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Keywords:

  • Adipose-derived stromal cell;
  • Immunomodulation;
  • Mesenchymal stromal cell;
  • Signal transduction;
  • T cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Mesenchymal stromal cells regulate immune cell function via the secretion of soluble factors. Cell membrane interactions between these cell types may play an additional role. Here, we demonstrate that subpopulations of allo-activated T cells are capable of binding to human adipose-derived stromal cells (ASC). The bound T-cell population contained CD8+ T cells and was enriched for CD4CD8 T cells, whereas the proportion of CD4+ T cells was decreased compared with the non-bound T-cell population. Bound CD4+ T cells had high proliferative activity and increased CD25 and FoxP3 expression. However, they also expressed CD127, excluding regulatory T-cell function. In CD8+ T cells, IL-2 sensitivity, as determined by the analysis of phosphorylated STAT5, was lower in the presence of ASC and even lower in bound cells. In contrast, IL-2-induced phosphorylated STAT5 levels were higher in bound CD4+ T cells than in non-bound CD4+ T cells. Additionally, pro-proliferative TGF-β signalling via endoglin and SMAD1/5/8 phosphorylation was detected in bound CD4+ T cells. Even after prolonged co-culture with ASC, the activated phenotype of bound CD4+ T cells persisted. In conclusion, these results demonstrate that the binding of lymphocytes to ASC represents an immunomodulatory mechanism in which CD8+ T cells are inhibited in their responsiveness to pro-inflammatory stimuli and reactive CD4+ T cells are depleted from the immune response.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Mesenchymal stromal cells (MSC) are multipotent progenitor cells that exist in a range of tissues throughout the body, including bone marrow and adipose tissue 1, 2. Their capacity to undergo multilineage differentiation has raised interest in the use of MSC for clinical therapy. This has been further strengthened by the advantageous immunological characteristics of MSC. MSC express low levels of co-stimulatory and HLA molecules 3 and may be less sensitive to cytotoxic attack than other cell types 4, which would allow transplantation of allogeneic MSC. MSC have, furthermore, immunomodulatory capacity, which is apparent in vitro by their inhibition of lymphocyte proliferation 5, 6, and in vivo by delayed allo-graft rejection 7 and amelioration of graft-versus-host disease after administration of MSC 8, 9. In the absence of an active immune response, however, MSC support the survival and proliferation of lymphocytes 10, 11, suggesting that MSC have a balancing effect on the immune system.

The mechanisms via which MSC bring about their immunomodulatory effects are not fully elucidated. The immunosuppressive effect of MSC is mostly attributed to the production and secretion of soluble anti-inflammatory factors. Multiple factors appear to be involved, including IDO 12 and TGF-β1 13. However, in addition to their repertoire of anti-inflammatory cytokines, MSC secrete chemokines 14 and express adhesion molecules that enable them to establish cell membrane interactions with lymphocytes 15. Suva et al. demonstrated that activated lymphocytes migrate under MSC, and suggested that this potentiates the inhibitory effects of soluble factors secreted by MSC 16. Proximity is particularly important for the effect of the short-lived nitric oxide, which is secreted by MSC and inhibits lymphocyte proliferation 14, 17. Whether attraction of lymphocytes by MSC merely serves to increase the efficiency of immunosuppression by soluble factors, or whether the association of lymphocytes and MSC comprises an immunomodulatory mechanism via cell membrane proteins, remains unclear.

In this study, we examined the establishment of cell contact interactions between adipose-derived stromal cells (ASC) and allo-activated lymphocytes. To investigate the effect of these interactions on T-lymphocyte function, responsiveness of T-cell subsets to the lymphocyte growth factor IL-2 was analysed by the measurement of phosphorylated levels of the downstream transcription factor STAT5 (P-STAT5). In addition, activity of the TGF-β signalling pathway was examined in T lymphocytes by measuring the levels of phosphorylated Smad. By measuring the activity of these intracellular signalling pathways, we aim to examine the impact of cell contact interactions between T lymphocytes and ASC on the immune response.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

ASC bind allo-activated PBMC

To study the occurrence of cell contact interactions between ASC and lymphocytes, PBMC were allo-activated for 6 days and added to monolayer cultures of ASC. The monolayers consisted of green fluorescent PKH67-labelled ASC that were autologous to PBMC and non-labelled ASC that were allogeneic to PBMC. After 1 or 24 h, non-adherent PBMC were removed and the remaining cells washed with PBS and photographed. Subpopulations of PBMC that were adherent to ASC were found after 1 and 24 h of co-culture (Fig. 1). PBMC bound to ASC of both allogeneic and autologous origin. Non-activated PBMC associated with ASC at a lower frequency, whereas anti-CD3 and anti-CD28 stimulation induced the attachment of the majority of PBMC to ASC (data not shown). These data confirm the earlier observations that activated PBMC associate with stromal cells 16.

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Figure 1. Bright-field microscopic images of ASC and PBMC co-cultures. PBMC were allo-activated for 6 days and added to monolayer cultures of ASC. After 24 h, non-adherent cells were removed and the remaining adherent cells photographed. (A) Image of PBMC with autologous (green fluorescent) and allogeneic ASC (non-fluorescent) and (B) detailed image of a single ASC with adherent PBMC. Bars represent 20 μm.

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ASC bind T-, B- and NK-lymphocyte subsets

To examine which PBMC subsets bound to ASC, 6-day allo-activated PBMC were added to ASC for 1 or 24 h. Non-adherent cells were then collected and adherent cells removed from the culture flasks by trypsinization. The cell fractions were analysed by flow cytometry and cells in the lymphocyte gate were immunophenotyped. The total lymphocyte populations were composed of, on average, 89.2% CD3+ T lymphocytes, of which 61.3% CD4+, 25.4% CD8+ and 2.5% CD4CD8, and of 5.1% NK lymphocytes and 5.7% B lymphocytes. After co-culture with ASC for 1 h, the bound lymphocyte population was significantly enriched for CD4CD8 T cells (30.7%, p=0.03) in comparison to the non-bound population, contained a reduced proportion of CD4+ lymphocytes (21.4%, p=0.03) and, furthermore, consisted of 11.2% B cells, 7.9% NK cells and 28.8% CD8+ cells (Fig. 2A). The non-adherent cell fraction contained 66.7% CD4+ cells, 24.4% CD8+ cells, 3.2% CD4CD8 cells, 2.0% B cells and 3.7% NK cells.

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Figure 2. Binding of lymphocyte subsets to ASC. Six-day allo-activated lymphocytes were added to ASC monolayer cultures. Controls were kept without ASC. The lymphocyte subsets composition was analysed in the bound and non-bound lymphocyte fractions after 1 h (A) or 24 h (B) of co-culture with ASC. Data shown are the means of six experiments. Statistical analysis is described in the text. (C) Representative dot plot demonstrating an increase of CD4CD8 T cells in the bound T-cell fraction. (D) Representative dot plot of TCR-γδand TCR-αβ expression in bound and non-bound CD4CD8 T cells. (E) Pre-treatment of ASC with 50 ng/mL IFN-γ for 6 days did not significantly affect the subset composition of the bound lymphocytes as determined by a paired t-test between the values of IFN-γ-treated and untreated populations. Bars represent the means of two experiments.

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After 24 h of co-culture with ASC, the composition of the adherent lymphocyte population was not significantly different from that after 1 h of co-culture. It contained 11% B cells, 9% NK cells and was significantly enriched for CD8+ cells (29.7%, p=0.009) compared with the non-bound population, whereas the proportion of CD4+ cells decreased to 27.1% (p<0.009) (Fig. 2B). The percentage of CD4CD8 cells remained significantly elevated compared with the non-bound lymphocyte population (23.2%, p<0.04) (Fig. 2B and C).

To further characterize the bound and non-bound T-lymphocyte populations, the expression of TCR-αβ and TCR-γδ was examined. Of the non-bound CD4CD8 T cells, an average of 46% expressed TCR-γδ and 54% expressed TCR-αβ. Of the bound CD4CD8 T cells, TCR-γδ was expressed by 15% of the cells and TCR-αβ by 85% (Fig. 2D). All CD4+ T cells expressed only TCR-αβ. A small percentage (2–3%) of the CD8+ cells expressed TCR-γδ, whereas the remaining CD8+ cells expressed TCR-αβ.

To examine whether up-regulation of HLA class II molecules on ASC would affect the binding of CD4+ T lymphocytes, ASC were cultured with 50 ng/mL IFN-γ for 6 days and allo-activated PBMC added. IFN-γ increased the expression levels of HLA class II on ASC, but had no significant effect on the proportion of bound CD4+ T lymphocytes (Fig. 2E).

Finally, annexin V and 7-AAD staining of the bound and non-bound cell populations after 24 h of co-culture with ASC showed that an average of 7.5% of the non-bound PBMC and 5.8% of the bound PBMC were apoptotic or dead, which was not significantly different (data not shown).

ASC bind activated T-lymphocyte subsets

Next, we examined the activation state of T lymphocytes that established cell contact interactions with ASC. PBMC were labelled with PKH and allo-activated for 6 days. They were then co-cultured with ASC for 1 or 24 h. The proliferative activity of bound and non-bound T cells was analysed by flow cytometric measurement of PKH dilution. CD4+ T cells that were bound to ASC were enriched for cells with high proliferative activity. Of the CD4+ T cells that were bound to ASC after 1 and 24 h, 54.5 and 86.9% had proliferated versus 2.6 and 5.6% of the CD4+ T cells in suspension (Fig. 3A and B). A difference in proliferative activity between bound and non-bound cells was not found with CD8+ and CD4CD8 T-cell subsets.

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Figure 3. Proliferative activity and expression of activation markers on allo-activated T-lymphocyte subsets bound and non-bound to ASC. PBMC were PKH labelled, allo-activated for 6 days and added to ASC for 1 h (A, C and E) or 24 h (B, D and F). Bound and non-bound cells were then analysed for proliferative activity by PKH measurement (A and B), for the expression of the activation marker CD25 (C and D), and for the activation and adhesion marker endoglin (E and F). Bars represent mean with SD of at least four experiments. * indicates p<0.05 and ** indicates p<0.001 as determined by paired t-test between non-bound and bound populations.

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The activation state of bound T cells and T cells in suspension was furthermore examined by the expression of the activation markers CD25 and CD69. After 1 h of co-culture with ASC, CD25 expression of bound CD4+ T cells was 14 times higher than that of CD4+ cells that remained in suspension. After 24 h of co-culture, bound CD4+ cells showed 44 times elevated expression of CD25 (Fig. 3C and D). There was no difference in CD25 expression between bound and non-bound CD8+ and CD4CD8 cells. The early activation marker CD69 was not expressed under the experimental conditions examined (data not shown). Furthermore, we examined the expression of endoglin (CD105) by T lymphocytes. Endoglin can be expressed by activated T cells and modulates TGF-β signalling. In addition, it has been indicated to play a role in cell adhesion 18, 19. After 1 h of culturing with ASC, adherent CD4+, CD8+ and CD4CD8 subsets showed higher endoglin expression than the corresponding cells in suspension (Fig. 3E and F). After 24 h of co-culture, endoglin expression was strongly increased in bound CD4+ lymphocytes, but not in the other subsets.

IL-2 induced STAT5 phosphorylation in T cells bound and non-bound to ASC

To examine whether bound and non-bound T cells showed differential regulation, we tested the responsiveness of T cells to the lymphocyte growth factor IL-2 in the presence and absence of ASC. IL-2 signalling is potentiated by CD25, the IL-2 receptor α-chain, which we found up-regulated on bound CD4+ cells. Activation of the IL-2 receptor results in the phosphorylation of JAK3, which in turn phosphorylates STAT5. Thus, PBMC were allo-activated for 6 days, added to ASC for 24 h and stimulated with IL-2 for 30 min. In the absence of ASC, P-STAT5 levels increased 16-fold in both CD4+ and CD8+ cells upon IL-2 stimulation (Fig. 4). There was a smaller effect of IL-2 on the levels of P-STAT5 in CD48 T cells (2.3-fold increase). In the presence of ASC, IL-2-stimulated STAT5 phosphorylation was not affected in the CD4+ T cells in suspension, whereas P-STAT5 increased 3.8-fold in the bound CD4+ cells. In contrast, IL-2-induced P-STAT5 levels were 1.6-fold and 2.8-fold reduced in CD8+ cells not bound and bound to ASC, compared with CD8+ cells in the absence of ASC. There was no significant effect of ASC on IL-2-induced P-STAT5 levels in CD4CD8 T cells.

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Figure 4. Effect of IL-2 stimulation on STAT5 phosphorylation in T-lymphocyte subsets in the presence and absence of ASC. PBMC were allo-activated for 6 days and added to ASC cultures for 24 h. The cells were then stimulated with 2000 U/mL IL-2 for 30 min and P-STAT5 analysed by flow cytometry in bound and non-bound cells. A representative example of STAT5 phosphorylation in CD4+ and CD8+ cells is shown in (A). Mean P-STAT5 levels in CD4+ (B), CD8+ (C) and CD48 (D) T-lymphocyte subsets with SD of five experiments are shown. ** indicates p<0.001 as determined by paired t-test between total and non-bound, total and bound, or non-bound and bound populations.

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SMAD1/5/8 phosphorylation in T cells bound and non-bound to ASC

We found highly elevated levels of endoglin on CD4+ T lymphocytes that were bound to ASC after 24 h of co-culture (Fig. 3F). Endoglin is a co-receptor for TGF-β and changes TGF-β intracellular signalling specificity from a SMAD2/3-dependent pathway to a pathway involving SMAD1/5/8 20. Although SMAD2/3 activation is associated with an inhibition of cell proliferation, SMAD1/5/8 has an opposite effect 21.

To examine whether endoglin expression on T cells would activate SMAD1/5/8 signalling, we analysed the levels of phosphorylated SMAD1/5/8 (P-SMAD1/5/8) in 6-day allo-activated T cells after 24 h of culture with or without ASC. P-SMAD1/5/8 levels were significantly higher in CD4+ T cells that were bound to ASC, compared with non-bound CD4+ cells or with CD4+ cells in the absence of ASC (Fig. 5). P-SMAD1/5/8 levels were not different in CD8+ and CD4CD8 T cells in the presence of ASC.

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Figure 5. Levels of phosphorylated SMAD1/5/8 in T-lymphocyte subsets in the absence and presence of ASC. PBMC were allo-activated for 6 days and added to ASC cultures for 24 h. (A) Representative example of P-SMAD1/5/8 expression in CD4+and CD8+T-cell subsets in the absence and presence of ASC. (B) Median P-SMAD1/5/8 levels in CD4+, CD8+ and CD48 T-lymphocyte subsets. Results represent the means with SD of four experiments. ** indicates p<0.001 as determined by paired t-test between non-bound and bound populations.

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Functional characterization of bound CD4+ T cells after prolonged co-culture with ASC

To further examine the functionality of the highly activated CD4+ T cells bound to ASC, IL-2 responsiveness of these cells was examined after 72 h of co-culture with ASC by the measurement of P-STAT5 levels. The CD4+ T cells bound to ASC after 72 h of co-culture showed higher responsiveness to IL-2 than non-bound, CD4+ T cells, and this effect was not significantly different from the results after 24 h of co-culture (Fig. 6A). This was in agreement with the expression level of CD25, which remained high after 72 h of co-culture in the bound compared with the non-bound CD4+ T cells (Fig. 6B). To investigate whether the bound CD4+CD25+ T cells had a regulatory phenotype, FoxP3 and CD127 expression was analysed after 24 and 72 h of co-culture with ASC. The bound CD4+CD25+ T cells expressed higher levels of FoxP3 compared with their non-bound counterparts (Fig. 6C and D). However, the activation marker CD127 was also elevated in the bound fraction (Fig. 6E).

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Figure 6. Effect of 72 h of co-culturing of allo-activated PBMC with ASC. PBMC were allo-activated for 6 days and added to ASC cultures for 24 and 72 h. Bound and non-bound CD4+ T cells were analysed for the levels of P-STAT5 (A), and expression of CD25 after stimulation with 2000 U/mL IL-2 for 30 min (B). A representative example of CD25, FoxP3 and CD127 expression is shown for bound and non-bound CD4+ T cells at 72 h (CD4+CD25+ T cells are depicted in dark blue) (C). Expression levels of FoxP3 and CD127 in bound and non-bound CD4+ T cells are shown at both time points (D and E). After addition of allo-activated cells to ASC for 24 h, non-bound cells were removed and kept in culture until 72 h. A proportion of the bound cells was found in suspension at 72 h. Both cell fractions were added to a new MLR at a ratio of 1:5 and proliferation was measured (F). Bars represent means with SD of four experiments. *indicates p<0.05 and ** indicates p<0.01, as determined by paired t-test between values at 24 and 72 h or between bound non-bound and populations.

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Finally, to examine whether bound and non-bound PBMC had immune regulatory capacity, non-bound PBMC were separated from ASC with bound PBMC after 24 h of co-culture and both fractions cultured for another 48 h. A proportion of the bound PBMC was released after 72 h. These released cells, and the cells that were non-bound at 24 h, were added to allo-activated PBMC at a ratio of 1:5 and proliferation measured after 7 days. Neither cell fraction had immunosuppressive capacity (Fig. 6F), confirming the finding that activated rather than regulatory lymphocytes bind to ASC.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

The immunosuppressive capacity of MSC has been demonstrated in several in vitro and in vivo studies 6, 7. However, there are also studies that show no, or even adverse, immune effects of MSC 22, 23. These contradictory results stress the fact that the mechanisms of interaction between MSC and immune cells are only partially revealed 24, 25. In particular, the relevance of cell membrane interactions between MSC and immune cells is a component of the mechanism that has been largely neglected.

In this study, we examined cell contact interactions between MSC derived from adipose tissue and allo-activated PBMC. Stimulation with allogeneic cells leads to the activation of a subpopulation of PBMC, whereas the majority of PBMC remains in a non-activated state. After 1 h of co-culture, we observed the establishment of cell membrane interactions between ASC and all lymphocyte subsets, of which the bound CD4+ T-cell subset was in a highly activated state. There was no evidence for scavenging of dead lymphocytes by ASC, as there was no enrichment of dead or apoptotic cells in the bound lymphocyte population. Binding of lymphocytes to ASC was furthermore independent of HLA class I, as interactions were established between PBMC and autologous and allogeneic ASC, and also of HLA class II, as HLA class II up-regulation by IFN-γ had no effect on the binding of T-lymphocyte subsets.

The subset composition of the lymphocytes that bound to ASC was different than that of the lymphocytes that remained in suspension, suggesting that binding is a specific process. Lymphocytes bound to ASC were enriched for B cells, CD8+ T cells and, in particular, for CD4CD8 T cells. Part of the bound CD4CD8 T cells expressed TCR-γδ and cells with this phenotype have been described as having regulatory capacity 26. However, the majority of bound CD4CD8 T cells expressed TCR-αβ. There is data demonstrating that CD4CD8TCR-αβ+ T cells can also represent regulatory T cells that are capable of inhibiting a variety of immune responses 27. Alternatively, the CD4CD8 T cells may represent non-mature T lymphocytes. This is supported by the previous finding showing that thymic CD4CD8T cells effectively bind to ASC 28. It is known for some time that stromal cells can establish interactions with haematopoietic precursor cells via the expression of cell surface adhesion molecules. These adhesion molecules include CD9, V-CAM and CD44 that bind to their respective ligands expressed on lymphocyte precursors 29–31 and biglycan, matrix glycoprotein sc1 and SIM that bind specifically pre-B cells 32.

The CD4+ T cells that bound to ASC showed high CD25 expression and proliferative activity. They furthermore expressed high levels of the regulatory T-cell marker FoxP3. They, however, also expressed high levels of CD127 and lacked immunoregulatory capacity. The bound CD4+ T cells can therefore not be regarded as regulatory T cells 33. It can be concluded that CD4+ T cells that bind to ASC are in an activated state.

Selective depletion of activated CD4+ T cells from immune cell suspensions involves a regulatory mechanism that will have impact on the immune response. In many experimental setups this effect is overlooked. For instance, when examining the effect of bone marrow or ASC on lymphocyte proliferation under cell contact conditions by flow cytometric methods, analysis is generally performed on lymphocytes in suspension.

The effect of ASC on the immunological activity of bound and non-bound T lymphocytes was examined by measuring the response to the lymphocyte growth factor IL-2. ASC had an inhibitory effect on IL-2 responsiveness of CD8+ T cells by reducing the increase in P-STAT5 levels. This effect was present in CD8+ T cells in suspension with MSC, but more pronounced in CD8+ cells that were bound to ASC. This result demonstrates that ASC have an immunosuppressive effect on CD8+ T cells by reducing their sensitivity to pro-inflammatory stimuli. The stronger effect of ASC on bound CD8+ cells suggests that cell membrane proteins are involved, or may result from the higher efficacy of soluble factors when cells are in close proximity. In contrast, ASC were not capable of inhibiting IL-2 responsiveness of CD4+ T cells. CD4+ T cells that were bound to ASC showed high levels of P-STAT5 even in the absence of IL-2, which was further increased upon IL-2 stimulation. The potent response to IL-2 in bound CD4+ cells correlated to the high CD25 expression in these cells. These results therefore indicate that ASC bind activated CD4+ cells, but are not capable of inhibiting their response to IL-2 after 24 h of binding.

After prolonged co-culture with ASC for 72 h, bound CD4+ T cells remained in an activated state, although there was a tendency for lower P-STAT5 and CD25 levels compared with 24 h. Nevertheless, it appears that CD4+ T cells remain in an active state when they are bound to ASC. It is possible that bound CD4+ T cells dissociate from the ASC upon returning to a resting state. This possibility needs further investigation.

Although IL-2 induces a stimulatory intracellular cascade in T lymphocytes, TGF-β signalling is considered a suppressive pathway that inhibits the proliferation of T cells. TGF-β signalling, however, can be modulated by TGF-β co-receptors, one of which is endoglin. Endoglin switches TGF-β signalling via the anti-proliferative SMAD2/3 pathway to the pro-proliferative SMAD1/5/8 pathway. After 24 h of co-culture with ASC, bound CD4+ T cells expressed high levels of endoglin. This appeared to be the result of induction of endoglin expression by ASC. This would suggest that bound CD4+ T cells adjust their TGF-β signalling pathways towards a more pro-proliferative pathway. Whether this is a result of the previous allo-activation stimulus or whether ASC play an active role in this process is not clear.

The findings of this study indicate that the binding of T lymphocytes to ASC can have biological significance in a clinical transplantation setting, where ASC are currently under investigation as cellular therapy for immunosuppression. In this setting, the allo-reactive subpopulation of lymphocytes may be selectively captured by ASC and be distracted from attacking the organ transplant, whereas non-allo-reactive T lymphocytes would be less affected. Analysis of chemokine expression profiles of ASC under inflammatory conditions shows strongly increased expression of the T-cell attractants CXCL9, CXCL10 and CXCL11 (unpublished results M. J. H.). This indicates that under inflammatory conditions the recruitment of T lymphocytes to the vicinity of ASC is optimized. In addition to the transplantation setting, the interaction between ASC and T lymphocytes could be biologically relevant in the recently proposed association between the infiltration of T cells in adipose tissue, which leads to local inflammation, and the development of obesity and type 2 diabetes 34, 35.

We can conclude from our study that binding of activated T cells to ASC has a role in the immune response. First, CD8+ T cells are inhibited in immune activity. Second, activated CD4+ T cells are depleted from the cell suspension compartment and are concentrated onto a stromal compartment.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Isolation and expansion of ASC

ASC were isolated from perirenal adipose tissue that became available as a waste product during kidney donation surgery, after written informed consent and approved by the Medical Ethical Committee of the Erasmus Medical Center Rotterdam (protocol no. MEC-2006-190), as previously described 36. In brief, adipose tissue of male and female donors between 29 and 79 years of age was mechanically disrupted and washed twice with PBS (5 min, 450 rcf). Pellet and intermediary fluid were removed and the fat digested with 0.5 mg/mL collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA) in RPMI 1640 (Invitrogen, Paisley, Scotland) for 30 min at 37°C. After addition of fetal bovine serum (FBS) containing α-MEM (Invitrogen) and centrifuging for 10 min at 450 rcf, the pellet was resuspended in 160 mM NH4Cl in PBS and incubated for 10 min at room temperature to lyse contaminating red blood cells. After washing, the cells were resuspended in α-MEM supplemented with 15% FBS and 100 U/mL penicillin and 100 U/mL streptomycin (Invitrogen) and filtered through a 70-μm cell strainer (BD Biosciences, San Jose, CA, USA), transferred to a 175-cm2 culture flask (Greiner Bio-One, Essen, Germany) and kept at 37°C, 5% CO2 and 95% humidity. Culture medium was refreshed twice weekly. The cells were subcultured at 90% confluency and reseeded at a density of approximately 1000 cells/cm2. ASC were used for experiments between passage numbers 2 and 5.

Characterization of ASC

Cells isolated and expanded from adipose tissue were immunophenotyped by flow cytometric analysis using an eight-colour FACSCanto II and FACS Diva Software (BD Biosciences). They showed an expression of CD90, CD105 (R&D Systems, Abingdon, UK), CD166 (BD Biosciences) and HLA-ABC (Serotec, Düsseldorf, Germany), and were negative for CD14, CD34, CD45, CD86, HLA-DR (all BD Biosciences) and CD80 (Serotec), characteristic of an ASC phenotype. The cells were furthermore capable of osteogenic and adipogenic differentiation and had the ability to inhibit lymphocyte proliferation in mixed lymphocyte reactions, as previously described in detail 36.

Mixed lymphocyte reactions

PBMC were isolated from heparinized peripheral blood of healthy donors and isolated by density gradient centrifugation using Ficoll Isopaque (δ=1.077, Amersham, Uppsala, Sweden) and frozen at −135°C until use.

PBMC were labelled with the cell membrane dye PKH67 according to the manufacturer's instructions (Sigma-Aldrich) and stimulated with γ-irradiated (40 Gy) HLA-A, HLA-B and HLA-DR mismatched PBMC at a ratio of 1:1 in RPMI with 10% heat inactivated FBS. The allo-activated PBMC were added on day 6 to a confluent layer of allogeneic ASC to examine the interaction between PBMC and ASC. To examine the responsiveness of PBMC to IL-2, cells were stimulated for 30 min with 2000 U/mL IL-2 (Chiron, Amsterdam, The Netherlands) in the absence or presence of ASC.

Microscopy

ASC-PBMC co-cultures were washed with PBS and viewed and photographed using an Axiovert 200 microscope (Carl-Zeiss, Germany), equipped with an AxioCam MRm; MR3 camera. Where indicated, ASC were labelled with PKH67 according to the manufacturer's instructions.

Separation and flow cytometric analysis of adherent and non-adherent PBMC

Allo-activated PKH67-labelled PBMC were cultured for 6 days and then added to ASC. After 1, 24 or 72 h, non-adherent PBMC were collected and washed with PBS. ASC and adherent PBMC were washed with PBS and incubated in 0.05% trypsin-EDTA (Invitrogen). Non-adherent cells were incubated in 0.05% trypsin-EDTA for an equal period of time. After removal of the adherent cells from the culture dishes, both cell fractions were washed with PBS and stained for the lymphocyte subset markers CD3, CD4, CD8, CD19, CD16/56, TCR-αβ, TCR-γδ and the activation markers CD25, CD69, CD105 and CD127 and analysed on a FACSCanto II flow cytometer (BD Biosciences). The expression of FoxP3 was determined using a staining set according to the manufacturer's instructions (eBioscience, CA, USA). Cell viability was analysed used an Annexin V-PE apoptosis detection kit (BD Biosciences) according to the manufacturer's description.

Phosphospecific flow cytometry

PBMC were fixed for 15 min at room temperature by adding formaldehyde (Polysciences, Warrington, PA, USA) to the culture medium to a final concentration of 4%. Pelleted cells were resuspended in 1 mL of ice-cold 90% methanol and incubated on ice for 30 min. Cells were washed twice in BD FACSFlow buffer (BD Biosciences) with 0.5% BSA. Phosphorylation of STAT5 in different lymphocyte subpopulations was determined by six-colour flow cytometry using the following mouse monoclonal antibodies according to the manufacturer's specifications: Phospho-STAT5(Y694)-PE, CD3-PERPC, CD8-APC, CD4-Pacific Blue and CD25-PE/Cy7 (BD Biosciences). For the detection of phosphorylated SMAD1/5/8, cells were incubated with CD105-FITC, CD3-PERPC, CD8-APC, CD4-Pacific Blue and CD25-PE/Cy7 (BD Biosciences) and an unconjugated rabbit monoclonal antibody against phospho-Smad1(Ser463/464)/Smad5(Ser463/465)/Smad8(Ser426/428) (Cell Signaling Technology, Danvers, MA, USA). Next, cells were washed twice with BD FACSFlow buffer with 0.5% BSA and incubated with a PE-conjugated goat-anti rabbit secondary antibody (Invitrogen). After washing in BD FACSFlow buffer, cells were analysed on an FACSCantoII flow cytometer (BD Biosciences) for data analysis. Ten thousand gated lymphocyte events were acquired from each tube. MFI values were generated by analysing the data with Diva 6.0 software (BD Biosciences).

Statistical analysis

Significant changes between the different populations, time points or treatments were determined by the paired t-test. Calculations were performed using GraphPad Prism 5 software. A p-value<0.05 was considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Conflict of interest: The authors have declared no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
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