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Biomedical subjects

Megan Sykes

Publications and source records attributed to Megan Sykes.

At least 73 records · Page 4Linked to original sources
PubMed2003

Donor lymphocyte infusion-mediated graft-versus-leukemia effects in mixed chimeras established with a nonmyeloablative conditioning regimen: extinction of graft-versus-leukemia effects after conversion to full donor chimerism.

BACKGROUND: We investigated an approach to separating graft-versus-lymphoma (GVL) effects from graft-versus-host disease (GVHD) in mice receiving a nonmyeloablative conditioning regimen allowing establishment of mixed hematopoietic chimerism. METHODS: We evaluated the ability of donor lymphocyte infusions (DLI) to mediate GVL effects without GVHD in mixed chimeras prepared with cyclophosphamide, anti-T-cell antibodies, and thymic irradiation. To examine the fate of GVH-reactive donor CD8+ T cells, we used the 2C T-cell receptor (TCR) transgenic mouse strain, which carries an Ld-specific transgenic TCR on the B6 background. RESULTS: Administration of DLI on day 35 post-BMT led to conversion from mixed to full donor chimerism and mediated a powerful GVL effect with complete protection (100% survival) against mortality induced by a host-type lymphoma (EL4) administered 7 days later (100% mortality in non-DLI controls; P<0.001). No GVHD occurred in DLI recipients. Rechallenging the surviving DLI recipients, which had converted to full chimerism, with the same tumor dose 17 weeks later led to rapid tumor mortality. Long-term DLI recipients had anti-host proliferative responses, but not CTL responses in vitro. When given as DLI together with wild-type spleen cells, marked expansion of GVH-reactive 2C CD8+ T cells was observed on day 10, followed by a marked decline in their numbers by week 10 post-DLI. CONCLUSIONS: Nonmyeloablative induction of mixed chimerism followed by administration of DLI can mediate powerful GVL effects. The late loss of DLI-mediated GVL effects may reflect the eventual loss of donor-derived GVH-reactive CTL, which occurs in association with conversion to full donor chimerism.

Animals↗
PubMed2003

Despite efficient intrathymic negative selection of host-reactive T cells, autoimmune disease may develop in porcine thymus-grafted athymic mice: evidence for failure of regulatory mechanisms suppressing autoimmunity.

BACKGROUND: CD4 T-cell reconstitution and xenogeneic tolerance is achieved in T cell-depleted, thymectomized C57BL/6 (B6) mice and nude mice by grafting of fetal pig thymus (FP THY). Sixty percent of grafted nude mice and 10% of grafted thymectomized B6 mice develop a clinical illness resembling chronic graft-versus-host disease. METHODS: Negative selection of mouse T cells in FP THY grafts was studied in "AND" TCR transgenic mice with a negative selecting MHC. Pathologic and immunohistochemical examinations and adoptive transfer assays were performed to determine the role of mouse CD4+ cells in the occurrence of autoimmune disease in this model. RESULTS: Marked clonal deletion of mouse thymocytes bearing a transgenic TCR ("AND"), which recognizes H2s expressed by host hematopoietic cells, was observed in FP THY grafts. Pathologic and immunohistochemical examinations of the liver, skin, lungs, and kidneys of mice with wasting syndrome showed marked mouse CD4+ T-cell infiltration without detectable pig cells. After adoptive transfer of splenocytes, but not of CD4+ cell-depleted splenocytes, from sick mice along with B6 bone marrow cells to lethally irradiated syngeneic B6 mice, the secondary recipients developed a similar autoimmune syndrome as the donors. Cotransfer of naïve syngeneic splenocytes prevented the occurrence of autoimmune disease in secondary recipients of splenocytes from healthy FP THY-grafted BALB/c nude mice. CONCLUSION: These results demonstrate a key role for mouse CD4+ T cells in causing autoimmune disease in this model and suggest the importance of regulatory mechanisms in addition to intrathymic clonal deletion for the maintenance of tolerance to recipient antigens.

Adoptive Transfer↗
PubMed2003

Antitumor effect of donor marrow graft rejection induced by recipient leukocyte infusions in mixed chimeras prepared with nonmyeloablative conditioning: critical role for recipient-derived IFN-gamma.

Some patients lose chimerism following nonmyeloablative hematopoietic cell transplantation (HCT), yet, surprisingly, enjoy sustained tumor remissions. We hypothesized that host-versus-graft (HVG) alloresponses might induce antitumor effects against recipient tumors. We explored this question in mice by administering recipient leukocyte infusions (RLIs) to mixed chimeras established with nonmyeloablative conditioning. Mixed chimeras were prepared in the B10.A (H2a)-->B6 (H2b) strain combination using depleting anti-T-cell monoclonal antibodies (mAbs), cyclophosphamide, and thymic irradiation. B6 myeloid leukemia cells (MMB3.19) were administered 7 days following donor lymphocyte infusion (DLI) or RLI on day 35. Conversion to full donor chimerism occurred without graft-versus-host disease (GVHD) following DLI, whereas RLI led to loss of chimerism. Both RLI and DLI significantly delayed tumor mortality. In another strain combination (B10.BR [H2k]-->BALB/c [H2d]), RLI-induced or spontaneous loss of chimerism was associated with antitumor effects against the host-type B-cell lymphoma A20. HCT was essential for the antitumor effect of RLI. RLI induced elevated serum interferon-gamma (IFN-gamma) levels, and recipient-derived IFN-gamma was critical for their antitumor effects. Thus, HVG reactions (spontaneous or induced by RLI) mediate antitumor effects against hematologic malignancies via a recipient-derived IFN-gamma-mediated mechanism. A novel approach to achieving anti-tumor effects without the risk of GVHD is suggested.

Animals↗
PubMed2003

NK cell tolerance in mixed allogeneic chimeras.

Alterations in inhibitory receptor expression on NK cells have been detected in mixed allogeneic chimeras and in mosaic MHC class I-expressing transgenic mice. However, it is not known whether or not NK cells are tolerant to host and donor Ags in mixed chimeras. In vitro studies have shown a lack of mutual tolerance of separated donor and host NK cells obtained from mixed chimeras. Using BALB/c-->B6 fully MHC-mismatched mixed chimeras, we have now investigated this question in vivo. Neither donor nor host NK cells in mixed chimeras showed evidence for activation, as indicated by expression of B220 and Thy-1.2 on NK cells in chimeric mice at levels similar to those in nonchimeric control mice. Lethally irradiated, established mixed BALB/c--> B6 chimeras rejected a low dose of beta(2)-microglobulin-deficient bone marrow cells (BMC) efficiently but did not reject BALB/c or B6 BMCs. In contrast, similarly conditioned B6 mice rejected both BALB/c and beta(2)-microglobulin-deficient BMCs. Thus, NK cells were specifically tolerant to the donor and the host in mixed allogeneic chimeras. The similar growth of RMA lymphoma cells in both chimeric and control B6 mice further supports the conclusion that donor BALB/c NK cells are tolerant to B6 Ags in chimeras. Administration of a high dose of exogenous IL-2 could not break NK cell tolerance in chimeric mice, suggesting that NK cell tolerance in chimeras is not due to a lack of activating cytokine. No reduction in the level of expression of the activating receptor Ly-49D, recognizing a donor MHC molecule, was detected among recipient NK cells in mixed chimeras. Thus, the present studies demonstrate that NK cells in mixed chimeras are stably tolerant to both donor and host Ags, by mechanisms that are as yet unexplained.

Animals↗
PubMed2003

Nonmyeloablative haploidentical stem-cell transplantation using anti-CD2 monoclonal antibody (MEDI-507)-based conditioning for refractory hematologic malignancies.

We initiated a clinical trial of nonmyeloablative haploidentical stem-cell transplantation (SCT) using MEDI-507, an immunoglobulin-G1 monoclonal anti-CD2 antibody. The trial was based on a preclinical major histocompatibility complex-mismatched bone marrow transplant model in which graft-versus-host disease (GVHD) was prevented and mixed chimerism as a platform for adoptive cellular immunotherapy was reliably induced. Twelve patients (three cohorts of four patients each) received cyclophosphamide, MEDI-507, and haploidentical unmanipulated bone marrow (n=8) or ex vivo T-cell-depleted peripheral blood stem cells (n=4) for chemorefractory hematologic malignancy. A two-dose regimen and schedule modifications of MEDI-507 were undertaken because of graft loss in the first cohort of four patients and GVHD in the second cohort. With ex vivo T-cell-depleted peripheral blood SCT, mixed chimerism occurred in all four patients without GVHD. Two patients, however, subsequently lost their grafts. Nonmyeloablative preparative therapy with MEDI-507 and haploidentical SCT have led to the reliable induction of at least transient mixed chimerism as a potential platform for adoptive cellular immunotherapy.

Adult↗
PubMed2003

Xenogeneic thymokidney and thymic tissue transplantation in a pig-to-baboon model: I. Evidence for pig-specific T-cell unresponsiveness.

BACKGROUND: The potential of xenotransplantation for clinical application will require overcoming barriers of humoral and cellular rejection, through strategies using immune suppression or tolerance induction. This laboratory has previously reported the induction of tolerance in the discordant xenogeneic model of pig-to-rodent thymic transplantation. We also have described a miniature swine model of fully mismatched allogeneic composite vascularized thymokidney transplantation that induced transplantation tolerance. We tested a combination of these approaches in a clinically relevant pig-to-primate model of xenotransplantation. METHODS: Composite thymokidney grafts were prepared 40 to 80 days before transplantation by the autologous implantation of thymic tissue under the renal capsule of human decay-accelerating factor transgenic swine. Baboons received xenotransplants of both human decay-accelerating factor composite thymokidneys and omental implants of thymic tissue. Recipients were treated with an immunosuppressive-conditioning regimen including thymectomy or thymic irradiation, extracorporeal immunoadsorption of anti-alphaGal antibodies and T-cell depletion. Recipients were followed for indicators of xenograft rejection, T-cell depletion and reconstitution, anti-alphaGal antibody levels, and mixed lymphocyte responses. Immunologic responses were studied in those animals that survived for more than 3 weeks. RESULTS: Thymokidney xenografts survived for up to 30 days, with evidence of viable thymic epithelium and Hassall's corpuscles under the renal capsule and in the omental implants, and with evidence of few host lymphocytes. Three animals demonstrated donor-specific unresponsiveness, while maintaining normal alloresponses, in mixed-lymphocyte-response assays performed after immunosuppression had been stopped. Rejected grafts demonstrated humoral damage without evidence of cellular infiltrates. After graftectomy, one animal maintained donor-specific cellular unresponsiveness and stable anti-alphaGal antibody levels for more than 2 months. CONCLUSIONS: We concluded that composite thymokidney and thymic-tissue xenotransplantation from swine to baboons can induce donor-specific cellular unresponsiveness and stable anti-alphaGal antibody levels, suggesting avoidance of sensitization after xenotransplantation. The presence of viable donor-swine thymic epithelium could have a role in the development of donor-specific T-cell tolerance. Further strategies to address humoral rejection could prolong graft survival and result in long-term tolerance to xenografts.

Animals↗
PubMed2003

Xenogeneic thymus transplantation in a pig-to-baboon model.

BACKGROUND: We have tested whether fetal porcine thymic tissue transplantation can lead to tolerance across a discordant (pig-to-baboon) xenogeneic barrier. METHODS: Six baboons underwent a conditioning regimen with thymectomy, splenectomy, and anti-monkey CD3 antibody conjugated to a diphtheria toxin binding site mutant (FN18-CRM9). Porcine fetal or neonatal thymic tissue was transplanted into three baboons. Three control baboons received either no transplanted pig tissue (n=1) or adult pig lymph node (n=2). Cellular responses and skin xenografts were used to test for tolerance. RESULTS: After T-cell depletion and thymic transplantation, recovery of thymus-dependent naïve-type CD4 cells (CD4/CD45RA ) and in vitro xenogeneic hyporesponsiveness were observed. No sensitization of alpha-galactosyl antibody responses was observed. The thymic grafts survived up to 48 days. Porcine skin xenografts were performed in two of these animals with survival of 22 and 24 days. Only two of these animals were completely T-cell depleted, and both failed to recover thymus-dependent T cells (CD4/CD45RA ). In one animal, general in vitro hyporesponsiveness was observed, with subsequent death from infection. The second animal demonstrated delayed recovery of T cells and prolonged general hyporesponsiveness in vitro. Neither animal demonstrated prolongation of porcine skin grafts compared with allografts (both rejected by day 13). CONCLUSIONS: Porcine thymic tissue is able to induce xenogeneic hyporesponsiveness. More efficient thymic engraftment may allow this approach to induce xenograft tolerance.

Animals↗
PubMed2003

Early host CD8 T-cell recovery and sensitized anti-donor interleukin-2-producing and cytotoxic T-cell responses associated with marrow graft rejection following nonmyeloablative allogeneic bone marrow transplantation.

OBJECTIVE: We developed a nonmyeloablative conditioning regimen for allogeneic bone marrow transplantation (BMT) followed by donor lymphocyte infusions (DLI) for treatment of chemotherapy refractory malignancies. Although the majority of patients who receive this regimen achieve lasting mixed or full allogeneic chimerism, approximately 30% show initial mixed chimerism followed by loss of the donor graft. These patients recover host hematopoiesis without significant cytopenias. To assess the role of immunologic rejection in graft loss, we compared T-cell recovery and in vitro alloresponses in six patients who lost their marrow graft to that in 16 concurrent patients with sustained donor chimerism. PATIENTS AND METHODS: Conditioning included pretransplant cyclophosphamide (150-200 mg/kg), thymic irradiation (700 cGy), and pre- and post-transplant equine antithymocyte globulin (ATG; ATGAM). HLA-identical related donor BMT was followed by DLI at approximately day 35 in patients without graft-vs-host disease. RESULTS: The group with transient chimerism showed significantly increased circulating host T-cell (median 416 cells/mm(3) vs 10 cells/mm(3), p<0.05) and CD8 T-cell numbers (354 cells/mm(3) vs 71 cells/mm(3), p<0.05) compared to the group with stable mixed or full donor chimerism within the first 100 days post-BMT. All DLI recipients who lost chimerism following DLI had greater than 80% recipient T cells at the time of DLI, whereas those with persistent chimerism had <60% host T cells. Graft rejection was associated with the development of a sensitized anti-donor bulk cytotoxic T-lymphocyte (CTL) response in 4 of 6 evaluated patients, compared to only 1 of 10 evaluated patients with sustained chimerism (p<0.05). Additionally, 3 of 5 evaluated transient chimeras showed high anti-donor CTL precursor frequencies in limiting dilution assays, and 3 of 4 evaluated transient chimeras showed high anti-donor interleukin-2 (IL-2)-producing T-helper (T(H)) cell frequencies. High anti-donor T(H) or cytotoxic T-lymphocyte precursors were not detected in sustained chimeras. CONCLUSION: These data indicate that loss of chimerism in patients receiving this nonmyeloablative regimen is due to immune-mediated rejection. This rejection appears to bemediated by recovering recipient cytolytic CD8(+) cells as well as IL-2-producing recipient T(H) cells. These data are the first to demonstrate sensitization of recipient anti-donor IL-2-producing cells in association with human marrow allograft rejection.

Bone Marrow Transplantation↗
PubMed2003

Stem cell activity of porcine c-kit+ hematopoietic cells.

OBJECTIVE: A marker for hematopoietic stem cells (HSCs) of pigs, which are considered to be the most suitable donors for clinical xenotransplantation, has not yet been identified. In this study, we examined the HSC activity of porcine c-kit+ bone marrow cells (BMCs). METHODS: The HSC activity of porcine c-kit+ BMCs was evaluated both in vitro using colony-forming unit (CFU) and cobblestone area-forming cell (CAFC) assays and in vivo in nonobese diabetic/severe combined immunodeficiency transgenic (NOD/SCID-Tg) mice carrying porcine cytokine transgenes. RESULTS: Purified c-kit+ BMCs were substantially enriched for both CFUs and CAFCs in vitro and their transplantation led to long-term porcine hematopoiesis in vivo in mice. Although porcine chimerism was detectable in the peripheral blood of NOD/SCID-Tg mice receiving porcine c-kit- BMCs at early time points after transplantation, the levels were markedly lower than those in mice receiving purified c-kit+ BMCs (0.2%+/-0.14% vs 7.7%+/-1.6% and 0.17%+/-0.17% vs 5.6%+/-2.1% at weeks 3 and 6, respectively). Importantly, all mouse recipients of porcine c-kit+ BMCs showed durable multilineage chimerism (>19 weeks), whereas no recipients of porcine c-kit- BMCs sustained long-term engraftment. Moreover, porcine HSCs that had engrafted for 19 weeks in the recipients of porcine c-kit+ BMCs gave rise to clonogenic progenitors in vitro and reconstituted porcine hematopoiesis in secondary recipients. CONCLUSION: The present study demonstrates that c-kit is an essential marker of both long-term-repopulating HSCs and progenitor cells with early engraftment capacity.

Animals↗
PubMed2003

NK cell recovery, chimerism, function, and recognition in recipients of haploidentical hematopoietic cell transplantation following nonmyeloablative conditioning using a humanized anti-CD2 mAb, Medi-507.

OBJECTIVE: Natural killer (NK) cells kill allogeneic cells that lack a class I MHC ligand for clonally distributed killer inhibitory receptors (KIR). Following HLA-mismatched hematopoietic cell transplantation (HCT), donor NK cells might mediate graft-vs-host (GVH) reactions that promote donor chimerism and mediate anti-tumor effects. Additionally, recipient NK cells might mediate donor marrow rejection. We have developed a nonmyeloablative approach to haploidentical HCT involving recipient treatment with a T cell-depleting mAb, Medi-507, that can achieve donor engraftment and mixed hematopoietic chimerism without graft-vs-host disease (GVHD). Donor lymphocyte infusions (DLI) are later administered in an effort to achieve graft-vs-leukemia/lymphoma (GVL) effects without GVHD. It is unknown whether NK cell "tolerance" develops in human mixed chimeras. METHODS: We have addressed these issues in 12 patients receiving Medi-507-based nonmyeloablative haploidentical HCT. RESULTS: NK cells recovered relatively early, despite the presence of circulating anti-CD2 mAb, but the majority of initially recovering cells lacked CD2 expression. These NK cells showed a reduced capacity, compared to those from normal donors, to kill class I-deficient targets. No association was detected between KIR mismatches in the host-vs-graft (HVG) or GVH direction and graft or tumor outcomes in this small series. NK cell chimerism did not correlate with chimerism in other lineages in mixed chimeras. NK cell tolerance to the host was not observed in a patient with full donor chimerism. One patient developed NK cell reactivity against donor-derived lymphoblast targets after loss of chimerism, despite the absence of an HVG KIR mismatch. CONCLUSION: Our results do not show an impact of NK cells on the outcome of nonmyeloablative, even T cell-depleted, HCT across haplotype barriers using an anti-CD2 mAb. Our data also raise questions about the applicability of observations made with NK cell clones to the bulk NK cell repertoire in humans.

Antibodies, Monoclonal↗
PubMed2003

Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-versus-host disease, and antitumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation.

In an attempt to capture graft-versus-tumor effects without graft-versus-host disease (GVHD), the authors initiated a trial of nonmyeloablative allogeneic bone marrow transplantation (BMT) in patients with advanced hematologic malignancies, with the majority of patients having chemotherapy-refractory disease. Forty-two patients received an HLA-matched related donor BMT after a cyclophosphamide and antithymocyte globulin-based conditioning that also included thymic irradiation for patients who had not received prior mediastinal radiotherapy. Prophylactic donor leukocyte infusion (pDLI) at a dose of 1 x 10(7) CD3(+) cells per kilogram were given beginning 5 weeks post-BMT to 16 patients with mixed chimerism (MC) but without GVHD, whereas 26 patients did not receive pDLI, either because of GVHD or early relapse. Twelve of 16 patients (75%) receiving pDLI had T cell chimerism at the time of pDLI >/=40%. These patients, by day 100 post-BMT, either converted to full donor chimerism (FDC) (n = 10) or had an increase in or stable donor chimerism (n = 2) after pDLI. Four of 4 patients whose T cell chimerism was </=20% at the time of pDLI, lost the graft. In contrast, only 5 of 18 evaluable patients (28%) not receiving a pDLI converted to FDC by day 100 post-BMT, 7 maintained MC, and 10 of an evaluable 22 lost the graft. Patients who had undergone a previous autologous stem cell transplant had a higher rate of conversion to FDC (69% v 31%) and higher incidence of GVHD (69% v 34%) compared with those who did not have a previous autologous SCT. Eleven of 16 patients (69%) who received a pDLI achieved a remission with 50% 1-year progression-free survival rate and 44% 3-year overall survival rate. Nineteen of 42 patients (45%) had >/=grade II acute GVHD, including 12 after BMT and 7 after pDLI. Approximately one third of patients, after having initial MC, eventually lost their donor graft. The authors conclude that (1) pDLI has the potential to convert MC to FDC; (2) sustained remissions can be achieved in patients with chemorefractory hematologic malignancies who receive a pDLI, albeit with a significant risk of acute GVHD; and (3) the degree of donor T cell chimerism at the time of pDLI is predictive of the fate of MC, ie, donor T cell chimerism >/=40% or </=20% at the time of pDLI correlates with conversion of MC or loss of the graft, respectively.

Adult↗
PubMed2003

Nonmyeloablative bone marrow transplantation: Infectious complications in 65 recipients of HLA-identical and mismatched transplants.

Infections are a common complication of allogeneic bone marrow transplantation and the leading cause of transplantation-related mortality. It had been hypothesized that transplantation following nonmyeloablative preparative regimens would result in fewer infections by causing less mucosal injury, less graft-versus-host disease, and allowing earlier immune reconstitution. We have retrospectively reviewed the infectious complications of 65 consecutive patients with advanced hematologic malignancies who underwent bone marrow transplantation using a novel preparative regimen consisting of cyclophosphamide, thymic irradiation, and in vivo T-cell depletion. Cytomegalovirus (CMV) infection occurred in 52% of cases in which the donor or recipient had evidence of prior CMV exposure. Using a strategy of preemptive therapy and secondary prophylaxis with ganciclovir, no CMV disease occurred. Infections with gram-positive bacteria predominated over the first 100 days after bone marrow transplantation. Thereafter, the relative proportion of gram-negative infections increased without a significant increase in episodes of neutropenia. The rate of bacterial infections was not influenced by relapse of the underlying malignancy. Seven patients developed infections with Aspergillus species, which was the most common infectious cause of death in these patients. Infections with viruses other than CMV (n=10) and with protozoan organisms (n=2) also occurred. The use of HLA-mismatched donors, the occurrence of grade II-IV acute graft-versus-host disease, and treatment with corticosteroids did not influence the risk of CMV or bacterial or fungal infections in patients who underwent transplantation following this preparative regimen. Overall, the incidence and spectrum of infections in this series was similar to the reported incidence of infections following conventional myeloablative allogeneic stem cell transplantation. We conclude that a quantitative T-cell deficiency in these extensively T-cell depleted patients may be a risk factor for infection, even in the absence of graft-versus-host disease.

Adolescent↗
PubMed2003

Position paper of the Ethics Committee of the International Xenotransplantation Association.

Xenotransplantation (XTx) provides a potential solution to the shortage of human organs and tissues, and has several advantages over other possible solutions to this problem. However, a number of scientific and ethical barriers exist, and need to be addressed in order to advance the field of XTx in a manner that optimizes its potential to benefit society and minimizes its risk. Some of the most pressing ethical issues are discussed, and the position of the Ethics Committee of the International Xenotransplantation Association is presented.

Animal Rights↗
PubMed2003

Fetal porcine thymus engraftment, survival and CD4 reconstitution in alphaGal-KO mice is impaired in the presence of high levels of antibodies against alphaGal.

Xenospecific T-cell tolerance can be induced among murine and human T-cells by porcine thymic grafting. However, anti-alpha 1,3-galactosyltranserase (alphaGal) (Galalpha1-3Galbeta1-4GlcNAc-R) natural antibodies (NAbs) pose a major barrier to porcine xenografts in humans. We used alphaGal knockout (KO) and muchain KO mice to explore the effect of natural anti-alphaGal and other xenoantibodies on porcine thymic engraftment and to examine the potential of thymic tissue to tolerize anti-alphaGal antibody-producing cells. Thymectomized [adult thymectomy (ATX)] non-immunized and rabbit red blood cell (RRBC) pre-transplant immunized alphaGal-KO (knockout), wild-type (WT) and mu chain KO B6 mice were treated with 3Gy total body irradiation (TBI), and T and natural killer (NK) cell depleting monoclonal antibodies (mAbs). These conditioned mice were grafted with fetal porcine thymus and liver (FP THY/LIV) tissue under the kidney capsule. Flow cytometric analysis was performed to follow CD4 reconstitution as a measure of FP THY engraftment and function. Only mice with >10% CD4+ peripheral blood lymphocytes (PBL) were considered successfully engrafted. Enzyme-linked immunosorbent assay (ELISA) was used to assess the kinetics of immunoglobulin M (IgM) and IgG anti-alphaGal antibodies. Anti-pig antibodies were monitored by flow cytometry (FCM). FP THY engrafted successfully in most of the immunoglobulin deficient mice (11 out of 12, 92%) and the outcome was similar in WT B6 controls (8 out of 12, 67%). Non-immunized alphaGal-KO mice grafted with FP THY had a similar success rate (7 out of 11) to that observed in non-immunized alphaGal-WT controls (2 out of 4). In contrast, alphaGal-KO mice immunized pre-transplant with RRBC, then grafted with FP THY/LIV, showed a significant reduction in the success of thymic grafting (2 out of 9, 22%) compared with pre-transplant immunized WT controls (4 out of 7; 57%) and non-immunized alphaGal-KO mice (7 out of 11, 64%). Anti-Gal and anti-pig antibody levels were not markedly augmented by porcine thymus grafts in mice with successful thymus grafts. FP THY engraftment is impaired in the presence of high levels of anti-alphaGal xenoantibodies. However, low levels of anti-alphaGal antibodies and other mouse anti-pig NAbs appear not to play a major role in the rejection of FP THY. Although grafting FP THY expressing the alphaGal epitope did not tolerize B cells producing anti-alphaGal antibodies in a T-cell independent manner, it prevented T-cell dependent sensitization by inducing T-cell tolerance to porcine antigens.

Animals↗
PubMed2003

B cell tolerance to xenoantigens.

Xenotransplantation of pig organs to humans is a possible solution to the shortage of donor organs for transplantation. Multiple immunologic barriers need to be overcome if pig-to-primate transplantation is to be successful. The presence, in humans, of natural antibodies (Abs) directed against Galalpha1-3Galbeta1-4GlcNAc epitopes on pig vascular endothelium provides the major barrier, as antibody-antigen binding initiates the process of hyperacute rejection. Even if hyperacute rejection is prevented, acute vascular rejection develops. Acute vascular rejection is also mediated, in part, by xenoreactive Abs and may be complement-independent. Efforts being made to overcome antibody-mediated rejection include depletion of antibody by extracorporeal immunoadsorption, prevention of an induced Ab response by pharmacologic reagents, B-cell and/or plasma cell depletion, depletion or inhibition of complement, and the use of organs from pigs transgenic for human complement regulatory proteins. The ultimate solution would be the induction of B-cell tolerance to xenogeneic antigens, which is being explored by attempting to induce xenogeneic hematopoietic chimerism. Here, we review the properties of the B cell types responding to xenoantigens and the strategies for tolerizing those B cells.

Animals↗
PubMed2003

Lack of role for CsA-sensitive or Fas pathways in the tolerization of CD4 T cells via BMT and anti-CD40L.

Anti-CD40L mAb plus bone marrow transplantation (BMT) and recipient CD8 T-cell depletion permits long-term mixed hematopoietic chimerism and systemic donor-specific tolerance to be achieved across full MHC barriers. Initial tolerance is characterized by peripheral deletion of donor-reactive CD4 cells. In regimens using costimulatory blockade without BMT to achieve allograft survival, cyclosporine inhibited graft survival, suggesting that the combination may not be clinically applicable. We assessed the role of cyclosporine-sensitive mechanisms and the mechanisms of T-cell apoptosis involved in the induction of early peripheral CD4+ T-cell tolerance by BMT with anti-CD40L. Neither a short course of cyclosporine (14 days) nor the absence of FAS-mediated activation-induced cell death (AICD) blocked the induction or maintenance of donor-specific tolerance. IL-2 production was not associated with tolerance induction, consistent with the lack of a role for Fas-mediated AICD. Mice in which passive T-cell death was impaired because of constitutive expression of a Bcl-xL transgene did not develop tolerance with this protocol. These data confirm that deletion of donor-reactive T cells is critical for the induction of mixed chimerism and tolerance. However, the mechanisms involved may differ from those involved in costimulatory blockade regimens that do not include BMT.

Animals↗
PubMed2003

Graft-versus-host disease can be separated from graft-versus-lymphoma effects by control of lymphocyte trafficking with FTY720.

Graft-versus-host disease (GvHD) mediated by donor T cells recognizing host alloantigens is associated with beneficial graft-versus-tumor effects in recipients of allogeneic hematopoietic cell transplants. Since leukemias and lymphomas reside largely within the lymphohematopoietic system, we have proposed that the desired graft-versus-leukemia or graft-versus-lymphoma effect can be separated from the complication of GvHD by confinement of the graft-versus-host alloresponse to the lymphohematopoietic tissues. Since the new sphingosine-1-phosphate receptor agonist immunosuppressive drug FTY720 leads to trapping of T cells in secondary lymphoid tissues, we evaluated the possibility that this drug could diminish GvHD, a disease involving epithelial target tissues, while permitting a beneficial alloresponse to take place within the lymphohematopoietic system, leading to graft-versus-lymphoma effects. We demonstrate here that FTY720 markedly reduces GvHD in a clinically relevant, haploidentical strain combination, while permitting antitumor effects against a T cell lymphoma of unshared host MHC haplotype to proceed unhindered. These results establish a potential new immunotherapeutic approach to separating graft-versus-leukemia effects from GvHD.

Animals↗