Effect of Ionizing Radiation on the Expression of p16, CyclinDI and CDK4 in Mouse Thymocytes and Splenocytes

Objective To investigate the effect of ionizing radiation on the expression of p16, CyclinDl, and CDK4 in mouse thymocytes and splenocytes. Methods Fluorescent staining and flow cytometry analysis were employed for the measurement of protein expression. Results In time course experiments, it was found that the expression of p16 protein was significantly increased at 8, 24, and 48 h for thymocytes (P<0.05, P<0.01, and P<0.05, respectively) and at 24 h for splenocytes (P<0.05) after whole body irradiation (WBI) with 2.0 Gy X-rays. However, the expression of CDK4 protein was significantly decreased from 8 h to 24 h for thymocytes (P<0.05,P<0.01) and from 8 h to 72 h for splenocytes (P<0.05-P<0.01). In dose effect experiments, it was found that the expression of p16 protein in thymocytes and splenocytes was significantly increased at 24 h after WBI with 1.0, 2.0, and 4.0 Gy (P<0.05-P<0.01), whereas the expression of CDK4 protein was significantly decreased with 2.0Gy for thymocytes (P<0.05) and 0.5-6.0 Gy for splenocytes (P<0.05-P<0.01). Results also showed that the expression of CyclinDl protein decreased markedly in both thymocytes and splenocytes after exposure. Conclusion The results indicate that the expression of p 16 protein in thymocytes and splenocytes can be induced by ionizing radiation, and the p16-CyclinD1/CDK4 pathway may play an important role for G1 arrest of thymocytes induced by X-rays.

Effects of an immunosuppressive active component of Periplocae Cortex (Periploca sepium Bge.) on positive selection of thymocytes in vitro

Objective:To determine the effect of an immunosuppressive active component (periploside A) isolated from the stem bark of Periplocae Cortex (Periploca sepium Bge.),a Chinese medicinal herb used in the treatment of rheumatoid arthritis for centuries in China,on positive selection of thymocytes in vitro.Methods:Female C57BL/6 mice at 6 weeks of age were housed in specific pathogen-free conditions.Double-positive thymocytes from C57BL/6 mice were induced into positive selection in vitro with or without periploside A treatment.Cell viability and expression of CD69,CD4,and CD8 were analyzed by flow cytometry.Results:Flow cytometric examination of thymocyte populations revealed that the percentage of CD8+ single-positive thymocytes was decreased by periploside A upon differentiation induced by an anti-CD3 antibody.However,the percentage of CD4+ single-positive thymocytes was decreased by periploside A upon differentiation induced by phorbol 12-myristate 13-acetate/ionomycin.Expression of CD69 plays a major role in prohibiting differentiation of thymocytes.Treatment with periploside A decreased CD69 expression in thymocytes.Conclusion:These results demonstrate that periploside A influences positive selection of thymocytes in vitro.

X-ray irradiation selectively kills thymocytes of different stages and impairs the maturation of donor-derived CD4^+CD8^+ thymocytes in recipient thymus

The aim of the present study was to determine whether the sensitivity of thymocytes to X-ray radiation depends on their proliferative states and whether radiation impairs the maturation of donor-derived thymocytes in recipient thymus.We assigned 8-week-old C57BL/6J mice into three treatment groups：1） untreated;2） X-ray radiation;3） X-ray radiation plus bone marrow transplantation with donor bone marrow cells from transgenic mice express-ing enhanced green fluorescent protein（GFP） on a universal promoter.After 4 weeks,the size of the thymus,the number and proliferation of thymocytes and ratios of different stage thymocytes were analyzed by immunohisto-chemistry and flow cytometry.The results showed that：1） CD4＋CD8＋ thymocytes were more sensitive to X-ray radiation-induced cell death than other thymocytes;2） the proliferative capacity of CD4＋CD8＋ thymocytes was higher than that of other thymocytes;3） the size of the thymus,the number of thymocytes and ratios of thymo-cytes of different stages in irradiated mice recovered to the normal level of untreated mice by bone marrow trans-plantation;4） the ratio of GFP-positive CD4＋CD8＋ thymocytes increased significantly,whereas the ratio of GFP-positive CD4＋ or CD8＋ thymocytes decreased significantly.These results indicate that the degree of sensitivity of thymocytes to X-ray radiation depends on their proliferative states and radiation impairs the maturation of donor-derived CD4＋CD8＋ thymocytes in recipient thymus.

RESPONSES OF HUMAN FETAL SPLENOCYTES AND THYMOCYTES TO INTERLEUKIN-2: LAK ACTIVITY AND PROLIFERATION

Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18-22 weeks). We observed that fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10-100 U ml) developed broad antitumor activity (LAK activity) although the kinetics and magnitudes of the responses were different. It indicated the LAK precursors are present in fetal spleen and thymus. Further, rIL-2 induced a strong proliferative response in splenocytes, but not in thymocytes. On the basis of the findings, we conclude that the responses of fetal splenocytes and thymocytes to IL-2 are different.

Functional maturation of mouse CD4^+CD8^- thymocytes induced by medullary-type thymus epithelial cells

Murine CD4+CD8- (CD4SP) thymocyte subset is a heterogeneous population, in which the Qa-2- cells are less functional, whereas the Qa-2+ cells are fully functional. Evidence is provided here that the transition from Qa-2- to Qa-2+ CD4SP thymocytes is an intrathymic process of differentiation induced by thymic medullary-type epithelial cells. The separated Qa-2-CD4SP could be induced to express Qa-2 molecules up to 84%- 89% of the total viable celb after cocultured for 3d with MTEC1 cells, a murine thymic medullary type epithelial cell line established in our laboratory. Kinetic study showed that both the percentage of Qa-2+ cells and the density of the expressed Qa-2 molecules on CD4SP thymocytes induced by MTEC1 were progressively increasing in 72-h cultures. The MTECl-induced Qa-2+CD4SP thymocytes were fully functional, which exhibited capabilities of proliferation and cytokine secretion in response to Con A stimulation as high as those of freshly isolated Qa-2+CD4SP thymocytes. The profile of

In vitro enhancing effects of thymic dendritic cells on apoptotic cell death of thymocytes

Using terminal deoxynucleotide transferase mediated dUTP digoxigenin nick end labeling (TUNEL) assay and propidium iodide DNA staining flow cytometry assay, the effects of mouse thymic dendritic cells (MTSC4) on the process of programmed cell death of thymocytes in vitro were investigated. It was noticed that thymocytes bound to MTSC4 used in this study. That the percentages of apoptotic nuclei of the bound thymocytes on MTSC4 were much higher than those of medium cultured thymocytes, while the bound thymocytes on mouse thymic epithelial cell (MTEC1) showed much lower percentages of apoptosis. FACS analysis quantitatively confirmed the observation. Phenotype analysis showed that MTSC4 induced the deletion of CD4+CD8+ cells and CD4+CD8 cells in 18 h of coculture. The results suggest that the negative selection of medullary thymocytes may be achieved by thymic dendritic cells through their enhancing effects on apoptosis.

Phenotypic analysis of the medullary-type CD4^-CD8^+ thymocytes in mice

Phenotypic analysis of the medullary-type CD4 CD8+ (CD8SP) thymocytes has revealed phenotypic heterogeneity within this cell population. The phenotype of mature peripheral CD8+T cells is TCRαβ+CD3+Qa-2+HSA3G11 6C10 , whereas in the medullary-type CD8SP thymocytes, 20% are Qa-2+; 33%, HAS ; 30%, 3G11 ; and 70% are 6C10 . The disparate expression patterns of these four cell surface markers suggest that medullary-type CD8SP thymocytes may undergo phenotypic maturation process. According to the distribution of these four celi surface markers, six subgroups of CD8SP thymocytes have been identified. The precursor-progeny relationship along with developmental pathway is postulated as follows: 6C10+HSA+3G11 Qa-2 - 6C10+HSA+ 3G11+Qa-2 6C10 HSA+3G11+Qa-2 -6C10 HSA 3G11 +Qa-2 -6C10 HSA 3G11 Qa-2 -6C10 HA S 3G11 Qa-2+, the cells inthe last subgroup exit the thymus and home into periphery.

Characterization of a murine thymic CD4^+ T cell subset-TCRαβ^+ 3G11^- 6C10^- CD4^+ CD8^- thymocytes

The presence of a relatively mature CD4 + CD8- （SP） T cell subset in mouse thymus has been demonstrated. Composing of 10% of total CD4SP thymocytes, this subset is defined by the absence of 3G11 and 6C10 expression with a phenotype of CD69+/-, HSAmed/lo and heterogeneous for Qa-2 expression. The proliferation capability of TCRαβ+ 3G11- 6C10- CD4+ CD8- thymocytes was high while using Con A stimulus. And Con A stimulation could result in secretion of IL-4, IL-10, IL-6 and a little amount of IFNγ. IL-2 was barely detectable. This is distinct from typical Th0 type cytokines. The cells of this subset were NK1.1 negative, but strongly expressed GATA-3 mRNA. The results suggest that the CD4+ subset of 3G11- 6C10- NK1.1- phenotype possesses immunocompetent cells with functions characteristic of Th2-like cytokines, which may indicate the cells at transitional status from Th0 to Th2, with a propensity to Th2.

PF18-3, a 35-ku molecule, expresses specifically on apoptotic subset of mouse thymocytes

PF18-3 monoclonal antibody (mAb), one of the rat mAbs against mouse thymic stromal cells (MTSC), has been found to inhibit thymocyte apoptosis induced by a mouse thymic dendritic cell line, MTSC4, in previous co-culture study. The aim of this research is to investigate the character of PF18-3 mAb recognized molecule ( PF18-3 molecule) and its role in MTSC4-induced thymocyte apoptosis. The characterization of PF18-3 molecule expression has been conducted by FACS analysis. PF18-3 molecules have been found to express on MTSC4 as well as on Con A activated but not freshly isolated thymocytes. Up-regulated expression of PF18-3 molecules has been also observed on thymocytes after being co-cultured with MTSC4 for 48 h. The results from FACS analyses by Pl staining for detecting apoptosis-related hypodiploid and by PF18-3 mAb staining reveal that PF18-3 molecules expresss specifically on the apoptotic subgroup of thymocytes with high hypodiploid content. The PF18-3 molecule expressed on apoptotic thymocytes

Liver X receptorβis required for the survival of single-positive thymocytes by regulating IL-7Rαexpression

Liver X receptors(LXRs)are known as key transcription factors in lipid metabolism and have been reported to play an important role in T-cell proliferation.However,whether LXRs play a role in thymocyte development remains largely unknown.Here,we demonstrated that LXRβdeficiency caused a reduction in single-positive(SP)thymocytes,whereas the transitions from the double-negative to SP stage were normal.Meanwhile,LXRβ-null SP thymocytes exhibited increased apoptosis and impairment of the IL-7Rα-Bcl2 axis.In addition,the LXR agonist T0901317 promoted the survival of SP thymocytes with enhanced IL-7Rαexpression in wild-type mice but not in LXRβ-deficient mice.Mechanistically,LXRβpositively regulated the expression of IL-7Rαvia direct binding to the Il7r allele in SP thymocytes,and forced expression of IL-7Rαor Bcl2 restored the survival of LXRβ-defective SP thymocytes.Thus,our results indicate that LXRβfunctions as an important transcription factor upstream of IL-7Rαto promote the survival of SP thymocytes.

Sin1-mTORC2 signaling drives glycolysis of devetoping thymocytes

Emerging studies highlight the import-ance of metabolic reprogramming in T cell development and function, although how these processes are regulated remains to be fully understood. Recent advances in dissecting the roles of Sinl-m T0RC2 signaling in early thymocyte development provide new in sight into the dynamic in terplay between immune signaling and cell metabolism, as well as the crucial role in directi ng thymocyte pro life ration and development.

Functional maturation of two murine medullary-type CD8SP thymocytes

TCRαβTCD4-CD8+ thymocytes are heterogeneity. They may undergo phenotypic and functional maturation within thymic medulla. Medullary-type CD8SP thymocytes were divided into seven subsets based on phenotypic analysis, and their precursor-progeny relationship along with the differential pathway was also delineated. To further testify the validity of the maturation pathway, we purified 6C10-CD69+ cells representing the early stage and 6C10-Qa-2+ cells representing the later stage among medullary-type CD8SP thymocytes and compared their functional maturation levels. CD8+ T cells of spleen were used as the control. It is shown that there is no obvious difference of proliferation ability among these three subsets; however, intracytoplasmic cytokine assay shows that there is a hierarchy of IFN-γ and TNFα secretion among these subsets, strikingly comparable to their phenotypic status among medullary type CD8SP thymocytes. The bioassays of IL-2 and IFN-γ in culture supernatant give the similar results.

The Possible Involvement of Apoptotic Decay of Terminal Deoxynucleotidyl Transferase-Positive Lymphocytes in the Reutilization of the Extracellular DNA Fragments by Surrounding Living Cells

The migrating TdT+ thymocytes can die in other tissues, promoting the surrounding cells’ renewing likes holocrine secretion does. To clarify the role of TdT-enzyme for this function of progenitor lymphocytes, their extracellular media with its components included by living cells analyzed in vitro before and after in vivo irradiation of donor rats. The nucleoid with DNase-sensitive (free) DNA and TdT activity discovered in extracellular media conditioned preliminary by spontaneous apoptotic death of a minor part of the thymocyte’s suspension in vitro. The penetration of labeled products of non-template synthesis with free DNA’ primers from media into cells by pinocytosis confirmed by exogenous polymeric DNA marked artificially. The DNA penetration into cells follows an increase of the cell’s viability and acceleration of spontaneous intracellular DNA-synthesis controlled with labeled thymidine uptake. Both phenomena are typical for either the lowest initial concentration of intact cells or their preliminary irradiation in vivo. The data point to possible involvement of apoptotic decay of TdT+ cells in the reutilization of the extracellular DNA fragments for reparation/regeneration of surrounding living cells.

The Effect of Tremella Fuciformis Spores Polysaccharides (TSP) on Immune Cellular Function of Mice in Vitro

TSP could markedly enhance the proliferative response of the murine splenocyte to LPS and induce the mitogenesis of the spleen cells.Furthermore,it was able to augment the activity of natural killer cell and ADGG;at a dosage of 25-250μg/ml,the ability of splenocytes to produce IL-2 induced by onA had been improved; at the concentration of 250μg／ml or more,TSP could inhibit the proliferative response of the murine lymphocyte to GonA and the ~3-HTdR spontaneous incorporation rate of thymocytes,and the inhibitory action ran in paralell with the increase in concentration of TSP.

Electrophoretic Analysis on RNA of Deltamethrin－resistant Culex Pipiens Pallens

TSP could markedly enhance the proliferative response of the murine splenocyte to LPS and induce the mitogenesis of the spleen cells.Furthermore,it was able to augment the activity of natural killer cell and ADGG;at a dosage of 25-250μg/ml,the ability of splenocytes to produce IL-2 induced by onA had been improved; at the concentration of 250μg/ml or more,TSP could inhibit the proliferative response of the murine lymphocyte to GonA and the ￣3-HTdR spontaneous incorporation rate of thymocytes,and the inhibitory action ran in paralell with the increase in concentration of TSP.

Effect of bilateral testicular resection on thymocyte and its microenvironment in aged mice

Aim: To observe the changes in thymocyte and its microenvironment in aged mice after bilateral testicular resection.Methods: In male old mice, at the 25th day after testicular resection, the peripheral blood and thymus were collect-ed . Blood and thymus suspension smears were prepared for quantitative histochemistry and immunohistochemistry studyunder light and electron microscopes. Results; In testes resected mice the size and the weight of thymus weremarkedly increased. The demarcation between cortex and medulla was clear. The cortex was thickened and the celldensity was increased. The ratio of cortex/medulla stereometry was increased. The total cell count, thymocyte count,the percentage of acid a-naphthyl acetate esterase (ANAE) positive thymocytes, nonlymphocytes and the rosette forma-tion of macrophages and thymocytes were all increased. The thymocytes surrounded closely to the light thymic epithelialcells, dendritic cells or macrophages. The lymphocytes, particularly the ANAE positive lymphocytes of peripheralblood were increased. Conclusion; After bilateral testicular resection, the thymus of aged male mice showed mor-phological regeneration and the thymocytes and its microenvironment appeared to be definitely improved. It is suggestedthat testicular resection may improve immune function. (Asian J Androl 2001 Dec; 3; 271 — 275)

Intermittent Fasting Ameliorates Delayed-Type Hypersensitivity in NC/Nga Mice

It is well documented that dietary restriction can prevent many different diseases and extend the life spans of different rodent species. In the previous study, we reported that intermittent fasting (IF) as well as moderate dietary restriction ameliorated the allergic dermatitis in ICR mice. In the present study, we demonstrated the ameliorative effects of IF on allergic dermatitis in NC/Nga mice, a strain known as a model for atopic dermatitis. Interestingly, the total number of CD4+CD8+ double positive thymocyte in mice after IF significantly decreased in comparison to that in mice fed ad libitum. Although it was reported that an immunosuppressive compound inhibited the contact allergic response by inducing the CD4+CD25+ regulatory T-cells, IF did not affect regulatory T cells in the present study. These results suggested that CD4+CD8+ double positive thymocytes play an important role in the regulation of allergy by IF in NC/Nga mice.

Mannosylated glycans impair normal T-cell development by reprogramming commitment and repertoire diversity

T-cell development ensures the formation of diverse repertoires of T-cell receptors(TCRs)that recognize a variety of antigens.Glycosylation is a major posttranslational modification present in virtually all cells,including T-lymphocytes,that regulates activity/functions.Although these structures are known to be involved in TCR-selection in DP thymocytes,it is unclear how glycans regulate other thymic development processes and how they influence susceptibility to disease.Here,we discovered stage-specific glycome compositions during T-cell development in human and murine thymocytes,as well as dynamic alterations.After restricting the N-glycosylation profile of thymocytes to high-mannose structures,using specific glycoengineered mice(Rag1CreMgat1fl/fl),we showed remarkable defects in key developmental checkpoints,includingß-selection,regulatory T-cell generation andγδT-cell development,associated with increased susceptibility to colon and kidney inflammation and infection.We further demonstrated that a single N-glycan antenna(modeled in Rag1CreMgat2fl/fl mice)is the sine-qua-non condition to ensure normal development.In conclusion,we revealed that mannosylated thymocytes lead to a dysregulation in T-cell development that is associated with inflammation susceptibility.

Analysis of the chemotactic activities of mouse chemokine MIP-2 to thymocyte subgroups

A mouse chemokine MIP-2(macrophage in-flamatory protein 2) is constitutively expressed not only by peritoneal macrophages, but also by fresh thymic stromal cells, based on RT-PCR detection. Moreover, the specific receptor of MIP-2 is expressed at different levels among four main subgroups of murine thymocytes including DN, DP, CD4SP and CD8SP. By the chemotaxis assays with Boyden chamber, we proved that the recombinant mouse MIP-2 can chemoattract the four main subgroups of thymocytes in different degrees, it mainly chemoattract the DP and SP subgroups. We firstly reported that MIP-2 is involved in the regulation of the directional migration of developing thymocytes.

Alterations in thymocyte populations under conditions of endotoxin tolerance

Background:Endotoxin tolerance(ET)is a protective phenomenon in which pre-treatment with a tolerance dose of lipopolysaccharide(LPS)leads to dramatically elevated survival.Accumulating evidence has shown that peripheral T cells contribute to the induction of ET.However,what happens to T cell development in the thymus under ET conditions remains unclear.The purpose of this study was to analyze the alterations in thymocyte populations(double-positive[DP]and single-positive[SP]cells)under ET conditions.Methods:Mice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups:a group examined 72 h after LPS injection(72-h group)and a group examined 8 days after LPS injection(8-day group).Injection of phosphate-buffered saline was used as a control(control group).Changes in thymus weight,cell counts,and morphology were detected in the three groups.Moreover,surface molecules such as CD4,CD8,CD44,CD69,and CD62L were analyzed using flow cytometry.Furthermore,proliferation,apoptosis,cytokine production,and extracellular signal-regulated kinase(ERK)pathway signaling were analyzed in thymocyte populations.The polymorphism and length of the T-cell receptor(TCR)P chain complementarity-determining region 3(CDR3)were analyzed using capillary electrophoresis DNA laser scanning analysis(ABI 3730).Results:Thymus weight and cell counts were decreased in the early stage but recovered by the late stage in a murine model of LPS-induced ET.Moreover,the proportions of DP cells(control:72.130±4.074,72-h:10.600±3.517,8-day:84.770±2.228),CD4+SP cells(control:15.770±4.419,72-h:44.670±3.089,8-day:6.367±0.513),and CD8+SP cells(control:7.000±1.916,72-h:34.030±3.850,8-day:5.133±0.647)were obviously different at different stages of ET.The polymorphism and length of TCR β chain CDR3 also changed obviously,indicating the occurrence of TCR rearrangement and thymocyte diversification.Further analysis showed that the expression of surface molecules,including CD44,CD69,and CD62L,on thymocyte populations(DP and SP cells)were changed to different degrees.Finally,the proliferation,apoptosis,cytokine production,and ERK pathway signaling of thymocyte populations were changed significantly.Conclusion:These data reveal that alterations in thymocyte populations might contribute to the establishment of ET.