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.     

Different subpopulations of developing thymocytes are associated with adherent (macrophage) or nonadherent (dendritic) thymic rosettes.

Thymic rosettes (ROS), structures consisting of thymic lymphoid cells attached to a central stromal cell, were isolated from mouse thymus by collagenase digestion and unit-gravity elutriation. The ROS were then separated into those where the stromal cells were either macrophage-like (M-ROS) or dendritic cell-like (D-ROS), on the basis of the differences in adherence properties or in the level of MAC-1 surface antigen. The ROS were then dissociated and the thymocyte content analyzed by immunofluorescent staining and flow cytometry. M-ROS and D-ROS differed in thymocyte composition, although the major component of both was the CD4+CD8+ cortical thymocyte. D-ROS were enriched in thymocytes expressing high levels of surface T-cell antigen receptor (TcR) and the associated CD3 complex, and these included both CD4+CD8-CD3++ and CD4-CD8+CD3++ mature thymocytes. M-ROS were enriched in CD4-CD8- thymocytes and had a reduced content of thymocytes expressing high TcR-CD3 levels; they nevertheless contained some mature thymocytes, but only of the CD4+CD8-CD3++ category. Several lines of evidence indicated that the mature thymocytes in ROS were cells recently formed in the cortex, and were not from the medullary pool. ROS-associated mature thymocytes expressed lower levels of H-2K than free, mature thymocytes. The CD4+CD8+CD3++ subpopulation, believed to be a developmental intermediate between cortical thymocytes and mature T cells, was present in both ROS populations. Further, late intermediates leading to both mature T-cell categories were evident in D-ROS, but only those leading to CD4+CD8-CD3++ T cells were evident in M-ROS. The results are compatible with a role for ROS in TcR-specificity selection and in the final maturation steps in the thymic cortex.     

Emigration of mature T cells from the thymus is inhibited by the imidazole-based compound 2-acetyl-4-tetrahydroxybutylimidazole.

The primary role of the thymus is to provide mature T cells for the peripheral immune system. The mechanisms involved in the cellular export processes are as yet unknown. In this study, we examined the ability of 2-acetyl-4-tetrahydroxybutylimidazole (THI), an agent widely used as a component of ammonia caramel food colouring, to inhibit T-cell export from the thymus. BALB/c mice were maintained on drinking water containing THI for 5 days. The mice showed a twofold increase in the total number of mature medullary thymocytes (CD4+CD8- and CD4-CD8+) as well as a slight decrease in the total number of immature double-positive cells (CD4+CD8+). The mature single-positive thymocytes were found to express high levels of the homing molecule L-selectin, suggesting that these potential emigrants were prevented from leaving the thymus. To confirm this, THI-treated mice were injected intrathymically with fluorescein isothiocyanate and the number of labelled T cells appearing in the lymph nodes and spleen was determined 16 hr later. A 10-fold decrease in the number of CD4+ and CD8+ recent thymic emigrants in the lymph nodes and spleen of THI-treated mice was observed. Previous studies have shown that THI does not affect other aspects of thymocyte development, such as proliferation and differentiation. Taken together, these results suggest that the immunosuppressive effects of THI may be due, in part, to preventing of the final step of T-cell export out of the thymus.     

Seeding of neonatal lymph nodes by T cells and identification of a novel population of CD3-CD4+ cells.

Mature T cells first appear in the thymus of the mouse a few days before birth, about 7-8 days after the thymus was colonized by stem cells. These mature cells are exported to the peripheral lymphoid organs beginning at about the time of birth, but because the number is very small at this stage, little is known about the phenotype or function of these early emigrants. We have examined the cells that accumulate in the peripheral lymph nodes (LN) during the first week of life to understand better the initial seeding of the periphery by T cells. Our studies showed that a high proportion of neonatal LN cells were CD4+, but that the majority of these were CD3- during the first few days of life. The CD3- population did not increase greatly in number after birth and rapidly diminished in proportion as the number of CD3+ cells increased. These CD3-CD4+CD8- cells were found to be Thy-1loCD44+ and to lack surface expression of heat-stable antigen. B220 and Mac-1. They had lymphoid morphology, did not phagocytose latex, and did not exhibit any precursor activity for cells of hemopoietic lineages. Their origin (intra- or extrathymic) as well as their function and physiological role, therefore, remains unknown. CD3+ T cells, both CD4+CD8- and CD4-CD8+, were present in low numbers during the first 1-2 days of life, but at post-natal day 3, a sharp increase in the accumulation of these cells occurred in both LN and spleen. By day 3 the CD4:CD8 ratio in LN was about 2:1, as in the adult. Crude estimates of the rate of export from the thymus from day 3 onwards gave values around 1% of thymocytes per day, i.e. close to our previous estimates for young adult thymus. We found no evidence of particularly high levels of emigration from the thymus during the first week after birth. Both CD4+CD8- and CD4-CD8+ T cell subsets were present in the LN as early as 1 day post-natally with CD4-CD8+ predominating among LN T cells, even though CD3+CD4+CD8- cells predominated over CD3+CD4-CD8+ cells in the thymus. By day 3 the ratio had changed to 2:1 (as in the adult). T cells, therefore, appear to emigrate from the thymus from about the time of birth with a dramatic increase around day 3 after birth.     

CD45RA expression on γδ and αβ T cells emigrating from the fetal and postnatal thymus

We have compared the expression of CD45RA on αβ and γδ T cells emigrating from the fetal and postnatal thymus. The fetal and postnatal thymus export both CD45RA⁺ and CD45RA^- T cells. The number of γδ⁺CD45RA⁺ T cells was remarkably constant regardless of stage of ontogeny or T cell maturity. Around 5--8% of γδ thymic emigrants, thymocytes and peripheral blood lymphocytes expressed CD45RA in both fetal and postnatal animals. In contrast to γδ T cells, up to one quarter of both fetal and postnatal αβ emigrants expressed CD45RA. Post-thymic maturation of CD45RA expression on αβ emigrants, which occurred both before and after birth, appeared to be antigen independent.     

Bone marrow-derived progenitor T cells convey the origin of maturational arrest from CD4+CD8+ to CD4-CD8+ thymocytes in LEC mutant rats.

A mutant strain of rats, LEC, shows a novel arrest of T cell maturation from CD4+CD8+ to CD4+CD8- but not to CD4-CD8+ cells in the thymus. Transplantation of LEC rat fetal thymuses into the subcapsule of the kidney of athymic nude rats resulted in a normal maturation of thymocytes in the thymus graft. Furthermore, both single-positive thymocytes and peripheral lymph node T cells expressed T cell receptor alpha/beta antigen, and lymph node T cells acquired the ability to produce interleukin 2 upon mitogen stimulation. Transplantation of fetal thymuses from LEA rats, which express the same major histocompatibility complex haplotype as LEC rats, into LEC rat kidney subcapsule resulted in the maturational arrest from CD4+CD8+ to CD4+CD8- cells in the thymus graft. These data strongly suggest that bone marrow-derived progenitor T cells carry the cause of maturational arrest and that the thymic stroma of LEC rats has a normal potential to nurse thymocytes.     

Characterization of constitutive and strain-dependent subsets of CD45RA+ cells in the thymus.

We have previously shown that the occurrence of CD45RA+ adult mouse thymocytes is strain-dependent, e.g. constituting approximately 0.6% in C57BL/Icrf and approximately 2.5% in BALB/c (Huby, R. and Goff, L., 1992. Eur. J. Immunol. 22:1659). Here we show that irrespective of strain, the thymus contains approximately 0.6% CD45RA+ cells which are composed of slg+ B cells (approximately 0.4%), slg- CD4-CD8- cells (< 0.2%), and CD4+ CD8+ cells (< 0.2%). In some strains an additional CD45RA+ population, representing up to approximately 2% of all thymocytes, is present and has a CD4-CD8+ phenotype. It is this CD4-CD8+CD45RA+ subset which is responsible for the observed strain difference. In BALB/c mice, this additional population comprises approximately 90% of the CD45RA+ thymic cells. They are larger than the majority of thymocytes, with a size typical of mature, single positive cells (CD4+CD8- or CD4-CD8+). Further phenotyping for co-expression of other maturation markers showed them to be distinctive; they are CD3int-hi, i.e. as bright as other CD8 single positives, which are dimmer than CD4 single positives. In addition they are CD44hi, MEL-14dim and hi, Thy-1lo, HSAlo/-, and PNAlo, suggesting them to be amongst the most mature cells in the thymus. This was corroborated by their phenotypic similarity to CD45RA+ lymph node T cells. Furthermore, in BALB/c adult thymus sections, CD45RA+ cells are localized mainly in the medulla, consistent with a mature phenotype. Comparable with most mature thymocytes, cell cycle analysis revealed this subset to be composed of resting (G0/G1) cells. The CD4-CD8+CD45RA+ cells are amongst the most mature thymocytes and yet are indistinguishable from peripheral T cell counterparts; the possibilities that they are mature thymocytes due to exit the thymus, or that they may represent recirculating peripheral T cells, are discussed.     

Cell generation within human thymic subsets defined by selective expression of CD45 (T200) isoforms

Although the thymus is the source of all mature peripheral T lymphocytes, the majority of thymocytes die intrathymically. Until recently, there has been no phenotypic marker to allow definition of the generative thymocyte lineage, thereby distinguishing those thymocytes committed to death from those which will evenually give rise to thymic emigrants. We believe that expression of the high-molecular-mass isoforms (p190, p205, and/or p220) of the leukocyte common antigen (CD45) distinguishes the thymic generative lineage from the vast majority of thymocytes expressing the low-molecular-mass isoform (p180) of CD45 and committed to die within the thymus. The thymocytes defined by their lack of CD45 p180, the low-molecular-mass isoform, comprise all thymocytes with clonogenic potential and include all major subsets defined by CD4 and CD8. We have proposed that a CD45 p180⁻ lineage exists in the human thymus and that this lineage results in the production of mature thymocytes and thymic emigrants. The objective of the present study was to determine by DNA analysis whether the degree of cell cycling in subsets of human thymus, defined by selective expression of high-molecular-mass isoforms of CD45, was sufficient to account for the generation of thymic emigrants. Multicolor immunofluorescence analysis of surface markers and 7-amino actinomycin D as well as propidium iodide staing was used to measure the DNA content of thymic subsets. Negative depletion methods were used to isolate and characterize human thymocyte subsets defined by CD45 isoform, CD3, CD4, and CD8, and subsequently to determine the cell cycle status of the isolated subsets by flow-cytometric analysis of cellular DNA content. CD3^−/lo thymocytes had a high number and CD1^−/lo thymocytes a low number of cycling cells, consistent with murine data. CD45 p 180⁻ cells, as well as the CD4⁻8⁻ and CD3⁻4⁻8⁻ subsets which express high molecular-weight CD45 isoforms, exhibited a significant number of cycling cells. Since CD45 p180- thymocytes exhibited a significant number of cycling cells, based on numerical arguments we conclude that this cycling thymocyte fraction is capable of generating the daily requirements of mature thymocytes and thymic emigrants.     

Cell death and thymic export during fetal life

In the fetus the peripheral T cell pool expands as the fetus grows, but the mechanisms that regulate T cell homeostasis during fetal life are unknown. Here, we show that the peripheral T cell pool in the sheep fetus is established by the export from the fetal thymus of twice as many CD8+ as CD4+ thymic emigrants every day. Clonal deletion of CD4+ thymocytes in the fetal thymus appeared to be more stringent than was the case for CD8+ thymocytes because only 1 in 35 single-positive CD4 (SPCD4) thymocytes was exported from the thymus whereas the majority (2/3) of the single-positive CD8 (SPCD8) thymocytes were exported from the fetal thymus each day. Furthermore, within the thymus, the number of apoptotic SPCD4 thymocytes was 40 times greater than the number of apoptotic SPCD8 thymocytes. A tissue-specific migration of CD8+ emigrants localizing in the spleen was also established in the fetus in contrast to CD4+ emigrants, which migrated randomly to spleen and LN.     

Functional maturation of recent thymic emigrants in the periphery: development of alloreactivity correlates with the cyclic expression of CD45RC isoforms.

The transition from fully developed CD4+CD8- single-positive (SP) thymocytes into fully mature recirculating peripheral T cells is both poorly understood with regard to the expression of restricted isoforms (CD45R) of the leukocyte common antigen and in terms of T cell function. The present investigation monitored the extrathymic development of CD4+CD8- SP thymocytes in euthymic recipients using allotype-marked donor cells and monoclonal antibody OX22 which recognizes an epitope on the C exon of rat CD45R. We established that donor-derived cells in the blood 1 day later bore the phenotype of the injected SP thymocytes (CD4+ Thy-1+ CD45RC-). T cells with the identical phenotype were also present in the thoracic duct lymph of uninjected rats, suggesting that the Thy-1+ CD45RC- T cells represent recent thymic emigrants (RTE) which have migrated to the periphery of their own accord. During extrathymic maturation donor-derived peripheral RTE lost Thy-1 within 3 days and expressed the CD45RC+ high molecular weight isoform by day 7; between days 8 and 14 a proportion (25%-30%) of the donor cells once again lost the high molecular weight isoform (CD45RC-). The transition of SP (CD45RC-) thymocytes to fully mature CD45RC+ CD4 T cells via intermediate peripheral RTE was accompanied at each stage by an increased ability of the maturing T cells to induce skin allograft rejection. Unexpectedly, the subsequent loss of the high molecular weight isoform, following presumed antigen encounter, was associated with a significant reduction in the ability of this Thy-1-CD45RC- subpopulation to effect graft rejection. The cyclic expression of CD45RC isoforms on both immature and mature CD4 T cells and the fact that the low molecular weight isoform was found in the periphery on both RTE (unquestionably naive) and antigen-experienced CD4 T cells, makes it unlikely that this isoform uniquely identifies memory T cells, at least in the rat.     

Definition of the thymic generative lineage by selective expression of high molecular weight isoforms of CD45 (T200)

Selective expression of CD45 isoforms distinguishes naive and memory T cells in peripheral blood. Paradoxically, although the most recent thymic emigrants are CD45R+ CD45 p180-, the majority of thymocytes are CD45 p180+. Speculating that the small subset of thymocytes selectively expressing only the high molecular weight isoforms of CD45 constitute the thymic generative lineage giving rise to peripheral T cells, we characterized the phenotypic and functional properties of CD45 p180- thymocytes. All cells bearing CD45 p180 were removed by rigorous depletion or all CD45R+ thymocytes were removed in a parallel depletion. CD45R- thymocytes were essentially the same in phenotype and CD4/CD8 subset distribution as unfractionated thymus, and dissimilar to naive peripheral blood lymphocyte (PBL) T cells. In contrast, CD45 p180- thymocytes, mainly CD45R+, were CD1- CD38- pgp 1+, corresponding closely to the phenotype of naive CD45R+ PBL T cells. This subset is enriched in CD4+ or CD8+ single positives, includes a high proportion of CD4-8- thymocytes which are predominantly CD3-, and appears to have a medullary location. Approximately 40%-50% of CD45 p180- thymocytes expressed a high density of CDw29 (4B4), which in the periphery is expressed at high density only on CD45 p180+ memory T cells and at low density on CD45R+ naive T cells. However, the expression of high density CDw29 in the absence of CD45 p180 indicates a close resemblance to fetal lymphocytes and suggests an essential role for CDw29 in both the least and the most mature of T cells. If CD45 p180- thymocytes constitute the generative lineage and CD45 p180+ cells are commited to intrathymic death, then the CD45 p180- subset should have enhanced proliferative potential. By combining depletion methods with a limiting dilution assay for clonogenic potential, we found that 100% of the clonogenic precursors present in unfractionated thymus were CD45R+ CD45 p180- cells. This indicates that the CD45 p180+ majority of thymocytes has a very limited capability for proliferation consistent with a commitment to intrathymic death. The clonogenic potential of CD45 p180- thymocytes indicates a greater functional resemblance to PBL T cells than to CD45 p180+ thymocytes. In so far as clonogenic potential in vitro reflects generative potential in vivo, expression of high molecular weight CD45 isoforms appears to define the generative thymic lineage. Our working hypothesis proposes that expression of CD45 p180 implements the mechanism for eliminating thymocytes with self-reactive receptor specificities.     

Origin and selection of peripheral CD4-CD8- T cells bearing alpha/beta T cell antigen receptors in autoimmune gld mice

We have analyzed the origin and development of unusual CD4-CD8- alpha/beta T cell receptor-positive peripheral T cells produced in large numbers by mice homozygous for the gld mutation (C3H-gld/gld). These mice may be an important model for investigating processes controlling T cell development. Bone marrow transfers demonstrated that the gld defect was intrinsic to bone marrow-derived cells. Clonal deletion of potentially autoreactive cells was observed in peripheral gld CD4-CD8-, CD4+CD8-, and CD4-CD8+ T cells, as well as mature thymocytes. This suggests that gld CD4-CD8- T cells have passed through the thymus in ontogeny and that gld autoimmunity does not result from a general defect in elimination of self-reactive thymocytes. These observations, combined with demethylation of the CD8 gene in the CD4-CD8- population, support prior expression of CD4 and/or CD8 in gld CD4-CD8- T cell ontogeny, perhaps at a CD4+CD8+ stage. Steroid sensitivity of gld thymocytes and CD4-CD8- T cells was normal. Therefore, we found no gross abnormalities in two major mechanisms of inducible cell death in the gld thymus, the clonal deletion process associated with tolerance and the steroid-inducible endogenous endonuclease thought to be involved in apoptosis of unselected thymocytes. The data suggest that if gld CD4-CD8- T cells arise via escape from normal elimination in the thymus, they must do so by a novel defect in thymic selection (perhaps related to aberrant positive signals) and/or are expanded by an extrathymic process which allows clonal deletion to occur.     

Immunosuppressant FTY720 inhibits thymocyte emigration

One major role of the thymus is to provide the peripheral immune system with mature T cells, but the mechanisms involving the cellular export are not fully understood. In this study, we examined the ability of a novel immunosuppressive reagent, FTY720, to inhibit T cell export from the thymus. Daily administration of FTY720 at a dose of 1 mg / kg resulted in a marked decrease in the number of peripheral blood T lymphocytes. In the thymus, long-term daily administration of FTY720 caused a three- to fourfold increase in the proportion of mature medullary thymocytes (CD4(+)CD8(-) and CD4(-)CD8(+)) as well as a slight decrease in the double-positive cell (CD4(+)CD8(+)) ratio. Phenotypic analysis (TCRalpha beta, H-2K(d), CD44, CD69 and CD24) revealed that these increased subsets represent possible peripheral recent thymic emigrants. High level expression of L-selectin by these subsets further suggests that they were prevented from leaving the thymus. By intrathymic labeling with fluorescein isothiocyanate, only one fourth of labeled cells could be detected in the lymph nodes and in the spleen of FTY720-treated mice compared to saline-treated control mice. Taken together, these results suggest that the immunosuppressive action of FTY720, at least in part, could be due to its inhibitory effect on T cell emigration from the thymus to the periphery.     

T cell receptor-negative thymocytes from SCID mice can be induced to enter the CD4/CD8 differentiation pathway

In order to investigate the role of T cell receptor (TcR) expression in thymocyte maturation, we have analyzed thymocytes from C.B-17/SCID mice, which are unable to productively rearrange their antigen receptor genes and fail to express TcR. Despite this defect, SCID thymocytes are functional as they produce lymphokines and proliferate in response to a variety of stimuli. Phenotypic analysis revealed that thymocyte populations from young adult SCID mice resemble thymocyte populations from normal embryonic mice in that they are large, Thy-1.2+, CD4-, CD8-, TcR- and enriched in CD5lo, IL2R+ and Pgp1+ cells. However, other TcR- populations normally present in adult mice (i.e., CD4-CD8+ cells and CD4+CD8+ cells) are absent from the thymus of TcR- adult SCID mice. To understand the basis of the developmental arrest of TcR- SCID thymocytes at the CD4-CD8- stage of differentiation, we analyzed thymi from the occasional "leaky" SCID mouse which possesses small numbers of TcR+ thymocytes. We found that the presence of TcR+ cells within a SCID thymus was invariably associated with the presence of CD4+ and/or CD8+ SCID thymocytes. Interestingly, however, the CD4+/CD8+ SCID thymocytes were not themselves necessarily TcR+. That is, emergence of SCID thymocytes expressing CD4/CD8 was tightly linked to the presence of TcR+ cells within that SCID thymus, but the SCID thymocytes that expressed CD4/CD8 were not necessarily the same cells that expressed TcR. Finally, we found that the introduction into TcR- SCID mice of normal bone marrow cells that give rise to TcR+ cells within the SCID thymus promoted the differentiation of SCID thymocytes into CD4-CD8+ and CD4+CD8+ TcR- cells. These data indicate that TcR+ cells within the thymic milieu provide critical signals which promote entry of CD4-CD8-TcR- precursor T cells into the CD4/CD8 differentiation pathway. When applied to differentiation of normal thymocytes, these findings may imply a critical role for early appearing CD4-CD8- TcR (gamma/delta)+ cells in initiating normal thymic ontogeny.     

Human intrathymic T cell differentiation

The human thymus develops early on in fetal gestation with morphologic maturity reached by the beginning of the second trimester. Endodermal epithelial tissue from the third pharyngeal pouch gives rise to TE3+ cortical thymic epithelium while ectodermal epithelial tissue from the third pharyngeal cleft invaginates and splits during development to give rise to A2B5/TE4+ medullary and subcapsular cortical thymic epithelium. Fetal liver CD7+ T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression on thymocytes of other T lineage surface molecules. Human thymic epithelial cells grown in vitro bind to mature and immature thymocytes via CD2 and CD11a/CD18 (LFA-1) molecules on thymocytes and by CD58 (LFA-3) and CD54 (ICAM-1) molecules on thymic epithelial cells. Thymic epithelial cells produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF--molecules that likely are important in various stages of thymocyte activation and differentiation. Thymocytes can be activated via several cell surface molecules including CD2, CD3/TCR, and CD28 molecules. Finally, CD7+ CD4-CD8- CD3- thymocytes give rise to T cells of both the TCRab+ and TCR gd+ lineages.     

Human thymocyte development in mouse organ cultures

A novel system to study human thymocyte development is described in which embryonic mouse thymic rudiments are seeded with human precursor cells in vitro. In these cultures human thymocytes proliferate extensively (greater than 20-fold increase in cell number) and mature, as evidenced by the accumulation of double and single positive (CD4+ and/or CD8+) cells. Data presented here suggest that the survival and ordered development of the mature human thymocytes in chimeric thymuses is dependent on human stromal elements. Immature CD4-CD8- human thymocytes failed to colonize or minimally recolonized mouse thymic lobes unless provided with high density (greater than 1.077 g/ml) human thymic cell fractions. These fractions contain multicellular complexes of epithelial/nurse cells, thymocytes, and dendritic cells/macrophages which dramatically enhanced the recolonizing capacity of purified CD4-CD8- thymocytes. The chimeric organ culture system described here provides not only a new approach for studying human T cell ontogeny but also a direct means for the future dissection of stromal interactions necessary for successful transition of precursor cells (CD4-CD8-) to immature double positive (CD4+CD8+) and mature single positive cells (CD4+ or CD8+) in the thymus.     

L-selectin expression on thymic emigrants defines two distinct tissue-migration pathways

We have studied the appearance and phenotype of recent thymic emigrants in blood, spleen and lymph nodes (LN) of neonatal lambs. Using in situ labelling of thymocytes with fluoroscein isothiocyanate (FITC), we examined the expression of the LN homing receptor L-selectin on alphabeta and gammadelta subsets of recent thymic emigrants 24 hr after labelling. There were marked differences in the proportions of CD4+, CD8+ and gammadelta T-cell receptor (TCR+) cells exported from the thymus to spleen compared to lymph nodes. Spleen was enriched in CD8+ and gammadelta TCR+ emigrants while LN were enriched in CD4+ emigrants. There were also marked differences in the expression of L-selectin by emigrants homing to spleen compared with those homing to lymph nodes. While the majority of thymic emigrants in LN expressed L-selectin, considerably fewer emigrants in spleen were L-selectin+. The presence of large numbers of CD8+ L-selectin- and gammadelta TCR+ L-selectin- thymic emigrants homing to spleen raises the possibility that unique homing receptor specificities underpin the migration of T cells to spleen as distinct from lymph nodes.     

Elevated apoptosis of peripheral T lymphocytes in diabetic BB rats

Thymocytes and peripheral lymphocytes of BioBreeding (BB) diabetes-prone (BBDP) and diabetes-resistant (BBDR) rat were analysed by fluorescence-activated cell sorter (FACS). The number of CD4- CD8-, CD4+ CD8-, CD4- CD8+ and CD4+ CD8+ subsets was not different between BBDP and BBDR rat thymocytes, whereas spleen and lymph nodes in BBDP rats undergo severe T-cell lymphopenia. Notably, mature CD4- CD8+ [T-cell receptor (TCR)-alphabeta+ and CD5+] cells are certainly present in the BBDP rat thymus, unlike some previous reports, suggesting that the differentiation of CD4- CD8+ from CD4+ CD8+ cells occurs normally in the BBDP rat thymus. As a cause of peripheral T-cell lymphopenia we suspected apoptosis of recent thymic emigrants. By FACS analysis with fluorescein isothiocyanate-labelled annexin V, elevated apoptosis was evident in BBDP rat peripheral lymphocytes. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) staining in BBDP rat splenic sections revealed that a number of TUNEL-positive cells were observed in the T-lymphocyte-rich area. From these results, we postulate that an abnormally elevated apoptosis of peripheral T lymphocytes, but not impaired thymocyte differentiation, is a cause of the peripheral T-cell lymphopenia in BBDP rats.     

Interleukin-4 induces expression of the CD45RA antigen on human thymocyte subpopulations

Interleukin-4 (IL-4) is a multifunctional lymphokine which promotes the growth and/or maturation of multiple cell types. We have examined the ability of IL-4 to promote the phenotypic maturation of subsets of human thymocytes. When cultured in serum-free medium supplemented with recombinant IL-4, a subset of immature CD3-CD45RA- human thymocytes ceased to express the CD1 common thymocyte antigen and acquired phenotypic features characteristic of relatively mature thymocytes, such as high-density expression of the CD3 antigen and de novo expression of the CD45RA isoform of the common leukocyte antigen family. These changes, which were not seen in cells cultured in medium alone, occurred over an 8-9 day period and were accompanied by a significant increase in cell size. The CD45RA+ cells that derived from these immature CD3-CD45RA- precursors were mainly CD4-CD8- or CD8+ cells, and a significant proportion of these cells expressed the T cell receptor delta chain. IL-4 also increased expression of the CD45RA antigen on the more mature CD3+ thymocyte population. However, the CD45RA+ cells derived from IL-4 stimulated CD3+ thymocyte precursors expressed either the CD4 or the CD8 antigen, and virtually all expressed alpha/beta TCR chains. Studies of cell viability and cell growth indicated that these findings were due to direct changes in the phenotype of responsive cells rather than the growth or selective survival of a small number of mature thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)