Long-term display of exogenous proteins on the cell surface may have important research and therapeutic implications. We report a novel method for the cell-surface display of proteins that involves generation of a chimeric protein with core streptavidin, biotinylation of cells, and "decoration" with the protein. A chimeric protein with the extracellular portions of FasL (SA-FasL) was efficiently displayed on the cell surface within 2 hr without detectable cellular toxicity. Biotin and SA-FasL persisted on the cell surface for weeks in vitro and in vivo. Immunomodulation with SA-FasL-decorated splenocytes effectively blocked alloreactive responses in naive and presensitized rodents and prevented the rejection of allogeneic pancreatic islets. This approach may serve as an alternative to gene transfer-based expression with broad research and therapeutic applications.

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Fas/FasL system and immune homeostasisThe immune system functions in complex and dynamic microenvironments, where decisions pertaining to the survival and/or physical elimination of antigen-specific T-cell clones are critical to the establishment and maintenance of functional immunity. Activationinduced cell death (AICD) is the primary homeostatic mechanism used by the immune system to control T-cell responses, promote tolerance to self-antigens, and prevent autoimmunity. [1][2][3] Following activation, T cells express Fas and Fas ligand (FasL) and upon repeated antigenic stimulation become sensitive to Fas/FasLmediated autocrine and paracrine apoptosis (Figure 1). [4][5][6] These 2 modes of apoptotic cell death serve to control the pool size of antigen-activated T-cell clones and regulate immune responses. 7 All lymphocytes, including CD4 ϩ and CD8 ϩ T, B, and natural killer (NK) cells as well as dendritic cells, monocytes, macrophages, and neutrophils are subject to Fas/FasL-regulated immune homeostasis. [8][9][10][11] Dysregulation of Fas/FasL-mediated AICD is associated with a series of pathophysiologic conditions, including degenerative and autoimmune disorders. 12 Mouse strains that lack Fas (lpr) or express a defective FasL molecule (gld) suffer from severe lymphoproliferative disorders and autoimmunity, 12,13 arising from the absence of antigen-induced clonal T-cell deletion in the periphery. 14,15 Similarly, the autoimmune lymphoproliferative syndrome in humans, an inherited disorder of lymphocyte homeostasis, is associated with the lack of AICD due to defects in Fas, FasL, and effector caspases. 16 Fas is a 45-kDa, type I cell surface protein with an extracellular domain that binds to FasL and a cytoplasmic domain that transduces the death signal. 17 Apoptosis is executed by the engagement and coaggregation of FasL with the Fas receptor on the cell surface followed by a series of intracellular molecular interactions that coordinate the hierarchical activation of caspases and cell death (Figure 2). Oligomerization of Fas following FasL engagement leads to the recruitment of Fas-associated proteins having death domains and the initiator procaspase-8 via its death effector domain into a death-inducing signaling complex (DISC). [18][19][20] Procaspase-8 undergoes autoproteolysis in the DISC complex to generate an active caspase-8, which in turn initiates extrinsic apoptosis by converting inactive effector procaspases-3, -6, and -7 into active enzymes via transproteolysis and intrinsic apoptosis via cleavage of Bid, release of cytochrome c, and activation of caspase-9. 18,21 Executioner caspases cleave various vital cellular substrates, which leads to cell death.FasL is a 40-kDa, type II cell surface protein that is inducibly expressed in lymphocytes, particularly T cells, 22 and constitutively expressed in cells present in immune privileged organs, such as Sertoli cells of the testis and epithelial cells in the eye. 23,24 Inasmuch as FasL-mediated apoptosis is critical to peripheral T-cell homeostasis...

Allogeneic islet transplantation is an important therapeutic approach for the treatment of T1D. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic T effector cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating T effector cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of FasL protein chimeric with streptavidin (SA-FasL), and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for over a week in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% C57BL/6 recipients. Tolerance was initiated and maintained by CD4+CD25+FoxP3+ T regulatory (Treg) cells as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of T1D.