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G C Starling

Publications and source records attributed to G C Starling.

32 records · Page 2Linked to original sources
PubMed1995

Activation of human peripheral blood dendritic cells induces the CD86 co-stimulatory molecule.

Maximal T lymphocyte responses require presentation of antigen by major histocompatibility complex molecules and delivery of one or more co-stimulatory signals. Interaction of the CD28 molecule on T lymphocytes with its ligands on antigen-presenting cells (APC) initiates a critical co-stimulatory pathway inducing T lymphocyte proliferation and cytokine secretion. Dendritic cells (DC) are potent APC for a primary T lymphocyte response and potential CD28/CTLA-4 ligands on DC are, therefore, of particular functional relevance. In these experiments, the expression and function of the CD28/CTLA-4 ligands B7.1 (CD80) and B7.2 (CD86) were examined on human blood DC. Resting DC populations directly isolated by immunodepletion of lineage marker-positive cells lacked cell membrane expression of CD80 and expressed little or no CD86, although CD86, but not CD80 mRNA was detected by reverse transcription-polymerase chain reaction analysis. In contrast, low-density DC isolated after culture in vitro strongly expressed CD86 surface protein, but expressed limited or no CD80, although mRNA for both molecules were detected. Short-term culture of directly isolated DC up-regulated both CD80 and CD86 expression. Analysis of the kinetics of CD28/CTLA-4 ligand induction showed that surface CD86 was present within 8 h, whereas CD80 antigen was first detected after 24 h of culture. The functional importance of CD28/CTLA-4 ligand up-regulation on DC during T lymphocyte interactions was demonstrated by the ability of both CTLA-4Ig and CD86 monoclonal antibodies (mAb), but not CD80 mAb, to block an allogeneic mixed lymphocyte reaction stimulated by DC populations initially negative for CD80 and CD86. These results demonstrate that CD86 is both the earliest and functionally the predominant co-stimulatory CD28/CTLA-4 ligand on DC.

Abatacept↗
PubMed1995

Intercellular adhesion molecule-3 is the predominant co-stimulatory ligand for leukocyte function antigen-1 on human blood dendritic cells.

Dendritic cells (DC) are potent stimulators of primary T lymphocyte responses to foreign antigen. The initial DC-T lymphocyte interaction involves the binding of the adhesion molecule leukocyte function antigen-1 (LFA-1; CD11a/CD18) on the T lymphocyte to an intercellular adhesion molecule (ICAM) on the DC. Although blood and tonsil DC express ICAM-1 (CD54) and ICAM-2 (CD102) on their surface, anti-ICAM-1 and anti-ICAM-2 monoclonal antibodies (mAb) have little inhibitory activity on the DC-stimulated mixed leukocyte reaction (MLR). We therefore examined the expression of the more recently identified LFA-1 ligand, ICAM-3 (CD50), in comparison to ICAM-1 and ICAM-2 on blood DC and sought a functional role for ICAM-3 in DC-mediated T lymphocyte responses. Resting blood DC expressed significantly more ICAM-3 than ICAM-1 or ICAM-2 as assessed by flow cytometry. Treatment of resting DC with interferon-gamma led to increased expression of ICAM-1; however, ICAM-2 and ICAM-3 levels remained relatively constant. Solid-phase recombinant chimeric molecules ICAM-1-, ICAM-2- and ICAM-3-Fc were able to co-stimulate CD4+ T lymphocyte proliferation in conjunction with suboptimal solid-phase CD3 mAb 64.1. However, the anti-ICAM-3 mAb CAL 3.10 inhibited a DC-stimulated MLR to a greater extent than anti-ICAM-1 or anti-ICAM-2 reagents and appeared to act by blocking the DC ICAM-3- T lymphocyte LFA-1 interaction. As ICAM-3 is the predominant LFA-1 ligand on resting blood DC, we postulate that DC may utilize ICAM-3 for initial DC- T lymphocyte interactions, and that ICAM-1, which is up-regulated upon DC activation, and/or ICAM-2, may contribute to DC migration or later phases of the T lymphocyte activation process.

Antigens, CD↗
PubMed1995

Role of tumour necrosis factor-alpha in dendritic cell-mediated primary mixed leucocyte reactions.

Tumour necrosis factor (TNF alpha) is a major inflammatory cytokine with potentiating effects on specific immune responses, including graft-versus-host disease. This study examined the contribution of TNF alpha to dendritic cell (DC)-mediated primary allogeneic T lymphocyte responses. Purified blood DC were shown to produce minimal amounts of TNF alpha mRNA but no significant TNF biological activity or secreted TNF alpha as measured by ELISA. Amplification of DC mRNA by PCR using oligonucleotide primers to CD120a (TNFRI, p55) and CD120b (TNFRII, p75) and probing with specific internal oligonucleotides, suggested that DC express the CD120b but little if any CD120a. These results were confirmed using monoclonal antibodies to the TNF receptors. Polyclonal antiserum specific for TNF alpha blocked the blood DC-stimulated allogeneic mixed leucocyte reaction (MLR). The addition of TNF alpha to suboptimal MLRs (limited DC stimulators), increased the proliferation of responding T lymphocytes. Having confirmed that T lymphocytes produce TNF alpha and express CD120b after stimulation, we sought to clarify whether the contributing effect of TNF alpha to the allogeneic MLR resulted from a TNF alpha-mediated signal stimulating DC activity, or as a result of autocrine stimulation of T lymphocytes. Pre-incubation of DC with TNF alpha did not increase DC stimulatory capacity and late addition of anti-TNF serum (up to 72 h) still had a significant inhibitory effect on the MLR. We conclude that TNF alpha is probably not involved in the initial DC-T lymphocyte interaction, but acts as an autocrine growth factor for DC induced T lymphocyte proliferation.

Antigens, CD↗
PubMed1994

A novel HLA-A*8001 allele identified in an African-American population.

HLA polymorphism varies among different racial origins. Certain antigens are restricted to a particular ethnic group, suggesting that genetic events might have generated further unique polymorphism following racial diversification. In this study, we have identified and characterized a novel HLA-A allele, officially designated A*8001. The class I molecule, HLA-AXBG, encoded by this allele could not be defined by commonly available HLA antisera. We have identified two alloantisera that appear to be monospecific for this new antigen. The observed frequency of AXBG antigen specificity is 2% in African Americans among the total of 254 tested, but it has not been found in 305 Caucasians tested. IEF and cDNA sequencing analyses on multiple individuals revealed that AXBG antigen defined by the two antisera are encoded by an identical HLA-A*8001 allele and it has a number of unique amino acid residues that have not been observed among other HLA-A alleles but are known to occur in certain human nonclassic class I genes and nonhuman primate class I genes. The ability to identify the new HLA-AXBG antigen will improve the precision of HLA-A typing in black populations and enable us to match a patient with a donor for this otherwise undefined antigen in transplantation.

Alleles↗
PubMed1994

Characterization of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells.

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) involved in the initiation of primary T-lymphocyte responses. However, despite their importance, no DC-specific surface marker has been identified in humans and many aspects of their ontogeny and the mechanisms underlying their potent functional activity remain unknown. In this report we describe a novel monoclonal antibody (mAb), CMRF-44, which recognizes an early activation antigen expressed by human DC and acts as a marker of allostimulatory activity within preparations of peripheral blood mononuclear cells (PBMC). The CMRF-44 antigen was expressed strongly on DC isolated from blood and tonsil by standard techniques, but was not detectable on Langerhans' cells within skin or on DC isolated directly from blood using a cell-sorting method which involves minimal DC manipulation/activation. Normal resting peripheral blood leucocytes did not label with CMRF-44, although weak staining of a small subpopulation (15%) of blood B lymphocytes was identified by double labelling. However, following overnight culture at 37 degrees, moderate staining of a subpopulation of PBMC was detected. Confirmation that CMRF-44 recognized an early marker of activated DC and hence allostimulatory activity was obtained by sorting cultured cell preparations on the basis of CMRF-44 reactivity. A marked enrichment of allostimulatory activity was observed in the CMRF-44-positive cellular population, whereas the CMRF-44-negative cells showed only minimal stimulatory activity. Activation studies established that the CMRF-44 antigen was an early activation marker, expressed constitutively on the majority of tonsil B lymphocytes, which can be induced on peripheral blood B lymphocytes and subpopulations of monocytes. Expression of the CMRF-44 antigen on cell lines was similarly restricted, CMRF-44 antigen being detected only on Hodgkin's disease-derived and B-lymphoid lines. The cellular distribution, expression kinetics and biochemical characteristics of the CMRF-44 antigen identify it as a new early marker of activated allostimulatory (DC) populations.

Animals↗
PubMed1993

Selection of an unrelated donor for marrow transplantation facilitated by the molecular characterization of a novel HLA-A allele.

Precise HLA typing is crucial in the selection of marrow donors for the treatment of patients with hematologic malignancy. This study was undertaken to characterize an unusual variant of HLA-A30, designated HLA-A30JS, identified in a patient with leukemia who was a candidate for unrelated donor marrow transplantation. IEF and cDNA-sequencing analyses revealed that A30JS is a novel variant differing from the IEF-defined subtype A30.1 (encoded by the A*3002 allele) by a single amino acid substitution. An unrelated marrow donor was identified who was matched with the patient for HLA-A3, B7, B18, DR2, and DR3, but mismatched within the A30 antigen family for the two distinct alleles A*3002 versus A30JS. These two alleles encode a single amino acid substitution, Arg versus Gly, at position 56 in the alpha 1 domain. Position 56 is located outside the antigen-binding cleft of the class I molecule, suggesting that this substitution may not be functionally significant. Transplantation from this donor was performed and the patient is surviving free of leukemia for more than 700 days after transplant. The maximum acute GVHD observed was scored as grade II, but immunosuppressive therapy is still required for control of chronic GVHD. This study demonstrates how the molecular characterization of a novel HLA-A allele in a patient could facilitate the selection of an unrelated donor. Lacking this information, it would not have been possible to select a donor for this patient, and thus apparently successful marrow transplant could not have occurred.

Adult↗
PubMed1993

B7/BB-1 is a leucocyte differentiation antigen on human dendritic cells induced by activation.

Activation of a primary T-lymphocyte response requires additional signals apart from interaction of the T-cell receptor (TcR)/CD3 complex with major histocompatibility complex (MHC) antigens on the antigen-presenting cell. The CD28 antigen on T lymphocytes provides an important co-stimulatory signal to T lymphocytes and we therefore searched for the presence of its ligand, the B7/BB-1 antigen, on blood and tonsil dendritic cells (DC). Blood DC, prepared from peripheral blood mononuclear cells with a minimal period of in vitro culture, did not stain with the monoclonal antibody BB-1 using flow cytometry analysis. In contrast, tonsil DC stained weakly for B7/BB-1 compared to positive control cell lines. Polymerase chain reaction (PCR) was used to amplify a 605 base pair (bp) fragment from human B7/BB-1 mRNA and demonstrated significant amounts of B7/BB-1 mRNA in tonsil DC but no specific product was obtained from blood DC, confirming the surface-staining results. Weak expression of B7/BB-1 antigen was detected by immunofluorescence analysis following culture of blood DC with either interferon-gamma (IFN-gamma) or granulocyte-macrophage colony-stimulating factor (GM-CSF). These data support the concept that blood DC give rise to tissue and/or lymphoid DC, which acquire co-stimulatory ligands as a result of activation and/or differentiation.

Antibodies, Monoclonal↗
PubMed1993

Expression of the CMRF-35 antigen, a new member of the immunoglobulin gene superfamily, is differentially regulated on leucocytes.

A new monoclonal antibody, CMRF-35, has been generated that recognized a 224 amino acid cell surface protein which is a novel member of the immunoglobulin gene superfamily. The antibody, raised against large granular lymphocytes (LGL), stains LGL, monocytes, macrophages and granulocytes but not platelets or erythrocytes. In addition, a subset of peripheral blood T lymphocytes (26.6 +/- 13.4% CD5+ cells) and B lymphocytes (13.7 +/- 6.8% CD20+ cells) stained with CMRF-35 but tonsil T and B cells were essentially negative. Expression of the CMRF-35 antigen (Ag) on different leucocyte populations was markedly influenced by stimulation of the cells with mitogens and cytokines. Activation of peripheral blood T cells with phytohaemagglutinin (PHA), or phorbol myristate acetate (PMA) and calcium ionophore (CaI) led to a decrease in the proportion of CMRF-35+ T lymphocytes. In contrast, PHA activation of tonsil T lymphocytes resulted in an increase in CMRF-35 Ag expression (47.1 +/- 1.5% CD5 cells at 6 days). An increase in CMRF-35 Ag was also seen on phorbol ester and CaI-activated tonsil B cells. No change in CMRF-35 expression on natural killer (NK) cells occurred following activation with interleukin-2 (IL-2) but the CMRF-35 Ag was down-regulated following Fc receptor stimulation. A moderate increase in CMRF-35 expression occurred during monocyte-macrophage differentiation and the expression of the Ag on monocytes was differentially regulated by interferon-gamma (IFN-gamma). This regulation of the CMRF-35 Ag on the leucocyte surface suggests that the molecule has an important function common to diverse leucocyte types.

Antibodies, Monoclonal↗
PubMed1992

Epstein-Barr viral DNA in acute large granular lymphocyte (natural killer) leukemic cells.

Serologic studies in a male Caucasian presenting with an acute hepatitis-like illness, associated with an increase in peripheral blood large granular lymphocytes (LGLs), suggested a chronic or reactived Epstein-Barr virus (EBV) infection. The LGL were shown to have a natural killer (NK) cell, CD3- CD16- CD56+ CD57- phenotype and mediated strong nonspecific major histocompatability complex-unrestricted (NK) cytotoxic activity. A progressive increase in the peripheral blood LGL count was associated with a rapid deterioration, hepatic necrosis, and death. Widespread organ infiltration with LGLs suggested a malignant lymphoproliferative condition, but no lymphoid (T-cell receptor or IgH) gene rearrangement or cytogenetic marker was detected. However, molecular analysis identified EBV genomic DNA present in a single episomal form within the LGL, establishing the clonal nature of the LGL proliferation. Confirmation that the EBV had infected the leukemic LGL was obtained by in situ hybridization studies that showed EBV RNA within the LGLs. Immunoblotting of LGL protein extracts established that, of the EBV gene products, EBV nuclear antigen-1 (EBNA-1) was expressed but EBNA-2 and the latent membrane protein (LMP-1) were not detectable in the leukemic cells. These results suggest that EBV may be involved directly in LGL cell transformation, in a manner similar to EBV-associated B-cell lymphomas, although other molecular changes probably contribute to the evolution of a fully malignant leukemic clone.

Antigens, CD↗
PubMed1990

CD45 molecule cross-linking inhibits natural killer cell-mediated lysis independently of lytic triggering.

The fact that certain CD45 [anti-leucocyte common antigen (LCA)] monoclonal antibodies (mAb) inhibit natural killer (NK) cell non-major histocompatibility complex (MHC)-restricted cytolysis led to the suggestion that these mAb block a 'trigger' for NK cell lytic activity. However, the discovery that the intracytoplasmic portion of the leucocyte common molecule has protein tyrosine phosphatase activity raises the possibility that the mAb initiate a direct inhibitory signal, independent of the triggering apparatus. To clarify this, we have tested the ability of CD45 antibodies to trigger NK cells and redirect cytotoxicity against mAb-producing hybridoma cells and autologous monocytes, an approach which has identified other cytotoxic trigger molecules. Peripheral blood NK cells failed to kill the CD45 antibody-producing hybridomas, although a CD3 antibody expressing hybridoma was susceptible to cytotoxic T-cell lysis. Furthermore, the CD45 mAb CMRF-12 + 26, 13.3 and HuLyM4 did not redirect lysis of autologous monocytes by NK cells, whereas the isotype-matched CD16 mAb did so. Bivalent CD45 antibody was necessary to block NK lysis of K562, as F(ab')2 but not F(ab') fragments of CMRF-12 + 26 antibody inhibited killing. Capping of the LCA appeared to correlate with the ability of the CD45 mAb to block killing, suggesting that cross-linking of LCA molecular isoforms on the NK cell surface is required for CD45 mAb to inhibit non-MHC-restricted cytolysis.

Animals↗
PubMed1989

Inhibition of non-MHC-restricted cytotoxicity by CD45 but not CD3 monoclonal antibodies in patients with large granular lymphoproliferative disease.

Nine patients with a lymphoproliferative disorder characterized by a persistent expansion of large granular lymphocytes (LGL) and an increased proportion of cells labelling with natural killer (NK) and T cell markers were identified. The six patients with an expansion of alpha beta CD3/TcR positive cells were shown to have rearranged T cell receptor (TcR) genes whereas three patients whose LGL lacked CD3/alpha beta TcR on the surface had no beta TcR rearrangement detected. Eight of the nine patients were shown to exhibit non-MHC-restricted cytotoxic activity against K562; this activity was inhibited by CD45 and CD45-associated monoclonal antibodies known to inhibit normal non-MHC-restricted cytotoxicity but not specific MHC-restricted cytotoxic T cell activity. In contrast, the CD3 monoclonal antibody OKT3 did not inhibit but redirected LGL non-MHC-restricted cytotoxicity against K562. Following modulation of the CD3 molecule, the LGL were still capable of cytolysis of K562 targets, but additional OKT3 could no longer redirect cytolysis. The data indicate that the CD3/TcR complex on the LGL clones in patients with large granular lymphoproliferative disease is not the receptor for antigen on K562 cells, although it retains functional capabilities. Thus the CD3/TcR positive subset appears to have bipotential cytotoxic characteristics involving additional unique receptors for non-MHC-restricted cytotoxicity.

Adult↗
PubMed1988

Hairy cell leukemia cells are relatively NK-insensitive targets.

The hypothesis that interferon alpha (IFN alpha) has its beneficial effects in hairy cell leukemia by activating natural killer cells against hairy cells was examined. Leukemic cells from patients with hairy cell leukemia were tested for their susceptibility to lysis by fresh and IFN alpha activated peripheral blood mononuclear (PBMC) cells from normal donors. All hairy cells tested were relatively insensitive to cytolysis by PBMC and IFN alpha activated PBMC. The low levels of 51Cr release obtained with a few donors was due to lysis of leukemic cells, not residual normal cells, and was mediated by a natural killer cell (T cell receptor independent) mechanism. Chronic lymphatic leukemic cells before and after treatment with phorbol ester were also resistant to cytolysis. Hairy cells were not susceptible to lymphokine activated killer (LAK) cells but were sensitive to lysis by antibody and complement. The insensitivity to cell mediated cytolysis against hairy cells was shown by cold target inhibition to be a lack of target recognition by NK cells.

Antibodies, Monoclonal↗
PubMed1987

Inhibition of natural killer-cell mediated cytolysis with monoclonal antibodies to restricted and non-restricted epitopes of the leucocyte common antigen.

Three monoclonal antibodies recognizing different epitopes of the leucocyte common molecule, CMRF-11 (against the restricted or B-220 leucocyte common molecule), CMRF-12 and CMRF-26 [each against a different epitope on the non-restricted or T200 leucocyte common (CD45) molecule], were tested for their effects on lymphocyte cytotoxicity. The individual monoclonal antibodies inhibited human natural killer cell-mediated cytolysis (NK-CMC) weakly, but a mixture of CMRF-11 + 12 + 26 antibodies inhibited cytolysis more consistently and to a greater extent. This mixture did not inhibit cytotoxic T lymphocytes derived from secondary mixed lymphocyte cultures. The CMRF-11 + 12 + 26 mix was shown to inhibit a post-conjugate formation stage of lysis at the effector cell level. Inhibition of NK-CMC of a wide range of target cells, including the T-cell lines Jurkat, HSB2 and Molt 4, was demonstrated.

Antibodies, Monoclonal↗