Tissue-specific segregation of TCRgamma delta+ NKT cells according to phenotype TCR repertoire and activation status: parallels with TCR alphabeta+NKT cells.

Whereas the majority of NKT cells in the mouse express an alpha beta TCR (NKTalpha beta cells), a small subset of NKT cells express a gamma delta TCR (NKTgamma delta). Here we have systematically analyzed the phenotype, TCR repertoire and activation status of NKTgamma delta cells in the thymus, liver, spleen and bone marrow of normal C57BL/6 mice. Our data indicate that NKTgamma delta cells segregate in a tissue-specific manner according to these parameters. While most NKTgamma delta cells in the thymus and liver have a recently activated CD62L(lo) phenotype and a TCR repertoire that is heavily biased to Vgamma1.1 and Vdelta6.3, the majority of NKTgamma delta cells in the spleen and bone marrow are CD62L(hi) and have a much less biased TCR repertoire. Moreover, expression of NK markers is high on NKTgamma delta cells in spleen and bone marrow but low in thymus and liver. Collectively our results reveal a tissue-specific segregation of NKTgamma delta cells that is strikingly similar to that recently described for CD1d-dependent and Cd1d-independent NKTalpha beta cells. We therefore propose that chronic TCR activation by tissue-specific endogenous ligands is a generic property of NKT cells of both the alpha beta and gamma delta lineages.

Developmentally regulated extinction of Ly-49 receptor expression permits maturation and selection of NK1.1+ T cells.

Clonally distributed inhibitory receptors negatively regulate natural killer (NK) cell function via specific interactions with allelic forms of major histocompatibility complex (MHC) class I molecules. In the mouse, the Ly-49 family of inhibitory receptors is found not only on NK cells but also on a minor (NK1.1+) T cell subset. Using Ly-49 transgenic mice, we show here that the development of NK1.1+ T cells, in contrast to NK or conventional T cells, is impaired when their Ly-49 receptors engage self-MHC class I molecules. Impaired NK1.1+ T cell development in transgenic mice is associated with a failure to select the appropriate CD1-reactive T cell receptor repertoire. In normal mice, NK1.1+ T cell maturation is accompanied by extinction of Ly-49 receptor expression. Collectively, our data imply that developmentally regulated extinction of inhibitory MHC-specific receptors is required for normal NK1.1+ T cell maturation and selection.

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.

Quantitation of endogenous mouse mammary tumor virus superantigen expression by lymphocyte subsets.

Superantigens (SAg) encoded by endogenous mouse mammary tumor viruses (Mtv) interact with the V beta domain of the T cell receptor (TcR-V beta). Presentation of Mtv SAg can lead to stimulation and/or deletion of the reactive T cells, but little is known about the quantitative aspects of SAg presentation. Although monoclonal antibodies have been raised against Mtv SAg, they have not been useful in quantitating SAg protein, which is present in very low amounts in normal cells. Alternative attempts to quantitate Mtv SAg mRNA expression are complicated by the fact that Mtv transcription occurs from multiple loci and in different overlapping reading frames. In this report we describe a novel competitive polymerase chain reaction assay which allows the locus-specific quantitation of SAg expression at the mRNA level in lymphocyte subsets from mouse strains with multiple endogenous Mtv loci. In B cells as well as T cells (CD4+ or CD8+), Mtv-6 SAg is expressed at the highest levels, followed by Mtv-7 SAg and (to a much lesser extent) Mtv-8,9. Consistent with functional Mtv-7 SAg presentation studies, we find that Mtv-7 SAg expression is higher in B cells than in CD8+ T cells and very low in the CD4+ subset. The overall hierarchy in Mtv SAg expression (i.e. Mtv-6 > Mtv-7 > Mtv 8,9) was also observed for mRNA isolated from neonatal thymus. Furthermore, the kinetics of intrathymic deletion of the corresponding TcR-V beta domains during ontogeny correlated with the levels of Mtv SAg expression. Collectively our data suggest that T cell responses to Mtv SAg are largely controlled by SAg expression levels on presenting cells.

CD4/CD8 lineage commitment in T cell receptor transgenic mice: evidence for precommitment of CD4+ CD8+ thymocytes.

CD4+ and CD8+ mature T cells arise from CD4+ CD8+ thymic precursors by a process of positive selection that ultimately requires interaction of the T cell receptor (TCR) with self major histocompatibility complex (MHC) molecules. The mechanism of commitment of immature CD4+ CD8+ thymocytes to CD4 or CD8 lineages is controversial. Using TCR transgenic mice, we present evidence that CD4+ CD8+ thymocytes are precommitted to either the CD4 or CD8 lineage, prior to positive selection and independently of TCR specificity for MHC. This lineage precommitment model places important constraints on signaling via CD4 or CD8 coreceptor molecules.

Clonal anergy to staphylococcal enterotoxin B in vivo: selective effects on T cell subsets and lymphokines.

Injection of bacterial superantigens such as staphylococcal enterotoxin B (SEB) in adult mice results in initial proliferation of SEB-responsive V beta 8+ T cells followed by induction of a state of non-responsiveness frequently referred to as clonal anergy. We show here that SEB-induced anergy involves selective changes in lymphokine production and that it affects CD4+ V beta 8+ and CD8+ V beta 8+ T cells in different fashions. Whereas both CD4+ V beta 8+ and CD8+ V beta 8+ cells from anergic mice exhibit strongly reduced proliferative capacity and interleukin(IL)-2 production upon restimulation with SEB either in vivo or in vitro the CD8+ subset from SEB-injected mice produces other lymphokines (such as interferon(IFN)-gamma) at normal or slightly increased levels in response to SEB. Changes in the levels of production of IL-2 and IFN-gamma protein correlated well with mRNA accumulation both in vivo and in vitro. Collectively these data suggest that superantigen-induced anergy involves selective changes in signal transduction and/or gene regulation in T lymphocytes.

Precommitment of CD4+CD8+ thymocytes to either CD4 or CD8 lineages.

CD4+ and CD8+ mature T cells arise from CD4+CD8+ precursors in the thymus. During this process, cells expressing T-cell receptors (TCRs) reactive with self major histocompatibility complex (MHC) class I or II molecules are positively selected to the CD8 or CD4 lineage, respectively. It is controversial whether lineage commitment of CD4+CD8+ thymocytes is controlled directly by TCR specificity for MHC (instructional model) or, alternatively, by processes that operate independently of TCR specificity (stochastic model). We show here that CD4+CD8+ thymocytes bearing a MHC class I-restricted transgenic TCR can be subject to two alternative developmental fates. One population of CD4+CD8+ cells is positively selected by MHC class I molecules to the CD8 lineage as expected, whereas the other CD4+CD8+ population rearranges endogenous TCR genes and is positively selected by MHC class II molecules to the CD4 lineage. Blocking TCR-MHC class II interactions in vivo does not interfere with the generation of CD4+CD8+ cells expressing endogenous TCRs but does prevent their subsequent maturation to CD4+ cells. These data support a version of the stochastic model in which CD4+CD8+ thymocytes are precommitted to the CD4 or CD8 lineage independently of TCR specificity for MHC and prior to positive selection.

In vivo responses of CD4+ and CD8+ cells to bacterial superantigens.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex (MHC) class II molecules and specifically activates T cells bearing V beta 8 T cell receptor domains. We have compared several aspects of the response of CD4+ and CD8+ T cell subsets to SEB in vivo. V beta 8+ cells in both subsets proliferated to a similar extent upon SEB injection. Furthermore, mRNA for interferon-gamma was induced in both subsets with similar kinetics and SEB dose-response. Finally CD8+ (but not CD4+) T cells from SEB-injected mice exhibited SEB-specific lysis of MHC class II-bearing target cells. Collectively, these data indicate that the CD4: MHC class II interaction confers no detectable selective advantage to CD4+ cells in the in vivo response to SEB. The observed effector functions of both subsets may contribute to SEB-induced immunopathology.

Clonal expansion precedes anergy and death of V beta 8+ peripheral T cells responding to staphylococcal enterotoxin B in vivo.

Staphylococcal enterotoxin B (SEB) selectively stimulates T cells bearing T cell receptor V beta 8 domains and hence provides a useful model to study immunity and tolerance in vivo. We show here that V beta 8+ T cells in both CD4+ and CD8+ subsets expand dramatically (fivefold) in lymphoid tissues of mice 2-4 days following injection with SEB. This initial clonal expansion, which is accompanied by a transient hyper-reactivity to SEB, is followed by a rapid decrease in V beta 8+ cells and a concomitant induction of specific non-responsiveness which persists for at least 30 days. Selective death of V beta 8+ cells occurs during this latter phase. Taken together, our data indicate that clonal expansion, anergy and death can occur as sequential stages of an immune response in vivo.

Does negative selection involve accumulation of self-reactive thymocytes in thymic rosettes?

Thymic rosettes, the natural associations between thymocytes and either macrophages or dendritic cells, were isolated from the thymus by collagenase digestion and unit-gravity elutriation. Rosettes from mouse strains where either the V beta 6-bearing thymocytes are deleted because of reactivity with products of the Mlsa allele of the minor lymphocyte stimulating locus, or where V beta 17a-bearing thymocytes are deleted because of reactivity with IE class II MHC molecules, were compared with rosettes from appropriate control strains to test if a selective association with stromal cells preceded deletion. Rosettes from an Mlsa-bearing strain were able to stimulate an Mlsa-reactive T-hybridoma, but much of this stimulatory activity was attributable to the few B cells associated with the rosette preparations; the stromal components of the rosettes appeared to be poor presenters of Mlsa gene products. There was no enrichment of thymocytes bearing high or low levels of V beta 6 TcR in the rosettes from the Mlsa-bearing strain, which might have reflected the poor presentation by the stromal cells. However, nor was there detectable selective association of thymocytes bearing C beta 17a in the rosettes from an IE-positive mouse strain. This argues against binding and immobilisation on stromal cells as part of the deletion process, but not against the stromal cells delivering a rapid signal during a transient association, leading later to deletion.

Neonatal tolerance to MIs-1a determinants: deletion or anergy of V beta 6+ T lymphocytes depending upon MHC compatibility of neonatally injected cells.

Recent investigations in mice revealed that natural immunological tolerance to endogenous minor lymphocyte-stimulating locus 1a (MIs-1a) antigen correlates primarily with deletion of MIs-1a-specific V beta 6+ T lymphocytes in the thymus. Similar mechanisms account for acquired tolerance in some instances since the neonatal injection of MIs-1a-expressing MHC compatible cells in neonatal mice within the first 24 h of life causes clonal deletion of V beta 6+ T cells. Here we demonstrate that V beta 6+ T cells are not deleted in mice neonatally treated with MIs-1a spleen cells expressing allogeneic H-2 molecules. However, when such non-deleted V beta 6+ T cells were tested in vitro, no interleukin 2 (IL-2) secretion or proliferation was observed after MIs-1a stimulation. This non-responsive state could be overcome by addition of exogenous IL-2, consistent with the fact that V beta 6+ cells enlarged and expressed IL-2 receptors upon MIs-1a stimulation. Furthermore, the same neonatally treated mice showed in vitro functional unresponsiveness of cytotoxic T cells but not of IL-2-secreting cells specific for the tolerated allogeneic MHC antigens. Taken together, our data indicate that neonatal tolerance to MIs-1a can be accomplished by either clonal deletion or clonal anergy, and that it does not necessarily correlate with tolerance to MHC determinants.

CD8+ T cells respond clonally to Mls-1a-encoded determinants.

T cell responses to the product of the minor lymphocyte stimulatory locus Mls-1a involve the selective use of TCR V beta domains (especially V beta 6 and V beta 8.1) and are generally considered to be restricted to the CD4+ mature subset. We show here that CD8+ (presumably MHC class I-restricted) T cells bearing V beta 6 or V beta 8.1 also respond preferentially to Mls-1a determinants either in vitro (in mixed leukocyte cultures) or in vivo (in an adoptive transfer system). In vitro responses of both CD4+ V beta 6+ and CD8+ V beta 6+ cells to Mls-1a were dependent upon the MHC haplotype of the stimulator cells, with I-E+ (H-2d or H-2k) alleles being much more stimulatory than I-E- (H-2q). These data strengthen the analogy between Mls gene products and other MHC class II-dependent superantigens such as the bacterial enterotoxins.

Programmed death of autoreactive thymocytes.

T lymphocytes bearing high-affinity T-cell receptors (TCR) for self-antigens are clonally deleted during thymus development. Several recent studies have identified variable domains of the beta-chain of the TCR that are specifically deleted in vivo in mouse strains that express major histocompatibility complex class II molecules in addition to poorly defined self-antigens, including those encoded by the Mls-1a and Mls-2a loci. Deletion of autoreactive cells in these systems occurs in the thymus, and antibody blocking experiments in vivo have implicated the phenotypically immature CD4+CD8+ 'cortical' subset as the target population for clonal deletion. Similarly, studies with transgenic mice bearing autoreactive TCR have provided independent evidence that clonal deletion occurs at the CD4+CD8+ stage of development. But none of these studies directly identified dying autoreactive cells, and the circumstances leading to deletion remain unclear. Here we report that neonatal thymus contains a significant population of phenotypically mature CD4+CD8- cells bearing autoreactive TCR. When placed in short-term culture, a large proportion (60%) of these autoreactive cells die selectively. Furthermore, their death can be prevented by inhibitors of macromolecule (RNA and protein) synthesis, as is the case for glucocorticoid-induced death of thymocytes. These data indicate that physiological clonal deletion of autoreactive cells involves 'programmed' cell death, and that it can occur in cells with a mature (CD4+CD8-) surface phenotype.

Selective expression of V delta 6 genes by B2A2- CD4- CD8- T cell receptor gamma/delta thymocytes.

Most CD4-CD8- adult murine thymocytes characterized by absence of the B2A2 (J11d) antigen express T cell receptors (TcR) alpha/beta and utilize preferentially V beta 8.2 segments. To a much lesser extent, B2A2-CD4-CD8- thymocytes also express TcR gamma/delta, as evidenced by biochemical and Northern blot analysis. We have now been able to exclude the possibility that these cells might co-express both types of TcR: V beta 8+ B2A2- CD4-CD8- thymocytes expressed no TcR delta mRNA whereas the corresponding V beta 8- subset transcribed full-length TcR gamma as well as delta mRNA. Furthermore, the TcR gamma/delta expressing B2A2- thymocytes were found to use preferentially V delta 6 genes. Conversely, they did not express V delta 5 genes which are most frequently used by other TcR gamma/delta-bearing populations such as B2A2+ CD4-CD8- thymocytes or CD4-CD8- peripheral lymph node cells. RNase protection experiments showed that two particular V delta 6 transcripts predominate in these gamma/delta populations, the most prominent V delta 6 sequence being highly homologous if not identical to V alpha 7.1. Our observations extend previous information on overlapping V alpha and V delta gene repertoires, particularly in the cross-hybridizing V alpha 7/V delta 6 gene family. Moreover, our data suggest that B2A2-CD4-CD8- thymocytes represent a developmentally unique subset in which both V delta and V beta segments are nonrandomly expressed.

Positive and negative selection of T cell receptor V beta domains controlled by distinct cell populations in the thymus.

The role of thymic MHC class II-bearing cells in the selection of the TCR repertoire has been investigated in allogeneic radiation bone marrow chimeras. Positive selection of mature CD4+ T lymphocytes expressing the V beta 6+ TCR domain was found to depend upon radioresistant (presumably epithelial) I-E+ thymic cells. On the other hand, negative selection of CD4+ V beta 6+ cells (which was additionally dependent upon expression of the Mls-1a gene product) was controlled by a radiosensitive I-E+ thymic component (most likely dendritic cells). These data argue in favor of a compartmentalization of positive and negative selection events during T cell development.

Postnatal disappearance of self-reactive (V beta 6+) cells from the thymus of Mlsa mice. Implications for T cell development and autoimmunity.

The postnatal ontogeny of potentially autoreactive T cells has been studied in a model system where a particular TCR beta chain variable domain (V beta 6) is correlated with reactivity to a minor antigen encoded by the Mlsa locus. Although absent among mature (CD4+ or CD8+) T cells in adult mice expressing Mlsa, brightly staining V beta 6+ cells were readily detectable in the thymus of neonatal animals, reaching a maximum after 4 d and decreasing rapidly thereafter. These V beta 6+ thymocytes were predominantly of the CD4+ phenotype and were localized in the medulla of the developing thymus. Furthermore, the intensity of TCR expression by these CD4+ cells was significantly (twofold) reduced as compared with age-matched Mlsb controls. A rapid disappearance of CD4+V beta 6+ cells (and corresponding decrease in TCR density) could also be observed in the thymus of Mlsb mice that had been injected neonatally with Mlsa spleen cells. Taken together, these results raise the possibility that some autoreactive T cells may persist after birth and that TCR downregulation may occur as a physiological response to tolerogenic signals in vivo.

Positive selection of CD4+ thymocytes controlled by MHC class II gene products.

The mature T-cell antigen receptor repertoire is characterized by lack of reactivity to self-components as well as by preferential reactivity to foreign antigens in the context of polymorphic self-proteins encoded within the major histocompatibility complex. Whereas the former characteristic (referred to as negative selection or tolerance) is associated with intrathymic deletion of T cells expressing T-cell antigen receptor beta-chain variable (V beta) domains, which confer a preferential reactivity to self antigens, the existence of the latter (referred to as positive selection or MHC restriction) has so far only been inferred indirectly from functional studies. We show here that intrathymic deletion of V+beta 6 T cells (reactive with a self-antigen encoded by the Mlsa locus) is controlled by polymorphic MHC class II determinants. Furthermore, in mice lacking expression of Mlsa, the same class II MHC loci control the frequency of occurrence of V+beta 6 cells among mature CD4+ T lymphocytes. These data are direct evidence for positive selection by MHC determinants in the thymus in unmanipulated animals.