CTLA-4 division of labour: Soluble isoform regulates immune tolerance

Immunological self-tolerance is a salient feature of the immune system, distinguishing self from non-self and preventing inappropriate immune responses. The cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) is a key regulator of this process, and a successful therapeutic target, but the mechanisms by which it mediates immune tolerance in vivo remain ill-defined.

CTLA-4 has been widely studied as a membrane-bound receptor isoform expressed on activated T-cells, or constitutively on regulatory T-cells, and acts as a negative regulator of immune responses. Whilst CTLA-4 can exert coinhibitory control on autonomous T-cell activation via engagement of negative signalling pathways, it can also, somehow extrinsically regulate distal cells populations.

This currently held perception of CTLA-4 is seemingly paradoxical, in that it is considered both an inhibitory self-regulator of the T-cell that expresses the molecule, but can also function, via an unknown mechanism, to extrinsically inhibit other T-cell populations. This begs the question- how is this receptor solely performing these dual inhibitory roles?

As opposed to the current paradigm, we propose a novel hypothesis that the inhibitory effects of CTLA-4, particularly those associated with cell-extrinsic regulation, are mediated by the alternatively-spliced soluble variant of CTLA-4 (sCTLA-4), and not the membrane-bound CTLA-4 (mCTLA-4) alone. sCTLA-4 is lesser studied aspect of immune tolerance and immunomodulation, primarily due to difficulty in differentiating the individual effects of soluble vs membrane isoforms and a tendency to observe and pursue (often inadvertently) the net effects of blocking/deleting both isoforms. This may have wrongly led to the assumption that CTLA-4 mediated immune tolerance mechanism only operates at the level of membrane-based interactions.

For example, CTLA-4 knockout mice (lacking both isoforms) succumb to a toxic phenotype within 3-4 weeks of birth, but it is not known how selective deletion of either isoform affects immune homeostasis and immune tolerance.

This proposal is based on the development of novel transgenic mouse strains that express either the membrane-bound or the soluble isoform of CTLA-4. Following discussions with PolyGene AG (Switzerland), we have designed two knockout strains using CRISPR-Cas9 technology to selectively knockout either of the CTLA-4 isoform. sCTLA-4 knockout (expressing only the membrane-bound isoform) will be achieved by CRISPR-Cas9 targeting of intronic DNA upstream of exon 3, that encodes the transmembrane domain.

The use of a fused exon 2-3 gene template for homology-directed repair will produce a copy of Ctla4 gene lacking endogenous splice sites required for alternative splicing and sCTLA-4 production. To generate mCTLA-4 knockout mice (expressing only the soluble isoform), CRISPR-Cas9 targeting of introns up- and downstream of exon 3 will enable excision of DNA encoding the transmembrane domain through non-homologous end-joining, eliminating inclusion of the transmembrane domain, only enabling expression of the soluble isoform.

Such strategy will be used to engineer C57Bl/6 embryonic stem cells which represent the ideal immunocompetent platform for the proposed work, in addition to being amenable to the generation of future crosses. We believe this to be the most appropriate system in which to address our aim of defining the regulatory roles for each CTLA-4 isoform.