Expression of interleukin-1 receptors in the later period of foetal thymic organ culture and during suspension culture of thymocytes from aged mice

Interleukin-1 has been reported to be involved in thymocyte development by exerting a variety of effects on immature CD4-CD8- double-negative (DN) thymocytes. In contrast to the well-documented involvement of IL-1 in thymocyte development, expression of IL-1 receptors (IL-1R) on thymocytes has not been well demonstrated. In the present study, expression of IL-1R on the developing thymocytes was investigated. Although normal thymocytes barely express IL-1R, expression of IL-1R (type I) substantially increased at days 12-15 of foetal thymic organ culture (FTOC), with an increase of the DN subset. The CD4/CD8 profile of the IL-1R (type I)+ cells showed that these cells were mostly restricted to the DN and CD4+CD8+ subsets. Interestingly, in vitro culture of the thymocytes from an aged mouse, but not those from young adult or newborn mice, revealed similar results to those of FTOC. In addition, half of the IL-1R+ cells that increased in the later period of FTOC were gammadelta thymocytes. These results demonstrate IL-1R expression on thymocytes during ex vivo culture and suggest that IL-1R is expressed in a certain environment during normal thymocyte differentiation.     

Asynchronism of thymocyte development in vivo and in vitro

Although fetal thymus organ culture (FTOC) has become widely used to investigate T-cell development, the differences between thymocyte development in vivo and in vitro (in FTOC) remain largely unknown. In this study, the viability and numbers of thymocytes recovered from embryonic thymus lobes in different gestation days (gd) mice or from 15 day embryonic thymus lobes cultured for different days in FTOC system were evaluated. The expression of CD3, CD4, CD8, CD95 ligand (CD95L), and CD69 on thymocytes were analyzed by FACS. The results showed that thymocytes, either in vivo or in vitro, could differentiate from double negative (DN) cells to double positive (DP) cells and to single positive (SP) cells. But the number of total thymocytes and the percentage of DP cells in vitro were less than that in vivo, and the expression of CD95L and CD69 on thymocytes in vitro was higher than that in vivo. Our results suggested that although thymocyte development in vitro could recapitulate thymic development in vivo, the proliferation of thymocytes in vitro was less intensive than that in vivo; the differentiation of thymocytes in vitro was delayed compared with that in vivo; and the apoptosis and activation of thymocytes in vitro were higher than that in vivo. In conclusion, FTOC is a useful system for the study of T cell differentiation, but it is necessary to interpret the results from in vitro studies carefully since the thymocyte development in vitro is asynchronous from that in vivo.     

Thymocyte LFA-1 and thymic epithelial cell ICAM-1 molecules mediate binding of activated human thymocytes to thymic epithelial cells.

We have investigated the binding in vitro of activated thymocytes to thymic epithelial (TE) cells, and studied the effect of up-regulation of TE cell surface intracellular adhesion molecule 1 (ICAM-1) and HLA-DR by IFN-gamma on the ability of TE cells to bind to both resting and activated human thymocytes. TE cell binding to activated and resting thymocytes was studied by using our previously described suspension assay of TE-thymocyte conjugate formation. We found that activated mature and immature thymocytes bound maximally at 37 degrees C to IFN-gamma-treated ICAM-1+ and HLA-DR+ TE cells and this TE-activated thymocyte binding was inhibited by antibodies to LFA-1 alpha-chain (CD11a) (68.1 +/- 5.6% inhibition, p less than 0.01) and ICAM-1 (73.9 +/- 7.7% inhibition, p less than 0.05). Neither anti-HLA-DR antibody L243 nor anti-MHC class I antibody 3F10 inhibited IFN-gamma-treated TE binding to activated thymocytes. As with antibodies to LFA-3 and CD2, antibodies to LFA-1 and ICAM-1 also inhibited PHA-induced mature thymocyte activation when accessory signals were provided by TE cells in vitro. Finally, LFA-1 and ICAM-1 were expressed early on in human thymic fetal ontogeny in patterns similar to those seen in postnatal thymus. Taken together, these data suggest that resting mature and immature thymocytes bind to TE cells via the CD2/LFA-3 ligand pair, whereas activated thymocytes bind via both CD2/LFA-3 and LFA-1/ICAM-1 ligand systems. We postulate that IFN-gamma produced intrathymically may regulate TE expression of ICAM-1 and therefore potentially may regulate TE cell binding to activated thymocytes beginning in the earliest stages of human thymic development.     

Ephrin-B1 is critical in T-cell development

Eph kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. In this study, we investigated the role of EFNB1 and the Ephs it interacts with (collectively called EFNB1 receptors) in mouse T-cell development. In the thymus, CD8 single positive (SP) and CD4CD8 double positive (DP) cells expressed high levels of EFNB1 and EFNB1 receptors, whereas CD4 SP cells had moderate expression of both. Soluble EFNB1-Fc in fetal thymus organ culture caused significant subpopulation ratio skew, with increased CD4 SP and CD8 SP and decreased DP percentage, while the cellularity of the thymus remained constant. Moreover, in EFNB1-treated fetal thymus organ culture, CD117(+), CD25(+), DP, CD4 SP, and CD8 SP cells all had significantly enhanced proliferation history, according to bromodeoxyuridine uptake. In vitro culture of isolated thymocytes revealed that EFNB1-Fc on solid-phase protected thymocytes from anti-CD3-induced apoptosis, with concomitant augmentation of several antiapoptotic factors, particularly in CD4 SP and CD8 SP cells; on the other hand, soluble EFNB1-Fc promoted anti-CD3-induced apoptosis, as was the case in vivo. This study reveals that EFNB1 and EFNB1 receptors are critical in thymocyte development.     

Changes in the thymus of peripubertal rats induced by centrally applied somatostatin-28

The aim of this study was to investigate the effects of centrally applied somatostatin-28 on morphometric characteristics of the thymus, the thymocyte subpopulations, as well as, on apoptosis and phases of cell cycle in thymocytes. For this purpose, peripubertal male rats were cannulated intracerebroventriculary and treated with repeated, nanomolar concentrations of somatostatin-28 (experimental group) or saline (control group). Animals were sacrificed and their thymuses were used for the analysis of thymocyte subpopulations, cell cycle and apoptosis by flow cytometry and for the evaluation of morphometric parameters by stereological analysis. Our results showed that somatostatin-28 caused decrease of the thymic mass and volume, as well as total thymocytes number. Stereological analysis revealed volume decrease of thymic cortex and medulla accompanied with cellularity decrease. Somatostatin in the deeper cortex decreased the number of thymocytes, per volume unit, while in outer cortex raised their number. A significant increase in the percentage of double-negative and both single-positive thymocyte subpopulations, in parallel with a diminished percentage of double-positive cells was found. The cellularity of double-positive and single-positive thymocyte subpopulations was decreased. Somatostatin-28 treatment augmented the percentage of apoptotic cells, while the percentage of the cells represented in phases of cell cycle was reduced. These results suggest that somatostatin-28 induce thymus hypotrophy as result of decreasing cortex and medulla volume and cellularity. Changes in the percentage and cellularity of thymocyte subpopulations and numerical density of thymocytes in outer and deeper cortex, indicate that somatostatin-28 evoked disturbance in transition of double-negative to double-positive thymocytes.     

Chronic alpha1-adrenoreceptor blockade produces age-dependent changes in rat thymus structure and thymocyte differentiation

In order to examine the influence of chronic alpha1-adrenergic receptor (alpha1-AR) blockade on the thymus structure and T-cell maturation, peripubertal and adult male rats were treated with urapidil (0.20 mg/kg BW/d; s.c.) over 15 consecutive days. Thymic structure and phenotypic characteristics of the thymocytes were assessed by stereological and flow cytometry analysis, respectively. In immature rats, treatment with urapidil reduced the body weight gain and, affecting the volume of cortical compartment and its cellularity decreased the organ size and the total number of thymocytes compared to age-matched saline-injected controls. The percentage of CD4+8- single positive (SP) thymocytes was decreased, while that of CD4-8+ was increased suggesting, most likely, a disregulation in final steps of the positively selected cells maturation. However, alpha1-AR blockade in adult rats increased the thymus weight as a consequence of increase in the cortical size and cellularity. The increased percentage of most immature CD4-8- double negative (DN) cells associated with decreased percentage of immature CD4+8+ double positive (DP) thymocytes suggests a decelerated transition from DN to DP stage of T-cell development. As in immature rats, the treatment in adult rats evoked changes in the relative numbers of SP cells, but contrary to immature animals, favoring the maturation of CD4+8- over CD4-8+ thymocytes. These results demonstrate that: i) chronic blockade of alpha1-ARs affects both the thymus structure and thymocyte differentiation, ii) these effects are age-dependent, pointing out to pharmacological manipulation of alpha1-AR-mediated signaling as potential means for modulation of the intrathymic T-cell maturation.     

Role of CTLA-4 in the activation of single- and double-positive thymocytes

CTLA-4, a homologue of CD28, is a negative regulator of T cell activation in the periphery and is transiently expressed on the cell surface after T cell activation. However, the role of CTLA-4 in T cell activation in the thymus is not clear. This investigation was initiated to determine the role of CTLA-4 in the activation of CD4(+)CD8(+) double-positive (DP) and CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes using fetal thymic organ cultures (FTOC) of MHC class II-restricted, OVA(323-339)-restricted TCR transgenic mice (DO11.10). We found that treatment of the FTOC with anti-CTLA-4-blocking Ab during activation with OVA(323-339) increased the proportion and number of DP thymocytes, but decreased the proportion and number of SP thymocytes compared with OVA(323-339)-stimulated FTOC without anti-CTLA-4 Ab treatment. In addition, anti-CTLA-4 Ab treatment inhibited OVA(323-339)-induced expression of the early activation marker, CD69, in DP thymocytes, but increased CD69 in SP thymocytes. Similarly, CTLA-4 blockage decreased phosphorylation of ERK in DP thymocytes by Ag-specific TCR engagement, but increased phosphorylation of ERK in SP thymocytes. CTLA-4 blockage inhibited deletion of DP thymocytes treated with a high dose of OVA(323-339), whereas CTLA-4 blockage did not inhibit deletion of DP thymocytes treated with a low dose of OVA(323-339). We conclude that CTLA-4 positively regulates the activation of DP thymocytes, resulting in their deletion, whereas blocking CTLA-4 suppresses the activation of DP thymocytes, leading to inhibition of DP thymocyte deletion. In contrast, CTLA-4 negatively regulates the activation of SP thymocytes.     

Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8(-). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.     

Effects of nicotine exposure on T cell development in fetal thymus organ culture: arrest of T cell maturation

There is evidence for both physiological functions of the natural neurotransmitter, acetylcholine, and pharmacological actions of the plant alkaloid, nicotine, on the development and function of the immune system. The effects of continuous exposure to nicotine over a 12-day course of fetal thymus organ culture (FTOC) were studied, and thymocytes were analyzed by flow cytometry. In the presence of very low concentrations of nicotine many more immature T cells (defined by low or negative TCR expression) and fewer mature T cells (intermediate or high expression of TCR) were produced. In addition, the numbers of cells expressing CD69 and, to a lesser extent, CD95 (Fas) were increased. These effects took place when fetal thymus lobes from younger (13-14 days gestation) pups were used for FTOC. If FTOC were set up using tissue from older (15-16 days gestation pups), nicotine had little effect, suggesting that it may act only on immature T cell precursors. Consistent with an increase in immature cells, the expression of recombinase-activating genes was found to be elevated. Nicotine effects were partially blocked by the simultaneous addition of the nicotinic antagonist d-tubocurarine. Furthermore, d-tubocurarine alone blocked the development of both immature and mature murine thymocytes, suggesting the presence of an endogenous ligand that may engage nicotinic acetylcholine receptors on developing thymocytes and influence the course of normal thymic ontogeny.