Association of some cell surface antigens of lymphoid cells and cell surface differentiation antigens with early rat pregnancy

Monoclonal antibodies and the immunoperoxidase technique were used to localize some cell surface antigens of rat lymphoid cells and cell surface differentiation antigens on cryostat sections of early rat pregnancies. The W3/13 leucocyte sialoglycoprotein was detected almost constantly on trophoblast. The immunoglobulins were more associated with mother's rather than with embryo-derived tissues. We were unable to detect considerable amounts of class I and class II major histocompatibility complex-derived antigens on trophoblast and adjacent decidual cells. The Ia+ cells of the lymphocyte type were occasionally detected in the sites exhibiting presence of immunoglobulins. The Thy-1 cell surface differentiation antigen was detected on the cells producing Thy-1+ material among decidual cells. Depletion of Thy-1 was followed by the regression of decidualized tissue. The OX-2 antigen, known as minor glycoprotein of rat thymocytes, was detected on trophoblast cells and endothelia of decidual vessels, the latter exhibiting also class I major histocompatibility complex-derived antigens. The non-pregnant uterine tissues, as well as the oviduct epithelium were also investigated. The possible role of some of these antigens in the maintenance of the 'immunologically privileged' stage of trophoblast, and in the control of the rearrangement of maternal tissues surrounding the embryo, is discussed.     

The presence of Thy-1 on the surface of rat lymphoid stem cells and colony-forming units.

By labeling Thy-1-positive cells of rats with xenoanti-Thy-1 or mouse alloanti-Thy-1 antibody and separating them from Thy-1-negative cells on a fluorescence-activated cell sorter, it was shown that early lymphopoietic stem cells all carry Thy-1-antigen. This was so for both young, adult bone marrow and near-term fetal liver cells. Two kinds of assay were used: long-term radiation chimeras employing B and T cell alloantigens to mark cells of donor origin, and in vivo colony-forming units in the spleens of irradiated recipients (bone marrow only). Thy-1-negative cells gave essentially no B or T progeny, even 6 to 12 months after reconstitution of the chimeras. The kinetics of appearance of Thy-1-positive cells in rat marrow, which peaked at 6-8 weeks of age, were also studied. In rats, Thy-1 is on the surface of pluripotent stem cells and on early progenitors of both B and T lymphocytes.     

Independence of HL-A antigens and immunoglobulin determinants on the surface of human lymphoid cells

Membrane-bound HL-A antigens and μ-chain determinants were studied on normal and leukemic lymphocytes and on a tissue culture cell line derived from a Burkitt's tumor, using membrane immunofluorescence on living cells. A redistribution of both antigens at the cell surface occurred when cells labeled in the cold by conjugated antisera were incubated at 37 °C. Double labeling experiments showed that HL-A antigens, either of the first or the second sublocus, were unaffected by the redistribution of IgM determinants and vice versa. This result strongly suggests molecular independence of these two systems. When redistribution was induced with a mixture of conjugated sera to HL-A and Ig, the caps showing each of the two specificities were located at the same pole of the cells.     

流式细胞术对不同肿瘤细胞表面所表达血小板免疫相关抗原的检测

目的：研究不同肿瘤细胞表面血小板免疫相关抗原的表达。方法：利用流式细胞术观察了ＰＧＣＬ３、ＰＡａ、ＰＧ－３、ＰＣ－３Ｍ、ＭＧＣ８０３、ＥＳＣＬ、ＢｅＬ、ＴＣＴ、ＫＢ、Ａ２７８０、ＣＣＡ８０１共１１种人肿瘤细胞,其中包括二对高低不同转移能力的人肺癌（ＰＧＣＬ３和ＰＡａ）和人前列腺癌（ＰＧ－３和ＰＧ－３Ｍ）细胞阳性表达不同血小板免疫相关抗原的细胞数及其肿瘤细胞表面抗原表达的平均荧光强度。结果：１１种人肿瘤细胞膜表面均有ＣＤ９、ＣＤ６３、ＣＤ４２ａ和ＴＳＰ,等血小板免疫相关抗原不同程度的表达,在ＭＧＣ８０３细胞膜表面发现有ＣＬ８６较强的表达,ＣＤ４１、ＣＤ４２ｂ、ＣＤ６１和ＣＤ６２均未发现表达。１１种人肿瘤细胞系中ＣＤ９＋、ＣＤ４２ａ＋、ＣＤ６３＋、ＴＳＰ＋细胞所占的比例各不相同,不同肿瘤细胞系每个抗原阳性细胞抗原表达的荧光强度也不同。与高转移ＰＧＣＩ３和ＰＧ－３Ｍ细胞相比,ＣＤ９在低转移细胞系ＰＡａ和ＰＧ－３上有较高的表达,ＣＤ４２ａ、ＴＳＰ有相对低的表达。ＣＤ４２ａ＋和ＴＳＰ＋细胞数和表达强度在高转移ＰＧＣＬ３和ＰＧ－３Ｍ细胞系均要高于低转移的ＰＡａ和ＰＧ－３;ＣＤ６３＋细胞数高转移ＰＧ－３Ｍ细胞系要比低转移     

流式细胞术对不同肿瘤细胞表面所表达血小板 免疫相关抗原的检测①

目的研究不同肿瘤细胞表面血小板免疫相关抗原的表达。方法利用流式细胞术观察了PGCL3、PAa、PG-3、PG-3M、MGC803、ESCL、Bel、HCT、KB、A2780、CC801共11种人肿瘤细胞，其中包括二对高低不同转移能力的人肺癌（PGCL3和PAa）和人前列腺癌（PG-3和PG-3M）细胞阳性表达不同血小板免疫相关抗原的细胞数及其肿瘤细胞表面抗原表达的平均荧光强度。结果11种人肿瘤细胞膜表面均有CD9、CD63、CD42a和TSP等血小板免疫相关抗原不同程度的表达，在MGC803细胞膜表面发现有CD36较强的表达，CD41、CD42b、CD61和CD62均未发现表达。11种人肿瘤细胞系中CD9     

Lentil-lectin affinity chromatography of surface glycoproteins and the receptor for IgE from rat basophilic leukemia cells.

Surface proteins and glycoproteins of RBL cells were labelled enzymatically with 125I and then solubilized with Nonidet P-40. Analysis by polyacrylamide-gel electrophoresis in SDS on 10% gel revealed 10 distinctive peaks ranging in molecular weights from 17,000 to 200,000 daltons. Mainly components of higher molecular weights were bound by lentil-lectin Sepharose and could be eluted with alpha-methyl mannoside. The receptor for IgE was clearly shown to bind to the lentil-lectin. A second cell surface component which previously had been shown to bind to IgE-Sepharose as well, was found to bind only slightly to lentil-lectin. Thus, it can be concluded that the receptor for IgE is a glycoprotein with mannose and/or N-acetylglucosamine in the carbohydrate moiety(s).     

EA rosette-forming lymphoid cells in chickens: specificity of the Fc receptor and its relationship to other surface antigens.

The receptor for erythrocyte-antibody (EA) complexes on the surface of chicken lymphoid cells was investigated using a rosette assay. The chicken EA receptor binds chicken immunoglobulin of the IgG class but not the IgM class. Binding to the EA receptor is dependent upon the Fc region of the immunoglobulin. No receptor for complement analogous to the mammalian C3b receptor was demonstrated on chicken lymphoid cells using the rosette assay. Inhibition studies utilizing immunoglobulins from several species demonstrated that chicken spleen cells do not bind mammalian immunoglobulin but may bind immunoglobulin of other avian species (turkey and duck) and a reptilian species (turtle). The chicken EA receptor is distinct from cell membrane bound immunoglobulin light chains, bursa-specific antigens and thymus-specific antigens. The receptor for EA complexes on chicken lymphoid cells is compared with the Fc receptor on mammalian lymphoid cells in the light of these observations.     

Immunoglobulin determinants on the surface of lymphoid cells of carps.

Immunoglobulin determinants were detected by indirect immunofluorescence on the surface of numerous lymphocytes of perpheral blood, spleen, and pronephros of craps. The most interesting finding was the high proportion (65–68 %) of Ig⁺ lymphocytes in the thymus of early adult carps, possibly related to thymus function.     

IgG2 and other immunoglobulin classes on the cell surface of rat lymphoid cells

The binding to rat lymphoid cells of the F(ab′)₂ fragments of purified rabbit antibodies specific for the Fc or Fd part of rat IgG₂ was studied. Both reagents heavily labeled about 15 % and 6 % of cells from spleen and cervical lymph nodes respectively of rats kept in a conventional animal house. In contrast to this there were very few IgG₂ positive cells in the thoracic duct lymph of any animals, or in the spleen of rats from an SPF animal house. Results on the quantitative binding of rabbit antibodies against rat Fab, IgM and IgA to rat lymphoid cells are also reported. IgM positive cells in the spleen bound much more anti-IgM relative to their binding of anti-Fab antibody, than IgM positive cells from thoracic duct lymph or cervical lymph node.     

Immunohistological demonstration of lymphocyte surface antigens in postmortem lymphoid tissues

Summary The postmortem stability of cell antigens has hardly been studied. Using monoclonal antibodies (mabs) we examined the postmortem detectability of lymphocyte surface antigens in different lymphoid organs by comparing two sensitive, immunohistological staining procedures.To quantify the probable degree of autolysis of the tissues a score system was applied by taking into consideration the postmortem age as well as the core temperature of the corpses.The antigens examined generally proved to be very resistant to autolytic influences. Differences were found when comparing different mabs and with regard to the type of lymphoid tissue. The loss of immunohistological reactions was most extensive in the spleen whereas tonsils showed almost no qualitative alterations in staining patterns. Reactivity of mabs with postmortem tissues decreased in the following order: Dako CD22 and anti-Leu 4, anti-Leu 3a, anti-Leu 7, Dako T8. The mabs anti-Leu 7 and Dako-T8 frequently failed to demonstrate their respective antigens but no correlation between the loss of staining and the degree of autolytic decomposition (our score) could be detected.In general, postmortem tissues as well as tissues shock frozen after delay are suitable for qualitative immunohistology of those cells characterized by the mabs applied.The APAAP-method proved unequivocally to be the superior staining technique.     

Expression of surface antigens during the differentiation of human dendritic cells vs macrophages from blood monocytes in vitro.

High expression of MHC antigens and adhesion/costimulation molecules is considered as one of the major characteristics qualifying macrophages (M) and dendritic cells (DC) as professional antigen presenting cells. Since accessory activity of M is known to be weaker than that of DC but both M or DC can differentiate from blood monocytes (MO) depending on culture conditions (i.e. GM-CSF vs GM-CSF/IL-4), we investigated the kinetics of expression of MHC antigens and several adhesion/costimulation molecules during the differentiation of DC or M from blood MO. Blood MO cultured with GM-CSF consistently induced M that showed adherence to plastic and CD14 expression. In contrast, MO cultured with GM-CSF/IL-4 rapidly became nonadherent, acquired DC morphology and lost CD14 expression. M but not DC proliferated as demonstrated by [H3]thymidine incorporation. MHC Class I was highly expressed in both M and DC. In contrast, MHC Class II molecules were significantly higher on DC compared to M. CD80 was upregulated on both DC and M but only on a subset of cells. CD80 expression peaked at day 3 on M and declined thereafter, while on DC expression increased significantly until day 10. CD86 was upregulated on the majority of DC and M. However, while M maintained stable expression of CD86 after day 3, DC progressively upregulated CD86 throughout the culture period. CD1a expression was initially low in both cell types and peaked at day 3 in M declining thereafter, while expression remained stable on DC until day 10. ICAM-1 expression was significantly upregulated on M when compared to DC at day 3. However, on M, ICAM-1 expression became undetectable by day 5 while on DC it increased through day 10. Similarly, CD40 was transiently expressed on M until day 5, while on DC it continuously increased until day 10. Finally, in contrast to other antigens, LFA-3 was always more strongly expressed on M than DC at all culture periods. Taken together, these data suggest that M showed a rapid but transient upregulation in the expression of adhesion/costimulation molecules, suggesting that maximal accessory ability is reached by M at an earlier time point than DC. Significant differences in surface antigen expression DC vs M were recognizable for MHC class II, CD86, CD80, CD1a, CD40 and ICAM-1. Specifically, major differences occurred for MHC class II, CD86, CD40 and ICAM-1. Therefore, the higher accessory ability of DC compared to M in naive T cell priming may be related to qualitative and quantitative differences in expression of these immunologically important surface molecules.     

Image analysis of lymphoid cell differentiation in rat thymus throughout development.

In order to identify subtle changes in cell morphology and nuclear pattern modification during thymus ontogenesis, cell image analysis using System for Analytical Microscopy in Biological Applications (SAMBA 200) was applied in 9 stages of rat thymus development. The morphometric and chromatin parameters made it possible not only to identify automatically between the two main cell populations in the thymus gland (lymphoid and epithelial cells), but also to classify automatically 5 lymphoid sub-populations (lymphoid stem cells, lymphoblasts, large lymphocytes, medium lymphocytes and small lymphocytes). The evaluation of the 18 parameters during the lymphoid cell differentiation was studied in detail. The nuclear texture parameters made it possible to discriminate, in each cell subpopulation, 4 phases of cell cycle (G0, G1, S-phase, and cells in G2). Evaluation of the nuclear parameters of the cell cycle in each lymphoid sub-population was studied in this investigation. The results illustrate the high majority of the lymphoid stem cells at the 14-day-old embryo stage while in the 20-day-old embryo the small lymphocytes become the main part of the whole lymphoid population. From the continuously renewed modification of lymphoid nuclear image analysis we discuss the origin of thymus lymphocytes. Lymphoid cells can be distinguished into different functional states and the striking morphological changes appearing during cell differentiation are related with drastic structural changes occurring in chromatin pattern from undifferentiated lymphoid stem cells to small lymphocytes. Terminal cell differentiation is associated with inhibited cell proliferation. The relative increase of chromatin condensation and nuclear pattern heterogeneity which reaches an extreme in small lymphocytes is accompanied by a progressive diminution of the nuclear area during the successive differentiation of the lymphoid population. Using one parameter of the nuclear texture features from the co-occurrence matrix (as LM or CON) and one parameter of the nuclear textures from the run-length section matrix (as GLD or RPC) the image analysis can discriminate between the different states of lymphoid cell differentiation.     

Production and characterization of monoclonal antibodies to guinea pig lymphoid differentiation antigens

We have prepared 2 mouse monoclonal antibodies which react with differentiation antigens on guinea pig lymphoid cells. Monoclone 5AB2 recognizes an antigen expressed on both T and B lymphocytes and absent on macrophages. It has proven useful in the preparation of populations of antigen presenting cells which are free of T and B lymphocytes. The second monoclonal, 8BE6, is specific for peripheral T cells and 10% of thymocytes. It reacts with a 68,000 dalton molecule which is also expressed on the guinea pig B cell leukemia, EN-L2C. 8BE6 has proven to be lytic for peripheral T cells in the presence of rabbit complement and has been used to deplete T cells from heterogenous cell populations.     

Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens.

Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with 125I-labeled anti-rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated 125 I-labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross-inhibition of binding of different monoclonal antibodies. It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat-stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major 125I-labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105 000. Five IgG-secreting clones identify the fourth antigen, a heat-stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross-inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.     

A demineralization procedure for immunohistopathological use. EDTA treatment preserves lymphoid cell surface antigens

An evaluation of the usefulness of EDTA treatment for decalcification of murine bone tissue in order to preserve both morphological details and immunologically intact cell surface antigens has been performed. The ABC immunohistochemical staining technique employing monoclonal antibodies to subsets of T-lymphocytes, B-lymphocytes and to Ia antigens was used on frozen sections. Treatment of mouse hindlegs with EDTA for 14 days resulted in an efficient decalcification and good preservation of morphological details. When lymphoid tissues were handled in the same manner monoclonal antibodies, defining Ly 1, Ly 2, L3T4, MAS 034 and Ia molecules, were shown to retain their reactivity comparable to that of directly frozen tissues. In contrast, formic acid, the commonly used decalcification agent, destroyed most of the antigenic reactivity. We conclude that EDTA treatment of non-fixed, bone-containing tissues provides a suitable demineralization procedure in the immunohistochemical study of, e.g., arthritis and periodontitis.     

Relationship between histocompatibility antigens, other surface determinants and the IgE receptor on rat mast cells.

Rat alloantibodies recognizing classical transplantation antigens (CTA) or non-H-1 determinants were able to compete effectively with monomeric IgE or IgG-coated sheep erythrocytes for receptor sites on the rat mast cell surface. Inhibitory capacity, however, was entirely confined to anti-CTA antibodies of the IgG2a subclass, whereas IgG1 antibodies lacked this ability. Analogously, F(ab')2 fragments of anti-CTA antibody consistently failed to affect IgE binding, but exposure of cell-bound F(ab')2 to anti-rat IgG restored its inhibitory capacity. From these results it was concluded that receptor sites recognizing the Fc portion of the anti-CTA molecule are involved in the inhibition process. Based on a cytotoxicity assay and on comparative absorption studies on alloantisera, the existence and relative amount of CTA and I region-associated antigens on purified rat mast cells and lymph node cells were analyzed. Whereas the CTA concentration per unit surface area on both cell types was very similar, rat mast cells consistently lacked Ia antigens.     

Different expression of Lyt differentiation antigens and cell surface glycoproteins by a murine T lymphoma line and its highly metastatic variant

Cloned lines of the methylcholanthrene-induced DBA/2 T lymphoma Eb and its highly metastatic variant line ESb were analyzed for differences in the expression of serologically detectable cell surface differentiation markers. Flow cytofluorographic analysis of cells stained with fluorescein isothiocyanate-conjugated monoclonal rate anti-mouse Thy-1, Lyt-1, Lyt-2 and complement-dependent cytotoxicity with mouse alloantisera against Lyt-3.2 and Ly-6.2 revealed, for the parental low metastasizing line, Eb, a phenotype of Thy-1+, Lyt-1-, Lyt-2+, Lyt-3+, Ly-6+, whereas the highly metastasizing variant line typed as Thy-1-, Lyt-1+, Lyt-2-, Lyt-3-, Ly-6-. Analysis of galactose oxidase/NaB3H4-labeled glycoproteins from Eb and ESb clones by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed further phenotypic differences. Selective binding of radiolabeled glycoproteins to Helix pomatia or Vicia villosa-Sepharose, respectively, allowed the identification of T130 to be expressed on Eb cells and T145 to be expressed on some ESb clones. The latter antigen is expressed on murine cytotoxic T lymphocytes. Immune precipitation analysis revealed that Eb and ESb bear different molecular forms of the T200 antigen. Comparisons of iodinated surface proteins derived from tumor cells either treated or untreated with tunicamycin indicated that many of the differences in membrane proteins between Eb and ESb cells could be attributed to differences in glycosylation. Our results, derived from a defined tumor system of lymphoid origin, show that the progression from a low to a high malignant tumor line can be associated with changes in the expression of various defined cell surface differentiation antigens. The question of a possible relationship between tumor progression and cell differentiation or dedifferentiation is discussed.