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E R Richie

Publications and source records attributed to E R Richie.

At least 19 recordsLinked to original sources

The CD4 T cell-deficient mouse mutation nackt (nkt) involves a deletion in the cathepsin L (CtsI) gene.

We recently reported a novel autosomal recessive mouse mutation designated nackt (nkt). Homozygous mutant mice have diffuse alopecia and a marked reduction in the proportion of CD4+ T cells in the thymus and peripheral lymphoid tissues. Here we show that the CD4 T-cell deficiency is due to a defect in the thymic microenvironment rather than the hematopoietic compartment. Furthermore, we identified the molecular basis of the mutant phenotype by demonstrating that the nkt mutation represents a 118-bp deletion of the cathepsin L (Ctsl) gene which is required for degradation of the invariant chain, a critical chaperone for major histocompatibility complex class II molecules. This finding explains the similarities in skin and immune defects observed in nkt/nkt and Ctsl -/- mice. The data reported here provide further in vivo evidence that the lysosomal cysteine protease cathepsin L plays a critical role in CD4+ T-cell selection in the thymus.

Animals↗

Transgenic expression of cyclin D1 in thymic epithelial precursors promotes epithelial and T cell development.

We previously reported that precursors within the keratin (K) 8+5+ thymic epithelial cell (TEC) subset generate the major cortical K8+5- TEC population in a process dependent on T lineage commitment. This report demonstrates that expression of a cyclin D1 transgene in K8+5+ TECs expands this subset and promotes TEC and thymocyte development. Cyclin D1 transgene expression is not sufficient to induce TEC differentiation in the absence of T lineage-committed thymocytes because TECs from both hCD3epsilon transgenic and hCD3epsilon/cyclin D1 double transgenic mice remain blocked at the K8+5+ maturation stage. However, enforced cyclin D1 expression does expand the developmental window during which K8+5+ cells can differentiate in response to normal hemopoietic precursors. Thus, enhancement of thymic function may be achieved by manipulating the growth and/or survival of TEC precursors within the K8+5+ subset.

Animals↗

Cyclin D2 overexpression in transgenic mice induces thymic and epidermal hyperplasia whereas cyclin D3 expression results only in epidermal hyperplasia.

In a previous report, we described the effects of cyclin D1 expression in epithelial tissues of transgenic mice. To study the involvement of D-type cyclins (D1, D2, and D3) in epithelial growth and differentiation and their putative role as oncogenes in skin, transgenic mice were developed which carry cyclin D2 or D3 genes driven by a keratin 5 promoter. As expected, both transgenic lines showed expression of these proteins in most of the squamous tissues analyzed. Epidermal proliferation increased in transgenic animals and basal cell hyperplasia was observed. All of the animals also had a minor thickening of the epidermis. The pattern of expression of keratin 1 and keratin 5 indicated that epidermal differentiation was not affected. Transgenic K5D2 mice developed mild thymic hyperplasia that reversed at 4 months of age. On the other hand, high expression of cyclin D3 in the thymus did not produce hyperplasia. This model provides in vivo evidence of the action of cyclin D2 and cyclin D3 as mediators of proliferation in squamous epithelial cells. A direct comparison among the three D-type cyclin transgenic mice suggests that cyclin D1 and cyclin D2 have similar roles in epithelial thymus cells. However, overexpression of each D-type cyclin produces a distinct phenotype in thymic epithelial cells.

Animals↗

Effects of calorie restriction on thymocyte growth, death and maturation.

We previously reported that calorie restriction (CR) significantly delays the spontaneous development of thymic lymphomas and other neoplasms in p53-deficient mice and their wild-type littermates. The purpose of the present study was to further characterize the anti-lymphoma effects of CR by assessing thymocyte growth, death and maturation in response to acute (6 day) and chronic (28 day) CR regimens. Male C57BL/6J mice fed a CR diet (restricted to 60% of control ad libitum intake) for 6 days displayed a severe reduction in thymic size and cellularity, as well as a decrease in splenic size and cellularity; these declines were sustained through 28 days of CR. Mice maintained on a CR diet for 28 days also displayed a significant depletion in the cell numbers of all four major thymocyte subsets defined by CD4 and CD8 expression. Analysis within the immature CD4(-)8(-) thymocyte subset further revealed an alteration in normal CD44 and CD25 subset distribution. In particular, CR for 28 days resulted in a significant decrease in the percentage of the proliferative CD44(-)25(-) subset. In addition, a significant increase in the percentage of the early, pro-T cell CD44(+)25(-) population was detected, indicative of a CR-induced delay in thymocyte maturation. Taken together, these findings suggest that CR suppresses (through several putative mechanisms) lymphomagenesis by reducing the pool of immature thymocytes that constitute the lymphoma-susceptible subpopulation.

Animals↗

TCR v(beta) repertoire restriction and lack of CDR3 conservation implicate TCR-superantigen interactions in promoting the clonal evolution of murine thymic lymphomas.

Thymic lymphoma development is a multistage process in which genetic and epigenetic events cooperate in the emergence of a malignant clone. The notion that signaling via TCR-ligand interactions plays a role in promoting the expansion of developing neoplastic clones is a matter of debate. To investigate this issue, we determined the TCR V(beta) repertoire of thymic lymphomas induced in AKR/J mice by either endogenous retroviruses or the carcinogen, N-methyl-N-nitrosourea (MNU). Both spontaneous and MNU-induced lymphomas displayed restricted V(beta) repertoires. However, whereas V(beta)6, V(beta)8 and V(beta)9 were expressed by a greater than expected frequency of MNU-induced lymphomas, V(beta)8, V(beta)7, V(beta)13 and V(beta)14 were over-represented on spontaneous lymphomas. The dissimilar TCR V(beta) profiles indicate that different endogenous ligands promote neoplastic clonal expansion in untreated and MNU-treated mice. Although the nature of these ligands is not clear, the lack of conservation in TCR beta chain CDR3 regions among lymphomas that express the same V(beta) segment suggests that endogenous superantigens (SAG), as opposed to conventional peptide ligands, are likely to be involved in the selection process. The biased representation of lymphomas expressing V(beta)6-, V(beta)7- and V(beta)9-containing TCRs that recognize endogenous SAG is consistent with this hypothesis. The finding that Bcl-2 is expressed at high levels in spontaneous and MNU-induced lymphomas suggests that preneoplastic thymocytes may be resistant to SAG-induced clonal deletion. A working model is presented in which preneoplastic clones expressing TCRs that recognize endogenous SAG are selectively expanded as a consequence of sustained TCR-mediated signaling.

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Interdependence of cortical thymic epithelial cell differentiation and T-lineage commitment.

Thymocyte and thymic epithelial cell (TEC) development are interdependent processes. Although lineage relationships among progressively maturing thymocyte subsets have been characterized, the developmental relationships among TEC subsets are obscure. Because epithelial cells express distinct keratin (K) species as a function of differentiation stage and proliferative status, we used K expression patterns to identify mouse TEC subsets and determine their lineage relationships. As expected, cortical and medullary TEC subsets express distinct K expression patterns in the normal thymus. However, we detected two distinct cortical TEC subsets, a major K8(+)K5(-) subset and a minor K8(+)K5(+) subset, which is highly represented at the cortico-medullary junction. Both cortical TEC subsets are also present in recombination activating gene 1 (RAG-1(-/-)) and TCRbetaxdelta-/- thymi in which T-cell development is blocked at the CD4(-)CD8(-)CD25(+)CD44(-) pre-T cell stage. In contrast, K8(+)K5(+) TECs predominate in the thymi of human CD3epsilon transgenic mice in which thymocyte development is blocked at an earlier CD4(-)CD8(-)CD25(-)CD44(+) stage. Transplantation of newborn human CD3epsilon transgenic thymi under the kidney capsule of RAG-1(-/-) mice results in the emergence of K8(+)K5(-) TECs concomitant with the appearance of CD25(+) thymocytes. Together, the data suggest that cortical TEC development proceeds from a K8(+)K5(+) precursor subset to a K8(+)K5(-) stage in a differentiation process concomitant with T-cell lineage commitment.

Animals↗

Cross-linking the TCR complex induces apoptosis in CD4+8+ thymocytes in the presence of cyclosporin A.

Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD4+8+ double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.

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Localization of a novel chromosome 7 locus that suppresses development of N-Methyl-N-nitrosourea-induced murine thymic lymphomas.

N-Methyl-N-nitrosourea (MNU) is a potent carcinogen that causes the development of murine thymic lymphomas. MNU-induced tumor incidence varies considerably among different inbred mouse strains. In particular, the AKR strain is highly susceptible, whereas the C57L strain is highly resistant to MNU-induced lymphoma formation. Crosses between AKR and C57L mice were established to investigate the genetic basis for the differential susceptibility of these inbred strains. A strong association between MNU-induced lymphoma development and coat color was observed in (AKR x C57)F2 and AKR x (AKR x C57)F1 progeny such that albino mice developed a higher tumor incidence than nonalbino animals. These data suggest that a locus on chromosome 7 influences tumor development. Analysis of four additional polymorphic loci (D7Rp2, Fes, Hbb, and Int-2) on chromosome 7 in AKR x (AKR x C57)F1 backcross mice revealed a significant linkage between high tumor incidence and homozygous inheritance of AKR alleles at the albino (tyrosinase) and Hbb loci. Thus, inheritance of at least one C57L allele at the albino or Hbb loci was associated with protection against MNU-induced lymphoma development. There was no association between tumor incidence and genotype at the D7Rp2, Fes, or Int-2 loci. Taken together, the data suggest that whereas C57L mice contain a dominant tumor suppressor gene on chromosome 7, in the AKR strain both alleles at this locus are defective resulting in enhanced susceptibility to MNU-induced lymphomagenesis.

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N-methyl-N-nitrosourea alters thymocyte subset distribution and targets immature CD4-8+ cells for lymphoma development.

The majority of N-methyl-N-nitrosourea (MNU)-induced lymphomas in AKR/J mice express a CD4-8+ phenotype. The CD4-8+ subset in normal thymus contains functionally mature medullary cells and immature cycling cells. This study demonstrates that MNU-induced lymphomas correspond to the immature CD4-8+ subset. In addition, specific changes in the distribution of thymocyte subsets defined by CD4 and CD8 expression were observed after MNU treatment. Cortical thinning and selective depletion of immature CD4-8+ and CD4+8+ subsets occur immediately after treatment. In contrast, immature CD4-8- progenitors and mature medullary CD4+8- and CD4-8+ subsets are relatively resistant to cytotoxicity. Normal thymic architecture and subset distribution are restored within 2 weeks after which selective expansion of the immature CD4-8+ subset occurs. The data suggest that MNU induces neoplastic conversion in progenitor cells corresponding to the CD4-8- or immature CD4-8+ stages of thymocyte maturation.

Animals↗

Influence of murine leukemia proviral integrations on development of N-methyl-N-nitrosourea-induced thymic lymphomas in AKR mice.

The AKR mouse strain is characterized by a high incidence of spontaneous thymic lymphoma that appears in older animals (greater than 6 months of age) and is associated with novel provirus integrations of ecotropic and recombinant murine leukemia viruses (MuLVs). Treatment of 4- to 6-week-old AKR/J mice with the carcinogen N-methyl-N-nitrosourea (MNU) results in thymic lymphomas that arise as early as 3 to 4 months of age and contain novel somatically acquired MuLV provirus integrations. The AKR/J strain develops MNU-induced lymphoma with a higher incidence and shorter latency than has been observed for other inbred mouse strains. To determine whether provirus integrations of endogenous MuLV account for the enhanced susceptibility of the AKR strain, the incidence and latency of MNU-induced lymphoma development was compared in AKR/J and AKR.Fv-1b mice. The restrictive b allele of the Fv-1 locus restricts integration and replication of endogenous N-tropic MuLV; therefore, AKR-Fv-1b mice have a very low incidence of spontaneous lymphoma. In contrast, AKR.Fv-1b mice develop MNU-induced lymphomas with an incidence and latency similar to those of the AKR/J strain. Furthermore, thymic lymphomas from both strains express an immature CD4-8+ phenotype, indicating neoplastic transformation of the same thymocyte subset. Southern blot analysis confirmed that lymphoma DNA from AKR.Fv-1b mice did not contain somatically acquired provirus integrations. These results demonstrate that provirus integration does not contribute to the predisposition of AKR mice to develop a high incidence of early MNU-induced lymphomas. Nevertheless, MNU treatment stimulated high-level expression of infectious ecotropic MuLV in AKR.Fv-1b as well as in AKR/J mice, suggesting that viral gene products might enhance lymphoma progression.

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Murine T-lymphomas corresponding to the immature CD4-8+ thymocyte subset.

N-methyl-N-nitrosourea induces murine CD4-8+ T-lymphomas that express high levels of J11d and low levels of CD5 antigens, a phenotype characteristic of immature CD4-8+ thymocytes. This assignment is supported by the fact that CD4-8+ lymphoma cell lines acquire CD4 expression after intrathymic (i.t.) transfer, a finding consistent with the established precursor potential of the normal immature CD4-8+ subset. CD4+8+ lymphomas recovered after i.t. transfer maintain a CD4+8+ phenotype in long-term culture. Northern blot analyses reveal that CD4 expression is regulated at the transcriptional level in immature CD4-8+ and CD4+8+ cell lines. CD4-8+ lymphomas express low levels of functional CD3/TCR complexes that mediate intracellular Ca2+ mobilization in response to CD3 or alpha/beta-TCR monoclonal antibody. These data suggest that the immature CD4-8+ subset contains cells capable of undergoing TCR-mediated signaling and selection events. In contrast to normal immature CD4-8+ cells, which comprise a heterogeneous and transient subset, the CD4-8+ lymphoma lines provide stable, monoclonal models of the immature CD4-8+ stage of thymocyte development.

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Nuclear events after activation of CD4+8+ thymocytes.

Functionally mature T cells respond to stimulation via the Ag receptor by secretion of IL-2 and/or other lymphokines and by proliferation. However, immature CD4+8+ thymocytes do not secrete IL-2 or proliferate in response to stimulation. We have analyzed murine thymocyte populations enriched for CD4+ and CD4+8+ cells as well as the functionally mature CD4+ lymphoma C6VL-B and the CD4+8+ lymphoma 1010 for their ability to express mRNA related to early products of T cell activation signals. When stimulated with the calcium ionophore (Ionomycin) plus PMA, all the cells, regardless of their phenotype, accumulated abundant levels of c-myc mRNA. However, in contrast to the CD4+ thymocytes and C6VL-B, which accumulated abundant levels of IL-2 transcripts, neither the normal CD4+8+ thymocytes nor 1010 expressed IL-2 mRNA before or after stimulation. We have also examined these cells for the presence of the murine equivalents of two nuclear DNA-binding factors, NFAT-1 and NFIL2-A, which have been shown to be involved in IL-2 gene expression in human T cells. Our results indicate: 1) NFIL-2A binding activity is constitutively expressed in both CD4+ and CD4+8+ thymocytes and lymphomas and 2) NFAT-1 binding activity is readily detected in CD4+ thymocytes and C6VL-B, but is detected in very minimal amounts in populations enriched for CD4+8+ thymocytes and in 1010 upon activation. These results suggest that the failure of CD4+8+ thymocytes to express IL-2 mRNA upon stimulation may be in part due to the lack of inducibility of NFAT-1 binding activity, and that functional maturation of this population might be associated with acquisition of the ability to induce NFAT-1 activity.

Animals↗

Genetics of N-methyl-N-nitrosourea induction of thymic lymphomas in AKR/J mice: assignment of a susceptibility gene to mouse chromosome 7.

Treatment of young AKR/J mice with N-methyl-N-nitrosourea (MNU) results in the induction of a high incidence of thymic lymphomas occurring between 4 and 6 months of age. The tumor incidence is higher and the latency period is shorter than that observed in other MNU-treated mouse strains. Analysis of tumor incidence in crosses of AKR/J with C57L/J mice indicates that several genes influence the incidence and latency of MNU-induced thymic lymphomas. One of these genes appears to be tightly linked to the albino locus of chromosome 7.

Albinism↗

Characterization of env gene recombination in x-ray--induced thymomas of C57BL/6 mice.

A role for specific recombination events at the env region of endogenous murine leukemia virus (MuLV) sequences in radiation carcinogenesis in C57BL mice has been suggested by a number of studies. We characterized env-related cell surface antigens from a primary, x-ray--induced, and several transplanted C57BL/6 thymomas of viral and radiation etiologies by immunoprecipitation and two-dimensional peptide mapping. DNA from lymphoma cells was also analyzed by Southern blotting for evidence of mink cell focus-forming (MCF) type env recombination events. Although gp70 molecules with novel structural determinants were found on all transplanted lymphomas examined, expression of novel env antigens was variable among these lymphomas, and there was a lack of correlation of characteristic MCF-type env recombination events in endogenous retrovirus DNA sequences with novel env antigens on lymphoma cell surfaces. Neither novel gp70 antigens nor MCF-type env provirus recombinant structures were consistent features of the C57BL/6 thymomas of radiation etiology examined in this study, even though MCF-type env recombination events have been suggested as etiologically significant in MuLV-mediated lymphomagenesis in both RadLV and x-ray--induced tumors in C57BL/6 mice.

Animals↗

Altered expression of lymphocyte differentiation antigens on phorbol ester-activated CD4+8+ T cells.

Altered expression of cell surface Ag is an early event accompanying Ag-, mitogen-, or phorbol ester-induced activation of mature T cells. In this report, phorbol ester-induced changes in the expression of several functionally significant cell surface molecules are explored on immature thymocytes and lymphoma cells presenting a cortical CD4+8+ double-positive (DP) phenotype. Both CD4 and CD8 expressions are down-modulated on DP cells incubated with PMA. Cell-surface expression of CD4 and CD8 remains depressed for 72 h in the presence of PMA, but is restored after removal of PMA from the culture medium. The PMA-mediated loss of Ag expression is associated with a rapid down-regulation of steady state CD4 and CD8 mRNA transcript levels in treated cells. The sustained loss of CD4 and CD8 surface expression on DP cells is a selective event because CD5 expression is enhanced, H-2 expression is unchanged, and CD3 expression is only transiently diminished by PMA stimulation. Other T cell-activating agents, including Con A, ionomycin, and anti-CD3 mAb, induce selected surface antigenic changes on DP cells, but do not mimic the pattern of altered Ag expression observed after PMA stimulation. These data demonstrate that, similar to mature subsets, T cells in the DP nonmature compartment undergo alterations in expression of functionally important cell-surface molecules in response to activating agents. Nevertheless, distinctions between mature and nonmature T cells regarding specific alterations in differentiation Ag phenotype suggest that the effect of PMA on expression of these molecules depends, in part, on the maturation stage of the target cell population.

Adjuvants, Immunologic↗