ES2320056B1 - USE OF NF-KB IN VITRO INHIBITORS TO INDUCE THE SELECTIVE DEPECTION OF ALORREACTIVE T-LYMPHOCYTES, PHARMACEUTICAL COMPOSITIONS DERIVED AND USES OF THE SAME. - Google Patents

Abstract

Use of NF-? B inhibitors in vitro to induce selective depletion of alloreactive T lymphocytes, derived pharmaceutical compositions and uses thereof.

The present invention relates to the use of NF-? B inhibitors in vitro to induce selective depletion of alloreactive T lymphocytes after mixed culture. In particular, the present invention relates to the in vitro use of NF-? B inhibitors, preferably proteosome inhibitors and more preferably bortezomib, in mixed lymphocyte cultures (MLR) to induce selective depletion of the alloreactive T lymphocytes and decrease the Th1 cytokine production. As well as pharmaceutical compositions and their use in therapy.

Description

Use of NF-? B inhibitors in vitro to induce selective depletion of alloreactive T lymphocytes, derived pharmaceutical compositions and uses thereof.

Technical field

The present invention relates to the use of NF-? B inhibitors in vitro to induce selective depletion of alloreactive T lymphocytes after mixed culture. In particular, the present invention relates to the in vitro use of NF-? B inhibitors, preferably proteosome inhibitors and more preferably bortezomib, in mixed lymphocyte cultures (MLR) to induce selective depletion of the alloreactive T lymphocytes and decrease the Th1 cytokine production. As well as pharmaceutical compositions and their use in therapy.

Background of the invention

The term of NF- \ kappaB is a collective definition that refers to the factors of dimeric transcription that belong to the Re1 family, and that regulate through exchanges between the cytoplasm and the nucleus, in response to cell stimulation (1). The family NF- \ kappaB has emerged as a key transducer of inflammatory signals involved in the maturation of dendritic cells and in the activation of T lymphocytes (2-4). Consequently, the various avenues of signaling located after TCR / CD3 converge on various key transcription factors, including the NFAT, AP-1 and NF-? B (5). The activation of the NF- \ kappaB factor is essential for immunogenic responses of T cells, but not for tolerogenic, while NFAT proteins perform functions both immunogenic and tolerogenic (6-9). In addition, the NF- \ kappaB factor activates multiple target genes, whose products can inhibit apoptosis, both activity being closely linked anti-apoptotic as the immunogenic response (10).

Bortezomib, a boronic acid dipeptide is a potent, selective and reversible inhibitor of protease.
ma (11-12). The proteasome is a multienzyme complex present in all cells. It degrades the proteins that regulate the progress of the cell cycle. The latter blocks the nuclear translocation and the transcriptional activity of the NF-? B factor. Bortezomib has shown cytotoxicity against a wide range of human tumor cell lines constitutively expressing the activity of the NF-? B (14,15) factor. In normal T lymphocytes, translocation of the NF-? B factor to the nucleus only occurs after contact with TCR / CD3 and other co-stimulatory molecules (3,16), while it is not activated in T lymphocytes in repose.

One of the main challenges in the transplantation of allogeneic hematopoietic stem or stem cells is to prevent the alloreactivity that leads to the graft reaction against the host (GVHD), while continuing to maintain the effect of the graft against leukemia (GVL) (16.17) Some in vitro data support the existence, within T cells, of responses limited to certain hematopoietic lines (18). Blocking the NF-? B factor could lead to the depletion of activated T lymphocytes in a mixed lymphocyte culture, thus selectively targeting the alloreactive T cells. In addition, blocking the NF-κB factor under in vitro activation conditions could induce overexpression of alternative intracellular pathways, thereby modifying the type of immune response. It is important to note that, in an earlier study, the demonstration that the use of bortezomib could prevent GVHD in a mouse model has been described (19). In the present work we demonstrate that, in human cells, bortezomib selectively targets the alloreactive T cells that induce apoptosis at the concentrations reached in the clinical environment. In addition, the behavior of cytokines is modified, since the drug induces a lower production of Th1 cytokines, which are those involved in the development of the GVHD reaction.

Brief Description of the Invention

A first aspect of the invention relates to the use of NF-? B inhibitors in vitro to generate selective depletion of alloreactive T lymphocytes. In a preferred aspect of the invention said NF-? B inhibitors are proteosome inhibitors. In an even more preferred aspect of the invention, said proteasome inhibitor is bortezomib.

A second aspect of the invention relates to use of the inhibitors of the invention to generate depletion Selective of the alloreactive T lymphocytes after mixed culture. In a preferred aspect of the invention said mixed culture comprises donor lymphocytes and receptor mononucleated cells. In a even more preferred aspect of the invention, said mixed culture comprises donor lymphocytes and dendritic cells of the receiver. In an even more preferred embodiment of the invention said mixed culture comprises donor lymphocytes and lineage cells Endothelial or epithelial receptor.

A third aspect of the invention relates to use of the product obtained after the use of inhibitors of the invention in mixed culture for the preparation of a pharmaceutical composition In a preferred aspect of the invention said pharmaceutical composition is used in therapy. In one aspect more preferred of the invention said pharmaceutical composition is used in the treatment of transplants, more preferably in Hematopoietic transplant treatment.

Description of the figures

Figure 1: Inhibition of induced growth by Bortezomib in T lymphocytes, evaluated by MTT. The results are express as a percentage of absorbance at 570 nM, using a spectrophotometer that considers 100% as the value corresponding to the sample incubated without bortezomib (0 nM). Be observed significant differences (p <0.01) in growth of cells between PHA-stimulated T lymphocytes, MLRs and the controls for the different concentrations of the drug (n = 11); when T cells were cultured with cells irradiated allogeneic dendritic (DC), T lymphocytes showed intermediate proliferative behavior between crops stimulated with PHA and MLR (n = 9); differences were observed significant (p <0.01), in terms of percent inhibition of growth, within T cells stimulated with PHA, Irradiated MLR or DC, depending on the concentration of bortezomib

Figure 2: Western blot analysis of the expression of NF-? B (A) and IkB (B) in control cells or those stimulated by PHA, exposed to 100 nM bortezomib for different periods of time.

Figure 3: Viability of the cells evaluated by flow cytometry through staining with Annexin V-PE and 7-AAD: (3A) between lymphocytes T stimulated with PHA (> 70% of CD25 + events in all cases), cell viability decreases significantly when the concentration of the drug increases; average percentage (more standard deviation) of negative CD3 cells with Annexin V PE and 7 ADF (viable T lymphocytes) for the different doses of Bortezomib in samples stimulated with PHA: 42.13 (7.03) at 0 nM; 46.14 (9.47) at 1 nM; 35.85 (12.24) at 10 nM; 21.2 (11.9) at 100 nM and 12.87 (7.72) at1000 nM; (3B) among resting T lymphocytes only a minority of cells express CD25 (<3% CD25 + events in all the cases). The viability of the cells evaluated as negative events to color with Annexin V-PE and 7-AAD is not left significantly affected by increases in concentration of the drug; average percentage (± standard deviation) of negative CD3 positive cells colored with Annexin V PE and 7 ADF (viable T lymphocytes) for the different doses of Bortezomib in unstimulated samples: 60.64 (6.03) at 0 nM; 65.6 (3.04) at 1 nM; 64.6 (3.37) at 10 nM; 63.21 (4.96) at 100 nM and 53.08 (4.32) at 1000 nM; (3C) Comparison of the percentage of CD3 positive cells and negative to color with Annexin V-PE and 7-ADF among T lymphocytes stimulated with PHA and not stimulated (control) at different doses of bortezomib

Figure 4: Samples were acquired with a calibrated number of Trucount ™ Tubes of fluorescent granules (BD) in order to calculate the absolute count of cells negative to color with Annexin V-PE plus 7-AAD between T cells at rest (CD25 -) and alloreactive (CD25 +). The numbers specified in the figures represent the number of granules of each assay and the events in the Annexin V-PE plus 7-AAD negative region, respectively.

Figure 5: Percentage of activated T lymphocytes, evaluated by staining with CD69 after secondary MLR: The mean percentage of activated T lymphocytes decreased significantly among previously cultured T cells with Bortezomib at 10 nM or 100 nM when stimulated again with T cells from the same donor (p = 0.05); on the contrary, the decreased T cell activation was not significant when T lymphocytes were stimulated again with T cells from third parties.

Figure 6: Western blot analysis of caspase 9 (A), caspase 3 (B) and bcl-2 (C) in control cells or stimulated with PHA, exposed to 100 nM bortezomib for different periods of time.

Figure 7: Samples cultured with different concentrations of bortezomib +/- Z-VAD-FMK were purchased with a calibrated number of Trucount MR tubes of fluorescent granule (BD) in order to calculate the absolute count of Annexin V positive cells -PE and / or 7-ADF using the following equation: (number of events contained in the Annexin V-PE region plus negative 7-ADF / number of events in the region of absolute number of granules) x (number of granules per test / test volume). The numbers specified in the figures represent the number of granules in each assay and the events in the Annexin V-PE and / or 7-ADF positive region, respectively . The absolute number of positive events to Annexin V-PE and / or 7 amino-actinomycin (7-AAD), which correspond to apoptotic cells, decreased by an average of 11%, 29% and 49% in the presence of the pancaspase inhibitor , after exposure to concentrations of 10, 100 and 1000 nM of bortezomib, respectively (p <0.05 for 100 and 1000 nM). When only Annexin V-PE positive cells were analyzed, similar values were observed.

Figure 8: (A) Intracellular expression IFN-? In CD3 + or CD4 + cells stimulated with PHA in the presence of different concentrations of Bortezomib (one of five similar experiments is presented) (B) Boxed of CD4 + IFN + cells for different doses of bortezomib of the 5 samples analyzed.

Figure 9: (A) Intracellular expression IFN-? In CD3 + CD25 + cells or CD3 + CD4 + CD25 + stimulated with PHA in the presence of different concentrations of bortezomib (one of five is presented similar experiments) (B) Traced in cell boxes CD4 + CD25 + IFN + for the different doses of bortezomib of the 5 samples analyzed.

Figure 10: IL-2 concentration in cultures for 48 h of non-T cell supernatants stimulated or stimulated with PHA or stimulated with anti-CD3 plus anti-CD28, incubated in presence of different concentrations of bortezomib.

Description of the invention

One of the main challenges presented by the allogeneic transplant approach is in the control of the graft reaction against the host, without altering the effectiveness of the procedure due to a decrease in the graft effect with respect to leukemia. Thus, numerous studies have been published that demonstrate a close relationship between GVHD and GVL (16,17,22) and the development of new approaches that allow the separation of both immune reactions is currently under investigation. In this regard, some in vitro data suggest that both GVL and GVHD are mediated by different T-cell clones. Thus, Michálek et al. (18) described the possibility of selective depletion of alloreactive donor T-cell clones afterwards. of a unidirectional MLR by an anti-CD25 immunotoxin, while the remaining cells could be stimulated with leukemic cells of the same patient. Unfortunately, although this is a promising approach, CD25 is also expressed in regulatory T lymphocytes (Treg) (23), and in vivo depletion of these Tregs could increase the risk of
GVHD (23.24). In order to overcome this problem, we have used other related membrane activation antigens such as CD69 to selectively reduce in vitro alloreactive T cells after
MLR (25).

In this invention, we have focused on the use of bortezomib as a new approach selectively aimed at alloreactive T cells. It has been shown that bortezomib exercises numerous biological effects, which include blocking the NF-? activation, thus inhibiting growth of different types of tumors with increased NF-? activity, such as myeloma multiple 11,12. Since the signaling pathways of T cell receptors (TCR) trigger the activation of NF- \ kappaB (26)}, we propose the hypothesis of that this property could promote apoptosis mediated by Bortezomib among activated T lymphocytes without affecting the rest of T cells that do not express NF-? activated. As demonstrated in MTT assays, in this invention we check that growth inhibition induced by Bortezomib was especially evident among T lymphocytes activated, compared to control. We confirm that, as described above (3,27), the expression of NF-? Increases in activated T lymphocytes, compared to resting T cells, thus becoming a target for the drug. In addition, growth inhibition was mainly related to the increase in apoptosis, evaluated by coloring with Annexin V-PE and 7 amino-actinomycin (7-ADF) by flow cytometry, and this effect was significantly superior among the alloreactive T lymphocytes after MLR, as it turned out further confirmed in secondary MLRs. Also, even though the alloreactive reaction was eliminated in the secondary MLR after exposure to the drug, this effect was much more evident when T lymphocytes were exposed to their cells donors, compared to third party donors, thus suggesting a very specific depletion of the alloreactive T cells, which allows to maintain the immune response when proceeding to the re-stimulation with other antigens.

As described above in different tumors and cell lines, the expression of several anti-apoptotic genes, such as FLIP, cIAPs, Bcl-2 or Bcl-XL, is regulated by NF-? B (27-29) } In this invention we confirm that, in activated human T lymphocytes, bortezomib decreases the expression of NF-? B. In addition, bortezomib caused the dissociation or excision of caspase 9 and caspase 3, generating truncated active fragments. This result clearly suggests that the mechanism of selective depletion of Bortezomib-stimulated T cells could involve the activation of proteolytic cascades orchestrated by caspases. In fact, treatment with pancaspase inhibitor Z-VAD-FMK prevented bortezomib-induced apoptosis, supporting an essential role of caspases in this effect. As regards caspases located after proteins, our data suggest that bcl-2 could participate in this proapoptotic effect. Thus, bcl-2 was overexpressed in activated T cells, compared to resting cells, as expected due to its dependence on NF-? B, and treatment with bortezomib caused its dissociation, generating a lower Mr fragment . Although bcl-2 with its full length is an anti-apoptotic protein, truncated forms have been shown to promote apoptosis (30). It is interesting to note that the apoptotic effect among the alloreactive T lymphocytes was evident at concentrations of the drug that varied from 10 to 1000 nM, which are those that are usually achieved with the standard doses currently recommended in the clinical setting. Sun and his collaborators published a previous study on a murine model (19) showing that bortezomib could prevent GVHD disease. Although in vitro studies using mouse T cells also showed a selective effect of the drug among activated T lymphocytes, it should be noted that the doses of the drug were significantly lower, varying from 1 to 4 nM (19). A possible explanation for these differences would be the existence of a different sensitivity to the effect of the drug between murine and human T lymphocytes. In addition, it should be noted that, in order to explain the in vivo effect on the prophylaxis of GVHD, not only the effect on activated T cells but also on dendritic cells should be taken into account (2,31) and, as regards Concerning prophylaxis, the latter could play a crucial role. Contrary to this result, delayed administration of bortezomib after allogeneic transplantation results in an accelerated morbidity of GVHD in a BMT mouse model not coincident with MHC, and this morbidity is mostly related to GVHD
intestinal (31). According to Sun and his collaborators, rising levels of TNF?, IL-1? And IL-6 were critical in the development of GVHD. In the present invention, we observed a decrease in Th1 cytokine secretion, but it is interesting that the cases that showed the highest apoptosis of activated T lymphocytes showed an increase in IL-2 levels in the supernatant, suggesting that lysis of activated T cells induced the release of cytokines into the medium. This could occur in mice with GVHD in a totally divergent model with MHC. In addition, bortezomib has moderate gastrointestinal toxicity (32) and, as we and other authors have described, toxicity to an organ could trigger GVHD in that specific organ (33).

As for the immunophenotypic assays performed in the present invention, it should be mentioned that, since the kinetics of the cell surface activation markers differ from each other (34), the analysis was performed at different intervals after activation of lymphocytes As indicated above, CD69 (24) has been used in conjunction with or instead of CD25 depletion, in order to avoid Treg depletion that could increase the risk of graft disease or rejection versus Guest. In our invention, bortezomib significantly reduced all-reactive T lymphocytes, as assessed by both early activation markers such as CD69 and CD25, suggesting a role for in vitro / in vivo purging with the use of this approach.

In addition to its antiapoptotic effect, NF-? Activates multiple target genes related to the immunogenic response (10). In this sense, NF-? Is activated not only through the TCR signaling pathway, but also through costimulatory molecules such as CD40L or CD28 (35), which are recognized as the main receptors that generate the full T cell activation. Other transcription factors such as AP-1 and the Activated T cell Nuclear Factor (NFAT) are fired following the TCR / CD3 (5)} activation cascade but, as an aspect interestingly, we could point out that the latter is involved not only in the immunogenic response, but also in the tolerogenic one (6). Since a competition between NFAT and NF-? B has been proposed to bind several target genes (36), bortezomib could alter this balance, thereby modifying the type of immune response. In this sense, in our study, blocking NF-? Not only induced apoptosis of activated T cells, but also reduced the production of Th1 cytokines, such as IFN-? And IL-2, evaluated by flow cytometry and the ELISA test, indicating that bortezomib modifies the behavior of cytokines. The fact that a tolerogenic response may or may not be obtained, as recently described, with the use of rapamycin (37) will require further studies, but it has already been demonstrated that the balance between NF-? And other factors Transcription as JNF / AP-1 plays a role in the immune response through the control of life and death of cells
dendritic ^ (31)}.

Therefore, bortezomib used at concentrations that are reached in the clinical environment, induces apoptosis among activated T lymphocytes, targeting selectively to alloreactive T cells after MLR. Further, the behavior of cytokines is also modified by NF-? B blockade that reduces the secretion of Th1 cytokines, such as IFN-? e IL-2

Therefore, a first aspect of the invention relates to the use of inhibitors of NF- \ kappaB in vitro to generate the selective depletion of alloreactive T lymphocytes. In a preferred aspect of the invention said NF-? B inhibitors are proteosome inhibitors. In an even more preferred aspect of the invention, said proteasome inhibitor is bortezomib. On the one hand, using bortezomib in vivo there is no certainty that there are no tumor cells in the recipient at the time of drug administration, which could cause depletion not only of alloreactive lymphocytes but also of antitumor lymphocytes. Moreover, this situation of persistence of residual disease before transplantation is quite likely in clinical practice. On the contrary, using in vitro bortezomib we can perform a mixed culture facing donor lymphocytes with recipient cells, being able to select with much greater safety healthy patient cells not contaminated with tumor cells, therefore the effect would be much more selective. On the other hand, the in vitro use of the drug allows its cytotoxic effect to be directed only against the donor's lymphocytes, thus avoiding its effect on other healthy cells and tissues of the recipient, such as the digestive tract or nervous system, thus avoiding the Drug toxicity, as in fact described in any of the previously mentioned in vivo models.

A second aspect of the invention relates to use of the inhibitors of the invention to generate depletion Selective of the alloreactive T lymphocytes after mixed culture. In a preferred aspect of the invention said mixed culture comprises donor lymphocytes and receptor mononucleated cells. In a even more preferred aspect of the invention, said mixed culture It comprises donor lymphocytes and receptor dendritic cells.  In an even more preferred embodiment of the invention said culture mixed comprises donor lymphocytes and lineage cells Endothelial or epithelial receptor.

A third aspect of the invention relates to use of the product obtained after the use of inhibitors of the invention in mixed culture for the preparation of a pharmaceutical composition In a preferred aspect of the invention said pharmaceutical composition is used in therapy. In one aspect more preferred of the invention said pharmaceutical composition is used in the treatment of transplants, more preferably in Hematopoietic transplant treatment.

Below the following examples and drawings they serve to illustrate but do not limit the present invention.

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Examples

Example one

Materials and methods 1.1. Cell cultures and proliferation assays

By centrifugation with density gradient, isolated peripheral blood mononuclear cells (PBMC) from leukocyte layers or white clots of voluntary donors, with the use of Ficoll-Paque solution, allowing its adhesion to tissue culture disc (Becton Dickinson, Franklin Lakes, NJ). After 2 hours at 37 ° C, the cells were collected that had not adhered, washed and resuspended in culture medium formed by RPMI 1640 L-glutamine (2 mM), penicillin (100 IU / mL) and streptomycin (10 mg / mL) plus one 10% human serum type AB (Sigma). More than 90% of monocytes / macrophages controlled by flow cytometry (data not illustrated) and the T cells were purified again with use of anti-CD3 magnetic granules (Miltenyi Biotec, Auburn, CA), if necessary.

In order to generate dendritic cells, the adhered cells were cultured for 7 days, incubating at 37 ° C with 5% CO2 in the middle of Dulbecco modified by Iscove (IMDM) supplemented with glutamine (2 mM), penicillin (100 IU / mL) and streptomycin (10 mg / mL) plus 10% human serum type AB. To the GM-CSF 1000 first culture vial was added U / mL (PeProtech) plus IL-4 1000 U / mL (R&D System), and for the last 48 hours of cultivation was added TNFα 20 ng / mL (R&D System). The analysis was carried out phenotypic, in order to ensure purity of dendritic cells of the sample greater than 90%, with the use of CD3 mAbs conjugated to FITC, CD56, CD14, CD19 (Exclusion Kit for cell studies dendritic, Cytognos) mAbs CD80 conjugated to PE, CD86 (BD Pharmingen), as previously described (20).

The inhibitory effect of bortezomib was evaluated in cell growth, measuring absorbance, by the cells, dye bromide 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium (MTT) To do this, 10 5 cells / 100 µL were placed in tripled in 96-well tissue culture discs in the middle of Bortezomib-free cultivation (kindly provided by Millenium Pharmaceuticals Inc) p at increasing doses of the drug: 1 nM, 10 nM, 100 nM and 1000 nM. The first or third days of the crop, was added Bortezomib, in order to allow the activation of T cells. fifth day the absorbance of the MTT dye was evaluated. For all these concentrations of the drug, lymphocytes were grown in the following conditions: (a) culture medium alone (control); (b) culture medium plus Phytohemagglutinin (PHA, 5 µg / mL); (C) culture medium plus anti-CD3 fixed to the plates (5 µg / mL) in combination with soluble anti-CD28 (2.5 µg / mL); (d) culture medium plus 0.5 x 10 5 lymphocytes Allogeneic irradiated T (15 Gy). Finally, and only for trials of proliferation, lymphocytes were also cultured in the middle of culture plus allogeneic dendritic (DC) cells irradiated in a 1:10 ratio. Although proliferation assays are carried out in all cases analyzed, the results described in the rest of the studies presented in this article they were repeated in a minimum of 5 cases, depending on the number of cells needed to Each experiment

T lymphocytes, co-cultured for five days, with or without bortezomib, plus T lymphocytes irradiated allogeneic were stimulated again in MLR cultures secondary, using T lymphocytes obtained from the same donor or from a third party. A mixed secondary MLF lymphocytes do not Bortezomib added. Seventy-two hours after the start of secondary culture immunophenotypic analysis of the activation markers

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1.2 Immunophenotypic analysis and coloration of Cytokines

5x10 5 non-adhered lymphocytes / well seeded in 48-well plates, grown, and grown in the medium alone or stimulated with PHA (5 µg / ml), anti-CD3 fixed to the plate (5 µg / ml) more soluble anti-CD28 (2.5 µg / ml) or 2.5x105 irradiated allogeneic T lymphocytes (15 Gy). Day 0 different concentrations of bortezomib (0.1 nM, 10 were added nM, 100 nM and 1000 nM) (for evaluation of the antigen at 24 hours) or the third day of the crop (for tests at five days). The cells were analyzed by direct immunofluorescence with the use of four-color coloring, with MoAbs conjugated to the following fluorochromes: Fluorescein FITC), Phycoerythrin (PE); Chlorophyll peridine - protein-Cyanine 5 (PerCP-Cy5) and allo-phycocyanin (APC). Specific antibodies were acquired from BD Bioscience Pharmingen, except for anti-CD25-PE, provided by Immunotech The following combinations were used: anti-CD3-FITC / anti-CD25-PE / anti-CD4-PerCP-Cy5 / anti-CD40L-APC; anti-CD25-FITC / IFN-PE anti-cytoplasmic / anti-CD8-PerCP-Cy5.5 / anti-CD4-APC; anti-CD25-FITC / anti-CD69-PE / anti-CD8-PerCP-Cy5.5 / anti-CD4-APC; anti-CD69-FITC / anti-cytoplasmic granzyme / anti-CD8-PerCP-Cy5.5 / anti-CD3-APC;  briefly, for surface coloration 100 µL of sample / tube for 30 minutes with the appropriate MoAb combination at room temperature (RT) in a dark environment. The cells are centrifuged (5 min at 540 g) and the granules were washed with 4 mL of PBS Finally, the cells were resuspended in 0.5 mL of PBS until the flow cytometer was analyzed. For coloring of the intracellular cytokine, brefeldin A (10 µg / mL) was added during the last four hours before withdrawal. The intracellular detection of IFN-? after coloration for surface proteins, with the use of a direct immunofluorescence technique. For this, the IntraStain Kit (Dako Cytomation, Denmark), following scrupulously the manufacturer's recommendations. After coloring of the intracytoplasmic antigens, the cells are washed and resuspended in 0.5 mL of PBS until Flow cytometer analysis. The acquisition of the data is performed on a flow cytometer FACSCalibur (BD, San José, CA), with the use of the CellQuest (BD) software program, analyzed with the Paint-A-Gate Pro (BD) software, except for IFN-? analysis intracellular, in which the software was used Cell Quest Samples were acquired and analyzed at different intervals after activation of the lymphocytes, taking into account the kinetics of the markers Superficial cells (21).

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1.3. ELISA

For the release test of IL-2, non-adherent lymphocytes were seeded as described above for immunophenotypic analysis. TO early concentrations of different concentrations of bortezomib The cells were incubated at 37 ° C, 5% CO2, and, at after 48 h, the supernatants were collected and the IL-2 secretion with the use of an assay Enzyme-linked immunosorbent, commercially available (ELISA) (Diaclone, Besancon Cedex, France).

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1.4 Evaluation of apoptosis

For the detection of apoptosis, they were performed cell cultures as described previously in the selection on immunophenotypic analysis, using the kit apoptosis detection Annexin V-PE / 7 BD Pharmingen amino-actinomycin. To expose it briefly, a minimum of 0.5x106 T lymphocytes were washed and resuspended in Binder Buffer (1:10 diluted in PBS), maintaining a cell concentration of 1 x 10 6 / mL. Be added both Annexin V-PE and 7 amino-actinomycin (7-AAD) 5 uL during 15 minutes. In order to identify T lymphocytes activated, it was also added anti-CD25-FITC. For each condition, 50,000 events were collected and analyzed. He calculated the Annexin V-PE percentage more 7-ADF of negative lymphocytes with the use of Paint-A-Gate Pro (BD) software.

In another series of experiments, they were isolated CD25 + dendritic cells after three days of a culture of mixed lymphocytes through positive selection with anti-CD25 magnetic microgranules directly conjugates (5 uL / 10 7 cells; Miltenyi Biotec, Auburn, CA) purifying itself in an LS + column. The negative fraction of CD25 cells were subsequently colored with the use of microgranules anti-CD3 magnetic coated with mAb (10 µL / 10 7 cells; Miltenyi Biotec) applying to a second magnetic column After purification, bortezomib was added to concentrations other than CD25 positive lymphocytes and negative for 48 hours, the evaluation of the apoptosis in both fractions as previously specified. To further evaluate the absolute number of Annexin V-PE plus 7-AAD negative cells within the different subpopulations of cells, in 5 cases the Samples were acquired simultaneously with the use of Tubes Trucount ^ (BD), which contain a calibrated number of fluorescent microgranules. Then you he made the absolute count of Annexin V-PE more 7-ADF of negative cells with the use of following equation: (number of events in the region that Contains Annexin V-PE plus negative cells 7-ADF / number of events in the region of absolute granule count) x (number of granules per test / test volume). Finally, in order to evaluate the role of caspase activation in apoptosis induced by bortezomib, pancaspase inhibitor was added Z-VAD-FMK (20 µM) (Sigma) a hour before adding bortezomib to samples grown during 24 hours with different concentrations of the drug. Finally calculated the Z-VAD-FMK in the count  Annexin V-PE absolute and / or positive cell 7-ADF

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1.5 Western Blot Analysis

10 6 cells were seeded in 25 bottles cm2 (Corning, NY), without stimulation or stimulated with 5 mug / ml PHA for 24 h. Then, means of 100 nM culture or bortezomib for 30 min, 1 h, 3 h, 6 h, 12 h and 24 hours. The cells were then washed with PBS (Gibco) and were lysed in ice-cold lysis buffer (140 mM NaCl, 10 mM EDTA, 10% glycerol, 1% Nonidet P-40, Tris 20 mM at a pH of 7.0, 1 µM pepstatin, 1 µg / ml of aprotinin, 1 µg / ml leupeptin, 1 mM sodium orthovanadate). The samples were centrifuged at 13,000 r.p.m. at 4 ° C and for 10 minutes, collecting the supernatants. Cell extracts they were subjected to 10% SDS / PAGE and dried over the membrane of PVDF (Miliporos). The membranes were incubated with anti-IkBα (1: 2000), anti-NF- \ papB p65 (1: 2000) (both from Santa Cruz Biotechnology, Inc.), bcl-2 (1: 2000), caspase 3 (1: 30,000) and caspase 9 (1: 1000) (of BioSciences). The first step antibodies fixed to the membrane were reacted with anti-rabbit (Bio-Rad) or anti-mouse (Amersham, Pharmacia) conjugated to horseradish peroxidase, and bands they were visualized with a luminol based detection system with intensification by p-iodophenol (15).

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1.6 Statistical analysis

For each variable, the mean values and their standard deviation (SD) as well as the range and the median were calculated, using the software program (SPSS 11.0, Chicago IL). The comparison between groups was made by analysis of variance (and then the Scheffé and Tukey tests were carried out to confirm the differences between groups). A Bidirectional Measurement of Repeated Multiple Analysis (MR-MANOVA bidirectional) was performed in order to compare the effect of the different doses of the drug within the different types of culture. P values less than 0.05 were considered significant.

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Example 2

Results 2.1 Activated T cells show an increase in bortezomib sensitivity

To investigate whether bortezomib affected the proliferation or survival of T lymphocytes, were performed MTT assays with resting T lymphocytes, stimulated with PHA or stimulated with MLR. Bortezomib treatment decreased the MTT absorption, especially at concentrations of 10 nM and superiors (figure 1). It is interesting to note that sensitivity to Bortezomib proved to be clearly superior in T cells stimulated with PHA or MLR, if compared with T cells in rest, as indicated by the inclinations of the curves of MTT absorption. In addition, when T lymphocytes were cultured in presence of dendritic cells (DC), the speed of proliferation showed intermediate values between PHA and MLR, and the growth inhibition in the presence of the drug presented also intermediate values between PHA and MLR (figure 1).

The NF-? Activity is controlled by the interaction with its inhibitor, IkB. The latter, in turn, is regulated by phosphorylation, which is directed to the IkB for proteolytic destruction. In order to evaluate the effect of bortezomib on the degradation of IkB and the expression of NF-? B, resting T cells or PHA stimulated were cultured. After 24 h, bortezomib was added and allowed to act for different periods of time. Western blot analysis confirmed that PHA-stimulated T cells overexpressed the NF-? B, compared to the control (Figure 2A). In addition, bortezomib induced a decrease in the expression of NF-? Within stimulated T lymphocytes, and this effect was more evident while increasing the incubation period. He also confirmed that, in PHA-stimulated T cells, IkB degrades, while the addition of bortezomib induced the accumulation of phosphorylated forms of IkB
(Figure 2B).

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2.2. Bortezomib decreases the viability of T cells activated or alloreactive

To analyze whether the decrease in absorption of MTT in response to bortezomib was due to a greater death of cells, we investigate the effect of the drug on T lymphocytes, using coloration with Annexin V-PE and 7 amino-actinomycin (7-ADF) by flow cytometry. As can be seen in Figure 3A, the percentage of negative events to Annexin V-PE and the 7 amino-actinomycin (7-ADF), which correspond to viable T lymphocytes, significantly decreased in samples stimulated with PHA at drug concentrations less than 10 nM. On the contrary, in T cells not stimulated, the viability was only poorly affected by Bortezomib (Figure 3B). Also, when we compare the effect of Bortezomib between resting T cells and stimulated with PHA, increased differences in terms of cell viability between both subgroups when the drug doses were increased (figure 3C). An important observation is that you are differences in terms of viability were also observed between T lymphocytes to reagents and at rest in MLR. So after all from three days of a mixed lymphocyte culture, they were cultured CD25 positive (alone active) and negative T lymphocytes (in resting), separated by classification of activated cells magnetically, in the presence of different concentrations of bortezomib In order to accurately quantify the number of viable cells in both subpopulations, samples were analyzed by flow cytometry with the use of TruCount MR tubes, with fluorescent granules, as specified in M&M. Is interesting to note that, as can be seen in figure 4, the viability of T lymphocytes decreased significantly in the subset of T cells with CD25 +, while the number of viable T lymphocytes remained virtually constant within the group of CD25-negative T cells at different concentrations of the drug. Thus, the average percentage of viability of T cells it was 75% versus 95% (p = 0.3) among CD25 + T lymphocytes and CD25- grown at 10 nM while it was 55% versus 93% (p = 0.003) at a concentration of 100 nM. Due to the small number of remaining events within the subpopulation of CD25 + cells at higher doses of bortezomib, the analysis could only be performed in 2 cases at 1000 nM. Consequently, we confirm that activated T lymphocytes become susceptible to the cytotoxic effect of the drug while the viability of resting T lymphocytes is not severely affected by bortezomib.

To further confirm the depletion selective of the alloreactive T cells after MLR, it performed secondary MLR reactions with use, such as cells effectors, of previously cultured T lymphocytes with or without bortezomib and, as stimulant cells, irradiated T lymphocytes from the same donor used in the primary MLR reaction or from a third party donor. The average percentage of activated T cells in the secondary MLR, evaluated by the expression of CD69, it was the 7.3% among T cells that had not previously been exposed to Bortezomib and 6.1% and 3.8% among previously grown with bortezomib at 10 nM and 100 nM, respectively, with p = 0.8. Is interesting to note that, when we analyze separately the CD69 expression after secondary MLR reaction between T lymphocytes co-cultured with T cells stimulants from the same donor, we check that the average percentage of CD69-positive T cells was 7.1%, 4.1% and 2.9% among previously cultured T lymphocytes without bortezomib or at a dose of 10 nM and 100 nM of the drug, respectively (p = 0.05).  On the contrary, when donor cells were used third in secondary MLRs as stimulatory cells, these figures were 7.5%, 7.6% and 4.7% (p = 0.5) (figure 5), indicating thus not only the selective apoptosis of T lymphocytes at reagents but also a highly specific depletion of the T lymphocytes to reagents against donor antigens primary. When activation was assessed through expression of CD25, similar values were obtained (data not shown).

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2.3 Mechanisms of bortezomib-induced apoptosis in activated T lymphocytes

In order to analyze the mechanisms of bortezomib-induced apoptosis between activated or alloreactive T lymphocytes, we performed the Western blot analysis, evaluating the effect of the drug on the activation of caspases, and the expression of the antiapoptotic and proapoptotic factors of the bcl-2 family. For this, non-stimulated or stimulated T cells were cultured with PHA, adding bortezomib and allowing it to act for different periods of time. As illustrated in Figures 6A and B, the activation of caspases 9 and 3, as indicated by the presence of dissociated subfragments, was observed among activated T lymphocytes exposed to bortezomib for 6 and 24 hours, respectively. Caspase 9 activation preceded that of caspase 3, as expected, since the former is in a position before caspase 3 (activation of caspase 8 was difficult to assess, since these cells expressed low quantities - data not shown). Interestingly, the expression of bcl-2 increased among activated T lymphocytes, compared to resting T cells. The addition of bortezomib caused a decrease in bcl-2 simultaneously with the appearance of a lower Mr form, resulting from the dissociation of bcl-2, as can be seen in Figure 6C. These results suggested that caspase activation and subsequent degradation of bcl-2 could mediate bortezomib-induced apoptosis among activated T lymphocytes.

To assess whether apoptosis induced by Bortezomib between stimulated T cells or not depended on the caspase activation, pancaspase inhibitor was added Z-VAD-FMK to T lymphocytes exposed to The drug for 24 hours. As illustrated in Figure 7, the Z-VAD-FMK rescued T cells from Bortezomib induced apoptosis.

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2.4 Bortezomib modifies the behavior of activation markers and cytokine secretion

So far we have shown that bortezomib induces cell death, particularly among T lymphocytes activated Since bortezomib regulates the NF- \ kappaB, and the latter intervenes in the activation of target genes linked not only to the anti-apoptotic activity but also to the immunogenic response, We also wanted to evaluate whether or not bortezomib could modify the cytokine activation and secretion model within the remaining viable activated T cells. First, we analyze the intensity of the expression of the activation markers of CD25 and CD69 T cells, in the presence or absence of bortezomib. How presented in table 1, the medium fluorescence channel (MFC) of CD25 between activated T cells decreased with the addition of Bortezomib in a dose-dependent manner. In addition, in crops stimulated with PHA and CD3 / CD28, we observed a decrease significant of the CFM of CD69, as well as a reduction in percentage of CD69 + T lymphocytes, which varied from 60% to 1 nM at 48% at 1000 nM.

In addition, we evaluate the intracellular expression of IFN-γ, as well as the percentage decrease in IFN-γ-positive T lymphocytes due to an increase in bortezomib doses, the average percentage (range) of INF + CD4 + cells being 11.42% (0.22-24.01%), 8.97% (0.4-18.97%), 3.39% (0.38-7.12%) and 1.2% ( 0.23-2.07%) in samples grown without bortezomib or a drug concentration of 10 nM, 100 nM and 1000 nM, respectively (p = 0.06, figure 8A and B). On the other hand, among the activated T lymphocytes identified by their FSC / SSC pattern as well as by the expression of CD25, the number of IFN-positive T cells also decreased significantly with a mean percentage (range) of CD25 + IFN + 32 T lymphocytes. , 88% (28.41-37.36%), 20.87% (15.45-26.3%) and 3.49% (3.06-3.93%) among samples grown without bortezomib or at 10 nM and 100 nM concentration of the drug, respectively (p = 0.033, figure 9A and B). In addition, in relation to CD8 + T lymphocytes, the average percentage of activated cells varied between 21.2% (5.7-53.17%), 5% (2.3-8.6%) and 1 , 7% (0-4.3%) of CD8 + CD25 + cells for cultures without bortezomib or at a concentration of 10 nM and 100 nM, respectively, (p = 0.06), while these figures were 39.3% (26.5-62.5%),
23.7% (8.5-34.6%) and 9.8% (0-20.4%) for CD8 + granzyme + T cells, respectively, (p = 0.003).

Finally, in 9 cases, the ELISA assays and we observed that IL-2 levels decreased in crop supernatants when increased the concentration of bortezomib, as can be seen in figure 10. It should be mentioned that in samples activated by 3 PHA, in which 1000 nM concentration was tested, found in the supernatant an increase in the level of cytokines. It is interesting to note that these cases are those that showed the most important apoptosis of T lymphocytes, suggesting that lysis of activated T cells Induced release to the cytokine medium.

So, bortezomib not only reduced the number of viable activated T lymphocytes but also modified the activation and behavior of cytokines between cells T activated remaining.

TABLE I

one

2

Expression intensity of CD25 and CD69 evaluated as a medium fluorescence channel (CFM) between T lymphocytes activated (*) p <0.05 compared to the respective value of the samples without bortezomib

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Claims (6)

1. Composition comprising a mixed culture of T lymphocytes obtained after generating the selective in vitro depletion of those T lymphocytes alloreactive with Bortezomib, for use as a medicine. 2. Composition according to the first claim, where said mixed culture comprises donor T lymphocytes and mononucleated receptor cells. 3. Composition according to the first claim, where said mixed culture comprises donor T lymphocytes and dendritic cells of the receptor. 4. Composition according to the first claim, where said mixed culture comprises donor lymphocytes and endothelial or epithelial lineage cells of the recipient. 5. Composition according to any of the previous claims, for use in the treatment of transplants 6. Composition according to any of the claims 1-4, for use in the treatment of hematopoietic transplants.