Only 2% of the human genome is protein coding. The rest of the 98% of it is noncoding. In order to better understand the complexity which prevails in gene regulation, cell differentiation and proliferation, we must also focus on the noncoding part of our genome, both in health and and in disease.
The noncoding RNAs, previously considered part of the junk material, has now taken a central stage in gene regulation. The principal aim of this proposal is to identify how Epstein-Barr virus, the ubiquitous herpesvirus contributes to lymphoma development by altering microRNAs which affect immune checkpoints like PD-L1 and ICOSL. We have identified that EBNA2, a virally encoded nuclear protein can increase PD-L1 by downregulating the PD-L1 targeting miR-34a. As an economical viable and faster alternative to murine models, we will test 3D microfluidic chip based assays to assess immunogenicity of EBV infected lymphomas. In the same 3D microfluidic models, we will investigate if combined delivery of immune checkpoint inhibitor antibodies and anti PD-L1 miRNA such as miR-34a reconstitutes the reduced immunogenicity of lymphoma cells. The 3D microfluidic system will be used to cocultivate the tumor cells reconstituted with immune checkpoint inhibitors and miRNAs with activated T lymphocytes and as the readout of activated T cell responses, IFN-g and caspase 3 expression will be monitored. It is out hope that we will be able to establish a reliable and fast approach to investigate the potential of RNA aided cancer immunotherapy in the 3D microfluidic chip based system.
Innovative aspects of the project:
EBV infected lymphomas with latency III (all nine virally encoded latency proteins present), are high PD-L1 expressors, but it is not known how EBV affects PD-L1 and other IC proteins. To this end, I will screen a vast number of in vitro established cell lines and identify EBV genes responsible for IC alteration. Such screening is undertaken for the first time which permits us to use isogenic cell lines with and without the virus to identify differentially expressed IC proteins.
Our preliminary data suggest that EBV infected U2932 DLBCL EBVGFP cl. B, which expresses all EBV latency proteins, is a high PD-L1 expressor. Instead, EBVGFP cl-A, which lacks EBNA2, has no PD-L1 induction. My host supervisor has one of the largest collection of EBV infected BLs, DLBCLs and PELs in Italy. These lines will be tested for PD-L1, CD80/86 and ICOSL expression by immunoblotting and flow cytometry. Additionally, the non-absolute association of the virus with lymphoma has helped our group to generate isogenic BL, DLBCL and PEL cell lines with and without the virus. This will permit us to understand the broader effect of EBV on IC proteins, across various types of B cell lymphomas. Immunoblotting and flow cytometry will be employed here to test IC protein expression in a recombinant EBV infected DLBCLs, BLs and PELs. The lack of PD-L1 in some cell lines with customary anti-PD-L1 antibodies could be due to recently described PD-L1 structural variants We will perform exome sequencing analysis in such cell lines to identify PD-L1 or other IC variants both in translated and un-translated regions.