Immunomodulation with genetically engineered cells expressing FasL has been extensively tested for the induction of tolerance to auto and alloantigens with reported efficacy in various preclinical settings (14
). However, the translation of this approach to the clinic remains to be realized primarily due to difficulties associated with efficient and rapid manipulation of donor primary cells under clinically applicable conditions to reproducibly express FasL and safety concerns of the gene therapy. Furthermore, FasL has pleiotropic effects on immune and nonimmune cells, and as such long-term stable expression of this molecule using gene therapy might have detrimental consequences. In the present study, we overcame these difficulties by transient display of a recombinant form of FasL protein with potent apoptotic activity (25
) on donor splenocytes in a rapid (~ 2 hours ) and efficient manner (~ 100% of the targeted cells). Systemic immunomodulation with FasL-engineered donor cells resulted in peripheral tolerance to cardiac allografts without the use of any additional immunosuppression.
Although, a series of studies using various antigen presenting cells genetically engineered to express FasL for immunomodulation reported alloantigen-specific immune nonresponsiveness, none of these studies demonstrated that such nonresponsiveness eventually leads to long-term allograft tolerance (11
). To our knowledge, this is the first study to demonstrate that systemic immunomodulation with donor cells engineered to express FasL on their surface induces long-term peripheral tolerance to cardiac allografts in a totally allogeneic rat strain combination. The mechanistic basis of the induced peripheral tolerance involves physical elimination of alloreactive cells by AICD early after transplantation and maintenance of tolerance by CD4+
Treg cells. The enhanced immunomodulatory effect of repeated splenocyte infusion reported in the present communication can be attributed to effective elimination of alloreactive immune effector cells at remote sites from the graft, robust induction/expansion of Treg cells, or both. T cells require antigen-specific activation and several rounds of sensitization to acquire sensitivity to Fas/FasL-mediated apoptosis (10
). Also, it is well-established that memory immune cells reside within non-lymphoid target tissues, plausibly to generate a rapid and effective secondary immune response to recurrent infection (41
). Therefore, systemic and repeated administration of engineered donor splenocytes may have the advantage of tolerazing compartmentalized memory responses defined as heterologous immunity that serves a major barrier for tolerance induction in the clinic (4
In our view, induction of CD4+
Treg cells using SA-FasL-engineered donor splenocytes is the most significant finding of the present study. The essential role of Treg cells in tolerance was demonstrated by adoptive transfer experiments where cells sorted from lymphoid organs of long-term graft acceptors transferred tolerance to naïve, unmanipulated secondary recipients (). Importantly, CD4+
T cells sorted from acutely rejecting animals did not prevent the rejection of donor grafts in naive secondary recipients following adoptive transfer. This may be because the sorted CD4+
Treg cells were not donor specific, contaminated with newly activated alloreactive Teff cells expressing CD25, or rat Teff cells transiently express FoxP3 following activation as reported for human Teff cells (42
). The presence of T regulatory cells, other than CD4+
Treg cells, in our model is consistent with our observations that Treg cell-depleted total splenocytes from SA-FasL-treated long-term graft survivors did not prevent the rejection of donor grafts upon adoptive transfer into secondary naïve graft recipients, but caused significant prolongation as compared with controls (). These regulatory T cells may include CD4−
T cells (43
T cells (44
), and/or newly described CD8+
T cells expressing IFN-γ (45
). These cell types are also implicated in allotransplantation achieved using donor-specific transfusion. For example, infusion of allogeneic donor splenocytes into graft recipients was shown to generate CD4−
T regulatory cells that used FasL to physically eliminate alloreactive T cells for tolerance induction (43
). Furthermore, tolerance to skin allografts disparate for H-Y antigen has recently been shown to require FasL on donor cells used for infusion and Fas in graft recipients (46
). Therefore, Fas/FasL system may serve as a common denominator of mechanisms responsible for tolerance achieved by immunomodulation of graft recipients using donor-specific transfusion in form of donor lymphocytes.
We envision three possible mechanisms for the induction/expansion of Treg cells by SA-FasL-engineered splenocytes. First, FasL interaction with Fas upregulated on the surface of Treg cells in response to donor antigens may transduce a mitogenic signal, leading to their expansion. Although Fas signaling was shown to be involved in the physiological process of T effector cell activation under selected conditions (47
), it remains to be determined if such a function also applies to Treg cells. Second, Treg cells may be less sensitive to Fas-mediated apoptosis as compared with T effector cells. Immunomodulation with SA-FasL-engineered splenocytes may preferentially eliminate T effector cells, thereby tipping the balance towards the expansion of Treg cells. However, the sensitivity of Treg cells to Fas/FasL-mediated apoptosis has been the subject of few studies with conflicting observations. While some studies reported that Treg cells are more sensitive to FasL-mediated apoptosis than T effector cells (49
), we (51
) and others indicated the exact opposite (52
). These conflicting observations may be due to the study designs, such as lack of comparative analysis of Treg and T effector cell sensitivity to FasL-mediated apoptosis in the course of an immune response to antigens under inflammatory conditions. Third, T effector cells undergoing apoptosis may give rise to the generation of adaptive Treg cells or expansion of naturally occurring Treg cells. Consistent with this notion are the observations that apoptotic lymphocytes release two cytokines, IL-10 and TGF-β, that play important roles in the generation and function of Treg cells (53
). Furthermore, apoptotic bodies from dying cells were shown to contribute to the generation of Treg cells through mechanisms that involved TGF-β and macrophages (54
). Further studies are needed to establish mechanisms that are responsible for the generation of Treg cells in our model and if these cells are of adaptive or natural type.
The immunomodulatory approach presented here has several attractive features with direct relevance to the clinic. First, it allows for rapid, efficient, and transient display of exogenous immunomodulatory proteins on the cell membrane with immediate function without a time lag required for gene transfer-based expression. Second, the transient display of immunomodulatory proteins with pleiotropic effects may minimize the potential undesired effects arising from their stable and long-term expression achieved by gene therapy. Third, several proteins with synergistic functions may be simultaneously displayed on the cell surface to maximize their therapeutic efficacy (Sharma and Shirwan, unpublished data). In particular, our approach may be incorporated into cell-based immunomodulation approaches, such as donor-specific leukocyte transfusion or hematopoietic stem cell transplantation, for tolerance induction in the clinic. Infusion of unmodified donor leukocytes or bone marrow cells into graft recipients for the purpose of immunomodulation has been extensively tested with observed beneficial effects in various preclinical and clinical settings (55
). The presence of SA-FasL on donor leukocytes and bone marrow cells may further improve their therapeutic efficacy by eliminating alloreactive pathogenic lymphocytes and/or inducing peripheral immunoregulatory mechanisms as shown by the present study. In conclusion, this protein display approach possesses the simplicity, safety, and efficacy required to make it a clinically relevant and practical alternative to gene therapy for immunomodulation with broad application to cell-based therapies, regenerative medicine, autoimmunity, and transplantation.