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 αβ heterodimer, specific for the male (H-Y) antigen in association with H-2D^b, was determined. The transgenic α chain was expressed on about 10% of the fetal thymocytes on day 14 of gestation. About 50% of day-15 fetal thymocytes expressed both α and β transchains and virtually all fetal thymocytes expressed the transgenicαβ heterodimer by day 17. The early expression of the transgenic TCR on CD4^-8^- thymocytes prevented the development of γδ 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 CD⁺4⁺8 and single positive thymocytes have the CD4^-8^- CD3^-J11d⁺ (or M1/69 ⁺) phenotype. Because of the early expression of the transgenic αβ 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.     

Co-ordinate expression of the pre-T-cell receptor complex and a novel immature thymocyte-specific antigen, IMT-1, during thymocyte development

Previously we described a monoclonal antibody (mAb) that reacted with a cell-surface antigen, immature thymocyte antigen-1 (IMT-1), which is expressed on thymocytes of late CD4- CD8- (double negative) to early CD4+ CD8+ (double positive) differentiation stages. In this study, we investigated the expression of IMT-1 on various cell lineages in thymus as well as in peripheral lymphoid organs. We found that IMT-1 is expressed on T-cell receptor (TCR)-betalo and TCR-deltalo thymocytes, but not on TCR-betahi, TCR-deltahi or natural killer (NK)1.1+ thymocytes, or on peripheral alpha beta or gamma delta T cells. We also investigated the kinetics of expression of IMT-1 during fetal thymocyte development and compared it with the expression of the pre-TCR complex, comprising CD3, pre-TCR-alpha (pTalpha) and TCR-beta. We found that expression of both was similar, starting at day 14.5 of gestation, peaking on day 16.5 and gradually decreasing thereafter. Furthermore, the expression of both IMT-1 and pTalpha was drastically reduced when DN thymocytes in recombination activating gene (RAG)-2-/- mice were challenged in vivo with anti-CD3 mAb. These results indicate that IMT-1 is expressed on not only immature thymocytes of alpha beta T-cell lineage but also on those of gamma delta T-cell lineage, and that the expression of IMT-1 and the pre-TCR complex is co-ordinately regulated during the alpha beta lineage thymocyte development.     

Generation of alphabeta T-cell receptor+ CD4- CD8+ cells in major histocompatibility complex class I-deficient mice upon activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Gene-targeted mice lacking the beta2 microglobulin gene (beta2m-/- mice), and hence functional major histocompatibility complex (MHC) class I molecules, do not develop CD4- CD8+ cells. We show here that both in vitro and in vivo treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a trans-activating ligand of the endogenous aryl hydrocarbon receptor (Ah-R), bypasses the need for MHC class I molecules for selection into the CD4- CD8+ cell pool. When beta2m-/- dams were given a single dose of 50 microg of TCDD, approximately 13% of CD4- CD8+ thymocytes could be detected in their newborn pups. In TCDD-exposed fetal thymus organ cultures of beta2m-/- mice, approximately 35% CD4- CD8+ thymocytes were detectable. About 16% of these CD4- CD8+ cells bore the alpha beta T-cell receptor (TCR) and approximately 33% bore CD3. Only a minority of the CD8+ cells were heat-shock antigen positive. The cells possessed killing activity as shown using the 51Cr-release assay comprising gamma delta TCR- CD4- CD8+ thymocytes from 3 to 4-day-old b2m-/- mice. Thus, TCDD leads to a significant increase of mature CD4- CD8+ thymocytes in relative and absolute numbers. High numbers of CD4- CD8+ thymocytes developed also in organ cultures from thymi, lacking both MHC class I and class II molecules, exposed to TCDD. A 10-fold transient increase of Notch1 mRNA in thymocytes from fetal thymus organ culture, exposed for 4 days to TCDD, was detected in CD4+ CD8+ cells compared with controls. We suggest that TCDD affects thymic selection and directs the lineage commitment of CD4+ CD8+ thymocytes towards CD4- CD8+ cells, possibly via up-regulation of the Notch1 gene.     

Effects of irradiation, glucocorticoid and FK506 on cell-surface antigen expression by rat thymocytes: a three-colour flow cytofluorometric analysis.

The expression of T-cell antigen receptor (TCR) alpha beta was investigated in rat CD4- CD8- thymocytes during thymic reconstitution after the exposure of animals to irradiation or glucocorticoid. The effect of the immunosuppressant FK506 on the expression of TCR alpha beta in rat CD4- CD8- thymocytes was also examined. The percentage of CD4- CD8- thymocytes constituted 2.6% of total thymocytes and that of CD4- CD8- TCR alpha beta high cells constituted 12.6% of CD4- CD8- thymocytes in normal adult Lewis rats. The percentage of CD4- CD8- TCR alpha beta high cells increased during thymic reconstitution after irradiation, and maximally constituted 28.6% of CD4- CD8- thymocytes on day 7. Similar results were obtained during thymic reconstitution after glucocorticoid treatment. In contrast, continuous treatment with FK506 for 7 days markedly decreased not only the percentages of CD4+ CD8- TCR alpha beta high and CD4- CD8+ TCR alpha beta high thymocytes, but also that of CD4- CD8- TCR alpha beta high thymocytes. These results indicate that rat CD4- CD8- thymocytes contain a subpopulation of mature (TCR alpha beta high) cells. The possible implications of the existence of this subpopulation with regard to thymocyte differentiation and maturation are discussed.     

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.     

Immunohistology of T cell differentiation in the thymus of H-Y-specific T cell receptor alpha/beta transgenic mice

We examined the immunohistological aspects of the H-Y specific T cell receptor (TcR) alpha/beta transgene expression in the thymus of male and female transgenic (Tg) mice. Virtually all thymocytes expressed the beta transgene in both the male and female thymus. Expression of accessory molecules (co-receptors) in Tg mice deviated from control mice. In the male Tg thymus, CD8 expression was either low or absent on both cortical and medullary thymocytes. In contrast, in the thymus of female mice, CD8+ cells were found both in the cortex and in the medulla. The majority of medullary thymocytes was bright CD8+. This is in clear contrast to the CD8 distribution in control B6 mice, where only a few percent of medullary cells are CD8+. Similarly, the proportion of cells expressing CD4 antigens was reduced in the cortex and medulla of the thymus from male Tg mice, as compared to the thymus of female Tg mice and B6 control mice. Comparative analysis of the stromal cell types of the thymic microenvironments in the three groups of mice revealed that the cortical thymic microenvironment of male Tg mice differed, compared to that of female Tg mice. In particular, the deep cortex showed a closely packed meshwork of epithelial reticular cells. Moreover, H-2Db molecules (which are the restricting elements for the Tg TcR alpha/beta) were abnormally expressed in the thymic cortex of male mice. The cortical microenvironment in female mice, on the other hand, appeared normal. Together, the data indicate that TcR alpha/beta transgene expression in male mice leads to an aberrant co-receptor expression in both cortical and medullary lymphoid cells as well as an abnormal composition of the cortical microenvironment. Both phenomena may be the consequence of "negative selection" of developing H-Y-specific T cells, as it occurs only in the male Tg thymus. The absence of the H-Y antigen, but presence of the restricting element H-2Db in the thymic cortex of female mice, leads to accumulation of CD8+ in the medulla, a phenomenon interpreted as "positive selection".     

Interferons alpha/beta inhibit IL-7-induced proliferation of CD4- CD8- CD3- CD44+ CD25+ thymocytes, but do not inhibit that of CD4- CD8- CD3- CD44- CD25- thymocytes.

Type 1 interferons (IFN-alpha/beta) have recently been shown to inhibit interleukin-7 (IL-7)-induced growth and survival of early B-lineage cells. The CD3- CD4- CD8- (triple negative; TN) thymocytes from normal mice strongly proliferated upon stimulation with IL-7 in suspension, culture. Such an IL-7-induced proliferation was suppressed by the addition of IFN-alpha/beta, but a fraction of the TN thymocytes still showed proliferation. The IL-7-induced growth of TN thymocytes from acid mice, which lack the CD44- CD25- subpopulation, was completely inhibited by the addition of IFN-alpha/beta. The IL-7 induced proliferation of CD4- CD8- thymocytes from T-cell receptor (TCR) transgenic mice, the majority of which are CD3+ CD44- CD25-, was resistant to IFN-alpha/beta-mediated suppression. In fetal thymus organ cultures (FTOC), the addition of IL-7 greatly increased the population of CD4- CD8- CD44+ CD25+ thymocytes and IFN-alpha/beta inhibited this IL-7-driven expansion. In contrast, the addition of IL-7 markedly decreased the percentages of CD4- CD8- CD3- CD44- CD25- cells, and IFN-alpha/beta reversed the effect and increased the subpopulations of CD44- CD25+ and CD44- CD25-. Finally, IFN-beta mRNA was found to be expressed in the thymus. The data suggest that type I interferons inhibit IL-7-driven proliferation of TN thymocytes, but do not block the normal differentiation process.     

Interleukin-7 treatment promotes the differentiation pathway of T-cell-receptor-alpha beta cells selectively to the CD8+ cell lineage.

In this report we have studied the influence of interleukin-7 (IL-7) on thymocyte differentiation by evaluating the effects of IL-7 on the generation of T-cell receptor-alpha beta (TCR-alpha beta) and TCR-gamma delta thymocyte subpopulations in rat fetal thymus organ culture. IL-7 enhanced the differentiation pathway of TCR alpha beta thymocytes, first increasing the numbers of immature CD8+ cells, and later those of both CD4+ CD8+ and mature thymocytes. The kinetics of thymocyte migration out of thymic lobes was also accelerated, and the average number of mature TCR-alpha beta phi emigrants per day was increased in the presence of IL-7. Moreover, mature CD4- CD8+ thymocytes were preferentially generated after IL-7 administration. This TCR-alpha beta hi cell population was not actively dividing, indicating that IL-7-promoted thymocyte differentiation was selective to the CD8 cell lineage. Distribution of some TCR-V alpha and TCR-V beta segments among mature thymocytes was also modified in IL-7-treated thymic lobes. On the contrary, the maturation of TCR-gamma delta was not affected by IL-7 addition during the first days of culture, but their numbers sharply increased by day 6 of culture. These results were confirmed with IL-7-treated cultures for 24 hr, showing that IL-7 responsiveness was acquired by TCR-gamma delta cells late in thymus ontogeny. The present results thus indicate a key role for IL-7 in the maturation of TCR-alpha beta thymocytes and the expansion of thymic TCR-gamma delta cells.     

CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.

T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.     

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.     

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.     

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.     

Expression and role of interleukin-2 receptor beta chain on CD4-CD8- T cell receptor alpha beta+ cells [corrected]

Using anti-murine interleukin-2 receptor beta chain (IL-2R beta) monoclonal antibody (mAb), we have examined the expression of IL-2R beta on murine thymocyte subpopulations. We found that it was constitutively expressed on 1%-4% of thymocytes in an almost mutually exclusive fashion with IL-2R alpha. The expression of IL-2R beta is developmentally regulated. While it is expressed mainly on T cell receptor gamma delta+ (TcR gamma delta+) cells during fetal age, the major subpopulation expressing IL-2R beta in adult mouse shifts to CD4-CD8-TcR alpha beta+ thymocytes. A considerable portion of CD4-CD8- TcR alpha beta+ cells in other organs, including spleen, bone marrow and liver, was also found to express IL-2R beta. In fetal thymus organ culture, the above thymocyte subset was induced to expand in response to exogeneous IL-2, and the expansion was inhibited by addition of anti-IL-2R beta mAb, suggesting that IL-2R beta is functional in this subpopulation. However, in vivo blockade of the IL-2/IL-2R pathway with the mAb did not exert any effects on the appearance of CD4-CD8- TcR alpha beta+ cells both in the thymus and the periphery. This indicates that the development of CD4-CD8- TcR alpha beta+ cells is not solely controlled by IL-2 but also by other complex elements.     

Selection of phenotypically distinct NK1.1+ T cells upon antigen expression in the thymus or in the liver.

Unlike the main TCR alphabeta T cell lineage in which deletion occurs at the CD4+ CD8+ double-positive (DP) stage upon TCR engagement by antigen in the thymus, some T cells appear to require such engagement for their selection, either in the thymus or extrathymically. We used a transgenic TCR (tgTCR) model which, as we previously showed, led to selection upon expression of the corresponding antigen H-2Kb (Kb) in the thymus, of tgTCR/CD3(lo) CD4- CD8- double-negative (DN) thymocytes that expressed the NK1.1 marker (NK T cells) (Curnow, S. J., et al., Immunity 1995. 3: 427). We now report that antigen expression on medullary epithelial cells of the thymus failed to select the NK T cells, whereas its expression on thymocytes did, although tgTCR DP thymocyte development was affected under both conditions. Antigen expression on hepatocytes (Alb-Kb mice) did not perturb tgTCR DP thymocyte development. No enrichment in tgTCR NK T cells was detected in the periphery, except for the liver of the Alb-Kb/tgTCR mice. When reconstitution of thymectomized and irradiated H-2k hosts expressing or not Kb was performed with bone marrow from tgTCR H-2k mice, an enrichment in tgTCR+ NK T cells was found in the liver, but not in the spleen, of the hosts which expressed Kb, either selectively on hepatocytes or ubiquitously. Surprisingly, the majority of the hepatic tgTCR+ NK T cells also expressed the CD8 alpha/beta heterodimer. These results indicate that thymus-independent NK T cells with unique phenotypic characteristics can be selected upon antigen encounter in the liver.     

Development of CD8 alpha/beta + TCR alpha beta intestinal intraepithelial lymphocytes in athymic nu/nu mice and participation in regional immune responses.

On the basis of the CD8 coreceptor expression, T-cell receptor (TCR)alpha beta-bearing intestinal intraepithelial lymphocytes (i-IEL) segregate into two populations. The CD8 alpha alpha + TCR alpha beta i-IEL develop thymus independently, whereas the CD8 alpha beta + TCR alpha beta i-IEL are generally considered to be thymus dependent. Flow cytometry analysis revealed a distinct population of CD8 alpha beta + TCR alpha beta i-IEL in individual athymic nu/nu mice. The i-IEL encompassing CD8 alpha beta + TCR alpha beta cells expressed potent cytolytic and interferon-gamma-producing activities. These findings demonstrate that CD8 alpha beta + TCR alpha beta i-IEL can develop in nu/nu mice independently from a functional thymus and suggest that these cells, directly or indirectly, perform biological functions in the gut.     

CD4-CD8- thymocytes that express the T cell receptor may have previously expressed CD8

Amongst CD4-CD8- (double negative) thymocytes there is a sizeable population (variable from strain to strain) of cells expressing surface T cell receptor (TCR). These TCR+ double negatives are predominantly non-cycling, have very little precursor activity, and, unlike the TCR-CD4-CD8- thymocytes, appear not to be part of the mainstream of thymocyte development. A unique feature of this population is the biased V beta-gene region usage. In CBA mice, 60-70% of TCR+ CD4-CD8- cells express receptors that utilize V beta 8 gene products, compared with peripheral T cells from the same strain which are only 20-30% V beta 8+. This suggests that the high V beta 8 usage may be the result of some selective process. A growing body of experimental data suggests that TCR specificity selection occurs at the CD4+CD8+ stage of thymocyte development. In order to gain some insight into the previous history of the TCR+ double negatives, in particular whether or not they have previously expressed CD8 and therefore been eligible for selection, we have determined the methylation state of the CD8 gene and compared it to other thymocyte populations. We show that the TCR+ CD4-CD8- thymocytes are demethylated at some sites in the CD8 gene, consistent with previous CD8 expression. However, the demethylation pattern is distinct from that seen on typical peripheral T cells or on mature thymocytes, suggesting that the TCR+ CD4-CD8- thymocytes are not derived from mature thymocytes or peripheral T cells which have returned to the thymus and downregulated CD8 expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of early expression of TCR alpha chain on thymocyte development

CD4(-)CD8(-) thymocytes expressing a transgenic T cell receptor (TCR) alpha chain have decreased capacity to give rise to CD4(+)CD8(+) thymocytes when compared with wild-type thymocytes. This inefficient CD4(-)CD8(-) to CD4(+)CD8(+) maturation is mediated by the transgenic TCR alpha chain pairing with endogenous TCR beta chain but not with endogenous TCR gamma chain. Comparison between TCR alpha chain-transgenic mice with or without a functional pre-TCR alpha (pT alpha ) chain reveals that the formation of transgenic alpha/endogenous beta TCR on CD4(-)CD8(-) thymocytes inhibits the formation of pre-TCR, but at the same time mediates CD4(-)CD8(-) to CD4(+)CD8(+) maturation in the absence of pre-TCR, albeit inefficiently. These results indicate that alpha beta TCR and pre-TCR provide different signals for thymocyte development. They also suggest that the precise regulation of the sequential rearrangements of TCR beta and alpha loci and the cellular expansion induced by the pre-TCR may both be evolved to ensure the efficient generation of mature alpha beta T cells.     

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.