Induction of calcium influx in immature T lymphocytes by anti-FT-1 monoclonal antibody

The functional role of FT-1 antigen was investigated by measuring changes in intracellular [Ca2+]i after the addition of anti-FT-1 monoclonal antibody to murine T leukemic cells and embryonic thymocytes. Anti-FT-1 induced a rapid and sustained increase in cytosolic free [Ca2+]i and this effect was completely blocked by EGTA and La3+. These results suggest that FT-1 molecule is functionally associated with a plasma membrane calcium ion channel in embryonic thymocytes.     

Detection and localization by the monoclonal anti-interleukin 2 receptor antibody AMT-13 of IL 2 receptor-bearing cells in the developing thymus of the mouse embryo and in the thymus of cortisone-treated mice

During embryonic development of the mouse, before expressing classical T cell markers, the blast cells colonizing the thymus react with the monoclonal antibody AMT-13 shown previously to detect interleukin 2 receptors. The proportion of AMT-13+ cells decreases as gestation time increases. On the other hand, the proportion of Thy-1+, Lyt-1+ and Lyt-2+ cells increases during ontogenesis. On the 19th day of gestation when the thymus architecture is comparable to the adult thymus, the AMT-13+ cells become localized in the subcapsular area of the cortex. In the adult thymus after cortison treatment the regenerating cells express the AMT-13 antigen. The AMT-13 antigen presumably the interleukin 2 receptor is the first marker of the early embryonic thymocytes reported until now that may be related to cellular function.     

Cell division-associated expression of an epitope, KH17, on early developing thymocytes.

A newly established monoclonal antibody, KH17, detects a unique epitope temporarily expressed on early developing CD3-thymocytes confined to a cycling stage. KH17 is detectable on a part of CD4-CD8-,CD4-CD8+, and CD4+CD8+ cells, but not on CD4+CD8- thymocytes. By four-color flow cytometry analysis using KH17, we were able to define the heterogeneity of immature CD4-CD8- thymocytes by the expression of KH17 and IL-2R. In Thy-1-congeneic bone marrow chimeras, the appearance of KH17-IL-2R+ thymocytes preceded the increase of KH17+IL-2R- cells. The antibody could also divide CD3-CD4-CD8+ cells into two subpopulations, KH17+ and KH17-, which showed a continuum. In the fetal thymus there was a rapid and dramatic increase of KH17-CD4+CD8+ thymocytes concomitant with a decrease of KH17+CD4-CD8+ thymocytes in later gestation days. KH17 is not expressed on resting peripheral T cells, but is expressed on a large proportion of Con A-activated blastic spleen cells. The KH17 molecules precipitated from Con A-activated spleen cells were 55 and 75 kd polypeptides, but different from IL-2R subunits.     

Estrogen Inhibits Fetal Thymocyte Development In Vitro

PROBLEM: Transient involution of the maternal thymus in mice is known to occur during pregnancy. We have previously reported that the hormone responsible for this involution is estrogen. Interestingly, although estrogen crosses the placenta, fetal thymus gland enlarges with advancing gestational age. It is not known if fetal thymocytes are resistant to estrogen or if there are other factors that prevent estrogen from exerting an effect on the development of fetal thymocytes. Therefore we studied the effect of estrogen on isolated fetal thymic glands in vitro. METHOD OF STUDY: Pregnant Balb/c mice were sacrificed at 15 days gestation and fetal thymic lobes were obtained from all fetuses. The glands were cultured in vitro using either control medium or medium to which estrogen was added in two concentrations of 0.5 mg/ 100 ml and 1.0 mg/100 ml. After 12 days of organ culture, total thymocyte counts and phenotypic analysis by three color flow cytometry were performed by using monoclonal antibodies to surface markers of T cell subsets. RESULTS: Estrogen treatment caused a marked suppression of the total number of fetal thymocytes. All CD4 and CD8 defined T cell subsets were reduced with a disproportionate loss of CD4+ single positive (SP), CD8+ SP; CD4+CD8+ double positive (DP) cells. The early thymocyte developmental stages, based on CD44 and CD25 expression, revealed the CD4-CD8-CD3- triple negative compartment (TN) to be composed of almost entirely the earliest population (CD44+CD25-) with the remaining maturational stages depleted. CONCLUSIONS: This study demonstrates that fetal thymus removed from the intact fetus is susceptible to the inhibitory effects of estrogen. Since the fetal thymus enlarges with advanced gestational age, it is clear that the intact fetus invokes a regulatory mechanism which neutralizes the anti-lymphopoietic action of estrogen observed in the adult female.     

Stage-specific expression of mouse BST-1/BP-3 on the early B and T cell progenitors prior to gene rearrangement of antigen receptor

Human bone marrow stromal cell antigen 1 (BST-1) was identifiedas a glycosylphosphatidylinositol-anchored ectoenzyme expressedon bone marrow stromal or synovial cell lines and having theability to facilitate pre-B cell line growth. The analysis ofthe expression of mouse BST-1/BP-3 on the surface of lymphoidcells in the bone marrow and thymus revealed that it was verytransiently expressed on both B and T cell progenitors undergoinggene rearrangement of the antigen receptor. Among CD45R⁺CD43+B cell progenitors in the bone marrow, BST-1 expression appearedon the CD24 (heat stable antigen)⁺, CD19⁺ or CD117 (c-kit)⁺population. In the thymus, BST-1 was expressed on CD4^-CD8^-CD3^-[triple negative (TN)] CD90 (Thy-1]+ cells. In TN thymocytes,the majority of CD25⁺ cells and CD44¹⁰ cells expressed BST-1.In fetuses, BST-1+ cells appeared In the thymus and liver atday 14 and 16 of gestation respectively. The expression levelof BST-1 by fetal thymus was maximal and >60% of thymocyteswere positive for BST-1 at day 15 or 16 and the proportion thengradually decreased during development. Among day 15 fetal thymocytes,BST-1 was negative on the CD44⁺CD25^- fraction, very slightlypositive on the CD44+CD25+ fraction, and strongly positive onthe CD44^10-CD25+ and CD44^-CD25^- fractions. These results showedthat murine BST-1 is a useful marker for lymphoid progenitorcells initiating gene rearrangement of their antigen receptors.     

Expression of early activation antigen (CD69) during human thymic development.

The novel early activation antigen, EA1, has been shown to be induced by mitogens, antigens and the tumour promoter, phorbol myristate acetate (PMA), on human lymphocytes. This antigen has been designated to be CD69. EA1 has also been shown to be expressed on thymocytes without exogenous activation stimuli. In order to characterize further the expression of EA1 on thymocytes, the ontogeny of its expression was studied. EA1 appeared between 7 and 9.5 weeks of gestation, after colonization of the thymic rudiment with CD7+ T cell precursors, but before the onset of compartmentalization of the thymus into cortical and medullary zones. After cortico-medullary differentiation, the majority of medullary thymocytes expressed EA1 while only a fraction of the cortical thymocytes expressed this antigen. In the fetal and post-natal cortex, EA1 expression appeared to cluster in the subcapsular cortex. EA1+ cells were also scattered throughout the inner cortex. By two-colour fluorocytometric analysis of post-natal thymocytes, it was shown that EA1 was expressed on 30 to 65% of thymocytes. EA1 was expressed on CD4+ CD8+ as well as on the more immature CD4- CD8- thymocytes. In contrast to circulating T cells, thymocytes were much less responsive to PMA stimulation for the expression of EA1. Molecular characterization showed that EA1 on thymocytes had the same structure as that of activated peripheral T cells. In addition, thymic EA1 was constitutively phosphorylated. Thus, EA1 expression is acquired early during thymic development after colonization of the thymic rudiment by CD7+ T cell precursors. However, the specific role that EA1 may play in the activation and function of developing thymocytes remains to be determined.     

Interleukin 7 preferentially supports the growth of gamma delta T cell receptor-bearing T cells from fetal thymocytes in vitro.

Murine fetal thymus cells were cultured with various interleukins (IL-1, 2, 3, 4, 5, 6, and 7) in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and it was found that only IL-4 and IL-7 induced a prominent proliferative response in the presence of PMA. A large proportion of cells grown in the cultures of fetal thymus cells (days 15 and 17 of gestation) stimulated with PMA plus IL-4 or with PMA plus IL-2 remained CD4-CD8-. In marked contrast, nearly 70% of the cells generated in the cultures of the same fetal thymocytes stimulated with PMA plus IL-7 expressed CD8 on their surface. Approximately 30% of these cells expressed TCR gamma, delta, whereas TCR alpha beta+ cells were virtually undetectable. The cells grown in cultures stimulated with PMA plus IL-7 comprised three populations: CD4-Lyt-2-3-, CD4-Lyt-2 + Lyt-3- and CD4-Lyt-2 + Lyt-3+, and that TCR gamma delta+ T cells were found in all three populations. It was also found that the addition of IL-7 in the culture of adult CD4-CD8- thymocytes on the monolayer of a thymic stromal cell line, which selectively promotes the generation of alpha beta T cells, resulted in the generation of gamma delta T cells. These results strongly suggest that IL-7 plays an important role in the development of gamma delta T cells.     

Morphological and immunocytochemical examinations on development of B-cells in human embryonic and fetal liver

Morphological characteristics of B cell in human embryonic and fetal livers during the first trimester of gestation were examined. Light microscopically, CD9+, CD10+, CD19+, and CD20+ cells of B cell lineage became detectable as small lymphoid cells from 8 weeks gestation. Tdt+ cells first appeared also as small lymphoid cells on the 43th day of gestation. Ia+ or CD34+ cells in embryonic livers between the 33th and 43th day of gestation were large blastic cells resembling myeloblasts while some of Ia+ or CD34+ cells after the 43th day of gestation as well as Tdt+ cells were similar to lymphocytes. Electron-microscopically, all Ia+, CD10+, and CD19+ cells existed solitarily in intercellular spaces of hepatocytes, but not in intravascular spaces. Ultrastructural aspects of these cells were distinguishable each other. These findings indicate that 1) B cells developed and differentiated in the fetal liver, but the fetal liver during the first trimester was not a lymphoid organ, 2) lymphohemopoietic progenitor cells were derived from Tdt+ cells in the livers between 43th and 56th day of gestation. Ia+ cells detected as a small lymphoid cell in the liver at the 50th day were considered to be progenitor cells of lymphocytic lineage.     

TLR在小鼠胸腺中的表达及其在胸腺细胞发育中的可能作用

检测体外培养和体内发育过程中,胎鼠胸腺处于不同发育阶段时Toll样受体(TLR)的表达,阐明TLR表达量与胸腺细胞发育相关性,为TLR和胸腺细胞发育分化相关研究提供基础数据。无菌取15d胎龄胎鼠胸腺进行体外培养(FTOC),在培养不同时间点(2d,4d,6d),检测处于不同发育期胸腺TLR的表达;同时在孕期不同天数(15~19d),分别取胎鼠胸腺,检测在体内发育过程中胸腺TLR的表达;在FTOC中加入二脱氧鸟苷培养6d以制备胸腺基质细胞,检测基质细胞与胸腺细胞TLR表达情况。结果:小鼠胸腺中检测到多种TLR。FTOC培养中:培养第2天(F2)开始检测到各种TLR,到培养第6天(F6),TLR1,TLR3,TLR6,TLR7,TLR8明显上调,而TLR4,TLR5保持低水平,TLR4在培养第6天又下降;体内发育过程中:TLR6表达量随胎龄增加有较明显上调,TLR1,TLR3-8保持低水平表达;TLR2,TLR9体内体外都未检测到明显表达。在对胸腺细胞与基质细胞TLR表达比较中发现TLR1,TLR5,TLR6,TLR7高表达于胸腺细胞。胎鼠胸腺表达某些TLR,并且在发育不同阶段表达量有所改变,提示TLR可能参与胸腺细胞的发育过程。     

Early deletion and late positive selection of T cells expressing a male-specific receptor in T-cell receptor transgenic mice

The ontogeny of T cells in T-cell receptor (TCR) transgenic mice, which express a transgenic alpha beta heterodimer, specific for the male (H-Y) antigen in association with H-2Db, was determined. The transgenic alpha chain was expressed on about 10% of the fetal thymocytes on day 14 of gestation. About 50% of day-15 fetal thymocytes expressed both alpha and beta transchains and virtually all fetal thymocytes expressed the transgenic alpha beta heterodimer by day 17. The early expression of the transgenic TCR on CD4-8- thymocytes prevented the development of gamma delta cells, and led to accelerated growth of thymocytes and an earlier expression of CD4 and CD8 molecules. Up to day 17, no significant differences in T-cell development could be detected between female and male thymuses. By day 18 of gestation, the male transgenic thymus contained more CD4-8- thymocytes than the female transgenic thymus. The preponderance of CD4-8- thymocytes in the male transgenic thymus increased until birth and was a consequence of the deletion of the CD4+8+ thymocytes and their CD4-8+ precursors. By the time of birth, the male transgenic thymus contained half the number of cells as the female transgenic thymus. The deletion of autospecific precursor cells in the male transgenic mouse began only at day 18 of gestation, despite the fact that the ligand could already be detected by day 16. The preferential accumulation of CD4-8+ T cells, which expressed a high density of the transgenic TCR, occurred only after birth and was obvious in 6-week-old female thymus. These data support the hypothesis that the positive selection of T cells expressing this transgenic heterodimer may involve two steps, i.e., the commitment of CD4+8+ thymocytes to the CD4-8+ lineage following the interaction of the transgenic TCR with restricting major histocompatibility molecules, followed by a slow conversion of CD4+8+ thymocytes into CD4-8+ T cells. In normal mice, the precursors of CD4+8+ and single positive thymocytes have the CD4-8- CD3-J11d+ (or M1/69+) phenotype. Because of the early expression of the transgenic alpha beta heterodimer, this population was not detected in adult transgenic mice. All CD4-8- M1/69+ cells expressed the transgenic receptor associated with CD3 and could be readily grown in media containing T-cell lectins and interleukin 2.     

Human thymic epithelium and T cell development: current issues and future directions

The human thymus develops early in fetal gestation with morphologic maturity reached by the beginning of the second trimester. TE3+ cortical thymic epithelium is most likely derived from endodermal third pharyngeal pouch, while A2B5/TE4+ medullary and subcapsular cortical thymic epithelium is likely derived from third pharyngeal cleft ectoderm. Fetal liver and yolk sac CD7+, CD4-, CD8-, surface(s) CD3- T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression of other T lineage surface molecules on developing thymocytes. CD7+, CD4-, CD8-, sCD3- thymocytes give rise to T cells of both the TCR alpha beta and TCR gamma delta lineages. Human thymic epithelial cells produce numerous cytokines including IL1, IL6, TGF alpha, leukemia inhibitory factor (LIF), M-CSF, G-CSF and GM-CSF- molecules that likely play important roles in multiple stages of thymocyte selection, activation and differentiation. Important areas for future research on human thymic epithelium include study of lymphoid and non-lineage differentiation potentials of CD7+, CD4-, CD8-, sCD3- T cell precursors in response to TE-cell produced cytokines, study of the triggering signals of cytokine release within the thymic microenvironment, and study of TCR-MHC mediated TE-thymocyte interactions.     

Immunocytochemical characterization of lymphocyte development in human embryonic and fetal livers

Lymphohemopoietic progenitor cells and the development of lymphocytes in human embryonic and fetal livers during the 4 to 11 weeks of gestation were examined immunocytochemically by using a panel of monoclonal antibodies. CD9+, CD10+, CD19+, and CD20+ cells of B cell lineage became detectable from the 8th gestational week. CD2+ and CD3+ cells of T cell lineage were observed from the 10th gestational week. Tdt+ cells first appeared on the 43rd day of gestation. Both CD34+ and Ia+ cells were observed in all examined livers, and these cells appeared morphologically as small lymphoid cells from the 43rd day of gestation. These seemed to suggest that B lymphocytes developed in fetal liver from 8 weeks of gestation and lymphohemopoietic progenitor cells were comprised in Tdt+ cells in liver during the 43rd to 56th day of gestation.     

Requirements for growth of immature thymocytes from fetal and adult mice in vitro

We report here defined culture conditions that allow reproducibly the growth of the majority of immature thymocytes from both fetal (14-15 days of gestation) and adult mice. The combination of phorbol myristate acetate (PMA), ionomycin and recombinant interleukin 2 (IL2) is both sufficient and necessary to induce growth of about 1/6.2 (range 1/3-1/9) and 1/4.3 (range 1/2-1/7) immature thymocytes from adult and fetal mice, respectively, in serum-free cultures. Several other combinations tested (e.g. PMA + IL2, concanavalin A + IL2) were poorly or not active. None of the agents tested alone (PMA, ionomycin, concanavalin A, pokeweed mitogen, IL2) had any effect. We found no evidence for a role of IL1 and IL3 on growth of these cells. The growth of activated immature thymocytes from either fetal or adult mice was inhibited by a monoclonal antibody against mouse IL2 receptors. Under the same conditions that stimulated growth of most immature thymocytes, they did not mature into cells expressing Lyt-2, L3T4 or T cell antigen receptor (KJ16) after 7 to 15 days of continuous proliferation in culture. Nor did they give rise to cells with cytolytic activity after 7-9 days of culture. In some but not all experiments cultures of immature thymocytes from adult mice but not from fetal mice generated cells (1 out of 120-310) with helper function for B lymphocytes. While we confirmed here that approximately 50-70% freshly isolated immature thymocytes express receptors for IL2, our results indicate that these cells need to be activated (by e.g. PMA + ionomycin) to respond to IL2. A possible mechanism to account for the expression of nonfunctionally competent IL2 receptors is proposed and our results concerning the maturation of immature thymocytes in vitro are discussed.     

Expression and function of CD2 during murine thymocyte ontogeny

CD2, originally recognized as the sheep erythrocyte receptor of human T cells, has been implicated in early T cell development in the thymus. In this report, we examined the expression and functional role of CD2 during murine thymocyte ontogeny by using monoclonal antibodies to murine CD2. Surface expression of CD2 was first detected in Thy-1+ fetal thymocytes at day 14 of gestation and it progressively increased during CD4-CD8- phenotype. Surface IL 2 receptor (CD25) expression was readily detected in surface CD2- cells at day 13 of gestation and the majority of CD2+ cells appeared to be generated from CD25+ cells thereafter. In adult CD4-CD8- thymocytes, the expression of CD2 and CD25 was mutually exclusive. These results indicate that surface CD2 expression is not a prerequisite for CD25 induction during murine thymocyte ontogeny. This was further confirmed by fetal thymus organ culture in which anti-murine CD2 mAb was included. The antibody treatment led to a suppressed CD2 expression on thymocytes; however, there was no effect on the appearance of CD25. Moreover, no influence on the development of mature CD3+ thymocytes was observed after fetal thymus organ culture in the presence of anti-murine CD2 mAb, and a substantial number of CD3+CD2- cells was demonstrated in fetal and adult CD4-CD8- thymocytes. These findings argue against the functional relevance of CD2 expression during early T cell development as proposed in humans.     

Characterization of the response of human thymocytes and blood lymphocytes to the synergistic mitogenicity of 12-O-tetradecanoylphorbol-13-acetate (TPA)-ionomycin

The combination of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore ionomycin is synergistically mitogenic for human fetal and infant thymocytes as well as peripheral blood lymphocytes. Optimal mitogenic stimulation is achieved when TPA and ionomycin are used at doses of 0.5-1 ng/ml and 0.5-1 microgram/ml, respectively. Phenotypic analysis and cell sorting show that the thymocytes responsive to the mitogen have a mature or medullary phenotype (T1+, T3+, T11+, T6-, HLA-A,B++, [TdT]-); similarly in blood the T cell subsets (T11+, T4+ and T11+, T8+) are selectively responsive to TPA-ionomycin. Both activated lymphocytes and thymocytes express HLA-DR antigens as well as activation antigens such as T9, T10 and T cell activation antigen. T cells activated by TPA-ionomycin can be grown for periods of up to 50 days without addition of exogeneous interleukin 2. The observations may have implications for the membrane-associated signals involved in T cell growth and proliferation.     

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.     

Appearance and Maturation of T-Cell Subsets During Rat Thymus Ontogeny

In previous papers, we have described the ontogenetical development of thymic stromal-cell components (epithelium, macrophages, dendritic cells) of Wistar rats. Here, we correlate those results with the maturation of rat T-cell precursors along the fetal and postnatal life. First T-cell precursors, which colonize the thymus anlage around days 13-14 of gestation, largely express CD45, CD43, CD53, and Thy 1 cell markers, and in a lesser proportion the OX22 antigen. Rat CD3^-CD4^-CD8^- thymocytes present in the earliest stages of gestation could be subdivided in three major cell subpopulations according to the CD44 and CD25 expression: CD44^-/+CD25^- → CD44⁺CD25⁺ → CD44⁺CD25^- On fetal days 17-18, a certain proportion of CD4^-CD8^-cells weakly,express the TcRβ chain, in correlation with the appearance of the first immature CD4^-CD8⁺ thymocytes. This cell subpopulation, in progress to the CD4⁺CD8⁺ stage, upregulates CD8α before the CD8β chain, expresses the CD53 antigen, and exhibits a high proliferative rate. First mature thymocytes arising from the DP (CD4⁺CD8⁺) cells appear on fetal days 20-21. Then, the CD4⁺:CD8⁺ cell ratio is ≤1 changing to adult values (2-3) just after birth. Also, the percentage of VβTcR repertoire covered in adult thymus is reached during the postnatal period, being lower during the fetal life. Finally, in correlation with the beginning of thymocyte emigration to the periphery a new wave of T-cell maturation apparently occurs in the perinatal rat thymus.     

Non-random migration of CD4+, CD8+, gamma delta + T19+, and B cells between blood and lymph draining ileal and prescapular lymph nodes in the sheep fetus

We have examined the circulation of CD5+, CD4+, CD8+, gamma delta + T19+, and B cells through ileal and prescapular lymph nodes in the sheep fetus in an environment uninfluenced by foreign antigen and ongoing immune responses or circulating immunoglobulins, and have contrasted this circulation with that occurring through the same tissue in 1-year-old sheep. The vast majority of lymphocytes circulating through fetal prescapular lymph nodes and fetal ileal lymph were T cells; however, there was a significantly higher concentration of B cells in ileal lymph compared to prescapular lymph. Furthermore, in contrast to 1-year-old sheep, there was an imbalance in the distribution of CD4+ cells and CD8+ cells in fetal prescapular and ileal lymph, with CD4+ cells enriched in prescapular lymph relative to other T cell subsets and CD8+ cells enriched in ileal lymph. Our results suggest that in the fetus either there is preferential migration of CD4+ cells through peripheral lymph nodes and/or CD8+ lymphocytes through the ileal gut, or newly formed CD8+ lymphocytes are being released from the ileum or ileal lymph node directly into ileal lymph.     

Human intrathymic T cell differentiation

The human thymus develops early on in fetal gestation with morphologic maturity reached by the beginning of the second trimester. Endodermal epithelial tissue from the third pharyngeal pouch gives rise to TE3+ cortical thymic epithelium while ectodermal epithelial tissue from the third pharyngeal cleft invaginates and splits during development to give rise to A2B5/TE4+ medullary and subcapsular cortical thymic epithelium. Fetal liver CD7+ T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression on thymocytes of other T lineage surface molecules. Human thymic epithelial cells grown in vitro bind to mature and immature thymocytes via CD2 and CD11a/CD18 (LFA-1) molecules on thymocytes and by CD58 (LFA-3) and CD54 (ICAM-1) molecules on thymic epithelial cells. Thymic epithelial cells produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF--molecules that likely are important in various stages of thymocyte activation and differentiation. Thymocytes can be activated via several cell surface molecules including CD2, CD3/TCR, and CD28 molecules. Finally, CD7+ CD4-CD8- CD3- thymocytes give rise to T cells of both the TCRab+ and TCR gd+ lineages.     

Expression of high molecular weight isoforms of CD45 by mouse thymic progenitor cells

We have studied the expression of isoforms of CD45 (leukocyte common antigen, LCA) among T cell precursors using the organ culture system of Jenkinson et al. (Eur. J. Immunol. 1982. 12: 583). These experiments show that cells capable of recolonizing alymphoid embryonic thymic lobes in vitro can be detected in the thymus of fetal and adult mice and are enriched when thymocytes are depleted of cells bearing CD4 or CD8. These data are consistent with results from in vivo experiments of Fowlkes et al. (J. Exp. Med. 1985. 162: 802) indicating that T cell precursors lie within the double-negative (CD4-CD8-) compartment. No precursors were detected among the reciprocal populations of cells bearing CD4 and/or CD8 (single and double positives). Double-negative cell fractions were then divided on the basis of reactivity with monoclonal antibodies RA3-2C2 and RA3-3A1. These antibodies recognize the high molecular weight species of the LCA or, more accurately, a product defined by exon A of the CD45 gene. Recolonizing cells were found predominantly in the CD45RA+ (RA3-2C2 and RA3-3A1 reactive) fraction of double-negative thymocytes; CD45RA- enriched populations had increased efficiency of recolonization and CD45RA- depleted populations had decreased ability to recolonize as compared with the whole CD4-CD8- fraction. To clarify whether progenitors enriched in the CD45RA+ fraction were capable of giving rise to mature CD4+, CD8+ and CD4+ CD8+ cells, we analyzed the progeny of lobes seeded with CD4-CD8-CD45RA+ fractions. After 7-9 days in organ culture the proportion of CD4+, CD8+ or CD4+ CD8+ cells had increased to 35.2%, 18.6% and 23.7%, respectively (mean of five experiments), indicating that progenitors among the CD45RA+ population were indeed multipotent. These results suggest that the majority of T stem cells in the thymus are among thymocytes that express the CD45RA molecule, an hypothesis supported by our finding that removal of CD45RA-expressing cells (using complement and antibody) eliminated recolonizing capacity of thymic cell fractions.