Lessons from NK Cell Deficiencies in the Mouse.

Since their discovery in the late 1970s, in vivo studies on mouse natural killer (NK) cell almost entirely relied on the use of depleting antibodies and were associated with significant limitations. More recently, large-scale gene-expression analyses allowed the identification of NKp46 as one of the best markers of NK cells across mammalian species. Since then, NKp46 has been shown to be expressed on other subsets of innate lymphoid cells (ILCs) such as the closely related ILC1 and the mucosa-associated NCR(+) ILC3. Based on this marker, several mouse models specifically targeting NKp46-expressing cell have recently been produced. Here, we review recent advances in the generation of models of deficiency in NKp46-expressing cells and their use to address the role of NK cells in immunity, notably on the regulation of adaptive immune responses.

Human natural killer cells promote cross-presentation of tumor cell-derived antigens by dendritic cells.

Dendritic cells (DCs) cross-present antigen (Ag) to initiate T-cell immunity against most infections and tumors. Natural killer (NK) cells are innate cytolytic lymphocytes that have emerged as key modulators of multiple DC functions. Here, we show that human NK cells promote cross-presentation of tumor cell-derived Ag by DC leading to Ag-specific CD8(+) T-cell activation. Surprisingly, cytotoxic function of NK cells was not required. Instead, we highlight a critical and nonredundant role for IFN-γ and TNF-α production by NK cells to enhance cross-presentation by DC using two different Ag models. Importantly, we observed that NK cells promote cell-associated Ag cross-presentation selectively by monocytes-derived DC (Mo-DC) and CD34-derived CD11b(neg) CD141(high) DC subsets but not by myeloid CD11b(+) DC. Moreover, we demonstrate that triggering NK cell activation by monoclonal antibodies (mAbs)-coated tumor cells leads to efficient DC cross-presentation, supporting the concept that NK cells can contribute to therapeutic mAbs efficiency by inducing downstream adaptive immunity. Taken together, our findings point toward a novel role of human NK cells bridging innate and adaptive immunity through selective induction of cell-associated Ag cross-presentation by CD141(high) DC, a process that could be exploited to better harness Ag-specific cellular immunity in immunotherapy.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-gamma. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-gamma production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-gamma production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-gamma production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I-specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

Different innate signatures induced in human monocyte-derived dendritic cells by wild-type dengue 3 virus, attenuated but reactogenic dengue 3 vaccine virus, or attenuated nonreactogenic dengue 1-4 vaccine virus strains.

DNA microarrays were used to assess the innate gene signature in human myeloid dendritic cells infected with chimeric dengue 1-4 vaccines, a wild-type dengue 3 virus, or a classically attenuated serotype 3 vaccine shown to be reactogenic in humans. We observed a very reproducible signature for each of the 4 chimeric dengue vaccines, involving stimulation of type I interferon and associated genes, together with genes encoding chemokines and other mediators involved in the initiation of adaptive responses. In contrast, wild-typeDEN3 virus induced a predominantly inflammatory profile, while the reactogenic attenuated serotype 3 vaccine appeared to induce a blunted response.

A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells.

Dendritic cells (DC) produce interleukin-12 (IL-12) in response to Toll-like receptor (TLR) activation. Two major TLR signaling pathways participate in the response to pathogens: the nuclear factor-kappaB (NF-kappaB)-dependent pathway leading to inflammatory cytokine secretion including IL-12 and the interferon (IFN)-dependent pathway inducing type I IFN and IFN-regulated genes. Here we show that the two pathways cooperate and are likely both necessary for inducing an optimal response to pathogens. R-848/Resiquimod (TLR7 ligand in the mouse and TLR7/8 ligand in human) synergized with poly(I:C) (TLR3 ligand) or lipopolysaccharide (LPS; TLR4 ligand) in inducing high levels of bioactive IL-12p70 secretion and IFN-beta mRNA accumulation by mouse bone marrow-derived DC (BM-DC). Strikingly, IL-12p70 but not IL-12p40 secretion was strongly reduced in BM-DC from STAT1(-/-) and IFNAR(-/-) mice. STAT1 tyrosine-phosphorylation, IL-12p35, and IFN-beta mRNA accumulation were strongly inhibited in IFNAR(-/-) BM-DC activated with the TLR ligand combinations. Similar observation were obtained in human TLR8-expressing monocyte-derived DC (moDC) using neutralizing anti-IFNAR2 antibodies, although results also pointed to a possible involvement of IFN-lambda1 (also known as IL-29). This suggests that TLR engagement on DC induces endogenous IFNs that further synergize with the NF-kappaB pathway for optimal IL-12p70 secretion. Moreover, analysis of interferon regulatory factors (IRF) regulation in moDC suggests a role for IRF7/8 in mediating IRF3-independent type I IFN and possibly IL-12p35 synthesis in response to TLR7/8.

Innate immune responses in human dendritic cells upon infection by chimeric yellow-fever dengue vaccine serotypes 1-4.

Dengue infection is an important public health issue worldwide. The ChimeriVax-Dengue (CYD) vaccine uses yellow fever (YF) 17D vaccine as a live vector. Dendritic cells (DCs) play a key role in initiating immune responses and could be an important primary target of dengue infection. We investigated in vitro the consequences of CYD infection of DCs on their activation/maturation and cytokine production. In CYD-infected DCs, we observed an up-regulation of HLA-DR, CD80, CD86, and CD83. Cells exposed to CYD secreted type I interferons, monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL-2), interleukin-6 (IL-6), and low amounts of tumor necrosis factor-alpha (TNF-alpha), but no IL-10, IL-12, or IL-1alpha. Parental dengue viruses induced a similar array of cytokines, but more TNF-alpha, less IL-6, and less MCP-1/CCL-2 than induced by CYD. Chimeras thus induced DCs maturation and a controlled response accompanied by limited inflammatory cytokine production and consistent expression of anti-viral interferons, in agreement with clinical observations of safety and immunogenicity.

Memory B and T cell responses induced by serotype 4 Streptococcus pneumoniae vaccines: longitudinal analysis comparing responses elicited by free polysaccharide, conjugate and carrier.

We have conducted a 1-year longitudinal study in mice vaccinated by free serotype 4 Streptococcus pneumoniae PS (PS4), the corresponding tetanus toxoid (TT)-conjugated vaccine, or the TT carrier alone. B and T cell immunity induced by these three types of antigen, were compared by monitoring the (i) long-term persistence of specific serum antibodies, (ii) frequency of memory B cell precursors in spleen, and (iii) T cell responses against the carrier. While PS4-specific antibody response appeared later than the anti-carrier response upon primary immunization, PS4-specific B memory and serum responses were quantitatively and qualitatively similar to the ones observed against TT upon immunization by either the free carrier or the conjugate. We also observed a parallel persistent carrier-specific T cell response in the spleen. These data indicate that the nature and long-term kinetics of the anti-PS4 antibody response induced by the conjugate vaccine are similar to "classical" T-dependent response elicited by conventional protein antigens.

Innate and adaptive cellular immunity in flavivirus-naïve human recipients of a live-attenuated dengue serotype 3 vaccine produced in Vero cells (VDV3).

VDV3, a clonal derivative of the Mahidol live-attenuated dengue 3 vaccine was prepared in Vero cells. Despite satisfactory preclinical evaluation, VDV3 was reactogenic in humans. We explored whether immunological mechanisms contributed to this outcome by monitoring innate and adaptive cellular immune responses for 28 days after vaccination. While no variations were seen in serum IL12 or TNFalpha levels, a high IFNgamma secretion was detected from Day 8, concomitant to IFNalpha, followed by IL10. Specific Th1 and CD8 responses were detected on Day 28, with high IFNgamma/TNFalpha ratios. Vaccinees exhibited very homogeneous class I HLA profiles, and a new HLA B60-restricted CD8 epitope was identified in NS3. We propose that, among other factors, adaptive immunity may have contributed to reactogenicity, even after this primary vaccination. In addition, the unexpected discordance observed between preclinical results and clinical outcome in humans led us to reconsider some of our preclinical acceptance criteria. Lessons learned from these results will help us to pursue the development of safe and immunogenic vaccines.