Allogeneic major histocompatibility complex-mismatched equine bone marrow-derived mesenchymal stem cells are targeted for death by cytotoxic anti-major histocompatibility complex antibodies

Abstract

Background: Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Controversy exists, however, over whether major histocompatibility complex (MHC)-mismatched MSCs are recognised by the recipient immune system and targeted for death by a cytotoxic antibody response.

Objectives: To determine if cytotoxic anti-MHC antibodies generated in vivo following MHC-mismatched MSC injections are capable of initiating complement-dependent cytotoxicity of MSCs.

Study design: Experimental controlled study.

Methods: Antisera previously collected at Days 0, 7, 14 and 21 post-injection from 4 horses injected with donor MHC-mismatched equine leucocyte antigen (ELA)-A2 haplotype MSCs and one control horse injected with donor MHC-matched ELA-A2 MSCs were utilised in this study. Antisera were incubated with ELA-A2 MSCs before adding complement in microcytotoxicity assays and cell death was analysed via eosin dye exclusion. ELA-A2 peripheral blood leucocytes (PBLs) were used in the assays as a positive control.

Results: Antisera from all 4 horses injected with MHC-mismatched MSCs contained antibodies that caused the death of ELA-A2 haplotype MSCs in the microcytotoxicity assays. In 2 of the 4 horses, antibodies were present as early as Day 7 post-injection. MSC death was consistently equivalent to that of ELA-A2 haplotype PBL death at all time points and antisera dilutions. Antisera from the control horse that was injected with MHC-matched MSCs did not contain cytotoxic ELA-A2 antibodies at any of the time points examined.

Main limitations: This study examined MSC death in vitro only and utilized antisera from a small number of horses.

Conclusions: The cytotoxic antibody response induced in recipient horses following injection with donor MHC-mismatched MSCs is capable of killing donor MSCs in vitro. These results suggest that the use of allogeneic MHC-mismatched MSCs must be cautioned against, not only for potential adverse events, but also for reduced therapeutic efficacy due to targeted MSC death.

Keywords: allogeneic; antibody response; complement-dependent cytotoxicity; horse; major histocompatibility complex-mismatched; mesenchymal stem cell.

Figure 1

Schematic of study design. Antisera obtained in the study by Pezzanite et al.9, was incubated with mesenchymal stem cells (MSCs) and peripheral blood leucocyte (PBL) target cells collected from equine leucocyte antigen (ELA)‐A2 donor horses and complement in microcytotoxicity assays. Eosin dye exclusion was used to estimate percent cytotoxicity of target cells.

Figure 2

10× images from Terasaki plate wells used for microcytotoxicity assays containing equine leucocyte antigen (ELA)‐A2 mesenchymal stem cells (MSCs) or ELA‐A3 MSCs and neat antisera collected on Days 0, 7, 14, or 21 post‐injection with either major histocompatibility complex (MHC)‐matched or MHC‐mismatched MSCs. Live cells appear round with a clear centre. Dead cells appear flat with a dark centre. Cell death was estimated to be <10% for MHC‐matched wells on all days and for MHC‐mismatched wells on Day 0 as shown in this figure. Cell death was estimated to be >80% for all MHC‐mismatched wells on Days 7–21 as shown in this figure.

Figure 3

Cytotoxicity scores of peripheral blood leucocytes (PBLs) and mesenchymal stem cells (MSCs) from the equine leucocyte antigen (ELA)‐A2 microcytotoxicity assays. A standard two‐stage microcytotoxicity assay was used to compare the cytotoxicity of ELA‐A2 haplotype PBLs and MSCs following incubation with antisera. Mean cytotoxicity scores and range for ELA‐A2 haplotype PBLs and MSC target cells following microcytotoxicity assay with n = 4 major histocompatibility complex (MHC)‐mismatched antisera neat a), 1:2 b), and 1:16 c) dilutions. *P<0.05, *^*P<0.01, *^**P<0.001 using repeated measures ANOVA and Holm–Sidak all pairwise multiple comparison procedures. Scores from the microcytotoxicity assay with MHC‐matched antisera are included as reference. Error bars indicate the range of scores above the mean.

Figure 4

Cytotoxicity scores of peripheral blood leucocytes (PBLs) and mesenchymal stem cells (MSCs) from the equine leucocyte antigen (ELA)‐A3 microcytotoxicity assays. A standard two‐stage microcytotoxicity assay was used to compare the cytotoxicity of ELA‐A3 haplotype PBLs and MSCs following incubation with antisera. Mean cytotoxicity scores for ELA‐A3 haploytpe PBLs and MSC target cells following microcytotoxicity assay with n = 1 major histocompatibility complex (MHC)‐mismatched antisera neat a), 1:2 b), and 1:16 c) dilutions. Scores from the microcytotoxicity assay with the same MHC‐mismatched antisera and ELA‐A2 target cells from Figure 3 are included as reference.