Antigen Shedding Research Articles

Expression and shedding of major histocompatibility complex product and blood group antigens by cells in monolayer cultures

The dynamic state of cell surface antigens is expressed not only by their lateral mobility, but also by their continuous biosynthetic turnover and shedding. Using a hemadsorption assay to visualize cell surface antigens, and a reverse plaque hemolytic assay to detect antigen release, we studied the expression and shedding of β 2-microglobulin ( β 2M), histocompatibility, and blood group antigens in monolayer cultures of fibroblasts (human and mouse) and epithelial cells (human colon carcinoma HT-29). It was found that antigen expression is related to age of the culture. β 2M, HL-A, and blood group substance A were abundantly expressed on young HT-29 cells, decreasing steeply with age of the culture. These antigens and the H-2 a antigen were only sparsely expressed on young human and mouse fibroblasts, but increased continuously with time after seeding. The media of these cultures showed marked antigenic activity for all these determinants, and there was a positive correlation between the levels of antigen expression on the cells and antigen shedding into the medium.

Correlation of cell cycle analysis with Duke's staging in colon cancer patients

The biologic potential of a tumor for growth and metastasis is relatively inaccurately mirrored by the present staging system in colon cancers [29]. Except for Stage IV disease, traditional morphologic criteria approaches only 50-75% accuracy for predicting recurrent disease. Reliable staging, however, has become more critical in the light of the current thrust in adjuvant therapy research. First, the patients receive toxic and expensive therapy. Second, and of even greater importance, a central tenet of adjuvant therapy research is that treatment and control groups under study are comparable. Inadequate staging not only undermines analysis, especially when small improvements of lo-20% may be expected, but also demands very large study groups to offset this defect. A significant amount of research has attempted to quantitate the metastatic potential of a tumor. In the animal model some factors such as cell adhesiveness, tumor angiogenesis, detachability, thromboplasticity, and shedding of membrane antigen have been associated with an increased propensity of a tumor to spread [23]. In the clinical forum, analysis of factors such as tumor grade [4, 71, vascular invasion [7], host immune status [ 1, 8, 91 have not been established as having a clear-cut prognostic value. Hopefully prospective studies, objective criteria, and more sophisticated statistical analysis [6] may yield less disappointing results in the future. In this respect, accumulating evidence indicates that the analysis of tumor kinetics will be a major determinant of the metastatic potential of a tumor. It is well established that kinetic data of tumor doubling time and labeling index (LI) is associated with length of survival [ 11, 18, 21, 271, but the effect of tumor doubling time on ultimate survival was reported by Joseph et al. [12, 161. In his report, patients with resection of solitary pulmonary metastasis with doubling times less than 40 days subsequently died of metastasis whereas 60% of the patients with tumor doubling time over 40 days were cured. Other measures of tumor proliferative activity, i.e, mitotic index in sarcomas 1281 and labeling index in breast cancer patients 1151 have recently been implicated as important prognostic determinants, and in the former instance has been incorporated into the A.J.C. stating system [20] as a factor influencing tumor grading. Finally, the development of flow cytometry [24] has now made the rapid and objective analysis of cell cycle kinetics an exciting clinical reality. In this study, the predictive potential of cell cycle analysis by flow cytometry was examined by comparing the proliferative activity of colorectal adenocarcinoma with Duke’s staging and modified Broder’s grading.

Turnover and Shedding of Ia Antigens by Murine Spleen Cells in Culture

Lactoperoxidase-catalyzed radioiodination was used to examine the metabolic fate of surface Ia antigens on murine spleen cells in culture. Ia antigens, detected predominately on splenic B lymphocytes, were lost from the cultured cells with biphasic kinetics: a 4 to 6 hr rapid phase, t 1/2 = 5 hr followed by slow release through 20 hr, t 1/2 = 30 hr. The rapid loss of Ia antigens observed was abolished by both harsh iodination conditions and nonphysiologic incubation conditions. The rapid decline in Ia activity was shown to be due to shedding of intact Ia antigens from the cell and to predominant release of IA subregion-coded proteins. Release of Ia antigens from the cell was accomplished by replacement at the cell surface, and thus reflected net membrane Ia turnover. Ia shedding was shown to be extremely temperature dependent, reflecting both a comparatively high activation enthalpy and entropy requirement for turnover.

Suppression of human T-cell colony formation during pregnancy

THE human fetus, as a natural allograft, escapes destruction by the maternal immune system. As a possible mechanism, Alexander1 proposed a rapid shedding of transplantation antigens by embryonic cells, resulting in a blindfolding of the otherwise fully functional maternal immune system. Other investigators have reported a suppressive activity of lymphoid cells from human cord blood on the proliferation of maternal lymphocytes2,3. Suppressor T cells are induced by α-fetoprotein in mice4, and the elevated levels of α-fetoprotein during pregnancy have been shown to exert an immunosuppressive influence in mice5,6 and in man7. Olding8 demonstrated a soluble factor from mitogen stimulated lymphoid cells of human newborns suppressing the proliferation of maternal lymphocytes. These observations point out that the embryo might protect itself by release of soluble factors, acting either themselves as lymphocyte suppressive agents, and/or by the induction of suppressor cells. However, no direct evidence has so far been presented to show the presence and action of T-cell suppressor cells in the immune system of pregnant women. Such an experiment would have to use components of the immune system of pregnant women solely, and therein demonstrate an immune suppression as compared to non-pregnant individuals. We present here results indicating that the ability of T cells from pregnant women to proliferate in soft agar cultures is strongly inhibited, regardless of the source of the serum being used in the assay, thus pointing rather to a cell-mediated suppressor mechanism.

Shedding of the glycoprotein from vesicular stomatitis virus-infected cells

In a culture of Chinese hamster ovary cells infected with vesicular stomatitis virus, there is specific shedding of viral antigens into the medium. This shedding appears to be unrelated to progeny formation or to cell lysis. Although all five of the virus-specific proteins are detected in the extracellular soluble fraction, the major antigen is the Gs protein. This protein has a molecular weight of 54,000. Indirect analysis of the content of sialic acid as well as peptide analysis of the Gs and G proteins of vesicular stomatitis virus suggest that the Gs protein is derived from the G protein by proteolysis. Both proteins are hydrophobic when analyzed by charge-shift electrophoresis. The presence of phenylmethylsulfonyl fluoride in the culture medium or the removal of serum from the culture medium partially reduces the shedding of Gs protein. Increased shedding of the Gs protein is seen when there is an unstable M or matrix protein synthesized by a temperature-sensitive mutant, tsG31. These results indicate that the G protein is cleaved at the cell surface, thus releasing Gs protein into the medium. Furthermore, the stability of G protein at the cell surface appears to be dependent on its association with the M protein.

Antigen shedding by human breast-cancer cells in vitro and in vivo.

Human breast cancer cells were adapted to chemically defined medium in order to recover naturally shed glycoproteins. Sephadex G-150 chromatography of these glycoproteins revealed 1 major and 2 minor peaks. When the cells were grown in the presence of 3H-leucine, the radioactive shed proteins had a Sephadex profile identical to the unlabelled shed glycoproteins. PAGE analysis of these proteins showed 5 major bands. Antigenically similar proteins were found in the serum of female nude mice bearing BOT-2 tumours, but not in controls.

The prevention of natural and experimental avian lymphoid leukosis with the androgen analogue mibolerone1

The development of lymphoid leukosis tumours induced by RAV-1, RAV-2, and field lymphoid leukosis viruses was prevented in chickens by feeding a diet containing the androgen analogue mibolerone (17beta-hydroxy-7a, 17-dimethylestr-4-en-3-one) at low levels during the first 7 weeks of life. Chickens fed mibolerone developed neutralising antibodies after inoculation with RAV-1, RAV-2, and after contact exposure to viraemic-tolerant chickens. Mibolerone did not affect the immunological tolerance status of chickens which were viraemic when hatched. Moreover, mibolerone did not seem to change the pattern of shedding of lymphoid leukosis viruses or group specific antigen in unincubated chicken eggs from infected hens. Mibolerone, thus, prevents the development of lymphoid leukosis tumours without interfering with the cycle of infection of lymphoid leukosis viruses.

Recent views on tumour antigens and their relation to host defence mechanisms.

Summary: Recent views on tumour antigens and their relation to host defence mechanisms.P. Hersey, Aust. N.Z. J. Med., 1977, 7, pp. 526–536. Tumour antigens on some human tumours can be detected by antibody produced against the tumour by the host. Tumour antigens defined in this way appear to be multiple and show diversity between different individuals. Several different categories of tumour antigens have been defined in animal studies which appear to be likely candidates for an equivalent role on haman tumour cells. These include altered histocompatibility antigens, oncogenic viral antigens and antigens similar to those on foetal tissue. The evidence for their expression on human tumours is reviewed. Problems in definition of these antigens as specific for tumours arise because of their expression on normal tissue under certain circumstances. It is suggested that previous exposure of human subjects to a number of these antigens on normal tissue produces a state of pre-sensitization to many of these antigens in both normal and tumour bearing subjects. This pre-existing state of sensitization has presented problems in the development of tumour specific assays of immune reactivity to monitor disease activity in human tumour bearing subjects. This problem appeared particularly marked in relation to assays of cell mediated immunity where it is thought that cytotoxicity to naturally occurring antigens may often obscure the “disease related” immune response. Information regarding the biological importance of the various tumour antigens in tumour rejection is reviewed. Preliminary evidence suggests that the role of the different tumour antigens in tumour rejection may vary. The factors which may determine the role of tumour antigens in immune rejection are poorly understood but may include the rate of shedding of antigens from the cell surface, the concentration of cell surface antigens and the number of pre-existing lymphoid cells which can recognise the tumour antigen. Although there are considerable deficiencies in our understanding of tumour antigens on human tumours, existing information appears to provide promising avenues for the development of diagnostic and immunotherapeutic procedures which will aid in the management of tumour bearing patients.

Antibody-Dependent Cellular Cytotoxicity against Murine Leukemia Viral Antigens: Studies with Human Lymphoblastoid Cell Lines and Human Peripheral Lymphocytes as Effector Cells Comparing Rabbit, Goat, and Mouse Antisera

A thymic lymphoblastoid cell line derived from a New Zealand Black mouse produces murine leukemia virus (MuLV) and was used as a target in model systems for the in vitro study of antibody-dependent cellular cytotoxicity (ADCC). Several human lymphoblastoid cell lines were investigated as potential effector cells. The most promising (Raji cells) bound to antibody-coated target cells but caused only modest levels of ADCC at 25:1 effector-to-target cell ratio with substantial lysis in the absence of antiserum. Human peripheral lymphocytes were active as effector cells in ADCC at a 5:1 ratio and produced no lysis in the absence of antibody. These cells were used to demonstrate that high dilutions of rabbit antisera to MuLV antigens p30, p15, p12, and p10 were capable of mediating lysis of MuLV-producing target cells but not of a virus-negative murine cell line. A murine antiserum to Thy 1.2 and three caprine antisera to MuLV antigens that were active in complement-mediated cytotoxicity functioned poorly in inducing ADCC; however, rabbit antisera to similar antigens were 16- to 512-fold more efficient in cell-mediated than in complement lysis. The inefficiency of goat antisera was not due to shedding of cell surface antigens or generation of blocking factors but rather to lack of lytic interaction of antibody-coated targets with the effector cells.

Galactosyltransferase Activity in Metastasizing and Nonmetastasizing Rat Mammary Carcinomas and Its Possible Relationship With Tumor Cell Surface Antigen Shedding 2

In order to study the mechanism of tumor cell surface antigen shedding, galactosyltransferase levels were compared in 5 spontaneously metastasizing and 3 nonmetastasizing rat mammary tumors. The enzyme activity both with or without exogenous acceptors was higher in the metastasizing group. This difference did not seem to be due to the variation in levels of degrading enzymes such as pyrophosphatase or beta-galactosidase found in these tumors. Little difference in the biochemical properties of the enzyme was found between the two groups. Most of the enzyme activity (60-70%) was recivered in the microsomal frctosyltransferase was assayed in "purified" plasma membrane fractions, 70% of the activity was associated with the plasma membrane vesicles, in which the enzyme was enriched by factors of 10-40. The number of galactose acceptor sites on the plasma membranes increased in parallel to the metastasizing capacity, indicating the presence of larger numbers of incomplete glycopeptides on their cell surfaces. These findings seemed to indicate that the greater turnover of glycoprotein in the spontaneously metastasizing tumor cell surface was caused by the shedding of surface antigens into the systemic circulation of the host.

Serum proteins in prostatic cancer VI. Reduction of the suppressive ("blocking"?) properties of serum on in vitro parameters of cell-mediated immunologic responsiveness following cryosurgery.

Preliminary studies of sera from prostatic cancer patients have indicated a reduction in the presence of suppressive ('blocking'?) properties of in vitro parameters of cell-mediated immunologic responsiveness induced by a non-specific mitogen (phytohaemagglutinin) in association with a decrease in the level of alpha2-globulin and favourable clinical response following cryosurgery. The origin of the immunosuppression factor(s) migrating on electrophoresis in the alpha2-globulin fraction of serum remains to be identified. Earlier demonstration of suppression of leucocyte migration by factors elaborated from tumour cells and recent observations of the suppression of lymphocytic reactivity by seminal plasma and coaguloprostatic fluid suggest that suppression and reduction or abrogation of the suppressive properties of serum following cryosurgical destruction of tumour may be attributed to a reduction in soluble prostatic tumour-associated antigen shed into the circlation by previously viable tumour. Such antigen while not at a sufficient concentration to engender an immunologic response in the aging and tumour-burdened host, may, however, have been sufficient to pre-empt the effector limb of cell-mediated responsiveness contributing to the observed suppression of lymphocytic reactivity. Cryosurgery, resulting in necrosis and cell death with depletion of the primary source of antigen might thereby have permitted a previously overwhelmed host to respond, viz., the favourable clinical response observed.

Insolubilization of protein antigens on polyacrylic plastic beads using poly-l-lysine

A new model system for quantitation of immunofluorescense on single cells is descibed using poly-L-lysine (PLL) coated polyacrylic plastic beads of approximately cell sizes as carriers for protein antigen. By increasing PLL concentration on the beads increased amounts of 125I labeled antigen were attached to the particles. Excess binding sites of PLL could be completely blocked by unrelated proteins. After staining with FITC-conjugated antibodies and quantitative fluorescence measurements of individual beads using a microspectrofluorimeter, strong correlations were found between antibody and antigen concentration on the beads. Neither repeated washings with PBS nor storage of the beads for two months caused detectable shedding of antigen—antibody complexes. There was a strong linear correlation between fluorescence intensity and the volume of beads, but the correlation between surface area and fluorescence was nonlinear. The described procedure was shown to be a simple method for quantitative and stable coating of particles with proteins. It can be applied as a useful model system for quantitative immunofluorescence studies on intracellular antigens.

Antibody-Induced Modulation and Shedding of Mammary Tumor Virus Antigens on the Surfaces of GR Ascites Leukemia Cells as Compared With Normal Antigens2

The distribution, antibody-induced redistribution, and shedding of murine mammary tumor virus (MuMTV) antigens and the surfaces of GR mouse ascites leukemia (GRSL) cells were studied by the immunoferritin technique and compared with the same activities of thy 1.2 and H-2.8 antigens. MuMTV antigens were redistributed easily and then largely shed from the cell surface; in contrast, H-2.8 antigen moved easily and probably was partially released from the plasms membrane and Thy 1.2 antigen moved slowly and was some what interiorized. The complement-dependent cytotoxicity test was used to study the possibility of antigenic modulation for these cell-surface antigens from the surface of the GRSL cells could be modulated by preincubation with anti-MuMTV serum, in contrast to H-2.8 and Thy 1.2 antigens. The results obtained with the immunoferritin technique and the cytotoxicity test correlated well and sug-ested that the shedding of MuMTV antigens from the cell surfaces may occur in vivo, providing the tumor a way to escape from the immune defense of the host. Thy 1.2 and H-2.8 antigens were present on the envolope of B and C particles, which suggested that these viruses do not select a Thy 1.2 or H-2.8-negative area of the GRSL cell surface as amaturation site.

Antigen recognition. IV. Discrimination by antigen-binding immunocompetent B cells between immunity and tolerance is determined by adherent cells.

Mouse spleen cells capable of specifically binding intrinsically tritium-labeled polymerized flagellin (POL) (labeling by biosynthesis of flagellar protein) via IgM receptors were found to comprise a distinct population of about 20-50 cells per 10(6) lymphocytes. Evidence is presented that the majority of mouse spleen cells binding tritium-labeled POL undergoes blastogenesis after antigen capping, antigen shedding, and receptor reformation. Under conditions of tolerance induction in vitro, however, loss of antigen from the cell surface was inhibited. Such inhibition of antigen redistribution and shedding was reversed by a short pulse of colchicine and new antigen receptors were formed. In spite of this, colchicine had no effect on the tolerant state. However, tolerance could be broken, regardless of presence or absence of the alkaloid, with radioresistant theta-negative accessory (A) cells (adherent cells) from normal but not from tolerant spleen cell populations. "Tolerant" A cells, although they were incapable of cooperating in a response to POL, were capable of participating in a response to a second unrelated antigen. It is concluded that tolerance to POL in vitro is induced by mechanisms other than the physical blocking of bone marrow-derived (B) cell receptors by antigen. Most likely, the discrimination by the B cell between a tolerogenic and immunogenic signal is mediated by A cells.

Immunogenicity of a rat leukaemia of spontaneous origin (SAL).

The SAL rat leukaemia, which resembles acute myeloblastic leukaemia, appeared initially to be non-immunogenic since resistance to an i.p. challenge with as few as 100 cells could not be obtained using stimulation of the RES or by immunization with SAL cells exposed to x-rays, nitrogen mustard, iodoacetate or glutaraldehyde. However, immunization with SAL cells exposed to low doses of mitomycin-C slowed the growth of the challenge inoculum. Cells treated with high doses of mitomycin-C did not immunize. The results are interpreted in terms of rapid shedding of a tumour-specific antigen from the membrane of SAL cells.

OROPHARYNGEAL EXCRETION OF EPSTEIN-BARR VIRUS BY RENAL TRANSPLANT RECIPIENTS AND OTHER PATIENTS TREATED WITH IMMUNOSUPPRESSIVE DRUGS

The excretion of Epstein-Barr (E.B.) virus was studied in eighty patients and staff in a hospital-based renal-disease unit. Virus shedding was measured by the capacity of throat washings to induce long-term proliferation of human umbilical-cord leucocytes and the appearance of E.B. virus nuclear antigen in the cultivated cells. The excretion-rates of E.B. virus, based on single throat samples, differed in the four groups of individuals tested: virus was recovered from 47% of patients with renal homografts, from 35% of other patients treated with immunosuppressive drugs, from 14% of patients with chronic uraemia and from 17% of healthy staff associated with the unit. Only individuals with antibody to E.B. virus capsid antigen shed virus. The excretion-rate for E.B. virus seropositive individuals on immunosuppressive drugs was 52% (seventeen out of thirty-three) compared with a rate of 18% (six out of thirty-four) for individuals not treated with these agents. Five seropositive patients received renal homografts during the study. None were E.B. virus excretors before transplantation; two were shedding virus 8 and 34 days after transplantation and institution of immunosuppressive therapy. The results support the hypothesis that immunosuppressive drugs (azathioprine and prednisone) are associated with reactivation of latent E.B. virus infections.