Fetal Thymus Cells Research Articles

Phenotypic characteristics of lymphoid populations of middle gestation human fetal liver, spleen and thymus

Mononuclear cells isolated from liver, spleen and thymus of fetuses between 18 and 24 weeks gestational age were stained for a number of lymphoid cell markers by indirect immunofluorescence and analyzed by flow cytometry. Studies were carried out on freshly isolated mononuclear cell preparations and on cultured cells after selective expansion in interleukin 2 (IL2). Many mononuclear cells in fresh isolates of liver and spleen could not be identified with antibodies to mature T- and B-cell markers. An average of 3% of isolated liver cells and 34% of isolated spleen cells stained positively for CD3, and 19% of liver cells and 37% of spleen cells stained positively for CD20. Lymphoid cells of the fetal thymus were an average 67% CD3 +, 76% CD4 +, 84% CD8 +, and showed greater CD45RO staining (93%) than mononuclear cells of other tissues. Propagation of liver and spleen cell populations in culture favored CD3 phenotypes and CD8 phenotypes. Propagated T cell populations of liver and spleen were primarily TCR α β + (81% in liver, 85% in spleen), suggesting a selective advantage in IL2 expansion of α/β T cells over γ/δ T cells. Propagated γ/δ T cells of liver and spleen were predominantly TCR γ δ 2 + . Whereas propagated cells of liver and spleen consisted of approximately 10% γ/δ + cells, thymus-derived cells expanded in culture were only an average of 2% TCR γ/δ +, demonstrating a rarity of IL2-responslve γ/δ T cells in middle gestation fetal thymus.

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 × 10-5) and fetal liver cells ( 1.7 × 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 × 10-2), CD4-8- adult thymocytes (1.6 × 10-2), and Thy-1 low positive adult thymocytes (3.3 × 10-4) retain the precursor activity.

Reproducible Procedures for Establishing Mouse Thymic Stromal Cell Lines

Mouse thymic epithelial cell lines (MTEC) were established from Day 14-18 fetal thymus by two novel protocols. The first protocol involved the selection of TEC by the formation of complexes with adult thymocytes after transformation with the helper-free Ad5.SVR4 recombinant virus. The second protocol involved the stimulation and selection of TEC in Ca 2+-free medium by the formation of complexes with Day 14 fetal thymocytes. The resulting TECs formed several types of thymic epithelial clusters to which Day 14 fetal thymocytes could bind. Many of these fetal thymocytes could deeply infiltrate, colonize, and proliferate within the clusters of NCAM high LFA-1 low TEC (MTSC-0420-1.4, MTSC-0420-1.5, and MTSC-0613-1.2 clusters), whereas very few could infiltrate the clusters of NCAM low LFA-1 high TEC (MTSC-0531-5.1 and MTSC-0531-5.2) and none bound to or infiltrated an NCAM neg fibroblast cluster (MTSC-fibro.). Hence, it is possible that the NCAM-positive epithelial cell lines are derived from the thymus cortex, where they may play an important role in intrathymic migration and the clonal growth of pro-T cells in fetal thymus.

A murine thymic stromal cell line which may support the differentiation of CD4 −8 − thymocytes into CD4 +8 − αβ T cell receptor positive T cells

A fibroblastoid cell line TSt-4 was established from fetal thymus tissue of C57BL/6 mice. When fetal thymus (FT) cells or CD4 −8 − (DN) cells of adult thymuses were cultured on the monolayer of TSt-4, a considerable proportion of lymphocytes expressed CD4 or both CD4 and CD8 within 1 day, and the CD4 +CD8 − cells were maintained further while the CD4 +8 + cells disappeared by Day 5. A large proportion of cells generated from DN cells but not FT cells was shown to express CD3 and T cell receptor αβ. Addition of recombinant interleukin (IL)-7 into the cultures resulted in a marked increase of cell recovery without virtual change in differentiation process of αβ lineage. The present work strongly suggests that thymic fibroblasts play an important role in T cell differentiation and IL-7 contributes to supporting proliferation of differentiated cells.

Differentiation patterns of [formula omitted] thymocyte subsets in cocultures of fetal thymus and lymphohemopoietic cells from c- fos transgenic and normal mice

This study examined the involvement of c- fos protooncogene in thymocyte development from lymphohemopoietic T cell progenitors, within the thymic microenvironment. We first analyzed the thymocytes developing in vitro in the fetal thymus from the c- fos transgenic mice and found a high proportion of CD4 + single positive (SP) cells. We then seeded either fetal liver or bone marrow (BM) cells from normal donors onto lymphocyte-depleted fetal thymus explants of c- fos transgenic mice. The results showed an increased proportion of mature CD4 + SP and decreased CD4 + CD8 + double positive (DP) cells. A similar pattern of CD4 CD8 thymocyte subsets was observed when either thymus or BM cells from c- fos transgenic mice developed within a normal thymic stroma. The kinetics of thymocyte development in organ culture (from Days 3 to 11) suggested that the SP cells obtained under these conditions may have bypassed the CD4 + CD8 + DP phase. It appears that the altered pattern of thymocyte development manifested in adult c- fos transgenic mice can be induced by the early embryonic thymic stroma, and may also involve cells in the lymphohemopoietic tissues.

Requirement of dendritic cells and B cells in the clonal deletion of Mls-reactive T cells in the thymus.

The present study was performed to identify cells responsible for the elimination of T cells reactive with minor lymphocyte-stimulating (Mls) antigens during T cell development. Experiments were carried out in a fetal thymus organ culture (FTOC) system. To examine the tolerance-inducing activity, various populations of cells from adult CBA/J (Mls-1a) mice were injected into deoxyguanosine (dGuo)-treated FTOC of C3H/He (Mls-1b) mice with a microinjector, and 2 d later, the thymus lobes were injected with fetal thymus cells from C3H/He mice as T cell precursors. After 14 d of cultivation, cells were harvested and assayed for the expression of the T cell receptor V beta 6 element. The absence or marked reduction of T cells expressing V beta 6 at high levels (V beta 6high) was regarded as indicating the deletion of Mls-1a-reactive T cells. T cell-depleted populations of thymic as well as splenic cells from CBA/J mice were able to induce clonal deletion. Further characterization of the effector cells was carried out by fractionating the spleen cells before injecting them into dGuo-FTOC. None of the dish-adherent population, dish-nonadherent population, or purified B cells alone were able to induce clonal deletion, whereas the addition of purified B cells to adherent cells restored tolerance inducibility. It was further shown that a combination of CBA/J B cells and C3H/He dendritic cells was effective in eliminating Mls-reactive clones. These results indicate that for the deletion of clones reactive with Mls antigens during T cell development in the thymus, both DC and B cells are required.

Tolerogenic ability of thymocytes in organ-cultured thymus lobes.

It is generally believed that macrophages and dendritic cells are the major cell populations that present tolerogenic self antigens to developing thymocytes. However, it is still controversial whether self antigens expressed on thymocytes themselves work as tolerogens in the thymus. To evaluate this possibility, Thy-1 bright cells were sorted out from fetal thymus cells on the 15th gestation day, and were colonized into 2'-deoxyguanosine-treated allogeneic thymus lobes. The repopulated thymus lobes were organ-cultured, and the allo-specific killer activity of thymocytes recovered from the lobes was examined. These cells were tolerant to class I but not to class II-MHC of the donor haplotype, indicating that class I molecules expressed on the thymocytes worked as tolerogen. Tolerogenic ability of Thy-1+ cells was also demonstrated in another system. Upon intimate contact with allogeneic thymus lobes on a polycarbonate filter, thymus lobes fused with each other and Thy-1+ cells co-migrated (Eur. J. Immunol. 19:1525-1530, 1989). In thymus lobes rendered parabiotic from day 5, CTL tolerance was achieved against class I but not to class II MHC. These data indicate that thymocyte-thymocyte interaction is sufficient to induce class I CTL tolerance in developing thymocytes.

Interleukin 7 preferentially supports the growth of γδ T cell receptor-bearing T cells from fetal thymocytes in vitro

Murine fetal thymus cells were cultured with various interleukins (IL-1, 2, 3, 4, 5, 6, and 7) in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and it was found that only IL-4 and IL-7 induced a prominent proliferative response in the presence of PMA. A large proportion of cells grown in the cultures of fetal thymus cells (days 15 and 17 of gestation) stimulated with PMA plus IL-4 or with PMA plus IL-2 remained CD4-CD8-. In marked contrast, nearly 70% of the cells generated in the cultures of the same fetal thymocytes stimulated with PMA plus IL-7 expressed CD8 on their surface. Approximately 30% of these cells expressed TCR gamma, delta, whereas TCR alpha beta+ cells were virtually undetectable. The cells grown in cultures stimulated with PMA plus IL-7 comprised three populations: CD4-Lyt-2-3-, CD4-Lyt-2 + Lyt-3- and CD4-Lyt-2 + Lyt-3+, and that TCR gamma delta+ T cells were found in all three populations. It was also found that the addition of IL-7 in the culture of adult CD4-CD8- thymocytes on the monolayer of a thymic stromal cell line, which selectively promotes the generation of alpha beta T cells, resulted in the generation of gamma delta T cells. These results strongly suggest that IL-7 plays an important role in the development of gamma delta T cells.

Equine infectious anemia virus derived from a molecular clone persistently infects horses

A full-length molecular clone of equine infectious anemia virus (EIAV) was isolated from a persistently infected canine fetal thymus cell line (Cf2Th). Upon transfection of equine dermis cells, the clone, designated CL22, yielded infectious EIAV particles (CL22-V) that replicated in vitro in both Cf2Th cells and an equine dermis cell strain. Horses infected with CL22-V developed an antibody response to viral proteins and possessed viral DNA in peripheral blood mononuclear cells, as determined by polymerase chain reaction assays. In addition, horses infected with CL22-V became persistently infected and were capable of transmitting the infection by transfer of whole blood to uninfected horses. However, CL22-V, like the parental canine cell-adapted virus, did not cause clinical signs in infected horses. Reverse transcriptase assays of CL22-V- and virulent EIAV-infected equine mononuclear cell cultures indicated that the lack of virulence of CL22-V was not due to an inability to infect and replicate in equine mononuclear cells in vitro.

Isolation of murine fetal thymus cell lines after infection with recombinant retroviruses containing the v-myc and v-Ha-ras oncogenes.

The mechanisms that regulate thymocyte proliferation and differentiation within the thymus are still poorly understood. As a novel approach to analyzing these problems, we have used a retroviral vector to insert oncogenes into fetal thymic cells, and construct a library of thymic cell lines. Infection with a double promoter recombinant retrovirus containing the v-Ha-ras and v-myc oncogenes resulted in the apparent immortalization of a range of cell lines derived from 13- and 14-day murine fetal thymus, and expressing both v-Ha-ras and v-myc mRNA at high levels. Cell lines were established from untreated thymic lobes (group 1), deoxyguanosine-treated lobes (group 2), and in the presence of IL-2 (group 3). Cell lines with a wide range of surface phenotypes were established. Group 1 were adherent cell lines expressing many antigens, including Mac-1, FcR, and Ia. Group 2 were also adherent cells but expressed very few Ag. Group 3 were IL-2-dependent lymphoid-like non-adherent cells, with a null or early thymocyte-like phenotype. None demonstrated full rearrangement and expression of TCR alpha-, beta-, or gamma-genes, although cell lines in group 2 expressed short beta and those in group 3, short beta and gamma gene transcripts. This library of immortalized cell lines appears to represent several types of stromal cell and early thymocyte precursors present in 13- and 14-day fetal thymus. These cell lines should prove invaluable in reconstructing the environment of the intact fetal thymic lobe, to study the cellular interactions that appear to govern thymocyte proliferation, development, and selection of Ag reactivity.

Antigen recognition by MHC-incompatible cells of a human mismatched chimera.

Tetanus toxin (TT)-specific T cell clones of donor origin were obtained from a patient with severe combined immunodeficiency (SCID) successfully reconstituted by transplantation of allogeneic fetal liver and thymus cells from two different donors performed 10 yr ago. A series of these clones recognized TT in the context of "allo" class II HLA determinants expressed by recipient APC. The restriction element of two T cell clones with the HLA phenotype of the first donor (HLA-DR1,8) and one T cell clone with the HLA phenotype of the second transplant (HLA-DR3,9) was HLA-DR4 of the recipient, whereas other T cell clones derived from the second transplant recognized TT in the context of HLA-DR5 of the recipient's APC. These latter T cell clones were not able to proliferate in response to TT when autologous APC were used. These data demonstrate that recipient and donor cells having different HLA phenotypes could cooperate across the allogeneic barrier and that MHC restriction of antigen (Ag) recognition is independent from the MHC genotype of the T cells but is influenced by the environment in which the T cells mature. We also isolated T cell clones that were able to recognize processed TT presented by all allogeneic EBV cell lines tested, indicating that the Ag specificity of these clones was not restricted by a particular class II MHC molecule. The Ag-specific proliferative response of one of these clones could be blocked by anti-class II MHC mAbs. These results demonstrate that in addition to Ag recognition in the context of specific class II MHC Ags, other types of Ag-specific responses may occur in this human chimera. It is not clear whether this "allo" plus Ag recognition is the result of education of transplanted fetal cells in the host thymus. Taking into consideration our previous findings indicating that alloreactive T cell clones specific for the recipient cells could be isolated in vitro from the PBL of the same patient, our data suggest that the mechanism for deletion of self-reactive clones and the generation of MHC-restricted responses are different.

Identification of CD2-/CD3+ T cells in fetal human tissue.

From 1 to 23% of fetal human spleen or thymus cells (from the 20th to 24th week of gestation) were found to display a previously unrecognized CD2-/CD3+ phenotype. IL-2-dependent, long-term clones of CD2-/3+ T cells did not react with a panel of anti-CD2 mAbs and did not form rosettes with sheep erythrocytes. These results show that (a) significant numbers of CD2-/3+ T cells are present in fetal human spleen and/or thymus; and (b) in contrast to the widely accepted view, expression of CD2 is not a prerequisite for the expression of the CD3 molecular complex on human T cells.

Relation between arachidonic acid metabolism and development of thymocytes in fetal thymic organ cultures.

Abstract Because products of arachidonic acid metabolism, particularly the PG, have been implicated as modulators of growth and differentiation of adult thymocytes, we investigated relations between metabolism of arachidonic acid and growth, as well as differentiation, of thymocytes during fetal thymic organ culture. Fetal thymic cells synthesized immunoreactive PGE2 during organ culture and were found to be capable of metabolizing exogenous arachidonic acid to products that cochromatographed with authentic 6-keto-PGF1 alpha, PGE2, PGF2 alpha. Synthesis of these products and growth and expression of Thy-1 and Lyt-1 Ag were inhibited by culture of fetal thymic lobes with indomethacin, a cyclooxygenase inhibitor, as well as meclofenamate and eicosatetraynoic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Only indomethacin inhibited expression of Lyt-2. Culture with eicosatetraynoic acid also inhibited the capacity of thymic lobes to synthesize 15-hydroxyeicosatetraenoic acid-like products. The inhibitory effects of indomethacin on growth and expression of Thy-1 were partially reversed by simultaneous addition of arachidonic acid. Thus, fetal thymic cells appear to require an intact cyclooxygenase, and possibly lipoxygenase, pathway of arachidonic acid metabolism for growth and differentiation. These data also provide evidence that Lyt-1 and Lyt-2 may be regulated by different requirements with respect to arachidonic acid metabolism.

Reactivity of antibody to ganglioside GD3 against enzootic bovine lymphosarcoma tumor cells.

The antibody to ganglioside GD3, which was invariably increased in tumor lymph node cells of enzootic bovine lymphosarcoma (EBL), was examined for its possible usefulness in detecting EBL tumor cells. The anti-GD3 antibody was prepared by immunization of chicken with GD3 from EBL tumor lymph node cells. The antibody to ganglioside GD3 reacted to ganglioside GD3 containing N-glycolylneuraminic acid from EBL tumor cells. The reactivities of anti-GD3 antibody to peripheral blood lymphocytes (PBL) from aleukemic and leukemic cattle and tissues of bovine fetuses were examined by complement-dependent antibody cytotoxicity test. The antibody to ganglioside GD3 had cytotoxic activity to PBL from EBL cattle, fetal bovine thymus and liver cells but not to PBL from healthy cattle irrespective of presence or absence of bovine leukemia virus infection. In addition, acctone-fixed EBL tumor cells showed a positive reaction to the antibody to ganglioside GD3 in an indirect fluorescent antibody test.

Analysis of CD1 Molecules on Thymus Cells and Leukemic T Lymphoblasts Identifies Discrete Phenotypes and Reveals That CD1 Intermolecular Complexes Are Observed Only on Normal Cells

AbstractWe looked at the surface expression of the three distinct human thymic cell surface differentiation antigens, CD1a, CD1b, and CD1c, that presently define the first cluster of differentiation (CD) on the cells from 34 patients with acute T cell malignancies. We also studied the expression of other T cell-restricted molecules, including the T cell receptors, on these cells. Our results confirm the extensive phenotypic heterogeneity of the cells from acute T cell malignancies, which contrast with the more limited phenotypic diversity of subacute or chronic T cell malignancies. Our study of normal children and fetal thymus cells shows that the extensive phenotypic heterogeneity of the malig- nant cells reflects the heterogeneity of the thymic subpopulations and shows that most of the phenotypes observed on malignant T cells have a normal counterpart, particularly in the fetal thymus. Moreover, we demonstrate that the CD1a molecules, which can form three different types of noncovalent intermolecular complexes on the surface of normal thymus cells, do not form any noncovalent intermolecular complexes on the surface of leukemic cells. We also show that CD1a molecules can form covalent intermolecular complexes with CD8 molecules on some but not all malignant cells.© 1987 by Grune & Stratton, Inc. 0006-4971/87/7003-0011 $3.00/0.

Analysis of CD1 molecules on thymus cells and leukemic T lymphoblasts identifies discrete phenotypes and reveals that CD1 intermolecular complexes are observed only on normal cells

We looked at the surface expression of the three distinct human thymic cell surface differentiation antigens, CD1a, CD1b, and CD1c, that presently define the first cluster of differentiation (CD) on the cells from 34 patients with acute T cell malignancies. We also studied the expression of other T cell-restricted molecules, including the T cell receptors, on these cells. Our results confirm the extensive phenotypic heterogeneity of the cells from acute T cell malignancies, which contrast with the more limited phenotypic diversity of subacute or chronic T cell malignancies. Our study of normal children and fetal thymus cells shows that the extensive phenotypic heterogeneity of the malignant cells reflects the heterogeneity of the thymic subpopulations and shows that most of the phenotypes observed on malignant T cells have a normal counterpart, particularly in the fetal thymus. Moreover, we demonstrate that the CD1a molecules, which can form three different types of noncovalent intermolecular complexes on the surface of normal thymus cells, do not form any noncovalent intermolecular complexes on the surface of leukemic cells. We also show that CD1a molecules can form covalent intermolecular complexes with CD8 molecules on some but not all malignant cells.

Limiting dilution analysis of the stem cells for T cell lineage.

Abstract Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers.

Fetal thymus and liver cell transplantation for immunodeficiency with short-limbed dwarfism

Five-year-old girl with immunodeficiency with short-limbed dwarfism is reported. She manifested deficient cell-mediated immunity but normal antibody-mediated immunity. Her T-lymphocyte had maturation arrest at stage I thymocyte.Two fetal thymus and fetal liver cell transplants were performed on her. Two weeks after the first transplant, mild graft versus host reaction was observed and after four and half months, new HLA antigen was detected. The transplanted fetal stem cells were considered to differentiate in this patient, but no clinical improvement or immunological reconstitution could be observed. The patient continued to deteriorate and expired with interstitial pneumonia five months after the transplantation.Mild GVH phenomenon and the detectin of new HLA antigen in this patient makes this therapeutic approach promising, and it will be reasonable when histocompatible donors are unavailable.

The ontogeny of antigen-presenting cells in fetal thymus evaluated by MLR stimulation.

This study demonstrates that cells with the characteristics of antigen-presenting cells (APC) are present in murine thymus from as early as 14 days gestation, around the time that T lymphopoiesis is initiated in the embryo. Thymic APC were identified as la+ adherent cells capable of presenting alloantigens to T cells in MLC. The possible role of these cells in thymocyte differentiation is discussed.

Can fetal antigens be used for prophylactic immunization?

Even if the inoculation of fetal tissue cells were a dependable and uniformly successful method of protecting experimental animals against chemically or virally induced tumours, it would for obvious reasons still not be feasible to use fetal tissues for such a purpose in human beings. Among possible substitutes syngeneic spermatozoa were tested on the grounds that they are the only adult cells that express T-alleles, but neither they nor teratocarcinoma cells protected mice against tumours raised by 3-methylcholanthrene. Testicular and thymic cells and tissue fragments have given effective protection in a number of experiments and it is noteworthy that fetal tissues, testicular cells and thymus cells are cross-reactive in respect of anti-embryo antibodies. Testicular cells probably act like fetal cells and, like fetal cells, are very prone to give rise to 'enhancement'. Thymic cells do not 'enhance' and may act quite differently. The variability of results--a source of grave concern--is attributed to the insensitivity of the test system which is ill-adapted to show up low degrees of protection.