Abstract
Our previous studies using hemophilia A and B mouse models have demonstrated that targeting FVIII or FIX expression to platelets under control of the aIIb promoter through lentivirus-mediated delivery to hematopoietic stem cells (HSCs) results in transgene protein expression and storage in platelet a-granules and that platelet-derived FVIII or FIX not only restores hemostasis but also induces immune tolerance in transduced recipients. In the current studies, we explored how immune tolerance is induced after platelet-specific gene therapy and whether this approach can be applied to induce immune tolerance to a non-coagulant protein. We used ovalbumin (OVA) as a non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the aIIb promoter (2bOVA). Since VWF propeptide (Vp) can reroute secreting proteins to a storage pathway, we designed another vector, 2bVpOVA, which contains Vp to secure OVA storage in platelet granules. We first confirmed that 2bOVA or 2bVpOVA lentiviral gene delivery to HSCs can induce anti-OVA immune tolerance in wild-type (WT) C57BL6 mice. 2bOVA or 2bVpOVA-transduced HSCs (CD45.2/B6) were transplanted into CD45.1/B6 recipients pre-conditioned with 6.6Gy total body irradiation (TBI). We found that 95% and 98% of OVA protein in whole blood was stored in platelets with an OVA protein level of 24.22±8.72 ng/108 platelets (n=10) and 1.41±0.73 ng/108 platelets (n=10) in 2bOVA and 2bVpOVA transduced recipients, respectively. Electronic microscope analysis demonstrated that the OVA transgene protein using both vectors was stored in transduced platelet a-granules. When the transduced recipients were immunized with OVA, anti-OVA antibody titers in both the 2bOVA group (560±68, n=10) and the 2bVpOVA group (320±34, n=10) were significantly lower than in untransduced controls (10424±2837, n=24), demonstrating that platelet-specific OVA gene delivery to HSCs can suppress the anti-OVA immune response. Of note, the titer of anti-OVA total IgG titer in 2bF8 (an unrelated control vector) transduced FVIIInull/B6 recipients without OVA immunization was 413±61 (n=12), which was not significantly different compared to the 2bOVA or 2bVpOVA group even after OVA immunization. In another unrelated control group, 2bGFP, anti-OVA titer was 84±17 (n=9), which was significantly higher than the data obtained from untransduced WT animals without immunization (33±7, n=24). Why there were various levels of anti-OVA antibody titers in unrelated vectors transduced recipients is still unclear and needed further investigation.
To explore how immune suppression is established after platelet-specific gene transfer, we transduced HSCs from OVA-specific TCR transgenic (OTII/CD45.2) mice with 2bOVA, 2bVpOVA, or 2bGFP (a control vector) and transplanted into CD45.1/B6 recipients preconditioned with 6.6Gy TBI. After BM reconstitution, the engraftments among the 3 groups were similar (86.4±2.3%, 86.2±2.2%, and 87.4±2.0%, respectively), but donor-derived CD45.2+ CD4+ T cells in the 2bOVA (0.2±0.1%, n=5) and 2bVPOVA groups (0.9±0.4%, n=6) were consistently significantly lower than in the 2bGFP group (3.1±0.9%, n=6) in peripheral blood during the entire study course. Similarly, donor-derived CD45.2+ CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA and the 2bVpOVA groups compared to the 2bGFP group. However, there were no differences in either percentage or total cell number of CD45.2+ CD4+ T cells in the thymus among the 3 groups, indicating that central tolerance may not play a role in platelet-targeted gene therapy. Notably, the frequency and total number of endogenous CD4 T cells were similar in the 3 groups. Annexin-V staining revealed that the percentage of apoptotic CD45.2+ CD4+ T cells in the 2bOVA and 2bVpOVA groups were significantly higher than in the 2bGFP group in both spleen and lymph nodes, but not in the thymus. The frequency of donor-derived regulatory T cells cells in the 2bOVA and 2bVpOVA groups were significantly higher than in the 2bGFP group in peripheral blood, spleen, and lymph nodes, but not in the thymus. Taken together, our studies demonstrate that platelet-specific gene therapy induces immune tolerance through peripheral antigen-specific CD4+ T cell clone deletion and regulatory T cell induction. Thus, platelet gene therapy can be a promising approach for immune tolerance induction.
Disclosures
Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..
292 The effect of pharmacological zinc on oral Salmonella vaccine efficacy
