Double-negative (CD4- CD8-) T cells with an alpha/beta T cell receptor. Non-MHC-restricted cytolytic activity and lymphokine production

T cell lines with a novel phenotype (CD3+ TCR-alpha/beta+ CD4- CD8-) were developed from the peripheral blood of a patient with a combined immunodeficiency and tissue injury resembling graft-vs-host disease. One of these IL-2-dependent T cell lines demonstrated non-MHC-restricted cytolytic function against tumor targets, syngeneic and allogeneic fibroblasts, and PHA blasts from allogeneic donors. The other cell line only became cytotoxic in the presence of lectin or anti-CD3 antibody. The two cell lines also differed in their expression of the T-200 gene products CD45RO (gp180) and CD45RA (gp220). Both cell lines produced tumor necrosis factor-alpha and -beta and IFN-gamma activity when activated with mitogens or PMA and IL-1. The in vitro functions of these T-cell lines suggest a potential role for alpha/beta double-negative T lymphocytes in tissue injury resembling graft-vs-host disease.     

人淋巴毒素基因5′端上游NF－KB类因子特异性结合位点的研究

淋巴毒素（Ｌｙｍｐｈｏｔｏｘｉｎ,ＬＴ）是由活化的Ｔ淋巴细胞分泌的一种糖蛋白。ＬＴ基因的表达调控主要是在转录水平上。ＰＨＡ＋ＰＭＡ诱导的Ｊｕｒｋａｔ细胞总ＲＮＡ点渍杂交发现,Ｊｕｒｋａｔ细胞的内源性ＬＴｍＲＮＡ的数量随诱导时间的延长而增加。凝胶迟滞电泳分析发现,ＰＨＡ＋ＰＭＡ诱导４小时的Ｊｕｒｋａｔ细胞核抽提物形成多种复合物,且在诱导不同时间后有明显变化。系列缺失片段的竞争反应表明,这些复合物的形成与ＬＴ基因５＇端上游──１２８ｂｐ──９３ｂｐ左右的序列相关。ＤＮａｓｅｌ足迹法及其竞争实验发现,ＬＴ基因５＇端上游─—１０４ｂｐ──８３ｂｐ（共２２ｂｐ）序列具有明显的蛋白质保护足迹。同源性分析发现此２２ｂｐ的序列中存在一个ＫＢ样结合位点：─１００ｂｐ──９０ｂｐ（５’－ＧＧＧＧＧＣＴＴＣＣＣ－３’）。ＮＰ－ＫＢ类蛋白质因子参与了此序列的ＤＮＡ－蛋白质的特异性结合,可能存在多种类型的ＮＰ－ＫＢ类蛋白因子参与了ＬＴ基因的表达调控。     

Differential regulation of fibronectin receptor subunit gene and cell surface expression in human peripheral blood T lymphocytes.

Members of the beta 1 subfamily of heterodimeric integrins, such as the fibronectin receptors alpha 5 beta 1 and alpha 4 beta 1, are expressed on human T lymphocytes. The presence of these two adhesion receptors on T lymphocytes suggests an involvement in cell-cell and cell-extracellular matrix interactions that may be important for the development of immune and inflammatory reactions. We have examined the cell surface expression of alpha 5, alpha 4, and beta 1 subunits on purified peripheral blood T lymphocytes before and after activation with Con A and PMA. Freshly isolated T lymphocytes contained distinct fractions expressing high or low levels of alpha 5 and beta 1. Only a high expressing T lymphocyte population was present after 72-h culture with Con A and PMA. Time course analysis indicated that the shift in alpha 5 and beta 1 expression occurred during the first 24 h after addition of activating agents and occurred in the absence of proliferation. In contrast to alpha 5 and beta 1, essentially all freshly isolated T lymphocytes expressed high levels of alpha 4. After 72-h culture with Con A and PMA, a wide distribution of alpha 4 expression was observed. Further experiments showed that after activation, a proportion of CD4-positive cells decreased their surface expression of alpha 4, but increased their surface expression of alpha 5 and beta 1. In contrast, most CD8-positive cells increased their surface expression of alpha 5, beta 1, and alpha 4 upon activation. An examination of mRNA levels in pan-T lymphocyte cultures after activation indicated that alpha 5 and alpha 4 mRNA expression decreased, whereas beta 1 mRNA expression was unchanged, in Con A/PMA-activated cells as compared to those cultured in medium alone. Our results indicate that T lymphocyte activating agents may differentially affect the expression of alpha 5 beta 1 and alpha 4 beta 1, thus providing a mechanism for the selective regulation of binding interactions that occur at sites of immune reactions.     

Human T-cell hybridomas producing lymphokines. II. Enhancement of lymphotoxin secretion from human T-cell hybridomas by phorbol myristate acetate

Human lymphotoxin (LT)-producing T-cell hybridomas were constructed by fusing concanavalin A-activated human peripheral blood lymphocytes with emetine-actinomycin D-pretreated human acute lymphatic leukemia cells. LT secretion from these hybridomas was considerably enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA) and concanavalin A or PMA alone. A study using cloned hybrid lines revealed that PMA/Con A acted directly on the LT-producing clones. Furthermore, PMA/Con A stimulated A-B9-24, one of the cloned hybridomas, and secreted fourfold larger amounts of LT under serum-free conditions than under serum-containing conditions. However, MIF/MAF and LT-producing cloned hybrid line E10-20 secreted rather decreased amounts of MIF/MAF when stimulated with PMA, while the LT secretion from the same hybridoma was enhanced with PMA.     

Regulation of the expression of IL-6 in human monocytes

IL-6 is a cellular regulatory molecule with various cell-dependent functions. We have studied the control of IL-6 expression in human monocytes because they play a key role in the production of this molecule. The effects of adherence and different cytokines including CSF-1, IFN-gamma, IL-1 alpha, and granulocyte-macrophage-CSF were tested on IL-6 expression. IL-6 mRNA was usually not detected in the starting population of PBMC. Adherence induced IL-6 gene expression in monocytes in less than 2 h and subsequently IL-6 secretion. Priming of monocytes by adherence was more efficient for IL-6 overinduction by CSF-1. In contrast, high level induction of IL-6 by IFN-gamma in unfractionated PBMC did not require adherence and in situ hybridization revealed that IL-6 mRNA was present in monocytes but not in lymphocytes. A similar phenomenon was observed for IL-1 alpha and granulocyte-macrophage-CSF. Two cell lines, HL-60 and U937, in which monocytic differentiation occurs after induction by PMA, were subsequently investigated. IL-6 was not constitutively detectable in these two cell lines, whereas PMA treatment induced IL-6 expression. This effect was rapid (30 min) and transitory in HL-60, whereas IL-6 mRNA was still detected after 72 h of induction in U937. Addition of human rIL-6 on U937 and HL-60 cells inhibited their proliferation and enhanced expression of HLA class I Ag.     

Expression of surface lymphotoxin and tumor necrosis factor on activated T, B, and natural killer cells.

The expression of membrane-associated forms of lymphotoxin (LT) and TNF were examined on cell lines of T, B, and myeloid origin, IL-2 dependent T cell clones, and peripheral blood lymphocytes. Inducible and constitutive patterns of surface LT expression were found on T cells as exemplified by the II-23.D7, a CD4+T cell hybridoma, and HUT-78, a T cell lymphoma. Phorbol ester induced surface LT expression on Ramos, an EBV transformed B cell line, but at a slower rate of appearance when compared to the II-23.D7. Secretion of LT was rapidly inducible by phorbol ester in II-23.D7 and also in HUT-78 but with slower kinetics; surface LT expression continued in both lines after secretion had ceased. Low levels of membrane TNF were transiently induced on II-23.D7 and HUT-78, but none was observed on Ramos. Peripheral blood monocytes and some myeloid tumor lines did not express surface LT. Several T cell clones expressed surface LT after Ag-specific stimulation, and expression persisted several days. Stimulation through the TCR or by IL-2 rapidly induced surface LT on resting peripheral T cells and CD56+ NK cells; pokeweed mitogen activation induced expression on CD20+ B cells. Consistent with previous results, immunoprecipitation with anti-LT mAb showed that LT was complexed with a distinct 33 kDa glycoprotein (p33) on cells that expressed surface LT, whereas secreted LT was not associated with p33. Surface and secreted modes of LT expression by activated T, B, and NK cells suggests that LT can be utilized as either a localized or diffusible mediator in immune responses.     

T cell differentiation model based on the expression of T cell receptor chains and genes--study in the human leukemia/lymphoma cell lines

A total of 33 human leukemia/lymphoma cell lines were classified into 4 groups with respect to the pattern of cell membrane (sm) expression of the CD3 and T cell receptor (TCR) molecules; (i) smCD3+TCR alpha beta (16 cell lines), (ii) smCD3+TCR beta delta (1 cell line), (iii) smCD3+TCR gamma delta (3 cell lines) amd (iv) smCD3-TCR- (13 cell lines), respectively. Using monoclonal antibodies (MoAbs) specific to CD3 (NU-T3), TCR alpha chain (alpha F1), TCR beta chain (beta F1), and TCR gamma chain (C gamma M1), respectively, cytoplasmic (cy) expression of these molecules was determined by immunofluorescence test. Expression of cyCD3 was present in all cell lines regardless of groups. In group (i), all 16 cell lines expressed both TCR alpha and beta chains. While only TCR beta chain was expressed in group (ii), TCR gamma chain was expressed in all 3 cell lines of group (iii). One (PEER) of the three in group (iii) expressed TCR beta chain as well. In group (iv), we found 8 cell lines with cyTCR alpha expression, 11 cell lines with cyTCR beta expression, and 10 cell lines with cyTCR gamma expression, respectively. For TCR genes, except 1 cell line all cell lines were found to present rearranged C beta gene and its mRNA, including all 3 TCR gamma/delta cell lines of group (iii). One of the TCR alpha beta cell lines exhibited rearranged C delta and J delta genes as well as its mRNA. Two cell lines of the 13 CD3-TCR- of group (iv) exhibited rearranged C delta and J delta and its mRNA. An NK-like activity and IL-2 production were induced in the TCR beta delta and gamma delta cell lines [group (ii) and (iii)] by treatment with PHA and PMA.     

Regulated expression of the Mac-1, LFA-1, p150,95 glycoprotein family during leukocyte differentiation

The regulation of Mac-1, LFA-1, and p150,95 expression during leukocyte differentiation was examined. LFA-1 was present on almost all cell types studied. Both Mac-1 and p150,95 were present on the more mature cells of the myelomonocytic series, but only p150,95 was detected on some B cell lines and cloned cytotoxic T lymphocytes. Phorbol myristate acetate (PMA) stimulation of B chronic lymphocytic leukemia cells dramatically increased p150,95 expression. The resultant Mac-1, LFA-1, p150,95 phenotype resembled hairy cell leukemia, a B cell plasmacytoid leukemia. The promonocytic cell line U937 and the promyeloblastic cell line HL-60 expressed only LFA-1. Monocytic differentiation of U937 cells was stimulated by PMA, and induced the concomitant expression of Mac-1 and p150,95, with more p150,95 induced than Mac-1. Granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulation of U937 cells gave similar results. PMA-stimulated monocytic differentiation of the HL-60 cell line also induced expression of both Mac-1 and p150,95. The number of p150,95 molecules on PMA-stimulated U937 and HL-60 cells were 5 X 10(5) and 3 X 10(5), respectively. Retinoic acid stimulated myeloid differentiation of HL-60 cells and induced expression of both Mac-1 and p150,95. These cells acquired a Mac-1, LFA-1, p150,95 profile that resembled that of granulocytes, with more Mac-1 than p150,95 induced. GM-CSF stimulation of HL-60 cells induced a similar Mac-1 and p150,95 phenotype. The contributions of Mac-1, LFA-1, and p150,95 to aggregation of PMA-differentiated U937 cells were assessed. Monoclonal antibodies to the beta subunit and the LFA-1 alpha subunit, but not those to p150,95 or Mac-1 alpha subunit, inhibited this homotypic adherence.