Dissociation of thymic positive and negative selection in transgenic mice expressing major histocompatibility complex class I molecules exclusively on thymic cortical epithelial cells.

Thymic positive and negative selection of developing T lymphocytes confronts us with a paradox: How can a T-cell antigen receptor (TCR)-major histocompatibility complex (MHC)/peptide interaction in the former process lead to transduction of signals allowing for cell survival and in the latter induce programmed cell death or a hyporesponsive state known as anergy? One of the hypotheses put forward states that the outcome of a TCR-MHC/peptide interaction depends on the cell type presenting the selecting ligand to the developing thymocyte. Here we describe the development and lack of self-tolerance of CD8(+) T lymphocytes in transgenic mice expressing MHC class I molecules in the thymus exclusively on cortical epithelial cells. Despite the absence of MHC class I expression on professional antigen-presenting cells, normal numbers of CD8(+) cells were observed in the periphery. Upon specific activation, transgenic CD8(+) T cells efficiently lysed syngeneic MHC class I(+) targets in vitro and in vivo, indicating that thymic cortical epithelium (in contrast to medullary epithelium and antigen-presenting cells of hematopoietic origin) is incapable of tolerance induction. Thus, compartmentalization of the antigen-presenting cells involved in thymic positive selection and tolerance induction can (at least in part) explain the positive/negative selection paradox.

A potential role for protein tyrosine kinase p56(lck) in rheumatoid arthritis synovial fluid T lymphocyte hyporesponsiveness.

Rheumatoid arthritis (RA) synovial fluid (SF)-T lymphocytes appear relatively inactive in situ and respond only weakly to diverse stimuli ex vivo. To characterize the molecular defects underlying this hyporesponsiveness we analyzed the expression level of several proteins involved in TCR-proximal signal transduction. As compared to peripheral blood (PB)-T lymphocytes, SF-T cells from some (but not all) of the patients analyzed expressed lower levels of TCRalphabeta, CD3epsilon, TCRzeta, p56(lck) and LAT, while p59(fyn), phospholipase C-gamma1 and ZAP-70 expression was unaltered. Semi-quantitative analysis of T cells from several patients revealed that the degree of TCRzeta chain and p56(lck) modulation correlated statistically significantly with the level of SF-T cell hyporesponsiveness. The differential reactivity of p56(lck) specific monoclonal and polyclonal antibodies in SF-T but not PB-T lymphocytes indicated that p56(lck) modulation consists of a conformational change rather than loss of expression. Our results indicate that multiple signaling molecules can be modulated in RA SF-T cells and show for the first time a direct quantitative correlation between T cell hyporesponsiveness and modulation of TCRzeta and of p56(lck), a critical protein tyrosine kinase required for T cell activation.

Interferon gamma gene polymorphism and susceptibility to, and severity of, rheumatoid arthritis.

A strong association between an interferon gamma (IFN-gamma) gene polymorphism and rheumatoid arthritis susceptibility and severity has been reported in a case-control study. We investigated this polymorphism in 103 patients with early rheumatoid arthritis and 130 controls. Severity of rheumatoid arthritis was measured after 4-year follow-up with a validated radiographic score. The median radiographic score in patients increased from 1 (IQR 0-4) to 11.5 (2-35) over the 4-year follow up. The distribution of IFN-gamma alleles did not differ between patients and controls, and the distribution of radiographic scores did not differ among patients carrying the different IFN-gamma alleles. We have failed to confirm the association between the IFN-gamma gene polymorphism and rheumatoid arthritis susceptibility or severity.

Protection from radiation-induced colitis requires MHC class II antigen expression by cells of hemopoietic origin.

Ulcerative colitis, an inflammatory bowel disease, is believed to result from a breakdown of dominant tolerance mechanisms that normally control intestinal immunity. Although CD4+ T lymphocyte subpopulations and expression of MHC class II molecules have been shown to play a role in the pathogenesis of the disease, the nature of the responsible mechanisms remains unclear. In this paper we describe a novel mouse model for inflammatory bowel disease, radiation-induced colitis, that occurs with complete penetrance 6-8 wk postinduction. A combination of high dose gamma-irradiation and lack of MHC class II expression on cells of hemopoietic origin results in development of colitis in C57BL/6 mice. Because of its versatility (due to susceptibility of mice of the widely genetically manipulated C57BL/6 background), high reproducibility, and 100% penetrance, radiation-induced colitis will be a useful mouse model for colitis and a significant tool to study dominant immunological tolerance mechanisms. Moreover, our data imply that tolerization to enteric Ags requires MHC class II mediated presentation by APC of hemopoietic origin.

Deficient T cell fate specification in mice with an induced inactivation of Notch1.

Notch proteins are cell surface receptors that mediate developmental cell specification events. To explore the function of murine Notch1, an essential portion of the gene was flanked with loxP sites and inactivation induced via interferon-regulated Cre recombinase. Mice with a neonatally induced loss of Notch1 function were transiently growth retarded and had a severe deficiency in thymocyte development. Competitive repopulation of lethally irradiated wild-type hosts with wild-type- and Notch1-deficient bone marrow revealed a cell autonomous blockage in T cell development at an early stage, before expression of T cell lineage markers. Notch1-deficient bone marrow did, however, contribute normally to all other hematopoietic lineages. These findings suggest that Notch1 plays an obligatory and selective role in T cell lineage induction.

In vivo T-lymphocyte tolerance in the absence of thymic clonal deletion mediated by hematopoietic cells.

Thymic negative selection renders the developing T-cell repertoire tolerant to self-major histocompatability complex (MHC)/peptide ligands. The major mechanism of induction of self-tolerance is thought to be thymic clonal deletion, ie, the induction of apoptotic cell death in thymocytes expressing a self-reactive T-cell receptor. Consistent with this hypothesis, in mice deficient in thymic clonal deletion mediated by cells of hematopoietic origin, a twofold to threefold increased generation of mature thymocytes has been observed. Here we describe the analysis of the specificity of T lymphocytes developing in the absence of clonal deletion mediated by hematopoietic cells. In vitro, targets expressing syngeneic MHC were readily lysed by activated CD8(+) T cells from deletion-deficient mice. However, proliferative responses of T cells from these mice on activation with syngeneic antigen presenting cells were rather poor. In vivo, deletion-deficient T cells were incapable of induction of lethal graft-versus-host disease in syngeneic hosts. These data indicate that in the absence of thymic deletion mediated by hematopoietic cells functional T-cell tolerance can be induced by nonhematopoietic cells in the thymus. Moreover, our results emphasize the redundancy in thymic negative selection mechanisms.

Thymic lineage commitment rather than selection causes genetic variations in size of CD4 and CD8 compartments.

During their development, immature CD4+ CD8+ thymocytes become committed to either the CD4 or CD8 lineage. Subsequent complete maturation of CD4+ and CD8+ cells requires a molecular match of the expressed coreceptor and the MHC specificity of the TCR. The final size of the mature CD4+ and CD8+ thymic compartments is therefore determined by a combination of lineage commitment and TCR-mediated selection. In humans and mice, the relative size of CD4+ and CD8+ peripheral T cell compartments shows marked genetic variability. We show here that genetic variations in thymic lineage commitment, rather than TCR-mediated selection processes, are responsible for the distinct CD4/CD8 ratios observed in common inbred mouse strains. Genetic variations in the regulation of lineage commitment open new ways to analyze this process and to identify the molecules involved.

Homeostasis limits the development of mature CD8+ but not CD4+ thymocytes.

The involvement of a variety of clonal selection processes during the development of T lymphocytes in the thymus has been well established. Less information, however, is available on how homeostatic mechanisms may regulate the generation and maturation of thymocytes. To investigate this question, mixed radiation bone marrow chimeras were established in which wild-type T cell precursors capable of full maturation were diluted with precursors deficient in maturation potential because of targeted mutations of the RAG1 or TCR-alpha genes. In chimeras in which the majority of thymocytes are blocked at the CD4- CD8- CD25+ stage (RAG1 deficient), and only a small proportion of T cell precursors are of wild-type origin, we observed no difference in the maturation of wild-type CD4- CD8- CD25+ cells to the CD4+ CD8+ stage as compared with control chimeras. Therefore, the number of cell divisions occurring during this transition is fixed and not subject to homeostatic regulation. In contrast, in mixed chimeras in which the majority of thymocytes are blocked at the CD4+ CD8+ stage (TCR-alpha deficient), an increased efficiency of development of wild-type mature CD8+ cells was observed. Surprisingly, the rate of generation of mature CD4+ thymocytes was not affected in these chimeras. Thus, the number of selectable CD8 lineage thymocytes apparently saturates the selection mechanism in normal mice while the development of CD4 lineage cells seems to be limited only by the expression of a suitable TCR. These data may open the way to the identification of homeostatic mechanisms regulating thymic output and CD4/CD8 lineage commitment, and the development of means to modulate it.

Quantitative impact of thymic clonal deletion on the T cell repertoire.

Interactions between major histocompatibility complex (MHC) molecules expressed on stromal cells and antigen-specific receptors on T cells shape the repertoire of mature T lymphocytes emerging from the thymus. Some thymocytes with appropriate receptors are stimulated to undergo differentiation to the fully mature state (positive selection), whereas others with strongly autoreactive receptors are triggered to undergo programmed cell death before completing this differentiation process (negative selection). The quantitative impact of negative selection on the potentially available repertoire is currently unknown. To address this issue, we have constructed radiation bone marrow chimeras in which MHC molecules are present on radioresistant thymic epithelial cells (to allow positive selection) but absent from radiosensitive hematopoietic elements responsible for negative selection. In such chimeras, the number of mature thymocytes was increased by twofold as compared with appropriate control chimeras This increase in steady-state numbers of mature thymocytes was not related to proliferation, increased retention, or recirculation and was accompanied by a similar two- to threefold increase in the de novo rate of generation of mature cells. Taken together, our data indicate that half to two-thirds of the thymocytes able to undergo positive selection die before full maturation due to negative selection.

Comparison of thymocyte development in normal and invariant chain-deficient mice provides evidence that maturation-related changes in TCR and co-receptor levels play a critical role in cell fate.

Cells of invariant chain-deficient mice show a substantial decrease in cell surface MHC class II protein expression, as well as a change in the occupancy of the expressed class II molecules. Taking advantage of recent advances in phenotypic identification of transitional populations of developing thymocytes, the effects of these changes in MHC class II on positive and negative selection were reanalyzed. A marked (approximately 6-fold) reduction in CD4 single-positive mature cells was seen in H-2b mutant mice, yet there was little change in the number of CD4hiCD8intTCRint cells, a population containing the cells from which mature CD4+ cells derive. In normal mice expressing I-E and MMTV-encoded vSAG, V beta-specific negative selection occurred at a later point in the maturation pathway for cells showing greater expression of CD8 than CD4. In invariant chain-deficient mice, vSAG-mediated negative selection was diminished in general and what deletion still occurred was seen in more mature populations as compared to wild-type mice. Taken together, the decrease in MHC class II expression in invariant chain mutant mice and these alterations in the timing of thymocyte deletion provide strong support for an avidity model of negative selection. Perhaps more importantly, they emphasize the importance of the increasing TCR expression, the changing co-receptor levels and the movement from one antigen-presenting cell to another that accompany T cell maturation in determining the fate of developing thymocytes.

Evidence for lineage commitment and initiation of positive selection by thymocytes with intermediate surface phenotypes.

Previous studies have shown that both MHC class I and class II molecules can stimulate CD4+CD8+ thymocytes to develop into TCRint cells with surface phenotypes, suggesting initial differentiation along either the CD4 or CD8 developmental pathways. In accord with the view that these cells have undergone lineage commitment, we show here that CD4intTCRint cells generated by recognition of either MHC class show the same partial down-regulation of CD8 from the level on precursor double positive thymocytes as do almost mature CD4intTCRhigh cells positively selected only by MHC class I recognition. As expected of cells undergoing positive selection, we find that either class I or class II MHC molecules on radioresistant thymic stromal cells alone are sufficient for generation of both types of transitional phenotype cells. Biases in V beta representation associated with allele-dependent positive selection are seen only in the TCRhigh and not the TCRint cohorts, however. These findings are consistent with the hypothesis that transitional phenotype thymocytes have begun but not completed positive selection, and that entry into the CD4 vs CD8 pathways is not uniquely determined by the specific coreceptor-MHC molecule interaction involved in precursor thymocyte activation.

The different roles of MHC class recognition in thymocyte CD4 versus CD8 lineage commitment and positive selection.

Commitment to the CD4+ or CD8+ T cell lineages and positive selection for full maturation of precursor T cells take place in the thymus. A detailed phenotypic analysis of differentiating thymocytes in normal and MHC-deficient mice has led to the identification of previously unappreciated subpopulations whose characteristics and dependence on major histocompatibility complex (MHC) class I versus class II molecule expression seem incompatible with a CD4/CD8 coreceptor-dependent 'instructional' model of thymocyte development. We suggest here that these and other recent data are most consistent with a model in which the TCR-mediated decision to enter the CD4 versus the CD8 lineage is independent of the class of MHC molecule recognized and is distinct from positive selection. This latter event appears to involve already lineage-committed cells and to require a match of the MHC class specificity of the lineage-defining, highly expressed CD4 or CD8 coreceptor and the TCR.

Development of mature CD8+ thymocytes: selection rather than instruction?

The role of major histocompatibility complex (MHC) molecules in T cell differentiation was investigated by comparison of thymocyte subpopulations in wild-type mice and beta 2-microglobulin (beta 2M) mutant mice deficient in MHC class I expression and mature CD8+ cells. On the basis of surface markers, glucocorticoid resistance, in vitro differentiation capacity, and absence in beta 2 M-l- mice, CD4intermediateCD8hi cells with high expression of alpha beta T cell receptor (TCR alpha beta) were identified as having been positively selected by MHC class I for development into mature CD8+ T cells. Activated CD4intCD8hi cells bearing intermediate rather than high amounts of TCR were present in both wild-type and beta 2M-l- animals. These data suggest that recognition of MHC class I molecules is required for full maturation to CD8+ T cells, but not for receptor-initiated commitment to the CD8+ lineage, consistent with a stochastic (selection) model of thymocyte development.

Defective major histocompatibility complex class II assembly, transport, peptide acquisition, and CD4+ T cell selection in mice lacking invariant chain expression.

We used gene targeting techniques to produce mice lacking the invariant chain associated with major histocompatibility complex (MHC) class II molecules. Cells from these mice show a dramatic reduction in surface class II, resulting from both defective association of class II alpha and beta chains and markedly decreased post-Golgi transport. The few class II alpha/beta heterodimers reaching the cell surface behave as if empty or occupied by an easily displaced peptide, and display a distinct structure. Mutant spleen cells are defective in their ability to present intact protein antigens, but stimulate enhanced responses in the presence of peptides. These mutant mice have greatly reduced numbers of thymic and peripheral CD4+ T cells. Overall, this striking phenotype establishes that the invariant chain plays a critical role in regulating MHC class II expression and function in the intact animal.

Endogenous gene and amplifiable cDNA construct both produce unstable t-PA mRNA in Bowes melanoma cells.

Bowes melanoma cells, which naturally produce tissue-type plasminogen activator (t-PA), were transfected with a plasmid containing a human t-PA cDNA under transcriptional control of the promoter/enhancer of the major immediate early gene of human cytomegalovirus (CMV) plus genes expressing geneticin (G418) resistance and dihydrofolate reductase (DHFR). In one of the initial geneticin-resistant transformants, t-PA mRNA transcribed from the chromosomally integrated plasmid had the same short half-life, 20-30 min, as did mRNA transcribed from the endogenous t-PA gene compared to 7-8 h for total poly(A)+ mRNA. After subsequent selection of such cells with methotrexate, a cell line was obtained in which the t-PA cDNA construct was co-amplified with the DHFR gene and which produced 10 times more t-PA protein than the original Bowes melanoma cells.

Allelic exclusion of a T cell receptor-beta minilocus.

A TCR-beta minilocus in germline configuration (beta M) has previously been shown to undergo rearrangement and expression in transgenic mice. To study allelic exclusion of TCR miniloci, several beta M transgenic mouse lines were generated and crossed with mice transgenic for a functionally rearranged TCR V beta 2 gene (beta R). PCR analysis of beta M beta R double transgenic mice revealed almost complete suppression of endogenous TCR V beta gene rearrangements, but blockage of minilocus V beta rearrangements was achieved with only one of five minilocus transgenic lines. This result cannot be explained by copy number or arrangement of the multiple miniloci integrated. It appears that the minilocus is not autonomously regulated which suggests that sequences flanking the integration sites influence accessibility of the minilocus for rearrangement and allelic exclusion. However, although productively rearranged genes were formed in double transgenic mice, surface expression of minilocus-encoded beta chains was not detected. This indicates that allelic exclusion may operate at a level after gene rearrangement.

Allelic exclusion at DNA rearrangement level is required to prevent coexpression of two distinct T cell receptor beta genes.

In mice double transgenic for functionally rearranged T cell receptor (TCR) V beta 2 and V beta 8.2 genes we found that most T lymphocytes express both TCR beta chains simultaneously. These T cells show no abnormality in thymic selection in vivo and their TCRs are capable of transducing activation signals in vitro. These results indicate that multispecific T cells may appear in the periphery if allelic exclusion of TCR beta genes is not established at the level of gene rearrangement.

T-cell specific rearrangement of T-cell receptor beta transgenes in mice.

To study rearrangement of T cell receptor (TCR) genes, transgenic mice were generated with a TCR beta minilocus in germline configuration, containing three V beta, two D beta, fourteen J beta and two C beta gene segments and the TCR beta enhancer. Using the polymerase chain reaction as an analytical tool both partial DJ as well as complete VDJ rearrangements were seen, indicating that the minilocus contained all sequence elements required for rearrangment. Rearrangements of minilocus gene segments were restricted to T cells in the thymus and the periphery and did not occur in B cells. V beta 8.3 and V beta 5 sequences encoded by the minilocus were expressed on the surface of peripheral T cells at high frequencies. Transgenic mice with TCR minilocus genes will be a useful system to identify DNA sequence elements required for regulation of rearrangement in vivo.