A novel role for CD28 in thymic selection: elimination of CD28/B7 interactions increases positive selection

While the importance of the CD28/B7 costimulation pathway is well established for mature T cells, the role of CD28 in thymocyte selection is less well defined. The role of CD28 in both negative and positive selection was assessed using H-Y-specific TCR-transgenic (Tg) RAG-2-deficient (H-Yrag) mice. Negative selection in male H-Yrag mice was not affected by deficiency in CD28 or B7. Surprisingly, absence of CD28 or B7 in H-Yrag females resulted in increased numbers of CD8 single-positive (SP) thymocytes. The CD8 SP thymocytes found in these females were mature and functionally competent. Furthermore, double-positive (DP) thymocytes from CD28-knockout (CD28KO) or B7.1/B7.2 double-KO (B7DKO) females had higher levels of both CD5 and TCR than those from WT females, consistent with a stronger selecting signal. CD28KO H-Yrag fetal thymic organ cultures also had elevated numbers of thymic CD8 SP cells, reflecting increased thymic differentiation and not recirculation of peripheral T cells. Finally, increased selection of mature CD4 and CD8 SP T cells was observed in non-TCR-Tg CD28KO and B7DKO mice, indicating that this function of CD28-B7 interaction is not unique to a TCR-Tg model. Together these findings demonstrate a novel negative regulatory role for CD28 in inhibiting differentiation of SP thymocytes, probably through inhibition of thymic selection.     

Impaired thymic selection in mice expressing altered levels of the SLP-76 adaptor protein

Intracellular signaling initiated by ligation of the TCR influences cell fate at multiple points during the lifespan of a T cell. This is especially evident during thymic selection, where the nature of TCR-dependent signaling helps to establish a MHC-restricted, self-tolerant T cell repertoire. The Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) adaptor protein is a required intermediate in multiple signaling pathways triggered by TCR engagement, several of which have been implicated in dictating the outcome of thymic selection (e.g., intracellular calcium flux and activation of ERK family MAPKs). To determine if thymocyte maturation and selection at later stages of development are sensitive to perturbations in SLP-76 levels, we analyzed these crucial events using several transgenic (Tg) lines of mice expressing altered levels of SLP-76 in the thymus. In Tg mice expressing low levels of SLP-76 in preselection thymocytes, the CD4:CD8 ratio in the thymus and spleen was skewed in a manner consistent with impaired selection and/or maturation of CD4+ thymocytes. Low SLP-76 expression also correlated with reduced CD5 expression on immature thymocytes, consistent with reduced TCR signaling potential. In contrast, reconstitution of SLP-76 at higher levels resulted in normal thymic CD5 expression and CD4:CD8 ratios in the thymus and periphery. It is curious that thymic deletion of TCR-Tg (HY) thymocytes was markedly impaired in both lines of Tg-reconstituted SLP-76-/- mice. Studies using chimeric mice indicate that the defect in deletion of HY+ thymocytes is intrinsic to the developing thymocyte, suggesting that maintenance of sufficient SLP-76 expression from the endogenous locus is a key element in the selection process.     

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.     

Developmental changes predispose the fetal thymus to positive selection of CD4+CD8- T cells.

Selection of a competent T-cell repertoire is dependent on complex interactions between immature thymocytes and components of the thymic stroma. These events may be preserved in vitro by excising developing thymus rudiments and maintaining them under carefully controlled conditions in fetal thymus organ cultures (FTOC). Using this approach, we have shown that the ability of C57B1/6 thymi to sustain positive selection of mature CD4+CD8- cells is profoundly influenced by the day of gestation on which they are excised: while thymocytes from day 14 rudiments fail to progress beyond the CD4+CD8+ stage of the developmental pathway, day 15 and day 16 thymi support the differentiation of CD4+CD8- thymocytes. Importantly, day 16 thymocytes transferred to day 14 deoxyguanosine-treated rudiments are likewise arrested at the CD4+CD8+ stage, suggesting that the thymic microenvironment of day 14 rudiments, rather than the state of differentiation of the thymocytes they contain, is responsible for the block in positive selection. Our studies of the stromal elements of day 14 rudiments have, however, revealed no obvious deficiencies in the cell types represented, or their expression of class II major histocompatibility complex (MHC) determinants. Furthermore, we have been unable to circumvent the blockage in positive selection by the addition of certain cytokines expressed late during gestation. These results suggest that subtle changes occurring at day 15 of ontogeny render the thymic microenvironment capable of positive selection.     

Development and function of autospecific dual TCR+ T lymphocytes

Recent studies have challenged the long held concept that each T lymphocyte expresses on its surface only a single, unique alphabetaTCR. Dual TCR+ T cells have been recognized, however, their origin and potential to escape screening for self-reactivity remain obscure. We now report the thymic generation of dual alphabetaTCR+ T cells in the H-2Db/H-Y-specific TCR transgenic (Tg) mouse. Dual TCR+ thymocytes were positively selected less efficiently than single TCR+ thymocytes, although a subset attained maturity. Importantly, when TCR Tg mice were bred onto a negatively selecting background, auto-specific cells survived central deletion and matured as CD4+ dual TCR+ cells. These cells were autoreactive when CD8 expression was restored. The existence of autospecific, dual TCR+ T cells may have implications for the maintenance of self tolerance.     

Alterations during positive selection in the thymus of nackt CD4-deficient mice

The T-cell repertoire is shaped by the positive and negative selection of immature CD4(+) CD8(+) double positive (DP) thymocytes. Positive selection of DP T cells to the CD4(+) CD8(-) and CD4(-) CD8(+) simple positive (SP) lineages is a multistep process which involves cellular interactions between thymocytes and stromal cells. Mutant nackt (nkt/nkt) mice have been shown to have a deficiency in the CD4(+) CD8(-) T-cell subset both in the thymus and in the periphery. The present report suggests that nkt/nkt mice present alterations in early steps of positive selection because they show decreases in the percentages of CD69(+) and CD5(+) cells within the DP subset. Experiments involving bone marrow transfer and thymic chimeras demonstrate that the thymic epithelium of nkt/nkt mice is involved in the alterations registered during positive selection and dictates the ultimate fate of CD4(+) SP cells.     

Extrathymic signals regulate the onset of T cell repertoire selection

The thymus is the principal site of T cell development, coordinating positive and negative selection of an immunocompetent repertoire. The ability of fetal thymi from C57BL/6 mice to support these selection events in culture is, however, critically dependent on the timing of their excision: whereas thymi from 16 day embryos generate mature CD4+8(-) and CD4(-)8+ thymocytes, 14 day thymi show a profound blockage in positive selection at the CD4+8+ stage. Here we show that this blockage is not due to intrinsic deficiencies in the ability of thymocytes to transduce positive selection signals, suggesting the defect to lie within the thymic microenvironment. Since vascularization of the thymus occurs at day 15 of gestation, we investigated whether the exclusion of plasma proteins from 14 day thymi was responsible. Accordingly, the addition of serum from 16 day embryos to organ cultures of 14 day thymi rescued mature CD4+8(-) and CD4(-)8+ subsets. Activity was found to reside in a low molecular weight fraction of serum, sensitive to proteolysis, which was present only transiently during ontogeny. Our data suggest that repertoire selection is initiated following "priming" of the thymic microenvironment by plasma proteins, thereby ensuring the onset of positive selection to be delayed until the entry of extrathymic proteins to which self tolerance must be established.     

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".     

Role of thymic selection in the development of thymic lymphomas in TCR transgenic mice

To investigate the role of antigen receptor-mediated interactions in lymphomagenesis we have analyzed the influence of alpha beta TCR- mediated selection on the development of spontaneous thymic lymphomas, which appear with a high (up to 50%) frequency in mice expressing a transgenic TCR specific for the male antigen (HY) in the context of H- 2Db molecules. To this end we compared the kinetics and the incidence of thymic lymphomas developing in females and males with selecting (H- 2b) and non-selecting (H-2k) MHC molecules. The kinetics of development of thymic lymphomas was similar in positively selecting (H-2b females) and non-selecting (H-2k females and males) environments but significantly slower (P < 0.01) in the negatively selecting environment (H-2b male). Injection of lymphoma cells derived from a H-2b female into the thymus of a H-2b male resulted in strong, antigen-specific inhibition of growth, indicating that the slower kinetics of lymphomagenesis in H-2b males could be due, at least partially, to the sensitivity of oncogenically transformed thymocytes to TCR-mediated negative selection. Phenotypic and functional analysis of lymphoma cells indicated that they originated from the stage of pre-TCR- dependent transition of immature CD4-CD8- to CD4+ CD8+ thymocytes, which in H-2b females and males developed into tumors under different environmental pressures. These results failed to provide convincing evidence for the role of positive selection but provided a strong indication that self antigen-induced negative selection, in addition to its well established role in self tolerance, can occasionally act as a tumor surveillance mechanism by eliminating or suppressing growth of thymocytes undergoing oncogenic transformation.      

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.     

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.     

Affinity of thymic self-peptides for the TCR determines the selection of CD8(+) T lymphocytes in the thymus

Experiments with synthetic antigen peptides have suggested that a critical parameter that determines the developmental fate of an immature thymocyte is the affinity of interaction between TCR and self-peptide/MHC expressed on thymic stromal cells. To test the physiological relevance of this model for thymocyte development, we determined the affinity of the anti-HY TCR (B6.2.16) expressed on CD8(+) cells for thymic self-peptide/H-2D(b) tetramers, then examined the ability of these self-peptides to determine the outcome of B6.2.16 CD8 cell selection in the thymus. The B6.2.16 TCR bound the male HY self-antigen with high affinity. Thymic self-peptides, which are highly abundant on the surface of thymic epithelial cells, bound the B6.2.16 TCR with low affinity. The ability of self-peptides to trigger positive or negative selection of B6.2.16 CD8 cells in cultured fetal thymi was determined by the relative affinity of self-peptide/H-2D(b) for the B6.2.16 TCR. High-affinity binding of the HY self-peptide resulted in B6.2.16 TCR complex zeta chain phosphorylation and the negative selection of B6.2.16 CD8 cells. Low-affinity binding of thymic self-peptides to B6.2.16 TCR resulted in the positive selection of B6.2.16 CD8 cells. Differences between the binding affinities of self-peptides to B6.2.16 TCR accounted for the self-peptide specificity of B6.2.16 CD8 cell positive selection. We conclude that the relative affinity of TCR for thymic self-peptide/class I MHC is a critical parameter in determining fate of CD8(+) cells during thymic selection.     

A cortisone sensitive CD3low subset of CD4+CD8- thymocytes represents an intermediate stage in intrathymic repertoire selection.

Two populations of CD4 single positive (SP) thymocytes were found in transgenic mice bearing class I-restricted Mls-1a reactive (V beta 8.1) TCR genes in the absence of the restriction element. CD3high CD4 SP cells were deleted in the presence of Mls-1a and were cortisone resistant, whereas CD3low CD4 SP cells were not deleted in the presence of Mls-1a and were cortisone sensitive. Intravenous transfer of CD3low CD4 SP cells into nude mice resulted in significant peripheral expansion of these cells with apparent upregulation of CD3. These data indicate that CD3low CD4 SP thymocytes represent an intermediate stage in the transition from CD3low double positive (DP) to CD3high SP thymocytes and raise the possibility that these cells may hve undergone positive but not negative selection events (at least to Mls-1a). Furthermore the fact that CD3high DP thymocytes were also deleted by Mls-1a in these mice suggests strongly that sensitivity to Mls-1a deletion is dependent upon stage of thymic maturation (as revealed by TCR density) rather than CD4/CD8 phenotype.     

In vitro positive selection of alpha beta TCR transgenic thymocytes by a conditionally immortalized cortical epithelial clone

Development of mature CD4 and CD8 single-positive T cells requires a process known as positive selection, which depends on the specific recognition of self-peptide-MHC complexes on thymic stromal cells by immature CD4+CD8+ thymocytes. We have used an in vitro reaggregate system to study the positive selection of thymocytes by conditionally immortalized thymic epithelial clones. Thymocytes from mice transgenic for the F5 alpha beta TCR, specific for a peptide from the influenza nucleoprotein in the context of H-2Db, are positively selected in the H- 2b MHC background, but fail to mature in mice expressing the H-2q haplotype. Development of embryonic day 15 F5 H-2q transgenic thymocytes was followed in reaggregate cultures supplemented with H-2b- expressing epithelial clones. A conditionally immortalized cortical epithelial clone, derived from H-2Kb-tsA58 transgenic mice, was found to be as efficient as freshly isolated thymic stromal cells in positively selecting CD8 transgenic thymocytes. In contrast, an H-2b- expressing kidney epithelial clone did not augment positive selection above background levels, implying that the effect of the thymic epithelial clone was not merely the presentation of selecting MHC molecules. Mature transgenic thymocytes generated in reaggregate cultures were able to differentiate into functionally competent cytotoxic T cells. This model provides an important in vitro system for the detailed study of the specific molecular interactions leading to positive selection of developing thymocytes.      

Involvement of CCR9 at multiple stages of adult T lymphopoiesis

The chemokine CCL25 is constitutively expressed in the thymus, and its receptor CCR9 is expressed on subsets of developing thymocytes. Nevertheless, the function of CCL25/CCR9 in adult thymopoiesis remains unclear. Here, we demonstrate that purified CCR9(-/-) hematopoietic stem cells are deficient in their ability to generate all major thymocyte subsets including double-negative 1 (DN1) cells in competitive transfers. CCR9(-/-) bone marrow contained normal numbers of lineage(-) Sca-1+c-kit+, common lymphoid progenitors, and lymphoid-primed multipotent progenitors (LMPP), and CCR9(-/-) LMPP showed similar T cell potential as their wild-type (WT) counterparts when cultured on OP9-delta-like 1 stromal cells. In contrast, early thymic progenitor and DN2 thymocyte numbers were reduced in the thymus of adult CCR9(-/-) mice. In fetal thymic organ cultures (FTOC), CCR9(-/-) DN1 cells were as efficient as WT DN1 cells in generating double-positive (DP) thymocytes; however, under competitive FTOC, CCR9(-/-) DP cell numbers were reduced significantly. Similarly, following intrathymic injection into sublethally irradiated recipients, CCR9(-/-) DN cells were out-competed by WT DN cells in generating DP thymocytes. Finally, in competitive reaggregation thymic organ cultures, CCR9(-/-) preselection DP thymocytes were disadvantaged significantly in their ability to generate CD4 single-positive (SP) thymocytes, a finding that correlated with a reduced ability to form TCR-MHC-dependent conjugates with thymic epithelial cells. Together, these results highlight a role for CCR9 at several stages of adult thymopoiesis: in hematopoietic progenitor seeding of the thymus, in the DN-DP thymocyte transition, and in the generation of CD4 SP thymocytes.     

Role of CD4 molecule in intrathymic T-cell development.

We have investigated the role of CD4 molecules in intrathymic T-cell repertoire selection. The administration of monoclonal antibody (mAb) to CD4 in organ culture of murine foetal thymus (FTOC) completely inhibited the development of CD4+8- cells, and additional treatment with anti-class II MHC (Ia) mAb caused no further effects on this inhibition. On the other hand, when the potentially autoreactive cells in Mls-1a mice were monitored by expression of the Mls-1a-reactive V beta 6 gene product of T-cell receptor alpha beta (TcR alpha beta), the treatment with anti-CD4 resulted in the appearance of V beta 6-bearing cells to some extent, but this effect was considerably reinforced by the combinatory use of anti-Ia mAb with anti-CD4. In a model system where the bacterial superantigen staphylococcal enterotoxin B serves as self-antigen to deplete V beta 8-bearing cells in FTOC, the depletion of V beta 8+ cells was restored partially by anti-CD4 alone but completely by the combination with anti-Ia. These results suggest that CD4 is indispensable for positive selection of all CD4+8- thymocytes, whereas participation of CD4 in negative selection is only partial. It was also observed that the development of TcR alpha beta-bearing cells in the CD4-8- population was inhibited by the treatment with anti-CD4 mAb. In Mls-1a mice, V beta 6-bearing cells were developed in CD4-8+, CD4+8+, and also in CD4-8- populations after anti-CD4 mAb treatment. It is suggested that TcR alpha beta-bearing CD4-8- cells are possibly originated from CD4+ cells and undergo CD4-mediated thymic selection.     

T cell developmental defects in 'viable motheaten' mice deficient in SHP-1 protein-tyrosine phosphatase. Developmental defects are corrected in vitro in the presence of normal hematopoietic-origin stromal cells and in vivo by exogenous IL-7

Defects in the gene that encodes SHP-1 protein tyrosine phosphatase result in multiple hematopoietic abnormalities and generalized autoimmunity in viable motheaten (me(v)) mice. These mice also exhibit early thymic involution and abnormalities in T cell development. Here, we describe the use of fetal thymic organ culture (FTOC) and bone marrow adoptive transfer to study the effects of SHP-1 deficiency on thymocyte development. Chimeric FTOC established with normal bone marrow placed onto deoxyguanosine-treated fetal thymic lobes or onto scid fetal thymic lobes generated T cells. Bone marrow from SHP-1-deficient me(v)/ me(v) mice generated decreased numbers of T cells in chimeric FTOC established using deoxyguanosine-treated thymi but generated normal numbers in chimeric FTOC established using scid thymi. However, scid fetal thymi seeded with me(v)/ me(v) bone marrow also exhibited morphological abnormalities and contained elevated numbers of macrophages. Addition of IL-7 to me(v)/ me(v) bone marrow-seeded scid FTOC led to increased cell numbers, particularly of macrophages. Intrathymic injection of IL-7 partially restored the ability of progenitor cells in me(v)/ me(v) bone marrow to populate the thymus of adoptive recipients. We conclude that abnormal T cell development in me(v)/ me(v) mice may in part be due to defects in the ability of bone marrow-derived accessory cells to provide bioavailable IL-7 to developing thymocytes.     

Gene expression analysis of thymocyte selection in vivo

Self versus non-self discrimination is a key feature of immunorecognition. Through TCR-activated apoptotic mechanisms, autoreactive thymocytes are purged at the CD4(+)CD8(+) double-positive (DP) precursor stage prior to maturation to CD4(+) or CD8(+) single-positive (SP) thymocytes. To investigate this selection process in vivo, gene expression analysis by oligonucleotide array was performed in TCR transgenic mice. In total, 244 differentially expressed DP thymocyte genes induced or repressed by TCR triggering in vivo were identified. Genes involved in the biological processes of apoptosis, DNA recombination, antigen processing and adhesion are coordinately engaged. Moreover, analysis of gene expression in thymocyte subsets revealed that TCR ligand-induced expression profiles vary according to their developmental stage, with 48 genes showing DP preference and nine showing SP thymocyte preference. Finally, our data suggest that both the extrinsic and the intrinsic apoptosis pathways are operating in thymic selection.     

Downregulated expression of Ly-6-ThB on developing T cells marks CD4+CD8+ subset undergoing selection in the thymus

Interaction of TCRs on CD4+CD8+ immature T cell with MHC-peptide complexes on stromal cells is required for positive and negative selection in the thymus. Identification and characterization of a subpopulation of CD4+CD8+ thymocytes undergoing selection in the thymus will aid in understanding the mechanisms underlying lineage commitment and thymic selection. Herein, we describe the expression of Ly-6 ThB on developing thymocytes. The majority of CD4+CD8+ thymocytes express Ly-6 ThB at high levels. Its expression is downregulated in a subset of CD4+CD8+ thymocytes as well as in mature CD4+CD8- and CD4-CD8+ T cells. More importantly, interaction of TCR/coreceptor with the self-MHC-peptide contributes to the downregulation of ThB expression on developing thymocytes. These findings indicate that downregulation of ThB on CD4+CD8+ thymocytes identifies a unique subset (CD4+CD8+ThBneg-low) of thymocytes that has received the initial signals for thymic selection but have not yet downregulated the CD4 and CD8 cell surface expression. In addition, these results also indicate that a high frequency (approximately 20-40%) of CD4+CD8+ immature thymocytes receive these initial signals during thymic selection.