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. 2011 Oct 18;108(42):17384-9.
doi: 10.1073/pnas.1103650108. Epub 2011 Sep 29.

Mesenchymal stem cells impair in vivo T-cell priming by dendritic cells

Sabrina Chiesa  1 , Silvia Morbelli, Sara Morando, Michela Massollo, Cecilia Marini, Arinna Bertoni, Francesco Frassoni, Soraya Tabera Bartolomé, Gianmario Sambuceti, Elisabetta Traggiai, Antonio Uccelli
Affiliations

Mesenchymal stem cells impair in vivo T-cell priming by dendritic cells

Sabrina Chiesa et al. Proc Natl Acad Sci U S A. .

Abstract

Dendritic cells (DC) are highly specialized antigen-presenting cells characterized by the ability to prime T-cell responses. Mesenchymal stem cells (MSC) are adult stromal progenitor cells displaying immunomodulatory activities including inhibition of DC maturation in vitro. However, the specific impact of MSC on DC functions, upon in vivo administration, has never been elucidated. Here we show that murine MSC impair Toll-like receptor-4 induced activation of DC resulting in the inhibition of cytokines secretion, down-regulation of molecules involved in the migration to the lymph nodes, antigen presentation to CD4(+) T cells, and cross-presentation to CD8(+) T cells. These effects are associated with the inhibition of phosphorylation of intracellular mitogen-activated protein kinases. Intravenous administration of MSC decreased the number of CCR7 and CD49dβ1 expressing CFSE-labeled DC in the draining lymph nodes and hindered local antigen priming of DO11.10 ovalbumin-specific CD4(+) T cells. Upon labeling of DC with technetium-99m hexamethylpropylene amine oxime to follow their in vivo biodistribution, we demonstrated that intravenous injection of MSC blocks, almost instantaneously, the migration of subcutaneously administered ovalbumin-pulsed DC to the draining lymph nodes. These findings indicate that MSC significantly affect DC ability to prime T cells in vivo because of their inability to home to the draining lymph nodes and further confirm MSC potentiality as therapy for immune-mediated diseases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
MSC inhibit LPS-induced cytokine production and modulate gene expression and phosphorylation of kinases involved in the TLR4 pathway. (A) Levels of intracellular IL-12 produced by CD83+ control DC (gray bars) and DC exposed to MSC during activation with 10 μg/mL of LPS overnight (black bars; P = 0.033). Results are representative of five independent experiments (mean ± SD). (B) TNF-α and IL-10 levels by ctr-DC (gray bars) and DC exposed to MSC during LPS activation (black bars), were quantified by ELISA (**P = 0.007 and *P = 0.03, respectively). Data are expressed as picogram per milliliter. Results are representative of four and seven independent experiments, respectively (mean ± SD). (C) After TLR4 stimulation, DC exposed to MSC (black bars) significantly down-modulated MyD88, MAP2k3, and NFkB1 compared with control DC (gray bars), as demonstrated using the RT2 Profiler PCR Array mouse Toll-Like Pathway. One of three independent experiments is shown. Gene expression is represented as relative mRNA amount (fold-induction) compared with the control sample. (D) Western blots of intracellular Akt, ERK1/2, p38 proteins from ctr-DC and MSC-conditioned DC, stimulated and not stimulated with LPS for 30 and 60 min, are shown. Anti–β-tubulin was used as control. One of three independent experiments are shown. (E) Histograms show the ratio between the activated phosphorylated p38 MAPK (Thr180/Tyr182) and total unphosphorylated p38 MAPK in ctr-DC (gray bars) and in MSC-conditioned DC (black bars) after 0, 15, 30, and 45 min of LPS stimulation. Data shown are representative of one of three independent experiments.
Fig. 2.
Fig. 2.
Gene expression of TLRs in MSC and inhibition of cytokines production by DC with MSC supernatants. (A) TLRs expression was assessed by quantitative RT-PCR on unstimulated (white bar) and LPS-stimulated MSC (10 μg/mL) (black bar). Mean ± SD from three independent experiments with unstimulated and LPS-stimulated MSC is shown. To magnify the differences among the expression of different TLRs, particularly in the case of TLR1, TLR8, and TLR9, two different scales have been depicted in the left and right graphs. (B) Secreted levels of IL12p70, IL-10, and TNF-α by ctr-DC (white bars), DC exposed to MSC (black bars), DC exposed to supernatants of MSC (light gray bar), and DC exposed to supernatants of LPS-stimulated MSC (dark gray bar), were quantified by ELISA (IL-12p70: ***P = 2.30931E-05, ***P = 4.53805E-05, ***P = 2.18932E-05, respectively; TNF-α: **P = 0.004, **P = 0.008, ***P = 9,42621E-05, respectively; IL-10: ***P = 0.0003, ***P = 0.0005, **P = 0.005, respectively). Data are expressed as picogram per milliliter. Results are the mean of three independent experiments (mean ± SD).
Fig. 3.
Fig. 3.
MSC interfere with antigen presentation to CD8+ and CD4+ T cells. (A) Percentage, at 48 h, of CD69+ CFSE-gated CD8+ T cells primed by ctr-DC (Left) or MSC-conditioned DC (Right) (DC:CD8 = 1:5). (B) Proliferating CD8+ T cells primed by ctr-DC (Left) or MSC-conditioned DC (Right), as depicted by CFSE dilution at 48 (Upper) and at 72 h (Lower); numbers indicate the absolute values of cells recovered in standardized acquisitions. One of three independent experiments is shown. (C) The expression of three APM-associated molecules, Delta, MB1, and LMP-10 on Raji cells (Top), control DC (Middle), and MSC-conditioned DC (Lower) is depicted. One three independent experiments is shown. (D) Inhibition of naive CD4+ OVA-specific transgenic DO11.10 T cells cultured with ctr-DC (Left) or MSC-conditioned DC (Right) pulsed with increasing concentrations of pOVA323–339 (0.02 μM; 0.2 μM; 2 μM) (DC:CD4 = 1:8). Absolute numbers indicate the absolute values of cells recovered in standardized acquisitions (shown at the top of the histograms) as described in Materials and Methods. One of three independent experiments is shown.
Fig. 4.
Fig. 4.
MSC impair in vivo CD4+ T-cell priming by DC. (A) The draining lymph nodes from a control (Left) and a MSC-treated mouse (Right) are shown. (Scale bar, 0.5 cm.) One of three representative experiments is depicted. (B) Histograms show the difference in the absolute cell number (×106) of the draining lymph node from control mice (gray bars) and MSC-treated mice (black bars); *P = 0.016. Results are representative of three independent experiments (mean ± SD). (C) The histograms show the percentage of positive cells (CD4+, CD4+/CD25+, CD8+, B220+, B220+/CD138+, CD3−/DX5+) expressed as absolute cell number for each cell subset in the draining lymph node of control mice (gray bars) and MSC-treated mice (black bars), *P < 0.05 for CD4+ T cells. Results are representative of three independent experiments (mean ± SD). (D) CFSE profiles of KJ1-26-gated transgenic DO11.10 CD4+ T cells demonstrate the absolute numbers of proliferating cells in the draining lymph node of control mice (Left) and MSC-treated mice (Right). Absolute numbers indicate the absolute values of cells recovered in standardized acquisitions (shown at the top of the histograms). One of three representative experiments is depicted. (E) Intracellular staining for IFN-γ, IL-2, and TNF-α on proliferating CD4+DO11.10 T cells from the draining lymph node of control mice (Left) and MSC-treated mice (Right) are depicted. The percentage of transgenic DO11.10 CD4+ T cells producing cytokines is shown at the top of plots. One of three representative experiments is displayed. (F) The percentage (Left) and the absolute number (Right) of CFSE+CD11c+ DC recovered from draining lymph nodes are shown. *P = 0.0232 (Left); *P = 0.0353 (Right) (G) The percentage (Left) and the absolute number (Right) of CFSE+CD11c+ DC expressing CCR7 and CD49dβ1 recovered from draining lymph nodes are depicted. Control mice (white bars) and MSC-treated mice (black bars). **P = 0.0013 (Left); *P = 0.0195, **P = 0.008 (Right).
Fig. 5.
Fig. 5.
MSC impair DC migratory features. Graphs represent the number of DC released by the site of injection (A) and recruited to the draining lymph nodes (B) as measured by the detection of radioactivity released by 99mTc-HMPAO–labeled DC. Labeled DC was followed for 60 min after injection by scintigraphic imaging. Four different experimental conditions were studied: (i) negative control group represents mice exclusively subcutaneously immunized with OVA-pulsed DC (○, 6 mice); (ii) positive control group (•, 7 mice) indicates mice intravenously injected with DO11.10 T cells and subsequently immunized with OVA-pulsed DC; (iii) in vitro MSC-conditioned DC group (□, 6 mice) represents mice intravenously injected with DO11.10 T cells and, subsequently immunized with OVA-pulsed in vitro MSC conditioned-DC; (iv) in vivo MSC–conditioned DC group (■, 4 mice) designate mice intravenously injected with DO11.10 T cells, subsequently immunized with OVA-pulsed DC and intravenously injected with MSC 15 min later (arrow).
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