Quantitative estimation of major histocompatibility complex antigens on live tumour cells

Quantitation of major histocompatibility complex (MHC) antigens on cells can accurately be done by using a flowcytometer. Since flowcytometer is not freely accessable an alternate, simple method for relative quantitation of MHC antigens has been devised. In this procedure, YAC lymphoma cells were first treated with a monoclonal anticlass I MHC antibody and then with a rabbit anti mouse Ig-antibody coupled to peroxidase, followed by colour development using a substrate of peroxidase enzyme. Various assay parameters have been optimized. The validity of the procedure was examined by assessing the enhanced MHC expression on YAC cells treated with a soluble rat spleen derived factor, by the new procedure as well as by the flowcytometer. Comparable results were obtained by using both techniques.     

Interferon-gamma induction of major histocompatibility complex antigens on cultured bovine luteal cells

To investigate immunological mechanisms that may be involved in luteal function, the presence of Class I and Class II major histocompatibility complex (MHC) antigens on cultured bovine luteal cells was examined. After 72 h in serum-free culture, Class I antigens were markedly expressed on luteal cells, as determined by indirect immunofluorescence, whereas expression of Class II antigens was limited. The expression of MHC antigens on luteal cells was increased by treatment with the T-lymphocyte factor, interferon-gamma (IFN-gamma). Class I and II antigens were elevated 25% and 370% above controls, respectively, after IFN-gamma exposure. Since the corpus luteum is regulated by luteinizing hormone (LH), luteal cells were treated with either hormone alone or hormone in addition to IFN-gamma, and antigen expression was determined. LH treatment attenuated IFN-gamma-induction of Class II antigens on bovine luteal cells. These observations are the first to demonstrate the presence of MHC antigens on bovine luteal cells and the modulation of antigen expression by the lymphokine IFN-gamma and by LH.     

The influence of major histocompatibility complex class I antigens on tumor growth and metastasis

The work described here demonstrates the importance of major histocompatibility complex class I antigens for the control of tumor growth and metastasis by the host's immune system. In certain murine tumor cells which have lost expression of H-2 class I antigens, a de novo expression of H-2 can be achieved by transfection with syngeneic class I genes. In contrast to the parental cells the transfected tumors do not grow any more in syngeneic mice, or in other cases they do not form metastases. The studies suggest that the de novo expression of the H-2 antigens renders the tumors highly immunogenic and leads to effective recognition of a tumor-associated antigen in conjunction with the transfected H-2 antigen. These conclusions were confirmed in other tumor systems. For example, separation of a heterogeneous tumor into clones expressing high or low amounts of H-2 showed that only the tumor cell with low H-2 grew well in syngeneic mice, whereas the H-2 high tumor clones were rejected. In other studies in vitro induction by IFN-gamma of H-2 antigen on H-2 negative tumors led to reduced tumor growth in vivo which was due to the increased immunogenicity. About 10% of human tumors are also low or defective for HLA class I expression and often these tumors appear to be more malignant. The class I negative tumors could either have arisen from class I low or negative tissues or are HLA loss variants which escaped the attack of the immune system. Altogether, our studies and the data of other laboratories demonstrate the important role of class I antigens for anti-tumor immunity and they suggest that modulation of class I expression by gene transfection or by induction with soluble mediators could be a useful tool for the manipulation of tumor immunity.     

Interactions between the receptors for platelet-derived growth factor and epidermal growth factor

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.     

Generation of major histocompatibility complex class I antigens

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of antigen-presenting cells is an effective extracellular representation of the intracellular antigen content. The intracellular proteasome-dependent proteolytic machinery is required for generating MHC class I-presented peptides. These peptides appear to be derived mainly from newly synthesized defective ribosomal products, ensuring a rapid cytotoxic T lymphocyte-mediated immune response against infectious pathogens. Here we discuss the generation of MHC class I antigens on the basis of the currently understood molecular, biochemical and cellular mechanisms.     

Degradation of endocytosed epidermal growth factor and virally ubiquitinated major histocompatibility complex class I is independent of mammalian ESCRTII

Models for protein sorting at multivesicular bodies in the endocytic pathway of mammalian cells have relied largely on data obtained from yeast. These data suggest the essential role of four ESCRT complexes in multivesicular body protein sorting. However, the putative mammalian ESCRTII complex (hVps25p, hVps22p, and hVps36p) has no proven functional role in endosomal transport. We have characterized the human ESCRTII complex and investigated its function in endosomal trafficking. The human ESCRTII proteins interact with one another, with hVps20p (a component of ESCRTIII), and with their yeast homologues. Our interaction data from yeast two-hybrid studies along with experiments with purified proteins suggest an essential role for the N-terminal domain of hVps22p in the formation of a heterotetrameric ESCRTII complex. Although human ESCRTII is found in the cytoplasm and in the nucleus, it can be recruited to endosomes upon overexpression of dominant-negative hVps4Bp. Interestingly, we find that small interference RNA depletion of mammalian ESCRTII does not affect degradation of epidermal growth factor, a known cargo of the multivesicular body protein sorting pathway. We also show that depletion of the deubiquitinating enzymes AMSH (associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)) and UBPY (ubiquitin isopeptidase Y) have opposite effects on epidermal growth factor degradation, with UBPY depletion causing dramatic swelling of endosomes. Down-regulation of another cargo, the major histocompatibility complex class I in cells expressing the Kaposi sarcoma-associated herpesvirus protein K3, is unaffected in ESCRTII-depleted cells. Our data suggest that mammalian ESCRTII may be redundant, cargo-specific, or not required for protein sorting at the multivesicular body.     

Three classes of epidermal growth factor receptors on HeLa cells

The kinetics of 125I-labeled epidermal growth factor (EGF) binding to receptors on HeLa cells were investigated. Scatchard analysis revealed the presence of 22,000 high affinity receptors (Kd = 0.12 nM) and 25,000 low affinity receptors per cell (Kd = 9.2 nM). The kinetic analysis of EGF binding to high affinity receptors was performed with cells pretreated with the monoclonal antibody 2E9, which prevents specifically EGF binding to low affinity receptors. The study of EGF binding to only low affinity receptors was performed with cells pretreated with the phorbol ester phorbol 12-myristate 13-acetate, which induces a conversion of high affinity receptors to low affinity receptors. This kinetic analysis of EGF binding to HeLa cells revealed the presence of three types of receptors. High affinity receptors were found to consist of one receptor type (type I) with a kinetic association constant (kass) of 6.2 x 10(5) M-1.s-1 and a kinetic dissociation constant (kdis) of 3.5 x 10(-4) s-1. The low affinity receptors were found to consist of two kinetic distinguishable sites: type II or fast sites with kass = 3.3 x 10(6) M-1.s-1 and kdis = 8.1 x 10(-3) s-1 and the type III or slow sites with kass = 3.2 x 10(4) M-1.s-1 and kdis = 1.6 x 10(-4) s-1. The regulatory mechanism which may determine the EGF binding characteristics is discussed.     

Distribution of the major histocompatibility complex antigens on different cellular components of human liver

Monoclonal mouse antibodies to the "framework" determinants of the class I and II molecules of the major histocompatibility complex (MHC) were used to demonstrate the presence of the MHC antigens in human liver. First, the localization of these antigens was demonstrated from frozen section histology with indirect FITC immunofluorescence and the cell component(s) binding the mouse antibody were identified by rabbit marker antisera and indirect TRITC immunofluorescence. Second, the antigen expression on the cell surface was analyzed by the Staphylococcus aureus rosette method from cytological cell smears. All antibodies reacted with cells in the liver sinusoids, both with the Kupffer cells and at least partially with the sinusoidal endothelial cells. The same antisera reacted also with the bile duct cells, though weaker, and with some stromal cells in close proximity of the blood vessels. The vascular endothelial cells of hepatic artery, hepatic vein, and portal vein displayed no reaction. Thus human liver differs strikingly from, e.g., human kidney, where the vascular endothelial cells contain large amounts of MHC antigens on the cell surface. This difference may be one explanation to why liver allografts are less promptly rejected than renal allografts in man.     

Specific epidermal growth factor receptors on porcine and human thyroid membranes

Specific, high affinity, saturable receptors for epidermal growth factor (EGF) have been demonstrated both on porcine and on human thyroid membranes. The binding affinities of porcine (Ka 3.0 X 10(-9) M) and human thyroid EGF receptors (Ka 1.75 X 10(-9) M) are very similar. TSH does not inhibit the binding of 125I-EGF to either membrane. These results suggest the possibility that EGF may be involved in the regulation of human as well as porcine thyroid follicular cell growth and function.     

Expression of major histocompatibility complex antigens on the bovine placenta

Immunohistochemistry was utilized to determine expression of the major histocompatibility complex (MHC) antigens on Day 8-9 hatched blastocysts and fetal membranes of mid- to late gestation cows and to examine the pattern of leucocytic infiltration into the gravid uterus. Hatched blastocysts were weakly positive for MHC class I antigens. In the mature placenta, chorioallantoic membranes in the interplacentomal area showed positive immunostaining for class I antigens on the chorionic epithelium but had no staining for class II antigens. There was an accumulation of lymphoid cells expressing class II antigens directly beneath the luminal epithelium of the endometrium. In addition, cells staining for leucocyte common antigen were present both within and beneath the luminal epithelium. Some cells positive for class II and leucocyte common antigen (CD45) were also associated with uterine glands. In the placentomes, class I antigens were expressed only on maternal caruncular septa. Fetal cotyledonary villi had no detectable immunostaining for class I and II antigens. No distinct pattern of leucocyte infiltration in the maternal caruncular tissue was observed; the caruncular septa contained some cells that were labelled for CD45 and a few class II-positive cells around blood vessels. The results indicate that the fetal placenta of the cow expresses MHC class I antigens in a regionally defined manner and there is a differential accumulation of lymphoid cells in the uterus.     

Interaction between glucocorticoids and epidermal growth factor in vitro in the growth of palatal mesenchymal cells from the human embryo

Previous studies of ours have shown that palatal mesenchymal cells from the human embryo (HEPM cells) are responsive both to the glucocorticoid dexamethasone (DEX) and epidermal growth factor (EGF) through mechanisms associated with cytoplasmic and cell surface receptors, respectively. HEPM cell growth was inhibited by DEX and was stimulated by EGF. In the present study, the interactions between DEX and EGF were investigated. DEX (10(-6) M) enhanced EGF-stimulated HEPM cell growth as assessed by an increase in cell number and ornithine decarboxylase activity under serum-free cell culture conditions. DEX also enhanced the specific binding of 125I-EGF to these cells, which was reflected in an increase in both the number and the affinity of EGF receptors. EGF (1 ng/ml), on the other hand, decreased the number of sites per cell which specifically bind 3H-DEX. EGF completely prevented the inhibition by DEX of HEPM cell growth. These results indicated that DEX and EGF interact with each other in the process(es) regulating HEPM cell growth. This interaction may be partially influenced by direct modulation of existing receptors for DEX and EGF present in the cells.     

Topography of human placental receptors for epidermal growth factor

These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.     

The major histocompatibility complex and human evolution

Many alleles at the human major histocompatibility complex (HLA) loci diverged before the divergence of humans and great apes from a common ancestor. This fact puts a lower limit on the size of the bottleneck in human evolution: the genus Homo must have been founded by no less than ten and probably by more than 10,000 individuals.     

Tolerance to limb tissue allografts between swine matched for major histocompatibility complex antigens

Transplantation of limb tissue allografts would greatly expand the realm of reconstructive surgery. However, the toxicity of chronic immunosuppression has adversely tilted the risk-benefit balance for clinical transplant. In this study, a procedure was sought to achieve host tolerance to limb tissue allografts through matching of the major histocompatibility complex (MHC) antigens between donor and host swine using only a 12-day course of cyclosporine. Massachusetts General Hospital (MGH) miniature swine were used as a large animal model with defined MHC, and musculoskeletal grafts from the donor hind limb were transplanted heterotopically to the recipient femoral vessels. Allografts from MHC-mismatched donors treated with cyclosporine (n = 4) were rejected in less than 6 weeks by gross inspection and histologic sections. Allografts from MHC-matched, minor antigen mismatched donors not treated with cyclosporine (n = 4) were rejected between 9 and 12 weeks. Allografts from similarly matched donors treated with 12 days of cyclosporine (n = 7) showed no evidence of rejection until sacrifice between 25 and 47 weeks. Thus allograft tolerance was achieved between MHC-matched swine using a limited course of cyclosporine. Demonstration of limb tissue allograft survival in a large animal model without long-term immunosuppression represents an important step toward clinical transplantation.     

Neuronal differentiation in response to epidermal growth factor of transfected murine P19 embryonal carcinoma cells expressing human epidermal growth factor receptors

The human epidermal growth factor receptor (hEGF-R) was introduced into murine P19 embryonal carcinoma (EC) cells, which do not express endogenous EGF-R. Undifferentiated stable P19 EC transfectants containing multiple copies of the hEGF-R complementary DNA were isolated. These cells express functional EGF-R, exhibiting characteristic biphasic EGF binding and intrinsic tyrosine protein kinase activity. Whereas normally EGF induces the expression of multiple nuclear protooncogenes, only junB expression is induced by EGF in the HER-transfected cells. This indicates that undifferentiated P19 EC cells contain at least part of a signal transduction machinery capable of coupling to the ectopically expressed hEGF-R. Interestingly, neuronal differentiation is induced in these cells in response to EGF under culture conditions resembling those during early preimplantation embryogenesis. These results indicate that neuronal differentiation of pluripotent P19 EC cells can be induced via activation of a tyrosine protein kinase signaling pathway.