MicroRNA manipulation of macrophage polarisation within the Lymphoma microenvironment

Abstract

The histological classification of B cell lymphomas includes a group of high-grade B cell lymphomas (HGBL) characterised by the presence of MYC, BCL2 and/or BCL6 rearrangements (double hit lymphomas). These subtypes have an aggressive clinical course and are poorly served by traditional immuno-chemotherapy with rituximab, cyclophosphamide, prednisolone, vincristine, and doxorubicin (RCHOP), which has led to recent research focusing on improving outcomes for this group of patients. In previous work at Southampton, CD163 positive tumour associated macrophages (TAM), were shown to make up a significant proportion of the tumour microenvironment (TME) in diffuse large B cell lymphoma (DLBCL) and were associated with poor response to immunochemotherapy. Targeting these TAMs to reverse immunosuppression is one potential approach for improving treatment response rates in patients with double hit DLBCL.
One means through which to target TAMs is by modulation of their microRNAs (miRNAs). Therefore, miRNAs differentially expressed in DLBCL and associated with poor response were identified using whole gene correlated network analysis of DLBCL datasets. To explore these, peripheral blood mononuclear cells (PBMCs) were obtained from normal blood donors and differentiated into macrophages following cell culture with MCSF. Macrophages were polarised using immune-suppressive cytokines IL4 plus IL13 or immunostimulating conditions LPS plus IFNγ and miRNA expression measured using RT-qPCR.
There was significant upregulation of miRNA 142-5p in macrophages polarised with IL4 plus IL13 compared to macrophages polarised with LPS plus IFNγ and non-polarised macrophages. To evaluate functional impacts, miRNA 142-5p was inhibited using a miRNA inhibitor, following establishment of a successful transfection protocol. To assess antibody dependent cellular phagocytosis (ADCP), macrophages were generated as above and transfected with miRNA inhibitors prior to co-incubation with target cells. Human Chronic Lymphocytic Leukaemia (CLL) cells from patient samples were used as target cells with Rituximab as the ADCP-inducing monoclonal antibody (mAb) and Herceptin as an isotype control. The percentage of Fc gamma receptor IIIA (FcγRIIIA)-expressing macrophages taking up target cells was determined using flow cytometry. Inhibition of miRNA 142-5p in the immunosuppressive macrophages led to a significant increase in FcγRIIIA, CD40 and CD47 expression resulting in enhanced phagocytosis of CLL cells opsonised with Rituximab plus anti-CD47 mAb when compared with controls.
Finally, to assess potential relevance in primary clinical samples, diagnostic biopsies from DLBCL patients were analysed for miRNA 142-5p and CD206 expression using miRNAScope in-situ hybridisation technology and immunohistochemistry (IHC). Analysis of 38 treatment naïve human DLBCL samples showed CD206 positive TAM infiltration in combination with high microRNA 142-5p expression predicted poor outcome following treatment with Rituximab plus chemotherapy in DLBCL (median PFS 12 months v NR p=0.0096), indicating the addition of a microRNA inhibitor targeting 142-5p to already established therapy may improve survival outcomes in patients with HGBL, whose disease will likely relapse early or be refractory to first line treatment. This could be achieved by conjugation of oligonucleotides to already established mAbs used in the clinic to allow cell specific targeting of miRNA inhibition. If successful, this new class of miRNA drug therapeutics could not only be translated across different cancer types to manipulate macrophage phenotype, but used in other diseases where macrophages are important in mediating the immune response such as multiple sclerosis and rheumatoid arthritis.

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ORCID for Jemma Longley: orcid.org/0000-0002-3829-0686

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