Evidence for major alterations in the thymocyte subpopulations in murine models of autoimmune diseases

Thymocytes can be divided into four major subpopulations: CD4+CD8+ (double-positive), CD4-CD8- (double-negative), CD4+CD8- (CD4+) and CD4-CD8+ (CD8+) cells. Recent studies have shown that T-cell development in the thymus progresses as: CD4-CD8(-)----CD4+CD8(+)----CD4+ or CD8+ cells. In the present study we investigated these and other subpopulations of thymocytes in autoimmune MRL(-)+/+, MRL-lpr/lpr, C57BL/6-lpr/lpr, BXSB and NZB mice before (1-month old) and after (4-6-months old) the onset of lymphadenopathy and autoimmune disease. All the autoimmune strains at one month of age and other H-2, sex and age-matched controls (C3H, DBA/2, and C57BL/6) demonstrated normal proportions of thymocyte subsets with approximately 75% double-positive cells, 5-7% double-negative cells, 11-15% CD4+ cells and 3-5% CD8+ cells. By 4-6 months of age, MRL(-)+/+ mice demonstrated a moderate increase in double-negative cells (approximately 13%) and a decrease in double-positive cells (approximately 46%). Interestingly, in the presence of the lpr gene, as seen in MRL-lpr/lpr mice, the double-negative cells increased to approximately 47% and the double-positive cells decreased to approximately 16%. In contrast, 4-6-month-old C57BL/6-lpr/lpr mice failed to demonstrate any alterations in the thymocyte subsets thereby suggesting that background genes, in addition to the lpr gene, played a role in the thymocyte differentiation. BXSB male mice with severe lymphadenopathy behaved very similarly to MRL-lpr/lpr mice, inasmuch as their thymus contained approximately 48% double-negative cells and only approximately 8% double-positive cells. In contrast to MRL-lpr/lpr and BXSB strains, NZB mice at 6 or 10 months of age had normal composition of thymocyte subsets. In MRL and BXSB animals, although there was a significant increase in CD4+ cells (approximately 23-33%), due to a consequent increase in CD8+ cells (approximately 11%), the ratio of CD4+:CD8+ cells remained 2-3:1, similar to that seen in normal mice. Furthermore, using the J11d marker expressed by the majority of the double-negative and all double-positive thymocytes but not by mature functional T cells, we confirmed the above findings and demonstrated further that MRL-lpr/lpr mice at 4-6 months of age had an increased percentage of J11d- double-negative cells and a decrease in J11d+ double-negative cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alterations in thymocyte subpopulations in Down's syndrome (trisomy 21)

To correlate the histologically observed thymic abnormalities with the cellular immunodeficiency found in Down's syndrome (DS), thymus fragments and thymocyte suspensions from 14 noninstitutionalized DS subjects were studied. Histologic examination and immunohistologic studies using an anticluster of differentiation (CD) 1 monoclonal antibody showed a contracted cortex due to cortical thymocyte depletion. When DS unselected thymocytes were phenotyped, a significant reduction of CD3-, CD1-, CD4-, and CD8-positive cells was found as compared to controls. To evaluate if the deficient expression of these markers was due to the reduction of thymocyte subsets identifiable on the basis of their physical properties, we separated DS unselected thymocytes into 10 fractions by continuous Percoll density gradient centrifugation. DS thymuses were almost completely devoid of high density thymocytes. Since in normal thymus, these cells correspond to small CD1+, CD4+, CD8+, and 50% CD3+ cortical thymocytes, their absence may explain the unrestricted reduction of markers on DS unfractionated thymocytes. Furthermore DS thymuses appeared to be enriched in CD1+ first fraction (Fr1) low density thymocytes of the Percoll gradient. Fr1 CD1+ cells constitute the main spontaneously proliferating pool in normal human thymus. When the spontaneous proliferating activity of DS Fr1 was compared to that of the control, a significant reduction was observed. This reduction associated with the absence of high density thymocytes, with the reduction of cells expressing alpha- and beta-chains of the T cell receptor and in conclusion with the lymphocyte depletion, suggests that in DS thymuses there is a deficient expansion of immature T cells resulting in a reduction of the various thymocyte subpopulations, including the thymocyte pool able to differentiate into functionally mature T cells.     

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.     

CD45RA is detected in all thymocyte subsets defined by CD4 and CD8 by using three-colour flow cytometry.

In the mouse, using three-colour flow cytometry, the presence of CD45RA+ cells is demonstrated amongst all of the thymocyte subsets defined by expression of CD4 and CD8, i.e. amongst the double negatives, immature CD8 single positives, double-positive blasts and CD4 and CD8 single positives. This evidence is compatible with the existence of a continuous lineage of T cells expressing CD45RA which would develop from double-negative to mature single-positive T cells.     

Differentiation of human mature thymocytes: existence of a T3+4-8- intermediate stage

A T3 complex-bearing subpopulation was characterized within an in vivo cycling T4-8- early thymocyte compartment which contains cells constitutively expressing interleukin 2 and transferrin receptors. We show differentiation in vitro of both mature subsets of thymocytes (T3+4+8- and T3+4-8+) from the above T4-8- compartment, their appearance being preceded by cells in a T3+4-8- intermediate stage. Furthermore, those mature thymocytes generated in vitro contain functionally competent cells which use T3, T4 and T8 structures for their cytolytic activity. The finding of T3+4-8- thymocytes in vivo, together with the observation that T3 antigen expression precedes that of T4 or T8 molecules in vitro, shows that T3 (and presumably Ti) is present early in ontogeny, and suggests that T3+4-8- cells constitute an "intermediate" stage relevant to the connection between early precursors and mature thymocytes during T lymphocyte ontogeny.     

Subpopulations of CD4-, CD8- thymocytes

A population of highly purified CD4-,CD8- thymocytes was analyzed by both flow microfluorometry and in situ hybridization in an attempt to further elucidate thymocyte subpopulations. The double-negative cells had a marked increase in expression of the c-myb and T cell gamma genes relative to unseparated thymocytes. Approximately 27% were Ly-24+, 8% Ly-6C+ and 6% 6B2+. All of the Ly-6C+ cells were also Ly-24+. A small population (6%) of the CD4-,CD8- thymocytes had surface expression of the T cell receptor for antigen (F23.1); all were bright Ly-1+ and half were Ly-24+. These studies demonstrate that there are further subdivisions of the CD4-, CD8- thymocytes based upon cell surface expression of markers previously found on bone marrow cells and their non-T cell progeny. Studies are in progress to determine whether these represent different stages of activation-maturation or different lineages of cells.     

Delineation of chicken thymocytes by CD3-TCR complex, CD4 and CD8 antigen expression reveals phylogenically conserved and novel thymocyte subsets.

To further define the relationship between thymocyte subsets and their developmental sequence, multi-parameter flow cytometry was used to determine the distribution of the CD3-TCR complex and the accessory molecules CD4 and CD8 on chicken thymocytes. As in mammals, adult thymocytes could be subdivided into CD3-, CD3lo, and CD3hi staining populations. CD4 and CD8 distribution on such populations revealed the presence of CD3-CD4+CD8- and CD3-CD4-CD8+ thymocytes, putative precursors to CD4+CD8+ cells, detectable in the adult and at high frequency during ontogeny. Of particular interest was the existence of CD3lo expression on CD4+CD8- and CD4-CD8+, and in some instances, on CD4-CD8- thymocytes. Such phenotypes are not easily detectable in the mammalian thymus but were readily observed in both adult and embryonic chicken thymus from 16 days of embryogenesis. Further analysis of the TCR lineage of these CD3lo cells revealed that they were essentially all of the alpha beta TCR type. Mature CD3hi thymocytes were found within the CD4+CD8+ and CD4+CD8- and CD4-CD8+ subsets. Both alpha beta and gamma delta TCR lineage thymocytes were detected within all CD4- and CD8-defined subsets, thus identifying novel thymocyte subsets in the chicken thymus, namely alpha beta TCR+CD4-CD8- and gamma delta TCR+ CD4+CD8- cells. Hence, this analysis of chicken thymocytes, while confirming the phylogenically conserved nature of the thymus, has revealed novel T cell subsets, providing further insight into the complexity of mainstream thymocyte maturation pathways.     

Activation of the Ras-related GTPase Rap1 by thymocyte TCR engagement and during selection

Signals mediated by activation of the small GTPase Ras play an essential role both in thymocyte development and in TCR-mediated activation of mature T cells. Given the critical requirement of Ras signaling pathways in thymocyte development, and recent indications that Rap1 may negatively regulate Ras-dependent signaling pathways, we examined the possible involvement of Rap1 in thymocyte TCR signaling. We find that Rap1 and proposed regulators of Rap1 (the proto-oncogene product Cbl, Crk family adaptor proteins, and the Rap1 guanine nucleotide exchange factor C3G) are expressed at equivalent levels in both double-negative and double-positive murine thymocytes. Rap1 was transiently activated following TCR stimulation of both total thymocytes and purified double-positive thymocytes, and this activation correlated with tyrosine phosphorylation of Cbl and Cbl association with CrkL. TCR-dependent Rap1 activation was enhanced by co-stimulation through CD28 and could be mimicked by treatment of thymocytes with phorbol ester and calcium. In contrast to mature peripheral T lymphocytes, Rap1 stimulation by CD3 ligation in thymocytes did not require intracellular calcium mobilization. Intriguingly, we found a clear elevation of activated Rap1 in thymocytes undergoing positive selection, suggesting a functional role for Rap1 in thymocyte development and selection.     

CD45 isoform expression during T cell development in the thymus.

Various isoforms of leukocyte common antigen, or CD45, are expressed differentially on T cells at different stages of development and activation. We report studies on CD45 isoform expression on various subsets of human T cells using two- and three-color flow cytometry and cell depletion. Bone marrow cells that were depleted of CD3+ and HLA-DR+ cells were CD45RA-RO-. The earliest CD3-CD4-CD8-CD19- thymocytes were CD45RO- with 20%-30% CD45RA+ cells. The most prominent population of CD4+CD8+ double-positive thymocytes were CD45RA-RO+. Even the CD4+CD8+ blasts were greater than 90% CD45RO+. About 80% of single-positive thymocytes (CD4+CD8- or CD4-CD8+) were also CD45RO+. Only 4.3% of CD4+ and 18% of CD8+ single-positive thymocytes were CD45RA+. In contrast, cord blood T cells which represent the stage that immediately follows single-positive thymocytes, contained 90% CD45RA+ cells. Thus, in terms of CD45 isoform expression, single-positive thymocytes are more like double-positive cells than cord blood T cells. These results suggest the following sequence of CD45 isoform switching during T cell development: CD45RA-RO- or RA+RO- (double-negative thymocytes)----RA-RO+ (double-positive and most single-positive thymocytes)----RA+RO- (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.     

Developmental regulation of TCR-CD3-dependent [Ca2+]i responses of individual normal and pp59fyn-deficient T lymphocytes.

The aim of this study was to ascertain whether different types of T-cell receptor (TCR)-mediated [Ca2+]i signals could begin to explain the different cellular responses of mature and immature T cells to ligation of the TCR-CD3 complex. Using a digital fluorescence imaging system, we measured and compared [Ca2+]i of individual cells from immature and mature murine T-cell populations following application of CD3-epsilon monoclonal antibody (mAb). Our approach revealed distinctions among developmental subsets which were not seen by previous measurements of [Ca2+]i in bulk cell populations. The CD3-mediated [Ca2+]i responses of individual thymocytes were very complex. Latencies to peak [Ca2+]i varied greatly among thymocytes, but the responses of splenic T cells were synchronized, novel evidence that the timing of [Ca2+]i responses may be an important informative parameter for TCR-CD3 signalling. In addition, among cells responding to CD3 mAb, higher peak [Ca2+]i responses correlated with maturity (CD4+ CD8+ thymocytes < single-positive thymocytes < splenic T cells). Examination of cells from pp59fyn-deficient mice showed that pp59fyn deficiency affects the amplitude and probability, but not the latency or synchrony, of CD3-mediated [Ca2+]i responses of CD4+ CD8+ and CD4+ CD8- thymocytes. All subsets showed equivalent receptor-independent mobilization of [Ca2+]i. These developmentally distinct [Ca2+]i features most probably reflect meaningful developmental changes in how the TCR-CD3 complex couples to intracellular signalling machinery including pp59fyn. By clearly showing how [Ca2+]i responses change during development, these results support the hypothesis that distinctive types of [Ca2+]i responses drive thymocyte differentiation.     

T cell receptor expression on immature thymocytes with in vivo and in vitro precursor potential

Immature CD8-CD4- double-negative (DN) thymocytes differentiate intrathymically into CD8+CD4- and CD8-CD4+ thymocytes and migrate to the periphery. This differentiation proceeds through several intermediate phenotypic changes in the expression of CD8 and CD4. We have recently established the existence of a CD8loCD4lo cell population in murine thymus that can repopulate the irradiated thymus in vivo and differentiate rapidly in vitro to CD8+CD4+ double-positive (DP) cells. The CD8loCD4lo cells score as DN upon direct cytofluorometric analysis, yet are distinct from true DN cells by various criteria. Experimental evidence strongly suggests that they are descendants of true DN in the maturation pathway. In the experiments presented here, we further characterize this CD8loCD4lo thymocyte population. Northern blot and RNA protection analysis reveal that these cells transcribe full length mRNA for the T cell receptor (TcR)alpha chain, unlike the less mature interleukin 2 receptor-positive DN thymocytes. Surface expression of the TcR-associated CD3 molecule occurs on approximately 15% of these cells at low levels characteristic of immature cells. In the course of in vitro differentiation a vast majority (approximately 80%) of these cells convert to CD8+CD4+ and significant numbers of the brightly staining DP convertants (11%-34% on day 1 and 48%-68% on day 2) express immature levels of CD3. Our results indicate that CD8lo, CD4lo cells might be the first thymic subset to rearrange TcR alpha chain genes and express TcR alpha/beta heterodimer on the surface at levels characteristic of immature cells. Furthermore, the surface expression of TcR persists on the in vitro progeny of these thymocytes.     

Interleukin-4 induces expression of the CD45RA antigen on human thymocyte subpopulations

Interleukin-4 (IL-4) is a multifunctional lymphokine which promotes the growth and/or maturation of multiple cell types. We have examined the ability of IL-4 to promote the phenotypic maturation of subsets of human thymocytes. When cultured in serum-free medium supplemented with recombinant IL-4, a subset of immature CD3-CD45RA- human thymocytes ceased to express the CD1 common thymocyte antigen and acquired phenotypic features characteristic of relatively mature thymocytes, such as high-density expression of the CD3 antigen and de novo expression of the CD45RA isoform of the common leukocyte antigen family. These changes, which were not seen in cells cultured in medium alone, occurred over an 8-9 day period and were accompanied by a significant increase in cell size. The CD45RA+ cells that derived from these immature CD3-CD45RA- precursors were mainly CD4-CD8- or CD8+ cells, and a significant proportion of these cells expressed the T cell receptor delta chain. IL-4 also increased expression of the CD45RA antigen on the more mature CD3+ thymocyte population. However, the CD45RA+ cells derived from IL-4 stimulated CD3+ thymocyte precursors expressed either the CD4 or the CD8 antigen, and virtually all expressed alpha/beta TCR chains. Studies of cell viability and cell growth indicated that these findings were due to direct changes in the phenotype of responsive cells rather than the growth or selective survival of a small number of mature thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective elimination of double-positive immature thymocytes by a thymic epithelial cell line

A cloned epithelial cell line, TEL-2, has been established from the stroma tissues of normal mouse thymus. Incubation of mouse thymocytes on TEL-2 cells resulted in the selective elimination of double-positive (CD4+CD8+) cells from the culture, whereas single-positive (CD4+CD8- or CD4-CD8+) thymocytes remaining in the culture were concentrated in non-integrated cell population. The CD3- or CD3 low-positive thymocytes were also eliminated by the TEL-2 cells from the culture, followed by the concentration of CD3 high-positive cells in the culture. Only intact viable thymocytes were integrated into TEL-2 cells. Electron microscopic examination showed that the integrated cells into TEL-2 cytoplasm were gradually degenerated. Mature single-positive T cells, mature B cells or double-negative thymocytes were not integrated into TEL-2 cells. The TEL-2 cell may provide information on the mechanism of selective disappearance of double-positive immature cells from the thymus.     

Analysis of clones derived from human CD7+CD4-CD8-CD3- thymocytes.

The differentiation of human thymocyte precursors was studied by analysis of clonal progeny of CD4-CD8-CD3- (triple negative or TN) thymocytes. Using a culture system of phytohemagglutinin, IL-2, and irradiated allogeneic lymphoid feeder cells, we found that 48% of clones (104 total) derived from TN thymocyte suspensions were TCR gamma delta cells, 12% of clones were TCR alpha beta cells, and 34% were CD16+CD3- cells. Importantly, 6% of clones were novel subsets of CD4+CD8-CD3- or CD4-CD8+CD3- thymocytes. The majority of TCR alpha beta, TCR gamma delta, and CD16+CD3- clones expressed low levels of CD4. Molecular analysis of freshly isolated TN- thymocytes prior to in vitro culture demonstrated that up to 40% of cells had TCR gamma, delta, and beta gene rearrangements, but were negative in indirect immunofluorescence assays for cytoplasmic TCR delta and beta. These data provide evidence at the clonal level for the presence of precursors of the TCR alpha beta and TCR gamma delta lineages in the human TN thymocyte pool. Moreover, a substantial proportion of freshly isolated human TN thymocytes had already undergone TCR gene rearrangement prior to in vitro culture. Whether these precursors of the TCR alpha beta and TCR gamma delta lineages mature from cells already containing TCR gene rearrangements into sTCR+ cells or differentiate in vitro from cells with TCR genes in germline configuration remains to be determined. Nonetheless, these data demonstrate that the predominant clone types that grow out of human TN thymocytes in vitro are TCR gamma delta and NK cells.     

Interplay between IL-2 and IL-4 in human thymocyte differentiation: antagonism or agonism

The effect of recombinant interleukin 2 (IL-2) and IL-4, as well as a combination of both lymphokines on human post-natal thymocytes at different maturation stages, was analyzed by culturing highly purified pro-T cells, pre-T cells, double-negative and double-positive thymocyte subsets in the presence of IL-2 and/or IL-4. Both IL-2 and IL-4 responsiveness are developmentally regulated in human thymocytes, since IL-2 and IL-4 responses decline with increasing thymocyte differentiation, double-positive T cells displaying far less proliferation than immature thymocytes. IL-2 and IL-4 may influence pro-T cell growth in both an antagonistic and additive fashion. At low doses, IL-4 inhibits IL-2-supported growth of pro-T cells, whereas, at higher concentrations, this inhibitory effect is masked by the ability of IL-4 to stimulate pro-T cell proliferation. In contrast to peripheral lymphocytes, IL-4 does not down-regulate the expression of the IL-2 receptor light chain on thymocytes. In pro-T cell cultures, IL-2 and IL-4 favour the differentiation of distinct cell populations, namely lymphocytes displaying preferentially a TCR alpha/beta+ and CD4+CD8- phenotype versus predominantly TCR gamma/delta+ and CD4-CD8+ cells, respectively. The effect of IL-2 dominates over that of IL-4, since the composition of cultures set up in the presence of IL-2 plus IL-4 resembles that of cells cultured with IL-2 alone. In synthesis, IL-2 and IL-4 exhibit reciprocal inter-relations in human thymocyte cultures, thus supporting the notion that these lymphokines are implicated in the complex regulation of a local cytokine network.     

Characterization of the subset of immature thymocytes which can undergo rapid in vitro differentiation

Recently, we reported that thymocytes expressing the CD8 molecule on their surface can give rise to CD4+CD8+ double-positive and CD4+ single-positive progeny following intrathymic transfer into an irradiated host mouse. Thymcoytes expressing a high density of CD8, referred to as CD8hi, and those expressing a low density of the molecule, CD8lo, were both able to differentiate in vivo. In this study we examined the ability of these CD8+ thymocytes populations and of CD4-CD8- double-negative thymocytes to change their phenotype during brief in vitro culture. CD8+ thymocytes were prepared by anti-CD4 plus complement lysis followed by positive selection of the survivors on anti-CD8-coated plates. After 16 h of culture, greater than 60% of CD8+ thymocytes became double-positive. Both CD8hi and CD8lo cells were able to show this in vitro change: about 30% of the former and about 80% of the latter became double-positive. In contrast to this, double-negative thymocytes which had been depleted of cells expressing low densities of CD8 did not show such a phenotypic conversion in vitro. Further panning experiments suggested that all of the CD8+ thymocytes actually express a low surface density of the CD4 molecule which is undetectable in our cytofluorometric assays.     

3, 3', 4, 4'-Tetrachlorobiphenyl Inhibits Proliferation of Immature Thymocytes in Fetal Thymus Organ Culture

The environmental pollutant 3, 3', 4, 4'-tetrachlorobiphenyl (TCB) leads to thymic atrophy and immuno-suppression, the former possibly causing the latter. TCB binds lo the cytosolic aryl-hydrocarbon receptor (AhR) and transforms it into a DNA-binding state. The development of fetal thymocyles is severely affected by TCB and other AhR-binding xenobiotics, leading to a skewed pattern of thymocyte maturation stages. Murine thymocyte proliferation after exposure to TCB was studied in fetal thymus organ culture (FTOC). C57BL/6 fetus thymic lobes from day 15 of gestation were explanted and grown for 2, 4, 6. and 8 days in organ culture in the presence or absence of 3.3 μM TCB. Subsets of thymocytes were defined by CD4 and CD8 surface markers, and their cell cycle was analysed by DNA staining with 7-amino-actinomycin D (7-AAD). Exposure of fetal thymi in vitro to 3.3 μM TCB significantly reduced the total number of thymocytes. and fewer thymocytes were in S/G₂M phase. The inhibition of cell proliferation induced by TCB treatment affected mainly the CD4⁻ CD8⁻ (double-negative, DN) and CD4⁻ CD8⁺ (single-positive, SP) subsets, and these inhibition appeared mainly in more immature thymocytes, i. e. DNCD3⁻ and CD8⁺CD3⁻ subpopulations, whereas no effect of TCB on CD4⁺CD8⁺ (double-positive, DP) cell proliferative activity was observed. Analysis of the relation of cell proliferation and development of subsets in differentiating fetal ihymocytes suggests that TCB enhanced thymocyte differentiation into mature CD8⁺ cells.     

Possible involvement of glial cell line-derived neurotrophic factor and its receptor,GFRalpha1, in survival and maturation of thymocytes

The glial cell line-derived neurotrophic factor (GDNF) and its receptors (GFR) play important roles in the promotion of survival and differentiation of central and peripheral neuronal populations. We show that GFRalpha1, a component of GDNF receptor, was expressed in thymocytes at an early stage of thymocyte-development and was involved in the survival of thymocyte precursors. GFRalpha1and GDNF were expressed in thymus, but not in spleen or lymph nodes in adult mice. During embryonic thymocyte development, GFRalpha1 was predominantly expressed on thymocytes from days 14.5 to 16.5 of gestation, and thereafter its expression gradually declined. In adult thymus, GFRalpha1 was expressed only on CD4(-)CD8(-) double-negative (DN) thymocytes, but not on CD4(+)CD8(+) double-positive or single-positive thymocytes. It was strongly expressed on RAG2(-/-) thymocytes arrested at the DN stage, and ist expression was reduced during their differentiation after in vivo anti-CD3 antibody stimulation. Additionally, fetal thymocyte precursors grew in serum-free medium of the fetal thymus organ culture system in the presence of recombinant GDNF (rGDNF), while the cells without rGDNF died. These results suggested that GDNF/GFRalpha1 are involved in the survival of both the nervous system and DN immature thymocytes.