Population Pharmacokinetics of Monalizumab in Patients With Advanced Solid Tumors.

Monalizumab is a novel, first-in-class humanized immunoglobulin G4 monoclonal antibody immune checkpoint inhibitor that targets the inhibitory CD94/NKG2A receptors. The objectives of this analysis were to develop a population pharmacokinetic (PK) model of monalizumab, evaluate the impact of clinically relevant covariates on monalizumab PK, and provide dose justification for clinical trials. We developed a monalizumab population PK model to characterize the PK properties of monalizumab in patients with advanced solid tumors or head and neck squamous cell carcinoma. Data from clinical studies D419NC00001 (NCT02671435) and IPH2201-203 (NCT02643550) were pooled for the analysis, resulting in a data set of 3066 PK samples derived from 507 subjects. The PK of monalizumab were reasonably described by a 2-compartment model with first-order elimination. Monalizumab generally exhibited linear PK over a dose range of 22.5-750 mg or 10 mg/kg every 2 weeks. The estimate of clearance was ≈0.255 L/day, and apparent volume of distribution was 6.36 L for a typical individual, consistent with previous findings for endogenous immunoglobulin Gs and other therapeutic monoclonal antibodies. Baseline albumin and body weight were identified as significant covariates of clearance; body weight, sex, and smoking status had a significant impact on volume of distribution; and none of these covariates had impact on peripheral volume of distribution. Although these covariates were identified as statistically significant, they are considered to be not clinically meaningful, as changes in monalizumab exposure were <30%. Therefore, no dose adjustments of monalizumab based on patient or disease characteristics are recommended.

CD73-Mediated Immunosuppression Is Linked to a Specific Fibroblast Population That Paves the Way for New Therapy in Breast Cancer.

BACKGROUND: Cancer-associated fibroblasts (CAF) are heterogeneous with multiple functions in breast cancer. Recently, we identified a specific CAF subpopulation (referred to as CAF-S1), which promotes immunosuppression and immunotherapy resistance. METHODS AND RESULTS: Here, by studying a large collection of human samples, we highlight the key function of CD73/NT5E in CAF-S1-mediated immunosuppression in breast cancer. We first reveal that CD73 protein level specifically accumulates in CAF-S1 in breast cancer patients. Interestingly, infiltration of regulatory T lymphocytes (Tregs) is significantly correlated with CD73 expression in stroma but not in epithelium, indicating that CD73 contributes to immunosuppression when expressed in CAF-S1 and not in tumor cells. By performing functional assays based on relevant systems using primary CAF-S1 isolated from patients, we demonstrate that CAF-S1 increase the content in both PD-1+ and CTLA-4+ Tregs. Importantly, the use of a blocking anti-CD73 antibody on CAF-S1 reduces CAF-S1-mediated immunosuppression by preventing expression of these immune checkpoints on Tregs. CONCLUSIONS: Our data support the potential clinical benefit of using both anti-CD73 and immune-checkpoint inhibitors in breast cancer patients for inhibiting CAF-S1-mediated immunosuppression and enhancing anti-tumor immune response.

Targeting MICA/B with cytotoxic therapeutic antibodies leads to tumor control.

Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.

IPH4102, a first-in-class anti-KIR3DL2 monoclonal antibody, in patients with relapsed or refractory cutaneous T-cell lymphoma: an international, first-in-human, open-label, phase 1 trial.

BACKGROUND: IPH4102 is a first-in-class monoclonal antibody targeting KIR3DL2, a cell surface protein that is expressed in cutaneous T-cell lymphoma, and predominantly in its leukaemic form, Sézary syndrome. We aimed to assess the safety and activity of IPH4102 in cutaneous T-cell lymphoma. METHODS: We did an international, first-in-human, open-label, phase 1 clinical trial with dose-escalation and cohort-expansion parts in five academic hospitals in the USA, France, the UK, and the Netherlands. Eligible patients had histologically confirmed relapsed or refractory primary cutaneous T-cell lymphoma, an Eastern Cooperative Oncology group performance score of 2 or less, were aged 18 years or older, and had received at least two previous systemic therapies. Ten dose levels of IPH4102, administered as an intravenous infusion, ranging from 0·0001 mg/kg to 10 mg/kg, were assessed using an accelerated 3 + 3 design. The primary endpoint was the occurrence of dose-limiting toxicities during the first 2 weeks of treatment, defined as toxicity grade 3 or worse lasting for 8 or more days, except for lymphopenia. Global overall response by cutaneous T-cell lymphoma subtype was a secondary endpoint. Safety and activity analyses were done in the per-protocol population. The study is ongoing and recruitment is complete. This trial is registered with ClinicalTrials.gov, number NCT02593045. FINDINGS: Between Nov 4, 2015, and Nov 20, 2017, 44 patients were enrolled. 35 (80%) patients had Sézary syndrome, eight (18%) had mycosis fungoides, and one (2%) had primary cutaneous T-cell lymphoma, not otherwise specified. In the dose-escalation part, no dose limiting toxicity was reported and the trial's safety committee recommended a flat dose of 750 mg for the cohort-expansion, corresponding to the maximum administered dose. The most common adverse events were peripheral oedema (12 [27%] of 44 patients) and fatigue (nine [20%]), all of which were grade 1-2. Lymphopenia was the most common grade 3 or worse adverse event (three [7%]). One patient developed possibly treatment-related fulminant hepatitis 6 weeks after IPH4102 discontinuation and subsequently died. However, the patient had evidence of human herpes virus-6B infection. Median follow-up was 14·1 months (IQR 11·3-20·5). A confirmed global overall response was achieved in 16 (36·4% [95% CI 23·8-51·1]) of 44 patients, and of those, 15 responses were observed in 35 patients with Sézary syndrome (43% [28·0-59·1]). INTERPRETATION: IPH4102 is safe and shows encouraging clinical activity in patients with relapsed or refractory cutaneous T-cell lymphoma, particularly those with Sézary syndrome. If confirmed in future trials, IPH4102 could become a novel treatment option for these patients. A multi-cohort, phase 2 trial (TELLOMAK) is underway to confirm the activity in patients with Sézary syndrome and explore the role of IPH4102 in other subtypes of T-cell lymphomas that express KIR3DL2. FUNDING: Innate Pharma.

Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies.

Immune checkpoint inhibitors have revolutionized cancer treatment. However, many cancers are resistant to ICIs, and the targeting of additional inhibitory signals is crucial for limiting tumor evasion. The production of adenosine via the sequential activity of CD39 and CD73 ectoenzymes participates to the generation of an immunosuppressive tumor microenvironment. In order to disrupt the adenosine pathway, we generated two antibodies, IPH5201 and IPH5301, targeting human membrane-associated and soluble forms of CD39 and CD73, respectively, and efficiently blocking the hydrolysis of immunogenic ATP into immunosuppressive adenosine. These antibodies promoted antitumor immunity by stimulating dendritic cells and macrophages and by restoring the activation of T cells isolated from cancer patients. In a human CD39 knockin mouse preclinical model, IPH5201 increased the anti-tumor activity of the ATP-inducing chemotherapeutic drug oxaliplatin. These results support the use of anti-CD39 and anti-CD73 monoclonal antibodies and their combination with immune checkpoint inhibitors and chemotherapies in cancer.

A phase 1 study of lirilumab (antibody against killer immunoglobulin-like receptor antibody KIR2D; IPH2102) in patients with solid tumors and hematologic malignancies.

PURPOSE: Anti-KIR monoclonal antibodies (mAbs) can enhance the antitumor responses of natural killer (NK) cells. We evaluated the safety of the anti-KIR2D mAb lirilumab in patients with various cancers. EXPERIMENTAL DESIGN: Thirty-seven patients with hematological malignancies (n = 22) or solid tumors (n = 15) were included in the study. Dose escalation (0.015 to 10 mg/kg) was conducted following a 3 + 3 design. Patients were scheduled to receive four cycles of treatment. In a second (extension) phase 17 patients were treated at 0.015 (n = 9) or 3 mg/kg (n = 8). RESULTS: No dose-limiting toxicity was recorded. The most frequent lirilumab-related adverse events were pruritus (19%), asthenia (16%), fatigue (14%), infusion-related reaction (14%), and headache (11%), mostly mild or moderate. Pharmacokinetics was dose-dependent and linear, with minimal accumulation resulting from the 4-weekly repeated administrations. Full KIR occupancy (>95%) was achieved with all dosages, and the duration of occupancy was dose-related. No significant changes were observed in the number or distribution of lymphocyte subpopulations, nor was any reduction in the distribution of KIR2D-positive NK cells. CONCLUSIONS: This phase 1 trial demonstrated the satisfactory safety profile of lirilumab up to doses that enable full and sustained blockade of KIR.

IPH4102, a humanized KIR3DL2 antibody with potent activity against cutaneous T-cell lymphoma.

Advanced cutaneous T-cell lymphoma (CTCL) remains an unmet medical need, which lacks effective targeted therapies. In this study, we report the development of IPH4102, a humanized monoclonal antibody that targets the immune receptor KIR3DL2, which is widely expressed on CTCL cells but few normal immune cells. Potent antitumor properties of IPH4102 were documented in allogeneic human CTCL cells and a mouse model of KIR3DL2(+) disease. IPH4102 antitumor activity was mediated by antibody-dependent cell cytotoxicity and phagocytosis. IPH4102 improved survival and reduced tumor growth in mice inoculated with KIR3DL2(+) tumors. Ex vivo efficacy was further evaluated in primary Sézary patient cells, sorted natural killer-based autologous assays, and direct spiking into Sézary patient peripheral blood mononuclear cells. In these settings, IPH4102 selectively and efficiently killed primary Sézary cells, including at unfavorable effector-to-target ratios characteristic of unsorted PBMC. Together, our results offer preclinical proof of concept for the clinical development of IPH4102 to treat patients with advanced CTCL.

Human XCR1+ dendritic cells derived in vitro from CD34+ progenitors closely resemble blood dendritic cells, including their adjuvant responsiveness, contrary to monocyte-derived dendritic cells.

Human monocyte-derived dendritic cell (MoDC) have been used in the clinic with moderately encouraging results. Mouse XCR1(+) DC excel at cross-presentation, can be targeted in vivo to induce protective immunity, and share characteristics with XCR1(+) human DC. Assessment of the immunoactivation potential of XCR1(+) human DC is hindered by their paucity in vivo and by their lack of a well-defined in vitro counterpart. We report in this study a protocol generating both XCR1(+) and XCR1(-) human DC in CD34(+) progenitor cultures (CD34-DC). Gene expression profiling, phenotypic characterization, and functional studies demonstrated that XCR1(-) CD34-DC are similar to canonical MoDC, whereas XCR1(+) CD34-DC resemble XCR1(+) blood DC (bDC). XCR1(+) DC were strongly activated by polyinosinic-polycytidylic acid but not LPS, and conversely for MoDC. XCR1(+) DC and MoDC expressed strikingly different patterns of molecules involved in inflammation and in cross-talk with NK or T cells. XCR1(+) CD34-DC but not MoDC efficiently cross-presented a cell-associated Ag upon stimulation by polyinosinic-polycytidylic acid or R848, likewise to what was reported for XCR1(+) bDC. Hence, it is feasible to generate high numbers of bona fide XCR1(+) human DC in vitro as a model to decipher the functions of XCR1(+) bDC and as a potential source of XCR1(+) DC for clinical use.

The toll-like receptor agonist imiquimod is active against prions.

Using a yeast-based assay, a previously unsuspected antiprion activity was found for imiquimod (IQ), a potent Toll-like receptor 7 (TLR7) agonist already used for clinical applications. The antiprion activity of IQ was first detected against yeast prions [PSI (+) ] and [URE3], and then against mammalian prion both ex vivo in a cell-based assay and in vivo in a transgenic mouse model for prion diseases. In order to facilitate structure-activity relationship studies, we conducted a new synthetic pathway which provides a more efficient means of producing new IQ chemical derivatives, the activity of which was tested against both yeast and mammalian prions. The comparable antiprion activity of IQ and its chemical derivatives in the above life forms further emphasizes the conservation of prion controlling mechanisms throughout evolution. Interestingly, this study also demonstrated that the antiprion activity of IQ and IQ-derived compounds is independent from their ability to stimulate TLRs. Furthermore, we found that IQ and its active chemical derivatives inhibit the protein folding activity of the ribosome (PFAR) in vitro.

Toll-like receptor 3 in Epstein-Barr virus-associated nasopharyngeal carcinomas: consistent expression and cytotoxic effects of its synthetic ligand poly(A:U) combined to a Smac-mimetic.

BACKGROUND: Nasopharyngeal carcinomas (NPC) are consistently associated with the Epstein-Barr virus (EBV). Though NPCs are more radiosensitive and chemosensitive than other tumors of the upper aero-digestive tract, many therapeutic challenges remain. In a previous report, we have presented data supporting a possible therapeutic strategy based on artificial TLR3 stimulation combined to the inhibition of the IAP protein family (Inhibitor of Apoptosis Proteins). The present study was designed to progress towards practical applications of this strategy pursuing 2 main objectives: 1) to formally demonstrate expression of the TLR3 protein by malignant NPC cells; 2) to investigate the effect of poly(A:U) as a novel TLR3-agonist more specific than poly(I:C) which was used in our previous study. METHODS: TLR3 expression was investigated in a series of NPC cell lines and clinical specimens by Western blot analysis and immunohistochemistry, respectively. The effects on NPC cells growth of the TLR3 ligand poly(A:U) used either alone or in combination with RMT5265, an IAP inhibitor based on Smac-mimicry, were assessed using MTT assays and clonogenic assays. RESULTS: TLR3 was detected at a high level in all NPC cell lines and clinical specimens. Low concentrations of poly(A:U) were applied to several types of NPC cells including cells from the C17 xenograft which for the first time have been adapted to permanent propagation in vitro. As a single agent, poly(A:U) had no significant effects on cell growth and cell survival. In contrast, dramatic effects were obtained when it was combined with the IAP inhibitor RMT5265. These effects were obtained using concentrations as low as 0.5 µg/ml (poly(A:U)) and 50 nM (RMT5265). CONCLUSION: These data confirm that TLR3 expression is a factor of vulnerability for NPC cells. They suggest that in some specific pathological and pharmacological contexts, it might be worth to use Smac-mimetics at very low doses, allowing a better management of secondary effects. In light of our observations, combined use of both types of compounds should be considered for treatment of nasopharyngeal carcinomas.

TLR3 as a biomarker for the therapeutic efficacy of double-stranded RNA in breast cancer.

The discovery of a targeted therapeutic compound along with its companion predictive biomarker is a major goal of clinical development for a personalized anticancer therapy to date. Here we present evidence of the predictive value of TLR3 expression by tumor cells for the efficacy of Poly (A:U) dsRNA in 194 breast cancer patients enrolled in a randomized clinical trial. Adjuvant treatment with double-stranded RNA (dsRNA) was associated with a significant decrease in the risk of metastatic relapse in TLR3 positive but not in TLR3-negative breast cancers. Moreover, we show the functional relevance of TLR3 expression by human tumor cells for the antitumor effects mediated by dsRNA in several preclinical mouse models carried out in immunocompromised animals. These 2 independent lines of evidence relied upon the generation of a novel tool, an anti-TLR3 antibody (40F9.6) validated for routine detection of TLR3 expression on paraffin-embedded tissues. Altogether, these data suggest that dsRNA mediates its therapeutic effect through TLR3 expressed on tumor cells, and could therefore represent an effective targeted treatment in patients with TLR3-positive cancers.

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.

Poly(I:C) induces intense expression of c-IAP2 and cooperates with an IAP inhibitor in induction of apoptosis in cancer cells.

BACKGROUND: There is increasing evidence that the toll-like receptor 3 (TLR3) is an interesting target for anti-cancer therapy. Unfortunately, most laboratory investigations about the impact of TLR3 stimulation on human malignant cells have been performed with very high concentrations--5 to 100 microg/ml--of the prototype TLR3 ligand, poly(I:C). In a previous study focused on a specific type of human carcinoma - nasopharyngeal carcinoma - we have shown that concentrations of poly(I:C) as low as 100 ng/ml are sufficient to induce apoptosis of malignant cells when combined to a pharmacological antagonist of the IAP family based on Smac mimicry. METHODS: This observation prompted us to investigate the contribution of the IAP family in cell response to poly(I:C) in a variety of human malignant cell types. RESULTS: We report a rapid, intense and selective increase in c-IAP2 protein expression observed under stimulation by poly(I:C)(500 ng/ml) in all types of human malignant cells. In most cell types, this change in protein expression is underlain by an increase in c-IAP2 transcripts and dependent on the TLR3/TRIF pathway. When poly(I:C) is combined to the IAP inhibitor RMT 5265, a cooperative effect in apoptosis induction and/or inhibition of clonogenic growth is obtained in a large fraction of carcinoma and melanoma cell lines. CONCLUSIONS: Currently, IAP inhibitors like RMT 5265 and poly(I:C) are the subject of separate therapeutic trials. In light of our observations, combined use of both types of compounds should be considered for treatment of human malignancies including carcinomas and melanomas.

Plasmacytoid dendritic cell-derived type I interferon is crucial for the adjuvant activity of Toll-like receptor 7 agonists.

There is a high demand for the development of adjuvants that induce cytotoxic T lymphocytes, which are crucial for the elimination of intracellular pathogens and tumor cells. Toll-like receptor (TLR) agonists are prime candidates to fulfill this role because they induce innate immune activation and promote adaptive immune responses. The successful application of the TLR7 agonist R837 for treatment of basal cell carcinoma shows the potential for exploiting this pathway in tumor immunotherapy. Imidazoquinolines like R837 and stimulatory ssRNA oligonucleotides both trigger TLR7-mediated immune activation, but little is known about their comparative ability to promote immunity induction. We investigated differences in innate immune activation and adjuvant activity between the imidazoquinoline R848 and the ssRNA TLR7 agonist polyUs21. In contrast to R848, polyUs21 induced detectable levels of intracellular interferon-alpha (IFN-alpha) in plasmacytoid dendritic cells (PDCs). In immunization studies, only polyUs21 led to robust priming of type 1 T helper cells and cytotoxic T lymphocytes, and it was more efficient in inducing antitumor immunity than R848. Notably, exogenous IFN-alpha augmented the adjuvant activity of R848, whereas depletion of PDC abrogated the adjuvanticity of polyUs21. This study, therefore, identifies sufficient IFN-alpha production by PDC as an important determinant of vaccine efficacy.

Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands.

Many cancer cells express Toll-like receptors (TLR) that offer possible therapeutic targets. Polyadenylic-polyuridylic acid [poly(A:U)] is an agonist of the Toll-like receptor TLR3 that displays anticancer properties. In this study, we illustrate how the immunostimulatory and immunosuppressive effects of this agent can be uncoupled to therapeutic advantage. We took advantage of two TLR3-expressing tumor models that produced large amounts of CCL5 (a CCR5 ligand) and CXCL10 (a CXCR3 ligand) in response to type I IFN and poly(A:U), both in vitro and in vivo. Conventional chemotherapy or in vivo injection of poly(A:U), alone or in combination, failed to reduce tumor growth unless an immunochemotherapeutic regimen of vaccination against tumor antigens was included. CCL5 blockade improved the efficacy of immunochemotherapy, whereas CXCR3 blockade abolished its beneficial effects. These findings show how poly(A:U) can elicit production of a range of chemokines by tumor cells that reinforce immunostimulatory or immunosuppressive effects. Optimizing the anticancer effects of TLR3 agonists may require manipulating these chemokines or their receptors.

Plasmacytoid dendritic cells mediate oral tolerance.

Oral tolerance prevents oral sensitization to dietary antigens (Ags), including proteins and haptens, and development of delayed-type hypersensitivity (DTH) responses. We showed here that plasmacytoid dendritic cells (pDCs) prevented oral T cell priming and were responsible for systemic tolerance to CD4(+) and CD8(+) T cell-mediated DTH responses induced by Ag feeding. Systemic depletion of pDCs prevented induction of tolerance by antigen feeding. Transfer of oral Ag-loaded liver pDCs to naive recipient mice induced Ag-specific suppression of CD4(+) and CD8(+) T cell responses to protein and hapten, respectively. Liver is a site of oral Ag presentation, and pDCs appeared to induce anergy or deletion of Ag-specific T cells in the liver relatively rapidly via a CD4(+) T cell-independent mechanism. These data demonstrate that oral tolerance relies on Ag presentation by pDC to T cells and suggest that pDC could represent a key therapeutic target for intestinal and systemic inflammatory diseases.

Macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce IL-10 in response to MyD88- and TRIF-dependent TLR signals, and TLR-independent signals.

We have previously reported that mouse plasmacytoid dendritic cells (DC) produce high levels of IL-12p70, whereas bone marrow-derived myeloid DC and splenic DC produce substantially lower levels of this cytokine when activated with the TLR-9 ligand CpG. We now show that in response to CpG stimulation, high levels of IL-10 are secreted by macrophages, intermediate levels by myeloid DC, but no detectable IL-10 is secreted by plasmacytoid DC. MyD88-dependent TLR signals (TLR4, 7, 9 ligation), Toll/IL-1 receptor domain-containing adaptor-dependent TLR signals (TLR3, 4 ligation) as well as non-TLR signals (CD40 ligation) induced macrophages and myeloid DC to produce IL-10 in addition to proinflammatory cytokines. IL-12p70 expression in response to CpG was suppressed by endogenous IL-10 in macrophages, in myeloid DC, and to an even greater extent in splenic CD8alpha(-) and CD8alpha(+) DC. Although plasmacytoid DC did not produce IL-10 upon stimulation, addition of this cytokine exogenously suppressed their production of IL-12, TNF, and IFN-alpha, showing trans but not autocrine regulation of these cytokines by IL-10 in plasmacytoid DC.

Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides.

Toll-like receptor 7 (TLR7) mediates innate responses by responding to viral RNA in endocytic compartments. However, the molecular pattern recognised by TLR7 and whether it differs between RNA of viral and self origin remains unclear. Here, we identify nucleic acids that act as TLR7 agonists for mouse and human cells. We show that uridine and ribose, the two defining features of RNA, are both necessary and sufficient for TLR7 stimulation, and that short single-stranded RNA (ssRNA) act as TLR7 agonists in a sequence-independent manner as long as they contain several uridines in close proximity. Consistent with the notion that TLR7 lacks specificity for sequence motifs, we show that it is triggered equally efficiently by viral or self RNA delivered to endosomes. Our results support the notion that TLR7 recognises uracil repeats in RNA and that it discriminates between viral and self ligands on the basis of endosomal accessibility rather than sequence.

Ikaros is required for plasmacytoid dendritic cell differentiation.

Plasmacytoid dendritic cells (pDCs) are specialized DCs that produce high levels of type I IFN upon viral infection. Despite their key immunoregulatory role, little is known about pDC ontogeny or how developmental events regulate their function. We show that mice expressing low levels of the transcription factor Ikaros (Ik(L/L)) lack peripheral pDCs, but not other DC subsets. Loss of pDCs is associated with an inability to produce type I IFN after challenge with Toll-like receptor-7 and -9 ligands, or murine cytomegalovirus (MCMV) infection. In contrast, conventional DCs are present in normal numbers and exhibit normal responses in vivo after challenge with MCMV or inactivated toxoplasma antigen. Interestingly, Ik(L/L) bone marrow (BM) cells contain a pDC population that appears blocked at the Ly-49Q- stage of differentiation and fails to terminally differentiate in response to Flt-3L, a cytokine required for pDC differentiation. This differentiation block is strictly dependent on a cell-intrinsic requirement for Ikaros in pDC-committed precursors. Global gene expression profiling of Ik(L/L) BM pDCs reveals an up-regulation of genes not normally expressed, or expressed at low levels, in WT pDCs. These studies suggest that Ikaros controls pDC differentiation by silencing a large array of genes.