Research ArticleTransplantation

Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation

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Science Translational Medicine  15 Nov 2017:
Vol. 9, Issue 416, eaam7828
DOI: 10.1126/scitranslmed.aam7828
  • Fig. 1. MSCs undergo in vivo apoptosis after infusion without affecting immunosuppression.

    (A) Luc-MSCs were injected intravenously into naïve, BM, and GvHD mice 3 days after transplantation. All animals were then injected intraperitoneally with Z-DEVD-aminoluciferin and imaged 1 hour later. n = 6 (one to three mice per group), grouped from three independent experiments. In each experiment, a different MSC expansion was used. White lines separate multiple photographs assembled in the final image. (B) TLS was measured from the images of mice in (A) and shown as mean ± SD. (C and D) Percentage of GvHD effector cells (CD8+Vβ8.3+) calculated in the lymphocyte gate (defined by the physical characteristics of the cells) in the spleen (C) and lungs (D) of GvHD mice (black circles) and GvHD mice treated with MSCs (black squares) 4 days after MSC injection. n = 15 (GvHD) and 13 (GvHD + MSCs) mice, grouped from four independent experiments. Means ± SD are shown. Statistics in (B): one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (**P < 0.01 and ***P < 0.001). Statistics in (C) and (D): unpaired t test (**P < 0.01). ns, not significant.

  • Fig. 2. MSC apoptosis is important for immunosuppression and requires functionally activated cytotoxic cells in the recipient.

    (A) The percentage of CD8+Vβ8.3+ cells in lung cell suspensions from naïve C57BL/6 male, BM, or GvHD mice was analyzed in the lymphocyte population. Means ± SD are shown. n = 12 (GvHD), 3 (BM), and 3 (naïve) mice, grouped from three independent experiments. (B) CD8+ cells were sorted from the lungs and spleens of naïve female Mh (gray bars) or GvHD mice (not treated with MSCs; white bars) 7 days after the transplant and tested for their ability to induce MSC apoptosis in vitro. The results show annexin-V+/7-AAD MSCs (mean ± SD) in three independent experiments (n = 10 per group; the black bar represents the value of apoptosis in MSCs cultured alone used as control (n = 3). (C) Luc-MSCs were infused in three independent experiments in GvHD (n = 7) and GvHDPerf−/− (n = 7) mice 3 days after transplantation. One hour later, mice were injected with Z-DEVD-aminoluciferin and imaged. White lines separate multiple photographs assembled in the final image. (D) TLS was obtained from (C) and expressed as mean ± SD. (E and F) The percentage of effector GvHD cells (CD8+Vβ8.3+) in the lymphocyte population was measured in the spleen (E) and lungs (F) of untreated GvHDPerf−/− (n = 16) and GvHDPerf−/− (n = 17) mice treated with MSCs (mean ± SD of four independent experiments). Statistics in (A) and (B): one-way ANOVA with Tukey’s multiple comparison test (*P < 0.05 and ***P < 0.001). Statistics in (D) to (F): unpaired t test (***P < 0.001).

  • Fig. 3. Cytotoxic activity against MSCs predicts clinical responses to MSCs in GvHD patients.

    (A and B) PBMCs obtained from healthy controls (HC) or patients with GvHD receiving MSCs in the following 24 hours were incubated in 24-well plates with MSCs at a PBMC/MSC ratio of 20:1 for 4 hours. Apoptosis was measured in MSCs assessing the percentage of annexin-V+/7-AAD cells by flow cytometry. (A) Representative plots for HC, clinical responders (R), and nonresponders (NR). Left panels show the background apoptosis of MSCs alone used in the corresponding cytotoxic assay. (B) Apoptosis was compared among HC (circles; n = 5), R (triangles; n = 5), and NR (squares; n = 12). Statistics: one-way ANOVA and Tukey’s multiple comparison test (***P < 0.0001).

  • Fig. 4. MSC apoptosis is mediated by activated CD8+ and CD56+ cytotoxic cells and is the result of a bystander effect.

    (A) PBMCs from healthy donors (each independent experiment used a different PBMC donor) were activated using phytohemagglutinin (PHA; PHA-aPBMCs) or MLR (MLR-aPBMCs). Resting PBMCs (gray bars), PHA-aPBMCs (black bars), or MLR-aPBMCs (dashed bars) were incubated with MSCs at the indicated ratios and MSC apoptosis (annexin-V+/7-AAD) calculated after 4 hours. ND, not done. (B) Apoptosis in MSCs cultivated with MLR-aPBMCs in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK (10 μM) or the corresponding concentration of its vehicle (dimethyl sulfoxide). (C and D) Apoptosis in MSCs cultivated with MLR-aPBMCs used as unfractionated or positively selected for CD11b+, CD4+, CD8+, or CD56+ cells (C) or depleted of CD56+, CD8+, or both (D). (E) Apoptosis in MSCs cultivated with MLR-aPBMCs in the presence or absence of the GrB inhibitor Z-AAD-CMK (300 μM) or the perforin inhibitor EGTA (4 mM). (F) Apoptosis in MSCs cultivated with PHA-aPBMCs in the presence or absence of neutralizing concentrations (10 and 100 μg/ml) of FAS-L monoclonal antibody anti-CD178. (G) Apoptosis in MSCs after culture with autologous (black bars) or allogeneic (gray bars) PHA-aPBMCs in the presence or absence of neutralizing doses of anti–HLA-A, anti–HLA-B, anti–HLA-C, or anti-HLA-DR antibodies. The white bar shows spontaneous apoptosis in MSCs plated alone. (H) Apoptosis in MSCs cultivated with MLR-aPBMCs in direct contact or in a Transwell. (I) Apoptosis in MSCs cultivated with PHA-aPBMCs in the presence or absence of escalating doses (10 to 75 μM) of PKCζ-PS (protein kinase Cζ–pseudosubstrate). In (B) to (I), the PBMC/MSC ratio was 20:1. Results represent the mean ± SD of three or six (H) independent experiments. Statistics: one-way ANOVA with Tukey’s multiple comparison test (*P < 0.5, **P < 0.01, and ***P < 0.001).

  • Fig. 5. MSCs do not compete with cytotoxic cell recognition of the cognate target.

    (A) Apoptosis in T2 cells after culture with 4D8 cells at a 4D8/T2 ratio of 20:1. Where indicated, increasing concentrations of MSCs (used as cold target) were added. Apoptotic T2 cells were identified as annexin-V+/7-AAD+ cells. (B) Apoptosis in K562 cultured with NK cells (NK/K562 ratio of 20:1). Where indicated, increasing concentrations of MSCs (used a cold target) were added. (C) Apoptosis in MSCs cultured with 4D8 cells (4D8/MSC ratio of 20:1). Where indicated, increasing concentrations of T2 cells (used as cold target) were added. (D) Apoptosis in MSCs cultured with NK cells at an NK/MSC ratio of 20:1. Where indicated, increasing concentrations of K562 (used as cold target) were added. In all experiments, apoptosis of MSCs, T2 cells, or K562 cells was assessed after 4 hours of coculture by flow cytometry. Results represent the mean ± SD of three independent experiments. Statistics: one-way ANOVA and Tukey’s multiple comparison test (*P < 0.05).

  • Fig. 6. Apoptotic MSCs exert in vivo immunosuppression in a TH2-type inflammation model in the absence of cytotoxic cells.

    (A) Luc-MSCs were injected into naïve (n = 3) and OVA + MSCs (n = 6) mice 1 hour after the last challenge. One hour later, mice received Z-DEVD-aminoluciferin and were imaged in three independent experiments. White lines separate multiple photographs assembled in the final image. (B) TLS was measured from (A) (mean ± SD). (C) Eighteen hours after MSC infusion, eosinophil infiltration was assessed in the BAL of naïve (n = 3) and those infused with MSCs (n = 3), OVA (n = 6), and OVA + MSCs (n = 6) in two independent experiments. Means ± SD are shown. (D) Eosinophil infiltration (mean ± SD) evaluated in BAL cytospin preparations from OVA-sensitized mice treated with apoMSCs. Groups were as follows: OVA without apoMSCs (n = 6), OVA treated with apoMSCs (1 × 106; n = 7), and naïve mice receiving apoMSCs (1 × 106; n = 2). Results represent the mean ± SD of three independent experiments. Statistics in (B): unpaired t test. Statistics in (C) and (D): one-way ANOVA and Tukey’s multiple comparison test (*P < 0.05).

  • Fig. 7. ApoMSCs exert immunosuppressive activity in GvHD and elicit IDO in engulfing recipient phagocytes.

    (A to D) The percentage of GvHD effector cells was assessed in the lymphocyte gate in the spleen (A and C) and lungs (B and D) of GvHD mice (black circles) and those treated with apoMSCs (black squares). ApoMSCs were infused intraperitoneally (ip; GvHD mice, n = 10; GvHD + apoMSCs mice, n = 8) (A and B), or intravenously (iv; GvHD mice, n = 9; GvHD + apoMSCs mice n = 7) (C and D). Results represent the mean ± SD of three independent experiments. Statistics: unpaired t test (*P < 0.05 and **P < 0.01). (E to K) MSCs were labeled using CellTrace Violet and subjected to apoptosis induction using GrB/anti-FAS (5 and 10 μg/ml, respectively). ApoMSCs were injected intraperitoneally (E, F, and J) or intravenously (G to I and K) into GvHD mice 3 days after the transplant. After 2 hours, animals were sacrificed, and mesenteric lymph nodes (LN) (E, F, and J) or lungs (G to I and K) were harvested. Cells engulfing apoMSCs were identified as Violet+ cells within the CD11b+ (E), CD11c+ (F), CD11bhighCD11cint (G), CD11c+CD11b (H), and CD11bhighCD11c (I) subpopulations. The corresponding subpopulations were gated in GvHD mice, which had not received Violet-labeled apoMSCs and used as controls. IDO expression was assessed in CD11c+ and CD11b+ (J) or CD11bhighCD11cint, CD11c+CD11b, and CD11bhighCD11c (K) cells positive for CellTrace Violet (engulfing apoMSCs) and compared with the corresponding populations in GvHD mice that had not received apoMSCs. Data are representative of similar results obtained from three mice in two independent experiments.

  • Fig. 8. Recipient phagocytes and IDO production are required for MSC immunosuppressive activity in GvHD.

    (A and B) GvHD mice were treated with liposomal clodronate 10 min after the transplant. Where indicated, MSCs were infused 3 days later. The percentage of GvHD effector cells (CD8+Vβ8.3+) was calculated in the lymphocyte gate in the spleen (A) or lungs (B) after 4 additional days. Mean ± SD was obtained by grouping three independent experiments with n = 12 (GvHD) and 10 (GvHD + MSCs) mice per group. (C and D) GvHD effector cell infiltration was studied in the spleen (C) and lungs (D) of GvHD mice treated with the IDO inhibitor 1-DMT. In the treated mice, MSCs were infused 3 days after the transplant (n = 11). Controls consisted of GvHD mice that did not receive MSCs (n = 9). Percentage of CD8+Vβ8.3+ cells refers to the lymphocyte population. Results refer to the mean ± SD of three independent experiments. Statistics: unpaired t test (*P < 0.05 and **P < 0.01).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/416/eaam7828/DC1

    Materials and Methods

    Fig. S1. MSCs can be traced in the lungs of mice after infusion.

    Fig. S2. Human MSC immunosuppression is not licensed by murine cytokines.

    Fig. S3. MSC apoptosis is induced by cytotoxic cells.

    Fig. S4. Cytotoxicity against MSCs varies among PBMC donors but is independent on the percentage of CD8+ or CD56+ in GvHD patients.

    Fig. S5. MSC killing is mediated by caspase 3 and effected by GrB and perforin.

    Fig. S6. Infused MSCs can be imaged in the lungs of mice with TH2-type lung inflammation.

    Table S1. Clinical features of GvHD patients.

    Table S2. Primary data.

    Video S1. Living-cell imaging of fluorescence resonance energy transfer (FRET)–MSCs plated alone.

    Video S2. Living-cell imaging of FRET-MSCs plated with PHA-aPBMCs.

    Video S3. Living-cell imaging of FRET-MSCs plated with resting PBMCs.

    Video S4. Living-cell imaging of FRET-MSCs plated with PHA-aPBMCs in the presence of the pan-caspase inhibitor Z-VAD-FMK.

    Video S5. Living-cell imaging of FRET-MSCs plated with PHA-aPBMCs in the presence of the GrB inhibitor Z-AAD-CMK.

    Video S6. Living-cell imaging of FRET-MSCs plated with PHA-aPBMCs in the presence of the perforin inhibitor EGTA.

    References (5557)

  • Supplementary Material for:

    Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation

    Antonio Galleu, Yanira Riffo-Vasquez, Cristina Trento, Cara Lomas, Luigi Dolcetti, Tik Shing Cheung, Malte von Bonin, Laura Barbieri, Krishma Halai, Sophie Ward, Ling Weng, Ronjon Chakraverty, Giovanna Lombardi, Fiona M. Watt, Kim Orchard, David I. Marks, Jane Apperley, Martin Bornhauser, Henning Walczak, Clare Bennett, Francesco Dazzi*

    *Corresponding author. Email: francesco.dazzi{at}kcl.ac.uk

    Published 15 November 2017, Sci. Transl. Med. 9, eaam7828 (2017)
    DOI: 10.1126/scitranslmed.aam7828

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. MSCs can be traced in the lungs of mice after infusion.
    • Fig. S2. Human MSC immunosuppression is not licensed by murine cytokines.
    • Fig. S3. MSC apoptosis is induced by cytotoxic cells.
    • Fig. S4. Cytotoxicity against MSCs varies among PBMC donors but is independent on the percentage of CD8+ or CD56+ in GvHD patients.
    • Fig. S5. MSC killing is mediated by caspase 3 and effected by GrB and perforin.
    • Fig. S6. Infused MSCs can be imaged in the lungs of mice with TH2-type lung inflammation.
    • Table S1. Clinical features of GvHD patients.
    • Table S2. Primary data.
    • Legends for videos S1 to S6
    • References (5557)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Video S1 (.avi format). Living-cell imaging of fluorescence resonance energy transfer (FRET)–MSCs plated alone.
    • Video S2 (.avi format). Living-cell imaging of FRET-MSCs plated with PHA-aPBMCs.
    • Video S3 (.avi format). Living-cell imaging of FRET-MSCs plated with resting PBMCs.
    • Video S4 (.avi format). Living-cell imaging of FRET-MSCs plated with PHA-aPBMCs in the presence of the pan-caspase inhibitor Z-VAD-FMK.
    • Video S5 (.avi format). Living-cell imaging of FRET-MSCs plated with PHA-aPBMCs in the presence of the GrB inhibitor Z-AAD-CMK.
    • Video S6 (.avi format). Living-cell imaging of FRET-MSCs plated with PHA-aPBMCs in the presence of the perforin inhibitor EGTA.

    [Download Videos S1 to S6]

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