CD34+CD38dim cells in the human thymus can differentiate into T, natural killer, and dendritic cells but are distinct from pluripotent stem cells

Recently we reported that the human thymus contains a minute population of CD34+CD38dim cells that do not express the T-cell lineage markers CD2 and CD5. The phenotype of this population resembled that of CD34+CD38dim cells present in fetal liver, umbilical cord blood, and bone marrow known to be highly enriched for pluripotent hematopoietic stem cells. In this report we tested the hypothesis that the CD34+CD38dim thymocytes constitute the most primitive hematopoietic cells in the thymus using a combination of phenotypic and functional analyses. It was found that in contrast to CD34+CD38dim cells from fetal liver and bone marrow, CD34+CD38dim cells from the thymus express high levels of CD45RA and are negative for Thy-1. These data indicate that the CD34+CD38dim thymocytes are distinct from pluripotent stem cells. CD34+CD38dim thymocytes differentiate into T cells when cocultured with mouse fetal thymic organs. In addition, individual cells in this population can differentiate either to natural killer cells in the presence of stem cell factor (SCF), interleukin-7 (IL-7), and IL-2 or to dendritic cells in the presence of SCF, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha(TNFalpha), indicating that CD34+CD38dim thymocytes contain multi-potential hematopoietic progenitors. To establish which CD34+ fetal liver subpopulation contains the cells that migrate to the thymus, we investigated the T-cell-developing potential of CD34+CD38dim and CD34+CD38+ fetal liver cells and found that the capacity of CD34+ fetal liver cells to differentiate into T cells is restricted to those cells that are CD38dim. Collectively, these findings indicate that cells from the CD34+CD38dim fetal liver cell population migrate to the thymus before upregulating CD38 and committing to the T-cell lineage.     

Flt3 ligand plus IL-7 supports the expansion of murine thymic B cell progenitors that can mature intrathymically

Flt3 ligand (flt3L) has potent effects on hemopoietic progenitors, dendritic cells, and B lymphopoiesis. We have investigated the effects of flt3L on intrathymic precursors. The addition of flt3L + IL-7 to lobe submersion cultures of murine fetal thymic lobes resulted in the expansion of an immature population of Thy-1(low), CD44(high), HSA(high) cells. This population contained cells with precursor activity, as determined by their capacity to repopulate deoxyguanosine-treated fetal thymic lobes. Upon reentry to the thymic lobe, flt3L + IL-7-cultured Thy-1(low), CD44(high), HSA(high) cells underwent expansion and differentiation into B cells. Two weeks after fetal thymic organ culture following thymic lobe reconstitution, intrathymic cells were Thy-1-, B220+, and a subset was sIgM+. The intrathymic B cells shared features of adult thymic B cells, including CD5 expression and proliferative responses to IL-4 + IL-5 + CD40 ligand, but not to LPS or soluble anti-IgM. Ig production was noted upon stimulation with IL-4 + IL-5 + LPS and IL-4 + IL-5 + CD40 ligand. In conclusion, we have demonstrated that flt3L + IL-7 supports the expansion of a subset of progenitors present in the fetal thymus. The cultured progenitors can repopulate a fetal thymic lobe and develop into mature functional B cells, demonstrating that the fetal thymus is able to support B cell as well as T cell development.     

Circadian rhythm in the hypothermic response to serotonin1A receptor agonist 8-OH-DPAT in rats

The gene encoding the CD2 mouse cell surface antigen was retrovirally transduced into cord blood CD34+ cells. On infection by culture at the contact of retrovirus-packaging cells, the mCD2 marker was expressed by 30-40% CD34+ cells, that included the most primitive stem cell-enriched Thy-1+ and CD38- subsets. Accordingly, sorted cord blood CD34+Thy-1+ cells could be directly infected in the same conditions. mCD2- transgenic cord blood CD34+ cells were then used to reconstitute human fetal thymus implanted in SCID mice. Five to 8 weeks later, the mCD2 antigen was detected on approximately 10% of the human thymocytes repopulating the thymus grafts and the transgene genome was detected in graft cell DNA by Southern blot. These results demonstrate efficient gene transfer into primitive cord blood hematopoietic cells endowed with lymphoid potential and suggest gene therapy schemes in neonates suffering inherited or acquired-such as HIV infection-disorders of the T-cell lineage.     

Comparisons of endothelial cell G- and F-actin distribution in situ and in vitro

Precursor of T lymphocytes undergo proliferation and maturation under the influence of the thymic microenvironment. In our study, we have attempted to determine the distribution of human postnatal thymocytes in division according to their stage of differentiation. Our data show that about 11.5% of all thymic cells are in S/G2/M phases, and that a subset of the cortical and precortical subpopulations contains most of the dividing cells. Rate of cell division is maintained at high levels from the prethymocyte precursor along the successive stages of differentiation represented by CD1+CD3-CD4-CD8- and CD1+CD3-CD4+CD8- cells. The percentage of dividing cells is maximal in an intermediate subset of CD1+CD3-CD4+CD8-CD45RO+ cells defined by the distinct expression of class I HLAdim/high molecules, which could contain cells in transit from prethymocytes to double-positive cortical cells. The CD3- fraction of the double-positive cortical cells contains most of the dividing thymocytes, although the rate of division within this subset is much less than that of the precursor CD1+CD3-CD4+CD8- cells. In a linear scheme of differentiation, cell division stops at or near the point of initiation of CD3 expression. These results suggest that in human thymus cell expansion takes place before the initiation of the positive selection process. According to this view the stringency of the selection process would require the previous generation of a large number of precursors to permit the production of sufficient numbers of mature T cells.     

Bone marrow-derived dendritic epidermal T cells express T cell receptor-alpha beta/CD3 and CD8. Evidence for their extrathymic maturation

The majority of murine Thy-1+ dendritic epidermal T cells (DETC) express virtually identical TCR encoded by V gamma 5 and V delta 1 genes and are derived from early fetal thymic precursors. However, not consistent with this notion is an early finding that DETC arise continuously from bone marrow (BM) precursors by a thymus-independent mechanism. To address this issue, donor-type DETC were characterized in lethally irradiated mice that were reconstituted by Thy-1-disparate BM cells with or without a thymus. The BM-derived DETC, unlike their normal TCR-gamma delta counterparts, were found to express the TCR-alpha beta/CD3 complex and CD8, and their migration to the epidermis dermis occurred independently of the thymus. The numbers of the BM-derived DETC increased with time and reached a plateau 6 mo after BM transfer, at which time the TCR-alpha beta/CD3 complex was expressed on a small fraction of the DETC in athymic BM chimeras. Although no further increase in the number was observed at later times, at 1 yr after transfer most of the BM-derived DETC came to express the TCR-alpha beta/CD3 complex in the absence of thymic influence. By contrast, most of BM-derived T cells in other lymphoid organs from athymic BM chimeras still failed to express the TCR-alpha beta/CD3 complex even at 1 yr after transfer. These results suggest that extrathymic differentiation of BM-derived DETC could occur with the epidermal microenvironment.     

Development of a method of thymocyte differentiation of bone marrow-enriched CD34+CD38- cells in postnatal allogeneic cultured thymic epithelia to evaluate immunodeficiency disorders

An in vitro model of CD34+CD38- stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC-CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple-positive CD3+CD4+CD8+, double-positive CD4+CD8+, and mature single-positive CD4+ and CD8+ T cells, which were TCR alpha beta+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single-positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow-derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC-CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple-negative and CD44+CD25-CD3-thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.     

Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line

In vitro exposure of murine hematopoietic stem cells (HSCs) to cell cycle-inducing cytokines has been shown to result in a defect in the ability of these cells to engraft. We used a porcine microvascular endothelial cell (PMVEC) line in conjunction with exogenous interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) to expand human HSCs that express the CD34 and Thy-1 antigens but lack lineage-associated markers (CD34+Thy-1+Lin- cells). Ex vivo expansion of hematopoietic cells was evaluated in comparison to stromal cell-free, cytokine-supplemented cultures. Cells expressing the CD34+Thy-1+Lin- phenotype were detectable in both culture systems for up to 3 weeks. These cells were reisolated from the cultures and their ability to engraft human fetal bones implanted into SCID mice (SCID-hu bone) was tested. HSCs expanded in PMVEC coculture were consistently capable of competitive marrow repopulation with multilineage (CD19+ B lymphoid, CD33+ myeloid, and CD34+ cells) progeny present 8 weeks postengraftment. In contrast, grafts composed of cells expanded in stroma-free cultures did not lead to multilineage SCID-hu bone repopulation. Proliferation analysis revealed that by 1 week of culture more than 80% of the cells in the PMVEC cocultures expressing the primitive CD34+CD38- phenotype had undergone cell division. Fewer than 1% of the cells that proliferated in the absence of stromal cells remained CD34+CD38-. These data suggest that the proliferation of HSCs in the presence of IL-3, IL-6, GM-CSF, and SCF without stromal cell support may result in impairment of engraftment capacity, which may be overcome by coculture with PMVECs.     

Defective T cells from gld mice play a pivotal role in development of Thy-1.2+B220+ cells and autoimmunity

The gld mouse represents a fascinating animal model of autoimmune disease, which is characterized by massive development of Thy-1.2+B220+ CD4-CD8- cells. These cells thus have double positive markers for T and B cells, but are double negative for CD4 and CD8 markers and are thus designated DN cells in the present context. An additional important feature in gld mice is a defect in expression of Fas ligand. To investigate the regulatory role of bone marrow-derived cells for the development of these DN cells and of gld autoimmunity, we constructed chimeric mice transplanted with fetal liver cells or fetal thymus from gld mice into nonirradiated severe combined immunodeficient (SCID) mice. These chimeric mice regenerated, developed both these DN cells and the gld autoimmune syndrome and also generalized lymphoproliferative disorders. However, when fetal liver cells from both gld and non-gld mice (C57BL/10 Thy-1.1 mice) were co-transplanted into SCID mice, the development of DN cells was apparently inhibited. Further, this inhibition was also seen in SCID mice that had been grafted with both gld and non-gld fetal thymus revealing the pivotal role played by T cells in development of DN cells. When B cells purified from non-gld (C3H+/+) mice were transplanted into SCID mice grafted with gld fetal thymus, the development of DN cells was not inhibited. Taken together, these findings indicate that T cells from non-gld mice inhibit the expression of gld features, e.g., lymphoproliferation, immune-based nephritic disease, and autoantibody production. These findings also suggest that the Fas ligand is selectively expressed on T cells.     

CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes

Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.     

HCA, an immunoglobulin-like adhesion molecule present on the earliest human hematopoietic precursor cells, is also expressed by stromal cells in blood-forming tissues

We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38(-/lo), Thy-1(+), rho123(lo), CD34(+) hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1(+) progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34(+) CD38(-) hematopoietic precursors, and CD3(+) CD6(+) peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34(+) rho123(med/lo) progenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.     

Influence of detergents on the function of cloned potassium channels

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.     

Early intrathymic precursor cells acquire a CD4(low) phenotype

C4Dlow cells are a population of lymphoid lineage-restricted progenitor cells representing the earliest precursors present in the adult thymus. Paradoxically, thymic progenitors with a similar phenotype in fetal mice and adult RAG-2-deficient (RAG-2-/-) mice lack this characteristic low-level expression of CD4. We now show that radiation-induced differentiation of CD4+ CD8+ double positive thymocytes in RAG-2-/- mice results in the appearance of low levels of CD4 on thymocytes that are phenotypically identical to C4Dlow progenitor cells present in the normal adult thymus. This suggests that CD4 surface expression can be passively transferred from double positive cells to early progenitor thymocytes. Analysis of mixed bone marrow chimeras, reconstituted with hematopoietic stem cells from both CD4-/- (CD45.2) and CD4wt (CD45.1) congenic mice, revealed a CD4low phenotype on cells derived from CD4-/- bone marrow cells. Furthermore, these CD4-/- -derived "C4Dlow" progenitors were capable of reconstituting lymphocyte-depleted fetal thymi, with all thymocytes displaying a CD4-/- phenotype. This directly demonstrates that genetically CD4-deficient thymic progenitor cells can passively acquire a C4Dlow phenotype. Moreover, CD4 expression on C4Dlow progenitor thymocytes is sensitive to mild acid treatment, indicating that CD4 may not exist as an integral cell surface molecule on this thymocyte population. Our findings demonstrate that low-level CD4 surface expression can be passively acquired by intrathymic progenitor cells from the surrounding thymic microenvironment, suggesting that other cell surface molecules expressed at low levels may also result from an acquired phenotype.     

High-level expression of a novel epitope of CD59 identifies a subset of CD34+ bone marrow cells highly enriched for pluripotent stem cells

To further define the hierarchy of human hematopoietic progenitor cells, we have attempted to identify antibodies to cell-surface molecules expressed on CD34+ progenitor cell subsets. Herein we describe the utility of a new monoclonal antibody, HCC-1, which binds to a novel epitope of CD59 differentially expressed among CD34+ progenitor cells. HCC-1 subdivides the adult marrow CD34+ population into HCC-1high and HCC-1low/- fractions of approximately equal size. Cobblestone area-forming cells (CAFC) in long-term bone marrow culture were enriched 10-30-fold in CD34+HCC-1high cells compared with CD34+HCC1-low/- cells and two-fold compared with CD34+ cells. When injected into fetal human bone fragments implanted in SCID mice, the CD34+HCC-1high population showed potent engrafting activity leading to the production of myeloid, lymphoid, and erythroid elements, as well as the retention of progenitor cell phenotype. These studies demonstrate that the CD34+HCC-1high population contains primitive pluripotent hematopoietic stem cells. No hematopoietic engrafting activity was detected in the CD34+HCC-1low/- population. Consistent with this finding, simultaneous five-color flow cytometric analysis revealed that HCC-1high cells include virtually all CD34+Thy-1+Lin- cells, a cell population previously characterized as highly enriched for primitive pluripotent hematopoietic stem cells. The ability of CD34+ cells divided into subsets by HCC-1 to produce T cells was assessed by transplantation of sorted cells into human fetal thymus implanted into SCID mice. A higher frequency of thymus-engrafting activity was observed in the CD34+HCC-1high than in the CD34+HCC-1low/- population. Consistent with the limited ability to engraft in the SCID-hu thymus model, the CD34+HCC-1low/- population was shown to contain a low frequency of CD34+CD10+ lymphoid progenitor cells. We conclude that the HCC-1 epitope is expressed at high levels on a subset of CD34+ cells that contain virtually all primitive pluripotent hematopoietic stem cells and that the population of CD59 molecules expressed on CD34+ cells is not homogeneous.     

The role of vitamin E in T-cell differentiation and the decrease of cellular immunity with aging

Spontaneously hypertensive rats (SHR) as a model for aging were used in this experiment and fed a regular (50 IU/Kg diet) or high vitamin E (500 IU/Kg diet) diet for 6 weeks. At 12 weeks old, they were killed and assayed. Although proliferation of thymic lymphocytes was significantly decreased in SHR fed the regular diet compared to Wistar Kyoto rats (WKY) fed the same diet, high vitamin E diet enhanced proliferation of thymic lymphocytes in SHR to almost the levels in WKY fed the regular diet. In addition, the expressions of both CD4 and CD8 antigens on CD+CD8+ T cells, immature T cells existing in thymic cortex, were also decreased in SHR, and significantly improved by high vitamin E diet. These results suggest that high vitamin E diet enhances thymic lymphocyte proliferation through increased T-cell differentiation in thymus. Then, the effect of vitamin E on T-cell differentiation in thymus was investigated by using male Fisher rats. Rats were divided into three groups; vitamin E-free, regular and high vitamin E groups and fed a diet containing various levels of vitamin E (0, 50 and 500 IU/Kg diet) for 7 weeks. Although the percentages of CD4+CD8- and CD4-CD8+ T cells in thymocytes were significantly greater in the high vitamin E group, the percentage of CD4+CD8- T cells inversely decreased in the vitamin E-free group compared to the regular group. We have tried to investigate the mechanism of the increased T-cell differentiation in thymus of rats fed the high vitamin E diet through cytokine production, and thymic epithelial cell (TEC) and macrophage functions. We have found that vitamin E enhances T-cell differentiation through the increase of not macrophage but TEC function in thymus, which is associated with the increased binding capacity of TEC to immature T cells via increased expression of adhesion molecule, ICAM-1. These results suggest that vitamin E is a potent nutrient for promoting health in the aged via the improvement of cellular immunity decreased with aging.     

Cardiac risk in surgery. A review and guidelines for risk evaluation and reduction of cardiac risk in general surgery

We report herein the phenotypic and functional analysis of human bone marrow and thymus derived early T cells. Commitment to T cell lineage is acquired during CD7 antigen expression by CD34+ precursors in human bone marrow and before thymus colonization. Early thymocytes show similar phenotypic characteristics as bone marrow T cells. They rapidly acquire CD4 before the dual expression of CD4 and CD8. Their expansion and differentiation is regulated by two major factors: thymic stroma and cytokines produced by these stroma cells or by thymocytes themselves. Among cytokines, IL1 and sCD23 produced by thymic epithelial cells support in vitro early T cell development.     

Intracellular immunization of rhesus CD34+ hematopoietic progenitor cells with a hairpin ribozyme protects T cells and macrophages from simian immunodeficiency virus infection

Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.