Induce T-cell Differentiation Research Articles

Plxdc2 Marks Hematopoietic Stem Cells and Th2 Cytokine-Producing Innate Lymphocytes in Adult Bone Marrow

Abstract 1278Hematopoietic stem cells (HSCs) have been highly enriched using combinations of more than 10 surface markers. However the simple method using a few positive markers is preferable to identify HSCs location in tissue section. We performed a stringent comparative gene expression profiling analysis to find genes preferentially expressed in the HSC population, and identified a total of 63 genes that are highly expressed in HSC among various hematopoietic cell population.In order to find HSC-specific marker we focused on genes encoding cell surface protein, and found that plexin domain containing 2 (Plxdc2) is highly expressed in CD34—c-Kit+Sca-1+Lineage−(CD34−KSL) HSC population using Plxdc2::GFP knock-in mice. Only 0.2% of whole bone marrow cells were Plxdc2+, and competitive repopulation assay clearly showed that all HSCs are included in the Plxdc2+ fraction. These results identify Plxdc2 as a new marker of HSCs. Plxdc2+ population contain not only HSCs but uncharacterized c-Kitlow/−Sca-1+Lineage−cells. To further purify HSCs, we investigated the additional positive marker. Throughout the screening of various known HSC-related marker, CD150 was selected. CD150 is already recognized as a positive HSC marker (Kiel, et al. Cell 2005). The Plxdc2+CD150+ fraction represented only 0.1%±0.002% in whole bone marrow, and 6% in c-Kit+Sca-1+Lineage− cells, respectively. To test whether the combination of Plxdc2 and CD150 with or without other markers can highly enrich long-term HSCs, we competitively reconstituted irradiated mice with single Plxdc2+CD150+ cells or single Plxdc2+CD150+c-Kit+Sca-1+Lineage− cells. One out of every 4.6 Plxdc2+CD150+ cells (22%), and one out of 2.2 Plxdc2+CD150+c-Kit+Sca-1+Lineage− cells (44%) engrafted and gave long-term multi-lineage reconstitution. The simple combination of Plxdc2 and CD150 significantly increased HSC purity. In addition, we found robust levels of PLXDC2 transcripts in purified human cord blood CD34+ HSCs.Next, we attempted to characterize the another Plxdc2+ fraction which is c-Kitlow/−Sca-1+Lineage−. Multicolor flowcytometric analysis revealed that Plxdc2+c-Kitlow/−Sca-1+Lineage− cells uniformly express CD45, IL7Rα, Thy-1.2, CD27, T1/ST2 (IL1RL1, a subunit of IL33R) and CD25. These cell surface phenotype indicated that this population is probably of lymphoid lineage. However, culturing Plxdc2+ c-Kit low/−Sca-1+Lineage− cells on OP9-DL1, which supports the development of T-cell progenitors to mature T-cells, did not induce T-cell differentiation. Plxdc2+c-Kitlow/−Sca-1+Lineage−cells also did not differentiate into B cells when co-cultured with OP9 stroma cell line. Furthermore Plxdc2+c-Kitlow/−Sca-1+Lineage− cells produce IL-5 and IL-13 in response to IL-33 or a combination of IL-2 and IL-25. These characteristics resemble that of “natural helper (NH) cells”, a recently identified cell population capable of producing large amounts of Th2 cytokines in fat-associated lymphoid clusters (Moro, et al. Nature 2010). Immunohistochemical staining of bone section to detect HSCs, and functional analyses to clarify why Plxdc2 specifically express in HSCs and bone marrow “NH cells” using Plxdc2-deficient mice are our ongoing tasks. Disclosures:No relevant conflicts of interest to declare.

Further Characterization of T-Cellular Precursors Generated From CD34+ Progenitors by Exposure to Immobilized Notch Ligand Delta-Like 4 In Vitro.

AbstractAbstract 3712Injection of donor derived T-cellular precursors has been proposed as a novel strategy to shorten delayed reconstitution of the T-lymphoid compartment following HSCT. In the past years, several research groups have successfully generated murine and human T-cellular precursors in vitro using Notchligand-based coculture systems such as OP9-DL1 or Tst-DL4. Murine T-cellular precursors generated in vitro, promoted reconstitution of the T-cellular compartment when applied in murine HSCT-models. In consistency, transfer of human T-cellular precursors, generated in vitro in coculture with OP9-DL1 or Tst-DL4 resulted in enhanced thymic repopulation in NOD/SCID/gc−/− mice. Yet, positive effects on peripheral T-cell reconstitution have not been reported. Moreover, clinical application of OP9-DL1 or Tst-DL4 coculture systems is limited, since they consist of murine stromal cells transduced with either DL1 or DL4. It has been described that exposure of CD34+ cells to immobilized DL4 induces T-cell differentiation in vitro and allows expansion human T-cellular precursors even in absence of stromal cell support. However, the hypothesis that DL4 alone can drive hematopoietic progenitors towards a T-cell fate in vitro, requires more evidence. Here, we further characterized the in vitro and in vivo potential of T-cellular precursors generated by single exposure to DL4.We exposed human CD34+ progenitors to immobilized DL4 in the presence of different cytokine combinations implicated in human haematopoiesis. Within 7 days, CD34+CD7+ and CD34−CD7++ T-cellular precursors emerged in the presence of DL4, but not under control conditions. After 7 days the CD34+CD7+ population subsequently declined while the CD34−CD7++ population further expanded. Two distinct progenitor subsets, CD5+ and CD5-, emerged within the CD34−CD7++ population. The CD34−CD7++CD5+ subset partially acquired CD1a, corresponding to a developmental stage between the early thymic progenitor (ETP) and the prethymocyte (pre-T) stage. Conversely to what observed in the OP9-DL1 system, T-cell development did not progress beyond the pre-T-stage. Indeed, we neither observed more advanced stages of T-cell development, such as immature single positive CD4+ cells, nor complete TCR-rearrangements.7-day exposure to immobilized DL4 induced a 90-fold increase of T-precursor frequency in CD34+ progenitors (1/8800 before culture vs. 1/90 after culture) as confirmed by limiting dilution assays on OP9-DL1. All T-cellular precursor activity was restricted to cells expressing CD34, CD7 or both (frequency: 1/9). In particular, elevated T-cellular precursor levels were found in the subsets expressing CD7 (CD34+/CD7+ and CD34−/CD7+), while the T-cellular precursor frequency in the CD34+/CD7− subset was equal to that seen in non-cultured CD34+ progenitors. In consistency the CD34−CD7− population did not contain any detectable T-cellular precursors.After 7 day exposure to DL4, cells phenotypically corresponding to T-cellular precursors were transferred into NOD/SCID/gc−/− mice. Within 2 months following HSCT, cells exposed to DL4 were able to reconstitute the recipients' thymus and partially gave rise to peripheral T-cells. When injecting non-cultured CD34+ progenitors, thymic reconstitution was likewise seen 2 months after HSCT. However, intrathymic T-cell development was less advanced and peripheral T-cells were absent. In contrast, cells cultured in presence of a control peptide did not retain any potential to repopulate the recipients' thymus.Our experiments provide further evidence that exposure DL4 induces early human T-cell development and allows generation of large numbers of T-cellular precursors in vitro. These precursors feature phenotypical and molecular properties corresponding to early precursors found in the human thymus. Furthermore, they have an increased potential to further differentiate into mature T-cells in vitro and when transferred into immunodeficient mice. Our preliminary data suggest, that injection of T-cellular precursors accelerates T-cell reconstitution after HSCT and provides further evidence for the feasibility of this novel strategy of immunotherapy. Disclosures:No relevant conflicts of interest to declare.

D-pinitol inhibits Th1 polarization via the suppression of dendritic cells

d-pinitol has been demonstrated to exert insulin-like and anti-inflammatory effects. However, the effects of the maturation and immunostimulatory functions of dendritic cells (DC) remain to be clearly elucidated. In this study, we have attempted to determine whether d-pinitol regulates surface molecule expression, cytokine production, endocytosis capacity, and underlying signaling pathways in murine bone marrow-derived DC. We also attempted to ascertain whether d-pinitol could influence Th1/Th2 immune response in vivo. The DC used in this study were derived from murine bone marrow cells, and were used as immature or LPS-stimulated mature DC. The DC were then assessed with regard to surface molecule expression, dextran-FITC uptake, cytokine production, capacity to induce T-cell differentiation, and underlying signaling pathways. d-pinitol was shown to significantly inhibit CD80, CD86, MHC class I, and MHC class II expression in the LPS-stimulated mature DC. The DC also evidenced impaired IL-12 expression and IFN-γ production. The d-pinitol-treated DC were found to be highly efficient in regards to Ag capture via mannose receptor-mediated endocytosis. d-pinitol was also demonstrated to inhibit LPS-induced MAPKs activation and NF-κB nuclear translocation. Moreover, the d-pinitol-treated DC manifested impaired induction of Th1 responses, and normal cell-mediated immune responses. These novel findings provide new insight into the immunopharmacological role of d-pinitol in terms of its effects on DC. These findings also broaden current perspectives concerning our understanding of the immunopharmacological functions of d-pinitol, and have ramifications for the development of therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.

Short exposure to Notch ligand Delta-4 is sufficient to induce T-cell differentiation program and to increase the T cell potential of primary human CD34 + cells

The Notch pathway plays a key role in cell fate choices and in T-cell development. The goal of our study was to evaluate whether a short in vitro stimulation of the Notch pathway may alter human progenitor cell behavior. CD34+ cord blood progenitors were exposed for 4 days to either immobilized Notch ligand Delta-4 or in control conditions. Phenotypic and molecular changes induced by the short stimulation were assessed at day 4. Next, long-term alteration of the fate of these progenitors was assessed in culture conditions suitable for B (coculture with MS5 stromal cells) and T (FTOC and OP9 stromal cells expressing Delta-4 systems) cell differentiation. Notch activation was sufficient to trigger immunophenotypic and molecular changes consistent with early T-cell lineage differentiation. Delta-4 induced, in 4 days, CD7+cytCD3epsilon+ cells. This paralleled at the gene-transcription level with de novo expression of several T cell-related transcription factors and TCRgamma rearrangement, while B cell transcripts were simultaneous silenced. As compared to non-Delta-4 primed cells, these early changes translated to long-term alteration of the potential of cells. Delta-4 priming led to an acceleration of T-cell development, including a completion of the TCR rearrangement, when cells were cultured in systems suitable for T-cell development while B-cell development was inhibited. A transient Notch activation is sufficient to promote T-cell differentiation from cord blood CD34+ cells. This system may be a useful tool for the amplification and the quantification of the T potential of CD34+ cells in various disease conditions.

Differential Activation of Notch1 and Notch2 by Delta1 and Jagged1 Determines Hematopoietic Cell Fate.

Spatially restricted in vivo expression of the Notch ligands Delta1 and Jagged1 suggests their differential roles in inducing hematopoietic cell fates, and studies have shown that each induces alternative fates during in vitro culture. We hypothesize that the ligands induce alternative fates via differential activation of Notch1 and/or Notch2. To address this, we assessed fate outcomes of Notch1- and Notch2-deficient murine bone marrow derived lin−Sca-1+c-kit+ Hoescht side population progenitors (LSKSP) after 14-days incubation with either purified Delta1 or Jagged1 in serum containing medium. Ligands consisted of purified Delta1 and Jagged1 extracellular domains fused to HumanIgG1 (Delta1ext-IgG or Jagged1ext-IgG). Equal amounts of ligand, as determined by ELISA, were immobilized to plastic surfaces of culture wells along with fibronectin. Notch deficient cells were generated by infecting either Notch1fl/fl or Notch2fl/fl LSKSP with lentivirus encoding cre recombinase. We show here that both ligands inhibit myeloid differentiation, since after 14 days, LSKSP incubated with either ligand generated multi-log increased numbers of immature progeny with significantly reduced percentages of GR1+ and/or F480+ cells compared to LSKSP incubated with control-IgG. However, only Delta1ext-IgG promotes T-cell progenitor differentiation, since a higher percentage of progeny cultured with Delta1ext-IgG expressed CD25 (37.0+/−0.6%) compared to Jagged1ext-IgG (1.7+/−1.0%; p=0.01). In contrast, Jagged1ext-IgG is less effective at inhibiting myeloid differentiation, since a higher percentage of progeny cultured with Jagged1ext-IgG (2.1+/−0.9%) expressed GR1 and F4/80 compared to Delta1ext-IgG (56.5+/−6.3; p=0.02). Furthermore, Delta1ext-IgG is more effective than Jagged1ext-IgG at inducing Notch activation as measured by increased expression of Notch target Hes1, since we found 3.3-fold more expression of Hes1 mRNA following incubation of LSKSP cells with Delta1ext-IgG compared to Jagged1ext-IgG. We further show that Notch2 is required to prevent myeloid differentiation, since Notch2 deficient LSKSP incubated with either Delta1ext-IgG or Jagged1ext-IgG generated cultures containing fewer numbers of cells and a higher percentage of GR1+ and/or F480+ myeloid progeny (83% with Delta1ext-IgGor 86% with Jagged1ext-IgG) similarly to those generated with control-IgG. Likewise, we found a reduced percentage of immature Sca-1+c-kit+ cells (19.3+/−4.4 or 13.0+/−5.2) than wild-type cells incubating with Delta1ext-IgG or Jagged1ext-IgG (92.2+/−3.0 or 71.0+/−15.0: p=0.004 or p=0.05). We found that Notch1 is required to induce T-cell differentiation, since Notch1 deficient LSKSP incubated with Delta1ext-IgG had a reduced percentage of CD25+ cells compared to wild-type cells (4.4+/−1.9% to 30.3+/− 3.3) even though myeloid differentiation was inhibited. In summary, we show that both Delta1ext-IgG and Jagged1ext-IgG induce signaling via Notch2 to prevent myeloid differentiation, whereas only Delta1ext-IgG induces signaling via Notch1 to promote generation of T-cell progenitors. Our results indicate unique Notch ligands differentially activate Notch1 or Notch2, resulting in alternative cell fate choices and lay a framework for investigating the mechanisms underlying differential activation, including determining the role of Notch modifiers such as Fringe.

Silibinin polarizes Th1/Th2 immune responses through the inhibition of immunostimulatory function of dendritic cells

Silibinin is the primary active compound in silymarin. It has been demonstrated to exert anti-carcinogenic effects and hepato-protective effects. However, the effects of silibinin on the maturation and immunostimulatory activities exhibited by dendritic cells (DCs) remain, for the most part, unknown. In this study, we have attempted to determine whether silibinin can influence surface molecule expression, dextran uptake, cytokine production, capacity to induce T-cell differentiation, and the signaling pathways underlying these phenomena in murine bone marrow-derived DCs. Silibinin was shown to significantly suppress the expression of CD80, CD86, MHC class I, and MHC class II in the DCs, and was also associated with impairments of LPS-induced IL-12 expression in the DCs. Silibinin-treated DCs proved highly efficient with regard to Ag capture via mannose receptor-mediated endocytosis. Silibinin also inhibited the LPS-induced activation of MAPKs and the nuclear translocation of the NF-kappaB p65 subunit. Additionally, silibinin-treated DCs evidenced an impaired induction of Th1 response, and a normal cell-mediated immune response. These findings provide new insight into the immunopharmacological functions of silibinin, especially with regard to their impact on the DCs. These findings expand our current understanding of the immunopharmacological functions of silibinin, and may prove useful in the development of therapeutic adjuvants for acute and chronic DC-associated diseases.

Overexpression of HES-1 is not sufficient to impose T-cell differentiation on human hematopoietic stem cells

AbstractBy retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs), we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here, we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction, human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development, it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary, a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells.

Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro

AbstractThe Notch signaling pathway plays a key role at several stages of T-lymphocyte differentiation. However, it remained unclear whether signals induced by the Notch ligand Delta-like 1 could support full T-cell differentiation from a defined source of human hematopoietic stem cells (HSCs) in vitro. Here, we show that human cord blood–derived HSCs cultured on Delta-like 1–expressing OP9 stromal cells undergo efficient T-cell lineage commitment and sustained T-cell differentiation. A normal stage-specific program of T-cell development was observed, including the generation of CD4 and CD8 αβ–T-cell receptor (TCR)–bearing cells. Induction of T-cell differentiation was dependent on the expression of Delta-like 1 by the OP9 cells. Stimulation of the in vitro–differentiated T cells by TCR engagement induced the expression of T-cell activation markers and costimulatory receptors. These results establish an efficient in vitro coculture system for the generation of T cells from human HSCs, providing a new avenue for the study of early T-cell differentiation and function.

In vitro generation of T lymphocytes from embryonic stem cell–derived prehematopoietic progenitors

AbstractEmbryonic stem (ES) cells can differentiate into most blood cells in vitro, providing a powerful model system to study hematopoiesis. However, ES cell–derived T lymphocytes have not been generated in vitro, and it was unresolved whether such potential is absent or merely difficult to isolate. Because the latter case might result from rapid commitment to non–T-cell fates, we isolated ES cell–derived prehematopoietic precursors for reconstitution of fetal thymic organ cultures. We found a transient Flk1+CD45– subset of these precursors generated T lymphocytes in vitro, and the use of reaggregate thymic organ cultures greatly enhanced reconstitution frequency. These findings reveal that ES cells can exhibit in vitro T-cell potential, but this is restricted to early stages of ES cell differentiation. Moreover, the results support the notion that the thymic microenvironment can induce T-cell differentiation from a subset of prehematopoietic progenitors and suggest deficient migration into intact thymi hindered previous attempts to generate T cells in vitro from ES cell–derived progenitors. These findings demonstrate that a defined subset of ES cells has the potential to generate T cells in vitro and could contribute to greater understanding of the molecular events of hematopoietic induction and T-cell lineage commitment.

Expression and function of histamine receptors in human monocyte-derived dendritic cells

Background: Histamine is a well-known mediator eliciting different responses in immune and nonimmune cells, but its role in modulating dendritic cell (DC) functions has been marginally investigated. Objective: The purpose of this investigation was to analyze whether human monocyte-derived DCs express functional histamine receptors according to their maturation stage. Methods: DCs were derived from monocytes and used as immature or LPS-differentiated cells. DCs were tested for histamine receptor expression, chemotaxis, cytokine release, and the capacity to induce T-cell differentiation in response to specific histamine receptor agonists. Results: Immature and mature DCs expressed the mRNA for H1, H82, and H3 histamine receptors. Histamine induced intracellular Ca2+ transients, actin polymerization, and chemotaxis in immature DCs. Maturation of DCs resulted in the loss of these responses. In maturing DCs, however, histamine dose-dependently enhanced intracellular cAMP levels and stimulated IL-10 secretion while inhibiting production of IL-12. As a consequence, histamine might contribute to the impairment of generation of allogeneic type 1 responses via maturing DCs. Specific histamine receptor agonists or antagonists revealed that Ca2+ transients, actin polymerization, and chemotaxis of immature DCs were due to stimulation of H1 and H3 subtypes. Modulation of IL-12 and IL-10 secretion by histamine involved the H2 and H3 receptors exclusively. Conclusions: Our study suggests that histamine has important biological effects on DC activities, opening the possibility that histamine released during inflammatory or immune responses could regulate DC functions and ultimately favor type 2 lymphocyte-dominated immunity. (J Allergy Clin Immunol 2002;109:839-46.)

Structure of interleukin 4 mutant E9A suggests polar steering in receptor-complex formation.

Interleukin 4 (IL-4) is a pleiotropic cytokine which induces T-cell differentiation and class switching of B cells. It therefore plays a central role in the development of allergies and asthma. An IL-4 variant in which Glu9 was mutated to alanine shows an 800-fold drop in binding affinity towards its high-affinity receptor chain. As shown by surface plasmon resonance measurements, this mostly arises from a decreased association rate. Here, the crystal structure of this mutant is reported. It reveals that the protein has a virtually identical structure to the wild type, showing that the unusual behaviour of the mutated protein is not a consequence of misfolding. The possibility that polar interactions in the encounter complex have a steering effect is discussed.

TP5 Triggers Signal Transduction Involving Mitogen Activated Protein Kinases in Monocytes

The pentapetide thymopentin (TP5) corresponding to the aminoacids RKDVY represents the residues 32 – 36 of thymopoietin (TP), which was originally isolated from bovine thymus. Both were observed to induce T-cell differentiation and maturation. Recently however it was shown, that TP represents the N-terminal 49 aa of the human thymopoietin (TMPO) isoforms TMPOα, β and γ, which are localized in the nucleus. TP5 was investigated in a variety of diseases and showed efficacy by improving the immune balance, whereby different cells increased in cell number or activity. Findings which support the assumption of multifunctional efficacy and a description of TP and TP5 modulating T cells lack any interpretation on molecular level. In the present study we investigated the binding of TP5 on white blood cells. We identified monocytes and neutrophils as TP5-binding cells by displacing fluorescein-labelled TP5 with an excess of unlabelled TP5 in competition assays. Binding of TP5 on cell surface proteins resulted in cellular signalling and we report here that TP5 triggers signal transduction involving mitogen activated protein kinases p42/p44 (MAPKs) in monocytes.

Electron microscope study of the secretory activity of epithelial cells in embryonic thymus

The possibility that intrathymic T-cell differentiation involves stem cell-lymphoid interactions in embryos led us to study the ultrastructure of epithelial cell in normal embryonic thymus. Studies in adult thymus showed that it produces several peptides that induce T-cell differentiation. Several of them have been chemically characterized, such as thymosin α 1, thymopoietin, thymic humoral factor or the serum thymic factor. It was suggested that most of these factors are secreted by populations of A and B-epithelial cells.Embryonic materials were obtained from inbred matings of Swiss Albino mice. Thymuses were disected from embryos 17 days old and prepared for transmission electron microscopy. Our studies showed that embryonic thymus at this stage contains undifferentiated and differentiated epithelial cells, large lymphoblasts, medium and few small lymphocytes (Fig. 5). No differences were found between cortical and medullary epithelial cells, in contrast to the findings of Van Vliet et al,. Epithelial cells were mostly of the A-type with low electron density in both cytoplasm and nucleus. However few B-type with high electron density were also found (Fig. 7).

Contrasting biological activities of thymopoietin and splenin, two closely related polypeptide products of thymus and spleen.

Thymopoietin, a 49 amino acid polypeptide hormone of the thymus discovered by its effect on neuromuscular transmission, was later shown to induce T-cell differentiation and to affect immunoregulatory balance. A radioimmunoassay for thymopoietin revealed a crossreaction with a product found in spleen and lymph node but not other tissues. This product, named splenin, differs from thymopoietin only in position 34, aspartic acid for bovine thymopoietin and glutamic acid for bovine splenin. Synthetic pentapeptides corresponding to residues 32-36, called thymopentin and splenopentin, reproduce biological activities of thymopoietin and splenin, respectively. Thus thymopoietin and thymopentin affect neuromuscular transmission and induce the phenotypic differentiation of T precursor cells in vitro while inhibiting phenotypic differentiation of B cells. Splenin and splenopentin, in contrast, do not affect neuromuscular transmission, and they induce both T- and B-cell precursors.

Effects of a nonapeptide FTS on lymphocyte differentiations in vitro

BACH et al.1,2 have reported the amino acid sequence of a putative thymic hormone isolated from pig serum3, a nonapeptide called FTS (facteur thymique serique). Natural and synthetic FTS showed high activity in the rosette test used as a bioassay1,2. Bioassays for thymic hormones4, however, can be spuriously triggered by many substances of non-thymic origin5, a finding probably related to mediation by a cyclic AMP second signal6. Early steps in B-cell differentiation are also mediated by cyclic AMP (refs 6–9) and comparison of induction of T cells and B cells from committed precursor cells in vitro (the dual induction assay) enables a distinction to be made between selective and non-selective inducing agents7,8. This is exemplified by induction studies in the mouse with thymopoietin10,11 and ubiquitin7,12,13. Bovine thymopoietin selectively induces T-cell differentiation and actually inhibits B-cell differentiation9. By contrast, ubiquitin is non-selective, inducing both T- and B-cell differentiation7. Ubiquitin at high concentrations does not induce differentiation whereas these high concentrations do not impair induction by thymopoietin or other agents8,14. Another feature of induction by ubiquitin is its inhibition by the β-adrenoceptor-blocking drug propranolol, which is without effect on induction by thymopoietin7. We have evaluated synthetic FTS in the dual induction system in the chicken to determine whether it showed inductive selectivity appropriate to a thymic hormone, and have found that it induced non-selective differentiation of both T cells and B cells and closely resembled ubiquitin in its activity.

Thymic factor in human sera demonstrable by a cyclic AMP assay

NORMAL human serum has been reported to contain thymus hormone activity as detectable by a rosette inhibition assay1. It has been suggested that this circulating hormone may act through stimulation of cyclic AMP synthesis2. In addition, other thymic hormones isolated from the thymus, such as thymosin3 and THF4, have been found to activate membrane adenylate cyclase and to increase cellular levels of cyclic AMP in thymocytes5,6. Thymopoietin, a thymic hormone the amino acid sequence of which has been established7, has also been claimed to induce T-cell differentiation by way of cyclic AMP8. Here we report on a human serum factor (SF) capable of increasing the level of cellular cyclic AMP in thymocytes; we also present evidence for the possible thymic nature of this factor.

INDUCTION OF T‐CELL DIFFERENTIATION IN VITRO BY THYMUS EPITHELIAL CELLS*

Thymus-reticular epithelial cells (TE-cells) were grown in a cell culture devoid of any lymphocytic elements. These cells were able to induce T-cell differentiation in spleen cells from T-dificient mice as expressed by con-A responsiveness and GvH reactivity. It was also shown that xenogeneic rat TE cells were as effective in the induction of T-cell differentiation in vitro as syngeneic TE cells. This system is therefore ideal for the study of T-cell development.

Induction of T-cell differentiation in vitro by thymin, a purified polypeptide hormone of the thymus.

Thymin, a purified polypeptide isolated from bovine thymus, was shown to induce the expression of differentiation antigens characteristic of thymocytes [TL and Thy-1 (theta)] when incubated in vitro with mouse bone marrow or spleen cells. This induction occurred in 5-10% of the cells from bone marrow after a 2-hr incubation with subnanogram concentrations of thymin. The induced cells expressed more TL and Thy-1 (theta) antigens than average normal thymocytes.