Induction of antibody responses to influenza virus in human lymphocyte cultures. I. Role of interleukin 2.
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The in vitro T cell-dependent antibody response of human lymphocytes to influenza virus X31 was used to study the role of T cell-derived lymphokines in antigen-specific responses. Supernatant from cultures of phytohaemagglutinin-stimulated, pooled human tonsil cells (PHA-MLR) was capable of replacing T cells and inducing T-depleted tonsil cells to secrete influenza-specific antibody. The T cell-replacing activity of PHA-MLR supernatant co-purified with interleukin 2 (IL 2) on Ultrogel AcA54 gel filtration and reversed phase-high performance liquid chromatography. PHA-MLR supernatant and IL 2 also enhanced B cell proliferation induced by anti-mu or Staphylococcal aureus strain Cowan I (SAC). A murine monoclonal antibody directed against the human IL 2 receptor (Mab 2A3) was used to completely block the enhancement of influenza-specific antibody production mediated by PHA-MLR supernatant, purified IL 2, and recombinant human IL 2. Mab 2A3 did not affect the T-independent B cell proliferation induced by anti-mu or SAC, but abrogated the enhancing effect of the PHA-MLR supernatant and IL 2 in this culture system. Immunofluorescence studies failed to demonstrate binding of Mab 2A3 to B cells activated by the X31 influenza virus and IL 2, or by SAC. By using Mab 2A3 to mask out IL 2 effects in the influenza-specific culture system, no other B cell differentiating activities were revealed in supernatants from lymphocytic cultures stimulated with a variety of mitogens. Thus, our results indicate that the production of influenza-specific antibodies by T-depleted human lymphocyte cultures is absolutely dependent on the presence of both antigen and IL 2.Keywords:
Phytohaemagglutinin
We have previously described a variant murine CTL clone that in contrast to all other clones tested, exhibited a novel capacity to produce IFN-gamma in response to IL-2. This alternative pathway of IFN-gamma induction differed from the conventional TCR complex-mediated pathway in that it was independent of elevated intracellular Ca2+ and insensitive to cyclosporine A. We report here the presence of an analogous pathway in the majority of T lymphocyte clones tested, when these clones are stimulated with IL-2 in the presence of syngeneic or third-party splenocytes. The accessory function of splenocytes in this alternative pathway is mediated by the MAC-1+ subpopulation and apparently involves cell-cell contact. However, the structure with which the MAC-1 antibody reacts probably is not involved directly. No involvement of Ag or the TCR for Ag could be demonstrated in this alternative pathway of lymphokine induction. The array of lymphokines induced by this alternative pathway is only a subset of those induced by antigenic stimulation. Finally, as with the previously described variant clone, IL-2-mediated induction of IFN-gamma production by the normal T lymphocyte clones is independent of normal extracellular Ca2+ levels and insensitive to cyclosporine A. Thus, this alternative pathway of lymphokine induction apparently constitutes a distinct signaling pathway in cloned T lymphocytes.
clone (Java method)
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Epstein-Barrウイルス特異的キラーT細胞活性に及ぼすinterleukin 2 (0.2U/ml), interferon α (500IU/ml), interferon γ (500IU/ml), OK-432 (0.01KE/ml), indomethacin (1μg/ml)の影響を正常ヒトリンパ球を用いて検討し,以下の結果を得た.キラーT活性の増強は測定前日にlymphokine, OK-432を添加した場合にもっとも強い傾向がみられた。10例について,キラーT活性測定8日, 4日, 1日前にinterleukin 2を添加したところ,添加時間に比例してgross % lysis値は上昇したが,非特異的キラー活性の増強が強く, net % lysis値でみた特異的キラーT活性の増強が得られたのは1日前にinterleukin 2を添加した場合だけであった.LymphokineとOK-432はキラーT活性測定前日に, indomethacinはキラーT誘導開始時に添加したところ, interleukin 2とinterferon αはキラーT活性を有意に増強させたが, interferon γ, OK-432, indomethacinには増強効果がみられなかった.同種リンパ芽球様細胞, K-562細胞を標的細胞とした場合,あるいは抗体陰性例のキラーT活性はlymphokineによって増強されなかった.以上のことから, interleukin 2, interferon αはEpstein-Barrウイルスに特異的なキラーT細胞活性を増強し得るものと老えられた.
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We have studied five patients with chronic lymphoeytosis consisting of large granular lymphocytes (LGL). The increased numbers of LGL in these patients had little or no natural killer activity, mediated antibody‐dependent cellular cytotoxicity, and were induced to kill tumour lines after culture for 3 days with interleukin 2 (IL‐2). Patients' LGL showed considerable reactivity with HNK‐1 and AB8.28 monoclonal antibodies (MoAb), whereas positivity for OKM1 and N901 was found m only two subjects, and only one patient reacted with B73.1. No appreciable reactivity has been found with anti‐Tac MoAb in the four patients tested. In the absence of stimulation, the patients’ LGL produced no IL‐2 and only minimal amounts of IL‐1 and interferon (IFN). On stimulation with lipopolysaccharides (for IL‐1) or phytohaemag‐glutinin A (PHA) (for IL‐2 and IFN), they produced IL‐1 and IFN in amounts similar to those produced by normal lymphocytes, but only modest levels of IL‐2. These results indicated that proliferating LGL, like normal LGL, have a secretory capacity. The lack of constitutive lymphokine production, the lack of Tac receptor expression, and the defect in IL‐2 production after PHA stimulation do not support the hypothesis of an autocrine proliferation sustained by a known growth factor.
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Lymphocytosis
Lymphoproliferative response
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T lymphocytes comprise a major class of lymphocytes and are themselves functionally heterogeneous. Some T lymphocyte functions are mediated by soluble products called lymphokines. Different lymphokines promote the activation, growth and differentiation of T and B lymphocytes, macrophages, and hemopoietic cells. Lymphokine production is associated with, but not limited to, helper T cells, and usually follows antigenic or mitogenic stimulation. The recognition that some lymphokines are produced after stimulation of neoplastic T cells has proved advantageous in the study of these molecules. T cell tumors are monoclonal, grow easily in vitro, and may produce fewer lymphokines than normal T cells. Thus, the purification and biochemical and biological characterization of some lymphokines have been facilitated by the availability of these tumors. More recently, T cell tumors have been used for evaluating the molecular structure of lymphokine-encoding genes. They have also provided information relevant to our understanding of the nature of T cell neoplasia.
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Phorbol myristate acetate (PMA), a reagent frequently used in vitro to bypass macrophage function and to elicit cytokines, has a variety of effects on the behavior of T lymphocytes in tissue culture. For example, PMA in concentrations greater than 10(-10)M is highly comitogenic with phytohemagglutinin (PHA) for C3H/HeJ thymocytes, and significantly potentiates lymphokine-induced proliferative responses of long-term MLC cells, cloned cytolytic T cells, and interleukin-2 (IL-2)-dependent T-cell lines. Since these activated T-lymphocyte populations are used to detect IL-2, PMA can interfere with accurate assessment of IL-2 concentration by quantitative bioassay. Further, if greater than 10(-10) M PMA remains as a contaminant in PMA-induced lymphokine preparations, it can mediate lymphokine-like biologic activity, and, therefore, obscure interpretation of experiments involving lymphocyte-lymphokine interactions.
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The ability of human lymphocytes to produce soluble mediator(s) with thrombocyte aggregating activity (TAA) was investigated in an in vitro technique. Isolated human mononuclear cells were stimulated with phytohaemagglutinin or purified protein derivative of tuberculin. The supernatants were investigated for lymphokine activity in a leucocyte migration inhibitory factor assay. Supernatants were then tested for their ability to aggregate human thrombocytes, and in contrast to the results of previous studies, we were not able to demonstrate any TAA. Most likely, lymphokines with TAA are not involved in the thrombotic processes seen in human cell‐mediated immune inflammatory reactions.
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The inhibition of migration of human peripheral blood cells in the presence of PPD was studied. It was found that migration inhibition of peripheral blood mononuclear cells (MN) from Mantoux-positive donors was far greater than the migration inhibition of peripheral blood leucocytes (PBL). Moreover, MN cells and T lymphocytes showed larger and more uniform areas of migration. In contrast, the migration of B lymphocytes and monocytes was poor. Further analysis using purified subpopulations of MN cells showed that PPD inhibited the migration of T lymphocytes but not of B lymphocytes and monocytes. Corresponding to these findings, lymphokine-containing supernatants also inhibited the migration of purified T cells from Matoux-negative donors. It was concluded that the T lymphocyte was the predominant cell in the MN cell population, which migrated, and was subject to inhibition by PPD or lymphokines. These results imply that the movement of human T lymphocytes may be influenced by soluble factors from antigen-activated sensitized cells.
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Numerous lymphocytes are recruited from the blood into cutaneous DTH reactions. Alpha/beta-interferon (IFN) and its inducers can recruit lymphocytes into the skin after i.d. injection, but activated T lymphocytes, which are responsible for DTH, produce very little IFN-alpha/beta. Our goal was to determine the major T cell lymphokine (LK) which could stimulate the migration of lymphocytes into the skin. Rats were injected i.d. with LK containing supernatants from activated T cells, and lymphocyte recruitment was measured by the accumulation of 111In-labelled lymphocytes in the skin. Large numbers of labelled cells migrated into sites injected with the LKs. The major portion of the recruiting activity of the LKs coeluted with IFN-gamma after hydroxylapatite and Affigel Blue chromatography, although a second recruiting factor was also found. Both the recruiting and IFN anti-viral activities were partially destroyed by pH 3. A monoclonal anti-IFN-gamma antibody inhibited up to 53% of the recruitment observed after 4 h and up to 43% after 20 h. Kinetic studies showed that maximal recruitment occurred 6 h after i.d. injection of the LKs. Recombinant rat IFN-gamma also stimulated lymphocyte migration into the skin. Histologically, sites injected with IFN-gamma showed a mononuclear cell infiltrate. It is suggested that IFN-gamma is the major mediator of lymphocyte recruitment produced by activated T cells.
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clone (Java method)
Macrophage-activating factor
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