Surface Of Lymphoid Cells Research Articles

Thymic non-lymphoid cells.

In formulating this summary of our simon-pure knowledge of the structure/function relationships in the thymus, we decided that the time may have come to introduce a suitable dose of cynicism to balance the sometimes hopeless optimism of the past. Are the non-lymphoid cells of the thymus necessary for thymic function? Probably, but not to the extent or uniqueness that some authors including ourselves have previously claimed; T cells can probably differentiate in other tissues but may acquire their preference for MHC class II in the thymus. Mouse thymic lymphoid cell traffic and surface phenotype has recently been summarized pictorally by Scollay and Shortman [95]. Briefly stated, within the thymus, cells are hatched, matched and then dispatched. Minimally, the non-lymphoid cells act either as scenically varied obstacles along the way, nurseries for newborn T cells, or as tombstones for life's disenfranchized, effete and autoaggressive thymocytes. Hassall's corpuscles are morphological structures unique to the thymus, which are most useful to medical students for identification of this tissue. Their function remains one of life's great mysteries. Morphologically, they are suitable companions to the more recently described strange multicellular complexes of lymphocytes and epithelial cells which might be functionally important. The thymus of the much studied inbred, environmentally mollycoddled, laboratory mouse has been often and majestically described. It is probably typical for that of man and most mammals. It may, however, be unrepresentative of the thymus of stressed and parasitized wild animals. Diseases of the thymus generally can be categorized as not having enough thymus, having a neoplastic thymus or having a thymus which does not work properly. The bottom line in our knowledge of thymic nonlymphoid cells is that if you are born without them, you get sick and die; unless, of course, you are a nude mouse in Omaha, in which case you just freeze to death.

Evidence from cDNA clones that the rat leukocyte-common antigen (T200) spans the lipid bilayer and contains a cytoplasmic domain of 80,000 M r

The leukocyte-common antigen (L-CA or T200) includes a family of lymphoid and myeloid cell surface glycoproteins with apparent molecular weights from 180,000 to 240,000. We report a partial protein sequence for thymocyte L-CA containing 1073 amino acids predicted from cDNA clones isolated using an oligonucleotide probe. Only one segment (residues 347–368) is likely to cross the membrane, and peptide data suggest that sequences N-terminal to this are outside the cell, with residues 369–1073 inside. The cytoplasmic domain includes possible phosphorylation sites and an internal homology between residues 385–671 and 676–986. Analysis of B lymphocyte cDNA clones suggests that B cell and thymocyte mRNAs are identical in 3′ sequences, but size differences in Northern blots suggest 5′ sequences may differ.

Simple immunocytochemical staining procedure for lymphoid cell surface markers done on cell smears.

Simple immunocytochemical staining procedure for lymphoid cell surface markers done on cell smears.

Binding of the Helix pomatia lectin to the surfaces of normal and leukaemic lymphoid cells

Binding of the <i>Helix pomatia</i> lectin to the surfaces of normal and leukaemic lymphoid cells

Cell receptors for the mammalian reovirus. II. Monoclonal anti-idiotypic antibody blocks viral binding to cells.

A monoclonal anti-idiotypic antibody directed against an idiotypic determinant on a monoclonal anti-reovirus type 3 hemagglutinin antibody with viral neutralization activity is capable of binding to the surface of lymphoid cells (BW5147, R.1.1, and R1.E) and of inhibiting viral binding to cell surface receptors. The inhibition of viral binding was specific for the anti-idiotype antibody; viral binding was not inhibited by antibodies bound to the H-2k and Thy-1.2 antigens on the surface. Viral binding to idiotype-negative cells (P-815, EL-4) was not inhibited by the anti-idiotypic antibody, although these cells are susceptible to type 3 reovirus infection. These data suggest that there are at least two structural classes of type 3 reovirus receptors on murine cells. It is probable that anti-idiotypic antibodies of this type will be useful in studying the structure and regulation of viral receptors on cell surfaces and for the purification of these receptors, and may provide a way to block viral infection.

Correlation of functional properties of human lymphoid cell subsets and surface marker phenotypes using multiparameter analysis and flow cytometry.

Correlation of functional properties of human lymphoid cell subsets and surface marker phenotypes using multiparameter analysis and flow cytometry.

Murine lymphocyte cell surface proteolytic activity is strain related

A/J and C57 Br/cdj mice bear the H-2 haplotypes which are generally associated with high responses to bovine gamma globulin and type 3 pneumococcal polysaccharide. A/J has been reported, however, to produce higher levels of antibody than C57 Br/cdj mice against the antigens. The two strains of mice were used as model systems in this study to determine whether the level of lymphoid cell surface proteolytic activity is also genetically controlled. The results of this study illustrate that the level of lymphoid cell surface proteolytic activity is strain related. Since A/J lymphocytes were found to have a significantly higher rate of proteolysis than C57 Br/cdj lymphocytes, a correlation between lymphoid cell surface caseinolytic activity and immune responsiveness is suggested.

Influence of proteolysis inhibitors and immunoadsorption technique on the composition of rabbit antigen-specific receptor preparations. Presence of Fc receptors.

Addition of proteolysis inhibitors during the isolation procedure of rabbit arsanylated bovine IgG specific receptors decreased significantly the amount of low-molar-mass proteins in all receptor preparations. Rabbit ARS-BGG-specific receptor preparations isolated by immunoadsorption technique contain molecules which do not react with antigen and antibodies against immunoglobulins and have identical molar mass and chymotryptic peptide composition as those of isolated Fc receptors. It is suggested that during isolation of antigen-specific receptors from the surface of lymphoid cells, Fc receptors react with complexes composed of antigen and Ig+ receptors on the surface of immunoadsorbent and are isolated together with antigen-specific receptors.

Interactions of lectins and monoclonal antibodies with human mononuclear cells. I. Specific inhibition of OKT4 and OKT8 binding by Ricinus communis agglutinin and wheat germ agglutinin.

We used flow cytometry to examine effects of lectins on interactions between human lymphocytes and the anti-T cell monoclonal reagents OKT4 (T helper-specific) and OKT8 (T suppressor-specific). Wheat germ agglutinin (WGA) inhibited OKT8 binding to lymphocytes by a mean 77% and Ricinus communis agglutinin (RCA-I) inhibited OKT4 binding by 66%. Inhibition was abolished in each case by appropriate carbohydrate hapten inhibitors of lectin binding, indicating it was mediated by the lectin saccharide combining sites. Neither WGA nor RCA-I inhibited binding of OKT3, a pan-T cell monoclonal reagent. In addition, a group of other lectins with a variety of nominal carbohydrate specificities did not inhibit OKT4 or OKT8 binding. Preincubation experiments and gel filtration indicated that inhibition in each case was due to competition between lectin and monoclonal for binding to cell surfaces, not to direct lectin-monoclonal antibody interactions. Treatment of lymphoid cells with OKT8 and complement reduced OKT8- and WGA-binding cells concurrently, whereas treatment with OKT4 and complement did not reduce percentages of either type of cell. Similarly, specific depletion of OKT8-binding cells abolished the mitogenic response to WGA but not that to PHA. Cell populations enriched for WGA-binding cells prepared by flow cytometry and cell sorting demonstrated parallel enrichment for OKT8-binding and depletion of OKT4-binding cells. Therefore, these data demonstrate specific inhibition of OKT4 and OKT8 binding by the lectins, RCA-I and WGA, respectively. Inhibition was mediated by lectin binding to lymphoid cell surfaces, perhaps directly to the T4 or T8 antigens. The observations indicate that lectins may prove useful for investigating structural features of some immunologic cell surface markers. Furthermore, they provide the possibility that certain in vitro effects of lectins on immune function may result from their interactions with molecules such as the T4 and T8 antigens.

T cell-mediated cytolysis: on the strength of effector-target cell interaction.

Allosensitized lymphoid cell populations contain T lymphocytes that can bind to target cells and lyse them. We asked whether there was a relationship between lymphocyte target cell-binding strength and occurrence of cytolysis. Using graded shear forces to dissociate effector-target cell conjugates, we found that (a) within an allosensitized lymphoid cell population derived from an heterogeneous mixed leukocyte culture, there were lymphocyte-target cell conjugates with binding strengths differing by a factor of at least 10(2), (b) even the minimal force required to release a significant amount of bound target cells could disrupt the plasma membranes of some tumor cells and (c) these tumor cells disrupted by shear forces were probably part of cytolysis-conducive rather than of non-cytolysis-conductive conjugates. We combined this approach with the use of cytolysis-inhibiting monoclonal antibodies (mAb), and found that antibody-induced decrease of cytolysis was correlated with a decrease in the percentage of strong or total conjugates, depending on the mAb used. When lectins were added to overcome the inhibitory effect of the mAb, reappearance of cytolytic activity correlated with reappearance of conjugates. This was especially striking using wheat germ agglutinin (WGA): the addition of WGA to irrelevant effector-target cell combinations did not lead to cytolysis; however, the addition of WGA to relevant effector-target cell combinations inhibited by mAb led to reappearance of cytolysis and of strong conjugates. Taken together, these and other results suggested that under our experimental conditions a threshold level of binding strength between effector and target cells might be important, although not sufficient, for T cell-mediated cytotoxicity. These results were not inconsistent with the involvement of mechanical factors in this process. Also, they were in line with the concept of nonantigen-specific lymphoid cell surface interacting molecules, detected by the mAb used and important for the establishment of strong, functional lymphocyte target cell interactions. Finally, they underlined the necessity of a quantitative estimate of cell-cell binding strength when investigating the effect of a given agent (e.g. a mAb) on lymphocyte target cell recognition.

Lymphoid cell surface interaction structures detected using cytolysis-inhibiting monoclonal antibodies.

We screened monoclonal antibodies obtained by xenogeneic immunization for their capacity to inhibit T cell-mediated cytolysis. These antibodies fell into two classes according to the cell structures they recognized, of 30-35 K and 94-180 K apparent molecular weight, respectively. The main features of these structures and of their interaction with the corresponding antibodies were reviewed. The inhibition of cytolysis by these antibodies was shown to occur mainly at the effector cell level, at the recognition stage of cytolysis, and to depend on the nature of target cells, effector cells, and link between these cells. T cell functions other than cytolysis were also inhibited by some of these antibodies. We considered various possible mechanisms to account for the inhibition of cytolysis by these mAb. We favor an hypothesis based on inhibition by these mAb of lymphoid cell surface interaction structures. This hypothesis was discussed within the general framework of cell interaction structures in immunological and non-immunological experimental systems.

Cell surface alpha 1-protease inhibitor on human peripheral mononuclear cells in culture.

We have studied expression of alpha 1-protease inhibitor (alpha 1-PI) by human mononuclear cells. alpha 1-PI was detected on 50% of freshly isolated peripheral mononuclear cells. Unless a proliferative stimulus was provided, alpha 1-PI subsequently disappeared from the cell surfaces. Plant mitogens, periodate, neuraminidase-galactose oxidase, or allogeneic cells all were effective stimuli of alpha 1-PI expression. Concanavalin A stimulated de novo synthesis of alpha 1-PI in cell cultures containing both lymphocytes and mononuclear phagocytes, and alpha 1-PI simultaneously appeared on surfaces of activated lymphocytes. Inhibition of protein synthesis by cycloheximide or monocyte depletion abolished de novo alpha 1-PI synthesis, but only monocyte depletion inhibited alpha 1-PI expression. Lymphocytes, but not monocytes, displayed saturable binding of radioiodinated alpha 1-PI. The data are consistent with the interpretation that human mononuclear phagocytes synthesize and secrete alpha 1-PI. When protein synthesis is inhibited, mitogenic stimuli may provoke release of previously synthesized alpha 1-PI from mononuclear phagocytes. Secreted alpha 1-PI then may bind to specific lymphocyte cell surface receptors. This pattern of alpha 1-PI synthesis, secretion, binding, and expression on lymphoid cell surfaces appears to be a common characteristic of many immunologic reactions in vitro.

Phenotypic characteristics of cell lines requiring interleukin 3 for growth.

A number of cell lines have been derived from bone marrow cultures in the presence of WEHI-3 conditioned media (CM) that continue to require WEHI-3 CM for growth in vitro. Because the WEHI-3 cell line has been shown to constitutively produce a lymphokine (IL 3) that induces the expression of 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of splenic lymphocytes from athymic mice, we examined whether these cell lines were dependent upon IL 3 for growth. The results demonstrate that the factor required for growth of these cell lines copurifies with IL 3 activity on G-100, DEAE cellulose, and CM cellulose column chromatography as well as in preparative isoelectric focusing and on hydrophobic supports in reverse phase high pressure liquid chromatography. The biologic activity of peak fractions in each case was similar in both types of assays. These results strongly suggest that the WEHI-3 CM-dependent cell lines are dependent on IL 3 for growth in vitro. All the cell lines have readily detectable levels of 20 alpha SDH but have differing cell surface phenotypes. The C3HSFFV line is devoid of conventional lymphoid cell surface markers with the exception of Lyt-1, whereas the FDC-P1 expresses Thy-1, Ly-5, and H-11. Other cell lines have intermediate phenotypes.

Translocation of Thy-1 antigen and a fluorescent lipid probe during lymphoblastoid cell interaction with Mycoplasma hyorhinis.

The translocation of membrane-associated components during in vitro surface interactions between BW5147 murine T-lymphoblastoid cells and Mycoplasma hyorhinis was investigated. Thy-1 antigen (shown to be selectively associated with mycoplasmas following their detachment from infected BW5147 cells) was found by immunoferritin techniques to be localized at the surface of organisms colonizing lymphoblastoid cells. These results are consistent with a transfer of Thy-1 to membranes of mycoplasmas on the lymphoid cell surface. The exchange of membrane lipid-associated components was further investigated with the fluorescent lipid probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Fluorescence polarization of DPH was much less pronounced in uninfected BW5147 cells than in mycoplasmas--a result indicating markedly different probe environments. The increase of fluorescence intensity and the characteristic polarization in mycoplasmal supernatant fractions obtained after incubation of organisms with DPH-labeled BW5147 cells indicated translocation of DPH from lymphoid cells to mycoplasmas. Incorporation of the probe into mycoplasmas using supernatants from DPH-labeled BW5147 cells (incubated alone) was not demonstrated; this observation is consistent with, but does not prove, a contact-dependent process of exchange. Direct monitoring of the exchange of surface antigens and lipophilic probes may be useful in studying the interaction of hydrophobic surface glycoproteins with membranes and the role of membrane components in mycoplasma-host cell interactions.

Functional relationships of lymphocyte membrane structures probed with cytolysis and/or proliferation-inhibiting H35-27.9 and H35-89.9 monoclonal antibodies.

The mechanism(s) of T lymphocyte “functions” such as cytolysis (1–4) or proliferation involve those cell surface structures that insure specific recognition and may involve other cell surface structures as well. Detection of these may be via the use of monoclonal antibodies (mAb) selected for their ability to inhibit lymphocyte functions. Indeed, anti-Lyt-2 mAb have been extensively studied as to their inhibitory effect on mouse T cell-mediated cytolysis, with repeated suggestions that Lyt-2 itself may be related to the T cell specific receptor (5–10). We have developed a range of xenogeneic rat anti-mouse mAb selected for their ability to inhibit T cell-mediated cycolysis (11,12). Three of them will be used in the present report: H35-17.2 mAb, which is most probably an anti- Lyt-2 mAb, as an experimental counterpoint to the two other mAb; H35-27.9 mAb, which differs from an anti-Lyt-2 mAb at least by the tissue distribution of the structures it recognizes; and H35-89.9 mAb, which immunoprecipitates from lymphoid cell surfaces two polypeptides of 180K and 94K molecular weight.

Inhibition of murine T cell-mediated cytolysis and T cell proliferation by a rat monoclonal antibody immunoprecipitating two lymphoid cell surface polypeptides of 94 000 and 180 000 molecular weight.

The monoclonal antibody methodology was use to identify membrane structures involved in T cell functions. To optimize chances to produce and detect relevant antibodies, a xenogeneic sensitization protocol was utilized and hybridoma supernatants were screened, on functional rather than structural grounds, for their ability to inhibit a given function. The test function was T cell-mediated cytolysis. Mouse cytolytic anti-allogeneic cell populations were used to sensitize a rat, the spleen cells of which were fused to produce hybridomas; the supernatants of the latter were screened for their ability to inhibit mouse T cell-mediated cytolysis in vitro. Several inhibitory antibodies were obtained, one of which, H35-89.9 monoclonal antibody, was studied in more detail. It inhibited specific and concanavalin A (Con A)-mediated cytolysis by T cells, by acting on the effector cells. It reversibly inhibited soluble antigen-, alloantigen and Con A-induced T cell proliferation (but not LPS-induced B cell proliferation), after the production of interleukin 2, by acting on the responder cells. It also had a desagglutinating effect on Con A and LPS blasts and on EL4 cells. In immunoprecipitated from thymocyte membrane preparations two structures of 94 000 and 180 000 apparent molecular weight, and recognized cell surface determinants on both T and B lymphocytes. Our findings suggest that several antibodies directed against distinct effector cell membrane structures inhibit cytolysis. The case of H35-89.9 monoclonal antibody, which exerts multiple functional effects and immunoprecipitates two membrane polypeptides, raises the problem of the various possible relationships between these structures and functions.

Phenotyping human leukemic T-cell lines: enzyme markers, surface antigens, and cytogenetics.

We have compared phenotypic markers for a series of established human leukemic T-cell lines collected from different laboratories. Cell lines were tested first for genetic markers using polymorphic enzymes and then for expression of T lymphoid cell surface differentiation antigens using monoclonal antibodies. Chromosomal analysis was used as an additional method for identification of selected cell lines. On the basis of enzyme markers, it was possible to assign each of the cell lines examined to one of nine different groups. With two exceptions, surface antigen phenotypes for each of 12 cell lines were clearly distinctive. Thus, some groups of cell lines indistinguishable by enzyme markers could be further subdivided by surface antigen phenotyping. However, significant quantitative variation in expression of individual antigens was observed. In addition, surface antigen expression was not uniform in different subcultures of one cell line studied in detail. These results indicate that leukemic T-cell lines cannot be used generally as simple models of surface antigen expression in normal T-cell differentiation.

Hematopoietic cells transformed in vitro by REV T avian reticuloendotheliosis virus express characteristics of very immature lymphoid cells

REV T (avian reticuloendotheliosis virus) is a replication defective acute leukemia virus (J. D, Hoelzer, R. B. Franklin, and H, R. Bose, 1979, Virology 93, 20–30) possessing a transformation-specific sequence designated rel. Hematopoietic cells transformed by REV T in vitro expressed cell surface markers of immature B lymphocytes. Some clones of transformed cells also expressed cell surface determinants of immature T lymphocytes. An antiserum specific for REV T-transformed cells reacted with the cell surface of lymphoid leukemia cells of both the B- and T-cell lineage as well as with a subpopulation of cells in normal spleen, thymus, and bone marrow. REV T-transformed cells did not express surface or cytoplasmic immunoglobulin chains nor terminal deoxynucleotidyl transferase, nor were they susceptible to infectious bursal disease virus. As expected, they were also negative for the expression of all erythroid and myeloid differentiation parameters tested.

Lymphoid cell surface receptor for Moloney leukemia virus envelope glycoprotein gp71. I. Binding characteristics.

The characteristics of Moloney leukemia virus (M-MuLV) gp71 binding on lymphoid cells have been studied by a very sensitive assay. Analysis of the results indicated the presence of 1 class of high affinity binding sites (Ka = 1.2 X 10(9) M-1) on BALB/c thymus cells. The total number of binding sites was estimated around 1.3 X 10(4) per thymus cell. No significative differences were observed for the binding of Moloney leukemia virus gp71 on thymus cells from different inbred strains of mice, suggesting that the genetic susceptibility to Moloney leukemia virus infection does not depend on the presence of gp71 receptor on the target cells. On the other hand, it seems that M-MuLV gp71 binds preferentially on T cells, which are the final target of Moloney leukemia virus-induced transformation. Very little binding was found on Moloney, Rauscher, or x-ray-induced lymphoma cells.

Lymphoid cell surface receptor for Moloney leukemia virus envelope glycoprotein gp71. II. Isolation of the receptor.

The lymphoid cell surface receptor for Moloney leukemia virus envelope glycoprotein gp71 was isolated by using anti-gp71 antibodies to immunoprecipitate the receptor-gp71 complex from detergent extract of radiolabeled murine thymus cells. Upon sodium dodecyl sulfate gel electrophoresis, a single molecule with an apparent m.w. of 190,000 was identified as the putative gp71-receptor. Analysis in nonreducing conditions indicated that the receptor may be composed of at least 2 subunits of 190,000 daltons.