Regulatory T Cells Enhance the Allogeneic Activity of Endothelial-Specific Cytotoxic T Lymphocytes – Protective Effect of Defibrotide

Damage to the vascular endothelium is the primary event of transplant related complications and often precedes loss of organ function. Depending on the amount of co-stimulatory signals, endothelial cells can either act as stimulating or inhibiting antigen presenting cells (APC). On the other hand, numerous data indicate that CD4+CD25+FoxP3+ T cells (Treg cells) can attenuate alloresponses of conventional T lymphocytes against classical APC and thus qualify for clinical use in various transplant settings. However, it is unknown whether Treg cells also influence T cell – endothelial cell interactions. Defibrotide (DF) is a polydisperse mixture of single-stranded deoxyribonucleotides with anti-thrombotic and anti-inflammatory activity, known to modulate the antigenicity of vascular endothelial cells.

CD8+ T lymphocytes (CTL) were isolated by magnetic microbead separation of peripheral blood mononuclear cells (PBMC) from healthy human blood donors and stimulated with mito-inactivated cells of a human microvascular endothelial cell line (CDC/EU.HMEC-1, further referred to as EC) and other primary and transformed micro- and macrovascular ECs for 7 days in the presence of interleukin 2 (IL-2). Treg cells from the CTL donor were prepared by CD4 (untouched) and a double CD25 microbead separation as well as a CD127^bright depletion, followed by anti-CD3/CD28 expansion in the presence of IL-2 and a phenotypic quality control. Treg cells were added to the CTL-EC co-culture (1:1:1) prior to ⁵¹Cr release or flow cytometric cytotoxicity assays. Additionally, Treg cells were also tested for their capacity to influence CTL lysis of Epstein-Barr-Virus-transformed B-LCL, which as classical APC were HLA-matched to the HMEC. Furthermore, EC targets were incubated in the presence or absence of DF (25μM) for 24 hrs to assess the drug's protective function on the allogenicity of EC.

EC-stimulated CTL showed a specific MHC class I-restricted target lysis. Addition of Treg cells prior to the cytotoxicity assay and during the afferent immune phase surprisingly increased EC lysis by CTL. In contrast, Treg cells alone did not show any lytic activity against EC. As a control, conventional CD4+CD25- T cells did not influence CTL activity either. Treg cell-mediated enhancement was endothelial-specific, since B-LCL lysis was not influenced. Further subpopulation analysis revealed the existence of CD8+/CD28-/CD57+ CTL, requiring cell-to-cell contact with Treg cells for their increased activity towards EC. Importantly, DF could almost fully protect EC against lysis by allogeneic CD28- CTL and the Treg cell-mediated enhancement. Of note, DF exclusively protected EC and did influence T cell function nor viability, suggesting a strong tropism for the endothelial cell type.

There is no doubt about the potential therapeutic efficacy of Treg cells to ameliorate outcome of allogeneic transplants, but the endothelium might require additional protective interventions to prevent specific alloreactivity, such as DF.

Eissner:Gentium, Sp.A.: Consultancy. Iacobelli:Gentium SpA: Employment.