Abstract
Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the 'eat me' engulfment signal for professional phagocytes. ERp57 is an interacting and co-translocating protein with calreticulin, but unlike surface calreticulin, surface ERp57 is not known to be immunogenic. Since calreticulin interacts with α-integrins via the conserved GFFKR motif within the integrin cytoplasmic domain, we assessed whether integrin function can modulate cell surface calreticulin and ERp57 levels in ICD. When stimulated to engage integrin substrates, leukemic T-lymphoblasts treated with an ICD-inducer exhibited decreased surface calreticulin and ERp57 compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin and ERp57 was recapitulated for cells in suspension and treated with various agents that stimulate integrin activation, including Mn2+ and the LIBS antibodies 9EG7 and HUTS21. Similarly, cells expressing a mutant truncated α-integrin bearing only GFFKR as the cytosolic tail, also exhibited low surface calreticulin and ERp57 when treated with ICD-inducers under non-adherent conditions. Furthermore, integrin β1 null cells with overall reduced α-integrin expression exhibited enhanced surface calreticulin and ERp57, consistent with an inhibitory role for integrins in ICD. We generated calreticulin or ERp57 null strains by CRISPR-Cas9, and show that ICD-induced surface calreticulin is ERp57-dependent, while surface ERp57 is not calreticulin-dependent. Using permeabilization techniques that distinguish between cytosolic and ER staining, we found that ICD-inducers promoted the accumulation of cytosolic calreticulin and ERp57 that originated from the ER. In similar fashion, ER to cytosolic trafficking for calreticulin is ERp57-dependent, while for ERp57, it is not calreticulin-dependent. We also showed that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation coupled with normal ER-cytosolic release, suggesting that activated integrins acts to sequester calreticulin within the cytosol. T-lymphoblasts co-treated with an ICD-inducer and integrin activators exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD-inducer. Our study reveals a previously uncharacterized function of integrins as negative regulators of immunogenic cell death by suppressing the presentation of cell surface calreticulin.
Disclosures
No relevant conflicts of interest to declare.
Integrins and ERp57 Coordinate to Regulate Cell Surface Calreticulin in Immunogenic Cell Death
