Depletion of plasmacytoid dendritic cells for the treatment of respiratory viral infections

Abstract

The present application relates to a method of treating a respiratory viral infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an agent that depletes plasmacytoid dendritic cells. Another object of the present application relates to a kit of part comprising said agent and at least one further therapeutic agent as a combined preparation for simultaneous, separate or sequential use in the treatment of a respiratory viral infection in a subject in need thereof.

Description

[0001] DEPLETION OF PLASMACYTOID DENDRITIC CELLS FOR THE TREATMENT OF RESPIRATORY VIRAL INFECTIONS

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular virology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Vertebrate antiviral immunity heavily depends on the cytokines type I and type III interferons (IFN-I / IIIs)1. IFN-I / nis promote all the three arms of antiviral immune defenses: intrinsic, innate and adaptive immunity. IFN-I / IIIs signal mainly through a major common pathway involving the activation by phosphorylation of three transcription factors, STAT1, STAT2 and IRF9, and their hetero-trimerization into the complex ISGF3 (interferon stimulated gene factor 3). ISGF3 binds to IFN-stimulated response elements (ISRE) in genomic promoter or enhancer regions, driving the expression of hundreds of genes called IFN-stimulated genes (ISGs). Many ISGs have direct antiviral functions: they act as viral restriction factors able to block specific phases of the life cycle of virus families in infected cells, thus being the effector molecules of antiviral intrinsic immunity. The expression of the receptors for IFN-I and IFN-III have a complementary, largely reciprocal expression, mostly on epithelial cells in all barrier tissues for the latter and in all other nucleated cells of the body for the former. Hence, IFN-I / IIIs protect the host from viral infection in a large part by reinforcing antiviral intrinsic immunity in putatively all nucleated cells of the body1. IFN-Is also contribute to the activation or functional polarization of immune cells, hence promoting protective innate and adaptive anti-viral immune responsesJ.

[0006] IFN-I / III responses are generally beneficial during viral infections. However, when these responses are deregulated, they can turn deleterious1. This is the case when they are inappropriately activated in certain inflammatory or autoimmune diseases, in the absence of detectable viral infection. It is also the case during certain viral infections, if their induction is not properly controlled in time and space, as the proinflammatory functions of IFN-I / IIIs can promote virally-induced immunopathology, contribute to immunosuppression, or increase susceptibility to secondary bacterial infections by preventing the induction of proper adaptive immune responses or delaying the healing of infection-induced tissue damage in epithelial barriers1, 2’3. One strategy to promote the beneficial functions of IFN-I / IIIs and limit their deleterious effects would be to determine whether and how this is linked to the modalities of their production. This includes identifying which cell types produce IFN-I / IIIs, when, where, with what functional consequences, and under the instruction of which sensing pathway. Indeed, if specific cell types or sensing pathways are driving deleterious IFN-I / III responses in certain viral infections or autoimmune / inflammatory diseases, their specific targeting would allow dampening detrimental inflammation or misfired immune responses while preserving protective antiviral immunity. This would improve disease treatment by avoiding the general immunosuppression and the associated infectious risks induced by broad anti-inflammatory drugs such as corticosteroids or JAK / STAT inhibitors.

[0007] Amongst cellular sources of IFN-I / III, plasmacytoid dendritic cells (pDCs) are special because they rapidly produce very high amounts of all subtypes of IFN-I / IIIs upon engulfment and endosomal sensing of nucleic acids derived from viral particles or material from infected cells, while being themselves highly resistant to viral infection1, 4. Specifically, pDCs express the endosomal TLR7 and TLR9 receptors that are respectively able to recognize viral single stranded RNA versus unmethylated CpG DNA sequences. TLR7 / 9 engagement induces IFN- I / III production via a MYD88-IRAK4-IRF7 signaling pathway1. In contrast, infected cells sense cytosolic or nuclear genome intermediates from the viruses replicating endogenously in their cytosol or nucleus, through specific helicases or the cGAS enzyme converging on the activation of a STING-IRF3 signaling pathway promoting the production of IFN-p and only few subtypes and low amounts of IFN-as, and with susceptibility to inhibitory viral immune-evasion mechanismsl’5. Hence, it is currently thought that pDCs generally play a critical and beneficial role in antiviral immunity as a major source of IFN-I / IIIs early after infection, indispensable for the reinforcement of intrinsic antiviral immunity and for proper orchestration of innate and adaptive immune responses5, 6, 7, 8. Yet, as we reviewed recently4and will summarize in the next paragraph, direct and robust experimental evidences supporting this dogma are scarce, in a large part because methodological bottlenecks are preventing rigorous and definite evaluation of the role of pDCs in antiviral immunity both in humans and in mice.

[0008] In humans, primary immune deficiencies abrogating pDC ability to produce IFN-I / IIIs, including loss-of-function mutations in TLR7, MYD88, IRAK4 or IRF7, have been associated primarily with a heightened susceptibility to Mycobacterium Tuberculosis or a few other pyogenic bacteria but not to viral infections, except with the respiratory viruses Influenza and SARS-Cov29’10, n’12, 13, 14, 15, 16, 17This contrasts with the much broader susceptibility to viral infections of patients suffering from inborn errors in genes necessary for the response to IFN- Is or IFN-IIIs, such as STAT1, STAT2, IRF9, IFNAR1 or IFNAR2 ’19. Hence, these data show that pDCs but not IFN-I / III responses are largely redundant for antiviral immunity in modern humans in the current hygiene and health care context. In mice, contrasting results were obtained regarding the consequences on antiviral immunity of the genetic inactivation of Tlr7 / 9 o Myd88, depending on the combination of the virus and mouse strains studied, on differences in the inoculum dose or route, as well as on the type of readout measured since the analysis of intrinsic antiviral immunity and side-by-side comparisons with mice deficient for IFN-I / III responses were seldomly performedL 20. In any case, whether in humans or mice, genetic inactivation of TLR7 / 9, MyD88, IRAK4 or IRF7 affects other cells and biological processes beyond pDC IFN-I / III production. For example, TLR7 and IRF7 are also expressed and functional for IFN-I induction in monocytes and macrophages, and Myd88 is required for the responses to IL-1 cytokine family members in many cell types4.

[0009] Several approaches have been developed to deplete pDCs in vivo in mice to assess the functional impact of their loss. However, most of these approaches are not specific enough, with off-target effects confounding rigorous interpretation of the contribution to phenotypes observed of pDCs as opposed to other cell types also directly impacted by the mutation or treatment, as summarized in21. For example, n Skaros'1mice22many cell types beyond pDCs are directly affected; this is the case for DC precursors and subpopulations of DCs and macrophages in Sz / cc / z-hDTR mice23, 24for subpopulations of DCs, macrophages and possibly B cells in ItgaxCre,-Tcf4~ / flmice23, 26‘27, 28, 29, for plasma cells, subpopulations of activated B cells, DCs, monocytes and macrophages in mice treated with anti-BST2 / PDCAl depleting antibodies. Studies performed using these tools supported that pDCs were a major source of IFN-Is in infected mice, but varied in the assessment of the effective contribution of pDCs to anti-viral immunity, from beneficial to dispensable or even detrimental, depending on the infection route and the combination of virus and mouse strains4. Currently, pDC depletion can be achieved with high specificity and efficacy in one mutant mouse model, the BDCA2- hDTR mice, upon diphtheria toxin (DT) administration30. However, triggering of hDTRon the pDCs from these mice induces their production of IFN-I before their death31, which might confound the interpretation of the requirement of pDCs in antiviral immunity or IFN-I responses at large when using this model Moreover, repeated DT injections in BDCA2-hDTR mice has been reported to induce a severe morbidity proposed to be dependent on chronic pDC IFN-I production31. Therefore, to determine rigorously whether and how pDCs modulate host antiviral defense, there is still an unmet scientific need for a mouse model allowing selective and sustained pDC depletion without any off-target or confounding effect. SUMMARY OF THE INVENTION:

[0010] The present invention is defined by the claims. In particular, the present invention relates to a method of treating a respiratory viral infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an agent that depletes plasmacytoid dendritic cells.

[0011] DETAILED DESCRIPTION OF THE INVENTION:

[0012] Plasmacytoid dendritic cells (pDCs) are major producers of type I / III interferons. Since these cytokines are crucial for antiviral defense, it is assumed to be also the case for pDCs. However, robust evidence supporting this dogma is scarce. Genetic mutations or pharmacological manipulations causing pDC loss or disrupting their interferon production affect other immune cells, which could confound interpretation. To overcome this bottleneck, the inventors engineered pDC-less mice, specifically and constitutively devoid of pDCs because expressing diphteria toxin under coordinated control by the Siglech and Pacsinl genes co-expressed only in pDCs. pDC-less mice mounted protective intrinsic and innate immune responses against systemic infection with mouse cytomegalovirus, and were more resistant to intranasal infection with influenza virus and SARS-CoV2. Thus, contrary to dogma, pDCs and their interferon production proved dispensable or deleterious during systemic or respiratory viral infections. pDC-less mice will enable rigorously revisiting the roles of pDCs in health and disease.

[0013] The first object of the present invention relates to a method of treating a respiratory viral infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an agent that depletes plasmacytoid dendritic cells.

[0014] As used herein, the term “subject”, “individual” or “patient" is used interchangeably and refers to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some preferred embodiments, the subject is a human.

[0015] As used herein, the term “respiratory viral infection” has its general meaning in the art and refers to an infection that is caused by a virus and that affects one or more different parts of the respiratory tract. Typically, the respiratory viral infection can be caused by various types of viruses, such as influenza virus, rhinovirus, coronavirus, adenovirus, respiratory syncytial virus, parainfluenza virus, and metapneumovirus. These viruses can affect different parts of the respiratory tract, such as the nose, throat, bronchi, or lungs, and cause symptoms such as fever, cough, sore throat, runny nose, sneezing, wheezing, shortness of breath, or chest pain. Respiratory viral infections can be transmitted from person to person through respiratory droplets, aerosols, or contact with contaminated surfaces.

[0016] In some embodiments, the viral infection is caused by a virus selected from the group consisting of influenza virus (e.g., Influenza virus A, Influenza virus B), respiratory syncytial virus, adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), rhinovirus, coxsackie virus, echo virus, herpes simplex virus, coronavirus (SARS-coronavirus such as SARS-Covl or SARS-Cov2), and smallpox. In some embodiments, the viral lung infection may be due to a member of the Pneumoviridae, Paramyxoviridae and / or Coronaviridae families are in particular selected from the group consisting of upper and lower respiratory tract infections due to: human respiratory syncytial virus (hRSV), type A and type B, human metapneumovirus (hMPV) type A and type B; parainfluenza virus type 3 (PIV-3), measles virus, endemic human coronaviruses (HCoV-229E, -NL63, -OC43, and -HKU1), severe acute respiratory syndrome (SARS) and Middle-East respiratory syndrome (MERS) coronaviruses. In particular, the method of the present invention is suitable for the treatment of Severe Acute Respiratory Syndrome (SARS). More particularly, the method of the present invention is suitable for the treatment of lung inflammation in patients suffering from COVID- 19.

[0017] In some embodiments, the patient suffers from a chronic respiratory viral infection. In some embodiments, the patient suffers from an acute respiratory viral infection.

[0018] As used herein, the term “chronic infection” refers to a long-term infection which may be an apparent, unapparent or latent infection.

[0019] As used herein, the term “acute lung infection” has its general meaning in the art and refers to a disease of the lungs characterized by inflammation and consolidation followed by resolution and caused by viral infection. The term is also known as “pneumonia”. Typically, acute lung infection is associated with lung inflammation that is the rapid onset of progressive malfunction of the lungs, and is usually associated with the malfunction of other organs due to the inability to take up oxygen. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc ]).

[0020] In particular, the method of the present invention is particularly suitable for the curative treatment.

[0021] As used herein, the term "curative treatment" refers to a type of treatment that aims to cure a disease or eliminate its cause, rather than just alleviate its symptoms or slow down its progression. As used herein, the term “plasmacytoid dendritic cell” or “pDC” has its general meaning in the art and refers to a unique subset of dendritic cells that circulate in the blood and peripheral organs and rapidly secrete large amounts of type I interferons (IFNs) following activation. They are activated in the presence of either viral or self nucleic acids, which are detected by the endosomal toll-like receptors 7 or 9 (TLR7 or TLR9), two pattern recognition receptors selectively expressed by pDCs. Prior to activation, pDCs display a plasma cell-like morphology and express low levels of MHC class II and co-stimulatory molecules. Following activation, they acquire a more characteristic dendritic cell-like morphology and upregulate MHC class II and co-stimulatory molecules, allowing them to prime naive CD4+ T cells, albeit relatively poorly in mice. In addition to secreting high levels of type I IFNs, pDCs can also secrete pro- inflammatory cytokines and chemokines including IL-6, IL- 12, CCL3, CCL4, CXCL8 and CXCL10. In mice, pDCs are commonly identified as Lin-CDl lc+B220 / CD45R+BST- 2 / CD317+Ly-6C+CD1 lb- cells that also express Siglec-H and SIRP alpha / CD172a. In humans, pDCs are typically identified as Lin-CDl lclowCD123 / IL-3 R alpha+BDCA- 2 / CD303+BDCA-4 / Neuropilin-l+ cells. Both mouse and human pDCs express high levels of E2-2, a master transcription factor required for pDC development and homeostasis, along with Spi-B and IRF8, two additional transcription factors required for pDC development.

[0022] As used herein, the term “type I interferon” or “type I IFN” has its general meaning in the art and refers to members of the type I interferon family of molecules that are ligands for IFNAR- 1 (i.e., members of the type I interferon family of molecules that are capable of binding IFNAR- 1). Examples of type I interferon ligands are interferon alpha 1, 2a, 2b, 4, 5, 6, 7, 8, 10, 14, 16, 17, 21, interferon beta and interferon omega. All type I IFNs bind to a specific cell surface receptor complex known as the IFN-a receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains.

[0023] As used herein, the term “depletion” with respect to pDCs, refers to a measurable decrease in the number of pDCs in the patient. The reduction can be at least about 10%, e g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the term refers to a decrease in the number of pDCs cells in the patient below detectable limits.

[0024] In some embodiments, the agent is an antibody having binding affinity for a cell surface marker of pDC. In some embodiments, the antibody binds to BDCA2.

[0025] As used herein, the term “BDCA2” has its general meaning in the art and refers to blood dendritic cell antigen 2, that is a type of cell surface marker that is exclusively expressed on human plasmacytoid dendritic cells (pDCs). BDCA2 is also known as CD303, which is the cluster of differentiation designation for this molecule. BDCA2 / CD303 is a C-type lectin receptor that can bind to self- and non-self-antigens and modulate the immune response of pDCs. BDCA2 / CD303 is involved in the regulation of interferon production and cytokine secretion by pDCs, and its activation can inhibit the maturation and function of pDCs..

[0026] As used herein the term "antibody" or "immunoglobulin" has the same meaning, and will be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four (a, 5, y) to five (p, s) domains, a variable domain (VH) and three to four constant domains (CHI, CH2, CH3 and CH4 collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site. CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H- CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. According to the present invention, the amino acid residues in the variable domain, complementarity determining regions (CDRs) and framework regions (FR) of the antibody or the antigen-binding fragment of the present invention are identified using the Immunogenetics (IMGT) database (http: / / imgt.cines.fr). Lefranc et al. (2003) Dev Comp Immunol. 27(l):55-77. The IMGT database was developed using sequence information for immunoglobulins (IgGs), T-cell receptors (TcR) and Major Histocompatibility Complex (MHC) molecules and unifies numbering across antibody lambda and kappa light chains, heavy chains and T-cell receptor chains and avoids the use of insertion codes for all but uncommonly long insertions. IMGT also takes into account and combines the definition of the framework (FR) and complementarity determining regions (CDR) from Kabat et al., the characterization of the hypervariable loops from Chothia et al., as well as structural data from X-ray diffraction studies.

[0027] As used herein the term “bind” indicates that the antibody has affinity for the surface molecule. The term “affinity”, as used herein, means the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of Biacore instruments.

[0028] In some embodiments, the the antibody is a fully human antibody, a humanized antibody or a chimeric antibody. As used herein, the term “fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.

[0029] As used herein, the term "chimeric antibody" refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody. In some embodiments, a “chimeric antibody” is an antibody molecule in which (a) the constant region (i.e., the heavy and / or light chain), or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. Chimeric antibodies also include primatized and in particular humanized antibodies. Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0030] As used hereon, the term “humanized antibody” refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody. In some embodiments, a humanized antibody contains minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof may be human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody / antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 : 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0031] Anti-BDCA2 antibodies are well known in the art and typically include those described in WO2012 / 080642, WO2014 / 09339 and WO2016 / 156450.

[0032] In some embodiments, the antibody of the present invention cross-competes for binding to BDAC2 with the monoclonal antibody BIIB059 (Litifilimab).

[0033] As used herein, the term “cross-competes” refers to monoclonal antibodies which share the ability to bind to a specific region of an antigen. In the present disclosure the monoclonal antibody that “cross-competes" has the ability to interfere with the binding of another monoclonal antibody for the antigen in a standard competitive binding assay. Such a monoclonal antibody may, according to non-limiting theory, bind to the same or a related or nearby (e g., a structurally similar or spatially proximal) epitope as the antibody with which it competes. Cross-competition is present if antibody A reduces binding of antibody B at least by 60%, specifically at least by 70% and more specifically at least by 80% and vice versa in comparison to the positive control which lacks one of said antibodies. As the skilled artisan appreciates competition may be assessed in different assay set-ups. One suitable assay involves the use of the Biacore technology (e.g., by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology. Another assay for measuring cross-competition uses an ELISA-based approach. Furthermore, a high throughput process for "binning" antibodies based upon their cross-competition is described in International Patent Application No. WO2003 / 48731. The variable heavy chain (VH) of BIIB059 has the following amino acid sequence: DVQLVESGGG LVKPGGS LRL SCAAS GFTFS TYTMSWVRQA PGKGLEWVAT ISPGDSFGYY YPDSVQGRFT I SRDNAKNS L YLQMNS LRAE DTAVYYCTRD IYYNYGAWFA YWGQGTLVTV SS (SEQ ID NO:2)

[0034] The amino acid sequences of the VH CDRs of BII059 are listed below:

[0035] VH CDR1 : GFTFSTYT (SEQ ID NO:3)

[0036] VH CDR2: ISPGDSFGY (SEQ ID NO:4)

[0037] VH CDR3: TRDIYYNYGAWFAY (SEQ ID NO:5)

[0038] The variable light chain (VL) of BIIB059 has the following amino acid sequence:

[0039] DIQLTQS PSS LSASVGDRVT ITCKASQSVD YDGDSYMNWY QQKPGKAPKL LIYAASTLES GVPSRFSGSG SGTDFTLT I S SLQPEDFATY YCQQANEDPR TFGQGTKVE IK (SEQ ID NO:6).

[0040] The amino acid sequences of VL CDRs of BII059 are listed below:

[0041] VL CDR1: QSVDYDGDSY (SEQ ID NO:7)

[0042] VL CDR2: AA (SEQ ID NO: 8)

[0043] VL CDR3: QQANEDPRT (SEQ ID NO: 9)

[0044] In some embodiments, the antibody of the present invention cross-competes for binding to BDAC2 with the monoclonal antibody comprising a VH domain having the H-CDR1 as set forth in SEQ ID NO:3, the H-CDR2 as set forth in SEQ ID NO:4 and the H-CDR3 as set forth in SEQ ID NO:5 and a VL domain having the L-CDR1 as set forth in SEQ ID NO:7, the L- CDR2 as set forth in SEQ ID NO:8 and the L-CDR3 as set forth in SEQ ID NO:9.

[0045] In some embodiments, the monoclonal antibody or the antigen-binding fragment of the present invention comprises VH domain having the H-CDR1 as set forth in SEQ ID NO 3, the H-CDR2 as set forth in SEQ ID NO:4 and the H-CDR3 as set forth in SEQ ID NO:5 and a VL domain having the L-CDR1 as set forth in SEQ ID NOY, the L-CDR2 as set forth in SEQ ID NO: 8 and the L-CDR3 as set forth in SEQ ID NO : 9

[0046] In some embodiments, the antibody or the antigen-binding fragment of the present invention comprises a VH domain having an amino acid sequence having at least 70% of identity with the amino acid sequence as set forth in SEQ ID NO:2 and a VL domain having an amino acid sequence having at least 70% of identity with the amino acid sequence as set forth in SEQ ID NO:6.

[0047] As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443 53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or polynucleotide sequence are identical irrespective of any chemical and / or biological modification. According to the present invention, a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

[0048] In some embodiments, the antibody or the antigen-binding fragment of the present invention comprises a VH domain having the amino acid as set forth in SEQ ID NO:2 and a VL domain having the amino acid sequence as set forth in SEQ ID NO:6. In some embodiments, the antibody suitable for depletion of pDC cells mediates antibodydependent cell-mediated cytotoxicity.

[0049] As used herein the term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refer to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. While not wishing to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs).

[0050] As used herein, the term “Fc region” includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the hinge between Cgammal (Cyl) and Cgamma2 (Cy2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va ). The “EU index as set forth in Kabat” refers to the residue numbering of the human IgGl EU antibody as described in Kabat et al. supra. Fc may refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. An Fc variant protein may be an antibody, Fc fusion, or any protein or protein domain that comprises an Fc region. Particularly preferred are proteins comprising variant Fc regions, which are non-naturally occurring variants of an Fc region. The amino acid sequence of a non-naturally occurring Fc region (also referred to herein as a “variant Fc region”) comprises a substitution, insertion and / or deletion of at least one amino acid residue compared to the wild type amino acid sequence. Any new amino acid residue appearing in the sequence of a variant Fc region as a result of an insertion or substitution may be referred to as a non-naturally occurring amino acid residue. Note: Polymorphisms have been observed at a number of Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and thus slight differences between the presented sequence and sequences in the prior art may exist. As used herein, the terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody. The primary cells for mediating ADCC, NK cells, express FcyRIII, whereas monocytes express FcyRI, FcyRII, FcyRIII and / or FcyRIV. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To assess ADCC activity of a molecule, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecules of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998).

[0051] As used herein, the term “effector cells” are leukocytes which express one or more FcRs and perform effector functions. The cells express at least FcyRI, FCyRII, FcyRIII and / or FcyRIV and carry out ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.

[0052] In some embodiments, the antibody suitable for depletion of pDC cells is a full-length antibody. In some embodiments, the full-length antibody is an IgGl antibody. In some embodiments, the full-length antibody is an IgG3 antibody.

[0053] In some embodiments, the antibody suitable for depletion of pDC cells comprises a variant Fc region that has an increased affinity for FcyRIA, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, and FcyRIV. In some embodiments, the antibody of the present invention comprises a variant Fc region comprising at least one amino acid substitution, insertion or deletion wherein said at least one amino acid residue substitution, insertion or deletion results in an increased affinity for FcyRIA, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, and FcyRIV, In some embodiments, the antibody of the present invention comprises a variant Fc region comprising at least one amino acid substitution, insertion or deletion wherein said at least one amino acid residue is selected from the group consisting of: residue 239, 330, and 332, wherein amino acid residues are numbered following the EU index In some embodiments, the antibody of the present invention comprises a variant Fc region comprising at least one amino acid substitution wherein said at least one amino acid substitution is selected from the group consisting of: S239D, A330L, A330Y, and 1332E, wherein amino acid residues are numbered following the EU index. In some embodiments, the glycosylation of the antibody suitable for depletion of pDC cells is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al. Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated or non-fucosylated antibody having reduced amounts of or no fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the present invention to thereby produce an antibody with altered glycosylation. For example, EPl 176195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation or are devoid of fucosyl residues. Therefore, in some embodiments, the human monoclonal antibodies of the present invention may be produced by recombinant expression in a cell line which exhibit hypofucosylation or non-fucosylation pattern, for example, a mammalian cell line with deficient expression of the FUT8 gene encoding fucosyltransferase. PCT Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al, 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al, 1999 Nat. Biotech. 17: 176-180). Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation pattern devoid of fucosyl residues (http: / / www.eurekainc.com / a&boutus / companyoverview.html). Alternatively, the human monoclonal antibodies of the present invention can be produced in yeasts or filamentous fungi engineered for mammalian- like glycosylation pattern and capable of producing antibodies lacking fucose as glycosylation pattern (see for example EP1297172B1).

[0054] In some embodiments, the antibody suitable for depletion of pDC cells mediated complement dependant cytotoxicity.

[0055] As used herein, the term “complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to initiate complement activation and lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santaro et al., J. Immunol. Methods, 202: 163 (1996), may be performed.

[0056] In some embodiments, the antibody suitable for depletion of pDC cells mediates antibodydependent phagocytosis.

[0057] As used herein, the term “antibody-dependent phagocytosis” or “opsonisation” refers to the cell-mediated reaction wherein nonspecific cytotoxic cells that express Fey Rs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

[0058] In some embodiments, the antibody suitable for depletion of pDC cells is a multispecific antibody comprising a first antigen binding site directed against a pDC cell surface marker (e.g.BDCA2) and at least one second antigen binding site directed against an effector cell as above described. In said embodiments, the second antigen-binding site is used for recruiting a killing mechanism such as, for example, by binding an antigen on a human effector cell. For example, monocytes, macrophages, which express FcRs, are involved in specific killing of target cells and presenting antigens to other components of the immune system. In some embodiments, an effector cell may phagocytose a target antigen or target cell. The expression of a particular FcR on an effector cell may be regulated by humoral factors such as cytokines. An effector cell can phagocytose a target antigen or phagocytose or lyse a target cell. Suitable cytotoxic agents and second therapeutic agents are exemplified below, and include toxins (such as radiolabeled peptides), chemotherapeutic agents and prodrugs. In some embodiments, the second binding site binds to a Fc receptor as above defined. Exemplary formats for the multispecific antibody molecules of the present invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to a specific surface molecule of ILC and another with a specificity to a second antigen; (ii) a single antibody that comprises two different antigen-binding regions; (iii) a single-chain antibody that comprises two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically- linked bispecific (Fab')2 fragment; (vi) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bi specific antibody that has two binding sites for each of the target antigens; (vii) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (viii) a so called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivaient bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (ix) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (x) a diabody. Another exemplary format for bispecific antibodies is IgG-like molecules with complementary CH3 domains to force heterodimerization. Such molecules can be prepared using known technologies, such as, e.g., those known as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A / S) technologies.

[0059] In some embodiments, the multispecific antibody is thus a bispecific antibody.

[0060] In some embodiments, the bispecific antibody is a BiTE. As used herein, the term “Bispecific T-cell engager” or “BiTE” refers to a bispecific antibody that is a recombinant protein construct composed of two flexibly connected single-chain antibodies (scFv). One of said scFv antibodies binds specifically to a selected NK receptor , the second binds specifically to another molecule such as CD3, a subunit of the T-cell receptor complex on T cells. In some embodiments, the BiTE antibodies are capable of binding T cells transiently to target cells and, at the same time, activating the cytolytic activity of the T cells. The BiTE-mediated activation of the T cells requires neither specific T-cell receptors on the T cells, nor MHC I molecules, peptide antigens or co-stimulatory molecules on the target cell.

[0061] In some embodiments, the antibody suitable for depletion of pDC cells is conjugated to a therapeutic moiety, i.e. a drug.

[0062] In some embodiments, the therapeutic moiety can be, e.g., a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immune stimulator, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as an "antibody-drug conjugates" or "ADCs"

[0063] In some embodiments, the antibody suitable for depletion of pDC cells is conjugated to a cytotoxic moiety. The cytotoxic moiety may, for example, be selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin- inhibitor such as maytansine or an analog or derivative thereof; an antimitotic agent such as monomethyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1 -dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analog or derivative thereof; an antimetabolite such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, a platinum derivative such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or an analog or derivative thereof; an antibiotic such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)); pyrrolo[2,l-c][l,4]- benzodiazepines (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules, ricin toxin such as ricin A or a deglycosylated ricin A chain toxin, cholera toxin, a Shiga-like toxin such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.

[0064] In some embodiments, the antibody suitable for depletion of pDC cells is conjugated to an auristatin or a peptide analog, derivative or prodrug thereof Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anti-cancer (US5663149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42: 2961-2965. For example, auristatin E can be reacted with para-acetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E). Suitable auristatins and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugation of auristatins to Abs, are described in, e.g., U.S. Patent Nos. 5,635,483, 5,780,588 and 6,214,345 and in International patent application publications W002088172, W02004010957, W02005081711, W02005084390, W02006132670, WO03026577, W0200700860, W0207011968 and W0205082023.

[0065] In some embodiments, the antibody suitable for depletion of pDC cells is conjugated to pyrrolo[2,l-c][l,4]- benzodiazepine (PDB) or an analog, derivative or prodrug thereof. Suitable PDBs and PDB derivatives, and related technologies are described in, e.g., Hartley J. A. et al., Cancer Res 2010; 70(17) : 6849-6858; Antonow D. et al., Cancer J 2008; 14(3) : 154-169; Howard P.W. et al., Bioorg Med Chem Lett 2009; 19: 6463-6466 and Sagnou et al., Bioorg Med Chem Lett 2000; 10(18) : 2083-2086.

[0066] In some embodiments, the antibody suitable for depletion of pDC cells is conjugated to a cytotoxic moiety selected from the group consisting of an anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, a PDB, or an analog, derivative, or prodrug of any thereof.

[0067] In some embodiments, the antibody suitable for depletion of pDC cells is conjugated to an anthracycline or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to maytansine or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to calicheamicin or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to duocarmycin or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to rachelmycin (CC-1065) or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 15 or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin E or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin F or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to pyrrolo[2,l-c][l,4]-benzodiazepine or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to irinotecan or an analog, derivative or prodrug thereof.

[0068] In some embodiments, the antibody suitable for depletion of pDC cells is conjugated to a nucleic acid or nucleic acid-associated molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease (RNase) or deoxy-ribonuclease (e.g., DNase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule) or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In some embodiments, the antibody is conjugated to an aptamer or a ribozyme.

[0069] Techniques for conjugating molecule to antibodies, are well-known in the art (See, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO 89 / 12624.) Typically, the nucleic acid molecule is covalently attached to lysines or cysteines on the antibody, through N- hydroxysuccinimide ester or maleimide functionality respectively. Methods of conjugation using engineered cysteines or incorporation of unnatural amino acids have been reported to improve the homogeneity of the conjugate (Axup, J.Y., Bajjuri, K.M., Ritland, M., Hutchins, B.M., Kim, C.H., Kazane, S.A., Halder, R., Forsyth, J.S., Santidrian, A.F., Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, J.R., Flagella, K.M., Graham, R.A., Parsons, K.L., Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, D.L., Li, G., et al. (2010). Engineered thio-trastuzumab-DMl conjugate with an improved therapeutic index to target humanepidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res.16, 4769- 4778.). Junutula et al. (2008) developed cysteine-based site-specific conjugation called “THIOMABs” (TDCs) that are claimed to display an improved therapeutic index as compared to conventional conjugation methods. Conjugation to unnatural amino acids that have been incorporated into the antibody is also being explored for ADCs; however, the generality of this approach is yet to be established (Axup et al., 2012). In particular the one skilled in the art can also envisage Fc-containing polypeptide engineered with an acyl donor glutamine-containing tag (e.g., Gin-containing peptide tags or Q- tags) or an endogenous glutamine that are made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). Then a transglutaminase, can covalently crosslink with an amine donor agent (e.g., a small molecule comprising or attached to a reactive amine) to form a stable and homogenous population of an engineered Fc-containing polypeptide conjugate with the amine donor agent being site- specifically conjugated to the Fc-containing polypeptide through the acyl donor glutamine- containing tag or the accessible / exposed / reactive endogenous glutamine (WO 2012059882).

[0070] As used herein, the term "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0071] Typically the active ingredient of the present invention (e.g. depleting agent of the present invention) is combined with pharmaceutically acceptable excipients, and optionally sustained- release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0072] In one embodiment, the agent according to the invention is administered by injection. Kit of part

[0073] Another object of the invention relates to a kit of part comprising the agent of the invention and at least one further therapeutic agent as a combined preparation for simultaneous, separate or sequential use in the treatment of a respiratory viral infection in a subject in need thereof.

[0074] As used herein, the term “therapeutic agent” or “active agent” or “active substance” or “active principle” or “active ingredient” relates to a chemical substance inducing an effect such as a therapeutic or a preventive effect. It may be a bioactive chemical compound from a drug or the drug itself. Active agent can be a single molecule or a mixture of several substances.

[0075] As used herein, the term “simultaneous use” denotes the use of the agent of the invention and at least one active agent occurring at the same time.

[0076] As used herein, the term “separate use” denotes the use of the agent of the invention and at least one active agent not occurring at the same time.

[0077] As used herein, the term “sequential use” denotes the use of the agent of the invention and at least one active agent occurring by following an order.

[0078] In one embodiment, active agents may be added to the pharmaceutical composition or used in combination with the agent of the invention in the case of the treatment of a respiratory viral infection.

[0079] In one embodiment, active agents used in combination with the agent of the invention comprising antiviral agents, antibacterial agents, bronchodilators or corticosteroids. Antiviral agents my be oseltamivir, baloxavir marboxil, zanamivir, nirmatrelvir, ritonavir, remdevisir, molnupiravir, ribavirin, nirsevimab or palivizumab.

[0080] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0081] FIGURES:

[0082] Fig. 1: The pDC-less mice allow selective and constitutive depletion of pDCs in different organs, a, Scheme illustrating the strategy used to generate pDC-less mice. LoxP is the sequence recognized by Cre recombinase. ‘STOP’ corresponds to a transcriptional stop sequence. DTA= Diphtheria Toxin Subunit A. b, Dot plots showing the absence of lin- CD11b- CDl lcmtBst2hlpDCs in cells isolated from indicated organs of control (CTR) vs pDC-less mice. pDC percentages (mean + / - s.e.m.) were pooled from two independent experiments (n=5 for each strain), c, Absolute numbers of total cells isolated from the indicated organs of CTR vs pDC-less mice (n= 9 for the spleen and lymph nodes (LN), n=5 for the bone marrow (BM) and liver), d-f, Percentages within CD45+ splenic cells of indicated lymphoid (d) and myeloid (e,f) lineages isolated from control vs pDC-less mice. RPM: red pulp macrophage cMo: conventional monocyte. The data shown (mean + s.e.m.) are pooled from two independent experiments (n = 9). An unpaired and nonparametric multiple t-test (Mann-Whitney) with Holm-Sidak method for correction was used for the statistical analysis.

[0083] Fig. 2: pDCs exert a dispensable role during MCMV infection. Control and pDC-less mice were infected (i.p) with 1x105PFU of MCMV Smith and analyzed at indicated time points after infection, a, Cytometric bead arrays was performed to quantify cytokine concentrations in the sera isolated from uninfected (0) and 36h infected (1.5) control and pDC-less mice. Each dot represents an individual animal. The data shown (mean + / - s.e.m.) are pooled from two independent experiments (n=3 for 0, n=7 for 1.5). An unpaired and nonparametric multiple t- test (Mann-Whitney) with Holm-Sidak correction method was used for the statistical analysis, b, Control and pDC-less mice were infected with 1.4 xlO5PFU of MCMV Smith and daily monitored for their survival, as shown in Kaplan-Meier curve (n= 10 for control, =8 for pDC- less). Log-rank (Mantel-Cox) test was used for statistical analysis, c, Viral titers were measured by RT-qPCR in the spleen, liver and lungs isolated from control and pDC-less mice 3 days after MCMV infection. Data shown are pooled from two independent experiments (9 CTR and 7 pDC-less for the spleen, 7 CTR and 6 pDC-less for the liver, n=8 for both strains for the lungs). Means are indicated by a horizontal bar. An unpaired and nonparametric multiple t-test (Mann- Whitney) with Holm-Sidak correction method was used for the statistical analysis, d, Expression levels of indicated genes was analyzed by RT-qPCR in indicated organs isolated from uninfected or MCMV-infected CTR, pDC-less and Ifriarl^0mice at indicated days after infection. Data were normalized to expression level of Actin gene. The data (mean + s.e.m.) shown are from two independent experiments (n=4 for each condition at each time points). A nonparametric One-Way ANOVA (Kruskal-Wallis test with Dunn’s correction) was used for the statistical analysis, e, The percentages of GranzymeB (GzmB)+, IFNY+and CD69+cells within splenic NK cells (mean + / - s.e.m.) were determined in control and pDC-less mice at indicated days after MCMV infection (n=4 for 0, =8 for 1.5, =7 for 2 and =10 for 3 days after infection for both strains of mice). An unpaired and nonparametric multiple t-test (Mann- Whitney) with Holm-Sidak correction method was used for the statistical analysis.

[0084] Fig. 3: Both pDCs and the response to IFN-I are detrimental during IAV infection. Mice were infected with 145 PFU (i.n). in a,b and in f, g, while in e, they were infected i.n. with IxlO6PFU of IAV strain H3N2A / Scotland / 20 / 74. lAV-infected mice were monitored daily to assess their morbidity and survival. BDCA2-DTR mice were inoculated with 15 ng / g of diphtheria toxin (DT) two days after IAV infection. Kaplan-Meier survival plot shows the percentages of survival of indicated mouse strains, a, n= 10 for both strains, b, n=9 for both strains, e, n= 10 CTR, 8 IfnarlOand 9 Statl ° mice, , n= 10 CTR, 9 77r7KOmice. Log-rank (Mantel-Cox) test was used for statistical analysis, c, Expression levels of IAV Ml gene was analyzed by RT-qPCR in lungs isolated from uninfected or lAV-infected CTR and pDC-less mice at indicated days after infection. Data were normalized to expression level of Actin gene. The data (mean + s.e.m.) shown are from two independent experiments (n=4 for each condition at each time points). n= 6 at 0, 3 at day 1, 5 at day 2, 14 at day 6 and 3 at day 8 after infection, d, Normalized expression (log2) of indicated genes based on Bulk RNASeq samples obtained by whole lung samples isolated from CTR and pDC-less mice at day 0 (n=3 / strain), 3 and 6 (n=4 / strain) after IAV infection.

[0085] Fig. 4: During IAV infection, pDCs increase in infected lungs and produce IFN-I. a-b, IJhb mice were infected (i.n) with 160 PFU of IAV strain H3N2A / Scotland / 20 / 74 and analyzed at different time points after infection, a, The percentages of eDCs, pDCs and tDCs within CD45+ cells (mean + / - s.e.m.) isolated from the lungs of infected Ifrib mice were determined at the indicated time points. One-Way ANOVA (Kruskal-Wallis test followed by Dunn’s post-hoc test) was used for the statistical analysis. The percentages of YFP+ pDCs (b) at the indicated days after infection have been represented. N=2 for day 0, =3 for day 2, =6 for days 4 and 6, =4 for day 8 after infection. A two-tailed non-parametric Mann-Whitney test was used for the statistical analysis, c, Cytometric bead arrays was performed to quantify IFNa and IFNP concentrations in the BAL isolated from CTR and pDC-less mice uninfected (0) or at indicated days after infection. Each dot represents an individual animal. The data shown (mean + / - s.e.m ) are pooled from two independent experiments (n=3 CTR and 4 pDC-less at day 0, 3 CTR and 2 pDC-less at day 2, 7 at day 3, 6 at day 6 and 7 at day 8 for both strains). An unpaired and nonparametric multiple t-test (Mann-Whitney) with Holm-Sidak method for correction was used for the statistical analysis, d-e, Representative images of histological sections isolated from the lungs of SCRIPT mice at day 4 after IAV infection. SOgm-thick lung sections were stained with anti-CD45 (white), anti -Hemagglutinin (HA) of IAV (orange), antiTomato (Tom, purple), anti-YFP (green). On each image acquired, the MFI of CD45 in highly inflamed areas was at least twice of that of lowly inflamed areas. The numbers of CD45+ cells , HA (IAV)+ cells , pDCs and YFP+ pDCs per mm2were quantified in highly and lowly inflamed areas in lAV-infected lungs. The data shown (mean + / - s.e.m.) are from n=5 at day 2, n=5 at day 3, n=10 at day 4 and n=4 at day 6, with two sections of the lung analyzed per mouse An unpaired and nonparametric multiple t-test (Mann-Whitney) with Holm-Sidak correction method was used for the statistical analysis.

[0086] Fig. 5: IFN-I produced by pDCs during IAV infection are deleterious, a, Scheme illustrating the generation of pDC Shield BM chimeric (SBMC) mice. Only hind limbs of pDC- less mice are irradiated, while the rest of the body is protected by a lead shield (gray oval). Upon local irradiation-induced distress, donor BM cells preferentially reconstitute immune cell types absent in the recipient, that is pDCs. b, Recipient CD45.1+ (gray) pDC-less mice are used as recipient of pDC SBMC reconstituted with donor CD45.2+ (black) BM cells. The percentage of donor (black) vs recipient (gray) cells was analyzed in indicated lineages 4 weeks after reconstitution (n= 4). c-e, pDC SBMC mice reconstituted with CTR, pDC-less or Myd88KOBM (c), with CTR or 7L7?7KOBM (d) or with CTR, pDC-less or Jrf7t)were infected (i.n). with 145 PFU of IAV strain H3N2A / Scotland / 20 / 74 lAV-infected mice and daily monitored to assess their morbidity and survival. Kaplan-Meier survival plots show the percentages of survival of indicated genotypes, c-d, Pool of two independent experiments (n=8 for each strain); e, Pool of three independent experiments (n=12 for each strain). Log-rank (Mantel -Cox) test was used for statistical analysis.

[0087] Fig. 6: During IAV infection, lung inflammation and cytokine production are reduced in the absence of pDCs. a-d, Representative images of H&E-stained sections of lungs of control (a-b) and pDC-less (c-d) mice isolated at day 8 after infection. Magnification is indicated on each image, e, Lung sections of all mice were semi-quantitatively scored as normal (0), mild

[0088] (1), moderate (2), marked (3) and severe (4) for inflammation in interstitium, intra-alveolar, peribronchial, intra-bronchial and perivascular areas, and as normal (0), focal (1), multifocal

[0089] (2), extensive (3) for hemorragic necrosis, emphysema and pleura. The data (mean + / - s.e.m.) shown are pooled from two independent experiments, n=8 CTR, 9 pDC-less mice. A two-tailed nonparametric Mann-Whitney test was used for the statistical analysis, f, Absolute numbers of all lung cells and of CD45+ cells isolated from the lungs of control and pDC-less mice at the indicated days after infection. Data are represented as mean + / - s.e.m. of the following numbers of individuals pooled from two independent experiments for each time point (n= 5 at day 0, n= 7 at day 3, n= 8 at day 6 and at day 8 for each mouse strain). Percentages of myeloid and lymphoid immune cells isolated from the lungs of control and pDC-less mice isolated at the indicated day after infection. For each mouse strain, n= 5 at day 0, n= 7 at day 3, n= 8 at day 6 and n= 4 at day 8, except n= 8 at day 8 for neutrophils, eosinophils, alveolar macrophages (AM) and monocytes (cMo). For fan unpaired and nonparametric multiple t-test (Mann-Whitney) with Holm-Sidak correction method was used for the statistical analysis, g, Control and pDC- less mice were infected with 145 PFU of IAV strain and treated with anti-Ly6G (1A8) antibody (triangle) or isotype control (IC, circle) at days 5 and 6 after infection. Survival and morbidity were daily monitored. Data are pooled from two independent experiments. Kaplan-Meier survival plot shows percentage survival of indicated conditions. For each mouse strain, n= 8 for mice treated with isotype control (IC) and n=9 for mice treated with 1A8 antibodies, h, Cytometric bead arrays was performed to quantify the concentrations of indicated cytokines in the BAL isolated from control and pDC-less mice at indicated days after IAV infection. Each dot represents an individual. Data shown (mean + s.e.m.) are pooled from two experiments. n= 3 CTR and 4 pDC-less mice at day 0, n= 3 CTR and 2 pDC-less mice at day 2, n= 7 at day 3, n= 6 at day 6 and n= 7 at day 8 for both mouse strain. An unpaired and nonparametric multiple t-test (Mann-Whitney) with Holm-Sidak method for correction was used for the statistical analysis.

[0090] Fig. 7: During IAV infection lung integrity and permeability are less affected in the absence of pDCs. a, Representative images of histological sections of lungs isolated from control and pDC-less mice uninfected (left) or at day 8 after IAV infection (right). 30pm-thick sections were stained with anti-CD45 (white) and anti-EpCAM (red) antibodies. EpCAM+ bronchi were classified as closed (red arrow) when EpCAM staining was uninterrupted and as opened (white arrow) when EpCAM was discontinuous, b-c, The percentages of the EpCAM+ area over the entire surface of each lobe (b) and the percentages of closed vs opened bronchi (c) were quantified in lung sections isolated from uninfected vs lAV-infected control vs pDC- less mice. The data (mean + / - s.e.m.) shown in b-c are from n=l at day 0 and n=4 at day 8 after infection for both mouse strains. We analyzed 2 lobe sections at day 0 and 9 lobe sections at day 8 for control mice, 3 lobe sections at day 0 and 8 lobe sections at day 8 for pDC-less. An unpaired and nonparametric multiple t-test (Mann-Whitney) with Holm-Sidak correction method was used for the statistical analysis, d-e, 8 days after IAV infection control and pDC- less mice were i.t. treated with fluorescein isothiocyanate (FITC-Dextran) (lOpg per mouse). Barrier permeability was measured as relative fluorescent units (RFU) of FITC-dextran leaked in the plasma 1 hour after i.t. FITC-dextran administration. Each dot represents an individual. Data shown (mean + s.e.m.) are pooled from two experiments (n = 2 for each strain and condition for uninfected mice, as well as for PBS-treated infected mice, n= 9 for FITC-treated infected CTR and 8 for pDC-less mice). An unpaired and nonparametric multiple t-test (Mann- Whitney) with Holm-Sidak method for correction was used for the statistical analysis.

[0091] Fig. 8: pDCs are not required for the resistance to SARS-CoV2 infection and can exert detrimental functions, a-c, K18:hACE transgenic mice bred or not with indicated mutant mice were infected with 200 PFU (i.n). of SARS-CoV2 strain. Infected mice were daily monitored to assess their morbidity and survival. Kaplan-Meier survival plot shows the percentages of survival of indicated mouse strains, a, n= 28 for female K18:hACE and K18:hACE pDC-less; n= 19 for male K18:hACE, = 26 for male K18:hACE pDC-less, b, n=10 for K18:hACE and plain Ifnarl^' , =15 for K18:hACE If arlO, c, n= 12 for female K18:hACE, n= 9 for female K18:hACE Tlr7^° n= 11 for male K18:hACE, n = 7 for male K18:hACE Tlr7KO. d, Normalized expression (log2) of indicated genes based on Bulk RNASeq samples obtained by whole lung samples isolated from transgenic K18:hACE mice infected with 1.1 10A5 pfu pfu of SARS-CoV2 at indicated days after infection. Data (mean + / - s.e.m.) shown are from n= 3 at days 0 and 1, = 5 at day 2 and 4, =2 at day 6 after infection, e, The percentages of YFP+ pDCs at the indicated days after infection have been represented. Data are pooled of three independent experiments. n=10 at day 0, =12 at day 1, =7 at day 2, =12 at day 4 after infection. A two-tailed non-parametric Mann-Whitney test was used for the statistical analysis., f, Expression levels of indicated genes was analyzed by RT-qPCR in lungs isolated from uninfected or SARS-CoV2-infected K18:hACE mice alone or bred with pDC-less or with IjharlOmice at indicated days after infection. Data were normalized to expression level of Actin gene. The data (mean + s.e.m.) shown are from two independent experiments (n=2 for all strains at day 0, n=10 for K18:hACE and K18:hACE pDC-less, =5 for K18:hACET^76zr / KOand Ifnarl^0at day 2, n= 7 for K18:hACE and K18:hACE pDC-less, =5 for K18:hACE Ifnarl^0and =3 for Ifriarl^0at day 5). A nonparametric one-way ANOVA (Kruskal-Wallis test followed by Dunn’s post-hoc test) was used for the statistical analysis. EXAMPLE:

[0092] Material & Methods

[0093] Mice

[0094] All animal experiments were performed in accordance with national and international laws for laboratory animal welfare and experimentation (EEC Council Directive 2010 / 63 / EU, September 2010). Protocols were approved by the Marseille Ethical Committee for Animal Experimentation (registered by the Comite National de Reflexion Ethique sur 1’Experimentation Animale under no. 14; APAFIS no. 21626-2019072606014177 v.4 and APAFIS #35098-2022020215455814 v4, APAFIS#26484-2020062213431976 v6). C57BL / 6J (B6) mice were purchased from Janvier Labs. Siglech1Cre(B6-Siglechtml(‘Cre)Ciphe'), Ifnarl-KO (B6.129S2-Ifnarltmlagt) and SCRIPT (Siglech1Cre;PacsinlLSL'tdT;^#7#EYEP) mice were previously described32, 477’tzc5zri7LoxP'STOP'LoxP'DTA(B6-PacsinltmI(D’IA)Ciphe, PacsinLSL'DTA) and Statl-KO (B6-Statltmld(EUCOMM)Ciphe) were generated by CIPHE. Homozygous PacsinlLSL'DTAand Siglech1Crewere bred together and their double heterozygous Siglech1Cre; PacsinlLSL'DTAprogeny was named pDC-less mice. Siglech1Creand PacsinLSL'DTAwere also bred with CD45.1 congenic mice, then interbred to generate 5.1 pDC-less mice used as recipient for Shield Bone Marrow Chimera (SMBC) generation. TLR7KO(B6-Tlr7tmIFlv) were kindly provided by L. Alexopoulou, CIML. B6.A2G-Mxl congenic mice were kindly provided by M. Le Bert, TAAM, Orleans, France. IR7KO(B6-IrptmlTtg) were kindly provided by C. Svanborg, Lund, Sweden. Ifti ™' (B6.129-IfnbltmlLky), BDCA2-hDTR (C57BL / 6-Tg (CLEC4C- HBEGF)956Cln / J) and heterozygous K18-hACE C57BL / 6J mice (strain: 2B6. Cg-Tg (KI 8- ACE2)2Prlmn / J) were obtained from Jackson Laboratories, USA. PacsinLSL'DTAand B6.A2G- Mxl mice were intercrossed to generate double homozygous mice, then bred with Siglech1Creto obtain as progeny Mxl+ pDC-less mice. Control mice used in all experiments were PacsinLSL'DTA, Siglech1Creor B6 mice. All mouse strains were on B6 genetic background and bred at the Centre d’lmmunoPhenomique (CIPHE) or the Centre dTmmunologie de Marseille- Luminy (CIML), under specific pathogen free-conditions and in accordance with animal care and use regulations. Mice were housed under a 12 h dark: 12 h light cycle, with a temperature range of 20-22 °C and a humidity range of 40-70%. All animals used were sex and age matched (8-12 weeks of age for all experiments, except for SBMC). Shield Bone Marrow Chimera (SMBC) generation

[0095] 5 weeks-old CD45.1+ pDC-less were anesthetized, then their hind legs were 9 Gy irradiated, while the rest of the body was protected with a lead shield. At the end of the irradiation, mice were intravenously injected with 15 x 10A6 cells ofBM isolated from indicated CD45.2+ donor mice. 4 weeks after BM engraft mice were bled to test the reconstitution with donor BM cells, then used for experimentation.

[0096] Viruses, viral infections and mice treatment

[0097] MCMV Smith stocks were prepared from salivary gland extracts of 3-week-old, MCMV- infected BALB / c mice. Mice were infected intraperitoneally with 105plaque-forming units (PFU) and sacrificed at the indicated time points. For survival experiments with MCMV, mice were infected with 1.4 x 105plaque-forming units. H3N2 A / Scotland / 20 / 74 IAV strain was produced and titrated in vitro by using Madin-Darby Canine Kidney (MDCK) cell line. Mice were anesthetized with ketamine / xylazine, then intranasally infected with 40pl of DMEM medium containing 160 PFU of IAV strain and euthanized at indicated time points. For survival studies mice were infected with 145 PFU of the same IAV strain, p CoV / France / IDF0372 / 2020 SARS-Cov2 strain was supplied by the National Reference Centre for Respiratory Viruses hosted by the Institut Pasteur (Paris, France). This strain was isolated from a human sample provided by the Bichat Hospital, Paris, France and corresponded to the original Wuhan / D614 SARS-Cov2 strain detected at the beginning of COVID19 pandemic. Infectious stocks were grown by inoculating Vero E6 cells and collecting supernatants upon observation of the cytopathic effect. Debris was removed by centrifugation and passage through a 0.22 mm filter. Supernatants were stored at -80 °C. Mice were anesthetized with 150pL of ketamine / xylazine, then intranasally infected with 30 pl of medium containing 200 pfu of CoV / France / IDF0372 / 2020 SARS-CoV2. For Bulk RNASeq studies mice were infected with an high dose (1.1 10 "A5 pfu) of SARS-CoV2 strain. lAV-infected and SARS-CoV2 infected mice were monitored daily for morbidity (body weight) and mortality (survival). During the monitoring period, mice were scored for clinical symptoms (weight loss, eye closure, appearance of the fur, posture, and respiration). Mice obtaining a clinical score defined as reaching the experimental end-point were humanely euthanized, according to experimental protocols approved by ethical committee. For pDC depletion in BDCA2-hDTR mice, mice were intraperitoneally injected with 15ng / g souris of Dyphtheria Toxin, Merck, at day 2 after IAV infection. For neutrophil depletion mice were intraperitoneally injected at day 5 and 6 after IAV infection with 500 qg / mouse of anti-Ly6G (1A8) or isotype control antibodies, both purchased from Biolegend.

[0098] Cell preparation

[0099] Mice were euthanized and perfused with PBS lx. Spleen and lymph nodes were digested for 25 min at 37 °C with Collagenase IV (Worthington biochemical) and DNase I (Roche Diagnostics). Organs were then mechanically crushed and filtered over 70-pm cell strainers (Corning). Bone marrow cells were flushed from mouse femurs. Livers and lungs were harvested, minced and mechanically digested with Collagenase IV and DNase I by using GentleMACS (Miltenyi, program 37_m_LIDK_l for the liver, 37_m_LDK_l for the lungs). At the end of the digestion, lung cell suspensions were filtered over 70-pm cell strainers and cells were pelleted by centrifugation. Liver cells were submitted to a 80:40 Percoll gradient. Small intestines were harvested, opened longitudinally, cut into 1-cm pieces and washed extensively with PBS lx, then incubated 3Xat 37 °C on shaking (200 r.p.m.) with PBS lxcontaining 2% fetal calf serum (FCS) and 5 mM EDTA. At the end of each incubation, supernatants were collected and centrifuged. Pelleted cells from the three incubations were pooled together and submitted to a 67:44 Percoll gradient. Bronchoalveolar lavages (BAL) were performed upon intratracheal injection of 1 ml of cold PBS, repeated once. BAL were then centrifuged, fluids harvested and frozen at -80°C until their use. RBC lysis was performed with RBC lysis buffer (Thermofisher). For the lungs, the left lobe was harvested for RNA extraction, while the other lobes were used for flow cytometry.

[0100] Flow cytometry analysis

[0101] Cells were first incubated with 2.4G2 mAb for 10 minutes at 4°C, then we performed extracellular staining in staining buffer, PBS l x supplemented with 2 mM EDTA (Sigma- Aldrich) and 2% FCS for 30 min at 4 °C. Dead cell staining (LIVE / DEAD Fixable Aqua Dead Cell Stain, Life Technologies) was performed in PBS lxaccording to the manufacturer’s recommendations. All antibodies used and their related dilution have been listed in the Reporting Summary of this manuscript. Before acquisition samples were fixed with Cytofix / Cytoperm BD IX (eBioscience) or with a 2% formaldehyde solution in PBS lx when using cells expressing EYFP protein. Samples were acquired with a FACS LSR UV (BD Biosciences) using BD Diva v.9.0. All data were analyzed with FlowJo v.10.8.1 software. RNA extraction and RT-qPCR

[0102] Harvested organs were incubated in RNAlater (Thermofisher) at 4°C overnight. Total RNAs from lung cells were extracted using RNeasy Plus Mini Kit (Qiagen) following the manufacturer protocol. Retrotranscription into complementary DNA (cDNA) was performed using the Quantitect Reverse Transcription Kit (Qiagen). The expression levels of the following murine genes were determined by quantitative PCR (qPCR) using the SYBR® Premix Ex TaqTM kit and analysed using the Prism 7500 Fast PCR System. Relative gene expression was calculated using the AACt method with Actinb as housekeeping gene for normalization. The primers used were as it follows: Actinb forward 5’-GGCTGTATTCCCCTCCATCG-3’(SEQ ID NO: 10) ; reverse 5 ’ -CC AGTTGGTAACAATGCCATGT-3 ’ (SEQ ID NO: 11); 1128b forward 5’— GGAGGCCC AGAGCAAGGA-3 ’ (SEQ ID NO: 12); reverse 5’- TTGAAACAGGTTGGAGGTGACA-3’ (SEQ ID NO:13); Ir forward 5’— CCACGCTATACCATCTACCTGG-3’ (SEQ ID NO: 14); reverse 5’- GCTGCTATCCAGGGAAGACAC-3’ (SEQ ID NO:15); Isgl5 forward 5’—

[0103] GGTGTCCGTGACTAACTCCAT-3’ (SEQ ID NO: 16); reverse 5’- TGGAAAGGGTAAGACCGTCCT-3’ (SEQ ID NO: 17); Mx2 forward 5’—

[0104] AGAGGGAGAATGTCGCCTATT-3’ (SEQ ID NO: 18) ; reverse 5’-

[0105] CGTCCACGGTACTGCTTTTCA-3’ (SEQ ID NO: 19) and Oas2 forward 5’— TTGAAGAGGAATACATGCGGAAG-3’ (SEQ ID NO:20) ; reverse

[0106] 5’GGGTCTGCATTACTGGCACTT-3’ (SEQ ID NO:21). Viral titration of MCMV was performed as previously described20. For viral titration of IAV, we performed RT-qPCR by testing the expression of Ml IAV gene, using the following primers: forward 5’ — AAGACCAATCCTGTCACCTCTGA-3’ (SEQ ID NO:22); reverse 5’- CAAAGCGTCTACGCTGCAGTCC-3’ (SEQ ID NO:23). For viral titration of SARS-CoV2, tissues were homogenized with ceramic beads in a tissue homogenizer (Precellys, Berlin Instruments) in 0.5 mL RLT buffer. RNA was extracted using the RNeasy Mini Kit (QIAGEN) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Amplification was carried out using OneGreen Fast qPCR Premix (OZYME) according to the manufacturer’s recommendations. Copy numbers of the RNA-dependent RNA polymerase (RdRp) SARS-CoV2 gene were determined using the following primers: forward 5’- CATGTGTGGCGGTTCACTAT-3’ (SEQ ID NO:24), reverse 5’- GTTGTGGCATCTCCTGATGA-3’ (SEQ ID NO:25). This region was included in a cDNA standard to determine the copy number determination down to 100 copies / reaction. SARS- CoV-2 copy numbers were compared and quantified using a standard curve and normalized to total RNA levels. An external control (mock-infected wild type animal) and a positive control (SARS-CoV2 cDNA containing the targeted region of the RdRp gene at a concentration of 104 copies / pl [1.94x104 copies / pl detected in the assay]) were used in the RT-qPCR analysis to validate the assay.

[0107] Bulk RNA-Seq

[0108] RNA was isolated from the lungs of uninfected and lAV-infected control and pDC-less mice (160 PFU, days 0, 3 and 6 after infection) or of uninfected and SARS-CoV2 infected K18:hACE2 mice (1.1 10A5 PFU, days 0, 1, 2, 4 and 6 after infection), as described in RNA extraction method. For Bulk RNASeq 500 ng of lung RNA / sample were used to prepare libraries for sequencing by using KAPA RNA HyperPrep Kits (Roche). Libraries generated with lAV-infected samples were sequenced by Macrogen, while libraries generated from SARS-CoV2 infected samples were sequenced at the Genomic facility of the CIML. All libraries were sequenced using Illumina platforms. Alignments of fastq files obtained after sequencing were generated with STAR 2.7.9a software using the GRCm38 reference genome. The number of reads mapped to each gene was determined with featureCounts v2.0.1 using Mus_musculus.GRCm38.100.gtf annotation file. Gene models (gene symbols of the type “Gmxxxx”) were them removed from the feature count file and TPM normalization was applied. Data obtained have been submitted as repository to GEO as

[0109] FITC-Dextran

[0110] To assess lung permeability, isoflurane-anesthetized mice were administered intratracheally with 10 pg / mouse (50pl of a solution at 200pg / ml in PBS lx) of FITC-dextran (Sigma) 8 days after IAV infection. 1 hour later, blood was collected from anesthetized mice from the retro- orbital sinus and the plasma was separated by centrifugation. Plasma were diluted 1 :2 in PBS IX. Dextran leakage in the bloodstream was measured as FITC fluorescence in the plasma. Fluorescence was read at 520 nm with excitation at 490 nm by using a Mithras LB 940 plate reader (Berthold Technologies).

[0111] Cytokine quantification

[0112] Cytokines and chemokines present in the sera of uninfected vs MCMV-infected mice and in the BAL of uninfected vs lAV-infected mice were analyzed using the LEGENDplex Mouse Anti-Virus Response Panel (BioLegend). Samples and standards were plated in technical duplicates and the assay was executed according to the manufacturer’s protocol. Data were collected using the CANTO II (BD Biosciences) and analyzed using the cloud-specific LEGENDplex Data Analysis Software Qognit (BioLegend).

[0113] Anatomopathological analysis

[0114] Lungs were harvested from control and pDC-less mice 8 days after IAV infection and kept with 10% buffered formalin (VWR Chemical) for 24 hours, then dehydrated and embedded in paraffin (Fischer Histoplast). 3,5pm-thick sections were cut using the microtome Leica RM2245. Hematoxylin-eosin staining was made automatically with Leica Autostainer XL. Finally, the slides were mounted with Entellan mounting medium (Merck) and kept at room temperature.

[0115] Immunohistofluorescence, confocal microscopy and image analysis

[0116] Mouse lungs were perfused with a mixture of 1 :2 of AntigenFix (Diapath, containing 4% paraformaldehyde) and 1 :2 of Optimal Cutting Temperature (OCT, SakuraFineteck), collected, fixed in AntigenFix for 4h at 4°C and then washed several times in phosphate buffer (PB; 0.025 M NaH2PO4 and 0.1 M Na2HPO4). Lungs were incubated in PB solution containing 30% Sucrose overnight at 4°C and then embedded in OCT freezing medium, snap frozen and stored at -80 °C. 30 pm -thick cryosections were performed using a microtome (Leica 3050s Cryostat) at temperatures between -24 °C and -20 °C.

[0117] For immunostaining, lung sections were blocked with PB, 0.1% Triton X-100 and 2% BSA for Ih at room temperature and then stained overnight at 4°C with primary antibodies diluted in PB, 0.1% Triton X-100 and 2% BSA. After several washings with PB, lung sections were then stained in PB, 0.1% Triton X-100 and 2% BSA with secondary antibodies for 2h at 4°C. To detect YFP signal, after the incubation with secondary antibodies, sections were washed and incubated with PB, 0.1% Triton X-100, 2% BSA and 5% rabbit serum for 45 min at room temperature. Sections were then stained with Alexa 488-conjugated anti-GFP antibodies for Ih at 4°C. Finally, lung sections were washed with PB and mounted with a coverslip and Prolong Antifade Gold mounting medium (Life Technologies). Stained lung sections were acquired using spectral confocal microscopes (Zeiss LSM880 or Zeiss LSM980) with xl0 / 0.45 or x20 / 0.8 objectives, respectively. Images were acquired using the ZEN blue software (Carl Zeiss Microscopy), in 16-bit format, in spectral mode to suppress autofluorescence for each fluorochrome used. Images were then analyzed using ImageJ software77For CD45 quantification areas expressing low versus high CD45 signal were qualitatively identified in each whole lung section, then a region of interest (ROI) was created for each area and CD45 mean fluorescence intensity (MFI) was measured. In highly inflamed areas CD45 MFI was at least twice of that detected in the lowly inflamed areas. We then used QuPath vO.4.378for cell quantifications. CD45+ cells, lAV-infected HA+ cells and Tom+ pDCs were quantified using the QuPath cell detection module. Then, using the QuPath classifier module, we quantified among the Tom+ pDCs, the IFN+ (Tom+ YFP+) pDCs expressing at least a threshold value of 2216 in YFP channel. For lung integrity analysis, EpCAM+ areas were defined as areas expressing at least a 5000 threshold for EpCAM channel, then their percentage was calculated on the whole lung surface analyzed. EpCAM+ bronchi were classified as closed when EpCAM staining was continuous and as opened when EpCAM was disrupted. Both types of EpCAM+ bronchi were manually quantified and corresponding percentages were calculated.

[0118] Statistical analysis

[0119] All statistical analyses were performed using GraphPad Prism 10 software. All results are expressed as mean ± s.e.m. All quantifications were performed with awareness of experimental groups, meaning not in a blinded fashion. Animals were matched in age and gender between experimental groups and each cage was randomly assigned to a treatment group. No animals or data were excluded under any circumstances. Statistical parameters including the exact value of n (number of biological replicates and total number of experiments) and the types of statistical tests are reported in corresponding legends. Comparisons between two groups were assessed using unpaired and non-parametric Mann-Whitney test. Multiple comparisons were assessed using a Kruskal -Wallis test followed by Dunn’s post-hoc test. Survival curves were analyzed using Log-rank (Mantel-Cox) test. Comparisons between groups were planned before statistical testing and target effect sizes were not predetermined. Each dot indicates an individual, p values have been specified in each panel.

[0120] Results:

[0121] The pDC-less mice allow constitutive and selective depletion of pDCs

[0122] We recently generated Siglech1Crex PacsinlLSLtdTomatodouble Knock-In mice, called pDC-Tom, in which tdTomato fluorescent protein was exclusively expressed in “bona fide” pDCs132. Specific pDC targeting was achieved using intersectional genetic approach based on the unique co-expression of SiglecH and Pacsinl genes in mouse pDCs. We adapted our two gene-based strategy by generating 7’6'C5zn / LoxP'STOP'LoxP'DTA(PacsinlLSL'DTA) knock-in mice, in which a floxed cassette containing the gene encoding the A subunit of the diphtheria toxin (DTA) was located at the 3’ of Pacsinl gene (Fig, la). PacsinlLSL'DTAwere then bred with Siglech1Cre, thus allowing the removal of STOP sequence and lethal expression of DTA exclusively in SiglecH+Pacsinl+cells, corresponding to pDCs. Indeed, pDCs were ablated in both lymphoid and non-lymphoid organs of double heterozygous Siglech1Cre; PacsinlLSL'DTAmice, hereafter called pDC-less mice (Fig, lb). pDC-less mice developed and grew normally (Data not shown). Moreover, the cell numbers isolated from lymphoid and non-lymphoid organs of control versus pDC-less mice were comparable (Fig. 1c). The development of several other lymphoid and myeloid lineages was not affected, as exemplified in the spleen (Fig. Id-e), thus confirming the selective ablation of pDCs. As pDCs and tDCs share a common hematopoietic precursor29, we extended our analysis by using an optimized staining panel allowing to discriminate “bona fide” pDCs from other eDCs and tDChland tDC10cell subsets expressing high or low levels of CD11c, respectively27, 29(Data not shown). We confirmed the lack of “bona fide” CD11c10Bst2hlLy6D+CX3CRlnegpDCs32in our pDC-less mice (Fig. If). cDC2s were unaffected, while cDCls were significantly increased in pDC-less mice, consistent with the previously reported enhanced expansion of cDCls when the development of pDC-committed precursors is inhibited33. Finally, tDClo / pDC-like were slightly, but significantly reduced, consistent with low, but detectable Tomato expression in late pDC-committed precursors isolated from pDC-Tom mice32, likely including recently described pro-pDC / tDC precursors29‘32. Altogether, our results demonstrate that pDCs are selectively and constitutively absent in pDC-less mice.

[0123] During MCMV infection pDCs are the main IFN-I source, but they are dispensable for cell-intrinsic and innate anti-viral immunity

[0124] During systemic infection by mouse cytomegalovirus (MCMV), a natural rodent pathogen, IFN-Is are mainly produced by pDCs30, 34, 35, 36. Indeed, both IFN-a and IFN-P were undetectable in the sera of MCMV-infected pDC-less mice at the peak of IFN-I production (1.5 days after infection). However, the production of other pro-inflammatory cytokines, such as IL- 12, TNF-a, IFN-y, CXCL10 and CCL2 was unaffected, thus supporting the existence of other sources of these cytokines redundant with pDCs (Fig. 2a). Splenic pDCs were also the main source of IFN-III during MCMV infection (Data not shown), consistent with enhanced expression of 1128a and 1128b genes in IFN-I-producing pDCs36. We then evaluated the impact of the loss of pDCs and, especially of their IFNs, on anti-viral immunity. pDC-less mice well survived MCMV infection, even at high viral loads (Fig. 2b) and efficiently controlled viral dissemination in all host organs analyzed (Fig. 2c). Next, we analyzed the kinetic of ISG expression during MCMV infection. In infected control mice Mx2 gene was induced at higher levels in the spleen than in the lungs and in the liver and its expression peaked in the spleen and the lungs at 1.5 days after infection, while it was delayed of 12 hours in the liver (Data not shown). When compared to uninfected mice, Irf7, Mx2, Isgl5 and Ifit2 expression was upregulated with similar kinetics in the organs of MCMV-infected control and pDC-less mice, but poorly in the organs isolated from infected IfnarlKOmice (Figure 2d) IFNs produced by pDCs during MCMV infection promote the activation of NK cells and eDCs, two innate immune lineages crucial for the induction of efficient antiviral responses during MCMV infection20, 30, 37, 38. However, granzyme B, IFN-y and CD69 were similarly upregulated in splenic NK cells isolated from MCMV-infected control and pDC-less mice, whatever time point after infection analyzed (Figure 2e). Moreover, splenic cDCl and cDC2 isolated from MCMV-infected control and pDC-less mice expressed comparable amounts of IFN-induced CD86 activation marker (Data not shown).

[0125] Altogether, our results thus demonstrate that during systemic MCMV infection pDCs are dispensable for mounting protective intrinsic and innate anti-viral immunity, despite being the main IFN source.

[0126] The pDCs and the response to IFN-I are both detrimental in local respiratory infection by influenza A virus pDCs were considered critical for enhanced susceptibility of IRF7 mutant patients to the respiratory influenza A virus (IAV) infection, as defective in IFN-I production in vitro upon TLR7 triggering12, 14In lAV-infected pDC-depleted mice the scenario appeared more complex, supporting evidence of dispensable, protective or even detrimental functions of pDCs39, 40, 41, 42,43’44However, in all mouse studies other cells, besides pDCs, were depleted, thus confounding the interpretation of the results obtained. Moreover, both mouse genetic background and viral strain differentially affected mice resistance to IAV infection. We thus investigated the specific contribution of pDCs by infecting control vs pDC-less mice with a IAV strain (H3N2 A / Scotland / 20 / 74), inducing severe pneumonia in mice on C57BL / 6 background45. When infected with LD50 of this strain, half of control mice died, while all pDC-less mice recovered from infection and survived (Fig. 3a). We extended our analysis to BDCA2-hDTR mice30. Upon DT administration, most of lAV-infected BDCA2-11DTR survived, while 80% of control C57BL / 6 mice died of lAV-induced immunopathology (Fig. 3b), thus confirming in two distinct mutant mice that pDCs were deleterious in this infection model. IAV replication in the lungs did not differ between control and pDC-less mice, as observed analyzing the expression of IAV Ml gene at different time points after infection (Fig. 3c). Moreover, Irf7, Oas2 and Isgl5 ISG genes were similarly induced in the lungs of infected control and pDC-less mice (Fig. 3d), as analyzed on bulk RNASeq data performed on RNA extracted from the lungs of control vs pDC-less mice at days 0, 3 and 6 after infection. Altogether, these data exclude a key role of pDCs in intrinsic anti-viral immunity induced during pulmonary IAV infection.

[0127] In most murine strains, including C57BL / 6, Mxl, the main virulence restriction factor against IAV, is defective, due to a genetic non-functional mutation. Yet, when C57BL / 6 mice express at least one functional Mxl allele, they become resistant to IAV, even at high viral loads, via a mechanism proposed to depend on TLR7-Myd8841. Lung viral titers were increased in IAV- infected Mxl+ mice when depleted of BST2+ cells, proposing a protective role of pDCs. We thus investigated the impact of selective pDC depletion in Mxl+ mice. To this aim, we bred our PacsinlLSL'DTAmice with B6.A2G-Mxl+ / +, by obtaining PacsinlLSL'DTAMxl+ / + mice, then we crossed them with SiglechlCremice, by obtaining Mxl+ / - pDC-less mice. Both Mxl+ PacsinlLSL'DTA, used as control, and MxH7- pDC-less mice survived to the infection with high viral loads of IAV and displayed low morbidity (Fig. 3e and Extended Data Fig. 3c), excluding an essential role of pDCs in lAV-infected Mxl+ hosts. Life-threatening lung immunopathology induced by pathogenic IAV strains was abrogated in Ifnar 7KO42, 4- supporting a deleterious role of the response to IFN-I. When infected with LD50 of H3N2 A / Scotland / 20 / 74 strain, 90 % of IfnarlKOmice survived to the infection, while all StatlKOmice succumbed by day 8 (Fig. 3f), confirming the detrimental role of IFN-I and revealing a protective role of IFN-II and / or IFN- III. The administration of synthetic TLR7 antagonists inhibited lAV-dependent lung immunopathology, thus suggesting that deleterious IFN-Is were mainly produced via a TLR7- dependent pathway44. Accordingly, TLR7KOwell survived to IAV infection (Data not shown). In conclusions, the infection with the pathogenic H3N2 A / Scotland / 20 / 74 IAV strain induces in mice a life-threatening pneumonia elicited by IFN-I / pDC / TLR7 -dependent mechanisms.

[0128] During IAV infection lung pDCs increase and are a major source of IFN-a

[0129] As both pDCs and IFN-Is were harmful during IAV infection, we wondered whether pDCs could be a detrimental source of these cytokines. We determined whether and, if yes, when lung pDCs produced IFN-I during IAV infection by using Ifnl ™ reporter mice46. pDCs, as well as tDCs and eDCs, were rare within CD45+ cells isolated from the lungs of uninfected mice, while their percentages increased starting from day 4 after infection (Fig. 4a) We analyzed YFP expression, correlating with IFN-I production36, 46, in these different DC subsets. Within DCs isolated from the lungs of lAV-infected Ijhb mice only pDCs contained YFP+ cells (Extended Fig.4a) that reached the plateau between day 4 and 6 after infection (Fig. 4b), confirming pDC contribution to IFN-Is produced in the lungs of lAV-infected mice. We then evaluated the impact of pDC loss on whole IFNs detectable in the lungs at the transcriptional and protein level. First, we analyzed the expression of genes encoding for IFN-I / IIIs in bulk RNASeq samples derived from the lungs isolated from control vs pDC-less mice at days 0, 3 and 6 after IAV infection (Data not shown). The expression of all genes encoding IFN-Is and IFN-IIIs was detectable exclusively in infected mice. Ifnbl, Ifna4, Ifril2 and Ifni 3 were expressed at comparable levels in both mouse strains, while we observed a tendency to a lower expression of Ifnal, Ifha2, Ifria6 and Ifna5 in the lungs of infected pDC-less mice (Data not shown). We then analyzed IFN-a and IFN-P protein contents in bronchioalveolar lavages (BAL) isolated from control vs pDC-less mice at different days after IAV infection (Fig. 4c). Consistent with our transcriptional data, both IFN-I subtypes were detectable in the BAL starting day 3 after infection. However, only IFN-a were significantly reduced in the absence of pDCs, while IFN-P levels were comparable between infected control and pDC-less mice (Fig. 4c). Taken together, these results confirm that pDCs are a main source of IFN-a in infected lungs, although other cells produce other IFN-I subtypes, as well as IFN-in. Breeding of Ifnl ™ reporter with our pDC-Tom reporter mice generated SCRIPT mice that discriminate IFN+ (Tom+ YFP+) from IFN- (Tom+ YFP-) pDCs in tissues32. We quantified the distribution of whole pDCs vs IFN+ ones in the lungs of lAV-infected SCRIPT mice. Heterogeneous CD45 staining allowed to distinguish CD45low(lowly inflamed) vs CD45hlgh(highly inflamed) areas, the latter ones ressembling to inflammatory foci (Fig. 4d-e). Highly inflamed areas contained also the highest numbers of infected cells expressing viral hemagglutinin (HA+) (Fig. 4d-e) Both CD45+ and HA+ cells increased during the infection. pDC frequency significantly increased in highly inflamed areas starting from day 4 after infection (Data not shown), consistent with our flow cytometry data (Fig. 4a). IFN+ pDCs (YFP+ Tom+) were detected only in highly inflamed areas and peaked at day 4 after infection (Data not shown). Indeed, during IAV infection lung pDCs produce IFN-Is and are preferentially located in highly inflamed and infected areas.

[0130] IFN production by pDCs is mainly responsible of their deleterious role during IAV infection pDCs are a major, but not the unique source of IFN-Is in the lungs during IAV infection (Fig. 4). Studying the specific role of IFN-I produced by pDCs in lAV-induced pneumonia would require the inactivation of genes involved in the TLR7 / Myd88 / IRF7 signaling cascade uniquely in pDCs. However, none of currently existing conditional Cre mice, including our Siglech1Cremice32’47, allows the selective targeting of pDCs. To overcome this roadblock, we developed a novel chimeric mouse model, the pDC Shield Bone Marrow Chimeras (pDC SBMC) mice (Fig. 5a) To achieve this purpose, we lethally irradiated the hind legs of pDC-less mice, while the rest of their body was protected by a lead shield. This created a local perturbation that favored the engraftment of donor BM cells, especially for cell lineages absent in recipient mice, in our case pDCs. Indeed, four weeks after reconstitution, pDC SBMC mice were replenished with pDCs derived from donor BM, while the other immune lineages were still mostly composed by recipient cells, as validated by using CD45.2 donor BM cells in CD45.1+ pDC- less mice used as SBMC recipient (Fig. 5b). pDC SBMC thus allow to study the impact of the loss of a candidate gene selectively in pDCs. As cytokine production by pDCs is Myd88- dependent47, we evaluated the resistance to IAV infection of pDC SBMC reconstituted with Myd88KOpDC. We generated CTR, pDC-less and Myd88KOpDC SBMC by reconstituting shield-irradiated CD45.1+ pDC-less mice with CD45.2+ C57BL / 6, pDC-less or Myd88KOBM cells, respectively. When infected with IAV, half of CTR pDC SBMC succumbed, while both pDC-less and Myd88KOpDC SBMC mostly survived (Fig. 5c). TLR7KOpDC SBMC were also resistant to IAV infection, when compared to infected CTR pDC SBMC (Fig, 5d). pDC- intrinsic lack of Myd88 completely abrogates TLR7 / 9-dependent cytokine and chemokine production, while pDC-intrinsic deficiency in IRF7 abolishes exclusively IFN production47We thus generated IRF7KO, as well as CTR, pDC-less pDC SBMC and infected them with IAV. All IRF7KOandpDC-less pDC SBMC survived, while 60% of CTR pDC SBMC died (Fig. 5e). Hence, selective inactivation of TLR7 / Myd88 / IRF7 signaling in pDCs was sufficient per se to restore the resistance to IAV infection and confirmed the detrimental contribution of IFNs produced by lung pDCs.

[0131] During IAV infection lung inflammation and cytokine contents are reduced in the absence of pDCs

[0132] During IAV infection the recruitment of immune cells to the infected lungs and their activation are essential to establish protective anti-viral immunity. However, unbridled and sustained inflammatory responses can lead to severe pneumonia and acute respiratory distress syndrome (ARDS). Therefore, we performed anatomopathological analysis of lung sections isolated from lAV-infected control and pDC-less mice to evaluate the impact of pDC loss on lung inflammation and tissue integrity. We harvested our samples 8 days after IAV infection, when survival and morbidity start to differ between these two mouse strains (Fig. 3a). Most of the lungs isolated from infected control mice showed a marked broncho-interstitial pneumonia (Fig. 6a), associated with bronchiolar filling with degenerated epithelial cell and high alveolar inflammation (Fig. 6b). In contrast, lungs isolated from infected pDC-less mice displayed milder pneumonia and inflammation (Fig. 6c-d). Consistently, semi -quantitative score of lung sections, taking in account inflammation and necrosis in interstitial, alveolar, bronchial and vascular areas (Data not shown), was significantly higher in control than in pDC-less mice (Fig. 6e). In lAV-infected mice the depletion of BST2+cells correlated with reduced recruitment of inflammatory leukocytes in infected lungs42. Absolute numbers of whole lung cells and, especially, of CD45+ cells increased during infection and peaked at 6 days p.i., following a similar trend in infected control and pDC-less mice (Fig. 6f). The percentages of most myeloid and lymphoid lineages isolated from infected control and pDC-less mice were comparable, except for pDCs, as expected (Fig. 6g). However, both neutrophils and eosinophils were significantly increased at days 6-8 after IAV infection in the absence of pDCs (Fig. 6g). Neutrophils were proposed to exert detrimental vs protective roles upon IAV infection44, 48. We thus determined whether neutrophils were required for enhanced resistance of pDC-less mice to IAV infection by injecting lAV-infected control and pDC-less mice with isotype control (IC) or neutrophil-depleting antibodies (1A8) 5-6 days after infection. Neutrophil depletion severely affected the survival of both control and pDC-less infected mice (Figure 6h), showing that in our infection model neutrophils were protective, but via pDC-independent mechanisms. Indeed, selective pDC depletion did not inhibit the recruitment of immune cells into infected lungs, but it is rather associated with neutrophilia and eosinophilia.

[0133] IFN-Is produced by pDCs activate both innate and adaptive immune cells1, by inducing the expression of activation molecules, such as CD86 and CD69. The expression of both these markers was induced during infection on lung myeloid and lymphoid cells, but it was only transiently affected in the absence of pDCs (Data not shown). Depletion of BST2+ cells was also shown to reduce the amounts of proinflammatory cytokines detected in bronchioalveolar lavages (BAL) of lAV-infected mice and, consequently, lung immunopathology42The analysis of our bulk RNASeq data showed that mRNAs encoding most cytokine and chemokines analyzed were similarly upregulated in infected control and pDC-less mice at days 3 and 6 after infection (Data not shown). BAL isolated from both strains of mice at days 2, 3 and 6 after infection did not differ in their content for proinflammatory, such as CXCL10, CCL2 and IFN- y, and anti-inflammatory cytokines, such as IL-10 (Data not shown). However, at day 8 after infection the amounts of most cytokines detectable in the BAL were significantly lower in the absence of pDCs (Data not shown). Hence, during late phases of IAV infection enhanced resistance of pDC-less mice correlates with reduced bronchioalveolar amounts of cytokines known to promote ARSD. The alterations of lung integrity and permeability induced upon IAV infection are less severe in the absence of pDCs

[0134] Unbridled cytokine-dependent inflammation induced by IAV infection can promote severe lung lesions, fluid leakage and defaults in respiratory capacity leading to ARDS. Reduced cytokine levels in the BAL of lAV-infected pDC-less mice could witness of reduced tissue damage and leakage in the bronchiolar tree in the absence of pDCs, consistent with milder histopathological score in the lungs of infected pDC-less mice (Fig. 6e). Bronchi were clearly identified in the lung sections of uninfected control and pDC-less mice by staining Epithelial Cell Adhesion Molecule (EpCAM), a marker of bronchiolar cells (Fig. 7a). However, while in uninfected lungs EpCAM staining was continuous and homogeneous in both strains (Fig. 7a), it was often disrupted in the lungs of infected mice (Fig. 7a). On the whole lung sections the percentages of EpCAM+ areas were significantly higher in infected pDC-less mice (Fig. 7b), while the percentages of bronchi with disrupted (open) EpCAM staining were higher in infected control mice (Fig. 7c), thus suggesting an increased breaching of the bronchiolar tree and, potentially, of fluid leakage in the presence of pDCs. Pulmonary permeability can be analyzed by measuring the leakage into the plasma of fluorescent proteins instilled in the airways (Fig. 7d)49. We thus intratracheally instilled lOKDa FITC-Dextran control and pDC-less mice uninfected or 8 days after IAV infection. While the fluorescence was absent in the plasma of uninfected mice, as well as of PBS-instilled lAV-infected mice, it was clearly detectable in the plasma of IAV- infected mice and significantly lower in pDC-less than in control mice (Fig. 7e). pDCs thus contribute to promote lung lesions and fluid leakage during IAV infection.

[0135] During mouse SARS-CoV2 infection pDCs are not required for mice survival and can exert detrimental functions

[0136] Patients harboring genetic defects in MYD88, IRAK4, IRF7 or TLR7 genes were highly susceptible to the infection by several respiratory viruses, including SARS-CoV213, 14, 15, 16, 17As blood pDCs of these patients were defective in vitro in TLR7-dependent IFN production, patient susceptibility was attributed to impaired pDC-dependent viral control14, 17. However, other studies proposed rather a detrimental role of pDCs during SARS-CoV2 infection, by promoting via their IFN-Is macrophage-dependent cytokine storm50, 51, 52, 53. We thus decided to address the specific role of pDCs by using our pDC-less mice. C57BL / 6 mice are resistant to SARS-CoV2 infection, as the virus cannot use mouse angiotensin-converting enzyme 2 (ACE2) as receptor for cell entry. However, ectopic expression of human ACE2 under the control of the cytokeratin 18 (K18) promoter render transgenic K18:hACE C57BL / 6 mice highly susceptible to SARS-CoV infections54. Hence, we first generated K18:hACE x PacsinlLSL'DTAmice, then we bred them with Siglech1Cremice. Offspring triple heterozygous mice, hereafter called K18:hACE pDC-less, as well as control K18:hACE mice, were infected with SARS-CoV2. Infected male K18:hACE survived less than females, consistent with higher morbidity and mortality observed in male SARS-CoV2-infected patients55(Fig. 8a). We did not detect any significant difference in survival when comparing infected females K18:hACE mice and K18:hACE pDC-less mice. In contrast, infected males K18:hACE pDC-less mice were significantly more resistant than gender-matched infected K18:hACE mice (Fig. 8a) Indeed, pDCs are not required for K18:hACE mice survival to SARS-Cov2 infection and exert detrimental functions in infected males.

[0137] The response to IFN-I is essential for human survival to viral infections, including to SARS- CoV215. However, IFN-I-responsiveness is deleterious role in IAV infected-mice (Fig, 3 and Fig. 5). When infected with SARS-CoV2, K18:hACE Ifnarl^0mice were even more susceptible than K18:hACE (Fig. 8b), thus supporting a main protective role of IFN-I in this infection model. In contrast, plain Iftiarl mice were resistant, as lacking hACE receptor. Patients with TLR7 mutations, especially males, were found highly susceptible to SARS-CoV2 infection16. We thus addressed TLR7 role in K18:hACE mice, by breeding them with Tlr7KOmice. TLR7 deficiency significantly worsened the susceptibility of K18:hACE mice to SARS- CoV2 infection and this independently of mouse gender, showing a protective role of TLR7 (Fig. 8c). Altogether, these results demonstrate that the survival of K18-hACE mice to SARS- CoV2 infection requires the response to IFN-I sources that are TLR7-dependent, but pDC- independent.

[0138] The timing and the magnitude of IFN production during SARS-CoV2 infection varies between patients developing moderate vs severe CO VID50. In mice infected with SARS-CoVl and MERS-CoV2, IFNs were beneficial in very early phases of the infection, but detrimental later on56, 57. ISG expression in SARS-CoV2-infected lungs was induced as soon as 1-2 days after infection, consistent with SARS-CoV2 detection in infected tissues (Fig. 8d-e) ISG induction was not affected in infected K18:pDC-less mice (Fig. 8e), according with their dispensable role for viral control. In contrast, ISG upregulation was affected at variable extent in infected K18:hACE IfnarlQ(Fig. 8e), likely due to partial compensation by other IFNs, such as IFN- II and IFN-III. We then analyzed the kinetic of IFN production in pDCs by infecting with SARS-CoV2 K18:hACE SCRIPT mice. pDCs isolated from the lungs of infected K18:hACE SCRIPT mice started to express YFP protein at day 1 after infection, but they reached their maximal peak later on, between day 2 and 4 (Fig. 8f). Hence, IFN production by pDCs was delayed with respect to ISG induction and sustained during late phases of the infection, potentially fueling IFN-dependent detrimental functions, as proposed in humans53.

[0139] In conclusion, both IFN-I and TLR7 were protective during SARS-CoV2 infection of K18:hACE mice, while pDCs were dispensable in female and detrimental in male infected mice

[0140] Discussion;

[0141] In this study, we reported the generation of a new mouse model, the pDC-less mice, allowing specific and constitutive ablation of pDCs. pDC-less mice developed normally and were healthy, thus excluding a critical role of pDCs for host fitness in homeostatic conditions, at least in an environment free of specific pathogens. We did not detect any major disruption of other immune cell types in our mice, consistent with our intersectional genetic approach specifically targeting late pDC-committed precursors without affecting other lineages32. Only tDC10were slightly but significantly reduced in our pDC-less mice, consistent with the identification of a shared pDC / tDC-committed bone marrow precursor29Hence, in our pDC-less mice, the specificity of pDC depletion is highly improved, as compared to previous methods used to target pDCs but also affecting DC precursors, tDCs and subsets of monocytes / macrophages or B cells21, including the administration of anti-PDCAl / BST2 antibodies or the use of other mutant mice encompassing CDl lc-Cre; Tcf4- / fl (CKO) animals25, 29or Szg / ec / z-hDTR animals23, 24In addition, pDC-less mice allow evaluating the long-term impact of pDC loss without the confounding side-effects caused by repeated DT injections, as observed in BDCA2- hDTR mice30, 31. Moreover, the use of pDC-less mice as recipient for the generation of shield BM chimeras allowed to inactivate candidate genes selectively in pDCs, for the first time to our knowledge, thus representing an excellent surrogate method to overcome the current lack of pDC-specific Cre mice. pDCs are strongly conserved in vertebrates, both in their molecular make-up58and in their functional specialization, namely professional production of IFNs, with rapid and massive release of all subtypes of these cytokines upon exposure to virus-type stimuli1, 47. This implies that pDC functions benefit vertebrates in a manner enhancing their reproductive fitness, which is currently thought to be via rapid IFN-dependent reinforcement of antiviral intrinsic immunity throughout the body to promote early viral control and host resistance upon primary acute infections6, 7. Yet, robust experimental data supporting this hypothesis are scarce, both in humans and mice. Indeed, contrary to patients suffering from a global defect in responsiveness to IFN-I / III18, 19, patients suffering from primary immune deficiencies due to loss-of-functions mutations in TLR7, MYD88, IRAK-4 or IRF7 do not exhibit enhanced susceptibility to most viral infections, except with Influenza or SARS-Cov2n’12, 13, 14, 15, 16, 17, but the role of the loss of pDC IFN production in this heightened susceptibility is unclear since other cell types and biological processes are also directly affected by the same mutations. In mice, although pDCs are a major IFN sources during many viral infections, their depletion or functional inactivation did not compromise host survival, except upon systemic infection by HSV-2 or ocular infection with HSV-159, 60. Here, we showed that during systemic infection by MCMV, a natural rodent pathogen, pDCs were the main source of both IFN-Is and IFN-IIIs, consistent with previous studies30, 34However, the selective ablation of pDCs compromised neither the IFN-dependent induction of ISG, nor viral control or host survival. These results are consistent with our previous work showing a greater impact of Ifnarl inactivation over Myd88 deficiency on ISG expression and host survival in MCMV-infected mice20. Moreover, we also showed that ISG were induced early in the lungs of mice infected by Influenza or SARS-CoV2, irrespective of the presence of pDCs, and that pDCs were not required for resistance to these infections. This was also true in mice expressing a functional allele of Mxl, the key restriction factor against IAV. Hence, the increase in IAV titers observed previously in Mxl+ mice depleted with anti- Bst2 antibodies41was unlikely to have been caused by pDC depletion but rather resulted from the perturbation of other cell types also targeted by this treatment. Thus, contrary to the current dogma in the field, our results demonstrate that pDCs are redundant with other cellular sources of IFN for boosting antiviral intrinsic immunity and promoting overall host resistance to several systemic or respiratory viral infections in mice, which is also likely the case in humans.

[0142] Although immune cell activation was impaired in the absence of pDCs in several models of viral infections, including those used in our study20, 30, 37, 59, 61, these defects were transient and had very limited impact on host resistance to infection, suggesting the existence of other redundant mechanisms able to preserve the induction of protective innate and adaptive antiviral immune responses in the face of the loss of pDC functions. Our findings thus emphasize the need to reevaluate whether and how pDC functions could benefit the host during viral infections The use of pDC-less mice and of shield BM chimeras derived thereof will certainly be key to address these questions in future studies.

[0143] In the present study, during IAV or SARS-CoV2 infections of mice, we not only showed that pDCs were dispensable but that they could actually be detrimental for host survival Specifically, in K18:hACE infected mice, pDCs were dispensable in females, but deleterious in males. This appears contradictory with the prominent hypothesis in the field that pDCs are protective against human respiratory infectionsn. However, the picture is more complex since the role of pDCs in respiratory Influenza and SARS-CoV infections is debated. In mouse models of Influenza infection, different studies have drawn contrasting conclusions, proposing that pDCs are beneficial41dispensable39, 40or deleterious42, 43, 44However, these studies used experimental strategies to deplete pDCs or impair their functions that were not specific enough and also targeted other cell types or biological processes, potentially confounding the interpretation of the results in terms of the contribution of pDCs to the phenotypes observed. For example, previous studies showed a deleterious role during murine IAV infection of Bst2+ cells42, 43, 44, encompassing not only pDCs but also inflammatory monocytes, activated DCs and B cell subsets. In patients with severe COVID, blood pDCs were numerically reduced and defective in IFN production upon ex vivo stimulationl6‘62 63‘64Moreover, an enhanced frequency of severe COVID was detected in patients with loss-of-function mutations disrupting the TLR7-to-MYD88-to-IRF7 signaling pathway that is essential for pDC IFN production. These observations led to the proposal that pDCs are protective against COVID in most patients and that disruption of their responses contribute to disease severity. However, alternate interpretations should be considered. The decrease in pDC numbers and the loss of their ability to produce IFN in the blood of severe COVID patients could reflect the recruitment and activation of these cells into the infected tissues where they could contribute to fuel inflammation by producing IFN locally, as has been reported to occur during infections of macaques or humans by immunodeficiency viruses65, 66. The protective role of the TLR7-to- MYD88-to-IRF7 signaling pathway observed in human respiratory viral infections could occur in other cells than pDCs, in particular in monocytes or macrophages that also express, and respond to, these molecules16, 67, 68. Indeed, other studies proposed that pDCs are a deleterious source of IFNs promoting exacerbated activation of monocytes / macrophages responsible of life-threatening immunopathology50, 51, 52, 53. In SARS-CoV2-infected K18:hACE infected mice, our results do show contrasting roles of pDCs (deleterious) versus of overall IFN production and responses (protective), which supports the hypothesis that, in humans, the beneficial role TLR7 and IFN responses against COVID occurs in other cells than pDCs, whereas pDCs may be redundant or even deleterious. Future studies are required to test this hypothesis, especially to compare the kinetics and spectrum of the TLR7-dependent production of IFN subtypes between pDCs and other cells in vivo during SARS-CoV2 infection. These experiments could be achieved in mice but also perhaps in macaques, as the animal model naturally susceptible to this infection that is the closest to humans. This has important implications as it has been proposed to boost pDC IFN production to treat severe human respiratory infections8, which could lead to an outcome exactly opposite to expectations if pDCs are deleterious in these diseases as we propose. On the contrary, understanding whether and how pDC IFN production could worsen disease may uniquely allow to disentangle the mechanisms underlying the beneficial versus deleterious effects of IFN responses in these diseases. This could ultimately allow designing intervention strategies to inhibit the deleterious effects of IFNs while still preserving their beneficial functions.

[0144] Most IFN-a subtypes, except IFN-a4, but not IFN-P, were reduced in the lungs of lAV-infected pDC-less mice. The production of most IFN-a subtypes requires IRF7 that is constitutively expressed in pDCs but not in most other cell types, whereas IFN-a4 / p can be quickly released by lAV-infected cells in an IRF3 -dependent IRF7-independent manner69, 70. Although all IFN- Is interact with the same IFNAR receptor complex, IFN-I subtypes differ in their affinity and duration of binding to IFNAR, leading to differences in the intensity, kinetics and array of ISG induction. In addition, differently from most infected cells, pDCs are highly motile, especially after activation61, thus being able to release their IFNs to specific cells in a precise time and location, as we previously showed during MCMV infection32, 36. Hence, a division of labor may exist between different types of IFN-producing cells, with infected cells being mainly involved in reinforcing intrinsic immunity in neighboring cells to control viral propagation, whereas pDCs could deliver IFNs at specific times to specific cell types to exert unique immunostimulatory functions that could be generally beneficial but would turn detrimental in specific contexts including during infections causing immunopathology or autoimmunity.

[0145] In conclusion, contrary to the dogma currently prevailing in the field, we show that, in mice, pDCs, appear to be a largely dispensable source of IFN-I for host resistance to primary acute viral infections, similarly to what had been proposed several years ago in humans9, 10, 71. These findings illustrate how redundancy contributes to the robustness of immune responses20, 72, including for the signaling pathways and cellular sources leading to the production of IFNs during viral infections which is vital for the host73. However, our results also raise the question of the identification of the exact benefit for the host of the evolutionary conservation of pDCs, especially considering that current state of knowledge supports their detrimental roles in both infection-induced and sterile inflammation, whereas rigorous experimental proof are scarce of any beneficial role of pDCs in the natural history of diseases. Several hypotheses can be formulated. First, pDCs might be required for the protection of specific immune-privileged organs unable to produce protective IFN otherwise, as recently shown for the eye in a mouse model of cornel infection with HSV-160. Second, rather than being required for resistance against a primary acute infection with a first virus, pDCs may be required to induce a bodywide state of antiviral resistance including in barrier tissues remote from the initial site of infection74, in order to prevent superinfections by other viruses that could otherwise constitute a second lethal hit for the host. Third, pDC IFN production during a first primary acute viral infection could improve the induction of adaptive immune memory to enhance host protection to secondary infections, explaining in part the enhanced immune resistance of mice exposed to a normalized microbial environment75. Further studies will be necessary to investigate these hypotheses, which will require the generation of a novel mouse model of conditional rather than constitutive pDC depletion, not suffering from the confounding side effects reporting for existing models31, 34, and complex experimental set-up of sequential exposure to different pathogens76.

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Claims

CLAIMS:

1. A method of treating a respiratory viral infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an agent that depletes plasmacytoid dendritic cells.

2. A kit of part comprising the agent for use according to the claim 1 and at least one further therapeutic agent as a combined preparation for simultaneous, separate or sequential use.

Images