Allo-antibody production after intraarticular administration of mesenchymal stem cells (MSCs) in an equine osteoarthritis model: effect of repeated administration, MSC inflammatory stimulation, and equine leukocyte antigen (ELA) compatibility

Abstract

Background: Antibody production after allogeneic administration of mesenchymal stem cells (MSCs) could impact their clinical application. Proinflammatory priming of MSCs can potentiate their regulatory ability in vivo but increased expression of major histocompatibility complex (MHC) might augment their immunogenicity, potentially leading to immune memory thus limiting repeated allogeneic administration. This study aimed at evaluating the production of cytotoxic allo-antibodies directed against donor's ELA (equine leukocyte antigen) in mismatched and halfmatched horses receiving repeated intraarticular administration of stimulated MSCs (MSC-primed) and unstimulated MSCs (MSC-naïve) in pathologic joints.

Methods: From available stored samples from a previous in vivo study, cells from one donor and serially collected sera (five time-points) from three groups of recipients were used based on their ELA haplotypes to perform microcytotoxicity assays: Group 1 recipients mismatched with the donor that received MSC-naïve (naïve-mismatched recipients); Group 2 recipients mismatched with the donor that received MSC-primed (primed-mismatched recipients); Group 3 recipients halfmatched with the donor (sharing 1/2 haplotypes) that received MSC-primed (primed-halfmatched recipients). Sera from recipients (neat, 1:2 and 1:16 dilution) were tested against target cells from the donor (cryopreserved and expanded MSC-naïve and MSC-primed) or from one animal presenting the same ELA haplotypes than the donor (fresh peripheral blood lymphocytes as control).

Results: One to three weeks after first MSC administration, all recipient groups produced allo-antibodies regardless of MSC received (naïve or primed) and matching degree with donor. However, secondary response after MSC re-exposure was less evident in halfmatched recipients (MSC-primed) than in mismatched ones (both MSC-naïve and MSC-primed). Recipients of MSC-primed (both mismatched and halfmatched) tended towards developing lower antibody response than MSC-naïve recipients in vivo, but MSC-primed were targeted to death in higher percentage in vitro in the microcytoxicity assay.

Conclusions: After first intraarticular allogeneic administration, the immunomodulatory profile of MSC-primed would have led to lower antibody production, but these antibodies would target more easily MSC-primed after second injection (re-exposure), likely because of their higher MHC expression.

Keywords: Allogeneic; Horse; Humoral response; Immunogenicity; Joint; MSC priming; Major histocompatibility complex (MHC).

Fig. 1

Schematic representation of the study design. From all the animals of the previous study, one donor (black), four recipients of MSC-naïve (all mismatched, dark gray), and six recipients of MSC-primed (three halfmatched, black; three mismatched, dark gray) were selected to assess humoral response against allogeneic mesenchymal stem cells (MSCs) based on their equine leukocyte antigen (ELA) haplotypes. Peripheral blood lymphocytes (PBLs), unstimulated MSCs (MSC-naïve), and MSCs pre-stimulated with tumor necrosis factor alpha and interferon gamma (MSC-primed) of the same ELA haplotype than the donor were used as target cells. Sera collected from the selected recipients at different time-points (T0, pre-administration of corresponding MSCs; T1, 1 week after first MSC administration; T2, 3 weeks after first MSC administration—just before the second MSC administration; T3, 1 week after second MSC administration; T4, 90 days after second MSC administration) were tested neat, 1:2 and 1:16 diluted against all the three types of target cells using two-stage microcytotoxicity assays

Fig. 2

Evolution of cytotoxic scores along the time in each recipient group comparing different types of target cells. Mean ± S.E.M. of cytotoxic scores (Y axis) assigned to neat sera (top row; a, b, c), 1:2 diluted sera (middle row; d, e, f), and 1:16 diluted sera (bottom row; g, h, i) from mismatched recipients of MSC-naïve (left column; a, d, g) and MSC-primed, halfmatched (middle column; b, e, h) and mismatched (right column; c, f, i), along the time (X axis; T0, pre-administration of corresponding MSCs; T1, 1 week after first MSC administration; T2, 3 weeks after first MSC administration—just before the second MSC administration; T3, 1 week after second MSC administration; T4, 90 days after second MSC administration), when assayed against different target cells: PBLs, peripheral blood lymphocytes (white bar, control); MSC-naïve, unstimulated mesenchymal stem cells (light gray bar); MSC-primed, mesenchymal stem cells pre-stimulated with tumor necrosis factor alpha and interferon gamma (dark gray bar). Asterisks (*) point out statistically significant differences among time-points (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) and hashes (#) indicate significant differences between different target cells at one particular time (# = p < 0.05, ## = p < 0.01)