Adult Mouse Bone Marrow Research Articles

Mice Transgenic for the Human CGM6 Gene Express Its Product, the Granulocyte Marker CD66b, Exclusively in Granulocytes

AbstractThe nonspecific cross-reacting antigen-95 (NCA-95/CD66b), is a member of the human carcinoembryonic antigen (CEA) family encoded by the CGM6 gene that is exclusively expressed in neutrophils and eosinophils. No murine counterpart is known to exist. We have analyzed a cosmid containing the complete CGM6 gene. The coding sequence is contained within six exons spanning a 16.5 kb region. The main transcriptional start site was mapped to a tight cluster between nucleotides -95 and -101 relative to the translational start site. As with other members of the CEA gene family, no typical TATA or CAAT-box sequences were found in the CGM6 gene. Transgenic mice were established with the cosmid insert. CD66b expression is first seen in the fetal liver on day 12.5 of mouse embryonic development, and it first appears in the bone marrow at day 17.5. Northern blot analysis showed that CD66b transcripts are confined to the bone marrow of adult mice, whereas immunohistochemistry also showed CD66b-positive granulocytes in the spleen, thymus, and lungs. FACScan analyses of bone marrow and spleen cells showed CD66b expression to be exclusive to granulocytes. Thus, all the elements necessary for regulating granulocyte-specific expression are present within this cosmid clone. These mice could provide a model for transplantation and for inflammation studies using CD66b as a granulocyte-specific marker.

Mice Transgenic for the Human CGM6 Gene Express Its Product, the Granulocyte Marker CD66b, Exclusively in Granulocytes

The nonspecific cross-reacting antigen-95 (NCA-95/CD66b), is a member of the human carcinoembryonic antigen (CEA) family encoded by the CGM6 gene that is exclusively expressed in neutrophils and eosinophils. No murine counterpart is known to exist. We have analyzed a cosmid containing the complete CGM6 gene. The coding sequence is contained within six exons spanning a 16.5 kb region. The main transcriptional start site was mapped to a tight cluster between nucleotides -95 and -101 relative to the translational start site. As with other members of the CEA gene family, no typical TATA or CAAT-box sequences were found in the CGM6 gene. Transgenic mice were established with the cosmid insert. CD66b expression is first seen in the fetal liver on day 12.5 of mouse embryonic development, and it first appears in the bone marrow at day 17.5. Northern blot analysis showed that CD66b transcripts are confined to the bone marrow of adult mice, whereas immunohistochemistry also showed CD66b-positive granulocytes in the spleen, thymus, and lungs. FACScan analyses of bone marrow and spleen cells showed CD66b expression to be exclusive to granulocytes. Thus, all the elements necessary for regulating granulocyte-specific expression are present within this cosmid clone. These mice could provide a model for transplantation and for inflammation studies using CD66b as a granulocyte-specific marker.

Identification of Clonogenic Common Lymphoid Progenitors in Mouse Bone Marrow

The existence of a common lymphoid progenitor that can only give rise to T cells, B cells, and natural killer (NK) cells remains controversial and constitutes an important gap in the hematopoietic lineage maps. Here, we report that the Lin −IL-7R +Thy-1 −Sca-1 loc-Kit lo population from adult mouse bone marrow possessed a rapid lymphoid-restricted (T, B, and NK) reconstitution capacity in vivo but completely lacked myeloid differentiation potential either in vivo or in vitro. A single Lin −IL-7R +Thy-1 −Sca-1 loc-Kit lo cell could generate at least both T and B cells. These data provide direct evidence for the existence of common lymphoid progenitors in sites of early hematopoiesis.

Sox-4 facilitates thymocyte differentiation.

The mouse Sry-like transcription factor Sox-4 is expressed in thymus, bone marrow, and gonads of adult mice. Sox-4-deficient mice die at embryonic day E14 due to cardiac malformation. In transfer experiments to irradiated recipients, B cell development was shown to be severely impaired in Sox-4-deficient progenitor cells. However, no drastic effects on T lymphocyte development were noted, despite the high level expression of the Sox-4 gene in the thymus of normal mice. Here, we report a detailed analysis of T cell development from Sox-4-deficient progenitors. Explanted fetal thymic organ cultures (FTOC) of Sox-4-deficient thymi yielded 10-50-fold fewer CD4 CD8 double-positive and single-positive cells than FTOC of littermates. This effect was T cell-autonomous, since similar observations were made when FTOC were performed by culturing of Sox-4-deficient progenitors in wild-type thymus lobes. When Sox-4-deficient fetal liver cells were injected together with normal cells intrathymically, they did not compete efficiently for reconstitution. It is concluded that Sox-4 facilitates thymocyte development.

Bar Mitzvah for B-1 cells: how will they grow up?

The discovery of CD5+ B cells by Hayakawa et al. (1) initiated a 13 year-long discussion about the origin and functional properties of these cells. The CD5+ B cells (B-1a) and their phenotypic CD5− “twins” (B-1b cells) differ from conventional peripheral B cells (B-2) by anatomical location, developmental origin, surface marker expression, antibody repertoire and growth properties (2–4). B-1 cells form a dominant population of B lineage cells in the peritoneal cavity, but are rare in the spleen and lymph nodes of adult mice (3–6). The progenitors of B-1a cells are abundant in the fetal omentum and liver but in contrast to the progenitors of conventional B-2 cells, are missing in the bone marrow of adult mice (3, 7–9). The progenitors of B-1b cells are present in the fetal omentum and liver as well as in the bone marrow of adult mice (10, 11). However, a supply of B-1b cells from the bone marrow appears to be restricted by a negative feedback mechanism according to which the entrance of newly generated cells into the adult peripheral pool of B-1 cells is prevented by the presence of the mature B-1 population (12). In the absence of a continuous supply of bone marrow–derived B-1 cells, the size of the B-1 cell population is kept constant due to the self-renewal capacity of B-1 cells (3, 13). Due to their ability to produce large quantities of multireactive IgM, IgG3 and IgA, B-1 cells are considered carriers of “natural immunity” (7). It seems that the maintenance of B-1 cell population at a stable level might be necessary to control the level of antibody production by these cells. Thus the anti– IL-10 antibody-induced ablation of B-1 cells is accompanied by a drastic reduction of serum immunoglobulin titers (14). On the other hand, the expansion of autoreactive B-1 cells is associated with the development of autoimmunity in mice and man (15).

Structural identification of the hematopoietic progenitor antigen ER-MP12 as the vascular endothelial adhesion molecule PECAM-1 (CD31).

The monoclonal antibody ER-MP12 was recently described to recognize an antigen present on cell subpopulations of adult mouse bone marrow, including pluripotent hematopoietic stem cells. In an effort to understand the function of ER-MP12 antigen in hematopoiesis, we used biochemical and physical methods to determine its identity. ER-MP12 antigen was isolated by immunoprecipitation from FDCP-1 cell membrane proteins, yielding a glycosylated 113-kDa band upon analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thirteen peptides derived from trypsinized ER-MP12 antigen were analyzed by electrospray ionization mass spectrometry and compared to a protein sequence database. The search revealed the identity of the ER-MP12 antigen as platelet endothelial cell adhesion molecule-1, CD31 (PECAM-1). This result was subsequently confirmed by Edman sequencing of a single ER-MP12 peptide fragment followed by comparison with PECAM-1 sequence. In addition, flow cytometric analysis of bone marrow and embryonic stem cells revealed highly similar profiles between ER-MP12 and CD31 (MEC 13.3 antibody)-stained cells. The presence of PECAM-1 on primitive hematopoietic stem cells supports the theory for the interaction of hematopoietic progenitor and stem cells with bone marrow stroma and transendothelial migration.

Lack of evidence for an increase in interleukin-6 expression in adult murine bone, bone marrow, and marrow stromal cell cultures after ovariectomy.

Interleukin-6 (IL-6) has been implicated as a mediator of postmenopausal bone loss. In vitro studies of bone and bone marrow cells have suggested that estrogen regulates bone turnover by controlling the production of IL-6, a potent stimulator of osteoclastogenesis and bone resorption. To investigate this hypothesis in an in vivo model, we examined the effect of ovariectomy or estrogen replacement on IL-6 mRNA and protein expression in adult mouse bone and bone marrow in vivo and in marrow stromal cell cultures. Eight-week-old CD-1 mice were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized and subcutaneously implanted with 21-day slow-release pellets containing 10 micrograms of 17 beta-estradiol (O + E). Placebo pellets were implanted in the SHAM and OVX mice. Uterine weights at 1, 2, or 3 weeks after surgery were significantly decreased (68-76%) in OVX animals compared with SHAM or O + E. In mice sacrificed at 1 or 3 weeks after surgery, we found by nonquantitative reverse transcribed polymerase chain reaction (RT-PCR), that SHAM, OVX, and O + E calvariae (CALV) constitutively expressed IL-6 mRNA. In contrast, IL-6 mRNA was either barely detectable or absent in the tibia (TIB) and bone marrow (BM). In the mice sacrificed 3 weeks after surgery, we determined by quantitative RT-PCR that IL-6 mRNA in the CALV from the OVX and O + E groups were decreased by 81 and 92%, respectively, compared with SHAM. IL-6 protein levels in the flushed bone marrow (BMSups) were detectable and were not significantly different among the groups. In bone marrow cells that were cultured for 1 week, basal levels of IL-6 protein were low and did not differ significantly among the SHAM, OVX, or O + E groups sacrificed 1, 2, or 3 weeks after surgery. After the addition of hrIL-1 alpha, IL-6 protein levels increased 100- to 1300-fold over control. IL-6 levels in cells from animals sacrificed 2 weeks after surgery were significantly lower in the hrIL-1 alpha-stimulated OVX and O + E groups than in hrIL-1 alpha-stimulated SHAM cell cultures. In conclusion, in this model we could find no increase in IL-6 production with in vivo estrogen withdrawal in calvaria, long bones, bone marrow, or marrow stromal cell cultures. If increases in IL-6 expression are involved in the effects of estrogen withdrawal on bone, the magnitude of these changes are relatively small and below the limits of detection of the assays that we employed.

Evidence of Both Ontogeny and Transplant Dose-Regulated Expansion of Hematopoietic Stem Cells In Vivo

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150-fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.

The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.

The low level of amphotropic retrovirus-mediated gene transfer into human hematopoietic stem cells (HSC) has been a major impediment to gene therapy for hematopoietic diseases. In the present study, we have examined amphotropic retrovirus receptor (amphoR) and ecotropic retrovirus receptor mRNA expression in highly purified populations of mouse and human HSC. Murine HSC with low to undetectable levels of amphoR mRNA and relatively high levels of ecotropic retrovirus receptor mRNA were studied. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, ecotropic provirus sequences were detected in 10 of 13 long-term repopulated animals, while amphotropic proviral sequences were detected in only one recipient. A second distinct population of murine HSC were isolated that express 3-fold higher levels of amphoR mRNA. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, 11 of 11 repopulated mice contained ecotropic provirus and 6 of 11 contained amphotropic provirus sequences, a significant increase in the amphotropic retrovirus transduction (P = 0.018). These results indicate that, among the heterogeneous populations of HSC present in adult mouse bone marrow, the subpopulation with the highest level of amphoR mRNA is more efficiently transduced by amphotropic retrovirus. In a related study, we found low levels of human amphoR mRNA in purified populations of human HSC (CD34+ CD38-) and higher levels in committed progenitor cells (CD34+ CD38+). We conclude that the amphoR mRNA level in HSC correlates with amphotropic retrovirus transduction efficiency.

Long-Term Lymphohematopoietic Reconstitution by a Single CD34-Low/Negative Hematopoietic Stem Cell

Hematopoietic stem cells (HSCs) supply all blood cells throughout life by making use of their self-renewal and multilineage differentiation capabilities. A monoclonal antibody raised to the mouse homolog of CD34 (mCD34) was used to purify mouse HSCs to near homogeneity. Unlike in humans, primitive adult mouse bone marrow HSCs were detected in the mCD34 low to negative fraction. Injection of a single mCD34(lo/-), c-Kit+, Sca-1(+), lineage markers negative (Lin-) cell resulted in long-term reconstitution of the lymphohematopoietic system in 21 percent of recipients. Thus, the purified HSC population should enable analysis of the self-renewal and multilineage differentiation of individual HSCs.

Transcription of the Interleukin-1 Receptor-related T1 Gene Is Initiated at Different Promoters in Mast Cells and Fibroblasts

The delayed early serum response gene T1 encodes glycoproteins of the immunoglobulin superfamily with significant sequence similarity to the type 1 interleukin-1 receptor. The T1 gene is transcribed in fibroblasts into an abundant 2.7-kilobase (kb) and a rare 5-kb mRNA in response to proliferation-inducing stimuli. It gives predominantly rise to the longer transcript in the bone marrow of adult mice and in cultured mast cells. Alternative 3' processing is responsible for the two mRNA forms. The short transcript encodes a secreted protein with marked similarity to the extracellular domain of the interleukin-1 receptor, whereas the long mRNA is translated into a protein with an additional putative transmembrane and an intracellular domain. Here we demonstrate that T1 transcription in mast cells and fibroblasts initiates at two different start sites which are 10.5 kb apart. The alternative first exons are both spliced to exon 2 which contains the translation start site. Northern blot analysis and primer extension experiments revealed that promoter usage is strictly cell type-specific. T1 transcription in mast cells is initiated exclusively at the distal promoter, whereas in fibroblasts both the short and the long T1 mRNA start at the proximal promoter. Two GATA-1 elements were identified in the 5'-flanking region of the mast cell-specific distal exon 1.

Induction of micronucleated erythrocytes by MEA, AET, WR-2721 and X-rays

The induction of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow of adult male Swiss mice treated with MEA (cysteamine HCl), AET (2-aminoethylisothiouronium Br.HBr), or WR-2721 (S-2-(3-aminopropylamino)ethyl phosphorothioic acid), at a dose of 200 mg/kg body weight, and/or exposed to 6 Gy X-rays. MEA, AET, or WR-2721 was given alone or 15 min prior to X-ray exposure, and the frequency of MNPCEs was determined 24 h after the aminothiol treatment and X-irradiation of mice. A genotoxic effect was shown for MEA, AET, WR-2721, and X-rays, as well as a protective effect of the aminothiols against X-ray-induced genotoxicity in the mouse erythropoietic system. The aminothiol drugs given alone, without subsequent X-irradiation, elevated the frequency of MNPCEs, and WR-2721 appeared to be less toxic than AET and MEA. After exposure of mice to X-rays, the number of MNPCEs was distinctly increased. MEA, AET, or WR-2721 administration prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei, but a stronger radioprotective effect was obtained following WR-2721 and AET treatment than after MEA application. So, the genotoxic and radioprotective effect of the aminothiols was dependent on the compound applied.

Engagement of the antigen-receptor on immature murine B lymphocytes results in death by apoptosis.

During their development B lymphocytes pass through a maturational stage in which encounter with Ag leads to tolerance rather than activation. At least four mechanisms for achieving B cell tolerance have been reported: deletion, anergy, receptor editing, and competition for follicular niches. Although turnover rates for immature B cells in the adult mouse bone marrow and several transgenic model systems suggest that a major process contributing to negative selection of B cells is deletion, a detailed study of the negative effect of Ag-receptor engagement on primary, immature B cell survival has never been undertaken. We have utilized an in vitro culture system to determine whether cross-linking sIgM on tolerance-susceptible sIgM+IgD- B cells results in deletion by apoptosis. In contrast to the effect of sIgM cross-linking on mature splenic B cells, treatment of immature, bone marrow-derived B cells results in significant levels of apoptotic death. Ag receptor-mediated apoptosis is detectable by 14 h after sIgM engagement. Moreover, IL-4 and cycloheximide, which have previously been shown to prevent B cell tolerance induction, specifically block the sIgM-induced apoptosis observed in the immature B cells. Similarly, immature B cells from the neonatal spleen are also susceptible to apoptosis after sIgM cross-linking, although they manifest somewhat higher levels of unstimulated apoptosis as compared with bone marrow-derived B cells. These studies are the first detailed demonstration of Ag receptor-mediated apoptosis of primary immature stage B lymphocytes.

Long-Term Repopulation of Irradiated Mice With Limiting Numbers of Purified Hematopoietic Stem Cells: In Vivo Expansion of Stem Cell Phenotype But Not Function

Hematopoietic stem cells were isolated from normal adult mouse bone marrow based on surface antigen expression (Thy-1.1(low)Lin(neg)Ly-6A/E+) and further selected for low retention of rhodamine 123. This population of cells (Rh-123low) could mediate radioprotection and long-term (greater than 12 months) repopulation after transplantation of as few as 25 cells. Transfer of five genetically marked Rh-123low cells in the presence of 10(5) normal bone marrow cells resulted in reconstitution of peripheral blood by greater than 10% donor cells in 64% (30 of 47) of recipient mice. Of 46 animals surviving after 24 weeks, 10 had over 50% donor-derived cells in peripheral blood. Two general patterns of long-term reconstitution were observed: one in which many donor-derived cells were observed 5 to 6 weeks after reconstitution and another in which donor-derived cells were rare initially but expanded with time. This result suggests that two classes of long-term repopulating hematopoietic stem cells exist, differing in their ability to function early in the course of transplantation. Alternatively, distinct anatomic sites of engraftment may dictate these two outcomes from a single type of cell. As an approach to measure the extent of self-renewal by the injected cells, recipients of five or 200 stem cells were killed 8 to 13 months after the transplants, and Thy-1.1(low)Lin(neg)Ly-6A/E+ progeny of the original injected cells were isolated for a second transplant. While a numerical expansion of cells expressing the cell surface phenotype of stem cells was observed, along with activity in the colony-forming unit-spleen assay, the expanded cells were vastly inferior in radioprotection and long-term reconstitution assays when compared with cells freshly isolated from normal animals. This result demonstrates that in stem cell expansion experiments, cell surface antigen expression is not an appropriate indicator of stem cell function.

Isolation of a cDNA clone encoding a novel membrane protein expressed in lymphocytes

By subtractive hybridization using single-stranded phagemids with directional inserts, we isolated a mouse cDNA clone, LSM-1, from temperature-sensitive Abelson virus-tranformed immature B cells whose differentiation was being induced after the shift from the permissive (35°C) to the non-permissive temperature (39°C). LSM-1, which encodes an as yet unknown peptide of 197 amino acids, has a putative signal sequence and a trans-membrane region, and is expressed in B- and T-cell lines, in spleen, thymus, and bone marrow of adult mice, and in embryos.

Properties of mouse CD40: cellular distribution of CD40 and B cell activation by monoclonal anti-mouse CD40 antibodies.

We describe here the derivation of a rat monoclonal antibody (mAb) against mouse CD40 (designated 3/23), which stains 45-50% of spleen cells of adult mice, approximately 90% of which are B cells. Interestingly, some 5-10% of both CD4+ and CD8+ T cells in the spleens of (some, but not all) adult, unimmunized mice are also CD40+, whereas CD40+ cells were not detectable in the thymus, even following collagenase digestion. Some 35-40% of lymphoid cells in the bone marrow of adult mice are CD40+ and virtually all of these are B220+, and hence of the B cell lineage: triple-color flow cytometry showed that CD40 is expressed at low levels on some 30% of pre-B cells, at intermediate levels on 80% of immature B cells and on essentially all mature B cells in the bone marrow. These results, therefore, suggest that in the mouse CD40 is expressed relatively late during the process of B cell differentiation. The mAb induced marked up-regulation of major histocompatibility complex class II molecules, CD23 and B7.2 antigens on mature B cells. It also stimulated modest levels of DNA synthesis in mature B cells by itself: this was markedly enhanced by suboptimal concentrations of mitogenic (but not non-mitogenic) anti-mu and anti-delta mAb, and moderately enhanced by co-stimulation with interleukin-4. Hypercross-linking of CD40 (using biotinylated mAb and avidin) also enhanced the proliferative response to anti-CD40.

Mouse fetal liver cells lack functional amphotropic retroviral receptors

We have been transducing mouse hematopoietic cells with the human MDR1 (MDR) gene in retroviral vectors to determine the optimal conditions for retroviral gene transfer as a model system for potential human gene therapy. In these studies, we have demonstrated transduction and expression of the human MDR gene using ecotropic and amphotropic MDR- retroviral producer lines. To obtain more mouse hematopoietic cells for detailed study, mouse fetal liver cells (FLC) have been used for MDR transduction and expression, and to reconstitute the ablated marrows of live adult mice. FLC contain hematopoietic cells that have a reconstituting capacity comparable to that of adult mouse bone marrow cells. However, to our surprise, FLC can only be transduced with ecotropic retrovirus and not with amphotropic virus. This restriction of transduction of FLC cannot be overcome by higher titer virus. The resistance to amphotropic transduction by FLC may be part of a changing developmental program that results in a different antigen repertoire on FLC as compared with adult bone marrow cells.

Mouse Fetal Liver Cells Lack Functional Amphotropic Retroviral Receptors

We have been transducing mouse hematopoietic cells with the human MDR1 (MDR) gene in retroviral vectors to determine the optimal conditions for retroviral gene transfer as a model system for potential human gene therapy. In these studies, we have demonstrated transduction and expression of the human MDR gene using ecotropic and amphotropic MDR-retroviral producer lines. To obtain more mouse hematopoietic cells for detailed study, mouse fetal liver cells (FLC) have been used for MDR transduction and expression, and to reconstitute the ablated marrows of live adult mice. FLC contain hematopoietic cells that have a reconstituting capacity comparable to that of adult mouse bone marrow cells. However, to our surprise, FLC can only be transduced with ecotropic retrovirus and not with amphotropic virus. This restriction of transduction of FLC cannot be overcome by higher titer virus. The resistance to amphotropic transduction by FLC may be part of a changing developmental program that results in a different antigen repertoire on FLC as compared with adult bone marrow cells.

Fetal liver and bone marrow JORO 75+ lymphocyte progenitors are precursors of CD4+8- TCR/CD3- early thymocytes.

We show here that cell sorter purified JORO 75+ lymphocyte progenitors from fetal liver or bone marrow of adult mice give rise in vitro to CD4+8- T cell receptor (TCR)/CD3- early thymocytes and CD4+8- TCR/CD3+ thymocyte subsets after coculture with the EH6 subcapsular thymic epithelial cell line, recombinant interleukin 7 (rIL-7) and F (supernatants from the FLS4.1 fetal liver stromal cell line). We find that in cultures that had additionally received rIL-2, CD4-8+ TCR/CD3+ cells were also generated. The results strongly suggest that fetal liver and marrow JORO 75+ lymphocyte progenitors are precursors to the early CD4+8- TCR/CD3- intrathymic population previously identified in the adult mouse. The EH6 subcapsular thymic epithelial cell line should facilitate the study of the molecular events responsible for very early stages of T cell development including T lymphocyte-lineage commitment.

Neuroregulation of natural suppressor cell activity.

The neuronal control of murine natural suppressor (NS) cell activity was studied in mice following lesion of the arcuate nucleus of the hypothalamus performed by the postnatal treatment with monosodium glutamate (MSG). The suppressor activity was evaluated as the ability of spleen or bone marrow cells (suppressors) to suppress the mitogen-induced lymphocytes proliferation of allogeneic Balb/c spleen cells (indicators). The study was carried out on three different inbred strains of mice (C57BL/6, DBA/2 and CBA). The results demonstrated that, in all strains used, the treatment of mice (12 day-old) with MSG led to a marked reduction of natural suppressor cell activity. This reduction was observed in both spleen and bone marrow, the lymphoid organs in which the NS cell activity is usually present. A reduced NS activity was also present in the bone marrow of MSG-treated adult mice, suggesting a permanent alteration of NS cell activity. These results provide additional support on neuromodulation of immune system.