Alterations of mesenchymal stem cells on regulating Th17 and Treg differentiation in severe aplastic anemia

Patients

Five patients with SAA and five controls were enrolled in this study. Clinical data of these patients were outlined in Table 1, and all bone marrow aspirates were collected at the time of diagnosis without concomitant medication. All patients were previously untreated and aged less than 18 years old. The average ages of SAA patients and controls were 12.5 and 13.7 years, respectively. For SAA patients, chromosome breakage analysis, flow cytometry, and cytogenetic studies were done to screen for Fanconi anemia, paroxysmal nocturnal hemoglobinuria, and myelodysplastic syndrome. Secondary aplastic anemia following drugs, toxic exposure, autoimmune disorders, and infections was excluded. In all of the five patients with SAA, no specific cause of pancytopenia and bone marrow failure was identified.

Characterization of MSCs

In vitro, MSCs displayed fibroblast-like morphologies and adhered to plates when maintained in culture conditions (Figure 1A). There was no obvious difference in cell size and shape between MSCs from patients with SAA and those from controls. MSCs revealed a consistent immunophenotypic profile which was negative for CD34 and CD45, and positive for CD44 and CD105 (Figure 1B and Supplementary Figure 1). No significant difference was noted in the expression of any single surface marker between the two groups, as shown by the detected mean fluorescence intensity (Figure 1C). MSCs in both groups could achieve osteogenesis and adipogenesis when exposed to differentiation induction medium. However, MSCs from patients with SAA had less robust osteogenic differentiation, compared to those from controls. Less extent of mineralization in the SAA group was demonstrated by less intense von Kossa staining (Figure 1D). SAA MSCs also gave rise to less lipid-containing cells after adipogenic induction. The intracytoplasmic droplets of neutral fat stained by Oil red O were less and smaller within a single adipocyte in the SAA group (Figure 1E). Nevertheless, these findings fulfilled the criteria of International Society for Cellular Therapy to define human MSCs [13].

Figure 1. Characterization of MSCs. (A) In vitro culture, MSCs in the control and SAA groups shared similar growth patterns and morphologies (×100; scale bar = 100 μm). (B) Using flow cytometry, these cells were negative for CD34 and CD45, and positive for CD44 and CD105. The red dashed line indicates the isotype control; the black area indicates the stained cells. (C) There was no significant difference in the expressed MFI of CD34-PE, CD45-FITC, CD44-APC, and CD105-PE between the SAA group and the control group. (D) Osteogenic differentiation was demonstrated by mineralized deposits stainable with von Kossa stain (×100; scale bar = 100 μm). (E) Adipogenic differentiation was confirmed by intracellular accumulation of lipid droplets stainable with oil red O (×100; scale bar = 100 μm). MFI: Mean fluorescence intensity; ns: not significant; SAA: Severe aplastic anemia.

Purification of isolated CD4+ T cells

CD4+ T cells were obtained from splenocytes of C57BL/6 mice. Using flow cytometry analysis, about 32.9% of splenocytes were CD4+ cells (Supplementary Figure 2A). Purified CD4+ cells were further isolated by negative selection, and the final purity was 94.9% (Supplementary Figure 2B). The purified cells were used as CD4+ T cells for the following experiments.

Impaired inhibitory effects of SAA MSCs on IL-17 and IL-1β production during Th17 differentiation

To assess effects of MSCs on T helper 17 (Th17) differentiation, mouse CD4+ T cells were cocultured for 3 days with control MSCs or SAA MSCs after 48 hours of differentiation induction. At the end of differentiation on day 5, concentrations of mouse IL-17 and IL-1β in the cell culture supernatant were measured. Compared to the basic group, IL-17 and IL-1β levels in the supernatant were lower in the control group (P < 0.01), suggesting that coculture of CD4+ T cells with control MSCs inhibited IL-17 and IL-1β secretion during the process of differentiation into Th17 cells (Figure 2). These data suggested that MSCs of human origin could express immunosuppressive activities on immune cells derived from mouse splenocytes. It is interesting to note that IL-17 and IL-1β levels in the SAA group were significantly increased compared to the control group (P < 0.01 and P < 0.05, respectively). These results indicated that inhibitory effects of SAA MSCs on the production of IL-17 and IL-1β during Th17 differentiation were attenuated.

Figure 2. IL-17 and IL-1β concentrations in the culture supernatant after 5-days Th17 differentiation induction. IL-17 and IL-1β levels were lower when CD4+ T cells cocultured with control MSCs. The inhibitory effects were significantly decreased when cocultured with SAA MSCs. Data are presented as the mean ± SD. n = 5 in each group.

Impaired inhibition of SAA MSCs on Th17 activation

Following 5-days Th17 differentiation, the cells were activated with phorbol 12-myristate-13 acetate and ionomycin. As shown in Supplementary Figure 3, mouse IL-17 and IL-1β in the supernatant were at similar levels in the basic, control, and SAA groups. It was explained by that cytokines produced by the activated cells were captured in the rough endoplasmic reticulum and Golgi apparatus by the protein transport inhibitor, Brefeldin A. Activation of Th17 cells was confirmed by intracellular staining for IL-17 with flow cytometry (Figure 3A). Compared to the basic group, there was no obvious change in the percentage of CD4+ T cells either in the control group or SAA group after the stimulation of the Cell Activation Cocktail, implicating that coculturing with MSCs did not affect the quantity of CD4+ T cell population (Figure 3B). Further analysis showed that a population of IL-17-secreting cells was documented in the basic group at the percentage of 12.9 ± 1.2% under Th17 skewing conditions. In the control group, the percentage of IL-17-secreting cells was significantly decreased (6.6 ± 0.4%, P < 0.01), suggesting that coculturing with control MSCs could inhibit functional activities of Th17 cells. Compared to the control group, the percentage of IL-17-secreting cells was significantly increased in the SAA group (9.1 ± 1.5%, P < 0.01), indicating impaired inhibition of SAA MSCs on activation of Th17 cells.

Figure 3. Assessment of Th17 activation after 5-days Th17 differentiation. (A) Activation of Th17 cells was confirmed by intracellular staining for IL-17 with flow cytometry. (B) The percentage of IL-17-secreting cells were lower in the control group, compared to the basic group (6.6 ± 0.4% vs 12.9 ± 1.2%, P < 0.01). The percentage of IL-17-secreting cells were significant higher in the SAA group, compared to the control group (9.1 ± 1.5% vs 6.6 ± 0.4%, P < 0.01). Data are presented as the mean ± SD. n = 5 in each group.

Aberrant effects of SAA MSCs on Treg differentiation

To evaluate effects of MSCs on regulatory T (Treg) differentiation, mouse CD4+ T cells were cocultured for 3 days with control MSCs or SAA MSCs after 48 hours Treg differentiation induction. At the end of differentiation on day 5, differentiation of CD4+ T cells into CD4+CD25+FoxP3+ Treg cells was confirmed by intracellular staining for FoxP3 by flow cytometry (Figure 4A). Compared to the basic group, no obvious alteration in the percentage of CD4+ T cells was observed either in the control group or SAA group after Treg differentiation induction, implicating that coculturing with MSCs did not affect the quantity of CD4+ T cell population (Figure 4B). Further analysis showed that a population of CD4+CD25+FoxP3+ Treg cells in the basic group was documented at the percentage of 4.3 ± 0.8% under 5-days Treg polarization conditions. In the control group, the percentage of Treg cells was significantly increased (5.8 ± 0.8%, P < 0.05). Consistently, transforming growth factor-β (TGF-β) concentrations in the cell culture supernatant were significantly increased and IL-1β levels were decreased, compared to the basic group (Figure 4C). These data indicated that coculture of CD4+ T cells with control MSCs could enhance the differentiation into CD4+CD25+FoxP3+ Treg cells under polarization conditions. Again, our results showed xenogeneic MSCs of human origin could have immunosuppressive effects on mouse immune cells. It is interesting to note that the percentage of Treg cells in the SAA group was significantly higher compared to the control group (8.1 ± 0.5% vs 5.8 ± 0.8%, P < 0.01), suggesting the promotive effect of SAA MSCs on Treg differentiation (Figure 4B). Inconsistent with the increased number of Treg cells, TGF-β concentrations in the culture supernatant were conversely decreased and IL-1β levels were elevated in the SAA group (Figure 4C). These results suggested that coculture of CD4+ T cells with SAA MSCs during differentiation into Treg cells may induce the production of more Treg cells but without adequate function.

Figure 4. Evaluation of Treg differentiation after 5-days differentiation induction. (A) Differentiation of Treg cells was confirmed by intracellular staining for FoxP3 with flow cytometry. (B) The percentage of CD4+CD25+ FoxP3+ Treg cells was higher in the control group, compared to the basic group (5.8 ± 0.8% vs 4.3 ± 0.8%, P < 0.05). The percentage of CD4+CD25+ FoxP3+ Treg cells was significantly increased in the SAA group, compared to the control groups (8.1 ± 0.5% vs 5.8 ± 0.8%, P < 0.01). (C) In the SAA groups, TGF-β concentrations in the culture supernatant were decreased and IL-1β levels were increased, compared to the control group (P < 0.01). Data are presented as the mean ± SD. n = 5 in each group.

Patients

Idiopathic SAA was defined as pancytopenia and hypocellular bone marrow after excluding any other underlying diseases. The inclusion criteria were bone marrow hypocellularity of less than 25% and at least two of following: absolute neutrophil count < 0.5 × 10⁹ /l, platelet count < 20 × 10⁹ /l, and reticulocyte count < 1% [3]. Control subjects were patients who received bone marrow examination for diseases other than hematological diseases with pathological proof of normal bone marrow. All participants were younger than 18 years of age. This study was approved by the Institutional Review Board of the Chung Shan Medical University Hospital (CS2-21056), and written informed consents were obtained from the parents.

MSC isolation

MSCs were collected, isolated, and identified as in our previous reports [8–10]. Bone marrow cells were collected from iliac crest aspirates, and mononuclear cells were isolated by Ficoll-Paque density centrifugation (1.077 g/ml; Amersham Biosciences, Uppsala, Sweden). Then, cells were seeded in low-glucose DMEM (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) and antibiotic/antimycotic, and were incubated at 37° C in a humidified atmosphere under 5% CO₂. The medium with suspension of non-adhered cells was discarded after 48 hours, and replaced twice a week thereafter. Upon reaching 80-90% confluence, cells were detached with trypsin-EDTA (Gibco, Carlsbad, CA, USA) and re-plated for subculture. Under the approval of the institutional review board, parts of the cultured MSCs were stored at -80° C. The stored cells were thawed and further cultured, and MSCs of passage 5 were used for experiments in the present study.

MSC identification

The criteria of International Society for Cellular Therapy were used to characterize MSCs [13]. To evaluate immunophenotypic expression, cultured MSCs were detached, washed, and resuspended in phosphate-buffered saline. After fixing and blocking, cells were immunolabeled with phycoerythrin (PE) mouse anti-human CD34 (Clone 8G12; BD Biosciences, San Jose, CA, USA), fluorescein isothiocyanate (FITC) mouse anti-human CD45 (Clone 2D1; BD Biosciences, San Jose, CA, USA), allophycocyanin (APC) mouse anti-human CD44 (Clone G44-26; BD Biosciences, San Jose, CA, USA), or PE mouse anti-human CD105 (Clone 266; BD Biosciences, San Jose, CA, USA) antibodies. Mouse IgG (BD Biosciences, San Jose, CA, USA) served as isotype control. Data were analyzed by flow cytometry (Accuri C6; BD Biosciences, San Jose, CA, USA). To assess differentiation potential, cultured MSCs were detached and replated in 60-mm dishes. For osteogenic induction, MSCs were grown in DMEM with 10% FBS, 10 mM β-glycerophosphate, 0.1 μM dexamethasone, and 0.2 mM ascorbic acid. For adipogenic induction, MSCs were grown in DMEM with 10% FBS, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 0.1 mM indomethacin, and 10 μg/ml insulin. After 2-week induction, osteogenesis and adipogenesis were demonstrated by von Kossa stain (Cedarlane, Ontario, Canada) and oil red O stain (Sigma, St Louis, MO, USA), respectively.

Preparation of CD4+ T cells from mice

Eight-week-old C57BL/6 mice were purchased from the National Laboratory Animal Center. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Chung Shan Medical University Experimental Animal Center (IACUC Approval No: 2488). Immediately after sacrifices, spleen tissue samples were obtained, ground, and passed through 40 μm strainer. MojoSort Mouse CD4 T Cell Isolation Kit (BioLegend, San Diego, CA, USA) was used for purification of the collected CD4+ cells by negative selection, according to the manufacturer’s instruction. The purity of CD4+ cells was measured with the Accuri C6 flow cytometer. The final purified cells were collected and cultured in RPMI-1640 (Gibco, Waltham, MA, USA) supplemented with 10% FBS and antibiotic/antimycotic. These cells were used as CD4+ T cells for the following studies.

Coculture of CD4+ T cells with MSCs during Th17 differentiation induction

CellXVivo Mouse Th17 Cell Differentiation Kit (R&D Systems, Minneapolis, MN, USA) was used to induce differentiation of Th17 cells from the preparation of CD4+ T cells isolated from mouse splenocytes, according to the manufacturer’s instruction. CD4+ T cells were differentiated for a total of 5 days under Th17 polarization conditions. To assess effects of MSCs on Th17 differentiation, CD4+ T cells were cocultured with control MSCs or SAA MSCs in 24-well dishes without physical separation after 48 hours of differentiation induction. The ratio of CD4+ T cells to MSCs was 100:1. In the coculture system, MSCs could interact with CD4+ T cells through both direct cell-to-cell contact and paracrine mechanisms. For baseline control, CD4+ T cells in the basic group were differentiated for 5 days under the same conditions but without coculturing with MSCs. At the end of differentiation on day 5, the cell culture supernatant was collected and cytokine secretion was determined using the Mouse IL-17A ELISA kit (BioLegend, San Diego, CA, USA) and the Mouse IL-1β ELISA kit (BioLegend, San Diego, CA, USA). The levels were determined in triplicate.

Assessment of Th17 activation

After a 5-days Th17 differentiation, the cells were stimulated with the Cell Activation Cocktail with Brefeldin A (BioLegend, San Diego, CA, USA) for 6 hours. Following activation of cells with this cocktail, the cell culture supernatant was collected and concentrations of IL-17 and IL-1β were measured by ELISA as previously described. For intracellular detection of the secreted cytokines, the activated cells were stained with APC anti-mouse CD4 antibody (Clone GK1.5; BioLegend, San Diego, CA, USA) and then treated with Intracellular Fixation and Permeabilization Buffer Set and FITC anti-mouse IL-17A antibody (Clone TC11-18H10.1; BioLegend, San Diego, CA, USA). Thereafter, the cells were examined by the Accuri C6 flow cytometer.

Coculture of CD4+ T cells with MSCs during Treg differentiation induction

CellXVivo Mouse Treg Cell Differentiation Kit (R&D Systems, Minneapolis, MN, USA) was used to induce differentiation of Treg cells from the preparation of CD4+ T cells isolated from mouse splenocytes, according to the manufacturer’s instruction. There was a total of 5 days for Treg differentiation induction. To evaluate influences of MSCs on differentiation of CD4+ T cells into Treg cells, CD4+ T cells were cocultured with control MSCs or SAA MSCs at the ratio of 100:1 after 48 hours of differentiation induction. As the procedure of Th17 polarization, MSCs could interact with CD4+ T cells in the coculture system through both direct cell-to-cell contact and secreting soluble factors. For baseline control, CD4+ T cells in the basic group were differentiated for 5 days under the same conditions but without coculturing with MSCs. On day 5 of differentiation, the cell culture supernatant was collected and cytokine secretion was determined using the TGF-β1 Mouse ELISA kit (ThermoFisher Scientific, Waltham, MA, USA) and the Mouse IL-1β ELISA kit. The levels were determined in triplicate.

Treg cell differentiation was further documented by analyzing marker expression via flow cytometry. The differentiated cells were stained with APC anti-mouse CD4 antibody and PE anti-mouse CD25 antibody (Clone 3C7; BioLegend, San Diego, CA, USA), followed by fixation and permeabilization with FOXP3 Fix/Perm Buffer Set (BioLegend, San Diego, CA, USA). Then, the cells were stained with FITC anti-mouse FOXP3 antibody (Clone 150D; BioLegend, San Diego, CA, USA) and analyzed by the Accuri C6 flow cytometer.

Statistical analysis

Data analysis was performed using GraphPad Prism 9.0 software. For continuous variables, Mann-Whitney U test was used to compare groups. A value of P < 0.05 was considered to be statistically significant.