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.     

Epithelial-thymocyte interactions in human thymus

Our data demonstrate that the epithelial component of the human thymic microenvironment is not an inert cell type, but rather is capable of being directly involved in the promotion of both early and late stages of T-cell maturation. Data from our laboratory [54,69], together with the work of Plunkett et al. [61] and Shaw et al. [70] suggest that an endogenous ligand for the CD2 molecule in humans is the LFA-3 molecule. Using an SV40 transformed human thymic epithelial cell line of subcapsular cortical origin, Mizutani et al. have confirmed that thymic epithelial cells bind thymocytes via a CD2/LFA-3 interaction [78]. The data reviewed in this paper suggest that within the thymus one endogenous ligand for the alternative pathway of thymocyte activation via the CD2 molecule is the LFA-3 molecule on TE cells. Following thymocyte binding to TE cells, immature thymocytes are directly activated to proliferate, and their response to both IL1 and IL2 is augmented. Also, following TE-thymocyte binding, TE-IL1 secretion is augmented and TE cell MHC class II antigen expression is induced. Moreover, while undergoing activation, thymocytes appear to be able to modulate their microenvironment milieu of MHC antigens and IL1. Further analysis of the sequelae of TE-thymocyte interactions using phenotypic characterization of thymocytes with anti-T-cell MoAbs, coupled with molecular analysis of thymocyte T-cell receptor genes, should allow for the determination of the precise sequential stages that immature T cells undergo enroute to functional maturity. Understanding these steps in T-cell maturation will be critical to our understanding of the events that transpire in the genesis of autoimmune, lymphoproliferative, and immunodeficiency diseases.     

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.     

Enhancement of IL-7 following irradiation of fetal thymus

The effect of ionizing radiation on intra-thymic T cell development was investigated using a fetal thymic organ culture (FTOC) method in vitro. When double-negative (DN) fetal (day 15) thymocytes were co-cultured with an irradiated (25 Gy) fetal (day 15) thymus in the absence of direct contact or mitogenic stimulation, the induction of TCRgammadelta+ T cells was observed. About 50% of the TCRgammadelta+ T cells developed after 4-day-co-culture with the irradiated fetal thymus, whereas only a few TCRgammadelta+ T cells developed after co-culture with the non-irradiated fetal thymus. About 50% of the TCRgammadelta+ T cells were CD8+ cells with alphabeta heterodimeric chains. Cultured supernatants of the irradiated fetal thymi also induced the differentiation from DN thymocytes to CD8+ TCRgammadelta+ T cells after 3-day-culture. To clarify the factor in the cultured supernatants, several neutralizing antibodies (Abs) were used. Only anti-IL-7-Ab inhibited the differentiation from DN thymocytes to CD8+ TCRgammadelta+ T cells. RT-PCR revealed the increased expression of IL-7 mRNA in the fetal thymus 24 hours after radiation. Electron microscope studies demonstrated proliferative epithelial cells in the irradiated fetal thymus. These findings strongly suggest that fetal thymic epithelial cells affected by irradiation proliferate and enhance the production of IL-7, which induces the differentiation of CD8+ TCRgammadelta+ T cells from DN thymocytes.     

Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) provide co-stimulation in positive selection along with survival of selected thymocytes

T-cell differentiation in the thymus depends on positive selection of CD4+CD8+ double positive (DP) thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows maturation of only those thymocytes that are capable of self-peptide-MHC recognition. Thymocytes that fail to bind self-peptide-MHC die by apoptosis. An important question in thymocyte differentiation is whether co-stimulation is required for positive selection and on which cells co-stimulatory molecules may be expressed in the thymus. The vascular cell adhesion molecule (VCAM-1) and the intercellular cell adhesion molecule (ICAM-1) are known to be potent co-stimulatory molecules in activation of peripheral T-cells by interacting with the integrins VLA-4 and LFA-1, respectively. We were prompted to investigate whether VCAM-1 and ICAM-1 may also act as co-stimulators during selection of thymocytes. By using recombinant proteins of murine VCAM-1 and ICAM-1 fused to the Fc region of human IgG1 (rVCAM-1, rICAM-1) we examined the capacity of VCAM-1 and ICAM-1 to act as co-stimulatory molecules in positive selection in vitro. Triggering the CD3/TCR complex together with co-stimulation applied by rVCAM-1 or rICAM-1 induced the generation of CD4+ single positive (SP) thymocytes from CD4+CD8+ DP thymocytes whereas either signal alone did not result in generation of CD4+ SP thymocytes. VCAM-1 and ICAM-1 act therefore as co-stimulatory molecules in thymocyte positive selection in vitro. The generation of CD4+ SP cells is accompanied by cell survival both when it was co-stimulated with rVCAM-1 and with rICAM-1. Importantly we show here that VCAM-1 expression in the murine thymus is restricted to cortical F4/80 positive hematopoietic antigen presenting cells (hAPC) present exclusively in the cortex whereas expression of ICAM-1 has been reported on the epithelium both in cortex and medulla. This suggests that not only the cortical epithelium may use the co-stimulatory molecule ICAM-1 to mediate positive selection, but also cortical hAPCs may contribute to positive selection of thymocytes by using the co-stimulator VCAM-1.     

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.     

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.     

Demonstration of phenotypic abnormalities of thymic epithelium in thymoma including two cases with abundant Langerhans cells.

A panel of monoclonal antibodies that phenotypically define stages of normal human thymic epithelial (TE) cell maturation was used to compare thymic epithelium of nine thymomas with hyperplastic thymic epithelium in myasthenia gravis (MG) and thymic epithelium of normal thymuses. It has been shown previously that normal thymic epithelial cells express antigens of early TE cell maturation (A2B5, TE-4) throughout thymic ontogeny and acquire antigens 12/1-2, TE8, and TE-15 at 14 to 16 weeks of fetal gestation. Hyperplastic MG thymic epithelial cells expressed TE antigens in phenotypic patterns similar to that seen in normal postnatal thymus, ie, TE in subcapsular cortex and medulla was TE4+, A2B5+, and 12/1 - 2+ and Hassall's bodies were reactive with antibodies TE8 and TE15. In contrast, thymic epithelium in primary mediastinal thymomas was TE4+, A2B5+, TE8-, and greater than 75% of thymoma epithelium was 12/1 - 2-, a thymic epithelial phenotype similar to that seen on normal fetal thymic epithelium at 14 to 16 weeks fetal gestation. In one subject with a mature epithelial histologic pattern, thymoma epithelium was found to be strongly TE8+, a phenotype suggestive of a later stage of TE maturation. Lymphocytes in five of seven thymomas with immature thymic epithelial cells predominantly expressed immature thymocyte phenotype while two thymomas with immature epithelial phenotype showed a predominance of Langerhans cells and surrounding lymphocytes expressing a mature phenotype. Lymphocytes in the thymoma with differentiated epithelial cells expressed a mature thymocyte phenotype. Thus, in thymomas of varying histologic types, phenotypic abnormalities of thymic epithelium are present; these phenotypic abnormalities may reflect abnormal thymic epithelial maturation.     

T-cell development in human thymoma.

Human thymoma is derived from thymic epithelial cells and often associated with a large number of cortical thymocytes. Since thymic epithelial cells play key roles in T-cell development in the normal thymus, we hypothesized that the neoplastic epithelial cells of thymoma may support T-cell differentiation. We attempted to reconstitute the T-cell development in vitro by using neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on a proportion of CD4-CD8- cells in thymoma. These CD34+CD4-CD8- cells also expressed both IL-7R alpha-chain and common gamma-chain. Purified CD4-CD8- cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4+CD8+ cells via CD4 single positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. The CD34+CD4-CD8- cells purified from a normal thymus also differentiated to CD4+CD8+ cells in an allogeneic co-culture with the neoplastic epithelial cells of thymoma. In addition, a pleural dissemination from thymoma contained a large amount of cortical thymocytes. These results suggest that the neoplastic epithelial cells retain the function of thymic epithelium and can support T-cell development in thymomas.     

Only dull CD3+ thymocytes bind to thymic epithelial cells. The binding is elicited by both CD2/LFA-3 and LFA-1/ICAM-1 interactions

In view of the necessity for thymocytes to interact with thymic epithelial cells to differentiate into mature T cells, this study analyzed the binding between human thymocytes, cultured thymic epithelial cells (CTEC) and the required adhesion molecules. Immediately after separation, thymic epithelial cells (TEC) readily expressed ICAM-1, which is one of the ligands of LFA-1 cell adhesion molecules. However, the ICAM-1 expression was gradually lost upon culture of TEC. IFN-gamma re-induced ICAM-1 on the CTEC, and the ability of CTEC to bind to thymocytes was also increased by IFN-gamma treatment. The increase in binding seemed to be caused by the LFA-1/ICAM-1 interaction, since it was inhibited by anti-ICAM-1 monoclonal antibody (mAb) and anti-LFA-1 mAb. This suggests that the LFA-1/ICAM-1 interaction is also involved in vivo with the binding of thymocytes to TEC, which have been shown to express ICAM-1. To better understand the nature of the cells involved in binding, thymocytes were sorted into CD3-, CD3dull+, and CD3bright+ subsets (which are supposed to represent the immature, intermediate and mature stages of differentiation, respectively), and were examined for their binding to IFN-gamma-treated CTEC. The result showed that only the CD3dull+ subset bound to CTEC. CD3-, CD3bright+ cells and peripheral blood T lymphocytes did not bind, but they were induced to bind by neuramidase treatment All these bindings were inhibited by anti-LFA-1 mAb and anti-CD2 mAb. These findings indicate that CD3dull+ cells can bind to TEC via CD2/LFA-3 and LFA-1/ICAM-1 interactions. Other cells seemed not to bind to TEC because of sialylation.     

Role of thymic cortex-specific self-peptides in positive selection of T cells

During T cell development in the thymus, a virgin repertoire of diverse TCRαβ recognition specificities in immature thymocytes is selected through positive and negative selection to form an immunocompetent and self-tolerant repertoire of mature T cells. Positive selection supports the survival of thymocytes that receive weak signals of low-avidity TCR engagement, whereas negative selection deletes potentially harmful self-reactive thymocytes upon high-avidity TCR engagement. Early studies have highlighted the role of TCR interaction with polymorphic MHC determinants in positive selection, while negative selection imposes TCR specificity to peptide antigens displayed by MHC molecules. However, recent advances in the biology of thymic stromal cells have indicated that the formation of an immunocompetent TCR repertoire requires positive selection by thymic cortical epithelial cells expressing a unique protein degradation machinery, suggesting the role of self-peptide repertoire specifically expressed by thymic cortical epithelial cells in the development of the acquired immune system.     

Treatment with anti-LFA-1 alpha monoclonal antibody selectively interferes with the maturation of CD4- 8+ thymocytes.

Maturation of T lymphocytes in the thymus is driven by signals provided by soluble factors and by the direct interaction between thymocytes and stromal cells. Although the interaction between T-cell receptor (TCR) and major histocompalibility complex (MHC) molecules on stromal cells is crucial for T-cell development, other accessory molecules seem to play a role in this process. In order to better understand the role of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) molecules in thymocyte maturation, mice were treated from birth with saturating doses of non-cytolytic-specific monoclonal antibodies. The effect of this treatment on thymocyte subpopulations and the expression of CD3 and TCR-alpha beta by these cells was investigated by flow cytometry. Our data demonstrated that the effective saturation of LFA-1 alpha chain in the thymus, but not ICAM-I or LFA-I beta chain, selectively interfered with the maturation of CD8+ T cells, as manifested by a marked reduction in the frequency of CD4-8+ thymocytes expressing high levels of CD3 and TCR-alpha beta. This selective reduction was also observed in peripheral blood mononuclear cells and spleen cells. The analysis of the frequencies of various V beta TCR showed that CD4-8+ thymocytes were globally affected by the treatment. These results underline the importance of the interaction between LFA-1 and its ligands in the maturation of CD8+ T cells and document the existence of different molecular requirements for the differentiation of CD4+ and CD8+ T cells.     

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.     

Expression of CD44 molecules and CD44 ligands during human thymic fetal development: expression of CD44 isoforms is developmentally regulated

It has recently been recognized that CD44 comprises a largefamily of alternatively spliced forms.In the thymus, CD44 hasbeen postulated to play an important role in immature T cellmigration and maturation. In this paper, we have studied theexpression of CD44 molecules and two CD44 ligands, hyaluronan(HA) and fibronectin (FN), during human thymic fetal development.We found that mAbs against all CD44 isoforms (A3D8 or A1G3)reacted with both thymic epithelial (TE) cells and thymocytesbeginning at the time of initial colonization of the human thymusby hematopoietic stem cells at 8.2 weeks of fetal gestation.However, mAbs specific for splice variants of CD44 containingmembrane-proximal inserts (11.24, 11.10 and 11.9) reacted onlywith terminally differentiated TE cells in and around Hassall'sbodies beginning at 16–19 weeks of fetal gestation. Studiesof differentiated versus undifferentiated TE cells in vitroconfirmed the selective expression of CD44 variant isoformson terminally differentiated TE cells. Expression of HA andFN was determined by fluorescence microscopy using either biotlnylated-HAbinding protein or an anti-FN mAb. We found that whereas FNwas present throughout the human fetal thymus beginning at 8.2weeks, HA was not present until 16 weeks of gestational age.These data demonstrate the differential expression of standardversus variant CD44 isoforms during thymic ontogeny and implicateCD44 interactions with ligands other than HA as important inthe earlier stages of humanthymus development     

Thymocytes can tolerize thymocytes by clonal deletion in vitro.

Clonal deletion of thymocytes bearing TCR for self antigens is one major mechanism of T cell tolerance induction. Peptide antigen-induced deletion of thymocytes from alpha beta TCR transgenic mice has been studied using single cell suspension cultures. The results show that antigen-presenting immature CD4+CD8+ thymocytes can tolerize antigen-reactive immature thymocytes in vitro by programmed cell death (apoptosis) 6-8 h after antigen exposure. Antigen-induced apoptosis of immature thymocytes was inhibited by antibodies specific for the alpha beta TCR, CD3, CD8, and LFA-1 molecules. This implies that clonal elimination of self-reactive CD4+CD8+ thymocytes does not depend on specialized deleting cell types in the thymus and occurs whenever the TCR of immature thymocytes bind antigen fragments presented by MHC molecules.     

Thymic microenvironment reconstitution after postnatal human thymus transplantation

A functional thymus develops after cultured thymus tissue is transplanted into subjects with complete DiGeorge anomaly. To gain insight into how the process occurs, 7 post-transplantation thymus biopsy tissues were evaluated. In 5 of 7 biopsies, the thymus appeared to be predominantly cortex with thymocytes expressing cortical markers. Unexpectedly, the epithelium expressed both cortical [cortical dendritic reticulum antigen 2 (CDR2)] and medullary [cytokeratin (CK) 14] markers. Early medullary development was suggested by epithelial cell adhesion molecule (EpCAM) reactivity in small areas of biopsies. Two other biopsies had distinct mature cortex and medulla with normal restriction of CK14 to the medulla and subcapsular cortex, and of CDR2 to cortex. These data are consistent with a model in which thymic epithelium contains CK14+ "progenitor epithelial cells". After transplantation these cells proliferate as CK14+CDR2+ thymic epithelial cells that are associated with cortical thymocytes. Later these cells differentiate into distinct cortical and medullary epithelia.     

Mouse thymic epithelial cell lines interact with and select a CD3lowCD4+CD8+ thymocyte subset through an LFA-1-dependent adhesion--de-adhesion mechanism

Differentiation of bone-marrow-derived precursor cells into mature mouse T lymphocytes occurs in the thymus and involves sequential interactions with MHC-positive hemopoietic and epithelial stromal cells. To study the in vitro molecular mechanisms at play during the lympho-epithelial cell adhesion, we derived thymic stromal cell lines which were shown to possess cytokeratin filaments and tight junctions. These mouse thymic epithelial (MTE) cell lines did not express the classical hemopoietic stromal cell surface markers (i.e. LFA-1, Mac-1, and CD45) but expressed ICAM-1, NCAM, J11d, CD44, and MHC molecules. A quantitative cell adhesion assay was used to evaluate the interaction of various lymphoid cell subsets with MTE cells. Two cell interaction patterns could be defined: first, a rapid adhesion of a fraction of CD4+CD8+ and of a few CD4-CD8- immature thymocytes to MTE cells was observed at 4 degrees C. The CD8 molecule was shown to be partially involved in this initial contact. The strength of adhesion between MTE cells and distinct thymocyte subsets was evaluated and found to be maximal with neonatal thymocytes. Second, a temperature-dependent adhesion step characterized by a rapid and active stabilization of the interaction of MTE cells with 20% of CD4+CD8+CD3low thymocytes was seen, followed by a more progressive de-adhesion step. This active process of engagement was highly LFA-1-dependent, involved the CD4 and CD8 molecules, and required protein kinase C activation and cytoskeletal integrity. The results are consistent with the involvement of LFA-1 in a transient and regulated cell adhesion under the control of the TCR-CD3 complex that progressively appears on maturing cells. This phenomenon might contribute to the selection of a subset of immature thymocytes by epithelial cells occurring during the process of maturation of these cells.     

Distribution of LCA protein subspecies and the cellular adhesion molecules LFA-1, ICAM-1 and p150,95 within human foetal thymus

The distribution of leucocyte common antigen (LCA) protein subspecies and the cellular adhesion molecules LFA-1 (CD11a), ICAM-1 (CD54) and p150,95 (CD11c) has been established within frozen sections of human foetal thymus. Whereas over 95% of foetal cortical thymocytes and approximately 85% of medullary thymocytes were CD45RO positive, CD45RA was only expressed by approximately 29% of medullary thymocytes. The majority of foetal thymocytes also expressed CD11a, whereas CD54 was expressed by thymic epithelial and accessory cells and also apparently by some cortical thymocytes adjacent to epithelial cells. The distribution of CD54 and the major histocompatibility complex (MHC) class II molecule HLA-DR, demonstrated with a monoclonal antibody to a monomorphic determinant, was similar. The CD11c molecule was present on a population of dendritic-type accessory cells, but was absent from the large, scavenger, KiM8-positive macrophages occurring throughout the thymic cortex.     

Mouse LFA-3 studied with chimeric soluble CD2 shows preferential expression on lymphoid cells.

Here we have used a soluble, polyvalent form of mouse CD2, i.e. CD2-hC mu, to detect its ligand LFA-3. Mouse LFA-3 is preferentially expressed on lymphoid cells unlike human LFA-3 which shows a wide tissue distribution. Mouse LFA-3 is one of the early surface molecules expressed on developing thymocytes appearing on the surface of fetal thymocytes on day 13 of gestation. Like human LFA-3, the mouse homolog could be shown to be a phosphatidyl inositol-linked membrane protein. Since mouse LFA-3 is preferentially expressed on lymphoid cells and since CD2 is expressed by both T and B lymphocytes, we would favor the view that the CD2/LFA-3 adhesion system in the mouse may play a role in interactions between lymphocytes (T-T and/or T-B) rather than in cell interactions between lymphocytes and non-lymphoid cells in the thymic, bone marrow or spleen microenvironments.     

Murine thymic nurse cell clone supports the growth of fetal thymocytes in the presence of interleukin 2.

To investigate the role of thymic nurse cells (TNC) in activation and differentiation of fetal CD4-CD8- (double-negative) thymocytes, we have co-cultured murine fetal thymocytes (14-15 days of gestation) with an established murine TNC clone. We show here that TNC induced the growth of the fetal double-negative thymocytes in the presence of recombinant interleukin 2 (rIL2). Activated fetal thymocytes markedly formed lymphocyte-TNC complexes and proliferated extensively after 5 days in the co-culture. The activated fetal thymocytes in this co-culture condition remained double negative after 10 days in culture. None of them gave rise to phenotypically and functionally competent lymphocytes during this period. TNC alone and the supernatant of TNC had no effect on activation. The presence of both TNC and rIL2 was necessary for the growth of fetal thymocytes in our system. The proliferation of fetal thymocytes was inhibited by a monoclonal antibody against mouse IL2 receptors (IL2R). The fetal thymocytes could be maintained further in this co-culture condition. The prolonged cultivation of fetal thymocytes resulted in the establishment of the fetal thymocyte line and its several clones. CD4 single-positive cells of activated fetal thymocytes first appeared 14 days after the onset of culture and their number increased, whereas CD8+ cells or CD4CD8 double-positive cells were not observed. These results indicate that fetal CD4-CD8- thymocytes underwent phenotypic change after long periods of culture. All established clones of fetal thymocytes are CD4 single positive showing lymphocyte-TNC interactions but do not express CD3 complex. Northern blot analysis detected mRNA for the gamma T cell receptor, but no messages for the delta, alpha or beta T cell receptor. Chemical cross-linking of 125I-labelled IL2 revealed that the 90-kDa band (presumably considered to be the IL2R beta chain) was clearly present in IL2-responsive fetal clones, whereas freshly isolated day 14-15 fetal thymocytes lacked the band. Taken together, TNC might be involved in the differentiation and/or expansion of murine fetal thymocytes by inducing IL2R beta chain, which forms the functional IL2R together with IL2R alpha chain and CD4, one of the T cell accessory molecules, on the cell surface through direct cell-cell interaction.