NK cell activation in visceral leishmaniasis requires TLR9, myeloid DCs, and IL-12, but is independent of plasmacytoid DCs

Natural killer (NK) cells are sentinel components of the innate response to pathogens, but the cell types, pathogen recognition receptors, and cytokines required for their activation in vivo are poorly defined. Here, we investigated the role of plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs), Toll-like receptors (TLRs), and of NK cell stimulatory cytokines for the induction of an NK cell response to the protozoan parasite Leishmania infantum. In vitro, pDCs did not endocytose Leishmania promastigotes but nevertheless released interferon (IFN)-α/β and interleukin (IL)-12 in a TLR9-dependent manner. mDCs rapidly internalized Leishmania and, in the presence of TLR9, produced IL-12, but not IFN-α/β. Depletion of pDCs did not impair the activation of NK cells in L. infantum–infected mice. In contrast, L. infantum–induced NK cell cytotoxicity and IFN-γ production were abolished in mDC-depleted mice. The same phenotype was observed in TLR9−/− mice, which lacked IL-12 expression by mDCs, and in IL-12−/− mice, whereas IFN-α/β receptor−/− mice showed only a minor reduction of NK cell IFN-γ expression. This study provides the first direct evidence that mDCs are essential for eliciting NK cell cytotoxicity and IFN-γ release in vivo and demonstrates that TLR9, mDCs, and IL-12 are functionally linked to the activation of NK cells in visceral leishmaniasis

Impaired functionality of antiviral T cells in G-CSF mobilized stem cell donors: implications for the selection of CTL donor.

Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells. CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry. G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF. To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production

Mean levels of multimer staining analyses in seropositive and seronegative donors.

<p>Mean percentages of WB, WBM, A and G samples per donor were calculated for each multimer separately. Donors were grouped according to their CMV and EBV serostatus, respectively, and the resulting groups were analyzed using the Mann-Whitney test. Significant differences between staining results in seropositive and seronegative donors are indicated in the figure and were determined for mCMV_pp50_A01, mCMV_pp65_A02 and mCMV_pp65_B07. For mEBV, no significance differences could be determined due to the small number or complete lack of seronegative donors for the individual multimers. Sample sources: (A) apheresis tubing set, (G) graft, (WB) whole blood, (WBM) mobilized whole blood on day of apheresis.</p

Study Cohort.

<p>Number of samples analyzed by multimer staining and ELISpot according to sample type, w/o excluded samples (168 donors).</p><p>Sample sources: (A) apheresis tubing set, (G) graft, (WB) whole blood, (WBM) mobilized whole blood on day of apheresis.</p

Effects of sample type on multimer staining results.

<p>For donors with 3 sample types (WBM, A, G), percentages of multimer-positive cells (sum of all matched alleles) in CD3⁺CD8⁺ T cells were compared for (A) CMV-seropositive (n = 14) and (B) EBV-seropositive (n = 11) donors. Inclusion criteria for the analysis were availability of serostatus and a staining result ≥0.1%. In the majority of samples, the staining results for each sample type remained in the same range. Sample sources: (A) apheresis tubing set, (G) graft, (WBM) mobilized whole blood on day of apheresis.</p

<i>In vivo</i> treatment of stem cell donors affects antiviral response to peptide pools for CMV, EBV and ADV.

<p>(A) Number of spot forming units (sfu) per well for untreated platelet and G-CSF-treated stem cell donors. (B) Number of sfu per 10,000 CD3⁺ cells for untreated and treated donors. (C) Response of pairs of stem cell donor cells to peptide pools of CMV, EBV and ADV before and after mobilization. Asterisks indicate significant differences (*p<0.05, **p<0.01, ***p<0.001). n.s. = not significant.</p

Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment

A dual role for hepatocyte-intrinsic canonical NF-kappaB signaling in virus control.

peer reviewedBACKGROUND & AIMS: Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver macrophages. However, also hepatocytes, the parenchymal cells of the liver, possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct anti-viral mechanisms employed by hepatocytes. METHODS: Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-kappaB signaling (IKKbeta(DeltaHep)) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-alpha/beta signaling-(IFNAR(DeltaHep)), or interferon-alpha/beta signaling in myeloid cells-(IFNAR(DeltaMyel)) were infected. RESULTS: Here, we demonstrate that LCMV activates NF-kappaB signaling in hepatocytes. LCMV-triggered NF-kappaB activation in hepatocytes did not depend on Kupffer cells or TNFR1- but rather on TLR-signaling. LCMV-infected IKKbeta(DeltaHep) livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8(+) T-cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKKbeta, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IKKbeta(DeltaHep) mice, enhanced hepatocytic LCMV accumulation was observed in livers of IFNAR(DeltaHep), whereas IFNAR(DeltaMyel) mice were able to control LCMV-infection. Hepatocytic NF-kappaB signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-alpha/beta-mediated inhibition of HBV replication in vitro. CONCLUSIONS: Together, these data show that hepatocyte-intrinsic NF-kappaB is a vital amplifier of interferon-alpha/beta signaling pivotal for early, strong ISG responses, influx of immune cells and hepatic viral clearance