Methods and systems for generating apoptotic monocytes

Abstract

The present invention relates to a method for producing apoptotic monocytes, comprising the steps of subjecting said blood component to ultraviolet radiation having a wavelength between 200 nm and 320 nm, said radiation being arranged so as to induce a difference between the rate of apoptosis of the irradiated cells and the non-irradiated cells greater than 15% after 48 hours of irradiation, thereby obtaining apoptotic monocytes capable of modulating the immune response of a patient.

Description

[0001] The present invention relates to a method, an irradiation device, and a system for generating apoptotic mononuclear cells contained in donor peripheral blood components and using these cells to treat conditions associated with immune system dysfunction.

[0002] The invention is applicable to the fields of cell therapy and immunotherapy, especially in vitro photoimmunotherapy.

[0003] Phototherapy, also known as extracorporeal photochemotherapy (ECP), is a cell therapy technique that has been used since Edelson first treated cutaneous T-cell lymphoma (CTCL) in the 1980s (Edelson et al., 1987). The invention involves drawing blood from the patient, separating the mononuclear cells (MNCs) from the blood by centrifugation, treating these cells with ultraviolet A (UVA) in the presence of a photoactivating intercalator called 8-methoxypsoralen (8-MOP), and then re-injecting the activated cells into the patient. The cells are processed outside the body.

[0004] ECP has proven its value in treating conditions related to immune system dysfunction, whether in autologous cases (specific patients), such as tumors or autoimmune diseases, or in allogeneic cases (donor-recipient relationships), such as solid organ or hematopoietic stem cell (HSC) transplantation, potentially associated with major complications such as graft-versus-host disease (GvHD).

[0005] The safety and effectiveness of this treatment are now prompting clinicians to expand the use of ECP, especially in the treatment of autoimmune diseases (Adamski et al., 2015).

[0006] The mechanism of action of ECP is not fully understood. However, it is believed that 8-MOP penetrates the cell membrane and embeds itself between the two DNA strands. Upon UVA irradiation, 8-MOP is activated, triggering a photoaddition process (Jeantet, 2004). This process inhibits DNA replication and transcription, leading to cell proliferation arrest and apoptosis. Among monocytes treated during CEP, T lymphocytes are the most affected by apoptosis (Heshmati, 2014). Apoptosis is a highly regulated programmed cell death; another type of cell death is necrosis, a so-called "accidental" death that occurs when the environment is disturbed.

[0007] After being re-injected into the patient, the T lymphocytes treated with ECP undergo apoptosis and are phagocytosed by antigen-presenting cells (APCs) (also known as dendritic cells), activating the immune system and causing immune tolerance (GvHD, transplant rejection) or immune activity (cutaneous T lymphoma).

[0008] More specifically, CEP stimulates monocytes to differentiate into dendritic cells, which then phagocytose apoptotic bodies of T lymphocytes. The role of dendritic cells is to present peptides of phagocytosed elements on their surface to signal the presence of a foreign substance, or to activate specialized T cells and induce immune responses associated with the release of pro-inflammatory or tolerance cytokines.

[0009] Currently, there are two types of ECP procedures, depending on the organization.

[0010] The "closed" or "online" technology proposed by Therakos refers to the use of a single system in a closed system (Cellex system) for processes such as collection, cell separation, processing, and re-injection.

[0011] Another approach is the “open” or “offline” technology proposed by Maco Pharma, in which the sampling / separation and processing stages are performed on different machines and require multiple operations.

[0012] However, the implementation of these two technologies remains relatively time-consuming and complex, and there is still a need to simplify CEP.

[0013] Document WO 2020 / 139495 proposes various systems and methods for ECP on small volumes (less than 500 ml), particularly by reducing the number of devices required for operation. However, these systems and methods still require the use of 8-MOP and UVA irradiation.

[0014] To offset the potential risk of malignant skin tumors following UVA-activated psoralen, document WO2015 / 162279 proposes using 5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin X, instead of 8-MOP. However, the steps required for this technique are the same as those for the 8-MOP / UVA ECP.

[0015] WO 2017 / 005700 discloses an alternative method to the ECP approach that does not use an apoptotic agent to obtain activated monocytes stimulated to differentiate into APCs. This method involves subjecting a patient blood sample containing monocytes to shear force by circulating the blood sample in a flow chamber.

[0016] WO 2016 / 170541 describes cell preparations that include enrichment and aggregation of apoptotic monocyte populations. Monocytes from different donors are induced to apoptosis by incubation with methylprednisolone, followed by gamma irradiation to inhibit cross-reactivity between different donor cells. Ultraviolet irradiation is cited as an alternative to gamma irradiation. These cell preparations can be used to treat immune, anti-inflammatory, or autoimmune diseases, particularly graft-versus-host disease.

[0017] A team of dermatologists, including Tuchinda and Chanisada, published a paper entitled "Comparison of activation of apoptotic pathways in human peripheral blood mononuclear cells by broadband UVB, narrowband UVB, broadband UVA, and UVA1" (Photodermatology, photoimmunology & photomedicine 23.1(2007):2-9). The paper concerns the induction of apoptosis in peripheral blood mononuclear cells by UVA or UVB radiation after the cells had been washed, i.e., without plasma and erythrocytes. Caspase activation was measured as a function of the type and dose of cell irradiation. The conclusion is that all ultraviolet radiation induces apoptosis, and it is suggested that apoptosis may play a role in certain UV-responsive inflammatory skin diseases, such as psoriasis.

[0018] In their article "Effects of Repeated Exposure to Low-Dose Ultraviolet Radiation on Peripheral Blood Monocyte Apoptosis" (Archives of Dermatology 145.2(2009):133-138), J. Narbutt et al. compared the effects of repeated whole-body or partial-body exposure to low-dose UVB (using a lamp emitting 54% UVB and 46% UVA) or simulated solar radiation (using a lamp emitting 4% UVB and 96% UVA) on peripheral blood monocytes in volunteers. Based on this study, the following conclusion can be drawn: UVA, but not UVB, induces lymphocyte apoptosis through photosensitized oxygen free radicals.

[0019] Finally, Buchele, Vera, and Holger Hackstein's "Simplified in vitro phototherapy procedure based on a single high-dose UVA irradiation shows similar in vitro efficacy." Transfusion (2020) proposed that by exposing blood mononuclear cells to high-dose UVA irradiation (5 J / cm²), 2 To achieve CEP without 8-MOP.

[0020] This invention proposes a simpler and faster method designed to mimic the effects of CEP on monocytes in the absence of photoactivators.

[0021] Therefore, according to a first aspect, the present invention proposes a method for generating apoptotic mononuclear cells contained in a donor peripheral blood component, wherein the plasma content of the peripheral blood component is between 30-50%, the process comprising the step of irradiating the blood component with ultraviolet light at a wavelength between 200 nm and 320 nm, wherein the irradiation is expected to induce a difference of more than 15% in the apoptosis rate between irradiated and non-irradiated cells after 48 hours of irradiation, particularly after 24 hours of irradiation, thereby obtaining apoptotic mononuclear cells capable of regulating the immune response of a patient.

[0022] According to a second aspect, the present invention relates to an irradiation apparatus for implementing the method based on the first aspect, the apparatus comprising an ultraviolet light source emitting radiation with a wavelength between 200 and 320 nm and a control unit configured to subject a peripheral blood component containing mononuclear cells to a predetermined dose of ultraviolet light to induce a difference of more than 15% between the apoptosis rate of irradiated cells and the apoptosis rate of unirradiated cells within 48 hours after irradiation.

[0023] According to another aspect, the present invention includes a system for generating apoptotic mononuclear cells containing donor peripheral blood components according to the method of the first aspect, said system comprising...

[0024] (a) An irradiation container for receiving peripheral blood components containing mononuclear cells, the irradiation container being permeable to radiation with wavelengths between 200 and 320 nm, and

[0025] (b) An irradiation apparatus according to the second aspect of the invention.

[0026] Another aspect of the invention relates to apoptotic mononuclear cells obtained by the method according to the first aspect for the treatment of conditions related to immune system dysfunction.

[0027] Other purposes and advantages are described in more detail below.

[0028] [ Figure 1 [ ] shows a schematic diagram of the irradiation bag according to the system of the present invention.

[0029] [ Figure 2 [ ] shows a schematic diagram of the irradiation device according to the system of the present invention.

[0030] [ Figure 3 [This indicates that in the presence of 8-MOP, different doses of UVA (J / cm²) were used.] 2 The difference in apoptosis rate (%) of JURKAT cells after 1 day and 2 days of irradiation.

[0031] [ Figure 4 [This indicates that in the presence of 8-MOP, different doses of UVA (J / cm) were used.] 2 The proliferation inhibition rate (%) of JURKAT cells after 3 days of irradiation.

[0032] [ Figure 5 [Indicates the use of different doses of UVB (J / cm²)] 2 The difference in apoptosis rate (%) of JURKAT cells after 1 day and 2 days of irradiation.

[0033] [ Figure 6 [Indicates the use of different doses of UVA (J / cm²)] 2The proliferation inhibition rate (%) of JURKAT cells after 3 days of irradiation.

[0034] [ Figure 7 [Indicates the use of different doses of UVC (J / cm²)] 2 The difference in apoptosis rate (%) of JURKAT cells after 1 day and 2 days of irradiation.

[0035] [ Figure 8 [Indicates the use of different doses of UVC (J / cm²)] 2 The proliferation inhibition rate (%) of JURKAT cells after 3 days of irradiation.

[0036] [ Figure 9 [This indicates that in the presence of 8-MOP, different doses of UVA (J / cm²) were used.] 2 The difference (%) in apoptosis rate of JURKAT cell suspension in plasma after 0, 1 and 2 days of irradiation.

[0037] [ Figure 10 [This indicates that in the presence of 8-MOP, different doses of UVA (J / cm²) were used.] 2 Irradiation, the proliferation inhibition rate (%) of JURKAT cell suspension in plasma after 3 days.

[0038] [ Figure 11 [Indicates the use of different doses of UVB (J / cm²)] 2 The difference (%) in apoptosis rate of JURKAT cell suspension in plasma after 1 day and 2 days of irradiation.

[0039] [ Figure 12 [Indicates the use of different doses of UVB (J / cm²)] 2 Irradiation, the proliferation inhibition rate (%) of JURKAT cell suspension in plasma after 3 days.

[0040] [ Figure 13 [] represents the difference (%) in apoptosis rate of JURKAT cell suspension in plasma after 1 day and 2 days of irradiation with UVA or UVB in the presence of 8-MOP in two different systems, based on the logarithmic scale of the irradiation dose.

[0041] [ Figure 14 This indicates UVA irradiation with 8-MOP, or with two different doses (J / cm). 2 The difference in apoptosis rate (%) of spleen cells after 1 day of UVB irradiation or culture in the presence of huperzine (an apoptosis inducer).

[0042] [ Figure 15 The result shows 0.007 J / cm 2The difference in apoptosis rate of spleen cells after 1 day and 2 days of UVB irradiation (%).

[0043] [ Figure 16 The result shows 2.5 J / cm. 2 The difference in apoptosis rate of spleen cells after 1 day and 2 days of UVC irradiation (%).

[0044] This invention proposes a process aimed at obtaining cellular responses comparable to those obtained by conventional ECP (8-MOP / UVA), which does not use photoactivators such as 8-MOP or porphyrin derivatives, but only relies on light.

[0045] According to a first aspect, the present invention relates to a method for generating apoptotic mononuclear cells comprising a component of donor peripheral blood. The process includes an ultraviolet irradiation step.

[0046] Peripheral blood mononuclear cells (PBMCs) are all peripheral blood cells with a single nucleus. These cells are composed of lymphocytes (T cells, B cells, NK cells) and monocytes. Red blood cells and platelets do not have nuclei, while granulocytes (neutrophils, basophils, and eosinophils) have multifidus nuclei.

[0047] Prior to implementing the method of this invention, a monocyte-containing component of donor peripheral blood is obtained via leukocyte ablation. This involves using a blood component separation device that draws blood from the donor, separates it into plasma, red blood cells, and white blood cells / platelet layer by centrifugation, reinjects the plasma and red blood cells into the donor, and separates a leukocyte-rich component containing the monocytes. The product of leukocyte ablation includes a peripheral blood component containing monocytes. This leukocyte-rich component has a volume between 150 and 200 ml. It comprises more than 90% monocytes suspended in plasma.

[0048] Another alternative method is to obtain a peripheral blood fraction containing monocytes by separating the leukocyte-platelet layer from a whole blood sample before implementing the process. The whole blood sample is then centrifuged to separate the blood into plasma, red blood cells, and the leukocyte-platelet layer. The separated leukocyte-platelet layer is the peripheral blood fraction containing monocytes suspended in the plasma. The initial volume of whole blood is typically between 100 and 500 ml. With a whole blood sample volume of 100 to 200 ml, the volume of the peripheral blood fraction containing monocytes is between 6 and 10 ml. With a whole blood sample volume of 450 to 500 ml, the volume of the peripheral blood fraction containing monocytes is between 40 and 50 ml.

[0049] Alternatively, peripheral blood components may be cryopreserved before thawing and ultraviolet irradiation. For example, monocytes may be stored in a cryopreservation solution at -80°C, which includes dimethyl sulfoxide (DMSO), serum albumin, and hydroxyethyl starch.

[0050] In particular, the hematocrit of peripheral blood ranges from 0-8%, typically between 1-4%, and sometimes even as high as 2%. The presence of red blood cells affects the amount of ultraviolet radiation absorbed by monocytes, forming a barrier against them.

[0051] To achieve acceptable hematocrit levels, we dilute peripheral blood components containing monocytes before irradiation.

[0052] Dilute with plasma and / or a biocompatible solution (such as physiological saline or a buffer solution). It is best to dilute with only physiological saline.

[0053] In one implementation, the volume of the peripheral blood component is between 6 and 200 ml, and the diluted volume is between 200 and 500 ml, approximately 300 ml.

[0054] The plasma content of peripheral blood fractions containing monocytes is between 30% and 50%. Excessive lipids and / or bilirubin in plasma can affect plasma clarity, thereby limiting the absorption of ultraviolet light by monocytes. Reducing the proportion of plasma in peripheral blood fractions through dilution and / or centrifugation can decrease the risk of excessive plasma absorption.

[0055] The peripheral blood fraction containing monocytes and exposed to ultraviolet light does not contain photosensitizers or apoptosis-inducing agents, such as methylprednisolone. Because these agents are not required, this method is simpler and safer than conventional ECP, as the process can be carried out entirely in a closed system once the peripheral blood fraction to be irradiated is obtained.

[0056] According to one implementation, the process is entirely autologous, meaning that the peripheral blood component containing monocytes is obtained from the patient scheduled to undergo the induced monocyte therapy. Therefore, the donor and recipient are the same person.

[0057] After separating the peripheral blood component from whole blood, the process of the present invention is performed in vitro. The peripheral blood component is placed in a container, separate and isolated from the donor.

[0058] The method according to a first aspect of the invention includes subjecting the blood component to ultraviolet light irradiation with a wavelength between 200 nm and 320 nm.

[0059] The wavelength of ultraviolet radiation is generally between 200 and 280 nm, specifically 255 nm. Alternatively, the wavelength of ultraviolet radiation is between 280 and 320 nm, around 310 nm.

[0060] According to a certain implementation plan, when peripheral blood components flow continuously in a tube or bag forming a serpentine flow channel, the peripheral blood components are irradiated with ultraviolet light.

[0061] In another embodiment, the peripheral blood components are intermittently irradiated with ultraviolet light while they are present in the container.

[0062] To ensure that the monocytes in the peripheral blood fraction receive uniform irradiation, the peripheral blood fraction is continuously agitated during the irradiation process.

[0063] According to the method of the present invention, after 48 hours of irradiation, especially after 24 hours of irradiation, it is expected that the difference in apoptosis rate between irradiated and non-irradiated cells induced by ultraviolet irradiation is greater than 15%.

[0064] The difference between the apoptosis rate of irradiated cells and that of non-irradiated cells is called the apoptosis rate difference. The apoptosis rate difference is determined by calculating the difference between the apoptosis rates of irradiated and non-irradiated cells.

[0065] In particular, the difference in apoptosis rate was greater than 15% after 24 hours of irradiation.

[0066] By irradiating with ultraviolet light with wavelengths between 200 and 320 nm, and primarily between 280 and 320 nm, apoptotic monocytes that can regulate the patient's immune response can be obtained.

[0067] Studies of apoptosis or programmed cell death often employ dual labeling with annexin-V, a FITC fluorophore, and propidium iodide (PI). The labeled cells are then analyzed using flow cytometry.

[0068] Annexin V is a protein with a high affinity for the cell membrane protein phosphatidylserine. When cells are alive, phosphatidylserine on the membrane is located on the inner side of the cell, preventing annexin V from binding. Once cells undergo apoptosis, these proteins are exogenously expressed on both sides of the membrane. Therefore, annexin V can be used to label apoptotic cells.

[0069] The combined use of PI (a DNA intercalating agent) allows for the differentiation of apoptotic cells from necrotic cells. In fact, cell necrosis is accompanied by a loss of membrane integrity, while apoptotic cells do not. This allows PI to penetrate cells and intercalate into DNA. This dual labeling makes it possible to distinguish between living, necrotic, and apoptotic cells: living cells are PI-negative and annexin-V-negative; necrotic cells are PI-positive and annexin-V-negative; early apoptotic cells are PI-negative and annexin-V-positive; and late apoptotic cells are PI-positive and annexin-V-positive.

[0070] Flow cytometry is a technique used to analyze the physical and biological characteristics of individual cells, thus distinguishing cell populations in suspension (Carmaux, 2008). Morphological analysis allows cell populations to be differentiated based on two parameters: size (forward scattering, FSC) and granularity (side scattering, SSC). The result is a scatter plot, with each point corresponding to a cell. Dense, homogeneous regions correspond to a cell population, and these regions can then be delineated using software such as Accuri. This allows for the identification and analysis of the desired cell populations.

[0071] Ideally, irradiated monocytes should continue to undergo apoptosis upon re-injection into the patient. Furthermore, the difference in apoptosis rate induced 48 hours after irradiation is expected to be greater than the difference in apoptosis rate 24 hours after irradiation.

[0072] To achieve immunomodulatory effects in patients, monocytes must be in an apoptotic state rather than necrotic. Therefore, in the process of this invention, irradiation is expected to induce a necrosis rate of less than 5%, preferably less than 1%.

[0073] Another effect of ECP on cells is the inhibition of cell proliferation, especially T lymphocyte proliferation.

[0074] In the method according to the first aspect, irradiation is expected to induce a cell proliferation inhibition rate of greater than 70% three days after irradiation. Preferably, the cell proliferation inhibition rate is greater than 90% three days after irradiation.

[0075] The cell proliferation inhibition rate was calculated by dividing the difference between the proliferation rates of non-irradiated control cells and irradiated cells by the proliferation rate of non-irradiated control cells. The cell proliferation rate was determined by dividing the final total number of cells by the initial total number of cells. The proliferation inhibition rate was determined 3 days after irradiation and after cell culture.

[0076] The amount of ultraviolet light required to achieve the appropriate apoptosis rate difference in monocytes depends on several factors. These factors include intrinsic factors related to the components of peripheral blood, such as hematocrit, plasma volume, and plasma transparency, as well as extrinsic factors related to the irradiation system, such as the shape of the irradiation container receiving the peripheral blood and its transparency to ultraviolet light, the configuration of the ultraviolet light source (unilateral or bilateral irradiation), the type of irradiation container and the stirring speed, and the thickness of the peripheral blood in the irradiation container.

[0077] In a particular implementation, a cell model consisting of JURKAT cells was used to determine the appropriate dose of ultraviolet light to be applied to peripheral blood components containing monocytes.

[0078] JURKAT cells are an immortalized human CD4 T lymphocyte line that was established in the late 1970s from the blood of a 14-year-old boy with leukemia.

[0079] The advantage of this strain is that it is both usable and a target of CEP, as it is composed of T cells. Similar to monocytes, JURKAT cells showed a gradual increase in cell death after treatment with 8-MOP and UVA irradiation (Cunderlíková, 2014; Lauhlé, 2019).

[0080] To determine the appropriate UV dose for application to peripheral blood components, JURKAT cells were irradiated with UV light at wavelengths between 200 and 320 nm, particularly between 280 and 320 nm. After 24 hours, the irradiation dose was determined to be sufficient to induce a 30-40% difference in apoptosis rates. More specifically, after 48 hours of irradiation, the irradiation dose was determined to induce a 60-70% difference in apoptosis rates.

[0081] According to the first implementation scheme, JURKAT cells are suspended in a saline solution of PBS type.

[0082] In another embodiment, JURKAT cells are suspended in a 2% blood cell solution containing plasma, specifically 30-40% plasma.

[0083] The wavelength of ultraviolet radiation is between 280 and 320 nm, specifically 310 nm, or between 200 and 280 nm, specifically 255 nm.

[0084] We determined that the irradiation dose was sufficient to induce an inhibition rate of more than 70% against JURKAT cell proliferation three days after irradiation.

[0085] According to another aspect, the present invention relates to an irradiation apparatus for performing the above-described process. The irradiation apparatus includes an ultraviolet light source emitting radiation with a wavelength between 200 and 320 nm, and a control unit configured to irradiate a peripheral blood component containing mononuclear cells with a predetermined dose of ultraviolet light to induce a difference of greater than 15% between the apoptosis rate of irradiated cells and the apoptosis rate of unirradiated cells within 48 hours after irradiation.

[0086] An irradiation device and an irradiation container for receiving peripheral blood components containing mononuclear cells together constitute a system, wherein the irradiation container is capable of transmitting rays with wavelengths between 200 and 320 nm, for generating apoptotic mononuclear cells contained in donor peripheral blood components according to the method of the first aspect of the invention.

[0087] Irradiation containers are designed to receive peripheral blood components containing mononuclear cells to be irradiated. They are particularly suitable for containing and / or transporting liquids to be irradiated. Irradiation containers are robust and durable.

[0088] In the case of continuous flow irradiation, the irradiation container may be a tubular type, a small bag forming a serpentine flow channel, or a flow box.

[0089] In cases of discontinuous irradiation, the irradiation container is preferably in the form of an irradiation bag.

[0090] Figure 1 An example of such an irradiation bag is shown. Irradiation bag 1 is made of a material that allows ultraviolet light to pass through, such as EVA. Figure 1 In this irradiation bag 1, there is an inlet 2 for introducing peripheral blood components into the bag and an outlet 3. The inlet 2 is connected to a conduit 4 terminating at a perforator 5. The perforator 5 is used to connect to a source bag containing a blood component containing mononuclear cells. The irradiation bag 1 also includes another inlet 6 for introducing diluent if necessary.

[0091] The irradiation device 7 is configured to be associated with the irradiation container 1.

[0092] according to Figure 2 The irradiation device 7 is configured to be associated with the irradiation bag 1. The irradiation device 7 includes a tray 8 on which the irradiation bag 1 is placed. The central portion 9 of the tray 8 has good penetrability to ultraviolet light with wavelengths between 200 and 320 nm, especially between 280 and 320 nm, so the irradiation bag 1 can be irradiated from either side. The central portion 9 of the tray 8 may be made of quartz.

[0093] Tray 8 is preferably a shaker tray. The shaker tray can rotate to ensure uniform irradiation of the contents of the irradiation bag 1.

[0094] The rotational motion of the tray 8 is powered by the motor 10.

[0095] The irradiation device 7 also includes an ultraviolet light source 11 that emits radiation with a wavelength between 200 and 320 nm, particularly between 280 and 320 nm, and more specifically in the range of 310 nm.

[0096] exist Figure 2 In the light source 11, there are multiple lamps that emit ultraviolet light with a radiation wavelength between 200 and 320 nm, especially between 280 and 320 nm.

[0097] exist Figure 2 In the irradiation device 7, there are two sets of six lamps arranged on both sides of the tray 8 to irradiate the irradiation bag 1 from above and below.

[0098] Alternatively, the light source 11 can consist of one or two groups of light-emitting diodes.

[0099] exist Figure 2The device includes a reflector 12 that reflects the light emitted by the light source 11 onto the irradiation bag.

[0100] The irradiation device 7 also includes a control unit configured to subject peripheral blood components containing mononuclear cells to an ultraviolet irradiation dose with a wavelength of 200 to 320 nm, and to predetermine that the difference in apoptosis rates between irradiated and unirradiated cells is greater than 15% after 48 hours of irradiation.

[0101] In particular, the irradiation device 7 provides the irradiation container with an ultraviolet irradiation dose with a wavelength between 200 and 320 nm, which is intended to induce a greater than 15% difference in the apoptosis rate of monocytes after 24 hours of irradiation.

[0102] Specifically, the wavelength of ultraviolet irradiation is between 280 and 320 nm. To control the irradiation dose supplied by the irradiation device 7 to the irradiation container, the irradiation device 7 includes one or more optical sensors disposed on the light source 11. These optical sensors detect the irradiation intensity emitted by the light source.

[0103] The control unit takes the form of an electronic and computer system, including, for example, a microprocessor designed to execute command programs. Executing these programs enables the control unit to control the ultraviolet light source, particularly depending on signals received, for example, by optical sensors. For instance, the control unit determines the irradiation time required to achieve a target dose, as a function of the light intensity determined by the optical sensors.

[0104] In one implementation, the irradiation dose is predetermined using the JURKAT cell model described above.

[0105] Alternatively, the response of JURKAT cells to the difference in apoptosis rates can be modeled using a square numerical model to determine the irradiation dose. Cells undergo apoptosis even without irradiation. When a high dose is applied, the apoptosis rate saturates. Between these points, the function exhibits monotonicity: the difference in apoptosis rates increases with increasing dose.

[0106] A method for operating the above system is described herein to generate apoptotic mononuclear cells contained in donor peripheral blood components.

[0107] The method of operating the system includes the following steps:

[0108] (a) Provide an irradiation container using peripheral blood components from a donor containing mononuclear cells.

[0109] (b) Placing the irradiation container in an irradiation device, which includes an ultraviolet light source emitting wavelengths between 200 and 320 nm;

[0110] (c) Irradiating the irradiation container with the ultraviolet light in the irradiation device, after 48 hours of irradiation, especially after 24 hours of irradiation, it is expected that the difference in apoptosis rate between irradiated and non-irradiated cells induced by ultraviolet irradiation will be greater than 15%.

[0111] Specifically, the ultraviolet light source wavelength is between 280 and 320 nm, and / or the difference in the expected apoptosis rate induced within 24 hours after irradiation is greater than 15%.

[0112] According to the method of the present invention, after ultraviolet irradiation, monocytes can regulate the patient's immune response.

[0113] Therefore, this invention relates to apoptotic mononuclear cells obtained according to the above method for the treatment of conditions related to immune system dysfunction. Such conditions include cutaneous T-cell lymphomas (CTCL), including Cezari syndrome, graft-versus-host disease (GvHD), solid organ transplant rejection, systemic scleroderma, atopic dermatitis, psoriasis, lupus erythematosus, and Crohn's disease.

[0114] In one particular implementation, apoptotic monocytes are cryopreserved after ultraviolet irradiation for future use. Example

[0115] To demonstrate that the conventional ECP technique is equivalent to 8-MOP / UVA in cellular response and to the method of this invention, several experiments were conducted.

[0116] 1. Cell model

[0117] Since it is difficult to obtain mononuclear cells from patients, the JURKAT T lymphocyte line extracted from human lymphoma was chosen as the cell model for the experiment.

[0118] 2. Acceptance Criteria

[0119] Used to validate conventional ECP (200-333 ng / ml 8-MOP and 2 J / cm) 2 The standard for the in vitro efficacy of UVA is the inhibition of T lymphocyte apoptosis and T lymphocyte proliferation.

[0120] For patients with monocytes, the acceptance criteria are:

[0121] Three days after ECP, the cell proliferation inhibition rate was greater than 70%, and

[0122] - The difference in apoptosis rate within 24 hours after ECP was greater than 15% (Taverna, 2015).

[0123] In the JURKAT cell model, the acceptance criteria are:

[0124] -ECP 3 days later, the cell proliferation inhibition rate was greater than 70%, and

[0125] - The difference in apoptosis rate within 24 hours after ECP was greater than 30% (Lauhlé, 2019).

[0126] 3. Preparation of cells for irradiation

[0127] To obtain the required number of JURKAT cells, they were cultured in RPMI 1640 medium supplemented with fetal bovine serum (10% v / v), 1% L-glutamine (Lonza), and 1% penicillin-streptomycin (Lonza). Cells were grown in culture flasks placed in an incubator at 37°C and 5% CO2. The doubling time for JURKAT cells was approximately 27 hours.

[0128] Based on the experimental setup, each irradiation container (petition dish or irradiation bag) used a 10x10 inch irradiation tank. 6 Or 400x10 6 JURKAT cells.

[0129] Cells were suspended in PBS (phosphate-buffered saline) solution or in a 2% blood cell solution containing 1 / 14 anticoagulant ACD-A (a 50 / 50 mixture of NaCl and plasma). The final volume was such that the cell suspension in the container was approximately 3 mm thick.

[0130] Under UVA irradiation, the cell solution also contains 200 ng / ml or 333 ng / ml of 8-MOP.

[0131] 3. Irradiation equipment

[0132] Three types of irradiation equipment were used.

[0133] The first is the Macogenic G2 irradiation device, manufactured by Maco Pharma (France), used to irradiate a bag. It consists of six lamps that provide UVA radiation, arranged on both sides of a UV-transparent quartz tray. The tray is shaken at 60 rpm to ensure uniform irradiation of the irradiation bag.

[0134] The second device is the Macotronic ultraviolet irradiation system from Maco Pharma (France), used to irradiate a bag. It consists of six lamps that provide UVC (254nm) radiation, arranged on both sides of an ultraviolet-transparent quartz tray. The tray is shaken at 110 rpm to ensure uniform irradiation of the bag.

[0135] The third type of irradiation device is from Opsytec Dr. The BS02 hood-type irradiator is used for irradiating culture dishes. This irradiator uses a removable lamp to provide a prescribed dose of irradiation, which can be housed inside the device. The lamps used are UVA with a peak wavelength of 352 nm, UVB with a peak wavelength of 311 nm, or UVC with a peak wavelength of 257 nm. A dosimeter controls the irradiation time based on the irradiation dose measured by sensors within the device. To ensure uniform irradiation of the cell solution, a rotary shaker is placed in the irradiation chamber of the device. The shaking speed is approximately 450 rpm.

[0136] 4. Cell characteristics: Apoptosis test and measurement of cell proliferation

[0137] At the end of the irradiation phase, the cell solution with a hematocrit level of 2% was washed to recover as many JURKAT cells as possible for culture and to reduce red blood cell contamination. Components were separated using a density gradient with Ficoll solution (GE Healthcare) for this purpose.

[0138] After JURKAT was isolated, the cells were washed three times to remove Ficoll solution. The first two washes were performed in PBS containing 2 mM EDTA (Thermo Fisher Scientific), and the third wash was performed in PBS. The cells were then resuspended in supplemented RPMI medium and counted using Vi-cell and ABX to estimate the number of isolated JURKAT and assess the number of residual red blood cells.

[0139] To monitor target parameters (apoptosis and inhibition of JURKAT proliferation), cells were cultured by cell counting. For this purpose, 5 x 10⁶ cells were cultured. 6 Processed and 5x10 6 One untreated cell was seeded in 10 ml of culture medium.

[0140] 4.1 Apoptosis assay

[0141] Apoptosis was determined using dual labeling of annexin-V with FITC fluorophore and propidium iodide (PI).

[0142] The difference in apoptosis rates is determined using the following formula:

[0143] [Mathematical Formula 1]

[0144] Difference in apoptosis rate = (Apoptotic cell rate after treatment - Apoptotic cell rate in untreated cells)

[0145] 4.2. Inhibition of cell proliferation

[0146] The initial quantity of JURKAT is 5 x 10 per culture flask. 6 Cells. Then Vi-cell is used to count the number of cells in each culture flask.

[0147] For this purpose, the cell-containing culture medium was centrifuged at 1500 rpm for 5 minutes, and the cell particles were resuspended in supplemented RPMI medium. Depending on the cell density, the cells could be diluted 1:10 in PBS.

[0148] The percentage of cell proliferation inhibition on day n (Dn) is calculated using the following formula:

[0149] [Mathematical Formula 2]

[0150] %PI(day n) = 100 - %P(day n)

[0151] Where %P(day n) is the cell proliferation rate on day n, calculated using the following formula:

[0152] [Mathematical Formula 3]

[0153]

[0154] Example 1: Comparison of 8-MOP / UVA, UVB and UVC treatments on JURKAT cells in PBS.

[0155] The purpose of this experiment was to determine the irradiation dose for each type of radiation so that (i) the difference in apoptosis rate of JURKAT cells was between 30% and 40% one day after irradiation, between 50% and 60% two days after irradiation, and (ii) the proliferation inhibition rate was greater than 70% three days after irradiation.

[0156] Initially, these experiments were conducted on cells suspended in PBS (without plasma or blood cells) to determine the minimum required radiation dose.

[0157] Will contain 10x10 6 Two ml of cell suspension per cell was placed in a culture dish. Only during UVA irradiation was 200 ng / ml of 8-MOP added to the cell suspension. The culture dish was then placed in the BS02 irradiation apparatus and irradiated using one of three modes: UVA, UVB, or UVC. The shaking speed was set to 450 rpm.

[0158] a.ECP 8-MOP / UVA

[0159] For UVA irradiation, the JURKAT cell suspension contained 200 ng / ml of 8-MOP. The first group tested 0.03 and 0.4 J / cm². 2 Several UVA irradiation doses were used in the second set of tests, based on the results of the first set of tests. Three doses (0.1 J / cm²) were employed. 2 0.13 J / cm 2 and 0.16J / cm 20.13 J / cm 2 The UVA dose met the acceptance criteria in terms of both the difference in apoptosis rate and inhibition of proliferation. Figure 3 and Figure 4 ).

[0160] For all doses of UVA radiation tested, the cell necrosis rate remained below 1%.

[0161] b. UVB irradiation

[0162] After testing from 0.5 mJ / cm 2 Up to 1J / cm 2 After the irradiation dose, the target dose was strictly controlled between 1 and 11 mJ / cm. 2 Between 7mJ / cm 2 The dosage was determined to meet the acceptance criteria in terms of the difference in apoptosis rate and inhibition of proliferation. Figure 5 and Figure 6 ).

[0163] After 24 hours of irradiation, for 7mJ / cm 2 At the given irradiation dose, approximately 20% of the cells undergo late apoptosis, and 40% undergo early apoptosis.

[0164] For 0.5 mJ / cm 2 and 1J / cm 2 For all UVB irradiation doses between these doses, the cell necrosis rate was less than 1%.

[0165] c. UVC irradiation

[0166] Tested at 0.5 to 3.5 mJ / cm 2 After the irradiation dose, the levels of 1 to 5 mJ / cm were retested. 2 Relevant dosage ( Figure 7 and Figure 8 Finally, three doses (2mJ / cm) were tested. 2 2.5 mJ / cm 2 and 3mJ / cm 2 ). 2.5 mJ / cm 2 The UVC dose was determined to meet the acceptance criteria in terms of the difference in apoptosis rate and inhibition of proliferation.

[0167] After 24 hours of irradiation, for 2mJ / cm 2 At the given irradiation dose, approximately 40% of the cells undergo late apoptosis, and 30% undergo early apoptosis.

[0168] It is worth noting that, unlike UVA / 8-MOP and UVB irradiation, for 2mJ / cm 2The difference in apoptosis rate between the first and second days did not increase with the dose of ultraviolet radiation.

[0169] For all doses of UVC tested, the cell necrosis rate remained below 1%. Example 2: Comparison of the effects of 8-MOP / UVA and UVB treatments on JURKAT cells suspended in plasma / saline solution with 2% blood cells.

[0170] The purpose of this experiment is to determine the exposure dose for each type of radiation to meet the following acceptance criteria:

[0171] The difference in apoptosis rate among JURKAT cells was 30% to 40% on day 1 and 50% to 60% on day 2.

[0172] - The proliferation inhibition rate was greater than 70% on day 3.

[0173] Prepare a 2 ml solution containing 10 x 10 6 JURKAT cells, with a blood cell ratio of 2%, 1 / 14 ACD-A, and a 50 / 50 mixture of saline and plasma. 333 ng / ml of 8-MOP was added only during UVA irradiation.

[0174] Will contain 10x10 6 Two ml of cell suspension per cell was placed in a culture dish, which was then placed in a BS02 irradiation device. This device is used for both UVA and UVB irradiation. The shaking speed was set to 450 rpm.

[0175] a.8-MOP / UVA

[0176] Regarding the difference in apoptosis rate and inhibition of proliferation, the dose required to achieve the acceptable standard for the difference in apoptosis rate is 4 J / cm². 2 The acceptable dose for achieving the proliferation inhibition rate is 3 J / cm³. 2 ( Figure 9 and Figure 10 ).

[0177] b.UVB

[0178] Regarding the difference in apoptosis rate, from 0.1 J / cm 2 The dosage began to reach the acceptable standard, and in terms of proliferation inhibition, it started from 0.025 J / cm². 2 Begin to meet acceptance criteria ( Figure 11 and Figure 12 ).

[0179] After 24 hours of irradiation, for 0.1 J / cm 2 At the given irradiation dose, approximately 40% of the cells undergo late apoptosis, and 25% undergo early apoptosis.

[0180] Example 3: Ultraviolet treatment of JURKAT cells suspended in a 2% blood cell saline / plasma solution.

[0181] In this experiment, JURKAT cells were suspended in a mixture of 2% hematocrit, 1 / 14 ACD-A, saline solution, and plasma at a 50 / 50 volume ratio. 400 x 10⁻⁶ cells were then placed in the solution. 6 A cell suspension of 100 cells with a volume of 330-400 ml was placed in a UV-permeable bag.

[0182] Irradiation was performed using a Macotronic ultraviolet light instrument from Macopharma (France). The intensity of 25 mJ / cm² was investigated. 2 and 50mJ / cm 2 Two dosages.

[0183] The results showed that the difference in apoptosis rate remained unchanged on the first and second days after irradiation, at an ultraviolet dose of 25 mJ / cm². 2 The ratio is approximately 50%, and the ultraviolet dose is 50 mJ / cm. 2 The ratio is approximately 70%.

[0184] Regarding the inhibition of cell proliferation three days after irradiation, a dose of 25 mJ / cm 2 The inhibition rate was approximately 65% ​​at a dose of 50 mJ / cm. 2 The inhibition rate is approximately 80%.

[0185] Example 4: Relevance to Maco Pharma's ECP Technology

[0186] Using the same matrix as in Example 2, JURKAT cells (330x10⁶ cells / cm²) were processed using the ECP system sold by Maco Pharma. 6 Irradiate 300 ml of 8-MOP (333 ng / ml) per cell.

[0187] Maco Pharma's ECP system consists of a UVA permeation bag and a Macogenic G2 irradiation device, designed with a concentration of 2 to 2.5 J / cm³ based on the hematocrit of the solution contained in the bag to be irradiated. 2 The irradiation dose. The irradiation cycle lasts approximately 12 minutes.

[0188] From 2J / cm 2 The dosage was started at the acceptable level, which was achieved by reaching the difference in apoptosis rate and inhibiting proliferation, consistent with the system's recommendations.

[0189] To simulate the cellular response to differences in apoptosis rates, the Sigmoidal digital model is suitable.

[0190] Figure 13 A graph showing the results obtained using a logarithmic scale for irradiation dose is presented.

[0191] Acceptance criteria depend on the combination of the irradiation container and equipment, as well as the optical configuration of the system. For example, the 2.1 J / cm² irradiation standard provided by the Macogenic G2. 2 The UVA dose is equivalent to 4 J / cm provided by a BS02 irradiator. 2 The UVA dose or 0.081 J / cm provided by the BS02 irradiator. 2 UVB dose.

[0192] Example 4: Mouse spleen cell model

[0193] As part of preparation for a study on a mouse GvH model, an experiment was conducted to examine whether a dose derived from irradiation with JURKAT (Example 1) could induce apoptosis in mouse spleen cells.

[0194] Will contain 5x10 6 A suspension of spleen cells (Immune InsighT, France) was irradiated using a BS02 irradiator at the following doses: UVA / 8-MOP 0.13 J / cm². 2 UVB is 7 mJ / cm 2 and 70mJ / cm 2 UVC is 2.5 mJ / cm 2 .

[0195] Under UVA irradiation, the cell suspension contained 200 ng / ml of 8-MOP.

[0196] Each dose tested induced apoptosis. Notably, the difference in apoptosis rates decreased after 2 days of irradiation, which was due to the high apoptosis rate measured in the control group. Figures 14 to 16 ).

Claims

1. A method for generating apoptotic mononuclear cells contained in a peripheral blood fraction of a donor, wherein the plasma content of the peripheral blood fraction is between 30-50%, the method comprising the step of exposing the peripheral blood fraction to ultraviolet light, characterized in that, The wavelength of the ultraviolet irradiation is between 200 nm and 320 nm. The irradiation is arranged to induce a difference of more than 15% between the apoptosis rate of the irradiated cells and the apoptosis rate of the unirradiated cells after 48 hours of irradiation, thereby obtaining apoptotic monocytes that can regulate the patient's immune response.

2. The method according to claim 1, characterized in that, The irradiation was arranged to induce a difference of more than 15% between the apoptosis rate of irradiated cells and the apoptosis rate of unirradiated cells 24 hours after irradiation.

3. The method according to claim 1 or 2, characterized in that, The irradiation was designed to further induce cell proliferation inhibition of more than 70% three days after irradiation.

4. The method according to claim 1 or 2, characterized in that, The wavelength of the ultraviolet irradiation is between 280 and 320 nm.

5. The method according to claim 1 or 2, characterized in that, The wavelength of the ultraviolet irradiation is 310 nm.

6. The method according to claim 1 or 2, characterized in that, The peripheral blood components were obtained by leukocyte separation and / or by separating the leukocyte and platelet layers from a whole blood sample.

7. The method according to claim 1 or 2, characterized in that, The peripheral blood component contains more than 90% mononuclear cells.

8. The method according to claim 1 or 2, characterized in that, Prior to the irradiation step, the method includes the step of diluting the peripheral blood components with physiological saline.

9. The method according to claim 1 or 2, characterized in that, The peripheral blood components contain a hematocrit level of 0 to 8%.

10. The method according to claim 1 or 2, characterized in that, The volume of the peripheral blood component is between 6 mL and 200 mL.

11. The method according to claim 1 or 2, characterized in that, The peripheral blood components containing monocytes are stirred during irradiation.

12. The method according to claim 11, characterized in that, Peripheral blood fractions containing monocytes do not contain photosensitizers or apoptosis inducers.

13. An irradiation apparatus for carrying out the method according to any one of claims 1 to 12, said irradiation apparatus comprising an ultraviolet light source emitting radiation with wavelengths between 200 and 320 nm, characterized in that, It further includes a control unit configured to subject a peripheral blood component containing monocytes to a predetermined dose of ultraviolet light to induce a difference of more than 15% between the apoptosis rate of the irradiated cells and the apoptosis rate of the unirradiated cells after 48 hours of irradiation.

14. The irradiation apparatus according to claim 13, characterized in that, It further includes a shaker tray and / or an optical sensor that detects the intensity of illumination emitted by the light source.

15. A system for generating apoptotic mononuclear cells contained in a peripheral blood fraction of a donor using the method according to any one of claims 1 to 12, said system comprising: - An irradiation container for receiving peripheral blood components containing mononuclear cells, the irradiation container being permeable to radiation with wavelengths between 200 and 320 nm, and - The irradiation device according to any one of claims 13 or 14.

16. Use of apoptotic mononuclear cells obtained by the method of any one of claims 1 to 12 in the preparation of a medicament for treating a condition associated with immune system dysfunction, said condition being cutaneous T-cell lymphoma (CTCL).

17. Use of apoptotic monocytes obtained by the method of any one of claims 1 to 12 in the preparation of a medicament for treating conditions associated with immune system dysfunction, including Cezari syndrome, graft-versus-host disease (GvHD), solid organ transplant rejection, systemic scleroderma, atopic dermatitis, psoriasis, lupus erythematosus, and Crohn's disease.

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