A cytophotometric and flow cytofluorometric approach to the differentiation of T lymphocytes in the thymus

Summary By cytophotometric and flow cytofluorometric DNA and protein determinations two main proliferating subpopulations of thymus lymphocytes with a different percentage of cells in the S phase could be distinguished. One subpopulation had a very low protein content, was cortisone sensitive and located in the cortex. Cells with comparable low protein contents were not found amongst lymphocytes of the peripheral blood. The other lymphocyte subpopulation had a higher protein content, was cortisone resistant and situated in the cortex around a group of epithelial cells and in the medulla. The protein content of these thymus lymphocytes appeared to be comparable to that of the peripheral blood lymphocytes. On the basis of the protein content per cell, it is possible to identify and isolate the more often described major subpopulation of cortisone sensitive thymus lymphocytes remaining and dying in the thymus, and the minor cortisone resistant subpopulation of thymus lymphocytes which is the source of the peripheral T lymphocyte.     

cytophotometric,flow cytofluorometric and morphometric demonstration of the existence of an antigen dependent thymus lymphocyte subpopulation

Summary Using absorption cytophotometry and flow cytofluorometrical DNA and protein estimation of single thymus lymphocytes we were able to establish that after injection of a large dose of antigen (ovalbumin) a subpopulation of lymphocytes arises in the thymus with high protein contents above that of those lymphocytes normally present, however, in small quantities in the thymus. By morphometrical analysis it was established that these lymphocytes are situated in the outermost cortex.     

Automatic search for model to simulate the differentiation of T lymphocytes within the thymus

The differentiation of T Lymphocytes within the thymus is an important biological phenomenon during wich these cell acquire their functions to further control the immune system. Numerous experiments under various conditions have been devised to understand the different mechanisms involved in this complex process. Nevertheless, interpretation of these experiments lead to still contradictory debatable hypotheses. Modelisation of this process through classical simulation methods cannot be envisaged because they are not adapted to modifications of the model structure, which is the point of interest. For these reasons, we proposed a new approach of automatic search for model. The program consists of four independent connected modules : The generator produces model, based on the rationale of formal grammars. Protocol and experimental data are stored in a set of experiments. The simulator using a protocol and a model provides simulated results. Finally, the supervisor by comparing simulated results and experimental data, adapts the model parameters to increase their fit and either chooses a new experiment to explore, or modifies the model structure. Change of the model structure is performed among still unexplored models according to their promise level, which is iteratively evaluated relatively to previously explored models through a proposed model distance. The generator is written in Prolog and the other modules in C++. The architecture of the program allows us to modify or complete a module without changing anything in the other modules. As a consequence, the proposed modeling approach conceived to study T lymphocyte differentiation within the thymus remains independent of this biological phenomenon and can be applied to other biological problems.     

Role of an insoluble thymus fraction in the differentiation of T lymphocytes]

An insoluble thymic fraction, free of both thymocytes and thymic hormone, was injected intraperitoneally to C57B1/6 mice. Following this injection, marked changes were noted in thymocyte populations: these consisted mainly in a significant increase of subcapsular prothymocytes, peaking at day 7. These observations were made by histological examination and confirmed by a study of physical characteristics of thymocyte populations. The possibility that this insoluble thymic fraction might have a biologically active component responsible for prothymocytes recruitment is examined.     

T cell differentiation in the thymus

T lymphocytes arise in the thymus and seed to peripheral lymphoid organs as fully functional cells at the time of exit. In humans, the thymus begins to function very early in ontogeny and releases large numbers of T cells before the time of birth. However, the vast majority of developing thymocytes (>95%) die within the thymus as a result of stringent selection processes. Positive selection imposes self-MHC-restriction on thymocytes and dictates the MHC-restricted repertoire of post-thymic T cells. Negative selection results in deletion of autoreactive cells. Both types of selection depend on cell to cell contracts and on the presence of appropriate growth factors which are still largely undetermined. Cell to cell contacts occur between developing thymocytes and cells of the thymic microenvironment (accessory cells), and are mediated by several receptor/ligand interactions which subserve the function of establishing and stabilizing these contacts. Besides MHC-TCR interactions, adhesion molecules are important for thymocyte maturation, selection and activation, and for the export and peripheral homing of mature T cells produced in the thymus. Here we describe a novel integrin involved in thymocyte-thymic epithelial cell interactions.     

The differentiation of T lymphocytes. I. Proliferation kinetics and interrelationships of subpopulations of mouse thymus cells

Differential quantitative cytotoxic assays were used to distinguish the major high θ population of mouse thymus from the minor low θ subpopulation. The low θ cells were isolated in good yield by killing all high θ cells with controlled anti-θ and complement treatment, followed by a damaged cell removal step. A third population of labile cells, subject to rapid death in culture, was distinguished as a variable component within the high θ category. The corticosteroid sensitivity and anatomical location of these subpopulations was briefly considered. Large and medium sized dividing lymphocytes were studied by pulse labeling with tritiated thymidine, followed by radio-autography of separated subpopulations. Both the high θ and low θ categories included large dividing cells. Kinetic studies under conditions of continuous labeling were used to explore precursor-product relationships among the small thymocytes. Both low θ and high θ small lymphocytes showed continuous and close to linear accumulation of labeled cells, with no evidence for a marked lag in labeling. The turnover time of high θ small lymphocytes was three–four times that of low θ elements. The results suggest largely independent pathways are involved in the development of the two antigenically defined subpopulations. They do not support a direct transfer of “immature” high θ, TL positive small thymocytes in mature, active, low θ,TL negative cells. Some alternative models of T cell development are discussed.     

Chromatin degradation during the exposure of thymus lymphocytes in vitro to differentiation inducers and gamma irradiation

Chemical inductors of differentiation were shown to cause chromatin degradation in thymus lymphocytes. This process was prevented by the protein synthesis inhibitors. The fragments formed after the effect of chemical differentiation inductors on thymocytes were fully identical to chromatin internucleosome degradation products formed in the exposed cells. Chromatin degradation under the effect of chemical differentiation inductors was most pronounced in a more radiosensitive thymocyte fraction.     

Ultrastructure and antigens in differentiation of thymus lymphocytes in human embryogenesis]

Thymus lymphocytes of 7--8-week human embryos have nuclei of irregular form with 1--3 distinct nucleoli characterized by absence of compact chromatin or heterchromatin. The electron-dense cytoplasm of these cells contains polysomes and an insignificant number of mitochondria. No receptors to sheep red blood cells and T antigen are revealed on their surface. In 11--12-week human embryos one can observe a decrease in the size of thymus lymphocytes, appearance of heterochromatin in their nuclei and receptors to sheep red blood cells (79%), and T antigen (60%) on the cell surface. Subsequently the quantity of compact chromatin in thymus lymphoid cells increases, and the cells acquire definitive properties and structure.     

Immunohistology of lymphoid and non-lymphoid cells in the thymus in relation to T lymphocyte differentiation

The present paper reports the distribution of lymphoid and non-lymphoid cell types in the thymus of mice. To this purpose, we employed scanning electron microscopy and immunohistology. For immunohistology we used the immunoperoxidase method and incubated frozen sections of the thymus with 1) monoclonal antibodies detecting cell-surface-differentiation antigens on lymphoid cells, such as Thy-1, T-200, Lyt-1, Lyt-2, and MEL-14; 2) monoclonal antibodies detecting the major histocompatibility (MHC) antigens, H-2K, I-A, I-E, and H-2D; and 3) monoclonal antibodies directed against cell-surface antigens associated with cells of the mononuclear phagocyte system, such as Mac-1, Mac-2, and Mac-3. The results of this study indicate that subsets of T lymphocytes are not randomly distributed throughout the thymic parenchyma; rather they are localized in discrete domains. Two major and four minor subpopulations of thymocytes can be detected in frozen sections of the thymus: 1) the majority of cortical thymocytes are strongly Thy-1+ (positive), strongly T-200+, variable in Lyt-1 expression, and strongly Lyt-2+; 2) the majority of medullary thymocytes are weakly Thy-1+, strongly T-200+, strongly Lyt-1+, and Lyt-2- (negative); 3) a minority of medullary cells are weakly Thy-1+, T-200+, strongly Lyt-1+, and strongly Lyt-2+; 4) a small subpopulation of subcapsular lymphoblasts is Thy-1+, T-200+, and negative for the expression of Lyt-1 and Lyt-2 antigens; 5) a small subpopulation of subcapsular lymphoblasts is only Thy-1+ but T-200- and Lyt-; and 6) a small subpopulation of subcapsular lymphoblasts is negative for all antisera tested. Surprisingly, a few individual cells in the thymic cortex, but not in the medulla, react with antibodies directed to MEL-14, a receptor involved in the homing of lymphocytes in peripheral lymphoid organs. MHC antigens (I-A, I-E, H-2K) are mainly expressed on stromal cells in the thymus, as well as on medullary thymocytes. H-2D is also expressed at a low density on cortical thymocytes. In general, anti-MHC antibodies reveal epithelial-reticular cells in the thymic cortex, in a fine dendritic staining pattern. In the medulla, the labeling pattern is more confluent and most probably associated with bone-marrow-derived interdigitating reticular cells and medullary thymocytes. We discuss the distribution of the various lymphoid and non-lymphoid subpopulations within the thymic parenchyma in relation to recently published data on the differentiation of T lymphocytes.     

A method for the differentiation of T and B lymphocytes and monocytes migrating under agarose.

This report describes alterations in the agarose lymphocyte migration technique which resulted in satisfactory differentiation of T and B lymphocytes and monocytes which have migrated as a monolayer for 1-3 days. The Wright-Giemsa staining used in the original method did not permit identification of individual migrating cell types. The most important modifications were changing from a plastic to a glass migration surface, and significantly reducing the overlying thickness of agarose which permitted a short fixation time and easy preparation of permanent slides stained for nonspecific esterase. The esterase staining of monocyte cytoplasm was intense and diffuse. One or two small, discrete areas of cytoplasmic esterase activity were identified in the majority of T lymphocytes. B lymphocytes showed either a trace or no evidence of esterase activity. The modified method should prove useful for the histochemical differentiation of migrating subpopulations of mononuclear cells.     

Effect of irradiation and chemical differentiation inducers on the survival of thymus lymphocytes in mice

Chemical inductors of differentiation were shown to produce a cytotoxic effect on thymus cells. The protein synthesis inhibitors prevented this effect. The toxic action of the inductors was more pronounced in a most radiosensitive thymocyte fraction. A combination of differentiation inductors and ionizing radiation did not produce the additive effect. This was observed after the effect of radiation and substances being not the differentiation inductors but toxic for cells.     

The differentiation of cytotoxic T lymphocytes in vitro

Summary Previous neurohistological studies have been extended to include the structures contained solely or mainly within the junctional esophageal segment which may play an important role in the sphincter mechanism. The main findings were: 1) a progressive cranio-caudal thickening of the muscularis mucosae; 2) a conspicuous thickening of the circular muscle layer; 3) abundant and close interconnections between the esophageal striated fibres and gastric smooth muscle cells; 4) presence of annulo-spiral elastic fibres coiled around bundles of striated musculature; 5) increase of the intramural nerve component, particularly Auerbach's plexus, which consisted of a continuous nervous layer containing twice as many neurocytes as found in the upper esophageal segments; 6) presence of numerous interconnected motor endplates often possessing ultraexpansional fibres and secondary endplates. The findings are discussed with emphasis on functional correlations in order to attain a unitary morpho-functional view.Abbreviations used LES lower esophageal sphincter - HPZ high pressure zone; mm: muscularis mucosae - CNS central nervous system - CCK-PZ cholecystokinin-pancreozymin Dedicated to Prof. Dr. Wolfgang Bargmann for his fundamental contributions to Comparative Morphology     

Interaction of thymus lymphocytes with histoincompatible cells. IV. Mixed lymphocyte reactions of activated thymus lymphocytes

CS7BL-activated CBA T cells (T.TDL) were obtained by thoracic duct cannulation of (CBA × C57BL)F₁ mice 4 days after heavy irradiation and injection of CBA thymus cells. T.TDL behaved differently from the TDL of normal CBA mice in unidirectional mixed lymphocyte culture in a number of respects: (a) the response of T.TDL was directed specifically against C57BL antigens, whereas normal TDL responded to both C57BL and BALB/c antigens; (b) the response of T.TDL was rapid but transient compared to that of TDL; (c) whereas only approximately 3% of TDL synthesized DNA specifically in response to C57BL antigens, as many as 25% of C57BL-activated T.TDL responded to these antigens. Evidence is presented which suggests that the T.TDL have a very limited capacity to proliferate. Most of the cells which responded to antigen synthesized DNA without subsequently entering mitosis.     

T cell differentiation antigens on lymphocytes in the human intestinal lamina propria.

Recently, T cell subpopulations presumably representing memory T lymphocytes have been described in vitro. Intestinal lamina propria T cells (LP-T) have characteristics resembling those of memory cells. We therefore investigated the expression of surface Ag associated with memory phenotype in vitro on lamina propria lymphocytes (LPL) and PBL and on the T cell subpopulations defined by the bright expression of CD45R0 by flow cytometric analysis of isolated cell populations. LPL had significantly increased percentages of CD45R0 and CD58 positive cells compared with PBL. Whereas PBL showed bimodal expression profiles of CD45R0, CD58, and CD2, the vast majority of LPL was bright for these Ag. Expression of CD45RA was significantly reduced in both frequency and intensity in LPL, and LPL had significantly reduced percentages of CD11a/CD18 and CD29 positive cells compared with PBL. The CD45R0 bright T cell subpopulations of both PBL and LPL were characterized by a lack of CD45RA. CD45R0 bright T cells from the peripheral blood (PB-T) were predominantly bright for CD2, CD58, CD29, and CD11a/CD18 whereas CD45R0 dim PB-T had bimodal expression profiles and CD45R0 negative PB-T were dim or even negative for these Ag. CD45R0 bright LP-T were also bright for CD2 and CD58 but had significantly reduced surface densities of CD11a/CD18 and CD29 compared with CD45R0 bright PB-T. The surface density of CD29 on CD45R0 bright LP-T corresponded to that of CD45R0 negative PB-T, and a significant proportion of CD45R0 bright LP-T was even negative for CD11a/CD18 and CD29. Additionally, CD45R0 bright LP-T in contrast to PB-T were characterized by a lack of 1-selectin and the expression of CDw49a and the mucosa-specific T cell Ag HML-1 on high percentages of cells. Our results show that the phenotype of CD45R0 bright T cells from the lamina propria clearly deviates from that of memory T cells in vitro and of CD45R0 bright T cells in the peripheral blood. We conclude that memory T cell populations in vivo undergo specific differentiation depending on their tissue localization, leading to unique phenotypic and presumably functional features.     

Qa alloantigen expression on functional T lymphocytes from spleen and thymus

The cell-surface expression of the class I alloantigen Qa-2 was analyzed on resting and activated spleen and thymus cells using cytotoxic elimination and immunofluorescence and flow cytometry. Spleen cells activated by mitogens or alloantigen were homogeneously positive for cell surface Qa-2, but activated splenic T cells expressed only about one-third as much Qa-2 per cell as did nonstimulated T cells. These data correlated with the ability to perform cytotoxic elimination with Qa-2-specific monoclonal antibodies (mAbs) in that cytotoxic T lymphocyte (CTL) activity was completely abrogated by pretreatment of spleen cells prior to in vitro culture but was only partially eliminated by treatment of CTL effectors. Qa-2-positive cells constituted only a small subpopulation of fresh normal thymocytes, but were enriched (>40% positive) among cortisone-resistant thymocytes (CRT). These Qa-2-positive CRT contained mature thymocytes as defined by Ly phenotype Ly-2^–, Ly-1^hi. When normal thymocytes were treated with Qa-2-specific mAb and complement prior to in vitro sensitization for generation of allogeneic CTL, CTL activity was completely abrogated despite the fact that the fraction of cells eliminated were undetectable as assessed by cell recovery. CTL effectors from alloantigen-stimulated thymocytes were also susceptible to cytotoxic elimination with Qa-2-specific mAb. These data suggest that the Qa-2 molecule may serve not only as a marker on resting and activated peripheral T cells, but also as a unique marker for functionally mature T cells in the thymus.     

EA-1, a novel adhesion molecule involved in the homing of progenitor T lymphocytes to the thymus

The mouse progenitor T lymphocyte (pro-T) cell line FTF1 binds in vitro to thymus blood vessels, the thymic capsule, and liver from newborn mice. A mAb, EA-1, raised against an embryonic mouse endothelial cell line, blocked adhesion. The antibody also interfered with pro-T cell adhesion to a thymus-derived mouse endothelial cell line; it had no effect on the adhesion of mature T lymphocytes and myeloid cells. The antigen recognized by EA-1 is located on the vascular endothelium of various mouse tissues and absent on pro-T cells. EA-1 antibody precipitates molecules with apparent molecular weights of 110,000, 140,000, 160,000, and 200,000. Immunoclearing and binding-inhibition studies with antibodies against known adhesion molecules suggest that the EA-1 antigen is a novel adhesion molecule involved in colonization of the embryonic thymus by T cell progenitors.     

Physical heterogeneity of mouse thymus lymphocytes.

Mouse thymocytes have been separated by velocity sedimentation in a density gradient. The resulting fractions have been analyzed using electrophoretic light scattering. The electrophoretic distributions of the individual sedimentation fractions reveal the presence of physically distinct subpopulations. Comparison of the mean mobilities of each fraction indicates that the faster-sedimenting cells tend to have a higher electrophoretic mobility.