Thymocyte Population Research Articles

Nature of nontargeted radiation effects observed during fractionated irradiation-induced thymic lymphomagenesis in mice

Changes in the thymic microenvironment lead to radiation-induced thymic lymphomagenesis, but the phenomena are not fully understood. Here we show that radiation-induced chromosomal instability and bystander effects occur in thymocytes and are involved in lymphomagenesis in C57BL/6 mice that have been irradiated four times with 1.8-Gy γ-rays. Reactive oxygen species (ROS) were generated in descendants of irradiated thymocytes during recovery from radiation-induced thymic atrophy. Concomitantly, descendants of irradiated thymocytes manifested DNA lesions as revealed by γ-H2AX foci, chromosomal instability, aneuploidy with trisomy 15 and bystander effects on chromosomal aberration induction in co-cultured ROS-sensitive mutant cells, suggesting that the delayed generation of ROS is a primary cause of these phenomena. Abolishing the bystander effect of post-irradiation thymocytes by superoxide dismutase and catalase supports ROS involvement. Chromosomal instability in thymocytes resulted in the generation of abnormal cell clones bearing trisomy 15 and aberrant karyotypes in the thymus. The emergence of thymic lymphomas from the thymocyte population containing abnormal cell clones indicated that clones with trisomy 15 and altered karyotypes were prelymphoma cells with the potential to develop into thymic lymphomas. The oncogene Notch1 was rearranged after the prelymphoma cells were established. Thus, delayed nontargeted radiation effects drive thymic lymphomagenesis through the induction of characteristic changes in intrathymic immature T cells and the generation of prelymphoma cells.

An In vitro Model for the Study of HIV Infection of Thymocytes

The study of human thymocytes requires an appropriate matrix to enable the proper function of thymocytes. Although OP9-DL1 cells have been well established as an ideal co-culture system for the generation of T-cells from their progenitors; their ability to support a mixed population of mature human thymocytes for in vitro HIV infection studies has yet to be established. We assessed the effects of co-culturing a heterogeneous population of mature human thymocytes with a mouse derived cell line (OP9) transduced with the notch ligand delta like 1 (OP9-DL-1) and compared this to standard co-culture with human thymic epithelial cells (TEC). Co-culturing thymocytes with OP9- DL1 cells resulted in higher viability and lower apoptosis when compared to TEC co-cultures. The subset distribution and CD127 expression of thymocytes varied slightly between conditions. Thymocytes co-cultured with OP9-DL1 cells had a lower proportion of CD3+DP cells and higher proportion of SP4 cells compared to TEC co-cultures. The mature CD3+CD4+CD8- (SP4) cells also had lower levels of CD127 expression in OP9-DL1 cultures when compared to TEC. Interleukin-7 stimulation of thymocytes resulted in a decrease in CD127 expression in OP9-DL1 co-cultures, as previously observed with TEC co-cultures. Thymocytes co-cultured with OP9-DL1 tended to have higher levels of IL-7 induced STAT-5 phosphorylation and had higher levels of Interleukin-7 induced Bcl-2 expression. OP9-DL1 cells provide a microenvironment which is permissive to HIV infection in thymocytes in vitro. Co-culturing thymocytes with OP9-DL1 will facilitate the study of human thymocytes and aid in the study of exogenous stimuli or infection on individual thymocyte subsets.

Acute Immunotoxic Effects of Perfluorononanoic Acid (PFNA) in C57BL/6 Mice.

otrganic perfluorochemicals (PFCs) have become an environmental concern due to widespread detection in human blood and experimental evidence for immune, developmental, and liver toxicity. Whereas the blood concentrations of many PFCs are declining, blood levels of Perfluorononanoic Acid (PFNA) are rising in the United States. The purpose of the present studies was to determine the effects of PFNA on lymphoid organs and immune cells of C57BL/6 mice. The present study demonstrates that PFNA produces immunotoxic effects in both male and female C57BL/6 mice as evidenced by splenic atrophy, decreased splenocyte numbers, and a marked reduction in thymocyte viability. The current study also demonstrates that the effects of PFNA on different leukocyte populations are not uniform. The CD4+CD8+ double-positive thymocytes were particularly sensitive to PFNA in which the proportion of this population was >95% decreased relative to the entire CD4+ thymocyte population in PFNA-treated mice. Interestingly, PFNA also markedly increased serum levels of TNFα in response to LPS in mice. Collectively, the present studies demonstrate that PFNA decreases lymphocyte viability and alters the immune response to LPS in C57BL/6 mice.

Thymocyte apoptosis and proliferation modeling during rat thymic involution is influenced by ovarian hormones in a thymocyte subset-specific manner

The study was aimed to define the putative role of ovarian hormones in shaping thymocyte apoptosis and proliferation during thymic involution. Thymocytes from young adult and middle-aged rats ovariectomized (Ox) before puberty were examined for apoptosis and proliferation. Apoptosis and proliferation were measured in fresh thymocyte suspensions and in their 18-hour cultures, and fresh thymocyte suspensions, respectively. The thymocyte population and the major thymocyte subsets were analyzed following triple staining using anti-CD4 and anti-CD8 monoclonal antibodies and 7-AAD to label apoptotic or proliferating cells. The frequency of apoptotic cells was lower in thymocyte suspensions and cultures from Ox rats of both ages. This reflected in a diminished frequency of apoptotic cells amongst CD4+CD8+ double positive (DP) and CD4+CD8- single positive (SP), and DP cells in young and middle-aged Ox rats, respectively. Additionally, in thymocyte cultures from Ox rats the frequency of apoptotic cells amongst CD4+CD8- and CD4-CD8+ SP cells decreased with age, but increased within DP and CD4-CD8- double negative (DN) subsets, reaching in the former subset from middle-aged Ox rats higher values than in age-matched controls. The frequency of proliferating cells was also lower in Ox rats than in controls. This reflected the lower frequency of cycling cells amongst CD4+CD8- SP and CD4-CD8+ SP thymocytes in young rats, and DP and CD4-CD8+ SP thymocytes in middle-aged rats. Besides, in both SP and DP thymocyte subsets from Ox rats the frequency of proliferating cells declined with age. In conclusion, thymocyte apoptosis and proliferation exhibit ovarian hormone-dependent thymocyte subset specific alterations during thymic involution.

Histological and histometrical evidences for phenol immunotoxicity in mice.

Phenol is a common industrial and ubiquitous environmental chemical which is used to synthesize resins and plastics. Due to its anesthetic and disinfectant properties, phenol is also widely used in pharmaceutical products. Since there were no adequate data about phenol immunotoxicity, the purpose of the present study is to investigate its toxic effects on the histological structures of the lymphoid organs in the mice. A total of 80 mice were randomly distributed into one control group and three experimental groups. The control group received only distilled water, whereas experimental groups were orally administered phenol at the concentrations of 80, 180, and 320 mg/kg/day, respectively. After 28 consecutive days, tissue samples were taken and histological changes of the spleens, thymuses, adrenal glands, and lymph nodes were examined using optical microscopy. The results showed that in the phenol treated animals; splenic megakaryocyte counts increased, the diameter of the splenic follicles decreased, the thymocyte population in both cortex and medulla reduced, the thickness of the reticular layers of adrenal gland increased and lymphatic cells populations in the lymph node were reduced, significantly (P < 0.01). Also, remarkable histological changes were noted in the various lymphatic organs of the treated mice. Overall, present findings give some histological evidences that selected qualitative and quantitative parameters of the lymphatic organs were significantly altered by phenol administration. In conclusion, the significant decreases of the immune cell populations together with histological alterations in the immunocompetent organs of the mice exposed to phenol indicate the immunosuppressive and immunotoxic properties of this chemical material.

Accumulation of tissue macrophages and depletion of resident macrophages in the diabetic thymus in response to hyperglycemia-induced thymocyte apoptosis

AimsWe investigated the dynamics and morphology of thymus macrophages in response to thymus involution caused by hyperglycemia. Thymus is an organ affected early and dramatically after the onset of diabetes, losing most of the thymocyte populations but diabetes's impact on the components of the thymus stroma is largely unknown. MethodsRats were injected with streptozotocin and thymus weight, body weight, and glycemia were measured at various time points. The dynamics and morphology of macrophages in the diabetic thymus were investigated by histology, immunohistochemistry, qPCR, electron microscopy and flow cytometry. ResultsIn hyperglycemic animals the involuting thymus is gradually infiltrated by tissue macrophages (ED1-positive) and depleted of resident macrophages (ED2-positive). While ED1 positive macrophages are scattered in both cortex and medulla the ED2 positive ones are limited to the cortex and cortico-medullary junction. CD4+CD11b+macrophages also accumulate. The TUNEL reaction that detects the degradation of the DNA from apoptotic thymocytes in the macrophages is enhanced. The thymic macrophages enlarge and accumulate lipid vacuoles and apoptotic bodies. qPCR measurements of the expression of macrophage markers showed a persistent increase in the diabetic thymus after the injection of streptozotocin. ConclusionsThymus involutes rapidly and persistently after the onset of hyperglycemia because of the elevated apoptosis in the thymocytes. Tissue macrophages accumulate in the thymus and the resident macrophages decrease. This results in an overall increase in macrophage activity in the diabetic thymus in response to the elevated apoptosis of thymocytes produced by hyperglycemia.

Expansion of Highly Suppressive FOXP3+ Regulatory T Cells from Pediatric Thymic Tissue

Introduction: Infant heart transplant (HTx) recipients have better graft survival than patients transplanted at older ages. Nonetheless, due to lifelong need for therapy, infant HTx recipients can expect to carry an immunosuppressive heavier burden, resulting in substantial morbidities from adverse drug effects. Development of a cellular therapy using regulatory T cells (Tregs) to suppress graft-directed immune responses would greatly benefit these infants. A major challenge for such a therapy, however, is generating a large quantity of stable, highly suppressive Tregs. Infants undergoing HTx usually have thymectomy in order to gain adequate exposure of the retrosternal operative field. We investigated the potential of explanted thymic tissue as a source for isolation and expansion of highly suppressive CD4+CD25+CD127lowFOXP3+ Tregs. Methodology: Thymic tissue (n=3) was obtained from thymectomy during pediatric cardiac surgery and thymocytes were recovered through mechanical dissociation. FOXP3+ cells were isolated by automated magnetic cell separation of CD4+CD127lowCD25+ thymocytes. CD4+CD127low-depleted cells were used as controls. Cells were expanded for two weeks by stimulation with anti-CD3, IL-2, rapamycin and CD32+ L cells. FOXP3 and intracellular cytokine staining was performed to define characteristics of expanded cells. The suppressive capacity after expansion was determined by co-culturing the expanded cells with PKH-labeled anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMC) and analyzing proliferative responses by flow cytometry. Results: CD4+CD25+CD127lowFOXP3+ cell frequency within the total thymocyte population ranged from 2.8 to 7.9%. Isolated CD4+CD25+CD127low cell populations were 65-84% positive for FOXP3. After two weeks of culture, we observed a 4 to 40-fold expansion of CD4+CD25+CD127low cells with >95% viability; 0.4 to 25-fold expansion was observed for control cells with 49-88% viability. The expanded CD4+CD25+CD127low cells were >95% FOXP3+ and produced no IL-2 or IFN-γ, whereas control cells were < 14% FOXP3+ and 58-65% produced IFN-γ. Moreover, in contrast to control cells, expanded CD4+CD25+CD127lowFOXP3+ cells were highly potent suppressors, efficiently suppressing the frequency of proliferating PBMC >80% even at a 1:20 ratio of Tregs:PBMC. Conclusion: In this preliminary study, we showed that highly suppressive FOXP3+ Tregs can be expanded from CD4+CD25+CD127low T cells isolated from pediatric thymic tissue, indicating that explanted thymuses may be a potential source for isolation and expansion of Tregs for cellular therapy. Future work includes defining the stability of expanded Tregs and their suppressive capacity of alloantigen immune responses.

Molecular and Cellular Mechanisms of Mycobacterium avium-Induced Thymic Atrophy

Thymic atrophy has been described as a consequence of infection by several pathogens and shown to be induced through diverse mechanisms. Using the mouse model of Mycobacterium avium infection, we show in this study that the production of NO from IFN-γ-activated macrophages plays a major role in mycobacterial infection-induced thymic atrophy. Our results show that disseminated infection with a highly virulent strain of M. avium, but not with a low-virulence strain, led to a progressive thymic atrophy. Thymic involution was prevented in genetically manipulated mice unable to produce IFN-γ or the inducible NO synthase. In addition, mice with a selective impairment of IFN-γ signaling in macrophages were similarly protected from infection-induced thymic atrophy. A slight increase in the concentration of corticosterone was found in mice infected with the highly virulent strain, and thymocytes presented an increased susceptibility to dexamethasone-induced death during disseminated infection. The administration of an antagonist of glucocorticoid receptors partially reverted the infection-induced thymic atrophy. We observed a reduction in all thymocyte populations analyzed, including the earliest thymic precursors, suggesting a defect during thymic colonization by T cell precursors and/or during the differentiation of these cells in the bone marrow in addition to local demise of thymic cells. Our data suggest a complex picture underlying thymic atrophy during infection by M. avium with the participation of locally produced NO, endogenous corticosteroid activity, and reduced bone marrow seeding.

Immature CD4+CD8+ Thymocytes Are Preferentially Infected by Measles Virus in Human Thymic Organ Cultures

Cells of the human immune system are important target cells for measles virus (MeV) infection and infection of these cells may contribute to the immunologic abnormalities and immune suppression that characterize measles. The thymus is the site for production of naïve T lymphocytes and is infected during measles. To determine which populations of thymocytes are susceptible to MeV infection and whether strains of MeV differ in their ability to infect thymocytes, we used ex vivo human thymus organ cultures to assess the relative susceptibility of different subpopulations of thymocytes to infection with wild type and vaccine strains of MeV. Thymocytes were susceptible to MeV infection with the most replication in immature CD4+CD8+ double positive cells. Susceptibility correlated with the level of expression of the MeV receptor CD150. Wild type strains of MeV infected thymocytes more efficiently than the Edmonston vaccine strain. Thymus cultures from children ≥3 years of age were less susceptible to MeV infection than cultures from children 5 to 15 months of age. Resistance in one 7 year-old child was associated with production of interferon-gamma suggesting that vaccination may result in MeV-specific memory T cells in the thymus. We conclude that immature thymocytes are susceptible to MeV infection and thymocyte infection may contribute to the immunologic abnormalities associated with measles.

Bacterial Cellulose: Long-Term Biocompatibility Studies

The bacterial cellulose (BC) secreted by Gluconacetobacter xylinus is a network of pure cellulose nanofibres which has high crystallinity, wettability and mechanical strength. These characteristics make BC an excellent material for tissue-engineering constructs, noteworthy for artificial vascular grafts. In this work, the in vivo biocompatibility of BC membranes produced by two G. xylinus strains was analyzed through histological analysis of long-term subcutaneous implants in the mice. The BC implants caused a mild and benign inflammatory reaction that decreased along time and did not elicit a foreign body reaction. A tendency to calcify over time, which may be related to the porosity of the BC implants, was observed, especially among the less porous BC-1 implants. In addition, the potential toxicity of BC nanofibres — obtained by chemical-mechanical treatment of BC membranes — subcutaneously implanted in mice was analysed through bone marrow flow cytometry and histological analyses. At 2 and 4 months post-implantation, the nanofibres implants were found to accumulate intracellularly, in subcutaneous foamy macrophages aggregates. Moreover, no differences were observed between the controls and implanted animals in thymocyte populations and in B lymphocyte precursors and myeloid cells in the bone marrow.

Notch signaling expands a pre-malignant pool of T-cell acute lymphoblastic leukemia clones without affecting leukemia-propagating cell frequency.

NOTCH1 pathway activation contributes to the pathogenesis of over 60% of T-cell acute lymphoblastic leukemia (T-ALL). While Notch is thought to exert the majority of its effects through transcriptional activation of Myc, it also likely has independent roles in T-ALL malignancy. Here, we utilized a zebrafish transgenic model of T-ALL, where Notch does not induce Myc transcription, to identify a novel Notch gene expression signature that is also found in human T-ALL and is regulated independently of Myc. Cross-species microarray comparisons between zebrafish and mammalian disease identified a common T-ALL gene signature, suggesting that conserved genetic pathways underlie T-ALL development. Functionally, Notch expression induced a significant expansion of pre-leukemic clones; however, a majority of these clones were not fully transformed and could not induce leukemia when transplanted into recipient animals. Limiting-dilution cell transplantation revealed that Notch signaling does not increase the overall frequency of leukemia-propagating cells (LPCs), either alone or in collaboration with Myc. Taken together, these data indicate that a primary role of Notch signaling in T-ALL is to expand a population of pre-malignant thymocytes, of which a subset acquire the necessary mutations to become fully transformed LPCs.

Prenatal cadmium exposure alters postnatal immune cell development and function

Cadmium (Cd) is generally found in low concentrations in the environment due to its widespread and continual use, however, its concentration in some foods and cigarette smoke is high. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports of immunomodulatory effects of prenatal exposure to Cd. This study was designed to investigate the effects of prenatal exposure to Cd on the immune system of the offspring. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl2 (10ppm) and the effects on the immune system of the offspring were assessed at two time points following birth (2 and 7weeks of age). Thymocyte and splenocyte phenotypes were analyzed by flow cytometry. Prenatal Cd exposure did not affect thymocyte populations at 2 and 7weeks of age. In the spleen, the only significant effect on phenotype was a decrease in the number of macrophages in male offspring at both time points. Analysis of cytokine production by stimulated splenocytes demonstrated that prenatal Cd exposure decreased IL-2 and IL-4 production by cells from female offspring at 2weeks of age. At 7weeks of age, splenocyte IL-2 production was decreased in Cd-exposed males while IFN-γ production was decreased from both male and female Cd-exposed offspring. The ability of the Cd-exposed offspring to respond to immunization with a S. pneumoniae vaccine expressing T-dependent and T-independent streptococcal antigens showed marked increases in the levels of both T-dependent and T-independent serum antibody levels compared to control animals. CD4+FoxP3+CD25+ (nTreg) cell percentages were increased in the spleen and thymus in all Cd-exposed offspring except in the female spleen where a decrease was seen. CD8+CD223+ T cells were markedly decreased in the spleens in all offspring at 7weeks of age. These findings suggest that even very low levels of Cd exposure during gestation can result in long term detrimental effects on the immune system of the offspring and these effects are to some extent sex-specific.

Role for Ets-2Thr-72 Transcription Factor in Stage-specific Thymocyte Development and Survival

Interference of Ras signaling deregulates thymocyte development in mouse models. However, the role of Ets-2, a transcription factor that is phosphorylated on a critical threonine residue (Thr-72) by the Ras/MAPK pathway in thymocyte development, has not been defined. Transgenic mice overexpressing a phosphomutant Ets-2 (T72A) in the thymus displayed reduced thymus size associated with a 60-80% reduction in thymocyte populations. The transgenic mice exhibited a 20-fold increase in a c-Kit(+) CD4(+) CD8(+) CD3(-) population and a 5-fold increase in a unique CD5(low) population associated with a partial developmental block at the DN2-DN3 stage of thymocytes. Transgenic thymocytes exhibited increased apoptosis, and overexpression of Bcl-2 rescued the hypocellularity and associated thymocyte developmental block in double transgenic mice. The observed defects in these mice are not dependent on Ets-1 expression. These studies implicate for the first time a stage-specific Ets-1-independent regulatory role for Ets-2 in early thymocyte development and survival.

Glut1-mediated glucose transport regulates HIV infection

Cell cycle entry is commonly considered to positively regulate HIV-1 infection of CD4 T cells, raising the question as to how quiescent lymphocytes, representing a large portion of the viral reservoir, are infected in vivo. Factors such as the homeostatic cytokine IL-7 have been shown to render quiescent T cells permissive to HIV-1 infection, presumably by transiently stimulating their entry into the cell cycle. However, we show here that at physiological oxygen (O(2)) levels (2-5% O(2) tension in lymphoid organs), IL-7 stimulation generates an environment permissive to HIV-1 infection, despite a significantly attenuated level of cell cycle entry. We identify the IL-7-induced increase in Glut1 expression, resulting in augmented glucose uptake, as a key factor in rendering these T lymphocytes susceptible to HIV-1 infection. HIV-1 infection of human T cells is abrogated either by impairment of Glut1 signal transduction or by siRNA-mediated Glut1 down-regulation. Consistent with this, we show that the susceptibility of human thymocyte subsets to HIV-1 infection correlates with Glut1 expression; single-round infection is markedly higher in the Glut1-expressing double-positive thymocyte population than in any of the Glut1-negative subsets. Thus, our studies reveal the Glut1-mediated metabolic pathway as a critical regulator of HIV-1 infection in human CD4 T cells and thymocytes.

Impact of mouse pregnancy on thymic T lymphocyte subsets

It has been reported that fetal lymphoid progenitor cells are acquired during gestation and are able to develop in the maternal mouse thymus into functional T cells. Moreover, previous pregnancies increase the number of fetal cells in the mother. In the present study, we investigated whether mouse pregnancy induces changes in T lymphocyte subsets in the maternal thymus. We determined the T lymphocyte subsets in two allogeneic cross-breedings, namely CBA/J×BALB/c (normal) and CBA/J×DBA/2 (abortion prone), and investigated the effects of the age and parity of the female, as well as pregnancy outcome, on thymocyte populations. In addition, hormonal effects were evaluated in a syngeneic combination (CBA/J×CBA/J). We found that during pregnancy both hormonal and allogeneic stimuli induced a reduction in the CD4(+)CD8(+) subset with an increase in the CD4(+)CD8(-) population. Only young females of the normal combination exhibited an increase in the CD4(-)CD8(+) population. All young mice showed an increase in CD4(+)CD25(+)FoxP3(+) T cells. Interestingly, the γδT thymus pool was increased in all females of the normal allogeneic pregnancy only, suggesting the participation of this pool in the observed beneficial effect of multiparity in this cross-breeding. Our results demonstrate that allogeneic pregnancies induce important variations in maternal thymocyte subpopulations depending on the age of the female and the male component of the cross-breeding.

CD248 expression on mesenchymal stromal cells is required for post-natal and infection-dependent thymus remodelling and regeneration

The role of mesenchymal stromal cells (MSCs) in regulating immune responses in the thymus is currently unclear. Here we report the existence and role of a MSC population in the thymus that expresses the pericyte and MSC marker CD248 (endosialin). We show using a CD248-deficient mouse model, that CD248 expression on these cells is required for full post-natal thymus development and regeneration post-Salmonella infection. In CD248−/− mice the thymus is hypocellular and regeneration is poorer, with significant loss of all thymocyte populations. This identifies the requirement of CD248 to maintain optimal thymic cellularity post-partum and infection.

Toward Quantifying the Thymic Dysfunctional State in Mouse Models of Inflammatory Bowel Disease

Inflammatory bowel disease is characterized by a number of immunological alterations, not the least in the T-cell compartment. Numerous animal models of colitis have revealed aberrant thymocyte dynamics associated with skewed thymocyte development. The recent advancements in quantitative methods have proposed critical kinetic alterations in the thymocyte development during the progression of colitis. This review focuses on the aberrant thymocyte dynamics in Gαi2-deficient mice as this mouse model provides most quantitative data of the thymocyte development associated with colitis. Herein, we discuss several dynamic changes during the progression of colitis and propose a hypothesis for the underlying causes for the skewed proportions of the thymocyte populations seen in the Gαi2-deficient mice and in other mouse models of colitis.

Identification of Novel Immunoregulatory Molecules in Human Thymic Regulatory CD4+CD25+ T Cells by Phage Display

Thymic CD4+CD25+ cells play an important role in immune regulation and are continuously developed in the thymus as an independent lineage. How these cells are generated, what are their multiple pathways of suppressive activity and which are their specific markers are questions that remain unanswered. To identify molecules involved in the function and development of human CD4+CD25+ T regulatory cells we targeted thymic CD4+CD25+ cells by peptide phage display. A phage library containing random peptides was screened ex vivo for binding to human thymic CD4+CD25+ T cells. After four rounds of selection on CD4+CD25+ enriched populations of thymocytes, we sequenced several phage displayed peptides and selected one with identity to the Vitamin D Receptor (VDR). We confirmed the binding of the VDR phage to active Vitamin D in vitro, as well as the higher expression of VDR in CD4+CD25+ cells. We suggest that differential expression of VDR on natural Tregs may be related to the relevance of Vitamin D in function and ontogeny of these cells.

Modulation of microRNA expression in human T-cell development: targeting of NOTCH3 by miR-150

AbstractOntogenesis of T cells in the thymus is a complex process whose molecular control is poorly understood. The present study investigated microRNAs involved in human thymocyte differentiation by comparing the microRNA expression profiles of thymocytes at the double-positive, single-positive CD4+ and single-positive CD8+ maturation stages. Microarray analysis showed that each thymocyte population displays a distinct microRNA expression profile that reflects their developmental relationships. Moreover, analysis of small-RNA libraries generated from human unsorted and double-positive thymocytes and from mature peripheral CD4+ and CD8+ T lymphocytes, together with the microarray data, indicated a trend toward up-regulation of microRNA expression during T-cell maturation after the double-positive stage and revealed a group of microRNAs regulated during normal T-cell development, including miR-150, which is strongly up-regulated as maturation progresses. We showed that miR-150 targets NOTCH3, a member of the Notch receptor family that plays important roles both in T-cell differentiation and leukemogenesis. Forced expression of miR-150 reduces NOTCH3 levels in T-cell lines and has adverse effects on their proliferation and survival. Overall, these findings suggest that control of the Notch pathway through miR-150 may have an important impact on T-cell development and physiology.

Arachidonic acid accumulates in the stromal macrophages during thymus involution in diabetes

Diabetes is a debilitating disease with chronic evolution that affects many tissues and organs over its course. Thymus is an organ that is affected early after the onset of diabetes, gradually involuting until it loses most of its thymocyte populations. We show evidence of accumulating free fatty acids with generation of eicosanoids in the diabetic thymus and we present a possible mechanism for the involution of the organ during the disease. Young rats were injected with streptozotocin and their thymuses examined for cell death by flow cytometry and TUNEL reaction. Accumulation of lipids in the diabetic thymus was investigated by histology and electron microscopy. The identity and quantitation of accumulating lipids was done with gas chromatography-mass spectrometry and liquid chromatography. The expression and dynamics of the enzymes were monitored via immunohistochemistry. Diabetes causes thymus involution by elevating the thymocyte apoptosis. Exposure of thymocytes to elevated concentration of glucose causes apoptosis. After the onset of diabetes, there is a gradual accumulation of free fatty acids in the stromal macrophages including arachidonic acid, the substrate for eicosanoids. The eicosanoids do not cause thymocyte apoptosis but administration of a cyclooxygenase inhibitor reduces the staining for ED1, a macrophage marker whose intensity correlates with phagocytic activity. Diabetes causes thymus involution that is accompanied by accumulation of free fatty acids in the thymic macrophages. Excess glucose is able to induce thymocyte apoptosis but eicosanoids are involved in the chemoattraction of macrophage to remove the dead thymocytes.