Tumor induction by an Lck-MyrAkt transgene is delayed by mechanisms controlling the size of the thymus.

Transgenic mice expressing MyrAkt from a proximal Lck promoter construct develop thymomas at an early age, whereas transgenic mice expressing constitutively active Lck-AktE40K develop primarily tumors of the peripheral lymphoid organs later in life. The thymus of 6- to 8-week-old MyrAkt transgenic mice is normal in size but contains fewer, larger cells than the thymus of nontransgenic control and AktE40K transgenic mice. Earlier studies had shown that cell size and cell cycle are coordinately regulated. On the basis of this finding, and our observations that the oncogenic potential of Akt correlates with its effect on cell size, we hypothesized that mechanisms aimed at maintaining the size of the thymus dissociate cell size and cell cycle regulation by blocking MyrAkt-promoted G(1) progression and that failure of these mechanisms may promote cell proliferation resulting in an enlarged neoplastic thymus. To address this hypothesis, we examined the cell cycle distribution of freshly isolated and cultured thymocytes from transgenic and nontransgenic control mice. The results showed that although neither transgene alters cell cycle distribution in situ, the MyrAkt transgene promotes G(1) progression in culture. Freshly isolated MyrAkt thymocytes express high levels of cyclins D2 and E and cdk4 but lower than normal levels of cyclin D3 and cdk2. Cultured thymocytes from MyrAkt transgenic mice, on the other hand, express high levels of cyclin D3, suggesting that the hypothesized organ size control mechanisms may down-regulate the expression of this molecule. Primary tumor cells, similar to MyrAkt thymocytes in culture, express high levels of cyclin D3. These findings support the hypothesis that tumor induction is caused by the failure of organ size control mechanisms to down-regulate cyclin D3 and to block MyrAkt-promoted G(1) progression.

CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection.

Thymocytes from mice lacking the CD3delta chain of the T-cell receptor (TCR), unlike those of other CD3-deficient mice, progress from a CD4- CD8- double-negative to a CD4+ CD8+ double-positive stage. However, CD3delta-/- double-positive cells fail to undergo positive selection, by which double-positive cells differentiate into more mature thymocytes. Positive selection is also impaired in mice expressing inactive components of the Ras/mitogen activated protein (MAP) kinase signalling pathway. Here we show that CD3delta-/- thymocytes are defective in the induction of extracellular signal-regulated protein kinase (ERK) MAP kinases upon TCR engagement, whereas activation of other MAP kinases is unaffected. The requirement for CD3delta maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3delta rescues both ERK activation and positive selection in CD3delta-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3zeta chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3delta-/- thymocytes may derive from defective tyrosine phosphorylation of CD3zeta in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.

Protection of CD3 delta knockout mice from lymphocytic choriomeningitis virus-induced immunopathology: implications for viral neuroinvasion.

For a virus to establish a neuronal infection, it must spread from its primary site of infection to the central nervous system (CNS) before immune-mediated clearance occurs. Lymphocytic choriomeningitis virus (LCMV) is a murine pathogen that can result in persistent neuronal infection in newborn mice and in adults that lack CD8(+) T cells. To determine the neuroinvasive capacity of LCMV in the presence of an existent, but compromised, cytotoxic T lymphocyte response, the course of LCMV infection was examined in mice that possess 10% of the normal complement of T lymphocytes, due to the lack of the CD3 delta (delta) subunit of the T cell receptor complex (CD3 delta KO mice). Unlike immunocompetent mice that produced a massive immune response that caused death by 6-7 days postinfection, CD3 delta KO mice mounted a weak response and survived. The presence of viral antigen gradually shifted from the class I MHC-positive meninges and ependyma to class I MHC-deficient CNS neurons 10-30 days postinoculation. The infected CD3 delta KO mice developed a delayed T cell response that suppressed virus replication in peripheral tissues but not in the CNS; subsequent adoptive transfer experiments supported the hypothesis that the lack of clearance from neurons was due to sequestration of LCMV in an immune-privileged cell type. Based on these results, we propose that a critical parameter in the pathogenesis of neurotropic viruses is the rate of immune activation; individuals with impaired T cell responses may be more vulnerable to persisting CNS infections.

Regulation of lineage commitment distinct from positive selection.

Developing alphabeta T cells diverge into the CD4 and CD8 lineages as they mature in the thymus. It is unclear whether lineage commitment is mechanistically distinct from the process that selects for the survival of T cells with useful T cell receptor (TCR) specificities (positive selection). In HD mice, which lack mature CD4+ T cells, major histocompatibility complex (MHC) class II-restricted T cells are redirected to the CD8 lineage independent of MHC class I expression. However, neither TCR-mediated signaling nor positive selection is impaired. Thus, the HD mutation provides genetic evidence that lineage commitment may be mechanistically distinct from positive selection.

Peripheral CD4+ T cell maturation recognized by increased expression of Thy-1/CD90 bearing the 6C10 carbohydrate epitope.

The SM6C10 IgM autoantibody recognizes a surface determinant, 6C10, that is highly expressed on all immature thymocytes. In contrast, its expression on peripheral T cells appears developmentally regulated, i.e., absent from most naive T cells in spleen of neonatal mice, but expressed on 40-80% of naive CD4+ T cells in adult. In this paper, we demonstrate that SM6C10 recognizes a carbohydrate epitope on the Thy-1 glycoprotein using immunoprecipitation analysis, by binding to affinity-purified Thy-1 in an ELISA, and by sensitivity to N-glycosidase-F treatment. Retroviral Thy-1 gene transduction experiments into Thy-1- variant T cell lines and a pro-B cell line provide evidence that 6C10 glycosylated Thy-1 expression is not restricted to T cells but depends on the recipient cell. Therefore, differences in 6C10 levels among Thy-1+ T cells in mice likely reflect developmental regulation of posttranslational modification of the Thy-1 glycoprotein. The ability of naive CD4+ T cells to respond to anti-Thy-1 stimulation increases from neonate to adult, and 6C10- naive cells from adult mice respond poorly compared with 6C10+ cells, similar to the cells in neonatal mice. These results suggest that there is functional maturation by peripheral CD4+ T cells that coincides with 6C10 glycosylated Thy-1 up-regulation, and natural autoantibody recognizes this 6C10 carbohydrate epitope.

Limiting TCR expression leads to quantitative but not qualitative changes in thymic selection.

Thymic selection is controlled in part by the avidity of the interaction between thymocytes and APCs. In agreement, the selective outcome can be modulated by altering the expression levels of selecting ligands on APCs. Here we test the converse proposition, i. e., whether changing TCR levels on thymocytes can alter the selective outcome. To this end, we have generated mice in which all thymocytes express two transgenic TCRs simultaneously (dual TCR-expressing (DTE) mice), the class I-restricted HY TCR and the class II-restricted AND TCR. Due to mutual dilution, surface expression levels of the two individual transgenic TCRs are diminished in DTE relative to single TCR-expressing mice. We find that thymic selection is highly sensitive to these reductions in TCR surface expression. Positive selection mediated by the AND and HY TCRs is severely impaired or abolished, respectively. Negative selection of the HY TCR in male DTE mice is also partly blocked, leading to the appearance of significant numbers of double positive thymocytes. Also, in the periphery of male, but not female, DTE mice, substantial numbers of single positive CD8bright cells accumulate, which are positively selected in the thymus but by a highly inefficient hemopoietic cell-dependent process. Overall our results favor the interpretation that the outcome of thymic selection is not determined solely by avidity and the resulting signal intensity, but is also constrained by other factors such as the nature of the ligand and/or its presentation by different subsets of APCs.

Altered functional responsiveness of thymocyte subsets from CD3delta-deficient mice to TCR-CD3 engagement.

CD3delta-deficient (delta degrees) mice are defective in alphabeta T cell development. Here we explore the capacity of TCR-CD3 signaling complexes expressed on delta degrees thymocytes to mediate the following functional outcomes in response to antibody cross-linking: (i) the transition from the CD4-CD8- to CD4+CD8+ stage, (ii) the transition from the CD4+CD8+ to CD4+CD8- or CD4-CD8+ stages and (iii) the induction of apoptosis. We provide evidence that CD3deltaepsilon complexes are dispensable for mediating the anti-CD3-mediated CD4-CD8- to CD4+CD8+ transition. On the other hand, CD3delta is critical at the CD4+CD8+ stage. We demonstrate that CD4+CD8+ thymocytes from delta degrees mice, unlike delta degrees CD4-CD8- thymocytes and wild-type CD4+CD8+ thymocytes, require prolonged or consecutive stimuli to elicit functional responses. Depending on the nature of the secondary stimulus, delta degrees thymocytes can be induced to undergo apoptosis or preferential maturation to the CD4-CD8+ stage. Taken together these results indicate that the signaling capacity of the TCR-CD3 complex is noticeably altered in the absence of CD3delta. The essential role of CD3delta at the CD4+CD8+ stage of development correlates with the onset of TCRalpha rearrangement, consistent with a critical structural and/or functional relationship between CD3delta and TCRalpha.

HD mice: a novel mouse mutant with a specific defect in the generation of CD4(+) T cells.

We have identified a spontaneous mutation in mice, which we term HD for "helper T cell deficient." This mouse is distinguished by the virtual absence of peripheral T cells of the CD4(+)8(-) major histocompatibility complex (MHC) class II-restricted T helper subset due to a specific block in thymic development. The developmental defect is selective for CD4(+)8(-) cells; the maturation of CD4(-)8(+) and gamma delta T cells is normal. The autosomal recessive mutation underlying the HD phenotype is unrelated to MHC class II, since it segregates independently of the MHC class II locus. Moreover, the HD phenotype is not caused by a defect of the CD4 gene. Bone marrow transfer experiments demonstrate that the defect is intrinsic to cells of the hematopoietic lineage, i.e., most likely to developing thymocytes themselves. The frequency of CD4(+)8(low) intermediate cells is markedly increased in HD mice, suggesting that class II-restricted thymocytes are arrested at this stage. This is the first genetic defect of its kind to be described in the mouse and may prove highly informative in understanding the molecular pathways underlying lineage commitment.

Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 delta is physically associated but not functionally required.

Maturation of immature CD4-CD8- (DN) thymocytes to the CD4+CD8+ (DP) stage of development is driven by signals transduced through a pre-T cell receptor (TCR) complex, whose hallmark is a novel subunit termed pre-T alpha (pT alpha). However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear. Moreover, progress in understanding pre-TCR function has been hampered thus far because previous attempts to demonstrate expression of pT alpha-containing pre-TCRs on the surface of normal thymocytes have been unsuccessful. In this report, we demonstrate for the first time that pT alpha-containing pre-TCR complexes are expressed at low levels on the surface of primary thymocytes and that these pre-TCR complexes comprise a disulfide-linked pT alpha-TCR-beta heterodimer associated not only with CD3-gamma and -epsilon, as previously reported, but also with zeta and delta. Interestingly, while CD3-delta is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-delta-deficient mice. The fact that any of the signaling components of the pre-TCR are dispensable for pre-TCR function is indeed surprising, given that few pre-TCR complexes are actually expressed on the surface of primary thymocytes in vivo. Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (gamma, delta, epsilon, and zeta), possibly because pT alpha itself possesses signaling capabilities.

CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage.

The CD3 complex found associated with the T cell receptor (TCR) is essential for signal transduction following TCR engagement. During T cell development, TCR-mediated signalling promotes the transition from one developmental stage to the next and controls whether a thymocyte undergoes positive or negative selection. The roles of particular CD3 components in these events remain unclear. Indeed, it is unknown whether they have specialized or overlapping roles. However, the multiplicity of CD3 components and their evolutionary conservation suggest that they serve distinct functions. Here the developmental requirement for the CD3 delta chain is analyzed by generating a mouse line specifically lacking this component (delta-/- mice). Strikingly, CD3 delta is shown to be differentially required during development. In particular, CD3 delta is not needed for steps in development mediated by pre-TCR or gamma delta TCR, but is required for further development of thymocytes expressing alpha beta TCR. Absence of CD3 delta specifically blocks the thymic selection processes that mediate the transition from the double-positive to single-positive stages of development.

T lymphocyte receptor deficiencies.

Signalling through the TCR is mediated by the cytoplasmic tails of the CD3 complex. Deficiencies in the expression of different CD3 components have lead to dramatic, yet dissimilar, effects on T-cell development and to selective deficits in peripheral T-cell subsets. Recent studies of human patients and animal models with CD3 deficiencies are providing insights into the redundant and unique roles of these molecules.

L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs.

cDNAs coding for the HLA class II DR and DQ alpha and beta chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded alpha and beta chains of the DR3 or DR4 haplotypes, as well as the trans-encoded alpha and beta chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR alpha/DQ beta or DQ alpha/DR beta) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most "pan-DQ" mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain alpha beta heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.

Surface expression of alternative forms of the TCR/CD3 complex.

T-cell antigen receptor (TCR) heterodimers of both the alpha beta and gamma delta types are expressed at the surface of T cells only in association with a complex of invariant chains called CD3. The requirement for individual CD3 components to achieve TCR surface expression was examined by cotransfection of a non-T-cell line with TCR alpha and beta, as well as CD3 delta, epsilon, gamma, and zeta, cDNAs. Both transient and stable transfectants expressing TCR and CD3 epitopes at the cell surface were generated. By transfection of TCR and CD3 components in different combinations, the TCR chains, as well as the CD3 epsilon and zeta chains, were each shown to be essential for reconstituting surface expression. On the other hand, CD3 delta and gamma chains could be used alternatively, providing evidence for two different types of TCR/CD3 complexes.

A novel method for generating stable high-level transfectants involving direct immunomagnetic selection for cell-surface epitopes: expression of HLA class-II genes in HeLa cells.

A versatile method that allows efficient detection and selection of both transient and stable transfectants expressing exogenous cell-surface molecules is described and used to generate stable HeLa transfectants expressing each of the human HLA class-II isotypes, specifically the DR1, DQw8 and DPw2 heterodimers. The method combines use of the strong mammalian expression vector, CDM8, and a highly efficient transfection protocol with the powerful technique of immunomagnetic selection. It offers significant advantages in comparison to standard procedures involving co-selection with drug-resistance markers. The transfection efficiency can be assessed 60 h after transfection rather than after three weeks of drug selection. Repeated rounds of immunomagnetic selection applied over the subsequent ten days result in homogeneous populations which express the surface marker of interest stably at high levels, making further subcloning or fluorescence-activated cell sorting unnecessary. Any number of surface products can be transfected into the same cell, the only limitation being the availability of specific monoclonal antibodies (a DP/DR double transfectant is described expressing four exogenous gene products simultaneously). The high sensitivity of immunomagnetic selection and its applicability to large samples allows rescue of transfectants present at very low frequencies. Finally, the technique can be used as a coselection procedure, by analogy with drug coselection, to achieve expression even of non-cell surface products.

Identification of a T-cell-specific enhancer at the locus encoding T-cell antigen receptor gamma chain.

The gamma delta and alpha beta T-cell antigen receptor (TCR) heterodimers are expressed in a lineage-specific, mutually exclusive manner. Regulation of expression occurs at the transcriptional level. A 13-kilobase (kb) stretch of DNA encompassing variable-joining-constant segments V gamma 4-J gamma 1-C gamma 1 of the murine gamma-chain gene was examined for the presence of transcriptional enhancing elements by a transient transfection assay. DNA fragments from this region were inserted into a test plasmid containing a heterologous promoter fused to the human growth hormone gene. An 1800-base-pair (bp) fragment located 3 kb 3' to C gamma exon III was found to display enhancing activity in several T-cell lines. Maximum enhancing activity could be localized further to fragments as small as 400 bp in some cell lines. Nucleotide sequence analysis of this 400-bp segment revealed homologies to previously described core enhancer elements and to other TCR gene enhancers. The TCR gamma-chain gene enhancer is active in both gamma delta and alpha beta T cells, indicating that it is not primarily responsible for lineage-specificity of expression, but it is inactive in non-T-cells.

Structural features of the cytoplasmic region of CD4 required for internalization.

CD4, the T cell surface antigen, is phosphorylated and internalized when T cells are activated or treated with a phorbol ester, PMA. The actual phosphorylation sites have been identified and the role of phosphorylation of each on CD4 internalization investigated. Seven different mutants, in each of which one, two or all three of the serine residues of the cytoplasmic region was modified to alanine(s) (CD4.SA mutants) and one mutant in which the whole amino acid sequence from Gln421 to the C-terminal Ile433 was changed (CD4.EP mutant) were constructed and used to determine the effect of phosphorylation on CD4 internalization. Ser408 was the most efficiently phosphorylated by PMA treatment, Ser415 next and Ser431 to a minor extent. The effect of mutation on internalization was well matched with the effect on extent of phosphorylation, i.e. Ser408 was the residue most important for internalization. However, complete inhibition of CD4 internalization was achieved only by mutating all three serine residues. Interestingly, the mutant CD4.EP in which Ser408 was present and phosphorylated was not measurably internalized, suggesting that phosphorylation of Ser408 induces CD4 internalization only when other structural features of the cytoplasmic domain remain intact. In addition, the data suggest the existence of an additional minor pathway for CD4 internalization which is phosphorylation independent.

Recognition of a self major histocompatibility complex TL region product by gamma delta T-cell receptors.

Ligand specificity of a murine gammadelta T-cell receptor-expressing hybridoma (KN6) derived from adult thymocytes has been analyzed in detail. The molecule recognized by the KN6 gammadelta T-cell receptor is expressed on syngeneic cells of various sources (peritoneal macrophages, thymocytes, spleen cells, and Abelson murine leukemia virus-transformed cell lines) and on transformed cells arrested at an early stage of development (e.g., PCC3 embryonal carcinoma cells). Linkage of the gene coding for the KN6 ligand to the major histocompatibility complex genes could be demonstrated by testing KN6 hybridoma reactivity to cells from congenic strains that differ only at H-2. In addition, analysis of recombinant strains indicates that the gene controlling the KN6 ligand is located in or distal to the TL region. Involvement of the KN6 gammadelta T-cell receptor in this recognition process could be directly demonstrated by transferring the KN6 TL specificity after introduction of the productively rearranged KN6 gamma and delta genes into an alphabeta T-cell clone or into the germ line in transgenic mice. These observations raise the possibility that at least some gammadelta cells regulate hemopoietic cell maturation and activation.

Structure and evolution of the HLA class II SX beta gene.

The class II major histocompatibility complex antigens are cell-surface heterodimers consisting of an alpha and a beta chain. Cosmid cloning has shown that the three families of class II antigens, DR, DQ, and DP, are encoded within the HLA-D region of chromosome 6 as a series of discrete gene clusters. The DP cluster contains two pairs of alpha and beta genes, one of which encodes the biochemically-defined DP antigen. In order to assess whether the other two genes, SX alpha and SX beta, are also expressed, potential coding regions have been subcloned and sequenced. The SX3 beta gene is shown to contain regions closely homologous to all six exons of DP beta. A 1 bp deletion in the beta 2 exon, also observed for the SX4 beta allele, causes a translational frameshift, suggesting that SX beta is a pseudogene. However, all the other exons, as well as their splice sites and the putative promoter region, appear to be intact.