In vitro effects of zinc on the cytokine production from peripheral blood mononuclear cells in patients with zinc allergy.

Metals, such as nickel, cobalt, chromium and zinc, are ubiquitous in the environment. Systemic reactions, including hand dermatitis and generalized eczematous reactions, can be caused by the dietary ingestion of metals. In this study, we aimed to determine whether the cytokine production from peripheral blood mononuclear cells (PBMCs) obtained from zinc allergy patients can be used as a sensitive marker to investigate zinc-allergic contact dermatitis. The diagnosis of sensitivity to metal was made based on the results of a metal patch test. The PBMCs were stimulated with various concentrations (5-100 μM) of zinc sulfate (ZnSO4) for 24 h. The culture supernatants were collected and analyzed using ELISA for measurement of the cytokine production. The levels of IFN-γ, TNF-α, IL-1β, IL-5, IL-13 and MIF were significantly higher in the zinc-allergic patients (n = 5) than in the healthy controls (n = 5) at 100 μM of ZnSO4 stimulation. Although, patch testing is considered as standard test to diagnose metal allergy but false-positive and -negative reactions may limit its use in conditions of existing dermatitis. Therefore, this study suggest that in support of patch testing the determination of cytokine production using PBMCs cultures would be helpful for making an early diagnosis of such conditions

Transduction of SIV-Specific TCR Genes into Rhesus Macaque CD8+ T Cells Conveys the Ability to Suppress SIV Replication

The SIV/rhesus macaque model for HIV/AIDS is a powerful system for examining the contribution of T cells in the control of AIDS viruses. To better our understanding of CD8(+) T-cell control of SIV replication in CD4(+) T cells, we asked whether TCRs isolated from rhesus macaque CD8(+) T-cell clones that exhibited varying abilities to suppress SIV replication could convey their suppressive properties to CD8(+) T cells obtained from an uninfected/unvaccinated animal.We transferred SIV-specific TCR genes isolated from rhesus macaque CD8(+) T-cell clones with varying abilities to suppress SIV replication in vitro into CD8(+) T cells obtained from an uninfected animal by retroviral transduction. After sorting and expansion, transduced CD8(+) T-cell lines were obtained that specifically bound their cognate SIV tetramer. These cell lines displayed appropriate effector function and specificity, expressing intracellular IFNγ upon peptide stimulation. Importantly, the SIV suppression properties of the transduced cell lines mirrored those of the original TCR donor clones: cell lines expressing TCRs transferred from highly suppressive clones effectively reduced wild-type SIV replication, while expression of a non-suppressing TCR failed to reduce the spread of virus. However, all TCRs were able to suppress the replication of an SIV mutant that did not downregulate MHC-I, recapitulating the properties of their donor clones.Our results show that antigen-specific SIV suppression can be transferred between allogenic T cells simply by TCR gene transfer. This advance provides a platform for examining the contributions of TCRs versus the intrinsic effector characteristics of T-cell clones in virus suppression. Additionally, this approach can be applied to develop non-human primate models to evaluate adoptive T-cell transfer therapy for AIDS and other diseases

Correction: Transduction of SIV-Specific TCR Genes into Rhesus Macaque CD8+ T Cells Conveys the Ability to Suppress SIV Replication.

[This corrects the article DOI: 10.1371/journal.pone.0023703.]

Efficient inhibition of SIV replication in rhesus CD4+ T-cell clones by autologous immortalized SIV-specific CD8+ T-cell clones

AbstractCD8+ cytotoxic T lymphocyte (CTL) responses play an important role in controlling the replication of primate lentiviruses. Induction of these responses is a key objective for most current AIDS vaccine approaches. Despite a variety of approaches for measuring properties and activities of CTL, the functions responsible for controlling viral replication in vivo have not been clearly identified. Assays measuring CTL-mediated suppression of viral replication in vitro are beginning to be used as possible correlates of in vivo virus suppressive activity, but the utility and interpretive value of these assays are typically limited by properties of the cells that have been used. We investigated the capacity of SIV-specific CTL clones (effectors), immortalized by transduction with human telomerase reverse transcriptase (hTERT), to suppress SIV replication in autologous hTERT immortalized CD4+ T-cell clones (targets). Immortalized and non-immortalized SIV-specific effector cells showed IFN-γ production and degranulation in response to viral antigen specific stimulation and significantly inhibited SIVmac239 replication (2 to 4 log decrease in viral RNA or cell-associated proviral DNA) (p<0.0005). Our in vitro assays of inhibition of viral replication, using T-cell clones as effectors and targets, provide a well-defined approach for evaluating possible mechanisms of CTL-mediated control of viral production which may involve direct killing of infected target cells and/or release of proinflammatory cytokines such as IFN-γ and TNF-α. The use of hTERT immortalized effector and target cells for such assays preserves relevant functional properties while providing a convenient, reproducible means of conducting studies over time

Diagram of TCR expressing retroviral vector and the mature TCR chains.

<p>The MSGV1 murine retroviral vectors sequences are displayed as black boxes and lines while the TCR expression cassette is in white. The fine structure of the TCR chain fusion cassette is presented below the vector with the different MamuA*01-restricted TCRs molecularly-cloned from DAJ T-cell clones that were inserted into the vectors indicated above the cassette. The sequences separating the TCR genes, the furin recognition sequence, KAKR, the S-G-S-G spacer, and the P2A fowl pox self-cleaving peptide, are shaded gray. The furin cleavage site and the P2A self-cleavage site are indicated below the cassette with arrows. The mature α and β chains produced by this vector are displayed at the bottom of the figure.</p

Flow cytometry analysis of transduced T cells.

<p>A, analysis of the TCR-transduced EZP cell lines for CM9 peptide/MHC tetramer and SL8 peptide/MHC tetramer is presented with that of the untransduced CD8⁺ control cell line from recipient animal EZP. B, tetramer analysis of two SIV-specific CTL clones isolated from donor animal DAJ is presented above tetramer-sorted TCR transduced CD8⁺ cell lines. The DAJ SL8–42 clone is the TCR gene donor for the SL8–42 TCR EZP cell line.</p

In <i>vitro</i> virus suppression assay of TCR-transduced cell lines.

<p>Flow cytometry analyses of mixed cultures consisting of effector CD8⁺ T-cell lines and a target autologous CD4⁺ T-cell clone that was untreated or exposed to either wild-type SIV_mac239 or SIV_myr- are presented. Effectors are labeled above each column and targets are labeled at the left of each row. The effector CD8⁺ T cells in the co-cultures were stained with CellTrace Violet® and excluded from the analysis so that only the target cells were counted.</p