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. 2006 Jun 15;107(12):4817-24.
doi: 10.1182/blood-2006-01-0057. Epub 2006 Feb 21.

Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma

Jennifer L Chan  1 , Katherine C Tang, Anoop P Patel, Larissa M Bonilla, Nicola Pierobon, Nicholas M Ponzio, Pranela Rameshwar
Affiliations

Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma

Jennifer L Chan et al. Blood. .

Abstract

Mesenchymal stem cells (MSCs) are mostly found around the vasculature system of the adult bone marrow (BM). They function as immune suppressors, express MHC-II, are phagocytic, and support T-cell cytotoxicity. We hypothesize that these contradictory properties of MSCs are important for BM homeostasis and occur partly through antigen presentation (antigen-presenting cells [APCs]) within a narrow window. Indeed, we have verified APC functions of MSCs to recall antigens, Candida albicans and Tetanus toxoid. The target cells have been identified to be CD4(+) T cells. APC assays with IFNgamma-knockdown MSCs and with anti-IFNgamma receptor confirmed that MHC-II expression requires autocrine stimulation by IFNgamma. During APC functions, as IFNgamma levels become elevated, there was a concomitant decrease in MHC-II on MSCs. This observation was correlated with flow cytometry studies showing a gradual decrease in MHC-II expression as IFNgamma levels were increased. The reduced levels of MHC-II correlated with losses in their allogeneic potential, as indicated in mixed lymphocyte reaction. In summary, endogenous and low levels of IFNgamma are required for MHC-II expression on MSCs, and for APC functions. APC functions occur during a narrow window before IFNgamma levels are increased. The study has implications for BM protection against infection and exacerbated inflammatory responses.

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Figures

Figure 1.
Figure 1.
Phagocytic and APC functions of MSCs. Macrophages and MSCs were treated with fluorescence microspheres for 2 hours and then examined by confocal microscopy. Three planes are shown in representative microscopic studies from 4 experiments, each performed with cells from different donors: (A) macrophages; (B) MSCs. Activated CD4+ cells were immunoselected from PBMCs that were challenged for 5 days with 10 μg/mL C albicans (C) or T toxoid, at 1:100 final dilution (D). Control studies were done in parallel in media alone (Unactivated CD4+). Activated CD4+ cells were added to autologous MSCs or macrophages, and exposed overnight to C albicans or T toxoid (Pulsed), or to media alone (Unpulsed). Cell proliferation was determined by [methyl-3H]-TdR incorporation, and the results were expressed as stimulation indices (SIs); mean ± SD (n = 6). Background disintegrations per minute (unactivated CD4+ and activated CD4+) were 308 ± 8 and 140 ± 4, respectively. *P < .05 versus unactivated CD4+ cells/pulsed and activated/unpulsed cultures. **P < .05 versus pulsed MSCs + activated CD4+ cells.
Figure 2.
Figure 2.
Effects of IFNγ concentrations on the expressions of MHC-II and IFNγRI on MSCs. Cells were stimulated with 10 U/mL or 100 U/mL IFNγ. After 4, 8, 12, and 16 hours, MSCs were studied for (A) MHC-II and (B) IFNγRI expression by flow cytometry. The results represent 4 experiments, each performed with MSCs from a different donor. The MFIs ± SD (n = 4) are shown for MHC-II in the panels. Solid histograms indicate isotype; open histograms, specific antibodies.
Figure 3.
Figure 3.
Role of endogenous IFNγ in MHC-II expression. (A) Northern blots for IFNγ using total RNA from MSCs derived from 3 different BM donors. Normalization was done with a cDNA probe for 18S rRNA. (B) Representative osteogenic and chondrogenic differentiation with MSCs, stably knocked down for IFNγ. (C) Representative flow cytometry for MHC-II in MSCs knocked down for IFNγ. (D) IFNγ (10 U/mL) was re-added to cultures of knockdown MSCs. After 24 hours, the cells were studied for MHC-II by flow cytometry. Figure represent 4 experiments, each with a different donor.
Figure 4.
Figure 4.
IFNγ regulates the allostimulator activity of MSC. MLR with MSC stimulators, pretreated with IFNγ (A), or knockdown for IFNγ (B). (A) MSCs from 7 different donors were incubated with 100 U/mL IFNγ for 8 hours and then used as stimulators in MLR. MSCs from each donor were studied with PBMCs from 2 different donors. (B) MSCs from 5 different donors were stably knocked down for IFNγ using wild-type or mutant siRNA, then used as stimulators in MLR. Control MLR comprised untransfected MSCs or those stably transfected with vector alone (pPMSKH1). *P < .05 versus untreated MSCs. **P < .05 versus other experimental groups. Results are expressed as mean SI ± SE.
Figure 5.
Figure 5.
IFNγ levels in MHC-II expression and APC function of MSCs. (A) APC assays were established as for Figure 1B, in the presence or absence of 1 μg/mL anti-IFNγRI or isotype control. At different times, culture media were determined for IFNγ levels and the CD4+ cells, and flow cytometry was done with the adherent cells colabeled with FITC anti-CD105 (for MSC) and PE anti–HLA-DR. Figure represents 4 different experiments. (B) Studies were set up as for Figure 1B, except that MSCs were pretreated with IFNγ-RI antibody or isotype control. The results are presented as the mean ± SD (n = 5). Background disintegrations per minute (unactivated CD4+ and activated CD4+) were 577 and 653, respectively. *P < .05 versus cultures of pulsed/activated/isotype control. (C) MSCs were incubated for 2 or 12 hours with IFNγ at 10 or 100 U/mL IFNγ. After this, cells were washed and then used in APC assays. *P < .05 versus unstimulated or 10 U/mL IFNγ;**P < .05 versus similar cultures with 100 U/mL IFNγ.
Figure 6.
Figure 6.
An image that summarizes the relative functional responses of MSCs in the presence or absence of C albicans or T toxoid. The studies show APC properties heightened during a small window when IFNγ levels are less than 25 pg/mL. As IFNγ levels are increased, the efficiency of APC functions is decreased. Based on previous studies, the reduced efficiency could correlate with the MSCs switching roles as immune-suppressor cells.
See this image and copyright information in PMC

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