A dual function of NKG2D ligands in NK-cell activation.

NK-cell killing requires both the expression of activating receptor ligands and low MHC class I expression by target cells. Here we demonstrate that the expression of any of the murine ligands for the NK-cell activating receptor NKG2D results in a concomitant reduction in MHC class I expression. We show this both in tumor cell lines and in vivo. NK-cell lysis is enhanced by the decrease in MHC class I expression, suggesting the change is biologically relevant. These results demonstrate that NKG2D ligand expression on target cells not only allows for activating receptor recognition, but also actively reduces expression of the inhibitory ligand, MHC class I, leading to enhanced recognition and killing by NK cells.

RAE1ε ligand expressed on pancreatic islets recruits NKG2D receptor-expressing cytotoxic T cells independent of T cell receptor recognition.

The mechanisms by which cytotoxic T lymphocytes (CTLs) enter and are retained in nonlymphoid tissue are not well characterized. With a transgenic mouse expressing the NKG2D ligand retinoic acid early transcript 1ε (RAE1ε) in ß-islet cells of the pancreas, we found that RAE1 expression was sufficient to induce the recruitment of adoptively transferred CTLs to islets. This was dependent on NKG2D expression by the CTLs and independent of antigen recognition. Surprisingly, the recruitment of CTLs resulted in the subsequent recruitment of a large number of endogenous lymphocytes. Whereas transgenic mice did not develop diabetes, RAE1 expression was sufficient to induce insulitis in older, unmanipulated transgenic mice that was enhanced by viral infection and pancreatic inflammation. These results demonstrate that the expression of an NKG2D ligand in islets is sufficient to recruit CTLs regardless of their antigen specificity and to induce insulitis.

IL-12 enhances CTL synapse formation and induces self-reactivity.

Immunological synapse formation between T cells and target cells can affect the functional outcome of TCR ligation by a given MHC-peptide complex. Although synapse formation is usually induced by TCR signaling, it is not clear whether other factors can affect the efficiency of synapse formation. Here, we tested whether cytokines could influence synapse formation between murine CTLs and target cells. We found that IL-12 enhanced synapse formation, whereas TGFbeta decreased synapse formation. The enhanced synapse formation induced by IL-12 appeared to be functional, given that IL-12-treated cells could respond to weak peptides, including self-peptides, to which the T cells were normally unresponsive. These responses correlated with expression of functionally higher avidity LFA-1 on IL-12-treated CTLs. These findings have implications for the function of IL-12 in T cell-mediated autoimmunity.

Costimulation through NKG2D enhances murine CD8+ CTL function: similarities and differences between NKG2D and CD28 costimulation.

Multiple studies have demonstrated that the NK cell activating receptor NKG2D can function as a costimulatory receptor for both mouse and human CD8+ T cells. However, it has recently been suggested that stimulation through NKG2D is insufficient for costimulation of CD8+ T cells. To aid in the delineation of NKG2D function in CTL responses, we investigated whether stimulation of NKG2D by the natural ligand RAE1epsilon was able to costimulate effector functions of a murine CTL line generated from DUC18 TCR transgenic mice. We found that NKG2D was able to costimulate DUC CTL responses and did so in a manner similar to CD28 costimulation. The T cells exhibited increased proliferation, IFN-gamma release, and cytotoxicity when presented antigenic peptide by P815 cells expressing RAE1epsilon or B7-1 compared with untransfected P815. In addition, both RAE1epsilon and B7-1 enhanced Ag-independent IFN-gamma secretion in response to IL-12 and IL-18 by DUC CTL. However, only costimulation through CD28 allowed for DUC CTL survival upon secondary stimulation, whereas ligation of NKG2D, but not CD28, induced DUC CTL to form an immune synapse with target cells in the absence of TCR stimulation. Understanding the outcomes of these differences may allow for a better understanding of T cell costimulation in general.