CD45 isoform expression during T cell development in the thymus.

Various isoforms of leukocyte common antigen, or CD45, are expressed differentially on T cells at different stages of development and activation. We report studies on CD45 isoform expression on various subsets of human T cells using two- and three-color flow cytometry and cell depletion. Bone marrow cells that were depleted of CD3+ and HLA-DR+ cells were CD45RA-RO-. The earliest CD3-CD4-CD8-CD19- thymocytes were CD45RO- with 20%-30% CD45RA+ cells. The most prominent population of CD4+CD8+ double-positive thymocytes were CD45RA-RO+. Even the CD4+CD8+ blasts were greater than 90% CD45RO+. About 80% of single-positive thymocytes (CD4+CD8- or CD4-CD8+) were also CD45RO+. Only 4.3% of CD4+ and 18% of CD8+ single-positive thymocytes were CD45RA+. In contrast, cord blood T cells which represent the stage that immediately follows single-positive thymocytes, contained 90% CD45RA+ cells. Thus, in terms of CD45 isoform expression, single-positive thymocytes are more like double-positive cells than cord blood T cells. These results suggest the following sequence of CD45 isoform switching during T cell development: CD45RA-RO- or RA+RO- (double-negative thymocytes)----RA-RO+ (double-positive and most single-positive thymocytes)----RA+RO- (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.     

Morphological analysis of the cellular interaction between thymocytes and a thymic stromal cell line (TEL-2)

In a previous study (Nakashima et al., Eur. J. Immunol., 20: 47-53, 1990), a cloned stromal cell line TEL-2 was established from Balb/c mouse thymus. Incubation of thymocytes with TEL-2 cells resulted in the selective elimination of CD4 and CD8 double-positive thymocytes from the culture. In the present report, both phase-contrast and scanning electron microscopes were used to examine, at various time intervals, TEL-2 cells cocultivated with thymocytes in order to elucidate the kinetic sequence of their cellular interaction. The thymocytes attached to the TEL-2 cell surface were more numerous at early times (30 min to 1 h), and their number decreased gradually with time. In contrast, the thymocytes that migrated into the TEL-2 cell layers were less abundant at early times, their number increasing with time thereafter. Destruction of the regular arrangement of TEL-2 cells was found at later than 1 h, suggesting active cellular interaction. The thymocytes adherent to the TEL-2 cell surface were found to be of various shapes and often showed variable profiles, e.g., extending small cytoplasmic processes along the surface of TEL-2 cells or appearing ameboidal. A remarkable feature of the TEL-2 cells was that they formed numerous "round spaces" at the surface of the TEL-2 cell layers. The thymocytes were often located around "round spaces," and some were seen migrating into TEL-2 cell layers through these round spaces. In addition, complementary examinations by transmission electron microscopy revealed that the internalization of thymocytes into TEL-2 cells occurs inside the TEL-2 cell layers after migration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two differential pathways from double-negative to double-positive thymocytes.

Murine thymocytes are divided into four major populations on the basis of expression of CD4 and CD8 antigens. The bulk of evidence favours the view that CD4-CD8- cells can develop into CD4-CD8+ and CD4+CD8- cells via the CD4+CD8+ stage in the thymus. However, CD4-CD8+ and CD4+CD8- thymocyte subsets contain not only CD3+ mature cells but also CD3- immature cells, which seem to be intermediate cells between CD4-CD8- and CD4+CD8+ cells. Here we demonstrate mouse strain differences in the proportion of immature single-positive thymocyte subsets in thymus at the steady or developing state. In C3H mice, immature CD4+CD8- is dominant in proportion over CD4-CD8+ in foetal thymus and in donor-derived thymocytes at an early stage of bone marrow transplantation. On the other hand, immature CD4-CD8+ is dominant over CD4+CD8- during T-cell development in the case of B10.BR mice. An intermediate pattern was shown in the case of F1 mice. Both of these immature single-positive subsets gave rise to double-positive cells after 24 hr culture. These results suggest that there exist two distinct differential pathways; one is from CD4-CD8- cells to CD4+CD8+ cells via CD4-CD8+ cells, and another is via CD4+CD8- cells, and that an application of the 'CD8 pathway' or 'CD4 pathway' seems to be genetically destined by BM-derived cells but not by thymic stromal cells.     

Developmental regulation of bcl-2 expression in the thymus.

An important factor in shaping the T-cell receptor (TcR) repertoire during thymocyte development is the susceptibility of double-positive (CD4+ CD8+) thymocytes to induction of apoptosis (negative selection) when the TcR is engaged by 'self'-antigens. Recent evidence has suggested that this susceptibility to apoptosis may be influenced by the expression of bcl-2, a proto-oncogene known to increase the resistance to apoptosis in various cell systems. Using a semi-quantitative polymerase chain reaction (PCR) technique in conjunction with staged embryonic material and purified thymocyte subpopulations we have investigated patterns of bcl-2 expression during normal T-cell development. Our results show that while bcl-2 alpha gene expression is readily detectable in immature CD3-CD4-CD8- thymocytes and in mature single-positive TcRhi cells, it is drastically reduced in TcR negative double-positive (CD3- CD4+ CD8+) cortical thymocytes of intermediate maturity. Careful mapping of bcl-2 alpha re-expression in relation to the onset of TcR expression within the population of embryonic thymocytes indicates that bcl-2 alpha is up-regulated as soon as TcR molecules are expressed on the surface of CD4+ CD8+ thymocytes. Therefore, thymocytes susceptible to apoptosis on TcR ligation express bcl-2 alpha mRNA suggesting that changing levels of bcl-2 expression are unlikely to be the only determinant regulating susceptibility to apoptosis in the thymus. The possible implications of these changes in bcl-2 expression regarding other facets of thymocyte development will be discussed.     

Characterization of the subset of immature thymocytes which can undergo rapid in vitro differentiation

Recently, we reported that thymocytes expressing the CD8 molecule on their surface can give rise to CD4+CD8+ double-positive and CD4+ single-positive progeny following intrathymic transfer into an irradiated host mouse. Thymcoytes expressing a high density of CD8, referred to as CD8hi, and those expressing a low density of the molecule, CD8lo, were both able to differentiate in vivo. In this study we examined the ability of these CD8+ thymocytes populations and of CD4-CD8- double-negative thymocytes to change their phenotype during brief in vitro culture. CD8+ thymocytes were prepared by anti-CD4 plus complement lysis followed by positive selection of the survivors on anti-CD8-coated plates. After 16 h of culture, greater than 60% of CD8+ thymocytes became double-positive. Both CD8hi and CD8lo cells were able to show this in vitro change: about 30% of the former and about 80% of the latter became double-positive. In contrast to this, double-negative thymocytes which had been depleted of cells expressing low densities of CD8 did not show such a phenotypic conversion in vitro. Further panning experiments suggested that all of the CD8+ thymocytes actually express a low surface density of the CD4 molecule which is undetectable in our cytofluorometric assays.     

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.     

Differential Effects of Interleukin-2 and Interleukin-7 on the Induction of CD4 and CD8 Expression by Double-Negative Human Thymocytes*

Expression of ceil surface CD4 (in the absence of CD3/T cell receptor) characterizes an early stage of intrathymic T cell development. Here, we investigated the appearance of CD4 and CD8 expression on highly purified CD4^?8^? double-negative human thymocytes in response to interleukin (IL)-7 and IL-2. While IL-7 preferentially promoted the appearance of CD4 single-positive and CD4⁺8⁺ double-positive thymocytes, I1-2 primarily induced CD8 single-positive thymocytes. A significant fraction of CD4 single-positive cells generated from double-negative thymocytes via IL-7 lacked cell surface CD3 expression. In contrast, the majority of CD8 single-positive cells generated from double-negative thymocytes via IL-2 coexpressed CD3. We conclude that IL-7 and IL-2 exert differential effects on the differentiation of early human T cell progenitors.     

3, 3', 4, 4'-Tetrachlorobiphenyl Inhibits Proliferation of Immature Thymocytes in Fetal Thymus Organ Culture

The environmental pollutant 3, 3', 4, 4'-tetrachlorobiphenyl (TCB) leads to thymic atrophy and immuno-suppression, the former possibly causing the latter. TCB binds lo the cytosolic aryl-hydrocarbon receptor (AhR) and transforms it into a DNA-binding state. The development of fetal thymocyles is severely affected by TCB and other AhR-binding xenobiotics, leading to a skewed pattern of thymocyte maturation stages. Murine thymocyte proliferation after exposure to TCB was studied in fetal thymus organ culture (FTOC). C57BL/6 fetus thymic lobes from day 15 of gestation were explanted and grown for 2, 4, 6. and 8 days in organ culture in the presence or absence of 3.3 μM TCB. Subsets of thymocytes were defined by CD4 and CD8 surface markers, and their cell cycle was analysed by DNA staining with 7-amino-actinomycin D (7-AAD). Exposure of fetal thymi in vitro to 3.3 μM TCB significantly reduced the total number of thymocytes. and fewer thymocytes were in S/G₂M phase. The inhibition of cell proliferation induced by TCB treatment affected mainly the CD4⁻ CD8⁻ (double-negative, DN) and CD4⁻ CD8⁺ (single-positive, SP) subsets, and these inhibition appeared mainly in more immature thymocytes, i. e. DNCD3⁻ and CD8⁺CD3⁻ subpopulations, whereas no effect of TCB on CD4⁺CD8⁺ (double-positive, DP) cell proliferative activity was observed. Analysis of the relation of cell proliferation and development of subsets in differentiating fetal ihymocytes suggests that TCB enhanced thymocyte differentiation into mature CD8⁺ cells.     

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.     

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.     

Clonal deletion of self-Mls-reactive thymocytes at the early stage in H-2-compatible but Mls-disparate radiation chimeras.

Clonal deletion of T cells capable of recognizing both host-type Mls and donor-type Mls occurred in the peripheral mature T-cell pool in radiation bone marrow chimeras of two H-2-compatible Mls-disparate strain combinations of AKR/J(H-2k,Thy-1.1,Mls-1a) and C3H/He(H-2k,Thy-1.2,Mls-1b). In order to determine further the stage at which the clonal deletion occurs in thymus, we examined the kinetics of thymocytes bearing V beta 6 capable of recognizing Mls-1a in both C3H/He----AKR/J and AKR/J----C3H/He chimeras. An almost complete replacement from host-derived cells to donor-derived cells occurred by Day 21 after reconstitution in both chimeras. At this stage, CD4+CD8+ double-positive thymocytes contained an appreciable number of cells that expressed V beta 6 on their surface, albeit at low intensity, whereas CD4 or CD8 single-positive thymocytes which expressed a high density of V beta 6 were virtually abolished in both C3H----AKR and AKR----C3H chimeras on Day 21. These results suggest that clonal deletion of self-Mls-reactive T cells begins at an early stage when the thymocytes interact with the early appearing donor-derived haemotopoietic cells and relatively radio-resistant host-derived cells in thymus of radiation bone marrow chimeras.     

Interaction of merosin (laminin 2) with very late activation antigen-6 is necessary for the survival of CD4+ CD8+ immature thymocytes

The laminin alpha2-chain is a component of merosin, a member of the laminin family molecules, which is mainly expressed in the basement membranes of striated muscle. It is known that laminin alpha2 gene (lama2) null mutant mice (dy3k/dy3k) exhibit congenital muscular dystrophy (CMD). Because the laminin alpha2-chain is also expressed in the thymus, the role of merosin in the thymus was examined. In association with the onset of muscular dystrophy, CD4+ CD8+ double-positive (DP) thymocytes disappear by apoptotic cell death, while CD4+ CD8- or CD4- CD8+ thymocytes remain. In order to study the mechanisms leading to the selective death of DP cells in the absence of merosin, the role of the interaction between very late activation antigen-6 (VLA-6), a candidate merosin ligand in the thymus, and merosin was examined. The in vitro survival of thymocytes from normal mice was maintained by the addition of either anti-VLA-6 monoclonal antibodies (mAbs) or merosin. Furthermore, when the normal thymocytes were cultured on thymic epithelial cell lines, viable DP cell recoveries on wild-type epithelial cells were better than on cells from null mutant mice. The results suggest that DP cells are more sensitive to an uncharacterized apoptotic death signal, and that survival is supported by the interaction between VLA-6 and merosin.     

Possible involvement of glial cell line-derived neurotrophic factor and its receptor,GFRalpha1, in survival and maturation of thymocytes

The glial cell line-derived neurotrophic factor (GDNF) and its receptors (GFR) play important roles in the promotion of survival and differentiation of central and peripheral neuronal populations. We show that GFRalpha1, a component of GDNF receptor, was expressed in thymocytes at an early stage of thymocyte-development and was involved in the survival of thymocyte precursors. GFRalpha1and GDNF were expressed in thymus, but not in spleen or lymph nodes in adult mice. During embryonic thymocyte development, GFRalpha1 was predominantly expressed on thymocytes from days 14.5 to 16.5 of gestation, and thereafter its expression gradually declined. In adult thymus, GFRalpha1 was expressed only on CD4(-)CD8(-) double-negative (DN) thymocytes, but not on CD4(+)CD8(+) double-positive or single-positive thymocytes. It was strongly expressed on RAG2(-/-) thymocytes arrested at the DN stage, and ist expression was reduced during their differentiation after in vivo anti-CD3 antibody stimulation. Additionally, fetal thymocyte precursors grew in serum-free medium of the fetal thymus organ culture system in the presence of recombinant GDNF (rGDNF), while the cells without rGDNF died. These results suggested that GDNF/GFRalpha1 are involved in the survival of both the nervous system and DN immature thymocytes.     

Proliferation and differentiation of immature thymocytes induced by a thymic stromal cell clone

The monolayer of our established thymic stromal cell clone (MRL104.8a) exhibited the capacity to maintain immature double-negative thymocytes. Such capacity was also expressed by a factor produced by the MRL104.8a monolayer. This factor designated as thymic stroma-derived T cell growth factor (TSTGF) was found to be distinct from IL-2 or IL-4 but similar to IL-7 of the previously described cytokines from the functional and molecular aspects. The MRL104.8a monolayer also exerted its differentiation-promoting effect on double-negative thymocytes. Culture for one day of purified double-negative thymocytes on the monolayer resulted in the induction of an appreciable per cent of CD3-4-8+ cells. This differentiation could also be induced by a semipurified TSTGF sample but not by recombinant IL-7, suggesting that the MRL104.8a cells elaborate a factor(s) responsible for initiating the differentiation of double-negative cells in addition to the growth-promotion factor identical or closely related to IL-7. When the culture period of double-negative thymocytes was extended to 2 or 3 days, an appreciable number of double-positive (CD4+8+) and single-positive (CD4+8-) cells were generated on the MRL104.8a monolayer. Thus, these observations provide strong support for the proposition that a specialized thymic stromal component plays an essential role in the intrathymic T cell development in the context of T cell growth and differentiation.     

Cross-linking of the T cell antigen receptor interferes with the generation of CD4+8+ thymocytes from their immediate CD4-8+ precursors

The majority of thymocytes are immature cells co-expressing the surface markers CD4 and CD8. About two thirds of these cells also express the T cell antigen receptor (TcR), though at a level distinctly lower than found on mature T cells. The direct precursors of these "double-positive" thymocytes are cycling cortical blast cells of the CD4-8+ phenotype. Using a new monoclonal antibody to a constant determinant of the rat TcR alpha/beta, it is shown here that (a) about 50% of these CD8 "single-positive" committed precursor cells already express the TcR alpha/beta, though at very low levels, (b) during short-term suspension culture in medium supplemented only with fetal calf serum they not only acquire CD4 but also TcR alpha/beta levels characteristic of CD4,8 "double-positive" thymocytes, and (c) cross-linking of the TcR during culture inhibits the acquisition of the CD4 antigen in the majority of these cells.     

Patterns of dual lymphocyte development in co-cultures of foetal thymus and lymphohaemopoietic cells from young and old mice.

Patterns of lymphocyte development in the thymus were analysed, focusing on newly emigrating bone marrow (BM) and resident thymic cells. We co-cultured foetal (Day 15 of gestation) thymic explants (FT, C57BL/Ka, Thy-1.1), with BM cells from young (2-3 months) or old (24 months) syngeneic, Thy-1 congenic (C57BL/6J, Thy-1.2) mice. When the FT was severely depleted [treated with either 2-deoxyguanosine (dGua) or exposed to an irradiation dose of 20 Gy] BM-type T lymphocytes were dominant, regardless of BM donor age. When the FT was only partially depleted of its proper lymphoid cells (by exposure to 10 Gy), the lymphocytes which developed were from both BM and FT origins, yet the level of donor-type thymocytes from the young mice was higher than that of the old. Under these conditions the proportion of FT-derived double-positive CD4+ CD8+ (DP) cells was higher, and that of single-positive CD4- CD8+ cells was lower, than in the BM-derived cells, irrespective of the BM donor age. The proportions of old BM-derived DP cells were lower than in the young. Co-cultures of thymus cells from young and old mice with partially depleted FT explants resulted in similar proportions of CD4/CD8 subsets from both donor and FT origins, with the exception that in the presence of old-thymus cells there was an increase in the level of FT-type CD4- CD8+ cells. Patterns of T-cell differentiation in the thymus thus seem to be determined by newly emigrating cells and the resident thymocytes.     

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.     

Evidence for major alterations in the thymocyte subpopulations in murine models of autoimmune diseases

Thymocytes can be divided into four major subpopulations: CD4+CD8+ (double-positive), CD4-CD8- (double-negative), CD4+CD8- (CD4+) and CD4-CD8+ (CD8+) cells. Recent studies have shown that T-cell development in the thymus progresses as: CD4-CD8(-)----CD4+CD8(+)----CD4+ or CD8+ cells. In the present study we investigated these and other subpopulations of thymocytes in autoimmune MRL(-)+/+, MRL-lpr/lpr, C57BL/6-lpr/lpr, BXSB and NZB mice before (1-month old) and after (4-6-months old) the onset of lymphadenopathy and autoimmune disease. All the autoimmune strains at one month of age and other H-2, sex and age-matched controls (C3H, DBA/2, and C57BL/6) demonstrated normal proportions of thymocyte subsets with approximately 75% double-positive cells, 5-7% double-negative cells, 11-15% CD4+ cells and 3-5% CD8+ cells. By 4-6 months of age, MRL(-)+/+ mice demonstrated a moderate increase in double-negative cells (approximately 13%) and a decrease in double-positive cells (approximately 46%). Interestingly, in the presence of the lpr gene, as seen in MRL-lpr/lpr mice, the double-negative cells increased to approximately 47% and the double-positive cells decreased to approximately 16%. In contrast, 4-6-month-old C57BL/6-lpr/lpr mice failed to demonstrate any alterations in the thymocyte subsets thereby suggesting that background genes, in addition to the lpr gene, played a role in the thymocyte differentiation. BXSB male mice with severe lymphadenopathy behaved very similarly to MRL-lpr/lpr mice, inasmuch as their thymus contained approximately 48% double-negative cells and only approximately 8% double-positive cells. In contrast to MRL-lpr/lpr and BXSB strains, NZB mice at 6 or 10 months of age had normal composition of thymocyte subsets. In MRL and BXSB animals, although there was a significant increase in CD4+ cells (approximately 23-33%), due to a consequent increase in CD8+ cells (approximately 11%), the ratio of CD4+:CD8+ cells remained 2-3:1, similar to that seen in normal mice. Furthermore, using the J11d marker expressed by the majority of the double-negative and all double-positive thymocytes but not by mature functional T cells, we confirmed the above findings and demonstrated further that MRL-lpr/lpr mice at 4-6 months of age had an increased percentage of J11d- double-negative cells and a decrease in J11d+ double-negative cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reactive oxygen species induced by the deletion of peroxiredoxin II (PrxII) increases the number of thymocytes resulting in the enlargement of PrxII-null thymus

In the thymus, CD4+ or CD8+ single-positive (SP) thymocytes develop and mature by positive and negative selection or undergo "death by neglect". CD4+ or CD8+ SP then circulate to other lymphoid tissues. We have investigated the role of reactive oxygen species (ROS) in thymocyte development using peroxiredoxin II (PrxII)-null mice. The level ofROS in PrxII-null thymocytes is higher than that in wild-type mice. Deletion of the PrxII gene leads to enlargement of the thymus in young (9 weeks) and old (64 weeks) mice. The increased number ofthymocytes in PrxII-null thymus is related to reduced hypodiploid cell formation. For mice on a normal diet, the ratio of SP to double-positive (DP) thymocytes in thymus of PrxII-null mice is lower than that in wild-type mice. After food restriction, which leads to increased ROS production, this ratio becomes much higher in PrxII-null thymus. The amount of apoptosis, induced by food restriction orby the injection of dexamethasone, is consistently lower in PrxII-null thymocytes than in wild-type thymocytes. In the presence of low serum concentrations, PrxII-deleted T cells proliferate more vigorously after stimulation with concanavalin A. Phytohemagglutinin- or OKT3-stimulated proliferation of human peripheral blood mononuclear cells is also higher in the presence of lower serum concentrations. Collectively, the results suggest for the first time that thymocyte maturations and proliferations are regulated by ROS levels induced by the deletion of PrxII gene in vivo.     

Developmental status of CD4-CD8+ and CD4+CD8- thymocytes with medium expression of CD3

In normal mice, more than 10% of thymocytes in the CD4+CD8- and CD4-CD8+ single-positive (SP) subsets express a medium level of CD3 on the cell surface. However, the fate of CD3medium cells is unclear. The CD3medium SP subpopulations might contain (i) cells in an immature stage of the pathways leading to CD3high cells, (ii) cells in developmental pathways that do not lead to CD3high cells, or (iii) cells that have been negatively selected. We found that sorted CD3medium CD4+CD8- thymocytes from adult mice up-regulated CD3 to high levels in reaggregation thymus organ culture. Unlike their CD3high counterparts, CD3medium CD4+CD8- thymocytes were unable to undergo chemotaxis towards the chemokines CCL19 and CCL21. CD3medium thymocytes of both CD4+CD8- and CD4-CD8+ subsets were also considerably more responsive than CD3high SP cells to apoptotic signals induced in vitro by ligation of CD95 (Fas/APO-1) or by dexamethasone. In both SP subsets, a higher frequency of thymocytes expressing forbidden Vbeta+ T cell receptors reactive with endogenous mammary tumor virus superantigens was found in CD3medium subpopulations than in CD3high subpopulations. These findings argue that the CD3medium SP thymocyte subpopulations contain apoptosis-susceptible precursor cells of CD3high SP cells and are subject to negatively selecting pressures.