Production and the characterization of monoclonal antibody against CD43, K06

A monoclonal antibody against human CD43 has been developed and designated as K06. Its reactivity in the lymphoid organs was different from that of known anti-CD43 monoclonal antibodies suggesting that this may recognize a novel epitope of human CD43 molecule. The CD43 epitope detected by anti-K06 monoclonal antibody was highly expressed in cortical thymocytes, platelets, and myeloid cells of normal peripheral blood, and its reactivity was comparable to that of known anti-CD43 monoclonal antibodies. However, the density of this epitope was lower in the medullary thymocytes. Biochemical studies indicated that anti-K06 monoclonal antibody could recognize glycosylated moiety of CD43 antigen. The expression profile of anti-K06 monoclonal antibody in several cell lines was somewhat different from that of known anti-CD43 antibodies. In addition, CD43 ligation through the K06 epitope appeared to induce apoptosis in human leukemic cell line, Molt-4. We therefore assume that K06 epitope of human CD43 might have some role in T-cell development.     

Death of T cell precursors in the human thymus: a role for CD38

Thymic T cell maturation depends on interactions between thymocytes and cells of epithelial and hematopoietic lineages that control a selective process whereby developing T cells with inappropriate or self-reactive receptors die. Molecules involved in this process are the TCR expressed on thymocytes together with the CD3 complex and MHC-peptide on accessory cells. However, other molecules may favor or prevent death of thymocytes, thus playing a role in selection. CD38 is expressed by the majority of human thymocytes, mainly at the double-positive (DP) stage. In contrast, CD38 is not found on subcapsular double-negative (DN) thymocytes and on a proportion of medullary single-positive (SP) thymocytes. CD38 enhances death of thymocytes when it is cross-linked by goat anti-mouse (GAM) antiserum or by one of its ligands, CD31, expressed by thymic epithelial cells or transfected into murine fibroblasts (L cells). As most thymocytes are at an intermediate (DP) stage of development, it is likely that these cells are most vulnerable to death mediated via MHC-peptide-TCR interactions that is increased by CD38 cross-linking. DN and SP thymocytes are refractory to CD38-induced apoptosis. Accessory molecules, e.g. CD38, are expressed during thymic cell maturation and their presence is relevant for the survival or death of DP T cells in the course of selection. Based on our data, CD38 enhances thymocyte death by interacting with CD31 expressed by accessory cells. In addition, CD28 expression on developing thymocytes also appears to play a role for their selection and it synergizes with CD38 to induce apoptosis of DP thymocytes.     

Thymocytes induced by antigen injection into the anterior chamber activate splenic CD8+ suppressor cells and enhance the antigen-induced production of immunoglobulin G1 antibodies

Injection of antigen into the ocular anterior chamber (AC) of a mouse eye (an immunologically privileged site) induces the activation of immunoregulatory NK1.1+, CD4- CD8-, T-cell receptor (TCR) alphabeta+ thymocytes. These thymocytes transfer the suppression of delayed-type hypersensitivity (DTH) when injected into mice sensitized to the same antigen but do not effect the suppression of DTH. On the other hand, the immunized recipients of these transferred thymocytes produce splenic CD8+ T cells that effect the suppression of DTH. However, it is unclear whether the thymocytes transferred from the AC-injected donor differentiate into and/or activate CD8+ T-splenic suppressor cells. We therefore sought to determine the origin of splenic suppressor cells produced in the recipients of immunoregulatory thymocytes transferred from donors that receive an injection of antigen into the AC. CD45.1+ thymocytes from mice that received an AC injection of 2,4,6-trinitrobenzene sulphonic acid (TNP)-bovine serum albumin (BSA) were transferred to congenic CD45.2+ TNP-BSA-immune recipients. Spleen cells from the recipients were then sorted based on anti-CD45.1 or -CD45.2 antibody binding and assayed for suppressor cells. This was done by the injection of separated spleen cells into the footpad of TNP-BSA-immunized mice, concurrent with the induction of footpad swelling (contact sensitivity) of the footpad elicited by an epicutaneous application of picryl chloride. The systemic distribution of antigen after the injection of antigen into the AC was demonstrated by the injection of fluorescein or 125I-labelled TNP-BSA into the AC. The results demonstrate that (i) splenic CD8+ T-suppressor cells produced in the immunized recipients of immunoregulatory thymocytes are derived from the CD45.2 recipient of the CD45.1+ thymocytes; (ii) the induction of recipient splenic suppressor T cells by the transferred immunoregulatory thymocytes requires that the recipient be immunized to the same antigen as that used to induce immunoregulatory thymocytes; (iii) antigen is introduced to the thymus after an injection of antigen into the AC; (iv) although the transfer of the suppression of DTH by regulatory thymocytes was not dependent on interleukin-4 (IL-4), CD4+ NK1.1- regulatory thymocytes from AC-injected donors enhanced the production of immunoglobulin G1 antibodies to TNP-BSA by an IL-4-dependent mechanism. These observations suggest that the adult thymus plays an active role in the induction and maintenance of anterior chamber-associated immune deviation as manifested by the generation of the suppression of cell-mediated immunity to exogenous antigen and the antigen-induced production of IgG1 antibodies.     

低剂量电离辐射诱导小鼠胸腺细胞蛋白的早期表达

采用双向电泳方法检测低剂量Ｘ射线全身照射诱导小鼠胸腺细胞核、细胞浆蛋白的早期表达。结果表明７５ｍＧｙＸ射线照射后４小时细胞核出现４个新蛋白点；５个增强蛋白点；ｌ个减弱蛋白点。胸腺细胞浆出现３个新蛋白点和３个增强蛋白点。这些早期基因表达产物可能参与细胞增殖、分化中的细胞信息传递过程和基因表达调控。     

Low rate of proliferation in immature thymocytes of the non-obese diabetic mouse maps to the Idd6 diabetes susceptibility region

Aims/hypothesis: The non-obese diabetic (NOD) mouse spontaneously develops T-cell-dependent autoimmune diabetes. This mouse strain has a number of immune dysfunctions related to T-cell development but so far there are no available data on the proliferation of NOD immature thymocytes. We therefore studied the thymocyte proliferation in the NOD mouse in discrete stages of T-cell development. Methods: We depleted thymocytes in vivo and analysed thymocyte proliferation during the thymus recovery from depletion. We used co-segregation analysis and quantitative loci trait analysis to investigate the genetic control of proliferation impairments in NOD thymocytes. Results: Immature thymocytes of female NOD mice proliferate with a relatively low rate compared to non-autoimmune C57Bl/6 mice. This aberrant proliferation was most pronounced in CD4^{– /lo} CD8⁺ cells differentiating from the CD4^–CD8^– to the CD4⁺CD8⁺ stage. A genetic mapping study using an F2 intercross between the NOD and the C57BL/6 strains showed that a major locus controlling this trait is linked to the insulin-dependent diabetes susceptibility locus Idd6. Conclusion/interpretation: Our results suggest that impairment of proliferation of immature thymocytes is one possible mechanism through which the Idd6 locus contributes to the pathogenesis of diabetes. [Diabetologia (2001) 44: 1054–1061] Received: 6 November 2000 and in revised form: 3 May 2001     

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.     

单侧迷走神经切断对大鼠胸腺细胞迁移的影响

目的探讨迷走神经在胸腺细胞输出过程中的作用。方法实验采取颈部右侧迷走神经切断术,应用免疫组织化学结合图像分析方法观察术后大鼠胸腺CD4／CD8阳性细胞表达及分布变化情况。结果CD4、CD8阳性细胞分布于大鼠胸腺特定区域,迷走神经切断后数量减少、染色光密度降低;分布区域变小。结论迷走神经切断术后,胸腺中未成熟T细胞减少,成熟T细胞数量相对增多。迷走神经在T细胞发育及输出过程中起重要作用。     

Mitochondrial and Nuclear Glucocorticoid-Sensitive Alkaline Proteases in Thymocytes

Glucocorticoid-sensitive alkaline proteases are localized in the nuclear and mitochondrial fractions of rat thymocytes. Dexamethasone load increased alkaline protease activity in the nuclear fraction and decreased or even eliminated it in the mitochondrial fraction. The inhibitory analysis showed that SH groups and disulfide bonds play a crucial role in the functioning of glucocorticoid-activated nuclear alkaline protease. Hence, this enzyme can be assigned to as cysteine proteases. Mitochondrial alkaline protease is a serine hydrolase, although it does not belong to the class of trypsin- or chymotrypsin-like enzymes. The role of alkaline proteases in apoptotic death of thymocytes is discussed.     

CD28 in thymocyte development and peripheral T cell activation in mice exposed to suspended particulate matter

The CD28:B7 signaling pathway is very important for the activity of mature peripheral T lymphocytes and thymocyte development. The proper development of thymocytes into mature single positive CD4⁺and CD8⁺ T cells is crucial for almost all immune functions. In naturally occurring conditions, T cells maturation in the thymus is influenced by environmental agents. The expression of CD28 and the distribution of CD28^low/high thymocytes have been examined at various stages of thymocyte development in BALB/c mice exposed to air-suspended particulate matter (ASM). Acute exposure to ASM resulted in the decrease of CD28 expression in the total thymocyte population. The increase of the percentage of CD28^low and the decrease of CD28^high thymocytes were observed, which may account for the acceleration of thymocyte development under the conditions of elevated risk resulting from the exposure of animals to environmental xenobiotics. ASM exposure resulted in the increase of the level of proliferation of lymph node T cells induced by anti-CD3 and anti-CD28 monoclonal antibodies activation despite normal expression of CD28 molecule. In contrast, the level of proliferation of spleen T cells was lowered or normal dependently of the concentration of stimuli used for activation. Results of these studies demonstrate that acute exposure of mice to ASM can result in the progression of two contrasting processes in the immune system: upregulation of thymocyte development, which contributes to the maintenance of peripheral T cell pool, and over-activation of lymph node lymphocytes, which may lead to uncontrolled immunostimulation.     

Cytometric analysis of adverse action of diphenyl ditelluride on rat thymocytes: cell shrinkage as a cytotoxic parameter

Despite the growing use of organotellurium compounds in the chemical and biomedical fields, there has been no great concern about their toxicity until now. To test the possibility that diphenyl ditelluride (DPDT) and tellurium chloride (TeCl2), organic and inorganic tellurium compounds, may exert adverse action on mammals, their effects on rat thymocytes were examined under in vitro conditions using a flow cytometer with fluorescent probes. Incubation of thymocytes with DPDT at 300 nM or more for 24 h significantly increased the populations of shrunken cells and of cells with hypodiploidal DNA. Z-VAD-FMK, a paninhibitor of caspases, greatly suppressed the DPDT-induced increase in the hypodiploidal cell population, suggesting the involvement of caspase activation in DPDT toxicity. Hence, it is possible that DPDT would increase the population of thymocytes undergoing apoptosis if the blood concentration in mammals reached at least 300 nM or more. TeCl2 was much less potent than DPDT in increasing the population of hypodiploidal cells.