A B220+ CD117+ CD19- hematopoietic progenitor with potent lymphoid and myeloid developmental potential

In this report, we identify in the bone marrow (BM) of normal mice a subpopulation of B220+ CD117+ CD19- NK1.1- cells with potent lymphoid and myeloid developmental potential. These cells represent 0.1-0.2% of nucleated BM cells. By limiting dilution analysis in the presence of the appropriate combination of stromal cells and cytokines, 1 in 5-10 sorted cells formed B cells, 1 in 10-15 formed T cells and 1 in 5-10 generated macrophages. When cultured on a mixture of OP9 stroma and OP9 stromal cells expressing the Notch ligand Delta-like-1, single cells generated both T and B cells. Following intravenous infusion, freshly sorted cells transiently reconstituted both the T and B cell progenitor compartments, generating cohorts of mature T and B lymphocytes. The relationship between B220+ CD117+ CD19- NK1.1- cells of wild-type mice and other multi-lineage BM progenitors is discussed.     

Regulation of Forssman antigen expression during maturation of mouse stromal macrophages in haematopoietic foci

During a trial to develop a monoclonal antibody (mAb) specific to stromal macrophages (M phi) in haematopoietic foci, we have created a mAb, designated F10, that stains the stromal M phi more selectively than any other mAb reported. As F10 was found to react with Forssman glycosphingolipid (GSL) specifically and to give clearer immunostaining than anti-Forssman GSL IgG, we have studied Forssman antigen expression during maturation of the stromal M phi in splenic haematopoietic foci using F10 and a system of allogenic bone marrow transplantation which allows us to know the turnover of the stromal M phi in vivo. C3H/He mice (H-2k) were lethally irradiated and intravenously infused with the bone marrow cells of BALB/c nu-nu mice (H-2d). Splenic frozen-sections and cytocentrifuge preparations of splenic haematopoietic clusters from the recipient mice were stained with F10 and with mAb against major histocompatibility class I antigens. H-2d-type stromal M phi began to appear in the haematopoietic clusters at Week 5 and they gradually replaced H-2k-type stromal M phi. The percentage of Forssman+ stromal M phi gradually decreased and reached a nadir at Week 6, when most stromal M phi were already of the donor type. At Week 8, however, Forssman+ stromal M phi levels returned to normal. The delayed expression of Forssman antigen on the stromal M phi in haematopoietic foci following genotypic conversion suggests that Forssman antigen is regularly expressed on the subpopulation of stromal M phi, which mature well under specific microenvironmental factors in vivo.     

B淋巴细胞在抗CD45RB抗体诱导的移植免疫耐受中的作用

 目的: 探讨B淋巴细胞在抗CD45RB抗体诱导的移植免疫耐受中的作用。方法: 抗CD45RB抗体对BALB/c裸鼠进行预处理后制备脾脏单细胞悬液,与BALB/c小鼠T淋巴细胞和C57BL/6小鼠脾细胞混合培养,流式细胞术分析Th1、Th2、Treg和Tm淋巴细胞。以B6.μMT^-/-小鼠为受体、BALB/c小鼠为供体建立皮肤移植模型,移植后向受体鼠腹腔注射抗CD45RB单抗,监测脾淋巴细胞CD3⁺CD45RB^hi细胞比例。在混合淋巴培养过程中加入抗CD45RB单抗,分离B细胞,建立以BALB/c小鼠为供体、B6.μMT^-/-小鼠为受体的心脏移植模型,通过尾静脉注射B细胞给B6.μMT^-/-小鼠,观察受体鼠生存期和B细胞分布。结果: 在裸鼠体内用抗CD45RB抗体处理过的B淋巴细胞,与T淋巴细胞混合培养时,可使Treg和Th2淋巴细胞比例明显升高,Th1淋巴细胞的比例明显下降,Tm细胞无明显变化。在体内B淋巴细胞缺失的情况下,抗CD45RB抗体依然能够降低T细胞表面CD45RB的表达,与对照组B淋巴细胞存在组相比,抗CD45RB抗体对T淋巴细胞表面CD45RB下调更为快速,但最终CD3⁺CD45RB^hi T细胞比例无明显变化。体外抗CD45RB抗体处理过的B淋巴细胞可以延长受体鼠的生存时间。B6.μMT^-/-鼠在接受抗CD45RB抗体处理的B细胞并进行同种异体心脏移植后,B细胞可向胸腺迁移。结论: 在抗CD45RB抗体诱导的免疫耐受中,B淋巴细胞可能通过介导各T淋巴细胞亚群比例发挥着重要作用,且在中枢耐受中也起到一定作用,但是仅靠B淋巴细胞无法形成完全耐受。     

Phenotypically distinct subpopulations of T cells in domes and M-cell pockets of rabbit gut-associated lymphoid tissues.

Follicle epithelium and domes of gut-associated lymphoid tissue (GALT) contain populations of lymphocytes which first contact antigen taken up from the intestine. In order to study the association of lymphocytes with M cells in follicle epithelium, monoclonal antibodies (mAb) were generated by immunizing BALB/c mice with lymphocytes populating GALT domes from NZW rabbits, and their specificity was assessed by immunohistochemistry and flow cytometry. mAb 3C10 (IgM) and 3B6 (IgG3) recognized subpopulations of intraepithelial lymphocytes associated with M cells. mAb 3C10 also identified macrophage-lymphocyte clusters in domes and tangible body macrophages in germinal centres of GALT but did not react with cells in T-dependent areas (TDA) or B cells in follicles. mAb 3B6 recognized lymphocytes in domes and B cells in follicles but not T cells in TDA of GALT. The distribution of 3B6+ cells overlapped with, but was more restricted than, that of Ia+ cells. Analysis of lymphocytes in follicle epithelium showed that greater than 95% of lymphocytes associated with M cells were Ia+. T cells represented approximately 95% of intraepithelial lymphocytes in the appendix and approximately 65% in Peyer's patches. A majority of intraepithelial lymphocytes was recognized by mAb 3B6, but mAb 3C10 identified only approximately 30%. Because neither 3C10 nor 3B6 recognized lymphocytes in TDA of GALT, these results indicate that most lymphocytes associated with M cells are a distinct phenotype of Ia+ T cells.     

Host origin of follicular dendritic cells induced in the spleen of SCID mice after transfer of allogeneic lymphocytes.

Follicular dendritic cells (FDC) are uniquely characterized by the ability to trap immune complexes. In a previous report, it was shown that functional FDC with the capacity to trap immune complexes via complement receptor emerged in the splenic follicle after transferring syngeneic lymphocytes into the severe combined immunodeficiency (SCID) mouse. In the present report, we have investigated whether FDC are derived from haematopoietic cells or surrounding stromal components, by transferring allogeneic lymphocytes into SCID mice. Transfer of allogeneic T and B lymphocytes (H-2k) into SCID(H-2d) mice, however, failed to induce the development of FDC in the splenic white pulp. This was due to a graft-versus-host reaction (GVHR) by allogeneic lymphocytes against host stromal cells, as revealed by the destruction of the splenic reticular meshwork. The GVHR was prevented in transfer experiments of T-cell-depleted allogeneic lymphocytes with daily administration of anti-Thy-1 antibody. This resulted in segregated lodgement of allogeneic B lymphocytes in the proper compartments and, thereafter, generation of FDC in the primary follicle of SCID spleen, as revealed by the trapped immune complexes via complement receptors. The H-2 of the newly generated FDC was examined by two-colour immunofluorescent staining. FDC were defined as the reticular cells stained with anti-CR1/2 or FDC-M1 antibodies. FDC carried host H-2, clearly indicating that newly generated FDC are host-derived. In addition, the FDC shared the BP-3 protein with the surrounding reticular cells, a specific marker of reticular meshwork in the murine lymphoid tissues, and formed a network continuous with the rest of the reticulum, suggesting that FDC and non-FDC reticular cells belong to the same cell lineage.     

Galactocerebrosides are required postnatally for stromal-dependent bone marrow lymphopoiesis

Galactocerebrosides (GCs) represent a major class of glycolipids in the nervous system. Here, we show that mice lacking the key enzyme to generate GCs, UDP-galactose:ceramide galactosyltransferase (CGT(-/-)), exhibit severe postnatal atrophy of all lymphoid organs, owing to a maturational arrest before the pro-B/T cell stage. This lineage-specific defect originates from the bone marrow (BM) stroma since it is not transplantable to irradiated wild-type recipients. Remarkably, CGT(-/-) long-term B lymphoid BM cultures displayed severe deficits in the number of CD45(neg)VCAM-1(pos) stromal cells and fibronectin matrix assembly, and produced floating macrophages rather than B lymphocytes. The fibronectin network was also altered in the CGT-deficient BM parenchyma. These results point to an essential role for galactolipids in the formation of fibronectin-enriched lymphoid-specific stromal niches in the BM.     

Contribution of 4–1BBL on radioresistant cells in providing survival signals through 4–1BB expressed on CD8+ memory T cells in the bone marrow

The persistence of memory lymphocytes is a critical feature of adaptive immunity. The TNF family ligand 4–1BBL supports the antigen‐independent survival of CD8+ memory T cells. Here, we show that mice lacking 4–1BB only on αβ T cells show a similar defect in CD8+ T‐cell recall responses, as previously shown in 4–1BBL‐deficient mice. We show that 4–1BB is selectively expressed on BM CD8+ but not CD4 + memory T cells of unimmunized mice. Its ligand, 4–1BBL, is found on VCAM‐1+ stromal cells, CD11c+ cells, and a Gr1^lo myeloid population in unimmunized mice. Adoptive transfer of in vitro generated memory T cells into mice lacking 4–1BBL only on radioresistant cells recapitulates the defect in CD8 + T‐cell survival seen in the complete knockout mice, with smaller effects of 4–1BBL on hematopoietic cells. In BM, adoptively transferred DsRed CD8+ memory T cells are most often found in proximity to VCAM‐1+ cells or Gr1+ cells, followed by B220+ cells and to a much lesser extent near CD11c+ cells. Thus, a VCAM‐1+CD45^? stromal cell is a plausible candidate for the radioresistant cell that provides 4–1BBL to CD8+ memory T cells in the BM.     

Monoclonal antibodies to stromal cell types of the mouse thymus

Seven hybridoma cell lines secreting monoclonal antibodies (mAb) to nonlymphoid cells of the mouse thymus have been prepared. These mAb clearly demonstrate the heterogeneity of the thymic stroma. Based on their anatomical distribution patterns observed with the immunoperoxidase technique on frozen tissue sections, they were subdivided into four groups. The first group of mAb, ER-TR1, 2 and 3, detects antigens encoded for by the I region of the major histocompatibility complex. These antigens are expressed on both stromal and lymphoid cells in lymphoid organs. mAb of the second category, ER-TR4, react with epithelial cells in the thymic cortex. mAb of the third group detect stromal cells of the thymic medulla. One antibody of this group, ER-TR5, exclusively reacts with medullary epithelial cells. ER-TR6, the other antibody of this group, reacts with medullary interdigitating cells and macrophages. The fourth type of antibodies, ER-TR7, detects the reticular fibroblasts of the thymus. The possible role of the thymic cell types detected by the present antibodies in T cell differentiation is discussed.     

In rat B lymphocyte genesis sixty percent is lost from the bone marrow at the transition of nondividing pre-B cell to sIgM+ B lymphocyte, the stage of Ig light chain gene expression

The cycling B precursor cells in rat bone marrow (BM) that carry the B220 antigen and no surface Ig daily produce 780 million new cells. The pool of recirculating B lymphocytes in the rat, however, renew at a rate of only about 40 million cells/day. To analyze at which stages in B lymphocyte genesis the cell loss occurs, we identified post-mitotic cells in the rat BM B lineage, and determined their renewal rates. We used 5-bromo-deoxyuridine (BrdUrd) to label DNA-synthesizing cells, identifying incorporated BrdUrd with the mouse monoclonal antibody BU-1. B lineage cell subsets were identified by the markers HIS24 antigen (rat B220), terminal deoxynucleotidyl transferase (TdT), Ig mu heavy chain, and complete Ig. By use of double and triple immunocytology, we determined the extent of BrdUrd incorporation in the various B lineage compartments [HIS24+TdT-Ig-, TdT+, cytoplasmic mu chain (c mu)+ surface (s) IgM- pre-B, sIgM+ B]. Both sIgM+ B lymphocytes and all B precursors with cell diameters less than 11-12 microns were virtually devoid of DNA synthesis, as indicated by S-phase indices below 2%. In contrast, S-phase indices of large B precursors ranged between 43%-66%. We established the renewal rates of nondividing BM B lineage cells by placing osmotic minipumps containing BrdUrd subcutaneously in the flank of rats. The nondividing BM B lineage cells all renewed rapidly at rates between 2.4% and 5.6%/h, representing average half-lives of 29 to 12 h. In absolute numbers, the renewal/day/whole body BM was 165 X 10(6) for sIgM+ B lymphocytes, 422 X 10(6) for small c mu+ sIgM- pre-B cells, 89 X 10(6) for small TdT+ cells and 35 X 10(6) for small HIS24+TdT-Ig- cells. Assuming that recirculating B lymphocytes in the periphery are the descendants of BM sIgM+ B lymphocytes, which in their turn are the progeny of small pre-B cells, the renewal data indicate the following. Of the 165 million potentially available BM B lymphocytes, only 40 million cells become incorporated in the pool of recirculating B lymphocytes, representing a loss of 75%. BM B lymphocytes, in turn, use only (165/422 X 100% = ) 40% of the potential output from their immediate precursors. The 60% loss that occurs here may reflect the extent of aberrant Ig light chain gene rearrangement in normal B lymphocyte genesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genesis of B lymphocytes in the bone marrow: Extravascular and intravascular localization of surface IgM-bearing cells in mouse bone marrow detected by electron-microscope radioautography after in vivo perfusion of 125I anti-IgM antibody

The role of mammalian bone marrow in generating surface IgM (sIgM)-bearing B lymphocytes is reviewed. Precursor cells in the marrow give rise to large, rapidly dividing cells bearing free cytoplasmic μ chains (cμ). The progeny of the large cμ⁺ cells form a population of small, nondividing cμ⁺ cells that mature into small lymphocytes, progressively expressing sIgM and other B-cell surface membrane components. Newly formed sIgM⁺ cells soon migrate through the bloodstream to the spleen and other lymphoid tissues, where they may die after a short lifespan or be activated to produce antibody molecules. The large-scale lymphocytopoiesis in the bone marrow thus maintains a population of rapidly renewed virgin B lymphocytes in the peripheral lymphoid tissues. This process continuously creates and selects B cell clones with the wide range of antibody specificites necessary to mediate primary humoral immune responses through postnatal life. A technique for perfusing radiolabeled anti-IgM antibodies in young mice has now permitted sIgM⁺ cells to be detected radioautographically in histological preparations of bone marrow under the electron microscope. Small sIgM⁺ lymphocytes are situated either singly or in small groups throughout the extravascular hemopoietic compartment of the bone marrow, often near sinusoid walls adjacent to late erythroblasts and reticular cells. Some regional concentrations of sIgM⁺ cells are apparent. sIgM⁺ cells also appear in transit through the sinusoidal endothelium and are markedly concentrated in the lumen of some sinusoids. Intrasinusoidal sIgM⁺ small lymphocytes have high densities of sIgM and long microvilli, on which sIgM molecules are concentrated. These studies reveal the localization and cell associations of specifically identified sIgM⁺ small lymphocytes in the extravascular marrow compartment and suggest that these cells may also undergo a transient intravascular storage and maturation phase. Use of this in vivo immunolabeling technique to detect other cell-surface markers may further elucidate the microenvironmental basis of B lymphocyte genesis in the bone marrow.     

Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue.

Two alternative integrins involved in mucosal homing (alpha 4 beta 7) or epithelial retention (alpha E beta 7) of lymphocytes were examined in the human gut. The distribution of the beta 7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by alpha E beta 7 + cells but weakly by alpha 4 beta 7 + cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T-cell receptor (TCR) gamma/delta +, and CD3- cells, expressed alpha E beta 7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B-lineage cells (CD19+) and macrophages (CD68+) were negative. In gut-associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (< 5%) cells were positive for alpha E beta 7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30-50%) expressed alpha 4 beta 7 (mAb Act-1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of alpha 4 beta 7 as in the lamina propria whereas relatively few B lymphocytes (< 50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for alpha 4 and the former three subsets as well as appendix CD8+ cells also for beta 7 while only lamina propria CD8+ cells had mRNA for alpha E. Together, the results suggested that alpha E beta 7 and alpha 4 beta 7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly alpha 4 beta 7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because alpha E beta 7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of beta 7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle-associated epithelium of GALT expressed alpha E beta 7, perhaps reflecting epithelial interaction during primary immune responses.     

Immunohistochemical localization of Forssman glycosphingolipid-positive macrophages and reticular cells in murine lymphoid tissue

Forssman (Fo) glycolipid antigen, as detected by a monoclonal antibody (mAb), is expressed by a subpopulation of murine macrophages in the spleen and peripheral lymph nodes. The histological distribution of Fo antigen in spleen and lymph nodes was studied by immunostaining of cryosections, and was compared with the staining pattern of four other mAbs known to recognize macrophage subpopulations: F4/80, Mac-1, MOMA-1, and ERTR-9. Fo+ macrophages were found exclusively in the red pulp of the spleen and the medulla of inguinal and axial lymph nodes. Macrophages in the other lymphoid organs were Fo-. Besides macrophages, reticular cells in T-dependent areas of spleen and lymph nodes were Fo+. Attempts to grow colonies of Fo+ macrophages from either bone marrow or spleen precursors were negative. While the usual number of F4/80+ colonies was obtained, only a few, small clusters of Fo+ macrophages were formed, which speaks against an early commitment of precursors to express Fo.     

GL7 defines the cycling stage of pre-B cells in murine bone marrow.

We have identified a novel subset of early B lineage cells in the mouse bone marrow (BM) by GL7 expression on cell surface. GL7(+)B220(low) BM cells have a large cell size and are CD43(-to low), CD95(-), Sca-1(-), I-A(low), IgM(-) and IgD(-), suggesting that they are large pre-B cells. These BM cells express lambda5 and VpreB but not terminal deoxytransferase (TdT) and Bcl-2, and approximately 50 % of them are in cell cycle. This fraction was not detected in BM cells of Rag-1-deficient and Scid mice, supporting that GL7(+)B220(low) BM cells belong to fraction C' and D according to Hardy's criteria or to an early large pre-B-II fraction according to Melchers-Rolink's criteria. Furthermore, GL7(+)B220(low) BM cells can differentiate into IgM(+) immature B cells in co-culture with stromal cells. These results suggest that B lymphocytes pass through the GL7(+) pre-B cell stage during differentiation in the BM. Thus, GL7 is the critical marker to define the proliferation stage of large pre-B cells.     

NZB serum factor (NZB-SF)-B precursor cell maturation factor. II. In vivo effects of NZB-SF or mAb against NZB-SF on B lineage cell populations.

In vivo effects of NZB serum factor (NZB-SF), which enhances the maturation of B precursor cells in vitro, were examined. Immunoaffinity-purified NZB-SF from young NZB mice was injected into B6 mice intraperitoneally twice weekly, five times total (5 micrograms/dose/mouse). Control mice were given 0.01% albumin. Then the B lineage cell populations defined phenotypically (sIg+ cells, B220+ cells, and AA4.1+ cells) or the numbers of colony-forming B lineage cells were examined. NZB-SF-treated B6 mice exhibited a decrease in the percentage of B precursor cells in marrow, even though the percentage of sIg+ cells in marrow or spleen did not differ from controls. In contrast, the frequency of colony-forming B cells in marrow and spleen, especially sIg- colony-forming B cells in marrow, increased significantly in NZB-SF-treated mice as compared to controls. In addition, monoclonal antibody (mAb) against NZB-SF was injected weekly for 9 weeks into NZB mice beginning at 7 weeks of age. mAb vs NZB-SF at a dose of 5 micrograms per mouse per injection, as stated above, prevented the decline of sIg- colony-forming B lineage cells which usually occurred in the adult NZB mice (greater than 16 weeks). This treatment also prevented, in part, the decline of the B220+ cell population which normally occurs in the marrow with increasing age. Thus NZB-SF impressively influences the composition of B lineage cell populations in normal B6 mice and may account for abnormal changes of B lineage cell populations observed in NZB mice.     

Effects of granulocyte/macrophage colony-stimulating factor on the development and differentiation of CD5-positive macrophages and their potential derivation from a CD5-positive B-cell lineage in mice.

In co-cultures of either the murine pre-B cell line J13, fetal liver cells, or adult peritoneal or bone marrow cells with ST2 mouse bone marrow stromal cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), the development of CD5+ macrophages was demonstrated by immunohistochemical staining and flow cytometry. Although CD5+ macrophages were not present in the peritoneal cavities of normal mice, approximately 30% of the peritoneal macrophages in viable motheaten (mev/mev) mice, deficient in SHP-1 protein tyrosine phosphatase, expressed cell surface CD5 and B220, markers for B cells. In the mev/mev mice, GM-CSF level in peritoneal fluid was increased significantly. At 5 days after daily intravenous injection with GM-CSF, many CD5+ macrophages appeared in the peritoneal cavity and in omental milky spots of normal mice but fewer in osteopetrosis (op) mutant mice, deficient in macrophage (M)-CSF. These results indicate that GM-CSF, in combination with M-CSF, induces the development and differentiation of CD5+ macrophages in the peritoneal cavity, particularly in the omental milky spots of mice. In the peritoneal cavity of GM-CSF-treated mice, the percentages of hematopoietic progenitor cells doubly positive for CD5 and CD34 or c-kit and of macrophage precursor cells doubly positive for CD5 and ER-MP58 or ER-MP20 were increased significantly during the development of CD5+ macrophages and CD5 B cells, suggesting that CD5+ macrophages and B cells may share a bipotential progenitor in vivo.     

A novel 26 kilodalton antigen expressed on the surface membrane of activated T cells

We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) 1B10, raised against an activated gammadelta T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb 1B10 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B10+. 1B10 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) alphabeta+ and gammadelta+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were 1B10+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb 1B10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb 1B10 from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb 1B10 recognizes a novel appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease.     

Monoclonal antibodies that block adhesion of B cell progenitors to bone marrow stroma in vitro prevent B cell differentiation in vivo

B cell differentiation requires adhesion of B cell progenitors to bone marrow (BM) or fetal liver stroma. We show that B lymphoid cells can adhere to the BM stroma cell line CS 1.3, in vitro. Two monoclonal antibodies, SAB-1 and SAB-2, inhibited the adhesion of a B220+ progenitor B cell line but did not interfere with the binding of cytoplasmic mu chain-positive pre-B cells or mature B cells to the BM stromal cell line. Injection of both SAB-1 and SAB-2 antibodies into pregnant mice reduced by 90% the number of B220+n B lineage cells in the livers of their embryos. Livers from such embryos also were virtually devoid of cells able to give rise to B cell colonies in soft agar cultures (CFU-preB). Either antibody separately had no effect. Flow cytometry analysis show that SAB-1 is present on CS 1.3 stroma cells and on a pre-B cell line while SAB-2 is present on pro-B and pre-B cell lines, but not on CS 1.3 stromal cells. SAB-1 and SAB-2 react with different molecules and neither antibody seems to recognize CD44, and adhesion molecule that may also participate in B cell differentiation. Proteinase K and trypsin can digest both SAB-1 and SAB-2 antigens from viable cells suggesting that both are cell surface proteins. We propose that antibodies SAB-1 and SAB-2 probably recognize novel cell-cell adhesion molecules, and that these molecules are involved in the interactions between B cell progenitors and stroma cells.     

Characterization of CD4+ T cells in mouse bone marrow. I. Increased activated/memory phenotype and altered TCR Vbeta repertoire

A significant proportion of memory B cells home to bone marrow (BM) which is a major site of anamnestic antibody responses in mice. We hypothesized that memory T cells likewise accumulate in BM perhaps to provide help for antibody production, and that the compartment of CD4+ T cells in BM of unimmunized mice would be enriched for memory phenotype cells that might have been activated by environmental antigens. The phenotype of activated/memory CD4+ lymphocytes has been defined as CD44hi CD45RBlo CD62L-. Conversely, the phenotype of immunologically naive cells is CD44lo CD45RBhi CD62L+. Flow cytrometric analysis of tissue from normal, adult C57BL/6 mice identified 1-2 % CD3+CD4+ cells in BM. Up to 40 % of CD3+CD4+ cells in the BM expressed the activated/memory phenotype compared with < or = 10% in the spleen and lymph nodes. Analysis of TCR Vbeta repertoire revealed that expression of Vbeta3 and Vbeta7 genes was increased as much as fourfold in BM compared to the periphery; most of this increase was within the CD44hi T cells. The accumulation of activated/memory T cells and clonotypic expansion(s) was not seen in the BM of germ-free mice, indicating that it reflects the history of the animal's exposure to antigens. Finally, immunization of mice which express a transgenic T cell receptor specific for ovalbumin peptide resulted in appearance of antigen-specific T cells with activated/memory phenotype in the BM.     

Neural cell adhesion molecule contributes to hemopoiesis-supporting capacity of stromal cell lines

To clarify mechanisms underlying cell-to-cell interactions between hemopoietic stem cells (HSCs) and stromal cells, we established a stromal cell line (FMS/PA6-P) from day-16 fetal bone marrow (BM) adherent cells using an anti-PA6 monoclonal antibody (mAb) specific for BM stromal cells. Importantly, this FMS/PA6-P cell line, showing homogenous fibroblastic morphology, is absent from hematolymphoid and endothelial lineage markers and maintains a high level of expression of PA6 molecule, recognized by the anti-PA6 mAb, for approximately 20 passages. Further, the cell line expressing a high level of PA6 molecule has a better hemopoiesis-supporting capacity in vitro than other stromal cell lines such as PA6 and MS-5. In fact, the PA6 molecule is closely related to the hemopoiesis-supporting capacity of the stromal cells because the proliferation of HSCs was suppressed to a great extent by the anti-PA6 mAb. Affinity chromatography and mass peptide fingerprinting revealed that the protein reacting with the anti-PA6 mAb is neural cell adhesion molecule (NCAM). The frequencies of long-term cobblestone area-forming cells and long-term culture-initiating cells were significantly suppressed by repression of NCAM in the FMS/PA6-P cells using NCAM small interfering RNA. Our findings clearly indicate that NCAM functions on the maintenance of HSCs.     

Notch1突变对HSC体外分化为T/B淋巴细胞的影响

目的:探讨Notch1信号传递水平对小鼠造血干细胞(HSC)体外分化为T/B淋巴细胞的影响。方法:应用表达Notch1配体Delta1的基质细胞OP9-DL与来源于小鼠胎肝和骨髓的Lin-HSC在体外共培养,检测Notch1基因突变对HSC在体外分化后B淋巴细胞或者CD4+CD8+细胞的比例。结果:Notch1基因点突变后Notch1受体结构发生改变,和特异性抗体以及配体的结合能力降低,导致信号传递能力下降。Notch1突变后,胎肝和骨髓HSC向B淋巴细胞分化过程未受影响,但是向T淋巴细胞的分化过程受到明显抑制,其中Notch1突变对胎肝HSC分化能力的影响高于骨髓HSC,分别被抑制44%和34%。结论:Notch1突变导致Notch1信号水平降低,对HSC向B淋巴细胞分化没有影响,但是能抑制向T淋巴细胞的分化。