Selective tolerization - Methods for selectively generating tolerogenic dendritic cells

Abstract

The present invention relates to a method for selectively generating tolerogenic dendritic cells. The present invention further relates to patient-specific tolerogenic dendritic cells obtained by the described method, which reduce the immunogenicity of the graft when administered prior to transplantation. The present invention also relates to patient-specific tolerogenic dendritic cells for use in reducing or preventing inflammatory conditions, such as graft-versus-host disease. In particular, the method can be used to reduce graft-versus-host disease. The tolerogenic dendritic cells of the present invention can also be used to treat autoimmune diseases.

Description

[Technical field]

[0001] The present invention relates to a method for selectively producing tolerogenic dendritic cells. The present invention further relates to a method for reducing the immunogenicity of a graft prior to transplantation by generating tolerogenic dendritic cells. The present invention also relates to tolerogenic dendritic cells, including the tolerogenic dendritic cells obtained by the described method. The tolerogenic dendritic cells reduce the immunogenicity of a graft when administered prior to transplantation. The present invention also relates to tolerogenic dendritic cells for use in reducing or preventing inflammatory conditions, such as graft-versus-host disease and / or autoimmune diseases. In particular, the tolerogenic dendritic cells can be used to reduce graft-versus-host disease. [Background technology]

[0002] Transplantation of organs, tissues or cells from one genetically distinct individual (donor) to another (recipient) remains the definitive treatment for several diseases, but is limited by the availability of organs and donors. Suitable donors are individuals with identical or nearly identical profiles of cell surface antigens known as major histocompatibility complexes (MHC) or HLA antigens. However, transplants from the same individual (autologous or autotransplants) are not always available, and the widespread application of transplants from different individuals (allogeneic or allogeneic transplants) is limited by differences in MHC or HLA antigens. Since there are many alternative forms (alleles) of each of the HLA antigens, it is highly unlikely that two unrelated individuals will have an exact HLA match. Side effects after transplantation of an organ or tissue from one genetically distinct individual to another can be very dangerous. The main side effect is the immunological rejection of the transplanted organ or tissue. This can be caused by the immune system of the recipient (organ, skin, etc.) attacking the graft. In addition, the graft can also attack the recipient (GvHD). To prevent or limit rejection, patients typically receive a combination of immunosuppressive drugs. These drugs are usually globally immunosuppressive, greatly increasing the recipient's susceptibility to serious infections. These side effects have prompted a search for therapies that can more selectively suppress rejection of transplanted tissues without harming other vital organs, while leaving the rest of the immune system intact. One approach to reverse transplanted organ rejection is the application of extracorporeal photopheresis (ECP), a process that involves the treatment of blood with a DNA crosslinking agent, such as 8-MOP, and UV light. One possible mechanism that explains the positive effects of ECP in treating graft-versus-host disease (GvHD) is that monocytes contained in blood samples differentiate into immunosuppressive dendritic cells when exposed to a combination of 8-MOP and UV light. These immunosuppressive dendritic cells are postulated to promote immune tolerance.However, it would be highly valuable to improve selective tolerization of allogeneic transplants in order to increase the pool of suitable donors, advance therapeutic approaches, and / or prevent autoimmune diseases, especially graft-versus-host disease, and also to elucidate possible mechanisms behind the immunosuppressive effects of ECP and ECP-like processes. Summary of the Invention

[0003] One object of the present invention is to provide a method for selectively producing tolerogenic dendritic cells. Another object is to provide a method for selectively producing antigen-specific tolerogenic dendritic cells. Another object is to provide a method for selectively producing tolerogenic dendritic cells that reduce the immunogenicity of a graft prior to transplantation.

[0004] Another object of the present invention is to provide tolerogenic dendritic cells, including ex vivo tolerogenic dendritic cells.

[0005] Another object of the present invention is to provide tolerogenic dendritic cells obtained by the method of the present invention.

[0006] Another object of the present invention is to provide tolerogenic dendritic cells for use in preventing or alleviating GvHD.

[0007] Another object of the invention is to provide tolerogenic dendritic cells for use in preventing or reducing rejection of organ transplants, for example, skin.

[0008] Yet another object is to provide a method for treating GvHD.

[0009] Another object of the present invention is to provide tolerogenic dendritic cells for use in treating autoimmune diseases.

[0010] Yet another object is to provide a method for treating autoimmune diseases.

[0011] These and other objects, as will become apparent from the following description, are solved by the subject matter of the independent claims. Some of the preferred embodiments of the invention form the subject matter of the dependent claims. Further embodiments of the invention can be seen from the following description.

[0012] The present invention, as illustratively described below, may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0013] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims.

[0014] The present invention is based in part on the data and clinical trials presented herein below, which provide the insight that apoptotic dendritic cells can tolerize the immune system of a future transplant recipient to allogeneic transplantation if they are incorporated into healthy dendritic cells. Surprisingly, the inventors have found that the apoptotic dendritic cells can be derived from the donor or the future recipient. Also, if the apoptotic dendritic cells are derived from a donor, the donor may or may not be HLA matched to the future recipient. Preferably, healthy dendritic cells are produced in vitro in the present context of the ECP-derived process. The inventors have observed that dendritic cells can be efficiently generated when a leukapheresed blood sample containing monocytes and platelets is passed through a plate. However, the blood sample must contain at least monocytes. Monocytes have been found to mature into healthy dendritic cells when subjected to shear stress. If platelets are present, the maturation process can be improved. Importantly, using this method, monocytes can be matured into healthy dendritic cells without the need for the addition of expensive cytokine cocktails. Because the above process mimics some of the aspects postulated to take place in vivo (see Han et al., 2020, “Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes”, Science Advances), dendritic cells generated by plate passage are hereafter referred to as “physiological dendritic cells” (phDCs).

[0015] It is therefore hypothesized that apoptotic dendritic cells derived from transplant donors or future recipients provide phDCs with a source of antigens from the future recipient, thereby initiating an efficient tolerogenic immune response. The present invention promotes and improves this process by directly contacting apoptotic dendritic cells with phDCs. By direct incubation, administration of selectively produced tolerogenic dendritic cells can improve the future recipient's tolerance to allogeneic transplantation, thus reducing or eliminating inflammatory conditions such as GvHD. The phDCs of the present invention are more efficient than dendritic cells produced by other methods, such as exposure to cytokine cocktails. Furthermore, phDCs can be produced in a standardized and reproducible manner, resulting in good control of the process of generating potent tolerogenic dendritic cells. Furthermore, phDCs can be used to treat autoimmune diseases.

[0016] The above-described selective generation of tolerogenic dendritic cells can be utilized in different ways, which are described below as first, second and third embodiments.

[0017] First aspect: A method for selectively generating tolerogenic dendritic cells in which donor dendritic cells undergo apoptosis In a first aspect, the present invention provides a method for producing a composition comprising the steps of: a) providing dendritic cells from a donor; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic donor dendritic cells of step b) with the physiological recipient dendritic cells from step c); The present invention relates to a method comprising the steps of:

[0018] In one embodiment, the method is performed prior to transplantation. In one embodiment, the method is for selectively reducing the immunogenicity of a graft or portion thereof prior to transplantation. In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed sequentially. Thus, phDCs from the recipient are not exposed to apoptotic factors. In one embodiment, phDCs from the recipient are not exposed to apoptotic factors at any time during the method.

[0019] In one embodiment, the dendritic cells in step a) are obtained from a donor. In one embodiment, the dendritic cells in step c) are obtained from the recipient. In principle, after the dendritic cells are obtained from the donor, the donor dendritic cells may or may not be viable. The donor dendritic cells may, for example, become apoptotic and be cryopreserved until they are combined with the physiological recipient dendritic cells from step c).

[0020] In one embodiment, the donor is allogeneic. In one embodiment, the donor is a haplo-donor.

[0021] In one embodiment, the present invention provides a) providing dendritic cells from a donor; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic donor dendritic cells of step b) with physiological recipient dendritic cells from step c); d1) co-incubating the mixture of step d); The present invention relates to a method comprising the steps of:

[0022] In one embodiment, step d1) of co-incubating the mixture is carried out for at least 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h or 6 h.

[0023] In one embodiment, step d) of combining the apoptotic donor dendritic cells with physiological dendritic cells from the recipient is performed within the recipient. In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed sequentially, thus, phDCs from the recipient are not exposed to apoptotic factors.

[0024] In step b) of the method, the dendritic cells obtained from the donor in step a) of the method are exposed to an apoptotic agent. In one embodiment, the apoptotic agent comprises psoralen and UVA, riboflavin phosphate and UVA, and / or aminolevulinic acid and light. Particularly preferred psoralens are 8-MOP and amotosalen. Most preferred psoralen is 8-MOP. In the most preferred embodiment, the apoptotic agent is a combination of 8-MOP and UVA. The embodiment should preferably be selected such that essentially all dendritic cells from the donor are contacted with the apoptotic agent. In the case of 8-MOP / UVA, essentially all dendritic cells from the donor should be contacted with 8-MOP and exposed to UVA light. A typical dose of 8-MOP and UVA is 1 J / cm combined with a concentration of 8-MOP between 100 ng / mL and 300 ng / mL. 2 ~3J / cm 2 The UVA is.

[0025] In a preferred embodiment, the dose of UVA is 3 J / cm 2 Below, 2J / cm 2 or less than 1J / cm 2 In other preferred embodiments, the dose of 8-MOP is less than or equal to 300 ng / mL, 250 ng / mL, 200 ng / mL, or 100 ng / mL. In particularly preferred embodiments, the dose of 8-MOP is 200 ng / mL and the dose of UVA is 1 J / cm. 2 It is.

[0026] In the context of the present invention, dendritic cells can in particular be obtained by plate passage of monocytes using a process derived from extracorporeal photopheresis (ECP).

[0027] Methods and apparatus for in vitro activation of monocytes and generation of dendritic cells therefrom are described in WO 2014 / 106629 A1, WO 2014 / 106631 A1, WO 2016 / 001405 A1, and WO 2017 / 005700 A1, each of which is incorporated by reference herein in its entirety. ECP refers to the process of activating and differentiating monocytes derived from a blood sample or fraction thereof into healthy, physiological dendritic cells, also referred to herein as phDCs, by exposing the monocytes to mechanical stress (e.g., shear force) and plasma components (e.g., platelets) or derivatives or mimetics thereof. ECP-derived processes, including differentiation of monocytes into ECPs and phDCs, can be performed in large-scale ECP devices, such as clinical ECP devices (e.g., THERAKOS® CELLEX® devices), or in small-scale ECP devices, such as the Transimmunization plates described in WO 2017 / 005700 A1; or in bags, such as plastic bags (e.g., plastic bags for blood, blood components, cell therapy, etc.).

[0028] The inventors have found that phDCs obtained by the above method are advantageous compared to DCs obtained by other methods, such as cytokines or direct isolation from the recipient, because phDCs are generated physiologically (without the need for chemicals such as cytokines) under precise in vitro laboratory conditions with greater reproducibility and controllability.

[0029] Therefore, in a particularly preferred embodiment, recipient phDCs are obtained by subjecting monocytes contained in blood sample to shear force by passing blood sample or a fraction thereof through the flow chamber of the device.Preferably, platelets are present in the flow chamber, which may be derived from recipient blood sample or a fraction thereof or provided separately.In addition or alternatively, plasma components may be present in the flow chamber, which may be derived from recipient blood sample or a fraction thereof or provided separately.However, generation of phDCs also works in the absence of platelets and / or plasma components.

[0030] The recipient's monocytes can be obtained by any suitable means, for example, from a blood sample or a fraction thereof. The blood sample fraction can be, for example, a buffy coat, which includes white blood cells and platelets. Alternatively, the blood sample fraction can be isolated peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated from the blood sample, for example, using centrifugation on a Ficoll-Hypaque gradient (Isolymph, CTL Scientific). In another example, the blood sample fraction can be a purified or enriched monocyte preparation. Monocytes can be enriched from PBMCs, for example, using one, two, or all three of the following: plastic adherence; CD14 magnetic bead positive selection (e.g., from Miltenyi Biotec); and Monocyte Isolation Kit II (Miltenyi Biotec).

[0031] Any suitable volume of blood may be used. The blood sample (e.g., the blood sample from which the fraction is derived) may be between about 1 μL and about 500 mL, e.g., between about 1 μL and about 10 mL, between about 1 μL and about 5 mL, between about 1 μL and about 1 mL, between about 1 μL and about 750 μL, between about 1 μL and about 500 μL, between about 1 μL and about 250 μL, between about 10 mL and about 450 mL, between about 20 mL and about 400 mL, between about 30 mL and about 350 mL, between about 40 mL and about 300 mL, between about 50 mL and about 200 mL, or between about 50 mL and about 100 mL. In some embodiments, the blood sample or fraction thereof, or the additional blood sample or fraction thereof, is about 100 mL or less (e.g., about 50 mL to about 100 mL).

[0032] In some embodiments, the ECP device is a small ECP device, such as a transimmunization (TI) plate. In some embodiments, the ECP device is a plastic bag. Those skilled in the art are familiar with how to distinguish dendritic cells, including phDCs, from monocytes, such as by evaluating gene expression.

[0033] Without being bound by scientific theory, the inventors currently assume that the remarkable effect of the present invention is due to damaged, especially dying DCs damaged by apoptotic agents such as psoralen and UVA (PUVA), especially the combination of 8-MOP and UVA, which provide antigens to the recipient's physiological DCs and provide tolerance signals to the recipient's immune system. If phDCs receive such tolerogenic signals from allogeneic PUVA-treated apoptotic DCs, phDCs can present antigens from allogeneic PUVA-treated apoptotic DCs on their surface (in addition to the tolerogenic signals they received), thereby enabling them to trigger antigen-specific tolerogenic responses. Similarly, the source of antigens can also be derived from immune cells, such as monocytes or lymphocytes. Thus, immune cells, such as monocytes or lymphocytes, can be apoptotic and combined with phDCs to generate antigen-specific tolerogenic responses. However, damaged DCs or related progenitor cells, such as monocytes, are preferred.

[0034] In one embodiment, the dendritic cells of step a) are derived from an ex vivo blood sample of the donor. In another embodiment, the dendritic cells of step a) are obtained by plate passage of PBMCs from the donor. Thus, the dendritic cells of the donor may also be phDCs. All the embodiments regarding the provision of an ex vivo blood sample and PBMCs described above for the recipient also apply to the donor.

[0035] In one embodiment, the recipient and the donor are mammals. Mammals include, but are not limited to, for example, humans, non-human primates, pigs, dogs, cats, horses, and rodents. In a preferred embodiment, the recipient and the donor are humans.

[0036] In one embodiment, the graft is a kidney graft, pancreas graft, liver graft, heart graft, lung graft, intestine graft, skin graft, bone marrow graft or stem cell graft.

[0037] In one embodiment, the stem cell graft is a hematopoietic stem cell graft.

[0038] All of the above embodiments may be performed in vitro.

[0039] The methods of the invention may be applied in combination with other therapies for the treatment of immune deficiencies associated with hematopoietic stem cell transplantation.

[0040] For the following embodiments of the first aspect, all of the above embodiments relating to the first aspect apply mutatis mutandis:

[0041] In one embodiment, the present invention provides a) exposing dendritic cells obtained from a donor to an apoptotic agent; b) combining the apoptotic donor dendritic cells of step a) with physiological dendritic cells obtained from the recipient. The present invention relates to a method comprising the steps of:

[0042] In the above embodiment, a coincubation corresponding to step d1) may be carried out as described for the further above embodiment. In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed continuously. Thus, the phDCs obtained from the recipient are not exposed to apoptotic factors. In one embodiment, the phDCs from the recipient are not exposed to apoptotic factors at any time during the method.

[0043] In one embodiment, the present invention provides a) providing immune cells, preferably lymphocytes, from a donor; b) exposing the immune cells, preferably lymphocytes, of step a) to an apoptotic agent; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic immune cells, preferably apoptotic lymphocytes, of step b) with the physiological recipient dendritic cells from step c); The present invention relates to a method comprising the steps of:

[0044] All of the embodiments of the first aspect apply mutatis mutandis to the above embodiments (i.e., the dendritic cells obtained from the donor are replaced by immune cells, preferably lymphocytes, obtained from the donor).

[0045] The method may also be based on dendritic cell-related cells, such as monocytes, which undergo apoptosis. Thus, in another embodiment, the present invention provides a method for the treatment of dendritic cell-related diseases, comprising: a) preparing monocytes from a donor; b) exposing the monocytes of step a) to an apoptotic agent; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic monocytes of step b) with the physiological recipient dendritic cells from step c); The present invention relates to a method comprising the steps of:

[0046] All of the embodiments of the first aspect apply mutatis mutandis to the above embodiments (ie donor-derived dendritic cells are replaced by donor-derived monocytes).

[0047] Second aspect: A method for selectively generating tolerogenic dendritic cells in which complementary haplodonor dendritic cells induce apoptosis In a second aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: a) providing dendritic cells from a recipient's complementary haplotype donor; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing physiological dendritic cells from a recipient haplodonor; d) combining the apoptotic complementary haplodonor dendritic cells of step b) with the physiological haplodonor dendritic cells of step c); The present invention relates to a method comprising the steps of:

[0048] In one embodiment, the method is performed prior to transplantation.

[0049] In one embodiment, the method is for selectively reducing immunogenicity in a graft or portion thereof prior to transplantation. In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed sequentially. Thus, phDCs from the recipient haplodonor are not exposed to apoptotic factors. In one embodiment, phDCs from the recipient haplodonor are not exposed to apoptotic factors at any point in the method. In one embodiment, the dendritic cells in step a) are obtained from a complementary haploidentical donor of the recipient. In one embodiment, the dendritic cells of step c) are obtained from the recipient's haplodonor. In principle, after the dendritic cells are obtained from the complementary haplodonor, the dendritic cells may or may not be viable. The complementary haplodonor dendritic cells may, for example, become apoptotic and be cryopreserved until they are combined with the physiological haplodonor dendritic cells from step c).

[0050] In one embodiment, the present invention provides a) providing dendritic cells from a recipient's complementary haplotype donor; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing physiological dendritic cells from a recipient haplodonor; d) combining the apoptotic complementary haplodonor dendritic cells of step b) with the physiological haplodonor dendritic cells of step c); d1) co-incubating the mixture of step d); The present invention relates to a method comprising the steps of:

[0051] In one embodiment, step d1) of co-incubating the mixture is carried out for at least 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h or 6 h.

[0052] In one embodiment, step d) of combining the apoptotic complementary haplodonor dendritic cells of step b) with physiological dendritic cells from the haplodonor is performed within the haplodonor.

[0053] In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed sequentially. Thus, phDCs from the recipient haplodonor are not exposed to apoptotic factors. In one embodiment, phDCs from the recipient haplodonor are not exposed to apoptotic factors at any point in the method. In step b) of the method, the dendritic cells obtained from the recipient's complementary haplodonor in step a) of the method are exposed to an apoptotic agent. In one embodiment, the apoptotic agent comprises psoralen and UVA, riboflavin phosphate and UVA, and / or aminolevulinic acid and light. Particularly preferred psoralens are 8-MOP and amotosalen. The most preferred psoralen is 8-MOP. In the most preferred embodiment, the apoptotic agent is a combination of 8-MOP and UVA.

[0054] The embodiment should preferably be selected such that essentially all dendritic cells from the recipient's complementary haplodonor are contacted with the apoptotic agent. In the case of 8-MOP / UVA, essentially all dendritic cells from the recipient's complementary haplodonor should be contacted with 8-MOP and exposed to UVA light. A typical dose of 8-MOP and UVA is 1 J / cm combined with a concentration of 8-MOP between 100 ng / mL and 300 ng / mL. 2 ~3J / cm 2 The UVA is.

[0055] In a preferred embodiment, the dose of UVA is 3 J / cm 2Below, 2J / cm 2 or less than 1J / cm 2 In other preferred embodiments, the dose of 8-MOP is less than or equal to 300 ng / mL, 250 ng / mL, 200 ng / mL, or 100 ng / mL. In a preferred embodiment, the dose of 8-MOP is 200 ng / mL and the dose of UVA is 1 J / cm. 2 It is.

[0056] As described with respect to the first aspect, dendritic cells in the context of the present invention can in particular be obtained by passage of monocytes through plates using a process derived from extracorporeal photopheresis (ECP), which activates and differentiates monocytes into healthy phDCs. All embodiments relating to the generation of phDCs described with respect to the first aspect also apply to the generation of phDCs in the second aspect.

[0057] Therefore, in a particularly preferred embodiment, haplodonor phDCs are obtained by subjecting monocytes contained in the blood sample to shear force by passing the blood sample or a fraction thereof through the flow chamber of the device.Preferably, platelets are present in the flow chamber, which may be derived from the haplodonor blood sample or a fraction thereof, or may be provided separately.In addition, or alternatively, plasma components may be present in the flow chamber, which may be derived from the haplodonor blood sample or a fraction thereof, or may be provided separately.However, the generation of phDCs also works in the absence of platelets and / or plasma components.

[0058] The haplodonor's monocytes can be obtained by any suitable means, for example, from a blood sample or a fraction thereof. The blood sample fraction can be, for example, a buffy coat, which contains white blood cells and platelets. Alternatively, the blood sample fraction can be isolated peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated from the blood sample, for example, using centrifugation on a Ficoll-Hypaque gradient (Isolymph, CTL Scientific). In another example, the blood sample fraction can be a purified or enriched monocyte preparation. Monocytes can be enriched from PBMCs, for example, using one, two, or all three of the following: plastic adherence; CD14 magnetic bead positive selection (e.g., from Miltenyi Biotec); and Monocyte Isolation Kit II (Miltenyi Biotec).

[0059] Any suitable volume of blood may be used. The blood sample (e.g., the blood sample from which the fraction is derived) may be between about 1 μL and about 500 mL, e.g., between about 1 μL and about 10 mL, between about 1 μL and about 5 mL, between about 1 μL and about 1 mL, between about 1 μL and about 750 μL, between about 1 μL and about 500 μL, between about 1 μL and about 250 μL, between about 10 mL and about 450 mL, between about 20 mL and about 400 mL, between about 30 mL and about 350 mL, between about 40 mL and about 300 mL, between about 50 mL and about 200 mL, or between about 50 mL and about 100 mL. In some embodiments, the blood sample or fraction thereof, or the additional blood sample or fraction thereof, is about 100 mL or less (e.g., about 50 mL to about 100 mL).

[0060] In some embodiments, the ECP device is a small ECP device, such as a transimmunization (TI) plate. In some embodiments, the ECP device is a plastic bag. Those skilled in the art are familiar with how to distinguish dendritic cells, including phDCs, from monocytes, such as by evaluating gene expression.

[0061] Without being bound by scientific theory, the inventors currently assume that the remarkable effect of the present invention is due to the damaged, especially dying DCs damaged by apoptotic agents such as psoralen and UVA (PUVA), especially the combination of 8-MOP and UVA, which provide antigens to the physiological DCs of the haplodonor and provide tolerance signals to the immune system of the haplodonor. If phDCs receive such tolerogenic signals from PUVA-treated apoptotic DCs from a complementary haplodonor, the phDCs can present antigens from PUVA-treated apoptotic DCs on their surface (in addition to the tolerogenic signals they received), thereby inducing an antigen-specific tolerogenic response in the haplodonor. Inflammatory conditions such as GvHD are reduced or eliminated in recipients who receive transplants from haplodonors treated as described in the second embodiment. Similarly, the source of antigens may also be derived from immune cells, such as lymphocytes or monocytes. Thus, immune cells, such as lymphocytes or monocytes, can be subjected to apoptosis and combined with haplodonor phDCs to generate an antigen-specific tolerogenic response, although injured DCs or related progenitor cells such as monocytes are preferred.

[0062] In one embodiment, the dendritic cells in step a) are derived from an ex vivo blood sample of the recipient's complementary haplodonor. In another embodiment, the dendritic cells in step a) are obtained by plate passage of PBMCs from the recipient's complementary haplodonor. Thus, the dendritic cells of the recipient's complementary haplodonor can also be phDCs.

[0063] In one embodiment, the complementary haplo donor and the haplo donor are mammals. Mammals include, but are not limited to, for example, humans, non-human primates, pigs, dogs, cats, horses, and rodents. In a preferred embodiment, the complementary haplo donor and the haplo donor are humans.

[0064] In one embodiment, the graft is a kidney graft, pancreas graft, liver graft, heart graft, lung graft, intestine graft, bone marrow graft or stem cell graft.

[0065] In one embodiment, the stem cell transplant is a hematopoietic stem cell transplant.

[0066] For the following embodiments of the second aspect, all of the embodiments described above relating to the first and second aspects apply mutatis mutandis:

[0067] In one embodiment, the present invention provides a) exposing dendritic cells obtained from the recipient's complementary haplodonor to an apoptotic agent; b) combining the complementary haplodonor dendritic cells of the apoptotic recipient of step a) with physiological dendritic cells obtained from the recipient's haplodonor; The present invention relates to a method comprising the steps of:

[0068] It should be understood that all method steps may be performed in vitro. In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed sequentially, such that phDCs obtained from the recipient haplodonor are not exposed to apoptotic factors. In one embodiment, phDCs from the recipient haplodonor are not exposed to apoptotic factors at any point in the method.

[0069] In one embodiment, the present invention provides a) providing immune cells, preferably lymphocytes, from a recipient's complementary haplodonor; b) exposing the immune cells, preferably lymphocytes, of step a) to an apoptotic agent; c) providing physiological dendritic cells from a recipient haplodonor; d) combining the apoptotic complementary haplodonor immune cells, preferably apoptotic complementary haplodonor lymphocytes, of step b) with the physiological haplodonor dendritic cells of step c); The present invention relates to a method comprising the steps of:

[0070] All of the embodiments of the second aspect apply mutatis mutandis to the above embodiments (i.e., dendritic cells obtained from the recipient's complementary haplodonor are replaced by immune cells, preferably lymphocytes, obtained from the recipient's complementary haplodonor). The method may also be based on dendritic cell-related cells, such as monocytes, which undergo apoptosis. Thus, in another embodiment, the present invention provides a method for the treatment of dendritic cell-related diseases, comprising: a) providing monocytes from a recipient's complementary haploidentical donor; b) exposing the monocytes of step a) to an apoptotic agent; c) providing physiological dendritic cells from a recipient haplodonor; d) combining the apoptotic complementary haplodonor monocytes of step b) with the physiological haplodonor dendritic cells of step c); The present invention relates to a method comprising the steps of: All of the embodiments of the first aspect apply mutatis mutandis to the above embodiments (i.e., dendritic cells obtained from the recipient's complementary haplodonor are replaced by monocytes obtained from the recipient's complementary haplodonor).

[0071] Third aspect: A method for selectively generating tolerogenic dendritic cells, in which recipient dendritic cells undergo apoptosis In a third aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: a) providing dendritic cells from a recipient; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c); The present invention relates to a method comprising the steps of:

[0072] In one embodiment, the method is performed prior to transplantation. In one embodiment, the method is for selectively reducing immunogenicity in a graft or portion thereof prior to transplantation. In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed sequentially. Thus, phDCs from the recipient (step c) are not exposed to apoptotic factors. In one embodiment, phDCs from the recipient of step c) are not exposed to apoptotic factors at any point in the method.

[0073] In one embodiment, the dendritic cells in step a) are obtained from the recipient. In one embodiment, the dendritic cells in step c) are obtained from the recipient. In principle, after the dendritic cells are obtained from the recipient (step a), the dendritic cells may or may not be viable. The dendritic cells may, for example, be in an apoptotic state and cryopreserved until they are combined with the physiological recipient dendritic cells from step c).

[0074] In one embodiment, the present invention provides a) providing dendritic cells from a recipient; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c); d1) co-incubating the mixture of step d); The present invention relates to a method comprising the steps of:

[0075] In one embodiment, step d1) of co-incubating the mixture is carried out for at least 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h or 6 h.

[0076] In one embodiment, step d) of combining the apoptotic recipient dendritic cells with physiological dendritic cells from the recipient is performed within the recipient. In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed sequentially. Thus, phDCs from the recipient (step c) are not exposed to apoptotic factors. In one embodiment, phDCs from the recipient of step c) are not exposed to apoptotic factors at any point in the method.

[0077] In step b) of the method, the dendritic cells obtained from the recipient in step a) of the method are exposed to an apoptotic agent. In one embodiment, the apoptotic agent comprises psoralen and UVA, riboflavin phosphate and UVA, and / or aminolevulinic acid and light. Particularly preferred psoralens are 8-MOP and amotosalen. The most preferred psoralen is 8-MOP. In the most preferred embodiment, the apoptotic agent is a combination of 8-MOP and UVA. The embodiment should preferably be selected such that essentially all dendritic cells from the recipient are contacted with the apoptotic agent. In the case of 8-MOP / UVA, essentially all dendritic cells from the recipient should be contacted with 8-MOP and exposed to UVA light. A typical dose of 8-MOP and UVA is 1 J / cm combined with a concentration of 8-MOP between 100 ng / mL and 300 ng / mL. 2 ~3J / cm 2 The UVA is.

[0078] In a preferred embodiment, the dose of UVA is 3 J / cm 2 Below, 2J / cm 2 or less than 1J / cm 2In other preferred embodiments, the dose of 8-MOP is less than or equal to 300 ng / mL, 250 ng / mL, 200 ng / mL, or 100 ng / mL. In a preferred embodiment, the dose of 8-MOP is 200 ng / mL and the dose of UVA is 1 J / cm. 2 It is.

[0079] As described with respect to the first and second aspects, dendritic cells in the context of the present invention can in particular be obtained by plate passage of monocytes using a process derived from extracorporeal photopheresis (ECP), which activates and differentiates monocytes into healthy phDCs. All embodiments relating to the generation of phDCs described with respect to the first aspect also apply to the generation of phDCs with respect to the third aspect.

[0080] Therefore, in a particularly preferred embodiment, recipient phDCs are obtained by subjecting monocytes contained in blood sample to shear force by passing blood sample or a fraction thereof through the flow chamber of the device.Preferably, platelets are present in the flow chamber, which may be derived from recipient blood sample or a fraction thereof or provided separately.In addition or alternatively, plasma components may be present in the flow chamber, which may be derived from recipient blood sample or a fraction thereof or provided separately.However, generation of phDCs also works in the absence of platelets and / or plasma components.

[0081] The recipient's monocytes can be obtained by any suitable means, for example, from a blood sample or a fraction thereof. The blood sample fraction can be, for example, a buffy coat, which includes white blood cells and platelets. Alternatively, the blood sample fraction can be isolated peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated from the blood sample, for example, using centrifugation on a Ficoll-Hypaque gradient (Isolymph, CTL Scientific). In another example, the blood sample fraction can be a purified or enriched monocyte preparation. Monocytes can be enriched from PBMCs, for example, using one, two, or all three of the following: plastic adherence; CD14 magnetic bead positive selection (e.g., from Miltenyi Biotec); and Monocyte Isolation Kit II (Miltenyi Biotec).

[0082] Any suitable volume of blood may be used. The blood sample (e.g., the blood sample from which the fraction is derived) may be between about 1 μL and about 500 mL, e.g., between about 1 μL and about 10 mL, between about 1 μL and about 5 mL, between about 1 μL and about 1 mL, between about 1 μL and about 750 μL, between about 1 μL and about 500 μL, between about 1 μL and about 250 μL, between about 10 mL and about 450 mL, between about 20 mL and about 400 mL, between about 30 mL and about 350 mL, between about 40 mL and about 300 mL, between about 50 mL and about 200 mL, or between about 50 mL and about 100 mL. In some embodiments, the blood sample or fraction thereof, or the additional blood sample or fraction thereof, is about 100 mL or less (e.g., about 50 mL to about 100 mL).

[0083] In some embodiments, the ECP device is a small ECP device, such as a transimmunization (TI) plate. In some embodiments, the ECP device is a plastic bag. Those skilled in the art are familiar with how to distinguish dendritic cells, including phDCs, from monocytes, such as by evaluating gene expression.

[0084] Without being bound by scientific theory, the inventors currently assume that the remarkable effect of the present invention is due to damaged, especially dying DCs damaged by a combination of psoralen and UVA (PUVA), which provide antigens to the recipient's physiological DCs and provide tolerance signals to the recipient's immune system. If phDCs receive such tolerogenic signals from PUVA-treated apoptotic autologous DCs, they can present antigens from the autologous PUVA-treated apoptotic DCs on their surface (in addition to the tolerogenic signals they receive), thereby triggering antigen-specific tolerogenic responses. Similarly, the source of antigens can also be derived from immune cells, such as monocytes or lymphocytes. Thus, immune cells, such as monocytes or lymphocytes, can be apoptotic and combined with phDCs to generate antigen-specific tolerogenic responses. However, damaged DCs or related progenitor cells, such as monocytes, are preferred.

[0085] In one embodiment, the dendritic cells in step a) are derived from an ex vivo blood sample of the recipient. In another embodiment, the dendritic cells in step a) are obtained by plate passage of PBMCs from the recipient. In one embodiment, the recipient is a mammal. Mammals include, but are not limited to, for example, humans, non-human primates, pigs, dogs, cats, horses and rodents. In a preferred embodiment, the recipient is a human.

[0086] In one embodiment, the graft is a kidney graft, pancreas graft, liver graft, heart graft, lung graft, intestine graft, bone marrow graft or stem cell graft. In one embodiment, the stem cell graft is a hematopoietic stem cell graft.

[0087] All of the above embodiments may be performed in vitro.

[0088] The methods of the invention may be applied in combination with other therapies for the treatment of immune deficiencies associated with hematopoietic stem cell transplantation.

[0089] For the following embodiments of the third aspect, all of the above embodiments relating to the third aspect apply mutatis mutandis:

[0090] In one embodiment, the present invention provides a) exposing dendritic cells obtained from a recipient to an apoptotic agent; b) combining the apoptotic recipient dendritic cells of step a) with physiological dendritic cells obtained from the recipient. The present invention relates to a method comprising the steps of:

[0091] In the above embodiment, a coincubation corresponding to step d1) may be carried out as described for the further above embodiment. In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed sequentially. Thus, phDCs from the recipient (step b) are not exposed to apoptotic factors. In one embodiment, phDCs obtained from the recipient in step b) are not exposed to apoptotic factors at any point in the method. In one embodiment, the present invention provides a) providing immune cells, preferably lymphocytes, from a recipient; b) exposing the immune cells, preferably lymphocytes, of step a) to an apoptotic agent; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic immune cells, preferably apoptotic lymphocytes, of step b) with the physiological dendritic cells of step c); The present invention relates to a method comprising the steps of:

[0092] All of the embodiments of the third aspect apply mutatis mutandis to the above embodiments (i.e., dendritic cells obtained from the recipient are replaced by immune cells, preferably lymphocytes, obtained from the recipient).

[0093] The method may also be based on dendritic cell-related cells, such as monocytes, which undergo apoptosis. Thus, in another embodiment, the present invention provides a method for the treatment of dendritic cell-related diseases, comprising: a) providing monocytes from a recipient; b) exposing the monocytes of step a) to an apoptotic agent; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic monocytes of step b) with the physiological dendritic cells of step c); The present invention relates to a method comprising the steps of:

[0094] All of the embodiments of the first aspect apply mutatis mutandis to the above embodiments (ie, dendritic cells obtained from the recipient are replaced by monocytes obtained from the recipient).

[0095] Fourth aspect: Tolerogenic dendritic cells obtained by the method according to the first aspect In a fourth aspect, the present invention relates to tolerogenic recipient dendritic cells obtained by a method according to the first aspect (including all embodiments described above). In one embodiment, the tolerogenic recipient dendritic cells obtained by the method according to the first aspect reduce the immunogenicity of a future graft from the donor.

[0096] Fifth aspect: Tolerogenic dendritic cells obtained by the method according to the second aspect In a fifth aspect, the present invention relates to tolerogenic haplodonor dendritic cells obtained by the method according to the second aspect (including all embodiments described above). In one embodiment, the tolerogenic haplodonor dendritic cells obtained by the method according to the second aspect reduce the immunogenicity of a future graft from the haplodonor.

[0097] Sixth aspect: Tolerogenic dendritic cells obtained by the method according to the third aspect In a sixth aspect, the present invention relates to tolerogenic recipient dendritic cells obtained by a method according to the third aspect (including all embodiments above). In one embodiment, the tolerogenic recipient dendritic cells obtained by the method according to the third aspect reduce the immunogenicity of a future graft.

[0098] Seventh aspect: Tolerogenic dendritic cells according to the fourth aspect for use in a method for preventing or reducing graft-versus-host disease. In a seventh aspect, the present invention relates to a tolerogenic dendritic cell according to the fourth aspect (including all embodiments of the first and fourth aspects above) for use in a method for preventing or alleviating graft-versus-host disease. In one embodiment, the tolerogenic dendritic cells obtained by the method of the first aspect are for use in the treatment of an allogeneic or haploidentical recipient in need of a transplant. When such treatment is administered prior to transplant, the risk of developing GvHD, and its severity if it does occur, is significantly reduced compared to not administering the tolerogenic dendritic cells prior to transplant.

[0099] Eighth aspect: Tolerogenic dendritic cells according to the fifth aspect for use in a method for preventing or reducing graft-versus-host disease. In an eighth aspect, the present invention relates to a tolerogenic dendritic cell according to the fifth aspect (including all embodiments of the second and fifth aspects above) for use in a method for preventing or alleviating graft-versus-host disease. In one embodiment, the tolerogenic dendritic cells obtained by the method of the second aspect are for use in the treatment of a haploidentical recipient in need of a transplant, where such treatment prior to transplantation significantly reduces the risk of developing GvHD, and the severity of GvHD if it does develop, compared to not administering the tolerogenic dendritic cells prior to transplantation.

[0100] Ninth aspect: Tolerogenic dendritic cells according to the sixth aspect for use in a method for preventing or reducing graft-versus-host disease. In a ninth aspect, the present invention relates to a tolerogenic dendritic cell according to the sixth aspect (including all embodiments of the third and sixth aspects above) for use in a method for preventing or alleviating graft-versus-host disease. In one embodiment, the tolerogenic dendritic cells obtained by the method of the third aspect are for use in treating an allogeneic or haploidentical recipient in need of a transplant, where such treatment prior to transplantation significantly reduces the risk of developing GvHD, and the severity of GvHD if it does develop, compared to not administering the tolerogenic recipient dendritic cells prior to transplantation.

[0101] Tenth aspect: Method for selectively generating tolerogenic dendritic cells In a tenth aspect, the present invention provides a method for producing a composition comprising the steps of: a) providing a first sample of dendritic cells obtained from a subject; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing a second sample of dendritic cells obtained from the subject; d) combining the apoptotic dendritic cells of step b) with the dendritic cells of step c); The present invention relates to a method comprising the steps of:

[0102] In one embodiment, the method relates to the selective generation of tolerogenic dendritic cells. In one embodiment, step d) of combining the apoptotic dendritic cells with the dendritic cells is performed in the subject. In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed sequentially. Thus, the DCs in step c) are not exposed to apoptotic factors. In one embodiment, the DCs in step c) are not exposed to apoptotic factors at any time during the method.

[0103] In one embodiment, the present invention provides a) providing a first sample of dendritic cells obtained from a subject; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing a second sample of dendritic cells obtained from the subject; d) combining the apoptotic dendritic cells of step b) with the dendritic cells of step c); d1) co-incubating the mixture of step d); The present invention relates to a method comprising the steps of:

[0104] In one embodiment, both samples of dendritic cells (steps a) and c)) are obtained from the same subject. In one embodiment, step d1) of co-culturing the mixture is performed for at least 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h or 6 h. In one embodiment, the steps of the above method are performed simultaneously. In one embodiment, the steps of the above method are performed sequentially, thus the DCs of step c) are not exposed to apoptotic factors.

[0105] In step b) of the method, the dendritic cells obtained from the subject in step a) of the method are exposed to an apoptotic agent.In one embodiment, the apoptotic agent comprises psoralen and UVA, riboflavin phosphate and UVA, and / or aminolevulinic acid and light.The particularly preferred psoralen is 8-MOP and amotosalen.The most preferred psoralen is 8-MOP.In the most preferred embodiment, the apoptotic agent is a combination of 8-MOP and UVA.

[0106] The embodiment should preferably be selected such that essentially all dendritic cells from the subject are contacted with the apoptotic agent. In the case of 8-MOP / UVA, essentially all dendritic cells from the subject should be contacted with 8-MOP and exposed to UVA light. A typical dose of 8-MOP and UVA is 1 J / cm combined with a concentration of 8-MOP between 100 ng / mL and 300 ng / mL. 2 ~3J / cm 2 The UVA is.

[0107] In a preferred embodiment, the dose of UVA is 3 J / cm 2 Below, 2J / cm 2 or less than 1J / cm 2 In other preferred embodiments, the dose of 8-MOP is less than or equal to 300 ng / mL, 250 ng / mL, 200 ng / mL, or 100 ng / mL. In a preferred embodiment, the dose of 8-MOP is 200 ng / mL and the dose of UVA is 1 J / cm. 2 It is.

[0108] As described with respect to the first and second aspects, dendritic cells in the context of the present invention, i.e. also with respect to the tenth aspect, can in particular be obtained by plate passage of monocytes using a process derived from extracorporeal photopheresis (ECP), which activates and differentiates monocytes into healthy phDCs. In one embodiment, the dendritic cells of step a) have been obtained by plate passage of PBMCs from the subject. In one embodiment, the dendritic cells of step c) have been obtained by plate passage of PBMCs from the subject. Thus, the dendritic cells of step a) and / or step c) may be referred to as physiological DCs. All embodiments relating to the generation of phDCs described for the first aspect also apply to the generation of phDCs for the tenth aspect. In one embodiment, the dendritic cells of step a) have been obtained from an ex vivo blood sample obtained from the subject. In one embodiment, the dendritic cells of step a) have been obtained from an ex vivo blood sample obtained from the subject and the dendritic cells of step c) have been obtained by plate passage of PBMCs from the subject.

[0109] Therefore, in a particularly preferred embodiment, the phDCs of a subject are obtained by subjecting the monocytes contained in the blood sample to shear force by passing the blood sample or a fraction thereof through the flow chamber of the device.Preferably, platelets are present in the flow chamber, which may be derived from the blood sample or a fraction thereof of the subject, or may be provided separately.In addition, or alternatively, plasma components may be present in the flow chamber, which may be derived from the blood sample or a fraction thereof of the subject, or may be provided separately.However, the generation of phDCs also works in the absence of platelets and / or plasma components.

[0110] The subject's monocytes can be obtained by any suitable means, for example, from a blood sample or a fraction thereof. The blood sample fraction can be, for example, a buffy coat, which includes white blood cells and platelets. Alternatively, the blood sample fraction can be isolated peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated from the blood sample, for example, using centrifugation on a Ficoll-Hypaque gradient (Isolymph, CTL Scientific). In another example, the blood sample fraction can be a purified or enriched monocyte preparation. Monocytes can be enriched from PBMCs, for example, using one, two, or all three of the following: plastic adherence; CD14 magnetic bead positive selection (e.g., from Miltenyi Biotec); and Monocyte Isolation Kit II (Miltenyi Biotec).

[0111] Any suitable volume of blood may be used. The blood sample (e.g., the blood sample from which the fraction is derived) may be between about 1 μL and about 500 mL, e.g., between about 1 μL and about 10 mL, between about 1 μL and about 5 mL, between about 1 μL and about 1 mL, between about 1 μL and about 750 μL, between about 1 μL and about 500 μL, between about 1 μL and about 250 μL, between about 10 mL and about 450 mL, between about 20 mL and about 400 mL, between about 30 mL and about 350 mL, between about 40 mL and about 300 mL, between about 50 mL and about 200 mL, or between about 50 mL and about 100 mL. In some embodiments, the blood sample or fraction thereof, or the additional blood sample or fraction thereof, is about 100 mL or less (e.g., about 50 mL to about 100 mL).

[0112] In some embodiments, the ECP device is a small ECP device, such as a transimmunization (TI) plate. In some embodiments, the ECP device is a plastic bag. Those skilled in the art are familiar with how to distinguish dendritic cells, including phDCs, from monocytes, such as by evaluating gene expression.

[0113] Without being bound by scientific theory, the inventors currently assume that the remarkable effect of the present invention is due to the damaged, especially the dying DCs from the subject that are damaged by the combination of psoralen and UVA (PUVA), which provide antigens to the physiological DCs of the subject and provide tolerance signals to the immune system of the subject. If phDCs receive such tolerogenic signals from the PUVA-treated apoptotic autologous DCs, the phDCs can present antigens, especially autoantigens, from the autologous PUVA-treated apoptotic DCs on their surface (in addition to the tolerogenic signals they receive), thereby causing antigen-specific tolerogenic responses. Similarly, the source of antigens can also be derived from immune cells, such as lymphocytes. Thus, immune cells, such as lymphocytes, can be apoptotic and combined with the phDCs of the subject to generate antigen-specific tolerogenic responses.

[0114] In one embodiment, the dendritic cells of step a) are derived from an ex vivo blood sample of the subject, hi another embodiment, the dendritic cells of step a) are obtained by plate passage of PBMCs from the subject.

[0115] In one embodiment, the present invention comprises the following steps (all the above embodiments also apply mutatis mutandis to the following embodiments): a) providing a first sample of dendritic cells obtained from a subject; a1) incubating dendritic cells with antigen molecules; b) exposing the incubated dendritic cells of step a1) to an apoptotic agent; c) providing a second sample of dendritic cells obtained from the subject; d) combining the apoptotic dendritic cells of step b) with the dendritic cells of step c); The present invention relates to a method comprising the steps of:

[0116] In one embodiment, the dendritic cells of step a) have been obtained by plate passage of PBMCs from the subject. In one embodiment, the dendritic cells of step c) have been obtained by plate passage of PBMCs from the subject. In one embodiment, the dendritic cells of step a) have been obtained from an ex vivo blood sample from the subject. In one embodiment, the dendritic cells of step a) have been obtained from an ex vivo blood sample from the subject and the dendritic cells of step c) have been obtained by plate passage of PBMCs from the subject.

[0117] In one embodiment, the antigen molecule is an autoantigen. In one embodiment, the autoantigen is associated with one or more autoimmune disorders. In one embodiment, the antigen molecule is derived from a natural source, chemically synthesized, or recombinantly produced. In one embodiment, the antigen molecule is derived from a cell.

[0118] Table A below provides a list of exemplary autoantigens associated with autoimmune disease and exemplary animal model systems that can be used to evaluate amelioration of autoimmune disease using tolerogenic phDCs (see also Experiments 7 and 8).

[0119] [Table 1-1]

[0120] [Table 1-2]

[0121] [Table 1-3]

[0122] [Table 1-4]

[0123] [Table 1-5]

[0124] [Table 1-6]

[0125] [Table 1-7]

[0126] [Table 1-8]

[0127] [Table 1-9]

[0128] [Table 1-10]

[0129] In one embodiment, the autoantigen is Rh blood group antigen, platelet integrin GpIIb:IIIa, non-collagenous domain of basement membrane collagen type IV, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex with or without hepatitis C antigen, pancreatic beta cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamic acid decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, immunogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IL-1), ... The autoantigen is selected from the group comprising IGRP), proinsulin, alpha-enolase, aquaporin-4, beta-arrestin, S100-beta, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein 39, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-beta-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, pancreatic islet cell antigen, cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL) and scleroderma antigen 70 (Scl-70). In one embodiment, the autoantigen is selected from myelin basic protein and collagen.

[0130] In one embodiment, the subject is a mammal. Mammals include, but are not limited to, for example, humans, non-human primates, pigs, dogs, cats, horses and rodents. In a preferred embodiment, the subject is a human.

[0131] All of the above embodiments may be performed in vitro.

[0132] The methods of the present invention can be applied in combination with other therapies for the treatment of autoimmune diseases. For example, the autoimmune disease can be any of the autoimmune diseases listed in Table A above. Other autoimmune diseases are known in the art.

[0133] Eleventh aspect: Tolerogenic dendritic cells obtained by the method according to the tenth aspect In an eleventh aspect, the present invention relates to a tolerogenic dendritic cell obtained by a method according to the tenth aspect (including all embodiments described above).

[0134] Twelfth aspect: Tolerogenic dendritic cells according to the eleventh aspect for use in the treatment of an autoimmune disease In a twelfth aspect, the present invention relates to a tolerogenic dendritic cell according to the eleventh aspect (including all embodiments of the tenth and eleventh aspects above) for use in the treatment of an autoimmune disease. In the treatment of autoimmune diseases, administration of tolerogenic dendritic cells is contemplated to provide several ameliorative effects, including improved quality of life; reduced severity of disease symptoms; reduced numbers of autoimmune cells; and increased survival time. Indicators of beneficial effect are well known in the art, and appropriate indicators for a particular application can be determined by one of skill in the art. Following such treatment, the severity of the autoimmune disease or symptoms associated therewith is significantly reduced compared to the absence of administration of the tolerogenic dendritic cells.

[0135] In one embodiment, the autoimmune disease is selected from the group comprising multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, Sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin-dependent diabetes mellitus (IDDM), Addison's disease, celiac disease, chronic fatigue syndrome, colitis, Crohn's disease, fibromyalgia, hyperthyroidism / Graves' disease, hypothyroidism / Hashimoto's disease, endometriosis, pernicious anemia, Goodpasture's syndrome, Wegener's disease and rheumatic fever.

[0136] In another embodiment, provided herein is a method of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of any of the tolerogenic dendritic cells described herein.

[0137] In another embodiment, provided herein is a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject an effective amount of tolerogenic dendritic cells, the tolerogenic dendritic cells comprising physiological dendritic cells that contain material derived from apoptotic dendritic cells obtained from the subject, an autoantigen, a fragment thereof, or a combination thereof.

[0138] In any of the aforementioned methods, the autoimmune disease may be selected from the group comprising multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, Sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin-dependent diabetes mellitus (IDDM), Addison's disease, celiac disease, chronic fatigue syndrome, colitis, Crohn's disease, fibromyalgia, hyperthyroidism / Graves' disease, hypothyroidism / Hashimoto's disease, endometriosis, pernicious anemia, Goodpasture's syndrome, Wegener's disease and rheumatic fever.

[0139] In any of the foregoing methods, the autoantigen is selected from the group consisting of Rh blood group antigens, platelet integrin GpIIb:IIIa, noncollagenous domain of basement membrane collagen type IV, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex with or without hepatitis C antigen, pancreatic beta cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamic acid decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, immunogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein. The antigen may be selected from the group comprising: interleukin-1 (IGRP), proinsulin, alpha-enolase, aquaporin-4, beta-arrestin, S100-beta, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein 39, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-beta-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, pancreatic islet cell antigen, cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL) and scleroderma antigen 70 (Scl-70).

[0140] Thirteenth Aspect: Ex Vivo Tolerogenic Dendritic Cells In a thirteenth aspect, the present invention relates to ex vivo tolerogenic dendritic cells comprising material derived from apoptotic dendritic cells obtained from a subject. In some embodiments, material derived from apoptotic dendritic cells obtained from a subject includes polypeptides, nucleic acids, organelles or parts thereof, or any other cellular contents. In some embodiments, the ex vivo tolerogenic dendritic cells comprise an autoantigen or a fragment thereof. Any suitable autoantigen may be included, including any autoantigen described herein (e.g., Table A). In the case of a polypeptide antigen, the fragment may be of any suitable size, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, or 1000 amino acids. In some embodiments, the ex vivo tolerogenic dendritic cells comprise one autoantigen or fragment thereof, hi other embodiments, the ex vivo tolerogenic dendritic cells may comprise two, three, four, five, ten or more different autoantigens or fragments thereof. In some embodiments, the autoantigen is Rh blood group antigen, platelet integrin GpIIb:IIIa, noncollagenous domain of basement membrane collagen type IV, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex with or without hepatitis C antigen, pancreatic beta cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamic acid decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, immunogen-38, insulin, insulinoma antigen-2 and 2beta, islet-specific glucose-6-phosphatase catalytic subunit-related protein. (IGRP), proinsulin, alpha-enolase, aquaporin-4, beta-arrestin, S100-beta, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein 39, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-beta-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, pancreatic islet cell antigen, cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL) and scleroderma antigen 70 (Scl-70). The subject may be suffering from an autoimmune disease, including any of the autoimmune diseases disclosed herein (eg, Table A).

[0141] Fourteenth embodiment: Composition comprising a sample of dendritic cells obtained from a subject In a fourteenth aspect, the present invention relates to a composition comprising: (a) a sample of dendritic cells obtained from a subject; (b) an apoptotic factor; and (c) an autoantigen or a fragment thereof. The composition may include any suitable apoptotic factor, including any apoptotic factor disclosed herein. In some embodiments, the apoptotic factor includes psoralen, riboflavin phosphate, 5-aminolevulinic acid, or a combination thereof. In some embodiments, the apoptotic factor is a psoralen. In some embodiments, the psoralen is selected from the group including 8-MOP and amotosalen. In some embodiments, the psoralen is 8-MOP. In some embodiments, the composition comprises an autoantigen or a fragment thereof.Can comprise any suitable autoantigen, including any autoantigen described herein (e.g., Table A).In the case of a polypeptide antigen, the fragment can be of any suitable size, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, or 1000 amino acids. In some embodiments, the composition comprises one autoantigen or fragment thereof, hi other embodiments, the composition may comprise two, three, four, five, ten or more different autoantigens or fragments thereof. In some embodiments, the autoantigen is Rh blood group antigen, platelet integrin GpIIb:IIIa, noncollagenous domain of basement membrane collagen type IV, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex with or without hepatitis C antigen, pancreatic beta cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamic acid decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, immunogen-38, insulin, insulinoma antigen-2 and 2beta, islet-specific glucose-6-phosphatase catalytic subunit-related protein. (IGRP), proinsulin, alpha-enolase, aquaporin-4, beta-arrestin, S100-beta, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein 39, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-beta-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, pancreatic islet cell antigen, cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL) and scleroderma antigen 70 (Scl-70). Dendritic cells can be obtained from a subject suffering from an autoimmune disease, including any of the autoimmune diseases disclosed herein (eg, Table A). [Brief description of the drawings]

[0142] [Figure 1] FIG. 1 shows the general design of the ECP leukemia haploidentical study. [Diagram 2] FIG. 1 shows the general design of the ECP leukemia haploidentical study. [Diagram 3] 1 shows the schematic design of the Balb / C→B6 fully mismatched GVHD system, with results shown. [Figure 4]Figure 1: Ex vivo psoralen UVA treatment (PUVA) of grafts ameliorates GVHD in a fully MHC mismatched model. Mice were injected subcutaneously with 2x105 MC38 tumor cells prior to implantation on day -4 or on the day of implantation on day 0. Mice were lethally irradiated with 950 cGy on day -1. On day 0, mice received Balb / c→B6 transplants. Mice received intravenous injections of 5x106 allogeneic T-cell depleted bone marrow (BM) cells along with 10x106 unmanipulated splenocytes or after ex vivo PUVA treatment of allo-stimulated grafts. As controls, groups of mice received syngeneic BM and splenocytes after tumor inoculation. (A, panels 1-3) Pooled data for mean body weight, GvHD score and survival rate for all groups. (B) Mean tumor volume. [Diagram 5] Kaplan-Meier survival curves of heart transplants. [Figure 6] FIG. 1 shows features of a subject. [Figure 7] FIG. 1 shows the grades and stages of GVHD. [Figure 8] Figure 1 shows the cumulative incidence of acute GVHD. Unrelated donor analysis includes one patient who had a 5 / 6 HLA-matched related donor. [Figure 9] FIG. 1 shows the cumulative incidence of extensive chronic GVHD. [Figure 10] Figure 1 shows overall survival. Unrelated donor analysis included one patient who had a 5 / 6 HLA-matched related donor. [Figure 11] Figure 1 shows the cumulative incidence of transplant-related mortality. Unrelated donor analysis included one patient who had a 5 / 6 HLA-matched related donor. [Figure 12] FIG. 1 shows the selection criteria for historical controls. [Figure 13] FIG. 1 : Characteristics of study and historical control subjects. [Figure 14] FIG. 1 shows the relative risk and 95% confidence intervals of transplant outcomes in multivariate analysis (the control group was used as the reference and assigned a relative risk of 1.0). [Figure 15] FIG. 1 shows the cumulative incidence of grades II-IV acute GVHD. [Figure 16] FIG. 1 shows adjusted probabilities of disease-free survival. [Figure 17] FIG. 1 shows adjusted probabilities of survival. [Figure 18] Figure 1 shows PD-L1 expression in fresh PBMC alone versus phDCs incubated with 8-MOP / UVA-injured syngeneic PBMC (PUVA syn PBMC) or 8-MOP / UVA-injured allogeneic PBMC (PUVA allo PBMC). [Figure 19-1] CFSE-labeled responder T cells (T cells) from one donor were co-incubated with gamma-irradiated stimulator PBMCs from the same donor (syngeneic cultures) or from unrelated donors (MLR). To suppress the MLR response, some cultures were additionally supplemented with syngeneic 8-MOP / UVA-treated PBMCs, and syngeneic TI plate-passed phDCs (MLR+PUVA syn.PBMC+phDCs). Proliferation of responder CD8 and CD4 T cells was assayed by measuring CFSE dilution by flow cytometry (FACS) (A,B). Activation status of responder CD8 and CD4 T cells was further assessed by FACS using CD44 and PD1 expression to detect activated T cells (C,D). N=number of blood donors analyzed; p-value=unpaired t-test with Welch correction. [Figure 19-2]CFSE-labeled responder T cells (T cells) from one donor were co-incubated with gamma-irradiated stimulator PBMCs from the same donor (syngeneic cultures) or from unrelated donors (MLR). To suppress the MLR response, some cultures were additionally supplemented with syngeneic 8-MOP / UVA-treated PBMCs, and syngeneic TI plate-passed phDCs (MLR+PUVA syn.PBMC+phDCs). Proliferation of responder CD8 and CD4 T cells was assayed by measuring CFSE dilution by flow cytometry (FACS) (A,B). Activation status of responder CD8 and CD4 T cells was further assessed by FACS using CD44 and PD1 expression to detect activated T cells (C,D). N=number of blood donors analyzed; p-value=unpaired t-test with Welch correction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0143] The following general definitions are provided:

[0144] When the term "comprising" is used in the present specification and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "consisting of". Hereinafter, when a group is defined to include at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of these embodiments.

[0145] For the purposes of the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtainable". Hereinafter, if for example an antibody is defined as obtainable from a particular source, this should also be understood to disclose the antibody obtained from this source.

[0146] When an indefinite or definite article is used when referring to a singular noun, such as "a", "an" or "the", this includes the plural of that noun unless something else is specifically stated. The term "about" or "approximately" in the context of the present invention refers to a range of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation of ±20%, preferably ±15%, more preferably ±10%, even more preferably ±5% from the indicated numerical value.

[0147] Moreover, terms such as "first," "second," "third," or "(a)," "(b)," "(c)," "(d)," or "(i)," "(ii)," "(iii)," "(iv)," and the like in this specification and claims are used to distinguish between similar elements and not necessarily to describe a sequential or chronological order. The terms so used are interchangeable under appropriate circumstances, and it should be understood that the embodiments of the invention described herein are capable of operation in sequences other than those described or illustrated herein.

[0148] When terms such as "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" or "(i)", "(ii)", "(iii)", "(iv)" relate to steps of a method or use or assay, there is no consistency of time or time interval between the steps unless otherwise specified, i.e. steps may be performed simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps unless otherwise specified in the application as set forth herein above or below.

[0149] Technical terms are used in their general sense. Where a specific meaning translates to a specific term, the definition of the term is given below in the context in which the term is used.

[0150] As used herein, a "transplant" refers to any sample of cells removed from a mammalian individual ("donor") and suitable for reintroduction, in whole or in part, into the same ("autologous") or different ("allogeneic") mammalian individual ("recipient"). Transplants may be freshly obtained, cultured, or frozen, but are maintained under conditions suitable to maintain sterility and promote viability. The term transplant is used interchangeably with the term graft.

[0151] The method of the present invention can be performed on strictly HLA-matched individuals who share all or nearly all of the class I and class II HLA antigens; haploidentical individuals, such as siblings who share half of the HLA antigens; or on unrelated, and therefore mostly HLA-mismatched individuals. In the context of the present invention, the term "haplo donor" refers to one genetic parent of the future recipient, while the term "complementary haplo donor" refers to the other genetic parent. Thus, the future recipient is the child. In other words, if the mother is the haplo donor, the father is the complementary haplo donor of the child (future recipient), and vice versa. In a preferred embodiment of the first aspect, the recipient and the donor are unrelated. In a preferred embodiment of the second aspect, each of the haplo donor and the complementary haplo donor has half of the HLA antigens of the future recipient.

[0152] The degree of HLA identity between individuals can be readily demonstrated by methods known in the art, including polymerase chain reaction, mixed lymphocyte reaction (MLR), and serological measurements.

[0153] As used herein, the term "antigen" refers to a compound, composition, or substance capable of stimulating the production of antibodies or a T-cell response in an animal, including compositions injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term is used interchangeably with the terms "immunogen" or "antigenic molecule." The term "antigen" includes all related antigenic epitopes. The terms "antigen," "antigenic molecule," or "immunogen" include fragments thereof that are still capable of acting as antigens. An "epitope" or "antigenic determinant" refers to a site on an antigen to which B cells and / or T cells respond. In one example, recipient antigens include antigens from dendritic cells, such as dendritic cells obtained from the passage of peripheral blood leukocytes (including monocytes or monocyte-derived cells) through a plate.

[0154] As used herein, the term "immunogenic" refers to the ability of a substance, such as an antigen, or a cell, or a portion thereof, to elicit an immune response in the human or animal body.

[0155] As used herein, the term "autoantigen" refers to a host antigen (or microbial superantigen) that is believed by those skilled in the art to be associated with autoimmune disease such that the presence of activated T cells specific for the autoantigen correlates with the development or progression of the disease.

[0156] The autoantigen can be a defined autoimmune target antigen, for example, a target antigen identified as myelin basic protein (MBP), MBP84-102, or MBP143-168 in multiple sclerosis; a pancreatic islet cell antigen; in uveitis, S antigen; or type II or other collagen in rheumatoid arthritis; in SLE, cytoplasmic linker protein-170 (CLIP-170); Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL); scleroderma antigen 70 (Scl-70); in Graves' disease, a thyroid receptor; and in myasthenia gravis, an acetylcholine receptor. The autoantigens of the present invention also include peptide mixtures eluted from MHC molecules known to be associated with autoimmunity, such as HLA-DQ and -DR molecules that confer susceptibility to some common autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis and multiple sclerosis, or HLA-B27 molecules that are known to confer susceptibility to reactive arthritis and ankylosing spondylitis. The autoantigens of the present invention may also be synthetic peptides that are predicted to bind to WIC molecules associated with autoimmune diseases. For other autoimmune diseases where the individual autoantigens have not yet been characterized, the autoantigens suitable for carrying out the method of the present invention may be cells or cell extracts from affected tissues (e.g., synovial cells in rheumatoid arthritis, skin lesions in psoriasis, etc.). The term autoantigen also includes fragments thereof that act as autoantigens.

[0157] As used herein, "immune cell" refers broadly to cells that are of hematopoietic origin and play a role in immune response.Immune cells include lymphocytes such as B cells and T cells; leukocytes; natural killer cells; and myeloid cells such as monocytes, dendritic cells, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0158] "Dendritic cells", also referred to herein as "DCs", are antigen-presenting immune cells that process antigenic material and present it to other cells of the immune system, particularly T cells. DCs function to capture and process antigens. When DCs endocytose antigens, they process the antigen into smaller fragments, usually peptides, that are displayed on the DC surface, which they present to antigen-specific T cells, for example, via MHC molecules. After ingesting antigens, DCs migrate to lymph nodes. During maturation, DCs are prompted by a variety of signals, including signaling through Toll-like receptors (TLRs), to activate and proliferate by cognate effector T cells (T eff ), thereby initiating a T cell-mediated immune response to an antigen. Alternatively, DCs can express costimulatory signals that induce T eff Antigens can be presented to antigen-specific T cells without providing a costimulatory signal (or while providing a costimulatory signal) so that T cells are not properly activated. Such presentation can, for example, cause death or anergy of T cells that recognize the antigen, or induce the activation of regulatory T cells (T reg The term "dendritic cells" includes differentiated dendritic cells, immature, and mature dendritic cells. These cells may be characterized by the expression of certain cell surface markers (e.g., CD11c, MHC class II, and at least low levels of CD80 and CD86), CD11b, CD304 (BDCA4)). In some embodiments, DCs express CD8, CD103, CD1d, etc. Other DCs may be identified by the absence of lineage markers such as CD3, CD14, CD19, CD56, etc. Additionally, dendritic cells may be functionally characterized by their ability to stimulate allorespons and mixed lymphocyte reactions (MLR).

[0159] "Tolerogenic DC" refers to a dendritic cell that can suppress immune responses, such as antigen-specific T cell-mediated immune responses, or generate tolerogenic immune responses, for example, by reducing effector T cell responses to a particular antigen, resulting in an increase in the number of antigen-specific regulatory T cells. Tolerogenic DCs can be characterized by the induction of antigen-specific tolerogenic immune responses ex vivo and / or in vivo. Such induction refers to the induction of tolerogenic immune responses to one or more antigens of interest presented by the induced tolerogenic dendritic cells. Tolerogenic dendritic cells have a tolerogenic phenotype that may be characterized by at least one of the following properties: i) they are capable of converting naive T cells into Foxp3+ T regulatory cells ex vivo and / or in vivo (e.g., they induce expression of FoxP3 in naive T cells); ii) they are capable of depleting effector T cells ex vivo and / or in vivo; iii) they retain their tolerogenic phenotype upon stimulation with at least one TLR agonist ex vivo (and in some embodiments, they increase expression of costimulatory molecules in response to such stimulation); iv) they do not transiently increase their oxygen consumption rate upon stimulation with at least one TLR agonist ex vivo; v) they have an increased expression of the expression marker PDL1, and / or vi) they have an increased expression of the expression marker GILZ. Items v) and vi) may be assessed by comparison with monocytes or PBMCs.

[0160] "Tolerogenic immune response" refers to any immune response that can result in immunosuppression specific to an antigen or a cell, tissue, organ, etc. expressing such an antigen. Such immune response includes any reduction, delay, or inhibition of an undesirable immune response specific to an antigen or a cell, tissue, organ, etc. expressing such an antigen. Such immune response also includes any stimulation, production, induction, promotion, or mobilization of a desirable immune response specific to an antigen or a cell, tissue, organ, etc. expressing such an antigen. Thus, a tolerogenic immune response includes the absence or reduction of an undesirable immune response to an antigen that can be mediated by antigen-reactive cells, and the presence or promotion of suppressor cells. Tolerogenic immune responses provided herein include immunological tolerance. "Generating a tolerogenic immune response" refers to the generation of any of the aforementioned immune responses specific to an antigen or a cell, tissue, organ, etc. expressing such an antigen.

[0161] A tolerogenic immune response includes any reduction, delay or inhibition of proliferation and / or activity of CD4+ T cells, CD8+ T cells or B cells. A tolerogenic immune response also includes a reduction in antigen-specific antibody production. A tolerogenic immune response also includes any reduction, delay or inhibition of proliferation and / or activity of CD4+ T cells, CD8+ T cells or B cells. reg cells, CD8+T reg cell, B reg It may also include any response that results in the stimulation, induction, production or recruitment of regulatory cells, such as cells. In some embodiments, a tolerogenic immune response is one that results in a conversion to a regulatory phenotype characterized by the production, induction, stimulation or recruitment of regulatory cells.

[0162] Tolerogenic immune responses also occur in CD4+ T cells. reg Cells and / or CD8+T reg It also includes any response that results in the stimulation, production, or mobilization of CD4+T cells. regThese cells express the transcription factor FoxP3 and can inhibit inflammatory responses and autoimmune inflammatory diseases (Human regulatory T cells in autoimmune diseases. Cvetanovich GL, Hafler D A. Curr Opin Immunol. 2010 December; 22(6):753-60. Regulatory T cells and autoimmunity. Vila J, Isaacs JD, Anderson A E. Curr Opin Hematol. 2009 July; 16(4):274-9). These cells also suppress T cell activity against B cells and induce tolerance to both self and foreign antigens (Therapeutic approaches to allergy and autoimmunity based on FoxP3+ regulatory T-cell activation and expansion. Miyara M, Wing K, Sakaguchi S. J Allergy Clin Immunol. 2009 April; 123(4):749-55). CD4+T reg CD8+ T cells recognize antigens when presented by class II proteins on APCs. reg The cells can also suppress the action of T cells against B cells, resulting in the activation of antigen-specific suppression inducing tolerance to both self- and foreign antigens. In some embodiments, the tolerogenic dendritic cells provided are capable of suppressing both types of responses (CD4+ T reg and CD8+T reg In other embodiments, FoxP3 can be induced in other immune cells, such as macrophages, iNKT cells, and the tolerogenic dendritic cells provided herein can similarly effect one or more of these responses.

[0163] Tolerogenic immune responses include T reginduction of regulatory cytokines such as cytokines; induction of inhibitory cytokines; inhibition of inflammatory cytokines (e.g., IL-4, IL-1b, IL-5, TNF-α, IL-6, GM-CSF, IFN-γ, IL-2, IL-9, IL-12, IL-17, IL-18, IL-21, IL-22, IL-23, M-CSF, C-reactive protein, acute phase proteins), chemokines (e.g., CCL-2, CXCL8, MCP-1, RANTES, MIP-1α, MIP-1β, MIG, ITAC or IP-10), production of anti-inflammatory cytokines (e.g., IL-4, IL-13, IL-10, etc.), proteases (e.g., MMP-3, MMP-9), leukotrienes (e.g., CysLT-1, CysLT-2), prostaglandins (e.g., PGE2) or histamine. These include, but are not limited to, inhibition of proliferation of effector T cells; inhibition of polarization towards Th17, Th1 or Th2 immune responses; inhibition of effector cell specific cytokines: Th17 (e.g., IL-17, IL-25), Th1 (IFN-γ), Th2 (e.g., IL-4, IL-13); inhibition of Th1-, Th2-, or Th17-specific transcription factors; inhibition of proliferation of effector T cells; induction of apoptosis of effector T cells; induction of tolerogenic dendritic cell specific genes; induction of FoxP3 expression; inhibition of IgE-induced or IgE-mediated immune responses; inhibition of antibody responses (e.g., antigen-specific antibody production); inhibition of T helper cell responses; production of TGF-β and / or IL-10; inhibition of effector functions of autoantibodies (e.g., inhibition of cell depletion, cell or tissue damage, or complement activation).

[0164] Any of the above can be measured in vivo in one or more animal models or can be measured in vitro. Those skilled in the art are familiar with such in vivo or in vitro measurements. Unwanted or tolerogenic immune responses can be monitored using, for example, methods that evaluate immune cell numbers and / or functions, tetramer analysis, ELISPOT, flow cytometry-based analysis of cytokine expression, cytokine secretion, cytokine expression profiling, gene expression profiling, protein expression profiling, analysis of cell surface markers, PCR-based detection of immune cell receptor gene usage (see T. Clay et al., "Assays for Monitoring Cellular Immune Response to Active Immunotherapy of Cancer" Clinical Cancer Research 7:1127-1135 (2001)), and the like. Unwanted or tolerogenic immune responses can also be monitored using, for example, methods that evaluate protein levels in plasma or serum, T cell or B cell proliferation and functional assays, and the like. In some embodiments, tolerogenic immune responses can be monitored by evaluating the induction of FoxP3.

[0165] Preferably, the tolerogenic immune response results in inhibition of the onset, progression or pathology of a disease, disorder or condition described herein, particularly GvHD. In some embodiments, the reduction of an undesirable immune response or the generation of a tolerogenic immune response can be assessed by determining a clinical endpoint, clinical efficacy, clinical symptoms, disease biomarkers and / or clinical scores.

[0166] As used herein, the term "animal" or "mammal" includes all mammals, including humans. Preferably, the mammal of the present invention is a human subject.

[0167] As used herein, the term "expose" refers to bringing into close proximity or direct contact with a state or condition.

[0168] The term "hematopoietic cell transplant" (HCT) is used herein to refer to blood and bone marrow transplant (BMT), a procedure that involves the infusion of cells (hematopoietic stem cells; also called hematopoietic progenitor cells) to reconstitute a patient's hematopoietic system.

[0169] As used herein, the term "autoimmune disorder" or "autoimmune syndrome" refers to a condition that occurs when the immune system mistakenly attacks and destroys healthy body tissues' own components. Autoimmune disorders may affect one or more organs or tissue types. Organs and tissues susceptible to autoimmune disorders include blood vessels, connective tissues, endocrine glands such as the thyroid or pancreas, joints, muscles, red blood cells, and skin.

[0170] In any method step of the invention that uses exposure to an apoptotic agent, the apoptotic agent includes psoralen and UVA, riboflavin phosphate and UVA, and / or aminolevulinic acid and light. Particularly preferred psoralens are 8-MOP and amotosalen. The following amounts are for orientation purposes. Those skilled in the art can easily find the concentrations and doses to be applied that achieve the effect of rendering the cells apoptotic. The concentration of riboflavin phosphate can be 1 mM to 100 mM. The concentration of amotosalen can be 50 pM to 500 pM. The light dose associated with the aforementioned riboflavin or amotosalen is 1 J / cm. 2 ~10J / cm 2 The corresponding light can be UVA or blue light. The concentration of 8-MOP can be 0.2 μM to 2.5 μM (or 43 ng / mL to 540 ng / mL). The associated light dose can be 0.5 J / cm. 2 ~5J / cm 2 The light may be UVA or blue light.

[0171] The method of the present invention, or certain steps of the method, may be carried out in a bag, such as a plastic bag. When plastic materials are considered, bags made of plastic films based on polyolefins, polyethylene, fluoropolymers, polyvinyl chloride, ethylene vinyl acetate copolymers, ethylene vinyl alcohol, polyvinylidene fluoride, or other plastic films approved for medical use may be used. In a preferred embodiment of the present invention, the bag is made of ethylene vinyl acetate copolymers. The bag may be made of a material that provides a degree of transparency so that the sample or cell mixture can be irradiated with visible or UV light.

[0172] The present invention will now be described with reference to some specific examples, which are for illustrative purposes and should not be construed as limiting.

[0173] experiment Experiment 1 - Generation of phDCs All studies were performed with blood donated by healthy human volunteers. Peripheral blood was collected in 1:100 5,000 U / mL heparin (McKesson Packaging Services) and platelet-containing PBMCs were isolated by density gradient centrifugation on Isolymph (CTL Scientific Supply Corp.) according to the manufacturer's protocol. Autologous plasma (which also contains platelets) was collected and stored. Washed PBMCs and platelets were resuspended in autologous plasma and incubated for 1 h in either a transimmunization (TI) chamber or clinical ECP plate.

[0174] In the TI chamber, cells were passed at a rate of 0.09 mL / min using a syringe pump. After passing through the plate, the cells were collected and the TI chamber was washed with 100% FBS at 0.49 mL / min while physically perturbing the plate surface by flicking or tapping to help detach any attached cells from the chamber. In the clinical ECP plate, cells were passed at a flow rate of 24 mL / min and then washed with human AB serum (Lonza BioWhittaker) at 100 mL / min while physically perturbing the plate surface by flicking or tapping to help detach any attached cells from the chamber. PBMCs that had passed through either the TI chamber or the ECP plate were collected, washed, and cultured overnight under standard conditions in RPMI without phenol red (Gibco, Carlsbad, CA) supplemented with 15% human AB serum (Lonza BioWhittaker), 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA), and 1% L-glutamine (Invitrogen, Carlsbad, CA). The following day, physiological dendritic cells were harvested (including harvesting of either adherent cells by scraping).

[0175] Study 2: ECP leukemia haploidentical transplant trial--outline design and expected results 1. The mother (future donor) becomes tolerant to paternal antigens by injecting the father's ECP-treated blood. 2. A check is made to see if the mother is actually tolerized to paternal antigens (reduced MLR response to paternal cells and no antibodies to paternal HLA type) 3. Transplant bone marrow from treated mothers to children with leukemia 4. Evaluate bone marrow engraftment in children 5. Study endpoints - Evaluate bone marrow engraftment, incidence and severity of GvHD, and cancer recurrence

[0176] The above procedure is illustrated in FIG.

[0177] When the above treatments are applied, the child does not develop GvHD or shows reduced symptoms of GvHD because the transplant was previously tolerized.

[0178] Study 3: ECP leukemia haploidentical transplant trial--outline design and expected results 1. Treat recipients with tolerogenic ECP prior to haplotype bone marrow transplantation or immunoablation conditioning (following Francine Foss's study design for fully matched grafts, see experiment 1) 2. Transplant is performed but the post-transplant cyclophosphamide dose (PTCy) is reduced. 3. Assess bone marrow engraftment 4. Study endpoints - Evaluate bone marrow engraftment, incidence and severity of GvHD, and cancer recurrence

[0179] The above procedure is illustrated in FIG.

[0180] Applying the above treatments allows for a reduction in PTCy to maintain antitumor immunity. PTCy may be omitted entirely and replaced with ECP.

[0181] Experiment 4: Prevention of GvHD by PUVA-treated dendritic cells material and method On days 1-4, future graft recipient mice (C57BL / 6, H2b MHC haplotype) are inoculated subcutaneously (flank) with C57BL / 6 tumors (MC38 colon carcinoma). On day 2-1, the future graft recipients (tumor bearing) are given a lethal dose (950 cGy) of gamma irradiation which eliminates the mouse's own immune system. 3 On the day of transplantation (day 0), prepare the tissue for transplantation. a. In non-graft controls, irradiated C57BL / 6 mice do not receive transplants. b. In syngeneic controls, the native C57BL / 6 immune system is reconstituted by the return of C57BL / 6 bone marrow and splenocytes. c. In allogeneic control grafts, recipients are reconstituted with fully mismatched bone marrow and splenocytes from Balb / c donor mice (H2d MHC haplotype). d. For allogeneic PUVA grafts, Balb / c bone marrow and splenocytes (including dendritic cells) were incubated with lethally irradiated C57BL / 6 splenocytes for 5 h and then irradiated with a very low dose of PUVA (200 ng / mL of 8-MOP, 0.1 J / cm) in petri dishes. 2 Treat with UVA. 4 The prepared tissue is transplanted into irradiated tumor-bearing recipients. 5 Recipients are then monitored for bone marrow engraftment (survival, confirmed later by blood analysis), GvHD, and tumor growth. Mice that are irradiated but do not receive grafts uniformly die by day 14. · Syngeneic control mice engrafted well, did not develop GvHD (as would be expected since the grafts were perfectly matched), and grew large tumors. Allogeneic control mice engrafted well and developed uniformly fatal severe GvHD at about day 25 (as would be expected since the grafts were completely mismatched), with tumor growth being relatively slow and mortality being rapid, making it difficult to assess. Allogeneic PUVA mice engrafted well and showed no signs of GvHD (day 47), and although tumors did grow, the growth rate appeared to be approximately 50% of that in syngeneic control mice, i.e., partially controlled by the GvT effect.

[0182] The results are shown in Table 1 below and in Figures 3 and 4. Figure 3 also shows a schematic of the above method.

[0183] result

[0184] [Table 2]

[0185] The inventors have surprisingly found that grafts treated with low doses of 8-MOP / UVA, i.e. according to the methods of the present invention, prevent GvHD, the rationale being that healthy dendritic cells incorporating dying dendritic cells provide tolerance to the recipient graft.

[0186] Experiment 5: Effect of pre-transplant infusion of donor splenocytes treated with extracorporeal photopheresis (ECP) on cardiac transplant survival rate The donor mice were BALB / c (H-2 d ), male, 8-14 weeks of age. Recipient mice were C57BL / c, male, 10-14 weeks of age (n=24). Splenocytes from donor mice were harvested and divided into two groups. Donor cells from group 1 were subjected to shear forces in a flow chamber ("Untreated" in Figure 5). Donor cells from group 2 were subjected to shear forces in a flow chamber and ECP ("ECP" in Figure 5). Ten recipient mice received untreated donor cells, while 14 recipient mice received ECP-treated donor cells.

[0187] Treatment of donor cells prior to injection in recipient mice was as follows:

[0188] The flow chamber was coated with platelet-rich plasma (PRP) by introducing 0.4 mL of the PRP fraction into the flow chamber for 60 min at 37°C. Donor splenocytes were injected into the flow chamber using a 60 mL syringe at a concentration of 400 million cells in 13.33 mL.

[0189] ECP was performed by adding 8-MOP to the flow chamber in a volume of 200 µL (200 ng / mL) in 20 mL of PBS per 100 million cells. Cells were exposed to approximately 20–22 mW / cm 2 The recipient mice were exposed to UVA for 200 seconds at 37 °C for 20 min. The recipient mice received the treated donor cells at approximately 50 million cells per recipient mouse.

[0190] Heart transplantation was performed 7 days after the injection of donor cells into the recipient mice. The outcomes are reported in Figure 5 as survival percentage in days after surgery. As can be seen from Figure 5, recipient mice given untreated donor cells (i.e., group 1, donor cells only subjected to shear force) die around day 9 after surgery, whereas recipient mice given ECP donor cells (i.e., group 2, donor cells subjected to shear force and ECP) ​​survived until day 29 after surgery. Thus, allogeneic transplants are significantly better tolerated in recipients if the recipients are given ECP-treated donor cells, such as ECP-treated splenocytes (spleen cells contain healthy dendritic cells) prior to transplantation. It can be concluded that pretreatment of recipients with ECP-treated donor cells results in long-term survival of allografts and donor-specific tolerance.

[0191] Study 6: Extracorporeal photopheresis for the prevention of acute GVHD in patients undergoing standard myeloablative conditioning and allogeneic hematopoietic stem cell transplantation material and method subject The study and consent forms were approved by the Institutional Review Boards or equivalent at all participating centers. All subjects signed the approved consent forms before starting study treatment. Eligibility criteria included subjects aged 18–60 years with hematological malignancy and organ function not precluding treatment with a myeloablative conditioning regimen and allogeneic HCT. Subjects were eligible for the study if they had been diagnosed with a hematological malignancy for which the treatment options were allogeneic bone marrow transplantation or PBSC transplantation. Subjects could be enrolled whether or not the disease was in remission or after a first or second relapse of the disease. Subjects were required to weigh at least 40 kg, have a platelet count of more than 20,000 / cmm, and have no known sensitivity to psoralens or citrate preparations. Subjects were required to have a related donor serologically or molecularly matched at all HLA-A, B, and DR loci, or a related donor mismatched at one HLA-A or -B locus but molecularly matched at the HLA-DR locus, or an unrelated donor molecularly matched at the HLA-A, -B, and DR loci. Enrollment occurred from October 2002 to January 2004, so HLA-C matching was not routinely performed. Conditioning regimens and prophylaxis for GVHD subjects included CY (60 mg / kg per day for 2 consecutive days) and TBI (10–13.5 Gy delivered in divided doses over 3 or 4 days). GVHD prophylaxis was CSP 3–5 mg / kg IV starting on D-1, adjusted to keep trough levels between 200 and 600 ng / mL. Except for subjects who relapsed or were intolerant to CSP, CSP was converted to oral administration if clinically tolerated and not tapered earlier than D100. Subjects receiving HCT from a matched related donor were administered MTX 10 mg / m on day 1. 2 IV on days 3, 6, and 11, whereas subjects with a mismatched related donor or a matched unrelated donor received MTX 15 mg / m on day 1. 2 , and 10 mg / m on days 3, 6, and 11 2MTX dosing was based on consensus among investigators to provide uniform prophylaxis against acute GVHD. Supportive care and prophylactic antibacterial treatment were given according to institutional guidelines at each study site.

[0192] Extracorporeal photopheresis ECP was performed using a UVAR XTS instrument (Therakos, Exton, PA, USA) as previously described (Miller et al., 2004). Collection of at least 1500 mL of buffy coat blood was typically performed for each treatment prior to the use of methoxsalen solution (UVADEX Therakos), which was infused into the recirculation bag of the ECP circuit after buffy coat collection was completed but before the photoactivation process. After completion of photoactivation, the treated buffy coat was reinfused into the subject. Patients underwent ECP on 2 consecutive days within 4 days prior to starting the conditioning regimen.

[0193] Grading of GVHD and adverse events Grading of adverse events was performed according to established criteria (Common Terminology Criteria for Adverse Events, version 3.0, December 12, 2003). Modified Seattle-Glucksberg criteria were used for staging acute GVHD (Glucksberg et al., 1974), and diagnostic criteria for limited and extensive chronic GVHD were as described by Schulman et al., 1980. To ensure uniformity of diagnosis of acute and chronic GVHD, investigators were trained on the diagnostic criteria before the start of the study. Each investigator employed the diagnostic method appropriate at his / her study site to determine the presence of acute or chronic GVHD.

[0194] statistics Study Analyses: The primary analysis was the incidence of grades II–IV acute GVHD in the first 100 days after transplantation, calculated using a cumulative incidence function to accommodate the competing risks of dying without developing acute GVHD. Similarly, the cumulative incidence method was used to calculate incidence rates for chronic GVHD, transplant-related mortality (TRM), and relapse to accommodate competing risks. Probabilities of overall survival (OS) and disease-free survival (DFS) were described using Kaplan-Meier product limit estimates with 95% confidence intervals. Treatment failure (death or relapse) was the event used in DFS evaluation. Descriptive statistics, such as median time to event, were also calculated.

[0195] Comparison with historical controls. Historical controls were identified using a database maintained by the CIBMTR. The CIBMTR is a research affiliate of the International Bone Marrow Transplant Registry (IBMTR) of the Medical College of Wisconsin and the National Marrow Donor Program, which consists of a volunteer working group of over 450 transplant centers worldwide that provide detailed data on consecutive allogeneic and autologous HCT to the Coordinating Statistical Center. Participating centers are required to report all transplants on an ongoing basis, and compliance is monitored by on-site audits. All patients in the database are followed longitudinally and include annual evaluations. Data quality is ensured by computerized error checking, physician review of submitted data, and on-site audits of participating centers. Observational studies conducted by the CIBMTR during this period were conducted with waiver of informed consent and in compliance with HIPAA regulations as determined by the Institutional Review Board and Privacy Officer of the Medical College of Wisconsin. The CIBMTR collects data at two levels: registry and research. Registry data include disease type, age, sex, disease stage and chemotherapy response before transplantation, date of diagnosis, graft type (bone marrow and / or blood-derived stem cells), high-dose conditioning regimen, disease progression and survival after transplantation, development of new malignancies, and cause of death. Requests for progression or death data for enrolled patients are at 6-month intervals. All participating CIBMTR centers provide registry data. Study data are collected on a subset of enrolled patients selected using a weighted randomization scheme to ensure representativeness and include detailed disease, pre-transplant and post-transplant clinical information. Historical controls for this study were obtained from the study database. ECP study subjects were transplanted between 2002 and 2004. CIBMTR controls were selected by applying the eligibility criteria for this study to subjects transplanted between 1997 and 2004.The longer time frame for the controls was used to ensure an adequate number of subjects for the adjusted comparison with reasonable statistical power. Outcome data for both study and control subjects were censored at 1 year to adjust for differences in follow-up duration. Because study subjects and controls were transplanted at two different time periods, the year of transplant (1997–1999 vs. 2000–2004) was examined for its potential effect on the outcomes of the controls. There were no differences in the 1-year outcomes of control subjects transplanted in 1997–1999 and those transplanted in 2000–2004. Study subjects and controls were compared using multivariate Cox regression analysis. Tests for proportionality were performed by adding time-dependent covariates. These tests showed that the assumption of proportionality was valid. Stepwise backward methods were used to identify significant covariates (other than ECP use) associated with outcomes. Variables considered in model construction were age, sex, race, donor relationship, HLA matching, graft type, disease type, disease status at time of transplant, and CMV serology status. Effect on treatment (ECP) was included in all steps of model construction. Tests for potential interactions between ECP treatment and other significant covariates did not reveal any significant interactions. One-year adjusted probabilities of overall and DFS were estimated from the final Cox models, stratified by treatment received, and weighted by the pooled sample proportion values ​​for each prognostic factor. These adjusted probabilities estimate the likelihood of outcomes in populations with similar prognostic factors.

[0196] result Subject and donor characteristics Sixty-six subjects were enrolled in the ECP study. Nine study centers enrolled 1–16 subjects each (average of 7 subjects per study center). However, three subjects did not receive study treatment with ECP, one patient withdrew consent for the study, one patient had delayed transplantation, and one center had mechanical problems with the ECP device. One subject received ECP but was not transplanted due to rapid disease progression. After study completion, 62 subjects were considered evaluable in the modified intent-to-treat population dataset. Two of these subjects received only one ECP treatment before starting the conditioning regimen due to mechanical problems with the subsequent ECP device. Subject, disease, donor characteristics, and disease status information are summarized in Figure 6.

[0197] engraftment One subject did not engraft and died of respiratory failure (cause unknown) on day 28. All other subjects had satisfactory neutrophil and platelet recovery, with no late graft failure. The median (range) time to reach a neutrophil count of 4500 / mL was 21 (14-39) days in subjects receiving bone marrow grafts and 14 (13-28) days in subjects receiving peripheral blood grafts. The corresponding times to achieve platelets of 420 000 / cmm were 14 (11-43) and 14 (6-56) days, respectively.

[0198] GVHD Grade II-IV acute GVHD developed in 22 of 62 subjects (36%), including 9 of 30 related-donor HCT recipients (30%) and 13 of 32 matched unrelated or one HLA antigen mismatched related-donor HCT recipients (41%) (Figure 7). Skin was the most frequent and most severely affected organ, with 38% of subjects having stage 2 or higher skin involvement, while 16% and 13% of subjects had stage 2 or higher gastrointestinal or hepatic involvement, respectively (Figure 7). The 100-day cumulative incidence of grade II-IV acute GVHD was 35% (95% CI, 23-48%) (Figure 8). Forty (66%) patients had biopsy-proven acute GVHD. No patients were described as having early chronic GVHD before day 100 or late acute GVHD after day 100. The median time to first diagnosis and maximum grade acute GVHD of grades II–IV was 35 days for both, ranging from 18–52 days for first diagnosis and 22–96 days for maximum grade. Fifty-three subjects were evaluable for chronic GVHD. Seven patients died before day 100 (three from acute GVHD, one from idiopathic pneumonia, and three from multiple organ system failure), and two patients had insufficient data collected to determine whether chronic GVHD developed. Chronic GVHD developed in 21 (40%) of the 53 subjects (limited: eight (15%), extensive: 13 (25%)). The 1-year cumulative incidence of limited and extensive chronic GVHD was 38% (95% CI, 21–47%) (Figure 9).

[0199] toxicity The most frequent serious adverse events occurring during the study were fever in eight subjects (13%), febrile neutropenia in four subjects (7%), and multiple organ system failure in three subjects (5%). Adverse events directly attributable to ECP included two subjects who experienced hypotension while undergoing ECP. CMV reactivation occurred in 17 subjects (27%), including two subjects with CMV disease in lung tissue demonstrated in biopsy specimens from bronchoscopy. Two subjects (3%) had systemic fungal infections during their participation in the study, one developing 19 days after transplantation and the other developing 9 months after transplantation.

[0200] survival rate The median follow-up of surviving patients was 371 days (range, 366–643 days), with 48 of 62 subjects (77%) still alive. Kaplan-Meier estimates of 100-day and 1-year survival after transplant were 89% (95% CI, 78–97%) and 77% (95% CI, 64–86%), respectively. The 1-year probability of OS for related and unrelated-donor HCT recipients was 89% (95% CI, 70–96%) and 66% (95% CI, 46–80%), respectively (Figure 10). The 1-year probability of DFS was 69% (95% CI, 64–86%) for all patients, 79% (95% CI, 59–90%) after related-donor HCT, and 60% (95% CI, 40–75%) after unrelated-donor HCT. Relapse occurred in 7 (11%) patients. Fourteen (23%) subjects died: 3 after related donor transplantation and 11 after unrelated donor transplantation. Causes of death were relapse (1), acute GVHD (3), chronic GVHD (1), infection (4), multiple organ system failure (3), and idiopathic pneumonia (2). The 1-year cumulative incidence of TRM was 21% (95% CI, 11-31%), with cumulative incidences after related and unrelated donor transplantation being 10% (95% CI, 1-20%) and 31% (95% CI, 18-50%), respectively (Figure 11).

[0201] ECP study subjects compared with CIBMTR controls The CIBMTR database was used to search for historical controls with similar characteristics to the study subjects. Control subjects had to meet the eligibility criteria defined in Figure 12. A total of 347 control subjects were identified. Their characteristics are compared with those of the study subjects in Figure 13. ECP-treated subjects were more likely to be over 40 years old, Caucasian, and have an unrelated donor than controls. The distribution of underlying diseases was also notably different. The potential impact of these differences was considered in the multivariate analysis. Multivariate analysis revealed a significantly lower incidence of grades II-IV acute GVHD in ECP-treated subjects compared with historical controls (relative risk [RR], 0.61; 95% CI, 0.38-0.97) (P=0.04) (Figure 14). This lower rate was due to a substantial delay in the onset of acute GVHD in ECP-treated subjects, rather than an absolute reduction in incidence (Figure 15). The probability of cumulative incidence of acute GVHD grades II-IV was 36% (95% CI, 25-48%) in the study patients and 39% (95% CI, 33-44%) in the historical control cohort (Figure 15). There was also a reduction in TRM in ECP-treated subjects compared with historical controls, but this was not statistically significant (RR, 0.55; 95% CI, 0.29-1.04) (P=0.065). There was no difference in veno-occlusive disease or interstitial pneumonia between the groups. However, opportunistic infections occurred in 85 (24%) of control patients and 5 (8%) of study patients (P=0.008). The adjusted probability of DFS and OS was significantly higher in ECP-treated subjects than in historical controls (Figures 16 and 17). The adjusted DFS rate at 1 year was 74% (95% CI, 62% to 82%) in ECP-treated subjects and 63% (95% CI, 58% to 67%) in the historical control cohort (RR for treatment failure [recurrence or death], 0.60; 95% CI, 0.36 to 0.99) (P=0.045). The adjusted OS rate at 1 year was 83% (95% CI, 72% to 90%) in ECP-treated subjects and 67% (95% CI, 62% to 71%) in the historical controls (RR for death, 0.44; 95% CI, 0.24 to 0.80) (P=0.007).

[0202] Consideration To put the results of this single Phase II study into perspective, collaboration with the CIBMTR was undertaken upon completion of the study to identify suitable historical controls for comparison with the ECP study subjects. Controls were selected using the eligibility criteria for patients in this study. However, as shown in Figure 13, there were some differences in the distribution of characteristics between the groups, with the ECP study subjects being older and more likely to receive unrelated donor grafts, both of which are associated with increased risk of GVHD. Despite the presence of these important demographic differences favoring the historical control group, acute GVHD developed more slowly in the ECP-treated cohort. Multivariate analysis, adjusting for differences in prognostic factors, revealed a significant difference in the rate of acute GVHD (grades II-IV) between the groups (Figure 15), with a significantly delayed time to onset of acute GVHD. Although the absolute incidence of acute GVHD was not lower with ECP, multivariate analysis revealed a trend toward less TRM, less treatment failure (relapse and TRM), and more overall and DFS in the ECP study cohort compared with the historical control cohort. Regimen-related toxicities were similar between groups, except that ECP-treated patients had significantly fewer opportunistic infections compared with historical controls. A later onset of acute GVHD may itself be beneficial, as it allows for more recovery from the conditioning regimen and transplant procedure, allows for a greater degree of immune reconstitution, and allows these patients to better tolerate the side effects of corticosteroids, suffer less end-organ damage, and overcome infections. Although the follow-up period for evaluation of recurrence was short, ECP did not appear to suppress the allogeneic immune-mediated graft-versus-malignancy effect, as recurrence rates were not significantly different between the two groups.

[0203] Experiment 7: Amelioration of autoimmune disease using tolerogenic phDCs In this example, an animal model is used to evaluate the amelioration of autoimmune disease using tolerogenic phDCs.

[0204] Many recognized animal models for autoimmune disease are available, and exemplary animal models are shown in Table A above. If necessary, autoimmune disease is induced prior to treatment with tolerogenic phDCs or controls. Animals are examined and scored according to clinical criteria relevant to the animal model. In some examples, animals are divided into four treatment groups as follows: a) Untreated animals b) Treatment with healthy phDCs presenting autoantigens c) Treatment with 8-MOP / UVA-injured apoptotic phDCs presenting autoantigens d) Treatment with healthy phDCs that had internalized 8-MOP / UVA-injured apoptotic phDCs containing autoantigens.

[0205] phDCs for the above treatment groups can be generated as follows: b) Healthy phDCs presenting self-antigens: Monocytes are obtained from healthy syngeneic animals, passed through TI plates as described (Ventura et al. J Vis Exp. 2019 May 17;147), and incubated overnight with autoantigen to ensure antigen uptake. c) 8-MOP / UVA injured apoptotic phDCs presenting self-antigens: Monocytes are obtained from healthy syngeneic animals, passed through TI plates as above, then exposed to 8-MOP and UVA at doses sufficient to induce cellular damage and incubated overnight with autoantigen. d) Healthy phDCs internalizing apoptotic phDCs injured with 8-MOP / UVA containing autoantigens: Cells from group c) are combined with an equal number of fresh monocytes, passed through TI plates as above, and co-incubated overnight to ensure uptake of autoantigen-containing 8-MOP / UVA-injured phDCs by healthy phDCs.

[0206] phDCs generated as described above are typically cultured at least three times at an appropriate dose (e.g., at least 1 × 10 6The cells are administered intravenously at 100x the dose (cells / animal) to appropriate groups of mice at a time interval appropriate for the animal model.

[0207] Animals are monitored (e.g., daily) for autoimmune disease for a period appropriate for the particular model. Additionally, animals can be sacrificed at different time points during the experiment: (i) before induction of disease, i.e., in healthy animals; (ii) after disease induction but without treatment, i.e., immunized animals; (iii) between the second and third vaccination, i.e., an intermediate time point; (iv) At the end of the experiment, i.e., the endpoint

[0208] After euthanasia, samples (e.g., spleen, and inguinal and axillary lymph nodes) are obtained from the animals, dissociated in single cell suspensions, and used to evaluate the immune response to immunization and treatment. Standard immune response assays include immune cell phenotyping by cell surface or intracellular flow cytometry, inflammatory or anti-inflammatory cytokine secretion assays (e.g., ELISA, Luminex, ELISpot), and T cell proliferation in response to autoantigen restimulation (e.g., carboxyfluorescein succinimidyl ester (CFSE) dilution).

[0209] Exemplary results include: a) Untreated animals: - Progressive disease consistent with that predicted by the model. b) Treatment with healthy phDCs presenting autoantigens: - Progressive disease, potentially more severe than predicted by the model due to the additional immune effects of healthy phDCs. c) Treatment with 8-MOP / UVA injured apoptotic phDCs presenting autoantigens: -Progressive disease, possibly resulting in less severity than predicted by the model due to some tolerogenic effect of 8-MOP / UVA-injured apoptotic phDCs being incorporated into healthy DCs from injected animals. d) Treatment with healthy phDCs that have internalized 8-MOP / UVA damaged apoptotic phDCs containing autoantigens: - The tolerogenic effect of healthy phDCs loaded with 8-MOP / UVA damaged apoptotic phDCs carrying self-antigens significantly reduces the disease, thus allowing for disease control and amelioration.

[0210] Experiment 8: Amelioration of autoimmune diseases including multiple sclerosis (MS) using tolerogenic phDCs In this example, a mouse model is used to evaluate whether tolerogenic phDCs ameliorate autoimmune diseases such as MS.

[0211] Experimental autoimmune encephalomyelitis (EAE) is a widely accepted mouse model of human MS that shares many similarities with the human clinical disease. The mouse EAE model is characterized by progressive paralysis, CNS inflammation, and demyelination, mediated primarily by myelin-specific CD4+ T cells, although CD8+ and B cells also play a role. Thus, EAE mice can be used to model tolerance induction by dendritic cells in an autoimmune disease setting.

[0212] 1. EAE is induced in 11-13 week old female C57BL / 6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG) peptides MOG35-55 or MOG1-125 in complete Freund's adjuvant (CFA) emulsion followed by administration of pertussis toxin (PTX) in PBS according to standard protocols known in the art.

[0213] 2.EAE is expected to develop 8-18 days after immunization. After disease induction, all animals are examined daily for health status and scored according to the following standard EAE clinical criteria: 0, asymptomatic; 0.5, loss of tone in distal half of tail; 1, loss of tone in entire tail; 1.5, hind limb weakness; 2, hind limb paralysis; 2.5, hind limb paraplegia; 3, forelimb weakness; 4, quadriplegia; 4.5, severe quadriplegia; 5, quadriplegia; and 6, death. Treatment is initiated on the first day that the mean clinical score exceeds 1.0, reflecting the onset of clinically relevant disease in the majority of mice.

[0214] 3. Divide the mice into four treatment groups of at least 10 animals each as follows: a) Untreated mice b) Treatment with healthy phDCs presenting MOG antigen c) Treatment with 8-MOP / UVA-injured apoptotic phDCs presenting MOG antigen d) Treatment with healthy phDCs that had internalized apoptotic phDCs injured with 8-MOP / UVA containing MOG antigen.

[0215] 4. phDCs for the above treatment groups are generated as follows: b) Healthy phDCs presenting MOG antigen: Monocytes were obtained from healthy syngeneic mice, passaged through TI plates as described (Ventura et al. J Vis Exp. 2019 May 17;147), and incubated overnight with MOG antigen to ensure antigen uptake.

[0216] c) 8-MOP / UVA injured apoptotic phDCs presenting MOG antigen: Monocytes are obtained from healthy syngeneic mice, passed through TI plates as above, then exposed to 8-MOP and UVA at doses sufficient to induce cellular damage and incubated overnight with MOG antigen.

[0217] d) Healthy phDCs internalizing apoptotic phDCs injured with 8-MOP / UVA containing MOG antigen: Cells from group c) are combined with an equal number of fresh monocytes, passed through TI plates as above, and co-incubated overnight to ensure uptake of 8-MOP / UVA-injured phDCs containing the MOG antigen by healthy phDCs.

[0218] 5. phDCs generated as described above (see 4.) are cultured at least three times at least at 1 × 10 6 Administer intravenously to mice in the appropriate groups (see 3.) at a dose of: - On the first day, the mean clinical score is above 1.0, reflecting the onset of clinically relevant disease in the majority of mice (e.g., day 13 post-immunization) - 4 days after the first treatment dose (e.g., day 17 after immunization) - 4 days after the second treatment dose (e.g., day 21 post-immunization)

[0219] 6. Mice are monitored daily for EAE clinical scores for at least 4 weeks post-immunization.

[0220] 7. Additionally, mice can be sacrificed at different time points during the experiment: (i) Before EAE induction, i.e., healthy mice; (ii) after EAE induction but without treatment, i.e., immunized mice; (iii) between the second and third vaccination, i.e., an intermediate time point; (iv) At the end of the experiment, i.e., the endpoint After euthanasia, spleens, as well as inguinal and axillary lymph nodes, are obtained, dissociated in single cell suspensions, and used to assess immune responses to immunization and treatment. Standard immune response assays include immune cell phenotyping by cell surface or intracellular flow cytometry, pro- or anti-inflammatory cytokine secretion assays (e.g., ELISA, Luminex, ELISpot), and T cell proliferation in response to MOG peptide restimulation (e.g., carboxyfluorescein succinimidyl ester (CFSE) dilution). Spinal cords are also harvested at the time of euthanasia, fixed in 4% paraformaldehyde (PFA), and used for further evaluation of disease by histology and immunohistochemistry.Typical histological analyses include the number of inflammatory foci, the number of apoptotic cells, and the extent of demyelination (immunohistochemistry for myelin basic protein).

[0221] Exemplary results include: a) Untreated mice: - Progressive EAE disease consistent with that predicted by the model. - Detection of inflammatory immune cells reacting to MOG antigens by immunological testing. - Histological examination showed signs of axonal injury and inflammatory damage consistent with the model.

[0222] b) Treatment with healthy phDCs presenting MOG antigen: - Progressive EAE disease, potentially more severe than predicted in this model due to the additional immune effects of healthy phDCs. - Increased detection of inflammatory immune cells responding to MOG antigens by immunological testing. - Signs of more severe axonal injury and inflammatory damage on histological examination.

[0223] c) Treatment with 8-MOP / UVA injured apoptotic phDCs presenting MOG antigen: -Progressive EAE disease, possibly less severe than predicted in this model due to some tolerogenic effect of 8-MOP / UVA-injured apoptotic phDCs incorporating healthy DCs from injected mice. - A slight decrease in the detection of inflammatory immune cells reacting to the MOG antigen by immunological tests. - Signs of low severity axonal injury and inflammatory damage on histological examination.

[0224] d) Treatment with healthy phDCs that had internalized apoptotic phDCs injured with 8-MOP / UVA containing MOG antigen: - The tolerogenic effect of healthy phDCs loaded with 8-MOP / UVA damaged apoptotic phDCs carrying MOG antigen significantly reduces EAE disease, thus allowing EAE disease to be controlled and ameliorated. - Immunologic testing demonstrated detection of tolerogenic immune cells, such as Tregs, and a significant reduction in inflammatory immune cells responding to the MOG antigen. - Significant reduction in axonal injury and inflammatory damage by histological examination.

[0225] Study 9: Amelioration of other autoimmune diseases using tolerogenic phDCs In this example, a suitable animal model (eg, an animal model described in Table A above) is used to evaluate whether tolerogenic phDCs ameliorate autoimmune disease.

[0226] For example, non-obese diabetic (NOD) mice are an art-recognized model of insulin-dependent diabetes mellitus (IDDM). In this example, NOD mice are evaluated using an approach similar to that described in Experiments 7 and 8.

[0227] Animals are examined and scored according to clinical criteria relevant to NOD mice. In some examples, animals are divided into four treatment groups as follows: a) Untreated animals b) Treatment with healthy phDCs presenting autoantigens (e.g., pancreatic β cell antigens) c) Treatment with 8-MOP / UVA-injured apoptotic phDCs presenting autoantigens (e.g., pancreatic β-cell antigens) d) Treatment with healthy phDCs that have internalized 8-MOP / UVA-injured apoptotic phDCs containing autoantigens (e.g., pancreatic β cell antigens).

[0228] phDCs for the above treatment groups can be generated as follows: b) Healthy phDCs presenting self-antigens (e.g., pancreatic β-cell antigens): Monocytes are obtained from healthy syngeneic animals, passed through TI plates as described (Ventura et al. J Vis Exp. 2019 May 17;147), and incubated overnight with autoantigen to ensure antigen uptake.

[0229] c) 8-MOP / UVA-injured apoptotic phDCs presenting self-antigens (e.g., pancreatic β-cell antigens): Monocytes are obtained from healthy syngeneic animals, passed through TI plates as described above, then exposed to 8-MOP and UVA at a dose sufficient to induce cellular damage and incubated overnight with an autoantigen (e.g., pancreatic β cell antigen).

[0230] d) Healthy phDCs that internalized 8-MOP / UVA-injured apoptotic phDCs containing self-antigens (e.g., pancreatic β-cell antigens): Cells from group c) are combined with an equal number of fresh monocytes, passaged through TI plates as above, and co-incubated overnight to ensure uptake of 8-MOP / UVA-injured phDCs containing autoantigens (e.g., pancreatic β cell antigens) by healthy phDCs.

[0231] phDCs generated as described above are typically cultured at least three times at an appropriate dose (e.g., at least 1 × 10 6 The cells are administered intravenously at 100x the dose (cells / animal) to appropriate groups of mice at a time interval appropriate for the animal model.

[0232] The animals are monitored for a diabetic phenotype. Additionally, animals can be sacrificed at different time points during the experiment: (i) before induction of disease, i.e., in healthy animals; (ii) after induction of disease but without treatment, i.e., immunized animals; (iii) between the second and third vaccination, i.e., an intermediate time point; (iv) At the end of the experiment, i.e., the endpoint

[0233] After euthanasia, samples (e.g., spleen, and inguinal and axillary lymph nodes) are obtained from the animals, dissociated in single cell suspensions, and used to evaluate the immune response to immunization and treatment. Standard immune response assays include immune cell phenotyping by cell surface or intracellular flow cytometry, inflammatory or anti-inflammatory cytokine secretion assays (e.g., ELISA, Luminex, ELISpot), and T cell proliferation in response to autoantigen restimulation (e.g., carboxyfluorescein succinimidyl ester (CFSE) dilution).

[0234] Exemplary results include: a) Untreated animals: - Progressive disease consistent with that predicted by the NOD model. b) Treatment with healthy phDCs presenting autoantigens: - Progressive disease, potentially more severe than predicted in the NOD model due to the additional immune effects of healthy phDCs. c) Treatment with 8-MOP / UVA injured apoptotic phDCs presenting autoantigens: -Progressive disease, possibly with less severity than predicted in the NOD model due to some tolerogenic effect of 8-MOP / UVA-injured apoptotic phDCs being taken up by healthy DCs from injected animals. d) Treatment with healthy phDCs that have internalized 8-MOP / UVA damaged apoptotic phDCs containing autoantigens: - The tolerogenic effect of healthy phDCs loaded with 8-MOP / UVA damaged apoptotic phDCs carrying self-antigens significantly reduces the disease, thus allowing the disease to be controlled and ameliorated.

[0235] Other autoimmune diseases can also be evaluated using approaches similar to those described above.

[0236] Experiment 10: Treatment of autoimmune diseases using tolerogenic phDCs Human patients with autoimmune diseases are treated using tolerogenic phDCs generated as described herein.

[0237] For example, a sample of dendritic cells is obtained from a patient suffering from an autoimmune disease (e.g., MS). The dendritic cells are exposed to an apoptotic agent (e.g., psoralen and UVA (PUVA), particularly a combination of 8-MOP and UVA). In some cases, an autoantigen is also added to the dendritic cells that have been exposed to the apoptotic agent. For example, in the case of MS, any suitable autoantigen, such as MBP and / or MOG, as described in Table A, can be added. The cells are exposed to the apoptotic agent for a period of time and under conditions that cause the cells to undergo apoptosis.

[0238] The resulting apoptotic dendritic cells can then be combined with physiological dendritic cells produced as described herein and co-incubated for, e.g., at least 0.5, 1, 2, 3, 4, 5, or 6 hours prior to administration to a subject. Alternatively, the combination can be administered directly to a subject without co-incubation. Treatment with phDCs is expected to result in amelioration of autoimmune disease.

[0239] Experiment 11: Increased PD-L1 expression in human phDCs incubated with PUVA-injured PBMCs Human phDCs were generated from blood of healthy volunteers using TI plates and incubated overnight with equal numbers of 8-MOP / UVA-treated syngeneic PBMCs (PUVA syn PBMCs) or allogeneic PBMCs (PUVA allo PBMCs). PD-L1 expression (reported as mean fluorescence intensity, MFI, of live CD14+ phDC fraction) was measured by flow cytometry at 18 hours and was found to be expressed at significantly higher levels on healthy phDCs, but not on precursor monocytes, after co-incubation with either allogeneic or syngeneic PUVA-treated PBMCs (Figure 18). N = number of blood donors analyzed; p-value = unpaired t-test with Welch's correction.

[0240] Experiment 12: Decreased PD1 expression on responder T cells in MLR assays An MLR (mixed lymphocyte reaction) assay was set up using blood from healthy volunteer donors. Briefly, 2 IgG from one donor were used. * 10 5 Purified CFSE-labeled responder T cells (T cells) were cultured in 4-well plates from the same donor (syngeneic cultures) or unrelated donors (MLR). * 10 5 To suppress the MLR response, some cultures were co-incubated with 100 μg of gamma-irradiated (3000 rad) stimulatory PBMCs. * 10 5 Syngeneic 8-MOP / UVA-treated PBMCs, and 1 * 10 5 Syngeneic TI plate-passaged phDCs (MLR+PUVA syn.PBMC+phDCs) were additionally supplemented. After 5 days of culture, proliferation of responder CD8 and CD4 T cells was assayed by measuring CFSE dilution by flow cytometry (FACS) (A,B). Activation status of responder CD8 and CD4 T cells was further assessed by FACS using CD44 and PD1 expression to detect activated T cells (C,D). For both CD8 and CD4 T cells, addition of healthy phDCs and PUVA-treated PBMCs significantly suppressed both proliferation and activation (Figure 19). N=number of blood donors analyzed; p-value=unpaired t-test with Welch correction.

[0241] Furthermore, the present invention relates to the following embodiments. 1. A method for selectively producing tolerogenic dendritic cells, comprising: a) providing dendritic cells from a donor; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic donor dendritic cells of step b) with the physiological recipient dendritic cells from step c); A method comprising: 2. The method according to 1, wherein after step d), a step of co-incubating the apoptotic donor dendritic cells of step b) with physiological recipient dendritic cells of step c) is performed. 3. The method according to claim 2, wherein the co-incubating step is carried out for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours. 4. The method according to 1, wherein step d) of combining the apoptotic donor dendritic cells with physiological dendritic cells from the recipient is carried out within the recipient. 5. The method according to 1, wherein the dendritic cells of step a) are derived from an ex vivo blood sample of the donor. 6. The method according to 1, wherein the dendritic cells in step a) are obtained by plate passage of PBMCs from a donor. 7. The method according to 1, wherein the apoptotic factors in step b) include psoralen and UVA, riboflavin phosphate and UVA, and / or 5-aminolevulinic acid and light. 8. The method of claim 7, wherein the psoralen is selected from the group including 8-MOP and amotosalen. 9. The method of claim 8, wherein the psoralen is 8-MOP. 10. The method according to 1, wherein the physiological dendritic cells in step c) are obtained by plate passage of PBMCs from the recipient. 11. The method of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein the donor and / or recipient is a mammal, preferably a human. 12. A method for selectively producing tolerogenic dendritic cells, comprising: a) providing dendritic cells from a recipient's complementary haplotype donor; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing physiological dendritic cells from a recipient haplodonor; d) combining the apoptotic complementary haplodonor dendritic cells of step b) with the physiological haplodonor dendritic cells of step c); A method comprising: 13. The method according to claim 12, wherein after step d), a step of co-incubating the apoptotic complementary haplodonor dendritic cells of step b) with the physiological haplodonor dendritic cells of step c) is performed. 14. The method of claim 13, wherein the co-incubating step is carried out for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours. 15. The method according to claim 12, wherein step d) of combining the apoptotic complementary haplodonor dendritic cells of step b) with physiological haplodonor dendritic cells of step c) is carried out within the haplodonor. 16. The method according to claim 12, wherein the dendritic cells of step a) are derived from an ex vivo blood sample of the recipient's complementary haplodonor. 17. The method according to claim 12, wherein the dendritic cells in step a) are obtained by plate passage of PBMCs from the recipient's complementary haploidentical donor. 18. The method of claim 12, wherein the apoptotic factors in step b) include psoralen and UVA, riboflavin phosphate and UVA, and / or 5-aminolevulinic acid and light. 19. The method of claim 18, wherein the psoralen is selected from the group including 8-MOP and amotosalen. 20. The method of claim 19, wherein the psoralen is 8-MOP. 21. The method according to claim 12, wherein the physiological dendritic cells in step c) are obtained by plate passage of PBMCs from the recipient's complementary haplodonor. 22. The method of any of 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, wherein the complementary haplodonor, the haplodonor and / or the recipient is a mammal, preferably a human. 23. A method for selectively producing tolerogenic dendritic cells, comprising: a) providing dendritic cells from a recipient; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c); A method comprising: 24. The method according to claim 23, wherein after step d), a step of co-incubating the apoptotic dendritic cells of step b) with physiological dendritic cells of step c) is carried out. 25. The method of claim 24, wherein the co-incubating step is carried out for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours. 26. The method according to claim 23, wherein step c) of combining the apoptotic dendritic cells of step b) with physiological dendritic cells of step c) is carried out within the recipient. 27. The method according to claim 23, wherein the dendritic cells in step a) are derived from an ex vivo blood sample of the recipient. 28. The method according to claim 23, wherein the dendritic cells in step a) are obtained by plate passage of PBMCs from the recipient. 29. The method of claim 23, wherein the apoptotic factors in step b) include psoralen and UVA, riboflavin phosphate and UVA, and / or 5-aminolevulinic acid and light. 30. The method of claim 29, wherein the psoralen is selected from the group including 8-MOP and amotosalen. 31. The method of claim 30, wherein the psoralen is 8-MOP. 32. The method according to claim 23, wherein the physiological dendritic cells in step c) are obtained by plate passage of PBMCs from the recipient. 33. The method according to any of 23 to 32, wherein the donor and recipient are mammals, preferably humans. 34. The method of any of 1 to 33, wherein the graft is an organ or stem cell transplant. 35. A tolerogenic dendritic cell obtained by the method according to any one of claims 1 to 11. 36. A tolerogenic dendritic cell obtained by the method according to any one of claims 12 to 22. 37. A tolerogenic dendritic cell obtained by the method according to any one of claims 23 to 34. 38. A tolerogenic dendritic cell according to 35 to 37 for use in a method for preventing or reducing graft-versus-host disease. 39. A method for selectively producing tolerogenic dendritic cells, comprising: a) providing a first sample of dendritic cells obtained from a subject; b) exposing the dendritic cells of step a) to an apoptotic agent; c) providing a second sample of physiological dendritic cells obtained from the subject; d) combining the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c); A method comprising: 40. The method according to claim 39, wherein after step d), a step of co-incubating the apoptotic dendritic cells of step b) with physiological dendritic cells of step c) is carried out. 41. The method of claim 40, wherein the co-incubating step is carried out for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours. 42. The method according to claim 39, wherein step c) of combining the apoptotic dendritic cells of step b) with physiological dendritic cells of step c) is carried out in a subject. 43. The method according to any of 39 to 42, wherein the dendritic cells in step a) are derived from an ex vivo blood sample of the subject. 44. The method according to claim 43, wherein the dendritic cells in step a) are obtained by plate passage of PBMCs from the subject. 45. The method according to any of 39 to 44, further comprising the step a1) of incubating dendritic cells with an antigen molecule. 46. ​​The method according to 45, wherein the antigen molecule is an autoantigen. 47. The method of 45 or 46, wherein the antigenic molecule is derived from a natural source, chemically synthesized, or recombinantly produced. 48. The method according to 45 or 46, wherein the antigen molecule is derived from a cell. 49. Autoantigens include Rh blood group antigens, platelet integrin GpIIb:IIIa, noncollagenous domain of basement membrane collagen type IV, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex with or without hepatitis C antigen, pancreatic beta cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamic acid decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, immunogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), and protease inhibitors. 47. The method of claim 46, wherein the antigen is selected from the group comprising: insulin, alpha-enolase, aquaporin-4, beta-arrestin, S100-beta, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein 39, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-beta-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, pancreatic islet cell antigen, cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL), and scleroderma antigen 70 (Scl-70). 50. The method of any of 39 to 49, wherein the apoptotic factors in step b) include psoralen and UVA, riboflavin phosphate and UVA, and / or 5-aminolevulinic acid and light. 51. The method of claim 50, wherein the psoralen is selected from the group including 8-MOP and amotosalen. 52. The method of claim 50, wherein the psoralen is 8-MOP. 53. A method according to any of 39 to 52, wherein the physiological dendritic cells in step c) are obtained by plate passage of PBMCs from the subject. 54. The method of any of 39 to 53, wherein the subject is a mammal, preferably a human. 55. A tolerogenic dendritic cell obtained by the method according to any one of claims 39 to 54. 56. The tolerogenic dendritic cell according to 55 for use in treating an autoimmune disease. 57. The tolerogenic dendritic cells for use according to 56, wherein the autoimmune disease is selected from the group comprising multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, Sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin-dependent diabetes mellitus (IDDM), Addison's disease, celiac disease, chronic fatigue syndrome, colitis, Crohn's disease, fibromyalgia, hyperthyroidism, Graves' disease, hypothyroidism, Hashimoto's disease, endometriosis, pernicious anemia, Goodpasture's syndrome, Wegener's disease and rheumatic fever. 58. A method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of a tolerogenic dendritic cell according to 55. 59. A method for treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject an effective amount of tolerogenic dendritic cells, the tolerogenic dendritic cells comprising physiological dendritic cells comprising material derived from apoptotic dendritic cells obtained from the subject, an autoantigen, a fragment thereof, or a combination thereof. 60. The method of claim 58 or 59, wherein the autoimmune disease is selected from the group comprising multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, Sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin-dependent diabetes mellitus (IDDM), Addison's disease, celiac disease, chronic fatigue syndrome, colitis, Crohn's disease, fibromyalgia, hyperthyroidism, Graves' disease, hypothyroidism, Hashimoto's disease, endometriosis, pernicious anemia, Goodpasture's syndrome, Wegener's disease and rheumatic fever. 61. The autoantigens are Rh blood group antigens, platelet integrin GpIIb:IIIa, noncollagenous domain of basement membrane collagen type IV, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex with or without hepatitis C antigen, pancreatic beta cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamic acid decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, immunogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proin 59 or 60. The method according to claim 59 or 60, wherein the antigen is selected from the group comprising surin, alpha-enolase, aquaporin-4, beta-arrestin, S100-beta, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein 39, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-beta-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, pancreatic islet cell antigen, cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL) and scleroderma antigen 70 (Scl-70). 62. Ex vivo tolerogenic dendritic cells comprising material derived from apoptotic dendritic cells obtained from a subject. 63. The ex vivo tolerogenic dendritic cell according to 62, further comprising an autoantigen or a fragment thereof. 64.(a) A sample of dendritic cells obtained from a subject; (b) an apoptotic factor; (c) their autoantigens or fragments A composition comprising: 65. The composition according to 64, wherein the apoptotic factor comprises psoralen, riboflavin phosphate, or 5-aminolevulinic acid. 66. The composition according to 65, wherein the psoralen is selected from the group including 8-MOP and amotosalen. 67. The composition of claim 66, wherein the psoralen is 8-MOP. 68. Autoantigens include Rh blood group antigens, platelet integrin GpIIb:IIIa, noncollagenous domain of basement membrane collagen type IV, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex with or without hepatitis C antigen, pancreatic beta cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamic acid decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, immunogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proin 68. The ex vivo tolerogenic dendritic cell of claim 62 or 63, or the composition of any one of claims 64 to 67, wherein the dendritic cell is selected from the group comprising surin, alpha-enolase, aquaporin-4, beta-arrestin, S100-beta, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein 39, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-beta-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, pancreatic islet cell antigen, cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL), and scleroderma antigen 70 (Scl-70).

Claims

1. 1. A method for selectively producing tolerogenic dendritic cells, comprising: a) providing dendritic cells from a donor; b) exposing the dendritic cells of step a) to an apoptotic factor; c) providing physiological dendritic cells from the recipient; d) combining the apoptotic donor dendritic cells of step b) with the physiological recipient dendritic cells from step c); A method comprising:

2. 2. The method of claim 1, wherein after step d), a step of co-incubating the apoptotic donor dendritic cells of step b) with the physiological recipient dendritic cells of step c) is performed.

3. 3. The method of claim 2, wherein the co-incubating step is carried out for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

4. 2. The method of claim 1, wherein step d) of combining the apoptotic donor dendritic cells with the physiological dendritic cells from the recipient is performed within the recipient.

5. 2. The method of claim 1, wherein the dendritic cells of step a) are derived from an ex vivo blood sample of the donor.

6. 2. The method of claim 1, wherein the dendritic cells in step a) are obtained by plate passage of PBMCs from the donor.

7. 2. The method of claim 1, wherein the apoptotic factors of step b) comprise psoralen and UVA, riboflavin phosphate and UVA, and / or 5-aminolevulinic acid and light.

8. 8. The method of claim 7, wherein the psoralen is selected from the group comprising 8-MOP and amotosalen.

9. 9. The method of claim 8, wherein the psoralen is 8-MOP.

10. The method of claim 1, wherein the physiological dendritic cells in step c) are obtained by plate passage of PBMCs from the recipient.

11. The donor in step a) is a complementary haplodonor of the recipient, and the dendritic cells in step a) are dendritic cells derived from the complementary haplodonor of the recipient, and 2. The method of claim 1, wherein the recipient of step c) is a haplodonor of the recipient, and the physiological dendritic cells of step c) are physiological dendritic cells derived from the haplodonor of the recipient.

12. 12. The method of claim 11, wherein step d) of combining the apoptotic complementary haplodonor dendritic cells of step b) with the physiological haplodonor dendritic cells of step c) is performed within the haplodonor.

13. The method described in claim 1, wherein the donor in step a) is the recipient and the dendritic cells in step a) are dendritic cells from the recipient.

14. 14. The method of claim 13, wherein step c) of combining the apoptotic dendritic cells of the recipient of step b) with the physiological dendritic cells of the recipient of step c) is performed within the recipient.

15. The method of claim 1 , wherein the donor and / or recipient is a mammal.

16. Tolerogenic dendritic cells obtained by the method of any one of claims 1 to 15.

17. A pharmaceutical composition comprising the tolerogenic dendritic cells of claim 16 for preventing or alleviating graft-versus-host disease.

18. 14. The method of claim 13, further comprising the step a1) of incubating the dendritic cells with an antigen molecule.

19. 19. The method of claim 18, wherein the antigenic molecule is an autoantigen.

20. 20. The method of claim 18, wherein the antigenic molecule is derived from a natural source, chemically synthesized, or recombinantly produced.

21. 20. The method of claim 18, wherein the antigenic molecule is derived from a cell.

22. The autoantigen may be Rh blood group antigen, platelet integrin GpIIb:IIIa, non-collagenous domain of basement membrane collagen type IV, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex with or without hepatitis C antigen, pancreatic beta cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamic acid decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, immunogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), protease inhibitors (PIs), or protease inhibitors (PIs).

20. The method of claim 19, wherein the antigen is selected from the group comprising insulin, alpha-enolase, aquaporin-4, beta-arrestin, S100-beta, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein 39, La antigen, nucleosome histones and ribonucleoproteins (snRNPs), phospholipid-beta-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, pancreatic islet cell antigen, cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL), and scleroderma antigen 70 (Scl-70).

23. Tolerogenic dendritic cells obtained by the method of any one of claims 18 to 22.

24. A pharmaceutical composition comprising the tolerogenic dendritic cells of claim 23 for treating an autoimmune disease.

25. 25. The pharmaceutical composition of claim 24, wherein the autoimmune disease is selected from the group comprising multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, Sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin-dependent diabetes mellitus (IDDM), Addison's disease, celiac disease, chronic fatigue syndrome, colitis, Crohn's disease, fibromyalgia, hyperthyroidism, Graves' disease, hypothyroidism, Hashimoto's disease, endometriosis, pernicious anemia, Goodpasture's syndrome, Wegener's disease and rheumatic fever.

26. Ex vivo tolerogenic dendritic cells comprising material derived from apoptotic dendritic cells obtained from a subject.

27. 27. The ex vivo tolerogenic dendritic cell of claim 26, further comprising an autoantigen or a fragment thereof.

28. (a) a sample of dendritic cells obtained from a subject; (b) an apoptotic factor; (c) with their autoantigens or fragments A composition comprising:

29. 29. The composition of claim 28, wherein the apoptotic factor comprises psoralen, riboflavin phosphate, or 5-aminolevulinic acid.

30. 30. The composition of claim 29, wherein the psoralen is selected from the group comprising 8-MOP and amotosalen.

31. 31. The composition of claim 30, wherein the psoralen is 8-MOP.

32. The autoantigen is selected from the group consisting of Rh blood group antigens, platelet integrin GpIIb:IIIa, non-collagenous domain of basement membrane collagen type IV, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex with or without hepatitis C antigen, pancreatic beta cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamic acid decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, immunogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin 28. The ex vivo tolerogenic dendritic cell of claim 26 or 27, wherein the antigen is selected from the group comprising: phospholipid, alpha-enolase, aquaporin-4, beta-arrestin, S100-beta, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein 39, La antigen, nucleosome histones and ribonucleoproteins (snRNPs), phospholipid-beta-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, pancreatic islet cell antigen, cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL), and scleroderma antigen 70 (Scl-70).

33. The autoantigen, wherein the autoantigen is Rh blood group antigen, platelet integrin GpIIb:IIIa, non-collagenous domain of basement membrane collagen type IV, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex with or without hepatitis C antigen, pancreatic beta cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamic acid decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, immunogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, α 32. The composition of any one of claims 28 to 31, wherein the antigen is selected from the group comprising enolase, aquaporin-4, β-arrestin, S100-β, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein 39, La antigen, nucleosome histones and ribonucleoproteins (snRNPs), phospholipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, pancreatic islet cell antigen, cytoplasmic linker protein-170 (CLIP-170), Sjogren's syndrome antigen A (SS-A / Ro), Sjogren's syndrome antigen B (SS-B / La), Sjogren's lupus antigen (SL), and scleroderma antigen 70 (Scl-70).

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