The production and delivery of functional perforin (PRF; PRF1 gene) by cytotoxic lymphocytes maintains immune homeostasis and tumour immune surveillance. In humans, co-inheritance of a common PRF1 polymorphism, 272C>T (alanine-91 to valine substitution, A91V) found in 8-9% of the Caucasian population, with another mutated PRF1 allele resulting in reduced PRF function, has been shown to result in hyper-inflammatory diseases and/or haematological cancers.
In this study, we sought to investigate the function of A91V on a wild-type perforin background. To achieve this, we screened and identified a cohort of healthy heterozygous carriers of A91V (PRF1A91V/+) and compared their natural killer (NK) cell cytotoxic function to healthy individuals who had inherited two wild type perforin alleles (PRF1+/+). We found that PRF1A91V/+ NK cells had a statistically significant reduction in cytotoxic function of between 35-50% compared to PRF1+/+ NK cells. To test for allelic expression imbalance between the wild-type and the A91V allele, we developed an assay that distinguished the relative levels of transcript from individual PRF1 alleles. We found that the A91V and WT alleles were expressed at similar levels, ruling out that allelic expression imbalance influenced NK function in PRF1A91V/+ individuals. In general, we found co-dominant transcription of PRF1 alleles, even in PRF1+/+ individuals. We then demonstrated that the A91V mutation results in protein misfolding and is likely the source of reduced NK activity in PRF1A91V/+ individuals. These results were remarkably consistent with in vitro studies showing a severe (>90%) loss of the PRF-A91V function, as well as a 50% reduction of cytotoxic activity in Prf1+/- mouse cytotoxic T-lymphocytes (CTL).
Overall, these studies demonstrate that perforin expression and activity are rate-limiting factors in NK/CTL function. We propose that in humans, homozygous inheritance of A91V may be the most common predisposing factor to immune mediated disease.