IFN-gamma production dominates the early human natural killer cell response to Coxsackievirus infection

Coxsackieviruses (CV) are important human pathogens that have been implicated in the pathogenesis of several diseases, including myocarditis and pancreatitis. How the human immune system recognizes and controls CV infections is not well understood. Studies in mice suggest that natural killer (NK) cells play a critical role in viral clearance and host survival, but the mechanism(s) by which human NK cells may contribute to the host anti-CV defence has not been investigated. Here we show that CVB3 infection markedly reduces HLA class I cell surface expression but does not increase the expression of the activating NK cell receptor ligands MICA/B and ULBP1-3 on human cells. We also demonstrate that the lowered target cell HLA class I surface expression does not correlate with an increased susceptibility to NK cell-mediated killing. However, NK cells responded with a robust production of interferon γ (IFN-γ) when peripheral blood mononuclear cells were cocultured with infected cells. In summary, this study shows that CVB3 interferes with the expression of NK cell receptor ligands on infected cells and indicates that IFN-γ production, rather than cytotoxicity, marks the early human NK cell response to CVB3 infection.     

Elevated numbers of Fc gamma RIIIA+ (CD16+) effector CD8 T cells with NK cell-like function in chronic hepatitis C virus infection

CTL are crucial in the defense against viral infections. In the course of investigating peripheral blood and intrahepatic CD8 T cells in patients with chronic hepatitis C virus (HCV) infection, we observed a significant population of CD8 T cells expressing the FcgammaRIIIA (CD16) receptor. This observation led us to characterize these cells with respect to their phenotype and function in a cohort of patients with chronic HCV infection as well as in healthy blood donors. On average, 10% of peripheral blood CD8 T cells from HCV-infected patients expressed CD16 compared with only a few percent in healthy donors. CD16(+) CD8 T cells displayed a late-stage effector phenotype with high levels of perforin. These cells exhibited a restricted TCR profile suggesting underlying clonal expansion. Stimulation of CD16 on CD8 T cells evoked a vigorous response similar to that of CD16 stimulation in NK cells. Our data suggest that CD8 T cells, during chronic HCV infection in humans, continue to differentiate beyond defined stages of terminal effector cells, acquiring CD16 and NK cell-like functional properties.     

A short-term dietary supplementation with high doses of vitamin E increases NK cell cytolytic activity in advanced colorectal cancer patients

Cancer patients with advanced disease display signs of immune suppression, which constitute a major obstacle for effective immunotherapy. Both T cells and NK cells are affected by a multitude of mechanisms of which the generation of reactive oxygen species is of major importance. Therefore, we hypothesized that two weeks of high-dose treatment with the anti-oxidant vitamin E may enhance NK cell function in cancer patients by protecting from oxidative stress. Seven patients with colorectal cancer (Dukes stage C and D) received a daily dose of 750 mg of vitamin E during a period of two weeks and the function, phenotype and receptor expression of NK cells were analyzed. The short-term vitamin E treatment significantly improved NK cell cytolytic activity in six out of the seven patients analyzed. The increased NK cell activity in patients’ PBMC was not due to increased numbers of NK cells or an increase in the proportion of the CD56^dim NK cell subpopulation. Furthermore, neither an increased perforin expression nor an enhanced ability of NK cells to produce IFN-γ was observed as a result of vitamin E treatment. Finally, vitamin E treatment was associated with a minor, but consistent, induction of NKG2D expression in all patients analyzed. In conclusion, this pilot study demonstrates that vitamin E may boost NK cell function in patients with colorectal cancer. Further studies are warranted to explore the potential of vitamin E as an adjuvant for immunotherapy against cancer and to determine the underlying mechanism(s) behind vitamin E induced NK cell activation.     

Effective immunotherapy against cancer

During the last decade, the breakthroughs in understanding of the molecular mechanisms responsible for immune activation and the advent of recombinant DNA technologies have changed the view on immunotherapy from a dream scenario to becoming a clinical reality. It is now clear that both cellular immunity comprising T and NK cells, as well as strategies based on antibodies, can provide strong antitumoral effects, and evidence is emerging that these strategies may also cure patients with previously incurable cancers. However, there are still a number of issues that remain unresolved. Progress in immunotherapy against cancer requires a combination of new, improved clinical protocols and strategies for overcoming mechanisms of immune escape and tumor-induced immune suppression. This review discusses some of the salient issues that still need to be resolved, focusing on the role of oxidative stress and the use of antioxidants to alleviate the immune hyporesponsiveness induced by reactive oxygen species (ROS).Abbreviations HLA Human leukocyte antigen - KIR Killer cell immunoglobulin-like receptor - NKR Natural killer cell receptor - ROS Reactive oxygen species - TAA Tumor-associated antigenThis work is part of the Symposium in Writing Tumor escape from the immune response, published in vol 53.     

Interactions of NK cell receptor KIR3DL1*004 with chaperones and conformation-specific antibody reveal a functional folded state as well as predominant intracellular retention

Variable interaction between the Bw4 epitope of HLA-B and the polymorphic KIR3DL1/S1 system of inhibitory and activating NK cell receptors diversifies the development, repertoire formation, and response of human NK cells. KIR3DL1*004, a common KIR3DL1 allotype, in combination with Bw4(+) HLA-B, slows progression of HIV infection to AIDS. Analysis in this study of KIR3DL1*004 membrane traffic in NK cells shows this allotype is largely misfolded but stably retained in the endoplasmic reticulum, where it binds to the chaperone calreticulin and does not induce the unfolded protein response. A small fraction of KIR3DL1*004 folds correctly and leaves the endoplasmic reticulum to be expressed on the surface of primary NK and transfected NKL cells, in a form that can be triggered to inhibit NK cell activation and secretion of IFN-γ. Consistent with this small proportion of correctly folded molecules, trace amounts of MHC class I coimmunoprecipitated with KIR3DL1*004. There was no indication of any extensive intracellular interaction between unfolded KIR3DL1*004 and cognate Bw4(+) HLA-B. A similarly limited interaction of Bw4 with KIR3DL1*002, when both were expressed by the same cell, was observed despite the efficient folding of KIR3DL1*002 and its abundance on the NK cell surface. Several positions of polymorphism modulate KIR3DL1 abundance at the cell surface, differences that do not necessarily correlate with the potency of allotype function. In this context, our results suggest the possibility that the effect of Bw4(+) HLA-B and KIR3DL1*004 in slowing progression to AIDS is mediated by interaction of Bw4(+) HLA-B with the small fraction of cell surface KIR3DL1*004.     

Characterization of natural killer cell phenotype and function during recurrent human HSV-2 infection

Human natural killer (NK) cell differentiation, characterized by a loss of NKG2A in parallel with the acquisition of NKG2C, KIRs, and CD57 is stimulated by a number of virus infections, including infection with human cytomegalovirus (CMV), hantavirus, chikungunya virus, and HIV-1. Here, we addressed if HSV-2 infection in a similar way drives NK cell differentiation towards an NKG2A(-)NKG2C(+)KIR(+)CD57(+) phenotype. In contrast to infection with CMV, hantavirus, chikungunya virus, and HIV-1, recurrent HSV-2 infection did not yield an accumulation of highly differentiated NK cells in human peripheral blood. This outcome indicates that human HSV-2 infection has no significant imprinting effect on the human NK cell repertoire.     

Physiological characteristics of the biocontrol yeast Pichia anomala J121

The yeast Pichia anomala J121 prevents mold spoilage and enhances preservation of moist grain in malfunctioning storage systems. Development of P. anomala J121 as a biocontrol agent requires in-depth knowledge about its physiology. P. anomala J121 grew under strictly anaerobic conditions, at temperatures between 3 degrees C and 37 degrees C, at pH values between 2.0 and 12.4, and at a water activity of 0.92 (NaCl) and 0.85 (glycerol). It could assimilate a wide range of C- and N-sources and produce killer toxin. A selective medium containing starch, nitrate, acetic acid, and chloramphenicol was developed for P. anomala. P. anomala was equally sensitive as Candida albicans to common antifungal compounds. Growth ability at a range of environmental conditions contributes to the competitive ability of the biocontrol yeast P. anomala J121.     

Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay

The activity of proteins is typically regulated by secondary modifications and by interactions with other partners, resulting in the formation of protein complexes whose functions depend on the participating proteins. Accordingly, it is of central importance to monitor the presence of interaction complexes as well as their localization, thus providing information about the types of cells where the proteins are located and in what sub-cellular compartment these interactions occur. Several methods for visualizing protein interactions in situ have been developed during the last decade. These methods in most cases involve genetic constructs, and they have been successfully used in assays of living cell maintained in tissue culture, but they cannot easily be implemented in studies of clinical specimens. For such samples, affinity reagents like antibodies can be used to target the interacting proteins. In this review we will describe the in situ proximity ligation assays (in situ PLA), a method that is suitable for visualizing protein interactions in both tissue sections and in vitro cell lines, and we discuss research tasks when this or other method may be selected.