Alterations in thymocyte subpopulations in Down's syndrome (trisomy 21)

To correlate the histologically observed thymic abnormalities with the cellular immunodeficiency found in Down's syndrome (DS), thymus fragments and thymocyte suspensions from 14 noninstitutionalized DS subjects were studied. Histologic examination and immunohistologic studies using an anticluster of differentiation (CD) 1 monoclonal antibody showed a contracted cortex due to cortical thymocyte depletion. When DS unselected thymocytes were phenotyped, a significant reduction of CD3-, CD1-, CD4-, and CD8-positive cells was found as compared to controls. To evaluate if the deficient expression of these markers was due to the reduction of thymocyte subsets identifiable on the basis of their physical properties, we separated DS unselected thymocytes into 10 fractions by continuous Percoll density gradient centrifugation. DS thymuses were almost completely devoid of high density thymocytes. Since in normal thymus, these cells correspond to small CD1+, CD4+, CD8+, and 50% CD3+ cortical thymocytes, their absence may explain the unrestricted reduction of markers on DS unfractionated thymocytes. Furthermore DS thymuses appeared to be enriched in CD1+ first fraction (Fr1) low density thymocytes of the Percoll gradient. Fr1 CD1+ cells constitute the main spontaneously proliferating pool in normal human thymus. When the spontaneous proliferating activity of DS Fr1 was compared to that of the control, a significant reduction was observed. This reduction associated with the absence of high density thymocytes, with the reduction of cells expressing alpha- and beta-chains of the T cell receptor and in conclusion with the lymphocyte depletion, suggests that in DS thymuses there is a deficient expansion of immature T cells resulting in a reduction of the various thymocyte subpopulations, including the thymocyte pool able to differentiate into functionally mature T cells.     

Developmentally Regulated Expression of the Transmembrane Adaptor Protein TRIM in Fetal and Adult T Cells

TRIM is a recently identified transmembrane adaptor protein which is exclusively expressed in T cells and natural killer (NK) cells. In peripheral blood T cells TRIM has been reported to coprecipitate, comodulate, and cocap with the T-cell receptor (TCR), suggesting that it is an integral component of the TCR/CD3/zeta complex. Here we investigate the expression of TRIM mRNAs and proteins in developing thymocytes. Two splicing isoforms with open reading frames are observed, namely a full length (TRIM) and a truncated version (DeltaTM-TRIM). The latter lacks the extracellular and transmembrane domains as well as the first 10 cytoplasmic aminoacids and is significantly expressed only as mRNA in early fetal thymocytes. TRIM mRNA is detected in all mainstream thymocyte subsets in adult mice. TRIM protein, in contrast, first appears in the DN2 (CD44+ CD25+) subset of adult double negative (DN) cells. In fetal thymocyte development, TRIM mRNA is seen from dg 14.5 onwards whereas TRIM protein appears first on dg 16.5. In contrast to the adult, the TRIM protein was seen in a subset of fetal DN1 cells. In fetal and adult thymocytes, TRIM protein expression was highest in DN2, DN3 (CD44-25+) and in DP cells, compatible with a functional role at or around phases of thymic selection.     

Double-negative thymocyte subsets in CD3′ chain-deficient mice: Absence of HSA+CD44−CD25− cells

Double-negative (DN) thymocyte subsets were examined in mice deficient in the CD3′ chain (ζ −/−). The HSA ⁺CD44⁻CD25⁻ subset was found to be missing, and DN thymocytes seemed to differentiate directly from HSA⁺CD25⁺CD44⁻cells to double-positive (DP) cells. When fetal thymic ontogeny was examined, we found a marked difference between ζ −/− embryos and heterozygous littermates from embryonic day 17.5, in terms of CD25, CD4 and CD8 expression, and thymus size. The ζ −/− thymocytes failed to down-regulate CD25 and to expand exponentially. The cell cycle status of adult thymocyte subsets indicated that although the HSA ⁺CD25⁻CD44⁻ subset was missing, the CD25⁺ DN population contained normal numbers of cycling cells, and the CD25⁺ DP cells (which were not detectable in normal mice) contained 5–10% cells in G2/M + S. Taken together these data suggest that the CD3′ chain might have a specific role in the control of proliferation of DN thymocytes during T cell development. Our data clearly show that one can dissociate the signal for a CD25⁺ DN cell to differentiate (which occurs in the absence of CD3′), from a signal to proliferate and from loss of cell surface CD25.     

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.     

Neuropilin 1 and CD25 co-regulation during early murine thymic differentiation

Neuropilin 1 (NP1) is a receptor for both semaphorin and vascular endothelial growth factor expressed by subpopulations of neuronal and endothelial cells. In the immune system, NP1 is present on dendritic and regulatory T cells. Here, we show that NP1 is expressed in the murine thymus, starting on day 12.5 of gestation. In the adult, NP1 is mainly expressed by CD4(-)CD8(-) double negative cells, CD4+CD8+ double positive cells, and CD4+CD25+ regulatory T cells but barely detected in single CD4+ and CD8+ positive thymocytes. Within the CD4(-)CD8(-)CD3(-) (triple-negative, TN) immature cells, NP1 expression starts in TN3 (CD44(-)CD25+) and increases in TN4 (CD44(-)CD25(-)) cells. In order to study the role of NP1 in thymocyte differentiation, we generated mice in which the np1 gene is selectively disrupted in the T-cell lineage. The mutant mice display normal thymocyte, peripheral, conventional and CD4+CD25+Foxp3+ regulatory T-cell populations. However, we observe a down-regulation of the CD25 expression between the TN3 and TN4 stages that is (i) correlated to increased expression of NP1 in control mice and (ii) altered in mutant mice, suggesting that NP1 is co-regulated with CD25 during early immature thymocyte differentiation.     

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)     

The Wnt signaling antagonist Kremen1 is required for development of thymic architecture

Wnt signaling has been reported to regulate thymocyte proliferation and selection at several stages during T cell ontogeny, as well as the expression of FoxN1 in thymic epithelial cells (TECs). Kremen1 (Krm1) is a negative regulator of the canonical Wnt signaling pathway, and functions together with the secreted Wnt inhibitor Dickkopf (Dkk) by competing for the lipoprotein receptor-related protein (LRP)-6 co-receptor for Wnts. Here krm1 knockout mice were used to examine krm1 expression in the thymus and its function in thymocyte and TEC development. Krm1 expression was detected in both cortical and medullary TEC subsets, as well as in immature thymocyte subsets, beginning at the CD25+CD44+ (DN2) stage and continuing until the CD4+CD8+(DP) stage. Neonatal mice show elevated expression of krm1 in all TEC subsets. krm1(-/-) mice exhibit a severe defect in thymic cortical architecture, including large epithelial free regions. Much of the epithelial component remains at an immature Keratin 5+ (K5) Keratin 8(+)(K8) stage, with a loss of defined cortical and medullary regions. A TOPFlash assay revealed a 2-fold increase in canonical Wnt signaling in TEC lines derived from krm1(-/-) mice, when compared with krm1(+/+) derived TEC lines. Fluorescence activated cell sorting (FACS) analysis of dissociated thymus revealed a reduced frequency of both cortical (BP1(+)EpCAM(+)) and medullary (UEA-1(+) EpCAM(hi)) epithelial subsets, within the krm1(-/-) thymus. Surprisingly, no change in thymus size, total thymocyte number or the frequency of thymocyte subsets was detected in krm1(-/-) mice. However, our data suggest that a loss of Krm1 leads to a severe defect in thymic architecture. Taken together, this study revealed a new role for Krm1 in proper development of thymic epithelium.     

Reversal of the abnormal development of T cell subpopulations in the thymus of autoimmune MRL-lpr/lpr mice by a polyamine biosynthesis inhibitor.

Polyamines--putrescine, spermidine, and spermine--are a group of positively charged organic molecules that are present in all living cells. They are important regulators of cell growth and differentiation, but the precise mechanism of their action is not known. Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines. Recent studies demonstrated that down-regulation of polyamine biosynthesis by irreversible inhibition of ODC with difluoromethylornithine (DFMO0 is a novel therapeutic approach for the treatment of murine lupus in autoimmune MRL-lpr/lpr mice. Since murine lupus in this strain is associated with a major alteration in thymic T cell subopulations, we questioned whether abnormal polyamine biosynthesis contributes to aberrant T cell maturation in the thymus of MRL-lpr/lpr mice. Thymocytes were analyzed for cell surface markers, CD4 and CD8 by 2-color flow cytometry using their respective monoclonal antibodies. The proportion of thymocyte subsets in disease-free mice (8-10 week of age) was approximately 72% double positive (DP; CD4+CD8+) cells, 5-7% double negative (DN; CD4-CD8-) cells, 11-16% CD4+ cells and 7-8% CD8+ cells. At 14 weeks of age, a stage of clinical disease expression, thymocytes were marked by the presence of approximately 40% DN cells and approximately 25% DP cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Role of the aryl hydrocarbon receptor in thymocyte emigration in vivo

The aryl hydrocarbon receptor (AHR) is a ligand-dependent member of the PAS-bHLH-family of nuclear receptors. Anthropogenic ligands include environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Over-activation of the AHR causes thymus atrophy and immunosuppression. Signaling via the AHR changes the thymocyte differentiation program at several checkpoints, in particular within the CD4-CD8- double-negative (DN) thymocyte subset. Here, we show that AHR over-activation led to the preferential emigration of DN thymocytes to the periphery and accumulation in the spleen. Some of these recent thymic emigrants (RTE) had a novel "activated immature" phenotype (CD3-TCRbeta-CD25+/intCD44-CD45RB+/intCD62L+CD69- cells). Gene expression profiling of DN RTE revealed 15 genes that were up-regulated more than threefold by TCDD, including the S100A9 gene. Exposure of S100A9 null mice to TCDD showed a role for this protein in AHR-mediated thymic egress.     

Thymopoiesis following chronic blockade of beta-adrenoceptors

The present study was undertaken in order to further clarify putative role of the adrenergic innervation in the regulation of the intrathymic T-cell maturation. For this purpose adult male DA rats were subjected to either 4-day- or 16-day-long propranolol treatment (0.40 mg propranolol/100 g/day, s.c.) and the expression of CD4/8/TCRalphabeta on thymocytes, as well as thymocyte proliferative and apoptotic index, was assessed in these animals by flow cytometric analysis. Propranolol treatment, in spite of duration, increased both the thymocyte proliferative and apoptotic index (vs. respective vehicle-treated controls). In 4-day-treated animals the thymus cellularity and thymus weight remained unaltered, while in 16-day-treated rats the values of both of these parameters were reduced (since increase in the thymocyte apoptotic index overcame that in the proliferative index). The treatments of both durations affected the thymocyte phenotypic profile in a similar pattern, but the changes were more pronounced in rats exposed to the treatment of longer duration. The relative proportion of the least mature CD4-8- double negative (DN) TCRalphabeta- cells was increased, those of thymocytes at distinct differentiational stages on the transitional route to the CD4+8+ double positive (DP) TCRalphabetalow stage decreased (all subsets of TCRalphabeta- in both groups of rats, and those with low expression of TCRalphabeta in rats subjected to 16-day-long treatment) or unaltered (all subsets of TCRalphabetalow cells in 4-day-treated rats). Furthermore, the percentage of CD4+8+ DP TCRalphabetalow cells was significantly elevated, as well as those of the most mature CD4+8- TCRalphabetahigh and CD4-8+ TCRalphabetahigh cells (the increase in the percentage of former was much more conspicuous than that of the latter), while the relative proportion of their direct detectable precursors (CD4+8+ DP TCRalphabetahigh) was reduced. Thus, the present study: i) further supports notion of pharmacological manipulation of adrenergic action as an efficient means in modulation of the T-cell development, and hence T-cell-dependent immune response, and ii) provides more specific insight into T-cell maturation sequence point/s particularly sensitive to beta-adrenoceptor ligand action.     

Estrogen Blocks Early T Cell Development in the Thymus

PROBLEM: Pregnancy and estrogen are known to suppress B lymphopoiesis as well as lead to thymic involution in the mouse. Additionally, estrogen deficiency by oophorectomy reportedly causes a selective increase in the B220+ B cells in the murine bone marrow. The purpose of this study was to determine if estrogens played a regulatory role in T cell development. METHODS: The first experimental group consisted of 5–6-week-old Balb/c mice that received subcutaneous pellets of placebo, estriol, estradiol, or progesterone. The thymus glands were examined 2–4 weeks after treatment. The second group consisted of 6-week-old Balb/c mice who underwent either bilateral oophorectomy or a sham procedure. Two weeks after the surgery, extensive phenotypic characterization of the thymus and spleen cells was performed by flow cytometry using monoclonal antibodies to surface markers of T cell subsets. RESULTS: Estrogen treatment causes a dramatic reduction of thymic size and cellularity. All defined T cell subsets of CD4 and CD8 were reduced, with a disproportionate loss of CD4+CD8+ double positive cells. Examination of the triple negative (CD3-CD4-CD8-) subset revealed a striking loss of TN developmental progression of the early precursor cells. Based on the expression of CD44 (pgp-1) and CD25 (IL-2Rα) markers, the TN thymic compartment was composed almost entirely of the earliest population (CD44+, CD25-), with the remaining maturational stages (CD44+, CD25+; CD44-, CD25+; CD44-, CD25-) depleted. In contrast, all T cell developmental stages in the thymus were found to be in normal proportions in the oophorectomized mice, with no differences in the splenic T and B cell subsets. CONCLUSIONS: The study demonstrates that estrogen but not progesterone blocks T cell development in the thymus. However, contrary to our expectation, estrogen deprivation by oophorectomy does not enhance T cell development.     

Age-related changes in the cellular composition of the thymus in children

BACKGROUND: T-cell development in the thymus is an extensively studied subject, mainly in mice. Nevertheless, the normal composition and cell numbers of the noninvoluted human thymus are largely unknown. OBJECTIVE: We aimed to gain insight into age-related changes in different thymic subpopulations and to provide reference values for the distribution of thymocyte subsets. The composition of the normal thymus may serve as a reference for thymi in pathological conditions and may aid diagnoses of immunodeficiency diseases. METHODS: Thymic lobes of 70 children (58 immunologically normal and 12 diseased), ranging in age from 8 days to 8 years old, were studied by 4-color flow-cytometric analysis. Detailed staining and gating strategies allowed us to dissect small subsets, including immature CD4(-) CD8(-) populations and thymic B, natural killer, and T-cell receptor gammadelta + cells. RESULTS: We demonstrate that distribution of thymocyte subsets changes with age and correlates with age-related fluctuations of T-lymphocyte counts in peripheral blood. Thymi of children 3 to 6 months old appear to be the most active: they have high numbers of total thymocytes, the highest percentage of double-positive cells, and large numbers of CD34 + progenitors in their thymi. Furthermore, we show that the human thymus is a site for B-cell development, because all B-cell progenitor stages that can be found in the bone marrow are also present in the thymus. CONCLUSION: We conclude that T-cell development in children is a dynamic process, answering the demands of a maturing and expanding immune system.     

Expression of CD11b (Leu15) antigen on CD3+, CD4+, CD8+, CD16+ peripheral lymphocytes. Estimation of CD3+8+11b+ and CD3+4-8-11b+ T-cell subsets using a single laser flow cytometer.

CD11b (Leu15) epitope is expressed on 20-30% of peripheral blood lymphocytes, including CD16+ large granular lymphocytes and CD8+ cells. This study confirms that 30% of CD8+ lymphocytes and virtually all CD16+ NK cells from healthy subjects express this determinant. In parallel, our data show that various proportions of CD3+4-8-, TCR-delta cytotoxic T lymphocytes and occasionally CD4+ lymphocytes subsets could also express this epitope. The CD8+11b+ phenotype is associated with suppression of T-cell proliferative response and has been extensively used to characterize suppressor T lymphocytes. Since about 25% of CD8 lymphocytes are non-T (CD3-) and express the CD16 NK antigen (CD8+16+3-), the expression of CD11b was also studied on CD8+3+ T-cell and CD8+16+ NK-cell subsets. To this end, we developed three methods using a flow cytometer equipped with a single laser and two fluorescence detectors. Results showed that T CD8+3+11b+ and NK CD8+16+11b+ lymphocytes account for 30% and 70% of CD8+11b+ cells respectively. Consequently, the CD8+3+11b+ phenotype would be more specific for suppressor T lymphocytes than the total CD8+11b+ phenotype which includes high proportions of CD16+ NK cells.     

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.     

Scheduled kinetics of cell proliferation and phenotypic changes during immature thymocyte generation

Precursor CD4-CD8- (DN) thymocytes rearrange their TCR-beta genes, and only those which succeed in beta-selection subsequently expand and differentiate into immature CD4+CD8+ (DP) thymocytes. The cell subsets corresponding to the successive steps of this transition can be defined in terms of CD44 and CD25 expression. We partially synchronized the differentiation process by eliminating cycling cells with the anti-mitotic agent demecolcine. Using in vivo pulse labeling with bromodeoxyuridine, we determined the order of entry into DNA synthesis of the different DN and transitory (CD4-/lo CD8+) cell subsets. Two independent proliferation phases were identified. The first cells to enter the cell cycle were CD44-CD25lo, and CD4/CD8/TCR-/BrdU four-color staining showed that they all expressed a low density of the TCR-beta chain, an element of the pre-TCR (the TCR-alpha locus is still in germ-line configuration at this stage). Cycling of CD44+CD25+ cells was detected later, and no starting point was observed at the CD44-CD25hi stage. CD8 expression was immediately detectable in cycling cells, but they took 24 h to reach the DP stage. The study of TCR-Calpha-deficient mice showed that beta gene rearrangement occurred once proliferation had ceased at the DP stage, and that it had no influence on the DN-DP transition. These data show that precursor thymocytes undergo two independent waves of expansion, and that the second wave is restricted to cells capable of pre-TCR expression.     

Loss of Bim results in abnormal accumulation of mature CD4-CD8-CD44-CD25- thymocytes

The process of thymopoiesis is tightly regulated by a series of selection events which ensure that only functional T-lymphocytes directed against foreign antigens are exported into the periphery. The adaptive immune response largely depends on the regulation of thymocyte development, and thymocytes which fail selection in the thymus are removed by apoptosis. However, the roles of specific apoptotic proteins in early T-lymphocyte development are poorly understood. Here, we report a novel function for Bim in thymocyte development. There is an accumulation of thymocytes in Bim(-/-) mice that lack expression of CD4, CD8, CD44, and CD25 but express CD3 and TCRbeta. Further, the CD4(-)CD8(-)CD25(-)CD44(-)CD3(+)TCRbeta(+) thymocytes are smaller and do not proliferate. These data suggest that these thymocytes are mature DN thymocytes that may have down-regulated the expression of CD4 and CD8. The DN thymocyte phenotype in Bim(-/-) mice is unaffected by the additional loss of Bak or Bax and is similar to the thymic phenotype in mice lacking both Bak and Bax. These data demonstrate that Bim functions to ensure the proper homeostasis of mature thymocytes during selection and thymic export.     

Delineation of chicken thymocytes by CD3-TCR complex, CD4 and CD8 antigen expression reveals phylogenically conserved and novel thymocyte subsets.

To further define the relationship between thymocyte subsets and their developmental sequence, multi-parameter flow cytometry was used to determine the distribution of the CD3-TCR complex and the accessory molecules CD4 and CD8 on chicken thymocytes. As in mammals, adult thymocytes could be subdivided into CD3-, CD3lo, and CD3hi staining populations. CD4 and CD8 distribution on such populations revealed the presence of CD3-CD4+CD8- and CD3-CD4-CD8+ thymocytes, putative precursors to CD4+CD8+ cells, detectable in the adult and at high frequency during ontogeny. Of particular interest was the existence of CD3lo expression on CD4+CD8- and CD4-CD8+, and in some instances, on CD4-CD8- thymocytes. Such phenotypes are not easily detectable in the mammalian thymus but were readily observed in both adult and embryonic chicken thymus from 16 days of embryogenesis. Further analysis of the TCR lineage of these CD3lo cells revealed that they were essentially all of the alpha beta TCR type. Mature CD3hi thymocytes were found within the CD4+CD8+ and CD4+CD8- and CD4-CD8+ subsets. Both alpha beta and gamma delta TCR lineage thymocytes were detected within all CD4- and CD8-defined subsets, thus identifying novel thymocyte subsets in the chicken thymus, namely alpha beta TCR+CD4-CD8- and gamma delta TCR+ CD4+CD8- cells. Hence, this analysis of chicken thymocytes, while confirming the phylogenically conserved nature of the thymus, has revealed novel T cell subsets, providing further insight into the complexity of mainstream thymocyte maturation pathways.