HLA-silenced Megakaryocytes evade allogeneic T-cell immune responses

Background

Clinical demand on platelets (PLTs) to treat thrombocytopenic patients is rising. Transfusion of in vitro produced megakaryocytes (MKs) may be used as an alternative to donor PLTs. HLA class I-silenced MKs effectively produced PLTs in the circulation of a refractory mouse model. However, their capacity to upregulate HLA class II may lead to harmful allogeneic immune responses. Hence, we investigated the potential beneficial effect of producing HLA class I and II-silenced MKs.

Methods

HLA class I or HLA- class I and II-silenced iPSCs were generated using lentiviral vectors encoding short-hairpin RNAs (shRNA) targeting β2-microglobulin (shβ2m) and CIITA (shCIITA) respectively. Nontransduced or with a nonspecific shRNA (shNS) encoding vector transduced iPSCs were used as control. Expression levels of β2m, CIITA and HLA class II were evaluated by RT-qPCR. MKs and PLTs were differentiated and phenotyping was performed by flow cytometry. Allogeneic CD4 and CD8 T-cell responses were assessed in CPD-based proliferation assays followed by flow cytometric analysis. Granular T-cell mediated cytotoxicity was measured by quantifying Granzyme B levels by ELISA. MK production was upscaled in an Eppendorf DasBOX Mini bioreactor.

Results

Gene expression levels of β2m and CIITA in HLA class I or class I and II-silenced iPSCs were significantly reduced (p<0.01). HLA-silencing had no effect on their differentiation capability. After incubation of primed T-cells with MKs, both HLA class I or HLA class I and II-silenced MKs led to significantly (p<0.001) lower CD8- and CD4 T-cell proliferation compared to the controls. T-cell granzyme B secretion levels were significantly reduced by 25% (p<0.01) after exposure to HLA class I-silenced MKs. Remarkably, release of Granzyme B-containing granules was even more pronouncedly reduced (52 %, p<0.01) when HLA class I and II-silenced MKs were used. In addition, MKs were successfully produced in a bioreactor.

Conclusion

This study reports the effect of silencing HLA class I and II antigens to prevent allogeneic T-cell-mediated responses towards allogeneic iPSC-derived MKs. Furthermore, the feasibility of upscaling MK production in bioreactors, to meet clinical needs was shown. Use of in vitro manufactured HLA-universal MKs may open new frontiers in the management of highly sensitized thrombocytopenic patients.

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