Credit: Simon Bradbrook/NPG

Therapeutic targeting of inhibitory receptors on T cells can boost immune responses to tumours and improve patient survival but, for reasons that remain unclear, such responses are only seen in a subset of individuals. Studies to identify the processes that govern immunosuppression of tumours have typically been conducted in vitro and have therefore not addressed the complex interaction among tumour cells, immune cells and the tumour microenvironment. Now, a study published in Nature has outlined an approach for the in vivo discovery of immunotherapy targets in melanoma using RNA interference technology, which could pave the way to modification of immune responses in diverse cancer subtypes.

In the current study, Zhou et al. focused on the response of cytotoxic (CD8+) T cells to B16 melanoma, an aggressive tumour, in mice. The researchers constructed two thematic libraries of short hairpin RNA (shRNA) molecules — one targeting genes associated with T cell anergy or exhaustion, the other aimed at genes encoding kinases and phosphatases. CD8+ T cells, each infected with an shRNA from these libraries, were injected into tumour-bearing mice. The T cells were engineered to express the receptor OT-1, and tumour cells expressed the cognate antigen ovalbumin, ensuring homing of the T cells to the tumour target site.

The rationale behind the approach was that shRNA molecules that were complementary to mRNA transcripts of upregulated, immunosuppressive genes in T cells would block the translation of these genes and, by definition, become enriched in tumours by enabling proliferation of these derepressed T cells. Seven days after injection, T cells were purified from tumour tissue as well as from the spleen (to act as a control tissue). Deep sequencing of the shRNAs was then performed to identify the upregulated genes responsible for T cell suppression in the tumour microenvironment.

the screen highlighted Ppp2r2d ... as being upregulated in T cells from tumours

Zhou et al. found 43 genes that were enriched in T cells from the tumour tissue, including several that were known to be inhibitors of T cell function, suggesting that the approach was valid. In addition to established targets, the screen highlighted Ppp2r2d (protein phosphatase 2 regulatory subunit B delta) as being upregulated in T cells from tumours. This gene encodes a regulatory subunit of the family of PP2A phosphatases that discourages T cell mitosis and regulates apoptosis. Biochemical assays revealed that shRNA-mediated knockdown of Ppp2r2d was associated with increased T cell proliferation and enhanced cytokine production by T cells.

Importantly, when the researchers injected melanoma-targeted T cells containing Ppp2r2d-specific shRNA into tumour-bearing mice, they observed significantly enhanced antitumour immunity in the form of increased melanoma cell death, reduced tumour burden and prolonged survival compared with mice treated with T cells containing lacZ control shRNA. The approach was also effective when Ppp2r2d knockdown was performed in CD4+ T cells, which suggests that broad immunomodulatory effects can be achieved by targeting this gene.

Together, the findings of this study not only highlight the potential therapeutic value of targeting PPP2R2D in T cells to treat melanoma but also provide a valuable approach to identify new targets for cancer immunotherapy.