European Journal of Immunology

doi: 10.1002/eji.1830141101pmid: N/A

McLaughlin‐Taylor, Elizabeth; Woodward, Jerold G.; McMillan, Minnie; Frelinger, Jeffrey A.

doi: 10.1002/eji.1830141102pmid: 6209147

By producing long‐term, stable, cytolytic T lymphocyte clones and utilizing targets expressing only a single gene product derived from the stimulator mouse strain, we have been able to directly demonstrate that T cells recognize distinct epitopes expressed on a single H‐2 molecule. These multiple determinants are distinguishable by inhibition patterns with monoclonal antibodies (mAb). When two T cell clones, P‐2.14 and P‐2.17, are tested on an L cell transfected with the Dp gene (λ12a), the T cells kill the transfected targets as well as blasts derived from Dp mouse strains. mAb 7‐16.10 inhibits recognition and killing of Dp targets by both P‐2.14 and P‐2.17. This mAb 7‐16.10, however, competees with H‐2.m22. Interestingly mAb 11‐20.3 which also recognizes the H‐2.m22 specificity inhibits clone P‐2.14 but not P‐2.17. The mAb 7‐16.10, however, competes with 11‐20.3 for binding to the surface of L cells expressing the Dp gene. Thus the two T cells must recognize an overlapping specificity. Other mAb which bind to the H‐2Dp molecule are unable to inhibit either of these two cytolytic T cell clones. Paradoxically, any of the mAb when tested individually are sufficient to inhibit the polyclonal response derived from in vitro mixed lymphocyte culture. Therefore, by using targets expressing only a single H‐2 molecule derived by DNA‐mediated gene transfer and cytolytic T cell clones we have been able to directly demonstrate the presence of multiple epitopes on a single molecule and define their inhibition with mAb directed to that same molecule.

Hughes‐Jones, Nevin C.; Gorick, Barbara D.; Miller, Nigel G. A.; Howard, Jonathan C.

doi: 10.1002/eji.1830141103pmid: 6209148

Two rat IgG2b monoclonal antibodies bound to different epitopes on a single RT1Aa antigen molecule synergize in their induction of complement‐mediated lysis of the red cells. The mechanism of this synergistic action has been investigated by determining the C1q: IgG relationship on red cells using the fluorescence‐activated cell sorter and making use of the considerable variation in antigen density between individual red cells. With a synergistic pair of antibodies, the C1q: IgG ratio was approximately 1 and independent of antibody density per cell, indicating that the antibody molecules are present as closely associated pairs, each pair binding two C1q molecules and thus forming a cyclic tetramer. When one of the antibodies was used alone, the number of C1q molecules bound relative to the amount of antibody was higher than expected from theoretical considerations based on a presumed random distribution of antigen‐antibody complex. This result can be explained if there is some mobility of the complex within the membrane. The interpretation of the results gives strong support to the hypothesis that C1q must bind bivalently to rat IgG2b complexes.

Paige, Christopher J.; Gisler, Roland H.; McKearn, John P.; Iscove, Norman N.

doi: 10.1002/eji.1830141104pmid: 6333989

A semi‐solid agar assay is described in which B cell progenitors, already present in day 12 fetal liver, generate colonies which contain antibody‐secreting cells. Panning experiments, in which cells which initiate colony formation are depleted on plates coated with monoclonal antibodies, suggest that by the 13th day of gestation they express the antigen recognized by the monoclonal antibody AA4.1 and by day 14 they also express the B220 form of Ly‐5 recognized by the monoclonal antibody 14.8. By similar criteria the precursor cells do not express μ, I‐A, I‐E or Lyb‐2. Growth of cells in this assay is dependent upon soluble products provided by either fetal liver adherent cells, bone marrow adherent cells or colony‐stimulating factor‐containing conditioned media derived from placenta cells, L929 cells, WEHI‐3 B(D−) cells, T helper cells or mouse lung cells. These experiments define two sets of growth conditions. In the first, when support is provided by fetal liver adherent cells, the limiting component appears to be the B cell precursor, allowing us to estimate the frequency of these cells during ontogeny. We find approximately 1 clonable pre‐B cell in 300000 fetal liver cells on day 12 of gestation and 1 in 6000 by day 16. Under the second set of growth conditions, when support is provided by bone marrow adherent cells or colony‐stimulating factor‐containing conditioned media, more than one cell, colony or cell product is limiting. Highly purified samples of granulocyte/macrophage colony‐stimulating factor, colony‐stimulating factor 1 and multilineage‐hematopoietic growth factor are effective in this assay suggesting that the colony‐stimulating factors are the active components under these conditions.

Savino, Wilson; Dardenne, Mireille

doi: 10.1002/eji.1830141105pmid: 6389155

The localization of the three best‐defined thymic hormones, namely, thymulin, thymopoietin and thymosin α1 was studied by immunofluorescence using antibodies directed against these three molecules. With both human thymus frozen sections and cultured cells, thymic hormones were found exclusively in the epithelial component (recognized by its keratin content), in normal as well as pathological thymuses. The double‐labeling experiments using the different anti‐thymic hormone antibodies showed that the same epithelial cells contained the three hormones. These results suggest that the production of different hormones in the thymus is accomplished by the same epithelial cells.

Miller, Richard A.

doi: 10.1002/eji.1830141106pmid: 6238832

This report presents a method for examining the behavior of individual antigen‐responsive helper T cell precursors (pHTL), following their repeated encounter with antigen. In our model system, lymph node cells from mice immunized with keyhole limpet hemocyanin (KLH) are cultured in vitro under limiting dilution conditions, using responder cell doses chosen to ensure that each culture well contains, at the outset, at most a single KLH‐reactive helper T cell precursor. Those wells which in fact did receive a KLH‐reactive pHTL are identified by the accumulation of the helper T cell product, interleukin 3 (IL 3), during a 6‐day culture interval. The lymphocytes generated during this culture interval are washed, divided into replicate aliquots, and then further cultured (with and without additional KLH) to see if the original pHTL has generated cells which are capable of further lymphokine production. Using this approach, we found a surprising amount of variation from clone to clone in two measurements. Clones differed greatly in “endurance”, that is in the ability of progeny cells to continue to secrete IL 3 in the absence of additional antigenic stimulation. Among 148 clones tested, 90 were found not to continue lymphokine secretion in antigen‐free secondary cultures, while IL 3 production among the other 58 clones varied from 1 to 128 arbitrary units. Individual clones also varied greatly with respect to their ability to secrete additional IL 3 in response to additional antigen, and in “burst size”, that is in the number of new, antigen‐responsive helper cell precursors which they can generate during the initial culture interval; 43% of the clones produced no new pHTL, while others produced as many as 800 new pHTL cells in an 8‐day period. Neither measure correlates strongly with the amount of lymphokine originally produced by the initial “parental” pHTL. This method will allow us to examine the factors which influence the allocation of clonal progeny cells into lymphokine producing effectors and antigen‐sensitive “memory” T cells.

Leoni, Patricia; Dean, Roger T.

doi: 10.1002/eji.1830141107pmid: 6238833

Freshly isolated and subsequently matured human monocytes secreted lysosomal hexosaminidase in response to exposure to IgG‐Sepharose, but not certain derivatized control Sepharoses. The cells bound selectively to the surface of IgG‐Sepharose (and not the control Sepharoses) but because of the large size of the particles, could not ingest them. Since the soluble IgG was covalently linked to the Sepharose and free soluble IgG was not an inducer of secretion, the secretion was thus induced directly at the cell surface. Zymosan, a yeast cell wall particle which contains a mannan, was also able to induce secretion in the various monocyte stages under study. It could even bind to the cell surface of fresh monocytes which lacked the receptor for mannose‐terminated glycoproteins, and induce secretion in these cells. The mannose receptor appeared as monocytes matured, and the Ir number on the surface was increased by the action of lymphokines. Although zymosan‐induced secretion could be inhibited by mannose and certain other sugars, these seemed to have some complex metabolic effects in human monocytes (which previous work with mouse macrophages has not revealed). Thus, it was not possible to demonstrate whether zymosan could initiate secretion directly by interaction at the monocyte surface mannose glycoprotein receptor.

Wikén, Margareta; Hellström, Ulla; Perlmann, Peter

doi: 10.1002/eji.1830141108pmid: 6238830

T cells from human peripheral blood were enriched in T4+ cells by lysis of T8+ cells with the monoclonal antibody OKT8 plus complement. The T4+ subset was separated into 4 fractions differing in avidity for the lectin Helix pomatia A hemagglutinin (HP). The fractions were studied for their capacity to help autologous B cells to differentiate and mature into immunoglobulin (Ig) synthesis and secretion after activation with tetanus toxoid (TT) in vitro. To ensure the antigen specificity of induction, very low doses of TT (1–100 ng/ml) were used for activation of the lymphocytes, all obtained from previously sensitized donors. The T4+ cells with low avidity for HP (fractions HP‐I and HP‐II) exerted little help for B cell differentiation. Removal of these cells enhanced the helper function of the remaining T4+ cells, indicating that fractions HP‐I and HP‐II contained suppressor cells. In contrast, efficient B cell help was provided by T4+ cells with high avidity for HP (fractions HP‐III and HP‐IV). However, these fractions differed in the quality of help provided. Thus, while HP‐III cells induced IgG secretion, HP‐IV cells mainly induced IgM secretion. Moreover, while the Ig secreted after help from HP‐III cells was TT‐specific antibody, the Ig secreted by B cells in the presence of autologous HP‐IV cells was polyclonal, probably reflecting induction of B cells, differing in their responsiveness to signals provided by different types of T cells. The results indicate that T4+ cells vary in their stage of differentiation as seen by differences in expression of the HP marker and that differences in HP‐marker expression appear to be associated with differences in cellular functions.

Muraoka, Shizuko; Ehman, David L.; Miller, Richard G.

doi: 10.1002/eji.1830141109pmid: 6238831

When added to a mixed lymphocyte culture, cells in T cell colonies grown from bone marrow (BM) suppressed the development of cytotoxic activity against H‐2 antigens shared by the colony cells and the stimulator cells, apparently by inactivating cytotoxic T lymphocyte precursor cells (CTLP). From the point of view of the added suppressor cells, the suppression was against self‐reactive cells. The suppressor cells were resistant to γ irradiation (1500 rds) but sensitive to UV irradiation. Inactivated CTLP separated from the suppressor cells by cell sorting could not be reactivated on being recultured with fresh stimulator cells, suggesting the suppression is irreversible. There was a critical time window, extending roughly from 20 to 40 h after culture initiation, during which the suppressor cell had to be present if CTLP were to be inactivated. During the first 20 h and after 40 h of exposure to stimulator cells, CTLP were resistant to the suppressor cell. Direct experimental evidence is presented against the possibility that the suppressor cells derived from BM colonies act by augmenting the production of Lyt‐2+ suppressor cells from the responder population which then produce the suppression, or that the suppressor cells interfere with an early interaction between CTLP and stimulator cells. We conclude that the suppressor cells in T cell colonies grown from BM act directly on activated CTLP and permanently inactivate them.

Leanderson, Tomas; Forni, Luciana

doi: 10.1002/eji.1830141110pmid: 6437843

Over a wide range of concentrations affinity‐purified rabbit anti‐mouse μ chain antibodies, or their F(ab′)2 fragments, inhibit the appearance of immunoglobulin‐secreting cells (plaque‐forming cells; PFC) in lipopolysaccharide‐stimulated murine spleen cell cultures without affecting proliferation. Both IgM and IgG PFC are inhibited although the number of blasts bearing surface IgG remains unaltered. The IgM and IgG PFC response could be reconstituted to normal levels in cell cultures suppressed by μ‐specific antibodies by the addition of supernatants from in vitro propagated helper T cell clones, or from EL4 lymphoma cells induced with phorbol ester. Interleukin 1‐containing P388 supernatant, or recombinant DNA‐derived murine interferon‐γ, did not reconstitute the PFC response in cell cultures suppressed by μ‐specific antibodies, indicating that other factors are responsible for these effects. When spleen cell cultures, pre‐activated with either lipopolysaccharide or monoclonal mouse μ‐specific antibodies coupled to Sepharose, were exposed to EL4 supernatants in the presence of soluble μ‐specific antibodies, maturation to secretion was inhibited while proliferation was not. The implications of these findings on assay systems for B cell growth and maturation factors are discussed.