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It is now well documented that the mode of primary stimulation in the mouse determines the ratio of Th1-, Th0- or Th2-type T cells obtained. In this paper we determine whether driving and cloning human peripheral blood mononuclear cells (PBMC) non-specifically with interleukin-2 (IL-2) and/ or IL-4 and mitogen, will differentially select T cells for IL-10 production. The presence of IL-2 during culture (with or without IL-4) yielded predominantly Th1-type clones (81% of clones with IL-2 alone and 77% with IL-2 + IL-4) and a low frequency of Th0-type clones (19% of clones with IL-2 and 10% of clones with IL-2 + IL-4), whereas IL-4 alone increased the yield of the IL-4 producing Th0 (35%) clones. The proportions of IL-2-producing clones did not alter with treatment; however, the combination of IL-2 + IL-4 altered the proportions of IL-10-producing clones. 47% of IL-2-cultured cells and 51% of IL-4-cultured cells produced IL-10 respectively, whereas only 22% of clones driven with IL-2 + IL-4 produced detectable levels of IL-10. Thus in the preliminary experiments described here we have demonstrated that the cytokines used to initially drive out human T cells and maintain the T-cell growth can skew the Th1/Th2/Th0 cytokine profiles of the clones obtained from mitogen-stimulated cultures. Moreover and unexpectedly the proportion of IL-10-producing cells is altered. These results are important in interpreting analysis of the cytokine profiles of human T cells and raise questions concerning how we should derive T-cell clones for a cytokine analysis that truly reflects the in vivo situation. [ABSTRACT FROM AUTHOR]

Summary T cells simultaneously producing multiple cytokines and possessing cytotoxic capacity termed polyfunctional cells (PFCs) are increasingly recognized as the immune correlate of protection against pathogenic viruses. We investigated co-expression of four cytokines (interferon-γ, macrophage inflammatory protein 1-α, tumour necrosis factor-α and interleukin-2) and degranulation capacity (CD107a surface expression) of Epstein-Barr virus (EBV) -specific CD4+ and CD8+ T cells upon stimulation by overlapping peptides of EBV lytic (BZLF1) and latent (EBNA1, EBNA3 and LMP2) proteins, in 20 healthy Chinese long-term carriers. Two patients with post-transplant lymphoproliferative disorder (PTLD), who had impaired T-cell immunity, were studied for comparison. Both EBV-specific CD4+ and CD8+ PFCs were readily generated in long-term carriers and showed immunodominance hierarchies of latent proteins (EBNA1 > EBNA3/LMP2 and EBNA3 > LMP2 > EBNA1 for CD4+ and CD8+ T cells, respectively), as evidenced by a higher proportion of PFCs generated by immunodominant EBV proteins than by subdominant viral proteins. In contrast, the proportion of EBV-specific PFCs was markedly decreased in patients with PTLD. The EBV-specific PFCs produced more cytokine per cell than single-functional T cells and comprised different subsets. Five-functional CD4+ and CD8+ T cells were detected and four-functional CD4+ T cells were mainly CD107a negative and expressed all four cytokines whereas four-functional CD8+ T cells were mainly CD107a positive and expressed three of the four cytokines (interleukin-2-negative). We conclude that EBV-specific PFCs are generated in much higher proportions in the long-term carriers than in the patients with PTLD and maintain the immunodominant characteristics of the virus. [ABSTRACT FROM AUTHOR]

Hepcidin regulates intracellular iron levels by interacting with and promoting the degradation of ferroportin, a membrane protein and the only known cellular iron exporter. Studies of hepcidin expression and regulation have focused on its effects in innate immunity and as a regulator of systemic iron metabolism. In the present study we characterized the expression of hepcidin messenger RNA (mRNA) in human peripheral blood mononuclear cells (PBMCs) with a focus on peripheral blood lymphocytes (PBLs). We found that (1) all human PBMCs analyzed express basal hepcidin mRNA levels; (2) hepcidin mRNA expression increases after T-lymphocyte activation; (3) expression by PBLs increases in response to challenge by holotransferrin (Fe-TF) and by ferric citrate in vitro; (4) the Fe-TF-mediated up-regulation of hepcidin decreases ferroportin expression at the cytoplasmic membrane of PBLs; and (5) silencing of tumour necrosis factor-α (TNF-α) abrogates the effect of Fe-TF. In summary, we show that hepcidin expression determines intracellular iron levels by regulating the expression of ferroportin, as described in other cells, and that inappropriately low expression of hepcidin impairs normal lymphocyte proliferation. The results establish hepcidin as a new player in lymphocyte biology. [ABSTRACT FROM AUTHOR]

We used various culture conditions to generate type 1 (Tc1) or type 2 (Tc2) cytotoxic T cells in vitro. T-cell receptor (TCR) transgenic T cells were cultured with antigen and spleen cells, or antigen and dendritic cells (DC), or anti-CD3 and anti-CD28. Tc1 cultures contained interleukin (IL)-2 and IL-6, and Tc2 cultures contained IL-2, IL-6 and IL-4. Tc2 cells generated in each culture condition acquired a CD62Llow CD44high phenotype, had high cytotoxic activity, and secreted IL-4, IL-5 and moderate amounts of interferon-γ (IFN-γ). In contrast, the phenotype and function of Tc1 cells varied depending on culture conditions. Tc1 cells from anti-CD3 and anti-CD28 cultures had high cytotoxic activity and were CD62Llow CD44high, while Tc1 cells from antigen and spleen cell cultures had low cytotoxic activity and were CD62Lhigh CD44low. Tc1 cells from antigen and DC cultures had an intermediate phenotype. All Tc1 cells secreted high amounts of IFN-γ, but only Tc1 from anti-CD3 and anti-CD28 cultures had antitumour activity in vivo. Differences were not caused by suboptimal culture conditions, as Tc1 cells divided at a similar rate whether cultured with antigen and spleen cells or with anti-CD3 and anti-CD28. We conclude that IL-4 not only induces‘type 2’ cytokine secretion in CD8+ T cells, but also affects their expression of surface markers and cytotoxic activity. [ABSTRACT FROM AUTHOR]

Since it was previously shown that protein antigens applied epicutaneously in mice induce allergic dermatitis mediated by production of T helper 2 (Th2) cytokines we postulated that this might induce suppression of Th1 immunity. Here we show that epicutaneous immunization of normal mice with a different protein antigen applied on the skin in the form of a patch induces a state of subsequent antigen-non-specific unresponsiveness caused by suppressor T cells (Ts) that inhibit sensitization and elicitation of effector T-cell responses. Suppression is transferablein vivobyαβ-T-cell receptor CD4+ CD8+ double positive lymphocytes harvested from lymphoid organs of skin patched animals and are not major histocompatibility complex-restricted nor antigen specific. Both CD25+ and CD25– CD4+ CD8+ T cells are able to suppress adoptive transfer of Th1 effector cells mediating cutaneous contact sensitivity.In vivotreatment with monoclonal antibodies showed that the cytokines interleukin (IL)-4, IL-10 and transforming growth factor-β (TGF-β) are involved in the induction of the Ts cells. Additionally, using IL-10–/– mice we found that IL-10 is involved in skin induced tolerance. Furtherin vitroexperiments showed that lymph node cells of skin tolerized mice non-specifically suppress[3H]thymidine incorporation by antigen-stimulated immune cells and this effect can be abolished by adding anti-TGF-β, but not anti-IL-4 nor anti-IL-10 antibodies. These studies indicate the crucial role of TGF-β in skin induced tolerance due to non-antigen-specific Ts cells and also show that IL-4, IL-10 and TGF-β play an important role in the induction of epicutaneously induced Ts cell suppression. [ABSTRACT FROM AUTHOR]

Summary Previous study suggested that MRL‐lpr/lpr mice treated with tamoxifen (TAM) had less severe proteinuria, reduced serum titre of anti‐dsDNA autoantibodies and an increased survival rate. To investigate further the regulatory mechanisms of TAM on MRL‐lpr/lpr female mice, a total dose of 200 µg per mice (5·5 mg/kg) was given every 2 weeks subcutaneously, while the control mice were injected with oil only. After being treated with TAM four times, the mice were killed and cellular functions were evaluated. The TAM‐treated groups had smaller sized spleen and lymph nodes. Flow cytometric analysis of splenocytes had a significantly lower percentage of cell number of T cells and double negative T cells (CD4– CD8– T cells). There was no difference in cytokine production (interleukin (IL)‐2, IL‐4, IL‐5, IL‐10 and interferon‐γ (IFN‐γ)) from splenocytes stimulated with concanavalin A (Con A) or cytokines (IL‐6) secreted by peritoneal exudate cells when stimulated with lipopolysaccharide (LPS). However, IL‐2 from lymph node cells was significantly higher on TAM‐treated mice. Finally, splenocytes or purified T cells stimulated with anti‐CD3 antibody plus cross‐linking immunoglobulin G (IgG) of the TAM‐treated group had higher 3H‐incorporation of proliferation assay compared with that of control groups. In vitro study further demonstrated that IL‐2‐activated proliferation of lymph node double negative (DN) T cells can be inhibited by TAM treatment in a dose‐dependent manner. Our finding demonstrated that TAM may potentially influence T cells and modulate the immune function, which offers a novel approach to explore the feasibility of hormone therapy for autoimmune diseases. [ABSTRACT FROM AUTHOR]