) and transforming growth factor β (TGF-β). The aim of the present study was to explore whether Sema3A expression on B reg and T reg cells from donors and/or recipients pre- or early post-transplantation can predict occurrence of aGVHD.
Methods: Thirty four consecutive patients referred to alloSCT were included in the first study cohort. Additionally, 10 donor/recipient (D/R) pairs were enrolled. All participants signed informed consent. 20 cc of fresh peripheral blood were drawn from recipients at day -7 pre-alloSCT and upon WBC engraftment, as well as from their corresponding donors. Mononuclear cells were isolated using ficoll gradient separation and subjected to FACS analysis using monoclonal antibodies evaluating the level of Sema3A expression on B reg cells (CD19/CD25high/ Sema3A), T reg cells (CD4/CD25high/Sema3A) and natural killer (NK) cells (CD3/CD56). Cutoff for positive expression of Sema3A on regulatory cells was considered only if ≥20% of cells expressed the above phenotype of T, B cell and NK population. The FACS results correlated with occurrence of clinical aGVHD grade II-IV. Descriptive statistical analysis and student t test are used to describe results.
Results: Overall, 44 recipients were analyzed. Median age at transplant was 49.9 (18-69), 34 were diagnosed with acute leukemia/MDS, 8 - lymphoproliferative and 2 - myeloproliferative diseases. All patients received peripheral mobilized stem cells. Myeloablative conditioning was administered to 33 and reduced intensity to 11 patients. GVHD prophylaxis consisted of standard cyclosporine and methotrexate. Twenty patients developed aGVHD grade II-IV, mostly grade II –III (80%). Recipient expression of Sema3A on B cells pre-transplant was higher in patients without aGVHD versus those with aGVHD (79.9% vs 69.5%, respectively; p<0.005) while pre-transplant expression of Sema3A on T cells was similar in patients without and with aGVHD (57.3% vs 58.6%, respectively) (fig 1). Post-transplant, recipient expression of Sema3A on B and T cells was similar in patients without and with aGVHD (B cells: 84.1% vs 79.1%, T cells 62.6 vs 58.4, respectively) (fig 2). In donors, the expression of Sema3A on regulatory T cells was higher if their recipients did not develop aGVHD as compared to the expression in donors with recipients who developed aGVHD (51.0% vs 36.2%). Sema3A expression on regulatory B cells was not different in donors, no matter whether recipients did not or did develop aGVHD (52.2% vs 55.0%, respectively) (fig 3). Sema3A expression on NK cells was below our determined threshold.
Conclusion: This is the first report of a correlation of Sema3A expression with the occurrence of aGVHD, opening a potentially important opportunity for early clinical intervention. Specifically, 1. Pre-transplant recipient Sema3A expression on B reg cells is significantly higher in patients who will not develop aGVHD. 2. Pre-transplant high expression of Sema3A on donor T reg cells is associated with a lower risk of aGVHD development in their corresponding recipients. 3. Sema3A expression on recipient T reg cells does not correlate with occurrence of aGVHD. Further studies with a larger patient cohort validation are needed to confirm the above findings.

Disclosures
No relevant conflicts of interest to declare.