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C LeGuern

Publications and source records attributed to C LeGuern.

At least 37 records · Page 2Linked to original sources
PubMed1997

Molecular and cellular events implicated in local tolerance to kidney allografts in miniature swine.

Long-term tolerance to class I-mismatched renal allografts can be induced in miniature swine by treatment with a short course of cyclosporine (CsA). Kidney recipients treated with CsA and untreated control kidney recipients both demonstrated infiltration of the transplanted kidney by mononuclear cells, which reached a maximum between postoperative days 8 and 11. Recipients that did not receive the tolerizing regimen rejected their grafts between postoperative days 8 and 12 in this model. The kinetics of cytokine gene expression, including interleukin (IL)-1alpha, IL-1beta, IL-2, IL-6, IL-10, tumor necrosis factor, and interferon-gamma (IFN-gamma), within the grafted kidney of rejector and acceptor animals, were determined using Northern blot hybridization. A strong correlation between rejection and up-regulation of the IFN-gamma gene was observed, whereas animals with long-term tolerance showed low levels of IFN-gamma, but high levels of IL-10 gene transcription. None of the other cytokine genes demonstrated a reproducible pattern of expression that correlated with acceptance/rejection of allografts. Analysis of transcription patterns of cytokine genes in mononuclear cells purified from renal grafts confirmed the initial observations made on biopsies. The phenotype of graft-infiltrating cells (GIC) showed a dominance of CD8+ cells, with an average of 66% single-positive cells and 19% CD4/CD8 double-positive cells, compared with 30% and 14%, respectively, for peripheral cells. Predominance of CD8+ GIC was dictated neither by the MHC antigen disparity nor the rejector/acceptor status. These results, therefore, suggest that GIC represent a regulated combination of mononuclear cells producing local immune mediators that, in part, control the fate of allografts in this large animal model.

Animals↗
PubMed1997

The mechanism of specific prolongation of class I-mismatched skin grafts induced by retroviral gene therapy.

In the present study, we examine the mechanism of specific hyporesponsiveness to major histocompatibility complex (MHC) class I-mismatched skin allografts induced by retrovirus-mediated gene transfer of an allogeneic class I gene into syngeneic bone marrow (BM). Using appropriate congenic recombinant mouse strains, we have mapped MHC determinants that are capable of restoring rapid rejection of Kb-bearing skin grafts. Our results indicate that either a single class I or a single class II alloantigen expressed on skin in association with Kb is able to restore the rapid rejection of Kb-mismatched skin grafts. These data suggest that third-party alloantigens expressed on skin in association with Kb abrogate hyporesponsiveness by providing T cell help. Consistent with this interpretation, spleen cells from mice reconstituted with Kb-transduced BM were unable to elicit a significant anti-Kb cytotoxic T lymphocyte response in vitro unless interleukin-2 was added to the culture medium. Skin graft survival was also analyzed on B10. AKM mice thymectomized 3-4 weeks post-reconstitution with Kb-transduced BM. Thymectomy did not result in significantly prolonged survival of B10. MBR skin grafts compared to euthymic controls, suggesting that even early after reconstitution, intrathymic deletion of Kb-reactive T cells must have been incomplete. Taken together, these data suggest that prolongation of skin allograft survival in this model is controlled at the level of T cell help.

Animals↗
PubMed1996

Long-term engraftment of precultured post-5-fluorouracil allogeneic marrow in mice conditioned with a nonmyeloablative regimen: relevance for a gene therapy approach to tolerance induction.

Introduction of MHC class I Kb cDNA via recombinant retrovirus into B10.AKM (Kk) bone marrow cells (BMC) has been shown to confer specific hyporesponsiveness to B10.MBR (Kb) allogeneic skin grafts in lethally irradiated B10.AKM recipients of the transduced syngeneic BMC. We have recently developed a nonmyeloablative conditioning regimen that allows engraftment of fully MHC-mismatched allogeneic bone marrow and the induction of donor-specific tolerance. We ultimately plan to adapt this nonmyeloablative regimen for the use of retroviral transfer of the Kb gene to syngeneic marrow. As a step toward this goal, we have assessed the effects of our current BMC transduction protocol on engraftment of class I mismatched marrow in mice prepared using the nonmyeloablative regimen. BMC from B10.MBR (KbIkDq) mice treated 2 days earlier with 5-fluorouracil (5-FU) were cultured for 4 days with rIL-3 and rIL-6, and then injected into B10.AKM (KkIkDq) recipients conditioned with anti-CD4 and anti-CD8 mAbs, 7 Gy of thymic irradiation and 3 Gy of whole body irradiation. Engraftment was comparable to that of freshly prepared normal B10.MBR marrow. All recipients of 10(6) precultured BMC developed long-term multilineage mixed WBC (white blood cells) chimerism, and six of seven of these animals showed long-term specific tolerance to B10.MBR skin grafts. Four of seven recipients of 2 x 10(5) precultured BMC showed long-term repopulation by the donor of > 1% of multiple WBC lineages and four of five recipients showed specific tolerance to B10.MBR tail skin. These data suggest that our previously described nonmyeloablative conditioning regimen could be applicable to the gene therapy approach for the induction of donor-specific transplantation tolerance.

Animals↗
PubMed1996

Culture and characterization of hematopoietic progenitor cells from miniature swine.

Miniature swine are being used as a large animal model in which cultured and retrovirus-transduced hematopoietic stem cells (HSC) can be tested in a reproducible manner for their long-term in vivo repopulating ability. As part of these studies, long-term bone marrow culture (LTBMC) and progenitor colony assay systems were developed and used to characterize the in vitro growth potential and in vivo frequency of hematopoietic progenitors in this species. We found that LTBMCs initiated with a single marrow inoculum produced myeloid colony progenitors continuously for at least 7 weeks. The sites of myelopoietic activity in these cultures were uniquely restricted to isolated, morphologically diverse germinal centers rather than more disperse cobblestone patches. We also used the progenitor assay to screen several human and murine recombinant cytokines for cross-reactivity to swine bone marrow cells, including interleukin-3 (IL-3), IL-6, Il-11, granulocyte and granulocyte-macrophage colony-stimulating factors (G-CSF and GM-CSF), c-kit ligand (also called mast cell growth factor [MGF]), and erythropoietin (Epo). With the exception of human and murine IL-3, each of the cytokines tested induced swine progenitor colony formation to varying degrees, with some combinations leading to the formation of primitive multilineage and high proliferative potential colonies. Finally, in an attempt to characterize alternative sources of HSC from swine, we compared the progenitor content of adult and juvenile swine bone marrow and fetal liver. The fetal liver samples were found to be highly enriched for both primitive and mature progenitors, while analysis of postnatal marrow samples revealed an approximately two-fold decline in overall progenitor frequency between the ages of 10 and 20 weeks. Taken together, these studies demonstrate the development and use of in vitro culture methods for characterizing hematopoietic elements from miniature swine and suggest a hierarchy of progenitor cell content in various hematopoietic tissues from the large animal model.

Animals↗
PubMed1996

Alloresistance to K locus class I-mismatched bone marrow engraftment is mediated entirely by CD4+ and CD8+ T cells.

Clinical application of approaches to inducing transplantation tolerance that involve bone marrow reconstitution will require achievement of engraftment without major toxicity to the recipient. These requirements are likely to vary according to the type of histoincompatibility between donor and recipient. We have attempted to determine the minimal conditioning required to achieve lasting mixed allogeneic chimerism and tolerance in the presence of a class I MHC disparity by evaluating the host elements that resist alloengraftment. We based our approach on a regimen that was shown to induce mixed chimerism in fully MHC-mismatched strain combinations. Recipient B10.AKM (KkIkDq) mice were treated with 7 Gy thymic irradiation (TI) and 3 Gy whole body irradiation (WBI) and received either anti-CD8 mAb alone or anti-CD4 plus anti-CD8 mAbs before transplantation of K locus-disparate B10.MBR (KbIkDq) marrow. All (27 of 27) animals receiving both mAbs showed lasting multi-lineage mixed chimerism and donor-specific tolerance. In contrast, five of 22 (23%) recipients pre-treated with anti-CD8 mAb alone in the same experiments failed to develop lasting multilineage mixed chimerism, suggesting that the CD4 T cell subset also participates in resistance to class I-mismatched marrow engraftment. We next attempted to determine whether or not host non-T cell elements resist allogeneic engraftment by comparing the minimum number of syngeneic vs allogeneic BMC required to achieve lasting multilineage mixed chimerism. Titrated numbers (10(6) to 10(7)) of B10.MBR (KbIkDq) bone marrow cells were administered to B10.AKM recipients treated with anti-CD4 and -CD8 mAbs, 3 Gy WBI and 7 Gy TI. All recipients of each marrow dose developed lasting multilineage mixed chimerism and showed specific tolerance to B10.MBR skin grafts. The level of donor-type repopulation in recipients of each dose was not lower than that observed in similarly irradiated recipients in an Ly5 congenic, otherwise syngeneic, BMT system. Together, our results suggest that CD4+ T cells contribute to resistance to K locus class I-mismatched marrow allografts and that resistance is mediated only by CD4 and CD8 T cells, with no role for non-T cell host elements.

Animals↗
PubMed1995

Molecular identification of porcine interleukin 10: regulation of expression in a kidney allograft model.

Clones encoding porcine interleukin 10 (IL-10) were isolated from a cDNA library produced from phytohemagglutinin-activated pig peripheral blood mononuclear cells. The porcine IL-10 nucleotide sequence was found to be highly homologous to the rat, mouse, and human IL-10 counterparts and to one of the open reading frames from the Epstein-Barr virus. In addition, pig IL-10 caused inhibition of gamma-interferon gene transcription as determined by a bioassay. To investigate the possible immunomodulatory role of IL-10, its expression during the induction of tolerance to kidney allografts by cyclosporin A in miniature swine was also investigated. Delayed expression and higher levels of IL-10 were observed in tolerant animals compared with animals rejecting their allografts. Since tolerance is achieved by a short course of cyclosporin A, we have also studied the in vitro effect of this drug on IL-10 gene transcription in blood mononuclear cells and have found that cyclosporin A inhibits IL-10 gene activation in T cells but does not interfere with IL-10 transcription in lipopolysaccharide-activated cells. These results suggest that the overexpression of IL-10, observed in cell populations infiltrating grafts from tolerant animals, may be a function of monocytes and/or B cells.

Amino Acid Sequence↗
PubMed1995

Specific unresponsiveness to a retrovirally-transferred class I antigen is controlled through the helper pathway.

To investigate the potential role for gene therapy in induction of tolerance to solid organ grafts, we used a murine model based on the introduction of an allogeneic MHC class I gene (H-2Kb) into hematopoietic cells of congenic animals differing only at the class I locus. The H-2Kb gene was placed into a retroviral vector under the control of regulatory sequences of the myeloproliferative sarcoma virus long terminal repeat. Transplantation of H-2Kb retrovirus-transduced autologous bone marrow into B10.AKM (Kk Ik Dq) recipients resulted in detectable levels of H-2Kb RNA and cell surface protein in lymphoid and myeloid lineages. H-2Kb expression was significant, although variable, in bone marrow Mac1+ cells, in splenic B cells, and in CD4+/CD8+ thymocytes 8 wk post-bone marrow transplantation. The recipients of the H-2Kb-transduced bone marrow showed specifically delayed rejection of B10.MBR (Kb Ik Dq) skin grafts disparate only for Kb. However, B10.MBR skin grafts were rapidly rejected when grafted simultaneously with skin grafts from B10 (Kb Ib Db) mice, a strain bearing additional third party alloantigens in association with Kb. Our experiments suggest that the hyporesponsive state induced by using the retrovirally mediated gene transfer model is characterized by the persistence of H-2Kb-specific cytolytic T cell precursors, which may be inactive because of deficient T cell help. Tolerance to Kb induced by this approach may therefore be restricted to T helper cell lineages.

3T3 Cells↗
PubMed1994

Bone marrow culture and transduction of stem cells in a miniature swine model.

Recombinant retroviral vectors, engineered to express the beta-chain gene of swine major histocompatibility complex class II DR, were developed for the genetic modification of swine hematopoietic stem cells (HSC). The expression of these vectors in swine bone marrow has been studied both in culture and after bone marrow transplantation. In addition, myeloid progenitor colony assays were performed on swine umbilical cord blood as part of a study to identify alternative sources of HSC for somatic gene transfer, revealing the presence of both granulocyte macrophage colony forming-units (CFU-GM) and CFU-Mix at frequencies comparable to those found in juvenile swine bone marrow.

Age Factors↗
PubMed1993

Extensive allelic polymorphism in the CDR2-like region of the miniature swine CD4 molecule.

MHC class II polymorphism is well documented whereas only minimal polymorphism has been reported for the CD4 molecule with which it interacts. We report on the structural basis of an allelic polymorphism of the CD4 molecule in miniature swine. Eleven of 13 nucleotide differences between the 2 alleles cause amino acid replacements. A majority of these replacements are clustered in a region protruding as a loop structure, termed Ig CDR2-like, from the surface of the amino terminal domain. This part of the human CD4 appears to comprise the binding site for the human immunodeficiency virus gp120 protein. The loop structure has also been implicated in the binding of CD4 to MHC class II, but this is currently a matter of some controversy. Our previous results have indicated that the porcine CD4 polymorphism, that we now show is situated in this loop, does not significantly affect the binding to class II. However, because the polymorphism appears to have been selected for and is situated in a very exposed part of the molecule, it is likely to be of functional significance.

Alleles↗
PubMed1993

Retrovirus-mediated transfer and expression of an allogeneic major histocompatibility complex class II DRB cDNA in swine bone marrow cultures.

The possibility of inducing transplantation tolerance by somatic gene transfer is under investigation in our miniature swine model. As a crucial step in this project, we have used a retroviral vector engineered to express both a drug-resistance gene (Neo) and a swine class II DRB cDNA to transduce porcine bone marrow (BM) cells. Analysis of cultured swine fibroblasts exposed to high-titer viral supernatants demonstrated that drug resistance had been conferred and that transferred vector sequences were transcribed appropriately. Similar transduction studies with swine BM demonstrated the transfer of drug resistance to as high as 14% of colony-forming unit-granulocyte-macrophage (CFU-GM). Using polymerase chain reaction (PCR) of cDNA, vector-derived allogeneic DRB transcripts were detected in colonies derived from primitive CFU-Mix and high proliferative potential-colony-forming cell (HPP-CFC), as well as in drug-resistant GM colonies grown from transduced bone marrow (BM) maintained in long-term BM cultures (LTBMCs) for up to 5 weeks. These results indicate that a significant proportion of both colony-forming progenitors and LTBMC-initiating cells were transduced with the DRB-recombinant retroviral vector and that both vector-derived genes were expressed in the differentiated progeny of these cells.

Animals↗