The myelin basic protein-specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential Vβ8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment

In the Lewis rat, myelin basic protein (MBP)-specific, encephalitogenic T cells preferentially recognize sequence 68–88, and use the Vβ8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP-specific T cell repertoire, we reconstituted severe-combined immunodeficient (SCID) mice with fetal (embryonic day 15–16) Lewis rat lymphoid tissue, and then isolated MBP-specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP-immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B¹), and none by I-A^d of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68–88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP-specific T cell clones used a seemingly random repertoire of Vβ genes without a bias for Vβ8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of Vβ8.2 was restored. The migration of liver-derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild-type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68–88 by MBP-specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.     

A model for personalized in vivo analysis of human immune responsiveness

Studies of human immune diseases are generally limited to the analysis of peripheral blood lymphocytes of heterogeneous patient populations. Improved models are needed to allow analysis of fundamental immunologic abnormalities predisposing to disease and in which to assess immunotherapies. Immunodeficient mice receiving human fetal thymus grafts and fetal CD34(+) cells intravenously produce robust human immune systems, allowing analysis of human T cell development and function. However, to use humanized mice to study human immune-mediated disorders, immune systems must be generated from adult hematopoietic cells. Here, we demonstrated robust immune reconstitution in mice with hematopoietic stem cells (HSCs) aspirated from bone marrow of adults with type 1 diabetes (T1D) and healthy control volunteers. In these humanized mice, cryopreservation of human leukocyte antigen allele-matched fetal thymic tissue prevented allogeneic adult HSC rejection. Newly generated T cells, which included regulatory T cells (T(regs)), were functional and self-tolerant and had a diverse repertoire. The immune recognition of these mice mimicked that of the adult CD34(+) cell donor, but the T cell phenotypes were more predominantly "na?ve" than those of the adult donors. HSCs from T1D and control donors generated similar numbers of natural T(regs) intrathymically; however, peripheral T cells from T1D subjects showed increased proportions of activated or memory cells compared to controls, suggesting possible HSC-intrinsic differences in T cell homeostasis that might underlie immune pathology in T1D. This "personalized immune" mouse provides a new model for individualized analysis of human immune responses that may provide new insights into not only T1D but also other forms of immune function and dysfunction as well.     

An embryonic source of Ly1 but not conventional B cells.

Ly1+ B cells differ from conventional B cells with respect to their anatomical localization, cell surface marker expression, and antibody repertoire suggesting that they may constitute a functionally distinct subset of B cells. To determine whether Ly1+ B cells also have a developmentally distinct site of origin we grafted various fetal primordia into adult severe combined immunodeficient (scid) mice and analyzed their potential to give rise to T and B cells. We demonstrated that fetal omentum, but not spleen or thymus grafts, reconstituted exclusively Ly1 B cells (including the Ly1 sister population) as well as a population of IgM and IgA producing plasma cells in the spleen and gut, respectively. Although thymus grafts regularly reconstituted T cells, thymus plus fetal omentum cografts gave rise to a population of Ly1+ B cells as well as T cells which were also derived from omentum. However, in neither omentum nor omentum plus thymus cografts were conventional B cells detected. These results provide the first evidence that Ly1 B cells but not conventional B cells are generated from the fetal omentum.     

Achievement of cellular immunity and discordant xenogeneic tolerance in mice by porcine thymus grafts

Specific cellular immune tolerance may be essential for successful xenotransplantation in humans. Thymectomized (ATX), T and NK ceil-depleted immunocompetent mice grafted with xenogeneic fetal pig thymic and liver tissue (FP THY/LIV) result in efficient mouse thymopoiesis and peripheral repopulation of functional mouse CD4^ T cell.Very importantly, the reconstituted mouse T cells are specifically tolerant to pig donor antigens. Studies demonstrated that porcine MHCs mediated positive and negative selection of mouse thymocytes in FP THY grafts, whereas mouse MHCs were involved in negative selection in grafts. Therefore, T cell tolerance to xenogeneic donor antigens could be induced by grafting donor thymus tissue. Xenogeneic thymic replacement might have a potential role in the reconstitution of cellular immunity in patients with AIDS or other immunodeficiencies caused bv thvmus dvsfunction.     

Toward development and production of human T cells in swine for potential use in adoptive T cell immunotherapy

Immunotherapy and vaccination for cancer or infection are generally approached by administration of antigen or stimulation of antigen-presenting cells or both. These measures may fail if the treated individual lacks T cells specific for the immunogen(s). We tested another strategy-the generation of new T cells from hematopoietic stem cells that might be used for adoptive immunotherapy. To test this concept, we introduced T cell-depleted human bone marrow cells into fetal swine and tested the swine for human T cells at various times after birth. Human T cells were detected in the thymus and blood of the treated swine. These cells were generated de novo as they contained human T cell receptor excision circles not present in the T cell-depleted bone marrow. The human T cells were highly diverse and included novel specificities capable of responding to antigen presented by human antigen-presenting cells. Our findings constitute a first step in a new promising approach to immunotherapy in which tumor- or virus-specific T cell clones lacking in an individual might be generated in a surrogate host from hematopoietic stem cells of the individual to be treated.     

Role of DOCK2 and DOCK180 in fetal thymus colonization

Fetal thymus colonization is initiated before the vascularization of the thymus primordium. This prevascular colonization of the fetal thymus by T‐lymphoid progenitor cells is guided by the coordination of CCR7‐ and CCR9‐mediated chemokine signals. However, the intracellular signals that mediate the prevascular migration of T‐lymphoid progenitor cells to the fetal thymus are unknown. Here we show that T‐lymphoid progenitor cells in fetal mice express two closely related CDM family molecules, DOCK2 and DOCK180. We found that the prevascular fetal thymus accumulation in vivo was significantly reduced in mice doubly deficient for DOCK2 and DOCK180 but not in mice deficient for either DOCK2 or DOCK180. Immature T‐lymphoid cells from mice doubly deficient for DOCK2 and DOCK180 were defective in their in vitro migration towards fetal thymus lobes. The T‐lymphoid progenitor cells generated in mice lacking DOCK2 and DOCK180 were capable of T‐cell development after their transfer into a fetal thymus environment, and the impaired fetal thymus colonization in mice deficient for DOCK2 and DOCK180 was not as severe as that in mice doubly deficient for CCR7 and CCR9. These results indicate that the combination of DOCK2 and DOCK180 plays a significant but not essential role in prevascular fetal thymus colonization.     

Human embryonic stem cells are prone to generate primitive, undifferentiated tumors in engrafted human fetal tissues in severe combined immunodeficient mice

Embryonic stem (ES) cells are uniquely endowed with the capacity of self-renewal and the potential to give rise to all possible cell types. Their differentiation potential has raised hope that these cells could be used as a renewable source for cell transplantation in severe degenerative diseases. However, progress in this direction is still limited. Using two human embryonic stem (ES) cell lines, H1 and HSF-6, and three types of human fetal tissues--thymus, lung and pancreas-we investigated whether engrafted human fetal tissues in severe combined immunodeficient mice (SCID) mice could provide a physiologically-relevant microenvironment for human ES cells to differentiate into mature cells of corresponding tissues. Surprisingly, we observed an aggressive growth of tumors when human ES cells were injected into engrafted human fetal tissues in SCID mice. These tumors displayed histological characteristics of primitive, undifferentiated tumors rather than differentiated teratomas. Additionally, these tumors exhibited a normal karyotype and did not express the characteristic antigens of embryonic carcinomas. We also found differences among human fetal tissue types in their abilities to support the growth of these primitive tumors. Our study supports and validates a previously reported phenomenon in mouse that tumorigenesis of ES cells is host dependent. Our study is also the first report to demonstrate that human ES cells are prone to generate primitive, undifferentiated tumors in human fetal tissue grafts in SCID mice and raises a potential safety concern for using human ES cell-derived cell products in humans.     

Murine T cells with invariant gamma delta antigen receptors: origin, repertoire, and specificity.

T cells bearing invariant gamma delta T cell antigen receptors localize to distinct epithelial sites in the adult mouse. These gamma delta T cells differ from the lymphoid alpha beta and gamma delta T cells in several ways. The epithelial gamma delta T cells appear to be the product of the earliest waves of TCR expressing cells in the fetal thymus. Additionally, the rearranged TCR junctions exhibit a distinct lack of diversity, possibly a result of the fetal origin and a specialized selection process. The use of a single invariant TCR and strict tissue localization suggest that these gamma delta T cells may provide a specialized function in the epithelial tissues distinct from that of the circulating alpha beta and gamma delta T cells.     

Mice Engrafted with Human Fetal Thymic Tissue and Hematopoietic Stem Cells Develop Pathology Resembling Chronic Graft-versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a significant roadblock to long-term hematopoietic stem cell (HSC) transplantation success. Effective treatments for cGVHD have been difficult to develop, in part because of a paucity of animal models that recapitulate the multiorgan pathologies observed in clinical cGVHD. Here we present an analysis of the pathology that occurs in immunodeficient mice engrafted with human fetal HSCs and implanted with fragments of human fetal thymus and liver. Starting at time points generally later than 100 days post-transplantation, the mice developed signs of illness, including multiorgan cellular infiltrates containing human T cells, B cells, and macrophages; fibrosis in sites such as lungs and liver; and thickened skin with alopecia. Experimental manipulations that delayed or reduced the efficiency of the HSC engraftment did not affect the timing or progression of disease manifestations, suggesting that pathology in this model is driven more by factors associated with the engrafted human thymic organoid. Disease progression was typically accompanied by extensive fibrosis and degradation of the thymic organoid, and there was an inverse correlation of disease severity with the frequency of FoxP3⁺ thymocytes. Hence, the human thymic tissue may contribute T cells with pathogenic potential, but the generation of regulatory T cells in the thymic organoid may help to control these cells before pathology resembling cGVHD eventually develops. This model thus provides a new system to investigate disease pathophysiology relating to human thymic events and to evaluate treatment strategies to combat multiorgan fibrotic pathology produced by human immune cells.     

T cells generated in the absence of a thoracic thymus fail to establish homeostasis

Cervical thymus mimics the thoracic thymus in supporting T‐cell development and exists in a subset of mice and humans. Importantly, it remains unknown whether the cervical thymus can generate T cells that are self‐tolerant in the complete absence of signals from the thoracic thymus. Using a fetal liver reconstitution model in thoracic thymectomized RAG^?/? mice, we found that T cells could be generated without contribution from the thoracic thymus. However, these mice had decreased T cells, increased proportions of effector memory T cells and Treg phenotype cells, increased serum IgG1/2b, and increased frequency of T cells expressing IFN‐γ, IL‐17 or IL‐10. Half of the mice that received a thoracic thymectomy and fetal liver cells, unlike sham surgery controls, developed substantial morbidity with age. Disease was associated with lymphopenia‐driven activation rather than inherent defects in the cervical thymus, as both thoracic and cervical thymocytes could generate disease in lymphopenic recipients. Administration of the homeostatic cytokine IL‐7 caused a rapid, transient increase in T‐cell numbers and reduced the time to disease onset. Together the data suggests that the cervical thymus can function in the complete absence of the thoracic thymus; however, the T cells generated do not establish homeostasis.     

HIV-1 pathogenesis and therapeutic intervention in the SCID-hu Thy/Liv mouse: a model for primary HIV-1 infection in the human thymus

The SCID-hu Thy/Liv mouse is a model for the analysis of human thymopoiesis. It has been constructed by engrafting fragments of human fetal liver and thymus into the immunodeficient C.B-17 scid/scid (SCID) mouse. The resulting ‘Thy/Liv’ organ promotes long-term differentiation of human T cells. Given the apparently normal physiology of the SCID-hu Thy/Liv organ, it has been used to explore the pathophysiologic mechanisms of HIV-1 infection in vivo, and to test therapeutic modalities such as anti-HIV-1 drugs and haematopoietic stem cell (HSC)-based gene therapy. In this review, I will summarise what we have learned from the SCID-hu Thy/Liv model, with a focus on recent findings in HIV-1 replication and therapy. Unique HIV-1 determinants have been identified which are required for replication in the Thy/Liv organ but not for replication in PBMC or in T cell lines in vitro. The mechanism of HIV-1 induced thymus depletion is not clear. It is correlated with high levels of HIV-1 replication. Both direct and indirect mechanisms may be involved. In addition to preclinical evaluation of anti- HIV-1 drugs, the SCID-hu Thy/Liv mouse has also been successfully used to test the feasibility of HSC-based gene therapy. A number of improved SCID-hu models have been constructed to meet different requirements. Using these SCID-hu Thy/Liv models, current/future efforts will provide insightful information for understanding pathogenesis and designing therapeutic interventions against HIV-1 infection in humans, especially in paediatric patients. © 1997 John Wiley & Sons, Ltd.     

The in vivo development of human T cells from CD34(+) cells in the murine thymic environment

There is increasing evidence that human hematopoietic stem cells can develop into lymphocytes expressing T cell surface markers in the organ culture of murine embryonic thymic lobes. If human T cells with functional maturity are inducible from human stem cells in the mouse, it may be a useful model to investigate human T cell development and the human immune response in vivo. To approach this, we produced a hybrid cluster of murine fetal thymic epithelial cells and human cord blood-derived CD34(+) cells (hu/m cluster) using reaggregate thymic organ culture, and subsequently implanted it under the kidney capsule of NOD/SCID mice. The implanted hu/m cluster grew in volume under the kidney capsule and contained increased numbers of CD4(+)CD8(+)cells as well as CD4 or CD8 single-positive cells with low CD1a expression. These lymphocytes were also shown to possess activity for producing IL-2 and IL-4. Characteristics similar to human T cells also developed in the thymus of newly established mice lacking NK activity from NOD/SCID mice. These results indicate that functionally mature T cells can develop in vivo from human hematopoietic progenitors in the murine environment composed of thymic epithelial cells.     

The human thymus during aging

The human thymusis required for establishment of a normal T cell repertoire in fetal development, as children born without a thymus (DiGeorge Syndrome) lack thymus-derived (T) and T cell immunity. While the function of the thymus in children for production of new T cells is clear, it has not been obvious that the adult thymus can produce significant numbers of new T cells. Until recently, no assays were available to directly evaluate postnatal thymic function. This paper reviews work on human thymic aging at Duke University School of Medicine and discusses the relevance of this work to devising new strategies for T cell immune reconstitution in man.     

The human thymus

The human thymus is a lymphoepithelial organ in which T cells develop during fetal life. After maturation and selection in the fetal thymic microenvironment, T cells emigrate to peripheral lymphoid tissues such as the spleen, gut, and lymph nodes, and establish the peripheral T cell repertoire. Although the thymus has enormous regenerative capacity during fetal development, the regenerative capacity of the human postnatal thymus decreases over time. With the advent of intensive chemotherapy regimens for a variety of cancer syndromes, and the discovery that infection with the Human Immunodeficiency Virus (HIV) leads to severe loss of CD4⁺ T cells, has come the need to understand the role of the human thymus in reconstitution of the immune system in adults. During a recent study of the thymus in HIV infection, we observed many CD8⁺ T cells in AIDS thymuses that had markers consistent with those of mature effector cytotoxic T cells usually found in peripheral immune tissues, and noted these CD8⁺ effector T cells were predominately located in a thymic zone termed the thymic perivascular space. This article reviews our own work on the thymus in HIV-1 infection, and discusses the work of others that, taken together, suggest that the thymus contains peripheral immune cell components not only in the setting of HIV infection, but also in myasthenia gravis, as well as throughout normal life during the process of thymus involution. Thus, the human thymus can be thought of as a chimeric organ comprised of both central and peripheral lymphoid tissues. These observations have led us to postulate that the thymic epithelial atrophy and decrease in thymopoiesis that occurs in myasthenia gravis, HIV-1 infection, and thymic involution may in part derive from cytokines or other factors produced by peripheral immune cells within the thymic perivascular space. An erratum to this article is available at .     

Interleukin-2 in the ontogeny of human lymphoid tissues

Cells from different fetal and adult lymphoid organs were tested for the capacity to (a) react to the T-cell growth factor interleukin-2 (IL-2) and (b) to produce IL-2 under appropriate conditions. IL-2 was determined as growth promoting activity for mouse T blasts. Optimal conditions for IL-2 production were: 5 × 10⁶ cells/ml; 4–6 µg mitogen, 24 h incubation time. Concanavalin A was preferable for fetal thymus, whereas phytohemagglutinin was the appropriate mitogen for lymphocytes from blood and lymph node. Fetal and adult spleen cells produced equal activities of IL-2 irrespective of the mitogen used. Cells from thymus and spleen of 17–21-week-old fetuses produced IL-2 activities like adult cells. Fetal liver cells from the same fetuses produced no IL-2. A comparison of IL-2 activities released from cells of 26 donors showed that within individual variations there was no decrease of the capacity to produce IL-2 with old age. An activity present in culture media of mitogen treated lymphocytes which increased thymidine uptake in fetal liver cells could be distinguished from IL-2 by its different molecular weight and by a heat treatment which abolished IL-2 but not fetal liver cell growth promoting activity. This latter activity is discussed to be colony-stimulating activity. It is concluded that IL-2 is produced by and reacts with T cells only after they have reached or passed the thymus.     

Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model

Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs.     

Noggin enhances dopamine neuron production from human embryonic stem cells and improves behavioral outcome after transplantation into Parkinsonian rats

Symptoms of Parkinson's disease have been improved by transplantation of fetal dopamine neurons recovered from aborted fetal tissue, but tissue recovery is difficult. Human embryonic stem cells may provide unlimited cells for transplantation if they can be converted to dopamine neurons and survive transplantation into brain. We have found that the bone morphogenic protein antagonist Noggin increased the number of dopamine neurons generated in vitro from human and mouse embryonic stem cells differentiated on mouse PA6 stromal cells. Noggin effects were seen with either early (for mouse, days 0-7, and for human, days 0-9) or continuous treatment. After transplant into cyclosporin-immunosuppressed rats, human dopamine neurons improved apomorphine circling in direct relation to the number of surviving dopamine neurons, which was fivefold greater after Noggin treatment than with control human embryonic stem cell transplants differentiated only on PA6 cells. We conclude that Noggin promotes dopamine neuron differentiation and survival from human and mouse embryonic stem cells. Disclosure of potential conflicts of interest is found at the end of this article.     

In vitro development of B cells and macrophages from early mouse fetal thymocytes

The developmental potentialities of early mouse fetal thymocytes were analyzed by in vitro culturing cell suspensions obtained from 12–15-gestational day thymus in contact with a bone marrow stroma clonal cell line that supports pre-B and myeloid cell differentiation. B cell and macrophage development from fetal thymocytes was observed at all fetal stages tested, but limiting dilution analysis revealed that the frequency of cells forming colonies on bone marrow stroma is the highest in the fetal thymus at day 12, then dramatically decreases until day 15. These observations suggest that the thymus rudiment is seeded by multipotential precursor cells which are not immediately committed to T cell development in the thymic cellular environment.     

Rat thymic epithelium positively selects mouse T cells with specificity for rat MHC class II antigens but fails to induce detectable tolerance in the mouse T cells to the rat MHC antigens.

BALB/c (H-2d) nude mice were grafted with allogeneic AKR/J (H-2k) or xenogeneic (ACI-N rat, RT1av1) fetal thymuses which were depleted of hemopoietic cells by incubating with 2'-deoxyguanosine (2'dGuo) in vitro prior to grafting. The nylon-wool-passed LN T cells from nude mice grafted with 2'dGuo-treated AKR/J thymus showed a poor proliferative response to B10BR (H-2k) stimulator cells, confirming that mouse thymic epithelium has the capacity to induce tolerance against the mouse MHC antigens on the thymic epithelium. On the other hand, the nylon-wool-passed LN T cells from nude mice grafted with untreated or 2'dGuo-treated ACI/N rat thymus showed significant proliferative responses to ACI/N, which can be blocked by anti-rat MHC class II mAb, whereas the nylon-wool-passed LN T cells from nude mice grafted with syngeneic thymus hardly responded to the xenogeneic stimulator cells. These results suggest that rat thymic stromal cells including thymic epithelium can not induce detectable tolerance in mouse T cells to rat MHC antigens; but rat thymic epithelium may positively select mouse T cells with specificity for rat MHC class II antigens, resulting in a mouse T cell repertoire with strong xeno-reactivity.