PubMed Health⌕ Search

Biomedical subjects

R I Lechler

Publications and source records attributed to R I Lechler.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1998

Assessment of the contribution that direct allorecognition makes to the progression of chronic cardiac transplant rejection in humans.

BACKGROUND: Two populations of T cells contribute to allograft rejection. T cells with direct allospecificity are activated after recognition of intact MHC alloantigens displayed at the surface of donor passenger leukocytes carried within the graft. In contrast, T cells with indirect allospecificity recognize donor alloantigens as processed peptides associated with self (recipient)-MHC class II molecules. In small animal models of transplantation, direct pathway T cells dominate the acute rejection process and are rendered tolerant to the graft after the loss of donor passenger leukocytes. It has been argued that indirect pathway T cells contribute substantially to continual graft damage after passenger cell loss. The purpose of this study was to determine whether donor-specific tolerance could be detected in T cells with direct anti-donor allospecificity in human heart transplant recipients after prolonged graft residence. METHODS AND RESULTS: Alloreactive helper (HTLf) and cytotoxic (CTLf) T cells were enumerated by use of limiting dilution analysis. These assay systems were refined to make them specific for the direct pathway of allorecognition and more sensitive in the case of the HTLf assay. Recipient:anti-donor frequencies were generated in 10 long-term recipients of heart grafts with progressive chronic rejection and compared with those against equivalently HLA mismatched recipient:third-party controls. For HTLf, direct pathway donor-specific hyporesponsiveness was detected in 5 of the 10 recipients (HTLf<1:100,000). Of these 5 recipients, 4 also had low anti-donor CTLf (<1:100,000). In the 5th recipient, although the CTLf was >1:100,000, it was significantly lower than that estimated against the third-party control. CONCLUSIONS: Donor-specific hyporesponsiveness is demonstrated in 50% of recipients in both the HTLf and CTLf compartments of the direct alloresponse. Direct allorecognition therefore appears unlikely to be responsible for the progression of chronic rejection, implicating indirect allorecognition as the predominant immunological driving force. Furthermore, these data have potential implications for graft outcome, adjustment of immunosuppression, and recipient monitoring.

Antibody Specificity↗
PubMed1998

In vitro accommodation of porcine endothelial cells by low dose human anti-pig antibody: reduced binding of human lymphocytes by accommodated cells associated with increased nitric oxide production.

Transplanted xenografts, protected from rejection by depletion of xenoreactive natural antibodies (XNA) and complement, can sometimes survive when complement levels and titres of anti-graft antibodies return to baseline; this phenomenon is called accommodation. We have previously reported that low concentrations of human IgG induce a change in the phenotype of immortalised porcine endothelial cells (IPEC) consistent with the development of accommodation. The cells acquired a resistance to lysis by human complement and showed a reduced expression of VCAM. In this study, we extend these findings by showing that VCAM expression falls on several IPEC clones and on primary porcine endothelial cells. Moreover, we show that these accommodated cells bind fewer human lymphocytes compared to controls, implying that leukocyte traffic through accommodated endothelium may be altered compared to that through normal endothelium. Finally we show that during the induction of accommodation, porcine endothelial cells produce greater amounts of nitric oxide than controls, due to the expression of inducible nitric oxide synthase (iNOS). We speculate that nitric oxide may be an important mediator in accommodation.

Animals↗
PubMed1998

T cell-mediated xenograft rejection: specific tolerance is probably required for long term xenograft survival.

T cell-mediated mechanisms of xenograft rejection appear resistant to standard immunosuppression protocols used to prevent allograft rejection and, consequently, higher doses of immunosuppressive drugs are required to promote xenograft compared to allograft survival. Evidence from recent studies suggests that porcine xenografts may be especially immunogenic in humans because of a prominent and vigorous indirect xenoresponse and because of the ability of porcine endothelium to activate human T cells. This has led to an anxiety that systemic immunosuppressives, used as the mainstay of therapy for clinical xenotransplantation, may not allow the long-term survival of porcine organs transplanted into human recipients. This article will review the biology of T cell xenoresponses, present the case for the development of novel graft-specific immunosuppressive regimes in clinical xenotransplantation, and review recent experimental progress in this area.

Animals↗
PubMed1997

Antigen presentation by epithelial cells induces anergic immunoregulatory CD45RO+ T cells and deletion of CD45RA+ T cells.

The immunoregulatory effects of alloantigen presentation by tissue parenchymal cells to resting peripheral blood CD4+ T cells was investigated. Coculture of CD45RO+ (memory) and CD45RA+ (naive) T lymphocytes with primary cultures of MHC class II-expressing epithelial cells rendered both populations of T cells hyporesponsive to a subsequent challenge by the same MHC molecule expressed on EBV-transformed lymphoblastoid B cell lines. However, the mechanisms responsible for the allospecific hyporesponsiveness were distinct. For the CD45RO+ T cells, responsiveness was restored by subsequent culture in the presence of IL-2; the addition of IL-2 had no effect on the reactivity of the CD45RA+ T cells. In contrast, the naive T cells were protected from the induction of nonresponsiveness by the presence of a neutralizing anti-CD95 Ab during the culture with thyroid follicular cells. In addition, the hyporesponsive CD45RO+ T cells effected linked suppression, in that they inhibited proliferation against a third-party DR alloantigen when the third-party alloantigen was coexpressed with the DR Ag against which hyporesponsiveness had been induced. These results suggest that recognition of Ag by T cells on tissue parenchymal cells plays an important role in the maintenance of peripheral T cell tolerance, inducing nonresponsiveness in naive and memory T cells by distinct mechanisms.

Animals↗
PubMed1997

Major histocompatibility complex class II-dependent unfolding, transport, and degradation of endogenous proteins.

We have analyzed the ability of major histocompatibility (MHC) class II molecules to capture proteins in the biosynthetic pathway and whether this may be associated with MHC class II-dependent antigen processing. When coexpressed with HLA-DR 4 molecules in HeLa cells, influenza hemagglutinin was inhibited from folding and trimerization in the biosynthetic pathway, targeted to endosomal compartments, and rapidly degraded. Due to the interaction with MHC class II molecules, therefore, unfolded forms of hemagglutinin were bypassing the quality control mechanism of the secretory pathway. More important, however, the transport, endocytosis, and rapid degradation of unfolded hemagglutinin in the presence of MHC class II molecules suggest that proteins captured in the endoplasmic reticulum by class II molecules may become substrates for antigen processing and presentation to CD4-positive T cells. In insect cells we show that this phenomenon is not restricted to a few proteins such as hemagglutinin. A highly heterogeneous mixture of proteins from the endoplasmic reticulum including coexpressed hemagglutinin can form stable complexes with soluble HLA-DR alpha and beta chains that were transported into the supernatant. This mechanism may gain biological significance in abnormal situations associated with accumulation of unfolded or malfolded proteins in the endoplasmic reticulum, for example during viral infections.

Biological Transport↗
PubMed1997

Expression of alpha-1,3-galactose and other type 2 oligosaccharide structures in a porcine endothelial cell line transfected with human alpha-1,2-fucosyltransferase cDNA.

The binding of xenoreactive natural antibodies to the Galalpha1-3Galbeta1-4GlcNAc (alpha-galactose) oligosaccharide epitope on pig cells activates the recipient's complement system in pig to primate xenotransplantation. Expression of human alpha-1, 2-fucosyltransferase in pigs has been proposed as a strategy for reducing the expression level of the alpha-galactose epitope, thereby rendering the pig organs more suitable for transplantation into humans. The aim of this study was to examine how the cell surface expression of alpha-galactose, H, and related fucosylated and sialylated structures on a pig liver endothelial cell line is affected by transfection of human alpha-1,2-fucosyltransferase cDNA. Nontransfected and mock-transfected cells expressed alpha-galactose, alpha-2,3-sialylated, and alpha-2,6-sialylated epitopes strongly, with low level expression of type 2 H and LewisX. By contrast, expression of the H epitope was increased 5-8-fold in transfected cells with a 40% reduction in the expression of alpha-galactose epitope and a 50% decrease in sialylation, as measured by binding of Maackia amurensis and Sambuccus nigra agglutinins. LewisX expression was reduced to background levels, while the LewisY neoepitope was induced in human alpha-1,2-fucosyltransferase-expressing pig cells. The activities of endogenous alpha-1,3-galactosyltransferase, alpha-1,3-fucosyltransferases, and alpha-2,3- and alpha-2, 6-sialyltransferases acting on lactosamine were unaffected. Our results show that a reduction in alpha-galactose epitope expression in porcine endothelial cells transfected with human alpha-1, 2-fucosyltransferase cDNA may be achieved but at the expense of considerable distortion of the overall cell surface glycosylation profile, including the appearance of carbohydrate epitopes that are absent from the parent cells.

ABO Blood-Group System↗
PubMed1997

Optimizing a limiting dilution culture system for quantifying the frequency of interleukin-2-producing alloreactive T helper lymphocytes.

BACKGROUND: The development of sensitive, specific, and reproducible techniques to quantify T cells with direct allospecificity has potential applications in the selection of bone marrow donors and in the monitoring of the antidonor alloresponse in patients after organ transplantation. Such data may provide an objective basis for altering existing immunosuppression, monitoring novel antirejection therapies, and predicting long-term graft outcome. We have previously published a correlation between donor antirecipient T helper frequencies (HTLf) and the severity of acute graft-versus-host disease after bone marrow transplantation. Using the same assay protocol, we have described the development of donor-specific hyporesponsiveness in a proportion of renal transplant recipients. However, several imperfections existed in the protocols used in these studies. Cellular interactions within the stimulator and the responder cell populations, and back stimulation of T cells within the stimulator cell population, could give rise to extraneous interleukin-2 and alter the validity or estimation of derived recipient antidonor HTLf. METHODS: Using peripheral blood mononuclear cells as the responding population and splenic mononuclear cells as the stimulating population, we have examined the possible effects of these cellular interactions on the results of limiting dilution analysis assays for HTLf measurement. RESULTS: These interactions have the ability to alter the validity or estimation of HTLf. We show that by depleting the responder population of HLA class II+ cells and depleting T cells from the stimulating population, these interactions are effectively abrogated. CONCLUSIONS: On the basis of the findings reported here, we describe an optimized HTLf assay which is sensitive, specific, and reproducible. This has obvious applications in the analysis of alloimmune responses in transplantation.

Bone Marrow Transplantation↗
PubMed1997

Quantitative but not qualitative variation in MHC class II alters CD4 interaction and influences T cell repertoire formation.

The influence of the interaction between CD4 and MHC class II molecules on selection of the T cell repertoire was studied in transgenic mice expressing human or human/mouse hybrid MHC class II beta chains. Either wild-type DR beta chains (DR1 beta) or hybrid beta chains comprising the beta1 domain of DR and the beta2, transmembrane, and intracytoplasmic domains of I-E (DRbeta 1Ebeta2) were introduced into and expressed in transgenic mice as a heterodimer with endogenous I-E alpha. Mice expressing low levels of DR1beta:I-E alpha or those expressing low or higher levels of the hybrid DRbeta 1Ebeta2:I-E alpha were studied. Immunization with a suboptimal dose of influenza nucleoprotein peptide exposed a fivefold lower frequency of DR-restricted, peptide-specific, IL-2-secreting T cells in the mice with low-level expression of DRbeta1 Ebeta2:I-E alpha when compared to mice expressing the same molecule at higher levels. The frequency in DRbeta wild-type mice was only twofold lower than that measured in mice with comparable levels of expression of DRbeta 1Ebeta2. These results suggest that positive selection is sensitive to quantitative variation in MHC class II density, unmasked when antigen is limiting, but is relatively insensitive to qualitative variation in the MHC class II: CD4 interaction.

Animals↗
PubMed1997

Dissociation of T cell anergy from apoptosis by blockade of Fas/Apo-1 (CD95) signaling.

Induction of anergy and deletion due to apoptosis are two of the mechanisms involved in peripheral tolerance. To clarify the relationship between these two phenomena we have used an in vitro system of T cell Ag presentation. The recognition of Ag displayed by MHC class II-expressing T cells (T-APC) induces partial signals in Ag-specific T cell clones. This leads to a blunted intracellular calcium flux, and the T cells become unable to proliferate in response to further challenge with professional APC. These T cells are unable to produce IL-2, but retain the ability to release IL-4. In the present study, we report that for some T cell clones, the predominant outcome of Ag recognition on T cells is cell death. For susceptible T cell clones, the number of cells that die is proportional to the peptide concentration. This cell death resulted from Fas/Apo-1 (CD95)/Fas-ligand interactions between the T cells, in that Fas ligand expression was detected following overnight culture of T cells with T-APC and neutralizing anti-CD95 Ab protected from death. Most notably, following anti-CD95-mediated protection from apoptosis, the rescued T cells remained unable to respond to rechallenge with Ag-pulsed, professional APC. These data suggest that anergy and apoptosis can be separated as consequences of partial T cell signaling.

Antibodies, Blocking↗
PubMed1997

Splicing by overlap extension by PCR using asymmetric amplification: an improved technique for the generation of hybrid proteins of immunological interest.

Major histocompatibility complex (MHC) proteins play a central role in the immune recognition of antigen. The generation of hybrid MHC molecules has been of great value in elucidating the structure: function relationships of these key glycoproteins. In this report, the generation of cDNAs coding for seven such hybrid proteins is described. We have used the technique of splicing by overlap extension by the polymerase chain reaction (SOE by PCR) [Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61-68] to generate intermediate products of each of the components of the hybrid, tipped with a small sequence of the other, and then mixed these products in a second-stage PCR to produce the final spliced product. Where we were unable to generate final product, we introduced an additional step of asymmetric PCR synthesis to generate an excess of those strands which would anneal in the final PCR and found this to be effective. We noted a significant but manageable mutation rate, possibly contributed to by the tendency of DNA polymerase to add additional non-templated nucleotides [Hu, G. (1993) DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment. DNA Cell Biol. 12, 763-770]. To avoid this, we modified our protocol to include a stage of blunting our intermediate products with T4 DNA polymerase prior to mixing them in the final PCR. We present this system as an effective mechanism to splice DNA.

Animals↗
PubMed1997

Multiple Cladosporium brain abscesses in a renal transplant patient: aggressive management improves outcome.

Infection remains one of the major complications of nonspecific immunosuppression in renal transplant patients and accounts for significant morbidity and mortality. The incidence of infectious complications has been shown to be related to the degree of immunosuppression and correlated with the total steroid dosage, use of antilymphocyte serum, and number of rejection episodes. We present the case of a patient who received a large cumulative immunosuppressive load as treatment for her original disease and for numerous rejection episodes following renal transplantation, and who later developed multiple brain abscesses. These were shown to be due to the saprophytic black fungus Cladosporium bantianum. This case emphasizes the importance of aggressively pursuing the diagnosis in immunosuppressed individuals--appropriate treatment may be instituted early and may save lives. There have been no previous cases of patients surviving this condition without neurosurgical resection of the lesions.

Adult↗
PubMed1997

Glaxo/MRS Young Investigator Prize. Xenotransplantation: immune barriers beyond hyperacute rejection.

1. The use of organs from animal donors (xenotransplantation) is a potential solution to the chronic shortage of allogeneic organs and currently the pig is thought to be the most suitable donor for man. However, porcine organs are rejected rapidly by a vascular process called hyperacute rejection which has so far prevented clinical xenotransplantation. Although it is likely that this barrier will be overcome in the near future by the application of novel strategies, probably involving the use of organs from transgenic pigs, data from animal models indicate that multiple other immune mechanisms will contribute to the rejection of xenografts. 2. We have described two aspects of these immune mechanisms. First, the phenomenon of 'accommodation', whereby xenografts acquire in vivo resistance to vascular rejection, has been explored in an in vitro model utilizing immortalized porcine endothelial cells. The results indicate that human anti-pig antibodies induce a concentration-dependent and time-dependent change in porcine endothelial cells compatible with the development of accommodation. 3. Secondly, the in vitro human anti-porcine T-cell response has been documented in detail, with particular emphasis on quantitative and qualitative comparisons with the in vitro T-cell alloresponse. The results of this work, which indicate that the response to porcine xenografts is likely to be significantly stronger than that against allografts, have important implications for the level of conventional immunosuppression that may be necessary to prevent xenograft rejection, and provide an important basis for the development of strategies to promote xenograft-specific immunosuppression and tolerance.

Animals↗
PubMed1997

Immobilized anti-CD3 mAb induces anergy in murine naive and memory CD4+ T cells in vitro.

The induction of non-responsiveness in resting murine CD4+ T cells was investigated using immobilized anti-CD3 mAb. Incubation of freshly isolated CD4+ T cells with immobilized anti-CD3 mAb led to apoptosis in 40-60% cells. The surviving cells were profoundly non-responsive to subsequent mitogenic stimulation. The non-responsive state was characterized by a lack of IL-2 production and hyper-responsiveness to added IL-2, but was not explained by further activation-induced cell death. The induction of non-responsiveness was not due to modulation of the TCR-CD3 complex, and required partial activation of the T cells in that it was accompanied by an increase in cell size and was inhibited by addition of cyclosporin A. Finally, analysis of anti-CD3-mediated responses in naive and memory CD4+ T cells, separated on the basis of CD44 expression, showed that both naive and memory T cells have similar sensitivity to immobilized anti-CD3 mAb-induced activation, apoptosis and anergy. These results demonstrate that TCR-CD3 engagement on freshly isolated resting CD4+ naive and memory T cells, In the absence of co-stimulation, as achieved by plastic-immobilized anti-CD3 mAb, induces both anergy and cell death.

Animals↗
PubMed1997

Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface.

Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation.

Animals↗