The activation of T cells is the fundamental on switch for the adaptive immune system. Ca(2+) signaling is essential for T cell activation and starts as initial, short-lived, localized Ca(2+) signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca(2+) signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca(2+) indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca(2+) signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca(2+) signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1(-/-) mice, either these early Ca(2+) signals were not detected or the number of signals was markedly reduced. Local Ca(2+) signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca(2+) signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca(2+) release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals.

The cytokine IL-6 plays a protective role in immune responses against bacterial infections. However, the mechanisms of IL-6–mediated protection are only partially understood. IL-6 can signal via the IL-6R complex composed of membrane-bound IL-6Rα (mIL-6Rα) and gp130. Owing to the restricted expression of mIL-6Rα, classical IL-6 signaling occurs only in a limited number of cells such as hepatocytes and certain leukocyte subsets. IL-6 also interacts with soluble IL-6Rα proteins and these IL-6/soluble IL-6Rα complexes can subsequently bind to membrane-bound gp130 proteins and induce signaling. Because gp130 is ubiquitously expressed, this IL-6 trans-signaling substantially increases the spectrum of cells responding to IL-6. In this study, we analyze the role of classical IL-6 signaling and IL-6 trans-signaling in the innate immune response of mice against Listeria monocytogenes infection. We demonstrate that L. monocytogenes infection causes profound systemic IL-6 production and rapid loss of IL-6Rα surface expression on neutrophils, inflammatory monocytes, and different lymphocyte subsets. IL-6–deficient mice or mice treated with neutralizing anti–IL-6 mAb displayed impaired control of L. monocytogenes infection accompanied by alterations in the expression of inflammatory cytokines and chemokines, as well as in the recruitment of inflammatory cells. In contrast, restricted blockade of IL-6 trans-signaling by application or transgenic expression of a soluble gp130 protein did not restrain the control of infection. In summary, our results demonstrate that IL-6Rα surface expression is highly dynamic during the innate response against L. monocytogenes and that the protective IL-6 function is dependent on classical IL-6 signaling via mIL-6Rα.

Robust cytotoxic CD8 + T-cell response is important for immunity to intracellular pathogens. Here, we show that the transcription factor IFN Regulatory Factor 4 (IRF4) is crucial for the protective CD8 + T-cell response to the intracellular bacterium Listeria monocytogenes. IRF4-deficient (Irf4 −/− ) mice could not clear L. monocytogenes infection and generated decreased numbers of L. monocytogenesspecific CD8 + T cells with impaired effector phenotype and function. Transfer of wild-type CD8 + T cells into Irf4 −/− mice improved bacterial clearance, suggesting an intrinsic defect of CD8 + T cells in Irf4 −/− mice. Following transfer into wild-type recipients, Irf4 −/− CD8 + T cells became activated and showed initial proliferation upon L. monocytogenes infection. However, these cells could not sustain proliferation, produced reduced amounts of IFN-γ and TNF-α, and failed to acquire cytotoxic function. Forced IRF4 expression in Irf4 −/− CD8 + T cells rescued the defect. During acute infection, Irf4 −/− CD8 + T cells demonstrated diminished expression of B lymphocyte-induced maturation protein-1 (Blimp-1), inhibitor of DNA binding (Id)2, and T-box expressed in T cells (T-bet), transcription factors programming effector-cell generation. IRF4 was essential for expression of Blimp-1, suggesting that altered regulation of Blimp-1 contributes to the defects of Irf4 −/− CD8 + T cells. Despite increased levels of B-cell lymphoma 6 (BCL-6), Eomesodermin, and Id3, Irf4 −/− CD8 + T cells showed impaired memory-cell formation, indicating additional functions for IRF4 in this process. As IRF4 governs B-cell and CD4 + T-cell differentiation, the identification of its decisive role in peripheral CD8 + T-cell differentiation, suggests a common regulatory function for IRF4 in adaptive lymphocytes fate decision.