In ~30% of cases, the severe immunodeficiency of HLH has been attributed to mutations in the PFN1
). Although mutations that cause a frame-shift or premature termination would be expected to abrogate perforin function, others result in single amino acid substitutions whose effect on perforin function is unknown. The appreciation that perforin mutations can result in immune dysfunction offers a unique opportunity to characterize perforin structure–function relationships. To date, however, such studies have been greatly hindered by a lack of appropriate methodologies for expressing perforin in cells capable of granule-mediated cytotoxicity. Perforin's capacity to disrupt lipid membranes means that few cell lines can accurately process and store perforin in secretory vesicles and then release it in a regulated fashion after conjugate formation. No appropriate human cell lines exist to attempt such an analysis. However, standard transfection methodologies in RBL cells have shown previously that perforin expression confers the capacity to lyse erythrocytes (16
), whereas coexpression of perforin and either granzyme A or B was necessary to effectively kill nucleated cells (17
). Despite this, perforin expression was not sufficiently stable in RBL cells to permit ongoing studies. To address these issues, we revisited perforin expression in RBL cells but applied efficient expression and cell sorting technologies to compare the function of WT and mutated perforin. For our analysis, we chose to study HLH Patient #5, a compound heterozygote with perforin missense mutations R225W and G429E (11
). The R225W mutation has also been identified in several unrelated individuals (13
). We had shown previously that overexpressing human perforin in rodent CTLs could disrupt the processing of endogenous perforin, suggesting that human and rodent perforin may be processed in subtly different ways (22
). Therefore, we introduced mutations equivalent to those of P5 into mouse perforin and compared the function of these molecules with WT mouse perforin.
We initially devised a rapid method for transient transfection of RBL cells, using the vector pIRES2-EGFP. 1 d after electroporation, fluorescent cells were sorted and immediately used in a 51Cr release assay with Jurkat target cells to which they were conjugated. The efficiency of electroporation was as high as 40%, and up to 106 GFP-expressing cells were obtained per electroporation. Although G429 is conserved in human, mouse, and rat perforin, R225 is not invariant and corresponds to T224 in mouse perforin. To confirm the functional equivalence of arginine and threonine at this position, we generated RBL cells expressing T224R mouse perforin and found they were as efficient in the 51Cr release assay as WT perforin-transfected cells. However, expressing perforin with tryptophan at the same position (T224W) resulted in complete loss of cytolytic function (). As expected, the WT protein had an apparent molecular mass of ~67 kD; however, the introduction of tryptophan resulted in the appearance of truncated (~45 kD) perforin (), suggesting the mutation facilitated proteolytic cleavage/ processing of perforin. Furthermore, immunohistochemistry analysis of transfected cells indicated mislocalisation of T224W, possibly due to a loss of putative signaling motif(s). Whereas WT perforin produced a punctate appearance consistent with packaging in secretory granules, T224W perforin produced diffuse staining throughout the RBL cell cytoplasm ( A).
Figure 1. Reduced cytotoxic activity and truncation of T224W mouse perforin expressed in RBL cells. Perforin-dependent 51Cr release from TNP-labeled Jurkat cells coincubated with transiently transfected, sorted RBL cells for 4 h in the presence of anti-TNP IgE. (more ...)
Figure 2. T224W and G428E perforin localize differently in RBL cells. (A) Immunohistochemistry of perforin-expressing RBL cells demonstrated with antiperforin antibody PI-8 and counterstained with eosin. (B) RBL cells either unlabeled or labeled with α-TNP–IgE (more ...)
When we similarly analyzed the effect of the G428E (G429E in humans) mutation co-inherited by Patient #5 (11
), we observed a reduced level of 51
Cr release compared with RBL cells expressing WT perforin (unpublished data). To accurately quantify this reduced activity, we produced cell lines that stably expressed WT and G428E perforin. Retrovirus-transduced RBL cells were analyzed on a flow cytometer, and the most highly fluorescent cells (0.2–5% of the total population) were sorted and expanded in culture resulting in >93% GFP-positive cells some days later. These cells expressed perforin at levels equivalent to IL-18/IL-21–activated mouse primary NK cells ( A). Perforin expression and cytotoxic function remained stable over many weeks of continuous culture (unpublished data). Consistent with our transient transfection experiments, RBL cells expressing WT perforin were efficient in lysing Jurkat target cells across a broad range of E:T ratios ( B). To determine the difference in cytolytic activity between WT and G428E perforin, the E:T ratios required to produce equivalent levels of 51
Cr release were compared. We found that RBL cells expressing similar levels of G428E were three to four times less efficient at inducing chromium release ( B).
Figure 3. Reduced cytotoxic activity but normal apparent molecular mass of G428E mouse perforin expressed in RBL cells. (A) Western blot showing perforin expression in stably transduced RBL cells compared with IL18/IL-21–activated mouse NK cells and empty (more ...)
We went on to investigate the reason for the reduced cytotoxicity of G428E perforin. As demonstrated by immunoblotting ( B), this was not due to protein cleavage or degradation. To rule out incorrect trafficking to secretory granules, we examined the intracellular localization of WT and G482E perforin in stably transduced RBL cells. Finding normal quantities of mutated perforin in the granules would further exclude a significant defect in gene transcription, mRNA stability or translation, or protein folding. When lysates of RBL cells expressing WT or G428E perforin were fractionated on a Percoll gradient and analyzed by Western blot, perforin was consistently localized in the fractions containing maximal β-hexosamidase activity, a marker of the lysosome-like secretory granules (21
) ( C). The correct targeting of perforin was also confirmed through immunohistochemical staining, as both WT and G428E perforin demonstrated indistinguishable punctate cytoplasmic staining ( A). G428E perforin was also released by exocytosis as efficiently as WT perforin upon RBL Fc
receptor cross-linking ( B).
Since G428E perforin was expressed at equivalent levels to WT perforin and correctly targeted to, and released from, granules ( and ), the mutation was likely to affect a postsynaptic function of perforin. To test this possibility, we generated and purified recombinant WT and G428E perforin using a baculovirus expression system and tested their ability to bind to sheep RBC membranes in a calcium-dependent manner. Whereas WT perforin displayed strong calcium-dependent plasma membrane binding with essentially all the added perforin bound, the binding of G428E perforin was markedly reduced (). Consistent with this observation, the cytolytic activity of the recombinant G428E mutant was ~5% of that of WT perforin (unpublished data). Although RBL cells have been used as a read-out of perforin function for many years, a perceived weakness of the model is that perforin exerts its cytolytic effects in the absence of granzyme B. Exposure of target cells to recombinant G428E-perforin with granzyme B did not rescue the perforin phenotype (unpublished data). Therefore, our findings strongly suggested that the diminished activity of G428E-perforin was due to diminished target cell membrane binding, rather than the absence of granzymes. Based on modeling studies and biochemical analysis of CTL clones, Uellner and colleagues found that perforin's COOH terminus strongly resembles that of calcium-dependent membrane-binding C2 family of protein domains, some of which are involved in vesicular trafficking at neuronal synapses (23
). Importantly, G428 is located in the C2 domain of perforin, immediately adjacent to one of five highly conserved aspartate residues believed to be essential for calcium binding (23
). We postulate that the G428E substitution may interfere with calcium binding to the C2 domain, leading to decreased affinity of perforin for lipids in the target cell membrane (23
). The apparent quantitative differences in G428E-perforin–induced cytolysis in the RBL-based and recombinant models probably reflects differences in the kinetics of perforin–target cell membrane interaction in the context of a cell conjugate (RBL model) and in solution (purified recombinant perforin).
Figure 4. The G428E mutation significantly reduces calcium-dependent membrane binding of soluble perforin. Equal quantities of recombinant WT and mutant perforin were tested for their capacity to bind to sheep erythrocytes in the absence (−) or presence (more ...)
This is the first study to successfully define the functional basis of naturally occurring perforin mutations that when co-inherited, lead to the catastrophic immunosuppression seen in HLH. Surprisingly, we demonstrated that partial loss of perforin function may be sufficient to bring about fatal disease. Whereas the T224W mutation (corresponding to R225W in humans) resulted in protein instability and complete loss of RBL cytotoxic function, G428E (G429E in humans) was only partially inactivating as RBL cells retained ~25–30% of WT lytic activity. It is not clear whether this substitution would lead to disease when inherited in the homozygous state; however, when combined with the completely inactive R225W allele, the overall cytolytic activity of perforin appears to be insufficient to rescue the patient. Based on the result of our RBL assays, one could predict that CTL expressing equal quantities of T224W- and G428E-perforin would have some residual but markedly reduced cytotoxic activity. In fact, the NK cells of Patient #5 did exhibit ~15% lytic activity of control samples (11
). The concordance of our data with the clinical findings in this case provides evidence that our experimental approaches should provide a robust basis for understanding other perforin mutations identified in HLH.