Mary E. White-Scharf

Pharmacologic immunosuppressive therapy and extracorporeal immunoadsorption in the suppression of anti-αGal antibody in the baboon

Abstract: The aim of this study was to deplete baboons of anti‐αgalactosyl (αGal] antibody and attempt to maintain depletion by pharmacologic immunosuppressive therapy (PI). In 12 experiments, involving nine baboons, repeated extracorporeal immunoadsorption (EIA) was carried out by plasma perfusion through immunoaffinity columns of synthetic αGal trisaccharide type 6. Five of the baboons were immunologically naive and four had undergone various procedures at least 6 months previously. All, however, had recovered lymphohematopoietic function and (with one exception) had levels of anti‐αGal antibody within the normal range. Eleven protocols included continuous i.v. cyclosporine (to maintain whole blood levels of approximately 1,600 ng/ml). In addition, in ten protocols, the baboon received one or more of the following drugs: cyclophosphamide (1–20 mg/kg/day), mycophenolate mofetil (70–700 mg/kg/day), brequinar sodium (1–12 mg/kg/day), prednisolone (1 mg/kg/day), melphalan (0.15–0.6 mg/kg/day), methylprednisolone (125 mg/day ×3), and antilymphocyte globulin (ATG) (50 mg/kg/day ×3). EIA was carried out on 1–9 occasions in each study and was temporarily successful in removing all antibody. When no PI was administered, antibody returned close to pre‐EIA levels within 48 hr. Cyclosporine alone delayed the rate of antibody return only slightly. While EIA was continuing on a daily or alternate day schedule, antibody levels (both IgM and IgG) were maintained at 20–45% of pre‐EIA levels. Once EIA was discontinued but PI maintained, IgM rose to 40–90% and IgG to 30–60% of pre‐EIA levels. In vitro testing demonstrated significant cytotoxicity to pig cells at these antibody levels. We conclude that i) EIA utilizing columns of αGal trisaccharide is successful in temporarily depleting baboons of anti‐αGal antibody, but ii) none of the PI regimens tested suppressed antibody production to levels which would be expected to prevent antibody‐mediated rejection of pig xenografts. Additional strategies will therefore be required if xenotransplantation is to become a clinical reality.

Effects of specific anti-B and/or anti-plasma cell immunotherapy on antibody production in baboons: depletion of CD20- and CD22-positive B cells does not result in significantly decreased production of anti-alphaGal antibody.

Abstract: Anti‐Galα1–3Gal antibodies (antiαGal Ab) are a major barrier to clinical xenotransplantation as they are believed to initiate both hyperacute and acute humoral rejection. Extracorporeal immunoadsorption (EIA) with αGal oligosaccharide columns temporarily depletes antiαGal Ab, but their return is ultimately associated with graft destruction. We therefore assessed the ability of two immunotoxins (IT) and two monoclonal antibodies (mAb) to deplete B and/or plasma cells both in vitro and in vivo in baboons, and to observe the rate of return of antiαGal Ab following EIA.

Clearance of mobilized porcine peripheral blood progenitor cells is delayed by depletion of the phagocytic reticuloendothelial system in baboons

Introduction. Attempts to achieve immunological tolerance to porcine tissues in nonhuman primates through establishment of mixed hematopoietic chimerism are hindered by the rapid clearance of mobilized porcine leukocytes, containing progenitor cells (pPBPCs), from the circulation. Eighteen hours after infusing 1–2×1010 pPBPC/kg into baboons that had been depleted of circulating anti-&agr;Gal and complement, these cells are almost undetectable by flow cytometry. The aim of the present study was to identify mechanisms that contribute to rapid clearance of pPBPCs in the baboon. This was achieved by depleting, or blocking the Fc-receptors of, cells of the phagocytic reticuloendothelial system (RES) using medronate liposomes (MLs) or intravenous immunoglobulin (IVIg), respectively. Methods. Baboons (preliminary studies, n=4) were used in a dose-finding and toxicity study to assess the effect of MLs on macrophage depletion in vivo. In another study, baboons (n=9) received a nonmyeloablative conditioning regimen (NMCR) aimed at inducing immunological tolerance, including splenectomy, whole body irradiation (300 cGy) or cyclophosphamide (80 mg/kg), thymic irradiation (700 cGy), T-cell depletion, complement depletion with cobra venom factor, mycophenolate mofetil, anti-CD154 monoclonal antibody, and multiple extracorporeal immunoadsorptions of anti-&agr;Gal antibodies. The baboons were divided into three groups: Group 1 (n=5) NMCR+pPBPC transplantation; Group 2 (n=2) NMCR+ML+pPBPC transplantation; and Group 3 (n=2) NMCR+IVIg+pPBPC transplantation. Detection of pig cells in the blood was assessed by fluorescence-activated cell sorter and polymerase chain reaction (PCR). Results. Preliminary studies: ML effectively depleted macrophages from the circulation in a dose-dependent manner. Group 1: On average, 14% pig cells were detected 2 hr postinfusion of 1×1010 pPBPC/kg. After 18 hr, there were generally less than 1.5% pig cells detectable. Group 2: Substantially higher levels of pig cell chimerism (55–78%) were detected 2 hr postinfusion, even when a smaller number (0.5–1×1010/kg) of pPBPCs had been infused, and these levels were better sustained 18 hr later (10–52%). Group 3: In one baboon, 4.4% pig cells were detected 2 hr after infusion of 1×1010 pPBPC/kg. After 18 hr, however, 7.4% pig cells were detected. A second baboon died 2 hr after infusion of 4×1010 pPBPC/kg, with a total white blood cell count of 90,000, of which 70% were pig cells. No differences in microchimerism could be detected between the groups as determined by PCR. Conclusions. This is the first study to report an efficient decrease of phagocytic function by depletion of macrophages with MLs in a large-animal model. Depletion of macrophages with MLs led to initial higher chimerism and prolonged the survival of circulating pig cells in baboons. Blockade of macrophage function with IVIg had a more modest effect. Cells of the RES, therefore, play a major role in clearing pPBPCs from the circulation in baboons. Depletion or blockade of the RES may contribute to achieving mixed hematopoietic chimerism and induction of tolerance to a discordant xenograft.

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.

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 patients 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.

Identification of swine and primate cellular adhesion molecules (CAM) using mouse anti‐human monoclonal antibodies

Abstract: A series of adhesion molecules of swine and Cynomolgus monkeys were identified by screening for cross‐reactivity with a panel of monoclonal murine anti‐human adhesion molecule antibodies obtained from the 5th International Workshop and Conference on Human Leukocyte Differentiation Antigens (Boston, MA, USA, 1993). Of 162 antibodies tested, 25 were found that cross‐react significantly with swine cells, while 67 were found to react strongly with cells of Cynomolgus monkeys. Cross reactivities to swine were found with antibodies to CD 18 (β2 integrin), CD29 (β1 integrin), β7 integrin, CD49d (α4 integrin) CD49b(α2 integrin), and to a lesser extent to CD62p(P‐selectin), CD62L(L‐selectin), CD102(ICAM‐2), CD11b, and CD49c (α3 integrin). Cross‐reactivities to primate cells also included CD18, CD29, CD49d, and CD49b. In addition, reactivities to Cynomolgus monkey cells were detected with antibodies to CD11 (a, b, and c), CD31, CD44, CD49e, CD49f, CD50, CD54, CD56, CD62p, CD 102 and CD56. The tissue distribution and molecular weight of the swine antigens are similar to their human counterparts. These findings provide a spectrum of monoclonal antibodies that react with shared epitopes on homologous adhesion molecules of human, swine, and monkey cells, thus facilitating study of the role of these molecules in the immunobiology of monkeys and swine. These reagents may be useful to dissect the role of adhesion molecules in both alio‐ and xenoreactivity.

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-&bgr;) and tumor necrosis factor-alpha (TNF-&agr;) 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-&agr; or TGF-&bgr; 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-&agr; and TGF-&bgr; to porcine cultures. Conclusions. Porcine cells may be more sensitive to inhibitory effects of TNF-&agr; and TGF-&bgr; 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.

Cryopreservation and mycophenolate therapy are detrimental to hematopoietic progenitor cells

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.

In vitro and in vivo investigation of a novel monoclonal antibody to plasma cells (W5 mAb)

Abstract:u2002 Natural antibodies (Abs), predominately anti‐Galα1–3Gal (Gal) Abs, in non‐human primates and human beings present a major hurdle to successful pig‐to‐primate xenotransplantation. Attempts to inhibit anti‐Gal Ab production in naïve baboons using non‐specific immunosuppressive or B cell‐specific reagents have failed. A new rat monoclonal antibody (W5 mAb) has been generated, which binds to all B cells, including memory cells, and to the majority of plasma cells, but not to T cells. It has been tested in vitro and in vivo. By immunoprecipitation, W5 mAb bound a human leukocyte antigen class II (HLA‐DR) determinant. Sorting splenic or bone marrow W5+ cells resulted in a highly enriched anti‐Gal Ab and total immunoglobulin (Ig)‐secretory population. In vivo studies in baboons demonstrated that W5 mAb was safe but, despite the concomitant administration of an anti‐CD154 mAb to inhibit sensitization, anti‐rat Abs were detected within 10u2003days and inhibited the effect of the W5 mAb. High levels of W5 mAb were able to completely deplete B cells in the blood, but not in lymphoid tissues. Enzyme‐linked spot‐forming assay (ELISPOT) demonstrated that only 50 to 60% of secreting cells (SC) were depleted in the bone marrow. No reduction in the serum levels of anti‐Gal Ab was observed. W5 mAb did not cause complete inhibition of anti‐Gal Ab production, probably as a result of its inability to completely deplete B and plasma cells from all lymphoid compartments.