Temperature sensitivity of human perforin mutants unmasks subtotal loss of cytotoxicity, delayed FHL, and a predisposition to cancer

Abstract

The pore-forming protein perforin is critical for defense against many human pathogens and for preventing a catastrophic collapse of immune homeostasis, manifested in infancy as Type 2 familial hemophagocytic lymphohistiocytosis (FHL). However, no evidence has yet linked defective perforin cytotoxicity with cancer susceptibility in humans. Here, we examined perforin function in every patient reported in the literature who lived to at least 10 years of age without developing FHL despite inheriting mutations in both of their perforin (PRF1) alleles. Our analysis showed that almost 50% of these patients developed at least 1 hematological malignancy in childhood or adolescence. The broad range of pathologies argued strongly against a common environmental or viral cause for the extraordinary cancer incidence. Functionally, what distinguished these patients was their inheritance of PRF1 alleles encoding temperature-sensitive missense mutations. By contrast, truly null missense mutations with no rescue at the permissive temperature were associated with the more common severe presentation with FHL in early infancy. Our study provides the first mechanistic evidence for a link between defective perforin-mediated cytotoxicity and cancer susceptibility in humans and establishes the paradigm that temperature sensitivity of perforin function is a predictor of FHL severity.

Fig. 1.

Patient mutations that adversely affect protein folding, as demonstrated by loss of antigenicity. (A) Mapping PRF mutations on the predicted perforin structure. The position of the mutated perforin residues are mapped onto the structure of C8α with the residues equivalent to hA91, hF193, hP201, and hR356 shown in yellow stick and labeled. Numbering is for human perforin with C8α numbering and amino acid identifier shown in parentheses (details are in Fig. S2). (B–D) PRF-transfected RBL cells were grown for 18–24 h at 37 °C or at permissive temperature of 30 °C before immunofluorescence microscopy or immunoblotting. (B) Immunofluorescence microscopy of wild-type (hWT) and mutant PRF expressed in RBL cells shows the recovery of hF193L and hR356W expression at 30 °C compared with 37 °C as detected by anti-hPRF δG9. Only sorted GFP-expressing cells were assessed for PRF expression. (C) Nonreducing immunoblot using P1–8 demonstrates the loss of antigenicity of hF193L and hR356W mutant PRF as the mutants are essentially undetectable at 37 °C, whereas the signal is recovered at 30 °C. (D) Reducing immunoblot using P1–8 and the same samples as in C demonstrates the recovery of the PRF signal for mutants hF193L and hR356W both at 30 °C and 37 °C.

Fig. 2.

Temperature sensitivity of PRF mutants is a general phenomenon. (A) The activity of PRF mutants hA91V, hF193L, hP201T, and hR356W, which are all clustered to the same subdomain of PRF (Fig. 1A), is recovered at 30 °C (compared with 37 °C as shown in Fig. S1). Shown is the mean relative ⁵¹Cr release ± SE of 3 independent experiments for each mutant and of 14 independent experiments for hWT and control (−ve). (B) PRF mutants hP39H, hG305D, hG317R, and hR410P demonstrate temperature sensitivity as their activity was recovered by culture at 30 °C. Shown is the mean relative ⁵¹Cr release ± SE of 2–3 independent experiments for each mutant and of 14 independent experiments for hWT and control (−ve). (C) PRF mutants hW95R, hH222Q, and hT435M (early onset FHL) show no recoverable function at 30 °C. The y axis scale is increased and hWT not shown for clarity. Shown is the mean relative ⁵¹Cr release ± SE of 2–3 independent experiments for each mutant and of 14 independent experiments for control (−ve). The table below shows Patients 24–28 identified in the literature, who inherited those PRF1 mutations and all had early onset FHL. (D) PRF mutants hV50M, hG149S, and hT450M (either early- or late-onset FHL) recovered low, but significant, levels of activity. The y axis scale is increased and hWT not shown for clarity. Shown is the mean of relative ⁵¹Cr release ± SE of 3–4 independent experiments and of 14 independent experiments for control (−ve). The table below shows Patients 29–41 identified in the literature, who had a variable (early or late) onset of the disease. FS, frame-shift mutations leading to premature stop-codons.

Fig. 3.

The recovery of cytotoxicity by mutant PRF following culture at 30 °C correlates with the onset of FHL or other pathologies. Shown is mean relative cytotoxicity of hWT and various hPRF mutants when expressed in PRF-deficient mouse primary CTLs and grown either at 37 °C or 30 °C for 18–24 h before their use in ⁵¹Cr release cytotoxicity assays, performed at 37 °C at an E/T ratio of 10. The mutants showing minimal recovery of cytotoxicity (<5% of hWT PRF levels) were invariably associated with early onset FHL. Mutants that have been consistently associated with late onset FHL or pathologies other than primary FHL had constitutive activity following culture at 37 °C or increased their cytotoxicity to >30% of hWT PRF levels when cultured at 30 °C. A further group of mutants with intermediate recovery of activity have been associated with either early or late onset FHL.