Thymocyte Emigration Research Articles

Thymocyte emigration is mediated by active movement away from stroma-derived factors.

T cells leave the thymus at a specific time during differentiation and do not return despite elaboration of known T cell chemoattractants by thymic stroma. We observed differentiation stage-restricted egress of thymocytes from an artificial thymus in which vascular structures or hemodynamics could not have been playing a role. Hypothesizing that active movement of cells away from a thymic product may be responsible, we demonstrated selective reduction in emigration from primary thymus by inhibitors of active movement down a concentration gradient (chemofugetaxis). Immature intrathymic precursors were insensitive to an emigration signal, whereas mature thymocytes and peripheral blood T cells were sensitive. Thymic stroma was noted to elaborate at least two proteins capable of inducing emigration, one of which was stromal cell-derived factor-1. Thymic emigration is mediated, at least in part, by specific fugetaxis-inducing factors to which only mature cells respond.

Measuring Emigration of Human Thymocytes by T-Cell Receptor Excision Circles

Recent advances in characterizing thymic function confirm the importance of thymus to T-cell diversity in the periphery of both children and adults during both health and disease. Lack of a marker to identify human recent thymic emigrants (RTEs) is the biggest hurdle to accurately characterizing and quantifying thymic output. T-cell receptor excision circles (TRECs) are used as an assay to measure RTE levels. Controversy exists, however, as to whether TREC concentrations reflect the number of RTEs or are mainly altered by peripheral T-cell division and death. In this review, we first summarize recent data on the human thymus and RTEs. On the basis of both experimental and mathematical analyses, we characterize factors that influence TREC dynamics in the periphery and elucidate primary elements that induce a decline in TREC concentrations during normal aging and HIV-1 infection. Our findings suggest that T-cell dynamics are key to the accuracy of TREC concentrations as a useful measurement of human RTEs.

Influence of breast cancer on thymic function in mice.

The thymus is the major site of T cell maturation; extensive proliferation, differentiation, and apoptosis occur in this organ. During mammary tumorigenesis, there is a profound involution of the thymus associated with a severe depletion of the most abundant subset of thymocytes, CD4+8+ immature cells. Experiments to investigate the mechanism of loss of the CD4+8+ population indicated that there was no increase in the systemic levels of glucocorticoids, no loss of bone marrow precursors, and no decrease in precursor seeding of the thymus. Likewise, no enhanced emigration of thymocytes from the thymus to the periphery was observed in tumor-bearing mice. A slight increase in apoptosis was found in tumor bearers' thymi, but there was no apparent decrease in the proliferation of early thymic precursors CD4-8- cells. Importantly, severely altered levels of subpopulations of the CD4-8- precursors, consistent with an arrest in differentiation at an early stage of development, were detected. Moreover, thymic stromal cell function appeared to become impaired during tumorigenesis, possibly due to the action of tumor-derived factors. Thus, downregulation of cell-mediated immune functions occurring at late stages of the disease may be causally related to the thymic involution occurring during mammary tumorigenesis.

Renewal of thymocyte progenitors and emigration of thymocytes during avian development

The avian thymus is colonized by three waves of hemopoietic progenitors during embryogenesis. An in vivo thymus reconstitution assay based on intrathymic injection of irradiated chicks showed that cells of para-aortic foci were able to differentiate into T lymphocytes, confirming their putative role in the first wave of thymus colonization. This assay was also used to detect and to characterize T cell progenitors from the bone marrow which are involved in the second and third wave of thymus colonization. In the bone marrow, progenitors that differentiated into T cells were found in a subpopulation that expressed the molecules HEMCAM, c-kit and c128. Engraftment of thymus lobes into thymectomized young chick recipients showed that T cell progenitors are replaced in the thymus by subsequent waves of progenitors after hatching. Finally, analysis of thymocyte differentiation suggested that γ δ and α β T cells migrate from the thymus to the periphery in alternating waves.

Appearance and maturation of T-cell subsets during rat thymus ontogeny.

In previous papers, we have described the ontogenetical development of thymic stromal-cell components (epithelium, macrophages, dendritic cells) of Wistar rats. Here, we correlate those results with the maturation of rat T-cell precursors along the fetal and postnatal life. First T-cell precursors, which colonize the thymus anlage around days 13-14 of gestation, largely express CD45, CD43, CD53, and Thy 1 cell markers, and in a lesser proportion the OX22 antigen. Rat CD3-CD4-CD8- thymocytes present in the earliest stages of gestation could be subdivided in three major cell subpopulations according to the CD44 and CD25 expression: CD44-/+CD25- → CD44+CD25+ → CD44+CD25- On fetal days 17-18, a certain proportion of CD4-CD8-cells weakly,express the TcRβ chain, in correlation with the appearance of the first immature CD4-CD8+ thymocytes. This cell subpopulation, in progress to the CD4+CD8+ stage, upregulates CD8α before the CD8β chain, expresses the CD53 antigen, and exhibits a high proliferative rate. First mature thymocytes arising from the DP (CD4+CD8+) cells appear on fetal days 20-21. Then, the CD4+:CD8+ cell ratio is ≤1 changing to adult values (2-3) just after birth. Also, the percentage of VβTcR repertoire covered in adult thymus is reached during the postnatal period, being lower during the fetal life. Finally, in correlation with the beginning of thymocyte emigration to the periphery a new wave of T-cell maturation apparently occurs in the perinatal rat thymus.

Thymocyte emigration in the chicken: an over-representation of CD4+ cells over CD8+ in the periphery.

We have examined the emigration of chicken thymocytes after intrathymic fluorescein isothiocyanate (FITC) labelling in situ. In this paper we show that in young birds about 0.7% and 0.4% of thymocytes emigrate from the thymus to the blood and the spleen, respectively, per day. This suggests that, as in mammals, most thymocytes die within the thymus. At 3 weeks of age gamma delta and alpha beta T cells leave the thymus in comparable levels to their appearance in the blood. The phenotype of recent emigrants in peripheral tissues is similar to that of mature T cells. Interestingly, recent emigrants contain relatively much higher numbers of CD4+ and fewer CD8+ cells than is observed in peripheral tissues in a steady-state situation.

Defect of thymocyte emigration in a T cell deficiency strain (CTS) of the mouse.

As bone marrow progenitors migrate to the thymus, differentiate into immunologically competent T cells, then leave the thymus and home to the peripheral lymphoid organs, each migration or homing is an essentially critical process for T cell differentiation. Homing of thymocytes into peripheral lymphoid organs has been extensively analyzed. On the contrary, little is known about the mechanism whereby mature thymocytes emigrate from the thymus to peripheral lymphoid organs. In this work, we show that the mutant T cell deficiency strain (CTS) of mouse, previously described as a peripheral T cell deficiency strain, has a novel defect of mature thymocyte emigration process. Flow-cytometric analysis demonstrated a marked decrease in T lymphocytes in peripheral lymphoid organs and an accumulation of mature thymocytes in the thymus. Histologic study revealed the high concentration of thymocytes within the giant perivascular space at thymic corticomedullary junction. These accumulating cells closely resemble normal peripheral T lymphocytes in terms of cell surface phenotype and responsiveness to mitogens or allogeneic cells. They were found to express the Mel-14 Ag, the homing receptor to lymph nodes, and had a high capability of homing to peripheral lymphoid organs when explanted thymocytes were injected i.v. The intrathymic labeling methods detected hardly any emigrating CTS thymocytes from the thymus. These results indicate that emigration defect lies in the thymus of CTS mice, and suggest that this mutation is due to abnormal releasing mechanisms occurring between the thymus and the bloodstream. Complete delineation of the precise mechanisms of this defect, however, requires further studies.

Modulating activity of mistletoe lectins 1 and 2 on the lymphatic system in BALB/c-mice

The galactoside-specific lectin (mistletoe lectin-1, VAA-1) and the N-acetylgalactosamine-specific lectin (mistletoe lectin-2, VAA-2) were purified from aqueous mistletoe extract and checked for their immunoactive potency. Regular subcutaneous administration of the optimal immunomodulating VAA-1 /VAA-2 dosage (1 ng lectin/kg body weight) could be shown to modulate thymocyte proliferation, maturation, emigration and activation in BALB/c-mice. Thus, the increase in thymocyte counts was statistically significant after VAA-1 treatment. However, analogue VAA-2 applications significantly decreased thymocyte proliferation. Determinations of lymphatic subsets revealed considerable upregulation (after VAA-1 treatment) and significant downregulation (after VAA-2 treatment) of immature L 3 T4(+)/Lyt-2(+) thymic cells. Counts of mature cells expressing helper/inducer (L3T4(+)) or suppressor/cytotoxic (Lyt-2(+)) phenotypes did not present remarkable differences after VAA-1/VAA-2 administration. Counts of BALB/c-mouse peripheral blood cells revealed evident (statistically non-significant) increases of lymphocytes (PBL) and monocytes (PBM) after VAA-1 treatment, however, cell counts after VAA-2 administration were comparable to non-treated control animals. The determination of activated PBL expressing interleukin IL-2 receptors proved that VAA-1 induced a potent immunostimulation, since these cells were significantly enhanced after VAA-1 administration but not after VAA-2 treatment.

Thymocyte emigration during chicken ontogeny: A key step for the development of the lymphoid system?

Thymocyte emigration during chicken ontogeny: A key step for the development of the lymphoid system?

Influence of Propionibacterium avidum KP-40 on the proliferation, maturation, emigration and activity of thymocytes and monocytes

Inactivated cells of Propionibacterium avidum KP-40 could be shown to induce thymocyte proliferation and maturation in BALB/c-mice after intraperitoneal administration of the optimal immunomodulating dosage (1 mg per mouse). The increase in thymus weight and thymocyte numbers per mg organ weight was most pronounced and statistically significant 10 days after P. avidum KP-40 administration. Determinations of lymphatic subsets revealed a considerable up-regulation of mature cells expressing helper/inducer (L3T4+) or cytotoxic/suppressor (Lyt-2+) phenotypes and immature cells presenting both L3T4+/Lyt-2+ antigens. Obviously, P. avidum KP-40 administration accelerated murine thymocyte proliferation and maturation. Counts of BALB/c-mouse peripheral blood lymphocytes (PBL) and monocytes (PBM) revealed statistically significant increases after P. avidum KP-40 administration with peak values after 6-10 days. The determination of activated PBL (expressing interleukin-2 receptors) or PBM (expressing MAC-3 antigens) proved that P. avidum KP-40 induced a potent immunostimulation since counts of these cells were significantly enhanced after P. avidum KP-40 treatment.

Cell adhesion molecules involved in intrathymic T cell development

During stem cell migration to the thymus, intrathymic maturation of T cells, and emigration of mature T cells out of the thymus, intercellular interactions of developing T cells with a myriad of cell types are required for normal T cell development. Intercellular interactions of T cell precursors with endothelial cells, thymic epithelial cells, fibroblasts, thymic macrophages and dendritic cells are all mediated by adhesion molecules on immature T cells binding to ligands on thymic microenvironment cells. While many receptor-ligand interactions that are important in intrathymic T cell development are known, the adhesion molecules that are important for migration of T cell precursors to the thymus and for emigration of mature thymocytes from the thymus are poorly understood. An emerging concept is that select adhesion molecules at discrete stages of T cell maturation participate in and regulate the complex processes of T cell development.

Demonstration of large-scale migration of cortical thymocytes to peripheral lymphoid tissues in cyclosporin A-treated rats.

Young adult Lewis rats were maintained on diets containing 0.015 or 0.027% cyclosporin A (CSA) for periods of up to 6 wk. All animals showed complete depletion of medullary thymocytes (CD4+8- and CD4-8+, T cell receptor [TCR] alpha/beta hi, Thy-1med/low, terminal deoxynucleotidyl transferase negative [TdT-]) and a 50% reduction in the number of TdT- cortical thymocytes (CD4+8+, TCR alpha/beta low, Thy-1med) within 1 wk of CSA treatment. In addition, about half of the animals displayed a 50% reduction in the number of TdT+ cortical thymocytes (CD4+8+, TCR alpha/beta low, Thy-1hi). These intrathymic changes were accompanied by a reciprocal increase in the number of double-positive (DP; CD4+8+) T cells in lymph nodes (LN) and spleens. To confirm that the latter T cells were recent thymic emigrants (RTE), CSA-treated rats were injected intrathymically with fluorescein isothiocyanate, and the phenotype of the labeled T cells appearing in LN was determined 16 h later. The results demonstrated that, in addition to those RTE exported in normal animals (> 90% medullary origin), the emigration of DP thymocytes, including large numbers of TdT+ thymocytes, was markedly increased. The presence of TdT+ cells, which normally do not leave the thymus, clearly identifies the DP RTE as originating from the thymus cortex. Intrathymic labeling studies also directly demonstrated that export of all thymocyte subsets ceases within 9 d of CSA treatment; and thymectomy experiments confirmed that the CSA-induced increase in phenotypically immature T cells resulted primarily from the disturbance of thymocyte maturation and emigration, rather than from a direct effect on preexisting T cells. These results suggest that a wave of cortical thymocytes, many of which presumably have not yet undergone negative selection, is released from the thymus during the first week of CSA treatment. The presence of these potentially unselected cells in peripheral lymphoid tissues may help to explain the increased frequency of autoreactive T cells observed in CSA-treated animals.

A pertussis toxin-sensitive process controls thymocyte emigration.

Although it is well known that essentially all peripheral T cells are derived from bone marrow progenitors that mature in the thymus, the mechanism whereby thymocytes gain access to peripheral compartments is obscure. We have learned that this process is sensitive to pertussis toxin (PT). Transgenic lck-PT mice were generated which express the catalytic subunit of PT in all thymocytes. In a previous study we observed that T cell receptor signaling is unimpaired in these cells despite the virtual elimination of their Gi protein signal transduction elements through endogenous PT activity. Here we demonstrate that mature T lineage cells accumulate in lck-PT thymuses and fail to populate peripheral lymphoid organs. The accumulating cells closely resemble normal peripheral T lymphocytes with respect to cell surface phenotype and responses to allogeneic spleen cells, yet perform poorly in in vivo homing assays. This migratory defect does not result from deficient expression of common homing receptors or alterations in intracellular cAMP concentrations. Based on these results, we propose that a novel PT-sensitive signaling pathway, almost certainly involving a Gi protein, is required for thymocyte emigration.

Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase.

Stimulation of the T lymphocyte antigen receptor-CD3 complex (TCR-CD3) causes T cell activation by a process associated with increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Evidence exists suggesting that GTP-binding (G) proteins, particularly the pertussis toxin (PT)-sensitive Gi proteins, participate in this signal transduction pathway. To clarify the role of Gi proteins in TCR-CD3 signaling, and to investigate other possible functions of Gi molecules in T cells, we expressed the S1 subunit of PT in the thymocytes of transgenic mice using the lymphocyte-specific lck promoter. Transgenic thymocytes contained S1 activity and exhibited profound depletion of Gi protein PT substrates in a manner suggesting their inactivation by S1 in vivo. Nevertheless, treatment of transgenic thymocytes with mitogenic stimuli provoked normal increases in intracellular free Ca2+ concentrations and IL-2 secretion, indicating that Gi proteins are not required for T cell activation. These normal signaling responses notwithstanding, mature thymocytes accumulated in lck-PT mice and did not appear in secondary lymphoid organs or in the circulation. Viewed in the context of the known features of Bordetella pertussis infection, our results suggest that a PT-sensitive signaling process, probably involving Gi proteins, regulates thymocyte emigration.

Lymphocyte emigration from the ovine thymus: characterization of lymphatic emigrants and venous emigrants.

Although it has been generally agreed that mammalian thymus produces lymphocytes of the T lineage and releases them as T cells into the general circulation, there have been differences in opinion expressed concerning the rate of thymocyte emigration compared to that of production and also concerning the maturity of emigrant cells. Cell traffic studies by the use of an intrathymic injection technique by Scollay and his associates indicated that, in the mouse thymus, only a small fraction of the cells produced in the thymus emigrate and that those emigrant cells are functionally mature (Scollay, Chen and Shortman, 1984). In contrast, experiments in other animal species often yielded results which indicated a high rate of thymocyte emigration with the emigrants being at least phenotypi-cally immature (Williams et al., 1971).

Cell kinetics in the fetal mouse thymus: precursor cell input, proliferation, and emigration.

The entry and differentiation of lymphoid precursor cells (LPC) in grafted mouse fetal thymuses and the emigration of explants thymocytes has been followed in a system in which donor and host lymphocytes could be distinguished on the basis of Thy-1 expression. It appears that LPC that invade the fetal mouse thymus between 10 and 13 days rapidly differentiate into Thy-1 positive thymocytes, giving rise to all of the lymphoid populations of both cortical and medullary locations until approximately the end of the first week after birth. Lymphoid precursor cells that enter the fetal thymus after 13 days of fetal life only differentiate into Thy-1 positive lymphocytes 6 or 7 days after birth, when they give rise to a second generation of thymocytes that grows exponentially and completely replaces the first generation in approximately 8 days. All cells leaving the thymus during the first 2 wk of life appear to be derived from the first wave of precursors.

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.

Variations in the expression of θ-C3H alloantigen on thymic and peripheral lymphocytes of newborn and young adult mice

The relative frequency of lymphocytes of mice showing varying degrees of surface θ-positivity (circumferential fluorescence) was recorded. Thymocytes were nearly 100% θ-positive. The relative proportions of θ-positive cells in Peyer's patches and lymph nodes of newborn mice varied in an almost identical fashion as a function of age. At 4 weeks of age and beyond, the relative numbers of θ-positive cells in Peyer's patches were consistently lower than in lymph nodes. As opposed to the predominance of thymocytes with complete rings, peripheralized thymic (T) lymphocytes showed a broad, age- and organ-dependent range of surface θ-positivity. These results suggest that surface θ may be lost rather rapidly upon emigration of lymphocytes from the thymus and/or that many θ-positive T cells with complete rings disappear within a short time. Variations in the relative proportion of complete rings on mesenteric lymph node cells on Days 1 and 4, were tentatively related to antigen-induced changes in the magnitude of thymocyte emigration. The pattern of surface θ-antigen of a given thymocyte or T cell with its size and DNA synthetic activity was compared. The findings suggest that incomplete ring fluorescence may especially be observed on proliferating lymphoblasts in the outer thymic cortex on their way to acquire the full complement of θ-antigen, and on medullary thymic lymphocytes or T cells having reentered mitotic activity, in response to antigenic and/or other microenvironmental stimuli. Our study yielded data consistent with the hypothesis that the progressive loss of surface θ-antigen does not represent a fully autonomous time-dependent process. Moreover, it is not clear if continued loss of θ-antigen by T cells below a certain threshold would render these cells undetectable by anti-θ sera.