Fetal Thymic Tissue Research Articles

Skin graft tolerance across a discordant xenogeneic barrier.

Specific T-cell tolerance may be essential for successful xenotransplantation in humans. Grafting of thymectomized, T cell-depleted normal mice with xenogeneic fetal pig thymus and liver (FP THY/LIV) tissue results in the recovery of functional CD4 antigen-positive cells. We have tested T-cell tolerance by skin grafting. Donor-matched pig skin survived permanently (> 200 days), whereas allogeneic mouse skin was rapidly rejected. Nontolerant control mice rejected pig skin within 26 days. Both porcine and murine histocompatibility class IIhigh cells were detected in long-term thymus grafts, and T-cell repertoire analyses suggested that tolerance to both donors and recipients developed, at least in part, by intragraft clonal deletion. This study demonstrates the principle that tolerance, measured by the stringent criterion of skin grafting, can be induced across a widely disparate species barrier.

Developmental aspects of prolactin receptor gene expression in fetal and neonatal mice.

The prolactin receptor (PRL-R), a member of the hematopoietin cytokine receptor superfamily, is widely distributed among mammalian tissues. To understand better the potential sites of action and onset of potential PRL responsiveness, the developmental distribution pattern of PRL-R mRNA expression in fetal and neonatal mice was examined. Fetal mouse tissues were collected at distinct stages from timed pregnancies. Following extraction of total RNA, onset of gene expression was evaluated via reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridization was employed for verification. Expression of PRL-R mRNA was first observed on day 14 in the liver and cranium and on day 15 in the kidney, lung and thymus gland. Pituitary and adrenal glands were positive for PRL-R at day 18 of gestation through to day 1 of postnatal life. Neither whole fetuses prior to day 14 (days 10-13) of gestation nor skin and bladder tissues from 2-day-old mice generated detectable RT-PCR signals for PRL-R. The presence of PRL-R mRNA in fetal thymus and spleen tissues suggests a possible role for PRL in the development of the immune system. Prolactin may act directly on the pituitary to influence its own secretion and/or that of other pituitary-derived factors, as evidenced by the presence of PRL-R mRNA in the pituitary glands of fetal and 1-day-old mice. These data are the first to show the presence of PRL-R gene expression in various organ systems in fetal mice and suggest that PRL is among several factors necessary to coordinate developmental activities.

Human T-cell leukemia virus infection of human hematopoietic progenitor cells: maintenance of virus infection during differentiation in vitro and in vivo

Human T-cell leukemia virus type I (HTLV-1) is the etiologic agent of adult T-cell leukemia and lymphoma and HTLV-1-associated myelopathy-tropical spastic paraparesis. We examined whether HTLV could productively infect human hematopoietic progenitor cells. CD34+ cells were enriched from human fetal liver cells and cocultivated with cell lines transformed with HTLV-1 and -2. HTLV-1 infection was established in between 10 and >95% of the enriched CD34+ cell population, as demonstrated by quantitative PCR analysis. HTLV-1 p19 Gag expression was also detected in infected hematopoietic progenitor cells. HTLV-1-infected hematopoietic progenitor cells were cultured in semisolid medium permissive for the development of erythbroid (BFU-E), myeloid (CFU-GM), and primitive progenitor (CFU-GEMM, HPP-CFC, or CFU-A) colonies. HTLV-1 sequences were detected in colonies of all hematopoietic lineages; furthermore, the ratio of HTLV genomes to the number of human cells in each infected colony was 1:1, consistent with each colony arising from a single infected hematopoietic progenitor cell. Severe combined immunodeficient mice engrafted with human fetal thymus and liver tissues (SCID-hu) develop a conjoint organ which supports human thymocyte differentiation and maturation. Inoculation of SCID-hu mice with HTLV-1-infected T cells or enriched populations of CD34+ cells established viral infection of thymocytes 4 to 6 weeks postreconstitution. Thymocytes from two mice with the greatest HTLV-1 proviral burdens showed increased expression of the CD25 marker and the interleukin 2 receptor alpha chain and perturbation of CD4+ and CD8+ thymocyte subset distribution profiles. Hematopoietic progenitor cells and thymuses may be targets for HTLV infection in humans, and these events may play a role in the pathogenesis associated with infection.

A novel putative helicase produced in early murine lymphocytes

DNA helicases (Hel) play a role in a number of processes involving DNA strand separation, including replication, repair, recombination and transcription. Rearrangement of receptor genes, which occurs in immature lymphocytes, could also be mediated by Hel. We report here the cloning from murine fetal thymus tissue of a novel putative Hel containing seven conserved Hel domains and belonging to the DEGH subclass of DNA Hel. We term the encoding gene lsh (lymphoid-specific Hel), since the gene is expressed in early thymocytes, but not in heart, liver, lung, muscle, brain or kidney, as judged by Northern analysis. Spleen cells expressed lsh following activation. T- and B-cell lines, at both the immature and mature stage, expressed lsh. To examine the earliest stages of lymphopoiesis, mouse embryonic tissues were examined; lsh was not detected in the yolk sac of day 12 of gestation, but was expressed in fetal liver and at high levels in fetal thymus at day 15 of gestation

Toxicity of 2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) to the Human Thymus After Implantation in SCID Mice

There are conflicting data with regard to the sensitivity of the human immune system to the toxic action of the highly toxic environmental pollutant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). SCID mice engrafted with human fetal thymus and liver tissue fragments (SCID-hu mice), which sustain normal human T cell differentiation in the thymus graft, were used to directly assess the sensitivity of the human thymus for TCDD. Wistar rats and SCID-hu mice were exposed to 1, 5, or 25 μg TCDD/kg body weight. Histopathologic effects were evaluated for rat thymus and transplanted human thymus on Day 4 after exposure. The relative size of the cortex showed a dose-dependent decrease in both the normal rat thymus and grafted human thymus (significant at 25 μg/kg). SCID-ra mice (SCID mice with a fetal rat thymus and liver graft) were used as an intermediate model between the normal rat and SCID-hu mice, and were exposed to the same dose levels of TCDD. However, 90% of the SCID-ra mice developed a cutaneous graft-versus-host reaction, associated with lymphodepletion of the rat thymus grafts, and hence a limited number of SCID-ra mice were available for evaluation of TCDD effects. The data obtained in SCID-ra mice were in line with those in normal rat and grafted human thymus. In gas chromatography/mass spectrometry analysis, TCDD tissue concentrations in the normal rat thymus and grafted human thymus were similar. We conclude that the human thymus serves as a target for TCDD, and that the human thymus and the Wistar rat thymus display a comparable sensitivity to the toxic action of TCDD.

The SCID-hu mouse as a tool in immunotoxicological risk assessment: effects of 2-acetyl-4(5)-tetrahydroxybutyl-imidazole (THI) and di- n-butyltin dichloride (DBTC) on the human thymus in SCID-hu mice

SCID mice engrafted with human fetal thymus and liver tissue fragments (SCID-hu mice) are currently considered as a new tool in human immunotoxicological risk assessment. Testing of various immunotoxicants exerting thymotoxicity via different intrathymic target cell types is necessary for validation of this model. Therefore, SCID-hu mice were exposed to 2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)-imidazole (THI), the immunotoxic component in the food additive, Caramel Colour III, or the organotin compound, di- n-butyltin dichloride (DBTC). Histopathological examination of the human thymus grafts of SCID-hu mice either exposed to THI or to DBTC showed a reduction in the relative size of the thymus cortex, an effect also described in rodents. These results indicate that the human thymus is a target for the immunotoxic action of both THI and DBTC. In addition, they indicate the promising potential of the SCID-hu mouse model as a tool for human immunotoxicological risk assessment.

Programmed differentiation of murine thymocytes during fetal thymus organ culture

Fetal thymus organ culture (FTOC) has become widely used to investigate the impact of immunomodulators on T cell development. However, these studies have given variable results among different laboratories. In this study, we have found that fetal tissue age and mouse strain differences can affect the development of T cell phenotypes in this system. T cell development in FTOC occurred in two ‘waves’, defined as peaks of cell recovery. The first wave consisted initially of CD4 −CD8 − double negative (DN) cells and CD4 −CD8 + single positive (SP) T cells expressing γδ T cell receptor (TCR). CD4 +CD8 + double positive (DP) cells expressing low levels of αβ TCR were produced soon thereafter; and these cells dominated the cultures for the balance of the first wave. Prolonged FTOC resulted in the production of another wave of T cells which were relatively enriched for CD4 or CD8 SP cells expressing high levels of αβ TCR, as well as DN cells and CD4 −CD8 + SP T cells expressing high levels of γδ TCR. As defined by cell number and differentiation of αβ TCR SP cells, development was delayed in FTOC using fetal thymus tissue from younger fetuses relative to that observed when older fetal thymus tissue was used. The degree of development of T cells in FTOC was also strain dependent. Organ cultures derived from 14 gestation days (gd) C.B-17 scid/scid fetal thymus did not generate TCR-bearing mature SP cells, but they did produce TCR-negative CD4 and CD8 SP cells likely to be precursors of DP thymocytes. Such cultures made from 18 gd tissue did not produce SP cells. Negative selection in FTOC was also evaluated. Mtv-specific V β3 cells were deleted in FTOC of C3H HeN tissue. Deletion occurred only in late FTOC, suggesting a late encounter between the Mtv deleting elements and susceptible T cells during ontogeny. These results show that while FTOC recapitulates normal thymic development by a variety of criteria, results can be influenced by the length of culture, as well as by the age and strain of fetal thymus tissue utilized.

In vitro construction of graded thymus chimeras

A simple in vitro approach is described for constructing chimeric thymi in which the degree of chimeric contribution can be tightly controlled. This is achieved by protease-assisted dissociation of fresh fetal thymic tissue, mixing the resulting cell suspensions at the desired ratios, and subsequent reaggregation. Analysis of these graded chimeras shows that stromal elements become evenly interspersed and form a microenvironment able to support the maturation of endogenous T cell precursors. Chimeras between normal and MHC-deficient thymi have been used to demonstrate the rescue of mutant, allotype-marked thymocytes by wild-type stromal cells. Other potential applications of the method include quantitative studies on positive and negative thymic selection, the functional role of defined stromal cell types in these processes, and the analysis of mutants (either natural or engineered) by complementation.

The concurrent maturation of mouse and human thymocytes in human fetal thymus implanted in NIH-beige-nude-xid mice is associated with the reconstitution of the murine immune system.

To determine whether the human thymus provides an environment for the maturation of murine T cells, human fetal thymus and liver (hu-thy/liv) were implanted into congenitally athymic NIH-beige-nude-xid (BNX) mice or C.B-17 scid/scid (SCID) mice. 3 mo after implantation, in contrast to the hu-thy/liv implant in SCID mice, which was populated only with human CD4/CD8 single- and double-positive thymocytes, the hu-thy/liv implant in BNX mice contained a chimeric population of human and mouse CD4/CD8 single- and double-positive thymocytes. Immunohistochemical staining of the hu-thy/liv implant in BNX mice indicated that the population of double-positive mouse thymocytes was localized to discrete areas of the human fetal thymus. Quantitative improvements in mouse T cell and immunoglobulin (Ig) G parameters were observed after grafting of the human fetal thymus and liver tissue into BNX mice. In addition, in contrast to the nonimplanted BNX mice, the implanted BNX mice were capable of mounting a keyhole limpet hemocyanin-specific IgG response and their peripheral T cells were responsive to stimulation with mitogens and antibodies directed to the T cell receptor. Furthermore, after in vivo priming, T cells present in lymph nodes of the implanted BNX mice were capable of mounting an antigen-induced in vitro T cell-dependent proliferative response. Thus, concurrent with the continued maturation of human T cells, murine T cells differentiated within the human fetal thymus implanted in the BNX mice and mediated the phenotypic and functional reconstitution of the murine immune system. Mice with a reconstituted immune system that contain a human thymic implant that is infectible with human immunodeficiency virus (HIV) should prove useful in the investigation of T cell maturation in the thymus and in the evaluation of potential HIV vaccines.

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.

Thymus transplantation and disease prevention in the diabetes-prone Bio-Breeding rat.

Bio-Breeding rat T lymphocytes proliferate poorly in response to alloantigen. Transplantation of Bio-Breeding rats with fetal thymus tissue from diabetes resistant rats leads to an improvement in the T cell proliferative response, but only if the thymus contains bone marrow-derived, radiation-resistant thymic antigen presenting cells of the diabetes-resistant phenotype. The current study provides evidence that thymus transplantation leading to the restoration of Bio-Breeding T cell proliferative function can also significantly reduce the incidence of insulitis and prevent the development of diabetes. It appears that a defect in the bone marrow-derived thymic APC population contributes to an abnormal maturation of Bio-Breeding T lymphocytes which in turn predisposes animals to insulitis and diabetic disease.

Human T cell antigen expression during the early stages of fetal thymic maturation.

Human thymus tissue was examined from 7 wk of gestation through birth for the expression of antigens reacting with a panel of anti-T cell monoclonal antibodies. Additionally, the reactivities of reagents against the transferrin receptor, against leukocytes, against low m. w. keratins, and against major histocompatibility complex antigens were studied on human fetal thymic tissue. Frozen tissue sections were evaluated by using indirect immunofluorescence assays. At 7 wk of gestation, no lymphoid cells were identified within the epithelial thymic rudiment; however, lymphoid cells reacting with both antibody 3A1, a pan T cell marker, and antibody T200, a pan leukocyte reagent, were identified in perithymic mesenchyme. After lymphoid colonization of the thymic rudiment at 10 wk of fetal gestation, fetal thymic tissue reacted with antibodies T1, T4, and T8. At 12 wk of gestation, antibodies T3, T6, A1G3 (anti-p80, a marker of mature thymocytes), and 35.1 (anti-E rosette receptor) all reacted with thymic tissue. Our findings indicate that T cell antigens were acquired sequentially on thymocytes at discrete stages during the first trimester of human fetal development. The 3A1 antigen was present on fetal lymphocytes before lymphoid cell colonization of thymic epithelium, suggesting that passage through the thymus was not required for the expression of the 3A1 antigen by T cell precursors. The appearance of mature T cell antigens, T3 and p80, on thymocytes by 12 wk of gestation implies that the T cell antigen repertoire may be established in the thymus during the first trimester. Thus, a critical period of T cell maturation appears to occur between 7 and 12 wk of human fetal gestation.

SUCCESSFUL BONE MARROW TRANSPLANTATION IN DIGEORGE SYNDROME

DiGeorge syndrome is a congenital immunodeficiency disorder with variable degrees of T cell immunity. Immunologic reconstitution with fetal thymic tissue or hormones has shown varied success, and appropriate tissue is difficult to obtain. A patient with DiGeorge syndrome presented at day 3 with hypocalcemia, seizures, Tetralogy of Fallot, and typical facies. Thymus shadow was absent on X-ray. Parathormone level was low. Evaluation at 26 weeks showed normal lymphocyte count, T cells 16% by E rosette, 1% by pan-T cell monoclonal antibody, B cells 65%, IgG 277 mg/dl, IgM 61 mg/dl, IgA 0, IgE 15 IU/ml, isohemagglutinins present at titer 1:1. Phytohemagglutinin (PHA), antigen and allogeneic stimulation were flat. Factor thymic serique was present at 1/4 (low),thymosin α1 was 700 pg/ml (normal). Because of the profound T cell defect with antibody defect, we performed bone marrow transplant (BMT) with 1.16 × 109 nucleated cells/kg from her HLA,A,B,C matched, DR mismatched, MLC non-reactive brother without conditioning. There was no evidence of GVH disease. PHA stimulated lymphocytes now show XY karyotype and T cells are 43% by E rosette. EB virus stimulated B cells are 29/30 XY, but DR type is unchanged. Immunoglobulins are normal, antibody and in vitro proliferative response to injected antigen are present. This experience indicates that little thymic function is necessary for successful BMT, but does not determine whether there has been central or peripheral reconstitution.

Characterization of the expression of cellular retrovirus genes and oncogenes in feline cells.

Expression of endogenous retrovirus genes and two different cellular oncogenes (c-onc genes) was examined at the transcriptional level in a variety of normal and lymphoma/leukemia tissues of the domestic cat. The two oncogenes, c-myb(related to avian myeloblastosis virus) and c-myc(related to avian myelocytomatosis virus) were selected for their association with the induction of hematopoietic malignancies, when present in the transforming retroviruses. Tissue-specific expression of endogenous feline leukemia virus (FeLV)-related genes was detected in cellular subpopulations of the cat placenta by in situ method of hybridization. Gel blotting analysis of placental poly(A)-selected RNA revealed that the FeLV-related RNA species were primarily subgenomic, representing the env gene region of the endogenous provirus elements. Like the endogenous retrovirus genes, c-myb and c-myc loci of the cat genomic DNA were also transcribed at differential levels in normal tissues of the cat. Dot-blot hybridization analysis showed that the expression of these two oncogenes was linked to growth and development as it varied with the gestational age of the fetus and from fetal to adult tissues. Among the major hematopoietic organs, spleen and bone marrow contained both c-myb and c-myc transcripts, while thymus preferentially expressed the c-myb gene. In contrast to the low level of c-myc expression in fetal thymus tissues, enhanced c-myc expression was detected in all five thymomas tested and also in several other neoplasms including two granulocytic leukemias. The feline c-myb gene was not very active in granulocytic leukemias or in three of the five thymomas. RNA gel blotting analysis of poly(A)-selected RNA of a thymoma and its lymph node metastasis showed identical pattern of c-myc transcripts.

Demonstration of steroid 17α-hydroxylase activity in human fetal kidney, thymus, and spleen

In previous investigations, steroid 21-hydroxylase activity was demonstrated in human fetal kidney, thymus, and spleen tissues. In the present investigation, steroid 17α-hydroxylase activity also was found in microsome-enriched preparations of human fetal kidney, thymus, and spleen tissues.

Human prothymocytes. Membrane properties, differentiation patterns, glucocorticoid sensitivity, and ultrastructural features.

Thymic precursor cells (prothymocytes) comprise a large proportion of the fetal thymic cell population, but are less frequently encountered in the postnatal thymus, where they compose < 1% of the entire population. In the present study we attempted to characterize a number of properties of the prothymocytes obtained from human fetal thymic tissues after depletion of the E-rosette thymocyes on a Ficoll-Hypaque gradient. The prothymocytes are larger than the thymocytes and show a different nuclear chromatin pattern. This subset of cells lacks the E-rosetting and natural-attachment capacities and, unlike thymocytes, does not bind the lectin peanut agglutinin. Human prothymocytes are highly sensitive to the in vitro cytolytic effect of hydrocortisone, whereas the thymocytes are resistant. Long-term in vitro culture of prothymocytes resulted in the expression of thymocyte characteristics together with a burst of mitotic activity. Results of this study indicate that the rate of the prothymocyte proliferation is regulated by the small thymocytes present in the same suspension.

Combination immunotherapy in chronic mucocutaneous candidiasis synergism between transfer factor and fetal thymus tissue

A child with chronic mucocutaneous candidiasis and hypothyroidism was found on immunological evaluation to have significant deficits of cell-mediated immunity. Skin anergy and absent lymphocyte transformation to specific antigens as well as failure to produce a lymphokine (macrophage migration inhibitory factor) by antigen-stimulated lymphocytes were observed. Immune responses to mitogens and allogeneic cells were normal. Two courses of combined transfer-factor therapy and amphotericin B did not result in clinical improvement or changes in immunologic function. A 20-week-gestation fetal thymus was transplanted intraperitoneally. Because of persistent deficits in cell-mediated immune function 3 months after thymus transplantation, transfer-factor therapy was reinstituted. Shortly thereafter, lymphocyte proliferative responses to specific antigens developed for the first time. After a partial clinical relapse, a second fetal thymus, 18-weeks gestation, was transplanted concomitant with vigorous transfer-factor therapy. Without further chemotherapy, delayed skin reactivity became positive with clearing of all candidal skin lesions. The success of the combination immunotherapy which resulted in clinical improvement and immunologic reconstitution may have been achieved by the synergistic interaction of transfer factor with the fetal thymus.

B CELL ONTOGENY IN RABBITS

The mammalian equivalent of the Bursa of Fabricius, the organ responsible for B cell maturation in avian species, has not been identified despite anatomic and ablative studies which suggest that the gut-associated lymphoid tissues (GALT) subserve this function. By analogy to the Bursa, the mammalian organ directing B cell ontogeny should be the site where IgM-bearing cells (B cells) are first identifiable. In this study, fluorescein-tagged heavy chain specific antirabbit IgM is used to localize initial sites of B cell appearance in rabbit fetal and neonatal lymphoid tissues. IgM-bearing cells are found 2 days before birth in the thymus and 1 day before birth in GALT. Immunoglobulin-bearing cells in spleen, lymph node, and bone marrow are undetectable until after birth. B cells bearing the IgM marked precede the appearance of IgG-bearing cells by 1 to 4 days in all instances. Intraperitoneal implantation of Millipore chambers containing immature fetal thymic tissue into neonatal hosts reveals that in situ development of IgM cells takes place independent of host cell traffic. The results suggest that B cell ontogeny in mammals is more complex than in avian species and demonstrates probable involvement of the thymus in the maturational process.

Blood: Fetal Thymic Transplant in Patients With Hodgkin's Disease

Abstract Five patients with Hodgkin’s disease were treated by transplantation of fetal thymic tissue. Clinical and immunologic studies, carried out for over 5 mo thereafter, revealed a prompt improvement in previously defective cellular immune functions, including a significant rise of absolute lymphocytes in the peripheral blood and a normalization of tuberculin skin sensitivity and of the response of peripheral blood lymphocytes to phytohemagglutinin. It is suggested that fetal thymic transplant into patients with Hodgkin’s disease appears at present the best tool of improving their immunologic deficiency.

THYMIC TRANSPLANTATION IN A CASE OF CHRONIC MUCOCUTANEOUS CANDIDIASIS

A 17-year-old male with chronic mucocutaneous candidiasis, Addison's disease, pernicious anaemia, and malabsorption complicated by multiple B-vitamin deficiencies was transplanted twice with fetal thymic tissue. Previously negative in-vitro blastogenic response to candida became positive within one week of the first transplant and was restored to normal by the second transplant. A positive candida skin test was seen for the first time in 4 years. The patient's clinical condition has improved and there has been clearing of oral candidal lesions and increased ability to mount a vigorous inflammatory response.