GPI-linked Molecules Research Articles

CD157 Can Replace CD24 and CD14 in a Single-Tube Flow-Cytometric Assay to Detect Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones on Both Neutrophils and Monocytes: A Prospective Study From North India.

IntroductionAs per current guidelines, detection of paroxysmal nocturnal hematuria (PNH) clones on leucocytes requires the demonstration of the loss of at least two glycosyl-phosphatidyl-inositol (GPI)-linked molecules on both neutrophils and monocytes by flow cytometry. CD24 and CD14 are GPI-linked molecules expressed on neutrophils and monocytes respectively, whereas another GPI-linked molecule, CD157, is expressed on both neutrophils and monocytes. This prospective study evaluated the ability of CD157 to replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes.Materials and methodsPNH clones were newly detected in 52 patients by an existing “standard” single-tube six-color flow-cytometric method, which was routinely performed in our laboratory at the time of undertaking this study. Six antibodies (CD45/CD15/CD64/CD24/CD14/FLAER) were used in this "standard" technique. Subjects were divided into two groups: (i) PNH disease (n=10), and (ii) aplastic anemia/myelodysplastic syndrome (AA/MDS) (n=42). Diagnosis of PNH disease and AA/MDS were made as per standard literature and guidelines. Results were compared with a single-tube five-color “test” assay using the antibodies CD45/CD15/CD64/CD157/FLAER by flow cytometry. Samples from 20 healthy control subjects were used to calculate cut-off values for the “test” assay.ResultsBy the "test" method, cut-off values for detecting PNH clones obtained from receiver operating-characteristic curve analysis were >0.4% for neutrophils (sensitivity=96.15%, specificity=95%), and >0.9% for monocytes (sensitivity=98.08%, specificity=95%). There was significant correlation between PNH clone sizes measured by both the “standard” and “test” assays in neutrophils (PNH disease: r=0.976, p<0.001; AA/MDS: r=0.980, p<0.001) as well as monocytes (PNH disease: r=0.806, p=0.005; AA/MDS: r=0.915, p<0.001). Bland-Altman analysis showed agreement between both assays in all the 52 patients and in individuals with AA/MDS. The cost of the test to the patients was about 15% less in the “test” method than the ”standard” technique, with improved technical efficiency.ConclusionCD157 can replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes, with reduced cost to the patients and improved technical efficiency.

Excellence in PNH in Canada (EPIC): A Single Centre Pilot Project Evaluating Disease Trajectory for PNH Patients Receiving Eculizumab

Introduction:Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease caused by mutations that impair formation of GPI anchors. Absence of GPI-linked molecules CD55 and CD59 renders blood cells sensitive to complement-mediated damage. The classic presentation includes intravascular hemolysis, thrombophilia, and marrow failure. Other symptoms, such as fatigue, dysphagia, and abdominal pain may also occur. Eculizumab inhibits complement protein C5 and has drastically improved outcomes in PNH. Despite greater awareness of PNH since eculizumab's approval, it remains a rare disease and patients may go years without a diagnosis. To investigate this, we reviewed our centre's experience to identify areas that could be improved upon.Methods:A retrospective review was completed of PNH patients followed at our centre over the last 5 years. Data was collected via chart review and interviews. Information collected included age of symptom onset, time from symptoms to assessment, hematology referral, diagnosis, and to start of therapy. Laboratory investigations were also recorded. Patients with small clones (<10%) were excluded as they would not qualify for eculizumab.Results:Nine patients were enrolled (6 male, 3 female). Table 1 summarizes their presenting features. For reference, the same categories are presented for Canadian patients in the International PNH Registry. Figure 1 shows symptom progression from onset, diagnosis, and start of therapy.Average age at symptom onset was 38.2±20.5 years. Cytopenia was the most common presentation (78%). Dark urine occurred in 88%. Three patients (33%) reported fatigue as their first symptom. One, with aplastic anemia (AA) and treated with immunosuppression, developed abdominal pain and dark urine 7 years later and was found to have PNH despite negative initial testing.Table 2 summarizes individual patient timelines for diagnosis and treatment. Average time from initial symptom to medical assessment was 2.7±6.6 years, though 7 (78%) presented within several weeks. One did not present until 20 years after anemia and jaundice occurred, as she was asymptomatic. Another patient waited 4 years before assessment because he occasionally experienced dark urine with exertion and attributed this to exercise.Median duration from presentation to diagnosis was 3 years (range: 0.05-30); however, 3 (33%) patients were diagnosed within one month. One was diagnosed when he presented with worsening anemia one year after initial presentation. Another presented in 1975 with renal dysfunction, underwent extensive evaluation, but not diagnosed with PNH for 30 years. Dark urine with anemia prompted evaluation in 79%. Three reported abdominal pain, 22% jaundice, 22% dysphagia, 11% dyspnea, and 67% were fatigued.Only one patient in our cohort developed thromboembolism (11%), compared to 19% of Canadians in the International PNH Registry. This patient was anemic and jaundiced for many years but only after developing hepatic vein thrombosis was she was evaluated for PNH.Patients saw a median of 4 health care providers (HCP) (range: 2-8) before diagnosis. Six saw general practitioners (GP) and emergency physicians before hematology and 3 saw other specialists first. One saw dermatology for a rash not initially realized to be thrombocytopenic petechiae. He was then referred to hematology after developing severe anemia as well. One saw several nephrologists for dark urine; he was diagnosed with glomerulonephritis, and this was not re-evaluated for 20 years. One saw urology and gastroenterology before hematology referral. Two were worked up for PNH after their GPs retired and were reviewed by new ones.Eight patients (89%) are currently on eculizumab. One does not meet funding criteria. The average time between diagnosis and initiation of therapy was 1.3±1.7 years.Conclusion:PNH is rare and can present in heterogeneous ways. Outside hematology, HCPs may rarely encounter patients, if ever. Time interval between onset of symptoms to formal diagnosis in our cohort had the greatest duration and variation. Once diagnosed, all patients in our cohort were initiated on therapy quite rapidly save for one who had a nine-year delay between diagnosis and availability of eculizumab in Canada. The greatest delay in this cohort was the time from symptom onset to diagnosis, suggesting that a focus on increasing awareness of PNH may be the area where most efforts should be placed. DisclosuresPatriquin:Octapharma: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support and is site investigator for clinical trials with the company; Ra Pharmaceuticals: Consultancy, Research Funding.

Tumor membrane vesicle (TMV)-based cancer vaccine induces dendritic cell maturation and downstream T cell activation

Abstract Breast cancer remains the most commonly diagnosed cancers among American women, with over 250,000 new cases expected per year. Breast cancer can be subdivided into different categories depending on the expression of receptors, most commonly into ER+, PR+, HER2+, and TNBC. Clinically, small molecule inhibitors of the hormone receptors and antibodies for HER2 have been developed to target these receptors and inhibit tumor signaling or eliminate these cells via phagocytosis and ADCC. Despite this, tumor cells that develop resistance to therapy remain a significant unmet need as they tend to be more aggressive and metastasize. We developed a novel immunotherapy approach that addresses the high degree of inter-patient and intra-tumor heterogeneity by making tumor membrane vesicle (TMV) vaccines out of individual tumor samples and modifying them with GPI-linked molecules like IL-12 and B7-1 to induce an antitumor immune response. In the present study, we investigated whether GPI-GM-CSF and GPI-IL-12 on TMVs from HER2+ murine breast cancer can target and activate antigen presenting cells. Here, we show that TMVs containing with GPI-GM-CSF and GPI-IL-12 can be phagocytosed by both macrophages and dendritic cells, induce upregulation of MHC-I, MHC-II and CD86, and inflammatory cytokine production in vitro. Further, when the DCs activated with our TMV vaccine were co-cultured with syngeneic T cells, they induced the upregulation of T cell activation markers. In summary, our results indicate that TMVs containing GPI-IL-12 and GPI-GM-CSF can enhance uptake and maturation of APCs and induce a strong T cell response downstream making it a potential therapeutic approach for HER2+ breast cancer.

Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry.

The identification of eosinophils by flow cytometry is difficult because most of the surface antigens expressed by eosinophils are shared with neutrophils. Some methods have been proposed, generally based on differential light scatter properties, enhanced autofluorescence, lack of CD16 or selective positivity of CD52. Such methods, however, show several limitations. In the present study we report a novel method based on the analysis of glycosylphosphatidylinositol (GPI)-linked molecules. The combination of CD157 and FLAER was used, since FLAER recognizes all GPI-linked molecules, while CD157 is absent on the membrane of eosinophils and expressed by neutrophils. Peripheral blood samples from normal subjects and patients with variable percentages of eosinophils (n = 31), and without any evidence for circulating immature myeloid cells, were stained with the combination of FLAER-Alexa Fluor and CD157-PE. A FascCanto II cytometer was used. Granulocytes were gated after CD33 staining and eosinophils were identified as CD157(-)/FLAER(+) events. Neutrophils were identified as CD157(+)/FLAER(+) events. The percentages of eosinophils detected by this method showed a very significant correlation both with automated counting and with manual counting (r = 0.981 and 0.989, respectively). Sorting assays were carried out by a S3 Cell Sorter: cytospins obtained from CD157(-)/FLAER(+) events consisted of 100% eosinophils, while samples from CD157(+)/FLAER(+) events were represented only by neutrophils. In conclusion, this method shows high sensitivity and specificity in order to distinguish eosinophils from neutrophils by flow cytometry. However, since CD157 is gradually up-regulated throughout bone marrow myeloid maturation, our method cannot be applied to cases characterized by immature myeloid cells.

The human NKG2D ligand ULBP2 can be expressed at the cell surface with or without a GPI anchor and both forms can activate NK cells

The activating immune receptor NKG2D binds to several stress-induced ligands that are structurally different. MHC-class-I-related chain (MIC) A/B molecules have a transmembrane domain, whereas most UL16 binding proteins (ULBPs) are glycosylphosphatidylinositol (GPI)-linked molecules. The significance of this variability in membrane anchors is unclear. Here, we demonstrate that ULBP2, but not ULBP1 or ULBP3, can reach the cell surface without the GPI modification. Several proteins are expressed at the cell surface as both transmembrane and GPI-linked molecules, either via alternative splicing or by the expression of linked genes. However, to our knowledge, ULBP2 is the first single mammalian cDNA that can be expressed as either a transmembrane or a GPI-anchored protein. The rate of maturation and the levels of cell surface expression of the non-GPI-linked form were lower than those of the GPI-linked ULBP2. Nonetheless, non-GPI ULBP2 was recognised by NKG2D and triggered NK cell cytotoxicity. These data show that differences in membrane attachment by NKG2D ligands are more important for regulation of their surface expression than for cytotoxic recognition by NKG2D and emphasise that detailed characterisation of the cell biology of individual NKG2D ligands will be necessary to allow targeted modulation of this system.

Using T. brucei as a biological epitope-display platform to elicit specific antibody responses

The African trypanosome (Trypanosoma brucei) is transmitted by the bite of the tsetse vector to the mammalian bloodstream where it exists as a completely extracellular parasite. As a result of this exposure, the parasite elicits a robust immune response that is almost exclusively antibody mediated, and is extremely specific to the trypanosome coat displayed on the surface. This coat is comprised of ~11million copies of a single gpi-linked molecule (the variable surface glycoprotein or VSG) and can therefore be used as a powerful platform for the immunogenic display of antigenic determinants. Here we describe a method to display repetitive, ordered arrays of linear epitopes on the surface of T. brucei and to then use the engineered organisms to generate specific anti-epitope antibody responses, upon injection into mice. This method offers an alternative approach to generating anti-peptide antibodies, and could be a useful option in cases where more traditional methods have failed.

Expression of human FcγRIIIa as a GPI-linked molecule on CHO cells to enable measurement of human IgG binding

The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human FcγRIIIa, the key receptor for ADCC, an attractive alternative method of assessment. Here, we describe the development of cell lines and assays for this purpose. The transmembrane receptor, FcγRIIIa, requires co-expression with signal transducing subunits to prevent its degradation, unlike the homologous receptor FcγRIIIb that is expressed as a GPI-anchored molecule. Therefore, to simplify the production of cell lines as reliable assay components, we expressed FcγRIIIa as a GPI-anchored molecule. Separate, stable CHO cell lines that express either the 158F or the higher-affinity 158V allotype of FcγRIIIa were isolated using fluorescence-activated cell sorting. The identities of the expressed receptors were confirmed using a panel of monoclonal antibodies that distinguish between subclasses and allotypes of FcγRIII and the cell lines were shown to have slightly higher levels of receptor than FcγRIII-positive peripheral blood mononuclear cells. Because the affinity of FcγRIIIa for IgG is intermediate amongst the receptors that bind IgG, we were able to use these cell lines to develop flow cytometric assays to measure the binding of both complexed and monomeric immunoglobulin. Thus, by choosing the appropriate method, weakly- or strongly-binding IgG can be efficiently compared. We have quantified the difference in the binding of wildtype IgG1 and IgG3 molecules to the two functional allotypes of the receptor and report that the FcγRIIIa-158V–antibody interaction is 3- to 4-fold stronger that the interaction with FcγRIIIa-158F. Overall, these robust assays should be valuable for batch-testing clinical material as well as providing tools for improving the design of therapeutic IgG.

Glycosylphosphatidylinositol (GPI)-anchored surface antigens in the allogeneic activation of T cells.

GPI-linked surface molecules have recently been described as structures with an activation potential for human T lymphocytes. To study the role of these molecules in T cell activation we analysed GPI-deficient or normal T cells from patients with paroxysmal nocturnal haemoglobinuria (PNH). On activation with allogeneic Epstein-Barr virus (EBV)-transformed B cell lines GPI-deficient freshly separated T cells or continuously growing T cell lines exhibited a significantly lower proliferation or cytokine production compared with their normal counterparts. In contrast, stimulation via the T cell receptor-associated CD3 structure resulted in a comparable response. There was no difference in activation of normal T lymphocytes when GPI-deficient B cells were used as stimulators compared with normal B cells obtained from the same PNH patient. We conclude from these data that GPI deficiency in PNH leads to a functional deficiency of GPI-deficient T cells. In contrast, no difference in activation of T lymphocytes for GPI-deficient cells on the stimulator cell level was observed.

Diagnosing PNH with FLAER and multiparameter flow cytometry

PNH is an acquired hematopoietic stem cell disorder leading to a partial or absolute deficiency of all glycophosphatidyl-inositol (GPI)-linked proteins. The classical approach to diagnosis of PNH by cytometry involves the loss of at least two GPI-linked antigens on RBCs and neutrophils. While flow assays are more sensitive and specific than complement-mediated lysis or the Hams test, they suffer from several drawbacks. Bacterial aerolysin binds to the GPI moiety of cell surface GPI-linked molecules and causes lysis of normal but not GPI-deficient PNH cells. FLAER is an Alexa488-labeled inactive variant of aerolysin that does not cause lysis of cells. Our goals were to develop a FLAER-based assay to diagnose and monitor patients with PNH and to improve detection of minor populations of PNH clones in other hematologic disorders. In a single tube assay, we combined FLAER with CD45, CD33, and CD14 allowing the simultaneous analysis of FLAER and the GPI-linked CD14 structure on neutrophil and monocyte lineages. Comparison to standard CD55 and CD59 analysis showed excellent agreement. Because of the higher signal to noise ratio, the method shows increased sensitivity in our hands over single (CD55 or CD59) parameter analysis. Using this assay, we were able to detect as few as 1% PNH monocytes and neutrophils in aplastic anemia, that were otherwise undetectable using CD55 and CD59 on RBC's. We also observed abnormal FLAER staining of blast populations in acute leukemia. In these cases, the neutrophils stained normally with FLAER, while the gated CD33bright cells failed to express normal levels of CD14 and additionally showed aberrant CD45 staining and bound lower levels of FLAER. FLAER combined with multiparameter flow cytometry offers an improved assay for diagnosis and monitoring of PNH clones and may have utility in detection of unsuspected myeloproliferative disorders.

Placental Expression of Glycophosphatidylinositol (GPI)‐anchored Proteins in Paroxysmal Nocturnal Haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal stem cell disorder in which a defect of glycophosphatidylinositol (GPI)-anchored proteins leads to higher morbidity and mortality because of intravascular haemolysis, haemoglobinuria, pancytopenia and an increased frequency of thrombotic events. We report here the clinical features of a pregnant woman with PNH and present an immunhistochemical analysis of complement regulators, leukocyte activation markers and placental alkaline phosphatase (PALP) on syncytiotrophoblasts and inflammatory cells in her placenta. Placental tissue from normal deliveries served as controls. The patient had severe PNH with haemolysis, thrombosis episodes and signs of bone marrow failure. Placental syncytiotrophoblasts and villous cells of fetal origin in both normal placentas and the placenta from the PNH patient expressed PALP and the complement regulators CD46, CD55 and CD59. Additionally, CD11b-positive leukocytes of presumed maternal origin were negative for CD15 in the PNH placenta, while they stained positive within the villous space and in normal placentas. These findings show that fetally derived cells in the PNH placenta expressed GPI-linked molecules that are known to be of importance for a successful pregnancy outcome.

Role of NKG2D in tumor cell lysis mediated by human NK cells: cooperation with natural cytotoxicity receptors and capability of recognizing tumors of nonepithelial origin

NKG2D is a recently described activating receptor expressed by both NK cells and CTL. In this study we investigated the role of NKG2D in the natural cytolysis mediated by NK cell clones. The role of NKG2D varied depending on the type of target cells analyzed. Lysis of various tumors appeared to be exclusively natural cytotoxicity receptors (NCR) dependent. In contrast, killing of another group of target cells, including not only the epithelial cell lines HELA and IGROV-1, but also the FO-1 melanoma, the JA3 leukemia, the Daudi Burkitt lymphoma and even normal PHA-induced lymphoblasts, involved both NCR and NKG2D. Notably, NK cell clones expressing low surface densities of NCR (NCR(dull)) could lyse these tumors in an exclusively NKG2D-dependent fashion. Remarkably, not all of these targets expressed MICA/B, thus implying the existence of additional ligands recognized by NKG2D, possibly represented by GPI-linked molecules. Finally, we show that the engagement of different HLA class I-specific inhibitory receptors by either specific antibodies or the appropriate HLA class I ligand led to inhibition of NKG2D-mediated NK cell triggering.

Role of NKG2D in tumor cell lysis mediated by human NK cells: cooperation with natural cytotoxicity receptors and capability of recognizing tumors of nonepithelial origin.

NKG2D is a recently described activating receptor expressed by both NK cells and CTL. In this study we investigated the role of NKG2D in the natural cytolysis mediated by NK cell clones. The role of NKG2D varied depending on the type of target cells analyzed. Lysis of various tumors appeared to be exclusively natural cytotoxicity receptors (NCR) dependent. In contrast, killing of another group of target cells, including not only the epithelial cell lines HELA and IGROV-1, but also the FO-1 melanoma, the JA3 leukemia, the Daudi Burkitt lymphoma and even normal PHA-induced lymphoblasts, involved both NCR and NKG2D. Notably, NK cell clones expressing low surface densities of NCR (NCR(dull)) could lyse these tumors in an exclusively NKG2D-dependent fashion. Remarkably, not all of these targets expressed MICA/B, thus implying the existence of additional ligands recognized by NKG2D, possibly represented by GPI-linked molecules. Finally, we show that the engagement of different HLA class I-specific inhibitory receptors by either specific antibodies or the appropriate HLA class I ligand led to inhibition of NKG2D-mediated NK cell triggering.

PNH cells are as sensitive to T-cell-mediated lysis as their normal counterparts: implications for the pathogenesis of paroxysmal nocturnal haemoglobinuria.

The mechanism responsible for the bone marrow failure that is almost invariable in paroxysmal nocturnal haemoglobinuria (PNH) is unknown. Based on the close association between PNH and idiopathic aplastic anaemia, a plausible pathogenetic model predicts that, in PNH, autoreactive T cells specific for haemopoietic stem cells (HSCs) cause depletion of normal HSCs, whereas PNH HSCs escape this T-cell-mediated attack. In this study, we addressed the hypothesis that PNH HSCs are resistant to the cytotoxic effect of T cells because they lack surface expression of one or more glycosylphosphatidylinositol (GPI)-linked molecules. We tested the sensitivity of normal and PNH Epstein-Barr virus (EBV)-transformed B-cell lymphoblastoid cell lines (BLCLs) to the cytotoxic effect of autologous EBV-specific T-cell lines and clones from a patient with PNH in an in vitro experimental system. We found that the PNH BLCLs were no less sensitive to T-cell-mediated cytotoxicity than non-PNH isogenic BLCLs, indicating that GPI-linked molecules on the surface of target cells are not required for killing by T cells. This suggests that the mechanism whereby PNH HSCs survive T-cell attack is not because of the lack of surface expression of one or more GPI-linked molecules. By implication, other mechanisms become more probable.

IMPLICATIONS OF RECENT INSIGHTS INTO THE PATHOPHYSIOLOGY OF PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA

IMPLICATIONS OF RECENT INSIGHTS INTO THE PATHOPHYSIOLOGY OF PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA

Human Red Cells With Paroxysmal Nocturnal Haemoglobinuria Phenotype Are Competent For The Growth Of Plasmodium falciparum

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired blood disorder characterised by acute intravascular haemolysis, due to increased susceptibility of red cells to complement -mediated lysis. Any red cell abnormality that would affect the invasion and growth of Plasmodium falciparum would serve as a useful tool for the future intervention of malaria infection in humans. The aim was to find out whether P. Falciparum can invade and infect these abnormal human red cells, and whether the parasite depends on the host cell for the synthesis of its GPI anchors. Patients with the PNH condition have a mixture of normal and abnormal red blood cells (RBCs) in their blood. In order to obtain a homogenous sample of abnormal (PNH) cells, PNH red cells were purified from patients with this condition by the process of 'panning'. The purified cells were used as hosts for the culture of P.falciparum in vitro. Results show that GPI-linked molecules on the red cell surface are not required for the efficient entry of the parasites, and that the PNH red cells are competent to sustain the growth of P.falciparum. Nigerian Quarterly Journal of Hospital Medicine Vol. 9, No. 2 (June 1999) pp. 138-142

Cross-linking of the CAMPATH-1 antigen (CD52) mediates growth inhibition in human B- and T-lymphoma cell lines, and subsequent emergence of CD52-deficient cells.

The CAMPATH-1H (CD52) antigen is a 21 000-28 000 MW glycopeptide antigen that is highly expressed on T and B lymphocytes and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The humanized CAMPATH-1H anti-CD52 antibody is extremely effective at mediating depletion of both normal and tumorigenic lymphocytes in vivo and has been used in clinical trials for lymphoid malignancy and rheumatoid arthritis. Cross-linking GPI-anchored molecules, including CD52, on the surface of T lymphocytes in the presence of phorbol 12-myristate 13-acetate or anti-CD3, results in cellular activation. In the present study we have investigated the functional effects of cross-linking CD52 on T and B tumour cell lines. Cross-linking CD52 on either a B-cell line, Wien 133, which expresses high levels of endogenous CD52 or Jurkat T cells transfected and selected to express high levels of CD52 resulted in growth inhibition. This effect showed slower kinetics and occurred in a lower percentage of cells than growth inhibition stimulated via T- or B-cell receptors. Growth inhibition of the Wien 133 line was followed by the induction of apoptosis, which appeared independent of the Fas/Fas L pathway. Wien 133 cells surviving anti-CD52 treatment were selected and cloned and found to have down-regulated CD52 expression, with a characteristic biphasic pattern of 10% CD52-positive, 90% negative by fluorescence-activated cell sorter analysis. Interestingly, surface expression of other GPI-linked molecules, such as CD59 and CD55, was also down-regulated, but other transmembrane molecules such as surface IgM, CD19, CD20, HLA-DR were unaffected. The present study and previous work show that this is due to a defect in the synthesis of mature GPI precursors. Separation of CD52-positive and negative populations in vitro resulted in a rapid redistribution to the mixed population. Injection of CD52-negative cells into nude mice to form a subcutaneous tumour resulted in a substantial increase in expression of CD52. These results suggest that the defect in the Wien 133 cells is reversible, although the molecular mechanism is not clear. These observations have relevance to the clinical situation as a similar GPI-negative phenotype has been reported to occur in lymphocytes following CAMPATH-1H treatment in vivo.

Role of Natural Killer Cells, Macrophages, and Accessory Molecule Interactions in the Rejection of Pig-to-Primate Xenografts Beyond the Hyperacute Period

Pig-to-primate cardiac xenografts surviving beyond the period of hyperacute rejection succumb after 3–4 days to a secondary immunologic response characterized by xenograft infiltration with NK cells and macrophages. Circulating baboon mononuclear cells contain NK cell precursors which mediate lysis of porcine endothelium by two distinct mechanisms: antibody-dependent cellular cytotoxicity and lymphokine activation. IL-2 activated NK lysis of porcine endothelium was 2.4-fold stronger than lysis occuring following engagement of FcRIII by xenoreactive IgG. IL-2 augmented NK lysis involved interactions between CD2 and CD49d on baboon NK cells and their respective ligands on porcine endothelium, since NK lysis was reduced either by using Mabs against CD2, CD49d, or porcine VCAM, or by treating endothelial cells with PIPLC to cleave GPI-linked molecules. These results imply that interactions between accessory molecule receptor-ligand pairs on primate NK cells, macrophages and porcine endothelium are of critical importance in delayed xenograft rejection.

In Vitro Incorporation of GPI-Anchored Proteins Into Human Erythrocytes and Their Fate in the Membrane

In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4°C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4°C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.

In Vitro Incorporation of GPI-Anchored Proteins Into Human Erythrocytes and Their Fate in the Membrane

AbstractIn many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4°C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4°C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.

Signal transduction via GPI-anchored membrane proteins.

Eukaryotic cells carry upon their cell surfaces a number of proteins which are anchored in the membrane via glycosylphosphatidylinositol (GPI) membrane anchors1 These proteins are attached to the cell membrane by hydrophobic attraction of the phospholipid tails of the GPI-anchor with lipids in the outer face of the plasma membrane bilayer. Consequently, they neither span the cell membrane nor do they interact directly with intracellular components. During intracellular transport, GPI anchors recruit specialized lipids and glycolipids(2,3,4). Acquisition of these lipids renders GPI-anchored proteins insoluble in a number of non-ionic detergents, and enables their enrichment in cell lysates by density-gradient centrifugation. These associated lipids, which include sphingomyelin and cholesterol, may alter the cell-surface distribution of GPI-linked molecules relative to their non-GPI-linked counterparts, and this is currently the subject of intensive study(5). These GPI-rich detergent-insoluble complexes also contain a number of other proteins which are not GPI-linked, and it is thought that these other molecules may play a key role in the functional properties of GPI anchors. It was shown using photobleaching recovery (FRAP) or single particle tracking (SPT) techniques(6) that GPI-linked molecules are not freely mobile in the cell membrane, an expected finding for a molecule which is associated only with the outer lipid leaflet of the plasma membrane. This finding implies that the mobility of Thy-1 is influenced by its association with immobile components of the membrane, and thus, indirectly, with the cytoskeleton. These observations implicate transmembrane molecules in the interactions of GPI-linked molecules with cytoplasmic and cytoskeletal components.