Method for inducing an Anti-inflammatory phenotype in t-cells using mesenchymal stem cell-derived mitochondria

Abstract

The inventors have demonstrated that incorporating mesenchymal stem cell-derived mitochondria using T-cells induces a significant increase in their anti-inflammatory properties. This innovation could reprogramme T-cells isolated from individuals with inflammatory and / or autoimmune diseases by acquiring specific mitochondria (MT) that induce an anti-inflammatory phenotype. The MT-T-cells obtained could be used as therapeutic agents to effectively reduce inflammation and therefore the symptoms of these diseases.

Description

[0001] Method for inducing anti-inflammatory phenotype in T cells using mitochondria derived from mesenchymal stem cells

[0002] DESCRIPTIVE MEMORY

[0003] Background of the invention

[0004] Mesenchymal stem / stromal cells (MSCs) are multipotent progenitor cells that can be isolated from almost any mesodermal tissue, such as bone marrow (BM), adipose tissue (AT), umbilical cord (UC), among others, with broad immunosuppressive capabilities. They are capable of suppressing proinflammatory functions of T cells and promoting the generation of regulatory T cells (Treg), resulting in beneficial effects in a variety of preclinical models of immune-mediated diseases, whether autoimmune or inflammatory, such as encephalomyelitis, graft-versus-host disease, rheumatoid arthritis, delayed-type hypersensitivity (DTH), type 1 diabetes, celiac disease, lupus, multiple sclerosis, myasthenia gravis, Addison's disease, Crohn's disease, ulcerative colitis, among others.

[0005] For this reason, MSCs have been widely proposed as the best candidate for the treatment of inflammatory and autoimmune diseases, leading to more than 90 clinical trials in autoimmune diseases registered on the NIH website. However, the results of these studies have been variable in preclinical and clinical models, suggesting that the immunoregulatory properties of MSCs require a deeper understanding to optimize their clinical application.

[0006] In this context, MSCs have been widely shown to respond to several mediators, including the cytokines interferon gamma (ZFNγ) and tumor necrosis factor (TNFγ), thereby enhancing their immunomodulatory capacities. Recent studies have revealed that these inflammatory signals are associated with changes in MSC metabolism, characterized by a shift from oxidative phosphorylation to glycolysis. These findings are consistent with the results obtained by the inventors, who demonstrated that metabolic reprogramming of MSCs toward glycolytic metabolism using Oligomycin, a well-recognized ATP synthase inhibitor, significantly increases their glycolytic bioenergetic status, regardless of the MSC source.

[0007] Furthermore, the results demonstrated that pretreatment of MSCs with oligomycin, which leads to glycolytic MSCs, enhanced their in vitro immunosuppressive activity in freshly isolated peripheral blood mononuclear cells (PBMCs) activated with phytohemagglutinin (PHA) to induce T cell activation / proliferation (Figure 1). This effect was also observed in helper T (Th) cells and in CD4 T cells differentiated into proinflammatory Thl and Thl7 cells.

[0008] Likewise, results obtained in in vivo experiments, the inventors have determined that, compared to control MSCs, glycolytic MSCs exhibit significantly higher therapeutic efficacy in both the murine model of delayed-type hypersensitivity (DTH) and graft-versus-host disease (GVDH).

[0009] Recent studies have shown that the metabolic status of MSCs is critical for their immunoregulatory and therapeutic efficacy. Based on this, the inventors hypothesized that the effect of MSCs also depends on their mitochondrial metabolic activity. Previous studies have shown that in co-cultures, untreated MSCs can spontaneously transfer mitochondria to CD4 T cells, inducing a Treg phenotype and enhancing GHVD progression. In light of these results, the inventors proposed a strategy of metabolic reprogramming followed by mitochondrial (MT) transfer. In this approach, for the first time, MT mitochondria isolated from MSCs, metabolically modified to enhance glycolysis—for example, by pretreatment with oligomycin—are used and transferred to immune cells, enhancing their activity.

[0010] DESCRIPTION OF THE FIGURES

[0011] Figure 1. Modification of the expression of immunosuppressive potential of T cells from PBMCs from healthy donors incubated with UC-MSCs under different metabolic conditions. PBMCs were isolated from healthy donors, activated with PHA and cultured alone or in the presence of control UC-MSCs (Ctl), or pretreated with 2-DG or oligomycin. A) CD4+ T cells. B) The percentage of CD4+CD25+FOXP3+ T-producing cells. C) The percentage of CD4+IFNy+ T cells. Results are represented as mean ± SD considering four independent experiments using at least seven different PBMC donors and four UC-MSC donors. Statistical analyses were performed using a two-way ANOVA test comparing paired samples (*p<0.05; **p<0.01; ***p<0.001, ****p<0.0001).

[0012] Figure 2. Modification of the expression of the immunosuppressive potential of memory CD4 T cells by artificial mitochondrial transfer (MitoT) from UC-MSCs. Memory CD4 T cells isolated from healthy donors were incubated with mitochondria isolated from UC-MSCs pretreated with 2-DG (oxidative phosphorylation metabolism) or oligomycin (glycolytic metabolism). The expression of different markers is compared between non-transformed controls (Mito -) and transformed with mitochondria (Mito +) from both conditions. The percentage of A) T-CD4 + CD25 + IFNy + producing cells (Al), T-CD4 + CD25 + IL17 + (A.2) and T-CD4 + CD25 + FOXP3 + (A.3) is shown. B) Shows the level of the anti-inflammatory cytokine, IL-10. Results are represented as the mean ± SD (standard deviation) of four independent experiments using at least seven different PBMC donors and four UC-MSC donors.Statistical analyses were performed using a two-way ANOVA test comparing paired samples (*p<0.05; **p<0.01; ***p<0.001, ****p<0.0001).

[0013] Figure 3. Modification of the expression of immunosuppressive potential of memory CD4 T cells from patients with RA by artificial mitochondrial transfer (MitoT) from UC-MSCs. Memory CD4 T cells isolated from donors with rheumatoid arthritis were incubated with mitochondria isolated from UC-MSCs pretreated with 2-DG (oxidative phosphorylation metabolism) represented by diagonal stripes or oligomycin (glycolytic metabolism) represented by dotted lines. The control is shown in horizontal stripes. The expression of different markers is compared between non-transformed controls (Mito -) and transformed with mitochondria (Mito +) from both conditions. The percentage of A) percentage of memory CD4 T cells that express ICOS and B) percentage of memory CD4 T-producing cells that express F0XP3+ CTLA4+ are shown.The graphs represent the mean ± SD and statistical analysis using the nonparametric Mann-Whitney test between two parameters (comparison between MITO+ or MITO- or MITO+CTL vs MITO+OLIGO). Each point represents a different HD with at least three different donors.

[0014] Figure 4. Effect of UC-MSC mitochondria in an in vivo model of hypersensitivity-mediated inflammation. A) Effect of CD4 T cells from a mouse model of hypersensitivity-mediated inflammation (HMI), which contain MSC mitochondria labeled with the fluorophore Dendra, and where the MSCs had been pretreated or not with Oligomycin before mitochondria isolation for mitoception. Inflammation of the animal's paw is measured in control conditions and with treatment of CD-4 T cells with Dendra-labeled MSC mitochondria (MitoDendra) and with treatment of CD-4 T cells with Dendra-labeled MSC mitochondria pretreated with Oligomycin (MitoDendra+Oligo). It is seen that treatment with T cells and MSC mitochondria significantly decreases inflammation, and that MSC mitochondria pretreated with Oligomycin have an even greater anti-inflammatory effect.B) Graph showing the mitoception of both types of mitochondria in CD4 T lymphocytes, those obtained from MSC (MitoDendra) and those obtained from MSC pretreated with Oligomycin (MitoDendra+Oligo).

[0015] DESCRIPTION OF THE INVENTION

[0016] The present invention proposes a method for enhancing the anti-inflammatory activity of T cells by acquiring functional mitochondria isolated or obtained from umbilical cord mesenchymal stem cells (UC-MSCs). These UC-MSCs preferably undergo a modification that induces an increase in their glycolytic metabolism before their mitochondria are extracted.

[0017] T cells modified with these mitochondria can be reimplanted into the individual with the pathology, where they will contribute to reducing inflammatory processes in diseases such as rheumatoid arthritis (RA) or in delayed-type hypersensitivity (DTH) pathologies or in graft-versus-host disease (GVDH), as well as in other pathologies related to immune imbalances, and other autoimmune and / or inflammatory diseases such as encephalomyelitis, type 1 diabetes, celiac disease, lupus, multiple sclerosis, myasthenia gravis, Addison's disease, Crohn's disease, ulcerative colitis, among others.

[0018] The inventors studied the immunoregulatory potential of mitochondria (MT) obtained from both unmodified UC-MSCs and those with activated glycolytic metabolism. This study sought to determine whether MTs derived from glycolytic MSCs induced greater differentiation of CD4+ T cells toward a regulatory phenotype (Treg) and greater suppression of Th17 cells (pro-inflammatory phenotype) compared to MTs obtained from unmodified MSCs.

[0019] To clearly determine the level of MT-dependent immunoregulatory impact without interference from other factors, the inventors used an artificial mitochondrial transfer approach called mitoception, in which purified MTs were incubated with memory CD4 T cells. They then assessed phenotypic, functional, and metabolic changes in the CD4 T cells post-mitoception.

[0020] In the functional assay, the inventors measured the expression of anti-inflammatory cytokines, such as IL-10, in memory CD4 T cells, analyzing their pro-inflammatory and anti-inflammatory phenotype by fluorescence-activated cell sorting (FACS). Furthermore, to induce glycolytic metabolism in MSCs, oligomycin was used. Initially, peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with UC-MSC cells pretreated with 2-DG (an inhibitor of glycolysis metabolism) or oligomycin, comparing both metabolic conditions. The results are shown in Figure 1, and indicate that oligomycin pretreatment increased the immunosuppressive effect of UC-MSCs on PBMC-derived T cells, in terms of reducing the proliferation of the latter.Subsequently, the inventors isolated mitochondria from UC-MSCs pretreated or not with 2-DG or Oligomycin, revealing even stronger immunoregulatory results than whole cells, although no significant differences were observed between mitochondria derived from UC-MSCs pretreated with 2-DG or Oligomycin in samples from healthy donors (Figure 2). MTs from both types of pretreatment decreased the expression of proinflammatory markers such as IFNy, IL-17 and CD25+FOXP3+ and increased the expression of IL-10, an anti-inflammatory cytokine, indicating an increase in the anti-inflammatory activity of T cells.

[0021] Preliminary results obtained with T cells derived from PBMCs of healthy donors indicate improved anti-inflammatory activity in T cells incubated with mitochondria isolated from UC-MSCs. A priori, no significant differences were observed between mitochondria from UC-MSCs with and without induced glycolytic metabolism. However, when using T cells from donors with inflammatory and autoimmune diseases, such as rheumatoid arthritis, differences were observed: mitochondria derived from glycolytic UC-MSCs showed a superior anti-inflammatory effect compared to T cells from these donors.

[0022] This result validates the researchers' initial hypothesis, demonstrating that UC-MSC mitochondria with induced glycolytic metabolism are more effective in enhancing the anti-inflammatory activity of T cells in individuals with inflammatory pathologies, such as rheumatoid arthritis (RA), delayed-type hypersensitivity (DTH), or graft-versus-host disease (GVDH); encephalomyelitis, type 1 diabetes, celiac disease, lupus, multiple sclerosis, myasthenia gravis, Addison's disease, Crohn's disease, ulcerative colitis, among others. These results offer a strategy to obtain peripheral blood T cells from individuals with an immune-mediated disease with an inflammatory component, such as RA, DTH, or GVDH, and incubate them with mitochondria isolated from UC-MSCs pretreated with oligomycin, thus inducing a glycolytic state before mitochondrial extraction.The treated T cells can be reimplanted into the same individual, functioning as an autologous treatment with increased anti-inflammatory capacity, which would help mitigate the symptoms of these diseases (RA, DTH, GVDH, lupus or other diseases with immune imbalance).

[0023] Since this procedure uses internally modified T cells, the risks associated with conventional biological therapies are reduced. Together, these results suggest that MSC-derived mitochondria, particularly when metabolically reprogrammed toward a glycolytic profile, represent a promising therapeutic approach for modulating proinflammatory memory CD4 T cells, intervening in immunoregulatory imbalances and autoimmune diseases such as RA, DTH and GVDH encephalomyelitis, type 1 diabetes, celiac disease, lupus, multiple sclerosis, myasthenia gravis, Addison's disease, Crohn's disease, ulcerative colitis, among others.

[0024] Thus, the invention relates to a method for inducing an anti-inflammatory or regulatory T cell phenotype in a T cell population, wherein this method comprises the following steps: a. Obtaining a culture of umbilical cord mesenchymal stem cells (UC-MSC); b. Extracting the mitochondria from the cells of the previous culture and resuspending them in a buffer solution at a concentration between 50 to 200 mg of mitochondria per mL; c. Obtaining the mononuclear cell fraction from an isolated peripheral blood sample (PBMC), and isolating the memory CD4 T cells by using a kit, and maintaining them in culture; d. Incubating the memory CD4 T cells obtained from step c) with the mitochondria obtained in step b) and maintaining them in culture; e. Obtain memory CD4 T cells that have incorporated the mitochondria of UC-MSCs into their cytoplasm, thereby modifying their metabolism toward a regulatory T cell phenotype with anti-inflammatory properties.Resuspend the obtained T cells in a buffer solution, obtaining a composition of T cells with an anti-inflammatory or regulatory T cell phenotype.

[0025] Where in stage b) mitochondria are removed from a quantity of around 10 5 to 10 7cells from the MSC culture from step a). And additionally in step d) the mitochondrial suspension obtained in step b) is carefully applied to the memory CD4 T-cell culture obtained from step c), and the culture plates are centrifuged at 1500 g for 15 min at 4 ° C. Subsequently, in step d) the memory CD4 T-cells already centrifuged with the mitochondria are incubated for 1 to 4 hours, at 37 ° C and subsequently subjected to a second centrifugation at 1500 g for 15 min at 4 ° C. Finally in step d) after the second centrifugation of the memory CD4 T-cells incubated with the mitochondria, they are cultured at 37 ° C for 8 to 14 hours.

[0026] In a second embodiment, the invention relates to the method described above, where, optionally, in step a) the umbilical cord blood mesenchymal stem cells (UC-MSCs) are pretreated with oligomycin at a concentration of 0.1 to 10 pg / ml for 12 to 48 hours; and then washed with a buffer solution.

[0027] In a third embodiment, the invention relates to the use of memory CD4 T cells obtained according to any of the above methods, wherein these modified cells are incorporated into an injectable solution comprising the cells and a pharmacologically acceptable buffer solution, wherein the T cells have anti-inflammatory or regulatory T cell phenotypic properties. Wherein the injectable solution is useful for mitigating the symptoms of a disease in cases of immune imbalances or autoimmune diseases. Specifically, immune or autoimmune imbalance diseases are rheumatoid arthritis (RA), delayed-type hypersensitivity (DTH) or graft-versus-host disease (GVDH), type 1 diabetes; celiac disease; lupus; multiple sclerosis; myasthenia gravis; Addison's disease; Crohn's disease; ulcerative colitis.In one embodiment, the T cells modified by the method of the invention are conveniently injected autologously into the peripheral blood donor.

[0028] It is clear to those skilled in the art that a method using live blood cells and mitochondria must always work with appropriate buffers to preserve cellular and mitochondrial integrity. The nature of the buffer solution may vary, as it is not relevant in itself. The inventors preferentially use buffered saline (PBS) as the buffer, and DMEM as the culture medium.

[0029] The invention will become clearer in light of the following illustrative examples.

[0030] EXAMPLES.

[0031] Example 1. Effect of UC-MSCs on PBMC T cells derived from healthy donors

[0032] First, the inventors conducted a study to establish whether glycolytic metabolism effectively enhances the immunosuppressive potential of UC-MSCs in T cells derived from PBMCs of healthy donors.

[0033] 1.1. Obtaining UC-MSC

[0034] Umbilical cord-derived mesenchymal stem cells (UC-MSCs) were cultured in DMEM medium supplemented with 10% FBS, Penicillin / Streptomycin and glutamine (DMEM-10) until reaching 80% confluence when the culture medium was removed and washed once with buffered saline (PBS).

[0035] UC-MSC Oligomycin

[0036] For glycolytic stimulation, fresh DMEM-10 medium supplemented with an oligomycin solution containing isomers A, B, C at a final concentration of 1 pg / ml was added to the UC-MSCs and they were incubated for 24 hr. Subsequently, the medium that could contain some traces of oligomycin was removed and the cells were washed with buffered saline (PBS), thus obtaining oligomycin UC-MSC cells that have an activated glycolytic metabolism.

[0037] UC-MSC 2-DG

[0038] For the control without glycolytic stimulation, fresh DMEM-10 medium supplemented with a 2-DG solution at a final concentration of 10 mM was added to the UC-MSCs and they were incubated for 24 hr. Subsequently, the medium containing 2-DG was removed and the cells were washed with buffered saline (PBS), thus obtaining 2-DG UC-MSC cells that have inhibited glycolytic metabolism.

[0039] 1.2.Co-culture of UC-MSCs and PBMC

[0040] Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors and stained with CTV (CellTrace Violet). They were also activated with phytohemagglutinin (PHA) (5 pg / mL) for 96 hours to induce T cell activation and proliferation. PBMCs were cultured alone or co-cultured with control UC MSCs, or pre-treated with 2-DG or oligomycin at a PBMC:UC-MSC ratio of 20:1, for 4 days under typical human cell growth conditions (37°C, humidified atmosphere with 5% CO2). After 96 hours of co-culture, the different phenotypes (pro- and anti-inflammatory) of T cells were evaluated by FACS (fluorescence-activated cell sorting). The results are shown in Figure 1.

[0041] CD4+ T cells (or T helper cells) were identified based on the expression of the surface marker CD4, and proliferation was measured as the decrease in CTV fluorescence intensity. The results are shown in (Figure 1 A). When PBMC were coincubated with UC-MSCs under different conditions, a decrease in the proliferation of these cells was observed compared to the control, which was stronger when UC-MSCs with glycolytic metabolism (oligomycin) were used.

[0042] When the result is broken down, it is observed that the percentage of CD4+CD25+FOXP3+ T cells, markers of regulatory T cells, is increased with the presence of UC-MSC incubated with Oligomycin (Figure 1 B). Meanwhile, the percentage of CD4+IFNy+ T cells, markers of Thl cells, does not show variations in the different conditions (Figure 1 C). Both types of T cells were measured by specific intracellular staining to identify IFNy and FOXP3 in PBMC. Overall, we can say that co-culturing T cells with UC-MSC slightly increases the percentage of regulatory T cells, which increases when the mesenchymal cells have been pretreated with Oligomycin.

[0043] Example 2. Effect of UC-MSC Mitochondria on T cells derived from PBMCs of healthy donors

[0044] Since an advantage of incubating with UC-MSC cells with glycolytic metabolism (pretreated with oligomycin) was established in Example 1, it was decided to study the effect of the mitochondria of these cells.

[0045] 2.1 Obtaining mitochondria from UC-MSC

[0046] The cells obtained in example 1: UC-MSC Oligomycin and UC-MSC 2-DG, in this case the mitochondrial donor cells, were labeled with a specific MT fluorescent probe (MitoTracker Green) and then their mitochondria were isolated. Mitochondria were isolated using a commercial mitochondrial isolation kit for cultured cells (Thermo Scientific) following the manufacturer's instructions. Preparations of isolated mitochondria were made using about 10 6MSCs. A yield of 100 mg of mitochondria per million MSCs was obtained. The isolated mitochondria were resuspended in 1 ml of MLR (Mixed Lymphocyte Reaction) medium, kept on ice, and immediately used for artificial transfer or mitoception.

[0047] 2.2 Transformation of T Cells by Mitoception of Isolated Mitochondria.

[0048] The transfer of mitochondria from UC-MSCs thus obtained onto memory CD4 T cells derived from PBMCs of healthy donors were combined at a ratio of 1 UC-MSC mitochondria to 20 memory CD4 T cells isolated from PBMCs. The mitochondrial recipient cells were maintained in MLR medium, and the mitochondrial suspension was slowly added near the bottom of the well, across the entire culture surface. The culture plates were then centrifuged at 1500 g for 15 min at 4 °C. They were then placed in an incubator at 37 °C and 5% CO2. The next day, cells were analyzed by FACS to check for mitoception. Memory CD4 T cells were sorted by FACS and classified as MitoTracker Green positive (Mito+) or MitoTracker Green negative (Mito-).Mito+ and Mito- memory CD4 T cells were then activated with CD3 / CD28 microspheres and IL-2 for 96 hours under typical human cell growth conditions.

[0049] 2.3 Results

[0050] After culture, the different properties of the treated CD4 T cells were evaluated by FACS. The results are shown in Figure 2. The percentage of CD4+CD25+FOXP3+ T cells, CD4+CD25+IFNy+ T cells, and CD4+CD25+IL17+ T cells were evaluated. The results are shown in Figure 2-A. Additionally, the level of the anti-inflammatory cytokine, IL-10, was evaluated using the ELISA technique. The results are shown in Figure 2 B.

[0051] The results show that in all cases, cells that acquired mitochondria (Mito +) behave in a similar way, regardless of whether the glycolytic metabolism of the UC-MSC was activated (Oligomycin) or not (2-DG). In all cases, a small decrease in the subgroups evaluated is observed, being lower for T- CD4 + CD25 + FOXP3 +, markers of regulatory T cells (Figure 2-A.3) Compared to T- CD4 + CD25 + IFNy + (Figure 2-Al) (Thl cells) and T- CD4 + CD25 + IL17 +, (Figure 2-A.2) (Thl7 cells) where a slightly more pronounced decrease is observed. On the contrary, a significant increase in the expression of IL-10, an interleukin with anti-inflammatory activity, is observed in memory CD4 T cells that acquired mitochondria (Mito+), compared to those that did not acquire mitochondria (Mito-).These results demonstrate a positive effect of UC-MSC-derived mitochondria on memory CD4 T cells in terms of an increase in their anti-inflammatory capacity, regardless of the metabolism of the cell of origin.

[0052] Example 3. Effect of UC-MSC Mitochondria on T cells derived from PBMC of donors with rheumatoid arthritis Since a treatment such as the one proposed in the present invention will be applied in individuals with some autoimmune and / or inflammatory pathology, the inventors evaluated how the proposed treatment of artificial mitochondrial transfer (mitoception) of mitochondria obtained from UC-MSC with different metabolisms affects CD4 T cells of patients, specifically in cells obtained from patients with rheumatoid arthritis (RA).

[0053] 3.1 Transformation of CD4 T cells from AR donors by mitoception of isolated mitochondria

[0054] Mitochondria were obtained from UC-MSCs pretreated with 2-DG or Oligomycin and CD4 T cells were transformed with these mitochondria in the same manner as in Example 2 (2.1 and 2.2), with the only difference being that donors were RA individuals. Memory CD4 T cells were classified as MitoTracker Green positive (Mito+) or MitoTracker Green negative (Mito-) using Cell sorting. Mito+ and Mito- memory CD4 T cells were then activated with CD3 / CD28 microspheres and IL-2 for 96 hours under standard human cell growth conditions.

[0055] 3.2 Results

[0056] After culture, the various properties of the treated CD4 T cells were evaluated by FACS. The results are shown in Figure 3.

[0057] The percentage of CD4+ICOS+ T cells, CD4+FOXP3+ T cells, and CD4+CTLA4+ T cells was assessed using intracellular FACS staining to identify FOXP3, ICOS, and CTLA4 in memory CD4 T cells. ICOS, FOXP3, and CTLA4 are classical markers expressed in Treg cells and associated with their immunosuppressive activity. The results are shown in Figure 3.

[0058] The results show that in all cases, cells that acquired mitochondria (Mito +) performed better in all these parameters of regulatory T cell (Treg) activity, such as the expression of ICOS, FOXP3 and CTLA4 +. However, mitochondria obtained from UC-MSCs with induced glycolytic metabolism (pretreated with Oligomycin) showed a significant increase in the expression of these molecules compared to mitochondria obtained from untreated UC-MSCs (Figures 3.A and 3.B).

[0059] These results demonstrate that transforming memory CD4 T cells with mitochondria from UC-MSCs is highly beneficial in transforming these T cells from a diseased individual into regulatory T cells with anti-inflammatory properties. The results are even more effective when using mitochondria derived from UC-MSCs with glycolytic metabolism, that is, pretreated with oligomycin.

[0060] Example 4. Effect of UC-MSC mitochondria-modified T cells in an in vivo model of hypersensitivity-mediated inflammation.

[0061] To study the effect of T cells modified according to the method of the invention, an in vivo study was performed using a murine model of hypersensitivity-mediated inflammation (DTH), where mice are induced to experience inflammation in one of their paws with an allergenic molecule.

[0062] 4.1 Transformation of CD4 T cells from murine donors by mitoception of isolated mitochondria

[0063] The obtaining of mitochondria from UC-MSCs pretreated or not with Oligomycin and the transformation of CD4 T cells with said mitochondria was carried out almost in the same manner as in example 2 (2.1 and 2.2), the main difference being that we worked in a murine model and that the mitochondria were labeled with the fluorophore Dendra. We worked with 6 to 7 animals per condition. Figure 4 B) shows that CD4 T cells mitocept in a similar proportion both the mitochondria obtained from UC-MSCs with their metabolism unchanged (MitoDendra) and those obtained from MSCs pretreated with Oligomycin (MitoDendra+Oligo). Proving the point that despite mitocepting in a similar percentage, mitochondria obtained from MSCs pretreated with oligomycin have a better decrease in inflammation, as shown in Figure 4 A). Each animal in the DTH model was injected with 200 uL of T cells with mitochondria incorporated in PBS buffer. The injections were left to act for 12 hours.

[0064] 4.2 Results

[0065] The results are shown in Figure 4 A). Inflammation is observed in the control condition and with treatment of CD-4 T cells with MSC mitochondria labeled with Dendra (MitoDendra) and with treatment of CD-4 T cells with MSC mitochondria labeled with Dendra and pretreated with Oligomycin (MitoDendra+Oligo). It is seen that treatment with T cells and MSC mitochondria significantly decreases inflammation, and that MSC mitochondria pretreated with Oligomycin have an even greater anti-inflammatory effect.

[0066] This demonstrates that the method and treatment proposed in the invention allows T cells to be modified towards an anti-inflammatory or regulatory T cell phenotype, which effectively allows inflammatory symptoms to be alleviated in vivo, without an increased risk associated with this treatment.

Claims

Claims 1. A method for inducing anti-inflammatory phenotype and / or regulatory T cell phenotype in T cells, CHARACTERIZED in that it comprises the following steps: a. Obtaining a culture of umbilical cord mesenchymal stem cells (UC-MSC); b. Extracting the mitochondria from the cells of the previous culture and resuspending them in a buffer solution at a concentration between 50 to 200 mg of mitochondria per mL; c. Obtaining the mononuclear cell fraction from an isolated peripheral blood sample (PBMC), and isolating the memory CD4 T cells by using a kit, and maintaining them in culture; d. Incubating the memory CD4 T cells obtained from step c) with the mitochondria obtained in step b) and maintaining them in culture; e. Obtain memory CD4 T cells that have incorporated the mitochondria of UC-MSCs into their cytoplasm, thereby modifying their metabolism toward a regulatory T cell phenotype with anti-inflammatory properties; f.Resuspend the obtained T cells in a buffer solution, obtaining a composition of T cells with an anti-inflammatory or regulatory T cell phenotype.

2. Method according to claim 1 CHARACTERIZED in that in step b) the mitochondria are extracted from an amount of about an amount of 10 5 to 10 7 cells from the MSC culture from stage a).

3. Method according to claim 1 CHARACTERIZED in that in step d) the suspension of mitochondria obtained in step b) is carefully applied to the culture of memory T-CD4 cells obtained from step c), and the culture plates are centrifuged at 1500 g for 15 min at 4 °C.

4. Method according to claim 3 CHARACTERIZED in that in step d) the memory CD4 T cells already centrifuged with the mitochondria are incubated for 1 to 4 hours at 37°C and then subjected to a second centrifugation at 1500 g for 15 min at 4°C.

5. Method according to claim 4 CHARACTERIZED in that in step d) after the second centrifugation of the memory CD4 T cells incubated with the mitochondria they are cultured at 37°C for 8 to 14 hours.

6. Method according to claim 1, CHARACTERIZED in that optionally in step a) the umbilical cord mesenchymal stem cells (UC-MSC) are pre-treated with oligomycin at a concentration between 0.1 to 10 pg / ml for 12 to 48 hours; and subsequently washed with a buffer solution.

7. Method according to claim 6, CHARACTERIZED in that in step b) the mitochondria are extracted from an amount of about 10 5 to 10 7 cells from the MSC culture from stage a).

8. Method according to claim 6, CHARACTERIZED in that in step d) the suspension of mitochondria obtained in step b) is carefully applied to the culture of memory T-CD4 cells obtained in step c), and the culture plates are centrifuged at 1500 g for 15 min at 4 °C.

9. Method according to claim 8, CHARACTERIZED in that in step d) the memory T-CD4 cells already centrifuged with the mitochondria are incubated for 1 to 4 hours at 37°C and subsequently subjected to a second centrifugation at 1500 g for 15 min at 4°C.

10. Method according to claim 9, CHARACTERIZED in that in step d) after the second centrifugation, the memory CD4 T cells cultured with the mitochondria are cultured at 37°C for 8 to 14 hours.

11. Use of memory CD4 T cells obtained in accordance with any of clauses 1 to 10 CHARACTERIZED in that they are suitable for incorporation into an injectable solution comprising the cells and a pharmacologically acceptable buffer solution, where the T cells have anti-inflammatory or regulatory T cell phenotypic properties.

12. Use according to claim 11 CHARACTERIZED in that the injectable solution is useful for mitigating the symptoms of a disease in cases of immune imbalances or autoimmune diseases.

13. Use according to claim 12 CHARACTERIZED in that the immune or autoimmune imbalance diseases are rheumatoid arthritis (RA), delayed-type hypersensitivity (DTH) or graft-versus-host disease (GVDH), Type 1 Diabetes; Celiac disease; Lupus; Multiple sclerosis; Myasthenia gravis; Addison's disease; Crohn's disease; Ulcerative colitis.

14. Use according to claim 13, CHARACTERIZED in that the T cells modified by the method of the invention are conveniently injected autologously into the peripheral blood donor.

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