Background
Disease recurrence is a major complication after allogeneic hematopoietic stem cell transplantation in AML. Inducing graft-versus-leukemia (GVL) effect can control recurrence but often aggravates graft-versus-host disease (GVHD). Previous studies showed high IL-15 expression in AML cells relates to GVL, but the mechanism remains unclear. We aimed to develop an AML cell-targeted IL-15 delivery system to induce GVL without exacerbating GVHD.
Methods
We constructed PEG-PLGA-COOH nanoparticles encapsulating human recombinant IL-15 protein (IL15NP) and conjugated anti-CD33 antibodies to their surface (CD33-IL15NP) using EDC-NHS coupling. Nanoparticle uptake, IL-15 expression, and HLA-ABC changes in AML cell lines (OCI-AML3, MOM-13, HL60, MV4-11) were assessed by laser scanning confocal microscopy and flow cytometry. Proteomic and transcriptomic sequencing identified key genes and pathways involved in MHC class I upregulation. We created ISG15 knockdown cell lines using lentivirus and monitored proteasome subunit activities with different substrates. Specific proteasome subunit inhibitors (PR924, ML604440) were used to confirm regulatory roles. A humanized GVL NSG mouse model evaluated the nanoparticles' therapeutic potential in vivo, using 2Gy irradiation and transfusion of healthy donor peripheral blood stem cells with OCI-AML3 cells (NC or ISG15sh).
Results
IL15NP uptake by leukemia cells was concentration and time-dependent. IL-15 expression peaked at 12 hours post-administration, normalizing at 24 hours. AML cell lines showed significant HLA-ABC upregulation after 24-72h IL15NP co-culture, unlike with extracellular IL-15 protein. Proteomics revealed a strong correlation between MHC class I and ISG15, a ubiquitin-like protein linked to the ubiquitin-proteasome system. HLA-ABC upregulation was reversed in ISG15 knockdown cells across four AML cell lines. Transcriptomics showed upregulation of innate immune pathways (mTOR signaling, C-type lectin receptor signaling) related to ISG15 upregulation triggered by targeted IL-15 delivery.
Proteasome activity assays indicated significantly enhanced immunoproteasome activity (β1i, β5i) regulated by ISG15. Only combined use of β5i inhibitor (PR924) and β1i inhibitor (ML604440) reversed IL15NP-induced HLA-ABC upregulation. High concentrations of proteasome inhibitors showed strong anti-tumor effects but significantly downregulated MHC I molecules, suggesting excessive proteasome inhibition may lead to immune evasion due to MHC I loss.
In vivo imaging 21 days post-transplantation showed that for NSG mice with NC-OCI-AML3, systemic tumor progression in the IL15pro group (IL-15 protein-given) was comparable to the NS group (saline-given). IL15NP and CD33-IL15NP groups showed significantly reduced leukemia cells compared to NS and IL15pro groups. However, the IL15NP group had inferior survival due to severe GVHD. For NSG mice with ISG15sh-OCI-AML3, the IL15NP group showed significant tumor reduction compared to NS and CD33-IL15NP groups, but with severe systemic GVHD. Tumor progression in the CD33-IL15NP group was similar to NS despite targeted nanoparticle administration under situation of ISG15 knockdown.
Conclusion
Targeted IL-15 delivery induces ISG15 expression through innate immune pathway activation, enhancing immunoproteasome activity and upregulating MHC I molecules on AML cells. This strategy increases leukemia cell surface IL-15 and HLA-ABC expression, triggering GVL without severely increasing GVHD. Our study provides a novel approach for post-transplantation relapse treatment and establishes a foundation for clinical translation, suggesting that despite their malignant origin, leukemia cells retain innate immunity qualities.
No relevant conflicts of interest to declare.
This feature is available to Subscribers Only
Sign In or Create an Account Close Modal