Oral mucosal progenitor cells are potently immunosuppressive in a dose-independent manner

Abstract

Oral mucosal lamina propria progenitor cells (OMLP-PCs) are a novel, clonally derived PC population of neural crest origin with the potential to differentiate down both mesenchymal and neuronal cell lineages. In this study we aimed to determine the immunological properties of OMLP-PCs and to establish whether they would be suitable candidates for allogeneic tissue engineering and in the treatment of immune-related diseases. OMLP-PCs demonstrated no inherent immunogenicity with insignificant expression of costimulatory molecules (CD40, CD80, CD86, CD154, and CD178) or human leukocyte antigen (HLA) class II. OMLP-PCs required 7 days of stimulation with interferon-γ (IFN-γ) to induce cell surface expression of HLA II. Mixed lymphocyte cultures and mitogen stimulation demonstrated the potent immunosuppressive capability of OMLP-PCs in a contact-independent manner. Complete inhibition of lymphocyte proliferation was seen at doses as low as 0.001% OMLP-PCs to responder lymphocytes, while annexin V staining confirmed that this immunosuppressive effect was not due to the induction of lymphocyte apoptosis. These data demonstrate, for the first time, that OMLP-PC immunomodulation, unlike that for mesenchymal stem cells, occurs via a dose- and HLA II-independent mechanism by the release of immunosuppressive soluble factors and suggests these cells may have wide ranging potential in future immune-related therapies.

FIG. 1.

Oral mucosal lamina propria progenitor cells (OMLP-PCs) constitutively express human leukocyte antigen (HLA) class I but not HLA class II. (a) Flow cytometry was performed on OMLP-PCs cultured in (i–ii) the absence and (iii–iv) presence of interferon-γ (IFN-γ) for 1 (solid line), 2 (dotted line), and 7 (dashed line) days for the expression of HLA class I and II. Ig controls are filled histograms. (b) Western blotting for HLA class II on expanded OMLP-PCs cultured in the presence/absence of IFN-γ for 1, 2, and 7 days.

FIG. 2.

OMLP-PCs suppress the proliferation of lymphocytes in a dose- and HLA-independent manner. OMLP-PCs (10%–0.001% to responder cells)±prestimulation with IFN-γ for 7 days were cocultured with peripheral blood lymphocyte (PBLs) for 5 days. Responder cells (A) were cocultured with irradiated (x) (a) A cells or (b) PBLs pooled from 5 different donors (Px) as baseline and positive controls, respectively. Data are expressed as average counts per minute (CPM)±standard deviation (SD) of the mean (n=4 clones); ***P≤0.001.

FIG. 3.

OMLP-PCs suppress the proliferation of lymphocytes stimulated by the T cell mitogen phytohemagglutinin (PHA) in a dose- and HLA-independent manner. OMLP-PCs±prestimulation with IFN-γ for 7 days were cocultured with PHA-stimulated PBLs for 3 days. PHA-stimulated controls have been set at 100% to account for variation between experiments. Data are expressed as average percentage proliferation compared with A+PHA controls±SD of the mean (n=3); ***P≤0.001.

FIG. 4.

OMLP-PCs suppress the proliferation of lymphocytes through the release of soluble factors. OMLP-PCs±prestimulation with IFN-γ for 7 days were cocultured with PBLs in direct contact and in Transwell systems for 5 days. Responder cells (A) or A cells cocultured with PBLs pooled from 5 different donors (Px) acted as baseline and positive controls, respectively. Data are expressed as average CPM±SD of the mean (n=3); ***P≤0.001.

FIG. 5.

OMLP-PCs do not immunosuppress through the induction of PBL cell death. (a) OMLP-PCs±prestimulation with IFN-γ for 7 days were cocultured with PBLs in a one-way mixed lymphocyte culture (MLC) for 5 days and PBL viability was assessed by trypan blue exclusion. Responder cells (A) cocultured with PBLs pooled from 5 different donors (Px) acted as the baseline control. Data are expressed as average percentage viable cells to control (A+Px)±SD of the mean (n=3). (b) OMLP-PCs±prestimulation with IFN-γ for 7 days were cocultured with PBLs and PHA for 3 days. Apoptotic and necrotic PBLs were detected by flow cytometry using an annexin V-FITC antibody and LIVE/DEAD^® fixable far-red dead cell stain kit (Invitrogen). Apoptotic cells were defined as annexin V⁺ LIVE/DEAD^®− and necrotic cells annexin V⁺ LIVE/DEAD^®+. Data are expressed as average percentage apoptotic/necrotic PBLs±SD of the mean (n=3); *P≤0.05.

FIG. 6.

OMLP-PCs induce the expression and activation of indoleamine 2, 3-dioxygenase (IDO) in response to stimulation with IFN-γ and/or coculture with PBLs. (a) Induction of IDO expression within OLMP-PCs by IFN-γ was assessed over 1, 2, and 7 days of stimulation by western blotting. (b) IDO activity was determined by measurement of l-kynurenine levels within the conditioned media of OMLP-PCs (±prestimulation with IFN-γ for 7 days) cultured±PBLs for 5 days in contact and Transwell systems. Data within (b) are expressed as average l-kynurenine (μM)±SD of the mean (n=3); **P≤0.01; ***P≤0.001.