Characterization of Human CD8(+)TCR(-) Facilitating Cells In Vitro and In Vivo in a NOD/SCID/IL2rγ(null) Mouse Model.

CD8(+)/TCR(-) facilitating cells (FCs) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPCs). Human FC phenotype and mechanism of action remain to be defined. We report, for the first time, the phenotypic characterization of human FCs and correlation of phenotype with function. Approximately half of human FCs are CD8(+)/TCR(-)/CD56 negative (CD56(neg)); the remainder are CD8(+)/TCR(-)/CD56 bright (CD56(bright)). The CD56(neg) FC subpopulation significantly promotes homing of HSPCs to BM in nonobese diabetic/severe combined immunodeficiency/IL-2 receptor γ-chain knockout mouse recipients and enhances hematopoietic colony formation in vitro. The CD56(neg) FC subpopulation promotes rapid reconstitution of donor HSPCs without graft-versus-host disease (GVHD); recipients of CD56(bright) FCs plus HSPCs exhibit low donor chimerism early after transplantation, but the level of chimerism significantly increases with time. Recipients of HSPCs plus CD56(neg) or CD56(bright) FCs showed durable donor chimerism at significantly higher levels in BM. The majority of both FC subpopulations express CXCR4. Coculture of CD56(bright) FCs with HSPCs upregulates cathelicidin and ß-defensin 2, factors that prime responsiveness of HSPCs to stromal cell-derived factor 1. Both FC subpopulations significantly upregulated mRNA expression of the HSPC growth factors and Flt3 ligand. These results indicate that human FCs exert a direct effect on HSPCs to enhance engraftment. Human FCs offer a potential regulatory cell-based therapy for enhancement of engraftment and prevention of GVHD.

Posttransplantation systemic immunomodulation with SA-FasL-engineered donor splenocytes has robust efficacy in preventing cardiac allograft rejection in mice.

Apoptosis induced by the engagement of FasL with Fas receptor on the surface of lymphocytes is an important immune homeostatic mechanism that ensures tolerance to self-antigens under normal physiologic conditions. As such, FasL has been extensively tested as a tolerogenic molecule with the use of gene therapy in settings of autoimmunity and transplantation with conflicting outcomes. Although the mechanistic basis of these contradictory observations is largely unknown, the use of wild-type FasL and the means by which the gene was expressed may provide an explanation. To overcome these complications, we generated a chimeric FasL protein with streptavidin (SA-FasL) having potent apoptotic activity and displayed this molecule effectively and rapidly on biotinylated biologic membranes for immunomodulation. In the present study, we displayed SA-FasL on the surface of BALB/c splenocytes and injected 5 × 10(6) cells intraperitoneally into C57BL/6 recipients of BALB/c heart grafts on days 1, 3, and 5 after-transplantation. To control initial graft-reactive immune responses and facilitate FasL-mediated apoptosis, rapamycin was used as an immunosuppressant at 0.2 mg/kg daily for a total of 15 doses immediately after heart transplantation. All mice injected with SA-FasL-engineered donor splenocytes accepted their grafts during the 100-day observation period. In marked contrast, immunomodulation with control streptavidin protein-engineered BALB/c splenocytes had minimal effect on graft survival (mean survival, 21.4 ± 1.5 d). Taken together, these results establish posttransplantation systemic immunomodulation with SA-FasL-engineered donor splenocytes under transient cover of rapamycin as an effective regimen in preventing cardiac allograft rejection in rodents with important clinical implications.

Mouse T cells engineered to display on their surface a novel form of FasL protein undergo apoptosis when stimulated with alloantigens: implications for graft-versus-host disease.

BACKGROUND: Allogeneic bone marrow transplantation as a therapeutic approach in the clinic suffers from graft-versus- host disease (GVHD) initiated and perpetuated by donor T cells responding to alloantigens in immunocompromised hosts. Although the depletion of mature T cells from bone marrow inoculum overcomes GVHD, this manipulation is associated with engraftment failure and early post-transplant infection complications. Therefore, approaches that specifically purge out alloreactive T cells in the bone marrow inoculum without major effect on alloantigen-nonreactive T cells may be effective in facilitating engraftment without complications of GVHD and infections. METHODS: Inasmuch as Fas/FasL-induced apoptosis plays a critical role in self-tolerance, we tested whether the direct display of a novel form of FasL (SA-FasL) protein chimeric with streptavidin (SA) on the surface of T cells induces apoptosis in such cells in response to alloantigens. BALB/c and C57BL/6 total lymphocytes or purified T cells were biotinylated under physiologic conditions and engineered with SA-FasL protein taking advantage of the high-affinity interaction between biotin and SA. RESULTS: All engineered cells displayed SA-FasL protein on their surface as determined by flow cytometry. When used as responders against irradiated, unmodified allogeneic stimulators, the SA-FasL-engineered T cells underwent apoptosis, which resulted in minimal proliferation. This effect was specific to SA-FasL; control SA protein-engineered T cells generated a potent proliferative alloresponse without significant apoptosis. CONCLUSIONS: Taken together, these results demonstrate the feasibility of purging out alloreactive T cells by the display of SA-FasL protein on their surface with important implications for the prevention of GVHD associated with allogeneic bone marrow transplantation.

Immunomodulation with SA-FasL protein as an effective means of preventing islet allograft rejection in chemically diabetic NOD mice.

Allogeneic islet grafts are subject to rejection by both auto- and alloimmune responses when transplanted into diabetic individuals. T cells play a critical role in the initiation and perpetuation of both autoimmunity and allograft rejection. T cells up-regulate Fas and become sensitive to FasL-mediated killing following antigenic stimulation. Therefore, we tested if immunomodulation with an apoptotic form of FasL chimeric with streptavidin (SA-FasL) is effective in preventing the rejection of allogeneic C57BL/6 islet grafts in chemically diabetic NOD mice. C57BL/6 splenocytes and pancreatic islets were biotinylated and engineered to display the SA-FasL protein on their surface. Female NOD mice (6-7 weeks old) were treated with streptozotocin to induce diabetes and transplanted 5 days later with C57BL/6 islets engineered with SA-FasL in conjunction with transient treatment with rapamycin (3.0 mg/kg daily for days 0-19). Graft recipients were also systemically immunomodulated by intraperitoneal injection of 5 × 10(6) donor SA-FasL-engineered splenocytes on days 1, 3, and 5 after islet transplantation. This regimen resulted in the survival of all allogeneic islet grafts for the 250-day observation period, compared with a mean survival time (MST) of 14.2 ± 3.9 days for the control group. The survival effect was SA-FasL specific, with all NOD mice transplanted with control streptavidin protein-engineered islet grafts and treated with SA-engineered splenocytes under transient cover of rapamycin rejecting their grafts with an MST of 39.8 ± 8.5 days (P < .01). Taken together, these data demonstrate that immunomodulation with SA-FasL-engineered allogeneic islet grafts and splenocytes is effective in overcoming rejection in female NOD mice with preexisting autoimmunity with important clinical implications.

Engineering of bone marrow cells with fas-ligand protein-enhances donor-specific tolerance to solid organs.

Effective immunomodulation to induce tolerance to tissue/organ allografts is attained by infusion of donor lymphocytes endowed with killing capacity through ectopic expression of a short-lived Fas-ligand (FasL) protein. The same approach has proven effective in improving hematopoietic stem and progenitor cell engraftment. This study evaluates the possibility of substitution of immune cells for bone marrow cells (BMC) to induce FasL-mediated tolerance to solid organ grafts. Expression of FasL protein on BMC increased the survival of simultaneously grafted vascularized heterotopic cardiac grafts to 90%, as compared to 30% in recipients of naïve BMC. Similar results were obtained for skin allografts implanted into radiation chimeras at 1 week after bone marrow transplantation. Further reduction of preparative conditioning to busulfan resulted in acceptance of donor skin implanted at 2 weeks after transplantation of naïve and FasL-coated BMC, whereas third-party grafts were acutely rejected. The levels of donor chimerism were in the range of 0.7% to 12% at the time of skin grafting, with higher levels in recipients of FasL-coated BMC. It is concluded that FasL-mediated abrogation of alloimmune responses can be effectively attained with BMC. There is no threshold of donor chimerism, but tolerance to solid organs evolves during the process of donor-host mutual acceptance.

Tumor cells engineered to codisplay on their surface 4-1BBL and LIGHT costimulatory proteins as a novel vaccine approach for cancer immunotherapy.

Primary tumor cells genetically modified to express a collection of immunological ligands on their surface may have the utility as therapeutic autologous cancer vaccines. However, genetic modification of primary tumor cells is not only cost, labor and time intensive, but also has safety repercussions. As an alternative, we developed the ProtEx technology that involves generation of immunological ligands with core streptavidin (SA) and their display on biotinylated cells in a rapid and efficient manner. We herein demonstrate that TC-1 tumor cells can be rapidly and efficiently engineered to codisplay on their surface two costimulatory proteins, SA-4-1BBL and SA-LIGHT, simultaneously. Vaccination with irradiated TC-1 cells codisplaying both chimeric proteins showed 100% efficacy in a prophylactic and >55% efficacy in a therapeutic tumor setting. In contrast, vaccination with TC-1 cells engineered with either protein alone showed significantly reduced efficacy in the prophylactic setting. Vaccine efficacy was associated with the generation of primary and memory T-cell and antibody responses against the tumor without detectable signs of autoimmunity. Engineering tumor cells in a rapid and effective manner to simultaneously display on their surface a collection of immunostimulatory proteins with additive/synergistic functions presents a novel alternative approach to gene therapy with considerable potential for cancer immunotherapy.