Developing thymocytes organize thymic microenvironments.
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Cell Communication↗
Biomedical subjects
W T Germeraad
Publications and source records attributed to W T Germeraad.
Thymic microenvironments, 3-D versus 2-D?
Lympho-stromal interactions in the thymus crucially de- termine the fate of developing T cells. Epithelial cells, inter- digitating reticular cells, macrophages and fibroblasts all play a role in the shaping of the T cell repertoire. Recently published evidence shows that lympho-stromal interaction acts bi-directional. Developing T cell themselves, at different stages of differentiation, control the microarchitecture of thymic microenvironments, a phenomenon designated as 'crosstalk'. This paper reviews experiments showing that developing T cells crosstalk to different thymic epithelial cells in a stepwise fashion. In this way, correctly organized thymic microenvironments guarantee normal thymopoiesis.
Animals↗
Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments.
In the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of such microenvironments and more particularly the nature of communicative signals in lympho-stromal interaction during T cell development, we have employed the phage antibody display technology, with the specific aim of isolating thymic stromal cell-specific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intact, mildly fixed thymic fragments as target tissue and lymphocytes as absorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4-4, TB4-20, and TB4-28. While TB4-4 and TB4-20 are both epithelium specific, TB4-28 detects an epitope expressed on both epithelial- and mesenchymal-derived stromal cells. TB4-4 reacts with all cortical epithelial cells and with other endoderm-derived epithelia, but this reagent leaves the majority of medullary epithelial cells unstained. In contrast, MoPhab TB4-20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm- and ectoderm-derived epithelial cells. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection of evolutionary conserved epitopes expressed on subsets of thymic stromal cells.
Animals↗
Gene transduction into murine primitive hematopoietic cells with 2-gene retroviral vectors using a Transwell coculture system.
The present study aims at expressing a reporter gene in hematopoietic cells in vivo by introducing it into primitive hematopoietic cells with a 2-gene retroviral vector. Various constructs of retroviral vectors containing the human IL-2 receptor alpha chain gene (TAC) as the reporter and the neomycin phosphotransferase gene (neo) as a selectable marker were engineered, and the effectiveness of these vectors for expression of the reporter gene was evaluated after transfection into the packaging cell line GP + E86. It was found that the highest levels of reporter gene expression were attained with constructs ordered 5' long terminal repeat (LTR)-TAC-internal promoter-neo-3' LTR. In experiments investigating the expression of a reporter gene in hematopoietic cells, we used the Escherichia coli beta-galactosidase gene (lacZ) instead of TAC, because a very sensitive detection method was available for lacZ. For transduction of hematopoietic progenitors, packaging cell lines producing recombinant viruses were cultured in a Transwell hung into a Dexter-type bone marrow (BM) culture. The BM cells were selected with G418, and transferred into irradiated recipient mice. LacZ enzyme activity was detectable in the peripheral blood lymphocytes (PBL) of recipients taken 8 wk after reconstitution.
Animals↗
BCR/ABL P210 and P190 cause distinct leukemia in transgenic mice.
DNA constructs encoding BCR/ABL P210 have been introduced into the mouse germ line using microinjection of one-cell fertilized eggs. Kinetics of BCR/ABL P210 expression in transgenic mice were very similar to those of BCR/ABL P190 constructs in transgenic mice. mRNA transcripts were detectable early in embryonic development and also in hematopoietic tissue of adult animals. Expression of BCR/ABL in peripheral blood preceded development of overt disease. P210 founder and progeny transgenic animals, when becoming ill, developed leukemia of B, T-lymphoid, or myeloid origin after a relatively long latency period. In contrast, P190-transgenic mice exclusively developed leukemia of B-cell origin, with a relatively short period of latency. The observed dissimilarities are most likely due to intrinsically different properties of the P190 and P210 oncoproteins and may also involve sequences that control transgene expression. The delayed progression of BCR/ABL P210-associated disease in the transgenic mice is consistent with the apparent indolence of human chronic myeloid leukemia during the chronic phase. We conclude that, in transgenic models, comparable expression of BCR/ABL P210 and BCR/ABL P190 results in clinically distinct conditions.
Animals↗
Efficient retrovirus-mediated gene transduction into murine hematopoietic stem cells and long-lasting expression using a Transwell coculture system.
The neomycin phosphotransferase (neo) gene was transduced into murine hematopoietic stem cells by culturing a recombinant retrovirus-producing cell line in a Transwell (Costar, Cambridge, MA) (bottomed with a porous membrane) hung into a Dexter-type long-term bone marrow (BM) culture. Gene transduction into stem cells retaining long-term reconstitution ability was successfully performed by using protocols of total 15 to 18 days of culture including establishment of the Dexter culture, transduction, and G418 selection. In the irradiated recipients of these cells, a large majority of the BM, thymus, and spleen cells as well as peripheral blood (PB) leukocytes were of donor origin and the neo gene was present in these organs up to 21 weeks after cell transfer. One third to two thirds of the in vitro colony-forming cells in the BM of the recipient mice were resistant to cultivation with G418. It was further found that the hematopoietic system of secondary recipients given BM cells from a primary recipient mouse was predominated by original donor-type cells. The transduced neo gene was detected in the PB, BM, thymus, and spleen cells of these secondary recipients. These results indicate that our procedure of retroviral vector-mediated gene transfer is highly effective in safely introducing a gene into pluripotent hematopoietic stem cells.
Animals↗
Nucleotide sequence of a cDNA encoding an alternative form of LEF-1.
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Animals↗
Frequency analysis of the T cell precursors in the thymus.
A frequency determination of the T cell precursors in murine adult and fetal thymuses as well as in the bone marrow and fetal liver was made. Cells were serially diluted and injected into deoxyguanosine-treated fetal thymus lobes with a microinjector, and the lobes were cultured for 12 to 21 days. The lobes in which T cell development did not occur were discriminated from those in which T cells developed, and the precursor frequency was determined by Poisson probability distribution analysis. The precursor cell frequencies in adult bone marrow cells (2.4 x 10(-5)) and fetal liver cells (1.7 x 10(-4)) were comparable to those determined previously in in vivo intrathymus transfer experiments. The present study further shows that only a small fraction of fetal thymus cells (0.9-5.0 x 10(-2)), CD4-8- adult thymocytes (1.6 x 10(-2)), and Thy-1 low positive adult thymocytes (3.3 x 10-4)) retain the precursor activity.
Animals↗
Involvement of IL-7 in the development of gamma delta T cells in the thymus.
The effect of IL-7 on the growth of thymic T lymphocytes was investigated by adding recombinant IL-7 into cell suspension cultures and submersion organ cultures (SOC) of murine fetal thymuses (FT) and newborn thymuses (NBT). FT and NBT were obtained from C57BL/6 mice at day 15 of gestational age and at day 3 after birth, respectively. In both cell suspension cultures and SOC, addition of IL-7 highly improved the cell recovery. In cell suspension cultures, addition of IL-7 resulted in the growth of gamma delta T-cells from FT-cells, whereas the same cytokine promoted the growth of both alpha beta and gamma delta T-cells from NBT-cells. These results may indicate that this cytokine is able to support the proliferation of T-cells of both alpha beta and gamma delta lineages. In marked contrast, in SOC, addition of IL-7 resulted in the growth of gamma delta T-cells not only in FT but also in NBT, despite the fact that the SOC of NBT without exogenous cytokine exclusively promoted the growth of alpha beta T-cells. A similar effect was also seen when IL-2 was added to NBT-SOC, though the skewing to gamma delta lineage was not so strong as in the case of IL-7. In addition, we found that IL-7 mRNA is expressed in the day 15 FT at a much higher level than in the adult thymus. These results strongly suggested that the production of a large amount of IL-7 synthesized in the FT is one of the major factors leading to the generation of gamma delta T-cells in FT.
Animals↗
Inhibition of human immunodeficiency virus replication in a human T cell line by antisense RNA expressed in the cell.
The effect of the expression of antisense RNA against the human immunodeficiency virus (HIV) genome in a human T-cell line CEM on HIV replication was investigated. A 2.7 kilobase (kb) fragment of the HIV genome, including tat and a part of rev and env, was cloned into the retroviral vector WB in the antisense orientation under the SV40 or H-2K promoter. CEM cells transduced with this antisense gene via recombinant retrovirus expressed the RNAs of three different molecular sizes containing the antisense construct. CEM cells and these transduced cells were infected with HIV. HIV replication was evaluated 4-10 days later by an immunofluorescence assay and by determining the reverse transcriptase activity in the culture supernatant. The results indicate that although the recombinant retrovirus WB strongly enhanced the HIV replication in CEM cells, the expression of antisense RNA in the cells was highly effective in impeding the replication of HIV. The inhibitory effect was especially high in CEM cells transduced with the antisense gene under the control of SV40 promoter. In this case, HIV antigen-positive cells and reverse transcriptase activity in the culture supernatant of transduced cells were reduced to 30-50% and 5-10% of those in CEM cells and in the CEM cells transduced with WB, respectively.
Blotting, Northern↗
Alterations in dendritic cell phenotype and function associated with immunoenhancing effects of a subcutaneously administered cyclophosphamide derivative.
A single systemic dose of cyclophosphamide (CY) has been shown to enhance cellular immunity in a variety of antigen models. The immunoenhancing effects of CY have been attributed to its ability to selectively abrogate suppressor cell function. Previous studies from our group have demonstrated that local administration of distinct cytostatic drugs at the sensitization site can induce a similar enhancement of delayed-type hypersensitivity as systemic CY, with the obvious advantage of avoiding systemic side-effects. In the present study we investigated the effects of local administration of an optimally immunopotentiating dose of the active CY-derivative Z 7557 and, in selected experiments, of etoposide (VP-16) and systemic CY on mononuclear cells in draining lymph nodes. Whereas CY caused a long-lasting and marked depletion of B-cell areas, locally administered Z 7557 and VP-16 relatively spared B cells and even induced an increase in B- and T-cell numbers in (keyhole limpet haemocyanin-) sensitized mice. At Day 4 the CD4/CD8 ratio was slightly reduced in drug-treated mice. Interestingly, drug treatment reduced the proportion of interdigitating cells staining with the monoclonal antibodies NLDC-145 and MIDC-8. Upon isolation, dendritic cells (DC) from sensitized, Z 7557-treated mice showed longer dendritic protrusions and an enhanced accessory cell function compared to DC from saline-treated controls. These findings suggest that immunoenhancing effects of cytostatic drugs may occur via an effect on DC.
Adjuvants, Immunologic↗
Enhancing effects of locally administered cytostatic drugs on T effector cell functions in mice.
It is well established that systemic treatment with cyclophosphamide (CY) can augment cell-mediated immunity by selective toxicity for suppressor cells. The present paper analyzes the effect of local administration of the cytostatic drugs Z 7557, an active CY-derivative, and VP-16 (etoposid) on the development of cell-mediated immunity to KLH in mice. Both drugs were found to induce a long-lasting state of enhanced DTH responsiveness. Induction of enhancement was antigen-specific but required a conjunction of local drug treatment and s.c. priming. This indicates that locally applied cytostatics interact with the sensitizing process in the draining lymph node. In support of this view, draining lymph node cells showed an enhanced capacity to transfer DTH reactivity to syngeneic recipients in vivo, and to proliferate and produce IL-2 and MIF in response to KLH in vitro. Kinetic studies showed a lag in appearance of antigen-specific T cell responsiveness in drug treated mice, which indicates that effector cells are also sensitive to the drugs. If local chemotherapy is properly scheduled, the effector cells recover quickly and, released from the putative inhibitory influence of suppressor cells, give rise to an augmented T cell response. As systemic treatment with cytostatic drugs is known to cause adverse side effects, restricting its use for immunopotentiating purposes, the present local chemotherapy protocol provides a new, versatile approach for enhancing cell-mediated immunity.
Animals↗
Ex vivo gene transfer into myocardium using replication-defective retrovirus.
Heart transplantation is the most effective therapy for chronic severe heart failure, but there is an extreme shortage of hearts available. We examined the possibility that cardiomyocytes can be modified genetically prior to being grafted to the heart. We used a replication-defective retrovirus carrying the beta-galactosidase (beta-gal) reporter gene. The beta-gal gene was transduced into murine fetal cardiac myocytes by culturing a recombinant retrovirus-producing cell line in a Transwell plate hung into the primary cardiomyocyte culture. The cultured cells were stained with the di-beta-D-galactopyranoside (FDG) and were sorted by fluorescence-activated cell sorting (FACS). FACS analysis showed that 25.5 +/- 4.3% of the cardiomyocytes in a primary culture were positive for beta-gal activity. These cells were transplanted into the hearts of syngeneic adult mice. Expression of the beta-gal gene in the grafted cells was demonstrated by staining with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside (X-gal). Gene expression was recognized as long as 6 mo after cell transplantation. Histologic analysis showed neither inflammation nor fibrous scar tissue on the host myocardium. This study demonstrated that genetically modified cardiac cells were transplantable to the heart.
Animals↗