Developmental regulation of thymocyte susceptibility to deletion by "self"-peptide.

The specificity of the T cell receptor (TCR) repertoire for foreign peptide bound to self-major histocompatibility complex (MHC) molecules is determined in large part by positive and negative selection processes in the thymus, yet the mechanisms of these selection events remain unknown. Using in vitro organ culture of thymi isolated from mice transgenic for a TCR-alpha/beta specific for cytochrome c peptide bound to I-Ek, we analyzed the developmental timing of negative selection (deletion). On the basis of the experiments described below, we conclude that all CD4+8+ thymocytes, and only CD4+8+ thymocytes, are susceptible to negative selection mediated by the cytochrome c peptide antigen. First, we found that deletion of thymocytes resulting from addition of the cytochrome c peptide to the thymic organ cultures can occur at the earliest stage of TCR, CD4, and CD8 coexpression. Second, we found that CD4+8+ thymocytes isolated from positively selecting or nonselecting MHC haplotypes were equally efficiently deleted in vitro, suggesting that positive selection is not a prerequisite for deletion. Third, we examined the effects of TCR/ligand avidity on the developmental timing of deletion by varying the concentration of cytochrome c peptide added to the organ cultures. We detected deletion only at the CD4+8+ stage: intermediate concentrations of peptide that resulted in partial deletion of CD4+8+ cells did not eliminate the appearance of mature CD4+8- cells. Finally, we found that CD4+8- thymocytes were resistant to deletion as well as activation by peptide antigen added to the intact organ cultures. Nevertheless, the CD4+8- thymocytes isolated from the peptide-treated organ cultures responded vigorously to peptide presented by spleen cells in vitro. Thus, the T cells were tolerant of (but not anergized by) self-antigen encountered in thymic organ culture. Together, these results indicate that thymocytes susceptible to negative selection are not developmentally distinct from those susceptible to positive selection, and further, that the thymic microenvironment plays a role in regulating the outcome of TCR/ligand interactions.

Distinct roles of prostaglandin H synthases 1 and 2 in T-cell development.

Prostaglandin G and H synthases, or cyclooxygenases (COXs), catalyze the formation of prostaglandins (PGs). Whereas COX-1 is diffusely expressed in lymphoid cells in embryonic day 15.5 thymus, COX-2 expression is sparse, apparently limited to stromal cells. By contrast, COX-2 is predominant in a subset of medullary stromal cells in three- to five-week-old mice. The isozymes also differ in their contributions to lymphocyte development. Thus, experiments with selective COX-1 inhibitors in thymic lobes from normal and recombinase-activating gene-1 knockout mice support a role for this isoform in the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP). Concordant data were obtained in COX-1 knockouts. Pharmacological inhibition and genetic deletion of COX-2, by contrast, support its role during early thymocyte proliferation and differentiation and, later, during maturation of the CD4 helper T-cell lineage. PGE2, but not other PGs, can rescue the effects of inhibition of either isoform, although it acts through distinct EP receptor subtypes. COX-dependent PG generation may represent a mechanism of thymic stromal support for T-cell development.

Altered aging-related thymic involution in T cell receptor transgenic, MHC-deficient, and CD4-deficient mice.

During aging in mice and humans, a gradual decline in thymus integrity and function occurs (thymic involution). To determine whether T cell reactivity or development affects thymic involution, we compared the thymic phenotype in old (12 months) and young (2 months) mice transgenic for rearranged alphabeta or beta 2B4 T cell receptor (TCR) genes, mice made deficient for CD4 by gene targetting (CD4(-/-)), mice made deficient for major histocompatibility complex (MHC) class I (beta2M-/-) or class II genes (A(beta)(b-/-) on C57Bl/6 background) or both. The expected aging-related reductions in thymic weights were observed for all strains except those bearing disruption of both class I and class II MHC genes. Therefore, disruption of MHC class I and class II appeared to reverse or delay aging-related thymic atrophy at 12 months. Immunohistochemical analysis of aging-associated alterations in thymic morphology revealed that TCR alphabeta transgenes, CD4 disruption, and MHC class II disruption all reduced or eliminated these changes. All strains examined at 12 months showed alterations in the distribution of immature thymocyte populations relative to young controls. These results show that aging-associated thymic structural alterations, size reductions, and thymocyte developmental delays can be separated and are therefore causally unrelated. Furthermore, these results suggest that the T cell repertoire and/or its development play a role in aging-related thymic involution.

Quantitative analysis of the efficiency of clonal deletion in the thymus.

One of the major mechanisms for establishing self-tolerance is the clonal deletion of self-reactive T cells during their development in the thymus. Using a TCR transgenic mouse model, we have established a quantitative ex vivo assay for examining the sensitivity and specificity of negative selection. Thymic organ cultures established from mice of varying MHC haplotypes were incubated with antigen, and the efficiency of clonal deletion assessed. We show here that clonal deletion of CD4+8+ thymocytes is sensitive to both the gene dosage and the allelic variation of MHC class II molecules expressed on thymic antigen-presenting cells. We also find that when epithelial cells in the thymic cortex are the only antigen-presenting cells expressing the appropriate MHC class II molecules, negative selection of CD4+8+ cells is as efficient as when antigen is presented on all thymic antigen-presenting cells. These studies demonstrate that the induction of self-tolerance via clonal deletion in the thymus is a function not only of antigen concentration, but also of MHC class II cell-surface density. In addition, together with the reports of others, these results confirm that cortical epithelial cells can mediate negative selection, and demonstrate that they do so in the intact thymic microenvironment.

Purification and characterization of retrovirally transduced hematopoietic stem cells.

We have developed a method for the purification of retrovirally transduced hematopoietic stem cells, based on a modification of the stem cell purification protocols developed by Spangrude et al. [Spangrude, G., Heimfeld, S. & Weissman, I. (1988) Science 241, 58-64] and Spangrude and Scollay [Spangrude, G. & Scollay, R. (1990) Exp. Hematol. 18, 920-926], that depends upon the use of bone marrow cells isolated from 5-fluorouracil-treated mice that have been subsequently cocultivated with recombinant retrovirus-producing cell lines. We found that purified cell populations bearing a Sca 1+ Lin- Thy 1- surface phenotype represent a 50-100% pure population of spleen colony-forming cells (CFU-S day 12). Animals injected with 300 or more purified cells were consistently radioprotected and reconstituted in multiple lineages with donor cells. Sca 1+ Lin- Thy 1- CFU-S day 12 stem cells were shown to be efficiently (100%) transduced by the recombinant retroviruses used in the study. Gene transfer into long-term reconstituting stem cells, as evidenced by Southern blot analysis of mature hematopoietic cell types 3 months after transplantation, was observed only in recipients injected with large numbers (approximately 4000-5000) of the purified cells. The development of methods for purifying retrovirally transduced stem cells should prove extremely useful for various studies in which it is of interest to characterize the activity of a specific gene product (e.g., growth factor, receptor, oncogene) specifically in primitive hematopoietic cell types.

Do the CD4 and CD8 lineages represent parallel pathways?

It is now well established that the progression of T cell development requires interactions between the surfaces of thymocytes and thymic stromal cells. For example, the maturation of CD4+8+ cells into functional CD4+ or CD8+ cells requires TCR/MHC interactions, which, depending on the specificity of the particular TCR, direct the thymocyte to the appropriate lineage. Beyond this, little is known about the molecular mechanism of this lineage choice. Here we describe our recent studies of CD4/CD8 lineage commitment using TCR transgenic mice expressing a well-defined MHC class II specific TCR. While the results of these experiments are inconsistent with a model in which CD4 versus CD8 lineage is determined by an initial TCR/MHC/co-receptor interaction, they also do not support a simple stochastic model of lineage commitment. Instead, we suggest that the CD4 and CD8 lineage may not represent equivalent pathways of T cell maturation. Additionally, we draw several parallels between stochastic models in positive selection and in hematopoiesis.

Enhanced T cell maturation and altered lineage commitment in T cell receptor/CD4-transgenic mice.

The two mature subsets of T lymphocytes, CD4+ and CD8+ cells arise from a common progenitor during development in the thymus. The differentiation of this progenitor cell into one of the two mature T cell subsets is determined by the specificity of the alpha beta TCR for MHC class I or class II molecules. Using a line of TCR-transgenic mice expressing an MHC class II-specific TCR, 2B4, we have examined the thymocyte subsets present in a selecting versus a nonselecting MHC background. Our results are consistent with the model that CD4 versus CD8 downregulation occurs stochastically. In an effort to confirm these findings, we examined T cell development in double-transgenic mice expressing high levels of a CD4-transgene plus the 2B4 TCR transgenes. Unlike the findings with MHC class I-specific TCR-transgenic models, peripheral T cells in these mice include a substantial fraction of MHC class II-specific (2B4+) T cells expressing CD8 plus the transgene-encoded CD4. In addition, analysis of both thymocytes and peripheral T cells in these double-transgenic mice indicate that CD4 overexpression also leads to a striking enhancement of T cell maturation in 2B4 TCR-transgenic mice. Together with the studies of others, these data support a stochastic model for CD4 versus CD8 lineage commitment of an MHC class II-specific TCR during T cell development in the thymus.

A peptide antigen antagonist prevents the differentiation of T cell receptor transgenic thymocytes.

The developmental fate of an immature T cell is determined in the thymus. Depending on the specificity of its TCR, a thymocyte receives signals to either die or differentiate. We have used fetal thymic organ cultures derived from TCR transgenic mice to examine the role of MHC/peptide ligands in T cell selection. Single amino acid substituted peptide analogues of the Ag recognized by the transgenic TCR were examined for their ability to enhance or interfere with positive selection. We have identified a nonstimulatory peptide analogue that interferes with the differentiation of transgenic CD4+8+ thymocytes into CD4+8- cells. We also show that this peptide, substituted in a TCR contact residue, is a competitive antagonist for activation of the T cell hybridoma expressing the same TCR. These observations demonstrate a novel mechanism for tolerance induction in the thymus.

T cell development in PU.1-deficient mice.

These studies address the role of PU.1 in T cell development through the analysis of PU.1-/- mice. We show that the majority of PU.1-/- thymocytes are blocked in differentiation prior to T cell commitment, and contain a population of thymocyte progenitors with the cell surface phenotype of CD44+, HSAbright, c-kitint, Thy-1-, CD25-, Sca-1-, CD4-, and CD8-. These cells correspond in both number and cell surface phenotype with uncommitted thymocyte progenitors found in wild-type fetal thymus. RT-PCR analysis demonstrated that PU.1 is normally expressed in this early progenitor population, but is down-regulated during T cell commitment. Rare PU.1-/- thymi, however, contained small numbers of thymocytes expressing markers of T cell commitment. Furthermore, almost 40% of PU.1-/- thymi placed in fetal thymic organ culture are capable of T cell development. Mature PU. 1-/- thymocytes generated during organ culture proliferated and produced IL-2 in response to stimulation through the TCR. These data demonstrate that PU.1 is not absolutely required for T cell development, but does play a role in efficient commitment and/or early differentiation of most T progenitors.

The COX-2 inhibitor NS-398 causes T-cell developmental disruptions independent of COX-2 enzyme inhibition.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the function of cyclooxygenases, COX-1 and COX-2, which catalyze the first step in the synthesis of inflammatory mediators (PGE2). We sought to understand the roles of cyclooxygenases and NSAIDs in T-cell development. Our data show no significant defects in T-cell development in fetal thymic organ cultures of mice disrupted in both or either COX genes or in mice disrupted in either EP-1 or EP-2 receptor genes. On the other hand, NSAIDs reproducibly caused thymocyte developmental defects. However, the specific effects of the COX-2 inhibitors were not correlated with their potency for inhibition of COX-2 activity. We focused on the NS-398 COX-2 inhibitor and showed that its effects could not be reversed by exogenous PGE2. Furthermore, NS-398 was inhibitory even when its target, COX-2, was absent. These data show that the T-cell developmental effects of NS-398 are COX-2 and PGE2 independent.

Alterations in CD4 dependence accompany T cell development and differentiation.

Several studies have indicated that the necessity for co-receptor engagement during T cell activation depends on the avidity of the TCR-MHC interaction under investigation. Using thymocytes, naive T cells and a long-term T cell line isolated from 2B4 TCR transgenic mice, we have examined the role of the CD4 co-receptor on cells expressing the identical TCR at multiple stages of T cell maturation. When anti-CD4 Fab fragments were used to block CD4-MHC class II interactions, we found decreasing CD4 dependence as T cells matured. As a second approach to examining the role of the CD4 co-receptor, we generated I-Ek mutants defective in CD4 interactions. In the course of this study, we identified a new potential site for CD4 interaction in the beta1 domain of I-Ek. The new beta1 mutation and a mutation in the previously described CD4 binding site in the beta2 domain both interfere with stimulation of 2B4 thymocytes, but not mature T cells. Together these data demonstrate that the role of the CD4 co-receptor depends on the state of maturation of the T cell.

Conserved transmembrane tyrosine residues of the TCR beta chain are required for TCR expression and function in primary T cells and hybridomas.

The T cell receptor (TCR) beta chain transmembrane domain contains two evolutionarily conserved tyrosines (Y). In this study, the functional basis for the evolutionary conservation is addressed by mutation of the residues, expression of the mutants in hybridoma and primary T cells, and examination of TCR signaling function. We find that the phenotype of the mutants, both surface expression and ability to signal for IL-2 production, is highly variable in different mouse T hybridoma lines. Although we have not been able to determine the basis for these differences in the hybridomas, expression of the mutants in primary T cells provides a definitive assessment of mutant phenotype. We show that mutation of the N-terminal Y to either leucine (L) or alanine (A) results in low surface expression in primary T cells, while mutation of both N- and C-terminal Y to A or L abrogates surface expression. However, the more conservative mutation of both transmembrane Y to phenylalanine maintained receptor surface expression and assembly while severely disrupting signaling in primary T cells. Our data demonstrate that TCR beta chain transmembrane Y are essential for TCR signal transduction as well as complex assembly. These findings suggest that protein-protein interactions involving membrane-spanning domains are likely relevant for TCR signal transduction mechanisms.

Differential expression and regulation of cyclooxygenase isozymes in thymic stromal cells.

Prostaglandins (PGs) are lipid-derived mediators of rapid and localized cellular responses. Given the role of PG in supporting thymic T cell development, we investigated the expression of the PG synthases, also known as cyclooxygenases (COX)-1 and -2, in the biosynthesis of PGs in thymic stromal cell lines. The predominant isozyme expressed in cortical thymic epithelial cells was COX-1, while COX-2 predominated in the medulla. IFN-gamma up-regulated expression and activity of COX-2 in medullary cells, in which COX-2 was expressed constitutively. In contrast, IFN-gamma down-regulated COX-1 activity, but not expression, in cortical cells. Stromal cells support T cell development in the thymus, although the mediators of this effect are unknown. Selective inhibition of COX-2, but not COX-1, blocked the adhesion of CD4+CD8+ and CD4+CD8- thymocytes to medullary cell lines. No effect of the inhibitors was observed on the interactions of thymocytes with cortical epithelial lines. These data further support the differential regulation of COX-1 and COX-2 expression and function in thymic stromal cells. PGs produced by COX-2 in the medullary thymic stroma may regulate the development of thymocytes by modulating their interaction with stromal cells.

PU.1 is required for myeloid-derived but not lymphoid-derived dendritic cells.

The ets-family transcription factor PU.1 is required for the proper development of both myeloid and lymphoid progenitors. We used PU. 1-deficient animals to examine the role of PU.1 during dendritic cell development. PU.1(-/-)animals produce lymphoid-derived dendritic cells (DC): low-density class II major histocompatibility complex [MHC-II(+)] CD11c(+) CD8alpha(+) DEC-205(+). But they lack myeloid-derived DC: low-density MHC-II(+) CD11c(+) CD8alpha(-) DEC-205(-). PU.1(-/-) embryos also lack progenitors capable of differentiating into myeloid DC in response to granulocyte-macrophage colony-stimulating factor plus interleukin-4. The appearance of lymphoid DC in developing PU.1(-/-)thymus was initially delayed, but this population recovered to wild type (WT) levels upon organ culture of isolated thymic lobes. PU. 1(-/-)lymphoid DC were functionally equivalent to WT DC for stimulating T-cell proliferation in mixed lymphocyte reactions. These results demonstrate that PU.1 is required for the development of myeloid DC but not lymphoid DC.

Transmembrane polar residues of TCR beta chain are required for signal transduction.

Mutagenic analyses have identified structural motifs important for TCR-mediated signaling in the antigen-binding chains, CD3 and zeta subunits of the TCR complex. In this study, we altered selected residues in the transmembrane and extracellular constant regions of the TCR beta chain and expressed the mutants in a T hybridoma line bearing endogenous receptor. We measured cytokine production and apoptosis in response to antigen or antibody. We found that mutation of one or both of the transmembrane tyrosine residues in the TCR beta chain caused a marked reduction in responsiveness. Mutation of the transmembrane serine to alanine also reduced responses, although less markedly. Immunoprecipitation analyses showed that the TCR beta mutations did not alter association with zeta. These experiments identify a signaling role for the transmembrane domain of the TCR beta chain.

The in vivo mechanism of action of CTLA4Ig.

A single dose of CTLA4Ig, an inhibitor of CD28-mediated T cell costimulation, given 2 days after transplantation induces specific unresponsiveness to alloantigens in vivo. However, the mechanisms responsible are unknown. Using pigeon cytochrome c as a model Ag, we monitored the effect of CTLA4Ig on the fate of Ag-reactive T cells in normal mice and on pigeon cytochrome c-specific TCR transgenic cells adoptively transferred into congenic mice. CTLA4Ig significantly inhibits immunization with pigeon cytochrome c. In particular, ELISA and ELISPOT assays indicate an 80 to 90% reduction in Th1 (i.e, IL-2 and IFN-gamma) cytokine production and in the numbers of cytokine-producing cells. Interestingly, despite this profound reduction in cytokine-producing cells, Ag-reactive T cells expand in CTLA4Ig-treated animals, although the degree of expansion is reduced by 50% compared with that in control Ig-treated animals. Thus, loss of Th1 cytokine production in CTLA4Ig-treated animals is not fully explained by the decreased expansion of Ag-specific T cells. These results suggest two mechanisms of action for CTLA4Ig in vivo: inhibition of expansion of Ag-reactive cells and induction of anergy in the residual population.

The 3' untranslated regions of two related mRNAs contain an element highly repeated in the sea urchin genome.

Two closely related cDNA clones, pSpec1 and pSpec2, specifying two developmentally regulated tissue specific mRNAs from sea urchin embryos were used to probe a sea urchin genomic lambda library. Screening 10,000 phage by plaque hybridization yielded several hundred positive signals. With more stringent wash procedures, only two to three phage were positive. Three of these phage, one isolated by stringent wash procedures and two isolated by standard wash procedures were further investigated by restriction analysis, RNA gel blots, and DNA sequencing. The phage isolated by the stringent wash procedure appears to be a gene coding for the Specl mRNA. The other phage contain only partial homology to pSpec1 and pSpec2, 150 to 200 base pairs of the 3' untranslated region of the Spec1 and Spec2 mRNAs. It is concluded that the Spec1 and Spec2 mRNAs contain a highly repetitive element near their 3' end. The element is present at 2000 to 3000 copies per genome and may be transcribed at some sites other than those coding for the Spec1 and Spec2 genes. The possible function and evolutionary origin of the repetitive element is discussed.