WO2012055541A1

WO2012055541A1 - Tgf-beta in the development of conventional dendritic cells and uses thereof

Info

Publication number: WO2012055541A1
Application number: PCT/EP2011/005381
Authority: WO
Inventors: Hubertus Hochrein
Original assignee: Bavarian Nordic A/S
Priority date: 2010-10-25
Filing date: 2011-10-25
Publication date: 2012-05-03

Abstract

The present invention relates to TGF-beta in the development of conventional dendritic cells and the production of IFN-lambda by such conventional dendritic cells. The invention also relates to corresponding therapeutic uses of TGF-β and of the specific dendritic cell type in the prevention and treatment of infectious diseases and cancer. The conventional dendritic cells according to the invention are CD8+ conventional dendritic cells (CD8+ cDCs) and equivalents thereof (eCD8+ cDCs) in mouse and human.

Description

TGF-BETA IN THE DEVELOPMENT OF CONVENTIONAL DENDRITIC CELLS AND

USES THEREOF

FIELD OF THE INVENTION

The present invention relates to the field of immunotherapy, in particular to the field of the development of conventional dendritic cells and the production of interferons (IF) by conventional dendritic cells. The invention relates to TGF-β in the development of a specific dendritic cell type and to the specific dendritic cell type for the production of IFN-lambda (IFN-λ) and for use as antigen-presenting cell. In particular, the present invention relates to in vivo and in vitro generation of CD8+ and/or eCD8+ conventional dendritic cells (cDCs) and uses thereof. Furthermore, the present invention relates to compositions and methods for the production of IFN-λ in vitro and in vivo. The present invention thus relates to therapeutic applications of induction of CD8+ and/or eCD8+ cDC development and use of these cells in therapy, including their use as IFN-λ producing cells. BACKGROUND OF THE INVENTION ,

Dendritic cells (DCs) are the most effective antigen presenting cells (APC) which play an important role in the induction of adaptive immune responses including cytotoxic T- cells (CTL), T helper cells (T_H) type, regulatory T-cells (T_reg) and direct and indirect B- cell responses. Furthermore, DCs are important as sentinels of the immune system detecting any type of pathogen or danger and for inducing innate immune responses such as activation of natural killer (NK) cells. DCs are very small populations of immune cells which can be subdivided into several subsets. Those subsets differ in phenotype, origin and function (Shortman, K. and Y.J. Liu. 2002. Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2:151-161 ). DCs are often categorized into plasmacytoid DC (pDC) and non-plasmacytoid DC, named conventional DC (cDC). pDC are important antiviral DC which have the ability to induce enormous amounts of Interferon (IFN) type I (consisting of several IFN-as and one band several other IFN-I members) and IFN type III (also called IFN-I or IL-28/29) upon stimulation of the Tolllike receptors (TLR) 7 or 9, which recognize ssRNA or DNA respectively. The IFNs are essential mediators of antiviral responses and thus pDC play an important role for the fight against viruses (Liu, Y.J. 2005. IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu. Rev. Immunol. 23:275-306). However pDC derived IFN-I has been implicated in the exaggeration of certain auto immune diseases.

The other category of DC is the cDCs which can be further subdivided into several cDC subsets. One subset which in the spleen of mice expresses CD8aa homodimers is especially important due to certain functional aspects (Vremec, D., J. Pooley, H. Hochrein, L. Wu, and K. Shortman. 2000. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. Immunol. 164:2978-2986). CD8⁺ cDCs have also been described in other organs such as lymph nodes, thymus or liver (Hochrein et al. 2001 ; Pillarisetty et al. 2004). But DC with functional and phenotypic attributes of CD8+ cDC do not necessarily express CD8 on their surface. Those cells are named equivalents of CD8⁺ cDC (eCD8+).

The development of DC subsets is regulated by specific transcription factors. However the conditions which induce the development of a hematopoietic stem cell or precursor cell into a certain DC subset are only poorly understood. The CD8+ and eCD8⁺ cDC subset has a unique functional profile important for the induction of important innate and adaptive immune responses it would be highly desirable to increase the amount and development of eCD8⁺ cDCs.

The IFN-lambda (IFN-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-lambdas (IFN-As) are potent immune-modulatory and anti-viral cytokines, recently implicated in clearance of Hepatitis C virus in humans. IL-28A (also named IFN-A2), IL- 28B (IFN-A3) and IL-29 (IFN-A1) are type III interferons that are class II cytokine receptor ligands. 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-A R1 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 dendritic cells (pDCs) (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.

Several cell types have been described to produce IFN-λ including monocyte derived dendritic cells (DCs) and plasmacytoid dendritic cells (pDCs), but the cellular origin of double-stranded (ds) nucleic acid-induced IFN-λ in vivo is still elusive (Coccia et al., 2004; Ank et al., 2008; Osterlund et al., 2005). Monocyte derived DCs are not CD8+ conventional DCs (CD8+ cDCs) or equivalents of CD8+ cDCs (eCD8+ cDCs) since eCD8+ cDCs involve Fms-related tyrosine kinase 3 ligand (Flt3)-ligand (FL), but not GM-CSF, for development. Monocyte derived DCs fully depend on GM-CSF for development, even though GM-CSF might be combined with other cytokines such as IL-4 or TNF-alpha (TNF-a). GM-CSF dependent DCs are not equivalents of steady state DCs because the lack of GM-CSF or the GM-CSF receptor has no influence on the presence of normal pDC or cDC subsets in lymphoid organs (Naik et al. 2008). If cells are generated in vitro with the combination of GM-CSF and FL, only GM-CSF DC develop, but not pDCs or eCD8+ cDCs (Gilliet et al. 2002).

Polyinosinic:polycytidylic acid (poly IC) is a mimic of viral double stranded (ds) RNA generated during viral infections 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 it is an excellent adjuvant for the induction of Th1 CD4 T cell responses in a DC-targeted vaccine model (Longhi et al., 2009).

Conventional dendritic cells (cDCs) are not only effective antigen presenting cells but are also known as an 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 functional feature of CD8+ cDCs is their capacity for cross-presentation (Shortman et al., 2009).

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 CD1 1 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).

DC subsets can be generated in vitro from bone marrow precursor cells in the presence of Flt3-ligand (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 CD1 1 b and CD172a and shows a similar expression profile of TLRs. Functionally, the eCD8+ DCs demonstrate 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+ DCs express CD8 on their surface (Naik et al., 2005).

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+ cDCs, in contrast to CD8- cDCs, hardly express the RLHs and as a consequence are unable to detect the single stranded (ss) RNA viruses Sendai or

Influenza virus (Luber et al., 2010).

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 DCs 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 CD1 1 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 CD1 1 b^low cDC of human thymus correlated with the mouse thymic CD11 b^0W DC with high IL-12p70 production (Vandenabeele et al., 2001 ; Hochrein et al., 2001).

It is an object of the present invention to provide the specific type of cDC, which is the major producer of ds nucleic acid-induced IFN-λ. SUMMARY OF THE INVENTION

The present invention provides the following items:

[1] A method for producing CD8+ and/or eCD8+ conventional dendritic cells (cDCs), comprising incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs.

[2] A method of ex vivo expanding CD8+ and/or eCD8+ conventional dendritic cells (cDCs), comprising incubating a population of cells comprising CD8+ and/or eCD8+ cDCs with TGF-β.

[3] The method of item [2], wherein the population of cells is generated by incubating undifferentiated hematopoietic stem cells and/or precursor cells with TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs.

[4] The method of item [2], wherein the population of cells is a population consisting of CD8+ and/or eCD8+ cDCs.

[5] The method according to any one of items [1] to [4], further comprising exposing the CD8+ and/or eCD8+ cDCs to an antigen and/or a double-stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs.

[6] CD8+ and/or eCD8+ conventional dendritic cells (cDCs) obtained according to the method of any one of items [1] to [5] for use in the prevention and/or treatment of an infectious disease or cancer, preferably a viral infection, or for use in inducing an immune response in a subject to an antigen.

[7] Autologous CD8+ and/or eCD8+ conventional dendritic cells (cDCs) for use in preventing and/or treating a subject suffering from an infectious disease or cancer, preferably a viral infection, or for use in inducing a cytotoxic NK cell-mediated immune, wherein said autologous CD8+ and/or eCD8+ cDCs are generated from autologous undifferentiated hematopoietic stem cells and/or precursor cells incubated ex vivo with TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs.

[8] The autologous CD8+ and/or eCD8+ cDCs of item [7], further comprising separating the CD8+ and/or eCD8+ cDCs from non-CD8+ and/or non-eCD8+ cDCs after the ex vivo incubation and/or exposing the CD8+ and/or eCD8+ cDCs to an antigen prior to use in preventing and/or treating a subject suffering from an infectious disease or cancer, preferably a viral infection.

[9] The CD8+ and/or eCD8+ cDCs of item [6] or the autologous CD8+ and/or eCD8+ cDCs of item [7] or [8], further comprising the use of a double-stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs.

[10] TGF-β for use in enhancing the level of CD8+ and/or eCD8+ conventional dendritic cells in a subject suffering from an infectious disease or cancer, preferably a viral infection.

[1 1] A combined preparation comprising TGF-β and/or a double-stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ conventional dendritic cells.

[12] The combined preparation of claim [1 1] for use in the prevention and/or treatment of an infectious disease or cancer, preferably a viral infection.

[13] The method according to item [1] or [3], or the autologous CD8+ and/or eCD8+ cDCs according to any one of items [7] to [9], wherein the undifferentiated hematopoietic stem cells and/or precursor cells are cells isolated from cord blood, mobilized peripheral blood or bone marrow.

[14] The CD8+ and/or eCD8+ cDCs according to item [6], the autologous CD8+ and/or eCD8+ cDCs according to any one of items [7] to [9] and [13], TGF-β according to item [10], or the combined preparation according to item [12], wherein the viral infection is a persistent viral infection, preferably a viral infection of the liver or a Herpes virus infection, more preferably a Hepatitis virus infection.

[15] An ex vivo method for identifying a compound expanding CD8+ and/or eCD8+ conventional dendritic cells (cDCs), comprising: (i) contacting ex vivo undifferentiated hematopoietic stem cells and/or precursor cells with Flt3 ligand or a M-CSF receptor- ligand for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs; (ii) contacting the population of cells of (i) with a test compound; (iii) contacting the population of cells of (ii) with a double-stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs; and (iv) measuring the level of IFN-λ produced by the population of cells of (iii), wherein an increase in the level of IFN-λ in the presence of the test compound, compared to the level of IFN-λ in the absence of the test compound, is indicative for the test compound expanding CD8+ and/or eCD8+ cDCs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that the addition of TGF-β to FL-driven DC development skews the development of eCD8+ cDCs and at the same time prevents the development pDCs. The culture system is independent of GM-CSF. Surprisingly it was TGF-β, which drastically increased the amount of eCD8+ cDCs within cultures. To find such a new feature for TGF-β was very surprising since in general TGF-β is regarded solely as an immune suppressing cytokine whereas eCD8⁺ cDC (which are increased by TGF-β) are also know for many immune inducing features such as the production of cytotoxic T lymphocytes (CTLs) via their unique ability to cross-present external antigen, the production of Th1 inducing IL-12p70. TGF-β has been previously used in combination with GM-CSF to drive monocytes or hematopoietic precursor cells into the development of Langerhans cell like DCs. Importantly this development was always based on the presence of GM-CSF. GM-CSF is known to prevent the formation of pDC even in the presence of FL.

The present invention is based on a readout (IFN-λ production in response to ds RNA) specific for the eCD8+cDCs combined with the detection of eCD8+ cDCs by phenotype which is not shared with other DC subsets such as pDC, eCD8- cDCs or GM-CSF derived cDCs. The present invention describes for the first time that TGF-β drives the development of CD8+ and eCD8+ cDCs and reduces the production of pDC in FL driven bone marrow cultures.

The methods and uses of the present invention allow for increased levels of CD8+ and eCD8+ cDCs after being applied to individuals in vivo. These cells can be used to defend against infections, preferably viral infections, and mount or direct immune responses. In addition, the invention provides for therapeutic and prophylactic treatments against proliferative disorders including cancers. The invention also encompasses TGF-β for use as a medicament. In various embodiments, the invention is directed to TGF-β for the treatment of viral infections and/or proliferative diseases such as cancer.

In particular, according to the present invention a method of GM-CSF independent generation of CD8+ and eCD8+ cDCs by .administering TGF-β to FL-cultured precursor cells is provided. According to this embodiment, growth factors other than FL may be present or administered with TGF-β to the precursor cell.

The hematopoietic precursor cells that can be induced are, but are not limited to, hematopoietic stem cells and progenitor cells as, for example, Common Lymphoid Progenitor (CLP). According to a preferred embodiment the precursor cell is a bone marrow cell.

In yet other embodiments of the invention, CD8+ and/or eCD8+ cDCs induced by TGF- β in vitro can be exposed to antigens to stimulate specific immune responses.

In further embodiments of the invention, the CD8+ and/or eCD8+ cDCs generated by TGF-β can be used to stimulate immune responses in other immune cells. Another embodiment of the invention is a method of increasing CD8+ and/or eCD8+ cDCs in an animal, comprising co-administering TGF-β with an antigen to the animal, wherein the co-administration results in an increase in the number of CD8+ and/or eCD8+ cDCs in the animal.

The tern "antigen" is meant to refer to any substance that is capable of raising an immune response. An antigen may raise, for example, a cell-mediated and/or humoral immune response in a subject organism. Alternatively, an antigen may raise a cellular immune response (e.g., immune cell maturation, production of cytokines, production of antibodies, etc.) when contacted with immune cells. In certain embodiments, the antigen can be any material capable of raising a THI immune response, which may include one or more of a T cell response, an NK T cell response, a gamma/delta T cell response, or a THI antibody response. Suitable antigens include but are not limited to peptides; polypeptides; lipids; glycolipids; polysaccharides; carbohydrates; polynucleotides; prions; live or inactivated bacteria, viruses or fungi; and bacterial, viral, fungal, protozoal, tumor-derived, or organism-derived antigens, toxins or toxoids.

The term "immune response" is meant to refer to how your body recognizes and defends itself against bacteria, viruses^', and substances that appear foreign and harmful to the body. An immune response can refer to any of innate immunity; humoral immunity; cellular immunity; immunity; inflammatory response; acquired (adaptive) immunity.

The term "immunostimulatory" or "stimulating an immune response" is meant to include stimulation of cell types that participate in immune reactions and enhancement of an immune response to a specific antigenic substance. An immune response can be a "TH 1-type" immune response or a "TH 2-type" immune response. Th1-type immune responses are normally characterized by "delayed-type hypersensitivity" reactions to an antigen and activated macrophage function and can be detected at the biochemical level by increased levels of TH 1-associated cytokines such as IFN-gamma, IL-2, IL-12, and TNF-beta. TH 2-type immune responses are generally associated with high levels of antibody production, especially IgE antibody production and enhanced eosinophils numbers and activation, as well as expression of TH 2-associated cytokines such as IL- 4, IL-5 and IL-13.

In various embodiments, the antigens are derived from tumors, viruses, bacteria, fungi, parasites, prions, plants, molluscs, arthropods, or vertebrate toxins. In various embodiments, the antigens are derived from tumors. In various embodiments, the antigens are derived from viruses. In various embodiments, the antigens are derived from bacteria or fungi. In various embodiments, the antigens are derived from parasites or prions. In various embodiments, the antigens are derived from at least one of plants, molluscs, arthropods, and vertebrate toxins.

In the present invention, the term "antigen" comprises both the singular and the pluaral form of "antigen". Likewise, in the present invention the term "antigens" comprises both the plural and the singular form of "antigen".

In various embodiments of the invention the animal is a mouse or a human. In other embodiments of the invention, TGF-β is administered in a poxvirus vector, including, but not limited to an MVA vector. In other embodiments of the invention, TGF-β is administered in another viral vector. In yet other embodiments of the invention, TGF-β is administered in a plasmid or via RNA. The invention also includes embodiments in which the TGF-β is administered to the cultured cells as a polypeptide or as a nucleic acid that is expressed in the cultured cells wherein the nucleic acid is DNA or RNA. The invention also includes methods wherein the TGF-β is administered to the cultured cells in a poxvirus vector, in particular in a vaccinia virus vector, including, but not limited to, a Modified Vaccinia virus Ankara (MVA) viral vector.

A further embodiment of the invention includes a method of inducing an immune response to one or more antigens in an. animal, comprising removing hematopoietic precursor cells from an animal, culturing the precursor cells, administering TGF-β to the cultured cells, generating CD8+ and/or eCD8+ cDCs; exposing the CD8+ and/or eCD8+ cDCs to antigens, harvesting the primed CD8+ and/or eCD8+ cDCs, and reintroducing the primed CD8+ and/or eCD8+ cDCs into the animal.

As mentioned above, in various embodiments of the present invention the antigens are derived from tumors, viruses, bacteria,^* fungi, parasites, prions, plants, molluscs, arthropods, or vertebrate toxins. In various embodiments, the antigens are derived from tumors. In various embodiments, the antigens are derived from viruses. In various embodiments, the antigens are derived from bacteria or fungi. In various embodiments, the antigens are derived from parasites or prions. In various embodiments, the antigens are derived from at least one of plants, molluscs, arthropods, and vertebrate toxins. Yet another embodiment of the invention is a method of treating a patient suffering from a proliferative disorder and/or an autoimmune disease, comprising administering TGF- β to the patient, and increasing the number of eCD8+ cDCs in the patient.

A further embodiment of the invention is a method of treating a patient suffering from acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and/or systemic lupus erythematosus (SLE), comprising administering TGF-β to said patient and increasing the number of DCs in the patient.

The invention also encompasses TGF-β and its use as a medicament. Specifically, the invention is directed to TGF-β for the treatment of proliferative diseases such as cancer or leukemia, in particular AML and/or ALL, and/or for the treatment of autoimmune diseases such as SLE.

Yet another embodiment of the invention is a method of stimulating an immune response, comprising culturing hematopoietic precursor cells, administering TGF-β to the cultured cells; generating CD8+ and/or eCD8+ cDCs, and exposing the CD8+ and/or eCD8+ cDCs to an immune cell, wherein the immune cell is stimulated to produce an immune response.

The CD8+ and/or eCD8+ cDCs are preferably generated as described herein, i.e., the CD8+ and/or eCD8+ cDCs should be generated without co-administration of a further growth factor known so far to induce DC generation, such as, for example, FL and/or M-CSF. In a further embodiment, CD8+ and/or eCD8+ cDCs are generated FL- independent, but may be generated by co-administering other growth factor, whereas CD8+ and/or eCD8+ cDCs may be generated without addition of other growth factors known to be involved in their generation such as, for example, FL and/or M-CSF. The immune cells may be T-cells (including, but not limited to regulatory T-cells, suppressor T-cells, and/or killer T-cells), T-helper cells (including, but not limited to, Th1 , Th2, and/or Th17 cells), B-cells, natural killer cells, or macrophages. Stimulation of an immune response can be achieved in vitro or in vivo. Furthermore, the immune response may be an anti-allergic immune response, an anti-septic immune response, an anti-graft immune response, an anti-tumor immune response, an anti-autoimmune response, a tolerogenic immune response, an anti-pathogen immune response, or a regulatory immune response.

A further embodiment of the invention relates to a recombinant poxvirus comprising a nucleic acid sequence coding for TGF-β. Specifically, said nucleic acid sequence is included in the viral genome of said poxvirus. Preferably, coding sequences of growth factors known to be involved in DC generation, in particular the coding sequences of FL and/or M-CSF, are absent in the recombinant poxvirus. The poxvirus includes but is not limited to Vaccinia virus, in particular Modified Vaccinia Virus Ankara (MVA). In a preferred embodiment, said MVA is characterized by having at least one of the following properties: (i) capability of reproductive replication in vitro in chicken embryo fibroblasts (CEF) but no capability of reproductive replication in the human keratinocyte cell line (HaCaT), the human embryo kidney cell line (293), the human bone osteosarcoma cell line (143B), and the human cervix adenocarcinoma cell line (HeLa), (ii) failure to replicate in a mouse model that is incapable of producing mature B and T cells and as such is severely immune compromised and highly susceptible to a replicating virus, and (iii) induction of at least the same level of specific immune response in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA- prime/vaccinia virus boost regimes. According to further embodiments of the invention, the MVA is characterized by having at least two or all three of the advantageous properties. In a particularly preferred embodiment, the MVA is an MVA Vaccinia virus as deposited at the European Collection of Cell Cultures (ECACC) Salisbury (UK) under number V00083008, and derivatives thereof. The virus as deposited is also known as MVA-BN.

The recombinant poxvirus as outlined above may also further comprise a heterologous nucleic acid sequence selected from a sequence coding for at least one antigen and/or antigenic epitope. The present invention aMso relates to pharmaceutical compositions or vaccines comprising such a recombinant poxvirus and, optionally, a pharmaceutically acceptable carrier, diluent and/or additive. In a further embodiment, the invention relates to the recombinant poxvirus comprising a nucleic acid sequence coding for TGF-β, preferably the recombinant as described above, for use as a medicament or as a vaccine. Furthermore, the invention also encompasses a recombinant poxvirus comprising a nucleic acid sequence coding for TGF-β as outlined herein for the treatment of proliferative diseases and/or autoimmune diseases. Proliferative diseases have already been specified hereinabove and include, but are not limited to cancer and leukemias. In a preferred embodiment, said type of leukemia is AML. Autoimmune diseases have also been specified in the present application and include, but are not limited to, SLE.

In another embodiment, the present invention relates to the use of the recombinant poxvirus and/or the pharmaceutical composition for the generation of CD8+ and/or eCD8+ cDCs from hematopoietic precursor cells.

As described herein, "CD8+ and/or eCD8+ cDCs" means an abbreviation for "CD8+ cDCs and/or eCD8+ cDCs". Furthermore, as described herein, "cDCs" means "conventional dendritic cells".

A further embodiment of the invention is a kit for inducing an immune response to an antigen in an animal, said kit comprising, preferably in a first vial, TGF-β, preferably in the manner as described above, i.e., without a growth factor known to induce DC generation, as FL and/or M-CSF, and/or comprising a recombinant poxvirus including a nucleic acid sequence encoding TGF-β, preferably the recombinant poxvirus as described above, and an antigen, preferably contained in a second vial. In a preferred embodiment of said kit, the recombinant poxvirus is administered to an animal for generating and/or increasing CD8+ and/or eCD8+ cDCs and said antigen is subsequently administered to said animal after CD8+ and/or eCD8+ cDCs have been generated and/or induced.

In various embodiments, TGF-β and/or antigen can be administered to cultured cells by introduction of a DNA or RNA that encodes TGF-β and directs its expression within the cultured cell. Techniques for this method of administration include, but are not limited to, techniques for transfection, lipofection, electroporation, and transduction. TGF-β can also be administered to a cell by infection with a virus that carries the genetic information to produce TGF-β. Non-limiting examples of such a virus are DISC-Herpes virus and poxviruses, including, but not limited to Modified Vaccinia virus Ankara (MVA).

The term "tumor antigen" refers to an antigen associated with certain tumor diseases. Tumor antigens are most often antigens encoded by the genome of the host that develops the tumor. Thus, in a strict sense tumor antigens are not foreign antigens. However, tumor antigens are found in significant amounts in tumors; whereas, the amount of tumor antigens in normal tissues is significantly lower, and most often no tumor antigens are found at all in normal tissue. In various embodiments, tumor antigens include gp75 antigen for melanoma papilloma virus proteins for cervical cancer, and tumor specific idiotypic proteins for B cell lymphomas.

In further embodiments of the invention, TGF^-generated CD8+ and/or eCD8+ cDCs are used to stimulate immune responses in other immune cells in vivo or in vitro. These immune cells include, but are not limited to, T-cells (including, but not limited to, regulatory or suppressor T-cells, Killer T-cells (CTLs), and T- Helper cells (including, but not limited to Th1 , Th2, and Th17), B cells, Natural Killer cells (NK cells), and macrophages. The stimulated cells can be introduced into an animal in vivo to mount an immune response. Such immune responses include, but are not limited to, antiallergic responses, anti-septic responses, anti-graft rejection responses, anti-tumor responses, anti- autoimmune disease responses, tolerogenic immune responses, anti- pathogenic immune responses, and regulatory immune responses.

In further embodiments of the invention, <TGF^-generated CD8+ and/or eCD8+ cDCs are used to stimulate immune responses in other immune cells in vivo or in vitro. These immune cells include, but are not limited to, T-cells (including, but not limited to, regulatory or suppressor T-cells, Killer T-cells (CTLs), and T- Helper cells (including, but not limited to Th1 , Th2, and Th17), B cells, Natural Killer cells (NK cells), and macrophages. The stimulated cells can be introduced into an animal in vivo to mount an immune response. Such immune responses include, but are not limited to, antiallergic responses, anti-septic responses, anti-graft rejection responses, anti-tumor responses, anti- autoimmune disease responses, tolerogenic immune responses, anti- pathogenic immune responses, and regulatory immune responses.

TGF^-generated CD8+ and/or eCD8+ cDCs can also be exposed to stimulatory agents, wherein "stimulatory agents" are proteins and other molecules that induce a specific response from CD8+ and/or eCD8+ cDCs, preferably a response resulting in the CD8+ and/or eCD8+ cDCs producing IFN-λ. Stimulatory agents of the invention include, but are not limited to, TLR-agonists, viruses, bacteria, fungi, plants, parasites or parts thereof, or cytokines including but not limited to IFN-I, IL-6, IL-10, IL-12 and TNF-a. Preferably, the stimulatory agent is a ds nucleic acid as described herein.

The term "animal" includes, but is not limited to vertebrates, most preferably mammals, including, but not limited to humans, horses, cows, pigs, sheep, goats, llamas, cats, dogs, mice, and rats.

In other embodiments of the invention, antigens can be co-administered with TGF-β. These antigens include, but are not limited to, antigens present on viruses (in non- limiting example, influenza, HIV, CMV, EBV, human papilloma virus, adenovirus, HBV, HCV and vaccinia), bacteria, fungi, parasites, prions, and tumor cells (tumor antigens), as well as toxin antigens from viruses, bacteria, fungi, parasites, mollosucs, arthropods, and vertebrates. In embodiments of the invention antigens can also include peptides from autoantibodies which can be antigens for the treatment of SLE, and peptides corresponding to the mutant forms of Flt3 or c-kit, which can be antigens for the treatment of AML.

TGF-β and/or antigen can be administered to an animal as a protein, DNA, RNA, or virus. Administration of a protein to an animal can be achieved by, but is not limited to, oral, transdermal, transmucosal administration, or by injection (parenteral). The dose administered can vary depending on which type of administration is used. Pharmaceutically acceptable formulations of TGF-β and antigen are known in the art. Carriers or excipients can be used to produce pharmaceutical compositions. Examples of carriers include, but are not limited to, calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible solvents. Examples of physiologically compatible solvents include, but are not limited to sterile solutions of water for injection (WFI), saline solution, and dextrose. TGF-β can be administered by different routes, including but not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular, oral, transmucosal, rectal, or transdermal. In vivo, TGF-β and/or antigen is administered to an animal at levels of 0.01 pg - 100 mg/day, 0.1 pg-100 mg/day, 1 pg-100 mg/day, 10 pg - 100 mg/day, 100 pg -100 mg/day, 1 mg-100 mg/day, 10 mg-100 mg/day, 50-100 mg/ day, 0.01 pg -10 mg/day, 0.1 pg-10 mg/day, 1 pg-10 mg/day, 10 pg-10 mg/day, 100 pg -10 mg/day, 1-10 mg/day, 10-50 mg/ day, 0.01 pg-1 mg/day, 0.1 pg-1 mg/day, 1 pg -1 mg/day, 10 pg -1 mg/day, 100 pg -1 mg/day, 1-10 mg/day, or 1-50 mg/ day. Levels of 1-20 pg/day are preferable and 10 pg/day most preferable for administration to rodents. Levels of 1 -50 mg/day are preferable, and 25 mg/day most preferable, for humans. TGF-β can also be administered to animals on a per weight basis, including, but not limited to, 0.5 pg-10 g/g weight/day, 1 pg-10 g/g weight/day, 10 pg-10 g/g weight/day, 100 pg-10 g/g weight/day, 1g-10 g/g weight/day, 0.5 pg-1 g/g weight/day, 1 pg-1 g/g weight/day, 10 pg- 1 g/g weight/day, or 100 pg -1 g/g weight/day, preferably 0.5 pg/g weight/day. Other dosages are contemplated by the invention, and can be determined using assays known to the skilled artisan.

Further embodiments of the invention include administration of TGF-β to precursor cells, preferably in the manner as described herein above, wherein said precursor cells have been isolated from an animal. These cells are induced by TGF-β in vitro, exposed to antigen, and returned to the animal for a therapeutic or prophylactic effect. Techniques for such "ex vivo" therapies are known to those in the art. Other techniques for ex vivo therapy are also contemplated for the invention.

To induce hematopoietic precursor cells in vitro the cells can be cultured and DCs harvested by techniques known to those of skill in the art. In this embodiment the DCs may be characterized by observing DC cell surface antigens by techniques known to those of skill in the art. These techniques include, but are not limited to, surface staining and fluorescence activated cejl sorting (FACS). Quantitation of cytokine production can also be used, including, but not limited to, IFN-I, IFN-a, IL-12 p70, IL-6, TNF-a, MCP-1 , IFN-γ and IFN-λ. This is achieved with techniques known to those of skill in the art. These techniques include, but are not limited to, ELISA. Accordingly, in the present invention the skilled person will not have any problems in carrying out incubation with TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs according to the present invention. Likewise, in the present invention the skilled person will not have any problems in carrying out incubation with a growth factor (like Flt3 ligand) and TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs according to the present invention. Based on techniques known in the art the skilled person can without any problems determine any incubation time according to the present invention that allows development of CD8+ and/or eCD8+ cDCs.

In embodiments of the invention involving ex vivo therapies, TGF-β and/or antigen can be administered to cultured cells as a protein in vitro. TGF-β protein can be produced by methods known to those of skill in the art, including, but not limited to, in vitro, prokaryotic, and eukaryotic expression systems.

In embodiments of the invention, TGF-β and/or antigen is administered in vitro to cultured cells at levels including, but not limited to, 1 -100 ng/ml, 1-75 ng/ml, 1-50 ng/ml, 1-25 ng/ml, 1-10 ng/ml, 10-100 ng/ml, 10-75 ng/ml, 25-100 ng/ml, 50-100 ng/ml, 75-100 ng/ml, 25-75 ng/ml, or 50-75, ng/ml, preferably at 10-50 ng/ml, and most preferably at 20 ng/ml.

TGF-β and/or antigen can also be administered to cultured cells by introduction of a DNA or RNA that encodes TGF-β and directs its expression within the cultured cell. Techniques for this method of administration include, but are not limited to, techniques for transfection, lipofection, electroporation, and transduction. TGF-β and/or antigen can also be administered to a cell by infection with a virus that carries the genetic information to produce TGF-β and/or the antigen. Non-limiting examples of such viruses are DISC-Herpes virus and poxviruses, including, but not limited to, Vaccinia virus, in particular Modified Vaccinia virus Ankara (MVA).

In another embodiment of the invention, a therapeutic regime for proliferative disorders is provided. Such proliferative disorders include cancer types such as leukemias. These leukemias include, but are not limited to, AML. AML and other leukemias are mediated by activation of Flt3, the receptor for FL. Thus, in this embodiment of the invention, administration of FL to a patient to induce development of DCs would aggravate the disease. In contrast, the invention provides for administration of TGF-β, along with a tumor antigen, to a patient with leukemia, including, but not limited to AML, so that DCs can be induced to provide an immune response against the tumor cells, without further stimulation of the tumor cells with FL. An inhibitor of Flt3 can also be used together with TGF-β to treat the leukemias. Inhibitors of Flt3 are known to the person skilled in the art. Embodiments of the invention are also directed to the treatment of other proliferative disorders including, but not limited to, hematopoietic neoplastic disorders involving hyperplastic/neoplastic cells of hematopoietic origin arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. These include, but are not limited to erythroblastic leukemia, acute promyeloid leukemia (APML), chronic myelogenous leukemia (CML), lymphoid malignancies, including, but not limited to, acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocyte leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to, non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

In addition, embodiments of the invention include, but are not limited to, the treatment of malignancies of epithelial or endocrine tissues, including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include, but are not limited to, those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.

Furthermore, embodiments of the invention include a dendritic cell generated from a hematopoietic precursor cell by TGF-β stimulation of said precursor cell.

The generated dendritic cell is a CD8+ and/or eCD8+ cDC. Stimulation of the precursor cell may occur in the presence of TGF-β and other growth factors excluding GM-CSF. The hematopoietic precursor cell includes, but is not limited to, bone marrow cells. Methods for generating dendritic cells by TGF-β stimulation are extensively described herein.

The invention further relates to a recombinant poxvirus comprising a nucleic acid sequence coding for TGF-β. Preferably, the coding sequence of growth factor known to induce DC generation, in particular the coding sequence of FL and/or M-CSF, is absent in the recombinant virus.

According to the present invention the poxvirus may be any poxvirus. Thus, the poxvirus may be any virus of the subfamily of Chordopoxvirinae and Entomopoxvirinae (see Fields Virology 3rd edition, Lippincott-Raven Publishers, Philadelphia, USA, Chapter: 83, ISBN 0-7817- 0253-4). Viruses from the subfamily Chordopoxvirinae are particularly preferred if the recombinant poxvirus is used to express genes in mammalian animals, including humans. Particularly preferred genera belonging to the subfamily Chordopoxvirinae are Orthopoxviruses, Parapoxviruses, Avipoxviruses, Capripoxviruses, Leporipoxviruses and Suipoxviruses. Most preferred are Orthopoxviruses and Avipoxviruses. Examples for avipoxviruses are canarypoxviruses and fowlpoxviruses. An example for an Orthopoxvirus is vaccinia virus. The vaccinia virus strain that may be used according to the present invention may be any vaccinia virus strain, such as strains Copenhagen, Wyeth, Western Reserve, Elstree, NYCBH and so on. Particularly preferred is Modified Vaccinia Ankara (MVA).

In one embodiment, the poxvirus according to the present invention comprises at least one heterologous nucleic acid sequence. The term "heterologous" is used hereinafter for any combination of nucleic acid sequences that is not normally found intimately associated with the virus in nature, such virus is also called "recombinant virus". Preferably, the heterologous nucleic acid sequence is a sequence coding for at least one antigen, antigenic epitope, and/or a therapeutic compound. The antigenic epitopes and/or the antigens can be antigenic epitopes and/or antigens of an infectious agent. The infectious agents can be viruses, fungi, pathogenic unicellular eukaryotic or prokaryotic organisms, and parasitic organisms. The viruses can be selected from the family of Influenza virus, Flavivirus, Paramyxovirus, Hepatitis virus, Human immunodeficiency virus, or from viruses causing hemorrhagic fever. The infectious agent can be bacillus anthracis.

According to still a further embodiment, but also in addition to the above-mentioned selection of antigenic epitopes, the heterologous sequences can be selected from another poxviral or a vaccinia source. Th£se viral sequences can be used to modify the host spectrum or the immunogenicity of the virus.

In a further embodiment the poxvirus according to the present invention may code for a heterologous gene/nucleic acid expressing a therapeutic compound. A "therapeutic compound" encoded by the heterologous nucleic acid in the virus can be, e.g., a therapeutic nucleic acid such as an antisense nucleic acid or a peptide or protein with desired biological activity.

According to a further preferred embodiment the expression of heterologous nucleic acid sequence is preferably, but not exclusively, under the transcriptional control of a poxvirus promoter, more preferably of a vaccinia virus promoter.

According to still a further embodiment the insertion of heterologous nucleic acid sequence is preferably into a non-essential region of the virus genome as, for example, into a host range gene and/or at a naturally occurring deletion site (disclosed in PCT/EP96/02926) of the poxvirus genome. In another preferred embodiment of the invention, the heterologous nucleic acid sequence is inserted at or into an intergenic region of the poxviral genome (disclosed in PCT/EP03/05045). Methods how to insert heterologous sequences into the poxviral genome are known to a person skilled in the art.

According to a further embodiment the invention concerns the recombinant poxvirus according to the present invention for use as vaccine or medicament. Preferably, said vaccine or medicament does not include an additional growth factor that is already known to induce DC development, as FL and/or M-CSF. The invention also relates to the recombinant poxvirus comprising a nucleic acid sequence coding for TGF-β as described herein for the treatment of- proliferative diseases and/or autoimmune diseases as specified hereinabove. Furthermore, the present invention also encompasses the use of the recombinant poxvirus comprising a nucleic acid sequence coding for TGF-β for the preparation of a pharmaceutical composition for the treatment of proliferative and/or autoimmune diseases.

In more general terms, the invention relates to a vaccine or pharmaceutical composition comprising a recombinant poxvirus according to the present invention. Methods are known to the person skilled in the art how the vaccine or pharmaceutical composition can be prepared and administered to the animal or human body. If the vector is a viral vector such as a poxvirus or vaccinia virus vector, in particular an MVA vector, it may also be administered to the animal or human body according to the knowledge of the person skilled in the art, e.g. by intravenous, intramuscular, intranasal, intradermal or subcutaneous administration.

The pharmaceutical composition or the vaccine may generally include one or more pharmaceutical acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers in addition to the promoter, expression cassette or vector according to the present invention. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like. For the preparation of pharmaceutical compositions or vaccines, the recombinant poxvirus according to the present invention, in particular a recombinant vaccinia virus such as recombinant MVA is converted into a physiologically acceptable form. For vaccinia viruses, in particular MVA this can be done based on the experience in the preparation of poxvirus vaccines used for vaccination against smallpox (as described by Stickl. H. et al. [1974] Dtsch. med. Wschr. 99,2386-2392).

For vaccination or therapy the lyophilisate or the freeze-dried product can be dissolved in 0.1 to 0.5 ml of an aqueous solution, preferably water, physiological saline or Tris buffer, and administered either systemically or locally, i. e. by parenteral, intramuscular or any other path of administration know to the skilled practitioner. The mode of administration, the dose and the number of administrations can be optimized by those skilled in the art in a known manner.

In a further embodiment, the invention relates to the use of the recombinant poxvirus or the pharmaceutical composition according to the present invention for the generation of CD8+ and eCD8+ cDCs from hematopoietic precursor cells, preferably in the manner as described herein above.

The invention also encompasses kits for inducing an immune response to an antigen in an animal. In an embodiment of the invention, the kit comprises a recombinant virus according to the invention and the antigen against which the immune response is to be induced. The virus may be a recombinant poxvirus, preferably a recombinant Vaccinia virus, in particular a recombinant MVA^' containing additional nucleotide sequences which are heterologous to the virus. In a preferred embodiment, the recombinant poxvirus is an MVA virus comprising a nucleic acid coding for TGF-β. Preferably, the viral genome does not include the sequence of further growth factors known to induce DCs, in particular not the sequence of FL and/or M-CSF. In a particularly preferred embodiment, the kit comprises a recombinant poxvirus according to the present invention comprising a gene encoding TGF-β in a first vial/container and an antigen as described hereinabove in a second vial/container. The kit also comprises instructions to administer, in a first step, the first vial comprising the recombinant poxvirus according to the present invention to an animal in order to increase and/or generate CD8+ and eCD8+ cDCs in said animal. The first vial may also be administered in vitro and/or ex vivo to hematopoietic precursor cells that have been removed from the animal. Methods to determine whether dendritic cells have been increased and/or generated after addition of TGF-β are extensively described herein. After said dendritic cells have been generated, the second vial comprising an antigen may be administered to the generated dendritic cells in vitro and/or ex vivo. Said exposed dendritic cells may then be reintroduced into the animal. Alternatively, the second vial comprising an antigen may be administered to the animal in vivo. It was also found that ds RNA induces IFN-λ production in CD8+ conventional DCs (CD8+ cDCs) and equivalents of CD8+ cDCs (eCD8+ cDCs), whereas it is known in the prior art that plasmacytoid DCs (pDCs) are responsible for IFN-λ production by a different mechanism.

It was also found that ds nucleic acids, as dsRNA or dsDNA, as well as synthetic ds nucleic acid analogs, such as poly IC, induce large amounts of IFN-λ in CD8+ conventional DCs (CD8+ cDCs) and equivalents of CD8+ cDCs (eCD8+ cDCs) but not in pDCs or in other cDC subsets. Contacting CD8+ or eCD8+ cDCs with ds nucleic acid or an analog thereof stimulates the production of IFN-λ.

Plasmacytoid DCs (pDCs) produce large amounts of IFN-λ under conditions that also induce large amounts of IFN-alpha (IFN-a). This production via pDCs is completely dependent on the presence of the Toll-like receptor (TLR) adaptor molecule MyD88. Using several knock-out mice, the present inventors were able to demonstrate that the IFN-λ production of CD8+ cDCs in response to a synthetic ds nucleic acid analog is independent of the adaptor molecule for TLRs, MyD88, and the adaptor molecule for Rig-like helicases, Cardif.

Mouse CD8+ and eCD8+ cDCs were identified as major producers of IFN-λ in response to ds nucleic acids (dsRNA or dsDNA) as well as synthetic ds nucleic acid analogs, such as poly IC, in vitro and in vivo. The nature of the stimulus and the cytokine milieu determined if CD8+ cDCs produced IFN-λ or IL-12p70. IFN-λ, but not IFN-a, 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. TLR3, but not RLHs, was shown to be involved in in vivo poly IC-induced IFN-λ production. IRF7, which is required for MyD88-dependent type I IFN production, was also shown to be involved in 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. CD8+ cDC equivalents in mouse and human have been identified as the major source of IFN-As in response to ds nucleic acids (dsRNA or dsDNA) as well as synthetic ds nucleic acid analogs, such as poly IC.

Within all species studied, dendritic cells are rare cells present in blood, skin, and all lymphoid organs. In the spleen, for example, they account for only about 1 % of total splenocytes. Yet, it is clear that these rare cells are crucial for normal immune responses. Mice depleted of DCs display defective immune responses to viral (Ciavarra et al., 2006), parasitic (Jung et al., 2002; Liu et al., 2006a), and bacterial infections (Jung et al., 2002). The most extensive studies of DC subtypes have been carried out in the mouse system. It is clear that within every mouse lymphoid organ and blood there are two distinct categories of DCs: conventional Cs (cDCs) and plasmacytoid DCs (pDCs). The same scenario exists in other mammalian species, including humans. Accordingly, the CD8+ and eCD8+ cDCs of the present invention can be further separated by phenotype, function and origin. Within the murine spleen three major cDC subsets have been defined (see Table 1 ). Based on their selective expression of the molecules CD8- alpha (CD8a) and CD4 they are named CD8+ DC (CD8^pos, CD4^neg), CD4+ DC (CD8^neg, CD4^pos) and double negative DN-DC (CD8^neg, CD4^neg).

TABLE 1. Differential expression of selected molecules on the cell surface of spleen cDC subsets.

The CD8+ and eCD8+ cDCs according to the present invention can be further characterized by the differential expression of selected molecules according to the above Table 1.

Beside the phenotypic differentiation several functional differences have been identified, e.g. the CD8+ DCs are the major cross-presenters, the major IL-12p70 producers and are able to respond to dsRNA via TLR3. In contrast they cannot respond to ssRNA due to the lack of the ssRNA receptors TLR7 and RIG-I.

Whereas pDC are known to produce IFN-λ in response to CpG-DNA or to Sendai Virus (SeV), the inventors of the present application have surprisingly found that CD8+ cDCs are the sole producers of IFN-λ in response to dsRNA.

Besides the isolation of DC subsets from the animal, DC subsets can be generated utilizing Flt3-ligand (or M-CSF receptor ligand) to drive mouse bone marrow precursors into cDC and pDC (Brasel et al., 2000; Brawand et al., 2002; Gilliet et al., 2002; Hochrein et al., 2002; Fancke et al., 2008). These systems generate high numbers of immature cDC and pDC and has been instrumental in defining the mouse pDC in particular.

Subsets of DCs in Flt3-ligand cultures: pDC and cDC subsets are defined with the help of surface markers as follows:

pDC: CD1 1 C^pos, CD1 1 b^l0W, B220^high, CD45RA^hi, CD24^|0W, Sirp-a^pos

cDC equivalents of CD8^neg DC (eCD8^neg DC): CD1 1c^pos, CD1 1 b^high, B220^neg, CD45RA^neg, CD24^low, Sirp-a^pos

cDC equivalents of CD8+ DC (eCD8+ DC): CD1 1c^pos, CD1 1 b^l0W, B220^neg, CD45RA^neg, CD24^high, Sirp-a^neg

The finding that CD8+ and eCD8+ cDCs are major producers of IFN-λ enables one to use this feature to identify CD8+ and/or eCD8+ cDCs in different mixed cell populations of different organs. In those mixed populations the IFN-λ production corresponds with the presence of CD8+ and/or eCD8+ cDCs and thus allows detecting the presence of eCD8+ cDCs via their specific cytokine they produce.

In the present invention, the IFN-λ can be IFN-A1 , IFN-A2, or IFN-A-3, which are also referred to as IL-29, IL-28A and IL-28B, respectively.

In the present invention, the term "ds" is equally used for the terms "double-strand" and "double-stranded", respectively. Likewise, the term "ss" is equally used for the terms "single-strand" and "single-stranded".

As described above, eCD8+ dendritic cells according to the present invention represent a subset of conventional DCs, and eCD8+ dendritic cells according to the present invention are thus named eCD8+ cDCs accordingly.

Poly IC is a mismatched ds RNA with one strand being a polymer of inosinic acid, the other a polymer of cytidylic acid. Poly IC is a synthetic double-strand RNA and, thus, can be considered as a synthetic analog of ds RNA. Poly IC is a common tool for scientific research on the immune system. In a preferred embodiment, the ds nucleic acid or analog thereof according to the present invention is poly IC. However, further synthetic analogs of ds nucleic acids are equally suitable according to the present invention as, for example, polyadenylic-polyuridylic acid (Poly AU), which is a synthetic ds RNA, signalling exclusively via TLR3 (Wang et al. 2002). Likewise, equally suitable is poly (ICLC), which is a poly IC complexed with carboxymethylcellulose and poly L- lysine (Longhi et al., 2009), or poly (dA:dT), which is a synthetic ds DNA of poly (dA- dT)^*poly (dA:dT) complexed with liposomes (Ishii et al., 2006). The further synthetic analogs of ds nucleic acids described in Wang et al. 2002, Longhi et al., 2009 and Ishii et al., 2006 are incorporated herein by reference as synthetic analogs of ds nucleic acids, which are equally suitable in the present invention. Also suitable are artificial ds oligonucleotides (sense and antisense), which may be provided in combination with transfecting reagents.

As used herein, the phrase "pharmaceutically acceptable diluent or carrier" is intended to include substances that can be co-administered with the active compound of the medicament and allows the active compound to perform its indicated function. Examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The uses of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use in the present invention falls within the scope of the instant invention.

The term "effective amount" in accordance with the present invention refers to the amount necessary or sufficient to realize a desired effect, in particular a medical and/or biological one.

In the present invention, the ds nucleic ^*acid or analog thereof that is stimulating or inducing the production of IFN-λ in CD8+ and/or eCD8+ cDCs is preferably ds DNA or ds RNA, including analogs thereof. Suitable dsDNA may comprise natural dsDNA such as genomic DNA which might be of prokaryotic or eukaryotic or viral origin, e.g. mitochondrial DNA, plasmid DNA, viral DNA or thymic DNA. To faciliate the uptake of the DNA, methods for enhanced uptake such as liposomes, electroporation, or nanoparticles may be employed.

In one embodiment, the ds nucleic acid or analog thereof according to the present invention is provided by a dsDNA virus, a dsRNA virus or an ssRNA virus. The dsRNA or dsDNA according to the present invention, including analogs thereof, can be provided by a dsDNA virus, a dsRNA virus, an ssDNA virus, or a positive ssRNA virus. Thus, in one embodiment, the analog of a ds nucleic acid according to the present invention is an ss nucleic acid, which is processed or can be processed to a ds nucleic acid.

In various embodiments, the virus is a positive ssRNA virus, such as a Togavirus, a Flavivirus, an Astrovirus, a Picornavirus, a Calicivirus, a Hepevirus, a Nodavirus, an Arterivirus, or a Coronavirus. In various embodiments, the virus is a dsRNA virus, such as Reovirus or a Birnavirus. In various embodiments, the virus is a retrovirus, such as an HIV-1 , HIV-2, or SIV. 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.

In various embodiments, the ds nucleic acid or analog thereof that stimulates the production of IFN-λ in CD8+ and/or eCD8+ cDCs is produced by a dsDNA virus or an ssRNA virus. In preferred embodiments, the virus is a Poxvirus, Herpesvirus, Togavirus, or a Coronavirus.

In various embodiments, the ds nucleic acid or analog thereof according to the present invention is recognized via toll-like receptor (TLR) 3 on cDCs.

In various embodiments, the method for producing CD8+ and/or eCD8+ conventional dendritic cells (cDCs) comprising incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs further comprises incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with a growth factor, preferably an inducer for DC generation.

Accordingly, in various embodiments the present invention provides a method for producing CD8+ and/or eCD8+ conventional dendritic cells (cDCs) comprising incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with a growth factor and TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs. Preferably, the growth factor is an inducer for DC generation. More preferably, the growth factor is Flt3-ligand or a M-CSF receptor ligand.

In various embodiments, the autologous CD8+ and/or eCD8+ cDCs for use in preventing and/or treating a subject suffering from an infectious disease or cancer, preferably a viral infection, or for use in inducing a cytotoxic NK cell-mediated immune, are generated from autologous undifferentiated hematopoietic stem cells and/or precursor cells incubated ex vivo with a growth factor and TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs. Preferably, the growth factor is an inducer for DC generation. More preferably, the growth factor is Flt3-ligand or a M-CSF receptor ligand.

In various embodiments, the method of ex vivo expanding CD8+ and/or eCD8+ conventional dendritic cells (cDCs) comprising incubating a population of cells comprising CD8+ and/or eCD8+ cDCs with TGF-β comprises (i) providing a population of cells comprising CD8+ and/or eCD8+ cDCs, and (ii) incubating the population of cells of (i) with TGF-β. In various embodiments, the said population of cells of (i) is generated by incubating undifferentiated hematopoietic stem cells and/or precursor cells with Flt3 ligand and/or a M-CSF receptor ligand for a time sufficient to allow development CD8+ and/or eCD8+ cDCs.

In various embodiments, in the expression "antigen and/or a ds nucleic acid or analog thereof the term "or analog thereof means "analog of the ds nucleic acid".

In various embodiments, the method forproducing CD8+ and/or eCD8+ conventional dendritic cells (cDCs), comprising incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs is a method for producing producing IFN-A-producing CD8+ and/or eCD8+ cDCs due to exposing the CD8+ and/or eCD8+ cDCs to a ds nucleic acid or analog thereof. Accordingly, the present invention provides a method for producing IFN-A-producing CD8+ and/or eCD8+ cDCs comprising (i) incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs, and (ii) exposing the so developed CD8+ and/or eCD8+ cDCs to a ds nucleic acid or analog thereof. The present invention also provides IFN-A-producing CD8+ and/or eCD8+ cDCs obtained according to the said method for producing IFN-A-producing CD8+ and/or eCD8+ cDCs comprising (i) incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs, and (ii) exposing the so developed CD8+ and/or eCD8+ cDCs to a ds nucleic acid or analog thereof for use in the prevention and/or treatment of an infectious disease or cancer, preferably a viral infection, or for use in inducing an immune response in a subject to an antigen. Here, the use may furthermore comprise the use of a double-stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs.

The present invention also provides autologous IFN-A-producing CD8+ and/or eCD8+ cDCs for use in preventing and/or treating a subject suffering from an infectious disease or cancer, preferably a viral infection, or for use in inducing a cytotoxic NK cell- mediated immune, wherein said autologous IFN-A-producing CD8+ and/or eCD8+ cDCs are generated from autologous undifferentiated hematopoietic stem cells and/or precursor cells incubated ex vivo in the presence of TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs, wherein the so developed CD8+ and/or eCD8+ cDCs are exposed to a ds nucleic acid or analog thereof. Here, the CD8+ and/or eCD8+ cDCs may be separated from non-CD8+ and/or non-eCD8+ cDCs after the ex vivo incubation prior to exposing the CD8+ and/or eCD8+ cDCs to a ds nucleic acid or analog thereof.

Still further, the present invention provides TGF-β for use in enhancing the level of IFN- A-producing CD8+ and/or eCD8+ cDCs in a subject suffering from an infectious disease or cancer, preferably a viral infection.

The present invention also provides a combined preparation comprising TGF-β and a ds nucleic acid or analog thereof targeting IFN-A-producing CD8+ and/or eCD8+ conventional dendritic cells. The present invention provides the use of said combined preparation in the prevention and/or treatment of an infectious disease or cancer, preferably a viral infection.

In the present invention, the undifferentiated hematopoietic stem cells and/or precursor cells are preferably cells isolated from cord blood, mobilized peripheral blood or bone marrow.

In the present invention, the viral infection preferably is a persistent viral infection, more preferably a viral infection of the liver or a Herpes virus infection, and still more preferably a Hepatitis virus infection.

In various embodiments, the undifferentiated hematopoietic stem cells and/or precursor cells are obtained from single individual, preferably from a single mammal, more preferably from a single human subject.

In various embodiments, the undifferentiated hematopoietic stem cells and/or precursor cells are obtained from two or more different individuals, preferably from two or more different mammals, more preferably from two or more different human subjects.

In various embodiments, the method for producing CD8+ and/or eCD8+ cDCs comprising incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo in the presence of (a) Flt3-ligand and TGF-β, or (b) a M-CSF receptor-ligand and TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs further comprises identifying the development of CD8+ and/or eCD8+ cDCs.

In various embodiments, the TGF-β used in the present invention is recombinant human TGF-β, or TGF-β produced from a recombinant viral or plasmid vector comprising a nucleic acid sequence encoding TGF-β, in particular TGF-βΙ , TGF^2 and/or TGF^3. Here, the viral vector is preferably a poxvirus vector, more preferably a Vaccinia virus vector, and even more preferably the MVA vector.

In various embodiments, Flt3-ligand and TGF-β are co-administered. In various other embodiments, M-CSF and TGF-β are co-administered.

The term "co-administration" refers to the administration of more than one substance to an animal or to cultured cells. Co-administration can occur simultaneously or in series, with one substance administered before the other. When administered in series, the second substance can be administered, but is not limited to, within at least one of: 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 8 hours, 12 hours, 24 hours, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days, and/or 10 days.

In various embodiments, the incubation with Flt3-ligand and TGF-β is done simultaneously. Preferably, simultaneous incubation means simultaneous incubation with Flt3-ligand and TGF-β at the beginning of cell culturing, i.e. at the beginning ex vivo incubating undifferentiated hematopoietic stem cells and/or precursor cells.

In various embodiments, the incubation with a M-CSF receptor ligand and TGF-β is done simultaneously. Preferably, simultaneous incubation means simultaneous incubation with a M-CSF receptor ligand and TGF-β at the beginning of cell culturing, i.e. at the beginning ex vivo incubating undifferentiated hematopoietic stem cells and/or precursor cells.

In various embodiments, the incubation with Flt3-ligand and TGF-β is done consecutively, i.e. first incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with Flt3-ligand, and then incubating the cells with TGF-β.

In various embodiments, the incubation with a M-CSF receptor ligand and TGF-β is done consecutively, i.e. first incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with a M-CSF receptor ligand, and then incubating the cells with TGF-β.

In various embodiments, the incubation with TGF-β is done during incubation with Flt3- ligand, i.e. during culturing undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with Flt3-ligand. In various embodiments, the incubation with TGF-β is done during incubation with a M- CSF receptor ligand, i.e. during culturing undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with Flt3-ligand.

In various embodiments, the FL cultures run from about 6 days to about 10 days. In various embodiments, the FL cultures run from about 7 days to about 9 days. In various embodiments, the FL cultures run about 8 days. In various embodiments, the FL cultures run from about 5 days to about 8 days. In various embodiments, the FL cultures run from about 6 days to about 8 days. In various embodiments, the FL cultures run from about 7 days to about 8 days. In various embodiments, the FL cultures run from about 4 days to about 6 days. In various embodiments, the FL cultures run from about 5 days to about 7 days.

In various embodiments, the FL concentration is between about 10 ng/ml and about 100 ng/ml. In various embodiments, the FL concentration is between about 20 and about 90 ng/ml. In various embodiments, the FL concentration is between about 30 and about 80 ng/ml. In various embodiments, the FL concentration is between about 40 and about 70 ng/ml. In various embodiments, the FL concentration is between about 50 and about 60 ng/ml.

In the present invention, there is generally no need to replenish the media or the Flt3 ligand.

In various embodiments, the M-CSF cultures run from about 6 to 8 days. In various embodiments, the M-CSF cultures run from about 7 days to about 9 days. In various embodiments, the M-CSF cultures run about 7 days. In various embodiments, the M- CSF cultures run from about 5 days to about 8 days. In various embodiments, the M- CSF cultures run from about 5 days to about 7 days. In various embodiments, the M- CSF cultures run from about 6 days to about 7 days. In various embodiments, the M- CSF cultures run from about 4 days to about 6 days. In various embodiments, the M- CSF cultures run from about 4 days to about 7 days.

In various embodiments, the concentration of M-CSF is from about 20 ng/ml to about 100 ng/ml. In various embodiments, the M-CSF concentration is between about 30 and about 90 ng/ml. In various embodiments, the M-CSF concentration is between about 40 and about 80 ng/ml. In various embodiments, the M-CSF concentration is between about 50 and about 70 ng/ml. In various embodiments, the M-CSF concentration is about 60 ng/ml.

In various embodiments, the M-CSF culture is replenished every 2^nd day half of the volume of the culture media with fresh media supplemented with M-CSF. In various embodiments, the M-CSF culture is replenished every day half of the volume of the culture media with fresh media supplemented with M-CSF. In various embodiments, the M-CSF culture is replenished every 3^rd day half of the volume of the culture media with fresh media supplemented with M-CSF. In various embodiments, the M-CSF culture is replenished every 4^th day half of the volume of the culture media with fresh media supplemented with M-CSF.

In various embodiments, TGF-β can be administered in vitro to cultured cells at levels including, but not limited to, at least one of 1 -100 ng/ml, 1-75 ng/ml, 1 -50 ng/ml, 1 -25 ng/ml, 1-10 ng/ml, 10-100 ng/ml, 10-75 ng/ml, 25-100 ng/ml, 50-100 ng/ml, 75-100 ng/ml, 25-75 ng/ml, or 50-75, ng/ml; preferably at 10-50 ng/ml; and most preferably at 20 ng/ml.

In various embodiments, the concentration of TGF-β is from about 0.1 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 0.2 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 0.3 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 0.4 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 0.5 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 0.6 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 0.7 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 0.8 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 0.9 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 1.0 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 1.1 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 1 .2 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 1 .3 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 1.4 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 1.5 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 1.6 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 1.7 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 1.8 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 1 .9 to about 2 ng/ml.

In various embodiments, the concentration of TGF-β is from about 0.1 to about 1.9 ng/ml. In various embodiments, the concentration of TGF-β is from about 0.2 to about 1.8 ng/ml. In various embodiments, the concentration of TGF-β is from about 0.3 to about 1.7 ng/ml. In various embodiments, the concentration of TGF-β is from about 0.4 to about 1 .6 ng/ml. In various embodiments, the concentration of TGF-β is from about 0.5 to about 1.5 ng/ml. In various embodiments, the concentration of TGF-β is from about 0.6 to about 1.4 ng/ml. In various embodiments, the concentration of TGF-β is from about 0.7 to about 1 .3 ng/ml. In various embodiments, the concentration of TGF-β is from about 0.8 to about 1.2 ng/ml. In various embodiments, the concentration of TGF-β is from about 0.9 to about 1.1 ng/ml. In various embodiments, the concentration of TGF-β is about 1.0 ng/ml. In various embodiments, the concentration of TGF-β is from about 1.1 to about 2 ng/ml. In various embodiments, the concentration of TGF-β is from about 1.2 to about 2 ng/ml. In various embodiments, the concentration of TGF-β is from about 1 .3 to about 2 ng/ml. In various embodiments, the concentration of TGF-β is from about 1.4 to about 2 ng/ml. In various embodiments, the concentration of TGF-β is from about 1.5 to about 2 ng/ml. In various embodiments, the concentration of TGF-β is from about 1.6 to about 2 ng/ml. In various embodiments, the concentration of TGF-β is from about 1.7 to about 2 ng/ml. In various embodiments, the concentration of TGF-β is from about 1.8 to about 2 ng/ml. In various embodiments, the concentration of TGF-β is from about 1.9 to about 2 ng/ml.

In various embodiments, TGF-β is added to the culture immediately, i.e. at the start of the culture. In various embodiments, TGF-β is added to the culture 1 day after start of the culture. In various embodiments, TGF-β is added to the culture 1.5 days after start of the culture. In various embodiments, TGF-β is added to the culture 2 days after start of the culture. In various embodiments, TGF-β is added to the culture 2.5 day after start of the culture. In various embodiments, TGF-β is added to the culture 3 days after start of the culture. In various embodiments, TGF-β is added to the culture 3.5 days after start of the culture. In various embodiments, TGF-β is added to the culture 4 days after start of the culture.

Dendritic cells (DCs) are a heterogeneous population of cells that can be divided into two major populations: (1 ) non-lymphoid tissue migratory and lymphoid tissue resident DCs and (2) plasmacytoid DCs (pDCs). The term "classic" or "conventional" DCs (cDCs) has recently been used to oppose lymphoid organ-resident DCs to pDCs. Non- lymphoid organ DCs, on the other hand are mainly called tissue DCs. While non- lymphoid tissue DCs are also different from pDCs, and primary non-lymphoid tissue DCs can be found in lymph nodes on migration but are not cDCs, the tern cDCs refers to all non-pDCs whether they are present in lymphoid or non-lymphoid tissues.

Within the context of the present invention, an eCD8+ dendritic cell is defined as a conventional, non plasmacytoid dendritic cell which does not depend on GM-CSF for its development. In one embodiment, dendritic cells according to the present invention are isolated as in Example 2. In one embodiment, dendritic cells are isolated as in Example 5.

In accordance with the present invention, precursor cells can be incubated with an agent enhancing CD8+ and/or eCD8+ cDC formation in vitro and in vivo. In a preferred embodiment, the agent enhancing CD8+ and/or eCD8+ cDC formation is a Flt3-ligand or an M-CSF receptor ligand. The addition of a Flt3-ligand can increase the numbers of CD8+ or eCD8+ cDCs 30-fold or more. The administration of a Flt3-ligand to increase CD8+ or eCD8+ cDCs can be combined with stimulation of the CD8+ or eCD8+ cDCs with a ds nucleic acid or analog thereof to increase the production of IFN-λ.

Furthermore, in accordance with the present invention, precursor cells can be incubated with a cytokine. Preferably, the cytokine is selected from the group consisting of IL-3, GM-CSF, IL-4, and IFN-y.

In one embodiment, dendritic cells according to the present invention are isolated using antibodies against CD8. In one embodiment, dendritic cells are isolated using antibodies against BDCA3. In various embodiments, dendritic cells according to the present invention are isolated using antibodies against Clec9A and/or Necl2. In various embodiments, dendritic cells are isolated using antibodies against Clec9A and/or Necl2 and/or CD205. In various embodiments,- dendritic cells are isolated using antibodies against Clec9A and/or Necl2 and/or CD205 and/or CD1 1c. In various embodiments, dendritic cells are isolated using antibodies against Clec9A and/or Necl2 and/or CD205 and/or CD11c and/or CD24. In various embodiments, dendritic cells are isolated using antibodies against Clec9A and/or Necl2 and/or CD205 and/or CD1 1c and/or CD24 and/or CD11 b. In various embodiments, dendritic cells are isolated using antibodies against Clec9A and/or Necl2 and/or CD205 and/or CD1 1 c and/or CD24 and/or CD1 1 b and/or CD172a. In various embodiments, dendritic cells are isolated using antibodies against Clec9A and/or Necl2 and/or CD205 and/or CD1 1 c and/or CD24 and/or CD1 1 b and/or CD172a and/or MHC-II. In various embodiments, dendritic cells are isolated using antibodies against Clec9A and/or Necl2 and/or CD205 and/or CD11 c and/or CD24 and/or CD1 1 b and/or CD172a and/or MHC-II and/or CD103.

Isolation of cDCs according to the present invention can be based on positive expressed surface antigens combined with negative or low expressed surface antigens. Among the highly expressed surface markers on eCD8+ cells are Clec9A, Necl2, CD8, CD103, CD24, CD205, CD36, CD97, CD162, MHC-I, MHC-II, CD11c, and BDCA3 (=CD141), whereas, negative or lower expressed surface antigens that can be used to discriminate DC subsets also from other immune cells are BDCA1 (=CD1c), BDCA2, BDCA4, CD3, CD1 1 b, CD14, CD19, CD20, CD45R, CD45RA, CD172a, PDCA1 , BST2, and F4/80 antigen.

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 characterize CD8+ cDC or their functional equivalents that may lack CD8 expression (eCD8+). Among CD11c⁺ 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 as mentioned herein above. Thus, in various embodiments, the CD8+ and eCD8+ dendritic cells according to the present invention are characterized by positive expressed surface antigens combined with negative or low expressed surface antigens as mentioned above. Furthermore, in various embodiments, the CD8+ and eCD8+ dendritic cells according to the present invention are characterized by the highly expressed surface markers as mentioned above. In a preferred embodiment, CD8+ and eCD8+ dendritic cells according to the present invention have a high expression of Clec9A. In another preferred embodiment, CD8+ and eCD8+ dendritic cells according to the present invention have a high expression of Necl2. In a still further preferred embodiment, CD8+ and eCD8+ conventional dendritic cells according to the present invention have a high expression of Clec9A and/or Necl2. In various embodiments according the present invention, the CD8+ and eCD8+ cDCs according to the present invention are human BDCA3+ dendritic cells.

In various embodiments according to the present invention, the CD8+ and/or eCD8+ cDCs have a high expression of Clec9A and Necl2. High expression of Clec9a and Necl2 can be detected as described in Hochrein et al., 2008, and Shortman et al., 2009, both of which are hereby incorporated by reference.

The present invention provides a method for the prevention and/or treatment of an infectious disease, preferably a viral infection, or cancer, comprising administering to a subject in need thereof a composition comprising a ds nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs. In other words, the present invention provides the use of a composition comprising a ds nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs in the manufacture of a medicament for the prevention and/or treatment of an infectious disease, preferably a viral infection, or cancer.

The present invention also provides a combined preparation comprising a ds nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs and an agent enhancing ds nucleic acid-based IFN-λ production. Thus, the composition as well as the combined preparation provided by the present invention are characterized in that the ds nucleic acid or analog thereof comprised by the composition or combined preparation is targeting CD8+ and/or eCD8+ cDCs. In order to target CD8+ or eCD8+ cDCs, the stimuli for IFN-λ production in those cells, i.e. ds nucleic acids or an analogs thereof, may be coupled to or integrated into carriers, together with one or more surface marker binding molecules for CD8+ and eCD8+ cDCs. Surface marker binding molecules for CD8+ and eCD8+ cDCs may be antibodies to, e.g., CD1 d, CD8a, CD1 1 c, CD24, CD36, CD40, CD49f, CD103, CD135, CD141 , CD162, CD205, CD207, Necl2, Clec9a, XCR1 , TLR10, TLR1 1 , TLR12, and/or TLR13. Thus, in a preferred embodiment, the composition as well as the combined preparation provided by the present invention may comprise a ds nucleic acid or an analog thereof coupled to or integrated into carriers together with one or more of such surface marker binding molecules for CD8+ and eCD8+ cDCs.

Other possibilities include natural or artificial ligands for the surface markers expressed by CD8+ cDCs or eCD8+ cDCs, e.g., glycolipids (for CD1 d), MHC-I (for CD8), fibronectin (for CD1 1 c), laminin (for CD49f), CD62P (for CD24), oxidized low-density lipoproteins (for CD36), CD40-ligand (for CD40), E-cadherin (for CD103), Flt3-ligand (for CD135), thrombin (for CD141 ), P-Selectin (for CD162), mannose, N-acetyl glucosamine or fucose containing molecules (for DEC207), Class-l-restricted T cell- associated molecule (CRTAM) (for Necl2), dead cells (for Clec9a), XCR1 -ligand (for XCR1), TLR10-ligand (for TLR10), toxoplasma antigen or profilin (for TLR1 1), TLR12- ligand (for TLR12), and/or TLR13 ligand (for TLR13). Thus, in a further preferred embodiment, the composition as well as the combined preparation provided by the present invention may comprise a ds nucleic acid or an analog thereof coupled to or integrated into carriers together with one or more of such natural or artificial ligands for the surface markers expressed by CD8+ cDCs or eCD8+ cDCs.

The CD8+ cDC selective binding molecules mentioned above may be directly or indirectly connected to the stimuli (ds nucleic acids or analogs thereof), e.g. by covalent linkage, adaptor molecule binding complexes (e.g., biotin-avidin complexes) binding to micropheres, nanoparticles, virus like particles, and/or liposomes.

Ds nucleic acids may also be applied in conjunction with dead cells, which are selectively recognized by CD8+ and eCD8+ cDCs via Clec9a and up to now unknown uptake receptors. Dead and dying cells after viral infection in vitro would be another targeted application of ds nucleic acids, which are generated by the cells before death, in conjunction with a selective CD8+ and eCD8+ cDC stimulation. Thus, viral infection of cells in vitro provides dead or dying cells loaded with ds nucleic acid provided by the infecting virus. Such dead and/or dying cells are selectively captured by CD8+ and/or eCD8+ cDCs and elicit IFN-λ production in said CD8+ and/or eCD8+ cDCs by stimulation with the ds nucleic acid provided by the infecting virus. The cells to be used for viral infection in vitro may be any cell as long as such cells are not immunogenic to the subject, to which the dead and/or dying cells loaded with ds nucleic acid of a virus are administered.

In a preferred embodiment, the combined preparation according to the present invention may comprise a ds nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs and an agent enhancing ds nucleic acid-based IFN-λ production, wherein said enhancing agent is a Flt3-ligand, a M-CSF receptor ligand, a TLR2 ligand, a TLR4 ligand, a TLR9 ligand, a TLR10 ligand, a TLR1 1 ligand, a CD40 ligand, IL-3, GM-CSF, IL-4, or IFN-γ. Since the inventors of the present application found that CD8+ and eCD8+ cDCs produce enhanced amount of IFN-λ by way of combination of ds nucleic acids and other stimuli, wherein the latter themselves do not induce IFN-λ production (e.g. certain TLR ligands (see Fig. 2A) or CD40 ligands), the ds nucleic acid may be applied together with an enhancing stimulus to increase the IFN-λ production. Thus, the linkage of, for example, a CD40 ligand and ds nucleic acid achieves both, targeting to CD8+ cDCs and eCD8+ cDCs, respectively, and enhanced production of CD8+ and/or eCD8+ cDCs-derived IFN-λ. Accordingly, in a preferred embodiment the above described method for the prevention and/or treatment of an infectious disease or cancer comprising administering to a subject in need thereof a composition comprising a ds nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs further comprises the administration of an agent enhancing ds nucleic acid-based IFN-λ production. More preferably, said enhancing agent is a Flt3-ligand, a M-CSF receptor ligand, a TLR2 ligand, a TLR4 ligand, a TLR9 ligand, a TLR10 ligand, a TLR1 1 ligand, a CD40 ligand, IL-3, GM-CSF, IL-4, or IFN-γ.

The present invention also provides a method for increasing the level of CD8+ and/or eCD8+ cDCs in a subject suffering from an infectious disease or cancer comprising administering to a subject in need thereof a Flt3-ligand or a M-CSF receptor ligand. In other words, the present invention provides a Flt3-ligand or a M-CSF receptor ligand for use in increasing the level of CD8+ and/or eCD8+ cDCs in a subject suffering from an infectious disease or cancer. The Flt3-ligand or M-CSF receptor-ligand is to be administered to the subject at a dosage sufficient to increase the level of CD8+ and/or eCD8+ cDCs in said subject. In a preferred embodiment, the M-CSF receptor ligand is M-CSF or IL-34. In various embodiments of the method for increasing the level of CD8+ and/or eCD8+ cDCs in a subject suffering from an infectious disease or cancer, a ds nucleic acid or analog thereof can be administered to the subject in addition to a Flt3-ligand or a M-CSF receptor ligand. Said additional administration of a ds nucleic acid or analog thereof stimulates the production of IFN-λ in the subject suffering from an infectious disease or cancer.

The present invention furthermore provides a method for inducing the production of IFN-λ in a population of cDCs comprising contacting ex vivo cDCs with a ds nucleic acid or analog thereof. In particular, for inducing said production of IFN-λ ex vivo, cDCs are obtained from a subject prior to contacting said cDCs with a ds nucleic acid or analog thereof. In the method for inducing the production of IFN-λ in a population of cDCs according to the present invention, the subject from whom the cDCs are obtained is preferably a subject in need of a treatment with cDCs induced to produce large amounts of IFN-A. Thus, the subject may be a subject in need of a prevention and/or treatment of an infectious disease, preferably a viral infection, or cancer. More preferably, the cDCs may preferably be obtained from a subject suffering from a persistent viral infection, more preferably a viral infection of the liver or a Herpes virus infection, still more preferably a Hepatitis virus infection. Following incubation ex vivo with a ds nucleic acid or analog thereof, the cDCs are harvested and resuspended in appropriate media for therapy, i.e. for being reintroduced into the subject from whom they were derived. Thus, in the method for inducing the production of IFN-A in a population of cDCs according to the present invention the cDCs are preferably autologous cDCs. The re-introduction to the subject in need thereof may be carried out by a number of commonly known approaches, like for example intravenous injection. Furthermore, the population of cDCs induced for production of IFN-A may be reintroduced in a variety of pharmaceutical formulations. Thus, the present invention provides a population of IFN-A producing cDCs obtainable by a method for inducing the production of IFN-A in a population of cDCs according to the present invention as well as a pharmaceutical composition comprising said population of IFN-A producing cDCs. As mentioned, a population of cDCs induced to produce IFN-A by contacting ex vivo cDCs with ds nucleic or an analog thereof may be administered to a subject in need thereof. Accordingly, the present invention provides a method for inducing a reaction against an infectious disease or cancer in vivo comprising contacting ex vivo cDCs with a ds nucleic acid or analog thereof and introducing them into a subject suffering from an infectious disease or cancer. In other words, the present invention provides a method for the prevention and/or treatment of a subject suffering from an infectious disease or cancer comprising administering to said subject IFN-λ producing cDCs generated by a an ex vivo method for inducing the production of IFN-λ in a population of cDCs, said method comprising contacting ex vivo cDCs with a ds nucleic acid or analog thereof. In one embodiment, the present invention provides a method for the prevention and/or treatment of an infectious disease or cancer comprising: (a) providing a subject suffering from an infectious disease or cancer; (b) obtaining cDCs from said subject; (c) contacting said cDCs ex vivo with a ds nucleic acid or analog thereof to generate a population of cDCs producing IFN-λ; and (d) re-introducing said population of IFN-λ producing cDCs into said subject so as to induce an in vivo therapeutic reaction against the infectious disease or cancer. Preferably, the population of cDCs is washed prior to re-introducing into the subject. In another preferred embodiment, the population of IFN-λ producing cDCs is resuspended in media suitable for administration to the subject in need thereof. The populations of IFN-λ producing cDCs may be re- introduced to the subject by a number of well-known approaches like, for example, intravenous injection.

In all embodiments according to the present invention, which concern and/or include contacting ex vivo cDCs with a ds nucleic acid or analog thereof for inducing the production of IFN-λ in a population of cDCs, preferably Flt3-ligand- and/or M-CSF receptor ligand-pretreated cDCs are contacted ex vivo with a ds nucleic acid or analog thereof. This means that a Flt3-ligand and/or a M-CSF receptor ligand is administered to a subject prior to obtaining the cDCs from said subject for inducing the production of IFN-λ by contacting ex vivo the obtained cDCs with a ds nucleic acid or analog thereof. This pretreatment with a Flt3-ligand and/or a M-CSF receptor ligand provides for increasing the formation/level of cCDs in said subject prior to obtaining such pretreated cDCs from said subject for contacting ex vivo said pretreated cDCs with a ds nucleic acid or analog thereof.

In the context of obtaining cDCs from a subject for contacting ex vivo cDCs with a ds nucleic acid or analog thereof for inducing the production of IFN-λ in a population of cDCs, methods for obtaining/isolating cDCs from a subject are well-known to the person skilled in the art. In the present invention, the terms "obtaining cDCs from a subject" and "isolating cDCs from a subject" have the same meaning.

In the various embodiments according to the present invention, which concern/include contacting ex vivo cDCs with a ds nucleic acid or analog thereof for inducing the production of IFN-λ in a population of cDCs, cDCs obtained/isolated from a subject can be further incubated with a TLR2-, TLR4-, TLR9-, TLR10-, TLR1 1 - or CD40-ligand. This incubation increases the expression of IFN-λ. In various embodiments, the ligand is Pam3Cys, LPS, CpG-ODN, profilin or a CD40-ligand. In various embodiments, the cDCs obtained/isolated from a subject can be further incubated with a cytokine, wherein the cytokine preferably is IL-3, GM-CSF, IL-4, or IFN-gamma (IFN-y).

In all therapeutic applications according to the present invention, the infectious disease is preferably a viral infection. More preferably, in all therapeutic applications according to the present invention the viral infection is a persistent viral infection. Still more preferably, the persistent viral infection is a viral infection of the liver or a Herpes virus infection. In a specifically preferred embodiment, said viral infection of the liver is a Hepatitis virus infection. Accordingly, in the methods for the prevention and/or treatment of an infectious disease or cancer as well as in the methods for increasing the level of CD8+ and/or eCD8+ cDCs in a subject suffering from an infectious disease or cancer, preferably the viral infection is a persistent viral infection, more preferably a viral infection of the liver or a Herpes virus infection, and still more preferably a Hepatitis virus infection. In the present invention, a Hepatitis virus infection includes a Hepatitis A virus infection, a Hepatitis B virus infection, a Hepatitis C virus infection, a Hepatitis D virus infection and a Hepatitis E virus infection, wherein the Hepatitis virus infection preferably is a Hepatitis C virus infection. In another preferred embodiment, in the present invention the persistent viral infection is a retroviral infection.

The subject according to the present invention includes animals and human. In accordance with the present invention, a "subject" shall mean a human or vertebrate animal including a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat, and mouse. In the various embodiments according to the present invention, the subject is preferably human and the eCD8+ cDCs are human BDCA3+ cDCs.

In various preferred embodiments of the present invention, the subject suffering from cancer is a subject suffering from a tumor disease. Preferably, the tumor disease is a carcinoma, i.e. a cancer or tumor of the epithelial cells or epithelial tissue in a subject. Preferably the carcinoma is a squamous, cell carcinoma or an adenocarcinoma. More preferably, the carcinoma is squamous cell lung cancer.

In the methods and therapeutic applications described above, a ds nucleic acid can be used alone or in combination with one or more other anti-cancer or anti-tumor therapeutic uses and methods, wherein such therapeutic uses and methods are preferably selected from anti-tumor chemotherapy and immunotherapy. Thus, a ds nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs according to the present invention, i.e. which is capable of stimulating or inducing IFN-λ production in CD8+ or eCD8+ cDCs, can be administered prior to, along with or after administration of a chemotherapy or immunotherapy to increase the responsiveness of the malignant cells to subsequent chemotherapy or immunotherapy.

Also provided by the present invention is a method for the production of IFN-λ in a subject comprising administering to said subject a ds nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs.

The present invention also provides a combined preparation comprising a ds nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs and an agent enhancing ds nucleic acid-based IFN-λ production. In a preferred embodiment, the agent enhancing ds nucleic acid-based IFN-λ production is a Flt3-ligand, a M-CSF receptor ligand, a TLR2 ligand, a TLR4 ligand, a TLR9 ligand, a TLR10 ligand, a TLR1 1 ligand, IL-3, GM- CSF, IL-4, or IFN-y.

In a preferred embodiment, the ds nucleic acid or analog thereof used for therapeutic applications is dsDNA or dsRNA. More preferably, the ds nucleic acid or analog thereof according to the present invention is provided by a dsDNA virus, a dsRNA virus, an ssRNA virus, or a positive ssRNA virus. Thus, in one embodiment, the analog of a ds nucleic acid is an ss nucleic acid, which is processed or can be processed to a ds nucleic acid.

The present invention provides a method for producing IFN-λ and/or generating or obtaining a population of IFN-λ producing CD8+ or eCD8+ cDCs, comprising the steps of: (a) providing a population of cells copriprising CD8+ and/or eCD8+ cDCs; and (b) contacting the cDCs with a ds nucleic acid or analog thereof. Contacting the cDCs with the ds nucleic acid or analog thereof stimulates the production of IFN-λ. In various preferred embodiments, said population of cells is incubated with an enhancer of IFN-λ production. More preferably, said enhancer is a TLR-ligand or a TNF-family member. Still more preferably, the TLR-ligand is a TLR2-, TLR4-, TLR9-, TLR10- or TLR1 1- ligand and the TNF-family member is ^"a CD40 ligand or a cytokine. Even more preferably, the cytokine is IFN-γ. The combination of a ds nucleic acid or analog thereof, for example poly IC, and an immunostimulatory CpG DNA, for example CpG- 1668, synergistically induces even larger amounts of IFN-λ by CD8+ cDCs.

In various embodiments of the above described methods for producing IFN-λ and/or generating or obtaining a population of IFN-λ producing CD8+ or eCD8+ cDCs, the population of cells is further incubated with a cytokine. Preferably, the cytokine is selected from the group consisting of IL-3, GM-CSF, IL-4, and IFN-γ. In various preferred embodiments, the above described methods further comprise a step of identifying and/or detecting IFN-λ produced by the ds nucleic acid-stimulated cDCs. In various preferred embodiments, the above described methods still further comprise a step of isolating and/or separating IFN-λ produced by the ds nucleic acid- stimulated cDCs. In other preferred embodiments, the above described methods further comprise a step of identifying and/or isolating and/or separating IFN-λ producing CD8+ and/or eCD8+ cDCs.

The IFN-λ produced by the CD8+ and/or eCD8+ cDCs can be detected and quantitated by techniques well-known in the art, such as those in the examples. The IFN-λ produced by the cDCs in accordance with the present invention can also be collected, isolated, and purified by conventional biochemical techniques.

In a preferred embodiment of the above described methods for producing IFN-λ and/or generating or obtaining a population of IFN-λ producing CD8+ or eCD8+ cDCs, the population of cells comprises more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% 98% or 99% CD8+ and/or eCD8+ cDCs. In various preferred embodiments, the cDCs are preferably human BDCA3+ cDCs. In one embodiment, the population of cells comprising CD8+ and/or eCD8+ cDCs comprises more than 50% eCD8+ cDCs. In another preferred embodiment, the population of cells comprising CD8+ and/or eCD8+ cDCs comprises more than 75% eCD8+ cDCs. In a further preferred embodiment, the population of cells comprising CD8+ and/or eCD8+ cDCs comprises more than 85% eCD8+ cDCs.

In one embodiment, the population of cells comprising CD8+ and/or eCD8+ cDCs comprises more than 50% human BDCA3+ cDCs. In another preferred embodiment, the population of cells comprising CD8+ and/or eCD8+ cDCs comprises more than 75% human BDCA3+ cDCs. In a further preferred embodiment, the population of cells comprising CD8+ and/or eCD8+ cDCs comprises more than 85% human BDCA3+ cDCs.

The present invention provides a population of IFN-λ producing CD8+ and/or eCD8+ cDCs or a cell line of an IFN-λ producing CD8+ and/or eCD8+ cDC, obtainable by the above described methods for generating or obtaining a population of IFN-λ producing CD8+ or eCD8+ cDCs. Furthermore, the present invention provides a pharmaceutical composition comprising a population of IFN-λ producing CD8+ and/or eCD8+ cDCs obtainable by the above described methods for generating or obtaining a population of IFN-λ producing CD8+ or eCD8+ cDCs. In various preferred embodiments, said a pharmaceutical composition optionally further comprises a pharmaceutically acceptable carrier or diluent.

IFN-λ production in response to a ds nucleic acid or an analog thereof, for example poly IC, can be used to detect, diagnose or screen for the presence of eCD8+ cDCs even in complex mixtures of different cells and even if the amount of eCD8+ cDCs is very low (see Fig. 3A). IFN-λ can be used as a marker for finding the CD8+ and/or eCD8+ subsets of cells, which thus can be targeted in certain situations, for example when it is desirable to increase the amount of CD8+ and/or eCD8+ cDCs. The present invention encompasses methods for detecting or screening for the presence of CD8+ and/or eCD8+ cDCs. In particular, the present invention provides an in vitro method for detecting or screening for CD8+ and/or eCD8+ cDCs, comprising the steps of: (a) providing a population of cells; (b) contacting the cells with a ds nucleic acid or analog thereof capable of stimulating or inducing the production of IFN-λ in CD8+ and/or eCD8+ cDCs; (c) detecting the production of IFN-λ; and (d) correlating the production of IFN-λ with the presence of CD8+ and/or eCD8+ cDCs. In various preferred embodiments, said method is a method for detecting or screening for the presence of CD8+ and/or eCD8+ cDCs in a biopsy, preferably a biopsy of an organ or blood. Thus, 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 a ds nucleic acid or an analog thereof. Since the production of IFN-λ is quite constant after induction, one can quantitate the amount of the specific CD8+ and/or eCD8+ cDCs in, for example, the body of a subject or cell culture. Thus, one can detect/diagnose and determine conditions where the amount of CD8+ and/or eCD8+ cDCs is increased or decreased.

In various embodiments, the method for detecting or screening for CD8+ and/or eCD8+ cDCs further comprises a step of separating and/or isolating IFN-λ producing CD8+ and/or eCD8+ cDCs. The methods may further comprise measuring the IFN-λ production from said separated and/or isolated IFN-λ producing cDCs.

The IFN-λ produced by the CD8+ and/or eCD8+ cDCs can be detected and quantitated by techniques well-known in the art, such as those in the examples. The IFN-λ produced by the dendritic cells in accordance with the present invention can also be collected, isolated, and purified by conventional biochemical techniques.

The compositions of the invention are used in the preparation of medicaments, for treating the conditions described herein. These compositions of the invention are administered as pharmaceutically acceptable compositions. The pharmaceutical compositions as described herein can be administered in combination with other pharmaceutical and/or immunostimulatory agents, as described herein, and can be combined with a physiologically acceptable carrier. The compositions may be administered by any suitable means, including, but not limited to, intravenously, parenterally or locally. The compositions can be administered in a single dose by bolus injection or continuous infusion or in several doses over selected time intervals in order to titrate the dose. In some embodiments, the pharmaceutical compositions are administered in conjunction with a composition comprising an antigen. The composition can also comprise and an adjuvant or other immunostimulatory agent. For example, the pharmaceutical compositions are administered with an emulsion of antigen and an adjuvant.

Further aspects of the invention are:

[1] A composition comprising a double-stranded (ds) nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs for use in the prevention and/or treatment of an infectious disease or cancer, preferably a viral infection.

[2] A combined preparation comprising a ds nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs and an agent enhancing ds nucleic acid-based IFN-λ production.

[3] The combined preparation according to item [2], wherein the agent enhancing ds nucleic acid-based IFN-λ production is a TLR-ligand, wherein the TLR-Ligand is preferably a TLR2 ligand, a TLR4 ligand, a TLR9 ligand, a TLR10 ligand or a TLR11 ligand; or a TNF-family member, wherein the TNF-family member preferably is a CD40- ligand or a cytokine, wherein the cytokine preferably is a Flt3-ligand, a M-CSF receptor ligand, IL-3, GM-CSF, IL-4, or IFN-γ.

[4] A Flt3-ligand or an M-CSF receptor ligand for use in increasing the level of CD8+ and/or eCD8+ cDCs in a subject suffering from an infectious disease or cancer, preferably a viral infection.

[5] The composition according to item [1], or the Flt3-ligand or M-CSF receptor ligand according to item [4], wherein the viral infection is a persistent viral infection, preferably a viral infection of the liver or a Herpes virus infection, more preferably a Hepatitis virus infection.

[6] An in vitro method for producing IFN-λ and/or generating or obtaining a population of IFN-λ producing CD8+ or eCD8+ cDCs, comprising the steps of: (a) providing a population of cells comprising CD8+ and/or eCD8+ cDCs; and (b) contacting the cDCs with a ds nucleic acid or analog thereof. [7] The method according to item [6], wherein the population of cells is incubated with an enhancer of IFN-λ production.

[8] The method according to item [7], wherein the enhancer is a TLR-ligand, wherein the TLR-ligand is preferably a TLR2 ligand, a TLR4 ligand, a TLR9 ligand, a TLR10 ligand or a TLR1 1 ligand; or a TNF-family member, wherein the TNF-family member preferably is a CD40-ligand or a cytokine, wherein the cytokine preferably is IL-3, GM- CSF, IL-4, or IFN-y.

[9] A pharmaceutical composition comprising a population of IFN-λ producing CD8+ and/or eCD8+ cDCs obtainable by any method described herein and, optionally, a pharmaceutically acceptable carrier or diluent.

[10] An in vitro method for detecting or screening for CD8+ and/or eCD8+ cDCs, comprising the steps of: (a) providing a population of cells; (b) contacting the cells with a ds nucleic acid or analog thereof capable of stimulating or inducing the production of IFN-λ in CD8+ and/or eCD8+ cDCs; (c) detecting the production of IFN-λ; and (d) correlating the production of IFN-λ with the presence of CD8+ and/or eCD8+ cDCs.

[1 1] The method according to item [10] for screening or detecting the presence of CD8+ and/or eCD8+ cDCs in a biopsy, preferably a biopsy of an organ or blood.

[12] A method for inducing the production of IFN-λ in a population of cDCs comprising contacting ex vivo a cDC with a ds nucleic acid or analog thereof.

[13] The method according to item [12], wherein Flt3-ligand- and/or M-CSF receptor ligand-pretreated cDCs are contacted ex vivo with said ds nucleic acid.

As described herein, terms like "embodiments", "various embodiments", "other embodiments" and "further embodiments" mean "embodiments of the present invention", "various embodiments of the present invention", "other embodiments of the present invention" and "further embodiments of the present invention".

BRIEF DESCRIPTION OF THE DRAWINGS

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.

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-γ + GM-CSF with the stimuli as indicated. Representative results of at least 2 independent experiments are shown. Data represent mean +/- SD of duplicate samples.

Figures 3A and B depict that FL is involved in the production of IFN-λ in vivo. (A) Isolated total non parenchymal liver cells 2.5 x 10⁶/ml were stimulated in the presence of IL-3+ IL-4 + IFN-γ + 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 100pg 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.

Figure 4 depicts that TLR3, IFN-AR and IFR7, but not MyD88 or Cardif, are involved in IFN-λ production to poly IC in vivo. Mice with the indicated genotype were injected i.v. with 100pg 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.

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) l OOpg/ml poly IC + 10pg/ml Pam3Cys + lOpg/ml LPS or with (donor 2 and 3) l OOpg/ml poly IC for 18-24 h. Supernatants were analyzed for IFN-A1 and IFN-A2. The experiments are shown for the individual donors and data represent mean +/- SD of duplicate samples.

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.

Figure 7 depicts splenic CD8+ cDCs 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-A.^" Data represent mean +/- SD of duplicate samples. 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.

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.

Figures 10A-D depict that TLR3 and IFN-AR, but not MyD88 or Cardif, are involved in IFN-λ production to poly IC by FLDC-derived eCD8+ cDCs. 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+ DCs stimulated with poly IC in the presence of IL-4 and IFN-γ. (B) WT and TLR3-KO eCD8+ DCs stimulated with poly IC in the presence of IL-3 + IL-4 + IFN-γ + 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.

Figures 11 A and B depict the production of IFN-λ 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 100pg 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.

Figure 12 depicts that the addition of TGF-β increases the percentage of eCD8 cDCs but decreases the percentage of pDCs in FL-DC cultures. Total bone marrow cells (1.5 x 10⁶ cells/ml) were plated with Flt3-L (FL) (50 ng/ml) with or without TGF-β (1 ng/ml). Cells were analysed after 8 days of cultures for the expression of CD1 1c, CD45R, CD172a, CD24, CD1 1 b and CD24. Dendritic cells as detected by the expression of CD11c were gated and separated into pDCs (CD1 1c^pos, CD45R^pos, CD1 1 b^l0W, CD172a^pos) or eCD8 cDCs (CD1 1 c^pos, CD45R^neg, CD11 b^l0W, CD172a^ne9, CD24^high) and percentage of eCD8 (Fig. 2A) and pDC (Fig. 2B) compared to the total amount of DCs of the cultures with or without TGF-D were determined. Circles represent individual parallel experiments and the columns represent the mean thereof.

Figure 13 depicts that the addition of TGF-β increases the expression of CD103 on eCD8 cDCs in FL-DC cultures. Total bone marrow cells (1 .5 x 10⁶ cells/ml) were plated with Flt3-L (FL) (50 ng/ml) with or without TGF-β (1 ng/ml). Cells were analysed after 8 days of cultures for the expression eCD8 cDCs (CD1 1 c^pos, CD45R^ne9, CD11 b^low, CD172a^neg, CD24^high) and in addition analysed for the expression of CD103. The percentage of eCD8 cDCs highly expressing CD103 in addition to the eCD8 cDC markers from cultures with or without TGF-β is shown. Circles represent individual parallel experiments and the columns represent the mean thereof.

Figure 14 depicts that the addition of TGF-D during FL-DC generation increases the eCD8 cDC dependent IFN-D production but decreases the pDC dependent IFN-a production. FL-DC were generated in the presence of TGF-β (FL+ TGF-β) or absence (FL only). Cells were counted and 1 x 10⁶/ml total FL-DC were stimulated with either poly IC (a stimulus which selectively induces eCD8 dependent IFN-D production) or Sendai Virus (at a concentration which selectively induces IFN-D production via pDC). Supernatants were analysed for IFN-λ or IFN-a by specific ELISA.

Figure 15 depicts that the addition of TGF-β in FL-DC cultures decreases the generation of pDC but increases the generation eCD8 cDCs and the eCD8 cDC dependent IFN-λ production during the whole kinetic of the DC development. FL-DCs were generated in the presence of TGF-β (FL+ TGF-β) or absence (FL only). FL-DC cultures were analysed on the culture days 3 till day 8 for cells expressing pDC markers (CD11 c^pos, CD45R^pos) (Figure 15A) or eCD8 markers (CD11 c^pos, CD45R^neg, CD24^hi9h, CD103^high) (Figure 15B). Additionally cultures were stimulated with poly IC on day 3 till day 6, supernatants were taken after 24 hrs and analysed by ELISA for IFN-λ.

Figure 16 depicts that the addition of TGF-β up to 4 days after beginning of the FL-DC cultures decreases the generation of pDC but increases the generation eCD8 cDCs. FL-DCs were generated and TGF-β (FL+ TGF-β) was added on day 0, day 1 , day 2, day 3 or day 4 and the corresponding FL-DC cultures were analysed on day 8 for cells expressing pDC markers (CD1 1 c^pos, CD45R^pos) (Figure 16A) or eCD8 markers (CD1 1 c^pos, CD45R^neg, CD24^high, CD103^high) (Figure 16B).

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references cited throughout this application are hereby expressly incorporated by reference. EXAMPLES

1. Mice

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.

2. Cells and Flow Cytometric Sorting

DC subsets were isolated from pooled mouse spleens as described (Vremec et al., 2007). Briefly, spleens were chopped, digested with collagenase (Worth ington 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-CD 9, ID3; anti-erythrocytes, TER1 19 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 CD1 1 c, CD45RA, CD4, CD8a and CD172a and pDCs were purified based on CD1 1 c, CD45RA, and CD172a (all BD Biosciences) expression. Cell sorting was performed on a FACS Aria instrument (BD Biosciences).

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, CD11 b, CD24, and CD172a or CD103 (all BD Biosciences).

3. In vivo challenge with poly IC

Mice were injected i.v. into the lateral tail vein with 100 pg 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. 4. In vitro stimulation and cytokine detection

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-γ (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-1d) 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.

5. Isolation and stimulation of human DC

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-γ (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-A 1 and IFN-A2 by ELISA according to manufacturer's recommendations (Tebu-bio).

6. CD8+ cDCs are the major producers of IFN-A in response to poly IC

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).

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-λ 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-λ to poly IC (Fig. 9 A). Thus, CD8+ cDCs and their in vitro equivalents are the major producers of IFN-λ in response to poly IC stimulation.

7. IFN-λ and IL-12p70 production by CD8+ cDCs depends on the type of stimulus and the cytokine conditions

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 2A). Combinations of poly IC together with Pam3Cys, LPS, CpG-ODN or profilin, ligands for TLR2, TLR4, TLR9, TLR 10 or TLR1 1 , respectively, synergistically increased IFN-λ production (Fig 2A). 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 2A). 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. 2A) (Napolitani et al., 2005).

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. 2B). Combining IL-12p70 and IFN-λ enhancing cytokines (IL-3 + GM-CSF + IL-4 + IFN-y) 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. 2C). 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).

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.

8. FL is involved in IFN-λ production to poly IC in vivo

FL is a growth factor involved in 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. 3A). 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. 3A). 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. 9A) (Yarovinsky et al., 2005). The concomitant abrogation of IFN-λ 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.

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. 3B). Application of recombinant FL into FL-KO mice not only restored, but even increased, their IFN-λ producing capacity above WT level (Fig. 1 1 A). 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. 1 1 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.

9. TLR3, IFN-AR and IRF7 are involved in IFN-λ production to poly IC in vivo

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 al., 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 involvement of TLR3 in vivo supports that the CD8+ and eCD8+ cDCs are the source of IFN-λ because this subset is particularly known for its high expression of TLR3 and to recognize poly IC in a 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 involvement of TLR3 or Cardif seems to be mutually exclusive. A similar involvement of 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 involvement of FL, strongly suggest that the IFN-λ production to poly IC in vivo largely depends on DCs of the CD8+ and eCD8+ subsets. 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. 10D).

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).

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

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.

11. eCD8+ cDCs are major producers of IFN-A in response to DNA viruses

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 HSV-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-λ was able to protect against mucosal infection with HSV and TLR dependent protection was largely IFN-λ dependent (Ank et al., 2008).

The family of poxviridae, also named poxviruses, represent 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 parapoxviruses 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).

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.

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.

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 (M-CSF, IL-34). The viruses themselves can be recognized by the enhanced numbers of eCD8+ cDCs which can induce antiviral IFN-A, thus restricting 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. 12. eCD8+ cDCs are major producers of IFN-λ in response to RNA viruses

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.

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. 7A). 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.

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).

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 Herpes viruses.

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.

13. The addition of TGF-β to FL-driven DC devlopment skews the development of eCD8+ cDCs and at the same time prevents the development pDCs. The culture system is independent of GM-CSF.

A large panel of cytokines and growth factors as well as different growth conditions was tested to optimize the yield of eCD8+ cDC development within FL driven bone marrow cultures (FLDC). As one of the readouts the eCD8+ cDC subset-specific IFN-λ production upon poly IC treatment was determined.

Experiments were done as follows:

Addition of TGF-β at the beginning of cultures of bone marrow cells with FL increased the development of eCD8+ cDCs (Figure 12A) and at the same time drastically reduced the amount of the pDC which developed. (Figure 12B)

Comparison of the phenotypes of eCD8+ cDCs from FL-only or FL+ TGF-β cultures revealed largely identical surface marker expression with the exception that more eCD8+ cDCs expressed CD103 (Figure 13) and EpCAM (data not shown).

Functionally total FL-DCs from the TGF-β FL cultures produced largely increased IFN-λ upon eCD8⁺ specific stimulation (Figure 4Α) but at the same time decreased IFN-a production upon pDC specific stimulation. (Figure 14B)

Addition of TGF-β at the beginning of the cultures but analysis of DC subset generation between day 3 or day 6 of culture revealed that TGF-β decreases the pDC development (Figure 15A) but increases the eCD8 cDC development (Figure 15B) already at early times of the culture. The production of eCD8 derived IFN-λ demonstrated the increased development of eCD8 cDCs during the kinetic of DC development (Figure 15C). TGF-β has been added in a narrow concentration range with high concentration (>= 10 ng/ml) resulting in low yield of developing DC and low concentrations (<= 0.1 ng/ml) being not effective any more in skewing the DC subset development into the direction of eCD8+ cDCs (data not shown)

Addition up to 4 days after beginning of the FLDC culture still had effects in skewing the DC subset into more eCD8 cDCs and less pDCs (Figure 16 A+B).

There are different forms of TGF-β named β1 , β2 and β3. We tested the different TGF- βε for their DC subset skewing effect within FLDC cultures and found that recombinant human TGF-βΙ , TGF- 2 and TGF^3 were all active in increasing eCD8+ and at the same time decreasing the amount of developing pDC. We also tested different recombinant TGF^s from different suppliers and all were bioactive (data not shown). Sorted eCD8+ cDCs of TGF-β FL cultures were still able to produce IFN-λ, IL-6, IL- 12p70, ΜΙΡ-αΜΙΡ-1 and RANTES upon corresponding stimulation, (data not shown)

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Claims

A method for producing CD8+ and/or eCD8+ conventional dendritic cells (cDCs), comprising the steps of:

(a) incubating undifferentiated hematopoietic stem cells and/or precursor cells ex vivo with TGF-β for a time sufficient to allow development of CD8+ and/or eCD8+; and

(b) identifying and/or separating and/or isolating the CD8+ and/or eCD8+ cDCs.

The method of claim 1 , wherein step (a) comprises incubating undifferentiated hematopoietic stem cells and/precursor cells ex vivo with TGF-β and a growth factor excluding GM-CSF for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs.

The method of claim 2, wherein the growth factor is an inducer of dendritic cell (DC) generation, preferably is Flt3 ligand or a M-CSF receptor ligand.

The method of any of claims 1 to 3, wherein the TGF-β is produced from a recombinant viral vector, preferably a poxvirus vector, more preferably a Modified Vaccinia virus Ankara (MVA) vector, comprising a nucleic acid sequence encoding TGF-β.

The method of any of claims 1 to 4, wherein step (b) further comprises measuring an IFN-λ production in CD8+ and/or eCD8+ cDCs in response to a double stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs.

The method of any of claims 1 to 5, wherein step (b) is carried out using the in vitro method of claim 19.

7. A method for producing IFN-λ and/or generating or obtaining a population of IFN- λ producing CD8+ and/or eCD8+ cDCs, comprising the steps of:

(a) providing a population of cells comprising CD8+ and/or eCD8+ cDCs; preferably produced according to the method of any of claims 1 to 6;

(b) contacting ex vivo the CD8+ and/or eCD8+ cDCs with a double stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs.

8. The method of claim 7, wherein said population of cells is incubated with an enhancer of IFN-λ production, preferably a TLR ligand or a TNF-family member.

9. The method of any of the preceding claims, further comprising exposing the CD8+ and/or eCD8+ cDCs obtained according to the method of any of claims 1 to 6, or IFN-λ producing CD8+ and/or eCD8+ cDCs obtained according to the method of claim 7 or 8 to an antigen.

10. CD8+ and/or eCD8+ cDCs obtained according to the method of any of claims 1 to 6, 9, or IFN-λ producing CD8+ and/or eCD8+ cDCs obtained according to the method of any of claims 7 to 9, for use in the prevention and/or treatment of an infectious disease or cancer, preferably a viral infection, or for use in inducing an immune response in a subject to an antigen.

11. Autologous CD8+ and/or eCD8+ cDCs for use in preventing and/or treating a subject suffering from an infectious disease or cancer, preferably a viral infection, or for use in inducing a cytotoxic NK cell-mediated immune response, wherein said autologous CD8+ and/or eCD8+ cDCs are generated from autologous undifferentiated hematopoietic stem cells and/or precursor cells incubated ex vivo with TGF-β and a growth factor excluding GM-CSF for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs.

12. Use of TGF-β for enhancing the level of CD8+ and/or eCD8+ cDCs in vitro.

13. TGF-β for use as a medicament for enhancing the level of CD8+ and/or eCD8+ cDCs in a subject suffering from an infectious disease or cancer, preferably a viral infection.

14. Recombinant viral vector, comprising a nucleic acid sequence encoding TGF-β, preferably a poxvirus vector, more preferably a Modified Vaccinia virus Ankara (MVA) vector, for use as a medicament for enhancing the level of CD8+ and/or eCD8+ cDCs in a subject suffering from an infectious disease or cancer, preferably a viral infection.

15. A combined preparation comprising TGF-β and a double stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs.

16. A combined preparation comprising a double stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs and an agent enhancing double stranded nucleic acid-based IFN-λ production, preferably a TLR ligand or a TNF- family member.

17. The combined preparation of claim 15 or 16 for use in the prevention and/or treatment of an infectious disease or cancer, preferably a viral infection.

18. The CD8+ and/or eCD8+ cDCs of claim 10, the IFN-λ producing CD8+ and/or eCD8+ cDCs of claim 10, the autologous CD8+ and/or eCD8+ cDCs of claim 1 1 , the TGF-β of claim 13, the recombinant viral vector of claim 14, or the combined preparation of claim 17, wherein the viral infection is a persistent viral infection, preferably a viral infection of the liver or a Herpes virus infection, more preferably is a Hepatitis virus infection.

19. An in vitro method for detecting or screening for CD8+ and/or eCD8+ cDCs, comprising the steps of:

(a) providing a population of cells; .

(b) contacting the cells with a double-stranded nucleic acid or analog therof capable of stimulating or inducing the production of IFN-λ in CD8+ and/or eCD8+ cDCs;

(c) detecting the production of IFN-λ; and

(d) correlating the production of IFN-λ with the presence of CD8+ and/or eCD8+ cDCs.

20. Use of the in vitro method of claim 19 for detecting or screening for CD8+ and/or eCD8+ cDCs in a biopsy, preferably a biopsy of an organ or blood.

21. Use of IFN-λ production in response to a double-stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs as a marker for detecting CD8+ and/or eCD8+ cDCs.

22. An ex vivo method for identifying a compound driving the development of CD8+ and/or eCD8+ cDCs, comprising:

(i) contacting ex vivo undifferentiated hematopoietic stem cells and/or precursor cells with Flt3 ligand or a M-CSF receptor ligand for a time sufficient to allow development of CD8+ and/or eCD8+ cDCs, and with a test compound;

(ii) contacting the population of cells of (i) with a double-stranded nucleic acid or analog thereof targeting CD8+ and/or eCD8+ cDCs; and

(iii) measuring the level of IFN-λ produced by the population of cells of (ii), wherein an increase in the level of IFN-λ in the presence of the test compound, compared to the level of IFN-λ in the absence of the test compound, is indicative for the test compound driving the development of CD8+ and/or eCD8+ cDCs.

23. The method according to any of claims 1 to 6 or 22, or the subject-matter of claim 11 , wherein the undifferentiated hematopoietic stem cells and/or precursor cells are cells isolated from cord blood, mobilised peripheral blood cells or bone marrow.