Alternative Splicing Of The Receptor SLAMF6 Reveals a Novel Regulatory Mechanism Of T Cell Activation And Can Be Used For Cancer Immunotherapy

Introduction: SLAMF6 is a homotypic receptor abundantly expressed on CD8+ T-lymphocytes and thus of interest for its role in anti-tumor response. Recently we evaluated two isoforms of the SLAMF6 gene: The `canonical` sequence, and SLAMF6^Δ17-65 ,missing part of exon-2. In this work we set to evaluate the immune modulatory role of the long and the short isoforms of SLAMF6 and test their effect on anti-tumor immunity.

Materials: SLAMF6 splice isoforms were identified by RT-PCR and in RNA-seq databases from human donors and lymphoid cell lines. Melanoma lines aberrantly expressing each isoform were produced to generate a model of trans-activation of T cells via SLAMF6. In parallel, selective expression of SLAMF6^Δ17-65 in lymphocytes was achieved in Jurkat cells using CRISPR-Cas9 genome editing. To identify transcript levels of SLAMF6 isoforms in both healthy individuals and cancer patients receiving PD1-inhibitors, samples were collected from patients before and during anti-PD1 therapy, cells were sorted to CD8+ subsets and qPCR was performed. Finally, to shift the splicing, antisense-oligonucleotides (ASO) were transfected into Jurkat cells using electroporation.

Results: Using existing databases and our-own human-derived T cell samples, we showed that all SLAMF6 isoforms are constitutively apparent on T-cells. However, different isoform ratios were found in CD8+ subsets, determined by their differentiation states. In melanoma patients receiving PD-1 inhibitory antibody, a transition was noted in isoforms ratio, favoring a rise in the shorter isoform, which was most emphasized in patients with auto-immune side effects (n=7).

Melanoma cells aberrantly expressing canonical SALMF6 had distinct inhibitory effect on cognate TILs. However, to our surprise the opposite was observed with SLAMF6^Δ17-65. Melanoma expressing this isoform enhanced IFN-γ production by cognate TILs significantly and reproducibly.
In line with this, the exclusive expression of the short isoform in Jurkat cells was associated with a three-fold increase in IL-2 secretion.

Lastly, using ASO designed to target SLAMF6 splice junctions, we managed to shift the splicing in Jurkat cells, increasing the short isoform on the account of the canonical isoform. As an outcome, cells expressing higher levels of SLAMF6^Δ17-6 had a significantly improved function.

Conclusion: We showed that SLAMF6^Δ17-65 isoform has a strong agonistic effect on T cell activation while its canonical isoform is an inhibitor. The change in isoform ratio observed during anti-PD-1 therapy may suggest a new regulatory mechanism that T cells adopt along their activation. The agonistic effect achieved by splice-diverting ASO may be exploited in the future for cancer immunotherapy.