Mechanisms Involved in the Binding of Thymocytes to Rat Thymic Dendritic Cells

The effects of monoclonal antibodies (mAbs) to cell-surface molecules, divalent cations, and various cell-signaling and metabolic inhibitors on the binding of thymocytes to rat thymic dendritic cells (TDC) were studied using a rosette assay. It was found that TDC/thymocyte adhesion was stronger and faster at 37°C than at 4°C. Flow cytometric analysis demonstrated that bound thymocytes were predominantly CD4⁺CD8⁺ and CD4⁺CD8^-, but in comparison to the phenotype of whole thymocytes, they were enriched in the mature TCRαβ^hi subset. The binding of thymocytes to TDC at 37°C was almost completely dependent on Ca²⁺ and Mg²⁺ and partly on an intact cytoskeleton and calmodulin-dependent protein kinase. The adhesion was independent of new protein synthesis and the activities of protein kinases A and C, tyrosine kinases, as well as phosphotyrosine protein phosphatases. The TDC/thymocyte adhesion at 37°C was partly blocked by anti-LFA-1 (WT.1), anti-CD18 (WT.3), and anti-ICAM-1 (1A29) mAb. MAbs to class II MHC (OX-3 and OX-6), CD4 (W3/25), CD8 (OX-8), and αβTCR (R73) stimulated the adhesion via an LFA-1-dependent pathway, whereas an anti-CD45 mAb (G3C5) stimulated the rosette formation independently of LFA-1. MAbs to CD2 (OX-34), CD11b (ED7), CD11b/c (OX-42), and class I MHC (OX-18) were without significant effects on the adhesion process.     

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.     

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.     

Bone morphogenetic protein-2/4 signalling pathway components are expressed in the human thymus and inhibit early T-cell development

T-cell differentiation is driven by a complex network of signals mainly derived from the thymic epithelium. In this study we demonstrate in the human thymus that cortical epithelial cells produce bone morphogenetic protein 2 (BMP2) and BMP4 and that both thymocytes and thymic epithelium express all the molecular machinery required for a response to these proteins. BMP receptors, BMPRIA and BMPRII, are mainly expressed by cortical thymocytes while BMPRIB is expressed in the majority of the human thymocytes. Some thymic epithelial cells from cortical and medullary areas express BMP receptors, being also cell targets for in vivo BMP2/4 signalling. The treatment with BMP4 of chimeric human-mouse fetal thymic organ cultures seeded with CD34+ human thymic progenitors results in reduced cell recovery and inhibition of the differentiation of human thymocytes from CD4- CD8- to CD4+ CD8+ cell stages. These results support a role for BMP2/4 signalling in human T-cell differentiation.     

Cell effects of xenon <Emphasis Type="Italic">n vitro</Emphasis> under hypothermal conditions

The effects of Xe on cell viability and redox balance in the culture of Wistar rat thymocytes were studied in vitro during 24-h storage under hypothermic conditions. The results indicate that after bubbling of cell suspensions (5×10⁶ cell/ml, 4 ml medium), the weight of Xe in flasks was 15–43 mg, whereas in cell-free medium no weight increment due to gas accumulation in the system was detected. The content of Xe in cell suspension slightly decreased over 24-h culturing at ambient temperature (by 10% of initial level). Xenon significantly improved cell survival during thermal exposure of all modes. The maximum cytoprotective effect of Xe was observed under rigorous thermal conditions associated with significant cell death without chemical protectors (3°C, −35°C). The effect of Xe was less pronounced at mild temperatures (23–37°C) or in the presence of chemical protectors (−35°C with dimethylsulfoxide). The mechanisms of the effect of the inert gas are determined by its antioxidant or prooxidant action. The capacity of Xe to improve cell survival under hypothermic conditions can be used for the development of new methods for transportation and storage of cell material. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 2, pp. 74–77, April, 2007     

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.     

Jak3 activation in human lymphocyte precursor cells

Although expression of the Jak3 tyrosine kinase in T lymphocytes has been thought to be restricted to mature, activated cells, mutations of Jak3 can lead to the development of a human severe combined immunodeficiency (SCID) characterized by an absence of peripheral T lymphocytes. We therefore examined in detail the expression of Jak3 throughout human T cell differentiation and show that Jak3 is in fact present throughout the entire developmental process, with high levels expressed in thymocytes. Jak3 is highly expressed in double negative (CD4⁻CD8⁻) cells, one of the earliest stages of thymocyte differentiation, and can be activated via the IL-7 receptor. IL-7 is known to stimulate thymocyte proliferation and initiate re-arrangement of the T cell receptor (TCR) β gene, suggesting that the failure of mutated Jak3 proteins to transduce this signal may be responsible for failures in T cell development. While Jak3 SCID patients possess mature peripheral B cells, we demonstrate that the Jak3 tyrosine kinase is also expressed in human pre-B cells and can be activated by the pre-B cell growth factor IL-7.     

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.