New Method

Abstract

In particular, the present invention provides an ex vivo method for obtaining tolerogenic antigen-presenting cells (APCs) capable of inducing tolerance to an antigen in the immune system, the method comprising: (a) isolating monocytes from a sample obtained from a mammal; and (b) culturing the isolated monocytes in a cell culture to induce differentiation of the monocytes into antigen-presenting cells having a tolerogenic phenotype, wherein the cell culture comprises: (i) retinoic acid and TGF-beta; (ii) retinoic acid, TGF-beta and an AhR agonist; or (iii) retinoic acid and an AhR agonist.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates inter alia to ex vivo methods for obtaining tolerogenic antigen-presenting cells (APCs) capable of tolerizing to an antigen, to the tolerogenic APCs obtained by the methods of the invention, to the tolerogenic APCs themselves, and their uses, as well as to methods for treating unwanted immune responses to antigens and for preventing immune rejection of allogeneic (allo)grafts. [Background technology]

[0002] BACKGROUND OF THE INVENTION Tolerogenic antigen-presenting cell (APC)-based immunotherapies, which exploit the mechanism of antigen presentation in a tolerogenic manner, represent a promising, nontoxic approach for treating immune disorders or preventing transplant rejection, such as allografts. They can be used as a monotherapy or as an add-on to other types of therapy, such as in combination with immunosuppressants or other immunomodulatory therapies. This strategy relies on the ex vivo manipulation and introduction of cell therapy products to circumvent immune disorders with the goal of inducing antigen-specific tolerance. Therefore, the ultimate goal of such APC-based immunotherapies is to induce tolerance in vivo by delivering inhibitory signals to effector cells and inducing and expanding regulatory T cells (Tregs). For example, patients with autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis may benefit from treatment based on such tolerogenic APC-based therapies.

[0003] Induction of antigen-specific immune responses requires the participation of professional APCs that express major histocompatibility complex (MHC) molecules, as well as membrane-bound costimulatory and secreted inflammatory molecules. Furthermore, such APCs must be able to internalize, process, and present antigens in association with MHC molecules.

[0004] Similarly, the induction of antigen-specific immune tolerance also requires presentation of antigen in the context of the MHC. However, unlike initiating an immune response, tolerance induction requires high cell surface expression of tolerogenic molecules and secreted anti-inflammatory mediators, combined with low expression of membrane-bound costimulatory molecules and secreted proinflammatory molecules.

[0005] The main types of professional APCs in the immune system are dendritic cells (DCs), macrophages, certain B cells, and certain activated epithelial cells. At their immature stage, APCs ingest extracellular antigens by phagocytosis or pinocytosis and process the antigens into peptides within endocytic compartments such as endosomes and phagosomes, where the peptides are bound to MHC class II molecules. They also possess the unique ability to load peptides from exogenous proteins into the MHC class I presentation pathway, a process called "cross-presentation." With appropriate differentiation signals, APCs can develop into either tolerogenic or non-tolerogenic APCs. Tolerogenic APCs can mediate downregulation or prevention of immune responses and are thought to play an important role in maintaining peripheral tolerance.

[0006] Hemophilia A (HA) is an X-linked bleeding disorder caused by various mutations in the F8 gene, which encodes factor VIII (FVIII), that interfere with translated protein expression or procoagulant function. FVIII is primarily expressed in the liver and endothelial vascular beds. Because HA patients lack sufficient procoagulant activity, they are prone to bleeding episodes and their sequelae, including increased morbidity and mortality. The FVIII database currently identifies 2,015 unique FVIII variants based on case reports from 5,472 individuals. This vast array of point mutations (66.5%), deletions (23.2%), and others (duplications, polymorphisms, insertions, indels, and hybrids) results in diverse clinical outcomes. Patients can be treated acutely (on demand) or prophylactically with either plasma-derived or recombinant FVIII.

[0007] Because the immune system is not fully tolerized to certain sequences of normal FVIII, a significant number of patients develop neutralizing antibodies to FVIII, called "inhibitors" or anti-drug antibodies (ADAs), which block the activity of administered FVIII. Inhibitor production is currently the most serious and prominent treatment complication seen in HA patients.

[0008] Currently, once inhibitors have formed, the only proven method for eradication is immune tolerance induction (ITI) via frequent high-dose FVIII infusions, but this treatment regimen fails in 20–40% of patients.

[0009] The problem of ADA is not limited to hemophilia patients with defective FVIII. ADA can also develop in patients treated with other biologics. ADA production is a T-cell-dependent process resulting from a lack of central T-cell tolerance. Tolerance to self-proteins is an important part of the development of the immune system; proteins encountered later in life are usually recognized as foreign, depending on the context in which they are presented, leading to the development of an antibody response.

[0010] The first step in the immune response to FVIII is thought to be the uptake of FVIII by APCs. Following endocytosis of FVIII by APCs, FVIII is processed into small peptides and loaded onto MHC class II molecules, and the MHC class II-peptide complexes are then transported to FVIII-specific CD4 +These T cells are transported to the cell surface for presentation to T cells. Activation of these T cells requires additional activation signals provided by APCs. These activation signals are membrane-associated interactions between costimulatory molecules, such as CD40, CD80, and CD86, on the plasma membrane of APCs and, for example, CD28, CD154, and CTLA-4 on T cells. In addition to these receptor / ligand interactions, APCs signal T cells through secreted cytokines, such as IL-12 or IL-10. The combination of signals determines the differentiation direction of activated T cells. T helper 1 (Th1) cells generally induce cytotoxic immune responses, Th2 cells induce B cell-mediated antibody responses, and Tregs can induce immunosuppression / tolerance by suppressing activated B and T cells as well as through other mechanisms. Finally, activated FVIII-specific T cells can activate FVIII-specific B cells and induce affinity maturation and class switching of immunoglobulin genes in B cells. This results in the generation of anti-FVIII antibody-secreting plasma cells and circulating FVIII-specific memory B cells, which produce antibodies upon re-exposure to FVIII.

[0011] Autoimmune diseases occur when an adaptive immune response specific to a self-antigen is mistakenly initiated. The resulting immune effector pathways cause chronic inflammatory damage to tissues, which can prove fatal. Autoimmunity can be initiated by the activation of autoantigen-specific T cells and the production of autoantibodies. Specific genes found within the MHC and other immunoregulatory loci likely play a key role in determining an individual's susceptibility to developing autoimmune disease, likely through their ability to regulate adaptive T cell and B cell immune responses. T cell responses to self-antigens can cause tissue damage through cytotoxic T cell responses, inappropriate activation of other effector cells, and inappropriate T cell help for B cells. Furthermore, an immune system that ceases to recognize one or more normal components of the body as "self" can lead to the production of pathological autoantibodies. Autoantibodies that recognize self-antigens as foreign initiate further immune responses, resulting in increased T cell infiltration, production of inflammatory cytokines, and widespread tissue damage. Diseases in which these functions of T cells and B cells may be important include rheumatoid arthritis, type 1 diabetes mellitus, and multiple sclerosis.

[0012] A graft from a genetically unrelated donor of the same species is called an allograft. Allografts are considered a last resort for the treatment of chronic organ failure. Despite advances in organ preservation and immunosuppression, rejection remains a major post-transplant complication. Despite pre-transplant tissue typing and blood analysis, rejection occurs and is seen to varying degrees in almost all transplant recipients. With the exception of hyperacute rejection, which occurs due to the presence of pre-existing antibodies (due to pregnancy, blood transfusions, and / or previous transplants), transplant rejection can be broadly divided into two types: acute and chronic. While acute rejection is considered solely an immunological response, chronic rejection involves both immunological and non-immunological mechanisms.

[0013] Allorecognition is the presentation of graft antigens (alloantigens) and can be divided into two major subtypes: direct and indirect. DCs and other professional APCs migrating from the graft initiate direct allorecognition, in which recipient T cells are directly activated by alloantigen-binding peptides. Recipient APCs then pick up alloantigens shed from the donor-derived graft or dying cells and present the processed alloantigens in combination with self-MHC to recipient T cells (indirect allorecognition). A third subtype, semidirect allorecognition, has also been proposed, which involves recipient APCs passively acquiring donor MHC on their cell surface while patrolling the graft through a process called trogocytosis. Naive CD4 + T helper cells are among the first immune cells activated after transplantation and play a key role in rejection. + T helper cells develop into either Th1 (pro-inflammatory) or Th2 (anti-inflammatory) subtypes. Each subtype orchestrates a characteristic immune response profile (each is mutually inhibitory). In the presence of transforming growth factor beta (TGF beta) and IL-6, naive CD4 + T helper cells can differentiate into Th17 cells, a subset of Th cells that secrete IL-17, which are further stimulated by IL-23.

[0014] The ex vivo generation of APCs with appropriate tolerogenic functions can be implemented as a therapeutic treatment for anti-drug responses, autoimmune diseases, and for the induction of transplantation tolerance. Efficient suppression of harmful immune responses is achieved by CD4 + and CD8 + This includes tolerance induction in both T cells and immune cells. Therefore, it can be expected that ex vivo generated tolerogenic APCs should have the same properties for in vivo anti-drug responses, treatment of autoimmune diseases, and prevention of graft rejection.

[0015] Retinoic acid, a vitamin A metabolite, plays an important role in cell proliferation, differentiation, organogenesis, and regeneration, and plays an important role in mucosal immune responses. Retinoic acid mediates the upregulation of Foxp3. + It has been reported that retinoic acid enhances the differentiation of inducible and IL-10-producing Treg cells and induces gut-homing specificity in T cells (Bakdash et al., 2015). In addition, due to its regulatory activity, retinoic acid has been reported to play an important role in controlling inflammatory diseases not only in the gut but also in other tissues (Oliveira et al., 2018).

[0016] Transforming growth factor beta (TGF-beta) is a pleiotropic cytokine present in vertebrate and invertebrate organisms, functioning in numerous physiological and pathological processes. TGF-beta affects all cells of the immune system, and of the three known TGF-beta isoforms, TGF-beta1 is the predominant isoform expressed in immune cells. TGF-beta1 is known to play a pivotal role in regulating all immune cell functions, particularly T cell development and in inducing immune tolerance in DCs. DCs regulate immune functions, including immunosuppression, by secreting TGF-beta (Esebanmen et al., 2017).

[0017] The aryl hydrocarbon receptor (AhR) is a transcription factor activated by several exogenous and endogenous ligands. Among its physiological effects, AhR contributes to immune homeostasis by promoting immunomodulatory actions. Activation of AhR via agonist ligands such as 6-formylindolo(3,2b)carbazole (FICZ) during DC differentiation and maturation has been shown to result in increased expression of the enzyme IDO and reduced production of proinflammatory cytokines such as IL-6 and TNF-alpha. One study showed that FICZ-treated DCs reduced the CD4 expression of naive T cells. + CD25 高 Foxp3 +They were able to induce differentiation into Treg cells, demonstrating that AhR activation in human DCs promotes a tolerogenic phenotype ( Jurado-Manzano et al., 2017 ).

[0018] There is a need to provide alternative methods for the generation of tolerogenic APCs with distinct tolerogenic phenotypes useful for treating unwanted immune responses to autoantigens in autoimmune diseases and biologics such as Factor VIII against alloantigens in transplanted grafts. Summary of the Invention [Problem to be solved by the invention]

[0019] (Summary of the Invention) The inventors of the present application have surprisingly identified a specific cell culture method involving the use of a specific combination of retinoic acid, TGF beta and an AhR agonist, which advantageously allows the generation of tolerogenic APCs with a unique profile derived from a starting cell population of monocytes. [Means for solving the problem]

[0020] The present inventors have discovered that tolerogenic APCs can be generated by a method involving culturing monocytes in cell cultures containing specific combinations of components.

[0021] Advantageously, it is expected that the tolerogenic APCs obtained by the methods of the present invention can be used for targeted immunotherapy in mammalian subjects having or at risk of having an unwanted immune response to an antigen. The antigen can be, for example, a biologic or an autoantigen. Furthermore, it is expected that the tolerogenic APCs obtained by the methods of the present invention can be used to prevent immune rejection of an allograft in a recipient subject.

[0022] Therefore, in a first aspect of the present invention, there is provided an ex vivo method for obtaining tolerogenic APCs capable of inducing tolerance to an antigen, the method comprising: (a) isolating monocytes from a sample obtained from a mammal; and (b) culturing the isolated monocytes in cell culture to induce differentiation of the monocytes into APCs with a tolerogenic phenotype; wherein the cell culture comprises (i) retinoic acid and TGF-beta, (ii) retinoic acid, TGF-beta and an AhR agonist, or (iii) retinoic acid and an AhR agonist.

[0023] In another aspect of the present invention, there is provided a tolerogenic APC or population thereof obtainable or obtained by the method of the present invention.

[0024] In yet another aspect of the present invention, there is provided a tolerogenic APC or population thereof, wherein the cell or population expresses CD103 in the unstimulated state or upon stimulation with an immunogenic stimulus such as LPS, and has high expression of CD141, GARP, and ILT3, and low expression of CD83 and CD86.

[0025] In a further aspect of the invention, there is provided a tolerogenic APC or population thereof according to the invention for use in a method of treating a mammalian subject having or at risk of having an immune response to an antigen, said method comprising administering to the mammalian subject the tolerogenic APCs, thereby establishing immune tolerance to the antigen.

[0026] In yet a further aspect of the invention there is provided a method of treating a mammalian subject having or at risk of having an immune response to an antigen, comprising: (i) obtaining a tolerogenic APC or a population thereof capable of inducing tolerance to an antigen according to the present invention, the APC or population being obtained from a sample of isolated monocytes from a mammalian subject; (ii) administering the tolerogenic APCs or population thereof back to the mammalian subject to establish immune tolerance to the antigen. The present invention provides a tolerogenic APC or population thereof for use in a method comprising:

[0027] In another aspect of the present invention, there is provided a tolerogenic APC or population thereof according to the present invention for use in a method for preventing immune rejection of a donor-derived allograft in a recipient subject, the method comprising administering to the recipient subject tolerogenic APCs obtained from monocytes isolated from a sample taken from the donor, thereby establishing tolerance to the allograft.

[0028] In yet another aspect of the present invention, there is provided a tolerogenic APC or population thereof according to the present invention for use in a method for preventing immune rejection of a donor-derived allograft in a recipient subject, the method comprising administering to the recipient subject tolerogenic APCs obtained from monocytes isolated from a sample taken from the recipient, thereby establishing tolerance to the allograft. [Brief explanation of the drawings]

[0029] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows the expression of the maturation marker CD83 on DCs cultured under various conditions (Figures (A) and (B)). DCs were either unstimulated (gray bars) or stimulated with lipopolysaccharide (LPS) (black bars) (see Example 1). [Figure 2] Figure 2 shows the expression of the activation marker CD86 on DCs cultured under various conditions (Fig. (A) and (B)). DCs were unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 2). [Figure 3] Figure 3 shows the expression of the tolerogenic marker ILT3 on DCs cultured under various conditions (Figures (A) and (B)). DCs were unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 3). [Figure 4] Figure 4 shows the tolerogenic index, defined as the ratio of ILT3 / CD86 expression (MFI) on DCs cultured under various conditions: DCs were unstimulated (Figure (A)) or stimulated with LPS (Figure (B)) (see Example 4). [Figure 5]Figure 5 shows the ability of DCs to stimulate CD4+ T cell proliferation in a mixed lymphocyte reaction (MLR). DCs were either unstimulated (gray bars) or stimulated with either LPS (Figure (A)) or a proinflammatory cytokine cocktail (Figure (B)) before co-culture with T cells (black bars) (see Example 5). [Figure 6] Figure 6 shows the frequency of CD141 and GARP co-expressing cells among DCs cultured under various conditions (Figures (A) and (B)). DCs were unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 6). [Figure 7] Figure 7 shows the production of IL-23 by DCs cultured under various conditions (Figures (A) and (B)). DCs were unstimulated (gray bars) or stimulated (black bars) with either LPS (Figure (A)) or a proinflammatory cytokine cocktail (Figure (B)) (see Example 7). [Figure 8] Figure 8 shows the expression of the marker CD103 on DCs cultured under various conditions (Figures (A) and (B)). DCs were either unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 8). [Figure 9] Figure 9 shows the expression levels of the tolerogenic markers MERTK (Panel A), BTLA (Panel B), LAP (Panel C), HLA-G (Panel D), and CD49b (Panel E) on DCs cultured under various conditions (see Example 9). [Figure 10] Figure 10 shows the ability of DCs to induce CD4+CD25highFoxp3+ Tregs in an extended MLR (Figures (A) and (B)). DCs were unstimulated (gray bars) or stimulated with LPS (black bars) before co-culture with T cells (see Example 10). [Figure 11]Figure 11 shows the expression of maturation marker CD83 (Figure A), activation marker CD86 (Figure B), tolerogenic marker ILT3 (Figure C), tolerogenic index defined as the ILT3 / CD86 expression (MFI) ratio (Figure D), frequency of CD141 and GARP co-expressing cells (Figure E), expression of the marker CD103 (Figure F), expression level of the tolerogenic marker LAP (Figure G), IL-23 production (Figure H), T cell proliferation (Figure I), and induction of CD4+CD49b+LAG3+Tr1 cells (Figure J) when DCs were generated from CD14+ monocytes isolated from the blood of healthy donors (black bars) or hemophilia patients (striped bars) and cultured in the presence of RA, TGFb, and AhR agonist (see Example 11). [Figure 12] 12 shows the viability (Fig. A), expression of the maturation marker CD83 (Fig. B), expression of the tolerogenic marker ILT3 (Fig. C), expression of the activation marker CD86 (Fig. D), the tolerogenic index defined as the ratio of ILT3 / CD86 expression (MFI) (Fig. E), expression level of the tolerogenic marker LAP (Fig. F), and expression of the marker CD103 (Fig. G) when DCs were cultured in the presence of RA, TGFb, and an AhR agonist and then frozen / thawed (dark gray bars). The phenotype of the frozen / thawed cells was compared to that of fresh cells (light gray bars) (see Example 12). [Figure 13] Figure 13 shows the expression of maturation marker CD83 (Fig. A), activation marker CD86 (Fig. B), tolerogenic marker ILT3 (Fig. C), tolerogenic index defined as the ILT3 / CD86 expression (MFI) ratio (Fig. D), expression of marker CD103 (Fig. E), frequency of CD141 and GARP co-expressing cells (Fig. F), T cell proliferation (Fig. G), and generation of CD4+CD25highFoxp3+ Treg cells (Fig. H) when DCs were cultured under various conditions. DCs were unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 13). [Figure 14] Figure 14 shows T cell proliferation when DCs cultured in the presence of RA + TGFb + AhR agonist were loaded with tetanus toxoid and subsequently cultured in the presence of T cells. DCs were either unloaded (black bars) or loaded with TT (gray bars) (see Example 14). [Figure 15] Figure 15 shows the expression of maturation marker CD83 (Figure A), the expression level of the tolerogenic marker LAP (Figure B), the expression of the marker CD103 (Figure C), the expression of the tolerogenic marker ILT3 (Figure D), the expression of the activation marker CD86 (Figure E), and the tolerogenic index (defined as the ILT3 / CD86 expression (MFI) ratio) of DCs cultured in the presence of RA, TGFb, and AhR agonist and subsequently stimulated with CD40L (black bars) or not (gray bars). Figure G shows the induction of T cell proliferation by unstimulated and CD40L-stimulated RA, TGFb, and AhR agonist-treated DCs (see Example 15). [Figure 16] Figure 16 shows antigen loading of DCs cultured in the presence of RA + TGFb + AhR agonist and antigen (Figures A-B), expression of CD11c after antigen loading (Figure C), expression of maturation marker CD83 after antigen loading (Figure D), expression of activation marker CD86 after antigen loading (Figure E), tolerogenic marker ILT3 after antigen loading (Figure F), expression of marker CD103 after antigen loading (Figure G), expression of tolerogenic marker GARP after antigen loading (Figure H), and expression level of tolerogenic marker LAP after antigen loading (Figure I) (see Example 16). [Figure 17] Figure 17 shows the induction of B regulatory cells in cultures containing RA+TGF beta+AhR agonist-treated DCs, B cells, and T cells (Figure A), the ability of RA+TGF beta+AhR agonist-treated DCs, B cells, and T cells to stimulate T cell proliferation (Figure B), and the ability of RA+TGF beta+AhR agonist-treated DCs, B cells, and T cells to induce T cell activation (Figure C). [Figure 18] Figure 18 shows the ability of RA+TGFbeta+AhR agonist-treated DCs to induce T cell proliferation when co-cultured with allogeneic PBMCs (Figure A) and the ability of RA+TGFbeta+AhR agonist-treated DCs to induce Tregs in a mixed lymphocyte reaction (MLR) with allogeneic PBMCs (Figure B). DETAILED DESCRIPTION OF THE INVENTION

[0030] (Detailed Description of the Invention) It is understood that different applications of the disclosed products and methods can be tailored to specific needs in the art. It is also understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. Additionally, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to an "antigen" includes two or more such antigens.

[0031] definition "Monocytes" are large mononuclear phagocytes of peripheral blood. Monocytes typically range in size from 10 to 30 μm in diameter. The nucleus-to-cytoplasm ratio typically ranges from 2:1 to 1:1. The nucleus is often band-shaped (horseshoe-shaped) or kidney-shaped (kidney-shaped). It folds on itself, thus exhibiting brain-like convolutions. Nucleoli are not visible. The chromatin pattern is fine, arranged in strands resembling chromosome threads. The cytoplasm is abundant and appears blue-gray with many fine azurophilic granules, giving it a ground-glass appearance with Giemsa staining. Vacuoles may be present. Expression of specific surface markers is often used to determine whether a cell is monocytic. For example, monocytes express CD14 (a monocyte marker) and do not express CD1c (a DC marker), CD56 (an NK cell marker), CD19 (a B cell marker), CD3 (a T cell marker), CD16b (a neutrophil marker), or CD66b (a neutrophil marker).

[0032] "Antigen-presenting cells" (APCs) are part of a heterogeneous group of immune cells that mediate cellular immune responses by processing and presenting antigens for recognition by specific lymphocytes, such as T cells. APCs present antigens complexed with MHC on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). Classical APCs include DCs, macrophages, Langerhans cells, and B cells.

[0033] "Dendritic cells" (DCs) are APCs that exist in vivo, in vitro, ex vivo, or can be derived from hematopoietic stem cells, hematopoietic progenitor cells, or monocytes. DCs and their precursors can be isolated from various lymphoid organs, e.g., the spleen, lymph nodes, as well as from bone marrow and peripheral blood. DCs have a characteristic morphology with thin sheets (lamellipodia) extending in multiple directions away from the DC body. DCs constitutively express both MHC class I and class II molecules, each expressing CD8. + and CD4 + They present peptide antigens to T cells. In addition, human skin and mucosal DCs also express CD1 gene family MHC class I-related molecules that present microbial lipid or glycolipid antigens. DC membranes are also rich in molecules that enable T cell adhesion (e.g., intercellular adhesion molecule 1 or CD54) or costimulate T cell activation, such as B7-1 and B7-2 (also known as CD80 and CD86, respectively).

[0034] "Tolerance" is the result of a tolerogenic mechanism of immune response to a particular antigen, resulting directly or indirectly in the prevention, silencing, downregulation, or down-regulation of adaptive immune responses to one or more antigens. A substance capable of inducing tolerance, i.e., a tolerogenic substance, is one that modulates APCs, e.g., DCs, to become tolerogenic, and the resulting tolerogenic DCs are capable of mediating tolerance induction.

[0035] "Tolerogenicity" refers to the ability of a cell or substance to induce tolerance mediated through mechanisms such as loss of antigen-specific T cell responses via induction of Tregs, induction of unresponsiveness or anergy, and active suppression. "Tolerization" refers to tolerance induced in the adaptive immune system to an antigen, and means that tolerogenic mechanisms are mediated to induce immunosuppressive effects.

[0036] "Immunogenic" means a cell or substance that is capable of directly or indirectly activating the adaptive immune response to an antigen.

[0037] "Tolerogenic APC" refers to APCs that have acquired the ability to induce tolerance through exposure to tolerogenic stimuli, which can be combinations of microbial origin, mammalian cell components, cytokines, hormones, vitamins, and other biological or pharmaceutical agents. Tolerogenic APCs have a reduced ability to induce immunogenic responses, but an increased ability to induce activated Treg and other tolerogenic responses.

[0038] "Tolerogenic DC" refers to DCs that have acquired the ability to induce tolerance upon exposure to tolerogenic stimuli, which can be combinations of microbial origin, mammalian cell components, cytokines, hormones, vitamins, and other biological or pharmaceutical agents. Tolerogenic DCs have a low ability to induce immunogenic responses but a high ability to induce activated Treg and other tolerogenic responses.

[0039] "Autoimmune disease" means a pathological condition in which the adaptive immune system is directed against self-antigens in a destructive manner.

[0040] "Self-antigen" (or "autoantigen") refers to any molecule or chemical group in a mammal (e.g., a human) that acts as an antigen in the induction of an unwanted immune response (e.g., an antibody or effector T cell response) in a disease state, but that is tolerated by the mammal's healthy immune system. The terms "self-antigen" and "autoantigen" should be considered synonymous and are used interchangeably herein.

[0041] By "stimulus" is meant exposure to any artificial or natural compound that results in cell signaling.

[0042] method The present invention provides a method for obtaining tolerogenic APCs capable of tolerizing the immune system to an antigen, the method comprising culturing isolated monocytes under specific culture conditions. Monocytes can be isolated from a variety of sources. APCs are obtained according to the present invention. APCs obtained according to the present invention include DCs and macrophages, preferably DCs. DCs obtained by the method of the present invention may be myeloid DCs. The method is typically performed under ex vivo or in vitro conditions, preferably under ex vivo conditions. The isolated monocytes are typically present in a sample collected from a mammal. The mammal from which the sample is collected is typically a human (Homo sapiens). The sample is typically a blood sample, more preferably a sample of peripheral blood mononuclear cells (PBMCs) prepared from blood. The sample typically contains mononuclear cells, preferably monocytic cells, and particularly monocytes. The method typically comprises isolating a population of monocytes from a sample, such as a PBMC sample. Preferably, the isolated cells are CD14 + The method further comprises culturing the isolated monocytic cells in a cell culture.

[0043] In one embodiment, the cell culture of the method of the invention comprises retinoic acid and TGF-beta. In another embodiment of the invention, the cell culture of the method of the invention comprises retinoic acid, TGF-beta, and an AhR agonist. In a further embodiment of the invention, the cell culture of the method of the invention comprises retinoic acid and an AhR agonist. The cell culture preferably comprises retinoic acid and TGF-beta, and even more preferably comprises retinoic acid, TGF-beta, and an AhR agonist.

[0044] Retinoic acid is a metabolite of vitamin A1. The cell culture contains retinoic acid. Retinoic acid can be present in any form in the cell culture, such as all-trans-retinoic acid, 13-cis-retinoic acid, and / or 9-cis-retinoic acid, particularly all-trans-retinoic acid. Retinoic acid can be provided as a retinoic acid precursor, such as retinol and / or a variant form of vitamin A that generates retinoic acid in situ, such as retinal.

[0045] The AhR agonist used in the cell culture of the present invention can be any AhR agonist, for example, any of the AhR agonists disclosed in WO2012 / 050500, the contents of which are incorporated by reference in their entirety. The AhR agonist can be a full agonist or a partial agonist. The AhR agonist used in the methods of the present invention is preferably N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxoquinoline-3-carboxamide (IMA-06201) (Mahiout et al., 2017), herein referred to as "C1." The AhR agonist may alternatively be 6-formylindolo[3,2-b]carbazole (FICZ), 2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE), N-acetyl-N-phenyl-4-acetoxy-5-chloro-1,2-dihydro-1-methyl-2-oxoquinoline-3-carboxamide ("C2"), N-(4-trifluoromethylphenyl)-1,2-dihydro-4-hydroxy-5-methoxy-1-methyl-2-oxoquinoline-3-carboxamide ("C3"), or N-acetyl-N-(4-trifluoromethylphenyl)-4-acetoxy-1,2-dihydro-5-methoxy-1-methyl-2-oxoquinoline-3-carboxamide ("C4"). Other potentially useful AhR agonists are described in Denison and Nagy et al., Ann. Rev. Pharmacol. Toxicol., 43:309-34, 2003, and the references cited therein, all of which are incorporated by reference in their entireties.

[0046] The TGF beta used in the cell culture of the present invention can be any TGF beta isoform, such as TGF beta 1, TGF beta 2 or TGF beta 3, preferably TGF beta 1. Any TGF beta isoform can be used alone or in combination with any other TGF beta isoform.

[0047] The cell culture typically contains GM-CSF and IL-4. Alternatively, the cell culture may contain GM-CSF alone, for example, GM-CSF without IL-4, or M-CSF. The cell culture preferably contains GM-CSF and IL-4. GM-CSF and IL-4 are added to the cell culture to induce differentiation of isolated monocytes into APCs, preferably DCs. M-CSF is added to the cell culture to induce differentiation of isolated monocytes into APCs, preferably macrophages.

[0048] Typically, GM-CSF and IL-4 or GM-CSF or M-CSF are added to the cell culture before or at the same time as any one of TGF-beta, AhR agonist, and retinoic acid is added to the cell culture. Preferably, GM-CSF and IL-4 are added to the cell culture before or at the same time as any one of TGF-beta, AhR agonist, and retinoic acid is added to the cell culture. Typically, the AhR agonist is added to the cell culture at a first dose at the same time as GM-CSF or M-CSF and IL-4 are first added to the cell culture. Typically, the second dose of TGF-beta and the AhR agonist is added to the cell culture after the first dose of the AhR agonist is added to the cell culture. More typically, retinoic acid is added to the cell culture after the second dose of TGF-beta and the AhR agonist is added to the cell culture. Typically, GM-CSF or M-CSF and IL-4 are added to the cell culture at a second dose. Preferably, the second dose of GM-CSF or M-CSF and IL-4 is added to the cell culture at the same time that the second doses of TGF-beta and AhR agonist are added to the cell culture.

[0049] GM-CSF or M-CSF may be added to the cell culture before or at the same time as any one of TGF-beta, AhR agonist, and retinoic acid is added to the cell culture, and IL-4 is added to the cell culture after the first dose of AhR agonist is added to the cell culture. Typically, the second dose of TGF-beta and AhR agonist is added to the cell culture after the first dose of AhR agonist is added to the cell culture. More typically, retinoic acid is added to the cell culture after the second dose of TGF-beta and AhR agonist is added to the cell culture. GM-CSF or M-CSF may be added to the cell culture at the second dose. Preferably, the second dose of GM-CSF or M-CSF is added to the cell culture at the same time as the second dose of TGF-beta AhR agonist is added to the cell culture.

[0050] The cell culture may further comprise an antigen or an epitope-containing fragment of the antigen. The antigen or epitope-containing fragment thereof may be bound to or expressed on isolated monocytes in the culture. The antigen or epitope-containing fragment thereof may be expressed on the cell surface of monocytes or APCs. The antigen or epitope-containing fragment thereof may be intracellular. The antigen or epitope-containing fragment thereof may be an antigen or epitope-containing fragment not native to monocytes, for example, monocytes may take up the antigen or epitope-containing fragment thereof from other cells before being isolated. The monocytes or APCs may be transfected with antigen-encoding mRNA to express the antigen or epitope-containing fragment thereof. Methods for transfecting cells with antigen-encoding mRNA are known in the art.

[0051] In the case of methods for inducing tolerance to an allograft, the antigen or epitope-containing fragment thereof can be bound to or expressed on monocytes isolated from a sample taken from a transplant donor, or expressed by APCs during or after culture using the methods of the invention. Monocytes isolated from a sample taken from a donor cultured using the methods of the invention allow tolerogenic APCs to be produced. Such tolerogenic APCs can be administered to a recipient to prevent immune rejection of the allograft from the donor.

[0052] In the case of monocytes isolated from a sample collected from a recipient, the monocytes may be cultured using the methods of the present invention to produce tolerogenic APCs. The tolerogenic APCs may be loaded with donor antigens so as to present donor-derived antigens. Alternatively, the tolerogenic APCs may not be loaded with donor antigens ex vivo. Such tolerogenic APCs, which may or may not present donor-derived antigens, can be re-administered to the recipient to prevent immune rejection of the allograft from the donor.

[0053] Alternatively, the antigen or epitope-containing fragment thereof is a component added to the cell culture. Thus, the cell culture further comprises the antigen or epitope-containing fragment thereof. Preferably, the antigen or epitope-containing fragment is added to the cell culture before, simultaneously with, or after retinoic acid is added to the cell culture. The antigen or epitope-containing fragment thereof may be added as part of a mixture of antigens; for example, cells, tissues, or samples containing one or more antigens may be added to the culture.

[0054] The antigen or epitope-containing fragment can be a single antigen or epitope-containing fragment thereof (e.g., in purified form), or a pool or multiple antigens and / or multiple epitope-containing fragments thereof. The antigen or epitope-containing fragment can be a cell, blood, tissue sample, or extract thereof. In the case of a method for inducing tolerance to an allograft, the antigen or epitope-containing fragment thereof can be a sample of the graft, or can be derived from a sample of the graft or a tissue, cell, or blood sample from another donor, and the antigen or epitope-containing fragment thereof can be added directly to the cell culture.

[0055] The antigen or epitope-containing fragment thereof may optionally not be added directly to the cell culture. Instead, the antigen or epitope-containing fragment thereof may be present in vivo. Thus, the tolerogenic APC may optionally not be loaded with the antigen or epitope-containing fragment thereof ex vivo, but instead have the ability to be loaded with the antigen or epitope-containing fragment thereof in vivo when the tolerogenic APC is administered. Thus, the tolerogenic APC recognizes and processes the antigen or epitope-containing fragment thereof in vivo.

[0056] The antigen or epitope-containing fragment thereof may be or be derived from a biologic, and thus the biologic may be used as all or part of the antigen (i.e., the part that contains the epitope).

[0057] The antigen or epitope-containing fragment thereof may be or be derived from an autoantigen.

[0058] Generation of regulatory T cells The method of the present invention may further comprise ex vivo culturing the obtained tolerogenic APCs with T cells, thereby inducing the generation of Tregs. Therefore, ex vivo culturing of the tolerogenic APCs of the present invention with T cells induces Tregs. The induced Tregs may be any Tregs, for example, CD4 + CD25 + Foxp3 +Treg and / or type 1 regulatory T (Tr1) CD4 + CD49b + LAG3 + cells, preferably CD4 + CD25 + Foxp3 + They may be Tregs. Tregs or populations thereof may be obtainable or obtained by the methods of the present invention.

[0059] Tregs or populations thereof may be used in methods of treating a mammalian subject, or a mammalian subject having or at risk of having an immune response to an antigen.

[0060] As shown in Figures 5(A) and 5(B), the tolerogenic DCs of the present invention exhibit low T cell proliferation induction capacity when unstimulated or when stimulated with immunogenic stimuli such as LPS or an inflammatory cytokine cocktail. The combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the combination of retinoic acid and TGF-beta and the combination of retinoic acid and an AhR agonist in reducing the T cell proliferation induction capacity.

[0061] Therefore, suitable tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated have a T cell proliferation induction ability that is 100% or less, for example, 90% or less, for example, 80% or less, for example, 70% or less of that of control cells when unstimulated. Furthermore, suitable tolerogenic APCs of the present invention (e.g., DCs of the present invention) when stimulated with an immunogenic stimulus such as, for example, LPS or an inflammatory cytokine cocktail, have a T cell proliferation induction ability that is 70% or less, for example, 60% or less, for example, 50% or less of that of control cells when stimulated with an immunogenic stimulus such as, for example, LPS. The control cells are APCs of the same type from the same subject that have not been exposed to a tolerogenic compound.

[0062] As shown in Figures 10(A) and 10(B), the tolerogenic DCs of the present invention have high Treg induction potential. The combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the combination of retinoic acid and TGF-beta or the combination of retinoic acid and an AhR agonist in inducing Tregs.

[0063] Therefore, suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated have a Treg induction capacity that is at least 130%, such as at least 150%, for example at least 175%, for example at least 200% of that of unstimulated control cells. Furthermore, suitable tolerogenic APCs of the present invention (e.g., DCs of the present invention) when stimulated with an immunogenic stimulus, such as LPS, suitably have a Treg induction capacity that is at least 130%, for example at least 150%, for example at least 200% of that of control cells when stimulated with an immunogenic stimulus, such as LPS. The control cells are APCs of the same type from the same subject that have not been exposed to a tolerogenic compound.

[0064] As shown in FIG. 11(J), the tolerogenic DCs of the present invention have high Treg induction ability when CD14+ monocytes are isolated from the blood of hemophilia patients.

[0065] Therefore, suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated have a Treg induction potential that is at least 130%, for example, at least 150%, for example, at least 175%, for example, at least 200% of that of unstimulated control cells. Furthermore, suitable tolerogenic APCs of the present invention (e.g., DCs of the present invention) when stimulated with an immunogenic stimulus such as, for example, LPS, suitably have a Treg induction potential that is at least 130%, for example, at least 150%, for example, at least 200% of that of control cells when stimulated with an immunogenic stimulus such as, for example, LPS. The control cells are APCs of the same type from the same subject that have not been exposed to a tolerogenic compound.

[0066] Tolerogenic antigen-presenting cells Monocytes differentiate into DCs. During differentiation, the proportion of DCs can be polarized toward a tolerogenic phenotype. The method of the present invention typically allows for the generation of tolerogenic APCs, such as tolerogenic DCs, that have tolerogenic cell surface marker expression.

[0067] CD83 is an integral membrane protein and an activation / maturation marker. Typically, unstimulated APCs, particularly DCs, show CD83 expression levels that do not differ significantly between tolerogenic and non-tolerogenic cells. However, upon stimulation with an immunogenic stimulus, such as LPS, APCs, particularly DCs, may show increased CD83 expression levels. Therefore, CD83 expression levels typically increase to a greater extent in stimulated non-tolerogenic cells than in stimulated tolerogenic cells. Desirable tolerogenic APCs, particularly DCs, have low CD83 expression levels even after stimulation. Desirable tolerogenic APCs, particularly DCs, are as resistant as possible to immunogenic stimuli, such as LPS stimulation, in terms of upregulation of CD83 expression.

[0068] Tolerogenic APCs of the invention (e.g., DCs of the invention) have the advantage, at least in some embodiments, that upon stimulation with an immunogenic stimulus, such as LPS, they express less CD83 than correspondingly stimulated control cells, which are APCs of the same type from the same subject that have not been exposed to the tolerogenic compound.

[0069] Suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated have an expression level of CD83 that is 100% or less, such as 90% or less, for example 80% or less, for example 70% or less, for example 60% or less, for example 50% or less, of that of unstimulated control cells.

[0070] Suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) upon stimulation with an immunogenic stimulus, such as, for example, LPS, have an expression level of CD83 that is 70% or less, e.g., 60% or less, e.g., 50% or less, of that of control cells upon stimulation with an immunogenic stimulus, such as, for example, LPS. As described in more detail in the Examples section and shown in Figure 1, when the tolerogenic compounds used were retinoic acid, TGF-beta, and an AhR agonist, the greatest reduction in the expression level of CD83 in stimulated APCs compared to corresponding control cells was obtained when the APCs of the present invention were produced by the method of the present invention. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta, which in turn was superior to the use of retinoic acid and an AhR agonist.

[0071] The tolerogenic APCs of the invention (eg, DCs of the invention) also have the advantage, at least in some embodiments, of having CD83 expression levels that are resistant to further upregulation upon stimulation.

[0072] Suitably, tolerogenic APCs of the present invention (e.g., DCs of the present invention) upon stimulation with an immunogenic stimulus such as LPS have a CD83 expression level that is 200% or less, e.g., 175% or less, e.g., 150% or less, e.g., 125% or less, of that of unstimulated corresponding cells. As described in more detail in the Examples section and shown in Figure 1, when the tolerogenic compounds used are retinoic acid, TGF-beta, and an AhR agonist, the greatest reduction in CD83 expression level in stimulated APCs compared to corresponding unstimulated cells was obtained when the APCs of the present invention were produced by the method of the present invention. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta, which in turn was superior to the use of retinoic acid and an AhR agonist.

[0073] CD86 is an integral membrane protein and an activation / maturation marker and costimulatory molecule. Typically, unstimulated APCs, particularly DCs, exhibit CD86 expression levels that do not differ significantly between tolerogenic and non-tolerogenic cells. However, upon stimulation with an immunogenic stimulus, such as LPS, APCs, particularly DCs, may exhibit elevated CD86 expression levels. CD86 expression levels typically increase to a greater extent in non-tolerogenic cells than in tolerogenic cells. Desirable tolerogenic APCs, particularly DCs, express a low level of CD86 upon stimulation. Desirable tolerogenic APCs, particularly DCs, are as resistant as possible to immunogenic stimuli, such as LPS, in terms of upregulation of CD86 expression.

[0074] The tolerogenic APCs of the invention (e.g., DCs of the invention) have the advantage, at least in some embodiments, of lower expression of CD86 upon stimulation with an immunogenic stimulus, e.g., LPS, compared to control cells upon stimulation, which are APCs of the same type from the same subject that have not been exposed to the tolerogenic compound.

[0075] Suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated have a CD86 expression level that is 100% or less, for example, 90% or less, for example, 80% or less, for example, 70% or less, for example, 60% or less, for example, 50% or less, of that of unstimulated control cells.

[0076] Suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) upon stimulation with an immunogenic stimulus, such as, for example, LPS, have a CD86 expression level that is 80% or less, for example, 70% or less, for example, 60% or less, for example, 50% or less, of that of control cells upon stimulation with an immunogenic stimulus, such as, for example, LPS. As described in more detail in the Examples section and shown in Figure 2, when the tolerogenic compounds used are retinoic acid, TGF-beta, and an AhR agonist, the greatest reduction in CD83 expression level in stimulated APCs was obtained when the APCs of the present invention were produced by the method of the present invention, compared to corresponding control cells upon stimulation. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta, which in turn was superior to the use of retinoic acid and an AhR agonist.

[0077] The tolerogenic state of DCs is characterized by low costimulatory potential and high expression of inhibitory molecules. ILT3 is an inhibitory cell surface receptor that can be expressed by tolerogenic APCs, preferably DCs. DCs that overexpress ILT3 exhibit lower phosphorylation levels of NF-kappaB and are unable to stimulate the full program of Th proliferation and maturation instead of Treg cell differentiation (Vlad et al., 2009). ILT3 expression and upregulation are typically independent of immunogenic stimuli such as LPS. Typically, non-tolerogenic and tolerogenic APCs, particularly DCs, exhibit ILT3 expression levels that do not differ significantly between unstimulated and stimulated conditions (e.g., with an immunogenic stimulus such as LPS). The level of ILT3 expression is typically much higher in tolerogenic cells than in non-tolerogenic cells. Desirable tolerogenic APCs, particularly DCs, have high levels of ILT3 expression that are not downregulated upon stimulation with immunogenic stimuli, such as LPS.

[0078] The tolerogenic APCs of the invention (e.g., DCs of the invention) have the advantage, at least in some embodiments, of having higher expression of ILT3 when unstimulated or stimulated with an immunogenic stimulus, e.g., LPS, compared to unstimulated or stimulated control cells, respectively, which are antigen-presenting cells of the same type from the same subject that have not been exposed to the tolerogenic compound.

[0079] Suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated and stimulated with an immunogenic stimulus, e.g., LPS, have an ILT3 expression level that is at least 150%, for example, at least 175%, for example, at least 200%, for example, at least 250%, for example, at least 300% of that of control cells when unstimulated and stimulated with an immunogenic stimulus, e.g., LPS, respectively. As described in more detail in the Examples section and shown in Figure 3, when the tolerogenic compounds used are retinoic acid, TGF-beta, and an AhR agonist, the greatest increase in ILT3 expression levels in unstimulated and stimulated APCs compared to control cells was obtained when the APCs of the present invention were produced by the method of the present invention. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta, which in turn was superior to the use of retinoic acid and an AhR agonist.

[0080] The tolerogenic potential of APCs, particularly DCs, can be determined by the ratio of ILT3 to CD86 expression levels. The ILT3 / CD86 ratio is typically much higher in tolerogenic cells than in non-tolerogenic cells. Desirable tolerogenic APCs, particularly DCs, have a high ILT3 / CD86 ratio whether unstimulated or stimulated with an immunogenic stimulus, such as LPS.

[0081] Tolerogenic APCs of the invention (e.g., DCs of the invention) unstimulated or stimulated with an immunogenic stimulus, e.g., LPS, have the advantage, at least in some embodiments, of having a higher ILT3 / CD86 ratio compared to control cells unstimulated or stimulated with an immunogenic stimulus, e.g., LPS, respectively, where the control cells are APCs of the same type from the same subject that have not been exposed to the tolerogenic compound.

[0082] Suitably, unstimulated APCs of the present invention (e.g., DCs of the present invention) have an ILT3 / CD86 ratio that is at least 150%, such as at least 200%, for example at least 250%, for example at least 300%, for example at least 350%, for example at least 400%, for example at least 500% of that of unstimulated control cells. As described in more detail in the Examples section and shown in Figure 4, the greatest increase in the ILT3 / CD86 ratio in unstimulated APCs compared to control cells was obtained when the tolerogenic compounds used were retinoic acid, TGF-beta, and an AhR agonist when the APCs of the present invention were produced by the method of the present invention. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta, which in turn was superior to the use of retinoic acid and an AhR agonist.

[0083] Suitably, the APCs of the present invention (e.g., DCs of the present invention) upon stimulation with an immunogenic stimulus, such as, for example, LPS, have an ILT3 / CD86 ratio that is at least 150%, such as at least 200%, for example at least 250%, such as at least 300%, for example at least 350%, such as at least 400%, for example at least 500% of that of control cells upon stimulation with an immunogenic stimulus, such as, for example, LPS. As described in more detail in the Examples section and shown in Figure 4, the greatest increase in the ILT3 / CD86 ratio in stimulated APCs compared to stimulated control cells was obtained when the APCs of the present invention were produced by the method of the present invention when the tolerogenic compounds used were retinoic acid, TGF-beta and an AhR agonist. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta, which in turn was superior to the use of retinoic acid and an AhR agonist.

[0084] When coexpressed, CD141 and GARP are markers of tolerogenic APCs. Tolerogenic APCs that coexpress CD141 and GARP have been shown to be associated with enhanced Treg induction capacity (Agrawal et al., 2016). CD141 and GARP can be coexpressed on the cell surface of both naive and stimulated APCs, particularly DCs. Coexpression and upregulation of CD141 and GARP on tolerogenic APCs may not depend on immunogenic stimuli, such as LPS. The frequency of CD141 and GARP coexpressing cells is typically much higher among tolerogenic cells than among non-tolerogenic cells. Desirable tolerogenic APCs, particularly DCs, have a high frequency of CD141 and GARP coexpressing cells when naive or stimulated with immunogenic stimuli, such as LPS.

[0085] Tolerogenic APCs of the invention (e.g., DCs of the invention), unstimulated or stimulated with an immunogenic stimulus such as, for example, LPS, have the advantage, at least in some embodiments, of having higher co-expression of CD141 and GARP compared to control cells, unstimulated or stimulated, respectively, which are APCs of the same type from the same subject that have not been exposed to the tolerogenic compound.

[0086] Suitably, unstimulated APCs of the present invention (e.g., DCs of the present invention) comprise a high frequency of CD141 and GARP co-expressing cells that is at least 400%, for example, at least 450%, for example, at least 500%, for example, at least 600%, for example, at least 700%, for example, at least 800%, for example, at least 900%, for example, at least 1000% of that of unstimulated control cells. As described in more detail in the Examples section and shown in Figure 6, when the tolerogenic compounds used are retinoic acid, TGF-beta, and an AhR agonist, the greatest increase in the frequency of CD141 and GARP co-expressing cells was obtained among unstimulated APCs when the APCs of the present invention were produced by the method of the present invention, compared to unstimulated control cells. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and an AhR agonist, which in turn was superior to the use of retinoic acid and TGF-beta.The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta.

[0087] Suitably, the APCs of the present invention (e.g., DCs of the present invention) upon stimulation with an immunogenic stimulus such as LPS have a frequency of CD141 and GARP co-expressing cells that is at least 200%, e.g., at least 250%, e.g., at least 300%, at least 400%, e.g., at least 450%, e.g., at least 500% of that of control cells upon stimulation. As described in more detail in the Examples section and shown in Figure 6, when the APCs of the present invention are produced by the method of the present invention, the greatest increase in the frequency of CD141 and GARP co-expressing cells among stimulated APCs compared to stimulated control cells was obtained when the tolerogenic compounds used were retinoic acid, TGF-beta, and an AhR agonist. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta.

[0088] IL-23 is a proinflammatory cytokine released by APCs, particularly DCs, during inflammation. This cytokine, produced by DCs and macrophages, promotes host protection against mucosal pathogens through the induction of IL-17 and related cytokines by lymphocytes. IL-23 is generally only released by APCs, particularly DCs, upon cell activation. Typically, unstimulated APCs, particularly DCs, produce IL-23 at levels that do not differ significantly between tolerogenic and non-tolerogenic cells. However, upon stimulation with immunogenic stimuli, such as LPS or a proinflammatory cytokine cocktail, APCs, particularly DCs, can exhibit increased levels of IL-23. IL-23 production levels typically increase much more in non-tolerogenic cells than in tolerogenic cells. Desirable tolerogenic APCs, particularly DCs, produce low levels of IL-23 upon stimulation with immunogenic stimuli, such as LPS or a proinflammatory cytokine cocktail. Desirable tolerogenic APCs, particularly DCs, are as resistant as possible to immunogenic stimuli, such as stimulation with LPS or inflammatory cytokine cocktails, with respect to IL-23 production.

[0089] The tolerogenic APCs of the present invention (e.g., DCs of the present invention) have the advantage, at least in some embodiments, that they exhibit reduced production of IL-23 upon stimulation with an immunogenic stimulus, e.g., LPS or an inflammatory cytokine cocktail, compared to control cells upon stimulation with an immunogenic stimulus, e.g., LPS or an inflammatory cytokine cocktail. They also exhibit reduced production of IL-23 upon unstimulation. The control cells are APCs of the same type from the same subject that have not been exposed to the tolerogenic compound.

[0090] Suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated have an IL-23 production level that is 100% or less, such as 90% or less, for example 80% or less, for example 70% or less, for example 60% or less, for example 50% or less, of that of unstimulated control cells.

[0091] Suitably, the APCs of the present invention (e.g., DCs of the present invention) upon stimulation with an immunogenic stimulus, such as, for example, LPS or an inflammatory cytokine cocktail, have an IL-23 production level that is 50% or less, for example, 40% or less, for example, 30% or less, of that of control cells upon stimulation with an immunogenic stimulus, such as, for example, LPS or an inflammatory cytokine cocktail. As described in more detail in the Examples section and shown in Figure 7, when the tolerogenic compounds used were retinoic acid and an AhR agonist, retinoic acid and TGF-beta, and retinoic acid, TGF-beta, and AhR agonist, when the APCs of the present invention were produced by the method of the present invention, the level of IL-23 production in the stimulated APCs was reduced compared to the stimulated control cells.

[0092] The APCs of the present invention (e.g., DCs of the present invention) also have the advantage, at least in some embodiments, of having levels of IL-23 production that are more resistant than control cells to further upregulation of production following stimulation with an immunogenic stimulus, such as LPS or an inflammatory cytokine cocktail.

[0093] CD103 is a cell surface marker associated with intestinal tolerogenic DCs. CD103-expressing tolerogenic APCs induce Tregs and CD8 + It has been shown that tolerance can be maintained by protecting against tissue infection through cross-presentation of foreign antigens to T cells (Scott et al., 2011). CD103 expression can be upregulated on the cell surface of tolerogenic APCs, particularly DCs. CD103 expression and upregulation may not be dependent on immunogenic stimuli, such as LPS. Typically, non-tolerogenic and tolerogenic APCs, particularly DCs, exhibit CD103 expression levels that do not significantly differ between unstimulated and stimulated conditions (e.g., with an immunogenic stimulus such as LPS). CD103 expression levels are typically much greater in tolerogenic cells than in non-tolerogenic cells. Desirable tolerogenic APCs, particularly DCs, have high levels of CD103 expression upon unstimulated or stimulated with an immunogenic stimulus, such as LPS.

[0094] Tolerogenic APCs of the invention (e.g., DCs of the invention) unstimulated or stimulated with an immunogenic stimulus, e.g., LPS, have the advantage, at least in some embodiments, of having higher CD103 expression compared to control cells unstimulated or stimulated with an immunogenic stimulus, e.g., LPS, respectively, where the control cells are APCs of the same type from the same subject that have not been exposed to a tolerogenic compound.

[0095] Suitably, unstimulated APCs of the present invention (e.g., DCs of the present invention) have a CD103 expression level that is at least 150%, for example, at least 200%, for example, at least 250%, for example, at least 300% of that of unstimulated control cells. As described in more detail in the Examples section and shown in Figure 8, when the tolerogenic compounds used were retinoic acid, TGF-beta, and an AhR agonist, the greatest increase in CD103 expression level in unstimulated APCs was obtained when the APCs of the present invention were produced by the method of the present invention, compared to unstimulated control cells. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and an AhR agonist, which in turn was superior to the use of retinoic acid and a TGF-beta. The use of a combination of retinoic acid, TGF-beta and an AhR agonist was superior to the use of retinoic acid and TGF-beta alone.

[0096] Suitably, the APCs of the present invention (e.g., DCs of the present invention) upon stimulation with an immunogenic stimulus, such as, for example, LPS, have an expression level of CD103 that is at least 200%, for example, at least 250%, for example, at least 300%, of that of control cells upon stimulation with an immunogenic stimulus, such as, for example, LPS. As described in more detail in the Examples section and shown in Figure 8, when the tolerogenic compounds used are retinoic acid, TGF-beta, and an AhR agonist, the greatest increase in the expression level of CD103 in stimulated APCs compared to stimulated control cells was obtained when the APCs of the present invention were produced by the method of the present invention. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta.

[0097] MERTK, BTLA, LAP, HLA-G, and CD49b are cell surface markers of tolerogenic APCs, particularly DCs. MERTK, BTLA, LAP, HLA-G, and CD49b can be expressed on the cell surface of tolerogenic APCs, particularly DCs. The expression levels of MERTK, BTLA, LAP, HLA-G, and CD49b are typically much higher in tolerogenic cells than in non-tolerogenic cells. Desirable tolerogenic APCs, particularly DCs, have high levels of MERTK, BTLA, LAP, and HLA-G expression when unstimulated. Desirable tolerogenic APCs, particularly DCs, have high levels of MERTK, BTLA, LAP, HLA-G, and CD49b expression when unstimulated.

[0098] Unstimulated tolerogenic APCs of the present invention (e.g., DCs of the present invention) have the advantage, at least in some embodiments, of having higher MERTK, BTLA, LAP, and HLA-G expression compared to unstimulated control cells. Unstimulated tolerogenic APCs of the present invention (e.g., DCs of the present invention) have the advantage, at least in some embodiments, of having higher MERTK, BTLA, LAP, HLA-G, and CD49b expression compared to unstimulated control cells. The control cells are APCs of the same type from the same subject that have not been exposed to a tolerogenic compound.

[0099] Suitably, the APCs of the present invention (e.g., DCs of the present invention) when unstimulated have an expression level of MERTK that is at least 120%, for example, at least 150%, for example, at least 175% of that of unstimulated control cells. As described in more detail in the Examples section and shown in Figure 9(A), when the tolerogenic compounds used are retinoic acid, TGF-beta, and an AhR agonist, the greatest increase in the expression level of MERTK in unstimulated APCs was obtained when the APCs of the present invention were produced by the method of the present invention, compared to unstimulated control cells. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta.

[0100] Suitably, unstimulated APCs of the present invention (e.g., DCs of the present invention) have an expression level of BTLA that is at least 120%, e.g., at least 150%, e.g., at least 200%, of that of unstimulated control cells. As described in more detail in the Examples section and shown in Figure 9(B), when the tolerogenic compounds used were retinoic acid, TGF-beta, and an AhR agonist, the greatest increase in the expression level of BTLA in unstimulated APCs was obtained when the APCs of the present invention were produced by the method of the present invention, compared to unstimulated control cells. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta.

[0101] Suitably, unstimulated APCs of the present invention (e.g., DCs of the present invention) have an LAP expression level that is at least 120%, for example, at least 150%, for example, at least 200%, for example, at least 250% of that of unstimulated control cells. As described in more detail in the Examples section and shown in Figure 9(C), when the tolerogenic compounds used are retinoic acid, TGF-beta, and an AhR agonist, the greatest increase in LAP expression level in unstimulated APCs was obtained when the APCs of the present invention were produced by the method of the present invention, compared to unstimulated control cells. The use of a combination of retinoic acid, TGF-beta, and an AhR agonist was superior to the use of retinoic acid and TGF-beta.

[0102] Suitably, unstimulated APCs of the present invention (e.g., DCs of the present invention) have an HLA-G expression level that is at least 120%, for example, at least 150%, for example, at least 200%, for example, at least 250%, for example, at least 300% of that of unstimulated control cells. As described in more detail in the Examples section and shown in Figure 9(D), when the tolerogenic compounds used are retinoic acid, TGF-beta, and an AhR agonist, the greatest increase in HLA-G expression level in unstimulated APCs was obtained when the APCs of the present invention were produced by the method of the present invention, compared to unstimulated control cells.

[0103] APCs (e.g., DCs) of the present invention when unstimulated suitably have higher expression of ILT3 and lower expression of CD83 and CD86 compared to control APCs when unstimulated. Furthermore, APCs (e.g., DCs) of the present invention when stimulated with an immunogenic stimulus such as LPS suitably have higher expression of ILT3 and lower expression of CD83 and CD86 compared to control APCs when stimulated with an immunogenic stimulus such as LPS. The control cells are APCs of the same type from the same subject but not treated with a tolerogenic compound according to the present invention.

[0104] The expression level of ILT3 in unstimulated APCs (e.g., DCs) of the present invention is suitably at least 150%, for example, at least 175%, for example, at least 200%, for example, at least 250%, for example, at least 300%, of that in unstimulated control cells. The expression level of CD83 in unstimulated APCs (e.g., DCs) of the present invention is suitably 110% or less, for example, 100% or less, of that in unstimulated control cells. The expression level of CD86 in unstimulated APCs (e.g., DCs) of the present invention is suitably 110% or less, for example, 100% or less, of that in unstimulated control cells.

[0105] For example, the expression level of ILT3 in APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as LPS is suitably at least 150%, such as at least 175%, such as at least 200%, such as at least 250%, such as at least 300% of that in control cells upon stimulation with an immunogenic stimulus such as LPS. For example, the expression level of CD83 in APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as LPS is suitably 70% or less, such as 60% or less, such as 50% or less of that in control cells upon stimulation with an immunogenic stimulus such as LPS. For example, the expression level of CD86 in APCs (e.g., DCs) upon stimulation with an immunogenic stimulus such as LPS is suitably 80% or less, such as 70% or less, such as 60% or less, such as 50% or less of that in control cells upon stimulation with an immunogenic stimulus such as LPS.

[0106] The APCs (e.g., DCs) of the present invention, unstimulated or stimulated with an immunogenic stimulus such as, for example, LPS, suitably express CD103, and most suitably have higher CD103 expression than control APCs, unstimulated or stimulated with an immunogenic stimulus such as, for example, LPS, respectively. The control cells are antigen-presenting cells of the same type from the same subject that have not been treated with the tolerogenic compound according to the present invention. The CD103 expression level in the APCs (e.g., DCs) of the present invention, unstimulated or stimulated with an immunogenic stimulus such as, for example, LPS, is suitably at least 200%, for example at least 250%, for example at least 300%, of that of the control cells, unstimulated or stimulated with an immunogenic stimulus such as, for example, LPS, respectively.

[0107] When unstimulated, APCs (e.g., DCs) of the present invention suitably express CD103 (most suitably, high CD103 expression), and have high CD141, GARP, and ILT3 expression, and low CD83 and CD86 expression, compared to control APCs when unstimulated. Furthermore, when stimulated with an immunogenic stimulus such as LPS, APCs (e.g., DCs) of the present invention suitably express CD103 (most suitably, high CD103 expression), and have high CD141, GARP, and ILT3 expression, and low CD83 and CD86 expression, compared to control APCs when stimulated with an immunogenic stimulus such as LPS. The control cells are the same type of APCs from the same subject that have not been treated with the tolerogenic compound according to the present invention. For example, the expression level of CD103 in the APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as LPS is suitably at least 200%, such as at least 250%, for example at least 300%, of that in control cells upon stimulation with an immunogenic stimulus such as LPS. For example, the co-expression level of CD141 and GARP in the APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as LPS is suitably at least 200%, such as at least 250%, for example at least 300%, at least 400%, for example at least 450%, for example at least 500%, of that in control cells upon stimulation with an immunogenic stimulus such as LPS. For example, the expression level of ILT3 in the APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as LPS is suitably at least 150%, such as at least 175%, such as at least 200%, such as at least 250%, for example at least 300%, of that in control cells upon stimulation with an immunogenic stimulus such as LPS. For example, the expression level of CD83 in the APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as LPS is suitably 70% or less, such as 60% or less, for example 50% or less of that in control cells upon stimulation with an immunogenic stimulus such as LPS.For example, the expression level of CD86 in the APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as LPS is suitably 80% or less, for example 70% or less, for example 60% or less, for example 50% or less, of that of control cells upon stimulation with an immunogenic stimulus such as LPS.

[0108] For example, unstimulated APCs (e.g., DCs) of the present invention suitably express one or more (e.g., two, three, or all four) of MERTK, BTLA, LAP, and HLA-G (and suitably, their expression is high) compared to unstimulated control APCs. For example, unstimulated APCs (e.g., DCs) of the present invention suitably express one or more (e.g., two, three, or all four) of MERTK, BTLA, LAP, HLA-G, and CD49b (and suitably, their expression is high) compared to unstimulated control APCs. The control cells are the same type of APCs from the same patient that have not been treated with the tolerogenic compound according to the present invention. The expression level of MERTK in unstimulated APCs (e.g., DCs) of the present invention is suitably at least 120%, for example, at least 150%, for example, at least 175%, of that in unstimulated control cells. The expression level of BTLA in unstimulated APCs (e.g., DCs) of the present invention is suitably at least 120%, for example, at least 150%, or at least 200%, of that in unstimulated control cells. The expression level of LAP in unstimulated APCs (e.g., DCs) of the present invention is suitably at least 150%, for example, at least 200%, or for example, at least 250%, of that in unstimulated control cells. The expression level of HLA-G in unstimulated APCs (e.g., DCs) of the present invention is suitably 120%, for example, at least 150%, for example, at least 200%, for example, at least 250%, or for example, at least 300%, of that in unstimulated control cells. The expression level of CD49b in unstimulated APCs (e.g., DCs) of the present invention is suitably 120%, for example, at least 150%, for example, at least 200%, for example, at least 250%, or for example, at least 300%, of that in unstimulated control cells.

[0109] For example, antigen-presenting cells (e.g., DCs) of the present invention, when stimulated with an immunogenic stimulus such as LPS, suitably produce less IL-23 than control APCs when stimulated with an immunogenic stimulus such as LPS or an inflammatory cytokine cocktail. The control cells are the same type of APCs from the same subject that have not been treated with the tolerogenic compound according to the present invention. For example, the level of IL-23 production in APCs (e.g., DCs) of the present invention, when stimulated with an immunogenic stimulus such as LPS or an inflammatory cytokine cocktail, is suitably 50% or less, e.g., 40% or less, e.g., 30% or less, of that in control cells when stimulated with an immunogenic stimulus such as LPS. For example, the level of CD86 expression in APCs (e.g., DCs) of the present invention, when stimulated with an immunogenic stimulus such as LPS, is suitably 80% or less, e.g., 70% or less, e.g., 60% or less, e.g., 50% or less, of that in control cells when stimulated with an immunogenic stimulus such as LPS.

[0110] Suitably, the tolerogenic APCs (e.g., DCs) of the present invention are phenotypically stable with respect to, for example, their cell surface marker presentation, high Treg inducibility, low T cell proliferation inducibility, and other advantageous properties described herein.

[0111] Expression of cell surface markers can be assessed using methods widely used and known in the art, such as flow cytometry analysis.

[0112] Dosage regimen The compounds in the cell cultures of the present invention can be added to the cultures at any suitable dose, and the actual dosage level of the compounds in the cell cultures of the present invention can be varied to obtain an amount of compound that is effective to achieve the desired tolerogenic effect on the cells without causing toxicity.

[0113] When retinoic acid is added to a cell culture, a suitable dose of retinoic acid may be, for example, in the range of about 0.5 μM to about 10 μM. For example, a suitable dose may be about 0.5 μM to about 8 μM, 0.5 μM to about 6 μM, 0.5 μM to about 5 μM, 0.5 μM to about 4 μM, preferably 0.5 μM to about 3 μM.

[0114] When an AhR agonist is added to a cell culture, a suitable dose of the AhR agonist can be, for example, about 1 nM to about 10 μM, typically about 5 nM to about 2 μM, e.g., about 5 nM to about 750 nM. For example, a suitable dosage can be about 5 nM to about 500 nM, 5 nM to about 250 nM, 5 nM to about 100 nM, preferably 5 nM to about 50 nM. When no AhR agonist is present in the cell culture, a suitable dose is 0 nM.

[0115] When TGF-beta is added to a cell culture, a suitable dose of TGF-beta can be, for example, about 1 ng / ml to about 200 ng / ml, e.g., about 5 ng / ml to about 200 ng / ml, typically about 5 ng / ml to about 150 ng / ml. For example, a suitable dosage can be about 5 ng / ml to about 125 ng / ml, 5 ng / ml to about 100 ng / ml, 5 ng / ml to about 75 ng / ml, 5 ng / ml to about 50 ng / ml, preferably 5 ng / ml to about 30 ng / ml. When TGF-beta is not present in the cell culture, a suitable dose of TGF-beta is 0 ng / ml.

[0116] When retinoic acid, TGF-beta, and an AhR agonist are added to a cell culture, a suitable dose of retinoic acid may be, for example, in the range of about 0.5 μM to about 10 μM. For example, suitable doses may be about 0.5 μM to about 8 μM, 0.5 μM to about 6 μM, 0.5 μM to about 5 μM, 0.5 μM to about 4 μM, and preferably 0.5 μM to about 3 μM. A suitable dose of the AhR agonist may be, for example, in the range of about 1 nM to about 10 μM, typically about 5 nM to about 2 μM, e.g., about 5 nM to about 750 nM. For example, suitable doses may be about 5 nM to about 500 nM, 5 nM to about 250 nM, 5 nM to about 100 nM, and preferably 5 nM to about 50 nM. An appropriate dose of TGF-beta may be, for example, about 1 ng / ml to about 200 ng / ml, e.g., about 5 ng / ml to about 200 ng / ml, typically about 5 ng / ml to about 150 ng / ml. For example, an appropriate dose may be about 5 ng / ml to about 125 ng / ml, 5 ng / ml to about 100 ng / ml, 5 ng / ml to about 75 ng / ml, 5 ng / ml to about 50 ng / ml, preferably 5 ng / ml to about 30 ng / ml.

[0117] When retinoic acid, TGF-beta, and an AhR agonist are added to a cell culture, a suitable dose of retinoic acid may be, for example, in the range of about 0.5 μM to about 3 μM. A suitable dose of the AhR agonist may be, for example, in the range of about 5 nM to about 50 nM, preferably about 20 nM. A suitable dose of TGF-beta may be, for example, in the range of about 5 ng / ml to about 30 ng / ml, preferably about 20 ng / ml.

[0118] When retinoic acid, TGF-beta, and an AhR agonist are added to a cell culture, a suitable dose of retinoic acid may be, for example, in the range of about 0.5 μM to about 3 μM, preferably about 2 μM. A suitable dose of the AhR agonist may be, for example, in the range of about 5 nM to about 50 nM, preferably about 10 nM. A suitable dose of TGF-beta may be, for example, in the range of about 5 ng / ml to about 30 ng / ml, preferably about 10 ng / ml.

[0119] Dosage regimen can be adjusted to provide the optimum desired response.For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.As used herein, dosage unit form refers to a physically discrete unit that is suitable as a single dose for the culture conditions used; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect.

[0120] The compounds used in the cell culture of the present invention can be administered alone or in combination with one or more other compounds. One compound can be co-administered with one or more other compounds. Two compounds can be co-administered with one or more other compounds.

[0121] Co-administration of two or more compounds can be accomplished in a number of different ways. They may be administered together in a single composition, or in separate compositions as part of a co-administration. For example, one or more compounds may be administered before, separately, after, or sequentially with one or more other compounds, or concurrently or simultaneously.

[0122] Treatment and prevention of immune reactions to antigens and prevention of immune rejection reactions The present invention provides tolerogenic APCs or populations thereof for use in treating a mammalian subject having or at risk of having an unwanted immune response to an antigen, which can be any antigen discussed herein, e.g., FVIII, FIX, an antibody, an antigen associated with a graft (e.g., an allograft), or an autoantigen.

[0123] The tolerogenic APCs generated by the culture method of the present invention can result in autologous tolerogenic APCs or populations thereof. Autologous tolerogenic APCs used for treatment can be obtained from autologous monocytes isolated from a sample taken from the same subject. Monocytes taken from the subject can be cultured with an antigen or an epitope-containing fragment thereof, after which autologous tolerogenic APCs are obtained. The autologous tolerogenic APCs then have the ability to induce tolerance to the antigen when administered back to the subject.

[0124] The present invention further provides tolerogenic APCs or populations thereof for use in methods for preventing allograft rejection in a recipient subject. The allograft may be a kidney, pancreas, liver, lung, heart, skin transplant, or blood cell transplant, such as a stem cell transplant. Thus, the present invention provides a treatment for transplant rejection. The tolerogenic APCs used in the treatment may be obtained from monocytes isolated from a sample collected from a donor. The monocytes collected from the donor may be cultured to obtain tolerogenic APCs capable of inducing tolerance to antigens present in an allograft derived from the same donor.

[0125] Tolerogenic APCs used in treatment can be obtained from monocytes isolated from a sample collected from a recipient. The monocytes collected from the recipient can be cultured with an antigen or antigen pool, followed by the isolation of tolerogenic APCs. The tolerogenic APCs then have the ability to induce tolerance to antigens present in the donor-derived allograft. This method can be useful when monocytes cannot be obtained from the donor, for example, when the donor is not a living donor. Tolerogenic APCs derived from monocytes isolated from a sample collected from a recipient according to the present invention can be used in methods for treating or preventing immune rejection of xenotransplants.

[0126] The tolerogenic APCs of the present invention may be used in a manufacturing method. The present invention encompasses the use of tolerogenic APCs or a population thereof in the manufacture of a medicament for treating a mammalian subject having or at risk of having an unwanted immune response. The method may comprise administering to the mammalian subject tolerogenic APCs or a population thereof, thereby establishing immune tolerance to the antigen. The present invention also encompasses the use of tolerogenic APCs or a population thereof in the manufacture of a medicament for treating immune rejection of an allograft in a recipient subject, the graft being derived from a donor. The method may comprise administering to the recipient subject tolerogenic APCs or a population thereof, thereby establishing tolerance to the allograft, wherein the tolerogenic APCs or a population thereof are obtained from monocytes isolated from a sample taken from the donor or recipient. Such manufacturing methods may also be applied to xenografts, where the tolerogenic APCs or a population thereof are derived from monocytes isolated from a sample taken from the recipient.

[0127] mammal Mammals from which the samples according to the method of the present invention are obtained and which may be treated by the method according to the present invention include, in particular, humans (Homo sapiens). Mammals which may be treated by the method of the present invention are preferably humans (Homo sapiens).

[0128] sample The method of the present invention may comprise isolating monocytes from any suitable sample from a subject. The sample may be a blood sample, a fractionated buffy coat sample, a leukapheresis material sample, or a PBMC sample. The sample used in the method of the present invention is preferably a PBMC sample. The sample may be autologous, allogeneic, or xenogeneic; preferably, the sample is autologous or allogeneic.

[0129] Obtaining APCs from mammals According to the present invention, monocytes are first obtained from a mammal. One suitable method is to collect PBMCs by apheresis. PBMCs collected by apheresis can be kept under temperature-controlled conditions, for example, at ambient temperature, e.g., 18°C ​​to 25°C, or can be frozen, for example, at temperatures below -4°C, -20°C, or -80°C. Alternatively, PBMCs can be isolated by density centrifugation. Monocytes can then be positively selected from PBMCs using a solid phase, e.g., plastic adhesion, or beads, e.g., anti-CD14 magnetic bead isolation.

[0130] Administration of cells to a subject The cells can be administered to a mammal by a variety of routes, for example, intravenously, subcutaneously or intradermally, intranodally, or directly back into an accessible lesion site for injection. The medium containing the cells suitably contains human albumin as a cytoprotective protein. Typically, 1-100 x 10 7 The amount of cells / dose is administered in 1 to 10 doses at weekly to biweekly intervals. Treatment can be extended and the dosing regimen can be modified until the desired tolerance is achieved.

[0131] antigen The antigen or epitope-bearing fragment thereof may be or be derived from a biological drug, such as a protein drug (particularly a drug that is a protein containing at least 50, or at least 200, or at least 1000 amino acid residues). The biological drug may include a polysaccharide component.

[0132] Examples of biologics include blood factors (including, for example, FVIII or factor IX), hormones (including insulin and EPO), growth factors (including EGF, IGF, KGF, HGF, and FGF), cytokines (e.g., interleukins), enzymes (e.g., imiglucerase, rasburicase, imiglucerase, agalsidase beta, alglucosidase alfa, laronidase, idursulfase, and galsulfase), and the like. Further examples include GCSF and analogs, such as filgrastim and pegylated versions thereof (such as pegfilgrastim), and interferons (e.g., interferon beta-1a). In one preferred embodiment of the invention, the drug is FVIII. In another embodiment of the invention, the drug is factor IX. For FVIII, a variety of recombinant drugs are available, including the commercial products ReFacto AF, Helixate NexGen, Kogenate Bayer, Kovaltry, Advate, NovoEight, Esperoct, Nuwiq, Beriate, Beriate P, Feiba, Haemoctin, Hemofil, Monoclate P, Octanate [LV], Optivate, and Recombinate. These products can be used as antigens and epitope-containing fragments that can be derived from any of these products.

[0133] Further exemplary biopharmaceuticals include engineered proteins (such as fusion and chimeric proteins) and recombinant proteins. In some embodiments, the drug may be an antibody. The drug may be a monoclonal antibody, such as a humanized or fully human monoclonal antibody. The drug may also be a protein construct comprising a fragment of an immunoglobulin. The term antibody also includes bispecific antibodies, antibody-drug conjugates, and antibody-nanoparticle conjugates, as well as pegylated or otherwise extended analogs. In some embodiments, the antibody may be a single light chain antibody, a domain antibody or antibody fragment, including a VHH, scFv, Fab, F(ab')2, or BiTE, as well as pegylated or otherwise extended analogs of such.

[0134] Exemplary antibodies include infliximab (chimeric antibody, anti-TNF alpha), adalimumab (human antibody, anti-TNF alpha), basiliximab (chimeric antibody, anti-IL-2), abciximab (chimeric antibody, anti-GpIIb / IIIa), daclizumab (humanized antibody, anti-IL-2), gemtuzumab (humanized antibody, anti-CD33), alemtuzumab (humanized antibody, anti-CD52), edrecoloma (murine Ig2a, anti-EpCAM), rituximab (chimeric antibody, anti-CD20), palivizumab (humanized antibody, anti-respiratory syncytial virus), trastuzumab (humanized antibody, anti-HER2 / neu (erbB2) receptor), bevacizumab (humanized antibody, anti-VEGF), cetuximab (chimeric antibody, anti-EGFR), eculizumab (humanized antibody, anti-complement protein C5), efalizumab (humanized antibody, anti-CD1Ia), ibritumomab (murine antibody, anti-CD20), muromonab-CD3 (murine antibody, anti-T cell CD3 receptor), natalizumab (humanized antibody, anti-α4 integrin), nimotuzumab (humanized IgG1, anti-EGF receptor), omalizumab (humanized antibody, anti-IgE), panitumumab (human antibody, anti-EGFR), ranibizumab (humanized antibody, anti-VEGF), These include I-131 tositumomab (humanized antibody, anti-CD20), ofatumumab (human antibody, anti-CD-20), certolizumab (humanized antibody, anti-TNF-alpha), golimumab (human antibody, anti-TNF-alpha), emicizumab (humanized bispecific antibody, anti-FIXa / FX), denosumab (human antibody, anti-RANK ligand), and concizumab (humanized antibody, anti-tissue factor pathway inhibitor).

[0135] Examples of biologic drugs that are fusion proteins include etanercept.

[0136] An extensive list of biological protein drugs in clinical development and approved products is disclosed in the 2013 PhARMA report "Biologics," which provides details of 907 biologics targeting over 100 diseases - https: / / web.archive.org / web / 20161011093352 / http: / / www.phrma.org / sites / default / files / pdf / biologics2013.pdf, which is incorporated by reference in its entirety. It is believed that the present invention may be used with these and other biological therapeutic agents that elicit an immune response in the treated subject.

[0137] Thus, an antigen or epitope-bearing fragment thereof for use in the methods described herein can be, for example, FVIII or a derivative or fragment thereof, or Factor IX or a derivative or fragment thereof, or an antibody or antibody fragment thereof.

[0138] The antigen or epitope-containing fragment thereof can be bound to the allograft. Tolerogenic APCs that present the antigen bound to the allograft can be used to treat allograft rejection.

[0139] The antigen or its epitope-containing fragment can be or be derived from an autoantigen. Tolerogenic APCs presenting autoantigens can be used to treat autoimmune diseases. The range of autoantigens involved in autoimmune diseases includes desmoglein 3, BP180, BP230 (pemphigus), dystonin and / or type XVII collagen (pemphigoid), myelin (multiple sclerosis), pancreatic beta cell protein (type 1 diabetes mellitus), nicotinic acetylcholine receptor (myasthenia gravis), neuronal surface protein (autoimmune epilepsy and encephalitis), 2-hydrolase (autoimmune Addison's disease), FceRI (chronic autoimmune urticaria) and acetylcholine receptor (myasthenia gravis), fibrillarin (scleroderma), and cardiolipin (systemic lupus erythematosus). These autoantigens, or their epitope-containing fragments, can be used as antigens in the methods of the present invention.

[0140] The antigen or epitope-containing fragment may be unknown. Therefore, an unknown antigen may be used in the culture method of the present invention. The unknown antigen may be bound to a tissue sample or an extract thereof taken from a subject.

[0141] The antigen can be delivered from the exogenous medium across the cell membrane to the intracellular compartment by any suitable antigen delivery method or vehicle. For example, the antigen delivery method or vehicle can be a nanoparticle. Thus, the antigen can be bound to a nanoparticle, i.e., an antigen-nanoparticle conjugate. The antigen-nanoparticle can be a FVIII-nanoparticle conjugate. The nanoparticle can be a lipid nanoparticle, such as a vesicle or a micelle.

[0142] Alternatively, the antigen delivery vehicle can be a cell-penetrating peptide. The antigen can be fused to a cell-penetrating peptide that allows transport across a lipid bilayer membrane from the extracellular environment to the intracellular compartment. The cell-penetrating peptide can be a Tat peptide from the HIV-1 transactivator protein Tat, located in the third helix of the homeodomain of the antennapedia protein, called penetratin or polyarginine. The cell-penetrating peptide can be cell-selective.

[0143] Immune Responses and Uses of Tolerogenic APCs of the Invention The tolerogenic APCs of the present invention are useful for inhibiting unwanted immune responses in a subject to which the cells are administered. Unwanted immune responses can include antibody responses and / or cellular responses.

[0144] The tolerogenic APCs of the present invention are suitable, for example, for the treatment of a mammalian subject in which an immune response occurs, including the production of ADA to any drug capable of producing said response, e.g., any biological drug, e.g., a protein drug.

[0145] The tolerogenic APCs of the present invention are useful for treating bleeding disorders (hemophilia A and B; deficiencies of FVIII and factor IX, respectively), growth factor deficiencies (deficiencies of EGF, IGF, KGF, HGF, FGF, etc.), hormone deficiencies (deficiencies of EPO), enzyme replacement therapy (e.g., imiglucerase (e.g., CEREZYME™), α-galactosidase A (a-gal The present invention is suitable for use in the treatment of mammalian subjects that develop an immune response to drugs that involves the production of ADAs in many drug treatment situations, such as ADAs directed against α-glucosidase (GAA) (e.g., agalsidase beta, FABRYZYME), acid α-glucosidase (GAA) (e.g., alglucosidase alfa, LUMIZYME™, MYOZYME™), and arylsulfatase B (e.g., ADAs directed against laronidase, ALDURAZYME™, idursulfase, ELAPRASE™, galsulfase, NAGLAZYME™), as well as inflammatory and autoimmune disorders (e.g., anti-TNF alpha monoclonal antibodies).

[0146] The tolerogenic APCs of the present invention are suitable for use in treating mammalian subjects suffering from an autoimmune disease and who have generated an immune response against an autoantigen capable of causing said response.

[0147] The tolerogenic APCs of the present invention are suitable for use in treating mammalian subjects suffering from or at risk of suffering from rejection of a transplant, e.g., an allograft, and who have developed or are likely to develop an immune response against antigens associated with the transplant that can result in said reaction.

[0148] autoimmune disease or disorder The tolerogenic APCs according to the present invention may be useful in the treatment of autoimmune diseases.Suitably the autoimmune disease or disorder is achlorhydria, acquired hemophilia, acute hemorrhagic leukoencephalitis, acquired thrombocytopenic purpura, Addison's disease, alopecia areata, anemia, ankylosing spondylitis, anti-glomerular basement membrane disease, antiphospholipid syndrome, aplastic anemia, atopic allergy, autoimmune atrophic gastritis, autoimmune hearing loss, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune lymphoproliferative, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy Autoimmune polyendocrinopathy, autoimmune sensory hearing loss, autoimmune syndrome type II, autoimmune uveitis, Behçet's syndrome / disease, celiac disease, Chagas' disease, chronic active hepatitis, chronic inflammatory demyelinating polyneuropathy, chronic lymphocytic thyroiditis, Churg-Strauss syndrome, Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, Schilder's diffuse cerebral sclerosis, epidermolysis bullosa acquisita, erythroderma, Felty's syndrome, glomerulonephritis, membranous glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis Graves' disease, Guillain-Barré syndrome, Hammann-Rich syndrome, idiopathic thrombocytopenic purpura, inflammatory bowel disease, insulin resistance type B, Lambert-Eaton myasthenic syndrome, lens-induced uveitis, lichen sclerosing and atrophicus, lymphopenia, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, corneal ulcer, mucocutaneous lymph node syndrome, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, transverse myelitis, myocarditis, narcolepsy, neuromyelitis optica, ocular cicatricial pemphigoid, oculovestibular-auditory syndrome, sympathetic ophthalmia, opsoclonus-myoclonus syndrome, pancreatitis, bullous pemphigoid, pemphigus foliaceus, pemphigus vulgaris, polyarteritis nodosa, polymyalgia rheumatica, polyradiculomyopathy, primary biliary cholangitis, primary biliary cirrhosis, psoriasis, Raynaud's disease, Reiter's disease, relapsing polychondritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, sclerosing cholangitis, Sjogren's syndrome, stiff-person syndrome, adult-onset Still's disease, Takayasu's arteritis, temporal arteritis, thyrotoxicosis, type B insulin resistance, ulcerative colitis, uveomeningoencephalitis syndrome, vitiligo, and Wegener's granulomatosis.

[0149] Suitably the autoimmune disease is type 1 diabetes mellitus, rheumatoid arthritis, chronic lymphocytic thyroiditis, multiple sclerosis and ulcerative colitis.

[0150] Additionally, diseases that may be partially related to autoimmune reactions are arteriosclerosis, Parkinson's disease, and Alzheimer's disease.

[0151] Cryopreservation, storage and thawing The tolerogenic APCs of the present invention can be produced manually, semi-automatically, or by a fully automated closed system. The cells of the present invention (e.g., DCs of the present invention) may be preserved as a single-cell suspension in a cryopreservation medium containing 2 to 10% DMSO (dimethyl sulfoxide). The cells are then washed with a physiological sodium chloride buffer solution, preferably containing human serum albumin, and cryopreserved in a cryopreservation medium at a concentration of preferably 0.5 to 20 × 10 6 The cells may be resuspended in the range of cells / ml and transferred to a cryovial. The cells may then be placed in a freezing container in a -80°C freezer. After 24 hours in the -80°C freezer, the cryovial may be transferred to a -150°C freezer or liquid nitrogen tank for long-term storage. Preferably, the cells of the invention (e.g., DCs of the invention) are frozen and stored at temperatures below -120°C.

[0152] Cells of the invention (eg, DCs of the invention) may be thawed in a cryovial before use until only a small chunk of ice remains.

[0153] Storage and transportation The cells of the present invention (e.g., DCs of the present invention) may be preserved as single cells or in multicellular aggregates. The single cells or multicellular aggregates may be entrapped or encapsulated in a hydrogel. Entrapment or encapsulation of cells in a hydrogel can maintain their functionality at hypothermia and can be used to effectively store and / or transport multicellular aggregates while maintaining mechanical protection and preserving cell morphology, integrity, viability, and function.

[0154] Single cells or multicellular aggregates may be encapsulated in a hydrogel in vitro. The cells typically have structurally intact cell membranes, are viable or living cells, and have a cellular morphology representative of the cells of the present invention (e.g., DCs of the present invention). The hydrogel may be a coating that completely or incompletely covers / surrounds at least the majority of the single cells or multicellular aggregates to entrap the cells or aggregates within the hydrogel. The hydrogel coating may be formed separately from the single cells or multicellular aggregates and then placed on the single cells or multicellular aggregates. The hydrogel coating may comprise a layer of cross-linked alginate formed separately (i.e., spatially separated) from the aggregates. Alternatively, the hydrogel coating may be formed in situ (i.e., in the presence of the single cells or multicellular aggregates).

[0155] Single cells or multicellular aggregates may be entrapped or encapsulated in a reversibly crosslinked hydrogel, such as an alginate hydrogel. A "reversibly crosslinked hydrogel" refers to a hydrogel formed by reversible crosslinking (i.e., the crosslinking can be reversed so that the hydrogel returns to solution). Reversal of the crosslinking allows the entrapped or encapsulated multicellular aggregate(s) to be released from the hydrogel (e.g., after their point of use / transport or storage is complete). Examples of reversibly crosslinked hydrogels are well known in the art, and suitable hydrogels can be readily identified by those skilled in the art.

[0156] The hydrogel may comprise a hydrogel-forming polymer having a crosslinked or network-like structure or matrix; and interstitial fluid. The hydrogel may inhibit or prevent cell differentiation in the aggregates encapsulated or entrapped therein. The hydrogel may be semipermeable. The hydrogel may be a "hydrogel-forming polymer" that can be crosslinked or form a network-like structure or matrix under appropriate conditions, and the interstitial fluid and multicellular aggregates may be retained within such a structure or matrix. The hydrogel may comprise internal pores.

[0157] The hydrogel-forming polymer may be alginic acid or an alginate salt of a metal ion. Preferably, the metal is a Group 1 metal (e.g., lithium, sodium, or potassium alginate) or a Group 2 metal (e.g., calcium, magnesium, barium, or strontium alginate). Preferably, the polymer is calcium alginate, sodium alginate, or strontium alginate. The hydrogel-forming polymer may be a cross-linked acrylic acid-based (e.g., polyacrylamide) polymer. The hydrogel-forming polymer may be a cross-linkable cellulose derivative, a hydroxyl ether polymer (e.g., poloxamer), pectin, or a natural gum.

[0158] In the case of multicellular aggregates, cells may be directly or indirectly adjacent to or connected to one another in a manner that forms a cellular aggregate. A matrix, substrate, or scaffold, collectively referred to as a "structure," may connect adjacent cells into an aggregate. The structure may be a synthetic or natural polymer. Preferably, the structure is biodegradable. The structure may be a polymer, including, for example, polylactic acid, collagen, nylon, e.g., nylon mesh, collagen, gelatin, alginate, cellulose, glass, or Matrigel.

[0159] Cells can be adjacent via an extracellular matrix (ECM), such as an Alvatex polystyrene scaffold for 3D cell culture. Alternatively, the multicellular aggregates can be structure-free.

[0160] The hydrogel-encapsulated single cells or multicellular aggregates can be packaged and sealed in a container for storage or transport from a first location to a second location. The hydrogel-encapsulated single cells or multicellular aggregates can have a storage stability time of at least 1 hour, at least 2 hours, at least 5 hours, at least 12 hours, at least 24 hours, etc. The hydrogel-encapsulated single cells or multicellular aggregates can be stored or transported within the hydrogel (and sealed container) at temperatures ranging from -80°C to 45°C, preferably 4 to 45°C, or at ambient temperatures, e.g., 10 to 25°C, preferably 15 to 20°C.

[0161] Single cells or multicellular aggregates encapsulated in hydrogels can be stored or transported under cell culture conditions (e.g., about 37°C, about 5% CO2, and about 95% humidity) or under chilled conditions, e.g., 4-6°C, preferably about 4°C. Single cells or multicellular aggregates encapsulated in hydrogels may be refrigerated during storage or transportation, e.g., 2-8°C or 8-15°C. Single cells or multicellular aggregates encapsulated in hydrogels can be stored or transported at controlled room temperature (CRT) (defined as 15-25°C). They can be stored or transported at refrigerated or CRT (i.e., 8-25°C). Single cells or multicellular aggregates encapsulated in hydrogels can be stored or transported at hypothermia (i.e., below about 35°C, typically in the range of 0-32°C).

[0162] Hydrogels containing multicellular aggregates can be frozen before storage and / or transportation. This can extend the time that cells in the multicellular aggregates remain viable after thawing and / or increase the effective transportation time. Thus, the hydrogel can be used as a post-cryoprotectant in this manner. For example, the temperature of the hydrogel containing the aggregates can be reduced to below 0°C, below -15°C, or below -80°C. During storage and / or transportation, the hydrogel containing multicellular aggregates can be defrosted or thawed, preferably at a gradual, controlled or uncontrolled rate of temperature increase, i.e., its temperature may or may not be raised above 0°C. In another example, the hydrogels of the present invention are not cooled or frozen.

[0163] The single cells or multicellular aggregates encapsulated in the hydrogel can be stored and / or transported for up to 10 or 20 weeks. Preferably, the single cells or multicellular aggregates are stored in the hydrogel for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks before being released from the hydrogel. More preferably, the single cells or multicellular aggregates are stored in the hydrogel for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days before being released from the hydrogel.

[0164] Single cells or multicellular aggregates can be released from the hydrogel by appropriate cell-compatible conditions, i.e., conditions that are not detrimental or significantly detrimental to the integrity of the cells and / or cell membranes. For example, the hydrogel can be dissociated by chemical degradation or dissolved, for example, using an appropriate alginate lysis buffer.

[0165] Storage and / or transportation for multicellular aggregates is described in WO2019142004, the contents of which are incorporated herein by reference in their entirety.

[0166] QC (Quality Control) Release Assay RA+TGFbeta+AhR agonist-treated DCs can be quality tested based on the expression of several different markers and their ability to reduce T cell proliferation in an MLR. For example, RA+TGFbeta+AhR agonist-treated DCs can express the markers CD11c, HLA-DR, CD80, CD83, CD86, ILT3, GARP, CD141, LAP, CD103, BTLA, HLA-G, and CD49b. RA+TGFbeta+AhR agonist-treated DCs preferably express at least CD11c, HLA-DR, CD80, CD83, CD86, and ILT3, more preferably at least CD11c, HLA-DR, CD80, CD83, CD86, and ILT3, even more preferably at least CD11c, HLA-DR, CD80, CD83, CD86, ILT3, GARP, CD141, and LAP. T cell proliferation can be measured by: 3 T cell proliferation can be assessed by H-thymidine incorporation, CFDA, CD25 expression and KI-67. 3 H-thymidine incorporation, more preferably CFDA, and CD25 expression 3 H-thymidine incorporation, and even more preferably CFDA, CD25 expression, KI-67 and 3 It can be assessed by 3H-thymidine incorporation. [Example]

[0167] (Example) Materials and Methods for Examples 1-18 and 20-25 Isolation of human monocytes and T cells PBMCs were isolated by density centrifugation using Lymphoprep™ (StemCell Technologies, Vancouver, Canada) or cell preparation tubes (BD Bioscience, San Jose, United States). Monocytes were positively selected from PBMCs using anti-CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4 + T cells were isolated using EasySep Human CD4 + Purification was performed using a T cell isolation kit (StemCell Technologies).

[0168] Generation of dendritic cells (DCs) CD14 + Monocytes were cultured at a density of 1.25 × 10 in GMP DC medium (CellGenix, Freiburg, Germany) containing HEPES, GlutaMAX, and penicillin-streptomycin solution (Thermo Fisher, Waltham, MA) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 ng / ml; PeproTech, London, UK) and interleukin 4 (IL-4; 100 ng / ml; PeproTech) for 7 days. 6DCs were cultured at 1000 cells / ml. Cells were replenished with fresh medium and cytokines on day 3. Control DCs were differentiated in GM-CSF and IL-4 without additional compounds and, optionally, on day 6, further treated with lipopolysaccharide (LPS; 0.5 μg / ml; Sigma-Aldrich, Saint Louise, MO) or a proinflammatory cytokine cocktail consisting of TNF-alpha (10 ng / ml; PeproTech), IL-1 beta (10 ng / ml; PeproTech), and prostaglandin E2 (PGE2; 1 μg / ml; Sigma) to generate stimulated immunogenic DCs. Tolerogenic DCs (tolDCs) were generated by treatment with various tolerogenic compounds: AhR agonist "C1" (C1; 20 nM; IMA-06201; Immunahr AB, Lund, Sweden) on days 0 and 3, TGF-beta1 (10 or 20 ng / ml; PeproTech) on day 3, and retinoic acid (2 μM; Sigma-Aldrich) on day 6. Control tolDCs for comparison were established by treatment with dexamethasone (Dex; 100 nM; Sigma-Aldrich) and vitamin D3 (VitD3; 100 nM, StemCell Technologies) on day 3. The phenotypic stability of TolDCs was examined during the final 24 hours of culture by adding LPS (0.5 μg / ml) or a proinflammatory cytokine cocktail consisting of TNF-alpha (10 ng / ml), IL-1 beta (10 ng / ml), PGE2 (1 μg / ml), and, in Example 24, also IL-6 (10 ng / ml). On day 7, DCs were harvested and washed extensively before phenotyping and functional assays.

[0169] DC phenotyping Cell surface expression was examined using the following fluorescently labeled antibodies: CD11c (B-Ly6), CD83 (HB15e), CD86 (BU63), ILT3 (ZM4.1), CD141 (M80), GARP (7B11), CD103 (B-Ly7), MERTK (590H11G1E3), BTLA (J168-540), CD49b (P1E6-C5), HLA-G (87G), and LAP (FNLAP) from BD Biosciences (Franklin Lakes, NJ), BioLegend (San Diego, CA), and Thermo Fisher Scientific. DCs were washed, resuspended in staining buffer (phosphate-buffered saline supplemented with 0.5% BSA and 2 mM EDTA), and incubated with antibodies for 20 minutes, after which they were washed, acquired on a MACSQuant 10 flow cytometer (Miltenyi Biotec), and analyzed using FlowJo software (BD Biosciences). Live cells were distinguished from dead cells using Fixable Viability Dye (FVD) from Thermo Fisher Scientific.

[0170] DC cytokine production IL-23 production was determined in supernatants from DC cultures with and without LPS or inflammatory cytokine stimulation using the Luminex platform (Invitrogen, Carlsbad, USA).

[0171] DC / T cell culture: MLR and Treg induction To analyze the T cell stimulatory capacity of the generated DC population, allogeneic MLR was performed. DC / T cell culture was performed in complete medium: RPMI-1640 (Thermo Fisher) containing fetal bovine serum (FBS; 10%; Thermo Fisher), HEPES, GlutaMAX, and penicillin-streptomycin solution. T cells (10 5 cells / well) were cultured with DCs at a T cell:DC ratio of 10:1 for 5–7 days. 3 Proliferation was determined by H-thymidine incorporation. + CD49b+ LAG3 + For the determination of Tr1 cells, cells were phenotyped as follows: CD25 高 Foxp3 + For determination of Treg induction, T cells from MLR cultures were rested for an additional 7 days in complete medium containing IL-2 (20 IU / ml; PeproTech) and then subjected to phenotyping as described below.

[0172] Treg phenotyping Cell surface and intracellular marker expression was examined using the following fluorescently labeled antibodies: CD4 (A161A1), CD25 (M-A251), CD49b (P1E6-C5), LAG3 (11C3C65), and Foxp3 (259D / C7) from BD Biosciences and BioLegend. T cells were washed, resuspended in staining buffer, and incubated with antibodies for 20 minutes before being washed, fixed, permeabilized, and stained intracellularly for Foxp3 using a Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, Thermo Fisher Scientific). Live cells were distinguished from dead cells using Thermo Fisher's Fixable Viability Dye (FVD). Flow cytometry analysis was performed as described above.

[0173] Freezing and thawing of DCs Wash DCs once and then incubate at 10-20 x 10 6The cells were resuspended in freezing medium at a cell concentration of 1000 cells / ml and transferred to cryovials. These vials were placed in a freezing container and placed in a -80°C freezer. The freezing container allowed for a controlled freezing rate of approximately -1°C / min. After 24 hours in the -80°C freezer, the cryovials were transferred to a -150°C freezer for long-term storage. Two different freezing media were tested: CryoStor (CS, Biolife Solutions) with 10% DMSO (CS10) and a conventional freezing medium with 10% DMSO, 50% human serum albumin, and 40% cell culture medium. DCs were thawed in the vials in a 37°C water bath, and the cell suspension was carefully transferred to a 15 ml tube containing cold GMP DC medium (CellGenix) and washed once. The cells were then stained according to the DC phenotyping method described above.

[0174] Example 1 - Effect of various compounds on DC expression of CD83 Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs, represented here as one of several factors by the low expression level of the DC maturation marker CD83, which, in addition, should be as resistant as possible to upregulation by immunogenic stimuli. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce CD14 + TolDCs were differentiated from monocytes. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic DCs. TolDCs, or their less optimal variants, were cultured with GM-CSF and IL-4 as described above and then differentiated into CD14 DCs by treatment with various tolerogenic compounds, either alone or in combination: 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 20 ng / ml TGF-beta on day 3, and 2 μM retinoic acid (abbreviated as "RA" in this example section and figures) on day 6. +The synergistic effect of the claimed combination was demonstrated by differentiation from monocytes, as shown in Figures 1(A) and 1(B). Comparative TolDCs were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3 (abbreviated as "Dex / VitD3" in this Example section and figures). On day 6, some samples were treated with LPS and tested for phenotypic stability (Figures 1(A) and 1(B); black bars). On day 7, all cells were harvested and stained for CD83 cell surface expression using a fluorescently labeled anti-CD83 antibody, followed by flow cytometric analysis.

[0175] The mean fluorescence intensity (MFI) values ​​in live cells are shown as mean ± SD in Figures 1(A) and (B).

[0176] The results in Figure 1(A) show that DCs treated with RA + TGFbeta and RA + TGFbeta + AhR agonist expressed lower levels of CD83 compared with the corresponding control DCs and Dex / VitD3-treated comparator tolDCs, regardless of whether they were stimulated with LPS. Treatment with RA + TGFbeta + AhR agonist was superior to treatment with RA + TGFbeta in reducing CD83 expression in DCs.

[0177] The results in Figure 1(B) show that DCs treated with RA + TGFbeta, RA + TGFbeta + AhR agonist, and RA + AhR agonist expressed lower levels of CD83 compared with corresponding control DCs when stimulated with LPS. Treatment with RA + TGFbeta + AhR agonist and RA + TGFbeta was superior to treatment with RA + AhR agonist, and was also superior to treatment with RA as a single agent in reducing CD83 expression in DCs.

[0178] Example 2 - Effect of various compounds on DC expression of CD86 Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs, which, among other factors, should contain low expression levels of the costimulatory molecule CD86 and be as resistant as possible to upregulation by immunogenic stimuli. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce CD14 expression. + Control DCs were differentiated from monocytes. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs, or their less optimal variants, were cultured with GM-CSF and IL-4 as described above and then differentiated into CD14 DCs by treatment with various tolerogenic compounds, either alone or in combination: 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 20 ng / ml TGF-beta on day 3, and 2 μM RA on day 6. + The synergistic effect of the claimed combination was demonstrated by differentiation from monocytes, as shown in Figures 2(A) and 2(B). Comparative TolDCs were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some tolDCs were treated with LPS and tested for phenotypic stability (Figures 2(A) and 2(B); black bars). On day 7, all cells were harvested and stained for CD86 cell surface expression using a fluorescently labeled anti-CD86 antibody, followed by flow cytometric analysis.

[0179] The MFI values ​​of live cells are shown as mean ± SD in Figures 2(A) and (B).

[0180] The results in Figure 2(A) show that DCs treated with RA + TGFbeta and RA + TGFbeta + AhR agonist expressed lower levels of CD86 compared with the corresponding control DCs and Dex / VitD3-treated comparator tolDCs, regardless of whether they were stimulated with LPS. Treatment with RA + TGFbeta + AhR agonist was superior to treatment with RA + TGFbeta in reducing CD86 expression in DCs.

[0181] The results in Figure 2(B) show that DCs treated with RA + TGFbeta and RA + TGFbeta + AhR agonist expressed lower levels of CD86 compared with corresponding control DCs, regardless of whether they were stimulated with LPS. RA + AhR agonist-treated DCs expressed higher levels of CD86 compared with RA + TGFbeta and RA + TGFbeta + AhR agonist-treated DCs when stimulated with LPS. RA-treated DCs were not resistant to CD86 upregulation upon LPS stimulation compared with stimulated control DCs. Treatment with RA + TGFbeta + AhR agonist was superior to treatment with either RA + TGFbeta or RA + AhR agonist, and furthermore, treatment with all three combinations was superior to RA as a single agent in reducing CD86 expression in DCs when stimulated with LPS.

[0182] Example 3 - Effect of various compounds on DC expression of ILT3 Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs, which, among other factors, should contain high expression levels of the tolerogenic marker ILT3 and be resistant to downregulation by immunogenic stimuli. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce CD14 + Control DCs were differentiated from monocytes. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs, or their less optimal variants, were cultured with GM-CSF and IL-4 as described above and then differentiated into CD14 DCs by treatment with various tolerogenic compounds, either alone or in combination: 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 20 ng / ml TGF-beta on day 3, and 2 μM RA on day 6. +The synergistic effect of the claimed combination was demonstrated by differentiation from monocytes, as shown in Figures 3(A) and 3(B). Comparative TolDCs were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some tolDCs were treated with LPS and tested for phenotypic stability (Figures 3(A) and 3(B); black bars). On day 7, all cells were harvested and stained for ILT3 cell surface expression using a fluorescently labeled anti-ILT3 antibody, followed by flow cytometric analysis.

[0183] The MFI values ​​of live cells are shown as mean ± SD in Figures 3(A) and (B).

[0184] The results in Figure 3(A) show that DCs treated with RA + TGFbeta and RA + TGFbeta + AhR agonist expressed higher levels of ILT3 compared with corresponding control DCs and Dex / VitD3-treated comparator tolDCs, regardless of whether they were stimulated with LPS. Treatment with RA + TGFbeta + AhR agonist was superior to treatment with RA + TGFbeta in inducing ILT3 expression in DCs.

[0185] The results in Figure 3(B) show that DCs treated with RA + AhR agonist, RA + TGF-beta, and RA + TGF-beta + AhR agonist expressed higher levels of ILT3 compared with corresponding control DCs, regardless of whether they were stimulated with LPS. Treatment with RA + TGF-beta + AhR agonist was superior to treatment with RA + TGF-beta or RA + AhR agonist, and treatment with RA + TGF-beta was superior to treatment with RA + AhR agonist in inducing ILT3 expression in DCs. Treatment with RA + TGF-beta + AhR agonist and RA + TGF-beta was superior to treatment with RA as a single agent in inducing ILT3 expression in DCs, regardless of whether they were stimulated with LPS. Treatment with RA + AhR agonist was superior to treatment with RA as a single agent in inducing ILT3 expression in DCs upon stimulation with LPS.

[0186] Example 4 - Effect of various compounds on DC tolerogenic index ILT3 / CD86 Optimal tolDCs should express both low levels of costimulatory molecules and high levels of tolerogenic molecules. Therefore, to take this combinatorial effect into account, the ratio between the expression levels of representative tolerogenic molecules and costimulatory molecules could be shown as a proxy for the tolerogenic potential of specific subsets of tolDCs. Figures 4(A) and (B) show such a tolerogenic index, defined as the ILT3 / CD86 expression (MFI) ratio, in DCs cultured under various conditions in the absence (Figure 4(A); gray bars) or presence (Figure 4(B); black bars) of LPS.

[0187] The MFI values ​​of live cells are shown as mean ± SD in Figures 4(A) and (B).

[0188] The results in Figure 4(A) show that DCs treated with RA + TGF beta and RA + TGF beta + AhR agonist had a higher tolerogenic index than control DCs and Dex / VitD3-treated comparator tolDCs, and furthermore, RA + TGF beta + AhR agonist-treated DCs had a higher tolerogenic index than RA + TGF beta-treated DCs.

[0189] The results in Figure 4(B) show the same pattern as those in Figure 4(A). In particular, Figure 4(B) shows that DCs treated with RA + AhR agonist, RA + TGFbeta, and RA + TGFbeta + AhR agonist have a higher tolerogenic index than stimulated control DCs. TolDCs treated with RA + TGFbeta + AhR agonist have a higher tolerogenic index than those treated with RA + TGFbeta, and a higher tolerogenic index than those treated with RA + AhR agonist. DCs treated with RA + TGFbeta + AhR agonist, RA + TGFbeta, and RA + AhR agonist all have a higher tolerogenic index than DCs treated with RA as a single agent.

[0190] Example 5 - Effect of various compounds on DCs and their ability to induce T cell proliferation Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs, which could translate into a reduced ability to induce T cell proliferation and, in addition, should be as resistant as possible to being upregulated by immunogenic stimuli. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce CD14 + TolDCs were differentiated from monocytes. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs, or their less optimal variants, were cultured with GM-CSF and IL-4 as described above and then induced CD14 expression by treatment with various tolerogenic compounds, either alone or in combination: 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 10 ng / ml TGF-beta (Figure 5(B)) or 20 ng / ml TGF-beta (Figure 5(A)) on day 3, and 2 μM RA on day 6. + The synergistic effect of the claimed combination was demonstrated by differentiation from monocytes, as shown in Figures 5(A) and (B). Comparative TolDCs were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some of the DCs were treated with LPS (Figure 5(A); black bars) or a proinflammatory cytokine cocktail consisting of TNF-alpha, IL-1 beta, and PGE2 (Figure 5(B); black bars) and tested for phenotypic stability under various conditions. On day 7, all cells were collected and CD4 + Co-cultures with T cells were set up in an MLR setting with a T cell:DC ratio of 10:1. At the end of the 7-day culture, T cell proliferation was assessed by 3 Determined by 3H-thymidine incorporation.

[0191] Counts per minute (CPM) values ​​from triplicate or more samples are shown in Figures 5(A) and (B) as mean ± SD.

[0192] The results in Figure 5(A) show that DCs treated with RA + AhR agonist, RA + TGF beta, and RA + TGF beta + AhR agonist exhibited a lower ability to induce T cell proliferation than corresponding control DCs, including after LPS challenge. Treatment with RA + TGF beta + AhR agonist was superior to treatment with RA + AhR agonist or RA + TGF beta in reducing the ability to induce T cell proliferation.

[0193] The results in Figure 5(B) show the same pattern as those in Figure 5(A). Notably, Figure 5(B) shows that DCs treated with RA + AhR agonist, RA + TGF-beta, and RA + TGF-beta + AhR agonist were less able to induce T cell proliferation than corresponding control DCs, Dex / VitD3-treated comparator tolDCs, and DCs treated with RA, TGF-beta, or AhR agonist as single agents, including after stimulation with an inflammatory cytokine cocktail. Treatment with RA + TGF-beta + AhR agonist was superior to treatment with RA + AhR agonist or RA + TGF-beta in reducing the ability to induce T cell proliferation.

[0194] Example 6 - CD141 + GARP + (Effect of various compounds on DC frequency in Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs, including, among other factors, co-expression of CD141 and GARP. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce CD14 + TolDCs were differentiated from monocytes. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs, or their less optimal variants, were cultured with GM-CSF and IL-4 as described above and then induced CD14 expression by treatment with various tolerogenic compounds, either alone or in combination: 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 10 ng / ml TGF-beta (Figure 6(A)) or 20 ng / ml TGF-beta (Figure 6(B)) on day 3, and 2 μM RA on day 6. +The synergistic effect of the claimed combination was demonstrated by differentiation from monocytes, as shown in Figures 6(A) and 6(B). Comparative TolDCs were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some tolDCs were treated with LPS and tested for phenotypic stability (Figure 6(B); black bars). On day 7, all cells were harvested and stained for cell surface expression using fluorescently labeled anti-CD141 and anti-GARP antibodies, followed by flow cytometric analysis.

[0195] CD141 in live cells + GARP + The frequencies (%) of these are shown as mean ± SD in Figures 6(A) and (B).

[0196] The results in Figure 6(A) show that DCs treated with RA+AhR agonist, RA+TGFbeta, and RA+TGFbeta+AhR agonist expressed higher frequencies of CD141 than control DCs, Dex / VitD3-treated comparator tolDCs, and DCs treated with RA, TGFbeta, AhR agonist, and TGFbeta+AhR agonist. + GARP + Treatment with RA + TGFbeta + AhR agonist was superior to treatment with RA + AhR agonist or RA + TGFbeta. DCs treated with RA + AhR agonist, RA + TGFbeta, and RA + TGFbeta + AhR agonist had a higher frequency of CD141 cells than DCs treated with TGFbeta or AhR agonist as single agents, or DCs treated with Dex / VitD3. + GARP + DCs treated with RA + AhR agonist and RA + TGF beta + AhR agonist showed higher frequencies of CD141 cells than DCs treated with RA alone. + GARP + Contains cells.

[0197] The results in Figure 6(B) show that DCs treated with RA + TGFbeta and RA + TGFbeta + AhR agonist showed higher frequencies of CD141 than the corresponding control DCs and Dex / VitD3-treated comparator tolDCs, including after LPS stimulation. + GARP + Treatment with RA + TGF beta + AhR agonist was superior to treatment with RA + TGF beta.

[0198] Example 7 - Effect of various compounds on DC generation of IL23 Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs, including, among other factors, low production of the cytokine IL-23 in response to immunogenic stimuli. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce CD14 + TolDCs were differentiated from monocytes. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs, or their less optimal variants, were cultured with GM-CSF and IL-4 as described above and induced CD14 expression by treatment with various tolerogenic compounds, either alone or in combination: 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 10 ng / ml TGF-beta (Figure 7(B)) or 20 ng / ml TGF-beta (Figure 7(A)) on day 3, and 2 μM RA on day 6. + The synergistic effect of the claimed combination was demonstrated by differentiation from monocytes, as shown in Figures 7(A) and (B). Comparative TolDCs were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some of the tolDCs were treated with LPS (Figure 7(A); black bars) or a proinflammatory cytokine cocktail consisting of TNF-alpha, IL-1 beta, and PGE2 (Figure 7(B); black bars). Supernatants were collected from DC cultures on day 7, and IL-23 levels were measured using the Luminex platform.

[0199] IL-23 concentration values ​​are shown as mean ± SD in Figures 7(A) and (B).

[0200] The results in Figure 7(A) show that DCs treated with RA + AhR agonist, RA + TGF-beta, and RA + TGF-beta + AhR agonist produced less IL-23 upon LPS activation compared with stimulated control DCs. RA + TGF-beta was superior to RA + TGF-beta + AhR agonist and RA + AhR agonist in reducing IL-23 production.

[0201] The results in Figure 7(B) show a similar pattern to Figure 7(A). Notably, Figure 7(B) shows that DCs treated with RA + AhR agonist, RA + TGFbeta, and RA + TGFbeta + AhR agonist produced less IL-23 upon stimulation with the inflammatory cytokine cocktail compared to matched stimulated control DCs and DCs treated with TGFbeta or AhR agonist as single agents, or DCs treated with TGFbeta + AhR agonist.

[0202] Example 8 - Effect of various compounds on DC expression of CD103 Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs, including the highly expressed marker CD103, among other factors. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce a CD14 + TolDCs were differentiated from monocytes. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs, or their less optimal variants, were cultured with GM-CSF and IL-4 as described above and then induced CD14 expression by treatment with various tolerogenic compounds, either alone or in combination: 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 10 ng / ml TGF-beta (Figure 8(A)) or 20 ng / ml TGF-beta (Figure 8(B)) on day 3, and 2 μM RA on day 6. +The synergistic effect of the claimed combination was demonstrated by differentiation from monocytes, as shown in Figures 8(A) and (B). Comparative TolDCs were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some tolDCs were treated with LPS and tested for phenotypic stability (Figure 8(B); black bars). On day 7, all cells were harvested and stained for cell surface expression using a fluorescently labeled anti-CD103 antibody, followed by flow cytometric analysis.

[0203] The MFI values ​​of live cells are shown as mean ± SD in Figures 8(A) and (B).

[0204] The results in Figure 8(A) show that DCs treated with RA + AhR agonist and RA + TGFbeta + AhR agonist expressed higher levels of CD103 than RA, TGFbeta, RA + TGFbeta, control DCs, and Dex / VitD3-treated comparator tolDCs. Treatment with RA + TGFbeta + AhR agonist was superior to treatment with RA + AhR agonist in inducing CD103 expression.

[0205] The results in Figure 8(B) show that DCs treated with RA + TGFbeta + AhR agonist, whether stimulated with LPS or not, expressed higher levels of CD103 than control DCs and Dex / VitD3-treated comparator tolDCs. Treatment with RA + TGFbeta + AhR agonist was superior to treatment with RA + TGFbeta in inducing CD103 expression.

[0206] Example 9 - Effect of various compounds on DC expression of MERTK, BTLA, LAP, HLA-G and CD49b Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs consisting of the expression of various tolerogenic markers. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce CD14 +TolDCs were differentiated from monocytes. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs were cultured with GM-CSF and IL-4 as described above and then treated with various tolerogenic compounds, as shown in Figure 9(A)-(E): 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 10 ng / ml TGF-beta (for MERTK and LAP, Figures 9(A), (C), and (E)) or 20 ng / ml TGF-beta (for BTLA and HLA-G, Figures 9(B) and (D)) on day 3, and 2 μM RA on day 6. + TolDCs were differentiated from monocytes. For comparison, TolDCs were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 7, all cells were harvested and stained for cell surface expression using fluorescently labeled anti-MERTK, anti-BTLA, anti-LAP, anti-HLA-G, and anti-CD49b antibodies, followed by flow cytometry analysis.

[0207] The MFI values ​​of live cells are shown as mean ± SD in Figures 9(A) to (D).

[0208] The results show that DCs treated with RA + TGFbeta and RA + TGFbeta + AhR agonist expressed higher levels of MERTK (Figure 9(A)), BTLA (Figure 9(B)), and LAP (Figure 9(C)) than control DCs and Dex / VitD3-treated comparator tolDCs. In all cases, treatment with RA + TGFbeta + AhR agonist was superior to treatment with RA + TGFbeta. In addition, RA + TGFbeta + AhR agonist-treated DCs expressed higher levels of HLA-G and CD49b compared to control DCs and Dex / VitD3-treated comparator tolDCs, as shown in Figures 9(D) and (E), respectively.

[0209] Example 10 - Effect of various compounds on DCs and their ability to induce Tregs Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs that could translate into enhanced induction of Treg cells upon T cell / DC coculture. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce CD14 + TolDCs were differentiated from monocytes. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs, or their less optimal variants, were cultured with GM-CSF and IL-4 as described above and then differentiated into CD14 DCs by treatment with various tolerogenic compounds, as shown in Figure 10(A) and (B): 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 20 ng / ml TGF-beta on day 3, and 2 μM RA on day 6. + TolDCs were differentiated from monocytes. For comparison, TolDCs were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some tolDCs were treated with LPS to test their phenotypic stability (Fig. 10(A); black bars). All cells were collected on day 7 and CD4 + Cocultures with T cells were set up in an MLR setting at a T cell / DC ratio of 10:1. At the end of the first 7 days of culture, T cells were washed and rested in IL-2-containing medium for an additional 7 days. All cells were then harvested and stained for cell surface expression using fluorescently labeled anti-CD4 and anti-CD25 antibodies, and for intracellular expression of Foxp3 using fluorescently labeled anti-Foxp3 antibodies, followed by flow cytometry analysis.

[0210] CD4 + CD25 in live T cells 高 Foxp3 + The frequencies (%) of these are shown in Figures 10(A) and (B) as mean ± SD.

[0211] The results in Figure 10(A) show that RA+TGFbeta+AhR agonist- and RA+TGFbeta-treated DCs, whether stimulated with LPS or not, possessed enhanced ability to promote Treg induction compared with matched control DCs. Treatment with RA+TGFbeta+AhR agonist was superior to treatment with RA+TGFbeta in inducing Tregs.

[0212] The results in Figure 10(B) show that RA + TGF beta + AhR agonist-treated DCs possessed enhanced ability to induce Tregs compared with control DCs, Dex / VitD3-treated comparator tolDCs, and TGF beta + AhR agonist-treated DCs. Furthermore, RA + AhR agonist-treated DCs possessed enhanced ability to induce Tregs compared with control DCs and TGF beta + AhR agonist-treated DCs. Treatment with RA + TGF beta + AhR agonist was superior to treatment with RA + AhR agonist in inducing Tregs.

[0213] Example 11 - Effect of RA+TGFbeta+AhR agonist on DCs from the blood of hemophilia A patients in relation to CD83, CD86, ILT3, ILT3 / CD86, GARP+CD141+, CD103, LAP, IL-23 and CD49b+LAG3+Tr1 cells The AhR agonist referred to in the examples is C1. The combination of RA + TGFbeta + AhR agonist was tested for its ability to induce a tolerogenic phenotype in DCs, represented herein by low expression levels of the DC maturation marker CD83, low expression levels of the costimulatory molecule CD86, high expression levels of the tolerogenic marker ILT3, a tolerogenic index defined as ILT3 / CD86 expression, co-expression of CD141 and GARP, expression of CD103, expression of LAP, low spontaneous production of the cytokine IL-23, a reduced ability to induce T cell proliferation, and enhanced induction of Treg cell generation. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce CD141 from healthy donors or subjects with hemophilia. +TolDCs, or their less optimal variants, were differentiated from monocytes by culturing them with GM-CSF and IL-4 as described above and treating them with a combination of RA, TGFbeta, and AhR agonists, as shown in Figures 11(A)-(J). + Differentiated from monocytes.

[0214] The MFI values ​​of live cells are shown as mean ± SD in Figures 11(A) to (G).

[0215] IL-23 concentration values ​​are shown as mean±SD in FIG. 11(H).

[0216] T cell proliferation, assessed as counts per minute (CPM) values ​​from triplicate or more samples, is shown in Figure 11(I) as mean ± SD.

[0217] CD4 + CD49b + LAG3 + The frequency (%) of Tr1 cells is shown as mean ± SD in Figure 11(J).

[0218] These results demonstrate that RA+TGFbeta+AhR agonist-treated DCs generated from CD14+ monocytes isolated from the blood of hemophilia patients acquire a tolerogenic phenotype and function similar to that obtained with DCs generated from healthy donor blood. These results further demonstrate that RA+TGFbeta+AhR agonist-treated DCs generated from CD14+ monocytes isolated from the blood of hemophilia patients exhibit the same reduced ability to induce T cell proliferation and increased regulatory T cell generation as DCs generated from cells derived from healthy blood.

[0219] Example 12 - Viability experiments of DCs treated with RA + TGFbeta + AhR agonist The AhR agonist referred to in the examples is C1. RA+TGFbeta+AhR agonist-treated DCs were generated and tested for their viability before freezing / thawing, expression levels of the DC maturation marker CD83, the tolerogenic marker ILT3, the costimulatory molecule CD86, the tolerogenic index defined as ILT3 / CD86 expression, LAP expression, and CD103 expression. DCs were frozen in either conventional freezing medium or CryoStor10 (CS, Biolife solutions). Control DCs were cultured with GM-CSF and IL-4 for 7 days to denaturate CD14. + Differentiated from monocytes.

[0220] The survival rates before and after freezing are shown as mean ± SD in Figure 12(A).

[0221] The MFI values ​​in live cells for CD83, ILT3, CD86, LAP, and CD103 are shown as mean ± SD in Figures 12(B) to (D) and (F) to (G).

[0222] The tolerogenic index, defined as the ILT3 / CD86 expression (MFI) ratio, is shown in Figure 12(E).

[0223] Table showing the viability and recovery of RA+TGFbeta+AhR agonist treated DCs frozen in CS10 compared to conventional freezing medium (conv). [Table 1]

[0224] The results presented in the table above demonstrate that cryopreservation and thawing of RA+TGFbeta+AhR agonist-treated DCs results in useful cell viability, with RA+TGFbeta+AhR agonist-treated DCs having higher viability compared to control DCs. These results also demonstrate that cryopreservation and thawing of RA+TGFbeta+AhR agonist-treated DCs results in similar expression of surface markers as before freezing, and that using CS10 results in better recovery and viability of RA+TGFbeta+AhR agonist-treated DCs compared to when cells are cryopreserved in standard freezing medium.

[0225] Example 13 - CD83, CD86, ILT3, ILT3 / CD86, CD141+GARP+, CD103, T cell proliferation and CD25 高 Effects of various compounds on DCs associated with Foxp3+ Treg cells Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs. TolDCs, or their less optimal variants, were cultured with GM-CSF and IL-4 as described above and CD14 expression was induced by treatment with various tolerogenic compounds, either alone or in combination: 10 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 10 ng / ml TGF-beta on day 3, and 2 μM RA on day 6. + The synergistic effect of the claimed combination was demonstrated by differentiation from monocytes, as shown in Figure 13. Control DCs were differentiated from CD14 DCs by culturing them with GM-CSF and IL-4 for 7 days. + DCs were differentiated from monocytes. Immunogenic control DCs were generated and tested for phenotypic stability by either unstimulated (gray bars) or stimulated with LPS (black bars) added 2 hours after RA on day 6. All cells were harvested on day 7 and stained for cell surface expression using fluorescently labeled antibodies followed by flow cytometric analysis.

[0226] The MFI values ​​in live cells for CD83, CD86, ILT3, and CD103 are shown as mean ± SD in Figures 13(A) to (C) and (E).

[0227] The tolerogenic index, defined as the ILT3 / CD86 expression (MFI) ratio, is shown in Figure 13(D).

[0228] CD141 + GARP + The frequency (%) of double-positive cells is shown as the mean ± SD in Figure 13(F).

[0229] T cell proliferation, assessed as counts per minute (CPM) values ​​from triplicate or more samples, is shown in Figure 13(G) as mean ± SD.

[0230] CD4 + CD25 + Foxp3 + The frequency (%) of Tregs is shown as mean±SD in FIG. 13(H).

[0231] These results, as seen in Figure 13C, show that RA-treated DCs have higher ILT3 expression and a higher frequency of GARP compared to control DCs. + CD141 + RA-treated DCs induce less T cell proliferation and more Tregs compared to control DCs, Figures 13G and H.

[0232] AhR agonist-treated DCs have lower CD83 and CD86 expression and higher CD103 expression compared to control DCs, as seen in Figures 13A, B, and E, respectively. AhR agonist-treated DCs induce slightly more T cell proliferation and slightly more Tregs compared to control DCs when not stimulated with LPS (Figures 13(G) and (H)).

[0233] TGF-beta-treated DCs have a slightly higher tolerogenic ratio (ILT3 / CD86) and CD103 expression compared to control DCs (Figure 13(D-E)). Furthermore, TGF-beta-treated DCs induce less T cell proliferation and more Tregs compared to control DCs (Figure 13(G)-(H)).

[0234] RA+TGFbeta-treated DCs have lower CD83 and CD86 expression and higher ILT3 expression compared to control DCs, Figures 13A, B and C, respectively. RA+TGFbeta-treated DCs also have a higher frequency of GARP compared to control DCs. + CD141 + cells (Fig. 13(F)). RA+TGF-beta treated DCs, when used in combination, induce less T cell proliferation compared to either one (Fig. 13(G)). In summary, TGF-beta enhances the tolerogenic effect of RA.

[0235] RA+AhR agonist-treated DCs exhibited lower expression of CD83, CD86, and higher expression of ILT3 and CD103 compared to control DCs (Figures 13A, B, C, and E, respectively). RA+AhR agonist-treated DCs also exhibited higher GARP expression compared to control DCs. + CD141 + The frequency of RA-treated DCs was higher than that of RA+TGFbeta or RA+TGFbeta+AhR agonist-treated DCs (Fig. 13(F)). RA+AhR agonist-treated DCs, when used in combination, did not induce significant T cell proliferation compared to either DC (Fig. 13(G)). In summary, AhR agonists enhance the tolerogenic effect of RA.

[0236] The use of AhR agonist together with RA + TGF beta for the generation of tolerogenic DCs resulted in a higher tolerogenic ratio and higher CD103 expression (Figures 13(D) and (E)). Furthermore, the use of RA + TGF beta + AhR agonist resulted in slightly higher Treg induction compared to RA + TGF beta (Figure 13(H)).

[0237] Example 14 - Effect of RA + TGFbeta + AhR agonist on DCs and their ability to suppress tetanus toxoid T cell proliferation The AhR agonist referred to in the examples is C1.

[0238] Additional Materials and Methods Generation of TT-loaded dendritic cells (DCs) CD14 + Monocytes were cultured at a density of 1.25 × 10 in GMP DC medium (CellGenix, Freiburg, Germany) containing HEPES, GlutaMAX, and penicillin-streptomycin solution (Thermo Fisher, Waltham, MA) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 ng / ml; PeproTech, London, UK) and interleukin-4 (IL-4; 100 ng / ml; PeproTech) for 7 days. 6 Cells were cultured at 1000 cells / ml. Cells were replenished with fresh medium and cytokines on day 3. Control DCs were differentiated with GM-CSF and IL-4 without any additional compounds. For antigen-loaded cells, TT (30 nM) was added to the cultures on day 6 4 hours before treatment with a proinflammatory cytokine cocktail consisting of TNF-alpha (10 ng / ml; PeproTech), IL-1 beta (10 ng / ml; PeproTech), and prostaglandin E2 (PGE2; 1 μg / ml; Sigma) to generate immunogenic DCs. TolDCs were generated by treatment with the tolerogenic compounds AhR agonist TGF-beta1 and RA, as previously described. For antigen-loaded cells, TT (30 nM) was added to the cultures on day 6 2 hours after RA addition. On day 7, DCs were harvested, washed extensively, and subjected to phenotyping and functional assays.

[0239] Freezing and thawing PBMCs PBMCs were resuspended in freezing medium containing 10% DMSO and transferred to cryovials. These vials were placed in a freezing container, which was then placed in a -80°C freezer. The freezing container allowed for controlled freezing at approximately -1°C / min. After 24 hours in the -80°C freezer, the cryovials were transferred to a -150°C freezer for long-term storage. PBMCs were thawed in the vials in a 37°C water bath, and the cell suspension was carefully transferred to a 15 ml tube containing cold RPMI supplemented with DNase A with 10% FCS and washed once before use.

[0240] Autologous DC / T cell culture To analyze TT-specific T cell responses, DCs were loaded with 30 nM TT and stimulated with TNF-α, IL-1β, PGE2, and IL-6. The TT-loaded, RA+TGFbeta+AhR agonist-treated DCs (mDCs) were cultured with autologous T cells at a T cell:DC ratio of 10:1 for 6 days. mDCs were used as controls with autologous T cells, with or without TT. DC / T cell cultures were performed in complete medium: CTS™ OpTmizer™ T cell proliferation medium with OpTmizer™ T cell proliferation supplement (Thermo Fisher) and penicillin-streptomycin solution. Proliferation was maintained for the final 18 hours of culture. 3 Determined by 3H-thymidine incorporation.

[0241] RA+TGFβ+AhR agonist-treated DCs were loaded with TT and cocultured with autologous TT-loaded mDCs and T cells to study the TT-specific reduction in T cell proliferation induced by RA+TGFβ+AhR agonist-treated DCs. Both unloaded cells, mDCs, and RA+TGFβ+AhR agonist-treated DCs were used as controls.

[0242] T cell proliferation assessed as counts per minute (CPM) values ​​from triplicate samples is shown in Figure 14 as mean ± SD.

[0243] These results demonstrate that TT-loaded RA+TGFbeta+AhR agonist-treated DCs suppress mDC-induced TT-specific T cell proliferation.

[0244] Example 15 - Effect of RA + TGFbeta + AhR agonist on DC and T cell proliferation associated with CD83, LAP, CD103, ILT3, CD86, ILT3 / CD86 after CD40L treatment Additional Materials and Methods Phenotypic stability and sustained tolerogenicity after CD40L stimulation The phenotypic stability and sustained tolerogenicity of TolDCs were examined by adding CD40L (100 ng / ml) after harvest on day 7. Cells were stimulated for 24 hours, after which DCs were collected, extensively washed, and subjected to phenotyping and functional assays.

[0245] These results demonstrate that RA+TGFbeta+AhR agonist-treated DCs exhibit a stable phenotype after stimulation with CD40L, mimicking DC:T cell contact (Figure 15(A)-(F)). These results further demonstrate that RA+TGFbeta+AhR agonist-treated DCs after stimulation with CD40L have the ability to induce T cell proliferation comparable to unstimulated RA+TGFbeta+AhR agonist-treated DCs (Figure 15(G)). In conclusion, these results demonstrate that RA+TGFbeta+AhR agonist-treated DCs exhibit a sustained phenotype and tolerogenicity after stimulation with CD40L.

[0246] Example 16 - Effect of RA + TGFbeta + AhR agonist on DCs to take up antigens of various sizes and maintain a stable tolerogenic phenotype The AhR agonist referred to in the examples is C1. Additional Materials and Methods Antigen loading of DCs On days 3, 6, or 7 of culture, 100 nM FITC-labeled dextran, 1–10 μg / mL AF488-labeled KLH, 100 nM AF488-labeled tetanus toxoid (TT), or 10–100 nM FVIII was added to cultures of RA, TGF-beta, and AhR agonist-treated DCs for antigen loading. Antigen uptake was detected by flow cytometry. For FVIII detection, after fixation and permeabilization, cells were stained with a FVIII-specific FITC-labeled antibody. DC phenotyping was performed on DCs loaded with 60 nM FVIII for 2, 4, 8, or 20 hours. The percentage of antigen-positive cells was compared with that of RA, TGF-beta, and AhR agonist-treated DCs cultured without antigen.

[0247] Percentage of FITC-dextran, AF488-KLH, or AF488-TT-positive CD11c+RA+TGFbeta+AhR agonist-treated DCs on day 7 after culture with fluorescently labeled antigen from day 3 to day 7 (Figure 16(A)). For FVIII uptake, RA+TGFbeta+AhR agonist-treated DCs were cultured in the presence of 10, 30, or 100 nM FVIII from day 3 to day 7 (Figure 16(B)). On day 7, cells were stained for CD11c and then permeabilized and fixed to allow intracellular staining with a FITC-labeled FVIII-specific antibody (Sanquin, the Netherlands).

[0248] RA+TGFbeta+AhR agonist-treated DCs were cultured in the presence of 60 nM FVIII for 2, 4, 8, or 20 hours on day 7, and the phenotype of the cells in the presence of FVIII was determined (Figures 16(C) to (I)).

[0249] These results demonstrate that RA+TGFbeta+AhR agonist-treated DCs have the ability to take up a diverse range of antigens of various sizes and chemical classes. These results also demonstrate that RA+TGFbeta+AhR agonist-treated DCs have the ability to take up antigens at different time points in culture and have a stable phenotype after being challenged with FVIII at different time points.

[0250] Example 17 - Effect of RA + TGFbeta + AhR agonist on DCs affecting Bregs, B and T cell levels, T cell proliferation and activation The AhR agonist referred to in the examples is C1.

[0251] Additional Materials and Methods B cell / T cell / DC culture: Breg induction On day 7, autologous B cells were isolated from PBMCs by negative selection using a human B cell isolation kit (StemCell technologies). 40,000 B cells were co-cultured with 40,000 autologous T cells and isolated using the EasySep human CD4+ T cell isolation kit (StemCell technologies) as described above. 10,000 RA+TGFbeta+AhR agonist-treated or control DCs were generated as described above. Selected cultures were stimulated with 0.25 μM CpG-OGN to enhance the response. Breg induction was assessed by intracellular IL-10 staining, T cell proliferation determined by KI67 staining, and T cell activation by CD154 expression. Staining for IL-10, KI67, and CD154 was performed as described above using the following fluorescently labeled antibodies: IL-10 (JES3-9D7) from Biolegend, and KI67 (B56) and CD154 (TRAP1) from BD.

[0252] These data suggest that IL-10 expression after co-culture of autologous DCs, B cells, and T cells + CD19 + We demonstrate that the frequency of Bregs, determined as cells, is increased in the presence of RA+TGFbeta+AhR agonist-treated DCs compared to co-culture with control DCs as shown in Figure 17(A). After co-culture of autologous DCs, B cells, and T cells, T cell proliferation and activation were determined by assessing KI67 (Figure 17(B)) and CD154 (Figure 17(C)), respectively.

[0253] Breg cells are immunoregulatory cells that have the ability to suppress the activity of other immune cells. Breg cells are also antigen-presenting cells. Induction of Breg cells activates another level of immunosuppression, and Breg cells can function synergistically with Treg cells. These results demonstrate that RA+TGFbeta+AhR agonist-treated DCs cultured with B cells and T cells induce more IL-10-producing Breg cells compared with DCs cultured with control DCs. Furthermore, these results demonstrate that RA+TGFbeta+AhR agonist-treated DCs cultured with B cells and T cells induce less T cell proliferation and activation compared with control DCs. Decreased T cell proliferation and activation indicates the induction of regulatory cells, Breg cells, Treg cells, or both.

[0254] Example 18 - Effect of RA + TGFbeta + AhR agonist on donor blood-derived DCs when cultured with allogeneic PBMCs DCs generated from monocytes isolated from four healthy donors (designated A–D) were treated with RA, TGFbeta, and AhR agonist. RA, TGFbeta, and AhR agonist-treated DCs showed the same expression profile as described in the previous example, which was a high tolerogenic index defined as low expression levels of CD83 and CD86, high expression of ILT3, CD103, and LAP, and ILT3 / CD86 expression.

[0255] RA+TGFbeta+AhR agonist-treated DCs from donors A to D were cultured with allogeneic PBMCs. For example, DCs from donor A were cultured with PBMCs from donors B, C, and D in an MLR. RA+TGFbeta+AhR agonist-treated DCs had different abilities to induce T cell proliferation when cocultured with allogeneic PBMCs (Figure 18(A)). Furthermore, RA+TGFbeta+AhR agonist-treated DCs had different abilities to induce Tregs in an MLR with allogeneic PBMCs (Figure 18(B)).

[0256] T cell proliferation assessed as the frequency of KI67+ cells for RA+TGFbeta+AhR agonist-treated DCs and control DCs is shown in Figure 18(A).

[0257] Tregs (CD4 + CD25 + Foxp3 + The frequency (%) of each is shown in Figure 18(B).

[0258] These results demonstrate that the majority of donor RA+TGFβ+AhR agonist-treated DCs reduced T cell proliferation and increased Treg induction when cocultured with allogeneic PBMCs. Furthermore, these results indicate that the level of HLA mismatch, which can be assessed by HLA typing, can affect the outcome of the tolerogenic response induced by RA+TGFβ+AhR agonist-treated DCs.

[0259] Example 19 - Clinical Trial Study The study will include two periods: an initial dose escalation portion (Part 1) and an expansion study portion (Part 2).

[0260] First Study Part (Group 1): Part 1 of the trial will evaluate the safety of escalating doses. Four subjects will be included. Additional subjects may be included upon safety review by the Internal Monitoring Board (IMB). Patient 1 will receive three cell doses, spaced two weeks apart. The IMB will determine whether dose escalation can occur after Patient 1 receives his or her final dose. Patient 2 will receive his or her first cell dose at least two weeks after Patient 1 receives his or her final dose, followed by two more doses spaced two weeks apart. Patient 3 will receive his or her first dose at least two weeks after the second patient's final dose, followed by two more doses spaced two weeks apart. Patient 4 will receive his or her first dose at least two weeks after the third patient's final dose, followed by two more doses spaced two weeks apart. If the production yield does not reach the target dose, the patient will receive all collected cells and another patient will be added to the study.

[0261] Expansion Study Part 2 (Group 2): Eight subjects. Expansion Study Part 2 will begin after completion of Dose Escalation Study Part 1. The IMB will evaluate Group 1 after the last dose before proceeding to Expansion Study Part 2 (Group 2). Once the safety of the escalating dose administered to study subjects in Group 1 has been established, eight patients in Group 2 (numbers 5, 6, 7, 8, 9, 10, 11, and 12) will receive three intravenous injections every two weeks at the highest tolerated dose determined in Group 1 or the highest product yield obtained without exceeding the highest tolerated dose determined in Group 1.

[0262] Procedure for generating and transporting active material to each study clinic: Peripheral blood mononuclear cells should be collected from patients via apheresis. Fresh leukapheresis material should be sent to a GMP facility (Radboud University Medical Center, Nijmegen, Netherlands), where monocytes are enriched and cultured with a mixture of compounds that differentiate monocytes into dendritic cells. A tolerance-inducing cocktail of Idogens then converts these dendritic cells into tolerogenic dendritic cells, which are then loaded with recombinant FVIII (Kovaltry®, Octocog alpha) as a final step. The cells are then cryopreserved and shipped back to each study site.

[0263] Route of administration: Intravenous administration of cells. Cryopreserved cell suspensions are thawed and administered intravenously directly at the respective clinical site.

[0264] When patients develop antibodies to FVIII, the first treatment option is ITI therapy with high doses of FVIII. However, ITI is not always successful. It is a very expensive treatment that typically requires daily injections over a year and fails in one-third of patients (Aledort 2019, Carcao 2019, Lacroix-Desmazes 2020, Ljung 2019). It is particularly burdensome for very young children, who often develop inhibitors. The increased morbidity and healthcare costs associated with inhibitors (e.g., bleeding into joints and muscles) and health care costs (D'Angiolella 2018, CDC June 3, 2019) underscore the urgency of identifying strategies to prevent inhibitor development.

[0265] Idogen's Phase I / IIa trial will include HA study subjects who have inhibitors to FVIII, an active immune response to FVIII, and are not suppressed by established treatment protocols for ITI. This patient category has no other standard treatment alternatives remaining to eradicate FVIII inhibitors. Although the risks of this study are unknown, published clinical trials with autologous tolerogenic dendritic cell therapy have not reported any serious safety issues (Bell 2017, Benham 2015, Dhodapkar 2001, Dhodapkar and Steinman et al. 2002, Giannoukakis 2011, Harry 2010, Hilkens and Isaacs et al. 2013, Jauregui-Amezaga 2015, Joo 2014, Ten Brinke 2015, Thomas 2011, Willekens and Cools 2018, Willekens 2019, Zubizarreta 2019), and the cells display a tolerogenic phenotype in in vitro experiments (Lee 2016, Gordon 2016). 2014, Lutz 2000, Steinbrink 1997, Bartosik-Psujek 2010, Huang 2001, Hussien 2001, Bellinghausen 2012, Boks 2012, Chu 2012, Raich-Regue 2012, Saito 2011). Nevertheless, the possibility of serious adverse events possibly related to treatment cannot be excluded. Prophylactic antihistamines are an alternative option for patients receiving ItolDC-028.

[0266] A potential risk in studies involving subjects with bleeding diathesis is the occurrence of bleeding during blood draws, leukapheresis, or cell infusion. However, bleeding after venipuncture is rare in experienced hands, and subjects are managed by personnel familiar with managing patients with bleeding disorders. If venipuncture is traumatic, digital pressure or a pressure dressing at the puncture site can prevent further complications. Subcutaneous, intradermal, and small intramuscular injections rarely result in hematomas if firm digital pressure is maintained for at least 5 minutes (Powell and Rodgers et al., 2013).

[0267] Example 20 - Effect of RA + TGFbeta + AhR agonist on DCs from the blood of patients with autoimmune diseases associated with the B cell generation and T cell regulatory functions of IL-10 RA+TGFbeta+AhR agonist-treated DCs are generated from monocytes from patients with autoimmune diseases caused by one or several well-characterized antigens. RA+TGFbeta+AhR agonist-treated DCs are loaded with one or several well-characterized antigens. The DCs may then be co-cultured with autologous T cells. T cells are less activated compared to T cells co-cultured with control DCs loaded with the same antigens. Furthermore, co-culture of RA+TGFbeta+AhR agonist-treated DCs and T cells will induce a higher frequency of regulatory T cells compared to T cells co-cultured with control DCs under the same conditions.

[0268] RA+TGFbeta+AhR agonist-treated DCs generated from patients with autoimmune disease are co-cultured with B cells, which will have increased frequencies of regulatory markers such as IL-10.

[0269] RA+TGFbeta+AhR agonist-treated DCs generated from patients with autoimmune disease are co-cultured with B cells and T cells, and the T cells will have increased regulatory function compared to B cells and T cells co-cultured with control DCs.

[0270] The induction of tolerogenic phenotype and function of both T and B cells after co-culture with RA+TGFbeta+AhR agonist-treated DCs is beneficial in autoimmune diseases, as these cells reduce the autoreactive activity of T and B cells.

[0271] Example 21 - Effect of RA + TGF beta + AhR agonist on DCs from the blood of patients with autoimmune diseases and treated with several unknown and / or complex antigens or tissue samples in relation to the B cell generating and T cell regulatory functions of IL-10 RA+TGFbeta+AhR agonist-treated DCs are generated from monocytes of patients with autoimmune diseases related to several unknown and / or complex antigens. The RA+TGFbeta+AhR agonist-treated DCs are loaded with several unknown and / or complex antigens or tissue sample extracts from the same patients and then co-cultured with autologous T cells. The T cells will be less activated compared to T cells co-cultured with control DCs loaded with the same antigens. Furthermore, co-culture of RA+TGFbeta+AhR agonist-treated DCs with T cells will induce a higher frequency of regulatory T cells compared to T cells co-cultured with control DCs under the same conditions.

[0272] RA+TGFbeta+AhR agonist-treated DCs generated from autoimmune disease patients are co-cultured with B cells, which will have a higher frequency of regulatory markers such as IL-10.

[0273] RA+TGFbeta+AhR agonist-treated DCs are co-cultured with B cells and T cells, which will have increased regulatory function compared to B cells and T cells co-cultured with control DCs.

[0274] The induction of tolerogenic phenotype and function of both T and B cells after co-culture with RA+TGFbeta+AhR agonist-treated DCs is beneficial in autoimmune diseases, as these cells attenuate the autoreactive activity of T and B cells.

[0275] Example 22 - Effect of RA + TGFbeta + AhR agonist on DCs from the blood of patients with autoimmune disease treated without antigen, in relation to Breg induction after in vivo administration RA+TGF-beta+AhR agonist-treated DCs are generated from monocytes of patients with autoimmune diseases related to one or several unknown antigens. RA+TGF-beta+AhR agonist-treated DCs are injected in situ without prior antigen loading or freezing, allowing the DCs to take up disease-associated antigens and present them to T cells, thereby inducing disease-specific regulatory T cells.

[0276] RA+TGFbeta+AhR agonist-treated DCs from patients with autoimmune diseases are cultured with one or several unknown antigens. The DCs are administered in situ. RA+TGFbeta+AhR agonist-treated DCs will induce Bregs, further promoting tolerance induction to transplantation and reducing harmful antibody production. This is in contrast to what happens when recipients receive DCs from immunogenic donors or no DCs at all.

[0277] Example 23 - Effect of RA + TGF beta + AhR agonist on T cell activation and Treg induction in DCs derived from the blood of hemophilia A patients and loaded with FVIII Hemophilia A patients who are resistant to treatment with exogenous FVIII have been found to produce FVIII-specific antibodies (inhibitors) that upregulate FVIII-specific effector T cells that have the ability to activate B cells.

[0278] RA+TGFbeta+AhR agonist-treated DCs generated from monocytes from a hemophilia A patient were loaded with FVIII according to the method described above, and the RA+TGFbeta+AhR agonist-treated DCs were cocultured in vitro with T cells from the same hemophilia A patient in a recall antigen assay. T cells were less activated compared with T cells cocultured with control DCs. Furthermore, T cells cocultured with RA+TGFbeta+AhR agonist-treated DCs induced a higher frequency of regulatory T cells than T cells cocultured with control DCs under the same conditions.

[0279] Example 24 - Effect of RA + TGFbeta + AhR agonist on allogeneic donor-derived DCs in relation to Treg and Breg induction RA+TGFbeta+AhR agonist-treated DCs are generated from allogeneic donor monocytes. When administered to transplant recipients, RA+TGFbeta+AhR agonist-treated DCs will induce autoregulatory T cells and tolerance to the transplant. This is in contrast to what occurs when recipients receive DCs from immunogenic donors or no DCs at all.

[0280] RA + TGFbeta + AhR agonist-treated DCs from the donor are administered to the transplant recipient. RA + TGFbeta + AhR agonist-treated DCs will induce Bregs, further promoting tolerance induction to the transplant and reducing harmful antibody production. This is in contrast to what happens when recipients receive DCs from immunogenic donors or no DCs at all.

[0281] Example 25 - Effect of RA + TGFbeta + AhR agonist on DCs from allograft recipients in relation to Treg and Breg induction RA+TGFbeta+AhR agonist-treated DCs are generated from monocytes from allogeneic transplant recipients. The RA+TGFbeta+AhR agonist-treated DCs are loaded with a mixture of donor-derived antigens before administration to the transplant recipient. Such a mixture of antigens may be derived from donor blood or a tissue sample from the donor, preferably a sample of donor cells, tissues, organs, or other grafts to be transplanted. When administered to a transplant recipient, the RA+TGFbeta+AhR agonist-treated DCs will induce autoregulatory T cells and tolerance to the transplant. This is in contrast to what occurs when the recipient receives RA+TGFbeta+AhR agonist-treated DCs that do not contain donor antigens or no DCs at all.

[0282] RA+TGFbeta+AhR agonist-treated DCs from the recipient are loaded with antigens from the donor and administered to the transplant recipient. RA+TGFbeta+AhR agonist-treated DCs will induce Bregs, further promoting tolerance to the transplant and reducing harmful antibody production. This is in contrast to what happens when recipients receive RA+TGFbeta+AhR agonist-treated DCs that do not contain donor antigens or no DCs at all.

[0283] (References) [Table 2]

[0284] (abbreviation) [Table 3]

[0285] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a stated integer, step, group of integers or group of steps, but not the exclusion of any other integer, step, group of integers or group of steps.

[0286] All patents and patent applications mentioned herein are incorporated by reference in their entirety. The present application provides the following aspects of the invention. (Aspect 1) 1. An ex vivo method for obtaining tolerogenic antigen-presenting cells capable of inducing tolerance to an antigen, comprising: (a) isolating monocytes from a sample obtained from a mammal; and (b) culturing the isolated monocytes in cell culture to induce differentiation of the monocytes into antigen-presenting cells having a tolerogenic phenotype; wherein the cell culture comprises (i) retinoic acid and TGF-beta, (ii) retinoic acid, TGF-beta and an AhR agonist, or (iii) retinoic acid and an AhR agonist. (Aspect 2) 2. The method of embodiment 1, wherein the cell culture comprises retinoic acid and TGF-beta. (Aspect 3) 2. The method of embodiment 1, wherein the cell culture comprises retinoic acid, TGF beta, and an AhR agonist. (Aspect 4) 2. The method of embodiment 1, wherein the cell culture comprises retinoic acid and an AhR agonist. (Aspect 5) The method of any one of aspects 1 to 4, wherein said AhR agonist is N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxoquinoline-3-carboxamide (IMA-06201). (Aspect 6) The method of any one of aspects 1 to 5, wherein the cell culture further comprises GM-CSF and IL-4 to induce differentiation of monocytes into antigen-presenting cells. (Aspect 7) The method of embodiment 6, wherein GM-CSF and IL-4 are added to the cell culture before or simultaneously with adding any one of TGF beta, AhR agonist, and retinoic acid to the cell culture. (Aspect 8) 8. The method of embodiment 6 or 7, wherein the AhR agonist is added to the cell culture at a first dose at the same time that the GM-CSF and IL-4 are first added to the cell culture. (Aspect 9) 9. The method of any one of aspects 6 to 8, wherein after the first dose of AhR agonist is added to the cell culture, a second dose of TGF beta and AhR agonist is added to the cell culture. (Aspect 10) 10. The method of any one of aspects 6 to 9, wherein the retinoic acid is added to the cell culture after the TGF beta and the second dose of the AhR agonist are added to the cell culture. (Aspect 11) 11. The method of any one of aspects 6 to 10, wherein the GM-CSF and IL-4 are added to the cell culture at a second dose. (Aspect 12) 12. The method of embodiment 11, wherein the second dose of GM-CSF and IL-4 is added to the cell culture at the same time that the TGF beta and the second dose of AhR agonist are added to the cell culture. (Aspect 13) 7. The method of embodiment 6, wherein the GM-CSF is added to the cell culture before or simultaneously with the addition of any one of TGF beta, AhR agonist, and retinoic acid to the cell culture, and the IL-4 is added to the cell culture after a first dose of the AhR agonist is added to the cell culture. (Aspect 14) 20. The method of claim 6 or 13, wherein after the first dose of AhR agonist is added to the cell culture, a second dose of TGF beta and AhR agonist is added to the cell culture. (Aspect 15) 15. The method of any one of aspects 6 or 13-14, wherein the retinoic acid is added to the cell culture after the TGF beta and the second dose of the AhR agonist are added to the cell culture. (Aspect 16) 16. The method of any one of aspects 6 or 13 to 15, wherein the GM-CSF is added to the cell culture in a second dose. (Aspect 17) 17. The method of embodiment 16, wherein the second dose of GM-CSF is added to the cell culture at the same time that the TGF beta and the second dose of AhR agonist are added to the cell culture. (Aspect 18) The method according to any one of aspects 1 to 17, wherein the tolerogenic antigen-presenting cells are dendritic cells. (Aspect 19) The isolated monocytes are CD14 + The method of any one of embodiments 1 to 18, wherein the cells are monocytes. (Aspect 20) The method of any one of embodiments 1 to 19, wherein the mammal is a human. (Aspect 21) The method according to any one of aspects 1 to 20, wherein the sample is a sample of peripheral blood mononuclear cells. (Aspect 22) 22. The method of any one of aspects 1 to 21, wherein the cell culture comprises the antigen or an epitope-containing fragment thereof. (Aspect 23) 23. The method of embodiment 22, wherein the antigen or epitope-bearing fragment thereof is bound to isolated monocytes. (Aspect 24) 23. The method of embodiment 22, wherein the antigen or epitope-bearing fragment thereof is added to the cell culture. (Aspect 25) 25. The method of embodiment 24, wherein the antigen or epitope-bearing fragment is added to the cell culture simultaneously with or after the retinoic acid is added to the cell culture. (Aspect 26) 26. The method of embodiment 24 or embodiment 25, wherein said antigens or epitope-containing fragments are a pool of antigens and / or a pool of epitope-containing fragments thereof. (Aspect 27) 27. The method of any one of aspects 1 to 22 and 24 to 26, wherein the antigen or epitope-bearing fragment thereof is or is derived from a biologic. (Aspect 28) 28. The method of aspect 27, wherein the biologic is Factor VIII or a derivative or fragment thereof. (Aspect 29) 28. The method of aspect 27, wherein the biologic is Factor IX or a derivative or fragment thereof. (Aspect 30) 28. The method of embodiment 27, wherein the biologic is an antibody or an antibody fragment thereof. (Aspect 31) The method of any one of aspects 1 to 22 and 24 to 26, wherein the antigen or epitope-containing fragment thereof is bound to an allograft. (Aspect 32) The method according to any one of aspects 1 to 22 and 24 to 26, wherein the antigen or epitope-containing fragment thereof is an autoantigen or is derived from an autoantigen. (Aspect 33) 33. The method of any one of aspects 1 to 32, wherein the tolerogenic antigen-presenting cells have high expression of ILT3 and low expression of CD83 and CD86 when unstimulated or when stimulated with an immunogenic stimulus such as, for example, LPS or a proinflammatory cytokine or a proinflammatory cytokine. (Aspect 34) 34. The method of embodiment 33, wherein the tolerogenic antigen-presenting cells express CD103. (Aspect 35) The method according to any one of embodiments 1 to 34, wherein the tolerogenic antigen-presenting cells induce regulatory T cells when cultured with T cells. (Aspect 36) The method according to any one of embodiments 1 to 35, wherein the tolerogenic antigen-presenting cells are further cultured with T cells, thereby inducing the generation of regulatory T cells. (Aspect 37) The regulatory T cells are CD4 + CD25 hi Foxp3 + or Tr1 regulatory T cells. (Aspect 38) The method according to any one of aspects 1 to 37, wherein the tolerogenic antigen-presenting cells have low ability to induce T cell proliferation when unstimulated or when stimulated with an immunogenic stimulus such as LPS, an inflammatory cytokine, or an inflammatory cytokine cocktail. (Aspect 39) The method according to any one of aspects 1 to 38, wherein the tolerogenic antigen-presenting cells have high regulatory T cell induction ability when unstimulated or when stimulated with an immunogenic stimulus such as LPS, an inflammatory cytokine, or an inflammatory cytokine cocktail. (Aspect 40) A tolerogenic antigen-presenting cell or a population thereof obtainable or obtained by the method according to any one of aspects 1 to 35. (Aspect 41) A tolerogenic antigen-presenting cell or a population thereof obtainable or obtained by the method according to any one of aspects 1 to 23. (Aspect 42) A tolerogenic antigen-presenting cell or population thereof that expresses CD103 and has high expression of CD141, GARP, and ILT3, and low expression of CD83 and CD86, either unstimulated or stimulated with an immunogenic stimulus such as LPS or an inflammatory cytokine or an inflammatory cytokine cocktail. (Aspect 43) Plus: (i) HLA-G; (ii) BTLA; (iii) MERTK; (iv) LAP 43. The tolerogenic antigen-presenting cell or population thereof according to embodiment 42, characterized by expressing one or more of: (Aspect 44) 44. An antigen-presenting cell or a population thereof according to aspect 42 or aspect 43, which exhibits low production of IL-23 when unstimulated or when stimulated with an immunogenic stimulus such as, for example, LPS or a pro-inflammatory cytokine or a pro-inflammatory cytokine cocktail. (Aspect 45) 45. The tolerogenic antigen-presenting cell or population thereof according to any one of Aspects 40 to 44, for use in a method of treating a mammalian subject having or at risk of having an immune response to an antigen, the method comprising administering the tolerogenic antigen-presenting cell to the mammalian subject, thereby establishing immune tolerance to the antigen. (Aspect 46) 45. A method of treating a mammalian subject having or at risk of having an immune response to an antigen, the method comprising administering to the mammalian subject a tolerogenic antigen-presenting cell or population thereof according to any one of aspects 40 to 44, thereby establishing immune tolerance to the antigen. (Aspect 47) 1. A tolerogenic antigen-presenting cell or population thereof for use in a method of treating a mammalian subject having or at risk of having an immune response to an antigen, said method comprising: (i) obtaining, by the method of any one of aspects 1 to 35, a tolerogenic antigen-presenting cell or a population thereof capable of inducing tolerance to the antigen, the tolerogenic antigen-presenting cell or a population thereof being obtained from a sample of isolated monocytes from the mammalian subject; and (ii) administering the tolerogenic antigen-presenting cells or population thereof back to the mammalian subject to establish immune tolerance to the antigen. The tolerogenic antigen-presenting cell or population thereof, comprising: (Aspect 48) 1. A method of treating a mammalian subject having or at risk of having an immune response to an antigen, comprising: (i) obtaining a tolerogenic antigen-presenting cell or a population thereof capable of inducing tolerance to the antigen by the method of any one of aspects 1 to 35, the antigen-presenting cell or a population thereof being obtained from a sample of isolated monocytes from the mammalian subject; and (ii) administering the tolerogenic antigen-presenting cells or population thereof back to the mammalian subject to establish immune tolerance to the antigen. The method comprising: (Aspect 49) 42. The tolerogenic antigen-presenting cell or population thereof of embodiment 41 for use in a method for preventing immune rejection of a donor-derived allograft in a recipient subject, the method comprising administering to the recipient subject tolerogenic antigen-presenting cells obtained from monocytes isolated from a sample taken from the donor, thereby establishing tolerance to the allograft. (Aspect 50) 42. A method for preventing immune rejection of a donor-derived allograft in a recipient subject, comprising administering to the recipient subject tolerogenic antigen-presenting cells according to embodiment 41 obtained from monocytes isolated from a sample taken from the donor, thereby establishing tolerance to the allograft. (Aspect 51) 42. The tolerogenic antigen-presenting cell or population thereof of embodiment 41 for use in a method for preventing immune rejection of a donor-derived allograft in a recipient subject, the method comprising administering to the recipient subject tolerogenic antigen-presenting cells obtained from monocytes isolated from a sample taken from the recipient, thereby establishing tolerance to the allograft. (Aspect 52) 42. A method for preventing immune rejection of a donor-derived allograft in a recipient subject, comprising administering to the recipient subject tolerogenic antigen-presenting cells according to embodiment 41 obtained from monocytes isolated from a sample taken from the recipient, thereby establishing tolerance to the allograft.

Claims

1. 1. An ex vivo method for obtaining tolerogenic dendritic cells capable of inducing tolerance to an antigen, comprising: (a) isolating monocytes from a sample obtained from a mammal; and (b) culturing the isolated monocytes in cell culture to induce differentiation of the monocytes into dendritic cells having a tolerogenic phenotype; Including, the cell culture comprises retinoic acid, TGF beta, and an AhR agonist; the AhR agonist is added to the cell culture at a first dose; after the first dose of the AhR agonist is added to the cell culture, the TGF-beta and a second dose of the AhR agonist are added to the cell culture; and The method, wherein the retinoic acid is added to the cell culture after the TGF-beta and the second dose of the AhR agonist have been added to the cell culture.

2. The method of claim 1, wherein the AhR agonist is IMA-06201 (C1).

3. 3. The method of claim 1 or 2, wherein the cell culture further comprises GM-CSF and IL-4 to induce differentiation of monocytes into dendritic cells.

4. The GM-CSF and IL-4 are added to the cell culture before or simultaneously with the addition of any one of TGF-beta, AhR agonist, and retinoic acid to the cell culture; or The method of claim 3, wherein the GM-CSF is added to the cell culture before or simultaneously with the addition of any one of TGF-beta, an AhR agonist, and retinoic acid to the cell culture, and the IL-4 is added to the cell culture after a first dose of the AhR agonist is added to the cell culture.

5. 5. The method of claim 3 or 4, wherein the GM-CSF and IL-4 are added to the cell culture in a second dose.

6. the second dose of GM-CSF and IL-4 are added to the cell culture at the same time that the TGF-beta and the second dose of AhR agonist are added to the cell culture; or 6. The method of claim 5, wherein the second dose of GM-CSF is added to the cell culture at the same time that the TGF-beta and the second dose of AhR agonist are added to the cell culture.

7. The isolated monocytes are CD14 + The method of any one of claims 1 to 6, wherein the cells are monocytes.

8. the mammal is a human, and / or The method of any one of claims 1 to 7, wherein the sample is a sample of peripheral blood mononuclear cells.

9. The method of any one of claims 1 to 8, wherein the cell culture comprises the antigen or an epitope-containing fragment thereof.

10. the antigen or epitope-containing fragment thereof is bound to the isolated monocytes; or the antigen or an epitope-containing fragment thereof is added to the cell culture, and / or 10. The method of claim 9, wherein the antigens or epitope-containing fragments are a pool of antigens and / or a pool of epitope-containing fragments thereof.

11. 11. The method of claim 10, wherein the antigen or epitope-containing fragment is added to the cell culture before, simultaneously with, or after the retinoic acid is added to the cell culture.

12. 12. The method of any one of claims 1 to 11, wherein the antigen or epitope-containing fragment thereof is or is derived from a biologic.

13. the biological agent is Factor VIII or a derivative or fragment thereof, or Factor IX or a derivative or fragment thereof, or an antibody or an antibody fragment thereof, or the antigen or epitope-bearing fragment thereof is associated with an allograft; or 13. The method of claim 12, wherein the antigen or epitope-containing fragment thereof is or is derived from an autoantigen.

14. 14. The method of any one of claims 1 to 13, wherein the tolerogenic dendritic cells have, unstimulated or stimulated, an expression level of ILT3 that is at least 150% of that of control cells, an expression level of CD83 that is 70% or less of that of control cells, and an expression level of CD86 that is 80% or less of that of control cells.

15. 15. The method of claim 14, wherein the tolerogenic dendritic cells are stimulated with an immunogenic stimulus.

16. The method of claim 15, wherein the immunogenic stimulus is LPS or an inflammatory cytokine or an inflammatory cytokine cocktail.

17. The method of any one of claims 14 to 16, wherein the tolerogenic dendritic cells express CD103.

18. the tolerogenic dendritic cells are cultured with T cells to induce regulatory T cells; and / or The method of any one of claims 1 to 17, wherein the tolerogenic dendritic cells are further cultured with T cells, thereby inducing the generation of regulatory T cells.

19. The regulatory T cells are CD4 + CD25 hi Foxp3 + or Tr1 regulatory T cells.

20. The method according to any one of claims 1 to 19, wherein the tolerogenic dendritic cells, when unstimulated or stimulated, have a T cell proliferation induction capacity that is 70% or less of that of control cells and / or a Treg induction capacity that is at least 130% of that of control cells.

21. 21. The method of claim 20, wherein the tolerogenic dendritic cells are stimulated with an immunogenic stimulus.

22. 22. The method of claim 21, wherein the immunogenic stimulus is LPS or an inflammatory cytokine or an inflammatory cytokine cocktail.

23. A tolerogenic dendritic cell or a population thereof obtained by the method according to any one of claims 1 to 22.

24. The cell or population thereof is: (i) HLA-G; (ii) BTLA; (iii) MERTK; (iv) LAP and / or The tolerogenic dendritic cells or population thereof according to claim 23, wherein the dendritic cells, when unstimulated or stimulated, exhibit a production level of IL-23 that is 100% or less of that of control cells.

25. 25. The tolerogenic dendritic cell or population thereof of claim 24, wherein the tolerogenic dendritic cell or population thereof is stimulated with an immunogenic stimulus.

26. 26. The tolerogenic dendritic cell or population thereof according to claim 25, wherein the immunogenic stimulus is LPS or an inflammatory cytokine or an inflammatory cytokine cocktail.

27. 27. The tolerogenic dendritic cells or population thereof of any one of claims 24 to 26, for use in a method of treating a mammalian subject having or at risk of having an immune response to an antigen, said method comprising administering said tolerogenic dendritic cells to said mammalian subject, thereby establishing immune tolerance to said antigen.

28. The tolerogenic dendritic cells or population thereof are obtained from a sample of isolated monocytes from the mammalian subject and are capable of inducing tolerance to an antigen by the method of any one of claims 1 to 22, and 28. The tolerogenic dendritic cells or population thereof for use according to claim 27, wherein the tolerogenic dendritic cells or population thereof are administered back to the mammalian subject, thereby establishing tolerance to the antigen.

29. 24. The tolerogenic dendritic cells or populations thereof of claim 23 for use in a method for preventing immune rejection of an allograft in a recipient subject, comprising: the allograft is donor-derived; the method comprises administering the tolerogenic dendritic cells to the recipient subject, thereby establishing tolerance to the allograft; The tolerogenic dendritic cells or population thereof, wherein the tolerogenic dendritic cells are obtained from monocytes isolated from a sample taken from the donor or the recipient.

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