Diverse effect of the splice isoforms of immune receptor SLAMF6 – a new regulatory mechanism

Introduction: SLAM family of receptors are highly conserved receptors of hematopoietic cells. Most of the family members are homotypic-binders, with the exception of SLAMF4. Family member SLAMF6 is abundantly expressed on CD8 T cells and thus of interest for its role in tumor rejection.

Recently we isolated and evaluated two isoforms of the human SLAMF6 gene: The `canonical` sequence, composed of 8 exons, and SLAMF6^Δ17-65 -missing part of exon-2, which constitutes the interacting V domain of the receptor. In this work we set to evaluate the immunological modulatory effect of the long and the short isoforms of SLAMF6.

Materials/Methods: SLAMF6 splice isoforms were identified by RT-PCR in T-lymphocytes.To characterize their net-effect, we aberrantly expressed each isoform in melanoma and co-cultured with cognate TILs (Tumor infiltrating lymphocytes). In parallel, selective expression of SLAMF6^Δ17-65 short isoform in lymphocytes was achieved by knocking-out the canonical SLAMF6 in Jurkat cells using CRISPR-Cas9 genome editing. For successive total isoform knockout, siRNA against the short isoform was electroporated in these SLAMF6-KO/SLAMF6^Δ17-65+ Jurkat cells. Finally, to shift the splicing, antisense-oligonucleotides were transfected into WT Jurkat cells using electroporation.

Results: Using RT-PCR we showed that all SLAMF6 isoforms are constitutively apparent on T-cells, regardless of their activation or differentiation state. However, a difference in the level of isoforms transcripts was found in CD8+ subsets in healthy donors. Specifically, SLAMF6 canonical isoform has relatively low expression in Naïve and Central-Memory cells, but much higher on Effector and Effector-Memory CD8+ T cells.

Melanoma cells aberrantly expressing canonical SALMF6 had inhibitory effect on cognate TILs, while the opposite was observed with SLAMF6^Δ17-65, melanoma cells expressing this isoform enhanced IFN-γ production by cognate TILs.

In SLAMF6-KO/SLAMF6^Δ17-65+ Jurkat cells, the exclusive expression of the short isoform was associated with a dramatic increase in IL-2 secretion. Knock-down of SLAMF6^Δ17-65 using siRNA abolished this effect.

Recently, using antisense-oligonucleotide we managed to shift the splicing in Jurkat cells, and increase the short isoform on the account of the canonical, the effect of altered splicing pattern will be discussed.

Conclusion: SLAMF6 undergoing alternative splicing, a difference in the level of isoforms transcripts was found in CD8+ T cell subsets. We showed that the short and the long isoforms has strong but opposing impact on T cell activation – the short isoform is an activator while the long is inhibitory. Therefore – a shift of the splicing towards the short isoform can be a possible immunotherapeutic cancer treatment.