Our previous studies have demonstrated that platelet-specific gene transfer under control of the αIIb promoter (2b) results in effective immune tolerance induction to FVIII and FIX as well as the non-coagulant protein ovalbumin (OVA) in the unprimed model. Here we explored how the reactivity of immune responses affects the efficacy of transgene expression and the potential immune tolerance mechanisms in platelet-specific gene transfer in the primed model.

We used OVA as a surrogate protein for this study. Recipient wild-type (WT) CD45.1 mice were primed with OVA protein. Platelet-OVA expression was introduced by 2bOVA lentivirus transduction of Sca-1+ cells from either WT/CD45.2 or OTII/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy irradiation. We found that pre-existing high-reactive immune responses devastate platelet-OVA expression in recipients in both the WT/WT and OTII/WT models. Using the WT/WT mouse model, 6 of 7 2bOVA-transduced recipients had sustained platelet-OVA expression with levels ranging from 4.53-8.84 ng/108 platelets. Anti-OVA total IgG titers declined with time in 2bOVA-transduced OVA-primed recipients even when rechallenged with OVA. In contrast, recall memory immune responses were elicited in 2bGFP and untransduced transplanted controls. Furthermore, full-thickness tail skin grafts from CAG-OVATg mice were successfully engrafted onto 2bOVA-transduced recipients and survived throughout the remaining lifetimes of the animals. In contrast, skin grafts were completely rejected in control 2bGFP-transduced and untransduced transplanted recipients. To ensure that the immune system was not inactive in 2bOVA-transduced recipients, animals were immunized with unrelated antigen recombinant human FVIII (rhF8) using a protocol known to induce anti-F8 immune responses even in WT mice. All 2bOVA-transduced recipients developed anti-F8 inhibitory antibodies after rhF8 immunization with no differences between the 2bOVA group and the 2bGFP and untransduced control groups.

Using the OVA-specific CD4 TCR transgenic model (OTII/WT), we showed that sustained platelet-OVA expression was achieved in 90% of 2bOVA-transduced recipients. OVA-specific CD4 T cells were deleted and Treg cells were expanded in peripheral lymphoid organs in 2bOVA-transduced OVA-primed recipients. The levels of platelet-OVA expression negatively correlates with the percentages of OVA-specific CD4 T cells, but positively correlates with Treg cells. Immune tolerance was also achieved in the OTII/WT model even though all donor-derived CD4 T cells are OVA-specific. However, our studies showed that high-reactive immune responses could influence the neoprotein expression in platelet-targeted gene therapy. Using the OVA-specific CD8 TCR transgenic model (OTI/WT), in which all donor-derived CD8 T cells are OVA-specific, we found that low levels of platelet-OVA expression were obtained at week 5 after transplantation (0.72±0.65 ng/108 platelets) and then dropped to barely detectable at subsequent time points. Flow cytometry analysis showed that there were 33.9±12.9% platelets positive with GFP in 2bGFP LV-transduced recipients, demonstrating that high transduction efficiency and successful transplantation were achieved. Of note, OVA-specific CD8 T cells in the 2bOVA group were 48% lower than in the 2bGFP control group, demonstrating that antigen-specific CD8 T cells were partially deleted in the OTI/WT model after platelet-targeted OVA gene transfer. Interestingly, we found that the percentage of the antigen-specific CD8+Foxp3+ cells in the 2bOVA group was significantly higher than in the 2bGFP group (0.19±0.14% vs. 0.05±0.01%, respectively). These results suggest that there is a counterpace process between immune reaction and immune tolerance after platelet-targeted gene therapy.

In conclusion, our studies demonstrate that platelet-targeted gene therapy can induce antigen-specific immune tolerance even when the immune response has been mounted via peripheral clonal deletion of antigen-specific CD4 and CD8 T cells and expansion of Tregs, suggesting that platelet-targeted gene therapy is a promising approach to induce immune tolerance for treatment of diseases with undesired immune responses or even with pre-existing immune responses, such as hemophilia A with inhibitors or autoimmune diseases.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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