Postnatal development of mitogen responsiveness of guinea pig lymphocytes.

Guinea pigs are believed to be immunologically mature at birth. There is, however, little data available to support this concept. In this study, the postnatal development of the lymphocyte responsiveness to Tand B-cell mitogens in the guinea pig was investigated. The results show that guinea pigs are not immunologically mature at birth as to the mitogenic responses of blood lymphocytes. The constant level of PHA response in the blood is achieved from the age of 1 to 2 months and that of Con A at the age of 3–6 months. Furthermore, the results support the concept that the emigration of thymocytes occurs also during postnatal life. The emigration of PHA-responsive thymic cells seems to precede and be greater than that of Con A-responsive cells. These findings provide important clues for studies on the ontogenetic development of cell-mediated immunity.     

Differences in prostaglandin formation between thymocyte subpopulations.

The concentration of prostaglandin E and the activity of prostaglandin synthetase were determined in mature and immature mouse thymocytes. Hydrocortisone resistant thymocytes, or thymocytes separated from the immature cell population after agglutination of the latter by peanut agglutinin served as a source of mature thymocytes. It was found that mature thymocytes contain a much higher concentration of prostaglandin E and have an increased activity of prostaglandin synthetase than immature cells.     

Studies on thymocyte subpopulations in guinea pigs. Differences in cell volume and proliferative ability in vitro of two populations separated by density gradient centrifugation with Percoll

Thymus cells from guinea pigs were separated according to buoyant density by centrifugation with PVP-coated colloidal silica particles (Percoll). By creating an S-shaped density gradient, two populations (referred to as peak-I and peak-II cells) were obtained which differed in size as well as ability to spontaneous proliferation in vitro. Peak I contained low density cells of large size and was highly enriched with DNA-synthesizing cells. These continued to proliferate in culture for at least 30 h as demonstrated by mitotic studies in the intervals 0-10 and 20-30 h. In order to grow in vitro, however, the cycling cells of peak I depended on the medium (RPMI 1640) being supplemented with L-alanine. The high density cells of peak II constituted 70% of the thymocytes and had a small and uniform volume. This population was depleted of proliferating cells. The simple and rapid separation of these two major populations is considered a useful first step for the further characterization of thymocyte subpopulations. We suggest that peak I primarily includes proliferating precursor cells from the cortex as well as mature, immunocompetent cells. Peak II consists largely of small cortical cells.     

Differences in prostaglandin formation between thymocyte subpopulations

The concentration of prostaglandin E and the activity of prostaglandin synthetase were determined in mature and immature mouse thymocytes. Hydrocorthisone resistant thymocytes, or thymocytes separated from the immature cell population after agglutination of the latter by peanut agglutinin served as a source of mature thymocytes. It was found that mature thymocytes contain a much higher concentration of prostaglandin E and have an increased activity of prostaglandin synthetase than immature cells.     

Thymocyte and macrophage interactions: separation of murine thymocyte subsets and enrichment of syngeneic cell-responding thymocytes by adsorption to macrophage monolayers

The spontaneous binding of murine thymocytes to macrophage monolayers was employed to separate thymocytes into macrophage-unbound and -bound subsets, and the functional reactivities of these two subpopulations were examined. Macrophage-unbound thymocytes were found to be enriched in functional subsets reactive to semi-allogeneic and allogeneic stimulating spleen cells by proliferation in mixed leukocyte culture (MLC). Furthermore, macrophage-unbound thymocytes were frequently found to respond to syngeneic spleen cells. This syngeneic proliferative response showed both memory and specificity upon subsequent restimulation and thus seems to represent a syngeneic mixed leukocyte reaction (SMLR). Syngeneic responding thymocytes were also found to produce interleukin 2 when cultured with syngeneic but not allogeneic stimulator cells. In contrast, macrophage-bound thymocytes showed greatly reduced proliferative responses to allogeneic stimulators and no responses to syngeneic stimulators. The macrophage-bound thymocyte subset was not enriched in detectable suppressive activity; proliferative responses of macrophage-unbound thymocytes to either allogeneic or syngeneic cells were neither suppressed nor enhanced when macrophage-unbound thymocytes were added to the cultures. Thus, the macrophage-unbound subset seems to be enriched in functionally mature thymocytes and the macrophage-bound subset appears to be enriched in functionally immature thymocytes. This functional separation of thymocytes by macrophage adherence also correlated well with thymocyte subpopulations separated by bovine serum albumin density gradients; the low density mature thymocytes showed enhanced responses to both allogeneic and syngeneic stimulators, whereas the high density immature cells were unresponsive. This correlation was supported further by binding studies in which T cell tumor lines derived from C57BL/6 mice were used. ERLD tumor cells, which are similar to cortical immature thymocytes in both enzymatic and surface antigenic markers, were found to bind readily to macrophages, whereas both EL-4 and E male G2 tumor cells, with characteristics of mature thymocytes, bound to macrophages poorly. The binding of thymocytes and ERLD tumor cells to macrophages was not genetically restricted. We speculate that thymocyte binding to macrophages may play a critical role during the functional maturation of thymocytes.     

Thymic shared antigen-1. A novel thymocyte marker discriminating immature from mature thymocyte subsets.

In a previous study, we raised a mAb (MTS 35) reacting with a plasma membrane Ag expressed on both cortical thymocytes and a subset of thymic medullary epithelial cells. In view of the shared expression of this molecule, we have defined it as thymic shared Ag-1 (TSA-1). Considering its selective reactivity with cortical, but not medullary thymocytes, the relevance of TSA-1 as a marker of immature T cells was investigated in detail in this study, using multicolor flow cytometric analysis. TSA-1 was found on all immature thymocyte subsets (CD3-4-8-, CD3-4+8-, CD3-4-8+, CD3-4+8+, CD3low4+8+). Conversely, CD3high4+8- and CD3high4-8+ thymocytes, early thymic migrants and peripheral T cells were TSA-1-. More refined gating and analysis of the transitional CD3intermediate/high4+8+ thymocytes, proposed candidates for negative selection, demonstrated that approximately one half were TSA-1-. In fact, there was a directly inverse relationship between TSA-1 and CD3 expression on thymocytes. In the periphery, TSA-1 was detected on B lymphocytes. TSA-1 is PI-linked and has a molecular mass of 17 kDa nonreduced, or 12 to 13 kDa reduced. Through cross-correlation analysis, this molecule was distinct from H-2K, PNA-R, CD5, CD11a/18, Thy-1, HSA, Ly6A/E, Ly6C, ThB, CD25, CD44. Hence TSA-1 appears to be a unique marker which exquisitely separates mature from immature thymocytes.     

Selective stimulation of human thymocyte subpopulations by recombinant IL-4 and IL-3

Human thymocytes and thymocyte subsets were examined for their proliferative response to recombinant interleukin-4 (IL-4) and interleukin-3 (IL-3) in serum-free cultures. IL-4 induced marked proliferation of thymocytes after PHA and TPA stimulation, in contrast to the marginal response of T cells from adult peripheral blood. However, depletion of thymocytes bearing the CD3 antigen diminished the IL-4-induced proliferation of thymocytes, indicating that the response of thymocytes to IL-4 is mainly mediated by the CD3-positive cells. Phenotypic changes after culture with IL-4 showed an increase in the percentage of total thymocytes expressing mature T cell antigens (CD3, CD5, and TCR-1) and a decrease in CD1-positive cells. In addition there was an increase in the percentage of CD4+8- cells in both nylon wool-separated thymocytes and CD3-depleted cells with the disappearance of most of the CD4+8+ cells. However, an increase in the percentage of CD4-8- cells was also observed. The IL-4-responding cells do, however, express the mature T cell antigen, CD5, in high density. The effect of IL-3 on the proliferation of human thymocytes was very low and detected only when the thymocytes were cultured in serum-free medium. Depletion of CD3-positive cells did not diminish the IL-3-mediated proliferation of thymocytes, indicating that IL-3-responsive thymocytes are more immature than the subset of thymocytes which responds to IL-4. These results suggest that IL-4 and IL-3 play different roles in the development of human T cells.     

The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs

The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma₁ + gamma₂ globulins, gamma₁ globulins, gamma₂ globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen.     

Expression and role of the T cell receptor in early thymocyte differentiation in vitro

Fetal thymus organ culture was used to study the expression and function of antigen-specific, major histocompatibility complex-restricted receptors on thymocytes. Receptor gene rearrangement and expression occurred de novo in organ culture indicating that these events are induced in the thymus itself, presumably in response to thymus-derived stimuli. During organ culture a population of immature thymocytes expressing low levels of receptors developed first, and then diminished as mature thymocytes with high levels of receptor expression appeared. Continuous culture with antireceptor antibody modulated receptor from the surfaces of immature thymocytes, but did not prevent their appearance or accumulation. By contrast, appearance of receptor-bearing mature thymocytes was prevented in the presence of antireceptor antibody. These results indicate that the receptor is not essential for the generation of immature thymocytes but is involved in the selection or maintenance of mature cells from this pool.     

So-called antireceptor sera

Experiments in delayed type hypersensitivity transfer were carried out with the aim of studying the ability of rabbit antisera against peritoneal exudate cells of rats sensitized with bovine gamma globulin or rabbit kidney tissue antigen to block peritoneal exudate cells of guinea pigs. In the serological test the antisera prepared against the cells of sensitized rats and tentatively named "receptor antisera", reacted not only with the cells of these rats, respectively, but also with guinea pig cells. In hypersensitivity transfer experiments in guinea pigs receptor antisera showed a blocking effect on the transferred cells, making them incapable of transferring hypersensitivity, i. e. rabbit antisera against rat peritoneal exudate cells reacted with guinea pig cells. This interaction was specific: the blocking effect was manifested only when guinea pigs whose cells were used in the transfer were sensitized with the same antigen as the rats against whose cells the receptor antisera had been prepared. The control antisera, taken for the treatment of the transferred cells in the same doses as the receptor antisera, had no blocking effect on the cells.     

Defective thymocyte maturation in horses with severe combined immunodeficiency

Six monoclonal antibodies, designated EqT2, EqT3, EqT6, EqT7, EqT12, and EqT13, which identify T lymphocyte antigens present at different stages of T cell maturation were used to examine T lymphocyte development in foals with severe combined immunodeficiency (SCID). Flow microfluorimetry demonstrated the presence of EqT12+ and EqT13+ prothymocytes and a few phenotypically mature EqT2+ and EqT3+ thymocytes within the thymic remnants of SCID foals. However, very few EqT6+ and EqT7+ resident cortical thymocytes were detected. The near absence of EqT6+ and EqT7+ cortical thymocytes was confirmed by immunofluorescence analysis of thymic tissue from SCID foals. Those cells present were larger than normal cortical thymocytes. Furthermore, their activities of adenosine deaminase, adenosine monophosphate-deaminase, and 5' nucleotidase differed from those of normal cortical thymocytes. The combined evidence of monoclonal antibody analysis, size parameters, and purine enzyme activities demonstrate the near absence of cortical thymocytes in horses with this genetically defined immunodeficiency disorder.     

Species heterogeneity of hepatic alpha 1-adrenoceptors: alpha 1A-, alpha 1B- and alpha 1C-subtypes.

alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but not in rat hepatocytes. Binding competition experiments also showed high affinity for 5-methyl urapidil in liver membranes from guinea pigs and rabbits and low affinity in those from rats. The data indicated that guinea pig hepatocytes express alpha 1A-, rat hepatocytes alpha 1B- and rabbit hepatocytes alpha 1C- adrenoceptors. This was confirmed by Northern analysis using receptor subtype-selective probes.     

Recognition of Heterologous Cells by Macrophages

The ability of macrophages to recognize homologous and various heterologous cells was studied in mice, rats, and guinea pigs, in terms of the in vitro phagocytosis of non-opsonized viable thymocytes by macrophages. Mouse, rat, and guinea pig macrophages were found to phagocytize actively thymocytes from certain heterologous animals, including chickens. For instance, mouse macrophages displayed conspicuous phagocytic activities against chicken and duck thymocytes, moderate activities against guinea pig and frog thymocytes and weak activities against rat and mouse thymocytes. On the other hand, guinea pig macrophages revealed a different behaviour: they ingested only chicken thymocytes. These observations strongly suggested that mammalian macrophages possess some ability to discriminate homologous from certain heterologous thymocytes. The results, however, did not necessarily support the idea that the degree of phagocytosis is simply related to the phylogenetic distance between the animal species from which thymocytes and macrophages originated, because of the apparent exception in the mode of phagocytosis by guinea pig macrophages. Evidence demonstrating that antibodies are not involved in this phenomenon will be presented in the accompanying paper.     

Fas is expressed early in human thymocyte development but does not transmit an apoptotic signal.

We investigated the expression and function of Fas on human thymocytes prepared from fetal and pediatric tissue specimens and from SCID-hu Thy/Liv grafts. Unlike mouse thymocytes, human thymocytes exhibited a pattern of Fas expression skewed to immature cells, in that the highest expression was seen on double negative thymocytes and on intrathymic T progenitor cells. Fas expression was intermediate on double positive human thymocytes, and low or negative on mature single positive CD4 and CD8 medullary thymocytes. In spite of this relatively abundant surface expression, cross-linking of Fas with agonist mAb was incapable of triggering an apoptotic signal in human thymocytes. Apoptotic signaling was not enhanced by treatment with cycloheximide, nor by restoring a cosignaling milieu by addition of thymic stromal cells. Mouse thymocytes were induced to apoptosis by cross-linked recombinant soluble human Fas ligand both in vitro and in vivo, though human thymocytes were also resistant to this mode of receptor ligation. Membrane-bound Fas ligand also induced apoptotic death in murine thymocytes but not in human thymocytes. Human thymocytes were as sensitive as Jurkat cells, however, to apoptosis induced by TNF-alpha, suggesting that these cells have a signaling defect before activation of the earliest caspases. These data demonstrate a durable and specific resistance of human thymocytes to apoptosis induced through Fas receptor engagement, and reveal significant species-specific differences in the biology of thymocyte-programmed cell death.     

Human thymic epithelial cells function as accessory cells for autologous mature thymocyte activation

Using human thymocytes and autologous thymic epithelial (TE) cells grown in vitro in long-term culture, we have found TE cells can function as accessory cells for mitogen-induced mature thymocyte activation. Tritiated thymidine incorporation, blast formation, and protein synthesis were all induced in accessory cell-depleted thymocytes by autologous TE cells in the presence of suboptimal concentrations of PHA. After 3 days of mitogen stimulation of thymocyte-TE cell cocultures in vitro, thymocyte blasts bound to TE cells and 77 +/- 4% (mean +/- SEM) of TE cells acquired expression of major histocompatibility complex (MHC) class II (DR) antigen. TE accessory cell function for thymocyte activation was dependent on the number of TE cells added to thymocyte cultures, was not dependent on TE cell division, but did require TE cell protein synthesis. In thymocyte separation experiments, the predominant cell type responding to PHA in the presence of TE cells was T6- mature (stage III) thymocytes. Thus, human TE cells are capable of providing signals that lead to mature thymocyte activation.     

Inhibition or acceleration of tumor growth by subpopulations of thymus cells separable by a peanut lectin.

The effect of different lymphocytes subpopulations on tumor growth in mice was investigated using an in vivo adoptive neutralization test (Winn test). Thymocytes from non-tumor-bearing mice accelerated the growth of the tumors tested [Lewis lung carcinoma (3LL), thymoma 40-127-299 and fibrosarcoma P-14] when injected into syngeneic or F₁ mice in a mixture with the tumor cells. The thymocytes were separated with the aid of peanut agglutinin into immunologically mature and immature subpopulations (Reisner Y., Linker-Israeli and Sharon N., Cell. Immunol. 25, 129, 1976). The immature thymocytes accelerated tumor growth to an extent similar to that of the unfractionated cells, whereas the mature subpopulation exhibited pronounced inhibitory activity. Our findings demonstrate that the murine thymus contains two thymocyte subpopulations with opposite activities on tumor growth and that the mature thymocytes have an inhibitory effect on tumor growth similar to that of spleen cells.     

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes.

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains. Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells. These finding is in line with earlier observed differences in the glycerol-induced clustering of intramembranous particles. Furthermore, the results of this study support the view that the fluorescence polarization technique applied to whole cells does not exclusively monitor the plasma membrane.     

Early events in thymocyte activation. I. Stimulation of protein synthesis by a thymus-dependent human serum factor.

Human serum contains a thymus-dependent factor that raises cyclic AMP levels in thymocytes. We found that this factor stimulates protein synthesis in thymocytes cultured in vitro. This activity of serum factor is thymus-dependent, because it is absent in sera from thymectomized donors; furthermore, this effect is predominantly found on precursors of mature T cells. Incubation of thymocytes with other agents that increase cyclic AMP, induces an increase in protein synthesis similar to that observed with serum factor. Most likely, the increase in protein synthesis is one of the events following stimulation of adenylate cyclase in thymocytes that leads to cell differentiation.     

A homing receptor-bearing cortical thymocyte subset: implications for thymus cell migration and the nature of cortisone-resistant thymocytes

The thymus exports a selected subset of virgin T lymphocytes to the peripheral lymphoid organs. The mature phenotype of these thymus emigrants is similar to that of medullary thymocytes and has been cited as supporting a medullary rather than cortical exit site. Using the monoclonal antibody MEL-14, we identify a 1%-3% subpopulation of thymocytes that expresses high levels of a receptor molecule involved in lymphocyte homing to peripheral lymph nodes. We present evidence that these rare MEL-14hi thymocytes are predominantly of mature phenotype and represent the major source of thymus emigrants. Surprisingly, MEL-14hi thymocytes are exclusively cortical in location, although their mature phenotype may allow them to masquerade as medullary cells in conventional studies. We also demonstrate that unlike medullary thymocytes, many cortisone-resistant thymocytes (CRT) are MEL-14hi. Thus, in contrast to current dogma, CRT do not represent a sample of medullary thymocytes as they are found in situ and their level of immunocompetence does not necessarily reflect that of the medullary population. Our findings refute the hypothesis that phenotypically and functionally mature cells are restricted to the medulla, and support our proposition that most thymus emigrants are derived from the MEL-14hi cortical subset.