Figure 1.
Mycoplasma-infected MSC effectively inhibit MLR.
(A) MSC generated from the BM of PVG rats, subsequently found to be infected with M. hyorhinis, were added in tenfold dilutions at the start of co-cultures of 2×105 PVG.7B LNC and 2×105 irradiated BN LNC (gray bars). No MSC were added to the positive control (MLR, black bar). The basal proliferation of PVG.7B LNC in the absence of stimulus is also shown as negative control (neg ctrl, white bar). Values on the x-axis denote the common logarithm (log) of MSC:LNC ratios, i.e. 1∶10 (−1) through 1∶100 000 (−5). [3H]TTP incorporation was abrogated depending on the number of mycoplasma-infected MSC added. (B) MSC were added either at the start of MLR (dark gray bars), after 24 h (light gray) or 48 h (white), respectively, at 1∶100 (−2) or 1∶10 000 (−4) MSC:LNC ratios, and co-cultured for a total of 4 d. No MSC were added to the positive control (MLR). Mycoplasma-infected MSC fully inhibited the assay with at least 3 d of co-incubation at the 1∶100 MSC:LNC ratio. Representative data from at least three independent experiments are shown as the mean plus the standard error of the mean of triplicates. Statistical difference to the respective positive controls, • P<.05, •• P<.01.
Figure 2.
MLR is inhibited by MSC culture supernatant.
Inhibition of lymphocyte proliferation was reversed either by (A) filtration at 0.22 µm, (B) serial centrifugation at 100 000 g, or (C) heat-treatment at 60°C. (A) MSC-conditioned supernatant (MSC-sn; gray bars) was added at 1∶10 (v/v) dilution (22 µL added to 200 µL per well) at the start of mitogenic stimulation of 2×105 PVG.7B LNC with Con A (positive control with no MSC-sn added, black bar). MSC-sn was added either unfiltered or filtered with the indicated cut-off sizes. (B) MSC-sn was sedimented with the indicated centrifugal forces and durations (min) and added at 1∶10 (v/v) dilution at the start of allogeneic MLR (cf. Figure 1). The pellet fraction (MSC-pt) was obtained after centrifugation twice for 60 min at 100 000 g, resuspended and added at 1∶10 (v/v) dilution to the MLR. (C) Unprocessed MSC-sn or the resuspended pellet fraction described in panel B were treated at 30°C (light gray bars) and 60°C (dark gray) for 30 min, respectively, before adding to MLR (dilution 1∶10). Representative data of at least two independent experiments are shown as mean values plus the standard error of the mean of quadruplicates. Statistical difference to the respective positive controls, •• P<.01, ••• P<.001.
Figure 3.
A single mycoplasma-infected MSC can completely block the MLR.
(A) Mycoplasma-infected MSC were added at limiting dilution conditions at the start of MLR cultures with 2×105 responder cells. Proliferation was assessed in 9 replicate wells for each dilution. Wells with more than 70% or less than 15% of the [3H]TTP incorporation observed in the positive control (MLR without MSC) are shown as open (○) and filled circles (•), respectively. The log of MSC:LNC ratios are denoted. (B) Mycoplasma was detected in the supernatant of seven out of nine wells at the −6.5 dilution shown in panel A, and in the same wells (C) complete inhibition of MLR was observed. Positive (MLR) and negative (neg ctrl) controls are also shown (mean of triplicates). Results are representative of three independent experiments.
Figure 4.
Mycoplasma-treated or previously uninfected MSC show a markedly reduced ability to inhibit lymphocyte proliferation.
(A) Infected PVG.1U MSC were treated with Mynox reagent to eradicate the mycoplasma infection. Tenfold dilutions of untreated (dark gray bars) and treated MSC (light gray) were tested in allogeneic MLR. Mycoplasma-treated MSC, which tested negative for mycoplasma by PCR, inhibited MLR at 1∶10 (−1) but not at higher dilutions. Mycoplasma-infected MSC, on the other hand, effectively inhibited up to a cell ratio of 1∶106 (−6). Previously uninfected PVG.7B MSC (B) or the rat colon carcinoma cell line CC531s (C) were intentionally infected with M. hyorhinis by transfer of cell culture supernatant from the infected PVG.1U MSC line shown in panel A. Infection was verified by PCR after passage. MSC were added at the start of MLR, and CC531s cells were irradiated to prevent spontaneous proliferation and added at the start of lymphocyte culture with Con A. Proliferation was effectively inhibited by addition of M. hyorhinis-infected cells, but not by uninfected CC531s cells. Values on the x-axis denote the log of MSC:LNC and CC531s:LNC ratios, respectively. When infected cells were added at the highest dilutions, individual wells showed either full inhibition or a normal proliferative response (cf. Figure 3); e.g. when infected CC531s cells were added to the Con A culture, proliferation was detected in one of three (log dilution −5) and two of three (−6) replicates. Representative data from at least three independent experiments are shown as the mean plus the standard error of the mean of (A, B) quadruplicates or (C) triplicates.
Figure 5.
Mycoplasma-infected MSC inhibit T lymphocyte proliferation in vitro as measured by CFSE dilution.
Previously uninfected (light gray histograms) and intentionally infected PVG.7B MSC (dark gray; cf. Figure 4B) were added to CFSE-labeled, Con A-stimulated PVG.7B LNC (values to the left denote the log of MSC:LNC ratios). LNC alone were cultured either with (black histogram) or without (white) Con A as positive and negative controls, respectively. CFSE dilution indicating cell divisions was measured by flow cytometry after 3 d of incubation. Histogram plots (percent of maximum count) are representative of triplicates and show the fluorescence intensity of lymphocyte populations gated on CD3+CD4+CD8− (CD4 T cells) and CD3+CD4−CD8+ (CD8 T cells). The potent inhibition of lymphocyte proliferation by infected MSC measured by CFSE dilution cannot be explained by degradation of [3H]TTP. For the highest dilution (−6) of mycoplasma-infected MSC, individual cultures displayed either full inhibition or full proliferation (cf. Figure 3); one replicate in which full lymphocyte proliferation was detected is shown. Data are representative of three independent experiments.
Figure 6.
Mycoplasma infection spreads rapidly in MLR cultures.
Mycoplasma-positive MSC were cultured either alone (right panel) or together with PVG responder cells and irradiated BN cells (left panel) for 3 d. Supernatants were collected at the indicated time points and tested by PCR. The log dilutions of MSC are denoted; ctrl, no MSC added. Mycoplasma was detectable at considerably lower MSC concentrations when co-cultured with lymphocytes.
Figure 7.
Mycoplasma-infected MSC alter the cytokine profile of MLR and inhibit IFNγ production by T cells.
(A) Mycoplasma-positive MSC (PVG) were added at the start of MLR of PVG.7B and irradiated BN LNC (1×106 cells each) at the indicated dilutions (log [MSC:LNC]). The concentration of various cytokines was determined in the supernatant at the end of 6 d co-culture. IL-6 was markedly increased, while interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) were reduced by addition of MSC compared to the control MLR. Representative data from three independent experiments are shown as the average of duplicates. (B) Mycoplasma-infected MSC (PVG.7B) were added at the start of Con A-stimulated culture of PVG.7B LNC at the indicated tenfold dilutions. The fraction of IFNγ-expressing CD3+ T cells was determined after 24 h by intracellular staining (triplicates were pooled) and flow cytometry, as shown in (C). Cells were gated on CD3+ lymphocytes, numbers represent the percentage of gated cells. Data are representative of three independent experiments.