Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells.

The technique of DNA transfer by electroporation was investigated in an effort to evaluate its utility for the identification of developmentally controlled regulatory sequences. Transient and stable gene expression was detected in a variety of lymphoid cell lines subjected to electroporation. No correlation existed between the levels of chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) expression and stable transfection frequency. In all lymphoid cell lines tested, the simian virus 40 early region was a better promoter than was the Rous sarcoma virus long terminal repeat.     

Acquisition of MHC:Peptide Complexes by Dendritic Cells Contributes to the Generation of Antiviral CD8+ T Cell Immunity In Vivo

There is an increasing body of evidence suggesting that the transfer of preformed MHC class I:peptide complexes between a virus-infected cell and an uninfected APC, termed cross-dressing, represents an important mechanism of Ag presentation to CD8(+) T cells in host defense. However, although it has been shown that memory CD8(+) T cells can be activated by uninfected dendritic cells (DCs) cross-dressed by Ag from virus-infected parenchymal cells, it is unknown whether conditions exist during virus infection in which naive CD8(+) T cells are primed and differentiate to cytolytic effectors through cross-dressing, and indeed which DC subset would be responsible. In this study, we determine whether the transfer of MHC class I:peptide complexes between infected and uninfected murine DC plays a role in CD8(+) T cell priming to viral Ags in vivo. We show that MHC class I:peptide complexes from peptide-pulsed or virus-infected DCs are indeed acquired by splenic CD8α(-) DCs in vivo. Furthermore, the acquired MHC class I:peptide complexes are functional in that they induced Ag-specific CD8(+) T cell effectors with cytolytic function. As CD8α(-) DCs are poor cross-presenters, this may represent the main mechanism by which CD8α(-) DCs present exogenously encountered Ag to CD8(+) T cells. The sharing of Ag as preformed MHC class I:peptide complexes between infected and uninfected DCs without the restraints of Ag processing may have evolved to accurately amplify the response and also engage multiple DC subsets critical in the generation of strong antiviral immunity.     

Cutting edge: adaptive versus innate receptor signals selectively control the pool sizes of murine IFN-γ- or IL-17-producing γδ T cells upon infection

γδ T lymphocytes are commonly viewed as embracing properties of both adaptive and innate immunity. Contributing to this is their responsiveness to pathogen products, either with or without the involvement of the TCR and its coreceptors. This study clarifies this paradoxical behavior by showing that these two modes of responsiveness are the properties of two discrete sets of murine lymphoid γδ T cells. Thus, MyD88 deficiency severely impaired the response to malaria infection of CD27((-)), IL-17A-producing γδ T cells, but not of IFN-γ-producing γδ cells. Instead, the latter compartment was severely contracted by ablating CD27, which synergizes with TCRγδ in the induction of antiapoptotic mediators and cell cycle-promoting genes in CD27((+)), IFN-γ-secreting γδ T cells. Hence, innate versus adaptive receptors differentially control the peripheral pool sizes of discrete proinflammatory γδ T cell subsets during immune responses to infection.     

Immunoregulation in the tissues by gammadelta T cells

For a T-cell subset to be classified as immunoregulatory, it might reasonably be predicted that in its absence, animals would experience pathological immune dysregulation. Moreover, reconstitution of the subset should restore normal immune regulation. So far, these criteria have been satisfied by only a few of the candidate regulatory T-cell subsets, but among them is the intraepithelial gammadelta T-cell receptor (TCR)+ subset of mouse skin. In this article, we look at immunoregulatory gammadelta T cells, and the growing evidence for tissue-associated immunoregulation mediated by both gammadelta T cells and alphabeta T cells.     

Detection in situ of foreign DNA in eukaryotic cells

A simple technique is described that allows mixed populations of eukaryotic cells to be screened for clones containing multiple copies of a particular DNA. Essentially, eukaryotic cells are transferred to either nitrocellulose of Whatman 541 filters, and their DNA is immobilised in situ. Exposure of the filters to a 32P-labeled DNA "probe" results in detectable hybridisation only at the positions of clones containing multiple copies of the DNA. Using Whatman 541 paper, a portion of the cells, evenly distributed throughout the mixed population is retained on the culture dish, and can be propagated further for subsequent cell cloning. The technique has allowed rapid distinction of clones of transformed rat cells that contain a single or only a few copies per cell of polyoma viral DNA from clones maintaining multiple copies. The technique has also been used to distinguish between clones of mouse L-cells containing multiple and only a few copies of 0X174 DNA. In this manner the technique allows rapid detection of cells amplifying a particular species of DNA. Finally, the method can be used to detect cells assimilating many copies of a foreign DNA, even in the absence of a co-transfected selectable marker.     

Gamma(delta) T cells: non-classical ligands for non-classical cells

A recent study describes direct binding between a gammadelta T-cell receptor and its ligand, T22, a non-classical class I major histocompatibility complex (MHC) molecule. A companion study, solving the crystal structure of T22, highlights the differences between this interaction and those of classical MHC molecules and alphabeta T cells.     

Distinct cytokine-driven responses of activated blood gammadelta T cells: insights into unconventional T cell pleiotropy

Human Vgamma9/Vdelta2 T cells comprise a small population of peripheral blood T cells that in many infectious diseases respond to the microbial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), expanding to up to 50% of CD3(+) cells. This "transitional response," occurring temporally between the rapid innate and slower adaptive response, is widely viewed as proinflammatory and/or cytolytic. However, increasing evidence that different cytokines drive widely different effector functions in alphabeta T cells provoked us to apply cDNA microarrays to explore the potential pleiotropy of HMB-PP-activated Vgamma9/Vdelta2 T cells. The data and accompanying validations show that the related cytokines, IL-2, IL-4, or IL-21, each drive proliferation and comparable CD69 up-regulation but induce distinct effector responses that differ from prototypic alphabeta T cell responses. For example, the Th1-like response to IL-2 also includes expression of IL-5 and IL-13 that conversely are not induced by IL-4. The data identify specific molecules that may mediate gammadelta T cell effects. Thus, IL-21 induces a lymphoid-homing phenotype and high, unexpected expression of the follicular B cell-attracting chemokine CXCL13/BCA-1, suggesting a novel follicular B-helper-like T cell that may play a hitherto underappreciated role in humoral immunity early in infection. Such broad plasticity emphasizes the capacity of gammadelta T cells to influence the nature of the immune response to different challenges and has implications for the ongoing clinical application of cytokines together with Vgamma9/Vdelta2 TCR agonists.