Introduction

Dysregulation of protein tyrosine kinases is involved in the pathophysiology of a number of pathologies, including cancer, cardiovascular, autoimmune, and inflammatory diseases. The group of tyrosine kinase enzymes has become one of the most important pharmaceutical targets since approximately 30% of drugs developed worldwide involve the action of tyrosine kinase proteins1. Ibrutinib (IB) is a tyrosine kinase inhibitor (TKI), capable of inhibiting Bruton's protein tyrosine kinase (BTK), which allowed a great advance in the treatment of Chronic Lymphoid Leukemia and other B cell malignancies, including mantle cell lymphoma, marginal zone lymphoma and Waldenstrom macroglobulinemia2.

Despite inhibiting BTK with high specificity, IB also has an inhibitory action on several other kinases, including interleukin-2-inducible T-cell kinase (ITK), tyrosine kinase expressed in hepatocellular carcinoma (TEC), bone marrow tyrosine kinase gene in chromosome X (BMX), epidermal growth factor receptor (EGFR), Erb‑B2 Receptor Tyrosine Kinase 2 (ERBB2), Janus Kinase 3 (JAK3), and B-lymphocyte kinase (BLK)3,4. The fact that such target proteins are found in several cells of the immune system, including dendritic cells, T-cells, NK-cells, macrophages, and neutrophils, renders IB important immunoregulatory functions5.

Evidence that mice which receive bone marrow transplants from BTK- or ITK-deficient donors did not develop chronic Graft versus Host Disease (cGVHD) suggested a possible role for IB in the treatment of chronic GVHD6. In fact, the results of a phase II clinical trial in which 420 mg/day of IB was administered orally culminated with the United States Food and Drug Administration (FDA)-approval of IB as second-line therapy of steroid-refractory or steroid-resistant cGVHD in 20177.

Mesenchymal stromal cell (MSC) infusion has also been explored as a second-line therapeutic alternative for the treatment of acute and cGVHD8. These cells are distributed throughout the body9 and in addition to having a supportive role in tissue regeneration, they are able to control the immune response through cell–cell-contact, the secretion of anti-inflammatory factors, and the support of regulatory T-cells (Tregs) generation10,11. The wide physiological distribution of MSCs and the possibility of clinical use of these cells combined with adjuvant drugs requires the development of studies that allow a better understanding of the influence that new drugs can exert on the functional properties of MSCs. In fact, the influence of immunosuppressive drugs such as mycophenolic acid, rapamycin, dexamethasone, and others on the immunomodulatory properties of MSCs12,13,14 and also of TKIs such as Ruxolitinib15,16 has been investigated. In this study, our objective was to evaluate whether IB could influence the proliferation, phenotype, viability, and the immunomodulatory properties of MSCs. Furthermore, we evaluated the possible molecular changes involved in the interaction of this TKI with such cells, focusing on the expression of adhesion molecules, the production of anti-inflammatory mediators, and the generation of Tregs.

Material and methods

MSCs culture and treatment

MSCs were obtained from three female healthy donors following a lipoaspiration procedure performed at the Clínica Carpaneda Cirurgia Plástica, Brasilia, Brazil. The cells were cultured in T75 flasks using alpha‐minimum essential medium (α‐MEM) supplemented with 15% FBS (GIBCO, Waltham, MA, USA), 2 mM glutamine, and 100 U/mL penicillin/streptomycin (Sigma‐Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO2. The medium was changed every 2 days, and the cells were split when they reached 80–90% confluence. MSCs between passages 3–6 were used for all experiments. These cells were treated with IB (Cayman chemicals, Ann Arbor, MI, USA) at concentrations ranging from 0.1 to 2 µM. The study protocols were approved by the Ethics Committee of the Faculty of Health Sciences of the University of Brasilia. All methods described in the study were carried out in accordance with the approved guidelines. All participants provided written informed consent.

Morphological and Immunophenotypic characterization

The morphology of MSCs was observed under an inverted optical microscope (Primo Vert, Carl Zeiss Microscopy, Germany). MSCs were phenotypically characterized at 3rd passage according to the minimal criteria proposed by the International Society for Cellular Therapy17, using the BD Stemflow hMSCs Analysis kit (Pharmingen, BD Biosciences, USA). Briefly, MSCs in the absence or presence of IB were incubated with CD105-PerCP-Cy5.5, CD73-APC, CD90-FITC, CD44-PE and with the negative cocktail markers, which included CD45/CD34/CD11b/CD19/HLA-DR antibodies, all conjugated with PE. Ten thousand events were recorded for each sample and data were acquired using a FACSCalibur Flow Cytometer. FlowJo software 10.0.7 was used for data analysis (FlowJo LLC, USA).

Proliferative assessment of MSCs

The effect of IB over MSC growth (proliferation / viability) was determined by MTT [3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. For this experiment, MSCs were treated with IB (0.5, 1.0, and 2.0 µM). Briefly, 2 × 103 MSCs were plated in 96-well plates. After cell attachment, fresh media containing IB at different concentrations was added and cell proliferation was evaluated at 1, 3, 5, and 7 days post plating. In these time points, 20 μL of MTT (5 mg/mL) was added in each well and the plates incubated for 3 h. Then, MTT and medium were removed and replaced by DMSO. The optical density was read on a Multiskan FC Microplate Photometer (Thermo Scientific, Waltham, MA, USA) at 570 nm.

Annexin V/PI assay

The effect of IB on MSC was determined by annexin V/ PI staining using Flow Cytometry. For this, MSCs were treated with IB (0.5, 1, and 2 µM) for 72 h. After this period, cells were washed with phosphate-buffered saline (PBS), resuspended in a binding buffer and stained with Annexin V and Propidium Iodide (PI), according to manufacturer’s instructions. Ten thousand events were recorded from each sample using a FACSCalibur Flow Cytometer. The annexin V−/PI− cell population was considered viable, while annexin V + /PI− and annexin V + /PI + cell populations were considered apoptotic. The analyses were performed using the FlowJo software 10.0.7 (FlowJo LLC, USA).

Assessment of mitochondrial membrane potential (ΔΨm)

To evaluate the mitochondrial membrane potential, MSCs were treated with increasing concentrations of IB (0.5, 1, and 2 µM) for 72 h. After this period, cells were dissociated with 0.05% Trypsin (GIBCO, Waltham, MA, USA), washed with PBS, and incubated with 5 µg/mL of Rhodamine 123 for 20 min at room temperature. Then, the cells were washed with PBS and immediately analyzed on the FACSCalibur Flow Cytometer. Ten thousand events were acquired from each sample and data analysis was performed using FlowJo 10.0.7 software (FlowJo LLC, USA).

T-cell suppression assay

To assess the effect of IB on the ability of MSCs to control T-cell proliferation, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of three healthy volunteers and separated using Histopaque 1077 (Sigma-Aldrich, USA). Then, PBMCs were stained with 2.5 μM carboxyfluorescein succinimidyl ester (CFSE, Sigma-Aldrich, USA) and activated with 5 µg/mL of phytohaemagglutinin (PHA, Sigma-Aldrich, USA). These cells were co-cultured with MSCs (10:1 ratio) in the absence or presence of increasing concentrations of IB (0.1, 0.5, 1 and 2 µM) for 72 h. In parallel, the antiproliferative effect of IB on PBMCs was also determined. RPMI medium supplemented with 10% FBS was used for cell cultures.

After 72 h, PBMCs were recovered, stained with anti-CD3 APC (Invitrogen, USA), and the proliferation of T-cells was determined by flow cytometry. Ten thousand events were acquired from each sample and data analysis was performed using FlowJo software 10.0.7 (FlowJo LLC, USA). Suppression of T-cells proliferation was determined as follows:

$${\text{T-cell}}\;{\text{suppression}}\left( \% \right) = 100\% - {\text{T-cell}}\;{\text{proliferation}}\;{\text{after}}\;{\text{coculture}}\;\left( {\% \;{\text{of}}\;{\text{PHA-activated}}\;{\text{PBMC}}} \right)$$

T-cell activation assay

The expression of activation markers CD38 and CD69 was evaluated on T-cells after exposing these cells to MSCs and IB. For this, PHA-activated PBMCs were co-cultured with MSCs (10:1 ratio) and treated or not with 0.5 µM of IB. After 24 h, PBMCs were recovered, stained anti-CD3 PerCP (Invitrogen, USA), anti-CD38 APC (Invitrogen, USA), and anti-CD69 FITC (Invitrogen, USA), and analyzed by flow cytometry. Ten thousand events were acquired from each sample and data analysis was performed using FlowJo software 10.0.7 (FlowJo LLC, USA).

Treg production

We evaluated the percentage of CD4+CD25highFOXP3+ and CD8+CD28- regulatory T-cells in PHA-activated PBMCs exposed to MSCs and IB. For this, PHA-activated PBMCs were co-cultured with MSCs (10:1 ratio) and treated or not with 0.5 µM of IB. After 72 h, PBMCs were recovered and stained with anti-CD4 FITC, anti-CD25 APC, and FOXP3 PE according to the manufacturer’s recommendations (FoxP3 Staining Kit, BD Biosciences). One hundred thousand events were recorded for each sample in a FACSCalibur flow cytometer. Additionally, the cells were also stained with anti-CD3 PerCP, anti-CD8 PE (Invitrogen), and anti-CD28 FITC (Invitrogen) for non-classical CD8+CD28-Treg quantification18. For this, twenty thousand events were recorded. Analyses were performed using FlowJo software 10.0.7 (FlowJo LLC, USA).

Inflammatory medium obtention

Considering that the administration of IB occurs in an inflammatory setting19, to evaluate the effect of IB directly on MSCs, we used the supernatant of 1 × 106 PBMCs activated for 72 h with 5 µg/mL of PHA. The supernatant was collected, centrifuged, and used for the following assays.

Enzyme-linked immunosorbent assay (ELISA)

The inflammatory medium obtained from PHA-activated PBMCs was characterized according to the concentration of IFN-γ and IL-10 by ELISA (ImmunoTools) and used to subject MSCs to an inflammatory stimulus.

To determine whether IB could modulate the production of cytokines, PHA-activated PBMCs were cultured with MSCs and treated or not with IB. For this, a transwell system with a membrane pore size of 0.4 µm (Greiner Bio-one) was used. After 72 h, culture media were obtained and IFN-γ, TNF-α, and IL-10 levels were determined by ELISA. The absorbance was measured at 450 nm using the Multiskan FC Microplate Photometer (Thermo Scientific, Waltham, MA, USA) and the concentration was determined using standard curve values.

VCAM-1 and ICAM-1 expression

VCAM-1 (CD106) and ICAM-1 (CD54) expression was analyzed on MSCs and MSCs treated with 0.5 µM of IB, after the culture of these cells in the inflammatory medium. For this, after 72 h of culture, cells were recovered and stained with anti-CD54 APC (BD Biosciences) and anti-CD106 FITC (BD Biosciences). Ten thousand events were recorded, and analyses were performed using FlowJo software 10.0.7 (FlowJo LLC, USA).

Real-time PCR

Initially, gene expression analyses were performed in MSCs and MSCs treated with 0.5 µM of IB, after the culture of these cells in the inflammatory medium for 72 h. Gene expression analysis was also performed in PHA-activated PBMCs cultured with MSCs and treated or not with IB. The culture of PBMCs with MSCs was performed using a transwell system with a membrane pore size of 0.4 µm (Greiner Bio-one). RNA extraction was performed using the TRI reagent, according to the manufacturer’s instructions (Sigma-Aldrich, USA). The amount and quality of RNA samples were determined using Nanodrop one (Thermo Scientific). Total RNA was converted to single-stranded cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied BioSystems). mRNA expression levels of TGF-β, PD-L1, TSG-6, IL-10, IDO, IFN-γ, IL-17, and FOXP3 (Supplementary Table 1) were determined by Real-time PCR (Applied Biosystems StepOnePlus™) with SYBR Green MasterMix (Thermo Fisher, USA) combined with primers specific to each gene. All reactions were performed in technical duplicates and the relative fold change was obtained by the 2−ΔΔCt method20.

Statistical analysis

Data were reported as mean ± SEM. The statistical significance was calculated using Student’s t-test analyses to compare the differences between the two groups and using ANOVA to compare three or more experimental groups. The value of p < 0.05 was considered statistically significant and significance levels were defined as * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. All analyses were performed using Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

Results

MSCs characterization

MSCs presented typical MSC immunophenotype, with positive expression of CD44 (100%), CD73 (100%), CD90 (100%), and CD105 (95.1 ± 0.6%) markers and negative expression of CD34, CD45, CD11b, CD19 and HLA-DR. Interestingly, treatment with IB increased the number of MSCs expressing CD105 (95.1 ± 0.6% to 99.4 ± 0.1%; p = 0.002). The median fluorescence intensity (MFI) of CD105 was also significantly higher in such cells (113 ± 3.0 vs. 97.3 ± 9.0; p = 0.04). All MSCs and MSCs treated with IB expressed CD90; However, the MFI of this antigen was higher in MSCs treated with IB (396.3 ± 16.5 vs. 439.3 ± 10.4; p = 0.01) (Fig. 1A).

Figure 1
figure 1

Characterization of MSCs and effect of IB on cell viability. (A) Representative flow cytometry histograms showing the expression levels of CD44, CD73, CD90, and CD105 on MSCs (blue line) and MSCs treated with IB (MSC + IB, yellow line). Black line corresponds to the isotype control. On the right side of figure A, the MFI of the specific antibody evaluated is shown. (B) Morphology of MSCs and MSCs treated with IB (10X objective). The images were captured on the seventh day of treatment. (C) ΔCt expression of BTK, ITK, ERBB2, EGFR, and JAK3 in MSCs. The values were expressed as percentage relative to 1/ΔCt of GAPDH gene. (D) Proliferation/viability of MSCs after 1, 3, 5, and 7 days of treatment with IB at doses 0 (control), 0.5 µM, 1 µM, and 2 µM. (E) Percentage of apoptotic (annexin V +) MSCs after their exposure to 0 (control), 0.5 µM, 1 µM, and 2 µM of IB. (F) Rhodamine 123 staining in MSCs after their exposure to 0 (control), 0.5 µM, 1 µM, and 2 µM of IB. On the right side, Representative Rhodamine 123 Histograms from MSCs treated with 0 (control), 0.5 µM, 1 µM and 2 µM IB are shown. Results are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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Interestingly, MSCs showed expression of BTK, ITK, JAK3, ERBB2 and EGFR transcripts, which encode target proteins of IB. After seven days of treatment, IB did not change the size or fusiform morphology of MSCs (Fig. 1B-C).

Prolonged exposure to Ibrutinib inhibits MSC proliferation/viability

On the third day of cell culture, it was noticed that 2 µM IB inhibits the proliferation of MSCs (p = 0.0001). On the fifth day of cell culture, the use of IB at doses of 0.5 µM, 1 µM and 2 µM inhibited the growth of MSCs (p < 0.0001). On the seventh day of the experiment, inhibition of MSC growth was also observed when IB was used at doses of 0.5 µM (p = 0.003), 1 µM (p = 0.001) and 2 µM (p = 0.0002) (Fig. 1D).

IB is toxic to MSCs

To evaluate whether IB was capable of inducing apoptosis of MSCs, these cells were cultured for 72 h with 0.5 µM, 1 µM and 2 µM of IB. Importantly, the treatment with IB for 72 h did not influence the viability of MSCs at concentrations of 0.5 µM and 1.0 µM. However, at a dose of 2.0 µM, IB induced the death of 15% of MSCs (mean) (p = 0.007) (Fig. 1E).

IB impairs the mitochondrial membrane potential of MSCs at high concentration

Loss of mitochondrial permeability is a key event in cellular apoptosis21. At concentrations of 0.5 µM and 1.0 µM, IB did not influence the mitochondrial membrane potential of MSCs. On the other hand, when MSCs were treated with 2.0 µM of IB, there was a significant decrease in the mitochondrial membrane potential of these cells (p = 0.0002) (Fig. 1F).

The association of MSCs and 0.5 µM IB guarantees a greater immunosuppressive effect on T-cells

As expected, MSCs significantly suppressed T-cell proliferation (p < 0.0001). Importantly, the immunosuppressive capacity of MSCs was not impaired by their treatment with IB regardless of the concentration used. However, when MSCs were used in association with 2 µM IB, the immunosuppressive effects on T-cells were not greater than those observed with the exclusive use of IB at 2 µM. It is possible that the cytotoxic effect of 2 µM IB on MSCs contributes to this result.

Treatment of PBMCs with IB promoted a dose-dependent inhibition of T-cell proliferation (p < 0.0001). The combination of 0.1 µM IB and MSCs promoted superior immunosuppression compared to the IB-treatment group (p < 0.0001), but similar to MSCs alone. Interestingly, the combined use of 0.5 µM IB and MSCs guaranteed a superior immunosuppressive effect on T-cells compared to the isolated use of 0.5 µM IB (p < 0.0001) or MSCs (p = 0.05) (Fig. 2A). To proceed with the functional evaluation of the effect of IB on the immunoregulatory mechanisms of MSCs, we treated these cells for 72 h with 0.5 µM IB, given that this was the lowest non-cytotoxic dose of IB that, in combination with MSCs, was capable of potentiating the immunosuppression of T cells.

Figure 2
figure 2

Effect of IB on the immunosuppressive potential of MSCs. (A) T-cell proliferation after their treatment with IB (blue) or after their concomitant treatment with IB and co-culture with MSCs (yellow). Treatment with IB occurred with concentrations of 0 (control), 0.1 µM, 0.5 µM, 1 µM, and 2 µM. (B-C) CD38 and CD69 expression on T-cells cultured with MSCs and in T-cells cultured with MSCs and IB at 0.5 µM dose. (D) Representative density dot plots from flow cytometry experiments show the expression of T-cell activation markers. Results are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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Expression of CD38 and CD69 activation markers on PBMCs

The expression of the T-cell activation markers CD38 and CD69 was determined by Flow Cytometry. Compared to PHA-activated PBMCs, MSCs were able to increase CD69 expression on T cells from 52.3 ± 1.1% to 62.3 ± 1.9% (p = 0.03). Interestingly, after exposure to IB, T-cells co-cultured with MSCs did not show significant changes in the expression of CD38 (80.4 ± 0.6% vs. 80.1 ± 4.9%) nor CD69 (62.3 ± 1.9% vs. 58.7 ± 6.1%) (Fig. 2B–D).

IB treatment modulated the production of classical and non-classical Treg cells

IB treatment promoted a slight reduction in the percentage of CD4+CD25highFOXP3+ among PHA-activated PBMCs that were co-cultured with MSCs (p = 0.03). In contrast, although we did not identify statistically significant differences (p = 0.059), the IB caused a slight increase in the CD8+CD28- Tregs, from 19.6 ± 2.6% to 23.3 ± 1.8% (Fig. 3A–C).

Figure 3
figure 3

Treg generation and VCAM-1 and ICAM-1 expression by MSCs. (A-B) Generation of CD4+CD25highFOXP3+ and CD8+CD28- Tregs cells by MSCs and MSCs treated with 0.5 µM IB. (C) Representative density dot plots from flow cytometry experiments show the expression of Tregs. (D) Levels of IFN-γ and IL-10 in the inflammatory medium produced by PBMCs activated with 5 µg/ml of PHA for 3 days. (E) ICAM-1 (CD54) expression by MSCs after their treatment with 0.5 µM IB in the inflammatory medium. The average percentage of CD54-positive MSCs in non-inflammatory medium is represented by the dashed horizontal line. MFI of CD54 staining in MSCs and MSCs + IB is shown below (Data are presented as ratio in relation to MSCs cultured in non-inflammatory medium). (F) VCAM-1 (CD106) expression by MSCs after their treatment with 0.5 µM IB in the inflammatory medium. The average percentage of CD106-positive MSCs in non-inflammatory medium is represented by the dashed horizontal line. MFI of CD106 in MSCs and MSCs + IB is shown below (Data are presented as ratio in relation to MSCs cultured in non-inflammatory medium). (G) Representative histograms showing the expression levels of CD54 and CD106 in MSCs (blue line) and MSCs + IB (yellow line). Black line represents the isotype control. Results are presented as mean ± SEM. *p < 0.05; **p < 0.01.

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IL-10 and IFN-γ concentration in the inflammatory medium

The molecular effects of IB on MSCs were characterized in an inflammatory environment generated by the supernatant of PHA-activated PBMCs. The presence of PHA at 5 µg/mL promoted a significant increase in IL-10 (0 to 0.08 ± 0.007 ng/ml; p = 0.03) in PBMCs, as well as a more pronounced in IFN-γ (0 to 2.9 ± 0.03 ng/ml; p < 0.0001) (Fig. 3D).

IB induces VCAM-1 expression on MSCs exposed to inflammatory medium

In non-inflammatory media, only 47.2 ± 2.6% and 2.68 ± 0.4% of MSCs expressed ICAM-1 and VCAM-1, respectively. In an inflammatory environment, the expression of ICAM-1 and VCAM-1 was found in 100% and 27.9 ± 0.34% of MSCs, respectively. To determine whether IB could modulate the expression ICAM-1 and VCAM-1 on MSCs exposed to inflammation, we treated the cells with the IB in addition to exposing them to different inflammatory conditions. The treatment of MSCs with IB in an inflammatory environment increased the percentage of the cells expressing VCAM-1, from 27.9 ± 0.3% to 32.7 ± 1.5% (p = 0.003). The MFI of VCAM-1 was also significantly higher in such cells, compared to MSCs cultured in an inflammatory medium without IB (p = 0.003). MSCs cultured in inflammatory media did not show significant changes in ICAM-1 expression after treatment with IB (Fig. 3E–G).

IB modulates transcriptional levels of immunosuppressive factors in MSCs and PBMCs

Treatment with IB did not change the transcriptional levels of TGF-β or IDO in MSCs exposed to the inflammatory medium. In contrast, under these conditions the cells showed elevated transcriptional levels of PD-L1 (p < 0.0001), TSG-6 (p = 0.01), and IL-10 (p = 0.002), with fold of increase of 3.3, 1.2, and 5.3, respectively (Fig. 4A–E). In PBMCs cultured with MSCs, IB was able to significantly inhibit INF-γ (0.44 fold; p = 0.0007) (Fig. 4F–I).

Figure 4
figure 4

Gene expression of MSCs and PBMCs, and cytokine quantification. (A) TGF-β1, (B) PD-L1, (C) TSG-6, (D) IL-10, and (E) IDO expression in MSCs exposed or not to IB in the inflammatory medium. The fold changes were determined by the 2− ΔΔCt method, using the median Ct value of control MSCs as a reference. (F) IFN-γ, (G) IL-10, (H) IL-17 and (I) FOXP3 expression in PHA-activated PBMCs cultured with MSCs and treated or not with IB. The culture of PBMCs with MSCs was performed using a transwell system. The fold changes were determined by the 2 − ΔΔCt method, using the median Ct value of PHA-activated PBMCs cultured with MSCs as a reference. Levels of secreted (J) IFN-γ, (K) TNF-α, and (L) IL-10 in PHA-activated PBMCs cultured with MSCs and treated or not with IB. Cytokine quantification was performed by ELISA, after 72 h of cell culture. Results are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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IB inhibits IFN-γ and TNF-α secretion and induces IL-10 production in PBMC- MSC co-cultures

The presence of IB in the culture of PBMCs with MSCs decreased the secretion of the pro-inflammatory factors IFN-γ from 1.34 ± 0.02 to 0.89 ± 0.08 ng/ml (p = 0.0005) and TNF-α from 0.73 ± 0.17 to 0.33 ± 0.15 ng/ml (p = 0.02). Furthermore, the presence of IB in the culture of PBMCs with MSCs increased IL-10 levels from 0.22 to 0.32 ± 0.02 ng/ml (p = 0.0005) (Fig. 4J–L).

Discussion

Considering that the effect of IB is unknown on tissue-resident MSCs and that there is also no information on the possibility of combined therapeutic use of this TKI with MSCs, in this study we investigated the cellular and molecular effects that IB promotes on MSCs. Interestingly, our results show that such association potentiated the immune control over T-cells, increasing the expression of adhesion molecules and immunoregulatory mediators produced by MSCs.

It is important to note that MSCs showed expression of all IB targets tested (BTK, ITK, JAK3, ERBB2, and EGFR), and that the MSC treatment with IB increased the expression of CD90 and CD105 on such cells. Interestingly, both CD90 and CD105 expression have been associated with the osteogenic capacity of MSCs obtained from the adipose tissue22,23, but CD105 also appears to have a role in the capacity for adhesion and proliferation in other cell types24,25.

Interestingly, IB has been shown to inhibit the division of tumor cells26,27,28, but may stimulate the proliferation of healthy immune cells26. Here, we noticed a reduction in the growth capacity of MSCs treated with IB. In 3 days of cell culture, IB reduced the viability of MSCs when used at a dose of 2 µM. After 3 and 7 days of culture, IB inhibited the growth of MSCs at all doses evaluated in this study.

Currently, the use of IB has been investigated for the treatment of a range of solid and non-solid tumors29. In CLL samples, it has been demonstrated that IB can alter mitochondrial membrane potential and induce mitochondria-dependent apoptosis30,31. The mitochondrial membrane potential is an essential component in the process of energy storage that plays a key role in mitochondrial homeostasis. The loss of mitochondrial membrane potential is associated with the onset of apoptosis, but may also be a consequence of this cell death process32,33. Our data show that IB altered the mitochondrial membrane potential and induced the apoptosis of MSCs only when used at a dose of 2.0 µM for 72 h. However, this is an important finding that prompts further investigation into the effect that a higher dose of IB would have on endogenous MSCs.

Dubovsky and colleagues showed that the treatment of CD4 T-cells from healthy donors with IB and anti-CD3 controls the immune response of these cells through the inhibition of ITK and attenuates the expression of CD6934. Interestingly, we did not identify changes in the expression of the activation markers CD38 and CD69 on T-cells after the addition of IB in the co-culture of PBMCs with MSCs. However, we identified that MSCs were capable of inducing the expression of CD69 on T-cells. This finding is in accordance with other studies that have demonstrated that MSCs from adipose tissue stimulate the expression of such activation marker35,36,37. Importantly, we observed that the use of different doses of IB in the co-culture between MSCs and PBMCs potentiated the immunosuppression exerted on T-cells. This data indicates that the use of MSCs for immunological control could benefit from the addition of this TKI. Based on this functional observation, we evaluate the effect of IB on the main immunosuppression mechanisms exerted by MSCs.

The generation of classical CD4+CD25highFOXP3+ Tregs constitutes one of the mechanisms of immunoregulation by MSCs38. Interestingly, we noticed that IB promoted a decrease in the generation of these cells. This same effect has been observed in patients with CLL, who have a reduced number of Tregs during IB therapy39,40. In contrast, we identified that IB was able to promote a slight increase in the population of CD8+CD28- Tregs in PBMCs co-cultured with MSCs. Furthermore, our data showed that the addition of IB to MSCs cultured in an inflammatory milieu promoted a significant increase in VCAM-1 on their surface. Of note, it was demonstrated that MSC-mediated immunosuppression is significantly reversed when the adhesion molecules are functionally blocked41. It is important to highlight that recent data show that in endothelial cells, IB can block VCAM-1 expression, via the inhibition of Tec kinase BMX42.

Importantly, IB was able to significantly increase the expression of PD-L1, TSG-6, and IL-10 in MSCs. The increased expression of such molecules by MSCs is particularly relevant, considering that both are key anti-inflammatory factors involved in the immune control exerted by MSCs43,44,45. Interestingly, IB inhibited the expression of IFN-γ in PBMCs cultured with MSCs. In fact, the secretion of this protein and TNF-α was inhibited by IB in the co-culture of PBMC and MSCs. In addition to reducing the production of pro-inflammatory factors, the anti-inflammatory action of IB was reinforced by the increased levels of IL-10 detected in the co-culture of PBMC and MSCs, after their exposure to this TKI.

For the development of cell therapy, it is essential to investigate the possible effects of immunomodulatory drugs on the functional properties of MSCs. In this sense, it has been reported that—unlike ciclosporin A46 and mycophenolate acid -, rapamycin, tacrolimus, and calcineurin inhibitors can impair the immunomodulatory effects of MSCs12,13. Recently, it was demonstrated that Ruxolitinib, a TKI inhibitor that has been used in the treatment of GVHD, inhibits IDO and PD-L1 production by IFN-γ-licensed MSCs15.

Here we demonstrate for the first time that prolonged exposure to IB can compromise the viability of MSCs. However, IB does not impair the immunosuppressive effect of MSCs and stimulates the production of anti-inflammatory factors by such cells, such as VCAM-1, PD-L1, TSG-6, and IL-10.

This work present some limitations, as follows: although the activation of PBMCs with PHA is associated with the production of several inflammatory mediators, such as IL-lβ, TNF-α, IL-6, IL-2, IL-10, IFN-γ, and GM-CSF47,48, the supernatant from PHA-activated PBMC used here to induce an inflammatory environment to stimulate MSCs was only characterized according IL-10 and IFN-Ɣ levels. Another point is that our results demonstrated that the use of 0.5 µM IB associated with MSCs was able to induce a stronger inhibition of T-cell proliferation compared to the isolated use of MSCs or 0.5 µM of IB. However, our experiments were developed with fixed doses of MSCs, which prevents us from evaluating the occurrence of synergy and additive effects between MSCs and IB.

Additional studies involving other experimental models and MSC sources are needed to validate the effects of the association of MSCs and IB in more complex scenarios. These findings may not only ensure a better understanding of the effects of prolonged IB therapy on resident MSCs, but also serve as a basis for the development of new therapeutic approaches for the treatment of immunological disorders.