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.     

Kinetics of thymocyte developmental process in fetal and neonatal mice

Kinetics of thymocyte development in vivo during embryogenesis was pursued. The early development of thymocytes in the fetal and neonatal BALB/c mice was discontinuous, with four waves of cell proliferation occurring at fetal day (Fd) 14 to 17, Fd 18 to day (D) 1 after birth, D 2 to D 5 and D6 thereafter. The first three proliferation waves coincided with the generation of CD4^hiCD8^hi (DP), TCR CD4^hiCD8^-/^loCD8^int/hi(CD4 SP), and TCR CD4^-/^loCD8^int/hi (CD8 SP) thymocytes, respectively. The transition from DN to DP cells was further investigated and it was found out that there were two differential pathways via im-mature single positive (ISP) cells in the BALB/c mice, each functioning at different fetal ages. One is via TCR^-CD4^-CD8^ cells, occurring between Fd 15 and Fd 17 and the other is via TCR^-CD4^ CD86-cells,occurring from Fd 17 until birth. In contrast, the TCR^-CD4^-CD8^ pathway dominated overwhelminglyin the C57BL/6 mice. These findings shed new light on the hypothesis that the differential pathway pref-erence varies with mouse strains. With respect to the shift in the intensity of CD4 and CD8 expression onthymocytes from fetal to adult mice, the TCR CD4^hiCD8^-/^lo, and TCR^ CD4^-/^loCD8^int/hi subsets might be equivalent to the medullary type TCR^ CD4/CD8 SP cells.     

Distinct patterns of lymphokine requirement for the proliferation of various subpopulations of activated thymocytes in a single cell assay

The frequency and capacity for clonal expansion of several murine thymocyte subpopulations responsive to various IL (fetal day 15, and adult CD4-8-, CD4+8- and CD4-8+) were investigated using a single-cell limiting-dilution cell culture system without filler cells. This assay requires the presence of PMA and ionomycin. The main conclusions of these studies are the following: 1) IL-4 is a better growth factor than IL-2 for immature thymocytes (fetal day 15 or adult CD4-8-). 2) IL-2 is a better growth factor than IL-4 for mature phenotype thymocytes (CD4+8- and CD4-8+). 3) IL-4 is a relatively poor growth factor for adult CD4-CD8- thymocytes and CD4+CD8- thymocytes, while it induced strong responses in fetal day 15 and CD4-8+ thymocytes. 4) IL-6 enhanced the response of CD4+8- thymocytes to either IL-2 or IL-4. 5) Cortisone-resistant thymocytes grown initially with IL-4 and then switched to IL-2 showed a significant decrease in cloning efficiency. No inhibitory effect was observed when cells were cultured first with IL-2 and then switched to IL-4. 6) Finally, supernatant from Con-A stimulated rat spleen cells induced maximal growth of all adult thymocyte populations tested, suggesting that unidentified thymocyte growth factor(s) remain to be characterized. These results indicate that the maturational stage of thymocytes determines their requirements for activation and proliferation.     

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance

This study shows that the normal thymus produces immunoregulatory CD25+4+8- thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8- thymocytes, which constitute approximately 5% of steroid-resistant mature CD4+8- thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8- thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8- or CD4-8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8- thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25-4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8- thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8- thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous alpha-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8- thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.     

Expression of an unusual T cell receptor (TCR)-V beta repertoire by Ly-6C+ subpopulations of CD4+ and/or CD8+ thymocytes. Evidence for a developmental relationship between Ly-6C+ thymocytes and CD4-CD8-TCR-alpha beta+ thymocytes.

A novel thymocyte subpopulation expressing an unusual TCR repertoire was identified by high surface expression of the Ly-6C Ag. Ly-6C+ thymocytes were distributed among all four CD4/CD8 thymocyte subsets, and represented a readily identifiable subpopulation within each one. Ly-6C+ thymocytes express TCR-alpha beta, arise late in ontogeny, and appear in the CD4/CD8 developmental pathway after birth in a sequence that resembles that followed by conventional Ly-6C- cells during fetal ontogeny. Most interestingly, adult Ly-6C+ thymocytes express an unusual TCR-V beta repertoire that is identical to that expressed by CD4-CD8-TCR-alpha beta+ thymocytes in its overexpression of TCR-V beta 8 and in its expression of some potentially autoreactive TCR-V beta specificities. This unusual TCR-V beta repertoire was even expressed by Ly-6C+ thymocytes contained within the CD4+ CD8- 'single positive' thymocyte subset. Thus, expression of this unusual TCR-V beta repertoire is not limited to CD4-CD8-thymocytes, and is unlikely to be a consequence of their double negative phenotype. Rather, we think that Ly-6C+TCR-alpha beta+ thymocytes and CD4-CD8-TCR-alpha beta+ are developmentally interrelated, a conclusion supported by several lines of evidence including the selective failure of both Ly-6C+ and CD4-CD8-TCR-alpha beta+ thymocyte subsets to appear in TCR-beta transgenic mice. In contrast, peripheral Ly-6C+ T cells are developmentally distinct from Ly-6C+ thymocytes in that peripheral Ly-6C+ T cells expressed a conventional TCR-V beta repertoire and developed normally in TCR-beta transgenic mice in which Ly-6C+ thymocytes failed to arise. We conclude that: 1) expression of a skewed TCR-V beta repertoire is a characteristic of Ly-6C+TCR-alpha beta+ thymocytes as well as CD4-CD8-TCR-alpha beta+ thymocytes, and is not unique to thymocytes expressing neither CD4 nor CD8 accessory molecules; and 2) Ly-6C+ thymocytes are developmentally linked to CD4-CD8-TCR-alpha beta+ thymocytes, but not to Ly-6C+ peripheral T cells. We suggest that Ly-6C+TCR-alpha beta+ thymocytes are not the developmental precursors of Ly-6C+ peripheral T cells, but rather may be the developmental precursors of CD4-CD8-TCR-alpha beta+ thymocytes.     

Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant-negative p21ras protein (H-rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck-H-rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR-derived signals in mature CD4+8- or CD8+4- thymocytes. In contrast, some TCR-derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant-negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H-Y-specific TCR and lck-H-rasN17 demonstrated that antigen-specific negative selection occurs normally in the presence of p21H-rasN17. Superantigen-induced negative selection in vivo also proceeded unhindered in H-rasN17 thymocytes. In contrast, positive selection of thymocytes in the H-Y mice was severely compromised by the presence of p21H-rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters intrathymic T-cell development in mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was administered to 2-4-week-old mice (5, 25, and 50 micrograms/kg body wt.) and to in vitro cultures (10(-9) M) of fetal thymi. By monitoring thymocyte populations with respect to the differentiation antigens CD4 and CD8, it was found that the cell number in all thymocyte populations except for CD8+ decreased significantly compared with controls. In vivo the most marked decrease occurred among double negative (DN) and double positive (DP) cells, whereas in vitro, the DP cells were most severely affected. The cell number had already decreased to some extent by day 1 after a dose of 50 micrograms/kg body wt. of TCDD, although a severe reduction did not become apparent until day 4. There was a clear dose/response relationship between 5 and 50 micrograms/kg body wt. Autoradiography and liquid scintillation counting studies showed that incorporation of [3H]thymidine in the thymus had already decreased 24 h after TCDD treatment, with the decrease being even more pronounced at 48 h. By 96 h, the rate of cell proliferation had returned to approximately normal values. The results show that TCDD has a long-lasting effect on thymocyte abundance together with a transient effect on cell proliferation. This indicates that in addition to the initial effects of TCDD on cell proliferation, it may also more permanently disturb the normal process of elimination by means of selection.     

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.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

Persistence of the irradiated host component in thymocyte populations from bone marrow radiation chimeras infected with lymphocytic choriomeningitis virus

The thymus of chimeras made using T cell-depleted donor bone marrow from Thy1.1+ mice and 950 rad Thy 1.2+ recipients is dominated initially by cells expressing the Thy 1.2+ phenotype of the irradiated host. The thymocyte population recovered at 2 weeks after reconstitution comprises 80% Thy 1.2+ cells (host), the remainder being Thy 1.1+ (donor). This situation is normally reversed within a further week, with the host Ty 1.2+ (donor). This situation is normally reversed within a further week, with the host Thy 1.2+ thymocytes being present at a frequency of less than 5% from Week 4. Infection with lymphocytic choriomeningitis virus (LCMV) at 1 week after reconstitution with bone marrow causes a profound and persistent drop in the total number of thymocytes. The decline is equivalent for all categories of donor-derived thymocytes defined by two-color flow microfluorometric analysis for CD4 and CD8. However, there is a partial compensation by the retention of cells originating from the Thy 1.2+ host, which constitute 30-40% of the total thymocyte pool as late as 8 weeks after administration of bone marrow in the LCMV-infected chimeras. These radiation-resistant precursors give rise to CD4-8-, CD4-8+, CD4+8-, and CD4+8+ thymocytes, with the latter category being present at increased frequency. The potential skewing of the mature T cell repertoire as a consequence of persistent virus infection is discussed.     

Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.

The maturation of CD4+8- and CD4-8+ thymocytes from CD4+8+ thymocytes is dependent on the mandatory interaction of their alpha beta TCR with selecting ligands expressed on thymic epithelial cells (TE). This is referred to as positive selection. The deletion of CD4+8+ thymocytes that express autospecific TCR (negative selection) is mediated primarily by bone marrow-derived cells. Previous studies have shown that TE is relatively ineffective in mediating the deletion of CD4+8- thymocytes expressing autospecific TCR but TE can render them anergic, i.e., nonresponsive, to the self Ag. The mechanism by which anergy is induced in these cells is unknown. In this study, we used thymocytes expressing a transgenic TCR specific for the male Ag presented by H-2Db class I MHC molecules to examine how expression of the deleting ligand by TE affects thymocyte development and phenotype. The development of female TCR-transgenic thymocytes was examined in irradiated male hosts or in female hosts that had received male fetal thymic epithelial implants. It was observed that the development of transgenic-TCR+ thymocytes was affected in mice with male TE. CD4+8+ thymocytes with reduced CD8 expression and markedly enhanced transgenic TCR expression accumulated in mice with male TE. Development of CD4-8+ thymocytes was also affected in these mice in that fewer were present and they expressed an intermediate CD8 coreceptor level. These CD4-8+ thymocytes expressed a high level of the transgenic TCR, retained the ability to respond to anti-TCR antibodies, but were nonresponsive to male APC. However, the maturation of CD4+8- thymocytes, which are also derived from CD4+8+ precursor cells, was relatively unaffected. In an in vitro assay for assessing negative selection, male TE failed to delete CD4+8+ thymocytes expressing the transgenic TCR under conditions where they were efficiently deleted by male dendritic cells. Collectively these results support the conclusion that male TE was inefficient in mediating deletion. Furthermore, expression of the deleting ligand on thymic epithelium interferes with the maturation of functional male-specific T cells and results in the accumulation of CD4+8+ and CD4-8+ thymocytes expressing a lower level of the CD8 coreceptor but a high level of the transgenic TCR.     

Selective support of a mouse thymus epithelial cell line （MTEC1） to the viability and proliferation of CD4＋CD8—,CD4^—CD8^—,and CD4^＋CD8^＋thymocytes in vitro

The MTEC1 cell line,established in our laboratory,is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constituteively produce multiple cytokines.The selection of thymic microenvironment on developing T cells was investigated in an in vitro system.Unseparated fresh thymocytes from Balb/c mice were cocultured with MTEC1 cells or/and MTEC1-SN,then,the viability,proliferation and phenotypes of cultured thymocytes were assessed.Without any exogenous stimulus,both MTEC1 cells and MTEC1-SN were able to maintain the viability of thymocytes,while only the MTEC1 cells,not the MTEC1-SN,could directly activate thymocytes to exhibit moderate proliferation,indicating that the proliferative signal is delivered through cell surface interatcions of MTEC1 cells and thymocytes.Phenotype analysis on FACS of viable thymocytes after coculture revealed that MTEC1 cells preferentially activate the subsets of CD4^ CD8^-,CD4^ CD^8 and CD^4- CD^8- thymocytes;whereas MTEC1-SN preferentially maintained the viability of CD4^ CD^8- and CD4^-CD8^ thymocyte subsets.For the Con A-activated thymocytes.both MTEC1 cells and MTEC1-SN provided accessory signal(s) to significantly increase the number of viable cells and to markedly enhance the proliferation of thymocytes with virtually equal potency,phenotyped as CD4^ CD8^-,CD4^-CD8^ ,and CD^4-CD8^-subests,In summary,MTEC1 cells displayed Selection of thymic epithelial cells on thymocyte subsets. selective support to the different thymocyte subsets,and the selectivity is dependent on the status of thymocytes.     

The role of LFA-1/ICAM-1 interactions during murine T lymphocyte development.

We have examined the expression and function of the cell adhesion molecules LFA-1 (CD11a/CD18), ICAM-1 (CD54), and ICAM-2 in murine fetal thymic ontogeny and in the adult thymus. On fetal days 14 and 15, 40 to 50% of thymocytes coexpress high levels of LFA-1 and ICAM-1, as determined by flow cytometry. By day 16, more than 90% of fetal thymocytes are LFA-1+ ICAM-1hi, and all IL-2R+ cells are located in this population. Although LFA-1 expression remains unchanged thereafter, ICAM-1 expression appears to be differentially regulated in different thymocyte subpopulations, with CD4+8+ cells being ICAM-1lo and CD4-8- thymocytes remaining ICAM-1hi. ICAM-2 surface expression is dull on both fetal and adult thymocytes. Surprisingly, the expression of ICAM-1 is differentially up-regulated on T cells having a mature phenotype in thymus and in peripheral lymphoid organs, with CD8+ T cells bearing the highest amount of surface ICAM-1. Addition of anti-ICAM-1 or anti-LFA-1 antibodies to fetal thymic organ cultures results in the impaired generation of CD4+8+ cells. These results indicate that LFA-1/ICAM-1 interactions facilitate murine thymic development and suggest that cell adhesion molecules mediate important events in T cell differentiation.     

A population of early fetal thymocytes expressing Fc gamma RII/III contains precursors of T lymphocytes and natural killer cells.

We have identified a dominant fetal thymocyte population at day 14.5 of gestation in the mouse that lacks CD4 and CD8 but expresses Fc gamma RII/III several days prior to acquisition of the T cell receptor (TCR) in vivo. If maintained in a thymic microenvironment, this population of CD4-CD8-TCR-Fc gamma RII/III+ thymocytes differentiates first into CD4+CD8+TCRlowFc gamma RII/III- thymocytes and subsequently CD4+CD8-TCRhighFc gamma RII/III- and CD4-CD8+TCRhighFc gamma RII/III- mature Ti alpha-beta lineage T cells. However, if removed from the thymus, the CD4-CD8-TCR-Fc gamma RII/III+ thymocyte population selectively generates functional natural killer (NK) cells in vivo as well as in vitro. These findings show that a cellular pool of Fc gamma RII/III+ precursors gives rise to T and NK lineages in a microenvironment-dependent manner. Moreover, they suggest a hitherto unrecognized role for Fc receptors on primitive T cells.     

Delayed thymocyte maturation in the trisomy 16 mouse fetus

Mouse fetuses with trisomy 16, an animal model for human trisomy 21 (Down syndrome), have severe defects in several hematopoietic stem cell populations and a marked reduction in thymocyte number. To determine whether there are other defects in the development of the trisomic thymus, the ontogeny of the cell surface antigenic determinants, Thy-1, Ly-1, CD3, CD4, CD8, and TCR v beta, was investigated. The trisomy 16 thymocytes were able to express all of determinants either during fetal life (days 14 to 19 of gestation) or in cultures of intact thymus lobes. However, in all instances (except for Thy-1, which already had a high proportion of expressing thymocytes by day 14), there was a delay in the time at which the determinants were first expressed, as manifested by reduced numbers of positively staining cells. Furthermore, there was also a delay in the rate at which the positively staining cells attained maximal Ag densities. Overall, there was an approximate 2 day lag in development of the fetal trisomic thymocytes. This lag permitted the identification of a large population of CD4-8+ cells prior to the appearance of CD4+8+ thymocytes. These findings are consistent with the identification of CD4-8+ as an intermediate stage between CD4-8- and CD4+8+ in fetal thymocyte ontogeny.     

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.     

Beta-adrenoceptor blockade alters thymocyte differentiation in aged mice.

The thymus is the primary site for generation of naive T-lymphocytes in the young animal. With age, the thymus progressively involutes and fewer mature T-cells are produced and migrate to the periphery. With thymic involution, increased density of sympathetic noradrenergic (NA) innervation and concentration of norepinephrine (NE) have been observed. To determine if the age-related changes in thymocyte differentiation are modified by NE signaling through beta-adrenergic receptors, 2-month (mo) and 18-mo old BALB/c mice were implanted subcutaneously with pellets containing the non-selective beta-adrenoceptor antagonist nadolol. Four and one-half weeks later, thymus and peripheral blood were collected to assess changes in thymocyte differentiation and naive T-cell output by flow cytometric analysis of T-cell subpopulations. In old mice, but not in young mice, thymocyte CD4/CD8 co-expression was altered by beta-adrenoceptor blockade. In nadolol-treated old mice, the frequency of the immature CD4-8- population was increased, and the intermediate CD4+8+ population was reduced. A corresponding increase in the frequency of mature CD4-8+, but not CD4+8- cells was observed. The increase in CD4-8+ cells is most likely not mediated by more CD4-8+ cells undergoing positive selection, because CD3hi expression in the CD4+8+ population was not altered by nadolol. The percentage of CD8+44low naive cells in peripheral blood increased in nadolol-treated mice, suggesting that more CD4-8+ cells were exported from the thymus to the periphery. These results indicate that the age-associated increase in sympathetic NA innervation of the thymus modulates thymocyte maturation. Pharmacological manipulation of NA innervation may provide a novel means of increasing naive T-cell output and improving T-cell reactivity to novel antigens with age.     

Stage specific phosphoinositides turnover capacity of human intrathymic T cells following CD2-triggering

Triggering of distinct CD2 epitopes on human T lymphocytes increases their phosphatidylinositol (PI) cycle-related metabolism. In this work, we investigated the relationship between this signal transduction pathway following surface CD2 antigen triggering and intrathymic T cell development. Therefore, various thymocyte subsets were incubated with co-mitogenic CD2I+III mAb. The cells were then tested for their various phosphoinositides levels as well as their ability to proliferate in response to recombinant interleukin-2 (rIL-2). Our results indicate that immature CD4- CD8- cells have high PI metabolism while more mature CD4+CD8+ and unfractionated thymocytes display significantly lower PI-turnover. Mature CD4+CD8- and CD4-CD8+ thymocytes regain this transduction capacity. Thus, PI-turnover following CD2- triggering is linked to the developmental fate of thymocyte subclasses.