Endometrial mesenchymal stem/stromal cell modulation of T cell proliferation

in Reproduction
Correspondence should be addressed to J A Deane; Email: james.deane@hudson.org.au

*(X Yang and M Devianti contributed equally to this work)

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Perivascular mesenchymal stem/stromal cells can be isolated from the human endometrium using the surface marker SUSD2 and are being investigated for use in tissue repair. Mesenchymal stem/stromal cells from other tissues modulate T cell responses via mechanisms including interleukin-10, prostaglandin E2, TGF-β1 and regulatory T cells. Animal studies demonstrate that endometrial mesenchymal stem/stromal cells can also modify immune responses to implanted mesh, but the mechanism/s they employ have not been explored. We examined the immunomodulatory properties of human endometrial mesenchymal stem/stromal cells on lymphocyte proliferation using mouse splenocyte cultures. Endometrial mesenchymal stem/stromal cells inhibited mitogen-induced lymphocyte proliferation in vitro in a dose-dependent manner. Inhibition of lymphocyte proliferation was not affected by blocking the mouse interleukin-10 receptor or inhibiting prostaglandin production. Endometrial mesenchymal stem/stromal cells continued to restrain lymphocyte proliferation in the presence of an inhibitor of TGF-β receptors, despite a reduction in regulatory T cells. Thus, the in vitro inhibition of mitogen-induced lymphocyte proliferation by endometrial mesenchymal stem/stromal cells occurs by a mechanism distinct from the interleukin-10, prostaglandin E2, TGF-β1 and regulatory T cell-mediated mechanisms employed by MSC from other tissues. eMSCs were shown to produce interleukin-17A and Dickkopf-1 which may contribute to their immunomodulatory properties. In contrast to MSC from other sources, systemic administration of endometrial mesenchymal stem/stromal cells did not inhibit swelling in a T cell-mediated model of skin inflammation. We conclude that, while endometrial mesenchymal stem/stromal cells can modify immune responses, their immunomodulatory repertoire may not be sufficient to restrain some T cell-mediated inflammatory events.

 

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    Purification and expansion of human perivascular SUSD2+ endometrial MSC. (A) The W5C5 antibody recognises SUSD2+ perivascular cells (red, arrows) in the human endometrium. g, gland. Inset shows an isotype control to demonstrate antibody specificity. (B, C and D) SUSD2 antibody-purified and cultured eMSC retain SUSD2 expression as detected by immunostaining (B, red), or by flow cytometry (D). An isotype control (C) demonstrates antibody specificity. Nuclei are stained with Hoechst in A and B. Scale bars = 50 µm.

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    Dose-dependent inhibition of lymphocyte proliferation in vitro by endometrial MSC. (A) Inhibition of lymphocyte proliferation by eMSC from a representative patient-derived culture. Black columns show ConA-mediated lymphocyte proliferation measured by incorporation of [3H]-thymidine. Basal levels of proliferation in the absence of ConA are shown by white columns. αIL-10R indicates the inclusion of an IL-10 receptor-blocking antibody and isotype indicates a control antibody. (B) Proliferation index (ConA+ cpm/ConA cpm) measured from five patient-derived eMSC cultures. Boxes show median and interquartile range; whiskers show minimum to maximum. *P < 0.05 and **P < 0.01 using Friedman’s test and Dunn’s post hoc test.

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    Lymphocyte colonies from cultured mouse splenocytes and the effects of eMSC, inhibition of prostaglandin synthesis and TGF-β signalling. Indomethacin was used to inhibit prostaglandin synthesis and A83-01 to inhibit TGF-β signalling. Mouse splenocytes cultured for 72 h without ConA (No ConA); with ConA (+ConA); with ConA and eMSC (+ConA +eMSC); with ConA, eMSC and indomethacin (+ConA +eMSC +indometh); with ConA, eMSC and A83-01 (+ConA +eMSC +A83-01). Images are representative of n = 7 for No ConA and +ConA; n = 5 for +ConA +eMSC) and +Con A +indometh 30 µM; n = 4 for +ConA +eMSC +indometh 60 µM, and n = 3 for +ConA +eMSC +A83-01.

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    CD4+ T cell proliferation and the effects of eMSC measured by CFSE dilution. (A and B) A gate for CFSE-labelled CD4+ T cells (rectangle in dot plot panels) was used to generate histograms of CD4+ counts vs CFSE intensity (lower panels). Arrows in histograms show peaks of undivided cells. (A) Splenocyte cultures without ConA (no ConA), with ConA (+ConA), or with ConA and eMSC (+ConA +eMSC 1:5). (B) Splenocyte/eMSC cocultures with ConA plus indomethacin or A83-01. Plots are representative of n = 7 for No ConA and +ConA; n = 5 for +ConA +eMSC 1:5 and +ConA +eMSC 1:5 +30 µM indometh; n = 4 for +ConA +eMSC 1:5 +60 µM indomethacin, and n = 3 for +ConA +eMSC 1:5 +A83-01.

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    Quantification of CD4+ T cell proliferation. CD4+ T cell expansion index (A) and replication index (B) were calculated from CFSE dilution experiments. Data are shown as average ± s.e.m. and open white columns show cocultures containing eMSC, 30µM indomethacin. n = 7 for untreated control without eMSC; n = 5 for eMSC, eMSC +30 µM indomethacin; n = 4 for eMSC +60 µM indomethacin, eMSC +A83-01, 60 µM indomethacin; n = 3 for A83-01. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 using one-way ANOVA and Tukey’s multiple comparisons test.

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    Quantification of regulatory T cells in splenocyte cultures. (A) CD4+FoxP3-GFP+ regulatory T cells (Treg) and CD4+FoxP3-GFP-conventional T cells (Tconv) were identified by flow cytometry. Spl, splenocytes; eMSC, endometrial mesenchymal stem cells. Plots shown are representative of n = 4 for each group. (B) The relative abundance of CD4+FoxP3+ Tregs is expressed as a percentage of the total CD4+ population including conventional T cells. Data are shown as average ± s.e.m. and open white columns show cocultures containing eMSC. n = 4 for all groups. Data were analysed using one-way ANOVA and Tukey’s multiple comparisons test. (C) eMSC vs eMSC +A83-01 analysed using a paired t test.

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    Profiling eMSC cytokines, chemokines and growth factors. (A) A human-specific membrane-based antibody array assay was used to assess factors secreted by two independent human eMSC lines (samples 1 and 2) cocultured with mouse splenocytes. Membrane arrays comparing eMSC/splenocyte cocultures from sample 1 without (left) or with ConA (right) are shown. Each factor is represented by a pair of antibody spots at a designated position on the array as labelled on the left panel. The pairs of spots on the top left, top right and bottom left are reference spots. (B) Spots from eMSC/splenocyte cocultures using samples 1 and 2 were quantified by measuring their integrated pixel density and each bar is an average of duplicate spots.

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    eMSC do not inhibit T cell-mediated ear inflammation. (A) A timeline of the mouse contact sensitivity model of ear swelling. (B) An uninflamed vehicle-challenged ear compared to an inflamed oxazolone-challenged ear. (C) Measurement of ear thickness difference between oxazolone and vehicle-treated ears. Data are shown as average ± s.e.m. and were analysed using an unpaired t test with P value shown for control vs eMSC. n = 13 for vehicle control and 11 for eMSC at 0–24 h. n = 11 for vehicle control and 8 for eMSC at 48 h.

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    Summary of the effects of eMSC and TGF-β signalling on lymphocyte proliferation. (A) TGF-β promotes the differentiation of regulatory T cells (Tregs) that inhibit T-lymphocyte (Tconv) proliferation. (B) eMSCs further inhibit lymphocyte proliferation via a mechanism independent of TGF-β signalling. (C) A83-01 increases lymphocyte proliferation by reducing TGF-β signalling-dependent Treg differentiation, but eMSCs continue to inhibit lymphocyte proliferation via a mechanism independent of TGF-β signalling. (D) Maximum lymphocyte proliferation occurs in the absence of TGF-β signalling-dependent Treg differentiation, and the absence of inhibition by eMSC.

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