In vivo administration of IL-1 induces thymic hypoplasia and increased levels of serum corticosterone

Administration of IL-1 alpha or IL-1 beta to normal mice induces a decrease in thymic cellularity, the magnitude of which depends on the number of injections and dose of IL-1. Twice daily injections of 200 ng of IL-1 alpha or -beta for 4 days results in a 90% decrease in thymic cellularity, which regenerated after cessation of treatment. Study of thymocyte subpopulations revealed that the number of CD4+/CD8+ thymocytes was dramatically decreased in IL-1-treated mice. Functional assessment of the CD4-/CD8- population from treated animals showed that these cells had adequate mitogenic responses in vitro and that the proportion of these cells in cycle was not different from control CD4-/CD8- cells. IL-1 treatment also prevented the regeneration of thymic cellularity after irradiation. The use of strains of mice differing genetically at the Ly 1 locus to construct radiation bone marrow chimeras demonstrated that bone marrow-derived thymocyte precursors were able to seed the thymus in the IL-1-treated animals. Again, however, the CD4+/CD8+ thymocyte population was significantly decreased. Thymic repopulation occurred upon cessation of IL-1 therapy. Finally, we determined that a single i.p. injection of IL-1 caused a three-fold increase in serum corticosterone levels, which peaked approximately 3 h after IL-1 administration. Thus, an IL-1-dependent increase in serum corticosterone levels may be responsible for the observed thymic hypoplasia.     

Steroid sensitivity of thymocyte subpopulations during intrathymic differentiation. Effects of 17 beta-estradiol and dexamethasone on subsets expressing T cell antigen receptor or IL-2 receptor

We have studied the effects of the steroid hormones, 17 beta-estradiol and dexamethasone, on the relative proportion of thymocyte expression of CD4 (L3T4), CD8 (Ly-2), TCR and IL-2R, used to identify different stages of thymocyte differentiation. After short-term in vivo steroid treatment, a significant decrease in the number and proportion of the CD4+/CD8+, double positive subpopulation was observed in parallel with a proportional increase in the percentage of the CD4+/CD8- single positive, of the CD4-/CD8-, double negative and, to a lesser extent, of the CD8+/CD4- subsets. Either steroid treatment increased the proportion of cells expressing either the epsilon-chain of the CD3 complex and/or the beta-chain of the TCR (beta-TCR) (TCR+/CD3+) and the 55 kDa protein of the IL-2R (IL-2R+), related to the increase of CD4+ SP thymocytes and of DN cells, respectively. Furthermore, the increased proportion of CD3+ cells could also be partially related to the increase of both the CD4+ and DN subsets. A decrease of the effect on either DN/IL-2R+ cells or CD4+ SP cells was selectively observed after long-term treatments with 17 beta-estradiol or DEX, respectively. It is concluded that after short-term administration, different steroid hormones mediate a similar selective depletion of DP, TCR-/CD3-, IL2R- cells presumably in an intermediate stage of differentiation. However, either steroid effects evolve differently in long-term treatment schedules, resulting in different effects on early (DN/IL2R+) and late (SP/IL2R-) steps of thymocyte differentiation.     

Soluble factor-mediated differentiation of CD4+CD8+ thymocytes to single positives in vitro

Although the thymic microenvironment provides the necessary elements for T-cell differentiation, the precise role of individual components remains to be determined. In this paper, attempts were made to address the possibility that CD4 or CD8 single-positive (SP) thymocytes could be developed from immature CD4+CD8+ (double-positive; DP) thymocytes in a suspension culture in the presence of soluble factors. We observed that IL-4 and IFN-gamma weakly induced DP cells to differentiate to CD4 cells, but not to CD8. In contrast, IL-2 weakly induced differentiation to CD8. Interestingly, Con A sup strongly induced differentiation to CD8 SP from the purified DP thymocytes prepared from C57BL/6 or LCMV TCRtg mice. In particular, it was found that thymocyte culture with Con A sup generated CD69+DP cells, and the CD69+DP differentiated to CD8 SP under the suspension culture with soluble factors. Thus, Con A sup or combinations of IL-2, IL-4 and IL-7 strongly induced differentiation of CD69+DP to CD8 SP, whereas individual cytokines did not. These results suggest that soluble factors like cytokines play an important role in the generation of SP thymocytes in the absence of thymic stromal cells, at least from a distinctive subpopulation like CD69+DP thymocytes, and perhaps from those of broader range when in conjunction with TCR/MHC interaction.     

Expression of VLA-4 on thymocytes. Maturation stage-associated transition and its correlation with their capacity to adhere to thymic stromal cells.

The present study investigates the expression of VLA-4 on thymocytes at various stages of maturation and their capacity to adhere to thymic stromal cells. Whole thymocytes were stained with anti-CD4 and anti-CD8, as well as anti-VLA-4 antibodies. Flow microfluorometric analyses revealed that a) most of CD4-8- (double negative DN) and CD4-8intermediate thymocyte populations expressed large amounts of VLA-4, b) the levels of VLA-4 were considerably and markedly reduced on CD4+8+ (double positive DP) and single positive (SP) (CD4+8- or CD4-8+) populations, respectively. This contrasted with an increase in the levels of LFA-1 along with thymocyte maturation. DN, DP, and SP subsets were isolated and examined for their capacity to express VLA-4 and to adhere to fibronectin (FN) molecules as well as thymic stromal cells expressing FN. DN, DP, and SP subsets were confirmed to express the respective high, low, and very low levels of VLA-4, respectively. Approximately 70% of DN thymocytes became bound to FN-precoated culture plates, whereas 30 to 40% of DP and only 10 to 20% of SP cells adhered to FN. Similar patterns of adhesion were observed between these thymocyte subsets and thymic stromal monolayers. The binding of the DN subset to FN-plates or thymic stromal monolayers was inhibited only marginally by the RGDS peptide, but was efficiently inhibited by V10 peptide (cell-binding sequence that is located in the V region on FN and reacts with the VLA-4 integrin) or anti-VLA-4 antibody. Anti-VLA-4 antibody plus RGDS peptide strongly inhibited DN cell binding to FN-coated plates and thymic stromal monolayers. These results indicate that i) VLA-4 expressed on DN thymocytes functions as an important integrin for interacting with thymic stromal cells; ii) the expression level of this integrin decreases with the progress of thymocyte maturation, and iii) most of the mature thymocytes (SP) are rendered less adhesive to thymic stromal cells by reducing the level of VLA-4 expression.     

Selection of CD4+CD8+ thymocytes by complex formation with medulla-derived epithelial cells

The role of lymphostromal complexes in T-cell differentiation is far from elucidated, mainly because a clear association of a particular stromal cell type with a distinct thymocyte subset has never been identified. Using an in vitro system, detecting the adherence of thymocytes to a thymic medullary epithelial cell line (E-5), we showed that the phenotype of these thymocytes was that of cortical type: Thy-1hi, LFA-1+, PNAhi, CD4+CD8+, MEL-14-/lo, IL-2R-, CD3-/lo, and TcR V beta 8-/lo. They were enriched in cells in G2/M at the time of complex formation, showed a higher basal proliferation in culture, and did not respond to PHA, IL-2 and only marginally to Con A. These data show that complex formation with mouse thymic medullary epithelium selects for CD4+CD8+ thymocytes, as shown by the marked decrease in CD4+CD8-/CD4-CD8+ thymocytes, and the incapacity of CD4-CD8- thymocytes to adhere.     

p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent

The involvement of the tumor suppressor protein, p53, in thymic epithelial cell-induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage.     

Preferential proliferation and differentiation of double-positive thymocytes into CD8(+) single-positive thymocytes in a novel cell culture medium

The identification of factors that regulate the proliferation and differentiation of double-positive (DP) into CD4(+) and CD8(+) single-positive (SP) thymocytes has proven difficult due to the inability of DP thymocytes to proliferate, expand, and differentiate into SP thymocytes in available cell culture media. Here we report on the ability of DP thymocytes to differentiate in a novel conditioned medium, termed XLCM, derived from the supernatant of mitogen activated human cord blood mononuclear cells. During a 5-day culture in XLCM in the absence of thymic stromal cells, DP thymocytes from normal mice and MHC double knockout mice (lack SP thymocytes) proliferate, expand, and differentiate into several (alphabetaTCR(+), NK1.1(+)alphabetaTCR(+), and gammadeltaTCR(+)) subsets of CD4(+) and predominantly CD8(+) SP thymocytes. These studies suggest that the use of XLCM may aid in the characterization of factors that regulate the differentiation of DP thymocytes into CD8(+) SP thymocytes.     

Differentiation patterns of CD4/CD8 thymocyte subsets in cocultures of fetal thymus and lymphohemopoietic cells from c-fos transgenic and normal mice.

This study examined the involvement of c-fos protooncogene in thymocyte development from lymphohemopoietic T cell progenitors, within the thymic microenvironment. We first analyzed the thymocytes developing in vitro in the fetal thymus from the c-fos transgenic mice and found a high proportion of CD4+ single positive (SP) cells. We then seeded either fetal liver or bone marrow (BM) cells from normal donors onto lymphocyte-depleted fetal thymus explants of c-fos transgenic mice. The results showed an increased proportion of mature CD4+ SP and decreased CD4+CD8+ double positive (DP) cells. A similar pattern of CD4/CD8 thymocyte subsets was observed when either thymus or BM cells from c-fos transgenic mice developed within a normal thymic stroma. The kinetics of thymocyte development in organ culture (from Days 3 to 11) suggested that the SP cells obtained under these conditions may have bypassed the CD4+CD8+ DP phase. It appears that the altered pattern of thymocyte development manifested in adult c-fos transgenic mice can be induced by the early embryonic thymic stroma, and may also involve cells in the lymphohemopoietic tissues.     

Thymic involution and thymocyte phenotypic alterations induced by murine mammary adenocarcinomas

A profound thymic atrophy has been observed in mice bearing large adenocarcinomas of the mammary gland. Only 2 to 5% of thymocytes remained 4 wk after tumor implantation. Although there is a slight decrease in the overall percentages of Thy-1+ cells in tumor bearers, the majority of the remaining cells are of a Thy-1 low phenotype. There was a lower percentage of double positive (CD4+, CD8+) cells, an increase of CD4+ CD8- thymocytes, similar percentages of CD4- CD8+ cells and double negative (CD4- CD8-) thymocytes in tumor-bearing mice. In addition, an increased percentage of CD3 cells could be detected in these animals. These results indicate that proportionally less immature thymocytes are present in the atrophic thymuses of mammary tumor bearers. Enhanced levels of glucocorticoids are known to produce similar effects on the thymus. However, adrenalectomy of mice followed by tumor implantation did not result in reversal of the thymic atrophy. Furthermore, a study of serum corticosterone levels in tumor bearers indicated no significant changes during tumorigenesis. A study of several parameters of bone marrow (BM) populations indicate that there is an increase in cells of the granulocyte-macrophage lineage and a decrease in lymphocytes induced by tumor-derived granulocyte macrophage-CSF. An alteration of prothymocytes in the BM is not the main cause of the thymic atrophy because BM cells from normal and tumor-bearing mice reconstituted irradiated normal mice equally well. There was no preferential recruitment of double positive cells to the spleen as indicated by no significant differences in the levels of T cells of immature phenotype including the CD4+ CD8+ population in the spleens of tumor bearers. Because no major changes were observed in tumor bearers, either at their capacity to repopulate the thymus or at the patterns of subsequent redistribution of thymocytes, it was postulated that the thymic atrophy may be caused by a direct or indirect effect of the tumor or tumor-associated factor(s). Intrathymic injections of tumor cells into young normal recipient mice resulted in a significant reduction of the thymus weight and cellularity. These data suggest that mammary tumors can secrete factor(s) that are capable of severely impairing the normal development of cells of the T cell lineage.     

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.     

Role of a thymic stromal cell clone in inducing the stage-specific differentiation of various subpopulations of double negative thymocytes

The monolayer of a thymic stromal cell clone termed MRL104.8a induced the differentiation of adult double negative (DN) thymocytes (CD3-4-8-) through a CD3-4-8+ intermediate into CD3- (or dull) 4+8+ stages. DN thymocytes were separated into three subpopulations depending on their cell-surface expression of Pgp-1 and IL-2R, namely, Pgp-1+IL-2R-, Pgp-1-IL-2R+, and Pgp-1-IL-2R-. The present study investigated the requirements of the MRL104.8a monolayer for inducing the differentiation of these DN thymocyte subpopulations. The following were revealed: i) the MRL104.8a monolayer failed to induce the differentiation of a Pgp-1+IL-2R- subpopulation; ii) whereas a Pgp-1-IL-2R+ subpopulation did not express either CD4 or CD8 Ag when cultured in medium, culturing this subpopulation on the thymic stromal cell monolayers resulted in the expression of CD8 but not CD4 Ag; and iii) a Pgp-1-IL-2R- DN subpopulation obtained through less extensive treatments with anti-CD4 and anti-CD8 antibodies in the presence of C before sorting procedures spontaneously differentiated into double positive cells in medium. In contrast, most of DN cells with the same phenotype obtained through extensive anti-CD4 and -CD8 treatments before sorting failed to express CD4 and/or CD8 Ag in medium but could differentiate through a CD3-4-8+ into more mature stages only when they were cultured on the thymic stromal monolayer. These results indicate differential requirements of thymic stromal cells for the differentiation of various DN subpopulations with qualitatively distinct phenotypes and different magnitudes (very low vs almost zero levels) of CD4/CD8 expression.     

Intrathymic radioresistant stem cells follow an IL-2/IL-2R pathway during thymic regeneration after sublethal irradiation

Sublethally irradiated mice undergo thymic regeneration which follows a phenotypic pattern of events similar to that observed during normal fetal development. Thymic regeneration after irradiation is the product of a limited pool of intrathymic radioresistant stem cells undergoing simultaneous differentiation. We show that in this model of T cell development, thymic regeneration follows a pathway in which the IL-2R is transiently expressed on CD4-/CD8- cells. IL-2R expression occurred during the exponential growth period of thymic regeneration, and IL-2R blocking prevented this explosive growth. Flow cytometry analysis revealed that the IL-2R blockade affected primarily the development of the immature CD3-/CD4-/CD8- (triple negative) cells and their ability to generate CD3+/CD4+/CD8+ or CD3+/CD4+/CD8- and CD3+/CD4-/CD8+ thymocytes. Thus, our findings demonstrate that blocking of the IL-2R resulted in an arrest in proliferation and differentiation by intrathymic radioresistant stem cells, indicating that the IL-2/IL-2R pathway is necessary for the expansion of immature triple negative T cells.     

Abnormal thymocyte maturation in spontaneously diabetic BB rats involves the deletion of CD4-8+ cells.

The object of this study was to further characterize the pathophysiology of the peripheral T lymphopenia in the BB rat. Towards this end, surface markers on unseparated thymocytes and purified thymocyte subsets from age- and sex-matched diabetes-resistant (BBn) and diabetes-prone (BBd) rats were analyzed by two-color flow cytometry. The proportions of thymocytes falling into each of the four main phenotypic subsets were comparable in BBn (n = 9) and BBd (n = 8) rats: respectively, 4.6 +/- 0.6% and 4.4 +/- 0.8%, CD4-8-; 68.1 +/- 1.9% and 71.1 +/- 3.2%, CD4+8+; 18.3 +/- 1.5% and 15.4 +/- 2.3%, CD4+8-; 9.1 +/- 0.9% and 9.1 +/- 1.0%, CD4-8+. In addition, absolute numbers of thymocytes were not significantly different. The levels of expression of CD4, TCR-alpha beta within each thymocyte subset were comparable in BBn and BBd animals as were the anti-TCR-induced proliferative responses of their CD4+8- and CD4-8+ thymocytes. However, phenotypic abnormalities within the CD4-8+ thymocyte subset of the BBd rat were found. A very significant (p less than 0.005) deletion of mature CD4-8+, TCR-alpha beta + thymocytes and a proportional increase (p less than 0.005) of immature CD4-8+, TCR-alpha beta low thymocytes. Moreover, a twofold decrease of CD8 expression by mature CD4-8+ thymocytes was observed in BBd animals. These results suggest that an impaired thymic maturation contributes to the peripheral T lymphopenia of the BBd rat.     

Phenotypic complexity of intraepithelial lymphocytes of the small intestine.

A detailed phenotypic analysis of intraepithelial lymphocytes (IEL) of the small intestine was performed using multicolor fluorescence flow cytometry. CD4+8+ IEL (double positives; DP) could be detected in significant numbers in preparations from several mouse strains. DP IEL expressed Tcr alpha, beta and Thy1. Comparison of Tcr alpha, beta levels of thymocytes and IEL revealed that whereas the majority of DP thymocytes expressed low Tcr levels, DP IEL expressed high, mature T cell levels of Tcr. In addition, DP IEL were generally Ly3- (CD8 beta), unlike their thymic counterparts, which are Ly3+. Ly3 was not present on Tcr gamma, delta IEL, whereas CD4-8+ Tcr alpha, beta IEL contained Ly3- and Ly3+ subsets. The Ly3- population in either Tcr-bearing subset could be further subdivided by Thy1 expression. Ly1 (CD5) expression was also examined, and none of the Tcr gamma, delta IEL were Ly1+. Based on Thy1, Ly3, and Ly1 expression, four CD4-8+ Tcr alpha, beta IEL subsets were detected. The results indicate the cellular complexity of the IEL compartment rivals that found in the thymus. These findings are discussed in light of recent data suggesting an extrathymic origin of some IEL.     

Developmental regulation of the intrathymic T cell precursor population

The maturation potential of CD4-8- thymocytes purified from mice of different developmental ages was examined in vivo after intrathymic injection. As previously reported, 14-day fetal CD4-8- thymocytes produced fewer CD4+ than CD8+ progeny in peripheral lymphoid tissues, resulting in a CD4+:CD8+ ratio of less than or equal to 1.0. In contrast, adult CD4-8- thymocytes generated CD4+ or CD8+ peripheral progeny in the proportions found in the normal adult animal (CD4+:CD8+ = 2 to 3). Here we have shown that CD4-8- precursor cells from the 17-day fetal thymus also produced peripheral lymphocytes with low CD4+:CD8+ ratios. Precursors from full term fetuses produced slightly higher CD4+:CD8+ ratios (1.1-1.6) and precursors from animals three to 4 days post-birth achieved CD4+:CD8+ ratios intermediate between those produced by fetal and adult CD4-8- thymocytes. Parallel changes in the production of alpha beta TCR+ peripheral progeny were observed. Fetal CD4-8- thymocytes generated fewer alpha beta TCR+ progeny than did adult CD4-8- thymocytes. However, peripheral lymphocytes arising from either fetal or adult thymic precursors showed similar proportions of gamma delta TCR+ cells. The same pattern of progeny was observed when fetal CD4-8- thymocytes matured in an adult or in a fetal thymic stromal environment. In contrast to fetal thymic precursors, fetal liver T cell precursors resembled adult CD4-8- thymocytes by all parameters measured. These results suggest that fetal thymic precursors are intrinsically different from both adult CD4-8- thymocytes and fetal liver T cell precursors. Moreover, they lead to the hypothesis that the composition of the peripheral T cell compartment is developmentally regulated by the types of precursors found in the thymus. A model is proposed in which migration of adult-like precursors from the fetal liver to the thymus approximately at birth triggers a transition from the fetal to the adult stages of intrathymic T cell differentiation.     

Caspases in T-cell receptor-induced thymocyte apoptosis.

Apoptosis eliminates inappropriate or autoreactive T lymphocytes during thymic development. Intracellular mediators involved in T-cell receptor (TCR)-mediated apoptosis in developing thymocytes during negative selection are therefore of great interest. Caspases, cysteine proteases that mediate mature T-cell apoptosis, have been implicated in thymocyte cell death, but their regulation is not understood. We examined caspase activities in distinct thymocyte subpopulations that represent different stages of T-cell development. We found caspase activity in CD4+CD8+ double positive (DP) thymocytes, where selection involving apoptosis occurs. Earlier and later thymocyte stages exhibited no caspase activity. Only certain caspases, such as caspase-3 and caspase-8-like proteases, but not caspase-1, are active in DP thymocytes in vivo and can be activated when DP thymocytes are induced to undergo apoptosis in vitro by TCR-crosslinking. Thus, specific caspases appear to be developmentally regulated in thymocytes.