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R I Lechler

Publications and source records attributed to R I Lechler.

At least 19 recordsLinked to original sources
PubMed2000

Significant frequencies of T cells with indirect anti-donor specificity in heart graft recipients with chronic rejection.

BACKGROUND: The purpose of this study was to determine whether T cells with indirect allospecificity could be detected in heart transplant recipients with chronic rejection. METHOD AND RESULTS: Human T-cell clones were used to determine the most effective way to deliver major histocompatibility complex alloantigens for indirect presentation. Seven allograft recipients with evidence of progressive, chronic rejection were selected. Four heart graft recipients with no evidence of chronic rejection were used as controls. Peripheral blood T cells and antigen-presenting cells from the recipients were cultured with frozen/thawed stored donor cells or major histocompatibility complex class I-derived synthetic peptides in limiting dilution cultures and then compared with controls using tetanus toxoid and frozen/thawed third-party cells with no human leukocyte antigens in common with the donor. In 5 of 7 patients analyzed who had chronic rejection, elevated frequencies of T cells with indirect, anti-donor specificity (iHTLf) were detected. No such elevated iHTLf were detected in recipients without chronic rejection. DISCUSSION: iHTLf can be obtained from human transplant recipients, which supports the contention that the indirect pathway is involved in chronic transplant rejection.

Animals↗
PubMed2000

HLA-G inhibits the transendothelial migration of human NK cells.

The expression of the non-classical MHC class I molecule HLA-G is normally restricted to the placenta during pregnancy, where it is found on fetal endothelial cells and on invasive cytotrophoblast cells, specifically those at the maternal / fetal interface. Its precise physiological role has yet to be defined. HLA-G may have nonimmune functions relating to angiogenesis and placentation, but most evidence suggests that it protects fetal cells from lysis by maternal uterine NK cells, which are found in large numbers around invading trophoblast cells. This effect is due to specific interaction with inhibitory receptors expressed on NK cells. We have examined the hypothesis that another function of HLA-G is to inhibit NK cell migration. Using an in vitro transmigration assay system, we present data to support this hypothesis. NK cell migration across porcine endothelial cells transfected with HLA-G1 was specifically inhibited compared to migration across HLA-A2-transfected monolayers. HLA- G1 had no influence on the migration of a control T lymphocyte line. These results support the idea that in vivo, HLA-G may inhibit NK cell traffic across the placenta.

Cell Movement↗
PubMed1999

Inhibition of expression of the Galalpha1-3Gal epitope on porcine cells using an intracellular single-chain antibody directed against alpha1,3galactosyltransferase.

The carbohydrate epitope Galalpha1-3Gal has been shown to be the major target of natural antibodies responsible for hyperacute rejection of porcine tissues transplanted into primates. We have sought to produce a phenotypic knockout of the alpha1, 3Galactosyltransferase enzyme that is responsible for generating this epitope, using an intracellular antibody approach. We have isolated high affinity anti-alpha1,3Galactosyltransferase single-chain antibodies from a semi-synthetic phage display library. Expression of a KDEL-tagged anti-alpha1,3Galactosyltransferase single-chain antibody in a porcine endothelial cell line resulted in the decreased expression of the Galalpha1-3Gal epitope and increased resistance to lysis by human serum.

Amino Acid Sequence↗
PubMed1999

Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins.

BACKGROUND: Thrombotic vascular occlusion resulting in infarction occurs during hyperacute rejection of allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. A similar process is also found in disorders of diverse etiology including atherosclerosis, vasculitis, and disseminated intravascular coagulation. METHODS: We have previously constructed two membrane-tethered anticoagulant fusion proteins based on human tissue factor pathway inhibitor and the leech anticoagulant hirudin and demonstrated their functional efficacy in vitro. These constructs have now been modified by the addition of a P-selectin sequence to the cytoplasmic tail to localize them in Weibel-Palade bodies. They have been transfected into Weibel-Palade body-positive endothelial cells isolated from the inferior vena cava of normal pigs. RESULTS: In resting endothelial cells, fusion protein expression colocalized with P-selectin and was confined to Weibel-Palade bodies. These cells had a procoagulant phenotype in recalcified human plasma. However, after activation with phorbol ester the anticoagulant proteins were rapidly relocated to the cell surface where they specifically inhibited the clotting of human plasma. CONCLUSIONS: Novel anticoagulant molecules may prove useful therapeutic agents for gene therapy in thrombotic disease and postangioplasty or for transgenic expression in animals whose organs may be used for clinical xenotransplantation. Expression in vascular endothelial cells may be regulated by inclusion of P-selectin cytoplasmic sequence, to restrict cell surface expression to activated endothelium.

Animals↗
PubMed1999

Lack of T cell proliferation without induction of nonresponsiveness after antigen presentation by endothelial cells.

BACKGROUND: After priming or reactivation in lymph nodes, T cells recirculate to sites of inflammation, and enter tissues by migrating across activated endothelium. Given that activated endothelial and tissue parenchymal cells express both class I and class II MHC molecules, it is probable that transmigrating T cells encounter cognate antigen on endothelial cells, and on tissue parenchymal cells once they have entered the tissue. METHODS: In this study the consequences of antigen presentation by endothelial and epithelial cells to human CD4+ T cell clones were analyzed and compared by a two-step culture system. RESULTS: T cell clones that required B7-mediated costimulation to be activated were found not to be able to proliferate to antigen presented by either endothelial or epithelial cells, unless trans-costimulation was provided by the addition of B7-transfected cells in the cultures. Furthermore, antigen presentation by epithelial cells induced nonresponsiveness in the T cell clones. In contrast, after cognate recognition on endothelial cells, the ability of the T cell clones to proliferate to a subsequent rechallenge with antigen presented on a specialized APC was unaffected. CONCLUSIONS: These data suggest that endothelial cells have unique properties as antigen-presenting cells, in that they do not influence the subsequent reactivity of cognate T cells.

Animals↗
PubMed1999

Recently activated T cells are costimulation-dependent in vitro.

While the prominent role of B7-mediated signaling in the activation of naive and resting T cells has been exhaustively demonstrated, it is unclear whether costimulation is required in the amplification of an initiated immune response. In this study we have developed a multistep culture system to investigate the costimulation requirements of recently activated alloreactive CD4(+) T cells and the outcome of allorecognition of B7-deficient, MHC class-II-expressing epithelial cells. The results show that following in vitro "priming" with allogeneic costimulation rich antigen presenting cells, T cells can be reactivated to proliferate only if B7-mediated costimulation is provided. Furthermore, recognition of antigen on B7-negative epithelial cells induced allospecific nonresponsiveness in the responder T cells. Finally, the nonresponsive state was not accompanied by IL-4 secretion and appeared to be reversible, since T cell reactivity could be restored by short-term culture in the presence of IL-2. These observations suggest that "primed" T cells remain B7-dependent in vitro and are susceptible to functional inactivation following costimulation-deficient antigen presentation.

Animals↗
PubMed1999

Inhibition of tissue factor-dependent and -independent coagulation by cell surface expression of novel anticoagulant fusion proteins.

BACKGROUND: Thrombotic vascular occlusion occurs in disorders of diverse etiology, including atherosclerosis, vasculitis, and disseminated intravascular coagulation. The same process results in hyperacute rejection of renal allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. METHODS: We have previously described the design and expression of several genetic constructs encoding novel fusion proteins with anticoagulant properties. They are based on two naturally occurring soluble anticoagulant proteins, human tissue factor pathway inhibitor (hTFPI) and the leech protein hirudin, which act early and late in the clotting cascade, respectively. We report the expression of human hTFPI-CD4 on the surface of immortalized porcine endothelial cells (IPEC), and show that it functions across the species divide as evidenced by the binding of membrane-expressed porcine tissue factor (pTF)-human factor VIIa complexes. RESULTS: Using a human plasma recalcification clotting assay, we distinguished between pTF-dependent and pTF-independent fibrin generation, and we have demonstrated that expression of hTFPI-CD4 on IPEC effectively prevented pTF-dependent clotting. Moreover, we show that when hTFPI-CD4 was co-expressed with the hirudin construct, the procoagulant properties of in vitro cultured, activated IPEC were almost completely abolished. CONCLUSIONS: These results suggest that these novel anticoagulant molecules may prove useful therapeutic agents for gene therapy or for transgenic expression in animals whose organs may be used for cliniCal xenotransplantation.

Animals↗
PubMed1999

Antigen recognition influences transendothelial migration of CD4+ T cells.

The functional significance of MHC class II expression by vascular endothelial cells remains obscure. In this study the possibility that Ag presentation by endothelial cells (EC) influences T cell transmigration, facilitating the recruitment of Ag-specific T cells into tissues, was investigated. The frequencies of T cells with specificity for an HLA-DR alloantigen, or for the recall Ag tetanus toxoid (TT), were measured in peripheral blood CD45RO+ (memory) CD4+ T cells before and after transmigration through gamma-IFN-treated EC monolayers. Frequencies of anti-DR17, IL-2-secreting T cells were fourfold higher in the T cells that transmigrated through a monolayer of DR17-expressing EC. Similar increases were seen in TT-specific, DR7-restricted T cells that transmigrated through TT-pulsed, DR7-expressing EC. To examine more directly the effects of cognate recognition of Ag presented by EC, T cell clones were used. For clones that proliferated in a costimulation-independent manner to Ag presented by EC, cognate recognition arrested transmigration. In contrast, Ag presentation by EC to B7-dependent T cell clones, which do not proliferate following cognate recognition of EC, enhanced the rate of transendothelial migration. These data suggest that Ag presentation by EC may serve to augment the recruitment of Ag-specific T cells into tissues and that proliferation and transmigration are mutually exclusive T cell responses.

B7-1 Antigen↗
PubMed1999

Biased T-cell receptor usage is associated with allelic variation in the MHC class II peptide binding groove.

A comprehensive analysis was carried out of the tri-molecular complex of peptide, major histocompatibility class II molecule, and T-cell receptor (TcR) involved in the recognition of the promiscuous HA (306-318) peptide, restricted by one of two closely related HLA-DR alleles, HLA-DRB1*0101 and HLA-DRB1*0103. These two DR molecules differ by only three amino acids at positions 67, 70, and 71, in the third variable region of the DRB1 chain. None of the HA (306-318)-specific T-cell clones restricted by these two DR molecules tolerated amino acid substitution at the peptide-binding position 71, despite the fact that the substitution did not interfere with peptide binding. The majority of the DRB1*0103-restricted clones tolerated substitution of the amino acid at the TcR-contacting position 70, while the DRB1*0101-restricted T cells did not. Based usage of TRVA and TRVB segments was observed for the DRB1*0103-restricted clones; in contrast, apparently random usage was seen in the DRB1*0101-restricted T cells. Finally, limiting dilution analysis revealed a lower frequency of T cells reactive with the HA peptide in a DRB1*0103 compared with a DRB1*0101 individual. Taken together these data suggest that biased TcR gene usage may reflect a relatively low precursor frequency of T cells, and the need for clonal expansion of a limited set of high avidity T cells.

Alleles↗
PubMed1999

Selective inhibition of beta-1,4- and alpha-1,3-galactosyltransferases: donor sugar-nucleotide based approach.

A combined rational and library approach was used to identify bisphosphonates (IC50 = 20 microM) and galactose type 1-N-iminosugar (IC50=45 microM) as novel motifs for selective inhibition of beta-1,4-galactosyltransferase (beta-1,4-GalT) and alpha-1,3-galactosyltransferase (alpha-1,3-GalT), respectively. Our results demonstrate that, though these two galactosyltransferases both utilize the same donor sugar-nucleotide (UDP-Gal), the difference in their mechanisms can be utilized to design donor sugar or nucleotide analogues with inhibitory activities selective for only one of the galactosyltransferases. Investigation of beta-1,4-GalT inhibition using UDP-2-deoxy-2-fluorogalactose (UDP-2-F-Gal), UDP, and bisphosphonates, also led to the observation of metal dependent inhibition of beta-1,4-GalT. These observations and the novel inhibitor motifs identified in this study pave the way for the design and identification of even more potent and selective galactosyltransferase inhibitors.

Animals↗
PubMed1999

Improving the therapeutic monitoring of cyclosporin A.

The narrow therapeutic window of cyclosporin A (CsA) and the variable pharmacokinetics of the traditional preparation, CsA-SIM (Sandimmune), have made it difficult to establish its optimal use. The introduction of a microemulsion preparation, CsA-ME (Neoral), with less variable pharmacokinetics, has made it possible to attempt to define its use more closely. One hundred and one renal allograft recipients were converted from CsA-SIM to CsA-ME. Absorption was monitored using a standard pharmacokinetic 'profile' measuring 'whole blood' CsA levels at several time points following drug administration. Areas under the resulting time-versus-CsA concentration curve (AUC) were calculated. Surprisingly, many patients showed very little fluctuation in 'whole blood' levels after administration of the conventional preparation: 31% had a difference between trough (to) and maximal levels of < 100 micrograms/L. On microemulsion cyclosporin, there was much better correlation between AUC and several parameters incorporating elements of trough and peak values. The best correlation was with t0 + (t1/4), where t1 represents the concentration at 1 h following administration, (r2 = 0.779 for microemulsion cyclosporin). The use of this parameter is a practical possibility in an out-patient setting. The very common, but under-recognised, pattern of almost flat absorption profiles in patients on conventional CsA suggests that the use of CsA in numerous clinical contexts should be reviewed, since CsA immunosuppression may previously have been inadequately monitored.

Absorption↗
PubMed1999

Antigen presentation by parenchymal cells: a route to peripheral tolerance?

T-cell activation and the development of efficient immune responses requires the delivery, by the antigen-presenting cell, of two distinct signals. The first results from the engagement of the TCR:CD3:CD4 complex, and the second from the interaction of CD28 with the B7 family of co-stimulatory molecules. In this context, the physiological significance and the functional consequences of antigen presentation by B7-deficient parenchymal cells, which express MHC class II molecules as a result of inflammation, remains a matter of debate. In this paper we have attempted to critically review the often conflicting reports on the functional effects of antigen presentation by epithelial and endothelial cells to T cells, both in vitro and in vivo. Our own findings are summarised in a model which is consistent with the suggestion of an important role for antigen presentation by parenchymal cells in the induction and the maintenance of peripheral tolerance.

Abatacept↗
PubMed1999

Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice.

Thymic atrophy is a prominent feature of malnutrition. Forty-eight hours' starvation of normal mice reduced the total thymocyte count to 13% of that observed in freely fed controls, predominantly because of a diminution in the cortical CD4(+)CD8(+) thymocyte subpopulation. Prevention of the fasting-induced fall in the level of the adipocyte-derived hormone leptin by administering exogenous recombinant leptin protected mice from these starvation-induced thymic changes. The ob/ob mouse, which is unable to produce functional leptin because of a mutation in the obese gene, has impaired cellular immunity together with a marked reduction in the size and cellularity of the thymus. We found that ob/ob mice had a high level of thymocyte apoptosis resulting in a ratio of CD4(+)CD8(+) (cortical) to CD4(-)CD8(-) (precursor) thymocytes that was 4-fold lower than that observed in wild-type mice. Peripheral administration of recombinant leptin to ob/ob mice reduced thymocyte apoptosis and substantially increased both thymic cellularity and the CD4(+)CD8(+)/CD4(-)CD8(-) ratio. In contrast, a comparable weight loss in pair-fed PBS-treated ob/ob mice had no impact on thymocyte number. In vitro, leptin protected thymocytes from dexamethasone-induced apoptosis. These data indicate that reduced circulating leptin concentrations are pivotal in the pathogenesis of starvation-induced lymphoid atrophy.

Animals↗
PubMed1998

Expression of hirudin fusion proteins in mammalian cells: a strategy for prevention of intravascular thrombosis.

BACKGROUND: Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation. METHODS AND RESULTS: An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti-hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4-P-selectin fusion proteins accumulated in adrenocorticotropic hormone-containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane. CONCLUSIONS: Hirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Role of donor and recipient antigen-presenting cells in priming and maintaining T cells with indirect allospecificity.

BACKGROUND: It has been suggested that the sensitization of recipient T lymphocytes against peptides derived from allogeneic major histocompatibility complex (MHC) antigens in the context of self-MHC molecules may contribute to the pathogenesis of chronic allograft rejection. The purpose of this study was to quantitate and characterize the indirect alloresponse in renal transplantation. METHODS: An HLA-A2-negative patient whose A2-positive kidney transplant failed as a result of chronic rejection was selected for this study. T-cell clones were raised using a cocktail of peptides corresponding to polymorphic regions of the A2 sequence and studied by measuring their proliferation using [3H]thymidine incorporation. The presence in vivo of HLA-A2-specific T cells was assessed using limiting dilution analysis. RESULTS: T-cell clones were specific for a single peptide of HLA-A2, residues 92-120, and restricted by HLA-DRB1*1502. The frequency of interleukin-2-secreting T cells specific for this A2 peptide was 1:86,000, only 2-fold lower than that measured against the recall antigen tetanus toxoid. Capitalizing on the similarity of the donor and recipient DR15 alleles (DRB1*1501 and 1502), the question was addressed as to how these T cells had been primed in vivo. Although the large majority of clones responded to A2 synthetic peptide presented by both DR15 alleles, only 3 of 10 clones responded to cells co-expressing DRB1*1501 and A2. CONCLUSION: These data suggest that antigen presentation by recipient APCs is responsible for maintaining T cells with indirect allospecificity in vivo and that, in the context of partial DR matching, indirect presentation by the parenchymal cells of the graft may serve to induce tolerance in T cells with indirect allospecificity.

Antigen-Presenting Cells↗
PubMed1998

Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression.

Nutritional deprivation suppresses immune function. The cloning of the obese gene and identification of its protein product leptin has provided fundamental insight into the hypothalamic regulation of body weight. Circulating levels of this adipocyte-derived hormone are proportional to fat mass but maybe lowered rapidly by fasting or increased by inflammatory mediators. The impaired T-cell immunity of mice now known to be defective in leptin (ob/ob) or its receptor (db/db), has never been explained. Impaired cell-mediated immunity and reduced levels of leptin are both features of low body weight in humans. Indeed, malnutrition predisposes to death from infectious diseases. We report here that leptin has a specific effect on T-lymphocyte responses, differentially regulating the proliferation of naive and memory T cells. Leptin increased Th1 and suppressed Th2 cytokine production. Administration of leptin to mice reversed the immunosuppressive effects of acute starvation. Our findings suggest a new role for leptin in linking nutritional status to cognate cellular immune function, and provide a molecular mechanism to account for the immune dysfunction observed in starvation.

Adult↗
PubMed1998

Association between interleukin-4-producing T lymphocyte frequencies and reduced risk of graft-versus-host disease.

BACKGROUND: We have previously developed and used limiting dilution analysis to measure frequencies of alloreactive cytotoxic T cell precursors (CTLp) and interleukin (IL)-2-producing T helper cells (IL-2/HTLp) to assess the risk of graft-versus-host disease in bone marrow transplantation (BMT). However, no test has been available to measure precursor frequencies of the important IL-4-secreting subset. METHODS: We have now established a limiting dilution analysis to measure the frequency of IL-4-producing T helper cells (IL-4/HTLp) using the IL-4-responsive indicator cell line CT.h4S and have applied this assay to measure alloreactive IL-4/HTLp frequencies in BMT donor-recipient pairs. These frequencies were then analyzed in the context of clinical data to assess the relationship between the number of donor anti-recipient IL-4-secreting T cells and disease outcome. RESULTS: Frequencies of IL-4/HTLp have been studied in HLA-identical siblings, HLA-"matched" unrelated, and HLA-mismatched combinations and found to range from approximately 1/500,000 in HLA-identical sibling pairs to -1/2,000 in HLA-DR-mismatched pairs. These frequencies were independent of those for IL-2/HTLp and showed a negative correlation with those for CTLp. Clinical follow-up of 30 patients showed that high IL-4/HTLp frequencies are associated with a reduced risk of severe graft-versus-host disease. High IL-4/HTLp frequencies may also indicate an increased risk of leukemia relapse. CONCLUSIONS: Our data suggest that measurement of IL-4/HTLp frequencies provides information distinct from that obtained with CTLp and IL-2/HTLp. This new assay provides a valuable additional method for optimizing donor selection in unrelated BMT.

Animals↗