Differentiation of hematopoietic stem cells in irradiated mouse thymic lobes. Kinetics and phenotype of progeny

To define cell populations which participate in the very early stages of T cell development in the mouse thymus, we enriched hematopoietic stem cells from mouse bone marrow and injected them into thymic lobes of irradiated Ly-5 congenic recipients. The progeny of the stem cells were identified and their phenotypes were determined by two-color flow cytometry for the expression of various cell surface differentiation Ag during the course of their subsequent intrathymic development. The majority of the differentiation which occurred in the first 10 days after intrathymic cell transfer was myeloid in nature; hence, this study demonstrates that the irradiated thymus is not strictly selective for T cell development. Further, the maximum rate of T cell development was observed after intrathymic injection of 200 stem cells. Donor-derived cells which did not express Ag characteristic of the myeloid lineage could be detected and their phenotypes could be determined by flow cytometry as early as 7 days after intrathymic injection. At this time, the cells were still very similar phenotypically to the bone marrow hematopoietic stem cells. Exceptions to this were the expression of stem cell Ag 2 and a decrease in the level of MHC class I Ag expression. After 9 days, the donor-derived cells expressed high levels of the Thy-1 Ag and proceeded to change in cell surface phenotype as differentiation continued. These cell phenotypes are described for the time frame ending 18 days after injection, when most donor-derived cells were phenotypically small CD4+ CD8+ (double-positive) thymocytes.     

Incomplete differentiation of antigen-specific CD8 T cells in tumor-draining lymph nodes

CD8 T cells lacking effector activity have been recovered from lymphoid organs of mice and patients with progressing tumors. We explored the basis for lack of effector activity in tumor-bearing mice by evaluating Ag presentation and CD8 T cell function in lymphoid organs over the course of tumor outgrowth. Early after tumor injection, cross-presentation by bone marrow-derived APC was necessary for T cell activation, inducing proliferation and differentiation into IFN-gamma-producing, cytolytic effectors. At later stages of outgrowth, tumor metastasized to draining lymph nodes. Both cross- and direct presentation occurred, but T cell differentiation induced by either modality was incomplete (proliferation without cytokine production). T cells within tumor-infiltrated nodes differentiated appropriately if Ag was presented by activated, exogenous dendritic cells. Thus, activated T cells lacking effector function develop through incomplete differentiation in the lymph nodes of late-stage tumor-bearing mice, rather than through suppression of previously differentiated cells.     

Normal tissue depresses while tumor tissue enhances human T cell responses in vivo to a novel self/tumor melanoma antigen,OA1

Antitumor T cells often recognize targets that are nonmutated "self" tissue differentiation Ags, but the relative impact of Ag expression by normal and transformed tissue for a human self/tumor Ag has not been studied. To examine the influence of self-tolerance mechanisms on the function of self/tumor-specific T cell responses in humans, we sought to identify an Ag that was expressed, processed, and presented in an MHC-restricted fashion by tumor cells, but for which there was the human equivalent of a "knockout." In this study, we report the first immunological characterization of a melanoma/melanocyte differentiation Ag, called OA1, which meets these criteria. This Ag, an X chromosome-encoded melanoma/melanocyte differentiation Ag, was completely deleted in a male patient. Using a newly identified HLA-A*2402-restricted epitope (LYSACFWWL) to study T cell tolerance, we found that OA1-specific T cell reactivity was more than five SD higher in the knockout patient that in normal controls. These data provide compelling evidence for T cell tolerance to OA1 in humans. Most surprisingly, we found elevated levels of OA1-specific T cells in patients with metastatic malignant melanoma, indicating that the tumor-bearing state partially reversed tolerance observed in normal (non-"knockout") individuals. Taken together, these findings indicated that tolerance can exist for self/tumor Ags in humans, and that this tolerance could be partially abrogated by the growth of the tumor, increasing the reactivity of tumor Ag-specific T cells. Thus, the tumor-bearing state reverses, in part, the tolerance of T cells that results from the normal expression of tissue differentiation Ags.     

An immunodominant murine lymphoma cell surface heterodimer marks thymic progenitor subsets

mAb 1C11 was raised against the cells of retrovirus-negative, radiation-induced thymomas of C57BL/Ka mice. MAb 1C11 binds to radiation- and RadLV-induced C57BL/Ka lymphomas, to lymphomas of other mouse strains and to B-lineage tumors. The 1C11 Ag is expressed on a subpopulation of normal thymocytes that is enriched in immature cells. After fractionated x-irradiation, this percentage increases gradually during the preleukemic period, hence mAb 1C11 appears to identify a transformation-related cell surface molecule. This conclusion is supported by experiments demonstrating that flow microfluorimetry-sorted, 1C11-expressing preleukemic thymocytes progress rapidly to full neoplasia following intrathymic injection, whereas nonexpressing cells do not. Most of day-14 fetal thymocytes are as strongly positive as thymic lymphomas for the 1C11 Ag whereas Ag-activated T cell lines express moderate levels. Multiparameter flow microfluorimetry analysis shows that 1C11 is expressed predominantly on CD3-/lo thymic blast cells of three phenotypically defined subsets: CD4-8-, CD4-8+, and CD4+8+, all of which contain thymic progenitors. By immunohistochemical staining, the Ag is also found in association with epithelial cells on a variety of normal, nonlymphoid tissue, but is not detectable on heart tissue. The 1C11 antibody immunoprecipitates a disulfide-linked heterodimeric protein of 85/37 kDa and the antigenic determinant is located on the H chain of the molecule. When analyzed by SDS-PAGE under nonreducing conditions, the molecule exists as a 130-kDa protein. Enzymatic digestion of the heterodimer indicates that the H chain, but not the L chain, has at least three N-linked glycosylation sites. We propose that this novel cell surface glycoprotein may be associated with processes of differentiation and lymphomagenesis.     

Constitutive versus cell cycle regulation of c-myb mRNA expression correlates with developmental stages in murine B lymphoid tumors.

The expression of c-myb mRNA is differentially regulated in murine B lymphoid tumors such that B cell lymphomas and plasmacytomas contain significantly less c-myb mRNA than pre-B cell lymphomas. To examine the low level of c-myb mRNA expression in the murine B cell lymphoma cell line BCL1, nonessential amino acid starvation was used to block these cells in a G1 state. When BCL1 cells were released from this block, a 7- to 10-fold increase in c-myb mRNA was detected in late G1 and S phase cells relative to that detected in exponentially growing BCL1 cells. This increase was not inhibited by aphidicolin. To determine whether cell cycle regulation of c-myb mRNA expression occurred during exponential growth in both murine pre-B cell lymphoma and B cell lymphoma cell lines, elutriation was used to separate exponentially growing cell populations. An increase in c-myb mRNA expression was seen in late G1 and S phase fractions from B cell lymphoma cell lines. In contrast, c-myb mRNA levels remained constant in elutriation fractions isolated from pre-B cell lymphoma cell lines. Expression of c-myb mRNA was not detected in exponentially growing or in Go serum-stimulated murine fibroblasts. These results indicate that constitutive vs cell cycle regulation of c-myb mRNA expression is related to the state of differentiation in murine B lymphoid tumors and suggest that a switch in regulation may occur during normal B cell development.     

Modulation of mouse mammary tumor virus production in the MJY-alpha cell line.

Implantation of the mouse mammary tumor virus (MMTV)-producing mammary tumor cell line MJY-alpha into isogeneic mice elicited both humoral and T-cell responses against MMTV virion antigens. The carcinosarcomas which developed from the implanted cells showed a significant decrease in MMTV synthesis, compared with cells remaining in culture, which was detectable as early as 7 days after implantation and for five transplant generations. Electron microscopic examination of thin sections of the tumors revealed that intracytoplasmic A particles, budding particles, and cell-free MMTV B particles were all affected. However, immunofluorescence assays of tumor sections demonstrated the presence of MMTV viral antigens in the cells. Cell cultures initiated from first-, third-, and fourth-generation tumors were morphologically identical to the original in vitro cell line, although virus production was barely detectable. Analysis of the cultures by electron microscopy revealed a significant increase in MMTV virions after in vitro passage 3. Polypeptide profiles obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virions purified from these cultures were identical to MMTV. Immunodiffusion demonstrated the cross-reactivity between these virions and MMTV particles obtained from mouse milk. In vitro treatment of MJY-alpha cell cultures with rabbit anti-MMTV antiserum resulted in a reduction of extracellular MMTV virions, as well as alterations in their sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide patterns.     

Detection of Mouse Mammary Tumor Virus RNA in BALB/c Tumor Cell Lines of Nonviral Etiologies

A complementary DNA (cDNA) probe to mouse mammary tumor virus (MMTV) RNA was synthesized using calf thymus DNA oligonucleotides as a random primer. This probe was then used to study the expression of MMTV RNA in cell lines from BALB/c tumors induced in vivo either spontaneously or in response to viral, chemical, or hormonal stimuli. The cDNA had a length of approximately 400 to 500 nucleotides and specifically hybridized to MMTV RNA and BALB/c lactating mammary gland RNA, but not to Moloney leukemia virus RNA. Calf thymus DNA-primed cDNA could protect 50% of iodinated MMTV RNA from S1 nuclease digestion at cDNA-RNA ratios of 1:1 and 90% of labeled viral RNA at ratios of 10:1. Thermal denaturation of MMTV RNA-cDNA hybrids yielded a T(m) of 88.5 degrees C, indicative of a well-base-paired duplex. Screening of mouse mammary tumor cells for MMTV sequences revealed that three out of five lines of BALB/c origin had undetectable levels of viral RNA (相似文献   

Establishment of early lymphoid organ infrastructure in transplanted tumors mediated by local production of lymphotoxin alpha and in the combined absence of functional B and T cells

Lymphoid organogenesis is a highly coordinated process involving orchestrated expression of a number of genes. Although the essential role of lymphotoxin alpha (LTalpha) for the normal development of secondary lymphoid organs is well established, it is not clear to which extent it depends upon cooperation with T and B lymphocytes for lymphoid neo-organogenesis. To determine whether LTalpha is sufficient to mediate recruitment of basic elements needed for lymphoid organogenesis, we made use of a LTalpha-transfected cell line as an experimental tool and established tumors in nude and SCID mice. Our data showed that high endothelial venules formed and follicular dendritic cells accumulated and differentiated in response to LTalpha in the absence of lymphocytes. A CD4(+)CD3(-)CD11c(+) cell population that is found in the secondary lymphoid organ was also recruited into tumors expressing LTalpha. Furthermore, in nude mice, B cells migrated in response to LTalpha and formed intratumoral follicles. These B cell follicles were structurally well equipped with follicular dendritic cell networks and high endothelial venules; however, they were not functionally active; e.g., those B cells specific for a surrogate Ag expressed by the tumor were found in the spleen, but not in the tumor. We show that, even in the absence of functional T and B lymphocytes, local expression of LTalpha in transplanted tumors induced typical stromal characteristics of lymphoid tissue, emphasizing that LTalpha is a critically important cytokine for formation of lymphoid organ infrastructure.     

Mouse mammary tumor virus with rearranged long terminal repeats causes murine lymphomas.

Mouse mammary tumor virus (MMTV) is a slowly transforming retrovirus associated primarily with the induction of mammary tumors. It is widely accepted that T-cell lymphomas of various mouse strains are associated with extra proviruses of MMTV. These extra proviruses showed site-specific rearrangements in the U3 region of long terminal repeats (LTRs), consisting of about 400 nucleotide deletions and occasional substitution resulting in unique tandem repeats. However, the question of whether these mutant MMTVs cause lymphomas has not been experimentally resolved. Here we present distinct evidence that they do. We constructed chimeric MMTVs by replacing the LTR of the recently constructed pathogenic MMTV provirus clone with rearranged LTRs of MMTV proviruses obtained from two DBA/2 mouse lymphoma cell lines, MLA and DL-8, and inoculated them into BALB/c mice. These mice developed lymphomas, but no mammary tumors, 4 to 11 months postinoculation, whereas the original pathogenic MMTV clone alone induced mammary tumors. These results showed that the tissue specificity of MMTV tumorigenesis is determined by the LTR structures.     

Absence of mouse mammary tumor virus proviral amplification in chemically induced lymphomas of RF/J mice.

RF/J mice are susceptible to the induction of thymic lymphomas by the carcinogens 3-methylcholanthrene and N-methyl-N-nitrosourea. Given the association of mouse mammary tumor virus (MMTV) with certain thymomas, we examined genomic DNA from chemically induced lymphomas of RF/J mice for new MMTV proviruses. Of 13 tissue culture lines derived from 3-methylcholanthrene-induced tumors, 5 had acquired new proviruses. MMTV amplification coincided with the appearance of viral mRNAs and proteins. However, no primary tumors or animal-passaged tumors contained new proviruses. These observations indicate that MMTV does not have a role in the tumor induction process, although it may become activated and amplified in tissue culture lines derived from tumors.     

B cell response after MMTV infection: extrafollicular plasmablasts represent the main infected population and can transmit viral infection

The immune response to mouse mammary tumor virus (MMTV) relies on the presentation of an MMTV-encoded superantigen by infected B cells to superantigen-specific T cells. The initial extrafollicular B cell differentiation involved the generation of B cells expressing low levels of B220. These B220low B cells corresponded to plasmablasts that expressed high levels of CD43 and syndecan-1 and were CD62 ligand- and IgD-. Viral DNA was detected nearly exclusively in these B220low B cells by PCR, and retroviral type-A particles were observed in their cytoplasm by electron microscopy. An MMTV transmission to the offspring was also achieved after transfer of B220low CD62 ligand- CD43+ plasmablasts into noninfected females. These data suggest that B220low plasmablasts, representing the bulk of infected B cells, are capable of sustaining viral replication and may be involved in the transmission of MMTV.     

Isolation of a pathogenic clone of mouse mammary tumor virus.

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.     

Developmentally regulated rearrangement and expression of genes encoding the T cell receptor-T3 complex

Human leukemic cells corresponding to the earliest identifiable stages of intrathymic T cell differentiation lack cell surface expression of the T cell receptor(TCR alpha/beta)-T3 complex but transcribe TCR beta mRNA from either germ-line configuration (1/13) or partially (DJ) or fully (VDJ) rearranged (12/13) genes. These cells do not produce TCR alpha mRNA, but do contain T3 delta and T3 epsilon mRNA and accumulate T3 polypeptides, primarily in the perinuclear envelope. Equivalent normal T cells isolated from thymus have a predominantly germ-line configuration of TCR beta but contain intracellular T3 proteins. T3 gene expression is therefore a very early event in T cell differentiation. TCR alpha chain production appears to be the limiting maturation-linked event in the transport, assembly, and cell surface membrane insertion of the TCR alpha/beta-T3 complex.     

Ontogeny and distribution of the murine B cell Fc gamma RII.

The distribution and expression of the IgG FcRII (Fc gamma RII) on normal murine B cells was examined. Using multicolor flow cytometry, spleens from neonatal mice of increasing age and adult bone marrow were analyzed for expression of the Fc gamma RII. In addition, B cells from peripheral lymphoid organs, as well as panel of B cell tumors, were tested. The results demonstrate that the Fc gamma RII is expressed on all pre-B cells and immature B cells in the neonatal spleen and adult bone marrow, on all mature B cells in peripheral lymphoid organs, and on switched B cells in Peyer's patches. Furthermore, the Fc gamma RII was found to be present on B cell tumors representative of all stages of B cell maturation and differentiation. Taken together, the results indicate that Fc gamma RII is expressed during the entire lifetime of the B cell. In addition, examination of spleen cells from neonatal mice revealed a large number of pre-B cells, phenotypically defined as B220+, IgM-. These pre-B cells were present at birth, peaked in number between 2 and 3 wk of age, and became a minor population by day 30. Further phenotypic analysis of these cells demonstrated the expression of the BLA-1 and BP-1 Ag, and the lack of T cell and NK cell markers, thus confirming their assignment to the B cell lineage. Finally, the Fc gamma RII present on these pre-B cells was shown to be functional, by virtue of its ability to bind aggregated IgG.     

Mechanism of cytolytic T lymphocyte killing of a low class I-expressing tumor

Many tumors have been shown to express minimal levels of class I MHC Ag, which makes them more resistant to recognition and lysis by cytolytic T lymphocytes. Line 1, a BALB/c spontaneous lung carcinoma, normally expresses very low levels of class I Ag, but expression can be increased 50-fold by treatment with agents such as DMSO or IFN-gamma. Because class I Ag serve as restricting elements for cytolytic T cell recognition of tumor Ag, we wished to determine if cytotoxic T lymphocytes could play a role in the immune response to this type of class I low, but inducible, tumor. After immunization in vivo and restimulation of splenic cells in vitro we were able to generate T cell clones that lysed line 1 cells induced to express high levels of class I, but did not lyse uninduced, low class I expressing line 1 cells in short term (6-h) 51Cr release assays. Paradoxically, incubation of the T cells with uninduced class I low line 1 cells for a few days resulted in complete destruction of the tumor cells. We demonstrate that the T cells, stimulated by the tumor cells, produce IFN-gamma, which in turn induces class I expression on the line 1 cells making them susceptible to lysis by the T cell clone. This suggests that a positive feedback reaction can occur in generating a response to this and perhaps other inducible tumor cell lines.     

Mammary tumor virus proviral DNA in normal murine tissue and non-virally induced mammary tumors

The Southern DNA filter transfer technique was used to study the involvement of the endogenous mouse mammary tumor virus (MMTV) in the development of mammary tumors of nonviral etiology. The presence of extra MMTV proviruses in the genomes of these non-virally induced mammary tumors would indicate an integration of the provirus of an activated endogenous MMTV. Acquisition of MMTV proviruses did not seem to be an absolute requirement for the development of hormone or carcinogenically induced mammary tumors in strain BALB/c nor for hormone-induced mammary tumors in mouse strains 020, C57BL, and C3Hf. In some hormone-induced mammary tumors we did observe extra MMTV proviruses in submolar quantities, indicating that reintegration may occasionally occur and that only a part of the tumor cells acquired new MMTV DNA information. Hormone-dependent and -independent primary mammary tumors of the mouse strain GR, which are controlled by the Mtv-2 mammary tumor induction gene, all acquired extra MMTV proviruses. Most of these extra MMTV proviral-DNA-containing fragments appeared present in submolar quantities, suggesting that only part of the tumor cells acquired extra MMTV proviral information. These findings indicate that the initially transformed mammary gland cells of non-virally induced mammary tumors do not necessarily acquire extra MMTV proviral DNA information, in contrast to the MMTV-induced mammary tumors, in which all tumor cells contain extra MMTV DNA information.