WO2012065755A1 - Production of ifn-lambda by b cells - Google Patents

Abstract

Dendritic cells have been established as a major source of IFN-λ in response to double-stranded nucleic acids. A second wave of IFN-λ in response to double-stranded nucleic acids has also been discovered that is not dependent on dendritic cells, but is dependent on B cells and Cardiff. The invention encompasses compositions and methods for the production of IFN-λ in vitro and in vivo.

Description

PRODUCTION OF IFN-LAMBDA BY B CELLS

[001] The invention relates to the field of the production of interferons by dendritic cells and by B cells. The invention relates to the discovery of a dendritic cell type and a B cell type responsible for the production of IFN-lambdas (IFN-λ) and methods for regulating this production. The invention further relates to pharmaceutical compositions and medical uses.

Background of the Invention

[002] IFN-lambdas, also termed IL-28/29, are potent immune-modulatory and antiviral cytokines, recently implicated in clearance of Hepatitis C virus in humans. Polyinosinic:polycytidylic acid (poly IC) mimics double stranded RNA generated during viral infections. It is recognized via toll-like receptor (TLR) 3 or Rig-like helicases (RLHs) and is an effective inducer of IFN-a and IFN-λ in vivo.

[003] The IFN-lambda (-A) 1 , 2, 3 cytokine family, also called IL-29, IL-28A, and IL- 28B respectively, has recently been identified (Kotenko et al., 2003; Sheppard et al., 2003). IFN-As are related to type I IFNs (IFN-ls) as well as the IL-10 family of cytokines and signal via a heterodimeric receptor, consisting of one chain unique for IFN-A (IFN-AR1 or IL-28Ra) and another chain (IL-10R2) which is shared with IL-10 related cytokines. IFN-As possess antiviral, antitumor and various immune modulating functions and in many ways resemble the function of IFN-ls (Li et al., 2009). In contrast to the ubiquitous expression of the IFN-I receptor, the expression of the IFN-A receptor is restricted to limited cell types including epithelial cells and plasmacytoid (p) DCs (Ank et al., 2008; Sommereyns et al., 2008). Exposure to viruses or analogues of nucleic acids such as poly IC or CpG-oligonucleotides (ODN), conditions known to trigger the production of IFN-ls, also induce IFN-As and largely depend on similar signaling components (Ank et al., 2008; Osterlund et al., 2007; Onoguchi et al., 2007). IFN-As play a role in toll-like receptor (TLR) induced protection against mucosal viral infections and recent reports link the IL-28B gene with an ability to clear and recover from Hepatitis C infection (Ank et al., 2008; Ge et al., 2009). It is thus of utmost importance to understand the cellular origin of IFN-As and the regulation of its production.

[004] Several cell types have been described to produce IFN-λ including monocyte derived DC and pDC, but the cellular origin of poly IC induced IFN-λ in vivo is still elusive (Coccia et al., 2004; Ank et al., 2008; Osterlund et al., 2005). Monocyte derived DC are not CD8+ cDCs or eCD8+ cDCs cells since eCD8+ DC depend on Fms-related tyrosine kinase 3 ligand (FL), but not on GM-CSF, for development. Monocyte derived DC fully depend on GM-CSF for development, even though GM- CSF might be combined with other cytokines such as IL-4 or TNF-a. GM-CSF dependent DC are not equivalents of steady state DC because the lack of GM-CSF or the GM-CSF receptor had no influence on the presence of normal pDC or cDC subsets in lymphoid organs. (Naik, 2008). If cells are generated in vitro with the combination of GM-CSF and FL, only GM-CSF DC develop, but not pDC or eCD8+ cDCs (Gilliet et al., 2002). [005] Poly IC is a mimic of viral double stranded RNA and it is recognized by TRIF- dependent TLR3 or Cardif (also known as IPS-1 , MAVS, VISA)-dependent Rig-Like Helicases (RLH) in vivo. It is commonly used as an immune stimulant and is an excellent adjuvant for the induction of T_H1 CD4 T cell responses in a DC-targeted vaccine model (Longhi et al., 2009). [006] Conventional DC (cDC) are not only effective antigen presenting cells but are also well known as an important innate source of cytokines. Among the mouse cDCs, a subset defined by the expression of CD8aa homodimers (CD8⁺) was identified as the major producers of IL-12p70 in various organs including spleen, lymph nodes, thymus and liver (Reis e Sousa et al., 1997; Hochrein et al., 2001 ; Pillarisetty et al., 2004). Another important functional feature of CD8⁺ cDCs is their outstanding capacity for cross-presentation (Shortman et al., 2009).

[007] The CD8⁺ cDCs are clearly a functionally distinct DC subset. However, these functional attributes may not always correspond with CD8 expression. Thus, apart from the CD8 molecule, other combinations of surface markers can be used to identify CD8⁺ cDC or their functional equivalents that may lack CD8 expression (eCD8⁺). Among CD11 c⁺ MHC Class II high cells, various combinations of high expression of CD205, CD103, Necl2, Clec9a, CD24 accompanied with negative or low expression of CD1 1 b and CD172a can be used (Hochrein and O'Keeffe, 2008; Shortman et al., 2009).

[008] DC subsets can be generated in vitro from bone marrow precursor cells in the presence of FL (FLDC) (Brasel et al., 2000). The FLDC cDCs lack expression of CD8 and CD4 but using markers described above, they can be divided into functionally distinct subsets that resemble the spleen cDCs. One FLDC subset has been identified as the eCD8⁺ since it depends on the same transcription factors for development as CD8⁺ cDC, expresses several characteristic surface markers, such as high expression of Clec9a, but low expression of CD11 b and CD172a and shows a similar expression profile of TLRs. Functionally, the eCD8⁺ DC demonstrated a similar TLR-ligand responsiveness, as well as high IL-12p70 production and efficient cross-presentation. Upon in vivo transfer and recovery in the spleen, eCD8⁺ DC expressed CD8 on their surface (Naik et al., 2005).

[009] Expression of the different nucleic acid sensing systems TLR3, TLR7, or TLR9 and the RLHs varies among DC subsets (Hochrein and O'Keeffe, 2008). The downstream functions after engagement of these receptors also differ among the different DCs. pDCs predominantly use TLR7 and TLR9 for nucleic acid sensing, resulting in the high production of IFN-I and IFN-As. Among cDCs, CD8⁺ cDCs highly express TLR3 but lack expression of TLR7 (Edwards et al., 2003). Furthermore, it has been found by proteomics that CD8⁺ cDC, in contrast to the CD8^" cDC, hardly express the RLHs and as a consequence are unable to detect the single stranded RNA viruses Sendai or Influenza virus (Luber et al., 2009).

[0010] CD8 is not expressed on human DC, whereas CD4 is expressed by all DC subsets, and thus other markers have to be employed to define human DC subsets and to possibly align the mouse and human counterparts. A set of antibodies designated BDCA1-4 has been established and is used to differentiate between pDCs and subsets of cDCs (Dzionek et al., 2000). Human BDCA3 positive DC have been proposed as the human eCD8⁺ DC since they, as the mouse eCD8⁺ DC, selectively express high levels of Clec9a and Necl2, but only low amounts of CD11 b (Shortman et al., 2009). Genome wide transcriptional analysis substantiated a close relationship of murine CD8⁺ cDC with human BDCA3⁺ cDCs (Robbins et al., 2008). As with the mouse eCD8⁺ cDCs, the human BDCA3⁺ cDCs have been found in various organs including blood, spleen, lung, tonsils, lymph nodes, colon and liver. Functional correlation between these human and mouse DC subsets are scarce although the CD11 b^low cDC of human thymus correlated with the mouse thymic CD11 b'°^w DC with high IL-12p70 production (Vandenabeele et al., 2001 ; Hochrein et al., 2001).

[0011] Miyake at al., 2009, describes that poly IC activates NK cells via IPS-1 and TRIF dependent ways. Both pathways were involved in B16 tumor suppression via NK cells. CD8a+ cDCs were identified as source of type I IFN (IFN-alpha/beta), IL-6 and IL-12p40 and responsible for the NK cell activation as measured by IFN-gamma production by NK cells.

[0012] Schulz et al., 2005, describes that, dsRNA present in virally infected cells is recognized by dendritic cells via TLR3. That, poly IC activates CD8a+ cDCs (increase of surface markers such as CD40, CD86, CD80 and gene activation of TNF-alpha, IL-6 and IFN-alpha/beta but only IL-6 protein could be detected). It was shown that TLR3 was necessary for this activation and that activated CD8a+ cDCs induced stronger CTL induction via cross-presentation.

[0013] Diebold et al., 2009, describes that replicon plasmid induce dsRNA intermediates which are detected by CD8a+ cDCs in a TLR3 dependent way. In contrast the activation of CTL was independent of TLR3.

[0014] WO 2006/054177 describes that certain tumors express TLR3 and that these tumors might be treated with TLR3-agonists such as poly AU. [0015] WO 2009/088401 describes that combinations of TLR ligands with one of them being a TLR3 agonist would induce increased (adaptive) immune responses especially antigen specific CD8 T-cell responses. The claims also include activation of dendritic cells with combinations of TLR3 agonists and other TLR agonists and claim enhanced CD8 T-cell responses including enhanced cytokines produced by the T-cells.

[0016] WO 2004/060319 describes that combinations of TLR agonists and TNF/R agonist increase the amount of an antigen specific immune response. These antigen specific responses were either from T-helper cells (CD4 T cells) or Killer T cells (CD8 T cells).

[0017] WO 94/28391 describes that ligands for FLT3 can be used for hematopoietic stem cell or other immune cell expansion. Different forms of Flt3-ligands are described.

[0018] WO 2008/131926 describes that M-CSF can be used independent of Flt3- ligands or GM-CSF to induce the generation of dendritic cells. In particular the production of pDCs was independent of FL and of cDCs independent of GM-CSF.

[0019] Ank et al., 2008, describes that many different cell types produce IFN-lambda to TLR ligands or viruses. It also analyses the IFN-lambda receptor expression and uses in vivo virus infection models. Local application (intra vaginal) of poly IC or CpG-ODN protected mice from lethal intra vaginal HSV-2 challenge. It describes also that cDCs, pDC, B-cells T-cells and macrophages from the spleen produced IFN-lambda mRNA in response to HSV-2.

[0020] Sheppard et al., 2003, describes the existence of the IFN-lambdas and that they are related to IFN-I and IL-10 family of cytokines. It shows mRNAs for IFN- lambdas (IL-28A, IL-28B, IL-29), IFN-alpha and IFN-beta of human PBMCs after poly IC treatment or EMCV infection. The mRNA of the 3 IFN-lambdas and IFN-alpha and IFN-beta were upregulated upon exposure to either poly IC or virus. [0021] O'Keeffe et al., 2002, describes the increase of DC subsets in response to various growth factors including showing the increase of CD8a cDCs in response to flt3-ligand. IL-12p40 and IL-12p70 production in response to CpG was analyzed and CD8a+ cDCs and after FL to ProGP (fusion protein of FL and G-CSF) CD8a^int cDCs were the major producers of IL-12p70.

[0022] However, none of the above cited documents and patent applications provides a clue about cells which are the source of IFN-lambda.

[0023] It is therefore an object of the present invention to provide cell types which are responsible for the production of IFN-λ. Summary of the Invention

[0024] Mouse CD8+ and eCD8+ cDCs were identified as major producers of IFN-λ in response to poly IC in vitro and in vivo during the first 9 hours after stimulation. B cells were identified as major producers of a second wave of IFN-λ in response to poly IC in vivo. [0025] The nature of the stimulus and the cytokine milieu determined if CD8⁺ cDCs produced IFN-λ or IL-12p70. IFN-λ but not IFN-q production to poly IC in vivo was abrogated in mice that lacked most DC due to a lack of Fms-related tyrosine kinase 3 ligand (FL). The in vivo poly IC-induced IFN-λ depended on TLR3, but not on RLHs. IRF7, required for MyD88-dependent type I IFN production, was also required for this IFN-λ production. The BDCA3⁺ human DC, proposed to be the equivalents of mouse CD8⁺ DCs, displayed the highest IFN-A1 and IFN-A2 production upon poly IC stimulation. Thus, CD8⁺ cDC equivalents in mouse and humans have been identified as the major source of IFN-As in response to poly IC.

[0026] The second wave of IFN-λ production appeared to reach a maximum level at approximately 15 hours after in vivo stimulation with poly IC, and remained at high levels until 20-30 hours in most cases. The second wave of IFN-λ production was not dependent on TLR-3, and was still seen in a TLR-3 KO mouse. However, the second wave was dependent on Cardif. Experiments with RAG1-KO mice indicated that the second wave was dependent on B-cells.

[0027] The invention encompasses methods for producing an interferon lambda.

[0028] In one embodiment, the method comprises comprising isolating a population of cells comprising B cells and contacting the B cells with a ds nucleic acid or analog thereof. Contacting the B cells with the ds nucleic acid or analog thereof can stimulate the production of an interferon lambda (IFN-λ). Preferably, the population of cells lacks or has been depleted of TLR3 positive cells. Preferably, the population of cells lacks or has been depleted of dendritic cells. Most preferably, the population of cells lacks or has been depleted of CD8+ and eCD8+ dendritic cells. Preferably, the population of cells lacks or has been depleted of NK cells and/or T cells. Preferably, the B cells are plasma B cells or memory B cells.

[0029] In preferred embodiments, the population of cells comprising B cells can comprise more than 50% B cells, more than 75% B cells, or more than 90% B cells. [0030] In various preferred embodiments, the ds nucleic acid or analog thereof that stimulates the production of an IFN-λ is poly IC or a ds DNA.

[0031] In other preferred embodiments, the ds nucleic acid or analog thereof that stimulates the production of an IFN-λ is poly AU.

[0032] In various embodiments, the ds nucleic acid or analog thereof that stimulates the production of an IFN-λ is produced by a dsDNA virus or an ssRNA virus. In preferred embodiments, the virus is a Poxvirus, Herpesvirus, Togavirus, or a Coronavirus

[0033] The method can further comprise detecting the expression of the IFN-λ and/or isolating the IFN-λ produced by the B cells. The IFN-λ can be IFN-A1 , IFN-A2, or IFN-A3. [0034] In a preferred embodiment, the dendritic cells are administering to an animal, preferably a human.

[0035] The invention encompasses methods of detecting the presence of B cells. In various embodiments, the method comprises isolating a population of cells, stimulating the cells with a ds nucleic acid or analog thereof, detecting the production of IFN-λ, and correlating the production of IFN-λ with the presence of B cells.

Detailed Description of the Invention

[0036] The invention encompasses compositions and methods for producing an interferon lambda. In various embodiments, the method comprises isolating a population of cells comprising B cells and contacting the B cells with a ds nucleic acid or analog thereof. Contacting the B cells with the ds nucleic acid or analog thereof can stimulate the production of an interferon lambda (IFN-λ). Preferably, the population of cells lacks or has been depleted of TLR3 positive cells. Preferably, the population of cells lacks or has been depleted of dendritic cells. Most preferably, the population of cells lacks or has been depleted of CD8+ and eCD8+ dendritic cells. Preferably, the population of cells lacks or has been depleted of NK cells and/or T cells. Preferably, the B cells are plasma B cells or memory B cells.

[0037] The population of cells can comprise more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% B cells. [0038] In one embodiment, the cells are plasma B cells. In one embodiment, the cells are memory B cells. In preferred embodiments, the cells are mouse or human cells.

[0039] In one embodiment, the ds nucleic acid or analog thereof that stimulates the production of an IFN-λ is recognized by Cardiff on the B cells. In one embodiment, the ds nucleic acid or analog thereof that stimulates the production of an IFN-λ is poly IC or poly AU. [0040] In various embodiments, the ds nucleic acid or analog thereof that stimulates the production of an IFN-λ is a dsRNA or a dsDNA. The ds nucleic acid or analog thereof can be a DNA or RNA molecule. The dsRNA or a dsDNA can be produced by a dsDNA virus, a dsRNA virus, an ssDNA virus, or a positive ssRNA virus. [0041] In various embodiments, the virus is a positive ssRNA virus, such as a Togavirus, a Flavivirus, a Astrovirus, a Picornavirus, a Calicivirus, a Hepevirus, a Nodavirus, a Arterivirus, or a Coronavirus. In various embodiments, the virus is a dsRNA virus, such as Reovirus or a Birnavirus.

[0042] In various embodiments, the virus is a retrovirus, such as an HIV-1 , HIV-2, or SIV.

[0043] In various embodiments, the virus is a ds DNA virus, such an Asfarvirus, an Iridovirus, a Polyomavirus, a Papillomavirus, a Papovavirus, an Adenovirus, a Herpesvirus, a Poxvirus, or a Hepadnavirus. In a preferred embodiment, the virus is a poxvirus, such as an Orthopoxvirus or a Parapoxvirus. Preferably, the poxvirus is a variola virus, a cowpoxvirus, a camelpoxvirus, or a vaccinia virus. Particularly preferred is a MVA virus. In various embodiments, the virus is a Herpesvirus, such as a Herpes simplex virus (HSV 1 or HSV 2), Varicella Zoster virus, human cytomegalovirus, Epstein-Barr virus, and Kaposi sarcoma-associated herpesvirus.

[0044] The interferon lambda can be an IFN-A1 , IFN-A2, or IFNA-3, which are also referred to as IL-29, IL-28A and IL-28B.

[0045] The IFN-λ produced by the cells can be detected and quantitated by techniques well-known in the art, such as those in the examples. The IFN-λ produced by the cells can also be collected, isolated, and purified by conventional biochemical techniques. In various embodiments, the invention includes isolating a population of cells comprising B cells and measuring the IFN-λ production from these cells. [0046] IFN-λ production in response to poly IC can be used to detect or diagnose the presence of B cells, even in complex mixtures of different cells. For example, a biopsy of an organ or blood can be checked for the presence of those cells via their unique IFN-λ production in response to poly IC. [0047] Thus, the invention encompasses methods of detecting or diagnosing the presence of eCD8+ cDCs or B cells. In various embodiments, the method comprises isolating a population of cells, stimulating the cells with a ds nucleic acid or analog thereof, preferably poly IC, and detecting the production of IFN-λ from eCD8+ cDCs or B cells. The production of IFN-λ can be correlated with the presence of eCD8+ cDCs or B cells. The different timing of the production of IFN-λ from the various cell types (i.e., first vs. second wave) allows for a discrimination between the cell source of the IFN-λ.

[0048] Isolated dendritic can be further incubated with a TLR2, TLR4, TLR9, or TLR11 ligand. This incubation can increase the expression of IFN-λ. In various embodiments, the ligand is Pam3Cys, LPS, CpG-ODN, or profilin. In various embodiments, dendritic cells can be further incubated with a cytokine. Preferably, the cytokine is IL-3, GM-CSF, IL-4, or IFN-λ. In a preferred embodiment, cells can be incubated with FL or an M-CSF receptor ligand, such as M-CSF or IL-34, to increase the formation of dendritic cells. [0049] IFN-λ producing cells can be administered to an animal. The invention encompasses IFN-λ producing cells for the treatment of an infectious disease. Preferably, the cells are isolated dendritic cells or B cells. The cells are derived from a patient, stimulated to produce IFN-λ, and administered to the patient. The invention encompasses the use of IFN-λ producing cells to treat an animal, preferably a human.

[0050] The invention encompasses the use of IFN-λ producing cells for the preparation of a medicament or pharmaceutical composition to treat an animal, preferably a human. In various embodiments, the animal has an infectious disease. In preferred embodiments, the animal is infected with a hepatitis virus or a herpes virus. Similarly, the invention encompasses methods of treatment of a virus-infected patient. Preferably, the method is for treating a patient with a dsDNA or RNA virus infection.

[0051] Additionally, a ds nucleic acid or analog thereof can be administered to the animal, such that the ds nucleic acid or analog thereof stimulates the production of an interferon lambda (IFN-λ). The ds nucleic acid or analog thereof that stimulates the production of an IFN-λ can be poly IC, a ds DNA molecule, a ds RNA molecule, etc. In various embodiments, the invention comprises a ds nucleic acid or analog thereof for treating a patient with an infectious disease and the use of a ds nucleic acid or analog thereof to prepare a pharmaceutical composition for treating a patient with an infectious disease.

[0052] In various embodiments, an increase in IFN-λ production in a patient treated with FL or an M-CSF receptor ligand and/or a ds nucleic acid or analog is detected. The animal can be infected with a DNA or RNA virus. [0053] The present invention is further illustrated by the following drawings and Examples, which in no way should be construed as further limiting. The entire contents of the references cited throughout this application are hereby expressly incorporated by reference.

Brief description of the drawings [0054] Figure 1 depicts splenic CD8⁺ cDC are the major producers of IFN-λ in response to poly IC. Highly purified splenic cDC subsets 5 x 10⁵/ml were stimulated in the presence of IL-3 and GM-CSF with the stimuli as indicated in the examples. After 18 hours, supernatants were analyzed for IFN-λ. Representative results of 3 independent experiments are shown. Data represent mean +/- SD of duplicate samples.

[0055] Figures 2A-C depict the production of IFN-λ or IL-12p70 by CD8⁺ cDCs depends on the stimuli and the cytokine conditions. Sorted splenic CD8⁺ cDC 5 x 10⁵/ml were stimulated and supernatants were analyzed after 18 hours for IFN-λ and IL-12p70. (A) Stimulation in the presence of IL-3 and GM-CSF with the stimuli as indicated. (B) Stimulation with a combination of poly IC+CpG-1668 with the cytokines as indicated. (C) Stimulation in the presence of IL-3+IL-4+IFN-A+GM-CSF with the stimuli as indicated. Representative results of at least 2 independent experiments are shown. Data represent mean +/- SD of duplicate samples.

[0056] Figures 3A and B depict the production of IFN-λ in vivo depends on FL. (A) Isolated total non parenchymal liver cells 2.5x10⁶/ml were stimulated in the presence of IL-3+IL-4+IFN-Y +GM-CSF with the stimuli as indicated. After 18 h supernatants were analyzed for IFN-λ and IL-12p70. Representative results of 3 experiments are shown. Data represent mean +/- SD of duplicate samples. (B) WT and FL-KO mice were injected i.v. with 100Mg poly IC. After 3-4 h sera were analyzed for IFN-λ and IFN-a. Circles indicate the results of individual mice and columns represent the mean thereof. [0057] Figure 4 depicts IFN-λ production to poly IC in vivo depends on TLR3, IFN- AR and IRF7 but not on MyD88 or Cardif. Mice with the indicated genotype were injected i.v. with 100 g poly IC. After 3-4 h sera were analyzed for IFN-λ and IFN-a. Circles indicate the results of individual mice and columns represent the mean thereof. [0058] Figure 5 depicts human BDCA3⁺ cDCs are major producers of IFN-λ upon poly IC stimulation. PBMC, PBMC depleted of BDCA1 and 3, or cells selected for BDCA1 or BDCA3 were stimulated in the presence of IL-3, GM-CSF and IFN-γ with (donor 1 ) 100pg/ml poly IC + l Opg/ml Pam3Cys + Ι Ομς/ml LPS or (donor 2 and 3) with 100pg/ml poly IC for 18-24 h. Supernatants were analyzed for IFN-A1 and IFN- K2. The experiments are shown for the individual donors and data represent mean +/- SD of duplicate samples.

[0059] Figure 6 depicts splenic CD8⁺ cDC are the major producers of IFN-λ in response to DNA viruses. Highly purified splenic cDC subsets 5 x 10⁵/ml were stimulated in the presence of IL-3 and GM-CSF with the stimuli as indicated. After 18 h supernatants were analyzed for IFN-λ. Representative results of 3 independent experiments are shown. Data represent mean +/- SD of duplicate samples.

[0060] Figure 7 depicts splenic CD8⁺ cDC are the major producers of IFN-λ in response to ssRNA viruses. Highly purified splenic cDC subsets 5 x 10⁵/ml were stimulated in the presence of IL-3 and GM-CSF with the stimuli as indicated. After 18 h supernatants were analyzed for IFN-λ. Data represent mean +/- SD of duplicate samples.

[0061] Figure 8 depicts splenic pDCs produce large amounts of IFN-λ to CpG-2216. Highly purified splenic pDCs 5x10⁵/ml were stimulated in the presence of IL-3 and GM-CSF with the stimuli as indicated. After 18 h supernatants were analyzed for IFN-λ. Representative results of 3 independent experiments are shown. Data represent mean +/- SD of duplicate samples.

[0062] Figures 9A and B depict sorted FLDC-derived eCD8⁺ cDCs are major producers of IFN-λ to poly IC. Sorted FLDC subsets 2.5x10⁵/ml were stimulated for 18 h and supernatants were analyzed for IFN-λ and IL-12p70. (A) Stimulated in the presence of IL-4 and IFN-γ with the stimuli as indicated. (B) Stimulated in the presence of poly IC+CpG-1668 with the cytokines as indicated. Representative results of 2 independent experiments are shown. Data represent mean +/- SD of duplicate samples. [0063] Figures 10A-D depict IFN-λ production to poly IC by FLDC-derived eCD8⁺ cDCs depends on TLR3 and IFN-AR but not on MyD88 or Cardif. Sorted FLDC eCD8⁺ 5x10⁵/ml from mice as indicated were stimulated for 18 h and supernatants were analyzed for IFN-λ. (A) WT and MyD88-KO eCD8⁺ DC stimulated with poly IC in the presence of IL-4 and IFN-γ. (B) WT and TLR3-KO eCD8⁺ DC stimulated with poly IC in the presence of IL-3+IL-4+IFN-y+GM-CSF. (C) WT and Cardif-KO eCD8⁺ DC stimulated with poly IC+CpG-1668 in the presence of IL-3 and GM-CSF. (D) WT and IFN-AR-KO eCD8⁺ DC stimulated with poly IC+profilin in the presence of IL-3 and GM-CSF. Representative results of at least 2 independent experiments are shown. Data represent mean +/- SD of duplicate samples. [0064] Figures 11A and B depict the production of IFN-γ in vivo can be increased with treatment of FL or M-CSF. FL-KO mice were treated for 7 consecutive days with 10pg of recombinant FL (A) or M-CSF (B) per day. The next day after growth factor treatment mice were injected i.v. with 100 g poly IC. After 3-4 h sera were analyzed for IFN-λ..^" Circles indicate the results of individual mice and columns represent the mean thereof.

[0065] Figure 12 depicts that poly AU induces IFN-λ but not IFN-a production in vivo. Mice were injected (i.v.) with poly IC (100pg) or poly AU (100 or 500pg). After 3-4 h sera were analyzed for IFN-λ and IFN-a. Circles indicate the results of individual mice and their total number (n) is indicated in the graph. The columns represent the mean of all mice used. Two independent experiments have been performed.

[0066] Figure 13 depicts that in vivo FL expanded CD8a⁺ cDCs and eCD8a⁺ cDCs selectively produce IFN-λ to poly AU in vitro. Highly purified FL expanded ex-vivo isolated splenic 5 x 10⁵/ml were stimulated in the presence of IL-3+GM-CSF+IL- 4+IFN-Y with either poly IC (100 g/ml) or poly AU (100 g/ml). After 18 h supernatants were analyzed for IFN-λ.

[0067] Figure 4 depicts that CD40 costimulation enhances poly IC induced IFN-λ production in vivo. Mice were injected (i.v.) with poly IC (100pg), anti-CD40 mAb (l OOpg) or the combination of poly + anti-CD40 (100pg each). After 3-4 h sera were analyzed for IFN-λ and IFN-a. Circles indicate the results of individual mice and their total number (n) is indicated in the graph. The columns represent the mean of all mice used. Three (IFN-λ) or two (IFN-a) independent experiments have been performed.

[0068] Figure 15 depicts that IFN-λ production to poly IC in vivo depends on IRF3 and IRF7. Mice with the indicated genotype were injected i.v. with lOOpg poly IC. After 3-4 h sera were analyzed for IFN-λ and IFN-a. Circles indicate the result of individual mice and their total number (n) is indicated in the graph. The columns represent the mean of all mice per genotype. Three independent experiments have been performed.

[0069] Figure 16 depicts that IFN-λ production to poly IC in vivo depends on hematopoietic cells, FL and IRF8. Mice with the indicated genotype were injected i.v. with 100pg poly IC and after 3-4 h sera were analyzed for IFN-λ (A) BM reconstituted mice as indicated; (B) WT, IL-15R-KO and RAG1-KO; (C) WT and FL-KO; (D) WT and IRF8-KO. Circles indicate the result of individual mice and their total number (n) is indicated in the graph. The columns represent the mean of all mice per genotype. (A) one (BM chimeras) , (B) two (WT and RAG-KO) or one (IL-15R-KO), (C) three (WT and FL-KO) and (D) two (WT and IRF8-KO) independent experiments have been performed.

[0070] Figure 17 depicts that IFN-λ production to poly IC injection in vivo separates with CD45R-/CD 1 c+/CD8a+ splenocytes. 1.5 - 2 h after i.v. injection of poly IC spleens were harvested and processed. Cell free supernatants were analyzed for IFN-λ after in vitro culture for 18 h. (A) 5x10⁶ cells/ml total spleen cells or cells separated by density centrifugation into light density cells or heavy density cells. (B) total spleen cells 25x10⁶cells/ml of WT or CD1 c-DTR-tg mice treated 2 days before with diphtheria toxin (DT). (C) Total spleen cells before separation or after magnetic bead separation into the denoted populations. The initial cell number of splenocytes added onto the column was 20 x 10⁶. Without further counting each fraction was distributed into 2 wells with 200 μΙ medium/well. Bars represent the mean ± SD of 2 independent experiments (A + C) or 1 experiment (B) using 2 mice per experiment.

[0071] Figure 18 depicts that the production of IFN-λ or IL-12p70 by CD8a+ cDCs depends on the stimuli and the cytokine conditions. Sorted splenic CD8a+ cDCs 5 x 10⁵/ml were stimulated and supernatants were analyzed after 18 h for IFN-λ and IL-12p70. (A) Stimulation in the presence of IL-3 and GM-CSF with the stimuli as indicated. (B) Stimuli and cytokines as indicated. Bars represent the mean ± SD of 2 independent experiments using a pool of at least 8 mice per experiment.

[0072] Figure 19 depicts that in vivo and in vitro FL generated CD8a+ cDCs and eCD8a cDCs are major producers of IFN-λ and IL-12p70. Highly purified (A) FL expanded ex-vivo isolated splenic or (B) generated in vitro from B with FL cDC subsets 5 x 10⁵/ml were stimulated in the presence of IL-3+GM-CSF+IL-4+IFN-y with the stimuli as indicated. After 18 h supernatants were analyzed for IFN-λ and IL-12p70. Bars represent the mean ± SD of 2 independent experiments each using a pool of at least 2 mice per experiment. [0073] Figure 20 depicts that in vivo and in vitro FL generated CD8a+ cDCs, eCD8a cDCs and pDCs are major producers of IFN-λ to HSV-1 and parapoxvirus. Highly purified (A) FL expanded ex-vivo isolated splenic or (B) generated in vitro from BM with FL DC subsets 5 x 10⁵/ml were stimulated in the presence of IL-3+ GM-CSF+IL-4+IFN-y with the stimuli as indicated. After 18 h supernatants were analyzed for IFN-λ. Bars represent the mean ± SD of 2 independent experiments each using a pool of at least 2 mice per experiment.

[0074] Figure 21 depicts that the IFN-λ production to HSV-1 injection in vivo separates with CD45R+ and CD45R-/CD8a+ splenocytes. Spleen cells 1.5 h after in vivo injection with DISC HSV-1 were separated with anti-CD45R and magnetic beads into positive and negative fractions. The CD45R negative fraction was further separated into cells positive or negative for CD8a. Separated cells were cultured in vitro for the next 18 h and cell-free supernatants were analyzed for IFN-Af Bars represent the mean ± SD of 2 independent experiments using one mouse per experiment. [0075] Figure 22 depicts the induction of a second wave of IFN-λ, but not IFN-a, in vivo with poly IC. [0076] Figure 23 depicts the production of the second wave of IFN-λ in vivo with poly IC in wild-type and in various KO mice.

EXAMPLES EXAMPLE 1 : Mice

[0077] MyD88-KO mice were from S. Akira (Adachi et al., 1998), Cardif-KO mice were from J. Tschopp (Meylan et al., 2005), TLR3-KO mice were from The Jackson Laboratory (Alexopoulou et al., 2001), IRF7-KO mice from Tadatsugu Taniguchi (Honda et al., 2005) ,and IFN-AR-KO mice were originally from Michel Aguet (Muller et al., 1994). C57BL/6 WT mice were purchased from Harlan Winkelmann.

EXAMPLE 2: Cells and flow cytometric sorting

[0078] DC subsets were isolated from pooled mouse spleens as described (Vremec et al., 2007). Briefly, spleens were chopped, digested with collagenase (Worthington Biochemical) and DNase (Roche) at room temperature, and treated with EDTA. Low- density cells were enriched by density centrifugation; non-DC lineage cells were coated with mAbs (anti-CD3, KT3-1.1 ; anti-Thy-1, T24/31.7; anti Gr-1 , 1A8; anti- CD19, ID3; anti-erythrocytes, TER119 and anti-NK cells, DX5) and depleted using anti-rat Ig magnetic beads (Qiagen). Dead cells were excluded by propidium iodide staining. cDC populations were sorted based on the expression of CD11c, CD45RA, CD4, CD8a and CD172a and pDCs were purified based on CD11c, CD45RA, and CD172a (all BD Biosciences) expression. Cell sorting was performed on a FACS Aria instrument (BD Biosciences).

[0079] FL bone marrow culture derived dendritic cells (FLDC) were prepared as described (Hochrein et al., 2004). pDCs and eCD8⁺ and eCD8^" cDC subsets were sorted based on the expression of CD11c, CD45R, CD 11 b, CD24, and CD 172a or CD103 (all BD Biosciences). EXAMPLE 3: In vivo challenge with poly IC

[0080] Mice were injected i.v. into the lateral tail vein with 100 ig poly IC (Axxora) and serum was collected 3-4 h after challenge. Sera were pre-diluted 1/5, IFN-a was analyzed by ELISA as described (Hochrein et al., 2004). IFN-λ was determined by an IFN-A3 (IL-28B) ELISA (R&D Systems). This ELISA is largely cross-reactive to IFN-A2 (IL-28A) and does not differentiate between these two mouse IFN-As.

EXAMPLE 4: In vitro stimulation and cytokine detection

[0081] Cells were stimulated in vitro with single TLR agonists or combinations thereof containing 10 pg/ml Pam3Cys (InvivoGen), 100 pg/ml poly IC (Axxora), 10 pg/ml LPS (E.coli; Sigma-Aldrich or Axxora), 10 pg/ml R848 (Axxora), 1 μΜ CpG- 1668 or CpG-2216 (TIB-Molbiol), 1 pg/ml profilin of toxoplasma (Axxora). The recombinant cytokines mouse-IL-3, mouse-IL-4, rat-IFN-y (PeproTech) and mouse- GM-CSF (Tebu-Bio) (10 ng/ml each) were added as indicated. The addition of IL-3 and GM-CSF was based on previous observations that GM-CSF promoted the production of IL-12p70 and that the combination of IL-3 and GM-CSF increased virus induced IFN-a production in pDCs and cDCs (Hochrein et al., 2000; Hochrein et al., 2004). As source of a parapoxvirus Zylexis, which is used for veterinary purposes was purchased from a pharmacy. HSV-1 , in replication deficient form known as disc HSV-1 (HSV-1 d) was used as described (Hochrein et al., 2004). IFN-λ in supernatants was analyzed by ELISA and IL-12p70 was determined by FlowCytomix bead assay (Bender Medsystems) according to manufacturer's protocol.

EXAMPLE 5: Isolation and stimulation of human DC

[0082] PBMC were prepared from peripheral blood of non-atopic blood donors by density gradient centrifugation and BDCA3⁺ DC were purified from PBMC using the BDCA3/CD141⁺ Dendritic Cell Isolation Kit (Miltenyi Biotech) on an AutoMACS™ separator. Subsequently, BDCA1 ⁺ DC were purified from the BDCA3-depleted PBMC using the BDCA1/CD1 c⁺ Dendritic Cell Isolation Kit (Miltenyi Biotech). Preliminary experiments with PBMC and DC enriched fractions of PBMCs have indicated that the addition of the recombinant human cytokines IL-3, GM-CSF and IFN-Y (all PeproTech) (10ng/ml each) enhanced the IFN-A 1 and IFN-A2 production and accordingly this combination of cytokines was added to all stimulations shown. After stimulation for 18-24 h the supernatants were analyzed for IFN-A1 and IFN-A2 by ELISA according to manufacturer's recommendations (Tebu-bio).

EXAMPLE 6: CD8⁺ conventional DCs are the major producers of IFN-A in response to poly IC

[0083] Poly IC, well know for its ability to induce large amounts of IFN-I, has also been described as a potent inducer of IFN-A (Kotenko et al., 2003; Sheppard et al., 2003). pDCs were identified as major producers of IFN-As in response to several viruses or to CpG-ODN stimulation but the cellular source of poly IC induced IFN-A remains elusive (Coccia et al., 2004; Ank et al., 2008).

[0084] Stimulation of fractionated spleen cells with a panel of TLR ligands revealed that the major lymphocyte fractions consisting of T- and B-lymphocytes were unable to produce IFN-A whereas all IFN-A production was confined to enriched preparations of DCs. Among highly purified splenic DC subsets the pDCs, as previously reported, were the major source of IFN-A in response to the A-type ODN CpG-2216 (Fig. 8). However in response to poly IC stimulation the CD8⁺ cDCs were the major producers, with pDCs and CD8^" cDCs being largely unable to participate in IFN-A production (Fig. 1 and Fig. 8). In vitro generated FLDC subsets were also examined. As for ex vivo isolated pDC and cDC subsets, the eCD8⁺, but not the eCD8^" cDCs or the pDC, produced IFN-A to poly IC (Fig. 9 A). Thus, CD8⁺ cDCs and their in vitro equivalents are the major producers of IFN-A in response to poly IC stimulation. EXAMPLE 7: IFN-A and IL-12p70 production by CD8⁺ cDCs depends on the type of stimulus and the cytokine conditions

[0085] CD8⁺ cDCs are well known for their exceptional capacity for IL-12p70 production. Since it was found that the CD8⁺ cDCs were also able to produce large amounts of IFN-λ, the conditions that would govern IFN-λ were compared to those governing IL-12p70 production. Using a panel of TLR stimuli, it was found that TLR- ligands known for their high IL-12p70 induction, such as CpG-ODN or profilin of toxoplasma (Hochrein et al., 2000; Yarovinsky et al., 2005), induced large amounts of IL-12p70, as expected, but surprisingly under these conditions the CD8⁺ cDCs did not produce any IFN-λ. In contrast, poly IC induced IFN-λ but not IL-12p70 production by CD8⁺ cDCs (Fig 2 A). Combinations of poly IC together with Pam3Cys, LPS, CpG-ODN or profilin, ligands for TLR2, TLR4, TLR9 or TLR11 respectively, synergistically increased IFN-λ production (Fig 2 A). In line with a lack of TLR7 and thus unresponsiveness of CD8⁺ cDCs to TLR7 stimulation, R848 was unable to support poly IC induced IFN-λ production (Fig 2 A). These data demonstrate a synergistic increase of poly IC induced IFN-λ with myeloid differentiation primary response gene 88 (MyD88) - dependent stimuli and confirm described synergistic effects on the production of IL-12p70 by CD8⁺ cDCs (Fig. 2 A) (Napolitani et al., 2005).

[0086] It has been previously shown that the cytokine milieu during stimulation is highly influential for IL-12p70 production in murine and human DCs, with IL-4 being a major enhancer for bioactive IL-12 production (Hochrein et al., 2000; Kalinski et al., 2000). Using a combinatory stimulus (poly IC + CpG-1668), which induced both IFN- λ and IL-12p70, it was found that IFN-γ enhanced the production of IFN-λ with little effects on IL-12p70 production, whereas IL-4 increased IL-12p70, but not IFN-λ production (Fig. 2 B). Combining IL-12p70 and IFN-λ enhancing cytokines (IL-3 + GM-CSF + IL-4 + IFN-γ) with single stimuli (poly IC or profilin) demonstrated that the stimulus-dependent mutually exclusive production of IFN-λ or IL-12p70 by CD8⁺ cDCs was preserved (Fig. 2 C). However, combinations of stimuli (poly IC + CpG- 1668 or poly IC + profilin) plus cytokines enabled the production of large amounts of IFN-λ and IL-12p70 at the same time (Fig. 2, B and C).

[0087] Compared to the ex vivo isolated splenic DC subsets, FACS-sorted pDC, eCD8⁺ cDCs and eCD8^" cDCs from FLDC demonstrated a very similar subset specificity as well as stimulus and cytokine dependence for IFN-λ production (Fig. 9). Thus as described for other functional parameters such as IL-12p70 production or cross-presentation, the IFN-λ production of eCD8⁺ cDCs from FL cultures demonstrates a high degree of functional similarity to ex vivo isolated CD8⁺ cDCs.

EXAMPLE 8: IFN-λ production to poly IC in vivo depends on Flt3 ligand [0088] FL is an essential growth factor for the development of DCs in the steady state and mice deficient for FL (FL-KO) have drastically reduced amounts of DCs including pDCs and CD8⁺ cDCs (McKenna et al., 2000). To define the role of DCs as a source of IFN-λ in organs other than spleen, liver cells were isolated from wild type and FL-KO mice and stimulated them under cytokine conditions for expression of both IFN-λ and IL-12p70 induction with either solely poly IC or profilin or a combination thereof. As found with sorted CD8⁺ or eCD8⁺ cDCs (Fig. 2 and Fig. 8 B), liver cells from WT mice produced IFN-λ to poly IC and IL-12p70 to profilin whereas the combination of both stimuli supported the production of IFN-λ and IL- 12p70 simultaneously (Fig. 3 A). In contrast, liver cells of FL-KO mice displayed a largely abrogated production of IFN-λ as well as IL-12p70 to this stimulation (Fig. 3 A). Since non-hematopoietic cells and most non-DC populations are believed to be normal in FL-KO mice, this suggests that DCs were the major source of the IFN-λ produced. CD8⁺ or eCD8⁺ cDCs, but not pDCs or other cDC subsets, selectively express TLR1 1 and thus are selectively able to respond to profilin and to produce IL- 12p70 (Fig. 2 and Fig. 9 A) (Yarovinsky et al., 2005). The concomitant abrogation of IFN- and IL-12p70 in FL-KO liver cells upon stimulation selective for CD8⁺ and eCD8⁺ cDCs strongly suggests that this cDC subset is the source of the IFN-λ produced and points to a prominent role for eCD8⁺ cDCs as a major source of IFN-λ in the liver in vivo. Thus, the IFN-λ production under those selective stimulatory conditions might serve as an indicator for CD8⁺ cDC, even in a complex mixture of different cell types.

[0089] To extend these observations to a direct in vivo challenge, the response of WT and FL-KO mice to poly IC injection was compared. Serum levels of IFN-λ in response to poly IC were easily detectable in WT mice as were the levels of IFN-a. In sharp contrast, in FL-KO mice the levels of IFN-λ were almost abrogated, whereas IFN-a remained easily detectable (Fig. 3 B). Application of recombinant FL into FL- KO mice not only restored, but even increased, their IFN-λ producing capacity above WT level (Fig. 1 1A). Application of M-CSF into FL-KO mice was also able to increase IFN-λ production to poly IC demonstrating that M-CSF is able to increase the number of IFN-λ producers to poly IC (Fig. 11 B). Along those lines, FL treated WT mice which display elevated DC numbers, including CD8⁺ cDCs, had a greatly increased systemic IFN-λ response to poly IC challenge. The FL dependence strongly suggests that the IFN-λ production to poly IC in vivo is largely mediated by DC. Moreover these data indicate that the CD8⁺ and eCD8⁺ cDC subsets are responsible.

EXAMPLE 9: IFN-λ production to poly IC in vivo depends on TLR3, IFN-AR, and IRF7

[0090] Poly IC is detected by the immune system in redundant ways and roles for RLH as well as TLR3 have been described (Alexopoulou et al., 2001 ; Gitlin et at., 2006). To determine the pattern recognition receptors involved in the poly IC induced IFN-λ production in vivo, poly IC was injected into mice deficient for various pattern recognition receptors or their adaptor molecules, specifically TLR3, MyD88 or Cardif and IFN-λ as well as IFN-a were measured in the corresponding sera (Fig. 4). Large amounts of IFN-λ and IFN-a were induced in WT mice and MyD88-KO, demonstrating that MyD88-dependent TLRs were not involved and suggesting that pDC, which largely depend on MyD88 for IFN production, did not likely contribute to the production of both cytokines under those conditions. However, deficiency of TLR3 resulted in abrogated IFN-λ production with no effect on the production of IFN- a. The TLR3 dependence in vivo supports that the CD8⁺ and eCD8⁺ cDCs are the source of IFN-A_> because this subset is particularly known for its high expression of TLR3 and to recognize poly IC in an exclusively TLR3 dependent fashion (Edwards et al., 2003; Schulz et al., 2005). In contrast, Cardif-deficiency revealed no effects on IFN-λ production but, consistent with previous reports, complete abrogation of serum IFN-a (Fig. 4; Gitlin et al., 2006). Thus, whereas poly IC induced large systemic levels of both IFN-λ and IFN-a in WT mice, the dependence on TLR3 or Cardif seems to be mutually exclusive. A similar dependence on TLR3 but not Cardif or MyD88 for the production of IFN-λ could be detected with eCD8⁺ cDCs generated in vitro from the corresponding KO mice (Fig. 10 A-C). These findings, together with the observed dependence on FL, strongly suggest that the IFN-λ production to poly IC in vivo largely depends on DCs of the CD8⁺ and eCD8⁺ subsets.

[0091] It has been described that optimal IFN-I production in vivo requires expression of a functional IFN-I receptor (IFN-AR). A role for IFN-AR has also been proposed for the production of IFN-λ in response to either Sendai Virus or Herpes simplex Virus (Ank et al., 2008). Here it was found, in line with the data of Ank and colleagues, that systemic production of IFN-λ and IFN-a in response to poly IC was largely dependent on the presence of IFN-AR (Ank et al., 2008). A similar dependence on the IFN-AR was detected using in vitro generated eCD8⁺ from either WT or IFN-AR-KO mice (Fig. 10 D). [0092] To shed further light on the regulation of IFN-λ production to poly IC in vivo, the response of IFN regulatory factor 7 (IRF7) deficient mice was analyzed. IFN-a production was almost abrogated in IRF7-KO mice (Fig. 4). An essential role for IRF7 has been demonstrated previously for MyD88 dependent IFN-a production by pDC and a participation of IRF7 in TRIF-dependent IFN-I production by DCs has been proposed (Honda et al., 2005; Tamura et al., 2008). It was found that the production of IFN-λ in the serum was largely reduced in the absence of IRF7 indicating a prominent role for IRF7 for the production of IFN-λ by eCD8⁺ cDCs (Fig. 4). The in vivo findings of a prominent role for IRF7 for the production of IFN-λ in response to poly IC are in line with previous promoter based studies proposing a role of IRF7 in the induction of IFN-a and IFN-λ (Osterlund et al., 2007).

EXAMPLE 10: Human BDCA3⁺ DC are major producers of IFN-As upon poly IC stimulation

[0093] In mice, the separation into several cDC subsets is well established and correlates with subset specific phenotype and function, such as the ability of CD8⁺ cDCs to produce large amounts of IL-12p70 or to cross-present antigens. Even though the evidence for a similar cDC subset discrimination in human has increased in recent years, this is mainly based on phenotypic similarities with only few functional analogies. It was found that the IFN-λ production in response to poly IC in mice is a CD8⁺ cDC subset specific feature. It was desirable to establish if this feature correlated to any human DC subsets. Based on phenotypic similarities, such as Clec9a and Necl2 expression, the BDCA3 positive human DCs have been proposed as potential human eCD8⁺ cDCs. In PBMCs and fractions of DC-enriched PBMCs, it was found that poly IC induced IFN-A1 (IL-29) and IFN-A2 (IL-28A). Separation of cDC subsets using the markers BDCA1 or BDCA3 revealed that the BDCA3 positive cells for all donors tested were the major producers of IFN-A1 , as well as IFN-A2 (Fig. 5). Thus, in terms of IFN-A production upon poly IC stimulation, the human BDCA3 cDCs functionally resemble the murine eCD8⁺ cDCs.

EXAMPLE 1 1 : eCD8⁺ cDCs are major producers of IFN-A in response to DNA viruses

[0094] Herpesviridae is a family of double stranded DNA viruses also named herpesviruses which cause persistent recurring infections and in human include important pathogens such as Herpes simplex virus (HSV) 1 and 2; Varicella zoster virus (VZV), human cytomegalovirus (HCMV), Kaposi's sarcoma-associated herpesvirus (KSHV) and Ebstein-Barr virus (EBV). Previously, it was found that HVS-1 is recognized by pDC via TLR9 via a MyD88 dependent way but that it is seen by cDC independent of MyD88 via a up to date unknown recognition pathway (Hochrein et al., 2004). IFN-A was able to protect against mucosal infection with HSV and TLR dependent protection was largely IFN-A dependent (Ank et al., 2008). [0095] The family of poxviridae, also named poxviruses, are double stranded DNA viruses which can be separated into several subfamilies such as orthopoxviruses, parapoxviruses and others. Among the poxviruses are important pathogens for human and animals such as variola viruses the causative agent of smallpox, cowpoxvirus, camelpox and Vaccinia viruses. Parapoxviruses are important pathogens for cattle and other animals. Orthopoxviruses and para poxviruses are recognized by DC via TLR9 dependent and independent pathways (Samuelsson et al., 2008; Siegemund et al., 2009). Some poxviruses encode for an IFN-λ binding protein and poxviruses encoding recombinant IFN-λ were highly attenuated, suggesting a role for IFN-λ in the protection against poxvirus infections (Bartlett et al., 2005; Bartlett et al., 2004).

[0096] To determine if the eCD8⁺ cDC are also producers of IFN-λ in response to DNA viruses, response of cDC subsets to HSV-1 and a parapoxvirus, representing the families of Herpesviruses and poxviruses, was tested. [0097] It was found that among ex vivo isolated cDC from spleen the CD8⁺ cDC were the major producers of IFN-λ in response to either HSV-1 or parapoxvirus (Fig. 6). Using in vitro generated cDC subsets, it was found that again the eCD8⁺ cDCs were the main producers of IFN-λ to HSV-1 and parapoxvirus. eCD8+ cDCs generated from mutant mice which lacked either Cardif, MyD88 or TLR3 revealed that neither the RLHs nor the TLRs were important for the generation of IFN-λ by eCD8⁺ cDCs in response to HSV-1 or parapoxvirus.

[0098] Since IFN-As seem to induce antiviral activity against herpesviruses and poxviruses and based on the novel knowledge of eCD8⁺ as a major source of IFN-λ this can lead to new therapeutic approaches such as induction of large numbers of eCD8⁺ cDCs with growth factors e.g. FL or M-CSF-R ligands (Ivl-CSF, IL-34). The viruses themselves can be recognized by the enhanced numbers of eCD8⁺ cDCs which can induce antiviral IFN-λ, thus restriction the growth of the pathogenic viruses. Alternatively, external stimuli such as mimics for DNA or RNA e.g. poly IC can be used to induce the IFN-λ production by eCD8+ cDCs in vivo. EXAMPLE 12: eCD8⁺ cDCs are major producers of IFN-λ in response to RNA viruses

[0099] Since it was found that double stranded (ds) RNA e.g. poly IC is inducing IFN-λ by eCD8+ cDCs, it was next determined if RNA viruses would induce IFN-λ also. It is known that dsRNA is not only present upon infection with dsRNA viruses but that dsRNA intermediates are produced upon infection with single stranded (ss) RNA viruses especially of positive ssRNA viruses. Positive ssRNA families, such as Picornaviruses Flaviviridae, Coronaviridae, Togaviridae, include human and animal pathogens such as West Nile virus, Dengue virus, Hepatitis C virus, SARS, Rubellavirus and others. To test different positive ssRNA viruses representing two different ssRNA virus families, Semliki Forest Virus (SFV) and Mouse Hepatitis Virus (MHV), representing Togaviridae and Coronaviridae respectively, were used.

[00100] Among ex vivo isolated cDCs, the IFN-λ response to SFV and MHV was restricted to the CD8⁺ cDC subset with no production of IFN-λ by the CD8^" cDC subsets (Fig. 7 A). Similar results were found for in vitro generated eCD8⁺ cDCs. With eCD8⁺ cDCs, it was found that the production of IFN-λ to SFV and MHV was still robust in the absence of MyD88, but that the IFN-λ production to those viruses was lost in the absence of TLR3. Thus, eCD8⁺ cDCs use TLR3 to produce IFN-λ in response to ssRNA viruses, presumably via dsRNA intermediates.

[00101] An important role for IFN-λ in the susceptibility and cure against Hepatitis C virus (HCV) has recently been implicated by genomic analysis (Ge et al, 2009; Suppiah et al., 2009; Tanaka et al., 2009; Thomas et al., 2009).

[00102] It was found that the eCD8⁺ cDCs produce IFN-λ in response to positive ssRNA viruses (Fig. 7). Furthermore, it was found that eCD8⁺ cDCs can be identified in the liver (Fig. 3 A). Importantly, eCD8⁺ cDCs do not depend on MyD88 or RLHs for the production of IFN-λ. HCV is known to inhibit signaling of the RLHs and thus inhibits IFN-a production of body cells including CD8^" cDCs which rely on RLHs for the recognition of HCV (Meylan et al., 2005). Since it was found that eCD8⁺ cDCs do not use RLHs but TLR3 for the detection of poly IC and positive ssRNA viruses, this can result in eCD8⁺ cDCs still able to produce the antiviral cytokine IFN- λ to HCV whereas other cells that rely on RLHs are inhibited. Increasing the amount of eCD8⁺ cDCs can drastically increase the amount of IFN-λ produced in response to viruses including ssRNA viruses and can be further enhanced by the application of external stimuli such as poly IC or replication deficient DNA viruses (e.g. HSV-1d). The application of eCD8⁺ cDCs or the in vivo enhancement via growth factors can, with or without combinations with standard therapies such as IFN-I therapy, increase the antiviral response to persistent viruses such as HCV or Herpesviruses. [00103] The production of IFN-λ upon poly IC is a novel hallmark function of eCD8⁺ cDCs, conserved among evolutionary distant species. It is likely that the production of IFN-As contributes to the excellent adjuvant effect of poly IC administration. Moreover, CD8⁺ cDCs and their equivalents, well known for their cross-presentation and IL-12p70 capabilities, are likely contributors to TLR3 mediated anti-viral responses through their high production of IFN-As. These new findings can be transferred into novel therapeutic approaches which can impact hard to treat persistent infections such as Hepatitis C Virus infections.

EXAMPLE 13: Poly AU Induction of IFN-λ

[00104] Double stranded RNA (dsRNA) is recognized via TLR3 or via Rig-like Helicases (RLH). However, the lengths, the composition or modifications of the RNA can influence the detection via the different RNA receptors. It was seen that in response to polyinosinic:polycytidylic acid (poly IC) the early production of IFN-λ fully depends on the presence of TLR3 and on certain DC subsets (CD8a+ and eCD8a cDCs) whereas the systemic production of IFN-a was independent of TLR3 and independent of CD8a+ cDCs but was fully dependent on the RLHs (as seen with Cardif-KO mice which lack an essential adaptor molecule for RLHs). The dsRNA polyadenylic:polyuridylic acid (poly AU) is another form of dsRNA and we tested if poly AU can be used to induce IFN-λ in vivo. Interestingly, poly AU injection induced IFN-λ in the sera of mice, but systemic IFN-a was not detectable. Thus, using certain form of stimuli it is possible to induce systemic IFN-I without the induction of systemic IFN-a.

[00105] To determine if the IFN-λ production to poly AU seen in vivo would correspond with the same cells as in response to poly IC we enhanced the amount of DC in mice with FL treatment and sorted CD8a+ cDCs, eCD8a cDCs and CD11 b+/CD172a+ cDCs. Those cells were stimulated with poly IC or poly AU. Indeed only CD8a+ cDCs and eCD8a cDCs, but not the other cDCs (CD11 b+/CD172a+ cDCs) were able to produce IFN-λ to poly IC and to poly AU alike. The results are shown in Figure 13. EXAMPLE 14: Induction of IFN-λ with poly IC and CD40 stimulation

[00106] Dendritic cells (DC) can be stimulated with pathogen associated molecular pattern (PAMPs) such TLR-ligands and respond with maturation and cytokine production. Beside PAMPS endogenous stimuli exist and one of best described activator mechanism is the interaction of CD40 with its ligand CD40- ligand. DC express CD40 and activated T-cells express CD40-ligand and the interaction of T-cells and DC activates DC. One of the consequences of this activation is the production of cytokines including IL-12p70. Since it was found that the combination of IL-12 inducers such as profilin or CpG-ODN together with poly IC induced in vitro a synergistic increase of IFN-λ production, it was determined whether the combination of poly IC and CD40-stimulation would affect the IFN-λ production in vivo. As a stimulus for CD40, a monoclonal antibody (mAb) to CD40, know to be stimulatory in vivo, was used. The results are shown in Fig. 14.

EXAMPLE 15: Second wave of IFN-λ production in vivo

[00107] In response to poly IC, CD8a cDCs and their equivalents produce large amounts of IFN-lambda. Systemic IFN-lambda levels measured 3-4 hrs after injection of poly IC in vivo depended on TLR3, but not Cardif. An analysis was next performed as a time kinetic and measured IFN-a and IFN-λ in the serum after 3, 6, 9, 15, 20 and 30 hours after poly IC injection (100Mg per mouse i.v.). Surprisingly, a second wave of IFN-λ production was discovered (Fig. 22). After 9 hours, the IFN-λ levels in serum were close to the detection limit, but 15 hours after injection the mice again produced large amounts of IFN-λ, even though the variation between mice was greater as for the first wave of IFN-λ production (measured after 3 hrs). EXAMPLE 16: Second wave of IFN-λ production is independent of TLR3, but dependent on Cardif and B Cells

[00108] To elucidate the source and the mechanism behind this 2^nd wave IFN- λ production, a large panel of gene deficient mice was analyzed 15 hours after poly IC injection (100pg per mouse i.v.) for their IFN-λ content in serum (Fig. 23). Surprisingly TLR3-KO mice, which as shown before do not produce substantial IFN-λ 3 hours after poly IC injection (see Fig. 4), produced IFN-λ similar to wild type mice 15 hrs after poly IC injection. FL-KO mice which lack the growth factor flt3-ligand and have drastically reduced dendritic cells and some B-cell and NK-cell defects demonstrated only a low production of low production of IFN-λ. Since IL-15R-KO mice demonstrated a normal IFN-λ response NK-cells seem not to be involved into this production. Most of the RAG mice tested (RAG mice lack T-cells and B-cells) showed a dramatic reduction in IFN-λ production after 15 hours. Since MHC-II-KO mice (mice lack CD4 T-cells) and b2m-KO mice (mice lack CD8 T-cells) were able to produce normal IFN-λ, T cells seem not to be essential for the 2^nd wave of IFN-λ. The normal 2^nd wave IFN-λ production of CD40L-KO mice (CD40L is used by T cells to activate other immune cells e.g. dendritic cells) also indicates T cells are not responsible for the IFN-λ produced after 15 hours. On the receptor level, mice that lack either IFN-IR (those mice cannot respond to type I interferons such as IFN-α/λ) or IFN-gR (those mice cannot respond to IFN-gamma) produce relatively low levels of IFN-λ after 15 hours. Interestingly, mice which lack the transcription factor IRF7 (IRF7-KO) and thus are unable to produce systemic IFN-a to poly IC were normal in the production of IFN-λ after 15 hours. Lack of the adaptor molecule Cardif (Cardif- KO), which is essential for intracellular RNA detection receptors Rig-I and MDA-5, showed a complete abrogated IFN-λ production after 15 hours. Interestingly, those mice had a normal IFN-λ production to poly IC 3-4 hours after injection (see Fig. 4). References

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Claims

Claims

1. A composition comprising a double-stranded (ds) nucleic acid or analog thereof for use in the induction of IFN-λ production in B cells.

2. A composition comprising a double-stranded (ds) nucleic acid or analog thereof for use in the induction of a first and a second wave of IFN-λ production.

3. The composition of claim 2, wherein the second wave of IFN-λ production is not dependent on toll-like receptor (TLR) 3 positive cells.

4. The composition of claim 2 or 3, wherein the second wave of IFN-λ production is dependent on Cardif positive cells.

5. The composition of any of claims 2 to 4, wherein the second wave of IFN-λ production is dependent on B cells.

6. The composition of claim 1 or of any of claims 2 to 5, wherein the ds nucleic acid or analog thereof is polyinosinic:polycytidylic acid (poly IC).

7. An ex vivo method for producing IFN-λ comprising:

(a) Providing a population of cells comprising B cells, and

(b) Contacting the B cells with a double-stranded (ds) nucleic acid or analog thereof.

8. An ex vivo method for generating IFN-λ producing B cells comprising: (a) Providing a population of cells comprising B cells, and

(b) Contacting the B cells with a double-stranded (ds) nucleic acid or analog thereof.

9. The method of claim 7 or 8, wherein the B cells are plasma B cells.

10. The method of any of claims 7 to 9, wherein the population of cells comprising B cells comprises more than 50% B cells, preferably more than 75% B cells.

1 1. The method of any of claims 7 to 10, wherein the B cells are memory B cells.

12. The method of any of claims 7 to 11 , wherein the ds nucleic acid or analog thereof is polyinosinic:polycytidylic acid (poly IC) or poly AU.

13. The method of any of claims 7 to 11 , wherein the ds nucleic acid or analog thereof is a ds DNA.

14. The method of any of claims 7 to 1 1 , wherein the ds nucleic acid or analog thereof is produced by a dsDNA virus.

15. The method of claim 14, wherein the virus is a Poxvirus or a Herpesvirus.

16. The method of any of claims 7 to 11 , wherein the ds nucleic acid or analog thereof is produced by an ssRNA virus.

17. The method of claim 16, wherein the virus is a Togavirus or a Coronavirus.

18. The method of any of claims 7 to 17, further comprising detecting the expression and/or production of the IFN-λ.

19. The method of any of claims 7 and 9 to 18, further comprising isolating the IFN-λ produced by the B cells.

20. The method of any of claims 8 to 18, further comprising isolating the IFN-λ producing B cells.

21. A composition comprising an IFN-λ producing B cell for use in the prevention and/or treatment of a dsDNA or RNA virus infection.

22. The composition of claim 21 , wherein the virus infection is a hepatitis or a herpes virus infection.

The composition of claim 21 or 22, wherein the IFN-λ producing B cell is produced according to the method of any of claims 8 to 18 and 20.

An ex vivo method for detecting the presence of B cells comprising:

(a) Providing a population of cells comprising B cells and lacking or being depleted of toll-like receptor (TLR) 3 positive cells,

(b) Contacting the cells with a double-stranded (ds) nucleic acid or analog thereof,

(c) Detecting the production of IFN-λ, and

(d) Correlating the production of IFN-λ with the presence of B cells.

The method of claim 24, wherein the population of cells comprising B cells lacks or is depleted of dendritic cells.

The method of claim 24 or 25, wherein the ds nucleic acid or analog thereof is polyinosinic:polycytidylic acid (poly IC).

Use of the method of any of claims 24 to 26 for detecting the presence of B cells in a biopsy, preferably a biopsy of an organ or blood.

Use of a double-stranded (ds) nucleic acid or analog thereof for the induction of IFN-λ production in B cells ex vivo.

The use of claim 28, wherein the ds nucleic acid or analog thereof is polyinosinic:polycytidylic acid (poly IC).

A method for inducing IFN-λ production in B cells in a subject in need thereof, comprising the step of administering to said subject a double stranded (ds) nucleic acid or analog thereof.

31. A method for inducing a first and a second wave of IFN-λ production in a subject in need thereof, comprising the step of administering to said subject a double stranded (ds) nucleic acid or analog thereof.

32. A method for the prevention and/ or treatment of an infectious disease, comprising the step of administering to said subject an IFN-λ producing B cell.