Equine Mesenchymal Stem Cells Influence the Proliferative Response of Lymphocytes: Effect of Inflammation, Differentiation and MHC-Compatibility

Abstract

Immunomodulation and immunogenicity are pivotal aspects for the therapeutic use of mesenchymal stem cells (MSCs). Since the horse is highly valuable as both a patient and translational model, further knowledge on equine MSC immune properties is required. This study analysed how inflammation, chondrogenic differentiation and compatibility for the major histocompatibility complex (MHC) influence the MSC immunomodulatory-immunogenicity balance. Equine MSCs in basal conditions, pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were co-cultured with either autologous or allogeneic MHC-matched/mismatched lymphocytes in immune-suppressive assays (immunomodulation) and in modified one-way mixed leukocyte reactions (immunogenicity). After co-culture, frequency and proliferation of T cell subsets and B cells were assessed by flow cytometry and interferon-ɣ (IFNɣ) secretion by ELISA. MSC-primed showed higher regulatory potential by decreasing proliferation of cytotoxic and helper T cells and B cells. However, MHC-mismatched MSC-primed can also activate lymphocytes (proliferative response and IFNɣ secretion), likely due to increased MHC-expression. MSC-chondro maintained their regulatory ability and did not increase their immunogenicity, but showed less capacity than MSC-primed to induce regulatory T cells and further stimulated B cells. Subsequent in vivo studies are needed to elucidate the complex interactions between MSCs and the recipient immune system, which is critical to develop safe and effective therapies.

Keywords: allogenic therapy; co-culture; flow cytometry; haplotype; horse; immunogenicity; immunomodulation; immunosuppression assay; mesenchymal stem cells; modified mixed leukocyte reaction.

Figure 3

Mean ± SEM of the relative frequency of different lymphocyte subsets in the immunomodulatory assays: (A) CD3+ T lymphocytes, (B) CD8+ cytotoxic T cells, (C) CD4+ helper T cells, (D) CD4+ CD25^high regulatory T cells, (E) CD3−Pan-Ig+ CD21+ B cells. Phytohemagglutinin (PHA)-activated PBLs were exposed in vitro to MSC-naïve (light grey bar), MSC-primed (medium grey bar) and MSC-chondro (dark grey bar). Co-cultures of MSCs and PBLs were autologous (n = 3) or allogeneic, matched (n = 8) or mismatched (n = 7) for the MHC. Data from each PBL donor is normalised over the positive control (CTRL+) consisting of PHA-stimulated PBLs alone from the same donor (value 1), to account for inter-individual variability. Significant differences of each condition over the CTRL+ (black bar) are represented by a cross (+) above the corresponding bar (+ = p < 0.05; +++ = p < 0.001). Significant differences over the negative control (CTRL−; non-activated PBLs alone, white bar) are represented by hashes (#) above the corresponding bar (# = p < 0.05; ## = p < 0.01; ### = p < 0.001). Significant differences between experimental conditions are represented by a squared line with an asterisk (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Figure 4

Mean ± SEM of the relative proliferation of different lymphocyte subsets in the immunomodulatory assays: (A) CD3+ T lymphocytes, (B) CD8+ cytotoxic T cells, (C) CD4+ helper T cells, (D) CD4+ CD25^high regulatory T cells, (E) CD3−Pan-Ig+ CD21+ B cells. Phytohemagglutinin (PHA)-activated PBLs were exposed in vitro to MSC-naïve (light grey bar), MSC-primed (medium grey bar) and MSC-chondro (dark grey bar). Co-cultures of MSCs and PBLs were autologous (n = 3) or allogeneic, matched (n = 8) or mismatched (n = 7) for the MHC. Data from each PBL donor is normalised over the positive control (CTRL+) consisting of PHA-stimulated PBLs alone from the same donor (100%, maximum proliferation) to account for inter-individual variability. Significant differences of each condition over the CTRL+ (black bar) are represented by a cross (+) above the corresponding bar (+ = p < 0.05; +++ = p < 0.001). Significant differences between experimental conditions are represented by a squared line with an asterisk (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Figure 5

Mean ± SEM of the relative frequency of different lymphocyte subsets in the immunogenicity assays (one-way modified mixed lymphocyte reaction): (A) CD3+ T lymphocytes, (B) CD8+ cytotoxic T cells, (C) CD4+ helper T cells, (D) CD4+ CD25^high regulatory T cells, (E) CD3−Pan-Ig+ CD21+ B cells. Non-activated PBLs were exposed in vitro to MSC-naïve (light grey bar), MSC-primed (medium grey bar) and MSC-chondro (dark grey bar). Co-cultures of MSCs and PBLs were autologous (n = 3) or allogeneic, matched (n = 8) or mismatched (n = 7) for the MHC. Data from each PBL donor is normalised over the negative control (MLR M−, matched MLR) consisting of responder PBLs from the same donor exposed to MHC-matched stimulator PBLs (value 1), to account for inter-individual variability. Significant differences of each condition over the positive control (MLR mm+, mismatched MLR consisting of responder PBLs exposed to MHC-mismatched stimulator PBLs; black bar) are represented by a cross (+) above the corresponding bar (++ = p < 0.01). Significant differences over the negative control (MLR M−, white bar) are represented by hashes (#) above the corresponding bar (# = p < 0.05; ## = p < 0.01). Significant differences between experimental conditions are represented by a squared line with an asterisk (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Figure 6

Mean ± SEM of the relative proliferation of different lymphocyte subsets in the immunogenicity assays (one-way modified mixed lymphocyte reaction): (A) CD3+ T lymphocytes, (B) CD8+ cytotoxic T cells, (C) CD4+ helper T cells, (D) CD4+ CD25^high regulatory T cells, (E) CD3−Pan-Ig+ CD21+ B cells. Non-activated PBLs were exposed in vitro to MSC-naïve (light grey bar), MSC-primed (medium grey bar) and MSC-chondro (dark grey bar). Co-cultures of MSCs and PBLs were autologous (n = 3) or allogeneic, matched (n = 8) or mismatched (n = 7) for the MHC. Proliferation of each PBL donor is normalised over the proliferation observed in the negative control (MLR M−, matched MLR) consisting of responder PBLs from the same donor exposed to MHC-matched stimulator PBLs (value 1), to account for inter-individual variability. Significant differences over the negative control (MLR M−, white bar) were not observed. Significant differences between experimental conditions are represented by a squared line with an asterisk (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Figure 7

Mean ± SEM concentration (pg/mL) of interferon gamma (IFNɣ) in co-culture supernatants from immunomodulatory (A) and immunogenicity (B) assays. Phytohemagglutinin (PHA)-activated PBLs (A) or non-activated PBLs (B) were exposed in vitro to MSC-naïve (light grey bar), MSC-primed (medium grey bar) and MSC-chondro (dark grey bar). Co-cultures of MSCs and PBLs were autologous (n = 3) or allogeneic, matched (n = 8) or mismatched (n = 7) for the MHC. Significant differences of each condition over the positive controls were not observed. Significant differences over the negative controls are represented by hashes (#) above the corresponding bar (# = p < 0.05; ## = p < 0.01). Significant differences between experimental conditions are represented by squared line with asterisk (* = p < 0.05; *** = p < 0.001).

Figure 1

Study design showing mesenchymal stem cells (MSCs) and peripheral blood lymphocytes (PBLs) donors to establish autologous and allogeneic major histocompatibility complex (MHC)-matched and MHC-mismatched co-cultures, to study immunomodulatory capacity and immunogenic potential. MSCs were assayed unstimulated (MSC-naïve), primed with cytokines (MSC-primed) or chondrogenically differentiated (MSC-chondro).

Figure 2

Mean ± S.E.M (n = 3) of the percentage of MSC before (MSC-naïve) and after being exposed to proinflammatory cytokines (MSC-primed) positive for the surface markers MHC type I and MHC-II determined by flow cytometry. Each colour dot represents the value for each donor (D1, green dot; D2, pink dot; D3, blue dot). These results corroborated the increased expression of both MHC type I and II in MSC-primed, and the inter-individual variability for MHC expression.