Addition of treosulfan to a nonmyeloablative conditioning regimen results in enhanced chimerism and immunologic tolerance in an experimental allogeneic bone marrow transplant model.

Treosulfan (L-threitol-1,4-bismethanesulfonate) is an alkylating agent with routine clinical application in the treatment of ovarian cancer. In this murine study we show that this drug also has the ability to deplete primitive hematopoietic stem cells in a dose-dependent manner as determined by the cobblestone area-forming cell assay and is similar to its parent compound busulfan. Because busulfan is frequently used as part of the conditioning regimen before stem cell transplantation, we investigated an alternative nonmyeloablative protocol in an allogeneic bone marrow transplantation model in which low-dose treosulfan was added to an immune-suppressive regimen consisting of T cell-depleting antibodies, fludarabine, and thymic irradiation. Although this treatment protocol produced minimal myelosuppression, the addition of treosulfan proved to be important for allowing stable multilineage and mixed chimerism in C57BL/6 recipients of major histocompatibility complex-mismatched B10.A bone marrow without evidence of graft-versus-host disease. Donor lymphocyte infusion performed at 10 weeks after bone marrow transplantation had the effect of transforming the state of mixed chimerism to full donor-type cells, again without evidence of graft-versus-host disease. Donor-specific immunologic tolerance in the mixed chimeric animals was indicated by the acceptance of donor-type and rejection of third-party skin grafts. Thus, low-dose treosulfan may be considered as a useful component of a truly nonmyeloablative conditioning protocol in providing for mixed hematopoietic chimerism and, consequently, in establishing a platform for adoptive immunotherapy.

Engraftment of retroviral EGFP-transduced bone marrow in mice prevents rejection of EGFP-transgenic skin grafts.

We have investigated whether a state of tolerance toward EGFP-expressing skin tissue can be induced by prior establishment of EGFP molecular chimerism by transplant of gene-transduced bone marrow in mice. Irradiated (10 Gy) C57BL/6J mice were transplanted with bone marrow cells transduced with two different retroviral vectors encoding EGFP. EGFP-transduced, mock-transduced, and age-matched control mice received skin grafts from both C57BL/6 EGFP-transgenic (B6-EGFP. Tg) and MHC-mismatched B10.A donor mice at 8, 29, or 39 weeks after bone marrow transplantation. Although 14 of 17 control mice rejected EGFP.Tg skin grafts within 100 days, 24 of 25 mice receiving EGFP-expressing bone marrow cells accepted their B6-EGFP.Tg grafts out to 200 days after skin grafting, including animals with undetectable levels of EGFP expression in blood cells. The EGFP-transduced animals rejected third-party grafts from MHC-mismatched mice within 20 days, indicating that acceptance of the EGFP-expressing skin grafts was the result of the induction of specific and operational immune tolerance. Thus, our data indicate that (a) EGFP-expressing tissue elicits an immunological rejection in C57BL/6 mice and (b) tolerance can be induced by engrafting relatively small numbers of EGFP-transduced hematopoietic cells. These experiments utilizing EGFP as an immunogen point to the wider therapeutic potential of employing transplantation of gene-transduced hematopoietic cells for establishing immunological tolerance and thereby preventing rejection of gene-corrected cells and tissues.

Nonmyeloablative conditioning is sufficient to allow engraftment of EGFP-expressing bone marrow and subsequent acceptance of EGFP-transgenic skin grafts in mice.

Immunologic reactions against gene therapy products may prove to be a frequent problem in clinical gene therapy protocols. Enhanced green fluorescence protein (EGFP) is commonly used as a marker in gene transfer protocols, and immune responses against EGFP-expressing cells have been documented. The present study was designed to investigate the effect of a pharmacologic, nonmyeloablative, conditioning regimen on the development of EGFP+ donor/recipient mixed bone marrow chimerism and ensuing tolerance to EGFP-expressing transplants. To this end, C57BL/6J (B6) mice were treated with soluble formulations of either busulfan (Busulfex) or the closely related compound treosulfan, followed by transplantation of bone marrow cells from EGFP-transgenic (B6-EGFP.Tg) donor mice. Such conditioning regimens resulted in long-term persistence of donor EGFP+ cells among various hematopoietic lineages from blood, bone marrow, and thymus. Stable hematopoietic chimeras transplanted at 10 to 17 weeks after bone marrow transplantation (BMT) with B6-EGFP.Tg skin grafts all accepted their transplants, whereas non-EGFP chimeric B6 control animals were able to mount rejection of the EGFP+ B6 skin grafts. Control third-party grafts from major histocompatibility complex (MHC)-mismatched mice were rejected within 20 days, indicating that acceptance of EGFP-expressing skin grafts was the result of specific immune tolerance induction by the transplantation of EGFP-transgenic bone marrow. Long-term tolerance to EGFP in chimeric recipients was confirmed by the absence of anti-EGFP-reactive T cells and antibodies. These results broaden the therapeutic potential for using hematopoietic molecular chimerism in nonmyeloablated recipients as a means of preventing rejection of genetically modified cells.

Reprogramming immune responses: enabling cellular therapies and regenerative medicine.

Recent advances in cellular therapies have led to the emergence of a multidisciplinary scientific approach to developing therapeutics for a wide variety of diseases and genetic disorders. Although most cell-based therapies currently consist of heterogeneous cell populations, it is anticipated that the standard of care will eventually be well-characterized stem cell lines that can be modified to meet the individual needs of the patient. Many challenges have to be overcome, however, before such "designer cells" can become a clinical reality. One of the major hurdles will be to prevent immune rejection of the therapeutic cells. A patient's immune system may react to genetically modified or allogeneic cells as foreign, leading to their destruction. We propose that specific reprogramming of the immune system to accept cellular therapies can be accomplished by establishing hematopoietic chimerism. Successful engraftment of hematopoietic stem cells (HSCs), which have the same origin as those cells intended for therapeutic use, should lead to a re-education of the immune system so that the donor cells are recognized as self and will not be rejected. Developing safe, nontoxic protocols for reprogramming the immune system is critical to the success of this approach. Two major requirements exist for achieving stable HSC engraftment: (A) depletion or displacement of host stem cells, and (B) adequate immune suppression. Available data indicate that an agent such as busulfan is effective in depleting stem cells and that immune suppression can be accomplished with monoclonal antibodies that specifically target immune-reactive cells in the periphery.

Cryopreservation and mycophenolate therapy are detrimental to hematopoietic progenitor cells.

BACKGROUND: The aim of the present study was to determine whether certain components of nonmyeloablative regimens for hematopoietic cell transplantation might compromise the growth of hematopoietic progenitors. METHODS: Porcine peripheral blood progenitor cells (PBPC) were cytokine-mobilized, collected by leukapheresis, and cryopreserved using 5% dimethyl sulfoxide and 6% hydroxyethyl starch. The influence of cryopreservation on PBPC was tested in vitro by enumeration of colony-forming units (CFUs) in methylcellulose and cobblestone area-forming cell (CAFC) subsets in stromal-associated long-term cultures on fresh and frozen PBPC. The effects of mycophenolate mofetil (MMF) on porcine PBPC and baboon and human bone marrow (BM) were tested in vitro by adding varying doses of MMF to the CFU assays. One baboon was treated with increasing doses of MMF (100-500 mg/kg per day continuously intravenous), and sequential BM aspirations were tested for CFU content. RESULTS: Fresh cytokine-mobilized PBPC had similar frequencies of progenitor cells when compared with porcine BM. Freezing-thawing of PBPC had no effect on porcine CFUs but reduced the recovery of CAFCs by more than 90%. In vitro, MMF completely inhibited the development of porcine and human CFUs at a concentration of 1 microg/mL and of baboon CFUs at levels between 10 and 100 microg/mL. Plasma-free mycophenolic acid levels of 10 to 30 microg/mL were associated with decreased CFUs in the BM. CONCLUSIONS: Cryopreservation and MMF potentially prevent engraftment of porcine PBPC by reducing the content or development of progenitor cells. These results indicate that the use of fresh PBPC might improve the induction of mixed hematopoietic chimerism and raise the possibility that use of high doses of MMF in the poststem cell transplant may compromise engraftment.

Porcine hematopoiesis on primate stroma in long-term cultures: enhanced growth with neutralizing tumor necrosis factor-alpha and tumor growth factor-beta antibodies.

BACKGROUND: Donor hematopoiesis is at a competitive disadvantage when bone marrow transplantation is across species barriers. This could present major limitations to xenogeneic stem cell transplantation as an approach to tolerance induction. An in vitro model of xenogeneic engraftment was established to identify inhibitors of porcine hematopoiesis in a primate environment. METHODS: Porcine bone marrow cells (BMC), in the presence or absence of primate CD34+ positive cells, were cultured for 4-6 weeks on primate stroma with porcine cytokines. Cellularity and growth of colony-forming cells were indicators of hematopoietic growth. Effects of soluble factors were determined by using Transwell inserts to separate porcine cells from stroma. Neutralizing antibodies for human transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) were added to cultures. RESULTS: Porcine hematopoiesis can be maintained in long-term cultures on primate stroma with pig cytokines. Adding BMC to the stroma below Transwell-containing porcine cells dramatically inhibited porcine hematopoiesis, showing an inhibitory role for soluble factors. Neutralizing antibodies against TNF-alpha or TGF-beta caused a modest enhancement of porcine hematopoiesis; however, the combination of both led to a substantial increase. Inhibitory effects of these cytokines were confirmed by adding TNF-alpha and TGF-beta to porcine cultures. CONCLUSIONS: Porcine cells may be more sensitive to inhibitory effects of TNF-alpha and TGF-beta than primate cells and are at a disadvantage when in a primate environment. Potential implications of this observation are discussed in the context of establishing specific immune tolerance via mixed chimerism to facilitate xenotransplantation.

Expression of xenogeneic MHC class II molecules in HLA-DR(+) and -DR(-) cells: influence of retrovirus vector design and cellular context.

We recently established that molecular chimeras of major histocompatibility complex (MHC) class II molecules, created via retroviral transfer of allogeneic class II cDNAs into bone marrow cells (BMCs), alleviated complications associated with mixed BMC chimeras while leading to T cell tolerance to renal grafts sharing the transferred class II. Initially demonstrated for allogeneic transplants in miniature swine, this concept was extended to T-dependent antibody (Ab) responses to xenogeneic antigens (Ags) in the pig --> baboon combination. Successful down-regulation of T cell responses appeared, however, to be contingent on a tight lineage-specific expression of transferred class II molecules. The present studies were, therefore, designed to evaluate the influence of construct design and cellular environment on expression of retrovirally transferred xenogeneic class II cDNAs. Proviral genomes for pig class II SLA-DR expression, differing only at the marker neo(r) or enhanced green fluorescent protein (EGFP) gene, showed increased membrane SLA-DR density on HLA-DR(-) fibroblasts as well as HLA-DR(+), TF-1 erythroleukemia cells. More importantly, HLA-DR(+) human B cell lines, although efficiently transduced with pig DR retroviruses, exhibited unstable surface pig DR. Surface pig DR- B cells, nevertheless, stimulated autologous human T cells pre-sensitized to pig Ags, a proliferation likely occurring through presentation of class II-derived peptides. Collectively, these data suggest that surface expression of transferred class II molecules is not related to the ability of recipient cells to synthesize xenogeneic class II molecules but rather to their Ag processing capacities.

Porcine endogenous retrovirus transmission characteristics of an inbred herd of miniature swine.

Here we report the identification of inbred miniature swine that failed to produce human-tropic replication-competent porcine endogenous retroviruses (HTRC PERVs), using in vitro coculture assays. When HTRC PERVs were isolated from transmitting animals, all were recombinant viruses, with the receptor-binding domain of PERV-A combining with PERV-C-related sequences.