Glucocorticoid (GC) sensitivity and GC receptor expression differ in thymocyte subpopulations

Positive and negative selection steps in the thymus prevent non-functional or harmful T cells from reaching the periphery. To examine the role of glucocorticoid (GC) hormone and its intracellular receptor (GCR) in thymocyte development we measured the GCR expression in different thymocyte subpopulations of BALB/c mice with or without previous dexamethasone (DX), anti-CD3 mAb, RU-486 and RU-43044 treatment. Four-color labeling of thymocytes allowed detection of surface CD4/CD8/CD69 expression in parallel with intracellular GCR molecules by flow cytometry. Double-positive (DP) CD4+CD8+ thymocytes showed the lowest GCR expression compared to double-negative (DN) CD4-CD8- thymocytes and mature single-positive (SP) cells. DX treatment caused a concentration-dependent depletion of the DP cell population and increased appearance of mature SP cells with reduced GCR levels. GCR antagonists (RU-486 or RU-43044) did not influence the effect of DX on thymocyte composition; however, RU-43044 inhibited the high-dose GC-induced GCR down-regulation in SP and DN cells. GCR antagonists alone did not influence the maturation of thymocytes and receptor numbers. Combined low-dose anti-CD3 mAb and DX treatment caused an enhanced maturation (positive selection) of thymocytes followed by the elevation of CD69+ DP cells. The sensitivity of DP thymocytes with a GCRlow phenotype to GC action and the ineffectiveness of the GCR antagonist treatment may reflect a non-genomic GC action in the thymic selection steps.     

Low glucocorticoid receptor (GR), high Dig2 and low Bcl-2 expression in double positive thymocytes of BALB/c mice indicates their endogenous glucocorticoid hormone exposure

Several studies have shown that of the four major thymocyte subsets, the CD4/CD8 double positive (DP) thymocytes are the most sensitive to in vivo glucocorticoid hormone (GC)-induced apoptosis. Our aim was to analyse fine molecular differences among thymocyte subgroups that could underlie this phenomenon. Therefore, we characterised the glucocorticoid hormone receptor (GR) expression of thymocyte subgroups both at the mRNA and protein levels by real-time PCR and flow cytometry, and correlated these features to their apoptotic sensitivity. We also investigated the time-dependent effects of the GC agonist dexamethasone (DX) with or without GC antagonist (RU486) treatments on GR mRNA/protein expression. We also analysed the expression of two apoptosis-related gene products: dexamethasone-induced gene 2 (Dig2) mRNA and Bcl-2 protein. We found that DN thymocytes had the highest GR expression, followed by CD8 single positive (SP), CD4 SP and DP thymocytes in 4-week-old BALB/c mice, both at the mRNA and protein levels, respectively. In DP cells, the Dig2 expression was significanty higher, while the Bcl-2 expression was significantly lower than in DN, CD4 SP and CD8 SP thymocytes. Single high dose DX treatment caused time-dependent depletion of DP thymocytes due to their higher apoptosis rate, which could not be abolished with RU486 pretreatment. After a single high dose DX treatment, there was a transient, significant increase of the GR mRNA and protein level of unsorted thymocytes after 8 and 16 h, followed by a significant decrease at 24 h, respectively. The time-dependent GR expression changes after DX administration could not be inhibited by the GC antagonist RU486. Twenty-four hours after exposure to high dose DX the DN, CD4 SP and CD8 SP cells showed a significant decrease of GR mRNA and protein expression, whereas the DP thymocytes, showed no significant alteration of GR mRNA or protein expression. The kinetical analysis of GR expression and apoptotic marker changes upon single high dose GC analogue administration revealed a two-phase process in thymocytes: early events, within 4–8 h, include GR upregulation and early apoptosis induction, while the late events appear most prominently at 16–20 h, when the GR is already downregulated and apoptotic cell ratio reaches its peak, with marked DP cell depletion. The low GR, high Dig2 and low Bcl-2 expression, coupled with the absence of homologous downregulation of GR after exogenous GC analogue treatment, could contribute to the high GC sensitivity of DP thymocytes. The downregulated GR and Bcl-2 together with the upregulated Dig2 level in DP cells indicates the significance of intrathymic GC effects at this differentiation stage. Since GR expression changes and apoptotic events could not be completely inhibited by GC antagonist, we propose the involvement of non-genomic GR mechanisms in these processes.     

PNA Lectin-Based Separation of Thymocytes into Mature and Immature Subpopulations: CD4-8- Double Negative Cells Display Characteristics of PNAlo Mature Thymocytes

Cortical (immature) thymocytes are widely reported to express intermediate to high levels of receptors for the lectin, peanut agglutinin (PNA). Light-scatter studies of murine fetal thymocytes stained with PNA or anti-mouse CD4 and CD8 monoclonal antibodies indicated, however, that the most immature CD4-8- (DN) thymocyte subpopulation binds levels of PNA commonly described as PNA^lo. Evaluation of the PNA binding characteristics of fetal thymocytes negative for the CD8 antigen confirmed the existence of a major population (approximately 20% of total cells) of CD4^-8^- PNA^lo fetal thymocytes. The majority of these DN thymocytes were subsequently found to bind sub-agglutinating levels of PNA, similar to mature CD4⁺ or CD8⁺ single positive (SP) thymocytes. Given this information, an immunomodulating compound (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) known to produce a maturational delay in murine thymocytes was tested for a possible concurrent effect on thymocyte PNA lectin binding. A TCDD- induced increase in DN thymocytes was found to be paralleled by an increase of equal magnitude in PNA^lo thymocytes. Taken together, these data provide evidence that acquisition of the PNA receptor may be a maturational event occurring during the DN stage of thymocyte ontogeny. Further, these results suggest that separation of thymocytes into mature (medullary) and immature (cortical) subpopulations by PNA agglutination may result in contamination of medullary cells by the most immature (DN) subpopulation of thymocytes.     

Comparison of CDR3 length among thymocyte subpopulations: impacts of MHC and BV segment on the CDR3 shortening

Thymocytes are thought to be selected on the basis of antigen specificity between TCR and peptide-MHC (pMHC) ligands. The specificity depends primarily on extensive diversities of complementarity determining region 3 (CDR3), whose specificity is considered to be determined through thymocyte selection. We examined the CDR3 length profiles with 20 BV segments in thymocyte subpopulations from C57BL/6 (H-2(b)), C.B10 (Balb/c congenic, H-2(b)) and Balb/c (H-2(d)) mice. The CDR3 length was shorter in both CD4 single positive (SP) and CD8SP than in double positive (DP), but not altered among DP, double negative (DN) 4 and DN3 subpopulations. The CDR3 shortened more prominently in CD4SP than in CD8SP for C57BL/6 and C.B10, but the shortening was only slight for Balb/c. Although the shortening varied considerably among different BV segments, the greater shortening was observed in most BV segments for CD4SP and in several for CD8SP, in particular, the extent was the greatest in BV1, BV2, BV15, BV16, BV23 and BV26 for CD4SP, and in BV13-1 and BV29 for CD8SP. Moreover, the extent and the pattern of CDR3 shortening were basically the same among highly homologous BV segments (e.g. BV12-1 and 12-2; BV13-1, 13-2 and 13-3). These results taken together indicate that (1) the CDR3 shortening occurred between the DP to the SP stages but never earlier, that (2) there would be the MHC class preference for the CDR3 shortening, that (3) it was in part influenced by MHC haplotype, and finally that (4) the primary structure of particular BV segments would possibly affect the CDR3 length in selected thymocytes. It could be deduced from these results that the CDR3 shortening might play roles in ensuring geometrical disposition of CDRs unique to each BV segment and consequently allow CDRs to intimately interact with pMHC ligands.     

Antigen and glucocorticoid hormone (GC) induce positive selection of DP thymocytes in a TcR transgenic mouse model

Thymocyte maturation in the thymus is controlled by stromal and humoral components. Among the humoral regulators locally produced glucocorticoids (GCs) seem to have a key role in the positive selection of thymocytes. Our previous studies have shown that the administration of GCs or the stimulation through the CD3 complex can induce apoptosis of double positive (DP) cells, but the combined presence of these stimuli induces positive selection. In this work our aim was to investigate the effects of antigen exposure and synthetic GC hormone (dexamethasone, DX) administration on the selection processes of DP cells in TcR transgenic mice. In our model, AND—pigeon cytochrome c (PCC)-specific I-E^k (MHC-II) restricted Vβ3, V11 TcR expressing transgenic mice were treated with PCC, with high or low dose DX, or with PCC and DX together, followed by the analysis of total thymocyte numbers, thymocyte composition, with regard to their CD69, Vβ3 and Annexin V expression. The administration of PCC and/or DX for 2 days resulted in a decreased DP cell number and a significantly increased CD4 SP cell ratio. However, in both cases the total thymocyte numbers decreased. CD69 expression increased on both DP and CD4 SP cells after PCC and/or DX treatments. We found that after DX or combined treatment, the percentage of Annexin V positive cells increased. The ratio of Vβ3 TcR bearing DP thymocytes showed no change after DX or PCC administrations alone, but it decreased significantly after combined treatment. MHC-II bound PCC peptides in the presence of GCs enhanced the maturation of Vβ3+ DP cells into CD4 SP stage, therefore, the Vβ3− cells remained mostly in the DP immature stage. These data indicate that both antigen and low dose GC alone are capable of inducing positive selection of DP cells, but together they gave a stronger effect in promoting positive selection. From these we conclude that GCs influence the maturation and selection processes of thymocytes.     

PNA Lectin-Based Separation of Thymocytes into Mature and Immature Subpopulations: CD4–8- Double Negative Cells Display Characteristics of PNAlo Mature Thymocytes

Cortical (immature) thymocytes are widely reported to express intermediate to high levels of receptors for the lectin, peanut agglutinin (PNA). Light-scatter studies of murine fetal thymocytes stained with PNA or anti-mouse CD4 and CD8 monoclonal antibodies indicated, however, that the most immature CD4–8- (DN) thymocyte subpopulation binds levels of PNA commonly described as PNA^lo. Evaluation of the PNA binding characteristics of fetal thymocytes negative for the CD8 antigen confirmed the existence of a major population (approximately 20% of total cells) of CD4^?8^? PNA^lo fetal thymocytes. The majority of these DN thymocytes were subsequently found to bind sub-agglutinating levels of PNA, similar to mature CD4⁺ or CD8⁺ single positive (SP) thymocytes. Given this information, an immunomodulating compound (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) known to produce a maturational delay in murine thymocytes was tested for a possible concurrent effect on thymocyte PNA lectin binding. A TCDD- induced increase in DN thymocytes was found to be paralleled by an increase of equal magnitude in PNA^lo thymocytes. Taken together, these data provide evidence that acquisition of the PNA receptor may be a maturational event occurring during the DN stage of thymocyte ontogeny. Further, these results suggest that separation of thymocytes into mature (medullary) and immature (cortical) subpopulations by PNA agglutination may result in contamination of medullary cells by the most immature (DN) subpopulation of thymocytes.     

T cell receptor expression on immature thymocytes with in vivo and in vitro precursor potential

Immature CD8-CD4- double-negative (DN) thymocytes differentiate intrathymically into CD8+CD4- and CD8-CD4+ thymocytes and migrate to the periphery. This differentiation proceeds through several intermediate phenotypic changes in the expression of CD8 and CD4. We have recently established the existence of a CD8loCD4lo cell population in murine thymus that can repopulate the irradiated thymus in vivo and differentiate rapidly in vitro to CD8+CD4+ double-positive (DP) cells. The CD8loCD4lo cells score as DN upon direct cytofluorometric analysis, yet are distinct from true DN cells by various criteria. Experimental evidence strongly suggests that they are descendants of true DN in the maturation pathway. In the experiments presented here, we further characterize this CD8loCD4lo thymocyte population. Northern blot and RNA protection analysis reveal that these cells transcribe full length mRNA for the T cell receptor (TcR)alpha chain, unlike the less mature interleukin 2 receptor-positive DN thymocytes. Surface expression of the TcR-associated CD3 molecule occurs on approximately 15% of these cells at low levels characteristic of immature cells. In the course of in vitro differentiation a vast majority (approximately 80%) of these cells convert to CD8+CD4+ and significant numbers of the brightly staining DP convertants (11%-34% on day 1 and 48%-68% on day 2) express immature levels of CD3. Our results indicate that CD8lo, CD4lo cells might be the first thymic subset to rearrange TcR alpha chain genes and express TcR alpha/beta heterodimer on the surface at levels characteristic of immature cells. Furthermore, the surface expression of TcR persists on the in vitro progeny of these thymocytes.     

Hedgehog signaling regulates differentiation from double-negative to double-positive thymocyte

The hedgehog (Hh) signaling pathway is involved in the development of many tissues. Here we show that sonic hedgehog (Shh) is involved in thymocyte development. Our data suggest that termination of Hh signaling is necessary for differentiation from CD4-CD8-double-negative (DN) to CD4+CD8+ double-positive (DP) thymocyte. Shh is produced by the thymic stroma, and Patched and Smoothened (Smo), the transmembrane receptors for Shh, are expressed in DN thymocytes. A neutralizing monoclonal antibody against Shh increases differentiation of DN to DP thymocytes, and Shh protein arrests thymocyte differentiation at the CD25+ DN stage, after T cell receptor beta (TCRbeta) gene rearrangement. We show that one consequence of pre-TCR signaling is downregulation of Smo, allowing DN thymocytes to proliferate and differentiate.     

Involvement of CCR9 at multiple stages of adult T lymphopoiesis

The chemokine CCL25 is constitutively expressed in the thymus, and its receptor CCR9 is expressed on subsets of developing thymocytes. Nevertheless, the function of CCL25/CCR9 in adult thymopoiesis remains unclear. Here, we demonstrate that purified CCR9(-/-) hematopoietic stem cells are deficient in their ability to generate all major thymocyte subsets including double-negative 1 (DN1) cells in competitive transfers. CCR9(-/-) bone marrow contained normal numbers of lineage(-) Sca-1+c-kit+, common lymphoid progenitors, and lymphoid-primed multipotent progenitors (LMPP), and CCR9(-/-) LMPP showed similar T cell potential as their wild-type (WT) counterparts when cultured on OP9-delta-like 1 stromal cells. In contrast, early thymic progenitor and DN2 thymocyte numbers were reduced in the thymus of adult CCR9(-/-) mice. In fetal thymic organ cultures (FTOC), CCR9(-/-) DN1 cells were as efficient as WT DN1 cells in generating double-positive (DP) thymocytes; however, under competitive FTOC, CCR9(-/-) DP cell numbers were reduced significantly. Similarly, following intrathymic injection into sublethally irradiated recipients, CCR9(-/-) DN cells were out-competed by WT DN cells in generating DP thymocytes. Finally, in competitive reaggregation thymic organ cultures, CCR9(-/-) preselection DP thymocytes were disadvantaged significantly in their ability to generate CD4 single-positive (SP) thymocytes, a finding that correlated with a reduced ability to form TCR-MHC-dependent conjugates with thymic epithelial cells. Together, these results highlight a role for CCR9 at several stages of adult thymopoiesis: in hematopoietic progenitor seeding of the thymus, in the DN-DP thymocyte transition, and in the generation of CD4 SP thymocytes.     

Aberrant development of thymocytes in mice lacking laminin-2

In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin-2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4- CD8-) and SP (CD4+ CD8-, CD4- CD8+) populations and a significant decrease in the DP (CD4+ CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice.     

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.     

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.     

Exogenous Thy-1.1 expression as a marker for thymocyte maturation in transgenic mice.

We established a line of transgenic mice carrying the exogenous mouse Thy-1.1 gene (8.2 Kb Eco RI genomic DNA fragment). In these mice, Thy-1.1 was expressed on thymocytes but not on peripheral T cells, presumably due to the lack of cis-acting element(s) on the microinjected genomic DNA. Even in the thymus, however, while most of the CD3/TCR- thymocytes were positive for the transgenic Thy-1.1 gene expression, the CD4+ or CD8+ single positive (SP) thymocytes were composed of two groups, one Thy-1.1+ and one Thy-1.1-, suggesting that the former belongs to cells at premature stages of terminal T cell differentiation. There was no difference in the amount of CD3/TCR complexes expressed on two such SP thymocyte subsets. In the double negative (DN) thymocytes, all the CD3/TCR+ cells were Thy-1.1-. These results suggest that the maturational process of CD3+ DN thymocytes differs from that of SP thymocytes. The unique distribution of Thy-1.1+ population among CD3+ thymocytes suggests that the transgenic Thy-1.1 gene expression can serve as a useful marker to examine terminal maturation processes of CD3+ thymocytes.     

Maturation of CD4-8- alpha/beta TCR+ T cells induced by CD2-stimulation in vivo and in vitro.

Newly generated ('virgin') rat thymocytes of the immature CD4+8+ double positive (DP) subset were treated in suspension culture for 2 days with the stimulatory pair of anti-CD2 monoclonal antibodies OX-54 and OX-55. Approximately 50% of the recovered cells had downregulated CD4 and CD8 and upregulated the T cell antigen receptor (TCR). CD2-stimulated, but not control thymocytes proliferated in response to TCR plus IL-2 stimulation. In vivo, postnatal injection of OX-54/55 led to a dramatic and selective increase in functionally mature CD4-CD8- double negative (DN) alpha/beta--TCR(high) thymocytes and peripheral T cells. These findings show that CD2 stimulation can promote T cell differentiation and suggest that DN TCR(high) thymocytes can be generated from DP thymocytes via alternative pathways of T cell maturation.     

Survival versus neglect: redefining thymocyte subsets based on expression of NKG2D ligand(s) and MHC class I

BALB/c thymocytes can be divided into three distinct subsets according to the expression of a ligand for the NK activation receptor NKG2D (NKG2D-L) and the expression of MHC class I (MHC-I). The first subset (MHC-Imid/NKG2D-Lhigh or "N+") is predominately CD4+CD8+ double positive (DP), comprises approximately 35% of thymocytes in a 6-8-week-old adult and contains uncommitted cells that have neither undergone selection nor are committed to death by neglect. The second subset (MHC-Ilow/NKG2D-Llow or "M-"), also mostly DP cells, comprises approximately 50% of thymocytes and consists of cells committed to death by apoptosis, likely due to neglect. By contrast, the third subset (MHC-Ihigh/NKG2D-Llow or "M+") is largely single positive (SP), represents approximately 15% of thymocytes and mostly contains more mature cells that have undergone successful positive selection. The major advantage of the analysis is that it splits DP cells into two subpopulations, one committed to death by apoptosis and the other subjected to selection. The analysis also suggests that NKG2D-L may play a role in thymocyte development.     

Accelerated transition from the double-positive to single-positive thymocytes in G alpha i2-deficient mice

Deletion of alpha i2 subunit of heterotrimeric G proteins induces a 2-4-fold increase in the proportions of CD4 and CD8 single-positive (SP) thymocytes as compared with wild-type littermates, but how G alpha i2 is involved in thymocyte development is unknown. To determine a role for G alpha i2 in a specific developmental stage of thymocyte differentiation, we studied the ontogeny of thymocytes in G alpha i2-deficient mice. Our data show that an accelerated transition from the double-positive (DP) to SP thymocytes, rather than impairment in thymic emigration, accounts for a high proportion of the SP thymocytes in the absence of G alpha i2. Lack of G alpha i2 greatly augmented a response of thymocytes to TCR-mediated stimulation, as evidenced by enhanced proliferation of the DP thymocytes upon ligation of the TCRs. The augmented response may be the reason behind the expedited transition from the DP to SP thymocytes in the animal. In accordance with this, effects of G alpha i2 deficiency on CD8 or CD4 SP thymocyte differentiation required engagement of the TCRs with either MHC class I or MHC class II molecule. The abnormal thymocyte development resulted in an increase in positive selection, altered usage of TCR Vbeta gene, aberrant development of CD4+ CD25+ T regulatory cells and untimely thymic involution, the contribution of which to colitis development in the animal is discussed. These findings reveal a previously unappreciated role for G alpha i2 protein in clonal selection and functionality of thymocytes.     

Development of autoreactive diabetogenic T cells in the thymus of NOD mice

Type 1 diabetes results from destruction of pancreatic beta cells by beta cell-specific autoreactive T cells in the nonobese diabetic (NOD) mouse. Defects in thymic negative selection are thought to result in failure to delete potential beta cell-reactive T cells, contributing to the development of autoimmune diabetes. We investigated this possibility by comparing the deletion profile of double-positive (DP) thymocytes in NOD mice with diabetes-resistant strains of mice after anti-CD3 Ab treatment to trigger the TCR-mediated signaling pathway. We found that immature NOD CD4+CD8+ DP thymocytes have a lower activation threshold than C57BL/6 and Balb/c thymocytes. This was confirmed by showing that NOD DP thymocytes have a higher level of ERK and JNK phosphorylation. The low activation threshold of immature thymocytes resulted in rapid deletion of strongly activated immature DP thymocytes by negative selection, whereas weakly activated immature thymocytes differentiated more efficiently into CD69+CD3high DP thymocytes by positive selection. SP thymocytes, particularly CD4-CD8+ T cells that were efficiently generated from activated DP thymocytes, could induce severe insulitis and diabetes in NOD.scid mice. We conclude that the development of autoreactive diabetogenic T cells results from inordinate positive selection due to the low activation threshold of DP thymocytes in NOD mice.     

Protein phosphatase subunit G5PR that regulates the JNK-mediated apoptosis signal is essential for the survival of CD4 and CD8 double-positive thymocytes

Early T lineage cells are selected in the thymus by the specific recognition of peptide components presented by MHC molecules on the surface of thymic epithelial cells and dendritic cells. As a potential regulator of the apoptotic and survival signals, the protein phosphatase 2A-component G5PR regulates Bim phosphorylation in B-cells. Here, we studied whether G5PR is involved in the regulation of the similar apoptotic pathway for cell survival during the selection of thymocytes. T-cell-specific G5PR knockout (G5pr(-/-)) mice displayed thymic atrophy, significant reduction in thymocyte numbers, particularly a 10-fold decrease in the number of CD4 and CD8 double-positive (DP) thymocytes and few mature single-positive (SP) cells. G5pr(-/-) thymocytes exhibited normal potential of proliferation and differentiation during the transition from double-negative (DN) to DP stage, but significantly increased susceptibility to apoptosis at the DP stage. G5PR deficiency did not affect on Bim activation in thymocytes, but caused hyper-activation of JNK and Caspase-3 with augmented Fas ligand (FasL) expression, indicating that G5PR regulates the thymocyte unique apoptotic signal involved in JNK-mediated Caspase-3 activation but not in Bim activation. G5PR is essential for the survival of DP cells during thymocyte development.     

Effects of 2-acetyl-4-tetrahydroxybutyl imidazole (THI) on the thymus of rats.

2-Acetyl-4-tetrahydroxybutyl imidazole (THI), a component of the food colouring ammonia caramel, has been shown to produce suppression of cell-mediated immunity and a reduction in circulating TH and TC/S lymphocytes in rats. Accordingly in this study the effects of THI on the thymus has been investigated. THI (1 mg/kg/day) was given for up to 7 days in the drinking water to Fischer 344 rats on a vitamin B6 deficient diet. No marked change in thymus weight was found but the cellularity was marginally decreased and flow cytometric analysis of the lymphocyte subsets revealed an increase in the number of CD4+CD8- and CD4-CD8+ single positive (SP) cells and a reduction in the number of CD4+CD8+ double positive (DP) thymocytes. This reduction was in agreement with histological findings of increased numbers of pyknotic cells in the cortex, mainly engulfed by macrophages. Mitogen-induced proliferation of thymocytes prepared from THI-treated animals was increased, concordant with the gradual increase in the percentage of mature SP cells. No change in normal proliferation of thymocytes cultured in vitro, or, in proliferation in vivo, detected as 5-bromo-2'-deoxyuridine (BrdU) incorporation, was found. It is concluded that THI produced an increase in death of immature DP cells. However, THI did not affect thymocyte proliferation or their differentiation into mature SP cells in the thymus, but rather impairs their migration into the circulation. The mechanism of action of THI appears to be indirect, but THI does not act through increasing the release of adrenal corticosteroids to supra-physiological levels as the same histopathological changes in the thymus were found in adrenalectomized rats.