Induction of Tolerance to Cardiac Allografts Using Donor Splenocytes Engineered to Display on Their Surface an Exogenous Fas Ligand Protein

Abstract

The critical role played by Fas ligand (FasL) in immune homeostasis renders this molecule an attractive target for immunomodulation to achieve tolerance to auto- and transplantation Ags. Immunomodulation with genetically modified cells expressing FasL was shown to induce tolerance to alloantigens. However, genetic modification of primary cells in a rapid, efficient, and clinically applicable manner proved challenging. Therefore, we tested the efficacy of donor splenocytes rapidly and efficiently engineered to display on their surface a chimeric form of FasL protein (SA-FasL) for tolerance induction to cardiac allografts. The i.p. injection of ACI rats with Wistar-Furth rat splenocytes displaying SA-FasL on their surface resulted in tolerance to donor, but not F344 third-party cardiac allografts. Tolerance was associated with apoptosis of donor reactive T effector cells and induction/expansion of CD4+CD25+FoxP3+ T regulatory (Treg) cells. Treg cells played a critical role in the observed tolerance as adoptive transfer of sorted Treg cells from long-term graft recipients into naive unmanipulated ACI rats resulted in indefinite survival of secondary Wistar-Furth grafts. Immunomodulation with allogeneic cells rapidly and efficiently engineered to display on their surface SA-FasL protein provides an effective and clinically applicable means of cell-based therapy with potential application to regenerative medicine, transplantation, and autoimmunity.

Killer Treg restore immune homeostasis and suppress autoimmune diabetes in prediabetic NOD mice

“…Fourth, FasL can be employed for immunomodulation in conjunction with other strategies aiming to sensitize, expand and enhance the suppressive activity of Treg to arrest autoimmunity [16,26e28]. FasL is a physiological constituent of apoptotic signals transduced by human and murine adaptive Treg cells [13] and has the significant advantage of selective and antigen-specific elimination of autoreactive and alloreactive effector cells [18,19,23,32,53]. Finally, the most prominent feature of CD25 þ -FasL immunomodulation is to create a substantial disease-free window of opportunity during which additional approaches to definitive arrest of autoimmunity might be implemented prior to institution of therapies to replenish the damaged tissue [54].…”

Immunomodulation With SA-FasL Protein as an Effective Means of Preventing Islet Allograft Rejection in Chemically Diabetic NOD Mice

“…Recombinant SA-FasL and streptavidin (SA) proteins were produced in the laboratory using the Drosophila Expression System (DES) expression system (Life Technologies). 8 …”

“…Spleens were harvested from 8–12-week-old C57BL/6 females or males, processed into single-cell suspensions, and engineered with SA-FasL as previously described. 8 Splenocytes engineered with an equimolar amount of SA protein served as control. The levels of SA-FasL and SA on the cell surface were assessed using fluorescein isothiocyanate–labeled antistreptavidin antibodies in flow cytometry.…”

A Clinically Feasible Approach to Induce Delayed Tolerance in Recipients of Prior Kidney or Vascularized Composite Allotransplants

“…Intracellular IFN-F expression of CD4 + and CD8 + T cells from the naive animals and the KTx or VCA recipients was tested as previously described (28).…”