A CD8 T cell/indoleamine 2,3-dioxygenase axis is required for mesenchymal stem cell suppression of human systemic lupus erythematosus

Dandan Wang, Xuebing Feng, Lin Lu, Joanne E Konkel, Huayong Zhang, Zhiyong Chen, Xia Li, Xiang Gao, Liwei Lu, Songtao Shi, Wanjun Chen, Lingyun Sun, Dandan Wang, Xuebing Feng, Lin Lu, Joanne E Konkel, Huayong Zhang, Zhiyong Chen, Xia Li, Xiang Gao, Liwei Lu, Songtao Shi, Wanjun Chen, Lingyun Sun

Abstract

Objective: Allogeneic mesenchymal stem cells (MSCs) exhibit therapeutic effects in human autoimmune diseases such as systemic lupus erythematosus (SLE), but the underlying mechanisms remain largely unknown. The aim of this study was to investigate how allogeneic MSCs mediate immunosuppression in lupus patients.

Methods: The effects of allogeneic umbilical cord-derived MSCs (UC-MSCs) on inhibition of T cell proliferation were determined. MSC functional molecules were stimulated with peripheral blood mononuclear cells from healthy controls and SLE patients and examined by real-time polymerase chain reaction. CD4+ and CD8+ T cells were purified using microbeads to stimulate MSCs in order to determine cytokine expression by MSCs and to further determine which cell subset(s) or which molecule(s) is involved in inhibition of MSC-mediated T cell proliferation. The related signaling pathways were assessed. We determined levels of serum cytokines in lupus patients before and after UC-MSC transplantation.

Results: Allogeneic UC-MSCs suppressed T cell proliferation in lupus patients by secreting large amounts of indoleamine 2,3-dioxygenase (IDO). We further found that interferon-γ (IFNγ), which is produced predominantly by lupus CD8+ T cells, is the key factor that enhances IDO activity in allogeneic MSCs and that it is associated with IFNGR1/JAK-2/STAT signaling pathways. Intriguingly, bone marrow-derived MSCs from patients with active lupus demonstrated defective IDO production in response to IFNγ and allogeneic CD8+ T cell stimulation. After allogeneic UC-MSC transplantation, serum IDO activity increased in lupus patients.

Conclusion: We found a previously unrecognized CD8+ T cell/IFNγ/IDO axis that mediates the therapeutic effects of allogeneic MSCs in lupus patients.

Trial registration: ClinicalTrials.gov NCT01741857.

Copyright © 2014 by the American College of Rheumatology.

Figures

Figure 1
Figure 1
Umbilical cord–derived mesenchymal stem cells (UC-MSCs) inhibit lupus T cell proliferation, but not via conversion to induced Treg cells. A, UC-MSCs inhibit proliferation of T cells from patients with systemic lupus erythematosus (SLE) and healthy controls (HCs). B, UC-MSCs inhibit proliferation of CD4+CD25− responder T (Tresp) cells from patients with lupus patients and healthy controls. In A and B, ∗∗∗ = P < 0.001 by one-way analysis of variance (ANOVA) followed by Bonferroni test. C and D, UC-MSCs promote, rather than inhibit, proliferation of CD4+CD25+ Treg cells from lupus patients (C) and increase the absolute number of CD4+CD25+ Treg cells (D). Symbols represent individual subjects; horizontal lines show the mean (n = 6 per group in C and D). ∗∗ = P < 0.01; ∗ = P < 0.05, by t-test. E, UC-MSCs markedly inhibit, rather than induce, differentiation of Treg cells from lupus patients and healthy controls. ∗∗ = P < 0.01 by one-way ANOVA followed by Bonferroni test. Bars in A, B, and E show the mean ± SEM (n = 4 per group). HLF = human lung fibroblast; CFSE = carboxyfluorescein succinimidyl ester; iTreg = induced Treg cells.
Figure 2
Figure 2
Indoleamine 2,3-dioxygenase (IDO) is key to UC-MSC–mediated inhibition of lupus T cell proliferation. A, T cell receptor–treated peripheral blood mononuclear cells from patients with active SLE (sPBMC) stimulate significant up-regulation of IDO gene expression by UC-MSCs. B, Levels of kynurenine in supernatant are increased in the presence of PBMCs from SLE patients. C, IDO protein levels produced by UC-MSCs are increased in the presence of PBMCs from SLE patients, as shown by Western blot analysis. Results are representative of 3 separate experiments. D, Treatment with 1-methyl-dl-tryptophan (DL-MT) (0.4 μM) significantly blocks UC-MSC–mediated inhibition of CD4+ T cell proliferation. Bars in A, B, and D show the mean ± SEM (n = 5 per group in A, 5 per group in B, and 4 per group in D). ∗ = P < 0.05; ∗∗ = P < 0.01, by one-way ANOVA followed by Bonferroni test. hPBMC = healthy control PBMCs (see Figure 1 for other definitions).
Figure 3
Figure 3
Lupus CD8+ T cells promote indoleamine 2,3-dioxygenase (IDO) activity in UC-MSCs. A, The CD8+ T cell subset from patients with active SLE is the most important factor involved in enhancing IDO mRNA expression in UC-MSCs. B, Supernatant kynurenine levels are increased in the presence of SLE CD8+ T cells, as assessed by high-performance liquid chromatography. C, Up-regulation of IDO mRNA expression in UC-MSCs is not dependent upon cell–cell contact. Bars show the mean ± SEM (n = 7 per group in A, 7 per group in B, and 4 per group in C). ∗∗∗ = P < 0.001 by one-way ANOVA followed by Bonferroni test. NS = not significant (see Figure 1 for other definitions).
Figure 4
Figure 4
Lupus CD8+ T cell–derived interferon-γ (IFNγ) promotes indoleamine 2,3-dioxygenase (IDO) production. A and B, Levels of IFNγ in supernatant are increased in the presence of UC-MSCs and SLE CD8+ T cells (A), while no change in levels of transforming growth factor β (TGFβ) is observed (B). Symbols represent individual subjects; horizontal lines show the mean (n = 7 per group). C, CD8+ T cells derived from patients with SLE produce much higher levels of intracellular IFNγ as compared to other cell subsets. D, Recombinant anti-human IFNγ antibody significantly abrogates SLE CD+ T cell–mediated IDO mRNA expression in UC-MSCs. E, Kynurenine levels in supernatant decrease in the presence of anti-human IFNγ antibody. F–H, In cocultured lupus peripheral blood mononuclear cells (PBMCs) and UC-MSCs, anti-human IFNγ antibodies (10 μg/ml) significantly inhibit kynurenine levels in supernatant (F), while the level of tryptophan is increased (G), and the ratio of kynurenine to tryptophan is decreased (H) (effects similar to those observed when 1-methyl-dl-tryptophan [1-DL-MT] is added). I, In the coculture system described above, UC-MSC–mediated inhibition of CD4+ T cell proliferation is abrogated by the addition of anti-human IFNγ antibody. Bars in C–I show the mean ± SEM (n = 7 per group in C, 8 per group in D, 8 per group in E, 5 per group in F–H, and 5 per group in I). ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by one-way ANOVA followed by Bonferroni test. PE = phycoerythrin; FITC = fluorescein isothiocyanate; APC = allophycocyanin (see Figure 1 for other definitions). Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38674/abstract.
Figure 5
Figure 5
Defective indoleamine 2,3-dioxygenase (IDO) production in lupus bone marrow–derived MSCs (BM-MSCs). A–C, Expression of IFNGR1 (A), JAK-2 (B), and STAT-1 (C) was examined by real-time quantitative polymerase chain reaction. D, STAT-1, STAT-3, STAT-5, and their phosphorylated forms were assessed by Western blot analysis after treatment with UC-MSCs alone, UC-MSCs with different cell subsets (CD4+, CD8+, and non-CD4/CD8 T cells), or recombinant human interferon-γ (IFNγ). E, IDO mRNA expression in UC-MSCs, BM-MSCs from healthy controls, and BM-MSCs from SLE patients was examined after stimulation with different cytokines for 48 hours. F and G, Peripheral blood CD8+ T cells derived from patients with lupus were purified and used to stimulate UC-MSCs and BM-MSCs from healthy controls or lupus patients. Forty-eight hours later, IDO gene expression (F) and kynurenine enzyme activity (G) were determined. H, The ability of BM-MSCs from healthy controls and lupus patients to inhibit CD4+ T cell proliferation was compared. Bars in A–C and E–H show the mean ± SEM (n = 7 per group in A–D, 6 per group in E, 3 per group in F and G, and 4 per group in H). ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by one-way ANOVA. TGFβ = transforming growth factor β; IL-6 = interleukin-6; PBMC = peripheral blood mononuclear cell; NS = not significant (see Figure 1 for other definitions).
Figure 6
Figure 6
Serum indoleamine 2,3-dioxygenase (IDO) activity increases after UC-MSC transplantation in lupus patients. Peripheral blood mononuclear cells were isolated from lupus patients and healthy controls, and levels of CD8+ T cell subsets and interferon-γ (IFNγ) were compared. A–C, The percentage (A) and total number (B) of CD8+CD4−/CD3+ T cells, as well as serum levels of IFNγ (C) are significantly increased in SLE patients. D and E, The percentage (D) and total number (E) of intracellular IFNγ+CD8+ T cells are also increased in SLE patients. F–H, Serum kynurenine levels are increased (F), tryptophan levels remain unchanged (G), and the ratio of kynurenine to tryptophan is increased (H) 1 month after UC-MSC transplantation in lupus patients. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. Symbols in A–E represent individual subjects; horizontal lines show the mean. See Figure 1 for other definitions.

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