Fusion of T and B cells.

Hybrid cells were prepared by fusin an immunoglobulin-secreting mouse myeloma lin e (B cell) with an allogenic T-cell lymphoma which expresses the surface antigen Thy 1. The resulting hybrids expressed H2 antigens of both parental cells and secreted the immunoblobulin of the myeloma parent but did not express the Thy 1 antigen of the lymphoma parent. Twenty-one hybrids were formed from fusion of the same myeloma line with TNP-SRBC-primed spleen cells. Most of the hybrid lines exhibited characteristics expected for the fusion of the myeloma to B lymphocytes. No hybrids between the myeloma line and spleen T cells were identified as none of the hybrids expressed the T-cell-specific antigen Thy 1. We discuss possible reasons for failure to produce hybrids with T-cell characteristics in these types of fusion.     

Isolation of rat bone marrow mast lineage cells using Thy 1.1 and rat stem cell factor.

Recent reports have shown that various marrow-derived cell populations respond vigorously to recombinant rat stem cell factor (rrSCF164), one form of the kit-ligand. In the present study, we isolated cell populations from rat bone marrow using the Thy 1.1 antigen (an antigen that in the rat is differentially expressed on primitive hemopoietic progenitor cells) and fluorescently conjugated rrSCF164 (rrSCF164-PE). We show that rrSCF164 only stimulates cells that are enriched in the brightest Thy 1.1 populations (Thy 1.1bright). Numerous cell lines were generated by serial passage in rrSCF164 containing medium, and the prototypic cell lines have been designated SRT002 and SRT003. Each cell line retains the Thy 1.1bright phenotype and does not respond to interleukins (IL) 1-8, IL-10, granulocyte (G) colony-stimulating factor (CSF), granulocyte macrophage (GM) CSF, M-CSF, or crude preparations of mitogen-stimulated T-cell supernatants. The Thy 1.1bright population of rat marrow was subdivided into a subset that binds rrSCF164-PE (Thy 1.1bright, rrSCF164+). The majority of these cells possess certain characteristics in common with marrow-derived mast cells and the Thy 1.1bright, rrSCF164 responsive cell lines, having similar granule morphology, being metachromatic, and reacting positively with alcian blue. Moreover, rats treated with rrSCF164 displayed significant increases in Thy 1.1bright, rrSCF164+ cells in the bone marrow. These studies show that the combination of Thy 1.1 and rrSCF164 makes possible the isolation of a unique subset of rat bone marrow cells that differentially express the Thy 1.1 antigen and the cell surface receptor c-kit, the majority of which are morphologically similar to marrow-derived mast cells.     

Antigen expression of mouse spleen × thymocyte heterokaryons

The expression of Thy 1.2 and thymic leukemia (TL) antigens by heterokaryons of spleen cells of strain A mice (A-S) and AKR thymocytes (AKR-T) was determined. The A-S parental cells do not express TL antigens, although strain A thymocytes are TL-positive. Approximately 25% of A-S cells express Thy 1.2 antigens; however, AKR-T cells express a different Thy 1 antigen (Thy 1.1) and are phenotypically negative for TL expression. AKR-T × A-S heterokaryons were prepared with the aid of inactivated Sendai virus. Identification of heterokaryons was facilitated by prior isotopic labeling of AKR-T but not A-S cells, and the finding by autoradiography of binucleated cells with one radioactively labeled and one non-labeled nucleus. Antigenic expression of these fused cells was determined by exposure of the cells to specific antiserum and complement prior to autoradiography. 24 hr after fusion, fused cells were resistant to the cytotoxic effects of TL antiserum and fresh complement. However, a large proportion of these cells was lysed by treatment with antiserum directed against the Thy 1.2 antigen.     

Multiple occurrence of spontaneous AKR/J lymphomas with T and B cell characteristics.

Three Thy 1.1-positive and surface IgM-positive (Thy 1+, SIg+) AKR/J lymphoma lines are described. These doubly marked tumors arose spontaneously in the peripheral lymphoid organs of 14- to 16-month-old AKR/J mice that either had spontaneous thymus atrophy or had been thymectomized at 1 month of age. All lines bore surface Thy 1.1, Ly,Ig(micron-chain) and Fc receptor (FcR), detectable by immunofluorescence. Immune response region (Iak) antigen was present on the two lines tested. Persistence of Thy 1.1 antigen and SIg after long-term tissue culture provided evidence that these markers were not passively acquired. One of these tumor lines, AkTB-1 always grows in lymph nodes as Thy 1.1-positive,SIg-negative tumors cells, whereas tumor cells growing in the spleen are initially Thy 1.1 positive, SIg negative, but they rapidly acquire SIg, FcR, and 1a between 18 and 21 days of passage.     

In vitro differentiation of mouse embryonic yolk sac cells

The embryonic yolk sac is the first site in the mammalian embryo in which cells are found that can carry out cell-mediated immune functions, yet the relation of cells of this primitive hematopoietic organ to the development of the mature immune system has not been established. We have initiated a series of experiments to determine the potential of cells of the mouse yolk sac to differentiate in vitro, in order to get an insight into the development of immunocompetence in this primary population of hematopoietic stem cells. The present paper describes the conditions promoting stem-cell differentiation and provides an initial characterization of cell surface phenotypes of the cell lineages established in vitro. Yolk sac cells obtained from 10- to 13-day mouse embryos were maintained in culture for more than 18 months, giving rise to a variety of cell types belonging to the hematopoietic lineages and culminating in the establishment of long-term cell lines. Supernatants of secondary mixed leukocyte cultures were found to be an effective source of growth factors promoting the initial differentiation as well as the maintenance of these cells. Flow-cytometric analysis showed that, in contrast to freshly obtained yolk sac cells, which had no detectable Thy 1 antigen, cells expressing significant levels of Thy 1 were obtained after 1 week or more of culture. Ly1 and Lyt 2 antigens were detected only rarely and the L3T4 (GK 1.5) antigen was never expressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of Thy 1.2 surface antigen increases significantly during the murine mesenchymal stem cells cultivation period

This study sought to investigate the absence or expression of some surface antigens on murine mesenchymal stem cells (mMSCs) during the cultivation period of primary culture to passage 3 (equivalent to about 15 or 16 population doubling number). For this purpose, bone marrow cells from 6-8-week-old mice (either NMRI or Balb/c) were cultivated in 75-cm(2) culture flask for three successive passages, in each of which the culture was examined for the expression of CD135, CD44, CD31, Thy1.2, CD11b, CD45, CD34, Vcam1, Sca-1, and c-Kit antigens, using flow cytometry. Passage-3 cells from each strain can easily be differentiated into bone and fat, which was indicative of their mesenchymal nature. Our results demonstrated that for each given antigen, the percentages of the cells expressing that antigen had been changed by subcultures. The statistical analysis showed that nearly all differences between the passages were statistically significant. In this term, the expressional changes of Thy 1.2 seemed to be very significant in such a way that the expression increased to about half of the whole population in passage 3. In conclusion, it seems that this antigen could be considered as an enriching antigen for mMSCs population from bone marrow adherent cell culture.     

Effect of gene dosage on cell-surface expression of Thy-1 antigen in somatic cell hybrids between Thy-1− abelson-leukemia-virus induced lymphomas and Thy-1+ mouse lymphomas

Hybrids between pseudodiploid Thy-1.1⁺ lymphomas and Thy 1.2^– pseudodiploid Abelson-leukemia-virus-induced (ALV-induced) lymphomas express Thy-1 glycoprotein on their cell surface. These Thy-1⁺ hybrids invariably express the Thy 1.1 allelic form of the glycoprotein and may be either Thy 1.2⁺ or Thy 1.2^–. Sublines expressing both Thy 1.1 and Thy 1.2 can be isolated from Thy 1.1⁺, Thy 1.2^– hybrids by cell sorting. In contrast to hybrids with pseudodiploid ALV-induced lymphomas, hybrids between Thy 1.1⁺ lymphomas and pseudotetraploid Thy 1.2^– Abelson-leukemia-virus-induced lymphomas do not express Thy-1 glycoprotein on their cell surface and Thy-1 glycoprotein cannot be detected in detergent extracts of these cells. Thy-1⁺ revertants were isolated from one of the Thy-1^– hybrids by cell sorting. — These results demonstrate a gene dosage effect for the expression of the Thy-1 glycoprotein in somatic cell hybrids. They are consistent with the idea that diffusable gene products regulate Thy-1-glycoprotein expression in these hybrids. They also suggest that there may be additional, apparentlycis-active, regulatory mechanisms which determine the ability of theThy-1 structural genes of the Abelson-leukemia-virus-induced lymphoma parent to be expressed in somatic cell hybrids.     

成年转基因小鼠嗅鞘细胞的培养、纯化及生物学特性

已有多项研究表明,嗅鞘细胞具有修复中枢及外周神经损伤的潜能。我们选用了表达增强型绿色荧光蛋白（enhanced green fluorescent protein,eGFP）的成年小鼠,分离其双侧嗅球嗅神经纤维层及嗅小球层细胞,体外原代培养并予以纯化。同时结合共聚焦、相差显微镜,细胞增殖分析及免疫组织化学鉴定等技术,对其生物学活性进行研究。结果表明：（1）原代培养转基因成年小鼠嗅球嗅鞘细胞（Olfactory ensheathing cells,OECs）15d后,主要存在两种不同形态和免疫组织化学特征的细胞。一种是带有长突起的双极或多极OECs,表达P75^NTR（P75 low affinity neurotrophic receptor）、S100和胶质原纤维酸性蛋白（glial fibrillary acidic protein,GFAP）。另一种则是对Thy 1．1抗体免疫反应阳性,呈扁平或内皮样形态的成纤维细胞。（2）根据不同类型细胞在未覆层的培养器皿上贴壁速度的差异,我们建立了一种简单易行、不需任何抗体或昂贵仪器的细胞纯化方法,获得了大量高纯度的OECs。（3）在连续纯化培养22d后,OECs仍能保持较高的增殖活性。本实验支持和丰富了OECs发育的相关理论,为进一步体内移植修复CNS损伤提供了理想的材料。     

Hepatic lineages isolated from developing rat liver show different ways of maturation

Immunocytochemical analysis revealed that different hepatic cell types exist during liver development: (i). cells co-expressing the stem-cell marker Thy1 and the hepatic lineage marker CK-18 and (ii). cells only expressing CK-18 (hepatoblasts). In this study we separated the different hepatic cells and analyzed gene-expression and phenotype. Fetal rat livers were digested by collagenase solution. OX43- and OX44-positive hematopoietic cells were depleted and Thy1-positive cells were enriched using Magnetic cell sorting. The different cell compartments were analyzed by RT-PCR and immunocytochemistry for Thy1, CK-18, AFP, and albumin. Hepatoblasts expressed albumin at all times and AFP in the early stages. Thy1-enriched cells expressed CK-18 at all times, albumin in the early, and AFP in the late stages. Thy1-positive cells from fetal livers express liver specific genes. The data suggest that Thy1-positive hepatic cells develop towards hepatic stem cells, and hepatoblasts develop towards mature hepatocytes of the adult liver.     

A new protein (J1-31 antigen, 30 kD) is expressed by astrocytes,Müller glia and ependyma

Monoclonal antibody (MAb) J1-31 raised using human brain homogenate as immunogen in mice can be used as a cell type marker for certain types of CNS macroglia, namely astrocytes, Müller cells and tanycytes as well as ciliated ependymal cells. Except for the ciliated ependymal cells, these types of macroglia express glial fibrillary acidic protein (GFAP). J1-31 antigen is an intracellular protein which has a MW of 30 kD under reducing conditions for gel electrophoresis (Singhet al., 1986). This protein is distinct from GFAP (MW 50 kD) and vimentin (MW 55 kD), the two core proteins of 10 nm IFs known to be expressed in the above types ofmacroglia. This conclusion is based on several criteria including temporal differences in the onset of expression of GFAP and J1-31 antigen during development of the rat cerebellum. Also, there is no detectable (by immunofluorescence microscopy) expression of J1-31 antigen in the prenatal CNS or outside the CNS where vimentin has been reported to be abundant. The most direct evidence that J 1-31 antigen and GFAP are distinct proteins comes from studies on the mature ciliated ependymal cells which do not express GFAP and yet show intense immunostaining for J1-31 antigen.     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

The mitogenic properties of rabbit anti-mouse brain serum

The mitogenic activity of heterologous rabbit anti-mouse brain sera (RAMB) was investigated. By complement-dependent cytotoxicity and indirect immunofluorescence, RAMB was T-cell specific. Mitogenic activity was assessed by determination of [³H]thymidine incorporation into DNA. RAMB was mitogenic for spleen cells for Thy 1.1- and Thy 1.2-positive mouse strains. Maximal mitogenic responsiveness to RAMB occurred on Day 3 of culture. The incorporation of [³H]uridine into RNA and [³H]leucine into protein and percentage of blast cells in culture were also significantly increased following RAMB stimulation. The mitogenic activity of RAMB was abrogated by adsorption of the sera with BALB/c or AKR thymocytes or brains or with RL♂ 1.3+, a Thy 1.2-bearing T-cell lymphoma of BALB/c origin. In contrast, the mitogenic activity was not removed when RAMB sera were absorbed with RL♂ 1.4?, a variant of RL♂ 1 which appears to specifically lack cell surface Thy 1 determinants. These data suggest that the mitogenic activity of RAMB is Thy 1 directed. RAMB mitogenicity is T-cell dependent. Spleen cells from normal and heterologous nu/+ mice respond to RAMB, while spleen cells from nu/nu mice do not respond. Normal thymocytes and cortisone-resistant thymocytes do not respond mitogenically to RAMB. The response of unseparated spleen cells to RAMB is also macrophage dependent. Nylon-wool column-purified splenic T cells respond to high concentrations of RAMB in the absence of exogenous macrophages but do not respond to lower concentrations of RAMB unless exogenous macrophages are added to the cultures. Nylon-wool-adherent cells, which are B-cell enriched and relatively T-cell depleted, also respond to RAMB, suggesting that in the presence of even small numbers of T cells, B cells can be recruited into the response.     

Neurothelin: an inducible cell surface glycoprotein of blood-brain barrier-specific endothelial cells and distinct neurons

The blood-brain barrier is characterized by still poorly understood barrier and transport functions performed by specialized endothelial cells. Hybridoma technology has been used to identify a protein termed neurothelin that is specific for these endothelial cells. Neurothelin is defined by the species-specific mouse mAb 1W5 raised against lentil-lectin-binding proteins of neural tissue from embryonic chick. In the posthatch chick, neurothelin expression is found on endothelial cells within the brain but not on those of the systemic vascular system. Injection of the monoclonal antibody in vivo leads to labeling of brain capillaries, indicating that the corresponding antigen is expressed on the luminal surface of brain endothelial cells. Transplantation of embryonic mouse brain onto the chick chorioallantoic membrane results in rodent brain vascularization by the avian vascular system. Subsequently, normally mAb 1W5-negative endothelial cells, originating from blood vessels of the chick chorioallantoic membrane, are induced to express neurothelin when they are in contact with mouse neural tissue. In contrast to differentiated brain neurons that do not express neurothelin, neurons of the nonvascularized chick retina synthesize neurothelin. However, neurothelin is not found on retinal ganglion cell axons terminating on 1W5-negative brain cells. 1W5 immunoreactivity was also found in the pigment epithelium that forms the blood-eye barrier. Putting epithelial cells into culture results in concentration of neurothelin at cell-cell contact sites, leaving other cell surface areas devoid of antigen. Therefore, the distribution of neurothelin appears to be regulated by cell-cell interactions. In Western blot analysis, neurothelin was identified as a protein with a molecular mass of approximately 43 kD. The protein bears at least one intramolecular disulfide bridge and sulfated glucuronic acid as well as alpha-D-substituted mannose/glucose moieties. The exclusive neurothelin expression in the posthatch chick on endothelial cells of the central nervous system but not on systemic endothelial cells makes neurothelin a marker specific for blood-brain barrier-forming endothelial cells. The spatiotemporally regulated neurothelin expression in neurons suggests an interaction between vascularization and neuronal differentiation.     

Down-regulation of murine collagen-induced arthritis by a T cell hybridoma

T cell hybridoma cell lines were generated by somatic cell fusion of BW 5147 myeloma cells and splenic cells from mice suppressed for collagen-induced arthritis (CIA). Two cell lines were characterized for their cell surface phenotype, antigen recognition and capacity to down-regulate the erythema and edema associated with CIA. Cell line T101N was determined to portray the cell surface phenotype Ly1+2- L3T4- Thy1+ by a direct binding assay. Cell line T104B1 was determined to express only the Thy1+ alloantigen. Panning studies, measurement of IL-2 production in vitro and the suppression of antibodies to type I and type II collagen in vivo indicate that the hybridoma cells are not isotype specific in their recognition of the polymorphic interstitial collagens. Down-regulation of the erythema and edema of CIA occurred on injection of 1 X 10(5) T101N cells but not T104B1 cells. Histology of the tarsus region of the hind paw of CIA mice 33 days after the administration of T101N cells showed contrasting histopathology compared to that of CIA mice. The joints of CIA mice given T101N cells showed aligned articular surfaces resembling normal joint structure and only residual pannus. The data indicate that collagen-specific cloned T cell lines can modulate the gross pathology and joint architecture of joints exhibiting CIA.     

草鱼、中华鳖T淋巴细胞表面抗原的研究

以草鱼脑组织、中华鳖脑组织和胸腺细胞为抗原制备兔抗草鱼脑血清（RACBS）、兔抗顺华鳖脑血清（RATBS）和兔抗中华鳖胸腺细胞血清（RATTS）。补体依赖性细胞毒试验和不同组织对RATBS、RATTS的吸收试验结果表明：中华鳖胸腺细胞和脑组织均存在Thy1抗原（亦称脑组织抗原或胸腺-脑组织T细胞抗原）;草鱼脑组织缺乏Thy1抗原。应用间接酶标免疫组化染色技术显示：Thy1抗原阳性反应物沉淀于中华鳖胸腺细胞和外周一部分淋巴细胞表面,进一步用抗人白细胞分化抗原CD4、CD8单克隆抗体进行免疫组化交叉反应提示中华鳖淋巴细胞膜上含有与人类T淋巴细胞表面抗原CD4、CD8类似结构的物质。本文讨论了Thy1抗原、CD抗原的出现及其意义。探讨了淋巴细胞异质性及RATBS、RATTS的特异性和作用。     

Immortalization of brain capillary endothelial cells with maintenance of structural characteristics of the blood-brain barrier endothelium

Summary Early passage bovine brain capillary endothelial cells were immortalized by transfection with the plasmid pSV3 neo. Cells from one clone, SV-BEC, expressed nuclear SV 40 large T antigen, displayed a contact-inhibited and anchorage-dependent proliferation, and a high sensitivity to the addition of exogenous basic fibroblast growth factor. SV-BEC cells are morphologically unaltered and express typical markers of endothelial cells: Factor VIII-related antigen, angiotensin-converting enzyme andGriffonia simplicifolia agglutinin binding site. Endothelium like immunoreactivity was detected in the conditioned medium from these cells. Moreover, SV-BECs present numerous intercellular tight junctions characteristic of the blood-brain barrier and possess functionalβ1- andβ2-adrenergic receptors, as observed on isolated bovine brain capillaries.     

Antiviral CD8 T cells recognize borna disease virus antigen transgenically expressed in either neurons or astrocytes

Borna disease virus (BDV) can persistently infect the central nervous system (CNS) of mice. The infection remains nonsymptomatic as long as antiviral CD8 T cells do not infiltrate the infected brain. BDV mainly infects neurons which reportedly carry few, if any, major histocompatibility complex class I molecules on the surface. Therefore, it remains unclear whether T cells can recognize replicating virus in these cells or whether cross-presentation of viral antigen by other cell types is important for immune recognition of BDV. To distinguish between these possibilities, we used two lines of transgenic mice that strongly express the N protein of BDV in either neurons (Neuro-N) or astrocytes (Astro-N). Since these animals are tolerant to the neo-self-antigen, we adoptively transferred T cells with specificity for BDV N. In nontransgenic mice persistently infected with BDV, the transferred cells accumulated in the brain parenchyma along with immune cells of host origin and efficiently induced neurological disease. Neurological disease was also observed if antiviral T cells were injected into the brains of Astro-N or Neuro-N but not nontransgenic control mice. Our results demonstrate that CD8 T cells can recognize foreign antigen on neurons and astrocytes even in the absence of infection or inflammation, indicating that these CNS cell types are playing an active role in immune recognition of viruses.     

Cell type specificity of a neural cell surface antigen recognized by the monoclonal antibody A2B5

Summary The monoclonal antibody A2B5 reacts with the surface membrane of most neurons in monolayer cultures of cerebellum, retina, spinal cord, and dorsal root ganglion of embryonic and early postnatal C57BL/6J mice maintained in vitro for culture periods of 2 to 10 days. A small percentage of astroglial cells also expresses A2B5 antigen in murine, chicken and rabbit cerebellum, in chicken retina, and in murine spinal cord and dorsal root ganglion. Less mature astroglial cells are stained for A2B5 antigen to a greater extent than the more mature astrocytes. Astrocytes from rat cerebellum and mouse retina were not found to express A2B5 antigen under the present culture conditions. Some of the less mature oligodendrocytes recognized by 04 antibodies express A2B5 antigen, while the more mature 01 antigen and galactocerebroside-positive oligodendrocytes were not found to be A2B5 antigen-positive. Fibroblasts or fibroblast-like cells do not express detectable levels of A2B5 antigen. After fixation of the cells with paraformaldehyde and ethanol, all cell types present in culture are labeled by the A2B5 antibody intracellularly.